Post on 09-Jan-2019
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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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