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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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February 6, 2009

Prep TLC: The Good Old Days Live On

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Posted by Derek

I did something in the lab the other day that I hadn’t done in several years: run some preparative TLC plates. I had some small reactions that needed to be cleaned up, and the HPLC systems were all in use, so I thought “Why not?” (I wrote here about the decline of analytical TLC in general in some labs, and I think it's fair to say that the larger-scale prep version has seen an even steeper drop in use over the years).

Prep TLC, for those of you not in the business, is a pretty simple technique. You take a square glass plate that’s been coated with a dry layer of ground silica, a white slurry that for this application is about the grittiness of flour or ground sugar. You then take your mixture of gunk, dissolve it up in a small volume of solvent, and deposit it in a line across the bottom of the plate, an inch or so up from one side and parallel to it. Then you take a large glass container and add some solvent to the bottom of it, and put your plate in so that the streaked line of material is near the bottom. Here's one running.

The solvent soaks into the layer of silica, and after it gets up an inch or so it hits your line of stuff. As it continues to move up, soaking further and further up the glass plate, the different components of the mixture will be carried along at different rates. The compounds that stick to silica gel (for one reason or another) will lag behind, while the ones that don’t will move out into the lead. After an hour or so, the solvent line will be up near the top of the plate, and your mixture will now be spread out across it into a series of bands. (The TLC page at Wikipedia has some useful images of this). Up at the top, running with the solvent, will the the nonpolar stuff that didn’t have anything to slow it down. Right down near the bottom, not far up from your original streak, will be the most polar stuff, especially any basic amines – silica gel is mildly acidic, so the amines will stick to it very tightly indeed. And in between will be the other components, divided out according to how they balanced out the pull of the silica gel support with the attraction of the solvent moving them along. Sometimes you can see them as colored bands on the silica plate, but more often you shine a UV light on the whole plate to see them. The silica we use has an ingredient that makes it fluoresce green under ultraviolet, and our compounds usually show up as dark blue or purple bands against the green. It’s a color combination known to every working synthetic organic chemist.

You can see that picking different solvents for this process can change things a great deal. A weak solvent (like hexane) will allow almost everything to stick to the silica. (A compound has to be mighty greasy to be swept along by just hexane; I doubt if there’s a drug in the business that you’d be able to clean up that way). A standard mix is some proportion of ethyl acetate mixed with hexane. You can go up to straight ethyl acetate, or even further by mixing in methanol or the like. And if you’re desperate, you can go to most any solvent mixture you like – three-solvent brews, toluene, acetonitrile, acetone, whatever works.

So how do you get the things off? By the lowest-tech method you can imagine. You mark the position of the band (or bands) you want, and then take a metal spatula and scrape the silica there off the plate. You them dump that into a flask and stir it with a strong solvent, then filter off the silica and wash it some more to rinse your compound out.

This used to be much more of an everyday technique, but automated column chromatography (same principle, pumped through a tube) has taken over. But prep TLC still has its appeal. Done with skill, it can provide very clean compounds, with quite good recovery. In fact, its low cost and power have made it a favorite technique at places like WuXi, the outsourcing powerhouse in China. I've had several first-hand descriptions of their prep TLC room, with rows of plates being run, marked, and scraped in assembly-line fashion. It's the sort of thing you'd only do in a cheap-labor market, because of the unavoidable hand work involved, but it is effective.

I don't know where WuXi gets its plates, but if you make your own, it's an even cheaper technique (discounting labor costs, naturally). You take up the silica gel powder in water, make a thick, well-mixed slurry out of it, and spread it across a square of glass, shaking and tapping it to get the air bubbles out. Back when I was doing summer undergraduate work, I poured a number of these things, although it's certainly nothing I've had experience with since the first Reagan administration. For all I know, that's how WuXi does it now. Perhaps they've found a low-cost supplier of their own, but the idea of a cheap supplier for a Chinese outsourcing company is an interesting one all by itself. . .

Comments (32) + TrackBacks (0) | Category: Life in the Drug Labs | Pharma 101


COMMENTS

1. processchemist on February 6, 2009 9:13 AM writes...

A decade ago I translated in a POS all the operations involved in the production of an intermediate for a radio labeled diagnostic. Batch size of the final product: 30 mg. The final purification was a preparative TLC. A nightmare, from a production point of view, with heavy fluctuations in yeld (depending from: the hour of the day, the wheather, the season, the operator etc). Few months after the final purification was replaced by a preparative HPLC and things got a lot better. But I suppose that still can be a great technique and not only in the medichem field (isolation of impurities, f.e.)

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2. Nick K on February 6, 2009 9:44 AM writes...

A great technique for a purifying a few hundred mg. But make sure your technique is sound before committing a precious compound to a home-made plate - sometimes the silica can fall off the backing plate at the worst possible time, while you're scraping the product band off.

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3. milkshake on February 6, 2009 10:20 AM writes...

the silica used for pouring prep plates is quite different from column silica - it is typically finer grade and it should contain a binder (typically plaster, about 5%) and preferably also the europium-based UV-fluorescent indicator.

Also the used plates have to be scrupulously de-greased, and the drying of the produced plates has to be done best in a room with controlled temperature and humidity.

Merck EMD plates are not that expensive so most people in industry do not bother with making them.

I mourn decline of Chromatotron usage - this was a seriously useful instrument (up to few hundred mgs). It is particularly beautiful with brightly colored compounds.

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4. NH_chem on February 6, 2009 10:21 AM writes...

Another lost technique that can get you the necessary mg's for that assay and NMR. USed it a bunch in my day. Analtech makes good plates. This can actually be faster than HPLC.

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5. RB Woodweird on February 6, 2009 10:37 AM writes...

In the good old days, we used to prep a Curie or so of 3H or 14C-labeled radiochemicals on 500 or 1000 um plates. Watch out for the dust!

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6. MikeyMedChem on February 6, 2009 11:03 AM writes...

We had some multichannel prep-TLC plates back at my old company. They had four tracks of extra-thick layers of silica gel, with a celite band at the bottom for loading. The plates were even scored in between channels. You could load a couple hundred mgs on each channel, without being too careful, and run four compounds at a time. The cool thing about the celite loading zone is that you didn't have to be very careful about how you loaded (ie narrow line of sample), except that the sample had to be above the solvent level in the TLC tank. The solvent would move the big fat band up until it hit the silica gel, and when it arrived at the silica it was a nice, tight band. Loved those plates. Analchem, I think? Anyone else seen them?

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7. Jim Hu on February 6, 2009 11:29 AM writes...

This probably doesn't work on large prep scales, but we used to recover 32P labeled oligonucleotides from TLC plates by dissolving a nitrocellulose filter in acetone (IIRC, it was decades ago), and applying it to the desired area with a pipet. When the acetone evaporated, you'd just use forceps to pick up the nitrocellulose with the silica gel embedded in it, and redissolve it and extract what you wanted.

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8. CMC Guy on February 6, 2009 11:31 AM writes...

milkshake I too found great utility (and fun!) with the Chromatotron after spending years in grad school making/using prep TLC plates (which as Derek points out is very labor intensive). With that if separation was good enough I could load samples on same plate in delayed series and pull off the ring-bands desired as single collective fraction. I purchased one when first started at one place then when lab was closed down I tried to buy it but was unable to acquire unfortunately because they where no longer made as far as I could determine.

processchem you actually note the isolation of impurities by prep TLC which I did much more in the process realm than in medchem efforts where focused on the main component with usually little consideration of anything else.

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9. RandDChemist on February 6, 2009 11:39 AM writes...

While I certainly understand the benefits and ease of use of prep TLC, why not use a flash chromatography system (Biotage/Flashmaster, Varian/AnaLogix, and Grace come to mind as manufacturers). SiliCycle and Grace make excellent columns (best I've used in my experience), but my preference is for SiliCycle right now. Ever since I used a Flashmaster system, I've done everything I can to get a Flash system in if my employer did not have one. They are quite powerful.

Of course, if cost is an issue, then acquiring and using one is another matter.

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10. SR on February 6, 2009 12:13 PM writes...

Coincidentally, I'm running a prep TLC at the moment (grad student in a group without a lot of high end equipment). It works wonders for working up reactions with only a few mg or so of reactant. If I want half a mg of a very precious material that is free of impurities for a NMR experiment, it works great.

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11. Jose on February 6, 2009 12:14 PM writes...

R&D- prep TLC is actually faster and more efficient- no TLC to run or fractions to rotovap. Not that I choose to do it that way....

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12. Tok on February 6, 2009 1:03 PM writes...

Prep TLC using analytical plates is wonderful for forefront natural product synthesis. We've got Combiflash's, but with 1-2 mg you can't beat loading on an analytical TLC. The trick is getting all the stuff off the silica.

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13. transmetalator on February 6, 2009 1:57 PM writes...

So what is the best way to get it ALL off? People in our group always have debates about PTLC recovery being low.

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14. andreew on February 6, 2009 2:44 PM writes...

Does the stuff which gives the fluorescence leach into your solvents? I've never done prep TLC, and on analytical TLC, it doesn't matter...

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15. Anonymous on February 6, 2009 3:09 PM writes...

Did one of these not too long ago when the prep-HPLC method was streaking quite a bit and impuritites were showing up in my final product. I like to scrape the bands up and wash in fritted funnel with ~20-30% methanol in DCM. (It tends to get most all the product off). Of course you have to filter again through something finer after concentating to eliminate dissolved silica due to methanol.
Really nice technique for when the other stuff fails. Though not my first line of purification as stated before- its really labor intensive.

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16. MTK on February 6, 2009 3:28 PM writes...

When I was in graduate school I did a bunch of prep TLC's. It's very good when you don't have much material or when you have a lot of bands that tend not to separate too well using other chromatographic methods.

One thing that really helped was that someone in the group before I joined had made a homemade spotter. At least I think it was homemade. It basically consisted of a reservoir with a thin stainless steel tube dipping into it. The reservoir was on a little stand which was could be placed flush against the edge of the lab bench. You would then place the bottom of the TLC plate parallel to the edge of the bench, fill your reservoir with the spotting solution, and run the little spotter back and forth. Capillary action through the tube would deposit the solution onto the plate. You'd get a nice thin even line everytime.

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17. CMC Guy on February 6, 2009 4:01 PM writes...

#13 transmetalator most people probably use the most polar solvent in plate development to extract so if used 4:1 Hex:EtOAc would extract with 100% EtOAc. Like most stuff depends on the compound and may have to try different sorts of solvents. A drop of AcOH or NaOH, unless sensitive material, on occasion seemed to help release. MeOH was only used in extremes but could pull out inorganic (CaCl3 binder and/or Si?) that gave "boost" to yield. Typical would throw scrapings into a Fine frit funnel without vac then add solvent, swirl a bit, suck down with vac and repeat 3-5 times.

#15 andreew good question that I never observed but used mostly used home-made plates that did not have the fluorescencing aid. Even if covalently attached seems possible with a strong solvent that leaching might happen and per Murphy's Law the dye would hit or interfere with the assay system.

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18. MTK on February 6, 2009 4:24 PM writes...

Speaking of the good old days...

I was running a separation about two years ago and while I could print out the chromatogram, somehow the integrator wasn't working, so I couldn't see what the ratio of the two peaks of interest was. I'm not very patient, so I went old school. I copied the chromatogram at about 2x size, cut out the peaks with a pair of scissors and weighed them to get the ratio. About three days later when the integrator was fixed I re-ran the sample and found that the cut and weigh method was only off by about o.1%. I had to laugh.

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19. The Inorganic Gardener on February 6, 2009 6:32 PM writes...

I run TLC on that kind of scale all the time - I don't have the luxury of an HPLC and the one I sometimes use in another lab is used by people who are scared of radiation so they don't like me to run 35S and 14C compounds on it. Wusses. So, I use TLC, stain the plates, scrape off the spots and count the radiation by scint counting.

Incidentally, I do make my own plates because I like to use thick glass (easier to handle) and I've never found a cellulose plate in recent years that gave as good results as a handmade one.

tig

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20. 1 on February 8, 2009 4:34 PM writes...

yes, ptlc does the job decent enough that some times makes me wonder of the use of costly analytical and prep hplc in the synthesis lab. I wish to see the money spend else where. i bet an lc/ms is more useful than hplcs in the lab.

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21. Anonymous on February 8, 2009 8:26 PM writes...

Comment 20 said: "ptlc does the job decent enough that some times makes me wonder of the use of costly analytical and prep hplc in the synthesis lab."

I think the major advantage of prep HPLC is that it does most of the busywork for you. This is especially evident when you are routinely making libraries of, say, twenty or so compounds.

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22. FWF on February 9, 2009 10:45 AM writes...

Fell asleep last night thinking, "perhaps we'll split off 50-100 mgs of crude material so I can demonstrate Prep-TLC to a new graduate student." Thanks for the timely post . . . and reminding me of that thought.

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23. Kevin on February 9, 2009 12:34 PM writes...

Yea Chromatotron. We have one, and I'm the only one who ever uses it. The resolution is not quite as good as prep TLC, but there's no scraping. It is especially relaxing to watch colored bands elute.

I used prep TLC often in grad school for purifying greasy homoallylic thiols that had poor retention on flash columns in straight pentane.

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24. molecular architect on February 11, 2009 4:33 PM writes...

I'm also a big fan of the Chromatotron. Much faster for purification of 20 to 1000 mg than is any prep hplc, if you include all the post-separation steps. They were still available from Harrison research as of 2004 and Analtech offered a chromatotron clone (was not nearly as well made) back then. When I arrived at my previous employer, there were several chromatotrons sitting unused in most of the synthesis labs. I started using ours and once others saw the benefits, they followed suit. Soon had to order a second one for my lab. Low tech but high quality and fast separations, as long as you can use non-aqueous eluants.

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25. milkshake on February 11, 2009 9:25 PM writes...

I was in the lab where they were making amino-substituted ortho-quinones and they purified their rather messy reactions on Chromatotron. They had all colors of rainbow spinning on that thing - including beautiful blues and greens. Such were the joys.

One concern with Chromatotron was that since the silica disks had to be regenerated, there was a nonzero chance of tailing and poorly soluble compound sticking the disk and leaching into the subsequent purifications. When one works with super-high-activity compound that are bricks (a comon scenraio in kinase projects), followed then by mediocre active ones, one has to watch for cross-contamination.

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26. pasupathy on March 7, 2009 5:43 AM writes...

What about the fluorescence indicator coated in the silica gel. Does it get leached. Most of the times u get a very tall and broad aliphatic signal at 1.25 ppm when purified by prep tlc. Can some one tell me what does this due to. This is very typical for those cmpds purified by prep tlc.

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27. James on April 6, 2009 12:55 PM writes...

Does anyone have any good resources for how to prepare your own plates? We have some of the silica gel with binder for the chromatotron, but I haven't found anyone in our labs that have made PTLC plates.

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28. Mido on April 11, 2009 4:04 PM writes...

pasupathy on March 7
I admire you on noting on the 1.25 ppm signal, I would say may be due to a some sort of an organic binder! I have noted it with ready made TLC plate, self-made plates don't release such signal???

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29. Mido on April 11, 2009 4:11 PM writes...

James on April 6
Dear .... try the chromatotron manual, I remember using this manual to make my own plates, it accurately describes how much silica and how much water you mix together to get the right slurry for every plate thickness, it also tell the preparation ritual for the plate. If you donot have it try to download it

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30. fad on November 21, 2010 9:24 PM writes...

hey, does anyone know where can i buy the instrument to make the square PTLC.... it will be a lot cheaper if I make it, our lab use it a lot.... i mean A LOT...

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31. Sumanta Ray on February 28, 2011 6:16 AM writes...

Respected Sir,


Sir I am a research scholar from Jadavpur University, India. Sir I have a small doubt and I will be grateful to you if you clear it.

Doubt: Sir when I am running the sample in readymade TLC plates they are showing 4-5 equidistant prominent spots but when I am running it in hand made preparative plates I am unable to visualize them in the UV chamber. When I am putting them in iodine chambers then they are showing 2-3 bands and the bands not so much prominent in naked eye. It becomes a bit tough to find and scratch them out. Sir is there is any problem in using readymade TLC plates? as because readymade TLC plates gives me very prominent bands as compared to handmade plates and will help me to cut out the bands appropriately. I need your help and if my thinking is wrong then sir kindly explain it.

I will be waiting for your reply.

Thanking you,
With regards
Sumanta Ray

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32. student on March 27, 2014 7:15 PM writes...

As an RNA structural biologist, this is one of the times that I have to say that the chemists get it easy. Want to take a guess at what the most widely used RNA purification technique in the field is? That's right, you pour a giant polyacrylamide gel (generally 32 cm x 40 cm x 4 mm) and run your RNA out on the gel. Then you mark the band, cut it out of the gel, crush it and elute. Pretty similar to prep TLC, but these gels take hours to pour correctly and 10-12 hours to run, and you might have to run lots of these to get enough RNA for an NMR spectrum or crystal tray. And if there is any RNase contamination in your 500 ml of gel, you are hosed, start over tomorrow (or tonight, whatever). No other technique gives you the resolution of a gel, not even a $5000 HPLC anion exchange column, so we are still doing the low tech thing for the foreseeable future.

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