The physical characterisation of polysaccharides in solution

The physical characterisation of polysaccharides in solution

Stephen HardingUniversity of Nottingham

Viscometry SEC-MALLsAnalytical Ultracentrifugation

Imaging

The physical characterisation of polysaccharides in solution

Stephen HardingUniversity of Nottingham

Physical characterisation

1. Viscosity, stability2. Heterogeneity, Molecular weight &

distribution, stability3. Conformation in solution4. Interactions



distribution3. Conformation in solution4. Interactions

1: Viscometry. 2: SEC-MALLs & analytical ultracentrifugation. 3: Viscometry, SEC-MALLs & analytical ultracentrifugation. 4. Analytical ulttracentrifugation & atomic force microscopy

1. Viscosity from precision viscometry

1. Viscosity by Precision viscometry

[η]Intrinsic viscosity, ml/g

Types of Viscometer:

Ostwald Viscometer

1. “U-tube” (Ostwald or Ubbelohde)


Ostwald Viscometer



Extended Ostwald Viscometer


2. “Cone & Plate” (Couette)


Couette-type Viscometer


2. “Cone & Plate” (Couette)

3. Pressure imbalance on-line viscometer


Water bath +0.01oC

Density meter

Coolant system

Solution

Auto-timer

For normal (Newtonian) flow behaviour:

τ = (F/A) = η . (dv/dy)

Definition of viscosity:

η = τ/(dv/dy) units: (dyn/cm2)/sec-1

= dyn.sec.cm-2. . = POISE (P)

At 20.0oC, η(water) ~ 0.01P

shear stress

shear rateviscosity

Viscosity of biomolecular solutions:

A dissolved macromolecule will INCREASE the viscosity of a solution because it disrupts the streamlines of the flow:

We define the relative viscosity ηr as the ratio of the viscosity of the solution containing the macromolecule, η, to that of the pure solvent in the absence of macromolecule, ηo:

ηr = η/ηo no units

For a U-tube viscometer, ηr = (t/to). (ρ/ρo)

Reduced viscosity

The relative viscosity depends (at a given temp.) on the concentration of macromolecule, the shape of the macromolecule & the volume it occupies.

If we are going to use viscosity to infer on the shape and volume of the macromolecule we need to eliminate the concentration contribution.

The first step is to define the reduced viscosity

ηred = (ηr – 1)/c

If c is in g/ml, units of ηred are ml/g

The Intrinsic Viscosity [η]

The next step is to eliminate non-ideality effects deriving from exclusion volume, backflow and charge effects. We measure ηred at a series of concentrations and extrapolate to zero concentration:

[η] = Limc⃗0 (ηred)

units [η] = ml/g

sometimes researchers use dl/gso 200 ml/g = 2 dl/g

c (mg/ml)

Kraemer

Huggins

[η]ηred or ηinh(ml/g)

irradiated (10kGy) guar

Viscosity probe: chitosan stability

Fee et al, 2006

2. Heterogeneity and molecular weight: SEC-MALLs

2. Heterogeneity and Molecular Weight: SEC-MALLs

Molecular Weight: Light scattering

transmittedbeam

photodiode detectors

incident beam

Molecular Weight: Zimm plot

1/Mw

sin2(θ/2) + kc (k=100)

106.(Kc/Rθ)

Rg from slope

Molecular Weight: SEC–MALLS

Molecular Weight: SEC–MALLS

Colonic mucin

Can also couple a “pressure imbalance”type of viscometer on-line…

c (mg/ml)

Kraemer

Huggins



c (mg/ml)

Kraemer

Huggins

Solomon-Ciutaηred or ηinh(ml/g)


Analytical Ultracentrifugation

Optima XLA/ XLI

Sedimentation Velocity

Air Solvent

Solution

conc, c

distance, r

Top view, sector ofcentrifuge cell

Rate of movement ofboundary sed. coeff

Centrifugal force

so20,w

Sedimentation coefficient, S

Chitosan G213, 0.5 mg/ml

meniscus Cell base

Chitosan G213, 0.5 mg/ml

Sedimentation “g(s*) plot”:

from analysis of the change with time of the whole concentration profile

Guar, 0.75 mg/ml

Wheat starch, 8 mg/ml

SAN02 Freeze-thaw (1.16mg/mL)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0 10 20 30 40 50

s(S)

g*(s

)

Control10cycles20cycles25cycles

……stability studies

Multi-Gaussian fit estimates proportions of each species too:


Sedimentation Equilibrium

2 types of AUC Experiment:

Air Solvent

Solution

conc, c

distance, r



Centrifugal force

conc, c STEADY STATEPATTERN

FUNCTION ONLY OF MOL. WEIGHT PARAMETERS

distance, r

Centrifugal force Diffusion

so20,w

1S=10-13sec

Mw,app

cell bottom

Extraction of Mw,app from sedimentation equilibrium and “MSTAR” analysis

Chitosan G213

Extraction of Mw,app from sedimentation equilibrium and “MSTAR” analysis

Mw= (5.9+1.1)x106g/mol

xanthan

3. Conformation in solution

Conformation in solution

1. Experimental data required• Molecular weight• [η], s, Rg

2. Modelling strategies • General conformation type (rod, coil or sphere etc.)• Measure of flexibility – the persistence length, Lp, • If ~ rigid then aspect ratio.

Conformation in solution

1. Experimental data required• Molecular weight: SEC-MALLs reinforced by sed. equilibrium• [η], s, Rg

2. Modelling strategies • General conformation type (rod, coil or sphere etc.)• Measure of flexibility – the persistence length, Lp, • If ~ rigid then aspect ratio.


Air Solvent

Solution

conc, c

distance, r



Centrifugal force

so20,w

Sedimentation coefficient, S

so20,w extraction

κ-carrageenan

1/s20,w(S-1)

c (mg/ml)

1/so20,w

slope=ks/so20,w

Molecular Weight: Zimm plot

1/Mw

sin2(θ/2) + kc (k=100)

106.(Kc/Rθ)

Rg from slope

c (mg/ml)

Kraemer

Huggins



Haug Triangle

Rg ~ M0.5-0.6Rg ~ M1.0Rg ~ M0.33

so20,w~ M0.4-0.5so

20,w ~ M0.15so20,w ~ M0.67

[η] ~ M0.5-0.8[η] ~ M1.8[η] ~ M0

CoilRodSphere

Mark-Houwink-Kuhn-Sakurada Power law plot

Galactomannansa=0.74+0.01

Change in Conformation

Rollings, 1992

Rg ~ M0.5-0.6Rg ~ M1.0Rg ~ M0.33

so20,w~ M0.4-0.5so

20,w ~ M0.15so20,w ~ M0.67

[η] ~ M0.5-0.8[η] ~ M1.8[η] ~ M0

CoilRodSphere

ks/[η] ~1.6ks/[η] <1ks/[η] ~1.6

Conformation Zoning:

Pavlov, Harding & Rowe., 1997

Conformation Zoning:

Extra-rigid Rod: Schizophyllan

Rigid-rod: DNA

Semi-flexible coil: cellulose derivatives, chitosans

Random coil: Dextran, glycoproteinsfrom mucus

Globular/Branched: Proteins/glycogen

Morris et al., 2007

Pectins:

Contour Length

Worm-like Chain

Flexibility parameter: Persistence length Lp

Kuhn-statistical length λ-1 = 2Lp

Worm-like Chain

Flexibility parameter: Persistence length Lp

Theoretical limits: Random coil Lp = 0Rigid rod Lp = infinity

Practical limits: Random coil Lp ~ 1-2nmRigid rod Lp ~ 200nm

[ ]2/1

2/13/1

03/1

0

3/12 2w

L

pL

w MML

BMAM

−

−−⎟⎟⎠

⎞⎜⎜⎝

⎛Φ+Φ=⎟⎟

⎠

⎞⎜⎜⎝

⎛η

( )⎥⎥

⎦

⎤

⎢⎢

⎣

⎡+⎟

⎟⎠

⎞⎜⎜⎝

⎛++⎟

⎟⎠

⎞⎜⎜⎝

⎛×

−=

−

....22

843.13

12/1

32

2/1

0

00

pL

w

pL

w

A

L

LMM

AALM

MNvM

sπη

ρ

“Bohdanecky” relation

“Yamakawa-Fujii” relation

Global plot: xyloglucan

2 4 6 8 10 12 14 16 18 20200

300

400

500

600

700

800M

L(g

. mol

-1. n

m-1

)

Lp (nm) Patel et al, 2007

….or if you know the mass per unit length

Lp (nm)

2 4 6 8 10 12 14 16 18 200.0

0.2

0.4

0.6

0.8

1.0

Targ

et fu

nctio

n, Δ

Patel et al, 2007

200Xanthan

180+30Scleroglucan

120-200Schizophyllan

45DNA

10-20Pectins

5-8Xyloglucan

1-2Pullulan

Lp (nm)Carbohydrate PolymerFlexibilities of carbohydrate polymers

4. Interactions

Atomic Force Microscopy: chitosan

Sedimentation coefficient so

20,w ~ 1S

Deacon et al, 2000

chitosan-mucin complex


20,w ~ 2000S

chitosan-mucin complex


20,w ~ 2000S

very strong, irreversible interaction

“bioactive” arabinoxylan

very weak, reversible interactionPatel et al, 2007

“bioactive” arabinoxylan

very weak, reversible interaction




Thanks to: Prof. Arthur Rowe, Drs. Gordon Morris, Yanling Lu, Trushar Patel (NCMH) & Professor Jose Garcia de la Torre (Murcia)

for a copy of this presentation …

http://www.nottingham.ac.uk/ncmh

The physical characterisation of polysaccharides in solution

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