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Study Notes: HPLC Troubleshooting

The technique of HPLC is inherently complex with many pieces of equipment interacting to analyse a sample and to produce a chromatogram as a result. In such a system the potential for things to go wrong is necessarily high if the operator does not know what they are doing or is less than careful in their approach to their work.

But even for experienced and meticulous technicians things can and do go wrong! This is where the ability to ‘troubleshoot’ is important.

The following table contains examples of errors/malfunctions and the likely outcome of such an occurrence. These examples will be useful when you work with the virtual HPLC later.

Error/Malfunction
Likely Outcome
Forgetting to inject the sample. No peaks on chromatogram apart from the solvent peak.
Injecting the incorrect sample. An incorrect chromatogram.
Injecting a mixed sample (often from a poorly rinsed syringe). A chromatogram that contains peaks from each sample.
Poor injection technique. Reproducibility between samples is poor.
Forgetting to use a gradient elution. No components may elute.
Mechanical failure, blockage including blocked pre-column filter. Low flow rate, high pressure, peaks elute slowly or not at all.
Leaks in the system. Low pressure, reduced flow rate, peaks elute slowly and/or change position.
Incorrect gradient elution conditions. Poor resolution of peaks.
Incorrect flow rate. Poor resolution of peaks.
Incorrect pressure. Poor resolution of peaks.
Blown lamp in the UV-vis detector. No trace from point of lamp failure.
Chart recorder runs out of ink. No trace from point of ink run out.
Setting baseline in chart recorder incorrectly. High base line, tops of large peaks run off the end of the chart.
Baseline moves up during the run. Due to gradient elution - not an error.
Set gain on detector too high. Peak size magnified and may run off the top of the trace.
Column getting old or ‘poisoned’. Poor resolution of peaks.
Pressure fluctuating. Pump failure.

Note that there may be other errors/malfunctions that may occur.

 

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