11


Solvent A


Solvent B


Snyder 0


Strength


“weak solvent” “strong solvent” Selectivity group (silica) Heptane


Toluene Ethanol


Ethyl Acetate tBME


Acetone DCM


Acetonitrile


VII II


VIa I


VIa V


VIb


0.22 0.65 0.36 0.32 0.53 0.3


0.52


Table 1: A list of the solvents used in the normal phase Flash screen. For use with basic analytes, additional methods can be selected that incorporate the base diethylamine with the other solvents.


Time (min) or % eluent A % eluent B


~column volume 0 2


17


95 95 5


5 5


95 20 55


Table 3: Reverse phase scouting gradient on a scaling Flash column. Since one minute approximately equals one column volume on the analytical scale, the same gradient method can easily be scaled to any preparative Flash column based on column volumes.


Sample A: This compound was a mixture of an intermediate from a drug substance synthetic route and contained one major desired product and three minor impurities that required purging. The sample was prepared by dissolving 5 mg in 1 mL of dichloromethane, and then analysed using the normal phase screening system.


Normal phase conditions: Column: Luna Silica (2) 50 x 3.0 mm, 3 micron Mobile Phase: See Table 1


Flow rate: 4 mL/min (3.5 mL/min if Toluene is used) Temperature: Ambient Equilibration: see Table 2 Overall screening run time (6 methods): 45 min


Reverse phase conditions:


Column: Biotage C18 250 x 4.6 mm, 60 micron Mobile Phase: See Table 3 Flow rate: 3 mL/min (~ 1 column volume/min, based on Biotage data) Equilibration: 5 min equilibration at starting conditions Temperature: Ambient Eluents: A= Water/TFA (0.1 % vol) or Water/Ammonium Acetate (10 mM) B= Acetonitrile or Methanol


Overall screening run time (4 methods): ~1.5 hours


Results and Discussion


Automated Normal Phase Flash method screening


An automated normal phase method screening system was built using standard HPLC instrumentation. Glajch et al.3


demonstrated that solvents can be categorized using three


Figure 2: An example of how to estimate the isocratic Flash-LC solvent composition by relating the gradient retention time of any particular analyte from it chromatogram to the gradient profile. The report is engineered so that the estimated solvent composition that it predicts results in that analyte having a retention factor under those isocratic conditions that is suitable for a preparative purification process.


Figure 1: Example chromatograms showing the selectivity obtained using different normal phase solvent systems on silica for Sample A - a four component mixture containing impurities 1, 2 and 3.


descriptors (non-localized, base localized and dipole localized). These descriptors led to the classification of solvents for normal phase chromatography into eight selectivity families (Synder et al2


). A comprehensive method


˚


Time (min) % solvent A % solvent B % Ethanol 0


0.4


2.3 2.7


Time (min)


95 50 95 50


0 0


% solvent A 0


100 100


0 0


% solvent B


00 100 0.2 0.3


95


100 5


Table 2: Gradients used for the normal phase Flash screen: on the right, the column conditioning and equilibration gradient; on the left the gradient method used to generate the data.


development screen should ideally include a solvent from each of the eight selectivity families. However, some solvents are unfavourable in practice for reasons relating to safety, the environment or silica incompatibility. The method screening system includes solvents from several of the selectivity families (i.e. I, II, V, Via, VIb and VII) and performs a gradient with a choice of either heptane or toluene as the weak eluent (Tables 1 and 2). For basic analytes, the same methods can be run with basic additive conditions (using a dedicated column) by selecting the appropriate method in the instrument software. The diverse selectivity that can be obtained using different normal phase solvent systems is illustrated in Figure 1 using a typical reaction product (Sample A) from our laboratories. Sample A is an intermediate in an exploratory drug substance synthetic route and is comprised of four components, three of which are undesired impurities (designated 1, 2 and 3) that required purging. Significant selectivity differences can be observed between different solvents systems, leading to beneficial changes in resolution and even to differences in elution order (Figure 1). This approach enables rapid determination of the best solvent choice for optimal selectivity as well as facile


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