High end Liquid Chromatography (HPLC) is an analytical tool that separates, identifies plus quantifies components in a sample. This is a commonly used system in analytical biochemistry and biochemistry fields. Basically, the device carries the sample using a solvent or mixture of solvents to the stationary phase, where separation of compounds occurs. A detector captures the separated compounds and signals are sent to the integrator to generate the graphic visual.
HPLC consists of the components below:
᾿ Mobile Phase – this is the solvent or usually a combination of solvents used to transport the samples through the whole system. The solvents have to be miscible in the mixture; else the immiscible solvents will cause stress build-up in the HPLC system. The particular ratios of each solvent component in the mobile phase affect the separation associated with compounds as well as analysis length.
᾿ Pump or solvent delivery unit – this component is to provide the mobile phase and examples throughout the system at a constant flow rate or pressure. Usually, to get analytical purposes, HPLC pump is set to operate at constant flow rate.
᾿ Injector Port or auto sampler – analytical samples are introduced through this component. Examples introduced through injector port have to be manually injected using an appropriate HPLC syringes. Auto sampler enables an analyst to load all the samples in to the HPLC system and the system can automatically select the correct sample to inject at preset conditions.
᾿ Stationary phase – also known as column. This part of the system is actually the center of separation. It is made of tightly packed material in a stainless steel column.
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Due to the compactness of the packed material, high pressure are required to pump or deliver solvents throughout the system, hence HPLC sometimes are term as High Pressure Liquid Chromatography. As the samples circulation through the column, the compounds in the sample will interact simultaneously with all the stationary and mobile phase within a different manner to yield different elution time of each compound. The objective of each analysis is to separate the particular peak of interest from other present compounds.
᾿ Detector – this unit detects the separated compounds in the sample. There are various detectors using various mode of detection such as ultra-violet, fluorescence, mass spectroscopy and refractive index.
᾿ Integrator – integrator turns the signals conveyed from the detector into visual output known as chromatograms. Nowadays integrators come in the form of computer systems instead of the conventional ones which use paper charts.