In this video beta-carotene is isolated from spinach by grinding the spinach in a mortar
and pestle and soaking in acetone. The acetone extract is added to a column of silica gel
and the beta-carotene is collected using a mobile phase containing 9-to-1 hexanes to
ethyl acetate. TLC is used to analyze the results.
0.5 grams of spinach was placed in a mortar and pestle along with 0.5 grams of anhydrous
magnesium sulfate. The magnesium sulfate should soak up most of the water that's present.
1 gram of sand was also added, which acts as an abrasive to break down the spinach.
The contents were ground to a paste and transferred to a test tube. 3 milliliters of acetone was
then added and mixed with the paste using a spatula. This was then left to sit for about
10 to 15 minutes. In the meantime, a column was set up by clamping a large glass pipet
to a ring stand. A small piece of cotton was placed in the bottom of the pipet using a
wood stick, which will prevent the silica gel from falling out. Using a piece of weigh
paper as a funnel, the column was then filled with the silica gel. About an inch and a half
was left at the top of column for solvent. A small amount of sand was placed on top of
the silica so that when the solvent is added the sand will buffer the impact and the even
layer of the silica will be less disturbed. After the 10 to 15 minutes of soaking in acetone,
the spinach paste settled to the bottom of the test tube and the acetone layer appeared
to be black, at least until it was transferred. A few drops of liquid was transferred to a
small vial to be later used as a crude extract reference. The remaining liquid was transferred
to a 50 mL beaker and a stream of air was used to evaporate most of the acetone to concentrate
the extract. The column was then flushed with hexanes to remove all the air pockets. A large
pipet bulb was used to push the solvent through the column faster. Hexane was continually
added in small increments so that the solvent line never got below the level of the sand.
If it did more air would get back on the column. At least 10 mL of hexane was used during this
equilibration process. A stream of air, which may or may not be available to you at this
time, was also used in lieu of the pipet bulb since it's a bit faster. Once the column was
equilibrated, about 1 mL of hexanes was added to the concentrated extract. The liquid was
then added to the column and partially pushed on the silica with a stream of air. The beaker
was rinsed one more time with an additional 1 mL of hexane and added to the column. An
effort was made to get all of the green/yellow extract on the column in the least amount
of solvent as possible, while always maintaining the level of solvent above the sand. When
the solvent at the top of column became clear, larger portions of hexanes were then added.
The silicon dioxide in column works the same way as the silicon dioxide on a TLC plate.
Polar compounds will stick more to column than nonpolar compounds. Beta-carotene is
a nonpolar hydrocarbon, so we would expect it to be one of the first compounds to elute
from the column. Also similar to TLC, if we change the mobile phase to something more
polar then all of the compounds will move faster. After about 5 mL or so of the hexane
was added to the column, a broad yellow band, which is the beta-carotene was noticeable.
To narrow the band and to get it to elute faster, the solvent was changed from pure
hexanes to a solution containing 10% ethyl acetate or 9-to-1 hexanes to ethyl acetate.
After a few milliliters of this mobile phase was added and as the yellow band reached the
bottom, a separate test tube was used to collect it. If we wanted to separate and collect the
other components we could continue to incrementally change the polarity of the mobile phase by
using 5-to-1 hexanes to ethyl acetate, 2-to-1 hexanes to ethyl acetate, and so on. These
are fractions collected from a prior column doing just that. For this lab though, we are
only concerned with the first yellow fraction. So here is beta-carotene standard, the yellow
fraction collected from the column, and the crude extract set aside earlier. Three dots
were lightly placed on a TLC plate with a pencil at the 1 cm mark. The beta-carotene
standard was spotted on the first dot. Once it briefly dried, it was spotted again to
make sure it was highly visible. The yellow fraction was spotted on the second dot. Since
it was not easily visible, it was spotted several more times until it was. An effort
was also made to keep the spot small. A better way to spot the yellow fraction may be to
first concentrate it by transferring it to a beaker and use a stream of air to evaporate
some of the solvent. In this case, the spot should be easier to see with only having to
spot it once and this should also keep the size of the spot small, which is preferred.
The crude extract was spotted on the third dot several times. While this spot is easy
to see, it contains several compounds, which will all show up better if it is concentrated
enough. However, if any spot is too concentrated, it will likely smear as it moves up the TLC
plate. After drying briefly, the TLC plate was added to a chamber containing 70% hexane
and 30% acetone. The TLC confirms or in the very least suggests the yellow fraction is
indeed beta-carotene, which is also a minor component in the crude extract, which contains
several other compounds.