At some point in school, many of us experimented with paper chromatography. By making a mark with a pen on a piece of filter paper and placing it upright in a dish of water, you can separate the different pigments in the ink.
Ink molecules are large and brightly colored, making them easy to see with the naked eye once they’re separated. But how can you modify this method if you need to separate particles smaller than ink molecules or if you need more exact results? When that’s the case, you’ll need to use high-performance liquid chromatography.
Read on to learn all about the HPLC technique, how it works, and how it’s used in everyday life
What Is High-Performance Liquid Chromatography?
High-performance or high-pressure liquid chromatography (HPLC) is a common method used by science labs around the world. It uses specialized equipment to analyze the unknown contents of a mixture.
Like paper chromatography, HPLC separates particles into a gradient by size. However, paper chromatography is a passive process. HPLC involves forcing liquid containing an unknown mixture through a filter tube at high pressures.
The smallest particles in the mixture will travel farthest through the filter, while the largest particles staying near the entry point. Once you have your results—images called chromatograms—you can compare them to HPLC runs of known substances and identify the contents of your unknown mixture.
To better understand how we get these results, let’s take a closer look at the HPLC process.
How Does HPLC Work?
If you look at an HPLC diagram you’ll see that it has quite a few moving parts, all connected by a series of high-pressure tubes. Let’s take a closer look at the equipment and solutions used in HPLC analysis.
The Sample
The whole point of running an HPLC analysis is to identify the contents of an unknown sample. The sample can be made up of almost anything you’re interested in identifying. Body fluids, water or soil, and microbe samples are common choices.
The Mobile Phase
The mobile phase is a liquid solution with known contents. It’s called the mobile phase because it’s ejected from its container at a certain pressure and moves through the tubes of the HPLC system.
The type of sample you’re investigating determines which solution you should use for the mobile phase. It can be water-based or use another solvent, most often methanol or acetonitrile.
The Injector
For the sample to reach the filter and get separated into its components, it first has to mix with the mobile phase. The injector shoots a metered amount of the sample into the mobile phase as it flows through the tubes. The new solution then continues down the system until it reaches the HPLC column.
The Column
The column is the most important piece of an HPLC machine. This is where filtration and separation happens. After the sample is injected into the mobile phase, it’s pushed through the column at a fixed rate for a set amount of time.
An HPLC column is a tube filled with packing material that’s made of tiny particles (smaller than 5µm in diameter). Because it stays in place as the mobile phase moves through it, this material is called the stationary phase.
The type of column you should choose depends on the properties of your mobile phase and sample. For more information on how to choose the right column for your experiment, make sure to check out this HPLC columns guide.
As mentioned before, the mobile phase passes through the column, leaving the sample behind. The differently-sized particles in the sample stay suspended and spread out in the tube, forming a gradient called a liquid chromatogram. The mobile phase flows out of the system and into a waste receptacle.
Unlike ink molecules in paper chromatography, this gradient isn’t brightly colored—it isn’t even visible to the naked eye. To interpret the results, you’ll need a detector.
The Detector and Computer Data Station
A detector is a device that, in combination with a computer data station, translates a liquid chromatogram into a format we can understand. It measures how much light passes through, how much UV light is absorbed, or how much fluorescence is given off by the chromatogram at different points. The computer takes this data and makes an image we can use to interpret the results.
Real-Life Uses for High-Performance Liquid Chromatography
While the technique itself is awesome, the most fascinating part of high-pressure liquid chromatography is what you can do with the results. Much of the time it’s used in biological and pharmaceutical research, but there are more applications than that. Here are a few real-life situations where identifying unknowns in a sample is useful.
Forensics
When the police need to identify a substance in someone’s bloodstream or an unknown chemical used in an arson case, they ask the forensics team for help. The forensic scientists run the sample through HPLC and compare it to a database of known legal and illegal substances. They can even use this process to identify the specific brand of a drug to help police track down the source.
Medical Diagnostic Tests
When you visit the doctor and they need to see if you have a certain disease, they’ll often order a blood test. Scientists in a medical lab use HPLC to run diagnostic tests on blood and other bodily fluids. This can help them identify disease markers like purines and pyrimidines.
Industry and Manufacturing
HPLC is extremely common in the manufacturing world. When you’re making thousands of solutions a day and selling them all as the same product, you need a way to make sure they’re identical. Manufacturers can compare each day’s batch of a solution with the “expected” content to ensure their product meets the standard.
HPLC Machines Make a Huge Impact
How many things in your life were made possible because of HPLC? If you take the time to think about it, the answer is far more than you might have imagined.
If you often find yourself needing to identify the exact contents of a solution, high-performance liquid chromatography is a technique you’ll use a lot. Whether you’re doing biochemical research, working on forensic investigations, or running diagnostic tests in a medical lab, HPLC makes it possible.
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