By Solvent -solvent extraction starting with the least in order of Polarity eg. by suspending the extract in water and partitioning with N-hexane,ethylacetate and N-butanol in that order.

But what about what will be at the interface? Do I take from the separating funnel down with water and take to new separating funnel where I add the ethylacetate?

You can check the nature of interface by simultaneously coTLC with both the solvents used for extraction. Mix it with the similar one. Normally it resembles the solvent other than water.

Above given suggestion very importance from my side add one suggestion your methanolic extract will dissolve in water and transfer to separating funnel after that add hexane and shake in only one direction clock or anti-clock wise three time and keep for 10 min after that water density high so it is settle down and hexane upper layer and open the tap and collect your fraction water portion same step you follow for other sovents such ethyl acetate and n-butanol other method for fractionate such as by column chromatography

In case of fractionation each researcher do it according to his own experience. Generally, I will go with Dr Ahmdu. Start from low polarity to higher polarity for partitioning. Then dry each extract and go for column chromatography of resin column followed by Silica, C-18 etc.

Gudluck

:)

I think one of the best way is to use Sephadex LH20.

Ii is better to use 2 immiscible solvents

I think the best method to use depends on the kind of isolate one is looking for. For flavonoids, dissolve the extract in distilled water and successively partition in hexane, chloroform, ethyl acetate and butanol. Either Tlc or column chromatography can be followed to separate the pure constituents. For alkaloids, the methanol extract must be acidified to convert them into salts followed by treatment with NH3 and fractionate in ether before subjecting the dried sample to column chromatography.

To avoid interface layer, which always create problem in partitioning, add some solution of NaCl, and then extrcat the aqueous layer by using solvents of your choice.

It depends on purpose of the separation. If you have Flash Chromatography, By developing a suitable solvent system for separation of different constituents you can get different fractions of respective peaks.

How about trying open glass column chromatography with normal silica gel and use chloroform:Petroleum ether (7:3) as a mobile phase?

The best way of fractionating methanolic extract is by first suspending the extract in ether or hexane, followed by chloroform, then with butanol. The fractionastion should be done at least 4 to 5 times in order to achieve effective separation. By this we get different fractions containing similar groups of compounds which are easier to work on further by column chromatography.

Silica on column. Add extract dissolved in a less polar solvente and increase polarity using a mixture of solvents until the last should be the more polar. The solvents should be chosen accordingly to the aim fraction(s).

If one has not decided yet the the class of compound to work on, then what will be better to work ahead?

Either consequent fractionation from same methanol extract or to prepare enriched fractions using fresh methanol extract every time?

Is your methanol extract completely dried (say in a rotavac)?

If you have a dry extract, then add sufficient quantity of coarse silica gel and then mix them by grinding using a mortar and pestle.

Pack this mixed (dry) sample+ silica mixture on top of a Vaccuum liquid chromatography column (this is a dry column packing; packed with fine silica gel). Elute with successively with solvents of increasing polarity (n-hexane, dichloromethane, acetone, ethyl acetate and methanol). VLC uses suction (with a vaccuum pump). You will have fractions of increasing polarity

Only two best methods are usually being used

1. liquid-liquid partition and 2. column fractionation

For liquid-liquid partition you need to suspend your dried extract in distilled water and then use the solvents from lower to higher polarity; such as hexane/pet. ether, DCM/CHCl3, EtOAc and finally n-butanol. These solvents will form separate layers in water.

In second method, a little wet extract can preadsorved with silica and pack with a column and fractions can obtain with various solvents from lower to higher polarity as used in first method but acetone is much suitable in place of butanol.

To remove butanol from our fraction you can add equal portion of water to make a azeotropic mixture.

As per my personal experience, the second method is most easy and convenient.

Simply dry the extract and then add solvents low polar to high polar and with each solvent, use ultrasonic bath to boost extraction. Using two immiscible solvents sometimes become tedious as an interface layer is formed, to remove which needs some extra efforts.

it depends on what class of compounds you work. There is no classical methods that is adapted to all polar compounds. For the fractionation only use liqui-liquid partition using solvents of different polarity

to get pure compound through fractionation of MeOH extract

1- con the extract then fractionate over diaion using water with increasing the amount of methanol

2- examine the collected fractions by tlc mainly you get 4 main fractions

3- water extract contains carbohydrates, saponins, iridoids and more polar compounds

4- 10% until 50% methanol containing high polar phenolics

5- with increasing amount of methanol you will get less polar compounds

As many people have written, there are some questions to ask before the activity:

1. Do you know the class of compound you are looking for?

2. Are you interested in "everything"?

3. Are you looking for a molecule with an acidic group (carboxylic acid?) or a basic group (amine?) or neutral (sugars?) - I am assuming organic molecules are of interest and there might be some simple inorganic salts.

4. How much material do you have to work with? Did you extract grams or milligrams of material? do you have milliliters of material on hand or liters of solution?

5. How clean do you need the material? How are you analyzing it? How many components do you expect in the initial extract? 5? 100?

All these questions and their answers direct you to techniques and processes. It's not just about a methanolic extract of ?

There are so many methods and techniques: pick more or less the right one(s) and you can save a lot of time and get clean material.

If you want answers that are useful, give a more information about your material that you are working with.

As described by Yogesh, you can fractionate by using partitioning principle (using two immiscible solvents) or by using silica gel parked in a column. You can also triturate the silica gel with a slurry of the methanol extract and shake in a closed bottle with solvents of increasing polarity.

firstly you must evaporate methanol and then, dissolved the dry extract in distilled water (say 500 mL). It will be successively extracted with solvents of increasing polarity (each 500 mL, three times) . There, you will have the aqueous and several organic layers.

In my opinion, you need to first remove the solvent by evaporation to get the extract residue. Then, you should resuspend the dried residue in distilled water followed by extraction with hexane (3 repetitions for each solvent partition). Following by the partition using the solvents with increasing polarities solvents, such as ethyl acetate and butanol. There, you will obtained the aqueous layer as well as several organic layers. Finally, evaporate the aqueous off the final partition, which you will obtain the methanolic extract.

To evaporate the MeOH to obtain a dry resin. Washing with hexane (or chloroform/CH2Cl2), EtOAc and MeOH, respectively. You could think it as fractionation between solid phase and liquid. Moreover, you would find this is a simple, saving, quickly and cleaning method.

I am completely agree with suggestions of Barry Parnas, You should have clear Idea about the constituents of interest and their polarity because selection of solvent for column chromatography or solvent partition technique is polarity based.

First with n-hexane, then chloroform followed by ethyl acetate and last with n-butanol

what about if diethyl ether and chloroform; could we start with diethyl ether then by chlorofrm or vice versa? (which one is more polar)?

interesting discussion

1). n- Hexane removes all non polar compounds

2). chloroform removes all pigments such as chlorophyll etc

3). ethyl acetate is where all biological active compounds are excepted such as flavonoids, anthraquinones, anthocyanine ,benzo pyrans etc

4). n-butanol extracts all other hydrocarbons

Can be fractionated as you said. And there is another way the so-called liquid-dry. It is necessary to mix the extract with silica gel and then wash it with a solvent, increasing their polarity.

The concentrated methanol extracts can be fractionated by liquid-liquid fractionation using solvents of increasing polarity viz. Hexane, Chloroform, Ethyl acetate, Methanol. Suspend your dried methanol extract in Water:MeOH (4:1) and start fractionating it with the non-polar solvent, Hexane in a separating funnel. Separate hexane dissolved portion, i.e Hexane Fraction (HF). Concentrate and dry hexane fraction by removing the solvent under reduced pressure. Likewise other solvent fractions can be obtained by using further solvents (non-polar-Medium polar-Highly polar) in the order of increasing polarity following eluotropic series.

Alternatively, you can make different fraction by placing your methanol extract in a silica-gel-G packed column and subsequently eluteing with different solvents in the order of increasing polarity.

https://www.frontiersin.org/articles/10.3389/fphar.2018.01418/full

Bioassay-Guided Isolation of Anti-Candida Biofilm Compounds From Methanol Extracts of the Aerial Parts of Salvia officinalis (Annaba, Algeria)