The Robots are Coming

Posting on Curly Arrow is already a very erratic affair and things will not be improving for a while. There is less than two months til the big move back to Denmark. I'm experiencing total organisational chaos and trying desperately to obtain some results in the lab before it is too late. So please be patient.

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I'm sure that the following paper from Journal of Medicinal Chemistry is great (I haven't read it). The title is certainly great.

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Designing Active Template Molecules by Combining Computational De Novo Design and Human Chemist's Expertise

Eric-Wubbo Lameijer, Reynier A. Tromp, Ronald F. Spanjersberg, Johannes Brussee, and Adriaan P. IJzerman

J. Med. Chem., 2007, 50, 1925-1932

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D!

The Invisible Phase Boundary

All organic chemists have experienced it. Your reaction is done and your transfer it to a separatory funnel with an aqueous and an organic phase. You do your shaky, shaky bit and the phases separate but it is impossible to tell where the phase boundary is. The problem is most commonly encountered when very dark phases are obtained but it can also occur with perfectly clear phases (see picture). The solution to this problem is very simple. Simply tip a clean NMR tube cap into the separatory funnel and observe as it settles on top of the bottom phase. Problem solved. D!

Dry Column Vacuum Chromatography (DCVC) - revisited

I get a lot of questions about Dry Column Vacuum Chromatography (DCVC) so I believe it is time for another post on the topic. DCVC is really taking off and the paper now has >70 journal citations.

Firstly, let me reiterate that like most things in life DCVC is best learnt by doing. Some of the questions I get are very detailed and specific and I can't provide clear cut answers. With experience you'll have an idea what to do and you'll improvise along the way and get it right.

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Q&A session with the true believers:

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(1) ...did you know that your paper had the"honor" of being "html-ed" by Rhodium....

- I didn't know that. I guess that everyone is purifying their illicit drugs in the garden shed using DCVC these days.

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(2) ...searching for a good source for the silica gel, right now it is about 4-5 times more expensive than our flash gel source...

- That is a huge difference. You have a very sweet deal on flash silica. In my case it costs about 40 % more but considering how little you use compared to flash it works out as a big saving. If you find a cheap source of DCVC silica please let us know.

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3) ...In the comments, you mentioned that you have done DCVC with 50-100 mg, and I was wondering what size of fritted funnel (frit diameter) you would use for that. Just calculating the size using the approximate amount of silica gel gives me something ridiculously small...

- The smallest sinter I use has a diameter of 1 cm. I have run 20 mg columns with 5 ml fractions on this column with no problems. Smaller than that would be impractical.

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(4) ...Also, just to help me chose the size [Column] if I'd get one or two funnels made, what diameter(s) would you use for say 500 mg to 5 g?...

- I currently have the following four columns: 1, 4, 6 and 8 cm. My favourite (that I use 80% of the time) is 4 cm. It's a good size to work with and it's good for 20 ml fractions. I'll do anything from 100 mg and up to 5 grams or more (depending on the separation) on that column. For your requirements I'd say get a 4 and 6 cm column.

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(5) ...frit porosities - the notation 1 - 4 is what is used in Europe, here in the US they have C, M and F, which actually don't directly correspond to the 1-4 sizes. "3" is a size where there is no direct equivalent, which is too bad because it seems ideal also to have in a flash column. The actual numbers are below, and I was a bit surprised because it seems that the P3 should clog up with time when using the Merck 15111 silica gel with its size range of 15-40 micron.

American standards - (Kimble & Corning, ASTM) nominal pore size, in microns, Medium 10-15 µm, Fine 4-5.5 µm

European standards - (Robu & Schott, ISO 4793) nominal pore size, in microns, P3 (P40) 16-40 µm, P4 (P16) 10-16 µm

- I had no idea about all this. Why on earth can't we just standardise these things. Anyway, thank you very much. I have always wondered exactly what the pore sizes represented.

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(6) ...What is a least polar couple of spots with delta Rf 0.05 (often called "eight", "8") which you would separate via DCVC?...

- I only do DCVC. If it fails the next stop is prep. HPLC. Fortunately it still hasn't failed. As with all normal phase chromatography super non-polar compounds separation sucks. That said I have often columned stuff (not super non-polar) that appears to be one compound by TLC and managed to get two compounds of the column. Behold the power of slow step gradient elution. I have achieved truly mind boggling separations over only 20 fractions. The worst column ever occurred 1.5 years ago. I kept getting a fair bit of each diastereoisomer clean but it took 5 columns to get it fully separated. Still it was easily done in one day by using the same column 5 times and only collecting 30 fractions per column.

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(7) Do you always start from heptane even for very polar mixtures?

- Yes! It gets the stuff off the Celite on to the silica and wets the column so that it runs well. Generally I do 4 x hexane, heptane or pet. ether first.

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(8) ...can I use toluene/ethyl acetate, chloroform/ethyl acetate or chloroform/methanol mixtures successfully?...

- Yes! You can even use really low boiling solvents such as acetone, dichloromethane, ether etc. but due to evaporation it is easier to work with higher boiling solvents. Where I work now hexane and heptane has been replaced with 40-60 petroleum spirit which I use for my columns without too much difficulty. Remember to have the pump exhaust in the fume hood, especially with the low boiling solvents.

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(9) ...Does it work substantially better [with Celite] than preadsorbtion on silica gel?...

-Yes! Celite is easier to handle and it doesn't affect resolution.

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(10) ...Does this trick with Celite work even if a sample is only sparingly soluble in an eluent?...

- It doesn't matter what your eluent is. Dissolve your compound in something polar that is easy to get rid of, for example ethyl acetate or methanol. Add Celite, concentrate in vacuo and load it on the column. Ensure that you have removed all solvent prior to loading. A lot of say methanol in the Celite will compromise resolution.

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I hope that helps. If you are new to this area please read the previous post and check out the following paper (a copy can be supplied upon request):

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Dry Column Vacuum Chromatography, D.S. Pedersen and C. Rosenbohm, Synthesis, 2001, pp. 2431-2434.

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Let me know if you have more comment, suggestions or questions. D!

Let's talk about TLCs Part 3 - Phosphomolybdic Acid (PMA) Stain

PMA Stain is a highly versatile general stain for developing TLC plates. The stain is easily prepared by dissolving 10 wt. % phosphomolybdic acid in ethanol. More PMA can be used however as stains go PMA is relatively expensive.

TLCs developed with PMA stain upon heating give green/dark green/dark blue spots on a yellow/green background (see picture).

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Phosphomolydic acid (PMA) Stain Recipe

100 ml container

100 ml Ethanol

10 g Phosphomolybdic acid, H3[P(Mo3O10)4] · xH2O

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Alternatively, you can go for the more expensive solution and buy ready made PMA stain from Aldrich (see picture). Where I've worked in the past we simply poured some of the ready made stuff into a jar and use it for our TLC plates. At the end of the day we poured it back into the Aldrich flask and rinsed with some ethanol. Using this method the stain lasts for a very long time. I never had any problems storing the stain at room temperature despite Aldrich's recommendation to refrigerate.

See under Vanilin Stain for general staining guidelines. D!

100% Edible

Finally, I'm returning to normal. I'm back at the University working at full force and I've got the energy to do a bit of blogging again. Let's start with a nice and easy post. One of the new people at the University turned up with the following Fluka product the other day. Pretty sweet and good quality chocolates too. Apparently, Fluka is a Swiss based company and once a year (around Christmas I believe) they give their customers a truck load of these containers.One of the guys in the lab immediately grabbed the phone and gave Fluka Australia a call to hear if they stocked this item too. Unfortunately, this appears to be a Switzerland only bonus. The product also isn't listed in their catalogues.

Also my "great" idea of introducing Google adds on Curly Arrow was short lived. A lot of the adds where completely irrelevant to this blog so it's all been deleted again. D!

Open Access Journals - Beilstein Journal of Organic Chemistry

It's amazing how unpredictable life is. Only four months after I started my job in biotech I'm heading back to academia. Not that there was anything wrong with the job but I've been given an opportunity to return to Denmark (which always has been the long term plan) so I'm going with the flow. I recently had a post on open access journals and was moaning a fair bit about how we don't have any decent chemistry open access journals yet. Well I was wrong. A colleague alerted my attention to Beilstein Journal of Organic Chemistry (BJOC). This is the best attempt I've seen yet. They are publishing a reasonable number of papers, the quality of the material is good, the journal gets indexed by all the important organisations and it is absolutely free. The impact factor is still low (ISI WoK: 0.35) but this should improve with time. So check it out.

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The past year I've been involved in setting two labs up and whilst searching for information on equipment etc. I've been quite pleased with the Google adds people have on their web sites. I found a fair bit of the stuff I needed this way so I've decided to give it a go here at Curly Arrow. So sometime in the very near future adds will start appearing on Curly Arrow that should be useful to people interested in purchasing lab equipment. D!

Catalytic Hydrogenation - now fire free

In my experience you should not do synthetic organic chemistry if:

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(A) You are drunk (or the following day when you have a hang over)

and/or

(B) You are in a hurry

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Recently, I violated Commandment B and decided to put a large scale catalytic hydrogenation on really, really fast. Bad idea!

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Now hopefully most of you realise that the addition of palladium on charcoal (or charcoal alone) to organic solvents can result in instant bonfire. Unfortunately the chemists hand is often right above the flask as the catalyst gets added and some very nasty burns can result. Even worse the whole thing may take off and you'll frantically be looking for a fire extinguisher (Always know where the fire extinguisher is. You will need it one day).

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There is an easy way to avoid this problem. If you first remove all atmospheric air inside your flask there will be no fire. In other words what you have to do is flush the flask with nitrogen or argon. I normally hook my flask up in such a way that I can evacuate it with a pump and fill it with nitrogen. I've shown one of my recent setups on the picture. You can obviously connect things in many different ways depending on what equipment you have available.

I normally evacuate the flask and fill it with nitrogen at least three times. Only then do I proceed to add my catalyst. After addition I repeat the evacuate the flask procedure but this time I flush the flask with hydrogen and at the end I attach a big fat balloon with hydrogen gas and stir the reaction mixture vigorously.

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The other day when I was in a big hurry I decided to skip the flushing with nitrogen step (for the first time ever) and as a consequence I had my first Pd on charcoal fire. I was very lucky and didn't burn myself because the weighing paper was shielding my hand. The drama was quickly over as I had my lab book handy and sealed the flask with it. The hydrogenation worked fine and I made it in time to the pub. However, next time I think I'll skip the first pint with the boys and flush that flask as I normally do. D!

Recycling Silica Gel

Insert usual excuse for infrequent posting here: .....

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I recently received the following email regarding recycling of silica gel:

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"In the past we used to regenerate SiGel with fuming nitric acid, much as you do here (refers to previous post - D!) .Place the coloured, used SiGel (which of course must be uncoated and completely dry) in a large beaker. Approximately a third full. Place it inthe fume cupboard and pour in the nitric acid so the SiGel is completely moistened. If necessary, stir it. After a possible initial fuming has stopped, heat it on a steam bath for 10 minutes - leave it overnight. Next day, fill up with water, stir and let it settle. Wash 3 more times. At this point, it should be colourless, if not, repeat with more nitric acid. Then wash with satd. sodium bicarbonate until neutral and filter on a Buchner funnel. Wash with water, methanol and acetone and let it suck completely dry. Finally, activate by heating it in an oven overnight at 100-120 C.This way, it is as good as new for most purposes."

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This may be of use to some chemists working at institutions where money is very scarce. However, considering the amount of time and large volumes of solvent required to do this I think it can be classified as historically interesting but not practically useful to most of us.

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Nevertheless thank you for the email. I have wondered exactly how you would go about recycling your silica gel.

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Speaking of silica gel and techniques that are disappearing from the chemists hood check one of the most recent posts at In the Pipeline out. Derek seems to think that TLCs are on their way out as LC-MS is becoming more and more common. I believe that it will take several decades before Derek's predictions come true (especially in academia) but he does have some very good points. D!

How to clean your sintered funnel

Lately when I have been cleaning my sintered funnels people have stopped and asked me what I was doing. To my surprise many chemists don't seem to know how to take a nasty, dirty sintered funnel and making it nice, white and shining again in about 15 minutes.
These days I'm doing lots of old school chemistry that involves heating the crap out of the components using for example conc. sulfuric acid as the solvent. Needless to say things are polymerising and decomposing left, right and centre and when you filter it through your nice white sinter it ends up looking nasty (see picture above). The stuff doesn't go anywhere with acetone, water, 2M sodium hydroxide or hydrochloric acid etc. so what should you do? Before I proceed please note that if you attempt any of the following you must:

(1) Wear a closed lab coat, safety glasses and plastic gloves

(2) Conduct the cleaning in a fume hood with the sash down at all times
(3) Ensure that all the glassware is clean and doesn't contain residual organic material such as acetone
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Please take the above advice seriously. People have had nasty accidents doing the following because they weren't careful.
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There are two common ways to get your sinter clean:
(1) Conc. nitric acid, or
(2) Conc. sulfuric acid and hydrogen peroxide
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Nitric acid is the easy solution and more often than not it does the trick. However, occasionally it is necessary to use more vigorous conditions. I have never had a sinter that didn't become white after treatment with conc. sulfuric acid and hydrogen peroxide and this is generally the method that I use because I know it works every time.
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Procedure:
(1) Fit the funnel to a Büchner flask attached to a vacuum that you can control easily
(2) Add a small amount of conc. sulfuric acid so that it covers the surface of the sinter
(3) Add a dash of hydrogen peroxide and stand back. Things get pretty hot, bubbly and exciting at this point (See picture above).

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(4) When the ingredients have been cooking away for a minute or so apply a very gentle vacuum briefly. This should be sufficient to suck the sinter dry (See picture above),
(5) Allow the cocktail to settle down and cool off and clean all the equipment with lots of water taking care not to pour the contents all over yourself. Your sinter will now look like this.
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Too easy but please do be careful guys. D!

Christmas decorations for chemists

As I was decorating the Christmas tree today I found some of my chemistry contributions to the festive season. First up is my carbohydrate paper cone that I received as a gift some years ago:
And then there's this very old distillation receiving flask that looks great on the Christmas tree: Notice how they have marked each of the receiving arms with small blue coloured glass bumps. Okay that really is it until next year. Merry Christmas and Happy New Year. D!