Chromatography Forum

Hi everyone,

I am using a Waters UPLC with the AccQ-Tag ultra method for amino acids. I have large NH3 peaks (that is not caused by contamination) that are masking the CYA (cysteic acid) peaks. I just put a brand new column on and the separation between these peaks is 0.1 mins so I can not see the small CYA peaks anymore because of the NH3 tails. I am able to see the CYA peak when that peak is larger or if I have a sample with a low NH3. With my older column the separation between these peaks was 0.15 mins and I could see all the smaller peaks. But that column is now unable to be used anymore.

When i did a CYA MDL i was able to see these smaller amounts, but then again it was when the separation was at 0.15min

Does anyone have any method modifications to help? Wavelength, buffer changes, temperatures, etc? I am willing to run each sample with 2 methods (standard to get the AAs and modified to get CYA and NVAL if needed)
OR know anyway to reduce/remove NH3 before derivation?
Or know if I concentrate my sample more than recommended if CYA and NVAL if develop fully? Then i can run again with more diluted sample to get the other AAs?

thanks for any advice.

Have you tried purging out the ammonia with a flow of inert gas ? What is the pH of the sample before you derivatize ?

Peter

the pH is between 8.2-10

"Have you tried purging out the ammonia with a flow of inert gas ?"
Not sure how I would do this. Can you elaborate? I've very new to this field, sorry.

At the pH of your sample the ammonia will be un-ionised; therefore volatile (you might even be able to smell it). If you gently bubble nitrogen (or helium but it is much more expensive) through the sample it will carry the ammonia away as a gas without affecting any of the amino acids that you are interested in. I am assuming aqueous samples here.

Peter

ok thanks. I will give it a try.

Hi Peter,
I don't think i'm doing it correctly, sorry.

I was at first bubbling nitrogen gas into my sample for about 5 minutes - nothing happened. But then i realized that the sample is acidic until i added the derivatizing buffer which brings up the sample to a pH above 8. So I then tried to bubble the nitrogen gas in that sample. I only bubbled the gas for 20 seconds or so because its such a small amount (10ul of sample and 70 ul of buffer). But it still again didn't reduce my NH3.

Am I supposed to bubble for a longer amount of time?

sorry again if this is such a basic concept that i should know.

jennifer

Purging out the ammonia was just a guess at a sensible approach to removing a volatile contaminant from a non-volatile sample. With such tiny volumes I would have though that the ammonia would have diffused out quickly anyway. Are you sure that the problem is caused by ammonia ?

Peter

oh ok - it did sound like something that should have worked.

All i know is in the chromatogram the NH3 peak is very large and is merging with my cysteic acid peak. With my previous column the separation was 0.05 min longer between these peaks than with my new column so I can't see the 2 peaks anymore. If there was a way to reduce/remove NH3 i figured i would have a nice cysteic acid peak that i could quantify.

How do you know that the "NH3 peak" is due to ammonia in the sample ?

Peter

I’m simply wondering about lc-method (uv-detector?) which would be able to separate and detect ammonia.

Please be so kind and add some basic information, like complete method-description, used instrument and chromatograms.