the tube is not properly sealed. The tube is then removed from the bath, cooled, opened, and the contents transferred t o a 250-mi beaker using water to effect complete transfer. The solution is taken t o dryness at which time 30 ml of 3 :1 isopropanol-water (v/v) is added with warming until all salts have dissolved. The solution is added t o the cation resin in the column and the effluent collected in a 100-ml volumetric flask. This solution is then scanned from 300 mfi to 250 mp, using 3 :1 isopropanol-water as a reference blank. The total amount of methylbenzimidazolone is determined from the predetermined absorptivity, reading the absorbance at the point of maximum absorption. RESULTS AND DISCUSSION
4-Methylbenzimidazolone exhibits two absorption peaks of nearly identical absorptivity at 278 mp and 282 m p ; 5-methylbenzimidazoline exhibits one peak at 285 mp with a weak shoulder at 290 mp. The peak maximum of mixtures of the two isomers varies from 282 mp to 285 mp, depending on isomer ratio. In practice, the absorption peak is read at peak maximum and the average molar absorptivity of the two isomers is used to calculate total methylbenzimidazolone content (Table I). The peak shape and position has been used in these laboratories to estimate qualitatively the approximate ratio of the two isomers. No attempt was made, however, to determine the precise ratio. During the development of the method, a study was conducted to ascertain the optimum conditions for complete hydrolysis. No improvement in recovery of methylbenzimidazolone isomers was found by increasing hydrolysis time beyond 1 hour or by raising the bath temperature beyond 180 O C . No interferences have been encountered provided removal of the amines is complete. The use of insufficient resin results in slightly colored solutions and strong interfering absorption in the ultraviolet region. Under these conditions, the solution is passed through a fresh resin bed which, in most cases, has been found to satisfactorily remove the interference. Recovery data, as shown in Table 11, were obtained by complete hydrolysis and separation of synthetic standards prepared by adding known amounts of each isomer to ortho-free 80/20 TDI.
Table 11. Recovery of Methylbenzimidazolone Isomers from 80/20 TDI Total recovered, Recovery, Isomer Added, mg mg 4-Methyl 1.20 1.105 91.6 1.13 94.2 1.16 96.7 5-Methyl 1.30 1.32O 101.5 1.30 100.0 1.36 104.6 Mixed isomers 0.80 (4-methyl) 2. lob 100.0 1.30 (5-methyl) 2.20 104.7 Mixed isomers 1.80 (5-methyl) 2.52b 96.9 0.80 (4-methyl) 2.56 98.5 Mixed isomers 2.70 (5-methyl) 5 . 05b 99.0 2.40 (4-methyl) 5.01 98.2 a Triplicate determinations. * Duplicate determinations.
z
Sample 1
Sample 2
Table 111. Repeatability of Methylbenzimidazolone Content in MBA found, mg 0.56 0.61 0.53 0.47 1.90 2.00 1.67 1.92
TDI Std dev h0.06
10.14
The method has been applied routinely to determination of methylbenzimidazolones in commercial 80/20 T D I at an average time of 21/2hours per sample. Table I11 shows the repeatability which can be expected. These data were obtained on two samples of commercial TDI containing two levels of methylbenzimidazolone. The data indicate that the method has applicability for routine determinations. RECEIVED for review June 16, 1970. Accepted August 31, 1970. The authors thank Olin Corporation for allowing us to publish this work.
Fast-Response Differential Amplifier for Use with Ion-Selective Electrodes M. J. D. Brand and G . A. Rechnitz Department of Chemistry, State University of New York, Buffaa[o,N . Y . 14214 DESPITETHE RAPID ADVANCES which have occurred in ionselective electrode technology in recent years ( I ) , few attempts have been made to measure electrode response times or to use membrane electrodes to follow fast reactions in solution. (1) R. A. Durst, “Ion Selective Electrodes,” National Bureau of
Standards Special Publication 314, Washington, D. C., 1969. c. Kugler, ANAL. CHEM.. 39. 1682 ~, (1967). (3) G. Johansson and K. Norberg, J. Elecfroanal. Chem., 18, 239 (1968). (4) F. J. W. Roughton and B. Chance, in “Technique of Organic Chemistry,” S. L. Fries, E. S. Lewis, and A. Weissberger, Ed., Interscience, New York, N. Y.,1963, Vol. VIII, Part 11, p 784. (2) G. A . Rahnitz and G.
Measurements of glass electrode response times have been made and values in the range 10-2 to 10-3 second reported (2, 3). Reliable measurements have not usually been made on other electrodes, but in most cases the response times are long (seconds to minutes) at low concentrations and become fast at the highest usable concentrations (0.1 to 1.OM). Kinetic studies of fast reactions in solution using glass ( 4 ) and liquid (5) membrane electrodes have avoided problems associated with the electrode response times by rapid mixing continuous flow techniques. ( 5 ) B. Fleet and G. A . Rechnitz, ANAL. CHEM., 42, 690 (1970).
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4 4 Figure 1. Schematic of differential amplifier, 1 1 5 V supply is obtained from a Model 902 power supply (Analog Devices). All resistors are selected 0.1% tolerance metal film. R trim supplied with 183 L amplifiers
Figure 3. Response of amplifier to a step input Upper trace output signal, lower trace input signal. Vertical sensitivity 5 mV/cm, horizontal sensitivity 1 1 secicm.
Figure 2. Gain and common Mode Rejection Ratio o as a function of frequency A major experimental difficulty in measuring rapid changes in electrode potential is in the design of a n amplifier having a fast response time together with the vanishingly small input current required for a potentiometric measurement. To a large extent these two conditions are mutually exclusive and it is necessary to adopt a compromise between them. Previously (2, 3), amplifier bandwidth has been sacrificed to allow sufficiently small input currents to be obtained and the possibility exists that the exponential response curves obtained represent RC charging in the amplifier. It would be preferable to use an amplifier having a response time at least two orders of magnitude faster than the anticipated response of the electrode. Measurement of the membrane dielectric relaxation times for a number of electrodes (6) suggest that potentiometric response times are unlikely to be much shorter than second and a suitable amplifier would therefore have a response time of 10 wsec. The effects of amplifier input current o n membrane response have not been evaluated quantitatively but an approximation can be made if it is assumed that the membrane is purely resistive. Glass membranes have resistances up to lo9 D and if the potential is to be measured accurately t o 1 0 . 1 mV, a (6) M. J. D. Brand and G . A. Rechnitz, Department of Chemistry, State University of New York, Buffalo, N. Y., unpublished results. 1970. 1660
1
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FREQUENCY
Figure 4. Input current as a function of frequency current of kO.1 pA is the greatest that can be tolerated, while 10.01 pA would be preferable. Many commercial p H meters do not achieve this ideal, and amplifier currents of 1-10 pA are not uncommon. For rapid response measurements, it is improbable that an accuracy of better than i1 mV would be achieved and many membranes have resistances lower than 106 by one or more orders of magnitude. An amplifier input current of 1-10 pA is therefore considered acceptable. The differential amplifier circuit described previously (7), suitably modified to provide fast response, offers several advantages for rapid electrode potential measurement. The electrical noise which inevitably is found in high impedance membrane cells tends to be eliminated by a differential amplifier. Also, the current through the cell is the difference between the currents at each input, and by careful selection of input amplifiers, the net current is less than that at a single input amplifier. This note describes a fast response differential amolifier for use with ion-selective electrodes. (7) M. J. D. Brand and G . A. Rechnitz, ANAL. CHEM.,42, 616 (1970).
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Amplifier Circuit. The amplifier circuit, shown in Figure 1, is basically similar to the differential amplifier described previously (7). I n order to achieve fast response, the input stage consists of two 143 A F E T amplifiers (Analog Devices, Cambridge, Mass.) specially selected to have bias currents of less than 5 PA. The overall circuit response time is improved and ringing eliminated by including a small trimming capacitor in the negative feedback loop of the differential amplifier stage. To allow small potential changes t o be observed at high sensitivity-e.g., on an oscilloscope-an offset of up to 100 mV is summed with the output of the differential stage. Greater offsets can be obtained by reducing the value of the 500 KO input resistor t o the output amplifier. The offset is derived from a high stability f 5 V supply, a 805-V5 voltage regulator (Beckman Instruments, Fullerton, Calif.) driven by the amplifier power supply. Amplifier Response. The gain-frequency response of the circuit, Figure 2, shows the bandwidth to be greater than 100 KHz. The common mode rejection ratio, adjusted to better than 10j:l at dc shows a decrease with increasing frequency at approximately 20 db/decade. The response of the amplifier t o a step input, Figure 3, shows the response time t o be
about 2 psec for a small signal. This is approximately 1000 times faster than for the previously described unit (7). Measurement of the amplifier input current is difficult because it is so small but the following method gave an estimate. An ac oscillator with a n output of 10 mV was connected in series with a screened lo9 O resistor across the amplifier input. The amplifier output was then measured o n a n oscilloscope as a function of frequency. Difference between the input and output voltage was equated t o a n IR drop in the lo9O resistor, thus allowing the input current t o be calculated. Figure 4 shows that a t low frequencies, the current is about 1 pA while at high frequencies it is about 10 PA. The properties of this amplifier should allow the experimental study of membrane electrode potential relaxation processes. I n addition, interesting possibilities exist for the use of membrane electrodes to follow fast reactions in solution by stopped-flow techniques. RECEIVED for review June 26, 1970. Accepted August 31, 1970. This work was supported by a grant from the National Science Foundation.
Gas-Liquid Chromatographic Determination of Amino-Glycoside Antibiotics: Kanamycin and Paromomycin Kiyoshi Tsuji and John H. Robertson Conlrol Analytical Research and Decelopment, The Upjohn Company, Kalamazoo, Mich. 49001
KANAMYCIN ( I ) , paromomycin ( 2 ) , and neomycin ( 3 ) are water soluble, basic antibiotics which are markedly similar in chemical, physical, and biological properties. Commercial preparations of these amino glycoside antibiotics contain various isomers (Figures 1 , 2 , 3). They may be differentiated by paper, thin layer, and column chromatography ( 4 , 5 ) and quantitated by microbiological assay methods (6). The gas chromatographic assay method for neomycin (7) is adapted in this paper for the separation and determination of kanamycin and paromomycin and their isomers. EXPERIMENTAL
Apparatus. F & M Model 400 with flame ionization detector was used. Gas flow rates: hydrogen 40 ml/min, air 600 ml/min, and carrier gas (helium) 70 ml/min. Chart speed 0.25 inch/min. Oven temperature: 290°C for paromomycin and 300 “C for kanamycin. (1) T. Takeuchi, T. Hikiji, K. Nitta, S. Yamazaki, S. Abe, H. Takayama, and H. Umezawa, J . Antibiot. Ser A, 10, 107 (1957). (2) J. W. Davisson, I. A. Solomons, and T. M. Lees, Antibiot. Chemother. ( Washiitgton, D.C.) 2,460 (1952). (3) E. A. Swart, D. Hutchison, and S. A. Waksman, Arch. Biochem., 24, 92 (1949). (4) R. T. Schillings and C. P. Schaffner, “Antimicrobial Agents
and Chemotherapy,” Braun-Brumfield, Inc., Ann Arbor, Mich. 1961, p 274. ( 5 ) E. Roets and H. Vanderhaeghe, Pharm. Tijdschr. Belg., 44, 57 (1967). (6) Code of Federal Regulations, Title 21-Food and Drugs, U. S. Government Printing Office, Washington, D. C., 1968. (7) K. Tsuji and J. H. Robertson, ANAL.CHEM., 41, 1332 (1969).
Column. Glass column, 3 mm X 1830 mm (6 ft) packed with (1) 0 . 7 5 x OV-1 o n Gas Chrom Q, 100/120 mesh (Applied Science Laboratories, Inc., State College, Pa.) for paromomycin and (2) 3 z OV-1 o n Gas Chrom Q, 100/120 mesh (Applied Science Laboratories) for kanamycin were used. The columns were non-flow conditioned at 330 “C for 1 hour, followed by a n injection of approximately 50 p1 of Silyl-8 (Pierce Chemical Co., Rockford, Ill.) with flow at 200 and 250 “C. Silylated samples of the antibiotics were injected a t 300 “C. The columns thus prepared had 210 and 250 theoretical plates per foot for silylated paromomycin I and kanamycin A, respectively. Internal Standard-Silylation Reagent. For paromomycin determinations, a solution containing 3 mg of trilaurin (Supelco, Inc., Bellefonte, Pa.) and 70 p1 N-trimethylsilyldiethylamine (Pierce Chemical) per ml of Tri-Si1 Z (Pierce Chemical) was prepared. F o r kanamycin determinations, a solution containing 8 mg of trilaurin and 25 pl of N-trimethylsilyldiethylamine per ml of Tri-Si1 Z was prepared. Reference Standard. A water solution containing 10.0 mg of paromomycin sulfate or kanamycin sulfate reference standard per milliliter was prepared. One-milliliter aliquots were pipetted in duplicate into 2-cm3 serum vials (No. 621133174, Kimble Glass, Div. of Owens-Illinois, Toledo, Ohio) and freeze dried. The vials were capped using red rubber closures (No. V-35, West Company, Phoenixville, Pa.) and aluminum retainers (No. 13-FA-003, West Co.). Sample. Samples were prepared in the same way as the reference standard. Silylation Procedures. One milliliter of the internal standard-silylation reagent was added to the sample and the reference standard vials using a tuberculin syringe. The
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