Best method of plant analysis

[bliindsniper]

What is the best method for getting an idea of what compounds are contained in a plant extract. hplc is good but it must be followed my mass spectrometry which is far too expensive for me. Tlc is decent but if two compounds have similar rf values it can have errors. Ftir seems to be the only way to go if you want to know all compounds contained within a given mixture. Any recommendations will be helpfull.

 

[John Cuthber]

FTIR isn't good with complex mixtures either.

[CharonY]

What is the best method for getting an idea of what compounds are contained in a plant extract. hplc is good but it must be followed my mass spectrometry which is far too expensive for me.

 

Welcome to the wonderful and expensive world of molecular biology. The moment you deal with complex mixtures, the cost skyrocket. With low complexity and well-known compounds you may get away using chromatographic methods with simple detectors.

[vampares]

IR spec or nmr spec would probably be the best for organics but I think the complexity of it is what the problem is. Besides don't you need isolated chemicals?

 

Do you have any idea what you will be looking for? In that case chromatography is the way to go. What sort of extractions are you going to be using?

There's a fully functional NMR on ebay for under $15K.

Depending on the substance and what sort of yield you get, you can alway use the CRC density, freezing/boiling point, solubility and a few basic tests.

Edited April 24, 2013 by vampares

[bliindsniper]

Thanks everyone for the answers. My goal is to isolate medicinal compounds out of things like turkey tail mushrooms and things of that nature. The samples i will be testing will be quite pure already from various extraction methods. I just need to analyze them to see what compounds exactly the extracts contain.

 

As for the issue of isolation i will probably end up doing something like hplc with a fraction collector. I am open to ideas when it comes to this because hplc spectrometers don't seem to be able to divide a liquid into fractions based on each of its individual rf values very accurately What i mean by that is i am not aware of a way that a hplc device with maby a uv detector could rotate the test tubes it is emptying into based on what the rf value is or what the uv is scanning

Most fraction collectors seem to changes test tubes solely on the volume of liquid collected. Like in this video

http://www.youtube.com/watch?v=XQJPhskzzak

 

 

which means you may get 1 or 2 pure test tubes but the others will contain different gradients of contamination

[vampares]

I just need to analyze them to see what compounds exactly the extracts contain.

 

Mushrooms. Hmm. Thinking like using something like a glycerol solution to dissolve the material and then dilute it gradually to achieve some precipitate.

 

I have no laboratory experience with analytical organic chemistry. I've never worked with HPLC. It sound like it would provide separation. It would provide accurate and reliable comparative analysis.

 

The mushroom thing is messy though. The Chinaman who preps this stuff...I'm thinking like a pot of tea buried in the ground for a month or something. Highclass stuff. Mushrooms liquefy. The product is dark. This is a phase of mushroom. The growth medium of the mushroom will effect the result to some extent (oh yeah).

 

Your "extract" is probably still alive. When it hits the column of silica it may react. The cellulose and dextran gels may work.

 

According the the wikipedia the extracts contain polysaccharide-K. If that information is correct, it shouldn't be difficult to verify ("beta-glucan β-1,4 main chain with β-1,3 and β-1,6 side chains"). NMR or some sort of saccharide specific reagent, check Fisher Scientific.

 

You then ;have the protein. Are you interested in having that sequenced?

Edited April 25, 2013 by vampares

[CharonY]

If you need to identify compounds in complex mixtures I do not think there is any better way than LCMS or GCMS. Preparative HPLC can give you very good fractionation, provided that the LC part is set up very accurately and the complexity of your mixture does not overwhelm its separation and fractionation capabilities. Unless there is further purification and prefractionation a complete plant extract would be highly complex, however.

[John Cuthber]

It is certainly possible to get fraction collectors for HPLC which monitor the output signal from the detector and move on to the next tube based on that signal.

 

GC is great for separating things but

they have to be volatile and

it really can't cope with anything but very small quantities of materials

so I don't think it would be much good for making usable amounts of plant (or fungi) extracts.

 

HPLC does a better job with involatile things and it can be scaled up more readily.

 

You would probably still need some sort of pre-purification.

[CharonY]

Well, GCMS is more for identifying metabolites, as many of the more interesting compounds tend to be volatile (terpenes, flavenoids and lipids come to mind). But isolating them would be difficult endeavor with that, I agree. It really depends on what you want to do, but for a metabolomic approach I would do an extraction and run it on a GCMS to see whether there is anything of interest to begin with (which can be coupled with pre-fractionation). Especially because a decent GCMS is generally way cheaper than a comparable LCMS.

[bliindsniper]

Thanks everyone for the replies. I have a few things to add, the mixtures i am attempting to separate and analyze will have around 13 compounds max.

I am looking into a flash chromatography setup with a uv vis detector and an auto fraction collector mainly because its more compact and easier to manage and controll than a hplc device. Do you guys think this would provide the same seperation as hplc?

An example of what im looking at is this.

http://www.ebay.com/itm/Analogix-Intelliflash-280-Flash-Chromatography-System-/221212944048?pt=LH_DefaultDomain_0&hash=item338151a6b0

 

Not this exact one of course but it is just an example. Then after i got the fractions i am leading to analyzing them with nmr.

[John Cuthber]

"Do you guys think this would provide the same seperation as hplc?"

No, that's why people use HPLC.

But it might be a very good pre-purification step.

[vampares]

I would only buy from ebay if the seller is willing to go "unconditional money back guarantee". Sellers are almost always third parties with equipment they know nothing about. But perhaps you don't either without having your hands on it. From experience only, it looks like the equipment is good, so probably like a rock (with software). You'de pay shipping twice on the retur. It doesn't look like a pallet you have to arraign, but don't be scared of these either. They can run 85 to 200. I'd avoid any Western Union or check transactions. Use insured transaction methods. In the end it is just a pain in the ass when you are done.

 

 

Is this the equipment good for the tests? It should provide separation and comparison data. If not send it back the seller to be relisted.

 

NMR can provide comparison data as well. You need charts to compare data to. My impression of NMR is that it is like a chemical oscilloscope. There is a lot of data that can be interpreted from readings. It also provides some "quisi-voodoo" info, like if you are just screwing around. And this is probably more relevant in analytic chemistry than in electronic circuit design because this is the stuff -- the chemicals -- which you are to qualify. Almost as if you were to smell some spaghetti sauce that was on the stove. Or wine tasting. You could say it is 'je ne sais quoi', too biter or very woody.

 

But well interpreted NMR data I've seen in:

Fessenden Fessenden, University of Montana. Organic Chemistry. Pacific Grove, California: Brooks/Cole Publishing Company, 1990.

 

Chapter 9 Spectroscopy I: Infrared and Nuclear Magnetic Resonance

 

They use IR and NMR combined throughout the book.