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Affinity Capillary Electrophoresis 325
improved and less interference from impurities is anticipated. Selection of the
analyte and ligand concentration ranges allowing a complete binding isotherm
to be constructed is to a large extent dependent on the affinity of the system.
However, due attention should be paid to the sensitivity of the detection system.
6.2. Binding Constants
6.2.1. Procedures for Mobility Shift ACE (87)
6.2.1.1. Materials and Instrumentation
1. Analyte solution containing non-interacting internal marker, e.g. a small synthetic
peptide, dimethylsulphoxide or other molecule that is not binding to the ligand.
2. Protect sample against evaporation by carefully overlaying 10–20 L light mineral
oil (Sigma M-3516).
3. Mobility shift ACE is best performed in instruments with good thermostatting
capabilities to ensure reproducible temperature conditions in each analysis.
6.2.1.2. Electrophoresis Buffer
For many ACE experiments, a phosphate electrophoresis buffer will provide
sufficient neutral pH-buffering capabilities and high enough ionic strength to
suppress unwanted electrostatic interactions (see Note 8), e.g. 0.1 M phosphate,
pH 7.4:
40.5 ml 0.2 M Na 2 HPO 4 (35.61 g/l of Na 2 HPO 4 2H 2 O).
9.5 ml 0.2 M NaH 2 PO 4 (27.6 g/l of NaH 2 PO 4 .H 2 O).
50 ml H 2 O.
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Fig. 7. detection 311 nm (200 nm for HSA blank); hydrodynamic injection for 100 s
(50 mbar). See Subheading 6.1 for explanation of (a)–(e). In contrast to warfarin, HSA
absorbs very little at 311 nm. It is observed that the migration time of warfarin alone (e)
is shorter than for free warfarin (a) in the HSA-containing sample. This is most probably
caused by adsorption of HSA to the capillary wall leading to decreased electroosmotic
flow and thus longer migration times for warfarin in the sample mixtures. (B) Electropherograms
of 200 M warfarin with or without 54 M HSA; free warfarin concentration
72 M. Experiments were performed on a Beckman P/ACE 5010 instrument.
Conditions: Uncoated fused silica (57 cm × 75 m i.d., 50 cm effective length); applied
voltage +15 kV; detection 200 nm; hydrodynamic injection for 60 s (0.5 psi) (•, HSA;
,warfarin sample; □, warfarin standard). Modified and reproduced from (9,114).