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Investigating the Relationship Between the Rheological Properties of Hyaluronic Acid and its Molecular Weight and Structure using Multi-detector SEC and SEC-MALS
Presented by Bassem Sabagh, PhD Technical Support Supervisor – Separations Malvern Instruments UK Bassem.Sabagh@Malvern.com Authors: John Stenson1, Mark R. Pothecary3 , Bassem Sabagh1, Paul Clarke1 , John Duffy1 , and Agata Papa2
1 Malvern Instruments, Enigma Business Park, Grovewood Road, Malvern, Worcestershire. UK 2 ALFATESTLAB, Cinisello Balsamo, Italy 3 Malvern Instruments, Houston, Texas, USA
Agenda
› Hyaluronic Acid (HA)
› GPC Analysis Molecular Weight by Light Scattering
Molecular Structure by Online Viscometry
› Comparison of Modified Structures Quantitative Analysis
Structural Analysis
› Microrheology by DLS
› Summary
Hyaluronic Acids
› Natural polysaccharide consisting of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine.
› Important role as structural and mechanical support for tissues Skin, Tendons, Muscles and Cartilage
› Physico-chemical Properties led to wide range of applications Cosmetic, Pharmaceutical, Medical
› Native HA has two limitations: rapid clearance in vivo and mechanical weakness.
› Cross-linked/derivatised to delay degradation and improve
mechanical performance
HA Chemistry
Samples analysed by GPC
› Linear HA – Also the starting material
› Crosslinked HA – auto-crosslinking via ester bonds using carbodiimide chemistry. Approximately 1% (molar percentage) of carboxyl groups of HA were activated.
› Crosslinked HA – APMA – Crosslinked in presence of nucleophilic agent APMA. The primary amines bind to the carboxyl group of the glucuronic acid introducing side chains into HA leading to a branched product. Branching competes with crosslinking
› Analysis gives Absolute Molecular Weight, Hydrodynamic Radius,
and Structure
SEPARATION: SEC
A Sample loaded on column. B Sample components separated
by hydrodynamic size. C and D Components elute from
column and pass through detectors.
› Solution based technique.
› Purely physical separation. Larger molecules elute first
› No interaction with column.
SAMPLE MIXTURE A B C D
DETECTORS
SOLVENT FLOW
CHROMATOGRAM
RETENTION VOLUME A B C D
POROUS PACKING
SIZE EXCLUSION CHROMATOGRAPHY
6
Multi-detection GPC
› Conventional GPC compares the retention volume of a sample with that of standards of known molecular weight using a single concentration detector Gives Relative Mw
› Advanced GPC adds more detectors to make more measurements of the sample as it elutes. Static Light Scattering – Absolute Molecular Weight Differential Viscometer – Molecular Density, Structure
SEPA
RAT
ION
D
ETEC
TIO
N
8
SEC Instrument Schematic
Triple Detection Tetra Detection
Viscometer Light Scattering
Refractive Index
UltraViolet - PDA
› Photons from an incident beam is absorbed by a macromolecule and re-emitted in all directions
› We can characterize this scattered light using different detector systems to measure different macromolecular properties
Light Scattering Detector
LIGHT SCATTERING THEORY
› The Rayleigh equation can be used to measure molecular weight by measuring the intensity of the light scattered by the sample if all the other parameters are known
10
2
0 '
×∝
dcdnCKMR w
11
WHAT IS INTRINSIC VISCOSITY?
Solute (polymer) dissolved in Solvent
When a solute is dissolved in the solvent, the ability of these sheets to flow over one another is changed. This contribution of the solute to the overall viscosity of the solution is known as the intrinsic viscosity of the solute.
VISCOMETERY
Traditional Solution Viscosity Measurements
crel
inh)ln(ηη =1−= relsp ηη
Set Volume
Capillary
Reservoir
00 tt
rel ==ηηη Solution Drop Time
Solvent Drop Time
Derived from relative viscosity
Ubbelohde Tube
12
13
HOW CAN WE RELATE IV TO STRUCTURE?
Intrinsic viscosity has the units:
dL/g
We can look at structure in these terms:
IV ∝ volume mass
Intrinsic viscosity is inversely proportional to molecular density:
Which of these two molecules with the same mass occupies the largest volume of space?
IV ∝ 1 C density
IP + -
DP - + GPC IN OUT
Solvent
Sample
HOW DO WE MEASURE IV? 4-capiliary viscometer bridge - The Wheatstone Bridge Concept The viscometer detects changes in pressure when the sample travels though the viscometer.
Relationship of the output from the pressure transducers and specific viscosity
Relationship of the specific viscosity and intrinsic viscosity
DPIPDP
sp 24−
=η IVC×=
14
0
0
ηηηη −
=sp
Quantitative Comparison of Modified HA Structures
› Typical Triple detection chromatogram for LHA SEC-MALS 20 and TDA
Quantitative Comparison of Modified HA Structures
› Auto-crosslinking (XHA) increases Mw, IV and Rh
› Reacting with APMA prevents crosslinking, but… (?)
Sample Id Mw (kDa) IV Rh (nm) Rg (nm)
LHA 263 6.47 29 45
XHA 483 7.85 36 49
XHA APMA 333 7.02 32 47
The Kuhn–Mark–Houwink–Sakurada equation
αη MwK ×= ][› [η] is the Intrinsic Viscosity (IV) › K and α are the MH parameters which depend on the nature of the
polymer & solvent › Mw is the weight average molar mass (molecular weight) › a describes the relationship between molecular weight and IV, K is
the intercept, describing the flexibility of the backbone.
The equation describes the dependence of the intrinsic viscosity of a polymer to molecular weight
6TH INTERNATIONAL CONFERENCE ON THE HISTORY OF CHEMISTRY Staudinger - Mark - Kuhn: Historical Notes from the Development of Macromolecular Chemistry
Mark Houwink plot - Linear vs. Crosslinked › Structural comparisons made using Mark-Houwink plot which
relates the Intrinsic Viscosity to the Molecular Weight Log [η] = Log K + aLogM
› IV measured across entire Mw range › Fine differences between samples can be measured › Any size calculations are based on assumptions of shape
1
2
10
20
100
Intri
nsic
Visc
osity
(dL/
g)
410 42x10 510 52x10 610 62x10 710 72x10Molecular Weight (Da)
LHA
XHA
Structural Comparison of Modified HA Structures
› Reaction has favoured the up-take of APMA on the substrate, preventing auto-crosslinking but allowing branching.
2
3
4
10
20
30
40
Intri
nsic
Vis
cosi
ty (d
L/g)
510
52x10
53x10
54x10
55x10
610
62x10
63x10
64x10
65x10
Molecular Weight (Da)
LHA
XHA
XHA-APMA
Conformation plot - Linear vs. Crosslinked › Plot of Radius of Gyration vs Mw › Rg calculated by MALS only for Anisotropic scattering materials
› Limited to larger molecules, so not all distribution measured › Fit model influences results › No assumptions about shape for size calculation
LHA
XHA
Rg
Agenda
› Hyaluronic Acid (HA)
› GPC Analysis Molecular Weight by Light Scattering
Molecular Structure by Online Viscometry
› Comparison of Modified Structures Quantitative Analysis
Structural Analysis
› Microrheology by DLS
› Summary
Measuring polymer solutions using Microrheology
› Microrheology is termed ‘micro’ since it measures rheology on very small (micro) length scales
› In microrheology we measure the motion of a colloidal probe particle or tracer embedded in the sample.
› From this motion we can calculate the same rheological parameters that we obtain from mechanical rheology since; Stress is related to the particle size and force acting over the
surface of the particle Strain is related to the displacement resulting from this
applied stress The relative phase difference is dependent on sample
viscoelasticity
› This can me made using a Zetasizer ZS or ZSP
Brownian Motion, Particle Size and Viscosity
› The smaller the particle, the more rapid the Brownian motion
› The larger the particle, the slower the Brownian motion
› Diffusion is also governed by solution viscosity…
› …so for the same particle size, diffusion will be slower the higher the viscosity D =
3 π η a kT
Where a = particle radius, k = Boltzmann’s constant, T = absolute temperature, η = viscosity and D = diffusion coefficient
Different HA samples, same concentration
5mg/ml XHA
5mg/ml LHA
5mg/ml HHA
All measured at ~ 5 mg/mL LHA and XHA show similar properties HHA G’ and G’’ overlapping Suggests entanglement of the polymers
Comparison with Kinexus
› Good agreement between viscoelastic data generated by DLS microrheology and Rotational Rheology (Kinexus)
› Microrheology extends rheology data to higher frequencies (not accessible by mechanical rheology)
Summary
› Different samples of hyaluronic acid were measured using multi-detector SEC with SEC-MALS
› Show a correlation between the measured molecular properties of the samples and properties
› Demonstrate the valuable nature of multi-detector SEC for hyaluronic acid characterization.
› DLS Microrheology Short term dynamics and elasticity
Thanks to: And YOU for Listening! Dr Bassem Sabagh Technical Support Supervisor – Separation Scientist E-mail: Bassem.sabagh@malvern.com
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