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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
; ACKNOWLEDGMENT The authors thank Harry Reichard of Princeton Applied Research Corporation for initial assistance. Many helpful discussions with Allan Rosencwaig of Lawrence Livermore Laboratory and Robert Somoano of Jet Propulsion Laboratory are gratefully acknowledged. LITERATURE CITED
1
I
300
400
5cO
600
702
€02
I 933
WAVELENGTH ( n m )
Figure 12. Normalized photoacoustic spectrum of 0.35% TPP mechanically dispersed in BaSO,
dispersed sample in the PAS analysis of a highly colored material also was demonstrated. For compounds with low extinction coefficients, relatively large quantities of powder sample can be used in the PAS measurement. It has been clearly demonstrated that PAS can be employed effectively in obtaining the optical information of a wide variety of materials. We believe that the new analytical technique should be very useful in analyzing unknown samples when it is used properly in conjunction with many known chromatographic techniques.
Tynall, J. Proc. R. Soc. London 1881, 31, 307. Rontgen, W. C. Phil. Mag. 1881, 1 1 , 308. Bell, A. G. Phil. Mag. 1881, 1 1 , 510. Rosencwaig, A. Opt. Cornrnun. 1973, 7 , 305. Rosencwaig, A. Anal. Cbern. 1975, 47, 592. Rosencwaig, A. Phys. Today 1975, 28, 23. Rosencwaig, A.; Gersho, A. J . App. Phys. 1978, 47, 64. (8) Hershberger, W. R.; Robin, M. B. Acc. Cbern. Res. 1973, 6 , 329. (9) Somoano, R. E. "Organometallic Polymers"; Academic Press: New York. 1978; 165. (10) Somoano, R. B. Angew. Cbern., Int. Ed. Engl. 1978, 17, 238. (11) Gray, R. C.; Fishman, V . A.; Bard, A. J. Anal. Cbern. 1977, 49, 697. (12) Adams, M. J.; King, A. A,; Kirkbright. G. F. Analysf(London) 1978, 101, 73. (13) Adams, M. J.; Beadle, B. C.; Kirkbright, G. F. Analysf (London) 1978, 101, 553. (14) Adams, M. J.; Kirkbright, G. F. Analyst(London) 1977, 102, 281. (15) Adams. M. J.; Beadle, E. C.; Kirkbright, G. F. Ana/yst(London) 1977, 102, 569. (1) (2) (3) (4) (5) (6) (7)
RECEIVED for review February 16, 1979. Accepted June 11, 1979. A portion of this work was reported at the 176th Meeting of the American Chemical Society, Miami Beach, Fla., September 1978.
Interference of Oxidants in the Determination of Phenol by the 4-Aminoantipyrine and Ultraviolet Ratio Spectrophotometric Methods George Norwitz, John Farino, and Peter N. Keliher" Chemistry Department, Villanova University, Villanova, Pennsylvania 19085
A study is made of the interference of the oxldants CIO-, C102-, S 2 O t - , and NO3- with the 4C103-, Mn04-, H202, Cr20:-, amlnoantipyrlne (4-AAP) and ultraviolet (UV) ratio methods for the determination of phenol, and a method Is described for minimizing this interference by the addition of sodium arsenite, followed by dlstlllatiin. The nature of the interference essentially Involves oxidation of the phend, causing low resulls. The application of the UV ratio method without distlllatlon is limited because of the absorbance of oxidants in the UV region. The results by the 4-AAP method without distillation show that CIO- and Mn0,- Interfere markedly, H,02 Interferes less strongly, C102- and S20a2-do not Interfere unless present In fairly large amounts, while C103-, Cr2072-,and NO3- hardly interfere at all. When the solutions are distilled (heated), the interference of C102-, H,O,, and S20:- increases markedly.
The 4-aminoantipyrine (4-AAP) and ultraviolet (UV) ratio methods are among the most common methods used for the determination of phenol. The 4-AAP method (1-19) depends upon the purplish red color obtained on reacting 4-AAP with phenol in the presence of potassium ferricyanide (oxidizing
agent). The UV ratio or difference method depends upon the bathochromic shift (from about 270 to 290 nm) obtained when a solution of phenol is made alkaline ( 7 , 15, 20-28). It has been stated briefly that oxidants can interfere with the 4-AAP method by oxidizing the phenol and that this interference may be overcome by adding ferrous sulfate or sodium arsenite before the distillation (16-19). Apparently, no detailed study has been made of the effect of different oxidants in the UV ratio method. It is the purpose of the present paper to make a comprehensive study of the interference of oxidants with the determination of phenol by the 4-AAP and UV ratio methods and investigate ways to eliminate this interference.
EXPERIMENTAL Apparatus and Reagents. A Bausch and Lomb Spectronic 20 colorimeter (1-cm cell) was used. A Fisher phenol analyzer (25-27) monitored the bathochromic shift by use of two conventional hollow cathode lamps which are modulated in a time ratio mode. The distillation apparatus consisted of a 1-L round-bottomflask and a Graham condenser (with jacket 40 cm in length and an outer 24/40 joint at top) joined together by a connecting tube (Fisher No. 15-323D)with thermometer opening sealed off by a 10/30 ground glass stopper. The flask was heated by an electric mantle.
0003-2700/79/0351-1632$01,00/0 0 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
Distilled water and reagent grade chemicals were used (the sodium hypochlorite solution, 4 4 % NaClO. was “laboratory grade”). All solutions were stored in glass. The methyl orange, ammonium hydroxide (1to l), and phosphoric acid (1to 9) were dispensed by 4-oz dropping bottles (Arthur H. Thomas Co. No. 2248-B,4) that gave drops of about 0.08 mL (the volumes of the drops are important for the UV ratio method). The sodium hydroxide (20%) was dispensed with a medicine dropper about 14 cm long (Arthur H. Thomas No. 2248-F, 4) that was inserted into a narrow-mouth bottle about 10 cm high (to prevent attack of the plastic top or bulb). Phenol solution No. 1 (1mL = 1.00 mg phenol) was prepared by dissolving phenol (1.000 g) in water and diluting to 1 L in a volumetric flask. Phenol solution No. 2 (1 mL = 0.10 mg phenol) was prepared by diluting phenol solution No. 1 tenfold (prepared fresh daily). 4-AAP solution (2%) was prepared fresh daily. Potassium ferricyanide solution (8%) was prepared fresh weekly. 4-AAP Method, Preparation of Calibration Curue. Portions of phenol solution No. 2 (0.00, 1.00, 2.00,3.00,4.00, and 5.00 mL) were transferred to 125-mL Erlenmeyer flasks that had been calibrated at 100 mL with a 100-mL pipet. The solutions were diluted to the 100-mL mark and 2.0 mL of ammonium chloride solution (5%) was added. The solutions were rendered alkaline to pH 10.0 f 0.2 with ammonium hydroxide (1to 1) using a pH meter (about 16 drops or 1.3 mL of the ammonium hydroxide was required). Then, 2.0 mL of 4-AAP solution (2%) and 2.0 mL of potassium ferricyanide solution (8%) were added, the solutions were mixed, and after 15 min the absorbances were measured at 510 nm against distilled water. The absorbance of the blank was deducted and the absorbances were plotted against mg of phenol. S t u d y o f Interference o f Oxidants without Distillation. Three-milliliter portions of phenol solution No. 2 were added to the calibrated 125-mL Erlenmeyer flasks containing water. Various amounts of oxidant solutions (NaClO, NaClO,, KC103, KMnO,, HzOz, KzCrz07,KZSzO8,and KNOB)were added, the volumes brought up to the 100-mLmark, and the solutions allowed to stand for about 30 min. The color was then developed as described in the preparation of the calibration curve. S t u d y of the Interference of Oxidants after Distillation without S o d i u m Arsenite. Fifteen-milliliter portions of phenol solution No. 2 were added to 1-L round bottom flasks (calibrated at about 550 mL) containing about 500 mL of water. Five milliliters of CuS0,.5Hz0 solution (10% ) were added to prevent bacteriological degradation of the phenol and eliminate sulfide interference ( I 7 , 18). Various amounts of oxidant solutions were added and the solutions allowed to stand about 30 min. Five drops of methyl orange indicator (0.05%) were added and the solutions immediately neutralized just to a pink color with phosphoric acid (1 to 9) (if the solutions were not neutralized immediately, the indicator sometimes was destroyed by the oxidant). The volumes were brought up to 550 mL and 500 mL of the solutions distilled into 500-mL Erlenmeyer flasks (previously calibrated at the 500-mL mark). Care was taken not to evaporate the solutions to dryness (this would cause the results for phenol in the presence of oxidants to be very low because of vapors that would be driven over). Sufficient amounts of the samples were transferred to 125-mL Erlenmeyer flasks to attain the 100-mL mark and the phenol was determined as described in the preparation of the calibration curve. NOTE. The 1-L flasks were cleaned with hydrochloric acid (1 to 1) between runs. S t u d y o f the Interference o f Oxidants after Distillation with S o d i u m Arsenite. The procedure was the same as distillation without sodium arsenite up to the point at which the solutions had stood for 30 min. Then, 10 mL of sodium arsenite solution (10%) and the methyl orange indicator were added, the phosphoric acid was added to the color change, the solutions were allowed to stand for 10 min more, and the distillations were performed. UV Ratio Method. Preparation o f Calibration Curue. Portions of phenol solution No. 2 (0.00, 1.00, 3.00, 5.00, 6.00, and 7.00 mL) were transferred to 100-mL volumetric flasks and the solutions were diluted to the mark. A portion of the solution was transferred to the cell, 1 drop of phosphoric acid (1to 9) was added with mixing and the instrument was set to 0% absorption. Two
1633
drops of sodium hydroxide solution (20%) were added with mixing and the percent absorption was read (normal sensitivity). The cell and stoppers were washed thoroughly with running hot tap water and the next sample was analyzed. The percent absorption was converted to absorbance using appropriate tables, the absorbance of the blank was deducted, and the absorbances were plotted against mg of phenol. S t u d y o f Interference o f Oxidants zuithout Distillation. Three-milliliter portions of phenol solution No. 2 were transferred to 100-mL-volumetricflasks containing water. Various amounts of oxidant solutions were added, the volumes brought up to the mark, and the solutions allowed to stand for about 30 min. The phenol was then determined as described in the preparation of the calibration curve for the UV ratio method. S t u d y o f Interference o f Oxidants after Distillation without S o d i u m Arsenite. The procedure was the same as described for the distillation without sodium arsenite in the 4-AAP method up to the point at which the solution was distilled and 500 mL of solution collected. A portion of the solution was then transferred to the cell and the phenol determined as described in the preparation of the calibration curve for the UV ratio method. S t u d y of Interference of Oxidants after Distillation with S o d i u m Arsenite. The procedure was the same as described for the distillation with sodium arsenite in the 4-AAP method up to the point which the solution was distilled and 500 mL of solution collected. A portion of the solution was then transferred to the cell and the phenol determined as described in the preparation of the calibration curve for the UV ratio method.
RESULTS AND DISCUSSION Calibration Curves. The calibration curve for the 4-AAP method was prepared by diluting to 100.0 mL and then adding ammonium chloride solution, ammonium hydroxide, 4-AAP solution, and potassium ferricyanide solution, since it is customary in the 4-AAP method to take a 100-mL aliquot after the distillation and then add the reagents (17, 18). For the UV ratio method, t h e addition of 1 drop of phosphoric acid (1t o 9) t o the cell to give a p H of about 2.5, followed by the subsequent addition of 2 drops of sodium hydroxide solution (20%) to give a pH of about 11.5, was found to be satisfactory. T h e addition of the 1 drop of phosphoric acid was a precaution; ordinarily, the same result for phenol was obtained if the acid was omitted (this is in keeping with the observations of Martin e t al. (24) who found that an initial p H of u p to 6 was satisfactory). T h e sodium hydroxide solution must be added in such a manner as to prevent attack of the rubber bulb or plastic top of dropping bottles; otherwise high results will be obtained from carbon compounds (dissolved from the rubber or plastic) which absorb in the UV range. In comparing the recommended dropwise additions with those suggested by previous investigators, it must be borne in mind that the drops obtained from different medicine droppers vary in size from about 0.035 to 0.08 mL. Also, various size cells have been used. The calibration curves obtained for the 4-AAP method and UV ratio methods followed Beer’s law u p to absorbances of about 0.6 and 0.55, respectively. An alternative approach to converting the percent absorption t o absorbance in the UV ratio method was t o plot percent absorption directly. T h e curve so obtained was essentially a straight line u p t o about 50% absorption (about 0.3 absorbance) and then deviated. This was in keeping with earlier work a t Villanova University which was concerned with the determination of traces of phenol by the UV ratio method (the instrument used in that work had a somewhat different readout system than the Fisher Phenol Analyzer) (25). Absorbance was used in both the 4-AAP and UV ratio methods in the present investigation to better compare the methods. T h e blanks obtained for the 4-AAP and UV ratio methods were low (absorbances of 0.01 and 0.00, respectively). Effect of Oxidants on the 4-AAP and UV Ratio Methods without Distillation. The oxidants studied were
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
NaC10, NaC1OZ,KC103, KMn04,H202,KzCrz07,KzS20s, and KNOB. All concentrations were finally expressed in millimoles per 100 mL. Preliminary work established that the UV ratio method for phenol (like UV methods for the determination of organic compounds in general) was not readily applicable to direct analysis of solutions containing oxidants or diverse anions because of absorbance of these substances in the UV region. I t is well known that many common anions (excluding such anions as sulfate, phosphate, phosphite, hypophosphite, perchlorate, borate, cyanide, and chloride in the absence of heavy metals) absorb in the UV (29). Insofar as the phenol analyzer was concerned, this absorbance interference manifested itself by preventing the instrument from being set to zero or by giving meaningless high results. In the case of CrzO:-, negative readings were obtained because of the change of CrzO: to (3-042-on the addition of NaOH. The results obtained for phenol by the 4-AAP and the UV ratio methods in the presence of oxidants are shown in Table I. The results obtained for the 4-AAP method covered a wide gradation of oxidant while the results for the UV ratio method are limited for the reasons indicated above. In general, the results by the 4-AAP and UV ratio methods are comparable when both methods are applicable. I t is obvious that the prime interference effect of oxidants in the determination of phenol involves oxidation of the phenol, causing low results. T h e results show that C10- and Mn04- interfere markedly, HzOzinterferes less strongly, ClOc and SzOz- do not interfere unless present in fairly large amounts, while C l o y and NO3do not interfere at all. The Cr20:- does not react with phenol, but large amounts cause high results due to the yellow color of the Crz072-. The oxidation of the phenol at room temperature required up to 30 min in some cases, However, there were no significant changes if the solution stood for 30 min to 4 h after adding the oxidant. A peculiarity of the behavior of large amounts of SzOi2-was the formation of a red color after the addition of the 4-AAP, before the addition of the potassium ferricyanide. When the potassium ferricyanide was subsequently added, the usual full color development was achieved but the color faded rapidly after 5 min (see footnote of Table I). The reason for the formation of the initial color in the presence of large amounts of Sz082-is that a t p H 10, SZOs2-behaves like potassium ferricyanide (oxidant) in forming the phenol 4-AAP dye complex. In fact, SzOs2-has been used, as a substitute for potassium ferricyanide in the 4-AAP method (10, 141. Experiments were conducted to ascertain whether an excess of other oxidants would behave like potassium ferricyanide or SzOs2-in producing a color with phenol and 4-AAP at a pH of 10. It was found that C10- produced a faint pink color and Mn04- apparently produced a brownish color which was masked by the brownish color of Mn4+. The other oxidants had no effect. T h e exact mechanism of the reaction of phenol with oxidants to cause low results is uncertain. Many oxidants (e.g., permanganate (30)),when present in large excess, oxidize phenol to carbon dioxide and water; however, when only a small amount of oxidant is present, it is probable that intermediate substances such as dihydric phenols are formed. The reaction of C10- in aqueous solution is complicated by Clz + H 2 0 the following equilibrium: HClO C1- H' (the reaction tends to go to the right in acid solution and to the left in basic solution). It has been shown that Clz and C10can react with phenol to produce 2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,5-dichlorophenol, 2,6-dichlorophenol, and 2,4,6-trichlorophenol (31-33). Probably, CIOz- behaves like CIOz,which reacts with phenol to produce
+
+
Table I. Results for Phenol by the 4-AAP and UV Ratio Methods in the Presence of Oxidants without Distillation (0.30 mg of Phenol Present per 100 mL)
oxidant none NaClO
NaC10,
mmol of oxidant per 100 mL 0 0.00250 0.00500 0.01 00 0.0250 0.100 0.500 0.00250 0.01 25 0.0250
mg of phenol found per 100 mL 4- A M uv 0.301 0.314 0.295 0.203 0.032 0.032 0.045 0.308 0.314 0.288
0.0500
0.125 0.250
KC10 ,
KMnO,
1.25 2.50 3.75 6.25 0.830 1.66 12.5 16.6 0.00100
0.00200 0.00400 0.0100 0.100
0.200 4
0
2
0.0100
0.0500 0,100 0.500 1.00 5.00 10.0
0.308 0.314 0.314 0.175 0.131 0.314 0.308 0.308 0.314 0.301 0.268 0.255 0.084 0.012b 0.012b 0.321 0.308 0.308 0.282 0.236 0.084 0.026 0.301
0.303 0.294 0.267 0.237 NUa NU NU 0.294 0.294 0.303 0.303 0.259 NU NU NU NU NU 0.303 0.303 0.303 0.303 0.285 0.285 NU NU NU NU 0.321 0.330 0.340 NU NU NU NU 0.294 0. 276c 0.237c 0.194c
0.000732 0.00146 0.00292 0.301 0.00732 0.0146 NU 0.0292 NU 0.732 0.321 NU 1.46 0.327 NU NU 3.66 0.492 0.313 0.285 0.321 K*S,O, 0.321 1.57 0.285 0.314 0.267 3.13 6.26 d NU KNO, 0.0337 0.301 0.303 0.303 0.0674 0.294 0,337 0.308 0.294 1.69 NU 2.53 0.295 3.37 0.308 NU 8.43 0.295 NU 0.295 33.7 NU ' NU = not useable because of absorbance of the oxidant in the ultraviolet range. MnO, precipitated o n addition of the 4-AAP and was filtered off prior to making the absorbance readings. Result not valid because of the change of Cr20,2-to Cr0,'- on the addition of NaOH. 0.314, 0.262, 0.228, and 0.210 mg of phenol was found after making the absorbance readings in 5 , 15, 30, and 6 0 min, respectively. K, Cr2 0 ,
1,4-benzoquinone, 2,6-dichloro-1,4-benzoquinone, and 2chlorophenol (34). Many of the substituted phenols react less readily with 4-AAP than does phenol and the colors produced may have different regions of maximal absorbance and molar absorptivities than phenol (1-8,1&15,17, 18). In the UV ratio method, the substituted phenols may have different batho-
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
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Table 11. Results for Phenol by the 4-AAP and UV Ratio Methods in the Presence of Oxidants after Distillation with and without Sodium Arsenite (0.30mg of Phenol Present per 100 mL1 mg of Dhenol found Der 100 mL mg of phenol found per 100 mL with NaAsO, with NaAsB, mmol of without NaAsO, (1 9) mmol Of without NaAsO, (1 g ) oxidant per oxidant aer oxidant 100 mLQ 4-AAP UV 4-AAP UV oxidant uv 4 -AAP uv 1 0 0 m L Q 4-AAP none NaClO
0 0.000200 0.000500 0.00200 0.00500 0.0100
0.0200 0.0500 NaC10,
0.000100
0.000250 0.000500 0.00100
0.295 0.288 0.27 5 0.262 0.228 0.157 0.150 0.000
0.288 0.275 0.275 0.262 0.210 0.177 0.051 0.045
0.294 0.303 0.294 0.237 0.259 0.223 0.187 0.092 0.294 0.294 0.285 0.267 0.223
0.301 0.294 0.308 0.303 0.288 0.285 0.262
0.267
0.223 0.208 0.051 0.071
0.295 0.294 0.282 0.294
KMnO
0.00800 0.0400
0.032
0.076
0.164 0.026
0.187 0.038
H,O,
0.000400 0.00100
0.288 0.275 0.262 0.228 0.071 0.019 0.005 0.005
0.285 0.303 0.285 0.223 0.092 0.038 0.143 0.023
0.295
0.294
0.288 0.295 0.288 0.194 0.194
0.303 0.294 0.285 0.187 0.168
0.0040 0.0100 0.0400 0.100
0.200 1.00 2.00 3.00 K, Cr20, 0.0146 0.293 0.146 0.293 1.46 2.20 2.93 0.00025 K2S,O, 0.00050 0.00075 0.0025 0.0050 0.0250 0.0626 0.250 0.626 KNO , 0.676 1.69 3.38 13.5
0.285 0.216e 0. 295 0.303 0. 143e 0.295 0.303 0.0100 e 0.282 0.303 0.100 0.203 0.230 e 0.282 0.252e 0.150 0.105 0.155 e e 0.282 b 0.250 0.038 0.019 0.008 e e 0.275 KClO, 0.00166 0.282 0.294 0.321 0.303 0.00332 0.282 0.312 0.285 0.0332 0.275 0.276 0.294 0.166 0.282 0.155 0.332 0.275 0.303 0.295 0.303 0.038 0.288 0.303 0.303 0.288 0.294 0.830 0.282 0.000 0.282 0.294 1.66 0.255 0.294 0.295 0.294 0.275 0.294 3.3 2c 0.125 >1.0d 0.282 0.285 0.000 0.008 0.202 0.223 0.0000800 0.282 KMnO, 0.285 0.076 0.119 0.000200 0.295 0.303 0.321 0.295 0.303 0.288 0.000400 0.255 0.244 0.288 0.285 0.285 0.282 0.285 0.295 0.000800 0.248 0.252 0.301 0.303 0.00200 0.164 0.187 0.262 0.244 0.295 0.303 0.282 0.285 0.00400 0.091 0.108 0.210 0.230 These figures were obtained by dividing the millimoles of oxidant added before the distillation by 5. A pink color and high absorbance readings were obtained on addin the NaOII. This was the maximum amount tested in the distillation procedures because of possible hazards. Distillate smelled of chlorine. e Result not valid because of interference from the small amount of Cr,O.*- that distilled. 0.00250 0.00500
b b b
chromic shifts and molar absorbtivities than phenol (7, 15, 20, 21, 23-26, 28). Effect of O x i d a n t s on the 4-AAP a n d UV Ratio Methods a f t e r Distillation without Sodium Arsenite. As can be seen from Table 11, excellent recoveries for phenol (average 98.7%) were obtained in the absence of oxidants, using the apparatus and distillation technique described. In accordance with the recommendations of the American Public Health Association (17) and ASThl (18),copper(I1) sulfate was added to the solutions before the distillation and 500 mL of solution was distilled. Copper(I1) sulfate was not added to the solutions that had not been distilled because Cu2+would interfere with the 4-AAP and UV ratio methods by precipitating as copper ferricyanide and copper hydroxide, respectively. The results obtained by the 4-AAP and UV ratio methods after distillation without sodium arsenite are shown in Table 11. The results by the two methods are usually comparable when both methods are applicable. I t can be seen from a comparison of Tables I and I1 that when the solutions are distilled (heated), the interference of C10-, C103-, and Mn0,increases slightly, the interference of ClOZ-, H2OZrand S208,increases markedly, while Cr,072- and NO3- still have little oxidizing effect. The 4-AAP method after the distillation tolerates more C1-20,'- than before distillation, because the error due to the intense yellow color of large amounts of Cr2O7'- is eliminated. The UV ratio method after the distillation is not applicable in the presence of more than about 0.0025 mmol of CIOz- per 100 mL; in the presence of more
0.288 0.288 0.288 0.282 0.295 0.288 0.275 0.308 0.295 0.228 0.097 0.019 0.051
than this amount of C10< the solution turns a faint pink on adding the sodium hydroxide and the absorbance increases rapidly. No significant blank was obtained on distilling ClO; without phenol. The UV ratio method after the distillation is not applicable in the presence of more than about 0.015 mmol of CrzOi2- per 100 mL because some Crz072-distills (as indicated by the faint yellow color of the distillate) and interferes in the manner previously noted in the nondistillation procedure. When using the 4-AAP method after the distillations, it was noted that a pink color was sometimes obtained on adding the 4-AAP (before the addition of the potassium ferricyanide). There are certain contradictory forces that affect the oxidizing capabilities of the different oxidants during distillation. First of all, it would be expected that C10-, C102-, HzOz,and S202-would be partially or completely destroyed during the prolonged boiling ( 3 5 ) . Secondly, it is probable that C10-, ClOz-, and H202would partially distill (the C10- as HC10, the CIOz- as CIOz produced by decomposition, and the HzOzas such) (35). Finally, C103-, MnOL, Cr,072-, and NO; would become stronger in their oxidizing capabilities as the solution decreases in volume; however, as the solution decreases in volume. the amount of phenol left in the distillation flask would also decrease. Effect of O x i d a n t s on t h e 4-AAP a n d UV Ratio Methods a f t e r Distillation w i t h S o d i u m Arsenite. Various reducing agents were tested for their applicability to the elimination of excess oxidant prior to the distillation. It was found that powerful reducing agents such as hydrazine
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
Table 111. Maximum Amount of Oxidant (mmol per 100 mL) that Could Be Present without Causing an Error Greater than tO.02 rng of Phenol in Solutions Containing 0.3 mg of Phenol per 100 mL oxidant NaClO NaC10, KCIO, KMnO, HzO, K, Cr, 0, KZSZoB
KNO,
without distillation 4 -AAP uv 0.0050 2.5 >16.6
0.0010 0.5 0.7 3.1 >33.7
distillation without NaAsO, 4 -AAP uv
0.0025 0.050 >16.6 0.0020 0.010 0.0010 1.6 1.7
0.00020 0.00010 0.8 0.00020
0.00050 0.00050 1.7 0.00020
0.00040
0.0040
2.2 0.00050 >13.5
Table IV. Amount of Oxidant (mmol per 100 mL) That Oxidized Approximately One Third of the Phenol in Solutions Containing 0.30 mg of Phenol per 100 mL distillation without without distillation NaAsO, oxidant 4-AAP 4-AAP NaClO 0.01 0.007 NaClO, 3.5 0.003 KCIO, >16.6 2.4 KMnO, 0.006 0.0015
H,O, K,Cr,O, K,S,O, KNO,
1.9 >3.66 >3.13a >33.7
0.01 5 >2.93
0.001 >13.5
See footnote of Table I.
distillation with NaAsO, (1g ) 4-AAP uv 0.025
0.10 >3.32 0.005 1.9 >2.93 0.25 >13.5
0.020 0.12 >3.32 0.007 1.9
>1.46b 0.30 ~13.5
See footnote of Table 11.
were not useable because they reacted with phenol. Ferrous sulfate, a moderate reducing agent, was not entirely satisfactory, probably because ferrous hydroxide precipitated in the nearly neutral solution. Sodium arsenite was found to be the most satisfactory reductant and it was found that 1 g of this substance would readily eliminate the interference of the amount of oxidant that might be found in water or wastewater. Green copper orthoarsenite (CuHAs03)precipitated when the sodium arsenite was added to the solution containing the copper sulfate but this precipitate dissolved when the solution was neutralized to methyl orange with phosphoric acid (1to 9). Sodium arsenite is an alkaline salt, so a fairly large amount of the phosphoric acid (3.8-4.0 mL) was required for the neutralization. Only about 2 or 3 drops of the phosphoric acid was required for neutralization in the absence of sodium arsenite. The results obtained for the determination of phenol by the 4-AAP and the UV ratio methods after distillation with sodium arsenite are shown in Table 11. The results by the 4-AAP and UV ratio methods are generally comparable except in the case of more than about 0.3 mmol of Cr20:- per 100 mL. In the presence of more than this amount of Cr20?-, the CrzO:- is not completely reduced by the sodium arsenite, even on heating, and a small amount distills and interferes with the UV ratio method in the manner previously described. It can be seen from Table I1 that the sodium arsenite treatment decreases the interference of C10-, ClO,, C103, MnOc, H20z, and SzOa2-(the effect on H2O2and SzOa2-is especially pronounced). Various means were considered to compare more explicitly the results for phenol obtained without distillation, distillation without sodium arsenite, and distillation with sodium arsenite. Initially, an attempt was made to prepare a series of curves but this did not prove feasible because of the diverse nature of the interference. Finally, two tables were prepared that together seemed to portray the overall effect of oxidants. The first table (Table 111) shows from the data in Tables I and I1 the maximum amount of oxidant that could be present
distillation with NaAsO, ( 1 g) 4 -AAP uv
0.015 0.00050 >13.5
0.00050 0.0025 3.3
0.00050 0.0025 3.3
0.00040
0.00040
1.0 2.2 0.025 >13.5
1.0 0.3 0.06 >13.5
without causing an error greater than *0.02 mg of phenol, as determined by the 4-AAP and UV ratio methods. The second table (Table IV) shows by extrapolation of the data in Tables I and I1 the amount of oxidant that would be expected to oxidize about one third of the phenol. For the latter table, results are given for the 4-AAP method for the nondistillation and distillation procedures and for the 4-AAP and UV ratio methods for the distillation procedure using sodium arsenite. It can be seen from Table IV that within reason C10-, C103-, Mn04-, and H,02 show about the same interference for samples that had been distilled with sodium arsenite as for samples that had not been distilled. In contrast, C l o y and S202-show considerably greater interference for samples that had been distilled with sodium arsenite than for samples that had not been distilled. The probable explanation of the latter finding is that the reduction of Cloy and S202-by the sodium arsenite does not go to completion until the solutions are heated; however, when the solutions are heated, more phenol is oxidized by the CIOz- and &Os2-.
ACKNOWLEDGMENT The authors are indebted to John D. Johnson, Spectrogram Corporation, North Haven, Conn., for his encouragement and advice. LITERATURE CITED Emerson (Eisenstaedt), E. J . Org. Chem. 1938, 3, 153-65. Emerson (Eisenstaedt), E. J . Org. Chem. 1943, 8, 417-19. Gottlieb, S.;Marsh, P. B. Anal. Chem. 1946, 78,16-19. Shaw, J. A. Anal. Chem. 1951, 23, 1788-92. Ettinger, M. 8.; Ruchhoft, C. C.; Lishka, R. 1. Anal. Chern. 1951, 23, 1783-68. Dennis, M. Sewage Ind. Wastes 1951, 23, 1516-22. Mohler, E. F., Jr.; Jacob, L. N. Anal. Chem. 1957, 29, 1369-74. LaCoste, R. J.; Venable, S. H.; Stone, J. C. Anal. Chem. 1959, 37, 1248-49. Gordon, G. E. Anal. Chem. 1980, 32, 1325-26. Ochynski, F. W. Analyst (London) 1960, 85, 278-81. Faust, S.D.; AM, 0. M. J . Am. Water Works Assoc. 1962, 54, 235-42. Faust, S. D.; Mikuiewicz, E. W. WaterRes. 1967, 7 , 405-18, 509-22. Faust, S. D.; Lorentz, F. G. Proceedings of the 11th Ontario Industrial Waste Conference, June 1964, pp 173-201. Goulden. P. D.; Brooksbank, P; Day, M. 6. Anal. Chern. 1973, 45, 2430-33. Afphan, 6.K.:Belliveau, P. E.;Larose, R. H.;Ryan, J. F, Anal. Chirn. A& 1974, 77, 355-66. Gales, M. E., Jr.; Booth, R. L. J . Am. Water Works Assoc., 1976, 68, 540-42. Standard Methods for the Examination of Water and Waste Water", 13th ed.; American Public Health Association: Washington, D.C., 1971; pp 501-10. ASTM Designation D-1723-70, Standard Methods of Test for Phenolic Compounds in Water, American Society for Testing and Materials: Philadelphia, Pa., 1978. "Methods for Chemical Analysis of Water and Wastes", STORET Method No. 32730, US. Environmental Protection Agency: Cincinnati, Ohio. 1974. Murray, M. J. Anal. Chem. 1949, 27,941-45. Schmauch, L. J.; Grubb. H. M. Anal. Chem. 1954, 26, 308-11. Smuilin, C. F.; Wetterau, F. P. Anal. Chem., 1955, 27, 1836-38. Wexler. A. S. Anal. Chem. 1963, 35, 1936-43. Martin, J. M., Jr.; Orr, C. D.; Kincannon, C. D.; Bishop, J. L. J . Water Pollut. Control Fed. 1967, 39,21-32. Fountaine, J. E.; Joshipura, P. 8.; Keiiher, P. N.; Johnson, J. D. Anal. Chern. 1974, 4 6 , 62-66. Smith, L. S. U.S. Environmental Protection Agency, Environmental and Monitoring Support Laboratory, Report EPA-600/4-76-048, "Evaluation of Instrument for the Determination of Phenol in Water"; Cincinnati, Ohio, Sept. 1976.
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979 (27) Fisher Scientific Co. Bull. No. 441/7-028-10, Fisher Phenol Analyzer. (26) Williamson, J. A. "Rapid Determinationof Phenol Content of Water", paper Dresented at International Water Resources Assoc. Seminar on Water Resources Instrumentation", Chicago, Ill., June 1974. (29) Buck, R. P.;Sirghadeja, S.; Rogers, L. B. Anal. Chem. 1954, 26, 1240-42. (30) Kolthoff, I.M.; Beicher, R. "Volumetric Analysis", Vol. 111; Interscience: New York, 1957; p 113. (31) Buttschell, R. H.; Rosen, A. A,; Middleton. F. M.; Ettinger, M. B. J . Am. Water Works Assoc. 1959, 51, 205-14.
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(32) Grimley, E. B.; Gordon, G. J . Phys. Chem. 1973, 77, 973-78. (33) Murphy, K. L.; Zaloum, R.; Fulford, D. Water Res. 1975, 9 , 389-96. (34) Gkbsz, U. Chem. Stosowana, Ser. A . 1966, 10,211-20,; Chem. Abstr. 1967, 66, 3 1 8 4 4 ~ . (35) Prescott, A. 6.; Johnson, 0. C. "Qualitative Chemical Analysis", 10th ed.; Van Nostrand: New York, 1933, pp 479, 480, 522, 531, 533.
RECEIVEDfor review April 2, 1979. Accepted June 4, 1979.
Catalytic Determinations of Enzymes and Metal Ions by Sample Injection in Closed-Loop Flow Systems S. M. Ramasamy, A. Iob, and Horacio A. Mottola" Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74074
Catalysts (enzymes or metal ions) can only be determined in closed-loop systems if, after signal detection, they are physically removed from the system or rendered inactive by an inhibitor. Successful removal of the enzyme glucose oxidase (by physical adsorption on phenoxyacetylcellulose traps) and copper ions (by controlled potential electrodeposition) are described as examples leading to the determination of these catalysts by sample injection in closed-flow systems. The catalytic determination of copper combines a unique electrochemical removal of catalyst and slmultaneous regeneration of the monitored species.
Interest, among analytical chemists, in continuous-flow analyses has increased steadily since 1975 as a result of work with unsegmented continuous-flow analyzers ( I ) . This trend has recently received an adequate forum in internationally attended meetings ( 2 , 3 ) . Several ancillary techniques, when implemented, add to those advantages already recognized in the use of unsegmented streams (4). Typical examples of these ancillary techniques are the use of closed-loop flow-through systems and main reagent regeneration (5-81, whenever their implementation is feasible. The main thrust of this paper is to demonstrate that catalytic determinations, either of enzymes or metal ions, are possible in closed-loop, continuous-flow analyzers. In open systems, in which the catalyst, unreacted reagents, and products are sent to waste after detection, no logistic difficulties can be expected for the determination of catalysts. On the other hand, in the use of continuously circulated reservoir solutions (5-8), accumulation of injected catalyst in the system results in fast deterioration of base line and fast consumption of reservoir solution, rendering the use of a closed system unattractive. Physical removal of the catalyst after signal detection or chemical inhibition (under kinetically controlled conditions) permits the catalytic determinations by sample injection into unsegmented closed-loop flows. As examples of this implementation, the repetitive determinations of the enzyme glucose oxidase (monitoring oxygen consumption from the oxidation of glucose) and the metal ion catalyst copper(I1) (by monitoring the Fe(III)-S2O3*-indicator reaction) are presented here.
DETERMINATION OF GLUCOSE OXIDASE ENZYME Apparatus. An experimental setup basically identical to the one shown in Figure 1 of reference 6 has been used for deter0003-2700/79/0351-1637$01.00/0
mination. The square sponge traps of phenoxyacetylcellulose were positioned in the vessel indicated as point B in the figure and kept suspended in the solution by adequate stirring. The flow rate from reservoir to detection zone was 23 mL/min, and the pumping rate was about 45 mL/min. The potential applied to the working electrode in glucose oxidase determinations was 4.60 V vs. the SCE. Reagents and Solutions. Glucose oxidase was Purified Type I1 from Aspergillus niger and catalase was purified powder from bovine liver, stock No. (2-40. Both were supplied by Sigma Chemical Co. (St. Louis, Mo.). All other chemicals were AR grade. Deionized water was found satisfactory for solutions preparation. Typical reservoir solutions consisted of about 100 mL containing 20 g/L of D-glucose and 7 g/L of NaCl in a phosphate buffer of pH 7.00 (0.10 M total phosphate). The injected sample contained the glucose oxidase enzyme and 2.2 X lo4 units of catalase per mL. Typical size of the injected sample was 10 wL. One unit of glucose oxidase corresponded to that amount needed to oxidize 1.0 r M of glucose per minute at pH 5.1 and 35 "C, determined following the supplier's indications (7). One unit of catalase corresponds to the amount of enzyme decomposing 1.0 Mmol of H202per minute at pH 7.0 and 25 OC, while the H202 concentration falls from 10.3 to 9.2 mM of reaction mixture. Procedure. Samples were injected at the beginning of the mixing coil by means of a Teflon needle (0.027-inch i.d. bore) located at the center of the tube constituting the coil. Injection was manual and effected with the help of a Hamilton gas-tight syringe and a Hamilton PB600-1 repeating dispenser (Hamilton Co., Reno, Nev.).
RESULTS AND DISCUSSION The cumulative effect of a catalyst in a continuously circulated reservoir solution can be seen in traces A of Figure 1. Base-line deterioration and depletion of one (or more) of the reactants soon makes determination difficult and the advantages of sample injection into closed-loop flow systems vanish. Successful determination of the catalyst requires the removal or inhibition of the catalyst; for obvious reasons physical removal appears more attractive. Considering enzyme-catalyzed reactions, removal of the enzyme catalyst by the so-called "immobilization" of the enzyme can be used to accomplish such a removal. As principal methods of immobilization we can cite: (1)containment by membrane, (2) entrapment, (3) covalent bonding, and (4) adsorption (9). The first three of these approaches are both too involved and too slow for implementation in continuously circulated systems. Adsorption (physical) is left as the only feasible solution to the problem. After evaluation of several possibilities, the best medium found for removal of the enzyme glucose oxidase was small square sponges of cellulose with their surfaces chemically modified to have adsorption walls of phenoxyacetylcellulose. 0 1979 American Chemical Society
