High Performance Liquid Chromatography (HPLC) is a liquid form of chromatography used in the separation of a mixture of compounds into individual components. This makes use of small packaging particles and high pressure and it is done based on particular physical and chemical properties. The compounds are first dissolved in a solution before going through separation. The history of chromatography dates back to 1900, in Russia, where it was initially for the separation of plant pigments like chlorophyll.
The chromatography process goes through two important phases, namely the Mobile Phase and the Stationary Phase. This takes place both in gas and liquid chromatography.
The Mobile Phase: This is the phase when there is a definite movement in a certain direction in the form of a liquid, gas or fluid. This phase consists of the sample being separated or analyzed and the solvent moving the sample through the column at high pressure. This is also the phase when the mixture to be separated is dissolved in a fluid.
The Stationary Phase: The mobile phase passes through the chromatography column, which is the stationary phase. This is where the sample gets to interact with the stationary phase and is separated. Thus, the stationary phase is a non-mobile surface, particulate in nature, which marks the region over which the compounds get separated.
Sample Types: The chromatography process makes use of samples that are gaseous and at ordinary temperatures. These samples vaporize when heated. The type of samples determine how easy or hard it may be to analyze them, for example odorous samples like petrochemicals and perfumes are easy to analyze, whereas high molecular weight compounds can be measured after pyrolysis. Liquid samples and solvent solid samples are also used. However, it is quite imperative to note that Liquid Chromatography has a wide range of measurement subjects compared to Gas Chromatography. High molecular weight compounds can still be analyzed, given that they are soluble in solvents.
The Components of an HPLC instrument
High Performance Liquid Chromatography (HPLC) instrument consists of two reservoirs which contain separate solvents. The solvents have to be degassed using Degassers to get rid of the bubbles, they are then added into a mixing vessel. Degassing is done using a Vacuum Pump. The High-pressure Pump forces the fluid through to the pre-column right into the flow splitter. The flow splitter has the capability to detect differentials. The sample to be separated is injected using a Sample Injection port after which the flow goes into the pressure gauge and lastly the Analytical Column.
How to separate the sample into its components
Migrating the sample component at a high speed will depend on whether the component has an affinity for the stationary or mobile phase. This appears via various actions like absorption, partition and ion exchange. But than it is good to note that components that have an affinity for the mobile phase compared to the stationary phase migrate more quickly.
1. PREPARATION: Chromatography can be used to prepare certain components for use in the future. Here the components are separated purely in the form of a purification process or outcome
2. ANALYSIS: This is normally done with smaller amounts of material, usually done to ascertain the presence or level of the proportions of analytes in a particular mixture.
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