UV Trace Drift in Gradients

Gradient elution mode is an invaluable tool for us as analytical chemists, but the slope of the UV trace that results can present issues. You can understand why this happens by considering how each of the solvent systems has its own UV absorption profile. Some of the drift is due to differences in the refractive indices between the two solvents as well. Because of these differences, the UV readout will change continuously over the course of the gradient, producing the slope that we observe. If it is too steep, it can obscure eluting peaks, reducing sensitivity. It would be ideal to have the slope as shallow as possible if we can do so.

                The other day, I was working on a method development project for the USP that called for low pH. We observed tailing for some of our peaks and speculated that the lower pH was needed for this reason. To this end, we tried an additive of 0.5% formic acid in the mobile phase solvents but found a rise in the UV trace of 200 mAU over the course of our 30 min gradient. In an impurity method, where low detection levels are of critical importance, this degree of noise would clearly not be practical. By using TFA instead, however, we were able to reduce the additive concentration by a factor of five and still obtain the good peak shapes that we sought. With the lower additive concentration, the trace slope had risen by about 20 mAU, a mere tenth of the slope obtained previously! This clearly represented a much more viable option for a prospective USP method.

                You just have to remember a few things with TFA, though. It can be prone to oxidation from the atmosphere, so extraneous peaks or a steep UV trace can result if it degrades. In that case, you would have the same problem as before! Your best bet is to try single-use ampules of TFA, since these will have minimal contact time with air. If you use a resealable bulk bottle, you can try adding a blanket of argon after every time it’s opened to ensure it is kept free of air. This oxidation is a slow process, so it only becomes an issue after storage of the opened bottle. Then of course, another thing to keep in mind about TFA is its MS-incompatibility, as it contributes significantly to ion suppression. So if you develop a nice method for UV-based analyses with TFA, just be aware that its applicability will be more limited than with something like formic acid. Even so, TFA can do wonders for some tailing peaks of basic compounds, so it is a good tool for the analytical chemist to keep at the ready.

                I hope these are some helpful tips for you to try in your own method development process. I was always a fan of TFA for those situations where symmetrical peak shapes may be difficult to obtain otherwise. The effect of the trace slope can be one of the more tricky aspects of a UV-based gradient method. And if you can, maybe also consider using a more gradual solvent gradient. This will have a direct effect on how steep the resulting UV trace is.