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5


Basic Guidelines for the Column Screening Kit


Defining a column screening kit for SFC is a difficult task given the aforementioned wide variety of SFC stationary phases available and the complexity of mixtures requiring separation. In addition, the screening kit will be used to guide preparative separations and as such it needs to be receptive to the requirements of preparative SFC chromatography. Firstly, the columns should be commercially available and manufactured using robust support materials, refined chemical bonding procedures, represent stable bonded phases and high performance column packing technology. Secondly, the columns should be engineered to endure the high pressure regime of both analytical and preparative SFC. Finally, any stationary phase chemistry identified for the screening kit must be scalable to larger column formats and different particle sizes. There have been SFC stationary phase optimised for analytical column dimensions and unfortunately can’t be easily scaled to accommodate larger column formats in an economical fashion. The phases reviewed (GreenSep™, ES Industries, NJ) have all been commercially developed and optimised for SFC and are completely scalable from analytical formats through all sizes of preparative columns. GreenSep™ versions of the historical phases such as diol, cyano and amino are also available.


The Approach to Screening Kit Design


The scientific approach that was developed to build the screening kit is based upon three published articles (9 - 11). These articles have helped to define and quantify how analytes interact with various stationary phases in SFC separations. Each one of the referenced studies informed and has directly influenced the columns selected for the kit. The referenced studies have to a large degree utilised various chemometric based approaches to analyse and postulate how different stationary phases interact with analytes in an SFC regime.


Designing the Screening Kit Study 1


West and Lesellier have published several papers (9, 12-18) to characterise available types of stationary phases and their potential use for a particular SFC separation. In these papers they compare stationary phases using a quantitative structure- retention relationship (QSRR) based on the


linear solvation energy relationship (LSER) that uses Abraham’s parameters as the solvation parameter model. In other words the retention factor (k) of a selected set of probes is experimentally determined using a set of careful chosen operating conditions (9). The log of the experimentally measured retention factor (k) is then related to specific interactions by the following equation:


Log k = c + eE + sS + aA + bB + vV


Log k the log of the measured retention factor c


the intercept term of the model, which is this case is dominated by the phase ratio


E


excess molar fraction as calculated from the refractive index and is related to polarisability contributions from n and π electrons


S solute dipolarity/polarisability


A & B solute overall hydrogen-bond acidity and basicity


V


McGowan characteristic volume ((cm3


/mole)/100)


(e, s, a, b, v are the system constants of LSER Abraham’s parameters calculated from the multi-linear regression analysis of the data)


Table 1 is a representative subset of the chemical probes used in West and Lesellier studies and is included to explain how the terms of the LSER model relate to actual molecules (the probes).


From Table 1 benzene, toluene and ethyl benzene, are small neutral non polycyclic aromatic hydrocarbons that have very low hydrogen bond acidity or basicity, weak polarisable contributions from n


and π electrons, weak solute dipolarity/ polarisability and a small McGowan characteristic volume (the molecular volume of one mole of a compound when the molecules are stationary divided by 100, basically, the molecular space occupied by one molecule, it is calculated from McGowan’s work (19)). While on the other hand, both pyrene and perylene are larger neutral polycyclic aromatic hydrocarbons that have very low hydrogen bond acidity or basicity, strong polarisable contributions from n and π electrons, strong solute dipolarity/polarisability and a large McGowan characteristic volume. Polar molecules that are hydrogen bond acceptors such as pyridine and caffeine have strong hydrogen-bond basicity. Pyridine is also a small molecule similar to benzene with weak polarisable contributions from n and π electrons and a small McGowan characteristic volume. Polar molecules that are hydrogen bond donor such as the phenols have strong hydrogen-bond acidity. They used a total of 109 test probes from their study (9) and acquired data from a large number of commercially available columns including classic HPLC stationary phases such as ODS (Octadecylsilane), PFP (Pentafluorophenyl) and Diol as well as stationary phases specifically designed for SFC such as EP (ethyl pyridine). All 109 test probes were tested on each column and the retention factor (k) was measured. Using the measured value of k for each test probe on each column, a LSER Abraham’s parameters solvation model was generated using multi- linear regression analysis.


Table 1: Subset of Chromatographic solutes and LSER descriptors used by West and Lesellier (8)


Compound Benzene Toluene


Ethylbenzene Pyridine Caffeine Phenol


Coumarine Resorcinol


Phloroglucinol o-Chlorophenol m-Chlorophenol p-Chlorophenol o-Nitrophenol m-Nitrophenol p-Nitrophenol Pyrene


Perylene E


0.610 0.601 0.613 0.631 1.500 0.805 1.060 0.980 1.355 0.853 0.909 0.915 1.045 1.050 1.070 2.808 3.256


S


0.52 0.52 0.51 0.84 1.60 0.89 1.79 1.00 1.12 0.88 1.06 1.08 1.05 1.57 1.72 1.71 1.76


A


0.00 0.00 0.00 0.00 0.00 0.60 0.00 1.10 1.40 0.32 0.69 0.67 0.05 0.79 0.82 0.00 0.00


B


0.14 0.14 0.15 0.52 1.35 0.30 0.46 0.58 0.82 0.31 0.15 0.20 0.37 0.23 0.26 0.29 0.44


V


0.7164 0.8573 0.9982 0.6753 1.3630 0.7751 1.0620 0.8340 0.8925 0.8975 0.8975 0.8975 0.9490 0.9490 0.9490 1.5850 1.9536


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