CH-180
Chromatography
Introduction
Green plants get their color from a large number of pigments. Usually, chlorophyll is the
compound that imparts the green color to leaves, but other colors -- reds, yellows, oranges, etc., -are also present; we see those colors in autumn when the chlorophyll disappears from leaves. Some
of the colored compounds contribute to a healthy diet: In such cases, extracting the compounds for
use as a color additive in foods is highly desirable.
An especially beneficial natural color additive is -carotene, a compound found in many
vegetables, including carrots and spinach. -carotene is converted into vitamin A in the human
body. Vitamin A is a close relative of retinal, which causes a protein called opsin to send signals
from the eye's retina, along the optic nerve to the brain. The similarity of -carotene and retinal has
given rise to the notion that eating carrots promotes good vision.
The idea of today's experiment is to explore ways in which we can use knowledge of the
properties of substances (in this case, solubility, the ability to adhere to silica, and the ways
molecules interact with light) to accomplish a goal. Another way of stating this is to say that we are
going to apply scientific knowledge to solve a problem. Check out what a dictionary has to say
about technology; it applies here. Our specific goals in this lab are to 1) separate and 2) identify
chlorophyll and -carotene present in spinach leaves.
Safety Notes: petroleum ether and acetone are very flammable. Do not use them if anyone is
using an open flame in the lab! (Look around!) Silica is a powdery solid that can become trapped
in the lungs, where it can lead to a lung disease called silicosis. Be careful not to breathe the silica
dust!
Experimental Procedure:
WORK IN TEAMS OF TWO FOR THIS EXPERIMENT
1. Chop or cut some spinach leaves into small pieces. Place the leaves, along with 25 mL
petroleum ether/acetone solution (the solution is already prepared for you and placed in its own
bottle – check labels for mention of BOTH petroleum ether AND acetone) into a mortar and
grind the leaves with the pestle until the liquid assumes a dark green color.
2. Pour the liquid from the mortar into a beaker, leaving the solid spinach behind. The spinach can
be put in a receiving tray in the wall-mounted fume hood when you clean up.
3. Place 6 mL of the liquid into a 13  100 mm test tubes test tube and an equal volume of water in
a second 13  100 mm test tube. Put the two test tubes on opposite sides of the centrifuge, and turn
on the centrifuge for two minutes.
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4. Put 50 mL petroleum ether into a 100 mL beaker. Place 50 mL acetone into a second 100 mL
beaker.
5. Carefully clamp a large cork holding a disposable Pasteur pipet to
a ringstand, tip down. The figure at left illustrates the next steps.
6. Ball up a small portion of glass wool (about the size of a small
pea) and soak the wool in petroleum ether. Insert the wool into the
Pasteur pipet. Use a wire to poke the wool securely down into the
top of the pipet's tip. Pour enough sand into the pipet to form a ¼ cm
layer above the wool.
7. Add silica to the pipet until you have a column 5 cm (>2 inches)
high. Use a rubber hose-tipped funnel to guide the silica into the
pipet. Tap the pipet to ensure that the silica settles uniformly in the
pipet. Add another ¼-cm layer of sand to the top of the silica
column.
8. Use a second Pasteur pipet to deliver enough petroleum ether to
fill the silica column. Check the rate at which the petroleum ether drips out of the column. If your
drip rate is not as follows,
3 drops/sec > your rate > 1 drops/sec
you will need to discard your column in the wall-mounted fume hood and build a new column. Be
sure to put a 150 mL beaker under the column to collect all drips.
9. When the column has dripped down to the top sand layer, quickly add enough spinach solution to
fill the pipet to the very top.
10. When the column has dripped so that the spinach solution is all in the top sand layer, refill the
column with petroleum ether. Continue to add petroleum ether so that the liquid level is always
above the top sand layer.
11. Continually observe the column as the dripping proceeds. Do you see two colored layers?
Allow the yellow layer to drip through the column into a clean 30 mL beaker.
12. After the yellow layer has been collected, allow the liquid level in the column to drop to the top
sand layer. Then, fill the column to the very top with acetone, and continue to add acetone to the
column until the green layer has dripped out of the column and into a second clean 30 mL beaker.
13. Place 1 mL petroleum ether into a 10  75 mm test tube. Add 1 mL water and shake. Place 1
mL acetone into a second 10  75 mm test tube. Add 1 mL water and shake. Make observations
on the solubility of petroleum ether and acetone in water.
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Now we will use a spectrophotometer to measure the ways that both -carotene and chlorophyll
respond to light. The procedure used depends on which spectrophotometer is available.
I. Spectronic 20 Series:
S1. Find a cuvette and fill (to 2 cm of the top) with petroleum ether. This cuvette will be your
blank for the yellow sample.
S2. Fill a second cuvette (to 2 cm from the top) with the yellow portion collected from the column.
S3. Set the spectrophotometer's wavelength to 700 nm and calibrate the spectrophotometer, using
the procedure outlined by the instructor.
S4. Place the sample into the spectrophotometer's sample compartment and read the absorbance.
S5. Repeat the previous two steps, using each wavelength listed in the table on the next page in
place of 700 nm.
S6. Find a third cuvette and fill (to 2 cm of the top) with acetone. This cuvette will be your blank
for the green sample.
S7. Fill a fourth cuvette (to 2 cm from the top) with the green portion collected from the column.
S8. Perform Steps S3 - S5 until the table on the next page is complete for the green sample.
II. Beckman DU 7000 Series:
Unlike the Spectronic 20, the Beckman DU 7000 spectrophotometer can scan the entire wavelength
range (400 nm to 700 nm, in this case) at one time. A computer in the Beckman unit assists us
with data collection. The instructor will assist you in communicating with the Beckman’s
computer, and saving your spectrum to a floppy disk. Be sure to record in detail what you did
here: you may need to clearly state your procedure when you write your report.
D1. Place your -carotene sample in a clean 13  100 test tube. Into a second clean 13  100 test
tube, place your chlorophyll sample.
D2. Use the Beckman’s sipper to place one of your samples in the sample chamber.
D3. After entering your information into the Beckman’s computer, collect a spectrum.
D4. Reverse the flow on the sipper to return your sample.
D5. Repeat steps D1 - D4 for the second sample.
Use the form provided at the Website to complete your report. The form is available at (http://cstlcsm.semo.edu/rodgers/ch180f2f2007F/)on any Web browser. You will not submit a paper report
for this lab.
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Raw Data for Absorbance Spectra (Spectronic 20 Procedure Only)
Wavelength (nm)
700
690
680
670
660
650
640
630
620
610
600
590
580
570
560
550
540
530
520
510
500
490
480
470
460
450
440
430
420
410
400
yellow sample Absorption
green sample Absorption