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


  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

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

  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.

  4. Or you could just use CombiFlash or analogs.

  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.

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

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

  7. Hi, nice post. what kind of lighter did you use? since the butane as lighter gas in our lab failed to melt the glassware.....

    1. Regular butane for us. Place the tube near the top of the flame.

    2. Propane also works pretty well