High Performance Liquid Chromatography (HPLC)

Hi friends, in this article we are going to learn about High Performance Liquid Chromatography in detail along with diagrams and working principle.

High Performance 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 performance 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 Performance Liquid Chromatography

Diagram:

high pressure liquid chromatography HPLC
high performance liquid chromatography HPLC

A high performance 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 performance 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 Performance 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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Liquid Chromatography Principle & its Types

Hi friends, in this post we are going to discuss about Liquid Chromatography and its classification. Later on we will discuss each type of liquid chromatography in detail.

The Liquid Chromatography is classified as follows:

  1. Thin layer chromatography
  2. Paper chromatography
  3. Ion exchange
  4. Column chromatography : Column chromatography is again classified as :
  • liquid/ liquid (Partition)
  • Liquid/ solid (Adsorption)
  • Gel permeation

Introduction to various types of Liquid Chromatography:

Now we will see all above types of Liquid Chromatography in detail as follows:

1)      Thin layer chromatography:

 Thin layer chromatography
Thin layer chromatography
Separation of black ink on a TLC plate (This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license.)

In Thin Layer Chromatography the stationary adsorbents are applied to a planar glass or plastic surface and the mobile phase (sample to be analyzed) is allowed to flow over the stationary phase. For separation of components in the sample anyone of the following techniques is used:

2)      Paper chromatography:

Paper chromatography
Paper chromatography (This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license.)
Paper partition chromatography makes use of strips or hollow cylinders of filter paper to hold the solid and liquid phases. In this system, the drops of the sample solutions are applied to number of parallel strips placed at few inches from the end of each test paper and allowed to dry. These strips of paper are then placed in a chromatographic chamber with a saturating and equilibrating vapor and hung from a solvent downward can be timed and the components can be measured.

3)      Column Chromatography:

a)      Liquid/ liquid chromatography: In this method, the liquid stationary phase is retained on the surface of packing by physical adsorption and the sample (mobile phase) is allowed to percolates through the column (stationary phase).

b)      Liquid/ solid (Adsorption) chromatography: In this method, the stationary phase is formed by a solid adsorbent, generally silica and alumina in powdered form. The sample solution is allowed to percolate through the stationary phase in the column.

c)       Gel permeation chromatography: In this method, the separation is based on molecular size and shape. The gel permeation column is packed with a stationary phase in the form of a gel contacting pores of a specific size. The sample solution is allowed to flow over the column bed then the sample penetrates the pores in the packing gel (depending upon the size and shape of the molecules). The large molecules don’t penetrate the gel and leave the column earlier. For more details on Gel permeation click here.

4)      Ion Exchange Chromatography:

In ion exchange chromatography, the exchange of ions between solution and solid insoluble in contact with solution takes place. The ion exchange process is reversible. In this process, when a sample is introduced at the top of the ion exchange column, the sample ions are displaced into solution again and then re-exchange on to the resin. This process continuous till the sample ions leave the column. Now the various sample ions corresponding to the different components in the sample are hold on to the resin to different extent. This cause the different time of passage through the column for different ions and separation of the sample components is achieved. For more details on Ion exchange chromatography click here.

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What is Gas Chromatography

This post provides basic information about what is gas chromatography, construction and working of gas chromatography and finally some applications of gas chromatography.

Definition of Gas Chromatography:

Chromatography is a physical method of separation of the compounds of a mixture by distribution between two phases out of which one is a stationary bed of large surface area and the other is a fluid phase which percolates through or along the stationary phase.

Construction and working principle of gas chromatography:

The basic gas chromatograph consists of following parts

  1. Carrier gas supply with pressure regulator and flow monitor
  2. Sample injection system.
  3. Chromatographic column
  4. Thermal compartment
  5. Detection system
  6. Strip chart recorder

Diagram of Gas Chromatography:

Gas Chromatography
Gas Chromatography

In gas chromatography, the carrier gas is stored in a cylinder at a controlled pressure. The carrier is stored in a cylinder at controlled pressure. The pressure regulator is fitted on the cylinder. This gas is passed to sample injection port at a fixed, controlled flow rate through a floe regulator. The sample injection port is maintained at a certain temperature T1. The temperature T1 is selected such that the rapid vaporization of solute takes place but it doesn’t have a thermal degradation.

The sample injector injects the gas/ liquid sample through syringe. The solute vapor mixes rapidly with the carrier gas and flows into the column. In the column, the different solutes in the vaporized sample are separated from each other depending upon their interaction with the column packing. The column is operated at temperature T2. The resolution efficiency obtained with a particular column is dependent of this temperature T2. A detector is placed at the end of the column. The separated solutes reach the detector individually.

The detector then produces the electrical signals corresponding to each solute quantity in the sample. This detector output is connected to the strip chart recorder which plots the signals amplitude versus time. This plot is known as gas chromatograph. From this gas chromatograph the components of the mixture and their concentration is identified.

A typical gas chromatograph is as shown below:

strip chart gas chromatograph
strip chart gas chromatograph

Gas chromatography Applications:

  1. Using gas chromatography, substances that vaporize below ca. 300 °C (and therefore are stable up to that temperature) can be measured quantitatively.
  2. Gas chromatography can be used to analyze content of chemical product like measuring the quality of products in the chemical industries.
  3. It can also be used for measuring toxic substances in soil, water or air.
  4. Gas Chromatography is used extensively in forensic science.

Related topics:

Liquid chromatography
Gas chromatography
Liquid Chromatograph (HPLC)
Flame ionization detector

Watch following video to understand what is gas chromatography? and Working principle of Gas chromatography. what is Gas chromatography gcse?

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