Phenolic acids

[tashian]

Hi,

I am trying to use HPLC to analyze phenolic acids in some plant samples.

Since this is just a preliminary experiment, and no budget is involved, we are looking in the plant for the ones we have standards for: ellagic and gallic, coumaric, cinnamic derivate, caffeic, quinic and chlorogenic acids.

We only have a RP-Hydro column, so we are bound to use only this one.

I have tried some mobile pahses from the literature, but apparently all standards co-elute at the same retention time, and very early.

I would love to have your idea of what mobile phase can I use in order to separate these standards with such column. Basically the plant is extracted in 80% EtOH, so the standards are also dissolved in this.

Many thanks!

[tom jupille]

I have tried some mobile pahses from the literature

That's not a lot of information to go by. What mobile phases (exactly) did you try?

[Hollow]

shouldn't be too hard to get most of them separated

to start try a gradient, about 20 column volumes, from 5->95% of ACN in acidified Water (formic acid 0.1% or H3PO4 0.1% V/V).
Detection somewhere around 325nm
then adjust the stepness/range to your needs.

it's important that the water is acidified. otherwise the compounds won't get good retention.
depending on your LC system, inject only small volumes or dilute your standards to lower the EtOH concentration if peak deformation occurs

[tashian]

Thank you for your helpful replies.

What I've already tried, according to literature, were several systems:
1. acidified water (0.1% formic) and ACN (0% to 85%).
2. acidified (0.1% formic acid) MeOH:water (80:20) and ACN (0 to 85%).
3. acidified water (0.5% acetic) and ACN (10% to 70%).
4. acidified water (2% acetic) and MeOH:ACN (50:50), from 5% to 100%.
5. acidified water (2% acetic) and MeOH:acidified water (0.5% acetic) 80:20, from 0 to 100%.

Should I dissolve the standards in another solvent instead of EtOH? would that make a difference?

Hollow, correct me if I'm wrong, it seems to me that what I've tried resembles what you suggested, however I do not get a separation.

[Gerhard Kratz]

Please try to dissolve your standards in your mobile phase.

25% aqueous and 75% ACN I would use for to start with.
But first check the application data bases of the column manufacturers. You will find a lot of applications and I'm sure also some valuable hints.
Good luck.
What detection you use?

[Hollow]

Hollow, correct me if I'm wrong, it seems to me that what I've tried resembles what you suggested, however I do not get a separation.

yes, seems they should have worked at least for some of your compounds.

So I wonder if you get retention for other coumpounds or if any substance behave just the same.
-> is your system working well (flow, gradient proportioning valve etc)?
-> what about some test substances like caffeine or parabens. Are they retained/separated well with some 0-95% gradient?
-> is your column fully wetted/equilibrated?
-- equlibrate with at least 5-10 column volumes of the initial mobile phase before injecting the samples

[mattmullaney]

Hi Tashian,

Along Hollow's and Gerhard's good suggestions--with a RP-Hydro from Phenomenex it's likely not to be a wetting problem (I've some experience with this phase) in my opinion, but I'd also see into diluting your sample with as much water as it can stand. 80% (v/v) EtOH for the sample solvent is pretty strong (I understand that you need to actually extract the compounds you're trying to analyze).

With the samples, you can add, say, one volume of water to one volume of sample extract...and just inject twice as much. In an similar way, you could see what happens to standard material(s) dissolved in solvent mixtures containing higher water content than 20% (v/v).

This may help the analytes gain some solubility into the stationary phase...and be retained a bit. Please, if you let us know the flow rate and column dimensions as well as the retention time of the "peak," this can help us troubleshoot.

Best Wishes!

[tashian]

Thank you very much for all your ideas.

Yesterday I was trying to use 2% TFA as a solvent, I tried an isocratic run (68:32 with MeOH)- in which I saw peaks but small and broad, or with a gradient of ACN (4-50% ACN), which didn't work.
Another thing I tried was dissolving some standards in water, and using an ACN gradient in 0.1% formic, which I think worked... This is basically somewhat like Mattmullaney suggested. I am detecting at 220, 280, 320 and 350.

Another thing- on of the peaks I thought was a standard was apparently EtOH, as it also appears in the blank sample (80% EtOH).

I will try repeating this and see what happens, will try adding some water to standatds and will be back with the column details and the resutls.

Many thanks!

[chrsdrake]

You dont seem to be doing anything very wrong, so probably it is something small that is causing you problems. Lots of good suggestions already.

We analyse condensed tannins (a different class of polyphenols) using a gradient from 2.5% ACN in 1% formic acid to 50% ACN in 1% formic acid, then wash off with a steep gradient up to 100% ACN. Our column is a C18 and I use a column temperature of 60C. At lower temperatures I need to start the gradient at higher ACN concentrations, but I dont get as good a separation. Detection is fine at 280nm.

My main concern with your method is the injection solvent/volume. Try making your standards up as you do and dilute 1 part in 10 into your acidified mobile phase A before injection. You could aim for a final concentration of 100ug/ml and inject 5ul. This should give good sized peaks!!! I presume you are using a 4.6mm id column at a flow rate of about 1ml/min?

More details of your method might give us some other ideas to suggest.

[g_wallace]

I've done quite a bit of HPLC of phenolic acids. The conditions you've tried should certainly separate something. As mentioned earlier you have to dissolve your samples in something more suitable, acidified mobile phase is ideal. Could it be your column that's the problem Could you post a chromatogram ?

[tashian]

Dear All,

Thank you so much for all your helpful information and ideas.
What eventually worked was what Mattmulaney suggested, i.e dilluting the samples (which were in 80% EtOH) 1:4 with water.
Even then, I still had to be very percise with the ACN (a gradient of 1% over ten minutes or so) to have them separated- but it worked well!

So thank you very much again for your precious help.

[Gerhard Kratz]

I agree with Matt and the other colleagues. Different HPLC system brands can have different flow cell volume, what could be the reason why you see different peak high and shape. Please give more information on the systems.