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Saturday, May 2, 2020

Principle and applications of HPLC

High-performance liquid chromatography (HPLC) is a separation technique of analytical chemistry that is used for the isolation, identity, and quantification of compounds present in a sample mixture. It is the most essential technique used in laboratories worldwide.
Principle of high-performance liquid chromatography (HPLC):
All types of chromatography, for example, column chromatography, thin-layer chromatography (TLC), and gas chromatography, etc. work on the same principle. They have stationary phase and mobile phase, the solvent moves through the stationary phase and bringing the analytes with it.
In the principle of HPLC, A sample is injected through the injector in a flow of mobile phase and travels through a stationary phase (column). The analytes in the sample mixture move with the flow of the mobile phase and interact with the solid support. The rate of movement relies on the affinity of the analytes toward the stationary phase. The strongly interacting analytes with the stationary phase travel gradually, while the less interacting compounds elute quickly.
The separation in the HPLC relies on the interaction of molecules with the stationary phase and mobile phase. Below is a description of the isolation mechanisms which are commonly used in HPLC.
Normal phase chromatography (NP-HPLC): In this technique the compounds are separates according to polarity, using the non-polar mobile phase and polar stationary phase.
Reverse-phase chromatography (NP-HPLC): This type of chromatography works on the principle of hydrophobic interactions therefore the more non-polar compound has the longer be retained. In this technique the stationary phase is non-polar and the mobile phase is polar.
Size-exclusion chromatography (SE-HPLC): In this technique, the separation of compounds depends on the size. The compounds are separated from the stationary phase based on their exclusion from pores.
Ion-exchange chromatography (IE-HPLC): The ion exchange chromatography is a widely used technique for purification of proteins and some charged molecules. In cation exchange chromatography, positively charged molecules are attracted to a negatively charged stationary. Ion exchange chromatography works in contrast.

The various applications of HPLC are as follows:
Environmental applications: The phenolic compounds in water can be determined by HPLC, and also used to monitor the pollutants.
Clinical applications: it is used for the clinical diagnosis of diseases, disorders, and analysis of antibiotics, bilirubin, and other drugs in the blood.
Pharmaceutical applications: The high-performance liquid chromatography is used in the quality control, to observe the stability of molecules and analysis of pharmaceuticals.
Food and beverage applications: The HPLC is used to determination of soft drinks and water quality, analysis of preservatives, sugars, and additives in foodstuffs
Forensics applications: It is used to find out the steroids, cocaine, and some abused drugs from the urine, blood, etc.
Some other applications of HPLC include:
  • HPLC is used for analysis in pharmaceutical laboratories as well as scientific research for discovery.
  • It is also helpful in the isolation and purification of chemicals.
  • It can use for the analysis of synthetic polymers.
  • It is used for standards control by the government.
  • It can use the manufacturing of highly pure goods.
  • It can use to control the microbiological process.
Commonly asked questions on HPLC chromatography are as follows.  

What are the types of HPLC chromatography?
There are different types of HPLC chromatography used in the process of separation such as normal phase HPLC, reverse phase HPLC, size-exclusion HPLC, ion exchange HPLC.
 
What is the principle of HPLC?
The separation principle of high-performance liquid chromatography (HPLC) depends on the distribution of solute amongst the stationary phase (column) and the mobile phase (solvent mixture).
 
How many types of detectors are available for HPLC?
Liquid chromatography uses various detectors such as UV/VIS detector, photodiode array detector (PDA), refractive index detector, mass detector (LCMS), fluorescence detector, electrochemical detector, conductivity detector, light scattering detector, and an infrared detector.
 
What is the difference between UV and PDA detector?
The major difference between the UV and PDA detector is that the photodiode array detector can detect the in the different range of wavelengths 200 to 800 nm, while UV detector can detect only at a specific wavelength.

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