Detectors for HPLC

Schematic diagrams of flow cell detectors for HPLC using (a) UVA/is absorption spectrophotometry and (b) amperometry.

Quantitative accuracy and precision (see Section 2.5 below) often depend upon the selectivity of the detector because of the presence of background and/or co-eluted materials. The most widely used detector for HPLC, the UV detector, does not have such selectivity as it normally gives rise to relatively broad signals, and if more than one component is present, these overlap and deconvolution is difficult. The related technique of fluorescence has more selectivity, since both absorption and emission wavelengths are utilized, but is only applicable to a limited number of analytes, even when derivatization procedures are used. 

To appreciate the ways in which mass spectral data may be processed to utilize fully the selectivity and sensitivity of the mass spectrometer as a detector for HPLC. 

The ultraviolet-visible spectrophotometer is the most widely used detector for HPLC. The basis of UV-VIS detection is the difference in the absorbance of light by the analyte and the solvent. A number of functional groups absorb... 

Roe, D. K., Comparison of amperometric electrochemical detectors for HPLC through a figure of merit, Anal. Letts., 16, 613, 1983. 

After passing through the column, the separated solutes are sensed by an in-line detector. The output of the detector is an electrical signal, the variation of which is displayed on a potentiometric recorder, a computing integrator or a vdu screen. Most of the popular detectors in hplc are selective devices, which means that they may not respond to all of the solutes that are present in a mixture. At present there is no universal detector for hplc that can compare with the sensitivity and performance of the flame ionisation detector used in gas chromatography. Some solutes are not easy to detect in hplc, and have to be converted into a detectable form after they emerge from the column. This approach is called post-column derivatisation. 

A large number of devices have been used as detectors for hplc. The characteristics of five of the more important types are described, and examples are given of the range of samples for which they can be used. The use of derivative preparation as an aid to detection is considered. 

FTIR is a natural for HPLC in that it (FTIR) is a technique that has been used mostly for liquids. The speed introduced by the Fourier transform technique allows, as was mentioned for GC, the recording of the complete IR spectrum of mixture components as they elute, thus allowing the IR photograph to be taken and interpreted for qualitative analysis. Of course, the mobile phase and its accompanying absorptions are ever present in such a technique and water must be absent if the NaCl windows are used, but IR holds great potential, at least for nonaqueous systems, as a detector for HPLC in the future. 

When compared to fluorescence detectors for HPLC, the design of a fluorescence detector for CE presents some technical problems. In order to obtain acceptable sensitivity, it is necessary to focus sufficient excitation light on the capillary lumen. This is difficult to achieve with a conventional light source but is easily accomplished using a laser. The most popular source for laser-induced fluorescence (LIF) detection is the argon ion laser, which is stable and relatively inexpensive. The 488-nm argon ion laser line is close to the desired excitation wavelength for several common fluorophores. The CLOD for a laser-based fluorescence detector can be as low as 10 12 M. 

UV/vis Electronic transitions Commonest detector for HPLC. Not very structurally informative Sensitive lack of specificity usually requires deconvolution techniques... 

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]. 

The most commonly used detector for HPLC is still the UV-Vis absorbance detector. The amount of light transmitted through a solution of concentration c in a flow cell with path length ( is given by the Beer-Lambert law. 

Riordon JR. Diode array detectors for HPLC— High performance across the spectrum. Analytical Chemistry 72, 483A--187A, 2000. 

Electrochemical detection of carbohydrates at nickel-copper and nickel-chromium-iron alloy electrodes has been reported for sorbitol, and has been used as a detector for HPLC analysis [36]. Oxidation of various carbohydrates at the electrodes was used for detection, and baseline separation was achieved for mixtures of sorbitol, rhamnose, glucose, arabinose, and lactose. 

Fluorescence detection, because of the limited number of molecules that fluoresce under specific excitation and emission wavelengths, is a reasonable alternative if the analyte fluoresces. Likewise, amperometric detection can provide greater selectivity and very good sensitivity if the analyte is readily electrochemically oxidized or reduced. Brunt (37) recently reviewed a wide variety of electrochemical detectors for HPLC. Bulk-property detectors (i.e., conductometric and capacitance detectors) and solute-property detectors (i.e., amperometric, coulo-metric, polarographic, and potentiometric detectors) were discussed. Many flow-cell designs were diagrammed, and commercial systems were discussed. 

Figure 25-20 Photodiode array ultraviolet detector for HPLC. (a) Dual-beam optical system uses grating polychromator, one diode array for the sample spectrum, and another diode array for the reference spectrum. Photodiode arrays are described in Section 20-3. (fc>) Reversed-phase chromatography (using C18-silica) of sample containing 0.2 ng of anthracene, with detection at 250 nm. Full-scale absorbance is 0.001. (c) Spectrum of anthracene recorded as it emerged from the column. [Courtesy Perkln-Elmer Corp.. Norwalk. Cl]...

The availability of commercial bench-top mass spectrometry detectors for HPLC is facilitating the development of HPLC-MS methods for many analytes. This is more common in pharmaceutical than food applications. As is generally the case, mass spectrometry is first being applied to standard solutions and relatively simple samples before being applied to more complex food matrices. A standard mixture of ten vitamers, AA, DHAA, PN, PL, PM, thiamine, nicotinic acid, nicotinamide, pantothenic acid and biotin, were recently determined by HPLC-particle beam... 

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