SHIVAJI UNIVERSITY, KOLHAPUR

B. Sc. Part-II Practical Examination, March/April, 2015

BIO-CHEMISTRY
Instructions to Candidates
1. Read the slip carefully and perform the experiment as asked therein.
2. Immediately after recording the reading get them signed by the examiner.
3. If there be anything in the apparatus that you do not know, ask the examiner or laboratory assistant to help
you.
4. Express all observations in a tabular form. It is also desirable that all intermediate Calculations and results be
entered as neatly and clearly as possible.
5. No numerical figures should be written over either in the preliminary or final Observations. If any figure is
sought to be discarded, it should be run through and the desired figure written near to it and same should be
got initiated by any one of the examiners.
6. Please, see that your table is in good order before you leave the laboratory.
Sd/-
Chairman
B. Sc. Part-II Bio-Chemistry Practical
Examination, March/April, 2015.
 SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II Practical Examination, March/April, 2015
BIO-CHEMISTRY PRACTICAL PROGRAMME

Day Session Time Sub Group A Sub Group B

First Morning 11.00 a.m. to Calorimetric estimation Paper chromatography and isolation
2.00 p.m.

Evening 2.30 p.m. to Paper Chromatography Colorimetric estimation.

5.30 p.m. & isolation.

Second Morning 11.00 a.m. to Volumetric estimation General tests for Carbohydrates
2.00 p.m. lipids Enzymes and Urine analysis.

Evening 2.30 p.m. to General tests for Volumetric estimation

5.30 p.m. Carbohydrates
Enzymes and Urine
analysis/blood group/
diastase activity.

The candidate should remain present 15 minutes before the commencement of practical examination. The
candidates should bring with him certifies journal, pen pencil and other required material.
The cyclostyled charts are allowed at the time of examination. Experiments from slips will be given to the
students. The students will perform the experiment according to the procedure mentioned in the slip or the
procedure of regular practical course.

Sd/-
Chairman
B. Sc. Part-II Bio-Chemistry Practical
Examination, March/April, 2015
 SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II Practical Examination, March/April, 2015
BIO-CHEMISTRY
The practical examination of B. Sc. Part-II in Bio-Chemistry will be of 12 hours duration and will be conducted
in TWO SUCCESSIVE days, 6 hours per day.
The total practical examination will be of 100 Marks. The distribution of marks will be as follows.
1) Colorimetric estimation Marks 14
2) Isolation of biological samples Marks 12
3) Paper Chromatography / separation methods Marks 12
4) Volumetric estimation Marks 15
5) Qualitative analysis of Marks 22
a) Carbohydrate,
b) Enzyme/Blood group detection
c) Normal / Abnormal Urine components
6) Oral Marks 10
7) Journal and study tour report Marks 15
TOTAL 100

The practical batch will be of maximum 16 candidates. The batch will be divided in two sub groups A and B.
Sub Group A will consist of 8 candidates while Sub Group B will have 8 candidates.
If the number of candidates in a batch is less than 16th number should be divided in two equal sub group A
and B. Any number remaining will be placed in Sub Groups B.
Practical Examination Timing : 11.00 a.m. to 2.00 p.m. and
2.30 p.m. to 5.30 p.m.
Recess : 2.00 p.m. to 2.30 p.m.

Sd/-
Chairman
B. Sc. Part-II Bio-Chemistry Practical
Examination, March/April, 2015.
 A-1
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Paper Chromatography (Amino Acids) Marks-12

Separate the given binary mixture of amino acids by paper chromatography and identify the amino acids
by their Rf values. (Standard Rf values will be supplied for comparison).
Present your results as follows.
Obs. No. Rf Value observed Amino acids detected

1)

2)

Attach the chromatogram to the answer book.

A-2
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Paper Chromatography (Sugars) Marks-12

Separate the given binary mixture of sugars by paper chromatography and identify the sugars by their Rf
values. (Standard Rf values will be supplied for comparison).
Present your results as follows.
Obs. No. Rf Value observed Sugars detected

1)

2)

Attach the chromatogram to the answer book.

 A-3
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Ion exchange chromatography Marks-12

Requirements : Strong cationic resin

NaCl Solution
0.1 N NaOH
Ion exchange column (25 x 1) cm
Procedure : Prepare the column containing given amount of strongly acidic resin. Then wash the column
with 2N HCl followed by distilled H2O until the pH of effluent is same as that of distilled H2O (test with litmus
paper). When the level of H2O in the column just reaches to the resin, load 10 cm3 of given NaCl solution &
allow the solution to flow into the column at controlled rate. When the level of NaCl is just above the top of
resin elute it with distilled H2O till about 50 cm3 of effluent is collected. Titrate the effluent with 0.1 N NaOH
using phenolphthalein indicator. Calculate the uptake of Na+ ions.
36.5 g HCl ≡ 58.5 g of NaCl
58.5 g of NaCl ≡ 23 g of Na+
Results :1) Uptake of NaCl = ............................... g.
2) Amount of Na+ exchanged = ............................... g.

A-4
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Immobilization of baker's yeast by entrapment for invertase activity Marks-12

Requirements : 1) Sodium alginate powder

2) Baker's yeast
3) CaCl2 solution 2% w/v in H2O containing 1% glucose.
4) Sucrose solution (1% w/v in H2O)
Procedure : Take about 50 mg of sodium alginate and dissolve it in 25cm3 hot H2O. Cool to room
temperature and add 2 gm lyophilised yeast grains and make homogeneous slurry by constant stirring.
Take about 100 cm3 2% solution of CaCl2 to another beaker and using glass tube add the above slurry
dropwise to CaCl2 solution with slow stirring. The beads of calcium alginate containing entrapped yeast cells
are formed. Allow the beads to harden in CaCl2 solution for one hour. The entrapped yeast cells get activated
due to the presence of glucose. After one hour decant CaCl2 solution and wash the beads in beaker for 3 to 4
times by distilled H2O. Fill the beads in column with glass wool at its bottom. Wash the beads in column by
distilled H2O till the effluent shows no positive test for glucose.
Then wash the column by 1% sucrose solution. Fill the sucrose solution in the column till its level remains
well above the column and wait for 5 minutes. Yeast entrapped in the beads will hydrolyze sucrose in to
glucose and fructose. Test the effluent by Fehling's test, Benedicts test and Seliwanoff's test.
Results : Immobilized yeast cells hydrolyze sucrose in to ......................... and ..................... .
 A-5
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Quantitative Estrimation of Amylase Marks-12

Perkorm rcaction mixture Assay & find (Calculate) the amylase activity by using given standased
graph.

Results :.............................................................mg/ml/min.
 B-1
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Isolation of starch from Potatoes Marks-12

Requirements : 1. Potato sample

2. Absolute alcohol.
Procedure : Clean and weigh the given potatoes. Cut them in to small pieces and soak in little water for 10
minutes. Homogenize thoroughly in a blender and transfer the mass completely into a beaker containing about
200 cm3 water. Stirr well and filter while stirring through a tea filter or cheese cloth. Resuspend the residue in
another 200 cm3 water and filter as before to collect filtrate. This process may be repeated once again. Pool the
filtrates, stirr well and allow to stand for 10-15 minutes, when starch settles down completely, Decant out the
suparnatant. Wash the residue twice with distilled water, and finally with absolute alcohol. Filter on Buchner
and dry.
Calculate the percent yield and characterise the starch isolated.
Result : Amount of starch = ........................ g/100 g potato.

B-3
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Isolation of albumin and globulin from egg. Marks-12

Requirements : i) Egg white.

ii) Ammonium sulphate
Procedure :
Egg is punctured and egg white is collected, in to a measuring cylinder. Egg yolk is transfered in to another
beaker. To the egg white (NH4)2SO4 is added (till half saturation). Mixture is shaken well at this stage
globulin gets precipitated. This is centrifuged and supernatent is removed in a beaker and ammonium
sulphate is added (till full saturation).
pH is adjusted to 4.8 with 10% acetic acid using bromocresol green indicator. Content of the beaker is
filtered, residue of albumin is collected dried & weighed. Calculate the % yield and characterise the
albumin and globulin isolated.
Result : 1) Amount of Albumin ............... g.
2) Amount of Globulin .............. g.
 B-2
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, April/May, 2015
Isolation of casein from milk Marks-12

Requirements : 1. Milk sample

2. 10% acetic acid
3. Bromo cresol green (BCG) indicator.
4. Acetone.
5. Absolute akohol
Procedure : Dilute with distilled water the given milk sample to double its volume. Heat to just warm
it. Now add 10% acetic acid dropwise with constant stirring. Transfer a drop of this mixture to a spot plate
containing BCG indicator drops and adjust the pH to 4.6 (grey-green colour). Fine granular precipitate is
formed, allow it to settle down. Filter through several layers of cheese cloth by decantation. Transfer the
residue into a beaker containing about 25cm3 distilled acetone, stirr continuously and vigorously on hot sand
bath. Repeat the process with 25cm3 of absolute alcohol. Decant out the supernatant. Repeat the washing twice
with 25cm3 of diethyl ether, Finally dry and weigh.
Calculate the percent yield and characterise the casein isolated.
Result : Amount of casein = ........................ g/1003 of milk.
 SHIVAJI UNIVERSITY, KOLHAPUR C-1
B. Sc. Part-II, Practical Examination in Biochemistry, April/May, 2015
Verification of Beer-Lambert's Law Marks-14
3
Requirements : CuSO4 Solution, (5 mg/cm ), 1 : 1 ammonia solution.
Procedure : In to clean and dry test tubes make the additions as follows.
Observation Table

Test tube Cm3 of Std. cm3 of cm3 of mg. of At 625nm

No. CuSO4 distilled ammonia CuSO4
water (1 : 1) %T A

Blank 0 5 5
1 1 4 5
2 2 3 5

wait for 10 minutes

3 3 2 5

Mix well and

4 4 1 5
5 5 0 5
(unknown) 6 2 (unkn) 3 5
(unknown) 7 2 (unkn) 3 5

Plot the graph of absorbancy Vs. concentration and comment on it.

Result : Amount of CuSO4 in given sample - .................... mg/100 cm3

SHIVAJI UNIVERSITY, KOLHAPUR C-2

B. Sc. Part-II, Practical Examination in Biochemistry, April/May, 2015
Estimation of Protein by biuret Method Marks-14
3
Requirements : Standard protein solution (5mg/cm )
Biuret reagent, Unknown sample
Procedure : Pipette out the solutions into clean, dry and labelled tubes as follows.
Observation Table

Test tube cm3 of Std. cm3 of cm3 of mg of At 540nm

No. Protein distilled biuret protein
water %T A

Blank 0 5 5
1 1 4 5
2 2 3 5
wait for 10 minutes

3 3 2 5
Mix well and

4 4 1 5
5 5 0 5
unknown 6 2 (unkn) 3 5
unknown 7 2 (unkn) 3 5

Plot the graph of absorbancy Vs. concentration and determine the concentration of unknown from graph.
Result : Amount of protein in the given sample - .................... mg/100 cm3
 SHIVAJI UNIVERSITY, KOLHAPUR C-3
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Estimation of inorganic phosphate in blood by Fiske & Subbarow method Marks-14
Requirements : Standard phosphate solution (8µ g/cm3)
10% Trichloroacetic acid,
Molybdate solution
ANSA reagent
Blood sample
Procedure : 1) Preparation of protein free blood filtrate : Pipette out 18 cm3 TCA and 2cm3 blood/serum into
clean dry test tube. Shake well and filter through Whatman filter paper, into a clean dry test
tube. Use 5.0 cm3 of this filtrate as sample.
2) Prepare the blank, standard and sample tubes as follows.
Observation Table
3
Test tube cm of cm3 of cm3 of At 670 nm
No. Distilled Molybdate ANSA
H2O %T A

%T Exactly after 10 minutes

1 5cm3 10% 3.6 1.0 0.4
(Blank) TCA

Mix well and Wait for

10 minutes Record
2 5cm3 3.6 1.0 0.4
(Standard) Standard
solution

3 5cm3 Protein 3.6 1.0 0.4

(Sample) free blood
filtrate
Result : Amount of inorganic phosphate = ................. mg/100 cm3 of blood.

SHIVAJI UNIVERSITY, KOLHAPUR C-4

B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Estimation of Glucose by DNSA method Marks-14
3
Requirement : 1. Std. glucose 500 µg/cm .
2. Distilled Water
3. DNSA.

Procedure : Pipette out Solutions in clean, dry and labelled test tubes as follows and construct a standard graph of
'A' Vs µg of glucose

cm3 of cm3 of At 530 nm

Test tube cm3 of std. µg of Glucose
distilled DNSA
Keep in bolinhg water bath

No. glucose %T A
water
for 10 minutes

Blank 0.0 2.5 2.5

2 0.2 2.3 2.5
3 0.4 2.1 2.5
4 0.6 1.9 2.5
5 0.8 1.7 2.5
6 1.0 1.5 2.5
7 1.2 1.3 2.5
8 (unknown) 0.5 2.0 2.5
9 (unknown) 0.5 2.0 2.5

Result - Amount of glucose in the 100 cm3 sample ...................................................................mg

 C-5
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Estimation of RNA by Bial's-orcinol method Marks-14
3
Requirements : 1) Standerd RNA (50µgm/cm )
2) Orcinol-Acid reagent (2cm3 of 10% FeCl3 . 6H2O in 400 cm3 conc. HCl.)
3) Orcinol-reagent (6% orcinol w/v in 95% ethenol)
Procedure : Prepare blank, standard and sample tubes as shown in observation table and record %T at 660 nm.
Observation Table

Test tube Orcinol Acid Orcinol At 660 nm

No. reagent reagent %T A

1 2.5 cm3 3.6 1.0

(Blank) distilled H2O

Keep in boiling water

2 2.5 cm3 of 3.6 1.0

bath for 20 min.

(Standard) Standard
Solution

3 2.5 cm3 of 3.6 1.0

(Sample) unknown
sample solution

Result : Amount of RNA in the given sample = ................. mg/100 cm3

 C-7
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Estimation of the amount of urea in blood by DAM method Marks-14
Requirements :
H2SO4 - H3PO4 acid reagent, Na-tangstate (10%), DAM (2%), H2SO4 (2/3 N), Thiosemicarbazide (1mg/cm3)
Std. urea (25mg/cm3)
Procedure :
1. Protein free blood filtrate: In to a clear, dry test tube pipette out 1.0 cm3 blood or serum, 15.0 cm3 distilled water.
Mix well and add 1.5 cm3 Na-tungstate (10%), 1.5 cm3 H2SO4 (2/3 N). Mix well and filter into dry test tube using dry funnel
and filter paper.
2. Prepare blank, standard and sample tubes as shown in observation table.
3. Record the % Transmittance and extinction at 480 mm.
Observation Table
3
cm cm3 cm3 cm3

Mix Well and keep in boiling

T. T. Absorbance of 480
of DAM acid Thiosemi-

water bath for 5 minutes

No. %T E
H2O solution reagent carbazide
1 2.0 cm3 3.4 0.4 1.6 0.6
Blank water
2 2.0 cm3 3.4 0.4 1.6 0.6
Std. Std. urea

3 2.0 cm3 3.4 4.0 1.6 0.6

Sample filtrate

Result :
1. Amount of blood urea = ................. mg/100 cm3
 V-1
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Formal titration of Glycine Marks-15

Requirements : Glycine solution, 0.1N NaOH, 40% formaldehyde, phenolphthalein.

Procedure : Dilute the given glycine solution in volumetric flask up to the mark.
Part A
1) Pipette out 25.0 cm3 of glycine solution into a conical flask add 2 drops of phenolphthalein. Then add
from burette 0.1 N NaOH dropwise till faint pink colour is obtained (neutralised glycine).
2) Take aproximately 40.0 cm3 of 40% formaldehyde, add 2 drops of phenolphthalein. Then add from
burette 0.1 N NaOH dropwise till faint pink colour is obtained (neutralised formaldehyde).
Part-B
Now, pipette out 10.0 cm3 of neutralised formaldehyde into the neutralised glycine solution and titrate
against 0.1 N NaOH till faint pink colour is obtained.
Enter the readings in the tabular form and calculate the amount of glycine in the given sample.
Results :1. 25 cm3 of neutralized glycine solution ≡ ....................... cm3 of 0.1N NaOH.
2. Amount of glycine in given solution = ........................ mg.

SHIVAJI UNIVERSITY, KOLHAPUR V-2 (A)

B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Estimate the amount of lactose in milk by Benedict's method Marks-15

Requirements : Milk sample

10% Sodium tungstate
2/ N H SO
3 2 4
Standard lactose solution (2.5 mg./cm3)
Benedict's quantitative reagent
Procedure :
Part A :
Standardisation of Benedict's reagent : Fill the burette with standard lactose solution. Take 5cm3 of
Benedict's reagent in an evaporating dish and add 2gm of crystalline Na2CO3 and about 10cm3 distilled water.
Heat to boil vigorously and run lactose solution rapidly from the burette until the blue colour fades down.
Further addition must be done dropwise till the blue colour just disappears completely and chalk white
precipitate is formed. (There should not be trace of blue/green colour at the end point. The Benedict's solution
must be kept vigorously boiling with stirring through-out the entire titration).
Part B :
Add 12 cm3 of 10% sodium tungstate and 12 cm3 of 2_ N H2SO4 to the given milk sample. Mix well and dilute
3
upto the mark with distilled water. Transfer the contents to a clean dry beaker and allow the precipitate to
settle. Filter through Whatman paper by decantation. Now fill the burette with this filtrate and titrate with
5cm3 of Benedict's reagent as before. Present your results in tabular form and calculate the amount of lactose in
given milk sample.
Results : 1) 5ml. of Benedict's solution ≡ ..................... g of lactose.
2) Amount of lactose in given milk sample = ..................... g.
 V-2 (B)
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Estimate of lactose in milk by Fehling's method/Benedicts method. Marks-15
Requirements : 1) Standard lactose solution (2.5 mg/cm3)
2) Fehling's A reagent.
3) Fehling's B reagent.
4) Milk sample.
5) Sodium tungstate (10%)
6) H2SO4 solution 2/3 N).
7) Indicator (Potassium ferrocynide).
Procedure :
Part A : Standardisation of Fehling's reagent : Fill the burette with standard lactose solution. Pipette out 5.0
cm3 of Fehling's B reagent into an evaporating dish. Heat the solution to boil and perform titration in boiling
condition. Run the lactose solution from the burette solwly. Intermitantly take a drop of reaction mixture and
filter it through English filter paper, add a drop of indicator along the side of reaction mixture. In the beginning
of the titration red colour will develop at the junction of two drops. Continue the titration till there is no red
colour at the junction of two drops. Take three readings.
Part B :
_
To the given milk sample add 13 cm3 of 10% sodium tungstate and 12cm3 of N H2SO4 mix well and dilute
to the mark. Transfer the contents to a clean, dry beaker and allow the precipitate 2 to settle. Filter through
Whatman paper by decantation. Now fill the burette with this filtrate and titrate 3 with Fehling's solution as
before. Present your results in tabular form and calculate the amount of lactose in given milk sample.
Results :1) 10 cm3 of Fehling's solution ≡ ..................... g of lactose.
2) Amount of lactose in given milk sample = ..................... g.

V-3
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Estimate the amount of chlorides in urine Marks-15

Requirements : Urine sample

Standard AgNO3 solution (0.1 N)
Ammonium thiocynate solution.
Procedure :
A) Preparation of standard ammonium thiocynate -
Fill the burette with given ammonium thiocynate solution and pipette out 10 ml. of AgNO3 solution in
conical flask. Titrate till reddish brown colour is obtained. Take three readings and calculate the normality of
ammonium thiocynate.
If the normality of ammonium thiocynate is not exactly 0.1 N, then adjust the normality to exactly to 0.1
N and prepare 50 cm3 of 0.1 N solution.
B) Estimation of chlorides :
Pipette out 10 cm3 of urine sample into 100 cm3 volumetric flask. Add to it approximately 25.0 cm3 of
distilled water and exactly 20 cm3 of standard AgNO3 solution. Dilute the contents to the mark. Allow the
chlorides to precipitate and filter through filter paper. Now pipette out 25cm3 of filtrate into a conical flask and
titrate with standard ammonium thiocynate until reddish brown precipitate/colour is obtained.
Results : Amount of chloride present in 1.0 dm3 of urine sample = ................... g.
 V-4
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Estimate the amount of Vitamin C in tablet/lemon/orange Marks-15

Requirements : Standard ascorbic acid (20 mg/100 cm3), 2, 6-dichlorophenol indophenol, glacial acetic
acid.
Procedure : Part 1) Standardisation of 2, 6 dichlorophenol indophenol dye.
Pipette out 5 cm3 standard ascorbic acid into conical flask. Add to it 1 cm3 of acetic acid. Titrate against dye
solution till faint pink colour persists for 15 seconds.
Part 2) Squeeze the biological sample to extract juice and filter if necessary. Immediately titrate 5.0 cm3
each of the juice separately against the dye solution as before
For tablet, dissolve and dilute the given tablet into 250 cm3 in volumetric flask. Use 5.0 cm3 for titration.
Present your observations in tabular form and calculate the amount of vitamin C in each of the sample.
Results : 1. 1 cm3 of dye solution ≡ ..................... g of Vitamin C.
2. Amount of Vitamin C in tablet/Orange = .................... g.
3. Amount of Vitamin C in 100 cm3 lemon = ................... g.

V-5
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Determine the saponification value of oil/fat Marks-15

Requirements : 1) Alcoholic KOH (0.5 mol/dm3)

2) Standard HCl (0.1 mol/dm3)
3) Oil/fat sample (10% w/v, in fat solvant)
Procedure :
Blank titration (standardisation of alcoholic KOH) :
Dilute 25.0 cm3 of alcoholic KOH to 100 cm3 in a volumetric flask using distilled water. Now pipette out
25.0 cm3 of diluted solution to a conical flask and titrate against standard HCl using phenolphthalein indicator.
Back titration :
Pipette out 10.0 cm3 of oil/fat sample to 250 cm3 round bottom flask and also 25.0cm3 of alcoholic KOH. Add
3-4 boiling chips and attach a reflux condenser. Heat the flask on a boiling waterbath for 45 minutes. Allow to
cool to room temperature and dilute to 100 cm3 in a volumetric flask. Pipette out 25cm3 and titrate with
standard HCl using phenolphthalein indicator.
Present your results in tabular form and calculate the saponification value.
Results : 1) Blank titration reading = ............................. cm3 of std. HCl
2) Back titration reading = ............................. cm3 of std. HCl
3) Saponification value of oil/fat = .......................
 V-6 (A)
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Determine the iodine number of oil/fat Marks-15

Requirements : 1) Iodine monochloride ( ∼ 0.2 mol/dm3)

2) Potassium iodide (10%)
3) Standard sodium thiosulphate (0.1 N)
4) Oil/fat sample (2% w/v in chloroform)
Procedure :
Blank titration (standardisation of ICl solution) : Pipetteout exactly 10.0 cm3 of chloroform and 25.0 cm3
of iodine monochloride solution into a stoppered bottle. Add about 50.0 cm3 distilled water and 10.0 cm3 KI
solution. Titrate immidietly the iodine liberated, against standard sodium thiosulphate using starch indicator.
Back titration : Pipette out 10.0 cm3 of fat solution into a stoppered bottle, add 25.0 cm3 of iodine
monochloride solution. Stopper the bottle, shake it carefully but throughly and leave to stand in dark for an
hour, with intermitant shaking.
Rinse the bottle necks with about 50.0 cm3 distilled water and 10.0 cm3 of KI solution. Titrate immidietly
the liberated iodine against standard sodium thiosulphate using starch indicator.
Present your results in tabular form and calculate the iodine number.
Results : 1) Blank titration reading = .......................... cm3 of std. Na2S2O3
2) Back titration reading = ........................... cm3 of std. Na2S2O3
3) Iodine number of Oil/fat = ............................

V-6
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Determine the Iodine number of Oil/Fat Marks-15

Requirements : 1) Oil/Fat sample in chloroform.

2) I2 (0.2 mole/dm3) in HgCl2
3) KI (10% W/V in H2O)
4) Sodium thiosulphate (0.1 mol/dm3)
5) Starch indicator.
Procedure :
a) Blank titration : Pipette out exactly 25cm3 of standard iodine in stoppered bottle. Add about 20 cm3 of
10% KI solution and titrate immediately the iodine liberated against standard sodium thiosulphate
using starch indicator.
b) Back titration : Pipette out exactly 10.0 cm3 of given oil sample into a stoppered bottle. Add exactly
25.0 cm3 of standard iodine solution. Shake the mixture well for 1 hour. Add 20 cm3 of 10% KI solution
and titrate immediately the iodine liberated against standard sodium thiosulphate solution using
starch indicator. Present your results in tabular form and calculate iodine number.
Results : 1) Blank titration reading ............ cm3 of standard sodium thiosulphate solution.
2) Back titration reading ............. cm3 of standard sodium thiosulphate solution.
3) Iodine number of oil/fat .....................
 Q-1
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Qualitative analysis of carbohydrate, lipid and urine. Marks-14
Analyse the unknown carbohydrate, enyme, blood group and urine samples given in test tubes
marked as C, L/E B and U respectively.
Results :-
1. Given carbohydrate is ........................................... 08 Marks

Structure of osazones

2. Given enzyme / blood group is 06 Marks

.................................................................................................................................................
 Q2 (B)
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Marks-08
1. Analyse the urine sample for Normal / Abnormal constituents.
Result :
The given urine sample contains the Normal / Abnormal
...................................... ......................................
...................................... ......................................
...................................... ......................................
...................................... ...................................... constituents.

Q2 (C)
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
Marks-08
Solve the given Problems on sequerce of DNA, RNA & Genctic Code
 SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
PAPER - I
All questions are compulsory Marks
Question No. 1 ....... 12
1. Chromatographic separation of binary mixture of amino acids.
2. Chromatographic separation of binary mixture of carbohydrates.
3. Uptake of Na+ - ions by cation exchange resin.
4. Immobilization of bakers yeast by gel entrapment for Invertase activity.
5. Quantitative estimation of Anylase actinty.
Question No. 2 ........ 12
1. Isolation and characterization of starch from potatoes.
2. Isolation and characterization of casein from milk.
3. Isolation and characterization of albumin and globulin from egg.
Question No. 3 ........ 14
1. Verify Lambert-Beer's law and determine the amount of CuSO4 in the given sample.
2. Estimate the amount of protein by biuret method.
3. Estimate the amount of inorganic phosphate in blood by Fiske-Subrow method.
4. Estimate the amount of reducing sugar Glucose by DNSA
5. Estimate the amount of RNA by Bial's orcinol method.
6. Estimate the amount of blood urea by DAM-method.

Question No. 4 ...... 15

Journal and study tour.
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 SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2015
PAPER - II
All questions are compulsory Marks
Question No. 5 ....... 15
1. Estimate the amount of glycine by formal titration
2. Estimate the amount of lactose in milk by Benedict's/Fehling's method
3. Estimate the amount of chlorides in urine by Volhard's method
4. Estimate the amount of vitamin-C by 2, 6-dichloro phenol-indophenol method
5. Determine of saponification value of oil or fat
6. Determine of iodine number of oil or fat.

Question No. 6 ........ 22

1. Identify the given carbohydrate sample. 08
2. Identify the given Enzyme / blood group. 06
3. Identify the normal/abnormal urine constituents 08
OR
3. Solve the problems on sequence of DNA, RNA & Genetic code. 08
Question No. 7
Oral 10
47