Chromatography Forum

I'm developing an LC-MS method for thioguanosine nucleotides and I have discovered that in order to achieve good peak shape I need a pH between 7 and 8 and a high concentration of ammonium acetate.

I'm believing that if I could find another buffert that is MS compatible and buffer in the desired range (7-8) I could reduced the concentration of buffer. The trouble here is that I am having trouble finding a suitable buffer.

I have stumbled across 4-methylmorpholine as a suitable buffer but there aren't too many methods around (I can only find one).

Does anyone have experience of working with 4-methylmorpholine or know of any other buffer that I could use?

I am not familiar with analysis of thioguanosine nucleotides, but just from looking at the molecule (lots of tertiary amines), I can understand that you will have tailing problems with most columns I guess that is why you need so much ammonium acetate to suppress the silanols.

What column are you using? It could be worth a try to use a Primesep 100 column, which could allow you to work with more moderate ionstrengths. Otherwise go for the most endcapped column you can find. There should be good solutions for your problem, but it is a matter of budget also.

Svante,

You are correct that there aren't too many VOLATILE buffers that work in the full range. This is an excellent range for phosphate (pK2 = 7.2).

You mention that you are using large amounts of ammonium acetate. The pK of acetate is 4.75, so the acetate is well outside of the buffering range. You may find that you can get by with lower concentrations of ammonium bicarbonate, which is a good buffer in the range from 6.4 to 7.4. (Use ammonium bicarbonate reagent to avoid the carbamate impurity present in most ammmonium carbonate.)

There is a real gap in the volatile buffers between the carbonate region and the ammonium region, which runs from about 8.3 to 10.3.

I don't know if you could get away with using TRIS (tris-hydroxymethylaminomethane, which provides buffering from 7.1-9.1, but isn't commonly used as a volatile buffer.

If you can stay at the upper end of the range, I think you will find the ammonium bicarbonate buffer will serve you well. If you need to work close to pH 8, I would like to hear what others would use as a volatile buffer in this region.

We have used triethylammonium acetate at pH 7 for oligonucleotides. It is not a buffer, but it works well. Gilar et al. J. Chromatogr. A 958 (2002) 167 or J. Chromatogr. A 1169 (2007) 139

Svante,

I do not have a LC/MS, but I am using an Evaporative Light Scattering Detector, so I am faced with the same challenges of find a suitable volitile buffer. Ammonium carbonate might do the trick ranges are 5.5-7.5 and 9.3-11.3.

Thank you for your help everybody

Actually I'm running the separation in HILIC mode on a ZIC HILIC column from SeQuant and at the moment I'm running it without any ion pairing agents since that is normally not needed in HILIC (according to my experience). I'm pretty stuck between pH 7 and 8 because the packing materail can't tolerate more than 8 and at lower pH the peaks look awful.

My problem is that I need to go down from 60 mM Acetate to be able to use it for an MS method.

I will try both ammonium carbonate and triethylammonium acetate and what it looks like. If that does not work I'll probably return to reversed phase with an ion pairing agent.

Thanks again for all the good suggestions.

If you add some ionic interaction you will be able to go lower in pH and reduce buffer to 5-10 mmol:
http://www.sielc.com/compound_296.html
or instead of reverse phase with IP reagent you can go with mixed mode column where IP reagent is attached to the surface of silica. Browse these applications and may be you will find similar compounds. You can always use ammonium formate or acetate at pH 3-5 instead of acids (in some of our applications):
http://www.sielc.com/Applications_By_Compound.html

Imtakt has an application of mononucleotides using 50mM NH4HCO3
on Unison UK-Amino:

http://www.silvertonesciences.com/files/TI363E.pdf

Have you tried post column dilution with ACN?

The first pKa of carbonate does not work (I tried). The "buffer" is loosing the carbonic acid by evaporation of carbondioxide.

Maybe you could try the ZIC p-HILIC from SeQuant. It should be stable using higher pH.

http://www.sequant.com/sn/images/sheet_8.pdf

Maybe my ignorance of nucleotides is obviating this thought, but why not use a pH of ~3?

I am trying to analyze some modified adenosine mono- di- and triphospates HPIPC/MS.

You have written that previously you used HPIPC/MS.

What IP agent have you used and what column? And why have you chosen HILIC over HPIPC ?

The column you currently using may have too much undesirable ion-exchange property so that you have to use high ionic strength and certain pH to make it work. I would try a diol column (which has minimal secondary interation). If not successful, a proper type of mixed-mode column might work.

hi! Svante
this is an area we worked in for quite some time. TEAA is used by Waters because it is a volatile anion ion pairing agent, thus you will have to evaporate the TEAA before LC-MS. Waters produce an excellent Oligonucleotide purification applications booklet aimed at persuading you to buy their specifically designed 2.5uM Xterra C18 columns. We have, and they do work very well.
Methyl morpholine between pH7 and 8 is the buffer Waters recommend instead of phosphate which seems to trash Xterra(slowly) and many other Reverse phase columns(quickly). I doubt its suitability for LCMS but I have not tried it.
If you are analysing for trace amounts of your compounds by UV in the first instance, you may well have to use the most inert C18 on the market (Gemini perhaps) and either a phosphate or morpholine buffer for the best resolution. (Ionic strength of eluant is crucial). Not much use for LCMS though.