How to Troubleshoot HPLC Columns
The column of a high performance liquid chromatography (HPLC) machine is the stationary phase of the extraction process. The liquid--typically a buffer solution--continually flowing over the column is the mobile phase. As chemicals are injected into the HPLC they flow with the mobile phase over the column and elute at different and specific times to produce a chromatogram. The height of the peaks on the chromatogram tells you the amount of the chemical in your analyzed sample. Column issues cause poor results, so troubleshooting is important.
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Things You'll Need
- HPLC machine
- Analytical standard in desired concentration
- Injection syringe (if needed)
- New column (if needed)
Instructions
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Start up the HPLC and allow it to equilibrate for the amount of time that is standard in your lab protocol. In the meantime, make up the analytical standard(s) used on your HPLC (this will vary depending on what your lab and HPLC analyzes). Once the machine has equilibrated, inject a standard onto the machine--this will either be done by hand using a syringe or by loading your machine's autosampler.
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Look at the chromatogram produced by the standard. This is your best indication for column problems. The following steps will pinpoint common column issues found from this chromatogram.
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Check the elution time--the time it takes for the standard peak to appear. Results will vary for different chemicals, but your lab should have a "best case scenario" time for a peak to appear. If the peak shows up very early, this indicates the material inside your column is breaking down and the column needs to be replaced. If the peak is very far out this can indicate the column was not prepared well. In this case, the column can still be used if the peak is still good enough for your lab's needs (the longer the elution time, the smaller the peak will be).
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Examine the shape of the peak. Ideally you want a peak that rises sharply and has a symmetrical shape. A wide "hill-like" peak indicates the material in the column is breaking down as does a peak that "tails"--has a longer slope at one end than the other. In both of these cases, the column can still be used if results are good enough for your needs. To extend the life of the column you may be able to "flip" it (removing the column and turning it to flow the opposite way). This can re-pack the material inside, but does not always work.
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Examine the baseline (the lines leading up to your peak). If the line is not smooth, this is termed "noise" in the baseline. Sharp, jagged noise is likely electrical, but random "bumpy" noise can indicate the column has contaminants. Try to run fresh mobile phase over the column to clean it. If it is still noisy, follow your lab protocol to recondition the column. If it is still noisy you will have to replace the column. Some noise can be tolerated as long as it doesn't interfere with your readings.
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Tips & Warnings
HPLC troubleshooting takes patience. Try one thing at a time to solve problems and allow the machine ample time to equilibrate between your attempts.
Always keep your mobile phase and injection syringe clean. Do not touch the needle of the syringe with your hands or you can introduce skin oils into the column.
Use standard lab safety protocols when working on HPLCs.
References
- Photo Credit glass beakers image by Mark Aplet from Fotolia.com
