2084
Anal. Chem. 1981, 53, 2084-2087
compounds, and as the ring system becomes more fused, the molecule becomes less alkylated. Basic Nitrogen Compounds from LAH Reduction. Compounds identified in the N3 sample are reported in order of GC retention times in Table 111. Two aromatic amines: N-ethyltetrahydroquinoline and N-ethyl-4-methyltetrahydroquinoline (parent peaks, P = 161 and 175, base peaks a t m / e 146 and 160) were tentatively identified. Possible precursors leading to these compounds are amides such as A or B. alkylquinolines were also detected in the N3 sample, suggesting a-carbonylquinolines as part of the nonbasic nitrogen compounds present in the first polar fraction. R
R
Possible nonbasic nitrogen compounds leading to structure C upon LAH reduction are pyridines with a carbonyl function at the a position. The carbonyl function may be in the form of ketones, esters, or carboxylic acids. The strong carbonyl bands at 1720 and 1690 cm-l in the infrared spectrum of the first polar fraction agree well with those of a-alkylcarboxyl and a-acyl- or a-carboxylpyridines. The basicity of a pyridine nitrogen is sharply reduced by the presence of an electronwithdrawing group such as esters, ketones, and acids in the a position.
ACKNOWLEDGMENT The authors thank Rong J. Hwang for the helpful discussion concerning the mass spectra data. LITERATURE CITED
7’0
7%
7%
CH3
GH3
CH3
B
A
R = H, CH,
Alkylpyridines were the major class of compounds found in the N3 sample. GC/MS/DS analysis indicated these compounds were pyridine homologues with molecular weights in the range 191-289 (C8-to CI6-pyridines). The base peaks a t m / e = 121,134,135,148,149,163,177,191, or 205 were observed in the mass spectra of these alkylpyridine with m / e = 121 or 135 in most cases. While mass spectra data do not allow rigorous assignments of the substitution patterns in this molecular weight range, a comparison with those of lower molecular weight homologues indicates most compounds have substitution at the a and y position. The base peak at m / e = 121 or 135 is expected for a McLafferty rearrangement ion from 2- or 4-substituted pyridines with three-carbon or longer side chains (17). On the other hand, /3 cleavage is the primary fragmentation pattern for 3-substituted pyridines but is considerably weaker in those of 2- and 4-substituted compounds (18). Structure C can be tentatively assigned as representatives of alkylpyridines present in ND R
C X = R, OH, OR Y = H or alkyl groups
(1) Jewell, D. M.; Weber, J. H.; Bunger, J. W.; Plancher, H.; Latham, D. R. Anal. Chem. 1972, 44, 1391. (2) Hlrsch, D. E.; Hopklns, R. L.; Coleman, H. J.; Cotton, F. 0.;Thompson, C. J. Anal. Chem. 1972, 44, 915. (3) Suatonl, J. C.; Swab, R. E. J. Chromafogr. Sci. 1976, 14, 535. (4) Jewell, D. M. In “Chromatography in Petroleum Analysis”; Akgek, K. H., Gouw. T. H..Eds.: 1979: DD 278. (5) McKay, J. F.; Weber, J. H.; ‘catham, D. R. Anal. Chem. 1976, 48, 891. (6) Poulson, R. E.; Frost, C. M.; Jensen, H. B. In “Shale 011, Tar Sands and Related Fuel Sources”; Yen, T. F., Ed. American Chemical Soclety: Washington, DC, 1976; Adv. Chem. Ser. pp 1-10, (7) Poulson, R. E.; Jensen, H. B.; Cook, G. L. Prepr., Dlv. Pet. Chem., Am. Chem. SOC. 1971, IS(1), A49. (8) Van Meter, R. A.; Balley, C. W.; Smith, J. R.; Moore, R. T.; Allbright, C. S.; Jacobson, I. A., Jr.; Hylton, V. M.; Ball, J. S. Anal. Chem. 1952, 24, 1756. (9) Brown, D.; Earnshaw, D. 0.; McDonald, F. R.; Jensen, H. 8. Anal. Chem. 1970, 42, 146. (IO) Jewell, D. M. In “The Role of Nonhydrocarbons In the Analysis of Vlrgin and Biodegraded Petroleum;” Petrakls, L., Welss, F. T., Eds; Arnerlcan Chemlcal Society: Washington, DC, 1980; Adv. Chem. Ser., pp 219. (11) House, H. 0. ”Modern Synthetic Reactlons”; W. A. Benjamln: New York, 1972; pp 45. (12) Mlcovlc, V. M.; Mlchallovlc. M. L. Red. Trav. Chim. Pays-Bas 1952, 71, 970. (13) Shue, F.-F.; Yen, T. F. Prepr. Pap.-Am. Chem. SOC., Dlv., Fuel Chem. 1980, 25(3),89. (14) Snape, C. E.; Ladner, W. R.; Battle, K. D. Anal. Chem. 1979, 51, 2189. (15) Alyar, A. N.; Houwen, 0. H.; Bachelor, F. W. Fuel 1980, 59, 276. (16) Schiller, J. E. Anal. Chem. 1953, 25, 968. (17) Sample, S. D.; Llghtner, D. A.; Buchardt, 0.; Djerassl, C. J. Org. Chem. 1978, 32, 997. (18) Biemann, K. “Mass Spectrometry”; McGraw-Hill: New York, 1962, pp 135.
RECEIVED for review April 14,1981. Accepted July 28,1981. This work is supported by U.S. Department of Energy under Contract No. 79-EV10017.000.
Fluorometric Determination of Atmospheric Sulfur Dioxide without Tetrachloromercurate(I1) Purnendu K. Dasgupta’ Californla Primate Research Center, Unlvers& of California, Davis, Callfornia 956 16
l-Naphthyiammonium chloride is substituted for pararosanlilne hydrochloride for a fluorometric version (excitation 342 nm, emission 442 nm) of the Schiff reaction used for the determlnatlon of sulfur dioxide. The sensitivity Is 2 orders of magnltude better than the colorimetric method; in all other aspects the two methods are analogous. ‘Present address: Department of Chemistry, Texas Tech University, Box 4260, Lubbock, Texas 79409 0003-2700/81/0353-2084$01.25/0
We have recently developed a method (1) for the determination of atmospheric SO2 in which a dilute (0.02%) solution of formaldehyde buffered lightly at PH 4 is used to &.orb and stabilize soz as hYdroxymethanesulfonate. It has been shown (2)that this system exhibits much greater thermal stability than the chlorosulfonatomercurate(I1) complex formed in the Federal Reference Method (FRM) (3). Results from field tests employing the formaldehyde absorber, along with some further refinements of the pararosaniline procedure 0 1981 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 53, NO. 13, NOVEMBER 1981
as developed by us (I),have been shown to compare well with parallel determinations by the FRM (4). In the present paper, we report a much more sensitive spectrofluorometric procedure employing the same absorber and a similar analytical reaction with a reagent containing a fluorophore (5). THEORY Although it is implicit in the original paper of Nauman et al. (6),the necessity of dealing with the complex canonical chromophoric structures of pararosaniline obscures the fact that the Schiff reaction can be viewed very simply as one involving an acid-base indicator. Specifically, the indicator is an amirre (RNH2)which is colored in the free base form and colorless (or differently colored) in the protonated ammonium ion (RNH3+)form. If the dissociation constant of the conjugate acid is KI and the total concentration of the amine in the final riolution is C , the blank absorbance (Ablank)is given by Ablank = ~ICKI/(K~ [H+1) (1) Where al is the absorptivity of the free amine at the wavelength of interest and [H+]refers to the final solution. The parameters Kl and al are fixed with the choice of the amine and C is generally optimized with respect to the applicable range of the method. The remaining adjustable and critical parameter, [H+],is so chosen that the amine remains largely in the conjugate acid form, thus ensuring a low blank value. Upon the addition of S(1V) (to attain a concentration of, say, S molar in the final solution) and excess HCHO, the amine is converted to an aminomethanesulfonic acid (1, 6). This compound displays the same indicator behavior as the parent amine but the sulfomethylation decreases the pK of the indicator. l k e to the decreased basicity of the amino nitrogen atom(s) in the product, unlike the parent amine, it is largely unprotonated at the same operating pH. The predominance of this aminomethanesufonic acid in the “free base” form results in regeneration of color, the characteristics of which, naturally, are close to that of the parent amine. Let the dissociation constant of the conjugate acid of the aminomethanesulfonic acid be K z and the absorptivity of the free base form a t the wavelength of interest be u p Let us assume that only a fraction f of the S(1V) added undergoes the reaction of interest, the rest being converted to the formaldehyde addition compound. Thus, the sample absorbance will be given by Asample =
(aiKi(C - f 8 / ( K i
+ W1))+ (a2KdS/(K2 + WI))
(2) Making the reasonable approximation that al N u2 N a and subtracting eq 1 from eq 2, one obtains
Asample - Ablank =
=
a f S ( K / ( K 2+
WI))- (Ki/(Ki + [H’I)))
(3) If we assume that within the pH range of our interest f is independent of pH, it becomes possible to compute the optimum pH for maximizing the net analyte signal. Differentiating eci 3 with respect to [H’], one obtains d14net - - afS(K2 - Kl)(Kl& - [H+I2) (4) d[H+] ([H+I2+ ( K , + K2)[H+] K1&J2 Here, a, f , and S are nonzero constants and K2> K1 > 0. If eq 4 is set equal to zero, corresponding to a maximum of Anet as a function of [H’I, the only possible solution is
+
[H+l =
(5)
which, therefore, is the optimum pH of our interest. Due to the limitations of the assumptions made in the deriving eq
2085
5, the experimental optimum pH may not be identical with the calculated value. Equation 5 nevertheless provides an excellent starting point for trial and error experiments. The absolute sensitivity of the method increases with increasing absorptivity “u” of the parent dye, with increasing extent of reaction “/”, and with increasing difference between Kz and K1. We have earlier suggested that if N,N’&’‘pentamethylpararosaniline, which displays a native absorptivity much greater than that of pararosaniline, is substituted for the latter reagent, a more sensitive method will result. While such a substitution produces an interesting variation of the pararosaniline method (resulting in green blanks and blue solutions in the presence of S(IV)), the sensitivity was actually less than the pararosaniline method, even after ctptimization. Even though “u” is higher, the difference between K2and Kl is much lower for this case, resulting in a decreased overall sensitivity. The sensitivity of such a method may of course be increased by changing the detection method. Fluorescent/nonfluorescent (or differently fluorescent) acid-base indicators of amine functionality should be utilizable instead of thleir colored/colorless analogues. All of the above treatment is also equally applicable aa long a fluorescence intensity (for a fixed instrumental condition) is substituted for absorbance and one operates in the region where fluorescence intensity is linearly proportional to concentration. Additionally, the parent and product amine should have comparable characteristics regarding excitation and emission marima and quantum efficiencies. Fortunately these latter criteria are automatically satisfied because the actual fluorophore is unaffected during the reaction.
EXPERIMENTAL SECTION Equipment and Reagents. A Perkin-Elmer 650-10s spectrofluorometer, equipped with a 20-pL quartz flow-through cell and a Gilson Minipuls 2 sipper pump was used for all fluorometric measurements. This instrument uses a 150-W Xenon lamp as the exciting source. All reported data were obtained with both excitation and emission slits set at 5 nm. The excitation and emission maxima for 1-aminonaphthalenewas determined experimentally in the media of interest and these values wore reasonably close to data reported by Berlman (7) for other solvents. The sample did not come in contact with any surfaces other than glass or PTFE prior to measurement. The fluorescence signal was recorded on a Houston Instrument recorder (Omniscribe B-5000)and/or directly read off a digital millivoltmeter (Precision 2000) connected to the instrument. All pH measurements were made with an Orion 701A pH meter equipped with a Model 91-02 research electrode and an automatic temperature compensator probe. Calibration was performed with potassium tetroxalate (pH 1.68) and glycine-HC1 (pH 1.20) standard buffers. 1-Aminonaphthalene(Aldrich)was dissolved in excess of hydrochloric acid and recrystallized repeatedly from aqueous 130lution. White needle crystals of 1-naphthylammoniumchloride were obtained in good yield and were stored protected from light, moisture, and heat. One hundred eighty milligrams of the pure chloride salt were dissolved in HCl(700 mL, 2.00 M) and made up to 1L. The resulting solution is 1mM with respect to the amine and 1.40 M in acid. Sulfamic acid (Fisher, 0.6 g) was dissolved in water (90 mL) and the pH of the solution adjusted to -4 by the addition of NaOH and then made up to 100 mL. The reagent should be prepared fresh every 2 weeks. truns-l,2-Cyclohexylenedinitrilotetraacetic acid (CDTA, Aldrich, 18.2 g) was added to NaOH (100 mL, 1M) and made up to 1L to prepare a 50 mM NazCDTA stock solution. The buffered formaldehyde absorber was prepared as a stock reagent by diluting formaldehyde solution (37%, 5.30 mL), potassium hydrogen phthalate (2.04 g), and NazCDTA (20 mL, 50 mM) to 1 L. It was diluted 10-fold before use. The following procedure is recommended for collection and analysis. Sulfur dioxide is collected by using 15mL of the buffered
2086
ANALYTICAL CHEMISTRY, VOL. 53, NO. 13, NOVEMBER 1981
1-
I
2
3
4
5
,
' 6
I
7
8
I
I
I
0
5
IO
9
PH
I
I
15 20 TIME ( m i n )
I
I
25
30
Figure 1. The pH dependence of fluorescence intensity: 1naphthylammonlum chloride, excitation 342 nm, emission 442 nm.
Figure 2. Development and decay of fluorescence wlth tlme, 100 ng SO,/mL.
formaldehyde absorber in a midget bubbler at a sampling rate not greater than 400 mL m i d and preferably at 300 mL min-' or less. The sampliig time should be so chosen that totalamount of SO2 collected does not exceed 2.5 pg. One milliliter of the sulfamic acid/sodium sulfamate reagent is added immediately after collection and prior to storage. The samples may be refrigerated if analysis is to be performed at a later date. The samples need to be warmed up to room temperature (-22 O C ) prior to analysis. For analysis, 1mequiv of NaOH (2 mL, 0.5 M) is added to the formaldehyde absorber (blank or containing SOz). The solution is then mixed by a vortex mixer or by inversion. This above alkaline solution is added to the 1-naphthylammoniumchloride reagent (2.5 mL, 1 mM in 1.4 M HCl) and mixed by inversion. The fluorescence intensity is read between 20 and 25 min after fiial mixing. Excitation and emission wavelengths are set at 342 and 442 nm, respectively. 1-Naphthylaminomethanesulfonicacid, the reaction product, was obtained by carrying out the analytical reaction as above in large scale (500 mL), extracting with 1-pentanol,and separating the desired compound from the parent amine by preparative TLC.
Table I. Sensitivity and Precision of the Fluorometric Method" fluorescence fluorescence SO, in signal SO, in signal final (mean f final (mean b solution, RSD %),& solution, RSD %),& mV ng/mL mV
RESULTS AND DISCUSSION Among fluorescent acid-base indicators listed in standard compilations (8), 1-aminonaphthalene and 2-aminonaphthalene are the two obvious amine-type compounds. In addition, the fluorescent/nonfluorescent transition occurs for both compounds at a fairly low pH. This restriction is important because the reaction of our interest takes place only in acid media ( I ) . Both compounds were found to be Schiff-positive, i.e., both reacted in exactly the same fashion as pararosaniline, when supplanted for the latter in the previously published method ( I ) , the reaction being followed fluorometrically. In contrast, N-(1-naphthy1)ethylenediamine (Marshall's reagent, Griess-Saltzman reagent) did not react in the same fashion. Further investigations involving product isolation and characterization showed that sulfomethylation does occur with this compound also but does so on the aliphatic primary amino group. This does not sufficiently alter the basicity of the nitrogen atom connected directly to the fluorophore and renders the reaction ineffective for our purposes. Since 2-aminonaphthalene is regarded to have a greater carcinogenic potency compared to 1-aminonaphthalene, all further experiments were conducted with l-naphthylammonium chloride. The present method was designed to complement rather than substitute the existing colorimetric pararosaniline procedure. For this reason, we directed our optimization efforts to samples containing less than 100 ng SO2mL-l in the final solution. A concentration of 100 ng SO2 mL-' results in a net
0 4 8 20
84 90 99 133
40
60 80 100
199 269 324 388
a Zero signal corresponds to a 0.2 N dilute HC1 solution. Full scale signal is 1 V. Both slits 5 nm, variable sensitivity X10.
analyte signal of -0.057 AU in the colorimetric procedure and thus, there is some overlap of the range of applicability of the two procedures. The pK value of the 1-naphthylammonium ion was determined to be 3.8 by studying the dependence of fluorescence intensity as a function of pH as shown in Figure 1. This is in good agreement with the value of 3.9 reported in the literature (8). 1-Naphthylaminomethanesulfonicacid, isolated as described in the Experimental Section, was subjected to a similar experiment so that the optimum pH of the analytical method can be evaluated. Unfortunately, we failed to obtain a reliable value. This pK value is very low (Cl); aside from the difficulty of calibrating on this pH region, the compound decomposes back to the parent amine at a rate that increases with decreasing pH, thus limiting the accuracy of the experimental data. Optimization of reaction pH was therefore carried out by trial, by varying the acid concentration in the amine reagent. subsequently, the amine reagent concentration was optimized for a maximum analyte concentration of 100 ng SO2 mL-l in the final solution. The recommended procedure results in a final solution pH of -0.9. The temporal development of fluorescence and its decay are shown in Figure 2, the similarity with the colorimetric procedure (compare Figure 3, Ref. 1) is remarkable. Although calibration data are dependent on instrumental characteristics for fluorometric methods, data are presented in Table I to indicate the sensitivity and precision of the recommended method. Plotting of these data results in a linear plot with a correlation coefficient better than 0.999, a Y intercept (theoretical blank) of 77 mV, and a slope of 3.1 mV/ng SO2 mL-l. As the disparity of the theoretical and the experimental blank indicates, there is curvature in response at the low end, nonlinearity is quite pronounced below 8 ng
2087
Anal. Chem. 1981, 53, 2087-2090
SO2 mL-l. According to the guidelines for data quality evaluation in environmental chemistry (9), these data indicate a limit of detection of about 2 ng SO2 mL-' or more conveniently, 1ppb S as S(1V). Correspondingly, limit of quantitation begins at about 6 ng SO2 mL-'. As in the colorimetric method, added nitrite (to simulate concurrent presence of NOz) interfered. The interference was completely eliminated by the sulfamic acid/sodium sulfamate addition step. However, unlike the colorimetric method, wherein a small but perceptible decrease in method sensitivity results due to the sulfamic acid addition, no reduction in sensitivity was apparent in this procedure. Without, any CDTA in the absorber, added Mn(I1) interfered as in the pararosaniline procedure. No interference due to 400 ng Mn(I1) mL-' was noted in the presence of the recommended amount of CDTA. Above 1% concentration, 1-aminonaphthalene is subject to OSHA regulations because of its reported carcinogenicity. From the known pK value of 1-aminonaphthalene,we estimate that the vapor pressure of this compound over the stock reagent (1 mM in 1.4 M HC1) is lo4 times that over the solution which begins to be subject to regulatory control. The fiial solution involved in the analytical procedure is less acidic but is also tliuted 10-fold, resulting in approximately the same vapor pressure. While these levels probably do not pose any health hazards, we are pursuing a search for alternative compounds which would be deemed safer. 4-Amino-lnaphthaleriesulfonic acid and 5-amino-2-naphthalenesulfonic acid are both Schiff positive, and both display good quntum
efficiencies. Also, both are much less volatile compared to 1-naphthylamine and therefore appear very promising.
ACKNOWLEDGMENT The author thanks the Department of Civil Engineering for the loan of the spectrofluorometer and K. B. DeCesare for experimental assistance. The help and encouragement of I). P. Y. Chang and 0. G. Raabe are gratefully acknowledged. LITERATURE CITED Dasgupta, P. K.; DeCesare, K.; Ullrey, J. C. Anal. Chem. 1880, 5:?, 1912-1922. Dasgupta, P. K.; DeCesare, K. B., submitted for publication In Anal. Chem. Fed. Regist. 1871, 36 (84), 8187-8191. Dasgupta, P. K. Air Pollut. Control Assn. J. 1981, 31, 779-782. Dasgupta, P. K. "Preprint Extended Abstract", Division of Environmental Chemistry, 181st National Meeting of the American Chemical SooL etv, Atlanta, GA. March 1981; American Chemical Society: Washington, DC 1981; ENVR 35. Nauman, R. V.; West, P. W.; Tron, F.; Gaeke, 0. C. Anal. Cheni. 1080. - - -, 32. - -, 1307-1311. -Berlman, I. B. "Handbook of Fluorescence Spectra for Aromatic Molecule", 2nd ed.; Academic Press: New York, 1971; pp 342-349. DeMent, J. I n "Handbook of Chemlstry and Physics", 57th ed.;Weaat, R. C., Ed.; CRC Press: Cleveland, OH, 1976; pp 0-138, D-148. American Chemical Society Committee on Environmental Improvement Anal. Chem. 1980, 52, 2242-2249.
RECEIVED for review May 1, 1981. Accepted July 27, 198:l. This research was supported partially by a contract from the U.S. Department of Energy (DE-AM03-76SF00472) and partially by a grant from the US.Environmental Protection Agency (T900800) with/to the University of California, Daviw
Identification of a Chromogen in the Assay of Hippuric Acid with Acetic Anhydride, Pyridine, and 4 4 Dimethylamino)benzaldehyde Kazuhiro Hirota, * Mikiko Ikeda, Michi Kawase, and Shinji Ohmori Faculty of Pharmaceutical Sciences, Okayama University, Tsushima-Naka- I, Okayama 700, Japan
A yellow chromogen, formed In the assay system for hlppuric acld which conslsts of acetic anhydrlde, pyridine, and 4-( dlmethylamlno)benzaldehyde, was Isolated, and the structure was deterrnlned to be 4 4 1-acetyld( 1H)-pyrldylidene)-2phenyi-2oxazolln5-one. The structure was conflrmed by the chemlcal converslon to known compounds as well as by NMR spectra. The converslon was accornpllshed by hydrolysls of the chromogen to 4 4 (benroylamlno)methyl)pyrldlne, 4(amlnomethyl)pyridine, and benzoic acid vla 5-hydroxy-2phenyl-4-( 4-pyrldyl )oxazoie. 44 Dlmethylamln0)benzaldehyde included in the assay system Increased the production of the chromogen. The formation mechanlm of the chromogen via an intermediate susceptible to oxldation was proposed.
The formation of a yellow chromogen, in the reaction mixture of hippuric acid (HA) with acetic anhydride and pyridine in the presence of 4-(dimethylamino)benzaldehyde (CDAB), is a sensitive and simple assay of HA in urine and liver homogenate (I). The quantitative conversion of glycine into HA is a specific method for the assay of glycine. This method has been used to determine the amount of glycine 0003-2700/81/0353-2087$01.25/0
present in amino acid mixtures and biological samples (2). In this paper, a predominant yellow chromogen has been isolated from the color reaction mixture, and the structure was determined to be 4-(l-acety1-4(1H)-pyridylidene)-Plphenyl-2-oxazolin-5-one(APPO) which does not include the component of 4-DAB. The mechanism for the formation of APPO is proposed. This mechanism accounts for the absence of APPO as a product when a benzoylamino acid other than HA reacts with acetic anhydride and pyridine. The role aC 4-DAB in the chromogen formation is also described.
EXPERIMENTAL SECTION Chemicals and Instruments. 4-((Benzoylamino)methyl)pyridine was prepared by the benzoylation (3) of 4-(aminomethyl)pyridine,purchased from Tokyo Kasei Kogyo Co. (Tokyo). Nitrogen had a purity of 99.999%. Other compounds and solvent used were of the best grade commercially available. Nuclear magnetic resonance spectra of samples in CF3COOD,
CDC13, and CD3SOCD3 solution containing internal tetramethylsilane (MedSi) were recorded with a JEOL FX-100 spectrometer. The quantitative spectra were obtained by using gated decoupling without NOE. Infrared spectra of crystalline samples were recorded as Nujol mulls with a Nipponbunko A-102 spectrometer. Mass spectra were obtained in a Shimadzu LKB Type 9000 spectrometer at an ionizing energy of 70 eV. Absorption 0 1981 American Chemical Society
