Journal of Chromatography A, 1256 (2012) 253–260 Contents lists available at SciVerse ScienceDirect Journal of Chromatography A jou rn al h om epage: www.elsevier.com/locat e/chroma Innovative green supercritical fluid chromatography development for the determination of polar compounds Amandine Dispas a,∗ , Pierre Lebrun a , Patrick Sassiat b , Eric Ziemons a , Didier Thiébaut b , Jérôme Vial b , Philippe Hubert a a Laboratory of Analytical Chemistry, CIRM, Department of Pharmacy, University of Liège, 1 Avenue de l’Hôpital, B36, B-4000 Liège, Belgium b Laboratoire des Sciences Analytiques, Bioanalytiques et Miniaturisation, CNRS UMR PECSA 7195, ESPCI Paris Tech, 10 Rue Vauquelin, F-75231 Paris 5, France a r t i c l e i n f o Article history: Received 21 May 2012 Received in revised form 13 July 2012 Accepted 16 July 2012 Available online 24 July 2012 Keywords: Supercritical fluid chromatography (SFC) Method development Design of experiments (DoE) Design space (DS) Polar compounds Green analytical chemistry a b s t r a c t In the context of green analytical chemistry, a supercritical fluid chromatography method was developed. In order to prove the potential of this technology, a worst case was selected, i.e. the separation of very polar compounds. For that purpose, an innovative methodology based on design of experiments (DoE) and design space (DS) was previously developed and successfully tested on liquid chromatography. For the first time, this methodology was applied to a supercritical fluid chromatography (SFC) separation. First, a screening design was used to select the stationary phase and the nature of the mobile phase based on a maximization of the number of peaks eluted and a minimization of the number of co-eluted peaks. Then, a central composite design with orthogonal blocks defined a set of experiments used to model the retention times of each peak at the beginning, the apex, and the end. The gradient slope, the isocratic plateau before the gradient, the temperature, and the concentration of trifluoroacetic acid (TFA) in the mobile phase were the potentially influential factors. The critical quality attributes (CQAs), i.e. the separation (S) between peaks of the most critical pair, and the analysis time were the criteria considered to assess the quality of the separation. The DS was computed as the multidimensional subspace where the probability for the separation and analysis time criteria to be within acceptance limits was higher than a defined quality level. The DS was computed propagating the prediction error from the modeled responses to the quality criterion using Monte Carlo simulations. The optimal condition was predicted at a gradient slope of 3.8% min -1 to linearly modify the modifier proportion between 5 and 40%, an isocratic time of 3 min, a concentration of TFA of 25 mM, and a temperature of 60.5 ◦ C. This optimal condition was experimentally tested to confirm the prediction. Furthermore, chromatographic conditions included in the DS and on the limits of the DS were experimentally tested to assess the robustness of the developed SFC method. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Nowadays, a few powerful separation techniques are used by analysts [1,2], such as high performance liquid chromatography, fused-core technology, high temperature liquid chromatography, and the greener chromatographic technique: supercritical fluid chromatography (SFC). Nevertheless, empirical strategy is gener- ally used to develop analytical methods. Pharmaceutical guidelines ICH Q8 (R2) recommend a more systematic approach called quality by design (QbD) [3]. QbD can include prior knowledge, design of experiments, the use of quality risk management, and the use of knowledge management for pharmaceutical processes. Moreover, these guidelines define the design space (DS) as: “the multidimen- sional combination and interaction of input variables (e.g. material ∗ Corresponding author. Tel.: +32 4366 4323; fax: +32 4366 4317. E-mail address: amandine.dispas@ulg.ac.be (A. Dispas). attributes) and process parameters that have been demonstrated to provide assurance of quality”. Thus, the DS is a subspace of the experimental domain in which the assurance of quality has been proved. In the present study, the DS could be defined as the space of chromatographic conditions that will ensure the quality of the separation. As previously described by Lebrun et al. [4,5], the min- imal expected quality might be described by acceptance criteria () that apply to some critical quality attributes (CQAs). CQAs are values providing some indications about the overall achievement of the analytical method: in chromatography [6], CQAs may be the resolution (R crit ) or the separation (S crit ) of a critical pair of peaks, and the run time of the method (t tot ), while the acceptance crite- ria  may be S crit > 0 or t tot < 45 min. In this context, a result given as a predictive probability that the CQAs will be within acceptance intervals establishes the assurance of quality. This leads to a risk- based definition of the DS that may be expressed as: DS = {x 0 ∈  : P(CQAs ∈ |x 0 , data) ≥ } 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.07.043