Chromatography Forum

Hi,

It is quite easy to understand why there can be peaks from previous injections in isocratic mode (the peak continously move through the column), but how does it work in gradient mode?

I have a method that contains a gradient from 3 - 30% acetonitrile. When I inject a sample, the end of the gradient looks nice and clean. But when I inject the next sample or blank, I get a couple of nice sharp peaks in the end of the gradient that must come from the previous sample. I don't think I have ever seen this in gradient mode? Any thoughts?

Do you have different viscosity for mobile phase and sample solution? If you use an autosampler please implement a wash step before next injection is done. Or you have a so called "extra column effect" which means somewhere you have a inbuild dead volume. If you do several blank injections do you see that the unexpected peaks are getting smaller or do even disapear? When you start your gradient with 3% Acetonitril what is the 97%? Buffer? Do you see Peeks or Spikes? Run isocratic with 3% Acetonitril and than one run with 30% Acetonitril. Also possible is that your sample sticks on the frit at low Acetonitril concentrations. If different viscosities are in the game do you use autosampler with needle injection or sample loop with switching valve? I would check at first the injection system! Yes, this can happen also with gradient mode.

There's no reason you can't get peak carryover from previous injections in gradient mode, especially given that your method doesn't go to a very high acetonitrile %age. If you have components in your first injection that are only slowly eluted, the time at high organic in your method may not be long enough to fully elute them from the column.

Try running your gradient profile and adding another fifteen minutes run time on the end of the run, making sure to programme the pump to return to starting conditions at the time when it usually would. You may well see your extra peaks being eluted then.

You could try adding a quick high % ACN step at the end of your gradient, ie. go from 30% ACN at the end of the current gradient to 90% for one or two minutes

Thanks for your replies,

You are absolutely right that this is very possible also in gradient mode, and I have now inserted a 2 min gradient to 75% acetonitrile in the end of the method. It seems to work perfect so far!

The extra peaks that I got in the next injection turned out to come from an orange flavour that is part of the product.