Anal. Chem. 1985, 57, 1009-1012
Table 11. Determination of Penicillins in Pharmaceutical Samplesc type of sample (manufacturer) tablets (E. R. Squibb & Sons, Inc., Princeton, NJ) injectables (Eli Lilly Co., Indianapolis, IN)
determined value 2.51 X
2.48 x 2.49 x 2.19 X 2.18 x 2.15 x fermentation broth (Eli Lilly 12.3 Co., Indianapolis, IN)
*
manufacturer's value
lo6 U 105 105
250000 U
lo5 U
105 200000 u 105 0.1 mg/mL 12.58 mg/mL" 12.19 mg/mLb
"Determined by a wet chemical method. *Determined by an HPLC method. Each result is the average of triplicate runs (1U = 0.60 pg of penicillin G ) . reference electrode reservoir is responsible for the noise.
Determination of Penicillins in Pharmaceutical Samples. The instrumental setup of Figure 1and the optimum conditions discussed through the text were used to prepare calibration curves for penicillin G and pencillin V (both from Sigma) and determine them in pharmaceutical samples. M (correlation Calibrations curves were linear up to 5.0 X coefficients 0.999). Penicillin G was determined in tablets and injectables (Table 11) and penicillin V in fermentation broths. Even though dilutions of 4000 times (for tablets), 1000 times (for injectables), and 100 times (for fermentation broths) were performed, reproducibility was on the order of 1% in the final result for at least triplicate independent determinations. Because of the presence of additives and buffering species in the pharmaceutical products, the pH of some samples was different from that of the carrier. Therefore, a blank was run by injecting these samples into a blank single bead string reactor (without immobilized enzyme) similar to the enzyme reactor in dimensions. Because of the sample dilution with buffer, the p H and buffer capacity of sample and carrier solution are reasonably close that the effect on the rate of the enzyme catalyzed reaction is negligible. The difference in
1009
signal height between sample and blank was used for interpolation in the working curve obtaining the results summarized in Table 11.
ACKNOWLEDGMENT The authors express their appreciation to K. S. Troxel and the Eli Lily Co. (Indianapolis, IN) for samples of penicillin broth and corresponding analytical results. Registry No. Penicillinase, 9001-74-5; penicillin G, 61-33-6; penicillin V, 87-08-1; (p-aminophenyl)trimethoxysilane,3397643-1; (3-aminopropyl)triethoxysilane, 919-30-2; [N-(2-aminoethyl)-3-aminopropyl]trimethoxysilane, 1760-24-3; p-[N-(2aminoethyl)aminomethyl]phenethyltrimethoxysilane,75822-22-9; penicillin, 1406-05-9.
LITERATURE CITED Mottola, H. A. Anal. Chim. Acta 1983, 745, 27-39. Hamilton Mlller, J. M. T.; Smith; J. T.; Knox, R. J . Pharm. Pharrnacol. 1963. 75.81-91. Hughes, D. W.; Vilim, A.; Wilson, W. L. Can. J . Pharm. Sci. 1976, 7 7 , 97-108.
Fairbrother, J. E. Pharm. J . 1977,2 7 8 , 509-513. Nilsson, H.; Akerlund, A.; Mosbach, K. Blochim. Biophys. Acta 1973, 320, 529-534.
Enfors, S.Enzyme Microb. Techno/. 1979, 7 , 260-264. Cullen, L. F.; Rusllng, J. F.; Schleifer, A.; Papariello. G. J. Anal. Chem. 1974,46, 1955-1961. Rusling, J. F.; Lutrell, G. H.; Cullen, L. F.; Papariello, G. J. Anal. Chem. 1976,b8,1211-1215. Mattiasson, B. f€BS Left. 1977, 7 7 , 107-110. Marshall, M. A.; Mottola, H. A. Anal. Chem. 1985,5 7 , 729-733. Iob, A.; Mottola, H. A. Clin. Chem. (Winston-Salem, N.C.)1981,2 7 ,
195. Roy, A. B. Anal. Biochem. 1981, 776, 123-128. Ruzicka, J.; Hansen, E. H.; Ghose, A. K.; Mottola, H. A. Anal. Chem. 1979,51, 199-203. Hou, J. P.; Poole, J. W. J . Pharm. Sci. 1972,67,1594. Mottola, H. A.; Wolff, Ch-M.; Iob, A,; Gnanasekaran, R. In "Modern Trends in Analytlcal Chemistry"; Pungor, E., Buzas, I., Eds.; Akademia Kiado: Budapest, 1984; Part A, Electrochemical Detection In Flow Analysis, pp 49-75. Van den Winkel, P.; Mertens, J.; Massart, D. L. Anal. Chem. 1974, 4 6 , 1765-1768.
RECEIVED for review November 14,1984. Accepted January 23,1985. This work was supported by the National Science Foundation (Grant No. CHE-8312494).
Application of a Nested Loop System for the Flow Injection Analysis of Trace Aqueous Peroxides Purnendu K. Dasgupta* and Hoon Hwang
Department of Chemistry, Texas Tech University, Lubbock, Texas 79409-4260
The novel concept of a nested loop valve system wherein one Injection valve Is inserted within the loop of another has some unique benefits. This is demonstrated for part-per-billion level differential flow Injection analysls of aqueous H,O, and organlc peroxides.
The multiport loop type injection valve is currently an essential component of both high- and low-pressure continuous flow analytical systems including HPLC and FIA. A recent review by Harvey and Stearns (1) has explored in depth the manifold applications of combination of multiport valves to accomplish unique types of flow stream switching. We wish to report here a novel configuration of a six-port injection valve installed within the loop of another six-port
injection valve. Further, the unique capabilities of such a system to produce analyte differentiated dual signals is demonstrated for parts-per-billion level analysis. In searching for an appropriate designation, we have borrowed the term nested loop from the terminology of computational algorithms. Not only is this nomenclature accurate in describing the operating configuration but hopefully it also points out that although the simplest nested loop (n = 2) is described here, the degree of nesting can be much higher in principle.
PRINCIPLES In this paper, we focus our attention solely to potential situations where the loop of the nested (inner) valve contains an immobilized packed reactor with differentiating action on the analyte components. Other applications such as creating merged zones in FIA (2) are possible.
0003-2700/85/0357-1009$01.50/00 1985 American Chemical Society
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a La ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
most important atmospheric organic peroxide, CH3H02,is essentially unaffected within the reaction time in a given system (4).While it is not particularly difficult to immobilize catalase on a suitable carrier (6),we chose to investigate the applicability of MnOz or a finely divided metal (Pt) which is known (7) to destroy HzOz. MnOz and Pt black were found to destroy HzOzvery fast, even a t trace levels, and both exhibited preferential destruction of HzOzover CHBHOz(and even more so over other organic peroxides).
L2
v2
v2
EXPERIMENTAL SECTION
t
t 1
PUMP IAl
PUMP 161
Flgure 1. A nested loop injection system. R is a reactor packed with MnO,. (A) Both outer valve ( V l ) and nested valve (V2) are In load mode. (B) Both V 1 and V2 are in inject mode.
Consider the flow scheme in Figure l a where the outer valve (heretofore designated as V1) is in sample loading mode and so is the nested valve (V2). As the sample is loaded, i t bypasses the loop of V2 and is loaded into the two halves of the loop of V1, L1A and L1B. At this time, the loop of V2, consisting of L2A, L2B, and a packed reactor R, is filled with the carrier liquid (from the previous operating cycle). When both valves are switched to the inject mode (this can be done simultaneously for electromethanically or electropneumatically actuated valves; if the valves are manually operated, V2 is switched first, followed by Vl) as shown in Figure l B , the contents of L1A pass through the reactor R while the contents of L1B proceeds directly to the system. L2A and L2B are merely tubing lengths employed to isolate the contents of L1A and L1B. Also, it is possible to use a single ten-port valve (1) instead of the two six-port valves. Let us consider that the injected sample contains two components of interest, A and B, both of which produces a detector response, perhaps after appropriate chemistry. The contents of the reactor is such that it retains or destroys B, without any effect on A. The resulting detector output is two separate sequential signals, the first representing A B (from LlB) and the second representing A only (from LlA). Because the content of L1A undergoes more dispersion, it needs to be proportionately longer than L1B if peaks of equal heights are desired for L1B and L1A for a sample containing only A. It is not necessary that B is completely destroyed by the reactor, as long as a reproducible constant fraction is destroyed each time.
+
ANALYTICAL SYSTEM We demonstrate the use of the nested loop injection system for a hydrogen peroxide/organic peroxide differentiating assay procedure, of particular interest to our research program. A highly sensitive method of assay of aqueous peroxides is the dimerization of nonfluorescent (p-hydroxypheny1)acetic acid to form a fluorescent product via oxidative hydrogen abstraction, mediated by the enzyme p. roxidase. The method, originally formulated by Guilbault et al. (3), has been developed further and adapted for automated segmented continuous flow analysis by Lazrus et al. (4)as well as for nonsegmented FIA by us (5). It is well-known that many substances destroy aqueous peroxides. Lazrus et al. (4)have taken advantage of enzymatic destruction of peroxides, using the enzyme catalase. Catalase will destroy both HzOzand organic peroxides but the latter react a t a significantly slower rate. Thus, it is possible to employ an optimized enzyme concentration which virtually completely destroys HzOz while the
Reagents. (p-Hydroxypheny1)aceticacid (Eastman Kodak) was twice recrystallized from hot water and decolorized by activated carbon treatment. Peroxidase (type 11, Sigma Chemical) exhibited an activity of -150 sigma units/mg. Platinum black, tert-butyl hydroperoxide, benzoyl peroxide (all from Aldrich Chemical),manganese dioxide (analyticalreagent grade granules, Mallinckrodt),hydrogen peroxide (30%,without added stabilizer, Fisher Scientific), and peracetic acid (35%, FMC Corp.) were obtained as indicated. Methyl hydroperoxide was synthesized as described (8). The purchased peracetic acid contained large amounts of HzOP A dilute stock solution was treated with platinum black and stirred for several minutes before filtering off the reusable catalyst. (Manganese dioxide is not suitable for this purpose because at large peracetic acid concentrations, it is immediately solubilized to form permanganate. In fact, this conversion appears to be more facile than classical methods employing bismuthate or Ag+/persulfate.) Methyl hydroperoxide as synthesized also contained a small amount of HzOz,which was similarly removed by MnOz treatment. The peroxide solutions were then standardized and diluted to desired levels by serial dilution immediately before use. All peroxide standards are relatively unstable at sub-part-per-million-levelconcentrations. Further, peracetic acid standards exhibited a pronounced tendency to slowly produce HzOz, easily noticed by the present method (vide infra). Iodometric methods were used to standardize HzOz(9) and all the other peroxides (IO). Somewhat more involved procedures are necessary for the organic peroxides to accelerate the otherwise slow rate of iodine liberation from iodide. Reagent water was freed from peroxide by adding 10 mL of a catalase (from bovine liver, Sigma Chemical) solution (0.5 mg in 100 mL of 0.5 M phosphate buffer, pH 7) per liter of distilled deionized water, allowing the catalase to remain for 1h, and then boiliig the mixture for 2 h to denature the protein, shortly before use. All reagents and standards were prepared in this water and direct exposure to sunlight was avoided. The fluorescence derivatization reagent was prepared by dissolving 1 g of purified (p-hydroxypheny1)aceticacid and 0.156 g of NazEDTA in 250 mL of 0.1 M NH3 followed by adjusting the pH to 9.5 with HC1. Five milligrams of peroxidase was added to the reagent immediately before use. The solution is usable for 24-48 h when stored at ambient temperature; the enzymatic activity decreases slowly, presumably due to degradation of the protein. Equipment. A ratio recording spectrofluorometer (NOVA, Baird Instrument Corp., Bedford, MA) equipped with a 3O-pL flow cell was used as the detector. The optimal excitation and emission wavelengths were determined to be 329 nm and 412 nm, respectively (5),and used with 10-nmslit widths. A 1s integration time was used for the recorder output. A multichannel Gilson Minipuls 2 peristaltic pump with a slow speed drive module was used at a pump setting of 550 to pump both the carrier stream (water, 3.16 mm i.d. PVC tubing, flow rate 1.33 mL/min) and the reagent (1.23 mm i.d. PVC tubing, flow rate 0.28 mL/min). Following the mixing of the reagent with the carrier stream at a mixing tee, the flow was directed into the detector via a knotted delay line (115 cm long 0.8 mm i.d. TFE tube, containing a total of 33 knots). The knotted arrangement provides for good mixing, further details are given in ref 5. The knotted line produces about 22 s delay, and the total delay from the time of injection to the apex of the first peak (LlB) is -30 s. The complete experimental arrangement is shown schematically in Figure 2. Manually actuated Rheodyne (type 50,O.a mni bore) and/or electromechanically actuated Hamilton six-port rotary valves were
NESTED VALVE i 3 3 m ~ m n
*ixlNG
20
YI
DELAY
DETECTOR
KNOTED DELAY COIL
WATER
PUMP CHANNEL 2
TO DRAIN
0 28 "Llrnl"
I . I . I . I I . l . l L I . I . l . l . L . I . I . I . l O b 8 1 2
0 4 B I 2 0 4 8 1 2
0 4 8 1 2
O b l U
0 4 8 1 2 1 6
TIME (min)
Flgure 3. System response to duplicate injections of samples containing H202 and/or CH3H0,. Each sample produces two peaks. The first (left)peak is due to H,Oz 4- CH3H02 and the second peak is due to CH,H02. The first group (A) contains H202 only and has no second peak. Samples are from left to right: (A) 191 ppb H,Oz, (6) 211 ppb CH3H0,, (C) 1:l A B , (D) 1:5 A:B, (E) 1:lO A:B, and (F) 1O:l A B . For each group, the first injection is at 0 mln and second injection is at 6 min.
between the mixing point and the detector. The other peroxides also undergo the analytical reaction; compared to H202, the reaction is perceptibly slower for CH3HO2 and slower still for the other peroxides. (A kinetic study of the rate of fluorescence development is a feasible, but not particularly attractive, way of differentiating between H20z and organic peroxides.) Under the present experimental conditions, the fluorescence development due to H202 is essentially complete. While nearly comparable responses for H202 and CH3HOzmay be obtained by increasing the reaction time, peracetic acid and tert-butyl hydroperoxide, respectively, produce only 59% and 0.22% of the H202response on a molar basis. Increasing the delay time to assure equal response in the latter case is clearly impractical. Sample group C in Figure 3 shows the results of the duplicate injection of a sample containing 95.5 ppb HzOz and 105.6 ppb CH3H02(equal volumes of samples A and B). In the basis of the data for A and B, the calculated values for the first peak (H2O2 + CH3H02) and the second peak (CH3H02alone) are within 2% of the observed values. Similarly, group D shows the duplicate injection of a sample containing 31.8 ppb HzOZand 176 ppb CH3H02 ( 1 5 A:B); group E shows a 17.4 ppb H202and 192 ppb CH3H02sample (1:lO A:B), and group F shows a 174 ppb H 2 0 and 19.2 ppb CH3H02 (1O:l A:B) sample. In all cases, the concordance between calculated and observed values is excellent. As the reproducibility of the duplicate injections indicate, the precision is also excellent. The relative standard deviations for each peak (n = 5 ) are below 1%. Further these data demonstrate, beyond the successful performance of the nested loop system and the reactor, the linearity of response of the analytical system for both Hz02and CHBH02which have been reported previously (5). Similar results are produced by combinations of HzOzwith other organic peroxides. Benzoyl peroxide was found to produce low and erratic responses; the solubility of this compound in water is too low to work with it conveniently. The nested loop concept is obviously applicable to FIA systems wherever suitable reactor chemistry can be incorporated. By use of a concentrator column in the nested loop,
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Anal. Chem. 1985, 57, 1012-1016
it can be used for preconcentration purposes; normal loop use is restored by keeping the nested valve in inject position. A large number of chemical analysis systems may benefit from the nested loop concept. Examples include the following: the differentiation of N02-/N03- with a cadmium reductor as a packed column (11) via the Griess reaction (12, 13);Cr3+/ Cr0:- with a packed column containing an ion exchange resin via atomic spectrometric detection; Fe2+/Fe3+with a packed column containing an anion exchange resin in Mn04- form and determining the Fe3+ by reaction with SCN- (14); NaOH/NaCl with a packed column containing a H+ form cation exchanger via conductivity detection (15).
Lazrus, A. L.; Kok, G. L.; Gitlln, S. N.; Lind, J. A,; McLaren, S. Anal. Chem. 1985, 5 7 , 917-922. Hwang, H.; Dasgupta, P. K. Anal. Chim. Acta, in press. Zaborsky, 0. R. "Immobillzed Enzymes"; CRC Press: Cleveland, OH, 1973. Partington, J. R. "A Text Book of Inorganic Chemistry", 6th ed.; MacMillan: London, 1963; pp 195-196. Rieche, A.; Hitz, F . Ber. Dfsch. Chem. Ges. 1920, 62, 2458-2474. Vogel, A. I."A Textbook for Quantitative Inorganic Analysis", 3rd ed.; Longmans Green, London, 1961; p 363. Wlbaut, J. P.; Van Leeuwen, H. B.; Van der Wal, 6.R e d . Trav. Chlm Pays-Bas 1954, 7 3 , 1033-1036. Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok, J. S.; Tannenbaum, S.R. Anal. Blochem. 1982, 126, 131-138. Sawicki, E.; Stanley, T. W.; Pfaff, J.; D'Amico, A. Talanfa 1983, 70, 64 1-655. Dasgupta, P. K. Anal. Letf. 1984, 17 (AIO), 1005-1008. Kolthoff, I. M.; Sandell, E. 6.;Meehan, E. J.; Bruckenstein, S. Quantitative Chemical Analysis", 4th ed.; MacMiilan: London, 1969; pp 1049-1052. Stevens, T. S.; Miller, T. E., Jr. Anal. Chem. 1980, 52, 2023-2025. US. Patent 4 199323, April 30, 1980.
ACKNOWLEDGMENT The authors deeply appreciate the help of V. K. Gupta toward the synthesis of methyl hydroperoxide. Registry No. HzOz, 7722-84-1; MnOz, 1313-13-9;CH,HOZ, 3031-73-0; (p-hydroxypheny1)aceticacid, 156-38-7.
LITERATURE CITED Harvey, M. C.; Stearns, S. D. I n "Liquid Chromatography in Envlronmental Analysis"; Humana: Clifton, NJ, 1984: pp 301-340. Ruzlcka, J.; Hansen, E. H. Anal. Chlm. Acta 1983, 745, 1-15. Gulbault, G. G.; Brignac, P.; Juneau, M. Anal. Chem. 1968, 4 0 , 1256- 1263.
Received for review December 12, 1984. Accepted January 28,1985. This research has been supported by the U.S. Environmental Protection Agency through Grant No. R810894-010. However, this report has not been subjected to Agency review and does not necessarily reflect the views of the Agency and no official endorsement should be inferred.
Liquid Core Optical Fiber Total Reflection Cell as a Colorimetric Detector for Flow Injection Analysis Kitao Fujiwara* and Keiichiro Fuwa
Department of Chemistry, University of Tokyo, Tokyo 113, Japan
A hollow fiber (250 pm 1.d.) was used as a colorimetric cell for detecting iodlne absorption. To attain total reflection of source light inslde the capillary, carbon disulfide was used as a solvent which constltutes the fiber core. A funnel-shaped glass was used for efflclently condensing the light source emission into an aperture of hollow flber; a low-power tungsten lamp was usable as the light source. With a 5-m cell, 0.1 pg of I/mL (10 ng of I ) can be detected based on the iodlne absorptlon at 540 nm when the solution was injected into the carbon dlsulfide flow. An automated detectlon system of iodide ion was also constructed.
To measure the small absorption of a sample, several techniques have been recently developed including thermal lensing colorimetry, optoacoustic spectrometry, multiphoton photoionization spectroscopy, etc. These spectroscopic techniques are based on the use of lasers; i.e., high-power lasers with precise optical devices are required to attain high sensitivity in these methods, which causes increased cost and labor for the instrumentation. In previous papers (I, 2 ) , we discussed the long capillary cell (LCC) as the sample reservoir in absorption spectrometry. According to Lambert-Beer's law, the sensitivity of absorption spectrometry is dominated by the light path length of a sample cell. A highly sensitive measurement for extremely low absorption was possible by extending the cell length with limiting
necessary volume of sample when capillaries of less than 1mm i.d. were employed. When the refractive index of the solvent exceeds that of the capillary material, a highly light-transmissible cell can be constructed (2). According to Snell's law, source light is propagated through the cell via successive total reflections at the boundary between the solvent and cell wall. In this case, the shape of cell is independent of transmission efficiency of source light. Furthermore, the absorption is enhanced up to 6-8 times with the cell length because of the elongation of light path length due to the multiple reflections inside the capillary, which applied also to the mirror surface cell discussed by Dasgupta (3). When a hollow fiber was used as the LCC, the sample solution (which possesses high refractive index) constitutes the core of optical fiber. As the result, a >lo4 absorption enhancement has been attained by the use of optical fiber type LCC of 50 m length, in which necessary sample volume was only a few milliliters. In spite of these advantages in the use of a total reflection cell, some difficulties still remain in the practical operation, i.e., hard base line stabilization during the change of sample solutions and low efficiency of source light introduction into the cell. In the previous paper, a high-power xenon lamp was required as the source light ( 2 ) . In the present paper, to overcome these drawbacks, the liquid core optical fiber type total reflection cell will be applied for the detector of flow injection analysis (4, 5 ) . Also, the condensatiopn method of the source light to the aperature of
0003-2700/85/0357-1012$01.50/00 I985 American Chemical Society
