Volume 64. number 3
`
`CHEMICAL PHYSICS LETTERS
`
`15 July 1979
`
`DYNAMICS OF MOLECULAR OXYGEN
`
`IN MICELLAR SOLUTIONS
`
`Nicholas J. TURRO, Masayuki AIKAWA
`CBern&ry Department. Columbia Universit_v. New York. Sew York IOO27. USA
`
`and
`
`Ahmad YEKTA
`Chemistry Department. Arya-Mehr Uni&esrity of TecImolog_v, Tehran. Iran
`
`Received 23 March 1979
`
`FIuorescence quenching by moIecuhr oxygen has been employed to estimate dynamic parameters and solubiIity charuac-
`teristics of molecular oxygen in miceile forming detergent solutions. A kinetic model which assumes that oubgen quenching
`occurs only in the micelIar phaz is employed to analyze the data.
`
`1_ Introduction
`
`hlolecular oxygen is a general and effective quencher
`of the fluorescence of aromatic hydrocarbons
`[l] _ The
`quenching process is commonly diffusion controlled
`in
`homogeneous
`solution, requiring that quenching within
`the “solvent cage” [2] occurs with an efficiency close
`to unity. In two (or multiphase) pseudo-homogeneous
`aqueous solutions (micelle containing detergent solu-
`tions, solutions containing protein membranes and
`other macro biomolecules)
`the observed efficiency and
`reactivity of oxygen quenching will depend on the par-
`titioning of molecular oxygen in the various phases, the
`lifetimes of the excited quencher and the dynamics of
`entrance, exit and quenching interaction of the oxygen
`molecuIe in an “effective quenching volume”
`[3] about
`
`(1) can be very effective if the oxygen molecuIe and
`quencher are “pre-complexed”
`and confined to a small
`volume in a given phase or (2) can become very inef-
`ficient if the quencher and the moIecular oxygen are in
`separate phases and volumes of space and *the rate of
`penetration of oxygen into an “effective quenching
`volume” is slow compared
`to the decay rate of the ex-
`cited quencher_
`fluorescence
`Previous work on aromatic hydrocarbon
`quenching by molecular oxygen in aqueous solutions
`
`in part because
`has been limited in scope, presumably
`of the low solubility of aromatic hydrocarbons
`and of
`molecular oxygen in aqueous solutions_ The maximum
`concentration
`of aromatic hydrocarbons
`attainable in
`micelie containing aqueous solutions of detergents is
`much greater than that attainable in water. The oxygen
`concentration may be enhanced by simply increasing
`the pressure of oxygen in the system
`[4] _
`Evidence has been presented that molecular oxygen
`readily penetrates micelle [5] * and membrane
`[7]
`boundaries_ The mechanism of penetration of molecular
`oxygen across such boundaries is potentially of great
`significance for important biolo@cal processes such as
`respiration and photodynamic
`effects
`[8] _
`The “observed”
`rate consiant for quenching of fluo-
`rescent probes by molecular oxygen
`in solutions con- taining proteins [9], DNA [lo], micelles [5,6] and
`vesicles [7] is smaller than that observed for the same
`probes in homogeneous
`solution_ These results have
`been interpreted
`in terms of a smaller “‘effective” con-
`centration of oxygen in the phase or aggregates con-
`taining the probe, in terms of a smaller rate constant
`for quenching interaction or some combination of both
`factor,. The ability of association
`to “protect” excited
`
`* It slso appears that singlet oxygen can readily penetrate mi-
`celles: see ref. [6] _
`
`473
`
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`Page 1
`
`the quencher_ Thus, in extreme situations quenching
`

`

`Volume 64. number 3
`
`CHEMICAL
`
`PHYSICS LEl-l-ERS
`
`0 O*
`
`probes from oxygen quenching is strikingly demon-
`strated by the ready observation of phosphorescence
`from organic moIecules in micelle containing detergent
`sohrtions [ 1 I] _
`Information on the solubiIization, entry and exit of
`moIecular oxygen into and out of micelles produced in
`detergent solution is potentially available from an in-
`vestigation of the time resolved measurement of fluores-
`cence Iife*tie of a micellized probe as a function of
`oxygen pressure_ We repor? here a study of the oxygen
`quenching of I J-dimetiryioaphtbalene fluorescence in
`micelIe containing detergent soIutions of hexadecyltri-
`methyI-ammonium bromide (HDTBr) and sodium
`dodecji sulfate (SDS)_ A kinetic analysis of our data
`aIIows an estimation of dynamic and sohrbility param-
`eters of molecular oxygen in this “two phase” system.
`
`2_ Experimental strategy arrd kinetic model
`
`The experimental method employed involves time
`correlated single photon counting 1121 measurement
`of the ffuorescence decay of the probe as a function of
`oxygen pressure [Y,lO]
`At low partial pressure (< 1
`atm) of oxygen, the observed fluorescence decay (Eg.
`Ia) is exponentid within experimental error and equal
`to that of the ffuorescence decay of the probe in
`thoroughIy degassed solution (fig_ lb)_ However, at
`higher pressures of oxygen e
`I atm) the fluorescence
`decay (figs. I c, 1 d and I e) is initially non-exponential
`(“short time decay”. r-J but eventuahy becomes ex-
`ponentird (“long time decay”, re). Significantly, the
`“long time decay” is simrter tluuz the decaJ7 in degassed
`solution_ We interpret the short time decay in terms of
`“static” fhrorescence quenching in micelles that contain
`both an oxygen moiecule and an excited probe moIe-
`cute at the end of the excitation pulse
`inter-
`pret the long time decay in terms of “dynamic” fluores-
`cence quenching that occurs at the end of the excita-
`tion puIse (t = 0) that is due to diffusion of oxygen
`molecules (InitiaIIy located in the aqueous phase) into
`a micelIe containing an excited probe moIecuIe.
`We handIe the experimental data by employing
`scheme 1 as a model. This mode1 assumes that a com-
`bination of static and dynamic quenching of probe fhro-
`rescence operates_ We relate the experimental data to
`the following mice&n parameters [4] I (O,), the aver-
`age occupancy number of oxygen moIecuIes in a micehe
`
`474
`
`15 July 1979
`
`•t
`
`0 0: Q, kt +Q, C 0 0.
`
`Q,
`
`Scheme 1. Schematic representation of the model for quenching
`of a luminescent probe by a quencher which is present in both
`aqueous (~xJ) and mice&r (Qhl) phases_
`
`(based on Poisson statistics); CY, the efficrency of quench-
`ing of the excited probe by an oxygen moIecuIe once
`it is in a micehe; kt, the rate of entry of oxygen into
`the miceile; and, kb, the rate of exit of oxygen from
`the micelIe by the following expression [ 131:
`
`In [r(t)lr(O)] = -(t/r
`
`f (O&Y2)
`
`+
`
`exp [-(k, + Qt]
`
`is known, then this equation can be solved for
`If (0,)
`a and (k, i- kI,)_ Knowledge of these quantities then al-
`
`_ (1)
`
`Fig_ 1. Fhrorescence decay modes of I.5CBIN under various
`02 pressures. 02 pressures are: (a) degassed (IO- Torr);
`(b>
`0.2; (c) 1.36; (d) 2-72; (e) 5.44 atm. respectiveIy_ Curve (f) re-
`pxsents the shape of D2 fIxI& lamp (half width is = 2.1 ns).
`
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`
`_
`(t = 0); we
`Q,
`k,
`(O,h”
`

`

`Volume 64, number 3
`
`CHEMICAL PHYSICS LETTERS
`
`lows evaluation of the individual rate constants k, k,,
`and
`From eq. (1) in the long time domain one obtains
`
`iln
`
`P(Nl(o)l 3t4_ = -(f/T + (O,hY’), (2) so
`that for this Iimit the slope of a plot of eq. (2) fur-
`nishes the fluorescence
`lifetime T in the presence of dif-
`fusional quenching and extrapolation
`to the intercept
`from long time to t = 0 yields <02>~z.
`versus oxygen
`The plots of the latter values of (02)o?
`pressure PO, are shown in fig. 2. The good linearity of
`the plot validates the assumption
`that ln [r(t)lr(O)]
`is
`proportional
`to <02)_ The values of (02)
`for HDTBr and
`SDS micelies as a function of PO2 were evaluated from
`solubility measurements of Matheson and King [4] _
`These authors demonstrated experimentally
`that the
`concentration of micellized oxygen
`[Oz] mie is given by
`
`[O?] mic =a( [HDTBr]
`
`-
`
`CMC)Po2, (3
`
`for (HDTBr) and 1.04 X
`where a = I.4 X IO” atm-’
`lo9
`atm-l
`for (SDS)_ The relationship between the
`average micelle aggregation number E and the total mi-
`celle concentration,
`[MI, alows eq. (3) to be rewritten
`as
`
`since (&) = [02] mic/
`Furthermore,
`the concentration
`of micelles, we have
`
`where
`
`is
`
`(02) =
`
`(5)
`
`so that the slope of the plot in fig_ 2 is identified as
`c&ii. From the reported values [ 14 ] of ii the repre-
`sentative values E = 80 for HDTBr and ii = 60 for SDS.
`
`Fig. 2. A plot of<Oz?rrf against 02 pressure for HDTBr and
`SDS mice&u solution_ The slape gives the Lalue of a’&
`
`We derive a v&e of az = 0.89 f 0.15 (HDTBr) and
`0.80 2 0.17
`(SDS).
`Rearrangement of eq. (1) yields
`
`r
`b
`I + In [z@)/z(o)l + ‘IT = exp [_(k (O,>cY’ + II_ )t]
`
`The logarithmic plots of the above function yield k, +
`f 0.7) X
`kb = (4.6 t- 0.5) X lo7 s-1 (HDTBr) and (53
`lo7 S-I (SDS) and are independent of pressure. Our
`results place an upper limit to the sum
`and
`ako limit the values of the individual rate constants
`(table 1). However, since the experimental error and
`scatter in reported values [ 141 of ii in determining Q
`is estimated
`to be =20%,
`ev
`ation of the values of
`
`Table 1
`Summary of parameters derived from fluorescence quenching in micellar solutions
`
`Parameter
`
`----.
`
`--
`
`entrance rate constant, X-f
`evit rate constant, kb
`residence time. rm
`“static quenching constant, Ic,
`quenching efficiency, P
`equiliirium constant.
`Keq = kflkb
`----
`
`_-
`
`MicelIes
`
`HDTBr
`
`SDS
`
`-
`
`s-1
`
`(1.3 * 0.2) x 10’0 w
`< (4.6 f OS) x 10’ s-’
`<22x
`10-s
`< (4.6 f 0.5) x IO7 s-’
`0.89 t 0.15
`(2.8 f 0.3) x 10’ M-’
`
`(IA 2 0.2) x 1o’O H’ s-1
`< (5.3 f 0.7) x IO7 s-r
`c 19 x lo”, 5
`< (5.3 + 0.7) x IO7 s-’
`0.80 f 0.17
`(2.6 + 0.34) x IO2 M-t
`
`___
`
`-
`
`475
`
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`IPR2019-00685, IPR2019-00688, IPR2019-00694
`Page 3
`
`k,.
`[M]
`[hi ]
`EPo* ,
`PO, tafml-
`’
`k, f k,,
`

`

`VoIume 64. number 3
`
`CHEMICAL
`
`PHYSICS LETI-ERS
`
`15 July 1979
`
`3- Discussion: comparison with related published data
`
`The question arises as to whether the numbers derive1
`rrom our analysis are reasonable and consistent with
`-he magnitudes of analogous parameters derived from
`h>ther systems_
`The value of the entrance rate constants 13 X lOlo
`M-1 s-1 (HDTBr) and 1.4 X lOlo M-1 s-1 (SDS) are
`comparable
`to those derived from fluorescence quench-
`ing by oxygen_ For example, values of 8.3 X 109 M-l
`s-r (HDTBr) and 9.2 X IO9 M-t s-l (SDS) were re-
`ported for the total “bulk” oxygen quenching of pyrene
`fluorescence_ The long inherent lifetime of
`monomer
`pyrene fluorescence
`(= 200 ns in HDTBr and -450
`ns
`in SDS, respectively) probably allows oxygen escape
`from the micelle to be competitive with oxygen quench-
`ing; therefore,
`the observed quenching constant
`is some-
`what lower than that for diffusion of oxygen into the
`micelIe_
`(quench
`for interaction
`The value of the rate constant
`ing) of excited 1,5dimethyl
`naphthalene
`is expected
`to
`be of the order of that for intramicellar encounter of an
`oxygen molecule and an excited naphthalene molecule_
`From the values of the reported microviscosities of
`HDTBr and SDS micelIes (39 and 9 cP, r zpectively)
`[ 1.51, a micelle radius of = 20 A and an effective radius
`of the probe of -3.0
`a, a value of (l-5)
`X lo7 s-* is
`calculated
`from diffusion theory for the encounter
`rate
`[ 163 _ An experimental value of z IO7 s-l has been
`evaluated for the intmmicellar encounter of two pyrene
`molecules in an anionic micelle [ 17]_ Finally, a value
`of = 6 X IO7 s-1 has been evaluated for the rate of in-
`tramolecular excimer formation of 1,3-diiu-naphthyl-
`propane in HDTBr micelles [18] _ All these data suggest
`micellar interaction
`rate constants of the order of I&‘-
`IO8 s-r for the systems studied. Our derived value of
`< 4.6 X lo7 s-r or s 53 X IO7 s-* is comparable
`to
`literature estimates_
`The magnitude of (Y = 1 is consistent with results
`from homogeneous
`solution where O2 is found to quencl
`the fluorescence of aromatic hydrocarbons
`at close to
`the diffusional Iimit.
`Finally, the limit of Krs of% 3 X IO2 M-l
`implies
`a higher solubility of 0,
`in the micellar phase relative
`to the aqueous phase (& a molar basis). This result is
`consistent with the observation
`that 0,
`is generally an
`order of magnitude more soluble in organic solvents
`than it is in water [S-7] _ The comparable value of Kes
`
`Fig_ 3. A piot of r& e,painst G-, pressure for ZiDTBr and SDS
`micelbr sohtion The sIope givivrs kqro_
`_
`
`is subject to a large inaccuracy_
`individual rate constants
`Nevertheless the limiting residence time of O7 in HDTBr
`and SDS micelles is >200 X 10-g and > 9 1 X 10-g s.
`respectively_ These correspond
`to limiting exit rates of
`<4_6 X lo7 s-l and < 53 X IO7 s-* for HDTBr and
`SDS. respeetively-
`The relation between the diffirsional limited fluores-
`cence lifetime and the oxygen concentration in the
`water phase is given by [IO,131
`
`7’
`
`=ii' +kf[OZ]w_ (7)
`
`that [O,]
`
`,~
`
`Since Matheson and King [4] demonstrated
`= 139 X 10m3 Po2 we have
`i-i =,-I o
`
`x 10-3)P,2_
`
`tqi39
`
`(S)
`Fig. 3 shows a plot of To/r versus PO., for the HDTBr
`and SDS systems The siopes yield th; forward rate
`constant of oxygen associrttion with the micelie kt =
`13 X IOr M-t s-l (HDTBr) and 1.4 X IO*O M-t s-l
`(SDS)
`Unlike the values of zib and II-,, upon which we can
`only place limits, the values of Xrt are available within
`a precision of = 20%_ Knowledge of the vahres of kf
`together with the Iimiting values of kb allow the esti-
`mation of a limiting value of KW ss k#b:
`
`KJHDTBr)
`
`2 2.8 X 10’
`
`KJSDS)
`
`Z 26 X 10” hi-’ _
`
`(9)
`
`(10)
`
`476.
`
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`
`M-’ ,
`

`

`Voiume 64. number 3
`
`CHEMICAL PHYSICS LETTERS
`
`15 July 1979
`
`for HDTBr and SDS is consistent with a recent investi-
`gation of the oxygen quenching of naphthalene
`fluores-
`cence in aqueous solutions of these two detergents
`[19].
`
`4. Experimental
`
`HDTBr and SDS were washed several times with
`ether and recrystallized
`from water three times. DMN
`was purified by sublimation
`in vacua. Oxygen
`(Airco,
`Inc.) and 40% oxygen/nitrogen
`(Matheson Gas Products)
`for calibration or for lower O2 pressure were used_
`Transient decay curves of micellar solution under
`high oxygen pressure were measured using the time-
`correlated single-photon counting
`technique
`[12] _ The
`flash lamp was operated at a hiavoltage
`of 4.5 kV and
`repetition of = IS kHz. Deuterium at l/2 atm pressure
`was used as a flash gas. The gas discharged flash lamp
`gave a pulse with a half-width of about 2.1-2.6
`ns_
`Interference band pass filters (Corion Instruments Corp.)
`were used to select wavelengths from the exciting light.
`These filters transmitted 30% of the incident radiation
`at its wavelength of maximum 3 11.2 or 304.2 nm with
`a 10 nm half-width. Sample fluorescence of appropriate
`wavelength was selected with an American ISA, Inc.
`l/l0 meter monochromator. A RCA photomultiplier
`Inc.
`(C3 1034A02) with refrigerated chamber (Research
`TE-104-RF) was used as the photodetector_
`Fluores-
`cence intensities were adjusted at 350-700
`counts/s_
`The dark noise (adjusted via a discriminator) was about
`15 + 10 counts/s.
`The high pressure apparatus was placed inside of a
`“dark box” and adjusted in the light path of the D,
`lamp. The apparatus consists of a thick-walled
`(1/2-m_)
`cylindrical stainiess steel vessel which has optical quartz
`windows on the three sides and has a brass jacket com-
`bined with-temperature-controlled
`water bath [IO] _
`A ceI1 hoider was mounted on the bottom of the vessel.
`The high pressure vessel rested on a variable magnetic
`stirrer, allowing the agitation by a magnetic stirring
`bar in the cell set in the cell holder to be controlled
`to
`externally_ An inlet line to the vessel was connected
`a gas manifold through
`needle valve allowing the vessel
`to be loaded with the gas desired. A Boudon gauge
`(Matheson), accurate
`to 0.25% of full scale, was used
`to record the loaded pressure. An exit IErie was also
`attached
`to the vessel through a needle vaIve and allows
`the gas to be released_
`
`Before Ioading the vessel with desired pressure, the
`air of the vessel was replaced several times with oxygen
`or the mixture gas by controlling
`the inlet and outlet
`needle valves_ Then the gas was introduced over the
`vessel at the desired pressure, and the sohrtion in the
`cell was allowed to equilibrate with the dense gas for
`a minimum of two hours with stirring_ The stirrer was
`shut off, and the transient decay curve of the solution
`was measured_
`To test the accuracy of the “nominal” pressure ob-
`served in running experiments,
`the rate constant
`for
`quenching of fluorescence of aqueous s?lutions of 1.5
`Dbih’ was measured_ Pressures of a mixture of 02/N2
`(= 40/60) up to 100 lb/in? were employed_ The decay
`of
`fluorescence
`in H,O consisted of a single
`5DMN
`exponential
`throughout
`the range of 0, pressures
`studied_ The solubility of molecular oxygen in water
`was reported as
`
`w,1,\r =bPo*, (11)
`
`Fig. 4 shows
`[4]
`where Lr = 1.39 X 10-j mol ‘2-l atm-l
`the reciprocal of 1.5DMN
`lifetime as a function of
`[01] _ From_ eq. (11) and the slope of fig_ 4, a quench-
`ing rate constant of oxygen in water k, = 1.43 X lOlo
`M-1 s-1 is derived_ This value 1s in good agreement wi*&
`the oxygen quenching rate constant of various fluoro-
`phores in aqueous solutions
`[IO]
`
`_ [oz] I 10-M - 0 IO
`
`I
`
`0
`
`20
`1
`
`30
`I
`
`40
`1
`
`I
`
`Fig- 4. Stem-Volmer plot for quenching of 1,5-DhlN fluores-
`cence in H20 solution as a fuxtion of 02 pressures. Degassed
`(I@ mm Hg) (01, atomospheric condition (01 znd 02/N2
`mixed gas (0).
`
`477
`
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`IPR2019-00685, IPR2019-00688, IPR2019-00694
`Page 5
`
`a
`1
`_
`

`

`VoIume 64. number 3
`
`CHEhfICAL PHYSICS LETTERS
`
`15 July 1979
`
`lW
`
`[131
`
`[8] T_ Ito. Photochem- PhotobioL25 (1977) 47. and referen-
`ces therein_
`[9] \V_hf_ Vaughan and C_ Weber, Biochemistry 9 (1970) 464.
`[IO] J-R- Lakowin
`llnd G. Weber, Biochemistry 12 (1973) 4161
`[ 1 I] N.J. Turro. K C. Liu. M--F_ Chow and P_ Lee. Photochem.
`Photobiot 27 <1978j 523;
`K. Kaiyanasundamm. F. Grieser and J.K. Thomas. Chem.
`Phyr Letters 51 (1977) 501;
`R_ Humphry-Baker, Y_ hforoi and &f_ Grztzel. Chem. Phyr
`Letters 58 (1978) 207_
`W-A. Ware. Creation and detection of the excited state.
`Vol. IA (Dekker, New York, 1971) p_ 213_
`A_ Yekta. hf. A&awn and NJ. Turro. Chem. Phys. Letters
`63 (1979) 543;
`P-P_ Infeita. Chcm. Phys- Letters 61 (1979) 88.
`R-C_ Dorrance and T-F_ Hunter, J. Chem- Sot. Faraday I
`70 (1974) 1572;
`P_ Ekuall, L. hlandell and P. Solyom, J. Colloid Interface
`sci 35 (1971) 519;
`NJ_ Turro and A_ Yekta. J_ Am. Chem. Sot_ 100 (1978)
`5951;
`H_ Coil, J. Phys- Chem. 74 (1970) 520.
`NJ. Turro. hl Aikaxsa and A. Yekta. J_ Am. Chem. Sot-
`lOl(1979) 772;
`J_ Emert. C_ Behrens and l\f. Goldenberg. J. Am. Chem.
`sot. lOl(l979)
`771;
`K. Zachariasse, Chem. Phys. Letters 57 (1978) 429;
`Y_ Kubota. %fM. Kodama and hf. Miura. Bull. Chem. Soc-
`J3pan46 (1973) 100.
`hf. GrZtzei and J-K. Thomas, J. Am. Chem. Sot. 95 (1973)
`6885.
`U_ Khuzm~ B_K_ Selinger and R_ XfcDonald, Australian J.
`Chem_ 29 (1976) I_
`[ 181 &I. Aikitu3 and NJ. Turro. unpublished results. Columbia
`University_
`[ 191 XV_ Bockstaele. J_ Gelan, H. Martens, J_ Put, J.C. Dederen.
`N. Boens and F-C. de Schrijver. Chem. Phys- Letters 58
`(1978) 211.
`
`[I41
`
`[151
`
`[I61
`
`[I71
`
`Acknowledgement
`
`The authors thank the National Institutes of Health
`for their generous support of this work (Grant GM-
`25523)_ A_ Yekta thanks the Arya-Mehr University of
`TechnoIo,T
`and the Iranian Ministry of Sciences for a
`Visiting Research Fellowship during 1977-1978.
`
`References
`
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`[3] F. Penin. Ann_ Phys_ 12 (1929) 169;
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`(1978) 46.
`[S] M-W_ Geiger and NJ_ Turro. Photochem. PhotobioL 22
`(1975) 273;
`SC- WaXace rmd J-K_ Thomas, Rndiat. Res. 54 (1973) 49.
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`[7] S. Cheng. &I_ GrStzeI. J-K_ Thorns and C-F. CuIpa. in:
`Radiation research, eds. G-E_ Adams, bf_ FieIden snd B.
`blichzeels (TayIor and Francis. London. 1973) p_ 193;
`M-W_ Geiger and NJ_ Turro. Photochem- PhotobioL 26
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`
`478
`
`Opiant Exhibit 2314
`Nalox-1 Pharmaceuticals, LLC v. Opiant Pharmaceuticals, Inc.
`IPR2019-00685, IPR2019-00688, IPR2019-00694
`Page 6
`
`