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Commonly used detector for HPLC

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The role 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, refractive index, and chemiluminescence.

1 UV absorption detector

Ultraviolet absorption (UV) detector is currently the most widely used detector for HPLC. Its working principle is Lambert-Beer law. This detector has high sensitivity, wide linear range, and is insensitive to flow rate and temperature changes, and can be used for gradient elution separation. Ultraviolet absorption detection requires that the component of the sample to be tested has UV-visible light absorption, and the mobile phase used has no absorption, or no absorption at the absorption wavelength of the component to be tested. Generally, the detection is performed at the wavelength where the analyte has the maximum absorption in order to obtain the maximum sensitivity and anti-interference ability. When there is no maximum absorption, end absorption can be used. The choice of detection wavelength not only depends on the composition and molecular structure of the substance to be tested, but also factors such as mobile phase composition and coexisting component interference must be considered. In particular, various solvents have a certain lower limit of the transmission wavelength, beyond this wavelength, the absorption of the solvent will become so strong that the absorption intensity of the substance to be tested cannot be measured well. The following table lists the lower limit of the transmission wavelength of some commonly used solvents in HPLC.


2 Photodiode array detector

Photodiode array detector (PDAD), also known as fast scanning ultraviolet-visible spectrophotometer, is a new type of light absorption detector. It uses a photodiode array as the detection element to form multiple channels and work in parallel. At the same time, it detects the optical signals of all wavelengths separated by the grating, and then incidents them to the array receiver, and then quickly scans the diode array to collect data and obtain the absorption value (A) is a three-dimensional chromatographic spectrum plot of retention time (tR) and wavelength (L) as a function. Therefore, the spectral data corresponding to the chromatogram of each component can be observed in time, so as to quickly determine the wavelength with the best selectivity and sensitivity. Computerized data processing can also perform chromatographic peak spectral similarity comparison, peak purity detection and sample retrieval using the spectral library, providing more abundant information for qualitative and quantitative analysis.

In the single-beam diode array detector, the light emitted by the light source first passes through the detection cell, and the transmitted light is dispersed by the holographic grating into multi-color light, and then hits the array element, so that the light of all wavelengths can be detected on the receiver at the same time. The optical signal on the array receiver is quickly scanned and extracted by electronic methods, and each image only takes 10 ms, which is much faster than the chromatographic flow rate of the peak, so it can be scanned with the peak.

3 Fluorescence detector

The fluorescence detector (FD) is a highly sensitive and selective detector that can detect compounds that can produce fluorescence. The principle of the fluorescence detector is the same as the fluorescence analysis method. After the compound is excited by ultraviolet light, it emits light with a longer wavelength than the excitation light, which is called fluorescence or emission light. Many drugs and life-active substances have natural fluorescence and can be directly detected. Some non-fluorescent substances can be chemically derivatized to generate fluorescent derivatives, and then perform fluorescence detection. The minimum detection concentration can reach 0.1 ng/mL, which is suitable for trace analysis. In general, the sensitivity of a fluorescence detector is about two orders of magnitude higher than that of an ultraviolet detector, but its linear range is not as wide as that of an ultraviolet detector. In recent years, the laser-induced fluorescence detector, which uses laser as the light source of the fluorescence detector, has greatly enhanced the signal-to-noise ratio of fluorescence detection, and therefore has high sensitivity, and has been widely used in trace and ultra-trace analysis.

4 Refractive index detector

The differential refractive index detector (RID) is a universal concentration detector that responds to all solutes. Certain components that cannot be detected by selective detectors, such as polymer compounds, sugars, and fatty alkanes, can be detected by differential detectors. The differential detector is based on continuously measuring the change in refractive index between the sample flow path and the reference flow path to determine the sample content. When light enters another medium from one medium, it will be refracted due to the difference in refractive index of the two substances. As long as the refractive index of the sample component and the mobile phase are different, it can be detected. The greater the difference between the two, the higher the sensitivity. The output of the detector is proportional to the solute concentration within a certain concentration range.

5 Electrochemical detector

Electrochemical detector (ECD) is a selective detector, mainly including conductivity detector, ampere detector, dielectric constant detector and potentiometric detector, etc., which can detect electroactive compounds. Detectors such as conductivity and potential have been widely used in ion chromatography; dielectric constant detectors are similar in performance to refractive index detectors; amperometric detectors can detect oxidizing substances and have a wide range of applications.

Advantages of electrochemical detectors:

①The sensitivity is high, and the detection volume is generally ng level, which can reach pg level.

②Good selectivity, which can measure extremely trace electroactive substances in a large number of non-electroactive substances;

③Wide linear range, usually 4 to 5 orders of magnitude;

④The equipment is simple and the cost is low;

⑤Easy to operate automatically.

6 Chemiluminescence detector

Chemiluminescence detector (CD) is a fast and sensitive new detector developed in recent years. It has the advantages of simple equipment, low price, and wide linear range. The principle is based on the chemical reaction of certain substances at room temperature to generate reaction intermediates or reaction products in an excited state. When they return to the ground state from the excited state, they emit photons. Since the energy of the excited state of a substance comes from a chemical reaction, it is called chemiluminescence. When the separated components are eluted from the chromatographic column, they are immediately mixed with appropriate chemiluminescent reagents to cause a chemical reaction, causing the luminescent substance to produce radiation, and its light intensity is proportional to the concentration of the substance.

This detector does not require a light source or a complicated optical system. As long as there is a constant flow pump, the chemiluminescence reagent is pumped into the mixer at a certain flow rate, so that it can be quickly and evenly mixed with the column effluent to produce chemiluminescence. , Through the photomultiplier tube to convert the optical signal into an electrical signal, it can be detected. The minimum detection volume of this detector can reach 10-12g.

7 Evaporative light scattering detector

The evaporative light-scattering detector (ELSD) is a new general-purpose mass detector that appeared in the 1990s. It is suitable for detecting components with lower volatility than the mobile phase. It is mainly used to detect sugars, advanced Fatty acids, phospholipids, vitamins, amino acids, triglycerides and steroids, etc., and unknown compounds are detected without standard products and the structural parameters of the compound are unknown. It has almost the same response to each substance, but its sensitivity is relatively low, especially for components with ultraviolet absorption. In addition, the mobile phase must be volatile and must not contain buffer salts, etc. Its universal detection principle overcomes the shortcomings of traditional HPLC detection methods, and has been increasingly used in HPLC, supercritical chromatography and countercurrent chromatography. Unlike UV and fluorescence detectors, the response of ELSD does not depend on the optical characteristics of the sample. Any sample with lower volatility than the mobile phase can be detected without being affected by its functional groups. The sensitivity is higher than that of the refractive index detector, it is not sensitive to temperature changes, and the baseline is stable. It is suitable for combined use with gradient elution liquid chromatography.

(1) ELSD detection principle

There are three processes in ELSD operation: the first is the atomization process, in which the effluent from the chromatographic column is atomized with inert gas or purified air; the second is the evaporation process, in which the mobile phase is volatilized in a heating tube (drift tube); the third is In the detection process, the light scattering of the remaining sample particles is measured. All commodity ELSDs use one or two modes to complete these three processes. The operation of mode A is that all the column effluent (aerosol) enters the straight drift tube, allowing the mobile phase to evaporate in it. In mode B, the aerosol is passed through an elbow tube, where large particles are deposited and flow into the exhaust gas. Tube, the remaining small particles enter the spiral drift tube. In the above two modes, the sample particles enter the light tube to scatter the laser light and be detected.

(2) Advantages and disadvantages of ELSD detection


①It has good versatility, and any sample with lower volatility than the mobile phase can be tested;

②Under the same chromatographic conditions, substances with similar physical properties can give consistent responses;

③ Compatible with gradient elution method;

④The sensitivity is higher than that of the refractive index detector and the ultraviolet end absorption detection method.

⑤The experimental method developed on ELSD does not need to be modified if it is transplanted to mass spectrometer.

The main disadvantages of ELSD detection are:

①The sensitivity is not ideal;

②The choice of mobile phase is limited;

③Some samples have a narrow linear range and so on.

(3) Basic factors affecting ELSD detection performance

①Operation mode selection. Selecting an appropriate operation mode can improve the sensitivity of the method. The selection of the operation mode depends on the volatility of the sample, the composition of the mobile phase and its flow rate.

②The mobile phase composition and flow rate selection, the better the volatility of the mobile phase, the higher the sensitivity of the method. The lower the flow rate of the mobile phase, the stronger the corresponding signal.

③The influence of drift tube temperature on baseline level and noise has no obvious regularity. The optimal temperature should be the lowest temperature at which acceptable noise is generated based on the basic volatilization of the mobile phase.

④ The carrier gas flow rate is a very important factor that affects the detection performance. The optimal carrier gas flow rate should be the lowest flow rate at which the maximum detection response value is generated based on acceptable noise (for example, 0.5mV).

(4) Data processing mode during ELSD detection

The most commonly used mathematical model for ELSD detection is lgy=algx+b (y is the response value, x is the injection volume or sample concentration, a and b are regression constants), and there is also a quadratic curve model (y=ax2+bx +c). Due to the non-linear relationship between the response value (y) and the injection volume (x), the data processing is different from the ultraviolet detection method. When ELSD measures the content of known substances, the accompanying standard curve method is generally used instead of the external standard method, because the intercept of the correction linear equation is not zero. For the establishment of a new drug benchmark product, except that the reference product is another substance with a known content and a similar structure, the data processing method is similar to the above. When ELSD measures the purity of a substance, since the response value is not linearly related to the injection volume, it is often corrected by drawing the accompanying standard curve of one or several substances. For the analysis of multi-component substances, the data processing is similar to the above except that the linear range of the accompanying calibration curve is different. Although there are some shortcomings, ELSD, as a new type of general-purpose mass detector, has many unique advantages, such as its versatility, consistency of response factors and compatibility with gradient elution, etc. The analysis of ultraviolet absorbing substances is playing an increasingly important role.

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