HPLC (high-performance liquid chromatography) is an analytical chemistry technique scientists use to separate compounds in liquid sample mixtures. It’s very popular in industries like food and beverage, environmental, and biopharma/pharma and can help with purification, characterisation, and quantitation of compounds.
How It Works
There are four components of a high-performance liquid chromatography instrument:
• A pump delivers the mobile phase.
• The autosampler injects the sample into the machine.
• A stationary phase column will separate the compounds within the sample.
• A detector measures the compounds.
Many times, you’ll have other elements, such as tubing and connective capillaries, to ensure a continuous flow of the sample and mobile phase through the system. You may also require a CDS package to control everything.
The Steps Involved in HPLC
When performing high-performance liquid chromatography analysis, you’ll follow these steps:
1. Flowing Mobile Phase – The pump must push the solvents/eluents through the system using a specific flow rate that you determine.
2. Sample Injection – The sample is injected into the mobile phase’s flow path, travelling with it to the head of the column from the injection point.
3. Compound Separation – The compounds are physically separated in the column stationary phase. Once eluted from the column, the components will move to the detector.
4. Analyte Detection – The specific properties generate an electrical signal to help the machine detect your target analytes.
5. Chromatogram Generation – The CDS translates the analyte signal into a chromatogram you can use to analyse the information received.
Who Developed High-performance Liquid Chromatography?
You can thank Mikhail Semyonovich Tsvet for inventing liquid column chromatography (1901). Richard Lawrence Millington Synge and Archer John Porter Martin improved on the initial findings, and now laboratories worldwide use high-performance liquid chromatography for liquid samples.
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