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6.3.3 Operation of UV-visible absorbance detectors
The user must set a few operating parameters to use the detector. The main controls are generally the operating wavelength, the absorbance range, an autozero and the detector rise time. Setting the rise time has already been discussed. The range switch usually only affects the output signal at the recorder terminal. This output is usually 0-10 mY, with lOmV corresponding to full scale on the selected range, and is used when connecting the detector to a strip-chart recorder. Most detectors have a second output for connection to a computer or integrator. This is typically 0-1V, corresponding to 0-1 or 0-2 absorbance units (a.u.), and the output voltage is unaffected by the range switch. In this case, adjustment
Table 6.2 Approximate UV cutoff wavelength and RI of commonly used solvents
Solvent UV cutoff RI
Acetone 330 1.359
Acetonitrile 200 1.344
Benzene 280 1.501
Carbon disulphide 380 1.626
Carbon tetrachloride 265 1.466
Chloroform 245 1.443
Cyclohexane 210 1.427
Diethyl ether 220 1.353
Dimethyl sulphoxide 270 1.477
Ethanol 210 1.361
Ethyl acetate 255 1.370
Hexane 200 1.375
Methanol 210 1.329
Pentane 200 1.358
1-Propanol 210 1.385
Tetrahydrofuran 215 1.408
Toluene 285 1.496
of the size of the signal appearing on the computer or integrator output is adjusted at the integrator itself. The autozero control is used to automatically adjust the detector output to OV. This is particularly useful when a drifting baseline threatens to take the signal out of the integrator or recorder range.
The wavelength must be selected to correspond to a chromophore of the compound(s) of interest. Having the UV spectra of the analytes is helpful, preferably measured in the same solvent as the HPLC mobile phase, and at a similar pH. The availability of a wavelength scanning HPLC detector is very useful for this. Alternatively, reference to handbooks such as the Merck Index often provides useful information. Many compounds have absorbance maxima with highest absorbance coefficients below 220 nm, but a longer wavelength absorbance with a weaker chromophore may give better results. This is because of reduced likelihood of interferences and reduced absorbance due to the mobile phase. Certain solvents are suitable for use at low wavelengths, for example acetonitrile-water mixtures may be used at 200 nm, but any traces of absorbing contaminants may cause high background absorbances and poorer sensitivity. Use of recirculated solvents may also be problematic. Table 6.2 shows the minimum useful wavelength for a variety of commonly used solvents. The problem of choice of solvent can be particularly acute when using gradient elution.
An operating wavelength chosen to correspond to an absorbance maximum is optimum not only because of increased sensitivity, but also because there is a relatively small change in absorbance with wavelength close to the maxima. If detection is made at a wavelength where the analyte’s absorption coefficient varies rapidly as a function of wavelength, then small variations in the selected wavelength between analytical runs could lead to significant errors in quantitation of the solute. This is not usually a problem unless a change in wavelength followed by readjustment to the original wavelength has taken place. Typically, modern variable wavelength UV detector specifications allow ± 1 nm for wavelength accuracy, and ±0.5 nm for wavelength reproducibility when returning to any given wavelength setting.
6.3.4 Spectrophotometric detectors
Over the last decade a number of manufacturers have introduced spectrophotometric detectors for HPLC, capable of producing real-time absorbance spectra of the column eluent. To be useful in HPLC the detector has to produce a number of spectra every second, so that even fast-eluting peaks can have a number of spectral snapshots taken. Spectrophotometric detectors offer advantages over conventional variable wavelength detectors, particularly for method development. With an
Figure 6.3 Block diagram of a diode-array UV-visible absorbance detector. S, light source; BS, beamsplitter; L, lenses; C, flow cell; M, mirrors; G, diffraction grating; PDA1, PDA2, photodiode arrays; RC, reference flow cell.
unknown sample, the spectrophotometric detector can be used for scanning over a wide wavelength range to be confident of detecting all the sample components. The spectral information obtained for each peak can be used along with k' for peak identification. By comparing the spectra obtained at the beginning and end of a peak, information on the peak purity can be gained. If a single peak has different spectra at the beginning and end of its elution, it is certainly comprised of more than one component.