Preparation of mixed-mode stationary phase for separation of peptides and proteins in high performance liquid chromatography

HPLC analysis

A special µLC set-up was constructed using LC pump (10AD Shimadzu, Japan), an injector with 50nL injection loop (Valco (USA) C14 W.05), membrane degasser (Shimadzu DGU-14A), a UV–VIS capillary window detector (UV-2075), and glass lined micro-column. Very narrow and short connecting tubing were used to minimize the effect of extra column band broadening. After packing, the 1/16″ outlet of the reducing union was installed with a capillary (50 μm i.d. 365 ith a capillary (50 μm) of the reducing union was installed. Data collection and chromatogram processing were accomplished with Multichro 2000 software. The UV absorbance of the test analytes was monitored at 254 nm. The chromatographic data was analyzed by OriginPro8 (Northampton, MA).

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