If you’re an HPLC guru, then you probably think that everyone should be using HPLC.  And you might have a point – HPLC is very powerful and has broad applications across many fields.  But it isn’t the answer to every problem.

HPLC (high-performance liquid chromatography) is used to separate mixtures of compounds based on their polarity. This is carried out by injecting your sample onto a HPLC column and eluting the compounds along a concentration gradient.

The literature is bursting at the seams with applications for HPLC.  Here are some of the most common uses, along with some hypothetical situations in which you might use them.

To test for the presence of a specific compound/metabolite

Let’s say you work for a large animal feed distributor. There have been reports of contaminated grain appearing elsewhere and a new toxin has shown up. You could use HPLC to test for the presence of this toxin in your samples. You could also routinely test your product as part of a quality control program.

Quality control within the pharmaceutical industry

The pharmaceutical industry routinely uses HPLC to monitor their products for shelf life, stability, impurities and other important properties.

To analyze all of the compounds in a complex mixture

This might be useful in microbiology and environmental monitoring. Let’s say that a new mold has been found growing in the corners of old homes. This could potentially pose a health threat, as certain molds are known to produce toxic metabolites. You could use HPLC to investigate the compounds that this species produces to create a metabolite profile for this species. This will help you to determine whether or not it is a threat.

In this situation, HPLC can probably aid in identifying some of the compounds through comparisons with the metabolite profiles of other well-known molds. However, for a meaningful comparison, all samples should be tested under exactly the same conditions (gradient, absorbance profiles, etc.).

To separate and purify compound(s) of interest

HPLC is sometimes used to purify compounds on a relatively small scale (~20–50 mg). This is useful in basic research where the researcher wants to characterize a novel compound that they have found or synthesized. HPLC can usually yield enough compound to facilitate an NMR (nuclear magnetic resonance) analysis to determine compound structure.

Monitor chemical reactions

HPLC is routinely used throughout chemistry to determine whether a given chemical reaction has reached completion.  In this context, HPLC may also reveal undesirable by-products.

Assay platform for detection of metabolites

HPLC can be used to monitor biochemical assays. For example, HPLC is sometimes used to monitor enzymatic reactions, as the modified substrate will likely elute differently from the column.

Compound identification

Some institutions have setups in which unknown compounds can be compared to in-house databases for a match of retention time and absorbance profile. This only works if all compounds are run on the same HPLC setup, and is considered to be a tentative form of compound identification. The availability of authentic standards greatly enhances such identification.

If you come across a completely novel compound that does not have a match in any database, you will not be able to elucidate the structure by HPLC alone. But HPLC will give you hints about the compounds properties. For instance, absorbance at 215 nm indicates the presence of a peptide bond, while absorbance at 254–280 nm indicates that the compound contains aromatic residues (for example aromatic amino acids such as phenylalanine, tyrosine, tryptophan). If you want absolute structural elucidation, you should look into other techniques such as mass spectrometry and NMR analysis.

Quantification

With the use of authentic standards of known concentrations, it is possible to quantify your compound using HPLC. This approach can be very robust, but often requires extensive and time-consuming optimization. For this reason, many researchers see it as a kind of last resort, and only turn to HPLC when they are dealing with very small amounts of compound.

 

While HPLC may not be an appropriate choice in all circumstances, it might be just the technique you need for your next experiment.

What do you use HPLC for? We’d love to hear from you 🙂