Hyaluronic acid (HA) is a naturally occurring, unbranched polysaccharide that consists of alternately repeating D-glucuronic acid and N-acetylglucosamine units. This biopolymer is present throughout all mammalian systems but occurs primarily in synovial (joint) fluid, vitreous humor, and various loose connective tissues (such as rooster comb) (1). HA is of enormous commercial interest for ophthalmic, medical, pharmacological, and cosmetic applications.
Hyaluronic 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 (M w), molecular weight distribution (also known as polydispersity index [PDI]), intrinsic viscosity ([η]), 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 M w 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 M w, 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), R g, by using a MALS instrument, provided the sample R g is greater than about 10 nm.
where Q is mass flow rate, Δp is the pressure drop across the capillary, R is the flow impedance through the capillary, and η is the fluid viscosity.
This article discusses on-line absolute characterization of HA properties using MALS, differential refractometry, and differential viscometry detectors in series. Utilizing differential viscometry is particularly advantageous as compared with single-capillary viscometry, as will be shown.
Materials and Methods Seven separate HA samples were used for this study. Sample root sources varied, and included HA from rooster comb, umbilical cord, and bacterial fermentation. Ovalbumin was obtained from Sigma (St. Louis, Missouri). All other chemicals were analytical grade.