High-Performance Liquid Chromatography (HPLC): Principle, Instrumentation, and Applications

Posted on Author Binod G C 0

High-performance liquid chromatography (HPLC; formerly referred to as high-pressure liquid chromatography), is a technique in biochemistry and analytical chemistry used to separate, identify, and quantify each component in a mixture of liquid samples. High Performance Liquid Chromatography principle instruments applications will be described in this article.

The components of an HPLC device are a mobile phase, pump/compressor, injector, column, and detector.

Due to the pressure needed to push the mobile phase and sample through the tightly packed columns, HPLC was initially known as high-pressure chromatography. Unlike traditional liquid chromatography, which depends on gravity, HPLC uses a pump to transport the mobile phase and sample through the column. Concentrations below the ppt threshold are simple to find.

HPLC usually uses a variety of stationary phases, a pump to transport the mobile phase(s) and analyte through the column, and a detector to provide an analyte’s distinctive retention time. Other characteristics, such as UV/V is spectroscopic readings for the analyte, may also be provided by the monitor if it is so outfitted. The ratio/composition of the solvent(s) used, the flow rate of the mobile phase, and the intensity of the contact between the analyte and the stationary phase all affect the analyte retention time.

What is retention time?

The retention time is the length of time it takes for a component to move from the injector to the detector.

Why is High performance Liquid Chromatography better than conventional column chromatography?

In HPLC, the greater pressure needed to force the mobile phase and analyte through the tightly packed column is supplied by a pump rather than gravity. The reduced particle diameters are the cause of the greater density. Compared to conventional column chromatography, this enables a superior separation on shorter columns.

Principle of High-Performance Liquid Chromatography (HPLC)

  • The fundamental principle behind HPLC, a sample is broken down into its individual components based on the respective affinities of various molecules for the stationary phase and mobile phase that are being used to perform the separation.
  • It uses two pumps to pass a solvent referred to as a “mobile phase (eluent)” and a sample mixture known as “stationary phase (packing material).” 

Mobile phase: solvent (e.g., water, acetonitrile, methanol etc.)

Stationary phase: solid adsorbent material, granular material made of solid particles (e.g., silica, polymers, etc.).

  • Depending on the chemical composition of the analyte, the molecules are delayed as they move through the stationary phase. The specific intermolecular contacts between a sample’s molecules and the packing substance control how long it stays “on-column.” As a result, the separation is accomplished because the components of a sample elute at various periods.
  • After exiting the column, each substance is identified by an appropriate detector, which sends an indication to the computer’s HPLC program.
  • A chromatogram is acquired in the computer’s HPLC program at the conclusion of this procedure or run.
  • The various compounds can be identified and quantified using the chromatogram.

Instrumentation for High Performance Liquid Chromatography (HPLC)

Instrumentation for High Performance Liquid Chromatography (HPLC)

Solvent reservoir

  • Mobile phase reservoir is another name for the solvent reservoir.
  •  The use of highly thick solvents is prohibited because they require high pressure and take a long time to pass through columns. It is best to avoid using these since they cause peak widening. 
  • The type of material and the detector’s sensitivity influence the solvent option.

What is peak widening?

Peak width is the time from the start of the signal slope to reaching the baseline following repetitive drops in the detector signal.

The Pump

  • The pump creates a discharge of eluent from the solvent tank into the system and is positioned in the liquid chromatography system’s uppermost stream.
  • High pressure is produced by the HPLC pump, allowing the mobile phase to move continuously and consistently throughout the HPLC system.
  • During the chromatography operation, a pump can give either an isocratic (constant mobile phase composition) or a gradient (rising mobile phase composition).
  • E.g., Reciprocating pump, syringe pump, and pneumatic pump.

Injector

  • Next to the compressor is an injector.
  • The liquid sample is introduced into the flow stream of the mobile phase using the injector. Sample volumes vary between 5 and 20 microliters. (L).
  • The use of the autosampler (auto-injector) device, which enables repeated injections at predetermined planned times, is also very common.

Degasser for HPLC 

It is a device for extracting gas from an HPLC mobile phase.

HPLC Column

  • It ensures that the sample components are properly separated. 
  • The length, type, and particulate size of the column packaging material, as well as the interior diameter and length of the column, are all related to separation effectiveness.
  • The materials used to make pipes are typically stainless steel, while silica and alumina particles are used as packaging materials.
  • Different packing materials support different separation mechanisms – common are materials for normal-phase, reversed-phase, size exclusion, ion exchange, affinity, chiral, or hydrophilic interaction HPLC.

HPLC oven

It is a tool used to regulate a column’s temperature.

Detector

  • The detectors job is to keep track of when and how much a material elutes from the column. 
  • The detector detects changes in the eluent’s makeup and transforms this data into an electrical indication that a computer can use to assess.
  • Depending on the structural features of the analyte, different detectors are used. Refractometric, UV/VIS, electrochemical, and fluorescence detectors are typical detector types.

Types of High-Performance Liquid Chromatography (HPLC)

Normal Phase HPLC: NP-HPLC uses a polar stationary phase and a non-polar mobile phase to sort molecules based on their orientation.

Reverse Phase HPLC: The concept of hydrophobic interactions underlies reverse phase chromatography, and as a result, the longer an analyte is held, the more nonpolar properties it possesses. The fixed phase is nonpolar in nature, while the movable phase is polar in nature.

Size-Exclusion HPLC: Size absence Chromatography (SEC) is a chromatographic procedure that only distinguishes between molecules based on their size. In this method, molecules are divided by the column packing material based on their absence from holes.

Ion-exchange HPLC: Based on their affinity to the ion exchanger, ions and polar compounds are separated using ion-exchange HPLC. The most widely used technique for removing charges from proteins and other charged substances is ion exchange chromatography.

Learn more:

Chromatography: Definition, Principle, Types and Application

What is  High-Performance Liquid Chromatography used for?

  • Pharmaceutical uses for quality control, tablet dissolution research, medication stable monitoring, and other purposes.
  • Environmental uses such as phenolic compound identification in potable water and bio-monitoring of pollutants.
  • Forensics application, to measure drug levels in biological samples, identify cocaine, steroids, and other illicit drugs in blood, urine, etc.
  • Clinical applications, to analyze urine, bilirubin, antibiotics, and other substances in blood.
  • Food and beverage application, to analyze polycyclic compounds in veggies, analyze preservatives, analyze sugar, and assess the quality of water and soft beverages.

Advantages of High-Performance Liquid Chromatography

  • Method for quickly, automatically, and accurately determining specific chemical components in a material
  • High resolution
  • simple and accurate quantitative and qualitative analysis
  • Can be upgraded to mass spectroscopy

Disadvantages of High-Performance Liquid Chromatography

  • Requires the use of numerous costly solvents, power sources, and regular maintenance.
  • Operation expertise is required, difficult for beginners. The mobile phase, sample, and correct system functioning all contribute to the HPLC separation process’ dependability.
  • HPLC does have low sensitivity for certain compounds, and some cannot be detected as they are irreversibly adsorbed.

References

Binod G C

I'm Binod G C (MSc), a PhD candidate in cell and molecular biology who works as a biology educator and enjoys scientific blogging. My proclivity for blogging is intended to make notes and study materials more accessible to students.

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