We investigated and compared the robustness of supercritical fluid chromatography (SFC) separations of the peptide gramicidin, using either isocratic or gradient elution. This was done using design of experiments in a design space of co-solvent fraction, water mass fraction in co-solvent, pressure, and temperature. The density of the eluent (CO₂-MeOH-H₂O) was experimentally determined using a Coriolis mass flow meter to calculate the volumetric flow rate required by the design. For both retention models, the most important factor was the total co-solvent fraction and water mass fraction in co-solvent. Comparing the elution modes, we found that gradient elution was more than three times more robust than isocratic elution. We also observed a relationship between the sensitivity to changes and the gradient steepness and used this to draw general conclusions beyond the studied experimental system.

To test the robustness in a practical context, both the isocratic and gradient separations were transferred to another laboratory. The gradient elution was highly reproducible between laboratories, whereas the isocratic system was not. Using measurements of the actual operational conditions (not the set system conditions), the isocratic deviation was quantitatively explained using the retention model. The findings indicate the benefits of using gradient elution in SFC as well as the importance of measuring the actual operational conditions to be able to explain observed differences between laboratories when conducting method transfer.

我们研究和比较了使用等度洗脱或梯度洗脱的肽短杆菌肽超临界流体色谱（SFC）分离的耐用性。这是通过在共溶剂部分，共溶剂中水的质量分数，压力和温度的设计空间中进行实验设计而完成的。洗脱液的密度（CO ₂ -MeOH-H ₂O）是使用科里奥利质量流量计通过实验确定的，以计算设计所需的体积流速。对于两种保留模型，最重要的因素是助溶剂中的总助溶剂分数和水质量分数。比较洗脱模式，我们发现梯度洗脱的稳定性比等度洗脱高三倍。我们还观察到了变化敏感性与梯度陡度之间的关系，并以此为基础得出了超出所研究的实验系统的一般结论。

为了在实际环境中测试鲁棒性，将等度分离和梯度分离都转移到了另一个实验室。实验室之间的梯度洗脱具有很高的重现性，而等度系统则不是。通过测量实际操作条件（而不是设定的系统条件），使用保留模型定量解释了等度偏差。研究结果表明在SFC中使用梯度洗脱的好处以及测量实际操作条件的重要性，以便能够解释进行方法转移时实验室之间观察到的差异。