Having troubles on HPLCs can be incredibly frustrating. You’re trying to get some analysis done, and your research can’t move forward until you figure out why your tools aren’t working.
There are some common problems of HPLC columns that pop up from time to time. Knowing what these are and how to fix them can save you hours of frustration. Read on to discover some of these problems and how to address them. We limitted this article in basic level. We would recommend receiving consultation with the manufacturer of your HPLC system.
There can be several factors that cause no peaks or very small peaks to show up on your HPLC outputs. Normal readings should have large, thin peaks that may vary somewhat in height. Small peaks or no peaks at all may mean your detector lamp is turned off, you have no mobile phase flow, your sample is missing or deteriorated, or there’s a problem with your detector, integrator, injector valve or recorder.
Start by making sure your detector is turned on, and then check all the electrical connections and cables. Make sure your autosampler vials have enough liquid and that there are no air bubbles in the sample, and recheck the system with a new standard solution. If that doesn’t work, check the attenuation or gain settings status, and auto-zero if you have to.
If you are getting absolutely no peaks on your output, you may have no flow in your HPLC column. This may mean your pump is off or the flow is interrupted or obstructed somehow. You may also have a leak or air trapped in the pump head.
Start the pump if it’s off, and check the mobile phase levels in the reservoir and flow throughout the system. Check the sample loop for any obstructions or air locks, and make sure the mobile phase components are miscible and the mobile phase is properly degassed.
From there, move on to checking the system for loose fitting and the pump for leaks or other issues. Disconnect the tubing at the guard column if you have one, and check for flow. If you’re still having problems, purge the pump at a high flow rate, prime the system, loosen any check valves your system may have, and, if all else fails, flush the system with 100 percent methanol or isopropanol.
If your pressure is lower than usual or you have no pressure, you may have a leak or air trapped somewhere in the system. You might also have a faulty check valve or an obstructed or interrupted mobile phase flow. If your system pressure is too high, there’s likely a problem in the pump, injector, in-line filter, or tubing, or you might have obstructed guard or analytical columns.
For low pressure, start by checking the whole system for leaks, loose fittings, faulty pump seals, or bad valves. If everything looks okay, run the same mobile phase check we discussed earlier and check for flow at the analytical and guard columns. If that doesn’t work, reconnect everything and try pumping solvent at double the flow rate.
For high pressure, start by removing the guard and analytical columns from the system and replacing them with unions to reconnect the injector to the detector. Run the pump at 2-5 mL/min, and work on isolating the cause, starting with the detector, then the in-line filter and working back to the pump. If the problem seems to be with the analytical column, reverse and flush the column while it’s disconnected from the detector, and change the inlet frit or replace the column if necessary.
If you start seeing peaks show up that dip down in the middle, making an M shape, you may have some contamination on your guard or analytical column inlet. You may also have a partially blocked frit or an uneven void at the column inlet. It’s also possible that your sample solvent is incompatible with the mobile phase.
If you think you have contamination at an inlet, reverse and flush the column and change the frit if needed. You can also repack the top of the column with pellicular particles of the same bonded phase functionality and continue using the column in reverse flow direction.
If you think the problem is with the sample, adjust the sample in the mobile phase. Difference of pH in the sample and mobile phase causes split peaks.
Tailing and Fronting (Leading) Peaks
You may notice that rather than coming up and down in straight lines, your peaks start to develop a slight slant at the front or back, called fronting or tailing. This usually means your guard or analytical column may be worn out or your column may be overloaded. Tailing may be caused by a contaminated or deteriorated mobile phase or interfering mobile components in the sample, while fronting may result from problems with the sample solvent.
If you have tailing peaks, start by removing the guard column and attempting analysis, replacing it if necessary. You may also need to restore or replace the analytic column. Be sure to check on the make-up of the mobile phase and the column performance.
If you have fronting peaks, try injecting a smaller volume or diluting the sample. If your solvent is the problem, adjust the solvent, and flush polar bonded phase columns with 50 column volumes of HPLC-grade ethyl acetate at two or three times the standard flow rate, and then with intermediate polarity solvent before you start analysis. You may also want to try another column type.
If you see negative peaks start showing up, it’s possible that your recorder leads are reversed. You may also have a refractive index of solute less than that of the mobile phase or a sample solvent and mobile phase that vary greatly in composition. Your mobile phase may also be more absorptive your sample components to UV wavelength.
Check the polarity of your recorder leads to make sure they aren’t reversed, and then try either reversing the leads or using a mobile phase with a lower refractive index. You can adjust or change your sample solvent, and it’s a good idea to dilute your sample in a mobile phase whenever possible. If the problem is with UV wavelength, change the polarity when you’re using indirect UV detection, or use a mobile phase that adsorb your chosen wavelength.
Learn How to Resolve Problems of HPLC Columns
Running down the problems of HPLC columns can be frustrating, but remember, it’s like any other problem you’re trying to solve. Think critically about what might be causing the problem, start eliminating possibilities, and follow the suggested solutions here. You’ll have your HPLC columns back up and running in no time.
If you’d like to find HPLC columns that will stand up to your analysis, check out the rest of our site at Develosil. We have a wide range of HPLC and UHPLC columns with various phase chemistries. Check out our FlexFire series, the column designed for scalability and transferability today.