Chromatography Forum

Dear Members,

I would like to hear from you a suitable solvent pair ratio for the analysis of Flavonoid glycosides from leaf and seed samples. I am using Shimadzu LC10A with PDA. I have tried with ACN:water 70:30, but no proper elution of peaks I could notice.

Please inform me any suitable method avialable for the analysis of crude leaf extract for flavonoid cojugates.

Best regards

Best approach would be to start with a gradient covering a wide range of ACN concentrations (e.g. 5 - 95%).

Here is an example of some examples of flavonoids
(Imtakt used a gradient with TFA as the acidic modifier):

http://www.imtakt.com/TecInfo/TI294E.pdf

You will want a gradient method. I did anthocyanin conjugates in bilberry with a 5-95% MeCN gradient and had peaks almost the whole way across. http://www1.dionex.com/en-us/flash/fullflash62071.html I also tried MeOH and got somewhat different selectivity. The choice of acid is not that critical as long as you can get down below pH 3. A PDA is really nice to have too.

Echo Mark's comments on pH and PDA, however the choice of acids might have a small effect on your sensitivity.

It won't bother your flavonoids so much, but other components that don't have strong chromophores and which might co-elute with your flavonoids will be easier to detect if using a PDA.

Phosphate absorbs less than acetate or formate in low UV so your gradient "ramp" will be less significant. If using an acetonitrile based MP, I might be tempted to use dilute phosphoric acid first to keep the baseline as flat as possible, especially across a broad ACN concentration.

Best of luck!

CJ

Also, a cool site for you to look at re: flavonoids:

http://www.metabolome.jp/software/FlavonoidViewer

We have used the following method in the analysis of flavonol glycosides.

Column: Phenomenex Prodigy ODS 5u (3) (250 x 4.6, 5 um) with pre-column Phenomenex Prodigy ODS 5u (3) (30 x 4.6, 5 um)

Detection at 270 nm and 370 nm with a PDA detector

Solvent A: water-THF-TFA (98:2:0.1)
Solvent B: ACN

Solvent gradient:

Time (min): proportion of solvent B (%)
0-2: 15
2-14: 15-25
14-19: 25
19-24: 25-60
24-28: 60
28-30: 60-90
30-35: 90
35-40: 90-15
30-40: 15

In my colleague´s experience, flavonol glycosides should not be dissolved in ACN before injection, since they are not stable in the solvent. you might want to try e.g. MeOH-water (1:1)

Here's another application

Quercetin glycosides separated on Unison UK-Amino:
http://www.imtakt.com/TecInfo/TI369E.pdf