High Pressure Liquid Chromatography (HPLC)

High Pressure Liquid Chromatography (HPLC):

In this post we are going to learn construction and working of high pressure liquid chromatography and some basic types of high pressure liquid chromatography (HPLC).

Basically a high pressure liquid chromatography (HPLC) is a method used to separate the components in a mixture to identify and quantify each component.

Construction and working of High Pressure Liquid Chromatography

Diagram:

high pressure liquid chromatography HPLC
high pressure liquid chromatography HPLC

A high pressure liquid chromatography consists of following major components:

  • 1)      A high pressure pumps system to force the liquid mobile phase through the column.
  • 2)      Gradient elution or solvent programmer.
  • 3)      The sample injection system.
  • 4)      Column in thermo stated oven.
  • 5)      Detector and recorder.

In HPLC (High Pressure Liquid Chromatography) the sample to be analyzed is injected in the column as the mobile phase. This mobile phase flows over the stationary phase in the column. This causes separation of the sample components. These components leave the column at different time and reach at the detector. The detector detects the components and gives the signal to the recorder. The recorder shows the chromatograph as shown in the figure.

The peak position determines the component and the peak amplitude determines the concentration of the compound in the sample.

Sample injection system in High Pressure Liquid Chromatography (HPLC):

There are three methods of sample injection as follows:

  • 1)      Syringe injection method.
  • 2)      Sampling loops method.
  • 3)      Automatic injection method.
  • Syringe injection method:

In this method the sample is introduced through a self sealing elastomeric septum, for this purpose micro syringe designed to withstand pressure up to 1500 psi are used. In stop flow injections, the flow of solvent is stopped momentarily and a fitting at the column head is removed. Then the sample is injected directly onto the head of column packing. After replacing the fitting, the system is again pressurized. This method is simple but the reproducibility of the result can’t be obtained.

  • Sampling loop method:

Sampling loop method
Sampling loop method

The diagram shows sampling loop configuration. These valve devices are the integral part of high pressure liquid chromatography (HPLC) equipment and have interchangeable loops providing a choice of sample sizes from 5 to 500ul. Sampling loops of this type permit the introduction of samples at pressure up to 7000psi. Micro sample injection valves with sampling loops having volumes of 0.5 to 5 ul are also available.

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