Chromatography is a process in which the components of a liquid are separated by being passed through a solid medium. It works because each component will interact differently with the solid medium in different ways and will therefore travel at different speeds through the medium. This phenomenon has uses in many applications as diverse as drug testing, health screening, pharmaceutical manufacturing, specialist chemical manufacturing, the water industry, and scientific research.
HPLC. Image Credit: khawfangenvi16/Shutterstock.com
High-Performance Liquid Chromatography, formerly known as High-Pressure Liquid Chromatography, is a process in which a liquid is passed through a column packed with a specified material to separate components of the liquid. The technique is relatively modern, first being used in the middle of the 20th century, but it has its roots in work done in the 19th century.
History of Chromatography
The earliest recognition of chromatography appears to be when Runge used blotting paper to separate the components of dyes in 1855, although it is not certain that he recognized this phenomenon as chromatography.
In the 1860s, Schonbein and Goppelsroeder attempted to study the speed at which various components moved through the blotting paper. They did not call the phenomenon chromatography, but capillary analysis, as they believed the movement was due to capillary action.
Between 1927 and 1943 Liesgange developed a method of separating discrete spots of samples. This method of paper chromatography was developed around the same time by Archer Martin and lead to the more common use of the technique.
The first use of column chromatography, using a calcium carbonate column to separate plant pigment was described by Mikhail Tsvet in 1901. It was not until the mid-20th century that Martin and Synge collaborated to combine the techniques of their predecessors to develop the process by using silica in the column whilst seeking to identify amino acids.
The first commercial HPLC machine was produced in 1969 by the Waters Corporation. Since then, there have been many developments in techniques and technology making HPLC an important tool in industry and laboratories. Today it is even possible to have a portable HPLC machine that links by USB or wireless connection to tablets or mobile phones.
High-Performance Liquid Chromatography requires high pressures to operate, typically up to 500 bar or over 493 atmospheres. In recent times, the development of Ultra-High-Performance Liquid Chromatography has extended the pressure range up to 1500bar. This means that the equipment required is specialized and requires good engineering design to make it work and to be safe. Typically containing and moving high-pressure liquid will require very high-quality components and materials of construction.
A typical HPLC column would be 2 – 5mm diameter and 30 to 250mm in length which means that very accurate manufacturing is required to produce relatively small components capable of handling very challenging conditions. Modern manufacturing techniques mean that the equipment continues to evolve and improve.
A typical HPLC system will include a reservoir for the sample (mobile phase), high-pressure pump, degasser, mixing valves, a column containing specified packing (stationary phase), detector or detectors, a recording device, and a collection reservoir for the treated sample.
The quantities of the sample analyzed in an HPLC system are often very small which means the equipment required must accurately handle very small volumes. These elements bring some complexity and challenge to linking the equipment together and making sure it all works as designed.
High-Performance Liquid Chromatography Techniques
In addition to handling mixtures of liquids, different techniques can be applied to HPLC to achieve specific results. Typically, the sample or mobile phase is mixed with a solvent such as water or methanol to enhance the separation. The packing in the column can be varied as well depending on what kind of separation is required.
Normal phase chromatography is where the column, stationary phase, is more polar than the liquid mobile phase. Typical solvents would be hexane, heptane, dichloromethane, and acetone. A normal phase is not normally used in pharmaceutical applications as molecules are polar and take a long time to be eluted.
Reverse Phase chromatography is where the mobile phase is more polar than the stationary phase. Typical polar solvents are water, methanol, and acetonitrile. Reverse-phase chromatography is good for pharma as it results in rapid elution of samples.
In addition to varying the solvent identity, the solvent strength can be varied. A sample with a constant composition is known as isocratic. Analyzing a sample with a constant solvent content is known as isocratic elution. This method would be most likely used in a manufacturing process aiming to isolate a particular component.
It is possible to vary the amount of solvent present, which is known as a gradient elution. This would be used if a sample has a mixture of polar and non-polar components.
In addition to varying the mobile phase, the stationary phase can also be varied according to the process requirements. The stationary phase is usually more polar in normal phase chromatography. The converse is true generally in reverse phase chromatography the mobile phase is polar and the stationary phase less polar.
Ion Exchange Chromatography
An ionic stationary phase is used with an ionic mobile phase and the stronger the charge the faster the elution. The mobile phase is usually a buffered aqueous solution.
Size Exclusion Chromatography
In Size-exclusion HPLC the stationary phase has precisely sized pore sizes and it is used to separate large-sized molecules.
Applications for HPLC
HPLC can be used to analyze or separate many different mixtures. It is used as an analytical tool in research to qualitatively analyze samples, identifying what molecules are present, and quantitatively to identify how much of a compound is present. This means it can be used in chemical, biochemical, biological, and medical applications to identify and measure specific compounds in a laboratory setting. The advent of mobile units has meant that fieldwork can be carried out as well. For example, field analysis of hemoglobin in Africa to detect sickle cell anemia.
It is also a useful tool in testing for pesticides, herbicides, fungicides, pharmaceutical products, illegal food additives, toxins, and other organic contaminants. It can also be used to detect illegal or performance-enhancing drugs in humans.
With increasing developments in detection and recording equipment and the development of more sophisticated media columns, the potential uses for HPLC are likely to continue to grow.
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