CHAPTER
4
PHOTOCHEMISTRY
Quantum Yield of a Photochemical Reaction
Roy H. POTTIER and David A. RUSSELL
The Royal Military College of Canada
Canada
INTRODUCTION
Photochemistry - The Foundation of Photobiology
It can be said that the foundation of photobiology is photochemistry. Photochemical
reactions involve "bimolecular" interactions between a light quantum and a molecule, as well
as the subsequent chemical and physical changes which result from this interaction. Light
is always one of the initial reactants in a photochemical process. Photophysical processes are
transitions which interconnect excited states with each other or excited states with the ground
state, via radiative and/or radiationless processes. Photochemical processes, on the other
hand, are transitions from an electronically excited state that yield structures of different configurations than the reactant ground state.
Basic Laws of Photochemistry
There are two basic laws of photochemistry, and these laws apply to all photochemical systems, be they simple gaseous atoms or complex biological molecules found in living
systems. The first law of photochemistry, also called the Grotthus-Draper law, can be stated
as: only the light which is absorbed by a molecule can be effective in producing photochemical changes in the molecule. From the first law, it is obvious that there must be some overlap
between the wavelengths of light entering the reaction cell (or biological system) and those
absorbed by the system under investigation, if a photochemical reaction is to take place. For
quantitative work, this law must be rigorously applied to all aspects of a given photochemical
system. This implies that, in addition to knowing the absorbance spectrum of the molecule
being photolyzed, one should also know the spectral characteristics of the light source,
filter(s) (or band width of the light emerging from the excitation monochromator), reaction
cell windows and solvent, as well as the spectral characteristics of the photolysis products.
The second law of photochemistry, sometimes refelTed to as the Stark-Einstein law,
is related to the quantization oflight absorption by matter, and can be stated as: the absorption
of light by a molecule is a one quantum process, so that the sum of the primary process
quantum yields must be unity. The quantum yield (<1>B) of photoconversion of reactant A into
photoproduct B is defined as:
Roy H. POTnER AND DAVID A. RUSSELL, Departtnent of Chemistry and Chemical Engineering
The Royal Military College of Canada, Kingston, Ontario, K7K SLO, Canada
Photobiological Techniques. Edited by D.P. Y3.Jenzeno et al.
Plenum Press, New York, 1991
45
<l)B = _~M......"o,""le""c...u""le""s<..,;o""f,-,B
......fi","orm.......,.e"",d~pe""r,-,u....n"-!i....t .!. vo"'"l'-"u""m..,e.:. ,pio<'e""r.....u...n""it....t...im"""""e~
Quanta of light absorbed by A per unit volume per unit time
[1]
The denominator is related to the absorbance characteristics of the reactant. The numerator,
on the other hand, is related to the fraction of excited molecules that lead to the product B in
question, with respect to all other competing processes which lead to the destruction or
deactivation of the excited molecule. It is the sum of all the primary process quantum yields
that must be unity. As such, one must always specify the process of a quantum yield (e.g.
dissociation, isomerization, fluorescence, phosphorescence, radiationless transitions). The
Stark-Einstein law also holds true for absorbance between a metastable state and higher
electronically excited states (such as in triplet-triplet flash photolysis), and for biphotonic
processes (successive absorption of a molecule to two electronic states via two photons).
The fIrst step in any photochemical reaction is the absorption process, sometimes
referred to as "photon-annihilation" (Rodgers, 1988). Of special significance to photochemicaVphotobiological reactions is the long lived triplet state (10-5 s to several seconds).
Molecules in this energetic metastable state, at room temperature in liquid solutions, may
undergo on the order of 109 collisions during their lifetimes.
Energy Surface Pathways
A convenient way to view a photochemical reaction is via an energy surface pathway,
as illustrated in Fig. 1. This figure can be used to illustrate three main distinctions between
photochemical and thermal reactions. First, the energy of activation of a photochemical
reaction is provided by the absorption of light, as compared to the absorption of heat in a
thermal reaction. Second, a photochemically activated molecule may differ signifIcantly
Excited state
/ / ' energy surface
,
>
~
~
w
ifi
,~
' ... ",'"
~,
"
... ... ~~'" ",-,,,,
X* /-......
/
B* ~//
~
G rou nd state
energy surface
NUCLEAR GEOMETRY REACTION COORDINATE
Figure 1. Comparison of thermal and photochemical reactions for reactant (A) converted
into product (B). Star (*) indicates excited species. X and Yare metastable intermediate
species. Vertical lines pointing up indicate absorption processes, those pointing down
indicate emission. In a heat induced reaction the path is A M4 Y~B, where L1H is the enthalpy
of reaction. In a photochemical reaction, the path is A J!.l4A *~B, where hu is the absorbed
photon energy. Spin-orbit coupling induces the passage from the excited state surface to the
ground state surface, indicated by the dotted line where the two surfaces are in close
proximity.
46
from that of a thennally activated molecule. The excited molecule is in fact an electronic
isomer of the corresponding ground state molecule. Third, product fonnation is far more
thennodynamically accessible from a photoexcited state than from a ground state. In fact,
certain photoreactions can occur at temperatures near absolute zero.
Energetic Requirements of Photoreactions
In order for a photochemical reaction to proceed, its energetic requirements must be
met. Since it is the photon that contains the energy required to produce the excited state, an
examination of typical photon energies that are transferred in the absorption process would
be instructive. This energy is given by ~ = hu where ~ is the energy difference between
the excited and ground state, h is Planck's constant (6.63 x 10-34 J.s) and u is the frequency
of the photon in sol. Since c = A.·u, where c is the speed oflight in a vacuum (3.00x 108 m·s- l)
and A. is the wavelength of the photon:
[2]
.6.E=hc/A.
The amount of energy transferred through the absorption of one mole of photons (an Einstein)
of 600 nm light is thus:
.6.E = (6.63 x 10-34 x 3.00 x 108 x 6.02 x l(23)/(600 x 10-9) = 200 Hmol- l.
Table 1 shows the energy per Einstein available from photons of different wavelengths.
Table 1
Available energy per Einstein at various wavelengths
A. (nm)
AE (kJ'mol- l )
200 300 400 500 600 700 800 900 1000 1100 1200 1300
599 399 299 240 200 171 150 133
120
109 99_8
92_0
The energy required to break chemical bonds is well within the range of bond energies
found in many organic biomolecules. Some of the weakest bonds, such as 0-0 bonds, have
typical bond energies of -160 kJ 'mo}-l, while the stronger bonds, such as 0- H bonds, require
-440 kJ·mor l. Since 250 nm radiation corresponds to 479 kJ·mol- l , one might conclude that
such photons could be used to photoreact virtually all common organic compounds. A
reexamination of Fig. }, however, reveals that the excited state must also cross energy barriers
(activation energy) in order to proceed to product fonnation. Further, the rate of the reaction
must also be considered, as well as the rates of all other competing processes. The rate of a
reaction can be expressed as a function of the activation energy:
Rate of reaction
=Ae-Ea/RT =A x 1O-Ea/ (O.0192T)
[3]
47
where A (S-I) is a measure of the probability of reaction from a state which possesses the
minimum energy for reaction and Ea (kJ.mol- l ) is a measure of the activation energy required
for the reaction to proceed. For unimolecular reactions, A is typically 10 13 s-l, and for
bimolecular reactions, it is _107 S-I. E a values range from a few kJ·mol- 1 to -170 kJ·mol- l .
For long lived triplet states of the order of 1 s, a unimolecular rate constant at room
temperature (298 K), having an activation energy of 25 kJ'mol- l , can be expected to have a
value in the vicinity of 108 S-I. Such a rate is favorable for product formation, but if the
activation energy is of the order of 80 kJ ·mol- I , the rate drops to _10- 1 s- I, indicating virtu all y
no reaction. For bimolecular photoreactions, where A-10 8 S-I, an activation energy of 25
kJ·mol- 1would have a rate of only -1Q3 S-I. Since the excited state lifetimes are usually much
shorter than one second, photoreactions are only favorable if the activation energy is less than
-80 kJ·mol- 1 for triplet states and less than -20 Hmol- I for singlet states.
Some important photoreactions in photobiology are: 1) Linear addition to an
unsaturated molecule (e.g. cysteine + thymine), 2) Cycloaddition of an unsaturated molecule
(e.g., thymine dimer formation in DNA), 3) Photofragmentation (e.g., riboflavin ~
lumiflavin), 4) Photooxidation (e.g., photooxidation of cholesterol into 3 ~-hydroxy - 5ahydroperoxy - !:J.6 -cholestene), 5) Photohydration (e.g., UV hydration of uracil), 6) Cistrans isomerization (e.g., all-trans retinal~ II-cis retinal) and 7) Photorearrangement (e.g.,
7 -dehydrocholesterol~pre-vitamin D3). For further details on basic molecular photochemistry, in depth coverage is provided by Turro (1978). Photobiological applications are
discussed by Grossweiner and Smith (1989).
Quantum Yields
One of the most useful quantities that can be experimentally evaluated for any
photochemical process is the quantum yield of reaction, also called the quantum efficiency.
The quantum yield is the probability that the absorption of one photon at a given wavelength
will lead to a single observed event, such as the photolysis of one molecule; or the emission
of one photon. Although conceptually simple, as indicated in Eq. 1, quantum yield determinations can be quite difficult, especially if the fraction of incident light absorbed is measured
directly. Multireflections inside the photolysis cell can lead to more light being absorbed by
the system than that which is measured. These difficulties have been covered in detail by
Calvert and Pitts (1966). A more accurate method of quantum yield determination relies on
the use of a secondary actinometer solution with an accurately known quantum yield. This
method requires that the fractions of light absorbed by reactant and actinometer solution be
the same. Regardless of the true value of the fraction of light absorbed by the reactant or the
actinometer solution, it must be the same, since any reflection, absorption, or scattering of
the light by the cell windows is identical in the two cases, and the number of quanta absorbed
by each solution must be identical if the incident light intensity is kept constant in the two
experiments. If the reactant is irradiated for ~ seconds and forms nB molecules of product
B, and the chemical actinometer solution forms nAmolecules of its product A when irradiated
for tA seconds, the quantum yield of product B must be given by
[4]
In this experiment the quantum yield of 7-dehydrocholesterol (a steroid, also known
as ergosterol or provitamin D) photoconversion into previtamin D3 will be determined. This
reaction is an important reaction that takes place in mammalian skin, and is photoactivated
by sunlight. Previtarnin D3 is thermally converted to vitamin D3 by body heat. Vitamin D3
48
in turn binds to a plasma vitamin 0-binding protein for transport into the circulation. Vitamin
03 prevents rickets, which is characterized by defective ossifications and disturbance of
calcium uptake, resulting in weakness and deformation of the bones, especially in growing
children who do not get adequate sunshine. The photochemical mechanism of this reaction
has been extensively studied [Holick, et al., 1980]. The primary photoproduct (previtamin
03) undergoes two side reactions to produce the isomers lumisterol and tachysterol.
49
EXPERIMENT 5:
DETERMINATION OF THE QUANTUM YIELD
OF FORMATION OF PREVITAMIN D3 FROM 7DEHYDROCHOLESTEROL
OBJECTIVE
This laboratory exercise is designed to illustrate the use of actinometry in determining
the quantum yield of a photochemical reaction.
LIST OF MATERIALS
7-Dehydrocholesterol (7-DHC), 2 mg
Previtamin D3 (p-D 3), 1 mg
Potassium Ferrioxalate, 3 g
1,lO-phenanthroline, 0.1 g
Methanol (spectroscopic grade), 33 ml
Sulfuric acid, 0.5 M, 460 ml
Sodium acetate, 1 M, 600 ml
295 nm narrow bandpass filter or excitation monochromator set at 295 nm
10 cm focal length, convex quartz lens (2)
1 cm x 1 cm x 4.5 cm quartz cuvettes (2)
100 ml red volumetric flasks (3)
1000 ml red volumetric flask
1 and 5 ml graduated pipettes
2 ml pipette
Pasteur pipettes
Stopclock
UV light source, such as a stabilized 75 W high brilliance Xe arc lamp. (A 250, or 500
W Xe arc will also do.)
Double beam ratio recording spectrophotometer capable of multicomponent analysis.
Dark room (or facilities to significantly reduce room lighting)
PREPARATIONS
PREPARATIONS AND THE EXPERIMENT MUST BE CARRIED
OUT IN A DARK ROOM, USING A RED PHOTOGRAPHIC
SAFELIGHT, OR UNDER SUBDUED LIGHT.
Potassium Ferrioxalate Actinometer Stock Solution (0.006 M): Dissolve 2.95 g of solid
potassium ferrioxalate, ~Fe(Cp4)3·3"z0' in 800 ml of distilled water in a 1 liter red
volumetric flask. Add 100 ml of 0.5 M "zSO 4' dilute to 1 liter with distilled water and mix
well.
NOTE: The ~Fe(Cp4)3· 3"z0 crystals should be green in color. Light exposure will cause
the pure green crystals to decompose, with a color change to a yellowish brown. Pure
solid ~Fe(Cp4)3·3"z0 may be prepared by mixing 3 volumes of 1.5 M ~CP4
solution with 1 volume of 1.5 M FeCl3 solution (reagent grade chemicals). The
precipitated ~Fe(Cp4)3·3I\O is then recrystallized 3 times from warm water and
air dried at 45°C. The solid can be stored in the dark for extended periods of time
without change.
50
1,lO-Phenanthroline Solution: Dissolve 0.1 g 1, 100phenanthroline in 50 mI distilled water,
dilute to 100 mI. This yields a 0.1 % (mass/volume) phenanthroline solution.
Buffer Solution: Mix 600 mI of 1 M sodium acetate (C~COONa) with 360 mI of 0.5 M
~SO4' Dilute with distilled water to 1 liter.
7-Dehydrocholesterol Solution: Dissolve 7.4 x 10-4 g of 7-dehydrocholesterol in 5 mI
methanol, dilute to 10 mI with methanol in a red volumetric flask. In a 1 cm pathlength
absorbance cuvette, this solution should have an absorbance value of near unity.
Preparation of Mixed Standard Solutions: Dissolve 9.0 x 10-4 g of previtamin D3 in 5 mI
methanol, dilute with methanol to 10 mI in a red volumetric flask. Dissolve 9.0 x 10=4 g of 7dehydrocholesterol in 5 mI of methanol, dilute with methanol to 10 mI in a red volumetric
flask. The following amounts of these solutions are then taken to prepare the mixed standards
in a 5 ml volumetric (red) flask. These standard solutions are then diluted to the 5 mI mark
using methanol. From the exact masses ofp-D3 and 7-DHC, determine the respective concentrations in each mixed standard.
Volume ofpre-D3..!mll
Volume of7-DHC (mI)
Standard 1
1
3
Standard 2
2
2
Standard 3
3
1
Photolysis System: The photolysis system used to successfully test this experiment is
illustrated in Fig. 2.
Spectrophotometer: In this experiment, the analysis of product formation is made by
spectral analysis of the irradiated solution. As such, the spectrophotometer used must be
capable of multicomponent analysis. Low priced commercial instruments are now readily
available with this capability. This experiment was successfully tested using a Shimadzu
UV-160 spectrophotometer.
------------,
uv
EXCITATION
LAMP
MONOCHROMATOR
-~~
I
I
I
___+---~~J
LIGHT TIGHT BOX
Figure 2. Schematic of photolysis system. The UV lamp was a 75 W Xe, high brilliance
arc, stabilized by the use of an optical feedback system (PTI LOS200X). The excitation
monochromator was a 0.2 meter, PTI, set at 295 nm. The monochromator may be replaced
by a 295 nm narrow bandpass filter, in combination with a heat reflecting mirror. It is
important that the entire volume content of the cuvette be irradiated.
51
EXPERIMENTAL PROCEDURE
Actinometer photolysis
1.
Pipette 5 ml of the actinometer stock solution into a 100 ml red volumetric flask. Dilute
to 100 ml with distilled water. Pipette 3.5 ml (V I) of this diluted actinometer solution
into a'l x 1 x 4.5 cm quartz cuvette. Place this cuvette at the photolysis site, and start
the stopclock. Irradiate for 5 min (t pe+2).
2.
After photolysis, pipette 2 ml (V2) of the irradiated actinometer solution into a 5 ml (V3)
red volumetric flask. Add 0.4 ml of 0.1 % 1, IO-phenanthroline solution and 1 ml of
buffer solution. Dilute to the 5 ml mark with distilled water. Mix well and let stand in
the dark for 1 h. Label this solution as "irradiated".
3.
While the irradiated solution is standing in the dark, prepare a similar parallel solution
(2 ml of the diluted, unirradiated actinometer solution + 0.4 ml of the 0.1 % 1,10phenanthroline solution + 1 ml buffer solution, diluted to 5 ml with water) and allow
it to stand in the dark for 1 h. Label this solution as "non-irradiated".
Photolysis of 7.Dehydrocholesterol
4.
Pipette 3.5 ml of the 7-DRC solution into a 1 x Ix 4.5 cm quartz cuvette and irradiate
for 15 min (tp_D/
Calibration of Spectrophotometer for Multicomponent Analysis
5.
While the 7-DRC solution is being irradiated, the spectrophotometer should be
calibrated using the three mixed standard solutions. Record a base line between 200
and 500 nm, with both reference and analytical cuvettes filled with methanol.
6.
Record the absorbance spectra of the three mixed standards between 200 and 500 nm,
and enter them into the memory of the spectrophotometer, along with the concentrations of7 -DRC and pre-vitamin D3 in each standard solution. The exact order in which
these solutions and their concentration values are entered into the instrument will
depend on the type of spectrophotometer used.
Analysis ofIrradiated 7·DHC Solution
7. At the end of the 15 min irradiation of the 7-DRC solution, place the cuvette containing
the irradiated 7-DRC into the analytical compartment of the spectrophotometer and
record its spectrum from 200 to 500 nm. Enter this spectrum into the spectrophotometer
memory and determine the amount of previtamin D3 formed during irradiation.
Analysis of the Actinometer Solution
8.
52
The number of moles of reduced iron (npe+2) formed during the photolysis of the
ferrioxalate actinometer can easily be determined by spectrophotometry. At the end of
the 1 h standing period, place the two actinometer solutions (irradiated and nonirradiated) into the analytical and reference positions (respectively) of the spectrophotometer. Measure the absorbance (A) at 510 nm. Since A=ecl, where e is the molar
extinction coefficient of the Fe+2-phenanthroline complex (1.11 x 104 1·mol-l ·cm-I ), c
is the concentration in moRl, and I is the pathlength (1.00 cm), the number of moles
of the Fe+2 complex can easily be calculated from:
Quantum Yield of Formation of Previtamin D3
9.
The quantum yield of photoconversion of 7-dehydrocholesterol into pre-vitamin D3
may be calculated via:
<I>p-D3 = (<I>pe+2 np- D3 tpe+2) / (npe+2 tp-,,:!
,,)
The quantum yield of the actinometer, <I>Pe+2, is equal to 1.24 (Calvert and Pitts, 1966) at 295
nm.
DISCUSSION
In this experiment, you have determined the quantum yield of formation of previtamin
D3 from 7 -dehydrocholesterol, at a wavelength of 295 nm. The quantum yield of reaction of
previtamin D3 (<I>P_D3) has been reported to be 0.23 (MacLaughlin, et al., 1982). This could
have been done by accurately measuring the number of photons absorbed by the reactant,
using a photometric approach. If such a method is to be used, care must be taken to assure
thatthe measured fraction oflight transmitted by the reactant solution can be used to calculate
the actual light absorbed by the reactant molecules. For example, scattering of incident light
by turbid samples and reflection at the reaction cell surfaces can easily lead to errors in the
determination of the number of photons absorbed. For precise quantum yield determinations,
correction factors have been determined for these effects (Calvert and Pitts, 1966).
Although quantum yield determinations by the use of chemical actinometers are
generally easier and can produce very accurate results, a proper choice of actinometer must
be made. A list of currently known chemical actinometers has recently been published
(Bra slav sky and Kuhn, 1987) and should be consulted for their advantages and applicabilities. When chemical actinometers are used, it is important to assure that the same number
of photons are absorbed by both the actinometer and the sample under study. This is easily
done for the initial reaction time, by adjusting the concentrations of the actinometer and
sample such that they both have the same absorbance. However, once photolysis begins, the
rate of change in absorbance of both solutions may differ significantly_ Depending on the
photoproduct(s) formed from each solution, this could lead to a different number of photons
absorbed by the solutions. In order to minimize this possible difference, it is best to limit the
extent of both reactions to less than 10% of the total reaction.
Subsequent photoreactions or dark reactions from the photoproduct being studied can
also lead to errors in the quantum yield determined. In the example studied, it is well
established that previtarnin D3 forms three isomers (lumisterol, tachysterol and vitamin D3)
(Jacobs et ai, 1977). In such a case two approaches can be taken to assure correct quantum
yield determination. One approach is to separate the various products formed, say by HPLC,
then to calculate the quantum yield based on the specific product of interest (previtamin D3
in our case). The other approach is to determine the fraction of the product of interest by
spectral analysis. This can only be done accurately if the spectral details of all the products
formed are known, and that spectral separation can be easily achieved. HPLC analysis is
normally more time consuming, but is a more reliable approach.
53
Competitive or consecutive reactions can sometimes be eliminated, or partially reduced,
by a proper choice of experimental conditions. For example, the successive thermal reaction
of previtamin D3 into vitamin D3 can be essentially eliminated by carrying out the 7dehydrocholesterol photolysis at low temperature (Holick et al., 1980). A proper choice of
excitation wavelengths can also be used to favor the desired photoreaction and reduce the
extent of side or successive reactions. Thus although previtamin D3 (and subsequently
vitamin D3) is normally prepared by irradiating 7-dehydrocholesterol at 254 nm (Hg lamps),
the formation of the two isomers (lumisterol and tachysterol) is greatly reduced by using 295
nm radiation (MacLaughlin et al., 1982).
For a rigorous determination of the quantum yield of photoconversion of 7-dehydrocholesterol into previtamin D3, the amount of lumisterol and tachysterol should have been
determined. In this experiment, these two secondary reactions were neglected. The
experiment could be made more rigorous by the preparation of mixed standards containing
known fractions oflumisterol and tachysterol, as well as the 7-DHC and previtamin D3. Since
spectrophotometer multicomponent analysis normally requires n + 1 mixed standard solutions, where n is the number of components to be analyzed, a minimum of five mixed standard
solutions would be required. While testing this experiment, «I> _D3values on the order of 0.200.25 were obtained, well within the range of the published \.alue of MacLaughlin et al.,
(1982).
The accurate determination of quantum yields is an important step in most photochemical/photobiological studies. This experiment has been somewhat simplified in order to
illustrate the principle involved during a relatively short laboratory period. Accurately
determined quantum yields may require considerably more time.
REVIEW QUESTIONS
1.
7-dehydrocholesterol, dissolved in ethanol, has a molar extinction coefficient of 15 000
l·mol-1·cm-1at 280 nm. Chlorophyll a, dissolved in carbon tetrachloride, has a molar
extinction coefficient of 80 000 l·mol-1·cm-1 at 665 nm. Calculate the photon energy,
in kJ·mor 1, that is absorbed by these molecules, at the given wavelengths. Per photon
absorbed, which molecule gains more energy? Per incident photon, which molecule
has the higher capacity to absorb photons?
2.
Assume that you have money to invest, and you wish to support the development of new
drugs. You are approached by a photochemist who needs capital to set up an industry
to produce a new anti-tumor drug by a photochemical reaction. The raw material is
readily available at low cost and dissolves readily in water. You are shown an
absorbance spectrum of the raw material (reactant). The compound absorbs at 600 nm
with an extinction coefficient of 100 000 l·mol-1·cm-1. Sunlight is to be used to
photoexcite the reactant, in order to produce the new drug. In order to check out your
investment, you consult the chemical literature on reaction rate data. You find that the
proposed reaction is a unimolecular reaction with a preexponential factor (A) of 1013
S-l and an activation energy (Ea) of 170 kJ.mol- 1. Calculate the rate of the proposed
reaction at 25°C. Would it be wise to invest in such a venture?
3.
Explain why most C-H bonds (bond energy -420 kJ.mor1) in organic biomolecules are
not quickly broken when irradiated with 250 nm radiation.
4.
The concentrations of the diluted actinometer solution and that of the 7-dehydrocholesterol were chosen such that at 295 nm, both solutions had equal absorbance
values (1.0). If a mistake is made in the preparation of the 7-dehydrocholesterol
54
solution, and an absorbance value of 1.2 is obtained, how would this affect the validity
of the actinometry method of quantum yield determination? Would this lead to a lower
or higher (or equal) value of <l> D?
p- 3
5.
7-dehydrocholesterol has its first vibronic absorbance band at 290 nm, and has its
corresponding phosphorescence at 390 nm. Tetracycline absorbs at 400 nm and
phosphoresces at 510 nm. Could tetracycline photosensitize 7-dehydrocholesterol?
Could 7 -dehydrocholesterol photosensitize tetracycline? The drawing of an approximate energy level (singlets and triplets) diagram will aid you in answering this question.
Remember that wavelength is not directly proportional to energy (but frequency is).
ANSWERS TO REVIEW QUESTIONS
1.
7-DHC. Photon energy:
ilE = (he)/(A)
=(6.63 x 10-34 x 3.00 x 108 x 6.02 x 1023)/(280 x 10-9) = 428 kJ·mol-1
Chlorophyll a. Photon energy:
ilE = (6.63 x 10-34 x 3.00 x 108 x 6.02 x 1023 )/(665 x 10-9) = 180 kJ.mol- 1
7 DHC - gains more energy per photon absorbed, since it absorbs higher energy photons
(UV).
ChI a - has a higher capacity to absorb photons (higher molar extinction coefficient).
2.
Rate
= Ax
=
1O-Ea/(0.0912T)
1013 x 10-170/(0.0192 x 298)
= 2 x 10-17 s-I (no reaction, not wise to invest).
3.
250 nm radiation corresponds to 479 kJ·mor l (ilE =heA). This is greater than the bond
energy of the C-H bond. However, activation energy must also be provided. If this
excitation energy is greater than 59 kJ·mor l , reaction cannot proceed. Further,
deactivation processes will compete with photochemical process.
4.
The chemical actinometry method of quantum yield determination is based on both
solutions (actinometer and reactant under investigation) absorbing the same number of
photons. If this is not the case, one solution will absorb more photons than the other,
and a direct comparison cannot be made. If the 7-DHC concentration was higher than
that required for equal initial absorbance, it will absorb more photons than the
actinometer solution, and thus convert a greater amount of P-D3' This would produce
a higher value of np-D'3 yielding an artificially high value of <l>p-D'3
5.
Convert wavelengths to frequency or kJ·moJ-l as shown in the table below.
(nm)
ilE (kJ.mol- l )
Transition
290
413
SO~Sj
600
390
307
T j ~So
500
400
299
So ~Sj
400
510
235
ilE
s-1
SI--
300
T
-1
200
100
o
s
• -7-DUC
s.-Tetracycline
55
Photosensitization nonnally occurs via triplet-triplet energy transfer. Tetracycline
cannot photosensitize 7-DHC, since the triplet energy of tetracycline is lower than that
of? -DHC. 7 -DHCcould photosensitize tetracycline, by exciting 7-DHC at 290 nm (413
kJ.mol-1), having singlet-triplet intersystem crossing to the 7- DHC triplet (307 kJ.mol-1),
then energy transfer to the lower lying triplet state of tetracycline (235 kJ·mol- 1).
However, the 290 nm radiation would most probably be absorbed directly by the
tetracycline via higher excited singlet states, followed by internal conversion to its Sl
state and possibly intersystem crossing to its triplet state. In order to prevent this from
occurring, a long pass filterremoving all radiation below 290 nm would have to be used,
which would also prevent the 7-DHC from absorbing any radiation. The relative energy
levels of typical photosensitizer/acceptor molecules is shown below.
s
-1
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Filter
s
-1
T
-1
T
-1
s
o --
s
o --
Sensitizer
Acceptor
ACKNOWLEDGEMENTS
Special thanks are expressed to Dr. Michael F. Holick, Boston University School of
Medicine, for supplying the 7-dehydrocholesterol and the previtarnin D3 that were used in the
development of this experiment, as well as his helpful comments on the optimization of the
experimental design.
SUPPLEMENTARY READING
Grossweiner, L.L and K.c. Smith (1989) Photochemistry, In The Science of Photobiology,
2nd edition (Edited by K.C. Smith), pp. 47-78. Plenum Press, New York.
Simon, lP. (1971). Photochemistry and Spectroscopy. Wiley-Interscience, New York.
Covers the essentials of photochemistry and spectroscopy of organic compounds.
Schulman, S.G. (1977). Fluorescence and Phosphorescence Spectroscopy: Physicochemical Principles and Practice. Pergamon Press, Toronto. Contains many applications
on biological systems.
Jacobs, H.J.C. and E. Hevinga (1979) Photochemistry of vitamin D and its isomers and of
simple trienes. In Advances in Photochemistry, Vol. 11 (Edited by IN. Pitts, Jr., G.S.
Hammond, K. Gollnick and D. Grosjean), pp. 305-373.
56
REFERENCES
Braslavsky, S.E. and HJ Kuhn (1987) IUPAC Commission on photochemistry: Project chemical actinometers.
lAPS Newsletter, May, pp. 27-45.
Calvert, J.G. and J.N. Pitts Jr., (1966) Experimental methods in photochemistry, Photochemistry, Chapter 7,
pp.686-814. John Wiley & Sons, Inc., New York.
Grossweiner,L.I. and K.C. Smith (1989) Photochemistry, In The Science o/Photobiology, 2nd edition, (Edited
by K.C. Smith),pp. 47-78. Plenum Press, New York.
Holick, M.F., J.A. MacLaughlin, M.B. Clark, S.A. Holick,J. T. Potts Jr., RR Anderson, LH. Blank, J.A. Parrish
andP. Elias (1980) Photosynthesis of previtamin D3 in human skin and the Physiologic Consequences.
Science, 210, 203-205.
Jacobs, HJ.C., F. Boomsma, E. Havinga and A. van der Gen (1977) The photochemistry of previtamin D and
tachysterol. Rec. J. Roy. Netherlands Chern. Soc. 94, 113-118.
MacLaughlin, J.A., RR Anderson and M.F. Holick (1982) Spectral Character of Sunlight modulates
photosynthesis of previtamin D3 and its photoisomers in human skin. Science, 216,1001-1003.
Rodgers, M.AJ. (1988) Primary photochemical processes. In Photosensitisation: Molecular, Cellular and
Medical Aspects. (Edited by G. Moreno, R.H. Pottier and T.G. Truscott), pp. 11-18. Sprioger-Verlag,
Heidelberg.
Turro, N.J. (1978) Modern Molecular Photochemistry. The Benjamin/Cummings Publishing Co., Inc. Don
Mills, Ontario.
57
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