Non polar and affinity monolithic stationary phases for HPLC of large and small molecules and their use in a multi column liquid phase separation platform for the capturing and fractionation of sialoglycoproteins from human serum

Erandi Prashani Mayadunne Monolithic columns are emerging as important chromatographic separation media. In this investigation, a novel monolithic column was fabricated by the in situ co-polymerization of glyceryl methacrylate (GMM) and ethylene glycol dimethacrylate (EDMA) incorporated with multiwalled carbon nanotubes (MWCNTs). The optimized GMM-EDMA-MWCNTs monolith proved useful for the separation of small molecules including alkyl benzenes, benzene derivatives, anilines and phenoxy acid herbicides. Also, chiral separation of pharmaceutically and environmentally important compounds was achieved on the optimized GMM-EDMA-MWCNTs monolith. When compared to the GMM-EDMA-MWCNTs monolith, the blank GMM/EDMA monolith (i.e., without incorporated MWCNTs) did not yield significant retention toward nonpolar solutes, indicating that the separation on the GMM-EDMA-MWCNTs is solely based on solute differential interactions with the incorporated MWCNTs. However, the GMM-EDMA-MWCNTs monolith was not suitable for separating large molecules such as proteins. In this regards, another novel monolith was prepared by the in situ co-polymerization of octadecyl acrylate (ODA) and trimethloylpropane triacrylate (TRIM) with the intention of separating proteins. The optimized ODA-TRIM monolith proved efficient for separating small molecules (e.g., alkyl benzenes) as well as proteins. The retentive properties of the optimized ODA-TRIM monolith toward small solutes and proteins were further improved by incorporating small amounts of MWCNTs into the ODA-TRIM monolith to yield the optimized ODA-TRIM-MWCNTs monolith. This monolithic was used into a multi column liquid phase separation platform for the capturing and fractionation of sialoglycoproteins from disease free and breast cancer sera. The multi column platform assembled affinity monolithic columns for he depletion of high abundance proteins as well as two lectin monolithic columns specific for sialic acids for the capturing of sialoglycoproteins by lectin affinity chromatography. The two immobilized lectins were Sambucus nigra agglutinin (SNA) and Maakia amurensis lectin (MAL). The sialoglycoproteins captured by the two-lectin columns from human serum were subsequently fractionated online on the optimized ODA-TRIM-MWCNTs monolithic column. The LC-MS/MS analysis of the collected fractions allowed the identification of a panel of differentially expressed sialoglycoproteins in breast cancer serum relative to the disease free serum. This panel of differentially expressed sialoglycoproteins can be considered as a panel of candidate biomarkers for breast cancer.