DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY
UNIVERSITY OF DHAKA
1st Year B.Sc. (Minor) Practical Class
SOME COLOUR TESTS FOR PROTEIN AND AMINO ACIDS
1. Ninhydrin reaction : Mechanisms –
O
O OH
R
2H2O
o
+ H2N-CH-COOH
O
O
O
R
=N-CH-COOH
O
OH
(C)
A (Indine – 1,2,3-trione)Ninhydrin
`
O
–
CO2
O
H
H
O
O
G
H2O
O
N=
H
H
N=
O
O
R
NH–CHO
N
NH2
O
O
R-CHO
O
F
D
E
O
Principle:
The reaction of ninhydrin with amino acids of the type RCH(NH2)COOH involve initial formation of (C), which
undergoes decarboxylation to Schiff base (D), which in-turn is readily hydrolyzed to the amine (E). This amine
condenses with another mole of (A) to form (F), which, on loss of a proton, gives the blue anion (G). Since this
reaction is quantitative and reproducible, it serve as a precise method for amino acid analysis in addition to the
qualitative use.
Procedure :
Place 1 ml of approximately 0.1% amino acid solution in a test tube, and five drops of 0.2% ninhyhdrin solution.
Boil the solution for 2 min. All amino acids will give blue colour, whereas protein will give yellow colour.
2. Ehrlich’s reaction : Mechanism -
CH3
O
C
H
N
H
H
C–
N–CH3
CH3
– N – CH3
N
H
H
Red Violet colour
Principle :
When indole/pyrrole ring containing compound containing compounds in presence of conc. HCl is treated with the
Ehrlich’s reagent (10% solution of p-dimethylaminobenzaldehyde in conc. HCl). They condensed to form a colour
complex.
Procedure :
Add 2 ml of Ehrlich reagent to 0.5 ml of 0.1% solution of Tryptophan or indole or even urea. A positive test is indicated by
the formation of red violet colour complex.
3. Biuret test for peptide bonds : Mechanism NH3
O
O
O
O
H2N–C–NH2 + H2N–C–NH2
H2N–C–NH–C–NH2
O
C
O
NH
NH
C
Cu++
NH
C
O
NH
NH
NH
C
O
Cu2+
Principle
When urea is heated to about 180oC, it decomposes to form biuret. Addition of a very dilute solution of
copper sulphate to an alkaline solution of a protein produces a red to violet colour. This reaction is due
to the presence of the grouping – CO – NH CHR–CO–NH–. At least two peptides linkages (-CONH–)
must be present (di-peptides do not give the test). The biuret test apparently is due to co-ordinationof
cupric ions with the unshared electron pairs of peptide nitrogen and the oxygen of water to form
coloured coordination complex, which may be represented as above.
Procedure:
Add 5 drops of 1% copper sulphate solution to 2 ml of a protein/amino acid solution followed by 2 ml
of 10 M NaOH solution. Peptides and proteins will give violet colour whereas amino acids do not.
Carbohydrate Tests :1. Molisch test : Mechanism –
Carbohydrates
Monosaccharides
CHO
O
Furfural
OH
H2O
-napthol
O C
O
H O
Red to violet colour
Principle
In strongly acid solution, carbohydrates are converted into furfural (pentose), hydroxymethyl furfural (hexose),
methylfurfural (methylphentose as rhamnose) or other aldehydic compound. The aldehyde formed by action of
the acid is then condensed with a phenol, like, -napthol to give the colour. This is a very sensitive general
reaction for carbohydrate.
Procedure:
Add two drops of -napthol solution to 2 ml of carbohydrate solution and then carefully pour 1 ml of conc.
H2SO4 down the sides of the tube. A purple/violet ring develops at the junction of two layers.
2.
Barfoed test : Mechanism –
Cu2+ + –CHO
Cu2O + –COO–
Red ppt.
Principle
When reducing sugars are boiled with Barfoed reagent, it is reduced and Cu2O separates, which is a red ppt.
This test is used to distinguish monosaccharides from reducing disaccharide. Here concentration of the sugar
solution is important, since a more concentrated disaccharide solution will reduce faster than a dilute
monosaccharide solution.
Procedure:
To a 2 ml reagent (containing 6% cupric acetate in 1% acetic acid) and 1 ml of the sugar solution and heat in
boilig water bath for 3 min. Monosaccharides will give red ppt. of cuprous oxide immediately whereas reducing
disaccharide may give positive test very slowly.
3. Iodine test : To a 2 ml sugar solution add 1 to 3 drops iodine solution 5 ml in 30% KI solution).
Iodine forms complex which imparts colour to the solution.
Starch – Blue colour : Glycogen – wine red colour
Dextrin – Red, blue or purple, depending on the type of dextrin.
Cellulose and Insulin – no colour.
4.
Fehling’s test: This is a general test for reducing sugar. Place 5 ml of Fehling’s solution in a test tube
and heat to gentle boiling. Add a solution of 100 mg of the carbohydrate in 2 ml of water and
continue to boil gently for a minute or two and observe the result. A yellow or red precipitate of
cuprous oxide indicates the presence of reducing sugar.
5.
Benedict’s test: This is a modification of Fehling’s test. To 5 ml of Benedict’s solution add 50 mg
carbohydrate, boil for 2 minutes and allow to cool spontaneously. If no reducing sugar is present, the
solution remains clear; in the presence of a reducing sugar, the solution will contain yellow to red
precipitate of cuprous oxide.
6.
Bial’s test: This is test of pentose sugar. Place 20 mg of the substance in a test tube containing 2 ml
water. Add 3 ml Bial’s reagent and heat the test tube over a bunsen flame until the solution, just
begins to boil. A blue-green colour will indicate the presence of pentose sugar.
Note: It is necessary to perform Barfoed’s, Fehling’s, Benedict’s and Bial’s test only when the unknown compound
responds positively towards Molisch’s test.
4.
Fehling’s test: This is a general test for reducing sugar. Place 5 ml of Fehling’s solution in a test tube
and heat to gentle boiling. Add a solution of 100 mg of the carbohydrate in 2 ml of water and
continue to boil gently for a minute or two and observe the result. A yellow or red precipitate of
cuprous oxide indicates the presence of reducing sugar.
5.
Benedict’s test: This is a modification of Fehling’s test. To 5 ml of Benedict’s solution add 50 mg
carbohydrate, boil for 2 minutes and allow to cool spontaneously. If no reducing sugar is present, the
solution remains clear; in the presence of a reducing sugar, the solution will contain yellow to red
precipitate of cuprous oxide.
6.
Bial’s test: This is test of pentose sugar. Place 20 mg of the substance in a test tube containing 2 ml
water. Add 3 ml Bial’s reagent and heat the test tube over a bunsen flame until the solution, just
begins to boil. A blue-green colour will indicate the presence of pentose sugar.
Note: It is necessary to perform Barfoed’s, Fehling’s, Benedict’s and Bial’s test only when the unknown compound
responds positively towards Molisch’s test.
mukesh/-