J. Chem. SOC., Faraday Trans. I , 1986, 82, 157-160 Aggregation in Aqueous Solution of the Dye Pyronine G John Gormally* and Susan Higson Department of Pure and Applied Chemistry, University of Salford, Salford A45 4 WT The aggregation in aqueous solution of the dye Pyronine G has been studied using spectrophotometry. A high-purity sample of Pyronine G was used, and it is shown that the dye spectra can be interpreted in terms of dimerization of the dye. Values of the dimerization constant at three temperatures are given. Pyronine G is a metachromatic xanthene dye with the structure shown in fig. 1. This dye has been used as a stain in microscopy,l as a fluorescent laser dye2 and in the spectrophotometric determination of platin~m.~ It has also been used in the study of dye-polyelectrolyte interactions4 and in a study of the kinetics of dye aggregation in s01ution.~ In spite of these activities, reliable data on the solution properties of this compound are rare6 owing to the low purity of most of the samples studied.For example, reported molar absorptivities for dilute aqueous solutions at 547 nm range from 1.2 x lo4 dm3 mol-1 cm-l [ref. (7)] to 10.7 x lo4 dm3 mol-1 cm-l [ref. (6)]. We present below some values of parameters which determine the spectrophotometric properties and association behaviour of Pyronine G obtained with a high purity sample of the dye. Experimental Commercially available samples of the dye (BDH Chemicals Ltd and Eastman Kodak Co.) were found to be very impure. A large proportion of the contaminating material was insoluble in methanol and could be removed easily.The dye extracted in this way had a molar absorptivity at 547 nm of 5.7 x lo4 dm3 mol-1 cm-l at a concentration of mol dm-3. Recrystallization from both methanol and ethanol was attempted, but was not successful owing to the high solubility of the dye in these solvents. The dye used in this study was synthesized using the method of Jacobsen et aL8 and had a molar absorptivity at 547 nm of 10.96 x lo4 dm3 mol-1 cm-l mol dm-3 dye in lov3 mol dmP3 HCl at 25 "C). This is very close to the value reported by Jacobsen et aL6 (10.7 x dm3 mol-1 cm-l) and suggests that our product is similar in purity to theirs. To our knowledge this is the highest value reported for Pyronine G and the purity of the substance prepared in this way is supported by microanalysis and n.m.r.investigations described in ref. (8). Absorbance measurements were made using a Pye Unicam SPS-300 spectrophoto- meter with thermostatting facilities. Sample cells had pathlengths of 40 mm, 10 mm and 2 mm and prior to use they were wetted with a 2% solution of dimethyldichlorosilane in trichloroethane (B.D.H. Chemicals Ltd) and then rinsed in distilled water. This treatment inhibits adsorption of dye onto the cell walls. Solutions were made up in mol dm-3 HCl to restrict the pH to ca. 3 as Pyronine G spectra are relatively insensitive to pH fluctuations in this r e g i ~ n . ~ ? ~ The presence of HCl also reduces the tendency of cationic dyes to adsorb onto the surfaces of glas~ware.~ Absorbances were measured at wavelengths from 500 to 570nm at 10nm intervals and at the a-band absorbance peak at 547 nm. The concentration range studied extended from (0.8 to 75) x mol dm-3.157158 d - I 5 52- "0 5 0 - 3 4 8 - 4 6 - 4 2 4 0 - Aggregation of Pyronine G I I I -6 - 5 - 4 4 4 + G * - $ - - - 100 " I P - 3 m E a I 0 - - 50 0 - 1 P 1 1 I I 450 500 A/ niii I 5 50 Fig. 1. The absorption spectrum of Pyronine G chloride (concentration 1.02 x lop5 mol dmp3 at 25 "C). The wavelengths at which absorbances were measured are indicated by vertical lines and examples of such data are given in fig. 2. - 6 4 Fig. 2. Plots of apparent absorptivity against dye concentration at ( a ) 550 and (b) 510 nm at a temperature of 30 "C. Data were obtained at nine different wavelengths and at three temperatures.Curvature of the plots indicates departure from the Beer-Lambert law and is attributed to dye aggregation.J . Gormally and S. Higson 159 Results Typical results showing the variation of apparent absorptivity with concentration are shown in fig. 2. Curvature in these graphs is characteristic of metachromatic dyes and is attributed to dye aggregation. At any wavelength, A, the apparent molar absorptivity can be written as \ n / where the E , are the absorptivities of species n (n = 1 for monomer, 2 for dimer, etc.) and A, are the corresponding concentrations. Atot is the total dye concentration and is n = A,+2K,A:+3K,K3AX+ . . . where K,, is the equilibrium constant for the association equilibrium (3) It is readily shown that if we consider aggregation only to the dimer stage that eqn (1) becomes Data such as are shown in fig.2 were fitted to this expression using the simplex methodlo and a minimum sum of squares criterion was used to find the values of E,, c2 and K , which gave the best fit. This was done for sets of data obtained at different wavelengths to check the wavelength independence of K,. The spread in values of K2 obtained is reflected in the error limits of the values given in table 1. Attempts to evaluate dimerization constants from data obtained at a single wavelength can lead to an optimistic assessment of the errors involved.ll The same procedure was adopted with data obtained at one wave- length, but at different temperatures. In this case consistency in the values of el and E , was sought.The values shown in table 1 indicate the consistency found. It was noticed that the value of the monomer molar absorptivity at 547 nm increased by ca. 0.3% per degree drop in temperature. This effect was observed in very dilute solutions in which the concentration of aggregates was immeasurably small, and it is too large to be accounted for by the increase in concentration which attends thermal contraction of the solution. A similar effect has been reported for solutions of xanthene dyes in ethano1.12 Aggregation was also considered to the trimer by retaining the third term in eqn (3) which then becomes a cubic in A,. This was solved for A, using the method in ref. (13) and the resulting expressions were used in conjunction with eqn (1) and (2) to determine &(A) in terms of E,,E,,E,,K, and K3.Unique values of K2 and K3 could not be found, indicating that whilst higher aggregates may exist in these solutions their concentration is too small to have a significant effect on our measurements. Conclusion Aggregation in aqueous solutions of Pyronine G within the concentration range studied can be adequately explained in terms of dimerization of the dye with the parameters given in table 1. In comparison with most other metachromatic dyes, Pyronine G is very highly absorbing in the visible region and has a relatively small dimerization ~0nstant.l~ From the values of Kz at different temperatures, estimates of various thermodynamic parameters can be obtained. Within the temperature range studied the values for AG and AH were found to be - 18.8 f 0.2 kJ mol-l and - 21 5 kJ rnol-l, respectively.The error in AH is large, but we consider this to be reasonable considering the very low concentration of dimer which exists in these solutions (for a dye concentration of mol dm-3 at 6 FAR 1160 Aggregation of Pyronine G Table 1. Examples of absorptivities and dimerization constants derived from an analysis of spectrophotometric dataa T "C E , (547) Cd547) Ed5 10) c2(5 10) ~ ~ / 1 0 3 25 1 1.4( 0.1) 0.7( 0.2) 4.3( * 0.1) 14.3 & 0.5) 2.0( * 0.1) _- ~ ~ ~ ~ _ _ _ _ 30 1 1.2( f 0.1) 0.75( & 0.2) 4.3( k 0.1) 14.5( k 0.5) 1.7( 40.1) 14.5 11.7( & 0.2) 0.95( 0.2) 4.4( k 0.1) 15.5( & 0.5) 2.7( 0.15) a Absorptivities (wavelength in parentheses) are measured in lo4 dm:3 mol-' cm-'.25 OC, the concentration of dimer is around 2 x lo-' mol dm-"). Further, we note that some of the reported values of thermodynamic parameters relating to dye aggregation imply a very optimistic assessment of accuracy as is evident from the wide range of values for the enthalpy and entropy of dimerization of acridine orange to be found in the literature [ref. (14)]. It seems possible that these quantities could be measured more accurately by use of the isoextraction method which Mukerjee and Ghosh applied to a study of aggregation in methylene blue.15 However, we feel that the values given above are realistic in that any more accurate determination, should this be required, would give results within the error limits specified. References 1 F. H. Kasten, Stain Technol., 1962, 37, 265. 2 B. I. Stepanov and A. N. Rubinov. Souiet Physics Uspekhi, 1968, 11, 304. 3 S. Jaya, T. P. Rao and T. V. Ramakrishna, Analyst (London), 1984, 109, 1405. 4 V. Vitagliano and L. Costantino, J . Phys. Chem., 1970, 74, 197. 5 W. Ohling, Ber. Bunsenges. Phys. Chem., 1984, 88, 109. 6 P. Jakobsen, H. Lyon and S. Treppendahl, Histochemistry, 1984, 81, 99. 7 K. Fujiki, C. Iwanaga and M. Koizumi, Bull. Chem. SOC. Jpn, 1962, 35, 185. 8 P. Jakobsen, A. P. Andersen, H. Lyon and S. Treppendahl, Microsr. Acta, 1983, 87, 41. 9 P. Mukerjee and A. K. Ghosh, J . Am. Chem. SOC., 1970, 92, 6403. 10 J. A. Nedler and R. Mead, Comput. J., 1965, 7, 308. 1 1 R. L. Reeves, M. S. Maggo and S. A. Harkaway, J . Phys. Chem., 1979, 83, 2359. 12 J. E. Selwyn and J. I. Steinfeld, J . Phys. Chem., 1972, 76, 762. 13 I. S. and E. S. Sokolnikoff, Higher Mathematics for Engineers und Physicists (McGraw-Hill, New York, 14 V. Vitagliano, in Aggregation Processes in Solution, ed. E. Wyn-Jones and J. Gormally (Elsevier, 15 P. Mukerjee and A. K. Ghosh, J . Am. Chem. SOC., 1970, 92, 6419. 1941), p. 86. Amsterdam, 1983), p. 276. Paper 51570; Received 3rd April, 1985