Katz

UV Spectrophotometric Analysis of Caffeine and Benzoate in Soft Drink

Abstract

The caffeine and benzoic acid concentration of 2 samples, containing 2.00mL

and 1.00mL of Mountain Dew, were determined to be 16.38mg/L and 33.60mg/L

for the more concentrated and 8.78mg/L and 14.12mg/L for the more dilute. These

concentrations were determined using UV spectrophotomery. Six caffeine and

benzoic acid standards were created to generate relatively linear calibration curves.

The data from these curves, as well as the observed absorbance for the Mountain

Dew samples, were utilized to calculate the caffeine and benzoic acid

concentrations.

Results

To determine the amount of caffeine and benzoate in Mountain Dew, caffeine

and benzoic acid standard solutions were made. Six standards were made for each

component, the concentrations were determined using the formula N1V1=N2V2. For

the most dilute caffeine standard, 1.00mL stock caffeine solution (0.1046 x 103

mg/L) was added to a 50.00 mL volumetric flask and diluted to the calibration mark.

N1V1=N2V2 (1)

(0.1046x103mg/L)(1.00x10-3L)=N2(50.00x10-3L) (2)

N2=1.046mg/L (3)

The absorbance for each standard solution was observed at both 230 and 275

nanometers. The concentration and absorbance for the caffeine standards are

displayed in table 1.

Table 1: Absorbance observed for six caffeine standard solutions

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λ 1.046mg/L 4.184mg/L 8.368mg/L 12.55mg/L 16.74mg/L 20.92mg/L

230nm 0.031 0.114 0.265 0.387 0.482 0.617

275nm 0.053 0.206 0.431 0.645 0.841 1.063

The concentrations of the benzoic acid standards were also calculated using

N1V1=N2V2. For the most dilute benzoic acid standard, 1.00mL of a stock solution

(0.1018x103mg/L) was placed in a 200.00mL volumetric flask and diluted to the

calibration mark.

N1V1=N2V2 (4)

(0.1018x103mg/L)(1.00x10-3L)=N2(200.00x10-3L) (5)

N2=0.509 mg/L (6)

The concentration for each standard solution and the observed absorbance at 230

and 275 nm are displayed in table 2.

Table 2: Absorbance observed for six benzoic acid standard solutions

λ 0.509mg/L 2.036mg/L 4.072mg/L 6.108mg/L 8.144mg/L 10.18mg/L

230nm 0.092 0.228 0.416 0.610 0.797 0.978

275nm 0.035 0.041 0.060 0.081 0.090 0.116

The data from table 1 and 2 was used to construct 4 calibration curves, which are

shown in figures 1-4.

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Figure 1: Calibration Curve for Caffeine at 275nm

Figure 2: Calibration Curve for Caffeine at 230nm

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The extinction coefficients for caffeine and benzoic acid at 230 and 275nm were

determined from the calibration curves. Because the curve depicts the relationship

between absorbance and concentration, the slope of the linear regression line is

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Figure 3: Calibration Curve for Benzoic Acid at 275nm

Figure 4: Calibration Curve for Benzoic Acid at 230nm

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equivalent to the extinction coefficient. The extinction coefficients for each

wavelength are recorded in table 3.

Table 3: Extinction Coefficients for Caffeine and Benzoic Acid

λ Caffeine Benzoic Acid

230nm 0.0294cm-1mg-1L 0.0921cm-1mg-1L

275nm 0.0508cm-1mg-1L 0.0084cm-1mg-1L

This information was then used to determine the concentration of caffeine and

benzoic acid in 2 samples of Mountain Dew. The absorbance for each of these

samples at 230 and 275nm are displayed in table 4.

Table 4: Absorbance for 2 Mountain Dew sample solutions

λ 2mL mountain dew/50.00mL

1 mL mountain dew/50.00mL

230nm 1.646 0.865

275nm 0.588 0.313

These absorbances and the extinction coefficients were substituted into the

following formulas to determine the concentration of caffeine and benzoic acid in

the mountain dew samples.

(7)

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(8)

The calculated concentrations are displayed in table 5.

Table 5: Concentrations of Caffeine and Benzoic Acid in 2 Mountain Dew solutions

2mL mountain dew/50.00 mL

1mL mountain dew/50.00 mL

Caffeine Concentration 16.36 mg/L 8.78 mg/L

Benzoic Acid Concentration

33.60 mg/L 14.12 mg/L

The limit of detection (LOD) and lower limit of quantitation (LLOQ) were

determined by conducting 5 analyses of the most dilute standard caffeine solution at

the wavelength maximum. The absorbances observed for the five trials, as well as

the standard deviation are shown in table 6.

Table 6: 5 analyses of 1.046mg/L caffeine standard solution at 275 nm

Trial Absorbance

1 0.053

2 0.074

3 0.076

4 0.079

5 0.082

Standard deviation 0.011

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The LOD was found to be 0.65 mg/L by dividing the standard deviation and the

slope of the line in figure 1 (0.0508cm-1mg-1L); the quotient was multiplied by 3.

The LLOQ was found to be 2.17 mg/L by dividing the standard deviation and the

slope of the line in figure 1; the quotient was multiplied by 10. Therefore

concentrations of caffeine less than 2.17mg/L and greater than 0.65mg/L cannot be

quantified, but can be recognized.

The method allowed for the generation of calibration curves with relatively

linear relationships. The correlation coefficients ranged from 0.98468-0.9999,

showing that a strong linear relationship exists between the data points. The

relatively low correlation coefficient of the benzoic acid calibration curve at 275nm

is likely due to the fact that benzoic acid only absorbs minimally at this wavelength.

The low signal could have affected the readings of the spectrophotometer.

Conclusion

The content of caffeine and benzoic acid in a sample containing 2 mL of

mountain dew was determined to be 16.38 mg/L and 33.60mg/L, respectively. The

caffeine and benzoic acid concentration in the sample containing 1 mL of mountain

dew was 8.78mg/L and 14.12mg/L, respectively. These concentrations were

determined using UV spectrophotometric analysis. This method allowed for the

generation of relatively linear calibration curves and a determination of the LOD

and LLOQ.

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