Monday, January 14, 2019

Technique primer: Test tube columns

As many of you remember, a little over a year ago Phil did a AMA on reddit. On that AMA, a bunch of people requested that we do a post on how to build/run disposable test tube columns:

Well, writing up this blog post somehow slipped through the cracks for a year (Sorry!!), but without further delay, here's how we do the technique.

Step 1: Building the glassware. 

We use these columns on crude reaction mixtures from ~10 to ~500 mg scale. For smaller scale than this, we use prep TLC; larger, just a standard reusable column.

To build the column, for 10-50 mg, we just use a Pasteur pipette as the column. For 50-200 mg, we build it out of a 16 X 150mm tube. For 200-500 mg, we build it out of a 25 X 150 mm tube.

For the latter two, the first step is to pull a bottom spout from the test tube. Basically, you heat the tube with a Benzomatic torch, until you see the bottom glow orange and "wilt" slightly. At this point, you grab the glass with a pair of tweezers and steadily pull to make a long stem. It's hard to explain, but easy to demonstrate:

It's a bit tricky the first time, but gets simple with practice.

After letting the tube cool to rt, snapping off the bulk of the stem gets you a nice looking column:

(If you're using a Pasteur pipette, you get to skip to this stage)

Step 2: Building/running the column.

From here, it's just a matter of packing a small chunk of Kimwipe into the bottom, and tamping it down, followed by adding a bit of sand, and then silica gel. With modern silica gel, we have found that even <3" column height is still plenty to do reasonable separations. After equilibrating with a column volume or two of solvent, here's what it looks like, ready to load and use as usual:


One nice thing about the column sizes is that the 16mm tube (loosely) accommodates a 14/20 inlet adapter, while the 25mm tube (loosely) takes a 24/40 adapter. This way, you can run it with positive air pressure exactly as you would any other flash column. This is a 16mm tube, and as you can see, the small inlet adapter fits it nicely:


When the column is finished, here at Scripps, our EH&S allows us to dispose of the whole thing, tube and all, into our silica waste stream. Check with your EH&S for what to do at your institution.

Regardless, the best part of this technique is there are no dishes to wash!

Hope that this helps aspiring chromatographers,

Baran Lab


7 comments:

  1. An extra piece of advice - when pulling the bottom of the test tube with tweezers or tongs, it's best to also heat the end of the tweezer/tong alongside the test tube. When I've done this in the past and haven't heated the tong, the glass cools too quickly upon contact and you can't get a nice pull to make a usable column. Just my two cents

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  2. and.....what kind of SiO2 do you use?

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  3. We use Acros brand, "Silica gel for chromatography, 0.035-0.070 mm, 60A"

    At other times depending on what's on sale, we have also used the analogous product from Merck KGaA and in our hands they perform the same.

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  4. Or you could just use CombiFlash or analogs.

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  5. One modification that I would suggest for pipette columns is to not use a positive pressure of air/nitrogen or what have you, it's too easy to dry out the silica if you're not careful, or cause an alarming buildup of pressure. Instead, put a SubaSeal on the back end (may need a little parafilm to seal), then poke a long needle through to feed solvent from a syringe, which will also create enough positive pressure to run the column at a reasonable rate (which you can easily control with your hand). I am sure this is what a lot of people already do and I learned it from a few people, but for anyone looking to try out pipette columns it is much better and way less hassle. Also in my opinion far superior to prep TLC.

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  6. Which brand of test tubes do you use? Cheaper test tubes made of limestone glass can not withstand such heating. Is it pyrex?

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    1. Yeah, we use borosilicate (Pyrex) tubes. Part number: 47729-528 from VWR.

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