new insights into the characterization of ‘insoluble black hcn polymers’

New Insights into the Characterization of �Insoluble Black HCN Polymers�

by Marta Ruiz-Bermejo*a), Jose L. de la Fuenteb), Celia Rogeroa), Cesar Menor-Salvana),Susana Osuna-Estebana), and Jose A. Mart�n-Gagoa)c)

a) Centro de Astrobiolog�a (Consejo Superior de Investigaciones Cient�ficas – Instituto Nacional deTecnica Aeroespacial (CSIC-INTA)), Carretera Torrejon-Ajalvir, Km 4, E-28850 Torrejon de Ardoz,

Madrid (phone: þ34-91-520-6402/6458; fax: þ34-91-520-6410; e-mail: [email protected])b) Instituto Nacional de Tecnica Aeroespacial (INTA), Carretera Torrejon-Ajalvir, Km 4,

E-28850 Torrejon de Ardoz, Madridc) Instituto de Ciencia de Materiales de Madrid (CSIC), C/Sor Juana Ines de la Cruz, 3,

E-28049 Cantoblanco, Madrid

The data presented here provide a novel contribution to the understanding of the structural featuresof HCN polymers and could be useful in further development of models for prebiotic chemistry. Theinterpretation of spectroscopic and analytical data, along with previous results reported by other authors,allowed us to propose a mechanism for the aqueous polymerization of HCN from its primary andsimplest isolated oligomer, the diaminomaleonitrile (DAMN) tetramer. We suggest that �insoluble blackHCN polymers� are formed by an unsaturated complex matrix, which retains a significant amount of H2Oand important bioorganic compounds or their precursors. This polymeric matrix can be formed byvarious motifs of imidazoles and cyclic amides, among others. The robust formation of HCN polymersassayed under several conditions seems to explain the plausible ubiquity of these complex substances inspace.

Introduction. – HCN is ubiquitous in the universe and is a significant product inprebiotic simulation experiments [1– 4]. HCN Polymers may be the major componentsof dark matter, which could be present in objects such as asteroids, moons, planets, and,in particular, comets [1] [5 – 7]. It has been proposed that the reddish haze (tholins)present in the atmosphere of Titan, the largest moon of Saturn, could be due to thepresence of HCN polymers [8]. In addition, it has been suggested that HCN polymersmay be important substances in the first stages of the chemical evolution of life [9]. Thishypothesis is based on the fact that HCN polymers are precursors of importantbioorganic compounds such as purines, pyrimidines, and amino acids, as well as otherbiological compounds such as oxalic acid and guanidine [10 – 13]. HCN can sponta-neously polymerize in the presence of bases such as NH3 and free radicals from ionizingradiation, and occurs over a wide range of temperatures and pressures in both polar(water) and non-polar (hydrocarbon) solvents and surfaces [1]. The HCN polymers,also known as HCN oligomers, �azulmic acid� or �azulmin�, are heterogeneous solidsranging in color from yellow or orange to brown or black, depending on the degree ofpolymerization and/or cross-linking processes. The structures of HCN polymers havenot been fully characterized and remain controversial due to their complex andheterogeneous nature.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 25

� 2012 Verlag Helvetica Chimica Acta AG, Z�rich

Several models attempting to explain the complex structure of HCN polymers havebeen proposed [14 – 19]. Scheme 1 compiles these models.

During the polymerization of HCN in aqueous environments, a H2O-soluble and aninsoluble solid product are formed. Our study is focused on the insoluble solid,commonly named �insoluble black HCN polymers� or �black azulmic acids�. Thesepolymers were prepared from solutions of equimolar amounts of NH4Cl and NaCN inpure H2O at concentrations of 1 and 10m using different reaction times. To further

Scheme 1. Different Models Proposed in the Literature for HCN Polymers

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)26

elucidate the structure of this polymer, we used the methodology we developedpreviously for the characterization of tholins [20] [21]. Data from the elementalanalysis, FT-IR, 13C-CP-MAS-NMR spectra, and GC/MS analysis of the �insolubleblack HCN polymers� were compared to results previously reported in the literature.Additionally, we used X-ray photoelectron spectroscopy (XPS) to quantify variouschemical species in powder samples [20 –22]. Additionally, to obtain complementaryinformation, the �insoluble black HCN polymers� were acid hydrolyzed. The hydro-lyzed supernatants and black insoluble residues were also analyzed. Scheme 2 outlinesthe general treatment of the samples, the separation in fractions and the techniquesused for this study.

Scheme 2. Reaction Conditions, General Treatment of Samples, Fractionation, and Techniques Used


The results presented herein complete the data reported in a previous paper [20]where some spectroscopic data were shown. The strength of HCN polymerizationreactions in an aqueous medium and the proposed structures for �insoluble black HCNpolymers� are discussed.

Results. – The results of the elemental analyses of the �insoluble black HCNpolymers� are compiled in Table 1. Our results are comparable to those reported byEastman et al. [23] and Labadie et al. [24] for short reaction times (1 – 10 d). For longerreaction times (30 d), black insoluble solids richer in oxygen were obtained. However,a longer reaction time did not have a significant influence on the yield of these insolubleproducts, 29�4%. In this work, the yields were calculated from the initial amount of Cin the NaCN used as a reactant. The estimated empirical formulae, C3H4N3O andC5H6N4O2, indicate the formation of highly unsaturated structures with a backbonebased on C and N.

The elemental analyses of the insoluble black residues after acid hydrolysisprovided the following results: C, 36.6�0.6%; H, 4.1�0.2%; N, 33.2�0.8%; and O,22.3�0.9%. Thus, the estimated empirical formula for the hydrolyzed residues wasC2H3N2O.

Determination of Functional Groups. The FT-IR spectra of the �insoluble blackHCN polymers� synthesized in our laboratory were similar, with no significantdifferences among them (Fig. 1,c). These spectra highly resemble those reported in theliterature for HCN polymers prepared under different experimental conditions.Quirico et al. [8] reported an IR spectrum of an HCN polymer prepared from pure NH3

and liquid HCN that was similar to the IR spectrum in this study. Similar IR data werereported by Liebman et al. [26] and Umemoto et al. [16] for HCN polymers obtainedfrom liquid HCN and Et3N in MeCN, and from gaseous HCN and aqueous NH3,respectively. The IR spectra do not provide sufficient information to identify the


Table 1. Experimental Conditions, Yields, and Elemental Analyses for the Series of �Insoluble Black HCNPolymers� Prepared in This Work and by Other Authors. The yields in this work were calculated from the initialamount of carbon in the initial NaCN. N.R., not reported; c, initial concentration; T, reaction temp.; t, reaction

time.

Reactants c [m] T [8] t Yield [%] C[%]

H[%]

N[%]

O[%]

Empiricalformula

Ref.

KCN (aq)þNH4Cl(aq) 1–1.5 70 80 h N.R. 36.5 4.2 39.2 15.7 C3H4N3O [23]HCN(l)þNH3(aq)þEt3N N.R. r.t. 3 d, after

water 30 dN.R. 35 N.R. 38 12 – [25]

HCN(g)þNH3(aq) 12.5 r.t. 4 d N.R. 39.6 4.0 46.1 10.4 C5H6N5O [16]HCN(aq)þNH4OH 1 90 16 h 31 38.77 3.96 40.85 16.42 C3H4N3O [24]HCN(l)þNH3(l) N.R. r.t. 30 d N.R. 44.5 4.0 52.5 N.R. – [18]HCN(aq)þNH3(aq) N.R. 40–50 5 h 51 40.2 3.8 41.8 N.R. – [19]NaCN(aq)þNH4Cl(aq) 10 38 3 d 31 36.2 4.3 40.8 17.7 C3H4N3O This workNaCN(aq)þNH4Cl(aq) 1 38 3 d 25 36.5 4.4 40.8 17.4 C3H4N3O This workNaCN(aq)þNH4Cl(aq) 1 38 10 d 27 36.5 4.4 41.9 17.2 C3H4N3O This workNaCN(aq)þNH4Cl(aq) 1 38 30 d 33 36.2 3.9 39.6 19.2 C5H6N4O2 This work

differences between HCN polymers obtained in H2O solutions and those prepared inaprotic mediums or without solvents. Therefore, the IR spectra must be deconvolutedto identify the functional groups in the HCN polymers. The features in the IR spectra ofour �insoluble black HCN polymers� can be assigned to N-containing groups: primaryand secondary amines (3444 cm�1, �NH� stretch; 3330 cm�1, �NH2 antisym. stretch,and 3191 cm�1�NH2 sym. stretch), CN and carbodiimide groups (2187 cm�1, C�N and


Fig. 1. Transmission FT-IR spectra of a) hydrolyzed supernatant, b) hydrolyzed insoluble black residue,and c) �insoluble black HCN polymers�

�N¼C¼N�), amides, urea and triazines (1655 cm�1, C¼O stretch (amide I band);1560 cm�1, NH deformation (amide II band) plus ring stretch in triazine compounds;1395 cm�1, C�N stretch (amide III band)), and azoxy groups (1490 cm�1, N¼N�Oantisym. stretch).

All of the insoluble black hydrolyzed residues presented similar bands in their IRspectra (Fig. 1,b). These IR bands were assigned to the following functional groups:3084 cm�1 (very broad), primary and secondary amines (OH groups of carboxylicacids, and ¼CH in aromatic and unsaturated hydrocarbons may overlap); 1725 and1665 cm�1, C¼O stretch in carbonyl and amide compounds; 1590 cm�1, NH2 primaryalkyl amide; 1515 cm�1, NH in secondary amides and triazine compounds; and1420 cm�1�OH in carboxylic acids and C�N in primary amides.

The IR spectra of the soluble hydrolyzed materials exhibit bands that maycorrespond to the NHþ

4 cation (3050 and 1410 cm�1), �CH3 and �CH2 groups inaliphatic compounds (2895 cm�1), and �C¼O in carbonyl, carboxylic, and ureacompounds (1735 and 1670 cm�1).

As in the FT-IR analysis, the 13C-CP-MAS-NMR spectra of all of our samples weresimilar and resembled spectra obtained by Garbow et al. [25] for the �water-insolublefraction� as well as spectra reported by Mamajanov and Herzfeld [14] [15] for an HCNpolymer prepared from gas HC15N and Et3N. The deconvoluted solid-state 13C-NMRspectrum of our �insoluble black HCN polymers� displayed the following resonances: i)resonance at 168 ppm, which may correspond to amide groups (�CONH2); ii)resonance at 159 ppm, which may correspond to imine and/or heterocyclic groups(�C¼N�); iii) a group of resonances at 154, 149, and 139 ppm, which may correspond toC-atoms of heterocyclic compounds containing N; iv) a group of low-intensityresonances between 130 and 100 ppm, which may correspond to alkenes( ==C¼C

=

= ) and nitriles (�C�N); and v) a third group of unresolved resonancesbetween 100 and 60 ppm, which may correspond to C-atoms bound to a heteroatom,such as C�(N) of amines (Fig. 2,b). The 13C-CP-MAS-NMR spectra of the insolublehydrolyzed residues (Fig. 2, a) exhibit resonances similar to those of the �insolubleblack HCN polymers�, but with a well-defined signal at 171 ppm, which may correspondto carboxylic acids, as was observed in IR analysis.

XP Spectra were recorded to obtain further structural data for the HCN polymers.XP Spectra of the prepared samples recorded from 1200 to 0 eV provided an overviewof the main elements in the �insoluble black HCN polymers�, as well as any tracecontaminants (data not shown). As expected, the HCN polymers consisted mainly of Cand N with a lower contribution of O. It is important to note that C and O were themain contaminants in samples prepared in air and were also the main contaminants ofthe KBr pellets prepared. On the other hand, the C bonds were relatively well-definedby 13C-CP-MAS-NMR and IR spectra. Therefore, only the high-resolution N 1s corelevel spectra were relevant for elucidation of the structures of the controversial N-containing functional groups in the �insoluble black HCN polymers�, which can beconsidered the distinctive fingerprint of the possible structures of the �insoluble blackHCN polymers�.

Fig. 3 and Table 2 present the binding energies of the different components of the N1s core level peak, as well as their assignment and quantitative results. The two maincomponents are centered at 397.6 and 398.7 eV, and can be associated with the�N¼C�


bonds (imine and/or heterocyclic groups) and the �CO�N

=

= group (amides),respectively. The small contribution detected at higher binding energies, 400.5 eV,corresponds to the�NH groups in pyrrolic structures, as well as CN and amine groups.This assignment of the binding energies for the N 1s core level peak was checkedagainst standard samples prepared in our laboratory [21]. The functional groupsidentified by XPS were in agreement with the IR and 13C-NMR assignments . Theadvantage of the XPS technique is that it provides quantitative information about eachN-containing group in the �insoluble black HCN polymers�. Additionally, the XPspectra of the insoluble hydrolyzed residues were recorded (Table 2). For the insolublehydrolyzed residues, the absence of a component signal at 400 eV, the increase in


Fig. 2. 13C-CP-MAS-NMR Spectra of a) hydrolyzed insoluble black residue and b) �insoluble black HCNpolymers�

signals for amide groups in the XP spectra, and the presence of resonances assigned tocarboxy C-atoms in the 13C-NMR spectra indicated that the component with the signalat 400.5 eV in the �insoluble black HCN polymers� can be assigned to nitrile groups.

Additionally, the UV/VIS spectra were recorded, and one sample is shown in Fig. 4.The UV/VIS spectra of the all samples showed two shoulders at ca. 345 and ca. 465 nmthat can be related to N-heterocyclic macromolecular systems with p-extendedconjugation [27] [28].

Identification of Small Molecules Absorbed in the �Insoluble Black HCN Polymers�.The ability of the �insoluble black HCN polymers� to retain H2O and other low-molecular weight molecules was confirmed by preliminary thermogravimetric (TG)and GC/MS analyses.

The TG measurements (Fig. 5) indicated that the amount of adsorbed H2O was 8 –10% depending on the reaction times. Long reaction times led to a greater amount ofadsorbed H2O in the matrix of the �insoluble black HCN polymers�, which wasconsistent with data from the elemental analysis. A complete thermal study of these

Fig. 3. High-resolution N 1s core level spectra of �insoluble black HCN polymers�. The dots correspond toexperimental data, the solid line corresponds to the fit, and the filled gray-scale curves correspond to the

curve components used for deconvolution of the spectra.

Table 2. X-Ray Photoelectron Spectroscopy (XPS) Data of the N 1s Core Level, Indicating the NitrogenChemical Environments in �Black HCN Polymers� and in the Hydrolyzed Insoluble Residue

Group BE [eV] BE (% of the component)

�Insoluble blackHCN polymers�

Hydrolyzedinsoluble residues

�N¼ (imines)/�N¼C� (heterocycles) 397–398 397.6 (47.4) 398.0 (51.9)�CO�N

=

= (amides) 399 398.7 (47.4) 399.4 (48.1)�NH� (amines)/�C�N (nitriles)/pyrrolic�NH 400 400.5 (5.1)


samples was described in an other publication, dealing with the structure�propertyrelationships in this complex polymeric system [29].

About 30– 40% of the �insoluble black HCN polymers� was released as H2O-solubleorganic material after acid hydrolysis. This is consistent with the results reported byLabadie et al. [24]. The dried residues of the hydrolyzed supernatants were analyzedfor polar organic compounds by GC/MS (Scheme 2). The chromatograms of all thesamples showed similar profiles (Fig. 6) independent of the reaction times. In general,


Fig. 4. UV/VIS Spectrum of HCN polymers in DMSO

Fig. 5. Thermogravimetry (TG) Curve for HCN polymers. Heating rate was 108/min under N2.


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all samples contained large amounts of oxalic acid, urea, and glycine, as expected[13] [24]. These three compounds comprised ca. 10 – 15% of the soluble hydrolyzedmaterial. The purines, such as adenine, hypoxanthine, and guanine, and thepyrimidines, such as uracil, 5-hydroxyuracil, 5-aminouracil, and orotic acid, werepreviously identified by Miyakawa et al. [10] in a low-temperature experiment at theeutectic point of HCN. Negron-Mendoza et al. [30] quantified the dicarboxylic,malonic, succinic, and maleic acids in experiments involving the irradiation of NH4CN.However, to the best of our knowledge, these acids were not previously reported inH2O polymerization experiments with HCN. Furthermore, for the first time, theorganic compounds pyrazine-2,5-diol, hydroxymalonic acid, aminomalonic acid, 2,4,5-trihydroxypyrimidine, and 2,4,7-trihydroxypteridine were detected in HCN polymer-ization experiments. Further analyses are in progress to identify organic compounds inthe HCN polymers, because the detection of these molecules seems to depend onsample preparation and analytical tools rather than experimental conditions ofpolymerization [31].

In summary, we can conclude that �insoluble black HCN polymers� are formed by ahighly unsaturated C�N matrix, which can absorb H2O and low-molecular-weight polarmolecules. This matrix likely consists of macrostructures with a high content of N-containing heterocycles and non-hydrolyzable amides. Temperature and reaction timesdid not seem to affect the synthesis of �insoluble black HCN polymers� in an aqueousmedium. Therefore, the formation of �insoluble black HCN polymers� is robust and isexpected to occur under diverse environmental conditions.

Discussion. – Proposal of Structures for �Insoluble Black HCN Polymers�. The initialsteps in the oligomerization of HCN are well-understood [9]. The rate-determiningstep is the nucleophilic attack of the CN ion at the C�N bond of HCN, which leads tothe formation of imino-acetonitrile. This step is followed by the stepwise condensationof HCN to form aminomalonitrile (AMN) and diaminomaleonitrile (DAMN). DAMNis readily formed at room temperature in 0.1 – 1.0m aq. HCN solutions [32]. The lowest-order oligomer is isolated from aqueous solutions. The mechanism of formation ofhigher order HCN oligomers is much less clear. In this study, we propose a possibleformation mechanism and structures for the �insoluble black HCN polymers� startingwith DAMN (Scheme 3 ; new structures are framed).

DAMN is a weakly basic amine, and its chemistry has been explored in detail [33].This reagent has been used extensively in the preparation of heterocyclic molecules,such as dicyanoimidazoles, dicyanopyrazines, and purines. Furthermore, Johnson et al.have used DAMN in the preparation of different polymeric systems [34] [35].

DAMN is an A2B2 monomer. The linear polyamine shown in Scheme 3, particular-ized as a 12-mer, could be obtained by an addition reaction between the amine groups,the nucleophilic agent, and the CN groups of the other molecule of this reactivemonomer, which acted as the electrophilic agent. Subsequent elimination reactions,such as decyanation (Pathway a) and/or deamination (Pathway b), for this polyaminemay allow formation of new extended-conjugation macrostructures. The formation ofstable five- or six-membered rings is possible from the decyanation of this linearpolyamine. Pathway a-1 is consistent with the recent work of Mamajanov and Herzfeld[14], who have studied the solid-state reaction of crystalline DAMN (Scheme 1). The



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linear polyamide (Pathway a-2) displayed a high tendency to form cyclic components,because the polymerization reactions in solution favor intramolecular cyclizations. Forthe first time, the resulting cyclic polyamide was considered as a motif of the �insolubleblack HCN polymers�. Taking into account the quantitative results of XPS, this cyclicpolyamide system appeared to be prevalent in the �insoluble black HCN polymers�.

On the other hand, the formation of five- and six-membered rings would also beexpected by a deamination reaction (Pathway b) from the polyamine and intra-molecular cyclization between CN groups, which may yield the heterocyclic polymerindicated in Scheme 3. This is, to the best of our knowledge, the first time that this novelmacrostructure based on pyrrole and dihydropyrazine rings has been proposed forHCN polymers.

These heterocyclic macrostructures indicate the presence of a non-hydrolyzablematrix for the �insoluble black HCN polymers�, according to the spectroscopic datapresented above. This polymeric matrix can be formed by different motifs of imidazolesand cyclic amides, as well as other groups.

Astrobiological Implications of HCN Polymers. The study of HCN polymers isrelevant to prebiotic chemistry and astrobiology. It has been suggested that primitiveEarth may have been covered by water and carbonaceous materials originating frombolide bombardment or from photochemical reactions in the atmosphere, includingHCN polymers, which would have supplied essential components for establishingprotein/nucleic acid life [1]. As mentioned above, it is well-known that HCN polymersrelease nucleic acid bases and amino acids. In addition to those species, dicarboxylicacids were detected in this work. Some of these acids could have been involved in aprimordial variant of the Krebs cycle [36]. Metabolic-type reactions could have playeda central role in the processes that led to the origin of life. Although the hypotheticalexistence of self-organizing proto-metabolic cycles is controversial [37], our prelimi-nary GC/MS analysis of the �insoluble black HCN polymers� demonstrated that thematerials required for the development of a proto-metabolic system, and structural andinformational materials could be synthesized simultaneously. Recently, polycyclicaromatic hydrocarbons (PAHs) have been suggested as energy transduction elements[38] in possible proto-metabolic systems, because they absorb light in the near-UV andblue region, and can capture light energy either by donating electrons to producemolecules with higher chemical potential or by generation of ionic gradients. Thus,HCN polymers may also be capable of capturing or transducing energy due to theunsaturated nature of their matrix (Scheme 3).

Conclusions. – This study offers new insights into the structural characterization ofHCN polymers based on the complementary use of several spectroscopic and analyticaltools. Based on the data reported here, we conclude that �insoluble black HCNpolymers� are formed by an unsaturated complex matrix, which can retain H2O, andimportant bioorganic compounds or their precursors. We propose that this insolublematrix is composed of several polyheterocyclic motifs, mainly imidazoles and cyclicpolyamides. HCN Polymerization in H2O yields many compounds considered to bepotential life precursors. Furthermore, �insoluble black HCN polymers� can beobtained under several environmental conditions, which explains their ubiquity in alarge diversity of space environments.


The authors used the research facilities of Centro de Astrobiolog�a (CAB) and were supported by theInstituto Nacional de Tecnica Aeroespacial �Esteban Terradas� (INTA), and the projects AYA2009-13920-C02-01 and BIO2007-67523 of the Ministerio de Ciencia e Innovacion (Spain). We thank M. T. Fernandezfor recording the IR spectra, I. Sobrados from ICMM (Instituto de Ciencia de Materiales de Madrid,CSIC, Spain) for recording the 13C-NMR spectra, and S. Veintemillas and G. Munoz-Caro for usefulcomments.

Experimental Part

Preparation of �Insoluble Black HCN Polymers�. �Insoluble black HCN polymers� were preparedaccording to a method described by Borquez et al. [31] using solns. of equimolar concentrations of NH4Cland NaCN in pure H2O (MilliQ grade) at concentrations of 1 and 10m. The 1m solns. were left to stand for3 d, one week, and four weeks, and the 10m soln. was left to stand for 3 d. All of the solns. were heated to388 for the duration of the reaction for comparison to previous work concerning the formation of tholinsunder a CH4 atmosphere and spark discharges [20] [21] [39]. After the reaction, the samples were filteredusing a glass fiber filter (0.25 mm) and washed with dist. H2O (4� ) to collect the black solids, which weredried under reduced pressure. The results of the elemental analyses and the XPS recordings indicated theabsence of salts in all of the samples. NH4Cl and NaCN were obtained from Sigma�Aldrich.

Hydrolysis of the �Insoluble Black HCN Polymers�. For hydrolysis, 6n HCl at 1108 for 24 h was used.The hydrolyzed samples were centrifuged. The insoluble black residues were collected, washed with H2O(3� ), dried under reduced pressure, and studied by the same techniques as the �insoluble black HCNpolymers�. The supernatants were collected, freeze-dried, and analyzed.

Standard Spectroscopy Techniques. UV/VIS Spectra were obtained using an Agilent 8453spectrophotometer. All spectra were recorded in DMSO. All samples were partially soluble. IR Spectrawere obtained using a Nexus Nicolet FT-IR spectrometer. The spectra were obtained in CsI pellets in thereflectance mode and were recorded from 4000 to 500 cm�1. 13C-CP-MAS-NMR Spectra were obtainedusing a Bruker Advance 400 spectrometer and a standard cross-polarization pulse sequence. Sampleswere spun at 10 kHz, and the spectrometer frequency was set to 100.62 MHz. A contact time of 1 ms anda period of 5 s between successive accumulations were used. The number of scans was 5000, and thechemical shift values were referenced to TMS.

Elemental Analysis. Elemental C, H, and N analyses were performed using a LECO CHNS-932elemental analyzer. For the elemental O analyses, a LECO VTF-900 analyzer was used.

XP Spectroscopy. XP Spectra were collected in an ultra-high vacuum (UHV) system equipped with amulti-channeltron hemispherical electron energy-analyzer (Phoibos, Specs GmbH) using an Al Ka X-raysource, and recorded at normal emission (incident angle 458). For the XPS experiments, 25% KBr pelletswere prepared for all the samples analyzed. During the recording, the samples showed a small chargeeffect induced by the X-ray radiation. Therefore, to maintain the energy position of the first scan, thepeaks were energy-corrected. High-resolution N 1s, C 1s, and O 1s core level spectra were recorded withan estimated resolution of 0.9 eV. The spectra were well described by the superposition of severalDoniach�Sunjic curve components. Data were analyzed with CASAXPS and FittXPS software. Theintensities of the XPS core levels were evaluated by the peak areas after subtraction of the standardbackground area according to the Shirley procedure. Assignment of the binding energy was performedusing standard spectra from the � Handbook of X-ray Photoelectron Spectroscopy� [40] according toprevious experimental data for which reference samples were used [21] [22] [41].

Organic-Compound Analysis by GC/MS. To identify polar organic compounds of biological interestin hydrolyzed supernatants, the following protocol was used: i) hydrolyzed samples were freeze-dried toremove H2O and HCl for 48 h; ii) ca. 6 mg of the dried residues in 100 ml of BSTFAþTMCS (N,O-bis(trimethylsilyl)trifluoroacetamide with Me3SiCl (Thermo Scientific)) were maintained at 608 over-night to obtain the corresponding TMS derivatives; iii) the derivatized samples were analyzed by GC/MSusing the following GC oven program: 608 (initial temp.) held for 1.5 min, heating to 1308 at 58/min andholding for 11 min, heating to 1808 at 108/min and holding for 10 min, heating to 2208 at 208/min andholding for 15, and heating to 3008 at 108/min and holding for 10 min. An injection volume of 2 ml was


used for each sample. The temp. of the injector was 2208, and the injections were performed in splitlessmode. The detector temp. was 3008. The flow rate was 9.2 psi. The GC/MS analyses were performed infull-scan mode on a 6850 network GC system coupled to a 5975 VL MSD with triple-axis detectoroperating in electron-impact (EI) mode at 70 eV (Agilent), using an HP-5 MS column (30 m�0.25 mmi.d.�0.25 mm film thickness) and He as a carrier gas. Identification of the GC/MS peaks was confirmedby comparison of retention times and mass spectra with those of external standards purchased fromSigma�Aldrich and Fluka.

Thermogravimetry (TG) Analysis. A Perkin-Elmer TGA-7 was used for TG measurements. Theinstrument was calibrated both for temp. and weight by standard methods. Non-isothermal experimentswere performed over a temp. range of 25–10008 at a heating rate of 108/min. The average sample weightwas ca. 10 mg, and the dry N2 flow rate was 100 cm3/min.

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Received February 2, 2011


new insights into the characterization of ‘insoluble black hcn polymers’

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