Method of the Month — HPLC
June 2021
While you could argue that high performance liquid chromatography (HPLC) is more chemistry than biology, it is incredibly useful in analyzing biological samples — everything from proteins to fatty acids can be separated and quantified using this method.
There’s something really satisfying and “scientific looking” about the machines that carry out the process of HPLC. They often have tubes twisting and spiraling out of them in every direction (as shown in the “figure” below).
Of course, the tubes aren’t there for their aesthetic value, and (sadly) the monocle is not necessary for the functionality of the instrument.
So, what is HPLC?
It’s a technique that allows researchers to separate mixtures of molecules by pushing them through separation columns at high pressure. There are many variations on this method, but the basic setup always consists of two components: the mobile phase and the stationary phase.
The mobile phase is your sample dissolved in a solvent of your choice. As you’ll see later, the choice of solvent depends on the chemical properties of the sample you’re analyzing and how you would like your sample to separate.
The stationary phase — contained in a metal column — is what the mobile phase is passed through to separate it into its constituent parts. An HPLC machine takes the mobile phase, combines your sample with that phase, and (using all its tubing) pushes it all through the stationary phase at high pressure.
Now here’s the part that makes this method so versatile. The column can be packed with whatever kind of stationary phase your experiment requires. The interior can be polar, nonpolar, ionic, or even contain tiny beads with microscopic holes, which allows the separation of products based on their size.
As your sample passes through the column, the components of the sample with greater affinity for the stationary phase will move more slowly than the rest of the mobile phase, separating the mixture. For a detailed treatment of the chemistry behind this separation process, click here
Finally, the pressurized liquid of the mobile phase is passed through some kind of detector. Most commonly, it detects the way the sample absorbs UV light. If your separation is effective, the individual components of the mixture will show up on your data collection software as individual peaks of absorbance over time. The absorption data collected by the detector, along with the time it takes for the sample to pass through the system (known as the retention time)can be used to extract a wealth of information about your sample including concentration, mass, and even chemical composition.
This short description barely scratches the surface of HPLC techniques and applications. While the fundamental idea behind it is simple, the vast array of columns and solvent choices allows researchers to adapt the method to separate, purify and analyze a wide variety of biological samples. If this method sounds exciting to you and you’ve got some extra cash, you can take a look at some used HPLC machines for sale here (no we don’t get a commission if you purchase one).
As always, you can find a cartoon of the method below. While there are lots of good diagrams available on google images, they tend to lack personality…so I added some here.
