specifically address the specific separation of diastereomers. Ebinger and Weller evaluated the separation of 33 synthetic research samples representing a diverse set of diastereomers mixtures against 12 different columns from various vendors. From this study they discovered that 91% of their diastereomers mixtures from their diverse set could be separated using a bonded pyrene stationary phase. They postulate that the good separation performance of the pyrene phase for diastereomer mixtures is attributable to the rigid planar pyrene ring, strong π-π and charge transfer interactions.

The commercial development of a pyrene bonded phase has been explored (ES Industries) however the phase was found to be unstable. A Naphthalene bonded phase - GreenSep™ Naphthyl was developed which contains many of the properties of the pyrene phase including rigid planar ring, strong π-π and charge transfer interactions, however it is more stable. GreenSep™ Naphthyl has been included in the screening kit as the sixth column.

Separation Examples Using the Column Screening Kit

The stationary phases selected for the current screening kit are shown in Table 2 and separations using the screening kit are shown in the examples that follow.

Table 2: The Six Columns Selected for the SFC Screening Kit

1.GreenSep™ Basic – imidazole based, best peak shape for amines

2. GreenSep™ Ethyl Pyridine – Good overall selectivity and excellent for acid mixtures

3. GreenSep PFP™ – pentafluoro phenyl, unique selectivity, electron acceptor

4. GreenSep Nitro™ – nitro aromatic based, unique selectivity

5. GreenSep Napthyl™ – naphthalene based, ridged structure, good for diastereomers separation and non-polar compounds, π-π interaction

6. GreenSep Diol™ – the selectivity of silica without reactivity of silica

These examples were chromatographed using the newly introduced Shimadzu Nexera UC SFC system. The Shimadzu Nexera UC system had a fixed wavelength UV detector and a 5 µL fixed injection loop. The operating conditions for the examples are contained in the figures.

Figure 2: Separation of Caffeine Analogue Mixture on GreenSep™ Basic

differentiated by methyl group number and placement. The caffeine mixture was chromatographed on all six screening kit columns using a simple mobile phase consisting of an isocratic mixture of methanol and carbon dioxide with no mobile additive. The only column in the screening kit that completely baseline separated the caffeine mixture was GreenSep™ Basic, the chromatogram shown in Figure 2. The next best column for this separation was GreenSep™ Ethyl pyridine, the chromatogram shown in Figure 3. GreenSep™ Ethyl pyridine did separate the mixture but not a baseline separation.

Figure 1: Caffeine Analogue Mixture Structures

The first example is a comparative example to illustrate the chromatographic selectivity of the screening kit for the separation of closely related caffeine analogues (Figure 1).

These analogues are structurally similar heterocyclic xanthine bases and are

All other columns in the screening kit failed to separate the caffeine mixture. We also analysed the caffeine mixture on GreenSep™ Silica (bare silica), the chromatogram is shown in Figure 4. Bare silica has been widely used as a stationary phase for SFC (5), but was not included in our screening kit because it exhibits severe tailing for amines without mobile phase additives. GreenSep™ Silica did not completely separate the caffeine mixture (caffeine and theophylline were completely eluted).

Figure 3: Separation of Caffeine Analogue Mixture on GreenSep™ Ethyl Pyridine

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