Aim: To characterize sample as Carbohydrate, Protein OR Miscellaneous compound.

First of all find out whether compound is Carbohydrate, Protein OR Miscellaneous compound by performing
Biuret test and Molisch’s test as follow.

Unknown Sample (Solution)

(Biuret Test)

Violet Color No Violet color (Protein Absent)

Protein present (Molisch Test)

Violet or Purple coloured ring No Violet or Purple coloured ring

at the junction of two liquid. at the junction of two liquid

Carbohydrate present Miscellaneous Compound

present

Test Observation Inference

Biuret Test: Violet or purple Color in 1st The given sample contains
Take two test tube. Label as 1st test tube (test) which is protein.
as test and 2nd as blank. clearly distinguishable from
Take 3 ml of the given solution color of the 2nd test tube
in 1st test tube and 3 ml distilled (blank).
water in 2nd test tube. Add 1 ml
40% NaOH solution to both test
tube mix and add 2 drops of
Biuret reagent (1% CuSO4
Solution) to both the test tube.
Molisch Test: To the 3 ml. of Violet or Purple coloured ring The give sample contains
the given solution add 2 drops at the junction of two liquid. Carbohydrate.
of Molisch reagent (1%
alcoholic α-napthol solution),
Mix in the test tube. To that add
3 ml conc. Sulphuric acid from
the side of the tube so as to
form two layers.

Note: When both carbohydrate and proteins are absent, than sample may contain miscellaneous

Compound.

Scheme for Biochemistry qualitative analysis Page 1

 Scheme for identification of carbohydrate.

Sugar Solution
(Molisch’s Test)
Purple colored ring at the junction of the two liquids
∴ Carbohydrate Present
Compound and add Distilled water
( If Solution given, check clarity of solution)

Soluble OR Given solution is clear Insoluble OR Given solution is not

clear
∴ Monosaccharide like glucose, fructose OR ∴ Polysaccharide like starch, glycogen
Disaccharide like sucrose, maltose or lactose or dextrin etc. may be present.
may be Present (Iodine Test)
(Benedicts test)
(Fehlings Test) Blue ∴ Starch
(Tommers Test)
Red brown ∴ Glycogen
Red, yellow or Green precipitates No Red, yellow or Green Purple Red ∴ Dextrin
precipitates
(Above test positive) (Above test Negative)
∴ Reducing sugar like glucose, ∴ Non Reducing sugar
fructose, maltose or lactose may be like Sucrose may be present
Present And Perform inversion test
(Barfoed’s Test)

Brick red precipitates (Test No Brick red precipitates (Test

Positive) Negative)
∴ Monosaccharide like Glucose, ∴ Disaccharide like Lactose,
Fructose may be present maltose may be present
(Rapid furfural test) (OsazoneTest)
(Seliwanoff test)
Small needles like ball of pricklles
Brick Red ppt or Deep No Brick Red ppt or ∴ Lactose may be present
purple color Deep purple color
(Above test positive) (Above test Negative) Plate like crystals appear like
∴ Ketose like Fructose ∴ Aldose like Glucose Sunflower ∴ Maltose may be
may present may present present

Scheme for Biochemistry qualitative analysis Page 2

TESTS OBSERVATION INFERENCES

1. MOLISCH`S TEST: To 3ml of Purple colored ring at the junction Carbohydrates like glucose,
given sugar solution add 2 drops of of the two liquids fructose, lactose , starch etc.
Molisch’s reagent (1% alc. α- present
Napthol ) mix in the tube and add
3ml conc. sulphuric acid from side
of the test tube so as to form two
layers.

NOTE: Do not shake it. Gently

rotate it between palm.

2. SOLUBILITY TEST: To 3ml of Clear solution Monosaccharide like glucose,

the water add .5 gm of sample and fructose OR Disaccharide like
shake. lactose , sucrose etc. present

3. BENEDICT`S TEST: To 5ml of Green, yellow or red precipitates Reducing sugars like glucose,
Benedict`s reagent add 0.5 ml of fructose etc. may present
given sugar solution, shake it and
boil.

4. FEHLING`S TEST: To 2ml of Red to yellow precipitates Reducing sugars like glucose,
Fehling`s reagent A & B each, add fructose etc. may present.
1ml of given sugar solution, shake
it and boil.

5. TOMMER’S TEST: To 2ml of Yellow or red precipitates Reducing sugars like glucose,
Tommers reagent add 3 ml given fructose etc. may present.
sugar solution shake and boilit for 2
minute amd cool it

6. BARFOED`S TEST: To 3ml of Brick red precipitates settles down Monosaccharides like glucose,
given sugar solution add 3ml of at the bottom of the test tube. fructose etc. may be present.
Barfoed`s reagent, shake and boil
for 3 minute, cool it.

7. RAPID FURFURAL TEST: To Deep purple color develops as Ketose like Fructose may be
1ml of given sugar solution add 6 soon as the mixture begins to boil. present
drops of Molisch`s reagent (1% alc.
α-Napthol) and add 5ml conc HCl
shake and boil.

8.SELWINOFF`S TEST: To 1ml Red or brown precipitates. Ketose like Fructose may be
of given sugar solution add 3ml of present
Selwinoff`s reagent shake and boil
for 2 minute.

9. OSAZONE TEST : To 5ml of Needle shaped crystals Glucose or fructose

given sugar solution add 100 mg of
phenyl hydrazine mixture (equal
parts of anhydrous sodium Small needles appear like ball of

Scheme for Biochemistry qualitative analysis Page 3

carbonate CH3COONa and prickles Lactose
phenylhydrazine HCl ). Shake well
and heat on the boiling water bath
for 30 minute. Allow the tube to Plate like crystals appear like Maltose
cool down slowly to room temp. sunflower.
Spread the suspension on a clean
slide of microscopic slide and
observe the crystals under low
power of a microscope.

10. IODINE TEST: To 3ml of Blue color Starch present

given sugar solution add 1 to 2
drops dilute iodine solution. Purple-red color Dextrin present

Red – brown Glycogen present

Colorless or light yellow Monosaccharide or disaccharide

present

11. SUCROSE: INVERSION The resulting solution will give Non reducing sugar like sucrose
TEST: To 5 ml of given sugar test of glucose and fructose, due to may be present.
solution add 5 drops of conc. hydrolysis of sucrose to glucose
Sulphuric acid. Shake and boil for 2 and fructose.
minute. Cool it and neutralize with
satd. Sodium bicarbonate solution
till no effervescence observes.

Scheme for Biochemistry qualitative analysis Page 4

 Scheme for Identification of Protein in given sample.

Boil the given protein solution in test tube on flame

Coagulation of protein No Coagulation of protein

∴ Albumin OR Globulin present ∴ Gelatin, Casein OR Peptone present

(Half saturation test) (Neumann’s test)

Violet color No Violet color No Yellow color / Yellow color /

Precipitates. Precipitates.
∴ Albumin present ∴ Globulin present ∴ Gelatin OR Peptone ∴ Casein present
present

Test Observation Inferences

1. Neumann’s Test: Precipitates Casein present
i) Take 3 ml of solution and add 2 drops of
CPR indicator. Then add 1% acetic acid drop
wise until pink color just disappear and
distinct yellow color appears.
ii) To above precipitates add 3 to 4 drops
conc. H2SO4 and 10-12 drops conc. HNO3. Shining yellow or canary Casein present
Heat until mixture is colorless. (Add more yellow precipitates obtained
HNO3 if necessary). Cool and add few drops
of ammonium molybdate solution.
2. Heller test: Take 3ml. of conc. HNO3 in A white ring at the junction of Albumin present
test tube and add 3ml of Protein solution, by two liquid.
means of pipette to the side wall of test tube,
in such a way that it forms separate layer.
3. Half saturation test: Take 3ml solution and a) Violte OR Purple color Albumin present
add 3ml of saturated Ammonium sulphate
(NH4)2SO4 solution. Keep aside for 5 minute. b) No Violte OR Purple color Globulin present.
Remove the precipitates by filtration. To the
filtrate add 2ml 40% NaOH solution and 5 c) No rosy pink color Peptone present
drops 1% CuSO4 solution and shake well.

4. Full saturation test: Take 3ml solution and No Violet purple or rosy pink Albumin present.
add Ammonium sulphate (NH4)2SO4 till color.
solution saturates. Keep aside for 5 minute.
Remove the precipitates by filtration. To the
filtrate add 2ml 40% NaOH solution and 5
drops 1% CuSO4 solution and shake well.

Scheme for Biochemistry qualitative analysis Page 5

 Scheme for identification of Miscellaneous compound:

Take 3 ml of Given solution in two test tubes and add 2 drops of following indicators and observe
the change in color and follow the following scheme accordingly.

Nature Indicator Methyl Red Phenolphthalein Litmus paper

Acidic Red No color Blue to red

Basic Yellow Pink Red to blue
Neutral Yellow No color No Change

Depending upon the nature of the sample, follow the below scheme.

Miscellaneous Compound
(Find nature of the sample)

Acidic Neutral Basic

(AgNO3 Test) (Schiff’s test)

White Ppts No White Ppts Black/Brown stain

∴HCl Present ∴Lactic acid Present ∴Uricacid present

Colored solution Color less solution

(Hay’s Sunflower Test)

(Alk. Sod. Hypobromate Test)

Powder sinks Powder floats No Effervescences Effervescence of N2

∴Bile salt Present ∴Blood Present ∴Urea Present
(Rothera’s test)

No Permanganate color Permanganate color

∴Ketone bodies like acetone Present
(Jaff’s test)

Orange color No orange color

∴Creatinine Present
(Silver nitrate test)

White Precipitates
∴NaCl Present

Scheme for Biochemistry qualitative analysis Page 6

Test Observation Inferences
Silver nitrate test:
Take 2-3 ml sample, add 2 ml dil. Nitric White ppts. Chloride ion containing
acid and 1 ml dil. Silver nitrate solution compound like HCl, NaCl
and shake well. present
Uffleman’sTest:
To 5ml. of Uffleman’s reagent add given Immediate change of Lactic acid present.
solution drop by drop. color from blue to yellow (Uffleman’s reagent must
be blue in color)
Gunzberg Test:
Evaporate to dryness 5 drops of Purplish red or pink color Hydrochloric acid present
Gunzberg’s reagent in a clean dry
evaporating dish on water bath or low
flame. Dip a glass rod in given solution
and draw the moist end through the dried
reagent.
Schiff’s Test:
Moisten a strip of filter paper with Black/Brown stain Uric acid present.
AgNO3solution and add to it a drop of
given sugar solution.
Hay’s Sunflower Test:
To the 5 ml of given test solution sprinkle Powder Sinks at bottom Bile salt present.
sublimed sulpher powder on surface.
Alkaline Sodium Hypobromate Test:
To the 3 ml of given solution add few Effervescence of N2gas. Urea present
drops of Alkaline Sodium Hypobromate
solution.
Rothera’s Test:
To 5 ml of given urine solution add Permanganate color Ketone bodies like acetone
ammonium sulphate [(NH4)2SO4] till present
saturate. To this add 2 drops of sodium
nitro prusside and 2 ml of liquid ammonia
solution from side of the test tube.
Jaffe’s Test:
To 5 ml of urine sample add 1 ml of Orange color Creatinine present
saturated solution of picric acid. To this
add 1 ml of 10% NaOH solution and mix
well by shaking.

Scheme for Biochemistry qualitative analysis Page 7