~~
~
Table 111. Analysisa of Tetracycline Hydrochloride b Spiked with 0.6% EATCc and 0.6% ATCc Spike Recovered, % Replicate No.
EATC
ATC
100.6 99.7 2 98.9 98.2 3 101.o 99.5 100.0 4 102.1 5 102.4 99.4 Mean * stand dev 101.0 * 1.4 99.4 ?: 0.7 UDetector at 429 nm, flow at 0.5 ml/min, column temperature at 35.0 “C.bQuadruplicate analysis of the unspiked sample gave 0.146 * 0.004% ATC hydrochloride and 0.007 ?: 0.002% EATC hydrochloride. c A s the hydrochlorides. 1
common tetracyclines using solutions of 0.5 mg/ml in order to avoid overloadingthe column. Detection at 359 nm afforded maximum sensitivity to TC with minimum response to either ATC or EATC. Methacycline (k’ = 0.87),oxytetracycline (k’ = 1.39), and doxycycline (h’ = 1.95) were well separated from TC (k’ = 7.3). ETC (k’ = 4.7) exhibited adequate resolution from TC ( R , = 1.0) and was completely eluted before the TC maximum was reached. Chlortetracycline (k’ = 5.8) was separated but not well resolved from TC (R, = 0.4). Potential usefulness as an assay for ETC and/or TC was also investigated. Both TC and ETC are slowly degraded in aqueous alkali (3),but solutions of either one in 0.01 N sodium hydroxide are suitable if analyzed within 5 min of preparation. For both ETC and TC, peak heights were determined once a t each of five concentrations in the range 0.1-0.8 mg/ml, and the data were analyzed by linear regression using the 0.8 mg/ml solution as the standard. Each epimer showed excellent linearity (T = 1.000),the concentrations found (mg/ml) being
given by the equations y = 1.003 x - 0.003 (ETC) and y = 1.004 x - 0.003 (TC);L D values were 0.06 pg and 0.07 pg, respectively. However, TC begins to elute slightly before the ETC maximum is reached, and low-level determination of ETC in TC would be somewhat inaccurate. Nevertheless, excellent quantitative results should be possible for TC with little or no modification of the HPLC system. ACKNOWLEDGMENT We wish to acknowledge the contribution of J. E. Wolleben, who carried out two of the linearity studies. LITERATURE CITED (1) V. C. Waiton, M. R. Howlett, and G. B. Selzer, J. Pharm. Scl., 59, 1160 (1970). (2) 21 CFR 436.309, Fed. Reglst., 39, 18965 (1974). (3) J. R. D. McCormick, S.M. Fox, L. L. Smlth, B. A. Bitier, J. Reichenthai, V. E. Origonl, W. H. Muller, R. Winterbottom, and A. P. Doerschuk, J. Am. Chem. Soc., 79, 2849 (1957). (4) R. G. Kelly, J. Pharm. Sci., 53, 1551 (1964). (5) W. W. Fike and N. W. Brake, J. Pharm. Sci., 61, 615 (1972). (6) B. W. Grifflths, R. Brunet, and L. Greenberg, Can. J. Pharm. Scl., 5 , 101 (1970). (7) K. Tsujl and J. H. Robertson, Anal. Chem., 45, 2136 (1973). (6)“Application Highlights”, No. 25, Waters Associates, Framingham, Mass. (9) K. TsuJl, J. H. Robertson, and W. F. Beyer, Anal. Chem., 46, 539 (1974). (10) J. H. Knox and J. Jurand, J. Chrometogr., 110, 103 (1975). (11) K. Tsujl and J. H. Robertson, J. Pharm. Sci., 65, 400 (1976). (12) A. G. Butterfield, D. W. Hughes, N. J. Pound, and W. L. Wllson, Antlmlcrob. Agents Chemother.,4, 11 (1973). (13) C. Manning, A k a Corp., Palo Alto. Calif., personal communication, 1972. (14) J. J. Kirkland, J. Chromatogr. Sci., I O , 129 (1972). (15) B. Hoener, T. D. Sokoloski, L. A. Mitscher, and L. Maispels, J. Pharm. Scl., 63, 1901 (1974). (16) K. D. Schiecht and C. W. Frank, J. Pharm. Scl., 64, 352 (1975). (17) L. R. Snyder and J. J. Kirkland, “Introduction to Modern Liquid Chromatography”, John Wiley and Sons, New York, N.Y., 1974.
RECEIVEDfor review January 22, 1976. Accepted June 21, 1976.
Separafion and Quantitation of Diazonium Salts as Heptanesulfonate Ion Pairs by High Pressure Liquid Chromatography Edward Fitzgerald GAF Corporation, 25 Oralid Road, Johnson City, N.Y. 13790
Quantitative analysis of diazonium salts in formulations is important for the quality control of diazo reprographic products. Quantitative separations of several diazonium salts were obtained by reverse phase high pressure liquid chromatography of the heptanesulfonateion pairs. Chromatography was performed with an octadecyi bonded phase column and a buffer-acetonitrile eluent using a uv detector. The precision was 0.2% standard deviation with good least squares linearity (0.9988 coefficient of determination) over a range of 0.07 to 0.14% diazonium salt. The chromatographic system provides good routine In-process quality control of diazonium salts and couplers in reprographicformulations.
system, especially in formulations containing more than one diazonium salt. Reverse phase high pressure liquid chromatography on octadecyl bonded phase columns has been reported to perform good separations for a variety of compounds (1, 2). This type of column yielded a good separation for a mixture of typical diazonium salts except for the formation of two peaks for one of the salts. This effect was not reproducible and was particularly troublesome in quantitative analysis of the diazonium salt. Reverse phase chromatography of organic ions paired with heptanesulfonate has been recently reported (3),and it was believed that this ion would form one peak with the diazonium ion in question. One reproducible peak was obtained using the heptanesulfonate permitting good separation and quantitation.
Separation and analysis of diazonium sensitizers in reprographic formulations requires an efficient chromatographic
EXPERIMENTAL
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ANALYTICAL CHEMISTRY, VOL. 48, NO. 12, OCTOBER 1978
Reagents. Acetonitrile was purchased f r o m Burdick and Jackson,
t
3
Y
z m 0
L 4 TIME IMINUTESl
6
2
4 TIME [ M I N U T E S )
,
6
i_
8
Figure 1. Separation of diazonium salts with acetonitrile-buffer eluent on pBondapak C-18
Figure 2. Separation of diazonium salts in acetonitrile-buffer with heptanesulfonate added
Peaks ( I , 2), p-NJVdiethylarninobenzenediazonium chloride; (3),3-methyl-4pyrrolidinylbenzenediazonium chloride; (4), 2,5di-f+butoxy-4-morpholinobenzenediazonium chloride
Peak ( l ) ,p-N-Ndiethylaminobenzenediazoniumchloride; (2), 3-methyl-4-pyrrolidinylbenzenediazonium chloride, (3) 2,5di-n-butoxy-4-morpholinoben-
Muskegon, Mich. The diazonium salts were commercial sensitizers supplied by Philip Hunt and ABM Chemicals Ltd. and recrystallized several times. Apparatus. Chromatography was performed on a Waters C-900 3000 PSI pumping system with a Valco CV-6-HPAX sampling valve and a Waters Model 440 ultraviolet detector. The column was a Waters 30 cm X 5 mm pBondapak C-18 column. Chromatograms were integrated with a Spectra Physics Minigrator. Procedure. One-percent solutions of the diazonium salts were made up in water and stabilized with 1%citric acid. They were diluted 550 in acetonitrile along with a 10?6 toluene internal standard diluted 250 and injected in the 10-pl sample loop. The buffer consisted of 2.5% potassium phosphate monobasic with phosphoric acid added to obtain a P H of 3.
zenediazonium chloride
0.14% diazonium salt after dilution. The quantitative data were obtained by area ratio of the peak to the toluene internal standard.
CONCLUSIONS The diazonium ion probably exists as an ion pair in this system and the double peak of the p-N,N-diethylaminobenzenediazonium salt may be the result of ion pairs with two anions. Formation of the heptanesulfonate ion pair resulted in one peak. The retention time of the last salt, which was well retained, was increased significantly when heptanesulfonate was added as expected, but the first two salts which elute RESULTS AND DISCUSSION rapidly were not significantly more retained as heptanesul2,5-Di-n-butoxy-4-morpholinobenzenediazonium chloride, fonates. % (zinc chloride) and 3-methyl-4-pyrrolidinylbenzenediazoThis system is also useful for separation of diazonium salts nium chloride, 3/2 (zinc chloride) yielded sharp quantitative and couplers in reprographic formulations. The degree of peaks on the octadecyl column using 45 acetonitrile-55 quantitation is excellent for quality control application. aqueous PH3 buffer as eluent at a flow rate of 1.3 ml/min. LITERATURE CITED However, p-N,N-diethylaminobenzenediazoniumchloride, 1/2 (zinc chloride) yielded two peaks, as in Figure 1.Addition (1) R. F . Borch, Anal. Chern., 47, 2437 (1975). (2) M. Dong, D. Locke, and E. Ferrand, Anal. Chem., 48, 368 (1976). of 0.005 M heptanesulfonic acid to the buffer-acetonitrile (3) D. P. Wittmer, N. 0. Nuessle, and W. G. Haney, Anal. Chem., 47, 1422 eluent resulted in one sharp peak, as in Figure 2. The precision (1975). was 0.2% standard deviation with good least squares linearity RECEIVEDfor review May 19,1976. Accepted July 6,1976. (0.9988 coefficient of determination) over a range of 0.07 to
Analysis of Solid Materials by Laser Probe Mass Spectrometry R. A. Bingham A N Scientific Apparatus Ltd., Barton Dock Road, Urmston, Manchester, England
P. L. Salter" Department of Applied Physics, University of Hull, Hull, Yorkshire, England
Three types of laser have been examlned as sources of Ion Production for trace element analysis of solid materials by mass spectrometry. ComParlson IS made between the lasers and with the conventional rf spark source. Their analytical qualities and potential are dlscussed for the examination of both conductlng and nonconductlng materlals.
The conventional means of ionizing solid materials for the analysis of trace elements in mass spectrometry is by the radio-frequency spark ( 1 ) .The laser has an appeal as a competitor because of its inert nature and because it provides the possibility of examining small regions of single specimens without the need for special preparation. In the case of the
ANALYTICAL CHEMISTRY, VOL. 48, NO. 12, OCTOBER 1976
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