A high-performance liquid chromotography (HPLC) machine analyses laboratory samples by injecting the sample into the machine. As the continually flowing mobile phase (or moving phase) solution of the HPLC carries the sample over the HPLC column, the molecules in the sample become separated based on size and polarity. The individual molecules exit the column and cause an electrical charge of varying intensity, depending on the concentration of the molecule. A computer attached to the HPLC reads the electrical charge. This shows up on the monitor as a peak. The collection of peaks from the sample is called a chromatogram. To use an Agilent HPLC effectively, you must know how to troubleshoot problems.
- Skill level:
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Things you need
- Agilent HPLC
- 50/50 aceotnitrile/water solution
- New column (optional)
- Chemical standard made according to your lab's protocol
- HPLC syringe or automated sample loader
Check the pressure of the HPLC pump. The pressure will vary depending on your lab's HPLC set-up, and you should know what the normal range is. If the pressure is higher or lower than normal, this indicates a problem.
Check the HPLC pump for leaks if the pressure is low. Leaks occur at any connector and might not be obviously dripping. Check for build-up of salts around any connector. This is a sign of a slow leak. Tighten any fittings or replace worn fittings to remedy low pressure problems.
Turn the pump down to zero pressure and remove the column if you experience high pressure problems. With the column off, turn the machine back up to the resting flow rate (the flow rate you leave the machine at overnight when not in use). If there is still pressure without the column, flush the pump with a 50/50 acetonitrile/water mix until the pressure goes down. If the pressure stays at zero without the column attached, recondition or replace the column per your lab protocol.
Turn on the computer for your HPLC and run the program that produces your chromatograms. If the baseline of the chromatogram is noisy (not smooth) without injecting any samples into the HPLC, it is either caused by dirty mobile phase or electrical interference. Make new mobile phase according to your lab protocol. Always use HPLC grade water for the mobile phase, and filter the mobile phase well. The detector of your HPLC must be keep closed at all times.
Inject your prepared standard into the HPLC. If there is noise only when a sample is injected, check that you are not injecting any air bubbles into the HPLC. Air bubbles result in large, jagged noise peaks. If you are certain you aren't injecting air bubbles, the problem is likely an unclean solution used to make the standard. Remake the solution in which you dilute your standard, then remake the standard.
Watch the baseline for noise that appears in a regular cycle. This is normally caused by an air bubble within the system. Turn the pump to zero pressure, remove the column, and use the purge function to force the bubble out. Reattach the column and turn the pump back to its running pressure. Avoid air bubbles by keeping the mobile phase at a constant temperature and degassing the mobile phase per your lab's protocol.
Check the baseline for continued noise. If the baseline shows a series of constant and jagged noise rather than a straight line, refresh your electrodes. Depending on the model of Agilent HPLC you have, the electrode configuration and maintenance can vary. Follow your lab's protocol for cleaning, polishing and refilling your HPLC's electrodes.
Inject the prepared standard into the HPLC. If the sample peak is not symmetrical, or if the peak splits (one peak appears as two), the column packing material has worn down or become separated. Refresh or replace your column per your lab's protocol.
Inject the prepared standard. If the chromatogram shows peaks that are smaller than normal, you have a sensitivity issue or your standards are old. Remake the standards and run them again. If the peaks are still small, increase your HPLC's sensitivity to obtain a larger peak. A decrease in sensitivity over time is normal and indicates you need to purchase new electrodes in the near future. Changing your mobile phase ingredients and regularly replacing the pump seals also help increase sensitivity.
Inject a prepared standard into the HPLC. If the peaks are broader than normal, this indicates that you need to recondition your column to repack the column packing material. Also, check that you are running the HPLC pump at the correct flow rate, since low pressure will cause peaks to spread out.
Tips and warnings
- Try one thing at a time to fix your HPLC. Each "fix" requires the machine to re-equilibrate before you can see if it helped. HPLC maintenance requires patience.
- Always keep extra HPLC parts, such as seals and columns, on hand for HPLC maintenance.
- Turn your pump to zero pressure any time you remove the column from the pump. A sudden decrease in pressure can destroy the pump seals.
- Before you remove electrodes, turn the HPLCs power off to avoid shock.
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