Chemically-Bonded Aminosilane Stationary Phase for the High-Performance Liquid Chromatographic Separation of Polynuclear Aromatic Compounds S. A. Wise," S. N. Chesier, H. S. Hertz, L. R. Hilpert, and W. E. May Institute for Materials Research, Analytical Chemistry Division, National Bureau of Standards, Washington, D.C. 20234
A mmercialty available, chemicaiiy-bonded aminosiiane liquid chromatographic packlng material, pBondapak NH2, was used as a substitute for silica or alumina for the normal-phase high-performance liquid chromatographic (HPLC) separation of aliphatic hydrocarbons and polynuclear aromatic compounds. The pBondapak NH2 provides a more distinct separatlon of polynuclear aromatic compounds based on the number of condensed rings than the classical adsorbents because the addhion of alkyl groups to the aromatic rings has very little effect on their retention. The retention characteristics of polynuclear aromatic compounds on pBondapak NH2, classical adsorbents, and a reversed-phase packing material were compared. The application of pBondapak NH, to Isolate polynuclear aromatic compounds according to the number of condensed rings prior to analyds by gas chromatography-mass spectrometry (GC-MS) and/or reversed-phase HPLC with fluorescence emission spectral identification is described.
A large number of laboratories are currently involved in the analysis of hydrocarbons in the environment. In the past, research has focused primarily on the determination of the low molecular weight (less than 300) aliphatic and olefinic hydrocarbons because of their relative ease of analysis by gas chromatography (GC). Of more recent interest are the polynuclear aromatic hydrocarbons (PAHs) because of their possible carcinogenic and mutagenic properties. The current increasing use of fossil fuels and the potential use of liquified coal and shale oil require the development of meaningful analytical methodologies for the determination of PAHs in the environment. Fossil fuels contain a wide range of aromatic hydrocarbons with the alkylated PAHs generally being more abundant than the unsubstituted parent PAHs ( I ) . Soils and recent sediments from presumably unpolluted environments have also been reported to contain series of alkylated aromatics (2). The complexity of such samples necessitates the combination of several chromatographic techniques to achieve suitable separations. T h e typical analytical scheme for hydrocarbon analysis in environmental samples involves extraction, fractionation of aliphatic and aromatic hydrocarbons by column chromatography on silica and/or alumina, and finally quantitative and qualitative analysis by GC and GC-MS. Several problems are encountered when using classical adsorbents such as silica or alumina. For example, the retention characteristics of these adsorbents with nonpolar mobile phases are strongly influenced by the water content of the eluent. Thus, small changes in the water content of the eluent result in nonreproducible separations ( 3 , 4 ) . In addition, the high adsorptivity of the classical adsorbents can result in the loss of trace components and the tailing of late-eluting components. These difficulties make the use of silica or alumina for routine fractionation of PAHs unreliable. Recently, Giger and Blumer ( 5 ) described an analytical procedure for isolating PAHs which utilized gel filtration on Sephadex LH-20, adsorption chromatography on silica gel2306 * ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
alumina, and charge-transfer complexation. Novotny et al. (6-8) employed Sephadex LH-20 for purification and bulk separations, but utilized a polar, chemically-bonded stationary phase (oxypropionitrile) instead of adsorption chromatography for further fractionation of the PAHs prior to GC-MS analysis. Other workers (9, IO) have described the combination of adsorption or gel chromatography and reversed-phase HPLC t o resolve complex mixtures. Recent work in this laboratory has involved the use of MBondapak "2, a polar chemically-bonded stationary phase, as a substitute for silica to remove interfering polar compounds in the analysis of hydrocarbons in marine biota (11). In the present paper the retention characteristics of MBondapak NH2 and a reversed-phase HPLC material for aromatic hydrocarbons are compared with literature data on retention by classical adsorbents. In addition, the use of KBondapak NH2 to fractionate PAHs which were extracted from a marine sediment is reported. The KBondapak N H 2 yielded separations according to the number of condensed rings. Each PAH fraction was subsequently analyzed by reversed-phase HPLC to separate the alkyl homologues within each fraction. Fluorescence emission spectroscopy and GC-MS were utilized for identification of the individual components. T h e application of MBondapak NH2 for bulk fractionation of PAHs in crude oil prior to analysis by GC-MS is also described.
EXPERIMENTAL Column Comparison. The retention characteristics of pBondapak NH2 and pBondapak C18for a number of aromatic hydrocarbons were determined. Hexane was used as the mobile phase for the pBondapak NH2 and mixtures of 50-70% acetonitrile in water were used for the pBondapak C18. Marine Sediment Sample. An extract of a petroleumcontaminated sediment was obtained by ultrasonic agitation (- 2 h) of the sediment (990 g) with ether. The extract was concentrated by evaporation, diluted with freshly distilled pentane, and gently heated to remove the remaining ether. The pentane solution was concentrated to approximately 1 mL, and the latter was injected onto a 30 cm X 4 mm i.d. pBondapak NH2 column (Waters Associates, Milford, Mass.). The PAHs were eluted using freshly distilled pentane as the mobile phase. A small volume of acetonitrile was added to the PAH fractions and the pentane removed by evaporation. The concentrated fractions were analyzed by reversed-phase HPLC on a 30 cm X 4 mm i.d. pBondapak CIS column utilizing a linear gradient of 50-100% acetonitrile in water in 30 min. The column effluent was monitored with a UV absorption detector at 254 nm (Waters Associates, Milford, Mass.) and a fluorescence spectrophotometer (Model FP-4, JASCO Incorporated, Easton, Md.) in series. The fluorescence spectrophotometer had been previously modified by decreasing the slits of the excitation monochromator and increasing the cell volume from 9 to 36 pL (12). In order t o obtain a fluorescence emission spectrum on an eluting component, the mobile phase flow was diverted (by means of a valve) at the maximum fluorescence response, thereby leaving the peak sample trapped in the flow cell. This procedure allowed continuous monitoring of the chromatogram with the UV absorption detector while scanning to obtain the fluorescence emission spectrum of the component trapped in the fluorescence spectrophotometer flow cell.
Table I. Comparison of Retention Indices (I)of Aromatic Hydrocarbons on Several Liquid Chromatographic Packing Materials Logarithm of the retention index (log I) pBondapak Compounds One-ring aromatics Benzene Toluene rn-Xylene 1,2,4-Trimethylbenzene n-Pentyl benzene Nonadecyl benzene Two-ring aromatics Naphthalene 2-Methylnaphthalene 2,3-Dimethylnaphthalene 2,6-Dimethylnaphthalene 1,5-Dimethylnaphthalene 2,3,6-Trimethylnaphthalene 1,4,6,7-Tetramethylnaphthalene 2-Ethylnaphthalene Biphenyl 3-Ethylbiphenyl Acenaphthalene Three-ring aromatics Fluorene Dibenzothiophene Anthracene 2-Methylanthracene 9,10-Dimethylanthracene Phenanthrene 1-Methylphenanthrene p-Terphenyl Four-ring aromatics Fluoranthene Benzo [ a ]fluorene Benzo[ b ]fluorene Pyrene Naphthacene Benzo[a]anthracene Triphenylene Chrysene Five-ring and larger aromatics Benzo [ a Ipyrene Perylene Benzo [g,h,i]perylene Indeno [ 1,2,3-c,d]pyrene Dibenzo [a,c ]anthracene Dibenzo[a,h ]anthracene Picene Benzo [ b Ichrysene
Polystyrene gel ( 1 5 ) pBondapak (waterC,, (acetonitrileAlumina ( 1 4 ) methanolwater) (pentane) diethylether)
*" ( hexane)a
Silica ( I 3 ) (pentane)
1.00
1.00
1.00
1.00
1.00
1.00 1.00 1.00 0.71 0.50
1.30 1.43
1.05 1.08 1.23 1.08
1.22 1.32 1.60 1.81
1.51 2.16 2.65 3.80
2.00 1.96 2.08 1.94 1.93 2.00 2.05 1.92 2.25 2.20 2.00
2.00 2.25 2.50
2.00 2.11 2.32
2.00 2.25
2.21 2.92
2.09 2.23
2.31 2.25
...
2.58
2.61 2.75 2.95 2.92 2.95 3.00 2.98 3.40
3.26
2.79
2.70
2.95 2.97
3.00
3.00 3.26 4.51
3.00
3.39 3.46 3.53 3.68 3.93 4.00 4.00 4.03
3.42 3.95 4.14 3.06 3.95 4.00 4.04 4.00
4.30 4.41 4.61 4.72 4.93 4.93 >5 5.00
4.11 4.20 4.21
... ... ...
... 2.32 ... ...
.*. ...
*.. ...
... 4.92 ...
5.07 5.00
...
... 2.04 ... ...
...
...
. , .
... ... ... ... ...
...
3.18
... 3.09 ... ... 3.00 ...
3.44
3.66
3.41
3.36
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...
3.31
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4.00 4.06
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... ... 3.45 ... 4.00 ...
3.95
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4.63
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5.00
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.
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5.00
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2.00 2.59 3.47 3.15 3.00 3.54 3.86 3.14 2.57 3.46 2.73 2.78 2.98 3.02 3.63 3.82 3.00 3.43 3.94 3.42 3.74 3.78 3.51
...
4.00 3.82 3.94 4.57 4.43 >5 >5 4.84 >5 4.93 5.00
a Hexane was used as the mobile phase because of poor flow reproducibility with pentane. However, both solvents produce identical chromatographic results using p Bondapak NH, for normal-phase HPLC.
Crude Oil Sample. A crude oil sample was diluted with pentane and was then fractionated on a 61 cm X 7 mm i.d. pBondapak NH2 column using freshly distilled pentane as the eluent. After the individual fractions were concentrated by gentle evaporation in a heated sandbath, GC analyses were performed on a glass SE-30 coated, SCOT analytical column (100 m X 0.65 mm i.d.). A gas chromatograph-mass spectrometer computer system (Model 5930, Hewlett-Packard, Palo Alto, Calif.) was utilized for GC-MS analysis of these samples.
RESULTS AND DISCUSSION FBondapak NH2 consists of a n aminopropylsilane chemically bonded to 10-Wm fully porous silica particles. The highly polar, strongly basic surface of the pBondapak NH2 material makes it useful for the separation of polar compounds. However, when a nonpolar mobile phase such as hexane is used, a hydrocarbon class separation similar to that obtained on a silica or alumina column is achieved, i.e., saturated
hydrocarbons elute before olefinic and aromatic hydrocarbons, and the elution volumes for the aromatic hydrocarbons increase with the number of condensed rings. The addition of approximately 10% of a more polar solvent such as methylene chloride to the mobile phase results in the elution of 5-7 condensed-ring PAHs as sharp peaks in less than 10 min. Logarithms of the retention indices (I) for a number of aromatic hydrocarbons on pBondapak NH2, silica ( I 3 ) , alumina ( I 4 ) , polystyrene gel ( 1 5 ) , and WBondapak CI8, a reversed-phase packing, are given in Table I. The retention indices were calculated as previously described by Pop1 et al. ( I 3 ) , with the hydrocarbon standards being assigned the following retention indices: benzene 10, naphthalene 100, phenanthrene 1000, benzo[a]anthracene 10 000, and benzo[blchrysene 100000. The retention index of a n aromatic hydrocarbon was then calculated in a manner analogous to the calculation of Kovgts indices for gas chromatography. ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
2307
Time
porous polystyrene gel and found that the presence of alkyl groups increased the retention approximately to the same degree as on silica. Streuli (16) found the retention of PAHs on Sephadex LH-20 to decrease slightly with alkyl substitution. T h e data summarized in Table I indicate that the presence of alkyl groups affects t h e retention of the PAHs more on silica than on alumina. Martin et al. (17) also observed this behavior for alkylbenzenes. T h e reversed-phase packing material, pBondapak CIS, provides a separation for unsubstituted PAHs similar to silica, alumina, and pBondapak NH2. However, the presence of an alkyl side chain on the aromatic ring significantly increases its retention on the reversed-phase material. Reversed-phase liquid chromatographic separations are largely dependent on the relative solubilities of the components in the polar mobile phase (18,19). The addition of an aliphatic side chain results in a significant reduction in the solubility and, as a consequence, a large increase in the retention volume (see Table I). In complex samples containing alkyl-substituted PAHs as well as the unsubstituted parent PAHs, the use of reversed-phase HPLC could therefore yield misleading results due to overlapping alkyl homologues; for example, 1,3,5trimethylnaphthalene, 1-methylphenanthrene, and fluoranthene elute simultaneously even though these compounds consist of two, three, and four rings, respectively (see Table
-t
Figure 1. HPLC analysis of a sediment extract on pBondapak NH,. Numbers refer to the fractions collected for subsequent analysis. Conditions: pentane at 3 mL/min, 1.0 absorbance unit full-scale (aufs), 1.0 mL injected With the classical adsorbents, the addition of alkyl groups to a n aromatic ring generally increases the retention of the compound. In contrast, when using pBondapak N H y the presence of an alkyl group on the PAH has only a slight effect on retention; for example in Table I, compare the retention of m-xylene, 2-methylnaphthalene, and 1-methylphenanthrene with the unsubstituted parent aromatic hydrocarbon on silica, alumina, and pBondapak NH2. The pBondapak NH2 provides a more distinct class fractionation than achieved with silica or alumina. In normal-phase HPLC with pBondapak NH2, t h e retention is based primarily on the interaction between the aromatic T electrons of the PAH and the polar amino group of the stationary phase. Since the addition of alkyl substituents to the aromatic rings would affect the T electron system only slightly, alkyl substitution would also affect the retention on pBondapak NH2 only slightly. Pop1 et al. ( 1 5 ) studied the retention characteristics of PAHs on a macro-
I). The combination of separations, first with pBondapak NH2 to achieve a PAH fractionation based on the number of condensed aromatic rings, and then with reversed-phase HPLC to obtain the separation of the alkyl homologues within each fraction, provides a powerful technique for the LC analysis of complex mixtures of PAHs. This combination was applied to the analysis of a marine sediment collected near a natural oil seep. Figure 1 is the liquid chromatogram obtained on pBondapak NH, of an extract of the sediment. Numbers on the chromatogram identify the fractions which were collected for subsequent reversed-phase HPLC analysis. A reversed-phase HPLC analysis of fraction 4 is shown in Figure 2. In addition to both the UV absorption and fluorescence emission response, Figure 2 also contains the fluorescence emission spectra obtained for several of the (D
'
Y
\
J
FLUORESCENCE o x : 270 om: 400
0
I
10
I
I
1
20
30
40
Time (min)
Flgure 2. Reversed-phase analysis of sediment fraction 4 (Figure 1). Conditions: 50-100% acetonitrile in water, linear gradient in 30 min at 2 mL/min., 0.05 aufs, 200 pL injected (-20% of fraction 4). Upper chromatogram: UV absorption detection at 254 nm. Lower chromatogram: Fluorescence emission detection at 400 nm with excitation at 270 nm. Spectra a thru 9: Fluorescence emission spectra at 270-nm excitation
(numbers indicate wavelengths in nm)
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
1
198 212
B
I
30
,
35
I
,
,
,
,
,
,
40
,
I
,
,
,
(
,
45
,
,
50
Time ( m ) i n )
Figure 3. HPLC analysis of crude oil on pBondapak NH,. (A) crude oil, (B) separation of a synthetic mixture of aromatic hydrocarbons. Conditions: pentane at 2 mL/min, 2.0 aufs, 1.0 mL injected
Figure 4. GC-MS analysis of crude oil fraction 2 (Figure 3A). (A) single ion records m / e 184, 198, and 212. (B) total ion chromatogram. Tenative GC-MS identifications: ( a ) fluorene, ( b , c, d and e ) methylfluorenes, ( f ) dibenzothiophene, (g,h , i , and k ) C,-substituted I , and m ) methyldibenzothiophenes, ( n , 0 ,p , q , r , and fluorenes, (j, s) C,-substituted dibenzothiophene. Column: lOOm X 0.65 mm i.d. glass SE-30 coated SCOT. Conditions: Helium flow at 5 mL/min., temperature program from initial temperature of 80 OC for 8 min to 275 O C at 4'Irnin
chromatographic peaks. Peak a is tentatively identified as chrysene based on the retention volume and the fluorescence spectrum. The spectra for peaks b thru g are similar in shape t o the fluorescence emission spectrum of chrysene, but the fluorescence maxima are shifted slightly to higher wavelengths. Since the addition of alkyl groups, particularly methyl groups, t o the aromatic ring produces a displacement in the fluorescence spectrum towards higher wavelengths (201, these fluorescence spectra and the chromatographic retention data indicate that a series of alkyl (probably methyl) substituted chrysenes is present in this fraction. The largest peak in Figure 1 (fraction 3) is in the three-ring PAH region. Reversed-phase HPLC/fluorescence emission spectroscopy and GC/MS indicated that the compounds in this fraction were primarily phenanthrene and C1-, C 2 - ,and C3-substituted phenanthrenes with no detectable amounts of anthracene or alkyl-anthrancenes. This is not unexpected as Pancirov and Brown ( I ) have reported that phenanthrene and alkyl-phenanthrene homologues are the most abundant PAHs in several crude oils, and other investigators (21, 22) have reported t h a t phenanthrenes are present in some crude oils in much greater quantities than anthracenes. In addition, GC-MS analysis indicated the presence of dibenzothiophene and C1- and C2-substituted dibenzothiophenes in fraction 3. The aliphatic hydrocarbons, which are often used as an indication of the presence of petroleum, were identified in fraction 1 by GC-MS analysis. T h e application of FBondapak NH2 to fractionate crude oil prior to GC-MS analysis was also investigated. Chromatograms obtained on pBondapak NH2 for a synthetic aromatic hydrocarbon mixture and a crude oil are shown in Figure 3. A GC-MS analysis performed on the fraction between the naphthalene and anthracene fractions is shown in Figure 4. This fraction was found to consist primarily of three-ring compounds containing a five-member ring (i.e.,
dibenzothiophenes, dibenzofurans, and fluorenes). Figure 4 contains a total ion chromatogram ilnd the single ion records for m / e 184, 198, and 212 which indicate the presence of dibenzothiophene, C1-substituted dibenzothiophenes, and C2-substituted dibenzothiophenes. A series of alkylated fluorenes and another of alkylated dibenzofurans were also identified. The predominant components of the naphthalene region (fraction 1 in Figure 3) were identified as C1- through C4-substituted naphthalenes. The most abundant compounds present in the anthracene region (fraction 3 in Figure 3) were identified by GC-MS as C1- through C4-substituted phenanthrenes/anthracenes. Thus, the pBondapak NH2 not only provides a separation according to the number of condensed aromatic rings, but is also capable of separating three-ring components containing a five-membered ring from three-ring components consisting only of six-membered rings. Three-ring nitrogen heterocyclic compounds are more polar than sulfur and oxygen heterocyclic compounds and, therefore, require a more polar mobile phase composition for elution from the pBondapak NH2. We are presently investigating the applicability of pBondapak NH2 for the separation of more polar compounds such as the N-heterocyclic compounds and the phenols, which are found in crude petroleum and other fossil-derived oils. High-performance liquid chromatography using pBondapak NH2 provides a versatile, convenient method for the isolation of various hydrocarbon classes prior t o analysis by other techniques. In addition to separating aliphatic hydrocarbons from the aromatic hydrocarbons, PBondapak NH2 provides a convenient, reproducible HPLC fractionation of PAHs according to number of condensed aromatic rings. The use of this material eliminates one of the major difficulties encountered with classical adsorbents, Le., nonreproducible retention due to small changes in the moisture content of the eluent.
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LITERATURE CITED (1) R. J. Pancirov and R. A. Brown, "Proceedings of the 1975 Conference on Prevention and Control of Oil Pollution", American Petroleum Institute, Washington, D.C., 1975, p 103. (2) W. W. Youngbiood and M. Blumer, Geochim. Cosmochim. Acta, 39, 1303 (1975). (3) L. R. Snyder, "Principles of Adsorption Chromatography", Marcel Dekker, New York. N.Y.. 1968. (4) T.-Doran and N: G. McTaggart, J. Chromatogr. Sci., 12, 715 (1974). (5) W. Giger and M. Blumer, Anal. Chem., 46, 1663 (1974). (8) M. Novotny, M. L. Lee, and K. D. Bartle, J. Chromatogr. Sci., 12, 606 ( 1974). (7) M. L. Lee, M. Novotny, and K. D. Bartle, Anal. Chem., 48, 405 (1976). (8) M. L. Lee, M. Novotny, and K. D. Bartle, Anal. Chem., 48, 1566 (1976). (9) J. A. Schmit, R . A. Henry, R. C. Williams, and J. F. Dieckman, J. Chromatogr. Sci., 9, 645 (1971). (IO) C. Golden and E. Sawlcki, Anal. Lett., 9, 957 (1976). (11) s. N. Chesler, B. H. Gump, H. S. Hertz, W. E. May, and S. A. Wise, manuscript in preparation. (12) W. E. May and R. G. Christensen, manuscript in preparation. (13) M. Popl, V. Dolanskq, and J. Mostecky, J. Chromtogr., 117, 117 (1976). (14) M. Popl, V. Doianskq, and J. Mostecky, J. Chromatogr., 91. 649 (1974). (15) M. Popl, V. Doianskq, and J. CGupek, J. Chromatogr., 130, 195 (1977). (16) C. A. Streuli, J. Chromatogr., 56, 219 (1971).
M. Martin, J. Loheac, and G. Guiochon, Chromtographia, 5, 33 (1972). D. C. Locke. J. Chromatogr. Sci., 12, 433 (1974). R. B. Sleight, J. Chromatogr., 83, 3 1 (1973). I. B. Berlman, "Handbook of Fluorescence Spectra of Aromatic Molecules", 2nd ed., Academic Press, New York, N.Y., 1971, p 70. (21) K. W. Bartz, T. Aczei, H. E. Lumpkin, and F. C. Stehiing, Anal. Chem., 34, 1814 (1962). (22) 8 . J. Mair, J. L. Martinez-Pico, R o c . Am. Pet. Inst., 42, 173 (1962). (17) (18) (19) (20)
RECEIVED for review August 9, 1977. ~ ~ September ~ 22, ~ 1977. The authors acknowledge partid financid support from the Office of Energy, Minerals, and Industry within the Office of Research and Development of the U.S. Environmental Protection Agency under the Interagency EnergyjEnvironmerit ~~~~~~h and ~~~~l~~~~~~program, identification of any commercial product does not imply recommendation or endorsement by the National Bureau of Standards, nor does it that the or equipment identified is neeessarily the best available for the purpose.
Determination of Some Thiourea-Containing Pesticides by Pulse Voltammetric Methods of Analysis Malcolm R. Smyth" and Janet G. Osteryoung Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523
The polarographlc behavior of thiourea, phenylthlourea, a naphthytthlourea and benzyl(kso)thlourea has been lnvestlgated In Brltton-Roblnson buffer and sodlwn hydroxlde solutlons. The waves obtained for these compounds In 1 M NaOH are recommended for analytical purposes. I n particular, dmerentlal pulse polarography has been used to resolve a mixture contalnlng thlourea, phenylthlourea or a-naphthylthlourea and benzyl(1so)thlourea. Thls technlque can be used to determlne concentratlons of thiourea and phenylthlourea down to 1 X M, a-naphthylthlourea down to 2 X lo-' M and benM under optlmum condltlons. zyl(1so)thlourea down to 5 X Slnce thlourea, phenylthlourea, and a-naphthylthlourea form Insoluble complexes wlth mercury, these compounds can also be determined by cathodlc strlpplng voltammetry at a hanglng mercury drop electrode. Thls method of analysis can determine concentratlons ot these compounds down to 1 ng mL-' and has been applled to the dlrect determlnatlon of thiourea In urlne.
During the past 20 years, thiourea TU; I) has found widespread use in a variety of industria and biological applications ( I ) . In agriculture it has b en employed as a fungicide (2) [although its use as such has been shown to be harmful in citrus growing areas ( 3 ) ] ,as an accelerator of sprouting in dormant tubers ( 2 ) ,and to decrease the content of nitrifying bacteria in the soil ( 4 ) . I t has been isolated as a urinary metabolite both of CS2 (5) and of a S-containing heterocycle (6). Its presence in urine has also been taken as a nonspecific indicator of cancer (7). Several derivatives of thiourea, i.e., phenylthiourea (PTU; 11) and a-naphthylthiourea (ANTU; 111) have also been used as pesticides and exhibit properties harmful to man. P T U exhibits both 2310
ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1 9 7 7
herbicidal (8) and rodenticidal (9) activity and causes chronic goitrogenic and other glandular difficulties in man (10). The rodenticidal activity of ANTU was first demonstrated in 1945 (11). These thioureas appear t o exert their toxic effects by disturbing carbohydrate metabolism (12) and they have been shown to produce a tolerance to their own toxic action (13). S H2N-!-NH2
HV-g-NH,
S HY-E-NH,
There are many published methods for the determination of TU and P T U based on titrimetric procedures (14, 15) but these are not applicable for the analysis of trace quantities of these compounds in biological fluids. Colorimetric methods have been described for the determination of PTU which are based on its reaction with Folin-Ciocalteau reagent (16, 17), but these methods are usually slow (it takes 1.5 h for the color to develop), offer little selectivity, and have high limits of detection (ca. 3 X M) (16). The electrochemical methods that have most been applied to the determination of T U include microcoulometric argentimetric titration ( I @ , ion selective electrode potentiometry (19),amperometric titration (20), and hydrogen overvoltage measurements (21). The latter procedure could be used to quantitatively determine TU down to 1 X lo-' M, but it is not foreseen that this procedure could provide a convenient method for the determination of T U in biological fluids. Although polarographic methods have also been applied to the determination of T U , these have been based either on the liberation of the S atom and subsequent measurement as H2S (22) or on the catalytic wave produced by T U in the presence of Cu2+ions (23). We have therefore studied the inherent polarographic behavior of TU, PTU, and
