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Chromatogr. Commun. 1984, 7, 13-18. (23) Later, D. W.; Lee, M. L.; Bartle, K. D.; Kong, R . C.; Vassilaros, D. L. Anal. Chem. 1981, 53, 1612-1620. (24) Castle, R. N.; Tedjamulia, M. L.; Tominaga, Y.; Pratap, R.; Sugiura, M.; Kudo, H.; Lee, M. L.; IwaO, M.; Thompson, R. D.; Martin, G. E.; Gampe, R. T., Jr.; Musmar, M. J.; Wlllcott, M. R., 111; Smith, S. L.; Layton, W. J . ; Hurd, R. E.; Johnson, L. F. I n “Lectures in Heterocyclic ChemistV”; Castle, R. N., Ed.; Hetercorporation: Tampa, FL, 1984; Vol. 7, pp 1-52. (25) Wright, B. W.; Peaden, P. A,; Lee, M. L.; Stark, T. J . Chromatogr. 1982, 248, 17-34.
(26) Lee, M. L.; Kuei, J. C.; Adams, N. W.; Tarbet, B. J.; Nishioka, M.; Jones, B. A.; Bradshaw, J. S. J . Chromatogr., in press.
RECEIVED for review August 6, 1984. ~ ~September ~ 24, ~ 1984. This work Was supported by the Department of Energy, Office of Health and Environmental Research, Contract N ~ , DE-AC02-79EV10237,and the National Science Foundation, Grant No. CHE-8314769.
Role of Thermal Dissociation in the Direct Gas-Li quid Chromatographic Determination of Amine Maleate Salts S. Edward Krikorian* and Grace E. Ukpo Department of Medicinal ChemistrylPharmacognosy, University of Maryland, School of Pharmacy, Baltimore, Maryland 21201 Alan P. Force
U S . Army Chemical Research and Development Center, Aberdeen Proving Ground, Maryland 21010 Curtis W. Edwards
Baltimore District Laboratory, U S . Food and Drug Administration, Baltimore, Maryland 21201
Thermoanalytlcal, GLC, and spectrometric data have provlded evidence that the observation of analytically useful peaks from the direct gas chromatographlc analysls of otherwise lnvolatlle malelc acld salts of a family of tertlary amines Is the result of thermal dlssoclatlon of the salts to thelr respectlve free base and free acld moieties. The efflclency of the dlssoclatlon achleved upon Injectlon of the salts Into “unmodlfled” GLC systems Is dependent upon the Injection port temperature (IPT), as deduced from measurement of the molar responses of the peaks arlslng from both of the eluted components. Optlmum response Is obtalned over only a narrow IPT range (near 200 O C In the case of the antlhlstamine salts tested). DTA and TGA experlments provlded data which proved to be rellable predictors of the GLC behavior of the salts.
Despite their relative involatility, certain classes of amine salts (alkaloids and nitrogenous drugs) have been found to yield analytically useful peaks upon direct injection into gas-liquid chromatographic (GLC) systems (1-9) without prior conversion to their free bases or the use of alkali-modified forecolumns, analytical columns, or carrier gases. Based on the retention times of the peaks obtained by using such unmodified systems, it has been postulated that thermal dissociation of the salts in heated portions of the GLC system may be occurring to liberate the conjugate free bases, which then elute in the normal manner (1, 3-5,lO). If so, however, the fate of the acid part of the salts, even where the acid may be organic, has not been accounted for experimentally. Also, the quantitative reproducibility of the postulated thermal conversion has been questioned (I1,12), although attempts to exploit the direct salt injection procedure for quantitative purposes have been reported (8, 13). In order to establish the general utility of determining amine salts via direct injection under nonextraordinary GLC con-
ditions, the experimental limitations of the technique as well as further proof of the mechanism responsible for the observed GLC behavior were sought. For this purpose, the thermoanalytical and GLC characteristics of maleic acid salts of a series of commonly available antihistamines, viz., pheniramine (l), chlorpheniramine (2), and brompheniramine (3), have been examined. The maleic acid salt of N,N-dimethyl-n-propyl-
HOOC-CH I t 11 C H2CH2NHICH312 - OOC-CH
1. X=H 2,X=cI
3,X=Br
amine (4) is also included in this study, since it serves as a simple experimental model for 1-3, representing the “side chain” of the latter devoid of the substituents on the w-carbon atom. The tertiary amine and organic acid which would be the products of the thermal dissociation of these compounds are readily detectable chromatographically. Detection and quantitation of the acid moiety as well as the basic portion of the salts under the GLC conditions employed would provide further confirmation of the thermal dissociation mechanism as the source of the GLC peaks obtained from these intrinsically involatile analytes. The thermoanalytical experiments were intended to provide a basis for explaining and predicting the observed GLC behavior.
EXPERIMENTAL SECTION Chemicals. The following chemicals were obtained from commercial sources and were used without further purification: pheniramine maleate (Hexagon Laboratories), chlorpheniramine maleate (H. Reisman Corp.), brompheniramine maleate (A. H. Robins), N,N-dimethyl-n-propylamine (Alfa Products), maleic acid (Alfa Products), maleic anhydride (J. T. Baker), propionic acid (Amend Drug Co.), octadecane (Chem Service), and hexadecane (Poly Science). Solvents employed were all reagent grade. The maleic acid salt of N,N-dimethyl-n-propylamine (4) was synthesized by mixing a solution of 0.050 mol of the acid in 10 mL of acetone with 5 mL of a solution containing 0.055 mol of
0003-2700/65/0357-0312$01.50/00 1984 American Chemlcal Soclety
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 1, JANUARY 1985
the amine in acetone. Slow evaporation of the solvent resulted in the formation of white crystals, which were collected by filtration, washed with ether, and air-dried: mp 69-70.5 "C; IR (KBr) 2680 (NH+), 1585 (carboxylate C=O) cm-'; NMR (CDC13) 6 6.2 (9, 2, CH-CH), 3.2-2.8 (m, 8, CH,N(CHJ2), 2.0-1.5 (m, 2, CCH2C),1.0 (t, 3, J = 7 Hz, CH,C). Anal. Calcd for C9H1,N04: C, 53.18; H, 8.43; N, 6.89. Found C, 52.98; H, 8.35; N, 6.79. Standard solutions of the antihistamine free bases were prepared by treating aqueous solutions of their maleate salts with excess aqueous ammonia, followed by exhaustive extraction with chloroform and dilution to a known volume with the solvent. Thermoanalytical Instrumentation and Procedures. Differential thermal analysis (DTA) and thermogravimetric analysis (TGA) of the salts were performed by using a Du Pont Model 990 thermal analyzer interfaced to the intermediate temperature DTA cell module and Model 951 TGA module, respectively, All experiments were carried out over the temperature range 30-330 "C at a programmed rate of 10 "C/min with nitrogen purging of the sample compartment. Aluminum or platinum boats filled with 5 mg of sample were used for the TGA analyses. Differential scanning calorimetry (DSC) was performed on 2-5-mg samples of the salts by using a Perkin-Elmer Model DSC-4 interfaced with the thermal analysis data station or BascomTurner Model 8120R integrating recorder for measurement of temperatures and heats of transitions. For experiments up to 230 "C (3-5 "C/min heating rate), aluminum volatile sample pans were used, while for runs up to 400 "C (3 "C/min heating rate), stainless steel high-pressure capsules were used for samples. GLC Instrumentation and Procedures. The GLC systems employed, both fitted with flame-ionization detectors, were Hewlett-Packard Model 7620A interfaced to a Model 3390A integrator and Varian Model 3700 interfaced to Spectra-Physics Model 4000 data handling system. The nitrogen or helium carrier gas, hydrogen, and air flow rates were adjusted to 25-30,30, and 300-350 mL/min, respectively. Injection port temperature was varied from 160 to 280 "C in 10 "C steps. The columns employed were all 180 cm X 2 mm i.d. glass packed with the following stationary phases: (A) 3% OV-101 on 80/100-mesh Supelcoport (operated at 180 "C) for detection and quantitation of the antihistamine free bases (tR3.3, 6.6, and 9.4 min for 1-3, respectively), (B) 10% SP-1200/1% H3P04on €@/IOO-rneshChromosorb W-AW (operated at 120 "C) for detection and quantitation of maleic acid (tR3.6 min), and (C) 10% Apiezon L on 80/100-mesh Chromosorb G (temp programmed 60 "C for 4 min, 10 "C/min to 90 "C and held) for simultaneous detection and quantitation and maleic acid (tR near 4 and of N,N-dimethyl-n-propylamine 9 min, respectively). For determination of the relative molar responses (RMR) (14) of free base peaks, 0.010 M solutions of the three antihistamine salts and their free bases in chloroform were prepared, containing 0.010 M hexadecane or 0.010 M octadecane as internal standard for pheniramine or for the two halogenated amines, respectively. For determination of the RMR of maleic acid peaks, analyte solutions were 0.020 M in each salt or maleic acid and 0.014 M in propionic acid as internal standard using acetone as solvent. One-microliter injection volumes of all solutions were employed throughout the study. In all cases, the internal standard eluted between the solvent and test compound with base-line resolution of the peaks. Spectrometric Measurements. Electron-impact mass spectra (ionization energy 70 eV) of the column effluent from a Varian Model 1400 gas chromatograph were obtained by using a Du Pont Model 21-490 instrument interfaced to a Hewlett-Packard Model 2100A computer. The chromatograph was equipped with either column A (temp 230 "C) or column B (temp 145 "C) (injection port temp 250 "C, 30 mL/min He) and was connected to the MS source (temp 190 "C) via a heated glass jet separator. Infrared spectra of CC14solutions of liquids were obtained in 0.5 mm path length NaCl cells using a Perkin-Elmer Model 237 spectrophotometer. RESULTS AND DISCUSSION The role that the injection port temperature (IPT) plays in converting the salts to satisfactorily elutable species is illustrated in the gas-liquid chromatograms of Figure 1 for one of the salts. The quality of peak response appears then
313
S
i I
6.6
6.6 TIME (rnin)
6.6
Figure 1. Gas chromatograms (column A, 180 "C) of CHCI, solutions containing equimolar concentrations of octadecane (IS) and of (a)
chlorpheniramine maleate (IPT 160 "C), (b) chlorpheniramine maleate (IPT 200 "C), and (c) chlorpheniramine free base (IPT 160 "C).
30
80
130 180 230 TEMPERATURE ( " C )
280
Flgure 2. DTA curves for the maleate salts 1-4 showing melting endotherms (M) and regions of decomposition (D).
to be dependent upon a thermally induced process occurring in the injection port of the gas chromatograph. Indeed, the retention time of the peak observed when either the salt or the free base is injected coincide, consistent with observations previously cited. To provide an explanation for the dependence of GLC response of these salts on a thermal process, a study of their behavior using thermoanalytical experiments was first undertaken. Thermoanalytical Studies. The DTA curves for the four salts (Figure 2) reveal not only the expected melting endotherms but also a reproducible base-line perturbation, indicative of decomposition, consistently observed in the temperature range 190-240 "C. Evidence that the decomposition of the sample is accompanied by the generation of volatile products is provided by the TGA experiments, which yielded smooth and well-defined weight-loss curves (Figure 3) with inflection point temperatures which correspond to the de-
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Table I. DSC Measurement of Heats of Fusion of the Four Maleate Salts
1
compd
mp,O "C
AH,,.kJ/mol
1 2 3
106.8 f 0.9
38.12 f 1.05 50.46 f 0.50 47.36 f 0.42 18.83 f 0.42
133.9 f 0.4 132.8 f 0.3 4 68.9 f 0.1 aMean f standard error ( n = 4).
I
50
1
I
100
I
I
150
I
I
200
I
I
250
I
I
300
TEMPERATURE ("C)
Flgure 3. TGA curves for the maleate salts 1-4. The hightemperature plateaus represent 10% of the starting weights for 1-3 and 5 % for 4.
composition regions noted in Figure 2. The similarity in the thermal behavior of the antihistamine maleates with that of 4 suggests a common mode of decomposition which, considering the differences in structure between the salts, would support the occurrence of a thermal process at the site of the ionic bond rather than within the carbon skeleton of the molecules. In support of this premise, an oily condensate with an amine-like odor always collected on cooler portions of the furnace tube in all the TGA experiments with the antihistamine maleates. At the conclusion of one such run involving 2, recovery of the oil with CCll rinses of the tube gave a solution whose infrared spectrum was identical with that of an authentic sample of chlorpheniramine (15) (prepared from the maleate salt), providing direct evidence that the intact free base is one of the volatile products of the thermal treatment of the salts. No such evidence for the concurrent liberation of maleic acid was able to be educed from these experiments, however. The more facile thermal breakdown of 4 compared to that of the antihistamine salts, which is apparent from Figure 3, is certainly a consequence of the greater volatility of N,Ndimethyl-n-propylaminerelative to the free bases derived from 1-3 and the effect this has on the dissociation equilibrium under the open, dynamic conditions of the TGA experiments. On this basis, since these experiments serve as a good model for the kind of treatment the samples would receive in a GLC system, one may predict that the salt 4 will be converted to a chromatographically elutable species under milder thermal conditions than the antihistamine salts will require, even though the mechanism operative (deprotonation of the amino group) may be identical. Given the well-defined melting endotherms which were observed in Figure 2, the heats of fusion of the four salts could be measured by using DSC (Table I). Efforts to obtain heats of decomposition corresponding to the nondescript base-line perturbations in the DTA curves at 190-240 "C were unsuccessful for the salts 1-3, due to rupturing of the sealed sample pans during the DSC runs. However, 4 produced a well-defined DSC endotherm spanning the temperature range 160-255 "C, which yielded values of AH (decomposition) of 84.5 and 84.9 kJ/mol in duplicate runs. These values compare to 188-209 kJ/mol reported for the decomposition of a series of simple primary amine hydrochlorides (16).
GLC Studies Related to t h e Thermal Dissociation Process. Based on the results of the thermoanalytical studies, dissociation of the salts to the free tertiary amines could be expected to occur in the injection port of a gas chromatograph at readily accessible temperatures, e.g., 200 "C. The analytical utility of the observed peaks should then depend upon the efficiency of the prerequisite thermal conversion. This was examined further under GLC conditions by (a) corroboration of the thermal dissociation mechanism through confirmation of the identity of the actual eluates, including determination of the fate of the acid moiety of the salts, and (b) determination of the limits of the thermal conditions necessary to achieve quantitative dissociation. Correspondence between the retention times of the peaks obtained from injection of the salts 1-3 (IPT=200°C) and of their respective free bases was demonstrated on two columns, column A and an OV-17 column. Careful temperature programming of the column oven elicited no additional peaks in the chromatograms indicative of further volatile thermal breakdown products, even using IPT's as high as 280 "C. (Maleic acid was never seen to elute from these columns.) Mass spectral analysis of the column effluent (GC/MS) from salt injection (column A) produced spectra of the eluates which matched the spectra of their corresponding free bases (17)also introduced via the GLC interface. This evidence, together with that from the TGA experiment previously cited, provides unequivocal proof that the intact conjugate free bases of the compounds 1-3 are products of the thermal treatment of the salts. When column B was employed for GLC analysis of the four salts (IPT = 200 "C) a single peak was obtained whose retention time and mass spectrum matched those for the peak obtained from injection of pure maleic acid. (The conjugate free bases of the salts did not elute from this column.) However, these data also match the retention time and mass spectrum (18)for pure maleic anhydride obtained under the same conditions. It thus appears that the peak which elutes from column B upon injection of the salts arises from a two-step process, viz., liberation of maleic acid by thermal dissociation followed by its dehydration (19). As final proof of the validity of the salt dissociation mechanism, the simultaneous liberation of the salt components in stoichiometrically correct amounts was demonstrated under GLC conditions (column C, 4 as the analyte). Area response ratios obtained for the peaks due to the amine and the acid from duplicate injections of a 0.05 M solution of 4 in acetone at ten IPT's (160-280 "C) varied randomly over the range 0.82-1.10 with a mean value of 0.973 f 0.072 (95% confidence limits). The consistency and the magnitude of near unity of these area ratios (both eluates exhibited similar molar responses) provide excellent confirmation of the proposed mechanism. In order to determine the thermal limitations which might affect the efficiency of the salt dissociation process, the quantitativeness of the conversion of the salts to their conjugate free acid and base components was tested by measuring the relative molar responses (RMR) of the eluates as a function of IPT. (Neither the type of column packing, its temperature,
315
Anal. Chem. 1985, 57, 315-319
the volume of sample injected, nor the solvent employed was found to affect the magnitude of the molar responses obtained from monitoring either of the two constituents.) When compared to the R M R s of the $eaks obtained from injection of the free bases or maleic acid itself, all four salts yielded eluates which exhibited optimum quantitative response (RMRs of 102-104% of theoretical) at IPT 200-230 OC. At temperatures below (down to 160 OC) and above (up to 280 “C) this range, the salt eluate RMR’s decreased to 70-80% of the injected free base or acid responses. Lower IPT’s apparently result in such slow rates of dissociation kinetically that yields of the products in chromatographically detectable amounts under the dynamic conditions of the GLC system are diminished. At temperatures higher than the optimum, on the other hand, reduced peak response is most likely due to partial thermal degradation of the salts to nonvolatile species, as evidenced by the appearance of black involatile residues and
