658 nm laser

Glycosaminoglycan polysaccharides (4 mg) were dissolved in 0.5 ml of ammonium bicarbonate (100 mM) for 24 hr at 4°C and then centrifuged at 12000g for 10 minutes prior to application to a Superose 6 10/300GL gel filtration column (GE Healthcare, Little Chalfont, UK) running at a flow rate of 0.75 ml/min in the same eluent. Samples eluting from the column at room temperature passed through an in-line DAWN HELEOS-II laser photometer with a 658 nm laser (Wyatt Technology Corp., Santa Barbara, USA), WyattQELS detector model number Q-03 and an Optilab rEX refractometer; a refractive index increment (dn/dc) of 0.16 mlg⁻¹ was used throughout.^{28 (link)} Light scattering and concentration during elution were recorded using ASTRA software (v5.3.4.13). Multi-angle laser light scattering was used to determine weight-averaged molecular masses (M) and radius of gyration (R_g) using analysis modules within the ASTRA software. For molecules too small to accurately determine R_g, quasi-elastic light scattering was used to measure hydrodynamic radii (R_h), again using analysis modules within the ASTRA software. Conversion of calculated R_g to R_h was achieved using the formula R_g = 1.5R_h, derived previously for a random coil.^{29 (link)}