High-Performance Liquid Chromatography (HPLC)

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High-Performance Liquid Chromatography (HPLC)

  • High performance liquid chromatography, also more commonly referred to in the industry as HPLC. It can be described as an analytical process employed to separate, identify or quantify the elements within a mixture.
  • The mixture is then separated by the fundamental method of column-chromatography, and afterwards, it is identified and quantified using spectroscopy.
  • In the 1960s, the column chromatography LC with its low-pressure compatible glass column was developed into it’s current form, the HPLC with its high-pressure-adjusted metal columns.
  • HPLC is, in essence, an improved version that uses column liquid chromatography. Instead of allowing a solvent to flow through a column by gravity, it is forced through it under intense pressures that can reach 400 atmospheres.

Principle of High-Performance Liquid Chromatography (HPLC)

The purification is performed in a separation column that is two phases: a stationary and mobile phase. A stationary stage is an amorphous substance that has tiny particles of porous material in the separation column. The mobile phase however is a solvent or solvent mix that is forced by extreme pressure into the column. Through a valve that has an attached sample loop, i.e. an elongated tube or capillary constructed of stainless steel, the sample is introduced into the flow of the mobile phase from the pump into the separation column with the Syringe.

Then, the various components of the sample travel across the column in various speeds due to being retained to different degrees through interactions that occur with the stationary phase. Once the column has been removed in the end, the different components are identified by a suitable detector , and transmitted as a signal into the HPLC software running on the computer. When the column is finished, process/run, a chromatogram within the HPLC software that runs on the computer is generated. The chromatogram can be used to identify and quantification of various substances.

Components and Steps of High-performance Liquid Chromatography (HPLC) Analysis
Components and Steps of High-performance Liquid Chromatography (HPLC) Analysis

Instrumentation of High-Performance Liquid Chromatography (HPLC)

Instrumentation of High-Performance Liquid Chromatography (HPLC)
Instrumentation of High-Performance Liquid Chromatography (HPLC) | Image Source: https://laboratoryinfo.com/hplc/

The Pump

  • The invention of HPLC has led to the development of pumps system.
  • The pump is located in the highest line of the system . It produces an eluent flow from the reservoir that holds the solvent through the system.
  • High-pressure generation is an essential “standard” expectation of pumps. It must also be able to deliver an unaffected pressure in any time and with a controlled and consistent flow rate.
  • The majority of pumps utilized in LC systems create the flow via back-andforth motion of a piston driven by motors (reciprocating pumps). Because of the piston’s motion it generates “pulses”.


  • An injector is attached to the pump.
  • The most straightforward method is to make use of the syringe and then the specimen is then introduced to the liquid an eluent.
  • The most popular injection technique is based on sampling loops.
  • The autosampler (auto-injector) system is extensively used to allow repeated injections within a set time-schedule.


  • The separation process is carried out within the column.
  • The latest columns are usually constructed in stainless steel enclosure, in lieu of glass columns.
  • The most commonly used packing material is polymer gels or silica in comparison with calcium carbonate.
  • The eluent that is used in LC differs from basic to acidic basic solvents.
  • The majority of column housings are constructed of stainless steel as it is resistant to a wide range of solvents.


  • Separation of the analytes happens within the column, and detectors are employed to determine the separation.
  • Its composition will be uniform when no analyte is present. In contrast, the presence of analytes alters the composition of fluid. The purpose of the detector is to determine these variations.
  • This is recorded in the form of the electronic signals. There are a variety of detectors that are available.


  • The change in the eluent that is detected by detectors takes place as an electronic signal and therefore, it’s not visible to the naked eye.
  • In the past writing with a pen (paper)-chart recorder was widely used. Today, a computer-based data processing (integrator) is becoming more popular.
  • There are a variety of data processors. They range starting with a basic one consisting of an integrated printer and word processor to they have software specifically created to work with an LC system that does not just data acquisition, but also features such as peak-fitting, baseline correction automatic concentration calculation molecular weight determination and more.


The eluent utilized for LC analysis can contain gases like oxygen, which are not visible to the naked eye.

  • If gas is present within the eluent, it is identified as noise, and can cause an unsteady baseline.
  • Degasser utilizes specialized polymer membrane tubing to eliminate gasses.
  • The many tiny pores on the surface the polymer tube permit air to pass through, without allowing liquids to pass through.

Column Heater

The LC separation is usually affected by the temperature of the column.

  • In order to ensure reproducible results, it’s important to maintain consistent temperatures.
  • For certain analyses such as sugar or organic acid, higher resolutions are possible at higher temperatures (50 to 80 degC).
  • So, columns are usually stored in the oven of columns (column heater).

Types of High-Performance Liquid Chromatography (HPLC)

  1. Normal phase: Column packing is the polar phase (e.g silica) and the mobile phase is not polar. It is utilized for water-sensitive compoundsas well as geometric isomers and cis-trans isomers, as well as chiral compounds.
  2. Reverse phase: Column packing is not polar (e.g C18) Mobile phase is miscible solvent that is water-based (e.g Methanol). It can be used to make Ionizable, polar, non-polar and Ionic samples.
  3. Ion exchange: Column packing is made up of Ionic groups, and the mobile phase is called buffer. It’s used to separate Cations and anions.
  4. Size exclusion: Molecules disperse into the pores of a porous medium . They are separated according to their size in relation to the size of the pore. Large molecules first elute and smaller molecules will elute later.

Applications of High-Performance Liquid Chromatography (HPLC)

HPLC has evolved into a method that is universally applicable. HPLC has been developed into an approach that is universally adaptable which allows its application in all areas of chemistry biochemistry and pharmacy.

  • Analyzing drugs
  • Analyzing synthetic polymers
  • Environmental analysis of pollutants analytics
  • The determination of biological matrices containing drugs
  • The isolation of valuable products
  • Quality control and purity of industrial products as well as fine chemicals
  • The separation and purification process of biopolymers, such as nucleic acid or enzymes
  • Purification of water
  • Pre-concentration of trace component
  • Ligand-exchange chromatography
  • Proteins using ion-exchange chromatography
  • High-pH anion-exchange chromatography for carbohydrate and Oligosaccharides

Advantages of High-Performance Liquid Chromatography (HPLC)

  • Quick
  • Efficiency
  • Accuracy
  • Very precise and flexible in finding and quantifying chemical compounds.

Limitations of High-Performance Liquid Chromatography (HPLC)

  • The cost: Even with its many advantages, HPLC can be costly that requires large quantities of costly organics.
  • Complexity
  • HPLC does not have a high sensibility to certain compounds and certain compounds are not detectable because they are permanently attracted to.
  • The separation of volatile substances is made using gas chromatography.
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