Pigments in Paprika
Objectives: To learn thin-layer chromatography (TLC) and column chromatography
techniques. To separate paprika into the 3 major classes of compounds (listed
below) by column chromatography. To use TLC to identify the 3 major bands to be
isolated by column chromatography and to check the purity of the fractions
after the column.
Overview of the experiment: In this experiment you will extract paprika with
diethyl ether. The major compounds that are extracted into the ether include 1)
fatty acid esters of capsanthin, 2) fatty acid esters of capsorbin, and 3)
β-carotene, but there are other “carotenoids” as well. These
compounds are shown below. Because of the extended conjugation (alternating
single and double bonds) present in these substances, they are colored. The
esters of capsanthin and capsorbin are red and β-carotene is yellow.
Since these compounds have different polarities they can be separated by
adsorption chromatography, and because they are colored it is easy to visually
follow the progress of the separation.
Procedure
One student in a pair should begin extracting paprika for TLC. The other
student should begin packing the chromatographycolumn at the beginning of the
lab period.
Extraction of the Pigments. To a dry 50 mL Erlenmeyer
flask add 15 mL of ether, 0.5 g of ground paprika, and 0.5-1.0 g of anhydrous
magnesium sulfate. Add a clean, dry, magnetic stir bar, stopper the flask, and
stir for 15 min. Gravity filter (Zubrick p. 106 and 117) the solution into a
clean, dry 50 mL round bottomed flask and evaporate to near-dryness with a
rotary evaporator. Do not apply much heat to the flask, for ether is very
volatile. To the concentrated extract, add 1 mL of 15% diethyl ether, 85%
heptane, stopper, and set aside in a dark place.
Thin-layer Chromatography. (Zubrick Ch. 28)The
adsorbent will be silica gel, the developing solvent is 15% diethyl ether, 85%
heptane, and the developing chamber will be a clean, dry, 250 mL beaker
containing a piece of filter paper and covered with aluminum foil (Zubrick Fig.
28.6, p. 227). Commercial silica gel sheets and micro capillary tubes for
spotting are available in the lab.
Using a micro capillary, spot the sheet with your paprika
extract, making sure that the spot will be slightly above the developing
solvent in the beaker. A 2 cm sheet is wide enough for two
chromatograms, so you could make a second spot using more (or less) extract
than before. Be sure to make a pencilmark to denote the starting position of
your spots. Carefully lower the TLC plate into the developing beaker and cover
the beaker with foil. When the solvent front climbs to within 1 cm of the top
of the sheet, remove the sheet and quickly mark the solvent front with a
pencil. After the sheet has dried, circle each spot with a pencil and make a
note of the color. (Spots will fade.) Sketch a picture of the TLC plate into
your lab notebook. Calculate and record the Rf value
for the three major spots and indicate which compound is present in each spot.
Column Chromatography. (Zubrick Ch. 29) We will
use a chromatography column that has an internal diameter of about 22mm. It
will be equipped with a short length of relatively inert tubing and a pinch
clamp to control the flow of eluate. The adsorbent will be silica gel, and the
solvent will be 15% diethyl ether, 85% heptane, changing to 50% ether, 50%
heptane as elution progresses. Please do not to take more organic solvents than
you actually need for the experiment!
If a TA has not already done so, begin by placing a small plug of cotton in the
narrow neck of the chromatography tube. This will keep the silica gel from
running out the bottom of the column. The plug is best inserted from the bottom
of the column, using a short length ofwire to position the plug just below the
wide portion of the tube. Be careful not to break the tube. If the plug
doesn’t easily slip into place, you are using too much cotton! Mount the
tube vertically using a ring stand and clamp, and close the bottom of the tube
with a pinch clamp. Fill the tube about one-third full with 15% ether, 85%
heptane, then add sand to form a uniform 5-10 mm layer at the bottom of the
tube. If some sand sticks to the walls of the tube, wash it down with a little
of the 15% ether solution.
Next, introduce the silica gel. In the hood, carefully pour 40-45 mL of silica
gel into a 50 or 100 mL clean, dry beaker. The exact amount is not important.
Be careful not to breathe the dust, or spill the fine powder all over the hood!
In another dry beaker place about 50 mL of the 15% ether, and make a slurry by
carefully (slowly) introducing about half of the silica gel with stirring. Pour
the silica gel into the solvent, not the other way around! Pour this suspension
into the chromatography tube; rap the tube with a rubber stopper to ensure that
the silica gel settles uniformly. Open the pinch clamp at the bottom of the
tube to drain off some of the solvent into a clean, dry, Erlenmeyer flask. Use
this solvent to make a slurry with the remaining
silica gel asbefore, and pour this suspension into the column with tapping as
before. The slurry will rapidly settle, so you will have to act fast. Once all
the silica gel is in the column, drain the solvent until it is only about 1-2
cm above the silica. Then add enough sand to make a 5-10 mm layer above the
silica gel. (See Zubrick Fig. 29.1) Open the pinch clamp and drain the solvent
into a clean flask such that no solvent remains above the sand.
Use a Pasteur pipet to transfer the concentrated paprika extract directly to the
sand. Try to avoid running the solution down the walls of the column. Carefully
open the pinch clamp to again lower the level of the liquid in the column such
that no liquid remains above the sand. Using a Pasteur pipet, carefully add a
little 15% ether to the column. If you accidentally ran some of the pigment
solution down the column wall, wash it on down to the sand with this first
portion of your eluting solvent. Open the pinch clamp to again bring the liquid
level down to the sand. Repeat this 2-3 more times. You should now have your
pigments below the sand, at the top of the silica gel. Carefully add 15% ether
solution to the column and commence the elution. The pinch clamp can probably
be removed at this point. The first eluate will be colorless, and it can berecycled through the column. As the elution progresses
you should notice the yellow β-carotene moving rather rapidly down the
column. The red components will lag behind. The elution order will be the same
as with the thin-layer run, so it will be most helpful to refer to that
chromatogram as you continue the experiment.
Collect the β-carotene fraction in a beaker or Erlenmeyer flask. Be sure
to label it ‘fraction 1’. At this point the less polar red
components should be moving slowly down the column, and other (unknown) colored
substances may be eluting. Feel free to discard any eluate after the carotene
and before the first red component. (Do not recycle solvent once components
begin eluting from the column.) You can hasten the progress of the separation
by switching the solvent to 30% diethyl ether, 70% heptane. Collect the red
component in a beaker or flask labeled ‘fraction 2’. If you
continue with 30% ether you will notice the elution of other minor, unknown,
components. Feel free to discard these. To hasten the elution of the second red
component, you should switch to 50% ether, 50% heptane. Collect it in another
beaker or flask labeled ‘fraction 3’. Concentrate each of the three
fractions to about one-third of its original volume with the rotary evaporator,
and placethese more concentrated, ether-free solutions back into their original
vials. Once you have collected the three main components, drain all remaining
solvent from the column, remove the column from the clamp, turn it upside down,
and shake the silica gel into an empty beaker. A waste bottle will be provided
for the solvent and also the silica gel.
Analysis of Separated Pigments
Chromatographic analysis. Spot a silica gel plate with fraction 1 on the
left, fraction 2 in the middle, and fraction 3 on the right. Lightly mark the
starting point with a pencil, and perform the thin-layer chromatography with
15% ether, 85% heptane as before. Compare the chromatograms with each other and
with the chromatogram of the original mixture. Was the separation successful?
Show this chromatogram to the TA before leaving the lab and before the spots
fade. Once you have finished your TLC analyses, you should place all organic
solutions in the appropriate waste bottle in the back of the lab.
Post-Lab Questions
1. Which red fraction is capsanthin and which is capsorbin? How can you
tell?
2. After the last red fraction, you probably noticed some coloration remaining
in the column. How does the polarity of this unknown substance compare to the
polarity of the three known compounds inthis experiment?
3. Why not use 100% diethyl ether as the eluant in this experiment?
4. What problem will ensue if the level of the developing liquid is higher than
the applied spot in a TLC analysis?
5. What will be the result of applying too much compound to a TLC plate?
6. What will be the appearance of a TLC plate if a solvent of too low polarity
is used for the development? too high polarity?
7. What would be the effect of collecting larger fractions eluting from the
silica column in this experiment?
8. Once the chromatographic column has been prepared, why is it important to
allow the level of the liquid in the column to drop to the level of the silica
gel before applying the compound to be separated?
9. Arrange the following in order of increasing Rf on
thin-layer chromatography: acetic acid, acetaldehyde, 2-octanone, decane, and
1-butanol.
Order of Solute Migration on Chromatography
Fastest
Alkanes
Alkyl halides
Alkenes
Dienes
Aromatic hydrocarbons
Aromatic halides
Ethers
Esters
Ketones
Aldehydes
Amines
Alcohols
Phenols
Carboxylic acids
Sulfonic acids
Slowest
Reference: Williamson, Macroscale and Microscale Organic Experiments, 4th ed.,
Houghton Mifflin, 2003.
