Hello!
In the aim of linking the result of a non destructive chlorophylle and flavonoids estimator (Dualex) to the chemical concentrations of different species' leaves, we would measure pigments concentration spectrophotometrically.
We have developped a protocol of methanol extraction, based on litterature. However, we will use a spectrophotometer with plates with different properties than the cuvette used in studies providing equations linking absorbance and concentration.
Which commercial products should we choose to calibrate the parameters of those equations? We want to obtain a measure of total chlorophyll content and a proxy of flavonoids concentration.
Thank you!
In terms of flavonoids, it depends what you mean - there are many different types, which all have different properties. But assuming you mean the flavonols and/or flavones, then using something like Rutin is fairly standard. You can order the standard easily, and it is used by most labs that quantify flavonols etc (e.g. as rutin equivalents). This would typically be a methanol based extract, and then a dilution used in methanol and measured at 350nm. The extinction coefficient for rutin is published for methanol. If you are using plates, then make very sure they are UV transparent! We have used plates for quantifying flavonols and anthocyanins (different solvents for measuring) and found it works pretty well as a screening tool.
For Anthocyanins, there are several published standards people have used (with extinction coefficient data etc). Note though, that the extract must be diluted in 0.1N HCl/Methanol. This is to shift the form of anthocyanins into their flavillium (coloured) form, allowing them to be measured at 530 nm.
Once you get these up and working reliably with the standards, you should be able to extend this to your real samples and then compare this with the alternative methods you are working with.
Good luck.
Thank you so much for your response!
I will follow your advices carrefully.
- Badges
- Science topic