E5 & E6

Extraction of Chlorophyll from Fresh

Spinach and Investigation of the
Photochemistry of Chlorophyll
Chlorophyll a Chlorophyll b
C2507 Intensive General Chemistry Spring 2002 E5 and E6: Extraction and Photochemistry of Chlorophyll
E5 - Extraction of Chlorophyll from Fresh Spinach
E6 - Investigation of the Photochemistry of Chlorophyll
The aim of this experiment is to investigate the photochemistry of chlorophyll. This
experiment will be performed in two lab periods. In the first lab period you will extract
chlorophyll, the green pigment in leaves, from spinach. In the second lab period you will
investigate the interaction of light with chlorophyll. Read in Appendix B the reference section
titled "lectronic !tructure of "atter" copied from reference # before performing experiment
investigating the photochemistry of chlorophyll.
BACK!"#$%
$hlorophyll is the green pigment responsible for the color of leaves. Its presence in
leaves is crucial for photosynthesis. %hotosynthesis can be defined as the process by which
plants, algae, and photosynthetic bacteria use light energy to drive the synthesis of organic
compounds. The photosynthetic process involves the removal of $&' from the atmosphere,
which is used to synthesi(e carbohydrates, and results in the release of &'. The energy to drive
the chemical reactions of photosynthesis comes from the sunlight absorbed by the chlorophyll
molecules. )ence, the first step in photosynthesis is the absorption of visible light from the sun
by chlorophyll molecules. The chlorophyll molecules then transfer the light energy to
chloroplasts, the reaction center of photosynthesis. In this way light energy is converted to
chemical energy for converting $&' into carbohydrates.
The overall reaction for photosynthesis is*
6CO
2
+6
2
+
6

12

6
+ 6
2
This overall e+uation for photosynthesis is deceptively simple. In fact, a series of complex
reactions must occur in a coordinated manner for the synthesis of carbohydrates. To produce a
sucrose molecule, $,)#'&,, plants re+uire about -. distinct proteins that wor/ within a
complicated membrane structure.
0ithout light photosynthesis cannot ta/e place1 and hence the absorption of light by
chlorophyll is the first step in photosynthesis
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C2507 Intensive General Chemistry Spring 2002 E5 and E6: Extraction and Photochemistry of Chlorophyll
&hat happens 'hen light stri(es an atom or molec)le
*
+
!ince atoms and molecules obey +uantum mechanics, an atom or molecule can possess
discrete values of energy i.e. the amount of energy that an atom or molecule can possess is
+uanti(ed. The amount of energy an atom can absorb depends on the energy separation of the
electronic levels of the atom. If light of fre+uency, , is incident on the atom, the atom will
absorb this light only if the energy of a photon of this light 5
E
photon
=
6 e+uals the energy
difference between two electronic levels of the atom.
The energy absorbed excites an electron from the lower level to the upper level resulting
in an electronic excitation. !ince each atom has uni+ue energy separations between electronic
levels, if the fre+uency of the incident light is varied and the fre+uencies absorbed by the atom
are monitored, an absorption spectrum is recorded which is characteristic of the atom. lectronic
excitation also occurs in molecules, and li/e atoms since the electronic structure of each
molecule is different, electronic absorption spectra of molecules are also characteristic of each
molecule.
A molecule, in addition to its electronic levels, can also store energy in vibrations of the bonds in
the molecules, and rotations of the molecule. As with the electronic energy, vibrational and
rotational energies are also +uanti(ed1 i.e. molecules vibrate and rotate with distinct energies and
hence at distinct fre+uencies characteristic of a molecule. "onitoring the fre+uencies that excite
a vibration or rotation in a molecule 5resulting in a vibrational or rotational absorption spectrum
of the molecule6 identifies the molecule. The fre+uency of light that typically excites electrons
in atoms or molecules is located in the visible or ultraviolet region. 7ibrational excitation
re+uires light in the infra red region and rotational excitation re+uires light in the microwave
region. )ence,
E
electronic
>

>

In this experiment we will focus on excitation of electrons.

&hat happens to a molec)le once it has a,sor,e- energy
*
+
There are many pathways via which the excited molecule can loose the energy it has
absorbed from the incident light. The molecule can re4emit the light 8 this is called fluorescence.
&r the energy can be used to excite vibrations and rotations within the molecule. &r if the
molecule collides with another molecule it can transfer the energy to the collision partner 8 this
is called quenching. &r sometimes the molecule can dissociate 5i.e. brea/ a bond6 if the energy
23, 4 -
C2507 Intensive General Chemistry Spring 2002 E5 and E6: Extraction and Photochemistry of Chlorophyll
absorbed is greater than a bond energy in the molecule. 9ollowing excitation, the molecule can
follow any one of these pathways 5or sometimes a combination of these pathways6. The :fate;
of the excited molecule depends on factors li/e the relative time scales of each event, the phase
of the sample 5solid, li+uid and gas6, the pressure or concentration of the sample. An excited
molecule, which looses energy through mechanisms that do not involve emission of light, is said
to undergo a non4radiative process.
Photochemistry of chlorophyll
0hen sunlight stri/es a chlorophyll molecule, the chlorophyll molecule absorbs light.
0hat happens to the excited chlorophyll molecules< The chlorophyll molecules could fluoresce,
re4emitting the light. )owever, if all the chlorophyll molecules fluoresce, then the energy
absorbed by the chlorophyll is lost and cannot be used to drive photosynthesis. Instead, the
excited chlorophyll molecules transfer energy to chloroplasts to initiate the chemical reactions
involved in photosynthesis. )ence the chloroplasts quench the fluorescent emission of
chlorophyll, trapping the light energy so that it can be used to drive the chemical reactions of
photosynthesis.
In this experiment you will first extract chlorophyll from spinach via column
chromatography in the first lab period. In the second lab period you will investigate the
photochemistry of chlorophyll. =ou will 5i6 record an absorption spectrum of chlorophyll to
determine the range of wavelengths of light absorbed by the molecule1 5ii6 record a fluorescence
spectrum to determine the range of wavelengths emitted by chlorophyll 5iii6 determine the
relationship between fluorescence intensity and concentration and 5iv6 +uench the chlorophyll
fluorescence which simulates the transfer of energy from chlorophyll molecules to the
chloroplasts.
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C2507 Intensive General Chemistry Spring 2002 E5 and E6: Extraction and Photochemistry of Chlorophyll
E.PE!I/E$0A1 P!"CE%#!E 2a-apte- from !eference *3
*
st
1a, Perio-4 Extraction of Chlorophyll from Spinach
E5)ipment an- !eagents
' large Buchner funnels $ommercial blender !pinach leaves
> 4 '2. m? rlenmeyers %asteur glass pipettes $otton
# 4 2.. m? rlenmeyer 9ilter paper !terili(ed sand
#... m? !eparatory funnel @lass funnel Acetone
' 4 2.. m? filter flas/s Thermometer %etroleum ther
> 4 #2. m? bea/ers Rotary evaporator "ethylene $hloride
' 4 '2. m? 7olumetric flas/s Anhydrous !odium !ulfate Alumina
-. 4 2 or #. m? vials !odium Iodide )eptane
"ethanol - 4 '2. m? round bottom flas/s
Preparation of a spinach extract
#. &ne4half bag of supermar/et spinach leaves should be placed in a commercial blender with
about '.. m? of water and ground thoroughly for 24#. minutes, or until all of the leaves are
blended.
'. !plit the extract into two and filter each using a large Buchner funnel into a 2..4m? filter
flas/. Ase additional filter flas/s and Buchner funnels as necessary and change the filter
paper in the funnel when filtration slows due to clogging. =ou may need to add excess water
to remove all of the spinach remnants from the blender.
-. "eanwhile, prepare -B2 m? of petroleum etherCacetone solution in a ratio of D*'. %our about
-.. m? of this solution into a #... m? separatory funnel along with an e+ual amount of the
filtered a+ueous extract. $ap and sha/e the funnel vigorously, remembering to occasionally
vent the system by opening the stopcoc/. Add another 2. m? of the solution and sha/e
again. An emulsion may form. Erain off the a+ueous layer 5bottom green layer6 and then
decant the organic layer 5light green layer6 into a '2.4m? rlenmeyer. Ase about B2 g
anhydrous sodium sulfate to dry the organic layer and to brea/ up any emulsions. Allow to
23, 4 2
C2507 Intensive General Chemistry Spring 2002 E5 and E6: Extraction and Photochemistry of Chlorophyll
dry for at least forty4five minutes with regular swirling. =ou may need to add more sodium
sulfate if the sodium sulfate layer loo/s carameli(ed due to wetness.
>. 9ilter a small amount of the combined organic extracts into a '2.4m? round bottom flas/
using a funnel. Ase a rotary evaporator to remove the solvent, then filter more organic
extract into the round bottom flas/ and continue the evaporation process. Eo not let the
temperature of the bath exceed >2 $, although a temperature of '24-2 $ should be
sufficient to evaporate the petroleum etherCacetone 5hot plate set between ' and -6. The
result is a dar/ green li+uid 5#4- m?6, which may be diluted with a small amount of D*'
%etroleum etherCacetone solution and placed immediately on columns using a %asteur pipette
5&ne partner should finish the evaporation process while the other is setting up the column6.
Part II 6 Col)mn Preparation for Chromatographic Separation
The steps that follow are for extracting chlorophyll from each extract. )ence, for each
spinach extract follow the procedure below. =our group will be running two columns1 the
chlorophyll extracted from one column being used for the fluorescence vs. concentration
experiments and the chlorophyll from the second column being used for the chlorophyll
+uenching experiments.

#. $lamp a #>.2 x ..2 cm glass %asteur pipette in a thermometer clamp. Eo not screw the
clamp too tightly or the pipette will brea/. "a/e sure the pipette is vertical and firmly
clamped.
'. %ac/ the column. In a #2.4m? bea/er, put about '. m? of petroleum ether and place it under
the column. Eip a small swab of cotton into the petroleum ether and gently insert it into the
column using another pipette or a wooden splint. %ac/ about #.. cm of cotton into the
narrow section of the pipette to prevent solid substrate from flowing through the column. Eo
not pac/ the cotton too tightly.
-. Add sterili(ed sand to the column to form a ..24#.. cm layer above the cotton. Asing a
pipette and bulb, rinse the sides of the column with a little petroleum ether to wash down any
sand stic/ing to the sides of the column.
>. Add about -. m? of petroleum ether to a second #2.4m? bea/er. Then add approximately 2
g of alumina 5aluminum oxide6 and swirl to ma/e a slurry. To transfer the slurry to the
23, 4 ,
C2507 Intensive General Chemistry Spring 2002 E5 and E6: Extraction and Photochemistry of Chlorophyll
pipette column, use a pipette with its narrow end bro/en off. Brea/ the pipette carefully in a
towel. The column will run +uic/ly at first, so in order to prevent the alumina from drying
out, hold a finger over the bottom of the column during the addition of the slurry. %ac/ , cm
of alumina evenly. =ou may wish to mar/ off the , cm beforehand. Avoid any air bubbles in
the pac/ing of the alumina, and maintain the petroleum ether level above the level of the
alumina.
2. In order to protect the surface of the alumina, add another #.24' cm of sand. At this point, the
pac/ing should be Fust below the nec/ of the pipette.
,. Pre-'eigh two clean, dry, '2. m? round bottom flas/s. To weigh them, place the flas/s on
top of a bea/er, and then subtract the weight of the bea/er. =ou will rotary evaporate your
carotene and chlorophyll extracts in these flas/s.
Part III 6 Chromatographic Separation of Extract
#. ?et the column begin to drip. 0hen the petroleum ether level approaches the top layer of
sand, pipette your spinach extract onto the column. Add two pipettes of spinach extract onto
the column. 0hen the column has reached the top sand layer, continue to add petroleum
ether. Gever allow the solvent level to be lower than the sand layer. &therwise, the column
will begin to dry out, air poc/ets or crac/s will form in the alumina, and you will get poor
separation. The 4carotene will begin to move down the column as a yellowCorange band or
strea/ with some green material 5chlorophyll6 remaining at the top of the column.
'. After the second addition of petroleum ether, or when the orange band stops moving down
the column, begin eluting with methylene chloride. $ollect several fractions in a '2.4ml
flas/ as the 4carotene comes off. $ontinue eluting until all of the orange color has
disappeared from the column and the li+uid coming off. $ombine the carotene extracts from
both of your columns, and place the 4carotene extract on a rotary evaporator and remove the
solvent 5&ne lab partner may want to rotary evaporate the carotene while the other finishes
eluting the chlorophyll6. Temperatures between '24-2 $ should be sufficient to evaporate
the methylene chloride. =ou will be left with a yellow4orange powder.
-. 0hen no more orange is seen in the cotton at the bottom of the column and the eluted fluid is
colorless, begin eluting with acetone. =ou should see the green band begin to move down the
column. If the band stops moving, add one pipette of methanol. $ontinue adding solvent
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C2507 Intensive General Chemistry Spring 2002 E5 and E6: Extraction and Photochemistry of Chlorophyll
until all of the chlorophyll has come off the column 5The column will have a light green
coloring at the end6. $ombine the chlorophyll extracts from both columns and use the rotary
evaporator to concentrate the green extract. Eo not let the temperature of the bath exceed >2
$, although a temperature of about '2 $ should be sufficient to evaporate the acetone. =ou
will be left with a dar/ green slimy substance, chlorophyll.
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C2507 Intensive General Chemistry Spring 2002 E5 and E6: Extraction and Photochemistry of Chlorophyll
Photochemistry of chlorophyll 27
n-
la, perio-3
In this experiment, you will excite a solution of the chlorophyll using the ,-2 nm
radiation of a diode laser. The laser you will use is a low power, continuous source laser. This
experiment does not re+uire a laser to be used to excite the chlorophyll molecules in solution.
)owever, the fact the laser light is intense, monochromatic 5i.e. pure in color6, and collimated
allows the use of a low4cost, low power laser in a relatively simple experimental setup, with
sufficient sensitivity to ma/e the measurements described below.
The setup is shown above. The laser beam stri/es a cuvette filled with the chlorophyll
solution. The chlorophyll molecules absorbs the ,-2 nm light 5chec/ your absorption spectrum6.
The excited chlorophyll molecules then fluoresce. At H.
o
to the direction of the incident light is
first filtered by a colored glass that absorbs light at ,-2 nm and only allows the fluorescent light.
23, 4 H
beamstop
diodelaser(635nm)
cuvettein
sampleholder
photodiode
Top View
diodelaser(635nm)
cuvettein
sampleholder
beamstop
Front View
cuvettein
sampleholder
photodiode
voltmeter
Side View
C2507 Intensive General Chemistry Spring 2002 E5 and E6: Extraction and Photochemistry of Chlorophyll
9luorescence is typically at longer wavelengths than the wavelength of light used to excite
molecules 5compare the absorption and fluorescence spectra you will record in %arts II and III
below6. The filtered light then stri/es a light detector called a photodiode 5the photodiode is
similar to the photodiodes used to measure the speed of light in the Introduction to ?asers
experiment6. The output of the photodiode is a voltage proportional to the light intensity stri/ing
it. In this case the photodiode output voltage is proportional to the fluorescence intensity. The
photodiode voltage is recorded by a voltmeter. The blac/ box enclosing the cuvette and
photodiode is to prevent room light from being detected by the photodiode.
Part I 6 /a(ing Stoc( Sol)tions
Eetermine the mass of each residue using the method you used previously to weigh the flas/s.
)aving weighed the chlorophyll samples, determine the volume of heptane 5solvent6 re+uired to
prepare a stoc/ solution of #.- x #.
4-
". %repare the solution in a #.. or '2. m? volumetric
flas/. The two stoc/ solutions should be capped and refrigerated until needed 5!ome of the
chlorophyll may need to be eluted with a small amount of acetone if it stic/s to the side of the
round bottom flas/6.
Repeat the same procedure with the 4carotene extract, preparing a #.- x #.
4-
" solution of the
4carotene.
Part II 6 #8 8isi,le A,sorption Spectr)m
=ou will record an absorption spectrum of chlorophyll using the stoc/ solution. An absorption
spectrum reveals the range of wavelengths at which the molecule absorbs light.
#. Turn on A7 7isible !pectrophotometer 5?ambda #H6 and computer.
'. &n the main menu, clic/ on IA7 0in ?abJ icon.
-. 0hen the methods page appears, select Ichloro.mscJ method. The parameters for recording
the absorption spectrum of chlorophyll have been entered into this method. Gote the start
and end wavelengths that will be scanned.
>. @o to the I!ampleJ tab. $hange the Isample infoJ to your email address.
2. Return to the I!canJ selection. $lic/ on the IsetupJ /ey on the toolbar. 0ait for the start
button to turn green and clic/ on it. $lic/ on Io/J if the Imethods infoJ box appears.
23, 4 #.
C2507 Intensive General Chemistry Spring 2002 E5 and E6: Extraction and Photochemistry of Chlorophyll
,. The program will as/ you to insert the sample Iblan/J. Insert reference and sample cuvettes
filled with heptane, into their respective slots 5The reference cuvette goes in the bac/ slot and
the sample cuvette goes in the front one6. If your cuvette is clear on two sides and frosted on
the other two sides, ma/e sure the clear sides of the cuvette are facing the open sides of the
holder in the spectrophotometer. This means that the frosted side of both cuvettes should be
facing you. %ress Io/J.
B. After the bac/ground correction with the blan/ is complete, the program will as/ you to
insert the sample. Remove the sample cuvette and discard the solvent 5heptane6. 9ill the
cuvette to about -C> level with the stoc/ solution. Return the cuvette to the sample slot, and
clic/ on Io/.J =ou should see the absorption spectrum of chlorophyll appear as the scan
progresses.
D. After the sample has been read, clic/ on IprintJ under the IfileJ selection. "a/e sure that the
printer switch is set to IBJ first.
H. Eo not discard the stoc/ solution in the cuvette. =ou will use this cuvette filled with the
stoc/ solution to record a fluorescence spectrum.
#.. Record an absorption spectrum of the 4carotene stoc/ solution, repeating the procedure
described above
Part III 6 Fl)orescence or Emission Spectr)m
Gow you will record a fluorescence or emission spectrum of chlorophyll. A fluorescence
spectrum is a plot of the range of wavelengths emitted by a molecule when it is excited at a fixed
wavelength. 9or you to be able to record a fluorescence spectrum, clearly the excitation
wavelength must correspond to a wavelength at which the molecule absorbs. 9rom your
absorption spectrum of chlorophyll determine the pea/ absorption wavelength, i.e. the
wavelength at which the molecule absorbs the strongest.
#. %lace the cuvette filled with the stoc/ solution in the sample holder in the fluorescence
spectrometer.
'. ?aunch the program 9? 0in
-. !elect the method file $)?&R&%."T)
>. Based on your absorption spectrum enter value for the excitation wavelength
2. $hoose a start wavelength about '. nm below the excitation wavelength.
23, 4 ##
C2507 Intensive General Chemistry Spring 2002 E5 and E6: Extraction and Photochemistry of Chlorophyll
,. nd the scan at D.. nm
B. $lic/ the green light to start the scan.
D. A fluorescence spectrum of chlorophyll excited at the wavelength that you entered should
appear as the spectrometer scans over the wavelength range.
H. &n the printer next to the absorption spectrometer, set the printer selection /nob to A.
#.. %rint the fluorescence spectrum.
##. In the experiments in %art I7 and 7 the photodiode will be detecting a fluorescence signal
over the range of wavelengths indicated by the fluorescence spectrum.
Part I8 6 Fl)orescence vs9 Concentration
This experiment investigates the relationship between fluorescence intensity and concentration.
#. Asing one of the stoc/ solutions prepare six dilutions of chlorophyll 5"0 H..6 of
concentrations ranging from #.- x #.
4-
" to >.. x #.
42
". %repare at least -m? of each
solution. 9or each concentration prepare T)R solutions 5for replicate measurements6.
'. 9ill the cuvette with the most dilute sample place it in the sample holder. Bloc/ the laser
light right before the entrance to the cuvette and record the reading on the voltmeter. This is
the bac/ground reading 5with no fluorescence signal6. Anbloc/ the laser beam so that the
cuvet is now illuminated by the laser. Record the voltmeter reading.
-. Repeat this procedure for all solutions. Be sure not to move the sample holder when
changing solutions.
Part 8 6 Fl)orescence :)enching
This experiment investigates the effect of a +uencher on the fluorescence intensity. In this
experiment the +uencher is sodium iodide 5GaI6. The GaI is a source for iodide ions 5I
4
6 which
perform the same tas/ as the cholorplasts in the photosynthetic cycle. Gamely collisions
between excited chlorophyll molecules and I
4
molecules results in energy being transferred from
the excited chlorophyll to the I
4
. The result is that the chlorophyll is 8de4excited and the I
4
excited. In leaves, the chlorophyll transfers its energy to the cholorplasts. The cholorplasts use
this energy to initiate the chemical reactions of the photosynthesis.
#. "a/e a #. m? sample of ..2 " GaI in acetone. Be sure to /eep the solution capped since
acetone is volatile at room temperature.
23, 4 #'
C2507 Intensive General Chemistry Spring 2002 E5 and E6: Extraction and Photochemistry of Chlorophyll
'. %repare , dilutions of GaI 5"0 K #>H.DH gCmol6 from the stoc/ ..2 " GaI solution. Leep
these samples capped at all times.
-. Asing the second stoc/ chlorophyll solution prepare solutions of chlorophyll :spi/ed; with
GaI 5the GaI will act as a +uencher for the chlorophyll fluorescence, the same way that the
choloroplats +uench chlorophyll fluorescence6. 9or each concentration of the GaI solution,
ta/e # m? of the GaI solution and add ' m? of the chlorophyll stoc/ solution. Also for each
GaI concentration prepare three spi/ed solutions.
>. "easure the fluorescent intensity for each solution, following step ' of %art I7.
%ata Analysis for Parts I8 an- 8
#. 9or each of the fluorescence signal readings subtract the corresponding bac/ground reading.
-. 9or a given concentration average the three readings.
>. 9or the fluorescence vs. concentration experiment, plot the signal vs. concentration.
2. 9or the +uenching experiment, plot the signal vs. concentration.
Iss)es to thin( a,o)t for the ;%isc)ssion< section of the la, report
#6 ?oo/ at the absorption spectrum of chlorophyll and compare this with an emission spectrum
of the sun

5you can find an emission spectrum of the sun in astronomy boo/s, or on the internet6.
0hy is chlorophyll ideal for absorbing light energy from the sun< 0hich regions of the
electromagnetic spectrum does chlorophyll absorb the strongest<
'6 0hy is ,-2 nm appropriate for exciting chlorophyll<
-6 9rom the absorption spectra of chlorophyll and 4carotene can you explain why chlorophyll is
green and 4carotene orange<
>6 0hy do you measure fluorescence intensity at H.
o
to the incident 5excitation6 radiation<
26 0hat does the trend in the plot of fluorescence vs. concentration indicate<
,6 0hat is the effect of GaI on the fluorescence intensity of chlorophyll< Relate this to the
relationship between chlorophyll and chloroplasts.
B6 9or photosynthesis, would you expect the rate of fluorescence to be faster or slower than the
rate of +uenching by chloroplasts< xplain your choice of answer.
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C2507 Intensive General Chemistry Spring 2002 E5 and E6: Extraction and Photochemistry of Chlorophyll
There maybe other issues you would li/e to discuss in your lab report. The above +uestions are
Fust to help you focus your discussion of the results of the experiment.
!eferences4
#6 %rinciples of "odern $hemistry1 &xtoby, E.0. et al, !aunders $ollege %ublishing, >
th
dition
5#HHH6, p. ,.. 4 ,#>
'6 ?aser xperiments for Beginners1 Mare, Richard G. et al., #HH2, Aniversity !cience Boo/s,
!ausalito, $alifornia.
23, 4 #>