HPLC - The Art of LC Trouble-Shooting, a General Approach

Norbert Reuter*, Global Technical Support for CSD, Middelburg, The Netherlands

The art of trouble-shooting is becoming oblivious to many operators. If problems occur ‑ and they are recognized as problems ‑ a phone call to the nearest Agilent office is the standard solution. But maybe some of these problems can be solved immediately without external help. This will be an (uncomplete) guide to trouble-shooting.

Introduction

Everyone dealing with HPLC equipment knows that he has to cope with disturbances and defects. Some of these can be directly attributed to the equipment itself (e. g. pump disturbances, thermostat out of order etc.) and other problems to less direct factors (e. g. blockages). These problems are not solely dependent on the HPLC technique, but can be of a more general character. HPLC equipment is diverse and high standards are set on the electronics, mechanics, and materials. The lifetime of these parts is obviously limited and consequently so wear and tear can eventually cause failure or instability.

Furthermore, many of the demands tend to conflict with each other, precision, sensitivity, durability, manageability, required power etc., so that within the array of variables a compromise has to be found. As this is very critical, any deviation leads rapidly to poor chromatographic results.

Every HPLC user should therefore know how to localize and solve disturbances and sources of trouble. Without this knowledge a lot of time will be wasted in unsystematic and arbitrary searches for possible causes of the problem.

Systematic Approach

You can see, that trouble-shooting can be described with the help of a control cycle. One of the most difficult parts in that is the connection from an existing problem to the recognition of it by the operator.

Recognition

If you do not recognize a problem, it will be unsolved and the analytical results are influenced in a certain way. For this step a good knowledge of chromatography and the system you are using. Chromatograms can tell a lot about what’s happening (see also the article: HPLC Troubleshooting ‑ Changing Retention Times and Peak-Splitting).

Analysis - Types of Problems

Problems can have different sources; each source or type can then be divided into different culprits. On the other hand every culprit can also belong to different sources, that’s what makes trouble-shooting sometimes difficult. In spite of all these different possibilities operators tend to blame the column first, but you see that there are more risks and they also can interact with each other. For example if a pump (electrical or mechanical problem) does not work well, the eluent flow (chromatographic problem) can change and you will see shifting retention times on your chromatogram (also a chromatographical problem).

The chromatograms cannot only be used in the problem recognition, but are vital in the analysis and determination of the problem type.

Correction

This is the part where often two human natures fight against each other. The first wants to solve the problem by fixing everything that’s possible in a single strike and the second nature that tries to follow the systematic protocol, which says:

• Check one single source of error only
• Check only one system component at once
• If a part is not defective return it immediately into the system (do not replace it)
• A defective part should be scrapped and not put into the laboratory’s collection drawer
• Make immediate minutes of the process (no double checking of components)

If you check or replace more than one component at a time you won’t be sure which one caused the error.

Control

This is the easiest part of the process; the system is reassembled again, defective parts are replaced and/or erroneous procedures are adapted. The test run is done and the result look ok, then the problem is solved. Otherwise you return to the recognition part of the cycle and start all over again until the control checks positive.

Things a Chromatogram Can Tell You - Examples

Example 1:         The detection technique used was refractive index detection, the left chromatogram was expected, but the run showed as the right chromatogram:

A RI detector is very sensitive towards changes in eluent compositions and temperature changes. Using a thermostat/column oven can prevent temperature changes and the first one can suggest that the column had not enough time to equilibrate.

Example 2:        The left chromatogram was expected, but the right one was obtained:

All peaks even the t₀ peak show broader peak shapes, this is a typical error caused by a dead/void volume at the column inlet. Maybe the packing bed collapsed a bit.

Example 3:        An isocratic blank run show the following pattern:

Spikes on a base line can occur if the degassing system is not properly working and air bubbles travel with the eluents into the detector cell. Also power fluctuations can cause spikes.

Conclusion

With the right chromatographical and technical knowledge and skills in chromatogram interpretation and the commitment to follow a systematic path every operator can do trouble-shooting. In case of the lack of one or more of these skills you can always contact your nearest technical helpdesk from Agilent.

LC Troubleshooting Guide/Poster