Determination of Caffeine Content in an Energy Drink by RP-HPLC
Date: 2025-10-12
Abstract
A validated reversed-phase high-performance liquid chromatography (RP-HPLC) method
was applied to quantify the caffeine content in a commercial energy drink. The method uses
a C18 column with methanol:water as the mobile phase and UV detection near 272–275 nm.
A calibration curve prepared from caffeine standards enabled accurate quantification.
Representative example data, calculations, and uncertainty estimation are provided.
Introduction
Caffeine is an alkaloid commonly found in beverages and pharmaceutical products.
Quantification of caffeine is important for quality control and regulatory compliance.
Reversed-phase HPLC with UV detection is a widely used, robust technique for caffeine
determination due to its specificity and sensitivity. The following procedure is adapted from
validated methods in the literature and is suitable as an assignment-ready experimental
example.
Materials and Reagents
- Commercial energy drink (250 mL can)
- Caffeine standard (analytical grade)
- Methanol (HPLC grade)
- Distilled water (HPLC grade)
- Syringe filters (0.45 μm) or SPE cartridges (optional)
- Volumetric flasks, pipettes, and glassware
- RP-C18 HPLC column (e.g., 150 × 4.6 mm, 5 μm)
- HPLC system with UV detector capable of measurement at 272–275 nm
Instrumental Conditions (recommended)
Column: Reversed-phase C18 (150 mm × 4.6 mm, 5 μm)
Mobile phase: Methanol : Water (40 : 60, v/v) (or 50:50 depending on method)
Flow rate: 1.0 mL·min⁻¹
Detector: UV at 272–275 nm
Injection volume: 20 μL
Run time: 5–8 min (caffeine retention typically ~2.5–3.0 min)
These conditions follow published HPLC procedures for caffeine analysis and are
appropriate for undergraduate labs.
Procedure
Prepare a series of caffeine standards by diluting the stock solution to cover the expected
concentration range (e.g., 0, 10, 20, 40, 60 mg·L⁻¹).
Measure the absorbance/response of each standard by injecting into the HPLC and record
peak area or height.
Open the energy drink can and degas by gentle stirring. Filter the sample through a 0.45 μm
syringe filter or perform solid-phase extraction (SPE) to remove particulates and matrix
interferents.
If necessary, perform liquid–liquid extraction with chloroform to isolate caffeine, evaporate
the solvent, and re-dissolve the residue in a known volume of water or mobile phase.
Dilute the prepared sample to bring the expected caffeine concentration within the
calibration range (a typical dilution is 1:10).
Inject the diluted sample into the HPLC system in triplicate and record peak areas.
Use the calibration curve (peak area vs. concentration) to determine the sample
concentration, then correct for the dilution factor to obtain the original concentration in
mg·L⁻¹.
Calculate total caffeine per can by multiplying concentration (mg·L⁻¹) by can volume (L).
Representative Data (example)
Calibration data (peak area vs. concentration) — example values:
Concentration (mg·L⁻¹): 0, 10, 20, 40, 60
Peak area (a.u.): 0, 1200, 2400, 4800, 7200
Calibration slope (area per mg·L⁻¹) = 1200 area units per 10 mg·L⁻¹ = 120 area·(mg·L⁻¹)⁻¹
Sample measurements (diluted 1:10) — example peak areas (triplicate): 3580, 3620, 3600
(area units).
Calculations
1. Mean peak area of diluted sample = (3580 + 3620 + 3600) / 3 = 3600 area units.
2. Using calibration slope: concentration (diluted) = mean area / slope = 3600 / 120 = 30.0
mg·L⁻¹.
3. Correct for dilution (1:10): concentration (original) = 30.0 × 10 = 300.0 mg·L⁻¹.
4. Total caffeine per 250 mL can: 300.0 mg·L⁻¹ × 0.250 L = 75.0 mg.
Uncertainty and Precision
Estimate the standard deviation from the triplicate areas: deviations = -20, +20, 0 → s_area
≈ 20 area units.
Convert to concentration uncertainty: σ_C = s_area / slope = 20 / 120 = 0.167 mg·L⁻¹
(diluted).
Apply dilution factor: σ_C(original) = 0.167 × 10 = 1.67 mg·L⁻¹. Convert to total mg: 1.67 ×
0.250 = 0.42 mg.
Final reported value: 75.0 ± 0.4 mg caffeine per 250 mL can (represents measurement
repeatability only).
Discussion
The example shows that HPLC provides a precise and specific method for quantifying
caffeine in beverage matrices. Potential sources of error include incomplete extraction,
matrix effects, pipetting errors, and instrumental drift. Improvements include using solid-
phase extraction for cleaner samples, internal standardization, and performing method
validation (linearity, accuracy/recovery, precision, LOD/LOQ) if the procedure is to be used
for official analysis.
Conclusion
A validated RP-HPLC method is suitable for determining caffeine in energy drinks. Using
example calibration and sample data, the calculated caffeine content of the tested can is
75.0 ± 0.4 mg per 250 mL, which aligns with typical values reported for many commercial
energy drinks.
References
1. Pokhrel P. et al., 'A simple HPLC Method for the Determination of Caffeine Content in Tea
and Coffee', Journal of Food Science and Technology, 2016. (methodology adapted).
2. Naveen P. et al., 'Method Development and Validation for the Determination of Caffeine by
RP-HPLC', International Journal, 2018.
Additional sources: review articles on caffeine analysis by HPLC and spectrophotometry.
