A facile and efficient strategy for one-step in situ preparation of hydrophobic organic monolithic stationary phases by click chemistry and its application on protein separation.

A simple one-step in situ "click" modification strategy was developed for the preparation of hydrophobic organic monolithic columns for the first time. The column morphology and surface chemistry of the fabricated monolithic columns were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The chromatographic performances of the C8/C18 "click" monoliths were evaluated through the separation of a mixture of five proteins such as ribonuclease A, soybean trypsin inhibitor, cytochrome c, bovine haemoglobin and bovine serum albumin. Compared with the blank column, the higher hydrophobicity stationary phases obtained from the "clicked" modification have longer retention times and higher resolution for the five proteins. The separation of five proteins mixture on click C18 monolith with gradient elution at different flow rates was also investigated, the baseline separation of five proteins could be achieved at three different flow rates.