Introduction

Chlorinated phenols (CPs), in general, have posed a serious environmental concern due to their high levels of production and toxicity to organisms at extremely low concentrations, earning them a notorious profile and their induction in the list of endocrine disruptor compounds (EDCs) [1]. Due to the wide usage of these compounds in industry as antiseptics, insecticides, wood preservatives, and as important intermediates in the production of pharmaceutical products [2–4], their derivatives as well as their degradation products can be found worldwide in surface and ground waters, bottom sediments, and atmospheric air and solids. In order to mitigate the impact of CPs in the environment and human health, their use has been subject to severe restriction policies and even banning in several countries [4]. Nevertheless, although their use has experienced a steady decline, these chemicals still remain in the environment due to their chemical stability aided by additional physical properties in the water, soil, or sediment that harbors them such as pH and temperature [5].

Methods for the extraction, detection, and monitoring of CPs using numerous analytical techniques exist. The majority of these rely heavily on their intrinsic UV absorption (e.g., LC-MS) [6, 7] while others depend on their semi-volatility brought upon by their proclivity for derivatization (GC-MS) [8]. Although the power of these aforementioned techniques is well established in the analytical chemistry realm, they still represent one-dimensional approaches for the study of these analytes. It is only when we dwell into analyses procured by two-dimensional methods and/or hyphenated GC and LC methods that we truly see their power in studying these species. An approach centered on the well-established and powerful NMR spectroscopy technique offers a multidimensional series of analysis that can all be carried out in one sample and in a non-destructive manner, an absent quality in the previously mentioned methods. However, a NMR approach for the analysis of even CPs could be demandingly challenging, particularly when a mixture of other similar species such as other types of alcohols and a matrix is present. Due to the ubiquitous presence of the proton (1H) in most organic molecules (including those in a given matrix), analysis of such a sample would represent a tedious and complex undertaking. Therefore, a method capable of selectively tagging CPs amid a mixture of other analytes would be an invaluable tool during the data deconvolution process of a given analysis. Furthermore, if this selective tag offers the means to analyze CPs in multiple NMR channels (e.g., 19F, 13C), it would tremendously aid in the unequivocal identification of each CP component in the mixture. After surveying several tagging functionalities for alcohols, particularly acidic phenols like CPs, we selected the difluoromethyl moiety (CF2H) for our studies. The choice of the difluoromethyl tag for labeling CPs was supported by several factors that can be directly attributed to the versatility of the NMR-amenable nuclei available in its structure. For example, using the difluoromethyl tag immediately introduces two readily detectable nuclei (1H and 19F) by NMR, which as a result of their natural abundance enjoy great sensitivity in this form of spectroscopic analysis (Fig. 1). In the NMR field, 19F constitutes the most abundant isotope of the element and, as mentioned briefly previously, it introduces NMR as a highly sensitive method for the detection of fluorine-containing species in a rapid manner (lowest possible number of scans for a given sample). In addition, the large chemical shift range exhibited by the 19F NMR channel allows for the clear resolution of structurally similar derivatized CPs. Lastly, the tag further benefits from possessing another reporting nucleus in the 1H species (CF2 H), whose chemical shift occurs between 6.7 and 6.9 ppm, located further upfield and well outside the resonances of aliphatic and olefinic 1H nuclei. Additionally, analysis of the aromatic 1H signals of the derivatized CP (except for pentachlorophenol, PCP) further aids in their structure determination and discrimination amongst isomers. Studying the derivatization using the 13C NMR channel, although possible as demonstrated in Table 1, would present additional challenges for this type of analysis due to the inherent low sensitivity of this nucleus in NMR spectroscopy. Thus, for the analysis of mixtures, we envision the combined use of 1H and 19F NMR to rapidly and qualitatively assess the presence of acidic phenols (including CPs), followed by further complementary analysis by GC-MS and/or LC-MS. The derivatization protocol described in this work efficiently achieves the difluoromethylation of CPs producing modified species that can be studied by 1H, 19F, and 13C NMR spectroscopy (Fig. 1).

Fig. 1
figure 1

Key features of the difluoromethylation strategy described in this work

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Table 1 Physical properties of chlorinated phenols used in this study and NMR chemical information for their difluoromethylated derivatives
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Materials and methods

All chemicals were purchased from commercial suppliers and used as received. Acetonitrile, methylene chloride, and the used chlorophenols were purchased from Sigma-Aldrich Chemicals (St. Louis, MO), and diethyl (bromodifluoromethyl) phosphonate was purchased from Matrix Scientific Inc. (Columbia, SC). Deuterated acetonitrile (CD3CN) was purchased from Alfa Aesar (Ward Hill, MA).

Derivatization protocol

The chlorinated phenol (0.08 mmol) was placed in a glass autosampler vial equipped with a small stir bar. The phenol was treated sequentially via pipette with deuterated acetonitrile [9] (CD3CN, 600 μL) and diethyl (bromodifluoromethyl) phosphonate (21.4 μL, 0.12 mmol, 1.5 equiv. to phenol). To the above solution, aqueous saturated potassium hydroxide (300 μL) was added via pipette. The vial was capped and stirred at ambient temperature for 5 min (see Electronic Supplementary Material (ESM), Fig. S1 and Table S1). After the stirring was done, the mixture was allowed to stand to reveal a biphasic mixture and 500 μL of the top layer (CD3CN) was aliquoted into another glass autosampler vial containing anhydrous sodium sulfate (Na2SO4, 50 mg). The dried, organic fraction was filtered into a 5-mm NMR tube through a cotton plug and treated with 100 μL of a 0.17-M solution of hexafluorobenzene in CD3CN (19F NMR internal standard).

NMR analysis

Spectra were obtained using a Bruker Avance III 600-MHz instrument equipped with a Bruker TCI 5-mm cryoprobe (Bruker BioSpin, Billerica, MA) at 30.0 ± 0.1 °C. 1H NMR (600 MHz), 19F NMR (565 MHz), and 13C NMR (150 MHz) were recorded in CD3CN. 1H NMR chemical shifts are calibrated with respect to residual partially deuterated acetonitrile centered at 1.94 ppm. 19F NMR chemical shifts are calibrated with respect to the singlet given by hexafluorobenzene at −164.9 ppm. Lastly, for 13C NMR, the septet centered at 1.79 ppm from CD3CN was used for the spectral calibration.

GC-MS analysis

A 6890 Agilent GC with a 5975 MS detector equipped with a split/splitless injector was used for the analysis. The GC column used for the analysis was an Agilent DB-5MS capillary column (30 m × 0.25 mm id × 0.25 μm i.f.). Ultra high purity helium was used as the carrier gas at 0.8 mL/min. The injector temperature was 250 °C, and the injection volume was 1 μL. The oven temperature program was as follows: 40 °C, held for 3 min, increased at 8 °C/min to 300 °C, held for 3 min. The MS ion source and quadrupole temperatures were 230 and 150 °C, respectively. Electron ionization was used with ionization energy of 70 eV. The MS was operated to scan from m/z 29 to m/z 600 in 0.4 s.

Results and discussion

The difluoromethyl (CF2H) moiety has been a key player in the pharmaceutical industry where its role as an isosteric group for the methyl moiety has been, and continues to be, exploited for its superior chemical attributes. Even though its size is comparable to that of the methyl group, its electronic properties are significantly different due to the presence of the fluorine atoms in its makeup. Due to the importance of this group for the aforementioned reason, it is no surprise that many methods have been developed for its introduction in molecular targets [10]. Although some of the methods thus far developed require the use of heating and long reaction times, a method that makes use of diethyl (bromodifluoromethyl) phosphonate (DBDFP) developed by the Zafrani group provides a rapid and environmentally benign option [11]. Due to its high reactivity under basic conditions (pH ∼12), reactions using DBDFP are often carried out at low temperatures (−78 °C) followed by warming up the mixture to room temperature once the reagent has been used to modify a phenolic moiety. The proposed mechanism for the overall transformation is outlined in Scheme 1. Thus, in the highly basic conditions that the reaction is carried out, DBDFP rapidly reacts with the base to produce a bromodifluoromethyl carbanion that spontaneously decomposes, yielding the bromide anion and difluorocarbene. Reaction of the fleeting difluorocarbene with the nucleophilic phenoxide ion produces an anionic intermediate that rapidly protonates to furnish the difluoromethylated phenol [12]. The ease of formation of the phenoxide ion, especially in the case of the chlorinated phenols [13], is greatly favored under these conditions due to their low pK a values (Table 1). Note that most unwanted products will be soluble in the aqueous phase (i.e., chlorophenoxide anion, diethylphosphate) and will not be carried onto the organic phase where the newly derivatized CP now resides. The organic phase can be directly analyzed by NMR and GC-MS after drying over Na2SO4, yielding information that is unique for each derivatized CP (Table 1). The fact that DBDFP can accomplish the efficient derivatization of PCP in a very short amount of time (5 min), compounded to its liquid state at room temperature, makes it an attractive alternative for the derivatization and study of CPs.

Scheme 1
scheme 1

Proposed mechanism for the difluoromethylation reaction

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After having evaluated the efficiency of the approach in derivatizing CPs, we proceeded to test its selectivity for tagging these species in the presence of other structurally diverse alcohols. To this end, we prepared a mixture in CD3CN consisting of PCP (0.08 mmol) with 12 other structurally diverse alcohols (each spiked at 0.08 mmol) and these included primary (2-methyl-1-butanol, 2-methyl-1-pentanol), secondary (pinacolyl alcohol), tertiary (1-methylcyclopentanol, 3-ethyl-3-pentanol), N-substituted β-amino alcohols (N,N-diisopropylaminoethanol, N,N-dimethylaminoethanol, N-methyldiethanolamine), long-chained alcohols (2-decanol, 1-nonanol), and thioether- and sulfone-containing alcohols (2,2′-thiodiethanol, 2,2′-sulfonyldiethanol) (ESM, Fig. S2 and S3). Once the mixture was treated sequentially with DBDFP and aqueous KOH solution (pH = 12.2), it was stirred for 2 min and the organic layer analyzed by 1H and 19F NMR after drying over Na2SO4. Treatment of the equimolar alcohol mixture with 1, 1.5, 2, and 4 equivalents of DBDFP resulted in the efficient and selective derivatization of only PCP. The use of excess of DBDFP does not cause a surge of interfering signals in the vicinity of the OCF2H-derived triplet at ∼δ = 6.76 ppm, but signals localized in the δ = 0.7–1.1 ppm range arising from the reagent’s hydrolysis and by-products begin to increase in magnitude (ESM, Fig. S2). This observation was further confirmed by the GC-MS analysis of the mixture where 1.5 equivalents of DBDFP was used for the derivatization. The GC chromatogram demonstrated that the only products arising from the procedure belong to PCP and its minor contaminant 2,3,4,6-tetrachlorophenol, while the 12 alcohols in the mixture remained underivatized (ESM, Fig. S3). During our initial assessment of the protocol, we carried out the derivatization for a space of less than 2 min leading to what appeared to be the quantitative derivatization of PCP by NMR analysis. However, it was found that running the procedure for <2 min did not result in the complete derivatization of PCP (ESM, Fig. S3). Therefore, after conducting optimization studies on the protocol, it was found that 1.5 equivalents of DBDFP along with stirring for 5 min is enough to secure the full derivatization of PCP (ESM, Table S1 and Figures S6 to S17). Lastly, the efficiency of the protocol was evaluated by testing it out in a mixture of five CPs. The five CPs used for this study were 4-chlorophenol, 3,5-dichlorophenol, 3,4,5-trichlorophenol, 2,3,4,5-tetrachlorophenol, and PCP. It was found that all five CPs smoothly undergo the derivatization and that each product retains its unique chemical shift (1H and 19F NMR) (ESM, Fig. S4 and S5). Using the chemical shift values for the derivatives in both NMR channels and adding the information that each CP’s aromatic signals provide (i.e., aromatic substitution), one can deduce the exact nature of the original CP in a given mixture. Lastly, an additional, strong attribute of the process is its biphasic nature allowing its adaptation for CP analysis on organic as well as aqueous samples once the derivatization has taken place.

Conclusion

Our experiments have demonstrated the efficacy of diethyl (bromodifluoromethyl) phosphonate at difluoromethylating the endocrine disruptor pentachlorophenol and related CPs for their detection and analysis by multinuclear NMR. The reagent is convenient to use as it is a liquid at room temperature, and once it has reacted, its products are environmentally benign. Furthermore, the derivatization reaction is expedient in nature (5 min) and can be conveniently carried out at room temperature to furnish difluoromethylated derivatives that exhibit unique 1H, 19F, and 13C NMR spectra. Due to its biphasic nature, the derivatization can be applied to both aqueous and organic mixtures. In addition, the protocol demonstrates that CPs can be selectively derivatized in the presence of various kinds of aliphatic alcohols, underscoring the potential superiority of the approach over other general derivatization methods that indiscriminately modify all analytes in a given sample. Due to the ease and mild conditions involved in its execution, this methodology should find wide applicability in the derivatization and qualitative analysis of not only CPs but other environmentally relevant phenolic compounds as well. Most importantly, and as briefly alluded to in the present work, the protocol lends itself as an additional, non-destructive technique for the analysis of acidic phenols in conjunction with other techniques such as GC-MS and LC-MS.