Several Common Detectors for High-Performance Liquid Chromatography
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The function of the liquid chromatography detector is to convert the changes in the composition and content of the sample in the column effluent into detectable signals. Commonly used detectors include ultraviolet absorption, fluorescence, differential refractive index, and evaporative light scattering detectors.
1 Ultraviolet Absorption Detector (UV)
The ultraviolet absorption (UV) detector is currently the most widely used detector in HPLC. Its working principle is the Lambert-Beer law. This detector has high sensitivity, a wide linear range, is insensitive to changes in flow rate and temperature, and can be used for gradient elution separation. Ultraviolet absorption detection requires that the sample components to be detected have ultraviolet-visible light absorption, and the mobile phase used has no absorption, or no absorption at the wavelength where the analyte absorbs. Generally, detection is performed at the wavelength where the analyte has maximum absorption to obtain maximum sensitivity and anti-interference capability. When there is no maximum absorption, terminal absorption can be used. The choice of detection wavelength depends not only on the composition and molecular structure of the substance to be measured but also must consider factors such as the composition of the mobile phase and interference from coexisting components. In particular, various solvents have a certain lower limit of transmission wavelength. Beyond this wavelength, the absorption of the solvent becomes very strong, making it impossible to accurately measure the absorption intensity of the substance to be tested. The table below lists the lower limits of transmission wavelengths for some commonly used solvents in HPLC.
2 Fluorescence Detector (FD)
The fluorescence detector (FD) is a highly sensitive and selective detector that can detect compounds that produce fluorescence. The principle of the fluorescence detector is the same as that of fluorescence analysis. When a compound is excited by ultraviolet light, it emits light with a longer wavelength than the excitation light, called fluorescence or emission light. Many drugs and biologically active substances have natural fluorescence and can be detected directly. Some substances that do not fluoresce can be converted into fluorescent derivatives through chemical derivatization and then detected by fluorescence. Its minimum detectable concentration can reach 0.1 ng/mL, making it suitable for trace analysis. Generally, the sensitivity of the fluorescence detector is about 2 orders of magnitude higher than that of the UV detector, but its linear range is not as wide as that of the UV detector. In recent years, the laser-induced fluorescence detector, which uses a laser as the light source for the fluorescence detector, has enhanced the signal-to-noise ratio of fluorescence detection, thus having very high sensitivity and being widely used in trace and ultra-trace analysis.
Differential Refractive Index Detector (RID)
The differential refractive index detector (RID) is a universal concentration detector that responds to all solutes. Some components that cannot be detected by selective detectors, such as high molecular weight compounds, sugars, and aliphatic alkanes, can be detected by the differential refractive index detector. The differential refractive index detector measures the sample content based on the continuous measurement of the change in refractive index between the sample flow path and the reference flow path. When light enters another medium from one medium, refraction occurs due to the difference in refractive indices of the two substances. As long as the refractive index of the sample component differs from that of the mobile phase, it can be detected. The greater the difference, the higher the sensitivity. Within a certain concentration range, the detector output is proportional to the solute concentration.
4 Evaporative Light Scattering Detector (ELSD)
The evaporative light-scattering detector (ELSD) is a new type of universal mass detector that emerged in the 1990s. It is suitable for detecting components with volatility lower than that of the mobile phase, mainly used for detecting sugars, higher fatty acids, phospholipids, vitamins, amino acids, triglycerides, and steroids, and can detect unknown compounds when there are no standard substances and the structural parameters of the compounds are unknown. It has almost the same response to various substances, but its sensitivity is relatively low, especially for components with ultraviolet absorption. In addition, the mobile phase must be volatile and cannot contain buffer salts. Its universal detection principle overcomes the shortcomings commonly found in traditional HPLC detection methods and has been increasingly applied in HPLC, supercritical fluid chromatography, and countercurrent chromatography. Unlike UV and fluorescence detectors, the response of ELSD does not depend on the optical properties of the sample. Any sample with volatility lower than that of the mobile phase can be detected, regardless of its functional groups. Its sensitivity is higher than that of the differential refractive index detector, it is insensitive to temperature changes, has a stable baseline, and is suitable for use with gradient elution liquid chromatography.
The photodiode array detector (PDAD), also known as the rapid scanning ultraviolet-visible spectrophotometer, is a new type of light absorption detector. It uses a photodiode array as the detection element, forming multi-channel parallel operation. At the same time, it detects the light signals of all wavelengths separated by the grating, then projects them onto the array receiver, and then rapidly scans the diode array to collect data, obtaining a three-dimensional chromatographic spectrum where the absorbance (A) is a function of retention time (tR) and wavelength (L). This allows timely observation of the spectral data corresponding to the chromatogram of each component, thereby quickly determining the wavelength with selectivity and sensitivity. Computerized data processing can also perform spectral similarity comparison of chromatographic peaks, peak purity detection, and sample retrieval using spectral libraries, providing richer information for qualitative and quantitative analysis.
In a single-beam diode array detector, light emitted by the light source first passes through the detection cell, and the transmitted light is dispersed into polychromatic light by a holographic grating, which then shines onto the array elements, allowing light of all wavelengths to be detected simultaneously on the receiver. The optical signals on the array receiver are rapidly scanned and extracted using electronic methods, with each image taking only 10 ms, far exceeding the speed of chromatographic peak elution, thus allowing scanning with the peak.
Note: The above information and technical parameters are compiled and released by Yangzhou Darui Electric Co., Ltd.
