SRI SHAKTHI INSTITUTE OF

ENGINEERING AND TECHNOLOGY

COIMBATORE 641 062

DEPARTMENT OF FOOD TECHNOLOGY

BT8261
BIOCHEMISTRY LABORATORY

LABORATORY RECORD BOOK

APRIL 2019

Name : ………………………………………………………………
Reg. No. : ………………………………………………………………
Degree : B. Tech (Food Technology)
Year/Sem. : I / II
 SRI SHAKTHI INSTITUTE OF ENGINEERING AND TECHNOLOGY
COIMBATORE 641 062

DEPARTMENT OF FOOD TECHNOLOGY

This is to certify that this BT8261 BIOCHEMISTRY LABORATORY record work is

done by Mr. / Ms. ____________________________________ for the course B.

Tech FOOD TECHNOLOGY during First Year / Second Semester of Academic

year 2018 – 2019.

Staff – in – Charge HoD / FT

Register No. …………………………………..

This record is submitted for SECOND Semester B. Tech Practical

Examinations of Anna University conducted on _____________

INTERNAL EXAMINER EXTERNAL EXAMINER

BT8261 Biochemistry Laboratory 2 / 33

 ANNA UNIVERSITY, CHENNAI
SYLLABUS
(R 2017)

BT8261 BIOCHEMISTRY LABORATORY LTPC

0042

AIM

To learn and understand the principles behind the qualitative and quantitative estimation of
biomolecules (proteins, carbohydrates, lipids, metabolites etc.,) and laboratory analysis of the same
in the body fluids.

EXPERIMENTS

1. General guidelines for working in biochemistry lab (theory)

2. Units of volume, weight, density and concentration measurements and their range in
biological measurements. Demonstration of proper use of volume and weight measurement
devices.
3. Accuracy, precision, sensitivity and specificity (theory)
4. Preparation of buffer –titration of a weak acid and a weak base.
5. Qualitative tests for carbohydrates – distinguishing reducing from non-reducing sugars and
keto from aldo sugars.
6. Quantitative method for amino acid estimation using ninhydrin – distinguishing amino from
imino acid.
7. Protein estimation by Biuret and Lowry’s methods.
8. Protein estimation by Bradford and spectroscopic methods.
9. Extraction of lipids and analysis by TLC.
10. Estimation of nucleic acids by absorbance at 260 nm and hyperchromic effect (demo).
11. Enzymatic assay: phosphatase from potato.
12. Enzymatic assay: estimation of glucose by GOD-POD method after hydrolysis of starch with
acid and specificity of the enzymatic method.

TOTAL: 60 PERIODS

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 INDEX

Expt Page Faculty

Date Title Marks
No. No. signature
General guidelines for working in biochemistry
1
lab (theory)

2 Study of unit and measurements

Accuracy, precision, sensitivity and specificity

3
(theory)
Preparation of buffer – Titration of a weak acid
4
and a weak base
Qualitative tests for carbohydrates –
5 Distinguishing reducing from non-reducing
sugars and keto from aldo sugars

Quantitative method for amino acid estimation

6 using ninhydrin – Distinguishing amino from
imino acid

7 Protein estimation by biuret and lowry’s methods

Protein estimation by bradford and spectroscopic
8
methods
9 Extraction of lipids and analysis by TLC
Estimation of nucleic acids by absorbance at 260
10
nm and hyperchromic effect (demo)

11 Enzymatic assay: phosphatase from Potato

Enzymatic assay: Estimation of glucose by GOD-
POD method after hydrolysis of starch with acid
12
and specificity of the enzymatic method

Expt. No: 1
GENERAL GUIDELINES FOR WORKING IN BIOCHEMISTRY
Date: LAB

AIM
To study the general guidelines for working in biochemistry laboratory.

BT8261 Biochemistry Laboratory 4 / 33

GUIDELINES
There are two major concerns to consider when working in a biochemistry laboratory.
First is safety: this can never be overemphasized. The second is efficiency in the laboratory work.
Although the latter very much depends on the individuals doing the experiments, there are general
rules students are advised to follow:
 Keep the benches and shelves clean and well-organized.
 Avoid contaminating the chemicals; use only clean glassware and spatulas; label glassware in
use.
 Plan your experiments before starting to carry them out.
 Pay attention to others in the laboratory.

SAFETY IN THE LABORATORY

Students working in a biochemistry laboratory must always be aware that the chemicals used are
potentially toxic, irritating and flammable. Such chemicals are hazards, however, only when they
are mishandled. Students who come to the laboratory session must have a complete understanding
of the laboratory procedures to carry out and be familiar with both the physical and chemical
properties of chemicals and reagents to be used. Since the carelessness on the part of one student
can often cause injury to other students, one must have a special concern for the safety of
classmates. Students must be familiar with general safety practices, facilities and emergency action.

SAFETY RULES IN GENERAL

 Do not work alone in the laboratory.
 Unauthorized experiments are not allowed.
 Eating, drinking and smoking in the laboratory are strictly prohibited.
 Become familiar with the location and the use of standard safety features in the laboratory. The
laboratory is equipped with fire extinguishers, eye washes, safety showers, fume hoods and
first-aid kits. Any question regarding the use of these facilities should be addressed to your
instructor.
 Special care for eye protection is required. Safety glasses must be used when certain procedures
are being carried out. The instructor will call the students' attention to those procedures. The use
of contact lenses is not recommended, since they reduce the rate of self-cleansing of the eye.

SPECIAL SAFETY RULES

 While heating a solution one should make sure not to overheat it; therefore, vigorous mixing of

5/33
 the solution by shaking or stirring is required. The mouth of the glassware containing the solution
to be heated should never be pointed toward anyone.
 Handling of strong acids and bases requires special attention. When diluting concentrated acids,
the acid should be poured into the water and never the opposite.
 The pipettes should never be filled with solutions of toxic substances, biological fluids, strong
acids and bases by mouth suction. Use either automatic pipette or pipette pumps.
 Volatile liquids and solids that are toxic or irritating should be handled under fume hoods.
 While handling flammable liquids such as ether, alcohols, benzene, naked flame (burners,
matches) must not be in use. The above liquids must not be stored near radiating heat sources,
such as the laboratory oven.
 Before using electrical appliances, make sure they are grounded.
 Flasks with flat-bottoms or thin walls should not be desiccated.
 Before leaving the laboratory, electrical equipment should be turned off, and gas burners
extinguished. No tap water should be left running.

RULES TO FOLLOW IN THE CASE OF ACCIDENTS AND INJURIES

CHEMICAL SPLATTERS INTO THE EYE: First the eyelid should be opened by using the
thumb and the pointing finger. Then, by using the eye wash kit, the eye should be rinsed with large
amounts of water. When an acid or alkaline solution gets into eye, the eye should be rinsed with 1
% NaHCO3 or 1 % boric acid, respectively. The victim should be taken to the doctor as soon as
possible.
BURNING: The burned spot on the skin should not be treated with water; rather, a special bandage
should be used. See doctor if necessary.
POISONING: Prompt medical treatment should be obtained.

RESULT
General guidelines for working in the biochemistry laboratory were studied.

BT8261 Biochemistry Laboratory 6 / 33

 Expt. No: 2
STUDY OF UNITS AND MEASUREMENTS
Date:

AIM
To study the units and its measurements.

DEFINITIONS
VOLUME: Volume is the space occupied by liquid, gas or a solid. The SI unit of volume is Cubic
meter.
WEIGHT: Weight is the measurement that gives the information about the force with which the
object is pulled by the earth. The SI unit is Kilogram(Kg).
MASS: Mass of an object is the amount of matter contained in that object. Mass is the invariant
property of all objects.
DENSITY: It is the quantity that relates mass and volume. Density(D) is defined as mass(m) per
unit volume(V)
D = m/V
UNITS OF MASS:
 Gram - gm
 Kilogram - Kg
 Milligram - mg
 Nanogram - ng
 Picogram - pg
 Microgram - µg
 1kg = 1000gm = 1000000mg
 1gm = 1000mg =100000 µg
 1mg = 1000ng =1000000pg
 1ng = 1000pg

UNITS OF VOLUME:
 1millimole = 10-3mole = 1micromole = 10-6mole
 1 nanomole = 10-9mole = 1picomole = 10-12mole
 1ml = 1000litre(micro litres) = 1000 µl

UNITS OF CONCENTRATION:
Solution: A Solution is a homogenous mixture of two or more substances.

7/33
 Quantity of solute
Concentration of a Solution = x 100
Volume of solution

PHASE OF EXPRESSING CONCENTRATION:

PERCENTAGE BY WEIGHT: It is the weight of solute as a percentage of total weight of
solution.
Weight of solute
Percentage by Weight of a Solution = x 100
Volume of solution
MOLE FRACTION: Mole fraction (X) of a solute is defined as the ratio of number of moles to
total number of moles of the solution.
Moles of the solute
Mole Fraction (X) =
Mol es of the solution

MOLARITY: Molarity of the solution is defined as the number of moles of solute per litre of the
solvent.
No :of moles of solute
Molarity =
Litre of solvent
MOLALITY: Molality of the solution is defined as the number of moles of solute per kilogram of
the solvent.
No :of moles of solute
Molality =
Kilogram of solvent
NORMALITY: Normality of the solution is defined as the number of equivalent of solute per litre
of the solution.
Equivalent weight of solute
Normality =
Volume of solution∈litre

RESULT

Units and measurements were studied.

BT8261 Biochemistry Laboratory 8 / 33

 Expt. No: 3
ACCURACY, PRECISION, SENSITIVITY AND SPECIFICITY
Date:

AIM
To study the definitions of accuracy, precision, sensitivity and specificity.

DEFINITIONS

ACCURACY: Accuracy is the closeness of the mean of a set of replicate analysis to the true value
of simple. It is expressed in terms of error which is the difference between true value and observed
value.
PRECISION: Precision is the reproducibility of a method. It extent to which a number of replicate
measurements of a sample agreed with one another and it is affected by the random error method.
Precision is expressed numerically in terms of standard deviation statistically is represented as co-
efficient of variation in which,
SD
V= x 100
Mean
where,
V = Variation
SD = Standard deviation
SENSITIVITY: Sensitivity is defined as the ability to detect small amount of test substance for
example the smaller reading after zero can be constantly detected and measured the slope of the
calibration error is conventional way of expressing sensitivity
SPECIFICITY: Specificity is defined as ability to detect only the test substance, the specificity
will result in the positive result. If the method is quantitative, it is important to appreciate that
specificity is often linked with sensitivity.

RESULT

9/33
 Expt. No: 4 PREPARATION OF BUFFER –TITRATION OF A WEAK ACID

Date: AND A WEAK BASE

AIM
To titrate the weak acid with strong base to detect the pKa values of weak acid. The weak
acid is 0.1N CH3COOH and base 0.1N NaOH.

PRINCIPLE
The pH is the unit to measure the activity of the solutions. It is defined as the negative
logarithm of hydrogen ion concentration.
pH = -log [H+]
According to Henderson – Hasselbalch equation,
pH = pKa+log¿ ¿
pKa can be obtained from the graph by taking half of the value form the point at which there is a
gradual increase of pH. Half the value after extending on X-axis is given pKa. When an acid is
titrated with a base, at the midpoint of titration, the concentration of proton donor and proton
acceptor and are equal

APPARATUS REQUIRED
 Pipette
 pH meter
 Beaker and a glass rod

REAGENTS REQUIRED
 0.1N CH3COOH
 0.1N NaOH

PROCEDURE
1. Standardize the pH meter. 20ml of 0.1N CH3COOH is pipette out into a beaker.
2. 2ml of 0.1 NaOH is added to the solution and stirred well. The pH is measured.
3. The procedure is repeated with addition of 2ml of 0.1N NaOH till it reaches pH 11 or there
is a sharp increase in pH.

BT8261 Biochemistry Laboratory 10 / 33

 4. A graph is drawn, plotting the measured values of pH along X-axis (titration curve)
5. From the titration curve, pKa value of the weak acid can be determined. At this point pH =
pKa (when titration is half complete)

The buffer described in the reactions are suitable for use either in enzymatic or biochemical studies.
The accuracy of the table is with ±0.05pH at 23˚c

CITRATE BUFFER
A = 0.1M solution of citric acid (21.01g in 100ml)
B = 0.1M solution of sodium citrate (29.4g of C6H5O7)NO3.H2O in 1000 ml.
The use of salt with in 5.5H2O is not recommended. X ml of A+y ml of B,
diluted to total 100ml.
X Y pH X y pH

ACETATE BUFFER
STOCK SOLUTION
A = 0.2M solution of acetic acid(11.5ml of 1000ml)

B = 0.2M solution of sodium acetate

(16.4g of C2H3O2 NaCO3, 27.2g of C2H3O2 Na.3H2O in 1000 ml.)
x ml of A+y ml of B diluted the total of 100ml
X Y pH X y pH

11/33
PHOSPHATE BUFFER
STOCK SOLUTION
A = 0.2M solution of monobasic sodium phosphate (27.8g of 1000ml)
B = 0.2M solution of dibasic sodium phosphate
(56.5g of NaHPO4.7H2O or 71.7g of Na3HPO4.2H2O)
x ml of A+y ml of B diluted to a total of 200ml.

X Y pH X y pH

SUCCINATE BUFFER
STOCK SOLUTION
A = 0.2M solution of monobasic sodium phosphate (23.6g in 1000ml)
B = 0.2M NaOH(sodium hydroxide solution)
25 ml of A+x ml of B diluted to a total of 100ml.
X pH X pH x pH

TRIS(HYDROXY METHYL) AMINE METHANE TRIS BUFFER

STOCK SOLUTION
A = 0.2ml solution of tris (hydroxyl methyl amine methane 211.2g in 1000ml)
B = 0.2M Hcl (hydrochloric acid solution)
50 ml of A+x ml of B diluted to a total of 100ml.

X pH X pH

BT8261 Biochemistry Laboratory 12 / 33

OBSERVATION
S.No Volume of Acetic acid Volume of NaOH pH
(ml) (ml)

13/33
RESULT
The pKa value of the weak acid is____________

BT8261 Biochemistry Laboratory 14 / 33

 Expt. No: 5 QUALITATIVE TESTS FOR CARBOHYDRATES -
DISTINGUISHING REDUCING AND NON REDUING
Date: SUGARS AND KETO FROM ALDO SUGARS

AIM
To identify the sugar present in the given sample by performing quality analysis for carbohydrates.
Also distinguish between reducing & non-reducing sugars and keto & aldo sugars.

QUALITATIVE TEST

BENEDICT’S TEST
PRINCIPLE: Reducing sugars have either a free aldehyde or ketone group as a path of molecular
structure. This free or potential free group of aldehyde or ketone is a mono or disaccharide is
readily oxidized by milky alkaline solution of cupric ions of Benedict’s reagent. They reduced Cu 2+
to Cu2O by allowing the brick red precipitate. Carbohydrate that can reduce oxidation are called
reducing sugars. All monosaccharides almost are reducing sugars like Lactose and Maltose are
reducing but sucrose is not. Benedict test is a simple test for reducing sugar.
REAGENT: Dissolve 100gms of Na2cl3 and 173gm of sodium citrate dehydrate in final volume of
850ml of H2O slowly with string. Add a solution of 17.3gms of CuSO 4 penta hydrate in 100ml of
litre water. Bring the volume to 1litre.
PROCEDURE: Perform the test on 1% Carbohydrate solution in the test tube. Add 2ml of
carbohydrate and 5ml of Benedict’s reagent and shake each test tube. Place it in water bath for 5-
6minutes. Add 2-3drops of 3M Hcl to 2ml of 1% sucrose solution and starch solution(1%)
separately and heated in boiling water bath for 5minutes for sucrose and 30 minutes per starch.
Perform Benedict’s test and note the colour change. Compare your results with and without
treatment and record the observation.

BARFOED'S TEST

PRINCIPLE: This reagent is weakly acidic and is only reduced by monosaccharides. Prolonged
boiling may hydrolyze disaccharides to give false positive reactions. Colour of Cu 2O is more brick
red than the orange brown obtained with Benedicts test.
METHOD: 1ml of test Solution is added with 2ml of Barfoed's reagent boiled for 1min. Brick red
colour is observed with monosaccharides. Polysaccharides answer this test only after hydrolysis.

BIAL’S TEST

15/33
PRINCIPLE: When pentoses are heated with con. HCl, furfural is formed which condenses with
resorcinol in the presence of ferric ions to give a blue green colour.
METHOD: To 1ml of test Solution, add 2-5 ml of the reagent and heat it to boiling. A blue green
colour indicates the presence of pentose.

SELIWANOFF'S TEST

PRINCIPLE: Ketose are dehydrated more rapidly than aldoses to give a furfural derivative which
then condense with resorcinol to form a red complex, prolonged heating must be avoided.
METHOD: 1ml of test Solution is added with 2ml of this reagent the contents are boiled in a water
bath for 1 min. The reaction is given by fructose, Sucrose and other fructose containing
carbohydrates.
TEST FOR SUCROSE: Sucrose is the only common non-reducing dissaccharide. So that it does
not reduce on alkaline solution or form an osazone. Therefore it is hydrolysed in acid Solution to
glucose and fructose, which are then tested.
HYDROLYSIS OF SUCROSE: 5ml of Sucrose is taken and 2ml of con. HCl is added heated for
5 min in boiling water. Then solid Na2CO3 is added till the effervescence cases. Then the reduction
test and the Seliwanoff's test are performed to the hydrolysed Solution.

IODINE TEST
PRINCIPLE: The use of Lugol’s iodine reagent is useful to distinguish starch and glycogen.
polysaccharides reacts with Iodine solution and monosachharides yields no colour with Iodine. The
test solution remain characteristic brown yellow colour. Starch and glycogen consists of branches
are made up of linear chains of glucose units which are in the form of helical coils. Iodine atom fits
into helices and starch-iodine or glycogen iodine complex. Complex starch in the form of amylase
3- amylopectin has fewer but lower brancehes than glycogen accommodating more iodine atom in a
branch. This accounts for the difference in colour in iodine agent.
LUGOL’S IODINE REAGENT: It is transparent brown liquid consisting of 10 parts of KI to
5parts of iodine to 8.5ml of distilled water. When completely dissolved, add deionised 0.5g of
iodine crystals. When iodine is completely dissolved and deionised water to final volume (25ml)
PROCEDURE: Add 2-3 drops of Lugol’s iodine solution to 2.5ml of saple to be tested. Starch
gives a purple colour. Brownish blue colour is positive test for glycogen. Brownish yellow colour
in negative

TABULATION

BT8261 Biochemistry Laboratory 16 / 33

Mark (+) sign for positive reactions and (-) sign for negative reaction

Seliwanoff's test
Benedicts test

Barfoed's test
Iodine test

Bial's test
Carbohydrate

Glucose
Fructose
Starch
Sucrose
Maltose

DISTINGUISH BETWEEN REDUCING AND NON-REDUCING SUGAR

PRINCIPLE
The cupric or copper present in alkaline copper sulphate solution is reduced to copper hydroxide
which is unstable and forms Cu2O which appears as a coloured precipitate. Sucrose is non- reducing
sugar on acid hydrolysis forms a mixture of reducing sugar.

PROCEDURE
1. Take 2-3ml of unknown sugar solution.
2. Then add 2ml of Benedict’s reagent.
3. Boil it for 5minutes in a boiling water bath and then cool it at room temperature.
4. Red precipitate is formed which indicates the presence of reducing sugar.

DISTINGUISH BETWEEN ALDOSE AND KETOSE SUGAR

PRINCIPLE
Hot Hcl reacts with ketose to form dehydrated compounds that reacts with resorcinol that condenses
with Selivanoff’s reagent to cause dark and reddish brown colour ketoses, from 5hydroxy methyl
furfurol which reacts with resorcinol to give colour within two minutes. Also, hexoses reacts with
the same to form the product but slowly.

17/33
PROCEDURE
Place2-3 drops of 1% carbohydrate solution to be tested in a separate test tube. Add 1ml
of seliwanoff’s reagent and leave it in a water bath for 1minute. Record the colour you observe in
each test tube at the end of 1minute and four minutes.
Ketohexoses seliwanoff’s reagent Cherry red colour
Ketohexoses seliwanoff’s reagent Bluish green colour
Ketohexoses seliwanoff’s reagent Pink colour slowly appears
Disaccharides seliwanoff’s reagent No colour change

RESULT
i) Sugar present in the sample was ________.
ii) Red precipitate is formed that indicates the presence of reducing sugar.
iii) Colour observed indicate the presence of aldhose or ketose

BT8261 Biochemistry Laboratory 18 / 33

 Expt. No: 6 QUANTITATIVE METHOD FOR AMINO ACID ESTIMATION
USING NINHYDRIN – DISTINGUISHING AMINO FROM IMINO
Date: ACID

AIM
To distinguish between amino and imino acid using Ninhydrin

PRINCIPLE
Ninhydrin acts as a powerful oxidizing agent. It causes oxidative decarboxylation and deamination
of amino acid produces CO2, ammonia and aldehyde. It gets reduced. Then the reduced form of
ninhydrin react with one molecule of ninhydrin to yield Ruhemas’s purple coloured complex.
However Proline and hydroxyl will give yellow colour which have the secondary amino group.

PROCEDURE
1. Take 2-5ml of amino acid solution in a clean glass test tube.
2. Add few drops of concentrated ninhydrin (1mg of 1000ml)
3. Heat the test tube at 100˚c for a few seconds.

RESULT
Amino acid appears Purple while imino acid appears yellow in colour.

19/33
 Expt. No: 7
PROTEIN ESTIMATION BY BIURET AND LOWRY’S METHODS
Date:

AIM
To estimate the amount of protein in given sample by Biuret method.

PRINCIPLE
Compound containing two or more peptide bond take characteristic purple colour when treated with
copper sulphate in alkaline medium. The purple colour complex is due to the formation of co-
ordination complex by copper atoms with nitrogen atom for each of the tow peptide chains and for
the formation of coloured complex about 1-25mg of peptide is required. The puple colour is
colorimetrically read at 540nm.

REAGENTS REQUIRED
I. STOCK SOLUTION (BSA) SOLUTION:
Dissolve 5g of BSA and make up the volume to 100ml with distilled water. The
concentration is 5g/100ml or 50mg/ml.
II. WORKING STANDARD SOLUTION:
10ml of stock standard solution is diluted to 100ml with distilled water. The
concentration is 5mg/ml.
III. BIURET REAGENT:
To a standard flask, Dissolve 1.5g of CuS04 and 6g of Nak- tartarate in about
500ml of distilled water. To this add 300ml of 10% NaOH. Make up the
volume up to the mark in a standard flask.
PROCEDURE
i. 0.2ml of the working standard solution is pipette out with concentration of 20-
100µg/ml into a series of test tubes labeled as S1 to S5.
ii. Make up the given unknown sample with distilled water from that 0.2ml and 0.4ml
unknown solution are taken in two test tube labeled T1 and T2.
iii. Make up the volume of all test tubes to1ml using distilled water.
iv. 1ml of distilled water is served as blank and labelled as B.
v. Pipette out 5ml of Biuret reagent into cells of the test tubes including blank and
incubating at 67˚c for 10 min.
vi. The purple colour developed was measured colorimetrically at 540nm

BT8261 Biochemistry Laboratory 20 / 33

 vii. The intensity of the colour formed is directly proportional to the amount of protein
present in it.

OBSERVATION
TEST VOLUME OF CONCN. OF DISTILLED BIURET OD
TUBE PROTEIN PROTEIN H2O REAGENT 540nm
WORKING X (µg) (ml)
STANDARD
(ml)

RESULT
The amount of protein present in 200ml of the given sample is ____________
AIM
To estimate the amount of protein by Lowry’s method.

PRINCIPLE

21/33
Proteins when treated with alkaline copper sulphate reagent leads to the formation of Cu 2+ions from
complex with protein to alkaline solution. The complex is between Cu 2+ions from complex with
protein to alkaline solution. The complex is between Cu 2+ and four nitrogen atoms, two from each
of the adjacent peptide chains. The tetra amine copper complex reduces Folin’s reagent by transfer
of electron to phosphomolybolic acid.
The addition of folin phenol reagent results in formation of intense blue colour through the
reduction of molybdate.
Mo6+ Mo4+
The reduction is brought by tryptophan and tyroxine residues in protein.

REAGENT’S REQUIRED
STANDARD PROTEIN SOLUTION: Weigh 100mg BSA(Bovine Serum Albumin), dissolve it and
make up to 100ml using distilled water.
WORKING STANDARD SOLUTION: 10ml of stock solution, standard protein solution is
dissolved in 100ml of distilled water.

OBSERVATION
S.NO REAGENTS B S1 S2 S3 S4 S5 T1 T2

INCUBATION AT ROOM TEMPERATURE FOR 10MINUTES

INCUBATION AT ROOM TEMPERATURE FOR 30MINUTES

ALKALINE COPPER SULPHATE REAGENT:

Reagent A : 1% CuSo4
Reagent B : 2% Sodium potassium tartarate
Reagent C : 2% Na2CO3 in 0.1N NaOH
98ml of reagent C, 1ml of reagent A and 1ml of reagent B were mixed for use is used as Alkaline
CuSo4 reagent.
FOLIN’S PHENOL REAGENT: The reagent is diuted in the ratio of 1:2 with distilled water freshly
just before use.

PROCEDURE

BT8261 Biochemistry Laboratory 22 / 33

The standard protein solution of concentration 0.2ml to 1ml was pipette out into test tubes labelled
as S1 to S5. Volumes in all the test tubes was made up to 1ml using distilled water. One of these test
tubes had 1ml of water alone and serves as blank.
4.5ml of alkaline copper sulphate reagent is added to all tubes and mix well. The test tubes are
incubated at room temperature for 10min. 0.5ml of diluted folin’s phenol reagent was added to all
test tubes, mixed well and incubated at room temperature for 30minutes. The intensity of blue
colour developed is measured at 640nm. The standard graph is calculated taking concentration on
x-axis and optical density on y-axis.

RESULT
The amount of protein present in 100ml of unknown solution was found to be_________

23/33
 Expt. No: 8 PROTEIN ESTIMATION BY BRADFORD AND

Date: SPECTROSCOPIC METHODS

AIM
To estimate the amount of protein present in the unknown sample by Bradford’s method.

PRINCIPLE
Protein involves in non-covalent binding of the dye coomassie brilliant blue which is an acidic dye,
binds to basic aromatic amino acids present in the proteins. The dye is pale red in colour and
transforms to blue when it binds with protein. The blue colour developed is colorimetrically
measured at 550nm.

REAGENTS REQUIRED
1. STOCK STANDARD BSA: Dissolve 100mg of BSA and make it upto 100 ml with
phosphate buffer saline solution. The concentration is 1mg/ml.
2. WORKING STANDARD: 10ml of stock solution is diluted to 100ml in phosphate buffer
saline solution, concentration is 100µg/ml
3. COOMASSIE BRILLIANT BLUE: Dissolve 100mg of coomassie brilliant blue in 50ml of
95% ethanol, 100 ml of concentrated orthophosphoric acid and the volume is made up to
200ml in distilled water and stored in amber bottles.
4. COOMASSIE BRILLIANT BLUE DILUTION: Mix 1 volume of concentrated dye into 4
volumes of distilled water.

OBSERVATION
Test Volume Concentration Volume Volume Volume Optical
Tubes of of Working of of H2O of Dye Mix the density
Working Standard unknown (ml) (ml) content
Standard (ml) solution well and
(ml) (ml) keep the
test
tube
at room
temp.

BT8261 Biochemistry Laboratory 24 / 33

 (10min)

PROCEDURE
Pipette out 0.2 – 1ml of working standard in concentration of 20-100µg in a series of test tubes.
Make up the volume upto 4ml in distilled water. 0.4 ml ofgiven unknown solution is also made up
to 4ml. 1ml of dye is added to all the tubes and are kept at room temperature for 10 minutes after
mixing. Measure the blue colour developed is directly proportional to amount of protein present in
it. Draw a standard graph by plotting concentration of protein(X-axis) Vs optical density(Y-axis)

RESULT
The amount of protein present in the unknown sample is ____________

25/33
 Expt. No: 9
EXTRACTION OF LIPIDS AND ANALYSIS BY TLC.
Date:

AIM
To separate and identify the lipids in egg yolk by thin layer chromatography.

MATERIALS REQUIRED
 TLC Plate
 TLC chamber
 Filter paper
 Capillary tubes
 Egg yolk
 Chloroform
 Methanol mixture(2:1)
 Chloroform methanol acetic acid mixture(25:15:4)
 Solvent tank

PRINCIPLE
The impelling force of mobile pH carrier with it the lipid molecule for which it has affinity is
favoured by solubility of lipid molecules in mobile phase. The retarding force at stationary phase
hold back the lipid molecules with which it interacts. The balance between the two forces differs
for the different molecules which implies different mobilities and hence the separation of
molecules,
Distance moved by analyte
Rf =
Distance moved by solvent

Rf values are used to compare the unknown sample with known standard and identify molecule
present in given sample Rf values differ for various solvent mixtures used. Lipids in biological
materials is present as complex mixture, under fractionation, it separates into number by a group of
solvent interaction.
Yolk is a rich source of variety of important compound like protein, lipids, glyceride, cholesterol,
phospolipis etc. Egg lipids belong to two major classes, phosopholipids belong to two major
classes, phospholipids and non phosopholipids.

BT8261 Biochemistry Laboratory 26 / 33

Resolution of compounds can be carried out using the TLC solvent is selected according to the lipid
membrane interaction under investigation but generally lipids are separated with non polar solvents.
A number of separating media can be used and actual choice of adsorbent depends on group of
lipids and solvent employed silica is well known adsorbent which has silica hydroxide group on its
surface and widely used in the separation of lipids. The basis of separation is adsorption and
partition. The silica has ability to hold molecules on its surface.
The adsorption process involves work of weak – non ionic forces, attraction force of hydrogen
bonding forces, attraction of weak bonding forces. Non ionic forces on silica gel plates, as mobile
phase moves across one edge to opposite side. It transfer any analyte placed on the layer at the rate
determined by disturbing co-effect between stationary and mobile phases. Analyte movements
ceases when plate is removed from mobile phase reservoir. The measure of analyte is expressed by
RJ.

PROCEDURE
SAMPLE PREPARATION: Take 2ml of yolk and add 6ml of 1% Nacl in a ependorf tube. Shake
well and centrifuge at 500 rpm for 5min. Discard the lower layer, collect 2ml of clear supernatant
in another tube and add 2ml of 1% NaCl and 2ml of methanol choloroform mixture and mix well
centrifuge at 500rpm for 5min. Discard the upper phase carefully and use the lower phase for
experiment. Spot the lower phase at origin (i.e) 1cm from the bottom of TLC using capillary tube

Distance moved by analyte

Rf =
Distance moved by solvent

Keep the plates in the solvent tank containing chloroform methanol acetic acid mixture as mobile
phase saturate, the tank using a filter paper dipped in solvent mixture to avoid irregular run. After
the run is over located the spot using iodine vapour. Note that R J value should not be greater than 1
Measure the solvent front and measure the substances of various spot from the origin and calculate
Rf value.
Rf value of various components are approximated as 0.9 for phosphotidyl ethanol amine, 0.5 for
phosphotidyl choline, 0.2 for sphingomylin 0.1 for lysophosphatidyl choline as per the standards.
Sometimes phosphotidyl choline cannot be detected, but seems to move.

RESULT
Thus the various lipids were separated and R f value was noted for each lipid using Thin layer
chromatography.

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 Expt. No: 10 ESTIMATION OF NUCLEIC ACIDS BY ABSORBANCE AT 260

Date: NM AND HYPERCHROMIC EFFECT (DEMO)

AIM
To estimate the amount of DNA present in the given unknown solution by diphenylamine method.

PRINCIPLE
When DNA is treated with diphenylamine under the acidic condition a bluish green colored
complex is formed which has an absorption peak at 595nm.This reaction is given by 2
deoxypentose in general. In acidic solution deoxypentose are converted into a highly reactive β
hydroxyl leavulinic aldehyde which reacts with diphenylamine gives bluish green colored complex.
The colour intensity was measured using a red filter at 595nm.

REAGENT REQUIRED
1. Stock Standard Solution: 50mg of DNA was dissolved in 50ml of Saline Sodium citrate buffer.
Concentration 1mg/ml

2 .Working Standard Solution: 5ml of stock solution solution was diluted to 50ml with distilled
water. Concentration 100µg\ml

3. Diphenylamine Reagent: 10g of pure diphenylamine was dissolved with 25ml of concentration
sulphuric acid which was made up to 1ml with glacial acidic acid the solution must be prepared
freshly.

4. Buffered Saline ph 7.4: 0.14N Sodium chloride and 0.02M sodium citrate.

5. Unknown Solution: The given unknown solution is made up to 100ml with distilled water.

PROCEDURE
 0.5-2.5ml of working standard solution is pipetted out into 5 test tubes labeled as s1-s5 where
concentration ranging from 50-250µg.
 1ml and 2ml of unknown solution is pipetted out into two test tube u1 and u2.
 The volume in all test tubes is made up to 3ml with distilled water and 3ml of distilled water alone
serve as a blank.

BT8261 Biochemistry Laboratory 28 / 33

 4ml of diphenylamine reagent was added to all the tubes. The tubes were kept in a boiling water
bath at 36°C for 20min. The tubes were than cooled and the bluish colour developed is read
at 595nm.
 A standard graph is drawn taking concentration of DNA on x-axis and absorption of y-axis. From
the standard graph the amount of DNA present in the unknown solution is calculated.

RESULT
The amount of DNA present in the given unknown solution is found to be

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 Expt. No:11
ENZYMATIC ASSAY: PHOSPHATASE FROM POTATO
Date:

AIM
To determine the acid phosphatase activity in potatoes using Sodium-β-Glycerophosphate as
Substrate

PRINCIPLE
Phosphatases liberate inorganic phosphate from organic phosphate ester. Acid phosphatase
hydrolases a number of phosphomonoesters and phosphor proteins. Acid phosphatases can be
assayed by measuring the amount of inorganic phosphorus released from Sodium-β-
Glycerophosphate. The phosphorous released is estimated by Fiske Subbarao method.

REAGENTS

1. Citrate buffer (pH 5.6):

1M Citric acid : Dissolve 5.25 gms of citric acid in distilled water and makeup to 250ml with
distilled water
1M trisodium citrate: Dissolve 14.7 gms of trisodium citrate in distilled water and makeup to
500ml
Mix 137ml of citric acid with 363 ml of trisodium citrate solution and set up pH 5.6buffer
solutions.
(0.02M Sodium-β-Glycerophosphate in citrate buffer.)

2. 10% TCA (Tri Chloroacetic acid)

ENZYME EXTRACT
Clean and wash the potatoes with distilled water and peel out the skin. Weigh about 200gms by
using rough balance. Cut it into small pieces then add 200ml of ice cold distilled water and
homogenized in a grinder. Filter the homogenate using a cloth and stored the filtrate in a
refrigerator.

PROCEDURE
Take 5ml of Buffer substrate clean and dry the test tube then preincubate for 30 minutes at 370C.
At the end of the incubation period add 2.5 ml of 10% TCA and mix well. Keep the test tubes for
another minute at room temperature and filter the contents. Take 1ml of the filtrate for the
denaturation of inorganic phosphorous by Fiske-Subbarao method

Set up a control simultaneously by adding ml of TCA to 5 ml of buffer substrate followed by ml of

enzyme and proceeds as per test.

To determine the protein content of the enzyme extract by biuret method. For this experiment, take
1 ml of extract adds 1 ml of water and add 3ml of biuret reagent and follows as per the biuret
estimation procedure.

BT8261 Biochemistry Laboratory 30 / 33

RESULT
The amount of inorganic phosphorous present in the given unknown sample is __________ mg of

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inorganic phosphate formed per 1 ml of enzyme / 30 minutes.

BT8261 Biochemistry Laboratory 32 / 33

Expt. No:12 ENZYMATIC ASSAY: ESTIMATION OF GLUCOSE BY GOD-POD
METHOD AFTER HYDROLYSIS OF STARCH WITH ACID AND
Date: SPECIFICITY OF THE ENZYMATIC METHOD

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