How to find the sample load of a column (Solid Core)

[Mike H.]

I'm new to solid core technology. I'm using a Poroshell 120 EC-C18 (4.6mmx 150mm, 2.7µm). I'm seeing a tailing factor for my main peak of USP 1.5. The sample load is 2.5µl*0.2 mg/ml=0.5µg. The compound has one pKa at ~8. I'm using a standard gradient 90:10 -20:80 of 10mmol potassium phosphate monobasic pH 3.3:ACN on an Agilent 1200. I called and asked the Agilent column tech what was the recommended sample load for this could and he could not tell me.

Bottom line. I'd like to get the tailing as close to 1.0 as possible. Could someone recommend some suggestions on how to achieve this?

[danko]

Mike H,
Why do you think the sample load causes the tailing you're observing?
Did you try to load less concentrated solution og you compound? If not try that and if the sample load is the problem you'll see less tailing of course.
However I don't think that is the issue.
So, here is what I'd try if I were you:

Adjust the buffer to pH 2.5 or something like that. Because right now it's not a buffer (at pH 3.3)
After you have corrected the buffer and examined the effect of that, you might try to double the buffer concentratio - fx. 20mM

I'm sure you'lle see some changes there

Best Regards