Chromatographers are cautioned to avoid gradient elution when isocratic elution will do. In this work, we compared the analytical properties of gradient and isocratic separations of a sample which can be done quite readily under isocratic conditions. We found that gradient elution gave a shorter overall analysis with similar resolution of the critical pair compared to isocratic elution without sacrificing repeatability in retention time, peak area and peak height or linearity of the calibration curve. We also obtained acceptable repeatability in peak area/height and linearity of calibrations curves for a sample that required gradient elution using a practical baseline subtraction technique. Based on these results and related work which show that columns can be reequilibrated by flushing with less than two column volumes of the initial eluent, we conclude that many of the reasons given to avoid gradient elution deserve serious reconsideration, especially for those samples which are easily separated isocratically. However, we believe isocratic elution will remain preferable when: (1) the sample contains less than 10 weakly retained components (i.e. the last peak elutes with k′ < 5) or (2) the gradient baseline impedes trace analysis.
Bibliographical noteFunding Information:
The authors acknowledge financial support from the National Institutes of Health (grant # 5R01GM054585-09). We also wish to recognize the impact of two very perceptive reviewers whose comments greatly benefited the final manuscript.
- Baseline subtraction
- Gradient elution
- Isocratic elution
- RPLC method development