column suggestion.

[theodora]

I need a column that can detect polar and non polar vitamin, vitamin B, C and D. I am currently using a shidmazu class vp HPLC with uv detector. I found out that Atlantis dC18 column can do so, but it doesn't provide technical paper on how to prepare and extract my sample. Any good suggestion on any column or relevant method for atlantis dC18 column?

[Alex Buske]

No column can detect analytes. That is what the detector is for.
Often vendors supply chromatographic methods for their columns - thats called an application. On the other hand you can develop your your own method.
As nobody (of the column vendors) knows your sample, nobody can provide you with detailed extraction and sample preparation procedures.
You may find a suitable method in the literature.

Alex

[Uwe Neue]

One will need to understand, what your matrix is from where you need to extract the vitamines. I can guide you, if you give more information about the nature of your sample.

[theodora]

My samples are wild edible mushroom / fungi.

[Mark Tracy]

When faced with unusual sample matrices like yours, it may be necessary to evaluate a number of column/mobile phase combinations in order to resolve the vitamins from the matrix.

You will need one of the "polar embedded" or "AQ" type columns because some vitamins are best resolved using 100% aqueous buffer for the mobile phase.

I did an application for water-soluble vitamins using our PolarAdvantage column, and it was satisfactory for some fruit juice drinks. The mobile phase was phosphate buffer, pH 3.41 isocratic to elute thiamine, ascorbate, pyridoxine, niacin and pantothenate, then a gradient to 20% acetonitrile to elute B12, folate, biotin and riboflavin. To extract samples, use the phosphate buffer. You will need multiple UV wavelengths: 210, 245, 265, 285 and 310. B12 and biotin are usually too low in concentration to detect by a simple extract-and-shoot method. You can use small pH adjustments to tweak the separation.

Vitamin D (and other fat-soluble vitamins) will be a separate extraction and analysis. Natural sources of vitamin D are too low a concentration to do a simple extract-and-shoot method. The PolarAdvantage column can also do fat-soluble vitamins with 98% methanol as the mobile phase.

Some of our worthy competitors have published applications similar in nature.

[Uwe Neue]

There is an application note for the Atlantis column that shows that you can separate all the vitamins in a single gradient run. If you have not seen it, let me know.

The difficult part is the sample prep. Usually the sample prep is directed toward a class of compounds with similar properties. This is not the case for your analytes. No matter what you do, you will end up with different fractions, which you either will recombine or analyse separately.

I would probably start off with homogenizing the mushrooms in water, then centrifuge, and take the clear liquid for further sample workup directed towards the water-soluble vitamines. I would then extract the solid with something like t-butyl ether to get at the fat soluble vitamins. One will then think about further sample clean, for example via SPE.

[Uwe Neue]

Actually, coming to think of it, I woudl do a literature search. I am sure that you are not the first one to try to get something out of mushrooms...

[Alex Buske]

Just a few remarks: I dont think your problem is the column. The Atlantis should work (also the PolarAdvantage).
But: Ascorbic acid is somewhat unstable in water. It tends to oxidise. With Vit Bs we had some nasty experiences with extraction in water: it worked in one place, at a second location we got terrible precision and recoveries. Use of buffer (somewht more acidic than analytes pKa) for extraction solved the problem.

The next question is somewhat philosophic: Do you really measure the naturally occuring concentrations? Fungi and plants are full of enzymes and as soon as you grind, dry or somehow extract them, these enzymes are released and start all kinds of funny reactions.