Hi,

This is a general question as you do not know what polyphenols you have, or you intend to isolate. From the extraction using hot methanol, you are dealing with highly polar compounds but bear in mind that you also have many non-polar compounds extracted by hot methanol as well. So, polyphenols isolation from this extract only indicated your intention not the sole components of the extracts. There are ids no doubt that you have polyphenols as well so let us consider you try to purify individual components.

What mater most is your capability as there are numerous approaches based on the nature of polyphenols you have, tannins, flavonoids, sugars/polysaccharides or glycosides or many sorts will be components in these extracts. You mentioned you have size exclusion (Sephadex) and adsorption (silica- normal phase) capability but also TLC and HPLC systems.

The silica gel you mentioned is normal phase which ideal for relatively non-polar compound. The Mesh refers to the particle size and how easy the elution would be without applying pressure. One limitation you have here is the polarity range not being suitable for highly polar polyphenols. You need to consider a reverse phase system for water soluble polar compounds.

Sephadex LH-20 is designed for relatively non-polar compounds up to methanol solubility for example. As polarity increases, say for tannins, elution them through is difficult and you will experience these compounds permanently bound with the Sephadex and hard even to recover it. Hence, you will lose your Sephadex LH-20, or the advantage of reusing this relatively more expensive resin. For water soluble materials, I would not use Sephadex as a first separation system and hence I recommend you to use it after passing your extract through another system say, after silica-based separation system. Consider other types, if you afford it, that are more suited for water soluble compounds.

TLC is for detection though you may use it on prep scale once you have fractions with few components in it. HPLC is good for detection and for large scale separation, it is not ideal as it takes time due limited sample size or injection volume/weight. If you have fraction collectors based on other system such as reverse phase separation, your life would be easier.

Based on what you said, I would say to start by establishing the profile of your mixture/crude extract by using HPLC. Use a reverse system HPLC column for this. Once you establish, the profile of the components, you can try the above fractionation methods. If you are interested on polyphenols only, first fractionate the crude methanol extract with solvents of increasing polarity. In this case, the non-polar or non-phenolic components first removed. Hexane/chloroform can do this job. Medium polarity and polyphenols can appear in ethyl acetate fraction and n-butanol and water fractions would have the bulk of your polyphenols. HPLC can be used for the last stage purification.

Good luck

Dear Aws Bunyan Polyphenols as phytochemicals have gained significant importance owing to several associated health benefits with regard to lifestyle diseases and oxidative stress. To date, the development of a single standard method for efficient and rapid extraction of polyphenols from plant matrices has remained a challenge due to the inherent limitations of various conventional extraction methods. The exploitation of polyphenols as bioactive compounds at various commercial levels has motivated scientists to explore more eco‐friendly, efficient, and cost‐effective extraction techniques, based on a green extraction approach. The following modern green extraction techniques—supercritical fluid extraction, ultrasound‐assisted extraction, microwave‐assisted extraction, pressurized liquid extraction, and pressurized hot water extraction—as alternatives to conventional extraction methods for polyphenol extraction. These techniques are proving to be promising for the extraction of thermolabile phenolic compounds due to their advantages over conventional, time‐consuming, and laborious extraction techniques, such as reduced solvent use and time and energy consumption and higher recovery rates with lower operational costs. The growing interest in plant‐derived polyphenols prompts continual search for green and economically feasible modern extraction techniques. Modern green extraction techniques represent promising approaches by virtue of overcoming current limitations to the exploitation of polyphenols as bioactive compounds to explore their wide‐reaching applications on an industrial scale and in emerging global markets. Future research is needed in order to remove the technical barriers to scale‐up the processes for industrial needs by increasing our understanding and improving the design of modern extraction operations.

Solomon Habtemariam thank you sir for your wonderful answer I will definitely establish my work according to your advice.

D. S. Bhagat thank you sir for your answer, I hope I can use the techniques you mentioned but unfortunately I'm dictated by my laboratory limitations.