I am fine with having a mixture of phenolic compounds but it must be free from other components. Is there a purification procedure I can use after extraction?
Phenolic compounds are well-known phytochemicals found in all plants. They
consist of simple phenols, benzoic and cinnamic acid, coumarins, tannins, lignins, lignans and flavonoids. The most common techniques to extract phenolics employ solvents, either organic or inorganic. Several parameters may influence the yield of phenolics, including extraction time, temperature, solvent-to-sample ratio, the number of repeat extractions of the sample, as well as solvent type. Furthermore, the
optimum recovery of phenolics is different from one sample to the other and relies on the type of plant and its active compounds. The choice of extraction solvents such as water, acetone, ethyl acetate,alcohols (methanol, ethanol and propanol) and their mixtures will influence the yields of phenolics extracted. For instance, a high yield of phenolics can be extracted from sorghum leaf using water , while extraction of phenolics from wheat bran requires 80% aqueous ethanol. In another example, an investigation into the effect of different solvents on extraction of phenolics from aerial parts of plants showed that 50% aqueous ethanol is more efficient than pure or 50% aqueous forms of methanol, and acetone. In contrast, the highest levels of phenolics are extracted from Vitis vinifera wastes and sunflower meal using pure methanol and 80% aqueous acetone, respectively. These differences could be due to the properties of the henolic components of the plants concerned. Phenolic acids generally exist in a free, esterified or glycosylated form in plants. For extraction of free phenolic acids in rice using 70% ethanol at room temperature followed by centrifugation. The extract is then treated with 4 M HCl to reduce the pH to 2–3 and the phenolic
fraction separated using ethyl acetate and dried with anhydrous disodium sulfate.
Ravi, Do you have a reference for the method you describe? Currently we are extracting free phenolic compounds with Methanol:Water:HCl and Acetone:Water:HCl. Then we extract hyrolyzable phenolics with Methanol:Sulfuric acid. After the extractions we bring the pH to 4.0 then use a rotary evaporator to remove organic solvents and then we freeze dry. However we are getting a lot of protein in our samples and we are not able to detect any phenolic compounds in the freeze dried sample. Thanks.
Kristin, you should be able to do the same this with open glass columns as well; Barbara Timmermann's group has also had good luck with using ion exchange resins as a batch mode rather than a column, although my experience that batch mode is less efficient (requires more time stirring). I suggest looking up her papers, though I think they were working with alkaloids and strong cation exchange columns.
You can use ion exchange or reversed phase chromatography after extraction. Depending on the composition of your extraction solvent if it is having 50% or more water you can use reversed phase adsorbent, where you will see the binding of phenolics and proteins will come in flow through fraction. While using ion exchange chromatography you have decide for anion/cation exchange depending on strength of HCl you are using for extraction. Chromatography is the best alternative after extraction to remove the impurities.