Chromatography Forum

we have a formulation that contains keltrol as a thickener. After several injections onto a column the peaks start to split. Our samples are clear before injection, but we suspect the keltrol as being the cause of the split peaks. Our mobile phase contains an ion pair. We have reverse washed the column with water at 50 degrees followed by phase but we still get split peaks. - does anyone know how we can remove the keltrol and clean the column up. desperate for suggestions.

Perhaps some details might help - such as manuafcturer, column type, size, mobile phase composition ( isocratic or gradient - if gradient your column's probably doomed ), ion-pair agent and concentration, and flow, sample prepartion and solvent, and injection volume.

Have you got a guard column in place?. If so, what happens when you replace it - does the pressure drop?. Did you monitor the pressure when you were injecting the samples?.

I'd expect the Xanthan gum to form a goo over the column if you tried a gradient, and the backpressure would climb rapidly, possibly forming a void in front of the column. If that's the case, it's unlikely to go away.

You need to test the column with the manufacturer's test mix, and compare resolution, peak shape and backpressure with the original certificate. Make sure you wash all the ion-pair agent from the column first, and carefully monitor pressures. Slow downward trends in pressure when reverse flushing may suggest you will be able to wash the gum off with persistence and warm water. If peak shapes are OK, it's worth persisting with flushing.

If it's a void ( test solution shows all peaks split, and backpressure is similar to CoA, not really high ) the best solution would be to purchase a new column, however the second best solution may be to take the column to a pressure about twice normal operating pressure using a high flow rate for about 10 minutes, gradually decrease flow to zero. Obviously don't exceed column's maximun specified pressure.

Carefully unscrew front fitting - if there's a void, carefully fill with material from the back end of a used guard column, or some other source of similar material. Repeat the pressurisation process, and refill if necessary. I've had columns go from 40% of orginal plates to 85% with this nethod, and got another six months of life.

Now, address the cause - change your sample preparation. You need to either trap the Xanthan on a resin, or perform a sample purification, possibly using SPE or Liquid-liquid extraction of your analyte into an organic solvent that the gum doesn't like, with teh solvent choice depending on your analyte. Also, ensure you have aguard column in place, they usually are a little cheaper to replace.

Please keep having fun,

Bruce Hamilton