Experimental Techniques Explained; High Performance Liquid Chromatography

This week I’ve decided to focus on a scientific experimental technique used quite frequently nowadays, and one that can still be a bit of an enigma to rising students and sometimes just those not familiar with it. I’m talking about High-Performance Liquid Chromatography or HPLC (although it sounds like some kind of narcotic, don’t ask for a gram of HPLC from any strange men, kids… a gram is far too large an amount for something like this).
HPLC as the ‘C’ suggests is a form of chromatography, everyone’s favourite technique for the separation and analysis of liquid samples of any kind, be them pure or jam-packed with different compounds. HPLC is used across many fields but it is heavily used in biochemistry, and I can guarantee you coming across it in about 20 minutes if you’re searching through scientific journals. The main reasons for its’ popularity stem from the fact that it is a very sensitive technique, with HPLC machines being able to detect compounds within the microgram ranges like a shark detecting blood. It is also a very flexible technique as virtually any form of Chromatography you can think of (Normal Phase, Reverse Phase etc.) can be performed in an HPLC machine but with a higher through-put capability. This particular fact is down to the “HP” in the name meaning High Performance or, previously, High Pressure (so no, it’s not meaning the brown sauce).
“But what do you mean, High Performance?” I hear you cry. Hold your horses, people. Scroll down and I’ll explain.

How It Works-

Like any column chromatography HPLC works on forcing a liquid sample through a column of solid material. Many different columns can be used for various effects and separation characteristics but in general they are packed with tiny resin beads which are continually washed with a liquid, or mobile, phase. Our liquid sample is injected along with this mobile phase and as it passes through the column the mixed components move through it at different speeds depending on the specific interactions that the compounds have with the resin of the column. Because of this some compounds wash off, or are “eluted”, from the column very quickly whereas others are bound or slowed down by the stationary phase of the column; the time at which a specific compound elutes is called its’ “retention time” and is an important identifying characteristic within HPLC use. Thus, using different columns in your HPLC machine has the same result as changing the hook and bait on a fisherman’s rod; it allows for the capture and isolation of specific compounds. And you thought fishermen caught fish.
Mobile phases typically consist of a combination of water and an organic solvent (methanol, acetonitrile, ether etc.) because many organic compounds, once they have been concentrated down from their original medium are insoluble in water and stay as precipitate at the concentrations generally needed for scientific analysis. Changing gradients of water and solvent can be used to help tease out different compounds in a sample and increase the resolution of a run (i.e; the time between each compound being eluted). Acids such as formic acid are also commonly added to the mobile phase to a final concentration of 1% as well to increase the resolution of a run.
Now HPLC is different from other chromatography types due to the “High Performance” in its’ name. This is achieved through a high-pressure pump forcing the mobile phase and sample through the column. This pressure increases the flow rate of the apparatus by a significant amount compared to other gravity-reliant methods, and as a result the efficiency and ‘through-put’ of the system. See Figure 1 for a general layout of an HPLC machine.

Layout and innards of an HPLC machine. No HPLC machines were harmed in the making of this image.

Layout and innards of an HPLC machine. No HPLC machines were harmed in the making of this image.







Why Use HPLC?-

HPLC can be used for two main reasons;
1) Analytically. HPLC Profiles of samples can be created and used for comparison across certain conditions. For example, a micro-organism or human cell can secrete a different set of compounds into its’ surroundings depending on the stimuli that it detects. See Figure 2 for a (pretty rough) example.

A rough example of an HPLC trace. Each peak corresponds to a different compound contained within the sample. "Peak picking" can be used to elute and collect each seperate peak.

A rough example of an HPLC trace. Each peak corresponds to a different compound contained within the sample. “Peak picking” can be used to elute and collect each seperate peak.

2) Purification/Fractionation. HPLC can be used to separate very concentrated, small samples (usually around 1ml). So basically like any Chromatography technique.








Wikipedia; High Performance Liquid Chromatography page, (Accessed at 22.51 on 9.11.14). http://en.wikipedia.org/wiki/High-performance_liquid_chromatography


My own knowledge. Not a very good reference for information, but garnered from experience. Feel free to contact me for any details or questions!


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