Can anything be done to correct for baseline drift in gradient separations?
This is the latest "LC Troubleshooting" instalment in a series focusing on gradient elution (1–4) in liquid chromatography
(LC). In an earlier column (4) we considered problems related to the system dwell volume. This month, we'll continue looking
at gradient problems with a focus on baseline drift. If you're just moving from isocratic separations to gradients, one of
the first observations you make when you examine a chromatogram is that the gradient baseline is often not flat. With both
isocratic and gradient separations, the baseline can drift when the column temperature is not stable, but if you use a column
oven and the laboratory temperature is relatively stable, this is usually not a problem. Drifting baselines under gradient
conditions are common. Usually the drift is minor, and you learn to live with it. In other cases, it may be possible to compensate
for the drift by adjusting the mobile phase. In still other cases, there isn't much you can do. Let's look at each of these
When ultraviolet (UV) absorbance is used for detection, it is common to find that the A and B mobile phases differ in their
UV absorbance at the detection wavelength. This difference means that the baseline will drift during a gradient run, as is
seen in the upper trace in Figure 1. In this case, a gradient is run from 100% water (A) to 100% methanol (B) at 215 nm. Because
methanol has significantly stronger UV absorbance at 215 nm than water, the baseline rises — approximately 1 absorbance unit
(AU) in this case. If the display setting is set to a range of <1 AU, the baseline will drift off scale during the run. This
is inconvenient, but with many detectors today, the detector range is >1 AU, so peak data will still be collected, even though
they do not appear on the computer monitor until the scale is changed. However, in the days of strip-chart recorders, before
computerized data collection was used, an off-scale baseline or peak meant that no data were collected under those conditions.
In any event, we would like to be able to see the entire chromatogram without having to change from one display scale to the
next. For this reason, drift, such as that observed for methanol in Figure 1, is unacceptable for most of us. From a practical
standpoint, methanol has sufficient absorbance at low wavelengths that full-range water–methanol gradients are seldom used
below approximately 220 nm.
Figure 1: Baselines obtained from linear gradients of water–methanol at 215 nm and water–acetonitrile at 200 nm.
Contrast the plot for methanol at 215 nm with that for acetonitrile at 200 nm in Figure 1. The water–acetonitrile gradient
baseline looks flat at the same display scale because acetonitrile has very low UV absorbance relative to water under these
conditions. This is one reason why acetonitrile is often the preferred organic solvent when low-wavelength (<220 nm) UV detection