a small volume of distilled water and the Cu completely stripped out. Finally, using an Eppendorf pipet, 25 ~1 of this solution were placed into the graphite furnace of the flameless AA attachment. This last step was repeated several times.
RESULTS In the first set of experiments, samples of the electrolytic solution were first purified by the above method and then spiked to various values [in the range of 0.5 to 1.0 pg 1.-' (0.8 to 1.6 X 10-9M)] of Cu, Cd, and Zn. We analyzed for Cu and found the correct values to within f5%. The next set of experiments was identical to the first, except that seawater was used. The third set of experiments consisted of measuring the concentrations of Cu in various samples of seawater. The results agreed, within the combined experimental errors of f1596, with those obtained by Fukai and Huynh-Ngoc using anodic stripping voltammetry on the same samples of seawater (2). The final set of experiments was a series of measurements of the concentration of Cu in one particular sample of seawater. We obtained the value 0.57 f 0.04 pg Cu 1.-l (9.0 f 0.6 X 10-9M) making a total of 8 independent measurements. After each measurement, the concentration of Cu in the then metal-free sample was determined again. These results were always below 0.03 pg CU1.-l (0.5 X 10-9M). DISCUSSION The result of the anodic reduction and stripping is the transfer of essentially all the available Cu from the seawater to the distilled water. The concentration factor is thus the ratio of the two volumes, which was typically 100. The reduction and stripping of 100% of the metal is important as it results in a considerable simplification in the method. If some constant fraction of the metal is to be reduced,
then the reduction must be timed and the conditions maintained constant from run to run. This imposes stringent conditions on the cell geometry, mechanism and rate of stirring, geometry of electrode, and even the nitrogen bubbling (7). These conditions are normally so variable that it is often customary to calibrate each sample by the method of standard additions (7). In our method, such variations merely change the half-time for reduction and/or stripping. Since we wait many half-lives in both steps, such changes are not significant. In fact, we do not use the method of standard additions a t all. We merely prepare a standard solution of concentration in the range of the expected result, i.e., 100 times more concentrated than the seawater sample. A single standard is sufficient since the flameless AA technique is linear in this concentration region.
ACKNOWLEDGMENT We would like to thank L. Huynh-Ngoc for his advice and assistance throughout the project. One of us (LLE) would like to express his appreciation to the International Laboratory of Marine Radioactivity in Monaco for the opportunity to work there for an extended period. (1) (2)
LITERATURE CITED J. D. Winefordner and R. C. Elser, Anal. Chem., 43 (4). 24A (1971). R. Fukai and L. Huynh-Ngoc, XXiV Congres Assoc. Wen. Monaco, 6-14 Dec. 1974.
(3) D. A. Segar and J. G. Gonzelez. Anal. Chim. Acta. 58, 7 (1972). (4) C. Fairless and A. J. Bard, Anal. Chem., 45, 2289 (1973). (5) C. Fairless and A. J. Bard, Anal. Lett.. 5, 433 (1972). (6) W. R. Matson, D. K. Roe, and D. E. Carritt. Anal. Chem.. 37, 1594 (1965). (7) R. G. Clem. G. Litton, and L. D. Ornebs. Anal. Chem., 45, 1306 (1973).
RECEIVED for review May 5, 1975. Accepted August 4, 1975.
Direct Potentiometric Measurement of Several Thiols Paul K. C. Tseng and W. F. Gutknecht Department of Chemistry, Duke University, Durham, N.C. 27706
The sulfide ion-selective electrode has previously been used for the analysis of various thiol compounds. In 1971, Gruen and Harrap used the sulfide ion-selective electrode and silver nitrate as a titrant to determine potentiometrically the concentrations of cysteine and glutathione (1). Pungor et al. made similar titration measurements with thioacetamide (2) and phenylthiourea and N,N-diphenylthiourea ( 3 ) . In these analyses, the ion-selective electrode was used to monitor the change in [Ag+] during the course of the titration. Some success has been attained in the direct potentiometric analysis of several simple thiols. In 1972, Guilbault and von Storp used the sulfide ion-selective electrode to monitor the production of various thiocholine salts ( 4 ) . Peter and Rosset used the sulfide ion-selective electrode for the direct measurement of several thiols in a benzeneethanol solvent mixture ( 5 ) . Their results did not show good agreement with the Nernst relationship. In this paper, work is reported wherein several thiols in aqueous NaOH solution have been measured by the direct potentiometric method using a Ag2S membrane electrode. The electrode responses to these various thiols are Nern) to a detection limit as stian (i.e., Emeasd = f(1n U A ~ + ) down predicted by a model developed by Morf et al. (6). Subse2316
quently, the slopes of the response curves ( E m e m d vs. log cthiol) have been used to determine the stoichiometry of the electrode response reactions and the apparent formation constants for the silver-thiol complexes formed as a part of these reactions. The potential measured with an electrode utilizing a silver salt membrane can be described by the equation
EA^+ = E ' A ~ ++ RT -In U A ~ + F
where a A g + is the activity of the silver ion at the sample solution-electrode membrane interface. In the absence of complexing agents, this activity is described as the sum of: 1) the silver ion activity in the sample solution; 2) the silver ion activity due to the dissolution of the electrode membrane; and 3) the silver ion activity due to interstitial or defect silver ions. For a membrane composed of compressed, polycrystalline Ag& the silver ion activity due to membrane dissolution is much less than the defect silver ion activity, which is about 10-5.5M (6). Now, in the presence of thiols (silver ion complexing agents), the reaction between the silver ion and the thiol is
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
Ag+
+ pRS-Y
= Ag(RS)p'-PY
Table I. Results of the E v a l u a t i o n of the Thiol R e s p o n s e C u r v e s Thiols
PK
(-W
Response slope, mV per decade
Detection limit, M
P I X ratio
(Agx(RS)pX-Py)
Possible complex formula
-loa 1.9“ Ag(-SCH,CH(NH,)C02-)z32.0 x 10-4121 (in 0.1121 NaOH) (2 .O)b Ag(-SCH,C02-)2310.67 1.0 x 10-4121 -110 1.9“ Thioglycolic acid (in lhl NaOH) (2.0)b -110 . 1.9“ Ag(BCH,CH,C0,-)233 -Mer c apto 10.27 2.0 x 1 0 - 4 ~ propionic acid (in 5M NaOH) (2.0)b -a4 1.4“ Ag, (-SCH,CH,OH),9.50 1.0 x 10-4,w 2-Mercaptoethanol (in 5M NaOH) (1.5) Calculated using the theoretical electrode response to [Agf], 58 mV per decade (temperature adjusted). Calculated using the experimental electrode response to [Agf], 55 mV per decade. 8.33
Cysteine
-
If a is the defect silver ion concentration a n d L is the total sample solution thiol concentration, then as t h e thiol concentration is usually m u c h larger t h a n a , and also, t h e formation constants of silver-thiol complexes are moderately large, most of t h e silver ion present will be found in t h e complex (Ag(RS)p’-PY) (defined as P). If t h e concentration of “free” thiol, (RS-y), at t h e membrane surface is defined as 6, t h e n
S=L-pP
-500 I
> I I
P p
4 0 0 I
I
and the “free” silver concentration C A ~ += a - P. As t h e “free” (Ag+) will be very small with L large, C A ~ +N 0 and a 31
I
p.
I
.
-100 I
0
Now CAgf
=
I
Ag(RS)p l-py
I
I
I
K{(RS-y)P =-
P
K(6P
-
a
K{(L - pa)P
Substituting for U A ~ +in t h e Nernst equation written above and redefining EA^+ as E R S - ~t h, e cell voltage with thiol present,
RT ERS-Y= E ’ A ~ + + -In F
a
-Kf’
PRT In (L - p a )
F
T + RIn Y A ~ + F
where YAg+ is t h e activity coefficient for the [Ag+]. An equivalent expression for p = 2 has been described by Morf et al. (Equation 35 i n (6)).W i t h L large, i.e., L >> pa, a plot of E m e a s d vs. In [ L ]will yield a line of slope -pRT/F from which p, t h e stoichiometric t e r m can be determined. a n d a , K / can thus be determined. Knowing E’A~+
EXPERIMENTAL The sulfide ion selective electrode used in this work was prepared in this laboratory. To prepare a membrane, 0.5 g of reagent grade AgzS is ground with a mortar and pestle and then compressed in a KBr die at 30000 psi. The resulting membrane has a thickness of 1 mm and is quite rugged. This membrane is attached to a glass tube using epoxy resin. Before using the electrode, the membrane surface is polished on Microcloth (Buehler Ltd.) using 0.05-fim alumina as the polishing agent. The filling solution of this electrode was 0.004M AgN03 and the internal reference electrode was a silver wire. The reference electrode was a saturated calomel electrode. Connection between the reference electrode and test solution was made with a fiber-tipped secondary junction filled with 1M KN03 solution. The laboratory-made sulfide electrode responded linearly to sulfide in NaOH solution over a concentration range of 0.1to 10-6M. All solutions were prepared with reagent grade chemicals. Cysteine was standardized using a bromination method (7). All other thiols were used as supplied by the manufacturers. The cysteine
solutions were 0.1M in NaOH, the thioglycolic acid solutions 1.OM in NaOH, and the 2-mercaptoethanol and 3-mercaptopropionic acid solutions either 1.OM or 5.OM in NaOH. These high concentrations of base were found necessary to obtain rapid, stable responses. As will be discussed below, oxidation of these thiols was not a serious problem under these conditions. In addition, the NaOH solutions used maintained both the pH and ionic strength of the solutions a t constant values. Water was purified by deionization followed by distillation. All the cell emf measurements were made with a Beckman Model SS-3 pH meter. Solutions were stirred during each measurement and the solution temperatures were ambient a t 20-21 O C . The final potential of each test solution was determined when the measured potential changed less than 0.5 mV over a 5-minute period. Replicate measurements were reproducible to within &1 mV if care was taken to lightly repolish the electrode surface before each measurement.
RESULTS AND DISCUSSION Figure l ‘ s h o w s plots of Emeasd vs. log [ L ] for t h e four thiols mentioned above. T h e detection limits a r e all about 10-4M. T h e s e detection limits occur when t h e bulk thiol concentrations a r e n o longer m u c h larger than a and subsequently a ~ changes ~ + rapidly with change in thiol activity. When the thiol concentration is very low, Emeasd levels off at a constant value as U A ~ + a (6). T h e overall response curve is analogous t o a reverse titration of a s e t a m o u n t of silver ion [a]with thiol. T h e slopes of these plots were determined and used t o calculate t h e formulas of t h e silverthiol complexes as explained above. T h e s e results a r e shown in T a b l e I. It is interesting t o note that t h e slopes of t h e Emeasd vs. log [ L ] plots for t h e 1:2 silver-thiol complexes average -109 m V per decade, which compares favorably with t h e value of -118 m V per decade predicted by
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
2317
Table 11. Apparent Formation Constants of the Silver-Thiol Complexes Ionic m e n th of the sample solution, td
Thiols
Cysteine Thioglycolic acid 3-Mercaptopropionic acid 2-Mercaptoethanol a
K;
a
0.1 1.o
3.2 x 1013 1.6 x 1014
1.o
2.6 x
1.o
6.2 x 1029
1014
C a l c u l a t e d u s i n g t h e theoretical electrode response t o
[Ag+], 58
mV p e r decade ( t e m p e r a t u r e adjusted).
I
I
I
0
5
IO T I M E , MIN
I 15
I 20
Flgure 2. Response time of the AglS membrane electrode for several different concentrations of cysteine
Morf et al. (6). This value of -109 mV per decade appears in a more favorable light when it is noted that the laboratory-made electrode used in this work yields a response to sulfide ion of -27.5 mV per decade. That is, the average response of the electrode to the thiols (which yield a 1:2 complex with [Ag+])is within 1 mV of four times the response to sulfide (which yields the 2:l product, Ag2S). The apparent formation constant, K(, can also be determined as described above. One first plots the responses of the electrode to [Ag+] and the thiol in question. Both these plots are then extrapolated to the concentrations of 1.OM. A t these points
RT F
EA^+ = E " A ~ ++ -In Y A ~ + and
+ RT F
a RT Kf' F The 7 ~ ~ lfor - k the two solutions can be assumed to be approximately equal in that the [Ag+] and thiol solutions were equimolar in KN03 and NaOH, respectively. Subtracting the latter equation from the former yields RT a EA^+ - E R S - =~ - -In F K( and Kf/ can thus be calculated. (In this calculation it is assumed that the defect [Ag+] concentration is equal to the defect [Ag+] activity, which a t present, is known only approximately (6)). Kf' values for the four thiols mentioned previously are listed in Table 11. As a test of the validity of this approach to calculating the Kf' values, such values were also determined for Ag(CN)2- and A g ( S 2 0 ~ ) 2 ~from the response of the AgzS membrane electrode to the li. so determined were gands CN- and S Z O ~ ~ -Values pK'f,Ag(CN)2-= -18.1 and ~ K ' ~ , A ~ ( S=~-12.4, O ~ ) ~which ~compare well with previously published values of -18.75 and -13.2, respectively (8). It should be noted that if the experimental [Ag+] response of the electrode (55 mV per decade) is used to calculate these values rather than the theoretical value of 58 mV per decade (temperature adjusted), the values would be pK'f,Ag(cNI2- = -19.3 and = -13.3. PK'f,~g(~203)23The electrode response time is a factor to be considered in any practical application. Figure 2 shows the electrode response to different concentrations of cysteine. The same response time of only several minutes is also noted with the other thiols tested. ERS-Y
2318
= E " A ~ + --In
-+ -In 7 A g +
Another concern is oxidation of the thiols under the high pH conditions used in this work. The four thiols discussed above did not show any rapid oxidation in that freshly prepared solutions were stable, Le., yielded the same Emeasd values, for a minimum of four hours. Two other thiols tested, 1,2-dithioethaneand thiophenol did not yield stable potentials. Solutions of these thiols showed the presence of white precipitates upon standing for a period of time. It was thought that these precipitates might be oxidation products as thiols can be auto-oxidized by molecular oxygen through a free radical mechanism (9); that is
The rate of oxidation depends upon the concentration of the catalysts (e.g., Fe, Hg, etc.), (10, I I ) , the pH (12), the oxygen tension (131, and the thiol structure (14, 15). The precipitates formed in solutions of these two thiols were analyzed by IR, NMR, and mass spectrometry. The mass spectrum indicated that the molecular weights of these two samples were 218 and 184. IR spectra indicated that these two precipitates must be sulfides or disulfides as there are no absorption peaks in the region of 2500-2600 cm-l (the SH stretching region). The precipitate from the thiophenol solution was further examined by NMR using CC14 as the solvent. The resulting spectrum matched that of phenyl disulfide (16). The melting point for this precipitate was found to be 57 "C, very close to that of phenyl disulfide (59-60 "C), and it has subsequently been concluded that the precipitate formed from the thiophenol was indeed phenyl disulfide. The precipitate from the 1,2-dithioethane is probably the dimer
I I -c-s-s-c-c-s-s-c-
I
I I I
A seventh thiol to be tested was thioacetic acid, which yielded a response slope of about -28 mV per decade. It is apparent that hydrolysis occurs easily for this compound ( 1 7 ) and the ion detected is the sulfide ion, S2-. LITERATURE CITED (1)L. C.Gruen and B. S. Harrap., Anal. Biochem., 42, 377 (1971). (2) M. K. Papay, K. Toth, V. Izvekov, and E. Pungor. Anal. Chim. Acta, 64, 409 (1973). (3) M. K. Papay. V. P. Izvekov, K . Toth, and E. Pungor, Anal. Cbim. Acta, 69, 173 (1974). (4)L. H. von Storp and G. G. Guiibault. Anal. Chim. Acta. 62, 425 (1972). (5) Francis Peter and Robert Rosset, Anal. Chim. Acta, 64, 397 (1973). (6)Werner E. Morf. Gunter Kahr, and Wilhelm Simon, Anal. Cbem., 46, 1538 (1974). (7) Yazuru Okuda. J. Biochem. (Jpn), 5, 201 (1925).
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
(8) L. R. Sillen and A. E. Martell, "Stability Constants of Metal-Ion Complexes", Spec. Pub/. No. 77, The Chemical Society, London, 1964. (9) R. Cecil and J. R . McPhee, Adv. Protein Chem., 14, 293 (1959). (10) L. Michaelis and E. S. G. Barron. J. Biol. Chem., 81, 29 (1929). (11) C. Voegtlin. J. M. Johnson, and S. M. Rosenthal, J. Biol. Chem.. 93,435 119.111 , . - - .,.
(12) J. S. Fruton and H. T. Clarke, J. Biol. Chem., 108, 667 (1934). (13) E. S. G. Barron. Arch. Biochem. Biophys., 58, 502 (1955). (14) E. S. G. Barron, 2 . E. Miller, and G. Kalnitsky, Biochern. J., 41, 62 ,_^
(15) R. Wade, M. Winitz, and M. P. Greenstein, J. Am. Chem. SOC.,78, 371 (1956). (16) "The Sadtler Standard Spectra", Sadtler Research Laboratories, N.M.R., 1, 286 (1966). (17) M. Cefola, Sr., Simon Peter, P. S. Gentile, and Rev. A. V. Celiana, Ta/anta. a. 537 (19621. ~'~~B1vB I UuL ' r * A r v v
"UL'r
(1Y 4 I ) .
I AIDS FOR ANALYTICAL CHEMISTS Rapid and Inexpensive Method for Detection of Polychlorinated Biphenyls and Phthalates in Air C. S. Giam,' H. S. Chan, and G. S. Neff Depatiment of Chemistry, Texas A&M University, College Station, Texas 77843
The total production of phthalic acid esters is about ten times larger than that of polychlorinated biphenyls (PCBs), which are recognized as environmental pollutants. In recent years, scientists have been concerned with the release of phthalate plasticizers into the environment ( I ) , but previously reported methods are often insufficiently sensitive, elaborate, or multistep procedures which introduce contaminants to the sample. Thomas ( 2 ) , using ethylene glycol as a trapping solvent, detected 300 ng/m3 of di(2ethylhexy1)phthalate (DEHP), 750 ng/m3 butylphthalyl butyl glycolate, and 700 ng/m3 di-n-butyl phthalate (DBP) in air samples taken a t the water works of the Municipality of Hamilton in Canada. Recently, gas chromatographic column packings or solid supports have been used to trap organics from air (3-10) and have proved to be more advantageous than other methods. Mieure and Dietrich (7) used 5% Dexsil300 on 80-100 mesh H P Chromosorb W to determine trace organics in air; the particular phthalate esters present could not be identified because of GC interferences. It is well established that environmental analytical chemistry needs simple, inexpensive, and sensitive analytical methods for routine monitoring. This paper describes a simple, inexpensive, and sensitive method for the determination a t low levels (ng/m3) of not only specific phthalate esters but also PCBs and DDT in air; Florisil was used for both sampling and separation of the mixture of pollutants.
EXPERIMENTAL Apparatus. A Tracor Model MT-220 gas chromatograph (GC) equipped with a Nickel-63 (10 mCi) electron capture (EC) detector in a dc mode was used for analyses. It was fitted with a 6-ft X '/4-in. o.d borosilicate glass column packed with 3% SE-30 on Gas Chrom Q (100-120 mesh); nitrogen was used as the carrier gas at a flow rate of 60 cm3/min. The injector, detector, and column temperatures were 250,275, and 200 "C, respectively. A Hewlett-Packard Model 5700A GC equipped with a 63Ni (15 mCi) EC detector and a 6-ft X Yd-in. 0.d. borosilicate glass column packed with 3% SE-30 on Chromosorb WHP (100-120 mesh) was also used for analyses. Methane 10%/argon 90% was used as the carrier gas at a flow rate of 60 cm3/min. The injector, detector, and oven temperatures were 200,300, and 200 "C, respectively. The EC detector responses were linear up to 100 ng DEHP per injection.
'Author to whom all correspondence should be addressed.
Second-column confirmation was performed on a Barber-Colman Model 5360 GC with a Tritium (300 mCi) EC detector in a pulse mode, equipped with a 6-ft X %-in. 0.d. borosilicate glass column packed with 1.5% SP-2250 and 1.95% SP-2401 on Supelcon AW-DMCS (100-120 mesh). The injector, detector, and column temperatures were 210, 210, and 195 "C, respectively. The operating sensitivity for the detectors was about 5 ng DEHP giving 50% full scale deflection (fsd). Reagents. Petroleum ether (Mallinckrodt Nanograde) was used as received. Anhydrous ether (Mallinckrodt) was freshly distilled immediately before use. Anhydrous sodium sulfate (granular, Mallinckrodt) and Florisil (60/100 PR grade, Floridin Chemical Co.) were heated at 320 "C for at least 24 hr prior to use. All solvents and reagents were checked for contamination before use by GC analysis of a concentrate of the solvent or solvent rinse of solid reagents. Procedure. A Pasteur disposable capillary pipet or a short section of conventional GC column (%-in.0.d.) was used as the collection column. Each column was packed with 0.3 g of deactivated Florisil (3% water W/W), followed by 1 g of granular anhydrous sodium sulfate and a small plug of glass wool. The column was connected to an oil-less vacuum pump with Teflon tubing. For example, we used a flow rate of about 2-4 l./min with the free air capacity of the pump being 90 l./min. Sampling times varied but as long I ~ R60 hr were used. The Florisil was eluted with 4 ml of petroleum ether followed by 4 ml of diethyl ether. The two fractions were collected in graduated centrifuge tubes, concentrated or diluted as needed, and analyzed by gas chromatography. The petroleum ether fraction contains the chlorinated hydrocarbons while the ether eluate contains the phthalate esters. Excellent blanks were obtained with background levels of 1 ng for DEHP, 0.1 ng for PCBs, and 0.1 ng for DDT.
RESULTS AND DISCUSSION Since the electron capture detector is sensitive to the presence of phthalates (11) as well as to the chlorinated hydrocarbons, we have been investigating electron-capture gas chromatographic methods for the trace analysis of these two groups of compounds in environmental samples. In developing these procedures, we found that airborne contamination in the laboratory would be a significant problem. Since "clean-rooms" are expensive and often not available in many laboratories, we sought a way to rapidly analyze and monitor the air in various working areas in order to select those areas that were optimal for our work. The solid sampling systems, such as the gas chromatographic packings or other adsorbents, appeared to offer the
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
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