VI SEM B.Sc.

BIOCHEMISTRY PRACTICAL -VII

Experiment
Name of the experiment
no.
01 Estimation of Creatinine by Jaffe’s Method
02 Estimation of Inorganic Phosphate by Modified Fiske – Subbarow Method.
03 Estimation of Lactose By Dinitro – Salicylic Acid (DNS) Method.
04 Estimation of Iron Using Ammonium Thiocyanate.
05 Estimation of Protein by Folin-Ciocalteaus method
06 Conductometric Titration of Amino Acid against HCl solution.
07 Conductometric Titration of Amino Acid against NaOH solution.
08 Estimation of serum cholesterol by Zak’s method
09 Extraction of DNA from onions
 ESTIMATION OF CREATININE BY JAFFE’S METHOD

AIM:
To estimate, the amount of creatinine present in the given solution using standard creatinine
solution containing 100 µg /ml.

PRINCIPLE:

Creatinine is a nitrogenous compound connected with protein metabolism. It is an anhydride of

creatinine. It is formed by spontaneous cyclisation of creatinine phosphate which is an
irreversible and non-specific reaction.

Creatinine gives orange red color on reaction with picric acid in a strong alkaline medium. The
color is due to the enolised tautomer of creatinine picrate. The intensity of red color is a
measure of creatinine present, which is read at an absorbance of 540 nm colorimetrically.

The experiment is performed with Creatinine solution of different concentration along with
unknown mixture of creatinine solution and blank containing no creatinine. The creatinine in
the unknown is then determined graphically.
 TABULAR COLUMN:
Volume
Test Vol. of Std Amount of Volume of Volume of Volume of Absorbance
of 10%
tube creatinine creatinine distilled Picric acid Distilled
NaOH 540 nm
no. (ml) (µg) water (ml) (ml) water(ml)
(ml)

1 Blank 0 1.0 2.0 0.2 5.8

Mix And allow it to stand for 15 minutes at

room temperature for color development
2 0.2 20 0.8 2.0 0.2 5.8

3 0.4 40 0.6 2.0 0.2 5.8

4 0.6 60 0.4 2.0 0.2 5.8

5 0.8 80 0.2 2.0 0.2 5.8

6 1.0 100 0.0 2.0 0.2 5.8

1.0
7 ? 0.0 2.0 0.2 5.8
(Unknown)

1.0
8 ? 0.0 2.0 0.2 5.8
(Unknown)
PROCEDURE:

1. Arrange the clean, dry and labeled test tubes 1 to 8 orderly on a stand.

2. Pipette out 0.2, 0.4, 0.6, 0.8 and 1.0 ml aliquots of the given standard creatinine solution
into test tube 2, 3, 4, 5 & 6 respectively. Test tube 1 is Blank. Make up the given
creatinine (unknown) in 100 ml standard flask with distilled water to the mark and shake
well. Pipette out 1 ml of this made up solution into each of the test tube 7 & 8.

3. Pipette out 1.0, 0.8, 0.6, 0.4, 0.2 ml of distilled water into test tube 1 to 5 respectively.

4. Pipette out 2 ml of picric acid in to each of the test tubes 1 to 8.

5. Pipette out 0.2 ml of 10% NaOH into each of test tube and mix well.

6. Allow the test tube to stand for 15 minutes at room temperature for development of
color.

7. Pipette out 5.8 ml of distilled water into each of the test tubes and mix well.

8. Set the colorimeter for zero absorbance for the blank solution in the test tube at 540 nm

9 Read the absorbance of the solution in the remaining test tube 2, 3, 4, 5, 6, 7 & 8

10. A graph is drawn plotting the amount of creatinine in µg along X-axis verses the
absorbance along Y-axis.

11. From the straight-line graph obtained, the amount of creatinine in the unknown
solution is determined.
CALCULATION:

Test Tube No. 7

Amount of creatinine present in 1 ml of unknown solution determined graphically =_____ µg

Amount of creatinine in 100 ml of unknown solution = __ x 100=_______ µg

Test Tube No. 8

Amount of creatinine present in 1 ml of unknown solution determined graphically =______µg

Amount of creatinine in 100 ml of unknown solution =__ x 100 =_________ µg

RESULT:

Test Tube No.7:

Amount of creatinine present in 1 ml of unknown solution determined graphically =_____ µg

Amount of creatinine present in 100 ml of unknown solution = _______ µg

Test tube No 8

Amount of creatinine present in 1ml of unknown solution determined graphically =_____µg

Amount of creatinine present in 100 ml of unknown solution =____ µg

 ESTIMATION OF PROTEIN BY FOLIN-CIOCALTEAUS (FC) METHOD/
LOWRY’S METHOD
AIM:
To estimate the amount of protein (Bovine Serum Albumin) in the given solution using standard
Bovine Serum Albumin solution containing 100 µg /ml.

PRINCIPLE:
The Lowry’s method is reasonably sensitive detecting 10 µg/ml of protein and the sensitivity is
moderately constant from one protein to another. When Folin reagent (mixture of sodium
tungstate and molybdate), together with the copper sulfate solution, blue color is produced
which can be quantified at an absorbance of 660 nm. This method is based on both the Biuret
reaction where the peptide bond react with Cu 2+ ions under alkaline condition producing Cu +
which react with Folin reagent involving reduction of phosphomolybdotungstate to
heteropolymolybdenum blue by the copper catalyzed oxidation of aromatic amino acid. The
resultant strong blue color is therefore dependent on tyrosine and tryptophan content of
protein sample.
The experiment is performed with protein (BSA) solution of different concentration along with
unknown mixture of protein solution and blank containing no protein. The protein in the
unknown is then determined graphically.
TABULAR COLUMN:
Vol. of
Vol. of Vol. of
Test Vol. of BSA Amount alkaline

Incubate the tubes at room temperature for 10 minutes.

distilled FC Absorbance

Incubate the tubes at room temperature for 30 minutes

Tube (ml) of BSA copper
water reagent at 660 nm
No. (µg) reagent
(ml) (ml)
(ml)

1 Blank 0 1.0 5.0 0.6

2 0.2 20 0.8 5.0 0.6

3 0.4 40 0.6 5.0 0.6

4 0.6 60 0.4 5.0 0.6

5 0.8 80 0.2 5.0 0.6

6 1.0 100 0.0 5.0 0.6

1.0 5.0
7 ? 0.0 0.6
(Unknown)

1.0 5.0
8 ? 0.0 0.6
(Unknown)
PROCEDURE:
1. Arrange the clean, dry and labeled test tubes 1 to 8 orderly on a stand.

2. Pipette out 0.2, 0.4, 0.6, 0.8 and 1.0 ml aliquots of the given standard BSA solution into
test tube 2, 3, 4, 5 & 6 respectively. Test tube 1 is blank. Make up the given BSA
(unknown) in 100 ml standard flask with distilled water to the mark and shake well.
Pipette out 1 ml of this made up solution into each of the test tube 7 & 8.

3. The volume in each test tube is made to 1 ml with distilled water.

4. Pipette out 5 ml of alkaline copper reagent into all the test tubes and shake well.

5. Incubate the test tubes at room temperature for about 10 minutes.

6. Pipette out 0.6 ml of FC reagent (also called phenol reagent) into each of the test tube
and mix well immediately.

7. Incubate all the test tubes at room temperature for about 30 minutes

8. Set the colorimeter for zero absorbance for the blank solution in the test tube at 660 nm.

9. Read the absorbance of the solution in the remaining test tube 2, 3, 4, 5, 6, 7 and 8.

10 A graph is drawn plotting the amount of BSA in µg along X-axis verses the absorbance
along Y-axis

11. From the straight line graph obtained, the amount of protein in the unknown solution is
determined.
CALCULATION:

Test Tube No.7

Amount of protein present in 1 ml of unknown solution determines graphically = µg

Amount of protein present in 100 ml of unknown solution = x100 =___ µg

Test Tube No.8

Amount of protein present in 1 ml of unknown solution determined graphically = µg

Amount of protein present in 100 ml of unknown solution = x 100 =____ µg

RESULT:

Test Tube No.7:

Amount of protein present in 1 ml of unknown solution determined graphically = ____µg

Amount of protein present in 100 ml of unknown solution = ____ µg

Test tube No 8

Amount of protein present in 1ml of unknown solution determined graphically = __ µg

Amount of protein present in 100 ml of unknown solution = ___ µg

 ESTIMATION OF INORGANIC PHOSPHATE BY MODIFIED
FISKE – SUBBAROW METHOD
AIM
To estimate the amount of inorganic phosphate present in the given solution using standard
potassium dihydrogen phosphate solution containing 1µmol/ml.

PRINCIPLE:
Phosphate reacts with molybdic acid to form phosphomolybdic acid. On treatment with p-
methyl amino phenol (Metol), phosphomolybdic acid is selectively reduced to a product with a
deep blue color (probably a mixture of lower oxides of molybdenum). The intensity of the blue
color is proportional to concentration of phosphate in the solution, which is read at an
absorbance 660 nm colorimetrically.
The experiment is performed with inorganic phosphate solution of different concentration
along with unknown inorganic phosphate solution and blank containing no inorganic
phosphate. The inorganic phosphate is unknown is then determined graphically.
PROCEDURE:
1. Arrange the clean, dry and labeled test tubes 1 to 8 orderly on a stand.

2. Pipette out 0.2, 0.4, 0.6, 0.8 and 1.0 ml aliquots of the given standard inorganic
phosphate solution into test tube 2, 3, 4, 5 & 6 respectively. Test tube 1 is Blank. Make
up the given inorganic phosphate solution (unknown) in 100 ml standard flask with
distilled water to the mark and shake well. Pipette out 1 ml of this made up solution
into each of the test tube 7 & 8.

3. Pipette out 1.0, 0.8, 0.6, 0.4, 0.2 ml of distilled water into each tube 1 to 5 respectively.

4. Pipette out 1 ml of acid molybdate reagent into each of the test tube 1 to 8.

5. Pipette out 1 ml of reducing agent into each of the test tube 1 to 8.

6. Allow the test tubes to stand for 30 minutes at room temperature for color
development.

7. Pipette out 7 ml of distilled water into each of test tubes and mix well.

8. Set the colorimeter for zero absorbance for the blank solution in the test at 660nm

9 Read the absorbance of the solution in the remaining test tube 2, 3, 4, 5, 6, 7 & 8

10. Draw the graph by taking the amount of inorganic phosphate on X-axis and absorbance
on Y axis. From the straight line graph obtained calculate the amount of inorganic
phosphate present in the unknown solution of test tube 7 & 8.
 TABULAR COLUMN:
Test Vol of Std Amount of Vol of Vol of acid Vol of Vol of Absorbance
tube inorganic inorganic distilled molybdate reducing Distilled at 660nm
no. Phosphate Phosphate water (ml) (ml) agent water(ml)
(ml) (µmol) (ml)

1 Blank 0.0 1.0 1.0 1.0 7.0

temperature for 30 minutes for color

2 0.2 0.2 0.8 1.0 1.0 7.0

Mix And allow it to stand at room

3 0.4 0.4 0.6 1.0 1.0 7.0

4 0.6 0.6 0.4 1.0 1.0 7.0

development
5 0.8 0.8 0.2 1.0 1.0 7.0

6 1.0 1.0 0.0 1.0 1.0 7.0

7 1.0 ? 0.0 1.0 1.0 7.0

(Unknown)

8 1.0 ? 0.0 1.0 1.0 7.0

(Unknown)
CALCULATION:
Test Tube No.7

Amount of inorganic phosphate present in 1 ml of solution determined graphically =_____µmol

Amount of inorganic phosphate present in 100 ml of unknown solution = ____ x 100

= ___ µmol

Test Tube No.8

Amount of inorganic phosphate present in 1 ml of solution determined graphically

=_____µmol

Amount of inorganic phosphate present in 100 ml of unknown solution = ____ x 100

=_____ µmol
RESULT:

Test Tube No.7

Amount of inorganic phosphate present in 1 ml of unknown solution determined

graphically =_____ µmol.

Amount of inorganic phosphate present in 100 ml of unknown solution = _______µmol.

Test Tube: No. 8

Amount of inorganic phosphate present in 1 ml of unknown solution determined

graphically=______ µmol.

Amount of inorganic phosphate present in 100 ml of unknown solution = _______µmol

 ESTIMATION OF IRON USING AMMONIUM THIOCYANATE

AIM:

To estimate the amount of Iron (Fe3+) present in the given solution, using Standard Ferric
Ammonium Sulfate solution containing 1mg/ml.

PRINCIPLE

Ferric sulfate reacts with ammonium thiocynate to give ferric thiocynate a blood red colored
complex. The color intensity is proportional to the concentration of Fe 3+ in solution. The
intensity of this red color complex is measured at an absorbance of 480 nm.

The experiment is performed with Fe 3+solution of different concentration along with the
unknown solution and the blank containing no Fe 3+. The Fe 3+ in the unknown is determined
graphically.

REACTION

Fe2 (SO4)3 + 2NH4SCN 2 [Fe (SCN)]SO4 + (NH4)2 SO4

Ferric thiocyanate
(Blood red- colored complex)
 TABULAR COLUMN

Test Vol. of Std Amount of Vol. of Vol. of Vol. of 2M. Vol. of Absorbance
tube Fe3+ (ml) Fe3+ (mg) distilled 4N.HNO3 NH4CNS distilled at 480nm
no. water (ml) (ml) (ml) water (ml)

1 Blank 0.0 7.0 1.0 2.0 10.0

2 1.0 1.0 6.0 1.0 2.0 10.0

3 2.0 2.0 5.0 1.0 2.0 10.0

4 3.0 3.0 4.0 1.0 2.0 10.0

5 4.0 4.0 3.0 1.0 2.0 10.0

6 5.0 5.0 2.0 1.0 2.0 10.0

7 5.0 ? 2.0 1.0 2.0 10.0

(Unknown)

8 5.0 ? 2.0 1.0 2.0 10.0

(Unknown)
PROCEDURE

1. Arrange the clean, dry and labeled test tubes 1 to 8 orderly on a stand.

2. Pipette out 1.0, 2.0, 3.0, 4.0, 5.0 ml of standard Fe3+ solution (1 mg/ml) into the test tube
2,3,4,5 and 6 respectively. Test tube is blank. Make up the given Fe 3+ solution in 100 ml
standard flask with distilled water to the mark and shake well. Pipette out 5.0 ml of this
made up solution into test tube 7 and 8.

3. Pipette out 7.0, 6.0, 5.0, 4.0, 3.0, 2.0 ml of distilled water into test tube 1, 2, 3, 4, 5 and
respectively. Also pipette out 2 ml of distilled water to each of test tube 7 and 8
respectively.

4. Pipette out 1 ml of 4N. HNO3, into each of the test tube and shake well.

5. Pipette out 2 ml of 2M.NH4CNS into each of the test tube and mix.

6. Pipette out 10 ml of distilled water into each of the test tube and mix.

7. Set the colorimeter for zero absorbance for the blank solution in the test at 480nm

8. Read the absorbance of the solution in the remaining test tube 2, 3, 4, 5, 6, 7 & 8

9. Draw the graph by taking the amount of Fe 3+ on X-axis and absorbance at 480nm on
Y-axis. From the straight line graph obtained calculate the amount of Fe3+present in the
unknown solution of test tube 7 & 8.
CALCULATION

Test Tube No.7

Amount of iron present in 5ml of unknown solution determined graphically=________mg.

Amount of iron present in 100 ml of unknown solution = X 100 =______mg

5
Test Tube No.8

Amount of iron present in 5ml of unknown solution determined graphically=________mg.

Amount of iron present in 100 ml of unknown solution = X 100 =______mg

5
RESULT:

Test Tube No.7

Amount of iron present in 5ml of unknown solution determined graphically=________mg.

Amount of iron present in 100 ml of unknown solution = ________ mg.

Test Tube: No. 8

Amount of iron present in 5ml of unknown solution determined graphically=________mg.

Amount of iron present in 100 ml of unknown solution = ________ mg.

 ESTIMATION OF REDUCING SUGAR (LACTOSE) BY DINITRO – SALICYLIC ACID
(DNS) METHOD

AIM:

To estimate the amount of lactose present in the given solution using standard lactose solution
containing (1000g/ml).

PRINCIPLE:
A reducing sugar reduces quantitatively 3, 5-dinitrosalicylic acid (DNS) to a red colored product
3- Amino- 5- nitro salicylic acid on heating in an alkaline solution. The color intensity is
proportional to the concentration of the red color product which in turn is proportional to
concentration of lactose in solution. The intensity of the red color is measure of reducing sugar
present. This method has no practical value as a routine qualitative test, but the reaction has
been used successfully as the basis of qualitative method for the determination of reducing
sugar.
The experiment is performed with lactose solution of different concentration, along with the
lactose unknown solution and blank containing no lactose. The lactose in the unknown is then
determined graphically.
TABULAR COLUMN:

Test Vol of Std Amt.of Vol.of Vol.of Vol.of Absorbance

Heat for 15 minutes on boiling water bathe and cool

Tube Lactose (ml) Lactose distilled DNS Distilled At 540 mm
No. (g) Water (ml) Reagent water(ml)
(ml)
1 Blank 0 2.0 1.0 7.0

2 0.2 200 1.8 1.0 7.0

3 0.4 400 1.6 1.0 7.0

4 0.6 600 1.4 1.0 7.0

5 0.8 800 1.2 1.0 7.0

6 1.0 1000 1.0 1.0 7.0

1.0 1.0 7.0

7 ? 1.0
(Unknown)
1.0 1.0 7.0
8 ? 1.0
(Unknown)
PROCEDURE:
1. Arrange the cleaned, dried and labeled test tubes 1 to 8 orderly on a stand.
2. Pipette out 0.2, 0.4, 0.6, 0.8 and 1.0ml of standard lactose solution (1000g/ml) into test
tubes 2, 3, 4, 5 and 6 respectively. (Test tube 1 is blank).Make up the given lactose solution
(unknown) in 100ml standard flask with distilled water to the mark and shake well. Pipette
out 1ml of this made up solution into each of the test tube 7 & 8.
3. Pipette out 2.0, 1.8, 1.6, 1.4, 1.2, and 1.0ml of distilled water into test tube 1, 2, 3, 4, 5 and
6,7 & 8 respectively.
4. Pipette out 1 ml of DNS reagent into each of the test tubes.
5. Heat the test tube in boiling water bath and cool under tap water.
6. Add 7ml of distilled water into each of the test tube and mix well.
7. Set the colorimeter for zero absorbance for the blank solution at 540nm.
8. Read the absorbance for the solution in the remaining test tube 2, 3, 4, 5, 6, 7 and 8
9. Draw the graph by taking the amount of lactose on X- axis and absorbance on Y-axis. From
the straight line graph obtained, calculate the amount of lactose present in unknown
solution of test tube 7 and 8.
CALCULATION:
Test Tube No.7

Amount of lactose present in 1 ml of unknown solution determined graphically=_____g

Amount of lactose present in 100 ml of unknown solution = ___ x 100 =_________g

Test Tube No.8

Amount of lactose present in 1 ml of solution determined graphically = _____ g

Amount of lactose present in 100 ml of unknown solution = _____ x 100=_____ g

RESULT:

Test Tube No.7

Amount of lactose present in 1 ml of unknown solution determined graphically =______g

Amount of lactose in 100ml of unknown solution = --------------------- g

Test Tube No.8

Amount of lactose present in 1 ml of unknown solution determined graphically=____g

Amount of lactose present in 100 ml of unknown solution =____g

 CONDUCTOMETRIC TITRATION OF AMINO ACID AGAINST HCL
AIM:
1. Determination of molarity of Amino acid solution using 0.1N HCl.

2. Estimation of Amino acid dissolved in the given solution.

PRINCIPLE:
In this titration, the Amino acid behaves as base. Initial conductance is large due to presence of
highly conducting H+. As the Amino acids react with HCl, the conductance is decreased due to
the replacement of highly conducting H+ by less conducting NH3+. Beyond the end point,
conductance, do not vary as ionization of weak base is suppressed.

PROCEDURE:

1 Make up the Amino acid solution given in 100 ml flask upto the mark using distilled
water and shake well.
2. Transfer 50 ml of 0.1M HCl solution into a 100ml beaker.
3. Calibrate the conductometer.
4. Immerse the conductivity cell in HCl solution taken in a beaker.
5. Add, 1 ml of Amino acid solution at a time, shake gently. Note down the readings per
each addition of Amino acid solution.
6. Continue the titration till the reading shows a sharp decrease in conductance. Take 3 to
4 readings extra.
7. Plot a graph taking volume of Amino acids on X-axis and conductance on Y-axis.
8. Find out the end point. This is a rough end point
9. Carry out fractional titration around the rough end point. For this, fresh 50 ml of 0.1M
HCl solution is titrated by adding 0.2 ml of Amino acid at a time around the rough end
 point. Note down the readings. Fractional titration shall be carried out after the addition
of Amino acid solution whose volume is already decided from the rough titration.
10. Plot more graph for fractional titration from which, find out the accurate end point.
11. Calculate the Molarity and weight of the given Amino acid dissolved in 100 ml of the
solution.

REPORT:
1. Molarity of the given Amino acid =________M

2. Weight of Amino acid per litre = __________g

3. Weight of the Amino acid per 100 ml = ______g

OBSERVATION & CALCULATION:
Rough titration

Beaker:- 50 ml of 0.1 M HCl

Pipette:- Amino acid solution (1ml at a time)

Volume of Amino Conductance

acid added
0 ml
1 ml
2 ml
3 ml
4 ml
5 ml
6 ml
7 ml
8 ml
9 ml
10 ml
11 ml
12 ml
13 ml
14 ml
15 ml
Fractional Titration

50 ml of 0.1 M HCl + Amino acid whose volume is already decided from rough titration and
titrated against Amino acid (0.2 ml at a time)

Vol. of Amino acid Conductance

added (0.2ml)

Calculation:

Maa = (VM)HCl = 50 x 0.1 =……………………………..M

Vaa Accurate end Point

Weight of Amino acid/l = Maa x Mol. weight of Amino acid = ……………………g

Weight of Amino acid /100 ml = Weight of Amino acid per litre =……………g
10
 CONDUCTOMETRIC TITRATION OF AMINO ACID AGAINST NaOH SOLUTION

AIM:
1. To determine the molarity of the given Amino acid solution

2. To estimate the weight of Amino acid dissolved in the given solution by conductometric
titration using standard NaOH.

PRINCIPLE:
Amino acids like Alanine and Glycine are weak acids. Conductivity of the Amino acids solution is
low. On adding alkali to the Amino acids, highly ionized sodium salt of Amino acids is formed
and the conductance is increased. At the end point, further addition of NaOH increases OH -
ions. So conductivity suddenly shows a sharp increase. The point of intersection of both the
lines gives end point.

PROCEDURE:

1. Make the Amino acid solution given in 100 ml flask upto the mark using distilled water
shake well.

2. Transfer 25 ml of the made up solution into an 100 ml beaker.

3. Calibrate the conductometer

4. Immerse the conductivity cell in the Amino acids solution taken in a beaker (if the
electrodes is not immersed add 10 ml of distilled water). Record the conductance.

5. Add 1 ml of 1 M NaOH at a time. Shake gently note down the reading for each addition
of NaOH solution.

6. Continue the titration till the reading shows a sudden increase. Take 3-4 readings more.

7. Plot a graph taking volume of NaOH on X-axis and conductance on Y-axis. Find out the
end point. This is a rough end point.
8. Carry out a fractional titration around the rough end point. For this fresh 20 ml of Amino
acid solution and water (if added earlier) is titrated by adding 0.2 ml of NaOH at a time
around the rough end point. Note down the readings. Fractional titration shall be
carried after the addition of NaOH solution whose volume already decided from the
rough titration.

9. Plot one more graph for fractional titration from which find out the accurate end point.

10. Calculate the molarity and the weight of given Amino acid dissolved in 100 ml of the
solution.

RESULT:

1. Molarity of given Amino acid = ……………………………M

2. Weight of Amino acid per Litre=…………………………….g

3. Weight of Amino acid per 100 ml=……………………………g

OBSERVATION AND CALCULATIONS
Rough titration

Beaker 25 ml of made up Amino acid solution

Pipette/Burette 1M NaOH

Volume of NaOH Conductance

solution added

0 ml

1 ml

2 ml

3 ml

4 ml

5 ml

6 ml

7 ml

8 ml

9 ml

10 ml

11 ml

12 ml

13 ml

14ml

15ml
Fractional Titration

Beaker: 25 ml made up solution + water + NaOH solution whose volume is decided by rough
titration

Pipette / Micro burette = 1 M NaOH

Vol of NaOH End Point

solution added

Calculation:

M Amino acid =VNaOH x MNaOH

VAa

= Accurate end Point x 1

25

= ________M

Weight Amino acid/L = Mamino acid X Mol Wt. of Amino acid

Weight Aminoacid/100 ml = Wt.of Aa/L

10

=_______________g