High Performance Liquid Chromatography HPLC is an analytical tool which separates, identifies and quantifies elements in a sample. It is a widely used system in analytical chemistry and biochemistry fields. Basically, the system carries the sample with a solvent or mixture of solvents into the static phase, where separation of compounds happens. A sensor captures the separated compounds and signals are sent to the integrator to create a graphic visual.
HPLC consists of the components below:
Mobile Stage – this is the Solvent or usually a combination of solvents used to transfer the samples throughout the entire system. The solvents need to be miscible in the mix; else the immiscible solvents will lead to pressure build-up from the HPLC system. The ratios of each solvent component in the mobile phase have an effect on the separation of compounds in addition to analysis length.
Pump or solvent delivery unit – this element is to deliver the mobile phase and samples through the system at a constant flow rate or pressure. Usually, for analytical purposes, HPLC pump is set to operate in constant flow rate.
Injector Port or auto sampler – analytical samples are introduced by means of this component. Samples introduced through injector interface need to be manually injected using a proper HPLC syringes. Auto sampler enables an analyst to load all of the samples to the HPLC system and the system will automatically pick the proper sample to inject at preset problems.
Stationary phase – This section of the system is truly the center of separation. It is constructed of tightly packed material in a stainless steel column. Because of the compactness of the packed material, higher pressure are expected to pump or send solvents through the system, hence HPLC occasionally are expression as High Pressure Liquid chromatogram. Since the samples flow through the column, the chemicals in the sample will interact concurrently with the static and mobile phase in a different way to yield distinct elution time of each chemical. The aim of every analysis is to separate the summit of interest from other existing compounds.
Detector – this unit finds the separated compounds in the sample. There are various detectors utilizing distinct manner of detection such as ultra-violet, fluorescence, mass spectroscopy and refractive index.
Integrator – integrator turns the signs conveyed from the sensor into visual output called chromatograms. Nowadays integrators come in the shape of computer systems rather than the traditional ones that use paper charts.