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The Who, What and Why of HPLC

Purple line drawing of three HPLC systems

What is HPLC, and how does it work?

HPLC stands for High Pressure Liquid Chromatography – Chromatography is the method of separating different parts of a mixture based on their affinity to a material (stationary phase) vs a liquid (mobile phase). Many people are familiar with the concept of chromatography, having done basic paper chromatography to separate inks or other mixtures in school science lessons. HPLC uses the same concept, but can be used to separate a great number of analytes, as well as identifying and quantifying the analytes that are separated.

Paper chromatography. The first image shows a single ink spot, while the second image shows the separation of different colours
The concept of HPLC is comparable to that of paper chromatography, though HPLC has a far greater range of practical applications

The theory of HPLC is surprisingly simple, though in practice it can take time and experience to become truly competent in HPLC analysis. HPLC systems use a chromatography column – essentially a tube packed with sorbent (the stationary phase). Different components (analytes) within a mixture will have varying levels of attraction to the stationary phase vs the mobile phase, and as a result will pass through the column at different rates. A well thought out chromatography method, using the right column and components, will result in optimum separation of the analytes, allowing them to be identified and quantified individually.

What are the most common applications for HPLC?

HPLC is a very versatile analytical technique, used for separating different components in a mixture. The components can be identified and quantified reliably without any additional equipment, making it an exceptionally powerful technique.

HPLC is widely used in industrial, environmental, pharmaceutical and food analysis and testing, alongside other techniques such as FT-IR, in order to check product purity. It is a powerful tool for detection and quantitation of substances and metabolites in blood and urine, making it ideal for medical diagnosis, medical research, and drugs testing. Within research and development, the possibilities are near endless with HPLC. If an analyte of interest is dissolved in solvent, or can be extracted into a solvent, it can likely be identified and quantified using HPLC. Common analytes include impurities, drugs, food additives, biomarkers, hydrocarbons, ions and salts.

HPLC system and computer in a laboratory

What are the different parts of an HPLC system?

Since HPLC can be used for such a wide range of applications, it follows that there will be varying configurations for the equipment to best suit the desired outcome. Research into the best setup and method for your analytes of interest is important.

Pump

The pump controls the flow of liquid (mobile phase) through the column. Isocratic pumps are the most basic, using a continuous steady flow to deliver the mobile phase through the column. For more complex methods a gradient pump may be preferred. This allows the user to vary the solvent composition of the mobile phase, providing better separation where there are more (or more complex) analytes within a mixture. Gradient pumps may be binary or quaternary pumps, which are able to mix specified ratios of two or up to four solvents respectively.

Degasser

Not all systems will use a degasser. These units are used (as the name suggests) to remove gases from the solvent before it is pumped to the column. This is particularly useful when mixing solvents, due to outgassing of dissolved gases. These can form bubbles, leading to pressure drops in the column and problems with elution times and peak separation.

Injector

The most basic HPLC systems will use manual injection – simply an injection port near the pump. For labs processing more than a handful of samples most users will prefer an autosampler, which allows samples to be prepared in advance and run automatically. Manual injection can take a lot of time out of the day, so some users may prefer an autosampler, even for a small number of samples.

Close up woman hand in a rubber gloves put sample in a glass vial with blue cap into the autosampler rack in HPLC system. High performance liquid chromatography for separation compounds
Many HPLC systems have an autosampler, allowing many samples to be prepared and run automatically

Column

The chromatography column is where analyte separation takes place. There are many options for columns, varying by length and stationary phase composition. Columns are typically chosen specifically for each method, to ensure optimum separation of specific analytes.

Column Oven

Also referred to as a column compartment, the column oven holds the column at a steady, controlled temperature. Chromatography methods can vary the temperature and time ramps to optimise separation of analytes and reduce the viscosity of the mobile phase.

HPLC column oven
The column oven is used to hold and heat the chromatography column. The temperature can be varied to optimise elution times and peak separation

Detector

Without a detector, there is no way to know when analytes are eluted from the column, or measure what they are. There are a range of detectors to suit different applications. UV/Vis detectors may be fixed or variable wavelength, depending on the needs of the lab. Diode array detectors allow for simultaneous monitoring of multiple wavelengths; these are great for method development. Similarly, fluorescence detectors can be single or multiwavelength, depending on the needs of the user.

For some users who are looking for a wide range of unknown analytes, it may be necessary to have a more powerful detection method, so HPLC systems can be coupled with additional external detectors such as mass spectrometers.

How can Richmond Scientific support your HPLC analysis?

We have extensive experience building bespoke HPLC systems for our customers, from the most basic systems designed for monitoring one simple analyte, to more complex builds for a wide array of compounds. Our in-house HPLC engineer is highly qualified and able to advise on system requirements, as well as refurbishing and servicing systems and individual units.

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