UHPLC problems: viscous fingering/tailing & peak overlapping

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Hello! I have a problem with my UHPLC sugar measurements and hope, some of you have any tips for me..

I want to quantify various sugar and sugar alcohols (sorbitol, glucose, fructose, lactose, isomaltose). I use a HILIC column with guard column, MeCN/H2O (80:20) as mobile phase, flow: 1 mL/min, samples diluted 1:1 with mobile phase, column oven temperature 35 °C, RI detector at 35 °C rise time: 0.5 s and data collection rate: 5.00 Hz, injection volume: 1 µL. The column is brand new (I also tested a older column with the same results) and the whole UHPLC system was already checked and cleaned.

The problem is now, that the peaks are not symmetric but more like tailing. I think the problem could be viscous fingering. I thought that diluting the samples should solve the problem, but it doesn't really. I've also tried to vary all the parameters (flow rate, ratio of mobile phase, different mobile phase, oven temperature, injection volume, ...) but had no positive effect.

A second problem: The problem above was observed with one substance samples. But when I want to analyse a mixture of sugars then it comes to peak overlapping. Also here I change various instrument parameter with no positive effect.

Does anybody have more ideas what I could try to solve problem 1 or 2? I was also in contact with the company but they could not help me really.

Kind regards!
Are your samples real fruit juices, or just standards?

Is it possible that you have a build-up of crud on the frit or at the head of the column?

Any increase of pressure?

If so, use the maker’s instructions for column cleaning in the reverse flow direction (put the column in backwards), and disconnected from the detector.
JMB wrote:
Are your samples real fruit juices, or just standards?

Is it possible that you have a build-up of crud on the frit or at the head of the column?

Any increase of pressure?

If so, use the maker’s instructions for column cleaning in the reverse flow direction (put the column in backwards), and disconnected from the detector.


I use just standards. There is no build-up of crud on the frit. How can I check it at the head of the column? The pressure is constant.
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