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The second part of this blog series about best practice for IC separation columns focuses on application related topics that have an impact on the column suitability and stability. First, there is the proper choice of the column that best suits the intended application. Then we turn to the operating parameters which can be modified in order to optimize the separation between analytes, and what the respective effects and possibilities are.
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Choice of column length and diameter
Metrohm offers a broad range of IC columns that contain different stationary phases and have different lengths and/or inner diameters. The choice of the stationary phase has a great impact on the selectivity between the individual analytes on the one hand, as well as the stability against different sample matrices on the other hand. Instead, the column length has no impact on the selectivity, but rather on the separation efficiency between the individual peaks.
Find out more about Metrohm’s wide selection of separation columns for ion chromatography in our Column Catalog.
Effects of column length
In the following chromatograms (Figure 1), the effect of the column length on the separation efficiency for the Metrosep A Supp 17 column series is shown. Whenever choosing a column length, you should take the complexity of the intended separation and the presence of matrix components that could disturb the ions of interest into account.
Effects of column diameter
In addition to providing different lengths of IC separation columns, Metrohm also offers most columns in both in 4 mm inner diameter and 2 mm inner diameter (known as «microbore») versions. With regard to this, there are several criteria to distinguish:
- If you use online systems in a continuous mode (i.e., systems which run unattended for several days in a row such as the Metrohm Process Analytics MARGA system – Monitor for AeRosols and Gases in Ambient air), we recommend using 2 mm IC columns. Due to the reduced flowrate for microbore columns (only 25% of the flowrate for 4 mm columns), the eluent and the regenerant solutions last much longer, which increases the time the instrument can be left unattended.
- There are applications that require hyphenated techniques such as IC-MS for higher analyte selectivity and sensitivity. In this case, the use of 2 mm columns is ideal. The low flowrate is optimal for the electrospray process, and thus no flow splitter is required before entering the mass spectrometer.
- Sometimes, only a limited amount of sample is available for injection. In these situations, 2 mm columns are preferred. This is because less dilution/diffusion occurs during the separation process and therefore higher signals are obtained.
- On the other hand, if your sample contains a high load of matrix components, then selecting a suitable 4 mm IC columns will be a better choice because of the higher capacity available to separate the desired analytes from the matrix.
Find out more about MARGA and its capabilities for continuous air quality monitoring in our blog post.
Optimizing the analyte separation
Next to the column itself, several other parameters can be modified to optimize the selectivity of the separation. These parameters include temperature, eluent components and strength, and organic modifiers.
Effects of modifying the temperature
One of the simplest ways to fine tune the separation selectivity in IC is by modifying the temperature of the analysis. This is accomplished by using the integrated column oven in the instrument (if available). Multiple effects can be observed, for instance in anion analysis. As an example, the impact of the temperature on the selectivity is shown in the chromatogram overlay (Figure 2) for the Metrosep A Supp 17 column line.
- The monovalent ions such as fluoride, chloride, nitrite, bromide, and nitrate are all accelerated with increasing temperature, indicating that fewer interactions with the stationary phase happen.
- The behavior of multivalent ions such as phosphate or sulfate is more complicated to describe and will vary with each stationary phase. In general, multivalent ions are retarded more at higher temperatures, which causes the retention times to increase, as can be seen for sulfate. Phosphate on the other hand behaves differently, because of the temperature induced change of the eluent pH in a range close to the pKa value of phosphate. Due to this pH change, the effective charge of the phosphate ion changes as well (in this example, the effective charge is reduced with increasing temperature).
- The peak shape of the polarizable ions such as nitrite, bromide, and in particular nitrate, is significantly improved at higher temperatures. The reason for this behavior is the reduction of secondary interactions with the stationary phase.
Effects of modifying the eluent composition and strength
Eluent composition and strength can be used to change the elution order of several analytes while using the same separation column. In cation chromatography, a retention model was developed by P.R. Haddad and P.E. Jackson, which allows researchers to predict retention times when changing the eluent composition [1].
Considering that the column remains identical in each determination, no change of ion exchange equilibrium and column capacity is to be expected. Therefore, when changing only the eluent concentration, the following correlation can be used:
Where:
- k’ is the retention factor of the analyte of interest
- c is a constant
- x is the charge of the analyte
- y is the charge of the eluent
- Ey+M is the concentration of the eluent in the mobile phase
If nitric acid is used as the eluent, y = 1, and the model can be simplified to:
Applying this formula to practical situations in the laboratory means the following: with increasing the eluent strength, alkaline earth metals are accelerated much faster (x = 2) in comparison with alkali metals (x = 1), and thus it is possible to elute magnesium before potassium. This effect is called electroselectivity.
Multivalent metal ions are capable of forming complexes with dedicated complexing agents. Therefore, selectivities can be modified by adding complexing agents to the eluent. As an example, dipicolinic acid (DPA) is often used to complex calcium, which leads to a reduction of the effective charge of calcium. As a consequence, the retention time of calcium is reduced and calcium elutes before magnesium in the chromatogram (Figure 3).
The retention of monovalent cations can be influenced by the addition of crown ether to the mobile phase.
Anion systems are more complex regarding the retention time model, although the same electroselectivity effect can be observed to some extent for anions. However, when changing the eluent strength, the eluent pH also frequently changes, leading to different deprotonation equilibria of multivalent anions (e.g., phosphate). This influences the effective charge of the analyte, and by doing so, also influences its retention in a similar way as previously described for the effects of changing temperature.
In some cases, the use of a small amount of an organic modifier such as methanol, acetonitrile, or acetone in the eluent can make sense:
- If bacterial contamination has been an issue before, the addition of 5% methanol to the eluent can help prevent future bacterial growth.
- When samples containing a lot of organic solvent(s) need to be injected and no sample pretreatment such as extraction or matrix elimination (MiPCT-ME) is possible, it is recommended to add a suitable organic modifier to the eluent to ensure that the organic solvent(s) can be properly flushed out of the chromatographic column
Metrohm Inline Sample Preparation (MISP)
- When using IC-MS, it is also recommended to add an organic modifier to the eluent to improve the electrospray process
Be aware that the addition of organic modifiers will also affect the separation selectivities. For the standard anions, the effect is similar to that observed with increased temperatures: the peak shapes of the polarizable ions such as nitrite, bromide, and nitrate are improved.
Organic acids on the other hand may react very differently compared to the standard anions, and their reaction also strongly depends on the type of organic modifier used. Sample chromatograms that show the effect of the organic modifier on retention of analytes are shown in the manual for the Metrosep A Supp 10 column.
Download the Metrosep A Supp 10 column manual below to see example chromatograms showing the effects of organic modifier on analyte retention time.
Reference
[1] Haddad, P. R.; Jackson, P. E. Ion Chromatography: Principles and Applications; Journal of chromatography library; Elsevier; Distributors for the U.S. and Canada, Elsevier Science Pub. Co: Amsterdam, Netherlands; New York: New York, NY, USA, 1990.
