HPLC & UHPLC Method Improvement
Improvement of an HPLC method involves the adjustment of separation and detection conditions in order to enhance the most essential method’s characteristics without switching to a different HPLC mode. In line with this approach, any validation characteristic such as robustness, specificity, LOD/LOQ, or any commercial characteristic such as cost-effectiveness and labor-effectiveness can be enhanced.
The main directions of an HPLC method improvement are the following:
- enhancing the chromatographic separation;
- reducing asymmetry of chromatographic peaks;
- increasing sensitivity (improving LOD/LOQ);
- improving method’s robustness;
- enhancing method’s throughput (reducing the analysis time);
- adjusting the mobile phase composition to prolong the HPLC packing lifetime;
- addressing sample preparation, and optimizing sample treatment conditions to remove as much of background matrix as possible.
In addition, there are several method improvement tasks, which require introduction of substantial changes into a chromatographic separation conditions:
- converting an ion-pair RP separation into the non-ion-par mixed-mode separation.
- converting a gradient RP separation into the isocratic mixed-mode separation.
- converting non-MS-compatible HPLC methods into MS-compatible ones;
- converting ‘traditional’ HPLC separations into ‘ultra-fast’ UHPLC ones.
Upgrading an HPLC method can be performed by changing the following chromatographic conditions:
- the mobile phase composition;
- column length and diameter;
- adsorbent particle size;
- chemistry of a chromatographic packing;
- flow rate;
- column temperature
- the solvent used as sample diluent;
- injection volume;
- detection technique, and detector settings.
Threshold values for parameters that reflect the progress achieved should be specified in the technical specification.
In case of legislative constraints imposed on variability of chromatographic parameters, which is relevant, for example, for compendial (USP, EP, BP) HPLC methods, the corresponding threshold values should be also mentioned in the technical specification.
Every HPLC method improvement procedure is followed by the method’s specificity verification. Verification procedure is carried out for each of the improved methods; verification protocols constitute a part of the package we report to our customers.