Chromatography Forum

Hi
I'm trying to separate the major sugars components in wines, liquors and food: Fructose, Glucose, Saccharose, Maltose and Lactose.
I have been working about 2 years with NH2 columns of different brands and RI detection.
It worked quite well excepted that Columns life was tipically 3-5 months and determinations under 2 gram/liter worked only with new columns. (dry liquors...)
Now I'm working with an ICS 3000 and carbopack PA-10 column (250 x 4 mm). The problem is that the column is damaged and it's so expensive (2400€) that I'm looking for other solutions like Xbridge.
But before the purchase I want to know if someone of you have worked with these columns and Sugars.
I've heared several opinions about the Xbridge stability not too pleasant.

Excuse my english

Any HILIC column will separate these sugars. What will be different on the amide (or any other HILIC column) compared to you amine column is that you will get separation of the anomers of reducing sugars. The mutarotation can be speeded up by adding an amine to the eluent.
All amine columns are notoriously unstable, the amine column also reacts with reducing sugars forming schiff bases.
I guess your high LOQ is from the detector, are you using RI?

We have these applications for sugars but that is using MS, I dont know what LOQ you would get with an RI detector.
http://www.sequant.com/files/documents/ ... actose.pdf

http://www.sequant.com/files/documents/ ... actose.pdf

Many Thanks per your interest.
Yes I'm using an RI detector.
Anyway with amino columns I've been abble to quantitate as far as 0.2 g/l. but it increases as the column is ageing. (wider peaks, shifting baseline etc...)
I have no experience with amido columns. My question is if they are more stable in this environement, means reducing sugars, ACN/H2O as eluent etc.

Hi Serene,

It is hard to separate Saccharose and Maltose. We also found NH2 column is better than any Carbohydrate column. But if you do want to try Carbohydrate type, Pb would be the most suitable form. I send related data to your gmail account. Please check and hope this can help your research.

thanks a lot

We have the BEH Amide column and use it for the analysis of derivatised oligosaccharides but not for simple sugars. But I don't expect the column to have a longer lifeime than the Dionex columns.

Once we talked to a guy from China who had used a PA1 column for over 10 years without any serious deterioration in performance ! I think we normally achieve around 2000 injections before we start to think about replacement

thankyou

WillyOne - Were you able to get the BEH Amide column validated for sugar analysis in food? I am attempted to do the same thing with simple sugars. I ran into some issues with short column life and overpressurization at aroun 100-200 injections usin ACN, Water and TEA as a mobile phase modifier. We based our initial method on Waters Beer fermentation analysis method. Did you run into any problems with column overpressurizing or plugging? Glad I found this post, eventhough it is an old one. I have my own post going as well. It is really hard to find people attempting to use the column for monomeric analysis. There are several, problem free references of using the column for oligosaccharide analysis. We just cannot figure out what what the difference would be between the oligosaccharide analysis and using the same methodology for simple sugars in fermentation broth. We haven't even run real-world samples yet and have already wrecked two columns!!

Any help you can provide would be greatly appreciated.

Jamie

Jamie: You are the first person I know interested on it.
I have also damaged 2 columns and I'm working with the third one.
It seems that, due to the alkalinity of eluent, proteins precipitate clogging the inner filter of the column.
I have tried to overpass this issue washing with citric acid 0.5 %w/v in water at the end of each day.
The problem was that high water content in eluent takes to really high pressures. So, to clean the column a 5 hours program.
It works but anyway I had an increase on working pressure smaller than the previously observed but always positive.
Finally I decided to install a precolumn of the same phase and cleaning the system with 50:50 water/ACN plus 0.1% v/v of Trifluoroacetic acid.
My idea was that if I have a clogging, to wash only the precolumn that should be easier.
But the procedure works so well that I have not had to dismount the column. I have not observed any pressure increase in my last four runs since I begun working with TFA. (two months ago)
My eluent is 20:80 water/ACN plus 0.2% of Triethylamine.
Next Monday I will post here the program I'm using.

About validation:
My needs are Fructose, Glucose, Succrose, Maltose and Lactose.
Separation between Maltose and Lactose is bad. It does not reach baseline in my last column.
Happily on my samples I never have both of them in the same sample.
Another issue, natural orange juice (not coming from concentrates) present a peak coeluting with maltose one.
Linearity is very good for 1 to 20 grams/liter, R2 better than 0.9995 and individual residuals smaller that 5%.
I work with RI detection, Flow 1 ml/min, column and detector kept at 35°C. sensitivity 256 (Waters RI), injection volume 10 µl.
Intermediate precision looks good but I can't give you a RSD value, not yet.
I can tell you that I have participated in interlaboratory test on wines, honey, soft drinks and liquors with good results.

I Understand that I still have some work to do on sugars, but I haven't had time to dedicate to that subject.

If you need further info ....

Regards

Guillermo



I

Ah, the column I use is BEH Amido column 250 x 4.6, 3.5µ particle size from Waters

Correction: I have not observed any pressure increasing over 300 injections.
At the end of the work and before stopping the equipment, the cleaning procedure is:
A 50:50 Water/ACN + 0.1% TFA
B: 20:80 Water/ACN + 0.2% TEA
C: Water
D: ACN
Flow: 1 ml/min
Time 0 0-0-20-80 (A-B-C-D)
Time 10: 0-0-50-50
Time 25: 0-0-50-50
Time 26: 100-0-0-0
Time 86: 100-0-0-0
Time 90: 0-0-50-50
Time 105: 0-0-50-50
Time 110: 0-0-20-80
Time 120: 0-0-20-80
Time 120 Flow 1.0 ml/min
Time 122 Flow 0.0 ml