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J. Phys. Chem. 1984, 88, 3946-3949
and the amplitude cancellation at zero time is even more evident. In Figure 2b negative amplitudes at low frequencies are still evident and a second positive component is visible in the major peak. Computer simulations gave good fits to curves a and b of Figure 2 using positive components at 3.0 and 3.5 H z and a negative component at 1.2 Hz, and with appropriate scaling of the frequencies also fit curves a and b in Figure 1. An ELS experiment was performed by switching off the writing laser and connecting the PMT output to the spectrum analyzer. One major peak was observed, and the shift was identical with that found for the major positive component in the E H R S experiment. The velocities of the components were obtained with the relation ui = 2a(frequency shift),/K where the magnitude of K was 1738 and 1763 cm-’ for the measurements shown in Figures 1 and 2, respectively. The mobility for the ith component is defined as vi/Edcwhere Edcis the amplitude of the applied electric field. The mobility associated with the large positive component is (2.6 f 0.1) X cm2/(V.s) at 20.0 OC from both our EHRS and ELS measurements, and the values for other components scale according to their frequency shifts. For comparison,*theliterature value of the mobility of bovine serum albumin (BSA) at pH 9.55
is 2.5 X lo4 cm2/(V.s).12 The identities of the minor components which are detected by EHRS have not been established. Also, the possible effects of photoexcitation of the label on the conformation or state of aggregation of the HSA remain to be determined. It is clear that the time decay in Figure l a is not sufficient to characterize the sample and that FT or other computer analysis methods are required. Also, we emphasize that the signal amplitudes and phases depend on the labels and their environments rather than the Rayleigh ratios of the various species. Therefore, small labeled molecules can be observed in the presence of large molecules. This makes chemical exchange processes involving labels or labeled species accessible to EHRS while being invisible to ELS. Another application of considerable interest is the measurement of mobility of labeled particles in gels.
Acknowledgment. This work was supported in part under National Science Foundation Grant CHE-8317243 to C.S.J. and a Whitaker Foundation Grant to D.A.G. (12) Ware, B.; Flygare, W. H. J . Colloid Interface Sci. 1972, 39, 670-5.
Electrophoretic Mobility by Electric Field Modulated Forced Rayleigh Scattering Hongdoo Kim, Taihyun Chang, and Hyuk Yu* Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706 (Received: May 14, 1984)
By effecting the forced Rayleigh scattering measurements under an electric field with charged molecules that are either photochromic by themselves or labeled with photochromic dyes, we show here the electrophoreticmobilities as well as translational diffusion coefficients of such molecules can be determined under the identical conditions. To demonstrate how the technique works, we have chosen a photochromic azophenol dye dissolved in aqueous solutions at pH 6.9 and 10.7, and bovine serum albumin (BSA), labeled with an azobenzene moiety, at different pHs. For the labeled BSA the isoelectric point is determined as pH 4.5-4.7 from the observed pH profile of the mobility which is in accord with pH 4.7 reported in the literature.
Introduction The method of forced Rayleigh scattering (FRS) has been used to study various condensed-phase transport phenomena, notably and mass diffusion^.^^ In the latter case, one requires either photochromic or photobleachable labels on the diffusing species of interest. If the species are charged, then the method can further be tailored to determine the drift velocity under an electric field, and thus to deduce the electrophoretic mobility. The purpose of this letter is to demonstrate how this can be done. We show how the FRS technique is “retooled” by imposing a transient optical grating and an electric field at the same time so that we can probe the field-induced phase modulation of the decay profile of diffraction signal arising from the grating erasure and its lateral shift due to mass diffusion and electrophoretic drift, respectively. We use two specific systems in different media to show that electrophoretic mobility p as well as the translational diffusion coefficient D of the labeled species can be determined by this method, which we call electric field modulated forced Rayleigh (1) Pohl, D. W.; Schwarz, S. E.; Irniger, V. Phys. Reu. Lett. 1973, 31, 32. (2) Eichler, H.; Salje, G.; Stahl, H. J . Appl. Phys. 1973, 44, 5383. (3) Cowen, J. A.; Allain, C.; Lallemand, P. J. Phys. (Paris) Lett. 1976, 37, 313. (4) Chan, W. K.; Pershan, P. S.Biophys. J . 1978, 23, 427. (5) Ltger, L.; Hervet, H.; Rondelez, F. Macromolecules 1981, 14, 1732. (6) Coutandin, J.; Sillescu, H.; Voelkel, R. Makromol. Chem. Rapid Commun. 1982, 3, 649. Antonietti, M.; Coutandin, J.; Griitter, R.; Sillescu, H. Macromolecules 1984, 17, 798. (7) Chang, T.; Yu, H. Macromolecules 1984, 17, 115. (8) Wesson, J. A.; Noh, I.; Kitano, T.; Yu, H. Macromolecules 1984, 17, 782. (9) Rhee, K. W.; Gabriel, D. A.; Johnson, C. S., Jr. J . Phys. Chem., in press.
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scattering (EFM-FRS) because the phase modulation of FRS signal is induced by an applied electric field. The technique differs from electrophoretic light scattering l o (EPLS) in two distinct aspects. Since EFM-FRS is a technique monitoring the specifically labeled species, it can focus on such in the presence of various other charged moieties whereas EPLS monitors all charged scatterers in the sample. Thus, in the first place, EFM-FRS can probe the mobility of small molecules which do not scatter much of the light because of their small sizes so that EPLS cannot be used to determine their mobilities. More importantly, EFM-FRS can tolerate the presence of other scatterers by virtue of its being a tracer method such that p and D of labeled species dispersed in gels and concentrated polymer solutions can be probed, which renders this technique rather distinct from EPLS. We emphasize that this method can determine p absolutely as with EPLS, not relative to any standard as in the case of the more customary macroscopic method, Le., zone electrophoresis.” Experimental Section Materials. A photochromic azophenol dye, 2’,5-dichloro-4hydroxy-3-(N,N-dimethylsulfamoyl)-5’[N-(3-hydroxypheny1)sulfamoyl]azobenzene (I), has been used as an example of the small molecule test system. It has been supplied by Kodak and used without further purification. Also, bovine serum albumin (BSA, Pentex Brand Monomer Standard, Miles Laboratories) as an example of a globular macromolecule has been used after (10) Ware, B. R.; Flygare, W. H. Chem. Phys. Lett. 1971, 12, 81. Ware, B. R. In “The Application of Laser Light Scattering to the Study of Biological Motion”; Earnshaw, J. C.; Steer, M. W., Eds., NATO Advanced Study Institute Series; Plenum Press: New York, 1983. (11) Shaw, D. J. “Electrophoresis”; Academic Press: New York, 1967.
0 1984 American Chemical Society
Letters
The Journal of Physical Chemistry, Vol. 88. No. 18, 1984 3947 electrode
A I
M H
c1
electrodes
I
labeling it with p-phenylazophenyl isothiocyanate (Pierce); a 5% solution of BSA in 8 m M phosphate buffer at pH 8 was mixed with a small volume (typically 1/100 of the protein solution) of the dye in acetone, which came to about a 2:l molar ratio of dye to protein, and incubated for 50 h at 4 O C . The labeling yield was found as about 1:l (dye:protein) from the amount of unreacted dye which remained insoluble in the reaction mixture. After passing it through a Sephadex (G-25 fine; Pharmacia) column, it was confirmed that no free dye was present in the supernatant. Method. The FRS instrument used here has been described elsewhere* and the diffusion coefficients of the dye and BSA have been determined by the standard FRS technique using a 2-mm path length spectrophotometer cuvette as the sample cell. For the EFM-FRS technique, a special cell was constructed, which has two compartments, one (A) having a pair of platinum plate electrodes and the other (B) without electrodes, separated by an optically transparent thin electrical insulator; a microscope cover slide glass is used for this purpose. The spacer electrode holder for A and spacer for B are both made of a KEL-F sheet and a 5-mm path length spectrophotometer cuvette serves as the cell body. A schematic drawing of the cell is shown in Figure 1A, where the three components of the cell assembly are displayed in the top part and a top view of the assembled cell with appropriate dimensions is drawn in the bottom. In Figure lB, an expanded top view of the cell assembly around the incident beam spot is shown with all beam axes identified, the transient optical gratings in the both compartments schematically illustrated (not to scale), and the direction of drift velocity of the transient optical grating in compartment A indicated by a large arrow. A given sample is filled in both compartments, A and B, and a dc electric field is applied to the electrodes (A) prior to the pulsing of writing beams from an argon ion laser (488.0 nm) to impose a transient optical grating while the reading beam (He/Ne laser) is turned on beforehand. The phase-modulated diffraction signal is acquired on a transient recorder (Biomation Model 805) and the data set is transferred to an Apple I1 computer before the next data set either at the same electric field strength or at another field strength. Normally, we effect the FRS measurements first (without the applied field to A), followed by EFM-FRS measurements. The ranges of the field strength E applied to A were 90-180 V/cm for the azophenol dye and 15-80 V/cm for BSA. Since the diffraction signal decays exponentially and the lifetime of photochromically excited state is long compared to the relaxation time of the diffusion process responsible for the transient grating erasure, we can represent the diffraction signal decay in FRS as5
P(t)= (Ae-t/r + B)2 + CZ
(1)
where A is the optical field amplitude of the diffraction signal, T the relaxation time of the optical field, B the coherent background, and C the incoherent background. The phase-modulated decay profile can, on the other hand, be represented by where ud is the uniform drift velocity of the charged label under the applied E, d is the fringe spacing of the grating, and all others have the same meanings as in eq 1 whereas they are distinguished by primes except 7 . The uniformly drifting grating in compartment A while being erased at the same time traverses one full
s p a c e r - e l e c t r o d e microscope holder cover g l a s s
(KEL-F)
spacer (KEL-F)
\A
2mm
Top v i e w o f o s s e m b l e d c e l l
'reading diffracted beam
B Transient optical grating ( n o t to s c a l e )
I I
Compartment B +Microscope cover glass Compartment A (electrodes not shown)
Drift Direction
u
writing beams
Figure 1. (A) Schematic diagram of EFM-FRS cell. (B) An expanded top view of the beam spot.
fringe spacing relative to the nondrifting grating in compartment B in a time t , t, = d / U d = d / p E (3) where p is the electrophoretic mobility of the charged label in the limit of linear dependence of vd on E. Thus, a period of modulation in an EFM-FRS signal is just the fringe transit time rp. By examining the E dependence of t,, one can easily determine the electrophoretic mobility p by plotting 1/tp against E according to l/tp = (cL/d)E
(3')
Results and Discussion In Figure 2 we show the results for azophenol dye (I). In the inset, we display two EFM-FRS signals acquired at pH 10.7 and 6.9, both at electric field strength of 176 V/cm. It shows clearly the pH dependence of the drift velocity which should arise from a difference in the extent of dissociation of the phenol groups at the two pHs. The E dependence of the modulation period t , is plotted according to eq 3' thereby the slope is just the ratio, p/d. Given the fringe spacing d = 56.5 pm, we obtain p = (4.5 f 1.5) X cm2 V-' s-l at pH 6.9 and (6.6 h 0.6) X cm2 V-' s-l at pH 10.7 where the uncertainties represent 95% confidence intervals. Next, we turn to the results obtained for BSA. First, we show in Figure 3A both FRS signal V ' ( t ) and EFM-FRS signal V ( t ) overlaid on top of each other after appropriately scaling the two. In the inset, we display the ratio V ( t ) / V ' ( t )which accentuates
3948
The Journal of Physical Chemistry, Vol. 88, No. 18, 1984
f
Letters
I
I
1
1
I
1
0.8
0
1.6
t/S B
I
I
I
100
150
200
E / V * cm-' 'z
Figure 2. Electric field dependence of the reciprocal modulation period for the azophenol dye at pH 6.9 (unfilled circles) and pH 10.7 (filled circles) both at the grating spacing d = 56.5 pm. Solid lines are the least-squares fits to eq 3', and the electrophoretic mobilities are deterand 6.6 X cm2 V-I s-' at pH mined from the slopes as 4.5 X 6.9 and 10.7, respectively, at 24 ' C . Two EFM-FRS signal profiles of the dye at the two pHs, both at 176 V cm-I, are displayed in the inset.
the modulation profile upon normalizing by the FRS decay profile V'(t). The data shown here are the results of four accumulations for p(t)and of a single run for V ( t ) ,which show the general quality of the EFM-FRS signals that we have been able to obtain in this work. It should be noted that the profile V ( t ) does not precisely conform to eq 2 because there is a slight drift in the baseline as noted by all four minima not quite coinciding to the pretrigger value. In addition, the two baseline parameters, B and C, in p(t)are not the same as the corresponding ones, B' and C', in V(t)so that the damped and asymmetric profile of the ratio V ( t ) / V ' ( t )is entirely to be expected. In Figure 3B are displayed the observed dependences of 1/ t pon the applied field E at different pHs. The progression of decreasing p so obtained is summarized in Figure 3C. The negative sign of the BSA electrophoretic mobility at high pH is fixed to be consistent with the anionic surface charges on any globular protein at such pHs. Once the sign at pH 10.5 is fixed, the positive sign at lower pH emerges naturally without knowing specifically where the net charge reversal from anionic to cationic takes place. The error bars on the data points given by unfilled circles represent two standard deviations of the slope determination in the l / t , vs. E plots. The two data points given by filled circles, however, were obtained only at a given E and the error estimates were made by several repeated runs at the same E. For comparison, we show the result of a potentiometric titration of BSA by Tanford et al.'* obtained for 30 m M ionic strength, who found the isoionic point of 5.5. We should emphasize that no rescaling or adjustment of any sort is made in the above comparison except for fixing of the negative sign of p at high pH. From the pH dependence of p, we surmise that the isoelectric point of BSA is about 4.5-4.7 which is in excellent agreement of the value obtained by other methods.13 Having thus concluded the presentation of the results, we close this Letter with several observations. First of all, the key to successful implementation of EFM-FRS is the double cell arrangement which makes use of the reference diffraction signal of compartment B. At the same time there are a number of questions raised with regard to this cell configuration, particularly (12) Tanford, C.; Swanson, S. A.; Shore, W. S . J. Am. Chem. SOC.1955, 77, 6414. (13) Aoki, K.; Foster, J. F. J . Am. Chem.SOC.1957, 79, 3385.
+ 8Q.
/
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T i t r a t i o n Charge vs. pH
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-
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.-CI 0 U
PH Figure 3. (A) FRS and EFM-FRS signals of BSA (0.5% solution) at pH 6.4and E = 41.65 V/cm, designated respectively as P(t)and V(t),are is displayed overlaid for comparison. In the inset, the ratio V(t)/P(ct) in order to accentuate the modulation profile. (B) Reciprocal of the modulation period 1 /tp at d = 51.6 pm vs. applied electric field E of BSA at four different pHs. (C) pH dependence of the mobility p of BSA determined by EFM-FRS as shown in B. All the measurements were performed with 5 mg/mL of solution of BSA in 2 mM ionic strength at 24 O C . For comparison the result of potentiometric titration of BSA by Tanford et al.lz is shown in the same plot without any adjustment.
in compartment A. For instance, the drift velocity profile relative to the distance from the cell face to the optical partition (cover slide) must have an effect of smearing the predicted harmonic modulation profile as given by eq 2. The observed EFM-FRS signals in Figures 2 and 3A must therefore represent an average yet to be fully specified of the drift velocity across the penetration depth of compartment A. At the same time, we have no way of assessing the electroosmosis effect14 in compartment A which must have taken place, giving rise to the depression of the velocity profile. Despite these misgivings, it appears clear that EFM-FRS signals give rise to the correct p values for BSA as determined (14) Adarnson, A. W. "Physical Chemistry of Surfaces"; Wiley-Interscience: New York, 1976; 3rd ed, p 211.
J. Phys. Chem. 1984, 88, 3949-3951 by EPLS.I5 Whether the agreement is due to fortuitous circumstances of possible mutual cancellation of the two effects remains to be examined by use of different cell configurations. Our main motivation for developing EFM-FRS is to study p and D of protein-surfactant complexes and nucleic acids in various viscous media including polyacrylamide and agarose gels which (15) Caflisch, G . B.; Norisuye, T.; Yu, H. J . Colloid Interface Sci. 1980, 76, 174.
3949
will constitute the principal subject of our next communication.
Acknowledgment. Acknowledgment is made to the donors of the Research Fund, administered by the American Chemical Society, for partial support of this research. This is also in part supported by a research grant from Eastman Kodak Company and by an N I H Grant EY01483. We are most grateful to Dr. Thomas H. Whitesides of Kodak for the generous gift of the azophenol dye, and thank our colleague, Daniel R *Spi%el, for the FRS measurements of the dye.
Dynamics of Multiphoton Ionization-Dissociation of 2,4-Hexadiyne by the Two-Color Picosecond Pump-Pump Mass Spectrometric Technique: Formation of C6H5+,C4H4+, and C4H,+ Ions D. A. Gobeli,+ J. D. Simon, and M. A. El-Sayed* Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90024 (Received: June 8, 1984) The two-color picosecond pump-pump mass spectrometric technique is used to determine the mechanism of formation of C&+, C4H4+,and C4H3+from 2,4-hexadiyne by the multiphoton ionization dissociation process. The results suggest that, under our experimental conditions of laser power, pulse width, and wavelength, fragmentation is occurring directly from the parent ion. This evidence of ionic ladder climbing is supported by the two-color effect and the interrelation between the characteristic times observed for the dynamical processes involved in the formation of the C4H3+and C4H4+ions. The apparent biphasic behavior of the C6HS+mass peak as a function of the relative delay between the two laser pulses is explained in terms of the involvement of two different electronic states of the parent ion, one at 14 eV (three 266-nm photons) and the other at 18.66 eV (four 266-nm photons) to produce two different ionic species with the formula C6H5+.
Introduction In a recent publication1 we demonstrated that rapid energy redistribution or dissociation processes which occur during the multiphoton ionization-dissociation process can be studied effectively using a time-resolved picosecond pumppump laser mass spectrometric technique. Using this technique, we are able to directly observe rates of rapid ( 10'2-109 s-l) dynamical processes involved in the mechanism of dissociation that are not detectable by conventional mass spectrometric techniques?~~ which are limited s). by the analysis time Our previous report centered on energy redistribution in the parent ion of 2,4-hexadiyne prior to the formation of C4H4+,a low-energy daughter ion fragment.' The appearance potential of this daughter can be exceeded through the absorption of three 266-nm photons (one-photon resonant, two-photon ionization followed by the absorption of an additional photon to the first excited state of the molecular ion, see Figure 1). Upon threephoton excitation, the molecular ion would contain approximately 5 eV of excess internal energy. In addition to C4H4+,appearance potential data indicate that fragmentation to form C6H5+,C6H4+, C5H3+,and C3H3+daughter fragments could occur from this le~el.~,~ Our results' on C4H4+indicate that energy redistribution from within the first excited state of the molecular ion occurs in approximately 20 ns. This result is consistent with the fluorescence quantum yield and lifetime of the first electronic state of the 2,4-hexadiyne parent ion.6 This supports the contention that Present address: Rockwell Science Center, Thousand Oaks, CA 91360. (1) Gobeli, D. A,; Morgan, J. R.; St. Pierre, R. J.; El-Sayed, M. A. J. Phys. Chem. 1984,88, 178. (2) Proch, D.; Rider, D. M.; Zare, R. N. Chem. Phys. Lett. 1981,81,430. (3) Hipple, J. A. Phys. Rev. 1947, 71, 594. (4) Momingy, J.; Brakier, M. L.; D'Or, L. Bull. Cl. Sci. Acad. R . Belg. 1962, 48, 1002. (5) Dannacher, J. Chem. Phys. 1978, 29, 339.
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C4H4+is produced as a result of the absorption of three UV (266 nm) laser photons by the parent molecule. In the present Letter we examine the mechanism of the formation of the C6H5+ and C4H3+daughters. The results suggest that their formation as well as that of C4H4+can be described in terms of a ladder, rather than a ladder-switching mechanism. Furthermore, the presence of more than one characteristic time for the energy redistribution leading to the formation of C6H5+ could suggest the presence of more than one chemical species with the same mass/charge ratio. These two species are produced from two different electronic states of the parent ion, one at the three UV photon and one at the four UV photon level, each with different energy redistribution times.
Experimental Section The second (532 nm, 0.5 mJ/pulse) and fourth (266 nm, 2-10 kJ/pulse) harmonic of a passively mode-locked Nd:YAG laser (Quantel YG400) provided pulses of approximately 25 ps (fwhm). The two pulses were separated by means of a Pellin-Broca prism. After the 532-nm pulse traveled a variable delay time (0-10000 ps) the two beams were recombined and focussed into the ionization region of a differential pumped time-of-flight mass spectrometer constructed in our laboratory. The laser intensity was adjusted so that the visible pulse alone did not result in the formation of any ion. The ion current was detected with a Channeltron electron multiplier (Galileo Electrooptics 4800 series), amplified with a video amplifier (Pacific Precision Instruments), and analyzed with a boxcar integrator (PAR Model 4420,4422) and signal processor (PAR Model 4402). The experiment involved recording the ion current for a particular mass peak as a function of the delay time between the 266- and 532-nm laser pulses. In (6) Allan, M.; Maier, J. P.; Marthaler, 0.; Kloster-Jensen, E. Chem. Phys. 1978, 29, 331.
0 1984 American Chemical Society
