Phase Diagram of the Ouzo Effect Using Temperature-Controlled UV-Vis Spectroscopy

Significance of the Topic

The construction of phase diagrams is central to understanding how mixtures transition between solubility and turbidity under varying conditions. The ouzo effect—a rapid emulsification observed when water is added to anethole-containing spirits—serves as a model for studying colloidal stability, solubility limits, and phase behavior in complex mixtures. Insights from this study guide formulation design in food, beverage, pharmaceutical, and materials science sectors.

Objectives and Study Overview

This application note aimed to characterize the solubility boundary of anethole in pastis liqueur as a function of ethanol concentration and temperature. Specifically, the study sought to:

• Measure onset temperatures for anethole precipitation across ethanol levels from neat (45 % v/v) down to 30 % v/v.
• Build a detailed solubility phase diagram correlating temperature and alcohol content.
• Demonstrate the efficiency of temperature-controlled, multizone UV-Vis spectroscopy for rapid phase boundary determination.

Methodology

Eight pastis solutions (45.0, 44.0, 42.5, 40.0, 37.5, 35.0, 32.5, and 30.0 % v/v ethanol) were prepared in quartz cuvettes with stir bars. Each sample cuvette was matched with a reference containing only ethanol and deionized water. The temperature cycle consisted of:

• Stage 1 (heating): Ramp from 25 °C to 55 °C at 5 °C/min, hold 10 min to fully dissolve any precipitate.
• Stage 2 (cooling): Ramp from 55 °C to –5 °C at 1 °C/min while monitoring light scattering at 750 nm.

Light scattering at 750 nm provided a direct measure of anethole precipitation, free from chromophore interference. Onset temperatures were determined via the first-derivative method on absorbance vs. temperature curves.

Used Instrumentation

The key instrument was the Agilent Cary 3500 UV-Vis spectrophotometer equipped with the Multicell Peltier sampling module. Features included:

• Xenon flash lamp and double out-of-plane Littrow monochromator (SBW adjustable 0.1–4.0 nm).
• Eight cuvette positions with independent Peltier temperature control (–5 °C to 110 °C) and stirring.
• Multizone function enabling four parallel sample/reference temperature-ramping experiments.
• Nitrogen purge to prevent condensation during subzero measurements.

Main Results and Discussion

Cooling curves showed a clear increase in scattering absorbance as temperature fell below each mixture’s solubility limit. Key observations:

• Onset temperature for anethole precipitation rose from ~13 °C at 45 % ethanol to ~34 °C at 30 % ethanol, illustrating decreased solubility with dilution.
• An irreversible scattering drop near –3 °C indicated coalescence and phase separation of water domains.
• Reproducible onset temperatures required slow cooling (≤ 1 °C/min) to avoid kinetic hysteresis.

These data yielded an eight-point phase diagram demarcating soluble vs. turbid regions as a function of alcohol content and temperature.

Benefits and Practical Applications

• Rapid construction of phase diagrams in under 1.5 h for eight conditions versus days by classical approaches.
• Simultaneous multizone analysis increases throughput and consistency across replicates.
• Enhanced control over subzero and elevated temperature studies without condensation artifacts.
• Applicability to diverse formulations requiring solubility and turbidity profiling, such as emulsions, pharmaceuticals, and process monitoring.

Future Trends and Opportunities

Advances in automated multizone temperature control combined with high-throughput spectroscopy will accelerate phase mapping in complex systems. Potential developments include:

• Integration with microfluidic sampling for rapid dilution and mixing gradients.
• Coupling with scattering size analyzers or Raman spectroscopy to elucidate mechanistic details of nucleation and coalescence.
• Machine learning models trained on spectroscopic phase data for predictive formulation design.

Conclusion

The use of the Cary 3500 UV-Vis system with Multicell Peltier modules and multizone capability enabled a time-efficient, robust determination of the ouzo effect phase diagram. This approach offers a powerful platform for studying solubility boundaries and turbidity transitions in a wide range of formulations.

References

1. United States Pharmacopeia. General Chapter {855} Nephelometry and Turbidimetry. USP–NF, 2024.
2. Sitnikova, N. L.; Sprik, R.; Wegdam, G.; Eiser, E. Spontaneously Formed trans-Anethol/Water/Alcohol Emulsions: Mechanism of Formation and Stability. Langmuir 2005, 21, 7083–7089.