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ANALYTICAL CHEMISTRY, VOL. 50, NO. 6, MAY 1978
Determination of Biphenyl and Eight Biphenols in Microbial Extracts by Gas Chromatography and Thin-Layer Chromatography Patrick J. Davis, Lisa K. Jamieson, and Robert V. Smith" Drug Dynamics Institute, College of Pharmacy, University of Texas at Austin, Austin, Texas 78712
range. We proposed that the HFB-GC procedure might be useful for the separation of potential phenolic biphenyl metabolites. In addition, TLC systems were investigated to extend the utility of this methodology to a more extensive array of potential hydroxylated products. T h e devised analyses were designed for application to microbial metabolism studies.
Thin-layer and gas chromatographic methods have been developed for the determination of biphenyl and eight of its mono- or dihydroxylated phenolic analogues in microbiological media and homogenates of the fungus, Cunninghamella elegans. Biphenyl is analyzed by gas chromatography following n-heptane extraction of alkalinized systems. The biphenols are extracted at pH 6 with ethyl acetate and chromatographed as their heptafluorobutyrate derivatives. Using naphthalene and 1-naphthol as internal standards for biphenyl and the biphenols, respectively, recoveries of 88 % or better were achieved with all but two of the compounds analyzed while standard deviations were less than 5 %. Six thin-layer chromatographic systems were developed for separation and detection of the biphenyl compounds in microbial extracts. Chromogenic spray reagents are described which assist in distinguishing between biphenols with similar chromatographic mobilities.
EXPERIMENTAL Apparatus. A Shimadzu Model GC-4BM gas chromatograph wit,h dual flame ionization detectors and temperature program capability was employed throughout. Oven temperatures were as specified for the various analyses and included the following: A, 130 "C for 24 min, increased to 150 "C until the end of analysis; B, 110 to 150 "C at 'L"/min (starting at solvent front) and held at 150" until the end of analysis; C, 120 "C for 17 min, increase to 150 "C at, 2"/min and held at I50 "C until the end of analysis. The detector was maintained at 310 "C. Gas flow rates were as follows: carrier gas (nitrogen), 40 mL/min; hydrogen, 30 mL/min; and compressed air, 350 mL/min. A silanized glass column (Shimadzu Model 4M63.0-20) 2 m X 3 mm i.d. was packed with 3% OV-17 on chromosorb W-HP, 100--120mesh (Analabs, New Haven, Conn.) and conditioned at 260 " C for 24 h with normal carrier gas flow. Reagents and Standard Compounds. Biphenyl, 4-HBP, 2,2'-DHBP, 4,4'-DHBP (Aldrich); 2-HBP, I-naphthol, 2-naphthol (Sigma);3,3'-DHBP, 3,4-DHBP (RFR Corp.); 3-HBP, 2,5-DHBP (Eastman); and naphthalene (Fisher) were shown to be homogeneous by TLC and GC and gave melting points within acceptable limits to literature values. 2,6-Dichloroquinone-Nchlorimide CDCQ) was purchased from Eastman and heptafluorobutyric anhydride (HFBA, 95%) was obtained from Aldrich. Solvents utilized for GC analyses (benzene, acetonitrile, heptane) were of spectral grade (Burdick and Jackson). All other solvents were analytical reagent grade. Thin-Layer Chromatography. TLC analyses were carried out on silica gel GF,,, plastic plates (Brinkmann) and were developed with one of the following solvent systems: A, benzene-acetic acid (51);B, diethyl ether-Skellg B (1:l);C, benzene-methanol (9:1), D, benzene-acetic acid (10:l) E, benzenemethanol--acetic acid (36:4:1); F, benzene -methanol-ammonium hydroxide (5870)(36:6:1). Following development, air dried plates were visualized by one or more of the following methods: A , observing quenching of 254-nm induced fluorescence; B, spraying then 5% NaOH in 50% ethanol, and with Pauly's reagent (61, finally heating at 100 "C for 5 min; C, spraying with sodium cobaltinitrite spray (13,14),and then heating at 100 "C for 5 min or spraying with 570 KaOH in 50% ethanol; D, spraying with Gibbs reagent (2,6-dichlorobenzoyuinone-Nchlorimide, 2 % in abs. ethanol) ( 1 5 ) ; followed by heating at 100 "C for 5 min. exposing the plate to vapors of concentrated ammonium hydroxide, and finally allowing the plate to remain exposed in the air overnight. With each spray reagent, colors Lvere noted after each successive step. Culture Conditions. Cunninghanieila elegcins iATCC 92453 \vas maintained on agar slants and grown in soybean meal glucose medium as described previously ( 6 ) . For extraction of reference compounds from cell homogenates, the entire contents of' single second-stage flasks were fully homogenized (Polytron PT-10, Brinkmann Instruments) and adjusted to 25.0 mL with deionized
Biphenyl is used in the citrus fruit industry as a fungistatic agent. I t has also been employed as a model xenobiotic in studying in vivo and in vitro metabolism in mammalian species (1-5). Recent interest in biphenyl metabolism stems from correlations of microbial and mammalian metabolism (61, and its proposed use as a screening tool for carcinogens (7). Studies of t h e microbiological metabolism of biphenyl in our laboratory required analytical methodology for the extraction and chromatographic separation of anticipated phenolic metabolites. Specifically, previous metabolism studies (1-7) and preliminary microbiological screening indicated the need to separate and quantitate biphenyl, the monophenols, 2-, 3-, and 4-hydroxybiphenyl (HBP),and the diphenols 2,2'-, 3,3'-, 4,4'-, 3,4-, and 2,5-dihydroxybiphenyl (DHB). Raig and Ammon ( 1 , 2 ) separated several hydroxylated biphenyls as their trimethylsilyl (TMS) ethers using gas chromatography (GC) with a nitrile silicone (XE-60) or methyl phenyl silicone (SE-52) column. Meyer and Scheline (3)( I -7) resolved the TMS-derivative of several hydroxybiphenyls on a n OV-1 column. T h e isomeric 2- and 3-hydroxybiphenyls while an are reportedly separable on a 3% SE-30 column (8), unspecified SE column has been employed to resolve 2 - , 3-. and 4-hydroxybiphenyl ( 9 ) . 4 number of authors have described thin-layer chromatographic (TLC) systems for separating several potential phenolic metabolites of biphenyl ( 1 , 6 , 8-10). We have recently found that isomeric phenolic aporphines are readily separable by GC as their heptafluorobutyryl (HFB) derivaties ( I I , 12). T h e particularly appealing aspect of the use of such derivatives is the apparent ease of derivatization (with H F B anhydride) even with sterically hindered phenols, and their potential for analysis by electron capture detection, thereby extending assay sensitivity into the nanogram/mL 0003-2700/78/0350-0736$01 0010
C
1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 6, MAY 1978
water. A 2.0-mL aliquot of this homogenate was used for individual extractions described as cell-homogenate studies. Derivatization Kinetics of Hydroxybiphenyls. A 100-pL aliquot of acetonitrile solution containing 0.1 mg each of the hydroxybiphenyls (except 2,5-DHBP), 1-naphthol,and biphenyl was added to 100 pL benzene in a freshly silylated 1.0 mL Reacti-vial (Pierce). Derivatization was initiated by the addition of 25 pL HFBA and the sealed vial was placed in a heating block set at 65 "C (Reacti-therm, Pierce). A 2-pL aliquot was removed through the Reacti-vial silicone septum at 15, 45, 7 5 , and 120 min and injected directly into the GC operating under temperature condition A. Since, 2,5-DHBP gave a retention time identical to biphenyl under these conditions, its derivatization was studied independently using 1-naphthol as the internal standard. Attempts at using amine catalysts for derivatization were conducted by substituting 0.25% triethylamine in benzene for both benzene and acetonitrile in the derivatization procedure (16). At the end of the appropriate reaction time, the solution was extracted once with 100 pL 1.0 M phosphate buffer (pH 6.5) to remove excess triethylamine, and 2-pL aliquots of the benzene layer were analyzed by GC. Routine Derivatization of Hydroxybiphenyls. For routine GC analyses, standard compounds or extracts were dissolved in 100 pL acetonitrile and placed in a 1.0-mL Reacti-vial. Benzene (100 pL) and 2,5-pL aliquots of HFBA were added and the vials were heated at 65 "C for 90 min (Reacti-therm. Pierce). Aliquots of 2 jiL were injected directly into the GC. Extraction of Biphenyl and Derivatives. All glassware was freshly silanized with 2 % trimethylsilyl chloride in benzene for 15 min and heated at 110 "C for 30 min prior to use. For hydroxybiphenyl analysis, duplicate samples were prepared consisting of 50 wg 1-naphthol (internal standard) and each hydroxybiphenyl derivative at 10, 25, 50, 75, and 100 pg in a total volume of 100 pL acetonitrile in 1-mL Reacti-vials. Following HFB-derivatization, 2-pL aliquots were injected into the chromatograph operating under temperature program B. For extraction analysis, 100-pLaliquots of a 5 mg/mL acetonitrile solution of each hydroxybiphenyl, biphenyl, and 1-naphthol (internal standard) was used to spike 2-mL aqueous samples (medium or cell homogenate). After addition of 3 mL of 0.2 M phosphate buffer (pH 6.0), the mixtures were extracted by rocking with 2-mL portions of nitrogen-purged ethyl acetate for 15 min. Following centrifugation, a 200-pL aliquot of the organic layer was transferred to a 1-mL Reacti-vial, taken to dryness under a stream of nitrogen, and derivatized with HFBA as indicated above. A 2.0-pL aliquot was then analyzed by GC using temperature program B. For hiphenyl analysis, duplicate samples were prepared consisting of 50 wg naphthalene (internal standard) and biphenyl at 10,25, 50, 7 5 , and 100 pg in a total volume of 100 pL acetonitrile in 1-mL Reacti-vials; 2-pL aliquots were analyzed by GC operating isothermally at 130 "C. For recovery determinations, 50-pL aliquots of a 10 mg/mL acetonitrile solution of biphenyl and naphthalene (internal standard) were spiked into 2.0-mL aqueous samples (medium or cell homogenate). After the addition of 1.0 mL of 10% NaOH (final pH 13.2) the samples were extracted by rocking for 15 rnin with 2.0-mL portions of ra-heptane. The phases were separated by centrifugation, and a 2-gL aliquot of the n-heptane layer was injected directly into the GC operating isothermally at 130 "C ( T ,naphthalene, 3.4 min; TI biphenyl. 9.6 min). The alkaline phase resulting after n-hexane extraction neutralized with 0.5 mL 5 N HCI, and the resulting acidic solution was extracted as described for hydroxybiphenyls to evaluate this hack extraction routine for composite analysis of biphenyl and its hydroxylated analogues (see Results and Discussion). Calculations. Peak height ratios were obtained by dividing the peak height of each hydroxybiphenyl-HFB derivative by the peak height o f the HFB derivative of 1-naphthol. Calibration curves were prepared using least squares regression to determine the best fit line for the data obtained from the standards and were expressed as the peak height ratios from the standards vs. the concentration of the hydroxybiphenyl in the final solution to be injected. Calibration curves for biphenyl using naphthalene were similarly generated. Calibration curves generated consistently gave correlation coefficients of 0.995 or better and were revalidated on a day-to-day basis by injection of two standard samples.
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Figure 1. Derivatization of hydroxybiphenyls by HFBA. Peak height ratios are generated using biphenyl as an unreactive internal standard. Compound abbreviations: (A) 1-naphthol, (B) 2,2'-DHBP, (C) 2-HBP, (D) 2,5-DHBP, (E) 3-HBP, (F) 4-HBP, (G) 4,4'-DHBP, (H) 3,4'-DHBP, (I) 3,3'-DHBP
2-Naphthol was used as an internal standard to control the GC step when absolute recovery data was generated on the hydroxybiphenyls and 1-naphthol. Values for unknown concentrations of the hydroxybiphenyls or biphenyl in aqueous medium or cell-homogenate samples were obtained by computer interpolation (Wang 600) from the calibration curves.
RESULTS AND DISCUSSION GC P r o c e d u r e s . Heptafluorobutyryl (HFB) derivatives were chosen for development of a GC procedure for the hydroxylated biphenyl compounds indicated in Table I. The choice was based on ease of derivatization of hindered phenols, relatively mild reaction conditions. and potential future analyses of these derivatives a t nanogram levels by electron capture detection (11, 12). Derivatization at 65 "C in a mixture of acetonitrile and benzene was found to proceed quantitatively in 15 min with all of the phenols but 2-HBP and 2,2'-DHBP (see Figure I) which is probably due to steric hinderance in the 2-substituted analogues. Surprisingly, however, 2,5-DHBP does not exhibit parallel resistance to acylation by H F B anhydride. Considering the composite reactivities of compounds represented in Figure 1, a minimum reaction time of 90 min was utilized to ensure complete derivatization of all components. T h e necessity for elevated temperature and prolonged reaction time was also noted by Raig et al. ( 2 ) in preparing t h e T M S ether derivatives of several hydroxylated biphenyls (with N,0-bis(trimethy1si1yl)acetamide). In our studies, elevation of temperature above 65 "C did not result in a significant decrease in reaction times nor did the use of an amine such as triethylamine significantly catalyze the reaction (16). Most of the HFB-derivatives of phenolic biphenyls and biphenyl itself could be separated through isothermal development on an OV-17 column. However, there were difficulties in resolving biphenyl and 2,5-DHBP, and 4,4'-DHBP possessed excessively long retention times a t temperatures which rapidly eluted the remaining hydroxylated compounds. Temperature program A eliminated the second problem, while the lack of resolution of biphenyl and 2,5-DHBP remained (see Figure 2). Temperature program B gave satisfactory resolution of all components with short analysis time (see Figure 3). For samples containing low levels of biphenyl, or if biphenyl is selectively removed prior to analysis, program B is considered satisfactory. However, it might be anticipated that higher levels of biphenyl would obscure any 2,5-DHBP present. It was felt that selective extraction of biphenyl under strongly alkaline conditions would resolve this problem ( I 7 ) . Unfortunately, the instability of several phenolic components
ANALYTICAL CHEMISTRY, VOL. 50, NO. 6, MAY 1978
738
Table I. Recoveries of Hydroxybiphenyls and Biphenyl from Medium and Cell Homogenate % Recovery (t SD)
Compound Medium Cell homogenate Hydroxybiphenyls' 2-HBP 98.5 (1.0) 71.1 (4.0) 3-HBP 98.0 (3.2) 100.2 (4.3) 4-HBP 98.0 (3.2) 100.4 (4.4) 2,2'-DHBP 94.0 (4.7) 58.7 (4.7) 3,3'-DHBP 96.0 (4.8) 93.9 (3.2) 4,4'-DHBP 94.5 (4.9) 93.6 (4.3) 3,Q-DHBP 97.5 (3.7) 103.5 (3.4) 2,5-DHBP 100 (4.9) 91.2 (2.2) Biphenyla 87.8 (4.6) 88.8 (4.0) ' Mean of 4 to 5 determinations; concentration of 250 Separately extracted and analyzed (see ExperHg/mL. imental section). 8
,
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l
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-
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1
,
20
10
0
30
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Figure 2. Gas chromatogram of hydroxybiphenyls and biphenyl using temperature program A. Peak assignments: (A) 1-naphthol, (6) 2,2'-DHBP, (C) 2-HBP, (D) biphenyl, (E) 2,5-DHBP,(F) 3-HBP, (G) 4-HBP, (H) 3,4-DHBP, (I) 3,3'-DHBP, (J) 4,4'-DHBP
Table 11. TLC of Hydroxybiphenyls and Biphenyl Compound
I iA
Biphenyl 2 -HBP 3-HBP 4-HBP 2,2'-DHBP 3,3'-DHBP 4,4'-DHBP 3,4-DHBP 2,5-DHBP
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Flgure 3. Gas chromatogram of hydroxybiphenyls and biphenyl using temperature program 8. Peak assignments as presented in Figure 2
w
VI
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a
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a Y
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+ V Y
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TIME
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Figure 4. Gas chromatogram of hydroxybiphenyls and biphenyl using temperature program C. Peak assignments as presented in Figure 2 during alkaline extraction (see below), including 2,5-DHBP, necessitated the development of a third program. Temperature program C, although more lengthy, does resolve each component of the mixture (see Figure 4). Thus, a single initial analysis using this program allows initial judgement as to the
presence of 2,5-DHBP and permits decision as to the use of the other two temperature programs, particularly A. T h e requirement for temperature programming to separate t h e TMS-ether derivatives of various hydroxybiphenyls by GC was observed previously (1-3). In our system, however, isothermal program A may be used when samples d o not contain 2,5-DHBP. Recovery experiments were designed for applicability to microbiological systems currently being evaluated for biphenyl metabolism. In a study reported previously ( 6 ) ,extractions with ethyl acetate a t p H 2.0 were utilized. Studies currently being conducted indicated t h a t such conditions effect coextraction of a number of complicating medium components, presumably organic acids produced by the cultures. A p H value of 6.0 was chosen to avoid this problem while maintaining the p H well below the pK,'s of the phenols. Standard curves generated for the hydroxybiphenyls using 1-naphthol as the internal standard were linear over the concentrations used (10 to 100 pg) with correlation coefficients of 0.995 or better. Recovery experiments for both medium and homogenized cells were conducted, and the results obtained are presented in Table I. Recoveries of all components from medium ( n = 4) were nearly quantitative while extraction from cell homogenates ( n = 5 ) gave lower recoveries, although acceptable accuracy and precision was maintained (see Table I). Recovery of 1-naphthol (the internal standard used in hydroxybiphenyl analyses) was analyzed using 2-naphthol as an internal standard to control the GC step. Since recoveries from medium (99 f 2.3%, n = 3) and cell homogenate (96 1.0% n = 3) were nearly quantitative, t h e values in Table I may also be considered absolute recoveries. I n the case of biphenyl, recoveries of 89% were obtained on repetitive analyses with acceptable precision. Failure to
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 6, MAY 1978
Table 111. TLC Visualization of Hydroxybiphenyls with Modified Gibbs ' Reagenta
Table IV. TLC Visualization of Hydroxybiphenyls with Modified Pauly 's Reagent (Diazotized Sulfanilic Acid)
Sequential treatment and color produced
Compound Gibbs, Heat 2-HBP re d-yello w salmon 3-HBP yellow yellowbrown 4-HBP rust salmon 2,2'-DHBP yellow yellowbrown 3,3' -DHBP red-yellow brown 4,4'-DHBP lavender tan purple 3,4-DHBP maroon brown 2.5-DHBP yellowgreen
Air Ammonia Exposure, 18 h vapors dark blue rust grey-blue purple tan blue
tan maroon
slate blue grey-green black green
purple grey-tan black green
2% 2,6-Dichloroquinone-N-chloroimide in absolute ethanol. a
obtain higher recoveries may be related to the strong affinity t h a t biphenyl exhibits for glass and plastics (18). A number of other gas chromatographic assays for biphenyl have been devised (18-20), although these usually involve vapor phase analysis or distillation of adsorbed biphenyl for fungiciderelated analyses. A comparison of the recoveries from medium and cell homogenate in Table I indicates that standard curves utilizing t h e internal standards will be required for other cultures or other biological media, though there is good indication t h a t t h e GC methods described herein should be applicable to a number of biological systems. Attempts were made to combine the two extraction procedures (biphenyl and hydroxybiphenyls) for the selective removal of biphenyl prior to analysis of hydroxybiphenyls analogous to t h a t described by Wiebkin et al. ( I 7) and for quantitation of residual biphenyl for mass balance experiments. Such a procedure would allow for the use of the essentially isothermal program A by removing biphenyl interference with 2,5-DHBP. By using a mixture of all hydroxybiphenyls, biphenyl, and internal standards (1-naphthol and naphthalene), it was found that biphenyl and naphthalene were exclusively extracted into the heptane layer at p H 13 with no contamination by phenols. Following neutralization of t h e aqueous layer and extraction with ethyl acetate, however, it was observed that both 2,5- and 3,4-DHBP were decomposed by the alkaline extraction step. Nitrogen purging of the entire extraction, bisulfite addition (150 mg/extraction), and a combination of the two were attempted to stabilize these potential metabolites without success. Thus, the use of the various temperature programs to evaluate the significance of 2,5-DHBP present will be necessary as described earlier. Biphenyl recoveries a t p H 13 are shown in Table I and are acceptable for conducting mass balance experiments. TLC Procedures. Thin-layer chromatography has been qualitatively useful in studies of the metabolism of biphenyl. Several solvent systems were developed previously (1,6,8-10) with a limited number of biphenols. These were evaluated and a number of new solvent systems were developed for separation of biphenyl and t h e eight biphenols indicated in Table 11. As noted by other workers ( I ) , 3-HBP and 4-HBP are difficult t o separate by TLC. One might anticipate analogous problems with 3,3'- a n d 4,4'-DHBP. However, one solvent system (E) was found to partially resolve t h e latter two compounds. A number of reagents previously used with phenols were utilized for t h e visualization of hydroxybiphenyls. Gibb's reagent (ZO), 2,6-dichlorobenzoquinone-N-chloroimide, was particularly useful in identifying poorly resolved compounds. This reagent has been used previously with some of these
739
Sequential treatment of color produced Compound
Paule y 's reagent
Base
Heat
2-HBP 3-HBP 4-HBP 2,2'-DHBP 3,3'-DHBP 4,4'-DHBP 3,4 -DHBP 2,5-DHBP
salmon norx yellow-green brown no rx green orange -brown green
orange-salmon faint yellow yellow orange-salmon faint yellow green orange-salmon green wl yellow edge
brown-green brown-green brown-green dark green brown-green brown orange-brown brown-green w/ green center
Table V. TLC Visualization of Hydroxybiphenyls with Sodium Cobaltinitrite Compound 2-HBP 3-HBP 4-HBP 2,2 ' -DHBP 3,3'-DHBP 4,4'-DHBP 3,4-DHBP 2,j-DHBP
Sequential treatment and Sodium cobaltinitrite red-brown faint yellow faint yellow grey-brown faint grey green ochre green
color produced Heat brown-gold brown-gold brown-gold brown-gold brown-gold brown-gold brown-gold green
derivatives (I). In this study, colors were found to be diagnostic ,5 min after spraying, after heating, after exposure to ammonia vapors, and finally after air exposure for 18 h (see Table 111). Diazotized sulfanilic acid (Paulg's reagent) was also useful (see Table IV) as described earlier for a limited number of derivatives (6). Colored products are also formed with sodium cobaltinitrite (see Table V) used previously for a number of phenols, including 4-HBP (13). These sensitive spray reagents alone, and in combination, allow for t h e qualitative identification of even poorly resolved biphenols such as 3,3-DHBP and 2,5-DHBP. T h e resulting T L C procedures, coupled with the GC analyses described herein, allow for the sensitive differentiation and quantitation of the hydroxybiphenyls examined a t levels analogous to those being examined in microbial cultures.
ACKNOWLEDGMENT T h e authors are grateful to David T. Gibson and his coworkers for helpful suggestions at the outset of the described studies. LITERATURE CITED V. P. Raig and R. Arnmon, Arzneim.-Forsch., 22, 1399 (1972). V. P. Raig and R. Arnmon, Arzneim.-Forsch.. 20, 1266 (1970). T. Meyer and R. R. Scheiine, Acta Pharmacoi. Toxicoi., 39, 419 (1976). T. Meyer, J. C. Larsen, E. V. Hansen, and R. S. Scheline, Acta Pharmacoi. roxicoi., 39, 433 (1976). T. Meyer, Acta Pharmacol. Toxicoi., 40, 193 (1977). R. V. Smith and J. P. Rosazza, Arch. Biochem. Biophys., 161, 551 (1974). F. J. McPherson. J. W. Bridges, and D. V. Parke. Biochem. J., 154, 773 (1976). D. T. Gibson, R. L. Roberts, M. C. Wells, and V. M. Kobai, Biochem. Biophys. Res. Commun., 5 0 , 211 (1973). D. Cateiani, C. Soriini, and V. Treccani, Expenentia, 27, 1173 (1971). R. V. Smith, J. P. Rosazza, and R. A. Nelson, J , Chromatogr.,95, 247 (1974). D. M. Baaske, J. E. Keiser, and R. V. Smith, J . Chromatogr., 140, 57 (1977). J. R. Miller, J. W. Blake, and T. Tobin, Res. Commun. Chem. Pathoi. Pharmacoi., 15, 447 (1976). I. S. Bhatia. K . L. Bajaj, A. K. Verma, and J. Singh, J . Chromatogr., 62, 471 (1971). R. V. Smith and M. J. Garst, Anal. Chim. Acta, 65, 69 (1973). H. D. Gibbs, J . Bioi. Chem., 72, 649 (1927). H. Ehrsson, T. Waiie, and H. Broteli, Acta Pharm. Suec., 8, 319 (1971). P. Wiebkin, J. R. Fry, C. A. Jones, R. Lowing, and J. W. Bridges, Xenobiotica, 6, 725 (1976).
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 6, MAY 1978
(18) A. W. Wells, S. M. Norman, and F. P. Atrops, J . Gas. Chromatogr., 1, 19 (1963). (19) P. L. Davis and K. A . Munroe, J . Agric. Food Chem., 25, 426 (1977). (20) H. Beernaert, J . Chromatogr., 77, 331 (1973).
RECEIVEDfor review December 8, 1977. Accepted January
30, 1978. This work was supported by grant F-690 from the Robert A' U'elch Foundation and a seed grant Obtained through the Biomedical Research Support Grant awarded from the National Institutes of Health to the University of Texas a t Austin.
Liquid-Liquid Extraction of Zinc from Aqueous Iodide Solutions with 4-(5Nonyl)pyridine in Benzene Shamas-ud-Zuha and Mohammad E j a z " Nuclear Chemistry Division, Pakistan Institute of Nuclear Science and Technology, Nilore, Ra walpindi, Pakistan
An investigation has been made on the system 4-(5-nonyl)pyridine-Zn( 11)-I. Optimal conditions for the extraction of zinc lave been given, based on a critical survey of the relevent factrapssuch as the effects of the acids, iodide ions, salting-out and complexing agents, and the metal concentration. The mechanism underlying these extractions is discussed on the basis of the results obtained from partition, slope-analysis and loading-ratio data. Distribution coefficients and separation factors of several elements relative to zinc have been reported for the optimum concentrations of hydrochloric, nitric, and sulfuric acid solutions containing 1 M potassium iodide. The presence of iron does not interfere with extraction. It has been shown that in neutral and weakly acid solutions, 445nony1)pyridine is a better extractant for iodide complexes of zinc than the commonly used aliphatic tertiary and quaternary amines, and carbon and phosphorus bonded oxygen-donor extractants.
High molecular-weight pyridine amines are an interesting and versatile class of extractants. Depending on conditions, these compounds behave as liquid anion exchangers and give salts with various acids ( 2 , 2 ) or are incorporated as ligands in t h e complex ions of elements, forming penetration complexes (3-5). Complex formation and extraction of some transition metal ions in a thiocyanate system using 4-(5nony1)pyridine and its analogues have been studied ( 3 , s ) . It has been shown that the weaker hydration of thiocyanate ions favors the formation of 4-(5nonyl)pyridine solvates whereas t h e strong hydrophilic character of common anions like chloride and nitrate does not. The present investigation makes use of hydrophobic iodide ions for the extraction of zinc by 445-nony1)pyridine dissolved in benzene from aqueous mineral acid solutions containing potassium iodide, instead of hydroiodic acid which decomposes on storage leading to a drop in iodide ion concentration. T h e use of these mixtures also makes it possible to control the selectivity of the separation by varying t h e concentration of the acids and alkali-metal iodide. Extraction of zinc from aqueous iodide solutions by liquid anion exchangers and oxygen-containing solvents has been studied (6-8). Our investigation reveals t h a t 4-(5nonyllpyridine extracts zinc from aqueous iodide solutions much more efficiently than the previously used extractants (6-8). In addition, the method has the advantage that zinc can be quantitatively extracted in a single extraction from 0003-2700/78/0350-0740$0 1 O O / O
water and a wide range of acid concentrations; a strict control of acidity is unnecessary.
EXPERIMENTAL Reagents. 4-(5-Nonyl)pyridine (NPy) was used as an extractant. The characteristics of this compound are reported elsewhere ( 5 , 9). Solutions of potassium iodide were made by AnalR). All dissolving anhydrous chemically pure salt (B.D.H. other chemicals were reagent grade or of the highest purity available. Deionized water was used for the preparation of all aqueous solutions. Procedure. Extractions were carried out at room temperature (23 & 3 "C) in 20-mL glass vials; equal phase volumes were used. The stock solution of the mineral acid containing trace amounts (
