Best practice for separation columns in ion chromatography (IC) – Part 3

Mar 21, 2022

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This article is Part 3 of a series.

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This is the third and final post in our series providing you with tips and tricks on the proper use of ion chromatography columns. In the first part, we mostly discussed the standard operation conditions as well as operational limits for columns, while in the second post, we focused on application related topics and what elution parameters can be changed to modify the separation performance. In the conclusion of this series we will take a closer look at the ways to assess the column performance during its lifetime and offer some troubleshooting tips which can help in fixing issues that may appear.

Click on a topic below to go directly to each section:

Essential column parameters

Let’s begin by looking at the main parameters that can be used to judge the column performance. Most of these reference values can be extracted from the certificate of analysis. The separation column certificates can be found in the Metrohm Certificate Finder. Reproducing the certificate chromatogram periodically to verify the column performance can be helpful to detect changes in performance early and to avoid irreversible damage of the column.

Metrohm Certificate Finder

Below, we will consider the most important parameters one by one.

Retention time and column capacity

The retention time of the individual analytes is a good measure of the column selectivity and capacity. When the retention times of the analytes do not match the retention times of the certificate, there are many possible reasons for this behavior.

Possibly, the eluent composition is wrong, e.g., because the eluent components are not present in the correct concentrations. In case the eluent strength is too high, the retention times will be reduced. This phenomenon usually occurs for all ions simultaneously and shifts all the peaks closer together. Thereby, the multivalent ions are accelerated to a stronger extent than the monovalent ions. This issue can be fixed by preparing a fresh eluent with the correct composition.

The eluent may not be fresh or not sufficiently protected from the surrounding atmosphere, e.g., with a CO₂ adsorber. Carbon dioxide from the ambient air can change the eluent composition (i.e., strength, pH, etc.) over time and this will affect the retention times of the analytes. Weak hydroxide eluents are particularly affected by this issue, since the elution strength of carbonate ions is much stronger than the one of hydroxide ions, leading to strong shifts in retention times. Therefore, multivalent ions are more affected by this than monovalent ones. This issue can be fixed by preparing a fresh eluent and by using a CO₂ adsorber. When working with hydroxide eluents, it is also important to verify the status of the hydroxide stock solution as it can absorb CO₂ from the air. Figure 1 shows the effect of CO₂ uptake from the air on the retention times of common anions.

Some samples may contain constituents that occupy the ion exchange groups of the column material. In that case, the number of available ion exchange groups on the column is reduced compared to the initial state, leading to an apparent smaller column capacity and shorter retention times. Depending on the type of contaminant(s), there are possibilities to reverse this effect and wash them from the column surface. These regeneration procedures are adapted individually to the different stationary phases and should be used as a last option to save a column.

The procedures as well as the operation limits are described in the respective manual of the column and can be found on our website. Please consult your Metrohm dealer before performing a regeneration procedure.

Manual for Metrosep A Supp 17

In case the contamination is from a multivalent ion, it is usually possible to wash them off by flushing the column with an eluent of a higher concentration. When doing so, do not forget the operation limits of the column, particularly regarding the eluent pH.
Sometimes, organic molecules can adsorb to the column stationary phase because of their strong affinity to it. This can lead to a blockage of the ion exchange groups and reduced retention times along with increased operating pressure. When this happens, it can help to wash the stationary phase with an eluent containing a fraction of an organic modifier. This will increase the affinity of the contaminant to the mobile phase and help to detach it from the stationary phase.

Usually, after such a regeneration procedure, the original column capacity can be restored.

An even better way to extend the lifetime of the separation column is by regularly exchanging the guard column. One of its purposes is to protect the separation column from contaminants by retaining them.

Make sure to exchange the guard column three to four times over the lifetime of the separation column, or even more frequently when dealing with very complex sample matrices such as dyes or food matrices. In these situations, we suggest using the Metrosep RP 2 Guard/3.5.

Metrosep RP 2 Guard/3.5

The chromatogram overlay in Figure 2 shows the effect of a defective guard column on the peak shape.

In some cases, the column capacity is reduced due to chemical modification of the stationary phase. This happens when a column is used in conditions outside of the operation limits—e.g., extreme pH situations. Extreme pH conditions can alter the chemical bonds of the ion exchange groups to the base particle substrate. This irreversible process leads to permanent column capacity loss. In this case, no regeneration procedure can restore the original column capacity.

Please note that this loss of ion exchange groups also happens slowly during the regular use of ion exchange columns. The strength of the chemical bond of the ion exchange groups is thoroughly tested during the development of Metrohm columns to provide a long column lifetime.

Theoretical plates and peak symmetry

The number of theoretical plates (TP) can be a useful tool to judge the column packing status. The higher the TP value, the better the packing of the column. For a meaningful judgement of the column packing bed, choose an analyte that does not elute too early in the chromatogram (these peaks can be affected by extra-column effects, in particular on 2 mm systems) and that is not affected by secondary interactions (i.e., avoid using nitrate). For cations, potassium is a good choice, while for anions, sulfate is a suitable choice.

Although the columns are packed at pressures higher than the normal operating pressure, the packing bed continues to further densify throughout the column lifetime with the continuous application of flowing mobile phase (eluent). While the packing bed of the stationary phase improves due to this effect, it may also lead to some dead volume at the entrance of the separation column. This dead volume can be responsible for peak broadening and reduced theoretical plates, particularly at the beginning of the chromatogram.

While this is a regular aging process of the column, it is possible to slow it down by taking good care of the column. Preventive actions include the slow initiation of the eluent flow rate and temperature at start-up as well as the proper and complete shutdown of the high-pressure pump before removing the column, as mechanical stress can be detrimental to the packing bed. Exchanging the guard column can often have a positive impact on the theoretical plates and peak shapes (see Figure 2).

Issues with the column packing bed often also become visible in the asymmetry factor values and the overall peak shapes. More pronounced fronting as well as peak broadening can be warning signs for channeling in the column or the guard column. Unfortunately, this kind of damage on the column is irreversible and requires an exchange of the separation column.

Before column replacement, it is important to check if the issues originated from the column or another connection in the IC system whenever peak broadening is observed in the chromatogram. Make sure that all capillaries in the high-pressure path have a diameter of ≤0.25 mm and that all capillaries have been installed and connected correctly without additional dead volume. Systems equipped with small inner diameter (2 mm, microbore) columns are more strongly affected by dead volume than those with 4 mm columns. This means that when using microbore columns, less dead volume is required for the peak broadening effect to become visible compared to when using 4 mm columns.

Column pressure

Another important parameter to regularly check throughout the column lifetime is the system pressure. High pressure is among the most frequent reasons leading to a column replacement. Whenever a pressure increase is noted, it is important to verify what part of the IC system is the cause.

If the sample contains particles and insufficient sample preparation is applied, the particles will accumulate at the entrance of the protective guard column and eventually lead to increased system pressure. In this case, the guard column performs its intended use of protecting the separation column and needs to be replaced. If the guard column is not replaced soon enough, then particulate contamination may break through and load onto the separation column. While there is no regeneration procedure for a guard column loaded with particles, separation columns contaminated with particles may be regenerated by rinsing the column at low flow rates in the reverse flow direction.

Please note that these regeneration procedures may not always be successful.

Avoiding particles from being injected into the high-pressure path of the IC system is a good way of protecting the guard and separation columns. Metrohm provides several automated sample preparation techniques which result in a positive impact on the column lifetime. The most common techniques for this purpose are Inline Ultrafiltration (Figure 3) and Inline Dialysis.

Learn more about Metrohm Inline Ultrafiltration in our related blog post.

When do I have to exchange the filtration membrane with Inline Ultrafiltration?

Column end of life

All of the parameters described above should be considered to judge the column (and the IC system) performance. Many of these parameters can be monitored closely in the MagIC Net software so that a potential issue can be detected as early as possible.

Even though Metrohm IC columns are designed and manufactured to have very long lifetimes, at some point performance will decrease and even regeneration procedures may not be able to restore the required column performance to solve the application needs. This represents the end of the column life and makes the exchange of the separation column unavoidable.

Metrohm separation columns cannot be recycled and can be disposed with normal waste. However, depending on the samples measured as well as the used chemical types and their associated hazards, it may be necessary to consider a proper disposal option.

Irrespective of the sample nature, do not open the column at any time.

Troubleshooting overview

Table 1 gives an overview about certain troubleshooting strategies when looking at your column performance behavior.

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 inlet (i.e., the opening next to the intelligent chip) in a beaker, as this is now the outlet path for the contaminants. 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 lnline Ultrafltration
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. lt 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.

Conclusion

This article explained how IC column performance can be assessed and monitored throughout the column lifetime and what measures can be taken to ensure a long column lifetime. With that, we now conclude the series «Best practice for separation columns in ion chromatography». If you have more questions, do not hesitate to contact your local Metrohm IC salesperson.