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1 Molecular weight distribution analysis –1. Molecular weight distribution analysis SEC MALLs and AUC
2. Conformation and flexibility – Viscometry, AUC Light scatteringAUC, Light scattering
Lecture 2:
Light Scattering & SEC MALLs
Part 1: Light scattering basic theory (overhead transparencies)
Part 2: Instrumentation, SEC-MALLs and “Triple” Detection
Molecular Weight: Light scattering
photodiode detectorsphotodiode detectors
incident beamincident beam
transmittedtransmittedbeam
Molecular Weight: Light scattering
photodiode detectorsphotodiode detectors
incident beamincident beam
transmittedtransmittedbeam
Molecular Weight: SEC–MALLS
MALLs d t tdetector
ConcentrationSEC - columns Concentration detector
SEC - columns
1.0 LS 90o Mw = 396000 g/mol (0.1 %)
[η] = 107.6 ml/g (1%)Mw = 120000 g/mol (0.1 %)[η] = 46.2 ml/g (0.3 %)
Mw = 620 g/mol (7 %)[η] = 2.8 ml/g (0.4 %)
Viscostar - pullulans
0.8
DRI DPV
nse
Injected mass = 18 μg Injected mass = 92 μg Injected mass = 423 μg
0 6r Res
po
0.6
Det
ecto
Mw = 13150 g/mol (0.6 %)[η] 12 1 ml/g (0 7 %)
0.4
mal
ised
[η] = 12.1 ml/g (0.7 %)Injected mass = 94 μg
0.2Nor
m
0.020 25 30 35 40 45
Elution Time (min)
….Viscostar - Fibrinogen
1.0 LS 90o
DRIChicken Fibrinogen
0.8
onse
DRI DPV
Chicken FibrinogenMw = 335000 g/mol (0.1 %)[η] = 27.7 ml/g (0.3 %)Injected mass = 383 μg
0.6
ecto
r Res
po
0.4
mal
ised
Det
e
0.2
Nor
m
10 15 20 250.0
10 15 20 25
Elution Time (min)
A bead-shell model of a mutant IgG3
A bead –shell model of the of IgG3 wild type Lu et al, Biophysical Journal, 2006, p y ,
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