Triple Detection GPC of Hyaluronic Acid


Usage: Hyaluronic Acid (HA) is a natural biopolymer found in the cell membrane of humans. HA is found in many commercial eye products and synthetic lubricants injected into joints to supplement natural lubrication which ultimately helps reduce pain. Characterization of HA may be difficult, because human HA can have a molecular weight (MW) of several million Da, but is required to predict the final product’s therapeutic effect in patients and optimize ingredients accordingly. Molecular weight and intrinsic viscosity of HA can be precisely determined by Low Angle Light Scattering and the viscometer respectively, using Triple Detection GPC. Knowledge of these two parameters is crucial as they are intimately tied to the properties from which HA’s usefulness stems: viscosity and viscoelasticity.

Manufacturing: HA forms naturally in the plasma membrane by a class of proteins called hyaluronan synthases by repeatedly adding D-glucuronic acid and D-N-acetylglucosamine to freshly generated polysaccharides. The structure can be seen in Figure 1.

Chemical Formula: (C14H21NO11)n

Chemical Structure

Structure of Hyaluronic Acid Monomer

Figure 1. Structure of Hyaluronic Acid (HA) monomer.


Three high MW HA samples were dissolved in 0.05M NaNO3 at a concentration of 1 mg/mL. Sodium nitrate is used to eliminate ionic interaction between the columns and samples. Analysis was completed on a Viscotek Triple Detector Array (TDA) 302 equipped with a Refractive Index (RI) detector, Viscometer, Right Angle (90o) Light Scattering (RALS) and Low Angle (7o) Light Scattering (LALS). Separation was performed using PolyAnalytik AquaGelTM columns (2 x PAA 207 and 1x PAA 206) in series. Samples were injected at a volume of 100 µL and eluted through the system at flow rate of 1 mL/min in 0.05M NaNO3. A temperature of 35oC was maintained during separation and detection.

Results and Discussion

The hydrodynamic sizes of the samples are larger than the separation range of current GPC techniques. To overcome this, a Flow Injection Polymer Analysis (FIPA) method was employed to obtain average molecular parameters. In FIPA, the sample is separated from low MW impurities and passes through the GPC detectors as in batch analysis. While FIPA does not provide a distribution of the sample, it does provide absolute MW, hydrodynamic size, intrinsic viscosity and concentration. A refractive index overlay of three different samples can be seen in Figure 2. All injections have a good signal to noise ratio. The peak shape is not symmetrical but this is expected because FIPA does not provide a distribution of the sample.

Overlay Plot of the Refractive Index Response Versus Retention Volume of Hyaluronic Acid

Figure 2. Overlay of RI of different HA sample injections.

Overlay Plot of the Detector Response Versus Retention Volume of Hyaluronic Acid Signals

Figure 3. An overlay of the RI, RALS, LALS and Viscometer response signals of sample A.

Using data from RI, light scattering and the viscometer, the absolute MW, radius of hydration and radius of gyration was determined and reported as an average from 3 injections, see Table 1. FIPA method allows for good repeatability of injections as can be seen from the % RSD in Table 1

Table 1. Molecular weight and size of HA derived from FIPA method

Table of the Molecular Weight and Size of Hyaluronic Acid


Characterization of high molecular weight HA, outside the separation range of GPC, was successfully achieved using a FIPA method. Three samples were analyzed and absolute MW in the millions of Da with a % RSD less than two percent were determined. The radius of hydration and radius of gyration were also determined and both had a % RSD less than one percent indicating reproducibility. The FIPA method allows for analysis of high MW polymers outside the separation range of GPC but still provides absolute Mw and size which are essential for predicting therapeutic effects within the body and for quality control of commercial products.

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