Chromatography Forum

Dear All,

I am observing a steep negative baseline drift as the concentration of acetonitrile rise in my gradient.
the method details are described below:

Mobile Phase A: Acetonitrile/25mM Phosphate Buffer pH 7.0 27.5:72.5 v/v
Mobile Phase B: Acetonitrile/25mM Phosphate Buffer pH 7.0 60:40 v/v
Standard Diluent: 27.5% Acetonitrile 72.5% 25mM Phosphate Buffer pH 3.0
Column: Luna C18 (2), 5µm, 250 mm x 4.6 mm.
Guard Column: C18 4 x 3.0mm cartridge contained in Security Guard  Universal Holder
Injection Volume: 100µL
Detection: UV at 239 nm
Flow Rate: 0.75 ml/minute
Column Temperature: 40°C
Gradient: Time, min. % A % B
0 100 0
30 60 40
30.1 0 100
44 0 100
44.1 100 0
52 100 0
I conditioned the column by running 3 balnks before starting my analysis, but the baseline negative drop is still there. I do not know the column history as this was supplied by a customer, and in their chromatogram the baseline look flat. I would expect the baseline to show a positive drift when the % of acetonitrile increase.
I would much appreciate if you could give me advice on why I observe this drift and suggestion on how to solve this issue.

Many Thanks

Emanuele Scollo

Try removing the column and replacing it with a meter or so of capillary tubing (0.1mm id), then run the same gradient.

If you see about the same amount of drift, then it's obviously not a column problem, and I would suspect that the flow cell in your detector is out of alignment.

If the baseline looks OK without the column, then it obviously *is* a column problem. Washing the column with 100% acetonitrile may clean it up, but if it is heavily contaminated, it may be more cost effective to simple replace it.

We put together a brief tutorial on gradient baseline issues (taken from our HPLC Basics, Equipment, and Troubleshooting course). You can view it on the LC Resources web site:
http://tinyurl.com/47gwd4c