Characterization of Hyaluronic Acid with On-Line Differential Viscometry, Multiangle Light Scattering, and Differential Refractometry

Address correspondence to J. Waters. yaluronic acid (HA) has been studied extensively by many groups in the past (1–7). The physiochemical behavior of HA has been tied closely to material characteristics such as the weight-average molecular weight (Mw), molecular weight distribution (also known as polydispersity index [PDI]), intrinsic viscosity ([ ]), and molecular conformation. Past studies of HA have included many size-exclusion chromatography (SEC) experiments. Traditional SEC involves chromatographically separating samples and monitoring the output with a concentration detector such as a refractometer or UV absorbance detector. SEC in this form is a purely relative measurement, because the chromatographic system must first be calibrated with a series of known Mw standards, collectively known as a calibration curve. Other SEC studies of HA have added multiangle light-scattering (MALS) devices in series with concentration detectors. This proves advantageous because MALS is an extremely sensitive technique for measuring absolute Mw, as it does not rely on calibration standards or a priori assumptions about the molecular conformation. One also can determine a sample’s root mean-square radius (erroneously, but frequently referred to as the radius of gyration), Rg, by using a MALS instrument, provided the sample Rg is greater than about 10 nm. The Mendichi group at the Istituto di Chimica delle Macromolecole (Milan, Italy) has performed a number of elegant experiments involving on-line SEC of HA utilizing MALS, concentration detection, and single-capillary viscometry (2,3). This combination of detectors yields not only all of the aforementioned material characteristics but also elucidates sample intrinsic viscosity and, using the Mark–Houwink–Sakurada (MHS) relationship, molecular conformation information. Single-capillary viscometry is inherently vulnerable to noise generated by system pressure fluctuations. The pressure associated with laminar fluid flow through capillaries is first order with respect to flow rate, as can be seen in Poiselle’s law