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Anal. Chem. 1981, 5 3 , 786-789
A further improvement in the analysis error can be possible by employing an automatic calculation method using a microcomputer to the interference-removal technique. Application of the spectrometer to much higher SOz concentration, namely, over 1000 ppm, is the subject for a future study. ACKNOWLEDGMENT The authors wish to thank R. T. Koike for his hehful discussion and critical reading of the paper before publicakon. The authors were favored to have the assistance of S. Sekino, M. Hamajima, N. Uno, and A. Shimizu who contributed their experimental skill.
LITERATURE CITED (1) Bonflglioii, G.; Brovetto, P. Appl. Opt. 1964, 3 , 1417-1424. (2) Hager, R. N., Jr.; Anderson, R. C. J . Opt. Soc. Am. 1970, 60, 1444-1449.
Paper, No. 71-1045; American Institute of Aeronautics and Astronautics; New York, 1971.
(3) Hager, Robert N., Jr. AIAA
(4) Hunt, R. D.; Williams, D. T. Am. Lab. (Falrfleld, Conn.) 1977, 9 (6),
10-23.
(5) Izumi, T.; Nakamura, K. J . Phys. €1981, 14, 105-112. (6) Stewart, James E. Appl. Opt. 1965, 4 , 609-612.
RECEIVED for review March 3, 1980. Accepted December 29, 1980. Financial support by the Japanese Government is gratefully acknowledged. The paper is published by permission of the Director, Air Pollution Control Division, National Research Institute for Pollution and Resources, to whom the authors are grateful. Presented in part at the 25th Spring Meeting of the Japan Society of Applied Physics (27a-L-1, -2, and -3, Tokyo, Japan, March 1978) [in Japanese].
Quantitative Determination of Carboxylic Acids and Their Salts and Anhydrides in Asphalts by Selective Chemical Reactions and Differential Infrared Spectrometry J. Claine Petersen” and Henry Plancher Laramie Energy Technology Center, P.0. Box 3395, University Station, Laramie, Wyoming 82070
A method for determining the concentratlon of carboxylic acids and anhydrldes In asphalts and asphalt fractions has been developed. I n addltlon, carboxylale salts are determined independently from the free acids. The method is based on the selective Interaction of trfphenyltln hydroxide wlth free carboxylic acids, the hydrolysls of acids and anhydrides wlth sodlum hydroxide, and the silylatlon of free aclds and thelr salts. Dffferentlal spectra are used to make the quantftatlve determlnations.
A quantitative method for determining several compound types in asphalts that absorb in the carbonyl region of the infrared spectrum was described in a previous paper (I). In the method, anhydrides and carboxylic acids in asphalts were reacted simultaneously with added sodium hydroxide (NaOH), and the total combined absorbance of these two functional types was determined from the band area of the differential spectrum of NaOH treated vs. untreated samples. For determination of the carboxylic acid absorbance, silyl esters were formed from the acids in untreated samples, and the absorbance was estimated from the area of the acid band obtained from the differential spectrum of silylated vs. untreated samples. The absorbance of the anhydrides was then determined by difference. This procedure often presented problems because of band overlap between the free acid and silyl ester bands, making necessary the approximation of the area of the acid band. Determination of carboxylic acids separately using potassium bicarbonate (I) sometimes also presented problems because of the reaction of bicarbonate with some of the more easily hydrolyzed anhydrides. The present paper describes a modification of the earlier method (1) that obviates the problems outlined above. Triphenyltin hydroxide (TPTH) is used; this reagent interacts quantitatively and selectively with the carboxylic acids in asphalts at ambient temperature without interaction with
other functional groups that absorb in the carbonyl region. Free carboxylic acids and anhydrides are then easily determined independently using differential spectra. Carboxylic acid salts can also be determined independently of the free carboxylic acids.
EXPERIMENTAL SECTION Materials. A large number of asphalts and asphalt fractions have been examined by the modified method. Two fractions were chosen to illustrate the method: (1) Wilmington asphaltenes precipitated by pentane (2) and (2) an asphalt fraction that had been strongly adsorbed (SA) on the aggregate taken from an aged (air oxidized) road core. The SA fraction, which represented less than 1% of the asphalt, was the asphalt fraction not removed by extraction of the aggregate with benzene but that was removed by extraction of the benzene-extracted aggregate with pyridine. Details of the isolation of the SA fraction are found elsewhere (3). Benzene was analyzed reagent grade from J. T. Baker Chemical Co. Tetrahydrofuran (THF) from Eastman Chemical Co. was passed through a column of dry, activated basic alumina to remove possible interfering amounts of water and oxidation products, stabilized with 0.025% butylated hydroxytoluene, and protected from moisture by storing over activated, Type 4A molecular sieve from J. T. Baker and Co. Hexamethyldisilizane (HMDS) and trimethylchlorosilane (TMCS) were “specially purified grade” from Pierce Chemical Co. Sodium hydroxide (NaOH) was analytical reagent grade. Triphenyltin hydroxide (TPTH) of about 90% purity was obtained from Pfaltz and Bauer, Inc. The TPTH reagent was prepared as follows. Residual water was removed from 0.32 g of TPTH by azeotropic distillation with about 10 mL of benzene followed by complete evaporation of the benzene. The dried TPTH was then diluted with 100 mL of THF and stored in a brown bottle containing 4A molecular sieve. The bottle was stoppered with an inert cap, and, thus prepared, the reagent was stable for several weeks. With time the reagent slowly develops an interfering infrared band at about 1775 cm-’. Model compounds used in the interaction with TPTH were standard laboratory grade. Sample Preparation. Triplicate samples of asphaltenes (0.125 g) or SA fraction (0.0312 g) were weighed into 25-mL Erlenmeyer
This article not subject to U.S. Copyright. Published 1981 by the
American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 6,MAY 1981
flasks. From this point, treatment of either set of triplicate samples was the same. Two-tenths milliliter of 0.5 N NaOH was added to one of the triplicate sample flasks followed by the addition of 7 mI, of benzene to all three flasks. After the addition of boiling chips, the benzene was evaporated on a hot plate having a surface temperature of about 145 "C. After evaporation of the benzene to remove any moisture present, the two flasks containing no NaOH were stoppered with a septum cap for further treatment. The flask containing the NaOH-treated sample should contain visible amounts of water following evaporation of the 7 mL of benzene. If not, 0.1 mL of water was added to the flask followed by 2 mL of benzene and the evaporation repeated to assure hydrolysis of the anhydrides. Residual water remaining on the sides of the flask after hydrolysis was removed by inserting a hypodermic needle attached to a vacuum line into the neck of the flask. Complete sample drying was then accomplished by evaporation of 3-4 mL of benzene from the NaOH-treated residue. The flask was removed from the hot plate and immediately stoppered with a rubber septum cap to prevent moisture pickup by the residual NaOH. If the atmosphere contained sufficient moisture to interfere with subsequent infrared determinations in THF ( I ) , all flasks were flushed briefly with dry nitrogen, using hypodermic needles, immediately following capping with the rubber septum. To the NaOH-treated sample and one of the untreated samples was added 2.50 mL of dry THF through the septum cap using a hypodermic syring while venting the flask with a hypodermic needle inserted in the septum. To the remaining untreated sample was added 2.50 mL of the TPTH reagent through the septum cap in a similar manner. A 2.50-mL quantity of this reagent has the capacity to determine 0.15 mol L-l of carboxylic acids in a 0.125-g sample of asphalt. A 2-mL portion of TPTH-treated sample was transferred with a hypodermic syringe to a 6-mL septum-stoppered vial and silylated by the addition of 0.015 mL of HMDS and 0.1 mL of TMCS followed by heating for 0.5 h at 40 "C. (The top of the source compartment of the spectrophotometer provided adequate heat.) A t this point four samples had been prepared-untreated; TPTH-treated; NaOH-treated; and TPTH-treated, silylated-and were ready for infrared analysis. Determination of Infrared Spectra. An explicit description of the spectral determinations is found elsewhere ( I ) . Briefly, solvent-compensated spectra were obtained while using absorbance-scale chart paper to facilitate quantitative determinations of band areas; spectra were obtained according to the sequences described in the "Results and Discussion" using 1.00-mm cells. A variable attenuator in the sample beam was required to position the spectra during differential determinations. Calculations. Functional group concentrations were calculated from their corresponding band areas using the appropriate apparent integrated absorption intensity, B (4),for each functional group. Values for B obtained with sets of model compounds, together with details on calculation procedures, have been reported (1).
RESULTS AND DISCUSSION Asphalt contains functional group types including carboxylic acids that absorb in the carbonyl region of the infrared spectrum. Oxidation produces significant amounts of new functional groups that also absorb in the carbonyl region, particularly ketones (5),anhydrides (6),and to a lesser extent additional carboxylic acids ( I ) . Band overlap in the carbonyl region of oxidized asphalts makes virtually impossible the quantitative, and often qualitative, identification of the functional group types by direct observation of the spectrum. In the previous method ( I ) , band overlap in differential spectra sometimes caused problems in the quantitative determination of carboxylic acids and anhydrides as outlined in the introductory section. The present method using TPTH overcomes this problem. General Description of Method. The carboxylates of triorganotin have been extensively studied (7,8),and infrared studies of their benzoates have been reported (9). We found triphenyltin hydroxide extremely useful in quantitatively determining carboxylic acids in complex petroleum asphalt
787
01
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03 0.4
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Flgure 1. Examples of the carbonyl reglon of infrared spectra used
in analysis of asphaltenes: (A) untreated, (8)TPTH treated, (C) NaOH treated, (D) TPTH treated vs. untreated, (E) TPTH reagent vs. solvent. mixtures without previous separation or concentration of asphalt components. We selected two asphalt fractions for illustration of the method utilizing TPTH because acids are concentrated in these fractions and in one fraction the acids occur partially as carboxylate salts. The underlying principle of the method is to react the anhydrides and carboxylic acids with selective chemical reagents to alter or shift their analytical adsorption bands so that the bands may be independently identified and measured. The identification and measurement are facilitated by the use of differential spectra which are determined in the T H F solutions to avoid dimerization of the carboxylic acids ( I ) . Reaction with NaOH causes a shift in the absorption frequency of the 1730-cm-l acid carbonyl band and the 1765- and 1725-cm-l anhydride carbonyl doublet to that of the corresponding carboxylate salt band at about 1580 cm-'. The free carboxylic acid absorption at 1730 cm-' in asphalts is shifted to about 1640 cm'-' upon treatment of the asphalt solution with TPTH. Silylation of the TPTH-treated sample selectively converts the carboxylic acids in the TPTH interaction product together with any carboxylate salts initially present in the asphalt to their silyl esters having an absorption band of about 1715 cm-'; this band is well separated from the carbonyl band of the T P T H interaction product a t about 1640 cm-'. Differential spectra of solutions of the various reaction products can then be used to determine the amounts of functional types present as will be discussed by referring to examples in the following section. Examples of Use of the Method. Figures 1 and 2 are illustrations of spectra derived from Wilmington (California) asphaltenes. Spectra A, B, and C in Figure 1 were obtained in T H F solutions using solvent compensation as explained in the Experimental Section. Spectrum A in Figure 1 is from a solution of the untreated asphaltenes. The peak in spectrum A at 1730 cm-' is that of the free carboxylic acid; the acid exists entirely in the monomer state in the basic solvent THF. In solutions of many whole asphalts, particularly those that have undergone oxidation, this peak is difficult to detect on the strong shoulder of the much more intense ketone band centering at about 1700 cm-l. Spectrum B of Figure 1 is the spectrum of the TPTH-treated sample. Note the loss of the band area attributed to carboxylic acids. Spectrum C in the figure was determined on the NaOH-treated sample and shows the loss of hydrolyzable material in the carbonyl region representing both the carboxylic acid and the anhydrides.
788
ANALYTICAL CHEMISTRY, VOL. 53, NO. 6, MAY 1981 o
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Flgure 2. Examples of the carbonyl region of infrared spectra used in analysis of asphaltenes: (A) NaOH treated vs. TPTH treated, (6) TPTH treated, silylated vs. TPTH treated. Spectrum D in Figure 1 is the differential spectrum with the TPTH-treated sample in the sample beam and the untreated sample in the reference beam of the spectrophotometer. The shaded area represents the free carboxylic acids in the asphaltene sample at about 1730 cm-l. The peak is well-defined, and from its area the concentration of carboxylic acids can be calculated. The carboxylic acid concentration of this particular asphaltene sample was 7.3 X mol L-l. The band at about 1640 cm-' is the carbonyl absorption of the TPTH derivative of the carboxylic acids in the asphaltenes. When using differential spectra where reagents have been added to a sample contained in one of the cells, one must always be concerned about interference from reagent absorption. Spectrum E, Figure 1,is a spectrum obtained with TPTH reagent in the sample beam and THF in the reference beam. Although there are small absorption bands from TPTH, these are insignificant in intensity and do not appear at the carboxylic acid carbonyl frequency. Thus, interference from T P T H reagent bands is insignificant. The infrared band used for the determination of the anhydrides is shown in the shaded area of spectrum A, Figure 2. This spectrum is the differential spectrum of the NaOHtreated asphaltene sample vs. the TPTH-treated sample. The anhydrides occupy a broad band between about 1700 and 1800 cm-l and usually appear as a doublet with the major and minor peaks at about 1725 and 1765 cm-l, respectively. The anhydride in this sample was 2.4 X mol L-l. Spectrum B of Figure 2 was derived from the silylated, TPTH-treated sample of the asphaltenes in the sample beam and the TPTH-treated sample in the reference beam. All carboxylic acids and carboxylate salts in the TPTH-treated, silylated sample are converted to silyl esters, and the carboxylic acids in the TPTH-treated sample are converted to T P T H derivatives. The absorption bands of the silyl esters (about 1715 cm-') and the TPTH derivatives (about 1640 cm-l) are separated sufficiently from each other so that they do not interfere. This spectrum serves two purposes. If no carboxylate salts are present in the original sample, the concentration of silyl esters should correspond with that of the free acids. In this sample, calculated concentrations were identical, showing the absence of carboxylate salts. If carboxylate salts are present in the original sample, the spectrum of the silylated sample can be used to calculate their concentration, as will be shown. Spectra obtained on an asphalt fraction that was previously strongly adsorbed on the mineral aggregate of an air-aged road
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Flgure 3. Examples of the carbonyl region of infrared spectra used in analysis of strongly adsorbed asphalt fraction desorbed from a mineral aggregate: (A) untreated, (B) TPTH treated, (C) NaOH treated, (D) TPTH treated vs. untreated, (E) TPTH treated, silylated vs. TPTH treated. core (not desorbed with benzene but desorbed with pyridine, SA fraction) are shown in Figure 3. Spectra A, B, and C in Figure 3 are solvent-compensated spectra of the original, TPTH-treated, and NaOH-treated samples, respectively. Note the absence of a well-defined free carboxylic acid band at 1730 cm-' in spectrum A. The carboxylic acid band in air-aged asphalts and asphalt fractions is seldom directly observable because of intense absorption at about 1700 cm-'. The free acid band, however, is clearly discernible in spectrum D, which is the differential spectrum of spectra A vs. B. The free carboxylic acid concentration was found to be 0.11 X mol L-l. If, however, carboxylic acid content is calculated from the silyl esters of the acids using spectrum E, Figure 3 (the differential spectrum of the TPTH-treated, silylated sample vs. the TPTH-treated sample), an apparent value of 0.43 X mol L-' is obtained. The difference is accounted for by the presence of 0.32 X mol L-' of carboxylate salts in the original sample that reacted readily with the silylation reagent under the experimental conditions used. Absorption of the carboxylate salt carbonyl can be seen in the spectrum of the TPTH-treated sample as a shoulder at about 1580 cm-l in spectrum E. Carboxylate salts should be suspected in this SA fraction because it was a fraction that was strongly adsorbed on a mineral aggregate surface. Although the differential spectrum is not shown, the difference in the carbonyl region between spectra B and C of Figure 3 results primarily because of the reaction of anhydrides and 2-quinolones (1)with NaOH. The differential spectrum if obtained could be used to determine the anhydride concentration of the SA fraction as previously described for the asphaltene fraction (spectrum A, Figure 2). Interactions of TPTH with Model Compounds. To support the interpretation of the TPTH interactions of this study, we treated several compounds of known composition that absorb in the carbonyl region with the TPTH reagent. The compound l,&naphthalic anhydride was treated with TPTH reagent under the conditions described in the Experimental Section, and no measurable interaction was observed. In a mixture of l,&naphthalic anhydride and asphalt, no TPTH reagent interacted with the model anhydride. This type of anhydride is the predominant type expected in asphalts (5). Interaction of the TPTH reagent with adipic acid was quantitatively complete at room temperature within the time it took to load the infrared cells and run the spectrum. Esters apparently are absent in asphalt oxidized below 130 O C (6); phenyl stearate was treated with the TPTH reagent
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Anal. Chem. 1981, 5 3 , 789-793
and no reaction occurred. These model compound studies support the analyses performed on the asphalt fractions and further justify the use of T P T H as a convenient reagent for quantitatively determining the carboxylic acids, carboxylate salts, and anhydrides in petroleum asphalts and similar bituminous materials.
LITERATURE CITED (1) Petersen, J . C. Anal. Chem. 1975, 47, II;!. (2) Barbour, R. V.; Petersen, J . C. Anal. Chem. 1974, 46, 273. (3) Plancher, H.; Dorrence, S. M.; Petersen, J . C. Asphalt Paving Techno/. 1977, 46, 151. (4) Jones, R. N.; Ramsey, D. A.; Keir, D. S.; Dubrinev, K. J. Am. Chem. SOC. 1952, 74, 80.
(5) Dorrence, S. M.; Barbour, F. A.; Petersen, J . C. Anal. Chem. 1974, 46, 2242. (6) Petersen, J. C.; Barbour, F. A.; Dorrence, S. M. Anal. Chem. 1975, 47, 107. (7) Poller, R. C. "The Chemistry of Organotin Compounds"; Logos: London, 1970; Chapter 14. (8) Sawyer, A. K. "Organotin Compounds"; Marcel Dekker: New York, 1972; Voi. 3, Chapter 12. (9) Mesubi, M. Adediran Spectrochim. Acta, Part A 1976. 32A, 1327.
RECEIVED for review November 24,1980. Accepted February 2, 1981. Mention of specific brand names or models of equipment is made for information only and does not imply endorsement by the Department of Energy.
Determination of pK, Values and Total Proton Distribution Pattern of Spermidine by Carbon- 13 Nuclear Magnetic Resonance Titrations Mary M. Kimberly and J. H. Goldstein" Department of Chemistry, Emory University, Atlanta, Georgia 30322
The 13C resonance assignments of the carbons of spermidine have been made over the pH range 5-13 for a series of concentrations. Analysls of the data has provided the three thermodynamic pK, values as well as the total proton dlstrlbution pattern,. The charge distributlon deduced from the data appears to be reasonable, reflecting the asymmetry of the molecule and the expected effects of charge repulsion.
Polyamines are found in bacteria, bacteriophages, and plant and animal tissues (1). They appear to have a variety of biological roles ranging from acting as growth factors in some microorganisms to exerting stabilizing effects on nucleic acids (I). They have been found as degradation products and/or precursors of some alkaloids, for example, lunarine and palustrin (2,3) and as inhibitors of enzymes such as adenylate cyclase ( 4 ) . Wright et al. postulate that polyamines are modulators of cell membrane function ( 4 ) . Elevated levels of the polyamine, spermidine, and an increased spermidine/spermine ratio have been found in the blood of patients with cystic fibrosis (5). It is believed that, spermidine and its metabolic degradation products may play important roles in the pathogenesis of membrane dysfunctions in this disease (6). (For more detailed discussions of the functions of polyamines, see reviews by Tabor and Tabor (1) and Theoharides (7) and references therein.) At physiological pH ranges, these molecules are polycations and, therefore, have a high affinity for cellular polyanions such as fatty acids, phospholipids, nucleotides, and nucleic acids (8-16). One of the main functions of the polyamines seems to be that of acting as protective or stabilizing agents by being involved in interactions with these anions. Some of the most interesting of the interactions are those involving the various forms of nucleic acids (including DNA, rRNA, and tRNA) (8, 9,11,12,14-16). At sufficiently high concentrations, the polyamines have the ability to precipitate these macromolecules. The complexes are formed by ionic 0003-2700/8 1/0353-0789$0 1.25/0
interactions between the cationic amine groups of the polyamine and the anionic phosphate groups of the nucleic acid. This report describes an investigation of the protonation of spermidine
NH,CH,CH,CH,NHCH,CH,CH,CH,NH, a
1
2
3
b
4
5
6
7
c
utilizing 13C NMR spectrometry at natural abundance. The NMR titration technique provides pK, values and, in this case, information concerning the total proton distribution pattern (17-20). The NMR parameters obtained can provide a basis for further studies of spermidine interactions in other environments. To date, only symmetrical polyamines have been studied by NMR titrations. The unsymmetrical nature of spermidine should be reflected in its proton distribution pattern and in the various interactions mentioned above.
EXPERIMENTAL SECTION Sample Preparation. Spermidine was purchased as the trihydrochloride (Calbiochem,La Jolla, CA) and was used without further purification. First, 1.5 mL of a stock solution was titrated
potentiometrically with a known concentration of KOH. The data from this titration were plotted as pH vs. mL of KOH. From this graph, estimates of volumes of KOH needed for a range of pH values were made. NMR samples were prepared by adding the estimated volumes of KOH from a buret to individual 25-mL volumetric flasks containing 1.5 mL of the stock solution. The volume was brought to 25 mL with deionized water. The concentrations used in the NMR studies were 0.337,0.051,0.078, and 0.160 M spermidine. NMR and pH Measurements. Natural abundance 13CNMR spectra were obtained on a Bruker WH-90 apectrometer. All resonances were referred to external dioxane (67.4 ppm downfield from Me4Si). External D20was used as the lock material. The 0.160 M samples required a sweep width of 1400 Hz and 50 scans in order to obtain spectra. The 0.078 and 0.051 M samples required a sweep width of 3000 Hz and 2000 scans. The 0.037 M samples required a 3000 Hz sweep width and 5000 scans. pH measurements were made on a Corning 109 pH meter equipped with a microelectrode. For the NMR titrations, spectra 0 1981 American Chemical Society
