6 August / September 2016

West and Lesellier have displayed and plotted the LSER generated results in various forms including a five-dimensional Spider diagram (18). They define the vectors of the Spider diagram as the system constants obtained from LSER Abraham’s parameters e, s, a, b and v. The calculated normalised model results for each stationary phase tested were plotted on the five- dimensional Spider diagram with the various stationary phases placed on the diagram using bubbles of varying sizes depending on the strength of the interactions from the chromatographic system. This Spider diagram can be analysed in many ways including where the stationary phases are positioned between vectors lines, distant from the centre of the diagram as well as the size of the bubble. Columns clustered next to each other on the Spider diagram have similar system constants and are therefore believed to be similar in chromatographic behaviour. Columns distant from each other on the Spider diagram have different system constants and are therefore believed to be different in chromatographic behaviour. The desire to make the screening kit as diverse as possible, to fit a wide variety of samples, entails the selection of columns distant from each other on the Spider diagram; by doing this hopefully different chromatographic behaviour would be seen from each column. Given this approach the Spider diagram is very useful in building the column screen kit. The analysis of the Spider diagram has led to select two of the six columns for the screening kit, GreenSep™ Diol and GreenSep™ PFP. GreenSep™ Diol is between the hydrogen-bond basicity and hydrogen-bond acidity vectors and is not close to the diagram’s centre. GreenSep™ PFP is between the solute dipolarity/ polarisability and excess molar fraction which is related to polarisable π electrons vectors and is not close to the diagram’s centre. We believe that columns associated with the McGowan’s volume vector would not be suitable for the screening kit and therefore columns surrounding this region were not selected for the screening kit.

The articles published by West and Lesellier related to stationary phase behaviour in SFC represent immense effort, dedication to the subject, are extremely through and utilise a sound scientific approach. However, to build a column screening kit for the preparative chromatographer based solely on their very well informed efforts would be to some extent deficient. The QSRR approach doesn’t emphasise Gaussian elution nor does it comment on the symmetry of the eluted analytes peaks. The ideal symmetry

or Gaussian behaviour of the resulting chromatographic peaks is of key importance to preparative SFC separations where the use of mobile additives is particular discouraged.

Designing the Screening Kit Study 2

The column screening kit that is being defined is specifically targeted to the SFC preparative chromatography community and as such the selected columns should produce ideal peak symmetries without the use of mobile phase additives. As a result of this stipulation a second published approach has been selected, the work of McClain and Przybyciel (10), a chemometric approach based on SFC chromatography without mobile phase additives for the separation of structural classes of compounds with a focus on peak symmetry as the key response criteria. The details of the work can be found in the reference; however, it is important to understand how the basic approach of this work informs to the selection of columns for the screening kit

McClain and Przybyciel used a large and structurally diverse building block library available at Merck, USA representing chemical space to obtain representative compounds in four distinct functional group classes – carboxylic acids, amines, alcohols, and amides. These four functional group classes are important reactive groups for the synthesis of larger molecules. Fifteen chemicals were selected from each functional group class for a total 60 chemical entities. The structure of these proprietary compounds, which served as test probes in the study were not disclosed, however a chromatogram and structures of commercially available amines was shown in the paper. In order to identify the 60 chemical entities, the chemical library was queried by chemoinformatic based computer program developed by Merck. This computer program can utilise various chemoinformatic techniques, for the study the Tanimoto dissimilarity (20) was used. The Tanimoto dissimilarity method is a chemoinformatic technique used to query a large chemical library and in this case Tanimoto dissimilarity index was used to identify chemicals that are structurally most diverse from each other thus yielding a molecular diversity model. The Tanimoto dissimilarity index relies on various chemical and physical parameters that are associated with the chemicals in the chemical library such as molecular mass, polar surface

area, hydrogen acceptors, and hydrogen donors to name of few of the parameters. The 15 chemicals representing each of the four chemical classes were chosen to have maximal Tanimoto dissimilarity index in other words they were structural most different from each other. Therefore, it was reasoned that chemical space occupied by the Merck building block library, at that time was represented by the selected test probes.

The referenced study identified four stationary phases, one for each chemical class as the ‘best’ from that study.

Acids - Non-endcapped Ethyl Pyridine Alcohols – Diethyl Amino Propyl (DEAP) Amides – Non-endcapped Nitro phenyl Amines – Non-endcapped GreenSep™ Basic (a bonded imidazole derived phase)

From the McClain and Przybyciel study three columns for the screening kit are selected GreenSep™ Ethyl Pyridine II, GreenSep™ Nitro and GreenSep™ Basic.

The McClain and Przybyciel study provides a novel approach for selecting columns for the preparative SFC separations based on chemical functional group. Fortunately, for that study they had access to a sophisticated computer program and a large chemical library; unfortunately, the use of a propriety computer program and a large propriety chemical library limits access to many preparative SFC chromatographers. When new stationary phases are introduced or multi-functional chemical compounds need to be purified the preparative chromatographer does not have access to this approach. However, there are computer programs available for statistical analysis and many of these commercially programs calculate Tanimoto index, Floersheim distance and various other similarity/dissimilarity factors; maybe these programs can be targeted to the analysis of chromatographic data in conjunction with open source chemical space projects (21), which may make the investigative technique of McClain and Przybyciel more approachable to the general chromatography community.

Designing the Screening Kit Study 3

The published work of Ebinger and Weller (11) provides insight into another pharmaceutically important separation challenge – diastereomers. No specific effort was made in either by West and Lesellier nor McClain and Przybyciel to

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