The separation of all chromatography techniques including high-performance liquid chromatography works under the identical basic principle. The molecules of the sample mixture are get separated due to the dissimilarity of their affinities for the stationary and mobile phase used in the chromatography system. High-performance liquid chromatography (HPLC) also has a stationary phase and mobile phase, HPLC is a highly advanced form of column liquid chromatography. In column chromatography, the solvent is passed with gravity through the stationary phase (e.g. Silica gel), but in the HPLC system, instead of under gravity the mobile phase and sample are run under the force at about 400-4000 PSI, which separates the molecules very rapidly. HPLC is a common technique for the separation of analytes in a complex mixture and which is used for the qualitative and quantitative study of molecules. Depending on the stationary phase system in the process, there are different types of HPLC, such as normal phase HPLC (NP-HPLC), reverse phase HPLC (RP-HPLC), size-exclusion HPLC, and ion-exchange HPLC.
The HPLC system consists of the following major components.
- Solvent Reservoirs:
It fulfills the storage of HPLC solvents in sufficient quantity for uninterrupted operation of the system and it delivered to the pump by Teflon tube through the online degassing and filters. Generally, there are at least two reservoirs in a system, which are usually glass bottles. The reservoir and its attachment would be made of glass, Teflon, or stainless steel material so that the mobile phase is not contaminated. Generally, as a mobile phase, we are using HPLC grade methanol, acetonitrile, water, and buffer.
- The pump of HPLC:
As the name, is required to be generated the pressure with reproducible and the constant flow of mobile phase through the system. The HPLC pump would be capable to take solvent with single or multiple reservoirs with pulse-free output at various flow rates to permit the controlled mixing of different solvents/mobile phases from multiple reservoirs. The reciprocating pump, syringe pump, and pneumatic pump are the types of HPLC pumps.
- The Injector of HPLC:
The introduction of a sample in HPLC is a technique to inject the samples without interrupting the pressure and flow rate of the HPLC system. HPLC's work pressure is sufficiently high so that we cannot introduce a sample in the mobile phase by injecting the syringe, that's why we require an injector that gives reproducible results and without disturbing the system pressure and flow rate. Nowadays, modern injectors are autosamplers, that permit programmed injection of various volumes of sample, which takes from the vials in autosampler trays. Rheodyne injector, septum injector, and stop flow injector are the types of the HPLC injector.
- The Column of HPLC:
The Column is the heart of the HPLC system, it actually generates a separation of molecules in the sample mixture. A column is situated after the injector where the mobile phase is in contact with the stationary phase, creating an interface with the enormous surface. The development of most chromatography in recent years has led to the design of many different approaches to increase this interfacial contact. Typically HPLC columns are available 30 to 250 mm long, 01 to 05 mm in diameter, and 03, 05 and 10 microns in the pore size. The column is filled with porous particles, which is made of polymer and bounded by a thin layer of silica and polystyrene (e.g. C8, C18 column)
- The Detector of HPLC:
A detector is a tool used to detect the sample and column effluent, which is eluted from the column. The detector converts data into an electrical signal and is recorded by the system. The most common detector, UV/VIS is to be used in pharmaceutical analysis, It allows continuous monitoring of UV absorption over a particular wavelength or a range wavelength (using a PDA detector). The presence of components in the detector flow cell causes the change of absorbance.
Here are some types of HPLC detectors mentioned.
UV/VIS and PDA detector, Refractive index detector, Mass spectrometry (MS (MS), Fluorescence detector, Electrochemical detector, Conductivity detector, Light scattering detector, Infrared detector
- Data Acquisition and Control System:
All parameters of the HPLC system are controlled through the computer-based system i.e. Run time, mobile phase composition, wavelength, flow rate, temperature, injection volume, sample sequence, etc. We can monitor the system continuously and check the pressure and other things using this system. Receives the data from the detector and monitors the system's performance and also allows integrating the obtained data.
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