© 2016 Nature America, Inc. All rights reserved. PROTOCOL NATURE PROTOCOLS | VOL.11 NO.2 | 2016 | 377 INTRODUCTION VOCs are a class of highly toxic, persistent and ubiquitous environmental contaminants 1 that pose considerable risks to aquatic ecosystems and human health 2 . They are typically present at low concentrations and mixed with other contaminants in aquatic environments, thus rendering their sensitive, rapid and quantitative detection difficult. Currently, chromatographic or spectroscopic detection methods are commonly used for VOC detection. Although these analytical techniques are certainly sensitive and accurate, these methods require complex and time-consuming preconcentration and extraction steps for the analysis of complex environmental samples 3 , and they are usually confined to laboratory use. For in situ monitoring of VOCs in aqueous environments and timely control and man- agement of associated risks, rapid and simple in situ detection protocols are in demand 4 . We have recently developed a versatile analytical platform based on fiber optic IR-ATR spectroscopy (Fig. 1), which is readily adaptable to the detection of a wide variety of environmental contaminants ranging from small organic constituents 5,6 to bio(macro)molecules 7 . Development of the protocol IR-ATR spectroscopy and sensing provides an innovative solution to VOC monitoring, and it has been increasingly used for in situ detection of various organic contaminants in aqueous environments 8–11 , usually at detection limits approaching p.p.m. levels and without the need for sample pretreatment. However, in some environments such as drinking water, VOCs are usually occurring at p.p.b. concentration levels. At such low concen- trations, the background signals of water and other molecules are signficant, thus making conventional IR-ATR spectroscopic detection difficult. To address this challenge, our collaborative research team has recently developed an advanced ATR waveguide sensing tech- nology 10,11 . The detection sensitivity of IR-ATR using Fourier transform IR (FTIR) spectrometers depends critically on the waveguide performance. Unlike previously reported IR-ATR waveguides, which are in either planar 12,13 or cylindrical 14 con- figurations, a planar fiber waveguide with cylindrical extensions at both ends was used in our study. Next to the analyte concentration, the fidelity and quality of the finally obtained analytical signal is affected by several factors, inclduing the waveguide material and structure, the light source and the type of detector. In particular, the waveguide geometry markedly affects the number of internal reflections and hence the signal intensity during evanescent field absorption studies (Fig. 2). Here, the unique geometric structure (Fig. 2b,e) enables more internal reflections of the IR light and more efficient light coupling into the waveguide, and hence it sub- stantially enhances the analytical signal and the achievable signal- to-noise ratio 15,16 . Moreover, the waveguide surface is coated with a layer of hydrophobic ethylene/propylene (60/40) co-polymer (Fig. 2c,f) to enrich VOCs and to reject water molecules (Fig. 2d), thereby further enhancing the spectral signatures of VOCs while reducing the background absorbance of water 17,18 . Hence, the combination of these strategies leads to improved detection sen- sitivity for VOCs in aqueous matrices with IR chemical sensors. Comparison with other methods This protocol outlines distinct advantages for VOC detection versus conventional methods. For conventional chromatogra- phy techniques, such as high-performance liquid chromatog- rahphy 19 , gas chromatography 20 and gas chromatography–mass spectrometry (GC-MS) 21 , off-line sample pretreatment and pre- concentration steps are usually required. Other methods such as High-sensitivity infrared attenuated total reflectance sensors for in situ multicomponent detection of volatile organic compounds in water Rui Lu 1,2,6 , Wen-Wei Li 1,6 , Boris Mizaikoff 3 , Abraham Katzir 4 , Yosef Raichlin 5 , Guo-Ping Sheng 1 & Han-Qing Yu 1 1 Chinese Academy of Sciences (CAS) Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei, China. 2 Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, China. 3 Institute of Analytical and Bioanalytical Chemistry, University of Ulm, Ulm, Germany. 4 School of Physics, Tel Aviv University, Tel Aviv, Israel. 5 Department of Applied Physics, Ariel University Center of Samaria, Ariel, Israel. 6 These authors contributed equally to this work. Correspondence should be addressed to B.M. (boris.mizaikoff@uni-ulm.de) or H-Q.Y. (hqyu@ustc.edu.cn). Published online 28 January 2016; doi:10.1038/nprot.2016.013 In situ detection of volatile organic compounds (VOCs) in aqueous environments is imperative for ensuring the quality and safety of water supplies, yet it remains a challenging analytical task. We present a high-sensitivity method for in situ analysis of multicomponent VOCs at low concentrations based on the use of infrared attenuated total reflection (IR-ATR) spectroscopy. This protocol uses a unique ATR waveguide, which comprises a planar silver halide (AgCl x Br 1-x ) fiber with cylindrical extensions at both ends to increase the number of internal reflections, and a polymer coating that traps VOCs and excludes water molecules. Depending on the type of VOC and measurement scenario, IR spectra with specific frequency windows, scan times and spectral resolutions are obtained, from which concentration information is derived. This protocol allows simultaneous detection of multiple VOCs at concentrations around 10 p.p.b., and it enables accurate quantification via a single measurement within 5 min without the need for sample collection or sample pretreatment. This IR-ATR sensor technology will be useful for other applications; we have included a procedure for the analysis of protein conformation changes in Supplementary Methods as an example.