Liquid Chromatography – System Pressure Issues

Introduction

We often reach out to column manufacturers asking whether the column needs to be replaced when the column pressure increases significantly. However, after inquiring about the problem, it often turns out that the system pump was reading the max pressure and stopped pumping due to set max pressure limit. It often happens that there was some other reason (such as blockage / clogging) and not the column itself. This month we will address some of these other possible causes of system blockages and give you few clues how to overcome such issues.

What is HPLC system pressure?

To better understand the subject, we need to spend a few minutes to discuss the pressure. You will understand how and why you should setup and use the maximum system pressure in you CDS software. Additionally, we will consider how the increased pressure can impact your HPLC system.

Setting the Solvent Delivery Unit’s Maximum Pressure

Primary goal in setting the maximum pressure within your pump tab in your instrument settings is to protect the HPLC column. Each column has a maximum pressure resistance listed within the column information leaflet. In most cases the system is capable of withstanding much higher pressures than the listed max column pressure.

Furthermore, in order to detect abnormalities early enough, the maximum pressure of the solvent delivery pump is typically set to approx. 80% of the column’s withstand pressure, i.e. 10 to 15 MPa for a 5um silica analytical column. In case if the additional accessories are fitted between the pump and autosampler or autosampler and column (resistance tube, a high-sensitivity damper or pre-column, etc.), you must remember to add the additional pressure value generated by the accessories to the maximum pressure you setup in your instrument method. Refer to Figure 1 below for an overview of additional system accessories – 1) resistance tube, 2) high-sensitivity damper and 4) guard column.

Figure 1. To establish the blockage source – disconnect the tubing sequentially DOWNSTREAM and check pressure readings each time until you find the immediate pressure drop.

Problems That May Occur with Abnormal Increases in Solvent Delivery Pressure

Now that we discussed the maximum system pressure it’s time to check which parts of the HPLC system can be affected when too high pressure is noted on the system. What will happen if too much pressure is applied to:

1. Column stationary phase – the material may crack or become flattened and be pushed inside (packed even more). This means that further increase of pressure can occur. If, due to stationary phase cracking, a gap opens at the inlet-filling section the peaks will exhibit abnormal (deformed) peak shape. When using size- exclusion chromatography, the pore size becomes smaller and so separation will be adversely affected. Pressure is applied downstream from the column, and if the pressure gradient of the column itself is on a lower end the damage will be relatively small.
2. Detector cell – there may be a liquid leakage from the detector cell and the cell may crack making detector unusable.

When the system capillaries or filters become clogged with insoluble matter, liquid (mobile phase) will not flow smoothly through the system tubing and compounds of interest may adsorb to the system parts. This will cause deformation of peaks in your chromatograms.

Finally, if pump pressure becomes too high the system flowrates might not be as accurate. On the other note higher pressure will negatively impact the service life of consumable parts of your HPLC system.

LC System Pressure Causes – flow line blockage

Closer look at the mobile phase(s)

First protocol would be to examine the mobile phase bottles on top of the system. In case if visible particles are floating in the bottle(s) it most probably is insoluble matter present in your mobile phase. This matter was pushed through the system lines and caused blockage(s). Although there is a suction filter at the end of each mobile phase line, this can easily occur because the downstream inline filters pores are often way smaller. Even if the mobile phase was previously filtered, deposition may occur later if there is little leeway in the solubility of the solute.

Even before introducing the mobile phase to your system, you can perform a small bench experiment. With the gradient elution of buffer solution and organic solvent, use a beaker or a flask and while stirring add organic slowly to your buffer solution and observe when the white precipitate starts forming. This way you will find the point at which buffer will start “crushing out” from the solution. Depending on the mixing ratio, white turbidity/precipitate may even occur when organic solvents are mixed together. If white turbidity or precipitate occurs you have two options:

• If you are not able to update / change the method you need to consider introducing additional rinsing steps every time the analytical run is completed, i.e. rinse method utilizing different mobile phase lines (like line C and D using the same mobile phase composition as your method but without the buffer in it)
• If you are lucky and are able to update the test method conditions you should reconsider the mobile phase composition.

On the other hand, using only purified water as mobile phase will present different challenge, propagation of bacteria growth.

One by One – finding the blockage on the system

In general, remove tubing in order from the downstream end when checking the flow line will help to identify which part of the system is blocked. Keep the system flowing and observe the pressure value, ensure the flow is set at a rate that will not reach the maximum pressure but will allow you to monitor the pressure value. Refer to figure 1 above for the system points to check. Remove the connection no (1) (Figure 1), if the pressure drops suddenly (by several hundred kPa or more), then this indicates blockage in the back-pressure tube. If it does not drop, it indicates that the clogging is further upstream. Therefore, proceed to connection no (2) (Figure 1), apply the same logic. After this, connections (3), (4), (5), (6) and (7) are investigated in the same way. In some cases, other connectors can be removed. When checking the tube, cell, filter, or injector, there is a problem if a pressure of several hundred kPa occurs at the part itself, because pressure drop of several hundred kPa may naturally occur in tubing for semi-micro systems. Blockage is liable to occur at filters and at the inlets to fine tubes and so these places require particular attention. See Figure 2 and Figure 3 below for the system “hot spots” prone to blockages.

Figure 2. System locations prone to clogging

Figure 3. Inline column filter – integrated option

What to do when the LC system is blocked

There are parts of the system flow line where blockages can be easily removed by simple rinsing to dissolve the blockage(s). Saying that, there are also system cavities nearly impossible to rinse / clean. Refer to table 1 below for some ideas on how to rinse the system when treating either soluble or insoluble matter blocking the HPLC system.

Table 1. Examples of Blockages and Solutions/Solvents which can be used for cleaning (dissolving)

Closer look at system Tubing

HPLC systems are sophisticated, well-engineered instruments, however you can still find the spots withing the system which are more prone to blockages, such as the junctions between tubes with a large inner diameter and fine tubing, and parts where the flow line bends, refer to Figure 2 above. There are many cases where the problem is solved by cutting a length of approx. 1 cm off the inlet end of the tubing, this way removing the blocked part of the inlet tubing and solving the issue. Therefore, it is convenient to use a hand-tightened PEEK male nut for the connector, were possible.

Autosampler / Injector and in-line Filters

Should you find the blockage within the autosampler space, the first protocol to rinse it is to reverse the flow or just disassemble the blocked part and properly clean using ultrasonic cleaning.

Column blockage

If the column pressure is quite high, you need to consider the column check. Most of the QC Lab already control the column performance noting the theoretical plate counts with every run, which is the best practice giving you all information about the column performance you need and making you aware of upcoming column problem. Measurement of the number of theoretical plates under the manufacturer’s specified conditions helps to investigate whether or not it satisfies the requirements of the intended use. If the pressure is extremely high, as a general rule, the column is typically replaced. Unless you are a mature scientist using the column guards. Checking and replacing column guard is the first thing you need to do. Once column guard is replaced run your analytical method again and check if the high column pressure still persists. If the pressure is still high, the blockage in the filter in the inlet side’s column end might be present. Even in this situation it may be possible to solve the problem using the method described below, although the success of this method is not definitive. Try to reverse the column and backflush with half the flow onto white paper/tissue, at the same time observe the tissue as potential dirt/blockage can be released from the column inlet side. Continue observing the system pressure when performing the backflush. Once finished, remount the column and assess your analytical run.

It’s time for the Detector Cell

In case of the high pressure observed within the detector UV cell, perform the UV cell rinse as recommended by the detector manufacturer. If in doubt, contact your local system Engineer to help you with this task to prevent cell damaging.

What about other possible HPLC system high pressure situations?

Situation 1 – pressure gradually increases each time sample is injected.

This phenomenon can be caused by insoluble matter present in the sample injected or a matter that dissolves in the sample solvent but not in the mobile phase. Unless you change the sample, composition there is not much you can do. When the method is validated, it is hard to justify the change in sample composition, but it might be easier to perform supplementary validation exercise and change the mobile phase solvent or solvents ratio.

Some chemicals when mixed can cause more trouble than expected. Refer to table 2 below for incompatible solvents. Always consider the possibility of incompatible solvents and effects of their mixing.

Table 2. Solvents which should never be mixed together

Situation 2 – pressure increases a little on injection and then decreases

Insufficient solubility of the compound of interest or an increase in the viscosity resistance pressure which occurs when the sample solvent and mobile phase are mixed can be the cause of this intermittent pressure increase. There is not much you can about this except to ensure that your method is working within the operational column pressure range, at maximum 75% of the allowable column pressure to prevent the system stopping and column damage when this pressure spike occurs.

Situation 3 – system is used after a long break

It is typical that a system can be put on hold for a prolonged time, some project is finished and awaiting another one to kick in or system is switched off for a long holiday period. When using the instrument after a long interval, or if the previous operating conditions are unclear (we don’t know what was run), the flow lines should be first rinsed without the column to ensure the system is best prepared for analysis.

Notes on Routine Daily Analysis

Filter the mobile phase and sample diluent.

In preparation for analysis, it is best practice to filter the mobile phase and particularly, the sample diluent with a membrane filter. There is wide variety of available filter to suit the solvent/mobile phase composition. Additionally, before placing the liquids on the system perform the simple experiment and mix together the organic and buffered mobile phase part to ensure no precipitation will occur on the system. This we already discussed above.

Keep the records – especially pressure value

Once all setup and system is running, ensure to keep the record of the system pressure value. Remember to record this number for future reference but don’t go too far and don’t make the pressure one of your system suitability checks, as this is not indication of your chromatographic performance, it rather is physical indication of column performance. When you start tracking the pressure it will help you to discover potential problems earlier and anticipate when there is still time to act.

Pressure records:

• Isocratic method – record the pressure from the same solution injected, i.e. from one of the standards run within system suitability; it must be injection which you can always repeat and indicative of a good stable system
• Gradient method – if you can, you should acquire the pressure trace for comparison in future and at the same time record the starting pressure of one of the standard solutions injections from system suitability.

Some chromatographic software records auxiliary information like pressure, temperature, etc. without you even knowing (like Shimadzu LabSolutions software) and other require you to record a pressure channel, which is easily enabled in you instrument method part.

Take appropriate measures for the tip of the detector’s outlet tube on completion of analysis

The last thing you will probably consider is the end of the detector’s outlet tube (back-pressure tube). This tube is technically open to air, meaning that the solvent present in the tube is susceptible to evaporation. Once the analysis is completed and system washed ensure that the tube is directed to waste. If the equipment is not going to be used for several days, remove the column and store as directed by column manufacturer. When using a mobile phase that may shorten the column’s service life, thorough rinsing of the column must be considered and performed before storage.

Summary

It can be concluded that the pressure subject can be tricky and give you few headaches before you reach the resolution of your problem. I hope that this months’ blog brings you a bit closer to effective tracking of system pressure to mitigate any potential issues early enough. Additionally, you are now equipped with few tools and considerations when you encounter the system pressure hike.

Liquid Chromatography – Master the Basics

This article is part of our “Liquid Chromatography – Master the Basics” series, your go-to resource for comprehensive and insightful updates on the world of liquid chromatography. Each month in 2024 we will dive into a Liquid Chromatography topic, offering content that is both accessible to beginners and beneficial for experienced scientists.

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