Scale-Up of an analytical HPLC method for steviol glycosides to a preparative approach

Significance of the Topic

This study addresses the transfer of a high-resolution analytical HPLC method for steviol glycosides to a preparative scale, enabling efficient purification of key sweeteners such as rebaudioside A and stevioside. Steviol glycosides offer calorie-free sweetness roughly 400 times that of sucrose, making scalable purification critical for the food and beverage industry.

Objectives and Overview of the Study

The primary goals were to:

• Convert an existing gradient analytical method to an isocratic separation using simulation software.
• Scale the isocratic method from a 4.6 mm ID analytical column to a 20 mm ID preparative column while maintaining chromatographic performance.
• Evaluate column overload capacity and matrix effects to establish optimal loading parameters.

Methodology

The approach comprised:

• Simulation of gradient-to-isocratic conversion using DryLab software, identifying a 30:70 acetonitrile/water isocratic condition.
• Analytical validation on a 250 × 4.6 mm, 5 μm C18 column at 1.2 mL/min, 30 °C, UV detection at 210 nm.
• Preparative scaling on a 250 × 20 mm, 10 μm C18 column at 22 mL/min, using the KNAUER ScaleUp Converter to preserve HETP.
• Overload experiments with injection volumes up to 2000 μL to assess peak resolution and matrix impact.

Instrumentation Used

• Analytical: KNAUER AZURA HPLC Plus with Vertex Plus column, UV detector.
• Preparative: AZURA P 2.1L pump with ternary LPG module, UVD 2.1L detector, ASM module, multi-port valves, Labocol Vario-4000 fraction collector.
• Software: PurityChrom5 Basic and KNAUER ScaleUp Converter.

Main Results and Discussion

The isocratic method achieved near-baseline separation of rebaudioside A and stevioside with improved peak shape. Scaling to preparative dimensions maintained retention characteristics, demonstrating:

• Constant HETP ensured by equal column length and adjusted flow rate.
• Maximum loading capacity identified before peak overlap, guiding sample throughput.
• Significant matrix signal increase at high loads, underlining the need for pre-purification.

Benefits and Practical Applications

The presented workflow allows rapid method transfer across scales, reducing development time and preserving separation quality. It supports high-throughput purification of steviol glycosides and can be adapted to other analyte classes, enhancing laboratory efficiency and product yield.

Future Trends and Potential Applications

Advances may include online sample cleanup integration (SPE), continuous processing, and expanded use of scale-up software for diverse compound families. Automation and predictive modeling will further streamline preparative HPLC development.

Conclusion

This work demonstrates a straightforward strategy for scaling an analytical HPLC method to preparative scale for steviol glycoside purification using simulation tools and the KNAUER ScaleUp Converter. It offers a template for fast, reliable method transfer in industrial and research settings.

References

1. Samuel P., Ayoob K. T., Magnuson B. A., et al. Stevia Leaf to Stevia Sweetener: Exploring Its Science, Benefits, and Future Potential. J Nutr. 2018;148(7):1186S–1205S.
2. KNAUER Wissenschaftliche Geräte GmbH. KNAUER ScaleUp Converter Product Documentation, Berlin, Germany.