This document was

compiled with the motive

of summarizing all the

TESTS ON
tests carried out in the lab.

CARBOHYDRATES
Biochemistry I Lab

AND PROTEINS
THE 20 AMINO ACIDS

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THE 20 AMINO ACIDS

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CARBOHYDRATE TESTS

Molish’s test is a general test for carbohydrates.

Principle: The test is based on the different rates of dehydration and
rearrangement in the sugar molecule to form furfural derivatives, which in turn
react with phenolic compound forming coloured complexes.
Procedure: Pipette 2 ml of test solution into test tube. Add 2 drops of the Molish’s
reagent. Mix thoroughly, incline the tube and pour 1-2 ml of conc. H2SO4 down the
sides slowly and carefully to form layers. Formation of a purple ring at the
interface between the acid and the sugar solution indicates the presence of
carbohydrate. A green colour is not a positive test.
NB: Molish’s reagent = α-naphthol in 95% ethanol

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Iodine test is a general test for starch and glycogen
Principle: The test is based on the formation of coloured starch-iodine or
glycogen-iodine complexes.
Procedure: Add 2-3 drops of Lugol’s iodine solution to the be tested. Starch gives a
blue-black colour while glycogen gives a brown-blue colour. A negative test is the
brown-yellow colour of the test reagent.
NB: Lugol’s reagent = IKI

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Benedict’s test is a test for reducing sugars
Principle: The test is based on the presence of aldehydes and ketones and the
reduction of alkaline metallic solutions with the oxidation of the sugar.
Precedure: Pipette 1 ml of test solution into test tube. Add 5 ml of Benedict’s
reagent. Place the test tube in a boiling water bath and eat for 3 minutes. Cool and
observe. The presence of reducing sugar is indicated by the formation of a yellow
to brick-red precipitate.
NB: Benedict’s reagent = Complex mixture of sodium citratre, sodium carbonate
and copper (II) sulphate pentahydrate.
Clinitest is a tablet which contains copper sulphate, citric acid, and anhydrous sodium
sulphate. Its use is similar to the Benedict’s test.

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Barfoed’s test is to differentiate between monosaccharides and disaccharides
Principle: The test is based on the reduction of cupric acetate by reducing
monosaccharides and reducing disaccharides.
Procedure: Add 1 ml of test solution to 3 mol of freshly prepared Barfoed’s reagent
in a test tube. Place the test tube in a boiling water bath and heat for 3 minutes.
Remove and allow to cool. The formation of a green, red or yellow precipitate is a
positive test for reducing monosaccharides.
NB: Barfoed’s reagent = Copper acetate in a dilute solution of acetic acid.
1. Precipitate isn’t as voluminous as that seen in Benedict’s test and tends to
adhere to the walls of the test tube.
2. Do not heat the tube for more than 3 minutes, as a positive test can be
obtained with disaccharides if they are heated long enough ( thus disaccharides
will hydrolyze into reducing monosaccharides).
Barfoed’s test is also a copper reduction test but unlike Benedict’s test, it is carried out
in an acidic medium.

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Bial’s test is used to distinguish between pentoses and hexoses.
Principle: This test is based on the principle that under hydrolysis, pentosans are
hydrolyzed into pentoses. Further, pentoses are dehydrated to yield furfural,
which in turn condense with orcinol to form a blue-green precipitate. In the
presence of hexoses, hydroxy furfural is formed instead of furfural which upon
condensation with orcinol forms a muddy brown colored precipitate.
Procedure: Pipette 2 ml of test solution into test tube. Add 3 ml of Bial’s reagent.
Mix and boil for 1 minute and observe. The presence of blue-green colour is a
positive test for pentoses. The formation of muddy brown colour is a positive test
for hexoses.
NB: Bial’s reagent = Mixture of orcinol, conc. HCl and FeCl3

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Seliwanoff’s test is used to distinguish between keto-sugar and aldo-sugar
Principle: The test is based on the principle that when heated, ketoses are
dehydrated and form furfurals more rapidly than aldoses. The furfural derivatives
react with phenolic compound forming coloured complexes.
Procedure: Pipette 1 ml of test solution into test tube. Prepare a blank tube with 1
ml of water. Add 5 ml of Seliwanoff’s reagent to each test tube. Mix and heat for
exactly 30 minutes in the boiling water bath. Observe and record the results at this
time. Continue heating for 5 minutes, recording any changes that occur in a table.
A cherry-red colour indicates the presence of ketohexoses and a blue-green
solution indicates the presence of ketopentoses. Aldoses and disaccharides give no
colour change.
NB: Seliwanoff’s reagent = Resorcinol in conc. HCl.

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PROTEIN TESTS

Biuret’s test is a test for the presence of peptide bonds.

Principle: The test is based on the biuret reaction in which a peptide structure
containing at least two peptide links produces a violet colour when treated with
alkaline copper.
Procedure: To the sample, add 2ml of 2N NaOH and 1-2 drops of 1% CuSO4
solution. (NB: Run a nonprotein control). A purple/ violet colour indicates the
presence of proteins. A more intense colouration indicates the presence of more
complex proteins (more peptide bonds)
NB: Biuret’s reagent = Copper sulphate, sodium hydroxide and potassium tartrate

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Million’s test is a specific test for tyrosine.
Principle: The test is based on the nitrification of the phenol group in tyrosine, which
then forms complexes with heavy metals like mercury.
Procedure: Add some few drops of Millon’s reagent to 2 ml of the sample in a test
tube. Warm gently to boiling. A positive result is indicated by a faint pink colour
which develops gradually into red colour.
NB: Millon’s reagent = Solution of mercuric and mercurous ions in nitric and
nitrous acid. (CAUTION: Miliion’s reagent is highly toxic and highly corrosive)

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Hopekin Cole’s test is a test for amino acids containing an indole ring
Principle: The test is based on the fact that the layering of conc. H2SO4 over
tryptophan-containing proteins with the Hopkin Cole’s reagent results in the
formation of a violet ring at the interface.
Procedure: Add 2ml of Hopkin Cole’s reagent to the sample in a test tube. Mix
thoroughly. Incline the tube and gently add 5 ml of conc. H2SO4. Heat the mixture
in a water bath for 2 minutes and allow to stand. The appearance of a violet ring
at the interface of the liquid is a positive test.
NB: Hopkin Cole’s reagent = Glyoxylic acid

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Xanthoproteic test is a test for amino acids containing phenolic or indole groups.
Principle: The Xanthoproteic test is based on the fact that aromatic groups in the
amino acids are nitrated by heating with concentrated HNO3 to yield intensely
yellow-colored nitro derivative. On addition of alkali, a salt is formed to change
the colour from yellow to orange.
Procedure: Add 2ml of conc. HNO3 to the sample and heat until any precipitate
formed dissolves. Cool the solution and add 6N NaOH in Excess. Formation of
orange colour indicates the presence of phenolic ring.
NB: Reagent = Conc. Nitric acid (HNO3)

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 POSSIBLE QUESTIONS FOR LAB QUIZZES AND LAB EXAMS
1. Write a brief note on carbohydrates
2. Classify the following as aldoses or ketoses and according to the number of carbon
atoms present (pentose, triose, hexose etc.)
i. Ribose
ii. Xylose
iii. Galactose
iv. Maltose
v. fructose
3. Mention two (2) functional groups that may be present in carbohydrates.
4. What are the components of the following regents
i. Fehling’s
ii. Benedict’s
iii. Molisch’s
iv. Seliwanoff’s
v. Millon’s
5. Distinguish oligosaccharides from polysaccharides.
6. Are all disaccharides reducing sugars? Defend your answer.
7. Give three (3) examples each of
i. Monosaccharides
ii. Disaccharides
iii. Hexoses
iv. Pentoses
8. Draw the parent structure of an amino acid and identify the functional groups.
9. State two (2) examples each of
i. Basic amino acids
ii. Neutral amino acids
iii. Acidic amino acids
10. Delali bought a 100ml can of milk for his projects on lactose content
determination. In the experiment, he mixed 50ml of the milk with 10% sodium
tungstate and ammonium sulphate in a 500ml volumetric flask and diluted it to the

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mark. He filtered and then titrated the filtrate against 30ml of Benedicts quantitative
reagent and had the following results.

Experiment 1 2 3
Initial / ml 14.95 23.45 0.00
Final / ml 23.45 32.45 9.0

Given that 10ml of Benedict’s quantitative reagent = 27g of lactose, calculate

i. The quantity of reducing sugar in the milk (%w/v)

ii. The molarity; given the molecular weight of lactose to be 342.3g/mol
iii. Why was Na2WO4 and NH2SO4 added to the milk solution before the
titration
11. Define denaturation
12. State the amino acids present in keratin
13. What are globular proteins? Give two (2) examples.
14. Give three (3) benefits of cholesterol to humans.
15. Which type of lipid is commonly found in the CNS?
16. What is the principle behind Seliwanoff’s test?
17. Using Phenylalanine as the parent material, draw and name the structure of any two
(2) amino acids
18. Mention any two (2) sulphur containing protein
19. Imagine an amino acid in an acidic medium. Explain the overall charge with equations
20. Explain the significance of albumin in heavy metal poisoning.
21. Explain why some disaccharides can test for reducing sugars and some cannot.
22. A substance tested negative for xanthoproteic test but positive for Millon’s test.
Comment on this result
23. Animals : glycogen as plants : ………..
24. Name one (1) protein that tests positive for reducing sulphur test.
25. Name two (2) proteins that test positive for Hopkin-Cole’s test.
26. Name one (1) amino acid that tests positive for Xanthoproteic test.
27. Explain what it means when the dissolution of rabbit fur occurs

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28. Explain using equations, the reason why protein in acidic medium stains with acidic
dye but not basic dye
29. State the number of carbon atoms and functional group in the following sugars (as in
pentose, ketotriose, etc)
i. Ribose
ii. Xylose
iii. Galactose
iv. Arabinose
30. Plant : starch as animal : ……….
31. Do all proteins test positive for Biuret’s test? Defend your answer.
32. What is meant by salting out in protein separation and characterization?
33. Define the following
i. Isoelectric point
ii. Zuither ion
iii. Transcription
iv. Translation
34. Which free amino acids give a positive Biuret’s test?
35. Certain tests were carried out two (2) unknown crystalline substances. Study
the results below and answer the questions that follow.
Key:
+ => positive test
- => negative test

Test Sample A Sample B

Molish test + +
Barfoed’s test + -
Bennedict’s test + +
Biure’s test - +

i. What conclusion will your draw out of the test results?

ii. Give an example each of what substance A and B may be.

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36. Why should excess CuSO4 not be used in Biuret’s test?
37. Why should the result for phenol be positive/negative in Xanthoproteic test?
38. Do you expect adrenaline to give a positive Xanthoproteic test?
39. Which type of protein do you think will react positively with Molisch’s reagent?
Give one (1) example.
40. Do all proteins give a positive result to Biuret’s test?
41. What is the significance of heavy metal precipitation of albumin in terms of
metal poisoning?
42. What is the significance of “salting-out” to a pharmacognocist who wants to
extract a glycosidic drug from a medium containing protein?
43. With equations, explain cationic and anionic precipitation of proteins.
44. Classify the following as reversible or irreversible.
i. Denaturation
ii. Cationic precipitation
iii. Anionic precipitation
iv. Dehydration

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