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Measurement of magnetic circular dichroism (MCD) on a nanosecond timescale
CHEMICAL
Volume 156, number 6
2 1 April I989
PHYSICS LETTERS
MEASUREMENT OF MAGNETIC CIRCULAR ON A NANOSECOND TIMESCALE
DICHROISM
(MCD)
Robert A. GOLDBECK, Timothy D. DAWES, Steven J. MILDER, James W. LEWIS and David S. KLIGER Deparrmenr
Received
of Chemistry,
17 February
University
of CaliJbmia,
Sawn Cruz, CA 95064, USA
I989
A novel technique for the time-resolved detection of magnetic circular dichroism is described. Its apphcation to the photoinduced bleaching of Soret band (390-440 nm) MCD and concomitant observation ofthe MCD of the lowest excited triplet state in zinc tetraphenylporphine in toluene solution is presented. A time resolution of I8 ns is achieved, five orders of magnitude faster than conventional techniques.
1.
Introduction
Natural circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies are both important tools for the analysis of molecular structure [ 1,2]. CD provides information about the asymmetry of a molecule and its environment, whereas MCD, which can be considered to be an extension of Zeeman spectroscopy to the overlapping bands of condensed phase absorptions, is sensitive to structural features that directly affect the energy levels of a chromophore [ 31. A novel technique for time-resolved CD spectroscopy (TRCD) has been developed in this laboratory and it has been shown to be capable of measurements with nanosecond time resolution [ 4-71. Techniques for the time-resolved measurement of CD and MCD permit these properties to be determined for short-lived species, such as electronic excited states and chemical intermediates, and allow the kinetics of processes that change these properties to be followed. The kinetic measurement of CD and MCD has previously been limited by the time resolution of conventional instruments, which is on the order of milliseconds. Applications of the new method to CD transients produced in the photolysis of carbonmonoxy hemoglobin-and myoglobin [ 4,8,9], tRNA [ 101, and the excited states of inorganic complexes [ 5,11- 13 ] have been reported. In this report, we de-
scribe the application of the kinetic CD method to time-resolved magnetic circular dichroism (TRMCD) spectroscopy and report nanosecond time-scale MCD transients observed on photolysis of zinc tetraphenylporphine (ZnTPP) in toluene solution.
2. Method Circular dichroism is difficult to measure directly because it typically requires the detection of a small difference between two large quantities. For a light absorbing transition with molar extinction coeffcient t and circular dichroism AC= E,_- +, where c,_ and Ed are the molar extinction coefficients for left and right circularly polarized light, respectively. AC/ t is typically lop3 to 10m4. Rather than measure eL and tK directly, the TRCD technique uses a quasinull method in which a light beam linearly polarized along a horizontal axis first passes through a fused silica plate to which a mechanical strain is applied to introduce a slight birefringence (fast axis at & 45 ’ from horizontal ) , This produces an elliptically polarized beam whose major axis is oriented along the horizontal axis. With lo of retardation produced in the silica plate, the major (horizontal) axis intensity is about 10“ times that of the minor (vertical) axis. This degree of ellipticity will be changed by a cir545
Volume 156. number 6
CHEMICAL
PHYSICS LETTERS
cularly dichroic sample. The change in ellipticity is detected by passing the beam through a vertical analyzing polarizer before directing it into the detection system [ 4,6]. A quasi-null method rather than true-null method is necessary because the linear birefringence of the strain plate must be much greater than the circular birefringence (CB) (associated by the KramersKronig relation [ 11 with the circular dichroism of the sample) in order that CB not interfere with CD detection [ 71. The advantage of this technique is that a At that is only 10m4c can produce a change of 10% in the minor axis of the polarization ellipse under typical conditions ( 1’ of retardance in strain plate). This puts less demand on probe lamp stability and, combined with the use of more intense flashlamp light sources than in conventional instruments, makes possible CD measurements with high time resolution [7]. The TRMCD apparatus (fig. 1) consists of a modified laser photolysis apparatus in which the sample and strain plate ( 1/ 16 inch fused silica microscope slide mounted in an anvil to produce a well-characterized strain [ 61) are placed between crossed polarizers (Glan laser prisms, Karl Lambrecht ). A frequency-doubled Nd : YAG laser (Quanta-Ray DCR1) excites the sample with a 532 nm, 30 mJ, 7 ns pulse. The probe source is a xenon flashlamp with a 2 ps fwhm duration. The probe beam is detected with multichannel analyzer a spectrograph/optical (OMA) instrument for spectral measurements [ 141, or with a monochromator/photomultiplier/transient-digitizer system for kinetic measurements. The
FL
21 April 1989
OMA consists of a Monospec 27 (Jarrell-Ash) spectrograph ( 100 pm slit, 150 grooves/grating) coupled to an EG&G 1420 gated detector controlled by an EG&G 146 1 detector interface and an IBM PC. The transient digitizer apparatus uses a 9876QB (EMIGENCOM) photomultiplier mounted on a MP10 18B (Pacific Precision lnstruments) monochromator (800 pm slit, 1180 grooves/mm grating), coupled to a Tektronix 7912AD programmable digitizer. In the spectral measurements, the OMA detection gate is opened for 4 ps, beginning 2 ps after the laser pulse. The gate encompasses most of the temporal profile of the probe flashlamp. In the kinetic measurements, the laser and digitizer are triggered at the peak of the probe flash. The sample solution flows through a 1 mm fused quartz cell placed within a 7 kG permanent magnet (JASCO PM-l ) with the magnetic field lines collinear with the probe beam. The signal measured is s= (Z, _ZL)I(ZR
flL,)
1
(1)
where Z, and ZL are the intensities of light transmitted through the crossed polarizers when the strain plate is oriented to produce right or left elliptically polarized light, respectively. The extinction of the beam by the crossed polarizers was of the order of 10d6 without the sample in the beam, and IO-’ with the sample in the beam. It can be shown that when the strain plate retardance S is much greater than the ellipticity (Y,but less than 1, where a=0.575 At cz, c is the concentration in mol R-‘, z is the pathlength in cm, and 6 is the strain plate retardance in rad, the signal is approximately [4]
M R
P”
L3
LASER -0 Fig. I. Apparatus for fast magnetic circular dichroism measurements of laser-induced transients. Light from the flashlamp (FL) is collimated by lens Ll( focused (L2) through a horizontal Glan-Taylor polarizer (P,,) and strain plate (SP), and passed through the pole pieces of a permanent magnet (M) containing the sample (S). The actinic laser pulse enters at 90” from rhe probe beam. A sucrose solution is used as a dextrorotatory rotator (R) to compensate for Faraday rotation of the probe beam polarization by the sample cell windows and solvent. The probe beam then passes through a vertically polarizing Glan-Taylor prism ( Py), It is then recollimated (L3) and focused (L4) into a detector (D), which is a monochromator with photomultiplier for single-wavelength kinetic measurements or a spectrograph with OMA for single-time spectral measurements.
546
CHEMICAL
Volume 156, number 6
S=2.3 At cz/6
(o
1) .
PHYSICS LETTERS
(2)
Although the TRCD and TRMCD techniques are experimentally identical in most respects, an important additional feature of the TRMCD experiment is that the Faraday effect induced in the transparent solvent and cell windows by the applied magnetic field rotates the plane of polarization of the probe beam. This can result in a large change in the amount of the probe beam that passes through the analyzing polarizer, which can overwhelm MCD signals of interest. Because MCD and MOR (magnetic optical rotation) are commuting optical operations, this was compensated for by rotating the beam polarization back again with a dextrorotatory sucrose solution placed before the analyzing polarizer. Although the MOR due to the cell and solvent is wavelength dependent, far from an absorption line the MORDs (MOR dispersions) of toluene and quartz have approximately the same wavelength dependence (= l/n’) as the ORD of sucrose. Thus, the sucrose concentration could be adjusted to give excellent compensation over the spectra1 region studied. For kinetic measurements at a single wavelength, it is also possible to compensate for the Faraday rotation by rotating the analyzing polarizer by an equal angle. Baseline drift was observed in the MCD signals for both ground and transient difference spectra, This was due to probe lamp intensity fluctuations over the course of collecting a spectrum ( x 1 h), and, for the transient spectrum, thermal effects due to laser excitation. Consequently, a baseline correction was added to the MCD spectrum by assuming the MCD signal was zero at wavelengths where there is no ground state or triplet state absorption. The zinc complex of meso-tetraphenylporphine (Sigma) was used as received and dissolved in spectral grade toluene to give a solution with optical density of I .O at 423 nm in a 1 mm path length cell. The solution was filtered through a fine fritted disc to remove light scattering particles and deoxygenated by bubbling with argon to reduce O2 quencing of the triplet state. The kinetics of triplet state decay in ZnTPP were studied by Pekkarinen and Linschitz [ f 5 ] and the following rate law was found: -dC*/dt=k,
C*+k;(C*)2+k3C*Cg,
where C* is the concentration
of the excited triplet
21 April 1989
state, C, the concentration of the ground state, and k,=800s-‘,k,=3x10v~mol-‘s-‘,andk~=2x107 II mol-’ s-l. Under the experimental conditions used in the present study (total ZnTPP concentration of 2x lop5 M and 90% conversion of singlet state to triplet state), the triplet decay is dominated at short times by k2. bimolecular triplet-triplet quenching. The time to 50% decay predicted by the rate law for triplet-triplet quenching is 20 us, which is much longer than our probe flashlamp pulse.
3. Results and discussion Fig. 2 shows the absorption difference spectrum obtained upon laser excitation of ZnTPP, the excited triplet state absorption spectra (corrected to 100% conversion), and the ground state absorption spectrum. The AOD spectrum was obtained with standard nanosecond photolysis techniques. By using the known extinction coefficients for the ground and triplet state transitions [ 15 1, the average extent of conversion of ground state to triplet during OMA detection was determined to be 60%. The transient OD spectrum shows bleaching in the Soret region (-420nm) and T,eT, absorption to the red of the 60 40
20 w I 0
0
x w -20
-40 -60
350
I
I
I
400
450
500
1 (nm) Fig. 2. The absorption spectra of ZnTPP in toluene for the ground (- - - ) and first excited triplet (corrected to 100% conversion ) (-) states, and the transient difference spectrum (- --) for 60% conversion of ground to triplet state during a 4 ps window immediately following laser photolysls. 547
Volume 156, number 6
CHEMlCAL
Soret band. The broad, featureless absorption spectrum shown here for the excited state is identical to the T,,tT, spectrum reported previously [ 151 for ZnTPP in toluene, if the single point near the peak of the Soret band is neglected in the previous work. The transient difference MCD spectrum, the excited triplet state MCD spectrum (corrected to 100% conversion), and the ground state MCD spectrum of ZnTPP are shown in fig. 3. The MCD spectra are the averages of 2000 measurements taken with the OMA system using a 4 ps detection window. Excitation conditions were the same as those used to measure the absorption spectra. A quasi A term of “normal” MCD signature [ 31 associated with the Soret absorption band is the main feature observed in the ground state MCD spectrum [ 161. Upon laser photolysis, bleaching of the ground state MCD is the main feature evident in the transient MCD. However, the slight offset of the transient MCD spectrum relative to the ground state (after baseline correction) suggests the presence of an additional contribution which may be associated with the T,+T,
PHYSICS LETTERS
transition that lies in this spectral region [ 15 1. The spectral overlap of the excited state MCD with the T,cTI absorption band supports the assignment of the MCD to the same T,+T, transition(s). The similar time constants for decay of the transient MCD and triplet absorptions, and the fact that reintroduction of oxygen extinguishes both transient MCD and triplet absorptions also suggest that the excited state MCD originates from T,. The excited state MCD spectrum shows more structure than the ordinary absorption spectrum and suggests that there may be at least two overlapping T,+T, transitions with opposite MCD signs contributing to the spectra in this region. Kinetic MCD traces with a nanosecond time resolution are shown in fig. 4 for ZnTPP at 4 18 and 430 nm. These wavelengths are near, respectively, the positive and negative ellipticity extrema of the ground v.1,
I
-0.0 7 :E
-0.2 -
? I? 2
-0.4 -
t
A 423nm
_
-0.6 -0.8
2
21 April 1989
-
L
0
3
418 nm
-0.06 -200
u 350
}__I
-0.08 400
450
500
A (nm) Fig. 3. The MCD spectra of ZnTPP in toluene for the ground (- - -) and first excited triplet (corrected to 100% conversion) (-) states, and the transient difference spectrum (---) for 60% conversion of ground to triplet state during a 4 ps window immediately following laser photolysis. MCD is reported here as the magnetically induced molar elhpticity per unit magnetic field, [0],=3300Ae/H(degPmol-‘m-l G-‘),whereAt=t,_--tRis the difference between the molar extinction coefficients for left and right circularly polarized light, and H is the strength in G of a uniform magnetic field oriented along the propagation vector of the light beam.
548
cm
-0.11 0
I 200
-9
]
I 400
' 600
800
time (ns) Fig. 4. (A) Time-resolved absorption of ZnTPP at 423 nm, and time-resolved MCD of ZnTPP in toluene at (B) 418 and (C) 430 nm. MCD is reported here as the signal defined in eq. ( 1) in the text. The arrow indicates the time when the sample is excited by a 7 ns, 30 mJ, 532 nm laser pulse.
Volume 156, number 6
CHEMlCAL
PHYSICS LETTERS
state. Bleaching of the ground state MCD upon laser photolysis is clearly evident in both traces. Also shown in fig. 4 is the kinetic OD trace taken at 423 nm, the peak of the Soret band. The OD trace shows that after laser excitation there is prompt conversion of approximately 90% of the ground state to the triplet state. This is consistent with the lower conversion seen in the OMA measurements, where the triplet states have partially relaxed to the ground state during the detection time. The time resolution of the kinetic measurements was 18 ns. This is the time constant of an exponential smoothing that was applied to the data to minimize interference from a small amount of fluorescence from the Soret region, which was initiated by sequential, resonant two-photon absorption of the actinic laser pulse. The use of crossed polarizers makes the TRMCD technique susceptible to interference from unpolarized emission and scattering, and ZnTPP is known to exhibit S,+S, fluorescence when irradiated with a high-intensity pulse of lowenergy photons [ 17 1,
4. Conclusions The present study has demonstrated the measurement of transient changes in the magnetic circular dichroism of a metalloporphyrin on a 10 ns timescale, and, to our knowledge, we report the first MCD spectrum for an absorption arising out of a molecular excited state. A time resolution of 2 ns would have been possible in the absence of fluorescence interference. This time resolution is an improvement of five to six orders of magnitude over conventional techniques. The biological importance of porphyrins, coupled with their intense MCD signals and the sensitivity of their MCD spectra to structural features that perturb the electronic structure of the chromophore, suggests that fast MCD detection should be useful for studying the kinetics of structural changes in hemeproteins [ 31. The spectroscopy of molecular excited states of MCD active chromophores should also benefit from the additional structure apparent in MCD spectra as compared to ordinary absorption spectra. Work is in progress to obtain the excited triplet state MCD spectra of ZnTPP and other porphyrins
21 April 1989
using larger applied magnetic fields available from pulsed magnets. These spectra, along with calculations of MCD using semiempirical molecular orbital methods [ 18 1, should allow a deeper understanding of the electronic nature of this class of molecules.
Acknowledgement Financial support for this work was provided in part by National Institutes of Health grant GM38549.
I :ferences
I ] L.D. Barron, Molecular light scattering and optical activity ’ (Cambridge Univ. Press, Cambridge, 1982). 2 ] S.B. Piepho and P.N. Schatz, Group theory in spectroscopy with applications to magnetic circular dichroism (Wiley, New York, 1983). 31 R.A. Goldbeck, AccountsChem. Res. 21 (1988) 95. [4] J.W. Lewis, R.F. Tilton, C.M. Einterz. S.J. Milder, I.D. Kuntz and D.S. Kliger, J. Phys. Chem. 89 ( 1985) 289. [ 5] S.J. Milder, J.S. Gold and D.S. Kliger, J. Am. Chem. Sot. 108 (1986) 8295. [ 61D.S. Kliger and J. W. Lewis, Rev. Chem. Intermed. 8 ( 1987 ) 367. [ 71D.S. Kliger, J.W. Lewis and R.A. Goldbeck, Proc. SPIE 1057 (1989), in press. [ 8) C.M. Einterz, J.W. Lewis, S.J. Milder and D.S. Kliger, J. Phys. Chem. 89 (1985) 3845. [9] S.J. Milder, S.C. Eijorling, I.D. Kuntz and D.S. KIiger. Biophys. J. 53 (1988) 659. [IO] S.J. Milder, P.S. Weiss and D.S. Kliger, Biochemistry 28 ( 1989), in press. [ I 1 ] J.S. Gold, S.J. Milder, J.W. Lewis and D.S. Kliger, J. Am. Chem. Sot. 107 (1985) 8285. I 121 S.J. Milder, J.S. Gold and D.S. Kliger, Chem. Phys. Letters 144 (1988) 269. [ 131 S.J. Milder and D.S. KIiger, J. Am. Chem. Sot., submitted for publication. [ 141 J.W. Lewis, G.G. Yee and D.S. Kliger, Rev. Sci. lnstr. 58 (1987) 939. [IS] L. Pekkarinen and H. Linschitz, J. am. Chem. Sot. 82 ( 1960) 2407. [ 161 J.D. Keegan, E. Bunnenberg and C. Djerassi, Spectrochim. Acta40A ( 1984) 287. [ 171 H. Kohayashi and Y. Kaizu, in: Porphyrins: excited states and dynamics, eds. M. Gouterman, P.M. Rentzepis and K.D. Stranb (Am. Chem. Sot.. Washington. 1986) p. 105. [IS] R.A. Gotdbeck, B.-R. Tolf, A.G.H. Wee. A.Y.L. Shu, R. Records, E. Buhnenberg and C. Djerassi, J. Am. Chem. Sot. 108 (1986) 6449.
549
Volume 156, number 6
2 1 April I989
PHYSICS LETTERS
MEASUREMENT OF MAGNETIC CIRCULAR ON A NANOSECOND TIMESCALE
DICHROISM
(MCD)
Robert A. GOLDBECK, Timothy D. DAWES, Steven J. MILDER, James W. LEWIS and David S. KLIGER Deparrmenr
Received
of Chemistry,
17 February
University
of CaliJbmia,
Sawn Cruz, CA 95064, USA
I989
A novel technique for the time-resolved detection of magnetic circular dichroism is described. Its apphcation to the photoinduced bleaching of Soret band (390-440 nm) MCD and concomitant observation ofthe MCD of the lowest excited triplet state in zinc tetraphenylporphine in toluene solution is presented. A time resolution of I8 ns is achieved, five orders of magnitude faster than conventional techniques.
1.
Introduction
Natural circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies are both important tools for the analysis of molecular structure [ 1,2]. CD provides information about the asymmetry of a molecule and its environment, whereas MCD, which can be considered to be an extension of Zeeman spectroscopy to the overlapping bands of condensed phase absorptions, is sensitive to structural features that directly affect the energy levels of a chromophore [ 31. A novel technique for time-resolved CD spectroscopy (TRCD) has been developed in this laboratory and it has been shown to be capable of measurements with nanosecond time resolution [ 4-71. Techniques for the time-resolved measurement of CD and MCD permit these properties to be determined for short-lived species, such as electronic excited states and chemical intermediates, and allow the kinetics of processes that change these properties to be followed. The kinetic measurement of CD and MCD has previously been limited by the time resolution of conventional instruments, which is on the order of milliseconds. Applications of the new method to CD transients produced in the photolysis of carbonmonoxy hemoglobin-and myoglobin [ 4,8,9], tRNA [ 101, and the excited states of inorganic complexes [ 5,11- 13 ] have been reported. In this report, we de-
scribe the application of the kinetic CD method to time-resolved magnetic circular dichroism (TRMCD) spectroscopy and report nanosecond time-scale MCD transients observed on photolysis of zinc tetraphenylporphine (ZnTPP) in toluene solution.
2. Method Circular dichroism is difficult to measure directly because it typically requires the detection of a small difference between two large quantities. For a light absorbing transition with molar extinction coeffcient t and circular dichroism AC= E,_- +, where c,_ and Ed are the molar extinction coefficients for left and right circularly polarized light, respectively. AC/ t is typically lop3 to 10m4. Rather than measure eL and tK directly, the TRCD technique uses a quasinull method in which a light beam linearly polarized along a horizontal axis first passes through a fused silica plate to which a mechanical strain is applied to introduce a slight birefringence (fast axis at & 45 ’ from horizontal ) , This produces an elliptically polarized beam whose major axis is oriented along the horizontal axis. With lo of retardation produced in the silica plate, the major (horizontal) axis intensity is about 10“ times that of the minor (vertical) axis. This degree of ellipticity will be changed by a cir545
Volume 156. number 6
CHEMICAL
PHYSICS LETTERS
cularly dichroic sample. The change in ellipticity is detected by passing the beam through a vertical analyzing polarizer before directing it into the detection system [ 4,6]. A quasi-null method rather than true-null method is necessary because the linear birefringence of the strain plate must be much greater than the circular birefringence (CB) (associated by the KramersKronig relation [ 11 with the circular dichroism of the sample) in order that CB not interfere with CD detection [ 71. The advantage of this technique is that a At that is only 10m4c can produce a change of 10% in the minor axis of the polarization ellipse under typical conditions ( 1’ of retardance in strain plate). This puts less demand on probe lamp stability and, combined with the use of more intense flashlamp light sources than in conventional instruments, makes possible CD measurements with high time resolution [7]. The TRMCD apparatus (fig. 1) consists of a modified laser photolysis apparatus in which the sample and strain plate ( 1/ 16 inch fused silica microscope slide mounted in an anvil to produce a well-characterized strain [ 61) are placed between crossed polarizers (Glan laser prisms, Karl Lambrecht ). A frequency-doubled Nd : YAG laser (Quanta-Ray DCR1) excites the sample with a 532 nm, 30 mJ, 7 ns pulse. The probe source is a xenon flashlamp with a 2 ps fwhm duration. The probe beam is detected with multichannel analyzer a spectrograph/optical (OMA) instrument for spectral measurements [ 141, or with a monochromator/photomultiplier/transient-digitizer system for kinetic measurements. The
FL
21 April 1989
OMA consists of a Monospec 27 (Jarrell-Ash) spectrograph ( 100 pm slit, 150 grooves/grating) coupled to an EG&G 1420 gated detector controlled by an EG&G 146 1 detector interface and an IBM PC. The transient digitizer apparatus uses a 9876QB (EMIGENCOM) photomultiplier mounted on a MP10 18B (Pacific Precision lnstruments) monochromator (800 pm slit, 1180 grooves/mm grating), coupled to a Tektronix 7912AD programmable digitizer. In the spectral measurements, the OMA detection gate is opened for 4 ps, beginning 2 ps after the laser pulse. The gate encompasses most of the temporal profile of the probe flashlamp. In the kinetic measurements, the laser and digitizer are triggered at the peak of the probe flash. The sample solution flows through a 1 mm fused quartz cell placed within a 7 kG permanent magnet (JASCO PM-l ) with the magnetic field lines collinear with the probe beam. The signal measured is s= (Z, _ZL)I(ZR
flL,)
1
(1)
where Z, and ZL are the intensities of light transmitted through the crossed polarizers when the strain plate is oriented to produce right or left elliptically polarized light, respectively. The extinction of the beam by the crossed polarizers was of the order of 10d6 without the sample in the beam, and IO-’ with the sample in the beam. It can be shown that when the strain plate retardance S is much greater than the ellipticity (Y,but less than 1, where a=0.575 At cz, c is the concentration in mol R-‘, z is the pathlength in cm, and 6 is the strain plate retardance in rad, the signal is approximately [4]
M R
P”
L3
LASER -0 Fig. I. Apparatus for fast magnetic circular dichroism measurements of laser-induced transients. Light from the flashlamp (FL) is collimated by lens Ll( focused (L2) through a horizontal Glan-Taylor polarizer (P,,) and strain plate (SP), and passed through the pole pieces of a permanent magnet (M) containing the sample (S). The actinic laser pulse enters at 90” from rhe probe beam. A sucrose solution is used as a dextrorotatory rotator (R) to compensate for Faraday rotation of the probe beam polarization by the sample cell windows and solvent. The probe beam then passes through a vertically polarizing Glan-Taylor prism ( Py), It is then recollimated (L3) and focused (L4) into a detector (D), which is a monochromator with photomultiplier for single-wavelength kinetic measurements or a spectrograph with OMA for single-time spectral measurements.
546
CHEMICAL
Volume 156, number 6
S=2.3 At cz/6
(o
1) .
PHYSICS LETTERS
(2)
Although the TRCD and TRMCD techniques are experimentally identical in most respects, an important additional feature of the TRMCD experiment is that the Faraday effect induced in the transparent solvent and cell windows by the applied magnetic field rotates the plane of polarization of the probe beam. This can result in a large change in the amount of the probe beam that passes through the analyzing polarizer, which can overwhelm MCD signals of interest. Because MCD and MOR (magnetic optical rotation) are commuting optical operations, this was compensated for by rotating the beam polarization back again with a dextrorotatory sucrose solution placed before the analyzing polarizer. Although the MOR due to the cell and solvent is wavelength dependent, far from an absorption line the MORDs (MOR dispersions) of toluene and quartz have approximately the same wavelength dependence (= l/n’) as the ORD of sucrose. Thus, the sucrose concentration could be adjusted to give excellent compensation over the spectra1 region studied. For kinetic measurements at a single wavelength, it is also possible to compensate for the Faraday rotation by rotating the analyzing polarizer by an equal angle. Baseline drift was observed in the MCD signals for both ground and transient difference spectra, This was due to probe lamp intensity fluctuations over the course of collecting a spectrum ( x 1 h), and, for the transient spectrum, thermal effects due to laser excitation. Consequently, a baseline correction was added to the MCD spectrum by assuming the MCD signal was zero at wavelengths where there is no ground state or triplet state absorption. The zinc complex of meso-tetraphenylporphine (Sigma) was used as received and dissolved in spectral grade toluene to give a solution with optical density of I .O at 423 nm in a 1 mm path length cell. The solution was filtered through a fine fritted disc to remove light scattering particles and deoxygenated by bubbling with argon to reduce O2 quencing of the triplet state. The kinetics of triplet state decay in ZnTPP were studied by Pekkarinen and Linschitz [ f 5 ] and the following rate law was found: -dC*/dt=k,
C*+k;(C*)2+k3C*Cg,
where C* is the concentration
of the excited triplet
21 April 1989
state, C, the concentration of the ground state, and k,=800s-‘,k,=3x10v~mol-‘s-‘,andk~=2x107 II mol-’ s-l. Under the experimental conditions used in the present study (total ZnTPP concentration of 2x lop5 M and 90% conversion of singlet state to triplet state), the triplet decay is dominated at short times by k2. bimolecular triplet-triplet quenching. The time to 50% decay predicted by the rate law for triplet-triplet quenching is 20 us, which is much longer than our probe flashlamp pulse.
3. Results and discussion Fig. 2 shows the absorption difference spectrum obtained upon laser excitation of ZnTPP, the excited triplet state absorption spectra (corrected to 100% conversion), and the ground state absorption spectrum. The AOD spectrum was obtained with standard nanosecond photolysis techniques. By using the known extinction coefficients for the ground and triplet state transitions [ 15 1, the average extent of conversion of ground state to triplet during OMA detection was determined to be 60%. The transient OD spectrum shows bleaching in the Soret region (-420nm) and T,eT, absorption to the red of the 60 40
20 w I 0
0
x w -20
-40 -60
350
I
I
I
400
450
500
1 (nm) Fig. 2. The absorption spectra of ZnTPP in toluene for the ground (- - - ) and first excited triplet (corrected to 100% conversion ) (-) states, and the transient difference spectrum (- --) for 60% conversion of ground to triplet state during a 4 ps window immediately following laser photolysls. 547
Volume 156, number 6
CHEMlCAL
Soret band. The broad, featureless absorption spectrum shown here for the excited state is identical to the T,,tT, spectrum reported previously [ 151 for ZnTPP in toluene, if the single point near the peak of the Soret band is neglected in the previous work. The transient difference MCD spectrum, the excited triplet state MCD spectrum (corrected to 100% conversion), and the ground state MCD spectrum of ZnTPP are shown in fig. 3. The MCD spectra are the averages of 2000 measurements taken with the OMA system using a 4 ps detection window. Excitation conditions were the same as those used to measure the absorption spectra. A quasi A term of “normal” MCD signature [ 31 associated with the Soret absorption band is the main feature observed in the ground state MCD spectrum [ 161. Upon laser photolysis, bleaching of the ground state MCD is the main feature evident in the transient MCD. However, the slight offset of the transient MCD spectrum relative to the ground state (after baseline correction) suggests the presence of an additional contribution which may be associated with the T,+T,
PHYSICS LETTERS
transition that lies in this spectral region [ 15 1. The spectral overlap of the excited state MCD with the T,cTI absorption band supports the assignment of the MCD to the same T,+T, transition(s). The similar time constants for decay of the transient MCD and triplet absorptions, and the fact that reintroduction of oxygen extinguishes both transient MCD and triplet absorptions also suggest that the excited state MCD originates from T,. The excited state MCD spectrum shows more structure than the ordinary absorption spectrum and suggests that there may be at least two overlapping T,+T, transitions with opposite MCD signs contributing to the spectra in this region. Kinetic MCD traces with a nanosecond time resolution are shown in fig. 4 for ZnTPP at 4 18 and 430 nm. These wavelengths are near, respectively, the positive and negative ellipticity extrema of the ground v.1,
I
-0.0 7 :E
-0.2 -
? I? 2
-0.4 -
t
A 423nm
_
-0.6 -0.8
2
21 April 1989
-
L
0
3
418 nm
-0.06 -200
u 350
}__I
-0.08 400
450
500
A (nm) Fig. 3. The MCD spectra of ZnTPP in toluene for the ground (- - -) and first excited triplet (corrected to 100% conversion) (-) states, and the transient difference spectrum (---) for 60% conversion of ground to triplet state during a 4 ps window immediately following laser photolysis. MCD is reported here as the magnetically induced molar elhpticity per unit magnetic field, [0],=3300Ae/H(degPmol-‘m-l G-‘),whereAt=t,_--tRis the difference between the molar extinction coefficients for left and right circularly polarized light, and H is the strength in G of a uniform magnetic field oriented along the propagation vector of the light beam.
548
cm
-0.11 0
I 200
-9
]
I 400
' 600
800
time (ns) Fig. 4. (A) Time-resolved absorption of ZnTPP at 423 nm, and time-resolved MCD of ZnTPP in toluene at (B) 418 and (C) 430 nm. MCD is reported here as the signal defined in eq. ( 1) in the text. The arrow indicates the time when the sample is excited by a 7 ns, 30 mJ, 532 nm laser pulse.
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state. Bleaching of the ground state MCD upon laser photolysis is clearly evident in both traces. Also shown in fig. 4 is the kinetic OD trace taken at 423 nm, the peak of the Soret band. The OD trace shows that after laser excitation there is prompt conversion of approximately 90% of the ground state to the triplet state. This is consistent with the lower conversion seen in the OMA measurements, where the triplet states have partially relaxed to the ground state during the detection time. The time resolution of the kinetic measurements was 18 ns. This is the time constant of an exponential smoothing that was applied to the data to minimize interference from a small amount of fluorescence from the Soret region, which was initiated by sequential, resonant two-photon absorption of the actinic laser pulse. The use of crossed polarizers makes the TRMCD technique susceptible to interference from unpolarized emission and scattering, and ZnTPP is known to exhibit S,+S, fluorescence when irradiated with a high-intensity pulse of lowenergy photons [ 17 1,
4. Conclusions The present study has demonstrated the measurement of transient changes in the magnetic circular dichroism of a metalloporphyrin on a 10 ns timescale, and, to our knowledge, we report the first MCD spectrum for an absorption arising out of a molecular excited state. A time resolution of 2 ns would have been possible in the absence of fluorescence interference. This time resolution is an improvement of five to six orders of magnitude over conventional techniques. The biological importance of porphyrins, coupled with their intense MCD signals and the sensitivity of their MCD spectra to structural features that perturb the electronic structure of the chromophore, suggests that fast MCD detection should be useful for studying the kinetics of structural changes in hemeproteins [ 31. The spectroscopy of molecular excited states of MCD active chromophores should also benefit from the additional structure apparent in MCD spectra as compared to ordinary absorption spectra. Work is in progress to obtain the excited triplet state MCD spectra of ZnTPP and other porphyrins
21 April 1989
using larger applied magnetic fields available from pulsed magnets. These spectra, along with calculations of MCD using semiempirical molecular orbital methods [ 18 1, should allow a deeper understanding of the electronic nature of this class of molecules.
Acknowledgement Financial support for this work was provided in part by National Institutes of Health grant GM38549.
I :ferences
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