Chromatography Forum

I am trying to develope an HPLC analysis method for doxycycline.

Mobile phase composition: 76 0.02M KH2PO4 adjusted to pH 2.5 using phosphoric acid 24ACN. Isocratic.
Injection vol: 50 ul at 1 ml/min
Column: Symmetry shield RP18 4.6 x 250 mm
wavelength: 355 nm
RT for the analyte is 7.4 min

So far, I am able to get the analyte peak. The only problem I get is the shift of RT as the concentration decreases. For example at 20ug/ml its RT is 7.4 min, 2.5 ug/ml the RT is 7.6 min while at 1 ug/ml, RT increased to like RT 7.8 min.


What causes the shift? Is it normal and acceptable? By the way, if I want to check pKa of the analyte, other than Merck Index, is there any other way I can get from the website? Any suggestions would be much appreciated.

It might be an overload effect, although that usually makes retention time increase as a function of sample load.

Was there any effect on peak shape (tailing or fronting)?

I am trying to develope an HPLC analysis method for doxycycline.

Mobile phase composition: 76 0.02M KH2PO4 adjusted to pH 2.5 using phosphoric acid 24ACN. Isocratic.
Injection vol: 50 ul at 1 ml/min
Column: Symmetry shield RP18 4.6 x 250 mm
wavelength: 355 nm
RT for the analyte is 7.4 min

So far, I am able to get the analyte peak. The only problem I get is the shift of RT as the concentration decreases. For example at 20ug/ml its RT is 7.4 min, 2.5 ug/ml the RT is 7.6 min while at 1 ug/ml, RT increased to like RT 7.8 min.


What causes the shift? Is it normal and acceptable? By the way, if I want to check pKa of the analyte, other than Merck Index, is there any other way I can get from the website? Any suggestions would be much appreciated.

well a + 0.1 shift in retention time is acceptable and the RSD of 10 injections should be less than 2%.

more so you are working with single analyte, a shift in RT is not significant as far as your analyte is getting eluted within reasonable time (less than 10 mins) with a capacity factor between 1 - 10.

try keeping your chromatographic conditions constant as far as possible with little varriation that due to random errors you may find the varriation of RT within limits and your variation is not much.

check the LCGC artlce over retention time changes. You may find the answer to your RT varriation

for pka you can refer some medicinal chemistry books or the particular literature related to analyte you may find the pka mentioned in them.

I agree with Tom that RT shift is caused by overload. Doxycycline I think is a base, which will be positively charged in the mobile phase with PH 2.5. I read a few articles talking about repulsion effect of charged analytes in the stationary phase which overloads the C18 column. It looks like tailing problem but is actually overload. This can explain why retention time increases when the concentration decreases. If smaller injection volume is used, it should improve RT shift. A better solution might be a change of mobile phase, like using ion pair agent.

Dear all,

Really appreciate all your valuable suggestions!
I manage to sort it out.

Thanks for all your assistance.

tlili,

Please tell us how you sorted out the problem. What was the answer?

Sure, Victor.

I am still using the same composition and conditions exactly as what I have stated in my early message.

The only difference was before i begin my run everyday, I actually flush the column with 100% MilliQ for an hour, 50 ACN_50MilliQ for 30 min, 70ACN_30MilliQ for another 30 min. After that, I will proceed flushing my column using 100% MeOH for 30 min follow by my mobile phase composition for 30 min.

For some reason, eversince I did that I notice that I don't encounter anymore fluctuation in my RT for all my different concentration up to 0.1 ug/ml. The RT for all the different concentration are ±0.02.

I am also not sure what is the rationale behind. Any suggestions would be appreciated.