TY - JOUR TI - Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode AU - Enmark, Martin AU - Glenne, Emelie AU - Leśko, Marek AU - Langborg Weinmann, Annika AU - Leek, Tomas AU - Kaczmarski, Krzysztof AU - Klarqvist, Magnus AU - Samuelsson, Jörgen AU - Fornstedt, Torgny T2 - Journal of Chromatography A AB - 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. DA - 2018/09/21/ PY - 2018 DO - 10.1016/j.chroma.2018.07.029 DP - ScienceDirect VL - 1568 SP - 177 EP - 187 J2 - J. Chromatogr. A SN - 0021-9673 ST - Investigation of robustness for supercritical fluid chromatography separation of peptides UR - http://www.sciencedirect.com/science/article/pii/S0021967318308719 Y2 - 2018/10/12/13:35:19 KW - Gramicidin KW - Method transfer KW - Peptide KW - Robustness KW - SFC KW - Water ER -