Extractions in water vs ethanol?

Hi, I am testing various plant materials for my thesis and I am using FRAP, TPA etc. Doing samples in both ethanol and water. However when I come to HPLC, the results are higher in water than they are in ethanol. It's strange. Anyway, the crux of my question, can water be a better solvent of plant material than ethanol?

SOL

That would depend on the plant material, but if what you are looking for is really polar phenols and the like then they may well be easily dissolved in water.

Also, how do you prepare your samples? it may be that the water breaks down the overall plant material better leading to a greater release of its contents into the solvent?

I don't know the answer though, I'm just speculating, if you told us all exactly the process you were using though we might be able to help...

SOL

SOL wrote: »

That would depend on the plant material, but if what you are looking for is really polar phenols and the like then they may well be easily dissolved in water.

Also, how do you prepare your samples? it may be that the water breaks down the overall plant material better leading to a greater release of its contents into the solvent?

I don't know the answer though, I'm just speculating, if you told us all exactly the process you were using though we might be able to help...

The plant is Ashwagandha. Also known as indian ginseng. It is high in antioxidants. I prepared the extracts in water by boiling for 30 mins at 100 degrees celsius. Then I filtered using filter paper and used HPLC to analylse. I made up the ethanolic extracts by mixing an 4:1 mixture of ethanol:water, sonicated for ten mins, then filtered using filter paper.

bduffy

I'd imagine that boiling the sample for 30 mins would always result in a higher release than a physical process. Other than than you could do a gravimmetric test and simply weigh the extract residues after evaporation (assuming you're not using any release agents).

SOL

There is your problem, I'd say boiling it for 30 mins is a lot more effective than room temperature sonication. Also, a quick google tells me tropine and cuscohygrine and things like this are found in your plant and they are water soluble.

Also, if you are boiling your water, are you taking account of the water lost or do you have a closed system?

Why not boil it in ethanol for a while? (use a reflux condenser)
Or even better, use one of my favourite pieces of glass ware if you can find one...

Also, does this not raise seriously questions about the repeatability and quantative value of your ananlysis if you can't be sure you are getting all the stuff you are measuring out of the sample...

AaronEnnis

There are a few different factors at work here.

1. Extraction solvent

The more water-soluble analytes (I'm guessing polyphenols etc) may extract better in water than in ethanol, possibly giving a higher extraction yield in water.

2. Instrument conditions

How constant is this increase in peak area? Could it have anything to do with improper mixing? What mobile phase are you using....could the mobile phase pH lead to deprotonation/protonation in more suitable solvents?

3. Peak area

What method of quantification are you using? Internal/external standard? The peaks are only a detector's response - as bduffy said, you should remove the solvents after extraction and weigh the solid extract.

4. Starting amounts

....I assume you're starting with a constant weight of plant material, and that you're comparing the same area of plant (i.e. stem, leaf, root) in 2 different solvents and not analysing two different types of plant tissue??

5. Sonication vs. Boiling

Sonication won't reach the same extraction efficiencies as Soxhlet or Supercritical fluid extraction the whole time. Also, sonication tends to prove a bit too extreme for some of the more labile flavonoids.

AaronEnnis

AaronEnnis wrote: »

There are a few different factors at work here.

1. Extraction solvent

The more water-soluble analytes (I'm guessing polyphenols etc) may extract better in water than in ethanol, possibly giving a higher extraction yield in water.

2. Instrument conditions

How constant is this increase in peak area? Could it have anything to do with improper mixing? What mobile phase are you using....could the mobile phase pH lead to deprotonation/protonation in more suitable solvents?

3. Peak area

What method of quantification are you using? Internal/external standard? The peaks are only a detector's response - as bduffy said, you should remove the solvents after extraction and weigh the solid extract.

4. Starting amounts

....I assume you're starting with a constant weight of plant material, and that you're comparing the same area of plant (i.e. stem, leaf, root) in 2 different solvents and not analysing two different types of plant tissue??

5. Sonication vs. Boiling

Sonication won't reach the same extraction efficiencies as Soxhlet or Supercritical fluid extraction the whole time. Also, sonication tends to prove a bit too extreme for some of the more labile flavonoids.

It was 1 gram of Ashwagandha powder in all parts of the experiment. We used six different manufacturers for comparison. Two manufacturers specified that it was the root of the ashwagandha plant. The rest just stated that it was ashwagandha powder. This was an issue as different parts of the ashwagandha have different composition and even the composition of the plant can change depending on weather, soil conditions etc where it is grown.

The difference in peak of between the water and ethanol extracts varies from 17mmol/g difference to as little as 2mmol/g difference. The water samples were always higher concentration of the compounds I was examining.

Any day that I was carrying out work on the HPLC, I ran a calibration curve first.

The mobile phase was 0.1% formic acid.

SOL

SOL wrote: »

There is your problem, I'd say boiling it for 30 mins is a lot more effective than room temperature sonication. Also, a quick google tells me tropine and cuscohygrine and things like this are found in your plant and they are water soluble.

Also, if you are boiling your water, are you taking account of the water lost or do you have a closed system?

Why not boil it in ethanol for a while? (use a reflux condenser)
Or even better, use one of my favourite pieces of glass ware if you can find one...

Also, does this not raise seriously questions about the repeatability and quantative value of your ananlysis if you can't be sure you are getting all the stuff you are measuring out of the sample...

I probably should clarify. I am doing my thesis as part of my pharmacy degree. We only had 5 weeks to conduct the experiment. I finished before christmas. I had a lot of practice based pharmacy stuff to do over the christmas so I really only been looking over the results/writing the thesis the last two weeks. The system wasn't closed. So that could be a reason.

franb7111

hi daz, it is possible that the compound(s) you were trying to extract were protonated ( usually these are nitrogen containing compounds that have reacted with an acid), and were more soluble in water than in ethanol. It is possible that if they were liberated into their free base form they would be more soluble in ethanol or other organic solvent, dfferent solvents can give much different extraction profiles.