To understand what HPLC is, one should understand the definition of chromatography first. Chromatography can be defined as the process or technique used to separate the various components of a mixture based upon their molecular structure and composition.
In this process, there is a stationary phase and a moving phase, out of which the stationary phase is solid mainly while the mobile phase is either a liquid or a gas. The moving phase is made to flow over the stationary phase, as it carries the mixture component with it.
The components of the mixture that interact with the stationary phase will start moving slower than those that interact least with it. This way, the particles are separated. Now, HPLC (High-Performance Liquid Chromatography) is an advanced version of column-based liquid chromatography.
In contrast to column liquid chromatography, where the solvent is allowed to flow through the columns under gravitational force, in High-Performance Liquid Chromatography, the solvent is made to undergo a forced flow-through column at pressures as high as 400 atm.
High-Performance Liquid Chromatography Detectors:
These detectors are used to detect the presence of a solute in the columns, what is their composition, what does it identify as, etc. There are several types of High-Performance Liquid Chromatography detectors, each responding to a specific type of compound, sensitivity difference, etc.
Following are a few properties that are expected out of a standard High-Performance Liquid Chromatography detector:
● It should not be sensitive to temperature variation and liquid flow rate.
● There should be no contribution from the detector to zone spreading.
● The device should have a low level of detector noise as well as a detection limit.
● The sensitivity of the device should be higher towards the solute than the moving phase.
● The device should have a linear response to the concentration of the solute.
● The reaction towards the solute should either be general or specific.
Types of Detectors:
● Mass Spectroscopic HPLC Detector: This detector has a very high level of sensitivity and selectivity. Its detection is done based on the molecular fragmentation that occurs due to the presence of electric fields. The separation of these particles takes place based on the mass to charge ratio of the fragmented molecules.
● VIS/ UV HPLC Detectors: This type of detector can further be split into three types – fixed wavelength, variable wavelength, and diode array. The UV/VIS detectors are mainly used because of their specific response time to a certain class of compounds or particular compounds of the mixture. This depends entirely on the functional group of the molecules that can absorb light, but certain compounds do not absorb light but post their interaction with the columns; they can be observed post derivatization to introduce light-absorbing particles.
● PDA HPLC Detectors: PDA (Photodiode Array) detectors have many diodes present that act as detectors. This makes it possible to monitor many absorbing particles of the component at varying wavelengths simultaneously. This type of detector allows for saving a lot of time and costs that would have been incurred otherwise.
● Fluorescence HPLC Detector: These detectors are more sensitive, selective, and specific, and the sensitivity offered is far more than the UV/ VIS High-Performance Liquid Chromatography detectors. It is pretty natural to have fewer fluorescent compounds than those that react with UV, hence only a limited number of solvents after the post-column derivatization. The fluorescent detectors can be segregated into three types– laser-induced fluorescent detector, single wavelength fluorescent detector, and multi-wavelength fluorescent detector.
● Electrochemical HPLC Detector: The electrochemical detector can be further classified into dynamic detectors and equilibrium detectors. These detectors respond to reducible and oxidizable substances. They are extremely sensitive to change in the flow rate of the mobile phase and the composition. The electrical signals produced are a result of the reaction of these particles taking place on the surface of the electrode. The suitability of this detector with the appropriate solute-solvent is because of the solute molecule’s volumetric characteristics present in the organic or aqueous mobile phase.
● Light-Scattering HPLC Detector: These detectors are of two types – multiple-angle laser light scattering detector and low angle laser light scattering detector. They work on the principle of measuring the amount of light scattered. They are hence used to detect molecules present in the eluent, which have a higher molecular weight than the other constituents of the mixture. This is further examined by passing it through a sensor cell, which depends on the size of the molecules detected.
A few other types of detectors available are IR detectors, aerosol-based detectors, transport detectors, etc.
