Purification of chamazulene by preparative HPLC and its scale-up

Importance of the topic

Chamomile blue oil contains a complex mixture of bioactive components, among which chamazulene exhibits significant anti-inflammatory and analgesic properties. Purifying chamazulene in high purity and yield is critical for pharmaceutical formulations, quality control standards, and further bioactivity studies.

Objectives and overview of the study

This work aimed to develop and scale up a preparative reversed-phase HPLC method to isolate chamazulene from commercially obtained chamomile blue oil. The approach included initial analytical confirmation, semi-preparative purification, and a larger-scale process adaptation to improve throughput and solvent efficiency.

Methodology and used instrumentation

Sample preparation involved diluting chamomile blue oil in methanol–water (90:10, v/v), followed by filtration. A chamazulene calibration curve (0.015–1.500 mg/mL) was established using a commercial standard. Analytical HPLC was performed on a 250 × 4.6 mm C18 column at 1.3 mL/min with a water–acetonitrile gradient and UV detection at 245 nm.

• Analytical system components: AZURA P 6.1L LPG pump, AZURA AS 6.1L autosampler, AZURA DAD2.1L diode-array detector, CT2.1 column thermostat, Eurospher II 100-5 C18 column.
• Preparative system components: AZURA P 2.1L pump with 250 mL head, LPG ternary module, UVD 2.1L detector, fractionation valve, Eurospher II 100-10 C18 columns (250 × 20 mm and 150 × 50 mm).

Main results and discussion

Chamazulene was identified at a retention time of 7.6 min by matching the UV spectrum (190–400 nm) against the standard. Semi-preparative purification (20 mm ID column) with a 2 mL injection achieved ~100% recovery and >99% purity. Upon scale-up using a 50 mm ID column, a 10 mL injection yielded 82% recovery under increased flow rate (150 mL/min) and reduced column length. Spectral and chromatographic analyses confirmed the absence of co-eluting impurities in the collected fractions.

Benefits and practical applications

• High-purity chamazulene suitable for pharmaceutical research and development.
• Scalable purification protocol enabling increased sample throughput.
• Versatile method transferable between semi-preparative and preparative scales.

Future trends and potential applications

Exploration of normal-phase preparative HPLC could further improve solvent removal and selectivity. Industrial-scale implementations may focus on green solvent systems, continuous fractionation, and process automation. Additional studies may investigate downstream crystallization or formulation of isolated chamazulene.

Conclusion

A robust reversed-phase preparative HPLC method was successfully developed to isolate chamazulene from chamomile blue oil with excellent purity and scalable recovery. The technique offers a reliable route for producing analytical standards and active pharmaceutical ingredients, with clear potential for further process optimization and industrial application.

References

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3. Salihovic M., Sapcanin A., Spirtovic-Halilovic S., Zavrsnik D.D. DFT structural analysis of chamazulene. Bull. Chem. Technol. Bosnia Herzegovina 46:27–32 (2016).