DryLab for Mixed Mode Separations

[Rob Burgess]

Has anyone tried DryLab for mixed mode separations with say the PrimeSep column?

We are trying to develop a relataively fast isocratic assay and we are having problems trying to attain enough retention for one of our analytes (a sugar ester) while achieveing resolution due to some unknown contaminat peaks and, manually we don't seem to be getting anywhere fast.

The parameters I have tried adjusting are the % B (AcN) and the level of modifier (% TFA) whilst trying to maintain the run time under 5 mins!

[SIELC_Tech]

Rob,

I believe that ACD software has now Primesep capabilities. Also you can try to change buffer nature to achieve your resolution. Check the following link (glucose pentaacetate and prednisolone):

http://hplcmethods.com/compound_189.php

I will try to see if we can do something about usind ACD for youyr separation.

Regards,

VO

[SIELC_Tech]

Rob,

Here is article on application of ACD software for Primesep columns
" Prediction of Retention Times for Compounds on Mixed Retention Mechanism Stationary Phases"

http://www.acdlabs.com/download/publ/20 ... ention.pdf

[tom jupille]

DryLab should work just fine. It has a pH/gradient model that lets you look at the combined effects of pH and mobile phase strength.

[Rob Burgess]

To Tom,

The PrimeSep columns operate from two variable parameters % AcN and % TFA (I don't believe its strictly a pH affect we're trying to model here but rather from the concentration of a specified additive perspective).

In DryLab I was thinking I may have to create a new mode (i.e. %B / % additive). I have tried to create a mode for ternary gradients i.e. (3 solvent composition) but didn't seem to get anywhere with a decent mode that appeared to work. Basically I'm after some tips and tricks for creating a new mode.

Assuming that there is some level of interaction between % AcN and % TFA for this kind of mixed mode separation, how many runs would I require? For our neutral solutes I'm assuming the solvophobic relationship will fit linear with respect to % B but perhaps not for our amine! Therefore if it could be modelled adequately, how many runs would I need to model this for a isocratic separation. I was thinking of performing the following:

40% AcN (0.05, 0.10 & 0.20 % TFA)
50% AcN (0.05, 0.10 & 0.20 % TFA)
60% AcN (0.05, 0.10 & 0.20 % TFA)

i.e 9 runs. Does this sound OK. Does DryLab have a technical helpline I could call if I get stuck with creating this "new" mode?

[tom jupille]

Shortly after DryLab 2000/plus shipped, we released a "more modes" collection that included a "peptides" mode modeling gradient steepness and TFA concentration. It looks like it has disappeared from the Rheodyne website, but fortunately I'm something of a pack rat.

Rob, I just sent you an e-mail with the gradient template file, as well as a modified version for isocratic calibrations.

To use these:
1. Open DryLab and start a new file, then click on the Data Entry button.
2. On the Data Entry screen, select File . . . Load Data, and then navigate to the appropriate .inp file.
3. Exit DryLab, and then click on "Yes" to save changes to the mode list.

The next time you open DryLab, the new mode will be available at the bottom of the drop-down mode list.

Both of these modes require 6 calibration runs (a 2 x 3 matrix) with two gradient times (or %B values) and three TFA concentrations. The gradient times should differ by a factor of about 3 (e.g., 15 and 45 minutes for a 150 x 4.6 mm column) and the TFA concentrations should vary by successive factors of 3 - 5 (e.g., 0.01%, 0.05%, 0.2%). In the isocratic model, the %B values should be about 10 - 15% apart.

If you need to extend the range of prediction or refine the accuracy, you can add addition calibration data, with three limitations:
1. The program can accommodate a maximum of 16 calibration runs (I can't imagine that you'd want to invest that much time, but . . . ).
2. The additional data must fill out a rectangular matrix (i.e., to add an additional TFA concentration, you would have to do runs at both gradient times or %B values to fill out a 2 X 4 matrix; to add an additional %B value, you would have to do runs at each TFA value to fill out a 3 X 3 matrix).
3. The gradient model is restricted to two values on the gradient axis.

Let me know if you have any questions or problems.

[tom jupille]

Oh, if anyone else wants copies of those mode templates, you can e-mail me privately at the address below