Scaling-up flash purification – as easy as 1, 2, 3, 4

For many chemists performing bench-scale organic synthesis flash column chromatography is the primary purification technique.  When your synthesis needs scaling to multi-gram levels, so does the flash purification. The logical approach is to just increase the flash cartridge or column size, but this is only part of the solution.

In this post I discuss the process of simplified flash purification scale-up.

Scaling-up flash chromatography separations can seem a daunting task if you haven’t done it before, but in fact it is a fairly straightforward process once you determine the following…

  1. What cartridge size is appropriate for my reaction scale?
  2. What load amount and loading technique should I use?
  3. What gradient should be run?
  4. What flow rate should I use with the larger cartridge?

1. Cartridge size

In its basic form, scaling flash purification is a simple numbers game of maintaining ratios. If you purified 100 mg of a crude mixture on a 10 gram flash column, then a 1 gram reaction mix (10x increase) can be purified on a 100 gram cartridge and 10 grams purified (100x increase) on a 1 kg cartridge, etc. Logical.

However, (there is always a “however,” isn’t there?) maintaining the other small-scale purification parameters such as concentration, loading technique, gradient length and shape, and linear velocity (not flow rate) are essential to scale-up success.

Biotage has developed a useful scale-up table which you can reference for scaling up flash purification, table 1.  The table below helps you determine which cartridge size should be used based on your purification scale need from a 10 gram to a 1500 g. The scale factors in the table also apply to the amount loaded (mass). 

Table 1. Scale-up factors for 10 gram through 1500 gram cartridges

Size (g)1025501003407501500

2. Loading the cartridge

Besides column size, another related area to address is choosing the loading method. 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, if you are scaling up larger mixtures it can be more difficult and even dangerous to perform liquid loading via manual syringe injection with sample spillage, backpressure, and potential precipitation a real concern.

Options then?

  • Load onto the cartridge using an external pumping device like a small peristaltic pump.
    • Using this technique allows you to control how much and how fast your fully dissolved mixture you load onto the cartridge. This is a safe and efficient process but the standard liquid sample loading caveats apply…
      • The mix must be completely soluble in the loading solvent
        • Particulates will wear through the peristaltic pump tubing generating leaks
      • The loading solvent must be chromatographically weak so it does not carry the dissolved compounds down the bed during loading
      • The concentration needs to be sufficiently high to minimize bandspreading during loading
  • 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. At this scale, as with smaller scale, maintaining a sample/sorbent mass ratio of 1:2 to 1:3 will provide the best results. Depending on the liquid volume to be loaded, the use of pre-filled sample loading cartridges can be very advantageous and eliminate the need to pre-mix your liquid sample with loose sorbent.

3. Gradient scale-up

Not only cartridge or column size and load size/technique need to be considered. You also need to address the gradient and flow rate. The gradient scale up is simple if you work in terms of column volumes (CV), which is the internal volume in the column not occupied by sorbent. If you scale up using the same media in larger cartridges then the media’s volume per gram will remain consistent and will scale linearly. So, if you have a 10 gram silica cartridge with a 13 mL CV then 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.

Each gradient segment needs to be scaled using the same number of CV on the larger column as with the smaller. This applies to step gradients as well as linear gradients and isocratic methods.

4.  Flow rates

Unlike cartridge size and gradient length, linear scaling does not hold for flow rates. To maintain the separation performance of the smaller scale flash column, you need to maintain the linear velocity of the solvent moving through the larger column. Linear velocity is determined by your flash cartridges dimensions, CV, and the flow rate; not the amount of media in the vessel. You can calculate the linear velocity if you know the cartridge’s CV, solvent flow rate (FR), and media bed depth (BD). 

Linear velocity = BD/(CV/FR)

  • Linear velocity is expressed in cm/min
  • BD is expressed in cm
  • CV is expressed in mL
  • FR is expressed in mL/min

These scale up guidelines should work with any flash  products. As an example I 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, figure 1, and then scaled it 5-fold.  Using table 2, I found that a 50 gram cartridge running at a linear velocity of 7.36 cm/min (60 mL/min) and following the same gradient profile would achieve my goal (400 mg load), figure 2.  

A 13 CV linear gradient created from TLC data separates 80 mg of a 5 component mixture at 20 mL/min.
Figure 1.  A 13 CV linear gradient created from TLC data separated 80 mg of a 5-component mixture at 20 mL/min on a 10 gram silica cartridge.

Table 2.  10 gram to 50 g cartridge scale-up parameters

10 g cartridge 50 g cartridge
Scale factor 1 5
Media amount (g) 10 50
Column volume (mL) 15 66
Bed depth (cm) 5.5 8.1
Load (mg) 80 400
Flow rate (mL/min) 20 60
Linear velocity (cm/min) 7.33 7.36


Direct scale-up from an 80 mg load on a 10 gram cartridge to a 400 mg load on a 50 gram cartridge. Both the gradient shape and linear velocity were maintained
Figure 2. Direct scale-up from an 80 mg load on a 10 gram cartridge to a 400 mg load on a 50 gram cartridge. Both the gradient shape and linear velocity were maintained as is the separation efficiency.

Remember, for successful flash purification scale-up choose the cartridge with the correct media mass from a scale up table (or do the simple math), maintain the same gradient profile, the same mass load/gram of media (sample concentration), and linear velocity.

Scaling up flash really is as easy as 1, 2, 3, 4.

As always, share your thoughts on flash chromatography or future blog subject matter ideas.


Published by

Bob Bickler

Technical Specialist, Biotage

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