General methods for flash chromatography using disposable columns Special report Marlon Vincent V. Duro McK group meeting July 6, 2015

How I went from this… …to this!

Mol. Divers. 2009, 13,

Flash chromatography “Rapid chromatographic technique for preparative separations with moderate resolution” (J. Org. Chem. 1978, 43, ) Ubiquitous technique within organic chemistry Automated pumps using disposable columns available among different brands (Teledyne Isco, Biotage, Silicycle, Yamazen)

Problems Questions about flow rates and use of gradients How much silica to use? How fast of flow rate? Isocratic, Stepwise or linear gradient? During translation from TLC to flash chrom. Pre-existing guidelines not translated well among technologies Resulting in enormous waste of silica and solvent (and more importantly, time!)

Chromatography theory Tenets Increasing quantities of analyte reduces resolution There exists an optimal flow rate based on silica quality and column geometry More homogeneous phases pack better – give better resolution Stationary phases with smaller particles afford better resolution Flash chrom. is not expected to match the performance of HPLC but the equations can be adapted for optimizing the flow rate for a given column.

Chromatography theory

Optimized flow rates SizeFlow rate (mL/min) IscoRef. 4G G G3525 (25G) 40G40 120G8555 (110G) For normal-phase disposable flash columns (Teledyne Isco RediSep™ Rf micron)

Solvent system choice

Optimization is achieved using multiple analytical TLCs with different ratios of solvents. To achieve appropriate value of R f (optimal retention) with appropriate ΔCV ( optimal selectivity) Figures adapted from Teledyne Isco, Inc.

Sample loading Liquid loading Minimize use of polar solvent (THF, acetone, DCM) For ΔCV = 2 and appropriate R f, sample loading 1:20 (sample:silica) Chase with 1-2 CVs of initial mobile phase. Empty solid loading Silica gel, Celite, cotton (even boiling chips and Kimwipes!) Use 2 to 10-fold excess by mass, recommended 1:40 silica(sample):silica(column) Place into solid load cartridge Pre-packed solid loading Load as a liquid on the cartridge and dry by suction

Sample loading Size Sample load ΔCV <1 (difficult separation, 1:200) 1< ΔCV < 6 (medium separation, 1:100 – 1:20) ΔCV >6 (easy separation, 1:10) 4G20 mg40 mg – 0.2 g0.4 g 12G60 mg120 mg – 0.6 g1.2 g 24G120 mg240 mg – 1.2 g2.4 g 40G200 mg0.4 g – 2 g4 g 120G600 mg1.2 g – 6 g12g For difficult separations, it is better to use Gold columns (20-40 micron spherical particles, column has greater N)

Isocratic elution For two components, a TLC system that affors ΔCV >2 is appropriate for translation to isocratic elution ΔR f = 0.1, the R f values must be between 0.1 and 0.3 ΔR f = 0.2, the R f values must be between 0.1 and 0.45 If R f = 0.5 for upper spot and ΔR f = 0.1 with a mobile phase with X% strong solvent, try using a solvent with X/5 to X/2.

Gradient elution Linear gradients offer better resolution while reducing solvent waste. Samples are likely to be eluted in a state of higher purity. Linear gradient program from Biotage: when analytes show ΔCV >2 with R f values between 0.1 and 0.5 in a system with X% strong solvent Set initial condition as X/4 (or pure weak solvent if X/4 approached zero) Was column with 1-2 CV of initial mixture Run a linear gradient of 10 CV from X/4 to 2X Hold 2X solvent for 1 CV.

Isco’s gradient optimizer tool Utilizing an isocratic “hold” which optimizes the slope of the gradient to maintain resolution A shallower gradient increases separation with the trade-off of band broadening.

Case study Reaction: ΔRf ≈ 0.1 but Δ CV Difficult separation ~100 mg material, used 12G column Liquid loading (in 0.5 mL DCM) target impurity

PeakTrak’s Gradient Optimizer

Take TLC’s and input data

gradien t isocrati c hold

Results Should have used 24G column Another separation technique (SPE) was used to remove amine impurity