Assignment 2021

DEPARTMENT OF BIOTECHNOLOGY
Techniques in Biotechnology
Assignment I: Numerical
(Last date for submission is 25/12/2021)
1. Describe preparation of 2litres of 0.4MHCl. The conc. HCl is 28% pure and its specific
gravity is 1.15.
2. What are the (a) H+ ion concentration, (b) pH, (c) OH- ion concentration and (d) pOH of a
0.001M solution of HCl?
3. What is the pH of 10-8M HCl?
4. The dissociation constant for lactic acid is 1.38 x 10-4. How much lactic acid must be
added to 2 g of NaOH to make 500 ml of buffer at pH 4.00?
5. The weak acid HA is 0.1% ionized (dissociated) in 0.2M solution (a) what is the
equilibrium constant for dissociation of the acid (Ka)? (b) What is pH of the solution? (c)
How much “weaker” is the active acidity of HA solution compared to 0.2M solution of
HCl? How many ml of 0.1N KOH would be required to neutralize completely 500ml of
0.2M HA solution?
6. To 2ml of a glucose solution, 1ml of a rxn mix containing excess ATP, NADP+,MgCl2,
hexokinase and G-6-P dehydrogenase was added. A340 of final solution increased to 0.91.
What was the [glucose] in the original solution? ε340 for NADPH is 6220
7. An ultra Centrifuge is operated at a speed of 58,000 rpm. Calculate

a. The angular velocity (ω in radians per second) and the centrifugal field at a point
equivalent to 6.2cm from the centre of rotation.
b. How many ‘time g’ is this equivalent to?
8. Catecholamines (epinephrine and nor-epinephrine) can be converted to fluorescent

compounds (lutines) by oxidation and alkali treatment. The concentration of
catecholamine was determined by excitation at 405nm and If at 495. The two
wavelengths do not discriminate the two compounds. The following were recorded:
Iu = 1.5ml urine treated to induce catecholamines = 52 units; Ib = 1.5ml urine treated
for non conversion of catecholamines to fl cmpnds = 6u; Is = 1.5ml urine treated to
which 0.25µg epinephrine std added = 85. Calculate (a) [catecholamine] in the sample
and (b) if 900ml of urine were collected in 24hrs, the total daily catecholamine excretion.
9. Calculate the relative centrifugal field (RCF) exerted at the top and bottom of a centrifuge
tube being centrifuged in a fixed-angle rotor, assuming that the rotor dimensions for the
mininmum radius (rmin) at the top of the tube is 4.8cm and for the maximum radius (r max)
at the bottom of the tube is 9cm and that the rotor is spinning at a speed of 12000 rpm.
10. A research paper describes a method to pellet certain molecule, using a fixed angle rotor
(rotor A) that has a capacity factor (k) of 225 in 4h at its maximum speed. But you wish
to use a rotor (rotor B) that has a k factor of 63. How long do you think the spin has to be
done with the rotor B at its maximum speed to pellet the same molecule? (Hint: t = k/s)
11. A protein was subjected to ultracentrifugation at 59780rpm and a temperature of 20 oC.

Using the technique of sedimentation velocity, measurements were taken of the radial
position of the sedimenting protein boundary as a function of centrifugation time:
-------------------------------------------------------------
Time (min) Radial position (r in cm)
---------------------------------------------------------------
8 6.089
16 6.179
24 6.270
32 6.362
40 6.454
48 6.549
----------------------------------------------------------------
(i) From the information provided plot a graph of log r (centimeters) against time
(seconds) and hence calculate the sedimentation coefficient of the protein.
(ii) In subsequent analyses, also at 20oC, the protein was found to have an average
diffusion coefficient (D) of 4.0 x 10-11 m2 s-1, and a partial specific volume of
0.734 x 10-3 m3 kg-1. Calculate the relative molecular mass of the protein. (Given:
Molar mass (Mr) = RTs/D(1-vρ) Kg mol-1; ρ of water 998 kg m-3; The gradient of
the plot of log r (centimeters) against time (seconds) = sω2/2.3; ω = 2Πrpm/60; R
= 8.314 J K-1 mol-1; 0oC = 273 K; Also relative molecular mass = Mr x 1000)
12. The following table shows the distance moved in an SDS-PAGE by a set of marker
proteins of known Mr. A newly purified protein (X) run on the same gel showed a single
band that had moved a distance of 45mm. Construct a calibration graph by plotting log
Mr versus distance moved for each of the marker proteins and hence determine the Mr of
the protein X.
Protein Mr Distance moved (mm)

Transferrin 78 000 6.0
Bovine serum albumin 66 000 12.5
Ovalbumin 45 000 32.0
G3PDH 36 000 38.0
Carbonic anhydrase 29 000 50.0
Trypsinogen 24 000 54.0
Soyabean trypsin inhibitor 20 100 61.0
β-Lactoglobulin 18 400 69.0
Myoglobin 17 800 69.0
Lysozyme 14 300 79.0
Cytochrome c 12 400 86.5
Answer page no 230 edition 8
13. Two compounds A and B were separated on a 25cm long column. The observed retention
times (tR) were 7min 20s and 8min 20s respectively. The base peak width for the
compound B was 10s. The reference compound, which was completely excluded from
the stationary phase under the given elution condition, had a tR ( = tM)of 1min 20s.
Calculate:
a. The adjusted retention time (t’R) for A and B. (Formula: t’R = tR -tM)
b. The capacity factor for the compounds. (K’ = tR/ tM)
c. The selectivity factor (α) for the two compounds. (α = K’B / K’A)
d. The number of theoretical plates (N) in the column. [N = (t’R/w)2],
(Where w = base peak width).
e. The resolution of the two compounds. (Rs = (√ N/4) (α-1/ α) (K’B/[1+ Kav])
f. The required column length to double the resolution. (H = L/N)
14. The relative molecular mass (Mr) of a protein was investigated by exclusion
chromatography using a Sephacryl S300 column and using some protein of known Mrs.
The following elution data was obtained. Plot the graph of Mr versus retention volume
and deduce the Mr of the unknown sample.
Protein Mr Retention
Volume (Vr)
Aldolase 158 000 22.5 cc
Catalse 210 000 21.4 cc
Ferritin 444 000 18.2 cc
Thyroglobulin 669 000 16.4 cc
Blue dextran 2 000 000 13.6 cc
Unknown ? 19.5 cc
15. Write chemical structure of products of the reaction involving N-terminal amino acid and
a. 2,4-dinitrofluorobenzene,
b. Dansyl chloroide,
c. Phenyl isothiocyanate

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DEPARTMENT OF BIOTECHNOLOGY

3. What is the pH of 10-8M HCl?

7. An ultra Centrifuge is operated at a speed of 58,000 rpm. Calculate

8. Catecholamines (epinephrine and nor-epinephrine) can be converted to fluorescent

11. A protein was subjected to ultracentrifugation at 59780rpm and a temperature of 20 oC.

Protein Mr Distance moved (mm)

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