Advantages of preparative online SPE compared to batch LC for stevia purification

Importance of the Topic

Steviol glycosides from Stevia rebaudiana serve as high-intensity natural sweeteners with up to 400-fold sweetness compared to sucrose. Their calorie-free profile and favourable taste make them valuable for diabetic diets and low-carbohydrate nutrition, driving the need for efficient purification strategies.

Objectives and Study Overview

This study compares preparative online solid phase extraction (SPE) with conventional batch liquid chromatography (LC) to purify the key glycoside rebaudioside A and minimize matrix contamination. The goal is to enhance purity, protect the main column, and evaluate yield implications.

Methodology

An analytical gradient method was adapted to an isocratic preparative scale using a scale-up converter and a 20 mm ID C18 column at 22 mL/min. Online SPE was implemented upstream of the main column to remove sample matrix: conditioning, re-equilibration, sample loading (10 mL), and washing phases precede the target compound transfer. Final elution used 30:70 acetonitrile/water at 210 nm and 25 °C. Fractions were analyzed by analytical RP-HPLC.

Used Instrumentation

• AZURA P 2.1L preparative pump with ternary LPG module
• AZURA UVD 2.1L UV detector (3 mm, 2 µL flow cell)
• AZURA assistant module with multi-position valves
• AZURA P 4.1S feed pump and Labocol Vario-4000 fraction collector
• Eurospher II C18 columns (analytical and preparative dimensions)
• PurityChrom5 Basic software for data acquisition

Main Results and Discussion

Batch overload tests showed substantial matrix peaks and incomplete separation of rebaudioside A and stevioside. The online SPE approach dramatically reduced matrix signal and protected the main column. Fraction analysis indicated that fractions 3–5 (~15 mL) contained >90 % rebaudioside A and <10 % stevioside, while later fractions enriched stevioside but still retained rebaudioside A. Despite improved purity, the yield of isolated rebaudioside A was limited by partial co-elution.

Benefits and Practical Applications

• Enhanced sample purity through automated matrix removal
• Extended column lifetime by minimizing fouling
• Increased loading of target glycosides with maintained chromatographic performance

Future Trends and Potential Applications

Advances may include novel stationary phases for improved glycoside resolution, multimodal SPE materials, continuous flow integration, greener solvent systems, and real-time process monitoring. Scale-up challenges and economic assessments will guide industrial adoption.

Conclusion

Preparative online SPE significantly enhances steviol glycoside purity and safeguards the main column, yet full separation of rebaudioside A from stevioside under current conditions remains challenging, resulting in lower isolated yields.

References

1. Samuel P, Ayoob KT, Magnuson BA, et al. Stevia Leaf to Stevia Sweetener: Exploring Its Science, Benefits, and Future Potential. Journal of Nutrition. 2018;148(7):1186S–1205S.
2. KNAUER Wissenschaftliche Geräte GmbH. Scale-up Converter: KNAUER Scale-up Converter.