Reflections on lab protocols 1. Cleaning and drying NMR tubes

How do you clean and dry your NMR tubes? Myths abound in chemistry laboratories.

Your definition of "clean" might change depending on whether you re running 100 micrograms of a peptide or natural product (600's left and right) or a concentrated sample of something synthetic on the walkup (400 middle)

Your definition of “clean” might change depending on whether you
are running 100 micrograms of a peptide or natural product (600’s left and right) or a concentrated sample of something synthetic on the walkup (400 middle)

Firstly a bit of a caveat about what I mean by “clean”. A natural product chemist wanting to get maximum possible resolution and best possible lineshape for running spectra on the 100 micrograms of compound they isolated will likely use the best quality tubes they can afford, cleaned with the utmost care. If they are especially well funded, the may well use a brand new tube. If you’re a medicinal chemist collecting data for 1 intermediate in the synthesis of a 50 compound screening library you’re likely a bit less fussy. Sure, your final tested compounds well get the extra treatment so as to have a nice PDF of a proton to stick in your supporting information, and you’ll likely want to minimise residual solvent peaks. But my point is, there is no one set goal here.

There’s been lots of discussion over the years about best practice for cleaning tubes but I’m particularly interested in what comes next: The drying of them. This post is a bit back to front in that I’m going to put my own personal cleaning protocol at the end and jump straight to the drying bit. There was also this paper on bulk cleaning tubes published in OPRD that attracted a lot of attention online. Some for, some against. clearly it struck a nerve. We do this A Lot. Unless you are rich and genuinely do use the econo tubes as disposables (shudder).

So lets assume you have a slightly damp but clean NMR tube. What comes next? What about putting them in the lab oven to dry? Many moons ago, in fact last century, I as an undergraduate (as an Honours student under the Australian system), was told from on high that the three commandments of NMR tube maintenance were:

“Thou shalt not put NMR tubes in the oven, for they will bend! And they shall never spin again!”
“If though must put them in the oven, lay them gently down, so the bending be minimised”
“Thou shalt not steal a lab colleagues clean NMR tubes from the lab oven”

But the budding scientist in me shouted quietly: “Why?, Where’s the evidence?” No evidence or references were ever provided. Is this a laboratory myth, perpetuated through the decades? So just recently I went looking for evidence.

There are several academic labs with guides for NMR users who warn against tube warping/deformation from ovens, for example, Oxford, Temple, UMich, UCSD, and Guelph. Also tube vendors themselves discourage heating: Wilmad, and Norell. Novell say you shouldn’t go above 60°C.

I next put out a call on Twitter for people’s opinions on this and I must thank everyone who responded – see a list below. It seems that in the main people are pretty happy putting them in reasonably hot (~100° C) ovens, although in one case flattening of a tube in a hotter (~120°C) oven has been seen. Most report that laying the tubes down is the preferred option. Low boron glass having a lower melting point was also mentioned.

All in all, I’m prepared to call bunkum on the lab oven issue, I just don’t believe that they will bend under normal lab oven conditions (<100°C). I’ve never seen a tube that couldn’t spin because of it. That’s usually just dirt on the outside from crappy cleaning technique. And besides, nearly all of my high field NMR since about 2000 has been non-spinning anyway. The other frequent problem that I’ve seen in some labs is that nothing in the lab gets stolen faster than clean glassware left in the oven. Just putting your name on a beaker hoping that people will respect it is sadly, often not enough. But of course if you heat tubes up like this over many cleaning cycles, and you have an unusually hot oven, you may eventually see some deformation, but I doubt it. Likelier is that your tube will be lost, broken or stolen long before that eventuality. On the other hand, putting your tubes in the oven may put them at extra risk of being accidentally broken by falling/rolling out if you use the lay down method.


So I use a variety of techniques depending on how urgent my need for a fresh clean dry tube is, and yes, I will use the oven. But mostly I don’t. I should point out that I am not the worlds best example to cite of a chemist doing good laboratory practice. Typically I would have around 50-80 dirty tubes at a time. So after my bulk cleaning method (see below) the bottom line is that I usually let them dry in the fumehood until they’re dry to look at. Then if I’m going to be in the lab for a while and can keep an eye on the oven, I’ll put them in, take them out, let them cool and lock them in my office filing cabinet.

If I’m in a rush I have been known to put a dozen into a vacuum schlenk flask on a manifold line attached to an oil pump. This is a good technique as casual theft is less likely and can get your residual solvent levels on the glass walls down pretty quickly. On other occasions if I’m really pushed for time, a long blunt ended needle carefully attached to a Luer lock line on a nitrogen manifold or better, flexible HPLC polyethylene tubing and a few minutes N2 flow can get the job done, just be careful you don’t launch your tube across the room! Also even using a blunt ended needle like this can scratch tubes which leads to a breakage risk. As someone who has had to clean the probe on the walkup NMR many times due to broken tubes, this is to be avoided!

If you’re still reading and wonder how I do bulk NMR tube cleaning read on. But if all you’re interested in is “Do tubes bend”, then the take-home message is no, at sensible lab oven temperatures, and (maybe) laying them down, they’ll be fine.


Aldrich NMR tube cleaners

There are many ways of cleaning tubes but for what it’s worth here’s my standard protocol. [I also won’t go into details on how to remove stubborn stained tubes with things like fuming nitric or aqua regia either. Your local safety rules might well come into play here as well]. We currently* use these vacuum cleaning devices from Aldrich. Usually model B in a B24 quickfit fitting, attached to a standard 500 mL vacuum filter flask.

They’re a bit expensive but worth it if you have lots of tubes to clean. You stick and NMR tube cap on the rounded end and put them in the vacuum apparatus and pull wash solvent through them. They are a bit breakable however, the small central glass capilliary is prone to snapping off if you drop a tube in under vacuum rather than placing it in.

My normal protocol for cleaning NMR tubes is to first soak them overnight in acetone standing up in the fumehood. The next morning these are quickly drained off and then each tube is rinsed in the above apparatus with about 4 good squeezes of the Nalgene acetone wash bottle, and placed in another clean beaker to the side. After all tubes have been done I then rinse down the outside of all the tubes quickly, just bunched up together to remove any stray drops of sample residue.

I then replace the acetone-filled filter flask with a fresh one and repeat with de-ionised water, same 4 squeezes etc. Unless I’ve been running a lot of peptides I usually skip the step of washing the outsides with water.

I then replace the water-filled filter flask with a fresh one and repeat with acetone. I then remove all the caps from the bottom of the tubes and rinse the NMR tube butts with a little bit of acetone, putting all the rinsings into a beaker, with all the caps (these get another round of washing later).

Brisbane can get damp...

Brisbane can get damp…

And here it gets tricky. you now have pretty clean tubes, but in climates like Brisbane they’re going to get condensation on them pretty fast. But more importantly, it’s time to ask yourself, “how clean is my acetone?” because we use so much of it for cleaning purposes, we often save $$$ by buying Tech grade. This often contains residues that you’d rather not have in your samples. Obviously if you’ve been reasonably thorough in washing your tubes you don’t want to compromise that buy putting other organics back in.

So once again it’s time to ask yourself, “what’s my sample worth?” If the next sample is going to be a medchem intermediate then I’d say you’re nearly done. But if you’re a natural product or peptide/protein person, you’ve got a ways to go. Personally, and this is just me, at this point I put each tube normal way up in a beaker and put a couple of mL of AR grade diethyl ether into each, and quickly drain them, and stand them upside down into the final beaker. This gets pushed to the side in the fumehood and left until the end of the working day. Assuming your fumehood isn’t full of clouds of noxious acids, they’ll be fine.

At the end of the day the clean and pretty much dry NMR tubes are taken out of the hood, in fact, out of the lab, and locked in my office filing cabinet. As required they return to the lab to be used and the cycle starts over. Or as I said above, in urgent cases, vacuum or nitrogen flush methods are useful.

And finally thanks to those who contributed to this conversation on Twitter: @pinkyprincess, @NotHF, @SuperScienceGrl, @BrandonDoughan@sarahdcady@lauravlaeren@tom_wilks@stephengdavey@TimEasun@AttoSci@lgamon@RealTimeChem@timwbergeron@ChemistCraig@volatilechemguy,

*The astute reader will also note that this post is written in a strange mix of past and present tense. Journalistic laziness on my part, or merely the writings of someone who is still coming to terms with no longer being an active researcher?

About martin

almost on holidays
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1 Response to Reflections on lab protocols 1. Cleaning and drying NMR tubes

  1. cfellows65536 says:

    I was told the urban/laboratory myth “they’ll get brittle and shatter more easily”, and so though I have been drying them in a (<100 C) over for 20 years now, I have always felt vaguely guilty about doing it, even though I've been buying them myself for most of that time so its really my own business how long they last. So thanks for setting my mind at rest!

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