LCMS Troubleshooting Course
1 - Basics of LCMS Systematic Troubleshooting
2 - Preventive Maintenance Measures
3 - Pressure Issues
4 - No Peaks Detected
5 - Changes in Peak Shape - Part 1
6 - Changes in Peak Shape - Part 2
7 - Ghost Peaks
8 - Peak Area Fluctuations
9 - Baseline Disturbances
10 - Retention Time Fluctuations - Part 1
11 - Retention Time Fluctuations - Part 2
12 - Changes in Chromatographic Resolution
13 - Changes in MS Response
14 - Undesired Fragmentation & Ion Source Settings
15 - Course Summary & Quiz
Pressure is a characteristic of the chromatographic conditions employed for the separation. It can be used as a diagnostic tool to understand causes of common issues, thus it is imperative to record the pressure trace.
In LabSolutions, you can display the pressure via a right-click in the chromatogram then select "Display Setting" – "Status". Even if it is not displayed, the pressure is always recorded with every data file and can be displayed during the data processing and evaluation.
The causes of pressure disruptions are varied. They can either be caused by the hardware, by contamination of the eluents, particles in the samples, crystallised buffer, or similar contaminants that clog the system. Even small contaminants can lead to a slow but steady increase in pressure. As already mentioned in Unit 2, temperature also plays a major role. If the temperature rises, the viscosity of the eluent decreases and consequently the pressure also decreases.
However, there are also pressure fluctuations that are completely normal and do not cause concern. For example, if a gradient is run, we actively mix two or more liquids of different densities and viscosities. The result is a uniform increase or decrease in pressure, which in most cases corresponds to the shifted course of the gradient.
This section of the course will investigate the most common pressure issues including fluctuations during the run, and unexpected increases or decreases in pressure.
Pressure fluctuations can have many different causes. The back pressure of an LCMS system is similar to a heartbeat: any irregularity is immediately visible in both the pressure trace and the MS signal (total ion chromatogram - TIC). Therefore, it is crucial to monitor the pressure profile during all runs and investigate abnormalities promptly. The TIC is the total signal intensity for all ions which are detected in a given timeframe.
In LCMS, pressure issues are more critical than in standalone LC because they can also disrupt ionisation stability and vacuum performance in the mass spectrometer.
Fig. 1 A characteristic ripple caused by an air bubble, which can impact on the retentivity and robustness of an analysis.
Fig. 2 A contaminated ion source with a non-volatile phosphate-based mobile phase, which can cause pressure issues, as well as sensitivity problems.
Too high pressures are usually due to the column or a blockage. A clogged inline filter of the pump can also cause high pressures. The best way to find the cause is to loosen the first connection at the pump and successively add the components. If the pressure rises sharply, the culprit is identified. Common causes of high back pressure are:
| Issue | Description | Solution |
|---|---|---|
| Flow Rate Set Too High | If the flow rate is incorrectly set too high in the method, this will cause the pressure to increase and cause peaks to elute earlier than expected |
Check the flow rate settings to ensure the velocity is correct. Amend as necessary
|
| Blocked Column or Guard Column | A column can become blocked with particulates, especially with dirty samples. This will build on the head of the column and cause the pressure to increase over the course of a batch |
If possible or recommended by the column manufacturer, the column might be backflushed to remove the particulates. However, the build up will occur again if the samples are not properly prepared (i.e. filtering / precipitation etc)
The most likely course of action to correct for a blocked column is to replace the column
Replace the blocked guard column
|
| Incompatible Mobile Phase | Immiscible solvents or certain buffers with too high organic modifier might precipitate, which can cause the pressure to increase in the system |
Use the correct mobile phase with compatible solvents
Flush the column out with a cleaning solvent. Ensure the column is fully equilibrated before first use.
|
| Incorrect Column Installed | The pressure is a function of the column dimensions and particle size. If the incorrect column was installed in the system, it could have a different pressure. It should be noted that pressure may differ between columns of the same dimension and particle morphology but it should be within a reasonable tolerance based on usage. |
Install the correct column dimensions and particle geometry
|
| Injector Blockage | Air bubbles in the solvent lines or pump heads are a frequent cause of unstable pressure and can also destabilise the ESI spray. |
Purge solvent lines thoroughly; use a vacuum syringe on purge valves if necessary
Ensure degasser is function
Use low surface tension solvents (e.g. methanol) to help remove stubborn bubbles
|
Low pressure may be caused by:
| Issue | Solution |
|---|---|
| Incorrect Flow Rate |
Check the method settings are correct
Check the flow rate accuracy using a calibrated flow rate meter. Alternatively, collect a specific volume and monitor the time it takes to acquire.
|
| Leaks |
Check for leaks and reconnect the affected part
Replace worn out or damaged pump seals
|
| Air Bubbles |
Purge all the lines to remove the bubbles
Purge the system with IPA and ensure the check valves are working correctly. Sonicate the check valves if necessary in IPA
|
| Disconnected or Bypassed Components (e.g. column not properly seated) |
Ensure the flow path is correctly installed
Document the setup of the system for future users to know the configuration
|
| Incorrect Method Settings |
Check the method for the correct temperature and solvent lines as this can impact on the pressure
Check the correct column is selected if using a column selection valve
|
In LCMS, pressure fluctuations affect not only chromatographic performance but also MS spray stability and sensitivity. By combining LC troubleshooting steps with MS-specific checks (leaks, interface contamination, spray monitoring), you can resolve most issues efficiently.
Things to consider daily:
Eluents
- Use LCMS grade solvents and buffers
- Filter at high salt concentration
- After the instrument has finished, the buffer should be flushed out of the system to prevent precipitation issues
- Flush the LCMS with a cleaning solvent mix (i.e. water/acetonitrile (50:50 v/v) at the end of each day to prevent buildup in the source
- Consume limited shelf-life eluents within the shortest time
Samples
- Filter or centrifuge the samples to remove particulates
- If necessary, also perform purification steps, such as protein precipitation.
- Verify compatibility with the eluents
- Flush the injector if samples with high salt concentration were used.
Instruments
- Monitor both LC backpressure and MS vacuum pressure during runs.
- Inspect and clean the ionisation source and capillary regularly
- Keep the temperature constant to avoid pressure fluctuations
- Keep common consumables in the lab to minimise any potential downtime
- Always display the pressure profile. You will learn a lot about the condition of your system. With a little experience, you will be able to recognise the error based on the pressure.
Fig. 3 Recommended LCMS solvents for Shimadzu Instrumentation.
In the next course unit, we will look at possible causes for missing peaks, ranging from the sample, through the LC and then on to the detector, with simple solutions to solve the problem.
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