Lesson 5: Detectors

1. Detectors for HPLC

In HPLC, the separation of individual components in a sample is performed using a column, but it needs to be converted into a visible form. Detectors are used to extract results of the performed separation as electrical signals. Table 5 summarizes the detectors commonly used for HPLC analysis.

 
 

Table 5 Detectors commonly used for HPLC analysis

Detector Sensitivity Selectivity Gradient Target Analytes
UV Detector
Visible Absorption Detector
Light absorbing substances
Photodiode Array Detector
(Diode Array Detector)
Light absorbing substances
Refractive Index Detector Substances with a different refractive index from the eluent
(most substances)
Conductivity Detector Ionic compounds
Fluorescence Detector Fluorescent substances
Electrochemical Detector Redox substances
Evaporative Light Scattering Detector (ELSD) Non-volatile substances
Charged Aerosol Detector Non-volatile substances
Mass Spectrometer Ionizable compounds
Multi-Angle Light Scattering Detector Polymeric compounds

◎: Very good   〇: Good   △: Moderate   ×: Not good

1-1. UV detector

Ultraviolet-visible detector (UV detector) is the most commonly used detector in HPLC analysis. When a substance is irradiated with light, a light-absorbing substance absorbs light at a specific wavelength. The wavelength and intensity of absorbed light (absorbance) vary depend on the structure of substance. In absorbance detection, the light from the light source is diffracted by a diffraction grating to specific wavelengths and then directed onto a flow cell (the detection region where the eluent passes) to measure the absorbance. As mentioned, since the wavelength and intensity of the absorbed light (absorbance) vary depending on the structure of substance, there can be significant sensitivity differences among substances. UV detectors detect the ultraviolet region (approximately 195-370 nm), while visible detectors (VIS detectors) detect the visible region (approximately 400-700 nm). UV and VIS detectors detect a set specific wavelength. In contrast, photodiode array detectors (PDA detectors) simultaneously detect multi-wavelength, enabling acquisition of chromatograms at specific wavelengths as well as absorption spectra of each peak. PAD detectors direct the light from a light source onto the flow cell, diffract it with a diffraction grating, and then detect it with 100-1024 photodiode array elements.

1-2. Refractive index detector

Have you ever experienced a phenomenon where the bottom of a round container appears to float, when water is poured into it? This is caused by a refraction of light. Refraction occurs when light bends as it transitions from one substance to another at their boundary. Refractive index detector (RI detector) detects changes in the refractive index of a substance. A RI detector cell consists of a reference cell, which contains only the eluent, and a sample cell, through which the eluent eluted from the column passes. When an eluent passes through a sample cell without any sample components, the liquids in both cells are the same, thus light travels in a straight path. On the other hand, when an eluent passes through a sample cell containing sample components, a difference in refractive index occurs between the reference cell and the sample cell. This leads the light to bend and enables a detection. RI detectors are highly versatile as they can detect substances regardless of their properties. They also have a characteristic of showing a high correlation between analytes peak sizes and their concentrations. However, RI detector has a lower sensitivity compared to other detectors and is not suitable for gradient analysis. They are commonly used in sugar analysis and SEC analysis.

1-3. Conductivity detector

Conductivity detector (CD) is commonly used as an ion chromatography detector. CD detects changes in electrical conductivity. Electrical conductivity, in a simple term is a substance’s ability to conduct electricity. When column elute contains ion components derived from a sample, the ion concentration increases and this makes it easier for electric current to flow, i.e., resulting in higher electrical conductivity. Since CD detects all ions present in the aqueous solution, the eluent alone exhibits electrical conductivity as an ion chromatography eluent contains electrolytes. An eluent with high electrical conductivity (background electrical conductivity) tends to show high noise and this leads to low sensitivity. Two methods, suppressor method and non-suppressor method, are commonly used with a CD to overcome the issue. A non-suppressor method uses an eluent with low electrical conductivity for analysis and directly introduces the eluent eluted from a column into CD. Non-suppressor method is simple as it only requires installing a CD on a general-purpose HPLC system, and thus its initial cost would be relatively inexpensive. On the other hand, a suppressor method uses a device called a suppressor. By placing a suppressor between column and CD reduces the electrical conductivity of the eluent eluted from the column before introducing it into a CD. In anion analysis, suppressor method provides high-sensitivity analysis as it not only reduces the background electrical conductivity but also increases peak response. However, it is more expensive than the non-suppressor method as it requires a dedicated ion chromatography equipment.

1-4. Fluorescence detector and electrochemical detector

Fluorescence detectors (FL) and electrochemical detectors (ECD) are both highly sensitive and selective detectors. Fluorescence is a phenomenon where a substance absorbs light at one wavelength and emits light at a different wavelength. The absorbed light (excitation wavelength) and emitted light (fluorescence wavelength) vary among substances. FL detects the analyte by setting the excitation maximum wavelength (Ex) and the fluorescence maximum wavelength (Em). On the other hand, ECD detects analytes by measuring generated current when the analyte is oxidized or reduced on an electrode with a constant voltage applied to the eluent.

1-5. Evaporative light scattering detector and charged aerosol detector

Evaporative light scattering detector (ELSD) and charged aerosol detector (CAD) both evaporate the eluent eluted from a column and atomize analyte particles. ELSD measures scattered light from analyte particles when illuminated with light. On the other hand, CAD uses electrically charged nitrogen gas (discharged via corona) and electrically detects the charged particles as they collide with analyte particles. ELSD and CAD are used for similar applications as RI detector, but they offer higher sensitivity and are capable of gradient analysis. However, it is important to note that both detectors require evaporating the eluent, which means non-volatile salts cannot be used in the eluent.

1-6. Mass spectrometer

Atoms and molecules have mass, and a substance is composed of atoms and molecules coming together. Mass spectrometer (MS) ionizes substances and detects ions after separating them based on their mass-to-charge ratio (m/z). MS provides information about the mass of substances and can be used for structural analysis. Additionally, MS has gained popularity as LC/MS which has an ability to achieve high sensitivity detection.

1-7. Multi-angle light scattering detector

When a polymer solution is irradiated with a light (laser), it scatters the light at the same wavelength as the irradiated light (Rayleigh scattering). The intensity of this light scattering is proportional to the size of polymer’s molecular weight. Multi-angle light scattering detector (MALS) utilizes this property to directly measure molecular weight of polymers. Typically, in molecular weight distribution measurements using SEC, the relative molecular weight is determined using a calibration curve obtained from measuring calibration standard samples. However, when combined with MALS, absolute molecular weight distribution measurements can be performed.

1-8. Other detectors

In addition to the mentioned detectors, there are other detectors available for specific applications. These include: polarimetric detector which measures the optical rotation of optically active substances and chemiluminescence detector which measures the light from a chemical reaction of excited substance returns to its ground state.

 

 

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