High Pressure Liquid Chromatography procedure steps

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Quantitative Analysis: In order to determine the amount/ concentration of a given component present in a mixture, quantitative analysis is carried out.

High Performance Liquid Chromatography (HPLC) is a vastly superior system of liquid chromatography where separation and determination of components within mixtures of non-volatile substances are done very sensitively, efficiently and at a very fast rate compared to Liquid Chromatography. This is due to a very high pressure of up to 400 atmospheres being applied into the HPLC system for the mobile phase to move through the column and carry the components within the mixture along with it. Here are the stages taken in determining the concentration of a component within a mixture using HPLC:

  1. Firstly, many standard solutions (pure samples) are prepared with various range of concentrations, using the actual sample component which is being examined to determine its concentration within the mixture. The mixture will also be examined finally before drawing a calibration curve to identify the concentration of the specific component in the mixture. Each of these standard solutions- including the mixture itself- could either be manually loaded into the injection port one after the other by using a hypodermic syringe or by using an auto-sampler. The hypodermic syringe allows inserting sample volumes which could be from 5 to 20 microlitres; this indicates how only very small quantities are required for the process. The auto-sampler will load each standard solution with the appropriate volume that is set, into the injection port without human intervention. (Fundamentals of HPLC - Agilent Technologies PDF Manual Download - Free User Manual Guide, no date)
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  1. A pump is used to force a liquid mobile phase from the solvent reservoir attached to the system, to flow through the liquid chromatograph. The mobile phase is generally a polar solvent- for example a mixture of water and methanol. (high performance liquid chromatography - hplc, no date)

 A very high pressure is applied through the liquid chromatograph; therefore the separation of the component will be at a very fast rate. This step is taken prior to injecting a pure sample. The pure sample will be introduced into the flow stream of the mobile phase. ...

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