*Columns week, now with math*
Flash chromatography has nowhere near the resolving power of even a poor HPLC column, and none of the automated systems. Flash is dominant in organic labs because it’s fast, adaptable, and cheap. Playing to these strengths, I work off a few rules of thumb, eliding time consuming measurements. The previous caveat about chemists’ opinions still applies.
Step 1. The Columns
The brunt of my flash work is on quantities less than one gram [1], so I asked the department’s glassblower to prepare a standardized set of columns for the lab. Each spans a specific mass range, in which a minimum of 30:1 ratio of silica to sample is maintained [2].
Column A
Load mass: 15mg to 100mg
Diameter: 1.2 cm
Length: 48 cm
Reservoir volume: N/A
Silica Mass: 4.56 g
Test tube: 8 mL (fill 40%)
Column B
Load mass: 100 mg to 300 mg
Diameter: 1.8 cm
Length: 32.5 cm
Reservoir volume: 250 mL
Silica Mass: 11.9 g
Test tube: 8 mL
Column C
Load mass: 250mg to 500mg
Diameter: 2.2 cm
Length: 32.5 cm
Reservoir volume: 250 mL
Silica Mass: 17.3 g
Test tube: 25 mL (fill 40%)
Column D
Load mass: 500 mg to 1.5 g
Diameter: 3.5 cm
Length: 32.5 cm
Reservoir volume: 500 mL (detached in image)
Test tube: 25 mL
Column E
Load Mass: 1.5 g to 2.5 g
Diameter: 4.5 cm
Length: 48 cm
Reservoir Volume: N/A
If I need to purify less than 15 mg I use a Pasteur pipette as the column. To purify more than 2.5 g I base my column selection on this equation:
load mass ≤ π*radius^2 * 15cm * 0.30g/cm^3 / 30 (radius is in cm).
If the RF difference between the compound of interest and its impurities is greater than 0.2 all columns are loaded with 15 cm of silica gel (6 inches). If the spots are closer the silica may be slightly increased to 22.5 cm (9 inches), but in most cases with closely spaced spots it’s better to move to a larger column. Flow rate never varies from 5 cm/min (2 inches/min).
The Pressure
We’re currently using these chemglass adapters to apply pressure (this may change, I like azmanam’s suggestion), with a green keck clamp securing the connection to the column [3]. The teflon plug is used to vary pressure if house air pressure is used, though the setup can be simplified to an inlet adapter if a nitrogen tank is available.
The target flowrate is 5 cm/minute (2 inches/minute), which in most columns will require 1-3 PSI. If a nitrogen tank is available it is the preferred source of pressure [4]. Drying the column post-purification requires approximately 10 PSI.
Note: According to Still, slower flow rates do not have improved separation (due to diffusion throughout the column). When starting out 5 cm/minute feels incredibly fast, but this is flash chromatography. A full column should require about fifteen minutes from first fraction to last.
The Test Tubes
Test tubes are designed to hold about thirty seconds worth of column flow. At an Rf of 0.3 the compound of interest usually elutes around the twelfth test tube, and is complete by the sixteenth [4]. This corresponds to roughly the third column volume, as in this range elution CV ≈ 1/Rf. If the product is still eluting past the sixteenth test tube I’ll usually increase the solvent polarity (tomorrow’s post).
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[1] This is partly due to the nature of my work, and partly because I’ve worked hard to eliminate flash chromatography from the early steps of my syntheses. Extractions and recrystallizations are underrated.
[2] The values below were selected in part due to convenience (ex. these test tubes were available). For an extensive (and somewhat time consuming) approach you can plug your TLC results into this spreadsheet (.xls) and learn the perfect silica/test tube size/etc. values (original publication).
[3] The green clamps seem to be rated to about 15 PSI. A metal keck clamp would be preferred, but I haven’t found a reliable source for them.
[4] At one point I had a dedicated tank for columns and inert gas balloons. The record lifespan was about 13 months.
Chemtips 
If my product has an Rf of 0.3 at 70 Hex 30 Ethyl Ac you say around 12 test tube you get it… that means even if you have impurities on top of it you start the column directly at 70 Hex 30 Ethyl Ac without using a slightly less polar mixture before?
Solvent selection is going to depend on the relative Rfs of my impurities. If the spots are close together I find a constant binary mixture is best, but for wildly divergent Rfs (0.3 vs 0.8) gradients work better.
Gradients are one of the topics I’ll be covering tomorrow. You’re a step ahead of the game right now 🙂
I usually use 5% less polar mixture to be sure impurities won’t come along and keep it constant ’till my product comes. If I have another one I change binary mixture without gradient correpsonding to RF 0.3 of second product. Funny I always thought gradients worked better for close together spots. I’ll be looking forward for your post tomorrow then 🙂
It has been quite a few years, but in Still’s lab we switched from 24/40 joints to ball joints. We had to use clamps, but there was significantly less chance of stuck joints and broken columns. The male half of the ball joint was attached to the air line with a teflon/glass widget for pressure control. We used to get some amazing separations. Really close separations could be visualized by spotting a wide TLC plate alternating early tubes with later, i.e. 10, 20, 11, 19, 12, 18, 13, 17, 14, 16, 15.
I wonder if running so many flash columns somehow impacted my epigenetic markers, leading my 2nd son to work as the Flash character at a theme park one summer. Hmmm???
@John McDonald: Huh.. alternating early and later tubes. Never thought of that. would increase visual resolution. I’ll have to keep that one in mind. I like it.
Yeah alternating can come in handy. When I have 5+ fractions it can be hard to tell if a new compound is eluting or if the spots are just on an angle.
John, how often did you get frozen joints on your columns? I’ve literally never had that happen. When it happens on rotovaps and the like, 98% of the time it’s due to a joint being set while wet or hot+cold glass meeting.
Regarding the source of pressure to push the column, we (here being University of Southampton, UK) use bellows (ie hand-pushed balloon-like pressure reservoirs) or fish pumps (the ones you can find in pet shops for fish tanks). These are fairly cheap and give a good rate of incoming air, without building up too much pressure. Adapted with a swan neck on the column top, they’re doing a very good job.
When it comes to drying, I was told a good tip that always work in my case. Instead of trying to dry off the remains of solvents stuck to the silica (it can be very long), you can add a small volume of diethyl ether on top of the silica, that will remove traces of less volatile solvents and the remains of ether dry very quickly (even with the low flowrate of a fish pump).
That trick with diethyl ether sounds awesome. Waiting for the column to dry is pretty annoying, and I’d love to chop the wait down to a couple of minutes.