Gradient HPLC: Is your mobile phase causing a ghost peak? Here's an easy way to find out...

Although not usually a problem in HPLC methods that use isocratic elution, ghost peaks resulting from the mobile phase can be a problem for gradient elution methods. Contaminants present in the mobile phase can behave like sample components and interact with the column during the gradient, leading to a ghost peak (or peaks) in the chromatogram.

However, it is easy to diagnose if your ghost peak is from the mobile phase. Simply run a blank gradient. It’s better if you can do this without performing an injection (to rule out the injection as the source of the ghost peak). In Empower, it is called ‘condition column’. If the peak is coming from the mobile phase then increasing the equilibration time at the beginning of the gradient will increase the size of the peak. For example, you could compare holding at the starting conditions for 5 minutes to holding at 10 minutes. The ghost peak will double in size if it is coming from the mobile phase.

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