Analysis of meso-tetrakis (4-sulfonatophenyl) porphyrin in aqueous solution by constant-wavelength synchronous fluorescence spectroscopy

Yao Minna, Li Yaoqun
(The Key Laboratory of Analytical Sciences of MOE and Department of Chemistry, Xiamen University, Xiamen, 361005,  China)

Received Apr. 21, 2003; Supported financially by the National Natural Science Foundation of China (No.29875023), Educational Ministry Foundation of China and the Natural Science Foundation of Fujian Government.

1 INTRODUCTION
It is important to analyze the behavior of water-soluble porphyrins in aqueous solution because of their signification in biological processes. The porphyrin selected for our study is meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS) (Fig. 1). Its molecular structure is fairly rigid and has extensive electron delocalization in the nucleus. The porphyrin ring system contains two pyrrole nitrogen atoms. The free form is called unprotonated TPPS (TPPS^4-), and when added two protons, it is called diprotonated TPPS (H₂TPPS^2-). They have the dissociation equilibria in aqueous solution as follows: H₂TPPS^2-=HTPPS^3-+H⁺ (pK_a1), HTPPS^3-=TPPS^4-+H⁺ (pK_a2). ^[1,2] H₂TPPS^2- and TPPS^4- are both highly fluorescent in aqueous solution, so they are widely used in fluorescence analysis. The monoprotonated TPPS (HTPPS^3-) existed in very short pH range and could not be observed by fluorimetry. However, the excitation spectra and emission spectra of the two forms (H₂TPPS^2- and TPPS^4- ) overlap seriously, people can only study each form respectively by conventional fluorescence spectroscopy, ^[3,4] and the pK_a values have been calculated by using spectrophotometry.^[5,6] As well known, fluorescence measurements are more sensitive and potentially more selective than absorbance measurements. Spectrofluorometry could provide a more sensitive method to investigate the dissociation equilibrium of TPPS in lower concentration range than spectrophotometry.
    Synchronous fluorescence spectroscopy is a good way to analyze multi-component system.^[7-11] It has been developed into several variants, i.e., constant-wavelength (CW), constant-energy (CE) and variable-angle (VA) synchronous fluorescence spectroscopy etc. Constant-wavelength synchronous fluorescence spectroscopy (CWSF) is the simplest and easiest to be realized.^[8,9] The excitation and emission monochromators are simultaneously scanned, separated by a constant wavelength interval, Dl. The resulting synchronous fluorescence spectra represent the intensity profile of a 45° section cut through the excitation-emission matrix (EEM). The spectra is simplified, and the bandwidth is narrowed with high selectivity and good sensitivity.^[12] In this paper, a quick and simple analytical method is developed to analyze the two forms of meso-tetrakis(4-sulfonatophenyl) porphyrin simultaneously without interference by using CWSF.

2 EXPERIMENTAL
2.1 Reagents
The stock solution of meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS) (Prophyrin Products Inc.) was prepared in distilled-deionized water. The buffer solutions were prepared by 0.01M potassium hydrogen phthalate (KHC₈H₄O₄) (A.R.). NaOH and HCl were used to adjust pH values. Other reagents were of analytical grade. Distilled-deionized water was used throughout.
2.2 Apparatus  
All spectra were obtained on a laboratory-constructed computer (IBM)-controlled spectrofluorometer that was similar to the one previously described.^[10,11] The spectrofluorometer was equipped with a 350W Xenon lamp, and the slit bandpasses of excitation and emission monochromators were set at 5nm. In addition to conventional fluorescence spectra, the apparatus could provide all kinds of synchronous spectra. The pH values were measured by using Delta320pH meter (Mettler Toledo). An optic glass cuvette with a pathlength of 1×1cm was used throughout. The ionic strength of the solution is controlled at 0.1M with NaCl.
2.3 Procedure                    
To a 10-ml volumetric flask was added a volume of 100ml TPPS with concentration of 1×10^-4M and 1ml NaCl with concentration of 1M. Then the solution was diluted to the mark with buffer solution of different pH value (2.40-7.50). The final concentration of 1×10^-6M for TPPS was used throughout. The suitable scan parameters for CWSF were selected according to the excitation and emission spectra of the two forms of TPPS. The fluorescence synchronous spectra of 1×10^-6M TPPS with pH from 2.40 to 7.50 were obtained with Dl=225nm. Scan rates of the excitation and emission monochromaters are set at 240nm/min.

Fig. 1 The molecular structure of TPPS. Protonation occurs on the two nitrogen atoms in the porphyrin ring.

3 RESULTS AND DISCUSSION
3.1 Fluorescence spectra            
The experiments showed that when pH<4, the main form in solution was H₂TPPS^2-, and when pH>6, the main form in solution was TPPS^4-, so we used pH=2.40 and pH=6.30 to study the basic information of TPPS. The excitation and emission spectra of H₂TPPS^2- at pH=2.40 and TPPS^4- at pH=6.30 were obtained (Fig. 2). The maximal excitation and emission wavelengths of H₂TPPS^2- were at 435nm and 670nm, whereas the maximal excitation and emission wavelengths of TPPS^4- were at 412nm and 642nm. The results showed that the fluorescence intensity of H₂TPPS^2- was about three times higher than that of TPPS^4-. As showed in Fig. 2, the spectra of the two forms of TPPS overlapped seriously, especially the emission spectra.

Fig. 2 Fluorescence excitation (a) and emission (b) spectra of H₂TPPS^2- (solid line) at pH=2.40 and TPPS^4- (dotted line) at pH=6.30. The concentration of TPPS was 1.0×10^-6M.

Fig. 3 The CWSF spectra of H₂TPPS^2- at pH=2.40 (solid line) and TPPS^4- at pH=6.30 (dotted line) with Dl=225nm.

3.2 Selection of the parameters of synchronous fluorescence spectroscopy           
In CWSF, the most important scanning parameter is Dl. Theoretically when Dl of synchronous fluorescence spectroscopy is the Stokes shift of a substance, the intensity will be the highest and bandwidth will be the narrowest, but in fact the selection of Dl is still a result of tests considering the interference from background signal or other coexisting components.^[12] A series of Dl values (223nm, 225nm, 228nm, 230nm, 233nm, 235nm) were tested.
    The peak wavelengths of H₂TPPS^2- and TPPS^4- red-shifted about 3nm, and their mutual interference of H₂TPPS^2- and TPPS^4- became lower when Dl changed from 235nm to 225nm. It seemed that results would be good with the decrease of Dl, but when Dl was decreased to 223nm, the interference became higher again. Since the mutual interference changed not too much from 230nm to 225nm, any value could be selected in this range. In this experiment the Dl value of 225nm was used. In comparison with Fig. 2, well-resolved synchronous fluorescence spectra (shown in Fig. 3) for H₂TPPS^2- and TPPS^4- were obtained with Dl=225nm. The synchronous fluorescence peak wavelength of TPPS^4- was at 413nm and that of H₂TPPS^2- at 437nm. The interference between each other was lower than 5%, and the spectral distinction of Dl=225nm was the best comparing with that of other Dl values.


Fig. 4 The CWSF spectra of H₂TPPS^2- (A) and TPPS^4- (B) changed with different pH (2.40—7.50). The concentration of TPPS was 1.0×10^-6M.



Fig. 5 The pH dependence of the synchronous fluorescence intensity of H₂TPPS^2- (A) and TPPS^4- (B).

3.3 Fluorescence behavior of H₂TPPS^2-and TPPS^4- in aqueous solution with different pH
TPPS in aqueous solution in the pH range from 2.40 to 7.50 were analyzed by using CWSF (Dl=225nm), and a series of synchronous fluorescence spectra were obtained (Fig. 4). With the increase of pH, the spectral peak at 437nm decreased and the peak at 413nm increased gradually, indicating the alternation of H₂TPPS^2- and TPPS^4- clearly. When pH was lower than 4.00, the synchronous fluorescence of TPPS^4- did not appear. There was an equal fluorescence intensity point at 423nm. When pH reached 6.00, the synchronous fluorescence of H₂TPPS^2- completely disappeared.
3.4 Calculation of dissociation equilibrium constant
The synchronous fluorescence intensities of H₂TPPS^2- and TPPS^4- versus pH showed Sigmoidal shapes, which were due to the dissociation equilibrium in aqueous solution. Spectrofluorimetric pH-titration curves showed that, the synchronous fluorescence intensity of the TPPS^4- was not pH dependent after 6.00, and the intensity of H₂TPPS^2- was not pH dependent before 4.00. By using Origin6.0 professional software to fit Sigmoidal, the values of dissociation equilibrium constant were obtained as pK_a1=4.76±0.02 (Fig. 5-A) and pK_a2=5.00±0.02 (Fig. 5-B). This result coincided with the other spectrophotometral data listed in Table 1.

Table 1. Comparison of the pK_a values