Fast construction of polymer monolithic columns inside fluorinated ethylene propylene (FEP) tubes for separation of proteins by reversed-phase liquid chromatography

• Published in: Talanta (Oxford) 2020-09, Vol.217, p.121063-121063, Article 121063
• Main Authors: do Nascimento, Fernando H., Moraes, Amanda H., Trazzi, Catharina R.L., Velasques, Caryna M., Masini, Jorge C.
• Format: Article
• Language: English
• Subjects: Animals; Cattle; Chickens; Chromatography, Reverse-Phase; Egg white; Fluoropolymer tubes; Horses; Liquid chromatography; Monolithic columns; Muramidase - chemistry; Muramidase - isolation & purification; Muramidase - metabolism; Myoglobin - chemistry; Myoglobin - isolation & purification; Ovalbumin - chemistry; Ovalbumin - isolation & purification; Photografting; Polymers - chemistry; Polytetrafluoroethylene - analogs & derivatives; Polytetrafluoroethylene - chemistry; Ribonuclease, Pancreatic - chemistry; Ribonuclease, Pancreatic - isolation & purification; Urine
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2020.121063

This paper describes the preparation of polymer monolithic columns in the confines of fluorinated ethylene propylene (FEP) tubes. These tubes are cheap, chemically stable, and widely used in flow analysis laboratories. UV-initiated grafting with 5 wt% benzophenone in methanol for 1 h activated the internal surface walls, thus enabling the further covalent binding of ethylene glycol dimethacrylate (EDMA) from a 15 wt% solution in methanol, also via photografting. Both steps used 254 nm radiation under a potency of 120 mJ cm2. ATR-FTIR measurements revealed the presence of carbonyl, alkyl and vinyl groups in the functionalized FEP. The density of vinyl groups was high enough to firmly attach a poly(lauryl methacrylate-co-ethylene glycol dimethacrylate) monolith in 120 × 1.57 mm i.d. tubes, prepared via photopolymerization. The total preparation lasts less than 2-h. The columns were permeable, (1.58 ± 0.06) × 10−13 m2, providing reproducible chromatographic parameters of retention times, retention factor, selectivity, and resolution. The monoliths were stable at flow rates of 500 μL min−1, collapsing only at flow rates >700 μL min−1, a condition that increased the backpressure over 1000 psi (experiments at the room temperature). The separation of proteins by reversed-phase liquid chromatography demonstrated the efficiency of the columns. Determination of egg white proteins (ovalbumin and lysozyme) and myoglobin in spiked urine proved the applicability to the analysis of real samples. [Display omitted] •FEP tubes were photografted with benzophenone and ethylene glycol dimethacrylate.•Micro bore (1.50 mm i.d.) FEP tubing used as support for poly(LMA-co-EDMA) monolith.•The monoliths were stable at flow rates of 500 μL min−1 and pressures of 400 psi.•Suitable separation of egg white proteins was demonstrated by RPLC.•Separation and UV-detection of myoglobin in urine sample by RPLC.