Investigating the Relationship Between the Rheological Properties of Hyaluronic Acid and its Molecular Weight and Structure using Multidetector SEC and SEC-MALS Presented by Bassem Sabagh, PhD Technical Support Supervisor Separations Malvern Instruments UK Bassem.Sabagh@Malvern.com Authors: John Stenson 1, Mark R. Pothecary 3, Bassem Sabagh 1, Paul Clarke 1, John Duffy 1, and Agata Papa 2 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
HA Chemistry Native HA has two limitations: rapid clearance in vivo and mechanical weakness. Cross-linked/derivatised to delay degradation and improve mechanical performance
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. SAMPLE MIXTURE SIZE EXCLUSION CHROMATOGRAPHY A B C D B Sample components separated by hydrodynamic size. C and D Components elute from column and pass through detectors. POROUS PACKING DETECTORS SOLVENT FLOW Solution based technique. Purely physical separation. Larger molecules elute first No interaction with column. CHROMATOGRAM A B C D RETENTION VOLUME 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
SEC Instrument Schematic SEPARATION DETECTION UltraViolet - PDA Refractive Index Light Scattering Viscometer Triple Tetra Detection 8
Light Scattering Detector 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 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 dn R M K' C 0 w dc 2 10
VISCOMETERY 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. 11
Traditional Solution Viscosity Measurements Ubbelohde Tube η ηrel = = η t 0 t 0 Solution Drop Time Solvent Drop Time Set Volume Capillary Reservoir Derived from relative viscosity η sp = η rel 1 η = inh ln(η c rel ) 12
HOW CAN WE RELATE IV TO STRUCTURE? Intrinsic viscosity has the units: dl/g 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 We can look at structure in these terms: IV volume mass 13
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. GPC IN IP + - - + DP Solvent η sp = OUT η η0 η 0 Sample Relationship of the output from the pressure transducers and specific viscosity η sp = 4DP IP 2DP Relationship of the specific viscosity and intrinsic viscosity = C IV 14
Quantitative Comparison of Modified HA Structures Typical Triple detection chromatogram for LHA SEC-MALS 20 and TDA
Quantitative Comparison of Modified HA Structures 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 Auto-crosslinking (XHA) increases Mw, IV and Rh Reacting with APMA prevents crosslinking, but (?)
The Kuhn Mark Houwink Sakurada equation The equation describes the dependence of the intrinsic viscosity of a polymer to molecular weight [ η ] = K Mw α [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. 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 100 20 LHA Intrinsic Viscosity (dl/g) 10 2 XHA 1 4 10 4 2x10 5 10 5 2x10 10 Molecular Weight (Da) 6 6 2x10 7 10 7 2x10 IV measured across entire Mw range Fine differences between samples can be measured Any size calculations are based on assumptions of shape
Structural Comparison of Modified HA Structures 40 XHA-APMA 30 20 LHA Intrinsic Viscosity (dl/g) 10 XHA 4 3 2 5 10 5 2x10 5 3x10 5 5 4x10 5x10 Molecular Weight (Da) 6 10 6 2x10 6 3x10 6 4x10 6 5x10 Reaction has favoured the up-take of APMA on the substrate, preventing auto-crosslinking but allowing branching.
Conformation plot - Linear vs. Crosslinked Plot of Radius of Gyration vs Mw Rg calculated by MALS only for Anisotropic scattering materials LHA Rg XHA Limited to larger molecules, so not all distribution measured Fit model influences results No assumptions about shape for size calculation
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 = kt 3 π η a 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 HHA All measured at ~ 5 mg/ml LHA and XHA show similar properties HHA G and G overlapping Suggests entanglement of the polymers 5mg/ml LHA
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