HPLC users: it’s time to get your fronting peaks back on track.

The most common causes for peak fronting are overloading the column (resulting in too much injection mass on-column) or a column installation error, such as fittings swaged to a port depth different than that of the column in use.

Another possible cause could be a matrix mismatch with the injection solvent (sample diluent) and the mobile phase. Such mismatches could include different pHs, immiscible solvents, or injection solvents of a significantly stronger elution strength than the mobile phase.

An installation error would tend to cause all peaks to front, while a difference in pH could potentially distort only specific peaks that correspond to some particular ionizable compounds.  If the injection solvent is of a greater elution strength than the mobile phase, the earliest eluting peaks would tend to be the most affected. Injecting immiscible solvents would tend to yield the most erratic effects, including random peak shape distortions, unpredictable retention time shifting, missing peaks, and baseline distortions.

The first chromatogram below is of a conditioning injection on a new column for an established reversed phase method:

chromatogram_1

The overall response appeared comparable to what was typically observed on that system for this method, and PEEK fingertight connective fittings were used—essentially ruling out overloading and installation errors.

However, it was uncovered that a normal phase method was being run on this system just prior, and after further examination, it was realized that the mobile phase lines were properly flushed prior to introducing reversed phase solvents. The injector and needle wash remained in an iso-octane based solution.

After purging the injector loop and replacing the needle wash with reversed phase solutions, the following chromatogram was obtained from the same sample in a subsequent injection:

chromatogram_2

This is still not an ideal chromatogram for this method, but it shows the improvement as the residual iso-octane—that was inadvertently introduced to the column—is eluting.

This can take extensive flushing and, in some cases, be impossible to completely remove.


 

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