Gold nanoparticles incorporated into cryogel walls for efficient nitrophenol conversion

Journal Pre-proof Gold nanoparticles incorporated into cryogel walls for efficient nitrophenol conversion Berillo Dmitriy PII:

S0959-6526(19)33959-9

DOI:

https://doi.org/10.1016/j.jclepro.2019.119089

Reference:

JCLP 119089

To appear in:

Journal of Cleaner Production

Received Date: 27 June 2019 Revised Date:

10 October 2019

Accepted Date: 28 October 2019

Please cite this article as: Dmitriy B, Gold nanoparticles incorporated into cryogel walls for efficient nitrophenol conversion, Journal of Cleaner Production (2019), doi: https://doi.org/10.1016/ j.jclepro.2019.119089. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical abstract

Gold nanoparticles incorporated into cryogel walls for efficient nitrophenol conversion Berillo Dmitriy1,2,3* 1

School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK

2

Department of Biotechnology, Center for chemistry and chemical engineering, Lund

University, P.O. Box 124, 22 100, Lund, Sweden. 3

Water Technology Center (WATEC), Department of Bioscience - Microbiology, Aarhus

University, Denmark *Corresponding author: [email protected]

Abstract The past several decades have illustrated an enormous interest in noble metal nanoparticles for their superior catalytic activity, however, their industrial use is very restricted due to inefficient recovery leading to potential contamination of products and the environment. Immobilised nanoparticles illustrate promising results for scaling up processes, and can be successfully applied for various catalytic processes including waste industrial water treatment. The aim of the study was to design and study an easy and economically efficient green chemistry approach of preparation of macroporous material, with well distributed gold nanoparticles within the wall of cryogel. Ionic cryogels containingchitosan and tetrachloroaurate complex was reduced by 1.1.3.3-tetramethoxypropane, concurrently allowing chemical cross-linking of the polymer. A mechanism of reduction of noble gold complexes within the cryogel walls by tetramethoxypropane is also studied. Properties of the composite cryogels were evaluated using; differential scanning calorimetry, FT-IR, NMR, compression tests, SEM, TEM), Energy-dispersive X-ray spectroscopy and low temperature nitrogen adsorption. The catalytic activity of the in-situ synthesised gold nanoparticles was comprehensively studied using a model reduction reaction of 4-nitrophenol. Four different

concentrations of gold nanoparticles within the cryogel structure were investigated. The turnover number and the turnover frequency parameters for cryogels containing four different concentrations of gold nanoparticles were calculated. The conversion degree of 4nitrophenol to 4-aminophenol for cryogel containing the lowest of gold nanoparticles reached 96.8 % at room temperature. The catalytic activity of incorporated gold nanoparticles for 4-nitrophenol conversion did not decline over 14 consequent testing cycles.

Keywords: gold nanoparticles, catalysis, cryogel, tetramethoxypropane, nitrophenol. Research highlights: Characterisation of composite macroporous material with nanoparticles Synthesis of spherical gold nanoparticles with fine distribution in volume Tetramethoxy propane as a reducing agent for gold complex Catalytic reduction of nitrophenol by supported gold nanoparticles in a dynamic mode

1. Introduction A considerable release of various pollutants into aquatoria has resulted in serious contamination of the environment owing to the fast industry development in developing countries(Shoueir, Kandil et al. 2019). From the other side, persistent organic pollutants (dyes, antimicrobial detergents etc.) and pharmaceutical derivatives from hospitals mixed with urban waste water, which makes the bioremediation treatment process less efficient and sometimes demand the use of combination with various catalytic processes(Fenton reaction, TiO2 and UV irradiation etc.)(Gaur, Narasimhulu et al. 2018, Berillo, Caplin et al. 2019). Once discharged to the surrounding media, all these artificial compounds in nano and picomolar concentrations creates serious threats to humanity and overall aquatoria. Heterogeneous catalysis is a promising method for controlled water remediation due to large

scale production, accessibility, physicochemical stability and reusability. However, the cost significantly depends on the support of the catalyst(Guibal 2005, Primo and Quignard 2010, Gaur, Narasimhulu et al. 2018, Teimouri, Khosravi-Nejad et al. 2018, Liu, Liu et al. 2019, Shoueir, Kandil et al. 2019). Chitosan (CHI) is a polymer that can be obtained from renewable sources via the deacetylation of the natural polymer chitin containing subunits of D-glucosamine with N-acetyl-D-glucosamine. Chitin is the second most widely spread natural polymer next only to cellulose. The CHI can be used for preparation of materials with various texture and structure such as nano/micro particles, films, hydrogels(Li, Jiang et al. 2017, Shoueir, Kandil et al. 2019, Sultankulov, Berillo et al. 2019) and macroporous hydrogels or so called cryogels,(Capitani, De Angelis et al. 2001, Abbasi 2017, Berillo and Cundy 2018) as well as fabrication of biodegradable packaging and disposable table ware (Leceta, Guerrero et al. 2013). For the past 30-40 years the cost of production of CHI significantly decreased down to 10 USD has been a main driving force of movement from fundamental to applied studies of CHI based materials,(Gkika, Liakos et al. 2019) the CHI based materials have been extensively studied and applied in various fields such as water purification from metals and dyes, regenerative medicine(Shukla, Mishra et al. 2013, Sultankulov, Berillo et al. 2019), biosensors(Di Carlo, Curulli et al. 2012) and catalysis(Qu, Wirsén et al. 1999, Varma, Deshpande et al. 2004, Guibal 2005, Sogias, Williams et al. 2008, Ngah, Teong et al. 2011, Elsabee and Abdou 2013, Berillo, Mattiasson et al. 2014), due to combination of such properties as stability to degradation, easy production and polycationic structure, and antimicrobial activity. Cryogels are macroporous materials with a unique structure of interconnected open pores providing high water and gas permeability they also possess a large surface area accecible for various reactions. Usual cryogels are prepared via physical or chemical

cross-linking

of

polymers

or

polymerization

reaction

at

subzero

temperatures(Gun’ko, Savina et al. 2017, Berillo, Caplin et al. 2019). It was shown that cryogels can be prepared in a variety of shapes and configurations. The cryogel production process may be scalable up to 0.4L of each unit, these much larger scaffold/sample volumes gives a high potential for practical engineering/environmental applications(Savina, Ingavle et al. 2016). Gold nanoparticles (GNP) can be formed either by heating gold salt in CHI solution, or by light irradiation (Hortigüela, Aranaz et al. 2010, Seoudi and Said 2011, Boufi, Vilar et al. 2013, Berillo, Mattiasson et al. 2014, Teimouri, Khosravi-Nejad et al. 2018) . The free GNP suspension is limited in various catalytic processes, because of their aggregation over time which causes further decline of catalytic activity. Another drawback is the necessity of the catalyst separation at the end of the process. These side effects can be overcome by application of a supported catalyst. One of such materials is CHI, which has been extensively studied as a stabiliser for GNP preparation(Adlim and Bakar 2010, Hortigüela, Aranaz et al. 2010, Berillo, Mattiasson et al. 2014). A commonly used method in catalysis preparation is the immobilisation or adsorption of noble metal complexes or nanoparticles(NPs) on a mesoor macroporous support such as an activated carbon, alumina, titania, silica or iron oxide followed by reduction process with sodium borohydride(Teimouri, Khosravi-Nejad et al. 2018). However, there is a necessity for the separation of NPs after the catalytic process. Sodium borohydride is highly flammable, which restricts scaling up of this process. There are a few simple methods that are available for noble metal NP immobilisation on macroporous matrices for catalytic applications without borohydride(Teimouri, Khosravi-Nejad et al. 2018). It has been reported that different organic acids can affect the size distribution of the CHI stabilised GNP, and the containing electroconductive films applied in biosensors (Di Carlo, Curulli et al. 2012). Shen et al. demonstrated the possibility of ligand exchange in a mixture

of tetrachloroaurate and alkylamines with following formation of GNP (Shen, Du et al. 2006). GNP were incorporated into CHI hydrogels and microparticles, prepared at 30° C with incubation for 24h at different conditions and CHI concentrations. These materials were characterized by various methods and applied for drug delivery or catalysis, such as a Sonogashira cross-coupling reaction, allowing to synthesize complex organic compounds in a straightforward way with a high yield (Primo and Quignard 2010, Ramasamy and Maliyekkal 2014). Previously, scaffold preparation required a two-step method based on the CHI via the incubation with the gold complex at 80oC followed by freeze drying. The catalytic activity of GNP of these scaffolds were studied using classic 4-nitrophenol(4NP) reduction reaction(Hortigüela, Aranaz et al. 2010). Recently, a two-step preparation of composite cryogel based on p(DMAEM-co-MAAc) and GNPs with large average size distribution 431±250nm, different shapes and inhomogeneously distributed GNPs within the volume of cryogel was applied for 4-NP reduction(Tatykhanova, Klivenko et al. 2016). Aforementioned drawbacks can be overcome by the use of a one-step preparation of CHI cryogels with in situ formed GNP possessing narrow average size distribution(8-10nm) utilised as a monolith reactor for oxidation of aromatic aldehyde at room temperature (Berillo, Mattiasson et al. 2014). This study illustrates the use of cryogel with in-situ formed GNP and its exploitation as a catalytic flow through monolith. Such systems can be successfully applied for various heterogeneous catalytic processes in the liquid phase as well as the gaseous phase. Ionic cryogel(IC) containing gold complex was prepared in one step in an apparently frozen state. I hypothesize that the treatment of the ionic cryogel (IC) with 1,1,3,3-tetramethoxypropane (TMP) will lead to one step preparation of covalently cross-linked cryogel with in-situ formed GNP. TMP is used as a reducing and stabilising agent during GNP formation within

the cryogel structure and a mechanism is proposed. Using 4-nitrophenol(4-NP) reduction, the catalytic activity of in situ synthesized GNP within a monolithic flow-through reactor is reported. Narrow size distribution and homogeneous population of GNPs within the supporting microporous material is explored. The advantage of this heterogeneous catalytic system is the reusability of catalyst without loss of GNP activity and the absence of inconvenient separation step of catalyst from substrate(avoiding organic solvent use), this highlights a possibility of use in a flow through mode. Various catalytic parameters and conditions of reaction are considered. Such type of catalyst has potential to be used for practical wastewater treatment and other catalytic processes within mild conditions. 2. Materials and Methods. Medium viscosity chitosan with 85% degree of acetylation was from (Aldrich, Steinheim, Germany), sodium borohydride 98% and 4-nitrophenol (Fluka, Steinheim, Germany), sodium acetate trihydrate and 25 % glutaraldehyde(Merck, Germany),1,1,3,3,-tetramethoxypropane (TMP) 96% and tetrachloroaurate HAuCl4 x nH2O 1.45M (Sigma-Aldrich). Epoxy propane and Taab 812 resin were obtained from (TAAB, Berks, UK). 2.1 Preparation of ionic cryogels and cryogels with in-situ formed GNP Ionic cryogels were prepared as previously described (Berillo, Mattiasson et al. 2014). Briefly, to 1.1 (% w/v) CHI in acetic acid (0.5v/v %) a certain amount of concentrated stock solution of H[AuCl4] (3.5, 7.0, 14 and 28µL) was added and vigorously mixed to obtain final gold concentrations of 0.5, 1.0, 2.0 and 4.0mM. It was transferred into a glass tubes(7 mm diameter ) to freeze at -12 ºC in liquid cryostat for 18 h. The synthesized IC samples containing 98.5, 196.9, 393.9 and 787.8 ppm of gold were thawed at room temperature (RT) and placed into the solution of TMP (5%) in phosphate buffer(PB) and incubated at RT for 24 h. These cryogels of CHI–TMP–GNP contained 98.5 ppm (CHI-TMP-GNP-1), 196.9 ppm

(CHI–TMP–GNP–2), 393.9 ppm (CHI–TMP–GNP–3) and 787.8 ppm (CHI–TMP–GNP–4) gold were washed with distilled water and kept at 4 °C 2.2 Characterization of composite cryogels. After washing with water, cryogels CHI-TMP and CHI-TMP-GNPs were frozen and lyophilised and then utilised for ATR-FTIR using a Burker-ALPHA-P system. CHI solution in acetic acid was frozen and freeze dried as a control FTIR spectrum. Spectra were acquired in the range of 4000-400 cm-1 with a resolution of 4 cm-1 over 24 scans. The content of free gold complex in solution after cryogel preparation was evaluated using inductively coupled plasma mass spectrometry (ICP-MS [Agilent 7900]). Two samples of cryogels with total volume of 1 mL (787.8 ppm Au) were washed with 20 mL of distilled water in dark conditions overnight (as light leads to change of chemical composition and phisico-chemical properties of material(Berillo, Mattiasson et al. 2014), then the solution was filtered through a 0.22 µm membrane. The Au complex content was analysed using ICP-MS. To avoid the contamination, 1 v/v % nitric acid was purged between different sample analyses. Ionic chromatography (Thermo Scientific Dionex ICS-1100, Thermo Scientific, Sunnyvale, CA, USA) was used to estimate the concentration of chloride ions due to the ligand exchange reaction for 25 min (injection volume of 5 mL). Colunm (Dionex IonPac TM AS23 for anions, RFIC TM , Analytical 4µm x 250 mm), elution buffer [4.5 mM Na2CO3 / 0.8 mM NaHCO3], flow rate [1 mL/min at 30 °C]. Regeneration settings: Dionex Anion selfregenereting suppressor, Dionex ASRS TM 300 4 mm Auto suppression TM recycle mode. Retention time of ions were acetic acid 5.7, fluoride 3.957, chloride 6.521, bromide 10.364 minutes, respectively. In order to preserve the initial structure of the cryogel for scanning electron microscopy, samples were freeze dried and cut thin discs of 1-2 mm thickness manually by hand and

sputtered with gold/palladium in proportion of 40:60) in automatic mode. The sputtered samples were examined by a SEM analysis. Transmission electron microscopy (JEOL JEM1400-Plus Transmission Electron Microscope operated at 120 kV equipped with a Gatan OneView camera (4k × 4k) (Jeol, Tokyo, Japan)) was used to analyse GNP within the cryogel wall. Samples were embedded in Taab 812 resin. Embedding was gradual, using two 5min rinses in epoxy propane and then amounts of Taab 812 in epoxy propane (33%, 50%, 100%) was increased every hour. Blocks were polymerized in an oven at 60°C for 24 h. Then a thin slice (0.12-0.2 µm) of the cryogel was cut by Reichert Ultracut E microtome(Reichert-Jung, Vienna, Austria). The slice was placed on a Quantifoil holey carbon copper grids (Quantifoil Micro Tools GmbH, Cu 200 R2/1, (Agar Scientific, Stansted, Essex, UK) and the specimen was incubated at RT overnight. The prepared slices were examined using TEM at magnifications of x5,000, x30,000 and 50,000). Note, the cryogel with incorporated GNP were significantly more stable to the electron beam irradiation compare to epoxyresin (0.12 µm thickness). A range of sections (0.08, 0.12, 0.15 and 0.18 µm) were prepared and the most stable, 0.18 µm, used for the following investigation. Different areas of the cryogel microphotographs were analysed by FIJI software (Schindelin et al., 2012) to estimate the projected areas on the TEM grids. The area of the various GNP shapes was used for a dependence calculation of the frequency (number) of particles vs GNP size. Control samples contained 5 µL of GNP suspension, in which particles were stabilized by TMP, placed on the copper grid and dried in air for 10-15 min. H1- and C13- Nuclear Magnetic Resonanse (NMR) spectra of 20 mg/mL GA or TMP dissolved in D2O were measured by NMR 400 MHz (Bruker). Five uL of H[AuCl4] stock solution (1g/L) dissolved in 0.5 mL of D2O and ten uL of GA with the concentration of 20 mg/mL or TMP (99.8%) mixed with D2O. The H1-NMR spectra of these samples were recorded. The addition of H[AuCl4] to TMP resulted in rapid colour shift from a transparent

to yellow. Five mg of sodium bicarbonate was added to the reaction mixture of H[AuCl4] and GA or TMP with D2O, then incubated at RT for 10-20 min. Emission of carbon dioxide and precipitate formation attributed to GNP was observed, which was removed by centrifugation. The remaining clear solution was used for H1-NMR spectra analysis. 2.3 Catalytic activity of chitosan-based cryogels containing GNP The catalytic activity of CHI-TMP containing different quantities of GNP (49, 98, 196 and 392 ug) was studied in batch mode at RT. A cryogel with a volume of 0.5 mL was placed into 7 mL of 0.05 M carbonate buffer at pH 10, and 0.5 mL of 0.05 M stock solution (4nitrophenol [4-NP] in ethanol) was added. Then, 0.15 mL of 0.2 M freshly prepared NaBH4 in carbonate buffer was added under gentle magnetic stirring with experimental set up shown on the Fig. A13. Reaction mixtures containing 4NP (3.5-65) mM and NaBH4 solution (4.0 mM) were continuously recirculated through a flow quartz cuvette(Quartz Flowing Cuvette, Type D 10 mm light path, 2 transparent windows) : Z(center height)=8.5 mm, Aperture dimension= 2 mm, Volume=30 uL. Tabulation dimensions: OD=3.2 mm, ID=1.8 mm) at a flow rate of 0.5 mL/min and absorbance was measured every minute at 400 nm using a UVvis spectrophotometer (Biowave II, Biochrom Cambridge, UK) in automatic mode. For testing of the next cycle of catalytic activity, the cryogel with GNP(49, 98, 196 and 394 µg) without preliminary washing was immersed into a freshly prepared solution of 4NP (20, 25, 50, 60, 70 mM) containing borohydride(4.0-20 mM). Cryogels were kept in carbonate buffer solution at 4oC overnight between experiments. Every catalytic cycle of reduction of 4-NP proceeded for 2 h. The reaction rate constants (k) were calculated under identical reaction conditions using plots of ln(At) vs. time t, which gives a straight line with a slope of minus. A control experiment without GNP was performed in batch mode at RT. 0.15 ml of 0.05 mM stock solution of 4NP was mixed with 6.85 mL of 0.05 M carbonate buffer at pH 10, and, then 0.15 mL of 0.1 M NaBH4 was added. The solution was continuously recirculated

through the blank cryogel CHI-TMP without GNP at a flow rate of 0.5 mL/min and connected to the flow through quartz cuvette. The absorbance measurement of 4NP were carried out at 400 nm in automatic mode every minute. The lifetime of the catalyst was expressed using the turnover number (TON), which is the number of moles of substrate to a mole of the catalyst GNP can convert before inactivation (Hagen 2015). The TON was calculated according to the (Eq.1): TON = [4NP] * [GNP]-1 * t

Eq.1

where [4NP] and [GNP] are the molar concentrations of the substrate and the catalyst, respectively, and it is the overall catalytic time. The turnover frequency (TOF) was calculated according to the equation 2: TOF = [4NP] * [conversion]*[GNP]-1 * t-1

Eq.2

3. Results and Discussion 3.1 Preparation and characterisation of ionic cryogels with gold complex A rapid mixing of H[AuCl4] with CHI solution then freezing at -12 oC leads to the formation of IC due to a ligand exchange mechanism(Boufi, Vilar et al. 2013, Berillo, Mattiasson et al. 2014). The obtained cryogel was stimuli-sensitive and can respond to ionic strength, temperature and visible light spectra. The solution was able to perform decomposition or dissolution with formation of a suspension of spherical GNP. The treatment of the IC with a cross-linking agent such as glutaraldehyde leads to the conversion into a robust macroporous material, concentrations of all components are in table A1. Cryogels with well distributed immobilised/fixed in the structure GNP could be used within a flow reaction. The advantage of the developed cryogels with in-situ synthesised GNP in comparison with free nanoparticles (NPs) is the absence of aggregation of NPs during exploitation, a catalyst and energy saving process. This means that GNP separation after catalytic reaction and a two step method for

preparation process are not necessary. Based on these properties, the developed material can find application as a catalyst for liquid phase and potentially for gaseous phases. Differential scanning calorimetry (DSC) was used to understand the physicochemical process that resulted in the ionic cross-linking of CHI under cryoconditions. For example, a glasstransition temperature (Tg), melting point (Tm) and decomposition temperature (Tdecom) of CHI with different concentrations of gold complex were evaluated. The initial CHI had an endothermic peak at 93.1 °C with a heat capacity of 232 J/g (50 °C), (Fig. A.1a) which is attributed to the evaporation of weakly bonded water. DSC data for control sample CHI is in agreement with published data. It is known that CHI is not a completely amorphous polymer(Ogura, Kanamoto et al. 1980). Tg of CHI was 159.8 °C, followed by the crystallisation of the amorphous fraction. The composition IC1 had a similar DCS profile with evaporation of water at 86 °C and heat capacity of 268 J/g (36.9 °C) (Fig. A.1a). It can be assumed that the crystallization of the amorphous part of the polymer started to occur at 149 °C,followed by decomposition/oxidation of functional groups of CHI and reduction of Au (III) to Au (0) at approximately 231 °C, this can be attributed to the oxidative properties gold as the Au (III) complex of E0[AuCl4]- / Au0 . has a high redox potential of 1.002 V. This observation was in agreement with the decomposition of various gold complexes under heat treatment of other studies (Nedoseykina, Plyusnin et al. 2010, Loseva and Ivanov 2014). The second and third heating/cooling cycles were carried out to confirm that the sample was decomposed at 250 °C in inert atmosphere during the first heating cycle. The similar thermal effect of the first cycle was not revealed for the 2nd and 3rd cycles. The colour of the sample was changed from white to black, which was related to carbonisation process due to high temperature. Similar phase transitions were observed in samples IC–3 and IC–4 (Fig. A.1c,d). However, the sample IC–2 had an endothermic peak of water evaporation at 92.7 °C with a heat capacity 361.2 J/g. Probably, the thermal event took place at a range of 173.9 – 207.6 °C

attributed to reduction reaction of Au (III) to Au (0). The following thermal event at 236.5 °C was related to the CHI decomposition that correlated with the lost weight registered by thermal gravimetric analysis (TGA). As expected, Tgs for the IC1–C4 were not observed due to cross-linked structure of CHI. One can observe that the increase of concentration of tetrachloroauric acid in the IC from 1 to 5mM led to the proportional increase of the heat capacity. This can be related to the larger amount of protonated aminogroups due to generated hydrochloric acid by ligand exchange process(Berillo, Mattiasson et al. 2014). Higher number of protonated groups resulted in an increased amount of solvated water. The increase of the content of the gold complex resulted in decreasing the decomposition temperature (Table A.1). 3.2 Synthesis and characterisation of composite cryogels with in-situ formed GNP The advantage of usage of in-situ synthesised GNP within the polymer architecture is the resistance to aggregation during storage (for several years) and the repeated catalytic reaction cycles. In-situ GNP formation within the walls of the cryogel was observed by the colour change from yellowish to red (Fig. 1). GNP had mostly spherical morphology that was also confirmed by TEM analysis GNP (Fig. 4). The control cryogel CHI without gold complex changed colour upon TMP treatment from transparent to yellowish. As expected, the increased GNP concentration led to a higher intensity of red (Fig. 1). The linkage of gold complex from the IC cryogel during the reduction process was monitored by ICP-MS indicating a negligible concentration of gold (0.696 µg/L; 3.55 nM/L). The washing of the ionic cryogel after preparation in 25 mM carbonate buffer led to 11% initial concentration, i.e. Au complex binding to CHI yielded in 89%, this was confirmed by ICP-MS analysis. The washing solution after IC preparation (787.8 ppm Au) indicated some increase of chloride ion concentration in comparison with initial solution, as analysed by ionic chromatography.

Fig. 1. Photographs of cryogels of from left to right: CHI–TMP, CHI–TMP–GNP–1, CHI– TMP–GNP–2, CHI–TMP–GNP–3, CHI–TMP–GNP–4 cryogels swollen in water at RT.

Previously, we have shown the reduction of noble metal complexes with CHI using glutaraldehyde (GA)(Berillo and Cundy 2018), however the control experiment with pure GA was not conducted. It is known, that GA has a tendency to aldol condensations or formation of hemiacetal over storage time(Tashima, Imai et al. 1991) confirmed by the presence of additional signals in 1H-NMR spectrum of GA at 4.8, 4.99 and 5.26 ppm (Fig. A.3b). Therefore, it might contain the mixture of oligomers at the same time. Aldehyde groups are sensitive to oxidation by oxygen of air leading to glutaric acid formation or partly oxidised oligomers of glutaraldehyde. The ratio of integrals for monomeric and polymeric form of GA at 2.5 and 4.8 ppm led to 68.65 % of polymeric form (Fig. A.3b). These mixtures of compounds for polymer cross-linking may activate the inconsistency of cross-linked GA material and poor reproducibility of results from batch to batch. Therefore, GA is not an ideal option to get a highly reproducible result. To overcome this drawback, the use of individual compound containing di- or tri-aldehyde groups is required. It is known, that TMP hydrolyses at acidic conditions tothe1.3-propanedial(Nair, O'Neil et al. 2001). An additional advantage of TMP as an individual compound compared to a mixture of oligomeric GA is its enhanced

ability to penetrate via 3D-polymeric network, due to lower molecular weight, resulting in even cross-linking of the material. Freshly formed 1.3-propanedial immediately reacted with amonogroups of CHI leading to the cross-linking forming Schiff’s base(eq. 3), which is characterised by a change of the colour from white to yellowish. Concurrently, an excess of aldehyde groups provides reduction and acts as a stabilising agent for the gold complex leading to the GNP formation. The mechanism of gold complex reduction is similar to formaldehyde and formic acid (Hou, Dante et al. 1999) and following GNP stabilisation is similar to GA(Berillo, Mattiasson et al. 2014). Briefly, the aldehyde group was oxidised to carboxyl groups reducing the gold complex to zero valent state and GNP formation(Eq 4-5 ). To support this hypothesis, the reduction of noble metal complexes and stabilising ability were performed by the cross-linking agent series of control experiments utilising pure GA and TMP. The reducing properties of GA and TMP were confirmed by 1H-NMR. Stabilised GNP were characterised by TEM and EDX analysis. The reduction of ion to zero valent state within the solution of GA in presence of gold complex at acidic conditions did not occur (Fig. A.2). The broad signal of aldehyde proton at 9.454 ppm indicated the presence of polymerised GA. A poor resolution of the

1

H-NMR spectra was related to partial

polymerisation of GA (Fig. A.2). The heating of the non-buffered solution at 60°C led to formation of microplates with gold crystals confirming a reduction reaction at acidic conditions. In order to use GA as a reducing and stabilising agent, the reaction was performed in a standard carbonate buffer solution keeping pH at the same level. The neutralised solution of GA and H[AuCl4]in carbonate buffer at standard conditions led to the change of colour of the reaction mixture from yellowish to violet. This was a marker of gold nanoparticle formation with plasmon resonance peak at 570 nm at the first step of the experiment. On the second step, the peak was

shifted to 650 nm due to partial aggregation of GNP stabilised by GA. A large fraction of GNP–GAs aggregated and precipitated overnight. GNP prepared in 5 % GA, when dissolved in phosphate buffer, had a Plasmon resonance peak at the range 546 – 558 nm. Also observed with TMP was a reduction reaction with emission of the CO2 due to formation of acid (Eq.3). (CH3O)2CH–CH2–CH(OCH3)2 + 2HN-R-NH2 + H2O => R-N=CH-CH=CH-NH-R + 4CH3OH

Eq.3

3 (CH3O)2CH–CH2–CH(OCH3)2 + 4 H[AuCl4] + 12 H2O => 4Au0 + 16Cl- + 16H+ + 12 CH3OH + 3 HOOC–CH2–COOH

Eq. 4

3(CH3O)2CH–CH2–CH(OCH3)2 + 3R–NH2 + 4H2O + 2[AuCl4]- => 4CH3OH + 2Au0 + HOOCH–CH=CH–NH–R + 8Cl-

Eq.5

Determining R –chitosan Some shift of proton signals in the range of 1.2 – 1.8 ppm was noticed in the GA–GNP 1HNMR spectrum due to formation of glutaric acid (Fig. A.3 and Eq. A.2), in agreement with 1

H-NMR predictor software. Moreover, the ratio of integrals for monomeric and polymeric

forms of GA were calculated relative to the signal at 2.5 ppm. Other peaks at 5.3 and 5.0 ppm were attributed to hemiacetal GA form with increased peaks in the next intervals: 0.96 – 1.48, and 0.50 – 0.65 ppm, (Fig. A.3 a,b) indirectly illustrating the consumption of the monomeric form of redox reaction. 1H-NMR spectrum of TMP solution in D2O had a singlet signal of aldehyde groups at 9.636 ppm and 8.44 ppm (Fig. A.5). Signals at 1.94 and 4.56 ppm corresponds to CH2CH(OCH3)2 and CH2CH(OCH3)2, respectively. The ratio of integrals for CH2CH/CH2 signals after the GNP formation reaction changed from 0.97 to 0.53 ppm. That meant that approximately 50% of TMP was still in its nonhydrolised form (Fig. A4-A5). The addition of H[AuCl4]to the TMP solution in D2O led to the colour change from transparent to yellow. However, the following addition of solid sodium hydrocarbonate resulted in CO2 emission and a colour changed to blue, which was confirmed by UV-vis spectroscopy. This is

a characteristic of a very broad GNP with Plasmon resonance peaks in the range of 500–800 nm due to GNP aggregation(Pal, Ghosh et al. 2004). As a consequence, most of GNP were precipitated due to instability. The solution was centrifuged and analysed by 1H-NMR, however additional proton signals of carboxyl groups were not observed. According to literature, various carbonic acids were utilised as stabilising agents including a well-known Turkevich method with citric acid of GNP preparation (Kimling, Maier et al. 2006, Adlim and Bakar 2010, Bastús, Comenge et al. 2011). Therefore, instant interactions of formed malonic acid with GNP took place, explaining its absence in solution.

Fig. 2. FTIR spectra of cryogels: A) CHI–TMP–GNP and B) CHI–TMP cryogels.

Comparative analysis of shift of functional group frequencies of CHI, IC, CHI-TMP and CHI–TMP–GNP cryogels is presented in table A2. The carbonyl stretching frequency of amide bond in CHI was confirmed by the peak at 1633.9 cm−1 (Fig. A.6b) (Hortigüela, Aranaz et

al. 2010).

The reaction between primary aminogroups of CHI and aldehyde of 1.3-propanedial

led to the Schiff’s base bond formation according to equation 4, and amide group frequency at 1547 cm-1 was overlapped with a higher intensity group of conjugated Schiff’s base having a signal at 1592 cm-1. The proposed mechanism of Schiff’s base group rearrangement to a conjugated structure is shown in equation 3, which implies the coupling of aminoacids with TMP(Zhou, Zhao et al. 2010). FTIR spectra of CHI-TMP-GNP contained a small intensity peak at 1715 cm-1 attributing to carboxyl group formation due to oxidation of aldehyde by gold complex whereas the second aldehyde group reacted with amino group (Eq. 5). A broad peak at 3270 cm-1 corresponds to hydroxyl and primary amino groups of CHI. Frequencies of functional groups of CHI were observed at 2929–2886 cm-1 (–C–H), 1548 cm1

(–NH2), 1404 cm-1 (the coupling of –C–N and –N–H), 1062 and 1026 cm-1 (–C–O), 896 cm-

1

(C–C ring). The acetic acid was validated by the peak at 1259.7 cm-1 (Fig. A.6b)

(Hortigüela, Aranaz et al. 2010). A primary aminogroup frequency of CHI was observed at 3270 cm-1(Fig. A.6b). A secondary amino group (CH=CH–NH–) had a signal at 3244 cm-1 which is in agreement with TMP aminoacid derivatives(Zhou, Zhao et al. 2010). Amide bonds at 1633.4 cm-1 was not present at CHI-TMP and CHI-TMP-GNPs due to overlapping with high intensity Schiff’s base groups at 1592.7 cm-1. Additional small intensity peak at 1461 cm-1 was associated to –CH2– bond of TMP (Fig. 2b).

Fig. 3. CLSM images of cryogels: A) CHI–TMP–GNP–1, B) CHI–TMP–GNP–2, C) CHI– TMP–GNP–3, D) CHI–TMP–GNP–4. 20 and 100x magnification were determined by Rhondamin B staining (green) in cryogels and black or dark represents voids(porosity).

The macroporous structure of the cryogels CHI–TMP–GNP was found to have thin walls, this is due to the utilisation of diluted CHI (1 %) and acetic acid solution, thus observing a small concentration of solutes(Fig. 3), . The dimensions and GNP distributions within the CHI–TMP–GNP cryogel structure were analysed by TEM (Fig. 4). GNP were well

distributed in cryogel volume and GNP was characterised to possess a spherical shape and a narrow size distribution of CHI–TMP–GNP–1, CHI–TMP–GNP–2, CHI–TMP–GNP–3, CHI–TMP–GNP–4 with an average diameter of 5.9 ± 2.1, 3.9 ± 2.3, 8.0 ± 2.0, 9.6 ± 2.5 nm, respectively (Fig. 5 and Table A.3). CHI–TMP–GNP also displayed spherical morphology and absence of aggregates (Fig. 4). Using the low temperature nitrogen adsorption analysis method, we evaluated how altering the gold concentration effected the dispersion of nanoparticles across the channels and pore structure in composite cryogels. This was necessary to estimate the surface area and the nanoporosity of the cryogels. Experimental results were obtained for CHI–TMP–GNP cryogels by applying three different adsorption models (isotherms) named as Brunauer– Emmett–Teller (BET), Barrett–Joyner–Halenda (BJH) and Density Functional Theory (DFT) are shown on Figures A.7–9. The surface area (BET) of the CHI–GA cryogel was approximately 8 m²/g, whereas CHI–TMP–GNP–1, CHI–TMP–GNP–3, and CHI–TMP– GNP–4 had a surface area of 79 ± 4.3, 97.5, and 76 ± 23 m²/g, respectively. For comparison, SBET of CHI-GA-PdNP 0.2 and 0.4mM were 22 ± 3 and 44 ± 9.(Berillo and Cundy 2018) SBET for core–shell structure, yolk–shell structure, and hollow structure with GNPs were estimated to be 67.6 m2/g, 88.1 m2/g, and 90.1 m2/g, respectively(Zeng, Zhang et al. 2013). In this case, GNP additional voids were created around the GNP. The pore diameters of the CHI–GA and CHI–TMP–GNP–1 (–2, –4) cryogels calculated using the BJH model which were equalled to 9.9, 4.2, 3.7, 4.9 nm, respectively (Fig. A.8). There was no clear correlation between an increase of GNP concentration in the cryogel structure and an increase of total pore volume of CHI–TMP–GNP–1 (–2, –3, –4) cryogels, found to be 0.164, 0.048, 0.117, and 0.229 cm3/g (Table A.5) albeit larger than blank CHI–GA cryogel(0.021 cm3/g).

Fig. 4. A) TEM images of cryogels: a) CHI–TMP–GNP–1, b) CHI–TMP–GNP–2, c) CHI– TMP–GNP–3, d) CHI–TMP–GNP–4 at magnification of 30,000 and 50,000 x and scale bars of 100 and 10 nm.

Fig. 5. Size distribution of GNP within the cryogel walls: a) CHI–TMP–GNP–1 with mean diameter of 6.2 ± 2.1 nm, b) CHI–TMP–GNP–2 with mean diameter of 3.9 ± 2.3 nm, c) TMP–GNP–3 with mean diameter of 8.0 ± 2.0 nm, d) CHI–TMP–GNP–4 with mean diameter of 9.6 ± 2.5 nm (n=8), dN/dx represents a number of GNPs in area of sample.

The elevation of gold complex content in the ionic cryogel created a larger GNP that did not affect the complex distribution or shape (Fig. 4 and 5). This phenomenon is related to the inhibition effect of chloride ions on the stabilisation of GNP, where chloride ions are released due to ligand exchange process (Berillo, Mattiasson et al. 2014). The higher concentration of H[AuCl4] increased the portion of free electrostatically associated gold complex, (CHI-NH3+ [AuCl4]-) (Eq. A.3)increasing the amount of protonated aminogroups (Eq. A.4).. This caused the gold species to become more flexible, favouring aggregation upon the reduction (Eq. A.2). The accumulation of chloride ion concentration from 2.16 to 8.89 ppm was confirmed by ICP-MS and ionic chromatography (Fig. A.10). The ligand exchange process was

accelerated during neutralisation process due to excess of production of non-protonated aminogroups, a similar in principal ligand exchange mechanism for tetrachloroaurate was also studied for amide complexes in EDTA(Cornejo, Castineiras et al. 2003). Elemental mapping of cryogel and GNP distributions were correlated with TEM data, confirming the absence of aggregates (Fig. A.11). CHI stabilized GNP was prepared by incubation of IC2 at 60 oC with a mean diameter of 9.0 ± 5.2 nm. GNP synthesized via heat treatment of the CHI–[AuCl4] solution at 60 oC had an average diameter of 10.2 ± 4.5 nm. GNP within CHI–TMP–GNP–2 with a diameter of 3.9 ± 2.3 nm had an advantage of TMP as the cross-linking and reducing agent. Reduction of IC4 by GA and TMP created the GNP with an average diameter of 7.3 ± 2.2 nm (CHI–GA–GNP– 4) and 9.6 ± 2.5 nm (CHI–TMP–GNP–4), respectively (Berillo, Mattiasson et al. 2014). Based on results of TEM image analysis of GNP density, GNP population in the range of 2,000–14,000 particles/µm3 was revealed (Table A.4.). 3.3 Mechanical properties of composite cryogels with GNP To estimate the effect of GNP on the mechanical properties of the material, compression tests were carried out and the elastic modulus of composite cryogels were calculated. The initial 10% of material deformation containing 98.5–787.8 ppm of GNP were associated with elastic deformation (Fig. A.12 and Table A.3). The subsequent compression of the cryogels was related to plastic deformation(Charron, Braddish et al. 2019). IC cryogels without GNP were softer, compared to cryogels with in-situ formed GNP (Fig. A.12). This phenomenon was related to additional cross-linked cryogel walls by GNP via electrostatic interactions between carboxyl and amino groups. An excess of TMP during the cryogel synthesis had a function of a stabilising and reducing agent (Eq. 4-5). The GNP were surrounded by carboxyl groups(Eq. 5). Therefore, functionalised GNP participated as chemical and physical cross-linkers for polymeric chains of CHI due to electrostatic interactions. As expected, the increase of GNP

content enhanced the elastic modulus for CHI–TMP, CHI–TMP–GNP–2, CHI–TMP–GNP– 3, and CHI–TMP–GNP–4 as 0.41 ± 0.08, 1.69 ± 0.35, 4.6 ± 1.3, and 6.2 ± 0.3 kPa, respectively, which wascomparable with elastic modulus of IC (98.5 – 787.8 ppm of [AuCl4]) (Table A.1). CHI-TMP-GNP cryogels were more elastic compared to brittle cryogels crosslinked by GA. All CHI–TMP–GNP samples withstood up to 60 % of compression. Depending on the application purpose of the material, the mechanical properties and the size distribution of GNP could be tuned by the use of crosslinking agent GA or TMP. 3.4 Catalytic activity of composite cryogels with GNP Properties of obtained cryogels in terms of their application was studied as a superior catalytic scaffold. A model reaction of 4-nitrophenol (4-NP) conversion was examined by flow through mode illustrated on the figure A.13. We had demonstrated previously the catalytic activity of the flow-through reactor with integrated GNP partially stabilised by GA for oxidation of N,N-dimethylaminobenzaldehyde (Berillo, Mattiasson et al. 2014). In the current study, the cryogels with impregnated GNP were tested as catalysts in terms of reduction process. The conversion of 4-NP to 4-aminophenol (4AM) with an excess amount of NaBH4 has been used by as a classic model reaction to examine the catalytic performance of metal NPs (Hortigüela, Aranaz et al. 2010, Fenger, Fertitta et al. 2012). The reproducibility of every catalytic reaction cycle was associated with the stability of the catalyst for 10 – 14 cycles without activity decline (Fig. 5 A – C). A slower converting reaction took place during the first cycle due to accumulation of adsorbed hydrogen on the GNP particles, also observed in our previous studies testing PdNPs within CHI–GA scaffold (Berillo and Cundy 2018). Another slower 4NP reduction reaction was observed after an overnight stop of the catalytic reaction showed on the Fig. 5A (cycle 5) and Fig. 5B (cycle 3). This was related to the catalyst saturation of hydrogen. The degree of conversion for several catalytic cycles (2nd, 6th, 9th etc.) of the same material was comparable (Fig. 5 and Tables 1 – 3). These parameters are

features of catalyst stability over time. This tendency was detected for all studied composite cryogels with GNP amounts of 49.2, 98, 196, 392 µg. This reported catalytic system possessessuperior stability and significantly higher rate of reaction compared to previously reported cryogels with immobilised GNP(Table 1).

1st 2nd 3rd

ln(At/A0 at 400nm)

Cycle #

4th

5th

6th

7th

8th 9th 10th

A

0 -0.05 -0.1 -0.15 -0.2 -0.25 -0.3 -0.35 -0.4 -0.45 -0.5 0

Cycle # 1st

200 2nd

400 3rd

600 800 Time, min 4th

5th

1000 6th

1200 7th

B

ln(At/A0 at 400nm)

0 -0.2 -0.4 -0.6 -0.8 -1 -1.2 0 Cycle #

1st

200 2nd

3rd

400 Time, min 4th

5th

6th

7th

600 8th

800 9th 10th

ln(At/A0 at 400nm)

0 -0.2 -0.4 -0.6 -0.8 -1 -1.2 0

200

400

600 800 Time, min

1000

C

Fig. 5. Kinetic curves of the 4-NP reduction by NaBH4 in the presence of immobilised GNP: 98 µg Au (A), 196 µg Au (B) and 392 µg Au (C) in cryogels GNP–CHI–TMP in 10 cycles: ln At/A0 (where At stands for absorbance at 400 nm at time (t) and A0 stands for absorbance at 400 nm at time 0) versus time for the conversion of 4-NP in solution catalysed by GNP– CHI–TMP was shown on the plots. A 4NP concentration three-fold lower than previous, in the reaction mixture led to a dampened degree of conversion by 30% and the kinetic rate by three-fold, respectively (Table 1(S1 and S2)). A two-fold higher GNP concentration was used for the 4NP reaction with concentration of 22 µMresulted in acceleration of catalytic activity by 2.65 times (Table 1(S1 and S3)). CHI–TMP–GNP–1 and CHI–TMP–GNP–2 was comparable to the conversion degrees of 60 µM of 4NP GNP(Table 1(S2 and S3)). The rate of over 10 repeated cycles without regeneration was decreased by 8–12% (Table 1(S3)). Table 1. The rate constants of 4-NP conversion to 4-AP catalysed by GNP in the cryogel CHI–TMP–GNP–2 (98 µg of Au) (samples 1(S1) and 2 (S2)) and CHI–TMP–GNP–1 (49.2 µg of Au) sample 3 (S3) and degree of conversion (DC) for the time point 120 min. Calculation of the kinetic rate was performed for the linear region of the kinetic curve for 80 min. [NPs]

S1, [4NP] 22 µM

Cycle #

k*10-3,

k*10-

[4NP]

min-1

min-1

3

µM

DC, % 8.5

60.6

2

57.9

3 4

S3

k*10-3,

DC, %

1

S2, [4NP] 60 µM

DC, %

71.0

,

min-1 19.7

27.3

(60min)

17.4

86.7

7.6

94.3

24.5

*52.56

24.5

35.4

61.5

9.7

97.5

32.0

96.8

22.9

42.1

66.4

9.8

92.3

22.2

96.7

27.5

49.4

5

*46

5.3

92.4

26.1

95.2

27.3

62

*6

51.3

6.0

*84.4

22.0

*85.57

15.4

66

7

54.8

7.0

87

24.9

88.33

18.2

63

8

54.2

7.6

95

26.1

90.62

19.7

64

9

55.5

7.2

88.6

17.8

64

10

*46.36

5.5

*83.45

14.6

66

11

52.45

6.3

92.2

20.7

60

12

91.5

18.2

65.3

13

92.15

19.8

63.2

14

96.5

23.8

62.1

* The catalytic reaction continued after an overnight stop (storage at 4 °C in the reaction mixture). The conversion of 4NP(22 µM) at 37, 50, 56 and 60 % were detected for CHI–TMP–GNP–2 with average GNP size of 3.9 ± 2.3 nm for time points at 80, 120, 150 and 180 min, respectively. Thereafter,any further increase in conversion is insignificant not cost effective for environmental application. Recently, Liu et al reported GNP(mean diameter 2.3 ± 1.3 nm) stabilised by dendronized 1,2,3-triazolyl-containing ferrocenyl polymers in the two phase system dichloromethane and water, revealing a 100% conversion of 4-NP without loss of activity up to 20 times(Liu, Liu et al. 2019, Liu, Liu et al. 2019). The increase of catalyst with concentration of 4NP (65 µM) resulted in 96 % conversion. , The CHI–TMP–GNP–4 contained an average size of 9.6 ± 2.5 nm for 66 µM of 4NP. CHI–TMP–GNP–2 at time points of 60, 120, 180 and 230 min had improved conversion degree reaching to 50, 72, 87, and 93 %, respectively compared to the same sample tested at lower 4NP concentration. Hence, most of following experiments were performed within two hours. The increase of GNP from 98 to 196 µg in the volume of the same size cryogel enhanced the conversion degree of 4NP (22 µM) by 12–15 % (Table 1(S1 and S2), Figure 6 A). This was related to larger distribution size from 6.2 ± 2 to 9.6 ± 2 nm, and, therefore insignificant elevation of

the catalyst surface area. Note, that the GNP population (concentration) within the wall of CHI–TMP–GNP–2 and CHI–TMP–GNP–4 were comparable (around 13,000 GNP/µm3) (Table A.2). The rate of the reduction reaction was calculated for 4NP (60 µM), and was found to increase by 1.7 fold with higher catalyst concentration from 49 to 196 µg. The rate constants and conversion degreesof CHI–TMP–GNP–1 (Table 1(S3)) and CHI–TMP–GNP–3 (Figure 6 B(S1)) are demonstrated. CHI–TMP–GNP–3 cryogel over 13 catalytic cycles did not have a decline of the rate (Figure 6 A). The increased catalyst content from 98 to 196 µg (CHI–TMP–GNP–2 and CHI–TMP–GNP–3, 4NP 25 µM) accelerated the reaction by 1.6 fold (Table 1 (S1) and Figures 6 b (S2)). Cryogels with GNP specimens S2 and S3(Table 1) had significantly higher kinetic constant compare to previously published GNP with kinetic rate(k) of 12.4 * 10-3 min-1, at experimental settings of reaction volume 3ml of 100µM 4NPs and 66µM NaBH4) (Srivastava, Yamada et al. 2013). Comparative analysis of different catalytic parameters for supported GNPs for 4NP reduction with current research is presented in table A6.

Conversion degree, %

Conversion degree,%

100

A

100 80 60 40 20 0 1

2

3

80 60 40 20 0

4

5 6 7 8 9 10 11 12 13 Cycle # S1, [4NP] 60 µM S2, [4NP] 25 µM

1

5 6 7 8 9 10 Cycle # S1, [4NP] 50 µM S2, [4NP] 70 µM

C

0.04

B

2

3

4

D

0.03

k, min-1

k, min-1

0.03 0.02 0.01

0.02

0.01

0

0 1

2

3

4

5 6 7 8 9 10 11 12 13 Cycle # S1, [4NP] 60 µM S2, [4NP] 25 µM

1

2

3

4

5 6 7 8 9 10 Cycle # S1, [4NP] 50 µM S2, [4NP] 70 µM

Fig. 6. The degree of conversion (DC120min)(for reaction time 2h) and rate constants for 4-NP reduction catalysed by GNP in the cryogel: A) and B) CHI–TMP–GNP–3 (196 µg of Au); C) and D) CHI–TMP–GNP–4 (394 µg of Au). Calculation of the kinetic rate was performed for the linear region of the kinetic curve for 80 minutes.

The average conversion degree of 4NP (60 µM) for 394 µg of GNP was in the range of 86 – 92.4 % (Figure 6 C). Although, 8 fold elevation of gold content in the experimental set up did not allow to achieve 99.9 % of conversion degree.

Table 2 TON and TOF parameters of CHI–TMP–GNP cryogels catalyst for reduction of 4NP (n=2). GNP-

Weight

Average

Number

[4-NP],

CHI-

of GNP,

size of

of

µmol

TON

TOF,

Total

h-1

converted

TMP

µg

1

49.0

2

98.5

GNP, nm

5.9 ± 2.1

4

cycles

mmol 60

18.3

0.619

4.57

11

22

1.92

0.073

1.00

9

60

5.74

0.365

2.87

12

60

5.14

0.255

5.14

8.0 ± 2.0

12

25

1.72

0.071

1.72

9.6 ± 2.5

10

50

1.40

0.066

2.81

10

70

2.11

0.087

4.22

197

394

[4-NP],

14

3.9 ± 2.3 3

tested

It can be concluded that CHI–TMP–GNP–1 cryogels had the best performance with TON and TOF of 18.3 and 0.619 h-1, respectively. Data in table 2 were comparable with features of composite magnetic particles based on SiO2/Fe3O4/carbon double-layered shell with immobilised GNP (d∼2 nm) with TOF value of 17.4 min−1 at experimental set up of reaction volume of 3mL of 66mM NaBH4, 16.4µM 4NP and 50µg of catalyst(Zeng, Zhang et al. 2013). The TON parameter for CHI–TMP–GNP–2 and CHI–TMP–GNP–3 scaffolds had comparable values with a 4NP concentration of 22 µM. This was related to the insignificant GNP surface area change GNP and two-fold increased mean diameter of GNP. Therefore, the utilisation of higher concentration of gold complex is not favourable (Table 2). The indicated turnover number TON and TOF were not the final data because the decline of the catalytic activity was not observed over the catalyst testing (Table 1 and Fig. 6 A-D). Usually, the ultimate values of the TON and TOF are achieved when the catalyst has lost its catalytic activity (Hagen 2015), which was not in our case. Comparison of TOF data revealed that CHI–TMP–GNP–1 was better catalyst compare to CHI–TMP–GNP–2 based on the gold

mass difference between samples that were two-fold more. However, the GNP population was 6.8 fold higher in comparison with CHI–TMP–GNP–1. It was concluded that this cryogel contained significant fraction of tiny GNP that were difficult to distinguish from the background via TEM image analysis. The control CHI–TMP cryogels had some 4-NP adsorption (Fig. A.14a). Insignificant reduction of 4-NP in the presence of the borohydride without catalyst was revealed in the control experiment(Fig. A.14b). Synthesized composite cryogels with in situ formed GNP have a potential to be used for many environmental applications such as selective adsorption of mercury, via the same mechanism by supported AgNP for successful adsorption of mercury which previously was shown (Katok, Whitby et al. 2012). The amalgam formation between AgNP and mercury resulting in release of silver ions, have to be removed from the purified water due to its toxicity. GNP application for water purification doesn’t have this drawback. Designed composite cryogel can be successfully utilised to for removal of oxygen from the sensor chamber in a cost efficient way, compare to utilised suspension of Pd on activated carbon combined with H2 (Meyer, Larsen et al. 2002), data is not shown. IC of CHI with tetrachloroaurate can be further transformed into the material containing gold complexes and used as a catalyst for various reactions, for example tret-butanol oxidation(Nasser and Puddephatt 2014). GNP formed in the macroporous structure may be a convenient alternative to conventional catalyst for Sonogashira or Heck cross-coupling reactions that have widely been applied for preparation of valuable chemicals(Primo and Quignard 2010, Ramasamy and Maliyekkal 2014. 4. Conclusion In this study, a new approach of gold nanoparticle preparation incorporated into cryogel was shown. The reductive properties of GA and TMP were confirmed by the control experiments and H1-NMR analysis. This material with in-situ produced nanoparticles can be potentially applied as a flow through catalytic system as for non-aqueous solutions at mild conditions.

The GNP distribution within the 3D polymer networks were homogeneous. The advantage of proposed GNP synthesised in-situ within the cryogel structure is the absence of GNP aggregates upon catalytic reaction. The higher concentration of gold complex in the ionic cryogel resulted in increase of an average size from 5.9 ± 2.3 to 9.6 ± 2.5 nm. The catalytic conversion of 4-NP catalysed by the GNP within the material can be applied in a static and dynamic mode. TON and TOF parameters for four GNP microgram amounts were calculated. The initial conversion degree of 4-NP was less for the first cycle compared to the following catalytic cycles that was related to an activation time needed for GNP saturation with hydrogen or to diffuse 4-NP via the 3D-polymeric network. Constant rate of 4-NP conversion was comparable to published data of free suspension of GNP stabilised by CHI. The conversion degree of 4-NP for GNP–CHI–TMP containing lowest concentration of gold(0.5mM) reached 96.8 % at RT. The catalytic activity of incorporated GNP for 4-NP conversions did not decline over 10–14 consequent testing cycles, indirectly confirming the absence of GNP leakage from the scaffold. The composite cryogel with GNP was successfully utilised for 4-NP conversion in a flow though mode and thus gives a perspective to be used in a cascade with different composite catalyst containing cryogels allowing to proceed multistep reactions in a dynamic mode.

Acknowledgements. I gratefully acknowledge the H2020 Marie Skłodowska-Curie Actions grant agreement N 701289. I also acknowledge Prof. Sergey Mikhalovsky for advice and Mr. Peter Lyons from the School of Environment and Technology at the University of Brighton for assistance with ICP-MS analysis and Dr. Pascale Schellenberger for sample preparation, advice and imaging at the Cryo-EM facilities (University of Sussex’s Electron microscopy imaging centre), funded by the School of Life Sciences, the Wellcome Trust (095605/Z/11/A, 208348/Z/17/Z) and RM Phillips Trust. I thank Dr. Claire Mitchell from Cardiff University

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