Chromatography – Extraction and purification of Chlorophyll
CHM 220
INTRODUCTION
Extraction and purification of naturally
occurring molecules is of the most common
methods of obtaining organic molecules.
Locating and identifying molecules found in
flora and fauna can provide a number of
different molecules. These molecules are
frequently used as feedstock for the
chemical industry or as components of
medicines and foods. Obtaining pure
samples of these molecules is essential to
our modern chemical and pharmaceutical
industry.
In this experiment you will use a common
method of extraction to obtain a mixture of
organic compounds. Extraction involves
separation of selected components of a
mixture through contact with a second
insoluble “phase”. The ability to separate
the desired components from the rest of
the mixture will depend on the affinity of
the desired components for one phase
versus the other.
Plants are known to produce many interesting and useful organic compounds; today we will be
extracting Chlorophyll from spinach. A significant portion of any cellular material is water
soluble; however the most of the useful organic components are more soluble in non-polar
solvents than in water. We will take advantage of this trait by breaking down the cellular
structure of a sample in the presence of an organic solvent. As a result, the components of
interest will preferentially be dissolved in the organic solvent, and subsequent “washing” of the
organic phase with water will remove any remaining water soluble components. We are then
left with a mixture of organic compounds dissolved in our solvent, which will require further
purification to isolate the desired compound(s).
To further purify our Chlorophyll we will employ chromatography, another common laboratory
technique. Chromatography involves use of a mobile phase, called the eluent, and a stationary
phase. The mobile phase, typically a gas or liquid, is moved through the stationary phase, most
often a solid, to separate the mixture. The more attracted a compound(s) is to the stationary
Chromatography – Extraction and purification of Chlorophyll
CHM 220
phase the slower it will move, while those more
attracted to the mobile phase will move through the
stationary phase much faster. Resulting in a
separation of our mixture based on the
characteristics of the compound(s) and that of the
stationary and mobile phases. Most often
compounds are separated based on their polarity,
but other characteristics such as chirality can also be
used. The measure of any particular compound’s
mobility in a chromatograph is Rf, Retention factor.
The Rf of any particular compound is a ratio of how
far/fast the compound has traveled over how far/fast
the mobile phase has traveled. Because Rf is a ratio
that is dependent on the nature of both the mobile
and stationary phases, it can be used for
identification purposes, provided the same mobile
and stationary phases are used.
In the case of spinach, our remaining organic solution will contain a mixture of carotenes,
Chlorophyll’s A and B, and Xanthophyll’s. We will begin by performing Thin-Layer
Chromatograph to determine the best mobile phase to use; in TLC the stationary phase is a
thin-layer of silica(or alumina) powder adhered to a glass or
plastic plate. Our mobile phase will travel up the plate (due
to capillary action) through the silica separating our
compounds. Once an optimum mobile phase is found we will
perform larger scale Column chromatography by filling a
burette tube with silica (or alumina) powder and flushing the
column with our mobile phase to separate our compounds
into fractions. A fraction is a set amount of mobile phase that
has been put through our column. Ideally our purified
compounds should now be collected in a few of our fractions,
separated by other fractions which contain only solvent. To
determine what fractions our desired compounds are in, we
will then perform a TLC on each fraction.
Chromatography – Extraction and purification of Chlorophyll
CHM 220
PROCEDURE
1) Place 5g of raw spinach, torn up into small pieces, into a mortar and pestle.
2) Add 25mL of Petroleum Ether and 10mL of Methanol, and grind the leaves well.
3) Using a Buchner funnel, vacuum filter the resulting pulp making sure no solid particulate
gets through the filter, rinse the solid with 10mL of Petroleum Ether and transfer the
liquid to a separatory funnel. Dispose of the remaining solid in the trash can after
observing the color.
4) Add 25mL of a saturated NaCl solution to the separatory funnel and stopper the funnel,
then invert and “vent” the funnel by opening the stopcock. Carefully shake the funnel
venting often.
5) Place the funnel in a ring-stand and drain the aqueous layer into a 100mL beaker and
repeat step four again draining the aqueous layer into same beaker.
6) Drain the remaining organic layer into a 50mL beaker and add a small amount of
anhydrous Na2SO4 (approx. 2g).
7) While the Organic layer dries, label and prepare 3 TLC chambers with about 5mL of the
following solvents(see image below)
a. Hexane/Acetone 90:10 (least polar mixture)
b. Hexane/Acetone 70:30
c. Hexane/Acetone 50:50 (most polar mixture)
Chromatography – Extraction and purification of Chlorophyll
CHM 220
8) Obtain 3 TLC plates (avoid touching the silica [dull
side] with your fingertips by holding the plates by the side).
Draw, in PENCIL, an origin line at the base of the plates
about 1cm from the bottom, your origin should be ABOVE
the level of your eluent (solvent) when the TLC is placed in
the chamber. Make two crosshatch marks on each plate
equidistant on the origin line. Each crosshatch represents
the starting point of your samples.
9) Using a small capillary tube, draw up some of your
Spinach Extract and carefully place a SMALL dot of your
solution on one of your crosshatches (with practice this can
be as small as 1-2mm). Reapply your solution to your plate
1-2 more times in the same location allowing the spot to
dry each time.
10) Repeat step 9 using the KNOWN CHLOROPHYLL
sample on the second crosshatch. Be sure to gently label
each crosshatch below your origin line.
11) Place one TLC in each of your chambers and allow
your TLC’s to develop undisturbed. Remove your plates when the solvent has traveled
up the plate within about 1cm from the top, and immediately make a line where the
solvent stopped. DO NOT allow the solvent to reach the top of the plate.
12) Circle each spot on the TLC plate and calculate the Rf for each. Identify which solvent
(eluent) system displayed the best separation AND which spot is Chlorophyll. (Our
compounds are easily visible; however, organic compounds often are not visible on a
TLC plate to the naked eye and additional equipment may be needed to make your
compounds visible, such as UV light, I2(g), or other chemical indicator.)
13) Obtain a prepared Chromatography Column from your instructor. Your column should
have a small glass wool plug at the bottom of a burette followed by a layer of sand, on
top of the sand is a column of silica gel with a small amount of sand on top of that.
There should be about 2-3 mL of solvent ABOVE the sand in your column.
14) Carefully place your column in your hood so that you can collect the eluent drained
from it in small test tubes when placed in a test-tube rack. Drain the column into a
beaker until the solvent just reaches the top of the FIRST sand layer. FROM THIS POINT
ON DO NOT LET THE ELUENT (SOLVENT) GO BELOW THE TOP LAYER OF SAND.
15) With a pipet add 1mL of your Spinach Extract to top sand layer (try to make sure it
doesn’t run down the sides of the column), and again drain your column to the top of
the FIRST layer of sand. Obtain 50mL of your selected eluent (solvent), add 1 mL of it to
your column, and again drain to the top of the sand.
Chromatography – Extraction and purification of Chlorophyll
CHM 220
16) Fill a test-tube rack with small test tubes to
collect your fractions (test-tubes). When collecting
fractions you will want to collect approximately 2-3 mL
of eluent per fraction.
17) Fill your column with as much of your eluent
(solvent) as you can and begin collecting fractions by
allowing the eluent to run into the test-tubes. When a
test-tube reaches 2-3 mL, quickly move to the next
fraction (test-tube). You will have to carefully add
more eluent (solvent) to your column until you have
used all 50mL of your chosen eluent (solvent). Again
DO NOT LET THE ELUENT (SOLVENT) GO BELOW THE
TOP LAYER OF SAND. It is very easy to watch your
fractions and not notice your solvent has run out. (You
should end with around 15-18 fractions.)
18) As your fractions are finishing, prepare a TLC
plate with 3 crosshatches. On the first crosshatch
place 1-2 spots of your Spinach Extract, on the others place 2-3 spots of your first two
fractions, respectively. Develop the plate in a TLC Chamber using your chosen eluent
(solvent). Prepare a similar plate for 3rd and 4th fractions and for any remaining
fractions. Using your developed TLCs to determine which fraction(s) contain only
purified Chlorophyll.