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📅 Last Updated: October 23, 2026
High-Performance Liquid Chromatography (HPLC) is a technique that separates a mixture by pumping a liquid mobile phase at high pressure (1000-6000 psi) through a column. In pharmaceuticals, it helps you identify the components of a drug mixture, find impurities and purify compounds.
High-Performance Liquid Chromatography (HPLC) is an analytical technique that separates the components of a drug mixture on a column under high pressure. You can then identify, measure and purify them. It suits thermal labile compounds, high molecular weight peptides and non-volatile compounds. To see how the two techniques differ, read our post on HPLC and LC-MS.
The main purpose of HPLC is to examine a sample for identification, impurities and purification. Pharmacopoeias such as USP <621> Chromatography, and ICH Q2 for method validation, are the standard references for HPLC methods.
A column can separate the compounds that it retains. The separation can rely on differences in these properties: polarity, size, shape, charge, a specific affinity for a site, stereo isomerism and optical isomerism.
Double reciprocating pumps deliver the mobile phase at 1000-6000 psi as a pulse-free stream. Before each run, degassing the mobile phase is a standard step.
HPLC can run with both isocratic and gradient elution. The particle size is generally 5um for analytical columns. A guard column usually sits before the main column to filter the impurities, and it has a particle size of 30um.
The sample can be introduced by an autosampler or manually. The autosampler uses an injection needle to draw the sample from a vial in the sample rack, which is part of the HPLC unit. For manual introduction, you inject the sample into the column. For day-to-day work, see the calibration and operation of HPLC and system suitability in HPLC.
HPLC system components in High-Performance Liquid Chromatography (HPLC)
An HPLC system has six main components, from the mobile reservoir to the sample collector. The list below names each one.
- Mobile Reservoir
- Reciprocating pump to build the pressure (1000 psi-6000 psi)
- Autosampler and injection system
- Column placed in the oven
- Detector
- Sample collector
Types of HPLC based on the component and their separation
HPLC columns are of four types: normal phase, reverse phase, ion exchange and size exclusion. Read more about the types of HPLC columns.
Normal Phase columns
Reverse phase columns
Ion exchange columns
Size exclusion columns
Normal Phase columns: The stationary phase is more polar than the mobile phase, because the packing material is silica, which is polar in nature. Water is more polar than silica, so don’t use water with normal phase columns. The mobile phase is methylene chloride, hexane, chloroform, or a combination of these with diethyl. Polar sample components interact strongly with the polar stationary phase, so they elute slower. Less polar components interact weakly with it, so they elute early.
Reverse Phase columns: Reverse phase columns use bonded C8 and C18 hydrocarbons as the non-polar stationary phase, with a polar water-methanol or water-acetonitrile mobile phase. Other non-polar hydrocarbons can also serve as the stationary phase. The separation of components occurs based on polarity. It works in the opposite way to the normal phase HPLC column, so it is called reverse phase chromatography.
Ion exchange column: You can analyze easily ionizable components with ion-exchange columns. The stationary phase is acidic or basic and carries a negative or positive charge, while the mobile phase is polar. Separation is based on the attractive ionic force between the molecules and the charged stationary phase, caused by the exchange of ions during separation. This separation is called ion-exchange chromatography.
Size exclusion columns: Size exclusion columns use a porous stationary phase to separate the components according to their size. Polysaccharides and silica are used as the stationary phase. Small molecules penetrate the pores of the stationary phase, while large sample molecules penetrate only partially. So, large molecules elute quickly and small molecules elute later.
Frequently Asked Questions
In pharmaceuticals, HPLC is used to analyze drug samples and their components. It helps you identify the components, find impurities and purify compounds. The technique suits thermal labile compounds, high molecular weight peptides and non-volatile compounds, which makes it useful for many drug samples.
An HPLC pump works at 1000-6000 psi. Double reciprocating pumps build this pressure and deliver the mobile phase as a pulse-free stream. In the system, the reciprocating pump comes after the reservoir and before the autosampler and injection system.
An HPLC system has six main components. The first three are the mobile phase holder, the pump that builds the pressure and the autosampler with its injector. The last three are the column placed in the oven, the detector and the unit that collects the sample. Each one has its own place in the system.
A normal phase column has a polar stationary phase, usually silica, and a less polar mobile phase. A reverse phase column works in the opposite way, with a non-polar stationary phase and a polar mobile phase. This is why it is called reverse phase chromatography.
The particle size is generally 5um for analytical columns. A guard column usually sits before the main column to filter the impurities. It has a particle size of 30um. This way, the main column is protected from impurities in the sample before they reach it.
Pankaj Sharma — Quality Control Specialist
Pankaj Sharma is a pharmaceutical QC professional who reviews our technical content for GMP accuracy, laboratory practices, and regulatory relevance.