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HPLC Principle and Instrumentation

In this HPLC Principle and Instrumentation post we have briefly explained about HPLC technique principle, components, procedure, applications, advantages and limitations.

HPLC Principle and Instrumentation

High Performance Liquid Chromatography (HPLC) is essentially a more advanced version of column liquid chromatography. Instead of allowing a solvent to flow naturally through a column, it is forced through at high pressures of up to 400 atmospheres. It becomes more faster as a result of this.

All chromatographic separations, including HPLC operate under the same basic principle; separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation.


The separation principle of HPLC technique is based on the distribution of the analyte (sample) between a mobile phase (eluent) and a stationary phase (packing material of the column). Depending on the chemical structure of the analyte, the molecules are retarded while passing the stationary phase. The specific intermolecular interactions between the molecules of a sample and the packing material define their time “on-column”. Hence, different constituents of a sample are eluted at different times. Thereby, the separation of the sample ingredients is achieved.

A detection unit (e.g. UV detector) recognizes the analytes after leaving the column. The signals are converted and recorded by a data management system (computer software) and then shown in a chromatogram. After passing the detector unit, the mobile phase can be subjected to additional detector units, a fraction collection unit or to the waste. In general, a HPLC technique system contains the following modules: a solvent reservoir, a pump, an injection valve, a column, a detector unit and a data processing unit. The solvent (eluent) is delivered by the pump at high pressure and constant speed through the system. To keep the drift and noise of the detector signal as low as possible, a constant and pulseless flow from the pump is crucial. The analyte (sample) is provided to the eluent by the injection valve.

Types of HPLC

Normal Phase HPLC

The polarity of analytes is used to separate them in this approach. A polar stationary phase and a non-polar mobile phase are used in NP-HPLC. Hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these are common stationary phases, while hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these are common mobile phases. As a result, polar samples stay longer on the polar surface of the column packing than less polar materials.

Reverse Phase HPLC

The stationary phase is nonpolar (hydrophobic), whereas the mobile phase is a polar liquid, such as water-methanol or acetonitrile mixtures. It functions on the basis of hydrophobic interactions, which means that the more nonpolar the material, the longer it will be maintained.

Size-exclusion HPLC

The column is filled with material with exact pore diameters, and the particles are separated by their molecular size. Larger molecules wash through the column quickly, while smaller molecules penetrate the porous packing particles and elute slowly.

Ion-Exchange HPLC

The stationary phase has an ionically charged surface that is charged in the opposite direction as the sample ions. Almost all ionic or ionizable samples are employed in this approach.

The higher the charge on the sample, the more attracted it is to the ionic surface, and the longer it takes to elute. The mobile phase is an aqueous buffer in which the elution time is controlled by both pH and ionic strength.

Instrumentation of HPLC

High Performance Liquid Chromatography (HPLC)

HPLC Principle and Instrumentation

1. Solvent Resorvoir

A glass resorvoir holds the contents of the mobile phase. In HPLC technique, the mobile phase, or solvent, is commonly a mixture of polar and non-polar liquid components, the quantities of which vary based on the sample composition.

2. Pump

The mobile phase is aspirated from the solvent resorvoir and forced through the system’s column and detecter by a pump. Operating pressures of up to 42000 kPa (approximately 6000 psi) can be achieved depending on a variety of factors such as column diameters, stationary phase particle size, flow velocity, and mobile phase composition.

3. Sample Injector

A single injection or an automated injection system might be used as the injector. An injector for an HPLC technique system should be capable of injecting liquid samples in the volume range of 0.1100 mL with good repeatability and under high pressure (up to 4000 psi).

4. Columns

Columns are usually made of polished stainless steel, are between 50 and 300 mm long and have an internal diameter of between 2 and 5 mm. They are commonly filled with a stationary phase with a particle size of 3–10 µm.

Columns with internal diameters of less than 2 mm are often referred to as microbore columns. Ideally the temperature of the mobile phase and the column should be kept constant during an analysis.

5. Detector

The HPLC detector, located at the end of the column detects the analytes as they elute from the chromatographic column. Commonly used detectors are UV-spectroscopy, fluorescence, mass-spectrometric and electrochemical detectors.

6. Data Collection

Signals from the detector may be collected on chart recorders or electronic integrators that vary in complexity and in their ability to process, store and reprocess chromatographic data. The computer integrates the response of the detector to each component and places it into a chromatograph that is easy to read and interpret.


  1. Applications of HPLC: Resolution, identification, and quantification of a substance are all things that HPLC technique can provide. Chemical separation and purification are also aided by it. HPLC has a variety of different uses, including:
  2. Pharma: HPLC technique in the industrial and analytical field that it is help in structure elucidation and quantitative determination of impurities and degradation products in bulk drug materials and pharmaceutical formulations.
  3. Environment: HPLC technique appears promising for the identification and determination of non-volatile or strongly polar compounds in air and in surface, waste, and drinking waters.
  4. Forensic Sector: HPLC technique is used in drug analysis, toxicology, explosives analysis, ink analysis, fibers, and plastics.
  5. Food industry: HPLC technique is used increasingly in the analysis of food samples to separate and detect additives and contaminants.


  1. HPLC technique offers a rapid, automated and highly precise method to recognize certain chemical components in a sample.
  2. High-performance liquid chromatography offers a fast and precise quantitative analysis.
  3. A gradient solvent system can be applied in certain methods.
  4. It is highly reproducible.
  5. HPLC technique can be upgraded to mass spectroscopy (MS).
  6. The HPLC is very rapid, efficient, and delivers high resolution as compared to other chromatographic techniques, such as TLC, column chromatography, and paper chromatography.
  7. Manages all areas of analysis to increase productivity.


  1. HPLC can be an expensive method, it required a large number of expensive organics, needs a power supply, and regular maintenance is required.
  2. It can be complicated to troubleshoot problems or develop new methods.
  3. The lack of a universal detector for HPLC, however, the UV-Vis detector only detects chromophoric compounds.
  4. The separation in High-performance liquid chromatography has less efficiency than GC.
  5. It is more difficult for the beginner.
  6. HPLC pump process reliability relies on of cleanliness of the sample, mobile phase, and proper operation of the system.

Further Readings