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*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 Sizing Chart

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.

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