By Duaa Mir (Visiting lecturer department of Zoology)

LABORATORY MANUAL
GENERAL
BIOCHEMISTRY
COURSE CODE: Zoo-350
BY
DUAA MIR
(VISITING LECTURER DEPARTMENT OF ZOOLOGY)
 By Duaa Mir (Visiting lecturer department of Zoology)

EXPERIMENT NO 1:
QUALITATIVE ANALYSIS OF CARBOHYDRATES.

TESTS ON CARBOHYDRATES:
1) Molisch’s Test:
Molisch’s Test is a sensitive chemical test for all carbohydrates, and some compounds containing
carbohydrates.
Procedure:
1-Place 2 mL of a known carbohydrate solution in a test tube, add 1 drop of Molisch’s reagent
(10% α-naphthol in ethanol).
2-Pour 1-2 mL of conc. H2SO4 down the side of the test tube, so that it forms a layer at the bottom
of the tube.
3-Observe the color at the interface between two layers and compare your result with a control
test.
4- Observe the red or purple-colored compound as positive result.
2) Fehling’s Test:
Fehling’s Solution (deep blue colored) is used to determine the presence of reducing sugars and
aldehydes. Perform this test with fructose, glucose, maltose and sucrose.
Procedure:
1-To 1 mL of Fehling’s solution A (aqueous solution of CuSO4) add 1 mL of Fehling solution B
(solution of potassium tartrate).
2-Add 2 mL of the sugar solution, mix well and boil.
3-Try to see the red precipitate of cuprous oxide that forms at the end of the reaction.
3) Barfoed’s Test:
Perform this test with glucose, maltose and sucrose.
Procedure:
1-To 1-2 mL of Barfoed’s reagent, add an equal volume of sugar solution.
2-Boil for 5 min. in a water bath and allow to stand.
3-You will observe a brick-red cuprous oxide precipitate if reduction has taken place.
 By Duaa Mir (Visiting lecturer department of Zoology)

4) Seliwanoff’s Test:
Seliwanoff’s Test distinguishes between aldose and ketose sugars. Ketoses are distinguished from
aldoses via their ketone/aldehyde functionality. If the sugar contains a ketone group, it is a ketose
and if it contains an aldehyde group, it is an aldose. This test is based on the fact that, when heated,
ketoses are more rapidly dehydrated than aldoses. Perform this test with glucose, fructose,
maltose and sucrose.
Procedure:
1-Heat 1 mL of sugar solution with 3 mL Seliwanoff’s reagent (0.5 g resorcinol per liter 10% HCl)
in boiling water.

2-In less than 30 seconds, a red color must appear for ketoses.
3-Upon prolonged heating, glucose will also give an appreciable color.
5) Bial’s Test:
Bial’s Test is to determine the presence of pentoses (5C sugars). The components of this reagent
are resorcinol, HCl, and ferric chloride. In this test, the pentose is dehydrated to form furfural
and the solution turns bluish and a precipitate may form. Perform this test with ribose and
glucose.
Procedure:
1-To 5 mL of Bial’s reagent, add 2-3 drops of sugar solution and boil.

2- Upon boiling, note the green-blue color formed.

6) Action of Alkali on Sugars:

Procedure:
1- Heat 1 mL glucose solution with 1 mL 40% NaOH for 1 min.
2-Cool and apply test for reducing sugars (e.g.; Fehling’s Test).
3-Apply a control test with glucose solution to observe the difference.
5) Iodine Test:
Iodine test is an indicator for the presence of starch. Iodine solution (iodine dissolved in an aqueous
solution of potassium iodide) reacts with starch producing a blue-black color. Apply this test to
all the polysaccharides provided.
 By Duaa Mir (Visiting lecturer department of Zoology)

Procedure:
1-To 2-3 mL of polysaccharide solution, add 1-2 drops of iodine solution.
2-Observe the different colors obtained for each of the polysaccharide solutions.

4) The Inversion of Sucrose:

Sucrose is a disaccharide, which means that it is a molecule that is derived from two simple sugars
glucose and fructose. Inverted sugar is obtained by splitting sucrose into these two components.
The splitting of sucrose is a hydrolysis reaction which can be induced simply by heating an aqueous
solution of sucrose. Acid also accelerates the conversion of sucrose to invert.
Procedure:
1-Add 5 mL of sucrose solution to two test tubes.
2-Add 5 drops of conc. HCl to one test tube.
3-Heat both tubes in boiling water bath for 10 min.
4-Cool and neutralize with diluted NaOH (use litmus paper).
5-Test both solutions for the presence of reducing sugar with Fehling’s Test.
 By Duaa Mir (Visiting lecturer department of Zoology)

EXPERIMENT NO 2:
QUALITATIVE ANALYSIS OF AMINO ACIDS.

Tests for amino aids:

1) Solubility Tests:
Amino acids are essentially soluble in water. Their solubilities in water, dilute alkali and dilute
acid vary from one compound to the other depending on the structure of their side chains. The
solubility of amino acids and proteins is largely dependent on the solution pH. Apply this test to
glycine, tyrosine, glutamic acid and cysteine.
Procedure:
1-Note the solubility of amino acids in water and alcohol by placing a small amount in a test tube,
adding a few mL of solvent and warming if necessary.
2-Determine the amino acid solution is acidic or basic by using a litmus paper while testing the
solubility in water.
3-Repeat the solubility test using dilute HCl and dilute NaOH.
2) Ninhydrin Test:
Ninhydrin (triketohydrindene hydrate) is a chemical used to detect amino acids. Amino acids react
with ninhydrin at pH=4. The product obtained from ninhydrin yield a blue colored substance.
WARNING: Avoid spilling ninhydrin solutions on your skin, as the resulting stains are difficult
to remove.
Procedure:
1-To 1 mL amino acid solution add 5 drops of 0.2% ninhydrine solution in acetone.
2-Boil over a water bath for 2 min.
3-Allow to cool and observe the blue color formed.
3) Specific Reactions for Individual Amino Acids:
a) Xanthoproteic Test:
Some amino acids contain aromatic groups that are derivatives of benzene. This reaction forms
yellow product. Apply this test to tyrosine, tryptophan, phenylalanine and glutamic acid.
Procedure:
 By Duaa Mir (Visiting lecturer department of Zoology)

1-To 2 mL amino acid solution in a boiling test tube, add 2ml of concentrated HNO3.
2-Heat over a flame for 2 minutes and observe the color.
3-Now cool thoroughly under the tap and add in sufficient 40% NaOH to make the solution
strongly alkaline.
3-Observe the yellow color of the solution.
b) Millon’s Test:
Millon’s reagent is concentrated HNO3, in which mercury is dissolved. As a result of the reaction
a red precipitate or a red solution is considered as positive test. Apply this test to tyrosine,
phenylalanine, glycine and β-naphtol.
Procedure:
1-To 2 mL amino acid solution in a test tube, add 1-2 drops of Millon’s reagent.
2-Warm the tube in a boiling water bath for 10 min.
3-A brick red color is a positive reaction.

c) Hopkin’s Cole Test:

Apply this test to glycine, tryptophan and tyrosine.
Procedure:
1-To a few mL of glacial acetic acid containing glyoxylic acid, add 1-2 drops of the amino acid
solution.
2-Pour 1-2 mL H2SO4 down the side of the sloping test tube to form a layer underneath the acetic
acid.
3-The development of a purple color at the interface proves a positive reaction.
d) Lead-Sulfide Test:
When cystine is boiled with 40% NaOH, some of sulfur in its structure is coverted to sodium
sulfide (Na2S). The Na2S can be detected by using sodium plumbate solution which causes the
precipitation of PbS from an alkaline solution. In order to apply this test, first the sodium plumbate
solution should be prepared. Apply this test to cysteine and cystine.

Procedure:
 By Duaa Mir (Visiting lecturer department of Zoology)

Sodium Plumbate Solution Preparation:

1-Add 5 mL dilute NaOH to 2 mL dilute lead acetate.
2-A white precipitate of lead hydroxide forms.
3-Boil until the precipitate dissolves with the formation of sodium plumbate.
Test:
1-Boil 2 mL amino acid solution with a few drops of 40% NaOH for 2 min.
2-Cool and add a few drops of the sodium plumbate solution.
3-A brown color or precipitate is a positive test for sulfides.

EXPERIMENT NO 3:
 By Duaa Mir (Visiting lecturer department of Zoology)

QUALITATIVE ANALYSIS OF PROTEINS.

Tests for Proteins:

a) Biuret Test:
The Biuret Test positively identifies the presence of proteins (not less than two peptides). Apply
this test to gelatin, casein and albumin.
Procedure:
1-To 2 mL protein solution, add 5-6 drops of dilute CuSO4.
2-Add 3 mL 40% NaOH solution.
3-Observe the color change.
b) Precipitation of Proteins:
The precipitation of a protein occurs in a stepwise process. The addition of a precipitating agent
and steady mixing destabilizes the protein solution.
• By salts of Heavy Metals:
Heavy metal salts usually contain Hg2+, Pb2+, Ag1+, Cd2+ and other metals with high atomic
weights. Since salts are ionic, they disrupt salt bridges in proteins. The reaction of a heavy metal
salt with a protein usually leads to an insoluble metal protein salt.
Procedure:
1-Treat 3 mL of the protein solution provided with a few drops of mercuric nitrate.
2-A white precipitate formation should be observed.

• By Acid Reagents:
Apply this test to all the proteins provided.
Procedure:
1-Treat 3 mL of protein solution provided with a few drops of trichloroacetic acid solution.
2-Note the protein precipitate formed.

EXPERIMENT NO 4:
 By Duaa Mir (Visiting lecturer department of Zoology)

QUALITATIVE ANALYSIS OF LIPIDS.

1)Acrolein test:
This test is used to detect the presence of fats or glycerin.
Procedure:
1-The fat is heated strongly in the presence of a dehydrating agent such as potassium bisulfate.

2-The glycerol portion of the molecule is dehydrated to form the unsaturated aldehyde, acrolein
(CH2=CH–CHO).

3- The peculiar odor of burnt cooking grease marks the positive result of test.
2)Sudan IV Test:
This test is used to detect the presence of lipids.
Procedure:
1-In this test the dark red Sudan IV chemical is added to solution along with ethanol to detect
lipids.
2-If the lipids ae present in solution, the Sudan IV will stain them red-orange which marks the
positive result of test.
3)-Copper acetate Test:
This test is used to distinguish between fats and oils or the saturated and unsaturated fatty acids
due to formation of copper salts at the end of reaction.
Procedure:
1-Take two test tubes and to both add ½ gram of oleic acid (unsaturated) and steaic acid (satrated).
2-Add 3ml of petroleum ether to each test tube and after that add an equal volume of 5% copper
acetate solution.
3-In case of oleic acid the upper layer of petroleum ether will become green and lower layer will
become less blue due to formation of copper oleate.
4-In case of stearic acid the upper layer of petroleum ether remains colourless while the lower
layer consists of pale green precipitates of copper steaate at bottom.
4) Liebermann - Burchard Test:
It is used to detect the presence of cholesterol.
Procedure:
 By Duaa Mir (Visiting lecturer department of Zoology)

1-Dissolve a few crystals of cholesterol in 2 ml of chloroform in a test tube.

2- Now add 10 drops of acetic anhydride.
3- Add 2-3 drops of conc. sulfuric acid.
4- A positive result is observed when the solution first becomes red then blue and finally bluish-
green in colour.
5) Unsaturation test:
It is used To indicates the degree of unsaturation or amount of double bonds in the lipid sample.
Procedure:
1-Four different types of lipid compounds including mustard oil, coconut oil and olive oil will be
used in this test.
2- Equally into 3 flasks add 10ml of chloroform and after that ad 10 drops of Hubl’s iodine reagent
in each flask.
3-Chloroform shows pink colour due to presence of iodine.
4- To each of the 3 test flasks add the oil sample drop by drop.
5-Shake the flasks vigorously for about 30 seconds after addition of oil until the pink color is
disappeared.
6-Count the number of drops of oils used in each case.
7-Compare unsaturation , it should be remembered that more the number of drops required to
discharge the pink color, the less is the unsaturation.

EXPERIMENT NO 5:
ISOLATION OF OF AMYLASE FROM DIFFERENT SOURCES.
 By Duaa Mir (Visiting lecturer department of Zoology)

Sources: Sweet Potato and Potato.

Reagents required: CaCl2 solution.
Apparatus required:
Beaker
Pestle & Mortar
Measuring cylinder
Centrifuge
Centrifuge tubes.
Procedure:
1. Take 2 g of given source and homogenize using 10ml 10mM CaCl2 solution.
2. Incubate the homogenate for 24 h at 4ºC.
3. Centrifuge the homogenate at 10000 RPM for 20 min.
4. Collect the supernatant and discard the pellets.
5. Measure the volume of supernatant obtained.
Conclusion:
6-Amylase enzyme will be present in supernatant.

EXPERIMENT NO 6:
TO DETERMINE THE SOLUBILITY OF LIPIDS IN ETHANOL.
 By Duaa Mir (Visiting lecturer department of Zoology)

Ethanol emulsion test for fas and oils:

Materials and apparatus:
Test tubes
Ethanol
Deionized water
Procedure:
For solid samples:
1-Crush food sample and place in a dry test tube.
2-Add ethanol to about 2cm3 above level of sample and shake thoroughly.
3-Allow the solid to settle for about 3 minutes to allow the lipid to be extracted.
4-Decant the ethanol into a new test tube.
5-Add 2cm3 of deionized water to the second test tube.
6-Make observations.
For liquid samples:
1-Add a few drops of liquid food sample to a dry test tube.
2-Add 2cm3 ethanol and shake it thoroughly.
3-Add 2cm3 of deionized water.
4- Make observations.
Results:
1-If the solution remains colourless then no emulsion is formed which means that
lipid are not present in sample or not soluble in ehanol.
2-A layer of cloudy suspension forms at the top of solution with tiny globules of
lipid suspended into it is called emulsion. The formation of emulsion shows that
lipids are present in sample and are soluble in ethanol.
EXPERIMENT NO:7
 By Duaa Mir (Visiting lecturer department of Zoology)

TO DETERMINE THE OPTIMUM PH OF AMYLASE AND ITS

EFFEC ON ENZYMATIC ACIVITY.

Procedure:

1. Set up a Bunsen burner, heatproof mat, tripod and gauze.

2. Place a beaker of water on the gauze and adjust the flame to keep the water at
about 35°C.

3. Now put two drops of iodine solution into each spot of a spotting tile.

4. Add 2 cm3 of amylase enzyme solution to a test tube.

5. Place 2 cm3 of starch solution into the same tube.

6. Finally add 1 cm3 of buffer solution to the tube. This will keep the pH constant.

7. Mix the solution in the test tube and place it into the beaker of water on the
Bunsen burner.

8. Use a pipette to remove a few drops of solution every 20 seconds from the test
tube and put them into a different well of the spotting tile.

9. Repeat until the iodine solution stops turning black.

10. Record the time this takes.

11. Repeat with different pH solutions.

 By Duaa Mir (Visiting lecturer department of Zoology)

Results

pH of solution Time taken before iodine did not change colour (s)

5 240

6 120

7 60

8 140

Conclusions

The enzyme amylase breaks down starch into glucose. If the enzyme is working effectively, this
will happen quickly. At pH 7 it took the shortest time before the iodine no longer changed colour.
This shows that the starch was broken down more quickly at this pH. The optimum pH for amylase
is therefore pH 7.

EXPERIMENT NO 8:
 By Duaa Mir (Visiting lecturer department of Zoology)

TO STUDY THE RATE OF ENZYME REACTIONS AT

DIFFERENT TEMPERATURES.
Apparatus
1. 2 electric waterbaths
2. 2 test tubes and racks
3. Thermometer
4. lipase solution
5. milk
6. pH meter

Procedure:
1. Set up two waterbaths, one at 30ºC and the other at 60ºC.
2. Place a beaker of lipase in each waterbath to allow them to come to temperature.
3. Add 5cm3 of milk to each test tube.
4. Add 1cm3 of lipase at 30ºC to test tube 1.
5. Add 1cm3 of lipase at 60ºC to test tube 2.
6. Place each test tube in the correct water bath.
7. Record the pH of each test tube every 10 minutes.

Conclusion
The pH gets lower as the fat in the milk is broken down to fatty acids (and glycerol). If the solution
reaches a low (acidic) pH quickly then the lipase is working to speed up the breakdown of fat.