High Performance Liquid Chromatography or HPLC uses diode arrays for recording the absorption spectrum of samples when ultraviolet and visible light passes through them. This enables the user to gather qualitative information about the samples. Major applications of HPLC diode array detectors include agriculture, environment, and industries such as petrochemical, energy, chemistry, life sciences, and pharmaceuticals.
Diode array detectors of HPLC have the advantage of the ability to select the best wavelength for analysis. Therefore, when selecting a diode array for use as a detector in HPLC, one should consider features such as resolution, wavelength range, near infrared ranges, baseline stability, low noise, and peak integration. Some vendors also offer the technique of detecting using a configurable light path formed from fiber optics.
An HPLC has a tungsten lamp emitting light in the visible range. This light enters a deuterium lamp that adds the UV to the visible light, forming a polychromatic beam. As this beam passes through a flow-cell, the sample in the flow-cell absorbs certain wavelengths. The output light then enters a grating, which splits the polychromatic beam into its constituent wavelengths and these pass through a slit before falling on an array of photodiodes, which measure their intensities.
As the diode array detectors measure all wavelengths simultaneously, it is able to acquire the spectra as well as the multiple single wavelengths at the same time by the different diodes in the array. The diode array detector has high selectivity, as it can identify different substances by their spectra.
One of the major advantages of the diode array detector is the tungsten lamp offering light in the extended visible wavelength. Additionally, by controlling the temperature of the optical unit of the diode array detector, its signal quality improves dramatically. Moreover, the diode array detector does not require a reference diode.
While other types of detectors use a diode for reference, for a diode array detector, there is no direct measurement of a signal when there is no absorption. Rather, the HPLC uses a detector balance. This happens automatically as the user switches the instrument on or just before conducting a measurement. The user achieves a detector balance by setting the absorption values for all wavelengths to zero. According to the Lambert Beer’s law, this allows the measurement of all intensities during an experiment to be made relative to this zero absorption intensity.
To cater to baseline changes or drift during a measurement, the diode array detector uses a reference wavelength. The user has to select a specific wavelength as reference, and make sure there is no absorption in the wavelength during the entire chromatography measurement. The user then uses the relative changes of the reference intensities for correcting the proportional changes occurring in other wavelengths.
Five factors majorly affect the measurements done by HPLCs using diode array detectors. These are the slit width, the bandwidth, the response time, and the flow cells. The user has to adjust all of them to obtain the best response from the diode array detector when testing the absorption of a sample. For instance, the slit defines the amount of light the detector measures. The bandwidth defines the window for the data acquisition. The response time defines the time resolution, and the flow cell defines the flow range.