By Bharat Agarawal on Wednesday, April 14, 2004 - 09:51 am:

does the concentration of an ion-pairing agent have any effect on retention time? I have been using ACN: water (90:10) and an ion-pairing agent( N-Octane sulfonic acid sodium salt; 1mM) for the analysis of a basic drug. The retention time is 1.6 min. When I used 0.1mM ion-pairing agent the retention time is 6.7 min with a broad and tailed peak. I am not able to understand why did it happen? Please expalin it to me.

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By Anonymous on Wednesday, April 14, 2004 - 11:55 am:

Yes generally ion pairing agent concentration should have effect on the retention. More the ion pairing agent more will be retention.

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By AllsepTech on Wednesday, April 14, 2004 - 08:27 pm:

Check the following link, it describes retention of polar compounds without ion-pairing reagent. LC/MS compatible conditions, simple mobile phase, good peak shape and efficiency

http://www.chromatographyonline.com/lcg ... rticle.pdf

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By MG on Thursday, April 15, 2004 - 06:08 am:

Regarding Figure 3 of that link, I predict the TFA would drastically reduce sensitivity for the acids in LC/MS (negative ion), versus formic or acetic acid modifiers. If you've done the experiment and shown otherwise, I'd be interested to hear it.

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By AllsepTech on Thursday, April 15, 2004 - 07:15 am:

Dear MG,
TFA is one of several additives you can use with our columns. You can also use ammonium formate, and ammonium acetate at pH 3-4, formic acid, acetic acid etc (different concentrations). Mobile phase can be from 0% organic to 100% organic. Our mostly common mobile phases are ACN-water 40/60 with 10-50 mmol of ammonium formate (pH=3), ACN-water-TFA=40/60/0.1 and ACN-water-HCOOH=40/60/0.1

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By HW Mueller on Thursday, April 15, 2004 - 07:36 am:

Now I am really confused, having considered that dearly hated TFA as an ion pairing agent all this time (in protein applications, etc.).

Also, aren´t these mixed mode phases a little like those vacuum cleaners which are convertible to a drink mixer (or so): Don´t work correctly in either function??

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By MG on Thursday, April 15, 2004 - 09:20 am:

AllsepTech, your link was to a chromatogram using TFA, with the statement that the separation is LC/MS compatible. I think the LC/MS compatibility is debatable in this case. From my experience with reverse phase, I would not expect the chromatogram to look the same with HCOOH in place of TFA. Otherwise, why not just use formic acid instead of TFA if LC/MS compatibility is desired?

HW Mueller, I recall some debate on this board in the past as to whether TFA is an ion pair reagent.

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By AllsepTech on Thursday, April 15, 2004 - 09:37 am:

DEar MG,
Please download our Primesep Brochures to see explanation for mechanisms and few examples of LC/MS compatible conditions (ammonium acetate, ammonium formate, formic acid). The method in the link was one of our first methods and now we are trying to redevelop some of our methods of "more friendly" LC/MS conditions (without TFA)

Dear HW Mueller,
We think that if people know how to use the mixed mode columns they have almost no limitations in separations. Once everybody realize the science behind our columns the method development becomes very fast and easy, we have developed several methods for our customers in very very short time (a few hours) and people struggled with these methods for months using conventional C18 or ion-exchange columns

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By AllsepTech on Thursday, April 15, 2004 - 10:22 am:

Missing link for the brochures
http://www.allsep.com/Brochures_Home.php

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By Zelechonok on Thursday, April 15, 2004 - 10:58 am:

Dear HW Mueller
Every HPLC column is a mixed-mode column. RP or ion-exchange is just a matter of quantity not absolute characteristic. We all know that silica based RP columns have ion-exchange properties and polymer based ion-exchange columns have reverse phase properties. You will not find on the market a single column that has clear only one type of interaction with all analytes possible.
Using your analogy every column that not so called “mixed-modeâ€