How to solve two challenges of UV detectors in chromatography

UV detectors are unquestionably the most popular choice for detection in chromatography. This type of detection offers distinct benefits, including sensitivity, affordability and ease of use that has helped it gain acceptance in chromatography. However, UV detectors are subjected to several limitations all users must be aware of. See my advice on how to overcome two of the disadvantages of UV detectors in this post.

A colleague of mine just came back from her ski vacation this week. There was obviously a lot of sun on the slopes, because she was completely sunburned with chapped lips. The normal human in me told her she needed to use more sunscreen, the chromatographer in me joked that with all that absorption, she would blow up my UV detector.

Jokes aside, UV detectors are by far the most frequently used type of detectors in flash chromatography and prep HPLC. Although they might not be very helpful in prevention of sunburns, they are very helpful in the detection of many types of fractionated compounds. Still, even with all their popularity, UV detectors have drawbacks that must be considered.

How do UV detectors work?

Such types of detectors measure the change in intensity of a UV light beam passing through a solution. The absorption of the light is related to the concentration of molecules in the solution. Mathematically, this relationship is expressed by the Lambert-Beer Law:

Lambert-Beer Law, Lambert-beer equation, light absorption, UV detection, flash chromatography, preperative chromatography, HPLC

E = Extinction [dimensionless]
ε = Extinction coefficient [M–1· cm–1]
c = Solution concentration [mol/l]
d = Path length of the light beam through the solution [cm]

What to do if your solvent absorbs at the same wavelength as your compound

Every solvent or mobile phase has a UV absorbance cutoff wavelength. At values below, the solvent itself absorbs all the light. Problems arise if you select a solvent that has significant UV absorption at the wavelength at which measurements are taken. In such cases, the signal of the substance and the solvent will overlap, leading to incorrect fractionation.

One way to avoid this issue is to choose another compatible solvent.

Five critical solvents and their UV cutoff wavelength are indicated in the table below:

SolventUV limit/cutoff (nm)

It is possible that you do not know the absorption spectrum of the compounds of interest. In this case, it is beneficial to use multiple wavelengths simultaneously or a diode array detector (DAD), which can record the whole UV spectrum. DAD bring the additional benefit of confirming the purity and compound identity by showing the absorption spectrum of each peak.

What to do if your compound is not suitable for UV detection

UV detectors are selective. The instruments can only measure substances which absorb light of a selected wavelength in the UV range (200 to 400 nm) or visible range (400 to 800 nm). Many substances fit this profile contributing to the popularity of this type of detection method. Compounds suitable for UV detection contain a chromophoric group, such as an aromatic ring, two conjugated double bonds, carbonyl group and others.

If you are uncertain if all compounds are in your mixture are UV active, it might be a good idea to combine UV detection with another detector, such as an evaporative light scattering detector (ELSD). This combination can help you detect all products in the reaction mixture: chromophoric and non-chromophoric.

In summary, UV detectors are the most popular choice of detection in chromatography systems because they are easy, reliable, affordable, relatively sensitive, non-destructive to sample and solvent-gradient compatible. But users must be wary of poor responses from compounds that lack good chromophoric groups and from using solvents limited by UV cutoffs. I hope that I’ve offered ideas on how to overcome the two latter limitations in this blog post.

If you are interested in learning more about detection methods in chromatography, check out our newly released Chromapedia on the topic.

This is also not the first time UV detection and ELSD have been topics on the blog. Find out more in a previous post on how ELSD can complement UV detectors.

Until then, keep the UV adsorption for your reaction mixtures and off your face on the slopes. Here is to a fun end to the winter season, as we spring into more chromatography topics.

Till next time,

The Signature of Bart Denoulet at Bart's Blog