aminoglycoside antibiotic.. rp-isocratic lc.. no retention? - (Apr/05/2008 )
i'm working on a rp-hplc method for quantitation of an aminoglycoside (lots of OH/- groups and a few NH2/3+ groups) antibiotic in my lysate. for now i've only tried running a standard.. i got a paper which used spherisorb waters ODS column.. 80angstrom.. 5 micron.. % acn, 5%methonol, with some tfa + tca in it.. its an rp-isocratic lc..
what i did was used the same stuff.. just that the column they mention wasnt' available.. so i used a normal ODS, 80 angstorm.. 5 micron.. not waters. what i see is no retention:( my std elutes with the buffer. i had the method for ELSD detection.. i tried higher amounts on RI.. i see a dose response so i suppose the std is not binding (and not not-eluting). with literature i figured this is not reversed phase binding on the column but ion-pairing.
i tired to lessen and then remove the an and methanol % so has to make more polar mobile phase.. increase tfa.. etc.. i dint see binding. m i missing out something? how do i retain my antibiiotic here!! plss suggestt.. thank you!
not a real expert in HPLC myself. this compound is very hydrophilic and cationic, rp may not be the most efficient column for that. just wondering if normal phase or anion exchange hplc would work better in your case.
normal phase is not the way to go.
when you tried to match the columns, did you check for end-capping? spherisorb ods2 and odsb are end-capped, ods1 is not.
did you compare carbon load%? ods2 and odsb are 11.5%, ods1 is 6.2%.
ligand coverage? ods2 and odsb are 2.98umol/m2, ods1 is 1.49umol/m2.
these are all factors that will affect retention of your antibiotic. when you change columns you need to try to match the characteristics or expect to see significant differences.
you may have needed to reduce the organics in your mobile phase (or, at least, one of them (acn?)).
i'm attaching waters' hplc troubleshooting guide, it may help.