Abstract: Pancreatic ribonuclease, trypsinogen and α-chymotrypsinogen were dissolved in 5 mM bis-Tris/0.2 M dextrose (buffer A), to give solution of 2.5-3.5 mg/ml and applied to a column (10 cm x 4.6 mm) of strong cation-exchange polymer-based packing operated with gradient elution (0.5 ml/min) with buffer A and 5 mM bis-Tris/0.5 M NaCl (buffer B) from 0-80% buffer B over 20 min with online detection using a low-angle laser light scattering photometer, a UV-visible detector at 280 nm and a laser-based differential refractive index detector at 670 nm connected in series with a PC for data collection and processing. The system was also used in the FIA mode to measure the molar absorptivities of the proteins. Off-line refractive index measurements were performed with a laser-based differential refractometer, at 633 nm. Online differential refractive index measurements were similar to those determined off-line and required less sample (3-4 mg as opposed to 200 mg). Molecular wt. determinations agreed with literature values to within 3% and protein recoveries ranged from 66-87%. Other biopolymers, such as DNA fragments can be characterized using the same conditions.

Abstract: The samples were injected into the post-column flow stream of an HPLC system as the stream was pumped past a diode-array detector set at 260 nm for nucleic acids and 280 nm for proteins. Response was rectilinear between 3 ng and 7 µg of DNA, 10 ng and 10 µg of RNA and 10 ng and 80 µg of bovine serum albumin. The method was simple, required only 5 to 10 µL of sample solution, and was rapid (2 samples min-1).