Chromatography Forum

Hi, forum

I have an ion pairing HPLC method with 258nm detection of a EDTA-Fe(iii) complex. Flow rate is 1 ml/min, injection volume 10 ul. Column is c18 reverse phase. Mobile phase is 0.4% tetrabutylammonium hydrogen sulfite water:ACN=98:2 (v/v).

The problem is that the six consecutive standard injections show the following upward drifting peak area. What could be the cause?

working std injection 1: 26933
working std injection 2: 29234
working std injection 3: 31697
working std injection 4: 35314
working std injection 5: 37989
working std injection 6: 39446

Thanks!

At first glance it appears to be a column equilibration issue. Ion pairing methods are notorious for requiring long column equilibration times before you start your analysis. How long did you equilibrate prior to injection?

Inject 10X concentration standard a few times, then your standard. See comments in previous post.

Thanks for reply.

I equilibrated the column (4.6mm x 250mm c18) 200 minutes with the mobile phase before injection anything. Here is another results of six consecutive standard injections. You can see retention time is drifting downwards and the peak area is fluctuating a lot. Is the column equilibration time still short?

standard 1, Retention time=4.053, Peak area=91233
standard 2, Retention time=4.039, Peak area=85959
standard 3, Retention time=4.031, Peak area=75219
standard 4, Retention time=4.012, Peak area=40440
standard 5, Retention time=3.997, Peak area=50516
standard 6, Retention time=3.984, Peak area=58809

Thanks.

The drifting retention times are looking awkward. About 4 minutes are not enough to speak about chromatography with this column dimensions. Nevertheless you have chosen very harsh conditions. So a drift of retention time is expected since your column is degrading during that time. Also 200 minutes of equilibration is not very much for this kind of method, but I do not think that this would be the reason for your fluctuating areas.

Maybe there is a problem with the Ferrum-concentration. You can add EDTA to the mobile phase to avoid this, but there is a reasonable possibility that this will destroy you instrument!

Some years ago I had similar difficulties with a comparable method. Solution was to simple use the full-loop injection mode instead of using partial loop mode. But I worry that your problem is not so simple.

Hi Jjin,

What is your column temperature. What does your baseline look like. Do you see much fluctuation in pump back pressure.