Analytical HPLC with Field Flow Fractionation and different detectors

Austrian Centre of Industrial Biotechnology (acib)

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This combination of devices is part of the infrastructure for the process development of production processes of bionanoparticles supported by the FFG as part of the R&D infrastructure funding.
The device combination for analysis of bionanoparticles consists of a HPLC system (Infinity II, Agilent) for field flow fractionation (Eclipse® AF4 flow field-flow fractionation, Wyatt) coupled to

• a static multi-angle light scattering (MALS)-detector (DAWN 8 Multi-angle light scattering System, Wyatt)
• a dynamic light scattering detector/static light scattering detector (DLS/SLS detector, DynaPro NanoStar, Wyatt)
• a refractive index (RI) detector (Optilab Differential Refractometer, Wyatt).

The system is used to determine the concentration, composition, structure and size of bionanoparticles. Field flow fractionation (FFF) allows separation of different particle types without chromatography column, in an analytical and semi-preparative range. With the help of FFF, an improved resolution and separation of bionanoparticles, macromolecules or aggregates can be achieved compared to chromatography. The HPLC detector package is suitable for the analysis of bionanoparticles (e.g. viruses, VLPs, exosomes, microvesicles, etc.) whose particle radius is between 10 nm and 500 nm.
The MALS detector is used to determine the gyration radius of bionanoparticles. The particle concentration can then be calculated using the particle radius.
The DLS on-line detector is a flexible system that can determine the hydrodynamic particle diameter. By combining MALS and DLS signals, a distinction can be made between different particle compositions of a similar size. Both light scattering signals are measured in the same sample volume to ensure the same sample composition.
The RI detector can be used to calculate the molecular weight of a particle, which is an important parameter for particle identification and structural reconnaissance. A refractive index detector provides the concentration of particles at known dn/dc regardless of their size. For UV detectors, the extinction coefficient changes with the size of the particle. In the case of viruses and VLPs, it is therefore essential to work with an RI detector for concentration and molecular mass determination.


Dr. Martin Trinker

Research Services

This device is not part of a service facility but is open for scientific collaboration.
acib offers extensive and reliable partnerships in national and international research projects, as well as the implementation of contract research.

Methoden & Expertise zur Forschungsinfrastruktur

The HPLC-FFF-MALS-DLS system is used for separation of viruses and virus-like particles (VLPs) and other process related impurities. By using the combination of these detectors, the particle diameters and concentrations can be determined. As the detectors are directly connected it is ensured that the same sample is measured in each detector cell.
For fast size distribution and particle concentration calculation the DLS detector is used in batch mode. Only 4 µL of sample is needed to fill the quartz cuvette.

Zuordnung zur Core Facility

Austrian Centre of Industrial Biotechnology (acib)

Dr. Martin Trinker
Director Business Development & Fundraising
+43 316 873 9316
The device can be used in research collaborations. Costs depend on duration of service.
The device is used in a cooperation project with a company partner in the field of vaccine development.
Bousse, T., Shore, D. A., Goldsmith, C. S., Hossain, M. J., Jang, Y., Davis, C. T., ... & Stevens, J. (2013). Quantitation of influenza virus using field flow fractionation and multi-angle light scattering for quantifying influenza A particles. Journal of virological methods, 193(2), 589-596.

Chuan, Y. P., Fan, Y. Y., Lua, L., & Middelberg, A. P. (2008). Quantitative analysis of virus‐like particle size and distribution by field‐flow fractionation. Biotechnology and bioengineering, 99(6), 1425-1433.

Klein, M., Menta, M., Dacoba, T. G., Crecente-Campo, J., Alonso, M. J., Dupin, D., ... & Séby, F. (2020). Advanced nanomedicine characterization by DLS and AF4-UV-MALS: Application to a HIV nanovaccine. Journal of Pharmaceutical and Biomedical Analysis, 179, 113017.