IP-RP-HPLC

[Eric Moore]

Hi, all -

Why is the distinction "IP-RP-HPLC" sometimes made and sometimes not? We use trifluoroacetic acid (TFA) as an ion-pairing agent in our peptide mobile phase (H2O+acetonitrile). This method (C18 column) is typically referred to as simply "RP-HPLC." However, in running oligonucleotides with a trietylamine-acetate (TEAA) mobile phase (again on a C18 column), I always see the method referred to as "IP-RP-HPLC," or Ion-pairing Reversed Phase HPLC.

Is there a distinction? Is TEAA a stronger IP agent, and that's why it's specified?

Thanks -

EM

[oleg krokhin]

Hi Eric,
in my opinion - there is no distinction. this is always ion-pairing mode. ions always present in your mobile phase and interact with your charged analytes, affecting as consequence interactions with stationary phase.

[Uwe Neue]

Both methods are strictly ion-pairing methods, but people sometimes forget or ignore that TFA is an ion-pairing agent.

A prerequisite of ion-pairing is not what happens in the mobile phase, but that the IP reagent is adsorbed onto the stationary phase.

[Kostas Petritis]

I spent a lot of time in the past calculating the amount of TFA and other ion-pairing reagents that is adsorbed in the columns at 100% aqueous conditions by plotting breakthrough curves (UV + conductivity detector) in different C18 columns. You would be surprised how little (if any) TFA is adsorbed on the column which might explain about some people calling/considering it an ion-pairing reagent and some not... but then again you can find in the literature papers where they call PO4- an ion-pairing reagent so go figure...

[HW Mueller]

Kostas, did you also compare acetic acid breakthroughs? If I remember correctly, a discussion on whether TFA or acetic is more retentive on a RP column was never settled.

[Chromatographer2010]

The problem with the TFA retention on the column with 100% water is that the TFA is barely retained on the column. You can test this by doing small injections of TFA on RP columns with aqueous mobile phase and there is no detectable retention. A traditional breakthrough test is normally dependant on the analyte being very close to fully retained.

But when the TFA is in the mobile phase (at much higher concentration) I get peaks from doing 1% step changes of organic up to about 40% ACN. If I do the same with formic acid I typically get peaks only up to about 10-15%.

You could do the same test with acetic acid if you wanted to compare it to TFA.

[Kostas Petritis]

Hans,

No, I only did all the "volatile" aliphatic perfluorocarboxylic acids up to pentadecafluoro octanoic acid. I do have published some papers with the amounts adsorbed in the different columns with the different ion-pairing reagents...

[Bryan Evans]

We have data for TFA & acetic acid on 'conventional' ODS (Unison UK-C18):

http://www.imtaktusa.com/site_media/fil ... TI523E.pdf

Experimenatal conditions: 80 / 20 = 100mM ammonium Phosphate / acetonitrile
Looks like there's a little bit of retention for both TFA & acetic acid

[HW Mueller]

What is Scherzo C-18?
Presumably the pH is such that all are ionized?

[grzesiek]

It is multi mode column, with anion and cation exchange integrated

[danko]

• It is multi mode column, with anion and cation exchange integrated

Then it’s obviously the anion exchange mechanism that is responsible for the given retention and not RP interactions. The high salt concentration (100 mM) supports the above as well.
If the salt concentration is reduced - say to 50 mM - and everything else kept unchanged, I would expect longer retention times for the chosen compounds. The only question that remains unanswered is: Why the acetic acid elutes unretained (or almost unretained)? And the answer probably lies in the pH. If the pH in the mobile phase is low (< 3.75) then the acetic acid is protonated and for that reason doesn’t interact with the anion exchanger part of the stationary phase.
Bryan, do you have the possibility for checking the utilized eluent’s pH and share the info with us? Also, can you confirm that Scherzo C-18 incorporates both anion and cation groups in addition to the C18 ligand?

Best Regards

[Bryan Evans]

Then it’s obviously the anion exchange mechanism that is responsible for the given retention and not RP interactions. The high salt concentration (100 mM) supports the above as well.
If the salt concentration is reduced - say to 50 mM - and everything else kept unchanged, I would expect longer retention times for the chosen compounds.

Correct. High salt concentration (100mM) is required to elute the anions. A decrease in salt concentration, as you stated, would increase retention. Eluent pH is ~ 4.6 for this data.

The only question that remains unanswered is: Why the acetic acid elutes unretained (or almost unretained)? And the answer probably lies in the pH. If the pH in the mobile phase is low (< 3.75) then the acetic acid is protonated and for that reason doesn’t interact with the anion exchanger part of the stationary phase.

Below is data for acetic acid at low pH (~ 2.1):
http://www.imtaktusa.com/site_media/fil ... TI109E.pdf

As you said, acetic acid is protonated, thus k' is larger on Unison UK-C18.

Also, can you confirm that Scherzo C-18 incorporates both anion and cation groups in addition to the C18 ligand?

Yes, Scherzo SM-C18 is a multi-mode ODS phase. It contains 2 particles (3um silica + ODS + anion ligand) and (3um silica + ODS + cation ligand).
blended to form homogenous packing material

Unison UK-C18 & Scherzo SM-C18 have similar ODS ligand density.
So, UK-C18 can be used as a control, and the difference in retention
will be mostly due to IEX.

[danko]

Hi Bryan,

Exciting stuff.

Thanks for info.


Best regards

[Vlad Orlovsky]

It almost impossible, to retain very weak acids (like acetic acid with pKa of 5) on trimodal columns by anion-exchange mechanism. At lower pH this hydrophilic acids are not ionized and at higher pH (when they ionized) ion-exchange is minimal due to compensation effect of residual silanols (have almost same pKa) or acidic fragment of the tri-modal phase. This might not be 100% true when you have a physical mixture of 2 or more silicas. When you mix two opposite silicas (cation-exchange and anion-exchange) you are diminishing capacity of each stationary phase because you mix it 50/50). TFA is different as it is ionized in a wide range from 2 and up. At lower pH you might see some retention attributed to very weak hydrophobic interaction, but majority of the retention for TFA in mixed-mode columns comes from anion-exchange.

P.S. …..and Danko, physical mixtures of silicas are known for at least 20 years, did not ever make a main stream due to problems with efficiency, reproducibility, different rate of hydrolysis/deactivation which changes selectivity of the separation

[Bryan Evans]

It almost impossible, to retain very weak acids (like acetic acid with pKa of 5) on trimodal columns by anion-exchange mechanism. At lower pH this hydrophilic acids are not ionized and at higher pH (when they ionized) ion-exchange is minimal due to compensation effect of residual silanols (have almost same pKa) or acidic fragment of the tri-modal phase. This might not be 100% true when you have a physical mixture of 2 or more silicas. When you mix two opposite silicas (cation-exchange and anion-exchange) you are diminishing capacity of each stationary phase because you mix it 50/50). TFA is different as it is ionized in a wide range from 2 and up. At lower pH you might see some retention attributed to very weak hydrophobic interaction, but majority of the retention for TFA in mixed-mode columns comes from anion-exchange.

A lot of this is explained in our Technical Report. I would simplify it by saying RP is main mode of retention on SM-C18. After that, ion exchange interaction.

In addition, Scherzo SM-C18 is multi-mode (not tri-mode):
1. RP + anion exchange + cation exchange OR..
2. NP + RP

Finally, there's more than just 'capacity.' There needs to be a balance of anionic and cationic interaction. Below is NaCl:
http://www.imtaktusa.com/site_media/fil ... TI520E.pdf

There's a chance alpha may be quite large for Na / Cl on certain phases (meaning cation exchange interaction >>>>>>>anion exchange interaction, or vice versa).

P.S. …..and Danko, physical mixtures of silicas are known for at least 20 years, did not ever make a main stream due to problems with efficiency, reproducibility, different rate of hydrolysis/deactivation which changes selectivity of the separation

Correct, and Imtakt has found a solution for these issues with Scherzo SM-C18.