- Email: [email protected]
A novel and rapid method for the spectrofluorometric determination of curcumin in curcumin spices and flavors
MICROCHEMICAL
JOURNAL
46,
209-214
(1992)
A Novel and Rapid Method for the Spectrofluorometric Determination of Curcumin in Curcumin Spices and Flavors’ FADHIL Department
of
Chemistry, Received
College February
JASIM' AND FATIMA ALI of Science,
University
of Baghdad,
12, 1991; accepted
October
Jadriya,
Baghdad,
Iraq
17, 1991
A sensitive and rapid spectrofluorometric method for determination of microamounts of curcumin in curcumin spices and related flavors has been developed. It involves dissolving curcumin samples in dry acetone, irradiating the resulting clear solution at X, = 424 nm, and measuring the stable intense green-yellow fluorescence at X, = 504 nm. The fluorescent solution shows no change in A, or hF or in fluorescence intensity for at least 1 month under ambient conditions. Beer’s law is followed over the range 0.0-500 ppb of curcumin. The sensitivity and detection limit (S/N = 2) are 4.7 and 0.34 ppb of curcumin per fluorescence unit, respectively. The RSD and recovery for a series of concentrations (0.01-0.3 ppm) are 1.13-2.03 and 98.9&100%, respectively. Temperature control is needed; pH adjustment and 0, removal from test solution are unnecessary. Under the specified conditions, water is the only quencher for curcumin fluorescence. Direct calibration determination is satisfactory and therefore there is no need to use a rather lengthy standard additions technique. B 1992 Academic
Press, Inc.
INTRODUCTION
Techniques for the separation and determination of curcumin and its derivatives in a variety of matrices have been described in a previous paper (I). Data have been published on its uses in food technology as a flavoring agent, because of its antioxidant properties; in the dying industry, for dying natural and synthetic silks and rayons (2); in cosmetics (3); and as an insect repellent, for protection of crops, particularly wheat, barley, and corn during storage (4). Recently, attempts have been made to use it as an antioxidant additive for lubricants and motor oils (5). A survey of the chemical literature revealed no adequate and comprehensive study of the spectrofluorometric determination of curcumin in curcumin spices and flavors. Therefore, this paper describes investigations carried out with the aim of establishing a simple, direct, rapid, and sensitive method for analysis of micro- and submicroamounts of curcumin using molecular spectrofluorometry. EXPERIMENTAL Apparatus
All spectrofluorometric measurements were conducted with a Perkin-Elmer Model MPF-44B scanning spectrofluorometer equipped with an Hittachi Model ’ Iraqi Patent No. 21%, dated July 7, 1989; registered * To whom correspondence should be addressed.
under
the auspices
of Baghdad
0026-265x/92
$4.00
University.
209 Copyright 0 1992 by Academic F’ress, Inc. All rights of reproduction in any form reserved.
210
JASIM AND ALI
056 X-Y recorder and matched l-cm silica cells. Instrument Table 1.
settings are shown in
Reagents
A standard curcumin solution of 100 ppm was initially prepared by dissolving 10 mg of recrystallized curcumin (BDH) in 10 ml of pure dry acetone and diluting to 100 ml with acetone. Dilute standard solutions were prepared from stock solution by appropriate dilution with acetone. Procedure for Cut-cumin Spice and Flavor Samples
Synthetic and commercially available curcumin spices and flavors were dried (100°C for 24 h) and carefully ground in an agate mortar for 20 min to obtain a fine well-mixed powder. A sample of 0.1 g of each was dissolved in a minimum volume of the pure acetone. Undissolved particles, if any, were removed by a short centrifugation. The clear centrifugate and combined washings were transferred to a loo-ml volumetric flask and diluted to volume with acetone. A volume, containing up to 0.5 ppm, of this solution was pipetted into a 50-ml volumetric flask and the curcumin determined in either of two ways: (1) Directly by dilution with acetone- the curcumin content was calculated from the direct linear calibration graph (Fig. 2.1). (2) Indirectly by curcumin standard (0.0-0.5 ppm) additions and dilution to volume with acetone-the concentration of curcumin was calculated from a linear standard additions (0.0-0.5 ppm) graph (Fig. 2.2). The average of triplicate fluorescence power measurements was calculated for samples (simulated and commercial) and standard curcumin solutions. The blank reads zero fluorescence power. Because both direct and analyte addition calibration graphs have identical slopes, the unknown curcumin contents of samples were obtained from the direct calibration graph (Fig. 2.1). TABLE 1 Instrument Settings Parameter Slit bandwidth (nm) Rate of scan (nm/min) AE (nm) A, (nm) Instrument response Energy mode Dynode voltage (DV) Signal gain Scale expansion Temperature (“C) Xenon arc lamp (W)
Setting 240 424 504 1.2 Direct -750 3x 25 150
SPECTROFLUOROMETRIC
DETERMINATION
OF CURCUMIN
211
RESULTS AND DISCUSSION
Effects of Solvents Curcumin is sparingly soluble in water, dichloromethane, pyridine, and cyclohexane. The absorptiometric parameters of curcumin in 12 polar and nonpolar organic solvents were discussed in a previous paper (6). Figure 1 depicts the excitation and fluorescence spectra of curcumin in pure acetone. It appears that the fluorescence spectrum (Fig. 1B) is highly symmetrical, thus revealing the symmetrical nature of the fluorescent curcumin-acetone complex (7,8). From the fluorescence spectra of curcumin with each of the 12 organic solvents (6), it appears that the curcumin-acetone system is the most sensitive and accurate with the lowest detection limit and longest duration. Conformity
to Beer’s Law
Figure 2 is an enlargement of the portion of the main graph near the origin. It is linear only to 0.5 ppm and single-valued (not shown for brevity) to about 6 ppm. Beyond this value, the curve falls off because of concentration quenching (innerfilter effect) due to excessive absorption of fluorescence by the solution (selfquenching). Duplicate and identical spectra were obtained at several concentra-
hi -424
101 10
hp504
/ -
0 O!/
320
360 400 -
440
460
520
560
600
h,nm---b
FIG. 1. Excitation and fluorescence spectra of the curcumin-acetone
system.
212
JASIM AND AL1
160 140 ;120 $00 ;
80 0 ii 60 ii $J 40 0 2 20 00 0.0
0.2 Curcumin
03 IPPm)
0.4 Added
FIG. 2. Direct (1) and standard additions (2) calibration graphs of the curcumin-acetone
tion levels (0.01~50 levels.
ppm) only, indicating
system.
the absence of quenching within these
Effect of UV Radiation Excessive exposures (40 min) of the curcumin-acetone complex to UV radiation from a xenon arc lamp have no detrimental effect on the value of A, or XF or the power of fluorescence, confirming the absence of photodecomposition of the complex. Effect of Oxygen Dissolved air oxygen (ground triplet state) has no quenching effect. This is shown by the constancy of An and XF after the test solution is degassed with a stream of argon. Therefore, degassing of oxygen is not required. Fluorescence power remains the same before and after degassing. Effect of Temperature A change in temperature (5-50°C) has no effect on the values of A, and A, (Fig. 1); however, the fluorescence power (Fig. 3) is affected appreciably. According to Shpol’skii, the effects of nonradiative transitions are less at lower temperatures, leading to better vibrational transitions and therefore to enhanced fluorescence power. Consequently, standard and sample solutions must be measured at the same temperature. The use of thermostated temperatures is not necessary (unless otherwise stated) and any room temperature (20-2X) can be used (Table 1). Effect of Water It has been observed (Fig. 4) that the presence of more than 0.01% (v/v) of water in the curcumin-acetone solution increases At+ i.e., 504 to 520 nm, and causes quenching which increases with the increase in water contents. This is attributed to a reaction between H,O (electron donor = D) and the fluorescent curcumin-
SPECTROFLUOROMETRIC
0OLFIG.
10
15
DETERMINATION
20
25
30 Temp.%
3. Effect of temperature on the fluorescence
35
OF CURCUMIN
40
45
213
50
power of the curcumin-acetone
system.
acetone complex (C*-), resulting in the formation of a nonfluorescent (9, 10) and more stable complex (C*- D+) with lower S, vibrational energy levels and therefore lower energy contents and longer hF, C*100
+ D+ *CD. H,O
Added
90 80
FIG. 4. Effect of various concentrations for water (ml) on A, and fluorescence power of the curcumin-acetone system.
214
JASIM AND AL1
This fact, therefore, necessitates the use of dry acetone for the determination of cm-cumin. Others (II) attributed this quenching to the presence of polarization forces or dipole moments between H,O and the complex which tend to confer more nonradiative transitions with subsequent quenching effects. Water has no effect on excitation or A,. The absence of Rayleigh and Raman scatters may be due to the overlapping effects of the strong excitation and emission spectra of the complex. Sensitivity, Detection Limit, and Linearity Sensitivity, defined by the slope of the calibration curve, i.e., the ppb of curcumin which give one fluorescence power unit, is found to be 4.7 ppb. The limit of detection, defined as the ppb of curcumin which give fluorescence power equal to three times the standard deviation (3s) of 10 replicate measurements with the same solution, is found to be 0.34 ppb. Figure 2 shows that the linear dynamic range, defined as the concentration of curcumin from 10 times the detection limit to the upper concentration limit where a 5% deviation from linearity results, is 0.0034-0.5 ppm, or approximately 0.0-0.5 mm. Precision, Accuracy, and Duration of Fluorescence The percentage RSD is calculated from 10 replicate measurements using solutions of 0.1 and 0.3 ppm because they fall within the linear range of the calibration graph; the corresponding values are 2.03 and 1.13%, respectively. The last two values indicate that precision is a complex characteristic of the concentration of the curcumin-acetone system. The percentages recovery of 0.1 and 0.3 ppm curcumin are found to be 98.96 and lOO.OO%, respectively, for standard and simulated samples. Spectrofluorometrically, the A,, A,, and fluorescence power remain constant when the curcumin-acetone solution is kept at 5-30°C; the fluorescence could last for more than 1 month. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Jasim, F.; Ali, F. Micro&em. .I., 1988, 38, 106-110. Motoi, M.; Tomako, F.; Yoshiko, Y. Gnknba Kiyo, 1981, 29, 97-103. Tonmesen, H. H.; Kavlsen, J. J. Chromatogr., 1983, 259, 367-375. Ghulam, J.; Helen, S. J. &on. Entomol., 1983, 76, 154-165. Sharma, 0. P. Biochem. Pharmacol., 1976, 25, 1811-1817. Jasim, F.; Ali, F. Microchem. J., 1989, 39, 156159. Guibault, G. Cl. Molecular fluorescence spectroscopy. In Comprehensive Analytical Chemistry (G. Svehla, Ed.), Vol. VIII, 1977. 8. Guibault, G. G. Practical Fluorescence: Theory, Methods and Techniques, Dekker, New York, 1973. 9. Chandross, E. A.; Thomas, H. T. Chem. Phys. Lett. 1971, 9, 397402. 10. Beens, H.; Knibbe, H.; Weller, A. J. Chem. Phys., 1967, 47, 1183-l 189. Il. Selinger, B. K.; McDonald, R. J. Aust. J. Chem. 1972, 25, 897-906.
JOURNAL
46,
209-214
(1992)
A Novel and Rapid Method for the Spectrofluorometric Determination of Curcumin in Curcumin Spices and Flavors’ FADHIL Department
of
Chemistry, Received
College February
JASIM' AND FATIMA ALI of Science,
University
of Baghdad,
12, 1991; accepted
October
Jadriya,
Baghdad,
Iraq
17, 1991
A sensitive and rapid spectrofluorometric method for determination of microamounts of curcumin in curcumin spices and related flavors has been developed. It involves dissolving curcumin samples in dry acetone, irradiating the resulting clear solution at X, = 424 nm, and measuring the stable intense green-yellow fluorescence at X, = 504 nm. The fluorescent solution shows no change in A, or hF or in fluorescence intensity for at least 1 month under ambient conditions. Beer’s law is followed over the range 0.0-500 ppb of curcumin. The sensitivity and detection limit (S/N = 2) are 4.7 and 0.34 ppb of curcumin per fluorescence unit, respectively. The RSD and recovery for a series of concentrations (0.01-0.3 ppm) are 1.13-2.03 and 98.9&100%, respectively. Temperature control is needed; pH adjustment and 0, removal from test solution are unnecessary. Under the specified conditions, water is the only quencher for curcumin fluorescence. Direct calibration determination is satisfactory and therefore there is no need to use a rather lengthy standard additions technique. B 1992 Academic
Press, Inc.
INTRODUCTION
Techniques for the separation and determination of curcumin and its derivatives in a variety of matrices have been described in a previous paper (I). Data have been published on its uses in food technology as a flavoring agent, because of its antioxidant properties; in the dying industry, for dying natural and synthetic silks and rayons (2); in cosmetics (3); and as an insect repellent, for protection of crops, particularly wheat, barley, and corn during storage (4). Recently, attempts have been made to use it as an antioxidant additive for lubricants and motor oils (5). A survey of the chemical literature revealed no adequate and comprehensive study of the spectrofluorometric determination of curcumin in curcumin spices and flavors. Therefore, this paper describes investigations carried out with the aim of establishing a simple, direct, rapid, and sensitive method for analysis of micro- and submicroamounts of curcumin using molecular spectrofluorometry. EXPERIMENTAL Apparatus
All spectrofluorometric measurements were conducted with a Perkin-Elmer Model MPF-44B scanning spectrofluorometer equipped with an Hittachi Model ’ Iraqi Patent No. 21%, dated July 7, 1989; registered * To whom correspondence should be addressed.
under
the auspices
of Baghdad
0026-265x/92
$4.00
University.
209 Copyright 0 1992 by Academic F’ress, Inc. All rights of reproduction in any form reserved.
210
JASIM AND ALI
056 X-Y recorder and matched l-cm silica cells. Instrument Table 1.
settings are shown in
Reagents
A standard curcumin solution of 100 ppm was initially prepared by dissolving 10 mg of recrystallized curcumin (BDH) in 10 ml of pure dry acetone and diluting to 100 ml with acetone. Dilute standard solutions were prepared from stock solution by appropriate dilution with acetone. Procedure for Cut-cumin Spice and Flavor Samples
Synthetic and commercially available curcumin spices and flavors were dried (100°C for 24 h) and carefully ground in an agate mortar for 20 min to obtain a fine well-mixed powder. A sample of 0.1 g of each was dissolved in a minimum volume of the pure acetone. Undissolved particles, if any, were removed by a short centrifugation. The clear centrifugate and combined washings were transferred to a loo-ml volumetric flask and diluted to volume with acetone. A volume, containing up to 0.5 ppm, of this solution was pipetted into a 50-ml volumetric flask and the curcumin determined in either of two ways: (1) Directly by dilution with acetone- the curcumin content was calculated from the direct linear calibration graph (Fig. 2.1). (2) Indirectly by curcumin standard (0.0-0.5 ppm) additions and dilution to volume with acetone-the concentration of curcumin was calculated from a linear standard additions (0.0-0.5 ppm) graph (Fig. 2.2). The average of triplicate fluorescence power measurements was calculated for samples (simulated and commercial) and standard curcumin solutions. The blank reads zero fluorescence power. Because both direct and analyte addition calibration graphs have identical slopes, the unknown curcumin contents of samples were obtained from the direct calibration graph (Fig. 2.1). TABLE 1 Instrument Settings Parameter Slit bandwidth (nm) Rate of scan (nm/min) AE (nm) A, (nm) Instrument response Energy mode Dynode voltage (DV) Signal gain Scale expansion Temperature (“C) Xenon arc lamp (W)
Setting 240 424 504 1.2 Direct -750 3x 25 150
SPECTROFLUOROMETRIC
DETERMINATION
OF CURCUMIN
211
RESULTS AND DISCUSSION
Effects of Solvents Curcumin is sparingly soluble in water, dichloromethane, pyridine, and cyclohexane. The absorptiometric parameters of curcumin in 12 polar and nonpolar organic solvents were discussed in a previous paper (6). Figure 1 depicts the excitation and fluorescence spectra of curcumin in pure acetone. It appears that the fluorescence spectrum (Fig. 1B) is highly symmetrical, thus revealing the symmetrical nature of the fluorescent curcumin-acetone complex (7,8). From the fluorescence spectra of curcumin with each of the 12 organic solvents (6), it appears that the curcumin-acetone system is the most sensitive and accurate with the lowest detection limit and longest duration. Conformity
to Beer’s Law
Figure 2 is an enlargement of the portion of the main graph near the origin. It is linear only to 0.5 ppm and single-valued (not shown for brevity) to about 6 ppm. Beyond this value, the curve falls off because of concentration quenching (innerfilter effect) due to excessive absorption of fluorescence by the solution (selfquenching). Duplicate and identical spectra were obtained at several concentra-
hi -424
101 10
hp504
/ -
0 O!/
320
360 400 -
440
460
520
560
600
h,nm---b
FIG. 1. Excitation and fluorescence spectra of the curcumin-acetone
system.
212
JASIM AND AL1
160 140 ;120 $00 ;
80 0 ii 60 ii $J 40 0 2 20 00 0.0
0.2 Curcumin
03 IPPm)
0.4 Added
FIG. 2. Direct (1) and standard additions (2) calibration graphs of the curcumin-acetone
tion levels (0.01~50 levels.
ppm) only, indicating
system.
the absence of quenching within these
Effect of UV Radiation Excessive exposures (40 min) of the curcumin-acetone complex to UV radiation from a xenon arc lamp have no detrimental effect on the value of A, or XF or the power of fluorescence, confirming the absence of photodecomposition of the complex. Effect of Oxygen Dissolved air oxygen (ground triplet state) has no quenching effect. This is shown by the constancy of An and XF after the test solution is degassed with a stream of argon. Therefore, degassing of oxygen is not required. Fluorescence power remains the same before and after degassing. Effect of Temperature A change in temperature (5-50°C) has no effect on the values of A, and A, (Fig. 1); however, the fluorescence power (Fig. 3) is affected appreciably. According to Shpol’skii, the effects of nonradiative transitions are less at lower temperatures, leading to better vibrational transitions and therefore to enhanced fluorescence power. Consequently, standard and sample solutions must be measured at the same temperature. The use of thermostated temperatures is not necessary (unless otherwise stated) and any room temperature (20-2X) can be used (Table 1). Effect of Water It has been observed (Fig. 4) that the presence of more than 0.01% (v/v) of water in the curcumin-acetone solution increases At+ i.e., 504 to 520 nm, and causes quenching which increases with the increase in water contents. This is attributed to a reaction between H,O (electron donor = D) and the fluorescent curcumin-
SPECTROFLUOROMETRIC
0OLFIG.
10
15
DETERMINATION
20
25
30 Temp.%
3. Effect of temperature on the fluorescence
35
OF CURCUMIN
40
45
213
50
power of the curcumin-acetone
system.
acetone complex (C*-), resulting in the formation of a nonfluorescent (9, 10) and more stable complex (C*- D+) with lower S, vibrational energy levels and therefore lower energy contents and longer hF, C*100
+ D+ *CD. H,O
Added
90 80
FIG. 4. Effect of various concentrations for water (ml) on A, and fluorescence power of the curcumin-acetone system.
214
JASIM AND AL1
This fact, therefore, necessitates the use of dry acetone for the determination of cm-cumin. Others (II) attributed this quenching to the presence of polarization forces or dipole moments between H,O and the complex which tend to confer more nonradiative transitions with subsequent quenching effects. Water has no effect on excitation or A,. The absence of Rayleigh and Raman scatters may be due to the overlapping effects of the strong excitation and emission spectra of the complex. Sensitivity, Detection Limit, and Linearity Sensitivity, defined by the slope of the calibration curve, i.e., the ppb of curcumin which give one fluorescence power unit, is found to be 4.7 ppb. The limit of detection, defined as the ppb of curcumin which give fluorescence power equal to three times the standard deviation (3s) of 10 replicate measurements with the same solution, is found to be 0.34 ppb. Figure 2 shows that the linear dynamic range, defined as the concentration of curcumin from 10 times the detection limit to the upper concentration limit where a 5% deviation from linearity results, is 0.0034-0.5 ppm, or approximately 0.0-0.5 mm. Precision, Accuracy, and Duration of Fluorescence The percentage RSD is calculated from 10 replicate measurements using solutions of 0.1 and 0.3 ppm because they fall within the linear range of the calibration graph; the corresponding values are 2.03 and 1.13%, respectively. The last two values indicate that precision is a complex characteristic of the concentration of the curcumin-acetone system. The percentages recovery of 0.1 and 0.3 ppm curcumin are found to be 98.96 and lOO.OO%, respectively, for standard and simulated samples. Spectrofluorometrically, the A,, A,, and fluorescence power remain constant when the curcumin-acetone solution is kept at 5-30°C; the fluorescence could last for more than 1 month. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Jasim, F.; Ali, F. Micro&em. .I., 1988, 38, 106-110. Motoi, M.; Tomako, F.; Yoshiko, Y. Gnknba Kiyo, 1981, 29, 97-103. Tonmesen, H. H.; Kavlsen, J. J. Chromatogr., 1983, 259, 367-375. Ghulam, J.; Helen, S. J. &on. Entomol., 1983, 76, 154-165. Sharma, 0. P. Biochem. Pharmacol., 1976, 25, 1811-1817. Jasim, F.; Ali, F. Microchem. J., 1989, 39, 156159. Guibault, G. Cl. Molecular fluorescence spectroscopy. In Comprehensive Analytical Chemistry (G. Svehla, Ed.), Vol. VIII, 1977. 8. Guibault, G. G. Practical Fluorescence: Theory, Methods and Techniques, Dekker, New York, 1973. 9. Chandross, E. A.; Thomas, H. T. Chem. Phys. Lett. 1971, 9, 397402. 10. Beens, H.; Knibbe, H.; Weller, A. J. Chem. Phys., 1967, 47, 1183-l 189. Il. Selinger, B. K.; McDonald, R. J. Aust. J. Chem. 1972, 25, 897-906.