Monolithic columns for analytical applications have attracted the researcher's attention. In this work, the laboratory-made organic-polymer monolithic column is modified with trypsin and further applied as a nanobiocatalyst microreactor and a stationary phase for separating chiral compounds by liquid chromatography. The monolith was synthesized by in-situ copolymerization of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) or trimethylolpropane trimethacrylate (TRIM) as a crosslinking agent, with porogen of 1,4-butanediol/propanol/water (4:7:1 v/v) and AIBN as the radical polymerization initiator inside PEEK and silicosteel tubings (1.0 mm i.d  100 mm) at 60 C for 12 h. A total monomer ratio (%T) and crosslinking agent (%C) of 40:25 and 28:12 were applied to prepare poly-(GMA-co-EDMA) and poly-(GMA-co-TRIM), respectively. The produced monoliths were further modified by introducing trypsin (10 mg/L) through the ring-opening reaction of the epoxide group existing in the monolithic column. The trypsin-immobilized poly-(GMA-co-EDMA) monolithic column was applied as the nanobiocatalyst microreactor for online/flow-through and rapid digestion of β-casein sample into its peptide fragments. The trypsin-immobilized poly-(GMA-co-TRIM) column has potential application to be used as the HPLC stationary phase for the separation of R/S-citronellal enantiomers.

An enantiomer molecule consisted of the chiral atom has different structure conformations, which exhibit different activities as well. Yet, its separation considerably difficult since ordinary separation could not separate both molecules. One of the popular enantioseparations which are often used was using organic polymer monolithic column modified by ethylenediamine-β-cyclodextrin (EDA-β-CD) as the enantioseparations site. The aim of this research was to produce chiral stationary phase column for enantioseparations of (±)-citronellal. It was conducted by preparing monolithic column using monomer glycidyl methacrylate (GMA), trimethylolpropane trimethacrylate (TMPTMA) as crosslinker, 1-propanol, 1,4-butanediol, and water as pore-forming agents (porogens) in the presence of α,α'-azoisobutyronitrile (AIBN) as radical initiator inside polyetheretherketone (PEEK) tubing. It was then modified with EDA-β-CD synthesized from β-CD. Finally, it was installed as a high-performance liquid chromatography column. The result shows the produced chiral stationary phase column could separate (±)-citronellal at a retention time of 44.76 and 45.71 min.

Preparation and utilization of monolithic column as HPLC stationary phase for alkyl benzene separation with low mobile phase usage

One of the long-known separation methods is chromatography, such as high performance liquid chromatography (HPLC). This separation essentially uses column containing particle-packed which generates high flow-resistance and low mass transfer. As matter of fact, it has an impact to high amount mobile phase usage. Meanwhile, organic polymer monolithic column has been widely used as alternative due to its high mass transfer. In addition, the resulting column has stability over wide pH range, high temperature, shrinkage and swelling of the reservoir. The aim of this research was to produce monolithic column which able to utilized for separation with low amount of mobile phase usage. Preparation of monolithic column begins with pretreatment of polyetereterketone tubing column inner wall. It conducted by activating the inner wall with H 2 SO 4 49% (v/v), followed by vinylization with glycidyl methacrylate. Furthermore, pretreated column filled by polymer mixture consisted of 30 wt% of monomers glycidyl methacrylate:trimetilolpropane trimethacrylate 4:1 (w/v), 70 wt% of pore-forming agents (1-propanol/1,4-butanediol/water 7:4:1 w/v), and azoisobutyronitrile 1 wt% of total monomers amount. The polymerization conducted at 60°C for 12 h. Produced column connected to HPLC then applied to separate toluene and amylbenzene. The result shows both compounds successfully n flow rate of acetonitrile:water (75:25) at 84 and 132 min respectively.