Before performing this test, the test for reducing sugars had to be performed. If the result is negative the following test was performed.
2cm3 of sample were added to 1cm3 dilute hydrochloric acid. The mixture was then heated in a water bath for 2 minutes. With the aid of pH paper, the mixture was neutralised with sodium hydrogen carbonate. The Benedict’s or Fehling’s tests were then carried out.
Test for starch
A few drops of iodine/potassium iodide solution were added to some of the sample.
Test for lipids
Sudan III – To 2cm3 of sample, an equal volume of water was added. Two drops of Sudan III were added and the mixture was shaken. The mixture was then allowed to settle.
If the test is positive for lipids, the following test was performed to confirm the result.
Emulsion test – To 2cm3 of the sample, an equal volume of absolute alcohol was added. The mixture was then shaken and an equal volume of cold water was added.
Test for Vitamin C (ascorbic acid)
A few drops of DCPIP were added to 2cm3 of the sample.
Test for cellulose
A few drops of Schultz’s stain were added to the sample.
There are two tests which confirm the presence of proteins:
Millon’s test – Millon’s reagent contains mercury acidified with nitric acid. The amino acid tyrosine contains a phenol group which reacts to give a red mercury complex on boiling.
Biuret Test – Biuret reagent is a light blue solution which contains dilute copper sulfate in alkaline conditions. Nitrogen atoms present in the peptide chain, form a purple complex with copper (II) ions.
The two tests which confirm the presence of reducing sugars are Benedict’s test and Fehling’s test. The chemical principal behind these two tests is the same. The red precipitate indicates the presence of a reducing sugar. This occurs because the reducing sugar, including monosaccharides, such as glucose and fructose and some disaccharides, reduces the blue copper (II) ions present in the Benedict's reagent to copper (I) ions. These precipitate out as copper (I) oxide, which is a red colour.
When performing the test for non-reducing sugars dilute hydrochloric acid is added to the sample and then the mixture is boiled. In doing so, a hydrolysis reaction occurs, where a disaccharide such as sucrose, is hydrolysed to its monosaccharides, in the case of sucrose into glucose and fructose. Sodium hydrogen carbonate is then added to the resulting solution to neutralise the solution since the solution would be acidic. Then either Benedict’s test or Fehling’s test are performed, where the reducing sugar result is obtained.
When performing the test for starch a colour change from deep red to blue black indicates the presence of starch. The reason for this change is that a polyiodide complex is formed with starch.
There are two tests which confirm the presence of lipids:
Sudan III – Lipids are stained red and since they are less dense than water, it forms a layer on the surface of the water.
Emulsion test – If lipids are present, a cloudy white suspension will form. This is because any lipid present dissolves first in the ethanol, then when the water is added (in which lipid is insoluble), the lipid / ethanol mixture disperses in the water to produce a cloudy white emulsion.
Test for Vitamin C (ascorbic acid) involves the addition of a few drops of DCPIP solution to the sample. DCPIP solution which is blue in colour will become colourless in the presence of ascorbic acid. This happens because DCPIP solution is reduced by ascorbic acid which is a strong reducing agent.
When a few drops of Schultz’s reagent are added to the sample and a purple blue stain is obtained, cellulose is present. This happens because Schultz’s reagent dyes the cellulose.
