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Dimer134 kDa

Trimer205 kDa

Monomer66 kDa

small molecules radius < 15 nm Pθ = 1 for all θ

large molecules radius > 15 nm Pθ = 1 for θ = 0°

Intramolecular interference produces a disymmetry in the scattered light.

Size of molecule/particle must be significant compared to wavelength of light

with

Angular Dependence

Radius [nm] 3 15 50 150

Low Angle Light Scattering - LALS

Proteins Mol. Weight (D) Rh (nm)

Ribonuclease 13,700 1.93 b-Lactoglobulin 35,000 2.67 Serum albumin 65,000 3.38 Hemoglobin 68,000 3.40 IgG 150,000 5,02 Catalase 250,000 5.38 Thyroglobulin 660,000 7.50

RALS ---------------------------------------------------------------------

LALS IgM 900,000 11.7 DNA 3 Mio 70

Polysaccharides/PEG

MW Rh (nm) 12,000 2.62 50,000 5.62 80,000 7.27 150,000 9.81 RALS -----------------------------------

LALS 270,000 12.74 410,000 15.07 670,000 18.45

Relationship between Size and Molecular Weight

0.0

119 .5

12.0

24.0

36.0

48.0

60.0

72.0

84.0

96.0

108.0

Rig

ht A

ngle

Lig

ht S

catte

ring

Res

pons

e (m

V)

0.0

56.7

6.0

12.0

18.0

24.0

30.0

36.0

42.0

48.0

Low

Ang

le L

ight

Sca

tterin

g R

espo

nse

(mV

)

10.50 16.60Retention Volume (mL)

11 .5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5

RALS LALS

Intrinsic viscosity is the concentration normalized viscosity of the sample in solution at infinite dilution.

ηsp is called the specific viscosity of the solution whose concentration is C. ηo is the Solvent Viscosity. η is the Solution Viscosity.

In GPC, concentrations of solutions are sufficiently dilute that the extrapolation to zero concentration is negligible.

Intrinsic Viscosity is inverse proportional to the density

Intrinsic Viscosity x Molecular weight is proportional to the hydrodynamic radius of a sample

Intrinsic Viscosity: from Viscosity Detector Molecular Weight: from Light Scattering Detector

IP

+ -

DP-+GPC IN OUT

Solvent

Wheatstone bridge concept M. Haney, 1983

Sample

BSA

Hydrodynamic Radius

6,2 nm 5,1 nm

3,8 nm

• • • • 

×× ×

× ×

× ×

× × ×

Monomer Dimer

Trimer

0,0

1472,0

82,0 164,0 246,0 328,0 410,0 492,0 574,0 656,0 738,0 820,0 902,0 984,0

1066,0 1148,0 1230,0 1312,0 1394,0

Ref

ract

ive

Inde

x R

espo

nse

(mV

)

4,000

7,000

4,200 4,400 4,600 4,800 5,000 5,200 5,400 5,600 5,800 6,000 6,200 6,400 6,600 6,800

Log

Mol

ecul

ar W

eigh

t

0,0

239,0

14,0 28,0 42,0 56,0 70,0 84,0 98,0 112,0 126,0 140,0 154,0 168,0 182,0 196,0 210,0 224,0

Rig

ht A

ngle

Lig

ht S

catte

ring

Res

pons

e (m

V)

0,0

36,0

2,0 4,0 6,0 8,0

10,0 12,0 14,0 16,0 18,0 20,0 22,0 24,0 26,0 28,0 30,0 32,0 34,0

Visc

omet

er D

P R

espo

nse

(mV

)

5,30 10,00 Retention Volume (mL)

5,6 5,8 6,0 6,2 6,4 6,6 6,8 7,0 7,2 7,4 7,6 7,8 8,0 8,2 8,4 8,6 8,8 9,0 9,2 9,4 9,6

Monomer

Dimer Trimer

High MW Aggregates (Mw > 1 Mio D)

Mw (D) IVw (dl/g) Rh (nm) Weight Fraction (%)

Monomer 66.430 0,056 3,88 87

Dimer 133.000 0,071 5,32 11

Trimer 201.000 0,095 6,69 1,5

Light Scattering Viscometer Refractive Index

BSA

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