SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II Practical Examination, March/April, 2016
BIO-CHEMISTRY- IDS
Instructions to Candidates
1.
2.
Read the slip carefully and perform the experiment as asked therein.
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.
Please, see that your table is in good order before you leave the laboratory.
Sd/-
6.
Chairman
B. Sc. Part-II Bio-Chemistry Practical
Examination, March/April, 2016.
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II Practical Examination, March/April, 2016
BIO-CHEMISTRY- IDS PRACTICAL PROGRAMME
Day
First
Second
Session
Time
Sub Group A
Sub Group B
Morning
11.00 a.m. to
2.00 p.m.
Calorimetric estimation
Paper chromatography and isolation
Evening
2.30 p.m. to
5.30 p.m.
Paper Chromatography
& isolation.
Colorimetric estimation.
Morning
11.00 a.m. to
2.00 p.m.
Volumetric estimation
General tests for Carbohydrates
lipids Enzymes and Urine analysis.
Evening
2.30 p.m. to
5.30 p.m.
General tests for
Carbohydrates
Enzymes and Urine
analysis/blood group/
diastase activity.
Volumetric estimation
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, 2016
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II Practical Examination, March/April, 2016
BIO-CHEMISTRY- IDS
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, 2016.
A-1
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2016
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, 2016
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
1)
2)
Attach the chromatogram to the answer book.
Sugars detected
A-3
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2016
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
3
with litmus paper). When the level of H2O in the column just reaches to the resin, load 10 cm of given
NaCl solution & allow the solution to flow into the column at controlled rate. When the level of NaCl is just
3
above the top of resin elute it with distilled H2O till about 50 cm 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, 2016
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)
3
Procedure : Take about 50 mg of sodium alginate and dissolve it in 25cm hot H2O. Cool to room
temperature and add 2 gm lyophilised yeast grains and make homogeneous slurry by constant stirring.
3
Take about 100 cm 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, 2016
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, 2016
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
3
beaker containing about 200 cm water. Stirr well and filter while stirring through a tea filter or
3
cheese cloth. Resuspend the residue in another 200 cm 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, 2016
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, 2016
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
3
residue into a beaker containing about 25cm distilled acetone, stirr continuously and vigorously on hot
3
sand bath. Repeat the process with 25cm of absolute alcohol. Decant out the supernatant. Repeat the
3
washing twice with 25cm of diethyl ether, Finally dry and weigh.
Calculate the percent yield and characterise the casein isolated.
3
Result : Amount of casein = ........................ g/100 of milk.
SHIVAJI UNIVERSITY, KOLHAPUR
C-1
B. Sc. Part-II, Practical Examination in Biochemistry, April/May, 2016
Verification of Beer-Lambert's Law
Requirements :
Procedure
:
Marks-14
3
CuSO 4 Solution, (5 mg/cm ), 1 : 1 ammonia solution.
In to clean and dry test tubes make the additions as follows.
Observation Table
Cm 3 of Std.
CuSO4
No.
3
cm of
distilled
water
cm of
ammonia
(1 : 1)
0
5
5
1
1
4
5
2
2
3
5
3
3
2
5
4
4
1
5
5
5
0
5
CuSO4
(unknown)
6
2 (unkn)
3
5
(unknown)
7
2 (unkn)
3
5
Mix
well
a
n
d
Blank
At 625nm
mg. of
%T
A
wait for 10 minutes
3
Test tube
Plot the graph of absorbancy Vs. concentration and comment on it.
Result : Amount of CuSO 4 in given sample - .................... mg/100 cm
3
SHIVAJI UNIVERSITY, KOLHAPUR
C-2
B. Sc. Part-II, Practical Examination in Biochemistry, April/May, 2016
Estimation of Protein by biuret Method
Requirements :
Procedure
:
Marks-14
3
Standard protein solution (5mg/cm )
Biuret reagent, Unknown sample
Pipette out the solutions into clean, dry and labelled tubes as follows.
Observation Table
3
cm 3 of Std.
Protein
cm of
distilled
water
cm 3 of
biuret
0
5
5
1
1
4
5
2
2
3
5
3
3
2
5
4
4
1
5
5
5
0
5
unknown 6
2 (unkn)
3
5
unknown 7
2 (unkn)
3
5
Mix
well
a
n
d
Blank
mg of
protein
At 540nm
%T
A
wait for 10 minutes
Test tube
No.
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 cm
3
SHIVAJI UNIVERSITY, KOLHAPUR
C-3
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2016
Estimation of inorganic phosphate in blood by Fiske & Subbarow method
Marks-14
Requirements : Standard phosphate solution (8µ
3
g/cm ) 10% Trichloroacetic acid,
Molybdate solution
ANSA reagent
Blood sample
3
3
Procedure : 1) Preparation of protein free blood filtrate : Pipette out 18 cm TCA and 2cm blood/serum
into clean dry test tube. Shake well and filter through Whatman filter paper, into a
3
clean dry test tube. Use 5.0 cm of this filtrate as sample.
2) Prepare the blank, standard and sample tubes as follows.
Observation Table
No.
3
1
(Blank)
5cm 10%
TCA
2
(Standard)
5cm
Standard
solution
3
(Sample)
5cm Protein
free blood
filtrate
3
3
3
3
cm of
cm of
Distilled
H2 O
Molybdate
ANSA
3.6
1.0
0.4
3.6
1.0
0.4
3.6
1.0
0.4
cm of
At 670 nm
%T
A
10 minutes Record%TExactlyafter10
minutes
Test tube
Mix
wellandWai
tfor
3
3
Result : Amount of inorganic phosphate = ................. mg/100 cm of blood.
SHIVAJI UNIVERSITY, KOLHAPUR
C-4
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2016
Estimation of Glucose by DNSA method
Requirement :
Marks-14
3
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
2.5
2.3
2.1
1.9
1.7
1.5
1.3
2.0
2.0
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
a
t
h
µg of Glucose
for 10 minutes
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.5
0.5
distilled
water
3
cm of
DNSA
in bolinhg
water
Blank
2
3
4
5
6
7
8 (unknown)
9 (unknown)
cm of std.
glucose
cm of
e
e
p
Test tube
No.
3
3
Result - Amount of glucose in the 100 cm3 sample ...................................................................mg
At 530 nm
%T
A
C-5
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2016
Estimation of RNA by Bial's-orcinol method
Requirements :
Marks-14
3
1) Standerd RNA (50µgm/cm )
3
3
2) Orcinol-Acid reagent (2cm of 10% FeCl3 . 6H2O in 400 cm 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
2.5 cm
distilled H2O
2
(Standard)
2.5 cm of
Standard
Solution
3
(Sample)
2.5 cm of
unknown
sample solution
3
3
%T
3.6
1.0
3.6
1.0
3.6
1.0
Result : Amount of RNA in the given sample = ................. mg/100 cm
f
o
bath r 20 min.
3
1
(Blank)
At 660 nm
Orcinol
reagent
i
n boiling water
Orcinol Acid
reagent
Ke
ep
Test tube
No.
3
A
C-7
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2016
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),
3
3
Thiosemicarbazide (1mg/cm ) Std. urea (25mg/cm )
Procedure :
3
3
3
cm
of
H2 O
T. T.
No.
3
1
Blank
2.0 cm
water
2
Std.
2.0 cm
Std. urea
3
Sample
2.0 cm
filtrate
3
3
3
cm
DAM
solution
3
cm
acid
reagent
3
cm
Thiosemicarbazide
3.4
0.4
1.6
0.6
3.4
0.4
1.6
0.6
3.4
4.0
1.6
0.6
Result :
3
1. Amount of blood urea = ................. mg/100 cm
water bath for 5 minutes Mix
Well and keep in boiling
1. Protein free blood filtrate: In to a clear, dry test tube pipette out 1.0 cm blood or serum, 15.0 cm
3
3
distilled water. Mix well and add 1.5 cm Na-tungstate (10%), 1.5 cm 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
Absorbance of 480
%T
E
V-1
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2016
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
3
1) Pipette out 25.0 cm 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).
3
2) Take aproximately 40.0 cm 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
3
Now, pipette out 10.0 cm 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, 2016
Estimate the amount of lactose in milk by Benedict's method
Marks-15
Requirements : Milk sample
10% Sodium tungstate
2
/3 N H2SO4
3
Standard lactose solution (2.5 mg./cm )
Benedict's quantitative reagent
Procedure :
Part A :
3
Standardisation of Benedict's reagent : Fill the burette with standard lactose solution. Take 5cm of
3
Benedict's reagent in an evaporating dish and add 2gm of crystalline Na2CO3 and about 10cm 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 :
2
3
3
Add 12 cm of 10% sodium tungstate and 12 cm of N H SO to the given milk sample. Mix well and dilute
3
2
4
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
3
with 5cm 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, 2016
Estimate of lactose in milk by Fehling's method/Benedicts method.
Requirements : 1)
2)
3)
4)
5)
6)
7)
Procedure :
Marks-15
3
Standard lactose solution (2.5 mg/cm )
Fehling's A reagent.
Fehling's B reagent.
Milk sample.
Sodium tungstate (10%)
H2SO4 solution 2/3 N).
Indicator (Potassium ferrocynide).
Part A : Standardisation of Fehling's reagent : Fill the burette with standard lactose solution. Pipette
3
out 5.0 cm 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 :
3
3
To the given milk sample add 13 cm of 10% sodium tungstate and 12cm of
to the mark. Transfer the contents to a clean, dry beaker and allow the precipitate to settle. Filter through
_
N H2SO4 mix well and dilute
2
Whatman paper by decantation. Now fill the burette with this filtrate and titrate3 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, 2016
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
3
to 0.1 N and prepare 50 cm of 0.1 N solution.
B) Estimation of chlorides :
3
3
3
Pipette out 10 cm of urine sample into 100 cm volumetric flask. Add to it approximately 25.0 cm of
3
distilled water and exactly 20 cm of standard AgNO3 solution. Dilute the contents to the mark. Allow the
3
chlorides to precipitate and filter through filter paper. Now pipette out 25cm of filtrate into a conical flask
and titrate with standard ammonium thiocynate until reddish brown precipitate/colour is obtained.
3
Results : Amount of chloride present in 1.0 dm of urine sample = ................... g.
V-4
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2016
Estimate the amount of Vitamin C in tablet/lemon/orange
Marks-15
3
Requirements : Standard ascorbic acid (20 mg/100 cm ), 2, 6-dichlorophenol indophenol, glacial
acetic acid.
Procedure : Part 1) Standardisation of 2, 6 dichlorophenol indophenol dye.
3
3
Pipette out 5 cm standard ascorbic acid into conical flask. Add to it 1 cm 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
3
5.0 cm each of the juice separately against the dye solution as before
3
3
For tablet, dissolve and dilute the given tablet into 250 cm in volumetric flask. Use 5.0 cm for titration.
Present your observations in tabular form and calculate the amount of vitamin C in each of the sample.
3
Results : 1. 1 cm of dye solution ≡ ..................... g of Vitamin C.
2. Amount of Vitamin C in tablet/Orange = .................... g.
3
3. Amount of Vitamin C in 100 cm lemon = ................... g.
V-5
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2016
Determine the saponification value of oil/fat
Marks-15
Requirements : 1) Alcoholic KOH (0.5 mol/dm3)
3
2) Standard HCl (0.1 mol/dm )
3) Oil/fat sample (10% w/v, in fat solvant)
Procedure :
Blank titration (standardisation of alcoholic KOH) :
3
3
Dilute 25.0 cm of alcoholic KOH to 100 cm in a volumetric flask using distilled water. Now pipette out
3
25.0 cm of diluted solution to a conical flask and titrate against standard HCl using phenolphthalein indicator.
Back titration :
3
3
3
Pipette out 10.0 cm of oil/fat sample to 250 cm round bottom flask and also 25.0cm of
alcoholic KOH. Add 3-4 boiling chips and attach a reflux condenser. Heat the flask on a boiling
3
waterbath for 45 minutes. Allow to cool to room temperature and dilute to 100 cm in a volumetric
3
flask. Pipette out 25cm and titrate with standard HCl using phenolphthalein indicator.
Present your results in tabular form and calculate the saponification value.
3
Results : 1) Blank titration reading = ............................. cm of std. HCl
3
2) Back titration reading = ............................. cm 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, 2016
Determine the iodine number of oil/fat
Requirements : 1)
2)
3)
4)
Procedure :
Marks-15
3
Iodine monochloride ( ∼ 0.2 mol/dm )
Potassium iodide (10%)
Standard sodium thiosulphate (0.1 N)
Oil/fat sample (2% w/v in chloroform)
3
3
Blank titration (standardisation of ICl solution) : Pipetteout exactly 10.0 cm of chloroform and 25.0 cm of
3
3
iodine monochloride solution into a stoppered bottle. Add about 50.0 cm distilled water and 10.0 cm KI
solution. Titrate immidietly the iodine liberated, against standard sodium thiosulphate using starch indicator.
3
3
Back titration : Pipette out 10.0 cm of fat solution into a stoppered bottle, add 25.0 cm of iodine
monochloride solution. Stopper the bottle, shake it carefully but throughly and leave to stand in dark
for an hour, with intermitant shaking.
3
3
Rinse the bottle necks with about 50.0 cm distilled water and 10.0 cm 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.
3
Results : 1) Blank titration reading = .......................... cm of std. Na S O
2 2
3
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, 2016
Determine the Iodine number of Oil/Fat
Marks-15
Requirements : 1) Oil/Fat sample in chloroform.
2)
3)
4)
5)
3
I2 (0.2 mole/dm ) in HgCl2
KI (10% W/V in H2O)
3
Sodium thiosulphate (0.1 mol/dm )
Starch indicator.
Procedure :
3
a) Blank titration : Pipette out exactly 25cm of standard iodine in stoppered bottle. Add about
3
20 cm of 10% KI solution and titrate immediately the iodine liberated against standard
sodium thiosulphate using starch indicator.
b)
3
Back titration : Pipette out exactly 10.0 cm of given oil sample into a stoppered bottle. Add exactly
3
3
25.0 cm of standard iodine solution. Shake the mixture well for 1 hour. Add 20 cm 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.
3
Results : 1) Blank titration reading ............ cm of standard sodium thiosulphate solution.
3
2) Back titration reading ............. cm 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, 2016
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, 2016
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, 2016
Marks-08
Solve the given Problems on sequence of DNA, RNA & Genctic Code
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May, 2016
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.
4.
Uptake of Na - ions by cation exchange resin.
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.
53
SHIVAJI UNIVERSITY, KOLHAPUR
B. Sc. Part-II, Practical Examination in Biochemistry, (New Course) April/May,2016
PAPER - II
All questions are compulsory
Question No. 5
Marks
....... 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