Preparation of 15 N-labeled Potassium Ferrocyanide for Tracer Studies

Isotopic labels are widely used to trace the fate and cycling of common environmental contaminants. Many of the labeled materials are not available commercially and, depending on the complexity of the substance, the label and the enrichment level, custom syntheses are costly. A simple, straightforward, and cost effective method for the preparation of a highly enriched, N-labeled potassium ferrocyanide (K4[Fe(CN)6]*3H2O) has been developed to meet the requirements of related tracer experiments and minimize their costs. In this case, the N label was used to quantify iron cyanide detoxification (biodegradation and/or transformation) within soil-plant-systems. N-labeled potassium cyanide (KCN) and a ferrous iron salt have been used for the synthesis. Extensive qualitative and quantitative analyses showed a product, entirely identical in its functional and elemental components to commercial non-labeled K4[Fe(CN)6]*3H2O and in its N enrichment to the KCN used for its synthesis. To investigate their behavior and fate in various environmental compartments, other labeled iron or metal cyanide complexes might be synthesized in analogous manner.


Introduction
Iron cyanide contamination in the environment is primarily of anthropogenic origin.One of the greatest iron cyanide sources are the sites of former manufactured gas plants and coke ovens, existing in a high number (>8700) in Europe and the United States (Wehrer, Rennert, Mansfeldt & Totsche 2011).Due to their high stability and complex behavior, iron cyanides might both be very persistent (Meeussen, Keizer, van Riemsdijk & de Haan 1992) or migrate from one environmental compartment to another (Meeussen, van Riemsdijk & van der Zee 1995;Theis, Young, Huang & Knutsen 1994) and pose serious wildlife and human health risks by releasing toxic free cyanide (Kjeldsen 1999).To better understand and predict the complex behavior of contaminants, isotopic tracers have increasingly been applied in the past decades, including in studies on biodegradation and detoxification of simple and complexed cyanide species within plant tissue (Ebbs, Bushey, Poston, Kosma, Samiotakis & Dzombak 2003;Ebbs, Piccinin, Goodger, Kolev, Woodrow & Baker 2008;Ebbs, Kosma, Nielson, Machingura, Baker & Woodrow 2010;Ebel, Evangelou & Schaeffer 2007;Samiotakis & Ebbs 2004).Yet, labeled complexed (including iron) cyanides are not commercially available and custom label costs are rather high, which might cause extensive study limitations.Therefore, a novel procedure has been developed for the synthesis of potassium ferrocyanide (K 4 [Fe(CN) 6 ]), incorporating the stable 15 N isotope of nitrogen.There exist a few K 4 [Fe(CN) 6 ] synthesis methods described so far, incorporating a 57 Fe complex label and intended for Mössbauer spectroscopy studies (Jaskula & Petlicki 1978;Ganguli, Das & Bhattacharya 1998).However, including a 57 Fe label in the complex requires several preparation steps, special laboratory conditions, and consumables.The synthesis method described in this paper is simple, straightforward, effective and can be conducted under standard laboratory conditions.Qualitative and quantitative analyses of the product showed that it is entirely identical in its functional and elemental components and 15 N enrichment to commercial products.This is the first summary of such a synthesis procedure.It saves time and costs, thus facilitating further research on the fate of iron (complex) cyanides in the environment.Particularly the low costs and the high yield allow for the application of the product in large scale studies or even directly under real field conditions.Both have so far not been possible.DIN EN ISO 14403:2002.The limits of detection (LOD) of the system are 0.01-1.10mg L -1 with limits of quantification (LOQ) of 0.02-1 mg L -1 .

Chemi
Total CN was measured following acid digestion and distillation of 5 ml of the standard solutions on a micro distillation system (MICRO DIST®, Lachat Instruments, A Hatch Company Brand, US) according to the Lachat Equivalent Method QuickChem 10-204-00-1-X.
Elemental Analysis.Iron (Fe 2+ ) and potassium (K + ) concentrations in the prepared standard solutions of the synthesized K 4 [Fe(C 15 N) 6 ]*3H 2 O and the commercial K 4 [Fe(CN) 6 ]*3H 2 O were determined on a Unicam iCAP 6000 Duo ICP-AES with a CID68 detector (Thermo Fisher Scientific, Germany).The concentration of chlorine (Cl¯) ions in the standard solutions was determined on a Dionex DX 500 and DX 120 IC system (Thermo Fisher Scientific, Germany).
Isotope ( 15 N) Analysis.The 15 N enrichment was analysed on a Thermo Delta V Advantage IRMS (Thermo Fisher Scientific, Germany) with a ConFlo III open split interface (Finningan, Thermo Electron Bremen, Germany) and coupled to a Vario EL III elemental analyzer (Elementar Analysensysteme GmbH Hanau, Germany).

Fundamental Vibrational Modes
The comparison of the synthesized and the commercial ferrocyanide complexes showed slight but not fundamental differences in the vibrational modes of the two substances.Klyuev (1965).
He explained the splitting with disrupted symmetry of the perfect octahedron of the [Fe II (CN) 6 ] 4-anion, caused by alteration of the valence state of the central atom, due to the small differences in the outer shell of the Fe 2+ and Fe 3+ ions (3d 6 in Fe 2+ and 3d 5 in Fe 3+ ).No explanation could be suggested for the absence of the 2062 cm -1 band in the spectrum of the K 4 [Fe(CN) 6 ] (Figure 2 b).In spite of the differences in the main vibrational band of the two potassium ferrocyanide substances, they both possessed the fundamental vibrational modes of the ferrocyanide ion.Hence, it could be concluded that the two products are qualitatively identical.
The single, very strong vibrational band at 2116 cm -1 in the spectrum of the commercial potassium ferricyanide Figure 2 c is characteristic for the ferricyanide ([Fe III (CN) 6 ] 3-) ion (Klyuev, 1965;Miller & Wilkins, 1952) and was not observed in the two potassium ferrocyanide spectra.Hence, oxidation of the ferrous to ferric iron during the synthesis was not likely to have taken place and the presence of ferricyanide (Fe III -C≡N) as an impurity could be excluded in both products due to the lack of the characteristic band at 2100-2120 cm -1 (Figure 2 a, b).
The spectrum in Figure 2 d was used as a reference to identify the presence of unreacted KC 15 N in the synthesized product.The absence of the 2077 cm -1 band in the spectrum in Figure 2 a and the very low levels of weak acid dissociable CN measured in the synthesized product (Table 1) were evident for the absence of KC 15 N in the synthesized product.These results indicate that the commercial and the synthesized labeled potassium ferrocyanide are qualitatively identical.

CN and Elemental Components
Table 1 shows the median concentrations and masses of the elemental components of K 4 [Fe(CN) 6 ] and K 4 [Fe(C 15 N) 6 ], calculated according to the mass of the substance used to produce the standard solutions.The concentration and mass of the weak acid dissociable CN fraction of the K 4 [Fe(C 15 N) 6 ] complex was considered equal to the median levels measured in the K 4 [Fe(CN) 6 ] solutions.That is, provided that all unreacted KC 15 N has been separated in the last steps of the K 4 [Fe(C 15 N) 6 ] synthesis, the measured weak acid dissociable CN (CN WAD , Table 1) levels in the standard solutions of both complexes should be identical.
The chemical purity of the commercial K 4 [Fe(CN) 6 ] complex also excludes the presence of high levels of Cl¯ in its standard solution.However, as a by-product of its synthesis (equation 3), Cl¯ might be present in the K 4 [Fe(C 15 N) 6 ] standard solution.The last two columns of Table 1 show the calculated maximum concentration and total mass of Cl¯ ions, which could theoretically be present in the standard solution, provided the KCl separation from the final product in the last steps of the synthesis was incomplete.
The measured concentrations and masses of the elements compiling the synthesized and commercial potassium ferrocyanide substances are shown in parentheses in Table 1.Although the measured total CN concentrations deviate from the set concentrations, the concentrations measured in the commercial and the synthesized products are identical.The mass of weak acid dissociable cyanide in the synthesized product is only slightly higher than that of the commercial product and thus comparable to it.The iron and potassium concentrations and masses in the standard solutions do not deviate much from the set values and are in the correct stoichiometric relation to the measured CN levels.
The actual Cl¯ content of the synthesized product was with ~1.6 mg less than the theoretical content (Table 1), which would have been measured, provided incomplete separation of the target product (K 4 [Fe(C 15 N) 6 ]) from the by-product (KCl) has taken place.Thus, the presence of high levels of Cl¯ impurity in the synthesized product could be excluded.The above-described results showed that although not of high chemical purity and with small deviations, the synthesized 15 N-labeled potassium ferrocyanide is quantitatively identical in its elemental components to commercial potassium ferrocyanide.

15 N Enrichment
The 15 N enrichment of the synthesized K 4 [Fe(C 15 N) 6 ]*3H 2 O was determined to ensure it was identical in enrichment to the 15 N-labeled KCN, used for the synthesis.In the K 4 [Fe(CN) 6 ]*3H 2 O sample both masses 28 ( 14 N 2 ) and 29 ( 15 N 14 N) could be detected, with the 28 signal being stronger than the 29 signal.Although the 15 N-enrichment of the commercial K 4 [Fe(CN) 6 ]*3H 2 O was with 0.3836 at% slightly elevated, it was close to that of the control (0.3662 at%).Hence, the enrichment of the substance was identical to the natural enrichment levels of 0.3663 at%.In the samples of the synthesized K 4 [Fe(C 15 N) 6 ]*3H 2 O and the commercial KC 15 N, the 28 mass could not be detected, e.g. the sample signals contained solely the 29 signal.Thus, it could be demonatrated that all nitrogen atoms in the two substances were present as the 15 N isotope.
Overall, the results from the qualitative and quantitative analyses of the synthesized potassium ferrocyanide showed that with small deviations, the product was, regarding its functional and elemental components, identical to the commercial potassium ferrocyanide and regarding its 15 N enrichment, identical to the KC 15 N used for its synthesis.

Conclusions
The above described synthesis method is simple, straightforward, effective and does not require special laboratory conditions, equipment or consumables.Nevertheless, it delivers a product, qualitatively and quantitatively comparable to commercial ones.In addition, the high custom costs for the label can greatly be reduced.To trace their behavior and fate in different environmental compartments, further labeled metal and/or iron cyanide species can be synthesized in similar manner to better facilitate cyanide contamination-related research.

Table 1 .
Median set concentrations and masses of the elemental components of the commercial and the synthesized 15 N-labeled potassium ferrocyanide (n=5).The values were calculated using the mass of the two substances used for the preparation of the standard solutions.Measured values are given in parentheses CN Fundamental vibrational modes: to identify the presence of the fundamental vibrational modes characteristic for ferrocyanide ions, the synthesized K 4 [Fe(C 15 N) 6 ]*3H 2 O was scanned on a Fourier transform infrared spectrometer (FTIR) and the resulting spectrum was compared to that of the commercial K 4 [Fe(CN) 6 ]*3H 2 O.In addition, commercial potassium ferricyanide (K 3 [Fe(CN) 6 ]) and the KC 15 N used for the synthesis were scanned to identify the presence of ferric iron or simply bound cyanide in the synthesized product.(ii)CN: the total water soluble (CN T H₂O ) and weak acid dissociable CN (CN WAD ) concentration and content of the synthesized K 4 [Fe(C 15 N) 6 ]*3H 2 O and the commercial K 4 [Fe(CN) 6 ]*3H 2 O were measured in standard solutions, prepared with the two substances and the results were compared.Small amount (~100 µg) of the prepared dry K 4 [Fe(C 15 N) 6 ]*3H 2 O and the commercial K 4 [Fe(CN) 6 ]*3H 2 O was scanned on a Tensor 27 FTIR spectrometer (Bruker Optik GmbH, Germany) in attenuated total reflection (ATR) mode and the resulting spectra were compared.
CN Analysis.Weak acid dissociable and total CN concentrations in the prepared standard solutions of the synthesized K 4 [Fe(C 15 N) 6 ]*3H 2 O and the commercial K 4 [Fe(CN) 6 ]*3H 2 O were determined spectrophotometrically on a flow injection analysis system (MLE FIA Compact, Gesellschaft für Analysentechnik HLS, Salzwedel, Germany) according to