Esther Dujardin,
Pierre Laszlo, The Chlorophylls
and David Sacks'
Universite de Liege
au s a r t - T ~ I ~ ~ ~
4000 Liege 1, Belgium
There are a number of hiomolecules in which a metal ion
is enclosed in a cavity inside the molecular structure. Some
examples of these are enzymes such as carbonic anhydrase
(Zn2+),antibiotics such as valinomycin or monensin
A
CH,COOH
which effect the active transport of the alkali metal cations
(K+, Na+) through the cell membranes, or a respiratory
pigment such as hemoglobin (Fez+).This paper describes a
series of experiments appropriate for an undergraduate
lahoratory in which the chlorophyll pigments extracted
from green bean leaves have their Mg2+ metal ions re-
moved and reolaced bv various other metal ions such as
CU" and Zn". The experiments are designed more tu ex-
nose the student to hioloeiral and metallic chemistr\r rather
than to provide a thorough program of research, although
during the laboratory questions are posed that could lead
to further topics of research.
The Chlorophylls
The chlorophylls are a very important group of metallic
hiomolecules since, as the pigments responsible for the
. .;,
. polarizahility, and are hard to oxidize (3). Soft acids and
,
..
i . i i
. .
Figure 1. ElecVon micrograph of an unrathin longitudinal section in a spinach
chloroplast (enlargement = 60.000:,courlesy of Professor R. Bronchart. Uni-
versity of Li6ge).
742 / Journal of Chemical Education
An experiment in bio-inorganic chemistry
green color of plants, they "trap" solar energy for us. Lo-
cated in intracellular organelles called chloroplasts, the
chlorophylls are attached to lipoproteins which form what
are known as the chloroplast lamellae. These are mem-
branes which enclose flattened "sacs" called thylakoids;
some of them extend along the full length of the chloro-
plast, while other smaller ones cohere together to form the
grana, which can be seen under an ordinary microscope as
small dark-green grains. I t is in the lamellae of the chloro-
plast that the primary processes of photosynthesis take
place, converting light energy into chemical energy. The
chlorophylls, bound to proteins, play an essential part in
this process.
The basic structure of the chlorophylls is as follows
A tetrapyrrolic ring system surrounds a magnesium
atom. The pyrroles are linked together by methene
=CH-hridges. The tetrapyrrolic ring is characterized by a
system of conjugated double bonds. Various suhstituents
can attach themselves to the carbon atoms 1-8, as well as to
the methene hridging carbons a, & r ,or 6. The more ahun-
dant chlorophyll, chlorophyll a, was first synthesized by R.
B. Woodward in 1960 (1).
yH-nq FH,
CWH,
H
CH,
IphytylllOC-H,C.H
The chlorophylls have a saturated bond between atoms 7
and 8, a fifth ring (ring V), and a long carbon chain joined
by an ester link to the propionic acid group on carbon 7.
CH, H,C
I
phytol =CH,ICH-CH&H2CH,h-C
I- a W C H O H
Hard and Soft Acids and Bases
The exchange of the central Mg2+ ion in chlorophyll for
other metal ions is interesting because of its relevance to
Pearson's theory of Hard and Soft Acids and Bases
(HSAB) (2). Hard acids, according to this theory, are those
that are small in size with a high positive charge and do not
contain unshared pairs of electrons in their valence shells,
while hard hases similarly have high electronegativity, low
bases exhibit the opposite behavior: lower electronegativ-
ity, greater electron density, greater polarizahility. Al-
'Visiting student from Princeton University, Summer 1974.
though theoretical explanations have been offered (4), the
importance of the HSAB theory lies in its premise that
hard acids prefer to hind with hard bases, while soft acids
prefer soft bases. While innate acid or base strength is still
important, the HSAB theory adds the parameters of hard-
ness and softness in determining acid-base complex stabili-
ty.
This theory applies very nicely to the chlorophyll reac-
tions described in this paper. While nitrogen is a hard base
in a saturated molecule, the presence of extensive electron
delocalization, as found in the unsaturated system of the
chlorophylls, permits sufficient polarizahility that the ni-
trogen becomes softer, moving into the "borderline" cate-
gory. Magnesium(II), however, being a small, doubly
charged ion, is classified as a hard acid. Obviously this hard
acid-soft base complex is sufficiently stable to exist, but
chlorophylls with a softer central ion, such as Cu(I1) or
Zn(II), form stabler complexes than the magnesium(I1)
chlorophyll. I t is for this reason, that the central magne-
sium ion is easy to remove from the tetrapyrrolic ring hut
very difficult to replace, while the copper and zinc analogs
have central ions that are difficult to remove but extremely
easy to insert. I t is found that a 10%acetic acid solution or
a N HCI solution are sufficient to remove the Mg(I1)
and form a pheophytin
(suhstituenta omitted for clarity) but strong hydrochloric
acid is needed to remove the Zn(I1) ion. For the removal of
Cu(I1) ion the even stronger acidic solution required oftep
destroys the chlorophyll molecule.
In addition to the hardness or softness of the cation, it is
important also to consider the ionic radius (Table 1).
Mg(I1) has an ionic radius of 0.65 A. It is expected that
other ions must be of comparable size to fit into the cavity
and form a stable complex. The above statement may be an
over-simplification: the metal ion need not be in the cavity,
as was pointed out for magnesium ion by J. L. Hoard.
From Table 1, i t appears that Co2+, Co3+, Cu2+, Fez+,
Fe3+, Ni2+, Pt2+, or Zn2+ could possibly be inserted. The Y
values of hardness and softness listed in Table 2 indicate
those ions which would be expected to form stable com-
plexes.
The stability sequence of the structurally related metal-
loporphyrins is experimentally found to be: Pt(II)>Pd-
(II)>Ni(II)>Co(II)>Cu(II)>Fe(II)>Zn(II)>Mg(II) (5).
This agrees with our results for the modified chlorophylls.
The stability of the Pt(I1) and Pd(I1) complexes is surpris-
ing in view of the relatively large size of the central ions
compared to Mg(I1). Apparently, the softness of these ions
permits a greater stability in the complex that far out-
weighs any destabilizing size effect^.^
The Laboratory Program
Depending upon time available for this laboratory ses-
sion, the replacement of Mg(I1) by Zn(I1) and Cu(I1) can he
attempted both indirectly and directly. The indirect meth-
od involves: the extraction of the chlorophyll pigments in
an organic solvent, the removal of the Mg(I1) ion and con-
sequent formation of the pheophytin, and then the inser-
21f rigorous square planarity of the molecule is not required, it is
possible that large, soft metal cations such as Pt2+and Pd2+can
maintain strong soft acid-soft base bonds while raising themselves
out of the tetrapyrrolic plane, thus diminishing the importance of
ionic size as a factor in complex stability.
Table 1. Ionic Radii
ion A ion A
Table 2. The Y Values of Metal Ions'
Type Range of Y Metal ions
H a r d acid 12.8 Li+ 10.36). N a + (0.931. Kt (0.92).
I C I ~ P Ia1 ~ ~ ' + ( 0 . 8 7ca2+
). (1.62). ~ n ' +
13.03). A I ~ +(0.70). I n w (2.241,
Fe"(2.37). Co" (2.561. 5r2+
Borderline 2.8 - 3.2
(2.08). cr3+ (2.70)
Fez+ (3.091. cox+12.961. ~ i ' +
M i 6 0 n 0 . M.. et al.. 3. hers Nuel. Chem.. 29. 2685 119671.
tion of the new metal ion into the pheophytin to form the
modified chlorophyll. While the initial extraction is simply
a crushing of the green bean leaves in 80%acetone followed
by decantation and filtration of the green solution, it is also
possible in a cleaner experiment to perform a thin-layer
chromatography to separate the various chlorophylls and
then perform reactions with each extract, noting any differ-
ences in reaction behavior.
Both the initial mixture of chlorophylls and the pheo-
phytins formed by addition of acid (-pH 3) emit a red flu-
orescence, clearly seen under blue light (about 410-440
nm). I t should he noted that this low energy frequency of
light absorbed is due to the extensive conjugation in the
tetrapyrrolic ring, providing low energy transitions be-
tween energy levels.
I t is interesting that while the Fez+ chlorophyll is green,
the Fez+ hemoglobin molecule, which is also a tetrapyrrolic
structure, is red. This is presumably due to extra conjuga-
tion in the chlorophyll due to the V ring.
The pheophytins thus formed are then treated with con-
centrated salt solutions of Zn(I1) and Cu(I1) or other metal
ions to form the modified chlorophylls, many of which are
also fluorescent.
The direct replacement in the leaves of the Mg(1I) by
- . , or ZuiII)
Cu(1II . . is a demonstration of the reaction that oc-
curs when vegetables are cooked in a copper saucepan in
the nresence of viueear. The leaves are heated in test tubes
containing aqueous"acetic acid (10%) in the presence of
copper or zinc acetate. The reaction can be verified by com-
paring the color changes with those observed previously. If
there is time and if separation by thin-layer chromatogra-
phy has been ~erformed,the spectra of the modified and
original chlorophylls before and after chromatography can
be compared in a spectrophotometer (Fig. 2).
Experimental
Extraction of the Chlorophylls
Cut green hean leaves into fine strips and crush them in a mor-
tar containing washed sand and 80% acetone. Collect by decanting
Volume 52, Number 11, November 1975 / 743

320350 4W 450 500 550 600 650 700 lnm
Fiowe 2. Absorotkm Soechum of un~urif'edextract from bean leaves. Ill a*
etone exnact from leaves (2)me Fame extract aner a ~ ~ d d ~ a n(31
o na COP
per sullate solullon has been aaaea Tne Cu-chloraphylls have men exma-
ed *nth peholeum elner (40'-6O0C1
the green solution into a test tube. In this way a mixture (impure)
of chlorophyll o and chlorophyll b is obtained. Observe the color
and fluorescence, In whieh part or parts of the visible spectrum do
the ahsorhed wavelengths lie? Chlorophyll can he regarded as an
acid-base complex. The Mg(I1) ion is s Lewis acid, coordinated
with the four nitrogen atoms of the porphyrin nucleus. Is it a
square planar complex? Is the 18 electron rule fulfilled? Does it
have to he fulfilled? What is the normal coordination of magne-
sium?
Separation of the Pigments
Extract the pigments from the green bean leaves using ethyl
ether rather than aqueous acetone, as the solvent, and filter. The
concentrated ether extract is placed in long narrow streaks of 3 4
cm on cellulose tlc plates up to 1em from the edge. The plates are
then placed in developing tanks containing the following mixture
of solvents: petroleum ether (bp 60-80°C), acetone, n-propanol in
the ratio of 90:10:0.45 by volume. The plates are developed in the
dark at room temperature for ahout 30 min. The chromatography
is stopped when the solvent front is about 2-3 em from the top
edge of the plate. Immediately after removal from the tank, the
plates are observed under natural and ultraviolet light. Of the five
hands observed, only the green hands, the fourth and fifth from
the top of the plate, are ehloraphylls. The fourth band, chlorophyll
b, and the fifth hand, chlorophyll o, are separately scraped from
the glass plate and placed in centrifuge tubes. The addition of 5 ml
of ethvl ether forms a susoension which is eentrifueed. After een-
trifu~ation,the clear green supernatan1 liquids are collected and
their ahsorpt~onsmeasured hetwccn 400 and 3UO nrn and 600 and
700 nm in a spectrophorcrnetrr.
Removal of the Magnesium Ion
To 2 ml of the chlorophyll extract add 0.2 ml of 0.1 N HCI. Heat
in the water hath to accelerate the reaction. Note the change in
color and the fluorescence. This corresponds to absorption in
whieh part of the visihle spectrum?
Introduction of a Zinc or Copper Atom into Pheophytin
To the pheophytin solution, add a salt of zinc or copper, using 1
ml of concentrated solution of the salt. Heat for at least 1 min in a
water hath, until the solution turns green. Note the change of color
and fluorescence and again determine the frequencies of absorbed
light. Write the overall reaction that is taking place and determine
whieh metal salts should " eive the best vields of the modified chln-
rophylls. How could the importance of this variable be measured?
For which metal ions would the effect be expected to he most or
least important?
Direct Replacement of Mg by Cu or Zn
Heat leaf strips in diluted acetic aeid (10%).Separately, heat
other leaf strips in diluted acetic aeid but in the presence of a cop-
per salt. Repeat this operation with a zincsalt. Ohserve the color
changes in the leaf strips in order to verify the reaction.
Removing Cu(l1)From Cu Chlorophylland Zn(l1) From Zn
Chlorophyll
Take the test tubes that contain copper chlorophyll and zinc
chlorophyll, respectively. With different, more or less concentrated
solutions of hydrochloric aeid try to remove the copper and zinc.
Cheek that pheophytins are obtained.
Addnlonal Questions That May Be Asked from the Class
Natural chlorophyll is apparently one of t h e most unsta-
ble forms of chlorophyll. B u t what is t h e distribution of
metals in the earth's crust? Does t h e chlorophyll molecule
need t o be very stable in order t o function properly?
Can one, by using a solution of pheophytins, in t h e pres-
ence of the removed Mg(I1) ions, perceptibly lower t h e con-
centration of a n aqueous solution of a copper salt? Is t h e
reaction immediate? Can a auick titration he verformed
with a calorimeter? ( ~ h l o r o ~ h ~ pheophytins,
lls, a n d metal
c h l o r o ~ h v l l sare soluble in oetroleum ether which is n o t
miscibie k i t h water).
T h e replacement of Mg(I1) by other ions is a n equilibri-
u m reaction. How could t h e given list of modified chloro-
phyll stabilities he verified?
T h e maximum light intensity from t h e s u n lies in t h e
visible range. Would t h e modified chlorophylls he as effec-
tive in light absorption a s natural chlorophyll?
Literature Cited
(11 Woodward, R. B., Ayor. W. A., Beaton. J. M. Bickelhaupt, F., Bonnett. R., Buch-
8chacher.P.. Class, G.L.,Dutler.H.. Hannah, J., Hauek,F. P.. It&S.,Langemsnn.
A,, Le coif,E., Leimgruber, W.. L w o ~ k i W.,
. sauer. J., Vale"*, 2.. and Vok. H.,
rl Amen Chem Soc, 82,38W 119601.
(21 Pearson, 8. G.. "Hard and Soft Acids and Bawr: John Wiley & Sans, Stroudsbuq,
1973.
(3) Pearson R. G., J. CHEM. EOUC.. 45,584 (19681.
(11 Pcsrnon R. G., J. CHEM. EDUC.,45.643 (19681.
(5) Phillips J. N..Comp Blochem., 9-70 11963).