Sciencemadness Discussion Board

size exclusion chromatography

loki_tiwaz - 2-3-2007 at 19:32

edit: i throw this idea into the arena without much real interest in the outcome. ok i lied, i am very curious. i never heard of SEC before but when i read it it seemed an immediate solution to water soluble polymer contamination of water soluble alkaloid salt sources. i know chromatography is a pain but SEC seems like the most painless form of chromatography i've ever read about. just pump the water through and collect roughly uniform volumes of at least 10 in number and there should be a reasonable resolution of molar weights between the given fractions which can of course be refined further if one wants to.

edit: size exclusion chromatography brings bigger particles down before little ones, or so sayeth wikipedia: http://en.wikipedia.org/wiki/Size_Exclusion_Chromatography

edit continued: and yes, I know this is a pain in the butt but the purpose is to get a high value compound separated from a bunch of low value water soluble polymers. the efficiency is the whole point. anyone can get 50-80% yield but to get 90%+ - that is the point of this investigation. The big plus is no nonpolar solvent is required.

a familiar, perhaps, problem to people here would be the adulteration of useful precursor chemicals with nasty polymers like hypromellose, povidone, polyethylene glycol and polysorbate.

there has been all manner of methods devised to defeat these contaminants but overall the results haven't been consistently usable for all partly due to the variation of starting materials and getting yields much above 50% takes a lot of refining of the practical work being done.

after seeing a foaf suffer the ignominy of a miserable 20% yield - stbIII previously afforded about 50% - while attempting SPD, the foaf begged me to find out a better way, preferably one that didn't involve any use of hot naptha because this foaf was in a nasty ignition once and hates the smell.

so i did some research for this poor chap, eventually deciding there was no practical simple chemical way to remove the polymers, for example, with a long nonpolar soak because the polymers would then carry the goods into the nonpolar.

chromatography was the subject area i looked at and wouldn't you know it, there is a specific method for separating molecules by size called size exclusion chromatography. it is used in biochem to isolate proteins by their sizes using various types of polymer gels, but also, generally with silica, in polymer chemistry to fractionate polymers by chain length. yes, the exact method that is used to make polymer chain length fractions of these evil chemicals in industry.

it's so simple it's retarded.

1. dissolve the water solubles of the products in water. for this perhaps an initial steam bath cooking of the ground up dry material until it visibly turns nice and brown with tasty (not) caramel (but nice for being much longer chains). then boil up the mixture for a while until one is satisfied all the solubles are good and dissolved. not too caramelised, tho, because really badly burnt sugars are insoluble in water and might trap some of the goodies by encapsulation.

2. settle and filter out all the insolubles - no point in clogging up our lovely glass wool packing in the column with chalk and other crap. charmin filter probably the thing to do here.

3. get your silica gel adsorbent column all set up and nice and settled and de-bubbled (i suppose sitting it on top of something that is vibrating would help) with glass wool plugs on each end. (it occurs to me that since this is just done with water and we aren't using uv activity to locate the section of the column with the bits we are looking for it might as well be a 1 inch pvc pipe capped at the bottom with a hole in the bottom to let the fluid fall out)

4. get your separatory funnel loaded with the extracted, filtered fluid, mount it at the top of the column and let it slowly drain into the column until one has hit the capacity of the column, then put water i dunno how much maybe 3-4x the column capacity in the sep and plug a small compressor output hose into the top of the sep sealed in with a stopper turn it on and collect the fluid that comes out into little containers all lined up from starting elution to finishing in suitably sized containers.

5. get your mandelin indicator and take samples from each container and find out at which point there is no more positive yellow-->green reaction.

theoretically at this point apart from the one or two fractions between the transition, there will be pure product dissolved in water, the transition fractions could be run through it again with a large volume of water a couple of times more if one wants to, probably well worth it, getting the stuff one wants out nice and pure is always the best outcome. it should be pointed out though if the caramelisation was done the contaminant at this phase will be caramels, being that, assuming one didn't go overboard, they will be fairly small compared to the 80+ chain synthetic polymers.

if it works like the theory seems to suggest yield might be pushed up towards the late 90s. seeing that mechanical losses are the only route of loss here 1-5% loss seems reasonable and very attractive.

thoughts?



a side-topic/question i've pondered over for several years regarding sulphate salts and conversion to hydrochlorides without involving an acid base and nasty smelly flammable solvents - titrating calcium chloride into the solution of sulphate salt.

the problem i forsee is the inevitable contamination of the product with either excess CaCl2 or remnant *.sulphate and i can't think of any way to eliminate this problem - having said that i think that there is nothing to be concerned about here because a small amount of CaCl2 will not inhibit or damage the reaction significantly. the calcium sulphate (gypsum) will fall out of the solution and filter out easily enough. possibly one could take a mathematical route and estimate likely molarity of the sulphate salt in the solution and shoot for 90% equimolar of CaCl3 and then filter/settle and repeat until no visible signs of precipitating CaSO4 forming occur. a slow addition of dilute sulphuric acid while observing for the foggy formation of calcium could work making the remnant contaminant HCl rather than CaCL2

eliminating the need for dangerous and suspicious smelling naptha or toluene or xylene would be desirable for those working in confined environments or those who are justifiably paranoid about boiling hexanes near even the safest lab heat source.

one other thing that is nice about this is the elimination of losses from the inevitable incomplete phase-transfer in acid/base extraction, this way the desired material is only filtered a few times at most leading to only a very small mechanical loss.

i don't see why it wouldn't work, tartaric acid is isolated from potassium bitartrate by pretty much this exact same mechanism, adding calcium to the mixture to precipitate calcium tartrate which is then converted to calcium sulphate which precipitates resulting in tartaric acid isolated from potassium.

[Edited on 3-3-2007 by loki_tiwaz]

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[Edited on 3-3-2007 by loki_tiwaz]

unionised - 3-3-2007 at 05:11

At work we use GPC to separate pesticide residues from food extracts.
The stuff you are looking at seems fairly similar.
If we could get away with nice simple silica gell columns we wouldn't be using $3000 GPC columns.
You might get away with it because the things you are looking at are much more water soluble.
If you heat sugars until they caramelise they will react with any amino groups in the solution- this may be unhelpful for your purposes.
http://en.wikipedia.org/wiki/Amadori_product

loki_tiwaz - 3-3-2007 at 17:49

GPC is in fact exactly the same, however if the materials one wishes to fractionate are primarily synthetic water soluble polymers one need only use fine silica gel.
Quote:
Polymer chemists typically use either a silica or crosslinked polystyrene medium under a higher pressure.


I read that as basically meaning flash chromatography on silica.
Quote:
This is usually achieved with an apparatus called a column, which consists of a hollow tube tightly packed with extremely small porous polymer beads designed to have pores of different sizes. These pores may be depressions on the surface or channels through the bead. As the solution travels down the column some particles enter into the pores. Larger particles cannot enter into as many pores. The larger the particles, the less overall volume to traverse over the length of the column, and the faster the elution.


In the case of the mixture of PEG, hypromellose, povidone, polysorbate, sucrose/lactose and the (in the case I am studying) sulphate salt of an amine, this should result in the polymers coming out of the column first and the sugars and amines last being that they more readily adsorb in the tiny pores of the gel matrix, thus causing them to come out last. Several passes through this process should allow the total isolation of the amine from the sugars and the polymers.

All of the polymers used in the binders are around 2000-4000g/mol, the sugars are around 350g/mol, and the desired amine salt is either 428 for the sulphate or 214 for the hydrochloride. I would assume therefore that if the salt were the hydrochloride it would elute last, if it were the sulphate, it would elute between the polymer fraction and the sugar fraction. for this reason conversion to the hydrochloride would probably simplify the fractionation, which brings me back to the use of calcium chloride to convert it in situ by ion substitution and the precipitation of calcium sulphate. separating the calcium chloride from the amine salt will naturally be much simpler than getting those sticky polymers off it, and certainly it would be preferable to not have to separate the sugars and amines bearing in mind what you said about the reactions that can occur.

The hole in my knowledge however is whether this flash chomatographic process will adequately separate the amines and sugars from the polymers, being that these polymers themselves adsorb the amines. The only thing for it is experiment I guess. Mandelin indicator should react even if there is polymers present, although it may slow the reaction.

[Edited on 4-3-2007 by loki_tiwaz]

Maya - 7-3-2007 at 03:33

First off , you can't use silica gel for SEC, you can use it for flash.

You'll pop the stopper off the flash column if you try that way, it has to be a screwed onto glass type attachment.

SEC is usually meant to separate very high MW ( >20Kda ) from smaller ones , so I don't think you have very much magnitude difference in your starting mixture even if you found the correct pore size column material.

You will find that since your analyte, the amine, is so small that it may be retained for a considerably longer time than you are thinking or it may not elute at all.

I would consider ion exchange chromatography or reverse phase or silica gel/ alumina for your "amine" B4 SEC.

loki_tiwaz - 11-3-2007 at 13:43

i was wondering about the idea of using a zeolite column, being that the desired material is more polar than the undesired.

of course after doing a bit of reading it seems not terribly simple to determine whether this will work or not, more expensive and powerful equipment certainly could do the job but still goes beyond practicality. perhaps some research will illuminate a means to selectively bind up the polymers and leave the amines in solution.

[Edited on 12-3-2007 by loki_tiwaz]

Maya - 12-3-2007 at 06:48

<< i was wondering about the idea of using a zeolite column, being that the desired material is more polar than the undesired.>>

I doubt that'll work

chemrox - 18-3-2007 at 13:54

I'd like to read more about the Amadori .... could you direct us to more specific sources than Google?

chemoleo - 18-3-2007 at 15:24

Well I am one of those that uses gel filtration aka SEC on a daily basis almost.

IT is great for seperating compounds of different molecular masses; the greater the difference, the better the separation.
There are different types of gel filtration resins, some that are good from the 0-6 kDa range, then from 0-20, and 0-100 kDa. Buffer exchange columns work by the same principle, and the resin is cheaper. Here, the solvent is retained, and anything larger than 1 kDa or so flows straight through. So this should be best for small MW compounds, i.e. buffer molecules etc are separated from larger crap, or vice versa.
Alternatively, try dialysis with a 500 Da membrane, the small compounds should diffuse out, retaining the large ones in the dialysis bag.

loki_tiwaz - 27-3-2007 at 04:29

Being that the molecular weight of the undesired compounds in this case are mostly > 2000 and generally around 3500, I would think a 1000 MWCO membrane would work equally as well as a 500.

Dialysis membrane tubing is pretty expensive though, although the amount required for an average small scale purification would only be about at most a foot long and the membrane can be refilled after several replacements of the dialyte (presumably distilled water).

Working on a small scale with a short quantity of tubing it should be more economical than SEC. Just need to find a supplier of 1000Da or less pore dialysis membranes in 2 foot lengths, which should be possible one would think.

[Edited on 3-4-2007 by loki_tiwaz]