The refractive index (RI) detector is unique among common liquid chromatography (LC) detectors because it is truly universal in its detection capabilities. LC detectors based on the absorbance of ultraviolet (UV) light are the most popular detectors because they are simple, reliable, sensitive, and respond to a wide range of sample compounds, but only if the analytes have sufficient UV absorbance to detect. Fluorescence detectors are much more selective and can be more sensitive, but compounds must fluoresce to be detected. Mass spectrometry (MS) detectors are increasing in popularity and can provide extremely sensitive and selective detection, but only if the sample can be ionized. RI detectors respond to a universal, bulk property of the analyte — its refractive index. Usually referred to as differential refractive index detectors, these detectors detect peaks based on the difference in refractive index between the analyte and the background mobile phase. This is a benefit that makes the detector universal, but also a problem in that the detector also is sensitive to any other factor that affects refractive index. The major factors are temperature, pressure, and mobile-phase composition. This month's installment describes how RI detectors work and discusses some good practices to follow to get the most out of this powerful detector.
How It Works
A change in environmental temperature can be a major problem with RI detectors, because the refractive index of a fluid is dependent on its temperature. For this reason, RI detectors are contained in an insulated compartment. Most commercial detectors can control the temperature above room temperature, typically 30–35 °C up to 50–60 °C, although some models can cool the detector as well. Also, the incoming mobile phase must be at the same temperature as the thermostated portion of the detector, so heat exchangers are included to stabilize the temperature of the mobile phase. Although flow-cell volumes are relatively small, typically 8–10 μL, the heat exchanger volume may be 5–10 times this, or even more. This added volume means that RI detectors usually generate broader peaks than their UV counterparts with smaller total detector volumes.
The inherent design and operating principles of RI detectors leave them susceptible to several problem areas. Specifically, anything that causes changes in the temperature, pressure, or mobile-phase composition will create corresponding changes in the refractive index of the mobile phase as it passes through the sample cell. If this is not compensated by the static mobile phase in the reference cell, baseline disturbances will occur. We'll look at each of these problem areas next.