Organic solvent extraction
Liquid-liquid extraction is a useful method to separate
components (compounds) of a mixture
Suppose that you have a mixture of sugar in vegetable oil (it
tastes sweet!) and you want to separate the sugar from the oil.
You observe that the sugar particles are too tiny to filter and you
suspect that the sugar is partially dissolved in the vegetable oil.
How about shaking the mixture with
water
Will it separate the sugar from the oil?
Sugar is much more soluble in water
than in vegetable oil, and, as you know,
water is immiscible (=not soluble) with
oil.
Did you see the result?The water phase is
the bottom layer andthe oil phase is the
top layer, because water is denser than oil.
*You have not shaken the mixture yet, so
sugar is still in the oil phase.
By shaking the layers (phases) well, you
increase the contact area between the two
phases.The sugar will move to the phase in
which it is most soluble: the water layer
Now the water phase tastes
sweet, because the sugar is moved to the
water phase upon shaking.**You extracted
sugar from the oil with water
**In this example,water was the extraction
solvent ;the original oil-sugar mixture was
the solution to be extracted; and sugar was
the compound extracted from one phase to
another. Separating the two layers
accomplishes the separation of the sugar
from the vegetable oil
Liquid-liquid extraction is based on the transfer of a solute
substance from one liquid phase into another liquid phase
according to the solubility.
Extraction becomes a very useful tool if you choose a suitable
extraction solvent.
You can use extraction to separate a substance selectively from a
mixture, or to remove unwanted impurities from a solution.
In the practical use, usually one phase is a water or water-based
(aqueous) solution and the other an organic solvent which is
immiscible with water.
The success of this method depends upon the difference in
solubility of a compound in various solvents. For a given
compound, solubility differences between solvents is quantified as
the "distribution coefficient"
At a certain temperature, the ratio of concentrations of a solute in each solvent
is always constant.ﾊAnd this ratio is called the distribution coefficient, K.
when solvent1 and solvent2 are immiscible liquids
For example,Suppose the
compound has a distribution
coefficient K = 2 between solvent1
and solvent2
By convention the organic solvent is
(1) and waater is (2)
(1) If there are 30 particles
of compound , these are
distributed between equal
volumes of solvent1 and solvent2..
(2) If there are 300 particles of
compound , the same distribution
ratio is observed in solvents 1 and
2
(3) When you double the volume of
solvent2 (i.e., 200 mL of solvent2
and 100 mL of solvent1), the 300
particles of compound distribute as
shown
If you use a larger amount of extraction solvent, more solute is extracted
Liquid-Liquid Extraction
• LLE is a separation technique based upon transfer of a
solute between two immiscible liquids.
•
The key principle underlying LLE is often that the
solubility of the solute differs significantly between the
liquids, providing a thermodynamic driving force for
transfer from one phase to the other.
•
Any two immiscible liquids may be used, but it is common
to use water and an organic solvent.
•
In a small-scale chemistry laboratory, batch LLE is often
performed using a separatory funnel:
The funnel is shaken vigorously, while taking care to
vent any gases or vapors formed.
The shaking enhances the surface area of contact between
the phases, while the agitation enhances mass transfer from one phase
to the other by providing convection.
The shaken mixture is allowed to stand until the two phases separate
into layers (hopefully without forming an emulsion!)
Care is taken to recover the extract phase, which is thought to
contain a high concentration of the solute. The extract phase may
be either the top or the bottom phase!
The extraction may be repeated by shaking the remaining
raffinate phase with more solvent two or three additional times
until most of the solute is extracted. The raffinate phase is usually
discarded at this point.
After possible additional washing or drying steps, the solute is
generally recovered by distillation of the solvent, or by
crystallization from solution followed by filtration, to yield a
concentrated solute.
An example of a lab-scale LLE procedure
Suppose we wish to extract caffeine (a solid) from a cup of tea
(mostly water) to determine the caffeine content of the beverage.
We might follow a procedure like this:
1) The (cooled) cup of tea is shaken with methylene chloride
(CH2Cl2), an organic solvent, in a separatory funnel.
2) Much of the caffeine goes into the CH2Cl2 phase, but other
organic compounds (e.g. proteins) tend to remain in the water
phase.
3) The CH2Cl2 phase (extract) is recovered and saved.
Needle-like caffeine crystals
4) The extraction of the raffinate (water) phase is done two
more times, and the CH2Cl2 phase is recovered and saved
each time.
5) The CH2Cl2 phase is dried over anhydrous sodium sulfate
three times to remove residual H2O.
6) The solvent is removed by rotary evaporation, and the
caffeine crystals (and possibly other compounds) are
recovered and dried in an oven.
Acetone precipitation of proteins
• A strategy for removing undesirable
substances is to add a compound that causes
protein to precipitate.
• After centrifugation the pellet contains the
precipitated protein, the supernatant
containing the interfering substance is
removed and the protein pellet is re-dissolved
in buffer
• Precipitation has an advantage over dialysis or
desalting methods in that it enables
concentration of the protein sample as well as
purification from undesirable substances.
• One disadvantage of protein precipitation is
that proteins may be denatured
Materials Required
• Cold (-20°C) acetone, a volume four times that
of the protein samples to be precipitated
• Centrifuge tube, made of acetone-compatible
material such as polypropylene and able to
hold five times the sample volume
• Centrifuge and rotor for the tubes used,
minimum 13,000 x g required
Protocol
• 1. Cool the required volume of acetone to -20°C.
• 2. Place protein sample in acetone-compatible tube.
• 3. Add four times the sample volume of cold (-20°C)
acetone to the tube.
• 4. Vortex tube and incubate for 60 minutes at -20°C.
• 5. Centrifuge 10 minutes at 13,000-15,000 x g.
• 6. Decant and properly dispose of the supernatant,
being careful to not dislodge the protein pellet.
Precipitation with a concentrated acid such
as Trichloroacetic acid (TCA) causes
extremely low pH resulting in precipitation.
To minimize the risk of acid hydrolysis of
peptide bonds the experiment should be
performed on ice and care should be taken
to remove residual acid by washing the
protein pellet with acetone.