Chromatography Forum

Hi,
How could I improve peak shapes when using 5mM DBAA ? They are not focused very well and tailing too. I do not want to use more than 5 mM IP (volatility). Could somebody help me, please?

A bit more detail would be helpful:
- analytes
- column
- mobile phase
- temperature

analytes: nucleotides
column: HyPurity Aquastar
mobile phase: ACN vs. 5mM DBAA pH=6
temperature: 30°C

I am not sure, if it is not peak shouldering. Or is it the fact, that I adjusted the pH of the buffer with acetic acid and therefore somehow changed the IP agnet?

Purine nucleotides have been separated on our Unison UK-C18 column
using 20mM ammonium acetate and 10mM DBA-Acetate:

http://www.imtakt.com/TecInfo/TI133E.pdf

I have got some more questions.
1. what is the pH of the buffer you use and how does the pH influence the separation and retention?
2. I have read that 10 mM DBAA supress the signal in MS. Have you tried this IP in lower concentration? (5mM)
3. Do you make this buffer by yourself or you buy it ? (I have bought it but I find it too expensive)
4. Don´t you ever observed loss of retention or backpressure elevation when using this IP agent for a longer period of time?

Hi -

As you know, nucleotides are highly polar and are difficult to analyze on LC-MS using ODS stationary phase.

1. ammonium acetate is a neutral buffer. It is used in this example to achieve consistent retention since the compounds being injected are ionizable.
2. Sorry, the only data we have is in 10mM
3. Loss in retention / increase in pressure I think is more of a result of inadequate sample clean up procudure. You may want to evaluate this (and / or use a guard column / cartridge).
4. Evalute other phases other than ODS if you are not happy with your results. Just make sure the supplier can provide you good batch to batch reproducibility data.

So you see no problem using 10 mM DBAA for a longer period of time?
And do you use the DBAA made of your own or you buy it as concentrate ?

Forgot to mention - the buffer can also improve peak shape.

Why don't you just try ammonium acetate instead of acetic acid,
see if that helps? Then you can determine if you still need more IP reagent.

Is this a biological sample?

I ll try it for sure. It is not a biological sample yet. I need to separate the standards first.

Well, I tried the separation in 10 mM ammonium acetate and it did not work. What should be the pH of the buffer ?

10 mM acetat ammonium + 5 mM DBAA of course

You can also try (NH4)2HPO4 as done on our Cadenza CD-C18:
http://www.imtakt.com/TecInfo/TI032E.pdf

What nucleotides are you trying to separate?

I cannot use phosphate buffer because it is not volatile. I am trying to separate N-6 substituted adenine nucleotides. Right now I am making experiments with pH of the DBAA buffer.

You are right, it's by no means the first choice for LC-MS, but ammonium phosphate can be used on an ESI source.

Lots of articles have been done on LC-MS of nucleotides - a google search will be very helpful.