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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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February 29, 2008

How Not To Do It: Column Chromatography

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Posted by Derek

I was mentioning chromatography last week, and I’ve been running several columns this week myself. I’m doing them the new-fangled-with-sufficient-funds way, which has been the standard in the drug industry for many years. You buy the columns of silica gel pre-packed and plug whatever size you need into a machine. Then you load your sample on, tell it what solvents you want to use, put in a rack of test tubes, hit the button and go do something else.

You can set the machine to collect all the solvent that comes off, or (if your compound absorbs ultraviolet light) to only collect when something UV-active starts coming out the other end. Twenty minutes or so later, you come back to a rack of fractions and a printed map showing which UV-active peaks are in which tubes. All this is very nice, and would have caused me to faint with desire if I’d seen it when I was in grad school – not that I could have, since plug-and-play systems like this weren’t on the market back then.

The standard way was (and is, in less well-funded environments) to slurry up your silica gel in solvent, pour that into a glass column, push the solvent through with a stream of air or nitrogen pressure from the top (usually holding it down by hand to keep things from getting out of control), loading your mixture, and eluting fractions into test tubes or Erlenmeyer flasks until you’re sure that your stuff is all out. I wouldn’t want to guess how many columns I’ve run by hand like that over my lab career, but it’s been several years since my last one, and I don’t see another one in my future, with any luck. (For those of you who want to see how it's done, and have twenty minutes to spare, the folks at MIT will tell you all about it).

It’s hard to mess up the automated systems, although you should never underestimate the ingenuity of the user base. But the hand-run columns can easily be loused up in all sorts of ways. Perhaps the most spectacular I’ve seen was when the guy across the hall from me in grad school, who I’ll call “Bob”, since that was his name, decided to run a big column using DMSO as the solvent.

Most of my chemistry readership will have just looked at the screen and said “He did what?” DMSO is a mighty odd choice for a chromatography column. It’s a strong, strong solvent, for one thing, and would mostly just be expected to dissolve everything and sweep it right out the other end. And it’s thick and viscous, too, compared to the solvents that reasonable people use, which means that it would be no fun to get it to come out that other end at a reasonable rate.

But that was Bob’s choice, and he was working on a bunch of nasty, insoluble stuff, so DMSO seemed like a good idea to him at the time. But he didn’t run his column as I’ve described above. He was of another school of setting up columns – apostates, if you ask me – which advocated packing the silica gel into the column dry and running solvent through it before loading the sample. (That always seemed to take longer and use up more solvent, as far as I could tell).

It was a particularly ill-suited method for running a big honking DMSO column. DMSO, as you’ve probably never had the chance to notice, has rather exothermic solvation behavior with silica. In non-PhD language, it gets very hot, very quickly, when it wets the dry powder. So when Bob started, against all odds and a lot of common sense, to force a big bolus of DMSO down his dry column, things shortly got out of hand. Next door, I heard a big “POW!” and ran over to see what it was this time.

There was Bob, staring with dismay at the remains of his column, which had cracked and spewed DMSO-soaked silica all over the bench. In retrospect, he’s lucky that it didn’t shrapnel all over the place. As it was, he had an awful mess to clean up. I never got around to asking him just what he was going to do with all the DMSO fractions he would have taken off the column - evaporating that stuff off is no joke, although it's another problem that yields to sufficient funding. But, then, if sufficient funds had been available back then, Bob never would have been running a column in DMSO in the first place. . .

Comments (24) + TrackBacks (0) | Category: How Not to Do It


COMMENTS

1. eugene on February 29, 2008 10:02 AM writes...

"He was of another school of setting up columns – apostates, if you ask me – which advocated packing the silica gel into the column dry and running solvent through it before loading the sample."

Yeah, well, it doesn't blow up if you don't use DMSO of course. I really hate the slurry since it's hard to get it off the walls, but the biggest reason is that it looks absolutely disgusting and I have some sort of mild phobia against it. As long as you throw away the first 100 mL. of ether that come off the column (since they are a little wet), you can recycle the subsequent ether until the column is set and you don't up wasting a lot of solvent if you have a giant column. Very little actually.

Anyways, I'm looking forward to working with an automated instrument in industry.

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2. George D. McCallion on February 29, 2008 10:10 AM writes...

As far as automated flash systems, the Teledyne Isco system is the unit of choice (IMHO). Another is the Biotage system, but this cannot go as large a column size (1.5kg) as Isco.

Isco Companion XL, to be exact. The detectors are good, but unless You fine tune them prior to your run, it should not treated as simply, "walk away".

Cheers!

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3. Matt on February 29, 2008 10:54 AM writes...

Let us not forget the 'over pressurization' method of how not to run a column. We often times try to achieve the highest pressures possible while running column chromatography because it speeds up the process and can impact the quality of the separation. However, there is a fine line that one has to walk being that the columns that hold the silica are more often then not made of glass, which can shatter due to the pressure of air/gas being forced into them (as Derek described in his exothermic example with DMSO).

I personally have never had a column shatter or explode on me. I'm a big fan of the hand pump (aspirator bulb attached to a piece of tubing and adapter) because you get a physical readout of how much pressure is in the column everytime you squeeze the bulb to push air through and you're less likely to pressurize a column to the point of shattering through sheer hand strength. Rarely have I heard column explosions happenning because I believe most people use a reasonble pressure, but at current post-doc position I've seen one of the senior grad students blow up 2 columns in the short time that I have been here, with one of the incidents sending him to the emergency room because a large piece of glass lodged itself in his had. He's a very good chemist, but for whatever reason likes to run his columns at really high pressures.

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4. milkshake on February 29, 2008 11:59 AM writes...

ISCO is lovely - we have four machines here in our two labs. We like the slightly cheaper version of the Companion without the touch screen - we control it from a PC. (A cheap desktop with a flat 17 in monitor is only few hundred bucks nowadays.)

The best ISCO-compatible silica columns are from Silicycle - they are even much better than the original ISCO columns. (Luknovia columns are inexpensive but their quality blows)

For big scale purifications (>120g of silica) I would rather pack my own column - is is much cheaper on scale and one does not have to deal with space limitations of the autosampler rack. Also on a big scale-purification one can usually see the band of main fraction eluting, by the changes in refractivity of the eluate.

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5. Squeak on February 29, 2008 3:06 PM writes...

I had a column explode on me about three weeks into my honours year... or maybe it was my undergrad project... so long ago that I don't remember (in fact I forgot about it until I read this post). I don't remember exactly how it happened, but I think maybe I let the silica dry out and then tried to put too much pressure through, but I was very green so I was pretty scared to use very high pressure anyway. I counted my self very lucky that (1) my boss was very understanding and didn't go nuts :) and (2) that the column exploded above my compound and mainly spewed petrol all over me and not too much silica and no glass injuries.

Towards the end of my lab time during my PhD we purchased some aquarium air pumps from a pet shop and rigged them up to our columns. Since we had no house nitrogen on our benches (which was one of a number of mistakes when they designed the labs in our new building - now about 3 years old - like natural gas on the benches but not in the fume hoods and N2 in the hoods but not on the benches... fancy consulting biochemists about lab design for a chemistry lab. Something I don't worry about now in IP thankfully.) this was a easier and cheaper alternative to lugging around N2 cylinders.

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6. SP on February 29, 2008 3:39 PM writes...

I once heard of a grad student who had compounds that could be visualized right on the column (similar to the standard undergrad ferrocene/acetylferrocene separation.) He would do a partial separation on the column but instead of wasting time eluting, he then blew the whole plug of silica back out of the top and sliced it like a sausage to collect the desired compounds for extraction from the gel.

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7. Anonymous on February 29, 2008 4:27 PM writes...

Seems like I remember a technique simlar to the above, whereby, rather than extruding the silica, you use a plastic column and slice it off lengthwise, thus freeing the packing - I honestly think I saw this in a sophomore chem lab text.

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8. Nadia Ahmad on February 29, 2008 5:51 PM writes...

'large piece of glass lodged itself in his had'

Is that 'head' or 'hand' ??

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9. psi*psi on February 29, 2008 9:43 PM writes...

Automated flash systems rock my world. We have a little Analogix that works pretty well (as an academic lab, I guess we're lucky to have anything at all).
I'm so stealing SP's idea. Everything we make is colorful.

Permalink to Comment

10. Ben T on February 29, 2008 10:03 PM writes...

Lucky that Bob wasn't drenched with the eluent when his column exploded, as it would have proceeded to carry anything dissolved in it straight through his skin and into his bloodstream...

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11. pi* on March 1, 2008 8:31 AM writes...

I ran a column once in supercritical diazomethane...can be useful for those hard to get separations

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12. WTF? on March 1, 2008 9:43 PM writes...

Comment Submission Error

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Permalink to Comment

13. Earl E. Riser on March 2, 2008 12:19 PM writes...

Anonymous is thinking of Dry Column Chromatography

http://www.saiadsorbents.com/dcc.htm

Permalink to Comment

14. Norepi on March 2, 2008 1:26 PM writes...

There was a girl in the hood next to me once that was a notorious multi-tasker; she'd be setting up a reaction whilst running a column, and thus she'd have multiple flasks and graduated cylinders of solvents/reagents in her fumehood at any given time...

One day she was running a column in (presumably) EtOAc/Hex, while tending to a reaction that required large amounts of H2SO4.

Shame she had them in identical grad cylinders and wasn't watching what she was grabbing, because she grabbed the wrong one and tried to force a "big bolus" of *98% H2SO4* down her column...

...KABLOOEY!

Most problems I've ever had have been with temperamental flash adapters and Keck clips.

Permalink to Comment

15. tom on March 2, 2008 4:31 PM writes...

Funding is a big problem in my undergrad lab, and the old timer dept chair refuses to let the glassblower blow a RBF on the old gravity columns we have. He says that flash columns offer no seperation advantage and complains "how are you gonna load anything onto the column!?!?!?!"

So I get the honor of taking rubber stoppers and boring holes through them for a compressed air inlet/exit and then stuffing them as tight as they will go into the column.

Permalink to Comment

16. sjb on March 2, 2008 5:06 PM writes...

pi* (11) "I ran a column once in supercritical diazomethane...can be useful for those hard to get separations"

I hope you meant di*chloro*methane, not di*azo*methane - would not like to vac that down on anything approaching scale...

S

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17. A-non-y-mous on March 3, 2008 6:12 AM writes...

I thought I was the only person to ever see someone run a column with DMSO!

When asked how in the world he decided to use DMSO he replied "I got separation on the TLC, so why not?" Removal of the solvent hadn't occurred to him yet. Unfortunately, this was probably the most normal thing he did in grad school.

Permalink to Comment

18. Matt on March 3, 2008 10:40 AM writes...

To #8, Nadia

"had" should have been "hand". I didn't notice the few typos I had in the post until after I submitted, but never thought that "had" could have been extrapolated to 'head' instead of 'hand'. Thanks for asking.

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19. Brooks Moses on March 4, 2008 8:51 PM writes...

You know, over in the mechanical engineering side of the research world, when we're putting high-pressure gasses into things that might go kablooey, we tend to put blast shields around them. :)

Might be an idea worth considering; I don't think it would take more than a lexan tube loose-fitted around the glass one. (Though I suppose then you'll complain -- and rightfully so -- that lexan's a fuel.)

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20. Sili on March 21, 2008 4:55 AM writes...

Yet another reason I never felt like a real chemist - before I just gave up.

I never actually ran a real column. Not least because my labskills sucked so much I rarely got any product.

I did once try to dilute conc. H2SO4 with DMSO - that went BOOM too. The glass survived, though.

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21. Anonymous on May 20, 2008 12:29 PM writes...

Chemistry Sucks

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22. Anonymous on February 5, 2009 8:35 PM writes...

What was bob thinking... tsktsk.

DMSO!!!
and im a first year undergrad in BIOLOGY.

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23. Andre on January 13, 2012 9:04 AM writes...

What's the big fuss with DMSO? I use it in columns to get out all those sticky compounds.. After that is either Rotavapor (yes, using a 300€ high vacuum cheasy pump) or precipitation..

Permalink to Comment

24. SSJadav on July 28, 2012 8:51 AM writes...

My compounds are soluble in DMF/DMSO and i want to purify them, so can i use DMF as solvent with compound for loading into column(DRY Packing).

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