By Stephan on Wednesday, July 2, 2003 - 07:42 pm:
I have scaled analytical methods from 4.6x50mm with success to the semi-preparative scale - 21.2x50mm. This was always done with 25mM trifluoroacetic acid as buffer. The results were consistent with the resolution and the retention times being similar.
I switched the buffer to 25mM Ammonium Acetate (pH = 6) to help resolve an aziridine from its corresponding amino-alcohol. The resolution was great on the analytical scale column but horrid at the semi-preparative scale.
The semi-preparative column was cycled through about 17 column volumes to equilibrate the column pH by running a blank injection prior to injecting the sample.
DI water was added to the residual sample in the injection vial and the pH was determined to be between 6 and 7. So I don't think the sample had any significant impact on the pH of the column.
The semi-preparative injection volume was 1mL of MeOH (about 1/42 of the flow rate) so I don't expect the injection solvent strength to play a significant role in the poor resolution.
The columns have the same stationary phase which is a non-endcapped C8 touted as extremely stable for a pH range from 1 to 6. The column are probably from different lots but I haven't checked.
One thing to note is that the amino-alcohol capacity factor increased more than the than the aziridine capacity factor.
By Uwe Neue on Thursday, July 3, 2003 - 03:16 pm:
I assume that you mean by "horrid" that your peaks are strangely distorted. If this is the case, it could be due to the fact that the sample is injected in methanol. On a standard analyticl system, the sample is often diluted significantly with mobile phase before it reaches the column. This may not be the case for the prep separation. I would try to see if the problem goes away when the sample is dissolved in mobile phase. If this is the case, then you have to work on the details how you inject the sample.
By Anonymous on Friday, July 4, 2003 - 03:03 am:
I think ammonium acetate is a very poor buffer at pH 6, whereas TFA is a pretty good buffer at around pH 2.3; I guess this is where you are at, unless you've added something else besides TFA. So I think you are probably drastically overloading the mobile phase in your semi-prep separation (IF you are injecting relatively more-although you don't tell us the quantities you inject in the analytical vs semi prep case). In the analytical case maybe you can get away with it.
See J. Chromatogr. A 987 (2003) 17