Different types of HPLC detectors are used to generate signal proportional to the amount of sample mixture that emerges from the HPLC column, allowing for quantitative sample analysis.
High-performance liquid chromatography is a potent technique for the isolation and quantitative determination of analytes in a sample mixture. HPLC mechanism is based on the principle of affinity chromatography it consists of two phases such as stationary phase (silica-based column) and a mobile phase (solvent, water/buffer). The sample solution is injected into the column through the injector and the mobile phase is pumped through a column under high pressure. The sample splits into its components when traveling through the column, analyzes that have a low affinity for the stationary phase are rapidly eluted from the column. A detector detects the analytes present in the element coming from the column.
The separation of each analyte in the sample mixture has been performed inside the HPLC column, although this separation should enable us to see. How, where, and how much separation occurs, we need this information visually and statistically. Hence, we use a detector for this purpose. It is used to monitor and electronically express the separated analytes. In high-performance liquid chromatography, detectors are used to detect the compound present in the eluent coming from the column. Different types of detectors are available for different types of HPLC chromatography, the selection of the detector being the most important thing in compound detection. The detector is selected based on the detector's sensitivity for that particular compound and, application of analysis.
Some important features required in HPLC detectors are as follows.
- A detector must respond to each molecule present in the mixture of the sample.
- The detector used in HPLC analysis must be non-destructive.
- It should not affect the response by variations in flow rate, temperature, mobile phase composition, and pressure of the system.
- It should not respond to the solvent or mobile phase used in the analysis.
- It should be able to detect high as well as low concentrations of solute.
- It should give a linear response to linear concentrations.
- This should produce reproducible and stable results.
- It should not affect the response of mobile phase composition in isocratic and gradient elution.
Different types of HPLC detectors are classified into two, namely solute and bulk property detectors.
A: Bulk property detectors: As compared to the mobile phase alone, the bulk property detector is used to determine differences in the physical property of the compound, for example, refractive index and conductivity detector. The application of bulk property detectors is usually universal, however, it has poor sensitivity and limited range. These kinds of detectors are not useful for techniques such as gradient elution since results are affected by small changes in the composition of the mobile phase.
B: Solute property detectors: These types of detectors are a response to a particular chemical or physical property. They are high sensitivity, most specific, and having a broad linear response range. Typically, they need to be used with very pure solvents or mobile phases that measure the property. The most widely used solute property detector is the ultraviolet (UV) detector.
The classification of HPLC detectors is as follows.
1. UV/VIS detector
a. Fixed wavelength
b. Variable wavelength
c. Diode array detector
2. Mass detector
3. Fluorescence detector
4. Refractive index detector
a. Deflection detector
b. Refractive detector
5. Electrochemical detector
6. Conductivity detector
7. Light scattering
8. IR detector
Solute property detectors:
1. UV/VIS HPLC detectors:
The ultraviolet or visible detector is the most widely used in HPLC, since provide good stability, easy to operate, and it has good sensitivity for light-absorbing molecules up to the ~pg level. There are three types of detectors in UV / Vis, namely fixed wavelength detector, variable wavelength detector, and a diode array detector.
Fixed wavelength: The detector that works on the fixed-wavelength, the 254 nm is the most commonly used.
Variable wavelength: Variable wavelength detectors determine the absorption of the sample at multiple wavelengths.
Diode array detector: PDA is the broadly used detector in HPLC to record absorbance in the range of the ultraviolet and visible (UV-VIS).
UV/Vis absorption is the determination of the attenuation of a light beam after passing through a sample solution. Absorption determination can be made over a single wavelength or a wavelength range.
2. Photodiode array (PDA) HPLC detectors:
PDA is also a UV detector. It can detect multiple wavelengths simultaneously because it contains many individual diodes, resolution elements, or pixels. This is a usual ideal detector for the entire spectrum in a UV/VIS dispersive spectrophotometer. After passing through the sample compartment a polychromatic beam from the source is irradiated onto the polychromator inlet slit. The polychromator disperses the spectrum's narrowband onto the diode array. The photodiode turns light into electrical signals and stores them for the time being. UV or VIS detectors show two dimensions of the result obtained, however, photodiode array adds the third dimension i.e. wavelength. It is appropriate to determine the most proper wavelength of the molecules without repeating the analysis.
3. Mass spectroscopic HPLC detectors:
MS is an ideal detector to provide molecular weights and structures of the component. A mass spectroscopy detector gives high sensitivity and selectivity. In which the detection is based on the molecular fragmentation by electric fields and separation is based on the mass to charge ratio of fragmented molecules. The compounds are separated by liquid chromatography, and the different sample species are sprayed into the atmospheric pressure ion source, in which they change into ions in the gas phase. The mass detector detects the ion’s mass to charge ratio by exposing it to a magnetic/electrical field that can change the ion movement, allowing them to sort the ions by their mass. The detector can then determine and amplify the ion current to calculate the number of ions sorted.
4. Fluorescence HPLC detectors:
The major advantage of the fluorescence technique is its high sensitivity for selective groups of components. The component atoms are excited by the use of a specific wavelength and then emit a light signal. The emitted light intensity is used to measure the concentration of the components. The components that have fluorescence, which can be determined by the fluorescence detector, for other components they don’t or have low fluorescence absorbance, fluorescence derivatives can be used to treat them. Many natural materials, pharmaceuticals, petroleum products, and clinical samples have fluorescent absorbance.
Bulk property detectors:
1. Refractive index HPLC detectors:
The refractive index detector measures the refractive index of the molecule that passes through the flow cell. It works on two principles which are deflection and reflection of light in solution. There are several types of RI detectors such as the thermal lens detector, the Christiansen effect detector, dielectric constant detector, and interferometer detector. These types of detectors are used to identify the nonionic analytes which in the UV region are neither fluoresced nor absorb.
2. Electrochemical HPLC detectors:
The electrochemical HPLC detector is used to determine the molecules exhibiting oxidation-reduction reactions, and measure the electric currents produce from these reactions. The electrochemical detector has high sensitivity and selectivity as the voltage required for the oxidation-reduction reaction depends on the molecule. The equilibrium and dynamic detectors are two types of ECD. These types of detectors respond to oxidizable substances and are sensitive to alterations in the mobile phase composition and slow rate of the system. The reaction occurs on the electrode surface which generates electrical signals. Their suitability in the aqueous or organic mobile phase may depend on the volumetric characteristics of the component. To perform the process, they require an auxiliary electrode, a reference electrode, and the working electrode.
3. Electrical conductivity HPLC detectors:
The conductivity detectors are used to determine the conductivity, so it is classified as a bulk property detector. The ion in liquid can carry an electrical charge under the influence of a potential gradient, and therefore if a voltage is used across two electrodes located in the liquid, a current travel among the solution and the electrodes. It is universal, reproducible, and has good sensitivity of the charged species and surfactants. The measured electronic resistance of the mobile phase or the sample is directly proportional to the concentration of ions present in the sample solution.
4. Light scattering HPLC detectors:
The light scattering detector is used to determine the scattered light emanating from the eluent. It is useful for those molecules having large molecular weight such as surfactants, lipids, and sugar. The types of light scattering detector are low angle laser light scattering detector and the multiple angle laser light scattering detector. The major advantage of a light scattering detector is that it is a universal detector, it can be used with gradient elution, and does not require one to be a chromophore in the analytes to detect it.
Other popular types of HPLC detectors are the IR detector, pulsed amperometric detectors, chiral detectors, aerosol-based detectors, and the transport detectors.
Commonly asked questions on HPLC chromatography are as follows.
What is the most commonly used detector in HPLC?
The UV detector is widely used in the analysis of HPLC since most of the compounds absorb in UV or visible region, it has good sensitivity, easy to operate, and provide good stability.
What is the tailing factor?
To determine peak tailing the tailing factor is used in the different types of chromatography such as HPLC, and GC. The tailing factor for peak should 0.9 to 1.4. Tailing 1.0 specifies a perfectly symmetrical peak shape.
What is the difference between UV and PDA detector?
The key difference between the UV and PDA detectors is that a UV detector can work on the specific wavelengths while the PDA detector works on the whole range of UV.
What is the guard column?
The HPLC column is protected from impurities and suspended solids by using a guard column. It is set up between the HPLC injector and the column. Guard columns are commonly used for prolonging the life of the analytical columns. It is usually about 2 cm in length and comes with an exchangeable cartridge and interchangeable packing design.
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