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sk five organic chemists for chromatography advice and you’ll get at least six contradictory opinions. Everyone has their own way of doing things.
With that said, I’m proud of my method. It’s designed to get things done in the bare minimum amount of time, without sacrificing resolving power. I’ve yet to find someone who looks forward to their weekly allotment of columns, so I think that’s about the best you can hope for.
For those familiar with flash chromatography, I slurry pack my columns, adding compounds via wet load. I learned the basics from Kyle Finchsigmate (1, 2, 3, 4, without swearing here), the Advanced Practical Organic Chemistry book, and the original Still publication. Today we’ll cover the essentials of loading and running a flash column, moving into the finer details later in the week. If you have an improvement or find something unclear let’s hear it about in the comments.
Setting up the Column
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Begin by measuring out fifteen centimetres (6 inches) of flash silica into an empty column. Pour the silica into a 250mL Erlenmeyer, then add in enough low polarity solvent to cover the silica [1]. With a long glass rod from the glassblower’s shop and a small piece of cotton, lightly plug the end of the column. This plug should have no effect on solvent flow rates, and is simply there to prevent sand/silica from flowing through the stopcock.
With clamps at the top and bottom, anchor the column to a stand or (preferably) the fumehood’s gridwork. Take care to ensure that the column is perfectly vertical; this will be important later. Add in enough coarse sand to cover the bell of the column, then fill the column roughly 5 cm high. Tap the outside of the column with a cork ring to resettle the sand [2]. While swirling the Erlenmeyer flask pour in your silica, then rinse the flask and add any stubborn residue to the top of the column. Put the flask under the column’s spout to collect solvent waste.
With 1-3 PSI of pressure bring down the surface of the solvent until it rests just above the layer of silica (use a pressure adaptor) [3]. If the silica is uneven or slanted correct the angle of the column and then tap the outside near the surface of the silica with a cork ring. In extreme cases you can use the long glass rod to agitate the top 2 cm or so of the silica.
Adding Your Sample
Dissolve your sample in the lowest polarity solvent (mixture) that it is soluble in (ex. 9:1 Hexanes/EtOAc for a EtOAc/Hexanes column). If the compound has only moderate solubility in the column solvent use DCM or toluene, provided the Rf in these solvents stays below 0.3. If those solvents give a high Rf you’ll have to dry load (check back tomorrow).
Using a long Pastuer pipette, slowly add your dissolved compound to the top of the column. To avoid perturbing the silica layer, bring the tip of the pipette as close to the silica layer as possible and rotate around the inner column wall during addition. To prevent the compound from sinking into the silica before it’s evenly dispersed keep the column’s stopcock closed, and start with a thin layer of pure solvent above the silica (1-2 mm). Once all the compound has been added let it absorb onto the silica under simple gravity flow, then rinse the flask and walls of the column twice.
Add a 5 mm layer of sand to the top of the column, then via pipette add solvent until you have a 3-10 cm (1-4 inches) head [4]. Once the solvent is high enough you can simply pore in more, as needed.
Running the Column
Under pressure elute the column at a rate of 5 cm (2 inches) of solvent per minute, collecting fractions every thirty seconds. Your compound should start eluting at around 1/Rf column volumes, where a column volume is height of your silica plug [5]. Spot fractions onto a TLC plate as you fill each test tube, and run plates two at a time in the TLC chamber. Track the elution of your compound(s) with either ultraviolet light or a staining solution.
When your compound has eluted, increase the pressure to ~10 PSI and allow the column to drain dry. As solvent-silica bonds are broken the silica will cool, and when the silica at the bottom of the column is cold (~15 min), silica will pour easily into the solid waste bin.
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[1] By “low polarity solvent” I mean the less polar solvent in your biphasic mixture. If you’re running a hexanes/ethyl acetate column this would be hexanes; dichloromethane/methanol, dichloromethane. The solvent is added early to prevent water from inactivating the silica, and may be overkill.
[2] If your column comes with a coarse glass frit there’s no need for the cotton/sand. The sand is there because in the bell portion of the column compounds traveling along the walls will have farther to travel than those in the middle, decreasing the effective resolution. Fritted columns aren’t as good for DCM/MeOH columns though.
[3] I’ve heard if allowed to settle slowly the silica will form bands of differing density, reducing resolution. I haven’t empirically tested this though.
[4] Larger columns require a higher head, while simultaneously taking significantly more solvent to get there. To speed things up put down a thick sand layer (~1.5 cm) and add solvent via wash bottle.
[5] Ie. One column volume is 15 cm of solvent. This is distinct from “void volume” the amount of solvent contained in the silica. Move of a rule of thumb than an exact measurement.
“pasture pipette”??? Seems that MS Word hates French people, even dead ones!
For my pressure regulator, I took an old Bunsen burner base that had lost it’s ‘smoke stack.’ I put a t-joint in my air line with one out going to the column and the other out going into the Bunsen burner base. adjusting the ‘intake rate’ of the Bunsen burner base sets the bleed rate of the air line and gives good control of the ‘flow rate’ of the column.
Ouch, no kidding. Fixed.
I’ll have to give that a try. The glass regulators are notoriously fragile, and cost more everything else put together.
I think we’ve got one sitting somewhere that’s been patched six times. It’s basically the ship of Theseus.
A poor man’s version of the pressure regulator is to use a 90° hose adapter on the top that’s *not* held in place with a Keck clip. The slight lateral pull of the hose is enough to keep the adapter in the same loosely fit position, which lets you control the air escaping out and so the pressure. You can easily convert the Stille cm/s flow rates into “seconds per X mL fraction” which to me is much easier set of units to deal with, especially if you’ve got a reservoir on top of your column.
What’s your resolution like on your columns? The loading seems like kind of a fat band to me…but if it works it works! How do you folks deal with residual sample left in you vial/RBF/whatever? A second rinse and load? Or just accept the loss of material in exchange for better resolution?
Test tube volume may be an easier method. Originally, I liked cm/min because I could use a permanent marker to measure movement of the solvent. After a while I just got good at eyeballing flow rates.
Resolution is usually ~0.12 Rf, better if I increase the amount of silica/column size. For a DCM wet load (as the pictured one was) I would do a second/third rinse and load; for a Hex/EtOAc it would depend on whether or not I was trying to separate closely aligned spots.
It seems that everyone has pressure line in the labs but here in Belarus we use hand pumps for blood pressure measurement apparatus
First, you push, then you add another portion of solvent and then again push… Push – add – push – add and so on.
By the way, can you please highlight what is “non-flash” chromatography? Is it about pressure in the column?
The closest analogue to flash chromatography would be simple gravity flow. A gravity column uses a different grade of silica, and takes ~3 hours to run properly. I’ve heard the separation can be better, but haven’t done a head-to-head comparison.
More broadly, chromatography is any process with a solid absorbent, impure compound and mobile phase. HPLC, TLC, Prep TLC, size exclusion columns, etc. are all examples of chromatography.
This approach to F.C. is so 1950. Please let your readers know about automated F.C. When I was in graduate school (organic synthesis). I wish BADLY someone had told me about, for example, Isco’s systems- I would have made my grad advisor get one of these systems, or failing that, I would have scraped together my own funds and bought one. After getting my first industry position after grad school, I learned just how much of my life I lost to the tedium of column packing, eluting, and fraction collecting. Arg.
With the proper equipment, you set it up, walk away, come back and pull your fractions and rotovap, e-z p-z. This is the Cadillac: http://www.isco.com/products/products3.asp?PL=101101005
But you can buy simpler, used ones. Old skool F.C. is just way too tedious and unnecessarily so. My advice is learn the theory but don’t ever actually do it by hand.
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