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BEL420 Analytical Methods in Bi

Major Exam Hnd semeste 2007-08
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'30- 0'1 - 2oD 8'
Time: 2 hours Max Marks: 50
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1. A key and often laborious element in analytical methods is the elimination
of/correction for errors. In isotope exchange gas chromatography coupled to mass
spectrometry analysis of a model biological system, list possible elTors and difficulties
and eXDlain,/
how they are corrected/overcome (in tabular form). (12)

Explain the principle of fluorescence microscopy. (6)

,/'. h) An analysis of cell contents shows their cytoplasm contains 5% by weight of

lutes: 1% is proteins of average molecular weight 45000; 1% is soluble lipids of
m~ecular weight ~ I % is sugars of mole~u'-ar weight 170~nd 2% is salts like !SCt.
What is the osmotic pressure TfiSide these cells relative ~ure watd at j t"C? If tEe
intracellular contents are released in a more controlled way (e.g. by a press) rather than Jr~ ell '(
by simple osmotic shock as above, what would a typical intracellular release vs. number
of passes profile look like? Show by a sketch. (6)+(6)

3. Two proteins have the same molecular weights. At pH 5.5 both have considerable
secondary structure and are at least 75% a helix. At pH 8.5, one of them loses all
structure and is a random coil; when returned to pH 5.5, its structure is restored. Devise a
procedure for separating these proteins. (3)

A procedure has been devised for purifying 1t particular enzyme using gel
. . chromatography. In an effort to increase the amount of material to be handled, you use a
s pie whose concentration of protein is ten times that normally used with this
procedure. The enzyme activity now elutes principally in the void volume. Explain what
happened. .

In general, to increase the amount of material to be handled, what physical parameters of

the column should one vary? (4)

~ J'Write five most important empirical rules of interpreting fluorescent spectra of

~ns. (3)

;J 2'(i) How ma~y ~pm are a~so~iatedwith 1yCi ?~radioa~tive material? lfthe instrume~t
. used for countmg IS 25% efficIent at detectmg dlSlntegratlOnevents, how many cpm w1l1
i yield? What is Cherenkov counting and for which radioactive material can it be
u~? 0r
(ii) What is the role of SDS and polyacryamide gel in SDS-P AGE electrophoresis?
What is the use of Fergueson's plot? (2+ 1)r-
(iii) Are there matrix peaks in the electrospray mass spectra and in MALDI? Explain.
Name two matrices used and their properties. (3)~