Chromatography Forum

Dear All,
I am running reverse phase with Eclipse XBD C18 0.1% TFA and ACN (10 to 100% over 30mins) - UV-Vis detector.
I have turned off the column oven and the peak shapes are much better than at 40degC.
I always assumed peak shape would improve with increased temperature.
WK

Dear WK, you may need an eluent preconditioner for you column compartment. Adding cold eluent to the column will cause a temperature gradient and distort peak shape.

A

Could also be that your compound is slightly degrading at temperature.

Bruce Hamilton

I would not 100% cancel out the possibility of a temperature gradient, nevertheless I personally experienced that 40 degrees is not critical yet.

Degradation may also be a reason.

Nevertheless, I would be careful with saying that the peak shape always improves. This is mostly apparent in cases when columns used like based on a zirconium resin, with strong ionic sites.

A possibility could also be that the change of pH and pKa has in this particular case a negative effect on your chromatography.

Maybe other members of this forum can either confirm or invalidate this.
or invalidate this.

Thank you for the replies - my eluent preconditioner (heater) is blocked and I had trouble with the fittings so I cannot use this to try unfortunately.

You can try to make a coil with 50cm 0.13 stainless steel tubing and use this as a preheater. It’s not very efficient, but you can check whether you see improvement in peakshape.

Normally peaks of poor preheated solvents do not look like a peak anymore, but have the shape of a bump.

Could also be that your compound is slightly degrading at temperature.

Bruce Hamilton

If you're using only increased temperature on your column (40 degrees), and you did't heat the analyses before injection, I would'n say that the improvement in peak shape is due to the degradation on higher temperature.
Why?
The compounds in the column during the separation are bonded with the stationary phase. In that kind of conditions even temparature sensitive compounds don't undergo degradation (because they are most of the time bonded by different intermollecular forces to the stationary phase)
Let us presume that a degradation really had occured. Wouldn't we expect to find unknown peaks in the chromatogram, peaks of those degradation products?

Zoran

I do not fully agree with you on this.

You will find different peaks when the mix before injection is degradated in different products. When the compound is degradating on-column, the properties of the molecule are changing during elution. This results in peaks with tailing, or other strange observations.

As far as I know there is only limited literature about this. Some groups reported about it, but I never saw detailed investigations.
.

If you're using only increased temperature on your column (40 degrees), and you did't heat the analyses before injection, I would'n say that the improvement in peak shape is due to the degradation on higher temperature.
Why?
The compounds in the column during the separation are bonded with the stationary phase. In that kind of conditions even temparature sensitive compounds don't undergo degradation (because they are most of the time bonded by different intermollecular forces to the stationary phase)
Let us presume that a degradation really had occured. Wouldn't we expect to find unknown peaks in the chromatogram, peaks of those degradation products?

Zoran

Nice theory. The mobile phase contains TFA. If acidic degradation of the main peak occurs as the product travels along the column, the impurity peak will be less resolved the further through column the main peak travels. Increasing the temperature will promote degradation.

The consequences of on-column degradation could be:-
- The impurity doesn't elute, so main peak just gets smaller.
- The impurity elutes on or near the main peak, so the main peak smears.
- The initially-resolved impurity will smear towards the main peak, but impurity peak height won't change. The main peak height can change, and will smear towards the impurity.

I'm sure there are other scenarios, but if you put a sample into an environment of 0.1% TFA, you should always consider the possibility of degradation.

Bruce Hamilton