Are you observing more chromatographic peaks than you expect compared to TLC or other assessment data? Well, it could be that your method is separating some isomers or, it could be that there is an actual method issue.
In this post I will discuss what could cause a method issue and suggest some ideas as to how to fix it.
In a previous post I talked about a related issue – split peaks – caused by a defective column or column components. The split peak issue is different than today's topic. With split peaks all eluting compounds show the issue. In today’s topic, only a few, perhaps even only one of the mixture's components, shows a distorted elution band (otherwise known as de-banding).
Let’s say you have performed your compound synthesis and then tested the reaction progress with TLC or LC-MS. The data shows you have product, perhaps some by-products, and one or more of the starting materials. You created a performed a flash purification. The results show an extra peak or two, Figure 1. Well, if your like me I'd ask - what’s happening? Is the peak real or is there a chromatography problem?
Figure 1. An example chromatogram showing severe peak de-banding. In this case, due to an improper injection solvent (ethyl acetate). Cartridge: Biotage® SNAP Ultra, 10 gram; injection volume: 0.3 mL; gradient 5-40% ethyl acetate in hexanes.
In this situation there is typically a chemistry issue related to either one or more reaction component’s mobile phase solubility and/or mass transfer during purification. Mass transfer, for those of us not fluent in chromatography speak, is the movement of analyte (i.e. the transfer of its mass) typically between the mobile and stationary phases. In the chromatogram above, it is a mass transfer issue that was caused by the injection solvent that is just too polar for the chromatography method employed. Specifically, in this case, the issue ended up being between the mobile phase of the method and the injection solvent. When I see this issue, I solve it by either altering my mobile phase or changing the sample dissolution solvent or sample injection technique.
In previous posts, I have discussed both the impact of dissolution solvent choice and the benefits of dry loading. Switching to a weaker dissolution solvent or using dry loading are your best options to eliminate the problem, Figure 2.
Figure 2. By changing sample solvent to DCM, chromatographic peak shapes have been restored. Injection volume: 0.3 mL; cartridge: Ultra Silica 10 gram; gradient: 5-40% ethyl acetate in hexanes.
Have you encountered this issue? If so, what did you do to correct it.
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