Separation of ATP and thymidine monophosphate

[chromeleon]

Hi all,

I am interested in the thy phos, but it has the same retention time (5m) as ATP in the existing solvent system (10mM KH2PO4/MeCN 95:5). also the ATP concentration is relatively large so the peak is HUGE, with some tailing.

I am guesiing HILIC would be more suitable to resolve these, albeit I'm not very experienced with that technique.

any suggestions? thanks

[unmgvar]

or maybe a polar embeded like we use from sepax
they have an application for something close
http://www.sepax-tech.com/application_notes/OD1001.pdf

[Bintang]

ATP generaly is easy to retain in HILIC mode but often shows poor peakshape.

We have this application note for ATP, ADP and AMP using our ZIC-HILIC column.
http://www.sequant.com/files/documents/ ... nd_ATP.pdf

And Thymidine mono phosphate is relatively simmilar to UMP which we have this note on, different pH but still
I will eat my lab coat if ATP and TMP co-elute.

http://www.sequant.com/files/documents/ ... nd_UTP.pdf

Also TMP will elute before the huge ATP peak.

[Andy Alpert]

You can separate TMP and ATP isocratically using the ERLIC mode. See Fig. 19 in the following paper: http://pubs.acs.org/doi/pdf/10.1021/ac070997p
If all you want to do is separate those two compounds, then you can use mobile phase conditions more convenient than the ones used in this figure.

[nschwartz]

Restek also has a polar imbedded C18 column that can work well for compounds such as this. Here is an example chromatogram that shows separation of ATP and TMP:

http://www.restek.com/chromatogram/view/LC_0128/IBD

Also some examples for related compounds:

http://www.restek.com/chromatogram/view/LC_0133/IBD
http://www.restek.com/chromatogram/view/LC_0132/IBD
http://www.restek.com/chromatogram/view/LC_0129/IBD

[Bintang]

I do not think a polar embedded RP phase will give enough separation if you have a huge ATP peak it will likely tail into the TMP (kind of looks that way on the links given by nschwartz). I think HILIC (or ERLICH, I will take Andys word for that it will work) is a better alternative.

[unmgvar]

chromeleon can you say which column c-18 you have used?
from all the application shown from start to end, it is interesting to see that peak shape of all peaks are "bad" since done mostly in an isocratic mode.
you will probably need to make a gradient run in order get a better peaks shape.
can you give us the ratio difference you have between the compounds?
can you show us your chromatogram?

[chromeleon]

Dear All,

sorry for the late reply. I appreciate the number of replies and attempts to resolve this (pun intended)

I attempted method development on Waters uBondapak, C18, 7.8 x 300 mm, 10 um particle size.
the reason for using these dimensions is that I intended to incubate cell homogenates with some compounds I'd synthesised. I was then taking the incubant and diluting with 1.2 mL mobile phase, followed by filtration.

Essentially 1 mL injected onto column.

it transpired after this that some of my chemistry wasn't working, so Im focused on that right now...
I will say that the target molecules (thymidine analogues) are quite lipophilic, so my reasoning is that even after deprotection of the thymidine scaffold, the molecules should still be lipophilic enough not to elute too early. I am almost certain that they will elute significantly later than ATP.

but first I must make them, and characterise them, before handing over to biologists!!

edit: it is quite late so I will return to this thread on monday and deal with the various comments/suggestions
I'm thankful for this community