Capillary poly(styrene-co-octadecene-co-divinylbenzene) monolithic trap columns for bioanalytical analysis

The increasing demand for faster and more efficient separations for biological samples have driven organic polymer-based monolithic columns back into the spotlight. The nature of the monolithic structure allows the use of higher flow rates without increasing the system backpressure. They are also easier to contain inside the column body, opposed to particle packed columns, which are held in place by frits. These are some of the advantages that monolithic columns offer compared to particle packed columns. Poly(styrene-co-octadecene-co-divinylbenzene) (PS-OD-DVB) monolithic columns have been prepared and characterized using the neuropeptide oxytocin, “the love hormone” as test compound. The columns, 50 µm inner diameter x 100 mm in length, were to be used as trap columns in a miniaturized liquid chromatography-mass spectrometry (LC-MS) column switching system for bottom-up proteomics analysis. The monolithic PS-OD-DVB columns were prepared in situ (inside the fused silica capillary), using a polymerization mixture consisting of an initiator (lauroyl peroxide), a crosslinker (divinylbenzene), monomers (styrene and 1-octadecene), a good porogen (N,N-dimethylformamide (DMF)) and a bad porogen (1-decanol). Polymerization time, polymerization temperature, initiator concentration, and monomer to porogen ratios were investigated in order to obtain a monolithic structure with a high surface area and good permeability. The best PS-OD-DVB monolithic column was prepared using a mixture of 1.9% lauroyl peroxide (weight percentage of monomer amount), 14.2% divinylbenzene, 15.2% monomers, 11.9% DMF and 57.5% 1-decanol. The column was polymerized for one hour at 73°C and gave a plate height of 62 µm, a backpressure of 23 bar at a flow rate of 500 nL/min with a mobile phase consisting of 20% B (acetonitrile (ACN), water, formic acid (FA), 80/20/0.1, v/v/v) and 80% A (water with 0.1% (v/v) FA) and a retention time repeatability of 2.6% (relative standard deviation). The repeatability of the columns made by various individuals was satisfactory. However, when preparing twenty-one columns by the same individual, the repeatability revealed to be questionable. Hence, further improvements in the polymerization procedure do seem necessary and might be able to enhance the repeatability of the columns