Journal of Chromatography A, 1030 (2004) 195–200 Comparison of styrene–divinylbenzene-based monoliths and Vydac nano-liquid chromatography columns for protein analysis Cristina Legido-Quigley a,∗ , Nicola Marlin a , Norman William Smith b a Centre for Analytical Sciences, Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AY, UK b Microseparations Group, Department of Pharmacy, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN, UK Abstract This report details the fabrication of polystyrene-based nano-LC monolithic columns for the separation of protein molecules. The report focuses on the practical advantages of monolithic columns when compared to conventional packed columns. Capillary columns were made to 100 and 50 m i.d. and used to analyse a mixture of proteins, these separations were then compared with a conventional protein phase under the same conditions. A second functionalised monolithic polystyrene-based column was also manufactured and compared for the analysis of proteins, under the same analytical conditions, to the standard non-functionalised bare polystyrene monolith. Nano-LC polystyrene monolithic columns were found to be advantageous to conventional phases for the analysis of protein molecules, with a one-step fabrication process, faster analysis times, lower limits of detection hence higher sensitivity. © 2004 Elsevier B.V. All rights reserved. Keywords: Monolithic columns; Stationary phases, LC; Poly(styrene–divinylbenzene); Proteins 1. Introduction Monolithic columns have been an area of extensive re- search within the past 5 years. These columns have many advantages, they are easy to manufacture, the monolith be- ing formed in situ, often via a one-step reaction process, and its properties such as porosity, surface area and functionality can be tailored. Many applications have been investigated including solid-phase extraction, sample pre-concentration, and separation analysis in fields such as pharmaceuti- cal, environmental, and more extensively, biomolecules [1–4]. In the last 3 years, the group of Premstaller et al. [5,6] have made big advances for the chromatographic separa- tion of biomolecules such as proteins and nucleic acids. The group synthesised a styrene-based monolith that enabled the highly efficient separation of biomolecules by reversed- phase micro-high-performance liquid chromatography (- RP-HPLC). Their key discovery was the use of a mixture of tetrahydrofuran and decanol as porogens for the fabrica- tion of a micropellicular poly(styrene–divinylbenzene) (PS– DVB) backbone. ∗ Corresponding author. E-mail address: c.legidoquigley@ic.ac.uk (C. Legido-Quigley). The poly(styrene–divinylbenzene) polymer is prepared by free radical cross-linking copolymerisation of styrene and di- vinylbenzene monomers in the presence of a diluent, which can be a solvent, a non-solvent or a linear polymer, and is the pore forming agent as shown in Fig. 1. In a solvating diluent, macroporous polymers are only produced when the divinylbenzene concentration is high, and the monomer con- centration is diluted. However, when non-solvating diluents are used, the macroporosity appears at lower concentrations of divinylbenzene and with less diluted monomers. Recently, a 200 m i.d. column manufactured from PS–DVB has been made available in the market for the analysis of peptides and proteins. Following this work, we have studied three new aspects of the styrene monolith. Our first study focused on the suc- cessful fabrication of columns with internal diameters of 100 and 50 m i.d. This is done due to the increase in the sensitivity of the analysis achieved as the column internal diameter decreases. In 1995, Ryan proposed a model to adapt normal flow in chromatography to micro-flow chromatography [7]. The theory states that when going from a column of diameter X to a smaller diameter Y the gain in sensitivity would be equivalent to the partition of the squares of the internal diameters [X 2 /Y 2 ]. Thus, in practical terms, when adapting a method from a 2.1 to a 0.5 mm i.d. column there would 0021-9673/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2004.01.003