High performance liquid chromatography (HPLC) is a widely used technique in analytical chemistry to separate, identify, and quantify the individual compounds in a mixture of chemicals. It has a wide scope of applications, from pharmaceuticals, forensics, environmental laboratories, and more!
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Principles of Chromatography
After a chemical reaction takes place, the product is typically made up of a mixture of compounds: the desired product (also called the analyte) , combined with other side products, impurities, etc. (also called the matrix). To separate the mixture, chromatography can be used. Chromatography separates a mixture based on differences in polarity and uses two different parts. The stationary phase is a nonmoving component- in HPLC, this is typically a column packed with small silica or polymer based particles. The mobile phase is typically some sort of solvent, and it runs through the column, pushing out the sample. The different compounds in the mixture experience different attractions to the stationary phase and the mobile phase due to differences in polarity. (Nonpolar compounds attract nonpolar compounds, and polar compounds attract polar compounds!) Thus, the compounds of the mixture will be pushed out (eluted) at different rates.
Components of an HPLC System
- Solvent Pump: the pump pushes the solvent (mobile phase) through the system at an extremely high pressure. Because of this high pressure, HPLC is much faster than other chromatography methods such as column chromatography!
- Autosampler: the autosampler ejects the sample into the flow of the system
- Column: the different chemicals in the sample are separated here, since they move through the column (stationary phase) at different rates
- Detectors: different HPLC systems may use various detectors. Common detectors include UV-Vis, mass spectrometers, conductivity, refractive index, etc. There are two main types of detectors: specific (detect properties of the specific compound in the sample), and bulk (detect properties based on both specific compounds in the sample and the solvents in the mobile phase).
- Chromatography Data System (CDS): a computer translates the detector signals into chromatograms that scientists can interpret
Variations of HPLC
- Normal Phase Chromatography: The column (stationary phase) is filled with polar particles, commonly silica. The solvent (mobile phase) is a relatively nonpolar solvent. Thus, nonpolar compounds will elute first because of their higher affinity towards the mobile phase.
- Reverse Phase Chromatography: The column (stationary phase) is filled with nonpolar particles, such as a C18 column (silica with a highly nonpolar 18-carbon chain attached). The solvent (mobile phase) is a relatively polar solvent. Since polar attracts polar, the polar compounds will elute first, opposite of normal phase chromatography! This is the most common type of HPLC used in labs across the world.
- Ion Exchange Chromatography: The column (stationary phase) is filled with particles with a charge opposite to that of the desired product. The sample is passed through, and since opposite charges attract, the desired product is trapped in the column while byproducts wash through. The compound can be released by increasing the ionic strength of the buffer or by changing the pH.
- Size Exclusion Chromatography: Instead of polarity, size exclusion chromatography separates molecules based on their size by passing the sample through a gel with various pores. Molecules smaller than these pores will be trapped, whereas larger molecules will be eluted first.
Advantages and Disadvantages of HPLC
With many different chromatography methods available to the typical scientist, why is HPLC so frequently used? The first reason is because HPLC tends to be highly automated, which leads to good precision and reduces the possibility of human error. This leads to experiments done with HPLC being easily reproduced, something that’s critical in the Scientific Method! Furthermore, the high pressure used by the solvent pump, combined with the efficiency of the machine, leads to experiments being completed faster. Additionally, HPLC is extremely sensitive. Therefore, it is able to detect trace amounts of impurities and byproducts that other methods may miss.
However, HPLC may be more expensive than other methods for the machines to be set up and maintained. Another expense of running HPLC is the large amounts of solvent the machines consume. Developing new methods or troubleshooting errors also tends to be more complex than other methods, requiring scientists with advanced or specialized training. Also, the samples must be prepared extremely carefully, since the validity of an HPLC method depends on the accuracy and purity of the sample, mobile phase, and operation of the system. Finally, analyzing the data generated by the Chromatography Data System (CDS) may require specialized software.