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132 Forde
5. BDGE is toxic (by inhalation, contact with skin or if swallowed) and should
be handled in a fume hood. A 2 3−1 factorial experiment was used to explore
the effects of three parameters on the total ligand density: time for glutathione
immobilization (24 and 48 h), temperature during immobilization (37 and 45°C)
and the length of the spacer arm (BDGE, a 10-carbon spacer arm, and hexane
diglycidyl ether, a 12-carbon spacer arm). It was found that all of the parameters
have a significant effect on ligand density, and the highest ligand density was
obtained for immobilization conditions of 37°C for 24 h using BDGE as the spacer
arm. Using a suitable spacer arm is important: binding capacities can be increased
by placing the ligand at some distance from the matrix as this helps to reduce
the effects of steric hindrance caused by the matrix (9). The ideal spacer arm will
have appropriate coupling functionalities on both ends and an overall hydrophilic
character (10). The length of the spacer arm is critical. If it is too short, the arm is
ineffective and the ligand fails to bind substances in the sample due to the steric
interference of the matrix. If it is too long, non-specific effects become pronounced
and reduce the selectivity of the separation as very long spacer arms can bind
substances via hydrophobic interactions. Non-specific hydrophobic interactions
are undesirable in chromatographic systems as contaminants may be co-purified.
6. This stage is required in order to remove water and create an environment that
is conducive for glutathione immobilization. NaHCO 3 acts as a buffer for this
purpose.
7. The orientation of the glutathione ligand attachment to the base matrix is determined
by the pH at which the coupling reaction is conducted. At pH 7.5–8.5,
the coupling occurs primarily through the thiol group of glutathione molecule,
which leaves the amine group exposed for adsorption of GST protein. This was
found to yield significantly higher capture of the GST compared with the opposite
case at pH greater than 9 where the ligand coupling was enabled via the amine
group of glutathione and GST adsorption was via the thiol group. Analysis of
the glutathione-Streamline adsorbent prepared according to the steps described in
Subheading 3 showed that over 95% of free binding groups are amine groups,
which indicates that ligand coupling was achieved predominantly via the thiol
group as desired.
8. Ninhydrin is toxic (by inhalation, contact with skin or if swallowed) and should be
handled in a fume hood. Ninhydrin is used to detect ammonia or primary amines.
When reacting with free amines, a deep blue or purple colour is evolved. In order
to generate the ninhydrin chromophore, the amine is oxidized to a Schiff base by
redox transfer from the ninhydrin moiety.
9. Concentration of dithiodipyridine in reagent (mg/ml) = (Mass dithiodipyridine
added to reagent (mg) – mass dithiodipyridine collected on filter paper
(mg))/reagent volume (ml).
10. Dithiodipyridine reacts with thiol groups forming a disulphide bond, which can
be monitored by means of the absorbance change at 343 nm. Knowing the ligand
density of an adsorbent enables calculations of ligand utilization to be made and
what effect the process parameters have on the ligand density. By measuring the