Carbohydrate analyses with Amino Columns and UV

[josebenjamin]

Dear Friends,

I would like to hear from anyone of you with comments and experience doing carbohydrates by NH2 column separations and low UV detection. I am trying to help some people who has horrible problems doing the "traditional" ion exclusion separations and RI work. That works fine in some cases but everything has to be in nearly perfect conditions (pump, mobile phases, detector, temperature control, etc).

Hopefully some of you can give us help. The problems to be worked out have no sample limitations and there is no possibility of doing derivatizations, or acquiring other detection modes (electrochemical, ELSD).

Thanks very much.

josebenjamin

[Noser222]

What carbohydrates and at what concentrations are you doing? I've used amino columns for neutral sugars using LC-MS detection. The main problem was the stability of the column in my mobile phase of 80% acetonitrile/20% water, since the amines create a basic environment and self-hydrolyze. The stationary phase bleed left my spray chamber coated with a nice white power, but it still worked
I've performed low UV so I can't speak to your experiences there. You might try a polymeric stationary phase such as the Shodex Asahipak. For the group of sugars I use the selectivity suffers a bit, but there is no column bleed.
If you consider doing pre-column derivitization, there is a reverse-phase ion-pair method that could work for you with detection at 303 nm.

[josebenjamin]

Dear Noser22,

The concentrations we will work can be adjusted to any practical level we wish. And unfortunately we do not hace MS detection.

We are mostly interested in common componentes such as, glucose, fructose, sucrose, etc. We are aware that column stability can be a problem, but we hope that some of them such as the Zorbax SB series could give us some good results.

As I mentioned before, we have no possibility of unusual detectors or derivatization techniques.

Thanks for your comments,

josebenjamin

[Noser222]

I guess you are getting the sensitivity you need with UV and RI. The only way I know of to improve your sensitivity would probably be to derivitize.

If you are going to buy a new amino column, I'd just recommend the Shodex Asahipak over the Zorbax.

[Uwe Neue]

I don't know about low-UV detection, but I have done sugar separations with RI detection on amino columns that were specially designed for these separations. Under these circumstances, the common problems - long baseline drift and bleed - can be eliminated. You may check the Carbohydrate Analysis column from Waters.

[josebenjamin]

Dear freinds,

Thanks for all the info. I will look into Asahipak and Waters columns.

josebenjamin

[Kostas Petritis]

You may also want to look at the Astec NH2 column which must be very similar to the Asahipak column (so try the cheaper one)...

[Rob Burgess]

Jose,
What sugars are you interested in and what is your matrix. Ligand exchange column methodology may be a good alternative to use. Take a look at Shodex's website for more info.

I developed a method for lactose a while back. What we did was simply chnage the dissolving solvent to pure water so we could achieve a higher concentration of dissolved sugars. Also the ligand exchnage columns use pure water as the mobile phase which gives less baseline noise for your RID application. In comparison RID was still more sensitive than UV @ 190 nm for this chromatographic system.

[josebenjamin]

Dear Friends,

Thanks for all the info. I will try to get my hands on the Asahipak columns. My concern is also that large ID columns may dilute too much the elution bands and kill any hint of detection by UV. If any of you has any experience on using narrow bore NH2 columns for this application I would like to hear about it.

thanks again,

josebenjamin