Chromatography Forum

The extraction pump has the same gradient as before. Washing with 10% MeOH until the valve switches at 2 min.
On the separation pump I am using gradient programs. Basicly they look something like this:

Time A(MeOH) B(H2O)
0-2 min 5-20%(MeOH) - 95-80%(H2O)
2-4 min gradient to 50-65% - 50-35%
4-13 min gradient to 80-90% - 10-20%
13-17 min equlibrate 5-20 - 80-95%

Running a couple of samples the RT times are getting shorter the higher the max organic gradient is set to.
Often the peaks come out at about 11-12 minutes, but sometimes (with just very slight modification to the gradients or equlibration time), the peaks may elute at only 7-9 minutes. There is something happening here that makes my method extremely sensitiv to subtle changes in MeOH. I presume it's because just a little higher MeOH content makes the column retain the analytes much less. Experiments shows this occurs at about 20-30% MeOH. Other times there are no peaks (or very low peaks). To help this I have doing additional equlibration of my analytical column (Zorbax Eclipce 3mmx25cm C18 5um particle), but even another 1-2 minutes does not give near constant RT, peak heights or peak withs. In total there is about 4+2+2=8 min equlibration time to lower the column organic content from 80-->10%. Adding another 7 minutes seems to stabilize this, and my analytes elute at about 12 minutes.

I have severeal analytes that I am interested in, about 6-8. Not all of them have sensitivity issues, but 2-3 of them have. They all elute at within 1-2 minutes of each other.

Arghh...now i think I understand. I think I have induced a "phase collaps" by equilbrating with to high water content(90-100%).

You won't get the dewetting phenomenon (what you called "phase collapse") with the Oasis packing (it was designed not to do that), but it is not impossible that this is a problem with your C18 column.

I've got it sorted out. I definitely had a problem with my Oasis extraction column. After I started to flush it for several minutes of an organic phase it started to work again. But if pure water flows through for about 6-7 it doesn't seem to retain anythng. Maybe not a dewetting phenomenon, but still it's 100% reproducible. Faulty column perhaps?

Anyway, things are working now and I've got nice, sharp peaks.
Precolumn is washed with 10% MeOH until the valve switches. The analytical column runs with a gradient:
0-8 min: 10-90%
8-13 min 90%
13-15 min: 10%

Peaks are about 12000 CPS, RT 11.37. Peak with at 50%: 0.1min. At baseline peak is about 0.5min wide. Some signs of tailing. This is a standard sample in water, injecting 100uL. - I can do up to 400uL inject.

I've been looking around for other columns that we have available, and I have several shorter and narrower. I might try a ID 2mm with 3 micron particle size, which as you say could boost the sensitivity by a 2-fold. Alltough I am concerned of loading to much sample(400u). Further, it may prove to be difficult to seperate my chemically very identical analytes. The 3x250mm C18 does seem to do a goog job here.

I am not sure about what is going on. Your observations contradict my own measurements. Contact me at my private email and we can discuss things further.

I am not concerned about the volume that you can load. You can put a lake through the precolumn, if you have enough time, enough retention of your analyte, and a low enough analyte concentration. If you still do not yet have enough MS sensitivity, you are miles away from overloading the precolumn.

You do not need a long column for getting good gradient separations. We can optimize the gradient later.