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Spectrophotometric Determination of Chocolate in Chocolate Products1
SPECTROPHOTOMETRIC
DETERMINATION
IN CHOCOLATE
OF CHOCOLATE
PRODUCTS 1
P. M. T. HANSEN Department of Dairy Technology, Institute of Nutrition and Food Technology The Ohio State University, Columbus ABSTRACT
A method was developed to determine the concentration of fat-free cocoa solids (true chocolate) in chocolate or chocolate-flavored milk and ice cream by the absorbanee of a trichloroaeetic acid (TCA) filtrate of the product at wave length 275 m~, using either chocolate or theobromine as the standard reference. Results were within ± 4.5% of the expected values based on chemical analyses. The mean error for a series of duplicate determinations was ± 0.22%. The influence of milk or ice cream solids was not significant, provided the amounts were maintained constant in test and reference samples. The true chocolate content in 19 commercial chocolate ice creams averaged 2.1.3% and ranged from 1.3 to 3.1%.
In conducting a study on the chemical nature of chocolate flavoring materials, observations indicated that certain of the procedures being used would lend themselves to the development of a method for determining the true chocolate (fat-free cocoa solids) content of chocolateflavored dairy or other food products. Such a method would appear to offer promise as a means of evaluating chocolate-flavored products on the basis of their absolute chocolate concentration. Therefore, attention was given to the development of a chocolate determination procedure and to its application, and results are presented in this paper. E X P E R I M E N T A L PROCEDURE
Inst~n~ment:
The measurements were performed on a ttitachi Perkin-Ehner spectrophotometer with digital read-out. Materials: a) TCA-reagent. Dissolve 67 g of trichloroacetic acid (A.R.) in water and dilute to one liter. b) :Filterpaper no. 42 Whatman, 11 Cln. TO ensure constancy of results, discard the first and last ten pieces of a fresh box and store the remaining in a covered g!a~s container until use (2). c) Theobromine, Eastman Kodak Co., Rochester, N. Y. d) Sodium Hydroxide, approximately 0.] Normal solution. (1) Procedure: a) Disperse 2.75-3.25 g of sample, accurately weighed, in approximately 25 ml of hot water, and dilute to 200 ml. To 5 ml of diluted sample add 15 ml of TCA reagent, mix thoroughly, and filter through the stipulated ~eceivcd for publication May 25, 1965. Article 5-65. The Department of Dairy Technology. Research supported in part by a grant from the American Dairy Association.
filter paper. Discard the first 8-10 ml of filtrate. Mix the remaining filtrate and transfer a pertion to a 10-ram quartz cell. b) Measure the absorbanee of the TCA-filtrate at wave length 275 m~, using a TCA-filtrate from a nonchoeolate-flavored sample as a blank, c) Determine the true chocolate concentration by comparison to a standard curve prepared either from chocolate flavoring materials of known composition or from theobromine. (2) Preparation of standard curve from chocolate. Obtain an unfortified chocolate sample of known fat, moisture, and ash content. Disperse an amount equal to 3 g of fat-free cocoa solids in 50 ml of water, heat the mixture to 90-100 C, and place it in a mechanical shaker for 2 hr. Dilute the dispersed chocolate to 100 ml with water. Mix the sample thoroughly and transfer l-5-ml portions containing 30-150 mg of true chocolate to volumetric flasks of 200-ml capacity. Add 3 g of milk or unflavored ice cream and dilute to 200 ml with water. Precipitate 5-ml portions with 15 ml TCA reagent and filter. Measure the absorbance of the filtrate at 275 mtL against a blank prepared from milk or ice cream, free of chocolate. Plot the absorbance versus the amounts of true chocolate in the filtrates (0.75-3.75 rag). Use this standard curve to convert the absorbanee reading of the sample to the amount of true chocolate in the 20-ml portion. To obtain the true chocolate content in per cent of the sample, use the equation : 4C True chocolate content ( %) = - -
1401
S,
where C is the number of milligrams of true chocolate in the 20-ml portion, and S is the
1402
r . 2¢I. T. JcIANSEN
weight in grams of the undiluted sample. (3) Preparation of theobromine standard curve. Dissolve exactly 50 mg of theobromine in 5 ml of 0.1 normal N a O H and dilute to one liter. To 1-5 ml portions containing 0.05-0.25 mg of theobromine, add 15 ml of T C A - r e a g e n t and adjust the volume to 20 ml with distilled water. Measure the absorbanee at wave length 275 mt~ against a blank of 5 ml water + 15 nfl of TCA-reagent. P l o t the absorbance versus the concentration of theobromine. Use this standard curve to convert the absorbanec reading of the sample to the corresponding number of milligrams of theobromine in the 20-ml portion. To obtain the theobromine standard value of the sample, use the equation: 4T Theobromine standard value = S, where T is the nmnber of milligrams of theobromine in the 20-ml portion, and S is the weight in grams of the undiluted sample. To convert the theobromine standard value to true chocolate content in p e r cent of the sample, nmltiply with the f a c t o r s : (a) for ice cream or milk 17.6 (b) f o r water suspensions 16.0 R E S U L T S AND D I S C U S S I O N
The developed method involves a measurement of the ultraviolet absorbance of a TCA-filtrate, p r e p a r e d f r o m the chocolate product a f t e r suitable dilution. The absorbanee readings are converted to chocolate concentration by comparison to a standard curve, obtained either from chocolate flavoring materials of known composition or from theobromine. Since it is a normal practice in the formulation of chocolate milk or ice cream to balance the amounts of chocolate to be used on the basis of the fatfree cocoa content (4), the results have been arbitrarily designated in terms of a true chocolate concentration, which represents the f a t - f r e e cocoa a f t e r correction for its content of moisture and ash. The absorption spectra for the TCA-filtrate of chocolate and f o r the 5 % TCA-reagent are shown in F i g u r e 1. Previous studies (3) revealed that chocolate contains dialyzable substances that absorb strongly in the ultraviolet spectrum and these substances are also present in a TCA-filtrate of chocolate products. AIthough the T C A - r e , g e n t absorbs ultraviolet light appreciably with a maximmu of 250 m/x, its interference with the maximum for the ehocolate filtrate at 275 m~ is sufficiently small to permit quantitative measurements at this wave
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, 325
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(mp.)
WAVELENGTH
:PIG. 1. Absorption spectra for (A) TCA-reagent vs. water, and (B) chocolate filtrate vs. TCAreagent. length. Values for the absorbance produced 'by several chocolates suspended in water, milk, or ice cream are presented in Table 1. Results for the milk and ice cream suspensions were not significantly different as revealed by the mean values and confidence intervals. They were, however, significantly higher for the water suspensions. This effect is also evident in F i g u r e 2. The slope f o r chocolate ( L A G ) in milk or ice cream is 10% lower than the slope for the water suspension, and the intercept is higher. The difference is the result of two factors: a) a contribution to the extinction by milk constituents, and b) a reduction in the extinction by a Langmuir-type absorption of chocolate material, probably tannins, to the precipitated milk protein. Therefore, it is necessary to maintain a constant amount of milk or ice cream in test and TABLE 1 Absorbance at 275 m~ of TCA-filtrates from different chocolate products dispersed in water, milk, oi' ice cream Choeo]ate material
(See Table 2)
Water
Milk
Ice Cream
ACC LAG LBL 3IC 3CG 3KD 4EH 3HH DRU 4DE
(Absorbance/mg "true chocolate~20 ml) .171 .151 .155 .160 .147 .149 .]69 .151 .159 .151 .139 .142 .162 .147 .151 .163 .147 .151 .154 .142 .144 .173 .153 .159 .172 .155 .156 .159 .141 .142
Mean
.163±.006" .147~+.004 a .151--+.005 "
Confidence interval (95%).
CI-IO C O L A T E
DETERMINATION
7C
.6C "~
AB~/~
50
,,~ ~o z
.20
.10
I
4 CONCENTRATION OF TRUE CHOCOLATE (mg/2Oml)
F I e . 2.
Si;andard curve f o r chocolate.
A: Water suspensions, Y = .162 X + .004. B: Milk or ice cream suspensions (10% nonfat milk solids), Y = .149 X-/- .021. Standard error of estimate: ± .0033. reference samples. From the regression equations in Figure 2 it was estimated that a variation of ± 0.25 g in the weight of milk or ice cream taken for the analyses could be tolerated, since the resulting uncertainty would not exceed the standard error of ---+.003 absorbance unit. This limit applies to milk or ice cream of 10% nonfat milk solids and of less than 5% true chocolate. An additional variation in the nonfat milk solids content of the products within 8-12% would not increase this estimated uncertainty to more than three times the standard error, which is still insignificant. Inspection of Figure 2 suggests that the error caused by variations in the nonfat milk solids beyond these limits would be important only at very low or at very high concentrations of chocolate, but not at moderate levels. This was verified by analyscs of products containing 3% true chocolate but varying in nonfat milk solids from 5 to 15%, for which the corresponding absorbance readings were .351 and .362. Other variables that might affect the results were also studied. ]~Iolding the filtrates for as long as 24 hr before making the determinations caused no error. Therefore, the time variable was unimportant. The concentration of TCA used in the procedure had a significant (5%) effect. This was caused by an insufficient precipitation of milk protein at low levels of milk or ice cream if less than 5% TCA in the final dilution was used; on the other hand, the 5% TCA-concentration yielded consistent results at all levels of milk solids investigated. :Higher concentrations of TCA (15% of the final dilution) were found to eliminate most of the inter-
1403
ference due to milk constituents but, in this case, the interference of TCA became pronounced. The 275 m/z absorption maximum for chocolate filtrate is due in part to theobromine, a natural constituent of chocolate, present at concentrations of 1-2% of the whole bean or approximately 3.5% of cocoa (1). In addition to theobromine, there are other components o f chocolate that absorb ultraviolet light and overlap the 275 rn/z wave length. F o r this reason, the measurements do not represent the theobromine concentration alone. However, theobromine may be used as a standard reference and the measurements may be expressed in terms of a theobromine standard value, i.s., the concentration of theobromine that would produce an absorbance at 275 m/z equal to that of the test sample. The use of theobromine for standard reference eliminates the need for a chemical analysis of a chocolate reference material and it avoids, furthermore, the possibility of selecting a fortified or otherwise unsuitable chocolate reference. The theobromine standard plot is shown in Figure 3 and is strictly linear. The theobromine standard value corresponding to I m g of true chocolate (average) was 0.0623 mg for the water suspension and 0.0567 mg for the milk and ice cream suspensions. Therefore, the theobromine standard values may be converted to true chocolate content by multiplication with the factors 16.0 or 17.6, respectively. A comparison of the true chocolate content by a chemical analysis and by the spectrophotometric method is shown in Table 2 for a number of chocolate products varying in bean variety and processing, as disclosed by the manufacturers. Individual recoveries were 0.7
0,6
0.5
~ o~ z ~
0.3
0.2
oJ
o.o
0.00
I
I
I
I
I
0.05
0,10
0.15
0,20
0.25
CONCENTRATION OF THEOSROMINE (mg/20ml)
FIG. 3. Theobromine standard curve, Y=2.59 X 4- .002.
P. M. T. H A N S E N
1404
TABLE 2 Chemical composition and spcctrophotometric analysis of ten chocolate samples Spectrophotometric procedure
Chemical composition
Code
Bean variety and process ~
Fat
Moisture
True Chocolate b
Ash
True choeolate ¢
Recovery
(%) ACC LAG LBL 3IC 3CG 3KD 4EH 3HH DRU 4DE
Accra Natural LaGuayra Natural LaGuayra Natural Basic Natural Basic Combination Basic Dutch Flavor A- basic Natural Flavor + basic Combination Not identified Not identified Not identified Dutch
17.91
1.48
5.98
74.63
78.45
105.1
20.98
1.04
5.12
72.86
71.74
98.5
41.92
1.04
3.88
53.16
55.23
103.9
12.82
1.71
5.76
79.71
75.23
94.4
40.96
2.71
4.27
52.06
52.10
100.1
34.07
0.38
4.56
60.99
61.26
100.4
34.11
1.95
4.70
59.24
56.46
95.3
38.94
3.96
4.62
52.48
55.97
106.6
41.26
3.61
3.98
51.60
54.70
106.0
22.66
3.37
8.10
65.87
64.59
Mean recovery Standard deviation
98.1 100.8 ± 4.5
Basic: Unidentified, basic bean product; Flavor: Unidentified, flavor bean product; Combination: Blend of Dutch and natural process products. b The mean error associated with the calculation of true chocolate from duplicate determinations of fat, moisture, and ash ± 0.26 ( s t a n d a r d deviation). The mean error of duplicate determinations was ± 0.22 (scandard deviation). within ~ 4 . 5 % ( s t a n d a r d d e v i a t i o n ) . T h e m e a n e r r o r f o r a series o f d u p l i c a t e d e t e r m i n a t i o n s w a s _+_ 0.22, s i m i l a r to the c o m p o s i t e e r r o r of -+-0.26% a s s o c i a t e d w i t h the e s t i m a t i o n o f f a t - f r e e cocoa solids f r o m the s e p a r a t e chemical d e t e r m i n a t i o n s o f f a t , ash, a n d m o i s t u r e . T h u s , the a b s o r b a n e e p r o d u c e d b y the f u n d a m e n t a l , f a t - f r e e chocolate m a t e r i a l , o r t r u e chocolate, v a r i e s to s o m e e x t e n t w i t h the source. H o w ever, r e s u l t s s u g g e s t the m e t h o d m a y h a v e a p -
plication for determination of true chocolate in dairy products containing' chocolate of an u n k n o w n source. F o r e x a m p l e , f o r a p r o d u c t o f an a c t u a l c o n t e n t of 2 . 5 % t r u e chocolate t h e d e t e r m i n a t i o n s would be e x p e c t e d to yield a v a l u e w i t h i n the r a n g e o f 2.35-2.65%, r e g a r d less o f t h e s o u r c e o f chocolate. I f , on the o t h e r h a n d , the chocolate m a t e r i a l u s e d in the p r o d u c t was a v a i l a b l e f o r r e f e r e n c e , a d e t e r m i n a t i o n on m i l k or ice cream w o u l d be as a c c u r a t e as a s e p a r a t e chemical d e t e r m i n a t i o n on the origi n a l chocolate. The d e v e l o p e d p r o c e d u r e w a s u s e d f o r t h e a n a l y s i s of several d i f f e r e n t c o m m e r c i a l chocolate ice c r e a m s . T h e c o n c e n t r a t i o n of t r u e chocolate in these s a m p l e s a n d r e s u l t s o f t h e
TABLE 3 ]~e]ationship between true chocolate content and flavor quality of commercial chocolate ice cream Coneentration Flavor of true score choco(maxilate " m u m 4 0 ) (%) 3.1
2.9 2.6
2.5 2.4 2.4 2.3 2.1 2.1 2.1 2.0 2.0 2.0 2.0 1.8 1.8 1.7 1.4 1.3 Estimated
38.0
39.0 38.0
Flavor character Harsh, stale S1. scorched Harsh Unnatural Unnatural Unnatural
35.0 37.0 36.0 40.0 37.5 Harsh Harsh 39.0 Fla*, lacks fine flavor 38.5 Unnatural 37.0 37.0 Lacks chocolate character 40.0 Lacks fine flavor 39.0 Lacks chocolate character 38.0 Lacks fine flavor 39.0 Lacks fine flavor 39.0 37.0 Unnatural Lacks chocolate character 38.5 from theobromine standard curve.
CHOCOLATE DETERMINATION o r g a n o l e p t i c e v a l u a t i o n by f o u r t r a i n e d j u d g e s are r e p o r t e d in Table 3. The t r u e chocolate c o n t e n t r a n g e d f r o m 1.3 to 3.1%, with a n average of' 2.13%. There was n o direct r e l a t i o n s h i p between the absolute chocolate c o n c e n t r a t i o n a n d the over-all flavor score s s s i g n e d to the samples, but there a p p e a r e d to be a d e p e n d e n c y of the flavor i n t e n s i t y u p o n the chocolate concentration. Most of the samples of less t h a n a v e r a g e contempt of chocolate were criticized f o r a lack of flavor~ w h e r e a s several of those of relatively high chocolate c o n t e n t were described as h a v i n g a h a r s h flavor. ACKNO:WLEDG),IENT
The author appreciates the assistance of Mrs.
~405
G. Clendening in the chemical analysis of the chocolate samples. REFERENCES (1) COOK, L. R. 1963. Chocolate Production and Use. p. 289. Magazines for Industry, Inc., 660 Madison Avenue, New York. (2) HANSEl, P. M. T. 1965. Observations on a Characteristic Distribution of Ultraviolet Absorbing Impurities Within Boxes of Analytical Filter Paper. J. Dairy Sci., 48: 497. (3) HANSEN, P. M. T., AND GOULD, I. A. 1964. Dialyzable Flavor Components of Chocolate Products. J. Dairy Sei., 47: 682. (4) RAMACHANDKAN, K. S., GOULD, I. A., AND LINDAI~OOD, J. B. 1961. Cacao, Cocoa, and Chocolate with Particular l~eference to Flavor in Ice Cream. (A Review.) Ice Cream Field, 78 (12) : 18.
DETERMINATION
IN CHOCOLATE
OF CHOCOLATE
PRODUCTS 1
P. M. T. HANSEN Department of Dairy Technology, Institute of Nutrition and Food Technology The Ohio State University, Columbus ABSTRACT
A method was developed to determine the concentration of fat-free cocoa solids (true chocolate) in chocolate or chocolate-flavored milk and ice cream by the absorbanee of a trichloroaeetic acid (TCA) filtrate of the product at wave length 275 m~, using either chocolate or theobromine as the standard reference. Results were within ± 4.5% of the expected values based on chemical analyses. The mean error for a series of duplicate determinations was ± 0.22%. The influence of milk or ice cream solids was not significant, provided the amounts were maintained constant in test and reference samples. The true chocolate content in 19 commercial chocolate ice creams averaged 2.1.3% and ranged from 1.3 to 3.1%.
In conducting a study on the chemical nature of chocolate flavoring materials, observations indicated that certain of the procedures being used would lend themselves to the development of a method for determining the true chocolate (fat-free cocoa solids) content of chocolateflavored dairy or other food products. Such a method would appear to offer promise as a means of evaluating chocolate-flavored products on the basis of their absolute chocolate concentration. Therefore, attention was given to the development of a chocolate determination procedure and to its application, and results are presented in this paper. E X P E R I M E N T A L PROCEDURE
Inst~n~ment:
The measurements were performed on a ttitachi Perkin-Ehner spectrophotometer with digital read-out. Materials: a) TCA-reagent. Dissolve 67 g of trichloroacetic acid (A.R.) in water and dilute to one liter. b) :Filterpaper no. 42 Whatman, 11 Cln. TO ensure constancy of results, discard the first and last ten pieces of a fresh box and store the remaining in a covered g!a~s container until use (2). c) Theobromine, Eastman Kodak Co., Rochester, N. Y. d) Sodium Hydroxide, approximately 0.] Normal solution. (1) Procedure: a) Disperse 2.75-3.25 g of sample, accurately weighed, in approximately 25 ml of hot water, and dilute to 200 ml. To 5 ml of diluted sample add 15 ml of TCA reagent, mix thoroughly, and filter through the stipulated ~eceivcd for publication May 25, 1965. Article 5-65. The Department of Dairy Technology. Research supported in part by a grant from the American Dairy Association.
filter paper. Discard the first 8-10 ml of filtrate. Mix the remaining filtrate and transfer a pertion to a 10-ram quartz cell. b) Measure the absorbanee of the TCA-filtrate at wave length 275 m~, using a TCA-filtrate from a nonchoeolate-flavored sample as a blank, c) Determine the true chocolate concentration by comparison to a standard curve prepared either from chocolate flavoring materials of known composition or from theobromine. (2) Preparation of standard curve from chocolate. Obtain an unfortified chocolate sample of known fat, moisture, and ash content. Disperse an amount equal to 3 g of fat-free cocoa solids in 50 ml of water, heat the mixture to 90-100 C, and place it in a mechanical shaker for 2 hr. Dilute the dispersed chocolate to 100 ml with water. Mix the sample thoroughly and transfer l-5-ml portions containing 30-150 mg of true chocolate to volumetric flasks of 200-ml capacity. Add 3 g of milk or unflavored ice cream and dilute to 200 ml with water. Precipitate 5-ml portions with 15 ml TCA reagent and filter. Measure the absorbance of the filtrate at 275 mtL against a blank prepared from milk or ice cream, free of chocolate. Plot the absorbance versus the amounts of true chocolate in the filtrates (0.75-3.75 rag). Use this standard curve to convert the absorbanee reading of the sample to the amount of true chocolate in the 20-ml portion. To obtain the true chocolate content in per cent of the sample, use the equation : 4C True chocolate content ( %) = - -
1401
S,
where C is the number of milligrams of true chocolate in the 20-ml portion, and S is the
1402
r . 2¢I. T. JcIANSEN
weight in grams of the undiluted sample. (3) Preparation of theobromine standard curve. Dissolve exactly 50 mg of theobromine in 5 ml of 0.1 normal N a O H and dilute to one liter. To 1-5 ml portions containing 0.05-0.25 mg of theobromine, add 15 ml of T C A - r e a g e n t and adjust the volume to 20 ml with distilled water. Measure the absorbanee at wave length 275 mt~ against a blank of 5 ml water + 15 nfl of TCA-reagent. P l o t the absorbance versus the concentration of theobromine. Use this standard curve to convert the absorbanec reading of the sample to the corresponding number of milligrams of theobromine in the 20-ml portion. To obtain the theobromine standard value of the sample, use the equation: 4T Theobromine standard value = S, where T is the nmnber of milligrams of theobromine in the 20-ml portion, and S is the weight in grams of the undiluted sample. To convert the theobromine standard value to true chocolate content in p e r cent of the sample, nmltiply with the f a c t o r s : (a) for ice cream or milk 17.6 (b) f o r water suspensions 16.0 R E S U L T S AND D I S C U S S I O N
The developed method involves a measurement of the ultraviolet absorbance of a TCA-filtrate, p r e p a r e d f r o m the chocolate product a f t e r suitable dilution. The absorbanee readings are converted to chocolate concentration by comparison to a standard curve, obtained either from chocolate flavoring materials of known composition or from theobromine. Since it is a normal practice in the formulation of chocolate milk or ice cream to balance the amounts of chocolate to be used on the basis of the fatfree cocoa content (4), the results have been arbitrarily designated in terms of a true chocolate concentration, which represents the f a t - f r e e cocoa a f t e r correction for its content of moisture and ash. The absorption spectra for the TCA-filtrate of chocolate and f o r the 5 % TCA-reagent are shown in F i g u r e 1. Previous studies (3) revealed that chocolate contains dialyzable substances that absorb strongly in the ultraviolet spectrum and these substances are also present in a TCA-filtrate of chocolate products. AIthough the T C A - r e , g e n t absorbs ultraviolet light appreciably with a maximmu of 250 m/x, its interference with the maximum for the ehocolate filtrate at 275 m~ is sufficiently small to permit quantitative measurements at this wave
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, 325
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WAVELENGTH
:PIG. 1. Absorption spectra for (A) TCA-reagent vs. water, and (B) chocolate filtrate vs. TCAreagent. length. Values for the absorbance produced 'by several chocolates suspended in water, milk, or ice cream are presented in Table 1. Results for the milk and ice cream suspensions were not significantly different as revealed by the mean values and confidence intervals. They were, however, significantly higher for the water suspensions. This effect is also evident in F i g u r e 2. The slope f o r chocolate ( L A G ) in milk or ice cream is 10% lower than the slope for the water suspension, and the intercept is higher. The difference is the result of two factors: a) a contribution to the extinction by milk constituents, and b) a reduction in the extinction by a Langmuir-type absorption of chocolate material, probably tannins, to the precipitated milk protein. Therefore, it is necessary to maintain a constant amount of milk or ice cream in test and TABLE 1 Absorbance at 275 m~ of TCA-filtrates from different chocolate products dispersed in water, milk, oi' ice cream Choeo]ate material
(See Table 2)
Water
Milk
Ice Cream
ACC LAG LBL 3IC 3CG 3KD 4EH 3HH DRU 4DE
(Absorbance/mg "true chocolate~20 ml) .171 .151 .155 .160 .147 .149 .]69 .151 .159 .151 .139 .142 .162 .147 .151 .163 .147 .151 .154 .142 .144 .173 .153 .159 .172 .155 .156 .159 .141 .142
Mean
.163±.006" .147~+.004 a .151--+.005 "
Confidence interval (95%).
CI-IO C O L A T E
DETERMINATION
7C
.6C "~
AB~/~
50
,,~ ~o z
.20
.10
I
4 CONCENTRATION OF TRUE CHOCOLATE (mg/2Oml)
F I e . 2.
Si;andard curve f o r chocolate.
A: Water suspensions, Y = .162 X + .004. B: Milk or ice cream suspensions (10% nonfat milk solids), Y = .149 X-/- .021. Standard error of estimate: ± .0033. reference samples. From the regression equations in Figure 2 it was estimated that a variation of ± 0.25 g in the weight of milk or ice cream taken for the analyses could be tolerated, since the resulting uncertainty would not exceed the standard error of ---+.003 absorbance unit. This limit applies to milk or ice cream of 10% nonfat milk solids and of less than 5% true chocolate. An additional variation in the nonfat milk solids content of the products within 8-12% would not increase this estimated uncertainty to more than three times the standard error, which is still insignificant. Inspection of Figure 2 suggests that the error caused by variations in the nonfat milk solids beyond these limits would be important only at very low or at very high concentrations of chocolate, but not at moderate levels. This was verified by analyscs of products containing 3% true chocolate but varying in nonfat milk solids from 5 to 15%, for which the corresponding absorbance readings were .351 and .362. Other variables that might affect the results were also studied. ]~Iolding the filtrates for as long as 24 hr before making the determinations caused no error. Therefore, the time variable was unimportant. The concentration of TCA used in the procedure had a significant (5%) effect. This was caused by an insufficient precipitation of milk protein at low levels of milk or ice cream if less than 5% TCA in the final dilution was used; on the other hand, the 5% TCA-concentration yielded consistent results at all levels of milk solids investigated. :Higher concentrations of TCA (15% of the final dilution) were found to eliminate most of the inter-
1403
ference due to milk constituents but, in this case, the interference of TCA became pronounced. The 275 m/z absorption maximum for chocolate filtrate is due in part to theobromine, a natural constituent of chocolate, present at concentrations of 1-2% of the whole bean or approximately 3.5% of cocoa (1). In addition to theobromine, there are other components o f chocolate that absorb ultraviolet light and overlap the 275 rn/z wave length. F o r this reason, the measurements do not represent the theobromine concentration alone. However, theobromine may be used as a standard reference and the measurements may be expressed in terms of a theobromine standard value, i.s., the concentration of theobromine that would produce an absorbance at 275 m/z equal to that of the test sample. The use of theobromine for standard reference eliminates the need for a chemical analysis of a chocolate reference material and it avoids, furthermore, the possibility of selecting a fortified or otherwise unsuitable chocolate reference. The theobromine standard plot is shown in Figure 3 and is strictly linear. The theobromine standard value corresponding to I m g of true chocolate (average) was 0.0623 mg for the water suspension and 0.0567 mg for the milk and ice cream suspensions. Therefore, the theobromine standard values may be converted to true chocolate content by multiplication with the factors 16.0 or 17.6, respectively. A comparison of the true chocolate content by a chemical analysis and by the spectrophotometric method is shown in Table 2 for a number of chocolate products varying in bean variety and processing, as disclosed by the manufacturers. Individual recoveries were 0.7
0,6
0.5
~ o~ z ~
0.3
0.2
oJ
o.o
0.00
I
I
I
I
I
0.05
0,10
0.15
0,20
0.25
CONCENTRATION OF THEOSROMINE (mg/20ml)
FIG. 3. Theobromine standard curve, Y=2.59 X 4- .002.
P. M. T. H A N S E N
1404
TABLE 2 Chemical composition and spcctrophotometric analysis of ten chocolate samples Spectrophotometric procedure
Chemical composition
Code
Bean variety and process ~
Fat
Moisture
True Chocolate b
Ash
True choeolate ¢
Recovery
(%) ACC LAG LBL 3IC 3CG 3KD 4EH 3HH DRU 4DE
Accra Natural LaGuayra Natural LaGuayra Natural Basic Natural Basic Combination Basic Dutch Flavor A- basic Natural Flavor + basic Combination Not identified Not identified Not identified Dutch
17.91
1.48
5.98
74.63
78.45
105.1
20.98
1.04
5.12
72.86
71.74
98.5
41.92
1.04
3.88
53.16
55.23
103.9
12.82
1.71
5.76
79.71
75.23
94.4
40.96
2.71
4.27
52.06
52.10
100.1
34.07
0.38
4.56
60.99
61.26
100.4
34.11
1.95
4.70
59.24
56.46
95.3
38.94
3.96
4.62
52.48
55.97
106.6
41.26
3.61
3.98
51.60
54.70
106.0
22.66
3.37
8.10
65.87
64.59
Mean recovery Standard deviation
98.1 100.8 ± 4.5
Basic: Unidentified, basic bean product; Flavor: Unidentified, flavor bean product; Combination: Blend of Dutch and natural process products. b The mean error associated with the calculation of true chocolate from duplicate determinations of fat, moisture, and ash ± 0.26 ( s t a n d a r d deviation). The mean error of duplicate determinations was ± 0.22 (scandard deviation). within ~ 4 . 5 % ( s t a n d a r d d e v i a t i o n ) . T h e m e a n e r r o r f o r a series o f d u p l i c a t e d e t e r m i n a t i o n s w a s _+_ 0.22, s i m i l a r to the c o m p o s i t e e r r o r of -+-0.26% a s s o c i a t e d w i t h the e s t i m a t i o n o f f a t - f r e e cocoa solids f r o m the s e p a r a t e chemical d e t e r m i n a t i o n s o f f a t , ash, a n d m o i s t u r e . T h u s , the a b s o r b a n e e p r o d u c e d b y the f u n d a m e n t a l , f a t - f r e e chocolate m a t e r i a l , o r t r u e chocolate, v a r i e s to s o m e e x t e n t w i t h the source. H o w ever, r e s u l t s s u g g e s t the m e t h o d m a y h a v e a p -
plication for determination of true chocolate in dairy products containing' chocolate of an u n k n o w n source. F o r e x a m p l e , f o r a p r o d u c t o f an a c t u a l c o n t e n t of 2 . 5 % t r u e chocolate t h e d e t e r m i n a t i o n s would be e x p e c t e d to yield a v a l u e w i t h i n the r a n g e o f 2.35-2.65%, r e g a r d less o f t h e s o u r c e o f chocolate. I f , on the o t h e r h a n d , the chocolate m a t e r i a l u s e d in the p r o d u c t was a v a i l a b l e f o r r e f e r e n c e , a d e t e r m i n a t i o n on m i l k or ice cream w o u l d be as a c c u r a t e as a s e p a r a t e chemical d e t e r m i n a t i o n on the origi n a l chocolate. The d e v e l o p e d p r o c e d u r e w a s u s e d f o r t h e a n a l y s i s of several d i f f e r e n t c o m m e r c i a l chocolate ice c r e a m s . T h e c o n c e n t r a t i o n of t r u e chocolate in these s a m p l e s a n d r e s u l t s o f t h e
TABLE 3 ]~e]ationship between true chocolate content and flavor quality of commercial chocolate ice cream Coneentration Flavor of true score choco(maxilate " m u m 4 0 ) (%) 3.1
2.9 2.6
2.5 2.4 2.4 2.3 2.1 2.1 2.1 2.0 2.0 2.0 2.0 1.8 1.8 1.7 1.4 1.3 Estimated
38.0
39.0 38.0
Flavor character Harsh, stale S1. scorched Harsh Unnatural Unnatural Unnatural
35.0 37.0 36.0 40.0 37.5 Harsh Harsh 39.0 Fla*, lacks fine flavor 38.5 Unnatural 37.0 37.0 Lacks chocolate character 40.0 Lacks fine flavor 39.0 Lacks chocolate character 38.0 Lacks fine flavor 39.0 Lacks fine flavor 39.0 37.0 Unnatural Lacks chocolate character 38.5 from theobromine standard curve.
CHOCOLATE DETERMINATION o r g a n o l e p t i c e v a l u a t i o n by f o u r t r a i n e d j u d g e s are r e p o r t e d in Table 3. The t r u e chocolate c o n t e n t r a n g e d f r o m 1.3 to 3.1%, with a n average of' 2.13%. There was n o direct r e l a t i o n s h i p between the absolute chocolate c o n c e n t r a t i o n a n d the over-all flavor score s s s i g n e d to the samples, but there a p p e a r e d to be a d e p e n d e n c y of the flavor i n t e n s i t y u p o n the chocolate concentration. Most of the samples of less t h a n a v e r a g e contempt of chocolate were criticized f o r a lack of flavor~ w h e r e a s several of those of relatively high chocolate c o n t e n t were described as h a v i n g a h a r s h flavor. ACKNO:WLEDG),IENT
The author appreciates the assistance of Mrs.
~405
G. Clendening in the chemical analysis of the chocolate samples. REFERENCES (1) COOK, L. R. 1963. Chocolate Production and Use. p. 289. Magazines for Industry, Inc., 660 Madison Avenue, New York. (2) HANSEl, P. M. T. 1965. Observations on a Characteristic Distribution of Ultraviolet Absorbing Impurities Within Boxes of Analytical Filter Paper. J. Dairy Sci., 48: 497. (3) HANSEN, P. M. T., AND GOULD, I. A. 1964. Dialyzable Flavor Components of Chocolate Products. J. Dairy Sei., 47: 682. (4) RAMACHANDKAN, K. S., GOULD, I. A., AND LINDAI~OOD, J. B. 1961. Cacao, Cocoa, and Chocolate with Particular l~eference to Flavor in Ice Cream. (A Review.) Ice Cream Field, 78 (12) : 18.