Mass Spectrometry & Spectroscopy
Expanding the Boundaries of Light Scattering for Macromolecules Sébastien Rouzeau, Tosoh Bioscience GmbH, Griesheim, Germany. Contact:
sebastien.rouzeau@
tosoh.com
In the past four decades, light scattering detection (LS) has become a widely used technique to obtain the supposed “absolute” or true molecular weight of macromolecules such as synthetic polymers, biopolymers, proteins, and antibodies. When coupled with size exclusion chromatography (SEC), molecular weight and size, as well as aggregation or branching can be investigated to provide analytical scientists with a powerful characterisation tool for these large molecules.
Whether new biopharmaceutical products or polymers with tailored properties, the macromolecules’ complexity increases, requiring even more extensive and in-depth characterisation for a full understanding of the material. With limited amounts of costly samples available for analysis, analytical scientists are facing the challenge of fi nding new technologies that help them improve and optimise their analytical characterisation.
The latest light scattering technology introduced by Tosoh Bioscience features an entirely new detector design that addresses the limitations and shortcomings of the current, state- of-the-art LS instruments.
Principles and theory of light scattering
When a beam of light illuminates molecules in a solvent, oscillating dipoles are generated within the molecules, re-emitting a small amount of light in all directions. This physical phenomenon shows two main properties. Firstly, the intensity of the scattered light is directly related to the molecular weight of the molecules: higher molecular weight molecules scatter more light. Secondly, the scattered light’s intensity is not identical in all directions, and this dissymmetric scattering pattern is related to the size and shape of the molecules.
Consequently, molecular weight information is obtained from the intensity of the scattered light, while molecular size is obtained by examining intensity changes with the angle of observation.
Rayleigh defi ned the theory and equations of light scattering by sub-micron particles. He established the relationship of the intensity of scattered light to the molecular weight and size of the molecules, the concentration of the solution, and the measurement angle [1].
Rayleigh determined the molecular weight of a molecule can be obtained directly from the intensity of the scattered light in the same direction as the incident beam (0° angle), regardless of the molecule’s size and shape.
However, due to the incident beam, measuring the amount of scattered light at 0° angle is technically impossible. Light scattering instruments nevertheless have to work around this technical challenge to provide the molecular weight information.
Traditional light scattering instruments and their limitations
The fi rst LS instruments, in the 1970’s, used a low angle light scattering approach (LALS) in which the angle of measurement is close enough to 0° to assume safely that the measurement at such a low angle is the same as the theoretical 0° measurement. This is still considered the most accurate molecular weight measurement method, as it requires no additional assumptions or extrapolations. However, those early LALS instruments were not easy to use and a low angle measurement alone does not provide size information since it does not measure the angular dependence of the scattered light intensity.
To achieve both easier molecular weight and size measurements simultaneously, multi- angle light scattering (MALS) detectors were developed to collect light at multiple angles. These measurements are extrapolated back to 0° for molecular weight (MW) determination, while the scattering pattern is mapped to obtain size information described as the radius of gyration (Rg).
MALS instruments typically consist of multiple photodiodes (detectors) arranged in the same plane, around a circular or cylindrical fl ow cell. There are several technical limitations to this design since the geometry of this design offers limited space available for the physical
location of the detectors around the cell. As a result, the lowest and highest measurable angles are some distance from 0° and 180°. Furthermore, due to the type of glass employed for the optical fl ow cell, refl ection of the scattered light inevitably occurs on the cell walls, thus creating stray light that interferes with the measurement. The resulting generated noise affects the lower and higher angles to a greater extent compared to the 90° angle. Consequently, it is common for those to be excluded from use in data processing.
What do these technical limitations mean for SEC-MALS users?
• MFirstly, the noise from stray light limits the overall sensitivity of traditional MALS detectors. For example, this affects their capability to detect low amounts of protein aggregates, increasing the amount of sample required for each analysis.
• Secondly, the lowest angle is critical to obtain an accurate molecular weight since the Rayleigh equation says that MW is proportional to the scattered light intensity only at 0°. The extrapolation may cause signifi cant errors in molecular weight determination when low angles are unusable.
Figure 1: LenS3
fl ow channel.
• Thirdly, having the lowest and highest possible angles is necessary for the size determination of smaller molecules by detecting the small differences in scattered intensity. Practically, traditional MALS detectors cannot provide angular dissymmetry for molecules smaller than 10-12nm (Rg).
Innovative LS instrument design
To address the highlighted limitations, a completely new approach to MALS instrument design has been developed. The LenS3
MALS detector (Tosoh Bioscience, King of Prussia,
PA, USA) combines a novel fl ow channel concept with improved optics to provide more signal intensity while reducing the core noise at each angle of measurement.
New fl ow channel confi guration
The traditional fl ow cell is replaced with an elongated fl ow path with a dual-cone shape, as shown in Figure 1.
This cell block consists of a black, non-refractive, inert polymeric material (PEEK) assembled with two optical lenses that seal the fl ow chamber and let the incident beam go through the chamber. The inlet fl ow splits in half at the centre of the fl ow path and exits from two outlets. The laser beam illuminates the sample in the entire fl ow path, maximising the scattering volume, hence the number of molecules that interact with the incident light and
INTERNATIONAL LABMATE - FEBRUARY 2021
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64
