Detectors for HPLC
As with gas chromatography, numerous detectors have been developed for use in monitoring HPLC separations.14 To date, the majority of HPLC detectors are not unique to the method, but are either stand-alone instruments or modified versions of the same.
The most popular HPLC detectors are based on spectro- scopic measurements, including UV/Vis absorption, and fluorescence. These detec- tors range from simple designs, in which the analytical wavelength is selected using appropriate filters, to essentially a modified spectrophotometer equipped with a flow cell. When using a UV/Vis detector, the resulting chromatogram is a plot of absorbance as a function of elution time. Instruments utilizing a diode array spec- trophotometer record entire spectra, giving a three-dimensional chromatogram showing absorbance as a function of wavelength and elution time. Figure 12.29a shows a typical flow cell for HPLC when using a UV/Vis spectrophotometer as a de- tector. The flow cell has a volume of 1–10 μL and a path length of 0.2–1 cm. One limitation to using absorbance is that the mobile phase must not absorb strongly at the chosen wavelength.
Table 12.3 lists the wavelengths below which UV/Vis ab- sorbance cannot be used for different mobile phases. Detectors based on ab- sorbance provide detection limits of as little as 100 pg–1 ng of injected analyte. Fluorescence detectors provide additional selectivity since fewer solutes are capable of fluorescing. The resulting chromatogram is a plot of fluorescence intensity as a function of time. Detection limits are as little as 1–10 pg of injected analyte.
Another common group of HPLC detectors are those based on electrochemical measurements such as amperometry, voltammetry, coulometry, and conductivity. Figure 12.29b, for example, shows an amperometric flow cell. Effluent from the column passes over the working electrode, which is held at a potential favorable for oxidizing or reducing the analytes. The potential is held constant relative to a downstream reference electrode, and the current flowing be- tween the working and auxiliary electrodes is measured. Detection limits for amper- ometric electrochemical detection are 10 pg–1 ng of injected analyte.
Several other detectors have been used in HPLC. Measuring a change in the mobile phase’s refractive index is analogous to monitoring the mobile phase’s thermal conductivity in gas chromatography. A refractive index detector is nearly universal, responding to almost all compounds, but has a poorer detection limit of 100 ng–1 μg of injected analyte. Furthermore, a refractive index detector is not useful for a gradient elution unless the mobile-phase components have identical refractive indexes. Another useful detector is a mass spectrometer. The advantages of using a mass spectrometer in HPLC are the same as for gas chromatography. De- tection limits are quite good, typically 100 pg–1 ng of injected analyte, with values as low as 1–10 pg in some situations. In addition, a mass spectrometer provides qualitative, structural information that can help identify the analytes. The interface between the HPLC and mass spectrometer is technically more difficult than that in a GC–MS because of the incompatibility of a liquid mobile phase with the mass spectrometer’s high vacuum requirement. Recent developments in mass spectrome- try, however, have led to a growing interest in LC–MS.
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