Not everything is about shape when it comes to chromatograms. To get faster and more confident at troubleshooting your HPLC technique, you need to learn how to recognize other indicators that tell you when there is a problem.

The first question you need to ask yourself is: how do chromatograms see? They *peak*, obviously.

Peaks were covered in this previous post which included ways to improve peak shape. This is often the first and most common sign of an issue. The second thing you can do is check your column performance by running the same separation as stated on your certificate of analysis (COA) and compare results. This might not even be needed to spot a common problem in HPLC.

The separation presented on your COA represents your column at time T0. As soon as a problem occurs, you can rerun that separation to compare and see how things may have changed. Ideally, you could run this exact separation first thing when you receive the column to compare future chromatograms with a T0 in your own conditions.

Retention Time Changes

Have you noticed that the retention times are different from what you expected, either in the COA separation or your routine separations? Small changes in the mobile phase can lead to large changes in retention times. Make sure that your mobile phase is not contaminated, and always use HPLC grade solvents. Degas the eluent to prevent bubbles from entering the system.

Water is the most common source of contamination in the mobile phase, so using distilled or deionized water is the best practice. Regardless of this precaution, deionizers sometimes introduce organic contaminants in water. If that is your case, you can remove them by passing your water through charcoal or C18 bonded silica gel. In addition, you should not prepare your mobile phase in advance because the aqueous buffers promote bacteria growth, and this is not something you want to introduce into your column.

Alternatively, the flow rate or temperature can fluctuate, making visible changes to the chromatograms. To maintain an even temperature, use a column oven and make sure to heat the eluant before it enters the column. This is especially important for ion exchange sorbents.

Moreover, the stationary phase might be degrading if using the column outside the recommended pH range. Bonded sorbents are stable in a range of pH from 2 to 10 and working outside of this will cause it to deteriorate prematurely beyond repair.

Volume overload could also be a cause. This happens when the injected sample volume carries the analyte through a significant portion of the column volume. In addition to longer retention times, this would cause shark fin type peaks. Concentrate your sample to inject a smaller volume of injection solvent.

You can also check for loose fittings that may cause leaks, an obstructed guard column due for a change, an incomplete equilibration or trapped air in the pump. As the column ages, these changes can appear. Make sure to care for your column by doing regular cleaning procedures, so it does not age prematurely, but it could be time to let it go and change for a new one.


The perfect chromatogram calls for a straight baseline. If the baseline is not perfectly flat, first check your detector and set the wavelength to UV absorbance maximum. The same small fluctuations that affect the retention times can have similar repercussions on the baseline. Make sure to avoid contaminants in your mobile phase and that it is heated before entering the column. Refractive indices and conductivity detectors vary with temperature, so you need to keep it constant.

This can also happen when equilibration is too slow. If you changed your mobile phase between two runs, it is crucial to equilibrate your column. Flush with an intermediate solvent and equilibrate with 10 to 20 column volumes of your new mobile phase.

Analytes or contaminants that are too strongly retained can also cause the baseline to drift. Using a guard column will keep these contaminants out of the column. Make sure to change your guard column routinely and to run cleaning procedures.

Air bubbles can also cause baseline drifts. To remove all bubbles from your system, purge the detector, degas the mobile phase, and flush the system with methanol or another strong solvent. If necessary, clean the mobile phase mixing chamber with nitric acid.

Pressure Is Too High

When the pressure is too high check the flow rate. It might be too high for the column size. You can use the table below to find the typical flow rate depending on your column’s internal diameter.

Internal Diameter (ID) Selection
Type of ColumnsID (mm)Typical Sample LoadTypical Flow RateTypical Applications
Narrow Bore2.10.04 - 1.5 mg0.1 - 0.3 mL/minUsed with low sample volumes or when more sensitivity and selectivity are needed over 3 mm ID.
3.00.08 - 3.0 mg0.2 - 0.6 mL/minUsed to reduce flow rate and solvent consumption over 4.6 mm ID. It is gaining popularity.
Analytical4.60.5 - 10.0 mg1.0 - 1.5 mL/minThis is the most common ID used for traditional quantitative analysis.
Semi-Preparative102.0 - 45.0 mg4.0 - 10 mL/minUsed for small-scale (mg) preparative purifications.
Preparative21.210.0 - 220 mg20 - 30 mL/minUsed for large-scale (hundreds of mg to gram) purifications. The higher the diameter, the greater the loading capacity.
3020.0 - 430 mg40 - 70 mL/min
5050.0 - 1,200 mg110 - 250 mL/min
10096 - 3,200 mg240 - 700 mL/min

Pressure issues arise if the solvents in the mobile phase are not miscible or the buffer precipitates. You should backflush the column and run a regeneration procedure before changing to a more adapted mobile phase. The mobile phase flow could also be interrupted, in that case check the mobile phase reservoir, sample loop or air lock for obstruction. It could be a leak or some salt buildup, trapped air or worn pump seal.

Something might be clogged: the frit, the guard column, the in-line filter, or something else. Try to change your guard column, then to backflush the system without the column connected to the detector and run a few column volumes. If the pressure is still too high, run a complete regeneration procedure.

Check all components for something obstructing the flow: leaks, kinked tubing, particles blocking the injector, or faulty seals or valves. If the system has a check valve, loosen it to allow some air to escape. Disconnect the guard and analytical column and purge the system. Purge the pump at high flow rate. If the pressure is still too high when you reconnect the system, flush it with 100% methanol.

If the problem persists, contact your system manufacturer. Do not keep working under such high pressure, take a deep breath, and consult references on how to reduce pressure in the workplace. Maybe do corporative yoga? No need to have a second system under high pressure.

Make sure to use a frit with pore size adapted to the particle size of your material so the sorbent does not block the frit. For particle sizes around 5 to 20 microns, the frit’s pores should be 2 microns and for particle sizes between 2 and 4 microns, use a frit with pores around 0.5 microns.

Low Resolution

A low resolution could be due to poor separation conditions. If this is a known separation and the resolution has changed since your previous runs, look at your mobile phase for signs of contamination and always use fresh mobile phase. You might also need to run a regeneration procedure.

In Conclusion

A good regeneration procedure can help with many of the problems you encounter in your HPLC run. Equilibration between the analyses and choosing the right (and freshly made) mobile phase can also help in many cases.

You should never neglect anything on your system or your chromatogram that tells you something is off. Do not forget to give some love to your columns by cleaning them regularly so they do not age prematurely. A good skin care routine is recommended. SiliCycle has HPLC experts on staff with up to 30 years of experience to help you with your method whenever you are unsure.

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Eliane, M.Sc

Eliane is the Scientific Content Specialist here at SiliCycle, with years of experience both in the lab and client support. She studied at Laval University, for both her Bachelor’s and Master’s in Chemistry. In fact, her thesis was in organic electronics in which purity is of the utmost essence, making her in-tune to the purification needs of chemists.