How to Scale-Up Normal-Phase Purification

For chemists performing bench-scale organic synthesis, flash column chromatography is often the primary purification technique.  When synthesis needs scaling to multi-gram levels, so does the flash purification. The logical approach is to increase the flash cartridge or column size, but this is only part of the solution. In this article we discuss the process of simplifying flash purification scale-up.

Scaling-up flash chromatography can seem a daunting task but it is a fairly straightforward process once the following parameters are determined:

-What cartridge size is appropriate for the reaction scale?

1. -What load amount and loading technique is applicable?

2. -What gradient should be run?

3. -What flow rate should be used with a larger cartridge?

1. Cartridge size

In its basic form, scaling flash purification is a numbers game, one of maintaining ratios. If 100 mg of a crude mixture has been purified on a 10 gram flash column, then a 1 gram reaction mix (i.e. a 10-fold increase) can be purified on a 100 gram cartridge and 10 grams purified (100x increase) on a 1 kg cartridge.

However, maintaining other purification parameters such as concentration, loading technique, gradient length and shape, and linear velocity (not flow rate) are also essential to scale-up success.

Included in an article published on our popular flash chromatography blog, is a handy scale-up table which you can reference for scale-up.  The table helps determine which cartridge size should be used based on your purification scale need from a 10 gram to a 1500 g. 

2. Loading amount and Technique

At small scale a liquid injection is often performed as the volume is small enough to inject with a disposable syringe. If the components to be separated are poorly soluble, then reaction mixes can easily be blended with and pre-adsorbed onto small volumes of an inert media for dry loading.

However, scaling-up larger mixtures can be more difficult and even dangerous to perform liquid loading via manual syringe injection with sample spillage, backpressure, and potential precipitation could be a real concern. There are options:

(1) Load onto the cartridge using an external pumping device like a small peristaltic pump.  (2) Use a dry loading method by pre-absorbing the liquid reaction mix onto an inert solid support followed by solvent evaporation. This technique adds some time to the purification process but will eliminate the concerns associated with liquid loading in the early development phases.  Development of dry-loading methods is supported for all our development scale cartridges.  Once at pilot or clinical (Kg) scale, dry loading methods are often designed out and swapped for liquid loading methods to then reduce further the risk of potentially harmful loose silica or API product contact by process operators.

3. What gradient for scale-up?

Gradient scale up is simple when considered in terms of column volumes (CV), which is the internal volume in the column not occupied by stationary phase. Scale-up using the same media in larger cartridges means the media’s volume per gram will remain consistent and will scale linearly. So, a 10 gram silica cartridge with a 13 mL CV, a 100 gram cartridge will have a CV of 130 mL and a 1000 gram cartridge will have a 1300 mL CV, and so on.  This means a linear gradient method using 10 CV on a 10 gram flash chromatography cartridge will use 10 CV on any other flash purification cartridge packed with the same media.
 

4.  Flow rates

Linear scaling does not hold for flow rates. To maintain the separation performance of the smaller scale flash column, the linear velocity of the solvent moving through the larger column must be maintained.  Linear velocity (in cm/min) is determined by the flash cartridge bed dimensions (BD), column volume (CV), and the flow rate (FR). 

Linear velocity = BD/(CV/FR)

BD is expressed in cm
CV is expressed in mL
FR is expressed in mL/min

These scale-up guidelines work with any flash technique.  In our quick example, we used the Isolera™ TLC to linear gradient tool to determine the gradient conditions and sample size for a 10 gram Biotage® SNAP KP-Sil cartridge, and then scaled it 5-fold to 50g.  A 50 gram cartridge running at a linear velocity of 7.36 cm/min (60 mL/min) and following the same gradient profile achieved the goal (400 mg load).  

Now that you've mastered normal-phase, continue on to read about how to use Scale-up in Reversed-phase applications:

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