High-performance liquid chromatography

High-performance liquid chromatography (HPLC) is a powerful tool that scientists use to separate, identify, and measure the different parts of a liquid mixture. It is often used in chemistry, biology, and medicine to study everything from small chemicals to large molecules like proteins and DNA. HPLC works by using high pressure to push liquids through a special column filled with tiny particles. This helps separate the different substances faster, more clearly, and more accurately than regular liquid chromatography. HPLC is very important in both research labs and industries, such as pharmaceuticals, where it helps check the quality of medicines and study how they work.^[1]

A modern self-contained HPLC

HPLC works by pushing a liquid sample through a very thin column using high pressure. The liquid that moves through the column is called the mobile phase, and the material inside the column that stays still is called the stationary phase. The stationary phase is usually made of tiny silica particles that have special chemicals attached to them. The way the sample moves through the column depends on how its parts interact with both the mobile phase and the stationary phase. As the different parts of the sample pass through the column, they move at different speeds based on their chemical properties like how big they are, how charged they are, how polar they are, or how much they stick to the stationary phase. These differences cause each part to come out of the column at a different time, which is called the retention time. By looking at when each part comes out, scientists can detect and measure the different substances in the mixture.^[1]

There are different types of HPLC, each designed to work best with certain kinds of samples. The most common type is called reverse-phase HPLC. In this method, the inside of the column (the stationary phase) is nonpolar, and the liquid moving through it (the mobile phase) is polar. This setup helps separate many kinds of chemicals, especially in water-based mixtures. Other types of HPLC include normal-phase HPLC, which does the opposite. It uses a polar stationary phase and a nonpolar mobile phase. There is also ion-exchange chromatography, which separates substances based on their electric charge; size-exclusion chromatography, which separates based on molecule size; and affinity chromatography, which separates based on how molecules stick to each other. Because there are so many ways to use HPLC, it can be applied to a wide range of samples. These include medicines, pollution in water or soil, food ingredients, and body fluids like blood or urine. HPLC is especially important in the pharmaceutical industry. It helps scientists check how pure a drug is, make sure it works correctly, and follow strict safety and quality rules during drug development and production.^[1][2]

In HPLC, once the sample is separated into its different parts, scientists need to detect and measure those parts. This is done using special detectors. Common detectors include UV-visible spectrophotometers, which measure how much light a substance absorbs, and fluorescence detectors, which measure the light given off by certain chemicals. There are also refractive index detectors, which look at how a substance bends light. These help figure out what the substance is. One of the most advanced tools used with HPLC is called LC-MS, which stands for liquid chromatography-mass spectrometry. LC-MS not only separates the sample, but also helps identify what each part is made of. It is very sensitive and can detect even tiny amounts of a substance. This makes it very useful in fields like proteomics (the study of proteins), metabolomics (the study of small molecules in cells), and forensic science. Modern HPLC systems are often fully automatic and run using computers. Scientists can control things like the pressure, temperature, flow speed, and mix of liquids very precisely. This makes HPLC faster, more accurate, and easier to use for studying complex samples.^[1][3]