Sample Purification Triggered with the Agilent 1260 Infinity Evaporative Light Scattering Detector

Importance of the Topic

This overview addresses the challenge of purifying and collecting non-UV-absorbing and low-UV-absorbing compounds in preparative chromatography. Conventional UV detectors fail to trigger fraction collection for these analytes. By integrating an evaporative light scattering detector (ELSD) with a flow splitter, preparative workflows gain universal detection capability, enhancing recovery and purity of target molecules that lack strong chromophores.

Objectives and Study Overview

The study aims to demonstrate an automated fraction-collection strategy using the Agilent 1260 Infinity ELSD coupled via a flow splitter to preparative-scale chromatography. A drug-like mixture containing both UV-active and non-UV chromophore compounds was purified. The key goals were to achieve ≥99 % purity and ≥90 % recovery while validating method reproducibility over multiple injections.

Methodology

A flow splitter downstream of the preparative column diverts a controlled portion of eluent to the ELSD while passing the remainder to a fraction collector. Detection thresholds and trigger slopes were set for both UV and ELSD signals to initiate fraction collection. A binary gradient (2 %–98 % acetonitrile in water with 0.1 % formic acid) at 25 mL/min was applied to a 21.2 × 100 mm C18 column. Six replicate injections were performed for precision assessment, and larger volume injections were used to evaluate recovery and purity.

Used Instrumentation

• Agilent 1260 Infinity Preparative-scale Purification System with Gradient Extension
• Agilent 1260 Infinity Evaporative Light Scattering Detector (ELSD)
• Agilent 1260 Infinity Multiple Wavelength Detector (MWD/DAD)
• Agilent 1260 Infinity Fraction Collector
• Agilent ZORBAX SB-C18, 21.2 × 100 mm, 5 µm prep cartridge
• Agilent OpenLAB CDS ChemStation software

Key Results and Discussion

Retention time RSD across six injections was 0.02 %, and peak area RSD averaged 2.74 %, demonstrating high reproducibility of the splitter–ELSD configuration. Fraction collection triggered by ELSD accurately isolated the non-UV-active compound, yielding 91 % recovery and 99 % purity. Overlay plots of UV and ELSD signals confirmed selective triggering by the ELSD detector for non-chromophoric analytes while UV triggers collected the UV-active peaks.

Benefits and Practical Applications

• Reliable detection and collection of compounds lacking UV chromophores
• High reproducibility and precision in preparative fractionation
• Automated workflow reducing manual intervention and impurity co-collection
• Applicability to natural products, peptides, and small molecule libraries

Future Trends and Opportunities

Advancements may include integration of ELSD triggers with mass spectrometric feedback, AI-driven peak recognition for dynamic thresholding, and modular splitter designs for higher flow rates. Scaling workflows from analytical to preparative scales can be further automated using predictive software.

Conclusion

The combination of Agilent 1260 Infinity ELSD and a tailored flow splitter offers a robust solution for preparative purification of low-UV analytes. This approach delivers high purity and recovery with excellent precision, expanding the scope of preparative chromatography workflows.

References

1. B. Schuhn, Performance Characteristics of the Agilent 1290 Infinity Evaporative Light Scattering Detector, Agilent Technical Overview, publication number 5991-2097EN (2013).
2. B. Schuhn, Optimizing the Performance of the Agilent 1290 Infinity Evaporative Light Scattering Detector, Agilent Technical Overview, publication number 5991-2176EN (2013).