A uthor: Jiawei Zhang Supervisors: Dr. Barbara Beckingham; Prof. Dr. Peter Grathwohl

1. Master thesis Visualization of the nile red distribution in polyoxymethylene and polyethylene during sorption processes Author: Jiawei Zhang Supervisors: Dr. Barbara Beckingham; Prof. Dr. Peter Grathwohl Center for Applied Geosciences; University of Tübingen, Germany Introduction Objective: Visualization of the cross-sectional polyoxymethylyne (POM) and polyethylene (PE) thin films during the absorption process of nile red as a fluorescent tracer in methanol solution as well as desorption of nile red from POM and PE in infinite water bath over time using Confocal Laser Scanning Microscopy (CLSM). Aims of the project are: 1. To prove whether nile red distributes evenly within POM and PE when they reach sorption equilibrium; 2. To determine first order rate constants modeled by fitting the nile red intensity during desorption and uptake. A strategy for using passive samplers under non-equilibrium conditions is to incorporate performance reference compounds (PRCs) into the sampler material and measure the loss, applying the assumption that the PRCs are lost from the sampler at the same rate as the uptake of environmental analytes. PRCs may appear to be released at a slower rate than analytes from solution are being taken up, known as hysteresis, if there is a physical change or deformation to the polymer phase resulting from the loading process or if the PRCs are not initially homogeneously distributed within the sampler (Kleineidam et al. 2004, Bouchard, 2003). Settings:Confocal microscope model LeicaDM500Q with 40x dry objective lens, 488 nm laser excitation, and analysis by LAS AF Lite and FIJI Image J softwares. XYZ stacks using reflectance (λemission: 488 nm) and fluorescence (λemission: 580-620 nm for POM, 520-590 nm for PE) channels. Materials: POM (76+/-5μm) and PE (50 μm+/-20%) obtained from CS Hyde (USA) and Goodfellow Plastics (UK), respectively. One side of POM was polished during the manufacture. Nile red Cw,sat = 0.18 μg/ml (EpiSuite) and Kpom=3.8 (Endo et al. (2011)) and Kpe=5.6 (Lohmann (2012)). Razor blades (EB 30-0303 Lutz-22) from LUTZ GmbH & CO.KG for cutting samples. Sample preparation and data analysis: Samples cross-section imaged by cutting with razor blades while sampler secured in a specialzed clamp-stand. Fluorescence was analyzed as a box area-average over the entire cross section. Samples were shaken on a horizontal platform shaker during desorption and uptake processes. All the fluorescence and reflectance images and data were obtained at depth of 8.34 μm from the sampler surface. Desorption: Nile red was loaded into samplers for either 35 d or 7mo from a 10mg/l nile red solution in 80:20 methanol/water. Time=0 taken after samples removed from loading solution; placed in deionized water with fine activated carbon and desorption imaged over time. Uptake: Imaged samples over time while loading in a nile red solution (8mg/l in 100% methanol) until equilibrium was reached. Experimental set-up Results Figure 3: PE desorption Figure 2: POM desorption Confocal images polished side Nile red is a hydrophobic probe. Fluorescence depends on its environment. More nile red is accumulated where the red color is brighter. Cross-sectional profiles-Figures 2, 3, 4, 5: Nile red is lost from PE within 24 h (Fig. 3), but fluorescence declines by only 53% in POM after 108 d (Fig. 2). Nile red is removed from POM by hexane:acetone (1:1,vol) after 36 h (e.g. not irreversible). Cross-sectional profiles at later times show characteristic desorption gradients(Fig. 2 and Fig. 3). No extreme accumulation of nile red at edges is observed in PE (Fig. 3) compared to POM at 0 h (Fig. 2), despite more uneven surfaces from cuts in PE. Not inculding edges, 30% more nile red accumulated at unpolished side at 0h (Fig. 2) but not at 15 d at uptake when nile red was fully loaded in POM (Fig. 4). Figure 5: PE uptake Figure 4: POM uptake polished side Figure 6 Figure 7 The nile red intensity during sorption vs. time-Figure 8, 9,10: Negligible loss of nile red from POM in early times (Fig. 6). POM and PE nile red uptake first order rate constants are 0.010 h-1(Fig. 7) and 0.294 h-1(at early times until 12 hours) (Fig.8). Rate constant of desorption in PE was 0.245 h-1(Fig. 8). Figure 7: POM Conclusions • POM: • Extremely high nile red accumulation found at edges in POM. • Hyp: More ordered crystalline structure in interior of POM than at the surface layers. (Plummer et al.1995). • 30% higher nile red at unpolished side during desorption process. • Hyp: Slightly more crystalline at unpolished side. • Faster initial sorption in PE than in POM. • Higher crystallinity in POM than in PE (H.P.T, 2006). • Methanol can enhance amorphous regions in POM. When the • methanol left from POM during the desorption process, the enhanced amorphous region was no longer available as a pathway for nile red to diffuse out of POM (Fujii et al. 2007) • PE: • Homogeneous nile red distribution over the entire cross section. Figure 8 Figure 9 Figure 10 Figure 9 and 10: One part of the whole cross-section of POM (Fig. 9) and PE (Fig. 10) fully loaded with nile red imaged with ‘auto focus’ shows homogenous nile red distribution in PE but not in POM. In POM, there is more nile red at edges than interior and variable intensity at edges over the length of the sampler. References Kleineidam, S., Rügner, H and Grathwohl, P.G (2004) Desorption kinetics of phenanthrene in aquifer material lacks hysteresis. Environ. Sci. Technol. 38:4169 Bouchard, D.C. (2003) Cosolvent effects of phenanthrene sorption-desorption on a freshwater sediment. Environ. Toxicol. Chem. 22:736. Plummer, C. J. G.; Kausch, H. H (1995) Real-time image analysis and numerical simulation of isothermal spherulite nucleation and growth in polyoxymethylene. Colloid & Polymer Science, 1995, Vol. 273, No. 8. Harper, C.A. edit-in-chef, Handbook of plastics technologies, the complete guide to properties and performances, McGraw-Hill Company(2006) Fujii, Y.; Shen, H. 2007. Relationship between fine structure of polyoxymethylene copolymer plates and methanol transport properties. Journal of Polymer Science: Part B: Polymer Physics, 2007, Vol. 45, 1234–1242. Contact Jiawei Zhang Universität Tübingen · School of Mathematics and Natural Sciences Center for Applied Geoscience jiawei_zhang@gmx.de