What’s in your library?

For a variety of reasons I’m taking stock of the various books I’ve purchased, appropriated from the group stores, or borrowed from the University’s library.    Most serve as long-term references, though the occasional book is for searching for research project inspiration.

My list:

Arpe – Industrial Organic Chemistry

Barton and Ollis – Comprehensive Organic Chemistry Vol 2: Nitrogen Compounds (1979)

Carey and Sundberg – Advanced Organic Chemistry A and B

Chan and White – Fmoc Solid Phase Peptide Synthesis – (Reviewed)

Collins and Ferrier – Monosaccharides: Their Chemistry and Their Roles in Natural Products

Dermer and Ham – Ethylenimine and other aziridines

Dodd – The ACS Style Guide

Feibelman – A PhD Is Not Enough! (Loaned out)

Fieser – Reagents for Organic Synthesis (vol 1)

Greene and Wuts – Protective Groups in Organic Chemistry

Hammesfahr and Stong – Creative grass blowing

Hudlicky – Oxidations in Organic Chemistry

Hudlicky and Reed – The Way of Synthesis

Kern and Di – Drug-like Properties: Concepts, Structure Design and Methods

Kuchner – Marketing for Scientists – (Reviewed)

Larock – Comprehensive Organic Transformations

Leonard – Advanced Practical Organic Chemistry – (Reviewed)

Levy and Fugedi – The Organic Chemistry of Sugars

Li – Modern Organic Synthesis in the Laboratory – (Reviewed)

Lodish – Molecular Cell Biology

March – Advanced Organic Chemistry

Narain – Chemistry of Bioconjugates

Nelson – Navigating the Path to Industry – (Reviewed)

Newman – Steric Effects in Organic Chemistry

Paquette – Encyclopedia of Reagents for Organic Synthesis (Vol 1) [1]

Pennington and Dunn – Peptide Synthesis Protocols – (Reviewed)

Seyden-Penne – Reductions by the Alumino- and Borohydrides in Organic Synthesis

Slonczewski and Foster – Microbiology: An Evolving Science

Stoddart – Stereochemistry of Carbohydrates

Tufte – The Visual Display of Quantitative Information

Weinstein and Wagman – Antibiotics: Isolation, separation and purification

 

My work is a blend of chemistry and biology techniques, so it’s interesting to see that the reference books lean heavily towards chemistry.  This may be in part due to how standardized the molecular biology techniques are, and wealth of online protocols.

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[1] This is online now.  I just like flipping through the hard copy.

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Preparing For Independent Research

“Nothing in the world can take the place of persistence. Talent will not; nothing is more common than unsuccessful men with talent. Genius will not; unrewarded genius is almost a proverb. Education will not; the world is full of educated derelicts. Persistence and determination alone are omnipotent. The slogan Press On! has solved and always will solve the problems of the human race.”
― Calvin Coolidge

 

Every lab has a multitude of research notebooks, physical or electronic.  As a student, the lab book is the most obvious record of your trials and tribulations: the experiments that worked and the many failed attempts. Alongside your thesis, lab books are also the most obvious sign of your passage.  They’re history in solid form.

Let’s talk about the future.

The tail end of a PhD isn’t just for sagely offering advice to incoming students and slowly reducing lab hours to focus on “writing.”  It’s also the time to buy a new notebook.  A nice one, faux leather or perhaps moleskin.  This book isn’t for your PhD, or the post-doc, if you’re planning on going that way.  Rather, it’s a place to collect all the ideas, potential projects, and marketable inventions that inspire you.  This is a book for your independent career [1].

Reading Ahead

To steal from Stephen King, if you want to be a scientist, you must do two things above all others: read a lot and write a lot. There’s no way around these two things that I’m aware of, no shortcut.

Great ideas require inspiration, and seeing the work of others inspires.  Over the past eighteen months I’ve read more papers than the past five years combined, even on one occasion skimming through the entirety of the the 1965 volume of the Journal of the Chemical Society (resumed) [2].  Voracious reading lets you link disparate observations into new ideas, and with your own experiences sometimes you see an experiment and wonder, “what would happen if I tried these conditions on this substrate?”  Conferences and seminars can have the same effect, but the literature is far broader and always available.

Being inundated with information, it’s important to view each paper in the context of your research interests [3].  This will skew your view, which means you just might see something the original authors didn’t [4].

Work It Out

Alright, let’s say you were browsing the JACS ASAPs, and you’ve hit on a connection to some 1980’s Tet. Lett. paper.  Quick, write it in the notebook!  Good.  Now, let’s see if it’s worth pursuing.

First, search the literature.  High impact research tends to be pretty obvious, so there’s a good chance that someone else has hit on the same connection.  A quick search on Scifinder, Web of Science, and Google can determine if the idea was worked to its logical conclusion long before you were born.

If you read enough, as the months go on you’ll start to accumulate a fair number of ideas, to the point where it’s not feasible to investigate them all.  To triage I focus on three core questions:

1. Is the idea novel?

Does it stand out from prior work?  If someone has hit on something related to your idea then there’s less to disclose and subsequently less impact.

2. Will this change how people work?

Does this meet an unmet need?  There are a lot of ways to making amides, but not too many for nitriles.  Nitriles are pretty useful, so that represents an unmet need that you could fill.

3. How can you demonstrate the impact?

If your idea is important, prove it.  If you have a new reaction, this generally means making an challenging natural product or therapeutic with ease.  If it’s a med. chem. project, you could demonstrate impact by testing against a whole cell or model organism (collaboration will probably be required; network early).  Keep the demonstration simple; the goal is not to wag the dog.

Show Your Work

Once you have about a half-dozen ideas, it’s time to take the best and start filling in the details.  Ultimately the goal is to get hired/get funded/start a company, and the only way to do that is to convince people that what you want to do will work.  Hope Jahren has put together an extremely good write-up on how to turn an OK proposal into a great one, so in the interest of brevity I’ll point you her way.  If you’re looking for a quick proposal outline, I wrote one up some time, and Kenneth Hanson covered his style as part of a larger series on getting an academic job.

Writing budgets and triple-checking for typos isn’t necessarily fun, but to paraphrase Hope Jahren, think of what you’re asking for.  Hiring and paying a new professor until tenure review costs any given university the better part of a million dollars in start up funds, overhead and salary.  If you had a million dollars lying around, how likely are you to give it to me?

I’d need to be pretty convincing.

Find the Time

Looking over everything, I’m asking for a pretty big investment of your time.  Say 20 hours per bolt of inspiration (~3 bolts per viable idea), another 20 to develop the idea, and anywhere from 20 to 80 hours to write and hone the proposal.  That’s almost a month of dedicated time, sandwiched in between all of your other responsibilities.

Like thesis writing, the best policy is to find a 1-3 hour block of time in your week that generally isn’t productive, and dedicate that to this effort.  For me this tends to be ~8-10 in the evenings (prime blog writing time, unfortunately), Sunday mornings, and Friday afternoons.  Your times may vary, but the point is to dedicate some period to producing excellent research ideas.  With persistent effort you can do anything.

 

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[1] Think of the notebook as a cliffs notes guide to your ideas, not a comprehensive guide.  You’ll also need to accumulate supporting references and eventually prepare formal proposals and talks, but there’s no need to carry those wherever you go.

[2] By the end I had fifty or so papers worth looking into a little closer, and one or two that gave me ideas of my own.  Other sighs of obsessiveness include the Dictionary of Natural Products, and Wikipedia’s List of Organic Reactions, all 700 or so entries.  Most of the reactions didn’t stick, but I now know at least a half–dozen different transformations that are really just variations on the Swern Oxidation.

[3] As a bit of an explanation, one of my interests is nitrogen.  Whether that entails nitrogen containing natural products or reactions, I’ve decided my work must involve this bit of the periodic table in some way.  So whenever I see an Aldol, Heck and Pinacol reaction I start adding in nitrogen atoms.  The first two cases give me the Stork-Enamine and Hartwig-Buchwald reactions, but that third…hmm.

[4] Knowing what to focus on is a post in and of itself, and one that’s proven stubbornly resistant to writing.

What To Look For When Joining A Lab

aka The Life Cycle of a PI aka The Five Types of Profs You Might Work For

 

Labour day is around the corner, and so is the start of the school year. To help those looking to join a research group I’ve put together a list of the major phases in most professors’ career.

First, a couple of qualifiers. The most important thing when looking for a potential supervisor is their personality. If you get along with a person (and love their research) you’ll likely do very well in their lab, all other factors being equal. That said, when looking for supervisors at the graduate and postdoctoral level I only considered labs that fit the following equation:

Number of papers per year / Number of students ≥ 0.75

That is, for every student in the lab the PI should publish at least 0.75 papers per year (preferably >1, maintained over the last 5 years). This is a high bar, but goes a long way to taking the luck out of a scientific career [0]. Publications are the currency of academia, and regardless of the quality of the lab an extended period of no publications will make it difficult for you to get scholarships, fellowships and grants [1, 2] .

 

1. The New Hire

Often mistaken for postdocs (or grad students), newly minted assistant professors are a high-risk, high-reward option. At least a plurality of the department believes their research ideas are red hot (otherwise someone else would have the job), but a new professor has no proven track record of independent research or lab management, and their chief source of funding is an ever diminishing start-up grant.

This is the one period where the interests of professor and student are perfectly aligned. New profs live or die by the research output of their students, and so they provide excellent one-on-one training. The first few hires in the lab (graduate or undergrad) will be ideally placed to gain many useful skills, and will reap the lion’s share of the early pubilcations. While a letter of reference from a junior faculty member may not carry as much weight as a professor near retirement, they are also much more likely to be effusive in their praise, and (provided the lab is successful), students will have a solid publication record to stand by.

For postdocs the view is less rosy. Close to the New Hire in experience, a postdoc gains far less from their intensive supervision. Recently postdocs themselves, new professors are also weak in many of the skills that postdocs lack (grant writing, lab management), and so can offer little in the way of training. Worse, the junior professor is just starting out in their independent career, and needs to show a strong ability to create original research. They may be less willing to share credit, and have not built up an extensive network of collaborators.

2. Tenure-track

After 4-6 years a prof can no longer be called a new hire, and working in their lab will be an entirely different experience. Startup funding has given way to early career grants, and the PI has a solid reputation for work in their particular field.. At this point official promotion from assistant to associate professor is either a foregone conclusion or has already happened (otherwise stay far, far away).

In the lab, the first hires are in the process of writing and defending their theses (if they are on track for an 8-year PhD that’s another red flag), and those red-hot ideas that got the professor hired are coming to a close.

The most important factor for the success of an incoming student will be the next round of ideas. Outside of the Ivy league, years 5-10 will be a period of growth for the lab, as initial successes lead to increased funding. It’s a good time to arrive, provided that there are some good ideas to soak up all this new funding [3] . Look for vision, and a clear idea of how the initial works are going to advance. A philosophy of “more of the same, but bigger” can lead to diminishing returns, but with steady funding good profs can strike out into more ambitious territory, with a corresponding boost in impact.

3. Stability Sam

By mid-career an academic has survived both the tenure process and at least one bitter departmental feud. Outside of the odd move, labs that reach this point often exist in a stable equilibrium for a decade or more, old students graduating and being replaced by the same people with different faces. The PI is unlikely to take that fateful trip to Stockholm, but they are well respected in their field, with a dense network of collaborations.

The quality of a mid-career lab can usually be determined from the publication to student ratio, normalized over the last five years or so. Provided the lab culture is good, most labs in this phase will be good to join, especially as a graduate student. One caveat: in this period the burden of ingenuity starts shifting to the student, especially in the experimental details. Time away from the fumehood can translate into a dulling of chemical intuition, and it’s during this period that input from the experienced members of the lab (graduate students or postdocs) becomes important.

4. The Bigwig (Chair, Director, VP Research, etc.)

Broadly speaking, two types of people head into administration: those who have lost much of the original fire that drove them to research, and those with so much fire that one lab is not capable of creating all the change they seek [4] .

Regardless of motivation, administration draws much of these academics’ time, and their research often suffers. If their labs remain productive through the administrative term (and even absent oversight momentum will carry a good publication rate for ~3 years), it is likely that there is a strong guiding hand or culture at the lab level, often either from senior graduate students, postdocs, or research associates. As always, talk to peoiple in the lab—and take the pessimistic ones reports with a grain of salt—and get a feel for the lay of the land.

In the event of an absent PI the lab may still be an excellent fit [5], especially for those with prior training and strong, self-directed work ethic. The chair has first pick of departmental resources, and a good letter of recommendation can carry a lot of weight, especially if they have a history of great research.

5. The Greyhair

Not yet emeritus, these form the old guard in many departments. Those still actively involved in research are adept at departmental politics (expect them to have the nicest offices), and have established a nice, productive routine.

In the lab this may translate into an incredible depth of knowledge, a walking treasure trove of reactions and papers stretching back to the ’60’s. This is excellent for problem solving, but can lead to research calcification. After all, what use are new methods or approaches when the old strategies work just as well? (Especially if the lab has previously developed their own solution.)

Success in this environment depends on working at the edge of the lab’s expertise, either on projects with little history or by completely in the wilderness. With subjects the PI is less comfortable in their wealth of understanding can lead to new insights, and the high impact publications that result. This of course forces you to become an expert in something slightly outside the lab’s speciality, which is great for future job prospects but can be demanding and time intensive without a proper background.

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[0] Beware large labs especially.  With 20+ people they will ALWAYS be publishing great work, but this is true even if only 30% of the students are productive.

[1] My favourite counterexample is the double-Nobel prizewinner Frederick Sanger. He published at a rate of 2-5 papers per year for his whole career, and did most of the work either himself or with the aid of a handful of technicians. A graduate student in that lab may not publish much, but they’d get incredible training.

[2] Beware a high concentration of papers with a single (student) lead author. It’s great to BE the golden boy/girl, but much less fun to be one of the many in the shadows.

[3] This is not to say that incoming students won’t have ideas of their own, or have nothing to contribute. However, a PI is committed by way of grants and interest to particular topics and projects. Students to greater or lesser degrees control HOW projects evolve, but it will be some years before most can propose an entirely new research focus.

[4] A third type is occasionally seen, and is best typified by the phrase, “Somebody had to do it.” Their research output tends to mimic those who have lost their fire.

[5] Ie. Expect one or two one-on-one meetings per year.

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 (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.

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[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.

Open Letter to a New Student

Tags

advice, career advice, grad school, lab techniques, mother liquor, motherlode

Stored alongside the welcome packages detailed previously was a lecture that David Collum apparently gives to new students each year.  Titled “Mother Liquors”, it’s a concentrated collection of good laboratory practices, progress expectations and reaction shortcuts.  I was already planning on putting together something similar, and so used his outline as a framework for this post.

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Welcome,

Graduate school has been an incredible experience, filled with some of my best–and worst–memories.  I was fortunate enough to work under a supportive supervisor who allowed me to find my own path, and through guided trial and error gained confidence in my techniques and my instincts.  Along the way I learned what worked for me and what didn’t, building a set of guidelines that helped me be both productive and content.  These guidelines are detailed below.  I hope you find them useful during your own career.

Regards,

Brandon Findlay

Progress and Time Management

Set up at least one reaction per day, completing more than ten reactions each week.  Complete in this case ranges from full characterization (NMR, MS, MP, etc.) for new, pure compounds to clean-up and lab book annotation for reactions which lead only to decomposition.

Never sit idle, especially when you are just starting research.  If you have a surplus of free time while your reactions are running clean dirty glassware, characterize purified compounds, catch up on literature reading or set up another reaction.

At the end of each day make a list of experiments and tasks planned for the next day.  At the beginning of each day start working through the list.

Project Planning

Always work on at least two projects at once, but never more than five.  Switch between projects as conditions demand (ie. when a needed reagent must be ordered or frustration mounts).  If necessary tape a to-do to your desk, to remind you what needs doing on a weekly or monthly basis.

Before starting a project, take the time to thoroughly examine the literature surrounding the planned synthetic scheme or reaction.  Look for alternative approaches to each intermediate (if any are published) and similar substrates which have undergone your proposed transformations.  Be on the lookout for potential side reactions and difficult to separate byproducts.  Two days of planning can eliminate a month of frustration.

When testing a hypothesis try to use experiments that give data for both positive and negative results.  Remember: absence of evidence is not evidence of absence.

Planning an Experiment

Stagger your reactions, such that when one is ready to purify another has just begun.  When preparing a series of compounds run the reactions and workup in parallel, while purifying compounds sequentially (unless you have an automated purification system)  Try not to tweak reactions or purification conditions from sample to sample.

Build from published procedures, modifying conditions as appropriate.  Even adapting a procedure designed for a completely unrelated substrate is better than building the synthesis from whole cloth.  Order reagents for 2-4 steps at once, to cut down on time wasted waiting for chemical orders to arrive.

Begin with a trial reaction, on a convenient scale (ie. 200 mg).  When scaling up never increase beyond a factor of 5, and never change the reaction conditions.

Ex.  Trial reaction: 200 mg.  Second reaction: 1g.  Third reaction: 5g.

When working at the “front” of the synthesis, use the smallest appropriate scale.  75 mg of material is far easier to workup and purify than five grams, and will consume your precious intermediates slower.  When preparing said intermediates work in bulk, to reduce the amount of non-productive time (repeating your work is not making progress).

Use appropriately sized glassware:

Reactions less than 750 mg:  4 mL screw cap vial, with a “flea” stir bar (8.5 mm x 1.5 mm).

Reactions less than 3 g: 20 mL screw cap vial.

Reactions greater than 3 g: Round bottomed flasks.

Never fill a reaction vessel more than 2/3 full with solvent.  Extract reaction mixtures weighing less than 3 g in the reaction vessel or another vial; use separatory funnels for larger quantities of material.

Selecting Reagents

When ordering chemicals consider the cost of your labour, in both money and time.  In general, spending an extra $800 is worthwhile if it saves a week spent preparing a literature compound or reagent (~$400 in wages, and equal part in reagents and solvents).  At the postdoc level the cost of wages almost doubles.  Within reason time is more valuable than money.

Most of the reagents stored in the lab since time immemorial are still of acceptable purity [1].  Unless you have reason to be suspicious don’t routinely check purity before a first reaction (do check if the reaction fails).  If a reaction does fail, always double-check your technique and reactants/reagents before heaping scorn on the published procedure.

Running a Reaction

Always monitor your reactions.  New reactions should be observed via TLC at a standard set of timepoints (ex. 5min/15 min/30 min/1 hr/4 hr/overnight), regardless of the published reaction time.  Run a new TLC after the reaction has been quenched and/or immediately before purification.

Clean glassware, set up flash columns, or otherwise occupy your time while TLC plates are running.  Visualize TLCs with a general purpose stain like vanillin/anisaldehyde/PMA/CAM, even if your compound is UV active [2].  Draw scale models of key TLC plates in your notebook, or photograph/scan the plates.

Anoxic and anhydrous conditions range from essential to overkill to counter-productive, depending on the reaction.  The first time you run a reaction use your discretion, but if low yields occur switch to strict anhydrous conditions (use a glovebox or glove bag, if necessary).

Working up the Reaction

Deviating from a published workup may increase the yield of a reaction, but for a new student should not be the first impulse.  Begin by replicating the author’s procedure, and deviate on the second attempt if the workup fails or gives low yields.

When devising a new procedure, these general rules may be helpful:

First remove your reaction from any sources of heat and allow it to cool to room temperature.  If the reaction is chilled quench it before allowing the flask to warm.

Strong bases can be quenched with a small quantity of saturated ammonium chloride in water or acetic acid, while acidic reactions are amenable to sodium bicarbonate or triethylamine.  Radical reactions can be quenched by sodium thiosulfate, and biphasic reactions will stop when stirring ceases.

If possible always add a quenching agent.  Simply removing the solvent will quench many neutral reactions, but during concentration side reactions may occur, decreasing yields.

If possible, use an extraction to remove otherwise difficult to separate impurities.  A basic fractionation between water and ethyl acetate will remove any salts from the crude, but by using acids (NH₄Cl, 0.1 M HCl), or bases (NaHCO₃, 0.1 M NaOH) amines,  carboxylic acids, phenols, and other functional groups can be removed as well.  Saturated lithium chloride will scavenge DMF from the organic layer, while a brine wash is essential when ethyl acetate is used as the extracting solvent.

Always keep the size of glassware to a minimum.  Phases separate faster in 20 mL vials than in separatory funnels, and give higher yields as well.  Weigh your compounds both before and after the workup, to ensure that large quantities are not lost in the water layers.  Do not take crude NMR spectra unless you have reason to suspect compound degradation during workup or have not observed your expected product in the TLCs.

Once the crude has been worked up either purify it immediately or store it in the dark at reduced temperatures.  Otherwise stable compounds can rapidly degrade when impure.

Purification

All intermediates must be pure-by-NMR before being used in subsequent reactions.  Garbage in, garbage out.

Carefully consider the impurities in your sample.  While flash chromatography is the most general purpose purification method it is not always the most suitable, especially for gram-scale quantities.  When working on an early step in a long synthesis take time to explore recrystallization, distillation, tandem extractions and other less common techniques.

See previous posts for information on flash chromatography.

Compound Characterization

Obtain all necessary data the first time you purify a given compound, not the fifth.  This limits subsequent compound characterization to a simple ¹H NMR, and ensures that during paper/thesis writing all spectral data is accounted for.

At a minimum collect ¹H NMR, ¹³C NMR, COESY, HSQC and LRMS.  High resolution mass spec data and/or elemental analysis should be obtained after the compound’s purity and structure are established.  Additional tests (optical rotation, melting point, IR, HPLC retention time, etc.) will be at the discretion of your supervisor and committee.

Key peaks in the NMR spectra can be used as a preliminary way to determine if the transformation was successful, for quick turn-around time between reactions.  The remainder of the spectra should be assigned weekly, and written up into a publishable format by the end of each month.  This limits the amount that has to be written immediately before a paper is published, and speeds up thesis writing.

Pure samples should be stored at reduced temperature, out of the light.  Most will likely be stable for months or more at room temperature, but one bad sample can ruin a week or more of work.

Data Management

The lab book is your index, even for digital information.  Cross reference your experiments with all compound characterization data (melting points, NMR, MS) noting date of analysis and sample names.

Back up all digital data on a regular basis, on a cloud server if possible (encrypt the data if necessary).  Lab books should be written in indelible (gel) ink and scanned once complete.  Publish your work as soon as possible, or failing that write up all necessary information for new compounds (if the spectra is recorded losing the sample in a far has less impact).  Be aware of the damage that a crashed hard drive or lab fire can have, and limit your exposure as much as possible.

Working Hours

Treat graduate school like any other job you care about.  Arrive at approximately the same time each day (I prefer to start between 8 AM and 9AM), take short lunches, and only the occasional break.  Work hard and efficiently, keeping time spent surfing the web or chatting to a bare minimum.

At the same time, do not allow graduate school to consume your life.  Long hours in the lab are not generally productive, and quickly degrade your mental health.  Leave the lab at a reasonable hour and go play sports, knit, photograph the scenery, and just generally unwind.  Above all, limit the time you work to 40-50 hours each week.  Productivity drops off rather sharply after a few weeks of 40+ hours, and the quality of the work suffers even more.

Beware burn-out.  Take time off when you need it, including at least a few weeks of vacation each year.  Graduate school is a marathon, and solid, steady work will always win out.

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[1] I once came across a half-empty bottle of anisaldehyde from 1969.  It was >99% pure by NMR.

[2] The universal stain will show impurities that may not have delocalized bonds.

“Potpourri” for $1000: Candidacy and Thesis Questions

Tags

candidacy, defence, prelims, quals, studying, tests, thesis, thesis defence

One of the more challenging aspects of graduate school is the shift in testing from writing (exams, essays) to oral evaluation by a committee including your supervisor.  The format switch can leave students a little in the lurch, so I’ve done what I can below to demystify the tests and help with preparation.

Quals/Prelims/Candidacy Preparation

Breadth, not depth, is the hallmark of this exam. Positioned either at the end of formal classwork, the rough midpoint of a PhD program, or just prior to thesis writing, the exam of many names is designed to ensure that students are familiar with their field as a whole, not just their own research. This generally means understanding the mechanism of every reaction that you have performed, key work from related labs (in synthesis heavy programs this encompasses most named reactions), and the theoretical underpinnings of the techniques you practice. At best the experience is like a single-contestant version of Jeopardy, at worst it’s two hours of hospital rounds, just for you.

Sample questions:

Please draw pulse sequence for the 1H proton and 2D COSY NMR experiments on the board/Draw all the 4f orbitals/Draw and name all five-member heterocycles containing oxygen, nitrogen or sulfur.

Given line broadening in an HPLC trace, walk us through the steps you would take to improve resolution, assuming multiple causative factors.

Draw the mechanism of the Wittig reaction and explain why unstabilized Wittig reagents give cis-alkenes.  Which three alkene-forming reactions give predominantly trans alkenes?

How are ethyl acetate, acetonitrile and N-methylpyrolidone produced on an industrial scale? (Evil)

 

Preparation

To handle the basic theory questions run through a couple of your sub-field’s intermediate level textbooks–the sort generally used by good 3rd or 4th year undergraduate [1]. Once you feel comfortable describing the inner workings of named reactions and NMRs, read up on your committee members’ research. Most of their questions are going to come from their field of interest, so if you’re familiar with a few of their recent papers and the basics of their sub-field you’ll have quite the edge.

Once the essentials are covered spend a day or two thinking up potential questions. If you have supportive labmates, ask them to quiz you en mass, to mimic the rapid switching between topics.  Bring a water bottle to the exam, and get plenty of sleep the night before.

Expect lots and lots of sketching during the candidacy.

I can guarantee that during the exam there will be questions asked that you can’t answer (including a few that have no known answer).  When this happens tell the committee that you’re uncertain, but offer your best guess and a follow-up experiment [2].  You don’t have to be right every time, but do your best not to be wrong.

The Thesis Defence

There is only one expert in your research, and that’s you.  In a small lab your advisor is probably steeped in the planning and execution of your work, but even their knowledge pales in comparison to what you’ve learned over years of dedicated study.  As a result, don’t expect your committee to dive into the minutiae of your work, looking for flaws in the experimental design and conclusions.  They know less than you (and are well aware of that fact), and generally aren’t eager for their ignorance to be exposed.

Outside of a few warm-up questions, most of the defence falls into one of two streams:

1) Back to the Fundamentals

The committee may not be comfortable in a back and forth on the fine details of your research, but they’re quite at home chipping away at the edges. Your thesis will be used as a springboard to broader questions on experiment design, troubleshooting, and basic chemical principles [3].

Sample questions:

The purity of your compounds was assessed via NMR, HPLC and HRMS. How did you quantify the concentration of various non-fluorescent salts, like sodium trifluoroacetate?

You state on page 173 of your thesis that compound 87 did not bind to the enzyme of interest. How was this determined? (Followup) How could you have assessed binding in the absence of fluorescence/ITC/NMR measurements? (Followup) What other means of assessing binding could you use?

How accurate are your yields? What would be required for statistical significance? (Evil)

2) What’s the impact?

Being a research scientist is (unfortunately) as much about promoting your research as doing it.  Regardless of whether your career lies in industry or academia a good committee will probe your ability to defend your work against attack, as well as see the see the larger picture of how it can benefit others and change our understanding of the world.

Sample questions:

How does your synthesis of everestane compare to previously published work? In which ways have you improved our access to this compound?

As the foremost expert in your research, how does it affect our understanding of the cell/environment/”Famous-name” process?

What is the most important next step for this project?

Explain your research to us using only non-scientific words (a common question long before up goer five became famous).

Preparation

Depending on the dedication of your committee you are likely to see a bias in questions stemming from the first few chapters of your thesis. If you have received feedback on your thesis, the comments they have made there are also likely to reoccur during the defence. My preparation focused on brushing up on the basic chemistry (ie. mechanisms for every interesting reaction), reviewing comments made by the committee at earlier stages in the PhD, and brainstorming potential questions.

Get plenty of sleep for 2-3 days before the defence.  Don’t try to cram every last factoid in the hours before the defence, and instead focus on getting plenty of sleep for at least 2-3 days beforehand.  Common sense and proactive problemsolving will get you through many of the more difficult questions, but like the candidacy expect to be faced with the unanswerable. A defence is meant to determine the limits of your knowledge, and the only true way to assess that is to stretch you to the breaking point.

Good luck.

 

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[1] Clayden is good for most programs, though synthesis heavy subfields may require Carey/Sundberg or March. Most books are too big to actually read in their entirety, so focus your attention on likely chapters and skim the rest.

[2] Ex. Q: What is the acid stability of allylic epoxides?

A: I’m not certain, but I believe that the alkene would increase acid susceptibility.  To verify this a diene could be reacted with stoichiometric mCPBA, then run on a 2D TLC.  The epoxide of an equivalent alkene could be used as a standard, and off diagonal spotting would clearly demonstrate acid-based decomposition.

[2] If you are at all uncertain about devising reaction mechanisms from scratch read “The Art of Writing Reasonable Organic Reaction Mechanisms.”  It’s the basic, 2nd year stuff that will trip you up.

BRSM Blog Party: The British Superpower

Tags

accents, advice, brsm, brsmblogparty, brsm_blog, fun

*This post is dedicated to BRSM, on the occasion of his move to the United States*

Let’s talk language. Time, geographical separation, and the odd rebellion have pulled the United States and the United Kingdom apart, and this has left it’s mark on the lexicon. Strange words are in use in North America, while familiar ones like queue, titfer, chinwag and yonks have fallen out of favour (or never gained it)  [1]. Accents are also a little…different in America.To put it mildly, be prepared for the words you say to have a little more authority than you’re used to.

While you may think that this increased authority is limited to high-class English accents, in truth most of the people you meet no idea what a high-class English accent sounds like. From the rolling R’s of Scotland to the most incomprehensible Welsh sing-song, all British accents are appreciated, and all can lend authority to your words. Use this power for good: teach the undergrads of your new lab proper safety protocols and thrill graduate students with strange and mysterious tales. However, beware the faculty. Most professors travel regularly and are quite inured to the powers of your accent.

Outside the lab, consider giving back to the community. Volunteer to record automated messages for airports and subways, where your words can ensure that baggage is not left unattended and travelers stay behind the painted lines. Spend time at the nearby clubs and bars, thrilling locals with tall tales and “traditional” drinking songs. And in your travels be tolerant of the strangers who crack jokes about British dental care [2] and insist on calling you “guv’nor”. The Irish have it far worse.

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[1] “Favour” has also fallen out of ‘favor’. You may as well throw half your u’s out now.

[2] This joke comes from an early Simpson’s episode. Memorize quotations from the first eight seasons of the Simpsons and you’ll blend in easily.

Discovered by Mistake: A Practical Synthesis of Indigo

Tags

BASF, dye, indigo, mercury sulfate, mistake, serendipity, thermometer

Here’s a neat addendum to the “Compounds Discovered Entirely by Mistake” post, courtesy of my new favourite non-fiction book “Eurekas and Euphorias: The Oxford Book of Scientific Anecdotes.”

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At the turn of the 1900’s synthetic organic chemistry was beginning to coalesce into a serious art.  Infrared and NMR spectroscopy were both more than half a century away, but combustion analysis was allowing relatively facile assignment of molecular formulae (molecular structure was a bit of a guessing game).

Of great interest at the time was the production of new synthetic dyes, as well as synthesis of dyes found in nature.  Of key concern was indigo, isolated from plants of the genus Indigofera.  Since ancient times indigo had been produced in India and sold to Europe, and over the past hundred years the East India Company had helped transform the humble dye into a major cash crop for the British [1].  Locked out of India and frustrated by the British prices, German companies began to produce the dye synthetically.

Given the limited techniques available, indigo was a challenging target for early chemists.  Much of the early work was completed by Alfred Baeyer, who over twenty years determined the true structure and developed an early, impractical synthesis (this work in part earned him the 1905 Nobel Prize in Chemistry).  One of the more promising commercial syntheses began with the readily available napthalene, which was oxidized to phthalic anhydride in fuming sulfuric acid (H2SO4/SO3).  The reaction was slow but effective, and the low cost of the starting material helped make the the final synthetic indigo competitive with natural sources (if barely).The breakthrough came when a worker at BASF’s indigo plant, Eugene Sapper, decided that one batch of oxidizing napthalene was in need of stirring.  Lacking a tool appropriate to the task he decide to use a mercury thermometer, which of course broke under the strain.  In the acidic, oxidizing environment the mercury was quickly converted to mercury (II) sulfate, a hitherto unknown but very effective catalyst for the oxidation of napthalene.

Other workers at BASF, notably Karl Heumann, were quick to capitalize on the discovery, and seven years later large quantities of synthetic indigo began to flood the market.  Fifteen years after that production in India had all but ceased, never to recover.

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[1] The Indian farmers did not benefit significantly from the profits collected by the EIC.

Compounds Discovered Entirely by Mistake

Tags

mistake, serendipity

Whether the product of poor lab housekeeping, clumsy researchers, or an inexplicable urge to taste everything, there are some discoveries that would never have been made on purpose.

1. Artificial Sweeteners

Until the introduction of neotame in 2002, every common artificial sweetener had been discovered by mistake, usually by a chemist accidentally ingesting one of their compounds [1]. This trend started with saccharin in the 19th century, when Constantin Fahlbert was working in the lab of Ira Remsen.  After a long day of creating new toluene derivatives, Fahlberg left for dinner without washing his hands.  Breaking open a dinner roll, he found the bread to be unnaturally sweet, and eventually traced the cause back to a chemical residue left on his hands.   Together with Remsen he purified the residue and together they published a paper titled, “On the Oxidation of ortho-Toluenesulfonimide” (in German of course), where the effect was detailed.  Several years later Fahlberg optimized the synthesis, patented the results, and began industrial production of saccharin—all without telling Remsen.  Saccharin has had an interesting history since then, and was the first product ever sold by Monsanto.

The Remsen-Fahlberg synthesis of Saccharin. Via wikipedia.

In almost a shot-by-shot repeat, almost ninety years later aspartame was discovered by James M. Schlatter. While synthesizing a tetrapeptide subsequence of the hormone gastrin his flask bumped, splashing aspartame (in methanol) over his hands. Unfazed, he continued lab work, until a little later he needed to pick up a piece of paper.  Licking one of his fingers, he initially thought his hand was covered in sugar, eventually tracking the taste back to his recrystallization flask.

Even among sweeteners the story of sucralose deserves special mention, as the only agent discovered accidentally-on-purpose. After hearing that his student Shashikant Phadnis had prepared the chlorinated sucrose as part of a search for new insecticides the (presumably Scottish) Leslie Hough said to “tæst it.” And so Phadnis did, dropping a small amount of the powder onto his tongue via spatula. He reported back to Hough, who was soon adding “serendipitose” to his morning coffee.

2. Polymers and other Materials

Polymer science also owes quite a bit to lab mishaps, starting with the (apochryphal) story of Charles Goodyear accidentally mixing rubber and sulfur on a hot stove. The saran in Saran Wrap first appeared as an off-green, impossible to remove residue on the inside of the vials and beakers at Dow Chemical, and was plucked from obscurity by Ralph Wiley, a college student hired to clean the glassware.

In another cleaning mishap, Patsy Sherman saw the promise of scotchgard when few drops of the polymer spilled onto the shoe of a research assistant. The stain on the shoe couldn’t be removed by soap or organic solvents, but also shrugged off dust and dirt. Together with Sam Smith the formulation was optimized, then marketed as a stain repellant.

Regardless of the original field of research, serendipitously discovered polymers usually have immediate applications. Such as it was for the invention of laminated (safety) glass, which almost literally fell into the lap of Edouard Benedictus.  Grabbing reagents from a high shelf via ladder, Benedictus accidentally knocked down an unrelated beaker, shattering it instantly.  Back on the ground,  he discovered the glass had largely retained it’s shape, held together by the cellulose nitrate stored inside. After a furious twenty-four hours of (rather expensive) experiments triplex was born.

3.Pharmaceuticals

Entire drug classes have grown out serendipitous side effects, with Viagra probably the best known. The list is a little too large to cover succinctly, so instead I’ll direct you to this classic paper on the subject.

Focusing on compounds discovered through poor lab technique, in 1976 Barry Kidston was a graduate student at the University of Maryland.  Kidston had a rather unhealthy interest in narcotics, and for several months had been synthesizing the analgesic desmethylprodine (MPPP). His technique was on a slow decline during this period, until in a rush one day he accidentally overheated the final reaction step.  Elimination occurred, contaminating the MPPP with MPTP.  Unaware of his mistake and electing not to recrystallize the product, he injected the mixture.  Within three days Kidston was in the hospital with what appeared to be an acute case of Parkinson’s disease. The condition proved largely permanent, though the role of MPTP wasn’t confirmed until several years later [2].  Like a true case of Parkinson’s disease his symptoms responded to treatment with levodopa, and MPTP eventually found a role in research, inducing model Parkinson’s in primates.

On the happier side of history lies LSD-25, lysergic acid diethylamide.  With an effective dose of just twenty micrograms, it’s actually a testament to the lab skills of chemist Albert Hofmann that when the chemical was first synthesized its effects as a psychedelic went completely unnoticed. Created during a hunt for new analeptics, early research in LSD showed little biological activity, save for “marked excitation” in some lab animals. Shelved for five years, it was during the second synthesis of LSD that Hofmann was accidentally dosed. When symptoms appeared Hoffman excused himself from work for the rest of the day (it was a Friday afternoon), and went home to rest. Lying on a couch at home he then experienced the world’s first acid trip, as documented in an incident report:

Last Friday, April 16, 1943, I was forced to stop my work in the laboratory in the middle of the afternoon and to go home, as I was seized by a peculiar restlessness associated with a sensation of mild dizziness. On arriving home, I lay down and sank into a kind of drunkenness which was not unpleasant and which was characterized by extreme activity of imagination. As I lay in a dazed condition with my eyes closed (I experienced daylight as disagreeably bright) there surged upon me an uninterrupted stream of fantastic images of extraordinary plasticity and vividness and accompanied by an intense, kaleidoscope-like play of colors. This condition gradually passed off after about two hours.

By Monday morning he was fully recovered and curious to discover the root of his illness. Assuming that one of the chemicals he had been working with was responsible, he started by ingesting a miniscule 0.25 mg of LSD, not realizing the drug has an effect at a tenth that dosage. The results were “rather dramatic.”

[…]I asked my laboratory assistant to accompany me home as I believed that I should have a repetition of the disturbance of the previous Friday. While we were cycling home, however, it became clear that the symptoms were much stronger than the first time. I had great difficulty in speaking coherently, my field of vision swayed before me, and objects appeared distorted like images in curved mirrors. I had the impression of being unable to move from the spot, although my assistant told me afterwards that we had cycled at a good pace…. Once I was at home the physician was called.

This Friday will be the 70th anniversary of Hofmann’s second dose, celebrated by some as “Bicycle Day.”

[1] Lead (II) acetate may be an exception to this, but I’m willing to bet that the Romans didn’t intentionally add crushed minerals to their food.

[2] Unfortunately, in 1982 another sloppy chemist started making MPPP, this time dealing the results to heroin addicts.  At least seven people developed severe Parkinson’s.

Academia and the Art of Haggling

Tags

assistant professors, career advice, negotiating, postdocs, winter reading list

Outside of the odd garage sale or car purchase, haggling has faded from everyday life in North America.  Research is no exception, but there is one brief window in an academic’s career where haggling is not only a good idea, but is expected.  In the small gap between when a university offers one of their applicants an assistant professorship and the applicant accepts some rather heated negotiations take place.  I alluded to this in the subnote of a previous post, and on reflection this is a topic that could use a little more oxygen.

Due to the investment of the hiring process, this is the brief moment in any academic’s career where they have true leverage, and in theory nearly any aspect of the an assistant professor’s life is up for negotiation.  In practice most of the back and forth is around a few key points:

Salary

Moving Costs

Spousal job opportunities (for academics with the two-body problem)

Early year teaching loads

Startup funds

The Negotiation in Context

The issue of salary usually gets top billing, but is generally difficult to negotiate.  It’s a simple matter to determine median salaries at universities in Canada (pdf) [1] and the US, but many faculties will refuse go give ground regardless of your negotiating position, or are forbidden from doing so by union contracts.

Of far greater importance is the issue of startup funds.  Early grant-free funding is key to establishing an effective research program, by making it easier to produce low-impact papers on the preliminaries of your work.  These early papers make it far easier to obtain the first external grant, which eventually leads to more personnel and greater funding.  During the negotiations the department head (and sometimes even the dean) is your ally, as they have a vested interest in your success.

Unfortunately, it’s difficult to know what to request in the negotiations.  The size of the startup package varies wildly from candidate to candidate, and unlike salaries there isn’t much information on “standard” packages.  Worse, online accounts vary from $50,000 to over $1,000,000 (Bio).  More prestigious universities will of course offer greater support, but greater transparency is obviously needed.  To put us all on the same page, I’ll put forward the best consensus I could establish.

Your startup package should fund all aspects of your first 2-3 years of research.

This includes all reasonable equipment, as well as salaries for 2-3 hires (graduate students, undergraduates and perhaps a postdoc).  Values in the US are generally around the $400-600k mark, depending on equipment and staffing needs.  Canadian packages tend to be about 3-4 times lower (ie. $100-200k), though they appear to be on the rise.

The gap comes from the nature of funding in Canada, which depends on several disparate agencies.  At some Canadian universities professors can make their “big-ticket” equipment purchases under Canadian Foundation for Innovation (CFI) grants, negating about a third of the US startup funds.  In the past staffing needs were also mitigated by early career NSERC discovery grant (about $20k/year at the 1 year mark, enough for a single graduate student), but the success rate has dropped to around 60% in recent years (pdf).  This is having an effect on the size of startup funding, as early-career researchers can no longer rely on early external funding.

Know What You Need

Prior to the job interview draw up a list of everything you will need in your first three years, itemized and priced out (example).  Prioritize your list, with a few nice-to-have items added on as bargaining chips, and be prepared to put every purchase in context, linking equipment back to the experiments it is needed for and cogently explaining why you need the funding to succeed.  Also, don’t be afraid to ask for the moon.  Obtaining funds once you’ve been hired can be difficult to impossible, and if you’re in demand anything is possible.

Things to Push For

• Support for two graduate students for two to three years.
• Enough funds to cover consumables for the above students (~$9k/year for organic chemistry research, closer to $20k for molecular biology).
• A waiver on teaching for the first year (two years if possible).
• Deferred or preferential committee assignments [2].
• Reduced teaching load through to tenure review (ie. 2 courses/year instead of 3).
• Time on shared equipment, and funds to cover said use.
• Support for an undergraduate student during the first summer.
• (US Only) Summer salaries for the first 2-3 years.
• (US Only) Big ticket equipment.

Things to Watch Out For

• The amount of money required to support a graduate student varies by university.  Make sure that your offer letter adjusts for this.
• Your salary/summer salary should not be drawn from the startup funds, unless this was built in during the negotiations.
• If you are offered time on shared equipment in lieu of an equipment purchase, ensure that money to cover usage fees is added to the startup funds.
• Beware hiring freezes.  A quick hiring process is far less susceptible than one that drags out for months.
• Funds should not come with conditions on their use (ie. $50k must go to item X).  If the department prefers to pay for specific items, the negotiations should focus on the items themselves, not dollar values.
• For accounting purposes startup packages usually come with a “spend by X date” clause.  Make sure you have 3-4 years to spend the funds, to secure against the possibility that your first grant application is unsuccessful.

And of course, the most important note.  If it isn’t in writing, it wasn’t offered.  Get everything written down, even if you need to offer to write the offer yourself.

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[1]  Like the long form census, Stats Canada is no longer collecting this information.

[2]  Proactive committee involvement is a requirement for tenure.  As a result, it can only be deferred, not waived.  Not all committees require equal time commitments though.