to n = 2 or 3 into Equation 26 for each of the 21 data sets, it was found that only for the model assuming 1 :1 and 1 :3 hafnium-chloranilic acid did any combination of parameters yield positive values for the a , coefficients with errors less than the a , coefficients in an acceptable curve fit (13). Questions concerning the missing intermediate species will be studied further using precision wide range spectrophotometers. The machine method of absorbance data interpretation presented here should be applicable t o many systems similar to the above, given good data over wide concentration and wavelength ranges.
The average of 21 values calculated for the overall concentration formation constants of Equations 5 and 6 were = (5.31 It 0.15) X lo3 and p3 = (4.27 =t0.18) X 10” where the errors are the standard errors of the mean. In an identical manner the calculations were made assuming the 1 :1 and 1 :2 hafnium :chloranilic acid complexes, obtaining as a result the value p2 = 2.2 X lo7 used in the following comparison calculation. Linear Regression Analysis. Equation 16 written in the form Eli
where n
X 11 =
+ PI&) + pn(L)nl
=
al(L)
+
(26)
ACKNOWLEDGMENT
2 or 3, is seen t o be linear in the coefficients if the
p constants are known. At each one of the 21 wavelengths
The advice and assistance of Richard M. Wallace, E. I. du Pont de Nemours & Company, Savannah River Laboratory, S. C., who provided the original matrix rank program, is gratefully acknowledged. Acknowledgment is made also t o W. V. Accola of the Oklahoma State University Computer Center for assistance in program modification.
studied here, 12 equations were set up in the form of Equation 26 and by a series of weighted least squares linear regression calculations using the program outlined in Reference (13), it was possible to determine which model best fit the experimental data, the model assuming one and three chloranilate ligands per hafnium ion or the model assuming one and two ligands. By systematically programming /3 constants corresponding
RECEIVED for review April 3, 1967. Accepted June 2, 1967. This work was supported, in part, by the Oklahoma State University Research Foundation. Abstracted from the M. S. thesis of Fred C. Veatch, O.S.U., May, 1966.
(13) L. P. Varga, W. D. Wakley, L. S. Nicolson, M. L. Madden, and J. Patterson, ANAL.CHEM.,37, 1003 (1965).
Sensitive Direct Spectrophotometric Determination of Fructose and Sucrose after Acid Degradation Edward R. Garrett and Jaime Blanch College of Plzarmacy, Unicersity of Florida, Gainescille, Flu.
32601
An ultraviolet chromophore,, , ,X 283 mp, is produced from fructose under well-defined conditions of acid concentration, temperature, and time, e.g. 9.25 hours at 80° C in 1.OM HCI, and is a direct function of the sugar concentration. The properties of the chromophore serve as a basis for a simple, inexpensive, and sensitive assay of the sugar. Sucrose hydrolyzed under the same conditions produces the same absorption band to provide an indirect method of assay. The specific acid-catalyzed generated chromophore is destroyed by alkali and the ,,,X, 286 mp in alkali vanishes by a first-order process. The chromophore is incompletely extractable with chloroform from the acid solution and can be differentiated from degraded 2-deoxy-D-ribose, which i s totally extractable. This assay is sensitive to concentrations as low as 10-5M.
SEPARATION TECHNIQUES such as paper or column chromatography ( I , 2) with analysis of the effluents ( I , 3) and dialysis based on differential kinetics (4) have been applied t o the analysis of sugars. Colorimetric procedures (5) are also available. The heating of sugars with concentrated mineral (1) L. Hough, “Methods of Biochemical Analysis,” I. D. Glick, Ed., Interscience, New York, 1954, p. 205. (2) E. F. Walborg, Jr., L. Christensson, and S. Gardell, Anal. Biochem., 13, 177 (1965). (3) E. F. Walborg, Jr., and L. Christensson, Zbid., 13, 186 (1965). (4) S. Siggia, J. G. Hanna, and N. M. Serencha, ANAL. CHEM., 36, 638 (1964). (5) Z . Dische, “Methods in Carbohydrate Chemistry,” R. L. Whistler and M. L. Wolfrom, Eds., Vol. 1, Academic Press, New York, 1962, p. 477.
acid forms degraded products which have ultraviolet spectral absorbances. The absorbances between 200 and 380 mp have been correlated with the sugar concentrations (6, 7, 8). The degradation products can also react with specific reagents t o produce colored derivatives to be assayed in the visible region and correlated to the sugar concentration (9,
IO). The desirability of a convenient analytical method specific for sucrose has been recognized for many years (11). The application of the isotope dilution principle to the determination of sucrose in sugar beets required 3-5 days and included sophisticated and complex operations. The procedure was modified so that only 24 hours were needed, but it is still a complicated one (11). The conventional assay methods for sucrose depend on polarization measurements but they are unreliable in the presence of other optically active substances and they are sensitive only to concentrations of 7.6 X 10-4M (12). Although chromophore production on strong (6) M. Ikawa and C. Niemann, J . Bid. Clzem., 180, 923 (1949). (7) R. M. Love, Biochem. J . , 55, 126 (1953). (8) F. A. H. Rice and L. Fishbein, J. Am. Chem. SOC.,78, 1005
(1956). (9) Z . Dische, Biochem. Z . , 189,77 (1927). (10) J. M. Webb and H. B. Levy, J . Biol. Chem., 213, 107 (1955). (11) M. J. Sibley, F. G. Eis, and R. A. McGinnis, ANAL.CHEM., 37, 1701 (1965). (12) G. B. Levy, “Standard Methods of Chemical Analysis,” F. J. Welcher, Ed., Part A, Vol. 111, Van Nostrand, Princeton, N. J., 1966, p. 266. VOL. 39, NO. 10, AUGUST 1967
1 109
1.0
0.9
0.8
0.7
0.6 0 V
c
4 0.5
I /n\
m 0
I
2 IO4 Y Fructotr
Figure 2. Degraded fructose absorbance as a function of concentration Curve A is the absorbance at Amax 283 mp of degraded fructose (10.0 hours at 80.0" C, in 1.OM HCI) as a function of fructose concentration Curve B is the absorbance at Xmaz 283 mp of 5 ml of the original solution after successive extraction with 3, 3, 2, and 2 ml of chloroform, as a function of fructose concentration Curve C is the absorbance at Xmal 278 mp of the combined 10-ml chloroform extracts of 5 ml of the original solution, as a function of fructose concentration
n
a
0.4
0.3 0.2
0.I 0.0 Wavelength, my
Figure 1. Ultraviolet spectrum of the final product of the degradation of 2.5 X 10-4Mfru~tosein 1.OMHCI Curve A is after 10.0 hours at 80.0 C Curve B is the spectrum of the aqueous solution after successive extractions with 3,3,2, and 2 ml of chloroform Curve C is the spectrum of the collected 10-ml chloroform extracts of 5 ml of the original solution Curve D is the spectrum of 5 ml of the final product immediately after adjustment to 0.2M KaOH and is equivalent to 1.55 X 10-4M fructose acid treatment of sugars is a well known phenomenon, more specific and sensitive analytical techniques may be produced by well-defined, moderate conditions. For example, a chromophore,, , ,A 261 mp, with specific properties can be generated from 2-deoxy-~-riboseunder well-defined conditions of acid concentration, temperature, and time (13). Several authors have applied this method t o the assay of isolated sugars and drugs ( 1 4 , mixtures of sugars (1.9, and biological materials (16). The chemical nature of the chromophores and the mechanism of their formation are also of interest (17). This present work reports on such analytical methods for fructose and sucrose which are simple, economical, and of high sensitivity. The properties of the acid-generated chromophores which may be useful in identification and separation from other analyzable substances are also described. (13) J. K. Seydel and E. R. Garrett, ANAL.CHEW,37, 271 (1965). (14) R. J. Doyle and H. M. Burgan, A i d Biochem., 17, 171
(1966). (15) E. R. Garrett, J. Blanch, and J. K. Seydel, J . Pharm. Sci., 56, (1967). (16) P. Byvoet, A m / . Biochem., 13, 170 (1965). (17) J. K. Seydel, E. R. Garrett, W. Diller, and K. J. Schaper, J. Pliarm. Sci., 56, 858 (1967). 1 1 10
0
ANALYTICAL CHEMISTRY
The analytical method is based on the treatment of fructose with 1.OM HCl a t 80.0" C. A chromophore, A,, 283 mp, is generated after 9.25 hours of heating (Figure 1). This chromophore is stable for 2 hours. When sucrose is hydrolyzed under similar conditions, a chromophore with and the same properties is formed. the same, ,A, EXPERIMENTAL
Apparatus and Materials. A Beckman Model D U spectrophotometer, slit width 0.1 mm, was used. Complete absorption spectra of the solutions containing the chromophores were obtained on a Cary recording spectrophotometer, Model 15. Sucrose, fructose, and 5-hydroxymethylfurfural were obtained from the J. T. Baker Chemical Co., Distillation Products Industries, and K & K Laboratories, Inc., respectively. All other chemicals were of analytical reagent grade. Spectrophotometric Assay of Fructose or Sucrose. The calibration curve for each sugar was prepared using concentrations between 3.0 X 10-4M and 3.0 X IO-jM in 1.OM HC1. The samples were maintained in a thermostated bath at 80.0" C. An aliquot of each solution was taken 10.0 hours after the degradation was initiated. The absorbances of the cooled aliquots were read a t 283 mp against a 1.OM HC1 blank. Absorbances were plotted against the respective sugar concentration as shown in Figure 2, Curve A for fructose. The absorbance values may be read between 9.25 and 11.25 hours under those conditions. Absorbance readings taken over these time intervals did not vary more than ~k0.008absorbance unit. A solution of the sugar of unknown concentration was prepared so that the absorbance after heating 10.0 hours a t 80.0" C in 1.OMHCI did not exceed 1.20 a t 283 mp. The solution may be diluted with 1.OM HC1 if necessary. The absorbance of the cooled aliquots was read after 10.0 hours a t 283 mp. The pertinent sugar concentration was read from the calibration Curve A , Figure 2. The method has a sensitivity of lO-5M and the standard deviation of an assay to is 2.6% in the sucrose concentration range of 3.0 X 3.0 x 10PM. If chloroform-extractable interferences are present in the sugar solution, they may be extracted with successive amounts
1.2
1
1
OC
I.o
ro
a (u
0
c
4 U .2
Hours
.o Hours
Figure 3. Absorbance a t 283 mp as a function of HCI concentration and time for the degradation of 3.0 X 10-4Mfructose at 80.0" C of 3,3,2, and 2 ml of chloroform. Absorbance of the aqueous solution was read at 283 mp against chloroform-equilibrated 1.OM HC1. The pertinent sugar concentration was read from the calibration Curve B, Figure 2. If interferences nonextractable with chloroform are in the sugar solution, the absorbance of the collected chloroform extracts may be read at 278 mp against a 1.OM HC1-equilibrated chloroform blank. The pertinent sugar concentration was read from the calibration Curve C, Figure 2. Kinetic Studies. The kinetics of the 283-mp chromophore development from 3.0 X lOP4M fructose were studied as a function of 0.14, 0.27, 0.55, 0.82, and 1.00M HC1 a t 80.0" C. The kinetics of this development from 2.5 X 10P4Mfructose were studied in 1.OM HC1 a t 60.0", 70.0", 75.0", and 80.0" C. The kinetics of destruction of the acid-generated 283-mw chromophore were followed spectrophotometrically at the 286-mp maximum in 0.2M NaOH at 38.0", 50.0", 60.0", and 77.5" C. Similarly, alkaline destruction was studied at 35.0" C in 0.06, 0.14, 0.22, and 0.30M NaOH. In all cases, the rate constants were invariant with fructose concentration or the magnitude of the chromophoric absorbance. Thin Layer Chromatography. Acid-degraded fructose and 5-hydroxymethyl furfural (HMF) were compared by thin layer chromatography. The chloroform extract of acid283 mp, and chloroform solutions of degraded fructose,, , A, H M F were applied to silica gel G5-254 (E. Merck) thin layer plates. Development was made in three solvent systems. The R, values were 0.63, (chloroform-methanol, 80:15); 0.44 (benzene-methanol, 5 :2); and 0.23 (benzene-methanol, 9 :1). No differences were apparent between H M F and the chloroform extract of acid-degraded fructose. The spots were revealed with 2,4-dinitrophenylhydrazine. For a mixture of H M F and the chloroform extract of equal absorbances, no separation or difference was observed after development on T L C plates. RESULTS AND DISCUSSION
Effects of Acidity and Temperature on Fructose Spectra. An ultraviolet chromophore,, , ,A 283 mp, (Curve A of Figure 1) appears with time (Figure 3) when fructose is acid-degraded under thermal conditions. An asymptotic value for the absorbance a t this wavelength is achieved with time after 9.25 hours of degradation in 1.OM HC1 a t 80.0" C.
Figure 4. Absorbance a t 283 mp as a function of time and temperature for the degradation of 2.5 X 10-4M fructose in 1.OM HCI This asymptotic value is maintained for 2 hours. The rate of appearance of the 283-mp chromophore is not strictly first order. Deviations from linearity are observed when log ( A , - A ) is plotted against time at initial time values, where A , is the value for the asymptotic absorbance and A is the absorbance at any time t. These deviations are apparently due to an induction period in the 283-mp chromophore development (Figures 3 and 4). The apparent first-order rate constant, k , can be obtained from the linear portion of such plots. Concomitant with the appearance of the 283-mp absorption band, a band at 228 mp appears by an apparent first-order rate process. The rate constants for the appearance of both chromophores are nearly the same. The, , ,A 228-mp chromophore is not preferred for analytical purposes as it has lessened absorptivity. The apparent first-order rate constants for the appearance of both chromophores are proportional to the activity of the hydrogen ion uH- at a given temperature, where U H + = f [HCl], the activity coefficient f is obtained from the literature (18), and kE+ = k / a E + . The apparent rate constant k was obtained from the linear portion of the plot of log ( A , A ) against time t , in accordance with log(A,
- A)
= (k/2.303)t
+ constant
(1)
The apparent first-order rate constants for the degradation of 3.0 x lO-*M fructose at 80.0" C, as determined from the rate of appearance of the 283-mp chromophore, were evaluated at different hydrochloric acid concentrations. The values are lo5 k 2 8 3 m p , [HCl], U H + : 8.59, 1.00, 0.788; 6.69, 0.82, 0.626; 3.91, 0.55, 0.381; 2.00, 0.275, 0.201; 0.89, 0.138, 0.105. The kH+ value is 1.04 X I O P 4 liter/mole/second. The apparent rate constants for the degradation of 2.5 X 10-4M fructose in 1.OM HC1 were evaluated at different temperatures. The values are "C, lo5 k 2 8 3 m g : 60.0", 1.02; 70.0", 2.70; 75.0", 5.14; 80.0", 8.59. The k values are given in second-'. The apparent heat of activation AH, for the rate of appearance of the 283-mp absorbance from fructose in 1.OM HC1 is 25.0 kcal/mol. The value for the heat of activation (18) H. S. Harned and B. B. Owen, "The Physical Chemistry of Electrolytic Solutions," 3rd ed., Reinhold, New York, 1958. VOL. 39, NO. 10, AUGUST 1967
1111
was calculated from a plot of log k , where the apparent firstorder rate constants k values were obtained in 1.OM HCl us. the reciprocal value of the absolute temperature T, in accordance with the expression log k
=
(AHa/2.303R) l / T
+ log P
where R is the gas constant (1.987 calories degree-' mol-') "C. The apparent log P value for the apand T = 273 pearance of the 283-mp absorbance was 11.4. The absorbance A at 283 mp, obtained at 10 hours of degradation in 1.OM HC1 at 80.0" C , is linearly related to fructose concentration F (Figure 2). The apparent molar absorptivity of fructose under these conditions is E = A / F = 3746 with a standard deviation of 94. Concentrations as low as lO+M can be assayed by this method. The strongest acid condition and the highest temperature gave the highest asymptotic absorbance and thus the greatest analytical sensitivity (Figures 3 and 4). However, upper limits of temperature and acidity are necessary because high acidities produce other products (6). Determination of Sucrose. When sucrose is hydrolyzed under the same conditions of acid concentration and temperature, an absorption band at 283 mp is also generated;
+
Sucrose
+
glucose
+ fructose
-+
degraded glucose
+
degraded fructose (3) Because the rate of hydrolysis of sucrose under these conditions is very fast, the rate-determining step in chromophore production is the degradation of the product fructose (19). When sucrose is degraded in 1.OM HC1 at 80.0" C, the asymptotic absorbance at 283 mp is reached also after 9.25 hours of degradation and is maintained for 2 hours. A small contribution to the apparent molar absorptivity of degraded fructose is made by the degraded glucose a t 283 mp. Equimolar glucose solution degraded in 1.OM HCl at 80.0" C produced very low absorbances; E ranges between 50 and 60 measured a t 9.25-1 1.25 hours after initiation of degradation. The contribution from degraded glucose at this wavelength can be subtracted from the absorbance values for the calibration curve of sucrose. When this is done, the curve is the same as for fructose (Figure 2). Properties of Acid-Degraded Fructose. Extraction of the 283-mp Chromophore of Degraded Fructose. The 283-mp chromophore can be partially extracted with chloroform from the acid solution. Five milliliters of acid-degraded fructose were extracted successively with 3, 3, 2, and 2 ml of chloroform. The combined extracts were collected and made up to 10 ml in a volumetric flask. The chromophore in the chloroform extracts,, , ,A 278 mp, is shown in Figure 1. The amount extracted of the, , ,A 283 mp chromophore is 45 f 2 % by this procedure. This simple operation provides an additional method for the differentiation of acid-degraded fructose from the chromophores generated from o-ribose and 2-deoxy-~-ribosewhich are quantitatively extractable from the acid solution (15). Acid-degraded fructose can be readily assayed in the presence of chromophores which are completely extractable in the organic phase. Applying this procedure and analyzing the acid-degraded fructose remaining in the acid phase after ex-
(19) W. W. Pigman and R. M . Goepp, Jr., "Chemistry of the Carbohydrates," Academic Press, New York, 1948, p. 206. 1 1 12
ANALYTICAL CHEMISTRY
traction, the sugar concentration is known from the calibration Curve B in Figure 2. Partitioning of Acid-Degraded Fructose between Equal Volumes of Solvent. The 283-mp chromophore at five different concentrations was partitioned between equal volumes of 1.OM HC1 and chloroform. The values obtained for the apparent partition coefficient Kappwere similar in all cases, with an average value of 2.88 for the ratio of the 283-mp absorbance in 1.OM HC1 and the 278-mp absorbance in chloroform. The true partition coefficient K defined as
K
=
A J ( A - A,)
(4)
was also calculated, where A , and Ab are the absorbances at 283 mp in 1.OM HC1 after and before the partitioning, respectively. A value of 2.74 was found for K . From Equation 4 and after replacing A = ec, where A is absorbance and c concentration, the ratio of the apparent absorptivity in chloroform at 278 mp and in HC1 at 283 mp was 0.95 (K/Kapp). From such results the absorptivity value for the 278-mp chromophore in chloroform is 3570. Alkaline Destruction of the 283-mp Chromophore. The absorption band obtained under conditions previously described can be destroyed by alkali. When the acid solution is made alkaline by addition of NaOH, the 283-mp band shifts to 286-mp and the absorbance at this wavelength disappears by a first-order process to a small residual absorbance. The apparent first-order rate constants for these disappearances a t 35.0" C calculated in accordance with Equation 1 are 10; k , [NaOH], uoH-: 1.65, 0.30, 0.213; 1.35, 0.22, 0.161; 0.89, 0.14, 0.106; 0.455: 0.06, 0.048. They are directly proportional to the hydroxyi ion activity Q ~ H -at a constant temperature. The bimolecular rate constant, ken- = k / uoH-,is 8.35 X 10-5 liter/mole/second. The apparent first-order rate constants for the destruction of the 286-mp chromophore of acid-degraded fructose (2.5 X 10-4M) in 0.2M NaOH are: "C, 105 k : 38.0", 1.46; 50.0", 4.19; 60.0", 10.5; 77.5", 34.3, where the k values are given in second-'. The heat of activation A H , for the destruction of the 286-mp absorbance is 17.3 kcal/mol (Equation 2). The log P value for the apparent rate constant k is 7.3. The apparent rate constants at a given NaOH concentration and temperature were invariant with degraded fructose concentration. The alkaline destruction of acid-degraded sugars permits differentiation. When acid-degraded ribose, , , ,A 277 mp, is made alkaline, the absorbance at that wavelength is unchanged. It slowly decreases and a specific chromophore 324 mp (15). This behavior is arises simultaneously at A,, different for acid-degraded fructose and acid-degraded 2deoxy-D-ribose, which shows an alkaline chromophore shift to 293 mp and extremely rapid destruction of the chromophore (13). Chemical Nature of the 283-mp Chromophore. Indications have been given in the literature (20, 21, 22) that H M F is one of the products of the acid degradation of hexoses and sucrose. Studies of some of the properties of H M F were made in order to compare with those observed for the acid-degraded fructose. H M F presents two absorption Dands ir. 1.OM
(20) F. A. H. Rice and L. Fishbein, J. Am. Chem. Soc., 78, 3731 (1956). (21) B. Singh, G. R. Dean, and S. M. Cantor, Ibid.,70, 517 (1948). (22) W. N. Haworth and W. G. M. Jones, J. Chem. SOC.,1944, p. 667.
HC1: one is observed at Amax 283 mp with a value of 17,000 and the other a t 228 mp of reduced absorptivity. These results are similar to those reported previously (23). Using the value of E ~ S ~ ~ ,the , , yield of H M F after 10 hours heating at 80.0" C in 1.OM HC1 is 2 2 z . Also, H M F can be observed in chloroform at 279 mp and at 286 mp in 0.2M NaOH. (23) G. MacKinney and 0 , Temmer, J . A,,,. Chem. sot., 70, 3586 (1948).
Thin layer chromatographic studies also confirmed the identity of H M F and the chromophoric compound resulting from the acid degradation of fructose. RECEIVEDfor review March 10, 1967. Accepted May 24, 1967. This investigation was supported in part by a n institutional grant to the University of Florida by the American Cancer Society and in part by grant GM-09864-04,05 from the National Institutes of Health, u. s.Public Health Service, Bethesda, Md.
Determination of Nitrogen Compound Types and Distribution in Petroleum by Gas Chromatography with a Coulometric Detector D. Kendall Albert Research and Decelopment Department, American Oil Co., Whiting, Ind.
A recently developed selective nitrogen detector was used with gas chromatography to determine quantitatively the nitrogen compound distribution in light catalytic cycle oil. The dominant nitrogen compound types-pyridines and quinolines, indoles, and carbazoles-were determined directly on the sample without prior separations. Application of the method, which requires about 2 hours for an analysis, to three cycle oils of varying origin indicated that the relative distribution of types was similar, although the absolute concentration levels of nitrogen differed. Combination of gas chromatography with chemical methods of separation and mass spectrometry was used for detailed nitrogen compound distribution studies on fractions of light catalytic cycle oil and light virgin gas oil. The latter contained quinolines, carbazoles, and benzocarbazoles as the dominant nitrogen compound types and minor amounts of benzoquinolines, pyridines, and indoles. Other minor nitrogen compound types-e.g., nitriles-were indicated in the oils studied but were not identified. Chemical separations included a modified perchloric acid extraction method to separate indoles from carbazoles. The reaction of perchloric acid with model compounds was studied with semiquantitative results varying with the nitrogen com pou nd type.
ADVERSE EFFECTS of nitrogen compounds in petroleum on many important catalytic processes and on product stability are well recognized. To surmount these effects, sensitive and accurate analytical determinations of the different types of nitrogen compounds-e.g., pyridines, quinolines, indoles, and carbazoles-are needed. Several methods, which generally comprise a combination of various analytical techniques, for separating, identifying, and determining nitrogen compounds in petroleum, have been described ( I , 2). For example, basic nitrogen compounds were isolated from a heavy gas oil by a combination of acid extractions, alumina adsorption, and paper chromatography (3). Benzoquinolines, quinolines, and other types were identified by ultraviolet, infrared, and mass spectrometry. Nonbasic nitrogen compounds-indoles, carbazoles, and benzocarbazoles-were quantitatively determined in cracked (1) H. V. Drushel and A. L. Sommers, ANAL.CHEM., 38,19 (1966). (2) L. R. Snyder and B. E. Buell, Zbid.,36,767 (1964). (3) D. M. Jewell and G. K. Hartung, J . Chem. Eng. Data, 9,297 ( 1964).
gas oils and straight-run distillates by linear elution adsorption chromatography ( 2 , 4). A combination of nonlinear and linear elution adsorption chromatography was used for qualitative analysis of nitrogen compounds (5). Basic compounds, indoles and carbazoles, were isolated from a light catalytic cycle oil by a combination of silica gel adsorption and acid extraction ( I ) . The extractants included sodium aminoethoxide in ethanolamine for a variety of weakly acidic compounds and 72% perchloric acid (6) for indoles and carbazoles. The latter types also were isolated from a hydrogenated furnace oil with 72% perchloric acid (6); and, in addition, phenazines and nitriles (7) were identified. Phenazines were identified in a black precipitate which formed during the perchloric acid extractions; nitriles were identified in the acid raffinate. Many of these methods, however, are not quantitative because of interferences-e.g., hydrocarbons, oxygen compounds, and sulfur compounds-and/or incompleteness of chemical reactions. In a recently published procedure (8), the gas chromatographic separations of the nitrogen compounds of a petroleum sample can be followed, even when non-nitrogenous components are present. The separation is monitored not by a conventional detector, but by the catalytic hydrogenation of the nitrogen compounds to ammonia, which is titrated coulometrically. Consequently, only nitrogen compounds can be detected. For calibration, the nitrogen types within each gas chromatographic fraction are identified by spectral techniques and, where possible, by the retention times of individual compounds. This procedure has now been applied directly to light catalytic cycle oil (LCCO) and to fractions of LCCO and light virgin gas oil (LVGO) obtained by acid extraction. Nitrogen compound distribution in LCCO can be quantitatively determined in about 2 hours. Except for the separation of indoles from carbazoles, the acid extraction scheme is conventional ( I , 6). Pyridines and quinolines are extracted from benzene solution with hydro(4) L. R. Snyder and B. E. Buell, Anal. Chim. Acta, 33,285 (1965). ( 5 ) L. R. Snyder, ANAL.CHEM., 38, 1319 (1966). (6) G. K. Hartung and D. M. Jewell, Anal. Chim. Acta., 26, 514 (1962). (7) Zbid., 27,219 (1963). ( 8 ) R. L. Martin, ANAL.CHEM., 38, 1209 (1966). VOL. 3 9 , NO. 10, AUGUST 1967
1 1 13
