Plant Metabolites

[thekid]

hello,
my college is planning to buy a HPLC and would like to know what kind of columns can be used to purify secondary metabolites from plants.
the HPLC is going to be mainly used to separate compounds from plant extracts made from solvents like chloroform, ether, isopropanol, 70% ethanol, acetone and water.
the procedure is to extract the plant substance using one of these solvents in step wise and then put the extracts through the HPLC to separate out the components. hence i would like to know what type of column would be preferable and what size of the column should be used.
any help in this matter would be greatly appreciated.

thank you in advance.
thekid

[Wen Jiang]

Hi,
THis is a wide topic. One column may not separate all the secondary metabolites from plant. Please give more detail information on your specifically interested compounds.

[Mark Tracy]

You might want to talk to Chromadex, they specialize in extraction and analysis of phytochemicals. www.chromadex.com

[Tobias Jonsson]

Plant extracts are complex samples, so you need columns of at least 150 mm, perhaps 250 mm length. You probably also need to run gradients to resolve the components. If you plan to use a mass spectrometer (which I hope) for quantification I would use a column of 1.0-2.1 mm ID.

Many metabolites are rather polar and often contain phosphate groups or glycosides. In my opinion you need either a reversed-phase C18 column, quite likely with polar embedded groups, or a HILIC column (such as ZIC-HILIC from SeQuant). I would recommend the latter, or ideally, both types, especially if you are carrying out a full characterisation or are looking for metabolites of widely different polarity.

With HILIC, the retention is higher the more polar the compound is. You also have the benefit of much higher MS sensitivity compared to reversed phase due to the usage of high acetonitrile concentrations (60-95%). I would think sample preparation could be easier for HILIC than reversed-phase for several of these extracts.

[thekid]

hello all,
thank you for the information you have provided. we will be looking for many different metabloites at first. the basic reason would be for example. we take a plant and extract from it all the compunds possible in different solvents as mentioned, then we would be checking in for antimicrobial activity and then which ever extract shows activity will be subjecting it to HPLC.
the HPLC will mainly be used to find the compounds present in the active extract and also to quantify it. hence the extract will be having all the compounds that can be dissolved in the particular solvent.

once the compounds have been identified, further purification of the particular extract will be carried out to elute out each compound in a larger more pure form ,so that the antimicrobial activity can be checked.

hence the prefernece of column is crucial and also the type of detector used to quantify each of the compounds from the random extracts.

[tom jupille]

I hate to sound negative, but you may be biting off more than you can chew.

Each of your extract will contain hundreds of compounds, with concentrations ranging over several orders of magnitude. A "reversed-phase" HPLC column (the most common type) has a "peak capacity" of about 200 (for Rs of 1, which is "partial" resolution). In practice, that means that if you run a random sample containing 12 compounds, there is a 50% probability that two peaks will overlap.

Running samples on several "orthogonal" columns (e.g., a reversed-phase, normal-phase, anion-exchange, cation-exchange) can help, but this is most effective if you collect fractions from the first column and run each fraction on the second, from which you collect fractions . . . etc.

The real answer is LC-MS/MS, but I hope your college has rich, generous alumni, because the price tag is an order of magnitude greater than that of a basic LC system.

If this is a intended as a student (undergraduate research?) project, a better initial approach might be thin layer chromatography (TLC). You could run your extracts first on "analytical" plates, then screen for activity directly on the plates. Run the same separation on a prep (thicker) plate, screen for activity on a small portion or lane of the plate and extract the corresponding portion from the rest of the plate. Repeat with a different type of plate and then go to the HPLC, characterizing the sample by different separation mechanisms as suggested above.

The catch is that there is no way to tell a priori exactly which columns will be required.

[Mark Tracy]

In this kind of work, your active substances may well be a small percentage of the total extract. This means that you will need to inject milligrams of extract to obtain micrograms of active substance. This is the realm of semi-preparative HPLC, that is 10mm i.d. and larger columns. The flow rates start at 5 mL/min and go up by the square of the diameter. This is going to be expensive.

Since you don't have any specific chromatography by which to judge columns, just pick a C18 column that you can afford from a reliable vendor, and standardize on it. Don't worry too much about competitive "specifications" that vendors use; the only characteristic that is helpful to you is loading capacity. Be sure to get a guard column and pre-column filter too. For a second column, get a silica column for normal-phase LC. (I can afford to give this advice because Dionex does not make semi-prep C18 columns.)

For detection, the big outfits use a PDA followed by MS, sometimes with evaporative light scattering. At minimum, get the PDA. UV and MS can't do blind quantification of mystery substances. ELSD can get you a rough estimate.

For semi-prep HPLC work, you may also want fraction collection, another pricey hardware/software option. Either that or you stand there with vial in hand.

[Uwe Neue]

A few years ago, a colleague did exactly the type of work that you are planning to do. First, you identify the solvent fractions that contain actives. Then you do HPLC with these fractions. In general, running slow gradients over a large solvent composition range allows you to collect many fractions from your HPLC instrument. Then you can use the bioassay to check for the active fractions. Once you know where they are, you can then do very specific preparative HPLC with very specific gradients on prep columns.

For your fraction collection gradient where you check for actives, I recommend that you collect all fractions and do the bioassay on all fractions, whether your UV chromatogram tells you that there is something or not. It could very well be that your compounds of interest are not UV active. Actually, the ELSD is a better choice as a detector, but you need to split the flow. Once you have experience with the inital setup, you can tailor specific gradients for specific solvent extractions, but I would start with a generic gradient from 0% to 100% organic.

I think that initially you should be able to get away with an analytical instrument and 7.8 mm i.d. columns. You should be able to load 5 to 10 milligram quantities on such a column without difficulty. You may want to use a special trick called at-column dilution for loading the samples. I can get you more info on this.

When my colleague did the work, he used in parallel UV, MS and ELSD. The MS helped in the identification of the actives.

Ultimately, you will need a prep instrument and prep columns to get larger quantities of the actives for identification, and you should keep this in mind. For the time being, you are doing OK with an analytical gradient HPLC.

[thekid]

hello all,
thank you for that really nice advice, after discussing it with some more of my collegues, going through all the sugegstions we thought it best by trying out using analytical gradient HPLC with either a reversed-phase C18 column or HILIC column with a MS to detect the peaks and then do a bioassay for it.
the column length would be around 10mm id. then once we are sure about the active compound go in for a prep column.
tom jupille's idea of using TLC first was quite reliable and workable and will give that a shot. and as by Mark Tracy will also look into the filters and injection that you have mentioned.
Uwe Neue you have mentioned that i could very well use a MS for detection and also that the column can be 7.8 mm id. i would like to know as to which column your collegue has used, and if possible give me more details.

well the HPLC will not only be used for undergraduate but also for research purposes by PhD guys. the lab is mainly into the extraction of active compounds against different microorganisms. it is well funded and is always in a lookout to get the best.
i would like to thank all for contributing to the discussion and also please do let me know if the choice i have made is right. it seems that we do not yet have a standard method for going through a extraction process for plants.

thekid

[Uwe Neue]

SymmetryPrep C18 5 micron, 7.8 mm i.d. x 100 mm length.

If you are using MS for detection and later identification, you want a large split into the MS for two reasons. For one, you want to catch nearly all compounds into your collection vial, but more importantly, the MS will go blind at the large concentrations that you are using. Even with a 100:1 split before the MS, you will need a make-up pump to dilute the sample for the MS.

I do not remember the details about the runs of my colleague, but for this column, I would suggest at least 2 mL/min, possibly better 4 ml/min with a 1 hour gradient from a 5% aqueous to 100% acetonitrile, each with about 0.2% formic or acetic acid for pH control.