Peak Fronting

[BradPrater]

I'm doing oligosaccharide working (fluorescently labeled) on a normal phase column. I'm looking at alternative sample prep methods to remove protein after oligo release with enzyme. With the established prep method, I get sharp, nicely resolved peaks. However, with my alternative prep method, all the peaks are fronting significantly. Based on peak intensities and reduced injection volumes, I can rule out overloading as the cause, since I'm actually loading less material with the alternative method. And since the control sample is fine, I'm ruling out column channelling and mobile phase as the culprit. Any thoughts on what might be causing this? I should note that the sample prep change results in the labeled oligosaccharide being eluted in 1/5 the volume of the standard method. Any help is appreciated!

Brad

[Uwe Neue]

Insufficient information!

Since you get different results from different sample prep methods, it would be useful to know the details of these methods.

Of course, the advice could be that the problem is your sample prep method, but I doubt that this will help you...

I suspect a problem with the final solvent that you end up injecting onto the column. The unknown sample solvent may be incompatible with the unknown mobile phase...

[Bruce Hamilton]

Insufficient information!
......
The unknown sample solvent may be incompatible with the unknown mobile phase...

Bravo! Correct and concise. .

Bruce Hamilton

[BradPrater]

Sorry. In the standard celanup method the sample is bound to a low volume nylon syringe filter (~300uL bed volume). To automate the process in the new method, sample cleanup is performed on an actual normal phase resin (~20uL bed volume). The wash and elution buffers are the same for both preps. Wash with 95% Acetonitrile and elute with 20% Acetonitrile. The difference being in the typical prep, the elution is done with 1mL and the new method the elution is with 200uL. The amount recovered is comparable between the 2 methods and that has me wondering if it might be a solubility thing in the lower volume elution. Thanks.

Brad