CHROMATOGRAPHY
MALS DETECTION T
THE MERITS OF
Patrick Endres discusses how light scattering technology with high sensitivity facilitates the analysis of synthetic and biopolymers
he multi-angle light scattering (MALS) technique has become the standard to
determine molecular weight (MW) and size – measured in radius of gyration (Rg) of proteins, biopolymers, synthetic polymers and polysaccharides. In contrast to some conventional light scattering detectors already available on the market, a new MALS detector – Tosoh LenS3 – is capable of directly determining absolute molecular weight and Rg without extrapolation. Tis is achieved by combining an extreme low angle (LALS – 10°) and an extreme high angle (HALS – 170°) with a right
Fig.1. LenS3 MALS detector design
100 120 140 160
20 40 60 80
0 20.5 21 21.5 22 22.5 23 23.5 Volume (mL) 24 24.5 25 25.5 26 7 8 1
5,000 10,000
6 5 4 3 2 1,000 500
Peak # 1
Theoretical MW 266
2370 3474 4 5 6 7 8
578 682 786 890 994
Mp from LALS 255 377 503 618 725 849 985
1105
Fig. 2. Accurate determination of A-500 polystyrene standard combining TSKgel SEC columns and LenS3 detector
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angle (RALS – 90°) to form a three-angle MALS detector (Fig.1.). As opposed to a conventional flow cell, the new extended “flow chamber” allows maximum interaction of the incident beam with the molecules of interest, resulting in significantly higher scattering intensity. Coupling this smart design feature with a lower laser wavelength, 514nm, as opposed to a typical 660nm, LenS3 offers excellent detection sensitivity. As well as this, in contrast to a typical optical flow cell composed of a glass-like material, the new flow chamber is made with PEEK, a non-refractive material, substantially reducing the destructive stray-light effect in light scattering detection. Hence, the quality and stability of signals at extreme angles (LALS and HALS) are significantly improved. Measurement at such angles on the detector allows MW determination as low as 200 Da (Fig. 2).
ADDRESSING OTHER LIMITATIONS
In addition, the LenS3 addresses the two major limitations of the Zimm method by a new angular dependence calculation using the “angular dissymmetry plot”. Tanks to this new underlying theory, the strategic positions of the angles, and the superior sensitivity, LenS3 can now determine Rg values at much lower size range compared to some existing MALS instruments without requiring solute concentration or refractive index increment (dn/dc) values. Even with many more angles of observation, conventional detectors can only determine Rg down to 10nm. With the LenS3 detector, determination of Rg as low as 2nm is now possible. In summary, the LenS3 MALS detector is a novel innovation in light scattering technology. Te new detector integrates the best of both MALS and LALS concepts to create a new paradigm in light scattering detection
Patrick Endres is a senior lab specialist with Tosoh Bioscience.
www.tosohbioscience.com
Molecular weight (Da)
RI (mV)
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