Detectors
The detectors in an HPLC are quite different than those for a GC. Whereas GC detectors must detect solutions of gases, HPLC detectors must detect liquid solutions. When there is no analyte component present, the mobile phase flows through the detector by itself. When an analyte component is present, the properties of the liquid mobile phase change. Detectors are designed to detect changes in the concentrations of substances in the mobile phase. The most popular detector for HPLC is a UV/visible spectrophotometer, usually a special type called a diode array detector (DAD). This detector measures the ultraviolet and visible spectrum of the solution as it flows through. Most mobile phases are not active in the UV or visible spectral range, so the detector does not respond to pure mobile phase. A DAD simultaneously measures all of the wavelengths of UV and visible absorption of the analyte, so there are many ways that the data can be presented. For example, a simple chromatogram of retention volume (similar to retention time) versus absorption can be plotted. This will look similar to a gas chromatogram. In addition, however, many HPLC systems are capable of plotting absorption versus retention volume versus wavelength. This “3-dimensional” (3-D) plot presents a great deal more information than a simple 2-dimensional plot. An example of a 3-D plot is given in Figure 6.12. There are also other detectors that can be used for HPLC. They are briefly discussed below.
• Fluorescence – This detector will detect only those substances that exhibit fluorescence, such as the illicit drug LSD. This limits its utility, but it is extremely sensitive. Ultraviolet or visible light is used as the source. A discussion of fluorescence spectroscopy can be found in Chapter 5.
• Conductivity – Most liquids will conduct electricity to a greater or lesser extent. A small electric current is continuously being passed into the mobile phase and the ability of the solution to conduct the electricity is measured. When an analyte, especially a polar one is introduced into the mobile phase, the conductivity changes, thus enabling detection of the analyte. This works best if the mobile phase contains at least some water because water is a very good conductor of electricity.
• Refractive index – The ability of liquids and other transparent materials to bend and decrease the velocity of light as it passes through, is called refraction. All liquids refract light differently. A mobile phase with an analyte dissolved in it will refract light differently than the mobile phase alone. A refractive index detector can measure this change.
• Mass Spectrometry – LC/MS has not been around nearly as long as GC/MS, in part because of some formidable engineering hurdles that had to be overcome. When the mobile phase and analytes reach the mass spectrometer the mobile phase is stripped away and the mass spectrum of each component of the analyte is measured. As in GC, this permits separations and identifications to take place in one step.
