The details I have are quite vague, but basically I need to find out why retention times of some compounds are seen to decrease with successive injections. The method utilises ion-pair reagents - specifically heptane sulphonic acid. I know that such methods are prone to slow equilibration and variability with temperature, but effort has been made to thermostat the column and the column has been allowed to equilibrate with a flow of mobile phase for several hours. One of the methods is for norepinephrine (a catecholamine). This problem does not appear to be an issue with any one phase or confined to any one manufacturer. I would appreciate it if anyone could lend me their expertise in this matter.

Thanks in advance.

Are you doing a gradient with ion-pairing? I have seen this cause a loss in retention due to lack of equilibration. Most people opt to not try a gradient with ion-pairing for this reason. I have worked with epinephrine for a couple of years now and initially folks in my group were using an ion-pairing method, but it ended up always having robustness issues. The best column for catecholamines I have come across is the Alltech Altima C18, has excellent retention without the use of ion-pairing. We use it for both assay and impurity methods. A quick assay method is 96/4 water/ACN with 0.05% TFA, pre-mix and run isocratic through one line at 1.8 mL/mn. column temp = 40C, and injection vol 5uL. Column dimensions are 250 X 4.6, 5um. It can be used with UV or fluorescence (285ex., 305em.) if you need added sensitivity. Injection volume can be increased to around 20uL if you have an all aqueous sample solution. We needed some organic for our application so we had to go down to 5uL. Give it a shot, you won't be sorry.

-Daren

Steve,
I forgot to add the run time for the method I described is 4 minutes. But run it out longer the first time and see where norepinephrine comes out. You will probably have to increase the ACN concentration if you want to keep a 4 minute run time since norepinephrine will retain better than Epi.

-Daren

For catecholamines, ion-pairing is probably the most well understood technique for retaining and separating these compounds by RP HPLC. The order of elution (when ion-paired at pH 3.0) is NE,E,DHBA (IS) and DA. If looking at acid metabolites, the order is NE, E, DOPAC, DA, 5HIAA, HVA and 5HT. This is with a C18 column, OSA (ion-pair) at pH 3.0.

Normally when a column is not equilibrated with the ion-pair, the RTs start to get longer until they equilibrate. You mention shorter RTs. A non-equilibrated column will not give shorter RTs.

If you are using an older column (you likely are if following a method) then the C18 ligand is likely monofunctionally bonded and is hydrolyzing at low pH. That is why you see shorter and shorter RTs. Another reason could be if you start your runs in the AM (when the lab is cool as the HVAC system kicks in), and your column is not thermostated, the lab is likely getting warmer as the day goes on. Catecholamines and acids are very susceptible to temp changes.

Try an Atlantis column from Waters. I believe they have an application for catecholamines although I question its applicability to real-world samples. Still, it'll give you better lifetimes than most columns out there and is very, very good at low pH.

Also, if you are looking at catecholamines you are likely using electrochemical detection. Call the manufacturer for mobile phase/column sugestions. If it is ESA, you are in luck. If it is Antec, good luck.

A non-equilibrated ion-pair system wiil give shorter retention times than a fully equilibrated one will. It has to, for compounds that participate in the ion pairing, unless you are using the ion pairing to reduce your retention, which would be news to me! We just examined a gradient IP method with inadequate reequilibration times: RT = 5 min, but with full reequilibration, RT = 15 min.

simple is usually the right answer. do you need to turn off your sparge? does pH of mobile change?