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[Home](https://www.sigmaaldrich.com/KR/en)[Solid Phase Extraction (SPE)](https://www.sigmaaldrich.com/KR/en/applications/analytical-chemistry/sample-preparation/solid-phase-extraction)Ion Exchange Methodology
# Ion Exchange Methodology
In order for electrostatic retention to occur, both analyte and sorbent functional groups must be in their ionizedIon Exchange Methodology form. This is done through strict pH control of the sample matrix. For basic analytes, the pH should be adjusted to at least 2 pH units below the molecule’s pKa. For acidic analytes, the pH should be adjusted to at least 2 pH units above the molecule’s pKa.
To elute, the opposite is true. By adjusting the pH of the eluant to at least two pH units above or below the analytes’ and/or sorbent’s pKa, one can effectively neutralize one or both functional groups disrupting the electrostatic interaction allowing for elution to occur.
Note: Because the kinetic exchange processes between sample and sorbent functional groups are considerably slower for ion-exchange than for normal and reversed-phase, flow rates should be drop wise (~1 drop/second). One may also need to increase elution and wash volumes, allowing sufficient residence time for the mobile phase and stationary phase to interact.
__Figure 1.__Ion Exchange Methodology
Retention Mechanism:Electrostatic attraction of charged functional groups of the analyte(s) to oppositely charged functional groups on the sorbent. Combination of reversed-phase and ion-exchange for mixed-mode
Sample Matrix:
Aqueous or organic samples of low salt concentration (< 0.1M)
- Biological fluids
- Solution phase synthesis reactions
Analyte Characteristics:
Analytes exhibiting non-polar functionalities
- Use anion-exchange for isolating acidic compounds: carboxylic acids, sulphonic acids, and phosphates
- Use cation-exchange for isolating basic compounds: primary, secondary, tertiary, and quarternary amines
Elution Scheme:
Electrostatic interactions disrupted via:
- pH modification to neutralize compound and/or sorbent functional groups
- Increase salt concentration (> 1M); or use a more selective counter-ion to compete for ion-exchange binding sites
Common Applications:
- Drugs of abuse and pharmaceutical compounds in biological fluids
- Fatty acids removal in food/agricultural samples
- Cleanup of synthetic reactions
- Organic acids from urine
- Herbicides in soil
Table 1.Ion-Exchange & Mixed-Mode SPE
## [](https://www.sigmaaldrich.com)Counter Ion Selectivity & Ion Exchange:
Counter ion selectivity is defined as the degree to which a counter ion is capable of competing with other counter ions for the functional group of an ion exchanger sorbent. Retention is facilitated by having a sorbent and/or sample matrix preequilibrated with a counter-ion that is less selective than the analyte functional group (minimum competition). Analyte elution is facilitated by using buffers with counter-ions more selective than analyte functional group.
For Cation Exchangers:
- Ca2+ > Mg2+ > K+ > Mn2+ > RNH3 2+ > NH4+ > Na+ > H+ > Li+
For Anion Exchangers:
- Benzene Sulphonate > Citrate > HSO4- > NO3- > HSO3- > NO2- > Cl- > HCO3- > HPO4- > Formate > Acetate > Propionate > F- > OH-
To change to a higher selective ion, pass 2-5 bed volumes of 1N solution of the new counter ion through sorbent. To change to a lower selective ion, pass 5-65 bed volumes of 1N solution of the new counter ion through sorbent.
__Note__: Number of bed volumes dependent on how much less selective the new counter ion is than the present one on the sorbent.
## [](https://www.sigmaaldrich.com)Basic Steps
1. __Sample Pre-treatment__ Salt concentration should be less than 0.1M. Dilute sample 1:1 with buffer of appropriate pH to ensure analyte functional groups are ionized.
Examples:
- Basic compounds: dilute with 10-25 mM buffer (e.g., potassium phosphate or ammonium acetate), pH 3-6
- Acidic compounds: dilute with 10-50 mM buffer (e.g., acetate buffer), pH 7-9
For interference laden samples (e.g., biological fluids) containing varying levels of salt concentration, use mixed-mode SPE technology.
2. __Conditioning/Equilibration__ If samples are in a non-polar solvent, the same solvent should be used to condition the SPE device. For aqueous samples, condition with 1-2 tube volumes of methanol or acetonitrile. Equilibrate with buffer similar/identical in pH and salt concentration to buffer used sample pre-treatment.
3. __Sample Load__ Apply sample (from step 1) at a consistent and reduced flow rate of ~1-2 drops/second to ensure optimal retention. Mass transfer kinetics of ion-exchange SPE are slower than reversed-phase and normal-phase. Reduced flow rate is critical for consistent recovery.
4. __Wash__ Adequate control of pH and ionic strength should be maintained to prevent premature elution of the analytes of interest. Use buffer of appropriate pH (e.g. buffer used in sample pre-treatment) to remove polar interferences. More hydrophobic interferences can be removed using up to 100% methanol diluted in sample pre-treatment buffer.
5. __Elution__ Elute at a consistent and reduced flow rate of ~1-2 drops/second to ensure optimal compound desorption. The most common elution strategy is by pH manipulation. Also, most ion-exchangers exhibit some mixed-mode behavior. Addition of organic modifier is necessary to disrupt secondary reversed-phase interactions.
Examples:
- Basic compounds: elute with 2-5% ammonium hydroxide in 50-100% methanol
- Acidic compounds: elute with 2-5% acetic acid in 50-100% methanol.
Other elution strategies:
- Use an SPE eluate of higher salt concentration (> 1M)
- Use a more selective counter-ion to compete for ion-exchange binding sites
6. __Eluate Post-treatment__ A number of elution strategies are available. Various elution strategies should be tested and optimized to minimize eluate post-treatment.
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- [Analysis of Drugs of Abuse in Urine](https://www.sigmaaldrich.com/KR/en/technical-documents/technical-article/analytical-chemistry/solid-phase-extraction/analysis-of-drugs-of-abuse-in-urine-after-cleanup-with-hlb)
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