15
Improved optics The LenS3
MALS detector also features
improved optics to provide even more intensity of scattered light and cleaner signals (Figure 2).
Using a green laser (λ0 =505nm), the Figure 2: LenS3 MALS enhanced optics, showing the low angle (LALS), right angle (RALS) and high angle (HALS) light scattering detectors.
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?
• Firstly, 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.
• 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 ultimately increasing the scattering intensities. The chamber’s non-refractive material prevents the scattered light hitting the wall from bouncing back, leading to stray light and subsequent noise. The conical shape defi nes the forward (10°) and backward (170°) angles of collection of scattered light, while the perpendicular (90°) measurement is made at the centre of the channel through a separate observation window equipped with a spherical lens.
=660nm) used in most LS instruments, as scattering is proportional to 1/λ0
scattered intensity increases by a factor of three, compared to the typical red laser (λ0
4.
The optical bench includes mirrors in both the backscattering and forward scattering positions, with a hole where the incident beam can travel through the mirrors so that the incident beam is effectively eliminated. Only the annulus of light at the desired angles 10° and 170° is collected.
Overall, the gain in performance and sensitivity of the LenS3
MALS detectors, as
compared to traditional MALS instruments, comes from the combination of the following elements:
• The wider angles of measurement:
o True, usable ultra-low angle (LALS at 10°) for accurate and direct MW determination without extrapolation
o Ultra-high angle (HALS at 170°) used in conjunction with the LALS and the 90° angle (RALS) to detect the smallest
difference in scattered intensity for Rg measurements of smaller molecules.
• The novel fl ow channel:
o Elongated conical shape to maximise scattering volume and thus signal intensity.
o Black, inert polymeric material eliminates stray light to reduce noise.
• The advanced optics:
o A green laser to increase the intensity of scattering by a factor of three.
Table 1: Experimental chromatographic conditions for the applications Monoclonal Antibody Oligonucleotide Columns
Mobile phase
TSKgel UP-SW3000 (2 µm, 4.6 mm x 30 cm)
100 mmol/L NaH2PO4
pH 6.8 + 100 mmol/L Na2
SO4 ,
TSKgel UP-SW2000 (2 µm 4.6 mm x 30 cm)
0.5 mol/L NaCl, 0.1 mol/L EDTA, pH 7.5 0.1 mol/L Na2 0.03% NaN3
SO4 , in 0.1 mol/L Flow Rate 0.35 mL /min
Temperature 25°C Detection Sample
MALS
phosphate buffer 0.3mL/min 30°C
UV@260 nm; MALS Herceptin biosimilar 20 bases custom
oligonucleotide with MW= 6141 Da (1 mg/mL)
1 mL/min 40°C
MALS Polystyrene standards Polystyrene
TSKgel GMHHR-N (5 µm, 7.8 mm x 30 cm)
Toluene
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