127.1 Expt 4

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

Caffeinewhite 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 CaffeineBenefits 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 Acidcolorless crystalline solid and the simplest aromatic carboxylic acid salts are used as a food preservative

Benefits and Harmful Effects of Benzoic AcidBenefits 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 LawA = ebcA = 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 %TransmittanceBLL shows the linear relationship betweeen absorbance and concentration

Limitations of the Beer-Lambert Law: Causes of Nonlinearitydeviations 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 AnalysisThe 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 +


= 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




for standard solutions of both X

ObjectivesTo 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: MethodologyProcedure 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


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


the slope of the calibration curve =


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

Calibration CurveCALIBRATION CURVESTable 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 Curve230nm 272nm

Slope (a R2






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 CurveTable 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 Curve230nm 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 Curvea230,benz = 0.0934 a272,benz = 0.0086A230,benz = 0.0934bCbenz A272,benz = 0.0086bCbenz

a230,caff = 0.0514 a272,caff = 0.0939A230,caff = 0.0514bCcaff A272,bcaff= 0.0939bCcaff

Sample Analysis: MethodologyProcedure 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 AnalysisTable 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


Dilution Factor 25



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



25 Sprite 50

272 229.5 272 229.5





25 7Up 50

272 229.5 272 229.5





Sample Analysis272,caff=0.0939 1

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

cm-1ppm-1 -1 -1 230,benz=0.0943 cm ppm230,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 ErrorUse 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 RecommendationsUV-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

ReferencesEuropean Parliament and Council Directive 95/2/EC (1995) on food additivies other than colours or sweeteners. (1995). Offic