127.1 expt 4
TRANSCRIPT
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
Temporary wards off drowsiness
Restores alertness
Ergogenic Hepatoprotective
properties
“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)
Benefits Harmful Effects
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
Treatment of fungal diseases
Topical antiseptics Inhalant
decongestant
Sodium benzoate : possible cause of DNA damage and hyperactivity
Excessive amounts may affect liver and kidney
Benefits Harmful Effects
Beer-Lambert’s Law
A = ebc Where 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. The higher the concentration, the higher the
absorbance Only applicable for dilute solutions
Beer-Lambert’s 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 λ1 and λ2 at which both compounds show significant absorbance,
Atot,λ1 = AX,λ1 + AY,λ1 and Atot,λ,2 = AX,λ2 + AY,λ2
Atot,λ1 = εX,λ1bcX + εY,λ1bcY Atot,λ2 = εX,λ2bcX + εY,λ2bcY
If all the molar absorptivities are known, system of two equations and two
unknowns, cX and cY
The four ε values can be determined from the slopes of four Beer's Law plots: A vs. c at λ1 and λ2 for standard solutions of
both X and Y
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: MethodologyProcedure Rationale
• 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
Will be used in constructing Calibration Curves -HCl was used to maintain the protonated form of the compounds
• UV baseline from 350 to 210 nm was recorded with distilled H2O in the sample and reference cuvets
To calibrate the UV-Vis spectrophotometer
• U V Spectra of the standards with distilled water as reference were recorded
Water was used as reference since it was the solvent
• The wavelength of peak absorbance for benzoic Acid and for caffeine ,λ’ and λ’’ respectively, were noted. Absorbance at each measured
To generate data for calibration curves
• Calibration Curves were prepared: abs vs. [S] of each compounds at each wavelength
To know the absortivity of benzoic acid and caffeine at λ’ and λ’’
• Each graph should go to zeroTo 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
From Beer-Lambert’s Law, Aλ = ελbc the slope of the calibration curve = ελ
In the experiment, it was used to know the absorptivity of caffeine and benzoic acid
Calibration Curve
Table 1. Concentrations of benzoic acid standards and their corresponding UV-Vis absorbance at the given wavelength
Conc (ppm)
Absorbance
230nm 272nm
2.00 0.223 0.020
4.00 0.369 0.033
6.00 0.560 0.052
8.00 0.742 0.069
10.0 0.932 0.086
Figure 1. Standard calibration curve for benzoic acid standard, showing absorbance readings from 350-210nm
CALIBRATION CURVES
Calibration Curve
Figure 2. Absorbance curve showing application of Beer’s Law at the absorbance of benzoic acid at wavelengths 230nm and 272nm. Y-intercepts set at 0 for 0 absorbance at 0ppm benzoic acid
230nm 272nm
Slope (aλ,benz) 0.0934 0.0086
R2 0.9958 0.9965
Calibration Curve
Table 2. Concentrations of caffeine standards and their corresponding UV-Vis absorbance at the given wavelength
Conc (ppm)
Absorbance
230nm 272nm
4.00 0.200 0.367
8.00 0.409 0.751
12.00 0.613 1.125
16.00 0.824 1.503
20.00 1.032 1.881
Figure 3. Standard calibration curve for caffeine standard, showing absorbance readings from 350-210nm
Calibration Curve
Figure 4. Absorbance curve showing application of Beer’s Law at the absorbance of caffeine at wavelengths 230nm and 272nm. Y-intercepts set at 0 for 0 absorbance at 0ppm caffeine
230nm 272nm
Slope (aλ,caff)
0.0514
0.0939
R2 0.9998
0.9999
Calibration Curve
a230,benz = 0.0934
a230,caff = 0.0514
a272,benz = 0.0086
a272,caff = 0.0939
A230,benz = 0.0934bCbenz A230,caff = 0.0514bCcaff
A272,benz = 0.0086bCbenz A272,bcaff= 0.0939bCcaff
Sample Analysis: MethodologyProcedure Rationale
Boil to remove CO2 - Removes the carbonic acid buffer system in the soft drink- Allows for later control of pH and ionic strength of analyte solution
Filter to remove particles - Ensures no solid particles in solution that would interfere with absorption reading (blocks transmitted light)
Prepare two dilutions of each sample (2:100, df=50, 4:100, df=25)
- Ensures that analyte would fall within calibration curve
Add 10mL 0.1M HCl - Ensures consistent pH and ionic strength of analyte solution
Each sample analyzed for absorbance at λ’ and λ’’
Sample Analysis
Figure 7. Absorbance readings for Mountain Dew sample at 2:100 and 4:100 dilutions from wavelengths 350nm-210nm
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
λ (nm) Abs Conc. Caffeine
(ppm)
Conc. Benzoic Acid
(ppm)
Percent Deviation*
Mountain Dew25 272 0.364 81.04 173.3 Caffeine: -48.45%
Benzoic acid: +0.7558%
229 0.814
50 272 0.187 82.86 182.5 Caffeine: -47.29%Benzoic acid:
+6.105%229 0.426
Sprite25 272 0.074 4.742 163.3 Caffeine: +4.742%
Benzoic acid:-5.058%
229.5 0.620
50 272 0.048 10.60 163.3 Caffeine: +10.60%Benzoic acid:
-5.058%229.5 0.316
7Up25 272 0.064 2.893 154.5 Caffeine: +2.893%
Benzoic acid:-10.17%
229.5 0.583
50 272 0.033 4.150 146.5 Caffeine: +4.150%Benzoic acid:
-14.82%229.5 0.278
Sample Analysis
ε272,caff=0.0939 cm-1ppm-1
ε272,benz=0.0086 cm-1ppm-1
ε230,caff=0.0514 cm-1ppm-1
ε230,benz=0.0943 cm-1ppm-1
Mountain DewDilution Factor=25 (4:100)A272= ε272,caffBCcaff + ε272,benzBCbenz
A230= ε230,caffBCcaff + ε230,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 ε 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-and-health/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.