Chromatography Forum

Hi all, I need advice about maintenance/troubleshooting of amine columns.

I got a new amine column which was performing good intially. I noticed though that the retention time of my compound has been shifting continuously to an earlier time (Eg. from 20min to 16min). My guess is that the column is degrading. It shouldn't be degrading though as I've only used it for 2 weeks with about 60 samples.

I've been following the column maintenance instruction which is to wash the column with the mobile phase (without the buffer) when leaving the column for a long period of time. I've also tried flushing it with 500mL of pure isopropanol to clean it out but the retention time just continued to shift earlier.

I also replaced the guard cartridge to make sure that any impurities is removed. Replacement of guard cartridge brought the column pressure down from 70bar to 50bar. However retention time is still becoming earlier (from 20min its now 15min).

My mobile phase is 80% acetonitrile (pH 5, buffer is NaOH/AcOH).

Is there another means of reviving an amine column? Is there anything that I'm doing wrong with column maintenance? Any help would be very much appreciated. Thanks!

Which amine column are you using? Most amine columns are known to degrade faster than other column types. They are particularly susceptible to Schiff base formation in the presence of carbonyls (aldehydes and ketones). I would worry that if your IPA had small amounts of acetone (the oxidation product of IPA), you could have further reacted the amine groups.

Reversing this reaction may be difficult. Your column may be dead. What sample type were you analyzing?

Silica-based amine columns in aqueous mobile phases have multiple problems. Schiff-base formation is commonly blamed for the issues, but it is in most cases very low on the list of causes.

So please confirm first that you have a silica-based amino column. Second, I expect that your buffer concentration is very low, since you are using only 20% aqueous. Please state the buffer concentration in the aqueous component of the mobile phase. I got a pretty good idea what might be happening, but I need this information to confirm.

Thanks very much to those who've replied!

I'm using an Agilent Zorbax Normal Phase NH2 column. According to the specs, it has boro-silica microbeads packed inside the column.

My buffer consists of 100mL of 0.02M acetic acid and 200 microliter of 0.1M NaOH.

The sample that I run through the column is a plant extract (also water-based).

I hope that there is still a way of recovering the column.

The next time, you either need to purchase a column that has been designed for use in an aqueous environment, or you need to start equilibrating it as soon as possible with a high (maybe 1M) buffer concentration in water before you go to your mobile phase. The issue is the very high concentration of the amine in the column, and the low buffer concentration, which means that you are effectively running your column for a long period of time at pH 9 or so, which means that you split off some of the bonded amine before you are in the final stage.

If you want to get around some of these headaches, I can give you advice on which column to buy. Contact me!

Below is durability data for Unison UK-Amino (3um silica based):
http://www.imtaktusa.com/site_media/fil ... no_web.pdf

The blue line in the data is what you see in conventional, silica-based NH2 columns.

Also, I'd recommend ammonium acetate. Increasing [NaOH] in such high % acetonitrile may cause some headaches.