Chromatography Forum

Hi all,

Has anyone have an experience of resolving sugar phosphates such as ribose-5-p / ribulose-5-p, Fructose-6-p/glucose-6-p by LC-MS? I 've tried to optimize the pH of amino HILIC to separate them, but I couldn't get the baseline separation either at pH 7.5 or 3.5. I am wondering if RP phase with ion paring like the tributylamine can work for them. Also I worried about tributylamine can contaminate the column and mass spectrometry as I work at an open instrument.

Thanks!

Song

Such analytes will be very well retained on a silica HILIC column, without a need to use modifiers in the mobile phase. Acetonitrile water as eluent on an Atlantis silica HILIC column. My guess is that you will need at least 70% water to elute these analytes.

Hi Song -

Below are applications for sugar phosphates on Unison UK-Amino:
http://www.imtaktusa.com/site_media/fil ... TI446E.pdf
http://www.imtaktusa.com/site_media/fil ... TI447E.pdf

We do have customers here in the U.S. using UK-Amino for LC-MS of sugar phosphates.
But I'd prefer to discuss it in private with you since I do not have
their data yet. Please email me - we can help!

Uwe -

Don't you think these solutes would achieve better peak shape with bufferred eluent?

Bryan: I do not think he needs a modifier on a silica column. the charge of the analyte and the charge of the silica are similar - if any. So the only retention mechanism is a rather strong partitioning into the water layer on the surface. On the other hand, with an amino column, you effectively have ion-exchange and you need to modify your mobile phase to get around the ion-exchange mechanism. This may not be the best idea, if he wants to use MS detection. On the other hand, if there is too strong a partitioning into the water layer, he can manipulate this easily by addition of formic aicd or TFA, which both are MS compatible. However, I would try first simply with water/acetonitrile.
One can also manipulate the strength of the interaction with the stationary phase by substituting the proposed Atlantis silica with an XBridge HILIC column.

Hi Uwe -

Interesting idea - thanks!

Hi Uwe,

I worried about ion pairing for MS. But I tired the luna amino column in multiple gradient conditions and couldn't get nice separation of isomer sugar phosphates. I doubted if Xbridge / Atlantis silica is able to resole them better than luna.

If conditions are such that both the silica and analyte are negatively charged one has to consider Donnan equilibrium, that is, ion exclusion. As an example, a mismatch of analyte solvent and mobile phase can then cause severe peak broadening. The organic content might ameliorate this effect, but it could be that one needs pH and/or ionic strength control.

If it is any consolation my experience has been the same: several flavours of hilic retain hexose phosphates beautifully but the variations in elution times between different hexose phosphates vary between uncomfortably small and totally non-existant. I notice there are a few literature references using hilic for broad-scale profiling of polar cell contents that group all the hexose phosphates together.

Here is publication on separation of sugar phosphates using our Primesep SB mixed-mode reversed-phase anion-exchange column:

Metabolite profiling of Calvin cycle intermediates by HPLC-MS using mixed-mode stationary phases. Jeffrey A. Cruz, Caroline Emery, Matthias Wüs David M. Kramer and B. Markus Lange. The Plant Journal , Volume 55 Issue 6, Pages 1047 – 1060

Abstract for this and other publications can be retrived here:
http://www.sielc.com/Literature_Publica ... rnals.html


Also, here is application on some other sugar phsophates:
http://www.sielc.com/compound_289.html

let me know by email if you need more information

Song jack... the separation mechanisms between ion exchange on an amino column and real HILIC on a silica column are so different that nobody can predict whether the separation will be better or worse. Since you are not as successful as you would like to be with the amino column, why not try the silica HILIC column - with all the advantages outlined in my last post.

here's hoping no one needs to measure G6P and F6P separately. You can, of course, do them in spectrophotometric enzyme-coupled assays relatively easily.

We've studied this extensively with plant-extracted sugar phosphates, and even demonstrated separation between G6P and F6P. The systems we evaluated were porous graphitic carbon (PGC) and ZIC-HILIC, both with MS-friendly modifiers. For refs that give running conditions, see J. Chrom. A, 1172:170-178 (2007) (PGC) and RCM 22:1399-1407 (2008).

regards
Tony

thanks for references!