Chromatography Forum

I am going to try and quantitate sugars with the Water's XBridge Amide column. Looking at the Water's website the applications they have for this use use either 0.1% NH4OH or 0.2% Triethlyamine to get single peaks.

In some places they list the column pH range as 2-11 and in other places 2-12. The NH4OH would be about pH 10, not sure about the TEA.

My question is is there any reason to think using one or the other of the modifiers would give longer column life? This method will be used for a lot of injections.

Thanks,
- Karen

I ran that column ONCE with TEA and it was toast. It developed ridiculoulsy high back pressure
at any flow. In fairness to Waters I was in a rush to try it and didn't spend enough time preparing
my HPLC. The machine had never run so basic before and there could have been all kinds of horrible things suddenly going into solution or coming out of it and precipitating on the column.

I ran my Tosoh amide for thousands of injections with no problem. The Waters column looked promising but I never really got to evaluate it cuz it died such an early death.

To Karen01:
I'm really interested in your experience with Waters Amido column.
I'm working in an Laboratory dependent of the Regional Gouvernement in Valencia, Spain.
Please e-mail me in order to do not bore the forum with the carbohydrate analysis again

I am very interested in the result on sugar analysis using amide HILIC columns, too. Please share your experience with the others on this forum if possible.
My questions: is a high pH mobile phase necessary to obtain good peak shape for sugars when using an amoide column? Does higher temperature have the same effect on this type of column?

XL:
Sugars with a reducing end exist in solution in either the alpha- or beta- anomer form, with interconversion occurring via the transient open-chain structure. When interconversion is slow, then the two anomers are separated in the HILIC mode and the sugar elutes in two separate peaks connected by a continuum. When interconversion is fast, then you get a singlet peak that's the time-weighted average of the two forms. High pH accelerates mutarotation (= the interconversion). When HILIC was introduced in 1975 with analysis of sugars with amino-silica columns, the microenvironment of high pH at the surface of the stationary phase was sufficient to insure fast mutarotation and elution in singlet peaks. When people subsequently started trying neutral columns for the purpose, the anomer separation was noted. You can solve the problem by adding 0.1% triethylamine to the mobile phase and sample solvent. This raises the pH enough for the purpose. HILIC columns with an amide functional group require addition of triethylamine or some other (any other) base for the purpose. If you want to read more about this, look up my 1994 paper in Journal of Chromatography on HILIC of complex carbohydrates.

High temperature might accelerate mutarotation enough to produce singlet peaks too. Try it! I take no responsibility for the longevity of your column if you do, though.

Andy,

Thanks for the explanation. I tried Tosoh amide column for sugar analysis at elevated temperatures (50 to 70 C). The separation and peak shapes were ok, but the column efficiency dropped after less than 100 injections of standards. It looks to me more of a bed stability issue than a chemical stability issue. I am not sure if silica HILIC column will hold at high pH (10 to 12) under high organic solvent mobile phases (say MeCN in 70 to 90% range). Can you shed some lights on this? Thanks! I will read the paper you suggested.

To Karen01:
I'm pushing my boss for buying a Waters Amido column. But I would be sure I'm not doing the wrong choice.
The matrix are dry and sweet wines and alcoholic drinks, honey, soft drinks and different kind of foods. The requirements are at the nutritional label, (Carbohydrate content: grams per liter, g/100 ml or simply % ww.
I would work with an RI detector.
May I have your opinion about?

I used amide UPLC columns in a food lab for few years. We did analysis on melamine in high sugar content samples on them. They last. The NH2 columns were not as good for our purposes to separate melamine and co eluting sugars.

We confirmed by HRMSLC that some peaks co eluting on HPLC and separated by UPLC are not melamines, but have the same [M+H]+ and even few fragments of melamine masses, but HR confirmed the difference.

I tried both NH3 and TEA, but at such high % you need it will not be compatible with MS; I used mM levels.

Alexandre - I noticed that this post is very old, but I am still hoping that you can help me. I am trying to use the BEH amide column for sugar analysis in fermentation samples. We are using Water's base beer fermentation method as a start: ACH, water and 0.1% TEA as a modifier. We are running into the same problem as some of the other users in this series of posts. We are running the system just as Water's describes and we end up with super high back pressure after ~100 injections or so of JUST sugar standards in the appropriate solvent. 50/50 ACN/Water.

You said that the columns last. I am curious how many injections you can get per column. I wonder what your trick is for a long column life, and I am wondering if you would be willing to share it. We are using the 2.1x100 mm BEH amide column with the VAnguard guard column before it. We have wrecked two columns, doing nothing more than running sugar standards on it.

Any thoughts you might have on the matter would be greatly appreciated.

Jamie

I ran that column ONCE with TEA and it was toast. It developed ridiculoulsy high back pressure
at any flow. In fairness to Waters I was in a rush to try it and didn't spend enough time preparing
my HPLC. The machine had never run so basic before and there could have been all kinds of horrible things suddenly going into solution or coming out of it and precipitating on the column.

I ran my Tosoh amide for thousands of injections with no problem. The Waters column looked promising but I never really got to evaluate it cuz it died such an early death.

So, it is safe to assume that you never figured out what caused the column to fail? We are experiencing the same issues with the 2.1 x 100 mm Amide column. We have only injected standards so far, no real-world samples. We wrecked two columns so far. What I find so interesting, is that there are several applications in the literature describing the use of the same column, under the same conditions analyzing oligosaccharides from beet cells walls et cetera. I am starting to wonder if monomers are actually the problem. . . . . . .

Any thoughts or additional information that you have would be greatly appreciated.

Against the possibility that the 0.1% TEA is attacking the silica or BEH material, try inserting a "saturator" column inline between the pumps and the injector valve. This is just an HPLC column that's packed with some cheap, uncoated silica (say, 15-20 micron, uncoated, totally porous silica). It will saturate the mobile phase with silica, thereby sparing the silica in the analytical column.

I had forgotten I started this thread!

These days we routinely use an Aquity 2.1 X150 BEH amide UPLC column with 0.2% TEA on an HClass, injecting fermentation broth. Injecting standards NEVER causes a problem and we can often do several hundred injections of samples. However SOME samples can kill it almost immediately (Something added to the fermentation causing an issue) - but typically NOT because of backpressure- the separations deteriorates rapidly with those types of samples.

Thanks, everyone. We were able to get ~500 injections of standards when we switched to NH4OH instead of TEA at our elevated temp. We are going to go back to low temp, NH4OH and see how many injections we can get on the column. If that works, we are then going to revisit TEA at 35C, all else being equal.

Karen,

Wondering if you can comment on how many samples you can inject onto the column at 35C with TEA?

Jamie_Koehler@cargill.com

Thanks again! We are making progress, slowly but surely

Hi Karen,

How is your work going? Did you get better results with Nh4OH than with TEA? at 35ºC, I will really aprecite you coments and I am working with lactose on an amide column and I am getting mad!!

Hi Karen,

How is your work going? Did you get better results with Nh4OH than with TEA? at 35ºC, I will really aprecite you coments and I am working with lactose on an amide column and I am getting mad!!

Stayed with the TEA... In general no issues. Lactose is not an issue for us.

What type of samples?
How do you prep them?
What concentration range are you looking at?
What type of detection are you using?

- Karen