Chromatography Forum

I am trying to develope a method for Tylosin oxytetracycline and doxycline.
But I have a problem with the right column choice.

I am hoping someone can tell me wich column is suitable for this method.

If it helps, there is a USP Monograph for both doxycycline and oxytetracycline. Each monograph lists an L21 column, which is, per USP "a rigid, spherical styrene-divinylbenzene copolymer, 3 to 30 μm in diameter." As you may know, USP methods can be less than optimal but depending on how robust you need your method to be, you can use this as a starting spot.

Thank you for youre replay.

At this moment i have found a method which gives a separtion that is good enough. The problem is that most peaks have tailing.

I am thinking of using a ion pare to give a better tailing.
Does anyone can advise me which one is best to use? and why?


The method

75% phosphate buffer 0.01m ph 3.0
25% acetonitril

If tailing is the major problem, try increasing the buffer concentration first. Ion pair methods are not particularly easy to develop and bring their own "baggage" of problems (e.g., equilibration time and baseline issues).

What concentration would be recommende for a buffer ?

I have tryed it with a buffer of 0.05m phosphate. ( there was no change in tailing factor)

If there is no change in tailing factor when going from 10mM to 50mM, I don't see any sense in going to even higher concentrations. Buffer capacity doesn't seem to be your problem.

Some thoughts:
-Which column are you using? Tetracyclins can AFAIK act as metal chelators. If you're using some kind of "old" (type A) silica column, residual metal ions might lead to tailing. If this is the case, switching to a more pure type B silica column and/or including EDTA in your eluent can improve the tailing.
-According to drugbank.ca, the pka of oxytetracycline is 3.27. A pH of 3 in your mobile phase does not promise a robust separation. You usually want to be at least 1 unit away from the pkA of your analytes.

Thanks for youre reaction.

Yes a I'm using a old column type ( lichrosphere) and that is a type A column.
( was also thinking of changing it )

I will also try to lower the pH of the mobile phase