The method under development at NIOSH uses a pH gradient to accelerate the elution of highly retained compounds, rather than the more common organic modifier gradient. This is made possible because isocyanate derivatives of 1-(9-anthracenylmethyl)piperazine62,89,90 (MAP) contain a highly basic tertiary amine group that is easily protonated. The degree of protonation, which is controlled by the mobile phase pH, has a very large effect on the retention of MAP derivatives, especially those containing multiple derivatized isocyanate groups. Several disadvantages associated with organic modifier gradients are minimized or eliminated with the pH gradient. Reequilibration time between runs is very short, baseline changes during the gradient are relatively small, and elution of artifacts originating from the mobile phase solvents is less likely because the gradient selectively accelerates compounds with amine functionalities. Similarly, if a MAP-isocyanate derivative should co-elute with a non-amine interferant, a small change in pH gradient will move the MAP derivative away from the interferant. With a nonselective organic modifier gradient, the separation of an analyte from a co-eluting interferant is not so straightforward since both compounds will respond to a change in the gradient. The UV response of MAP-derivatized isocyanates is only minimally affected by the changing pH. The fluorescence (FL) response of MAP derivatives is greatly affected by mobile phase pH, but this is readily corrected by lowering the pH with post column addition of acid. Disadvantages of pH gradients are that they are limited to ionizable compounds and these ionizable compounds are more prone to problems associated with adsorption in the chromatographic system. These problems may include tailing peaks, reduced peak heights and areas, and carry-over. Special procedures or equipment (such as inert fluid paths in the HPLC or base-deactivated analytical columns) are necessary to avoid adsorption problems. Another type of gradient that has been used for total isocyanate analysis is a mobile phase flow gradient.91 Instead of changing mobile phase composition, the flow rate is increased to reduce the retention time of lateeluting compounds. The advantages of this procedure are that only one mobile phase is required, there is minimal disturbance of the baseline, and no mobile-phase related artifacts are eluted. One of the disadvantages is that, although peaks are narrower, UV or FL detector responses decrease with increasing flow rate. Also, since the acceleration of the analytes is directly proportional to the flow rate increase, pressure limitations make the accelerating power of this gradient modest compared to organic modifier or pH gradients. Capillary-zone electrophoresis (CZE) has been investigated for isocyanate analysis.92 It has several advantages relative to HPLC — low solvent consumption, relatively short analysis time, higher resolution, and a greater certainty that all analytes will reach the detector. Moreover, there is a degree of selectivity associated with the technique because only charged analytes migrate to the detector. The major disadvantage of CZE is the relatively poor concentration sensitivity owing to the extremely small injection volumes used. Both 1-(2methoxyphenyl)piperazine (MOPP) and MAP have been found to be useful derivatizing reagents for CZE analysis.93 One advantage that MAP derivatives possess is that they are more basic than MOPP derivatives. As a result, they are protonated in the solutions of relatively high organic content needed for dissolving oligomeric isocyanate species. f.
Identification
In typical chromatographic analyses of environmental contaminants, analytical standards exist for the analyte of interest. The analyte is identified as such in a real sample if its chromatographic retention time matches that of the analytical standard. However, for isocyanate species, pure analytical standards generally exist only for derivatized monomers. Moreover, in many environments, monomers contribute very little to the total isocyanate group present. The analysis of a derivatized bulk of prepolymeric isocyanate product can be very useful in identifying non-monomeric isocyanate species in real samples collected during use of that product. There are limitations to using such products as analytical standards for identification and quantification. Not 1/15/98
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