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Monitoring and maintaining column performance

One of the basic requirements for ensuring reliable chromatographic analyses is a high-performance separation column. Ion chromatography (IC) users should regularly check the performance of their column. This way, if a drop in performance becomes apparent, steps can be taken with enough time to restore or maintain the proper functioning of the column, reducing downtimes in sample throughput. In this blog post, we explain how you can assess column performance, which parameters you should monitor, and which measures you can take to ensure excellent column performance.

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First-time use of a new separation column

When you use a column for the very first time, we recommend that you check its initial performance. The Certificate of Analysis (CoA), which you receive with every purchase of a Metrohm column, is your source of reference here. Record a chromatogram and use the analysis conditions specified in the CoA: these include flow rate, temperatureeluent (mobile phase)analyte concentrationsample loop size, and suppression.

You can evaluate the column’s performance by comparing some of the result parameters with the values listed in the CoA (e.g., retention time, theoretical plates, asymmetry, resolution, and peak height).

Regular monitoring of column performance

Columns that are already in use should be monitored regularly, too! We recommend carrying out these tests with check standards under the application conditions you normally use, because performance varies depending on the type of analysis and associated analysis conditions as well as the instrumental setup. If a reduction in performance is observed, the requirements of the application are crucial to determine whether it can still be used.

Below, we explain how to determine your column performance based on five performance indicators. You will also find out how you can prevent or rectify a decline in performance.

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Monitor the backpressure: When you use your new column for the first time, save the backpressure value under the analysis conditions of your application as a reference («common variable» in MagIC Net). Then use the user-defined results to monitor the difference between the initial backpressure and the one displayed during the current determination.


If you identify an increase in the backpressure in comparison with the saved initial value, this indicates that particles have been deposited in either the guard column or separation column. If the measured increase is greater than 1 MPa, action must be taken. First, you should check which of the columns is affected (guard vs. separation). If the guard column is contaminated, it should be replaced, as this is its primary function. If the separation column is affected, remove it from the system, turn it around and reinstall, and then rinse it for several hours in this reversed flow direction. If this doesn’t help, we strongly recommend that you consider replacing the column. This will be essential if the maximum permitted backpressure for the column is reached.

Learn more about MagIC Net software here

Retention time

To track changes in the retention time (which signal a decrease in column performance), the retention time of the last analyte peak is monitored in the chromatogram. Sulfate, for example, is suitable for this, as it usually elutes right at the end of standard anion chromatograms. Here too, work with a common variable in MagIC Net to save the initial value.

Unstable retention times can be caused by carbon dioxide introduced from the ambient air or from air bubbles present in the eluent. Luckily, these problems can be resolved easily (see Table 1).

Table 1. Preventing and correcting performance loss in IC columns.

Indicator Cause Preventive and corrective measures
Increasing counterpressure Particles on the guard column Replace the guard column.
Particles on the separation column

Rinse out the separation column in the reverse flow direction:

  • Place the column outlet in a beaker.
  • Rinse out the separation column for approx. one hour.
  • Reinstall the separation column in the flow direction.
Particles in the sample Sample preparation, e.g., remove particles through Inline Ultrafiltration
Shortened retention time Carbonate in the eluent

Carbon dioxide from the air affects the carbonate/hydrogen carbonate balance in the eluent. A carbonate/hydrogen carbonate eluent weakens over time; a hydroxide eluent strengthens.

  • Always tightly seal eluent bottles and bottles containing eluent concentrate.
  • Always use a CO2 adsorber.
Air bubbles in the eluent

Air bubbles make the eluent flow unstable. The counterpressure is an indicator of unstable flow. It should remain stable within a range of ± 0.1 MPa.

  • Deaerate the high-pressure pump.
  • Use an eluent degasser.
Capacity loss in the column due to high-valency ions Regenerate the column as per the column leaflet to remove any inorganic deposits.
Resolution loss Eluent too old or produced incorrectly Eluents should be freshly prepared. Make sure that they are produced correctly and particularly that carbonate and hydrogen carbonate are not confused.
Adsorptive effect of the contamination deposited in the guard column Replace the guard column.
Adsorptive effect of the contamination deposited in the separation column Regenerate the column as per the column leaflet to remove any organic or inorganic deposits.
Loss of theoretical plates Guard column contaminated Replace the guard column.
Separation column contaminated Regenerate the column as per the column leaflet to remove any organic or inorganic deposits.
Separation column overloaded

The separation column can be overloaded by factors such as a high salt content in the sample matrix.

  • Dilute the sample.
  • Inject less sample.
Dead volume in the IC system
  • Check that all capillaries have a diameter ≤ 0.25 mm; if they don't, replace the capillaries.
  • Check that all of the capillaries have been installed correctly. The installation process is described step by step in the IC Maintenance» multimedia guide.
Asymmetry Dead volume or contamination on the guard column Replace the guard column.
Separation column contaminated Regenerate the column as per the column leaflet to remove any organic or inorganic deposits.

The column may also have lost some capacity. This capacity loss can be caused by the presence of high-valency ions which are difficult to remove due to their strong attraction to the stationary phase. The column should then be regenerated in accordance with the column leaflet to remove any contamination. If this doesn’t lead to any improvement, then consider replacing the column depending on the requirements of the application, particularly in the event of progressive capacity loss.

Capacity loss can also occur if the functional groups are permanently detached from the stationary phase. In such a case, the column cannot be regenerated and must be replaced.


Monitor the chromatographic resolution by comparing measurements from a predefined check standard with an initial reference value. If the resolution is R > 1.5, the signal is considered baseline-separated (see illustration below). However, in cases involving highly concentrated matrices and for peaks that are more widely spread, the resolution value must be higher to ensure baseline separation.


If a loss of resolution occurs, first make sure that it is not caused by the eluent or the IC system. Once these have been ruled out, it is possible that the adsorptive effect of contaminations in the guard column or separation column may be responsible. A contaminated guard column should be replaced. If the cause of the problem is found to be the separation column, this should be regenerated in accordance with the column leaflet to free it from any organic or inorganic contamination. If the loss of resolution progresses, a column replacement is inevitable.

Theoretical plates

Save the initial number of theoretical plates in MagIC Net as a common variable, as mentioned earlier for other parameters. Usually, the last eluting peak is used – in anion chromatograms, sulfate would yet again prove to be a suitable candidate. Theoretical plates also depend on the analyte concentration. Therefore, it is ideal to monitor this parameter during check standard measurements and not during sample measurements. You can track the development of any changes to the number of theoretical plates via the user-defined results in MagIC Net.

A decrease in the theoretical plates can suggest dead volume in the IC system (Table 1). A low number of theoretical plates may also be observed if the column has been overloaded by a high salt concentration in the sample matrix, for instance. If the theoretical plates decrease by more than 20%, this indicates that column performance is declining. Depending on the requirements of the application, action may need to be taken.

If the guard column is the reason for the drop in performance, it should be replaced. If the problem is with the separation column, we recommend regenerating the column in accordance with the column leaflet to eliminate any organic or inorganic contamination. If this doesn’t help, you should consider replacing the column, particularly if a trend toward lower theoretical plates is observed.


Determine the initial asymmetry of the analytes by measuring a predefined check standard under the analysis conditions of your application. Save it as a common variable, then track the user-defined results to observe the development of asymmetry over time. The maximum acceptable values for the asymmetry vary depending on the analyte. For example, calcium and magnesium peaks initially present relatively high asymmetry values.

Asymmetry is defined as the distance from the centerline of the peak to the descending side of the peak (B in the figure below) divided by the distance from the centerline of the peak to the ascending side of the peak (A in the figure below), where both distances are measured at 10% of the peak height. Some pharmacopoeia may use other figures – please check to be sure of the requirements in your country.


AS >1 means a peak has tailing, and AS < 1 equates to peak fronting. Optimum chromatography is achieved with peak asymmetries as close as possible to 1. As a general rule, column performance is considered in decline when the asymmetry is AS >2 or AS <0.5. Depending on the requirements of the application, measures have to be taken in this case in order to improve symmetry and to enable better integration.

The reason for high asymmetry values may be down to the ion chromatograph – due to dead volume, for example. If this is not the case, it is important to find out whether the asymmetry is caused by problems with the guard column or with the separation column. If the guard column causes the asymmetry, it should be replaced. If it is the separation column, it should first be regenerated in accordance with the column leaflet to remove any organic or inorganic contamination. If this doesn’t help, you should consider replacing the column. If a trend toward higher asymmetry values can be observed, replacement is unavoidable.


There are many ways in which you can estimate the performance of the column and track concrete figures over its lifetime. Proper maintenance can extend the lifetime of the separation column, as well as always using a guard column for extra protection. For more information, check out our other blog articles on this topic!


Dr. Alyson Lanciki

Scientific Editor
Metrohm International Headquarters, Herisau, Switzerland