David M. Goodall,' Paul W. Harrison, and John H. M. Wedderburn The University of York
Heslington, York, England
Flash Photolysis Experiments for Teaching Kinetics and Photochemistry
T h e exciting developments in reaction kinetics for which Professors Eigen, Norrish, and Portcr shared the 1967 Nobel Prize for Chemistry are now introduced in most kinetics and photochemistry textbooks, and havc been lucidly surveyed in recent reviews ( 1 3 ) . We feel that experiments illustrating flash photolysis and relaxation kinetics are worthy of a place in the pllysical chemistry practical syllabus of every college. In this article we describe a series of cxpcriments in flash kinetic spectrophotometry which complement the study of iodine at,om recom(4, 5). bination prcviously described in THIS JOURNAL A subsequent paper (6) deals with relaxation kinetics. Examples chosen illustrate bchavior in rigid and fluid media, reversible and irreversible systems, and utilize molecules of interest to both chemists and biochemists. Study is restricted to the millisecond to second timc range so that an inexpensive, safe apparatus built around a standard photographic flash gun may be used. Altcrnatively, a commercial apparatus now available specifically for teaching purposes may be adopted.= The experiments train the student in fast reaction methodology-thc use of an oscilloscope, kinetic analyses, sample handling by the freeze-pump-thaw tcchniqne, ctc.-and also teach the physical principles of photochemistry. Flexibility has been born in mind always; chemistry and biochemistry students sharing the same laboratory can get optimal use out of the apparatus. Some suggestions for further study are included: these may prove helpful for teachers planning project work for the more able students. Review of Experiments
of a solution of riboflavin in a polymer matrix produces the triplet state, and the decay to the ground state is monitored by triplet-triplet absorption or E-type delayed fluoresccnce. Photopinacolisation of Benzophenone. The mechanism of this well-known irreversible photochemical reaction is investigated in aqueous isopropanol, by following the second order decay of the intcrmediate radicals over thc pH range 4-13. Freeze-pump-tha~~ cycles are necesssry to deoxygcnate the solutions used. Photodissociation of Carboxymyoglobin. Rlyoglohin is the protein responsible for oxygcn storagc and transport in mammalian muscle. Carbon monoxide can bind to the heme grouping in placc of oxygen, and thc Fe(I1)-CO link is split by photolysis. The rate of recombination of myoglobin and carbon monoxidc is studied as a function of the pressure of carbon monoxide above the solution. Instrumentation
A very cffectivr iucxpcnsivr flash kinetic sprctrophotometer has recently been described by Porter and Wcst (7). This avoids the high voltagc powrr supplies and rather elaborate procedures for fabricating flash lamps which characterizcd prcvions designs (4, 5). Wc havr found the use of any standard photographic flash gun to be quitr satisfactory: all havc half peak widths 1-2 ms, and a model with 60J output or more is dpsirablc (e.g., Mctz Mccablitz 184). The plastic covcr above thc flash tube must be removed, as this absorbs all light below 360 nm. A simple trigger circuit using a phototransistor is used to initiate the oscilloscope trace when the flashgun is fired. For
Triplet Decay of Catacmdensed Aromatic Hydrocarbons in Polymethylmethacrylate. The student is introduced to the apparatus with an experiment on the decay rates of a series of aromatic hydrocarbon triplet states in polymethylmethacrylate matrices. Excellent first-order decay curves are immediately obtainable with the polymer samples, ~vhichare stable for many months. Both phosphorescence and triplet-triplet absorption are used to monitor triplet decay, and the decay rates are correlated with triplet en~rgiesin accordancc with the theories of radiationless electronic transitions. The Triplet State of RiboJEavin. Flash photolysis
' Author to whom correspondence regarding this article should be addressed. %AppliedPhotophysics Ltd. (20 Alhemarle Street, London W l X 3HA, England) produce both a flesh photolysis apparatus and sets of polyrner solutions.
pn*o1yb
cim*
~mn-istcc
cimn
Wl3d
Figure 1.
Apparatus for millisecond flmh photolyris studies.
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the monitoring beam we use a 50-W 12-V quartz iodine projector lamp running off a stabilized supply (A. C. Farnell, 500 pV p-p ripple), and a standard optical train (4, 7) terminating in a variable interference filter (Schott, model Veril S 60) and a phototube (Mullard QOAV). The signal to noise ratio over most of the wavelength range is better than 5 X 103:l. Figure 1 summarizes the apparatus used. Our outgassing apparatus resembles that of Yamanashi and Nowak (d), with the omission of the filter jacket around the 6 cm long photolysis cell-a gelatin filter or rectangular filter cell can readily be interposed between the flash gun and the photolysis cell. An oscilloscope with a 10 mV cm-I Y scale is required for these experiments, and one having 1 mV cm-I sensitivity allows the capability of the apparatus to be fully realized. 1,2,5 switching is necessary on both horizontal and vcrtical scales. The oscilloscope is by far the most expensive item in this experimental arrangement, and should be chosen carefully. Storage oscilloscopes are becoming cheaper every year: the extra cost for storage should be weighed against the convenience and saving in film entailed. Decays can be recorded by tracing from thc oscilloscope screen onto plastic film (5); this procedure can also be used with a cheap oscilloscope having a P7 phosphor, if it is convenient to house the apparatus in a darkened room. Experiments Triplet Decay of Catacondensed Aromatic Hydrocarbons in Polymethylmethacrylote
The photochemistry of aromatic molecules is discussed in all the standard photochemistry textbooks (8-11), and has recently been reviewed in depth by Birks (13). When a solution of anthracene is exposed to a flash the first excited triplet state is produced in quantum yield of 0.7 by intrrnal conversion from the excited singlet initially generated. The triplet state has strong absorption a t 424 nm (decadic extinction coefficient = 7 X lo4 1 mole-' em-'), and concrntrations as low as 10-8 mole I-' can readily be detected using our apparatus. In a polymethylmethacrylate matrix at room temperatures triplet anthracene decays to the ground state in an exponential fashion with rate constant -30 s-'. The triplet lifetime, defined as 7 = l/k, is -30 ms, and excellent first order
0
L50
U
Figure 2. State diogrom for anthrorene. The following abbreviations ore used. Singlet state 61. Triplet state (TI. Absorption I A b d Fluorescence IFll. Phosphorescence IPh). Intersystem crossing (ISCI. Ground vibrotianol levels of electronic energy stater are given in the diogmm. Relaxation to there levels from excited vibrational levels populated in absorption, emission, and intersystem crossing is always rapid lthe rate conrtont 1 0IP1-1 is probably correct lo an order of magnitude for molecvier in a condensed phase). Other rate constants are derived from lifetime ond quantum yield studies, with the phorphorercence rote constant inferred b y analogy with other ommoticr.
plots are obtained from monitoring the disappearance of the triplet-triplet absorption at 424 nm. The state diagram for anthracene (Fig. 2) indicates that the preferred route for the triplet-singlet transition is a non-radiative process. In this experiment the triplet state decays are followed for a series of hydrocarbons, and the rates of the radiationless TI + So transitions which are determined are correlated with the triplet energies ET. Molecules for study are chosen from those listed in the table. The lifetimes determined by a number of workers (15-15) are summarized in the central column. Provided that the polymer solutions are prepared carefully according to literature procedure^,^ there is no difficulty in obtaining decay rates which agree well with those in the table. If the samples arc stored in an oxygen-free atmosphere they are stable for many months. The time resolution of our apparatus does' not permit study of naphthacene, though this is accessible using the arrangement with 200 ps flash time described by Porter and West (7). Samples in the latter half of the table may be studied both by phosphorescence and triplet-triplet absorption. The phosphorescence quantum yield is given by the expression
Triplet-triplet Absorption Maxima, Fluorescence Maxima, Triplet Lifetimes, and Triplet Energies for Selected Catacondensed Aromatic Molecules
Molecule 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
670
Naphthacene Perylene Anthracene 3,4-Bensopyrene Pyrene 1,2,5,6Dibenzsnthracene Naphthalene Phenanthrene Coronene Triphenylene
/
Wavelength of most intense (b) phospho(a) T-T res,cence absorption emlsslan in visible (16) (14, 17) x/nm hlnm ,461 ... 488 ... 424 ... 470 ... 416 594 538 546 415 500 490 495 465 563 450 464
Jaurnol o f Chemical Education
Triplet lifetime in PMMA at 298K (15-16) ./a
0.0008 0.006 0.02-0.035 0.07 0.4 1.1 1.3-1.5 1.3-2.5 6.5-6.8 5.3-9.4
-Triplet energy---(a) from phospho(b) Huckel rescence (1%) calc. (82) v/103 em-' E/6 0.59 10.3 0.69 12.6 0.83 14.7 0.74 14.7 0.89 16.9 0.95 18.3 1.24 21.3 1.21 21.6 1.08 19.1 23.1 1.36
Na
+
NH Nc 0.400 0.400 0.418 0.375 0.385 0.389 0.445 0.417 0.333 0.400
where 41 is the quantum yield for triplet state production, r is the triplet lifetime, and r, the phosphorescence contribution to this lifetime. Values for 61 lie between 0.1 and 1, and r, is around 30 s (18). Checking the values of r from the table, it is easy to calculate that 6, falls from >0.1 for triphenylene to aliphetic C-H Triplet benzophenone carries 290 kJ mole-' of excitation energy, and this is greater than the activation energy for abstraction of hydrogen from bonds which have energy less than 400 kJ mole-'. The a-hydrogen in isopropanol is transferred to the reactive earbonyl group of benzophenone, forming the protonated ketyl (CeH&COH. Dimerization of these radicals gives the reaction product, benzpinaeol. I n a competing reaetion pathway, favored at pH values higher than 8, the benzpinaeol is formed from the ketyl present in equilibrium with its protonated form. At high benzophenone concentrations the quantum yield for reaction is 2, as the acetone ketyl produced in step ( 3 ) generates additional benzophenone ketyl by hydrogen atom transfer to benzophenone. 672
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+CaHscO- + H + (K = 6 X 10-10 male 1-1
(5)
I n this experiment values for both kl and kl are determined. I n the pH range 1-7 all the benzpinacol is produced by reaction ( 6 ) , and the decay of the protonated ketyl is seeond order with rate constant k,. I n the pH range 10-13 all the benzpinacol is produced by reaction ( 7 ) , and the decay of the ketyl is second order with rate constant k 2 [ H + ] / K . I n the intermediate pH range reactions (6) and ( 7 ) compete, and the rather rapid deeay is more difficult to follow and interpret (27). Benzophenoue is recrystallized from ethanol, and a 5 X 10WSmole 1-' solution in analytical grade isopropanol is prepared and mixed with an equal volume of 0.01 mole I-' sodium hydroxide solution. Oxygen is removed from the solution by a series of freeze, pump, and thaw cycles using a rotary vacuum pump and a standard vacuum line (4). Flash photolysis produces benzophenone ketyl, and the relatively s l o ~seeond order deeay is followed a t 630 nm-the wavelength of maximum absorption for the intermediate (28). A typical oscilloscope record is shown in Figure 6. For evaluation of the decay curve it is essential that the baseline be recorded along with the deeay profile, and the voltage difference for "light on" and "light off" through the solution must be knovn in order to determine how the absorbance varies throughout the run. If the absorbance at time t is A , then the concentration of ketyl c may be calculated using Beer's law. log,, I-, = A = ccl
I
1 is the cell path length, and the molar deeadic extine-
Figure 6. Second order decay of benzophenone ketyl in 50% v:v iropmpanol water. [OH-] = 5 X male I-', A = 630 nm, T = 293-K, Y scale = 5 mV c ~ - ' . Vo = 110 mV. Ttmebore = 200 m l cm-I.
lo-'
*
Figure 7. p H dependence of k.a.. T = 293 2°K. Doto in olkoline wlution from the decoy of benrophenone ketyl a t 630 nm. Data in ocid from tho pmtonated ketyl a t 5 4 5 nm. Interference filter half width = 1 5 nm, and slit width 1.5 mm. Open circler-results from reference (27). Filled circle-resulbobtained with the teaching flash photdysis apparatur
tion coefficient r has the value 5 X loS 1 mole-' em-' a t 630 nm. A graph of l/c versus t should give a good linear plot, with gradient the second order rate constant
The experiment is repeated at various hydroxide ion concentrations, and then conducted in buffer solutions in the acid range where & = kl. The decay of the protonated ketyl is observed at 545 nm, with t = 5 X lo3 1 mole-' cm-I. Figure 7 presents the variation of the observed rate constant with pH. It can be seen that the results are in good agreement with the original data of Beckett and Porter. k2 is determined from the data in alkaline solution using the value of K from eqn. (5), which is obtained using a pH scale for the solvent where pH is defined as 14.0 log [OH-].
+
The Photodissociation of Corboxymyoglobin (29)
The studies of Kendrew and his associates on sperm whale myoglobin (50) provided the first triumph of protein X-ray crystallography. The functionally important part of the protein is the heme grouping, with a planar porphyrin ring system co-ordinated to Fe(I1) via its four nitrogens. The fifth co-ordination position of the iron is linked to a histidine in the globin chain, and the last position is available for binding the ligand-normally oxygen, though many species (CO, NO, isocyanides, nitroso compounds, etc.) can also bind there. Deoxymyoglobin has the sixth coordination site vacant (31). The equilibrium constant for the reaction Mb
+ CO sk~ MbCO k-1
where Mb stands for deoxymyoglobin, is 4 X 10' 1 mole-'. This is a factor of 40 higher than the formation constant for oxymyoglobin. The same order of binding strength is found for hemoglobin. Carbon monoxide poisoning is due to displacement of oxygen from these storage and carrier proteins, thus depriving the mitochondria of the essential substrate for controlled oxidation of biochemical substrates, and oxidative phosphorylation (38). Since myoglobin must respond rapidly to change in the oxygen level in muscle, its equilibration reaction can only be followed using fast reaction techniques.
Figure 8. Rate of reaction of carbon monoxide with deoxymyoglobin as a function of carbon monoxide pressure. T = 299 & 1 OK. Value of kcdetermined from slope = 9 X 10" mole-' r-1.
The reaction with carbon monoxide is also rapid and a value for the rate constant kl is determined in this experiment. Gibson found that flash photolysis of carhoxymyoglobin caused photodissociation in high quantum yield (89). The return to equilibrium may be followed as the carbon monoxide and deoxymyoglobin recombine: the Soret band of deoxymyoglohin has A,, = 435 nm, r = 12 X lo4 1 mole-' cm-', and there is a significant shift in absorption for carboxymyoglobin with A,., = 424 nm, = 21 X lo4 1 mole-' em-' (33). A solution of the protein having concentration approximately 1 X lo-# mole 1-' is prepared in 0.1 mole 1-I phosphate buffer at pH 7.0. After adding a small quantity of sodium dithionite to ensure that any Fe(111) metmyoglobin is reduced to the Fe(I1) form, the solution is transferred to the outgassing flask, cooled in an ice bath, then pumped out. After flushing the system with carbon monoxide and pumping out again, carbon monoxide is introduced at a measured pressure, around 3 X 10%N m-2 (1 cm Hg = 1.3 X lo3 N m-2). The solution is decanted into the photolysis cell and flashed a t room temperature, with the recombination monitored at 435 nm and recorded on the oscilloscope. Thc photolysis apparatus is then returned to the vacuum line and carbon monoxide a t pressure 6 X 103 N m-z introduced. The rate of reaction should now have doubled. Altogether, the rate of reaction should be measured at six or seven pressures in the range 3-30 N m+. A plot giving the observed rate constant as a function of carbon monoxide pressure is given in Figure 8. k~ is determined from the slope of the graph, using Henry's law as [CO] = P, to convert from pressure to concentration of carbon monoxide in solution. The solubility of the gas a t room temperature and atmospheric pressure (1.0 x lo5 N m-?) is 1.0 X 1 0 4 mole I-'. The value of kl may he compared with the figure of 7 X 105 1 mole-' s-I a t 296°K from Gibson's original paper (!29), and 5 X lo5 1 mole-' s-I at 293°K from recent work on horse myoglobin (33). Related projects which may be investigated by the student are: (1) Flash photolysis studies of the photodissociation of myoglobin combined with other ligands, e.g., isocyanides (34). (2) Flash photolysis studies of co-operative effects Volume 49, Number 10, Odober 1972
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in the binding of carbon monoxide to hemoglobin, a multi-subunit protein (53,56). (3) Can carboxymyoglohin be photolysed by selective excitation of the visible region bands at 579 and 540 nm?
. .
..
and previous papers in this series.
(14) BALDWIN. B. A,. A N D OFFEN, H. W.. J. Chem. Phyr.. 46,4509 (1967). M. I..MCCALLOM, K. I.. WOODO.R. J.. A N D FORMOS~NHO. (15) WEST, J.. Trans. Forodoy Soc.. 66,2135 (1970). n . A N D W i ~ n s o n hl. , \T.. Pioc. Roy. Soc., A 245,238 (1958). (16) P o n ~ ~G.. (17) CL*n. E.,a m ZANDER, M., Citcm. Be?.. 89,749 (1956). , E., A N D BENNETT, R. G., J . Chem. Phur., 41,3042 (1964). (18) K ~ m o c oR. (19) HENRI,D. R., A N D KASHA,M., Ann. Rcu. Phys. Chem., 19, 161 (1968). (20) R o n r ~ s o aG. , W..A N D Fnoac", R. P.. J. Chem. I'hps., 37, 1962 (1962). (21) Slrsn*ro, W., J. Chem. Phys., 44, 4055 (1966): 47, 2411 (1967). , AND C O D ~ ~ O C.NA,, . "Diction&ry of x-Electron (22) S ~ n s r ~ w l s s s nA,. Calculations," Pergamon Press, Oxford. 1965. ~ , Z..A N D PORTER, G., Pioe. Roy. Soc.. A 268, 46 (1962). (23) H a r ~ a * M. (241 P o n ~ ~G.. n . AND W I ~ K I N S O N F.. . P ~ cROV. . S m . . A 264. 1 (1961). iz5j ~ * n a ~ C. n ;A., ~ a u o nphotohem.. . Z , 305 i m 4 ) . n PENSEA,G. R.. AND RADDA.6. K., E w . J . (26) O o n o o ~ - W * ~ a =A., Biochem.. 13,313 (1970). (27) BBCRETT.A., A N D PORTER,G., Tian*. Foraaay Sor., 59, 2038 (1'363). x . Tmns. Farodoy Soc.. 57. 1686 (1961). (28) Ponmn. G.. *so W r ~ n ~ a s oF.. (29) GIBSON.Q. H.. J.Physio1.. 134, 112 (1956). (30) KENDREW. J. C., Sci. Amer. 205, 98 (1961). K r ; ~ n n ~ J. w ,C., *no IVATSON, H. C., Natwe. 209. 339 (31) Nonss. C. (1966). (32) An excellent disousaion of the physiological role of the oxygen carrier ~ . AND proteins is hvnilahle in the hook by T n r r e . A,, H A W D L EP., Snmn, E. L.. "Prinoiples of nioehemiatry" (3rd ed.), MeGraru-Hill. New York. 1968. (33) ANTONINI, E., Phydol. Rcu., 45, 123 (1965). G m s o ~ Q. , H.. Aun RoncxTon. F . J. W., P ~ c ROY. . (34) A w r r o m n . Soe.. B 152, 331 (1960). (35) Gmsow, Q. H.,P~oor.Rcat.Kinct.,2,321 (1964).
S.
Acknowledgment
The authors would like to thank Dr. M. A. Wcst for assistance in the choice of experiments, and the loan of polymer solutions of aromatic hydrocarbons. Dr. G. It. P ~ n z c rsuggested the experiment on riboflavin. literature Cifed
S.
(1) C ~ ~ ~ s s o x(Edit04 . "Nobel Symposium +Fast Reactions and Primary Processes in Chemical Kinetics," Almqvist and Wiksell (Intersoienoe), Stockholm, 1967. (2) A s x ~ o nP.G.. ~ , UA~TON F. . M. (Edilors), "Photoohemistry and Reaction Kinetics." Cambridge University Press. Cambridge. 1967. . Science. 160, 1299 (1968). (3) P o m ~ n G., (4) YAM EN ASH^, B. A N D NOWAX, A. V,,J. CHEM.EDUC.. 45.705 (1968). . A,, Bnnxs. G. A N D CXANO, K., J. CXEM.EDUC.,46. 745 (5) B ~ n n n I. (1969). (6) GOODALL. D. M., HAEATION,P. W.. HARDY.M. J., AND KIRX,C. J.. J. C a w . Eouc.. 49,675 (1972). (7) PORTER. G . , A N DW ~ TM. . A , . Educ. Chem. 7,230 (1970). " Wilev 61 (8) C * L v z n ~ ,J. G., m n PITTS.J. N.. " P h ~ t o ~ h e m i a t r y ,John Sons, Ine. New York, 1966. (9) Tonno. N. J., "Molecular Photoohemistry," Benjamin. New York. 1967.
S.,AND Snoo=w.T.
S..
Nelson, Lon(10) Cnno*~', R. B.. AM, Gir;e~nv,A.. "Photochemistry: don. 1970. (11) W n r m . R. P.. "Photoohemistry," Butterwartha. London. 1970. . B.. "The Photoohvsios of Aromktio Moleeules." \Irilev-Inter1121 B l n ~ sJ. soience. ~ k n d o n ,1970. (13) K ~ b ~ a o R. o . E.. AND T Y E T ~ N. . C.. J . Chem. Phvs.. . 45.. 3156 (1966).
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