Chromatography Forum

Is it normal for peaks to tail as column temperature increases?

I am analyzing sugars using a HILIC column and notice significant tailing as I increase column temperature from 25C,30C, 35C, 40C.

Andy Alpert will probably give you the right answer when he notices that this is a Hilic-related thread. But I'd ask how good your column oven and solvent pre-heater are? Hilic is very dependent on things like pH, and pH is very dependent on temperature, and temperature on the outside of a column isn't necessarily the same as that on the inside of the column, if the solvent isn't perfectly pre-heated. If the solvent bottles are sitting at room temperature, and the column oven is set to 25, then everything's more or less the same temperature and there will be only very small temperature gradients, no matter how bad the column oven and preheater. As you raise the temperature, the oven and preheater have more work to do to make sure everything stays coordinated, and temperature differentials will get bigger, as will any problem they cause.

I defer to LMH regarding the instrumental issues. As to the chromatography:

1) What column are you using for HILIC?
2) What's your mobile phase(s)?
3) Exactly which sugars are tailing? Are any sugars not tailing? If so, then which ones?

Some sugars have two or more forms and you might see this. We have an approach to address this issue. Contact me through our website and I will advise you on what to do.

I defer to LMH regarding the instrumental issues. As to the chromatography:

1) What column are you using for HILIC?
2) What's your mobile phase(s)?
3) Exactly which sugars are tailing? Are any sugars not tailing? If so, then which ones?

1) Luna Omega SUGAR 3um 250x4.6mm
2) 75% ACN in H2O
3) All of them slightly but Galactose more than others: fructose, glucose, sucrose, maltose, lactose. Galactose tailing increases significantly as column oven temperature increases.

Andy Alpert will probably give you the right answer when he notices that this is a Hilic-related thread. But I'd ask how good your column oven and solvent pre-heater are? Hilic is very dependent on things like pH, and pH is very dependent on temperature, and temperature on the outside of a column isn't necessarily the same as that on the inside of the column, if the solvent isn't perfectly pre-heated. If the solvent bottles are sitting at room temperature, and the column oven is set to 25, then everything's more or less the same temperature and there will be only very small temperature gradients, no matter how bad the column oven and preheater. As you raise the temperature, the oven and preheater have more work to do to make sure everything stays coordinated, and temperature differentials will get bigger, as will any problem they cause.

Don't have a solvent pre heater, but do have a column oven. Will it make significant difference if the column temperature fluctuates between 25-27 degrees due to room temp?

I wouldn't have thought 25-27 would be a problem! But I defer to Andy on the subject of Hilic chemistry! I still think it's magic... (but amazingly helpful magic)

My first thought was that the tailing was a sign of partial separation of anomers. Reducing sugars exist in two anomers. That's probably the two forms that Vlad Orlovsky is referring to. HILIC separates them. Partial separation can look like tailing, while good separation results in two peaks with a continuum between them (consisting of the intermediate open chain forms).

All of the sugars in your list are reducing sugars except for sucrose. Does the sucrose peak tail as well? If it does, then we'll have to look elsewhere for the reason. If it's the only one that doesn't, though, then we can safely assume that the problem involves anomer separation. Now, at high pH, anomer interchange (mutarotation) is so fast that one gets a singlet peak that's the time-weighted average of the two anomers. You can accomplish that in two ways:
1) Add 0.1% of an amine (usually triethylamine) to the mobile phase;
2) Use a stationary phase that has an amine on the surface. The locally high pH microenvironment then accomplishes the same thing. This is the basis of the "amine silica" columns in use for HILIC of sugars since 1975.

The Luna Omega SUGAR column does have amines on the surface but it also has a mix of other functional groups. It's possible that that dilutes the concentration of the amine groups to the point that the local pH isn't quite high enough to accelerate mutarotation sufficiently, at least at your elevated temperatures. Try adding 0.1% triethylamine to your mobile phase to see if that improves the tailing.