Need a little help with a purification paper/project

[soisports]

Hi, I am working on a term paper involving the purification and characterization of an unknown substance and would like to ask for some help and advice as to whether I am on the right track or not.

The substance is an unknown compound obtained from plant leaves (aqueous extract).

Are there simple analytical techniques to determine the nature of a constituent detectable by bio-assay? For example, if you are looking for a compound within the extract that displays a bio-activity, what would be a good technique(s) to separate the components into organic versus inorganic, protein/non-protein, steroid/non-steroid if you have an established bio-assay procedure to test the component(s) at each stage/separation?

I have also proposed, regardless of the known nature of the compound, that a solid-liquid extraction be performed on the initial extract using rotational planar extraction with increasingly polar solvents and RPMs, bio-assay of the fractions to determine which one gets to be carried on.

Fractionation of those extracts into two fractions: polar and non-polar using a 3 phase countercurrent liquid-liquid chromatography technique where the phases are placed into a column and separate out (from top to bottom) into non-polar mobile, stationary and polar mobile. unknown sample is injected into the stationary while an external source of the non-polar mobile is injected (from below) into the polar mobile and vice versa for the polar mobile (injected from the top) into the non-polar mobile. Of course constituents are eluted from both the top (non-polar ones come out) and the bottom (polar ones come out). From here, the fractions can again be bio-assayed and chosen for continued purification...

The next steps are where I am having more difficulty. I would think using RP-HPLC for the polar and NP-HPLC for the non-polar constituents but what would be the best methods to optimize mobile phases before jumping straight into HPLC? Is first testing on TLC applicable? Or is it ok to trouble shoot using HPLC straight away?

Finally, after these steps to finally purify and structurally characterize the compound, I am proposing using hyphenated HPLC methods coupled to Mass Spec and NMR.

I am sure there are many many ways to go about this but I just want some feedback and comments as to whether I am on the right track. More importantly, I am curious about simple ways to intitially characterize a crude extract as being organic/inorganic, protein/non-protein etc... I initially thought simple phase (organic/aqueous) separation but you may stil have inorganic molecules in the aqueous phase, no?

Anyway, any help would be greatly appreciated!!!

Thank you in advance,

Sois

[DR]

This is pretty far from my area of moderate competence, but here's my take:

TLC may be a good preliminary step to help you see approximately how many things you have left in a fraction.

For a bio assay to work, don't you have to know what you're dealing with? I think bio assays are extremely specific.

A series of SPE columns may yield similar groups of extracts with less effort than planar extraction (maybe cull proteins, water soluble organics & "other" stuff out into groups via SPE, then RPE each group w/ different classes of solvents)?

[Kostas Petritis]

I agree with DR that you can achieve the fractionations that you mentioned earlier with a series of SPE.

Personally, as more chromatography oriented, I would probably just do a C18 preparative chromatography and separate my mixture in about 20 fractions and see where my compound with biological activity is. If it is in the void volume I would probably repeat the fractionation but this time I would use a HILIC approach.

Once I identified the fraction(s) witht the biological activity, the I would procceed with efforts for it's identification. My next steps would depend on the complexity (number of compounds) in the matrix. But for sure, I would try to have MS/MS, accurate mass and NMR to help me determine the structure of my compound...

There is more on this (I am really an expert on this field) that you can do but if this is just a student paper, I think that what you described is already pretty advanced...

[soisports]

Thank you very much for the replies.

The compound I am looking for has inhibitory effects in cell cultures (growth and inflamatory). I am asked to provide preliminary tests to characterize the compound as either organic or not, protein or not, hormone or not... and then outline a methodology to purify and characterize the substance. Since each fractionation and separation have to be preparative to allow testing of the substances in cell culture, this is what I am worried about in developing a method that will allow easy dialysis or evaporation of the extract/fraction for testing.

I think both the SPE and fractionating on C18 are good. C18 is best for small molecules and peptides, correct?

I am able to fabricate some preliminary results as to the nature of the compound which would clearly help in the purification. However, other than tests like heating the sample and adding proteases to eliminate proteins and then testing the residual sample (after some cleanup) for inhibitory activity. I guess steroids, being lipophilic should be easy to rule out... are there safe ways to defat a complex mixture using chromatography?
Even more fundamental I can't come up with a simple (and bioactivity preserving) way of separating organic from inorganic compounds.

Sorry for all the questions, I am thinking as I type here.

What I would like help with the most is if there are any sound techniques to definitively separate biochemical compounds like proteins, carbohydrates, lipids or hormones from a complex mixture and still ensure they retain their bioactivity?

Thanks again so much.

Sois

[Kostas Petritis]

I think that the only compounds that you have to worry about bioactivity is proteins as they might denaturate if you add an organic modifier etc...
You can have a look here about how to handle proteins:

http://www.bi.umist.ac.uk/users/mjfssrf ... efault.asp

Maybe what you need to do is to do Size exclusion chromatography (using an SPE PD-10 type column) to separate small from large molecules.

Test for biological activity, if it is the high molecular weight substance then it can either be proteins, or polysuccharides or somekind of other biopolymer...

If it is the fraction with the low MW then it can be one of the other choices...

Now if it is inorganic, when you'll do a C18 fractionation, it will probably elute in the void volume along with some other hydrophilic organic compounds. So you can find out if it is organic or inorganic that way (I would assume that is organic). This won't tell you if it is 100% inorganic but it will tell you 100% if it is organic...

Well if these are the only choices you have, by elimination you can say that it is an hormone if is not protein and it is not inorganic (although there are plenty of other possibilities).

I hope that you make sense from my message as I wrote it pretty fast...

[soisports]

Thanks again for all your help

Here is what I came up with. Let me know if it sounds believable to you…

Source is a collection of plant leaves picked off the ground, chopped into small bits, dried at room temp for ~5 days, followed by crushing and extraction with 37 degree celcius deionized H20 at a gentle shake for 5hrs (250g of dry matter litre). Solution was then filtered and the filtrate lyophilized (powder).

Lyophilized powder was shown to have inflammation inhibitory bioactivity in cell culture by established bioassay.

Preliminary tests:

1) Resuspended powder in suitable buffer/h20, spit into two aliquots and treated one with proteinase K to degrade proteins. Proteinase K digest failed to show bioactivity, undigested did.

2) Resuspended powder in h20, mixed 1:1 with n-hexane and collected organic phase. Evaporated and resuspended organic phase exhibited bioactivity. Aqueous phase did not.

3) Size exclusion chromatography with G25 (or LH20) sephadex (~5000 Da cut-off) using hexane(if possible), maybe chloroform. eluent

Preliminary studies suggest organic, non-protein, hydrophobic (non-polar) small molecule, potentially a steroid

To fully characterize:

1) Reversed phase SPE on octadecyl(C18)-bonded silica. Column primed with hexane, methanol, then h20. Powder resuspended in h20 and added to column 15-20 tube volume fractions taken with a methanol-chloroform gradient, with final hexane elution. Assay fractions and continue purification with bioactive fraction(s).

2) RP C18 SPE again of bioactive fraction or pooled fractions (after evaporation and resuspension in h20). Optimize organic solvent for better fractionation if needed.

3) RP-HPLC fractionation and optimization of mobile and stationary phases for peak resolution. Possible C18 or C4 columns

4) RP-HPLC with optimized phases and column coupled with DAD-MS-NMR for final purification and elucidation of unknown chemical structure.

Well, do you think the preliminary studies make sense for initial rough characterization and are believable? I think it may be worth mentioning or including in the purification to go back to a dried plant leaf powder and extract a few times with hexane rather than just the water to obtain more of the desired hydrophobic small molecule. Any comments would again be greatly appreciated.

Thanks!

Sois