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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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May 11, 2006

A Day at the Rota-Vap

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Posted by Derek

I've been spending a good part of the last couple of days rota-vapping down toluene. Why would I do such a thing, you ask? Because I ran a big column in the stuff, first time I've ever done that, I think. The chemists in the audience will guess that I examined many alternatives before settling on this one, and they are correct. Toluene's not especially toxic or smelly, but it is rather high-boiling compared to most of the solvents that we use for purifying things. You have to turn your water bath up and allow for plenty of time when you're taking a lot of it off. In this case, it turned out to be by far the best solvent for separating two closely running compounds.

Here's how I got there - it's a good illustration of a day-to-day chemical problem. I wandered through all sorts of common (and uncommon) solvent mixtures, checking each by thin-layer chromatography (a quick way to see if things separate). In ethyl acetate/hexane, the standard brew, you could just tell that there were two things in there after running a TLC plate three times. Switching to ether/hexane, which sometimes does the trick, was no help. Straight dichloromethane gave too fast-runnig a spot, and mixtures of dichloromethane-hexane didn't separate anything. Chloroform/hexane, on the other hand, wasn't too bad - not as good as toluene, but at least you could see some daylight in between the two spots. Isopropanol/hexane did no good at all.

I was trying here to run a bunch of different solvent types. Hexane is a common theme, since it's pretty much the plain vanilla of solvents. More polar stuff is added to it, and you see what happens. In this case, an ester co-solvent did a little bit of good, but ether and alcohol additions did nothing. Chlorinated solvents showed some promise - well, at least chloroform did. But it's an oddity, more polar than the others of its kind. Toluene was the only aromatic solvent I tried (it's really the only convenient one for chromatography), and something about its flat shape and electron clouds did the trick. This stuff is brutally empirical.

So toluene it was, with a little ethyl acetate at the end to speed the latter spot along. I got some mixed fractions, naturally, but quite a few clean ones, certainly enough for my purposes. (If I get bored or desperate, I can go back and re-run the mixed ones).

Comments (41) + TrackBacks (0) | Category: Life in the Drug Labs


COMMENTS

1. secret milkshake on May 12, 2006 3:59 AM writes...

Apart from the teflon pump, I have an oil pump hooked up to my Buchi. Puls DMF, DMAc, even DMSO nicely, at decent temperatures. The rotavap condenser is dry-ice cooled (I have additional Savant electricaly-chilled cold trap before the pump). I get down to about 0.5 Torr with the rotavap spinning.

But I won't use oil pump for toluene - toluene goes with (good) teflon pump from 30C bath, at about 5 Torr.

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2. George Laszlo on May 12, 2006 8:16 AM writes...

Now, I'm not a chemist and you can tell me to shut up if that makes sense. But, has no-one else already perfected this method so you don't have to play around? I'm asking this because it begs a question about the level of cooperation going on in the scientific community. Certainly the Interet is a big help (as the comment streams on these blogs show) but there are also good ways to collaborate with peers within your own company. Is this happening? If so, is it at the appropriate scale? What are the factors that hold back collaboration? Technology, willingness, corporate support, vanity, NIH syndrome?

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3. tom bartlett on May 12, 2006 8:21 AM writes...

I wish benzene didn't have that nasty tendency to cause cancer! Great solvent; lower boiling and less viscous than toluene, but just too toxic to use!

I've contemplated trying fluorobenzene for columns on occasion (bp=85), but it would be too costly for the scale-up guys down the hall.

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4. Derek Lowe on May 12, 2006 9:24 AM writes...

Tom B., I'd never thought of that one. Fluorobenzene would probably be a great chromatography solvent - just a bit of polarity, all those pi-electrons. I'll have to try it for TLCs, at any rate. But you're right, it's not the sort of things that scales up (not that the scale-up guys want to hear about tough chromatographys to start with. . .)

And for George L., it's strange but absolutely true, which is a big reason I wrote about this. Chemistry is full of things like this which are empirical right down to the tiniest detail.

There is truly no decent way to predict which combination of solid phase support and liquid phase eluent will be optimal for a given separation. You can make some broad statements and some educated guesses for starting points, but you just have to get in there and mess with it. No amount of co-operation will help with stubborn, atomized, every-one-unique problems, and we sure do have a lot of 'em.

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5. Atompusher on May 12, 2006 9:31 AM writes...

I've used Toluene/Acetonitrile mixtures with great success on polar organic and organometallic molecules. Good at dissolving stubborn molecules, but it takes forever to remove the solvents. I once heard of a friend of a friend of a friend who was running 60/40 benzene carbon tetrachloride columns for his project.

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6. qetzal on May 12, 2006 10:11 AM writes...

Derek,

You mentioned the solid phase support in comment #4, but the original post only talked about different solvents.

Did you try different supports? Or is that not usually as productive in your field?

I'm curious, because my (fairly limited) chromatography experience is with biomolecules (proteins, nucleic acids). In that area, it seems like there's much more emphasis on trying different solid phases, and perhaps less emphasis on the mobile phase (other than to vary the concentrations of standard components).

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7. Chemist of Sorts on May 12, 2006 10:17 AM writes...

I was curious what combinations people like to use for very polar compounds? The standard is 10% methanol/chloroform (or DCM) sometimes with ammonia hydroxide. I have had poor luck getting good resolution with that system so that if I need to do a tight separation, I am SOL. I have had a little better time if I swap out the CHCl3 with 1:1 aceteone:isopropanol. TLC is easier since you can use systems like 3:1 toluene:acetic acid.

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8. Derek Lowe on May 12, 2006 10:36 AM writes...

We have some other solid phases around, but I figured that I could get away with good old silica. It took less time and effort to mess around with solvents and silica than it would have to round up the other solid-phase possibilities (and their associated TLC plates to see if they worked). It's quite possible that there would be a much better solution lurking in there, but I went with "good enough". As we often do.

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9. Milo on May 12, 2006 10:37 AM writes...

Derek, First time with toluene eh? Welcome to the club ;-)

I have spent a great deal fo time running toluene and toluene/pyridine (10:1) columns. I find that for close running aryl compounds, you just can't beat toluene mixtures (has to be aryl-aryl interactions...). I even keep a bunch of premixed toluene based TLC solvents on the bench.

Interestingly, I have found that a certain set of aromatic compounds I have recently made run well in toluene/ether and hexane/DCM. However, the order of elution is completely opposite (!). My product comes off first in the toluene system and second in the DCM.

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10. Demosthenes by day on May 12, 2006 10:42 AM writes...

One of the little tricks I use for very polar compounds is if they're acidic I add 0.1% acetic acid to my solvent or if basic, 0.5% TEA. I've found it really helps to sharpen up the bands. If that doesn't work I will run very gradual gradients to tease the spots apart. Sometimes as low as 0.5% of the polar component of the eluent every 4 column equivalents. Time consuming as well but it allows me to use easy to evaporate solvents. My time is spent in front of the column instead of the rotovap. It all depends on which poison is more to your liking. Like crystallization, chromatography is where we are more alchemist than chemist.

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11. Milo on May 12, 2006 10:46 AM writes...

Re #8:

I routinely use (MeOH/NH3)/DCM for very polar compounds. The trick is to not use ammonium hydroxide (the H2O screws things up), but to bubble dry NH3 into dry MeOH. I think at room temp MeOH can be at most 9M in NH3. The beauty of this system is that you can run 100% MeOH/NH3 and not dissolve the silica gel (to any real extent). If you run straight MeOH (or even 30%MeOH/DCM), you'll dissolve lots of silica. Also, pretreating the silica with 1% (MeOH/NH3)/DCM removes the "solvent front" effect (the solvent front pushing everything down quickly).

For carboxylic acids, I always try DCM (or chloroform)/MeOH/HOAc at something like 90:7:3 (by vol).

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12. anon on May 12, 2006 11:06 AM writes...

I like the methanolic ammonia/dcm system as well. Aldrich sells a 2M sol'n of ammonia in methanol, which is a bit more convenient than making it yourself.

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13. Mark on May 12, 2006 11:07 AM writes...

Milo:

Being aware of the reversal of spots when going to halogenated tlc systems is one of many gems my doctoral adviser turned me on to in grad school.

*does anyone out there have a good rationale for this behavior?

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14. secret milkshake on May 12, 2006 12:32 PM writes...

Spot order reversal: If you have your typical hexane-ethyl acetate mix rich in hexane, many compounds will not be greatly soluble in it (and you may have to apply the mix in small amout of dichloromethane or toluene). So with hexane-rich mixtures, you get separation ased on polarity and on relative solubility also. In most cases you won't have the solubility factor when using the dichloromethane-based mixtures. So your order of elution can reverse.

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15. JC on May 12, 2006 12:54 PM writes...

Tri-nary mixtures that have worked for me in the past include DCM/Ether/hexanes 1:1:2

but mostly EtOAc/hexanes, DCM/MeOH (from between 0.05 to 20%) or chloroform/acetone

& for the record 2,2,4-trimethylpentane offers no noticeable advantage over hexanes

Toluene sounds a little nutty but so is recrystallizing from DMF (and that worked the other day awesomely) :)

I find that when loading the column and eluting the first few 100's of mL's that if I let it proceed slowly dropwise at first the separation is much improved

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16. Milo on May 12, 2006 1:42 PM writes...

Milkshake,

Good point with the solubility. The extreme is when your stuff crashes out mid-column, like what happend to me about 20 minutes ago... grrr...

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17. Chrispy on May 12, 2006 2:05 PM writes...


Does no one recrystallize stuff anymore? That used to be one of my favorite parts of synthetic chemistry. Now it seems everyone just does a flash step because it's faster. Seems inelegant, though. Of course, I'm sure the scaleup folks look very hard at recrystallization.

We used to bubble ammonia through methanol in an iced graduated cyl. When the volume stopped increasing you knew it was saturated. Careful, though, as the stuff in a tightly closed bottle can be a bomb.

Works great for cleaning glass, too!

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18. jose on May 12, 2006 2:39 PM writes...

>The beauty of this system is that you can run 100% >MeOH/NH3 and not dissolve the silica gel (to any >real extent).

Just to clarify, it is the binders and fluorescent indicator you are dissolving, and not the silica. After all, at the beach, the ocean doesn't dissolve the sand; it seems pretty unlikely that MeOH could dissolve silica.

Permalink to Comment

19. secret milkshake on May 12, 2006 3:08 PM writes...

I think the "dissolving silica' in MeOH is a thermal effect. MeOH strongly binds to silica and it produces considerable local heating. If you use only few % of MeOH on your column, MeOH first accumulates on top of your column. You often get a zone of MeOH-enriched front - which tend to push everything in front of it because it is warm and polar . If you dry-pack and do not compress the silica too much, you can get few cloudy fractions too, as it picks up the silica due to the turbulence. "Dissolving silica" is much lesser problem if you slurry-pack with the same solvent mix you will use for elution and if you have a decent medium-porosity sintered bottom on your column.

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20. Mark on May 12, 2006 3:11 PM writes...

**The column chromatography gurus are starting to come out of the woodwork!

Does anyone have any modified silica stories. Here's mine:

In order to purify a long sought after total synthesis target I had to prepare silver (I)-doped silica gel. It worked beautifully to separate my desired enediyne from several other regioisomeric compounds. But I had to run the column at midnight in near darkness and elute the samples into tubes that contained brine!

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21. Derek Lowe on May 12, 2006 3:16 PM writes...

I'm getting some good ideas for column solvents here, I have to say.

I'm with JC on the slow-drip technique at first - I do exactly the same thing. I think it helps because of the viscosity of the concentrated loading solution. If you put the pressure to it right away, that stuff fingers down the column randomly, but a slow gravity drip for a while gives everything a chance to even out.

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22. Derek Lowe on May 12, 2006 3:25 PM writes...

Silver-doped silica - that's a trick I haven't thought of in years. I've never run a column with the stuff, but I did make some TLC plates in grad school to try to resolve a couple of alkene isomers. They came the closest of anything I tried, but still didn't do it. I had to use a spinning band column, for the first - and I fervently hope only - time in my life.

As for Jose's comment about the beach, it reminds me of A. E. Housman:

"The toil of all that be
Helps not the primal fault.
It rains into the sea,
And still the sea is salt."

Okay, yesterday was Joyce.

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23. JC on May 12, 2006 4:04 PM writes...

Loading the goods on the column is always key.

Often I will dissolve it in a min of EtOAc/hex/DCM

MeOH and Acetone are to be used sparingly because they will cause excessively fast elution impairing separation.

Girth is better than length in these column situations (but long length (10 inches height for a 1 in diameter) column is not unusual)) And anything over an inch in diameter I don't 'flash' but use gravity exclusively, & dropwise at that.

Mostly I drip the concentrated loading solution (leave it spinning on the rotovap in a warm bath, nothing is worse than having the supersaturated solution crash out while you are preparing the column) directly onto the silica gel column, load it slowly, then once it is on add a cm or so of sodium sulfate to the top of the column. I use sodium sulfate not sand, always! Often if the loading mixture is full of polar solvents the first elution volume might be just hexanes; I am more in favor of isocratic elution tha gradients, it takes longer but re-running is a big time waster IMHO.

I often take multiple racks of fractions & for large columns my hood will be just filled with 125 mL erlenmeyer flasks. Easier to combine than re-run.

As far as loading the material to be purified onto silica gel via MeOH/Acetone/DCM then putting that solid on the top of the column, sometimes (infrequently) it works but mostly it's bunk, at least in my hands.

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24. secret milkshake on May 12, 2006 4:31 PM writes...

to avoid streaking, I prefer to load somewhat diluted sample solution (say, 1g of mix /10mL) in things like toluene or dichloroethane onto a column slurry-packed with a slower mix, then do isocratic elution with slightly faster system.

When your column fals and the product tails and you have 50 test tubes of mixed fractions and the material still did not completely came off the column, this Donne's verse summes it all:

Well, I may now receive, and die
My sinne indeed is great
But I have beene in a purgatorie
Such as fear'd hell is
A recreation to and scant map of this

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25. Mark on May 12, 2006 4:34 PM writes...

Milshake:

doesnt that verse nearly sum up the grad school experience itself?

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26. heeler on May 12, 2006 4:37 PM writes...

Dry loading on to silica with low solubility samples always worked great for me; once I switched to biotage or isco type systems (the isco was more user friendly IMO). I almost exclusively used this method instead of trying to set up a chromatography only to find I could not get my stuff to dissolve in the eluent.

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27. LNT on May 12, 2006 4:46 PM writes...

Since everyone seems to be putting in thier 2 cents worth, here goes mine:

I nearly always dissove my material in DCM/MeOH and evaporate it onto silica gel in order to load it onto my column. I can avoid most solubility issuses that way.

As for very polar compounds, DMC/MeOH/and anhydrous NH3 works wonders. But here's something that hasn't been mentioned yet: REVERSE PHASE. That's the best way to purify polar compounds. You can easily purify a couple grams of material using a Gilson HPLC and a medium size prep column. Has anyone used a reverse phase column on Biotage/Isco type systems?

It always amazes me to see people still using those crazy glass columns when we have plenty of the automated Isco/Biotage systems around -- so you can just load your material on and walk away from them and come get your fractions an hour later. In my experience I actually get BETTER seperation using an Isco than by the old manual glass column method.

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28. Milo on May 12, 2006 5:17 PM writes...

Re #18...

Jose, I was referring to a silica column. I normally collect the baseline gunk by running 9M MeOH/NH3, and don't see any silica upon rotovapping. When I use 100% MeOH, I can certainly see SiO2 in my flask.

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29. Milo on May 12, 2006 5:24 PM writes...

LNT,

Here in the Great White Tower of Academia, the toys you speak of are fantasy!

I agree 100% with dry loading a compound. I get incredibly tight bands that way.

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30. Luke, I am your fungus! on May 12, 2006 5:35 PM writes...

The newly redesigned ISCO Combiflash Companion(after Teledyne bought ISCO) is great - if you have $20k to purchase it and extra money for these disposable silica columns. Once you start using it, there is no going back - you get the gradient control, programmable UV detection and automatic fraction collectionm with general easiness of use. I would only do a glass column for a big purification (>10g of crude) because the biggest available column for ISCO is 120g of silica and the fraction collector space is somewhat limited. Another reason would be if I needed to go with a slurry-packed column (like NEt3-desactivated silica for acid-labile compounds).

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31. BCP on May 12, 2006 10:46 PM writes...

This is a great thread - many comments ring painfully true; who hasn't been there as the sun's going down as their bands merge together and tail to infinity...

One question to throw out -- process chemists I've worked with in the last couple of years have actually been quite big fans of using trifluorotoluene as a subsitute (when all else fails) for DCM in snarky med chem routes. It's alleged to offer a similar polarity/dielectric. But one factor that makes it attractive is that by virtue of it's CF3 group it's significantly easier to distil away - it's also available in plant scale quantities. Anyone tried flashing with that?

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32. ss ( Poor grad student) on May 13, 2006 6:29 AM writes...

I was under the impression that guys in big pharma always used those snazzy chromatography systems from ISCO or Argonaut with automatic fraction collector etc.etc. the whole works. Didnt know that even big pharma people have to sweat it out .

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33. heeler on May 13, 2006 11:23 AM writes...

LNT,
We'e used the ISCO reverse phase columns with good success. you can recycle them. we also would dry load on to C18, just like a regular column. One watch out is for a new column is to condition it prior to running. We would run pure ACN and the run it up to the starting aqueous amount we wanted. It's kind of like conditioning a RP-SPE cartridge.

As to trifluoromethyl toluene, I can remember copying a paper in the late nineties about being a replacement for DCM, but never actually using it. I think it was claimed to be useful in combichem operations.

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34. Canuck Chemist on May 13, 2006 11:26 AM writes...

A couple of comments based on my fairly extensive experience with chromatography:

-The method of loading your column is critical, because the narrower the starting band, the better the separation will be. It's a good idea to run a quick TLC with the solvent system you'll be using to dissolve your crude and load the column with, to make sure your compound doesn't run down the column with this solvent (or solvent system). People often make the mistake of loading their non-polar compounds as DCM solutions, which in fact may push the stuff halfway down the column before they've even started. For this reason, dry-loading is often a good option, though it's a bit of a pain. Just be sure to use just the minimum amount of silica to adsorb all of your compound. For small samples this is best done in a small vial which you can evaporate on the rotovap.

-The shape of your column is also important, because you want that starting band to be as narrow as practically possible. For this reason I err towards using a shorter, wider column. The narrower columns often require a greater height of silica gel to get the same separation (because your starting band will be thicker), and your compound will take longer to elute, and you will probably see more tailing and waste more solvent.

-Whoever recommended isocratic elutions (keeping the same solvent mix for a better separation) is incorrect. It's almost always best to go with a gradient. For example, instead of running 5% of the polar solvent, you'll get the same (or better) separation in less time with less solvent waste by starting off at e.g. 2% and increasing gradually to 10%.

-The automated systems are far superior to anything you can do by hand-- I highly recommend them if you can get your hands on one. I've used the newer Isco and Biotage systems and they are generally excellent. You can easily program a custom gradient which is tailored to your particular separation, and you can use this method every time you have the same (or a similar) compound. They also often obviate the need for TLCs because they have UV detectors which tell you what is in every fraction. I was able to convince my academic boss to buy one, and it's been inexpensive to use because we simply pop the tops off the cartridges and refill them with our own bulk silica gel. Isco will sell you refillable cartridges with their system. It's such a waste of time to sit in your fume hood changing test tubes and mixing solvent by hand (unfortunately I must do this in my new lab...)

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35. Anonymous on May 13, 2006 7:05 PM writes...

JC, why Na2SO4 and not sand? Is that common?

Also: Are there any rules of thumb out there for Rfs/ volumes when eluting over a gradient? My habit is just to peg the Rf at .35 and slam 5 volumes down the column. It's quick but I worry that it may be too dirty.

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36. Jordan on May 15, 2006 9:26 AM writes...

This is an awesome thread. I'm going to print it out.

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37. Milo on May 15, 2006 10:34 AM writes...

Anon (#35)

I also use sodium sulphate, especially with the more hydrophillic solvents like MeOH... I am working off the theory that is tends to dry the solvent a little bit before it hits the silica. Of course, like everything in chromatography, this may be considered voodoo :-)


I agree with Jordan (#36), this is one of the best threads I have seen.

Permalink to Comment

38. JC on May 15, 2006 11:02 AM writes...

Sodium sulfate works well for me. I use it exclusively.

A guy I used to work with was using sand on his hydroxamic acid purification; turns out sand has iron (& who knows what else) traces in it, that in this case chelated his product.

I don't run a lot of columns lately (because the project is a 'brick factory') & we currently have a purification group to HPLC final compounds.

Isocratic or gradient I believe it is compound and chemical class related, through trial and error expertise with a series can be established; it's whatever works for you, at the end of the day. I go isocratic (or extremely shallow if the TLC runs at 0.3 in 1:1 H:E then set up and elute first with 6:4 H/E then go to 1:1 (for example) use gravity (medium fast dropwise at first) and take lots of fractions, this methodology works well in most cases, first time.


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39. Sean on May 16, 2006 11:46 AM writes...

Does anyone else use Celite for dry loading? (I use an older ISCO system.) I find the compounds come right off of the Celite and make a nice tight band on the top of my column. The only disadvantage is that I need to pre-wet the sample on the Celite inside the solid load cartridge so the system doesn't push a lot of air onto my column while the compound is loading.

I've been meaning to run a comparison between Celite loading and silica gel loading but haven't gotten around to it yet. My guess is that the silica gel loading gives a broader band than Celite.

Permalink to Comment

40. Milo on May 16, 2006 1:21 PM writes...

Sean,

I have used celite in the past, but now I use course (gravity style) silica for dry loading then the really fine flash grade for the column. Seems to work well, nice tight bands etc...

Permalink to Comment

41. Charlie on August 20, 2006 2:20 AM writes...

Eric,
I believe crystallization can work wonders sometimes. I wonder what Solka-Floc you use, maybe a catalog no. of some sort? And how much charcoal do you generally add? (an example will be highly appreciated.) I mostly do a short column to get rid of the base line then to try the crystallization.
Thanks

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