5-DimethylaminonaphthaIene-1-SuIfonic Acid (DANS Acid) as Standard for Quantum Yield of Fluorescence Chester M. Himel and Richard T. Mayer Department of Entomology, University of Georgia, Athens, Ga. 30601
As PART of a study of the mechanism and mode of action of biologically active molecules, an extensive series of fluorescent probe molecules have been designed and synthesized in these laboratories. Fluorescent probes are defined (1-3) as fluorescent molecules whose spectral response (quantum yield and fluorescence excitation and emission) vary as a function of the biophysical environment with which the probe is associated. The expanding use of fluorescent probe spectroscopy creates a n important need for well defined techniques for the measurement of quantum yields and the availability of new quantum yield standards. Quinine sulfate is a generally accepted standard for the determination of quantum yield. It is not soluble in organic solvents and precise analysis of the quinine content of its hydrated sulfate and bisulfate salts is subject to important errors. Anthracene, another quantum yield standard, is soluble in organic solvents but is insoluble in water, has narrow spectral bands and is subject to severe oxygen quenching. The dansyl moiety from 5-dimethylaminonaphthalene-1sulfonic acid (DANS Acid) (I) is of increasing importance in many biologically oriented fluorescence spectroscopic studies. The sulfonyl chloride of (I) is readily available commercially and is adaptable to a wide variety of synthetic procedures for introduction of the dansyl moiety into organic molecules.
&”” Stereochemically designed molecules containing the dansyl moiety have been synthesized in these laboratories and used as histological tracers, fluorescent probes for enzyme conformation and dynamics, and as fluorescent alternate substrates in enzyme-substrate research. Concurrent with that research, a study was made of the NMR and fluorescence spectroscopy of (I) and an extensive series of its derivatives. This paper is concerned with the suitability of DANS Acid (I) as a quantum yield standard and also reports the quantum yield of p - N methyl, N(5-dimethylaminonaphthalene-1-sulfonamido) phenol (DMP) (II), a new fluorescent probe model compound. D M P (11) is photolytically stable in many organic solvents but is rapidly degraded in aqueous solvents when irradiated in the ultraviolet range.
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DANS Acid (I) is photolytically stable, has adequate solubility in water over a wide pH range and is soluble in a series of alcohols to the extent required for optical density determinations (10-4 M ) . It is commercially available, easily purified and readily analyzed. The fluorescence spectral characteristics of DANS Acid suggests its use as a quantum yield standard. The quantum yield of DANS acid has been studied ( 4 , 5). It is not subject to oxygen quenching. Chen (4) has reported the quantum yield in O.1M NaHCO8 to be 0.36. In acid solutions, the emission peak at 335 nm has been assigned to the protonated excited state (6). The well documented emission peak at 515 nm corresponds to the deprotonated excited state. Other aminonaphthalene sulfonic acid isomers have an emission maximum in the 400-430 nm range. The larger than expected bathochromic shift in the emission of the deprotonated excited state of DANS Acid may represent contribution of a stabilized 1-5 quinoid structure. In the acid pH range, DANS Acid exhibits excitation and emission characteristics strongly dependent on pH. These complications indicate that it would not be a satisfactory quantum yield standard under acid conditions. The 335 nm emission disappears in the pH 4-6 range and the quantum yield, emission maximum, and photolytic properties are stable in the pH 7-9 range. This range is also very convenient experimentally. The 0.1M N a H C 0 3 (pH = 8.5) conditions suggested by Chen appear to be excellent, for general use. An extensive NMR and fluorescence spectral study will be reported separately. Although quantum yields can be obtained by absolute methods (7, S), it is more convenient to use comparative methods in which compounds of known quantum yield are used as reference standards. Many studies have been made of the experimental problems inherent in the determination of quantum yields, these include : the fluorescence artifact present during the determination of optical density (9-11) ; concentration quenching of fluorescence; oxygen quenching of fluorescence ; temperature; purity and concentration of solutions ; solvent fluorescence; excitation wavelength (where the quantum yield is not independent of wavelength); pH (where absorption and/or emission spectra change with pH); instrumental problems ( 9 , I I ) 12, 14); effect of refractive (1) G. M. Edelman and W. 0. McClure, Accounts Chem. Res., 1(3), 65 (1968). (2) L. Stryer, Science, 162,526 (1968). (3) R. F. Chen, “Fluorescence”, G. G . Guilbault, Ed., Marcel Dekker, Inc., New York, 1967, p 443. (4) R. F. Chen, Nature, 209,69 (1966). (5) G. Weber and F. W. J . Teale, Trans. Faraday SOC.,53, 646 (1957). (6) D. Lagunoff and P. 0. Ottolenghi, Compt. Rend. Trau. Lab. Carlsberg, 35, 63 (1966). ( 7 ) W. H . Melhuish, J. Phys. Chem., 65,229 (1961). (8) J. W. Eastman, Photochem. Photobiol., 6, 55 (1967). (9) G. K. Turner, Science, 146, 183 (1964). (10) A. N. Fletcher, Photochem. Photobiol., 9, 439 (1969). (11) G. K. Turner Assoc., Palo Alto, Calif., Bull. 8885 (1966). (12) C. A. Parker and W. T . Rees, Analyst, 85, 587 (1960). (13) D. M. Hercules and H. Frankel, Science, 131, 1611 (1960). (14) R. F. Chen, Anal. Biochem., 20, 339 (1957).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 1, JANUARY 1970
Table I. Quantum Yield Data Compound Anthracene Anthracene Anthracene Anthracene Anthracene Anthracene Anthracene Anthracene Dans-Acid Dans-Acid Dans-Acid
Excitation (A nm) 254 366 365 366 365 365 340 376 366 ,..
...
Conc. ( M )
Temp., “C
... x 10-3
...
...
... ...
... ...
...
3 1.5 X 5.6 x 10-6 1.5 X 1.5 X 1,5x
...
25 25
25 25 25 23 23
Dans-Acid Dans-Acid
366 320
...
1.5 x
20 25
1,8 ANS 1,8 ANS DMP
365 365 340
1 . 0 x 10-6 1.5 X 2.0 x 10-6
20 25 25
Solvent Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Water Water
6 0.30 0.270
...
0.28
0.30 0.31 0.31 0.31 0.53 0.37 0.36
0.1M
Bicarbonate Water 0.1M Bicarbonate +Butanol n-Butanol isopropanol
index (1.5-18). The effect of refractive index when the reference standard and the unknown are measured in two different solvents can be appreciable. This factor is one basis for the search for quantum yield standards with a wide range of solubility in organic solvents and in water. Quantum yield data are collected in Table I. EXPERIMENTAL
Apparatus. All spectra were run on a “Spectro 210” (G. K. Turner Associates, Palo Alto, Calif.). This absolute spectrofluorometer presents corrected emission spectra and excitation spectra at constant energy. In the absorption mode, the instrument minimizes the fluorescent artifact present in many absorption spectra (9). The importance of the fluorescence artifact in quantum yield determination has been discussed by Fletcher (10) and by Turner (11). Chemicals and Solvents. Anthracene was Eastman White Label, recrystallized three times from benzene and dried in Gacuo. Quinine sulfate was obtained from Columbia Organic Chemicals. It was recrystallized four times from water, then dried in GLZCUO at 100 “C. Product purity was determined by direct oxygen analysis, this being the single most significant analysis related to purity and composition. 8-Anilinonaphthalene-1-sulfonicacid was obtained from Aldrich Chemical Co., recrystallized twice from H 2 0 and dried in cacuo at 100 “C. Analysis was by neutral equivalent. 5-Dimethylaminonaphthalene-1-sulfonic acid (DANS acid) was prepared from the sulfonyl chloride (Peninsular Chemresearch Inc.). The sulfonyl chloride, mp 69-71 “C, (0.5 g) was added to 0.0037 moles N a H C 0 3 in 50 ml of HrO. After reflux (12 hr) the reaction was cooled and acidified t o p H 4.2 with 0.1N HCl. The white crystalline material was recrystallized three times from H?O dried in caciio and analyzed for water of crystallization by neutral equivalent. Solvents were fluorescent grade from Matheson, Coleman and Bell, Harleco, or U. S. Industrial Chemicals, Co. Water was double distilled and was found to have low intrinsic fluorescence. -
( I S ) A. N. Fletcher, J. Mol. Spectry., 23, 221 (1967). (16) E. H. Gilmore, G. E. Gibson, and D. S. McClure, J. Chem. Phys., 23, 399 (1955). (17) A. Shepp, ibid., 25, 579 (1956). (18) A. N. Fletcher, U. S . Naval Ordance Test Station, China Lake, Calif., personal communication, 1969.
0.29
Reference
... ... 0.306 ... ...
(5) (4) (15) (12) (13)
0.32 0.32 0.32
This Paper This Paper This Paper
... ...
(5) (4) (4)
...
...
0.36
0.36
0.56 0.66 0.41
0.72 0.43
.*.
(21)
This Paper (20)
This Paper This Paper
Methods. Quantum yield determinations were made using Equation 1, given by Turner (11) and modified to include the effect of refractive index as suggested by Fletcher (15).
4Il where A D d
X
=
AuDsdsAs(nu>2
” AsD,d,X,(n,)2
(1)
Emission peak area corrected for solvent blank
= Optical density a t the excitation wavelength =
= = n = s, u =
+
=
Dilution factor in the dilution of the sample used for the optical density measurements, to the concentration used in the fluorescence measurements Excitation wavelength Quantum yield Refractive index of solvent at A,, emission subscripts denoting standard (3) and unknown (U)
Use of this method eliminates weighing errors in the preparation of solutions and the effect of any nonfluorescent solvent of crystallization (product composition) on the quantum yield. Use of Equation 1 requires quantitative dilution techniques and adherence to the above considerations concerning reference standards. All optical density and quantum yield determinations were made with emission and excitation bandwidths set at 2.5 nm. Fluorescent solutions subject to oxygen quenching were purged at least 20 minutes with special purity Nr and transferred via nitrogen pressure through a glass capillary tube into a fluorescence cell purged with Nr. The fluorescence cells were fluorescence quality quartz with ground and fitted stoppers. Time for effective purge of anthracene solutions was determined by measurement of the degree of quenching after purge times ranging from 1 minute to 3 hours. The fluorescence well in the “Spectro 210” was temperature controlled to 25 + 0.01 “C. When necessary the fluorescence well was blanketed with a slow flow of high purity Np. Purged samples of anthracene solutions in stoppered but unblanketed cells showed 5 % quenching after 20 minutes and 7 % quenching after 35 minutes. Parker and Rees (12) have previously emphasized the importance of oxygen quenching. Solutions used in the fluorescence measurements were prepared from solvents stored under nitrogen in the absence of light. Fluorescence measurements were made on solutions in the range of 1 X 10-6 molar to minimize concentration quenching and were routinely checked and found to have a n optical density less than the range of 0.03.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 1, JANUARY 1970
131
50
>.
40
(3
w K
z w
30
W
-I-> 3
20
W 0:
I
w
>
F 4
10
250
300
350
400
450
500
550
600
NANOMETERS
Figure 1. Corrected fluorescent spectra of 1.5 X 10-6M DANS acid in 0.1MNaHC03 solution Curve No. 1, excitation spectrum, emission at 515 nm. Curve No. 2, emission spectrum, excitation at 320 nm. (Excitation and emission bandwidths fixed at 2.5 nm, temperature 25 “C) RESULTS AND DISCUSSION The quantum yield of quinine sulfate has been reported at a variety of excitation wavelengths. Fletcher (19) has discussed the effect of rotatable auxochromes in quinine as related to its fluorescence response at different excitation wavelengths. In this study, quinine sulfate in 0.1N H2S04, excited at 365 nm and having a quantum yield of 0.55 was used as the standard. Anthracene in ethanol was studied with excitation at 376 nm, 365 nm, and 340 nm. Quantum yields reported in Table I were constant at these different excitation wavelengths, however, significant advantages accure from the use of peak maxima (340 nm, 376 nm) for excitation because of the increased fluorescence response. 5-Dimethylaminonaphthalene sulfonic acid (DANS Acid) appears to be an excellent standard for quantum yield at
(19) A. N.Fletcher, J. Phys. Chem., 72, 2742 (1968).
132
pH = 7-9. Data presented herein, duplicate the quantum yield of 0.36 in 0 . 1 M N a H C 0 3as previously reported by Chen (4). The excitation wavelength was 315-320 nm corresponding to the broad absorption maximum when p H = 7-9. Excitation spectra at constant energy and corrected emission spectra for DANS acid in 0.1M N a H C 0 3 are given in Figure 1. 8-Anilinonaphthalene-1-sulfonic acid (1,8 ANS) was strongly quenched by oxygen. The quantum yield of 0.66 (or 0.72) in n-butanol (after purging with NS and transfer to a N2 purged and blanketed fluorescence cell) was found to be significantly higher than the value reported by Stryer (20). The quantum yields reported in this paper were calculated with and without the effect of refractive index. Fletcher (18) has pointed out that ideally such a correction factor should be based on the actual refractive index as a function of the total emission scan. The apparent effect of the refractive index correction is presented in Table I in a separate column. Quantum yield data have been obtained for anthracene in ethanol, 8-anilinonaphthalene-1-sulfonic acid in n-butanol, 5-dimethylaminonaphthalene-1-sulfonic acid (DANS acid) in 0.1N NaHC03, and p-N methyl, N(5-dimethylaminonaphthalene-1-sulfonamido) phenol in isopropanol. All data were obtained at 25 “Cwith the use of 2.5-nm bandwidth for excitation and emission in both the fluorescence and absorption modes of the “Spectro 210.” Quantum yield data obtained in this study are collected in Table I along with corresponding values from the literature. All values in this research are an average of not less than six determinations whose range was kO.01. RECEIVED for review August 18, 1969. Accepted October 20, 1969. Research supported in part by grants from the National Institutes of Health (ES 00207) and the Agricultural Research Service (12-14-1 00-91 48( 33)).
(20) L.Stryer, J. Mol. Biol., 13,482 (1965). (21) E.J. Bowen and D. Seaman, “Luminescence of Organic and Inorganic Materials,” H. P. Kallmann and G. M. Spruch, Eds., John Wiley and Sons, Inc., New York, N. Y.,1960,p 157.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 1, JANUARY 1970
