BIOLOGY Volume 1 - A Guide to General Biology - 2004

3. CHEMICAL COMPONENTS OF LIVING MATTER

3.7. Identification of Biomolecules

In this section, we describe several simple experiments that can be used to identify various substances that play crucial biological roles within Cells. More sophisticated Methods for the identification and Separation of cellular components also exist. Foremost among these are Chromatography and Electrophoresis, which we will discuss in the Appendix (Vol. 3).

It is advisable to first master the analytical Procedures by working with pure samples of the substances to be tested. Once you have mastered the technique and learned to recognize the corresponding color changes, you can then proceed to investigate various Tissues.

Experiment 3.1. Identification of Pure Biomolecules

CAUTION! In all the analyses described here, heating should be carried out in a boiling Water bath. Direct heating of test tubes over an open flame is strictly prohibited.

Materials and Equipment

Litmus paper

Test tubes

Test tube rack

Bunsen burner

Pipettes

Spatula

Syringe (1 ml)

Iodine-potassium iodide solution

Benedict's reagent

Dilute sulfuric acid

Sodium bicarbonate (baking soda)

Sudan III

Millon's reagent

Fehling's reagent

5% potassium hydroxide solution

1% copper sulfate solution

Dichlorophenolindophenol (DCPIP) solution

1% starch solution (preferably from corn flour)

Carbohydrades

REDUCING SUGARS. Reducing sugars include all Monosaccharides, such as glucose and fructose, and some Disaccharides, such as maltose. Among the most common sugars, the only non-reducing sugar is sucrose (a disaccharide). Use 0.1–1% sucrose solutions for the analysis.

Procedure

Observed result

Explanation

Benedict's test

Pour 2 ml of the reducing sugar solution into a test tube. Add an equal volume of Benedict's reagent. Shake and gently bring to a boil, continuing to shake constantly to prevent the liquid from spitting out of the test tube

The blue color of the solution turns green, then yellowish, and finally a brick-red precipitate forms

Benedict's reagent contains copper sulfate. Reducing sugars reduce the soluble blue copper sulfate, containing copper(II) ions [Сu²⁺], to an insoluble brick-red copper(I) oxide precipitate. The copper oxide precipitates out

NOTE. The test is semi-quantitative, meaning it provides only a rough estimate of The amount of reducing sugar present in the sample. The color of the precipitate shifts from green to yellow, orange, and brick-red as the amount of reducing sugar increases. (The green color results from the mixing of the newly formed yellow precipitate with the blue copper sulfate solution.)

Procedure

Observed result

Explanation

Fehling's test

Pour 2 ml of the reducing sugar solution into a test tube. Add 1 ml of Fehling's solution A and 1 ml of Fehling's solution B. Bring to a boil while shaking

The initial blue color of the mixture turns green, then yellow, after which a brick-red precipitate forms

Same as for Benedict's test

NOTE. Fehling's test is less convenient than Benedict's test because solutions A and B must be stored separately prior to the analysis. Its sensitivity is also lower.

NON-REDUCING SUGARS. The most common non-reducing sugar is sucrose, which is a disaccharide. If it is known that reducing sugars are absent in the test solution (i.e., if the previous test gave a negative result for this solution), the appearance of a brick-red precipitate in the test described below indicates the presence of a non-reducing sugar. However, if it has been established that the test solution contains reducing sugars, the reaction described below will yield a more abundant precipitate than the previous test due to the presence of a non-reducing sugar as well.

Procedure	Observed result	Explanation
Pour 2 ml of sucrose solution into a test tube. Add 1 ml of dilute Hydrochloric acid. Boil for 1 min. Carefully neutralize with sodium bicarbonate, checking with litmus paper (caution is required as the liquid may boil over). Perform Benedict's test	As in Benedict's test	Upon boiling with dilute hydrochloric acid, the disaccharide is hydrolyzed into monosaccharide units. Sucrose is broken down into glucose and fructose. Both are reducing sugars and give the corresponding positive results in Benedict's test

STARCH. Very sparingly soluble in water, forming colloidal Suspensions. The analysis can be carried out using either a suspension or dry starch.

Procedure

Observed result

Explanation

Iodine test

Pour 2 ml of 1% starch solution into a test tube. Add a few drops of I₂/KI solution

Alternative method: apply the solution to dry starch

Upon Treatment with the I₂/KI solution, starch turns dark blue

A polyiodide-starch complex is formed

Cellulose AND Lignin. See Appendix 2 (staining) in Vol. 3.

Lipids. Lipids include oils (such as corn and olive oil), fats, and Waxes.

Lipids are immiscible with water. When water is added to the alcoholic lipid solution, an emulsion is formed; tiny lipid droplets suspended in water scatter light, making the emulsion appear white and opalescent

Procedure	Observed result	Explanation
Sudan III test Sudan III is a red dye. Add 2 ml of oil to 2 ml of water in a test tube. Add a few drops of Sudan III and shake	A red-stained oil layer floats on top of the water. The water remains unstained	Fat globules stain red and float because their density is lower than that of water
Emulsion test Add 2 ml of fat or oil to a test tube containing 2 ml of absolute ethanol. Shake vigorously to dissolve the lipid. Add an equal volume of cold water	A cloudy white suspension forms
Grease spot test Place a drop of the test sample on paper. Wait a while to allow any water present to evaporate. The paper may be gently warmed to speed up the process	A translucent spot remains on the paper

Proteins

Chicken egg albumin is very well suited for these analyses

Procedure

Observed result

Explanation

Millon's test

Pour 2 ml of protein solution or suspension into a test tube. Add 1 ml of Millon's reagent and boil.

CAUTION! Millon's reagent is toxic — handle with care!

A white precipitate forms, which coagulates upon boiling and turns red or orange-pink

Millon's reagent is a solution of mercury(II) in nitric acid containing traces of nitrous acid. The amino acid Tyrosine contains a phenyl group, which reacts with Millon's reagent to form a red mercury(II) complex. This is a non-specific reaction characteristic of all phenols. Proteins usually coagulate upon heating, forming a dense precipitate. Among all proteins used for this analysis, gelatin is the only one that lacks tyrosine

Biuret test

Pour 2 ml of protein solution into a test tube. Add an equal volume of 5% potassium hydroxide and mix. Add 2 drops of 1% copper sulfate solution and mix. No heating is required

A pinkish-violet or purple color slowly develops

This is a test for compounds containing peptide bonds. In the presence of a dilute copper sulfate solution under alkaline conditions, the nitrogen atoms of the peptide chain form a purple-colored complex with copper(II) ions [Сu²⁺]. Biuret (a urea derivative) also contains the —CONH— group and therefore gives this reaction

Vitamin C (ascorbic acid)

This method can be used for quantitative determination if necessary. In this case, the specified volumes must be measured very precisely. A suitable source of vitamin C is fresh orange or lemon juice mixed with distilled water (1:1). Commercially available vitamin C tablets can also be used.

Procedure	Observed result	Explanation
A 0.1% ascorbic acid solution serves as the standard. Pour 1 ml of DCPIP into a test tube. Draw 0.1% ascorbic acid solution into a 1 ml syringe. Add the ascorbic acid to the DCPIP drop by drop, gently stirring with the syringe needle. (Do not shake¹.) Continue this until the blue DCPIP solution is decolorized. Note the volume of ascorbic acid consumed	Disappearance of the blue color — the solution becomes colorless	DCPIP, a blue dye, is reduced by ascorbic acid (a strong reducing agent) to a colorless compound
¹ Shaking the solution may cause atmospheric oxygen to oxidize the ascorbic acid. You can test for yourself how shaking and boiling affect the experimental results.

DNA

See Table 5.5.

3.15. How can the concentration of ascorbic acid in the obtained sample be determined?

3.16. You are given three sugar solutions: one contains glucose, the second contains a mixture of glucose and sucrose, and the third contains sucrose.

a) How would you determine which sugar is present in each of these solutions?

b) Briefly outline the subsequent procedures that can be used to verify the correctness of your answer (assuming that the necessary equipment is available and that time permits such determinations).

3.17. How can you prepare 100 ml of a 10% glucose solution?

3.18. You have at your disposal a 10% glucose solution and a 2% sucrose solution as stock solutions. How can you prepare from them 100 ml of a mixture with a final concentration of 1% sucrose and 1% glucose?

Experiment 3.2. Detection of biomolecules in tissues

A biochemist often has to detect the presence of specific biomolecules or determine their quantity in living tissues, i.e., perform qualitative or quantitative analysis. Sometimes these determinations can be carried out directly on the tissue itself, but they are frequently preceded by some extraction or purification process.

It is helpful to practice on common food products or plant material, identifying the biomolecules discussed in Experiment 3.1. Where possible, we suggest an extraction procedure that yields a clear, colorless solution for analysis. Once students grasp the rationale behind such procedures, they will be able to devise Structure/131.html">Similar Methods themselves when necessary.

Materials and Equipment

Everything listed in Experiment 3.1 (from the beginning up to the DCPIP solution)

Mortar and pestle

Microscope

Microscope slides and coverslips

Razor blade

Watch Glass

Solution

Schulze's

Phloroglucinol + concentrated hydrochloric acid

Potato tuber

Apple

Cotton wool

Lignified stem

Seeds/nuts

Soaked peas

Beans

Microscopic examination of thin tissue sections

This method is suitable for observing reserve deposits that can be seen under a microscope, such as starch grains in a potato tuber.

Microscopic examination of sections following appropriate staining or other chemical treatment

The method is suitable for detecting the substances listed below.

Reducing sugars. Place the section in a few drops of Benedict's reagent and heat gently to boiling; add water if necessary to prevent drying out.

Starch. Place in dilute I₂/KI solution.

Protein. Place the section in a few drops of Millon's reagent and heat gently to boiling; add water if necessary to prevent drying out.

Oils and fats. Stain the material to be tested, such as seeds, with Sudan III, then wash with water and/or 70% alcohol. Prepare sections and mount in an appropriate medium.

Cellulose, lignin, etc. See Table 5.5.

Investigation of clear aqueous solutions

Bleach the tissue if necessary. Pigments present in the tissue may interfere with colour reactions, but these pigments can usually be easily removed from the tissue using organic Solvents, such as 80% ethanol or 80% propanone (acetone) (keep away from naked flames). It should be remembered, however, that these solvents may remove lipids and soluble sugars from the tissue. This technique is suitable for extracting chlorophyll from leaves.

Homogenisation of material. Sugars and proteins. Grind the pieces of the test material into a paste with a small amount of water using a pestle and mortar or a blender. Strain the crushed material through several layers of fine cheesecloth or nylon, pre-wetted with water, and either filter or centrifuge to remove solid particles. This may not be necessary if the suspension is highly dispersed and practically colourless. Analyse the clear solution as usual, or prepare appropriate dilutions if required. The solid residue, if of interest, can also be analysed.

Lipids. Grind the material, transfer to a test tube, and boil. Lipids separate as oil droplets. Perform the Sudan III stain. Alternatively, prepare an emulsion from finely shaved nut kernels or other food products (which may also be coloured) and perform the emulsion test.

The described procedure is suitable for detecting the substances listed below in various materials.

Fruits (e.g. apples or oranges)	(vitamin C, sugars)
Nuts	(oils)
Castor oil seeds	(oil)
Pea seeds	(protein)
Pine seeds	(protein, oil)
Potatoes	(starch, vitamin C)
Eggs	(protein)

The materials under investigation can be separated into fractions so that each fraction—such as seeds, fruit pulp, peel, or juice—can be examined individually.

Last update: 06/08/2026

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