Amino (NH2) column: How to keep high performance?
Introduction
Amino columns, also known as NH2 columns, are widely used in both normal-phase and reversed-phase chromatography due to their unique selectivity and versatility. However, their distinct properties also bring challenges, such as declining column efficiency, increased backpressure, retention time shifts, and abnormal peak interferences. These challenges stem from the distinct properties of amino columns, particularly their susceptibility to hydrolysis.
Understanding the best practices for usage and maintenance is key to prolonging column life and ensuring reliable results.
Understanding Amino Columns
The bonded phase of an amino column consists of aminopropyl functional groups. Unlike conventional reversed-phase columns such as C18, C8, or C4, amino groups are more prone to hydrolysis, leading to a shorter lifespan. Proper handling and maintenance strategies can mitigate these issues and extend the column's usability.
Amino columns can function in both normal-phase and reversed-phase chromatography. However, since solvents in these modes are not mutually soluble, an appropriate transition solvent (such as isopropanol) should be used when switching between them. Below are essential guidelines for optimizing amino column performance in both modes.
Reversed-Phase Mode
In reversed-phase mode, amino columns are particularly sensitive to pH and water content in the mobile phase. Hydrolysis is accelerated under acidic conditions (pH < 3) or when the water content is high. To mitigate this, the ideal pH range for the mobile phase should be maintained between 3 and 7.
After analysis, the column should be flushed and stored appropriately. If the mobile phase contains no acid or salt, flushing with 100% acetonitrile is sufficient. However, if salts are present, first flush with a 60% acetonitrile in water for 40 minutes, then switch to 100% acetonitrile, flush again before storage.
In cases of high backpressure, peak shape abnormalities, or reduced column efficiency, flush the column sequentially at the standard flow rate using the following solvents: 100% acetonitrile → 100% methanol → 100% isopropanol → 100% acetonitrile, each for 30 minutes. Reduce flow rates during isopropanol flushing due to its higher viscosity.
Normal-Phase Mode
Post-analysis cleaning in normal-phase mode starts with flushing with 100% hexane, followed by storage in a 90/10 hexane/isopropanol mixture.
In normal-phase chromatography, the water content in both the stationary and mobile phases plays a crucial role in retention time stability and separation performance. Fluctuations in retention times are often attributable to variations in water content. To address this, the column can be dehydrated using a solution of 2.5% dimethoxypropane and 2.5% glacial acetic acid in hexane, flushed at 30 column volumes.
For mobile phase preparation, a "half-saturated" approach is recommended. This involves mixing anhydrous non-polar solvent with a water-saturated portion, stirring for an hour, and removing excess water before recombining the two phases. This ensures consistent water content and improves reproducibility.
If column performance declines, a cleaning sequence with 100% isopropanol → 100% methanol → 100% isopropanol (each for 40 minutes) can restore functionality. Again, the flow rate should be adjusted for isopropanol due to its viscosity.
Special Considerations for Lactose Analysis Columns
Xtimate® Lactose-NH2 is a specialized amino column designed for lactose analysis, of which maintenance protocols differ. These columns should be flushed and stored with 100% acetonitrile. If performance issues arise, sequentially flush with 100% methanol → 100% acetonitrile → 100% isopropanol → 100% acetonitrile, each for 3 hours.
To ensure optimal performance with lactose-specific columns, additional precautions are necessary.
- The mobile phase should be thoroughly mixed, vacuum-filtered, and degassed by sonication for 10 minutes.
- Avoid inline filters and use a single mobile phase channel to prevent fluctuations.
- Maintain injection volumes at 10 μL, as larger volumes increase peak broadening. Switching to a 10 μL loop minimizes extra-column dead volume.
- Additionally, since refractive index detectors (RIDs) are sensitive to temperature fluctuations, the column compartment should be insulated with dry materials like cotton or towels to stabilize the baseline.
Conclusion
The aminopropyl functional group’s polarity and susceptibility to hydrolysis make amino columns more challenging to maintain than conventional phases. However, by adhering to proper usage guidelines—such as controlling pH, managing water content, and implementing rigorous cleaning protocols—users can significantly extend the lifespan of these columns and maintain their high performance. Whether in reversed-phase, normal-phase, or specialized applications, understanding and addressing the unique needs of amino columns is key to achieving reliable and reproducible results.