We are all familiar with the importance of HPLC as a critical analytical tool in the food industry. Â Among the various modules of an HPLC, the detector is the one that finally detects the analyte after its separation through the HPLC column. Â Some of the common detectors used with the HPLC include:
- UV/UV-VIS detector
- Diode array detector (DAD, PDA: Photodiode Array Detector)
- Fluorescence (FL) detector
- Differential refractive index (RI) detector
- Conductivity detector
- Electrochemical detector (ECD)
- Evaporative light scattering detector (ELSD)
- Corona® Charged Aerosol Detector
- Mass detctors (not covered in this article)
UV/UV-VIS detector
UV/UV-VIS detectors are most frequently used to measure components showing an absorption spectrum in the ultraviolet or visible region. Â A UV detector employs a deuterium discharge lamp (D2 lamp) as a light source, with the wavelength of its light ranging from 190 to 380 nm. Â If components are to be detected at wavelength longer than this, a UV-VIS detector is used, which employs an additional tungsten lamp (W lamp).
Figure 1 shows the optical system. Light from the lamp is shone onto the diffraction grating, and dispersed according to wavelength. For example, when the measurement is performed with a wavelength of 280 nm, the angle of the diffraction grating is adjusted so that 280 nm light can shine on the flow cell. By monitoring the reference light divided from the light in front of the flow cell, the difference in light intensity can be determined between the back and front of the flow cell, and this is output as absorbance.
A many components have an absorption in the ultraviolet or visible region. However, attention should be given to the fact that different components have a different spectrum. Components with a large molar extinction coefficient can show a large peak even in small amounts. Thus, the concentration cannot be determined from peak size. Typically, the measurement is performed at a certain fixed wavelength. Â If all of the components of a sample are to be detected with high sensitivity, the time program function can be used to measure each component along with its maximum absorption wavelength during the analysis.
Diode array detector (DAD, PDA: Photodiode Array Detector)
Photodiode arrays (semiconductor devices) are used in the detection unit. A DAD detects the absorption in UV to VIS region. While a UV-VIS detector has only one sample-side light-receiving section, a DAD has multiple (1024) photodiode arrays to obtain information over a wide range of wavelengths at one time, which is a merit of the DAD.
The idea is that spectra are measured at intervals of 1 second or less during separation by HPLC with continuous eluate delivery. If the measurement is performed at a fixed wavelength, components are identified from only their retention time; thus, a minor deviation in retention time can make identification of components difficult. In such a case, the DAD can be used to identify components by a comparison of the spectrum.
Figure 2 shows a DAD optical system.
DADs differ from UV-VIS detectors in that light from the lamps is shone directly onto the flow cell, light that passes through the flow cell is dispersed by the diffraction grating, and the amount of the dispersed light is estimated for each wavelength in the photodiode arrays. Â Compared with a UV-VIS detector, the DAD has the following disadvantages: noise is large because the amount of light is small; the DAD is also susceptible to various changes, such as lamp fluctuations, because the reference light cannot be received. However, the DAD has recently been improved to reduce its difference in performance from UV-VIS detectors.
The results of a measurement with a DAD are shown in the contour map as in Figure 3. Convenient functions are provided, including a peak purity check and library search, as well as quantitative analysis with a specified chromatogram.
Fluorescence (FL) detector
A UV/UV-VIS detector monitors the absorption of light with a specified wavelength. However, some substances absorb light at one wavelength, and then emit light called fluorescence at another wavelength. This is a phenomenon in which a substance absorbs light to reach a high-energy level and then emits light to return to its original level. Such a substance has specific wavelengths of light that it absorbs (excitation wavelengths) and emits (emission wavelengths). As familiar examples, fluorescent paints and highlighters emit fluorescence with a clear color.
Figure 1 shows a FL detector optical system. While a UV/UV-VIS detector detects light that has passed through the flow cell, an FL detector detects fluorescence emitted in the direction orthogonal to the exciting light.
Fluorescence detection is suitable for trace analysis because of generally having high sensitivity and selectivity (not detecting impurities).
There are not many components that originally emit fluorescence (natural fluorescence). However, amino acids, etc. can be detected as fluorescent substances, after reaction with a fluorescence reagent (derivatization). This method makes it possible to measure various components with high sensitivity.
Differential refractive index (RI) detector
An RI detector detects components based on the refraction of light in solution.
As shown in Figure2, the flow cell of an RI detector is divided into the sample-side and reference-side cells. Prior to analysis, eluate is introduced into either cell, until the flow of the eluate becomes equilibrated. The reference-side cell is filled with eluate, and the column eluate is introduced into the sample-side cell through the changed flow channel. When components are eluted from the column, the chemical composition changes in the sample-side solution, which changes its photorefractive level.
As a result, the amount of light received by the light-receiving section changes, showing a peak which can be detected. Any component in the eluate can be detected; thus, the RI detector is often called a “universal detector”.
Conductivity detector
Electric conductivity measurement of a solution is a method of detecting ions in the solution.
After the targeted ions are eluted, the change in electric current is detected, with a constant voltage imposed between the electrodes. A conductivity detector is employed as a detector in an ion chromatograph, which is a system dedicated to measuring ions. This detector is used mainly to measure inorganic ions and small organic substances, including organic acids and amines.
The items shown below are often measured. Some of these items can be measured by atomic absorption analysis. However, the ability to analyze negative ions simultaneously is a particular merit of the ion chromatograph.
Negative ions; F–; Cl–, NO2-, NO3-, Br-, SO42-, PO43–
Positive ions; Na+; K+, NH4+, Mg2+, Ca2+
The conductivity detector is highly sensitive, but very susceptible to the effect of temperature variation (a change of 1℃ in solution temperature causes a change of roughly 2% in electric conductivity). Various methods of avoiding temperature variations have been devised, such as constant-temperature cells.
Suppressor and non-suppressor
In the ion chromatograph system, the electric conductivity of a solution is measured. A higher electric conductivity of an eluate produces larger noise. Two measures for this fact can be considered:
An eluent is used that originally has low conductivity.
→ Non-suppressor method
The electric conductivity of the eluate after column separation is reduced.
→ Suppressor method
The suppressor method reduces the electric conductivity by replacing Na+ with H+ or replacing SO42- with OH– in the eluate through ion exchange resins or membranes. The non-suppressor method is easy and inexpensive, because a conductivity detector is simply added to the typical LC system. Usually, the non-suppressor method can be used to measure samples with a concentration of roughly 0.1 ppm or more.
Electrochemical detector (ECD)
An ECD is used to measure components displaying oxidation-reduction reactions, and detects electric currents generated by these reactions. The ECD has high selectivity, because the necessary voltage to cause an oxidation-reduction reaction depends on the component. The ECD has also high sensitivity, and is often used for measurement of biogenic substances such as catecholamine.
Evaporative light scattering detector (ELSD)
An ELSD atomizes the column eluate, shines light on the resulting particulate components, and detects the resulting scattered light. Theoretically, an ELSD can detect any nonvolatile component. An ELSD has a sensitivity roughly 10 times higher than an RI detector, but has a slightly low sensitivity to low molecular components due to their small size. An ELSD is used mainly to detect non-UV-absorbing components. Attention should be given to the fact that nonvolatile salts cannot be used as the eluent.
Corona® Charged Aerosol Detector
Like an ELSD, a Corona® CAD® atomizes the column eluate to make the sample components particulate. However, a Corona® CAD® detects them electrically by ionizing them with charged N2 gas. A merit of the Corona® CAD® is the ability to detect components with a sensitivity higher than that of an ELSD, with a sensitivity which depends only slightly on component. The eluent for a Corona® CAD® is similar to that for ELSD.
The merits of the major detectors are summarized in Table 1.
Table 1. Merits of the various detectors
| Detector | Selectivity | Sensitivity | Merits | |
|---|---|---|---|---|
| Optical detection | UV/UV-VIS detector | 2 | 3 | A wide variety of substances can be detected that absorb light from 190 to 900 nm. Sensitivity depends strongly on the component. applications include vitamins, lipids, antibiotics |
| Diode array detector (DAD, PDA) | 2 | 3 | A wide variety of substances can be detected that absorb light from 190 to 900 nm. Sensitivity depends strongly on the component. The spectrum can be confirmed for each component. applications include colours, sweetners, preservatives, flavours, antioxidants | |
| Fluorescence (FL) detector | 3 | 4 | Components emitting fluorescence can be detected selectively with high sensitivity. This is often used for pre-column and post-column derivatization. Â applications include mycotoxins, vitamins, amino acids, carbamates, glyphosates | |
| Differential refractive index (RI) detector | 1 | 1 | Any component that differs in refractive index from the eluate can be detected, despite its low sensitivity. Cannot be used to perform gradient analysis. Â applications include carbohydrates, sugar profiling | |
| Evaporative light scattering detector (ELSD) | 1 | 2 | This detector atomizes the column eluate, and detects the scattered light of the resulting particulate components. Non-UV-absorbing components are detected with high sensitivity. Â The ELSD detector is 10 – 100X more sensitive than RI detectors for all samples and under all mobile phase conditions. Â Detects all semi- and non-volatile analytes in your sample, including those transparent to other detectors, like sugar profiling etc. | |
| Electrical detection | Conductivity detector (CD) | 2 | 3 | Ionized components are detected. This detector is used mainly for ion chromatography. Â applications include ions, organic and inorganic acids |
| Electrochemical detector (ECD) | 3 | 4 | Electric currents are detected that are generated by electric oxidation-reduction reactions. Electrically active components are detected with high sensitivity. Â applications include vitamins, ions, carbohydrates | |
| Corona® Charged Aerosol Detector | 1 | 3 | This detector atomizes the column eluate and electrically detects the resulting particulate components treated with corona discharge. UV-nonabsorbing components can be detected with sensitivity higher than that of ELSD. rarely used, but considered as a universal detector | |
4 = Excellent, 3 = Good, 2 = Moderate, 1 = Poor
Refer to Figure 3 to select detectors.
