Solid Phase Reactions for Derivatization in Hplc (HPLC-SPR)

Publisher Website
Google Scholar
Abstract
As in all other forms of chromatography, high performance liquid chromatography (HPLC) has come to rely quite heavily on derivatization of individual analytes for improved qualitative and quantitative identifications (1–13). Virtually all of this work has involved the use of homogeneous type derivatizations, wherein the sample solution to be injected, in the pre-column approach, or the HPLC effluent, in the post-column approach, are fully mixed with the derivatization reagents in solution. Clearly, homogeneous type derivatizations can be done off-line or on-line, in either the pre-or post-column modes, but in general, pre-column methods have been done offline, and post-column approaches have been done both on-line and off-line. Online type derivatizations, in either the pre-or post-column modes, appear to offer some very significant advantages. Major among these is the ability to perform derivatization-separation-detection or separation-derivatization-detection following injection of the sample mixture. The other off-line approach requires an initial derivatization off-line, then injection-separation-detection, in the pre-column mode. Off-line, post-column methods require injection-separation-derivatization off-line, then detection. Ideally, for any derivatization method, be this pre-or post-, on-line or off-line, one would like to be able to avoid any additional sample pre-treatment before injection, and any extra sample/analyte handling after the point of injection. Unfortunately, on-line, pre-column derivatizations are very rare in the existing literature, and this has to do with the major problem of using solvents for the derivatization step that will be compatible with the separation needed via the HPLC step. Most recently, various instrument manufacturers have attempted to automate off-line, pre-column derivatizations with automated sample injections-separation-detection of a large number of samples. Automated on-line, post-column derivatizations have also now become available, wherein a homogeneous reaqent solution is mixed, after the analytical column, with the HPLC effluent, in a small dead volume, high mixing efficiency chamber before the elevated temperature reaction coil. The final, derivatized eluent solution with unreacted derivatizing reagent are then passed into the detector for the final analyte detection/identification.