Online Hydrodynamic Size Measurement - Integrating Dynamic Light Scattering with Chromatography

By AZoNano.com Staff Writers

Topics Covered

Introduction
Limitations of SEC Systems
Methodology
Results and Discussions
Conclusion
About Avid Nano

Introduction

Dynamic light scattering (DLS) technique offers excellent speed and sensitivity and proves suitable for online applications such as chromatography of proteins. For molecular characterization and purification, Size Exclusion Chromatography (SEC) separation systems are generally employed.

Figure 1. Typical chromatography system

Avid Nano has developed the W130i DLS system, which can be equipped with a low volume flow cell and joined to any chromatography system to offer rapid online measurement of scattering intensity and hydrodynamic radius (Rh). It can also be used for detecting aggregates.

Figure 2. Compact size of W130i system makes hardware placement within cold cabinet possible

Limitations of SEC Systems

The SEC system utilizes UV detection to determine the elution times of each size component, and thus provides an indication of the aggregate content and size distribution. Although the addition of (static) light scattering imparts molecular weight measurement capability, this requires calibration with standards.

However, Dynamic Light Scattering (DLS) does not require calibration to calculate Rh. Besides measuring intensity and Rh, the molecular weights of globular species can be consistently estimated by utilizing the proprietary algorithm built within the i-Size 2.0 software from Avid Nano.

Methodology

The W130i DLS system flow cell tubing is joined inline after the UV detector or separation column before going to waste or fraction collection. Once the sample is injected or as soon as the UV peak starts to appear, DLS acquisitions are commenced until all the peaks have passed through.

Subsequently, the UV and DLS data obtained is directly exported for additional analysis and correction for inter-detector volume, if required.

Figure 3. SEC – DLS of protein complex

Results and Discussions

When Rh and scattering intensity are directly measured every few seconds, it offers a plot which can be easily superimposed with the UV chromatogram. The Rh measurements can be averaged across each peak.

Also, molecular weight estimate can be ascertained by utilizing the Concentration Calculator utility integrated within the i-Size 2.0 software.

Conclusion

Avid Nano’s W130i DLS system can be integrated with a low volume flow cell and joined to any chromatography system to offer direct online measurement of Rh and scattering intensity.

About Avid Nano

Avid Nano produce the most exciting and innovative new dynamic light scattering products. Avid Nano pursue the highest levels of performance, know-how, technology, convenience and reliability for all our products, used in laboratory applications requiring fast, accurate DLS measurements in the nano-metre size range.

The W130i dynamic light scattering system is a breakthrough, high sensitivity instrument for size and molecular weight measurement of proteins, bio- molecules and sub-micron particles. The W130i combines ultimate measurement performance with an economical benchtop footprint, rugged durability and supreme user convenience. It's extraordinarily compact dimensions are the result of miniaturized 'diamond geometry' S.A.B.Re fibre optic technology.

This information has been sourced, reviewed and adapted from materials provided by Avid Nano.

For more information on this source, please visit Avid Nano.

Date Added: Aug 17, 2013 | Updated: Aug 20, 2013
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