EXPERIMENT 4: SPECTROPHOTOMETRIC ANALYSIS OF CAFFEINE AND BENZOIC ACID IN SOFT DRINKS

Group 4: Lopez, Palmario, Sibug

Soft drinks
Non-alcoholic beverages typically containing carbonated water, caffeine, soduim benzoate, sweeteners, and flavoring agents soft in contrast to hard drinks (alcoholic beverages)

Caffeine
white crystalline xanthine alkaloid and psychoactive stimulant acts as a central nervous system (CNS) stimulant most widely consumed psychoactive substance contains diuretic properties

Benefits and Harmful Effects of Caffeine

Benefits Harmful Effects caffeinism : nervousness, irritability, anxiety, hyperreflexia, insomnia, heart palpitations Increases production of stomach acid Caffeine induced psychiatric disorders: caffeine intoxication, caffeine-induced anxiety disorder, caffeine-induced sleep disorder, and caffeine-related disorder not otherwise specified (NOS)

Temporary wards off drowsiness Restores alertness Ergogenic Hepatoprotective properties

Benzoic Acid
colorless crystalline solid and the simplest aromatic carboxylic acid salts are used as a food preservative

Benefits and Harmful Effects of Benzoic Acid

Benefits Harmful Effects

Treatment of fungal diseases Topical antiseptics Inhalant decongestant

Sodium benzoate : possible cause of DNA damage and hyperactivity Excessive amounts may affect liver and kidney

Beer-Lamberts Law
A = ebc
A = absorbance, e = molar absorptivity, b = pathlength (in cm), c = concentration in moles/liter The amount of light absorbed by a chemical is directly related to the concentration of the chemical in a solution.
Where


The higher the concentration, the higher the absorbance

Only applicable for dilute solutions

Beer-Lamberts Law vs %Transmittance

BLL shows the linear relationship betweeen absorbance and concentration

Limitations of the Beer-Lambert Law: Causes of Nonlinearity

deviations in absorptivity coefficients at high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity scattering of light due to particulates in the sample fluorescence or phosphorescence of the sample changes in refractive index at high analyte concentration shifts in chemical equilibria as a function of concentration non-monochromatic radiation, deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band stray light

Dual Beam Spectrophotometer

Multicomponent Analysis
The spectrum of a mixture of two compounds X and Y is just the sum of the spectra from the individual components X and Y. At any wavelength at the spectrum of the mixture,

Atot = AX + AY

Choosing two wavelengths 1 and 2 at which both compounds show significant absorbance,

Atot, 1 = AX, 1 + AY, 1 and Atot, Atot, 1 = X, 1bcX + Atot, 2 = X, 2bcX +

,2

= AX, 2 + AY, Y, 1bcY Y, 2bcY

If all the molar absorptivities are known,

system

of two equations and two unknowns, cX and cY

The four values can be determined from the slopes of four Beer's Law plots:
A

vs. c at and Y

and

for standard solutions of both X

Objectives
To be able to create or produce absorption spectra of soft drinks and standards using dual beam spectrophotometer To be able to determine the concentration of caffeine and benzoic acid in soft drinks using spectrophotometric methods

Results and Discussion

Calibration Curve: Methodology

Procedure Benzoic Acid Solution: 2, 4, 6, 8, 10mg/L in 0.010M HCl Caffeine Solution: 4, 8, 12, 16, 20 mg/L in 0.010M HCl UV baseline from 350 to 210 nm was recorded with distilled H2O in the sample and reference cuvets U V Spectra of the standards with distilled water as reference were recorded The wavelength of peak absorbance for benzoic Acid and for caffeine , and respectively, were noted. Absorbance at each measured Calibration Curves were prepared: abs vs. [S] of each compounds at each wavelength Each graph should go to zero Rationale Will be used in constructing Calibration Curves -HCl was used to maintain the protonated form of the compounds To calibrate the UV-Vis spectrophotometer Water was used as reference since it was the solvent To generate data for calibration curves To know the absortivity of benzoic acid and caffeine at and To set 0 absorbance at 0 concentration of benzoic acid and caffeine

Calibration Curve
CALIBRATION CURVES
a

graph showing how the experimental observable (the absorbance in this case) varies with the concentration of known standard solution Beer-Lamberts Law, A = bc

From


the slope of the calibration curve =

In

the experiment, it was used to know the absorptivity of caffeine and benzoic acid

Calibration Curve
CALIBRATION CURVES
Table 1. Concentrations of benzoic acid standards and their corresponding UV-Vis absorbance at the given wavelength

Conc (ppm) 2.00 4.00 6.00 8.00 10.0

Absorbance 230nm 0.223 0.369 0.560 0.742 0.932 272nm 0.020 0.033 0.052 0.069 0.086

Figure 1. Standard calibration curve for benzoic acid standard, showing absorbance readings from 350-210nm

Calibration Curve
230nm 272nm

Slope (a R2

,benz)

0.0934

0.0086

0.9958

0.9965

Figure 2. Absorbance curve showing application of Beers Law at the absorbance of benzoic acid at wavelengths 230nm and 272nm. Y-intercepts set at 0 for 0 absorbance at 0ppm benzoic acid

Calibration Curve
Table 2. Concentrations of caffeine standards and their corresponding UV-Vis absorbance at the given wavelength

Conc (ppm) 4.00 8.00 12.00 16.00 20.00

Absorbance 230nm 0.200 0.409 0.613 0.824 1.032 272nm 0.367 0.751 1.125 1.503 1.881

Figure 3. Standard calibration curve for caffeine standard, showing absorbance readings from 350-210nm

Calibration Curve
230nm 272nm

Slope (a ,caff) R2

0.0514 0.9998

0.0939 0.9999

Figure 4. Absorbance curve showing application of Beers Law at the absorbance of caffeine at wavelengths 230nm and 272nm. Y-intercepts set at 0 for 0 absorbance at 0ppm caffeine

Calibration Curve
a230,benz = 0.0934 a272,benz = 0.0086
A230,benz = 0.0934bCbenz A272,benz = 0.0086bCbenz

a230,caff = 0.0514 a272,caff = 0.0939

A230,caff = 0.0514bCcaff A272,bcaff= 0.0939bCcaff

Sample Analysis: Methodology

Procedure Boil to remove CO2 Rationale - Removes the carbonic acid buffer system in the soft drink - Allows for later control of pH and ionic strength of analyte solution - Ensures no solid particles in solution that would interfere with absorption reading (blocks transmitted light) - Ensures that analyte would fall within calibration curve - Ensures consistent pH and ionic strength of analyte solution

Filter to remove particles

Prepare two dilutions of each sample (2:100, df=50, 4:100, df=25) Add 10mL 0.1M HCl Each sample analyzed for absorbance at and

Sample Analysis

Figure 7. Absorbance readings for Mountain Dew sample at 2:100 and 4:100 dilutions from wavelengths 350nm210nm

Figure 8. Absorbance readings for 7up sample at 2:100 and 4:100 dilutions from wavelengths 350nm-210nm

Figure 9. Absorbance readings for Sprite sample at 2:100 and 4:100 dilutions from wavelengths 350nm-210nm

Sample Analysis
Table 3. Different soft drinks brands and their corresponding UV absorbance readings at given wavelengths. The concentrations of the analyzed sample and overall samples itself were determined along with percent deviation from average values obtained from literature. *Mountain Dew: 157.2ppm caffeine, sprite: 0ppm caffeine, 7Up: 0ppm caffeine 172ppm benzoic acid for all soft drinks

Brand

Dilution Factor 25

(nm)

Abs

Conc. Caffeine (ppm) 81.04

Conc. Benzoic Acid (ppm) 173.3

Percent Deviation*

272 229

0.364 0.814 0.187 0.426 0.074 0.620 0.048 0.316 0.064 0.583 0.033 0.278

Mountain Dew 50

Caffeine: -48.45% Benzoic acid: +0.7558% Caffeine: -47.29% Benzoic acid: +6.105% Caffeine: +4.742% Benzoic acid: -5.058% Caffeine: +10.60% Benzoic acid: -5.058% Caffeine: +2.893% Benzoic acid: -10.17% Caffeine: +4.150% Benzoic acid: -14.82%

272 229

82.86

182.5

25 Sprite 50

272 229.5 272 229.5

4.742

163.3

10.60

163.3

25 7Up 50

272 229.5 272 229.5

2.893

154.5

4.150

146.5

Sample Analysis
272,caff=0.0939 1

cm-1ppm-1 -1 272,benz=0.0086 cm ppm

cm-1ppm-1 -1 -1 230,benz=0.0943 cm ppm

230,caff=0.0514

Mountain Dew Dilution Factor=25 (4:100) A272= 272,caffBCcaff + 272,benzBCbenz A230= 230,caffBCcaff + 230,benzBCbenz

Ccaff,final = 25(3.242 ppm)= 81.04 ppm Cbenz,final = 25(6.931 ppm)= 173.3 ppm

Sources of Error
Use of slightly lower wavelength (229 or 229.5nm) instead of maximum (230nm) for sample analysis

Absorbance readings would be slightly lower than if they were measured at 230nm
 

Caffeine would appear to be higher Benzoic acid would appear to be lower

Caffeine has greater at 230nm, so lower apparent absorbance would have caffeine contribute greater absorbance via higher concentration

Sample Preparation Instrumental Error

Conclusions and Recommendations

UV-Vis spectrophotometry can be a reliable method for determination of independently absorbing species in samples at trace amounts Could have a spiked sample to be analyzed
Add

known amounts of caffeine and benzoic acid and check if UV-Vis analysis could accurately return the data

References
European Parliament and Council Directive 95/2/EC (1995) on food additivies other than colours or sweeteners. (1995). Official Journal of the European Communities L61, 18.3.95, (pp. 1-40). Hensrud, D. (2011). Nutrition and healthy eating - Coffee and health: What does research say? Retrieved August 2011, from Mayo Clinic: http://www.mayoclinic.com/health/coffee-andhealth/AN01354 Howell, J. A. (1997). Ultraviolet and Visible Molecular Absorption Spectrometry. In F. Settle, Handbook of Instrumental Techniques for Analytical Chemistry (pp. 481-506). New Jersey: Prentice Hall. Lino, C., & Pena, A. (2010). Occurrence of caffeine, saccharin, benzoic acid and sorbic acid in soft drinks and nectars in Portugal and subsequent exposure assessment. Food Chemistry , 121 (2), 503-508. Wibbertmann, A., Kielhorn, J., Koennecker, G., Mangelsforf, I., & Melber, C. (2005). Concise International Chemical Assessment: Benzoic Acid and Sodium Benzoate. World Health Organization. Hanover: United Nations Environment Programme. Wilson, J. (2011). Caffeine Content of Popular Drinks. Retrieved August 2011, from http://wilstar.com/caffeine.htm Yucesoy, C. (2000). Determination of some parameters which affect accuracy and precision in UV-Vis spectrophotometry. J. Fac. Pharm., Ankara , 29 (2), 7-18.