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, PhDTechnical Support Supervisor – SeparationsMalvern 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. UK2 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

ASample loaded on column.

BSample components separated by hydrodynamic size.

C and DComponents elute from column and pass through detectors.

› Solution based technique.

› Purely physical separation.Larger molecules elute first

› No interaction with column.

SAMPLE MIXTUREA B C D

DETECTORS

SOLVENT FLOW

CHROMATOGRAM

RETENTION

VOLUMEA 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

SE

PAR

ATIO

ND

ETE

CTI

ON

8

SEC Instrument Schematic

Tetra Detection

ViscometerLight 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( 1 relsp

Set Volume

Capillary

Reservoir

00 tt

rel 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 ∝ volumemass

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 Cdensity

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

IVC

14

0

0

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

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

Vis

cosi

ty (d

L/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 65x10Molecular 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

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 akT

Where a = particle radius, k = Boltzmann’s constant, T = absolute temperature, = 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 microrheologyand 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

Thank you!

Useful links:

Malvern GPC instruments: www.malvern.com/gpcLight scattering explained: www.malvern.com/slsMolecular weight: www.malvern.com/mwMicrorheology www.malvern.com/mr

Dr Bassem SabaghTechnical Support Supervisor – Separation ScientistE-mail: Bassem.sabagh@malvern.com