Chromatography Forum

Dear list Members,

Has anyone experience with heptafluorobutyric acid ion-pairing on
mixed-phase type Primesep-200, pH 2?

C18 - phases is sensitive, but Phenomenex Synergy Polar-RP acts very long
(>1000 injections) using MeCH:H2O: HFBA (95:5:0.2). Low pH is necessary
for detection and HFBA is best for making pH and ion-pairing simultaneously, not need weighting for preparing mobile-phase.

I have just obtained 2 Primesep-200 columns and would like to experience
first with HFBA because I have no any TFA, actually.

Dear Kazimierz,

Are you analyzing HFBA or using it is ion-pairing reagent? Can you provide us with more details-we can assist you more efficiently. What do you mean by resistance? If you are retaining basic molecules you don't need HFBA with PS-200. If you method requires pH=2 you might need Primesep 100 as at pH-2 Primesep 200 is partially suppressed (pKa value of the acid on the column is 2)
You can send your details to mail@sielc.com

Kind regards,
Vlad

Dear Vlad,

According to http://hplcmethods.com/application_013.php
I deduce, that PS-200 phase requires also extra ion-pair acid TFA of 0.2%,
for quanidine-like compounds in serum samples.
Fluorimetric detection require such low-pH as possible, usualy pH 2-3.

The question is - whether HFBA could be also good, or I need to find other
similar acid, like chloroacetic acid, or not ion-pairs-acid only?

TFA is actually not available in this year, but I have HFBA enough.
PS-100 columns could be bether, but I will able to buy theirs on the next
year.

sincerely
KHK

Kazimierz,

You don't need HFBA, you can for low UV you can use sulfuric, phosphoric or methanesulfonic acid. All three acids (at 0.1%) will give you different retention for guanidine type of compounds (phosphoric providing the longest retention). You can aslo use traditional buffers (sodium phosphate, sodium sulphate, etc.)
For LC/MS/ELSD you can use TFA, ammonium acetate, ammonium formate and formic acid. In both cases you need to create certain ion-strength of your mobile phase. You guanidine (if it does not have any hydrophobicity) will elute faster with lower pH and higher concentration of buffer (using the same buffer).

We recently have developed a method for guanidine type of compound and used Primesep 200 and ACN-water-phosphoric acid. Guanidine itself elutes at 2.5 minutes (4.6x150 mm) but guanidine-based product elutes at 10 minutes due to additional hydrophobicity.
Use guard column to trap proteins from serum; I don't know how many injections the guard will survive, but I would recommend this to protect the column. You can try to recover guards while running your analysis-guard is attached to the column though Agilent valve (http://www.sielc.com/pdf/SIELC_September_2004.pdf).

Vlad