High Performance Liquid Chromatography | HPLC 

High-Performance Liquid Chromatography is commonly known as HPLC chromatography, is an analytical technique used to separate, identify or quantify each component in a mixture. The mixture is separated using the basic principle of column chromatography and then quantified and identified by spectroscopy. In the 1960s, column chromatography with a glass column adapted to low pressure and further developed to HPLC with a metal column adapted to its high pressure. HPLC is thus basically a highly advanced form of column liquid chromatography. Instead of allowing a solvent to drip through a column under gravity, it is forced through it under high pressures of up to 400 atmospheres.

HPLC Chromatography Principle| Principle of HPLC Chromatography | Principle of High-Performance Liquid Chromatography 

The principle of HPLC chromatography is based on the purification takes place in a separation column between a mobile phase and a stationary phase. The stationary phase is a granular material consisting of very small porous particles in a separation column. On the other hand, the mobile phase is a solvent or solvent mixture that is forced through the separation column at high pressure. Through a valve with an attached sample loop, i.e. a small tube of a capillary is made from stainless steel, the sample is injected into the mobile phase flow from the pump to the separation column using a syringe. Thereafter, different components of the sample migrate through the column at different rates as they are retained to varying degrees by interactions with the stationary phase. After leaving the column, the separated substances are detected by a suitable detector and passed as a signal to the HPLC software on the computer. At the end of this operation/run, a chromatogram is obtained in the HPLC software on the computer.

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Chromatogram allows identification and quantification of different substances. Molecules with higher affinity are adsorbed for a longer time, which reduces their speed of movement through the column. However, molecules with low affinity move at a faster rate, thus allowing the molecules to separate into distinct fractions.


HPLC chromatography diagram


Working method of HPLC

High pressure generation pumps require a “standard” requirement; in addition, it should also be able to provide a consistent pressure and a controlgic and reproductive flow rate in any situation. An injector is placed next to the pump. The simplest method is to use a syringe, and the sample is introduced to the flow of eluent. The most widely used injection method is based on the sampling loop.

The separation is done inside the column. Recent columns are often outfitted in stainless steel housings instead of glass columns. More commonly used packing materials are silica or polymer gels than calcium carbonate. The eluent used for LC changes from acidic to basic solvents.

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The separation of the analyte is done inside the column, while a detector is used to visualize the separation obtained. When no analyte is present the composition of the eluent is consistent. While the presence of the analyte changes the composition of the eluate. This difference is monitored as an electronic signal.

The change in the eluent detected by a detector is in the form of an electronic signal, and thus is still not visible to our eyes. In the olden days, pen (paper)-chart recorders were popularly used. Nowadays, computer-based data processors (integrators) are more common. There are different types of data processors; From a simple system with an in-built printer and word processor, while with software that is specifically designed for an LC system, which allows not only data acquisition but also peak-fitting, baseline correction, automatic concentration calculation, molecular weight There are features like scheduling, etc.,

Degasser:- The eluent used for LC analysis may contain gases such as oxygen which are not visible to our eyes. When gas is present in the eluent, it is detected as noise and causes an unstable baseline. The degasser uses special polymer membrane tubing to remove the gases. Many very small holes in the surface of the polymer tube allow air to pass through while preventing any liquid from passing through the holes.

Column Heater:- LC separation is often substantially affected by column temperature. To obtain repeatable results, it is important to maintain constant temperature conditions. Also for some analytes, such as sugar and organic acids, better resolutions can be obtained at higher temperatures (50 to 80 °C). Thus columns are usually placed inside a column oven (column heater).

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Types of high performance liquid chromatography

There are four types of High Performance Liquid Chromatography.

  1. Normal phase:

The column packing is polar (such as silica) and the mobile phase is non-polar. It is used for water-sensitive compounds, cis-trans isomers, geometric isomers and chiral compounds.

  1. Reverse phase:

The column packing is non-polar (eg C18), the mobile phase is a water soluble solvent (eg methanol). It can be used for polar, non-polar, ionic samples and ionizable.

  1. Ion exchange:

The column packing consists of ionic groups and the mobile phase is the buffer. It is used to separate the anion and the cation.

  1. Size exclusion:

Molecules diffuse into the pores of the porous medium and separate according to their size relative to the pore size. Large molecules elute first and small molecules elutes later.

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HPLC chromatography detectors

 There are different types of HPLC chromatography detector

  1. Ultraviolet/visible spectroscopic detectors
    1. Fixed Wavelength Detector
    2. Variable Wavelength Detector
    3. Diode array Detector
  2. Refractive-Index Detector
    1. Deflection Detector
    2. Refractive Detector
  3. Evaporative Light Scattering Detector
  4. Multi-Angle Light Scattering Detector
  5. Optical Rotation Detector
  6. Electro Chemical Detector
  7. Mass spectrometric

Application of high performance liquid chromatography | HPLC chromatography uses

  • Application of High Performance Liquid Chromatography can be used in almost all areas of biochemistry, chemistry and pharmacy.
  • High-performance liquid chromatography is used in the analysis of pollutants present in environmental samples and drugs analysis.
  • This is done to maintain product purity and quality control of various industrial productions.
  • This technique can also be used to separate various biological molecules such as proteins and nucleic acids.
  • The increased speed of this technology makes the process more effective and faster. 

Advantages of High-Performance Liquid Chromatography | Advantages of HPLC

The main advantages of high-performance liquid chromatography are as follows :-

  • The advantages of HPLC i.e. it can test both raw materials and finished products and It can determine product stability.
  • It helps to solve the problems of product failure.
  • It can detect contaminants and other impurities.
  • The test can also be performed with only a small sample size.
  • This enables you to modify the test based on the required quantification level.
  • The results produced by it are reliable.
  • It helps in developing better products.
  • This allows you to get a better understanding of competitor’s products.

Disadvantages of High-Performance Liquid Chromatography | Disadvantages of HPLC

  • Cost: Despite its advantages, HPLC can be expensive, requiring large quantities of expensive organics and its complexity.
  • HPLC has low sensitivity for some compounds, and some cannot be detected because they are irreversibly adsorbed.
  • Volatile substances are not better separated by liquid chromatography whenever it is better separated by gas chromatography.

Examples of HPLC

High-performance liquid chromatography has been performed to test the efficiency of various antibodies against diseases such as Ebola.

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