What Is High-Performance Liquid Chromatography (HPLC)?

In order to fully understand High-Performance Liquid Chromatography or HPLC for short, it is necessary to have a basic understanding of Liquid Chromatography.

Liquid Chromatography is a technique often performed in a lab, that is used to separate a mixture in order to measure its components, as well as test for potency of a particular substance. How this process works is somewhat complicated, however, it put it simply, the mixture is first dissolved in a fluid. Once the mixture has been dissolved into liquid form, it becomes mobile, hence this is known as the mobile phase. At this point, the liquid is then transferred through a machine that contains another material, which is used to separate the components of the mixture. As the liquid passes through the separating material different compounds begin to move at different rates. This is what’s known as the stationary phase, as the separating material remains stationary while the liquid mixture passes through it. When the different compounds in the mixture travel through at different speeds it causes them to separate. HPLC is based directly on this technique.

HPLC is different from traditional liquid chromatography because high pressure is required. Ordinary liquid chromatography generally uses gravity to pass the mobile phase through a column in order to separate the mixture. The earliest experiments done in this way date back to the 1900’s. The process is time-consuming, and also limited, due to restrictions in the size of particles that can be used for separation using gravity alone.

It was not until the 1960s, when it was predicted by Cal Giddings, Josef Huber, and others, that LC could be operated in a high-efficiency mode by using pressure to increase velocity in the mobile phase. After extensive experimentation with pressure, High-Pressure Liquid Chromatography or HPLC was born. The name has since been changed to High-Performance Liquid Chromatography due to further technological advances, but the acronym remains the same. Comparatively to LC, HPLC is superior in its ability to separate different compounds in mixtures, which makes it a more powerful technique.

HPLC is currently one of the most popular methods used in analytical chemistry to separate, identify, and measure different compounds present in samples. This technique will work on almost anything, so long as it can be dissolved into a liquid.

In the modern world, the technology has advanced to such a degree, that trace amounts of a compound can be detected in a mixture even as low as parts per trillion (or ppt). Today, HPLC can be applied to just about anything, including food, pharmaceutical drugs, nutraceutical supplements, cosmetic products, forensic samples, and industrial chemicals. HPLC is also used in the manufacturing of certain products, for drug testing purposes, and even in scientific and medical research.

HPLC instruments come in many forms, but generally all include a degasser, sampler, pumps, and a detector. The sampler brings the mixture into the mobile phase stream, which carries it into a column where the stationary phase is located. The pumps then deliver the desired pressure necessary to push the mobile phase through the column. The detector is needed to see the separated compounds as they exit from the column. It then generates a signal that is directly proportional to the amount of mixture emerging from the column, which allows the different compounds in the sample to be measured. Often, a computer is used control the HPLC instrument, and for data analysis of the sample. The data that is generated by the detector is then transferred to the computer which creates a reading. This reading is known as a chromatogram. Many HPLC instruments also have an oven that allows for the adjustment in temperature, if necessary, to perform the separation of certain materials. The choice of which components to use in the mobile phase vary. Additives like salts or acids are sometimes used based on the nature of the sample if it helps to break it down. Often a series of trial runs are performed with a sample in order to find the best HPLC method for separating a particular substance. When all is said and done, the mobile phase exits the detector and is either sent to waste management, or collected as desired.

Obviously, this explanation of HPLC is very basic and further research can be done if one is generally interested in learning more about the various aspects of, and techniques involved with chromatography. There are methods that are even more advanced such as Ultra High Performance Liquid Chromatography or UHPLC, which has a higher throughput rate as well as faster run times than traditional HPLC. Many laboratories now have a combination of both HPLC and UHPLC systems for their liquid chromatography needs.

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