​High Performance Liquid Chromatography (HPLC) is a form of column chromatography used extensively in biochemistry and both analytical and preparative chemistry. The basic principle of HPLC is the separation of compounds at a molecular level due to differences in their distribution via a liquid “mobile phase” and the distinct affinity of each component in the compound for a stationary phase, most often silica gel. This allows scientists to examine, identify, and quantify each component within a compound.

HPLC begins with sample injection onto the column. The mobile phase then transports the sample through the packed column, where band broadening and longitudinal diffusion mechanisms separate the compounds based on their specific chemical properties. Detection then follows, with the system recording an elution peak for each separated compound. The area under each peak is then used to determine the concentration of each compound within the sample.

  • ​HPLC pump: These are employed to generate a flow of the mobile phase. The pump's precise control of flow rates is key to the entire chromatography process.
  • Sample injector: Functions to introduce the sample onto the column in a reproducible and precise manner.
  • HPLC column: This is where the separation of compounds occurs. The column is generally packed with a stationary phase, with the sample being resolved due to differences in interaction with the mobile phase.
  • Detector: Identifies and quantifies the separated components after elution from the column. The type of detector employed is dependent upon the specific requirements of the analysis.

​While the goal of most chromatography methods, including HPLC, is the same -- to separate the individual components of a compound -- the steps they take to achieve this can vary significantly. The most striking difference lies in the way the mobile phase is transported through the system.

In HPLC, high pressure drives the mobile phase through the column, thus separating the components more quickly and efficiently. In contrast, in conventional liquid chromatography, the column is generally longer and the separation process is usually slower, as it is driven by gravity.

HPLC often affords higher resolution due to the use of smaller particles in the stationary phase, for faster analyses and improved separation efficiency. The use of high pressure also gives HPLC a significant advantage in terms of speed and resolution over gravity-fed methods.

​The importance of HPLC is evidenced by its wide-ranging applications. One of the most prevalent uses of HPLC is in the pharmaceutical industry. It aids in the development, production, and quality control of medications. It identifies and quantifies active pharmaceutical ingredients, identifies impurities, and ensures the uniformity of drug content.

HPLC also is pivotal in environmental testing, where it detects and quantifies pollutants and other hazardous substances in our environment. Its role extends to the food industry as well, where it tests for additives, preservatives, and contamination.

HPLC in Pharm​aceutical Analysis

In pharmaceutical analysis, HPLC is utilized for various purposes, including the quantification of drugs, identification of drug impurities, and analysis of drug metabolism. It is particularly useful in assessing the physiochemical properties of drugs, including their stability and solubility, which are integral for dosage form design. The rigorous testing delivered by HPLC ensures the safety, efficacy, and quality of pharmaceutical products.

Environmental Testing using​ HPLC

HPLC is used widely in environmental monitoring and assessment. These include analysis of water to identify pollutants and hazardous substances, soil analysis, and air quality assessment. ​HPLC also is frequently employed in the analysis of pesticides, providing detailed and accurate results that aid in preserving the health of our ecosystem.

​HPLC in Food ​Analysis

HPLC offers a dependable means of detecting and quantifying food additives, quality markers, and potential contaminations. For example, it can help detect mycotoxins in grains and nuts, antioxidants in fruit, or determine the sugar content in honey. Thus, HPLC helps ensure the safety and quality of our food supplies.

​Frequently Aske​​d Questions 

What is the difference between normal phase and reversed phase​​ HPLC?

Normal p​hase HPLC means the stationary phase is polar and the mobile phase is non-polar; reversed phase means the stationary phase is non-polar and the mobile phase is polar.

What is t​he difference between High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography?

The difference between traditional LC and HPLC is that the solvent in LC travels by the force of gravity.

What types of HP​LC columns are there?

Three types of HPLC columns are Normal phase, Reverse Phase, and Ion Exchange.