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RECEIVED for review July 29, 1982. Resubmitted February 2,1983. Accepted April 21,1983. This work received financial support from the U.S. Army Research Office, Contract No. DAAG-29-81-C-0023.
Microbore Liquid Chromatography with Flow Cell Fourier Transform Infrared Spectrometric Detection Robert S. Brown and Larry T. Taylor* Department of Chemistry, Virginia Polytechnic Instltute and State University, Blacksburg, Virginia 2406 1
Fourier transform Infrared spectrometry (FTIR) has been used as a detector for mlcrobore high-performance llquld chromatography (HPLC) through the use of a low volume transmlsslon flow cell. Characterlstlcs of the system have been determined and compared with analytlcal scale HPLC separations with FTIR detectlon. Improvement In sensltlvtty Is reported for the mlcrobore scale system over the analytlcal scaie system. Direct comparlson of the detection llmlls conslderlng equal quantttles Injected for 2,6dCferf-butylphenoI Is made for both systems.
The potential of Fourier transform infrared spectrometry (FTIR) as a highly specific and almost universal detector for high-performance liquid chromatography (HPLC) has been noted by several authors (1-4). Two basic approaches exist to utilize FTIR as an HPLC detector. One method makes use of a solvent removal-sample concentrationsystem with spectra being produced by diffuse reflectance measurements of collected fractions off-line (5). The second and more straightforward method involves the direct on-line sampling of the chromatographic effluent in a “flow cell” by standard transmission measurements with the solvent compensated for by a ratioing method (3). The second approach which we have adopted, although easy to implement, has several sometimes severe restrictions. To date it has found its biggest use with size exclusion separation (1,3, 6) where the eluting solvent normally has large IR windows and does not play a role in the separation. The major problem of using FTIR flow cell detection with normal- and reversed-phase separations lies in the incompatability of most solvents for both simultaneous chromatographic and spectroscopic measurements. In addition, gradient elution has yet to be demonstrated as feasible with on-line FTIR detection. Many solvents have reasonable IR transparency in the mid-IR (4000-400 cm-l) range at pathlengths of 1mm or less. These include Freon 113, CC14, CHCl,, and CDC&to name a few. To these solvents can be added small percentages of polar additives such as CH&N without loss of IR transparency. These neat or binary solvents have therefore proven to be reasonably good normal phase solvents for isocratic normal phase separations employing flow cell FTIR detection (2, 7,8). A t 1.0-0.2 mm flow cell pathlengths, sensitivity is poor compared to the more conventional
UV detection. The increased information content of IR over UV is, however, very substantial and if detection limits could be improved for FTIR detection, more general applications of this technique could be made. It is particularly appealing for complex mixture analysis where retention times alone are not adequate for compound identification. It is generally not practical to increase the pathlength employed beyond 1 mm since spectral transparency is rapidly lost even for good IR solvents. However, by increasing the concentration of eluting compounds, one can obtain better IR spectra of separated species with less than 1mm pathlength cells. Although this arrangement does nothing to enhance the absolute sensitivity of the FTIR, the relative HPLC/FTIR sensitivity is increased. Preparative columns have been employed (7) in this regard which have produced higher S I N spectra at the expense of chromatographic resolution. More recently significant success has been reported with microbore HPLC columns coupled with FTIR detection (7,8). Several papers describing the advantage of microbore HPLC have appeared (9, IO, 11). We see three basic advantages to their use with FTIR detection. First, the approximate 20-fold increase in eluent concentrationfor microbore columns (1mm i.d.) over analytical scale columns (4.6 mm i.d.) (11) aids us greatly in our attempt to increase the detectability of species as a function of sample amount injected. This is especially important given a limited sample size. Second, the low solvent consumption of microbore columns (typically
