he miniaturization of columns for chromatography and electrophoresis is an T important and rapidly developing area in the field of analytical chemistry.
However, every advance in column technology must be accompanied by simultaneous improvements in detection technology. The primary goal of detection improvement is to reduce the detection volume, generally from the microliter to the nano-or picoliter range, without sacrificing detection sensitivity or linearity. Unfortunately, these objectives are in direct conflict for the detection methods most commonly used in chromatography and electrophoresis. It is, therefore, worthwhile to consider new conceptual approaches to detection technology that offer the opportunity to achieve this goal without compromise. Although the examples cited herein will emphasize spectroscopic methods, the general concepts and principles are also applicable to other detection techniques.
Four promising approaches can be identified. First, it is desirable to develop those detection techniques in which the analyte response is proportional to the radiant power of the source. Under these circumstances, the loss in sensitivity resulting from reduction of the optical path length or volume can be fully compensated for by a proportionate increase in the source intensity. The detection techniques that clearly benefit from the use of high source power include thermo-optic and thermoacoustic methods, fluorescence and phosphorescence, photoionization and photoconductivity, and certain light-scattering measurements. For each of these detection techniques, some maximum source power level will be attained beyond which no further enhancement in signal-to-noise ratio is possible. Under ideal conditions, this power level is dictated by the saturation of the observed transition for the analyte molecule, when the signal reaches a maximum and remains relatively independent of power fluctuations. Under most practical circumstances, however, the maximum useful power level is limited by the background noise originating from the solvent or flow cell or by thermal effects such as optical distortion, dielectric breakdown, or sample degradation.
A second promising approach is to develop those detection techniques in which the response is independent of or only nominally dependent upon the path length or volume of the flow cell. Using this approach, sensitivity can be maintained as the detector flow cell is further miniaturized. For example, any spectroscopic technique in which a pump laser is used to induce a transient photochemical or photophysical event and a second probe laser is used to interrogate that event is dependent on the intersection volume of the lasers rather than on the flow-cell volume. Thus, thermo-optic techniques such as thermal lens calorimetry, thermal diffraction, and thermal deflection, as well as certain nonlinear Raman scattering techniques, often exhibit detection limits that are relatively independent of flowcell dimensions. In addition, spectroscopic techniques in which the response arises