0.05 cm sec-1 (*2a) from these data. This result is in excellent agreement with the measurements quoted in the previous paragraph, particularly those of Rohko and coworkers. The agreement is particularly impressive, considering that the time scales of the earlier measurements were approximately three orders-of-magnitude smaller than those of Figure 1. It is also worth noting that the heterogeneous charge transfer kinetic contribution to the data in Figure 1 ranges from 2% to only about 10% of the diffusional contribution. Calculation of the k,-value from the phase angle cotangent response a t the admittance peak does not require knowledge of the a-value, as indicated by Equation 8. No attempt was made to quantitatively ascertain the magnitude of a from fundamental harmonic data ( e . g . , from the cot #J - Edc profile), although it was evident that a was significantly larger than 0.5. Although use of the cot #J E& profile is usually a convenient and satisfactory means for calculating a , the large k , and a-values characterizing the iron oxalate couple lead to a rather shallow, asymmetric profile (6). As a consequence, normally useful features of the cot #J - Edc profile, such as its peak potential, represent inaccurate bases for calculating 01 in this case, even with the excellent data precision provided by the Fourier transform approach. The iron oxalate couple a-value is much more intelligently calculated from observations of nonlinear faradaic response components which can be obtained simultaneously ( I , 5, 15). In our laboratory, even low-frequency second harmonic faradaic current measurements provided a convenient and reasonably accurate means of ascertaining CY for the iron oxalate couple, yield0.06) ( 1 5 ) which was in excellent ing a result ( a = 0.80 agreement with the published values (9, 13). Returning to the subject of k , measurement, if one more or less assumes that the smallest acceptable cot #J slope must have a magnitude exceeding the noise level by four standard deviations or more, then the smallest measureable slope would be set a t 8 X unit for the conditions extant in obtaining the data of Figure 1. This would correspond to a k , of approximately 20 cm sec-1 for D O and DK values in the neighborhood of 0.5 x cm2 sec-1, which is quite encouraging. Further, the 100-pass ensemble average used to generate the data of Figure 1 did not require an inordinately long data acquisition period. Consequently, it is not impractical to consider the possibility
of higher measurement precision and a correspondingly higher rate constant upper limit by simply averaging more data ensembles. Whether this would yield the statistically-expected improvement is open to question, as we have not attempted handling larger numbers of measurement replicates. Further, there is a suggestion of a systematic error contribution to the data scatter in Figure 1 in the fact that the low and high frequency points tend to lie above the least squares line, while some intermediate points fall below it. However, even the worst deviation (lowest frequency point) is only 0.005 cot unit, or 0.15", from the least squares value. Consequently, these error magnitudes are a t a level where we totally lack experience in their interpretation and only speculation can be given a t this point. It is possible that the slight upward curvature a t high frequencies might be due to uncompensated ohmic resistance (6, 7) in the fractional ohm range ( a distinct upward curvature is noted when 2 ohms are deliberately left uncompensated). The low frequency positive deviations might arise from leakage effects ( 4 , 26) due to small sidebands generated by mercury drop growth during the 0.1-second waveform acquisition period ( 1 ) . Other possibilities are small effects of faradaic nonlinearity ( 4 ) , and cell-potentiostat network imperfections. This speculation regarding the possibility of small systematic error contributions should serve as a warning that efforts to reveal small kinetic contributions of interest by enhancing data precision might, in addition (or instead), turn up measurement artifacts which normally are too small to be of consequence a t normal precision levels. Such words of caution notwithstanding, we feel that the example presented in Figure 1 provides a compelling demonstration that high levels of data precision provided by Fourier transform faradaic admittance measurements can be converted into a substantially greater sensitivity to kinetic manifestations of interest. When the enhanced precision capability is used to complement the more common strategy of employing high frequencies, upper limits of accessible rate constants might be advanced considerably, relative to presently accepted levels.
(15) D E Glover, Doctoral Dissertation, Northwestern University, Evanston. Ill , 1973
(16) R K Otnes and L Enochson. Digital Time Series Analysis Wiley-lnterscience New York N Y 1972
*
#J
Received for review April 25, 1973. Accepted July 5 , 1973. This work was supported by National Science Foundation Grant GP GP-28748X. S.C.C. was an NSF Trainee 197172 and an NDEA Fellow, 1970-71.
Quantitative Determination of Theophylline in Blood by Differential Spectrophotometry Ramesh C. Gupta and George D. Lundberg Section of Laboratories and Pathology, Room 2900, LAC-USC Medical Center, 1200 N. State Street, Los Angeles, Calif. 90033
Theophylline is used in treatment of conditions such as hypertension, asthma, and cardiac and nephrotic edema. An overdose of theophylline could be toxic and has occasionally proved fatal ( 2 3 ) . Toxic effects are characterized (1) B. H. White and C. W. Daeschner, J. Pediat., 49, 262 (1956) (2) V. J. Rounds, J. Pediat., 14, 528 (1954). (3) G. A . Merril, J. Amer. Med. Ass., 123,1115 (1943).
by frequent vomiting and unusual thirst, and in severe cases are followed by convulsions, shock, and death. The drug is administered alone, together with a barbiturate, or in combination with a barbiturate and ephedrine or papaverine. Although methods for quantitative determination of theophylline in tablets and capsules have been published
A N A L Y T I C A L CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
2403
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Wavelength (mu) Figure 1. Differential absorption curve of solution containing pg/ml theophylline in 0 . 2 N sodium hydroxide
40
Table I. Recovery of Theophylline after Addition to Blood Theophylline Added to blood, pg/mi
Detected in blood, p g / m l
20.0
18.0 26.1 36.8 44.0 54.4 65.2 68.5 40.9
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Recovery, Yoa 90.0 87.0 92.0 88.6 90.6 93.2 85.6 9 1 .o
-4.1 Average recovery, 89.7%. Percent standard deviation from the mean Dercent recovery, f6.12%.
( 4 , 5 ) , an accurate procedure for determining concentration of this drug in blood is lacking. This article describes a spectrophotometric method for detecting quantities of theophylline in blood when present alone or in combination with barbiturates, ephedrine, or papaverine. A distinguishing feature of this method is that the solution of the extracted theophylline is placed in both sample and reference cuvettes of a spectrophotometer a t two different pH’s, and differential absorption is recorded as shown in Figure 1. The UV absorption that may be caused by impurities extracted from blood is thus eliminated, and a n absorption maximum produced by theophylline a t 285 nm is obtained.
EXPERIMENTAL Materials a n d Reagents. Ratio recording UV spectrophotometer; chloroform, spectral grade; 0.2N sodium hydroxide; 4 . 4 N sodium hydroxide: 4N hydrochloric acid; and sodium dihydrogen phosphate solution 30 gm/100 ml were used. Extraction Procedure 1. This procedure was used for extraction of theophylline when barbiturate was not present in blood. Five milliliters of blood were extracted with 60 ml of chloroform in a separatory funnel. The chloroform extract was filtered into a (4) F. Yokoyama and M. Pernarowski, J. Pharm. Sci.. 50, 953 (1961). (5) F. H . Preston, E. C. Frank, and D. J. Campbell, J. Amer. Pharm. Ass., XLVI, 644 (1957). 2404
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Figure 2. Standard curve of theophylline extracted from blood. Varying amounts, 1 0 , 20, 30, 50, and 60 pg/ml theophylline were added to blood and carried through the procedure
second separatory funnel containing 10 ml of 0.2N sodium hydroxide, and shaken for 5 minutes. The alkaline layer was separated and centrifuged. Extraction Procedure 2. This procedure was used for extraction of theophylline when a barbiturate was present in blood. Five milliliters of blood were extracted with 60 ml of chloroform in a separatory funnel. The chloroform extract was filtered into a second separatory funnel containing 5 ml of 4.ON hydrochloric acid, and shaken for 5 minutes. The acid layer was separated and centrifuged, and 4 ml of this acid extract were mixed with 4 ml of 4 . 4 N sodium hydroxide. Spectrophotometry. Three ml of the alkaline extract obtained from Extraction Procedure 1 or 2 were placed in both sample and reference cuvettes. One-half milliliter of sodium dihydrogen phosphate solution was added to the reference cuvette, and 0.5 ml of 0.2N sodium hydroxide to the sample cuvette. UV absorption was recorded between 340 to 220 n m . Absorbance a t 285 n m was measured and the quantity of theophylline present in blood sample was estimated from a standard graph, Figure 2, prepared from known amounts of theophylline added to theophylline-free blood and carried through the same procedure.
RESULTS AND DISCUSSION Known amounts of theophylline were added to barbiturate-free and barbiturate-containing blood samples and extracted according to Extraction Procedures 1 and 2, respectively. UV absorptions were directly proportional to concentration over a range of 0.0 to 100 Fg/ml. For plotting the standard graph, each blood sample spiked with a known quantity of theophylline was carried through the procedure four times, and mean value was used for each point on the graph. The absorbance obtained in four trials on each sample did not vary more than 2.1% from the mean. Recovery of theophylline from blood using either extraction procedure was 85 to 9370,with a n average recovery of 89.7% (Table I). With appropriate reduction in volume of the final alkaline extract, the method is capable of detecting less than 2 Fg/ml of theophylline in blood. Normal theophylline concentrations in blood after administration of therapeutic amounts are in a range of 2-4 pg/ml ( I , 6, 7 ) . In overdose cases, blood theophylline concentrations may be 20 pg/ml (6) J . Schluger, M . T. Joseph, and H. J. Douglas, Amer. J. Med. Sci., 233, 296 (1957). (7) E. B. Truitt, V. A. McKussick, and J. C. Krantz, J. Pharmacol. Exp. Ther., 100, 309 (1950).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 14, DECEMBER 1973
or more, and these can be determined accurately by the described procedure. Absorptions due to natural impurities extracted from blood are eliminated by this method. Ephedrine and papaverine, frequently present with theophylline, do not interfere because they show no differential absorption. Barbiturates, which are likely to interfere if present, are completely separated from theophylline using Extraction Procedure 2. Theophylline gives a maximum a t 272 nm when scanned against sodium hydroxide as reference, but it shows maxima a t 285 and 240 nm by differential spectrophotometry. The absorption maximum a t 285 nm was used for estimating the amount of theophylline because no
interference by impurities was observed in this region of the spectrum. Of the several common drugs examined, morphine alone interfered with this determination. If morphine and theophylline both are present in a sample, they must be separated by thin layer or column chromatography before quantitation for theophylline can be attempted by this method. However, since morphine disappears from blood so rapidly, only in a very rare instance may the two drugs be found together in a blood sample. Received for review March 29, 1973. Accepted June 4, 1973.
Atomic and Molecular Absorption Measurements by Intra-Cavity Quenching of Laser Fluorescence H. W. Latz, H. F. Wyles, and I?.B. Green Clippinger Graduate Research Laboratories, Department of Chemistry, Ohio University, Athens, Ohio 45701
The interest in laser emission from fluorescent organic compounds, commonly referred to as dye lasers, has been steadily increasing because of the capability of tuning the laser emission over all wavelengths of the normal fluorescence band. Physicists have emphasized the use of these devices as sources of monochromatic radiation for a variety of spectroscopic applications whereas our interest has been in the utilization of the stimulated emission as an analytical signal in itself. Attempts to obtain stimulated emission from naturally occurring coumarins of analytical interest by flashlamp pumping have been unsuccessful to date. This led to a study of solvent effects on the lasing properties of 7-diethylamino-4-methylcoumarin,a compound which has been lased by many workers using flashlamp pumping. The results observed are predictable from normal fluorescence measurements but the effects are amplified by the spectral narrowing achieved with laser fluorescence. These same studies produced some anomalous results which are attributed to a cavity defect and led to a promising analytical technique for measuring trace absorptions of light by atoms and molecules. The purposeful introduction of NO2 into the resonant cavity of our laser produced a well defined absorption spectrum of this gas. A search of the literature revealed that workers a t the National Bureau of Standards had previously observed this phenomenon and had reported on the intra-cavity absorption of a pulsed rhodamine 6G laser by sodium vapor ( I ) and by solutions of E u ( N 0 3 ) ~( 2 ) . In each case, the wavelength absorbed from the broad-band laser emission corresponded to characteristic absorption wavelengths of the metals. More recently, selective absorption of a continuous wave rhodamine 6G laser by iodine vapor was reported by Hansch, Schawlow, and Toschek (3). The results of our studies with NO2 are reported here along with preliminary data on laser quenching due to cavity absorption by metal atoms in flames and by an organic compound in solution. N. C. Peterson, M . J. Kurylo, W. Brown, A . M . Bass, and R. A. Keller, J . Opt. SOC.Amer., 61, 746 (1971). R. A. Keller. E . F. Zalewski, and N. C. Peterson, J. Opt. SOC.Amer., 62, 319 (1972). T. W . Hansch, A . L. Schawlow, and P. E. Electron., QE-8, 802 (1972).
Toschek, I € € € J. Quantum
EXPERIMENTAL A Chromabeam 1050 Laser (Synergetics Research, Inc., Princeton, N.J.) was used for the solvent studies and for molecular absorption measurements. In the latter case, a 10- or 15-cm quartz absorption cell (Cary Instruments, Monrovia, Calif.) was positioned in the resonant cavity of the laser between the laser cell and t h e output mirror (Figure 1). The laser beam was directed into a spectrograph with a dispersion of 1 2 A/mm (Applied Research Laboratories, Inc., Glendale, Calif.). The spectra were recorded on Kodak Tri-X P a n film, one pulse a! the laser being sufficient, usually requiring a neutral density filter to diminish its intensity. Atomic absorption studies were conducted using a laboratory constructed laser with a co-axial flashlamp based on the design reported by Sorokin et d,( 4 ) . A laminar flow atomic absorption burner (Jarrell-Ash Division, Fisher Scientific Go., Pittsburgh, Pa.) modified to accept a three-slot burner head was positioned before the output mirror within the laser cavity. This provided a flame wider than the laser beam. A 0.5-meter Jarrell-Ash Monochromator was modified to accept a 35-mm camera body for recording of the laser output on Kodak Panatomic-X film. The monochromator has a dispersion of 16 A/mm.
RESULTS Solvent Effects. The typical broad-band laser output of rhodamine S in ethanol is shown in Figure 2. The tuning to lower wavelengths is achieved by lowering the concentration of the lasing compound and thereby reducing the self-absorption a t these lower wavelengths. Figure 3 shows the range over which 7-diethylamino-4-methylcoumarin (DEAMC) can be concentration-tuned as well as the significant shift in output wavelength that results from changing the solvent. The shift to longer wavelengths is attributed to increased hydrogen bonding between the solvent and the lasing compound. The total shift is the same as that observed with normal fluorescence but the incremental changes are more obvious due to the narrower bandwidth of the laser emission. This large shift can be achieved continuously by the addition of water to an ethanol solution of DEAMC as shown in Figure 4. The lasing in this case is totally quenched when the water content is approximately 40%. The same shift and subsequent quenching occur when dihydroxy solvents are added to (4) P. P. Sorokin, J. R. Lankard, V. L. Moruzzi, and E. C. Hammond, J. Chem. Phys., 48, 4726 (1968)
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