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Life in the Drug Labs

Dig the New Breed

You know you’re getting older when techniques that you used to use constantly are in danger of becoming lost arts. The one that I’m thinking about today is thin-layer chromatography, or TLC. This is a classic lab method, taught to generations of undergraduates and used by untold hordes of working organic chemists. And it’s slowly on the way out.
Before we go into what’s killing it, a brief bit of background for the non-chemists in the crowd. To do a TLC, you take a plate of glass (or something else stiff, like thick aluminum foil) that’s been coated with a thin layer of some finely ground solid. The usual choice is silica gel, which is basically very pure, very finely ground sand. In its powdered state, it resembles a slightly grittier form of corn starch. In the old days, you’d spread this stuff out on the plates yourself, but it’s been twenty years since I saw anyone do that. During my whole career, you’ve been able to buy them premade, in all sorts of variations.
Then you take a drop of your mixture and put it on the silica layer, down near the bottom of the plate. Once it dries, you stand the thing up in a beaker or jar that has some solvent in the bottom – the idea is to wet the plate at the bottom, but not so far up that it rinses off your spot. As the silica gel layer wets, the solvent creeps up the plate. And (as in all the other forms of chromatography), the various compounds in your mixture will travel faster or slower, depending on their interactions with the silica versus with the solvent. A strong polar solvent (methanol) will tend to whip everything up with the solvent front, and a wimpy one (hexane) will tend to leave everything back at the start. Adjusting the solvent mixture can give you a spread of spots up and down the plate once you’ve let it run for a bit, and you can see those with a UV lamp, or by dipping the plate in some reagent that will generate colored material from your compounds. Excellent pictures of the process can be found here.

TLC is cheap, fast, convenient, and can be run in untold different variations. So what’s killing it? Something even faster, more convenient, and more powerful: liquid chromatography/mass spectrometry. That was just barely possible when I was in grad school, and was expensive and tricky when I was in my early years in the industry. But now the machines, while still not cheap, are everywhere, and they’re used in walkup mode. Just enter your data – or link it over from your electronic lab notebook – put your sample vial in the rack, and go away. What you get back is a better separation than TLC can give you, and every peak/spot now can be checked for the masses of the compounds in it. You can ask all the possible questions, such as “which peak has the mass I’m looking for?”, or “What the heck is the main mass in that peak, anyway?”. The mass spec gives you more information than you can deal with, and it’s all stored digitally for your later perusal and second thoughts.
This trend has been coming on for years now, but it’s reached a very noticeable point. Even a comparatively old-school guy like me hardly runs TLC plates any more. Once in a while, I’ll need to, but mostly, it’s just “throw it on the LC/MS”. And I get the impression that people coming through grad school now are losing the finer points of TLC completely. And why not? They’ve never had to worry about them at all. . .

17 comments on “Dig the New Breed”

  1. tom bartlett says:

    You still need TLC to monitor what’s coming off your Flash columns. Until they invent super-ultra high speed LC-MS, TLC is in no danger of disappearing.

  2. Chrispy says:

    Seems like the IR gathers a lot of dust, too. Everyone seems to jump right to NMR except for things which IR is really good at (e.g. azides).

  3. medchemist says:

    Who still does flash columns??

  4. weirdo says:

    Flash columns? Those are disappearing, too. Our last hold-out gave those up a year ago.

  5. Jose says:

    Not to mention 2-D TLC to assess the stability to silica.

  6. VT-Ken says:

    I wish I had the toys you industry folk apparently have. In all but the most well funded academic labs, TLC is still prevalent as is flash chromatography.
    Any industry folk have a LC/MS or hell just a ESI-MS they’d care to donate? We would love you…
    Grad student Ken of VT

  7. startup says:

    I laughed so hard after reading this I almost cried. All I ever hear from our analytical guy is “your sample is no good” or “I wish you talked to them about buying more columns”.

  8. mosqismimi says:

    I beg to differ. After 7 yrs as medicinal chemist in a big pharma, I still run TLC everyday to check my reaction using different stains. Not everything is visible in LC/MS.

  9. anon says:

    Has anyone around here used a µHPLC (my HTML code should render as ‘Greek letter mu’HPLC) system? (Last I looked there was one commercially available from Eire (anyone got the Co name? and no, I’m not shilling for ’em). (The electroosmotic pumps are particularly cool; it’d be worth buying the instruments to cannibalise it for the pumps).
    Similarly, µGC has been around since the 1970s; anyone got a link to the picture of the GC column etched into silica which is also part of the GC oven? It’s about the size of a penny. Anyway, I’m not aware whether or not this instrument is commercially available. Anyone around here know different?
    In short – can anyone comment on their experiences / knowledge etc of micro analytical systems in a commercial environment.
    (I’d be interested in whether or not they are ‘plant compatible’ for in-process checks. Whether or not people have done pharmacopeoal (sp?, ie USP, EP) analysis with these instruments. And anything else that may be of interest).

  10. DrSnowboard says:

    Prep TLC still used at WuXi I believe…
    And I totally agree that you can get a crap TLC from a single peak LC-MS…and the NMR usually follows the TLC. 2.5min LC-MS runs are a compromise, something most people conveniently ignore.

  11. LNT says:

    As long as normal phase chromatography is in use, TLC will not go away. Yes, we in industry seldom run traditional flash columns. However, we use Isco and Biotage type automated silica gel chromatography systems all the time. I don’t think too many people will throw their material onto any sort of silica column (automated or not) until they have run a TLC to get a rough idea of its retention on silica gel.
    I completely agree, however, that TLC for monitoring reactions is essentially dead.
    The technique that is more dead than TLC: distillation (especially microdistillation)

  12. TNC says:

    All these techniques are alive and well in academia. I have done all of these in the US in the last 2-3 years. Hell, I did a distillation yesterday in my pharma lab.
    LC/MS is great, granted. But it will only gain strong prevalence in academic organic chemistry labs when it’s very robust and maintenance-friendly and doesn’t have a squad of technicians to worry over it. Your average grad student doesn’t want to change her pump oil — this gal’s going to run tuning solution every 3 months?

  13. eugene says:

    “don’t think too many people will throw their material onto any sort of silica column (automated or not) until they have run a TLC to get a rough idea of its retention on silica gel.”
    I don’t see those kinds of people around me though. I do just that all the time and I don’t have a regular TLC setup anymore. After you’ve done about 100-200 columns, you have a pretty good idea of what polarity solvent mix you’ll need for a column of a certain size to get your product out well separated. TLC is just a waste of time for me now; I know what an extra oxygen (whether ether, ketone, or alcohol) will do to my retention times without it. I teach all my students to do the same — unless they have a lot of fractions after a column and it’s a simple separation and time can be saved with a quick TLC analysis. I actually do more distillations (well, on a 3 ml scale at least) than I do TLC now…
    The thing I was wondering about though, is in drug discovery, a lot of it is still based on small compounds, so why does HPLC have such a big advantage over cheaper GC systems? I love injecting stuff from a fraction I get off the column into a GC that’s turned on in the morning and seeing the result ten minutes later with much better accuracy than what I would get from a TLC. You can get a lot of information from a GC even before it’s attached to an MS detector since boiling points are very predictable and peak shape can tell you about the compound’s nature. And in a company, I’d imagine prep GCs would not be a problem?

  14. George D. McCallion says:

    As far as TLC goes, I insist upon using it. Many times it has saved the day (and an entire project, for that matter). Can’t get much easier when monitoring reactions, that’s for sure!
    A really good technique is QUANTITATIVE TLC. Oh yes, it can be done. The key to QTLC is making a suitable standard at the lowest possible level of detection.
    Also, another point that may be overlooked is the notion to have the SAME silica gel on the plate as that which would be used in the flash column.
    And YES, flash columns are still performed these days. Both manual & Teledyne Isco systems. I have used Teledyne and they are very good (btw, max size is 1.5kg). The suppliers are few (3 that I know of: Teledyne, Silicycle, and Grace-Davison).
    But this technique should not be discarded; rather one should keep the practice in-check by doing (simple) pasteur pipet columns. Although in the Kilo-Lab, this is considered as the ‘technique of last resort’.
    Perhaps a re-examination of the infamous W.C. Still article:
    Rapid chromatographic technique for preparative separations with moderate resolution
    W. Clark Still, Michael Kahn, and Abhijit Mitra
    J. Org. Chem.; 1978; 43(14) pp 2923 – 2925
    http://pubs3.acs.org/acs/journals/doilookup?in_doi=10.1021/jo00408a041
    This is worth a good read!
    Cheers!

  15. Jose says:

    My favorite part of the Still and “Con” paper is the time estimates- 10g in 10-15 minutes, all inclusive!
    Even after a decade plus of practice, I think it takes me at least that long to TLC the fractions…..

  16. zt says:

    Re Tom Bartlett (post 1): But flash columns these days have built in UV detectors to monitor what is coming off of them. Put your compound on, walk away, come back later, concentrate the fractions that the computer tells you to. I only use TLC when I have broad, poorly resolved peaks and I want a clearer picture of which compound is in which fraction.
    Prior to running a column, I will check the TLC if I am unfamiliar with the class of compounds and how they will behave on the column. If they are similar to other compounds that I already know separate well on a standard hexanes-EtOAc or CH2Cl2-MeOH linear gradient, I will rarely bother checking.
    Otherwise, I only use TLC when running reactions with compounds that are not UV active (which is not very often), or that are otherwise difficult to follow by LC-MS (e.g., they have similar retention times and the MS does not show a mass peak).
    Of course, my comments are from someone in a situation similar to that of the original post–an industrial setting where we have all these expensive toys. Grad students have to do it the hard way, but that just makes life so much better when you finally get the resources to do it the easy way.

  17. Stephen says:

    The price of of an LC-MS is prohibitive to just about every academic lab. For the same price, you can hire a post-doc for a year, or buy a microwave reactor set up; or maybe both, the way that microwave reactor prices are dropping back to earth.
    There is also the issue of bench space. Now I know the LC-MS doesn’t take up all that much room, but compared to a jar with a TLC plate in it….

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