Why Does My Baseline Go Negative When Switching Gradients?
Question:
When I run gradients on my HPLC and switch from one gradient to another, my baseline appears negative. If I auto-zero, it becomes positive for the remainder of the run, but when I start again, it goes negative. What can I do?
Explanation
Negative baselines during gradient runs are not uncommon. If your method uses components that absorb UV—even moderately—at the detection wavelength, it is challenging to perfectly balance absorbance between channels.
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Acetic acid is particularly problematic in this regard.
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The lower the wavelength, the harder it is to manage.
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For example, achieving a smooth gradient with peptides at 214 nm using TFA illustrates this difficult
