Chromatography Forum

As you have "totally unknown compounds", it's quite likely that you don't yet know the compounds you are going to purify further. A couple of extra points to consider...

Try to have a clear idea of the separations you want the column to perform, and don't hesitate to ask the vendor to confirm the column is suitable , perhaps even by running samples or providing an equivalent analytical column.

I've found it's alway a good strategy to negotiate the price of the preparative column, and then ask the vendor to provide guard/analytical columns at extremely-discounted rates.

The more you can fractionate unwanted junk out of your extracts before loading, the more good stuff you will be able to load each time. Watch the pressure changes during loading/running in analytical-scale runs, as repeat preparative runs can magnify increases.

Unless your extracts are really clean, I would strongly recommend purchasing a guard column with significant capacity to retain any really-sticky gunk from your natural product extracts. Guards are always cheaper to replace than columns of the same packing.

Depending on your natural products and extract, it may be preferable to perform a preliminary HPLC run to crudely fractionate the extract, and then a second run to resolve and purify closely-eluting compounds.

You can get good indications of sample cleanup and mobile phases from literature reviews using similar natural products.

Please keep having fun,

Bruce Hamilton

i have used waters atlantis prep column 250x22 mm RP-18 for 4 years and never had any problems...and most of the analytical columns i had were from waters..so my experience with waters are really good..they are little expensive but robust..thats why i was going with waters columns..since my compounds will be totally unknown i wanted to know general columns..i will be mostly using acn,meoh with some formic acid or TFA

thanks bruce for all your suggestin ..i do clean my junk with HP-20 resin first then only i load in prep columns..and do have guard column and filters for the current prpe col i have

You are in the natural compound buissiness - want to consinder two points

The concentration of the target compount can be very low, you might need a big coulum with a matching prep system in order get enough substance

With an crude plant extract, you can easily destroy every column very fast
so you can do some pre-cleaning or another good way is to use a packing station in order to repack the column when it died. The packing procedure is not difficult with modern DAC stations.

In my experience, liquid-liquid-partition chromatography (FCPC) is the best way to seperate natural compounds. generally spoken, its a column with a liquid stationary phase. You can change ( or repack) it just by pumping the phase into the column. Also you have 100%recovery (by pumping the stationary phase out of the system )

Best regards
Chris

mk12, what do you mean by "totally unknown"? You don´t know whether there is something useful there? Whether potentially useful compounds are hydrophobic or not, base, acid or neutral? Macro or small? Stability? . . . . . . . . . . .

You're already a Waters fan, so a futile flame fest followed... what fun, enlivens a boring day

Hans, the molecules are not "totally unknown", as they will have adsorbed onto a Diaion HP20 resin, which, IIRC, is a synthetic PS-DVB. That means they will be sparingly soluble organics and/or be aromatic, but small enough to fit into the pores. I think it's a product of Mitsubishi Chemical.

My only use of it was to scavenge target small molecule drugs from active fermentation broths. It's very popular in the fermentation area, as you can add it to broths instead of tedious solvent extractions.

Please keep having fun,

Bruce Hamilton

Bruce, I went back to re-read the original question and tried to understand why that was asked.

mk12, what do you mean by "totally unknown"? You don´t know whether there is something useful there? Whether potentially useful compounds are hydrophobic or not, base, acid or neutral? Macro or small? Stability? . . . . . . . . . . .

yes I think so. With plant extracts you're really fishing in the dark

That's propably the reason mk12 want to do the prep work, to fractionate/isolate the extracts and see if some of the fractionates exhibits some value and what molecule it is.

Therfore he wants a general purpose column, so he is able to run it at low pH for acidic and neutral components but also at high pH if there are some bases (e.g. alkaloids) in there.

It's the same reason I've bought a XBridge prep column some time ago, but unfortunately didn't get the chance to use it as much as I would like to.

But there have been some people getting Nobel Prices for this sort of work. It might help to see what their methods were.

HW..Yeah I dont know what type of compound it is ..only on the basis of polarity i can guess its hydrophilic or hydrophobic..i have no idea about its stability until i isolate it..our workflow is crude ext shows activity..use 96 well plate method to nail down active peaks and go from there to purify those active peaks..

SEC, IEC, RP, etc... all types are available in Prep from Sepax: http://www.sepax-tech.com/products.php

Hope this helps!