APC LAB
Chromatography of Natural Pigments
Teacher Prep Sheet
Each group will need
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Spinach extract
mortar and pestle
(4) test tubes that will fit into centrifuge
(2) 100 mL beakers
(1) 250 mL beaker for waste
disposable pipet
ring stand
clamp to hold disposable pipet
large paper clip
small funnel and tube setup used to pour silica into disposable pipet
Place on Each Riser:
1. 225 mL of 80:20 mixture of petroleum ether : acetone (each group will need 50mL)
2. 225 mL of petroleum ether (each group will need 50 mL)
3. 225 mL of acetone (each group will need 50 mL)
4. small piece of glass wool
5. small container of sand
6. small container of silica
Have extra on hand:
80:20 petroleum ether : acetone
petroleum ether
acetone
silica
sand
Spectrophotometer or laptop with probe
Cuvettes
Kim-Wipes
Chromatography Separation Column Supplement
-Carotene
Acetone
Petroleum ether = a mixture of hydrocarbons (it's not really even an ether!)

Beta carotene is polar / nonpolar

Chlorophyll is
polar / nonpolar

Acetone is polar / nonpolar

Petroleum ether is
polar / nonpolar
Remember:
"like" dissolves "like
Polar solvents dissolve polar
substances
Nonpolar solvents dissolve
nonpolar substances
How Can Spectrophotometry Tell
Me About the Purity of My Sample?
Remember how a prism will bend
incoming "white" light and
separate it out into it's component
colors?
A spectrophotometer works in a
similar way.
1. Light is focused on a sample
2. The sample absorbs certain
frequencies of the light.
3. The rest of the light is
transmitted and detected by a
sensor.
Incident
light
Cuvette
with
your
sample
The
sample
absorbs
some
light
Transmitted
light
Seeing Colors
"White" light is a mixture of all visible wavelengths (380 nm -- 800 nm). When we
"see" a color, we are actually seeing something called a color complement. We are
seeing white light minus the wavelength absorbed by the substance we are looking
at. The table below lists the wavelengths for each color and it's corresponding color
complement.
Wavelength (nm)
(of absorbed light)
380 – 435
435 - 480
480 - 490
490 - 500
500 - 560
560 - 580
580 - 595
595 - 650
650 - 800
Color
(what is absorbed)
Violet
Blue
Green/blue
Blue/green
Green
Yellow/green
Yellow
Orange
Red
Color Complement
(what we see)
Yellow/green
Yellow
Orange
Red
Purple
Violet
Blue
Green/blue
Blue/green
Example:
Stick dude with one big hand is wearing a
red shirt. That means we are seeing the
color complement red.
Look at the table above...for us to see red,
stick dude's shirt must be absorbing
blue/green waves (wavelength of 490-500
nm)
So, white light minus blue/green waves
appears red to us.
So how can we use spectrophotometry to test the purity of our betacarotene and chlorophyll samples?
It's easy!!! Beta-carotene appears yellow, so we'll test a sample in
the spectrophotometer. When we measure the absorbance of beta
carotene, we would expect it to absorb blue waves (approximately 450
nm). So we'll measure the absorbance of the sample at different
wavelengths (we'll start around 400 nm and measure every 10 nm all
the way up to 700 nm). We hope to see a single peak (a lot of
absorbance) around 450 nm proving that our beta carotene sample is
pure.
The same thing can be done for chlorophyll. It appears green, so we
would expect to see major absorbance around 400 nm and again
around 650 nm)
The absorbance readings the spectrophotometer gives us are based on a
formula known as Beer's Law.
A = abc
Where
A = absorbance
a = absorptivity constant ()
b = path length (width of cuvette)
c = concentration of sample
Chromatography of Natural Pigments
From Wiley: Experience the Extraordinary Chemistry of Ordinary Things
Objectives
This experiment will introduce you to column chromatography and the separation of a
few of the natural pigments contained in spinach. You will also identify the purity of the
separated pigments by spectrophotometric analysis.
Background
The dark green color of spinach is actually a combination of many naturally colored
substances. This includes -carotene, which is one of many ingredients in spinach
beneficial to humans. This chemical is considered a vitamin A precursor because it is
converted into vitamin A in our bodies. -carotene is so successful as a natural color
additive that the American food industry has adopted it as a dye for producing various
shades of red and yellow in foods. This in turn is beneficial to consumers because it
eliminates the need for a truly artificial color. The organic molecular “stick structure” of
-carotene is shown below. The alternating double bonds in the long chain that connects
the two rings help to give its color. See Figure 6.1.
When isolated from a solution of petroleum ether, -carotene’s crystals are red; however,
when still dissolved in dilute solutions of this solvent, it exhibits a yellow color. Beta
carotene is one of the natural pigments that you will isolate from spinach in this
experiment. You will examine the color of this substance (while it is dissolved in a
solvent) using your eye and an instrument called a spectrophotometer.
Figure 6.1 -carotene molecular structure
Other colored components of spinach include a group of molecules called chlorophylls,
and the second component that will be separated in this experiment is a mixture of these
compounds. Chlorophylls are organic molecules that coordinate (hold on to) magnesium
in the center of a relatively complex chemical ring system. Besides the important part
chlorophylls play in the photosynthetic cycle of green plants, they are also extracted from
plants and used to dye leather and as deodorants; however, their deodorant ability
apparently has little effect on spinach.
Procedure
A. Chromatographic separation of -carotene and chlorophylls
CAUTION: Petroleum ether and acetone are very flammable liquids. Use caution.
Silica should not be breathed.
1. Place your room temperature (freshly thawed is best) spinach in about 50 mL of a
80:20 mixture of petroleum ether/acetone in a mortar. Grind with the pestle until
the liquid is dark green. Decant the solvent (separate the liquid from the solid by
slowly pouring it into a beaker) and place 5 mL of this extract in a test tube and
centrifuge for 2 minutes. (Check with your lab instructor as to the size test tube
that fits your centrifuge.) Measure the 5 mL using a small graduated cylinder. If
you have not been instructed in the use of the centrifuge then ask the lab
instructor to demonstrate. Remember to use a counter weight tube for proper
balance in the centrifuge. Set the centrifuged liquid aside for Step 6. Put 50 mL
of petroleum ether and 50 mL of acetone from the stock containers into two
separate 100 mL beakers. You can use the markings on the beakers to estimate
the volumes.
2. Secure a disposable Pasteur pipet in a buret clamp attached to a ring stand, taking
care not to clamp and break the fragile tip of the capillary. Use a rubber band if
necessary.
3. Soak a swab of glass
wool (balled up to be
about the diameter of a
dime) in petroleum
ether and then, with a
piece of wire, push the
wool through the top of
pipet down to the
beginning of the tip of
the Pasteur pipet, just
where the body of the
pipet starts to narrow.
See Figure 6.2. Put
enough sand in the pipet
secured to the ring stand
to make a layer about
0.25 cm over the glass
wool.
4. Add silica to this
chromatographic column
with a spatula until you have
Figure 6.2 Chromatographic column
a layer about 5 cm high. You may consider using a small funnel. Take
precaution when handling silica. Do not breathe this solid! Above the 5 cm (2
inches) column of silica, place another 0.25 cm (1/8 inch) layer of sand, again
using the funnel set up.
Fill the column with petroleum ether using a Pasteur pipet. At this point the
column should drip at about 1 drop per second. If the dripping rate is
unsatisfactory (3 drops per second is probably too fast and 1 drop every 5 seconds
is too slow), stop, empty the solid material into an appropriate waste beaker and
construct another column starting at step 2.
5. Allow the chromatographic column to drip petroleum ether until the solvent level
drops down to the top of the upper sand layer but no farther. Immediately go to
step 6. Collect all of the waste in this experiment in a 250 or 150 mL beaker and ,
at the end of the lab, empty it into a suitable waste crock or class waste container.
6. Quickly add enough spinach extract to fill the column to the top of the pipet.
7. Allow the column to drip until the spinach extract level falls to the top of the
upper layer of sand. At this point fill the column again with petroleum ether (not
more spinach extract) and keep the level of this solvent above the upper sand level
throughout the steps described below. Watch carefully so that the solvent never
drops below the topmost sand layer.
8. Observe the chromatographic column as your chromatogram develops. Two
colored bands will separate from the original green spinach mixture. As the
lower, yellow band moves downward and then exits the tip of the column, collect
this fraction in a 100 mm (4 inch) test tube. (You can use a small beaker if you
wish. Clearly label whatever you use with wax pencil). This yellow band is carotene. When the petroleum ether level drops down to the upper sand layer and
after the yellow fraction has been collected, fill the column to the top with
acetone instead of petroleum ether as before. Continue replacing the solvent at
the top of the column with acetone until the green band has eluted and been
collected in another similarly labeled test tube or small beaker. The green band is
the chlorophyll fraction.
B. Spectrophotometric determination of -carotene and chlorophyll
1. Use a USB cable to connect the Vernier Spectrophotometer to the computer.
2. Start Logger Pro 3.4.5 or later on your computer.
3. Allow spectrophotometer to warm up for 3 minutes after plugging in to USB port.
4. We will first measure the absorbance spectrum of -carotene.
a. In order to have enough volume of liquid to measure, we will need to pool
together each group’s sample of -carotene which they collected from
their separation column.
b. Place all the group’s sample into one test tube and then put about 4 mL of
this in one of the test tube-like cuvettes that come with the
spectrophotometer. Always wipe your newly filled cuvettes with a KimWipe.
c. Put about 4 mL of petroleum ether in another cuvette. This is the blank.
d. Place the cuvette containing the blank in the observation cell of the
spectrophotometer. Make sure that the smooth surface on the cuvette
always faces the light source when you put the cuvette in the cell. Click
“Calibrate”, “Spectrometer”, “Finish Calibration”.
e. Take the blank out of the spectrophotometer and replace it with the cuvette
containing the class’ sample of -carotene. Click “Collect”. Print the
absorbance spectrum after you have added a title.
5. Next, measure the absorbance spectrum of chlorophyll.
a. Pool together the class’ chlorophyll samples into a test tube, then put
about 4 mL of this sample into a clean cuvette.
b. Create a blank cuvette by adding 4 mL of acetone to a clean cuvette.
c. Repeat the process from step 4 by first calibrating the spectrometer with
the blank in, then replace it and measure the absorbance spectrum of
chlorophyll.
d. Add a title to the graph and print a copy of the chlorophyll absorbance
spectrum.