High-Performance Liquid Chromatography (HPLC)

By Sushmita Baniya •  Updated: 07/20/22 •  5 min read

High-performance liquid chromatography (HPLC) is a chromatographic technique for the identification and purification of the compounds of a mixture. High-performance liquid chromatography is a modified and improved type of column liquid chromatography and uses high pressure. HPLC is used in biochemistry and analytical chemistry. This technique was developed in 1969 by Kirkland and Huber.

Parts of the HPLC

The different parts of the HPLC are:

Solvent delivery system 

The solvent delivery system has a pump to deliver the solvent, which is the mobile phase. The mobile phase acts as the carrier of the sample. The pump can deliver solvent from the reservoir to the detector. The pump can pump more than 50 ml/min of solvent at pressures up to 10,000 Pascals.

Sample injector

The injector is positioned after the pump to introduce the sample into the mobile phase. Syringes are the most typical sample injectors. In the auto-injector, injection of the sample occurs automatically at the predetermined time.

Degasser helps remove the air bubbles that may be formed in the mobile phase. The formation of the gas causes fluctuation in the baseline. It uses a special polymer membrane tube having numerous small pores to remove the gases.

Column  

The separation of the individual components in the mixture takes place in the stationary phase in the column. Instead of the glass column, it is prepared in stainless steel. Generally, silica or polymer gels are used as stationary phases. It is durable under intense pressure. The length of the column is 10-30 cm, and the diameter is 4-5 mm.

The size of the particles and the mechanical strength of the packing materials are the two key factors that affect column packing. The particle can be packed and dried if larger than 20 mm, but if smaller than 20 mm, it must be suspended in the appropriate solvent. The slurry is then packaged.

Detectors

Different types of detectors used in HPLC are refractive index detectors, UV detectors, and fluorimetry detectors.

Recorder and data analyzing devices

Integrator is the computer-based data processor used to record the electronic signal. Simple to specially designed software is developed for HPLC. 

How does an HPLC work?

The solvent delivery system consists of a pump, through which solvent (mobile phase) is delivered at a controlled flow rate. If air gets dissolved in the mobile phase, it may create air bubbles that fluctuate the flow rate. Degassing unit is present, which removes such air bubbles. The sample solution is injected into the mobile phase by the sample injector system. Then it is delivered into the column.

The sample separation occurs in the column for which temperature needs to be constant. So to maintain the constant temperature, a column is placed in the column oven. The interaction of the individual components and the stationary phase start to occur. If the stationary phase and the individuals have the same nature, i.e., both are polar, then the polar compound will interact with it for a long time. Due to this, it will be eluted later only in the detector. But if the individual component and stationary phase are different, i.e., having different polarity, then the component will be eluted faster in the detector. The time taken for the components to elute in the detector is called retention time. Then the signals from the detector are processed, and a chromatogram is obtained. Based on the chromatogram, quantitative and qualitative analyses are done.

Types of HPLC

Normal phase HPLC

In the normal phase chromatography, a column is polar, and the mobile phase is non-polar. Silica is widely used in the normal phase.

Reverse-phase HPLC

It is the reverse of the normal phase chromatography. The stationary phase is non-polar, and the mobile phase is polar. In the stationary phase, bounded hydrocarbons like C8, C18, and other non-polar hydrocarbons are used. In the mobile phase, water-acetonitrile is used.

Ion exchange HPLC

It is used to separate the cations and ions. Solute ions and the stationary phase in the column have their charge. If the charges among them are opposite, they are retained in the column, which is further eluted. 

Size-exclusion HPLC

In the column, a porous stationary phase is present. Based on the size, the components are separated. In this type of chromatography, large molecules are eluted first than smaller molecules. 

High performance liquid chromatography (HPLC)
High-Performance Liquid Chromatography (HPLC)

Applications of HPLC

Pharmaceutical industry

  • HPLC is used to study the stability and dissolution of drugs.
  • HPLC is used to determine and quantify drug doses. For e.g.: To determine paracetamol in panadol tablet.

Forensic 

  • HPLC is used to separate the steroid hormones in blood and urine.
  • HPLC is used in determining the drug concentration in the blood serum.     
  • HPLC is used to analyze the drugs like the heroine in the abusers.  

Food industry

  • HPLC is used for the sweetener analysis in fruit juice and preservative analysis in sausage.

Environment

  • HPLC is used to detect the presence of phenolic compounds in drinking water.  

Limitation of HPLC

  • HPLC is an expensive technique.
  • HPLC has low sensitivity for some compounds.

References:

  • Blum, F. (2014). High-performance liquid chromatography. British Journal of Hospital Medicine, 75(SUPPL. 2). https://doi.org/10.12968/hmed.2014.75.Sup2.C18

Sushmita Baniya

Hello, I am Sushmita Baniya from Nepal. I am a postgraduate student of M.Sc Medical Microbiology. I am interested in Genetics and Molecular Biology.

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