i am trying to separate GTP (Guanosine triphosphate) and c-di-GMP molecule by HPLC using Water's Xterra RP-C18 column (4.6X250mm). i used isocratic or gradient both solvent system of water and methanol. i am getting both peak very close together less thn one min difference.
GTP (molecular weight 523.18 g/mol).
c-di-GMP (mw 690.09 g/mol).
Try adding 50mM Triethylammonium acetate (TEA) pH 7 to the water with 2mM EDTA. The EDTA chelates divalent metal ions like calcium, the TEA provides ionic strength to help with the resolution. I typically made 50mM acetic acid in water and brought it to pH 7.0 with triethylamine then added 372mg/L ETDA. I ran a shallow gradient starting at 0% to 15% methanol over 30 minutes to separate GMP and GDP from GTP.
Thank you Bruce Levison Sir.....surely i will try these modification in my protocol.....thanks :)
You also could change to ion exchange chromatography. See, as example,
Acta Chemica Scandinavica 8 (1954) 1877-1879
http://www.dionex.com/en-us/webdocs/7441-AN162_LPN1631.pdf http://www.dionex.com/en-us/webdocs/88518-Bro-ICS-5000-Bioanalyses-11Oct2010-LPN2585.pdf
Best regards
Thanks Sebastian....... i want to separate or detect only GTP and c-di-GMP which is in mixture give peak close together..... other NTP (CTP, TTP, ATP) are not present in my reaction mixture
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