BIOLOGY PAPER 3 GUIDE(AS-LEVEL): Benedict's Test for Reducing Sugars The Benedict's Test for Reducing Sugars is used to determine the presence of reducing sugars. sugars . There is a Benedict's Test for non-reducing sugars as well. Benedict's solution is the principle reagent in the Benedict's Tests. The Fehling's Tests for reducing as well as for non-reducing sugar are alternatives alternatives to the Benedict's Test. Although based on the same principle, the Fehling's Test is less sensitive. It is also less convenient as the Fehling's Reagents - Fehling's A and B - have to be kept separate until the test is conducted.
What is a Reducing Sugar? Sugars are classified as reducing or non-reducing based on their ability to act as a reducing agent during the Benedict's Test. A reducing agent donates electrons during a redox reaction and is itself oxidized. The aldehyde functional group is the reducing agent in reducing sugars. Reducing sugars have either an aldehyde functional group or have a ketone group - in an open chain form which can be converted into an aldehyde. Reducing sugars are simple sugars and include all monosaccharides and most disaccarides. Some examples of monosaccharides monosaccharides are glucose, fructose and galactose.Examples galactose.Examples of reducing disaccharides are lactose and maltose. Note that the disaccharide sucrose is not a reducing sugar. sugar . In fact, sucrose is the most common non-reducing sugar.
Procedure 1.A liquid food sample does not need prior preparation except dilution if viscous or concentrated. For a solid sample prepare a test solution by crushing the food and adding a moderate amount of distilled water. Decant the suspension to remove large particles. Use the decanted liquid as the test solution. 2.Add 2 cm3 of the sample solution to a test tube. 3.Add an equal volume of Benedict's solution to the test tube and swirl or vortex the mixture. 4.Leave the test tube in a boiling water bath for about 5 minutes, or until the colour of the mixture does not change. 5.Observe the colour changes during that time as well as the final colour. 6.To prepare a control, repeat steps 3-5 using 2 cm3 of distilled water instead of sample solution.
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Observations and Interpretation OBSERVATION (FINAL COLOUR CHANGE)
INTERPRETATION
No colour change (mixture remains blue)
No reducing sugar present
Green
Trace amounts of reducing sugar present
Yellow
Low amounts of reducing sugar present
Orange
Moderate amounts of reducing sugar present
Brick Red
Large amounts of reducing sugar present
During the experiment, the colours of the mixture transition in this order: blue--> green--> yellow--> orange--> brick red
The final colour may be any of the colour above, depending on the quantity of reducing sugar present. If you do no observe the brick-red colour, it does not mean that your experiment has not worked out well. It is important to note that the Benedict's Test for Reducing Sugars is not specific to any one type of reducing sugar, and that the colour corresponds to the total reducing sugar present.
Principle of the Benedict's Test for Reducing Sugars Benedict's Solution contains copper(II) sulphate, sodium carbonate and sodium citrate. The blue copper(II) ions from copper(II) sulphate are reduced to red copper(I) ions by the aldehyde groups in the reducing sugars. This accounts for the colour changes observed. The red copper(I) oxide formed is insoluble in water and is precipitated out of solution. This accounts for the precipitate formed. As the concentration of reducing sugar increases, the nearer the final colour is to brick-red and the greater the precipitate formed. Sodium carbonate provides the alkaline conditions which are required for the redox reaction above. Sodium citrate complexes with the copper (II) ions so that they do not deteriorate to copper(I) ions during storage. 2
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Benedict's Test for Non-reducing Sugars The Benedict's Test for Non-reducing Sugars determines the presence of non-reducing sugars - sugars which do not have an aldehyde functional group. Many students confuse the Benedict's Test for Non-Reducing Sugars with the Benedict's Test for Reducing Sugars, perhaps because some of the steps in the procedure are similar. Benedict's solution is the principle reagent in both tests. The test for non-reducing sugars is often conducted on a food sample which tested negative for reducing sugar. If reducing sugars have been shown to be present, a heavier precipitate is often observed when the test for non-reducing sugar is conducted. The Fehling's Test for Non-reducing sugar is an alternative to the Benedict's Test. However it is less popular as it less sensitive and requires tha the reagents - Fehling's solutions A and B be kept separate until the experiment is carried out.
What is a Non-Reducing Sugar? Sugars can be classified as either reducing or non-reducing based on their ability to reduce copper(II) ions to copper (I) ions during the Benedict's Test. Non-reducing sugars do not contain an aldehyde group - the reducing species. Reducing sugars are simple, disaccharide sugars. Sucrose is the most common disaccharide non-reducing sugar.
Procedure 1.A liquid food sample does not need prior preparation except dilution if viscous or concentrated. For a solid sample prepare a test solution by crushing the food and adding a moderate amount of distilled water. Decant the suspension to remove large particles. Use the decanted liquid as the test solution. 2.Add 2 cm3 of the sample solution to a test tube. 3.Add 1 cm3 of dilute hydrochloric acid and boil for one minute. 4.Allow the tube to cool and then neutralize the acid with sodium hydrogen carbonate. Exercise caution due to 'fizzing' or effervescence. Check with blue litmus paper or pH paper. Do not worry if the resulting solution is slightly alkaline; Benedict's solution is also alkaline since an alkaline medium in needed for oxidation of the copper (II) ions by the sugars Then carry out Benedict's Test as for reducing sugars: 5.Add an 2 cm3 of Benedict's solution to the test tube and swirl or vortex the mixture. 6.Leave the test tube in a boiling water bath for about 5 minutes, or until the colour of the mixture does not change. 7.Observe the colour changes during that time as well as the final colour. 8.To prepare a control, repeat steps 2-6 using 2 cm3 of distilled water instead of sample solution.
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Observations and Interpretation OBSERVATION (FINAL COLOUR CHANGE)
INTERPRETATION
No colour change (mixture remains blue)
No non-reducing sugar present
Green
Trace amounts of non-reducing sugar present
Yellow
Low amounts of non-reducing sugar present
Orange
Moderate amounts of non-reducing sugar present
Brick Red
Large amounts of non-reducing sugar present
Whilst boiling the colours of the mixture transition in this order: blue--> green--> yellow--> orange--> brick red
The final colour may be any of the colours above, depending on the quantity of non-reducing sugar present. If you do no observe the brick-red colour, it does not mean that your experiment has not been successful. It is important to note that the Benedict's Test for Non-reducing Sugars is not specific to any one type of non-reducing sugar, and that the colour corresponds to the total reducing and non-reducing sugar present.
Principle of the Benedict's Test for Non-reducing Sugar Disaccharides are hydrolyzed to their constituent monosaccharides when boiled in dilute hydrochloric acid. The monosaccharides are reducing sugars are they contain the aldehyde group which is the reducing species. Neutralization of the mixture by sodium hydrogen carbonate is necessary as the reduction of the copper(II) ions will not take place in acidic conditions - of excess acid is present. Benedict's Solution contains copper(II) sulphate, sodium carbonate and sodium citrate. The blue copper(II) ions from copper(II) sulphate are reduced to red copper(I) ions by the aldehyde groups in the reducing sugars. This accounts for the colour changes observed. The red copper(I) oxide formed is insoluble in water and is precipitated out of solution. This accounts for the precipitate formed. As the concentration of reducing sugar increases, the nearer the final colour is to brick-red and the greater the precipitate formed. Sodium carbonate provides the alkaline conditions which are required for the redox reaction above. Sodium citrate complexes with the copper (II) ions so that they do not deteriorate to copper(I) ions during storage. 4
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Iodine Test for Starch The Iodine Test for Starch is used to determine the presence of starch in biological materials. At this level, you will be testing for the presence of this complex carbohydrate in foods, or in leaves as part of a photosynthesis experiment. If you study chemistry as well, you should pay attention principle of the test, as it helps explain how starch acts as an indicator for Redox Titrations or in the 'Iodine Clock Reaction'. The sole reagent required for the test is bench iodine solution 0.1 M potassium triiodide solution.
Procedure Solid Sample'
1.Peel off the skin of any vegetables e.g. potato and fruits as these are often impermeable. Use a clean spatula to remove samples of powdered food. Avoid cross contamination with other foods. 2.Add a few ( 2-3) drops of bench iodine solution potassium to a piece of solid food on a white tile. 3.Make observations. Liquid Sample
N.B. The iodine test for starch cannot be performed on very dark liquids which do not permit the obervation of a colour change. 1.Add 10 cm3 of the liquid food sample to a clean, dry test tube. 2.Add about 5 drops of iodine solution to the test tube. 3.Note any colour changes. 4.To prepare a control, perform steps 1 -3 for de-ionized water.
Observations and Interpretation TEST
POSITIVE
OBSERVATION
A blue-black colour develops
INFERENCE
Starch is present
NEGATIVE No change/ Iodine remains brown Starch is not present
Recall that starch is a storage molecule found only in plants. Only plants and plant-based foods should test positive for the presence of starch.
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Principle of the Iodine Test for Starch Starch is a polysaccharide, consisting of glucose units joined together by glycosidic bonds. The chains formed during the condensation reaction are either linear or highly branched molecules. Linear - both straight and helical - molecules of starch are referred to as Amylose
whereas branched molecules are called Amylopectin.
Iodine on its own (small non-polar molecule) is insoluble in water.Therefore Potassium triiodide solution - Iodine dissolved in potassium iodide solution - is used as a reagent in the test. To be more specific, potassium iodide dissociates, and then the Iodide ion reacts reversibly with the Iodine to yield the the triiodide ion. A further reaction between a triiodide ion and an iodine molecule yields the pentaiodide ion. Since molecular iodine is always present in solution, the bench iodine solution appears brown; the iodide and triiodide pentaiodide ions are colourless. Foods which are high in amylose have more intense blue-black colour. As the Beer-Lambert Law is obeyed spectrometric analyses can quantify the quantity of amylose in starches.
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Ethanol Emulsion Test for Fats and Oils The Ethanol Emulsion Test is a food test which determines the presence of a broad group of naturally occurring compounds known as lipids. Lipids consist of fats and oils. Other lipid tests include the Grease Spot Test and the Sudan Stain Test. The Grease spot test is performed on fats - lipids which are solid at room temperature. Sudan stain colours lipids red, but is a less common bench reagent than ethanol. The Ethanol Emulsion Test is the most common test amongst the three.
Procedure Soild sample : 1.Crush the food sample and place in a dry test tube. 2.Add ethanol to about 2 cm3 above the level of the sample and shake thoroughly. 3.Allow the solid to settle (about 3 min) to allow the lipid to be extracted. 4.Decant the ethanol into another test tube. 5.Add 2 cm3 of deionized water to the second test tube. 6.Make observations.
Liquid sample: 1.Add a few drops of the liquid food sample to a dry test tube. 2.Add 2 cm3 ethanol and shake it thoroughly 3.Add 2 cm3 of deionized water. 4.Make observations.
Results and Interpretation OBSERVATION
INTERPRETATION
POSITIVE TEST
A layer of cloudy white suspensionforms at the top of the solution. (Upon close inspection you can see the tiny globules of fat suspended in the solution. This an emulsion. Foods Lipids are present with high lipid content have a ‘higher’ layer than foods with less).
NEGATIVE TEST
Solution remains colourless. No emulsion is formed.
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Lipids are not present
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Principle of the Ethanol Emulsion Test The solubilities of lipids and ethanol are exploited in this test. Lipids are non-polar organic compounds. Hence they are soluble in organic solvents such as ethanol (alcohol), but insoluble in water. Ethanol is an organic substance and so dissolves other organic substances; it is frequently used as an organic solvent. However ethanol is also miscible in water due to the presence of thehydroxyl (OH) functional groups and the shortness of its chain (2C). The hydroxyl group participates in hydrogen bonding with water (see below).
The hydrophobic interaction of the carbon in the short chain with water is not great and is overcome by the hydrogen bonding. Ethanol extracts the lipid from the crushed solid sample. As ethanol is miscible with lipids no change is seen upon its addition to the solid and liquid samples. The lipid spontaneously comes out of solution when water is added and is dispersed as micelles (small droplets) throughout the solution of ethanol and water.( This happens as hydrophobic portion of the lipid molecules project inwards and excludes the aqueous environment; the hydrophilic portion (-COOH) group faces the aqueous environement.) A layer is formed at the top as lipids are less dense than water. The droplets diffract light, appearing cloudy white.
A Conclusive Test A positive test shows conclusively that lipids are present - and not the other major biological molecules: 1. Carbohydrates
Reducing sugars and non- reducing sugars - slightly soluble in ethanol and soluble in water
Starch – insoluble in both ethanol and water
2. Proteins – insoluble in ethanol (the addition of ethanol is used to precipitate proteins) 3. Nucleic Acids – are insoluble in ethanol and soluble in water No change is seen in a negative test as there are no lipids to come out of solution.
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Experimental Skills The Ethanol Emulsion Test procedure is suitable for assessment of the Measurement and Manipulation Experimental skill. Below are some criteria your teacher may use to assess you. He will be scrutinizing the steps you take to prevent contamination , including cross contamination from other food samples. CRITERIA Preparation of a Solid Sample: 1. Completely crushes adequate amount of food with a clean and dry mortar and pestle. 2. Transfers an adequate amount of crushed food sample to a clean test tube. 3. Adds 2cm3 ethanol above the food sample. 4. Shakes the test tube thoroughly and safely (using a vortex mixer if available). 5. Allows the solid to settle for about 3 min. 6. Decants the ethanol carefully, into a clean, dry test tube without transference of food particles. 7. Adds 2cm3 of de-ionized water to the test tube. 8. Compares each result with the negative control. 9. Compares the heights of the emulsion layer of all the samples.
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Biuret Test for Proteins The Biuret Test is often used to determine the presence of peptide bonds in protein. At this level you will be testing for the presence of protein in foods. The test may also be extended to quantitatively measure the concentration of total protein. ➔ Instead of the Biuret Reagent, the following may be used: •Fehling’s •Sodium
Solutions A and B
hydroxide and copper (II) sulphate solutions
Procedure 1.
Add 2 cm3 of the liquid food sample* to a clean, dry test tube
2. Add 2 cm3 of Biuret Reagent. Alternatively: 3. Use sodium hydroxide solution and copper sulphate solutioninstead. Add 1 cm3 of sodium hydroxide solution (40% or bench solution) and 1% copper (II) sulphate solution dropwise – drop by drop - to the food sample 4. Use Fehling's A and B solutions instead. Fehling's A and B should be freshly prepared. Fehling’s A is copper (II) solution and Fehling's B is a mixture of sodium potassium tartrate and sodium hydroxide solution. 1cm3 of each solution – A and B – should be added to the food sample. 5. Repeat steps 1 and 2 with de-ionized water to prepare a negative controland with albumin (egg white) to prepare a postive control. 6. Shake well and allow the mixture to stand for 5 minutes. 7. Observe any color change. * Prepare liquid samples from solid foods. Crush the solid food, add a little de-ionized water and decant the liquid. This liquid should be used for the food test. The quantity of food crushed and water used depends on the number of tests to be conducted.
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Observations and Interpretation
TEST
POSITIVE
NEGATIVE
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OBSERVATION
The solution turns from blue to violet( purple) The solution turns from blue to pink No change /The solution remains blue
INFERENCE
Proteins are present Peptides are present ( Peptides or peptones are short chains of amino acid residues) No protein is present
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Drawing Biological Diagrams
Guidelines for Biological Drawings Biological drawings large, clear, neat and contain all the relevant structures, labels and annotations. Here are some guidelines for drawing them. 1.Use a sharp pencil . Do not use a soft, black-marking pencil as it will smudge and look untidy. The standard writing pencil - HB - will suffice. I highly recommend using 0.5 mm mechanical pencils. As they reduce on sharpening time and make drawing 'lines of even thickness' a breeze. Ensure you have spare leads in an exam. 2.Use a clean and soft eraser . I recommend soft vinyl erasers, as they erase more cleanly than standard pink erasers and are non-abrasive, so will not tear the paper. 3.Use white, unlined paper for biological drawings.
4.Decide how large your diagram should be , and where you will place it on the page, so that structures are clearly seen and there is adequate space for labeling. A Drawing should occupy at least half the page. A4 paper is often used for drawings. 5.Move your specimen around , observing its general features from all angles. As you draw, keep referring to your specimen to capture the detail accurately. Use a magnifying glass to observe fine detail. Use one eye to look through a microscope and the other to draw. Observe the specimen under different magnifications. 6.Draw continuous lines. 7.Draw structures proportionately. 8.Avoid shading and hatching , as well as excess detail. Include distinctive features. Draw only one complete structure e.g. A cell if the neighboring cells are similar. You may have to exaggerate the thickness of structures if they are thin. Add an explanatory note if you do. 9.Use conventions e.g. a double line to show cut edges 10.Draw horizontal labeling lines , ensuring that they do not cross. Labeling lines in biological drawings should be on the right. Ensure that the labeling lines touch the structures. Do not include arrowheads or dots on the lines. 11.Print correct labels. For drawings, all the letters need to be lower case. Check for correct spelling. Do not use the plural form of the word, if you are pointing to a single part.
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Drawing Biological Diagrams 12.Remember to include annotations , especially if they are required. Annotations are brief notes next to labels which are either descriptive or state the function of the structure. These can help you earn marks in an exam. 13.Centre the title and print it in capitals at the bottom. The title should include the name of he specimen and the view. Scientific names are capitalized in the title. If appearing elsewhere, convention is followed.
14.The magnification should be included at the end of the tile , to 1 decimal place. The magnification is obtained by dividing the length of drawing, by the length of the specimen. When using a microscope, calculate the combined magnification of the eyepiece and objective lenses. If you continue Biology beyond secondary level, you will have to add a scale to the drawing, by making a rough estimate of the field diameter using a transparent ruler on the stage, or a graticule.
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Vascular Bundle
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stomata
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