Optical detector, HPLC

Fig. 15. Schematic layout of the HPLC chip a layout of the channel system, b cross section along the channel axis, c cross section along the detector cell axis. IS sample and mobile phase inlet, S split injector, OS outlet for rejected sample and mobile phase, C separation channel, F frit, D optical detector cell, OD outlet to waste, P positioning grooves for optical fibers (reprinted with permission from [84]. Copyright 1995 John Wiley)...

Supercritical fluid chromatography is compatible with both HPLC and GC detectors. As a result, optical detectors, flame detectors and spectroscopic detectors can be used. The FID is the most common detector used. However, the mobile phase composition, column type and flow rate must be taken into account when the detector is selected. Some care must also be taken such that the detector components are capable of withstanding the high pressures of SFC. 

Detectors. A wide variety of detection methods for CE have been reported many of which are directly comparable to those employed in HPLC. Optical detectors including ultraviolet, photodiode array, ultraviolet-visible and fluorescence have proved to be the most popular and all are commercially available. With these detectors the optically opaque polyimide layer must be removed and the somewhat limited sensitivity has been significantly improved by modifying the internal geometry of the capillary to give an increased pathlength. 

Improvement of optical detection limits can be achieved by either increasing the signal or decreasing the noise. In an ideal optical detector, noise is determined by the fundamental "shot or statistical noise of the photon flux incident on the photodetector. However, this ideal situation is not always realized in HPLC optical detectors, for which both electronic and thermal noise sources can exceed optical shot noise, degrading the signal to noise level Inherent to the optical design of the detector. 

Reduction of "non-optlcal noise sources below the shot noise limit of the detector is thus critical in extending the performance of HPLC optical detectors. One must understand these noise sources prior to optimizing the design of a detector, and prior to applying new technology such as that of the linear photodiode array to HPLC detection. 

What about the future We have tried to indicate already some of the areas where we and others are continuing to develop detection approaches for HPLC and FIA, and how those developments might proceed in specific regards. What else is there to be done in the future, aside from the use of lasers in optical detectors, which some of our colleagues are already developing or have successfully developed ... 

Detectors provide another route to optimizing selectivity by enhancing detectability of various classes of compounds. Discussion is included that describes the noise sources in optical detectors that allow optimization of such detectors. Two additional chapters deal with new and potential detectors including laser fluorimetry for HPLC, microcolumn HPLC, and FIA. 

The fluorescence HPLC detector (HPLC FL) is similar to the UV-absorp-tion HPLC detector (HPLC UV) in that a source of UV radiation is made incident to a micro-flow cell in whieh the chromatographically resolved analyte passes through. However, a photomultiplier tube (PMT) and associated optics are positioned at right angles relative to the incident UV radiation. Lakowicz has articulated just what molecular luminescence is (93) ... 

In the chiral analysis of many drugs, pharmaceuticals and agrochemicals by HPLC, detection is mostly achieved using the UV mode [1-7], and hence this detection mode has also been used for the chiral resolution of some environmental pollutants [8], However, some organochlorine pollutants are transparent to UV radiation hence, the UV detection mode is not suitable and some other detection devices have to be used in chiral HPLC, the most of which are MS, optical detectors and so on. 

To avoid extra-colximn band broadening, a split injector, a packed column, a frit and an optical detector cell have been integrated onto a silicon chip (see Figure 9). Internal volumes are 0.5 [iL for the empty column, and 3.5 nL for the total of the dead volumes on the chip. A single capillary of the frit had a defined volume of only 59 fL (see Figure 10). Preliminary results indicated that these HPLC chips could be operated at pressures up to 120 atm, with the limiting factor not the chip itself, but the interface with the peripheral system. It was possible to pack the capillary column on the assembled chip, and a separation of two components could be obtained [47]. 

FTIR instrumentation is mature. A typical routine mid-IR spectrometer has KBr optics, best resolution of around 1cm-1, and a room temperature DTGS detector. Noise levels below 0.1 % T peak-to-peak can be achieved in a few seconds. The sample compartment will accommodate a variety of sampling accessories such as those for ATR (attenuated total reflection) and diffuse reflection. At present, IR spectra can be obtained with fast and very fast FTIR interferometers with microscopes, in reflection and microreflection, in diffusion, at very low or very high temperatures, in dilute solutions, etc. Hyphenated IR techniques such as PyFTIR, TG-FTIR, GC-FTIR, HPLC-FTIR and SEC-FTIR (Chapter 7) can simplify many problems and streamline the selection process by doing multiple analyses with one sampling. Solvent absorbance limits flow-through IR spectroscopy cells so as to make them impractical for polymer analysis. Advanced FTIR... 

Although this section provides a brief description of most commonly nsed detectors for HPLC, most of the focus is on a few detection modes. Optical absorbance detectors remain the most widely nsed for HPLC, and are discnssed in some detail. We also focns on flnorescence, condnctivity, and electrochemical detection, as these methods were not widely nsed for HPLC in the past, bnt are especially well suited to micro- and nano-flow instrnments becanse of their high sensitivity in small sample volumes. Mass spectrometry has also come into wide and rontine nse in the last decade, but as it is the subject of another chapter, it will not be fnrther discnssed here. Miniaturization has been particularly important for capillary and chip-based electrophoresis, which often employs sub-nanoliter detection volnmes [36,37]. 

---