Chromatography Forum

Hi All,

Have been getting a lot of HPLC knowledge thru the Forum, so first let me thank all the input.

I am developing a HPLC method for WS vitamins.

I manage to separate all 9 vitamins (B1,B2,B3,B5,B6,B12,Biotin,Folic acid and Ascorbic acid).

My replicates for standards are stable and no shifts at all. But when i inject the samples, the replicate injection will have all peaks shift late. And when go on to the next sample it gone back to the normal RT.

My HPLC condition: 5uL injection volume. 0.6ml/min. room temp. gradient mobile phase of phosphate buffer and acetonitrile. (max at 50% acetonitrile). I have a 10 min condition at the starting mobile phase concentration before injections. So I don't think it's the gradient change that cause the peak shifts (and I assume it only cause peak shift early anyway).

Thank you for the help.
Regards.

Most often with a gradient run, differences in retention times from injection to injection can be traced to be resulting from a too-short amount of equilibration time (really: solvent volume) after the run has ended to allow the column to equilibrate to the starting mobile phase. Just because the mobile phase pumping has the starting composition, the column needs to equilibrate.

Typically 5 to 10 column volumes is used for equilibration. Volume is the volume of the tubing and the space inside the column. Oftentimes this works out to 5 or 10 minutes.

If a longer equilibration time helps, then you needed it !!!

Hi, thanks for the reply.

But the problem is my replicate injections (3 times each for 5 different levels) on my standards is OK. This only happened to my samples and I already have allowed a 10 minute of run time at the initial mobile phase condition before all the replicates (both standards and samples).

And the only problem is on the 2nd injection of sammple, 1st injection always normal in peak RT compare to the standards.

Hi, I think I may have idnetify my problem by reading thru the archive.

I was adding NaOH in my standard in order to dissolve the Folic Acid (but only dropwise). But in my sample preparation, I added excess amount of 1M NaOH (10ml to 200ml) so the solution now is basic. instead of the buffer pH 5.5.

Is this why I have the shift in retention times of my samples peak but consistent retention time in my standard?

I guess I'll have to adjust my sample preparation.

Thank you again for the help from this Forum.

Thanks for getting back to us with the solution!

I was adding NaOH in my standard in order to dissolve the Folic Acid (but only dropwise). But in my sample preparation, I added excess amount of 1M NaOH (10ml to 200ml) so the solution now is basic. instead of the buffer pH 5.5.

Aha !! You held back some information !!!