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between a stationary liquid phase and a mobile gas phase of the volatilised analytes as

they are carried through the column by the mobile gas phase. Its use is therefore

confined to analytes that are volatile but thermally stable. The partition coefficients

are inversely proportional to the volatility of the analytes so that the most volatile

elute first. The temperature of the column is raised to 50 300 oC to facilitate analyte

SS

N

SSP

Matrix

Immobilised 2 ́-pyridyl ligand

Thiol-containing

protein

Covalently attached

protein Thione form

Detect at 343 nm

2-Thiopyridine

(i) Elute non-bound proteins

(ii) Remove unreacted thiopyridyl

group with 4 mM dithiothreitol

(iii) Displace bound protein with 20–50 mM

dithiothreitol

Released purified

protein

2,2 ́-Dipyridyldisulphide

Regenerated ligand

Matrix

Matrix SH + P SH

Matrix SS

+

NSH S

H

N

SS

NN

N

PSH

Fig. 11.11Principle of purification of a protein (P-SH) by covalent chromatography.

471 11.9 Gas chromatography