UNDERGRADUATE FIRST YEAR PRACTICALS MANUAL

RAMAKRISHNA MISSION VIVEKANANDA COLLEGE EVENING COLLEGE, CHENNAI-4

DEPARTMENT OF BIOCHEMISTRY

Quantitative Analysis Expt 1:Estimation of Amino Acid by Sorenson’s Sorenson’s Formol Titration AIM: To estimate the amount of amino acid ac id present in the whole of the given solution.

PRINCIPLE: The acid group present in the glycine glycine can be titrated with NaOH. It is not easy in this case because the amino amino group present will interfere interfere at the end point. To prevent it exces excesss of form formal alde dehy hyde de is used used by whic which h the the amin amino o grou group p is block blocked ed by the the formation of methylene methylene glycine. Then it is titrated with NaOH NaOH using phenolphthalein indicator.

REAGENTS REQUIRED: (i) (ii) (iii) (iv) iv) (v)

Standard oxalic acid 0.1 N Link solution NaOH Formaldehyde Pheno henolp lpht htha hale lein in indi indica cato tor r Amino acid solution

PROCEDURE: Standardisation of NaOH Rinse a clean burette first with distilled water and then with the link solution of NaOH. NaOH. Rinse Rinse a clea clean n 20ml 20ml pipette first with distilled water and then with the given standard standard oxalic acid. acid. Pipette Pipette out 20ml of oxalic acid acid into a clean conical conical flask. flask. Add a few drops of phenolphthalein indicator and titrate titrate against NaOH taken in in the burette. The end point is the appeara appearance nce of perman permanent ent pale pink colour colour. Repeat Repeat the titrat titration ion for concordant values. Estimation of Amino acid Make Make up the the give given n amin amino o acid acid solu soluti tion on with with dist distil ille led d wate waterr to 100ml 100ml in a volumetric volumetric flask, observing observing the usual precautions. precautions. Shake the solution solution well for uniform concentration. Pipette out 20ml of amino amino acid solution into a clean conical flask. Add 5 ml of HCHO and keep it for 2 minutes. Then titrate it against the NaOH solution solution taken in the burette. Phenolphthalein is used as the indicator. indicator. The end point is the appearance of permanent pale pink colour. colour. Repeat the titration for concordant values.

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Blank titration Pipette Pipette out 20ml of distilled distilled water in a clean conical flask. flask. Add 5ml of HCHO and keep it aside aside for a few minutes. Add 1-2 drops of phenolphthalein indicator. indicator. Titrate it against against the NaOH solution solution taken in the burette. burette. The end point is the appearance appearance of permanent pale pink colour. colour. Repeat the titration titration for concordant values. Result: The amount of amino acid present in the whole of the given solution = _______ g EXPT 1 Titration I Standardisation of NaOH Concentration of Oxalic acid = 0.1 N Standard oxalic acid Vs NaOH Volume of Burette reading S.No. Oxalic acid (ml) Initial Final

Concordant Value (ml)

1. 2. 3. Calculation Volume of oxalic acid, V1 = Normality of oxalic acid, N1 = Volume of NaOH, V2 = Normality of NaOH, 2N=

ml N ml N

Titration II Estimation of amino acid Amino acid + HCHO Vs NaOH Contents in Burette reading S.No. conical flask Initial Final 1.
















Calculation Volume of NaOH, (Test value) = ml Volume of NaOH, (Blank value) = ml Volume of NaOH used to titrate amino acid = Test value – Blank value =

ml

Titration III Blank titration Standard NaOH Vs HCHO + H2O Contents in Burette reading S.No. conical flask Initial Final


1. 2. 3. Calculation Volume of amino acid, V1 = ml Normality of amino acid, N1 = N Volume of NaOH, V2 = ml Normality of NaOH, N 2N= Strength of amino acid = V(NaOH) x N(NaOH) = V(amino acid) x N(amino acid) Amount of amino acid present in 100ml of the given solution = Equivalent weight x Normality x 100 1000















Expt 2: Estimation of glucose by Benedict’s quantitative reagent AIM: To estimate the amount of glucose present p resent in the given unknown sample by Benedict’s method. PRINCIPLE: Benedict’s quantitative reagent is the modified modified form of qualitative reagent. reagent. It consists of cupric sulphate, sodium carbonate and sodium citrate, potassium thiocyanate, and potassium ferrocyanide. The alkali present in the Benedict’s reagent enolises the sugar, thereby causing them to be a strong reducing agent. Ferrocyanide serves to dissolve the copper hydroxide while thiocyanate helps to convert the red cuprous oxide to whit crystals cr ystals of cuprous thiocyanate, which gives the clear end point. REAGENTS REQUIRED (i)Benedict’s (i)Benedict’s quantitative reagent reag ent (BQR) (a)Cupric sulphate, (b)Sodium carbonate, (c)Sodium citrate, (d)Potassium thiocyanate, (e)Potassium ferrocyanate. (ii)Anhydrous sodium carbonate (iii)Working (iii)Working standard glucose solution (iv)Porcelain beads. PROCEDURE: Titration I: Standardisation of Benedict’s Benedict’s Qualitative Reagent Accurately pipette out 5ml of Benedict’s quantitative reagent into a clean conical flask. Two spatula full of 2g of sodium carbonate was added into the conical flask. Few pieces of porcelain beads were also added in order to avoid bumping. The contents were brought to temperature approximately 60-70 oC. The contents were titrated against the standard glucose solution with regular regular shaking until the blue colour disappeared. disappeared. The end point is the appearance of chalky white white precipitate. The titrations were repeated for concordant values. Titration II: Estimation of Glucose The given unknown sample solution was made up to 100ml 100 ml with distilled water in a standard flask. It was shaken well for uniform concentration. The burette was filled with this unknown solution and the titrations were performed as mentioned above till the appearance of chalky white precipitate. The titrations were repeated for concordant values.















EXPT 2 Titration I Standardisation of Benedict’s Quantitative reagent Std Glucose Vs Benedict’s Benedict’s Quantitative Quantitative reagent

Content Contentss in Burette reading S.No. conical flask Initial Final (ml)

Indicator : Self Self


1. 2. 3.

Titration II Estimation of Glucose Standard Benedict’s Quantitative reagent Vs Unknown Glucose Indicator : Self Contents Burette Concordant S.No. in conical reading Value flask (ml) (ml) Initial Final 1. 2. 3.

Calculation 100 of the unknown glucose solution solution contains = Standard value x 100 unknown value















Expt: 3 Estimation of Ascorbic acid by Titrimetric Method AIM: To estimate the amount of ascorbic acid present in the given solution. PRINCIPLE: Ascorbic acid is a strong strong reducing agent. It gets oxidized to dehydro dehydro ascorbic acid by 2, 6 dichlorophenol indophenol dye. At the same time the dye dye gets reduced to a colourless compound. So the reaction with end point can easily be determined.

REAGENTS REQUIRED (i)Std (i)Std Indophenol solution : 42mg of sodium bicarbonate and 52mg of 2, 6 dichlorophenol indophenol were dissolved in 50ml of water and diluted to 200ml. The solution was filtered filtered and stored. stored. (ii)Std (ii)Std Ascorbic acid : acid : 50mg ascorbic acid was dissolved and made up to 250ml using 0.6% oxalic acid. Preparation of test solution : Given solution was made up to 100ml with 0.6% (iii) Preparation oxalic acid. PROCEDURE: Titration Titration I: standardisation of dye The burette was rinsed and filled up to the convenient mark with d ye solution. 10ml of std ascorbic acid solution was pipetted out into into a clean conical flask. It was titrated against the dye dye taken in the burette. The end point was the appearance of permanent pale pink colour which remains for 10 minutes. The titrations were repeated for concordant value. The dye equivalent of std ascorbic ascorbic acid was calculated. Titration Titration II: II: Estimation of Ascorbic acid 10ml of made up solution was pipetted out into a clean conical flask. flask. It was titrated against the dye taken in the burette, till the permanent pale pink colour was obtained. The titrations were repeated for concordant value. Using the dye equivalent of ascorbic acid, the amount of ascorbic acid in the given solution was calculated.

















EXPT 3 Titration I Standardisation of dye Standard Ascorbic acid Vs Dye Volume of Burette S.No. Ascorbic reading acid (ml) Initial Final (ml) (ml)

Indicator : Self Concordant Value (ml)

1. 2. 3. Concentration of standard = 100µg/ml Titration Titration II Estimation of Ascorbic acid Standardised dye Vs Ascorbic acid Volume of Burette S.No. Unknown reading ascorbic acid Initial Final (ml) (ml) (ml) 1.

Indicator : Self Concordant Value (ml)

















Expt 4 : Determination of acid number of an edible oil AIM: To determine the acid number nu mber of the given edible oil. PRINCIPLE: The Acid number is defined as the number of milligram of KOH required to neutralize the free fatty acid present in one gram of oil or fat. Fat may become rancid during the the storage for a long time. time. Fat or oil are hydrolysed by micro organisms with the formation of free fatty acids. The amount of free fatty acid present in the oil in the indicator of the age and quality of that oil. Thus the high acid number will indicate that the oil is old and rancid. REAGENTS REQUIRED: (i)Oxalic acid (0.1N) (ii)KOH (iii)Fat solvent (Ethanol and ether in 1:1 ratio) (iv)Phenolphthalein PROCEDURE: Titration Titration I: Standardisation of KOH 10ml of the standard oxalic acid (0.1N) is pipetted out into a clean conical flask. Add 2 drops of phenolphthalein indicator and titrate against KOH taken in the burette till the appearance of permanent pale pink colour. Repeat the titration for concordant values. Titration Titration II: II: Determination of Acid number Weigh around 1g of the oil and transfer into a clean conical flask. Add 20ml of lipid solvent to dissolve dissolve the oil and shake well. Add a few drops of phenolphthalein

















EXPT 4 Titration I Standardisation of KOH Standard oxalic acid Vs KOH Indicator : Phenolphthalein Volume of Burette Concordant S.No. unknown reading Value oxalic (ml) acid Initial Final (ml) (ml) (ml) 1. 2. 3. Calculation: Volume of oxalic acid, V1 = Normality of oxalic acid, N1 = Volume of KOH, V2 = Normality of KOH, N2 = V1x N1 = V2x N2

ml N ml N

Titration Titration II Determination of acid number Standard KOH Vs Oil Indicator : Phenolphthalein Contents Contents of Burette Concordant S.No. the conical reading Value flask (ml) (ml) Initial Final (ml) (ml)

















Titration III Blank titration Determination of acid number Standard KOH Vs lipid solvent Indicator : Phenolphthalein Volume of Burette Concordant S.No. lipid Reading Value solvent (ml) (ml) Initial Final (ml) (ml) 1. 2. 3. Acid Acid number number = KOH x Volume of KOH consumed x Equivalent weight of KOH x 1000 Weight of oil x 1000

Strength Strength of

















Expt 5 :Determination of Saponification number of an edible oil AIM: To determine the saponification number of the given edible oil. PRINCIPLE: Saponification value is defined as the number nu mber of milligram of KOH required to neutralise the fatty acid resulting in the complete hydrolysis of 1g of oil o r fat. It gives an indication of the nature of the fatty acid present in the fat. On refluxing the fat or oil with alkali, the ester of glycerol is hydrolysed into glycerol and potassium salt of free fatty acid.

If the length of the free fatty acid chain c hain is longer, the acid is lesser in the utilization of KOH is less and vice-versa. A weighed amount of fat is saponified with known amount of a lcoholic KOH, excess alkali is back titrated with standardized acid and the saponification value of the oil is determined. REAGENTS REQUIRED: (i)Alcoholic KOH 0.5N (ii)Hydrochloric acid 0.5N (iii)Sodium carbonate 0.5N (iv)Phenolphthalein (v)Methyl orange PROCEDURE Titration Titration I: Standardisation of Hydrochloric acid 10ml of standard sodium carbonate is is pipetted out into a clean conical flask. Add 2 drops of methyl orange indicator and titrate it against HCl taken in the burette. burette. End



















The saponification number of the given edible oil is ___________mg EXPT 5 Titration I Standardisation of hydrochloric acid Standard Sodium carbonate Vs HCl Indicator : Methyl orange Volume of Burette Concordant S.No. sodium reading Value carbonate (ml) (ml) Initial Final 1. 2. 3. Calculation Volume of sodium carbonate, V1 Normality of sodium carbonate, N1 Volume of HCl, V2 Normality of HCl, N2 V1x N1 = V2x N2 Titrati

II

= = = =

ml N ml N

















Titration III Blank titration Lipid solvent+KOH Vs HCl Indicator : Phenolphthalein Contents of Burette Concordant S.No. the conical Reading Value flask (ml) (ml) Initial Final 1. 2. 3.

















AIM: To estimate the amount of chloride present p resent in the given solution. PRINCIPLE: When a solution of silver nitrate is added to a solution of sodium chloride, white precipitate of silver chloride is formed. Potassium chromate is used as an indicator and produces the silver chromate. The end point is the change from yellow to reddish brown due to the formation of silver chromate.

REAGENTS USED: (i)NaCl (0.29g of sodium chloride dissolved in 100ml of water in a 100ml standard flask. (ii)Silver nitrate (0.85g of silver nitrate dissolved in 100ml of water) (iii)Indicator : Potassium chromate solution (2%) PROCEDURE: Titration Titration I: Standardisation of silver nitrate

















Titration I Standardisation of Silver nitrate Standard AgNO3Vs unknown Chloride Indicator : Potassium chromate Volume of Burette Concordant S.No. NaCl reading Value (ml) (ml) Initial Final 1. 2. 3. Calculation Volume of NaCl, V1 = ml Normality of NaCl, N1 = N Volume of HCl, V2 = ml Normality of HCl, N2 = N V1x N1 = V2x N2 Titration Titration II Estimation of Chloride Standard AgNO3Vs unknown Chloride Indicator : Potassium chromate Volume of Burette Concordant

















To estimate the amount of chloride present p resent in the given solution. PRINCIPLE: Silver nitrate reacts with potassium thiocyanate to form a precipitate of silver thiocyanate.

When all silver nitrate has been converted conve rted to silver thiocyanate, this results in dark brown colour due to the formation of ferric thiocyanate in the prescence of ferric alum. From this the chloride chloride equivalent to silver nitrate nitrate in terms of potassium potassium thiocynate, can be calculated.

REAGENTS USED (i)Silver nitrate 0.05N (ii)Potassium thiocyanate 0.05N (iii)Ferric alum (iv)6N nitric acid PROCEDURE: Titration Titration I: Standardisation of potassium thiocyanate The link solution potassium thiocyanate thiocyanate is taken in the burette. burette. 10ml of standard



















S.No. Contents in in the conical flask (ml)

Indicator : Ferric alum Burette Concordant reading Value (ml) Initial Final

1. 2. 3. Calculation Volume of sodium chloride, V1

=

ml

















PRINCIPLE: Iodine number is defined as the number of grams of iodine taken up by 100g of oil or fat. In this case, addition reaction reaction takes place across the double bonds of unsaturated fatty acids present in the fat by the add ition of a halogen, such as iodine. Thus, the iodine number gives the indication of the degree of unsaturation u nsaturation of fats. Iodine value is directly proportional to the degree of unsaturation. unsaturation. Determination of iodine number is used for the assessment of its purity. purity. In Hanu’s method, the oil is treated with Hanu’s reagent. (Iodine bromide in chloroform) for a period of time. The unreacted iodine is titrated titrated against standardized thiosulphate solution.

















Contents in S.No. the conical flask (ml)

Burette reading Initial


Final

1. 2. 3. Calculation Volume of potassium dichromate, V1 = Normality of potassium dichromate, N1 =

ml N

















1. Moli Molisc sch’ h’ss reage reagent nt 5% α naphthal in alcohol, i.e., 5g of αnaphthal dissolved in 100ml of ethanol. 2. Iodi Iodine ne solu soluti tion on 0.005% in 3% KI, i.e., 3g of KI dissolved in 100ml water and then 5mg of iodine is dissolved. 3. Bene Benedic dict’ t’ss solu soluti tion on 17.3g of sodium citrate and 10g of sodium carbonate are dissolved in 75ml of water. 1.73g of CuSO4.5H2O is dissolved in in 20ml of water. water. Mix the CuSO4 solution with alkaline citrate with constant stirring, finally the whole volume is made up to 100ml with water. water. 4. Barf Barfoe oed’ d’ss reage reagent nt 13.3g of copper acetate in 200ml of water and add 2ml of glacial acetic acid. 5. Bial Bial’’s reag reagen entt

















Barfoed’s reagent is weakly acidic and it is only reduced by monosaccharides. Prolonged boiling may hydrolyze the disaccharide to give false positive test. 5. Bial’s test When pentose is heated with con.HCl, furfural, which condenses with orcinol in the prescence of ferric ion to give a blue green colour. 6. Seliwanoff’s test Ketoses are dehydrated more rapidly than aldose to give a furfural derivatives, which then condenses with resorcinol to form a red colour complex. 7. Muci Mucic c aci acid d ttes estt



















S.No.

1.

EXPERIMENT Molisch’s test To 1ml of test solution, add 2 drops of Molisch’s reagent.

OBSERVATION

INFERENCE

Violet Violet coloured ring is formed at the

Prescence of carbohydrate.

































a) b) 2)

Water Dilute NaOH solution Precipitation by neutral

Colloidal solution is formed. Partially soluble.

Presence of protein. Presence of protein.

















10)

1% sulphanilic acid and Cool it in an ice bath. ba th. Then add 1ml of 5% NaNO2 and 1ml of 1% Na2 CO3. Millon’s test:

protein.

















add few drops of 20% NaOH .Then add 1ml of sodium – nitroprusside followed by the addition of 1.5ml of 1% Glycine.Boil it for few

in the protein.



































































































































































UNDERGRADUATE FIRST YEAR PRACTICALS MANUAL

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