A UV/Visible detector detects components that absorb light between 190 and 900 nm. For example, aromatics, pigments, proteins, and drugs can all be measured. By selecting the measurement wavelength, it is possible to measure the sample while suppressing the influence of interfering components (Fig. 1). In addition, the sensitivity can be improved by performing measurements at the wavelength where the maximum absorption occurs. By measuring the UV/Visible absorption spectrum at the elution peak and searching a library, it is possible to predict the components that are present. The purity can also be checked from the absorption spectrum.
A fluorescence detector detects components that emit light between 220 and 900 nm. Fluorescence detectors are more selective than UV/Visible detectors since the excitation and emission wavelengths are both specific for a particular substance. The excitation and emission wavelengths can be switched programmatically, making it is possible to simultaneously detect fluorescent substances with different wavelengths if the elution time is different (Fig. 2).
Refractive Index (RI) Detector
A Refractive Index (RI) detector detects components based on their refractive index relative to the mobile phase’s refractive index. Most compounds have a different refractive index to that of the mobile phase, so any component can be detected. However, refractive index variations also occur due to changes in temperature and solvent composition, so it is necessary to perform measurements at constant temperature and in isocratic solvent ratio mode.
Comparison of Detectors
Table 1 summarizes the characteristics of UV/Visible, fluorescence, and RI detectors. UV/Vis and fluorescence detectors are both highly sensitive as well as selective. A gradient elution method can be used with either of these detectors since they are less sensitive to temperature. A RI detector has the advantage of being able to detect a wide range of components; however, it is sensitive to temperature, so the gradient elution method cannot be used. A RI detector has lower sensitivity compared to a UV detector but can detect components that an UV detector cannot.
Although they are somewhat expensive, Evaporative Light Scattering Detectors (ELSD) are available that can detect a variety of components with high sensitivity in gradient elution mode.
|Gradient Elution Method
Figure 3 shows the lower sensitivity of the RI detector, but that detects components that the UV detector cannot.