209
Anal. Chem. 1980, 52, 209-211
are obtained with the quicker method B. However, with this procedure, t h e losses are of about 10% and t h e relative standard deviations between 10 and 12%. These values may at first seem rather high when compared t o those obtained with a reference sample such as dried bovine liver (for which we had recoveries of close t o 100% and relative standard deviations of much less t h a n 10% (5)). But, if we consider only the 11foodstuffs, marked with a n asterisk in Table I, and which have a basic composition similar t o t h a t of bovine liver, we obtain t h e recoveries and relative standard deviations, respectively, given below: 95.6% and 6.2% for lead 95.3% and 4.7% for cadmium 94.5% and 12.3% for copper Thus we have found that a calcination temperature as high as 750 "C can be used in a single-step dry ashing procedure if sulfuric acid is added as an ashing aid, when lead, cadmium,
and copper are to be determined in foodstuffs. As a result of this study, we have been able t o develop a method particularly well suited to the determination of heavy metals in protein-rich biological products such as meat and fish.
LITERATURE CITED Crosby, N. T. Analysf (London) 1977, 102, 225-68. Holak, Walter. J , Assoc. Off. Anal. Chem. 1977, 6 0 , 239-40. Gorsuch, T. T. Analyst(London) 1959, 8 4 , 135-73. Menden, E. E.; Brockman, D.; Choudhury, H.; Petering, H. G. Anal. Chem. 1977, 4 9 , 1644-45. Feinberg, Max; Ducauze, Christian. Bull. SOC. Chim. France 1978, 4 19-25. "Official Methods of Analysis". Association of Official Analytical Chemists, 12th ed.; A.O.A.C.: Washington, 1972; Chapter 25. Feinberg, Max. Doctorate lhesis, Paris, 1979; Chapter 2. Hinchen, John D. "Practical Statistics for Chemical Research"; Associated Book Publishers: London, 1969; Chapter 4
RECEIVED
for review June 11, 1979. Accepted September 20,
1979.
Assay of Thioacetamide and Thiobenzamide with Chloramine-T N. M. Made Gowda, V. M. Sadagopa Ramanujam, Norman M. Trieff," and Marvin S. Legator Division of Environmental Toxicology, Department of Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, Texas 77550
Thioacetamide is widely used as a substitute for hydrogen sulfide (because of its lower toxicity and less offensive odor ( I ) ) for qualitative and quantitative determinations of heavy metals both in solution (2-7) and in pharmaceutical preparations (8, 9). T h e methods available in t h e literature for thioacetamide include: gravimetric precipitation using t h e Hg(I1)-ammine complex ( I O ) , indirect complexometric titration using the Zn(I1)-EDTA system (II), oxidimetric titrations using oxidants such as sodium hypobromite (12) and ammonium vanadate (13),and potentiometric titrations with silver nitrate (14) and Hg(I1) (15, 16) solutions. The relative errors involved in these methods usually range from 1-3%. T h e oxidimetric methods described in t h e literature are timeconsuming and less precise since t h e oxidant hypobromite requires a t least 1 h at room temperature for the completion of the reaction (12) and ammonium vanadate requires at least 60 "C for the titration (13). In our continuing efforts to explore t h e usefulness of sodium N-chloro-4-methyl benzene sulfonamide, commonly called chloramine-T (CAT), as an analytical reagent (17-21), we have recently developed both direct and indirect titrimetric methods for the assay of thioacetamide (TAA) and thiobenzamide (TBA). To our knowledge, no report of any quantitative estimation of the latter compound appears in t h e literature. T h e methods presented in this article for analyses of TAA and TBA are simple, reproducible, less time-consuming, and more accurate (99% based on active chlorine titration. Approximately 0.1 and 0.02 N solutions were prepared in demineralized water and standardized by the iodometric procedure (24). Reagent-grade chemicals were used in preparing other solutions. Direct Titration. To the solution of thioamide (TAA in pH 1 aqueous buffer or TBA in alcohol + 1 mL of 4 N HC1) was added 2 mL of starch-KI solution (19~starch and 0.1- 0.4 g of KI in the case of TAA; for TBA, the amount of K I is not critical) and sufficient water to dilute to 25 mL. The solution was then titrated with 0.02 N CAT to a stahle pale blue end point. Calculations were performed according to the method of Mahadevappa and Gowda (19). Back Titration. Prelimmar> Studies. In preliminary experiments, known amounts of the thioamide solution (TAA 0.13 mmol; TBA 0.08 mmol) prepared in the appropriate buffer or solvent were added to a known excess of CAT solution (1.0 m o l ) in a glass-stoppered Erlenmeyer flask at room temperature (-24 "C). The reaction mixture was set aside for various intervals of time, with occasional shaking. Then the unreacted CAT was determined iodometrically by back titration. A typical set of results for the extent of oxidation of TAA or TBA after 10-min standing with an excess of CAT is given in Table I. Oxidation of TAA is nonstoichiometric a t pH 3-8, attaining a maximum in moles CAT/mole TAA at pH 4. There is, however, a reproducible 5-electron change for the oxidation of TAA in HC1-KC1 buffer at pH 1. Oxidation of TBA is slow in the absence of acid hut in the presence of hydrochloric acid (0.1-0.5 N) the reaction is rapid and stoichiometric with a reproducible 5-electron change. 1979 American Chemical Society
210
ANALYTICAL CHEMISTRY, VOL. 52, NO. 1, JANUARY 1980
Table I. Observed Stoichiometry of Thioacetamide (TAA) and Thiobenzamide (TBA) in Different Buffer and Solvent Media with Excess of Chloramine-F thioacetamide medium 0.1 N HCl 0.5 N HCl PH 1 PH 2 PH 3 PH 4 PH 5 PH 6 PH 7 PH 8 0.1 N NaOH alcohol
3.11 3.01
2.76 2.86 2.75 2.42 2.48
RESULTS AND DISCUSSION
thiobenzamide
mmol CAT used b/mmol TAA taken medium 2.41 2.55 2.50 2.55 2.61
respectively. Both these samples were identified as benzoic acid by comparison of the fragmentation pattern with that of a standard mass spectrum' of benzoic acid (25).
n o acid 0.01 N HC1 0.10 N HC1 0.25 NHC1 0.50 N HCI -
mmol CAT usedb /mmol TBA taken 2.23 2.58 2.50 2.50 2.50 -
TAA taken = 0.13 mmol; TBA taken = 0.08 mmol (in Elecalcohol); CAT taken = 1 . 0 mmol; time = 1 0 min. tron change per molecule of TAA or TBA is merely the ratio: (mmol CAT/mmol thioamide) x 2.
For the direct titration, the results for thioacetamide and thiobenzamide are accurate and reproducible within h0.5% relative error between 2 and 40 mg of compounds. T h e direct oxidation of TAA and TBA with CAT involves a two-electron change which can be represented as oxidation (Equation 1)and reduction (Equation 2) halves of the couple, thus: S
0 II
I1
+
R'-C-NH, + 2 H 2 0+ R'-C-OH
R-NClNa
+ 2H+ + 2e
-
+
NH,'
S + 2e
H'
+ Na+ + C1-
R-NH2
(1)
(2)
where R = p-H3C-C6H,-SO2 and R' = CH3for thioacetamide and R' = CsH5 for thiobenzamide. Addition of Equations 1 and 2 results in Equation 3: S It
R'-C-NH2
I
R-NClNa
+
2H20+ H + +
0
Recommended Procedure. A solution containing the thioamice (-2 mg/mL) in buffer or alcohol (TAA in pH 1 aqueous buffer; TBA in alcohol) was prepared. An aliquot of the solution was added to 25 mL of 0.1 N CAT in a glass-stoppered Erlenmeyer flask (enough 2 N HCl was added to make the overall acid concentration 0.1-0.5 N for TBA), the mixture was shaken occasionally, and after 10 min, 10 mL of 2 N HCl and 10 mL of 20% KI solutions were added, the liberated iodine being titrated with 0.05 N thiosulfate solution (VJ. The experiment was repeated without the addition of the thioamide (VI). Product Analyses. Product analyses were carried out under two different conditions using: (i) stoichiometric amounts of TBA and CAT (direct titration; reaction mixture A) and (ii) TBA and excess of CAT (back titration; reaction mixture B), both in alcohol/water = 4060 (v/v) mixtures containing HCI. Each of these reaction mixtures were found to contain yellow colloidal sulfur as a product. The precipitate obtained from the reaction mixture A was filtered and separated by preparative TLC using silica gel plates and a toluene/dichloromethane/methanol, 25:lO:l (v/v), solvent system. The sample gave two spots (visualized under short wave UV light) with Rf = 0 (a yellow spot, probably sulfur, which does not move in this solvent system) and 0.68 (whose Rf corresponded to that of a pure sample of benzoic acid). This material with Rf = 0.68 was eluted with ethanol, evaporated to dryness, and recrystallized in water-alcohol mixture to give white needles melting a t 122 "C (uncorrected). This sample was further characterized by mass spectrometry (see below). The filtrate (from the reaction mixture A) gave a positive qualitative test for ammonium ion (giving a brown precipitate with Nessler's reagent). The control solutions such as CAT, p-toluenesulfonamide, and TBA themselves are negative to this specific test for NH4+ion run under experimental conditions a t room temperature. The p-toluenesulfonamide (the reduction product of CAT) in the filtrate was identified by paper chromatography using benzyl alcohol saturated with water as the solvent yielding a spot with Rf = 0.91 (identified with vanillin (0.5%)-HC1 (1%)in ethanol spray reagent (19). The precipitate obtained from the reaction mixture B also gave two spots with Rf = 0 and 0.68 in the toluene/dichloromethane/methanol, 25:lO:l (v/v), solvent system. The purified material corresponding to R f = 0.68 melted at 122 O C and was further characterized by mass spectrometry. The filtrate from the reaction mixture B was shown to contain NH4+ (positive Nessler's test), S042- (white precipitate with barium chloride), and p-toluenesulfonamide, as previously described. Mass Spectrometry. Mass spectra of the oxidation product of thiobenzamide isolated from the reaction mixtures A and B were obtained on a Du Pont 21-291 mass spectrometer using 70-eV electrons with source and probe temperatures at 250 and 50 "C,
II
R'-C-OH
+
R-NH,
+
NH,'
+
Na'
C1-
t
1-
S (3)
There is no effect of acid or potassium iodide on t h e stoichiometry of t h e thiobenzamide reaction. There is, however, some effect of acid and potassium iodide on the end point of t h e reaction for thioacetamide. Hence t h e titration was carried out with t h e TAA solution at p H 1 buffer having a n optimum concentration of potassium iodide (0.1-0.4 g). Since it has been established t h a t thiourea, cysteine, glutathione, and methionine undergo a direct oxidation with CAT (19), it is likely that they will interfere in t h e direct titration of TAA and TBA, whereas, on the basis of the same reasoning, urea, glutamic acid, leucine, glutamine, serine, glycine, threonine, alanine, valine, proline, arginine, histidine, and cystine should not interfere. The results for the back titration method indicate a relative error of *0.6% or less for sample sizes of TAA a n d TBA between 2 and 27 mg. T h e five-electron change observed suggests that the oxidation of thioamides can be represented as oxidation (Equation 4) and reduction (Equation 5) halves of t h e couple thus: S I1 2R'-C-NH2 + 8H,O + 6H' -+ 0 I/
2R'-C-OH
+
2NH,+ + SO,'.
+ 10H' + 10e
5R-NClNa
-
- 16"
5R-NH2
t
S + 10e (4)
+ 5Na+ + 5C1(5)
Hence, t h e over-all reaction in this instance is
S It
2R'-C-NH2
+
5R-NClNa
+
8H,O
-+
2R'-C-OH + 5R-NH' 5Na' + 5C1- + S
+
0 I1
2NH,+
+
SO,'-
+ (6)
Since it has been shown that cysteine, cystine, glutathione, serine, threonine, and methionine undergo oxidation in t h e back-titration procedure (21),i t is likely that they will also interfere in the back-titration of TAA and TBA with CAT.
ACKNOWLEDGMENT We thank D. J. McAdoo, Marine Biomedical Institute, Galveston, Texas, for his help in obtaining t h e mass spectra of t h e products.
21 1
Anal. Chem. 1980, 52, 211-213
LITERATURE CITED H. E. Christensen, T. T. Luginbyhl, and B. S. Carroll, Eds., "Suspected Carcinogens. A Subfile of the NIOSH Toxic Substances List". USDHEW. Public Health Service. US. Government Printing Office, Washington, D.C., 1975. A. M. Amin. Chemist-Analyst, 44, 66 (1955). A. M. Amin and M. Y. Farah, Chemist-Analyst, 44, 62 (1955). E. H. Swift and E. A. Butler, Anal. Chem., 28, 146 (1956). A. M. Amin. Chemist-Analyst, 45, 95, 101 (1956). E. A. Butler, D. G. Peters, and E. H. Swift, Anal. Chem.. 30, 1379 (1958). E. H. Swift and F. C. Anson, Adv. Anal. Chem. Instrum., 1, 293 (1960). J. Jue and C. L. Huyck, J. Pharm. Sci., 51, 773 (1962). M. A. Ghafoor and C. L. Huyck, J . fharm. Sci., 51, 894 (1962). A. Cyganski, Talanfa, 23, 868 (1976). K. Lesz, H. Wieczorkiewicz, and T. Lipiec, Chem. Anal. Warsaw, 6 ,
(17) N. M. Trieff, V. M. Sadagopa Ramanujam. and G. Cantelli Forti, Talanta, 24, 108 (1977). (18) N. M. Trieff, V. M. Sadagopa Ramanujam, and G. Cantelli Forti, Microchem. J . , 22, 222 (1977). (19) D. S. Mahadevappa and N. M. M. Gowda, Tabnta, 22, 771 (1975). (20) N. M. M. Gowda and D. S. Mahadevappa, J. Indian Chem. Soc., 53,
705 (1976). (21) N. M. M. Gowda and D. S. Mahadevappa, Taalanta, 24, 470 (1977). (22) N. A. Lange and G. M. Forker, in "Handbook of Chemistry", Handbook Publishers, Sandusky. Ohio, 1956,p 952. (23) T. Higuchi, K. Ikeda, and A. Hussain, J. Chem. Soc. 8 , 546 (1967). (24) E. Bishop and V. J. Jennings. Tabnta, 8. 697 (1961). (25) E. Stenhagen, S. Abrahamsson, and F. W. McLafferty, Eds., "Atlas of Mass Spectral Data", Volume I, lnterscience Publishers, New York,
1969.
1033 (1961). A. Cbeys, H. Sion, A. Campe, and H. Thun, Bull. Soc. Chim. Belg., 7 0 ,
576 (1961). C. G.Ramachandran Nair, S. Geetha, and P. T. Joseph, Indian J. Appl. Chem., 30, 60 (1967). E. Bovalini and M. Piazzi, Ann. Chim. Roma, 49, 1067 (1959). M. K. Papay, K. Toth, V. Izvekov, and E. Pungor, Anal. Chim. Acta, 64,
409 (1973). T. P. Hadjiioannou and E. A. Piperaki, Anal. Chim. Acta, 90, 329 (1977).
RECEIVED for review June 15,1979. Accepted September 19, 1979. We are grateful to the Robert A. Welch Foundation (Grant No. H-416 to N.M.T.) and Electric Power Research Institute (Grant No. A-12022 to M.S.L.) for supporting the present work.
Steam Distillation Apparatus for Concentration of Trace Water Soluble Organics T. L. Peters Analytical Laboratories, The Do w Chemical Company, Midland, Michigan 48640
Direct injection of aqueous solution is widely used in the gas chromatographic analysis of water soluble organics. The technique is straightforward, rapid, and minimizes sample handling. However, samples containing dissolved solids can rapidly plug the injection port with salts. In addition, when organic concentrations are below the detection limits of the instrument, a preconcentration technique is needed. For many cases, an adsorption concentration technique is useful. In this procedure, an aqueous injection of up to 500 p L is made onto a Tenax GC precolumn a t ambient temperature. After venting off the water, the precolumn is rapidly heated to flash the organics onto an analytical column ( I ) . Another concentration technique currently being used is the volatile organic analysis system (2, 3 ) . Here the volatile organics are purged from a water sample with an inert gas onto a n adsorbent trap. The trap is then thermally desorbed to drive the organics onto an analytical column. Static headspace analysis techniques offer another possibility for determining relatively volatile materials in the presence of water ( 4 ) . However, the described methods are generally not suitable for effective concentration of low-molecular-weight alcohols, nitriles, ketones, and aldehydes. These types of compounds, classified as volatile polar organics (VPOs), have previously been concentrated for determination by a distillation/ headspace/gas chromatographic analysis technique ( 5 ) . One hundred milliliters of sample was heated and the first 1.5 mL of distillate collected. This distillate was saturated with sodium sulfate and the static headspace sampled a t elevated temperature. Detection limits claimed were from 4 to 8 parts-per-billion for the VPOs in the original sample. For simplicity and versatility, an easier procedure is desired for concentration of VPOs, one that employs a minimum of equipment and sample handling. This work describes a small all-glass distillation-concentration system that is convenient 0003-2700/80/0352-0217$01.OO/O
to operate and fulfills the above requirements.
EXPERIMENTAL Distillation Apparatus. A diagram of the all-glass distillation-concentration system is shown in Figure 1. It consists of a distillation pot, condenser, a condensate collection (distillate) chamber, steam/water contact column, and an overflow return tube for a portion of the condensate to return to the pot. The system was built by the Dow Glass Fabrication Laboratory. Gas Chromatography. Gas-liquid chromatography was performed on a Varian Model 1400 gas chromatograph equipped with a flame ionization detector. The column used for the determination of acrolein and acrylonitrile was a 1.8 m X 2 mm i.d. glass column packed with 60/80 mesh Tenax GC adsorbent. The temperatures of the injection port and detector were 180 and 230 "C, respectively. The column temperature was maintained at 95 "C. The carrier gas was nitrogen at 15 mL/min flow rate. The sample size used was 2 pL. For the remainder of the components examined, the column was 1.8m X 2 mm i.d. glass packed with 0.1% SP 1000 over 80/100 mesh Carbopack C. The injector, column, and detector temperatures were the same as before. The nitrogen carrier gas flow rate was 30 mL/min. Procedure. For this study, 300 mL of sample was placed in the 500-mL round bottom flask. Several boiling stones were added and the sample was heated sufficiently for a distillation rate of 2 mL/min. After refluxing for an appropriate interval of time, 2 p L of the distillate from the top chamber was withdrawn and injected into the chromatograph using conditions suitable for the determination of compounds of interest.
RESULTS AND DISCUSSION In practice, the VPOs will azeotropically distill into the distillate chamber and be preferentially retained there. Condensate overflow back to the pot is stripped by the rising steam and W O s are recycled back into the distillate chamber. Since such dilute solutions are being used, each component behaves independently of the others. This results in co-dis0 1979 American Chemical Society
