Can I perform batch experiments with a cuvette instead of my flow cell in DAWN?

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Introduction

Using a MALS instrument to measure unfractionated samples, often referred to as batch mode analysis, can be an invaluable characterization method. This type of measurement provides the weight-average molar mass, the z-average RMS radius, and the second virial coefficient (A2) of a sample and its unfractionated components. You may not be able to distinguish between individual constituents in these measurements; however, batch mode can be a quick, convenient complement to SEC-MALS or FFF-MALS.



When to use batch measurements

Batch measurements are used in a variety of instances, including when:

  • There is no SEC method available (e.g., the column is not suitable for your sample or is not compatible with your mobile phase).
  • Fractionation may affect the sample (e.g., your column may remove, shear or degrade sample components.)
  • You want to monitor a reaction or perform a time-dependent analysis.
  • You want to measure the second virial coefficient (A2) or determine equilibrium constants and kinetics. (These experiments can be automated in CG-MALS with the Calypso® when using the flow cell.)
  • You need to validate a SEC-MALS measurement to ensure that aggregate content has not been reduced by dilution or column shear, by comparing the weight-average molar mass determined by both techniques.


The microCurvette accessory

Batch measurements can be performed by injecting the sample into a DAWN® or miniDAWN® flow cell. If you own a DAWN, batch measurements may also be carried out with a microCuvette™ (p/n WMCW). The microCuvette can be purchased as an accessory kit that includes everything you need to run batch experiments. It comes with a quartz cuvette and a cuvette holder, which is easily installed in place of the flow cell manifold, as shown below.

The microCuvette uses detectors 7, 9, 11, and 13 in the DAWN and detectors 3-6 in the DAWN 8 to make angular measurements of light scattering. (ASTRA automatically enables the correct detectors when you select the microCuvette as the sample cell). This allows for RMS radius determination from 10 to 50 nm (up to 150 nm with shape-specific models) as well as molar mass determination between 200 Da to 10 MDa. It is also compatible with the WyattQELS™ module when the DLS fiber is installed at the 90° detector angle, enabling simultaneous acquisition of MALS and DLS data, and expanding the size range to Rh = 0.5 nm to 1 µm.

The microCuvette has a number of advantages over the flow cell and can be the perfect tool when:

  • Your sample is precious and needs to be recovered. The microCuvette for DAWN uses as little as 30 µL of volume, while flow cell batch injections take a minimum of 1 mL per sample to fill the MALS flow cell and all the connecting tubing.
  • Your sample may be too viscous for flow through the inlet and outlet tubing connected to the flow cell manifold.
  • You intend to study time-dependent characteristics of samples—such as stability or self-assembly kinetics.
  • Your sample may foul the flow cell glass and be challenging to clean (the microCuvette is a quartz cuvette that can be easily remove and cleaned compared to the flow cell).
  • You are using CG-MALS to measure highly concentrated or viscous proteins not amenable to Calypso operation. The microcuvette can be used with manual dilutions and the CALYPSO™ software for data acquisition and interaction analysis.

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Conclusion

The Wyatt Support Center offers a number of technical notes and tutorials for running batch experiments with ASTRA. The microCuvette accessory kit can be purchased from the Wyatt Technology Store.

Do you have a question? Contact our experts here in Customer Support. We’re happy to help! Call +1 (805) 681-9009 option 4 or email support@wyatt.com.