DISTANCE EDUCATION SELF LEARNING MATERIAL PROGRAMME
: M.Sc. Chemistry
YEAR
:
FINAL
PAPER
:
VB
TITLE OF PAPER
: ANALYTICAL CHEMISTRY
MADHYA PRADESH BHOJ (OPEN) UNIVERSITY BHOPAL (M.P.)
Course Name -M.Sc. Chemistry
PAPER-
DISTANCE EDUCATION SELF LEARNING MATERIAL
MADHYA PRADESH BHOJ (OPEN) UNIVERSITY BHOPAL (M.P.) FIRST EDITION
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2013
UNIVERSITY
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M. P. Bhoj (Open) University,Bhopal .
PROGRAMME
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M.Sc. Chemistry
TITLE OF PAPER -
ANALYTICAL CHEMISTRY
BLOCK .1 UNIT WRITER-
(1) (Name) (Address)
DR. ANJU SAXENA, DIRECTOR SUNDER DEEP GROUPE OF ENGINEERING COLLEGE , DASANA, GHAZIABAD
EDITOR-
(2) (Name)
DR.VINAY PRABHA SHARMA,LECTURER
(Address)
DEPARTMENT OF CHEMISTRY, MEERUT COLLEGE, MEERUT
(Name)
DR.MAHESH SRIVASTAVA,D.Sc.
(Address)
DEPARTMENT OF CHEMISTRY, MEERUT COLLEGE, MEERUT 250001
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M. P. Bhoj (Open) University, Bhopal.
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ALL RIGHTS RESERVED No part of this publication may be reproduced in any form, by mimeograph or any other means, without permission in writing from M.P. Bhoj (Open) University. The views expressed in this SLM are that of the author (s) & not that of the MPBOU. The cost of preparation and printing of Self-Learning Materials is met out of DEC grant. Further information on the MPBOU courses may be obtained from the University’s office at Raja BhojMarg, Kolar Road, Bhopal (M.P.) 462016 Publisher: Registrar, M.P. Bhoj (Open) ( Open) University, Bhopal (M.P.) Phone: 0755-2492093 Website: www.bhojvirtualuniversity.com.
Course Name - M.Sc. Chemistry (FINAL) DISTANCE EDUCATION SELF LEARNING MATERIAL
BLOCK 1:--
VB-ANALYTICAL CHEMISTRY
UNIT I UNIT II UNIT III UNIT I V UNIT V
(TITLE) INTRODUCTION (TITLE) ERRORS AND EVALUATION (TITLE) FOOD ANALYSIS (TITLE) ANALYSIS OF WATER POLLUTION (TITLE)ANALYSIS OF SOIL FUEL ,BODY FLUIDS AND DRUGS
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Paper (Number) Title VB Block (Number) – 1 (1 TO 5)
BlockIntroduction BlockIntroduction
ANALYTICAL CHEMISTRY
2012 UNIVERSITY
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Madhya Pradesh Bhoj (Open) University Bhopal ( M.P)
PROGRAMME
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M.Sc. Chemistry (Final)
PAPER
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V-B
TITLE OF PAPER
-
Analytical Chemistry
BLOCK NO.
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1
UNIT WRITER
UNIT-I, II, III & IV
–
PROF.(Dr.) Anju Saxena
Director Sunderdeep Group of Engineering College, (Dasna), Ghaziabad. UNIT-V
-
Dr. Vinay Prabha Sharma
Lecturer in Chemistry Meerut College, Meerut EDITOR
-
Dr. Mahesh Srivastava M.Sc.,Ph.D., D.Sc.
Department of Chemistry Meerut College, Meerut-250001
BLOCK-I UNIT-I ANALYTICAL CHEMISTRY
Role of analytical chemistry. Classification of analytical methodsclassical and instrumental. Types of instrumental analysis. Selecting an analytical method. Neatness and cleanliness. Laboratory operations and practices. Analytical balance. Techniques of weighing, errors. Volumetric glassware cleaning and calibration of glassware. Sample preparations dissolution and decompositions. Gravimetric techniques Selecting and handling of reagents. Laboratory notebooks. Safety in the analytical laboratory.
UNIT-I ANALYTICAL CHEMISTRY
1.0 Introduction 1.1 Ojectives 1.2 Analytical Chemistry and its Role. 1.3
Classification
of
Analytical
Methods
Instrumental Methods.
1.3.1 Classical Methods. 1.3.2 Instrumental Methods. 1.3.3 Advantages of Instrumental Instrumental Methods. 1.4
Types of Instrumental Analysis.
1.4.1 Volumetric Analysis. 1.4.2 Gravimetric Analysis. 1.4.3 Optical Methods. 1.4.4 Separation Methods. 1.4.5 Electrical Methods. 1.5 Selecting an analytical Methods. 1.6 Methods & Cleanliness. 1.7 Laboratroy Note Books. 1.8
Safety in the Analytical Laboratory.
1.9 Laboratory operations & practices.
1.9.1 Filtration.
-
Classical
and
1.9.2 Drying 1.9.3 Measuring Volume 1.9.4
Graphs.
1.9.5
Concentration.
1.9.6
Percentage Solute.
1.9.7
Activity.
1.9.8
Standards.
1.9.9
Sampling.
1.9.10 Drying 1.9.11 Weighting 1.9.12 Precipitation 1.10 Analytical Balance .
1.10.1
Technique of weighing.
1.10.2
Weighing Errors.
1.10.3
Electronic Balance.
1.11 Cleaning and Calibration of Glassware. Glassware.
1.11.1
Leaning of Glassware.
1.11.2
Calibration of Glassware. Glassware.
1.12 Selecting and Handling of Reagents. 1.13 Sample Preparations. Preparations.
1.13.1 Dissolution and Decomposition. Decomposition. 1.13.1 (a) Decomposing samples with Inorganic acid acid in open vessels.
(b) Microware Decomposition. Decompositio n. (c) Combustion our an open flams dry ashing. (d) Procedure of fusion.
UNIT-I ANALYTICAL CHEMISTRY 1.0 Introduction Chemistry could be divided into five main areas analytical, biochemical inorganic, organic and physical. The analytical chemistry it is necessary for growth and development of science and technology and it must be integrated with other scientific and chemical disciplines .
1.1 Objectives Analytical chemistry chemistry may be defined as the science and art of determining determining the composition composition of materials in terms of of the elements or compounds compounds contained contained in them. Analytical chemistry chemistry is the science of chemical identification identification and determination of the the composition of substances and materials their chemical structure, analytical aim can be achieved, analytical methods can be divided into identification or detection methods, methods for measuring the content of an element in a sample and methods of determining the molecules composition of materials.
1.2 Analytical chemistry and its Role Analytical chemistry is concerned with the chemical characterization of the matter. It plays a imp. role in nearly all aspects of chemistry, for ex.-clinical, agricultural,
environmental,
forensic,
manufacturing,
metallurgical,
and
pharmaceutical concerns.Food must be analysed for contaminants (eg: pesticide residence) and for essential (eg: vitamin) contents.
The quality of manufactured products often depends upon proper chemical proportions and measurement of the constituents is a necessary part of quality control. The above description of analytical chemistry provides an overview of the discipline of analytical chemistry. An analytical analytical chemist tries to serve the needs of many fields. fields.
(1)
In medicine, analytical chemistry is the basis of clinical laboratory laboratory tests which help physicians to diagnose diseases and chart progress in recovery.
(2)
In industry, analytical chemistry provides the means of testing raw materials and for assuring the quality of finished product whose chemical composition is critical.
(3)
Environmental quality
is often
evaluated evaluated
by by
testing testing
for suspected
contaminants contaminants using the techniques of analytical chemistry. (4)
The nutritional value of food is determined by chemical analysis for major components and trace components such as vitamins and minerals.
(5)
An analytical chemist also makes imp. contribution to fields as diverse as forensics, archaeology and space science.
1.3 Classification of Analytical Methods - Classical and Instrumental Methods The goal of a chemical analysis is to provide information about the composition of a sample of matter. Modern analytical chemistry has at its disposal a multitude of the most diverse methods and techniques of analysis for conducting quantitative determination. Their classification is based on different principles, but most frequently they are divided into two large classes (i) classical methods (ii) Instrumental methods.
1.3.1 Classical Methods These methods generally involved measurement of mass of a substance or the volume of resulting solution.
These are also called chemical methods and according to quantity measured, are classified as gravimetric and volumetric methods.
(i)
Gravimetric Chemical Methods These methods depend on the conversion of the substance concerned to a
reaction, product and an accurate determination of its mass.
(ii)
Volumetric or Titrimetric Analysis
It is related to the measurement of the volume of reagent of exactly known concentration used up in the titration.
In this method, the equivalence point, the moment when the amount of standard solution added is equivalent to that of the substance being determined, should be detected correctly.
Volumetric analysis include a wide variety of the types of chemical reactions such as neutralization reaction, oxidation - reduction, precipitation reaction and complex formation reactions.
A direct titration is a more frequent technique where an unknown solution is titrated directly with a standard solution and the desired constituent is determined by measuring the volume of the standard solution required to react completely with the constituent.
Back titration is used when no indicator is suitable for a titration or the
reaction is slow and does not involve a sharp change in concentration at the equivalence point. In these titration two standard solutions are employed.
Indirect method of titration are used when the substance to be determined
does not react directly with standardised solution or reacts with it in a nonequivalent amount. In this method, the substance under titration, with the help of auxillary reagent, is converted into an another compound, which is titrated with the standard solution. eg. in the titration of K 2Cr2O7 with Na2S2O3, the K 2Cr2O7 is first titrated with excess KI, as a result of which I 2 evolved in an amount equivalent to the K 2Cr2O7 content, is titrated with Na2S2O3 standard solution using starch as an indictor. Cr 2O72 6 I 14 H 3 I 2 6S 2O22
3
2Cr
2
3S 4O6
1.3.2 Instrumental Methods
6I
3 I 2 7 H 2O
The methods which involved the measurement of a physical parameter of the system which is functionally related to the amount of the component being determined are called instrument methods.
(i)
These further can be (i)
Physicochemical Methods
(ii)
Physical Methods
Physicochemical Methods
These methods are concerned with the measurement of certain physical parameters of a chemical system that are dependable on the nature of the system components and vary in the process of the reaction. The parameters include: (a) Potential value in potentiometry, and (b) optical densities of coloured complexes in spectrophotometry spectrophotometry etc.
Physicochemical methods may be direct or indirect, depending on the procedure of the determination. determination. In direct methods, a substance is determined directly by measuring some property of a system. In indirect methods, a change in a property is used for detecting the end point of a chemical reaction, i.e., it serves as a peculiar sensitive indicator.
The classification of physical and physicochemical physicochemical methods generally depends upon the character of the measured properties of the system. These methods are generally classified into two broad groups : spectral (optical) and electrochemical methods.
The optical methods are based on the relation between optical properties of a system and its composition. The electrochemical methods are based on the interdependence of electrochemical properties properties and composition of the system. In addition to these groups, radiometric, mass spectral number is fast growing day by day.
(ii)
Physical Methods
These methods do not use chemical reactions. The composition of a substance is determined by measuring characteristic physical properties of material such as density, viscosity, surface tension of solution etc. or its components. Spectral analysis is one of the most widely used physical methods of analysis.
Physical properties are of two types, namely, specific and non-specific. A direct measurement is however, possible if the measured property is specific
for the substance being measured. If no specific property is available, the measurement of two or more non specific properties may allow correction for interfering substances. 1.3.3 Advantages of Instrumental Methods
These methods may be used by the analytical chemist to save time, to avoid chemical separations or to obtain increased accuracy. The time saving features can be realized in routine analysis, or where a considerable number of determinations are to be made. The accuracy of some of the instrumental methods depends upon the accuracy with which the classical or wet chemical analysis can be made. In other words, we can say that an improvement in the classical methods of analysis will mean further improvement in the accuracy of instrumental methods of analysis. Table-1 Properties and Methods based on them Physical property measured
Analytical
methods
based
on
measurement of property
1. Mass
Gravimetric.
2. Volume
Volumetric.
3.
Electrical
potential,
electrical Conductometry,
conductance,
electrical
current, voltammetry,
quantity of electricity etc.
potentiometry chronopotentiometry,
polarography,
amperometry
and
coulometry,
(electrometric
or
electroanalytical electroanalytical methods). 4. Absorption Absorption of radiation
Spectrophotometry Spectrophotometry Visible,
IR,
absorption, resonance
(X-ray, (X-r ay,
colorimetry, nuclear and
UV, atomic
magnetic
electron
spin
resonance). 5. Emission of radiation
Emission
spectroscopy, spectroscopy,
photometery,
flame
fluorescence,
radiochemical methods 6. Scattering of radiation
Turbidimetry, Turbidimetry, nephelometry. Raman
Specroscopy 7. Refraction of radiation
Refractometry, Refractometry, interferometry
8. Rotationof Rotationof radiation
Polarimetry, interferometry
9. Diffraction of radiation
X-ray electron diffraction methods
10. Mass to chare ratio
Mass spectrometry
11. Thermal properties
Thermal conductivity and enthalpy methods
1.4
Types of Instrumental Analysis
Following techniques are used in analytical chemistry 1.
Volumetric analysis
2.
Gravimetric analysis
3.
Optical methods
4.
Separation methods
5.
Electrical methods
1.4.1 Volumetric Analysis
In these methods measurements are made in volume. In this techniques a gas volume is measured or a volume of a titrant is measured. The procedures in which volume of a titrant is measured is called a titration. In a titration, a solution of known concentration is prepared as one of the reactants and it is known as the titrant. It is then titrated against the sample solution or unknown solution using a suitable indicator for detecting the end point at which the stoichimetric reaction between the titrant and the sample is complete. From the volume of the sample solution needed for a known volume of titrant of known concentration (standard solution), the amount of the reactant in the sample can be calculated. Volumetric titration are of four types and each type depends type of reaction.
For example, acid base titration involve a neutralization reaction. Thus acids are determined by titration or neutralization with a standard base solution and based by titration with a standard acid solution. For example,
H+ + Cl- + Na+ + OH+ +
Na+ + Cl- + H2O
In oxidation reduction titration , a change in oxidation state of the
substances and the titrant is involved. For example, 6Fe2+ + Cr2O2-7 + 14H+ 6Fe3+ + 2Cr3+ + 7H2O
In precipitation titrations , a stoichiometric amount of titrant as-
precipitating precipitating agent is added and volume required for the complete reaction is measured. The amount of desired constituent is then calculated because the precipitating precipitating agent is used as standard solution. Ag+ + NO-3 + H+ + Cl- AgCl +H+ + NO-3
In complexometric titrations, a complex is formed by adding the titrant (a
complexing agent) to the sample solution. For example, titration of Cu (II) with EDTA. HO2CCH2 Cu2+ +
CH2CO2H –N N - CH2 - CH2 –
Na+O-2CCH2
CH2CO-2Na+ Cu EDTA + 2Na+ + 2H+
Ethylenediaminetetraacetic Ethylenediaminetetraacetic acid disodium salt (EDTA)
It should be noted that all complexing agents can be used as titrants in complexometric titrations. 1.4.2 Gravimetric Analysis
It involves the separation of a substance from the solution of the weighed sample into a pure weighable form of a stable compound of known percentage composition. This analysis may be carried out by precipitation electrodeposition and volatilization.
Gravimetric analysis by precipitation is the chemical analysis in which the constituents of the substances in solution are determined by the measurement of weight of the corresponding precipitate. The various operations in obtaining a pure sample by precipitation are: (1)
Precipitation Precipitation of the desired constituent
(2)
Filtration
(3)
Drying
(4)
Weight of the precipitation
In electrodeposition, the desired constituent is deposited or isolated at an
electrode by the passage of an electric current. The weight of the desired constituent is then calculated by the difference in weight of the electrode before and after the Electrodeposition. Electrodeposition.
In volatilization , the sample is decomposed by a known stoichiometric
reaction in which one of the products is volatilized. The amount of vapourized constituent is then calculated by the difference in weight before and after volatilization. In this respect the process is similar to the electrodeposition method. 1.4.3 Optical Methods
Optical methods are based on how the sample acts towards electromagnetic radiation. The most important optical properties which can be co-related with concentration are: (1)
The absorption or emission of radiant energy.
(2)
The bending of radiant energy.
(3)
The scattering of radiant energy.
(4)
The delayed emission of radiant energy.
These measurements are made making use of instruments which involve the use of lenses, mirrors, prisms and gratings. There are some other instruments which are included in this general classification but do not have optical parts and still depend on electromagnetic electromagnetic radiation. The most important techniques are nuclear magnetic resonance, electron spin resonance and mass spectrometry.
1.4.4 Separation Methods
Separations are necessary to simplify the sample by removing the interferences prior to the final measurement. However, separation is not only associated with interference removal. Qualitative or Quantitative separation of components of a mixture is useful for purification or needed for concentration one or
all the components. Separation methods are used in many industrial processes of isolating metals, organic compounds and other metals. 1.4.5 Electrical Methods
Electrical methods involve electronic instruments that are used to measure or produce electrical phenomena. Current flow as a function of time, potential developed or required, ability to pass a current and resistance, etc., are the important properties which are related to the reaction taking place or/are causing a reaction to take place. The fundamental measurements then are resistance, current, potential and time. 1.5
Selecting an Analytical Method
It is an imp. task for the analyst to select the best procedure for a given determination. This will require careful consideration of the following features :(1)
The type of analysis required : - elemental or moleculal, routine or occasional.
(2)
Problems arising from the nature of the material to be investigated eg: radioactive substances, corrosive substances, substances affected by water.
(3)
The concn. range which needs to be investigated.
(4)
The accuracy required.
(5)
The facilities available.
(6)
The time required to complete the analysis, this will be particularly relevant when the analytical results are required quickly for the control of a manufacturing process. This may mean that accuracy has to be a secondary rather than a primary consideration as it may require the use of expensive instrumentation.
(7)
The number of analyses of similar type which have to be performed.
(8)
Nature of the specimen.
(9)
Magnitude of the sample available.
(10)
Kind of information sought.
1.6
Methods & Cleanliness
For an analyst it is very imp. to be aware of the procedures, handling of instruments and basic techniques of analytical operations.
(1)
The bench must be kept clean and a bench-cloth must be available so that any spillage of solid or liquid chemicals can be removed immediately.
(2)
The container vessels or bottles must be labeled, so that the contents can be readily identified.
(3)
To prevent contamination of the contents by dust, air and moisture, the the vessels should be covered immediately after use.
(4)
Bark corks should not be used to cover the vessels because they invariably tend to shed some dust.
(5)
Reagent bottles must never be allowed to accumulate on the bench, bench, they must be placed on the reagent shelves immediately immediately after use.
(6)
All determinations should be performed in duplicate.
(7)
The graph paper a printout obtained from a printer of modern instrument, should be attached to the observation page of the laboratory record book.
(8)
It should be regarded as normal practice to perform a rough rough calculation to confirm the right order of printed results.
(9)
Safety procedure must be observed in the laboratory at all times.
(10)
Poisonous chemicals must be handed very carefully.
(11)
All laboratory workers workers should familiarize themselves themselves with local safety safety requirements.
(12)
In some laboratories laboratories the wearing wearing of safety spectacles and gloves gloves must be compulsory.
1.7
Laboratory Note Books
The laboratory note book is a record of the work of an analytical chemist. It is the source for reports, publications and regulatory submission the note book is a record of original ideas. Same good rules are given below for a well-maintained note book. (1)
A hard hard covered note book book of A-4 size must be used for for recording experimental observations observations as they are made.
(2)
Never use loose leafs.
(3)
Number the pages consecutively.
(4)
Always records only in ink.
(5)
Never tear out pages. If page is not used, put a line through the page.
(6)
Each page should be dated and signed properly.
(7)
Name of the project should be written on the people space.
(8)
All the data should be recorded on the same day, when they are obtained.
(9)
In a note book, a double page should be devoted to each determination, determination, title of which must be clearly indicated.
(10)
The record must conclude conclude with the calculation calculation of the result of the analysis and in this connection the equations & reactions involved in the determination should be shown together.
(11)
Finally, appropriate appropriate comments should should be made made upon the degree of the accuracy and the precision achieved.
(12)
The printout printout from the printer printer should be permanently permanently attached to the observations observations page of the laboratory note book.
(13)
It should should be regarded as a normal practice practice to to perform a "rough" "rough" calculation calculation to confirm that the printed result is of the right order.
1.8
Safety in the Analytical Laboratory
The General safety rules in the analytical laboratory laboratory are given below:(1)
In the laboratory always clean up split chemicals.
(2)
Broken on chipped glassware should not be left on the bench a shelves.
(3)
Chemical bottles and apparatus should be placed properly after use.
(4)
One should neutralize the acid spills with sodium bicarbonate bicarbonate and alkali spills with boric Acid.
(5)
Mercury spills should be vacuumed up with a section flask or dusted with sulphur powder.
(6)
Analyst should clean up the mercury thoroughly becok mercury vapours from five droplets are highly toxic.
(7)
If required, one should wear protective glasses, while working n the the laboratory.
(8)
A person working in a chemical laboratory should locate fire extinguishers, exits, safety showers, eye foundations and fire blankets.
(9)
Any dangerous or potentially dangerous laboratory situation should be brought immediately to the notice of the laboratory supervisor.
(10)
One should should perform only the the authorized authorized experiments, experiments, and should not work work alone in the laboratory. laboratory.
(11)
When working working with with volatile volatile chemicals, as when heating acids or when using organic solvents, use the fume hood.
(12)
One should use a safety shield, shield, when working with potentially potentially dangerous reactants.
(13)
Special care should be taken taken when working with organic solvents.
(14)
Many chemicals are inflammable inflammable and many many have been identified identified as as acute acute or chronic toxic substances frequently carcinogenic. Use rubber gloves when possible and avoid breathing in fumes.
1.9
Laboratory Operations & Practices
For a quantitative analysis there are many laboratory operations and techniques. These are described as follows. 1.9.1 Filtration
Filtration is the separating process of solid phase from the liquid phase which is the in its contact solution. The filtering process use diff. types of filter support to collect the solid such as:(A)
Filter Papers :- Munktels (Swedish filter paper), Schull (German)
(B)
Crucibles :- Gooch crucibles, Aluminum Gucibles, Silica Gucible.
(c)
Filter pulp Mats
(d)
Asbestos
(e)
1.9.2
Filter Membranes
Drying
For drying purpose desicator is used. It allows to cool the hot crucible in a dry atmosphere, before desicator provides a dry and moisture free atmosphere for storing a sample.
1.9.3
Measuring Volume
In analytical chemistry solutions are measured by graduated glass wares such as volumetric flaks, pipette, burette etc. People handling and use of glass wares in volumetric analysis needs an accurate reading of the liquid a solution level. 1.9.4
Graphs
In analytical chemistry, the experimental observations are analysed and measured by calibration and titration curves. 1.9.5
Concentration
In stoichionetry reactions, conc n is generally defined in terms of (i)
Molarity M
(ii)
Volune of soluti solution on (in lt )
Formality F
(iii)
Moles Moles
Gram formula formula weight Litre Litre of Solution
Normality N
1.9.6 Percentage Solute
Gram equivalent weight Litre Litre of Solution
The percentage of solute may be defined as percentage by weight or percentage by volume. When percentage percentage solute is expressed as
percentage by by
weight, it means the parts of the total solution weight that is participated by the solute.
1.9.7 Activity
Ideal concentration like normality molarity, or formality, corresponds to what was originally added and true or effective concn. which makes into account the various interaction, is called the activity. a = c 1.9.8
Standards
For all types of determent of a standard or references point is necessary. Standard
Primary Standard
Secondary Standard
In chemistry, a substances where purity has been analysed may be primary standard substances. 1.9.9 Sampling
Sampling is an imp. operation to analyse a material or substance. It is a difficult task to get a proper and homogeneous sample. A lot of time and efforts will be involved in the analysis of an improper sample. Sampling is of two types. (a)
Statistical Sampling
(b)
Random Sampling
1.9.10
Drying
After getting getting the sample, it is imp. to know whether sample sample was to be be used as such, or it has to be dried. Some samples are hygroscopic in nature a may contain moisture. Hence there are 2 types : (a)
Received Basis
(b)
Dried Basis
1.9.11 Weighing
Samples are weighted after the drying process. Generally are analytical balance is used for employed in triplicate. 1.9.12 Precipitation
If the main goal is quantitative precipitation, the whole process is known as gravimetry. In this procedure the measurement is weight or weight change. (a)
The sample being analysed is weighed accurately.
(b)
The pH should be adjusted by using buffer solution.
(c)
Precipitation is carried out in hot dil. solution.
(d)
The ppt is then separated form the mother liquor by filtration.
1.10 Analytical Balance
Weighing is an integral part of almost any analysis, both for measuring the sample and for preparing standard solutions. Analytical balance gives the accurate measurement of mass i.e., it compares an unknown mass to a known mass under the same gravitational force. A chemical analyst really deals with mass rather than weight. For most analytical purposes a balance with a maximum load of 100-200g and an ability to weigh an object to 0.1 is required. 1.10.1
Technique of Weighing
There are two general techniques to weigh a sample.
In first technique, the entire sample, placed in a weighing tube is weighted, subsequently the weighing tube is taken out from the balance pan and sample is transferred to another container with the help of a spatula. The empty weighing tube is again weighted and the weighted of the sample is determined by the difference between the two. This technique is known as weighing by difference. This procedure can be repeated for additional sample.
In an another technique, the sample is directly weighed on the pan of the balance and collected on a weighed paper or watch glass. Once the sample is
weighed, it is transferred to another vessel. (It is not advisable to keep the removing sample until a desired weight is transferred) 1.10.2
Weighing Errors
While using an analytical balance, there are five main sources of errors. These are : (a)
Static Errors
These can be imparted to the balance from the operator or from the objects placed on the pan. Semimicro and microbalances of equal arm type are particularly susceptible to this type of error. (b)
Defective Balance
Errors due to balance construction or operation of the weights are possible. These errors are difficult to discover in single pan balance, where the working parts are enclosed. Defects may also occur because of corrosion, chipped knife edges, dust and magnetic damping errors. (c)
Temperature Effects
These may be caused by temperature gradient in the balance case, because convection currents will be present due to temperature gradient. Small temperature gradient may cause draft against the pan and lead to erroneous weights, while large temperature gradient may cause the beam lengths to change because of expansion and thus lead to an error. Hence hot objects should never be weighted and balance should be protected from sources of heat or draft.
(d)
Operative Errors
Occur as a result of carelessness and improper handling of the balance and they can easily be corrected and controlled. For example. (1)
Spillage of chemicals may lead to etching of the the pan or other parts of the the balance.
(2)
Sudden jarring or adding or removing weights or the sample from the pan can damage the knife edges in the balance.
(3)
Misreading the weight scale, spilling of weighted samples, poor handling of the object during weighing are also operative errors.
These kinds of errors may permanently affect the accuracy as well as sensitivity of the balance. In addition, volatile contaminents may also cause errors. For example:- H 2O or CO2 may be absorbed by the objects from the atmosphere during weighing. As a result, object will change weight during weighing. Even containers holding the sample is capable of this action. Operative error may be minimized by placing the balance in a low humidity, controlled temperature room or in a dry box. Sometimes a small amount of descant is placed in the balance. It is also desirable to dry the sample in an oven before weighing at a temperature that is high enough to remove water, but not high enough to cause sample decomposition. Before weighing the sample and its container are cooled to room-temperature room-temperature in desiccator. (e)
Buoyancy Effects
Buoyancy effects arise when an object is placed on the balance pan, the net downward forces on the pan is due to the mass of the object minus the force due to the buoyancy of air on the object. At balance. m0 = mw + (Buoyancyo - Buoyancy w) 1.10.3
Electronic Balance
Modern electronic balances offer convenience in weighing and are subject to fewer errors, a mechanical failure, than the mechanical balances. (1)
There are no weight or knife edges as in mechanical balances.
(2)
In this, pan sits on the arm of a movable hanger & this movable system is compensated by a constant, electromagnetic force.
(3)
The position of the hanger is monitored by an electrical position scanner which brings the weighing system back to the zero position.
(4)
The compensation current is proportional proportional to the weight placed on the pan which appears as a digital display.
(5)
These balances use the principle of electromagnetic electromagnetic force compensation.
(6)
The balance is "zeroed" or calibrated, with a known weight.
(7)
A single control bar is used to switch the balance balance on and and off, to to set the the display to zero, and to trace a container automatically on the pan.
(8)
Electrochemical quartz balances are available with 100 g range that can detect ng (10-9g) changes.
1.11
Cleaning and Calibration of Glassware
1.11.1 Cleaning of Glassware
Glassware must be perfectly clean & free from grease, other wise the result will be inreliable. Various methods methods are available available for cleaning of glassware. (1)
Decon 90
Many commercial detergents are available which are suitable for this purpose. eg: Decon '90' effective in moving contamination due to radioactive materials. (2)
Teepol
Teepol is relatively mild and inexpensive detergent which may be used for cleaning glassware.
(3)
CARE
A method which is frequently used, consists of filling up the apparatus with "Chromic Acid Cleaning Mixture (CARE)", a nearly saturated solution of powered sodium dichromate (or potassium dichromate) in conc n sulphuric acid, and allowed to stand for overnight. The acid is then poured off, the apparatus thoroughly rinsed with distilled water and allowed to drain until dry. (4)
Mixture of Sulphuric Acid Nitric Acid
This may be used when the vessel is very greasy & dirty. 1.11.2 Calibration of Glassware
The calibration procedure involves determination of the weight of water contained or delivered by the particulars piece of apparatus. The temp. of water is observed, and from the known density of water at that temp, the volume of water can be calculated. In all calibration operations, apparatus to be calibrated must be carefully cleaned and allowed to stand adjacent to the balance which is to be used together
with the supply of distilled deionized water, so that they acquire the temp. of the room. Graduated Flask
In the clean dry flask a small filter funnel is inserted into the neck and distill water is added slowly. The funnel is then removed and then using a dropping tube later is added dropwise until the meniscus stands on the graduation mark. Now the flask is weighted and the temperature of water is noted.
Pipette
The pipette is filled with distilled water above the mark. Remove the excess e xcess of water the pipette is then allowed to discharge into a clean weighted stopped flask. The receiving flask is weighted & the temp. of the water is noted. Burette
The calibration of a burette is similar to the procedure for a pipette, except that several volumes will be delivered. To calibrate a burette, first make satisfaction about its (i) leakage and (b) delivery time. 1.12
Selecting and Handling of Reagents
The purest reagents should be used for quantitative analysis, the analytical reagent (AR) quality is generally employed. While handling the reagents following points should be kept in mind. (1)
Liquid reagents should be poured from the bottle, a pipette should never be inserted into the reagent bottle.
(2)
Particular care should be taken to avoid contamination contamination of the stopper of the reagent bottle. When a liquid is poured from a bottle, the stopper should never be placed on the shelf or on the working bench.
(3)
All reagent bottles should be kept scrupulously clean particularly ground of the neck on mouth of the bottle.
(4)
It there is any doubt about the purity of the reagents used, they should be be tested by standard methods for the impurities, which right the cause of errors in the determination. determination.
(5)
Reagents which are used in analytical laboratory should be of analytical analytical reagent grade.
(6)
Special grades to solvents for special purpose should be used e.g., spectral grades or chromatographic grades.
(7)
Whenever possible, pick the smallest bottle that will supply the desired quantity.
(8)
Unless specifically directed, never return any excess reagent to a bottle.
(9)
Unless directed otherwise, never insert spatulas, spoons or knives into a bottle that contains a solid chemical.
(10)
Keep the the reagent shelf and the laboratory laboratory balance clean & neat clean up spills immediately, even though some are waiting to use the same chemical or reagent.
(11)
Observe local local regulations regulations concerning the disposal disposal of surplus reagent reagent and solutions.
1.13
SAMPLE PREPARATIONS
1.13.1
Dissolution and Decomposition
Most of the analytical determinations are performed in aqueous solutions of the analytic. It is very difficult to select a people reagent and technique for decomposing and dissolving an analytical sample particularly when refractory substance is involved or the sample is present in trace quantity. There are four types of common methods of decomposing & dissolving an analytical sample. (1)
Heating with aqueous strong acids or bases in open vessel.
(2)
Microwave heating with acids.
(3)
High temp ignition in air or oxygen.
(4)
Fusion in molten salt media.
1.13.2(a) Decomposing Samples with Inorganic Acids in Open Vessels
The mineral acids are generally used to decompose the inorganic samples in open vessel. Usually, a suspension of the inorganic sample is prepared in the mineral acid and then it is heated by flame or a hot plate until the dissolution is confirmed to be complete by the total disappearance of a solid phase. In this process the temp of decomposition is adjusted at the boiling point of the used acid reagents. The following acids can be used for this purpose (a)
HCl
(b) HNO3
(c)
H2SO4 (d)
(e)
Oxidising mixture of acids (f)
(b)
Microwave Decomposition
Perchloric Acid
Hydrofluoric acid
Microwave decomposition can be carried out in both closed vessels because higher pressure and temperature can be achieved in them. The decomposition of compounds in microwave oven is very fast and speedy, even in case of typical and difficult samples it takes only. 5 to 10 minutes. In contrast, the same results require several hours when sample is heated over a flame or a hot plate. (c)
Combustion over an Open Flame (Dry Ashing)
To determine the cations of an organic sample, it has to be heated till red hot in an open dish or crucible once a flame so that all the carbonaceous matter gets oxidized and converted into volatile components follows dissolution of the residual solid. In addition, voltaic metallic compounds may be lost during the ignition process. Although dry ashing is the simplest method of decomposing organic compounds, it is often the least reliable. (d)
Procedure of Fusion
(1)
Firstly, the sample is prepared by grinding and fine power is obtained.
(2)
Mixing of flux and sample is carried out in a crucible.
(3)
After fusion, the formation of a clear melt indicates the completion of decomposition.
(4)
The mass is allowed to cool, before solidification.
QUESTIONS
1. What is analytical chemistry ? Classify the analytical methods and describe the factors affecting the choice and selection of an analytical. 2.
What are the important points to remember before cleaning an analytical laboratory?
3.
How will you differentiate between: (a)
Classical and instrumental method
(b)
Electronic balance and single-pan balance
(c)
Parts per thousand and parts per million
(d)
Stoichiometric Stoichiometric and non-stoichiometric non-stoichiometric
(e)
Normality and Formality
(f)
Molarity and Normality
(g)
Wet ashing and dry ashing
4.
Write a short essay on laboratory operations techniques.
5.
Write short notes on the following: (a) (b)
Laboratory Laboratory note book Analytical balance balance
(c)
Semi-micro and micro balance
(d)
Cleanliness and neatness in laboratory
(e)
Selecting and handling of reagents
BLOCK-I UNIT-II ERRORS AND EVALUATION
Definition of terms in mean and median. Precision standard deviation, deviation, relative standard deviation. Accuracy-absolute error, relative error. Types of error in experimental data-determinate (systematic), indeterminate (or random) and gross. Sources of errors and the effects upon the analytical results. Methods for reporting analytical data. Statistical evaluation of data-indeterminate errors. The use of statistics.
UNIT-II ERRORS AND EVALUATION 2.0 Introduction. 2.1 objectives. 2.2 Mean. 2.3 Median. 2.4 Precision.
2.4.1 Standard Deviation. 2.4.2 Variance. 2.4.3 Co-Efficient of Variation. 2.5 Accuracy.
2.5.1 Absolute Error. 2.5.2 Relative Errors. 2.6
Types of Errors.
2.6.1 Determinate Errors. 2.6.2 Indeterminate or random Errors. 2.6.3 Gross errors. 2.7 Reporting of Analytical Data. 2.8 Statistical Evaluation of Data. 2.9
The uses of Statistics.
UNIT-II ERRORS AND EVALUATION 2.0 Introduction
When numerical data and numerical result are measured with the greatest Exactness that the instrument method and observer are capable of, it has been observed that the results of successive determination differ among themselves to a great or lesser extent. The average value of a series of measurements is accepted as the most probable value. 2.1 Objectives
In all method the reliability of the result depends upon the magnitude of the difference between the average value and the true value. In some cases difference may be small, and in others it may be so large that the result is unacceptable. All the measurement from the true value. The average value of these observations is then considered to be the most probable value. The difference a between most probable value and the true mean value the absolute error. The absolute error in the measurement may be beyond the permissible limits and the probable average value.
2.2
Mean
Mean (Arithmetic Mean of ungrouped data) x 1,x2,x3 …..xn, are n values of a variable x1 then the arithmetic mean or simply the mean of there value is denoted by
x is
defined as. The arithmetic mean of a set of observation is equal to their sum
divided by he total number nu mber of observations. Ex.
If the mean of 6, 7, 4, and 10 is 8. Find the value of . 8
6 4 7 10 5
, 40=27+
13 = Arithmetic Mean of grouped Data. In a discrete frequency distribution distribution the arithmetic mean may be computed by the any one of the following methods. (1)
Direct Method
(2)
Shortcut Method
(3)
Step Diviation Method Here, we shall study the computational arithmetic mean by direct method
only. Direct Method X
f i xi
N
, i =1 and N = f i =f 1+f 2+f 3 …..f n
Ex.
Find the mean of the following distribution. x4
6
9
10
15
f 5 10
10
7
8
Sol n
Calculate the Arithmetic Mean x i
f i
f x i i
4
5
20
6
10
60
9
10
90
10
7
70
15
8
120
N= f i=40
N= f i=360
Mean Mean x
2.3
f i xi f i
360 40
9
Median
The median is the middle result when replicate data are arranged according to increasing or decreasing. For on odd no. of result the median can be evaluated directly. For an even number, the mean of the middle pair is used. Median of an ungrouped Data
It the values xi in the raw data are arranged in order of increasing or decreasing magnitude then the middle most value in the arrangement is called the median. 2.4
Precision
The precision the responsibility of the result accuracy without precision is impossible. Precision describe the reproducibility of measurement in their words the closeness of results that have been obtained in exactly the same way. Three terms are used to describe precision. (i)
Standard deviation
(ii)
Variance
(iii)
Co-efficient of variance
2.4.1 Standard Deviation
Standard deviation has been found to be more reliable than the near deviation or relative mean deviation. The standard deviation of single measurement can be obtained by extracting the square root of quotient obtained by dividing the sum of the square of the individual deviations of the no of measurement made.
( xi )
2
n
xi = (Mn-M) Mn = No. of observation M = Mean n
=
Number of measurements.
When the no. of determinations is small.
s
( xi )
2
(n 1)
(n-1) is the No. of independent deviations. From the mean which arises prove in determination. 2.4.2 Variance
The square of standard deviation is called varience. It is devoted by S 2. 2.4.3 Co-efficient of Variation
The co-efficient of varience is an accurate measure of the precision. CV
S 100%
S = Standard deviation
= data set mean value.
Relative Standard Deviation
When standard deviation is devided by the mean value
called relative
standard deviation. Relative Standard deviation
S x
if the result is expressed in parts per thousand RSD RS D in ppt
2.5
s x
100 ppt
Accuracy
The accuracy indicate the closeness of the measurement to the true or accepted value and is expressed by the errors. Accuracy is often more difficult to determine because the true value is usually unknown. Accuracy is expressed expressed in terms of of (i)
Absolute error
(ii)
Relative error
2.5.1. Absolute Error
The absolute error E is the measurement of a quantity x which which is given by
E=x i - x t x t = true of accepted value x i = individual value of x making up a set of m replicate
measurement 2.5.2. Relative Error
The percentage relative error is given by the expression E r
xi
xt
xi
100
Relative error can also be expressed in parts per thousand. 2.6
Types of Errors
(1)
Determinate or Systematic or constant error's
(2)
Indeterminate or random errors
(3)
Groos errors
(4)
Errors in measurements
(5)
Other errors
2.6.1. Determinate Errors
These are the errors which can be avoided and whose Magnitude can be determined and the measurements rejected. It affects to the same degree the results of a series of determination. Clarification of Determinate errors. These are clarified into following categories. (a)
Personal errors
(b)
Operational errors
(c)
Instrumental and reagent errors
(d)
Methodic errors
(e)
Additive and proportional errors
(a)
Personal Errors
These are due to the factors for which the individual analyst is responsible and are not connected with the method or procedure. 1.
Some persons are unable to judge colour changes sharply in visual titrations may result in a slight over stepping of the end point.
2.
Mechanical loss of Material in various step of analysis.
3.
Errors in reading a burette.
4.
Improper washing of precipitate.
5.
Insufficient cooling of crucible in weighing.
6.
Using impure reagents.
7.
Ignition of precipitate at incorrect termperature.
8.
Allowing hygroscopic materials to absorb moisture before or during weighing.
(b)
9.
Failure to apply buoyancy correction when required.
10.
Errors in calculations.
Operational Errors
These errors are mostly physical in nature and occur when sound and proper analytical technique is not followed e.g. non-representative sampling. (c)
Instrumental and Reagent Errors
Due to improper instruments and reagents. (1)
Faulty construction of balances.
(2)
Use of improperly calibrated weights.
(3)
Graduated glassware and other instruments.
(4)
The
attack attack
of
reagents
upon upon
glassware glassware
introduction introduction of foreign materials.
(d)
(5)
Valatilisation Valatilisation of platinum at very high temperatures.
(6)
Use of reagents containing impurities.
Methodic Errors
resulting
in
the
These originate form incompleteness of a reaction. In gravimetric analysis errors may arise owning to appreciable solubility of precipitates, co-precipitation and post-precipitation, decomposition or voltalisation of weighing forms on ignition and precipitation of substances other than the intended ones. In titrimetric analysis error may occurs owning to failure of reaction to proceed to completion. (e)
Additive and Proportional Errors
The absolute value of an additive error is independent of the amount of the constituent present in the determinant. Loss in weight of a crucible in which a precipitate is ignited and errors in weights are the well known examples of additive errors. 2.6.2 Indeterminate Errors
These errors are accidental and quite intangible over which the analyst has no control. These errors are revealed by the small differences in the successive values of a measured quantity when the measurement are made by the same analyst. Types (a)
Variation within determinate errors
(b)
Erratic Errors
(a)
Variation within Determinate Errors
These errors cannot be prevented from variation i.e. igniting a precipitate of xl (OH)3 to constant weight, an analyst may obtain. Successive values which vary without a definite trend. This variation could be due to varying amount of water in the weighed residue to xl 2O3. (b)
Erratic Errors
The analyst has no control over erratic errors weighing with a sensitive balanced subjected to variation will show erratic errors. (1)
Small errors occur more frequently than large ones.
(2)
Positive and Negative errors of the same numerical
equally likely occur.
magnitude
are
(3)
Narrow packed curve with steep slope indicate a relatively high degree of precision.
(4)
X broad curve indicates a relatively lows degree of precision.
2.6.3. Gross Errors
The grass errors are due to carelessness of the analyst.
2.7
(1)
Use of numerically incorrect conversion factors.
(2)
Wrong selection of method.
(3)
Unsuitable storage of samples.
Reporting of Analytical Data
In expressing an experimental data, the following points and rules should be kept in mind : (1)
Eliminate all digits that are not significant. Never retain more than one doubtful digit.
(2)
Express only one uncertain figure. For example, a values which is known to
be between 25.5 ml and 25.7 ml should be written as
25.6 ml, but not as 25.60 ml. (3)
In rounding off quantities to the correct number of significant figures. Last figure retained if the following figure is 5 or above. This is known as rounding up. Thus the number 8.856 has been rounded up to 8.86. If the last digit discarded is less than 5, leave the next digit unchanged. This is called rounding down.
(4)
In addition or Subtraction. There should be in each number only as many significant figure as there are in the last accurately known number.
(5)
In multiplication multiplication or division, retain in each factor one or more significant figures than contained in the factor having the largest uncertainty. The percentage precision of a product or quotient can not be greater than the percentage precision of the least precision factor entering into the calculation. Thus the multiplication of 1.26 x 1.336 x
0.5834 x 25.8652 should be done using the valued 1.26 x 1.336 x 0.583 x 25.87 and and the result should should be expressed in three three significant figures. (6)
Computation Computation involving a precision not greater than one fourth of 1% should be made with a inch slide rule. Slide rule is a good method for checking the calculations made by logarithms.
Relative error can also be expressed in parts per thousand. 2.8
Statistical Evaluation of Data
1.
Defining a numerical interval around the mean of a set of replicate analytical result within which the population mean can be expressed to lie with a certain probability the interval is called as confidence interval.
2.
Determining the No. of replicate measurement required to ensure that an experimental means falls within a range certain range with a given leaves of probability.
3.
Estimating the probability that an experimental means and a true value or two experimental means and different.
4.
Determining at a given probability level within the precision of two sets of measurement differs.
5.
Comparing the mean of more than two earnest to determine weather difference in mean is real or it is the result of random errors. This process is known as analysis of variance.
6.
Deciding with a certain probability weather an appointment outlive in a set of replicate measurement is the result of a gross error and this care be rejected.
2.9
The Uses of Statistics
Statistics is an essoutiae tool for the analyst. The use of statistical methods can prevent judgment being mode on the basis of limited information in addition there is rapiding developing subject of cheneometries which may be broadly defined as he application of Mathematical and statistical methods to design or to appomise measurement procedure and to prove the chemical information by analyzing relevant data.
QUESTIONS
1.
Explain the difference between (a)
Determinate and indeterminate error
(b)
Mean and Median
(c)
Absolute error error and relative relative error
(d)
Random and systematic error
(e)
Personal and proportional error.
2.
What do you know about the terms precision and accuracy?
3.
What is an error ? Describe different types of errors.
4.
Name three types of systematic. Suggest some sources of random error in measuring the width of a 3-m table with with a 1-m metal rule?
5.
Define (a) Variance (b) Coefficient of variation (c) Relative standard deviation?
BLOCK-I UNIT-III FOOD ANALYSIS
Moisture, ash, crude protein, fat, crude fibre, carbohydrates, calcium potassium, sodium and phosphate. Food adulteration-common adulterants in food, contamination of food stuffs. Microscopic examination of foods for adulterants. Pesticide analysis in food products. Extraction and purification of sample. HPLC Gas chromatography chromatography for organophosphates. organophosphates. Thin layer chromatography for identification of chlorinated pesticides in food products.
UNIT-III
FOOD ANALYSIS 3.0 Introduction 3.1 Objectives 3.2 Moisture Analysis in Foods.
3.2.1 Form of water in Foods. (a)
Free Water.
(b)
Absorbed water.
(c)
Water of hydrogenation. hydrogenatio n.
3.2.2 Procedure for Moisture Analysis. (a)
Drying Method.
(b)
Oven Drying Method.
(c)
Distilaltion Procedure Procedure for Spices and condiments.
(d)
Chemical Method for low moisture Foods.
(e)
Determination of KFR water equivalence (KF-Reg.)
3.3
(f)
Coulmetric Tirtation.
(g)
Physical Methods.
Ash Analysis
3.3.1 Ash Content in Foods
3.3.2 Methods for Ash Analysis 3.4
Analysis of Protein
3.4.1 Protein (Casein) Content in Milk. 3.4.2 Protein (Casein) Content in Butter. 3.4.3 Analysis of cruds Protein Content of food. 3.4.4 Ultra violet absorption Method. Method. 3.4.5 Birret Method 3.5
Analysis of Fat
3.5.1 Discontineous Solvent Extraction Method. 3.5.2 Non Solvent Kit Extraction Method. 3.5.3 Detergent Method of Milk Fat. 3.5.4 Refractive Index Method 3.5.5 Instrumental Method. 3.6
Analysis of Cruds Febric
3.6.1 Gravimetric Method. 3.6.2 Chemical Methods for the Analysis of Fibre. 3.7
Analysis of Carbohydrates
3.7.1 Methods of Analysis. 3.7.2 Analysis of Reducing Sugars. (Before Inverse) 3.7.3 Analysis of Reducing Sugars. (After Inversion) 3.7.4 Physical Methods for the Analysis of Carbohydrats Syrups. 3.7.5 Enzymetic Methods.
3.7.6 Modern Analytical Methods. 3.8 Determination of Calcium. 3.9
Analysis of Potassium By Flame Photometric Method.
3.10 Common Adulteration in foods.
3.10.1 Harmful Effects of food Adultrants. 3.11 Contamination of food stuffs.
3.11.1 Organisms Causing food contamination. contaminatio n. 3.11.2
Food Spoilages Spoilage s above pH 4.5.
3.11.3
Food Spoilages Spoilage s below pH 4.5.
3.11.4
Miscellaneous Miscellane ous Micro organisms causing food
contamination. 3.12 Microscopic Examination Examination of food. 3.13 Analysis of organophosphats in food by HPLC. HPLC. 3.14 Gas Chromatography for organophosphate in food. 3.15 Thin Layer Chromatography for chlorinated pesticides in food.
UNIT-III FOOD ANALYSIS 3.0
Introduction
Chemical analysis of food is done to determine be acceptability, nutritive value equality, composition and authencity of the food products. Major steps in the analysis included (i)
to select and prepare samples,
(ii)
to perform the assay,
(iii)
to calculate and interpret the data.
3.1 Objectives The food materials contain organic as well as in organic constituents. The organic compounds comprise carbohydrates, proteins, fats, oils, and nitrogenous compounds. The food analysis involves the determination of percentages of moistures, ash, crude fat or ether extract, crude protein, sugars and crude fibers etc. all of these important of our life.
3.2
Moisture Analysis in Foods
(1)
Moisture is used used as a quality factor for Jams, filling, sugar syrups & it is a quality factor in the preservation of food products.
(2)
Reduced moisture is used for convenience in packing of concentrated milks and fruit juice, liquid sugarcane sweetener.
(3)
Moisture is an inexpressive filler.
3.2.1 Form of Water in Foods (a)
Free Water : Free water acts as the dispersing agent for caller and the
solvent for salts. (b)
Absorbed Water : This water is held slightly to protein or is occluded in all
walls or protoplasm.
(c)
Water of hydration : This water
is bound chemically i.e. lactose
monohydrate. 3.2.2 Procedures for Moisture Analysis (a)
Drying Method's
The dry matter that remain after moisture removal is referred to as total solids. The food samples can be dried in forced draft oven, vaccum oven or microwave oven etc. (b)
Oven Drying Methods
Samples is heated and the loss of weight is used to calculate the moisture content of the sample. The moisture content value obtained is highly dependent on the type of oven used, condition in the oven, time and temperatures of drying. % Moisture Moisture ( w / wt )
Weight of water in sample
% Moisture Moisture ( wt / wt )
Weight of wet wet sample sample
wt of wet wet sample sample wt of d ry sample sample
% T otal Solids ( wt / wt )
(c)
100
wt of wet wet sample wt of d ry sample sample wt of wet wet sample sample
100
100
Distillation Procedure for Spices and Condiments
Direct and reflux distillation techniques involve Co-distilling the water in a food sample with a high boiling point solvent that is immiscible in water. Measuring the volume of water. Distillation methods casue less thermal decomposition of some foods than oven drying at high temperature. Water in measured directly in the distillation procedure but reading the meniscus of a receiving tube to determine the volume of water is less accurate than a weight measurement.
(d)
Chemical Method for Low Moisture Foods
Chemical methods are used for low moisture foods like dried fruits & vegetables roasted coffee coils, fats, sugar or protein. The records involve reduction of I2 by SO 2 in the presence of of water. water. 2H2O+SO2+I2 H2SO4+2HI
Titration procedure
Iodine and SO2 are added to the sample in a closed chamber protected from atmospheric moisture. Excess of iodine can't react with H 2O can be determined visually. The colour in red brown. Karl Fischer Reagent (KFR) is added directly as the titrant if the water in the sample is accessible. If water in the solid sample is in accessible to the reagent, the moisture extracted from the food with Methanol. The Methanol extract is then titrated with KFR. Determination of KFR water Equivalence (KF Req.)
The KFReq. value represents the equivalent amount of water that reacts with 1 ml of KFR. The KFReq. KFReq. can be established established with pure water, water, a water-in-Methanol standard or Sodium tartrate dehydrate. KF Re q. (mgH 2O / mL)
369 H 2O / MolNaC MolNaC 4 H 4O6 .2 H 2O S 1000 230.08 g / mol A
KFR eq. = KFR water equivalence, S=wt of sodium titrate dehydrate (g) A=ml of KFR required for titration of sodium tartrate dehydrate. % moisture content
KF Re q Ks S
100
Source of Error in the Karl Fisher titration method in : 1.
Atmospheric moisture must not be allowed to infiltrate the reaction chamber.
2.
Moisture adhered to the walls of glassware must of drived.
3.
Owing to incomplete water extraction.
4.
Certain food constituents may interfere.
(f)
Coulmetric Titration
It is ideal for samples with very low levels of moisture, from 0.03% to ppm levels. In this method, I 2 which is electrolytically generated to titrate the water, is determined by the current needed to generate the iodine. (g)
Physical Methods
(1)
Electrical Method
(a)
Dielectric Method : Moisture content in certain foods can be determined by
measuring the change in capacitance or resistance to an electric current passed through a sample. (b)
Conductivity Method : The conductivity of an electric current increase with
the percentage of water in the sample. (c)
Hydrometry :
The moisture moisture content content in salt brines, brines, beverages beverages and sugar
solutions can be calculated by measuring specific gravity or density by pycnometer or hydrometers. (d)
Refractonetry's Moisture: In liquid sugar product and condensed milks
can be determined determined by by using a baume hydrometer hydrometer a refractometer, refractometer, or by gravimetric means. Refractive index, n
3.3
sin of incident ray angle sin of refracted ray angle
Ash Analysis
Ash refers to the inorganic residues remaining after complete oxidation of organic matter in a food stuff. 3.3.1 Ash Content in Foods
The ash content of of most fresh food rarely exceed 5% 5% while dried
may
contain 11.6% ash (wet weight basis). 3.3.2 Methods for Ash Analysis Sample Preparation (i)
Fat and Sugar Products : Animal products, species and syrups require
treatment prior to cashing because of high fat and moisture or high sugar content (forming) result in loss of sample. Bean, Sugars and syrups need to be evaporated to dryness. (ii)
Plant Materials: Plant materials are dried prior to grinding. The sample may
be used for multiple determination. Fresh stem and leaf tissues should be dried in two stages stages to prevent afrifact afrifact lignin. Dry Ashing Principle
Dry ashing is incineration at high temperature (550 0C) in muffle furnance. Ashing time is reduced with microwaving. Water and volatiles are vaporised and organic substances are burned in air to CO 2 and oxides of N 2. Elements such as Pb, Fe, Se, Mg may partially volitilise. Procedure
-
Weight 5 to 10 g sample in a tared crucible
-
Place crucible in muffle. Ignite at 550C for 12 to 18 hours.
-
Open the door of muffle furnance carefully to avoid leasing ash that may be fulfill.
-
Quickly transfer the crucible to a desiccator for cooling and weigh it.
Calculation % ash (dry basis)
2.
wt after ashing tare wt . of crucible wt . of Sample dry matter coefficien t
Wet Ashing
Wet ashing or wet digestion is a procedure for dissolving minerals and oxidizing substances with high fat content (meatsite) using oxidizing agents. Procedure
-
Add 1g of dried, powdered sample is placed in 150 ml Griffin beaber.
-
Add 10 ml HNO3 and allow it to soak overnight.
-
Add 3 ml of 60% HClO4 and heat upto 350 0C until fronting stops and HNO 3 is almost evaporated.
-
Continue boiling until per chloric reaction occurs. Place watch glass on beaker. Sample should be colourless.
-
Cool the beaker wash watch glass with minimum devonised water. Add 10 ml 50% HCl.
-
Transfer to 50 ml volumetric flask and dilutes with deinoibled water.
-
Wash the hood precautiausly after last sample.
Preparation for Iron and Analysis in a Meat
Boil 2g sample in 30 ml HNO 3 at 3500C on host plate until 10 ml remain. Add 10 ml of 60% HClO 4 and continue boiling until copious fumes occurs. Advantages of Wet Ashing
-
Minerals usually remain in solution.
-
There is little or no loss form volatilization volatilization because of lower temperature.
-
The oxidation time is short and requires a hot plate, food, tongs and safety equipments.
Disadvantages
-
Wet ashing requires constant operation attention.
-
Corrosive reagents are necessary.
-
Only a few samples can be handled at one time.
-
Per chloric acid react with iron to form ferrous perchlorate.
3.
Modified Dry-Wet Ash Oxidation
-
Evaporate moist sample (25 to 50 ml) at 1000 C over might or in a microwave oven.
-
Heat on a hot plate until smoking classes.
-
Ash at 5250C for 3 hours.
-
Coal and wet with devosed distilled water and 3 ml HNO 3.
-
Dry and incinerate at 5250C for 1 to 2 hours.
-
Weight sample after cooling in desiccatar.
4.
Low Temperature Plasma Ashing Principle- Low temperature plasma ashing refers to a specific type of dry
ashing method whereby foods are oxidised in a partial vaccum by nascent oxygen formed by radio frequencies electromagnetic field generator. Instrumentation
The equipment consists of a glass system with a variable number of chamber for sample that may be evacuated by a vaccum pump. Procedure
The ground material is inserted into individual glass boats which separate glass chamber. The chamber is sealed and a vaccum is applied. A small flow of O 2 or air is introduced into the system maintaining minimum vaccum. The frequency generator is less than 14 MHZ and adjusted by the amount of wattage applied (30200 ks)to control undation. Advantages
- There is less change of lasing trace elements by volatilization. - The low temperature (150 0C) used with plasma as hers keeps the microscopic and crystalline structure unaltered. Disadvantages
The major disadvantages are small sample capacity, expense of the equipment and operator's time. Other Ash Measurements 1.
Soluble and Insoluble Ash in Water
Weigh the total ash
Add 10 ml distilled water, water, cover the crucible and heat to boil.
Filter on ashless filter paper and rinse with hot distilled water five to six times.
Dry and re-ash filter paper for 30 min.
Weigh and calculate as percent water-insoluble ash.
2.
Ash Insoluble in Acid
This ash determination is used to measure surface contamination (silicates) of fruits, vegetables, wheat, and rice coatings.
Add 25 ml of 10% HCl to to total ash ash or water insoluble insoluble ash.
Boil for 5 min. filter on ashless filter paper and wash with hot distilled water.
Re-ash dried filter paper and residue for 30 minutes.
Weigh and calculate as percentage.
3.4
Analysis of Protein
Protein is a common ingredient of all food materials analysis of protein is required to know.
Total protein content. Amino acid composition.
Protein content during isolation and purification.
Non protein nitrogen
Nutritive value
3.4.1. Protein (Casein) Content in Milk
Milk protein (casein) can be separated from milk and analysed by the following methods : Procedure
Dilute 200 ml milk to one litre with distilled water in a 26 beaber. Add 1g glacial glacial acetic acid when when a white precipitate precipitate settles down.
Decent off the aqueous layer and wash the precipitate with water.
Grind the precipitate with a little 0.1% NaOH solution to neutralise the acid.
Filter the resultant suspension through muslin clatn by pressing it hard.
Add it’s the filterate by adding glacial acetic acid so that the solution contains
0.1% of it.
Wash the precipitate obtained from decanted water neutralist with 0.1% NaOH solution and filter.
Repeat the process of precipitation and washing. Again filter the the precipitate with with alcohol and then with to to remove fats. fats.
3.4.2. Protein (Casein) Content in Butter
It can be calculated as --------Percentage of protein = (Percentage of moisture+lactic moisture+lactic acid+fat-100)
3.4.3. Analysis of Cruds Protein Content of Food Kjeldahl Method Sample Preparation- Solid foods are ground to pass a 20 mesh screen.
Sample should be homogenous.
Procedure
Add H 2SO4 and catalyst (HgO, Cu 3 in 3:1) for complete breakdown of organic matter. During digestion, protein N is liberated to form NH 4+ ions H2SO4 oxidises organic matter matter and combines combines with ammonium formed formed carbon and hydrogen hydrogen are converted to CO 2 and H2O. Pr otein ( N )
H 2 SO4 Catalys
( NH 4 ) 2 SO4
Neutralisation and distillation's Alkali containing sodium thiosulphate is added to nutralise H2SO4. Sodium thiosulphate helps to release N from Hg which tends to bind NH4+. The ammonia so formed is distilled into boric acid solution containing indictors methylene blue and methyle red. (NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O. NH3 + H3BO3 NH4+ + H2BO3- (Boratic ion) Titration Borate anion is titrated with standardized HCl. H3BO3- + H+ H3BO3. Calculations
Moles HCl = Moles NH 3= Moles Ni The sample and reagent blank should be run to subtract reagent N from the sample N. % N NHCl
Concentrat ed acid volume g of sample sample
14 g Mole Mole
100
3.4 4. Ultra violet absorption Method Principal
At 280mm due to tryptophan and tyrosine resides in proteins. Since the content of tryptophan and tyrosine in each protein is constant the absorbance at 280mm could be used to estimate the concentrate of protein, using Beer's laws. Procedure
Protein are solublised in alkali or Buffer. Absorbance of protein solution in read at 280nm again a reagent blank.
Absorbance, x=abc, where =absorptive, b= cell or white path length, c= concentration. Advantages
-
method is used to determine protein content of milk and meat products.
-
The method is rapid, sensitive non destructive and used widely in part-column detection of protein.
Disadvantages
-
has not been used widely in food systems.
-
Nucleic acid also absorb at 280mm. The absorption 280mm/260mm ration or pure protein and nucleic acids are 175 and 0.5. One can correct the absorption of nucleic acids at 280mm is the ratio of 280/26mm is known.
3.4.5. Biuret Biuret Method
A violet purple colour is obtained when cupric ion are complied under alkaline conditions. Absorbance Absorbance of colour occurs at 540nm. Colour intensity is proportional to the is proportional to the protein content of the sample. Other methods protein can also be analysied by measuring the physiochemical properties of proteins by lowery method, ninhdrin method, turbidimetric method idye binding method and Bradford method. 3.5
Analysis of Fat
Fats are esters of fatty acids with teracyleglycerols. An accurate analysis of lipids in foods is important for nutritional labeling to determine whether the food with the standard of identify and is uniform. Methods
1.
Solvent extraction methods.
2.
Non-solvent wet extraction methods.
3.
Refractive index method.
4.
Instrumental method.
5.
Calorimetric method.
6.
Mojonnier Method for the analysis of milk fat.
3.5.1. Discontineous Solvent Extraction Methods
Principle fat is extracted with a mixture of ethyl ether and petrollium ether. Extracted fat is dried to a constant c onstant weight and expressed as percent fat by weight. Proportion of samples weight or measure the test portion of a homogenous milk sample. If lumps of cream do not disperse, warm the sample 38 0C and cool the warmed sample to 20 0C.
First Extraction
-
Weight 10 g milk into a mojonnier fat extraction flask.
-
Add 1.5 ml NH4OH and shake vigaroulsy. NH 4OH neutralizes acidic and dissolves protein.
-
Add 10 ml of 93% ethanol to prevent gel formation and shake for 1 minutes.
-
Add 25 ml ethyl ether to dissolve the lipid and shake well.
-
Add 25 ml petroleum ether and shake. It remove moisture from the ethyl ether extract and dissolves more nonpolar lipid.
-
Centrifuge for 30s 600 rpm.
-
Decant ether solution from the flaks into the previously wughed mojonner fat disk.
Second Extraction
-
Add 5 ml of 95% ethanol and shake vegarously for 15s.
-
Add 15 ml ethyl ether and shake for 60s.
-
Add 1 ml petroleum ether and shake for 60s.
-
Centrifuge for 3s at 600 rpm.
-
Decent solution into the same mojonneir disk.
Third Extraction
-
Add 15ml ethyl ether and 15mL petroleum shake for 60S.
-
Centrifuge for 30S at 600 rpm and decent solution into the same mojonnier disk.
-
Evaporatis the solvent in the disk on hot plate at 100 0C in a food.
-
Dry the disk and fat to a constant weight in a forud air oven at 100 0C.
-
Cool the disk to room temperature and weight.
Calculation
% Fat = 100 x [(wt. of disk+fat)] - [(wt. of disk)] - ( av wt. of blank reside) (wt. of sample) Mojonnier Method of Fat in Flour
-
Mix 2 gm sample and 3 ml ethanol in a 50 ml beaker.
-
Add 10 ml HCl heat the beaker at 800 C in water bath with stirring for 30 minutes for hydrolysis.
-
Add 10ml alcohol and cool. The acid hydrolysed flour is extracted by a mixture of ethyl ether and petroleum petroleum ether as described in the Mojonnier Mojonnier method for milk fat.
3.5.2. Non solvent wt Extraction Methods Gerber Method for Milk Fats
Principles Sulphuric acid and anayl alcohol are added to a known volume of milk H2SO4 digests proteins and carbohydrates, releases fat and generates heat. Procedure
-
Add 10 ml of H2SO4 at 200C into a Gerber milk battle.
-
Accurate measure milk sample 11 ml into a Gerber bottle using a Gerber pipette. Add issonyl alcohol.
-
Heat the bottle in a water bath at 60 0C for 5 min. Read the fat content from the graduations on the bottle neck.
Applications
Gerber method is simpler, faster and has wider application to a variety of dairy products.
3.5.3. Detergent Method for Milk Fat
An anionic detergent, dioctyle sodium phosphate, is added to disperse the protein layer to liberated fat. In case of other food products, a strong hydrophilic nonionic polyoxy ethylene detergent, sorbitan monolauete is added to separate fat. The percent fat is measured volumetrically and expressed as percent fat. 3.5.4. Refractive Index Method
The Refractive index is characteristics of each kind of fat and the values vary with Degree and type of nusatcoation oxidation heat treatment temperature and fat content. 3.5.5. Instrumental Methods Infrared method for milk fat- infrared method is based on adsorption of
infrared energy by fat at a wave length of 5.73 .The more the energy absorption at 5.73 the higher the fat content of the sample. This method is used to determine the fat content. 3.6
ANALYSIS OF CRUDE FEBRIC
Dietary Fibre in defined as lignin plus plant poly sacchrides that cannot be digested by human enzymes. 3.6.1 Gravimetric Methods (i)
Crude Fibre:- The crude fibre is analysed by sequential extraction of the
sample with 1.25% H 2SO4 and 1.25%. NaOH. The The insoluble residue is dried, weight and head to correct for mineral contamination of the fibre residue. (ii)
Total, Insoluble and soluble fibre :- Ruplicate samples of try fact
extracted ground foods are enzymatically digested analysis dnyloglucosidare and protease to remove starch and protein. Insoluble fibre is collected by filtration. Soluble fibre is precipitated precipitated by by adding 78% ethanol filtrate filtrate fibre Residue weight - (Weight of protein + dist) This (sos/method can be used to determine fibre content of all foods. 3.6.2 Chemicals Methods for the Analysis of Fibre
Chemicals procedure collect macromolecules in the amylase amyloglu cosidase digest by filtration with or without ethanol precipitation. The poly
saccharides in the precipatute are hydrolysed with H 2SO4 and quantitated calorimetrically or chromatographically (GC or HPLC). Thander - Marlet Methods Principle
Free Sugar and lipids are extracted with ethanol and hexane starch is removed by enzymatic digestion and insoluble fibre is separated from soluble fibre. Fibre protections are hydrolysed with H 2SO4. Procedure
1.
Dry ground sample of food.
2.
Sonicate and extract with ethanol and then hexane (2 times)
3.
Filter between extraction with Whatman No. 65 paper.
4.
Extracted residues are dried and weighed to determine Sugar and lipid loss.
5.
Dry sample 4 to 59.
6.
Digest starch with termamyl in 75 ml of 0.1 m acetate buffer, pH 5.0 with 70 ppm Ca2+ at 960C.
7.
Digest starch with amyloglucarisdase amyloglucarisdase for 16 hours.
8.
Centrifuge and filter to separate soluble and insoluble fibre.
Filtrate
(a)
Collect soluble polysachracides polysachracides ppt with ethanol or by dialysing filtrate and freeze-drying the diolysate.
(b)
Hydrolyse soluble fibre with in H 2SO4 (3h, 100%)
(c)
Soluble fibre, amalyse sugar Uronic acids are anaylysed colorimetrically, colorimetrically, Neutral Sugars are measured by HPLC or GC.
Pellet and Filter Retentate
(a)
Wash with ethanol, acetone and dry overnight under vaccum (40 0C)
(b)
Hydrolyse cellulose with 12N H 2SO4 (1hr)
(c)
Dilute acid to 1N and hydroluse non cellulose insoluble polysaccharides for 3 hrs.
(d)
Centrifuge and vaccum filter. Wash filter retentate with water.
3.7
ANALYSIS OF CARBOHYDRATES
Carbohydrates play an important role in human nutrition. Carbohydrtes analysis of raw materials and processed foods can be used to provide a wealth of information. The fingerprint oligosaccharide patterns can be used to detect food adulteration. 3.7.1 Methods of Analysis
1. Chemicals methods methods for the analysis of Monosaccharides Monosaccharides and oligasaccharides. oligasaccharides. (i)
Munson and Walker method for the analysis of reducing sugars carbohydrates are oxidized on heating with an excess of cupric sulphate and alkaline tartrate in basic medium to keep copper as copper (Cu++) hydroxide.
-
Upon heating, water is driven off and copper oxide is converted to cuprous oxide.
-
Cuprous oxide precipitates precipitates as the carbohydrates carbohydrates are oxidized and can be determined by following methods. (i)
Gravimetric Method.
(ii)
Electrolytic deposition from HNO 3 where the copper oxide
dissolved in HNO 3 and then deposited on Pt
electrodes.
is
The weight gain
the electrode electrode is related related to the reducing Sugar content. content. (iii)
By titration with sodium thiosulphate Cuprous oxide is dissolved in nitric acid. KI is added and the iodine is oxidized to I 2.
(iv)
By titration with KMno 4 cuprous oxide is related with ferric sulphate Fe3+ is reduced to Fe 2+. Ferrous ion is then titrated with KMno4 resulting in a colour change. Cu2O+Fe2(SO4)3 FeSO4+CuSO4+Cuo
Modified Munson and Kialber Method for the Analysis of Glucose Fructose and Invert sugar is modified method involves to use of an excess of
alkaline copper Nitrate and Na 2CO3. Following deduction, the excess copper Nitrate is reacted with excess KI and the liberated I 2 is titrated with sodium thiosulphate to amalyse carbohydrates. carbohydrates.
3.7.2. Analysis of Reducing Sugars (Before Inverse)
Take 1% reducing Sugar solution (e.g. honey) is aressed burette. Pipette out accurately 5 ml of fehling solution A and 5 ml of fehling solution B into a 100 ml conical flask and dilute it with 40mL of distilled water. -
Fehling solution A, Dissolve 69.278g CuSO 4 in distilled water and dilute to 1000 ml.
-
Fehling Solution B, Dissolve 346g of Rochelle salt and 200g of NaOH in distilled water and dilute to 100ml.
-
Heat the mixture of conical flask on hot plate.
-
Run down 1% honey solution from the Burette into the mixture of conical flask till the solution terms brick red in colour.
-
Add 1mL of 0.2% methylene blue indicator solution.
-
Again add honey solution till the end point is indicated by the colour change from blue to red.
-
Note the reading of the burette.
Calculation Total Reducing Sugar
Strength Fehling solitor 50 0.98 Titro value of the noney Solution
Calculation of Strength of Fehling Solution
Strength of Fehling solution is obtained by titration against 0.5% glucose (0.5 g in 100 ml of water) solution. If x ml of 0.5% glucose solution is required for complete reduction of 10 ml of Fehling solution A and B . Strength of Fehling Solution
0.5
x
100 10
Conversion factor from glucose (mol. wt. 180) to starch (mol. wt 162). 10 parts of glucose are equal to 9 parts of starch.
Conversion
from glucose to Starch
162
180
9 10
0.9
3.7.3. Analysis of Reducing Sugars (After Inversion)
-
Pipitte out 1 ml of 10% none (reducing Sugar) in 100ml conical flask.
-
Add 2 ml glacial acetic acid and heat to bail keep it for 2 hours.
-
Neturalise at with Na2CO3.
-
Make up the solution to 100 ml with water in a measuring flask.
-
Titrate this solution against 10 ml of Fehling solution A and B as done in previous experiment.
Calculation Reducing Sugar (after inversion)
Strength of Fehling Solution x 50 x 0.98 Titrate Value of the noney Solution
3.7.4. Physical Physical Methods for the Analysis of Carbohydrate Syrups
Physical methods like polarimetry ,refractometry and specific gravity are useful as rapid quality control techniques for pure carbohydrate syrups and juices. 3.7.5. Enzymatic Methods (i)
Total change method
Sufficient enzyme is added to convert av of the substrate in the food sample to product. The amount of substrate in the sample is than determined from one total change in the sample either as substrate disappearance or as product formation. (ii)
Rate as say per Kinetic Method
First the initial role of the enzyme substrate reaction is determined.From the relation the concentration of enzyme, substrate, activator or inhibator may be determined. 3.7.6. Modern Analytical Methods
(i)
Modern
Methods
afford
accurate
carbohydrates carbohydrates at trace concentration. (i)
HPLC
analysis
of
structurally
similar
3.8
(ii)
Microscale HPCL
(iii)
High performance capillary elecrophoresion.
(iv)
Micullar performance capillarty chromatography. chromatography.
(v)
Capillary gas chromatography. chromatography.
(vi)
Supercritical fluid chromatography. chromatography.
(vii)
Mass and NMR spectroscopy. spectros copy.
Determination of Calcium
Reagent Required
-
Dilute HCl 2 volumes of Conc. HCl dilutes with 5 volumes of water.
-
NH4OH and saturated ammonium oxalate solution.
-
Standard 0.1 KMno4 solution. Standardised against sodium oxalate.
-
Dilute H2SO4 1 volume of Conc. H 2SO4 diluted with 4 volumes of water.
-
Acetic Acid volume of glacial acetic acid diluted with 2 volumes of water.
-
Bromo cresol green indicator solution Grind 0.1g bromo 10 Cresol green with 14.3 mL of 0.01N NaOH is an agate mortor.
Procedure
-
Weigh 2 to 4 g of the material and obtain its total ash.
-
Digest the ash in dish with Dil. HCl Evaporates to tryness. Treat the residue with dilute HCl and again evaporate to dryness on a water bath. Treat the residue with 10 ml Conc. HCl add 30 ml of water and and filter in a 250 ml beaker.
-
Wash to the residue with not water and collect the washing in the same beaker.
-
Add to the solution in the beaker 0.5 ml of bromocresol green indicator and then NH4OH till the colour changes to blue.
-
Filter the solution wash with hot water Called the washing in the same beaker and heat to boil.
-
Add Saturated ammonium oxalate solution dropwise till the precipitate precipitate appears and then add excess solution heat to boil.
-
Digest for 3 hours.
-
Pour 20 ml of hot water on the precipate remaining on the filter paper by washing with hot dilute HCl into the original beaker. Wash the filter paper with hot water.
-
Reprecipitate Reprecipitate by adding NH 4OH and a little ammonium oxalate solution.
-
Digest for 3 hours filter. Through the some filter paper. Wash with hot water until it is chloride free.
-
Perforate the apix of the of the filter Con. Wash the filter paper with not dilute H2SO4 and titrate with standard KMno 4 solution at 70 0C.
Calculation Calcium perc p ercen entt by mass
2.8 NV M
where
N
=
Normality of standard KMno4 solution
V
=
Volume (ml) of the standard standard KMno4 solution used
for
titration. M
=
Ca mg/100g
3.9
Mass (g) of the material taken for the test analysis. Titre x 0.2 x Total Volume of ash soletic x 100
Volume taken for estimation x Kit of sample taken for ashing
Analysis of Potassium By Flame Photometric Method
Principle
Potassium in solution is atomized into an oxyhydrogen flame. The flame execitesatoms of potassium causing them to must radiation limited is measured on a spectrophotometer. Reagent
(1)
KCl stock solution Dissolve 1.90 g of KCl in Distilled water and make up the volume to 1 litre.
(2)
Standard solutions measure 150 ml stock standard solution and 5ml HCl into a flask and make solution to 1 litre. In order to compensate for minute
interferences caused by other ions in the determination of potassium. It is necessary that the standard solution be augonented with equivalent concentration of those ions that occurs in highest proportion in the sample being analysed. Standard Curve
Draw a allegation of standard solution from 0 to 150 ml atomise sitting the top standard at 100% trasmittance. Note the luminosity of the flame for each concentration. Draw a standard curve by plotting concentration on abscissa and the percentage luminosity on the ordinate. Procedure
Dilution aliquotes of ash solution so that it contains less than 150 ppm potassium. Add HCl so that the concentration of acid is same as that in the standard solution. Atomise the diluted extract in a calibrated flame photometer with the wavelength dial set at 768 nm and transmittance set at 100% for the top standard solution of potassium. Calculations Kng/100g
PPm found from standard conce x volume made up x dilut dilut ion x 100 K Weight of Sample x 1000
3.10 Common Adulteration Adulteration in Foods
Milk the addition of starch skimmed milk water of removal of fat. -
Milk powder Starch, Moisture, fat deficiency.
-
Pure Ghee- Vanaspati Ghee, Animal Fat, Rancid Stuff, excess moisture.
-
Vanaspati Ghee- Animal body fat, rancid fat, argemon oil, seami oil,
prohibited colours as well as flavour. -
Vegetable oils- Minerals oil, rancid oil, argemone oil, rubber seed oil, tea,
seed oil, watermelon seed oil. -
Butter- Animal fat, starch excess moisture, rancid stuff, Vanaspati ghee,
prohibited colours. -
Pulses- Massoor, gram,Khesari dal, sand dirt, coal tar dyes.
-
Dal- Arhar Khesary dal, coloured with netainl yellow.
-
Wheat flour- Atta, sujji, maida,sand dirt, soapstone, excess bram, chalk
powder. -
Mustard - Argenoa seed
-
Common Salt- fine white sand, excess moisture excessive salt.
-
Vinegar- mineral acid, coal tar dyes, Synthetic Vinegar Vinegar sold as malt,
or
wine, less amount of acetic acid. -
Ajwain- fine sand and foreign seed.
-
Coriander- Cow dung, saw dust, house dung, powered bran, foreign starch,
-
Chilles- Brick powder, coloured saws dust talcum powder, foreign starch,
powdered bran. -
Cardamon (Choti ilachi) Exhausted spices
-
Cinnamon (Dalchini) - Canis bark.
-
Garam Masala (powdered mixture of various species)
-
Tealeaves - Saw dust.
-
Coffee powder used coffee chicory roasted date seeds, tamarind husk starch
-
Sweets, Metanil yellow.
-
Ice Cream- Artificial sweetner, starch and no permitted colour.
-
Soft drink - Prohibited colours, flavours ,sweetner, Saubarine, mineral acids, bacterial and metal contamination. contamination.
-
Processed foods - Prohibited food additive solvent residue and microbial contamination.
-
Ginnamon - Carnica burk.
3.10.1 Harmful Effect of Food Adultrants
(1)
The consumption of adulterated foods has a slow pairing effect. The victims of adiable oil adulterated with argemone oil show epidemic lepopsy.
(2)
Mixture of turmeric powder with lead chromate causes lead poisoning, anaemia and even abortion.
(3)
An excessive intake of khesaridal in pulses causes permanent paralysis of limb.
(4)
The use of culture and non-permitted colours in dals, sweets or tea leaves.
(5)
The consumption of meat from antibiotic fed animals causes multiple structure resistance hardening of arteries and caronary heart disease.
(6)
Gossypolain cotton said flour and panallidine in mushrooms causes cancer in human.
3.11 Contamination of Food Stuffs
Under processing the failure to destroy all bacteria capable of subsequent growth in the product during the heat process. Leakage is due to the contamination of the product after an adequate heat process due to faulty seam or damage to the can after sealing. 3.11.1 Organisms Causing Food Contamination
(1)
(2)
Thermophilic group such as (i)
Flat sour
(ii)
Thermophilic anaerobes
(iii)
Sulphide spoilage microbes
Mesophilic groups include (i)
Putrefractive anaerobes
(ii)
Butyric anaerobes
(iii)
Aciduric flat saws
(iv)
Lactobacilli
(v)
Yeasts
(vi)
Moulds
3.11.2 Food Spoilages above pH 4.5
-
Bacillus stero thermophilic cause flat saur spoilage in low acid foods (ph 5.3) like peas corn, potatoes.
-
Bacillus coagulants contaminate in medium acid (ph 4.6) canned products such as corn, spinach asparagus.
-
Mesophilic anaerobes including clostridium behefinura disintegrate solid food products produce Odour, CO 2, H2.
-
Butyric acid anaerobes, Cl butyricum/ateterorate butyricum/ateterorate vegetables, Corns, soups and sauces.
-
Cocciymould and yeasts casue frothy permentation in liquars and brines due to leakage.
3.11.3 Food Spoilage Below pH 4.5
-
Bacillus Coagulants (B. thermoacidurans) thermoacidurans) causes occasional spoilage in tomato juice dew to under processing.
-
Moulds causes softening or disintlgration disintlgration in canned fruits due to leakage of containers.
-
Cl. pasteurianum and Cl. butyricum, mesophilic obligate anaerobes spoil pears, figs, pine apple due to spare forming anaerobes.
-
B. polymyxa group (B. macerans) contaminate citrus juices, prunes jack fruit and peach etc.
3.11.4 Miscellaneous Micro-Organisms Causing Food Contamination
-
Zearalemone, an oestrogenic mycotoxin produced by Fusarium readily colonise wheat, barely, rice, maize, cerals and bread.
-
Listeria contaminate contaminate with dairy products (Chease, yoghurt, /meat and vegetables).
-
Dconynivalenol (DON or vemitoxin-) DON is related to compound known as tirchathocens formed by fusarium graminearum and F. Culmorum.
-
Aspergillus floves and A. parasiticus produce alfatoxine that damage a variety of foods including nuts, dry fruits, cereals and herbs.
3.12 Microscopic Examination of Food 1.
Plate Method
Adding enriched food samples to a micro titrate plate a highly specific monoclonal antibody conjugate is added to it which finds to microbe antigens
forming an immune complex. The presence or absence of microbe is determined by the addition of a colourless substrate which produces a coloured product in presen ce of a particular micro-organism. 2.
Coliform Count Method for Examining Fresh Fruits and Vegetables
Reagent required is ringer solution. It is used at one-quarter the original strength, and is prepared prepared by dissolving dissolving 2.15 g NaCl, 0.075 g KCl, 0.12 g CaCl 2 / / dry/and 0.5g Na 2S2O3.5H2O in 1000 mL of Distilled water. Methods Shake 100 g of the vegetables with 200 mL of Ringer solution. Allow it to sand for 20 min and decant. Test positive tubes for the presence of coli. 3.
Total Bacteria Count for Examination Frozen Fruits and Vegetables
Preparation of dextrose- Tryptone broth or Agar culture medium. The ingredients are 10g tryptone, tryptone, 5g dextrose, dextrose, 0.04g bromo cresol purple 12g and 1 l water. Steam the ingredients until dissolved. Procedure
Add 1g of inoculum to the sterlises petridish and pour 10mL of melted agar medium. The growth of organisms in the broth is indicated by the change of colour from purple to yellow. The colonies appear on the surface with a typical spot in the centre. Count total bacteria growing at 7 0C in 5 days also count lactic acid bacteria, moulds and yeasts. Slime usually contains large number leuconostocs which gives them a yellow appearances but sometimes heavy growth by coryneforms produces ammonia and this neutralizes the acid formed by Luconostoc count on frozen vegetables are low (10 6 /g).
4.
Microbial Count in Vegetables and Fruits
Culture and inoculate dextrose - tryptone agar, tomato juice and incubate at 300C aerobically and anaerobically. a naerobically. Counts for lactic acid bacteria, flat sour thermophiles, mould, yeast and H 2S producing anaerobes.
In both carbonated and non-carbonated drinks the microbial counts dimisshed in time.
In fruit juices, lactic acid and acetic acid bacteria may grow at pH 4. Fruit juices of oranges, lemon and grapes fruit used for soft drinks and beverages must be sterile and give a negative result.
5.
Direct Microscopic Examination of Food
Take the food content in a sterile container for subculturing. Make smears with a sterile inoculating loop stain with methylene blue and gram stain. The presence of gram positive rod suggests underprocessing while E. cocci and yeasts etc. Suggests Leakage Spoilage
Pesticides Analysis in food product chromatographic techniques used for the analysis of pesticides in food stuffs are HPLC, GLC and TLC. General Extraction and Purification of the Food Samples
Extract 20g of finally ground food sample with dichloromethane dichloromethane in the soxhlet exractor for 3 hours. Transfer it to a Kudema Damish flask. If the cereals extract it cloudy pass it through a column containing 20g of anhydrous Na 2SO4 wash the column with 40mL of lexane or isopropyl alcohol and mix the washing to extractor before transferring it to the vaporator. Add 3 drops of liquid paraffin heat the contents on a steam bath until the volume becomes 3 ml.
3.13 Analysis of Organophosphates Organophosphates in Food by HPLC
Organophasphate insectides present in food products may be parathion, malathion, methyl paratnion, diazenon, tritnion etc. Reagents
(i)
H2SO4
(ii)
Acetone
(iii)
Hexane
(iv)
Dichlaromethane
(v)
Methanol as eluting solvent
Experimental Techniques Extraction of the Food Sample
Extract the sample (10) in a separately funnel and evidety to pH with H 2SO4. Add 60 ml of acetone and shake. Then extract with 60 ml dichloromethane dichloromethane and hexane (1:1) in the original sample container and organic phase is collected in kudema Danish flask. Extraction is repeated twice with 50 ml each of dichlaro methane and hexane and solvent is treated as a above. Solvent extract volume. Then concentrates is injected injected to HPLC for the analysis analysis of organophosphates. organophosphates. Standard Conditions for Operating HPLC
The column of the HPCL is packed with 5 m c-8 bonded phase particles.
The width and height dimension of column are 4.5 x 250 mm.
The flow rate of eluting solvent method is maintained of 2mL per minute.
A UV detector detector is attached to the instrument. instrument.
Process
The organophosphates organophosphates are separated by using the gradients elution system of mobile phase and the monitored the UV detector at 254mm chromatogram showing the peaks of various. Organophosphates in food sample. Calculations
Calculations are based on peak height measurement of the sample and the standard for determing the concentration of species. Analysis of Chlorinated Insecticides Insecticides of Milk Milk by HPLC
Automated HPLC pesticides analysea can be used for the analysis of chlorinated insecticides in milk by injecting raw milk on to a shot slica precolumn where the fat was retuned and the pesticides eluted with hexane. The less polar fat eluting organo chlorines such as DPT, DDE, and -BHC stared at the top of a larger analytical column. 3.14 Gas Chromatography Chromatography for Organophosphates Organophosphates in Food
Organophosphates Organophosphates such as malathinon parathion, diazenon, chearthion metasystox in food stuffs can be analysed by gas chromatography. chromatography. Analytical Steps Sample preparation
It involves chapping, grinding armacreating the food sample containing argamophasphate. Extraction and Purification
A 250g of the copper or macerated composite sample is blended with acetonitirile aracetone.
Anhydrous salt (NaCl or Na 2SO4) can be added to absorb water or water can be added added so that
the crude extract can can be purified purified with with a subsequent
partitioning steps with an organic solvent.
Sometimes emulsion are formed. Emulsion formation can be minimized by adding a small quantity of saturated salt solution or or with a fews fews drops of alcohol.
For matrices like Butter fats and animal tissue sufficient water is added to the sample to obtain at total of 100g of water.
Acetonitrile (200ml) and dichlaromethane dichlaromethane (150ml) or acetone (200ml) and petroleum ether (150ml) are added to water amended sample together with NaCl.
The mixture is blended at high speed for 2 min.
The dilution reconcentrations steps are repeated to ensure the complete removal of those extraction solvents that can affect the operations of the detector.
Standard Conditions for Operating Gas Chromatography Chromatography
Capillary column pached column megabore column may be used. The column of GC is packed with chronosurb WHp capilay GC columns are fabricated from fused silica.
Active stationary phase is usually permanently bonded to the inner surface of the column.
The temperature of the gas chromatograph kept at 200 0C for 10 minutes the temperature of the injector injector is maintained maintained at 2250C.
N2 is used as carrier gas and its flow rate is maintained at 25ml per minutes.
Procedure for the Analysis
An autoinjector can chromatograph.
deliver a present volume of sample to the
Components partition between the stationary phase and a carrier gas according to their relative affinities for the two phase.
Now a cilliary devices are used in automation gas chromatographs chromatographs to increase the degrade of automation of the separation, detection and quantitation of chromatograms chromatograms of pesticides in foods.
Micro computer based GC software packages can be used to compare test chromatogram chromatogram with a control of the same food matrix.
3.15 Thin Layer Chromatography for Chlorinated Pesticides in Food Products
Chlorinated pesticides the economic poison employed to regulate the impact of various pests in agriculture/ contaminate in crops and food stuffs. These are DDT, BHC, aldrin, alteration, lindane. Extraction and purification of the food sample follow the sample produce procedure as employed in HPLC. Procedure for the Analysis
Prepare the glass plate by coating silica gal slurry or alumina layer. In adsorption TLC is to performed the layer of sorbent is activated by heating to 200 0C for 4 hours. The food sample sample containing containing chlorinated pesticides is applied applied as aspect aspect near one end of the plate. The plate is placed in a closed chamber saturated with hexane. The chromatogram is developed with chromogenic reagent 0.2% AgNO 3 or 0.5% solution of p-methylamino p-methylamino hydrochloride hydrochloride made in sodium ethnoxide. ethnoxide. Moisture the plates by spraying distilled water. R F values of Some Pesticides are aldnin, 079, DDT, 0.60, BHC 0.4, dieldrin 0.17, and methoxychlor 0.12. HTPLC
High performance thin layer chromatography is a new techniques in which TLC plates are coated with smaller, more uniform particles of controlled porosity. This permits better detection of organochlorines in food stuffs.
QUESTIONS
1.
Why is food analysis important ? Discuss the safrty of food.
2.
What do you know about the moisture content of food ? how will you determine moisture in vegetable oils and spices?
3.
How will you analyse total ash, water-insoluble ash and acid-insoluble ash in a food sample?
4.
How will you determine or organophosphate insecticide in a food materials?
5.
What are the common food adulterants? How will you analyse crude fibers as adulterant in food products.
BLOCK-I UNIT-IV ANALYSIS OF WATER WATER POLLUTION POLLUTION
Origin of waste water, types, water pollutants and their effects. Source of water pollution-domestic, industrial, agricultural soil and radioactive wastes as sources of pollution. Objectives of analysis-parameter for analysis-colour turbidity, total solids, conductivity, acidity, alkalinity, hardness, chloroide sulphate, fluoride, silica, phosphates and different forms of nitrogen. Heavy metal pollution-public health significance of cadmium, chromium, copper, lead zinc, manganese, mercury and arsenic. General survey of instrumental technique for the analysis of heavy metals in aqueous systems. Measurements of DO, BOD, and COD. Pesticides as water pollutants and analysis. Water pollution laws and standards.
UNIT-IV
ANALYSIS OF WATER WATER POLLUTION 4.0 Introduction 4.1 Objectives 4.2
Water Pollution.
4.3
Water Pollutants.
4.4
Origin of waste Pollutants.
4.5 Effects of water Pollutants.
4.5.1 Physico-Chemical Effects. 4.5.2 Biological Effects. 4.5.3 Toxic Effects. 4.5.4 Pathogenic Effects. 4.6
Source of water Pollution.
4.7
Water Analysis.
4.7.1 Colour 4.7.2 Turbidity. 4.7.3 Conductivity. 4.7.4 Alkalinity. 4.7.5 Hardness. 4.7.6 Chlorides. 4.7.7 Sulphate. 4.7.8 Fluorides 4.7.9 Silica 4.7.10 Phosphates
4.8
Analysis of Different forms of Nitrogen
4.8.1 Ammonia 4.8.2 Nitrite 4.8.3 Nitrate 4.9
Heavy water pollution
4.10 Dissolved oxygen (DO) 4.11 Biochemical oxygen demand (BOD) 4.12 Chemical oxygen demand (COD)
UNIT-IV ANALYSIS OF WATER WATER POLLUTION POLLUTION
4.0
Introduction
Pollution:- The term pollution has been derived from the Latin word 'Pollution',
meaning 'defilement from polluere' to soil or defile (make dirty). In recent times the word pollution is used to denote the contamination of water soil or air. 4.1 Objectives
Water pollution is harmful for our life. Water pollution may be divided into ground water pollution , surface water pollution ,river water pollution, lake water pollution , sea water pollution. Water is an indispensable need of life. It is extremely necessary to develop a suitable technology to protect at lest, the quality of drinking water not only against biological hazardou pollutants and biodegradable biodegradable organics. 4.2
Water Pollutions
1.
Water pollution means the presence of any substance in water which affects temporarily or permanently the quality of its usefulness.
2.
The presence of any foreign material which is undesirable and objectionable either solid liquid or gas in water causes water pollution.
3.
Water is polluted when its basic properties are changed it becomes harmful for human health.
4.
Any toxic material in water which changes either chemical or physical properties causes water pollution.
5. 6.
Addition of any unwanted substance which changes the composition. composition. Presence of radioactive substance in water which alters its basic properties gives rise to water pollution".
7.
Any unusual activities of man, which make water unfit for all living being directly or indirectly, causes water pollution.
8.
Reduction of oxygen, addition of pathogens and increase toxicity in water.
4.3
Water Pollutants
A water pollutant pollutant can be defined as a physical, physical, chemical or biological factor causing aesthetic changes or making it detrimental to aquatic life and those who consume water. 1.
In the form of chemicals:- Which remain dissolved in water.
2.
Physical factors:- Among the physical pollutants, heat and radiation are
important which have a marked effect on organisms. 3.
Biological factors:- Certain micro organisms present in water, specially
pathogenic species, cause diseases of man and animal. Classification of pollutant
1.
Common ions, salts or fixed fix ed dissolved soils.
2.
Heavy metals and inorganic toxicants.
3.
Organic material or BOD (Biological oxygen Demand)
4.
Coliforms and other other bacteria and and viruses.
5.
Nutrients particularly, N,P,K.
6.
Pesticides other toxicants.
4.4
Origin of waste Pollutants.
The origin of waste water can be traced to its natural occurrence on the earth formation by transformation and concentration of natural substances. The generation of sewage and waste containing agrochemicals, certain pesticides and surfactants. Many chemicals do not occur in nature and pollution caused by them is entirely man-made. For ex. synthesis of various pesticides, surfactants, radionuclide. 4.5
Effects of water Pollutants
4.5.1 Physico-chemical effects:- A large number of pollutants imparts imparts colour,
taste and order and even unfit for domestic consumption. consumption. The changes is oxygen content, temperature temperature and pH value affect the chemistry of water. The direct addition of nutrients through various sources enhances the algae and other biological growth which when die and decomposes, they further deplete the oxygen. 4.5.2 Biological effects:- The addition of pollutants leads to the shift s hift in flora and
fauna due to homeostatic factors operating in the aquatic system. Most of the fresh water algae are highly sensitive to pollutants and their elimination modifies the preypredatory relationship relationship by breaking down the food chain.This result in change of the whole plants and animals communities. The varieties of organisms decreases leaving presence of only a few tolerate forms in the polluted conditions. 4.5.3 Toxic Effects:- These are caused by pollutants such as heavy metals,
biocides, cyanide and other organic and inorganic compounds. These chemical (heavy metals, pesticides) are toxic to the aquatic organisms and many specially non-biodegradable, non-biodegradable, accumulate in the body of organisms and bio-magnify along the tropic levels causing long lasting effects. 4.5.4 Pathogenic effects:- Besides the chemical substances, a few water like sewage, also contain several pathogenic and non-pathogenic microorganisms microorganisms and viruses. The clostridium porfringers and and streptococcus faecalis causes various various type of food poisoning. Water born disease like cholera typhoid, colitis. 4.6
Sources of Water Pollution
1.
Domestic Sewage:- Sewage consist of water born wastes of the community
and contain about 99% water and 1% of soil. It contain 65% are protein, 25% are carbohydrates, 10% of fats. The major problems associated with sewage spread of enteric diseases, besides organic pollution which leads to oxygen depletion and fish kill. Sewage also contain huge quantities of nutrients in the form of nitrogen and phosphorus even after the secondary treatment that often causes eutrophication. eutrophication. 2.
Agricultural Agricultural wastes:- Agricultural wastes usually originate in the form of
run off from the agricultural fields and animal farms. Modern agricultural uses a large number of chemicals, called agrochemicals, in the form of fertilizers, organic manure, pesticides, nutrients solution and others.
3.
Industrial wastes:- The Industrial waste have the greatest potential for
polluting the recipient water. The nature and composition of industrial waste depends upon the raw materials, processes and operational factors. Metal plating industries, release substantial quantities of heavy metals and cyanide in their wastes. The chemical industries release wastes with highly variable composition composition which are often acidic or alkaline in nature. 4.
Radioactive sources:- The radioactive substance are used for power
industry, heating homes, preserving food, fuelling, transport and as medicines for curing disease. They can also be used to prepare nuclear bomb & nuclear power plant. The wastes from atomic reactors, hospitals etc. are most dangerous because their radioactivity can't be destroy easily at human level. These wastes destroy the aquatic plant and animals to a great extent. They generally cause gene mutationionization of body fluids and chromosomal mutation. mutation. 4.7
Water Analysis
4.7.1 Colour
Pure water has no colour. The presence of humic acids, fulvic acids, metallic ions, industrial effluents may give colour to natural water. Colour can be detrermined by following two methods: 1. 2.
Platinum cobalt Method. Forel-ule colour scale method. 1. Platinum cobalt method
This method gives a quantitative measure of the dominant spectral colour (blue). Colour Standard:- Dissolve 1 gm of crystalline cobalt chloride and 1.245 gm of
potassium chloro platinate platinate in a small quantity of water. Then add 100 ml of conc. then diluted to 1 litre. This solution has a colour value 500 colour unit. Prepare standard by tilting 5.0, 1, 1.5, 2.0 etc. of above solution with distilled water to 50 ml in standard nessler tubes. This solution has colour value of 5, 10, 15 respectively. Indicate the colour value on each tube and protect them. Methods:-
1.
Centrifuge the sample at high speed to remove suspended matter.
2.
Fill standard nessler tube with sample to the same level as that of standard (50 ml)
3.
Compare the colour of sample with that of various standard tubes held vertically above a white surface and find one standard with same colour as that of sample and read the colour value. Note: If the sample shows a colour more than 70 units it should be diluted with distilled water and the colour estimated is multiplied with dilution factor.
2. Forel-ule colour colour scale me that :- This method gives a qualitative recognition
of colour of sample. Materials:- 1. Solution S-I: add 0.5g of CuSO 4.5H2O to 5 ml strong NH 4OH and dil to 100 ml with, distilled water. Solution S-2: Add 0.5 g of K 2CrO4.5H2O to 5 ml of strong NH 4OH and dilute to 100
ml with distilled water. Solution S-3: Add 5g of CaSO 4 .7H2O to 5ml of strong NH 4OH and dil. to 100 ml
with distilled water. Empirical Forel: Ule colour scale is prepared by mixing different proportions of the
above solution (Solution I, II, III). Method
1.
centrifuge the sample so as to remove the suspended matter.
2.
Fill the sample in clean tube and compare its colour with 22 mixtures of the forel-use colour scale.
4.7.2 Turbidity
Turbidity in water is caused by suspended matter like clay, silt, organic matter, phytoplanptons phytoplanptons and other micro scopic organism. Turbidity when caused (i) Largely because as phytoplankton, is considered as an index of productivity, put on the contrary, when caused (ii) because of suspended matter other than phytoplankton, it restricts the light penetration in water resulting in reduced primary production (photosynthesis). (photosynthesis). Material
1.
Nephelometer (Turbiditymeter) (Turbiditymeter)
2.
Sample Tubes: Made of colour said scratch-free glass.
3.
Standard turbidity suspension : Dissolve 1g of hydrazine sulphate in distilled water to prepare 100 ml of solution: (a) Dissolve 10g of hexamethylene tetramine in distilled water to prepare 100 ml of solution. (b) Mix 5 ml of each of the solution (a and b) in 100 ml volumeric flask and allow to stand for 24 hours at about 25 C. Dilute it with distilled water to the mark. This is a suspension having 400 NTU (Nephelometric turbidity unit) and can be stored for about one month.
Dilute 10 ml of above stock solution (of 400 NTU) to 100 ml with distilled water. This standard solution has 40 NTU and can be stored for a week. Method
1.
Set the Nephelometer at 100 using 40 NTU standard suspension. In doing so percent of the scale will be equal to 0.4 NTU turbidity.
2.
Shake the sample thoroughly and let the air bubbles subside.
3.
Take the sample in nephelometer sample tube and find out the value on scale. If the sample has turbidity more than 40 NTU, dilute it, so that its turbidity can be read on the same scale.
Calculation = Turbiding = Mephelometer reading x 4 x dilution factor.
4.7.3 Conductivity
The cell of the conductivity meter or salt bridge is filled with water sample and the electrical conductivity (EC) is measured. It is expressed as Ds/m at 25 0C, and mhos/Cm at 25 0C. The Relationship between conductivity and salt concentration varies some what depending on the ionic composition of the solution the electrical conductivity provides a rapid and resorably accurate estimate of solute concentration. Conductance is the reciprocal of resistance unit is ohm -1 or mhos or siemens(s). The EC is directly proportional to the area (surface area) and inversely proportional to the length (distance). EC [Electrical conductivity] and a/l EC= k a/l a=area, l=lengtn k= proportionality constant called specific conductance. In case a=1 cm2, l-1cm / then conductivity =k Measurement of Electrical conductivity
The instrument consist of an AC salt bridge or electrical resistance bridge and conductivity cell having electrodes coated with platinum black. Principle: A simple wheatstone bridge circuit is used to measure EC by null method.
The bridge consist of two known and fixed resistance r 1,r2 and variable standard resistance r4 and the unknown r 3. The variable resistance r 4 is adjusted until a minimum or zero current flows trough the AC galvanometer. galvanometer. At equilibrium,
r 1 r 2 OR
r 3
r 3 r 4 r 1 r 2
r 4
Since conductivity is reciprocal of resistivity it is measured with the help of r 3
Equipment and reagents 1.Conductivity 1.Conductivity meter and cell 2.Beaker Standard potassium chloride solution (0.0 1m)
0.7456g of dry AR AR grade KCl is dissolved in freshly freshly prepared double distilled distilled 0 water and made to one litre. At 25 C it gives an electrical conductivity of 1.413 in mhos/cm/(ds/m). mhos/cm/(ds/m). This instrument is to be checked and calibrated with this solution. Methods
1.
Take sample in 925 ml beaker.
2.
Warm up the instrument for 20 minutes.
3.
Use 0.01M KCl solution to calibrate the meter.
4.
Rinse the conductivity cell with distilled water and then with the sample.
5.
Temperature and cell constant corrections are adjusted on the conductivity meter, if provided.
6.
Connect the conductivity cell to meter and dip in the sample. Pass the current and adjust the current by rotating the dial in such a way that maximum sensitivity is obtained.
7.
Read the conductivity value in ds/m. Direct reading may be obtained in digital types of meters. Observed values of EC are multiplied by the cell constant (usually given on conductivity cell) and a temperature factor to express results at 25 0C, if instruments are provided with temperature compensations in which reading directly comes at 25 0C. Operating manual must be read before the operation of the instrument.
Sometimes water water shows acidity acidity due to presence of uncombined CO 2, Salts of strong acids and weak bases and mineral acids. For determing the acidity of water, it is titrated with standard solution of strong base by using a suitable indicator. Apparatus :
1.
Conical flask
2.
Pipette
3.
Burette
Reagents :
1.
Phenolphthalein and methyl orange in indicator.
2.
N/100 NaOH Solution
Procedure :
1.
Take 100 ml sample in a conical flask and add 2 drops of phenalphthalum indicator, and titrate it with N/100 NaOH Solution.
2.
Note down the volume used of N/100 NaOH Na OH from burette.
Calculation : Total Acidity as CaCO 3 (mg/litre)
=
used volume of NaOH XN 100 1000 Volume of sample sample taken(ml )
N=Normally of NaOH solution 4.7.4 Alkalinity
Water shows alkalinity due to presence of salts of weak acids, and strong bases the alkalinity in water is caused due to presence of (1) carbonates (CO 3--) (2) Bicarbonates (HCO3-) (3) hydroxides (OH -) Alkalinity can easily be determined by titration using Phenolphathalein Phenolphathalein (work in alkaline pH range above 8.2) or methyl orange indicator (work in acidic pH range below 6.0) Reagents
1.
Phenolphthalum Phenolphthalum Indicator: 0.25% solution in 60% ethyl alcohol.
2.
Methyl Arrange Indicator: 0.5% solution in 95% alcohol.
3.
Standard Sulphuric Acid: Dil. 2.8 ml of Conc. H 2SO4 to 1 litre with distilled
water, dilute 200 ml of this solution to again 1 litre for getting .02 NH 2SO4 standardize this solution. Method :
1.
In a porcelain dish 5 ml of the sample is diluted with distilled water to about 25 ml.
2.
Pink colour produced with a few drops (2-3) of phenolphathalein indicates presence of carbonates and it is titrated with 0.02N sulphuric acid until the colour just disappears because of alkali carbonate being converted to bicarbonate. This burette reading its designated as (Al)
3.
To the colourless from this titration (or to the original sample of water its there was no colour with phenopthalein) phenopthalein)
1-2 drops of methyl orange or or
methyl red indicator are added and the titration continued till the colour changes from yellow to rose red. 4.
Record the final reading. Suppose A ml of acid used for titration with phenolphthalein. B=ml of acid used for total titration (with phenolphthalein and methyl orange both). The observation can be summarised in the following ways. (a) When A=B A 1000 Total Alkalinity of hydroxide (mg/litre) = ml of sample
(b)
When A
1 2
B
Total Alkalinity (mg/litre)=
B 1000 ml of sample
(a) When A=1/2B Total Alkalinity (mg/litre)=
B 1000 ml of sample sample
carbonate alkalinify
1 (b) When A B 2 Total alkalinity, (mg/litre)=
B 1000 ml of sample
Carbonate alkalinity, (mg/litre)= (c)
2A 1000 ml of sample
When A=0 Total alkalinity of Bicarbonate =
B 1000 ml of sample
4.7.5 Hardness
(a)
Temporary Hardness : It is due to the presence of bicarbonates of Ca 2+
and Mg2+ . It may be removed by boiling the water. (b)
Permanent Hardness : This hardness can't be removed by boiling the
water. It is due to the salts of Ca, Mg, Fe, Strontium.
Reagents
1.
Ericherome Black T indicator: This is prepared by mixing 0.5g of erichrome black and about 100g of NaCl in 20 ml of water by warming. The solution is stable for 100 days.
2.
Ammonia Buffer: Take Take 16.9g of NH4Cl in 143 ml. of liquor ammonia and dilute to 250 ml with distilled water. In presence of metallic ions, use borate buffer. Take 20g of barax (Na 2B4O7 10H2O) in 400 ml of distilled water. Dissolve 5g of NaOH and 2.5g of sodium supplied in 50 ml of distilled water, mix with borex solute on and dilute to 500ml with distilled water.
3.
Standard EDTA Solution (0.01N): Ethylene Diaminetetra-acetic acid
disodium salt is prepared by dissolving 2g in distilled water to which 0.05g of magnesium, Chloride is added and diluted to 1 litre. Method:
1.
Take 100 ml of sample in a conical flask and add 1 ml. of ammonia buffer and 2 drops of Erichrome black T indicator. Shake the solution well.
2.
Titrate it with standard (0.01 N) EDTA. The colour Charge is from wine red to blue or bluish green.
3. 4.
At the end point no tinge tinge of red colour colour should remain. remain. Note the volume used for EDTA solution from burette. This is the end point reading.
Calculations:- Hardness (mg/litre) as CaCO 3 can be obtained by
following formula: Hardness (mg/litre) =
Volume of EDTA used (ml) XN 1000 ml of sample taken
N= Normality of EDTA solution. 4.7.6 Chlorides
Chloride is in variably present in small amounts in almost all natural waters. The estimation of chloride may be carried out by Mohr's method, when the electrical conductivity of water sample is greater than one ds/m at 25 0C Reagents
1.
0.02 N sodium chloride 1.170g of NaCl is dissolved in double distilled water and made to 1 litre.
2.
0.02N silver nitrate 3.40g of silver nitrate is dissolved in double distilled water and made up to one litre. This to be standardized against the standard NaCl solution and stored in amber (brown) coloured bottle away from light.
3.
Potassium chromate indicator indicator 5.1 of aq. solution of pure K 2CrO4
Methods :
1.
Take 5 ml of sample in a porcelain dish and dilute it to about 25ml with distilled water .
2. 3.
Add 5 to 6 drops of K 2Cr2O4. Titrate with standard AgNO 3 solution with stirring till the first brick red tinge appears.
4.
Note down the volume of AgNO 3 solution used from burette. Calculation : Chloride in multi equivalent/litre
=
Normalit Normality y of Ag Ag NO3 Volume of Ag AgNO NO3 1000
Chlorides in gram/litre
ml of sample taken
=
Normalit Normality y of Ag AgNO NO3 Volume of Ag AgNO NO3 eq. weight cl- (35.5) ml of sample sample taken
4.7.7 Sulphate
Sulphates are generally found in hard water. Suphate can be determined gravimetrically, gravimetrically, Colourimetrically and Turbdionetrically. Turbdionetrically. The procedure given here is based on EDTA titration described by Jackson (1973) . Reagents
1.
0.02N MgCl2, BaCl2 and EDTA AR grade BaCl 2 (2.44 g/l) can be directly weighed out and dissolved. MgCl 2 being highly hygroscoic, the required quantity of AR magnesium metal of MgCo 3 may be dissolved in a little excess of dilute HCl and made up to the volume. The EDTA solution must be standardized against 0.02N CaCl 2.
2.
Buffer solution : This consist of 8.25g of NH 4Cl plus 5 ml of NH 4OH (Sp. gr. 0.88) in a litre. the amount of NH 4OH may be so adjusted that 10 ml of this solution added to 50 ml of water sample can give pH of 10.
3.
Standard CaCl 2 solution : 0.02N of solution is prepared by dissolving 1.001g of dried CaCO3 (AR) in minimum quantity of dilute (1+3) HCl and made upto 1 litre with water.
4.
Erichrome Black T indicator: 0.5g of the indicator and 4.5g of hydroxylamine hydrochloride dissolved dissolved in 100 ml of 75% ethyl alcohol.
Method:
Take 100 ml of sample and add a few drops of methyl orange indicator and slightexcess of HNO3. Boil the mixture to remove dissolved CO 2. Add 10 ml of standard CO2. Add 10 ml of standard BaCl 2 solution in the boiling solution. Allow to Cool down and make the volume up to 150 ml of clean supernatant liquid into a beaker. Add 1 ml of Buffer solution and some amount of Erichrome Black T indicator titrate with EDTA solution until a permanent blue colour is produced indicating indicating end point. Calculation: Suppose 25ml of sample is taken for precipitation of sulphates then the following formula can be used. 10ml of BaCl2 solution = 10gm of CaCo 3 = 10ml EDTA solution.
SO-24 (mg/litre)=
Titrate value in hardness + value equivalent volume estimated
of BaCl2 used Titre value insulphate determination
0.98
1000 25
Note:
If the sulphate in water is more than 100 mg/litre then volume of barium chloride should be in creased. 4.7.8 Fluorides
Fluorides are more commonly found in ground water than in surface water. The main source of fluoride in water are apatite and mica. The maximum permissible limit of fluoride in drinking water is recommended to be 1.5 mg/kg by WHO. Materials and Reagents:
1.
Spectro photo meter
2.
Alizarin red solution:- Dissolve 354 mg of zirconyl Chloride octahydrate in 600 ml of destilled water. Add slowly 33.3 ml of con. sulphuric acid followed by 100 ml of con Hcl. Cool and further add distilled water to make the volume 1 litre.
3.
Standard Fluroide solution :- Disolve 221 mg of sodium fluoride distilled water and make the volume 1 litre. This stock solution contains 10 mg F/l. Prepare a series of standard fluorides solutions by taking 0,10,15, 20 .......ml stock solution in volumetric flask and diluting it to 100 ml. These contain 0,1,1.5,2 .... mg F/l respectively.
Method:
1.
Take 100 ml of sample in a flask and add 5 ml each of alizarin red solution and zirconyl acid solution.
2.
Wait for 1 hour and then note the absorbance on spectrophotometer at 520 nm.
3.
Run blank using distilled water.
Preparation of Standard Curve:
1.
Run the standard of various concentrations in similar manner and record.
2.
Plot standard curve between concentration and absorbances obtained for standard solutions.
3.
Determine the fluoride content of the sample by comparing its absorbance with standard curve and express the result as mg f/l.
4.7.9 Silica
Silica in water is present as silicate. In concentration in natural water is considerably high. Silica is an important structural constitunt for diatoms and many sponges. The assimilation of silica and subsequent sedimentation by diatoms is the major source of silica in water.
Materials and Reagents:
1.
Hydrochlaric Acid (50%) : Add 50 ml concentrated hydro chloric acid to
50 ml of distilled water. 2.
Ammonium Molybdate Solution (10%) : Dissolve 20g of ammonium
molybdate in distilled water and make the volume to 200 ml. Adjust the pH between 7 and 8 by adding ammonium hydroxide keep the solution in a polyethylene bottle. 3.
Oxalic acid solution (10%) : Dissolve 20g of oxalic acid in distilled water
and make the volume up to 200 ml. 4.
Standard silica solution: Add 0.6714g of sodium fluorosilicate in a little
distilled water and heat to dissolve. Further add distilled water to make the volume 1 litre. This stock solution contains 100mg Sio 3-Si/litre. Dilute the stock solution with distilled water to prepare a series of standard silica solution, (take 0, 5, 10 .... and 50ml) stock solution in 100ml of volumetric flask and make the volume with distilled water. This will give working standards having 0, 5, 10 .... 50 mg Sio 3- Si/litre. Method
1.
Take 50 ml of sample in on Erlenmeyer flask and add 1 ml of HCl and 2 ml of ammonium molybdate solution.
2.
Wait for about 10 min and add a dd 1.5 ml of oxalic acid solution.
3.
Mix thoroughly and record the absorbance on spectrophotometer at 410nm.
4.
Carry out blank with distilled water.
5.
Run standard silica solution in similar manner and record absorbance readings. Plot a standard curve between absorbance and concentration of standard solutions. Deduce the silica content of sample from the standard curve.
6.
Express results in mg Sio 3-Si/litre.
4.7.10 Phosphates Materials and Reagents :
1.
Spectrophotometer.
2.
Per chloric acid 70%.
3.
Phenolphthalien indicator. Dissolve 1.0g of phenolphthelien in 100ml ethyl alcohol and 100ml of distilled water.
4.
NaOH Solution (1 N): Dissolve 4g NaOH in distilled water and make volume
to 100ml.
5.
Reagent A: Weight 1g of ammonium molybdate and 0.02g of potassium
antimony Tartrate in 1000ml volumetric flask. Add 16 ml of concentrated H2So4 slowly by touching outlet point of measuring cylinder to the inner neck of the flask. Add distilled water slowly, shake and make the volume upto the mark. 6.
Reagent B: Weight 0.88g of ascorbic acid and dissolve in 1 litre of reagents A. It should be prepared fresh.
7.
Standard phosphate solution: Dissolve 2.19g of dried anhydrous potassium hydrogen phosphates in Distilled water and make upto 500ml mark. Take 10 ml of this solution and add distilled water to make 1 litre of stock containing 1 mg P/I. Prepare standard phosphorus solution of various strengths by diluting the stock solution with distilled water.
Methods:
1.
Take 25 ml sample in an Erlenmeyer flask and evaporate to dryness.
2.
Cool and dissolve the residue in 1 ml of perchloric acid.
3.
Heat the flask gently, so that the content be comes colourless. Cool and add 10 ml distilled water and 2 drops of phenolphthalic in indicator.
4.
Titrate against sodium hydroxide solution until pink colour appears. Make up the volume to 25 ml by adding distilled water.
5.
Transfer this into 50 ml volumetric flask and 10 ml of reagent B.
6.
Make the volume to 50 ml with distilled water and let the blue colour develop. Wait for 30 minute and record the absorbance on spectrophotometer at 660 nm.
7.
Run simultaneously a distilled water blank in similar manner.
Calculation : P mg/l = 4.8
mg P in 50ml 1000 Volume of sample
Analysis of Different forms of Nitrogen
Nitrogen (Ammonia/Nitrile/Nitrates (Ammonia/Nitrile/Nitrates and organic Nitrogen) Ammonical –Nitrogen is determined as volumetrically as described below:
4.8.1Ammonia:
Colorimetrically as well
Colormetric Method Material and reagents :
1.
Spectrophotomer.
2.
Phenol nitrogen prusside solution: Dissolve 30g of phenol in 1000ml of distilled water. Add 2ml of freshly prepared 1.5% w/v aqueous solution of sodium nitroprsside.
3.
Alkaline hypochlorite, hypochlorite, solution: Dissolve 20g of sodium hydroxide in some distilled water and add 5.4 ml of 10% solution of hypochlorite. Make the volume to 1000ml with distilled water.
4.
Standard ammonium chloride solutions: Dissolve 1.91 g of anhydrous ammonium chloride in distilled water an make volume to 500 ml. This stock solution contain 1 g NH 4+ /L or (1.22g NH3 /L.). /L.). Prepare range of standard solution by taking stock solution as o,1,2,3,4 .... and 6 ml and diluting it with distilled water to 100 ml.
Method:
1.
Take 40ml sample in a 50ml volumetric flask add 4 ml each of phenolnitroprusside phenolnitroprusside solution and alkaline hypochloric solution.
2.
Make up the volume of contents to 50 ml by adding ammonia-free distilled water.
3.
Keep it in a dark place at 25 0C for about 1 hour.
4.
Record the absorbance on spectrophotometer at 62 nm use distilled water as blank.
5.
Process the standard ammonium chloride solution of different concentration in similar way and record the absorbance for each.
6.
Plot the concentration of ammonium ions in sample in mg NH 4+ /L against arrogance and prepare standard curve. Calculate concentration of unknown with the help of curve.
Volumetric Method: Method: Materials and Reagents Micro-Kjeldhal Micro-Kjeldhal distillation assembly.
1.
Hydrochloric acid (0.01N): Dilute 8.34 ml of 12N conc. HCl with a distilled
water to prepare 100ml of 1.0 N HCl. Dilute 10ml of this HCl with Distilled water to prepare 1 litre of 0.01 HCl. 2.
Boric acid cum Indicator solution: Dissolve 4 g of boric acid in 100 ml of
warm distilled water. Prepare 0.5% bromocresol green solution and 0.1% methyl red solution in ethyl alcohol mix Bromo cresol green and methyl red solutions in the ratio of 2:1 to make a mixed indicator. Add 5 ml of this mixed indicator to 100 ml of Boric acid solution. If the colour of solution becomes blue add 0.01N HCl until it terms faint pink to brown. 3.
Borex buffer solution : Add 4g of borax crystals to 100 ml of distilled water
and heat to dissolve. Method
1.
Take 50 ml of sample in micro-kjeldhal distillation flask and 1 ml of borax buffer solution.
2.
Put 5 ml of Boric acid cum indicator solution in a conical flask. Place it below the condensor so that the dip of outlet of condenser is dapper in contents of conical flask.
3.
Heat the kjeldhal flask containing water sample
4.
Continue distillation until about 40 ml of destillate is collected in the conical flask.
5.
Remove the conical flask having distillate which turns blue due to dissolution on of ammonia.
6.
Titrate the distillate in conical flask against 0.01N hydro chloric acid. turning of blue colour to taint pink brown indicates the end point.
7.
Run a blank with distilled water in a similar way.
4.8.2 Nitrite
Nitrites can be determined colourimentrically by EDTA method and sulphonilamide method. The EDTA method in the followings. Materials photometer.
1. 2.
Spectrophotometer. EDTA solution: Dissolve 0.5g of disodium salt of EDTA in distilled water to
prepare 100 ml of solution. 3.
Sulphanilic acid solution : Dissolve 600 mg of sulphuric acid in about 70
ml of concentrated HCl and make the volume of content to 100 ml by further adding distilled water. Naphthylamine Naphthylamine HCl solution : Dissolve 0.6g of Naphtylamine HCl in a 4. little distilled water. Add 1ml of conc. HCl and make the volume to 100 ml by further adding distilled water. 5. Sodium acetate solution : Dissolve 27.2g of sodium acetate in distilled water to make volume to 1 litre. Methods :
1.
Take 50 ml filtered sample in an Erelenmeyer flask and add 1ml of each EDTA Soln Sulphonic acid and naphthylamine HCl one after the other appearance of wine red colour indicates the presence of nitrites.
2.
Record the absorbance of this solution on spectrophotometer at 520 nm.
3.
Carry out blank with distilled water.
4.
Run standard nitrite solution in similar way and record the absorbance for different conc of standard solutions and deduce the nutrite nitrogen content of sample by comparing its absorbance with the standard curve.
4.8.3 Nitrate Phenol desulphonic acid method Materials and Reagents:
1.
Spectrophotometrs
2.
Hot water bath
3.
Phenol disulphide acid: Dissolve 25g as white phenol in 150 ml of Conc. H 2So4 then again add 85 ml of conc. H 2So4. That it for about 2 hours on a water bath, cool and keep the solution in a dark bottle.
4.
Liquor ammonia (Lr grade) : It is diluted with equal volume of water.
5.
Standard nitrate solutions: Dissolve 0.722g of anhydrous patassium nitrate in distilled water to prepare prepare l litre of stock solution. solution. This This stock solution solution contain 100mg NO3 /l.
Method:
1.
Take 25ml of sample in a porcelain dish 58 and vaporate it to dryness on a hot water bath.
2.
Add 3 ml of phenol disulphuric acid and dissolve the latter by rotating the dish.
3.
After 10 minute 15 ml of distilled distilled water is added and stirred with a glass rod.
4.
Add ammonia (1:1) slowly s lowly with mixing till the solution is alkaline as indicated by the development of yellow colour due to the presence of nitrate. Then add another 2ml of ammonia and the volume made up (100 ml) with distilled water.
5. 6.
Intensity of yellow colour is red in the colorimeter at 420nm (blue filter). Add 5 ml of distilled water and 1.5 ml of potassium potassium hydroxide solution.
Total Organic Nitrogen (TON) Materials:
1.
Micro – Kjeldahl distillation assembly.
2.
Digestion mixture. Dissolve 16.25g of potassium sulphate in 200ml of distilled water. Add 0.4g of mercuric oxide and slowly 25ml of conc. H 2SO4 further add distilled water to make the volume 250 ml
3.
Hypo solution: Dissolve 50g of sodium hyroxide in 200ml of distilled water and add 10g of sodium thiosulphate. Make up the volume to 250 ml by adding distilled water.
4.
Boric acid solution : Dissolve 1g of boric acid in distilled water to make 100 ml of solution.
5.
Mixed Indicator : Prepare 0.1% methyl red solution and 0.5% Bromo cresol green solution in 95% ethyl alcohol. Mix the methyl red and bromo cresol green solution in 1:2 ratio.
6.
Hydrochloric acid (0.01N): Dilute 8.3 ml of conc. HCl with distilled water to prepare 100ml of 1.0 N HCl. Dilute 100ml of this 1.0 NH 4 with distilled water to prepare 1 litre of 0.1N HCl. Take 100ml of 0.1N HCl and dilute to 1 litre with distilled water to get 0.01 HCl.
Method:
1. 2.
Take 200ml of sample in an evaporating dish and evaporate to dryness. Add 4ml of digestion mixture to the residue and dissolve it in about 20ml of distilled water.
3.
Heat the solution to fuming for over 15 min and cool.
4.
Transfer the digest to micro kjeldahl distillation assembly and add about 3-5 ml of hypo solution.
5.
Take 5ml of Boric acid solution containing 2-3 drops of mixed indicator in a conical flask and place the flask below the condensor that the tip of outlet of the condenser is dipped in contents of conical flask.
6.
Heat the Kjeldahl flask: Continue distillation for about 10 mm. Remove the concial flask having. destillate.
7.
Titrate the the distillate against against HCl end point point is determined by change of blue blue colour to pink colour.
8.
Also run a blank blank using distilled distilled water in similar way.
Calculation T.O.N (g/k) =
(T B) N 1000 14 Volume of aliquot
Where T= Volume of titrant HCl used against sample (ml) B= Volume of titrant (HCl) used a against blank (ml) N= Normality of titrant (0.0) The atomic weight of N is 14 4.9
Heavy Metal Pollution
Water pollution problem exists every where in the country and are increasing day-by-day around inertial and urban countries. Metal and their toxicity: The power of toxicity varies from metal to metal. The
term toxicology can be defined as a branch of science which deals with the study of adverse and harmful effect of chemical agents on any biological system.
Types of Toxicology: (a)
Clinical Toxicology
(b)
Industrial Toxicology
(c)
Forensic Toxicology
(d)
Environment Toxicology
(e)
Economic Toxicology
1.
Cadmium (Cd): Cadmium does not exist free in nature and there is no
specific are from which it can be obtained. Cadmium is obtained from refining of zinc, and copper copper as by product. product.
Uses:
1.
Cadmium is used in Industries as protective Coating for iron, steel and copper.
2.
In electronic equipments Ni-Cd batteries are used. Toxicity and diseased cadmium dust, fumes and the atmosphere. Cadmium toxicity of ever 50 mg quantity causes various disease in man such as.
3.
Cadmium toxicity is responsible for vomiting, loss of consciousness.
4.
It cause retardation of growth, diformity in bone hypertension impaired kidney functioning, impaired reproductive function and formation of tumor.
5.
Inhaling of cadmium dust or fumes causes chocking of nose, coughing.
2. Chromium : Chromium occurs of chrome iron ore.It is also found in soil and plants. The quality of chromium in a human is almost about 6mg chromium attached with the protein B-glabuline and disturbed in different parts of body like spleen, tests, liver, brain,Heart. Uses:
(a)
Steel manufacturing, Photographic work.
(b)
Production of jet engines
(c)
Preparation of paints, electric cells matches
3.Copper Occurance:In nature, copper copper occurs in sulphide sulphide ores copper is present present in liver and
brain in blank pepper its is present as at 53 ppm whereas in oysters its quantity is 137 ppm. toxicity and disease copper is an industrial health nazard. More than 470 mg copper in human body is toxic and causes many disorders in the body. 4.
Lead
Occurance: ores are galena, and its sulphide. Lead is present in the bones the
concentration of lead increases with age lead is not an essential metal for mammalians. Lead is usually deposited in bones and some soft tissues. It is also retained by animal in lever. Kidney muscle etc. Under some Specific conditions conditions lead becomes Stimulatory and enhances (a) protein synthesis (b) DNA Synthesis (c) cell replication. Toxicity and Disease: About 800 mg lead creates toxicity inhuman beings resulting in lead poisioning. Due to lead paisioning a number of body disorders are caused. Generally, lead toxicity is due to the concentration of diffusion of Pb in soft tissues Another possible common mechanism. For Pb toxicity is formation of metallotheonic. 5.
Zinc
Occurance: Zinc is not found in free form, it is present in ores. These are
(a)Sulphide (b) Silicate ore. In human body (i)
Muscles – 65% Zinc
(ii)
Bones – 20% Zinc
Toxicity and diseased : More than 165 mg zinc causes some disorders in human
body. It cause Vomitting, cramps, Nausea. 6.
Manganese
Occurance: Manganese occurs in nature as oxide as
(a) pyrolusite (b)
Magnatite
(c)
Braunite
(d) tephroite Manganese is also found in:1.
Sea-water-an amount of | ppb.
2.
In human body about 12 mg. Mn is used in industry in the formation of (a) Alloys (b) Dry cell Batteries (c) Fire works (d) glass and cermanic Industries
Mn is less toxic but Mn is toxic when its in concentrate form. it is more than 100 ppm in human body it causes following disorder and Blindness, fever etc. 7.
Mercury
Occurance: Mercury occurs as native metal mixed with its ores. The human body
contain about 13 mg mercury 70% of which present in muscles tissues. In human body mercury is found in kidney, lever, Intestinal and colon walls, brain, heart and lungs. Toxicity and disease mercury and its salts are severe health hazards. It is toxic more than 100mg causes body disorder. Inorgonic form of mercury is also injurious to health. Mercury is converted into methyl mercury salt and dimethyl mercury by micro organisns, which escape into atmosphere. Blood serum protein from complex are responsible for killing of fish in rivers and oceans. From Industries, CH 3Hg Settle as sediment at the bottom of water bodies from where fish trap. Mercury salts. If such types of mercury contaminate fish are used in food they cause neutrotoxic minanata disease. Body tissue retention is greater for CH 3Hg+ than for Hg2+ salts. The toxic action is due to crowding of Hg 2+ ions around the immediately available thiol groups of proteins and delay in distribution of these ions among rest of thiol group throughout the body. 8.
Arsenic
Occurance: Arsenic occurs in nature as a brittle metal. It is present in following
forms: (a)
in marine water – 5 ppb
(b)
in earth's crust- 2 ppb
(c)
in human body - about 18 mg found in tissue
(d)
in blood- about 25 gm.
Arsenic is found in living body particularly particularly in hair and spleen, in erythrocytes, erythrocytes, here arsenic binds globin part of naemoglobin. Compounds of arsenic are absorbed easily by skin of human being. Toxicity and Disease : Arsenic trioxide AsO 3 is more toxic than As 2O5. Its large quantity in human body (more than 25mg) cause many disease, like diarrhea, Vomiting, Nausea, Nausea, Skin Eruption, Eruption, Inflammation, Death. limits of some heavy metals in drinking water are prescribed by the Government of India. Metals
Chronium Copper Manganse Zinc Iron
Permissive limit Excessive limit – 50
1000 5000 – 300
3000 15000 200 1000
4.10 Dissolved Oxygen (DO)
Dissolved Oxygen (DO) in water is an index of physical and Biological processes going on non-polluted surface waters are generally saturated with dissolved oxygen.
1.
Diffusion from air/absorption from air
2.
Photosynthetic activist within water by vegetation etc. Oxygen is consider to be a limiting limiting factor, especially in lakes lakes and in waters with a heavy load of of organic material material organisms have specific oxygen requirement. requirement. Low Low dissolved oxygen may prove fatal for many organisms for their survival.
Oxygen meter method: Material :
1.
Oxygen meter with dissolved oxygen probe
2.
Electrical stirrer
3.
5% sodium sulphite solution
Methods
Read the operation manual carefully and adjust the instrument accordingly. Dip the Do probe in 5% Sodium sulphate solution with constant stirring. Now dip the D.O. probe in water. Sample being constantly stirred and record the dissolved oxygen in mg/litre from the scale. Winkler's Method Principal : Oxygen combines with Mn(OH) 2 and form higher hydroxides which on
subsequent acidification in the presence of iodine, liberate iodine in an amount equivalent to the original dissolved oxygen content of the sample. Materials and reagents:
1.
BOD bottles (100-300ml)
2.
Manganous sulphate solution: Dissolve 100g of manganous sulphate in 200ml of previously boiled distilled water and filter the solution.
3.
Alkaline potassium iodine solution. Weight 50g of KI, and 100g of potassium hydroxide. Dissolve the chemicals in 200ml of previously boiled distilled water.
4.
Sodium thiosulphide solution (0.025N): Dissolve 6.205g of sodium, in one litre previously boiled distilled water and add a pallet of NaOH as a preservative. Keep it in coloured bottle.
5.
Starch Indicator: Dissolve 1g starch in 100 ml warm distilled water and add a few drops to toluene as preservative.
6.
Concentrated sulphuric Acid: Sp. gravity 1.84.
Method :
1.
Take a glass stoppered BOD bottle of known volume (100-300ml) and fill it with sample avoiding any bubbling. No air should be trapped is bottle after the stopper is placed.
2.
Open the bottle and pour is each 1 ml of manganese sulphate and alkaline potassium Iodide solution using separate pipettes. If the volume of sample is over 200 ml add 2 ml of each reagent instead of 1 ml.
3.
A precipitate will appear. Place the stoper and shake the bottle thoroughly. Sample at this stage can be stored for a few days, if required.
4. 5.
Add 2ml of of sulphuric acid to dissolve the the precipitate, precipitate, shake thoroughly. thoroughly. Transfer gently avoiding bubbling whole content, or a known part of it in a conical flask put a few drops of starch indicator. Titrate against sodium trisulphate solution and note the end point when initial blue colour disappears.
Calculation :
(i)
If whole content is used for titration D mg/l =
(ii)
V1 N 8 1000 V2 V3
If a fraction of the content is used for titration D mg/l =
V1 N 8 1000 V V4 (V2 3 ) V2
Where D.O - Dissolved Oxygen V1 = Volume of titrant ( ml 1) 1) V2 = Volume of sampling sampling bottle after placing placing the stopper stopper (in ml) V3 = Volume of manganous manganous sulphate + potassium potassium iodide added V4 = Volume of fraction fraction of the content used for filtration (ml) N = Normality of titrant (0.025) the equivalent weight of oxygen
is 8.
4.11 Biochemical oxygen Demand (BOD)
As the amount amount of oxygen required required by micro-organisms micro-organisms to to stabilize biologically biologically decomposable decomposable organic matter in the waste water under aerobic conditions. Tests of BOD : This can be evaluated by measuring oxygen con. in 9 sample
idometrically before and after incubation in the dark at 20 0C for 5 days. Excess dissolved oxygen must be present during the whole incubation. Sometimes a culture of bacteria is also added so that more of the organic matter is used up during the incubation. Materials and Reagents:
1. 2.
BOD incubator, BOD bottles All reagents used in determination of dissolved oxygen as discussed in foregoing pages.
3.
B.O.D. free water: Pass the deionized glass distilled water through a column of activated carbon and redistilate.
4.
Phosphated buffer solution : Dissolve 42.5g potassium dihydrogen phosphate in 700 ml BOD free water and add 8.8g NaOH. Adjust the pH at 7.2 add 2g ammonium sulphate and dillute to 1 litre with BOD free water.
5.
Magnesium sulphate solution: Dissolve 82.5g of magnesium sulphate in BOD free distilled water to prepare 1 litre of solution.
6.
Calcium chloride solution : Dissolve 27.5g of anhydrous calcium chloride in BOD free distilled water to prepare 1 litre of solution.
7.
Ferric chloride chloride solution: solution: Dissolve Dissolve 0.25g of ferric chloride in in 1litre of BOD free distilled water.
8.
Sulphuric acid (1N): Add 2.8 ml of conc. sulphuric acid to 100 ml of BOD free distilled water.
9.
Sodium hydroxide solution (1N): Add 4g of sodium hydroxide in BOD free distilled water and make the volume 100 ml.
10.
Aruglthiourea solution : Dissolve 100 mg of Aruglthiourea in distilled water and make the volume 1 litre.
Method
1.
The required volume of BOD free distilled water in a glass container by bubbling compressed air for 1 to 2 days to attain dissolved oxygen saturation. After saturation it is kept at 20 0C for at least one day. Add per litre of this water 1 mL each of phosphate buffer solution. If required, add requisite amount of seed (sewage) also.
2.
Dilution of sample : Adjust the pH of sample to neutral. Dilute the sample with appropriate amount of water according to the expected BOD current of the sample.
3.
Fill two sets of BOD Bottles and add 1 ml of Aruglthiourea solution to each bottle. As for as possible avoid entrapping air bubbles in BOD bottles.
4.
Stopper the bottle immediately.
5.
Determine the dissolved oxygen content. Take out the battles after 5 days and determine immediately their dissolved oxygen content (Ds) Calculations : BODs (mg/l ) = (D.O-Ds) ×Dilution ×Dilution factor Dissolve Dissolve oxygen after 5 days days sample factor.
4.12 Chemical Oxygen Demand (COD) The amount of oxygen required for
oxidation of organic compound which are present in water by means of reaction. Substances such as potassium dichromate and potassium permangate, potassium dichromate ion is most suitable oxidant.
Principle : Organic matter decomposes poses and produces carbon dioxide and
water when it is boiled with a mixture of potassium dichromate and sulphuric acid. Amount of potassium potassium dichromate dichromate in the sulphuric sulphuric acid medium and the excess dichromate is titrated against ferrous ammonium sulphate (FAS).
Material and Reagents:
1.
COD reflux unit consisting of flat bottom flask with ground glass Mouth and leibig's condenser.
2.
Hot water bath or heating mantle.
3.
Potassium dichromate solution (0.25N): Dissolve 12.25g of LR grade potassium dichromite previously dried at 103 0C in distilled water.
4.
Dry powder of silver sulphate.
5.
Conc. H2SO4.
6.
Ferroin Indicator solution: Dissolve 0.695 g of ferrous sulphate and 1.485g as 1110-phenethroline in distilled water to make 100 ml.
7.
Standard ferrous ammonium sulphate solution (0.25N): Dissolve 98g of ferrous ammonium sulphate in Distilled water, dilute add 20 ml of sulphuric acid, cool and dilute to 1 litre by further adding distilled water to standardize this solution/dilute 25 ml of potassium dichromate solution to about 250 ml with distilled water, add 20 ml of sulphuric acid and cool it.
Normality of FAS =
Volume of K 2Cr 2 O7 0.25 Volume of FA FAS S (mL)
Methods :
1.
Take 20 ml of sample in the flask of reflux unit and add
10 ml of
patassium dichromate solution, pinch of each silver sulphate and mercuric sulphate and 30 ml of H 2SO4 acid. 2.
Attach to the moush of flask and heat the flask on a hot water bash or heating mantle mantle for at least 2 hours to reflux the contents. contents.
3.
Cool the flask, detach from unit and dilute its contents to about 150 ml by adding distilled water.
4.
Add 2-3 drops of forroin indicator solution and titrate against ferrous ammonium sulphate solution. At the end point blue green colour of contents changes to reddish blue. Calculation : COD (mg/Le)=
(B T) N 1000 8 Volume of sample (mL)
QUESTIONS
1.
What is the origin of waste water ? Describe the water pollutants and their effects.
2.
What are the sources of water pollution? Give details about industrial wastes as source of pollution.
3.
How do the pesticides and radioactive wastes pollute drinking water ?
4.
How can the BOD be evaluated e valuated in a water sample?
5.
Determine the presence of heavy metals in water by atomic absorption spectrophotometer.
BLOCK-I UNIT-V ANALYSIS OF SOIL, FUEL, BODY FLUIDS AND DRUGS
(a)
Analysis of Soil: Moisture, pH, total nitrogen, phosphorus,
silica, lime
magnesia, manganese, sulphur and alkali salts. (b)
Fuel Analysis : Solid, liquid and gas. Ultimate and proximate analysis
heating values-grading of coal. Liquid fuels-Flash, aniline point octane number and carbon residue. Gaseous fuels-producer gas and water gas-calorific value. (C) Clinical Chemistry : Composition of blood-collection and preservation of
samples. Clinical analysis. Serum electrolytes, blood glucose, blood urea nitrogen, uric, albumin, globulins, barbiturates, acid and alkaline phosphatases. Immunoassay : principles of radio immunoassay (RIA) and applications. The blood gas analysis - trace elements in the body. (D) Drug Analysis : Narcotics Narcotics
Screening
by
measurements.
gas
and
and dangeorous drugs. Classification of drugs.
thin-layer
chromatography
and
spectrophotometric
UNIT-V ANALYSIS OF SOIL FUEL, BODY,FLUIDS AND DRUGS 5.0 Introduction 5.1 Objective 5(a) Analysis of Soil
5a.1 Introduction 5a.2 Determination of moisture 5a.3 pH measurement 5a.4 Analysis of total nitrogen 5a.5 Analysis of phosphorus 5a.6 Analysis of silica 5a.7 Analysis of lime 5a.8 Analysis of Magnesium 5a.9 Analysis of Manganese 5a.10 Analysis of Sulphur 5a.11 Analysis of Alkali-salts 5(b) Fuel Analysis
5b.1 Solid, Liquid & Gaseous Fuels 5b.2 Gaseous Fuels 5b.2.1
Producer Gas
5b.2.2
Water Gas
5b.3 Analysis of Coal 5b.4 Flash Point 5b.5 Aniline Point 5b.6 Octane number 5b.7 Calorific Value 5(c) Clinical Chemistry
5c.1 Composition of blood 5c.2 Collection and and preservation preservation of samples samples of blood 5c.3 Analysis of Serum electrolytes 5c.4 Estimation of serum proteins (Albumin and globulin) 5c.5 Estimation of glucose 5c.6 Estimation of blood blood urea nitrogen 5c.7 Estimation of Uric Acid in serum serum 5c.8 Determination of barbiturates 5c.9 Estimation of serum serum alkaline phosphatase 5c.10 Immunoassay 5c.10.1
Principles of Radio Immunoassay Immunoassay (RIA)
5c.11 Blood gas analysis anal ysis 5c.12 Trace Elements in the body
5d
Drug Analysis
5d.1 Narcotics and dangerous drugs 5d.2 Classification of drugs 5d.3 Screening of drugs by gas and thin-layer thin-layer chromatography and spectrophotometric measurements. 5c.3.1
Spectrophotometric Spectrophoto metric determination determinati on
5c.3.2
TLC determination
5c.3.3
Gas Chromatographic Chromatogra phic determination determinati on
5.2
Let us sum up
5.3
Check your progress : The Key
5.4 References 5.5 Activity
UNIT-V ANALYSIS OF SOIL FUEL, BODY FLUIDS AND DRUGS
5.0
Introduction
Soil, fuel, body fluids and drugs are non-separable parts of human-beings & their livlihood since since time immortal. Without Without body fluids existence of of human body is not feasible. Soil forms the base upon which we live. Food we eat is produced on the soil. Fuels are the need for cooking our food, for providing energy to us, for the transportation, for generating electricity. Fuels are needed to heat boilers for the industries. Drugs cure ailments occurring in body. Studies in these fields are a must. 5.1
Objective
Clinical analysis are needs to check proper functioning of body. Any imbalance in the proportion of constituents of body fluids leads to abnormal condition called disease. Therefore, analysis of total blood, plasma and serum is need for the diagnosis of diseases. Soil analysis should be done to make up deficiencies of soil to increase productivity. Fuel analysis helps us to chose good fuel & is needed for safety needs. Drugs analysis of synthetic as well as natural drugs helps us to chose proper drug for disease. Analysis of natural drugs leads to way for the discovery of new and safer drugs. Taking into consideration importance of soil, fuels and drugs as well as body fluid constinents, their study and analysis is essential, which will form subject matter of this unit-V, i.e. will be the objective of our study. 5a.
Analysis of soil
5a.1 Introduction
Soil is as much essential as the food. We draw our food either from plants or from animals. Plants grow in soil, and animals also depend upon plants and indirectly draw their food from soil. Main components of soil are moisture, nitrogen, phosphorus, silica, lime, magnesia, manganese, sulphur and alkali metal salts etc. Thus soil may be acidic or basic. A brief study of origin of soil may reveal its
components, therefore, its composition. Before proceeding to analysis of soil a conceise study of soil & hence its composition will be useful. Formation of Soil
Soil might have formed mainly by three important process occurring in rocks :
1.
1.
Physical weathering
2.
Chemical weathering
3.
Biological weathering
Physical Weathering
Physical weathering i.e. disintegration of soil exerts mechanical effect on the rocks as a result of which rock might have broken down into particles of smaller size. Physical weathering does not bring any chemical transformation of rock minerals. This process occurs in deserts, at high altitudes, high latitudes and in topographic relief as well as the rocks where vegetation is poor. The temperature, water, ice, gravity and winds are some climatic factors responsible for physical weathering. e.g. temperature may bring about the break down of heterogenous rocks. Water may be responsible for mechanical weathering of rocks by rain water. Freezing and melting of ice causes weathering of rocks by frost fr ost action and glacier formation. Landslides and rock slippages due to earthquake fall in the category of gravitational weathering where rocks are disintegrated by abrasion and forces of
impact. Stromy winds carry suspended sand particles & may cause abrasion of exposed rocks. 2.
Chemical Weathering
Exposed rocks undergo chemical weathering which causes chemical decomposition or transformation of parent mineral into new mineral or secondary minerals. For instance, chemical weathering converts Feldspar (Primary mineral of aluminium and silicon) into clay (a secondary mineral). It is worth noting that chemical weathering is not effective in deserts as moisture and air are essential for chemical weathering. Few processes processes involved in chemical weathering are are delineated below : (i)
Formation of solution
Water soluble minerals like lime stone and gypsum etc., get weathered by the solvent action of water which is enhanced in presence of CO 2 and organic acids. Later come into existence by the decay of organic remains of animals and plants. Solutions of these minerals are either absorbed on the surface of negatively charged colloidal particles or are removed by leaching. (ii)
Hydrolysis
Hydrolysis involves chemical action of water over strong bases and produces hydroxides of iron, aluminium, magnesium & calcium etc. e.g. K 2 Al Al2Si6O16+CO2+2H2O 4SiO2+Al2O3.2SiO2.2H2O+K 2CO3 Primary Mineral
Kaolin (Sec. clay mineral)
Hydrolysis releases Na, K, Ca, Mg and silicates into the solution. These are responsible for the growth of plants. (iii)
Carbonation
Carbonation involves the combination of CO 2 and H2O to carbonic acid which combines with hydroxides of Ca & Mg as well as other minerals of rocks leading to the formation of carbonates and bicarbonates. e.g. CO2 + H2O
H2CO3
Ca (OH)2 + CO2 CaCO3 + H2O CaCO3 + H2O + CO2 Ca (HCO3)2 (iv)
Oxidation
Oxidation involves reaction of minerals with oxygen leading to the formation of oxides which get dissolved in water & weaken the rock. This process bring about the weathering of rock e.g. 4 FeO + O 2 2 Fe2O3 Oxides and sulphides of iron, manganese and aluminium are easily oxidized and lead to chemical weathering of rocks. (v)
Reduction
Fe2O3 (red ferric oxide) can be reduced to FeO (grey ferrous oxide). Reduction generally occurs in deep zones of earth crust. 2Fe2O3 4 FeO + O2 (vi)
Hydration
During hydration water gets attached to rock material as water of crystallization. This process leads to softening as well as increase in volume of original material. Hydration also lead to weathering. 2 Fe2O3 + 3 H2O 2FeO2.3H2O 3.
Biological Weathering
Micro-organisms like bacteria, fungi, protozoa, lichens and mosses transform rocks into a dynamic system where energy is stored & organic material is synthesised. This process changes physical structure as well mineral composition of rocks. From the process of soil formation it is clear that chief constituents of soil are moisture, oxides, hydroxides, silicates, lime, salts of magnesium, aluminium, alkali metals etc. It contains organic compounds & may be rich in nitrogen & sulphur. Moreover, soil may be acidic as well as basic. 5a.2 Determination of moisture
Methods generally used for the determination of moisture contents in soil are : (i)
Gravimetric method
(ii)
Volumetric method
(iii)
Electrical conductivity
(iv)
Gamma rays attenuation
(v)
Neutron scattering
(vi)
Soil moisture tension
(vii)
Time domain refractromety Volumetric and gravimetric methods are simplier and are more popular
methods which are discussed here :
(1)
Volumetric Method
Volumetric determination of moisture contents in soil may be done by (i) Soil core (ii) Bulk density of soil (i)
By Soil-core
Requirements:
(1)
Sample tube
(2)
Physical balance
(3)
Moisture boxes
(4)
Drying oven
Procedure:
1.
Place the sample of soil in sampling tube whose volume (v) is known.
2.
Weigh the sample is moisture box (A).
3.
Dry it in oven to constant weigh (B) at 105 C and again weigh it.
Calculation:
Moisture percentage ( Mv)
A B dw V
100
Here, dw = density of water Volume of sampling sampling tube = r2h r = radius h = height N.B.
1.
For better results take more than 50 C.C. in size and greater than 200 g of soil.
2.
Analysis of 10 samples/hectare samples/hectare is enough during investigations.
(ii)
Bulk-Density Method
Moisture percentage on volume basis (Mv) = Mw Bd
Mw = Moisture percentage on dry wt basis Bd = Bulk density of soil (mg/m 3) (2)
Gravimetric Method
For gravimetric estimation of moisture content soil sample is kept at 105 is oven and is dried to constant weight. Difference in weight of moist soil and dry soil gives wt of moisture. Formula used: % moisture
Loss in weight dry weight soil
100
Requirements:
(1)
Sample auger
(2)
Physical balance
(3)
Moisture cans
(4)
Drying electrical oven
(5)
Desiccator
(6)
Drying agent.
Procedure:
1.
Weight the empty moisture can.
2.
Transfer about 100 g of sample in the sample can with the help of auger.
3.
Close the moisture can immediately immediatel y to prevent loss of moisture through evaporation.
4.
Weight the moisture can immediately.
5.
Remove the caps of moisture can and heat in oven at 105 to constant weight. It takes about 45 hrs.
Precaution:
After each heating cool the moisture cans in desiccator containing drying agent.
Observations:
(i)
Wt of empty moisture can = X gm
(ii)
Wt of moisture can + Moist soil sample = Y gm
(iii)
Wt of moisture can + dry soil = Z gm
Calculations:
Moisture contents in soil = Y - Z Wt of dry soil = Z - X Perce Percenta ntage ge of moisture
(Y Z ) ( Z X )
100
Result:
Soil sample under investigation contains _______ % of moisture. 5a.3 "pH Measurement"
pH is negative logarithm of hydrogen ion concentration. pH =-log [H+] pH of soil ranges from 0 to 14 accordingly soil may be acidic or alkaline. Soil with pH 1 to 7 is acidic, with exact 7 is neutral and soil with pH range 7 to 14 is alkaline. Word pH derives it origin from French word" pouvoir hydrogen" mean hydrogen power. pH of soil provides important information about properties of soil. It affects solubility of soil as well as plant growth as later depends upon activity of micro-organisms. Nitrogen fixing bacteria of soil responsible for growth of legumes are not very active in acidic acidic soil. Bacteria causing causing decomposition decomposition of organic organic matter of soil are ineffective in strong acidic medium. However, fungi survive in acidic medium. Soil of pH range 4-5 have toxic concentrations of manganese and aluminium. Tea, blue berries, Green berries, few confers as well as rhododandrons grow is strongly acidic soil. But beans, alfa, beets and barley grow in slightly acidic to moderately basic soil on account of high calcium demand and little tolerance of aluminium. Minerals are soluble in acidic soil as compared to basic or neutral soil.
Measurement of pH
Two important methods for determination determination of pH of soil are : (1)
pH - meter method
(2)
Colorimetric method
(1)
is generally used.
pH-Meter Method
For measuring pH of soil pH meter used which is a glass electrode pH meter with calomel reference electrode with salt-bridge. Modern digital pH meters are single electrode pH meters which require calibration before use with buffer solutions of known pH. A glass surface in contact of H + ions pH of which is to be measured acquires an electrical potential & gives H + ion concentration i.e. pH of solution. Requirements:
(i)
Glass electrode pH-meter
(ii)
Beaker
(iii)
Glass rod
(iv)
Distilled water (D.W.)
(v)
Standard buffer solution
Preparation of buffer solutions of standard pH
These solutions may be of pH 4.0, 7.0 or 9.2 in distilled water. To prepare them tablet available in market may be used. A single Tablet is dissolved in double distilled water and volume is make up to 100ml. In case, standard buffer is not available saturated solution of potassium hydrogen tartarate (A.R.) may be used which gives a pH of 3.6 at 25 C. Determination of pH of soil:
pH of soil can be determined in three forms of soil solutions: (i)
pH in Saturated Soil Paste
1.
Prepare the paste of the soil in small amount of distilled water in a beaker with the help of a spatula.
2.
A saturated paste glisters & flows upon tilting the beaker.
3.
After preparing saturated paste cover it & allow it to stand for approximately four hours.
4.
Now there should be no free water on the surface of soil and the paste should not stick or loose its glistening. From this paste pH can be determined.
5.
If soil loses it shinning more soil may be mixed to paste.
(ii)
pH in 1:2 Soil Water Suspension
1:2 soil-water suspension may be prepared by adding 80 ml of distilled water to 40g of soil in 250ml Erlenmeyer flask & mixing the contents on reciprocating shaker for 1 hr. During the process as well as after preparing the suspension flask should be stoppered. (iii)
pH in 1:2 Soil and Calcium Chloride Solution Suspension
1.
Place 20g of air dry soil in a 100 ml beaker.
2.
Add 40 ml of 0.01 M-CaCl2 solution to it. CaCl2 solution can be prepared by dissolving 14.7g CaCl 2.2H2O in 10 litre water. pH of this solution must be checked which should be between 5.0-6.5. This pH may adjusted with Ca (OH)2 or HCl.
3.
Now leave the contents so that CaCl 2 solution get absorbed by the soil. It is worth mentioning do not stir the contents during absorption process. But after the absorption has taken place stir thoroughly for 10 seconds with the help of glass rod.
4.
Do further stirring four or five times after 5 minutes gaps.
5.
Allow the suspension to settle down for 30 minutes.
Procedure:
1.
Place saturated solution of soil or 1:2 soil-water or soil-CaCl2 suspension in a beaker or conical flask.
2.
Set the temperature compensating compensating knob of pH-meter and also ensure if the electrode is completely filled with saturated KCl solution. Let the pH-meter to warm for 15 minutes so that asymmetric potential of instrument get eliminated.
3.
Take standard buffer solution say that of a pH of 7 in a beaker & immerse both the electrodes or one electrode in case of combined electrode into the buffer solution. With the help of knob adjust instrument reading at the pH of buffer (7 in this case). Now remove the buffer & wash the electrode thoroughly & carefully with distilled water. Similarly, adjust the pH-meter at the other pH say that of 9.2. After adjusting pH-meter at 9.2 again electrode is washed well with distilled water.
4.
Now read the pH of soil paste or 1:2 soil-water or 1:2 soil-CaCl2 suspension with the pH - meter calibrated c alibrated between 7.0 and 9.2 in step-3.
5.
After step-4 again electrodes/electrode electrodes/electrode are washed with distilled water & then immersed in distilled water. To maintain electrodes in working condition they should be kept immersed in distilled water.
5a.4 "Analysis of Total Nitrogen"
Nitrogen is major plant nutrient of soil and is >90%. It exists in soil as ammonium, nitrate as well as in organic matter and is essential for plant growth. Total nitrogen of soil can be determined by Autoanalyzer. Theory:
Soil sample is first digested with H 2SO4 so that organic nitrogen get converted in NH+4 ions. N-N and N-O nitrogen can't be converted completely by Kjeldhahl digestion. However, in soil very little nitrogen is in this form. But, it contains nitrities and nitrates. Requirements:
1.
Digestion block [20- place block digester with tractor auto temperature controller].
2.
A 250 ml digestion tube [295 x 40 mm in dimeter].
3.
Conc. H2SO4[18M], 96%.
4.
Kjeltab : Each tablet contains 3.5g K 2SO4 and 0.4 g CuSO 4.5H2O.
Procedure:
1.
Place ~ 2g of mineral soil with low content of nitrogen (60 mesh) into digestion tube after accurate weighing and add 10 ml. of conc. H 2SO4 with shaking.
2.
Now heat the digestion tube in digestion block until very black [for about 30 minutes]. Digestion block should be loaded in fume-cupboard to avoid irritation.
3.
Add one Kjeltab to the contents. Heat till Kjeltab dissolves at 200 C which takes about 15 minutes.
4.
Increase the temperature to 300C and heat for 30 minutes.
5.
Now, let the temperature rise to 375 C and continue heating till sample turns turquoise which may take upto 45 minutes.
6.
Take out digestion tube from block and allow to cool for 5 minutes. Cooling should not be done in heating block, because ammonia formed from ammonium sulphate formed during the digestion may be lost by the action of heat.
7.
Mix the sample with 50 ml of water till sample goes into the solution.
8.
Analyse as per the instructions of analyser.
Alternative Method
In Kjeltc Auto 1030 Analyses method ammonia liberated by the distillation of digest with strong alkali is absorbed in unstandardized H 3BO3 to get Ammonium borate. Borate is titrated back to H 3BO3 by titration against standard acid (HCl).
Requirements:
1.
Kjeltec Auto 1030 Analyser
2.
Distillation apparatus
3.
Titration Apparatus
4.
40% NaOH; 10g NaOH in .25 litre H 2O
5.
H3BO3 solution : 100g H 3BO3 is 10 litre water.
6.
Receiving Solution : To above
H3BO3 solution add 10 ml
Bromocresol solution solution (100g in 100 ml CH 3OH). Add 70ml 7 0ml methyl red to it (100mg in 100 ml CH 3OH). To this solution also add 5 ml of 4% NaOH. 7.
Standard HCl (0.01m).
Procedure:
1.
Make up digest to 100ml
2.
Follow instructions of operation of Kjeltc 1030 Analyser.
3.
Set Alkali pump to deliver 30ml of 40% NaOH.
4.
Titrate with 0.01M-HCl
Calculations:
% of nitrogen in sample = 1.401 (T B) Molarit Molarity y of standard HCl Weight of oven dry sample sample ( g )
Here, T
=
Total Volume (ml) of standard HCl used for the titration of sample
B
=
Standard HCl for titration in blank (ml)
Check your progress-I Notes:
(a)
Write your answer in the space give below.
(b)
Compare your answer with those given at the end of the unit. (i)
Can moisture in soil be determined gravimetrically. gravimetrically.
(ii)
pH of acidic soil is 10. Ture/False.
(iii)
To make the soil basic _____________ is added.
(i)
_________________________________________________
(ii)
_______________________________________________
(iii)
_________________________________________________
5a.5 "Analysis of PHOSPHORUS"
Phosphorus is present in the soil to the extent of 0.01 to 0.3% and is present in different forms. Primary minerals of apatite group are the original source of soil phosphorus. It is found as hydroxy-phosphate of Fe, Al, Ca, Mg, Mn and Ti. Phosphates of Ca, Fe and Al are most important quantitatively. Out of total soil phosphorus only a little amount is available to plants. Out of various chemical methods available to determine available P Olsen method is most important. Olsen's Methods
In this method NaHCO 3 of pH 8.5 is used as an extractant. Reaction between phosphate and ammonium molybodate is the basis of this method: H3PO4+12H2MoO4 H3P (MO3O10)4 + H2O Molybdophosphoric acid [H 3P(Mo3O10)4] formed by the reaction of phosphate and ammonium molybdate is then reduced to blue coloured complex through the reaction with ascorbic acid. Now, absorbance is recorded at 730 nm on a spectrophotometer. A standard curve prepared by using standard solutions is used to determine concentration of phosphates in the sample. Requirements: (i)
0.5 M NaHCO 3 solution (Extracting solution)
To prepare it dissolve 42 g of NaHCO 3 in 1 litre of distilled water (D.W.) and adjust the pH at 8.5 with 1M- NaOH solution. (ii)
Dacro G-60 or equivalent grade phosphorus free Charcoal
80 g of Dacro G-60 or phosphosrous free Charcol is used to prepare slurry in D.W. It is kept overnight in 1 lt. of 6 M- HCl in 60 mm diameter columns (250ml capacity) and then deionized water is added to it till leachate is free from chloride. Then, these contents are dried at 110 C. (iii)
Ammonium Molybdate Solution
Prepare it dissolve 40g of ammonium molybdate in 1 lt. of D.W. (iv)
Ascorbic Acid Solution
Dissolve 26.4g of L-ascorbic acid in 500 ml of D.W. (v)
Sulphuric Acid (2.5M)
Add 140 ml of conc. H 2SO4 in 1000 ml of D.W.
(vi)
Antimony Potassium Tartrate Solution
Dissolve 1.454 g of Antimony potassium tartrate in 500ml D.W. (vii) Murphy-Riley Murphy-Riley Developing Solution
To prepare it mix 75ml Ammonium molybdate solution, 250 ml of 2.5M H2SO4, 50 ml L-ascorbic acid solution and 25 ml of antimony potassium tartrate in 100 ml D.W. (viii) p-Nitrophenol p-Nitrophenol Indicator (ix)
Standard solution of Phosphorus [Stock solution]
Dissolve 0.439gm of KH 2PO4 in 500ml D.W.; and 25 ml of 7 N-H 2SO4 to it and make up the volume to 1 lt. with D.W. This solution is 100 ppm stock solution of phosphorus 5ml of it are diluted to 100 ml in volumetric flask to get 5 ppm phosphrous solution. (x)
Spectrophotometer.
Procedure:
1.
Take 2.5 gm accurately weighted air-dried soil sample in 125 ml flask.
2.
Add to it a little phosphorus free Darco G-60.
3.
Now add 50 ml of NaHCO3 solution at 25 C and shake the contents for 30 minutes.
4.
Now apply steps 1-3 to blank [without soil].
5.
Filter the extract through whatman 42 or 40 filter paper.
6.
Pipette out 10 ml of aliquot in 50 ml volumetric flaks & add 10 ml of D.W. to it followed by 1 drop of p-nitrophenol indicator. Acidify contents to the pH 5.0 using 2.5 M-H 2SO4 drop by drop till colour disappears.
7.
Add 8 ml Murphy - Riley solution & make up the volume to 50 ml with D.W. Now, keep the contents form 15 minutes at R.T. & record the absorbance of blue coloured solution on spectrophotometer or colorimeter at 730 mm.
Calculations:
Wt of soil sample = 2.5 g Volume of NaHCO NaHCO3 extractant added = 50 ml. Vol. of extract extract taken to develop develop colour = 1o ml. Reading of colorimeter or spectrophotometer = X Concentration Concentration of phosphorus in 50 ml extract = C/10g/ml. In 1 g of soil concentration of phosphorus C
50
10 2.5
g / ml
Hence, available phosphorus in Kg/ha
C 10
50 2.5
2.24
= C 4.48 ppm in Kg/ha. or Available p
C vol . of extrac tan t / aliquol 2.24 wt of soil
Here, C = g P in aliquots (obtained (obtained from curve) Preparation of Standard Curve :
For the preparation of standard curve take 1, 2, 3, 4 and 5 ml of 5 ppm solution in 50 ml of volumetric flasks & making up the volume. Record the absorbance on colorimeter or spectrophotometer as given in procedure above. Prepare standard curve by plotting absorbance Vs concentration of phosphorus in standards. 5a.6 "Analysis of Silica"
It is reported that silicon contents enhance length and number of stems of rice plants as well as their fresh and dry weights. Perhaps they also increase productivity productivity of barely and cucumber. Silicon is second most important element and is present in most of the minerals. Soil contain large number of silicates. Gram and to
some extent other plants also contain silicon. Analysis of total silicon contents involves following steps. 1.
Digest soil with perchloric and nitric acids followed by heating for 15 minutes.
2.
Dilute the digest and filter silica through whatmann no. 41 filter paper. Wash it with warm and dil HCl (1:20) as well as with D.W.
3.
Evaporate the filtrate [containing washings as well] to dryness and heat the residuce at 110C for an hour. This step recovers small amount of silicic acid present in solution.
4.
Now extract the residue with dil HCl, filter and wash as given as step 2.
5.
Ignite the combined residue along with filter paper in a weighed platinum crucible in muffle furnace till constant weight and find out the weight of Crude silica. From the weight of silica amount of Si present in soil can be determined by multiplying with the factor 0.4672. Analysis of water soluble Silicon : Quantity of water soluble silicon in soil
can be determined by molybdenum blue method. Requirements:
1.
Standard silicon solution
2.
10% W/v ammonium molybdate solution
3.
0.25 M-HCl
4.
Na2SO3 solution containing 17g dm 3 Anh. Na 2SO3.
5.
Citric acid.
Procedure:
1.
Extract soluble Si from soil with water and filter.
2.
Pipette out 25 ml of clear filtrate in 15 ml conical flask and add to it 15 ml of dil HCl.
3.
Add 15 ml of ammonium molybdate molybdate solution after 6 minutes to the contents of flask followed by 2 ml of citric acid & 25 ml of Na 2SO3 solution.
4.
Mix the contents well and record optical durity (O.D.) at 650 mm exactly 1 minutes after adding reductant.
5.
Standard curve needed to estimate Si can be prepared to cover the range 0-5
g/ml of Silicon. 5a.7 "Analysis of Lime"
Lime increases pH of soil. Optimum degree of soil acidity requirements vary from crop to crop. Lime in soil increases supply calcium to plants. Lime requirement of soil can be determined by shoe maker method (also known as Buffer method). Shoe Maker Method Requirements:
(i)
p-Nitrophenol p-Nitrophenol - 1.8 g
(ii)
Triethanol amine - 2.5 ml
(iii)
K 2CrO4 - 3.0 g
(iv)
(CH3COO)2Ca - 2.0 g
(v)
CaCl2.2H2O - 40.0 g
To prepare Buffer dissolve above salts in 800 ml of D.W. and adjust pH at 7.5 with the help of dil HCl or NaOH solution. solution. Dilute it to to 1 litre with D.W. Procedure:
1.
Add 10 g of soil to 20 ml of above Buffer and mix well for 10 minutes by stirring.
2.
Record pH of suspension and determine lime requirement of soil from observed decrease in pH of Buffer using curve prepared from the data of shoemaker. For the initial quantity of soil taken as 50g divide the result by 5.
5a.8 "Analysis of Magnesium"
Magnesium can be titrated with EDTA using Eriochrome black T dye as indicator at pH -10 in presence of NH 4Cl and NH4OH buffer. Completion of titration is indicated by the change of colour from wine red to blue or green. Requirements:
(i)
EDTA - 0.01 N.
(ii)
NH4Cl - NH4OH buffer : To prepare it dissolve 67.5 g NH 4Cl in 570 ml of conc. NH3 and make up the volume to 1 lt.
(iii)
Eriochrome black T indicator
(iv)
2% NaCN solution.
(v)
Carbamate
Procedure:
1.
Pipette out in a 150 ml conical flask 25 ml aliquot containing not more than 0.1 ml of Ca+ Mg. If solution in more concentrated it should be diluted.
2.
Add 2-5 Crystals of Carbamate & 5 ml NH 4Cl - NH4OH buffer
3-4 drops of
Eriochrome black - T indicator. 3.
Titrate the solution in conical flask with 0.01N EDTA till colour charges from wine red to blue or green.
Calculations:
If N1 & V1 are normality and volume of aliquot taken and N 2 & V2 are normality & volume of EDTA used. Then, N1 V V1 = N2 V V2 N 1
or
N 1
N 2V 2 N 1
Volume of EDTA EDTA Normalit Normality y of EDTA Volume of aliquot tkaen
N1 = Normality = equivalent of Ca 2+ + Mg2+ in one lt.
Milli equivalents of Ca2+ + Mg2+ /lt.
Volume of EDTA Vo Vol l . of EDTA1000 Volume of aliquot tkaen
Milli equivalents (Me) of Mg 2+ =Me[Ca 2+ + Mg2+] - Me of Ca 2+ + Mg2+ g/lt.
Normality of EDTA vol. of EDTA Eq. wt. of Ca 2+ + Mg2+ = Vol. of aliquot aliquot (ml) taken taken Since , Me/lt. eq. wt. / 100 = g/ lt g/lt of Mg 2+ = g/lt of Ca 2+ + Mg2+ - g/lt of Ca 2+ Importance of Mg:
Mg is known as secondary micronutrient for plant growth & is required from the beginning in small amount. It is found dissolved in water as soluble salts. Mg is integral part of chlorophyll and is also involved in enzymatic reactions. It helps to increase vitamin, sugar, starch and insulin in root crops. Its deficiency causes chlorosis.
5a.9 "Analysis of manganese (Mn)"
Mn is micronutrient and is determined by using DTPA (Diethylene Triamine Pantaacetic Acid) which is a chelating agent for the combination of free metal ion present is pollution. Requirements (Apparatus)
(i)
100 ml capacity narrow mouth polyethylene stopped bottles.
(ii)
20 ml pipette
(iii)
Reciprocating Reciprocating electric shaker.
(iv)
Whatman No. 1 or No. 42 Filter paper.
(v)
Funnels.
(vi)
Atomic Absorption Spectrophotometer Spectrophotometer (AAS)
(vii)
Hollow cathode lamp of Mn.
(viii) Analytical balance. Reagents Required: (i)
Extracting Solution
0.005 -M DTPA + 0.01-M CaCl 2.2H2O+ 0.1 M TEA (Triethamol (Triethamol amine with pH = 7.3) (ii)
Stock Solution
Standard stock solution should be prepared by dissolving 0.1 g foil or wire of Mn in dil HCl (1:1) and making up the volume to 1 lt. (iii)
Working Standard Solutions
These are prepared by transferring 0, 1, 2, 3, 4, 6 and 8 ml of strock solution to 100 ml volumetric flask and diluting each to mark with DTPA extracting solution. These solutions contain 0, 1, 2, 3, 4, 6, & 8 g/ml (ppm). Procedure:
1.
Weigh 10 g air dried soil sample.
2.
Transfer it to 100 ml narrow mouth Polyethylene bottle or conical flask Add 20 ml DTPA solution.
3.
Stopper them & shake at 25 C for 2 hrs.
4.
Filter the contents through whatman No. 1 or No. - 41 filter paper.
5.
Filtrate is used for analysis.
6.
Keep a blank (without soil) for analysis also.
7.
Set zero of instrument with blank.
8.
Place standards in AAS to standardize it and read absorbance.
9.
Feed DTPA extract & record absorbance or concentration of Mn.
10.
Repeat above steps.
Calculations:
Wt of soil taken = 10 g. Vol of DTPA solution added added = 20 ml. Dilution = 2 times. Absorbance shown shown by AAS = A.
Concentration of micro nutrient (Mn) read from standard curve against Absorbance A = C. Mn is soil sample = C 2 mg/kg or ppm or g/ml. N.B. Standard curve is drawn between readings of absorbance & concentrations of
Mn in ppm on graph paper.
Importance of Mn
It is a constituent of enzyme & is involved in protein synthesis. It increases translocation of sugars through stems and leaves of cotton plant to improve the fibre strength of cotton. Mn also favours the accumulation of ascorbic acid in grape leaves and of sugar in grapes. Excess of Mn leads to chlorosis and nacrosis of leaves. 5a.10 Analysis of Sulphur
For the determination of S, heat soluble sulphur, 0.15% CaCl 2 extractable sulphur and phosphate extractable sulphur methods are used. Heat soluble sulphur method is more common. Requirements:
(i)
1% NaCl solution
(ii)
BaCl2.2H2O solution : 20-30 mesh crystals.
(iii)
Stablizing Solution : 75 g Nacl, 30 ml Conc. HCl, 100 ml absolute alcohol,
50 ml glycerol and 250 ml D.W. (iv)
Standard Sulphate Solutions : 1.080 g K 2SO4 dissolved in D.W. and made
up the volume to 100 ml. (v)
3% H2O2.
Procedure:
1.
Place 5.0 g accurately weighed soil in a silica basin and add 20 ml D.W. to it.
2.
Keep the basin in gently boiling water and evaporate to dryness. Then, heat it in hot air oven at 102 C for 60 minutes.
3.
Cool the contents and transfer the soil in 50 ml centrifuge tube. Extract it with 33 ml of 1.0% NaCl solution.
4.
Pipette out 25 ml of aliquot in silica basin and evaporate to dryness with 2 ml of 3% H2O2. Again heat it in hot-air oven at 102 C for 60 minutes.
5.
Cool the residue and add to it 25 ml D.W., transfer again to centrifuge tube and centrifuge centrifuge to remove suspended particles. From From this aliquot sulphur is determined.
6.
Place 10 ml aliquot in 250 ml flask and add to it 20 ml D.W.
7.
Now, add 2.5 ml stabilizing solution, 0.2 g BaCl 2 crystals by a spatula to the standard and extract. Shake the flask for 1-minute.
8.
After about 3 minutes measure the turbidity in colorimeter using blue filter or in spectrophotometer at 340 nm.
9.
Prepare standard curve by plotting absorbance of standard solutions against their concentrations and read the concentration corresponding to absorbance of soil sample.
Calculations:
SO4 - S mg/Kg
S (mg / l ) in aliquot volume of extract Vol Vo l . of aliquot Wt of oven day soil
Estimation of S by Colorimetic Method
[Palaskar et al. (1981) method] In this method S is extracted in 0.15% CaCl 2.2H2O solution in 1:5 soil to solution ratio. 10-20 g of soil is used and mixture is shaken for 30 minutes. S is estimated in 5-10 ml aliquot obtained by extract filtered through whatmann No. 42 filter paper. Requirement from p-36 of original. Procedure:
1.
Pipette out 5 ml of 0.1 N Barium chromate in 150 ml volumetric flask. Add 1.2 ml 5N-NH4OH to it which reduces free acidity to 0.05N. This solution should be clear.
2.
Aliquot of soil extract is added to it to keep SO 4-S content below 2000 g. Shake the contents and keep for half an hour.
3.
Now add 1 ml NH 4OH to precipitate unreacted barium chromate & make upto 100ml with D.W.
4.
Stopper the flask and make upside down 3-4 times. Filter the contents through whatmann - 42 filter paper. Reject first few ml fitrate.
5.
Record absorbance of yellow solution in colorimeter using deep blue (420 mm) filter and determine concentration of S using standard curve.
N.B.
1.
pH of test solute should be around 2-3 before addition of Ba-chromate. If solution is alkaline it should be adjusted with 6N-HCl.
2.
For the preparation of standard curve 0, 4, 8, 16 and 20 ml of 100 ppm solution of SO 4-S is made up to 100 ml in volumetric flasks with D.W. These solutions have 0, 4, 8, 16 and 20 ppm S concentrations. Color intensity of these solutions is recorded on photoelectric colorimeter using deep blue (420 mm) filters and intensity of blank is also recorded. Intensties are plotted Vs concentration and concentration of sulphur is determined.
Important Information
S is present in soil in both organic as well as inorganic forms, but only a fraction is available to plants. Inorganic sulphur is in the form of SO 4--. 5a.11. Analysis of Alkali-Salts Alkali-Salts
Alkali salts make the soil saline or alkaline. In aqueous solution they are present in their ionic concentrations. Water soluble alkal-salts are determined in Soil by:
(1)
(1)
Saturation extracts
(2)
1 : 2 soil water extracts.
Saturation Extract Method
Procedure:
1.
Weigh 200-400 g of soil accurately.
2.
Take it in weighed flask and add sufficient D.W. to it with mixing to prepare saturated solution.
3.
Allow to stand for 4 hrs. If soil has stiffness and does not glistens, add more D.W. and mix well.
4.
Weigh the container and contents. Record increases in weight which corresponds to amount of water added.
5.
Calculate the saturation percentage (SP) gravimetrically. gravimetrically. SP
6.
Water content of Saturated Soil Pa Paste ste Oven day wt of soil
100
Now transfer the soil to Buchner funnel with highly retentive filter paper & collect the extract - by applying vacuum till air passes through filter. Add one drop of 1.0% (NaPO 3)6 solution per 25 ml of extract to check the precipitation of CaCO3.
7.
Store the extract at 4C till it is analysed.
(2)
1:2 Soil-Water Extract Method
Procedure:
1.
Weight 25 g of soil in `100ml beaker.
2.
Add 50 ml D.W. to it & shake on shaker for 30 minutes.
3.
Filter the suspension through whatman No. 1- filter paper & collect the filtrate.
4.
This filtrate is used for the determination determination of alkali metal salts.
N.B.
(1)
The cations [Ca2+, Mg2+, Na+, K +] are normally determined by compleximetric titration, AAS or atomic emission spectrometric methods.
(2)
Primary anions in soil extracts are normally SO 42-, CO 2-2, Cl -, NO 3- and HCO3-. These are determined by titrimetric or Atomic emission spectrometric methods.
Check your progress-II Notes:
(a)
Write your answer in the space give below.
(b)
Compare your answer with those given at the end of the unit. (i)
Can moisture in soil be determined gravimetrically.
(ii)
pH of acidic soil is 10. Ture/False.
(iii)
To make the soil basic _____________ is added.
(i)
_________________________________________________
(ii)
_________________________________________________
(iii)
_________________________________________________ 5b.
"FUEL-ANALYSIS"
5b.1 Solid, Liquid and Gaseous Fuels
The materials which when burnt produce heat energy are known as fuels. For instance; Coal, charcoal, wood, LPG, Kerosene, petrol and diesel etc. Fuels are concentrated store houses of energy which is released as heat when fuels are burnt. They are infact, most important sources of energy in daily life; for example, in homes, transport and industry. Important Important uses of fuels are: 1.
In homes for cooking & other heating purpose. Fuels like LPG, Wood, Coal, Kerosene, Cow-dung & Charcoal etc., are used for this purpose.
2.
For transporting men and material from are place to other through motor cars, trains and aeroplanes etc. Petrol and diesel are used for this purpose.
3.
Fuels used in industry are coal, fuel oil or natural gas which are used for heating the boilers.
4.
For generating electricity coal or natural gas are used in thermal power plants.
5.
In rockets used for exploring space: Special fuels known as propellants are the fuels for this purpose. For instance, synthetic rubber and liquid hydrogen are rocket fuels. Most of the fuels are carbon compounds containing hydrogen. Therefore,
when a fuel is burnt, it combines with O 2 of air and produces CO 2, water vapours and a lot of heat. Some fuels like Coke and Charcoal are mainly carbon. Three main sources of natural fuels are; coal, petroleum and natural gas.
Classification of Fuels
Fuels can be classified in following ways. 1.
As solid, liquid and gaseous fuels.
2.
As natural or raw fuels and manufactured or processed fuels.
3.
As Primary and secondary fuels.
(I)
Classification of Fuelss as Solid, liquid & Gases
This classification is based on physical state. Examples of each are:-
1.
2.
3.
Solid Fuels
Liquid Fuels
Gaseous Fuels
Wood
Coal
Coke
Charcoal
Paraffin Wax
Kerosene
Petrol
Diesel
Alcohol
Liquid hydrogen
Liquid fuels
Natural gas
LPG
Coal gas
Producer gas
Gobar gas
Acetylene
Hydrogen gas
Other gaseous fuels
Merits of Liquid & Gaseous fuels over solid fuels
1.
No residue is left after burning liquid & gaseous fuels, but solids leave behind ash.
2.
Liquid and gaseous fuels can be transported through pipe - lines.
3.
Ignition temperatures of gaseous and liquid fuels are low, therefore, they burn easily as compared to solid fuels.
4.
Liquid & solid fuels have high calorific values, hence, produce more heat on burning than equal amounts of solid fuels.
5.
Liquid and gaseous fuel produce no or little smoke.
6.
Handling of liquid and gaseous fuels is easier than solid fuel. For instance, it is easier to handle kerosene than coal.
(II)
Classification of Fuels as Natural Natural or Raw Fuels and Manufactured Manufactured or Processed Fuels
Fuels that are used in the same form as they occur in nature are called natural fuels or raw fuels. e.g., Wood, Coal, natural gas, petroleum,
animal drug and agricultural wastes.
On the other hand, fuels which are prepared by natural fuels by various physical and chemical processes are known as manufactured or processed fuels e.g. fuels like charcoal, coke, petrol, diesel, kerosene, coal gas, producer gas & water water gas etc. etc. Charcoal is prepared by heating natural natural fuel wood by heating in absence of air.
(III) Classification of Fuels as Primary Primary Fuels and Secondary Fuels: Fuels:
Fuels which are used directly to produce heat are known as primary fuels e.g., Wood, Coal, Petroleum, natural gas, agricultural wastes etc. Infact, primary fuels are raw fuels.
The fuels which are prepared from primary fuels are known as secondary fuels. Infact, they are processed fuels e.g., producer gas, coal gas and water
gas etc.
Choice of fuel depends upon factors like cost, calorific value, convenience, side effects like pollution as well as availability. Most of the fuels we use come from bio-mass and fossils and are known as Bio-mass fuels and fossil fuels,
respectively. It is worthnoting waste materials of living objects (like cattle dung) and dead parts of living objects (like animals, plants and trees) are Bio-mass. But, fossils are remains of pre-historic animals or plants, buried under the earth, millions
of years ago. (i)
"Solid Fuels"
Ingredients of solid solid fuels are to to two types: types: (i) Combustible Combustible organic matter matter and (ii) Incombustible matter. Combustible organic matter includes carbon, hydrogen and sulphur; but non-combustible part of solid fuel involve moisture and minerals like silicates, phosphates, carbonates, sulphides of Ca, Fe, Mg, Al, K etc. Non-combustible materials of solid fuels are responsible for the formation of ash. Solid fuels are classified into: (1)
Natural solid fuels.
(2)
Artificial solid fuels.
(3)
Industrial solid fuels.
(1)
Natural Solid Fuels
Some example of natural solid fuels are
Wood Coal Natural Solid
Li nite Ba asse Peat
(i)
Wood
Wood is the fuel found in plenty in India. It contains cellulose, lignin, resin, inorganic material and water [to the extent of 60%]. Approximate composition of dry wood is C=50%, O=35%, N=70%, H=6%, & ash=2% Calorific value of Wood (dried) varies from 4000 - 6400 BTU/pound. Following products are obtained by the destructive distillation distillation of dry wood. Product
Percentage by weight
1.
Charcoal
23.5%
2.
Pyroligneous acid
44.3%
3.
Wood tar
5.3%
4.
Gases: CO, H2, CH4 and CO2, N2 etc.
26.9%
Dry wood is better fuel on account of its low ignition temperature and low ash contents. Advantages of wood as a fuel are:
1.
As it gives bright flame, it can be used for burning other fuels.
2.
It is easily available.
3.
It is suitable for domestic purpose.
4.
Wood ash is rich in potassium contents, hence, can be used in agriculture.
5.
Because, wood gives smaller amounts of ash and soot it can be used in some industries like glass, firing of porcelain etc.
Disadvantages of wood as fuel are:
1.
High moisture contents keep its calorific value low. So, it is rarely used in industry.
2.
It's smoke is harmful for the health of eyes.
(ii)
Coal
Coal is complex mixture of compounds containing carbon, hydrogen, oxygen and also contain some free carbon. Some nitrogen & sulphur compounds are also f ound in coal. Coal is found in deep coal mines beneath the surface of earth. India is rich in coal in states like Bihar, West Bengal, Orissa and Madhya Pradesh.
Jharia and Bokaro are rich coal mines found in Bihar, whereas Ranigang is in west Bengal. Coal is still an important source of energy in our country, therefore, it is regarded as backbone of energy sector of our national economy. Coal can be converted into other sources of energy like : electricity, synthetic petrol, coal gas etc. It is a rich source of organic compound which can be converted into dyes, drugs, polymers like fibres, explosives, detergents and paints etc. Original of Coal
It is thought that coal was formed millions of year ago by the decomposition of large land plants and trees buried deep inside earth. Forests might have been buried under the surface of earth due to Volcanoes and earthquakes etc and in due course they were covered with sand, clay and water. At high temperature and pressure and in absence of air inside the earth, wood might have been converted into coal. This slow chemical process of conversion of wood into coal is named carbonization. Carbonization is very slow process and may have taken
thousands of years to take place. As carbonization is slow process several intermediate products of carbonization exit. They are: anthracite, bituminous, lignite and peat. Anthracite is last step of coal formation. These products differ in percentage of carbon. Anthracite is richest in carbon. Type of Coal
Based upon the concentration of carbon following variation of coal are formed depending upon the extent of carbonization: carbonization: Type of Coal
Percentage of Carbon
a.
Peat
60%
b.
Lignite (Soft coal)
70%
c.
Bituminous (House hold coal)
80%
d.
Anthracite (Hard coal)
90%
(a)
Peat
Peat is formed by the gradual decomposition of plant materials under moist conditions. This is the initial state of transformation during the coal formation. Peat is jelly like and is brown colored. Calorific value of peat is low (6000-9000 BTU) hence, it is not of any economical value. In dry peat ash contents are 2.5-6.0%. Peat is sometimes used as fertilizer as well as packing material. Deposits of peat are
found in England, Scotland, Iceland, Northern Europe, Germany, Sweden & Russia etc. In India peat reserves are found in Nilgiri Hill, Palani Hills, in west coast of Tamil Nadu and in Kashmir state. In dry condition c ondition composition composition of peat is C
=
57%
H
=
6.1%
O
=
34.9%
Non-combustible mass or ballast of peat contains appreciable moisture contents 60-70%. On destructive distillation, it yields (NH 4)2SO4, fuel gas, tar, wax and hydrocarbon oils. (b)
Lignite (Soft Coal)
It is also known as brown coal and its composition is between peat and bituminous coal. Lignite is amorphous, fibrous or woody in texture. Moisture contents are high in it and calorific value is 4000-6000 cals/Kg. It is used to heat boilers & evaporating pans in industry & homes. Its reserves are in Assam coal field & Neyveli. Neyvali lignites are compact and are used in thermal power generation and also in the manufacture of fertilizers. Whereas Rajasthan liginte is less compact & fibrous. Its non-combustible material (ballast) are high. On exposure to air it absorbs oxygen readily & ignites spontaneously. Upon carbonization it yields tar, synthetic petrol by high pressure hydrogenation. hydrogenation. It is superior to peat. (c)
Bituminous Coal
Formation of Bituminous Coal is third stage of coalification. Bituminous coal is of two types: (i) Sub-bituminous coal (ii) Bituminous coal (i)
Sub-Bituminous Sub-Bituminous Coal
Sub-bituminous coal is also known as black lignite as it is black is colour. Its calorific value is 7000-15000 BTU. Moisture content of Sub-bituminous coal ranges from 10-25%. It ignites readily.
(ii)
Bituminous Coal
It is hard, black & dense. Bituminous coal is of two types: low volatile coal & high volatile coal. Fuel ratio of low volatile at coal is < 2. Bituminous coal burns with yellow smoky flame. High volatile variety have long flames and find use in gas industry, coal-tar distillation and in the manufacture of glass. Light grade bituminous Super-bituminous coal. Its calorific value range is 12000-15000 coal is known as Super-bituminous
BTU. Super-bituminous coal have higher fuel ratio as compared to bituminous coal & have best heating power among all coals. Bituminous coal can be converted to coke. (d)
Anthracite
Anthracite contains highest percentage of carbon [~ 90%]. It is hard, compact & black with sub-metallic lusture and is of fossil origin. Anthracite is last product of carbonization process. It is sulphur-less is low-ash coal & burns with smokeless flame. Calorific value range of anthracite is 14000-15000 BTU and fuel ratio is 9.0-10.2. Anthracite is used in metallurgy, in slow combustion stoves as well as for domestic purpose. (e)
Pulverised Coal
Pulverised coal is obtained after grinding the fossil coal. It burn quickly & its combustion temperature temperature is very high. Burning capacity of this coal depends upon the fineness of particles. Pulverised coal has low ignition temperature because of presence of volatile material in it. The pulverization procedure includes crushing, drying & grinding of coal. (iii)
Bagasse
Bagasse (the residuce of sugar industry) is used as fuel for generating steam in the boilers of sugar industry as well as in some other industries like paper.
(2)
Artificial Solid Fuels
Artificial fuels are obtained by the processing of natural fuels. Some artificial solid fuels are describe below :
Briquette Artificial
(a)
Briquette
Briquette is compressed fuel. It is prepared from coal, peat, lignite or coke in presence of binding material like tar-pitch, asphalt pitch or molasses (5-8%) at an applied pressure of 1800-3000 psi. It is dried to 50% moisture contents & in further dried by blowing through tubular driers. Briquette is domestic fuel but sometimes is used in industries also. It's calorific value in 8000 BTU/pound. Brequetting can also be carried out without binder under a pressure of 12 tonnes or more per square inch. (b)
Charcoal
Charcoal can be prepared by the destructive distillation of wood or heating the wood in limited supply of air. It can also be made in Kilns and in India charcoal is prepared generally by this method. Generally hard wood is carbonised and gas, tar and aqueous liquors are obtained, in addition to charcoal. Yield of charcoal is 3035%. Gases obtained are 20-23%; tar and oil are 5-7%. Aqueous distillate contains acetone, methanol and acetic acid. Charcoal contain litte volatile matter hence gets easily ignited & burns with non-smoky flame. It is good fuel containing small amount of sulphur and phosphorus. It is used in metallurgy as well as for domestic purpose & also for making carbon disulphide. Its caloric value is 6000-8000 Cals/Kg. (c)
Coke
Coke can be prepared by the destructive distillation of coal in coke-oven at about 500-750C. Low temperature carbonization produces semicoke with volatile matter 5-15%. Semicoke is used for domestic purpose as such or after brequtting or in the form of pulverized coal. High temperature carbonization (900-1400 C) produces hard-metallurgical coke with high mechanical strength but low calorific value. Best coke contains about 90% carbon and less than 8% ash.
(3)
Industrial Solid Fuels
Fuels used in industry are known as industrial fuels. Some of them are:
Fossil Fossil coal coal Peat Peat Industrial Industrial Fuels Fuels
Fur Fu Feun Feunac rnace naace cee SSla Sla la ss Oil Oil Shales Shales Meta Me Meta Metall tall llur llur ur ur ical ic ical ical al Coal Co Coal Coal al
2.
Liquid Fuels
(i)
Kerosene
The composition of Kerosene oil is between C 10 to C12 hydrocarbons with the boiling range 170-250 C. It is used as a domestic fuel in stoves for cooking and for lighting purpose in lanterns. Kerosene oil is also used for making gaseous fuel " The carburetted water gas."
(ii)
Petrol
It is also known as gasoline. Petrol is a mixture of C 5 to C10 hydrocarbons and its boiling range is 40 C to 170 C. Petrol is a fuel for motor cars, motor cycles, scooters and other light vehicles. Petrol is also a good solvent & is also used in drycleaning as well as in making petrol gas. Its octane No. ranges 60-70. (iii)
Diesel
Diesel is a mixture of C 13 to C15 hydrocarbons and its boiling range is 250350C. It is a fuel for heavy vehicles like trucks, buses, ships and diesel enzines etc.
Diesel is also used to run water pumps for irrigation as well generators which generate electricity on small scales for houses, agriculture and industry. (iv)
Fuel oil
Fuel oil contains hydrocarbon containing 15-18 carbon atoms. Boiling range of fuel oil is 350-400 C. It is used in furnaces as well as to heat boilers. Advantage of using oil fuel is that it leaves no residuce.
(v)
Alcohol
Power alcohol is used as fuel when blended with petrol in internal combustion combustion enzymes. Ethanol can't be used as such as a fuel but is used as an additive. Octane number of alcohol is 95. When alcohol is mixed mix ed with petrol it increases its Octane No by 0.9 unit for percentage volume of alcohol. Therefore, it is blended with petrol by using blending reagents like benzene, ether and tetralin. Blending can't be done without blending reagent. Alcohol is blended because of its anti-knocking property & can be used in enzines with higher compression ratio. However, alcohol reduces calorific value of petrol.
5b.2 Gaseous Fuels
Some gaseous fuels are : Natural Gas Producer Gas Gaseous Fuels
Coal gas Blue Water gas Semi water gas Carburetted Water Gas Ethyene Aectylene
(i)
Natural Gas
Natural gas consists of mainly methane with small quantities of ethane & propane. It contains about 95% of methane. Natural gas occurs deep under the crust of earth either alone or alongwith oil above the petroleum deposits. Thus, some well dug into the earth produce only natural gas while others produce natural gas as well s petroleum oil. In latter case, natural gas is by-product of petroleum mining. In India, we have a number of gas fields. Some recent ones are located in Tripura, Jaisalmer, off-shore area of Bombay and in Krishna Godvani delta. Natural gas is formed under earth by the decomposition of vegetable matter lying under water. Decomposition occurs through anaerobic bacteria in absence of oxygen. Natural gas obtained from oil wells is of two types: (a)
(a)
Dry Natural Gas
(b)
Wet Natural Gas
Dry Natural Gas
It is obtained from the wells which contain no crude petroleum. It is composition composition is CH4=96%, C2H6=0.8% and N 2=3.2% (b)
Wet Natural Gas
This type of natural gas is obtained from oil wells and contains high boiling hydrocarbons hydrocarbons like n-propane, n-butane, iso-butane and iso-pentane also. Sometimes helium gas is also found is natural gas. Calorific value of natural gas ranges 950-1100 BTU/cubic ft. This gas is also formed by the fermentation of organic matter in sewage which is mixture of : CH 4 = 70% & CO 2=30% and calorific value of which is 62.5 BTU/cubic ft. Natural gas obtained by the fermentation of cow-dung is used as fuel gas also.
Properties of Natural Gas
It buns with smoky flame in ordinary burners & it can be liquified under pressure & cooling to 121 C. Its 200 volumes can be absorbed on 1 Vol. of fuller's earth at -121C. Advantages of Natural Gas
1.
Natural gas being a complete fuel in itself can be used directly for heating purposes in home & industries. Nothing needs to be added to it.
2.
It is a good quality fuel with high calorific value. It leaves behind no residue & also burns with smokeless flame. Moreover, gases produced by burning it are not poisonous.
3.
A great advantage of natural gas is that it can be supplied directly from the gas well to homes and factories for burning through undergrounds pipelines. This eliminate the need for additional storage and transport. The construction of pipelines for natural gas was earlier thought to be
expensive, but it is economical in long run. Once, a pipeline has been laid, there is no need to spend money on the construction of storage tanks for gas or for transporting the gas by rail or road. Earlier, pipeline for natural gas was constructed in Baroda city. Uses of Natural gas
1.
It is used as domestic as well as industrial fuel.
2.
Natural gas is used as a source of hydrogen needed in fertilizer industry. Upon heating the natural gas strongly methane present in it decomposes to give carbon & hydrogen. Strong CH 4
C 2 H 2
heating
[Present in natural gas] Hydrogen gas thus obtained in used to prepare ammonia which reacts with acids to give fertilizers. 5b.2.1
(ii)
Producer Gas
Producer gas is a mixture of carbon monoxide and nitrogen. It can be obtained from low grade coal, hence is a cheaper gas. Wooden wastes, peat, spent tar bark and other carbonaceous fuel , are used to prepare it; but fuel generally used are coke and coal. For its preparation restricted supply of air is allowed through burning solid carbonaceous fuel in a special type of furnace known as producer. Temperature of the furnace is kept at 1000-1400C. Producer gas obtained from coal has a calorific value of 145 BTU/cubic ft. Another gas produced with producer gas is semi-water gas. Combined Manufacture Manufacture of Producer Gas and Semi-Water Gas
Two types of manufacturing units are used for this purpose: (i)
Pressure type Unit (ii)
(i)
Pressure Type Unit
Suction type unit.
In pressure plants air is pumped through hot fuel. A steam-air mixture can be forced through hot fuel bed by steam pressure.
(ii)
Suction Type Units
Suction type units are used in case of gas-enzines. Here air is forced by the strokes of enzine or by the means of suction fan. Characteristic Characteristic feature of such as plant are:
1.
Pressure type plant has a diameter of 10 feet and its height in 12ft.
2.
Plant is made up of steel shell with refraction linking.
3.
Fuel is kept on stationary crate at the bottom of shell.
4.
Consumption of coal in it is 1500-2000 Kg/hr.
5.
In pressure type plant steam- air mixture is forced through fuel with the help of pipe at the bottom & semi-water gas or producer gas passes out from the top through outlet pipe. Composition Composition of producer gas and semi water gases are:
(a)
(b)
Producer Gas from Coke
CH4
=
0.4%
C2H4
=
0%
H2
=
13.2%
CO
=
25.3%
CO2
=
5.4%
N2
=
55.2%
O2
=
0.5%
Calorific value
=
137 BTU/cubic ft.
Producer Gas from Coal
CH4
=
2.5%
C2H4
=
0.4%
H2
=
12.0%
CO
=
27.0%
CO2
=
2.5%
N2
=
55.2%
(c)
O2
=
0.3%
Calorific value
=
145 BTU/cubic ft.
Composition of Semi-Water Gas
CH4
=
2.0%
C2H4
=
0.0%
H2
=
34.0%
CO
=
27.0%
CO2
=
8.0%
N2
=
29.0%
O2
=
0%
Calorific value
=
245 BTU/cubic ft.
Formation of Producer and Semi-Water Gas
1.
First CO2 is formed in restricted supply of air. 2C + O2 2CO - 57,000 cals. C + O2 CO2 - 96,500 cals.
2.
At higher temperature CO2 reacts with hot carbon to give carbon monoxide gas. C + CO2 2 CO + 38,700 38,700 cals. cals.
3.
N2 of air remains unaffected.
4.
Some H2 and CO are formed by the action of steam and carbon. C + H2O CO + H2 + 39,900 cals.
5.
Some CO2 escapes reduction.
6.
Some CH4 and C2H4 are formed by the destructive distillation of coal.
7.
For the formation of semi-water gas 7 volumes of air and 1 volume of steam is passed.
N.B.
(1)
Producer gas is mainly a mixture of carbon monoxide & Nitrogen. [CO + N2]
(2)
Temperature of the furnaces is initially 1000 C which gradually increases to 1400C.
(3)
1400C temperature is maintained by regulating the supply of air steam mixture composition. composition.
5b.2.2 Water Gas
Water gas is mainly of two types : (i)
Carburetted water gas
(ii)
Blue water Gas Water gas is mainly mixture of CO gas and H 2 gas.
(i)
Carburetted Water Gas
Carburetted water gas is produced by mixing oil gas & permanent gases produced by cracking kerosene oil. Oil gas contains considerasle amount of ethylene. It's calorific value is 444 BTU/cubic ft. Manufacturing unit for carburetted water gas consists of :
(ii)
(a)
Generator
(b)
Carburetter
(c)
Steel cylinder with lining of refractory bricks.
(d)
Super heater
(e)
Purifiers
(f)
Scrubbers
(g)
Condensers.
Blue Water Gas
Water gas is known as blue water gas as it burns with the blue Flame. Blue water gas can be prepared by passing steam through hot bed of coke at 1000 C1400C. The reaction is endotheremic, hence temperature falls down from 1400 C. Little carbon dioxide is also formed and its amount becomes considerable at 1000 C.
H2O+C CO + H 2 + 38,700 Cals. 2H2O + C 2H2 + CO2 - 19,000 Cals. CO2 has no calorific value, hence it is undesirable. The forma formation tion of water gas depends upon the decomposition of steam by carbon. At 1125 C composition of water gas becomes H 2 = 50.7%, CO=48% and CO 2 = 1.3% Actually, rate of decomposition of steam and formation of water gas are maintained by alternate run and blow periods. As soon as, temperature falls below 1000 C, the steam blast is stopped and an air blast is passed through red hot coke to raise the temperature of coke to ~1400 0C. When temperature is reached air blast is stopped & steam blast is resumed. As air forms explosive mixture with water gas, latter remaining in the generator is removed by passing steam for one minute. Steam sweeps off water gas from generator. The steam passed to remove water gas is known as purage steam (~ 1minute). The period for which air is passed is known as hot blow & for which steam is passed is called cold blow. Calorific value of blue water gas is 330 BTU/cubic ft. Compositions Compositions of water gas & carburetted water gas are: CH4 = 0.1%, H 2 = 51.5%, CO = 41.0%, CO 2 = 4.0%, N 2 = 3.4%, and CH 4 = 9.0%, C2H6 = 1.5%, C 2H4 = 7.4%, H 2 = 37.3%, CO = 34.8%, CO 2 = 3.7%, O 2 = 0.4%, N2 = 5.9%, respectively. Check your progress-III Note:
(a)
Write your answer in the space give below.
(b)
Compare your answer with those given at the end of the unit. (i)
Gaseous fuel leaves no residue. True/False
(ii)
Main constituents of water gas are _____________ .
(iii)
What shall happen if diamond is thrown in fire.
(i)
_________________________________________________
(ii)
_________________________________________________
(iii)
________________________________________________
5b.3 "Analysis of Coal"
[Ultimate and Proximate Analysis] Coal is formed from animal and plant organic matter which were buried inside the earth thousands of years ago. As it was formed from organic matter it contains elements of organic compounds i.e. C, H, N, O and S besides non-combustible inorganic matter. Quality of coal is assessed by two types of analysis : 1.
Ultimate Analysis
2.
Proximate Analysis
Ultimate Analysis
Estimation of carbon, hydrogen, nitrogen, sulphur and ash in the coal is carried out in the ultimate analysis of coal. This analysis is needed to calculate the heat balances in any process for which coal is used a fuel. 1.
Estimation of Carbon and Hydrogen
Approximetely 2.0 gm of accurately weighed coal sample is used for this purpose. It is burnt in a combustion apparatus which converts C & H into CO 2 and H2O, respectively. CO 2 is absorbed in pre-weighed KOH tube & H 2O in pre-weighed anh. CaCl2 tube. Percentage of C & H is calculated from the increase in weight of absorption tubes. C + O2 CO2 12
44
H2 + ½ O2 H2O 2
18
2 KOH + CO 2 K 2 CO3 + H2O CaCl2 + 7 H2O CaCl2.7H2O Increase Increase in wt of KOH tube
(i)
% age of C
(ii)
% age of H
2.
Estimation of Nitrogen
wt of coal taken Increase Increase in wt of C aCl aCl 2 tube wt of coal taken
12 44
100
2 18
100
For the determination determination of nitrogen in coal 1 gm of coal sample is taken in Kjeldahl flask alongwith H 2SO4, K 2SO4 & HgSO4. They are
heated to convert
nitrogen into ammonium salt. The sample solution in than made alkaline with NaOH and liberated ammonia is distilled into definite volume of standard acid solution, in which it gets absorbed. Unused acid is determined by titration with standard NaOH solution. From the volume of acid used with ammonia liberated is used to calculate percentage of nitrogen. % of Nitrog Nitrogen en
3.
Vol Vo l . of acid used Normalit Normality y of acid 1.4 wt of coal taken
Estimation of Sulphur
Sulphur is estimated from the washings of known mass of coal used in Bomb calorimeter to determine calorific value. Sulphur in washings is converted into sulphate for which washing are treated with BaCl 2 solution and BaSO 4 is precipitated. Precipitate is filtered through whatman filter peke no. 42, ignited & heated to constant weight. % age of sulphur sulphur
4.
Wt of Ba BaSO SO4 obtained
32
Wt of coal and in bomb colorimeter 233
100
Estimation of Ash
For the determination of ash 1-2 gm powered coal is taken in platinum crucible & are heated carefully on a Bunsen burner. Then lid is removed & strong heating is done to burn any tarry material. The lid is kept in desiccator. Heating of the coal in crucible is continued at 800 C in muffle furance till coal is completely burnt leaving behind ash only. Crucible is now cooled is desiccator & weighed to constant weight. % age of Oxygen
5.
Wt of ash l eft Wt of coal take t aken n
100
Estimation of Oxygen
%age of oxygen = 100 - [%age of C+H+N+S+ash]
Results of ultimate analysis of peat and anthracite are given below:
S.
Type of Coal C
No. 1
Peat
H
O
N
S
%
%
%
%
%
60
06
32
1.5
0.5
Caloric Value
8000
BTU/
Pound 2
Anthracite
94
03
02
01
00
15000
BTU/
Pound 2.
Proximate Analysis
It records moisture, volatile carbon matter, ash & fixed carbon percent of original wt of coal sample. 1.
Determination of Moisture
1-2 gm of air-dried powder of coal sample is taken in a silica crucible which has been heated to constant weight. This crucible is then heated in electric oven for 1 hr at 107 2C. Crucible is cooled in desiccator and weighed again. Amount of weight loss is equal to amount of moisture. % age of moisture
2.
Loss in wt Wt of coal taken
100
Determination of Volatile Carbonaceous Matter
Dried coal left in moisture moisture determination determination is covered covered with lid lid & heated heated in Muffle furnace at 925 2C for 7 minutes. Now, crucible is taken out and cooled first in air, then in desiccator and is weighed again. Loss in weight gives volatile matter. % age volatile matter
3.
Loss of wt due to removal of volatile matter Wt of coal sample taken
100
Determination of Ash
As given in ultimate ultimate analysis. analysis. 4.
Determination of Fixed Carbon
The residue left behind in the determination of volatile carbonaceous is matter fixed carbon + ash. Fixed carbon is left behind after destructive distillation of coal. Weight of ash is detected from weight of residue & % age of fixed carbon can be calculated as
% fixed carbon = 100 [Percentage of moisture + Ash + volatile carbonaceous mater] Usefulness of Proximate Analysis
It helps in the assessment of quality of coal in following ways: 1.
If moisture contents of coal are low, it is good quality fuel. Moisture contents are lower the calorific value of fuel. Because, it takes some of the liberated heat in the form of latent heat of evaporation. 10% moisture contents produce uniform fuel bed & less fly - ash.
2.
High Volatile carbonaceous matter is undesirable as they escape unburnt. Such a fuel burns with long sooty flame and have low calorific value.
3.
Ash in non-combustible non-combustible material reduces calorific value of coal. Presence of ash causes early wear out of furnace walls & burning apparatus. Thus, high ash content lower quality of coal.
4.
High percentage of fixed carbon increases calorific value of coal, it also decreases volatile matter and therefore indicates quality of coal is good.
Proximate Analysis of Peat & Anthracite
S.
Type of coal
No.
Moisture
Volatile
Fixed
%
matter %
carbon %
Ash %
1.
Peat
20
50
21
03
2.
Anthracite
01
08
88
03
N.B.
Calorific value of peat & anthracite are 7700 & 15,000 15,000 BTU/pound. BTU/pound. Grading of Coal
Different qualities of coal have following grading on the basis of (i) Ultimate analysis (ii) Proximate analysis (iii) Calorific value determination; (1)
Peat (2) Lignite and brown coal (3) Sub-bituminous
Bitumnous coal (5) Super - bituminous coal (6) Anthracite Characteristics Characteristics of Good Go od Quality Fuel
coal (4)
1.
It should should have high calorific value.
2.
Should have low non-combustible matter.
3.
Should burn smoothly.
4.
Moisture contents should be low.
5.
Should not burn spontaneously.
6.
Should be of low cost.
7.
Should be easily transportable. transportable.
8.
Should have low ash contents.
9.
Should burn at moderate rate.
10.
Should burn without smoke.
11.
It should not pollute atmosphare. atmosphare . Gases like CO2, SO2, H2S, PH3 etc. should not form when it burns.
5b.4 "Flash Point"
Use of Kerosene oil containing lower boiling fractions is dangerous in lamps, stoves etc.; because there are chances of explosion. Therefore, it is needed that Kerosene or other oil used for heating and illuminating purpose should not be as much volatile as to form explosive mixture with air at ordinary temperature. The minimum temperature at which an oil gives sufficient amount of vapours to form explosive mixture with air is known of flash point. For safety
purpose flash point of oil is fixed by law of the country, depending upon its climate. For instance, In India, flash point has been fixed 44 C. In England and France, flash points are 22.8 C and 55C, respectively. At slightly higher temperature, temperature, the heat from the flash becomes sufficient to evaporate more liquid and maintains the combustion. The minimum temperature [Usually 5-40 higher than flesh point] at which oil gives off sufficient vapours when ignited to continuously burn it for at least five seconds, is known known as fire point of that oil. Determination of Flash Point
Flash point of an oil can be determined by using Abel's apparatus. The oil to be tested is placed in a small metallic cup surrounded by water bath & is slowly
heated on water both. Cover (lid) of the metallic cup is opened from time to time & vapous of oil are ignited by tiny flame introduced momentarily by the means of an automatic device. The temperature at which the oil vapor burn with slight explosion is noted which is the flash - point. The heating of the sample is done at the rate of 5 F/minute and speed of stirring paddle is kept approximately one revolution per second. As the temperature of the oil reaches within 15 F of probable flash point first application of the flame is made by pulling sliding shutter outwords when the test flame drips into the central opening in the lid & comes in contact contact
with the ascending ascending vapour-air vapour-air mixture;
Subsequently, test flame is applied at every 2 F rise in temperature. When the application of the flame first produces a distinct blue flash in the interior of the oil cup, the temperature on the test thermometer (T 1) is recorded and heating is discontinued. Now, oil is allowed to cool down (if cooling is slow some cold water may be added to water bath by funnel). When temperature comes within 10 F of T1; test flame is again applied after every 2 F fall of temperature & the lowest temperature at which flash is produced is recorded. This is T 2. Flash Flash po int of oil sample sample
T 1 T 2 2
0
F .
Precautions
1.
Moisture affects the flash point, hence, all parts of the cup & its accessories should be properly dried before filling oil in it.
2.
No oil should be left between sliding and fixed plates before convering the cup.
3.
Oil should be filled so as it just prevents wetting of the cup above the pointed tip.
4.
For oil with low flash point sample as well as cup may be cooled in melting ice before filling.
5.
For the determination of flash point fresh oil should be used each time. Used oil gives higher flash point.
6.
Thermometer should be kept dipped in oil.
7.
While applying flame, slide should be drawn open slowly & closed quickly.
8.
Stirring should be stopped while applying flame.
Significance Significance of Flash - Point Determination
1.
It is helpful in providing safeguards against hazards during storage, transport & handling of oil.
2.
Liquids having flash point < 140 F are are known known as flammable & those with > 140F are combustible.
3.
Volatility of oils increases significantly when heated to or above flash point. Hence, a low flash point indicates appreciable evaporation evaporation loss & possibility of formation of gas lock in fuel pipe of spark ignition enzines .
4.
Flash & fire points are used to detect the solvent contamination contamination and to determine the approximate extent of dilution of lubricating oils.
5b.5 Aniline Point of Fuels
Aniline is easily soluble in lubricants which are rich in aromatics including napthelinic compounds. In paraffin rich lubricant dissolution of aniline takes place at higher temperature. The Tendency of lubricants to mix with aniline is expressed as its Aniline point . Aniline point is also known as standard aniline
point. Aniline point with respect to petroleum oils is
the lowest
temperature at which oil is completely soluble with equal volume of freshly distilled aniline . In other words, aniline point is the minimum equilibrium
solution temperature for equal volumes of aniline and sample of lubricant. Mixed Aniline Point
Some lubricants (with very higher aromatic compounds) when mixed with equal volumes of aniline, may remain completely soluble and separation into different phases may not be visible even upon solidification. For the determination of such low aniline points 1 Volumes of sample is mixed with two volumes of aniline & one volume of suitable dilutent [n-hexane or n-heptane]. Here, dilutent lowers the solubility of aniline with lubricant. Hence, with decrease in temperature, separation of phases may be visible. Equilibrium solution temperature observed under these condition is known as mixed aniline point which has same significance as standard aniline point . Determination of Aniline Point of Fuel
For it equal volumes of sample & aniline are heated till dissolution and are then cooled under controlled condition. The temperature at which two phases separate i.e. cloudiness appears throughout the solution is regarded as the aniline point of the sample. Methodology
1.
Whole apparatus is cleaned & dried at 100-110 C.
2.
5-10ml of pure aniline [dried over KOH pellets, filtered & freshly distilled] & exactly same volume of sample [dried over one Na 2SO4] are taken in 2.5 x 15cm test tube of heat resistant glass.
3.
Test tube is corked with Cork carrying stirrer and thermometer of suitable range. The bulb of thermometer is kept 5mm above the bottom of test-tube.
4.
This tube is inserted into air-jacket [4x175cm] which is also made up of heat resistant glass.
5.
Contents of tube are stirred to get a homogenous solution. If clear solution is not formed at R.T. jacket holding tube may be immersed in a hot-bath.
6.
Stirring is continued and heating through bath is done till dissolution is complete. Now hot bath is taken off & temperature is allowed to fall with stirring at a rate below 1 C/minute.[ A cold bath may be used if needed].
7.
The temperature at which cloudiness appear is noted. It is aniline point of the sample. This is 1-2 C below the temperature at which turbidity first appeared.
Precautions
1.
Apparatus should be completely dry. Even traces of moisture give high results.
2.
Aniline is hygroscopic. Hence, water should not be used even in hot or cold baths.
3.
If aniline point is expected to be below the dew point of atmosphere, space over sample-aniline mixture should be filled with dry nitrogen.
4.
Aniline shoul be measured by pipette with rubber suction bulb or aspirators as it is highly toxic.
5.
Stirring speed should be such that there is no splashing or formation of bubbles.
Significance Significance of Aniline Point
1.
It is a measure of aromatic contents of oil.
2.
Low aniline point i.e. high aromatic content make oil attack rubber gaskets, seals etc.
3.
Lubricants with high aniline point are useful for systems where rubber seals are used.
4.
Determination of rubber attacking tendency is easier & takes few hrs as compared to testing by dipping rubber in oil.
5b.6 "Octane Number"
Octane number is index of Knocking power of gasoline (petrol) in internal combustion enzines. Modern high speed enzines need high compression for
efficiency. But, a straight run air-gasoline mixture mixture ignites prematurely prematurely under high high compression & burns in an explosive & dis-orderly fashion producing a sharp metallic sound known as Knocking . Knocking leads to loss of power, because part of energy is converted into rattle. This rattling leads to high fuel consumption & damage to enzines. Therefore, some anti-knowing agent is added to gasoline. Edger (1927) observed the normal straight chain alkanes knock badly but branehed ones much less. Efficiency of a fuel is rated in terms of Octane numbers. n-Heptane is given octane number zero. But, iso-octane [2, 2, 4-trimethlylpentane] is given octane number 100. CH3 | CH3-CH2-CH2-CH2-CH2 | n-Heptane
CH2 | CH3
H3C-C-CH2-CH-CH3 |
|
CH3
CH3
Iso-octane
Octane Number of a given fuel is defined as percentage of iso-octane in a
mixture of iso-octane and n-heptane which has same knocking properties as the fuel under examination in a standard one cylinder enzine operated under standard conditions. Higher the octane number, higher will be aniknocking property. It is observed that in a alkane series the octane number decreases as the carbon chain is lengthened & increases with branching of chains. Octane number increases as double bond is shifted towards centre of molecule. Octane number of benzene is 106 and that of alcohol is 95. The aviation gasoline generally has
octane number > 100. It should be noted: (i)
Gasoline from paraffin crudes have low octane number.
(ii)
Octane number of normal gasoline in 74.
(iii)
The hydrocarbon 2,2,3-trimethyl butane (Triptane) is superior to iso-octane with octane number 124. Normally, quality of spirit blend containing TEL (Commonly used fuel] have
different colours which indicate their value. Their octane rating is expressed as: Octane No
Powe Po wer r number 100 3
100
N.B.
Power No. is proportional to power supplied by engine. Check your progress-IV Notes:
(a)
Write your answer in the space give below.
(b)
Compare your answer with those given at the end of the unit. (i)
In ultimate analysis ___________ the estimated.
(ii)
Describe determination of aniline point.
(iii)
Octane No. of good fuel is low/high.
(i)
_________________________________________________
(ii)
_________________________________________________
(iii)
_________________________________________________
5(b).7
"CALORIFIC VALUES"
Calorific value
of a fuel is the amount of heat produced by the
complete combustion of a unit mass of fuel. Actually, this is the number of
parts of water which get heated by the complete combustion of one unit weight of fuel at 1C provided whole heat is absorbed by the water at atmospheric temperature and pressure. Units of caloric values are 1.
Calorie
It is unit of heat in C.G.S. system & is defined as amount of heat that raises the temperature of 1 gm of water by 1 C [15-16C].
1 Calorie = 4.185 Joule = 4.185 x 10 -7 erg. 2.
Kilo Calories
Kilocalories is the amount or heat require to raise the temperature of 1 Kg of water by 1 C. It is unit of heat is M.K.S. system. 1 K. Cal. = 1000 Cals. 3.
Centigrade heat Unit (CHU)
It is the quantity of heat that raises the temperature of one pound of water by 1C. 1 K. Cal.
4.
=
2.2. CHU.
=
3.968 B.t.u.
British Thermal Unit (B.T.U.)
B.t.u. is the quantity of heat that that raises the the temperature temperature of 1 pound pound of water by 1F (60-61F). 1 B.T.U.
1 K. Cals.
=
252 Cals
=
=
1.054 Joule
=
1.054 x 107 erg.
=
3.968 B.t.u.
0.252 K. Cals
Net Calorific Value
"Amount of heat evolved when one unit of fuel gets burn completely and combustion products products are allowed to escape is known as net calrofic value". It is rarely feasible that combustion products of fuel are cooled to R.T., water vapours escape as such alongwith hot combustion gases and are not condensed. Net Calorific value = Gross calorific calorific value - Latent heat of of water vapours formed Net calorific value is also known as low calorific value .
Gross Calorific Value
"Amount of heat evolved when one unit of fuel is burnt completely and product of combustion are cooled to R.T. [15 or 60F] is known as Gross calorific value". For the determination of Gross calorific value all hydrogen present in fuel is converted into steam. When the product of combustion are cooled to R.T. latent heat of steam also get induced in the measured heat. This total value is known as Higher or Gross calorific value.
"Determination of Calorific Value" Object: To determine calorific value of unknown sample. Principle
The Calorific value is determined by the use of bomb calorimeter. Calorific values of solid and liquid fuels can be determined by it. Bomb-calorimeter consist of following parts : 1.
Combustion Bomb :
It is made up of chromium - nickel mohybdenum steel and is resistant to acids as well as corrosion. It weighes approx - 3 Kg. and its capacity in 250-300 ml. Bomb is provided with an air- tight screw cap to which a couple of stainless steel electrodes and release valve is attached. To electrode is attached a small ring that supports crucible containing fuel.
2.
Copper Calorimeter
Copper calorimeter is a vessel that is polished outside and it can contain 2litre of water when bomb is placed inside. Bombs should be sub-merged in water. A space should be there between water and cover. 3.
Outer Water Jacket
It is made in the from of a wide annular vessel around calorimeter vessel.
4.
A Mechanical Stirrer
Mechanical stirrer is fitted for stirring water in calorimeter to stir at uniform rate. 5.
A Beckman thermometer
6.
A Crucible
It is made up of nickel, stainless steel or fused silica. Procedure
Place weighed quantity of fuel in crucible. Keep the crucible containing fuel at its position supported over the ring. A fine Pt- wire is tightly stretched across the pole piece of the bomb and one end of the piece of sewing cotton thread is tied around the wire. Loose end of cotton thread is so arranged to be in contract of with the fuel (e.g. coal). Now, 10 ml of distilled water is placed inside the bomb by the means of pipette to absorb vapours of H 2SO4 and HNO3 during combustion. Oxygen is forced into the bomb till a pressure of 25 atm is reached. Now, bomb is placed inside the copper calorimeter that contains a known amount of water in which bomb is submerged, but terminal remains above the water level. Make the electrical connection and place the lid and start stirring through mechanical stirrer. Note down down initial temperature of of water by thermometer. Connect the electrodes electrodes with battery & complete the circuit so that sample burns and heat is liberated. Uniform stirring of water is done and maximum temperature attained is noted by Beckman thermometer. Now, bomb is left for at least one hr. so that all the acid mist formed settles down. Dismantle the assembly & bomb is placed on holder. Release the oxygen from bomb carefully. Open the bomb to examine internal portion
if any unburnt fuel content. If combustion is complete wash the contents with distilled water quantitatively & transfer the washing in beaker. HNO 3 and H2SO4 can be estimated from these washings (which are formed during combustion) which are used for corrections.
Calculation of Calorific Value
Rise in temperature of water in which bomb is submerged can be used to determine gross calorific value : G.C.V. =
( w w)(t 2 t 1 t c ) (C S C N C F C C ) m
Here ; G.C.V. = Gross calorific Value w
=
Weight of water in calorimeter
w
=
Water equivalent of calorimeter
m
=
Mass of fuel burnt
t1
=
Initial temperature of water in calorimeter .
t2
=
Final temperature of water in calorimeter.
CS
=
Correction for H2SO4
CN
=
Correction for HNO3
CF
=
Correction for fuse wire
CC
=
Correction for cotton thread.
Net calorific calorific value or lower calorific value, L.C.V, L.C.V, can also be calculated by L.C.V. = G.C.V. (0.09 x 587) cals/gm or K.Cals/Kg. Hence,
587 cals/g = Latent heat of steam.
H
Percentage of hydrogen in fuel.
=
Corrections :
1.
Acid Correction :
Fuels that contain sulphur and nitrogen get oxidized to H 2SO4
,
HNO3
,
respectively at high temperature and pressure. S + 2H + 2O 2
H2SO4 + Heat
2N + 2H + 3O 2
2HNO3
These acids can be estimated from the washing of bomb through titration. For each ml of N/10 - H 2SO4, 3.6 cals should be subtracted and for each ml of N/10 HNO3 1.43 calories are minused from the actual value of G.C.V. or L.C.V. 2.
Cotton thread correction
These corrections are made by the weight of cotton thread used for firing. Calorific value of cellulose is 4140 cals/gm. 3.
Fuse wire correction
This correction is made for the amount of heat given out by the ignition of fuse wire burnt in the combustion experiment. Heat liberated for each fuse wire burnt in the experiment is to be subtracted from total heat evolved as per the instructions of fuse wire manufacturer . 4.
Cooling correction
Time for cooling of water from maximum temperature to R.T. should be recorded . From the rate of cooling and actual time/min (t/min) the cooling correction of dt x t is added to the rise is temperature. Hence Caloric Value
=
( w W )(t 2
Acid Fu Fuse se co rrection ) t 1 CoolingCor retion ) ( Acid Mass of Fu Fuel elBu Burnt rnt
Result : Calorific value of unknown sample (Fuel) is _______ cals/gm. Experiment : Determination of calorific value of wax without the use of Bomb calorimeter [A rough & simplified method] Methodology :
1.
Weigh a wax candle (W1)
2.
Take 500 ml Borsil beaker and fill it half with water .
3.
Before filling with water as well as after filling with water weight of beaker should be taken. taken. Difference of there two readings is mass of of water (m)
4.
Now a thermometer is placed in the beaker and initial temperature (t 1) of water is determined .
5.
Now candle is burnt below the beaker as a result beaker of water get heated & temperature gradually increase. When temperature of water increases by 150 or 200C candle is extinguished to stop heating & exact temperature (t 2) is recorded immediately .
7.
Extinguished candle is weighed (w2). The difference is weight of candle (w 1w2) is the weight of wax burnt.
Observation & Calculation
Initial wt of candle =
W1gm
wt after candle burnt
=
1. wt of burnt candle =
W1-W2gm
2. Mass of water
mgm
=
W2gm
3.(i)
Initial temperature of water
=
t1
(ii)
Final temperature of water
=
t2
Rise in temperature of water
=
t2-t1
4.
Heat absorbed by water = Mass x specific heat x rise in temperature =
M x S x (t2-t1)
This heat is produced by (w 1-w2) gm of wax. Calorific Value
=
=
Here, S
Heat Heat produce produceed ed Man of wax burnt m s (t 2
t 1 )
w1 w2
Joule Jouless / gm / 0 C
= Specific heat of water = 4.2 Joule /gm/00C
Result : N.B. :
Calorific value of wax's is __________ J/g/0C Calorific value determined by this method is rough because :
(i)
Heat absorbed by the beaker has been neglected .
(ii)
Heat lost to sounding is neglected.
(iii)
Loss of heat from beaker & water is neglected.
(iv)
Incomplete combustion wax of may have been .
(v)
Wrong method of ignition of wax.
5.C
"CLINICAL CHEMISTRY"
5c.1 "Composition of Blood"
Utility of blood for normal health and ailing persons is known since time immortal. Blood is a mixture of different constituents each of which is needed in definite amount. A test of blood gives indication of certain deficiencies and excess which lead to ailing condition. Hence, it essential to know composition of blood for a clinical chemists. Blood constituents can be broadly divide into. (1)
Blood Plasma
(2)
Cellular elements.
[1]
Blood Plasma
Blood plasma is the liquid part of circulating blood. It contain (a) serum and (b) Fibrinogen. Later is responsible for the clotting of blood. Upon clotting of blood fibrinogen is removed removed along with with cells & serum is left behind. Most of the blood blood tests are performed on serum. -
Normal range of some clinically important constituents of blood and plasma
(Serum) are summarized below : (A)
Blood
(i)
Amino acid nitrogen =
4 - 6 Mg/dL
(ii)
Ammonia
=
40 - 125 g/dL
(iii)
Non-Protein nitrogen
=
25-40 mg/dL
(B)
(iv)
Blood Sugar (Glucose)
=
65-90 mg/dL
(v)
Urea nitrogen
=
up to 20 mg/dL
Serum
(i)
Albumin
=
4-5 g/dL
(ii)
Amylase
=
upto 150 mg/dL
(iii)
Bilrubin
=
Direct upto 0.4 mg/dL Total upto 1.0 mg/dL
(iv)
Calcium
=
4.5-5.5 meq/L
(v)
CO2
=
25-32 meq/L
(vi)
Chloride
=
100-108 meq/L
(vii)
Total Cholesterol
=
140-250 mg/LB
(viii) Cholesterol (ester)
=
50-65% of total
(ix)
Creatinine
=
0.7-1.7 mg/dL
(x)
Lipase
=
upto 1.5 unit
(xi)
Lipids (Total)
=
350-800 mg/L
(xii)
Fatty acids
=
200-400 mg
(xiii)
Globulins (Total)
=
2.5 - 3.5g/dL
-1 ……………..
=
0.1 - 0.4 g/dL
-2 …………….. ……………..
= =
……………….
0.3 - 0.7 g/dL
0.4 - 0.9 g/dL =
0.6 - 1.3 g/dL.
(xiv) Iron
=
50-190 g/dL
(xv)
=
1.5-2.5 meq/L
Mg
(xvi) Phosphatase (Acid) =
upto 4 (Gutman units)
(xvii) Phospholipids
100-250 mg/dL
=
(xix) Phosphorus (inorganic)
=
3 - 4.5 mg / dL
(xx)
K (Potassium)
=
3.8 - 5.6 meq/L
(xxi) Protein (total)
=
6.5 - 8.0 g/dL
(xxii) Protein bound iodine
=
3.5-8.0 g/dL
(xxiii) Sodium
=
138 - 146 meg/L
(xxiv) Transaminase (SGO)
=
upto 40 units.
(xxv) Uric Acid
=
3 - 6 mg/dL
B
= Varies with age.
A brief study study of components components of blood plasma is discussed ahead: (A)
Water
=
91 - 92%
(B)
Other Solids
=
8% - 9%
(I)
(i)
Organic Substances
(ii)
Inorganic substances
(iii)
Respiratory gases.
Organic Substances
These are 7.1 - 8.1% They include (i)
Proteins (=7.0%) : Regulate blood volume, viscosity & osmotic pressure.
e.g.
(a)
Albumin
(b)
Globulin
(c)
Fibrinogen
(d)
Prothrombin
Fibrinogen is responsible for the clothing of blood. (ii)
Hormones : are secreted by endocrime glands.
(iii)
Enzymes : Amylase, Lipase, sucrase etc.
(iv)
Antibodies : Maintain viscosity & immune system.
(v)
Neutral Fats : e.g. Cholesterol, pentoses, Phospholipids etc.
(vi)
Non-Protein Nitrogenous Substances e.g: urea, ammonia, amino acids
and creatinine etc (II)
Inorganic Substances
NaCl, Ca, K, iodine, iron, bicarbonates etc. (III) Respiratory Gases
O2 and CO2.
[2]
Cellular Elements
Following types of cells are suspended in liquid portion of blood:
(a)
(a)
Red Blood Corpsules (R.B.C.)
(b)
White Blood Corpsules (W.B.C.)
(c)
Platelets (Thrombocytes) (Thrombocytes)
R.B.C.
These are also known as erythrocytes and are produced in bone marrow of flat bones, ribs, vertebrae, heads of humerus and femur. Their size is 7.2 micron
diameter. They They contain no nucleus. R.B.C. R.B.C. contain red pigment haemoglobin haemoglobin & their life is 120 days. R.B.C. are responsible for the transportation of O 2 & CO2 from one tissue to other. (b)
W.B.C.
These are also known as Leucocytes and are of five types : (i)
Neutrophil polymorph polymorph
(ii)
Eosinophil
(iii)
Basophil
(iv)
Lymphocytes
(v)
Monocytes.
(i)
to (iii) are also called granulocytes. (iv) and (v) are non-granular
leucocytes. There is only one W.B.C. for every 500 R.B.C. In adults they count 5,00010,000 per cm. of blood. They do not contain pigment. They constitute defence systems of body against infections by phagocytes. Their function is also production of antibodies by lymphocytes. (c)
Platelets
These are non-nucleated parts of cytoplasm & are also called thrombocytes. [Thrombo = Clottings, cyte=cell], they cause clotting of blood & are produced in spleen and bone-marrows. Size of platelets is 2-4 micron (diameter) and they count 2,50,000 to 5,00,000/cc of blood. Functions of Blood
(1)
It transports food from one tissue to other & provides connection link between individual cells of distant organs and tissues.
(2)
It carries oxygen from lung to other parts by combining with haemoglobin haemoglobin & removes CO2 from tissues through lungs to air.
(3)
Blood regulates body temperature & maintains water contents of body.
5c.2 Collection and Preservation of Samples of Blood
For the analysis of blood samples for glucose, cholesterol, uric acids, lipid profiles, R.B.C., W.B.C., platelets etc collection and preservation of blood samples is needed. Blood for some distinct purpose is collected after fasting over night. Collection of Blood
The blood is collected aseptically from the median cubital vein, in front of the elbow, into a sterile container containing an anti-coagulant solution. During collection the bottle is shaken gently to ensure that the blood and anti-coagulant are mixed well, therefore, preventing the formation of fibrin-clots. Not more than 420 ml of blood is taken at single attendance. Now container is sealed immediately and is cooled to 4-6 0C. For taking blood the equipment used is made up of plastic & is disposable. Container Container is also made made up of plastic. In America plastic plastic bags are used. Anti-Coagulants Anti-Coagulants (1)
Citrates
The solution most often used as anticoagulant of blood is Acid-citrate dextrose (ACP) which has following composition : Sodium acid citrate =
2.10 - 2.5 g
Dextose
=
Water for injection =
upto 120 ml.
3.0 g
The citrate prevents clotting of blood by binding to Ca 2+ ions as unionised calcium citrate. Earlier, trisodium citrate was used as anti-coagulant which has very alkaline pH & caused conisiderable darkening of dextrose during sterilization and the two solutions need to be autoclaved separately. Acid citrate gives a pH of 5 & gives very little or no caramelisation. Also, it is less likely to induce flaking of the glass of container. High concentration [2.5 g/20ml] is generally used as it more efficiently reduces the clot - formation. Dextrose delays haemolysis of electrolyte in vitro & prolongs their life after transfusion. 2.
Heparin
Heparin is naturally occurring blood anti-coagulant made by the mast cells of connective tissue surrounding the blood-vessels. It inhibits clotting of blood in circulatory system. Heparin is used in the blood transfusion when more amount of
blood is to be given to the patient and corresponding amount of citrate would be harmful e.g. in cardic surgery. It quickly loses activity in blood in vitro & normal quantities are effective for about a day. Heparin is expensive may continue its action after transfusion, needing the administration of neutralizing agents like protamine sulphate. 3.
Disodium Edetate
It is preferred when preservation of platelets are necessary. This is a chelating agent because of strong affinity for divalent metal like Ca. Collection of Blood of an HIV Infected Patient
The collection & handling of blood requires great care as some transferable disease constituents like HIV or AIDS may be present in it. To collect blood of such patient gloves and masks should be used. Such persons should not be handled without expert advise. Collection of Blood for Plasma or Whole Blood Analysis
Blood for this purpose is collected in tubes containing anti-coagulant. Anticoagulants used have already been discussed. However, potassium oxalate (about 1mg/ml blood) may also be used as anti-plasma coagulant. Pot. oxalate precipitates blood calcium which is required in clotting. Potassium Potassium oxalate causes R.B.C. to shrink shri nk & intracellular water diffuses into plasma. Collection of blood for serum Analysis
Blood for this purpose is also collected in dry & clean tube to prevent haemolysis & contamination. Haemolysis is destruction of R.B.C. & release of haemoglobin etc into serum or plasma. As haemolysis occurs, serum becomes red instead of normal straw colour. Therefore, blood sample is centrifuged as soon as possible to separate serum or plasma from cells. Collection of Blood for Glucose Analysis :
NaF is widely used as a preservative for the samples to be analysed for glucose alongwith anti-coagulant. NaF is enzyme inhibitor & prevents enzymatic break-down of glucose. 1 mg of NaF/ml of blood is enough. Collection of Blood for CO2 Analysis
Blood collected for this purpose should be kept anaerobically & mineral oil must be added added to collection tube tube which being ligther ligther covers the blood. A rubber stopper should not be used to such sample which swells up. Storage of Samples of Blood
Apart from period of transport and examination which much not exceed 30 minutes, blood should be stored at 4-6 C until required for use. Even, at his temperature some deterioration may occur. Leucocytes disintegrate in few hrs and platelets in few days. R.B.C. show fall in ATP and other organic phosphates, a reduction in oxygen carrying capacity and partrial loss of lipid from their membranes & increased fragility. Storage at R.T. even for a day seriously reduces posttransfusion survival
of R.B.C.. R.B.C.. Haemolysis Haemolysis makes blood unfit for use. Complete
haemolysis, especially if it occurs rapidly is a sign of bacterial infection but its absence is not confirmation of sterility since some bacteria predominatly pseudomonas & bacteria of coli-aerogenes group, can grow in blood at refrigerator temperature without causing haemolysis. Few organisms isolated from contaminated blood use citrate as their source of carbon which eventually leads to clotting. 5c.3 "Analysis of Serum Electrolytes"
Electrolytes are the substances which dissociate into free ions when dissolved or are in molten state. They constitute electricity conducting medium. Electrolytes generally exist as acids, bases bases salts. Electrolytes present in blood are are : Sodium, Sodium, Potassium, Calcium, Chloride, bicarbonates etc.
"Estimation of Sodium & Potassium" Po tassium"
In blood sodium and potassium are found in ionic state. Plasma contains 90% of the total blood sodium. But, potassium in present mainly in cells. Concentrations of Na+ & K + present in serum can be determined by the flame photometry or by chemical analysis based on precipitation of metal complex. Potassium in serum can be estimated by precipitation precipitation of of potassium as followed by photometric photometric or titrimetric titrimetric method. Methodology
Flame photometric method involves following steps:
1.
Sample in solution form is introduced in the form of fine continuous spray into non-luminous gas using either acetylene, propane or butane gas. The light is emitted by the use of colour filter or diffraction grating.
2.
The emitted light of a wave length characteristic of ion being analysed is isolated and focused on a photoelectric cell.
3.
The electrical response to photoelectric cell is measured on suitable meter which is already calibrated to convent electrical impulse to ion concentration either by the direct reading or by reference to calibration curve.
Range
(i)
Normal range of serum Na+ ions is 300-350mg/100 ml or 130-154 mEq./lt
(ii)
Concentration Concentration range of K +ions in serum is 14-22 mg/100ml or
3.6-5.6
mEq./lt. Inferences from Na+ and K+ion concentrations
-
Increase in serum potassium causes Addison disease, acute bronchial asthma & fevers.
-
Decrease in concentration of serum potassium is due to diarrhoea or vomiting during familial periodic paralysis.
-
Sodium ions maintain acid-base balance in the body as well as the smotic pressure.
"Estimation of Calcium Ions"
Calcium is present in serum as : (i)
Calcium bound to protein.
(ii)
Calcium bound to other organic substances like citrate.
(iii)
Ionized calcium. Physiological functions of calcium are due to ionic calcium. But in routine work total calcium is estimated. Commonly used method for the estimation of calcium is Clark & Collip Method.
Principle
Calcium present in serum can be precipitated with ammonium oxalate in the form of calcium oxalate. This precipitate is washed with ammonia in order to remove excess of oxalate and then calcium oxalate precipitate is treated with H 2SO4 to convert oxalate into oxalic acid. Oxalic acid is titrated with standard KMnO 4 solution is usual manner. Requirements (i)
Ammonium Oxalate 4%
Prepared by the dissolution of 4g ammonium oxalate in water & made up to 100 ml in volumetric measuring flask with D.W. (ii)
2% Ammonia
2 ml of H 2SO4 diluted to 100 ml ml with distilled water. water.
(iii)
1N-H2SO4
28 ml of H 2SO4 diluted to 1000 ml with distilled water (iv)
Stock KMnO4 0.1 N Solution
23.159 gm of KMnO 4 is dissolved in water & made up to 100 ml. This solution is allowed to stand for a week & then is filtered through asbestos (v)
0.01 N - Sodium Oxalate
0.67 gm of sodium Oxalate is dissolved in water & 5 ml of conc. H 2SO4 is added to it. The volume is made upto 1000 ml with water. Methodology
1.
Place 2 ml of serum, 2 ml of water and 1 ml of 4% ammonium oxalate solution in centrifuge tube. Mix them thoroughly and allow to stand for about half an hour.
2.
Mix again and centrifuge at 1500 g for about 15 minutes.
3.
Take our supernatant liquid. Drain the centrifuge tube by inverting it on a filter paper for few minutes.
4.
Add 3 ml of ammonia [2%] and shake, centrifuge again drain as in step 3.
5.
Add 2 ml of 1N-H 2SO4 and shake.
6.
Keep the tube in boiling water-bath with occasional shaking till precipitate completely dissolves.
7.
Titrate the warm solution with 0.01N-KMnO4 till pink colour develops and persists at least for one minutes.
8.
Find out the volume [ml] of 0.01N-KMnO4 used. Let it is Xml
9.
Take a blank reading with 2 ml of 1N-H 2SO4 by titrating it against 0.01 NMKnO4. Let volume used is Y ml.
Calculation
Serum calcium [mg/dL] ( X Y )
0.2 2
100
= (X-Y) 10
Range
Range of calcium is serum varies from 9-11 mg/DL in a healthy person.
Inference from the Concentration of Serum Calcium
-
Serum
calcium
increases
in
hypervitaminosis-D, hypervitaminosis-D,
hyperparathyroidism, hyperparathyroidism,
polycytharmia, infantile hypercalcemia, sarcoidosis, multiple myeloma, extensive metastatic involvement of bones etc. -
Serum calcium decreases in hypoparathyroidism, hypoparathyroidism, rickets, renal failure, acute pancreatitis, starvation, naphrotic syndrome, osteomalacia.
"Estimation of Chloride in blood"
Chloride is an important electrolytic ion present in body. It is responsible for osmotic pressure control, chloride - bicarbonate shift, acid-base balance etc. Onethird of total chloride is present in R.B.C. and 2/3 is serum. It can be estimated by Schales and Schales method. Principle
Chloride ion gets converted into un-ionized mercuric chloride when mercuric nitrate solution is added to them. 2Cl- + Hg (NO 3)2 HgCl2 + 2NO3Endpoint is indicated by the appearance of bright blue - violet colour in presence of diphenyl carbazone indicator. Titration can be done directly with serum. But highly coloured or jaundiced jaundiced sample, sample, may may suffer from clarity of end point. For such sample, a Folin Wu tungstate filtrate is prepared and titrated. Requirement
(i)
10% Sodium tungstate.
(ii)
2/3 N-H2SO4
(iii)
Hg (NO3)2 Solution : To prepare it dissolve 3g of Hg
500 ml of D.W. Add 30 ml of conc. HNO 3 to it, shake
and
(NO 3)2 in make
up
the
volume to 1 litre. (iv)
1% solution of diphenyl carbazone in CH 3OH or C2H5OH.
[Indictor].
(v)
Standard NaCl Solution : To prepare it dissolve 585 mg of NaCl in 1
lt D.W. Concentration of this solution is 10 m Eq./litre. Methodology 1.
Standardization Standardization of Hg (NO 3)2 Solution
Taking 2 ml of standard NaCl solution in 50 ml conical flask, add 3-4 drops of diphenyl carbazone to it. Now titrate it with Hg (NO 3)2 till colour change occurs. Note down the volume used. 2.
Titration of Sample
Taking 1 ml of serum or plasma or blood and add 7 ml of D.W. to it. Add 1 ml of 10% sodium tungstate & 1 ml of 2/3 N-H 2SO4 also. Stir the contents and filter. Taking 2 ml of this filtrate in 50 ml conical flaks titrate it with Hg (NO 3)2 solution. Note down the volume used. Calculation
Volume of serum has been diluted 10 times. If the concentration of Cl ion in serum is C, then
C
10
Vol. of Hg(NO3 ) 2 used with sample vol. of Hg(NO3 ) 2 used with standard
or C m Eq/l
10
Vol. of Hg(NO3 ) 2 used with sample Vol of Hg(NO3 ) 2 used with standard
100
Range
Rage of Cl- ion (i)
in Serum plasma
=
98-105 m Eq/lt
(ii)
in whole blood
=
78-85 mEq/lt
Inference from the Concentration of Cl - Ions in Plasma
-
Increase in concentration of Cl- ions in plasma takes place in nephritis. Under such conditions salt in take should be restricted.
-
Decrease in Cl- ion concentration in plasma is indication of gastrointestional disturbances associated with diarrhoea & vomiting.
-
Decrease in concentration of Cl- ions also occurs during Addison disease & pneumonia.
"Estimation of HCO3- ions"
Bicarbonate (HCO3-) ions in blood can be determined by adding an excess of 0.01 M-HCl to volatilize the HCO 3- ions to CO 2 & swirling to allow the CO 2 to escape. The excess of HCl is back-titrated with 0.01M-NaOH. The 0.01 M-HCl and NaOH solutions are prepared by diluting standard 0.1 M solutions. HCO3- + H+ H2O + CO2 H+ + -OH H2O. Requirements
(i)
1% saline (NaCl) solution in CO 2-free water.
(ii)
Phenol red solution 0.1% in 0.003 M-NaOH
(iii)
Anti foam A
(iv)
Standard 0.1 M-HCl & 0.1 M-NaOH.
0.01M-HCl and 0.01M-NaOH should be freshly prepared in saline solution. Saline helps in the volatilization volatilization of CO 2 from acidified solution. Methodology
1.
To prepare sample take 10-15ml of fresh blood in a sample
tube.
2.
Add NaF to it in order to prevent glycolysis [decomposition [decomposition of glucose], which may lead to pH change.
3.
Keep the tube anaerobically, for which stopper the sample tube to keep CO 2 out. Sample should be prepared at the time of analysis only.
4.
Take 6 ml of 1% saline in 25 ml Erlenmeyer flask and add 0.1 ml of serum or plasma.
5.
Now add 2-3 drops of phenol red indicator to it, stopper the flask and mix gently. End-point of this titration is in the pH range of 8.4-6.7 by the colour change from yellow to red.
6.
The pooled plasma or serum sample is prepared by touching end of stirring rod to the Antifoam Antifoam A & rotating it. This This prevents excessive foaming. foaming.
7.
Place 0.1 ml of plasma or serum in 25 ml capacity Erlenmeyer flask & add 1 ml of 0.01 M-HCl, 4 ml of 1% saline to it. Shake the flask for 1 minute so that CO2 gas goes out.
8.
Now add 2-3 drops of indicator and titrate with 0.01M-NaOH till pink colour appears.
Calculation
0.1ml of blood sample was taken & 0.26 ml of 0.01 M-HCl might have been consumed if the normal value of calcium [26 m Eq/l] was present in blood. As 1 ml of 0.01 M-HCl was taken, 0.74 ml of it should have been left un-reacted & back titration might has consumed about 0.7 ml of 0.01M-NaOH. From latter reading (X ml) amount of HCO 3- ion in blood can be calculated and expressed in meq/lt. Conc of HCO3- in meq/lt = 1 ml of 0.01 M-HCL - X ml of 0.01M-HCl Check your progress-V Notes:
(a)
Write your answer in the space give below.
(b)
Compare your answer with those given at the end of the unit. (i)
O2 reacts with blood to give ________________ .
(ii)
Why heparin (Sod. slat) & potassium oxalate are added to
(iii)
Write application application of Radioimmunoassay. Radioimmunoassay.
(i)
_________________________________________________
(ii)
_________________________________________________
(iii)
_________________________________________________
blood.
5c.4 Estimation of Serum Proteins
[Albumin and Globulin] Principle
Albumin protein present in serum binds with bromocresal green at pH of 4.1 to give green coloured complex. Intensity of colur is proportional to the concentration of albumin, therefore, it can be determined colorimetrically at 640 nm (or using red filter).
Chemicals Required (i)
Albumin reagent
It is prepared by mixing following chemicals in 900 ml of distilled water: (a)
Succinic acid = 8.85 g
(b)
Bromo Cresol Green = 108 mg.
(c)
Sodium azide =
100 mg
Brij 35
4.0 ml
(d)
=
pH of the solution is adjusted to 4.1 using NaOH and the volume is made up to 1 litre.
(ii)
Albumin Standard : Contains 4.0 gms/dl in normal saline alongwith 0.1
gm/dl sodium azide. Methodology of Estimation : 5 clean and dry test tubes are taken and numbered
as 1, 2, 3, 4 and 5. Reagent and their quant
ities added to each test tube are
given in the following table: Reagent (ml)
(i) (ii)
Albumin reagent reagent Serum (1:10 dilution)
(iii) Albumin
Standard
(1:10
dilution)
Test tube
Standard
Blank
(1)
(2)
(3)
(5)
5.0
5.0
5.0
5.0
0.5
0.5
5.0
-
-
-
0.5
-
-
-
(4)
0.5
–
(iv)Distilled (iv)Distilled water -
Absorbance of of all the test tube solution solution is recorded at 640 mm.
Calculations :
Serum albumin (g/tube) [0.05 ml of serum]
ODt ODs
0.002 [Conc.
of standard tube]
Serum albumin (gm/dl)
ODt 0.002 100
ODs
ODt ODs
0.05
4
Normal value of serum albumin = 3.3-4.8 gm/dl Serum Globulin (g/dl) = Total protein-Albumin A/G ratio =
Serum albumin(g/ dl) Serum globulin(g /dl)
0.5
Clinical Diagnosis
Higher value of serum albumin is noted during dehydration due to hemoconcentration. Decrease in serum albumin is noted during severe malnutrition, renal diseases, pregnancy, liver inefficiency and burn. However, serum globulin is increased during chronic infections, autoimmune diseases, multiple myeloma & macroglobulinemia. macroglobulinemia. Estimation of serum protein by Biuret method of o f Reinhold. Principle
Biuret method (most commonly used method) of determination of serum protein is based on the principle that subrtances subrtances which contain contain two-CONH 2 groups or more groups joined together or those containing peptide linkages give purple colour with alkaline CuSO 4. One Cu-atom complexes with four molecules of biuret & central nitrogen is involved in linkage. The amount of colour varies with different proteins and carbohyhate contents in complex protein. Chemicals required (i)
Biuret Reagent Stock :
This stock is prepared by dissolving 45 gms of sod. potassium tartarate in approx. 400 ml of 0.2 N-NaOH and adding 15 gms gms of CuSO 4 to it with continuous stirring. 5.0 gms of KI is also added to it and volume is made upto 1 litre with 0.2 NNNaOH. (ii)
Standard protein solution :
Bovine serum albumin 0.5 g/100 ml. (iii)
Biuret Reagent
200 ml of stock reagent is diluted to 1 litre with 0.2 N-NaOH containing 5.0 gm of KI/litre. Methodology of estimation
In 6 dry & clean test tubes different solutions are added in the quantities given in following Table:
Reagent (ml)
(i)
Serum (1 : 10 dilution)
(ii)
Standard Protein solution
(iii)
Distilled water
(iv)
Biuret reagent
Test
Standard
(1)
(2)
1.0
1.0
–
–
2.0 2.0 3.0
3.0
(3 (4)
_ 1.0
_
Blank (5)
_ 1.5
(6) –
2.0 –
2.0 1.5 1.0 3.0 3.0 3.0
3.0 3.0
Mix the solution in each test tube; keep on water-bath at 37 0C for 10 minutes & record the absorbance at 540mm calorimetrically using green filter. Calculations
Draw calibration curve & find out total protein present in serum by reading absorbance of sample from the curve. Normal value of serum protein is between normal 6-8%. An increase in normal value is noted due to dehydration and decrease is due to excess of water intake.
Hyporproteinemia:
Is due to increased globulin synthesis, chronic liver fever, chronic infections like Kalazar, bone disease like multiple myeloma, collagen discease etc.
5c. 5 "Estimation of Glucose" Estimation of glucose in the given sample colorimetricall colo rimetrically. y. Chemicals Required (i)
Standard sugar solution
To prepare it dissolve 25 mg of glucose in water and make up the volume to 100 ml. This solution contains 250 g glucose/ml. For obtaining calibration curve 0.1 to 1.0 ml of this solution are used. reagent : (ii) Anthrone reagent (0.2 % anthrone in 70% H2SO4). It is always prepared fresh & is allowed to stand for 30-40 minutes before use. Principle :
Anthrone reagent reagent upon heating heating with glucose glucose gives green or or blush green color. color. Hence, colorimetric method can be used. Methodology of estimation
Pipette out 10 ml of freshly prepared anthrone reagent in a test tube. Chill it in ice cold water. Take 1 ml of sugar extract and dilute to 10 ml with water. Take out 1 ml from this diluted sugar extract and layer on anthrone reagent. Cool it for 35 minutes and mix the contents thoroughly thoroughly while still immersed in ice-cold ice -cold water. Heat the contents in boiling water-bath for 10 minutes. Then immediately immediately cool in cold water. Record absorbance at 526 nm against blank. From calibration curve sample absorbance may be determined.
Estimation of blood sugar (glucose) calorimetrically calorimetrically Principle
Astor and king method (1954) of estimation of glucose is based upon the fact when blood is kept into isotonic sodium sulphate-copper sulphate solution no further copper sulphate is required. Upon addition of sodium tungstate to it copper tungstate if formed & proteins as well as non-glucose reducing substances are preipitated. Results obtained obtained by this method are very close to glucose concentration. From the filtrate concentration of glucose is determined by the use of its property to reduce alkaline cupric (Cu2+) ions producing Cu 2O (cuprous oxide). Cu 2O reduces phosphomolybdic phosphomolybdic acid to molybdenum blue which can be measured photometrically. photometrically.
Reagents required
(i)
Isotonic Na2SO4 CuSO4 Solution –
To prepare it dissolve 30gms of Na 2SO4. 10 H2O and 6 gms of CuSO 4. 5H2O in distilled water & make up the volume to 1 litre. (ii)
Sodium tungstate solution :
It is prepared by dissolving 10 gms of sod tungstate (Na 2WO4. 2H2O) in water & making up the volume to 1 litre. Tartarate Solution (iii) Alkaline Tartarate For it 25 gms of NaHCO N aHCO3 is dissolved in approx. 600 ml of water in 1 litre beaker. 20 gms of Na 2CO3 is added to it with shaking to dissolve 18 gms of potassium oxalate is dissolved separately in warm water and carbonate mixture is added to it. 12 gms of sod-potassium tartarate is also dissolved separately separately in small quantity of water & it is mixed to previously prepared solution & volume is made upto 1 litre. Phosphomolybdic acid reagent (iv) Phosphomolybdic 35 gms of molybdic acid, 5 gms of sodium tungstate and 20 gms of NaOH are boiled with 300 ml of DW in 1000 ml beaker for 20-40 20 -40 minutes to expel ammonia present in molybdic acid. Cool the solution & transfer in 500 ml volumetric flask. Now add 125 ml of conc. Orthophosphoric Orthophosphoric acid (85% H 3PO4) and make up the volume to 500 ml with DW. Standard Glucose Solution (Stock Solution) (v) For it dissolve 100 mg of pure glucose in 100 ml of isotonic Na 2SO4-CuSO4 solution. (vi)
Working Glucose Standard (2 mg%)
To make it pipette accurately 2.0 ml of stock glucose solution in 100 ml volumatrie flask & make upto mark with isotoric Na 2SO4-CuSO4 solution. 1 ml = 0.02 mg glucose. Methodology for Estimation
Pipette out 3.8 ml of isotonic solution in centrifuge tube. Add 0.1 ml of blood serum or plasma & mix it. Add 0.1 ml of 10% Sod. tungstate, mix and centrifuge after 5 minutes. Transfer the supernatant into clean tube & precipitate is discarded. Following amounts of different reagents are added to 1-5 clean & dry test tubes.
Reagent (ml)
Test (1)
(2)
Standard
Blank
(3)
(5)
(4)
(i)
Supernatant
(ii)
Standard glucose
(iii)
DW
(iv)
Isotonic
(v)
1.0
Alkaline tartarate tartarate
1.0
_
_
–
–
–
1.0
1.0
–
–
– –
–
1.0
1.0
1.0
2.0
2.0
1.0 1.0
1.0 2.0
1.0 2.0
Mix the solution & heat on water - bath for 10 minutes. Cool, add to test tube 3.0 ml g phosphomolybdic acid. Mix & record absorbance after 5 minuts at 680 nm. Calculations
mg of glucose in test/0.25 ml of blood/plasma/serum ODt ODs
0.02(conc
of standard/tube).
Blood/serum/plasma Blood/serum/plasma glucose (mg/ml)
ODtt OD ODss OD
0.02 0.025
100
ODtt OD ODss OD
80
Normal values (fasting)
Venious or capillary capillary whole blood blood = 60-100 mg/dl mg/dl Renal threshold for glucose
= 180 mg/dl
Plasma or serum
= 70-110 mg/dl
Clinical diagnosis
Blood
sugar
increases
during
Diabetes
mellitus,
hperthyroidism,
hyperadrenalism, hyperpituitarism. however, it decreases during Addison's disease, hyperinsulinism & glycogen storage storage disease. In old age level is higher. 5c6: Estimation of blood urea nitrogen
Blood urea nitrogen means amount of nitrogen in blood coming from urea. Urea is secreted is body by liver and removed from blood through kidney. It can be estimated flame photometricaly as well as colorimetrically.
Increase in blood urea nitrogen occurs in body during dehydration or shock and also during gastrointestinal haemorrhage. Decrease in blood urea nitrogen is of little importance but it leads to liver problems & poor poor nutrition. Over dehydration probably probably from intravenous intravenous fluids also leads to low blood urea nitrogen. 5c.7 Estimation of Uric acid in Serum
Uric acid is the product of urine metabolism in primates, birds and reptiles. Nucleic acids degrades in presence of nucleases to give free purine & pyrimidin bases. Estimation of uric acid is based upon its reaction with phosphotungstic acid. Important method for the estimation of uric acid is due to Heny et al. (1957). Principle
Uric acid gets gets oxidised with phophotungstic phophotungstic acid in alkaline alkaline medium. medium. In the process phosphotungstic acid gets reduced into a blue complex which can be determined calorimetrically between 650-700nm. Requirements
1.
10% sodium tungstate.
2.
2/3 N-H2SO4 : To prepare it add 18.8 ml of conc. H 2SO4 to 500 ml of D.W. and dilute to 1 lt.
3.
70% Na2CO3 solution .
4.
Phosphotugstic acid regent : It can be prepared by dissolving 40gm Sod.
tungstate in 300 ml water. Add to it 30 ml of orthophosphoric acid and glass beads. Refluxing is done for 2hrs. cool it to R.T. & dilute to 1lt. Dissolve 32 gms lithium sulphate in the reagent and mix thoroughly. Store the reagent in refrigerator. 5.
Stock solution of uric acid : To prepare it add 100 mg of accurately
weighed uric acid and 60 mg of lithium carbonate into 100ml volumetric flask. Mix 100 ml of D.W. in it and warm to 60 0C. Cool to R.T. and make the volume upto the mark. This solution contain 1mg/ml. 6.
Standard Uric Acid solution: (0.02mg/ml). It can be prepared by diluting
2.0 ml of stock solution to 100 ml with D.W.
Methodology
1.
Dilute 1ml of plasma with 8 ml of D.W., 0.5 ml of 10% sodium tungstate tungstate and 0.5 ml of 2/3 N-H2SO4. Mix the contents well and centrifuge . Uric acid is tested from the supernatant.
2.
To test uric acid take 5 test tubes and lebel 1-5; 1 & 2 contain sample; standard (0.04mg/tube) (0.04mg/tube) are in 3 & 4 and t.t. 5 is for blank reading. Compositions Compositions in test tubes 1-5 are as given in following table :
Reagents (ml)
Sample
Standard
Blank
t.t.1
t.t.2
t.t.3
t.t.4
t.t.5
1. Supernatant
2
2
-
-
-
2. Uric Acid (Standard )
-
-
2
2
-
3. D.W.
-
-
-
-
2
4. Na2CO3 Solution
2
2
2
2
2
5.Phosphotungstic 5.Phosphotungstic Reagent
3
3
3
3
3
3.
Mix the contents of each tube thoroughly allow and the tubes to stand at R.T. for 15 minutes.
4.
Read the O.D. against blanks [t.t.5] at 700-710 nm (Red filter)
Calculation
mg of Uric acid/tube i.e. 0.2 ml of blood =
ODT × 0.04(Conc. of sandard / t.t. ) ODS
Serum Uric acid (mg/dl)
=
=
OD T ODS ODT ODS
×
0.04 0.2
× 20
×100
Range
Range of uric acid in serum of a healthy pession in 2-6 2 -6 mg./dL. Inference from the concentration of Uric acid
-
Increase in uric acid [6.5-12mg./100ml of Serum] in an indication of gout. Gout is a disease of joints identified by the depositions of urates as cryratals.
-
Malfunctioning of kidney may also lead to increase in uric acid contents in blood ranging from 4-20mg/100ml. 4-20mg/100ml. However, However, extra renal functions also have effect on serum level.
-
Elevation in uric acid in serum level may also be seen in the case like leukemia, lobar pneumonia, large abcesses, toxemia of pregnancy etc.
-
Increase in uric acid in urine is also accompanied by uric acid increase in blood.
-
Decrease in concentration of uric acid has been recorded during Wilson's disease and fanconi syndrome.
5C.8 Determinations of Barbiturates Barbiturates Exercise 1
Determination of Phenobarbital (Luminal) Methodology
Dissolve in a conical flask accurately weighted 0.1g of sample in 5ml of pyridine and add 0.25 ml of thymolphtalein solution as well as 10 ml of AgNO 3 – Pyridine reagent to it. Titrate the contents with 0.1 N-NaOH (alcoholic) till blue color appears. Calculation
1.0 ml of 0.1 N-NaOH - 0.011610g of Phenobarbital. Exercise -2
Determination of Cyclobarbitone Cyclobarbitone calcium. Methodology
as in Ex-1
Calculation
1 ml of 0.1 NN- NaOH - 0.02553 g. g. of cyclobarbitone cyclobarbitone Calcium. Exercise -3
Determination of Quinobarbitone Quinobarbitone sodium. Methodology
Same as Ex -1 Calculations
1ml of 0.1 N- NaOH = 0.02603g of Quinobarbitone sodium. 5C.9 Estimation of Serum Serum Alkaline Phosphatase
Phosphates are the enzymes that catalyse the removal of phosphates form certain monophosphoric esters. This enzyme is most active at the pH-10. Phosphatases are intercellular but are also present in smaller amounts in plasme certain diseases their amount increases in plasma. Most popular method to determine concentration of alkaline phosphatases in plasma in King and King (1954) method.
At a pH of 10, phenyl phosphate phosphate is hydrolysed to phenol when incubated with alkaline phosphatases. phosphatases. The phenol formed condenses with with 2-amino antipyrine. antipyrine. The product formed, after the oxidation with alkaline potassium ferrocyanide gives red colored complex which can be estimated at 520 nm. Requirements 1.
0.1N-Disodium phenylphoshpate phenylphoshpate : To prepare it 2.18g of disodium
phenyl phosphate is dissolve in a lt. of D.W Boil the solution, cool it and preserve with 1ml of chloroform at 4 0C. This solution rest for 3-4 weeks. 2.
0.5N-NaOH : To prepare it dissolve 20 g of NaOH in 1lt. of D.W.
3.
O.1N-Carbonate buffer : It is prepared by the dissolution of 6.36 g of
anhydrous Na2CO3 and 3.36g of NaHCO 3 in distilled water. This solution is made up to 1lt. with D.W. The pH of this buffer is 9.5-10.0 4.
0.5N-NaHCO3: For this solution solution 4.2 4.2 g of NaHCO NaHCO 3 is dissolved in 100 ml of
D.W.
5.
0.6% 4-Amino antipyrine: To prepare it dissolve 600 mg of 4-amino
antipyrine in 100 ml of DW. 6.
K 4[Fe(CN) 6] : 4% Potassium ferrocyanide is prepared by dissolving 4g of it
in D.W. & diluting it to 100 ml with D.W. This solution should be prepared fresh and must be stored in brown bottle. 7.
1mg/ml solution is prepared by
Standard stock solution of phenol:
dissolving 100 mg of pure phenol in 100 ml of 0.1 N-NaOH.
Methodology
1. Four test tubes are lebelled as 1,2,3 & 4. They contain sample, control, standard and blank. They are filled with following solution:
S.No. Reagents
1
2
3
4
1.
Disodium Phenyl phosphate phosphate
1.0
1.0
-
-
2.
0.01N-Carbonate 0.01N-Carbonate buffer
1.0
1.0
1.1
1.1
0.1
-
-
-
Incubate in water bath for 15 minutes at 37 0C 3.
Serum
4.
Standard phenol soln.
-
-
1.0
-
5.
D.W.
-
-
-
1.0
Again incubate incubate in water bath bath for 15 minutes at 370C 6.
0.5N-NaOH
0.8
0.8
0.8
0.8
7.
Serum
-
0.1
-
-
8.
0.5 N-NaHCO3
1.2
1.2
1.2
1.2
9.
4-Amino antipyrine
1.0
1.0
1.0
1.0
10.
K 4[Fe(CN6)]
1.0
1.0
1.0
1.0
Thus total volume in each tube is 6.1 ml.
2.
O.D. of solution in each test tube is measured at 520nm against blank .
Calculation
Serum alkaline phosphatases phosphatases (In king Armstrong unit) =
O. D.T O. DC O. DS
10
King Armstrong unit: 1 mg of phenol liberated by 1dl of serum under assay
conditions. 1K.A. Unit = 7.1 U/L Range: Range of serum phosphatases in a normal adult is 3-14 KA- Unit or 22-92
U/L. Level of alkaline phosphates is about 2.5 times in actively growing children. Inference from the level of alkaline serum phosphates
1.
Increase in level have been found during rickets, osteomalacia and osteoblastic osteoblastic tumors etc.
2.
Serum alkaline phosphatases phosphatases activity also increases during disease of liver & bialary tract : like all types of jaundice except homolytic jaundice.
3.
Its level decreases during severe anaemia, scurvy, hypophosphatemia hypophosphatemia
&
cretinism. 5C.10 "Immunoassay"
Immunoassay is a technique for the determination of drugs, hormones and vitamins and some other compounds at nanogram and still smaller scale. It involves a reaction between analyte antigen and a specific antibody to give a precipitate or complex. 1.
Antigen :
Antigen is a foreign substance that can induce formation of antibody in the body and is capable of combining with it. Antigen is a macromolecule like protein, hormone etc. 2.
Antibody:
Antibody is a protein which is capable ca pable to recognize a foreign substance & can have stereospecific association with it. e.g. Virus and bacteria. Globulin protein with high mol. wt of about 1,50,000 is an antibody. This protein exhibiting activity of antibody in known as immunoglobulin (Ig) . Five main immunolglobulins in human blood are IgA, IgD, IgE, IgG and IgM. But, IgG is most abundant. The Ig consists of three polypeptide polypeptide clains. Two are of 240 amino amino acid residues and third of 430 animo acid residues which are linked through disulphide bridge. When Ig is treated with papain (an enzyme) three fragments (each of mol. wt approximately 50,000) are formed. Two are identical and can combine with antigen and are referred to as Fab – Fragment, antigen binding. Third can't combine with antigen and is fragment crystallizable crystallizable [Fc] It is worth- noting all antibodies are similar in structure but have variable antigen binding proportions of fab. Antigen-Antibody Antigen-Antibody association
Antibodies are produced in organism only after organism has at least one exposure to antigen through vaccination etc. For immunoassay antibody is produced by injecting antigen into animal and by recovering the serum that contains resultant antibody. This serum is known as antiserum. Antigen- Antibody reaction gives antigen-antibody complex. Forces binding them are called affinity. Infact, affinity is intrinsic association constant between antibody and univalent antigen. Here, is
another term avidity which refers to the overall binding energy between antibodies and a multivalent antigen. Overall binding reaction can be written as : Ab + Ag
=
Ab.Ag
Formation constant (K)
=
[Ab.Ag ] [Ab ][ Ag ]
Formation constants are of the order of 108-1010. The binding forces are weak Vender waal force, electrostatic and hydrophobic force. The bonds are broken by the addition of salts, increasing temperature, pH or polarity of solvent. 5.c10.1
Principles of Radio Immunoassay [RIA]
RIA takes into account sensitivity of radiochemistry , fluorescence and enzymatic tag with specificity of immunology. Latter is study of antigen-antibody reaction. Immunoassays [RIA] are based upon the discovery of Rosalyn yalow & Berson that low concentrations of antigen hormone insulin can be detected radiochemically due to their ability to combine with radiolabelled insulin (I 131). For this discovery they were awarded Noble Prize in 1977. The determination of unknown concentration concentration of antigen is based upon upon the fact that radiolabelled radiolabelled antigen and unlabelled antigen (from the standard sample) compete physiochemically for the binding sites on antibodies. For RIA following operations are carried out : 1.
Antibody solution (Ab antiserum) labelled antigen (Ag)* and serum sample containing unlabelled antigen antigen sample sample to be determined [Ag] are taken is reaction vessel.
2.
Contents of step-1 are incubated as a result of which antigen- antibody complex [Ag.Ab] is formed. In absence of unlabelled antigens, certain fraction of labelled antigen [Ag]* bind to give Ag* - Ab. Upon increasing the amount of Ag, the limited binding sites of antibody are progressively saturated and antibody binds less of the Ag*.
3.
After incubation, bound antigens are separated from free antigens & labelled portion of either or both phases is measured by measuring radio-activity or fluorescence in order to determine the percent bound to Ag*.
4.
A Calibration Calibration curve is prepared using antigen standards of known concentration by plotting either the percent bound to free as a function of unlabelled antigen concentration. From calibration curve concentration of antigen in the sample can be determined .
Drawbacks of RIA 125
I and
131
I emit -radiation which needs special counting equipment .
1.
Both
2.
The body concentration of I-atoms radioactive or non-radioactive in thyroid gland gets incorporated in thyroxin.
Application of RIA
It is an important important technique technique for clinical laboratories laboratories & is used in : 1.
Determination of plasma levels of : a.
Certain abused drugs
b.
Hormones
c.
Digitoxin or digoxin is patients receiving these drugs.
2.
Anti-DNA antibodies in systemic lupus erythematosus erythematosus [SLE]
3.
Blood banking.
4.
Diagnosis of allergies.
5.
Presence of Hepatitis B surface antigen in blood.
5C.11 Blood Gas Analysis
Blood gas analysis measures the amount of CO 2 in blood as well the pH(acidity) of blood. It evaluates evaluates the efficiency with with which lungs are delivering delivering O 2 into blood & are eliminating eliminati ng CO 2 out side. It also indicates the efficiency with which lungs & Kidneys are interacting to maintain normal blood pH i.e. acid base balance. Blood gas analysis analysis in done to access certain disease conditions conditions of lungs & kidneys. It measures the partial pressure of O 2 –contents, O2 saturation, bicarbonate contents and blood pH. It also indicates how much oxygen is combined with haemoglobin through
the analysis of oxygen saturation which compares
the amount of O 2
actually combined combined with haemoglobin to the total amount amount of O 2 which can combine with haemoglobin. haemoglobin.
CO2 dissolves in blood to form bicarbonate and carbonic acid that maintain pH of blood at which cellular functions go on normally. Lungs and kidneys maintain balance of carbonic acid and HCO 3 in blood, respectively. Normal Ranges of Blood gas Analysis :
1.
Partial pressure of Oxygen =
75-100 mm Hg.
2.
Partial pressure of CO2
=
35-45 mm Hg.
3.
Oxygen content
=
15-23 %
4.
Oxygen saturation
=
94-100%
5.
HCO-3
6.
pH
=
22-26 mEq./lt. =
7.35-7.45
Conclusions from blood gas analysis
-
Abnormal results indicate respiratory, Kidney or metabolic problems & also during trauma which effects breathing, results are abnormal specially during head & neck injuries. .
-
Abnormal results are also seen is disorders like anaemia which affects O 2 carrying capability of blood & gives abnormally low O 2 contents.
5C.12 Trace Elements in the body
Trace elements are found is very small amounts in body but are absolutely essential for different functions of body. There are seven essential trace elements for man : 1.
Copper
2.
Chromium
3.
Cobalt
4.
Iron
5.
Selenium
6.
Zinc
7.
Iodine
1.
Copper (Cu)
Cu is essential part of some enzymes which inactivate free radicals. Thus, it helps in anti-oxidant protection. It plays role in Iron-Metabolism. Enzymes containing Cu are involved in immune function. It is used for making blood cells. Increase in copper contents may lead to liver failure. Its recommended dietary allowance is 2-3 mg which is found in daily multivitamins.
2.
Chromium (Cr)
It is needed by cells in the glucose intake process. It's deficiency leads to increased blood sugar as well as increased cholesterol & triglyceride levels. Yeast, cheese & meet are chief source of Cr. Its recommended dietary allowance is 50200lg. Sometimes, its deficiency is supplemented through multivitamins which contain 15-100 µg of it. Cr is used up in exercise, infection & injuries etc. 3.
Cobalt [Co]
It is a part of Vitamins - B 12 . 4.
Iron (Fe)
It is part of RBC and have anti-oxidant activity. Fe is frequently used in the body to prevent its use by bacteria as a source of fuel. Iron gets attached to proteins & reaches to other parts of body through them. Vitamin-C promotes its functions. Deficiency of Fe leads to anaemia. Chief sources are liver & red metals. Recommended dietary allowance is 7-14 mg./day.
5.
Selenium (Se)
Se is anti oxidant. It marks immune body function which are like functions of vitamin-E. Deficiency of Se leads to heart diseases and anaemia. Its deficiency have also been found in HIV affected persons. Recommended dietary allowance of Se is approx 50 g from one to four times a day. 6.
Zinc (Zn)
Functions of Zn involve the wound healing & maintenance of membranes. It is also involved in anti-body production. During stess it is shifted from blood to tissues. Hence, plasma levels do not reflect their actual concentrations. Its deficiency impairs immune response and causes hair loss.
Other diseases like slow proteins
metabolism, diarrhoea, change of taste, loss of appetite, disturbance in absorption, low levels of testosterone etc have been found in Zn-Deficient persons. HIV patients also have low level of Zn. Recommended diectony allowance of Zn. is 15 mg. Multivitamins Multivitamins also contain is 15 mg of of Zn. 7.
Iodine(I)
It plays important role in the thyroid metabolism. It's supplementation is not the need. However, iodized salt fulfil the demand. 5 (d) Drug Analyisis
The word 'drug' is derived from French word 'drogue' which means a dry herb. It may be defined as a substance used in the prevention, diagnosis, treatment or cure of disease in animals including man. Characteristics Characteristics of Drug
1.
The action of an ideal drug should be localized at the site where it is desired to act. However, there is hardly any drug that have this activity.
2.
It should be non-toxic.
3.
It should have minimum side effects.
4.
Drug should not injure host-tissue or physiological process.
5.
Drug should be efficient.
6.
It should not make the host cells resistant to drug after its use for some time. However, most of the drugs do not possess this characteristic.
5 d. 1 "Narcotics & Dangerous drugs"
Narcotics is the term used to describe the substances which induce sleep. In legal context narcotics refer to opium or opium derivatives and their synthetic or semisynthetic substitutes. Cocaine and Coca-leaves are classified as Narcotics in the U.S. controlled substances Act. But, they are not narcotics technically. Infact, narcotic is a broad term; medical professionals use term opiod for natural, synthetic & semisynthetic substances which are pharmacologically like morphine. These are also known as narcotic analgesics. Narcotic analysis tends to produce euphoria that is an important factor in their addictive property which limit their use. Other limitations include include respiratory respiratory depression, increased increased biliary tract pressure, pressure, prunities due to histamine release & decreased gastro-intestinal motility leading to constipation. Effects of Narcotics
Effects of narcotics depend upon (i) dose (ii) Mode of administration (iii) previous exposure to the drug (iv) expectation of the user. Main drawbacks of use of narcotics are: 1. Drowsiness 2. Inability to concentrate
3. Lessened physical activity 4. Apathy 5. Constipation 6. Constriction of pupils 7. Nausea and vomiting 8. Respiratory depression 9. Dialation of subcutaneous blood vessels leading to flushing of face & neck. As dose increases, increases, subjective, analgesic and toxic effects become become pronounced. Except the cases of intoxication, there is no loss of motor co-ordination or slurred speech as with other depressants. Route of Administration Administration
1. Orally 2. Intravenous (though injections) 3. transdermally (skin patches) Complications
1. Hepatitis 2. Skin, lungs and brain abcesses 3. Endocarditis 4. AIDS etc. 5. They are habit forming. A chronic drug user needs more & more doses to attain desired effect like decrease in sedation, euphoria etc. Withdrawal Symptoms
Withdrawal symptoms from morphine & /or morphine like addictions are experienced before the time of next scheduled dose e.g. 1. Watering of eye 2. Running nose 3. Yawning & sweating 4. Irritability 5. Restlessness. 6. Loss of appetite 7. Increased heart beat & B.P. 8. Vomiting 9. Tremors and severe sneezing 10. Pain in muscles & back – bone 11.Severe depression
Any time during withdrawal withdrawal symptom admnistration of narcotic reverses the symptoms. Uses of Narcotics
These are used in the treatment of : 1. Pain, acute diarrhoea & cough supression. 2. Reduces tension, anxiety and agression & hence produces sense of well – being. Dangerous Drugs
Dangerous drugs are the drugs that are used illegally. Thay are dangerous in the sense that they cause addition as well as some harmful chemical and physical effects. Dangerous drugs can cause dangerous behaviour. They are good for none but are most dangerous for kids and teenagers who are at growing stage. These drugs can damage brain, heart and other important organs. For example, cocaine can cause heart attack even to kids & teenagers. Some examples, of most dangerous drugs are : (i) Methamphetamines (ii) Heroine (iii) cocaine (iv) Barbiturates (v) LSD (vi) Benzodiazepine (vii) Marijuana (viii) opium (ix) caffiene & nicotine (x) Hashish. 5d.2 "Classification "Classification of Drugs"
Most important classification of drugs is on the basis of their therapeutic actions. On this basis they are classified into two main classes: 1. Chemotherapeutic Chemotherapeutic agents 2. Pharmacodynamic Pharmacodynamic agents (1)
Chemotherapeutic Chemotherapeutic agents
These drugs are used to cure specific diseases like microbial infections, maleria, tuberculosis, syphillis etc. Chemotherapeutic agents attack and destroy the invading organisms without any adverse effect on infected organism. Chemotherapy is defined as the treatment of diseases by chemicals which selectively inhibit the growth of infecting organisms. First chemotherapeutic agent was an anti-syphillis agent discovered by Ehrlich (1910). This class has been divided into following types : (i) Antibacterials Antibacterials (ii)
Antimalarials Antimalarials
(iii)
Antiprotozoals Antiprotozoals
(iv)
Antifungals
(v)
Antiseptics
(vi)
Anthelmintics
(vii) Antineoplastic Antineoplastic agents (viii) Antitubercular & antileprosy antileprosy drugs. (ix) (2)
Organometallic Organometallic therapeutics
Pharmacodynamic Pharmacodynamic agents
These drugs have characteristic action on infected organisms but are not specific remedies for particular disease. These drugs are further classified into following classes: 1.
Anaesthetics
2.
Sedative, Hypnotics, Anticonvulsants
3.
Tranquillisers.
4.
Antipsychotic Antipsychotic drugs.
5.
Anti-anxiety drugs.
6.
CNS (Central nervous system stimulants)
7.
Pschotogenic drugs.
8.
Hypo and Hypertensive agents.
9.
Anti coagulants
10.
Adrenergic stimulants
11.
Cholinergic & anti-cholinergic agents.
12.
Diureties
13.
Antipyretics and analgesics.
14.
Antispasmodics
15.
Anti-histamines
16.
Anti-inflammatory Anti-inflammatory drugs
The medicinal value value of a drug is is expressed in "therapeutic "therapeutic index" which is the ratio ratio of amount necessary to kill the patient i.e. maximum tolerated dose to maximum curative dose. Therapeuti c index
Maximum Maximum tolerated dose Maximum Maximum curative dose
The therapeutic index of 10 indicates that 10 times of the dose used for curative purpose will kill the patient as well as to the parasite. Some important classes of both the major classes are being discussed here:
(1)
Anti-bacterials Anti-bacterials : These are of two types:
(A)
Sulpha – drugs
(B) (A)
Sulpha
Antibiotics drugs
[Sulphonamides]: Sulpha drugs are derivatives of
sulphanilamide. These were first synthetic compounds effective against bacteria that cause pneumonia, tuberculosis, gonorrhoea, diptheria, scarlet fever etc. Some examples are: sulphathiazole, sulphate acetamide, sulphapyridine sulphapyridine etc. (B)
Antibiotics : An antibiotic is a compound produced by one micro-organism
which is toxic to other micro-organism. Some examples are penicillin, streptomycin, streptomycin, tetracyclins, chloramphenicol etc. Antimalerials : They cure malaria (2) Antimalerials malaria caused caused by malaria parasite plasmodium vivax & & others. e.g. Quinine, chloroquine, chloroquine, camoquine, pamaquine, pamaquine, paludrine paludrine etc. (3) Antifungals : Anti-fungals cure fungal infutions in animals and plants. e.g. Clotrimazole, Clotrimazole, salicylic acid etc. (4) Antiseptics : Antiseptics may kill bacteria or may only prevent their multiplication. multiplication. They do not harm living cells hence are used on cuts & wounds. Few examples are : Dettol, savalon, gentian violet, acriflavin, mercurochrome, KMnO4, boric acid etc. (5) Anthelmintic : They kill worms e.g Ascaridol. (6) Antineoplastic: Thes are anticancer e.g fluoroceracil.
(7)
Antitubercular Antitubercular and anti-leprosy drugs: Anti-tubercular Anti-tubercular drugs are used to
cure tuberculosis-e.g. streptomycin, para-amino salicylic acid (PAS), isoniazid. Anti-leprotic agents are used in the treatment of leprosy caused by Mycobacterium leprae , e.g. sulphones like dapsone [DDS].
(8)
Organometallic Therapeuties : Examples of organometellic therapeutic
agents are Atoxyl, stovarsol. Generally they are effective against protozaon infections. (9)
Anaesthetics : The term anaesthetics is derived from Greek word,
anaesthesia means insensibility. Hence, anaesthetics may be defined as drugs which bring about the insensibility to the vital functions of all types of cells specially those of nevous system. Anaesthesia are of two types :
(A)
General Anaesthetics: These anaesthetics depress CNS to such an extent
that all the sensitivity to pain or feeling is lost. They cause unconsciousness all over the body e.g. Halothene, Pentothal sodium. (B)
Local Anaesthetics : Instead of affecting the whole body, these
anaesthetics make a particular organ insensitive e.g. Banzocaine, procaine
(10) Sedative, Hypnotic Hypnotic and Anti-convulsants: Anti-convulsants:
These are non-selective CNS modifers i.e. antidepressants. -
Hyprnotics are CNS depressants that produce sleep resembling the natural
sleep. e.g. Luminal -
Sedatives are CNS- depressants that reduce nervous tension and promote
relaxations without producing sleep. e.g. Diazepam Anticonvulsants are those CNS depressants depressants which prevent prevent or supress supress convulsions. Thus, these three differ only in degree of action. Large amount of hypnotics may produce anaesthesia & even death in some cases. (11)
Tranquilizers: These drugs are used in the treatment of mental disorders.
These are mood elevators and restore peace of mind without producing sleep. e.g. Amphetamin. (12) CNS-Stimulants CNS-Stimulants : These drugs stimulate central nervous system and are
anti-depressants. anti-depressants. e.g. Amphetamine. (13) Psychotogenic drugs : They induce behaviourial abnormalities resembling
psychosis. e.g. LSD [Lysergic acid diethyl amide] & mescaline [3,4,5trimethoxyphenyl trimethoxyphenyl ethylamine]. (14) Hypo and Hypertensive agents : Hypotensive agents increase B.P. &
hypertensive elvevate it e.g. methyldopa is used in hypertensions & reduces B.P. -
Sodium nitropruside is short acting hypotensive agent & is mostly employed as vasodialator in emergency treatments of hypertensive h ypertensive crisis.
Anticoagulants : Anticoagulants are the substances that interfere with blood (15) Anticoagulants
coagulation thus prolong coagulation time. e.g. Heparin & warfarin (a coumarin derivative).
Adrenergic & Cholinergic agents : The drugs that mimic stimulation of (16) Adrenergic
sympathetic nerve are called adrenergic drugs and the compounds that mimic the parasympathetic system are known as cholinergic drugs. Examples: are Adrenaline and ephedrine of former former and Acetylcholine Acetylcholine and Muscarine Muscarine of latter. (17)
Diuretics: compounds that increase urine volume output by kidneys are
called diuretics e.g. Caffeine, Acetazolamide. Antipyretics and Analgesics : Antipyretics bring down body temperature (18) Antipyretics
and analgesics provide relief from pain. e.g. Aspirin, paracetamol. Antispasmodic Agents : Control contraction of muscles. e.g. Khellin. (19) Antispasmodic Antihistamines : They also control contraction of muscles. e.g. Phenargan (20) Antihistamines
(promethazine). (21) Anti-inflammatory agents : They are used against swelling and
inflammation. e.g. during arthritis. Examples are : Phenylbutazone, oxyphenylbutazone phenylbutazone etc. 5d.3 "Screening of Drug by Gas & Thin-layer chromatography and spectrophotometric measurements"
Analysis of drugs drugs is being being discuss ahead under following three heads. (i)
Spectrophotometric Spectrophotometric determinations
(ii)
Thin-layer chromatographic (TLC) determinations
(iii)
Gas chromatographic chromatographic determinations.
5d. 3.1 Spectrophotometric Spectrophotometric determinations (UV/VIS)
Ultraviolet and visible spectrophotometry is a technique for the quantitative estimation of compounds and is also an auxillary tool for structural elucidation. Ultraviolet-visible region extends from 185-760 nm. Range of UV region is 185-400 mm & visible region is from 400 to 760 mm. Only coloured compounds absorb in visible region & colorimetry is concerned with the visible region of spectrum. In spectrophotometric analysis a source of radiation is used which gives radiation of 180-400 nm & the instrument used is known as spectrophotometer. But is colorimetric analysis determination of quantity of substance is made on the basis of colour changes which depends upon concentration of substance. The instrument used for colorimetric analysis is known as colorimeter.
Advantage of colorimetric and spectrophotometric spectrophotometric methods is that very minute quantities of substance present is solution can be determined by them. Upper limit of colorimetric determinations determinations is less than 1 or 2% Wave lengths of colours
Violet 400-450 nm Blue
450-500 nm
Green 500-570 nm
Yellow
570-590 nm
Orange
590-620 nm
Red
620-760 nm
In colorimetry colour may be either due to constituent itself or may be produced by the addition of suitable reagent & intensity of colour may be compared with that obtained by treating a known amount of substance in the same manner. Principles of spectrophotometry and colorimetry
When a monochromatic or heterogeneous beam of light falls on a heterogenous medium, a part of incident light is reflected, a part of it is absorbed within the medium & rest of it is transmitted. If the intensity of incident light is I o, that of absorbed light I a & transmitted light is It & reflected light is I r. I0 = Ia + It + Ir Absorption Absorption of light depends upon thickness of medium & is governed by Beer's & Lambert's laws which are : According to Lambert's law ln
or or
I t I o
I 0 I t
kI
It = IO. e-KI I t I 0
10
KI
is the fraction of incident light transmitted by thickeness l of the medium
as is known as Transmittance(T).
I0 It
= opacity
Absorbance of the medium A also known as optical density D or Extinction is given by formula :
A log
I0 It
According to Beer's Law : = Io. e-Kc.
It
= Io. 10-acl or
log
I0 It
= acl
combined Beer-Lambent law is : log
I 0 I t
cl
or = A/cl Instrumentation
Instrumentation of U.V.-visible spectroscopy is discussed ahead. Essential Features of simple filter colorimeter are : (i) Light source (ii) filter to select suitable wave length (iii) cuvettes (iv) photocell to receive transmitted light (v) a meter to measure the response of photocell. Double Beam Spectrophotometer
Modern UV-Visible spectrophotometer are double beam type & cover the range 200800 mm. In these instruments a monochromatic beam of light from tungstendeuterium lamps is divided into two identitical beams, one of which is allowed to pass through reference cell and other through sample cell. The absorption through reference cell is automatically subtracted from sample cell by the instrument & net signal corresponding to absorption for the component is recorded. The splitting and recombination operations on beam are carried out by two rotating sector mirrors which are geared to same electric motor so that they work in unison. The microprocessor which operates the instrument automatically corrects the dark current of photocell. Origin of Absorption Spectra
UV-Visible spectra are obtained as a result of excitation of electrons to higher energy levels. Lower energy excitations show absorption in visible region whereas higher energy excitations give absorption bend in UV-region. Electronic energy changes are accompanied by vibrational energy changes i.e. why instead of sharp absorptions lines, broad absorption bands are obtained. Absorptions are of different types : - *, n - *, - *, n - *. These are responsible for different absorption bands. Preparation of Standard Curve
When determinations are made by using spectrophotometer or filter photometer, it is essential to prepare standard curve, also known as calibration curve or reference curve, for the constituent to be determined. For it, determinations for suitable quantities are made in the same way as for the sample by measuring absorbance (or transmittance) at optimum wave length. Preferably absorbance (
log
Io It
) is plotted against the concentration & a straight line is obtained if Beer's Law
is obeyed. When absorbance is directly proportional to concentration few points are needed to prepare the line, but if relation is not linear greater number of determinations are needed to be done to get the line. In case, filter photometer is being used characteristics of light source as well as filter may change with time. While using transmittance, transmission of 100% is assigned to blank solution (reagent & water) which represents Zero concentration of the substance to be estimated. It is worthnoting that few coloured solutions have appreciable temperature co-efficient of transmission, therefore, temperature should remain constant through out the determination. determination. Selection of Solvent
To be a good solvent during spectrophotometric or colorimetric estimations it should have following characteristics : (i)
It should be a good solvent for the constituent to be determined.
(ii)
It should not react with the substance.
(iii)
It should not absorb at the wavelength used in determination. determination.
For inorganic substances water is the best solvent. But, for organic estimations organic solvent is needed. Polar organic solvents like alcohols, ketones, esters water etc suffer from drawback that they obliterate fine structure of spectrum due to vibrational effects. Therefore, hydrocarbons like cyclohexane (e.g. in case of
phenol) give better spectrum with three sharp peaks as compared to water in which a single broad band is obtained. Wavelengths of absorption of some good solvents are given below: Solvent
nm
Solvent
nm
Water
190
Cyclohexene
212
Hexane
199
Dichloromethene
247
Heptane
200
Chloroform
257
Diethylether
205
Benzene
280
Ethanol
207
Pyridine
306
Methanol
210
Acetone
331
Methodology of UV-Visible spectrometric & Colorimetric Determination (I) Cells should be washed with distilled Water
If solvent used is not miscible with distilled water after use cells should be washed with that solvent thoroughly. Finally they are rinsed with ethanol & dried. Drying may may be done done in vaccum vaccum
desiccator also. also. The cells which have become become
contaminated may be cleaned with detergents like teapot followed by proper drying. (II)
Preparation of Solutions
All the solutions needed should be prepared accurately. In UV-instruments direct measurements may be made without adding reagents. Solutions which are unstable should be prepared the moment, they are to used. The test solution should be dilute to the extent that absorbance lies is the region 0.2-1.5, otherwise they should be further diluted.
(III) Calibration Curve Preparation
Calibration curves are plotted by measuring absorbance at 5.0, 7.5, 10.0 and 15.0 ml of standard solutions which are diluted to 100 ml in graduated flasks & plotting them against concentrations. But, if same estimations are routine procedure, already prepared calibration curves may be used. use d. (IV) Calculation of Concentration Concentration of Unknown Unknown Solution Solution
During UV measurements concentration of unknown solution is calculated by the formula : Molar extinction coefficient ( ) = A/cl. Where, A = Measured absorbance of solution at concentration concentration C. l = Path length. During few measurements is unknown (e.g. during natural product 1cm measurements) because relative masses are unknown; then E 10 / 0 value is used
which is the absorptivity. absorptivity. Experiment
Estimation of phenacetin, caffeine and Aspirin in the given mixture of drugs. Principle
A mixture of phenacetin, caffeine and Aspirin may be analysed quantitatively by UV spectrophotometric method. Phenacetin shows absorption maxima ( max) at 250 nm, caffeine at 275 nm and aspirin at 277 nm. First they are separated & then analysed spectrophotometrically spectrophotometrically :
Chemicals Required :
(i)
Dichloromethane (CH2 Cl2)
(ii)
4.0% NaHCO3 Chilled
(iii)
Conc. HCl
(iv)
1M – H2SO4
Preparation of Standard Solution
Standard solution of each drug is prepared in CH 2Cl2. The concentration for solution are 10 mg/lt for caffeine, 20 mg/lt for phenacetin & 100 mg/lt for aspirin. They may be prepared by dissolving 25 mg of each drug in 100 ml of CH 2Cl2 & diluting 1 ml and 2 ml of these solution in 25 ml measuring flask to get 10 mg/lt & 20 mg/lt of solutions. As aspirin decomposes in the solution, hence, its solution should be prepared just before using it. Methodology
Accurately weigh
about 400 mg. of tablet in a beaker. Add to it 20 ml of
CH2Cl2 and transfer the solution to 60 ml separatory funnel. Extract aspirin from CH2Cl2 extract with two 10ml portions of chilled 4.0% NaHCO 3 solution. Add to extract 5 ml water containing two drops of HCl. Wash the extract with three 10 ml portions of CH 2Cl2 & add the washings to original CH 2Cl2 layer. Leave the aqueous extract is separatory funnel. Now filter the CH 2Cl2 solution through the filter paper impregnated with CH2Cl2 in a 50 ml volumetric flask & make up the volume. Acidify NaHCO3 layer with 6 ml 1M-H 2SO4 solution to avoid hydrolysis of aspirin. pH at this point should be 1-2. Extract this acidified solution with eight separate 10 ml portions of CH 2Cl2 and filter through filter paper already impregnated with CH2Cl2 in a 100 ml volumetric flask and make up volume upto the mark. Now, dilute 5 ml of this solution to 25 ml with CH 2Cl2 in a volumetric flask [Ca: 25 ml]. Record the absorbance of this solution on at 277 nm. Absorbance of standard solutions of aspirin is recorded under exactly same conditions at 277 nm from which concentration of aspirin in given mixure of drugs can be calculated. Concentration Concentration of aspirin is used to calculate its percentage. Similarly, concentrations concentrations and hence percentages of phenacetin and caffiene in the tablet (mixture of drugs) can be determined at 250 and 275 mm, respectively.
Result
Given
tablet
is
a
mixutine
of
drugs
:
.......................%
Aspirin;
...................... .........................% ...% phenacetin and ......................% ......................% caffeine.
5 (d)3.2 "TLC Determinations" –
This Layer Chromatography (TLC)
(i)
In this technique separation of components of mixture or identification of organic or inorganic compounds is made on a thin film of adsorbent which
acts as stationary phase. Adsorbent adheres to glass plate by virtue of its binding ability, for instance, calcium sulphate which is incorporated incorporated acts as a binding agent. This film prepared on glass is known as Chromaplate. As this technique uses thin film of adsorbent that is why it is known as thin layer chromatography (TLC). Some workers call it spread column chromatography,
open
column
chromatography
or
surface
chromatography. TLC Technique –
Infact TLC is adsorption chromatography and is based upon the difference in adsorption of the components of mixture on the adsorbent. Distribution coefficients of components between stationary and mobile phase play an important role in TLC – separations. Stationary phase in TLC is adsorbent coated on glass plate and mobile phase is the solvent system which moves on adsorbent thin layer. On the basis of difference in adsorbitivity different components get adsorbed at different distances and are hence separated. The process by which different components move upward through continuous flow of mobile phase is known as elution and solvent is known as eluents. The components which move upward are known as eluates. Entire TLC technique can be studied under following steps: (i)
Selection of adsorbent
(ii)
Preparation of TLC plates
(iii)
Choice of solvent
(iv)
(1)
Application Application of sample
(v)
Development of plates
(vi)
Location of spots
(vii)
Determination of R f f.
Selection of Adsorbent:
While selecting adsorbent polarities of sample to be separated or purified and that of solvent should be matched. Adsorbent should be completely insoluble in solvent. The particle size should be 200-300 mesh. Adsorbent should not enter in chemical reaction with sample as well as solvent. Earlier, silica gel, alumina (basic) and neutral Kieselghur were used as adsorbent. But, now a days a number of materials are available (with or without fluorescent compound) liked ZnS to view compounds under U.V. lights for this purpose. (2)
Preparation of TLC Plates (Preparation of chromato plates)
Before coating glass plates with adsorbent they should be properly cleaned with laboratory detergents using test-tube brush to remove adhering particles and washed thoroughly with distilled water. Plates should be allowed to drain and dried in oven. Plates should be handled from edges or the side which is not to be coated. Otherwise a mechanically unstable layer is formed. If glass plates have some greasy material over them, they should be cleaned with with chromic acid. Generally Generally 20×5 cm glass plates or microscopic slides are used to prepare chromatogram. First, glass plates of even thickness are selected. A slurry of adsorbent in suitable solvent is prepared. For example, 25g silica gel containing 13% plaster of paris is made into slurry in 50 ml of distilled water by slow addition and stirring with glass rod or spatula. Then a thin coating of this slurry is applied on glass plates by means of spreader. The slurry is transfered immediately to spreader which is pulled across with uniform motion over row of glass plates of even thickness. Thickness of thin layer of adsorbent adsorbent is controlled with the help of clearance clearance by the means of two two screws present. Generally a layer of 0.25 mm thickness is suitable. Alternatively, slurry may be prepared in dichloromethane also. For instance, 30g of adsorbent (usually silica gel or alumina) are slowly added to 100 ml of dry dichloromethane contained in wide mouth bottle with continuous stirring and bottle is capped. For preparing the plates pair of microscopic slides is held together and dipped in slurry, then slowly taken out and allowed to drain for a while over the mouth of bottle. Slides are separated carefully, placed horizontally and allowed to dry for 10 minutes. Adsorbent lying on the edges of slides is removed wih the help of a razor. After coating and drying plates are activated in oven at 110 0C for half an hour. Cellulose and polyamide plates are allowed to dry at room temperature and stored in dust-free cabinet; they don't need activation. (3)
Choice of Solvent
Choice of solvent depends upon the nature of sample to be separated as well as nature of adsorbent. In adsorption chromatography polarity of solvents follow the following order (Increasing order): Petroleum ether (or hexane) > carbon tetrachloride > Benzene (or toluene) > Dichloromethane > chloroform > Diethylether > Dimethyl formamide > Ethyl acetate > Pyridine > Acetone > Ethanol > Methanol > Formamide > water > Glycerol.
Generally, polarities of sample and solvents are matched and then choice is made. More polar solvents make more migration and give better separation. A combination of two solvents also gives better separation as compared to single solvent. Best position of the spot of compound is half way between line and solvent front. (4)
Application of Sample
1-3 mg of substance is dissolved in 0.1-0.6 ml of a solvent in which it dissolves well and which evaporates rapidly. Dichloromethane, diethylether, chloroform or light petroleum are best for this purpose (b.p.- 40-60 0c). Quick evaporation gives smaller spot which results in better separation of components in chromatographic development. Thin end of TLC capillary spotter is dipped in the solution which rises in the capillary. About 1 microlitre of the solution is applied on the already marked base line which is at a height of 1-20 mm from the bottom of chromoplate just by the touch of fime end of TLC spotter. A finish line is also marked at about 10 cm from base line. Size of the spot applied should be as small as possible (1-3 mm) and if the solution is very dilute first this spot is allowed to dry and solution should be applied twice or thrice to make up the concentration. Each time size of the spot fromed should be same. Finally, spot may be dried in hot air. Base line may also be marked by making a small notch which help in making the measurements at the later stage. Notch at starting line capillary
Finish line
Adsorbent
Base line Glass plate
Fig: Putting a spot of substance on TLC- Plate P late
(5)
Development of Plates
Chromoplate of 20 × 5 cm. size may be developed in cylinderical glass jar. Rectangular plates of 20 ×10 cm may require a rectangular glass tank of appropriate appropriate
dimensions. But microslides can be developed in 150 ml beaker or in a wide mouthed screw-capped glass bottle. Inside any of the above mentioned chambers is lined with filter-paper leaving a gap to view chromoplate. Filter paper is saturated with chosen solvent system for developing the plates by closing the jar containing solvent system and filter-paper for 10 minutes. A nonpolar eluent (solvent) forces nonpolar compound upwards, whereas polar eluents force both polar as well as nonpolar substances upward. These facts help in choosing the solvent system. Properly choosen solvent system is taken in the chamber upto the height of approximately 1 cm only; thins is enough to saturate the environment of chamber as well as to stop the evaporation of eluent from the plate. Spot of the plate should always be above the level of eluent, otherwise spot will dissolve in solvent. In such a event throwing away of the solvent and cleanring of the chamber will be required and fresh plate will be needed for running TLC. Thus, spotted TLC plate is placed inside the saturated chamber in slanting position in such a way that spot is above the level of solvent for the purpose of development and eluent is allowed to run up to finish line. When solvent reaches the finish line plate is immediately removed. Solvent is drained and air is blown over the plate to help evaporate the solvent so that plate becomes dry. Precaution is taken that elvent is not enhaled. (6)
Lacation of Spots: Following locating agents can be used for the the
visualization visualization of spots : (i)
Iodine : Iodine forms coloured and loose complexes with a number of
compounds. Iodine can be used for visualization either by spary of iodine is an organic solvent or by placing the developed plate in chamber containing iodine vapours for 1-5 minutes. This can locate a number of compounds but not all the compounds present in mixture. (ii)
Concentrated sulphuric acid : Concentrated sulphuric acid alone or
a solution of concentrated sulphuric acid (4 ml) in methanol (100 ml) spray also gives good results. After this spray plate is heated in oven to about 2000C until organic compounds appear as charred spots. Most of the components become visible by this method. (iii)
It has been found that sulphuric acid mixed with aromatic aldehydes, powerful oxidising agents like potassium permanganate, chromic acid, nitric acid and ceric sulphate also give good results.
(iv)
Other spray reagents like potassium permanganate (2%) in aqueous solution of sodium hydrogen carbonate (4%) or phosphomolybdic acid (10%) in ethanol may also be used.
(7)
Determination of Rf
Retention factor or Rf value which is characteristic of compound in a particular solvent system can be determined by the formula : Rf =
Distance travelled by componentfrom origin line Distance travelled by solventfrom origin line
As distance moved by solvent (i.e. solvent front) is always more than that travelled by component, Rf is always lesser than one. It is worth noting that Rf is constant for each component under identical conditions. Following Following factors affect Rf value : (i)
Quantity of adsorbent used
(ii)
Thickness of adsorbent layer
(iii)
Activation grade grade of adsorbent adsorbent
(iv)
Quality of solvent
(v)
Particle size of adsorbent
(vi)
Saturation of chamber by solvent vapours.
Precautions during TLC:
1.
Spot should not be too concentrated otherwise spot will smear out. If this happens spotting solution should be diluted.
2.
Spots should not be too close to each other otherwise spots start bleeding into each other and it becomes difficult to tell which spot came from which origin.
3.
Spot should not be close to edge, otherwise inaccurate Rf Value is obtained. As such a spot is not surrounded much by adsorbent and solvent, unequal forces work which donot give proper separation.
Exercise : Analysis of drug samples through TLC : Preparation of Sample
Take 125 mg of sample in a test tube and dissolve it in 4 ml of metharoltoluene (1:1) solvent system. Reference standards are prepared of strength 25 mg/ml.
Samples like cocaine, heroin & methamphtamine may be used. Application of Sample Commercially prepared fluorescent silica gel TLC – plates
may be used which are coated on plastic sheets of 8.5×3.5 cm dimensions. These plates have been activated at 120 0C for 20 minutes. All the four solutions [three references and one unknown] are carefully spotted on a single plate. Distance between two spots should be about .75 cm. Spots should not be larger than 2-3 mm. Development of chromatogram
Plate is placed in solvent system [prepared from Toluene [60 ml] + ether (30 ml) + CH3COOH (9 ml) + MeOH (0.5 ml)] about 5 ml of which is contained in TLC chamber. Chromatogram is developed till solvent reaches about 1 cm below the top of the plate. Remove the plate immediately & mark the solvent front. Allow the plate to dry. A hair-drier speeds up solvent evaporation process. Visualization Visualization : Colourless spots are visualized by illumination of plate under U.V.
lamp. Generally, aromatic compounds show bright fluorescence with characteristic colour. TLC-plates contain traces of fluorescent dye. Fluorescent compound show bright spot on light background, others show dark. spots since they quench to fluorescence of background dye Spots are encircled with a pencil & their colours are noted. Visualization may also be done with I 2 placed in a chamber. Plates may be placed in I2- chamber for 1-2 minutes. Observation & conclusion :
1. Rf values of all the spots are calculated. 2. From the number, positions & appearance of spots in reference compounds, the identity of unknown compounds may be determined. 5d. 3.1 "Gas Chromatographic Determination" (Drug Screening using Gas chromatography) chromatography)
Drug screening can also be done using Gas chromatograph [e.g. Perkin Elmer gas chromatograph] or through GC/MS. GC/MS have gas chromatograph combined with mass spectrometer. Here separation of sample is done using gas as an eluent moving over adsorbent filled column. An example of such identification is discussed below:
A gas chromatograph chromatograph of blood plasma extract from drug over dose of a patient may contain two peaks. One of which matches retention time of glutethimide (1). The GC/MS mass spectrograph show a peat at M/z 217 corresponding to mol wt. of this compound (1). Several other peaks are also present due to fragmentation of molecular ion [M + = 217]. These observations identify glutethimide glutethimide in sample. - Retention time of peak-2 does match with drug. But, mass spectrum of this
compound shows M+ at m/z 233 i.e. 16 mass unit higher than drug. This indicates presence of a metabolite differing 16 mass unit higher from drug in sample. This compound may be 4- hydroxy metabolite which is also indicated by the presence of fragments in mass spectra of two compounds. Check your Progress VI –
Notes : (a)
Write your answers in the space given below.
(b)
Check your answers from the key given in the end. (i) (ii)
Narcotics are habit-forming
True/False
Anti-neoplastic Anti-neoplastic agents are used to cure ................ .................. ..
(iii)
What is Therapeutic Index.
(i)
...................... ................................... ...................... .........
(ii)
...................... ................................... ...................... .........
(iii)
...................... ................................... ...................... ......... ........................ .................................... .................... ........ ...................... ................................... ...................... ......... ...................... ................................... ...................... .........
5.2
Let us Sum up
Our objective was analysis of various components of soil, fuel, body liquid as well study and identification of various drugs as they all are integral part of our life. Study can be summarized through following points: Soil is a natural medium for the growth and development of plants & it provides mechanical support to vegetation. Hence, presence of essential components Viz. moisture, nitrogen, phosphorus, silica, lime, magnesia, manganese, sulphate and alkali metal in proper proportions should be in it as required for growing the particular plant. If they are deficient it particular ingredient it should be added. Further more few plants grow well in acidic soil while others in basic soil, hence, pH of soil is important which should be maintained by adding fertilizer fertilizer or lime to it. Each ingredient mentioned above play important role is plant metabolism Thus, analysis and maintenance of soil is of of utmost importance. importance.
Methods of analysis of each constituent of soil are discussed is unit 5a. Moisture can be determined gravimetrically as well as volumetrically. For the determination of pH- electrical pH- meter method is best. Total nitrogen can be determined by autoanalyser. Olser method is generally used for the estimation of phophorus. Lime is determined by Shoe maker method & Mg by EPTA-titration. Mncan be estimated spectrophotometrically and sulphur by Palaskar et al. method. Fuel is essential to fulfill energy e nergy needs for different purposes like: (i)cooking (ii) Transport (iii) Industrial (iv) Space vehicles -
Characterization Characterization of fuel is necessary to choose good quality fuel for which (i) Proximate analysis & (ii) Ultimate analysis are used. Furthermore, fuel can be solid, liquid or gaseous. Among solid fuels coal like anthracite & charcoal are widely used. Important liquid fuels are diesel & petrol. Liquid fuels are characterized by the determination of calorific value, flash point and aniline point.
-
Gaseous fuels like LPG, natural gas, producer gas and water gas are widely used.
-
Fuels are characterized by ultimate analysis and proximate analysis, former estimates elements C, H, N 1, S1, O & ash. But latter determines (i) moisture contents (ii) volatile carbonaceous matter (iii) Ash (iv) fixed carbon. Anti-Knocking property of of gasoline (petrol) (petrol) is expressed in octane number. number. Clinical chemistry is related to the study of chemical aspects of human health.
For proper metabolism which is essential for good health all ingredients of body should be in normal range. Their concentration in blood, plasma, serum etc are determined chemically and spectroscopically. Different methods of analysis which help is the diagnosis have been described in unit 5(c). Preservation and collection methods related to blood, plasma and serum have been described. For preservation blood anticoagulants like heparin are added. Immunoassay and principle of redioimmunassay have been discussed.Later is based on antigen-antibody reaction to give complex which can be estimated spectrophotometrically. Blood gas analysis is done to check respiratory & kidney diseases. It also indicates pH of blood is 7.33-7.45.
Subunit 5d. deals with the study and screening of drugs. Dangerous and narcotic drugs have been differentiated. Classification of different drugs have been done on the basis of their therapeutic actions into (i)
Chemotherapeutic Chemotherapeutic agents.
(ii)
Pharmacodynamic Pharmacodynamic agents.
21-sub-classes of these two main classes have been described with examples of each. Screening of drugs through spectrophotometric and chromatographic methods (Gas & TLC) have been described.
5.3
Check your progress : ........... ........... The Key
(I)
(i)
Yes
(ii)
False
(iii)
Lime
(i)
Olsen method
(ii)
See 5a. 8 [Importance of Mg]
(iii)
Chlorolysis
(i)
True
(ii)
CO + H2
(iii)
It will be consumed.
(i)
Carbon, Hydrogen, nitrogen, sulphur and ash.
(ii)
See determination of aniline point in the unit.
(iii)
High.
(II)
(III)
(IV)
(V)
(VI)
(i)
Oxy-haemoglobin Oxy-haemoglobin
(ii)
These are anti-coagulants
(iii)
See RIA in unit 5(c).
(i)
True
(ii)
Cancer
(iii)
See Therapeutic index in unit 5d [classification of
drugs]
5.4
References
1.
Sharma, B.K; Engineering Chemistry, Krishna Prakashan
India (P)
Ltd,
Meerut (1999) 2.
Jackson, M.L; Soil chemical Analysis, Prentice Hall of India, Pvt. Ltd., New Delhi (1973)
3.
Chopra S.L., Kanwar J.S., Analytical Agricultural Chemistry, Kalyani Publishers, New Delhi, Ludhiana (1991).
4.
Hesse, P.R., Soil Sampling and Methods of Analysis, CBS- Publisher & distributors, distributors, New Delhi (1994).
5.
Evett S.R., Encyclopedia of water science, Mercel Decker (2003).
6.
Gartley, K.L., Recommended Soil Testing Procedures for the North Eastern united state (2011).
7.
Finar, I.L., Organic Chemisty, Vol. I, ELBS.
8.
Fieser, L. & Fieser, M., Organic chemistry.
9.
Roger's Manual of Industrial Chemistry, Vol I & Vol II
10.
Price, C.P. and Newmann, D.J., Principles and Practice of Immunoassay, Macmillan (U.K.) [1997]
11.
Samson Wright's Applied Physiology, Revised by Keele, C.A. and Neil, E., ELBS.
12.
Cooper & Gunn's Tutorial Tutorial Pharmacy, Ed. by S.J. Carter, CBS Publishers & Distributors, Distributors, New Delhi (2000)
13.
Kar, A., Medicinal Chemistry, New Age International (P) Ltd., New Delhi (2000)
14.
Gupta, A., Analytical Chemistry, Pragati Prakashan, Meerut (India) (2009)
15.
Agarwal O.P., Synthetic Organic Chemistry, Goel Publishing House, Meerut (India) (1999).
Books Suggested
1. 2.
Analytical Chemistry, Chemistry, G.D. Christian, J. Wiley. Fundamentals of Analytical Chemistry, D.A. Skoog, D.M. West and F.J. Holler, W.B. Saunders.
3.
Analytical, Chemistry-Principles, J.H. Kennedy, W.B. Saunders. Saunders.
4.
Analytical ChemistryChemistry- Principles and and techniques, L.G. Hargis, Hargis, Prentice Hall.
5.
Principles of Instrumental Analysis, D.A. Skoog and J.L. Loary, W.B. Saunders.
6.
Principles of Instrumental Analysis, S.M. Khopkar, Wiley Eastern.
7.
Quantitative Quantitative Analysis. R.A. Day, Jr. and A.L. Underwood, Wiley Eastern.
8.
Basic Concepts of Analytical Chemistry, S.M. Khopkar, Wiley Eastern.
9.
Handbook of of Instrumental techniques for for analytical Chemistry, Chemistry, F. Settle, P.H. P.H.
