We now have three solutions containing dissolved molecules - the 30% fraction containing fairly polar molecules, the 75% fraction containing slightly polar molecules and the 100% fraction containing molecules of very low polarity. The next step is to remove the water from these solutions so that we can obtain the dissolved compounds. This will be done using a rotary evaporator. This process will involve removing some of the air above the solution to create a partial vacuum. With the 30% and 75% fractions this is going to cause a problem as mixtures of water and acetone are unstable under those conditions and will tend to jump and spurt as we try to dry them down. To get around this we need to first use a process called
back-loading.
Back-loading a fraction involves reloading it onto a HP20 column. To encourage molecules back onto the non-polar column, the volume of the solution is doubled by adding water, thus making the solvent more polar. For example, my 75% fraction of
P. microcladioides had a volume of 240 mL so I added 240 mL of water to it before passing it through the HP20 column. This caused many of the molecules of low polarity to attach to the column.
In the photo to the right my 75% fraction of
P. angustum is being back-loaded onto the left-hand column while my 75% fraction of
P. microcladioides is being back-loaded onto the right-hand column.
Once the solution has passed through the column, the solvent is made still more polar by again doubling its volume by adding water. So I added a further 480 mL of water to my 75% fraction of
P. microcladioides. It is then passed through the column again and the moderately polar molecules will now attach to the column.
As the volumes become larger, passing the solution through the column can become very time consuming. You also have to watch out for compounds beginning to
crash out of solution. This can happen when you are adding water and the solution becomes too polar for some of the compounds to remain dissolved. These compounds then begin to precipitate out of solution. As soon as you see this begin to happen you need to stop adding water and start passing the solution through the column.
The picture to the left shows my 75% fraction of
P. angustum part way through the back-loading process. You can see that some orange coloured compounds have made their way to the bottom of the column while some green coloured compounds have attached further up. While the coloured compounds are the interesting ones to look at, many of the compounds will actually be colourless.
Once the molecules have been back-loaded onto the column they are then removed by passing acetone through the column. The eluate collected contains the molecules and the solution can now be dried down on the rotary evaporator as the solvent is acetone rather than a mixture of acetone and water.
Solutions in small conical flasks, or dried down fractions in vials, are kept in a fridge. Not only is it dark in the fridge (yes the light does go off when you shut the door), but it is also cold. As you know, reactions happen at a much slower rate at low temperatures, so any potential reactions that could occur will hopefully be slowed right down.
