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Anal. Chem. 1982, 54, 242-246
LITERATURE CITED
lasers, the maximum heat which can be deposited into a grating is approximately equal to the product of the laser power and time constant, which is only 15 pJ for the largest laser power applied in this work. If the pulse duration of a high energy pulsed laser used for excitation is less than the grating time constant, then all of the pulse energy is available for deposit into the grating. Although many laser sources produce pulses of much higher energy than the 15 CLJ figure above, the selection of excitation source must include criteria in addition to pulse energy. The spatial coherence of the beam must be excellent in order to develop fringes of high contrast; a single transverse mode of low order is optimal, e.g., TEM,. In addition, the temporal coherence of the beam must be sufficient to allow for small path differences in the interferometer; emission line widths of the order of 10 cm-l would be as large as one could conveniently tolerate. Within these constraints fall many powerful pulsed laser systems, which should prove valuable as excitation sources for thermal diffraction measurements of trace level absorbers.
Dlebold, G. J.; Zare, R. N. Sclence 1977, 196, 1439-1441. Hlrshfeld, T. Appl. Opt. 1978, 15, 2965-2966. Mathews, T. G.; Lytle, F. E. Anal. Chem. 1979, 51, 583-585. Hu, C.; Whinnery, J. R. Appl. Opt. 1978, 12, 72-79. Hordvlk, A. Appl. Opt. 1977, 16, 2827-2833. Whinnery, J. R. Acc. Chem. Res. 1974, 7, 225-231. Boccara, A. C.; Fournler, D.; Jackson, W.; Amer. N. M. Opt. Left. 1960, 5, 377-379. Elchler. H.; SalJe, G.; Stahl, H. J . Appl. Phys. 1978, 44, 5383-5388. Elchler, H.; Enterlein, G.; Glozbach, P.; Munschan, J.; Stahl, H. Appl. Opt. 1972, 1 1 , 372-375. Eichler, H. J. Opt. Acta 1977, 24, 631-842. Salcedo, J. R.; Siegman, A. E.; Dlott, D. D.; Fayer, M. D. Phys. Rev. Left. 1978, 41, 131-134. Eichler, H. J.; Klein, U.;Langhans, D. Appl. Phys. 1960, 21, 215-219. Damon. R. W.; Maloney, W. T.; McMahon, D. H. I n "Physlcal Acoustics"; Mason, W. D., Thurston, R. N., Eds.; Academic Press: New York, 1972; Vol. 7, Chapter 5. Harris, J. M.; Lytle, F. E.; McCaln, T. C. Anal. Chem. 1976, 48, 2095-2099. RCA, "Photomultiplier Tubes", Publlcatlon PIT-7OOC; RCA; Lancaster, PA, 1976. Sollmlni, D. J. Appl. Phys. 1966, 37, 3314-3315.
RECEIVED for review July 28, 1981. Acceptoed October 13, 1981. This material is based upon work supported by the National Science Foundation under Grant CHE79-13177. Additional funding by the University of Utah Research Committee is acknowledged.
ACKNOWLEDGMENT The authors acknowledge the assistance of N. J. Dovichi in the design of the signal processing electronics.
Reaction-Rate Spectrophotometric Method for Analysis of Gossypol in Cottonseed Extracts Floyd W. Crouch, Jr.,* and Melton F. Bryant' Department of Chemlstty, Unlversiv of Georgia, Athens, Georgia 30602
A flxed-tlme klnetlc spectrophotometrlc measurement technlque for the determlnatlon of gossypol extractable from cottonseed samples Is descrlbed. Both worklng curve and standard addltlon methods are utlllred. The klnetlc method Is based on an equlllbrlum spectrophotometrlc analysis of gossypol In mixed feeds whlch Involves the reactlon of gossypol with 1,3,5-trlhydroxybenrene (phloroglucinol) In concentrated hydrochlorlc acld. The reactlon produces a product whlch has an absorbance maximum at 550 nm. The wlthln day preclslon of the worklng plot method, obtalned at a flxed tlme Interval of 1.5 mln, Is approxlmately 1% relatlve standard devlatlon. The day-to-day preclslon of the standard addltlon method Is approxlmately 4 % relatlve standard devlatlon. The h e a r dynamlc range of both methods Is approxlmately 1 X I O 4 M to 8 X lo4 M gossypol. The standard addltlon method Is more rellable when conslderable amounts of lnterferlng gossypol analogues are present.
Gossypol and its analogues are found primarily in pigment glands of cottonseed but are also present in other parts of the cotton plant. The structures of the three tautomeric forms of gossypol itself are illustrated in Figure 1. Analogues of Current address: Conoco, Inc.,
City,
OK
74601.
P.O. Box 1267, RB 237, Ponca 0003-2700/82/0354-0242$01.25/0
gossypol have a similar structure and can either occur naturdy in cottonseeds or form as a result of storage and processing of cottonseed. The actual pigment composition of cottonseed pigment glands varies with the genetic type of the cotton plant and with storage and processing of cottonseed. These pigments are interesting in that they have exhibited both beneficial uses and harmful physiological effects. Gossypol has been found useful in industrial (1-4), analytical (5)) and clinical (6-8) applications. The toxicity of gossypol, however, limits its usefulness in many areas (9-11). It is assumed that bound gossypol, i.e., gossypol that is bound to protein, is less physiologically active than free gossypol (12) but there is still some controversy over this assumption (13). Analysis of gossypol and its analogues in cottonseeds, cottonseed meal, cottonseed oil, mixed feeds, various animal tissues, and other parts of the cotton plant have been performed (14). Values reported for the gossypol content of cottonseed and its products include percent free gossypol, percent bound gossypol, and percent total gossypol by weight. The distinction between bound and free gossypol is usually determined by which extraction solvent is used (15). Methods used for the analysis of gossypol include spectrophotometric methods (16-20), an NMR method (21))chemiluminescent methods (22),gas-liquid chromatographic methods (23,24), optical microscopy (%), near-IR reflectance (26),polarography, thin-layer chromatography, and paper chromatography (27). Most of these methods suffer from interferences due to the presence of the other pigments. 0 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982 CHO
OH
OH
CHO
A HO
A
CH3
CH3
A
CH3
tH
nCH3 CH3
A
CH3 CH3
0
HOHC-0
CH3
-CHOH
HO
C HO
A
CH3
CH3
A
CH3
Flgure 1. The three tautomeric forms of gossypol: dehyde, (B) cyclic carbonyl, (C) lactol.
CH3
(A) hydroxy
al-
This report deals with the development of a reaction-rate method for the analysis of gossypol that is based upon the reaction of gossypol with 1,3,5-trihydroxybenzene or phloroglucinol (PG) in highly acidic solution to form a product whose absorbance is monitored at 550 nm. No study of the mechanism of this reaction or of the structure of the final product of this reaction has been reported. The increase in the absorbance of the reaction mixture with time is measured spectrophotometrically. A fixed time data reduction technique was used in the development of the analysis. Results are given for the within day and the day-to-day precision of the method. The analysis of cottonseed samples for percent free gossypol by weight using both a working curve and a standard addition technique has been evaluated. EXPERIMENTAL SECTION Reagents and Standards. Standard gossypol acetic acid (GAA) (Sigma Chemical Co., St. Louis, MO) and spectral quality acetone (Baker Chemical Co., Phillipsburg, NJ) were used to prepare all GAA standard solutions. Standard solutions of GAA M to 7.9 X lob4M. Hywere prepared in the range 1.1 X drochloric acid (HC1) solutions were prepared from Baker Analyzed Reagent concentrated HCl (Baker Chemical Co., Phillipsburg, NJ) and distilled deionized water (DDW). The 6.82 X M phloroglucinol (PG) solution for the working curve reaction-rate method was prepared by dissolving phloroglucinol dihydrate (Eastman Kodak Co., Rochester, NY) in 100% ethanol and diluting to 500 mL with 100% ethanol in a volumetric flask. A 7.46 X M PG solution was used for the standard addition reaction-rate method. These solutions were also used as reagents in the equilibrium phloroglucinol method (20) (i.e., the method from which the reaction-rate method was developed) for means of comparison of results of percent free gossypol with working curve and standard addition reaction-rate analysis. All PG solutions were stored in brown bottles due to their susceptibility to decomposition upon exposure to light. For the extraction of gossypol from cottonseeds 0.2 M sodium hydroxide and 0.05 M sodium dithionite solutions were prepared from sodium hydroxide pellets (Baker Chemical Co., Phillipsburg, NJ), solid sodium dithionite (Fisher Scientific Co., Fair Lawn, NJ), and DDW. Baker Analyzed Reagent concentrated HCl and
243
anhydrous diethyl ether (Baker Chemical Co., Phillipsburg, NJ) were also used in the extraction procedure. Apparatus and Equipment. A Cary-16 spectrophotometer was used for absorbance measurements. The Cary-16 was equipped with an adjustable absorbance range recorder interface (Cary Model 1626 recorder interface). Absorbance vs. time plots were taken on a Linear strip chart recorder (Linear Instruments Model No. 355). One centimeter pathlength glass cuvettes (Precision Cells Inc., Hicksville, NJ) contained the reaction solutions. Procedures. Extraction of Gossypol from Cottonseed Meal. Fifteen grams of delinted and dehulled cottonseed was placed in a Varco grinder (Varco Inc., Bellville, NJ) and ground for 20 s. Portions for analysis were weighted by addition into 125-mL Erlenmyer flasks. A Teflon-coated magnetic stirring bar and 100 mL of anhydrous diethyl ether were then added. The portions were stirred on a magnetic stir plate for 8 h and filtered through a medium frit sintered glass funnel. The filtrate was extracted three times, each time with 50 mL of 0.2 M sodium hydroxide solution. The combined aqueous extracts, containing sodium gossypolate, were mixed with 75 mL of 0.05 M sodium dithionite in order to prevent oxidation of the gossypolate. Concentrated HC1 was added to the solution until the solution became acidic, as tested by litmus paper. The gossypol was extracted with two 60-mL portions of anhydrous diethyl ether. The diethyl ether extracts were combined and the ether was evaporated with a rotary evaporator. The solid residue was dissolved and diluted to either 250 or 200 mL with spectral quality acetone in a volumetric flask. Equilibrium Phloroglucinol Method. The reaction-rate procedure for the analysis of gossypol is based on the equilibrium phloroglucinol method of analysis for gossypol in mixed feeds (20). Working curves were prepared by using GAA standard solutions. Two milliliters of each standard or sample solution was pipetted into a 25.0-mL volumetric flask and 1.0 mL of a PG solution was added to the flask. The solutions were mixed and then 2.0 mL of concentrated HCl was pipetted into the flask. The entire contents were mixed and allowed to react for 20 min, after which the flasks were diluted to 25.0 mL with 100% ethanol. For the blank solution pure ethanol was substituted for the phloroglucinol solution in the flask. Reagent Mixing Procedure for the Reaction-Rate Methods. Two mixing methods were evaluated in terms of precision for use in the reaction-rate methods. The first method, which involved mixing reagents by use of a Teflon-coatedmagnetic stir bar and a stir plate in a 30-mL beaker, was deemed unsuitable for further use due to poor within day and day-to-day precision obtained by use of this mixing method. The second mixing method consisted of using a small electric motor (Micromo Electronics No. 1516E-012SL)and gearbox (Micromo Electronics No. 50/3) to drive a 3 in. long Teflon stir rod which mixed the reagents in a 13 X 100 mm test tube. This mixing device was used for all reaction-rate work. Reaction-Rate Measurement Procedures for Fixed Time Working Curve and Fixed Time Standard Addition Analysis. Constructionof working c w e s using the fixed time data reduction technique consisted of the following sequence: Seventy-five milliliters of each GAA standard solution was pipetted into a 100-mL volumetric flask, and the flasks were diluted to volume M PG solution. A 3.5-mL aliquot of the with the 6.82 X resulting solution was pipetted into a 13 X 100 mm test tube by an adjustable macropipet (Universal Scientific, Atlanta, GA). Next, 3.5 mL of a 6 M HCl solution was pipetted into the test tube with the adjustablepipet. The contents of the test tube were then stirred using the stir rod assembly for 15 s and a portion of the resulting solution was transferred to a sample cuvette. The cuvette was placed inside the Cary-16 sample compartment 1min after the HC1 solution was pipetted into the test tube, and at 1.5 min after the reaction was initiated, the absorbanceof the mixture was monitored vs. time on the recorder. Data reduction was performed by determiningthe change in absorbance (AA)at time intervals (At) of 0.5,1.0, and 1.5 min. The three At values were evaluated for their analytical usefulness. Working curves using each At values were obtained by plotting AA values vs. GAA standard solution concentrations. Extracted samples were subjected to exactly the same sample handling procedure as each of
244
ANALYTICAL CHEMISTRY, VOL. 54,NO. 2, FEBRUARY 1982
I
0.10
0,624
0.08
0.468
0.06
;I
2 0.312
0.04
0.156
0 02
0 1 0
HCL CONCENTRATION (Y)
2 0
0,00016
30
Figure 2. Effect of the concentration of HCI on AA at Af = 0.5 min.
the standards. AA values at each of the three At values were obtained for the sample solutions, and the concentration of gossypol in the samples was calculated from the standard data. For the standard addition analysis of samples essentially the same procedure was used. Ten milliliters of the extracted sample solution was pipetted into each of three 25.0-mL volumetric flasks. Next, 10 mL of spectral quality acetone was pipetted into the first flask, 10.0 mL of a 7.79 X lop6M GAA standard solution was pipetted into the second flask, and 10.0 mL of a 3.89 X lo-' M GAA standard solution was pipetted into the third flask. Each M PG solution. flask was diluted to volume with a 7.46 X These three solutions were then subjected to the same mixing and recording techniques that were applied in the analysis of gossypol in extracted samples by the fixed time working curve method. AA values were determined at At = 1.5 min for each absorbance vs. time recording. Gossypol concentrationsin the extracted samples were determined from plots of AA vs. the concentration of GAA standard added.
RESULTS AND DISCUSSION Preliminary Studies. Plots of rate of reaction vs. concentration of GAA standard indicated that the reaction is pseudo first order with respect to the concentration of GAA. The instrumental system used for these rate measurements, however, is not conducive to rapid rate measurements. Therefore it was not deemed appropriate to consider using an initial rate data reduction technique for absorbance vs. time plots. Ingle and Crouch have demonstrated (28) that with an instrumental system in which a linear concentration-signal relationship exists, a fiied time reduction technique is superior to a variable time data reduction technique if the reaction is f i t order or pseudo f i s t order. Since the instrumental system being used does provide a linear concentration signal relationship, a fixed time data reduction technique was deemed the most appropriate for use in the development of the rate method for the analysis of gossypol in cottonseed extracts. In order to maintain pseudo-first-order reaction conditions the concentration of HCl and Pg in the reaction mixture was always maintained at least at a 10-fold excess over the analyte concentration. The effect of the concentration of HCl on the rate of reaction can be seen in Figure 2. The x axis represents the HC1 concentration in the reaction mixture and the y axis the change in absorbance (AA)with a time interval (At) of 0.5 min. It can be seen that AA values do not vary in a linear manner with HC1 concentration. When the HC1 concentration in the
0,00032 0.00048 GAA STANDARD CONCENTRATION (M)
0,00064
Figure 3. Working plots at (A) Af = 1.5 min, (B) Af = 1.0 min, and (C) At = 0.5 min. C.C. refers to correlation coefficient.
Table I. Comparison of the Slopes of the Working Plots of the Reaction-Rate Method at Different A t Values and of the % RSD of the Slope Averages
day 1 2
3 % RSD
slopes at indicated A t values, absorbance L/mol 0.5 min 1.0 min 1.5 min 41 9.2 444.7 399.3 5.40
788.9 847.1 751.1 6.07
1099 1190 1040 6.81
reaction solution was 4.8 M the absorbance reading was off scale, even with the recorder interface on its highest range; Le., 2 absorbance units. In order to optimize the sensitivity and the linear dynamic range of the analytical procedures, we chose 6 M HCl as a reagent for use in all reaction rate analyses, giving a final HC1 concentration in solution of 3 M. The temperature of the reaction mixture was determined by the heat of mixing of the hydrochloric acid solution with the solution containing the cottonseed extracts. Development of Reaction Rate Analysis Procedure. A of 0.5,1.0, and 1.5 min was used to reduce the absorbance vs. time plots from the instrumental system. Typical working plots which utilize each of these At values can be seen in Figure 3. Each point on the plot represents the average AA for each concentration which was obtained from three AA values. The percent relative standard deviations (% RSD) for a fiial GAA M (illustrated standard solution concentration of 1.19 X as "a" in Figure 4) ranged from 13.9% to 4.13% when At values changed from 0.5 to 1.0 to 1.5 min. The % RSD values obtained for a final GAA standard solution concentration of 2.96 X 10" M (illustrated as "b" in Figure 4) ranged from 4.72% to 0.313% when At values changed from 0.5 to 1.0 to 1.5 min. Thus as At increases the precision of measurement improves. This supports statements by Carr (29) that as At increases the precision of measurement of the signal which corresponds to the product concentration improves for a first-order reaction. However, his statements were based upon knowledge of the effect that random variations of the rate constant can have on measurement precision, and it must be kept in mind that other factors may be influencing the pre-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982
Table 11. Percent Free Gossypol by Weight Values Obtained by Use of Different Methods % free gossypol by weight official equilibrium reaction-rate methods sample AOCS phloroglucinol portion wt, g method method A t 0.5 min A t 1.0 rnin 1 2 3 av % RSD
3.3759 3.5190 2.7487
1.06 1.06 1.06
0.686 0.710 0.731 0.709 3.18
0.608 (3.03%) 0.626 (2.78%) 0.580 (2.62%) 0.605 3.83
cision of these measurements. The day-to-day reproducibility of the slopes of the working plots was also of interest since for precise and accurate sample analysis the slope and intercept of the working plot should not vary considerably each time a new working plot is constructed. The % RSD for the slopes of the working plots were high, as seen in Table I, but were deemed acceptable for use in sample analysis. The correlation coefficients of the standard curves for the rate method and for the equilibrium phloroglucinol method were compared and both found to be suitable for routine sample analysis. Sample Analysis. One cottonseed sample was analyzed for percent free gossypol by weight using both the rate method and the equilibrium phloroglucinolmethod. The cottonseed sample had been ground and three portions of the sample subjected to the extraction and analysis procedures. All cottonseed samples were obtained from Harmon H. Ramey (Cotton Quality Laboratories, University of TennesseeAgricultural Campus, Knoxville, TN). The percent free gossypol by weight in these samples had previously been determined by using AOCS Official Method Ba 7-58 (16). The results of these analyses can be seen in Table 11. Each of the values in Table I1 under each one of the reaction rate headings is the mean of three percent free gossypol values obtained in one day's set of analyses. The number in parentheses to the right of these values is the % RSD for the three values. The two numbers at the bottom of each column for reaction-rate analysis represent the average of the three means of the values of percent free gossypol and the %RSD of the values, respectively. This latter quantity represents the precision of the entire analytical procedure, including extraction. There were apparent discrepancies between percent free gossypol by weight values obtained by use of the official AOCS method, the equilibrium phloroglucinol method, and the rate method. The discrepancy between the official AOCS method and all methods performed in our laboratory could be attributed to differences in extraction procedure and sample handling techniques. The discrepancies between the equilibrium results and the rate results could be due to interferences which may vary the rate of reaction or which may result in products which absorb at the analytical wavelength. There was an appreciable difference between the average of the means a t At = 0.5 min and the averages of the means at At = 1.0 rnin and At = 1.5 min. This discrepancy as well as the fact that as At increased the within day precision of analysis improved again lends support to the statement by Carr that fixed time data reduction sample analysis improves as At increases. It is apparent that the extraction procedure was quite precise. Since there may have been interferences in extracted samples that cause inaccurate sample analysis, a standard addition reaction-rate method of analysis for gossypol in cottonseed extracts was evaluated. The data reduction technique used was fixed time a t At = 1.5 min. One cottonseed sample had been ground and the three portions of this sample weighed and subjected to the extraction procedure and standard addition reaction-rate analysis. These results are compared with
0.806 (3.40%) 0.790 (2.78%) 0.813 (4.32%) 0.803 1.47
245
A t 1.5 min
0.803 (1.73%) 0.805 (0.513%) 0.819 (1.63%) 0.809 1.08
Table 111. Analytical Results of the Determination of Percent Free Gossypol by Weight in Cottonseed Samples % free gossypol by weight equilibrium official phlorostd addition por- sample AOCS glucinol reaction-rate method method method tion wt, g 1 2 3
2.71174 2.21718 1.88956
0.640 0.640 0.640
0.358 0.367 0.274 20.6%
0.373 (4.18%)'" 0.384 (1.05%)'" 0.302 (4.43%)'" 12.6%
a % RSD values for three separate determinations of % free gossypol by weight on three different runs. % RSD for the three % free gossypol by weight values of the three portions. Represents precision of the entire analytical procedure.
Table IV. Comparison of the Slopes of the Reaction-Rate Method Working Plots to the Slopes of the Standard Addition Plot for Portion 1 (absorbance L/mol) working curve std addition reaction-rate reaction-rate method method (for portion 1) (at = 1.5 min)
av slope
1099 1190 1040 1110
1248 1348 1209 1268
results from the official AOCS method and with results from the equilibrium phloroglucinol method in Table 111. Each standard addition reaction-rate result corresponds to an average of three values, each obtained on a different day. Therefore, the % RSD values shown in parentheses to the right represent day-to-day precision. The day-to-day precision of the standard addition reaction-rate method was quite acceptable. It can be seen in Table I that the % RSD values of the slopes at At = 1.5 rnin is 6.81%. Since this represents the day-to-day precision of the working plot reaction-rate method, the precision was somewhat improved by using the standard addition method. The errors of the mean values obtained for percent free gossypol by use of the standard addition reaction-rate method relative to the values obtained by use of the equilibrium phloroglucinol method are 4.2% for portion 1,4.6%for portion 2, and 10% for portion 3. Relative errors compared to the reported values obtained by using the official AOCS method were 42% for portion 1,40% for portion 2, and 53% for portion 3. However, it must be remembered that the extraction procedure as well as the analysis procedure is different in the official AOCS method. It was desired to compare the slopes of the reaction-rate working plots at At = 1.5 min to the slopes of the plots of AA vs. concentration of GAA standard added for the standard addition reaction-rate method for portion 1. The results are tabulated in Table IV. When an F test was conducted, it was
246
Anal. Chem. 1062, 54, 246-249
found that there was no significant difference between variances. Therefore, a t test was conducted to determine if there was any significant difference between the averages of the slopes. It was found that there was a significant difference. This finding plus the fact that the standard addition slopes are larger than the working curve slopes may indicate that interferences are present in extracted samples whose effect may be compensated for by use of the standard addition method.
(I 1)
(12) (13) (14) (15)
ACKNOWLEDGMENT The authors thank J. McGee, G. Birth, and G. Dull, Russell Research Center, USDA, Athens, GA, for providing some standard GAA and some very useful information. Also they thank H. Ramey, Cotton Quality Laboratories, University of Tennessee Agricultural Campus, for supplying cottonseed samples. They also are deeply indebted to G. Boyd, Department of Chemistry, University of Georgia, for the use of the Cary-16 in his laboratory.
(16) (17) 118) . .
(19) (20)
Markman, A. L.; Rzhekhin, V. P. “Gossypol and Its Derlvatlves”; Israel Program for Sclentlfic Translations: Jerusalem, 1968; p 178. Qlan, Shao-Zhen; Xu, Ye; Chen, Zhao-Cong; Gao, Lu-Mln; Sun, Sheng-Gang; Tang, Xi-Can; Wang, Yu-Ee; Shen, Ll-Ying; Zhu, MlngKang Yao Hsueh Hsueh Pa0 1070, 14, 513-520; Chem. Abstr. 1080, 92. 1917342. Qlan, Shao-Zhan; Xu, Ye; Jlng, Guang-Wel. Yao Hsueh Hsueh Pa0 1070, 74, 116-119; Chem. Abstr. 1080, 92, 33909f. Clark, E. P. J. Blol. Chem. 1028, 76, 229-235. Llener, I. E. “Toxlc Constltuents of Plant Food-Stuffs”; Academlc Press: New York, 1969; p 243. Pons, W. A. J. Assoc. Off. Anal. Chem. 1077, 60, 252-259. Llener, I . E. “Toxlc Constltuents of Plant Food-Stuffs”; Academic Press: New York, 1969; pp 232-235. American Oil Chemlsts Society “Offlclal and Tentatlve Methods”; American Oil Chemlsts Society: Champaign, IL, 1978; Offlclal Mathod Ba 7-58. Pons, W. A.; Quthrle, J. D. J. Am. 011 Chem. SOC. 1048, 26, 671-676. Mathur. J. M. S.: Sharma. N. D.: Slnah. M. J . Food Scl. Technol. 1072, 9 , 136-140. Boatner. C. H.; Caravella, M.; Kyame, L. Ind. Eng. Chem. Anal. Ed. 1044, 16, 566-572. Storherr, R. W.; Hollay, K. T. J. Agrlc. Food Chem. 1054, 2 ,
-
745-747. . .- . .. . (21) Walss, A. C.; Chan, B. G.; Benson, M.; Lukefahr, M. J. J. Assoc. Off. Anal. Chem. 1078, 61, 146-149. LITERATURE CITED (22) Aver’yanov, A. A.; Merzlyak, M. N.; Rubln, 6. A. Bio&him/ya 1078, 43, 1594-1601; Chem. Abstr. 1078, 89, 211079h. Askarov, M. A.; Dzhalllov, A. T.; Fatkhullaev, E.; Sukhlnlna, L. A. Ot(23) Raju, P. K.; Cater, C. J. Am. 011 Chem. SOC. 1087, 44, 465-468. kfYt/yi3, Izobret., Prom. Obrastsy, Tovarnye ZnakllO80, 8 , 99-100; (24) Huang, Ruey-Shlang, M.S. Thesls, Unlverslty of Georgia, Athens, GA, Chem. Abstr. 1080, 92, 216236d. 1981. Belley’ A‘ “Cottonseedand Cottonseed Products”; Interscience: New (25) Yun, S. P.; Krakhmalev, V. A.; Kharmats, D. E. Uzb.8/01. Zh. 1070, York, 1948; p 148. 6 , 23-26. Bickford’ w’ “; Pack’ F’ ”; Mack’ c’ J ’ Am‘ Oil (26) Blrth, G. S. Russell Research Laboratory, USDA, Athens, GA, personal Chem. SOC.1054, 31, 91-93. communication, 1980. Ram, A.; Pandey S 016aglneux 1050, 5 , 301. Prakash, 0.; (27) Markman, A. L.; Rzhekhin, V. P. “Gossypol and Its Derivatlves”; IsraMarkman, A. L.; Rzhekhln, V. P. “Gossypol and Its Derivatives"; Israel Program for Scientific Translatlons: Jerusalem, 1968 Chapter 8. el Program for Scientific Translatlons: Jerusalem, 1968 p 146. (28) Ingle, J. D.; Crouch, S. R. Anal. Chem. 1071, 4 3 , 697-701. Wang, NaI-Gong; Lei, Hal-Peng Chung-Hua I Hsueh Tsa Chlh 1070, P. W. Anal. Chem. 1078, 50, 1602-1607. (29) Carr, 59, 402-405; Chem. Abstr. 1080, 92, 174961d. Vermel, E. M.; Kruglyak, S. A. Vopr. On&ol. 1083, 9 , 39. Llener, I. E. “Toxlc Constltuents of Plant Food-Stuffs”; Academlc Press: New York, 1969; p 255. RECE~VED for review July 7,1981. Accepted October 26,1981.
Determination of Methyltin(IV) and Tin( IV) Species in Water by Gas ChromatographyIAtomic Absorption Spectrophotometry Y. K. Chau,” P. T. S. Wong, and 0. A. Bengert Canada Centre for Inland Waters, Burlington, Ontario; Canada L7R 4A6
The highly polar and solvated methyltln, dlmethyltln, trlmethyltln, and Sn( IV) species are extracted Into benzene containing tropolone from water saturated with sodlum chloride. These compounds are butylated In the extract to the tetramethylbutyltlns, Me, SnBu4-, , which have sufflclent volatlllty to be separated and analyzed by the GC-AAS system. Large volumes of water sample can be handled. Under normal laboratory conditions, detection llmlt of 0.04 pg/L can be achieved with 5 L of water sample. Absolute detection llmlt of the GC-AAS for Sn Is 0.1 ng. Volatlle organotln compounds such as Me4Sn and methyltln hydrides can also be analyzed by this method.
There is an ever increasing demand for analytical techniques capable of speciating organotin compounds in environmental studies for two obvious reasons. First is the increasing use of inorganic and organotin compounds in many industrial, chemical, and agricultural areas, very little being known about their environmental fate; secondly there is a great difference 0003-2700/82/0354-0248$01.25/0
in toxicity of the various organotin compounds according to the variation of the organic moiety in the molecules. We have special interest in the biotic and abiotic methylation of tin compounds (1-3) and the fate of some industrial organotins in the aquatic ecosystems. One possible route as discussed by Brinckman (4) is the dealkylation of the trialkyltin species eventually to Sn(IV),and the microbial methylation of Sn(1V) to the various methyltin species. Increasing methyltin concentrations with increasing anthropogenic tin influxes has been noted in the Chesapeake Bay (4). For environmental studies, sensitive and species-specific methods are required to determine the volatile tetraalkyltin of R4Sn type and the polar and solvated alkyltin R,Sn(4-fl)+ species in solution. Presently available methods include the conversion of these compounds to their volatile hydrides which can then be separated and detected in an element-specific detector (5,6);an alternate method is to further alkylate the alkyltin species with a selected Grignard reagent to convert them to the tetraalkyltin forms which have lower boiling points. Butyltin(1V) compounds have been determined by GC-MS after methylation (7) and by GC after pentylation (8). 0 1982 Amerlcan Chemical Soclety
