Testing for reducing sugars, non-reducing sugars, starch, lipids and proteins in unknown substances: Our aim: was to find out which substances from our five samples have reducing sugars present to determine what they may be for example; they could be monosacchrides or disaccharides. This was carried out by using Benedict’s test. To find out which substances from our five samples were non-reducing sugars, since some disaccharides are reducing sugars and this would be carried out by using Benedict’s test.
To find out which substances had starch present using iodine To find out which substances were lipids using the emulsion test To test for proteins in the substances using the biuret test Context of the experiments: The context of this experiment was that we were interested in finding out what biological compounds we were dealing with as we had no idea what each substance was and all samples looked the same therefore we had to carry out a series of tests to figure out what structures they were made of. Some simple sugars, including glucose, can be made to reduce blue copper sulphate to red copper oxide.
This reaction can be used as a test for some sugars. Copper sulphate is mixed with other chemicals to be used as Benedict’s solution which is used in the test. This was the test for reducing sugars. If the reducing sugar test comes out as negative (no colour change), the non-reducing sugar test can be done. The non-reducing sugar test works because if there is any sucrose present (which is a non-reducing sugar, that we are testing for), it is broken down into those monosaccharides, which can be tested for using the ordinary reducing sugar test. A positive result therefore means non-reducing sugars are present on the original sample.
Starch is easily detected in substances by its ability to change the colour of the iodine in potassium iodide solution from yellow to blue-black. Lipids do not dissolve in water, but do dissolve in ethanol. This characteristic is used in the emulsion test. Therefore this could apply to samples that are oils. Proteins are detected by a colour change when using biuret solution. Biuret reagent, which contains sodium hydroxide and copper sulphate, and is pale blue in colour, is added to the sample. These chemicals react with the peptide bonds found in proteins, which results in a colour change to lilac Evaluation:
Most of the experiments that were carried out were successful as they showed what actually was present in each substance however there was a mistake in the experiment for testing for proteins. This was likely to be down to contamination of the substances therefore for future reference, solutions should be done one at a time and test tubes should be clean before use. Test for reducing sugars: Equipment: Food sample in liquid form Test tube Benedict’s reagent Boiling water Stopwatch Measuring cylinder Method: Add 2cm2 of the food sample to be tested to a test tube.
Add an equal volume of Benedict’s reagent – this is an alkaline solution of copper (II) sulfate Heat the mixture in a gently boiling water bath for 5 minutes (to speed up the chemical reaction), record observations of any colour change Results: Sample 1 had no colour change when reacting it with Benedict’s reagent, therefore there is no chance of it being a reducing sugar Sample 2 had no colour change Sample 3 did change colour in the solution to orangey/red showing that a precipitate was formed and that it was a reducing sugar Sample 4 had no colour change Sample 5 had no colour change Conclusion:
What we can withdraw from the results is that out of all the five samples, only one sample which is sample 3 is a reducing sugar. We know this because this substance donated electrons to Benedict’s solution causing a reduction reaction and only a reducing sugar will react this way with the reagent, causing the formation of a precipitate and change of colour (only seen in sample 3). Test for non-reducing sugars Equipment: Food sample Test tube Measuring cylinder Hydrochloric acid Water bath or could use the Bunsen burner with heat proof matt, tripod, gauze and boiling beaker Sodium carbonate solution
pipette Method: If a substance does not react with Benedict’s solution, this test is used: Boil 2cm3 sample with 1cm3 hydrochloric acid – this hydrolyses any sucrose present, splitting sucrose molecules to give glucose and fructose (see below) Cool the solution and neutralise it by adding sodium carbonate solution drop by drop (an alkali solution) Carry out the reducing sugar test (Benedict’s test) again: if there were non-reducing sugars present in the original sample, the test will now come out as positive (as they have been broken down into reducing sugars glucose and fructose) Results:
Only sample 4 came out positive Conclusions: Sample 4 was sucrose therefore our experiment was successful since sucrose is a non-reducing sugar. Disaccharide can be hydrolysed to its monosaccharide constituents by boiling with dilute hydrochloric acid. Sucrose is hydrolysed to glucose and fructose, both of which are reducing sugars and give the reducing sugar result with the benedict’s test. Test for starch: Equipment: Food sample Test tube Iodine solution Pipette Method: To approximately 2 cm? of test solution add two drops of iodine/potassium iodide solution.
A blue-black colour indicates the presence of starch as a starch-polyiodide complex is formed. Starch is only slightly soluble in water, but the test works well in a suspension or as a solid. Results: Only sample 1 tested positive for starch Conclusions: Sample 1 had the presence of starch in it as a polyiodide complex is formed causing the change in colour. The rest of the samples had no starch in them. This makes sense since sample 1 was in fact a starch solution. Test for lipids: Equipment: Food sample Test tube Ethanol Water Method: Add 2cm3 fat or oil to a test tube containing 2cm3 of absolute ethanol.
Dissolve the lipid by shaking vigorously. Add an equal amount of cold water. A cloudy white suspension is evidence for lipids being present Results: Samples 2 and 5 both produced a white cloudy suspension and were the only ones to out of the five samples. Conclusion: Clearly, samples 2 and 5 are going to be substances with fat in them thus suggesting they may be oils. This was quite true since sample 5 was sunflower oil which does have a lot of lipids in and sample 1 was egg albumen, although this isn’t usually high in lipids, it had been surrounded by egg yolk which is very high in lipids.
Test for proteins Equipment: Food sample Test tube Sodium hydroxide solution Copper (II) sulfate solution Pipette Method: Place a sample of the solution to be tested in a test tube and add an equal volume of sodium hydroxide solution at room temp. Add a few drops of very dilute (0. 05%) copper(II) sulphate solution and mix gently A purple coloration indicated the presence of peptide bonds and hence a protein, if no protein is present, the solution remains blue Results:
Only sample 2 and sample 3 had been tested positive for protein, the rest of the solutions remained blue. Conclusion: Clearly, sample 2 and 3 tested positive. This makes sense for sample 2 as it is egg album therefore is high in proteins as it is part of an egg, and would have peptide bonds. However this does not make sense for sample 3 as it was glucose. Glucose is a sugar – more specifically a monosaccheride i. e. a single unit sugar. It does not have any peptide bonds – that is why it does not react. Therefore something clearly went wrong with this experiment.