High end Liquid Chromatography (HPLC) is an conditional tool that separates, identifies plus quantifies components in a sample. This is a commonly used system in analytical chemistry and biochemistry fields. Basically, the machine 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 usually sent to the integrator to generate a 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 pressure build-up in the HPLC system. The ratios of each solvent component in the mobile phase affect the separation associated with compounds as well as analysis length.
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᾿ Pump or solvent delivery unit – this component is to provide the mobile phase and examples throughout the system at a constant stream rate or pressure. Usually, for analytical purposes, HPLC pump is placed to operate at constant flow rate.
᾿ Injector Port or auto sampler – analytical samples are usually introduced through this component. Samples introduced through injector port have to be manually injected using an appropriate HPLC syringes. Auto sampler enables an analyst to load all the samples to the HPLC system and the system will automatically select the correct sample in order 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 firmly packed material in a stainless steel line. Due to the compactness of the packed materials, 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 flow through the column, the compounds within the sample will interact simultaneously using 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 within the sample. There are various detectors using different mode of detection such as ultra-violet, fluorescence, mass spectroscopy and refractive index.
᾿ Integrator – integrator turns the signals conveyed through the detector into visual output known as chromatograms. Nowadays integrators come in the shape of computer systems instead of the conventional ones which use paper charts.