BE 484 Analytical Methods in Biotechnology

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Major Exam (2ndsemester 2003-2004)

AttemQLillJ ..9uestions

Theory Max Marks: 40

Time: Total 2 hours

Q. I What is the basic principle of chromatographic method? Explain the difference

between open and closed column chtomatography. Between HPLC and FPLC, which
method has high resolving power and why? I + 1+ 2 = 4
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Q. 2 In a protein there are two solvent accessible tryptophan residues, one is on the
~4 surface and the other is in the interior surrounded by charged residues. How do you
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selectively quench the tryptophan on the surface?Explain with principle the experiment. 2

Q. 3 State and define various parameters that are used to evaluate the performance in
chromu~0graphicseparations. 2
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Q. 4 Distinguish between g:-oupspecific ligand affinity chromatography and immobilized'
metal affinity chromatography. Illustrate your answer with suitable examples. 3

Q. 5 How does the polarity of the solvent affect the absorrtiuD maxima of the protein?
How can the solvent rJerturbation method be' used to find the position of a particular
an1inoacid in native protein? 3

Q. b a. What is the principle on which zonal and isoUichl'phoresis electrophoretic

systems are based? Gi"r. an application for each category. 3

t... What is the function of the stacking and C separating gel 4\ystemsin DISC '.

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electrophoresis? .2
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c. What are gradient gels and what might be these used for? 2

Q. 7 a. Derive the expression relating velocity to mass-to-charge ratio in the MS

method. An ion of mass m = 100 amu and charge z = 1, is accelerated with a potential of
I kV. Calculate its velo<-ityl'l m/sec. The same ions encounter a magnet;..:field, the
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geometry of which requires a trajectory with radius r = 10 cm for the ions to reachthe
I exit slIt. What must be the magnetic field strength? 1+ 1+ 1= 3

b. Why is it essential thC1f

the ion-source be evacuated in the MS method?

c. Distinguish between electron-impact ionization and chemical ionization. For

which specific applications...would you use these
. two different methods of ionization? 2
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Laboratory Questions Max. Marks: 20 l
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I Q.1 a. While checking the punty nf a protein, which chromatographic system (FPLC or
I HPLC) would you prefer and why? 2

b. Proteins A and B interact in aqueous medium. How can you show with the HPLC
experiment that they are binding to each other. Explain with thc drawings of the
chromatogram. 2

c. You have bC0ngiven two different concentrations of a pure protein for recording
CD spectra. Would you get same results while caJculating its secondary structure? I
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d.What is the need for having a pure protein in CD studies?

Q.2 a. Why is methylation of a triglyceride carried out prior to GC analysis?

b. State the reaction calalyzed by phospolipase A2. ...

c.What is the principle of Folin-Lowry method for estimation of proteins in a

fermentation (cell-free) broth?
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d. Distinguish between transmittance and absorbance?
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e. Name different parts of a typical double beam UY/visible spectrophotometer.

Q.3 a. Proteins A and B having a molecular weight of 16,500 and 35,400 move 1.3 cm
and 4.6 cm, respectively. whcn clecfrophore~ed through a gcL What is thc molecular
weigh~of a protein C, which moves 2.8 cm in the saplp,gel? 2

b. Wny is electrophoresis dOI1P;0 solutions of low sait concentration?

c. A -,mallenzyme has a sedimentation coefficient:Jf 3.4 S. Whcn it bindS'to its

substrate (a small organic molecule), its s value changes to 2.9 S. Explain this change. I

d. If a boundary moves halfway down a centri(uge cell in 20 min at 20,000 rpm,

how long w',:Jit take to reach the same position if the speed ;s 40.000 rpm ? 2

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d. Show the ions being formed with methaneas a reagent gas. Would these be
calledas the primaryor secondaryionsand why?Whatis theirfunction? 2+ 1+ 1 = 4

Q.8 What is the difference between boundary and zonal sedimentation and under which
!conditions(for what kind 0f data generation) would you use these two methods?
; 2+1= 3
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'Q.9 a. The specific activity of a sample ofe2p] ATP is 5.3 Ci/mmol on Jan 23. What will
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the specific activity be on Feb 18 of the same year~given the half-life of32p is 14.2days?

b. In an experiment to measure DNA synthesis, would you use eH] thymidine or

e2p] nucleotide and why? 1

c. You have in your lab a Geiger counter with an efficiency of 22% for 14Cand a
background of 6 cpm and a scintillation counter with an efficiency of 72% for 14Cand a
background of 38 cpm. In an experiment with 14(:,you expect your sample to have very
low activity-that is, from 75 to 100 cpm. Which counter should you chose andwhy? -3

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