QUANTITATIVE STUDIES OF DIFFERENT INJECTION SYSTEMS IN CAPILLARY ELECTROPHORESIS

For a rigorous assessment of the precise amount of sample loaded, for quantitation purposes, different sample injection systems were evaluated with two commercially available units, Waters Quanta 4000 and Beckman P/ACE 2100. In the first system, sample introduction by hydrostatic means (i.e., placing the sample vial at some height, usually 10.1 cm, above the other capillary end) was evaluated. It was found that in this system there is a constant positive bias, i.e. the amount of sample loaded lies on a curve parallel and above the theoretically predicted loading curve. However, the excess of mass loaded was constant along the injection times explored (covering from 5 to 35 s) and, for a 75 mu m capillary, was found to be of the order of +6 nL (above the expected injected value). Thus it is easy to correct for this sample bias. In the electrokinetic mode, a very good correlation between expected and predicted sample loads was obtained for both units. In the pressure system (by positive pressure from a nitrogen tank, Beekman unit) a substantial discrepancy was found between experimental and predicted values (13.5% overload). Since the manufacturer claims a constant pressure of 0.5 psi, i.e. 3447.5 Pa, it would appear that the injection pressure is higher than the given value. Other causes for variation in sample load (e.g., as caused by diffusion of sample in the capillary just prior to injection, by the sudden insertion of the capillary tip into the sample vial, etc., lumped together in a general term as ''extraneous injection'') have been evaluated and found to contribute to an additional sample volume injection of the order of 1-2 nL, i.e. quite negligible. In conclusion, it is felt that both the hydrostatic and electrokinetic injection modes are highly reliable. The positive pressure mode can fail due to leakage of O-rings, valve malfunctioning and to the typical problems of mechanical systems. Thus, the latter injection system should be checked frequently for potential mechanical failures.