{"id":3549,"date":"2023-04-10T00:00:00","date_gmt":"2023-04-10T00:00:00","guid":{"rendered":"https:\/\/www.thermofisher.com\/blog\/analyteguru\/?p=3549"},"modified":"2026-09-05T00:46:17","modified_gmt":"2026-09-05T00:46:17","slug":"hplc-basics-what-you-should-know","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/analyteguru\/hplc-basics-what-you-should-know\/","title":{"rendered":"HPLC Basics: What You Should Know"},"content":{"rendered":"
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You would be hard-pressed not to find a high-performance liquid chromatography<\/a> (HPLC) instrument in today\u2019s analytical laboratory, given its critical role in a range of fields, from pharmaceutical to food and beverage, manufacturing and environmental safety.<\/p>\n

This blog post explains what HPLC is, how it works and the different techniques liquid chromatographs use to purify mixtures.<\/p>\n

\"\"What is HPLC?<\/strong><\/h3>\n

HPLC is a broad analytical chemistry technique used to separate, identify and quantify compounds in a chemical mixture. These separations utilize the pressure-driven flow of a mobile phase through a column packed with a stationary phase.<\/p>\n

The mobile phase carries a liquid sample through the column to the detector, and compounds \u2014 or analytes \u2014 separate due to varying degrees of interaction with the stationary phase.<\/p>\n

A detector measures the analytes after elution off the column, and a chromatography data system (CDS) translates the detected signal.<\/p>\n

The translated data output of an HPLC analysis is called a chromatogram, where the x-axis shows time and the y-axis is a specific signal generated by the detector.<\/p>\n

\"\"Figure 1. Example of an HPLC chromatogram.<\/span><\/span><\/p>\n

How does HPLC work?<\/strong><\/h3>\n

An HPLC instrument generally has four major hardware components: a pump, autosampler, column and detector. Additional elements include solvents and a CDS package plus connective capillaries and tubing to allow the continuous flow of the mobile phase and sample through the system.<\/p>\n

Every HPLC analysis includes the following steps:<\/p>\n

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  1. Mobile phase begins to flow<\/strong> \u2014 The pump pushes the eluents through the system at a specified flow rate.<\/li>\n
  2. Sample<\/strong> injection<\/strong> \u2014 After injection into the mobile phase, the sample travels with the mobile phase from the injection point to the head of the column.<\/li>\n
  3. Compound separation<\/strong> \u2014 Physical separation of the compounds happens on the column stationary phase. After elution from the column, the separated sample components travel to the detector.<\/li>\n
  4. Analyte detection<\/strong> \u2014 Detection of specified analytes based on an electrical signal generated by specific properties.<\/li>\n
  5. Chromatogram generation <\/strong>\u2014 Translation of the detected analyte signal by the CDS into a chromatogram of analyte signal versus time.<\/li>\n<\/ol>\n

    \"\"Figure 2: HPLC instrument diagram.<\/span><\/span><\/p>\n

    What factors affect HPLC separations?<\/strong><\/h3>\n

    Many factors like the mobile phase composition, column chemistry, and temperature can influence HPLC separations. Successful separation only occurs if the analytes have differing affinities for the column, so selecting the appropriate stationary phase for your compounds is crucial.<\/p>\n

    The main factors influencing the overall separation process are:<\/p>\n