Separation of native types I and III collagens and denatured chains by reverse-phase high-performance liquid chromatography

Abstract

A simple and effective high-performance-liquid-chromatography system has been developed for the separation of the native forms of Types I and III collagens. This separation is achieved on a commercially available 330-A-pore-size C18 reverse-phase support using a solvent system consisting of ammonium bicarbonate (0.05 M) and trifluoroacetic acid (0.4%) with tetrahydrofuran as the eluting solvent. This simple system can also be applied to the separation of mixtures of the denatured chains of both collagen types.

Defining the Roles of Collagen and Collagen‐Like Proteins Within the Proteome

“…There have been numerous studies describing the analysis and purification of collagen and collagen-derived fragments by RP-HPLC using cyanopropyl, [120][121][122] diphenyl, [123,124] C 4 , [124] C 8 , [123,125] and C 18 [120,122,[125][126][127][128][129][130] columns, unique pyridine-propanol gradients [121,123] or more ''conventional''…”

“…[120,122,128] The earliest studies compared pore sizes (100 Å vs. 300 or 500 Å ) and determined that large-pore columns reduced collagen chain peak widths [123] and improved collagen fragment resolution; [120] consequently, later collagen RP-HPLC studies used large-pore columns (300-500 Å ) almost exclusively. [122,124,125,127,129] Large-pore (300 Å ) columns have been utilized effectively for characterization of novel collagens. [131,132] However, switching from a large-pore (250 Å ) silica-based column to a nonporous polymeric column improved resolution of type VI collagen chains.…”