A Novel Automated Liquid/Liquid Extraction Technique for the Determination of Caffeine in Coffee

Importance of the Topic

The quantification of caffeine in coffee is crucial for beverage manufacturers, regulatory compliance, and consumer awareness. Accurate analysis supports quality control, flavor profiling, and ensures consistent stimulant levels across products.

Objectives and Study Overview

This study employed an automated liquid–liquid extraction method to determine caffeine content in various coffee blends, brands, and brewing methods. Light, medium, dark, and flavored variants of Brand A were evaluated, along with medium blends from Brands A, B, and C, and a comparison between single-cup and brewed-pot preparations of Brand C.

Methodology

An automated extraction workflow utilized methylene chloride to selectively partition caffeine from aqueous coffee samples. Two milliliters of brewed coffee were transferred into reaction vials and processed in triplicate to assess reproducibility. Calibration was performed with a 5000 ppm caffeine standard in water, generating a calibration curve for quantification.

Instrumentation

• EST Analytical FLEX autosampler with 250 µL liquid syringe for automated sample handling
• Agilent 7890 GC coupled to a 5975 mass spectrometer for separation and detection
• Titan PTV large-volume injector in pulsed split mode (250:1) with cryogenic cooling
• Restek Rxi-5 Sil MS column (30 m × 0.25 mm I.D., 0.25 µm film)
• Helium carrier gas at 1.4 mL/min and GC oven program from 40 °C to 310 °C over 14 min

Key Results and Discussion

Caffeine concentrations varied from 27.4 mg in decaffeinated coffee to 123.5 mg in medium roast B, with mocha-flavored roast exhibiting higher levels (119.8 mg) likely due to added chocolate components. Dark and medium roasts of Brand A showed nearly identical caffeine contents (~110.6 mg). Brand-to-brand differences among medium roasts were minimal. A brewed pot of Brand C contained substantially more caffeine (181.4 mg) compared to a single-cup serving (102.6 mg) due to the larger coffee-to-water ratio. The extraction also effectively removed flavor compounds such as vanillin and ethyl vanillin, with mocha extracts showing higher vanillin content than vanilla extracts.

Benefits and Practical Applications

The automated LLE approach streamlines sample preparation, reduces manual handling, and delivers high reproducibility, making it well-suited for routine quality control in coffee production, research laboratories, and regulatory testing.

Future Trends and Opportunities

Emerging developments may include coupling automated extraction with LC-MS for broader analyte coverage, integrating greener solvents, extending the method to other bioactive compounds in beverages, and implementing real-time inline monitoring for process control.

Conclusion

The FLEX automated extraction system provided a reliable and efficient workflow for caffeine analysis, yielding reproducible results across diverse coffee samples. The method’s robustness and automation potential make it valuable for industry and research applications.

References

Caffeine standard obtained from Sigma-Aldrich.