All You Need to Know About an HPLC and How it Works

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What is an HPLC?

An HPLC, or High Performance Liquid Chromatography, is a chromatographic process that is used to separate a mixture in analytical and biochemistry needs, to classify, measure, and/or clarify the individual components within the mixture.


How does an HPLC work?

agilent hplcSolvent/Mobile Phase Reservoir
A reservoir holds the components or solvent, which then moves to a high-pressure pump.

Pump Solvent Manager Solvent Delivery System (Mobile Phase)
The pump generates and meters the specified flow rate for the solvent, usually measured in milliliters per minute.

    Types:

  • Isocratic – one type of mobile phase used
  • Binary – two types of mobile phases used
  • Ternary – three types of mobile phases used
  • Quaternary – four types of mobile phases used

Sample/Injector AutoSampler Sample Manager
An injector is then used to retrieve and add the sample into the solvent stream that transports the sample into the HPLC column.

    Types:

  • Vial Trays
  • Syringes

waters hplcHPLC Column (Stationary Phase)
The chromatographic packing material, also known as the stationary phase, within the column then assists with the separation of the materials.

Detector
A detector is used to collect data and see the separated compound bands as they elute, or are removed, from the HPLC column.

    Types:

  • MWD – Multi Wavelength Detector
  • UV/VIS – Ultraviolet Index Spectrometer
  • DAD – Diode Array Detector
  • PDA – Photodiode Array Detector
  • FL – Fluorescence Detector

Waste
The solvent then exits the detector and is either sent to waste or collected through a fraction collector if needed.

Fraction Collectors
The fraction collector gathers the sample components for further analysis after the HPLC process. In this process, the liquids are moved from one vial to another and can be set up for timed events.


Additional Components

waters hplc

Degasser
The degasser removes gas throughout the HPLC process.

Column Heater
The column heater heats the HPLC column during the HPLC process.

Interface Cards
Interface cards are used to communicate between a computer and the HPLC equipment

    Types:

  • NIC – Network Interface Card
  • HPIB – Hewlett Packard Interface Bus Card
  • GPIB – General Purpose Interface Bus Card

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