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The generation of a micro-gradient for micro HPLC capillary columns is described. The system used is a modified and improved version of a split flow gradient elution device. Gradient tests have been performed and have shown good reproducibility under all conditions. The system has been successfully coupled with a particle beam LC-MS interface and the analysis of a mixture of twenty seven basic or neutral pesticides is reported.

The purpose of the study was to develop a micro HPLC system with physically changeable selectivity. This was done using two series-coupled beds of chemically differing stationary phases in a packed fused silica column. The selectivity change, in fine repeatable steps, was possible by the stepwise moving of a tube oven, covering only 50% of this “tandem column”. Each step of selectivity started with a position change of the oven, thus heating up a fresh position of the more polar and the non-polar column bed. As the chromatogram is thus repeated under these new selectivity conditions, multiple chromatograms result. For this reason we call the technique “Multi Micro HPLC”. This very simple mode of operation provides an analytical tool for developing separation conditions and which allows optimizing for a given special sample and checking for one of the most critical problems in HPLC: unidentified peak overlapping. The selectivity changes are nonlinearly correlated with the oven position with regards to temperature field distribution and are greater than is possible by simple change of column temperature. Micropacked columns are ideal for quick temperature changes. Elevated temperatures in HPLC help to shorten analysis time. The concepts discussed above provide an explanation of how a normal, formerly chemically homogeneous, column can become very temperature sensitive after detoriation at the inlet. In fact the column has become an unintentional “tandem column” and peak positions tend to move erratically as its selectivity changes drastically by inhomogeneous temperature changes along the separation path.

The retention and selectivity behaviour of some antiepileptic drugs were studied by micro high-performance liquid chromatography on nine types of phenyl-modified glasses, prepared with xylene solution containing phenyldimethylchlorosilane (P), diphenylmethylchlorosilane (D) or triphenylchlorosilane (T), using three types of glass with various mean pore diameters and/or specific surface areas. From elemental analysis data for carbon, the maximum number of accessible phenyl surface groups per 100A2 of glass (mean pore diameter: 335A, specific surface area: 69m2/g) in P, D and T gel was calculated to be 2.38, 1.58 and 0.76, respectively. Using various CH3CN−0.01 M KH2PO4 mixtures as eluents, the antiepileptic drugs were separated on all the glasses studied, but with different degrees of resolution. With an increase in the specific surface area, the k′ values of some antiepileptic drugs also increased.

The advantages of short microbore columns packed with a sorbent having a particle diameter of 3µm, such as the speed of analysis, the high solute concentration in the effluent and the detectability of small quantities are experimentally verified. The broadening in the valve is eliminated by the peak compression technique reached by adding a ion-pairing agent into the sample. The examples of catecholamines show the dependence of the height equivalent to a theoretical plate on the velocity of the mobile phase. The optimum is h=3 for a column 30mm long, i.d. 0.7mm, particle diameter 3µm. The minimal detectable quantity with a two-electrode amperometric detector with a gold electrode is from 30 to 40fmol for catecholamines.

Utilization of micro-HPLC is described for the direct measurement of the partition coefficients needed in studying the quantitative structure — activity relationships (QSAR). The method involves analyzing the solutes in acetonitrile — water as the mobile phase on commercially available reversed-phase stationary phases.

The separation of polycyclic aromatic hydrocarbons (PAH) on various reversed-phase columns (phenyl, ethyl, octyl and octadecyl) have been compared. The correlations between the logarithm of capacity factor (log k′) and several descriptors, which show the molecular size, shape and physical property of a solute, were determined. The results indicated the high possibility to predict the retention of PAH in reversed-phase high performance liquid chromatography.