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

Jun 01, 2005

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.

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

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.

Figure 1: Schematic of the differential viscometer design.
This means that even slight changes in flow rate can lead to vastly increased baseline noise. Such flow rate fluctuations are virtually omnipresent in commercial chromatography pumps in the form of pump pulsations. Increased baseline noise leads to a lower signal-to-noise ratio (S/N) and thus lowered experimental accuracy. Using elaborate pulse dampeners, pump pulses can be reduced but, unfortunately, not eliminated. Even using a pulse-free pump and Fourier-transform data filtering, single-capillary viscometry detector S/N of only approximately 125:1 has been shown (8,9). Another drawback of this technique is its reliance on universal calibration. Before use, the single-capillary viscometry device must first be calibrated using a large number of known standards.

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.