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There is an urgent need for detection technologies that enable accurate and precise quantification of solutions containing small organic molecules in a manner that is rapid, cheap, non-labor-intensive, readily automated, and without a requirement for specific analyte standards. We provide a theoretical analysis that predicts that the logarithmic nature of the working domain of the evaporative light-scattering detector (ELSD) will normally bias toward underestimation of chromatographically resolved impurities, resulting in an overestimation of analyte purity. This analysis is confirmed by experiments with flow injection analysis (FIA) and gradient reversed-phase high performance liquid chromatography (RP-HPLC). Quantification is further compromised by the dependence of response parameters on the matrix composition and hence on the retention time of the analyte. Attempts were made to ameliorate these problems by using the response surface of a single compound to calibrate throughout the HPLC gradient. A chemiluminescent nitrogen detector (CLND) was also used in a similar manner, and the performance of the two techniques were compared against those of each other and that of a reference standard technique. A protocol for this purpose was developed using proton nuclear magnetic resonance (1H NMR) and the ERETIC method to enable quantification by integrating proton signals. The double-blind comparison exercise confirmed molar nitrogen CLND response to be sufficiently stable and robust across a methanol gradient to be used with a single external nitrogenous calibrant to quantify nitrogen-containing compounds of known molecular formula. The performance of HPLC-CLND was very similar to that of NMR, while that of HPLC-ELSD was seen to be significantly worse, showing it to be unsuitable for the purpose of single-calibrant quantification. We report details and experience of our use of RP-HPLC-CLND-MS to characterize and quantify small amounts of solutions of novel compounds at nominal levels of 10mM in microtiter plate (MTP) format.

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Capillary electrochromatography (CEC) was coupled with electrospray (ESI) mass spectrometry and evaluated as an on-line high resolution technique for the separation and characterization of some typical pharmaceutical compound mixtures. Dispersion effects were observed and are discussed in the context of an optimized CEC/MS interface.

A prototype comb& capillary electrocbromatography/electmpray mass spectrometry interface (CEC/ESE MS) bas beem designed, built and evaluated for rapid, electricallydriven chromatographic analyses which facibtes high separation effkiincies with mass spectrometric detection. The system has an integral electrokineti iqjectien system and separation is performed in the interface probe using short columns with high field strengths. The system is eqdky capable of performing on-line capillary electrophoresis mass spectrometry (CEFrS) Capillary electrochromatography (CEC) has been slow to develop since it was first demonstrated by Pretorius et al. ' in 1974 as an alternative method to hydrostatic pressure for effecting a solvent flow in a packed capillary column. The flat flow profile resulting from electrically-driven systems, such as CEC, is in contrast to the parabolic Poiseuille flow of pressure-driven systems and results in enhanced chromatographic performance and the opportunity of using a very small particle diameter with no theoretical restriction on column length. It was not until 1981 that Jorgenson and Lukacs' revived interest in the technique when they described the separation of neutral molecules in packed capillaries using electroosmosis (EO) to drive the flow. The current resurgence of interest in the technique stemmed from reports by Knox and Grant3v4 which were instrumental in providing solutions to some of the practical problems of CEC including the suppression of bubble formation, thought to be induced by Joule heating, by the use of a pressurised electrochromatography system. ' Recently, new improved methods of reproducibly packing more robust columns697 have greatly increased the reliability of the technique and subsequently provided the opportunity to begin developing and optimizing a mass spectrometry interface for true CEUMS.8 Verheij et a1.9 and Huegener et a1." have previously reported the coupling of pseudo-electrochromatography, a combination of both pressure-driven and electroosmotically-driven chromatography, with mass spectrometry where the flow profile approaches that in a pressure-driven system." Gordon et al. have reported true CEC/MS coupling to both electrospray (ESI)" and continuous-flow fast-atom bombardment mass spectrometry'' and note a loss of chromatographic resolution as Author for correspondence. a result of post-detection window dispersion in the length of unpacked capillary necessary for coupling to the mass spectrometer in their system. We recently reported similar dispersion problems consistent with a length of unpacked capillary used to couple the CE to the mass spectrometers but also demonstrated that if fully packed capillaries are used, electrophoretic integrity is maintained into the mass spectrometer and high separation efficiencies are realized.It is our belief that the maintenance of the high separation efficiencies possible with CEC should be the goal in an optimized combined system using true CEC/MS with short columns fully packed with small diameter particles ...