HPLC- Definition, Principle, Parts, Types, Uses, Diagram


High-performance liquid chromatography (HPLC) is a technique in analytical chemistry used to separate, identify or quantify each component in a mixture. It is based on the principle of column chromatography, where a pressurized liquid (called the mobile phase) containing the sample mixture is passed through a column filled with a solid material (called the stationary phase). The different components of the sample have different affinities for the stationary phase and thus move at different speeds through the column. This results in the separation of the components as they elute from the column and are detected by a suitable detector.

HPLC is an advanced form of liquid chromatography that was developed in the 1960s to overcome the limitations of low-pressure glass columns. HPLC uses high-pressure metal columns that can withstand higher flow rates and provide better separation efficiency, versatility and speed. HPLC can also use a variety of stationary phases and mobile phases to suit different types of samples and analytical objectives.

HPLC has a wide range of applications in various fields such as pharmaceuticals, biotechnology, environmental analysis, food science, forensics and clinical chemistry. HPLC can analyze samples that are complex, thermally unstable, volatile or non-volatile, polar or non-polar, ionizable or non-ionizable. HPLC can also be coupled with other techniques such as mass spectrometry (MS) or nuclear magnetic resonance (NMR) to provide more information about the structure and identity of the analytes.

HPLC is composed of several components such as a pump, an injector, a column, a detector and a data processor.

The pump delivers a constant and controlled flow of the mobile phase through the system. The injector introduces the sample into the mobile phase stream using a syringe or an autosampler. The column contains the stationary phase that separates the sample components based on their interactions with it. The detector measures the changes in the properties of the mobile phase as it passes through the column and generates an electronic signal. The data processor records and analyzes the signal and displays it as a chromatogram, which shows the peaks corresponding to each component in the sample.

In this article, we will discuss the principle, instrumentation, types, applications, advantages and limitations of HPLC in detail.