Recycle your peaks – A comparison of two recycling methods

Importance of Recycling Chromatography

The ability to separate compounds that coelute or have very similar retention behavior is essential in analytical and preparative applications, including purity control, chiral separations and natural product isolation. Recycling chromatography offers a cost-effective way to simulate an infinitely long column, thereby enhancing peak resolution without the need for physically longer stationary phases.

Objectives and Study Overview

This work compares two recycling techniques under identical semi-preparative HPLC conditions for a model mixture of steviol glycosides (Rebaudioside A and Stevioside). The goal was to evaluate performance in terms of achievable resolution, cycle efficiency, system dead volume, solvent usage, and operational complexity.

Methodology

Two approaches were tested on the same instrument and with the same gradient:

• Recycling through pump: A T-fitting dilutes peaks into a closed loop via a 2-port-6-position fractionation valve downstream of the detector. Cycles repeat until resolution gain is offset by peak broadening.
• Alternate pumping: Two identical columns are linked by a 2-position-6-port recycling valve. After elution from the first column, the analytes enter the second column, and the valve toggles to return them to the first column in consecutive cycles.

Sample concentration was 0.2 mg/mL each of Rebaudioside A and Stevioside in 30:70 acetonitrile/water. Injection volume was 100 µL and flow rate 3.5 mL/min.

Instrumentation

• Pump: AZURA P6.1L HPG with 50 mL stainless steel pump head
• Assistant: AZURA ASM 2.2L with valve drives VU 4.1 and UV detector UVD 2.1S
• Recycling valves: 2-position-6-port and multiposition-6-port
• Columns: Vertex 3 Eurospher 100-10 C18, 125 × 8 mm (two in alternate pumping)
• UV detection at 210 nm, data rate 2 Hz
• Software: PurityChrom 5.09.069

Main Results and Discussion

Both methods increased peak resolution with each cycle, but differed in efficiency:

• Recycling through pump: Maximum resolution of 1.13 achieved after seven cycles before peak broadening limited further gains. Signal height decreased and peak width grew rapidly due to larger dead volume and mixing.
• Alternate pumping: Achieved superior resolution of 1.29 in just six cycles under similar runtime. Smaller system dead volume reduced remixing and maintained peak shape. No sample contact with pump head prevented contamination.

Alternate pumping requires two columns and a more complex valve network, and online monitoring is only possible with an additional detector outside the recycling loop.

Benefits and Practical Applications

• Enhanced separation of closely eluting or chiral compounds without longer columns.
• Reduced solvent consumption in closed-loop recycling.
• High sample purity for preparative or QA/QC tasks.
• Flexible integration into existing HPLC systems with minimal additional hardware.

Future Trends and Possibilities

Advances may include integration with multidimensional LC workflows, real-time peak detection for automated cycle control, miniaturized valve manifolds to further reduce dead volume, and broader application to peptide, lipid and chiral separations.

Conclusion

Recycling chromatography effectively enhances resolution for difficult separations. The alternate pumping method outperforms closed‐loop pump recycling by achieving higher resolution in fewer cycles and avoiding peak mixing and pump contamination, at the cost of greater instrument complexity.

References

• Menke J, Monks K. Recycle your peaks – A comparison of two recycling methods. KNAUER Wissenschaftliche Geräte GmbH; VTN0007.
• KNAUER application note VFD0170. Scale-Up of an analytical HPLC method for steviol glycosides to a preparative approach.
• KNAUER application note VFD0171. Evaluating preparative online SPE for the purification of stevia leaf extracts.