Chromatography Forum

Hi all,

We are looking for a suitable column for separation and quantitation of sugars (monosaccharides -fucose and galactose, trisaccharides and derivatives of these). We are using a Varian 385-LC ELSD for detection.

Currently a phenomenex Luna NH2 column is being used and we have achieved good separation, but have been seeing very poor S/N and poor peak shapes (they look like hedgehogs!). We think this is due to column bleed /phase hydrolysis. Can anyone suggest a more stable column for quantitation work?
We have considered Calcium columns but we are not sure if the high mobile phase water content required for these columns will lead to detector noise (our ELSD evaporator only goes up to 85ºC).

Attempts at using Hilic columns have lead to poor retention - no separation.

Is there anything we could try with our current column to improve S/N?

Thanks in advanced for any help!

You may want to look at the BEH Amide column from Waters. If you are interested, I can send you my presentation from the HPLC 2009 conference.

You can find references and links to scientific papers and other information related to analysis of sugars, sugar alcohols and other carbohydrates in different type of formulation and matrices using our HILIC columns at:

http://www.sequant.com/sugars

If you want to try a different amino column, I'd recommend Shiseido's CAPCELL PAK NH2. It seems to be more stable than other silica-based amino columns and works well for sugars in the HILIC mode.

Have you considered just using the bog standard RI detector for these mono-sachs?

I presume you can just dissolve up at high mg/ml your analytes in water? If so with a high enough injection volume, the RI should be able to give you the sensitvity you need.

Depending on what mon-units you are looking at will determine which cation phase to look at (Phenomenex and Shodex) have a lot of info on these types of columns.

Thanks for all your replies.

We unfortunately don't have an RI detector in house so that option is out. I'm starting to think that an RI detector would have been the better option for accurate quantitative work.

We may have to put up with noisy amine columns or try a BEH amide as Uwe suggested. Since the quantitation is primarily for leachate-tox studies we may not need this higher level of accuracy anyway.

You will be not happy with a Calcium column and your ELSED detector. Please try to heat your mobile phase to avoid any temperature gradient on your column. That will help to reduce the S/N ratio and might help to get better peak shape. For sure there are many HILIC columns on the market but I guess you have not the time to test all of them. Andy's recommendation is very good. I would like to add the Ashahipak NH2 column if you would like to look for an alternative NH2 column. In addition please isolate your tubing between column outlet and detector inlet to avoid any temperature losses. Good luck and have a nice weekend.