SESSION: 2023-2024

A laboratory Manual for

Biochemistry

(BP- 209P)

PREPARED BY-
MS. KAJAL CHAUDHARY

Name of Student …………………………………………………………………………….

Class …………………………………………………………..…. Batch …………….…….
Roll No. ……………………………………..
 Certificate
This is to certify that Mr./Ms………………………………………... Roll No
……………………………….of first year B. Pharm has completed the laboratory
work in Biochemistry (BP- 209P) for the academic year 2023- 2024.

C and scored Marks…………

He/She has attended the ………. experiments

(Subject Teacher) (Principal)

(External Examiner) (Seal of Institute)

 Instructions to Students
1. Students should note the following essential requirements for pharmaceutical

Analysis Practical.

Lab manual, observation note book, laboratory Apron, butter paper, permanent

marker, soap, etc.

2. Read the write up of each experiment to be performed a day in advance.

3. Organize the work and arrange the experiment by procuring required apparatus and

chemicals.

4. Follow time management and complete all experiment in time.

5. Understand the purpose of experiment and its practical implications.

6. Students may record the initial observations in rough notebook. However all

observations shall be recorded in the manual and obtain initials of the teacher. Finally

after calculations and conclusion the student shall obtain the signature of the teacher.

7. Students should maintain discipline in the laboratory.

8. Students should handle hazardous chemicals carefully.

9. Students should wash their hands thoroughly with soap, before leaving laboratory.
 List of Experiments Session (2023-2024)

S.NO EXPERIMENT

1.
To prepare the various standard buffer solution and measure the pH.
2. To perform the qualitative analysis for unknown sample of carbohydrates.

3. To determine quantity of glucose in urine sample.

4. To study the effect of different temperatures on the activity of salivary amylase

on starch.

5. To study the effect of substrate concentration on salivary amylase activity.

6. To study the enzymatic hydrolysis of starch.

7. To study the determination of salivary amylase activity.

8. To perform qualitative analysis for known sample (s) of lipids.[b]

9.
To perform qualitative analysis for known sample (s) of amino acids.

10.
To perform qualitative analysis for Proteins.

11.
To perform the qualitative analysis of urine sample for normal constituents.

12. To perform the qualitative analysis of urine sample for Abnormal constituents.
13. To perform the quantitative test for proteins by biuret reagent
14. To perform quantitative determination of cholesterol in blood sample.

15. To perform the separation of amino acids by Thin layer Chromatography.

 EXPERIMENT-1

Aim - To prepare standard buffer solution of citrate, phosphate and carbonate and measure its
pH.

Reference: Indian pharmacopoeia [noted by students]

Requirements:

Apparatus required: Volumetric flask, Pipette, glass rod

Chemical required: Sodium carbonate, sodium bicarbonate, citric aid, sodium dihydrogen
phosphate, Dibasic sodium phosphate, sodium citrate

Principle:
The pH meter measures at electrical potential developed by pair of electrode pins in a
solution. For measurement of pH, an electrode system sensitive to change in H+ ion
concentration of solution is taken. The electrode system consists of sequence of electrode
whose potential raise with pH (H+ concentration of the solution).
A buffer's main purpose is to control the solution's pH. Buffers can also play secondary roles
in a system, such as controlling ionic strength or species solving, perhaps even affecting the
structure or activity of protein or nucleic acid. Nucleic acids, nucleic acid-protein complexes,
proteins, and biochemical reactions are stabilized by buffers (whose products might be used
in subsequent biochemical reactions). Complex buffer systems in electrophoretic systems are
used to control the pH and to establish the pH gradient. Weak acids and bases are made up of
buffer solutions that make them comparatively resistant to pH change. Theoretically, buffers
offer a ready source of both acid and base to either supply additional H+ if the process
consumes H+ or if a reaction produces acid, combine it with excess H+.

Citrate buffer:
Reagent s required:

 Citric acid: Dissolve 2.101 gm of citric acid in 100ml distilled water.

 Sodium citrate solution (0.1 M): Dissolved 2.941gm of sodium citrate in

100ml distilled water.
Procedure:
46.5ml of citric acid with 3.5ml of sodium citrate solution and upto 100ml with
distilled water. It corresponds to 0.1 M citrate buffer and standardised with pH meter
and measures the pH of the prepared solution. This gives citrate buffer at pH 2.5.
Result:
Citrate buffer was prepared and the pH observed was 4.8 which was adjusted to 2.5
using 1N HCl and 5N NaOH.
Carbonate- bicarbonate buffer:
Reagents required:

 Sodium carbonate solution 0.2M: Dissolve 2.12gm of anhydrous sodium

carbonate in 100ml Distilled water.

 Sodium bicarbonate solution: Dissolve 1.68gm of sodium bicarbonate in 100ml of

distilled water.
Procedure:
Pipette out exactly 27.5ml of sodium carbonate (Na 2CO3) solution. To this add 22.5ml of
sodium bicarbonate solution and made up to 100ml with distilled water which corresponds to
0.2 M sodium carbonate and bicarbonate buffer.
Standardize pH meter and measure the pH of required buffer. This gives the Carbonate-
bicarbonate buffer pH 10.2.
Result:
Carbonate bicarbonate buffer was prepared and pH observed was 7.5 which was adjusted to
10.2 using 1N HCl and 5N NaOH.

Phosphate buffer:
Reagents required:

 Monobasic sodium dihydrogen phosphate: Dissolve 2.78gm of sodium

dihydrogen phosphate in 100ml of distilled water.

 Dibasic sodium phosphate (0.2M): Dissolve 5.3gm of disodium hydrogen

phosphate or 7.17 gm sodium hydrogen phosphate in 100ml distilled water.

Procedure:
39 ml of dihydrogen sodium phosphate is mixed with 61 ml of disodium hydrogen
phosphate This made up to 200ml with distilled water .This gives phosphate (PO 4)2- buffer of
0.2M.
Standardized pH meter with standard buffer. Washed electrode with distilled water and
introduced it into phosphate buffer prepared. The pH of the solution is 6.8.

Potassium phosphate buffer:

Reagents required:

 Dipotassium hydrogen phosphate

 Potassium dihydrogen phosphate

Procedure:
174.18 g/mol dipotassium hydrogen phosphate and 136.09 g/mol potassium dihydrogen
phosphate was taken and made up to 200ml using distilled water. This gives the potassium
buffer.
Standardized pH meter with standard buffer. Washed electrode with distilled water
and introduced it into potassium buffer prepared. The pH of the solution is 6.5.

Result:
Dipotassium hydrogen phosphate (K2HPO4) and potassium dihydrogen phosphate (KH2PO4)
solution were prepared and the pH was measured to be …………….respectively, the solution
were made using 1N HCl and 5N NaOH respectively and the pH was found to be
……………
 EXPERIMENT-2

Aim- To perform the qualitative analysis for unknown sample of carbohydrates

Reference- Kaushik GG, Sharma Neha, Dabeer Sabira, Jindal Ruchi “A Practical book of
biochemistry” CBS Publishers & Distributors Pvt Ltd; First edition 2020: Page no. 3.

Requirements: -

Chemicals: - Molisch’s reagent, Iodine reagent, Benedict’s Reagent, Selivanoff’s Reagent,

Barfoed’s Reagent, fructose, Conc. H2SO4

Apparatus: - Test Tube, Test Tube Stand, Boiling Tube, Test Tube Holder, Beaker,
Measuring Cylinder, Filter Paper, heating mantle, pipette, glass rod

Theory:-

 Carbohydrates are aldehyde or ketone derivatives of polyhydroxy alcohols

 Their main function is to provide energy
 Glucose is the main form of carbohydrate absorb from the gut
 Human can synthesize glucose from non- carbohydrate sources like lactate,
glucogenic amino acids, propionic acid, glycerol, pyruvate by a process known as
gluconeogenesis.
 Oligosaccharides: made up of less than ten monosaccharide subunits.

Classification of carbohydrates:-

Carbohydrates
Monosaccharide Disaccharides Polysaccharides Oligosaccharides

Cannot be Made up of two Made up of more than Made up of less

hydrolyzed monosaccharide ten monosaccharide than ten
further based on units , e.g subunits monosaccharide
the number of subunits
carbon atoms Sucrose= Homopolysaccharides
further divided Glucose+fructose Made up of one type of
into: trioses, monomeric subunits,
tetroses, pentoses, Lactose= e.g cellulose is a
hexoses and polymer of glucose
Glucose+Galactose
heptoses, glucose,
fructose, galactose Maltose= Heteropolysaccharides
are biologically
Glucose+glucose Made up of different
important
types of monomeric
monosaccharides
 in human subunits

e.g glycosaminoglycans

Procedure: -

(1) Molisch’s test: - This is the general reaction for all carbohydrates. To a 3 ml of sugar
solution add 4-5 drops of Molisch’s reagent followed by 3 drops of conc. H 2SO4 along
 the sides of tube. Formation of violet ring at the junction of the two liquids indicates the
presence of carbohydrate.

(2) Iodine test: - Take 2 ml of sugar solution add 2 drops of iodine solution.Does’not show
change in solution.In case of starch shows positive result.

(3) Benedict’s test: - This test is used for distinguish reducing and non reducing sugar.

To 5 ml of Benedict’s reagent in a test tube, add 5ml of sugar solution and boil for few
minutes. A light yellow coloured precipitate is formed indicating the presence of reducing
sugars.

(4) Barfoed’s test: - This test is used for distinguish reducing monosaccharide or
disaccharides.

To 1 ml of sugar solution, add 2 ml of Barfoed’s reagent and boil the contents. Formation
of a red colour solution within two minutes indicates the presence of monosaccharide.

(5) Selivanoff’s test: - This test is useful distinguish fructose and glucose.

To 3 ml of sugar solution, add 10 drops of Selivanoff’s reagent and heat the contents. red
cherry coloured solution is obtained if fructose is present.

(7) Solubility test:-Take few gm of sample and add distilled water in it.If the compound is
soluble shows Monosaccharide/ Disaccharide present and insoluble shows
Polysaccharides present.

Result- The following test shows the presence of fructose in the given unknown sample.
 Experiment No. -3

Aim:- To determine quantity of glucose in urine sample.

Reference:- Lovhare B. Rahul, Usman Md. Rageeb, Ahirrao, Pawar D. Yogesh, “ A Practical
book of biochemistry and clinical pathology” Published by S.Vikas & Company (Medical
publishers); Edition 2022: Page No.- 111.

Requirement:-

Chemical requirements :- Copper sulphate, sodium bicarbonate, sodium or potassium

citrate, potassium thiocyanate, potassium ferrocyanide, sodium carbonate.

Glassware:- Conical flask , burette, volumetric pipette, porcelain pieces etc.

Theory:-

Benedicts solution which is copper sulphate, in alkaline solution is reduced by glucose. The
benedicts quantitative reagent consist of copper sulphate, potassium thiocyanate and other
chemical in alkaline media. In these copper sulphate is reduced to the cuprous oxide by
glucose. The potassium thiocyanate reacts with cuprous oxide and forms white ppt of cuprous
thiocyanate instead of usual red precipitate of cuprous oxide. The disappearance of blue color
from solution indicate complete reduction of copper sulphate.

2 CU(OH)2 CU₂O + 2H₂O

Preparation of Benedicts qualitative reagent

Weigh copper sulphate 18 gm, sodium bicarbonate 200gm, sodium or potassium citrate
200gm, potassium thiocyanate 125gm, potassium ferrocyanide (5%) 5 ml with the aid of heat
to dissolve carbonate, citrate and thiocyanate in enough water to make about 800ml of the
mixture.

Procedure:-

1) In 100 ml of conical flask, pipette out 10 ml of benedicts quantitative reagent. Then add 2-
3 gm of anhydrous sodium carbonate a pinch of pumice powder or porcelain pieces is
incorporated to avoid bumping of solution and add 15 ml of distilled water. To adding a
distilled water for prevent the loss of water due to evaporation process.

2) Place the conical flask on the burner and boil the contents. Once the solution starts boiling
add

urine solution from the burette drop by drop till the blue color disappears. The color changes

from blue to colorless which marks the end point of the titration and finally not the volume of
urine consumed.

Observation table:-

S.No Initial reading of burette Final reading of burette Volume of urine

. consumed

01

02

03

Calculation:-

10 ml of benedicts reagent = Reduced by 20 mg of glucose

10 ml of benedicts reagent = 0.02gm of glucose

Now it means 'x' ml diluted sample of urine = 0.02 glucose I'x' - burette reading which
reduces 10 ml of benedicts reagent)

100 x 0.02

100 ml diluted sample of urine = --------------

X ml

Percentage of glucose in diluted urine = Y%

now calculate gm% glucose in given sample of urine(undiluted)

Percentage of glucose in given urine sample = % glucose in diluted urine (Y%) x Dilution
factor Percentage of glucose in given urine sample = Y % x dilution factor

= Z% glucose

Normal Value:-

Normal value of glucose excretion in urine is 2 to 10 mg glucose/100ml or 78.5 mg/day.

Result :-

1. Quantity of glucose in given sample of urine is ………… . g/100ml i.e……… g%

2. As glucose content of given sample of urine is ……….[less/more ] than normal value

Experiment-4

Aim: To study the effect of different temperatures on the activity of salivary amylase.

Reference:- Devhare L.D, Hiradeve M. Sachin, Chilate Vikrant, Jondhale D.S. “ A Practical
book of biochemistry and clinical pathology” Published by Global education limited; First
edition 2019: Page No.-139.

Requirement:-

Chemicals:- lodine solution, 1% ,Starch sol

Glassware:- Beaker, Test Tube, Test Tube Holder, Measuring Cylinder, Bunsen burner,
Test tube Stand, Tripod stand, Water Bath, Ice cubes, Butter Paper, Dropper, Match Box

Theory:
Salivary amylase, formerly known as ptyalinis, a glucose-polymer cleavage enzyme that is
produced by the salivary glands, breaks down starch into maltose and isomaltose. Amylase,
like other enzymes, works as a catalyst. All catalysts are enzymes, but not all enzymes are
catalysts. It comprises a small portion of the total amylase excreted, which is mostly made by
the pancreas. Amylases digest starch into smaller molecules, ultimately yielding maltose,
which in turn is cleaved into two glucose molecules by maltase. Starch comprises a
significant portion of the typical human diet for most nationalities. Given that salivary
amylase is such a small portion of total amylase, it is unclear why it exists and whether it
conveys an evolutionary advantage when ingesting starch.

Procedure :-
1) Take beaker containing 10 ml of 1% starch solution.
2) Than perform the identification test of starch by adding the few drops of iodine solution
in it and observe the blackish blue color as positive test of starch presence.
3) Than, take two test tube add 10ml of starch solution,3ml of saliva and few drops of
iodine solution in both the test tube.
4) Mark the test tube A,B.
5) A test tube will be kept at room temperature at 37 C and observe the color change after
30 mint.
6) Similarly,take the test tube B and heat it on water bath for few mint than observe the color
change.

Result:- It takes less time to reach achromic point at 37°C, as the enzyme is maximum active
at this temperature, while at higher temperatures more time is taken to reach the achromic
point.

Conclusion
All enzymes are proteinaceous in nature at higher temperatures, the enzyme is denaturated.
Therefore, more time will be taken by enzyme to digest the starch at lower and higher
temperatures. At 37° C, the enzyme most active, hence, takes less time to digest the starch.
Experiment-5

Aim-To study the effect of substrate concentration on salivary amylase activity.

Reference- Sadasivam S, Manickam A (2008). Biochemical Methods, New Age International

Publishers, 3rd Edition, ISBN: 978-81-224-2140-8

Requirements-

Reagents: Starch solution, Stock enzyme solution, Iodine

Apparatus: Test tube rack, Colorimeter tubes, Four Erlenmeyer flasks, 1 ml pipette,
Calorimeter

Theory:

Salivary amylase, formerly known as ptyalinis, a glucose-polymer cleavage enzyme that is

produced by the salivary glands, breaks down starch into maltose and isomaltose. Amylase,
like other enzymes, works as a catalyst. All catalysts are enzymes, but not all enzymes are
catalysts. It comprises a small portion of the total amylase excreted, which is mostly made by
the pancreas. Amylases digest starch into smaller molecules, ultimately yielding maltose,
which in turn is cleaved into two glucose molecules by maltase. Starch comprises a
significant portion of the typical human diet for most nationalities. Given that salivary
amylase is such a small portion of total amylase, it is unclear why it exists and whether it
conveys an evolutionary advantage when ingesting starch.

Procedure

Arrange a test tube rack having total 4 rows with each row having 10 colorimeter tubes.1 In
each row the first tubes should be labeled as 1:2, 1:4, 1:8 and 1:16 depending on the dilutions
of the starch. Now take Erlenmeyer flask and label it depending on the respective dilutions
and then to each flask add 50ml of distilled water. Now to each flask add 50 ml of starch
solution. Mix it properly and from this transfer 50 ml of the mixed solution to the second
flask and then again mix it properly. Repeat same with the third and fourth flask. Then from
fourth flask discard 50 ml of the solution. Now tale 1 ml pipette and transfer 0.2 ml of the
enzyme solution to all flask. Mix it properly and transfer 3 ml of the solution from each flask
to colorimeter tube. To each tube add 3 drops of iodine and allow it to mix properly. Then
place the tubes in the colorimeter for measuring absorbance at 540 nm. Calculate the
transmittance percent. Do this for every flask with 2 minute gap.

Result- The effect of substrate concentration on salivary amylase activity was performed
successfully.
 Experiment -6

Aim: To study the enzymatic hydrolysis of starch.

Reference- Devhare L.D, Hiradeve M. Sachin, Chilate Vikrant, Jondhale D.S. “ A Practical
book of biochemistry and clinical pathology” Published by Global education limited; First
edition 2019: Page No.-142

Requirements-

Chemical required- Starch solution – 1%, Phosphate buffer solution, Saliva , Iodine solution

Apparatus required- Water bath, Watch glass, Test tubes

Theory-

Enzymes are proteins that act as catalysts for metabolic reactions. They increase the rate of
the reaction, but do not influence the kind or amount of products formed. In general, each
metabolic reaction has to be catalyzed in the living organism by its own special enzyme.

Starch

The nutritional reservoir in plants is starch, which is actually a mixture of two

polysaccharides. Amylose, the unbranched type of starch, consists of glucose residues in -
l,4 linkage.

Amylases

Hydrolysis of starch is the process of digestion. Enzymes called amylases catalyze only the
hydrolysis of -1,4 glycosidic linkages in the amylose and amylopectin components of
starch. The hydrolysis does not proceed directly from polysaccharides to monomer units;
rather, partial hydrolysis products of intermediate size are obtained. These products are
maltose and dextrin. Maltose consists of two glucose units in -1,4 linkage; dextrin is made
up of several glucose units joined by -1,6 linkage in addition to -1,4 linkages. Amylase
enzymes are present in saliva, so the digestion of carbohydrates begins in the mouth.
Digestion continues briefly in the stomach until the pH drops too low, and then is completed
in the intestines by the attack of another amylase.

Procedure-

Effect of Enzyme Concentration

1. Collect approximately 5 mL of saliva in a medium test tube.

2. Place 2 droppers full of 1 % starch solution in each of 5 medium test tubes. Number the
test tubes 1-5.
3. Place the tubes in a 37-40 °C water bath. After 5 minutes, add the following amounts of
saliva to the test tubes as quickly as possible, mixing each solution thoroughly, and then
returning the tubes to the water bath. Do not overheat the bath or you will inactivate the
enzyme.

4.Prepare a spot plate for testing for the presence of starch in the samples by placing one drop
of iodine reagent in each of five depressions on the spot plate.

5. Two minutes after the addition of saliva, transfer one drop from each test tube (using a
different pippet for each tube) to a separate drop of iodine in the spot plate. Note the color
produced for each. Remember that the complex formed by starch and iodine is an indigo
blue. If the color of the iodine solution remains red or gold after adding the starch solution,
the starch has been completely hydrolyzed.

6. As soon as one of your starch solutions has hydrolyzed, use it to begin preparing the
solutions for Parts B and C below. Continue to test the remaining starch solutions that have
not yet hydrolyzed.

7. Clean the spot plate and then prepare it for the next testing by placing one drop of iodine
reagent in each of five depressions.

8. Repeat the testing at 5 minutes after the addition of saliva, and at 5 minute intervals there
after. Continue testing for 20 minutes, or until the blue-black color no longer appears for each
sample.

9. Record the time required for the hydrolysis of starch in each sample.

Result-
 Experiment-7

Aim: To study the determination of salivary amylase activity.

Reference- Devhare L.D, Hiradeve M. Sachin, Chilate Vikrant, Jondhale D.S. “ A Practical
book of biochemistry and clinical pathology” Published by Global education limited; First
edition 2019: Page No.-150

Requirements-

Chemical required- Toluene, starch paste, 0,02N iodine solution

Apparatus required- Beakers (100ml), pipettes, test tube, glass-stoppered bottles, plain
cavity slides, Staining tube, cavity blocks

Theory-

Salivary amylase, formerly known as ptyalinis, a glucose-polymer cleavage enzyme that is

produced by the salivary glands, breaks down starch into maltose and isomaltose. Amylase,
like other enzymes, works as a catalyst. All catalysts are enzymes, but not all enzymes are
catalysts. It comprises a small portion of the total amylase excreted, which is mostly made by
the pancreas. Amylases digest starch into smaller molecules, ultimately yielding maltose,
which in turn is cleaved into two glucose molecules by maltase. Starch comprises a
significant portion of the typical human diet for most nationalities. Given that salivary
amylase is such a small portion of total amylase, it is unclear why it exists and whether it
conveys an evolutionary advantage when ingesting starch.

Salivary amylase acts at a temperature of 37◦ C and at pH of 6.6 (acidic). When iodine
solution is mixed with starch, blue colour is obtained. When starch is first hydrolyzed with
amylase and then mixed with iodine solution, blue colour is not obtained because starch has
been broken into glucose and maltose.

Procedure-

Prepare 0.02N iodine solution and 1% starch solution (1 gm starch + 100 ml DW).

For collection of saliva, rinse own mouth with warm water quickly. Then take 20-25 ml of
water in mouth, rotate water with tongue for 2-3 min and collect saliva solution in a beaker.
This contains salivary enzyme amylase

In another beaker mix 5 ml of 1% starch solution and 5 ml of saliva solution. Incubate the
beaker in an oven set at 37◦C for one hour.
Take two cavity blocks or staining tube and mark them A and B respectively.

In each cavity block or staining tube, add 2 drop of iodine solution,

In cavity block A add drop of starch-saliva mixed solution with pipette.

With another pipette add a drop of starch only in cavity block B.

Result:-

In cavity block B, solution become blue white in cavity block A it remains colourless as
starch has been hydrolyzed into glucose and demonstrating activity of enzyme amylase.
 Experiment-8

Aim:- To perform qualitative analysis for known sample (s) of lipids.

Reference:- Kaushik GG, Sharma Neha, Dabeer Sabira, Jindal Ruchi “ A Practical book of
biochemistry” CBS Publishers & Distributors Pvt Ltd; First edition 2020: Page no. 12.

Requirement:-

Chemical Requirement:- Ether, chloroform, benzene, CCl4, CaCl2, Na2CO3, Bile salt
solution, oil, Borax solution, phenolphthalein indicator, glycerol, KHSO 4, Conc. H2SO4,
Cholestrol, Acetic anhydride, Iodine reagent , Mercuric chloride, ethanol, CHCl 3, Hubl’s
reagent.

Glassware:- Test tube, dropper, measuring cylinder, beaker, pipette, etc.

Theory:-

These organic compounds are nonpolar molecules, which are soluble only in nonpolar
solvents and insoluble in water because water is a polar molecule. In the human body, these
molecules can be synthesized in the liver and are found in oil, butter, whole milk, cheese,
fried foods and also in some red meats.

Lipids are a family of organic compounds, composed of fats and oils. These molecules yield
high energy and are responsible for different functions within the human body. Listed below
are some important characteristics of Lipids.

1. Lipids are oily or greasy nonpolar molecules, stored in the adipose tissue of the body.
2. Lipids are a heterogeneous group of compounds, mainly composed of hydrocarbon
chains.
3. Lipids are energy-rich organic molecules, which provide energy for different life
processes.
Procedure:

S. No Test Observation Inference

1 Solubility test: To small Completely soluble Indicates

quantities of fat /fatty acid taken in all our solvents a presence of fat or
in a separate test tube add the –d oil in the given
solution
following organic solvents
separately:

1. a) Chloroform,b) ether,c) ii) Indicates

benzene,d) hexane (3 mL/test presence of fat or
tube) oil in the given
solution
Found small floating
2. Distilled water droplets on water.
2 Saponification test: -Procedure: Appearance of Indicates the
Take 5 drop oil and 5 ml of foamy solution presence of oil or fat
KOH in test tube. Keep test
tube in boiling water bath for
15-20 mint and boil it till the
solution become soapy

3 Grease spot test

reagent:- Ether evaporates and Grease nature of
(Ether) leaving behind a lipid
translucent spot on
procedure: Take 3 ml of ether paper
in test tube +

Add 5 drop of oil to it shake

well and put 1-2 drop of solution
on filter paper Wait 5 min

4 Test for glycerol: Release of pungent Indicates Presence

odor of glycerol
Acrolein test: Reagents- solid
KHSO4

Procedure: Take dry test add +

1-2 drop of glycerol
add pinch of KHSO4 heat it
5 Dunstan's test Reagents: Medium is alkaline- Presence of glycerol
(borax solution + pink color disappear.
phenolphthalein indicator) Medium is acidic
than color will
Procedure:- 3 ml of borax reappear
solution in test tube + add drop
of phenolphthalein indicator
pink color produced indicate
medium is alkaline

+ add 20% glycerol drop by

drop until pink color disappears
indicate medium is acidic. Heat
again until pink color reappears
that indicate medium has
become alkaline again

6 Test for cholesterol: Observes formation Indicates presence of

of blue green color cholesterol
Salkowski test: Take 2 mL of at the junction of
cholesterol in a test tube and two liquids
dissolve in 1 mL chloroform to
 this add equal volume of
concentrated sulphuric acid
along the walls of the tube. (Do
not mix the contents).

7 Liebermann Burchard test: Formation of an Confirms the

emerald green color presence of
Take approximately Indicates presence of cholesterol
100 mg of cholesterol cholesterol Confirms
in a test the presence of
tube, dissolve by cholesterol
adding 1 mL of
chloroform add
equal volumes of
acetic anhydride
followed by drop
wise addition
(along walls) of
concentrated
sulphuric acid.

Result: The qualitative analysis for known sample (s) of lipids was done successfully.

Experiment-9

Aim: To perform qualitative analysis for known sample (s) of amino acids.

Reference:- Kaushik GG, Sharma Neha, Dabeer Sabira, Jindal Ruchi “ A Practical book of
biochemistry” CBS Publishers & Distributors Pvt Ltd; First edition 2020: Page no. 19.
Requirement:-

Chemical Requirement:- Amino acid solution,alkali solution,ninhydrin reagent,nitric

acid,sodium hydroxide solution,lead acetate solution,sodium nitroprusside
solution,ammonia,million reagent,sodium nitrite.

Glassware:- Test tube, dropper, measuring cylinder, beaker, pipette, etc.

Theory:-

Amino acids are organic compounds containing the basic amino groups (-NH2) and carboxyl
groups (-COOH). The ingredients present in proteins are amino acids. Both peptides and
proteins are long chains of amino acids. Altogether, there are twenty amino acids, which are
involved in the construction of proteins.

Amino Acids are the organic compounds that combine to form proteins, hence they are
referred to as the building components of proteins. These biomolecules are involved in
several biological and chemical functions in the human body and are the necessary
ingredients for the growth and development of human beings. There are about 300 amino
acids that occur in nature.

General properties of Amino acids

 They have a very high melting and boiling point.

 Amino acids are white crystalline solid substances.

 In taste, few Amino acids are sweet, tasteless, and bitter.

 Most of the amino acids are soluble in water and are insoluble in
organic solvents.
Procedure:

S.No Test Observation Inference

1 Ninhydrin test: Take Appearance of Presence of

2 mL of amino acid purple to blue amino acids
solution in a test color.
tube and add 5-7
drops of ninhydrin
reagent and mix the
contents. Place the
test tube in a boiling
water bath for 5 min.
Later remove and
cool to room
temperature.
2 Xanthoproteic test: After adding the Presence of aromatic
Take 2 mL of amino alkali, the color amino acids
acid solution in a test changes to
tube and 2ml of conc. nitric acid orange.
and mix well. Boil the
contents over a
bunsen flame, for
few minutes. After that cool it
under tap water and than Add
few
drops of alkali(30% NaoH).

3 Lead acetate test: Appearance of a Presence of cysteine

Take 2 mL of amino black precipitate of or cystine
acid solution and add lead sulphide
1ml of sodium
hydroxide (40%) to 2
mL of the amino acid
solution taken in a
test tube and add
few drops of 10%
lead acetate solution., and boil
the contents for 5-10
min over a Bunsenflame. Cool
the contents

4 Sodium Appearance of Presence of cysteine

nitroprusside test: intense purple
Take 2 mL of amino colour
acid solution and add
2ml of sodium
nitroprusside reagent
and mix well and add 0.5 ml of
the concentrated sodium
hydroxide.
mix well and heated.
5 Millon’s test: Take 2 Appearance of Confirms the
mL of amino acid red color presence
solution in a test of tyrosine
tube and add few
drops of Millon’s
reagent and mix well.
Boil the contents
 over a bunsen flame
for 3-5 min. Later
cool the contents
6 Sullivan and Appearance of red or Indicates presence of
McCarthy’s test: green colour methionine
Take 1 mL of amino
acid solution in a test
tube, add few drops
of sodium hydroxide
(5 N), followed by
addition of few drops
of glycine (2%) and
10% sodium
nitroprusside solution
and mix well. Place
the test tube in a hot
water bath, maintained at 40o C,
for 15
minutes. Cool the
tube in ice cold
water for 5 minutes
and add 0.5 mL of 6
N HCl. Mix the
contents and allow to
stand for 15 min at
room temperature.

Result- The qualitative analysis for known sample (s) of amino acids was successfully
performed.

Experiment 10

Aim: To perform qualitative analysis for Proteins.

Reference: Gupta PP, Gupta N. Qualitative Tests for Proteins and Amino Acids. Essentials
of Practical Biochemistry.2017.26-28

Requirements: -

Apparatus required: - Test Tube, Test Tube Holder, boiling test tube, glass rod, test tube
stand, funnel, volumetric flask, pipette, measuring cylinder, burner, electronic balance and
Water Bath

Chemical required: Biuret Reagent, Millon’s Reagent, albumin, Sodium Potassium

Tarterate, CuSO4, Potassium Iodide, NaOH Solution, Ninhydrin Reagent, Acetone, Mercury,
Conc. HNO3, Glacial Acetic Acid, Lead Acetate Solution.

Procedure: -

(1)Biuret Reagent: - Dissolve 45 gm of Sodium potassium tarterate, in 400 ml of 40%

NaOH solution, add 15 gm of CuSO 4 and stir continuously. Add 5 gm of potassium
iodide and make up the volume upto 1 liter with 40% NaOH solution.

Principle: - Proteins are polymers of amino acids (poly peptides). The amino acids of protein
are linked through “Peptide bond” (-CO-NH-). Biuret test is answered by
compounds containing peptide linkage. When protein solution is treated with NaOH
solution and a drop of CuSO4, a pink or violet colour is obtained. This is due to
formation of a coloured co-ordination complex between the Cu 2+ions and the nitrogen
of the peptide bond and the oxygen of water.

Biuret Test: - Take 2 ml of Protein sample and add 2 ml of biuret reagent. Formation
of a purplish violet colour indicates the presence of protein.

(2) Ninhydrin test: - Ninhydrin test is the most sensitive test is the most sensitive test
answered by the proteins. Ninhydrin reagent reacts with the proteins to give blue or
purple coloured complex. The reaction involved in this test are as follows:
hydrolysis
Protein amino acids.
on heating
Amino acid + ninhydrin keto acid + NH3 + CO2 + Hydrindantin

Hydrindantin + NH3 + Ninhydrin Blue or Purple Colour.

Ninhydrin test can be performed as follows:

To few ml of protein solution, add few drops of ninhydrin solution and heat the contents.
Observe the formation of blue or purple coloured solution.

(3) Millon’s Reagent: - Digest 1 part (By weight) mercury with 2 parts (by weight) nitric
acid and dilute the resulting solution with 2 volumes water.
 Principle: - Phenol and phenolic compounds, when mixed with Hg(NO 3)2 in nitric
acid and traces of HNO2, a red colour is produced. Tyrosine present in the sample is
responsible for the test.

Millon’s test: - To 3 ml of the protein solution add few drops of Millon’s reagent. A
red colour is obtained.

(4) Xanthoproteic Test: - Proteins in general, show positive xanthoproteic test.

Treatment of protein solution with conc. HNO 3 produce yellow colour is
Xenthoproteic test. The reason for this is the nitration of benzene ring of Tyrosine and
Tryptophan present in all proteins. The nitrated derivatives obtained are yellow in
colour and changed to orange on addition of alkali.

This can be performed as follows:

To 2 ml of protein solution add 1 ml of conc. HNO 3 heat the solution for about 2
minutes and cool under tap water. A yellow colour is obtained due to nitration of
aromatic ring. Add few drops of 40% w/v NaOH solution, the yellow colour obtained
initially changes to orange.

(5) Lead Acetate Test: - It is known as the test for sulphur containing amino acids.The
sulphur containing amino acids are cysteine, cystine and methionine.

Principle: - Sulphur containing amino acids liberate hydrogen sulphide on boiling with
alkali. Lead acetate forms black precipitate of lead sulphide which is insoluble in
dilute HCl.

S (Protein) + 2 NaOH Na2S

Na2S + (CH3COO)2Pb PbS (black or brown ppt) + 2CH3COONa

Cysteine and cystine respond to this test but the sulphur present in methionine is not
released by the above treatment, casein and gelatin which contain methionine but
practically negligible amounts of cysteine and cystine give negative test. This test is
given by both free and combined sulphur containing amino acids.

Procedure: - Take 2 ml of given protein solution and add 2 ml of 40% NaOH, boil
for about 3-4 minutes. Cool and add 2 ml. of glacial acetic acid followed by 1 ml of
lead acetate solution. Appearance of grey or black precipitate indicates presevce of
sulphur containing amino acids example cysteine, methionine, cystine.

(6) Sakaguchi test:- Take few ml of test solution in a test tube. Add 1-2 drops of 40%
NaOH and 3-4 drops of α- naphthol and 8-10 drops of bromine water and mix. Observe
the formation of red colour. It indicates the presence of arginine.

Confirmatory test for albumin:-

 Heat coagulation test:- Take 5 ml of given sample in a test tube and heat the upper
half of the tube over a flame. Appearance of white coagulum indicates albumin.

Result-
 Experiment-11

Aim-To perform the qualitative analysis of urine sample for normal constituents.

Reference:- Kaushik GG, Sharma Neha, Dabeer Sabira, Jindal Ruchi “ A Practical book of
biochemistry” CBS Publishers & Distributors Pvt Ltd; First edition 2020: Page no. 28.

Requirement:-

Chemical Requirement:- Sodium tungstste, arsenic pentoxide, phosphoric acid, HCl, HNO 3,
Ammonium hypobromite, Ammonium oxalate, anhydrous sodium carbonate, benedict’s
reagent, Acetic acid, HNO3, Ammonium sulfate, Sodium Nitroprusside, Ammonia,
Ammonium sulfate, sulfur, Fouchet’s reagent, P-dimethyl aminobenzaldehyde, sodium
acetate benzidine, H2O2 etc

Glassware:- Test tube, dropper, measuring cylinder, beaker, pipette, etc.

Theory:- Urine is the excretory product of body, formed by kidneys. It is made up of water
and water soluble waste products . examination of urine gives us idea of renal function.

Normal Constituents of Urine :-

1 Water 90-95%

2 Urea 25 - 30 gm/day

3 Creatinine 1 - 1.2 gm/day

4 Uric acid 0.4 – 0.7 gm/day

5 Sodium 3 – 3.5 gm/day

6 Potassium 2 – 2.5 gm/day

7 Chloride 10 - 15 gm/day

8 Calcium 0.1 - 1.3 gm/day

9 Phosphate 0.7 - 1.2 gm/day

10 Sulfate 1 - 1.2 gm/day

11 Ammonia 0.6 – 0.8 gm/day

Physical Examination of Normal Urine

1 Appearance Freshly void urine is clear, it becomes turbid on standing due to
precipitation of phosphates

2 Odor Aromatic odor, which becomes ammonical on standing

3 Color Straw color

4 Volume 1-2 L/day, depends upon the intake of water

5 pH Usually acidic, pH ranges from 4.5 to 8 post meal, pH of urine is alkaline; it

is called alkaline tide

6 Specific 1.010-1.025, measured with the help of urinometer, it tell us about the
gravity concentrating ability of kidneys

Procedure & Observations :-

S.N Test procedure Principle Observation Inference

0

Urea:-

1. Specific urease test Urease in soybean meal Presence of urea

reagents: Soybean meal, acts on urea and liberate
phenol red NH3, which makes the
medium alkaline. In
Procedure:- 4-5 ml of alkaline medium
urine + pinch of soybean phenolphthalein gives
meal + 1-2 drop of phenol red color
indicator mix and wait 10
mins

Sodium hypobromite
2. Effervescenc
test reagents:
Breakdown of urea and e due to
Presence of urea
Alkaline hypobromite release of nitrogen production of
Procedure: 4-5 ml sample produce effervescence nitrogen
+ 2 ml alkaline
hypobromite + mix.
3. Calcium test reagents:-
White ppt of Presence of
Ammonium oxalate
calcium calcium
Procedure: Take 2-3 ml Formation of calcium oxalate

ammonium oxalate + oxalate

 1-2 ml urine

4, Phosphate test reagents: Formation of phospho Canary Presence of

Ammonium molybdate molybdate yellow ppt phosphate
Procedure: Take 1-2 ml
urine + add conc HNO3 +
heat + ammonium
molybdate

5. Creatinine test Jaffe's Formation of creatinine Orange red Presence of

test remagents Alkaline picrate color creatinine
picrate

Procedure 3-4 ml urine +

1 ml

Alkaline picrate

6. Uric acid Benedict's test In alkaline medium, Blue color Presence of uric
reagents Sodiam tungstste arsenophosphotungstate develop acid
anec penioxide, of Benedict's uric acid
phosphoric and HCl regent is reduced to blue
anhydrous sodium colored
carbonate. arsenophosphotungstate:

Procedure: 4ml urine +

pinch of sodium carbonate
+ mix+ 4-5 drop of above
reagent + mix

7 Ammonia test reagents: Ammonia is alkaline in Tip of rod Presence of

Phenolphthalein indicatior phenolphthalein turns pink ammonia
Procedure: 9 ml urine + indicator pink
drop of phenolphthalein
indicatior + drop of 0.1 N
NaOH hold glass rod at
the mouth of tube

Physical Examination of pathological urine:-

 S. Normal Pathological
No
.

Appearance Clear a.Turbid in UTI

Odor Aromatic a. Fruity in diabetic ketoacidosis, starvation due to

presence of ketone bodies

b. Mousy odor in phenylketonuria

c. Foul smell in UTI

Color Straw a. Red in hematuria

color
b. Brown to black in alkaptonuria

c. Brown in hemoglobinuria

Volume 1-2 L/day a. Oliguria <300 ml/day

b. Polyuria >2500 ml/day seen in diabetes mellitus and

diabetes insipidus

c. Anuria <100 ml/day

pH 4.5-8 a. pH decreases in metabolic acidosis

b. Increase in pH is seen in metabolic alkalosis and UTI

Specific 1.010- a. Isothenuria SG is fixed at 1.010 seen in chronic renal

gravity 1.025 failure

b. SG 1.030 hyperosmolar urine, seen in diabetes adrenal

insufficiency and nephrotic syndrome.

c. SG <1.008 hypos molar urine, seen in diabetes

insipidus, compulsive polydipsia

Result:

Experiment-12
Aim: To perform the qualitative analysis of urine sample for Abnormal constituents

Reference:- Kaushik GG, Sharma Neha, Dabeer Sabira, Jindal Ruchi “ A Practical book of
biochemistry” CBS Publishers & Distributors Pvt Ltd; First edition 2020: Page no. 28.

Requirement:-

Chemical Requirement:- Sodium tungstste, arsenic pentoxide, phosphoric acid, HCl, HNO 3,
Ammonium hypobromite, Ammonium oxalate, anhydrous sodium carbonate, benedict’s
reagent, Acetic acid, HNO3, Ammonium sulfate, Sodium Nitroprusside, Ammonia,
Ammonium sulfate, sulfur, Fouchet’s reagent, P-dimethyl aminobenzaldehyde, sodium
acetate benzidine, H2O2 etc

Glassware:- Test tube, dropper, measuring cylinder, beaker, pipette, etc.

Theory:- Urine is the excretory product of body, formed by kidneys. It is made up of water
and water soluble waste products . examination of urine gives us idea of renal function.

Normal Constituents of Urine :-

1 Water 90-95%

2 Urea 25 - 30 gm/day

3 Creatinine 1 - 1.2 gm/day

4 Uric acid 0.4 – 0.7 gm/day

5 Sodium 3 – 3.5 gm/day

6 Potassium 2 – 2.5 gm/day

7 Chloride 10 - 15 gm/day

8 Calcium 0.1 - 1.3 gm/day

9 Phosphate 0.7 - 1.2 gm/day

10 Sulfate 1 - 1.2 gm/day

11 Ammonia 0.6 – 0.8 gm/day

Abnormal constituents of urine (Pathological Urine)

S. Test Principle Observation Inference
No.

1 Reducing sugar: Benedict Reducing sugars Color Con Presence of

test have free aldehyde reducing
or ketone group, Green 0.5-1% sugar
Reagents: Sodium citrate + which reduces
sodium carbonate + copper metallic Yellow 1-1.5%
ions.
sulphate Benedict reagent Orange 1.5-2%
Procedure: 5 ml reagent + 7-8 has cupric ion Brick red >2%
drop of urine sample + boil 2- which is reduced to
3 min + cool cuprous ion Blue No sugar

2 Protein heat coagulation Denaturation of Coagulum is Presence of

protein by heat formed at upper protein
test- part
Procedure: Fill 3/4 test tube
with sample + heat upper part
+ coagulum formed + add 2-
3% CH3COOH If coagulum is
dissolves, indicate presence of
phosphate

Heller's test-reagents: Nitric acid cause Protein

HNO3 precipitation of White ring formed present
protein at junction of two
Procedure: 3 ml of sample + liquid (Fig. 4.8b)
3-4 ml of HNO3 along the
side wall of tube

Bence Jones proteins: Heat-ppt disappear Presence of

cool reappear Bence Jones
Procedure: Heat 40-60°C ppt proteins
+ heat 100°C-dissolve ppt +
cool again reapper

3 Ketone bodies: Rothera's Ketone bodies form Purple ring formed Presence of
a purple colored at the junction of ketones
nitroprusside test reagents: complex with two liquid bodies
Ammonium sulfate, sodium sodium
nitroprusside, liquor ammonia nitroprusside in
Procedure: 5 ml urine alkaline medium.
saturate with ammonium beta-
sulfate + sodium nitroprusside hydroxybutyrate
+ shake + liquor ammonia does not give this
side of tube test

4 Bile salt Hay's sulfur test Bile salt reduces the Sulfur powder sinks Presence of
reagents-sulfur powder surface tension of towards bottom
Procedure: 4-5 ml of sample water Bile salts
in tube + add pinch of sulfur
powder on surface

5 Bile pigments Foucher's test BaCl2 , reacts with Green blue color Presence of
reagent: (FeCl3+TCA) 6 ml sulfur to form appear
 Result-The Qualitative analysis of abnormal constituents of urine sample was successfully
performed.

Experiment -13

Aim: To perform the quantitative test for proteins by biuret reagent

Reference: Gupta PP, Gupta N. Qualitative Tests for Proteins and Amino Acids. Essentials
of Practical Biochemistry.2017.30

Requirements:

Chemicals: Biuret Reagent, Copper sulfate (CuSO4) solution, Sodium potassium

tartrate ,Sodium hydroxide (NaOH) or potassium hydroxide (KOH) solution

Apparatus: Test tubes Dropper Test tube stand,PPE and other general laboratory equipment

Theory:

Proteins and peptides are polymers of amino acids. They are chains of amino acids as well as
other biomolecules or ions or compounds. The amino acids are covalently bound to each
other by a covalent bond, called a peptide bond, between the carbon number one (C 1) of one
amino acid and nitrogen number two (N2) of adjacent amino acid. The formation of a peptide
bond is a condensation reaction.

Principle of Biuret Test

The reaction in the biuret test is a colorimetric reaction where the result is indicated by a
color change from blue to purple or violet. In an alkaline environment, the cupric (Cu+2) ions
in the biuret reagent bind to the nitrogen atoms in the peptide bonds of proteins forming a
violet-colored copper coordination complex. The formation of purple color indicates the
presence of peptide bonds in the sample. The intensity of the developed purple color is
directly proportional to the concentration of peptide bonds present in the solution.

Preparation of Biuret Reagent

 Dissolve 1 gram of CuSO4 crystals in 100 mL of distilled water.
 Add 1.2 grams of sodium potassium tartrate to the mixture. (it stabilizes the Cu+2 ions)
 Dissolve 10 grams of NaOH pellet in 90 mL of distilled water to make a 10% NaOH
solution.
 Add 10 mL of the 10% NaOH solution to 100 mL of 1% CuSO4 solution.

Procedure of Biuret Test

Label three test tubes as ‘test’, ‘positive’, and ‘negative’.

In the test tube labeled as ‘test’, dispense 1-2 mL of sample, in the test tube labeled as
‘positive’, dispense 1-2 mL of albumin solution, and in the test tube labeled as ‘negative’,
dispense 1-2 mL of distilled water.

In each tube, add an equal volume of (1-2 mL) of Biuret reagent.

Shake well and let it stand at room temperature for 5 minutes.

Observe the tubes for the development of violet color in the suspension.

Result and Interpretation of Biuret Test

Positive Biuret Test: Formation of purple color after the addition of Biuret reagent. (Tube
with albumin solution will turn purple.)

Negative Biuret Test: No formation of violet/purple color (or formation of blue color)
solution after the addition of Biuret reagent. (Water will turn to blue color.)

Accordingly, if the color of the sample solution turns to violet/purple after the addition of the
Biuret reagent and incubation, report the sample positive for proteins/peptides.
If the color of the sample doesn’t change i.e. remains blue even after 5 minutes of the
addition of Biuret reagent, report the sample negative for proteins/peptides.
 Experiment 14

Aim: To perform quantitative determination of cholesterol in blood sample.

Reference:- Kaushik GG, Sharma Neha, Dabeer Sabira, Jindal Ruchi “ A Practical book of
biochemistry” CBS Publishers & Distributors Pvt Ltd; First edition 2020: Page no. 12.

Requirements: -

Apparatus required: - beaker, Test Tube, Test Tube Holder, test tube stand, boiling test
tube, pipette, glass rod, electronic balance.

Chemical required: Chloroform, Conc. H2SO4, Acetic Anhydride, Ferric Chloride and
Acetic Acid, cholesterol.

Theory:

Cholesterol circulates in the blood. As the amount of cholesterol in your blood increases, so
does the risk to your health. High cholesterol contributes to a higher risk of cardiovascular
diseases, such as heart disease and stroke. That’s why it’s important to have your cholesterol
tested, so you can know your levels.The two types of cholesterol are: LDL cholesterol, which
is bad, and HDL, which is good. Too much of the bad kind, or not enough of the good kind,
increases the risk cholesterol will slowly build up in the inner walls of the arteries that feed
the heart and brain.

Cholesterol can join with other substances to form a thick, hard deposit on the inside of the
arteries. This can narrow the arteries and make them less flexible – a condition known
as atherosclerosis. If a blood clot forms and blocks one of these narrowed arteries, a heart
attack or stroke can result.

Procedure: -

Salkowski Reaction: - To the few crystals of cholesterol in a test tube add chloroform 1-2
ml and 2 ml of conc. H2SO4 and gently shake the contents. The solution obtained consists of
an upper red layer and lower yellow coloured layer. It indicates the presence of cholesterol.

Liebermann-Burchard Test: - To the few crystals of cholesterol in chloroform add 5-6

drops of acetic anhydride followed by few ml of conc. H 2SO4. The solution becomes emerald
green colour. It indicates the presence of cholesterol.

Zak’s Reaction: - To the few crystals of cholesterol in chloroform add a solution containing
FeCl3 in acetic acid followed by little conc. H 2SO4. The solution obtained red in colour. It
indicates the presence of cholesterol.
Acrolin Test:- It shows the presence of glycerine and fats. It is a important test for glycerol.
Take a clean and dry test tube and add 0.5ml of a glycerol in it and 0.5 ml of Potassium
bisulfate and then heat the content on water bath for 10 minutes and the turbid solution will
became clear.

Result- The identification test of cholesterol was successfully performed in a lab.

Experiment No.-15

Aim :- To perform the separation of amino acids by thin layer chromatography.

Reference:- Kaushik GG, Sharma Neha, Dabeer Sabira, Jindal Ruchi “ A Practical book of
biochemistry” CBS Publishers & Distributors Pvt Ltd; First edition 2020: Page no. 110.

Requirement:-

Lead pencil, TLC Plate, Chromatography chamber , Capillary tube, oven.

Chemicals:- Amino acid samples, Solvent mixture (Butanol: Acetic acid: DW), Ninhydrin.

Glassware:- Beaker, measuring cylinder, pipette.

Theory:-
Chromatography :- This technique is used for the separation of closely related compounds,
present in a mixture solution like carbohydrates, lipid, amino acids, vitamins, etc. The term
was given by Mikhail Tswett.

Principle: Separation of mixture by chromatography depends on Stationary and Mobile

phase. There movement of solute mixture in Mobile phase, which move on Stationary phase.
This interaction, results in separation of compounds.
Migration of substance is measured as R (ratio of front) value

Rf = Distance travelled by substance (solute)

Distance travelled by solvent

Classification :-
1.Partition ( Paper, gas, liquid )
2.Adsorption
3. Ion exchange
4. Gel filtration
5.Affinity
6. HPLC
Procedure :-
1.Make 2% solution of amino acid (0.2 gm in 100 ml).
2.Cover beaker with aluminum foil to prevent it from oxidation and protection from direct
sunlight and air.
3.Make mobile phase solution 4: 1:5 with the help of n-butanol: acetic acid : water. Take a
clean TLC chamber.
4.Prepare the tlc plate by coating it with absorbent Silica gel-G.Dissolve 5gm of absorbent in
12.5 ml of distilled water and no lumps should be present in it.Than,spread the slurry on a
glass plate uniformly and dry in a hot air oven.
5.Mark three point on horizontal line. With the help of capillary put first solution on 1st
mark.
7.Then dry it with the help of dryer.
8.Repeat it with more solutions
9.Keep buffer solution in TLC chamber.
10.Put TLC paper into the chamber along the side wall such that the horizontal line get
completely emerged into the solution
11.Then wait for 40 minutes for allowing into stand After 40 minutes take TLC out of
chamber, and dry.
12.Dry for 20 minutes in oven, or help of dryer. After 40 minutes in oven , or help of dryer
After that stain will appear on the paper
13.Spry ninhydrin on the stain. Mark it with lead pencil.
14. Measure the distance from starting of horizontal line to the end point and also measure
horizontal-vertical line
15. Calculate Rf value. According this substance can be found out.

Result:-

Calculate R value

Rf = Distance travelled by substance (solute)

Distance travelled by solvent