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

“MALLs” detectorwith 120mW laser operating at 658 nm

Molecular Weight: Light scattering

photodiode detectorsphotodiode detectors

incident beamincident beam

transmittedtransmittedbeam

Molecular Weight: Zimm plot

106 (K /R )106.(Kc/Rθ)

1/MwRg from slope

sin2(θ/2) + kc (k=100)

Molecular Weight: Light scattering

photodiode detectorsphotodiode detectors

incident beamincident beam

transmittedtransmittedbeam

Molecular Weight: Light scattering

sity

Inte

ns

Molecular Weight: SEC–MALLS

MALLs d t tdetector

ConcentrationSEC - columns Concentration detector

SEC - columns

Molecular Weight: SEC–MALLS

ColonicColonic mucin

Fogg FJJ et al, Biochemical Journal.1996

Molecular Weight: SEC–MALLS

Vi itViscosity detector

P I b l Vi tPressure Imbalance Viscometer:

ΔP Pi

η = 1 + {(4ΔP) (Pi -2ΔP)}ηr 1 + {(4ΔP).(Pi 2ΔP)}

P I b l Vi tPressure Imbalance Viscometer:

ηr = 1 + {(4ΔP).(Pi - 2ΔP)}

Wyatt Technology Viscostar System

Viscotek System

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)

ViscotekIgG3wt

[η] = 9.9ml/g

IgG3m15[η] = 5.7ml/g

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 ,

Follow up bibliography:

1. On-line tutorials from: Wyatt Technology and Viscotekcorporation (see their web sites)

2. Harding, S.E., Sattelle, D.B. & Bloomfield, VA. Eds(1992) Laser Light Scattering in Biochemistry Royal Soc. Chem. Cambridge – Particularly chapters by W tt d R lliWyatt and Rollings