Pressure differences in HPLC systems

Written by Eva Arnold Posted in High performance liquid chromatography

In HPLC systems everything is associated with each other. There is not one common solution for one specific problem. Of course, pressure is completely normal for high pressure liquid chromatography (HPLC). This is the backpressure of the eluents, which are pumped through very narrow capillaries onto a tightly packed column. So, what can go wrong? Quite a lot!

Changes in the pressure of an HPLC system can have several different reasons and are often associated with other typical HPLC problems. For this reason, fixing a pressure problem is not that easy, especially as it firstly must be identified as such. The occurrence of peak broadening, double peaks, ghost peaks, intense baseline noise, or altered retention times is very often related to a pressure issue. Thus, it's always worth keeping an eye on the pressure. Regular pressure checks might therefore prevent the occurrence of these problems.

Particularly creeping pressure changes can only be detected in case the pressure is monitored regularly. The back pressure of the HPLC system should therefore be noted at regular intervals, at the latest each time the guard column is changed. The use of a guard column is generally always recommendable, even when examining "clean" samples: The normal material abrasion of the HPLC pumps may be enough to impair the durability of columns. The best way to determine the back pressure is a standardized HPLC run, which is not changed. In principle, you should always pay attention to the pressure, even - or especially - in routine measurements. To prevent pressure-related column or device damage, a maximum pressure should be set in the HPLC software, beyond which the pump switches off automatically. In this case, the pressure limit of the HPLC system and of the column used should be respected (see the manufacturer's instructions).


So, you have pressure fluctuations in your HPLC system?

Especially when you are routinely working, you may overlook the obvious. Thus, check at first if the system is properly connected (are all capillaries connected, are there any leaks?). Is the mobile phase empty and may air have been sucked in? Leaks and air in the pump heads can account for a very low to no pressure. Another simple explanation can be a flow rate set too low, as well as a flow rate being too high will cause a too high pressure. Depending on the column dimension, there is a specific ideal flow rate that is best suited. A flow rate being too high can damage the column.


Some pressure fluctuations within one run are quite normal

Especially during gradient runs, changes in pressure can occur, which can be explained by the viscosity of solvents and their mixtures. Gradients of water to methanol or tetrahydrofuran (THF) show a steep increase in pressure when mixed. This pressure should not be underestimated (more than twice as high for 50% methanol/50% water compared to 100% methanol) and should be well monitored when establishing new methods to prevent damage to the equipment or column. Always make sure that the eluents used are miscible with each other! Determine the gradient-typical pressure changes by performing a run without sample injection and recording the pressure fluctuations. Often, a baseline drift can also be seen which is quite normal for some gradients.

In some cases, just after the sample injection, a spike in pressure is observed. With larger injection volumes, this can be normal. The newly added volume at injection results in a short overpressure. In case it is a solvent of higher viscosity, the pressure can rise more than double for a brief period. If possible, use as sample solvent the same composition as the solvent used (at the beginning of the run).

Inject the sample solvent ("blank") to measure the pressure changes. If injection of a sample results in significantly higher-pressures values than the blank alone, the sample should be filtered or purified beforehand.


Where do sinusoidal pressure changes in isocratic systems come from?

We have just clarified pressure changes in gradient runs. In gradient runs, the pressure can increase during the run and decline towards the end. This pattern of pressure changes in gradient systems is very different from the regular “sinusoidal” pattern majorly observed in isocratic systems. Often a sinusoidal pattern of the baseline appears as concomitant. When raising the flow rate, the frequency of the pressure oscillation is also increased. Thus, the problem is due to the pumps. There may be the possibility of air bubbles being trapped in the pump heads. This air could be released by a simple procedure called venting. If venting is not successful in solving the problem, a lack of back pressure can be the reason for these fluctuations. A missing back pressure leads to pressure fluctuations due to the pump movements. Therefore, modern HPLC devices are often equipped with a pulse damper.

If the problems still exist, the valves and seals of the pumps may have become leaky due to wear and should be replaced. But before placing an expensive order, you should have a look on the column: a long-standing, unused column could dry out during storage and lead to such pressure fluctuations when reconnected.


A pressure too high?

If the pressure is considerably too high, you should check at first if the pre-column needs to be replaced. Additionally, you should check which samples were run last. Possibly, the samples have incorporated suspended particles and impurities, which now lead to blockage of the sample injection system or individual capillaries. In this case, you should consider filtration, centrifugation, or pre-purification (e.g., solid-phase extraction) of the samples if you do not want to risk blockage of the capillary system or guard column whenever few samples were run. The solvent used may also be the cause: especially self-mixed, aqueous phases must be well filtered to reduce bacterial growth and to remove suspended particles. For additives such as salt, make sure that everything is well resolved and that nothing is precipitating, especially in case of gradient runs with solvents.

Before starting to disassemble the whole system, let’s also check the obvious things: is the flow rate set too high? Is a high viscosity solvent mixture used (e.g., methanol/water 50:50)? Even a too low temperature of the column oven can lead to increased pressure.

If the replacement of the precolumn didn’t help, there is probably a blockage in the HPLC system. For the localization of the affected component it is best to exchange individual components by going backwards: Start with the outlet capillary, which leads from the detector to the waste canister, then replace the capillary between the column and the detector. If you have a new column available, then also exchange the column. Then bridge the injection valve or autosampler. If the pressure is still too high until here, the problem is due to the pumps and you should consult a technician.


What to do in case of a pressure too low?

Especially, small decreases in pressure are often not taken seriously. In case of poor maintenance, a too low pressure can be masked by a combination with a clogged capillary or guard column and thus remain undetected. This often appears in other issues such as poor reproducibility and recovery, as well as an intense baseline noise. If the base pressure is regularly checked and noted, a slight pressure drop is easily observed.

In any case, in the event of a pressure drop, all connections should be checked for leaks. A pressure being constantly too low can be a sign of a small leak or air bubbles in the pump system. Do not forget the obvious reasons, such as a too low flow rate or different column temperature. Even a blocked suction filter in the solvent bottle can lead to reduced pressure. If leaks can be excluded, there is probably air in the system, often appearing in combination with baseline noise and missing peaks.

If there is a strong, suddenly appearing pressure drop, you should immediately check whether the pumps have sucked in air or a capillary has slipped out of a connection and caused a large leak.


Trouble shooting HPLC could be time consuming and expensive, we know that from our own experience. We hope that with this blog post on one of the most important topics of HPLC troubleshooting, we have been able to help you solve a problem or at least give you an idea for finding a solution.