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Enhancing fluorescence intensity of Ellagic acid in Borax–HCl–CTAB micelles
Spectrochimica Acta Part A 78 (2011) 1013–1017
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Enhancing fluorescence intensity of Ellagic acid in Borax–HCl–CTAB micelles Feng Wang a,∗ , Wei Huang a , Shuai Zhang a , Guokui Liu a , Kexiang Li a , Bo Tang b a b
Department of Chemistry and Chemical Engineering, Zaozhuang University, Zaozhuang 277160, PR China College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 8 January 2010 Received in revised form 7 December 2010 Accepted 10 December 2010
Ellagic acid (C14 H6 O8 ), a naturally occurring phytochemical, found mainly in berries and some nuts, has anticarcinogenic and antioxidant properties. It is found that fluorescence of Ellagic acid (EA) is greatly enhanced by micelle of cetyltrimethylammonium bromide (CTAB) surfactant. Based on this effect, a sensitive proposed fluorimetric method was applied for the determination of Ellagic acid in aqueous solution. In the Borax–HCl buffer, the fluorescence intensity of Ellagic acid in the presence of CTAB is proportional to the concentration of Ellagic acid in range from 8.0 × 10−10 to 4.0 × 10−5 mol L−1 ; and the detection limits are 3.2 × 10−10 mol L−1 and 5.9 × 10−10 mol L−1 excited at 266 nm and 388 nm, respectively. The actual samples of pomegranate rinds are simply manipulated and satisfactorily determined. The interaction mechanism studies argue that the negative EA–Borax complex is formed and solubilized in the cationic surfactant CTAB micelle in this system. The fluorescence intensity of EA enhances because the CTAB micelle provides a hydrophobic microenvironment for EA–Borax complex, which can prevent collision with water molecules and decrease the energy loss of EA–Borax complex. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
Keywords: Ellagic acid CTAB Micelle enhancement Fluorescence spectroscopy
1. Introduction Ellagic acid, [2,3,7,8-tetrahydroxybenzopyrano [5,4,3cde]benzopyran-5,10-dione], is a polyphenol antioxidant found in numerous fruits and vegetables. The antiproliferative and antioxidant properties of Ellagic acid have spurred preliminary research into the potential health benefits of Ellagic acid consumption [1–5]. The structure of Ellagic acid is as follows: O
C
O
OH OH
OH HO
O
C
liquid chromatography [6,7], paper chromatography [8], capillary electrophoresis [9] and UV–Vis spectrophotometry [10] have been employed to determine Ellagic acid are present in a variety of matrices such as grape juice, strawberries and pomegranate rinds. But these current methods have a limited sensitivity because they are based on absorption measurements. Furthermore, the fact that Ellagic acid has poor solubility makes the determination of trace amount of Ellagic acid in aqueous solution difficult. In this paper, a study of the fluorescence of Ellagic acid in several micelle systems has been carried out. It is found that the fluorescence intensity of Ellagic acid can be greatly enhanced by the cationic surfactant cetyltrimethylammonium bromide (CTAB). Based on the above property, the sensitive quantitative analysis of Ellagic acid in aqueous solution is established. This method has the advantages of high sensitivity, selectivity and stability.
O
The highest levels of Ellagic acid are found in raspberries, strawberries, and pomegranates, especially when freeze-dried. Red raspberry leaves, which also contain Ellagic acid, are available in capsule, powder and liquid forms. But the correct dosages of these preparations are not known. Methods such as high performance
∗ Corresponding author at: Department of Chemistry and Chemical Engineering, Zaozhuang, Shandong 277160, PR China. Tel.: +86 632 3786735. E-mail addresses: [email protected], [email protected] (F. Wang).
2. Experimental 2.1. Apparatus Fluorescent spectra were obtained using F-4600 spectrofluorometer (Hitachi, Japan). Absorption spectra were recorded using UV-2401PC (Shimadzu, Japan) spectrophotometer. Electrical conductivities were measured by DDS-11A conductivity meter (Leici Shanghai). All acidity measurements were performed with a Delta320-S acidity meter (Mettler Toledo, Shanghai).
1386-1425/$ – see front matter Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2010.12.030
1014
F. Wang et al. / Spectrochimica Acta Part A 78 (2011) 1013–1017
100 80
a
100
1
40
1
80 60
60
If
b
4
If
4
40
20
20
0
0 250
300
350
400
400
Wavelength(nm)
450
500
550
600
Wavelength(nm)
Fig. 1. Excitation spectra (em = 454 nm) (a) and emission spectra (ex = 388 nm) (b). (1) Borax–HCl–EA–CTAB; (2) Borax–HCl–EA; (3) EA–CTAB; (4) EA. Conditions: EA: 4.00 × 10−5 mol L−1 ; CTAB: 5.00 × 10−3 mol L−1 ; pH 7.00 (Borax–HCl).
2.2. Reagents A stock solution of Ellagic acid (EA) (4.00 × 10−4 mol L−1 ) was first prepared by dissolving EA in methanol, and then diluted to proper concentration with methanol. A series of Borax–HCl buffer solutions (0.05 mol L−1 ) were used for the pH adjustment. A solution of surfactant cetyltrimethylammonium bromide (CTAB) (0.050 mol L−1 ) was prepared by dissolving CTAB in deionized water. All the chemicals used were of analytical reagent grade and deionized water was used throughout. 2.3. Experimental procedures In a 10 mL test-tube, solutions were added in the following order: 2.00 mL Borax–HCl (pH 7.00), 1.00 mL CTAB (0.050 mol L−1 ), 2.50 mL methanol and definite standard EA (or sample solution). The mixture was diluted to certain volume by water. The fluorescence intensity is measured at ex /em = 388 nm/454 nm in a 1 cm quartz cell and with slit at 10.0 nm for the excitation and emission.
CTAB, which causes a blue-shift to 454 nm. This indicates that EA is solubilized in the micelle of CTAB. 3.2. Effects of pH and buffers The effect of pH on the fluorescence intensity of the system is shown in Fig. 2. It can be seen that the maximum intensity is obtained in the pH 7.00 buffer. Experiments indicate that different buffers also have very different effects on fluorescence intensity (If ) of the system. The If for Na2 HPO4 –citric, NaH2 PO4 –Na2 HPO4 , KH2 PO4 –Na2 HPO4 , Tris–HCl, BR (Briton–Robinson)–NaOH, HMTA–HCl, Borax–HCl at pH 7.00 is 0.70, 0.73, 0.73, 0.60, 35.8, 0.54, 100.0, respectively. The results indicate that the Borax–HCl is the best in the buffers tested and the BR–NaOH is the second, so the Borax–HCl buffer (pH 7.00) is selected for the assay and the optimum volume of Borax–HCl buffer is 2.00 mL. The reason for the enhancement of the EA fluorescence is that the H3 BO3 in the two buffers of BR–NaOH and Borax–HCl can coordinate ortho-dihydroxy group which is present in the structure of EA to form a chelate [12].
2.4. Sample treatment Ellagic acid was extracted from real samples according to previously reported procedure: the samples spices (pomegranate rinds, acquired from drugstore) were dried at 100 ◦ C for 24 h and then ground to fine powder. A weighed comminuted pomegranate rind was refluxed at 85 ± 5 ◦ C in 100% methanol for 2 h and then the solvent was separated by filtration. The collected extraction was evaporated in a rotary evaporator at 35 ◦ C until no further droplet came out [11]; the concentrated solution and combined methanol washings were transferred into a volumetric flask and diluted to 50 mL with methanol. An appropriate volume of this solution was pipetted into a 10 mL flask and its Ellagic acid content was determined by standard addition method according to the procedure described above. 3. Results and discussion
3.3. Effect of surfactants The effects of different surfactants on the fluorescence intensity were tested and the results are shown in Table 1.
100
80
If
60
40
3.1. Fluorescence spectra 20
The fluorescence spectra of (1) Borax–HCl–EA–CTAB, (2) Borax–HCl–EA, (3) EA–CTAB, and (4) EA systems are shown in Fig. 1. It can be seen that EA in aqueous solution has very weak fluorescence with two excitation peaks of 266 nm and 388 nm and an emission peak at about 450 nm, whereas it has enhancement and red-shift in the Borax–HCl buffer. And the fluorescence peak can be further greatly enhanced by the addition of a cationic surfactant
0 3
4
5
6
7
8
9
pH Fig. 2. The effect of pH on the fluorescence intensity. Conditions: EA: 4.00 × 10−5 mol L−1 ; CTAB: 5.00 × 10−3 mol L−1 ; pH 7.00 (Borax–HCl).
F. Wang et al. / Spectrochimica Acta Part A 78 (2011) 1013–1017 Table 1 Effects of surfactants on the fluorescence intensity.
1015
100
Surfactants
CTAB
SDBS
OP
SDS
CPB
Without surfactant
If
100.0
14.6
14.7
15.5
0.31
14.6
80
If 100
60
80
40 60
If
10
20
40
30
40
50
methanol(V%) Fig. 4. The effect of methanol on the fluorescence intensity. Conditions: EA: 4.00 × 10−5 mol L−1 ; CTAB: 5.00 × 10−3 mol L−1 ; pH 7.00 (Borax–HCl).
20 0 0
2
4
6 -3
8
15 min was chosen as the standard for all the fluorescence measurements.
10
-1
CTAB(10 molL ) Fig. 3. The effect of the concentration of CTAB on the fluorescence intensity. Conditions: EA: 4.00 × 10−5 mol L−1 ; pH 7.00 (Borax–HCl).
In Table 1, it can be seen that anionic and nonionic surfactants have no obvious effect on the fluorescence intensity of EA, whereas the cationic surfactant CTAB has synergistic enhancement effect. But the cationic surfactant CPB (cetylpyridinium bromide) has quenching effect on the fluorescence intensity of EA. As it is known, EA is soluble in pyridine for the formation of chargetransfer complexes. So we assume that EA and the pyridine of CPB formed the charge-transfer complex in EA–CPB system. In Fig. 3, experiments clearly show that fluorescence intensity reaches maximum and no further change at CTAB concentration >0.002 mol L−1 . Therefore, CTAB at the concentration of 0.005 mol L−1 is selected for the assay. 3.4. Effect of methanol As EA dissolved in methanol, the quantity of methanol would influence the fluorescence of the system. The effect of methanol on the fluorescence intensity is shown in Fig. 4. It can be seen that the maximum fluorescence intensity of the system is obtained when the quantity of methanol is 35%. 3.5. Stability Tests show that the fluorescence intensity reached a maximum within 15 min after reagents had been added and remained stable for at least 2 h. So the assay has good stability. In this paper,
3.6. Effect of foreign substances The interferences of various ions including common anions, cations, amino acids and glucides were tested according to the standard procedure when the concentration of EA was fixed at 1.0 × 10−9 mol L−1 . In Table 2, it is found that these foreign substances have no or little effect on the determination of EA under the permission of ±5% errors, implying high selectivity for the proposed method. 4. Analysis of sample 4.1. Analytical parameters Under the optimum conditions defined, a linear relationship is obtained between the fluorescence intensity and the concentration of EA. The analytical parameters for this method are given in Table 3. It can be seen that the detection limits of this method are 3.2 × 10−10 mol L−1 and 5.9 × 10−10 mol L−1 for EA excited at 266 nm and 388 nm, respectively. 4.2. Sample determination The standard addition method was used for the determination of EA in real samples. The pomegranate rind samples were treated according to the sample preparation mentioned in Section 2 and determined using the proposed method. The results are shown in Table 4. As what can be seen, the results obtained by this method agree with the UV–Vis spectral methods.
Table 2 Interference from foreign substances. Foreign substances K+ , Cl− Na+ , SO4 2− Zn2+ , Cl− NH4 + , Cl Fe2+ , SO4 2− Fe3+ , SO4 2− Mg2+ , Cl− K+ , NO3 − Mn2+ , SO4 2− Ca2+ , Cl− Cu2+ , Cl− Na+ , Cl−
Concentration (10−7 mol L−1 ) 5 10 2 2 2 4 2 2 2 2 2 3
If (%) 4.082 4.32 4.863 2.933 5.292 4.25 2.394 4.359 4.288 2.827 2.959 4.139
Conditions: EA: 10−9 mol L−1 ; CTAB: 5.00 × 10−3 mol L−1 ; pH 7.00 (Borax–HCl). a 10−11 g mL−1 .
Foreign substances Sucrose Fructose Glucose l-␣-Ala l-Leu l-Arga l-Pro l-Glu l-Asp l-Trp DNAa RNAa
Concentration (10−7 mol L−1 ) 2 2 2 2 3 20 2 0.4 0.4 0.04 0.02 0.2
If (%) 4.139 4.204 3.925 4.029 2.006 5.268 3.678 3.09 4.096 3.618 6.706 4.621
1016
F. Wang et al. / Spectrochimica Acta Part A 78 (2011) 1013–1017
Table 3 Analytical parameters of this method. Excitation wavelength (nm)
Linear range (mol L−1 ) −10
−5
8.0 × 10 –4.0 × 10 8.0 × 10−10 –4.0 × 10−5
388 nm 266 nm
Linear equation
Correlation coefficient (r)
Limit of detection (mol L−1 )
If = 2.41 × 10 C + 0.344 If = 2.66 × 106 C − 2.15
0.9972 0.9857
5.9 × 10−10 3.2 × 10−10
6
Table 4 The results of samples determination.
5.2. Absorption spectra
UV absorption method (g/g, %) [10]
Proposed method (g/g, %)
Average (g/g, %)
RSD (%)
7.06 9.48
7.10, 7.17, 7.05, 7.06, 7.08 9.48, 9.49, 9.42, 9.51, 9.39
7.09 9.46
0.67 0.54
The above results indicate that the accuracy and precision of the method are satisfactory.
5. The interaction mechanism From Fig. 1 it is apparent that CTAB can greatly enhance the fluorescence of EA. Furthermore, it can be seen that the emission peak of EA shifts to a shorter wavelength, which indicates that the microenvironment of the system has changed. This change can also be proved by both the absorption spectra and the character of the micelle.
5.1. Define CMC In order to understand the present form of the surfactant in this system, the critical micelle concentration (CMC) of CTAB in the presence of EA is measured by determining its conductivity under this experimental condition as shown in Fig. 5. It can be seen from Fig. 5 that the CMC of CTAB in this system is 1.0 × 10−5 mol L−1 while the concentration of CTAB in this system is 0.005 mol L−1 , which is greatly higher than the CMC. Generally speaking, spherical micelle is easy to form in surfactant when its concentration reaches CMC with the shape and size of micelle not distinctively changed in the concentration of 1–10 CMC. But when the concentration of surfactant reaches 10 CMC or much higher, its shape turned into rodlike micelle in EA–CTAB system [13].
It is known that EA has four rings representing the lipophillic domain and four phenolic and two lactone groups in the Ellagic acid formula. Fig. 6 indicates that absorption spectra of EA in aqueous solution containing 10% methanol at varying pHs. The dissociation constant of a weak acid can be determined according to the formula [14]: pKa = pH +
A − AEA− , AEA − A
where A represents the absorption of system including unionized form EA and the deprotonated EA at a particular pH, and Ka is the dissociation constant of EA, AEA and AEA− is the absorption of unionized form EA and deprotonated EA, respectively. Experimentally generated profile was 5.6, which is in agreement with the theoretical profile based on the reported pKa value of 5.6 [15]. EA is a weak acid with four phenolic groups and theoretically, should exhibit four acid dissociation constants [16]. Below pH 5.6, EA exists in a mono-deprotonated form [15] whereas, at pH higher than 5.6, deprotonation occurs at two hydroxyl group positions. Therefore, it is assumed that in this system, EA and EA–Borax complex exist as anion formation. Fig. 7 is the absorption spectra of this system. It is shown that EA exhibits two weak absorption band centered at 253 nm and 365 nm, which all contains –* transition [17]. When EA is mixed with Borax, the characteristic absorption peak is enhanced and redshifted. The reason for this phenomenon is that the large and stable conjugate structure is formed through Borax combining with phenolic hydroxyl of EA. Furthermore, the fluorescence intensity of EA–Borax is also enhanced (Fig. 1b). The typical absorption peak of EA redshifts more and the fluorescence intensity is greatly enhanced when CTAB is added into EA–Borax system. This indicates that EA–Borax complex can be solubilized in the micelle of CTAB. 5.3. The effect of the ionic strength The influence of ionic strength on the fluorescence intensity of this system is tested as shown in Fig. 8. Experiments indicate that the fluorescence intensity of the system has obviously decreased
2.0
ABS
1.6
4 5 1
1.2
3 0.8
2 0.4 0.0 200
250
300
350
400
Wavelength(nm) Fig. 5. Conductivity curve of CTAB. Conditions: EA: 4.00 × 10−5 mol L−1 ; pH 7.00 (Borax–HCl).
Fig. 6. pH dependence of electronic absorption spectra of EA in mixture MeOH:H2 O = 1:9. pH: (1) 2, (2) 3, (3) 5, (4) 8 5, and (5) 9. Conditions: EA: 4.00 × 10−5 mol L−1 .
F. Wang et al. / Spectrochimica Acta Part A 78 (2011) 1013–1017
90
EA–Borax complex combines with the positive NH4 + group when EA–Borax is added into CTAB micelles. Therefore EA–Borax complex molecules presents at the outer shell of the cationic micelles contracts the charge of CTAB [19]. So, the “space” around the EA–Borax complex becomes smaller. Therefore, it is assumed that EA solubilized in the CTAB micelle as its rotational ability decreases while the viscosity of the solution and the fluorescence polarization of EA increases. Therefore, the CTAB micelle can provide a hydrophobic microenvironment for EA–Borax complex, which prevent collision with water molecules and thus decreasing the energy loss of EA–Borax complex. And it then improves the fluorescence quantum yield from 0.0032 to 0.025 (excited at 266 nm) and from 0.033 to 0.116 (excited at 388 nm), so that the fluorescence intensity of EA gets enhanced. But in the EA–Borax–CPB system, the charge-transfer complexes form because EA combines with pyridine ring which is in the outer layer of the micelle. So, the viscosity of the solution and the fluorescence polarization of EA reach the maximum value. Therefore, it is difficult for the fluorescence intensity to enhance because EA can be adsorbed to the outer layer of the micelle but not solubilized in the CTAB micelle.
80
6. Conclusion
1.5
2
1
1.0
A 0.5
3 4
0.0 250
300
350
400
450
Wavelength(nm) Fig. 7. The absorption spectra of EA. (1) Borax–EA–CTAB; (2) Borax–EA; (3) EA; (4) Borax–CTAB. Conditions: pH 7.00 (Borax–HCl); EA: 4.00 × 10−5 mol L−1 ; CTAB: 5.00 × 10−3 mol L−1 .
100
If
1017
70 60 50 0.0
0.2
0.4
0.6
0.8
1.0
NaCl(molL-1) Fig. 8. The effect of the ionic strength on the fluorescence intensity. Conditions: pH 7.00 (Borax–HCl); EA: 4.00 × 10−5 mol L−1 ; CTAB: 5.00 × 10−3 mol L−1 .
when NaCl is added into the system. So, it can be assumed that the interaction of EA, Borax and CTAB in this system forming the ternary complex is through electric attraction. At high concentration of salt, the fluorescence of this system decreases because charges could be screened, so that the interaction via electric attraction will be weakened.
In this work, a sensitive and convenient method for the determination of Ellagic acid is proposed. The method is used for the determination of the Ellagic acid in pomegranate rind samples, and the results are satisfactory. The interaction mechanism studies found that the negative EA–Borax complex is formed and solubilized in the cationic surfactant CTAB micelle. Ellagic acid is practically insoluble in water, but quite soluble in micelles, and in this medium it also fluoresces appreciably. Therefore, this work is valuable for the understanding of the interaction mechanism between Ellagic acid and surfactants, and potentially this method can apply to analyses of some drugs and cosmetics. Acknowledgement The Natural Science Foundations of Shandong Province Y2008B36 supported this work.
5.4. The fluorescence enhancement of EA–Borax–CTAB system References According to Perrin equation, the microviscosity of the microenvironment can be estimated using the fluorescence polarization of fluorescence probe [18]. A large value reflects a larger microviscosity than a lower. The polarizer accessories are installed at the F-4600 spectrofluorometer, and the system polarization spectroscopy are measured in the optimum conditions and then calculated according to the polarization formula P = (I − GI⊥ )/(I + GI⊥ ), G = i⊥ /i . The results are shown in Table 5. In Table 5, it can be found that the fluorescence polarization increases gradually from no surfactant, anion surfactant to cationic surfactant. The core of the cationic surfactant CTAB micelle consists of the combining hydrophobic group; and the outer layer is the charged groups of CTAB, namely NH4 + . It is believed that the negative Table 5 The fluorescence polarization of different surfactant systems. Surfactant
Without surfactant
SDBS
SDS
CPB
CTAB
P
0.03999
0.04201
0.04007 0.41292 0.11738
OP
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]
0.0465
D.A. Vattem, K. Shetty, J. Food Biochem. 29 (2005) 234. N.P. Seeram, L.S. Adams, S.M. Henning, et al., J. Nutr. Biochem. 16 (2005) 360. Madal, Shivappurkar, Galati, Stoner, Carcinogenesis 9 (1988) 1313. Mandal, Stoner, Carcinogenesis 11 (1990) 55. M. Falsaperla, G. Morgia, A. Tartarone, R. Ardito, G. Romano, Eur. Urol. 47 (2005) 449. I. Bala, V. Bhardwaj, S. Hariharan, M.N.V. Ravi Kumar, J. Pharm. Biomed. Anal. 40 (2006) 206. D. Venkat Ratnam, V. Bhardwaj, M.N.V. Ravi Kumar, Talanta 70 (2006) 387. T.Y. Lin, R.P. Vine, Food Sci. 55 (1990) 1607. B.H. Zhou, Z.H. Wu, X.J. Li, J. Zhang, X.M. Hu, Phytochem. Anal. 19 (2008). J.H. Chen, D.M. Wu, Y.M. Wang, Z.S. Wu, Biomass Chem. Eng. 41 (2007) 18. S.H. Häkkinen, S.O. Kärenlampi, H.M. Mykkänen, I.M. Heinonen, A.R. Törrönen, Eur. Food Res. Technol. 212 (2000) 75. O.S. Wolfbeis, P. Hochmuth, Monatsh. Chem. 117 (1986) 369. A. Malliaris, J. Lang, R. Zana, J. Colloid Interface Sci. 110 (1986) 237. H.Q. Peng, G.Y. Shi, M. Liu, J.M. Xu, J. East China Normal Univ. (Nat. Sci.) 27 (2002) 54. M. Hasegawa, M. Terauchi, Y. Kikuchi, A. Nakao, J. Okubo, T. Yoshinaga, H. Hiratsuka, M. Kobayashi, T. Hoshi, Monatsh. Chem. 134 (2003) 811. R.E. Press, D. Hardcastle, J. Appl. Chem. 19 (1969) 247. T. Sato, M. Kataoka, J. Heterocycl. Chem. 34 (1997) 665. G.Z. Chen, X.Z. Huang, J.G. Xu, Z.Z. Zhen, Z.B. Wang, Fluorescence Analytical Methods, third ed., Science Press, Beijing, 2006, pp. 184. G.X. Zhao, B.Y. Zhu, Principles of Surfactant Action, China Light Industry Press, 2003, pp. 591.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Enhancing fluorescence intensity of Ellagic acid in Borax–HCl–CTAB micelles Feng Wang a,∗ , Wei Huang a , Shuai Zhang a , Guokui Liu a , Kexiang Li a , Bo Tang b a b
Department of Chemistry and Chemical Engineering, Zaozhuang University, Zaozhuang 277160, PR China College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 8 January 2010 Received in revised form 7 December 2010 Accepted 10 December 2010
Ellagic acid (C14 H6 O8 ), a naturally occurring phytochemical, found mainly in berries and some nuts, has anticarcinogenic and antioxidant properties. It is found that fluorescence of Ellagic acid (EA) is greatly enhanced by micelle of cetyltrimethylammonium bromide (CTAB) surfactant. Based on this effect, a sensitive proposed fluorimetric method was applied for the determination of Ellagic acid in aqueous solution. In the Borax–HCl buffer, the fluorescence intensity of Ellagic acid in the presence of CTAB is proportional to the concentration of Ellagic acid in range from 8.0 × 10−10 to 4.0 × 10−5 mol L−1 ; and the detection limits are 3.2 × 10−10 mol L−1 and 5.9 × 10−10 mol L−1 excited at 266 nm and 388 nm, respectively. The actual samples of pomegranate rinds are simply manipulated and satisfactorily determined. The interaction mechanism studies argue that the negative EA–Borax complex is formed and solubilized in the cationic surfactant CTAB micelle in this system. The fluorescence intensity of EA enhances because the CTAB micelle provides a hydrophobic microenvironment for EA–Borax complex, which can prevent collision with water molecules and decrease the energy loss of EA–Borax complex. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
Keywords: Ellagic acid CTAB Micelle enhancement Fluorescence spectroscopy
1. Introduction Ellagic acid, [2,3,7,8-tetrahydroxybenzopyrano [5,4,3cde]benzopyran-5,10-dione], is a polyphenol antioxidant found in numerous fruits and vegetables. The antiproliferative and antioxidant properties of Ellagic acid have spurred preliminary research into the potential health benefits of Ellagic acid consumption [1–5]. The structure of Ellagic acid is as follows: O
C
O
OH OH
OH HO
O
C
liquid chromatography [6,7], paper chromatography [8], capillary electrophoresis [9] and UV–Vis spectrophotometry [10] have been employed to determine Ellagic acid are present in a variety of matrices such as grape juice, strawberries and pomegranate rinds. But these current methods have a limited sensitivity because they are based on absorption measurements. Furthermore, the fact that Ellagic acid has poor solubility makes the determination of trace amount of Ellagic acid in aqueous solution difficult. In this paper, a study of the fluorescence of Ellagic acid in several micelle systems has been carried out. It is found that the fluorescence intensity of Ellagic acid can be greatly enhanced by the cationic surfactant cetyltrimethylammonium bromide (CTAB). Based on the above property, the sensitive quantitative analysis of Ellagic acid in aqueous solution is established. This method has the advantages of high sensitivity, selectivity and stability.
O
The highest levels of Ellagic acid are found in raspberries, strawberries, and pomegranates, especially when freeze-dried. Red raspberry leaves, which also contain Ellagic acid, are available in capsule, powder and liquid forms. But the correct dosages of these preparations are not known. Methods such as high performance
∗ Corresponding author at: Department of Chemistry and Chemical Engineering, Zaozhuang, Shandong 277160, PR China. Tel.: +86 632 3786735. E-mail addresses: [email protected], [email protected] (F. Wang).
2. Experimental 2.1. Apparatus Fluorescent spectra were obtained using F-4600 spectrofluorometer (Hitachi, Japan). Absorption spectra were recorded using UV-2401PC (Shimadzu, Japan) spectrophotometer. Electrical conductivities were measured by DDS-11A conductivity meter (Leici Shanghai). All acidity measurements were performed with a Delta320-S acidity meter (Mettler Toledo, Shanghai).
1386-1425/$ – see front matter Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2010.12.030
1014
F. Wang et al. / Spectrochimica Acta Part A 78 (2011) 1013–1017
100 80
a
100
1
40
1
80 60
60
If
b
4
If
4
40
20
20
0
0 250
300
350
400
400
Wavelength(nm)
450
500
550
600
Wavelength(nm)
Fig. 1. Excitation spectra (em = 454 nm) (a) and emission spectra (ex = 388 nm) (b). (1) Borax–HCl–EA–CTAB; (2) Borax–HCl–EA; (3) EA–CTAB; (4) EA. Conditions: EA: 4.00 × 10−5 mol L−1 ; CTAB: 5.00 × 10−3 mol L−1 ; pH 7.00 (Borax–HCl).
2.2. Reagents A stock solution of Ellagic acid (EA) (4.00 × 10−4 mol L−1 ) was first prepared by dissolving EA in methanol, and then diluted to proper concentration with methanol. A series of Borax–HCl buffer solutions (0.05 mol L−1 ) were used for the pH adjustment. A solution of surfactant cetyltrimethylammonium bromide (CTAB) (0.050 mol L−1 ) was prepared by dissolving CTAB in deionized water. All the chemicals used were of analytical reagent grade and deionized water was used throughout. 2.3. Experimental procedures In a 10 mL test-tube, solutions were added in the following order: 2.00 mL Borax–HCl (pH 7.00), 1.00 mL CTAB (0.050 mol L−1 ), 2.50 mL methanol and definite standard EA (or sample solution). The mixture was diluted to certain volume by water. The fluorescence intensity is measured at ex /em = 388 nm/454 nm in a 1 cm quartz cell and with slit at 10.0 nm for the excitation and emission.
CTAB, which causes a blue-shift to 454 nm. This indicates that EA is solubilized in the micelle of CTAB. 3.2. Effects of pH and buffers The effect of pH on the fluorescence intensity of the system is shown in Fig. 2. It can be seen that the maximum intensity is obtained in the pH 7.00 buffer. Experiments indicate that different buffers also have very different effects on fluorescence intensity (If ) of the system. The If for Na2 HPO4 –citric, NaH2 PO4 –Na2 HPO4 , KH2 PO4 –Na2 HPO4 , Tris–HCl, BR (Briton–Robinson)–NaOH, HMTA–HCl, Borax–HCl at pH 7.00 is 0.70, 0.73, 0.73, 0.60, 35.8, 0.54, 100.0, respectively. The results indicate that the Borax–HCl is the best in the buffers tested and the BR–NaOH is the second, so the Borax–HCl buffer (pH 7.00) is selected for the assay and the optimum volume of Borax–HCl buffer is 2.00 mL. The reason for the enhancement of the EA fluorescence is that the H3 BO3 in the two buffers of BR–NaOH and Borax–HCl can coordinate ortho-dihydroxy group which is present in the structure of EA to form a chelate [12].
2.4. Sample treatment Ellagic acid was extracted from real samples according to previously reported procedure: the samples spices (pomegranate rinds, acquired from drugstore) were dried at 100 ◦ C for 24 h and then ground to fine powder. A weighed comminuted pomegranate rind was refluxed at 85 ± 5 ◦ C in 100% methanol for 2 h and then the solvent was separated by filtration. The collected extraction was evaporated in a rotary evaporator at 35 ◦ C until no further droplet came out [11]; the concentrated solution and combined methanol washings were transferred into a volumetric flask and diluted to 50 mL with methanol. An appropriate volume of this solution was pipetted into a 10 mL flask and its Ellagic acid content was determined by standard addition method according to the procedure described above. 3. Results and discussion
3.3. Effect of surfactants The effects of different surfactants on the fluorescence intensity were tested and the results are shown in Table 1.
100
80
If
60
40
3.1. Fluorescence spectra 20
The fluorescence spectra of (1) Borax–HCl–EA–CTAB, (2) Borax–HCl–EA, (3) EA–CTAB, and (4) EA systems are shown in Fig. 1. It can be seen that EA in aqueous solution has very weak fluorescence with two excitation peaks of 266 nm and 388 nm and an emission peak at about 450 nm, whereas it has enhancement and red-shift in the Borax–HCl buffer. And the fluorescence peak can be further greatly enhanced by the addition of a cationic surfactant
0 3
4
5
6
7
8
9
pH Fig. 2. The effect of pH on the fluorescence intensity. Conditions: EA: 4.00 × 10−5 mol L−1 ; CTAB: 5.00 × 10−3 mol L−1 ; pH 7.00 (Borax–HCl).
F. Wang et al. / Spectrochimica Acta Part A 78 (2011) 1013–1017 Table 1 Effects of surfactants on the fluorescence intensity.
1015
100
Surfactants
CTAB
SDBS
OP
SDS
CPB
Without surfactant
If
100.0
14.6
14.7
15.5
0.31
14.6
80
If 100
60
80
40 60
If
10
20
40
30
40
50
methanol(V%) Fig. 4. The effect of methanol on the fluorescence intensity. Conditions: EA: 4.00 × 10−5 mol L−1 ; CTAB: 5.00 × 10−3 mol L−1 ; pH 7.00 (Borax–HCl).
20 0 0
2
4
6 -3
8
15 min was chosen as the standard for all the fluorescence measurements.
10
-1
CTAB(10 molL ) Fig. 3. The effect of the concentration of CTAB on the fluorescence intensity. Conditions: EA: 4.00 × 10−5 mol L−1 ; pH 7.00 (Borax–HCl).
In Table 1, it can be seen that anionic and nonionic surfactants have no obvious effect on the fluorescence intensity of EA, whereas the cationic surfactant CTAB has synergistic enhancement effect. But the cationic surfactant CPB (cetylpyridinium bromide) has quenching effect on the fluorescence intensity of EA. As it is known, EA is soluble in pyridine for the formation of chargetransfer complexes. So we assume that EA and the pyridine of CPB formed the charge-transfer complex in EA–CPB system. In Fig. 3, experiments clearly show that fluorescence intensity reaches maximum and no further change at CTAB concentration >0.002 mol L−1 . Therefore, CTAB at the concentration of 0.005 mol L−1 is selected for the assay. 3.4. Effect of methanol As EA dissolved in methanol, the quantity of methanol would influence the fluorescence of the system. The effect of methanol on the fluorescence intensity is shown in Fig. 4. It can be seen that the maximum fluorescence intensity of the system is obtained when the quantity of methanol is 35%. 3.5. Stability Tests show that the fluorescence intensity reached a maximum within 15 min after reagents had been added and remained stable for at least 2 h. So the assay has good stability. In this paper,
3.6. Effect of foreign substances The interferences of various ions including common anions, cations, amino acids and glucides were tested according to the standard procedure when the concentration of EA was fixed at 1.0 × 10−9 mol L−1 . In Table 2, it is found that these foreign substances have no or little effect on the determination of EA under the permission of ±5% errors, implying high selectivity for the proposed method. 4. Analysis of sample 4.1. Analytical parameters Under the optimum conditions defined, a linear relationship is obtained between the fluorescence intensity and the concentration of EA. The analytical parameters for this method are given in Table 3. It can be seen that the detection limits of this method are 3.2 × 10−10 mol L−1 and 5.9 × 10−10 mol L−1 for EA excited at 266 nm and 388 nm, respectively. 4.2. Sample determination The standard addition method was used for the determination of EA in real samples. The pomegranate rind samples were treated according to the sample preparation mentioned in Section 2 and determined using the proposed method. The results are shown in Table 4. As what can be seen, the results obtained by this method agree with the UV–Vis spectral methods.
Table 2 Interference from foreign substances. Foreign substances K+ , Cl− Na+ , SO4 2− Zn2+ , Cl− NH4 + , Cl Fe2+ , SO4 2− Fe3+ , SO4 2− Mg2+ , Cl− K+ , NO3 − Mn2+ , SO4 2− Ca2+ , Cl− Cu2+ , Cl− Na+ , Cl−
Concentration (10−7 mol L−1 ) 5 10 2 2 2 4 2 2 2 2 2 3
If (%) 4.082 4.32 4.863 2.933 5.292 4.25 2.394 4.359 4.288 2.827 2.959 4.139
Conditions: EA: 10−9 mol L−1 ; CTAB: 5.00 × 10−3 mol L−1 ; pH 7.00 (Borax–HCl). a 10−11 g mL−1 .
Foreign substances Sucrose Fructose Glucose l-␣-Ala l-Leu l-Arga l-Pro l-Glu l-Asp l-Trp DNAa RNAa
Concentration (10−7 mol L−1 ) 2 2 2 2 3 20 2 0.4 0.4 0.04 0.02 0.2
If (%) 4.139 4.204 3.925 4.029 2.006 5.268 3.678 3.09 4.096 3.618 6.706 4.621
1016
F. Wang et al. / Spectrochimica Acta Part A 78 (2011) 1013–1017
Table 3 Analytical parameters of this method. Excitation wavelength (nm)
Linear range (mol L−1 ) −10
−5
8.0 × 10 –4.0 × 10 8.0 × 10−10 –4.0 × 10−5
388 nm 266 nm
Linear equation
Correlation coefficient (r)
Limit of detection (mol L−1 )
If = 2.41 × 10 C + 0.344 If = 2.66 × 106 C − 2.15
0.9972 0.9857
5.9 × 10−10 3.2 × 10−10
6
Table 4 The results of samples determination.
5.2. Absorption spectra
UV absorption method (g/g, %) [10]
Proposed method (g/g, %)
Average (g/g, %)
RSD (%)
7.06 9.48
7.10, 7.17, 7.05, 7.06, 7.08 9.48, 9.49, 9.42, 9.51, 9.39
7.09 9.46
0.67 0.54
The above results indicate that the accuracy and precision of the method are satisfactory.
5. The interaction mechanism From Fig. 1 it is apparent that CTAB can greatly enhance the fluorescence of EA. Furthermore, it can be seen that the emission peak of EA shifts to a shorter wavelength, which indicates that the microenvironment of the system has changed. This change can also be proved by both the absorption spectra and the character of the micelle.
5.1. Define CMC In order to understand the present form of the surfactant in this system, the critical micelle concentration (CMC) of CTAB in the presence of EA is measured by determining its conductivity under this experimental condition as shown in Fig. 5. It can be seen from Fig. 5 that the CMC of CTAB in this system is 1.0 × 10−5 mol L−1 while the concentration of CTAB in this system is 0.005 mol L−1 , which is greatly higher than the CMC. Generally speaking, spherical micelle is easy to form in surfactant when its concentration reaches CMC with the shape and size of micelle not distinctively changed in the concentration of 1–10 CMC. But when the concentration of surfactant reaches 10 CMC or much higher, its shape turned into rodlike micelle in EA–CTAB system [13].
It is known that EA has four rings representing the lipophillic domain and four phenolic and two lactone groups in the Ellagic acid formula. Fig. 6 indicates that absorption spectra of EA in aqueous solution containing 10% methanol at varying pHs. The dissociation constant of a weak acid can be determined according to the formula [14]: pKa = pH +
A − AEA− , AEA − A
where A represents the absorption of system including unionized form EA and the deprotonated EA at a particular pH, and Ka is the dissociation constant of EA, AEA and AEA− is the absorption of unionized form EA and deprotonated EA, respectively. Experimentally generated profile was 5.6, which is in agreement with the theoretical profile based on the reported pKa value of 5.6 [15]. EA is a weak acid with four phenolic groups and theoretically, should exhibit four acid dissociation constants [16]. Below pH 5.6, EA exists in a mono-deprotonated form [15] whereas, at pH higher than 5.6, deprotonation occurs at two hydroxyl group positions. Therefore, it is assumed that in this system, EA and EA–Borax complex exist as anion formation. Fig. 7 is the absorption spectra of this system. It is shown that EA exhibits two weak absorption band centered at 253 nm and 365 nm, which all contains –* transition [17]. When EA is mixed with Borax, the characteristic absorption peak is enhanced and redshifted. The reason for this phenomenon is that the large and stable conjugate structure is formed through Borax combining with phenolic hydroxyl of EA. Furthermore, the fluorescence intensity of EA–Borax is also enhanced (Fig. 1b). The typical absorption peak of EA redshifts more and the fluorescence intensity is greatly enhanced when CTAB is added into EA–Borax system. This indicates that EA–Borax complex can be solubilized in the micelle of CTAB. 5.3. The effect of the ionic strength The influence of ionic strength on the fluorescence intensity of this system is tested as shown in Fig. 8. Experiments indicate that the fluorescence intensity of the system has obviously decreased
2.0
ABS
1.6
4 5 1
1.2
3 0.8
2 0.4 0.0 200
250
300
350
400
Wavelength(nm) Fig. 5. Conductivity curve of CTAB. Conditions: EA: 4.00 × 10−5 mol L−1 ; pH 7.00 (Borax–HCl).
Fig. 6. pH dependence of electronic absorption spectra of EA in mixture MeOH:H2 O = 1:9. pH: (1) 2, (2) 3, (3) 5, (4) 8 5, and (5) 9. Conditions: EA: 4.00 × 10−5 mol L−1 .
F. Wang et al. / Spectrochimica Acta Part A 78 (2011) 1013–1017
90
EA–Borax complex combines with the positive NH4 + group when EA–Borax is added into CTAB micelles. Therefore EA–Borax complex molecules presents at the outer shell of the cationic micelles contracts the charge of CTAB [19]. So, the “space” around the EA–Borax complex becomes smaller. Therefore, it is assumed that EA solubilized in the CTAB micelle as its rotational ability decreases while the viscosity of the solution and the fluorescence polarization of EA increases. Therefore, the CTAB micelle can provide a hydrophobic microenvironment for EA–Borax complex, which prevent collision with water molecules and thus decreasing the energy loss of EA–Borax complex. And it then improves the fluorescence quantum yield from 0.0032 to 0.025 (excited at 266 nm) and from 0.033 to 0.116 (excited at 388 nm), so that the fluorescence intensity of EA gets enhanced. But in the EA–Borax–CPB system, the charge-transfer complexes form because EA combines with pyridine ring which is in the outer layer of the micelle. So, the viscosity of the solution and the fluorescence polarization of EA reach the maximum value. Therefore, it is difficult for the fluorescence intensity to enhance because EA can be adsorbed to the outer layer of the micelle but not solubilized in the CTAB micelle.
80
6. Conclusion
1.5
2
1
1.0
A 0.5
3 4
0.0 250
300
350
400
450
Wavelength(nm) Fig. 7. The absorption spectra of EA. (1) Borax–EA–CTAB; (2) Borax–EA; (3) EA; (4) Borax–CTAB. Conditions: pH 7.00 (Borax–HCl); EA: 4.00 × 10−5 mol L−1 ; CTAB: 5.00 × 10−3 mol L−1 .
100
If
1017
70 60 50 0.0
0.2
0.4
0.6
0.8
1.0
NaCl(molL-1) Fig. 8. The effect of the ionic strength on the fluorescence intensity. Conditions: pH 7.00 (Borax–HCl); EA: 4.00 × 10−5 mol L−1 ; CTAB: 5.00 × 10−3 mol L−1 .
when NaCl is added into the system. So, it can be assumed that the interaction of EA, Borax and CTAB in this system forming the ternary complex is through electric attraction. At high concentration of salt, the fluorescence of this system decreases because charges could be screened, so that the interaction via electric attraction will be weakened.
In this work, a sensitive and convenient method for the determination of Ellagic acid is proposed. The method is used for the determination of the Ellagic acid in pomegranate rind samples, and the results are satisfactory. The interaction mechanism studies found that the negative EA–Borax complex is formed and solubilized in the cationic surfactant CTAB micelle. Ellagic acid is practically insoluble in water, but quite soluble in micelles, and in this medium it also fluoresces appreciably. Therefore, this work is valuable for the understanding of the interaction mechanism between Ellagic acid and surfactants, and potentially this method can apply to analyses of some drugs and cosmetics. Acknowledgement The Natural Science Foundations of Shandong Province Y2008B36 supported this work.
5.4. The fluorescence enhancement of EA–Borax–CTAB system References According to Perrin equation, the microviscosity of the microenvironment can be estimated using the fluorescence polarization of fluorescence probe [18]. A large value reflects a larger microviscosity than a lower. The polarizer accessories are installed at the F-4600 spectrofluorometer, and the system polarization spectroscopy are measured in the optimum conditions and then calculated according to the polarization formula P = (I − GI⊥ )/(I + GI⊥ ), G = i⊥ /i . The results are shown in Table 5. In Table 5, it can be found that the fluorescence polarization increases gradually from no surfactant, anion surfactant to cationic surfactant. The core of the cationic surfactant CTAB micelle consists of the combining hydrophobic group; and the outer layer is the charged groups of CTAB, namely NH4 + . It is believed that the negative Table 5 The fluorescence polarization of different surfactant systems. Surfactant
Without surfactant
SDBS
SDS
CPB
CTAB
P
0.03999
0.04201
0.04007 0.41292 0.11738
OP
[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]
0.0465
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