Chromatography Forum

Hello,
We have a problem with amino columns - poor resolution after few analysis. We followed the manufacturers instructions for regeneration and storage. It is saying to store with MeOH:Water. But when we use this column again it is not working good.
Does somebody have experience with amino column? And how to handle it?

e_georgieva,

I know this isn't what the manufacturer recommends but I would suggest that you make sure that the phase isn't in the free base for when storing the column. The free base form should be prone to autohydrolysis reactions. I would add a bit (a few mM) of acetic acid to the storage solution to prevent problems.

hi Chris Pohl,
it is my first time to hear about that amino column be stored with a bit acetic acid to prevent autohydrolysis ...it is quite interesting, is there any literature/article about that? or only deduction or experience? thanks!

hi e-georgieva,
did you once inject acid sample(s) to the amino column such as juice, etc? I was once told that might ionize or charge partial amino groups and thus affect the resolutions. to reverse this, try to wash the column with 0.5%NH3 in 50/50 acetonitril/water about 10 column volume.

if you did analyze acid sample and then the amino column was down, and all regeneration methods you've tried can not help to restore the column, maybe you should try the above.

meanwhile, before we say there might be something wrong with amino groups, you should also check whether it is ok with the packing(is there void in the head) or cleaness(is that contaminated by sample) etc.

kiknos,

This is based on experience with various weak base anion exchange materials

Careful...

If the column is used for carbohydrate analysis, I do not recommend to acidify the column. The basic environment in the stationary phase is an integral part of a carbohydrate separation.

If this is not the analysis that the column is used for, feel free to follow Chris's recommendation.

We use it for lactulose analysis.

kiknos,

This is based on experience with various weak base anion exchange materials

thanks, Chris!

those materials silica-based or polymer-based?

Kiknos,

All your questions point to the analysis of carbohydrate or sugar samples.

For this type of analysis, it is important to stay in a basic, at least a mildly basic environment. If you acidify the mobile phase, you impede the interconversion of the anomers, and you get double peaks. This can look like a dead column, but it really has nothing to do with column performance. It just has to do with the (reversible) status of the amino groups on the surface.

hi Uwe, many thanks for your kind reply! any resource for that?

by the way, i also met one case before: amino column is used for trigonellinelline, certain herb ingredient, should be zwitterion compound.
amino column gave out very good shape without any modifer in the mobile phase(acetonitrile/water), but later shows retention change(quite much) between batches. that time i thought buffer should help fix the retention on columns of different batches(same vendor), but i didn't do the tests. however, the two amino columns were kept in different solvents(one in n-hexane/EtOAc 95/5 and another in IPA in stock and shipment).

we also found 2-3 cases that the stocking solvents would look like change the retention on certain polyol sugars. but we didn't do tests to locate that.

any comments for all the above? thanks!

The retention change is due to a (slow) dissolution of the amino bonded phase in the mobile phase that you were using. This is due to the alkaline pH in water acetonitrile created by the amine. The result is that the next column that you get is at a different stage of hydrolysis, and you can't get the same results.

Waters has a developed a Carbohydrate Analysis Column that does not have this problem. You may want to look at this column for your next assay that requires the use of an amino column in an aqueous mobile phase.

kiknos,

FYI: My earlier suggestion to acidify the storage solution related only to storage conditions not to use conditions. Of course for separation of carbohydrates amino phases (as Uwe suggested above), the column must be in the free base form in order to accomplish the separation. It's still possible to store the column in the more stable salt form if one is interested in maximizing stability of the phase. One can convert the acetate form back to the free base form simply by washing with an eluent containing a mixture of water and an organic solvent although the conversion process won't be exceedingly rapid.

One other thought on how to reduce autohydrolysis: store your columns in anhydrous solvents. Since the hydrolysis reaction by definition requires the presence of water, it would seem to follow that storage in anhydrous solvent should help reduce hydrolysis.

Another thought regarding possible causes of variability from column to column after use. This might be related to partially converting the column to the salt form due to anionic components in your sample. The method mentioned above should ultimately convert the column back into the free base form but may take a fair amount of time if the counteranion is a strong acid. You might want to try treating suspect columns with a water-acetonitrile mobile phase overnight in order to assess whether this might be the cause.

thanks, Uwe and Chris! your suggestions offer some points for me to try next time when i use amino column.

by the way, i always perferred to keep the amino column in 100% organic solvent.