TRIPLET-SINGLET EMISSION IN FLUID SOLUTION - The Journal of

2.506

TRIPLET-SINGLET EMISSION I N FLUID SOLUTION BY C. A. PARKER AND C. G. HATCHARU Royal Naval Scientijic Service, Admiralty Materials Laboratory, HoUon Health, Poole, Dorset, England Receiaed M a y d6, I963

Downloaded by NEW YORK UNIV on September 10, 2015 | http://pubs.acs.org Publication Date: December 1, 1962 | doi: 10.1021/j100818a043

Using a photoelectric spectrophosphorimeter of novel design, the long-lived luminescence from solutions of two dyestuffs and two aromatic hydrocarbons has been invmtigated. Solutions of eosin and proflavine hydrochloride in ethanol or glycerol give rise to two bands of long-lived luminescence which are attributed, respectively, to the triplet-singlet radiative transition and to delayed fluorescence arising from triplet-upper singlet thermal activation. A study of the band intensities aa a function of temperature and solvent can provide information about the probabilities of the three intersystem crossing processes. Triplet-singlet emission has been observed from phenanthrene in ethanol a t room temperature. At lower temperature weak delayed fluorescence also is emitted. Phenanthrene solutions containing trace quantities of anthracene give sensitized anthracene delayed fluorescence. Solutions of pure anthracene also give anthracene delayed fluorescence. The observations of delayed fluorescence from the hydrocarbons are discussed only briefly. They are described in detail elsewhere.

Introduction Some measurements of the phosphorescence of The investigation of molecules in the triplet state eosin solution made with this instrument have been in solution is normally made by the method of flash reported previously.2 It is our purpose here to photolysis. By using an intense flash of sufficiently review these results and to describe some prelimishort duration a relatively high concentration of nary results obtained with other compounds. The Spectrophosphorimeter.-The instrument was built triplet molecules can be produced, their absorption spectrum can be measured, and the kinetics of the round two Hilger D247 quartz prism monochromators, one isolation of the appropriate frequency of exciting light subsequent changes which they undergo can be for (from either a 1 kw. high pressure mercury lamp or a 375 w. followed in detail. The effect of medium and tem- xenon lamp) and the second for analysis of the fluorescence perature upon the rate of the intersystem crossing or phosphorescence emission from the specimen. The detecan E.M.I. 9558 photomultiplier, the output from process from triplet to lower singlet can thus (in tor waaafter amplification, was fed to one arm of a ratio reprinciple a t least) be investigated. Unfortunately, which, corder, the other arm of which was fed from a fluorescent it is dificult to obtain by this method similar in- screen monitors situated in the beam of exciting light. formation about the equally important transition The recorded output thus was automatically compensated from excited singlet state to triplet state because for fluctuationo in light intensity. princi le of the Becquerel phosphoroscope waa used it is difficult to determine precise quantum effi- to The distmguisg between fluorescence and delayed luminesciencies for triplet formation. Information about cence, but the inconvenience of having mechanically the latter has so far been obtained mainly by coupled sectors in the beams of exciting light and luminesmeasurements of the tripletsinglet phosphorescence light waa avoided by arranging for each of the sector to be driven by a synchronous motor. The disks could cence of solutes in rigid media where triplet quench- disks then be put in, or out of, phase by simply turning one of the ing is often small and high phosphorescence effi- motors. With the disks in phase, the detector recorded the ciencies can be observed. The pioneering work of sum of the fluorescence and delayed emission spectra. With Lewis and co-workers’ in this field also showed the disks out of phase, only the delayed luminescence waa observed. I n the latter position the fluorescence light leakthat thermal excitation from triplet to upper singlet age aat the sectors waa less than 1 part in 106. can occur and can be detected by measuring the T i e rate of chopping by the sectors waa 800 c./sec. The “delayed fluorescence’’which results from it. size of the slots in the first sector were adjusted so that the The same method can in principle be applied to specimen was irradiated for 111 of a complete cycle. The waa viewed through the second sector for the investigation of upper singlet-triplet conversion luminescence of a complete cycle. Thus, with the sectors in phase, the and triplet-upper singlet activation in solution. whole of the fluorescence emission was received by the In practice, however, the phosphorescence intensity detector, and with the sectors out of phase of the longis often very low owing to the long radiative life- lived luminescence waa received, assuming that the lifeof the latter waa long compared with the chopping time of most molecules in the triplet state and the time time (1 8~ sec.). For lifetimes comparable with the chopping comparatively high rate of the triplet-lower singlet time, t e long-lived luminescence decayed appreciably before radiationless transition. (The experimental difi- observation. The relationship between the intensity of culties are of course accentuated by the suscepti- luminescence observed and its lifetime is given by the expresbility of the triplet molecules to quenching by sion trace impurities in the solution-particularly oxygen.) A phosphorimeter therefore is required capable of measuring phosphorescence and delayed fluorescence intensities which may be one where thousandth or less of the normal fluorescence inP D = luminescence received in the out-of-phase position tensity from the same solution. To facilitate the P = total luminescence emitted per cycle measurement of such a weak phosphorescence specT = lifetime of luminescence trum and to measure the ratio of its intensity to tl = period of illumination that of the much more intense fluorescence, we te = period of darkness f a = period of viewing through second sector have set up a photoelectric spectrophosphorimeter sec. t, = period of cycle = (tl + t 2 ) = capable of measuring phosphorescence/fluorescence in favorable cases. ratios down to values of

i

(1) G.N. Lewis, D. Lipkin, and T. T. Magel, J . Ana. Cham. Soc., 63’ 3005 (1941).

(2) C. A. Parker and C. G. Hatchard, Trans. Faraday Sac., 57, 1894 (1961). (3) C. A. Parker, M a t w e , 182, 1002 (1958).

TRIPLETSISGLET EMISSTON IN FLUID SOLUTIOS

Dec., 1962

2507

For tl = tc/4 and t3 = t c / 3 , the following relationship between T and PD/Pis obtained 7

(msec.)

pD/p 7

(msec.)


pD/p

55 0.333 0.15 0.070

1 0.316 0.10 0.022

0.5 0.25 0.271 0.165 0.07 0.005

The “resolution time” of the instrument (i.e., the lifetime for which the observed intensity is reduced to one-half of the value correfiponding to infinite lifetime) is 0.25 msec., and for the lifetimes shorter than this, the observed intensity decreases very rapidly. It is feasible to reduce le by a factor of 5 . The corresponding resolution time would then be 0.05 msec. and the scope of the instrument would be greatly increased, although a greater “fluorescence leakage” past the choppers probably would have to be accepted. Long luminescence lifetimes (e.g., those observed with many solutes a t liquid nitrogen temperature) could readily be determined by recording the decay of luminescence (with sectors out of phaae) when the exciting light was cut off. For measuring shorter lifetimes down to 0.5 msec. the 800 c./sec. sectors were replaced by 100 c./sec. sectors. The photomultiplier output was fed to an oscilloscope so that, with the sectors out of phase, the luminescence was viewed for a period of about 5 msec. during the “dark” periods. The lifetimes then were estimated by visual or photographic observation of the oscilloscope traces. To obtain sufficient intensity, wider slits had to be used on the spectrometer than wm necessary for recording the luminescence spectra.

Phosphorescence of Eosin.-Typical spectra measured in glycerol and ethanol at various temperatures have been reported previously.2 Two bands are present, the relative intensities of which are strongly temperature-dependent. The visible band (-1.8 p - ‘ ) has a contour identical with the fluorescence band in the same solution. (This is the visible “phosphorescence” originally observed by Boudin4 in glycerol solutions, and has an intensity about l/*O0th of the fluorescence a t room temperature.) To interpret our results we assume that this band is the result of thermal activation from the triplet to the upper singlet level followed by radiative transition from there to the ground state, and we therefore call this the delayed fluorescence band. We assume that the far red band (-1.4 p - l ) corresponds to a direct transition from the triplet level to the ground state and we call this the phosphorescence band. If it is further assumed that the dyestuff is present as only one species and that no dissociation or association occurs in either of the excited states, then it can be simply shown2that

where #f, #, and #* are the quantum efficiencies of normal fluorescence, delayed fluorescence, and phosphorescence, ke is the rate constant for thermal activation from the triplet to the upper singlet level, and k, is the reciprocal of the radiative lifetime ( T ~ of ) the triplet state. The ratio of the intensities of the two delayed emission bands thus should be completely independent of triplet formation efficiency (#J and of all triplet quenching processes. Since k, represents a thermal activation (4) 9. Boudin, J . chim. phys., 27, 285:(1930).

where A is a frequency factor and A E is the activation energy, which should be equal to the energy difference between the triplet and the upper singlet levels as determined by the frequency difference between the fluorescence and phosphorescence bands. Plots of In (#e/#*) against 1/T were found to be linear and the derived value of AE (10 kcal.) agreed within experimental error with that determined from the spectroscopic data.2 The values obtained for A were 1.0 X lo9 see.-’ for glycerol and 7.2 X lo7 set.-' for ethanol. The process he consists of a thermal activation to an upper vibrational level of the triplet state (rate = kl exp(-AEIRT)) followed by an intersystem crossing (rate = k2). If we make the reasonable assumption that energy is equilibrated on every collision with solvent molecules, then k-1 will be equal to 2 X 10l2 set.-' a t room temperature. Of those molecules which acquire sufficient energy, some return to the lower level (rate = k4) and some cross over to the upper singlet (rate = k 2 ) . If all the activated molecules crossed over, A would be equal to kl. I n fact A is less than one thousandth of this (for glycerol) and hence h-2/h4 -10-3. The rate k4 of degradation of vibrational energy in the triplet state is uncertain. Intermolecular vibrations occur a t a rate of l O I 3 = lo1* sec.-l but a complex molecule such as eosin probably would have to execute a large number of vibrations before losing its excess of vibrational energy. Assuming kq = 1 O l 2 set.-', values of the intersystem crossing rate ( k z ) can be calculated (see Table I). The rate in glycerol is more than ten times that in ethanol, in spite of the lower viscosity of the latter.

TABLE I APPROXIMATE RATESOF INTERSYSTEM CROSSING FOR EOSINDISODIUM SALT Process

k3(S*-P t) kz(t -L S*) kb(t + s)

In ethanol

I n glycerol

4 X IO7 sec.-l 5 x 108 2 . 4 x lo* (-20’)

1 X 107sec.-1 4 x 107

2.5

x

10’ (-20’)

Comparison of the rates for the reverse intersystem crossing (from upper singlet to triplet) can be obtained by calculating #t from the absolute values of #p together with the corresponding phosphorescence lifetimes a t various temperatures.2 The relevant data are shown in Table 11. It is of particular interest that the yield of triplet in one solvent remains fairly constant when the temperature and viscosity vary over a wide range. In changing from glycerol to the more fluid ethanol, however, a reduction in the yield of triplet is observed. This is reflected in the difference between the rates of intersystem crossing from upper singlet to triplet (Table I). A t -20’ the lifetime in both solvents was 81)proximately the same, as was the lifetime at 7 7 O K . (assumed to be the natural radiative lifetime T ~ ) . In view of the high viscosity of glycerol at -20’ it is reasonable to assume that impurity quenching is negligible, and the same therefore must have been

C. A. PARKER AKD C. G. HATCHAIII)

2508

Tol. GG

I

I .0

2-0

1.6

2.0

1.8

1.6

Y--'

P-'


A

TABLE I1 QUANTUMEFFICIENCIES OF TRIPLET FORMATION AT SELECTED TEMPERATURES Note: The first result of each pair refers to an eosin concentration of 7 X 10-5 $1, the second t o 1.5 x 10-5 31 OC.

-196

-

70

-

20

+ 25 + 70

---Glycerol r , msec.

solutions-

dp

4t

10.7 0.0643 0.065 10.8 ,0553 ,055 5 . 5 ,0328 ,064 ,056 6 . 0 .0312 3.0 ,0160 ,058 3 . 0 ,0120 .043 ,0124 ,052 2.6 ,0091 .036 2.7 ,0047 ,056 0.9 1.0 ,0039 ,042

--Ethanol msec.

solutions4p 4t

8.9 9.3 3.9 3.5 2.6 2.9 1.4 1.7 0.7 0.6

0,0237 0.024 ,0224 ,022 ,0099 ,023 ,0099 ,026 ,0070 ,024 ,0070 ,022 ,0039 ,025 .0039 ,021

7.

,0015

,020

.0015

.023

true for ethanol a t this temperature. Comparison of T a t -20' with r 0 thus gives the value of the rate of intersystem crossing (kh) from triplet to ground state (see Table 11) which is apparently independent of viscosit,y a t this temperature (a result which also has been suggested recently by Livingston and co-workers5 for anthracene). At

lower temperatures, the quenching rate in glycerol falls sharply and this may be due to a rapid increase in the rigidity of the glycerol. Possibility of Excited Dimer Formation in Dyestuff Solutions.-It has been suggested6 that the weak phosphorescence observed by Kautskyl in many dyestuff solutions is due to the formation of long-lived excited dimers, and the question therefore arises whether the phosphorescence of eosin is produced by the same mechanism. The evidence presented here and previously2 seems to be con. clusive that it is not. Thus the delayed fluorescence clearly arises through thermal activation from the energy level responsible for the far red emission band, and the latter is most intense in rigid media at 77"K., where the formation of excited dimer by a collisional process is ruled out. We have commenced an investigation of some other dyestuffs and we present below some preliminary results obtained with solutions of proflavine hydrochloride which show that the mechanism of delayed luminescence with this dyestuff (5) G.Jackson, R. Livingston, and A. C. Pugh, Trans. Foradnu S o r . , 66, 1635 (1960).

(6) R Stevens, Nature, 192,725 (1961). (7) H.Kautshy, Ber., 68, 152 (1935).


Dee., 1962

TRIPLETSINGLET EMISSION ISFLUID SOLUTION

2509

Fig. 2.-Delayed emission spectra of M phenanthrene in ethanol. The left hand sections of the spectra (delayed fluorescence) all were recorded a t a sensitivity approximately 1000 times greater than that used for curve (1) (normal fluorescence). The right hand sections of the curves (phosphorescence) were recorded a t the following sensitivities: (2) -107” a t X 60; (3) -80’ a t X 300; ( 4 ) -70” a t X 600; (5) -48” a t X 1OOO; (6) -32” a t x 1000; (7) -14’ a t X 1000; (8) 4- 13’ a t X 1000.

(at the concentrations so far investigated) is analogous to that of eosin. Our results do not of course eliminate the possibility of excited dimer formationbut show that the majority of the luminescence does not arise by this mechanism. Phosphorescence of Proflavine Hydrochloride.Proflavine in its monoprotonated form is interesting for two reasons. First, it is one of the amino acridines which most easily undergo photoreduction in aqueous solution and the kinetics of the process at pH 4 have been investigated.8 Second, its triplet state has a long radiative lifetime (3.4 sec.) compared with thal of eosin (10 msec.). It might be expected therefore that at room temperature in fluid solut,ion its triplet state would be particularly susceptible to quenching and that phosphorescence would be weak. In fact this is not so. Quite intense delayed fluorescence is observed in glycerol solution at room temperature, having a lifetime of about 70 msec. (see Fig. 1) and even in ethanol at, room temperature the intensity of delayed fluorescence has the same order of magnitude as that observed for eosin. Proflavine thus must be particularly resistant to solvent quenching processes and warrants more detailed investigation than we have yet been able to give. The preliminary results (Fig. 1) are qualitatively similar to those obtained with eosin. The de(8) F. Rfillich and G . Oster, J . Am. Chem. Soc., 81, 1357 (1959).

layed emission spectra consist of a “delayed fluorescence” band and a “phosphorescence” band, the ratio of the band intensities being highly temperature-dependent. The log ratio of the band intensities is (within experimental error) a linear function of 1 j T and the activation energy derived from the slope of this plot (-8 kcal.) is close to that corresponding to the frequency difference between the two bands. The “delayed fluorescence” therefore arises by thermal activation from the level responsible for the low frequency band and since the latter persists at 77’K. in EPA it corresponds to a metastable level of a species already present in the ground state and not to a dimer formed by a collisional process in the excited state. Our present data are not sufficiently accurate nor extensive to justify detailed calculations. I t may, however, be noted that the frequency factor in glycerol is -10’ sec.-l, ie., about one hundredth that for eosin in the same solvent. This would imply that the probability of intersystem crossing (t 4S*) is lower than €or eosin. It should be emphasized that the interpretation of our results in terms of tripletsinglet and singlettriplet transitions depends on the assumption that the dyestuffs were present in solution as only one species (the monomer) and that no protolytic reactions occurred on excitation. The results do not entirely rule out the possibility of excitation of a

C. A. PARKER AND C. G. HATCHARD

2510 2.8

2.6

2.4

2.2


I

2.0

1.8

1.6

I

Fig. 3.-Delayed fluorescence from 5 X IO-* M anthracene in ethanol. Intensity of exciting light was approximately 1.4 X 10" einstein sec.-l a t 2.73 p - l ( 3 6 6 mp). Half-band width of analyzing monochromator was 0.05 p-l at 2.5 p-l. (1) Normal fluorescence spectrum (2) delayed fluorescence spectrum a t 380 timea greater sendtivity (spectra are distorted by self-absorption).

dimer already present in the ground state although in the case of eosin the comparatively small dependence of &,/& on concentration suggests that this is unlikely. A full interpretation of the effects requires an investigation of the dependence of both luminescence and absorption spectra on pH, concentration, solvent, and temperature. For example, the position of the peak absorption of proflavine monohydrochloride in ethanol (458 mp) is shifted from that in water (443 mp); the fluorescence band shifts in the opposite direction. Again, Millich and Osters observed an increased quantum yield of photoreduction, and strong self-quenching of fluorescence on increasing the concentration of proflavine in aqueous solution. They interpreted their results in terms of a transition of the excited singlet species to a long-lived state induced by the dye itself. If the same is true in ethanol, i t would imply an increased delayed luminescence as the concentration is increased. Preliminary measurements in ethanol suggest that there is little change in or in r , for concentrations in the range 10-4-10-6 14. Delayed Emission from Solutions of Aromatic Hydrocarbons.-The observation of long-lived luminescence from vapors of aromatic hydrocarbons (e.g., phenanthrene and anthracene) has been attributed to excited dimers formed from an excited singlet molecule and a normal molecule. The blue structureless band emitted by concentrated solutions of pyrene also has been shown to be due to an excited dimer,'O although in this case the majority of the luminescence has a short lifetime." Long-lived emission so far has not been (9) R. Williams, J . Chem. Phgs., 2 8 , 677 (1958). (10) Th.Fbrster and K. Kasper, Z . Elektrochen., 69, 977 (1955). (11) C. A. Parker and C . G. Hatchard, Nature, 190, 165 (1961).

T'ol. 66

reported from fluid solutions of other hydrocarbons

at room temperature and the results shown in Fig. 2 which refer to a deaerated solution of phenanthrene in ethanol are therefore of particular interest. At 77'K. in EPA phenanthrene shows a strong phosphorescence band in the region of 2.0 p-1 with well resolved fine structure, for which &/& was found to be about unity.12 At -107' this band still appears at high intensity (about one quarter of that observed in liquid nitrogen). The intensity decreases rapidly as the temperature is raised but the phosphorescence can still be observed a t room temperature (4-13') where its intensity is about l/loooth of that observed a t liquid nitrogen temperature. Unfortunately, to record the low values of phosphorescence shown by some of the curves in Fig. 2, wide slits had to be used and vibrational structure in both the fluorescence and phosphorescence bands is largely lost. However, there is little doubt that this band is due to the triplet-lower singlet radiative transition and this is the first time, so far as is known, that such an emission has been observed from an aromatic hydrocarbon in fluid solution a t room temperature. The point of special interest in Fig. 2 is the appearance, as the temperature is lowered, of delayed fluorescence emission, although admittedly at low intensity. To test whether this could be due to thermal activation from the triplet level, the log ratio of the band intensities was plotted against 1/T. The plot was not linear, but from the maximum slope an activation energy of 2-3 kcal. was derived. This is so much less than the energy difference between the two bands (-20-25 kcal.) that activation from the triplet level is ruled out. We have observed a more intense delayed fluorescence from carefully deoxygenated solutions of anthracene in ethanol. Typical results for 5 X ill anthracene are shown in Fig. 3, where the quantum efficiency of delayed fluorescence was almost 1% of that of the normal fluorescence, We also have observed quite intense delayed emission from solutions of phenanthrene containing very small amounts of anthracene. The spectral distribution of this emission was characteristic of normal anthracene fluorescence and it therefore must have been produced by sensitization of the anthracene by a long-lived phenanthrene species. The detailed results of our investigation of anthracene and phenanthrene solutions are presented elsewhereI3 but it may be mentioned that the delayed fluorescence efficiency is in both eases proportional to the intensity of the exciting light and it cannot be explained in terms of an excited dimer similar to that postulated to explain the delayed fluorescence observed in the vapor pha~e.699,'~ The results can, however, be explained by a mechanism in which triplet-triplet quenching produces a molecular species carrying the energy from two triplet molecules. We have some eyidence that a similar process can occur with solutions of pro(12) C. A. Parker and C. 0. Hatchard. Analyst, 87,664 (1962). (13) C. A. Parker and C. C. Hatchard, Proc. Chem. SOC., 147 (1962);Proc. Roy. Soe. (London), A269,574 (1962). (14) B. Stevens, E.Hutton, and G. Porter, Nature, 185,917 (1960).

Dec., 1062

STEPWISEPHOTOREVERSIBLE DYE SYSTEMS

flavine hydrochloride at high light intensities, although a t the light intensities used to obtain the results shown in Fig. 1 the delayed fluorescence produced by this mechanism was small compared

2511

with that produced by thermal activation from the triplet level. This paper is published with the permission of the Superintendent, Admiralty Materials Laboratory.

STEPWISE PHOTOREVERSIBLE DYE SYSTEMS’*2 BY GISELAKALLMANN OSTER,GERALDOSTER,AND CECILYDOBIN Department of Chemistry, Polytechnic Instituie of Brooklyn, Brooklyn, N . 1’. Received May 66,1962


In oxygen-free Eiolution phenosafranin bound to polymethacrylic acid is photoreduced by green light when EDTA is present as the electron donor for the light excited dye. The leuco species is converted by ultraviolet light to a fluorescent yellow species. This intermediate is insensitive to light and is insensitive to oxygen. The yellow species can be thermally decomposed to give predominantly leuco dye. Leuco dye in the presence of oxygen is readily converted to the normal red dye. Arguments are given against the yellow intermediate being a semiquinone. Sample cella containing a gas inlet and outlet were illuIntroduction minated with white light from a 500-watt tungsten lamp One problem which arises in the photoreduction slide projector with a pale yellow filter to cut off light below of dyes is whether the reduction is a two-step or a 400 mp. To follow the disappearance of a particular colored one-step procesa. Certainly the leuco form of the species an a paratus was employed4 using a Bausch and Lomb monoc[romator in conjunction with a photomultiplier dye bears two more electrons than the original dye whose output was recorded continuously. and is identical with the leuco dye produced by Near ultraviolet light of predominantly 365 mp wm obreaction in the dark with powerful reducing agent.s. tained with an A H 4 mercury lamp fitted with a Wood’s If the photochemical reduction proceeds by the glass filter. Absorption spectra were obtained with a Cary Model 11 two-step process proposed by Michaelis3 for purely recording spectrophotometer. For temperatures above room chemical reductions then one might be able to temperature a cell housing was employed whereby heating detect semiquinone intermediates during the photo- liquid from a temperature bath could be circulated. Paper electrophoresis was carried out in a Model E-800chemical reaction. With this in mind we have made a search for colored intermediates in photore- 2B Research Specialties Company apparatus. The solutions 0.1 M acetate buffer at pH 5.3) were applied to strips of ducing systems. One dye which seemed to show (in Whatman No. 1 paper and a potential of 250 volts was appromise is phenosafranin. In the presence of an plied. Equilibrium dialysis was carried out using Visking seamelectron donor for the light excited dye the red dye is photoreduced to its leuco form. If the reaction less sausage casings closed at both ends. The dialysis sacks containing 20 ml. of solution (dye and polymer) were placed is carried out slowly or if the dye is bound to a in 100 ml. of buffer and allowed to equilibrate for three days. high polymer and if a polychromatic light source is The amount of dye bound was calculated from the concenused, a yellow intermediate is observed. We tration of dye, determined colorimetrically, which dialyzed should like to demonstrate that this intermediate out of the sack. In all cases the amount of dye bound to the sack (determined when no polymer wm present) was does not occur, however, in the photoreduction step dialysis taken into account. but arises from the leuco species. Results Experimental There are a number of criteria to demonstrate Materials .-Phenosafranin (3,8-diamino-12-phenyl henazinium chloride) was Histological Grade, obtainecf from that phenosafranin is bound to polymethacrylic Fisher Scientific Co. The chelating agent, disodium ethyl- acid a t pH 5.3. When polymer is in excess so that enediaminetetraacetic acid (EDTA), Analytical Grade, was all the dye is bound the maximum in absorption of obtained from the same source. Acrylamide was obtained from American Cyanamide Co. and was purified by repeated the dye at 518 mp is shifted to 530 mp. In Table crystallization from methanol. Polymethacrylic acid was I are given the spectral data on bound and free made by Bofors Co., Sweden, and, for the sample used, had a phenosafranin. Incidentally, there is an absorpmolecular weight IUfound by diffusion and ultracentrifuga- tion maximum for phenosafranin also a t 273 mp tion measurementx (carried out by Mr. B. Palm of Uppsala University) of 5 ;K lo4. Prepurified nitrogen (Airco) was but this is unchanged on addition of polymer. purified further by passing through a freshly prepared solu- Binding of the dye also is accompanied by an intion of chromous chloride. This removes traces of oxygen crease of orange fluorescence. KO spectral shifts which may be present. Procedure and Apparatus.-All the dye solutions were are observed above pH 6 although the polymer is made up in 0.1 M’ acetate buffer a t pH 5.3. Prior to illu- fully charged. This is in contrast to nucleic acids mination the solutions were deaerated by passing bubbling (both RNAS and DNA6) as substrates where nitrogen through the solution for 30 min. binding of phenosafranin occurs even in basic media (up to pH 11.9). Also the spectral shift is (1) Supported by the United States Air Force through the 4 i r Force Cambridge Research Laboratories under Contract No. AF 19(604)somewhat greater for the nucleic acids; to 540 8056 and by the United States Atomic Energy Commission under mp when the dye is fully bound. For polymethContract No. AT(30-1)-2206. (2) Taken in part from Master of Science thesis of Cecily Dobin, Polytechnic Institute of Brooklyn, 1962. (3) For reviews, see for exaniple, L. Michaelis and M. P. Schubert, Cham. Rea.. 22, 437 (1938), and L. Michaelis, A n n . N . Y . Acad. Sci., 40, 39 (1940).

(4) N. ‘Kotlierspoon and (2. Oster. J. Am. Chem. S o c . , 79, 3993 (1927). ( 5 ) L. Michaelis, Cold S p r i n g HarboT Sump. Quant. Bid., 12, 1J1 (1947). (6) G. Oster and H. Grimseon, Arch. Biochem., 24, 119 (1949).

TRIPLET-SINGLET EMISSION IN FLUID SOLUTION - The Journal of

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