Vibrational Spectroscopy 66 (2013) 14–18 Contents lists available at SciVerse ScienceDirect Vibrational Spectroscopy j ourna l h om epa ge : www.elsevier.com/locate/vibspec Distribution of chlorosulfonyl groups in the subsurface of polystyrene substrates. Analysis by means of vibrational spectroscopy Anselmo del Prado a , Nerea Briz b , Rodrigo Navarro a , Mónica Pérez a , Alberto Gallardo a , Helmut Reinecke a,∗ a Institute of Polymer Science and Technology (ICTP-CSIC), Juan de la Cierva 3, E-28006 MADRID, Spain b Tecnalia, Mikeletegi Pasealekua, 2, Parque Tecnológico, E-20009 San Sebastián, Spain a r t i c l e i n f o Article history: Received 27 July 2012 Received in revised form 12 November 2012 Accepted 16 January 2013 Available online 31 January 2013 Keywords: Surface functionalization Polystyrene Confocal Raman microscopy FTIR-ATR a b s t r a c t In this work the activation of transparent PS substrates by chlorosulfonation is described and their distribution in the subsurface region is analyzed using vibrational spectroscopies. Confocal Raman microspectroscopy is shown to be insufficiently surface selective and only the highest modified sam- ples can be analyzed using a mathematical procedure for the correction of diffraction effects on the depth profile. On the other hand, FTIR-ATR spectroscopy carried out using different internal reflection elements and varying angles of incidence allows discrimination between the different modification pro- files including those with low modification degrees obtained at low treatment times. The results show that the electrophilic aromatic substitution of polystyrene in pure chlorosulfonic acid is extremely quick with the complete surface covered by chlorosulfonic groups after only 10 min reaction time at -10 ◦ C. It is further demonstrated that the reaction is very surface selective and that even after reaction times as long as 3 h the modification is limited to a layer with a thickness of less than one m. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Polystyrene is one of the most used polymer materials in the bioanalytical sector because it has excellent optical clarity, is easy to mold and relatively inexpensive. Slides and multiple-well plates from this material have gained widespread acceptance in part because pipetting, washing, and signal detection are easily auto- mated [1–3]. It has recently been shown [4,5] that PS substrates can be activated by a simple and economic wet-chemical treatment in chlorosulfonic acid at low temperatures providing the surface selectively with a tunable number of chlorosulfonic groups that can be used to create or anchor in a second step selectively a great variety of functional groups or biomolecules to the surface without losing its transparency. The pre-activated chlorosulfonated sub- strates are hydrolytically stable in ambient conditions and can be stored for months without losing their activity. The approach has been shown to be applicable to commercial multiple-well plates from PS that could be modified selectively with amino or carboxylic groups. These systems have successfully been probed for ELISA (Enzyme-linked Immuno Sorbent Assays) as cheap alternative to commercial materials. ∗ Corresponding author. Tel.: +34 91 5622900; fax: +34 91 5644853. E-mail address: hreinecke@ictp.csic.es (H. Reinecke). In the present work we have carried out a detailed study of the chlorosulfonation reaction of transparent PS surfaces. Our partic- ular aim here was to gather information concerning the number and distribution of the created functional groups on the air-film interface and in the subsurface region. For this purpose we used on the one hand a Raman microscope in the confocal mode [6–9] and on the other hand an FTIR spectrometer equipped with an ATR device that allowed changing the angle of incidence and the total reflection element. 2. Materials and methods 2.1. Materials Transparent Polystyrene sheets of 1 mm thickness and a surface of 8.0 cm × 2.5 cm were purchased from Resopal S.A., Madrid, Spain. Chlorosulfonic acid and concentrated sulphuric acid from Sigma–Aldrich were used. Chlorosulphonic acid was distilled under reduced pressure prior to use. Sodium p-vinylphenylsulfonate was purchased from Sigma–Aldrich and used without further purifica- tion. 2.2. Preparative aspects In a thermostatable reactor rectangular transparent PS samples (dimensions 2.5 cm × 8.0 cm × 0.1 cm) are brought in contact with 0924-2031/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vibspec.2013.01.006