CBSE Class 12 - Investigatory Project
Title: Estimation of Relative Amount of Caffeine in Different Brands of Tea (Comparative Study)
Student Name: _______________________
Class / Roll No.: ____________________
School: _____________________________
1. Aim
To estimate and compare the relative amount of caffeine present in different commercially available tea
brands using solvent extraction followed by UV-Visible spectrophotometry (calibration curve method).
2. Apparatus & Chemicals
• UV-Visible spectrophotometer (wavelength ~ 273 nm) or access to a school/college lab
• Analytical balance (±0.001 g)
• Conical flasks, volumetric flasks (10, 25, 50, 100 mL)
• Separatory funnel (250 mL) or liquid-liquid extraction apparatus
• Pipettes, micropipettes, graduated cylinders
• Beakers, funnels, filter paper
• Soxhlet apparatus (optional)
• Magnetic stirrer (optional)
• Caffeine standard (analytical grade) or pure caffeine powder
• Dichloromethane (DCM) or chloroform (extraction solvent) — handle with care
• Sodium carbonate (Na2CO3) or sodium hydroxide (to make solution basic)
• Distilled water, ethanol (for dissolving standard if needed)
• Gloves, goggles, lab coat — personal protective equipment (PPE)
3. Theory
Caffeine is an alkaloid soluble in organic solvents. The amount of caffeine in tea can be estimated by
extracting it into an organic solvent (e.g., dichloromethane) after making the aqueous tea solution slightly
basic. The extracted caffeine shows characteristic UV absorption (λmax ≈ 273 nm). Using a calibration
curve prepared from known concentrations of caffeine standard, the concentration in tea extracts can be
determined from measured absorbances by interpolation.
4. Procedure (step-by-step)
Preparation of tea sample: Weigh accurately 2.00 g of dry tea leaves (for each brand) and add 100 mL of
boiling distilled water. Allow to steep for 5 minutes with stirring. Filter to obtain the aqueous tea extract. Let
cool.
Make alkaline: Add small amounts of saturated sodium carbonate solution to the cooled aqueous extract
until slightly basic (pH ~ 8–9).
Liquid-liquid extraction: Transfer the alkaline extract to a separatory funnel and extract with 3 × 20 mL
portions of dichloromethane (DCM) or chloroform. Combine the organic layers.
Drying: Dry the combined organic layer over anhydrous sodium sulfate, then filter.
Evaporation: Evaporate the solvent gently under reduced pressure or in a fume hood to obtain crude
caffeine residue. Re-dissolve the residue in a known small volume (e.g., 10.00 mL) of ethanol or methanol
to make the final analyte solution.
Calibration curve: Prepare standard caffeine solutions of known concentrations (e.g., 2, 4, 6, 8, 10
µg/mL) by dissolving accurately weighed caffeine in solvent and dilute to volume. Measure absorbance at
λmax ≈ 273 nm.
Measurement: Measure absorbance of each re-dissolved tea sample at the same wavelength and
determine concentration from the calibration curve.
Blank & repetitions: Run a solvent blank and perform each measurement in triplicate for accuracy.
5. Data & Observations
A) Calibration data (example table). Replace sample values with your measured values:
Std No. Concentration (µg/mL) Measured Absorbance (273 nm)
1 2 _____
2 4 _____
3 6 _____
4 8 _____
5 10 _____
B) Tea samples: Record absorbances and compute concentration from calibration curve.
Brand Mass of dry tea Final
used volume
(g) of extract
Measured
(mL) Absorbance
Calculated
(273 nm)
caffeineCaffeine
(µg/mL) per g tea (mg/g)
Brand A 2.00 10.00 _____ _____ _____
Brand B 2.00 10.00 _____ _____ _____
Brand C 2.00 10.00 _____ _____ _____
Brand D 2.00 10.00 _____ _____ _____
6. Calculations (example)
1. From the calibration curve (Absorbance vs concentration), obtain the concentration of caffeine in the
final solution (µg/mL). 2. If final solution volume = V_final (mL) and concentration = C (µg/mL), total
caffeine mass in extract = C × V_final (µg) = (C × V_final)/1000 (mg). 3. Divide by mass of dry tea used (g)
to get mg caffeine per g tea. Example: Measured concentration from calibration = 6.5 µg/mL, V_final = 10
mL → total = 65 µg = 0.065 mg. If tea mass = 2.00 g → caffeine = 0.0325 mg/g. (Note: example numbers
here are illustrative; real extraction yields are larger.)
7. Precautions & Safety
• Perform solvent handling (DCM/chloroform) in a fume hood. These solvents are harmful if inhaled and
are volatile.
• Wear PPE — gloves, goggles, lab coat.
• Dispose solvent waste according to your school's chemical waste rules.
• Do not heat closed containers; evaporate solvents under controlled conditions.
• Use analytical balance carefully and avoid spills.
• If UV-Vis instrument is not available, consult a college lab or teacher for access — do not attempt unsafe
improvisations.
8. Alternatives & Notes
If a UV-Vis spectrophotometer is not accessible, alternative (but less quantitative) approaches include: -
Thin layer chromatography (TLC) with a caffeine standard for qualitative comparison of relative caffeine
amounts. - Coordinating with a nearby college lab for HPLC or UV-Vis measurements (HPLC is more
accurate but requires institutional access).
9. Conclusion
After completing measurements and calculations, compare caffeine content (mg/g) across brands and
comment on relative differences, sources of error, and reliability of the method.
10. References
• Skoog, D.A., Holler, F.J., & Crouch, S.R. (Analytical Chemistry textbooks) — for extraction & calibration methods.
• Standard caffeine UV spectra resources and reagent suppliers' safety data sheets (SDS).
• CBSE practicals and investigatory project guidelines (follow school instructions for report format).
Appendix A — Plotting Calibration Curve
Plot absorbance (y-axis) vs standard concentration (x-axis). Fit a straight line (y = mx + b) using linear
regression. Use the equation to calculate unknown concentrations from measured absorbances. Example
steps to plot: 1. Use graph paper or Excel/Google Sheets. 2. Enter standard concentrations and measured
absorbances. 3. Draw best-fit straight line and calculate slope (m) and intercept (b) or use spreadsheet
regression. 4. For unknown absorbance A_unknown, concentration C_unknown = (A_unknown - b)/m.
Appendix B — Sample Risk Assessment (brief)
• Hazard: Volatile organic solvents (DCM/chloroform) — Risk: inhalation, skin contact. Control: Work in fume hood,
PPE, avoid open flames.
• Hazard: Hot water/boiling — Risk: burns. Control: handle carefully with tongs, allow cooling.
• Hazard: Chemical spills — Control: follow spill procedures, inform teacher.