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.