Chromatography Forum

Hi

Recently I read an article in LCGC describing a about ION-pair chromatography, which explained that ion pairing agents added attach themselves to stationary phase and undergoes ionic interaction with the analyte thus helping in retention.

In our lab we popularly use ACN +0.1 % TFA and Water +0.1 %TFA as mobile phase (we work a lot on proteins and peptides)..i would like to know in this case TFA functions as described above or does it prevent non-specific (or unwanted ,in case on RP this can be ionic interaction with stationary phase ) between proteins and stationary phase..?

Thank you

This is a simplistic model (not necessary correct) but easy to understand:

The amount of ion pairing reagent attached to the column it will depend on the hydrophobicity of the side chain. In the case of TFA that is very small and a lot of people do not even consider TFA being an ion pairing reagent.

Anyways, the simplistic model goes that TFA or other similar ion pairing reagent they are mainly in the mobile phase and they work by attaching with the analyte on the mobile phase, they form something more hydrophobic which is then retained stronger in the column.

In the case of more hydrophobic ion pairing reagents they attach in the stationary phase much stronger (that is why you need longer times for the column to be equilibrated) and they work as hydrophobic dynamic ion exchangers.

Have a look also in this article:

PATTHY M
EXPLANATION OF THE SELECTIVITY DIFFERENCES BETWEEN REVERSED-PHASE ION-PAIR CHROMATOGRAPHIC SYSTEMS CONTAINING TRIFLUOROACETATE OR HEPTAFLUOROBUTYRATE AS PAIRING ION
JOURNAL OF CHROMATOGRAPHY A 660 : 17 1994

The TFA is quite weak as an ion-pair reagent. While I am not an expert on protein separations, my understanding of TFA is that it serves to "mask" positively charged site on the molecule allowing it to interact with the stationary phase by hydrophobic interaction.

All of the above.

At the risk of being long-winded, I'll recite a general reference:

The Parable of the Blind Men and the Elephant
John Godfrey Saxe

It was six men of Indostan
To learning much inclined,
Who went to see the Elephant
Though all of them were blind,
That each by observation
Might satisfy his mind.

The First approached the Elephant
And, happening to fall
Against his broad and sturdy side,
At once began to bawl:
`God bless me, but the Elephant
Is very like a wall!'

The Second, feeling the tusk,
Cried, `Ho! what have we here
So very round and smooth and sharp?
To me 'tis very clear
This wonder of an Elephant
Is very like a spear!'

The Third approached the animal
And, happening to take
The squirming trunk within his hands,
Thus boldly up he spake:
`I see,' quoth he, `The Elephant
Is very like a snake!'

The Fourth reached out an eager hand,
And felt about the knee:
`What most the wondrous beast is like
Is very plain,' quoth he;
`Tis clear enough the Elephant
Is very like a tree!'

The Fifth, who chanced to touch the ear,
Said, `Even the blindest man
Can tell what this resembles most;
Deny the fact who can:
This marvel of an elephant
Is very like a fan!'

The Sixth no sooner had begun
About the beast to grope
Than, seizing on the swinging tail
That fell within his scope,
`I see,' quoth he, `the Elephant
Is very like a rope!'

And so these men of Indostan
Disputed loud and long,
Each in his own opinion
Exceeding stiff and strong.
Though each was partly in the right,
They all were in the wrong!

The TFA is quite weak as an ion-pair reagent. While I am not an expert on protein separations, my understanding of TFA is that it serves to "mask" positively charged site on the molecule allowing it to interact with the stationary phase by hydrophobic interaction.

Well, also (and maybe you didn't mention this since it is fairly obvious), it's such a strong acid that it works well to keep the pH low enough to keep the charge on your peptide a positive one.

The only thing that bothers me about using TFA is that the pH of a 0.1% solution is around 2.0, while the pKa of the C terminus of any amino acid is around 2.1-2.3 when part of a peptide. It seems like you'd have a much more effective separation at around 1.5 or 3.5 (with a good basic ion pair reagent like TEA) than you'd get at 2.0

I know older columns weren't stable at pH lower than 2, but it's not hard to find good columns these days, so why not use something like 0.5-1.0% TFA if you're analyzing at 280, or HFBA if you're in the 210-230 range?

Below is a link to an interesting paper which shows that the main function of TFA is its strongly acidic nature. The paper shows that HCl can be substituted for TFA particularly if it is required to isolate the peptide, as it is much easier to remove from the purified peptide.

http://aem.asm.org/cgi/reprint/68/10/4803.pdf

I've recently come across an HPLC method that calls itself "ion exchange chromatography," yet uses a C8 column. The mobile phase is Hexafluoro-2-Propanol and triethylamine with a methanol gradient. In this case, is the ion-pairing so strong that this mechanism is a bigger influence on separation than hydrophobicity? I guess I'm wondering why it is called an ion-exchange method but uses a (typically) RP column. (I'm new to IEX, so maybe I just don't understand.)

Thanks.

Eric

It is similar to other ion-pairing methods. HFIP is an acid.

I suspect that the term "Ion Exchange" is the problem.
It is frequently applied to both silanole supression and to a mechanism of retention which may (or may not) both be at work in the same separation.

Although the term is not correct, the method is probably applied to compounds that are very hydrophilic (maybe inorganics or small ionic compounds) where they would normally be eluted in void volume under reversed phase conditions (even at 100% aqueous conditions). In this case, the retention would come solely from the dynamic ion exchange due to the IPC...

That is probably why the authors coined it ion-exchange, but again the term is not correct whatever the justification...

In the above mentioned application with triethylamine and HFIP, the "ion pair" reagent is the triethylamine. In the application, HFIP is acting as a chaotrope. From my view, triethylamine is not acting as a true ion pair reagent since adsorption onto C18 of the reagent is minimal except in 100% aqueous eluents (and therefore the oversimplified ion exchange description is incorrect in this case). Instead, it increases retention by decreasing the solubility of the analyte in the mobile phase.