Getting through the “black tar phase”

Lately I’ve been stuck in synthetic limbo, a single transformation consuming months of work.  With ample time to think I’ve tried to optimize my workflow, and the broad strokes are outlined below.  I’d be (very) interested to hear from anyone who’s found a different way through.

Fail early, fail often

An implicit assumption in this plan is that the bulk of what I do is wasted effort. Almost all of the reactions are going to fail, while the few that succeed may be low yielding or give products that rapidly decompose, even in the reaction vessel. Depending on the compound common analytical techniques like mass spec. and NMR may be unreliable or exceedingly difficult to interpret. In short, it’s going to be a bumpy ride.

Mentally, the key for me has been to dissociate the ego from the chemistry. It’s easy to get excited when something works and sad when it doesn’t, but the only way out involve a lot of bad news.  Since failure can’t be avoided it has little bearing on your skill as a chemist–you aren’t the first to go through a bit of a rough patch and won’t be the last.

From Baran's Welwitindolinone A synthesis.  At least they got cake.

From Baran’s Welwitindolinone A synthesis (link)At least they got cake.

What’s a win, anyway?

Let’s start by defining success.  The goal is compound XXX, but the various reaction vials aren’t going to shout out their contents.  Purifying each and subjecting the contents to full NMR/MS analysis is incredibly time consuming, and if each run requires a column you’re going to have plenty of time to memorize the treaty of Westphalia.

So ideally we’ll look for the compound of interest in the crude, after either the workup or quench. If you’re lucky enough to have a standard of the material you need to make (and it’s stable), TLC is simple, fast, and informative. I have a preference for multi-coloured stains like anisaldehyde or vanillin, which discriminate spots based on structure as well as Rf.

Crude NMR also sits in the “simple, but powerful” bracket. While I generally don’t run crude NMRs during routine synthesis, a quick scan of the proton spectra will reveal the presence or absence of key peaks, again saving you the time and effort of a tedius workup/purification. This works best for reactions that give >5% yield, as otherwise the peaks of interest tend to be buried in the baseline (longer scans will reduce throughput, which may or may not be a concern).

If you don’t already have a sample of your compound of interest, a different approach is required. Mass spectrometry is the easiest way of spotting a single compound of interest among dozens of byproducts, and nearly every lab has access to at least a basic instrument. GC(-MS) and LC(-MS) both go one step further, giving information on the yield and removing contaminants which might otherwise quench the MS signal. The ideal system may be an automated LC-MS, which is more useful than TLC [1].

Measure twice, cut once

Once you can definitely say if a reaction is successful, it’s time to find a few dozen to try. As the project goes on expect to spend significant amounts of time on Scifinder/Reaxys digging up obscure 1970’s protocols, looking for new conditions and reagents. Don’t worry about trying the obscure and implausible, as presumeably you’ve already tried the conditions most likely to work [2].

Once you have 5-20 papers picked out, start setting up reactions. I’m a big fan of vial chemistry at this stage, for the simple fact that multiple round bottomed flasks don’t fit on stir plates very well. Your starting material is likely precious, so restrict quantities to the smallest amount needed for analysis [3].

Take the time to do things properly. Work only with pre-dried glassware, maintain an inert environment, control the temperature, add reagents slowly, and mix well. Every reaction is new, and it’s hard to know in advance which require special care to work properly. The only thing worse than running 100 reactions without success is running 200 because one that should have worked failed due to a temp. fluctuation or math error.

Limit break

Most reagents will fail to react, while the broad remainder will decompose the starting material. Double your reaction load and try to make every reagent do both, by chilling the reactions that decomposed and heating/forcing those that did nothing at all. If you don’t see product under both gentle and forcing conditions, move on. In the absence of a confirmed hit (ie. a good NMR or LC-MS spectra) I haven’t found it useful to try a full suite of conditions for each reagent, but exploring the limits on either end gives information quickly and cleanly.

Learn from your successes

Eventually you’ll move forward, with one or more reagents forming the compound of interest. With several results the next step is clear: take the highest yielding or most accessible reagent and optimize the conditions, varying the temperature, time, solvent, proton source, catalyst equivalents, etc..  Again, yield by NMR or GC/LC/HPLC traces will save time over a full purification.

If the best reaction remains low yielding after optimization, in my limited experience it’s time to get a better understanding of what’s going on in the flask.  Either set up the reaction in an NMR tube, or if that’s not possible quench the reaction at various timepoints and do a set of crude NMRs or GC/LC/HPLC runs. Either approach gives qualitative information on the kinetics of the reaction(s), which can be used to determine if the low yield is due to transformation of the starting material into various side products or simply the result of decomposition of the final product.

Side product formation is probably the more challenging of the two situations, and can occasionally be (further) reduced by adding pathway-specific inhibitors. Modifications to the reagent itself may also be fruitful, but presumabely if this is the first hit in 100 reactions you’ll be relatively limited on that front [4].

With an unstable product the road forward is somewhat simpler. Overoxidation and the like can be partly controlled via slow addition of reagents (syringe pump), or if the expertise/equipment is available flow chemistry. In the worst case the crude NMR can be used to determine yield over time, and a mid-reaction quench can give good yield based on recovered starting material.

None of these are like the others

I’ll close with a quick disclaimer.  No two projects are the same, and the problems you face will likely be unique as well. Above all pick what works and discard the rest.


[1] And far more expensive, outside of the range of most academic labs. One can dream.

[2] Few obscure conditions are fully tested for substrate scope. I once took an alkylation procedure used on a single non-aromatic primary alcohol and adapted it to a system with four protected hydroxyls, four protected amines and one very hindered secondary alcohol. It worked quite well.

[3] Standard solutions are useful here. Dissolve your compound of interest in a low molarity carrier solvent (DCM/ether/pentane/etc.), add via syringe, then remove the solvent under an inert gas stream if necessary.

[4] It may be possible to elaborate on the reagent, for example moving from proline to a more complex proline-based scaffold (ex). However, unless the other reagents are commercially available or you are in pilot plant setting this route will probably take far longer than is practical.

Praise for the Kugelrohr

I don’t always do a small scale distillation, but when I do I use the Kugelrohr.

Kugelrohr Labeled

One of the lab’s kugelrohrs.

Distilling 1 mL or less has a number of complications, mostly due to condensation outside of the receiving flask.  In the standard setup the compound of interest boils from a RBF, liquifies over what is essentially a water jacketed condenser, then drips into the final receiving flask.  At small volumes there just isn’t enough liquid to form drops, and instead a fair bit of the yield is lost as dew on the condenser (which will also evaporate and leave the system via the vacuum line).

The kugelrohr condenses compounds directly in the collection flask, with the shortest practical distillation gap (about 2.5 cm/an inch).  Very little sample is lost, (if any), and the entire process generally takes less than 20 minutes or so.

Using the Kugelrohr

Transfer your sample of interest to a small round bottom flask, then attach at least two kugelrohr bulbs (female ground glass joint on top, male on bottom).

Place all but one bulb in the air furnace and chill the last bulb to at least 0C [1].  Slowly spin the bulbs (about 1 rotation per second) and gradually increase the heat.

Compounds will distill slowly, appearing first as a thin film then as single droplets.  When two or more samples are being distilled there will generally be a noticeable gap when the volume in the collection bulb does not increase.  At this point slide one of the bulbs in the air furnace out, and cool.

Repeat as needed.  If necessary collection bulbs can be swapped out for fresh glassware, and 3+ compounds can be collected.

KR - PipetWhen finished, allow the bulbs to cool.  The easiest way to drain out samples is to use a curved pipette tip (~30 degree angle, made by holding the pipette at an angle over a bunsen burner).  Use a bit of solvent to rinse the walls, then condense to final volume on a rotovap or with an inert gas stream.

Disassemble the kugelrohr.  Store glassware in the kugeldrawer.

Finding a Kugelrohr

There are now a number of commercial kugelrohr kits, but the physical requirements of the process lend themselves well to ad hoc construction.  The best examples are immortalized in the pages of J. Chem. Ed.,  in loving hand-drawn detail.  The hardest part to source is the heat source, though the air furnace can be replaced by upside-down variacs or spare rotovap.  For the discerning chemist I recommend the work of Guida and Gawley, as they “have found that commercially available “toaster-ovens” suite the purpose beautifully.” [2]


KR - Bath[1] The mode of cooling is a major point of departure in the kugelrohr guides I’ve seen.  I use a a 20 mL vial filled with crushed dry ice/acetone (~9:1 mix), which is loosely held above the outer bulb with a 3-prong clamp.  With good glass-glass contact this cools the collection bulb to -15C or so, far below what you can get with a stream of acetone or DCM.

[2] Urban legend has it that the first Alrich Kugelrohrs were the remains of stray coffee pots, combined with stripped down windshield wiper motors (bonus low-rent personal design).

Amphoteros – A Blogging Professor

For the past six months, Professor Andrei Yudin at the University of Toronto has been on sabbatical, gaining experience with protein crystallography at Toronto’s half of the Structural Genomics Consortium.  Like most chemists branching into biology, he’s mostly been thinking about all the chemistry he could be doing [1].  Rather than keeping these thoughts to himself he set up his own blog, and has been writing nearly every day.  The posts range from useful papers/topics to interesting unsolved research problems, with a few travelogues and biological insights thrown in for good measure.

The Yudin lab does some great work, and these posts are a glimpse into the planning behind each project.  I’d highly recommend taking a look through the archives yourself (the whole thing takes about 2 hours to go through), but in case you’re strapped for time here are some of my favourites:

1) Taking risks in chemistry

The best question to ask at the start of any research project is, “Is this worth the time and effort?”, while the most crucial question to ask periodically thereafter is, “Is it time to pull the plug?”  I’ve never had a dearth of projects or ideas, but feature creep and the sunk cost fallacy are always a problem.

2) Show me the numbers

What can we draw from a 30% yield?  Without the proper analysis the answer is generally, “not much.”  This post emphasizes the importance of a examining the crude reaction via a quantitative assay (HPLC, GC-MS, etc.).  The assay results can be used to determine an assay yield [2], which will show if the low experimental yield is the result of poor technique, poor conversion, or poor selectivity.

3) Empowering students with strong synthetic skills

How good is your microscale technique?  The University of Minnesota likes to challenge students with a MCPBA-based partial epoxidation of geraniol, on the 1 mg scale.  My organic labs heavily relied on microscale kits, but even we didn’t go this low.

 

[1] I kid, I kid.

[2] Assay yield = Selectivity x Conversion.  Selectivity is determined by the amount of product formed, while conversion is 1 minus the amount of starting material remaining.

Fieser and Fieser: A Labful of Tips

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It’s hard to look back and not wonder what we’ve lost.  Organic chemistry is not immune to fickle trends and fashion, and more than one darling of yesteryear lies forgotten in the dusty pages of Tet. Lett. [1].  While I’ve no desire to go back to preparing platonic solids, there’s a few tricks I feel happier to have up my sleeves.

1) Ethylene dibromide/1,2-Dibromoethane is a Grignard Entrainer/Initiator

Grignard reactions are notoriously finicky, which is why they are a standard feature in second-year organic chemistry labs.  The key lies in initiating the reactions.  Exposure to oxygen leaves a thin film of magnesium oxide on the surface of the metal, which weak bromides are unable to pierce.

Ethylene bromide is far stronger than your average electrophile, and will rapidly clear the magnesium oxide.  The product is ethylene gas, allowing both a clear sign that the grignard is proceeding and ensuring that no new grignard reagents are added to the system.

2) Toluenesulfonyl Chloride Purification (original unavailable)

TsCl is one of those chemicals that no organic lab will ever be without, in large part due to the fact that someone bought 250 g over a decade ago.  Unfortunately, by now what’s left in the bottle has at least partially decomposed to TsOH and HCl.

The cost of regenerating TsOH may be greater than a fresh bottle, but all is not lost.  Simply dissolve the entire bottle in about 2.5 mL chloroform per gram of material, then dilute five-fold in petroleum ether.  Filter off the resulting impurities, remove any fine impurities with activated carbon, and concentrate off the solvent.  The resulting “fine white crystals of analytically pure tosyl chloride, m.p. 67.5-68.5 [degC]” can go back in the bottle, while the TsOH collected during the first filtration can be kept elsewhere for future use.

2b) Potassium tert-Butoxide Purification

A reagent of 1001 uses, KOtBu rapidly hydrolyzes in air to form potassium hydroxide and butanol.  Unfortunately this process has no effect on the appearance of the off-white powder, and it can be almost impossible to tell if bottle has gone off.  While not mentioned in Fieser and Fieser [2], here’s a neat trick for purifying the mixture:

Dissolve the powder in a small amount of dry tetrahydrofuran.  While potassium t-butoxide has magnificent solubility in THF (25 g/100 mL, according to Fieser and Fieser), potassium hydroxide is so inert it can be used as a drying agent.  After filtration of the KOH the solvent can be removed under stringent anhydrous conditions (perhaps an Ar stream), or an aliquot can be mixed with water and titrated to determine the effective KOtBu concentration for a stock solution.

3) Sodium Azide and Iodine

Here’s a nice use for an old reagent.  Thiols and thioketones catalyze the reaction of sodium azide and iodine, and the liberated nitrogen can be used as a spot test to detect sulfur compounds.

4) Tetramethylguanidinium Azide

Nucleophilic substitution with sodium azide followed by palladium reduction is a beautiful way to convert alkyl halides to primary amines, but unfortunately sodium azide has very poor solubility in non-polar solvents.  The usual solution is to use dimethylformamide and/or water, but this reagent makes for a nice acetonitrile-soluble alternative [3].  The corresponding halide salt is insoluble in diethyl ether, and can be crashed out following completion of the reaction.

5) The DABCO-Bromine Complex

DABCO binds to bromine quite strongly, and the resulting DABCO-Bromine complexes are good oxidizing agents for the conversion of sulfides to sulfones.  Interestingly, this seems to have been rediscovered some 40 years later, with a recent publication in Tet. Lett. on alcohol oxidations.

6) Lithium Dimethylcuprate (Gilman’s Reagent)

I’m hesitant to call this reagent “forgotten”, but it is less well known than I would expect.  Effectively a soft carbanion, lithium alkyl cuprates replace good leaving groups with inversion of stereochemistry, convert acyl chlorides to the corresponding ketones, and add 1-4 to enones.  As an added bonus, these organocopper compounds will also reduce disubstituted alkynes to cis-alkenes.

7) Dimethylsulfoxide and Acetic Anhydride

This combination somewhat surprisingly converts secondary alcohols to ketones.

8) Zinc Dust and Dimethylformamide

Zinc causes rapid bromine elimination, converting alkyl bromides to alkenes.  This effect is so strong that it will even break aromaticity, converting o-xylene to a (rather unstable) tetraene.

Zinc Dust9) Formic Acid Cleaves Many Protecting Groups

The go-to conditions for cleavage of hindered acid-labile protecting groups is HCl in MeOH, but occasionally that just isn’t strong enough.  Pure formic acid has a bit more kick, and will remove carbamates and acetals (and presumably silyl ethers) much more readily.

10) The Vilsmeier Reagent (Dimethylformamide-Thionyl Chloride)

Ever wonder why chlorinating reactions work so well with a little DMF in the flask?  Thionyl chloride, oxalyl chloride, phosphorus pentachloride and phosgene all react with dimethylformamide to form a charged chloroforminium species through elimination of SO2.

Chloroforminium PreparationThis compound has been isolated (pdf), and is a (relatively) mild chlorinating reagent, reacting with alcohols and carboxylic acids to give alkyl chlorides and acyl chlorides, respectively.  It also formylates electron rich aromatic rings, via the Vilmeier-Haack reaction.


[1] Pre-Elsevier’s purchase, of course.

[2] Or Purification of Laboratory Chemicals, oddly enough.

[3] Fieser suggests chloroform as a solvent for the azide substitution reaction, which isn’t something I would ever recommend.

Edited to add schemes and Vilsmeier link.

Fieser and Fieser: Once Upon a Time in Chemistry

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The past is a foreign country: they do things differently there.
-L. P. Hartley

Imagine a world without flash chromatography, HPLC, high resolution MS or high field NMR, though if you ask nice you may be able to get time on the department’s new 60 MHz NMR (1D proton spectroscopy only, of course).

In this environment, how did chemists ever determine what lay in the bottom of their flasks?  IR is an excellent method for determining which functional groups are present, but like elemental analysis it says very little about how the bonds are put together.  Closely related compounds and isomers would be nearly impossible to distinguish by these methods, and so changes in physical properties became key.

While presently chief paperweight [1], tracking physical properties was what role that the CRC handbook was made for.  Older versions of the book had even less of the spectroscopic properties we now rely on for compound characterization, instead focusing on tests we may no longer even routinely perform.  Chief among these is of course melting point, but density and optical rotation also receive special mention [2].

Necessity drove innovation in purification techniques as well.  Distillation at reduced pressure can do wonders with low molecular weight oils, and with enough time almost any solid can be recrystallized [3].  Recrystallization solves many of the above mentioned structural assignment problems as well, but unfortunately could not be applied to every case.  Oils are nearly impossible to recrystallize and analyze (some do solidify at low temperatures), and many solids have enough disorder to highlight the fine crystallographic distinction between “theoretically possible” and “incredibly difficult.”  To combat these beasts several classes of recrystallization aids were devised.

Sodium bisulfite is likely the best known crystallization agent, and the most generally applicable.  Aldehydes combine with bisulfite to form stable sulfonic acid/alcohol adducts that are at best sparingly soluble in water.  Filtration of the resulting precipitate removes water soluble impurities, while alcohol/ether washes draw off the more organic soluble contaminants.  A final exposure to weak acid or base regenerates the original aldehyde, which is then collected via simple liquid/liquid extraction.

Somewhat less amenable were reagents like p-phenylazobenzenesulfonyl chlroide and p-phenylazobenzoyl chloride, which react with free amines and alcohols respectively to form brightly coloured azo dyes.  These compounds could be readily isolated by paper or silica chromatography–no stains required [4]–and recrystalized to allow for X-ray analysis, but only through destruction of the original material.  Carboxylic acids and amine salts which could do the same were highly prized, and there are far too many to list here.  One interesting example is dehydroabieyylamine, which selectively recrystallizes various penicillins.  Recrystallization aids fell out of favour in the research lab with the advent of flash chromatogrpahy, but these agents are still very useful for resolving mixtures of chiral compounds.


[1]  The CRC handbooks were giants, because in the pre-computer age they had to be to contain all relevant compounds. The 1963 edition was 3600 pages long.

[2] Augmenting IR and providing more detailed structural information was of course a multitude of functional group tests.  In the 1960’s though their sun was setting, so I’ll cover them another time.

[3] Cfi. The entire field of protein crystallography.

[4] Few of the TLC stains we take for granted seem to be common at this point.  Instead compounds could be tracked on column, provided one worked with specially prepared morin-stained alumina.

On Location in Kyoto, at ICBS 2013

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For the past few days I’ve been at the International Chemistry Biology Society‘s annual conference.  True to form I blogged about the talks, highlighting the research I found most interesting (with links, as much as possible).

Obligatory.

Notes from ICBS- Day 2 - Day 3

If you’d prefer the cliff notes version, together with Margaret Johns I also livetweeted the conference.  Check out the storify link here.

Conference Call

I’m headed to Kyoto this week for the International Chemical Biology Society’s annual meeting.  While there I’ll be livetweeting as @Chemtips alongside Maggie Johns, as well as posting highlights of interesting research to the ACS Chemical Biology community.

If you’re going to be at ICBS 2013, come say hi!  This will be my first trip to Kyoto, so I’ll be easy to spot–I’m the one frantically flipping through a Japanese phrasebook.

Fieser and Fieser: The Toxic and Terrifying

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AKA the Good, the Bad, and the Ugly.

Organometallics aside, the greatest advance since the days of Fieser and Fieser isn’t the development of new chemical transformations [1].  Rather, it’s the gradual replacement of hazardous or toxic reagents with more benign variants.  We do much the same work as before, but with greater substrate scope and less risk.

That said, it’s no surprise that water reactive workhorses like SOCl2/PCl3/POCl3/PCl5 have extensive write-ups in Fieser and Fieser.  We may no longer regularly run reactions in refluxing benzene (never mind the CCl4 recrystallizations), but with proper technique these are perfectly safe and highly effective chlorinating reagents.

PCl5 Synthesis - From Inorg. Syn. 1: 109 (1939).  The tank contains chlorine gas.

Inorg. Syn., 1, 99 (1939). A contains chlorine gas.

That figure up above is from a reference article, and it shows the lengths chemists used to go to for a little PCl5.  Chlorine gas from a lecture bottle is bubbled through sulfuric acid to remove any residual water, then reacted with PCl3 dripping down from an addition funnel.  Cl2 is no treat to play with, but it’s a common trend in F2 for even relatively benign reagents to require hazardous preparations [2].

Pertrifluoroacetic acid is a good example.  A powerful oxidizing agent with dozens of uses, CF3CO3H readily converts amines and oximes to nitro groups, installs phenols in mildy activated aromatic rings, and is an excellent Baeyer-Villiger reagent.  Unfortunately pertrifluoroacetic acid decomposes on standing, and preparation in situ requires 90% HOOH (ie. propellant grade).  Reactions using 30% peroxide and higher generally require the use of an explosion proof screen, and I’m not certain how the 90% solution could be safely stored in a lab filled with organic vapours.

Rocket fuel makes for stressful afternoon, but chemists occasionally get a little desperate.  There are limits though, and this last would probably force me to hang up my lab coat and take a week of unplanned vacation.

Oxygen difluoride, better behaved cousin of FOOF, is prepared by passing fluorine gas through a 2% solution of sodium hydroxide.  The resulting vapor must first be purified to remove the residual F2 and roughly full equivalent of byproduct O2 produced during the reaction.  This is done by cooling the mixture in a bath of liquid oxygen, then fractionally distilling out the desired gas [3].  Once pure, OF2 can be stored at room temperature in large glass bulbs, though it will react violently with silica and molecular sieves.  It is described as having a peculiar, foul odor.

OF<sub>2</sub> Generation.  From Inorg. Syn., 1, 109 (1939).

Inorg. Syn., 1, 109 (1939).

First water, then dry ice/acetone, then liquid oxygen.

First water, then dry ice/acetone, then liquid oxygen.  Oxygen difluoride collects in the third trap.

Once formed the gas is bubbled into trichlorofluoromethane (Freon 11, now banned by the Montreal protocol) at -78 degC, where it will oxidize alkenes and acetylenes to the corresponding mono- and di- alphafluoroketones.  Primary (aliphatic) amines are smoothly converted into the corresponding nitrosos (two equivalents of the amine are lost as the fluoride salt).  The whole setup from start to finish is an ugly beast, and I’m happy to say to those looking for a nitroso prep that there are better ways.


[1] Yes, that’s a pretty big aside. It’s been forty-six years.

[2] Given the plethora of hazardous preparations I was surprised at how cautious Fieser and Fieser are with diazomethane.  Five separate preparations are given, listed in order of preference.  Bis-(N-methyl-N-nitroso)terephthalamide is their preferred precursor, but this is 25 years before the first report of TMS-CN2.

[3] To quote the original authors,

Further purification can be accomplished by pumping
on the trap containing the liquid while it is still immersed
in liquid air.  A water pump is best, since the gas coming
off (principally oxygen at first) will eventually cause loud,
but not damaging, explosions if it comes in contact with the
oil of an oil-filled pump.

Fieser^2

Fieser and Fieser - Cover PageThe 1960’s were a heady time.  The Varian A-60 made it’s debut early, marking the beginning of routine NMR analysis,  while research by Woodward and others demonstrated the power and adaptability of organic chemistry (for which he received the Nobel in 1965).  The literature was booming, and the fourth volume of Organic Syntheses was larger than the first three combined.

If structural complexity was limited only by one’s imagination, work in the lab was considerably more challenging.  Though founded in the mid-fifties, the rise of Aldrich was still well over a decade away, and fine chemical manufacturing was in it’s infancy [1].  Aside from the few hundred bulk chemicals used in industry, if a reagent was needed for a particular transformation the only recourse was to prepare it in the lab.

The Fiesers loved their cats.  And pyridine.

The Fiesers loved their cats. And pyridine.

Chemists of the period turned to Fieser and Fieser’s “Reagents for Organic Synthesis.”  Essentially a chemistry cookbook, the twenty-six volumes of Fieser2 explain the use and preparation of the sort of mid-level reagents we take for granted, like sodium borohydride and acetyl chloride.  Volume 1 in particular has a reputation for sound advice and useful tips, even now, and has been on my to-read list for quite a while.  Unfortunately there’s far too much information to list everything in a single blog post, so I’ve divided things up into broad categories.  Click through for the first post in the series;

Fieser and Fieser: The Toxic and Terrifying.


[1] J. T. Baker, Eastman (Kodak), and Fisher were the big three, but crowd favourite Columbia Organic Chemicals (pdf) had already recovered from their first fire by this point.