Combining chromatography with Dynamic Light Scattering (DLS) - Case Study

The ‘flow-mode’ option for Malvern’s market-leading Zetasizer Nano particle characterization system, enables its use as an in-line chromatography detector for protein applications. A significant new development, ‘flow-mode’ permits the coupling of dynamic light scattering with size exclusion chromatography, conferring the ability to perform Absolute Size Exclusion Chromatography (ASEC). This allows protein size to be measured directly rather than relying on column calibration and means that molecular weight standards are no longer required to identify oligomeric species or determine protein purity.

In summary:

Separation using size exclusion chromatography is combined with the simplicity and accuracy of dynamic light scattering.

To use the Zetasizer Nano as an absolute light scattering detector it simply needs to be connected after the last detector in a size exclusion chromatography (SEC) system. The Zetasizer Nano can be used with any SEC system; no column calibration is required and the Zetasizer Nano’s software makes the entire set-up and measurement process simple and straightforward.





For the majority of protein separations in aqueous media, no parameter input is required. Size is plotted in real time as material is eluted from the column. Averages across each peak are calculated automatically at the end of each measurement. Here, as an example of an Antibody sample, the software detects the fragment as well as the Antibody peak without user intervention:





The sample consisted of rat antibody containing fragments, prepared at 1 mg/mL, 500μL of sample was injected, the buffer was PBS, flow rate 0.5 mL/min. The result can subsequently be viewed in a special chromatography report, summarising the peak analysis including the start and end volumes of the individual peak fractions.

	Presentations:
	On demand presentation on Absolute Size Exclusion Chromatography Chromatography and dynamic light scattering are common techniques in many biochemistry laboratories. View a short presentation about how the two may now be combined to measure size and intensity in flow mode.
	Application note - Absolute Size Exclusion Chromatography: BSA containing monomer, dimer, trimer Size exclusion chromatography (SEC) and dynamic light scattering (DLS) are two technologies common to protein laboratories.  SEC is routinely used for purification, identification, and quantification of protein mixtures, while batch dynamic light scattering is routinely used for pre-column size & polydispersity measurements, along with aggregate quantification.  The coupling of dynamic light scattering & SEC technologies has been difficult in the past, due in large part to the fact that eluted protein sample concentrations tend to be well below the detection limit for most dynamic light scattering instruments.  In this application note however, dynamic light scattering has proven capable of handling this low protein concentration challenge due to the sensitivity of the Zetasizer Nano S. The monomer, dimer, and trimer fractions of bovine serum albumin (BSA) were identified.
	Application note - Absolute Size Exclusion Chromatography: Carbonic Anhydrase The single peak encompassed an average size of 4.81nm diameter, corresponding to an estimated molecular weight of 26.2 kDa for a globular protein. The peak start and end volume are automatically identified as 18.0 and 18.6 mL. The molecular weight for this protein is 29 kDa, the result obtained here is in reasonable agreement with the expectation. The simple size measurement confirmed that the main elution peak was the monomer of Carbonic Anhydrase.
	Application note - Absolute Size Exclusion Chromatography: Antibody IgG4 A Superdex 200 column (GE Healthcare, Uppsala, Sweden) was used for separation of an Antibody (IgG4) of expected molecular weight near 150 kDa. The protein was prepared in 0.1 M ammonium bicarbonate buffer (pH not adjusted) at a concentration of 7 mg/mL. 100µL were injected, i.e. the column was loaded with 0.7 mg of protein. The system was run at a flow rate of 0.5 mL/min, and the dynamic light scattering accumulated continuously and analysed every 3 seconds. The software recorded all correlation functions and intensity values.