Polymeric monolithic columns have been valuable tools in bioanalysis by efficiently separating intact proteins, viruses, nucleic acids, etc. Monoliths were first prepared by free radical thermal polymerization in 4.6 - 8.0 mm i.d. inox tubes to separate proteins at high flow rates. The growing demand for the hyphenation of liquid chromatography with mass spectrometers motivated the columns' miniaturization. Nowadays, analytical monoliths are primarily prepared in fused silica capillaries (> 100 µm i.d.), requiring a drastic reduction of extra-column volumes of the chromatographic system. There is a growing interest in semi microcolumns (0.5 to 2.0 mm i.d.) to fill the gap between the capillaries and regular 4.6 mm i.d. columns. Here, we describe the construction of polymer monolithic columns inside 2-mm i.d. fluorinated ethylene propylene (FEP), polypropylene (PP), and fused silica stainless steel (FSSST) tubes for separation of proteins by reversed-phase (RP)1, ion exchange (IEX)2 and Immobilized Metal Ion Affinity Chromatography (IMAC)3. Columns prepared inside FSSST were based on the copolymer formed by free-radical polymerization of glycidyl methacrylate (GMA, functional monomer) and ethylene glycol dimethacrylate (EDMA, crosslinker) in a mixture of 1-propanol and 1,4 butanediol as the porogenic solvents using azobisisobutyronitrile (AIBN) as initiator. The polymerization proceeded for 24 h at 60 C. After the polymerization and washing out of the unreacted monomers, the free epoxy groups in the P(GMA-co-EDMA) were functionalized with Na2SO3 and iminodiacetate (IDA), forming strong and weak cations exchangers. The P(GMA-co-EDMA)-IDA column saturated with Cu(II) formed columns to separate proteins by IMAC. While the free radical thermal polymerization takes several hours to achieve suitable yield and reproducibility, UV-assisted polymerization takes only 20 min. Exploring UV-polymerization, lauryl methacrylate (LMA) was used to construct P(LMA-co-EDMA) monoliths inside 2 mm FEP tubes for RPLC and P(GMA-co-EDMA) columns modified with Na2SO3 and IDA inside PP tubes from ink pens for IEX. All tubes (FSSST, FEP and PP) require vinylization of the internal walls to bind the monolith covalently, thus keeping stable the chromatographic bed at backpressures at around 7 MPa without the need for retention frits. The separation of ovalbumin and lysozyme in egg-white samples by RPLC and IEX and myoglobin in urine by RPLC demonstrated the applicability of the columns to complex matrices.
1 F.H. do Nascimento, A.H. de Moraes, C.R.L. Trazzi, C.M. Velasques, J.C. Masini, Talanta 217 (2020) 121063
2 F.H. do Nascimento, C.R.L. Trazzi, A.H. de Moraes, C.M. Velasques, D.M. de Souza Costa, J.C. Masini, Journal of Separation Science 43 (2020) 4123-4130
3 F.H. do Nascimento, J.C. Masini, Analytical Letters 53 (2020) 522-535