Chromatography Forum

I am trying to develop a more simplistic HPLC analysis method for doxycycline than the one described in the USP.

I found this post: viewtopic.php?f=1&t=2155&hilit=doxycycline
Changed it to:

Mobile phase composition: 80 0.02M KH2PO4 adjusted to pH 2.5 with H3PO4 + 20 ACN. (Isocratic)
Injection vol: 10 ul at 1 ml/min
Column: Symmetry shield RP18 3.9 x 15 mm 5um
wavelength: 355 nm
RT doxycycline = 8.4 min

Like tlili I get the analyte peak. But after the injection point the baseline slopes upward until the analyte peak. After the analyte peak the baseline is back to zero.
(see image: https://docs.google.com/open?id=0B57F_5 ... lJSWlNiSG8)


What is causing this phenomena and how do I prevent it?

I suspect that the increasing absorbance is due to the doxycycline being eluted not as a single chemical entity, but possibly as a mixture of forms; e.g. protonated/non-protonated, or some other equilibrium species.

You might change (separately) pH and column temp. to shift any eqlbm.

Please post your results, whether it works or not.

Is the blank (diluent) chromatogram is good, only analysis of doxy, sample chromatogram has this baseline drift
check different diluents like acn, methanol, etahnol in pure form as well mixed with water based on the solubility profile and always compare with your blank chromatogram of the same diluent.
also mention whether you are working with API or Formulation product.

Sorry I haven't had the time to do further testing.
I'll come back on this when the next batch of doxycycline is delivered.

The picture of the chromatogram is an API doxycycline hyclate (100%).

I have the exact problem as artsjeroen, and non of the above solutions worked. This kind of baseline slope is more pronounced in higher concentrations. So it has to do something with Doxycycline itself. Does anyone else have any other ideas?
I would like to know, how artsjeroen managed the problem?

Try using a Progeny C8 column and 0.05 M (50 mM) pH 3 Potassium Hydrogen Phosphate Buffer.