In class we have looked at covalent bonds having different degrees of polarity depending on the difference in electronegativity of the two atoms involved in the bond. Some bonds are not polar at all (non-polar) while others may be only slightly polar or very polar. We have tended to classify molecules as either non-polar or polar and left it at that. However molecules also have varying degrees of polarity. For example, water is more polar than methanol which is more polar than acetone. We are going to use the relative polarities of these three solvents to help us to separate our mixture of molecules.
To begin with the second extract is passed through a column filled with 'HP20'. HP20 is comprised of small white beads of polystyrene divinylbenene. These beads provide a very non-polar surface that molecules can attach themselves to. Molecules will do this if their polarity is closer to that of the HP20 than it is to the polarity of the solvent they are in. So molecules in the mixture that have a low polarity will come out of the moderately polar methanol solvent and attach themselves to the HP20.Solution that passes through the column is collected in a conical flask at the bottom. The flow rate is about one drop per second so it can be quite a slow process. Once the second extract has passed through the column, it is the turn of the first extract which is loaded into the top of the column. Eventually both extracts have passed through the column and the solution containing all the molecules that have not attached to the column is in the conical flask at the bottom.
We now want to encourage some of the slightly more polar molecules onto the column. To do this we need to make the solvent more polar. This is achieved by doubling the volume of the solution by adding distilled water. Remember that water is more polar than methanol so the solvent (now a mixture of water and methanol) is more polar than it was before. The solution is put through the column again and this time some of the slightly more polar molecules attach to the column. This process is repeated one more time to make the solvent even more polar and to cause more molecules to attach to the column.
Molecules of high polarity will not, of course, attach to the column as they will prefer to remain in the polar solvent. This is not a problem for us as the molecules that we are hunting for do not tend to be of high polarity. However, just in case something important is still in the solution, we will keep it in the meantime.
Hi Boss,
ReplyDeleteDo you need to rinse the HP20 beads in between extracts or are you getting the same sorts of non-polar molecules from both extracts?
The beads don't get rinsed between the two extracts as we are wanting the molecules from both extracts to accumulate on the column. There should be similar molecules from the two extracts going onto the column although there may be some slightly more polar molecules in the first extract and some slightly more non-polar molecules in the second extract. The reason for this is that the algae contains water so the solvent for the first extract invariably ends up being a mixture of methanol and water (but mainly methanol). By the second extract the algae is considerably 'drier' and the methanol therefore doesn't tend to get diluted with water. The solvents for the first and second extracts therefore have slightly different polarity so the range of molecules in each may not be exactly the same.
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