Biochemistry Practical Note Book

First semester, Pharm.D

KUST, Kohat

Practical No.1

Titration of a strong acid with a strong base

Materials: Hydrochloric acid (0.1 N), Potassium hydroxide (0.1N), burette, beakers, pH meter

Procedure: Pipette 20 ml of 0.1 N HCl into a 100 ml beaker and measure the pH. Titrate this
solution with 0.1 N KOH and measure the ph after addition of 1ml aliquots of alkali. Nearing the
end point, reduce the increments of alkali added to give a reasonable change in ph.

Results/Calculations:

1. Calculate the theoretical ph:

I. of the initial solution

II. after addition of 10 ml of 0.1 N KOH

III. after addition of 19.9 ml of 0.1 N KOH

IV. after addition of 20.1 ml of 0.1 N KOH

2. Plot the titration curve by using your values and compare it with that calculated from

theory.

Practical No. 2

Determination of pKa

Materials:

I Solutions of the following acids (1.49g/l)

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 Acids pKa

Acetic acid 4.8

Benzoic acid 4.2

Lactic acid 3.9

p-Nitro phenol 7.1

II Potassium hydroxide (0.02M)

III Phenolphthalein (0.25% in ethanol).

Reagent bottles, dropping bottle, conical flask, pipette, burette

Procedure: Pipette out 10 ml of unknown solution into a 25 ml conical flask, add five drops of
phenolphthalein indicator and titrate it with 0.02 N alkali until pink colour is obtained. Carefully
note down the volume of alkali used. Then again pipette out 10 ml of same unknown solution
into 25 ml conical flask and add half the amount needed for complete neutralization of the acid
and measure the ph of the solution. This is the pKa of the acid.

Results: With the help of data, identify the acid as far as possible using the list given under
materials.

Practical No. 3

Prepare 50 ml 0.04 M Phosphate Buffer, pH 7.4, using Potassium Dihydrogen Phosphate

(KH2PO4, Mol Wt 136) and Disodium Hydrogen Phosphate (K2HPO4, Mol Wt 174.18). The
pKa of acid is 6.9.

Materials:

Disodium hydrogen phosphate and Potassium dihydrogen phosphate

Weighing paper, spatula, beaker 100 ml, cylinder 100 ml and glass rod

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Procedure: Weigh required amount of salt and acid; dissolve both separately in distilled water.
Mix the two, make up to given volume and check the pH. This would be the exact required pH.

Question:

i. How the buffer system works?

ii. What do you understand by buffering capacity, pH and pKa?

Practical No. 4

Prepare 100 ml Tris-HCl Buffer, pH 7.2. The Molecular Weight of Tris is 121.

Materials: Tris (hydroxyl methyl amino methane), HCl, beaker 100 ml, cylinder 50 ml 100 ml,
pipette 1 ml 2 ml. glass rod.

Procedure: Weigh out the required amount of Tris and dissolve in distilled water (approx 75 ml)
and also prepare 1 N HCl (approx 2.5 ml). First check the pH of Tris solution and then add 1 N
HCl drop wise until the pH of buffer reaches up to 7.2. Then raise the volume with distilled
water up to the required amount.

Question:

Prepare a list of ten important buffers used in biochemical experiments, mentioning the acid and
base used, pKa and effective pH range.

Practical No. 5

Tests for detection of Proteins.

Biuret Test:

Materials: 10% NaOH, 0.5% CuSO4, and 0.1% unknown samples, pipette 2 ml, reagent bottle
25 ml test tubes, test tube holders, test tube stands

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Procedure: Take 2 ml of unknown sample in a test tube. Add 2 ml of 10 % NaOH. Then add drop
wise 0.5% CuSO4 solution till violet colour appears, indicating the presence of peptide bond or
protein.

Xanthoprotic Test:

Materials: Concentrated HNO3 and NH4OH, test tubes, test tube holders, test tube stands,
pipettes 2 ml 5 ml, reagent bottle 25 ml, spirit lamp

Procedure: Take 2 ml of concentrated HNO3 in a test tube. To it add 4 ml of unknown solution.

White precipitate will be formed. Heat it gently till the precipitate disappeares and yellow colour
appears. Add 2 ml of NH3 solution. At the junction of the two layers, mustard ring will appear.

Ninhydrin Test:

Materials: Ninhydrin (0.2% freshly prepared), test tubes, test tube holders, test tube stands,
pipettes 2 ml 5 ml, reagent bottle 25 ml, spirit lamp

Procedure: Take 2 ml of unknown solution in a test tube. Add 4 drops of ninhydrin to it. Boil it
for one minute. Appearance of blue colour indicates the presence of amino acids while yellow
colour indicates the presence of imino acids.

Practical No. 6

Tests for detection of Proteins:

Million-Nasse`s Test:

Materials: Mercuric sulfate (15% solution of mercuric sulfate in 6N sulphuric acid), Sodium
nitrite (1% solution in water, freshly prepared), test tubes, test tube holders, test tube stands,
pipettes 2 ml 5 ml, reagent bottle 25 ml, spirit lamp

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Procedure: Take 3 ml of unknown solution in a test tube. Add 1 ml of mercuric sulfate solution
to it. Mix and boil for 2 minutes. Cool and add 1 ml of sodium nitrite. Warm gently. A red colour
indicates the presence of tyrosine.

Aldehyde Test:

Materials: Dilute formalin (40% formalin in 500 ml distilled water), mercuric sulphate solution
(same as Million Test) and concentrated sulphuric acid, test tubes, test tube holders, test tube
stands, pipettes 2 ml 5 ml, reagent bottle 25 ml, spirit lamp

Procedure: Take 3 ml of unknown solution in a test tube. Add 1 drop of dilute formalin. Mix and
add 1 ml of mercuric sulphate solution. Mix again. Incline the test tube and add 3-4 ml of
concentrated sulphuric acid along the wall of the test tube. Make the test tube upright and rotate
between palms. A radish violet colour at the junction of the two fluids indicates the presence of
tryptophan.

Sakaguchi Test:

Materials: Alpha-naphthol solution 5% in alcohol (freshly prepared), sodium hydroxide 10%

solution and sodium hypobromite/hypochlorite (Add 10 ml of bromine to 100 ml of 40% sodium
hydroxide with cooling and constant shaking), test tubes, test tube holders, test tube stands,
pipettes 2 ml 5 ml, reagent bottle 25 ml

Procedure: Take 3 ml of unknown sample in test tube. Add 2 drops of alpha-naphthol solution
and 2 ml of NaOH solution. Mix and add 2 drops of sodium hypobromite/hypochlorite. A red
colour indicates the presence of arginine.

Practical No. 7

Tests for detection of Carbohydrates:

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Molish Test:

Materials: Molish reagent (5% naphthol in alcohol, freshly prepared), conc. H2SO4, reagent
bottle, dropping bottle, test tubes, test tube holders, test tube stands, pipettes 2 ml 5 ml, reagent
bottle 25 ml

Procedure: Take 3 ml of given unknown solution one by one in the test tubes. Add a few drops of
Molish reagent in it, mix it well by shaking. Then add very carefully drop by drop approximately
3 ml of conc.H2SO4 along the side of the test tube. Purple ring appears at the junction of two
layers. This is the indication of presence of carbohydrate in unknown solution.

Iodine Test:

Materials: I2 solution (0.05 N I2 in 3% KI), conc. H2SO4, 10% NaOH, test tube stands, pipettes 2
ml 5 ml, reagent bottle 25 ml, dropping bottles.

Procedure: Take 2 ml of unknown solution one by one in the test tubes, add a drop of I2 solution.
Colour will appear at once. Blue colour means starch present, red represents dextrin while purple
colour is the indication of glycogen.

Barfords Test:

Materials: Barfords reagent (13.3 g Cu-acetate in 200 ml of distilled water and add 1.8 ml
glacial acetic acid), test tube stands, pipettes 2 ml 5 ml, reagent bottle 25 ml

Procedure: Take 3 ml of unknown solution, add 2 ml Barfords reagent and place the test tube in
water bath and heat it for 3-5 minutes. Red precipitate appears at the bottom of the tube,
indicates the presence of monosaccharide reducing sugars (glucose, galactose, fructose or
mannose). If no precipitate appears then reducing monosaccharides are absent. Now take 3 ml of
unknown solution in another test tube, add 2 ml of Barfords reagent and heat the tube for 7-12
minutes. Red colour precipitate appearance indicates the presence of reducing disaccharides

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(maltose/lactose). If no appearance of precipitate, this means presence of non-reducing
disaccharide (sucrose).

Practical No. 8

Tests for Detection of Carbohydrates:

Benedict Test:

Materials: Benedict reagent (34.6 g sodium citrate and 20 g sodium carbonate dissolved in 170
ml distilled water; 3.46 g CuSO4 separately dissolved in 20 ml distilled water. Mix the two and
make up to 200 ml. Test tube stands, pipettes 2 ml 5 ml, reagent bottle 25 ml, spirit lamp

Procedure: Take 5 ml of Benedict reagent and add 8 drops of unknown solution. Boil it for one
minute. Colour produced may be yellow, green, red or brisk red depending upon the quantity of
sugar present in the unknown solution.

Green colour indicates 0.1%

Yellow 0.5%

Red 1.0%

Brisk red 1.5%

Fehling Test:

Materials: Fehling solution No 1 (7% CuSO4 in distilled water), Fehling solution No 2 (25%
NaOH solution containing 35% K-Na tartarate), test tube stands, pipettes 2 ml 5 ml, reagent
bottle 25 ml, spirit lamp

Procedure: Take 1 ml Fehling solution 1, add 1 ml Fehling solution 2, mix and boil it for two
minutes. On heating there will be no change in the blue colour of solution. Add 1 ml of unknown

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solution and boil it for 1 minute. Red precipitate will appear and this is the indication of presence
of reducing sugar.

Silver Mirror Test:

Materials: AgNO3 (1%), NaOH (10%), liquid NH3, test tube stands, pipettes 2 ml 5 ml, reagent
bottle 25 ml, water bath

Procedure: Take 5 ml of AgNO3 in a test tube and add drop wise 10% NaOH till the precipitate
is formed. Now add liquid NH3 drop by drop till the precipitate dissolves. This solution is called
Tollens reagent. Add 1 ml of unknown solution and keep the test tube in hot water bath for 10
minutes. After completion of time silver mirror is deposited on the sides of the test tube indicated
the presence of reducing sugar.

Practical No. 9

Tests for detection of carbohydrates.

Seliwanoffs Test:

Seliwanoffs reagent (0.05 g resorcinol, 33 ml conc. HCl and distilled water to make 100 ml, so
the final concentration of HCl is about 12% as the strength of conc. HCl is 36%). test tube
stands, pipettes 2 ml 5 ml, reagent bottle 25 ml , spirit lamp.

Procedure: Take 3 ml of Seliwanoffs reagent and add 1 ml of the unknown solution in a test tube.
Boil for 30 seconds. The appearance of a red or pink colour indicates the presence of a
ketohexose.

Osazone Test:

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Materials: Osazone mixture (thoroughly mixed one part of phenylhydrazine chloride and two
parts of sodium acetate by weight), glacial acetic acid, test tube stands, pipettes 2 ml 5 ml,
reagent bottle 25 ml, dropping bottle, glass slide, cover slip, water bath, microscope.

Procedure: Take 5 ml of each given sugar solutions in corresponding labeled test tubes, add 0.3 g
of osazone mixture and 5 drops of glacial acetic acid into each tube. Warm gently to dissolve the
solids. Keep all the test tubes in a boiling water bath. Yellow crystals will appear in the tubes
containing glucose and fructose within 5 minutes. Remove these tubes from the water bath and
allow them to cool. Remove tubes after 30-45 minutes and let them cool. With the help of a glass
rod, take out some crystals on a glass slide, cover them with cover slip and observe under a
microscope.

Practical No. 10

Tests for Detection of Glycerol

Dunstans Test:

Materials: Borax solution (0.5% sodium borate in distilled water), glycerol solution (20%),
phenolphthalein indicator, test tube stands, pipettes 2 ml 5 ml, reagent bottle 25 ml

Procedure: Take 3 ml of borax solution in a test tube. Add a drop of phenolphthalein indicator. A
pink colour is produced indicating that the medium is alkaline. Add glycerol solution drop by
drop until pink colour disappears indicating that the medium has become acidic. Heat the
solution until the pink colour reappears indicating that the medium has become alkaline again.

Acrolein Test:

Materials: Potassium hydrogen sulfate, test tube stands, pipettes 2 ml 5 ml, reagent bottle 25 ml,
spirit lamp

Procedure: When glycerol is heated with potassium hydrogen sulphate, glycerol is dehydrated to
form an unsaturated aldehyde, acrolein that has got a characteristic pungent odour.

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