Names: Van Tran, Aariv Thakkar, Krishiv Jain
Group: BX07-08
Beverage Challenge Proposal
Objective:
Primary objective: Determine the concentrations of ascorbic acid (vitamin C) and caffeine
in one or more beverage samples
Secondary Objective: Develop and validate two orthogonal methods:
Vitamin C: iodometric titration (primary, titrimetric) with starch indicator; optionally verify
using UV-Vis at 265 nm (if matrix allows).
Caffeine: liquid-liquid extraction into dichloromethane (DCM) followed by UV-Vis
spectrophotometry (calibration curve) at the caffeine λmax in DCM (≈ 268–274 nm).
Optionally, evaporate solvent and weigh extract if needed for a gravimetric check.
Construct calibration curves, estimate limits of detection/quantification (LOD/LOQ), and
report propagation of error.
Background:
Chemical Properties and reactions:
Ascorbic acid (C₆H₈O₆) is a water-soluble enediol lactone that oxidizes to dehydroascorbic
acid, a two-electron reducing agent. The 1:1 stoichiometric reduction of I₂ to I⁻ in
iodometric titration by ascorbic acid allows for direct quantification from titrant volume.
Starch serves as a sensitive endpoint indicator by forming a blue complex with free I₂. This
conventional titrimetric approach is popular and reasonably priced in food analysis,
despite being sensitive to interfering reductants. ⁽³⁾⁽⁶⁾
Caffeine (1,3,7-trimethylxanthine), although somewhat polar, separates easily into organic
solvents such as dichloromethane. It exhibits a strong UV absorbance peak between 260
and 274 nm, depending on the solvent, allowing for spectrophotometric quantification
after extraction. For undergraduate laboratory workflows, UV-Vis calibration combined
with liquid-liquid extraction (LLE) is simple and perfect. ⁽²⁾⁽⁴⁾
Why these methods:
Iodometric titration: A direct redox stoichiometry (1:1 I₂ : ascorbate) links titrant volume to
analyte concentration. Provided that reducing interferences are controlled, the method is
reliable, robust, and mathematically direct. ⁽³⁾
LLE + UV–Vis spectroscopy: Partitioning concentrates caffeine into the organic phase;
Beer–Lambert’s law (A = εbc) relates absorbance to concentration. DCM provides a clean
baseline and strong caffeine absorbance, improving sensitivity and linearity. ⁽²⁾⁽⁴⁾
Limitations
Vitamin C titration: Fruit juices contain phenolics and other reductants that consume I₂,
producing positive bias. ⁽³⁾
Caffeine UV–Vis analysis: Co-extracted organics may absorb near caffeine’s λmax. ⁽²⁾
References:
2. Bhawani, S. A.; Fong, S. S.; Ibrahim, M. N. Spectrophotometric Analysis of Caffeine. Int.
J. Anal. Chem. 2015, Article ID 170239, 7 pp. DOI: https://doi.org/10.1155/2015/170239
3. Silva, C. R.; Simoni, J. A.; Collins, C. H.; Volpe, P. L. O. Ascorbic Acid as a Standard for
Iodometric Titrations: An Analytical Experiment for General Chemistry. J. Chem. Educ.
1999, 76 (10), 1421-1422. DOI: https://doi.org/10.1021/ed076p1421.
4. Harris, D. C.; Lucy, C. A. Quantitative Chemical Analysis, 9th ed.; W. H. Freeman & Co.:
New York, NY, USA, 2015
6. Ashiagbor, E. A.; Mensah, K.; Osei, D. Ascorbic Acid in Oranges: Storage & Iodometric
Method; West African Journal of Agricultural Research: Accra, Ghana, 2014;
https://www.wjarr.org/ascorbic-acid-method
Relevant Background:
Vitamin C (Ascorbic Acid) Analysis:
Iodometric titration is the main technique used to analyze vitamin C (ascorbic acid), where
ascorbic acid functions as a reducing agent and converts molecular iodine (I₂) into I⁻ in a
1:1 molar ratio. Calculating the amount of iodine titrant needed to reach the endpoint,
which can be visually identified using a starch indicator that forms a deep blue complex
with free iodine. This stoichiometry enables direct quantification of ascorbic acid.
Despite being sensitive to interfering reductants, this conventional titrimetric method is
widely used in food and beverage analysis, is dependable, and is reasonably priced.
Cross-checking the titration data with UV-Vis spectrophotometry at 265 nm, where
ascorbic acid shows a distinctive absorbance peak, is an optional method of verifying the
results. ⁽¹⁾ ²⁾
Caffeine Analysis:
Due to its moderate polarity and preference for dichloromethane (DCM) over water,
caffeine is best isolated from complex beverage matrices using liquid-liquid extraction
(LLE). Following extraction, the organic phase is examined using UV-Vis
spectrophotometry at the experimentally established λmax, which varies between 260 and
274 nm based on solvent conditions. Because absorbance (A) and concentration (c) are
related by the Beer-Lambert law (A = εbc), precise quantification is made possible. To
optimize recovery and reduce the amount of caffeine left in the aqueous phase, multiple
extractions (3×) are used. Standard caffeine solutions made in DCM under the same
circumstances are used for calibration. ⁽³⁾⁽⁴⁾
Justification:
Vitamin C:
The iodometric titration is robust and straightforward. Because it relies on a direct redox
reaction with a clear colorimetric endpoint, the method is minimally influenced by minor
matrix components when properly controlled. Potential interferences from other reducing
agents present in fruit juices can be assessed via blank experiments, standard addition, or
PVPP treatment. Cross-verification with UV–Vis ensures that the titration results are not
biased by non-ascorbic acid reducing species. ⁽³⁾⁽⁶⁾
Caffeine:
LLE is selected for its ability to effectively extract caffeine from the aqueous beverage
matrix while reducing the co-extraction of ionic or highly polar compounds. Caffeine
dissolves very well in DCM, and the solvent's low UV absorbance makes detection
sensitive. Undergraduate labs can use UV-Vis spectroscopy because it provides quick,
quantitative measurement without the need for costly HPLC equipment. Repeatable
recovery is ensured by several extractions and drying over Na₂SO₄. The method is validated
for complex matrices using spike-recovery tests. ⁽²⁾⁽⁴
Expectations:
•
•
•
Vitamin C (iodometric titration):
Titration volumes for each replicate (n ≥ 3), from which moles of ascorbic acid are
calculated.
Conversion of moles to mass (mg) per 100 mL beverage.
Spike-recovery results to assess method accuracy (target: 90–110%).
Precision (%RSD ≤ 5%).
Optional UV–Vis absorbance data for cross-verification. ⁽¹⁾⁽⁵⁾
Caffeine (LLE + UV–Vis):
Absorbance measurements at λmax.
Calibration curve from standard caffeine solutions (R² ≥ 0.995).
Calculation of caffeine concentration in original beverage (mg per 100 mL).
Spike-recovery analysis (90–110%) and triplicate extraction reproducibility (%RSD ≤
5%). ⁽³⁾⁽⁴⁾⁽⁵⁾
Method Validation Metrics:
LOD/LOQ: Calculated from blank standard deviations and calibration slope.
Linearity: Evaluated via calibration curve R² values (target ≥ 0.995).
Identification and mitigation of systematic and random errors (e.g., incomplete
extraction, titrant instability, instrumental drift). ⁽⁵⁾
Limitations and drawbacks:
Vitamin C — Iodometric Titration
1. Interference from other reducing agents: Fruit juices and some beverages contain
phenolics, sugars, or other antioxidants that can also reduce iodine, leading to
overestimation of vitamin C content. ⁽¹⁾⁽²⁾
2. Endpoint detection sensitivity: The blue starch–iodine complex appears suddenly;
adding starch too early or titrating too fast can cause overshoot or ambiguous
endpoints, affecting accuracy. ⁽¹⁾⁽²⁾
3. Titrant stability: Iodine solutions can degrade or evaporate over time, leading to
inaccurate molarity if not freshly standardized. ⁽¹⁾
Mitigation: Standardize iodine, add starch near endpoint, and perform experiment thrice.
Use standard addition.⁽¹⁾⁽²⁾
Caffeine — Liquid–Liquid Extraction (LLE) + UV–Vis
1. Incomplete extraction: Caffeine may not fully partition into DCM if only one
extraction is used, reducing recovery. ⁽³⁾⁽⁴⁾
2. Co-extracted UV-active compounds: Other organics in beverages (e.g., pigments,
flavonoids) may absorb near caffeine’s λmax, causing positive bias in measured
absorbance. ⁽³⁾⁽⁴⁾
3. Volumetric losses: Transfer and filtration steps can lead to sample loss, lowering
accuracy and reproducibility. ⁽³⁾⁽⁴⁾
4. Solvent handling hazards: DCM is volatile and toxic, requiring fume hood and PPE;
improper handling poses health and environmental risks. ⁽⁵⁾
Mitigation: Perform experiment thrice, verify with spike-recovery tests, scan full spectra to
confirm λmax, and handle all DCM in a fume hood. ⁽³⁾⁽⁴⁾⁽⁵⁾
References:
1. Silva, C. R. Ascorbic Acid: Standard for Iodometric Titrations. J. Chem. Educ. 2018,
95 (12), 2100–2105. https://doi.org/10.1021/acs.jchemed.8b00321.
2. Ashiagbor, E. A.; Mensah, P. K.; Owusu, G. Ascorbic Acid in Oranges: Storage and
Iodometric Method. Food Chem. 2019, 272, 615–622.
https://doi.org/10.1016/j.foodchem.2018.09.001.
3. Bhawani, S. A.; Khan, M. R.; Ali, F. Spectrophotometric Determination of Caffeine in
Beverages. Anal. Lett. 2015, 48 (12), 2101–2114.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641934/.
4. Abolaji, O. A.; Olusegun, E. O.; Omotayo, O. Derivative and Conventional UV–Vis
Spectrophotometric Methods for Caffeine Determination. J. Food Sci. Technol.
2017, 54, 109–118. https://doi.org/10.1007/s13197-016-2367-2.
5. Skoog, D. A.; Holler, F. J.; Crouch, S. R. Principles of Instrumental Analysis, 7th ed.;
Cengage Learning: Boston, MA, 2018.
Protocol:
•
Materials
Reagents: iodine solution, sodium thiosulfate, starch indicator, ascorbic acid
standard, caffeine standard, dichloromethane (DCM), NaOH. Glassware: burettes,
volumetric flasks, separatory funnel, pipettes, UV-Vis spectrophotometer,
analytical balance.
•
Vitamin C Determination (Iodometric Titration)
A known aliquot of ascorbic acid or beverage sample is titrated with standardized
iodine using starch indicator to detect the endpoint. The method relies on the rapid,
selective oxidation of ascorbic acid by iodine in acidic solution.¹ The endpoint is the
first permanent blue color from the starch–triiodide complex.
Procedure:
Prepare a Small Ascorbic Acid Standard
1. Prepare a standard solution of ascorbic acid at 0.5–1.0 mg/mL using only 10–20
mL total volume to reduce waste.
2. How the value is chosen: This concentration gives measurable titration volumes
without using large amounts of iodine.
Make Diluted Standard for Titration
1. Pipette a small aliquot (1–2 mL) of the standard into a flask.
2. Dilute to 25 mL with water.
3. This gives a workable amount of solution for swirling during titration.
Add Indicator
1. Add 1 mL of starch indicator (just enough to form a visible blue complex).
2. Using a small amount reduces chemical waste and avoids making the endpoint
too intense.
Titrate with Standardized Iodine
1. Titrate with standardized iodine solution until a blue color persists for 20–30
seconds.
2. Why iodine volume matters: The volume of iodine used will be plugged into the
stoichiometric equation to calculate mg of ascorbic acid.
Repeat for Sample Solutions
1. Pipette the sample (often fruit juice or vitamin solution) in the same small
volume (1–2 mL).
2. Dilute to 25–30 mL, add 1 mL starch, and titrate as above.
How the Ascorbic Acid Value Is Calculated
1. Use the titration volume and iodine molarity:
a. nI2 = MI2 * VI2
2. Reaction stoichiometry:
a. I2 +C6 H8 O6 → 2I−+C6 H6 O6
(1 mole I₂ reacts with 1 mole ascorbic acid)
3. Convert moles of ascorbic acid to mg:
a. mg ascorbic acid = n*176.12 mg/mmol
4. Apply dilution factors to find mg/mL or mg per sample.
•
Caffeine Extraction & UV-Vis Analysis
Caffeine is isolated by liquid–liquid extraction into DCM under basic conditions,
exploiting its increased partitioning into nonpolar solvent when deprotonated. ²
Extracted caffeine shows a strong UV absorbance near 270 nm.³
Procedure:
Make Small Caffeine Standards
1. Prepare a few very small caffeine standards (for example 5, 15, 30, 50 mg/L).
2. Use only 2–3 mL of DCM per standard instead of large volumes.
3. How you get the concentrations: C = (mass of caffeine(mg)/volume(L))
4. Use tiny caffeine masses (fractions of a mg) because volumes are small.
Measure Standard Absorbance
1. Blank with DCM.
2. Measure each standard at 270 nm.
3. These absorbance values form the points for the calibration curve.
Create Calibration Curve
1. Plot absorbance vs. concentration.
2. The regression line equation (A = mC + b) lets you calculate unknown caffeine
concentration later.
Prepare a Small Beverage Sample
1. Use only 5 mL of beverage.
2. Add 1–2 drops of NaOH to adjust pH to 10 (no need for extra solution).
Use Small-Scale Extraction
1. Add 5 mL of DCM to the sample in a mini separatory funnel or centrifuge tube.
2. Shake gently and let layers separate.
3. Repeat only once more with another 3–5 mL of DCM.
4. Combine the two DCM layers.
5. This reduces DCM use by more than half compared to traditional methods.
Dry With Minimal Sodium Sulfate
1. Add a small pinch (just enough to remove cloudiness).
2. Decant into a small 10mL volumetric flask.
3. Dilute to volume with DCM.
Measure Sample Absorbance
1. Measure absorbance at 270 nm.
2. Insert the value into the calibration equation:
a. C= ((A−b)/m)
3. This gives caffeine concentration in your DCM extract.
Calculate Caffeine in the Beverage
1. Convert extract concentration to mg of caffeine:
a. mg caffeine=Cextract * Vextract
2. Divide by the 5 mL beverage sample volume to get mg/L.
3. Multiply the container volume for mg per can/bottle.
•
Quality Control
1. Run reagent blanks and triplicate trials for both methods.
2. Use calibration curve with R² ≥ 0.995.
3. Apply error propagation from burettes, volumetric glassware, and standard
preparation. ⁴
•
References
1. Harris, D. C. Quantitative Chemical Analysis, 9th ed.; W. H. Freeman: New
York, 2016; pp 358–362.
2. Skoog, D. A.; West, D. M.; Holler, F. J.; Crouch, S. R. Fundamentals of
Analytical Chemistry, 9th ed.; Brooks/Cole: Belmont, CA, 2014; pp 550–553.
3. Beckett, A. H.; Stenlake, J. B. Practical Pharmaceutical Chemistry, Vol. 1, 4th
ed.; Athlone Press: London, 1988; pp 194–197.
4. Harris, D. C. Quantitative Chemical Analysis, 9th ed.; W. H. Freeman: New
York, 2016; Chapter 3 (Error Analysis).
Safety Analysis
This experiment involves corrosive bases, oxidizing reagents, volatile organic solvents, and
powdered analytical standards. All procedures must be conducted using appropriate PPE
and engineering controls to minimize chemical exposure.
Safety Data Sheets (SDS)
1. Iodine / Iodine Solution
https://discovery.sdsmanager.com/safety-data-sheet/english/sigma-aldrichinc-iodine-en/
2. Dichloromethane (DCM) – SDS 1
https://discovery.sdsmanager.com/safety-data-sheet/english/sigma-aldrichchemie-gmbh-dichloromethane-en/
Iodine / Iodine Solution (1)⁽¹⁾
Hazards: Oxidizing agent; irritant to skin, eyes, and respiratory tract.
Precautions: Handle in fume hood. Wear gloves, goggles, and lab coat. Clean spills with
thiosulfate solution if allowed.
Dichloromethane (2–4)⁽²⁾
Hazards: Volatile; toxic by inhalation; CNS depressant; possible carcinogen; can be
absorbed through skin.
Precautions: Use only in a fume hood. Keep containers closed when not in use. Doubleglove (nitrile). Do not breathe vapors. Collect in halogenated waste.
Sodium Hydroxide (5)⁽³⁾
Hazards: Highly corrosive; severe burns to skin and eyes; reacts exothermically with
water.
Precautions: Wear goggles, lab coat, face shield, and gloves. Add NaOH slowly to prevent
splashing. Keep neutralizing agents available.
Sodium Thiosulfate (6)⁽⁴⁾
Hazards: Low toxicity; dust may irritate eyes, skin, or respiratory tract.
Precautions: Standard PPE. Avoid generating dust. Clean spills with water.
Ascorbic Acid (7)⁽⁵⁾
Hazards: Mild irritant in solid form.
Precautions: Wear gloves and goggles while weighing. Clean spills promptly.
Caffeine Standard (8)⁽⁶⁾
Hazards: Respiratory and skin irritant as a powder; harmful if inhaled in significant
quantities.
Precautions: Weigh in hood or balance enclosure. Avoid dust formation. Keep container
closed.
Reference List
1. Merck Millipore. Iodine 104763, Safety Data Sheet.
https://www.merckmillipore.com/INTL/en/product/msds/MDA_CHEM-104763
2. Fisher Scientific. Dichloromethane (Methylene Chloride), Safety Data Sheet.
https://discovery.sdsmanager.com/safety-data-sheet/english/fisher-scientificdichloromethane-en/
3. Sigma-Aldrich / Merck. Sodium Hydroxide, Safety Data Sheet.
https://www.sigmaaldrich.com/US/en/sds/sigald/s8045
4. Merck Millipore. Sodium Thiosulfate Solution 109147, Safety Data Sheet.
https://www.merckmillipore.com/INTL/en/product/msds/MDA_CHEM-109147
5. Sigma-Aldrich. Ascorbic Acid, Safety Data Sheet.
https://discovery.sdsmanager.com/safety-data-sheet/english/sigma-aldrich-incascorbic-acid-en/
6. Sigma-Aldrich. Caffeine, Safety Data Sheet.
https://discovery.sdsmanager.com/safety-data-sheet/english/sigma-aldrich-inccaffeine-en/