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Ion-Pairing Flow Injection Analysis of Phenobarbiturate with Piezoelectric Detection
Microchemical Journal 62, 251–258 (1999) Article ID mchj.1999.1695, available online at http://www.idealibrary.com on
Ion-Pairing Flow Injection Analysis of Phenobarbiturate with Piezoelectric Detection Zhihong Mo, 1 Xiaohui Long, Yifeng Xu, and Xudong Zhou College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, People’s Republic of China New developments of ion-pairing flow injection with piezoelectric detection have been made for the analysis of the anionic drug phenobarbiturate. A cationic surfactant, hexadecyl trimethyl ammonium bromide, was used, and its adsorption and interfacial ion-pair formation with phenobarbiturate on the gold surface of piezoelectric quartz crystal were investigated. Modifications of the analytical system have been developed to improve its sensitivity, using reference and working detectors with higher frequencies, buffered reagent and sample solutions, and a short connecting tube. Influences of surfactant concentration and operating parameters were demonstrated. Under optimization for the determination of phenobarbiturate, the calibration curve was linear in the range 0 –1 mg ml 21, with a detection limit of 0.44 mg ml 21, a recovery of 99.%, an RSD of 0.11% and a sampling frequency of 60 h 21. The method has been satisfactorily applied to the determination of phenobarbiturate in pharmaceutical formulations. © 1999 Academic Press
INTRODUCTION A number of ion-pairing methods for drug evaluation using automated flow injection solvent extraction have appeared since the initial work of Karlberg and Thelander on the determination of caffeine in acetylsalicylic acid preparations (1, 2). Previous methods have adapted detection systems of a variety of types, which included piezoelectric quartz microbalance as well as atomic and molecular spectroscopy, turbidimetric, and electrochemical techniques (3–9). However, the separation of the ion-pair from the carrier in solvent extraction required necessitates incorporation of a segmenter and consumption of organic solvent, so that the applicability of this method is greatly limited. Recently, a new method, ion-pairing flow injection with piezoelectric detection, not using liquid–liquid extraction or phase separation, has been developed and applied to determination of several pharmaceuticals (10 –11). This method, based on in situ mass sensing of the adsorption of surfactant on a piezoelectric crystal surface, detects ion-pair formation simultaneously with desorption of the surfactant on the crystal surface. In previous work, cationic drugs were determined using surfactant of anionic type. In this paper, a cationic surfactant, hexadecyl trimethyl ammonium bromide, is used to determine an anionic drug, phenobarbiturate. Fundamental considerations on adsorption and interfacial ion-pair formation on electrode surfaces were treated theoretically and verified experimentally using a modified flow-injection system with a reference piezoelectric detector, buffered reagent, and sample solutions, and a short connecting tube. The determination of phenobarbiturate in pharmaceutical preparations was carried out satisfactorily by the developed system in comparison with chromatographic methods using spectral, photochemical, and electrochemical detection as well as titrimetry (12–16). 1
To whom correspondence should be addressed. 251 0026-265X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
252
MO ET AL.
FIG. 1. A schematic diagram of ion-pairing flow-injection with piezoelectric detection. D r, D w, reference and working detector, respectively; P, speed modulation pump; V, four-way automatic injection valve; FC, frequency counter; OM, oscillator and mixer circuit; PC, computer; c, surfactant solution as carrier; s, sample solution; w, waste solution.
EXPERIMENTAL Apparatus The flow-through detector, similar to those previously designed (10), had a capacity of 25 ml and was mounted with a 27 MHz AT-cut gold-plated piezoelectric crystal that was the resonator in a TTL-IC oscillator. Piezoelectric crystals with higher frequency than previously used to enhance the mass sensitivity. In this research, two detectors were set at the front and the rear of the injection valve, as shown in Fig. 1. The one preceding the valve was used as a reference detector, and the one following the valve was used as a working detector. The resonant frequencies of the two detectors were transmitted to a digital mixer that output the heterodyned frequency. The two oscillator circuits and the mixer circuit were built from standard parts. Measurements of the two resonant frequencies and the heterodyned frequency were taken at 1-s gate times from three SS7200 universal counters (SJZ No.4 Electronic Factory, China) by GPIB connection to a 486 computer running a graphical program. The heterodyned frequency, in directing frequency shift with respect to the frequency of the referencing crystal without ion pairing, directly demonstrates the desorption of the surfactant on the working crystal and eliminates frequency shift due to change in the environment temperature. Thus, the heterodyned frequency is measured in analytical applications of ion-pairing flow injection with piezoelectric detection. The frequency counter can achieve a resolution of 1 Hz for a 27-MHz signal with a 1-s gate. By measurement of the heterodyned frequency, which ranges from 0 to 10 kHz, this same resolution, 1 Hz, is acquired with a gate less than 1 ms. Thus, a 1000-Hz sample rate is possible (although the maximum GPIB transmission rate is 200 Hz). An additional advantage of measuring the heterodyned frequency is that unsophisticated and economical single-chip or plug-in card counters may be used to measure these low frequencies without compromising performance. In the flow injection system, an LP-2A speed modulation pump and a V-16A four-way
ION-PAIRING ANALYSIS OF PHENOBARBITURATE
253
multifunctional automatic valve fitted with a 50-ml bypass coil (Xintong Scientific Instrument Co., China) were used. Flow lines were of PTFE tubing (0.5 mm i.d.). The length of connecting tube between the valve and the working detector was 200 mm, chosen to be short enough to avoid significant distribution of the sample. Reagents Buffer solutions (0.1 M) with pH ranging from 3.0 to 8.5 were prepared using sodium phosphate and citric acid. A solution (0.02 M) of hexadecyl trimethyl ammonium chloride (HTAC) and a stock solution (5 mg ml 21) of sodium phenobarbiturate (SPB) were prepared in buffer solutions and stored frozen in a dark bottle. From these, more dilute solutions were prepared as required. Both reagent and sample solutions were buffered to eliminate changes in physiochemical properties of solutions as the ample solution was injected into the flowing reagent solution. Changes in physicochemical properties of solutions cause changes in resonant frequency of the piezoelectric detector. All reagents were of analytical grade. Ion-exchange - distilled water was used. RESULTS AND DISCUSSION Adsorption of HTAC on Gold Solutions of HTAC in the range from 0 to 0.5 mM were passed through one flowthrough detector at flow rates of 0.1, 1.0, and 5.0 ml min 21. The frequency of each detector was found to decrease significantly and become stable in a time proportional to the concentration of HTAC and inversely proportional to the flow-rate. The frequency shift relative to water was found to increase gradually to a limit when the concentration of HTAC increased to 0.25 mM, and then decrease with more HTAC presented in water, as shown in Fig. 2. In comparison among curves at different flow-rates in Fig. 2, maximum frequency shifts were nearly the same; however, the initial slope at lower concentrations of HTAC increased with increased flow-rate, and was nearly unchanged at flow-rates higher than 1.0 ml min 21. The change in frequency was assumed to be caused by the small change in mass at the surface of gold due to the adsorption of HTAC, based on the interaction of gold with hydrophobic groups of free surfactant molecules. It can be seen from the curves in Fig. 2 that the adsorbed amount of HTAC on gold increases and decreases before and after the maximum as the amount of HTAC in solution increases. The decrease probably results from the formation of micelles among surfactant molecules. Response to Injection of Phenobarbiturate As shown in Fig. 3, sharp positive peaks occurred on the graph of heterodyned frequency versus time, responding to injections of SPB. It was shown that the resonant frequency of the working detector increased as SPB passed through. The peak height was proportional to the concentration of SPB in the range 0 –1 mg ml 21. The increase in frequency corresponds to the decrease in adsorbed amount of HTAC at the gold surface, which should be caused by interfacial ion-pairing as SPB passed through and by the decrease in concentration of HTAC in the passed solution. The latter, however, was found to be much smaller when a blank solution was injected, showing that desorption of HTAC on the surface because of the decreased concentration of HTAC in the solution
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MO ET AL.
FIG. 2. Relationship of frequency shift (DF) to concentration of HTAC (C R) at various flow-rates: (F) 0.1, (■) 1.0, and (}) 5.0 ml min 21.
was too slow to keep pace with the passing injection band. Hence, in the linear adsorption regime of HTAC, the peak height responding to injection of SPB can be obtained as f p 5 ak ftKC RC S,
(1)
where a is the sensitivity constant of the piezoelectric detector’s response to the change in mass at surface, K is the adsorption constant of HTAC on gold affected by the flow-rate, k f and t are rate constant and reaction time for the interfacial ion-pair formation, and C R and C S are concentrations of HTAC passed and SPB injected, respectively. Hereby, the peak height is proportional to the injection concentration of SPB and can be applied to the determination of SPB concentration with an ascertained flow injection system using a fixed solution of HTAC as a carrier. The dependence of peak height on HTAC concentration and operating parameters were derived from the above equation and verified by experimental results as follows. Effects of Buffer pH and Concentration of HTAC Effects of the pH of the buffer and of the concentration of HTAC on peak height are shown in Fig. 4. A flow-rate of 1.0 ml min 21 and an injection concentration of 0.200 mg ml 21 were used in the experiment. The peak height was found to increase with increased buffer pH in the range 3.0 – 6.5, and to decrease with higher buffer pH (.6.5). As pH
ION-PAIRING ANALYSIS OF PHENOBARBITURATE
255
FIG. 3. FIA response of frequency vs. time to injection of phenobarbiturate using HTAC as carrier. Injection concentration of SPB, (1) 0.100, (2) 0.200, and (3) 1.00 mg ml 21; concentration of HTAC, 0.20 mM; flow-rate, 1.0 ml min 21; and length of connecting tube, 200 mm.
increases in the range 3.0 – 6.5, the increase in the concentration of free phenobarbiturate anion is greater than the decrease in free ammonium cation, so the peak height increases. When pH is over 6.5, the peak height decreases, since the concentration of ammonium cation declines seriously. The peak height was found to increase before achieving a maximum when concentration of HTAC was 0.20 mM, and subsequently to decrease with higher surfactant concentration. Within the linear range of the adsorption isotherm, according to Eq. (1), the peak height increases with increased concentration of HTAC, C R . The decrease of peak height at higher HTAC concentrations results from the decrease of the adsorption constant, K, as can be seen in Fig. 2. Hence, the pH of the buffer was selected to be 6.5 and the concentration of HTAC to be 0.20 mM to give the greatest sensitivity for the determination of SPB. Effect of Operating Parameters As seen in Eq. (1), the peak height is proportional to the reaction times (t). In the detector cell, the reaction time is proportional to the injection volume and inverse of flow-rate. In the following experiments, concentrations of HTAC and SPB were 0.20 mM and 0.200 mg ml 21, respectively. With the increase of flow-rate, the peak height was found to increase and decrease when the flow-rate was below and over 1.0 ml min 21, respectively. The former indicates that the dilution of SPB by diffusion in the tubing is significantly reduced with the increase of flow-rate in the slow flowing. In addition, slower flow-rates below 0.1 ml min 21 resulted in wider peak widths since HTAC readsorbed more slowly on the surface from the carrier.
256
MO ET AL.
FIG. 4. Effect of pH of HTAC solution on peak height (f p) for injection of phenobarbiturate at various concentrations of HTAC: (F) 0.10, (Œ) 0.15, (■) 0.20, and (}) 0.25 mM. Injection concentration of SPB, 0.200 mg ml 21; flow-rate, 1.0 ml min 21; and length of connecting tube, 200 mm.
With shorter connecting tubes and larger injection volumes, the peak height was found to be greater. However, plateaus occurred when the length of the connecting tube was less than 200 mm at injection of 50 ml SPBs and when the injection volume was larger than 50 ml at connecting tube of 200 mm. It indicated that no more HTAC was presented at the surface as the excessive injection band of SPB passed through the detector, and the peak height was no longer proportional to concentration of SPB injected. Hereby, the flow-rate, injection volume, and length of connecting tube were chosen to be 1.0 ml min 21, 50 ml, and 200 mm for determinations to get the highest sensitivity. Calibration Graph and Reproducibilty As optimized above, the ion-pairing flow injection system with HTAC concentration of 2.5 mM, a flow rate of 1.0 ml min 21, a connecting tube of 200 mm, and an injection volume of 50 ml was used for the determination of SPB. A linear calibration graph of relative peak height with respect to the blank (water) versus the concentration of SPB was obtained in the range from 0 to 1.00 mg ml 21, with a slope of 2.59 kHz ml mg 21 and a correlation coefficient of 0.999. The standard deviation for the blank injection (10 replicates) was 0.38 Hz. The detection limit (3 3 noise), calculated as 3 3 0.38/2.59, was 0.44 mg ml 21. The relative standard deviation of 0.200 mg ml 21 SPB (10 replicates) was 0.11%. The sampling rate was about 60 h 21.
ION-PAIRING ANALYSIS OF PHENOBARBITURATE
257
TABLE 1 Determination and Recovery of Sodium Phenobarbiturate in Pharmaceutical Preparations
Sample a
Nominal value (mg ml 21)
1
0.2
Added (mg ml 21)
Found by titrimetric method b (mg ml 21)
Found by FI method b (mg ml 21)
0.19 6 0.003
0.197 6 0.001 0.101 6 0.001 0.477 6 0.001 0.248 6 0.001 0.982 6 0.002 0.502 6 0.001
0.100 2
0.5
3
1.0
0.48 6 0.005 0.250 0.98 6 0.008 0.500
a b
Recovery (%)
101.0 99.2 100.4
Injections supplied by three manufactures. Average of three determinations 6 standard deviation.
Interference The influence of foreign compounds that commonly accompany SPB in pharmaceutical preparations was studied by preparing solutions containing 0.200 mg ml 21 SPB and increasing concentrations of the potential interferents up to 1.00 mg ml 21 or by adding an amount to give an error of 3%. The errors were determined by comparison with the peak heights given by a solution of analyte containing no foreign substances. Up to 1.00 mg ml 21 of glucose, lactose, sucrose, asparate, benzoate, citrate, oxalate, salicylate, and tartrate 0.200 mg ml 21 of tetraphenyl borate and benzenesulfonate, and 0.020 mg ml 21 of dodecyl sulfate and dodecyl benzenesulfonate were tolerated in the determination of 0.200 mg ml 21 SPB. Analysis of Pharmaceutical Preparations The proposed method was satisfactorily applied to the determination of SPB in pharmaceutical preparations. Commercially available formulations were analyzed and the results obtained are summarized in Table 1. As can be seen, for all formulations the assay results were in good agreement with values obtained by the nonaqueous titrimetric method (16). The recoveries obtained by adding SPB to each pharmaceutical formulation are also given in Table 1. CONCLUSIONS Ion-pairing flow injection with piezoelectric detection has been based on ion association between a cationic surfactant and a anionic drug. In comparison with conventional approaches using ion pairing, an important aspect of this method is the elimination of separation between the free ion-pairing reagent and the ion pair. The sensitivity, accuracy, and precision of this method have been improved by using reference and working detectors with higher frequencies, a buffer, and a short connecting tube. Moreover, developments would be an advance in gradient ion-pairing flow injection with piezoelectric detection used in multicomponent analysis in pharmaceuticals.
258
MO ET AL.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Karlberg, B.; Thelander, S. Anal. Chim. Acta, 1978, 98, 1. Catalayud, J. M.; Garcia Mateo, J. V. Pharm. Technol. Int., 1992, 4, 17. Alwarthan, A. A.; Al-Obaid, A. M. J. Pharm. Biomed. Anal., 1996, 14, 579. Johansen, G.; Grini, K.; Langseth-Manrique, K.; Karstensenb, K. H. Talanta, 1996, 43, 951. Emara, S.; Razee, S.; El-Shorbagi, A.-N.; Masujima, T. Analyst, 1996, 121, 183. Perez, R. T.; Martinez, L. C.; Tomas, V.; Siddrach, C. Analyst, 1995, 120, 1103. Alcada, M.; Lima, J.; Montenegro, M. J. Pharm. Biomed. Anal., 1995, 13, 459. Perez, R. T.; Martinez, L. C.; Tomas, V. J. Pharm. Biomed. Anal., 1994, 12, 1109. Wei, W.; Hu, C.; Zhu, W.; Yao, S. Anal. Lett., 1993, 26, 2371. Mo, Z.; Zhang, M.; Li, M.; Xia Z. Anal. Lett., 1997, 30, 663. Mo, Z.; Luo, J.; Li, M. Analyst, 1997, 122, 111. Mitchell, P.; Clark, B. J. Anal. Proc., 1993, 30, 101. Lima, J.; Montenegro, M.; Silva, A. J. Pharm. Biomed. Anal., 1990, 8, 701. Rollmann, B.; Lefebure, B.; Goetemans, A.; Telquin, B. J. Pharm. Belg., 1990, 45, 245. Wolf, C.; Schmid, R. W. J. Liq. Chromatogr., 1990, 13, 2207. Chinese Pharmacopoeia, Part II, p. 402. Chemical Industry Press and People Hygiene Press, Beijing, 1995.
Ion-Pairing Flow Injection Analysis of Phenobarbiturate with Piezoelectric Detection Zhihong Mo, 1 Xiaohui Long, Yifeng Xu, and Xudong Zhou College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044, People’s Republic of China New developments of ion-pairing flow injection with piezoelectric detection have been made for the analysis of the anionic drug phenobarbiturate. A cationic surfactant, hexadecyl trimethyl ammonium bromide, was used, and its adsorption and interfacial ion-pair formation with phenobarbiturate on the gold surface of piezoelectric quartz crystal were investigated. Modifications of the analytical system have been developed to improve its sensitivity, using reference and working detectors with higher frequencies, buffered reagent and sample solutions, and a short connecting tube. Influences of surfactant concentration and operating parameters were demonstrated. Under optimization for the determination of phenobarbiturate, the calibration curve was linear in the range 0 –1 mg ml 21, with a detection limit of 0.44 mg ml 21, a recovery of 99.%, an RSD of 0.11% and a sampling frequency of 60 h 21. The method has been satisfactorily applied to the determination of phenobarbiturate in pharmaceutical formulations. © 1999 Academic Press
INTRODUCTION A number of ion-pairing methods for drug evaluation using automated flow injection solvent extraction have appeared since the initial work of Karlberg and Thelander on the determination of caffeine in acetylsalicylic acid preparations (1, 2). Previous methods have adapted detection systems of a variety of types, which included piezoelectric quartz microbalance as well as atomic and molecular spectroscopy, turbidimetric, and electrochemical techniques (3–9). However, the separation of the ion-pair from the carrier in solvent extraction required necessitates incorporation of a segmenter and consumption of organic solvent, so that the applicability of this method is greatly limited. Recently, a new method, ion-pairing flow injection with piezoelectric detection, not using liquid–liquid extraction or phase separation, has been developed and applied to determination of several pharmaceuticals (10 –11). This method, based on in situ mass sensing of the adsorption of surfactant on a piezoelectric crystal surface, detects ion-pair formation simultaneously with desorption of the surfactant on the crystal surface. In previous work, cationic drugs were determined using surfactant of anionic type. In this paper, a cationic surfactant, hexadecyl trimethyl ammonium bromide, is used to determine an anionic drug, phenobarbiturate. Fundamental considerations on adsorption and interfacial ion-pair formation on electrode surfaces were treated theoretically and verified experimentally using a modified flow-injection system with a reference piezoelectric detector, buffered reagent, and sample solutions, and a short connecting tube. The determination of phenobarbiturate in pharmaceutical preparations was carried out satisfactorily by the developed system in comparison with chromatographic methods using spectral, photochemical, and electrochemical detection as well as titrimetry (12–16). 1
To whom correspondence should be addressed. 251 0026-265X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
252
MO ET AL.
FIG. 1. A schematic diagram of ion-pairing flow-injection with piezoelectric detection. D r, D w, reference and working detector, respectively; P, speed modulation pump; V, four-way automatic injection valve; FC, frequency counter; OM, oscillator and mixer circuit; PC, computer; c, surfactant solution as carrier; s, sample solution; w, waste solution.
EXPERIMENTAL Apparatus The flow-through detector, similar to those previously designed (10), had a capacity of 25 ml and was mounted with a 27 MHz AT-cut gold-plated piezoelectric crystal that was the resonator in a TTL-IC oscillator. Piezoelectric crystals with higher frequency than previously used to enhance the mass sensitivity. In this research, two detectors were set at the front and the rear of the injection valve, as shown in Fig. 1. The one preceding the valve was used as a reference detector, and the one following the valve was used as a working detector. The resonant frequencies of the two detectors were transmitted to a digital mixer that output the heterodyned frequency. The two oscillator circuits and the mixer circuit were built from standard parts. Measurements of the two resonant frequencies and the heterodyned frequency were taken at 1-s gate times from three SS7200 universal counters (SJZ No.4 Electronic Factory, China) by GPIB connection to a 486 computer running a graphical program. The heterodyned frequency, in directing frequency shift with respect to the frequency of the referencing crystal without ion pairing, directly demonstrates the desorption of the surfactant on the working crystal and eliminates frequency shift due to change in the environment temperature. Thus, the heterodyned frequency is measured in analytical applications of ion-pairing flow injection with piezoelectric detection. The frequency counter can achieve a resolution of 1 Hz for a 27-MHz signal with a 1-s gate. By measurement of the heterodyned frequency, which ranges from 0 to 10 kHz, this same resolution, 1 Hz, is acquired with a gate less than 1 ms. Thus, a 1000-Hz sample rate is possible (although the maximum GPIB transmission rate is 200 Hz). An additional advantage of measuring the heterodyned frequency is that unsophisticated and economical single-chip or plug-in card counters may be used to measure these low frequencies without compromising performance. In the flow injection system, an LP-2A speed modulation pump and a V-16A four-way
ION-PAIRING ANALYSIS OF PHENOBARBITURATE
253
multifunctional automatic valve fitted with a 50-ml bypass coil (Xintong Scientific Instrument Co., China) were used. Flow lines were of PTFE tubing (0.5 mm i.d.). The length of connecting tube between the valve and the working detector was 200 mm, chosen to be short enough to avoid significant distribution of the sample. Reagents Buffer solutions (0.1 M) with pH ranging from 3.0 to 8.5 were prepared using sodium phosphate and citric acid. A solution (0.02 M) of hexadecyl trimethyl ammonium chloride (HTAC) and a stock solution (5 mg ml 21) of sodium phenobarbiturate (SPB) were prepared in buffer solutions and stored frozen in a dark bottle. From these, more dilute solutions were prepared as required. Both reagent and sample solutions were buffered to eliminate changes in physiochemical properties of solutions as the ample solution was injected into the flowing reagent solution. Changes in physicochemical properties of solutions cause changes in resonant frequency of the piezoelectric detector. All reagents were of analytical grade. Ion-exchange - distilled water was used. RESULTS AND DISCUSSION Adsorption of HTAC on Gold Solutions of HTAC in the range from 0 to 0.5 mM were passed through one flowthrough detector at flow rates of 0.1, 1.0, and 5.0 ml min 21. The frequency of each detector was found to decrease significantly and become stable in a time proportional to the concentration of HTAC and inversely proportional to the flow-rate. The frequency shift relative to water was found to increase gradually to a limit when the concentration of HTAC increased to 0.25 mM, and then decrease with more HTAC presented in water, as shown in Fig. 2. In comparison among curves at different flow-rates in Fig. 2, maximum frequency shifts were nearly the same; however, the initial slope at lower concentrations of HTAC increased with increased flow-rate, and was nearly unchanged at flow-rates higher than 1.0 ml min 21. The change in frequency was assumed to be caused by the small change in mass at the surface of gold due to the adsorption of HTAC, based on the interaction of gold with hydrophobic groups of free surfactant molecules. It can be seen from the curves in Fig. 2 that the adsorbed amount of HTAC on gold increases and decreases before and after the maximum as the amount of HTAC in solution increases. The decrease probably results from the formation of micelles among surfactant molecules. Response to Injection of Phenobarbiturate As shown in Fig. 3, sharp positive peaks occurred on the graph of heterodyned frequency versus time, responding to injections of SPB. It was shown that the resonant frequency of the working detector increased as SPB passed through. The peak height was proportional to the concentration of SPB in the range 0 –1 mg ml 21. The increase in frequency corresponds to the decrease in adsorbed amount of HTAC at the gold surface, which should be caused by interfacial ion-pairing as SPB passed through and by the decrease in concentration of HTAC in the passed solution. The latter, however, was found to be much smaller when a blank solution was injected, showing that desorption of HTAC on the surface because of the decreased concentration of HTAC in the solution
254
MO ET AL.
FIG. 2. Relationship of frequency shift (DF) to concentration of HTAC (C R) at various flow-rates: (F) 0.1, (■) 1.0, and (}) 5.0 ml min 21.
was too slow to keep pace with the passing injection band. Hence, in the linear adsorption regime of HTAC, the peak height responding to injection of SPB can be obtained as f p 5 ak ftKC RC S,
(1)
where a is the sensitivity constant of the piezoelectric detector’s response to the change in mass at surface, K is the adsorption constant of HTAC on gold affected by the flow-rate, k f and t are rate constant and reaction time for the interfacial ion-pair formation, and C R and C S are concentrations of HTAC passed and SPB injected, respectively. Hereby, the peak height is proportional to the injection concentration of SPB and can be applied to the determination of SPB concentration with an ascertained flow injection system using a fixed solution of HTAC as a carrier. The dependence of peak height on HTAC concentration and operating parameters were derived from the above equation and verified by experimental results as follows. Effects of Buffer pH and Concentration of HTAC Effects of the pH of the buffer and of the concentration of HTAC on peak height are shown in Fig. 4. A flow-rate of 1.0 ml min 21 and an injection concentration of 0.200 mg ml 21 were used in the experiment. The peak height was found to increase with increased buffer pH in the range 3.0 – 6.5, and to decrease with higher buffer pH (.6.5). As pH
ION-PAIRING ANALYSIS OF PHENOBARBITURATE
255
FIG. 3. FIA response of frequency vs. time to injection of phenobarbiturate using HTAC as carrier. Injection concentration of SPB, (1) 0.100, (2) 0.200, and (3) 1.00 mg ml 21; concentration of HTAC, 0.20 mM; flow-rate, 1.0 ml min 21; and length of connecting tube, 200 mm.
increases in the range 3.0 – 6.5, the increase in the concentration of free phenobarbiturate anion is greater than the decrease in free ammonium cation, so the peak height increases. When pH is over 6.5, the peak height decreases, since the concentration of ammonium cation declines seriously. The peak height was found to increase before achieving a maximum when concentration of HTAC was 0.20 mM, and subsequently to decrease with higher surfactant concentration. Within the linear range of the adsorption isotherm, according to Eq. (1), the peak height increases with increased concentration of HTAC, C R . The decrease of peak height at higher HTAC concentrations results from the decrease of the adsorption constant, K, as can be seen in Fig. 2. Hence, the pH of the buffer was selected to be 6.5 and the concentration of HTAC to be 0.20 mM to give the greatest sensitivity for the determination of SPB. Effect of Operating Parameters As seen in Eq. (1), the peak height is proportional to the reaction times (t). In the detector cell, the reaction time is proportional to the injection volume and inverse of flow-rate. In the following experiments, concentrations of HTAC and SPB were 0.20 mM and 0.200 mg ml 21, respectively. With the increase of flow-rate, the peak height was found to increase and decrease when the flow-rate was below and over 1.0 ml min 21, respectively. The former indicates that the dilution of SPB by diffusion in the tubing is significantly reduced with the increase of flow-rate in the slow flowing. In addition, slower flow-rates below 0.1 ml min 21 resulted in wider peak widths since HTAC readsorbed more slowly on the surface from the carrier.
256
MO ET AL.
FIG. 4. Effect of pH of HTAC solution on peak height (f p) for injection of phenobarbiturate at various concentrations of HTAC: (F) 0.10, (Œ) 0.15, (■) 0.20, and (}) 0.25 mM. Injection concentration of SPB, 0.200 mg ml 21; flow-rate, 1.0 ml min 21; and length of connecting tube, 200 mm.
With shorter connecting tubes and larger injection volumes, the peak height was found to be greater. However, plateaus occurred when the length of the connecting tube was less than 200 mm at injection of 50 ml SPBs and when the injection volume was larger than 50 ml at connecting tube of 200 mm. It indicated that no more HTAC was presented at the surface as the excessive injection band of SPB passed through the detector, and the peak height was no longer proportional to concentration of SPB injected. Hereby, the flow-rate, injection volume, and length of connecting tube were chosen to be 1.0 ml min 21, 50 ml, and 200 mm for determinations to get the highest sensitivity. Calibration Graph and Reproducibilty As optimized above, the ion-pairing flow injection system with HTAC concentration of 2.5 mM, a flow rate of 1.0 ml min 21, a connecting tube of 200 mm, and an injection volume of 50 ml was used for the determination of SPB. A linear calibration graph of relative peak height with respect to the blank (water) versus the concentration of SPB was obtained in the range from 0 to 1.00 mg ml 21, with a slope of 2.59 kHz ml mg 21 and a correlation coefficient of 0.999. The standard deviation for the blank injection (10 replicates) was 0.38 Hz. The detection limit (3 3 noise), calculated as 3 3 0.38/2.59, was 0.44 mg ml 21. The relative standard deviation of 0.200 mg ml 21 SPB (10 replicates) was 0.11%. The sampling rate was about 60 h 21.
ION-PAIRING ANALYSIS OF PHENOBARBITURATE
257
TABLE 1 Determination and Recovery of Sodium Phenobarbiturate in Pharmaceutical Preparations
Sample a
Nominal value (mg ml 21)
1
0.2
Added (mg ml 21)
Found by titrimetric method b (mg ml 21)
Found by FI method b (mg ml 21)
0.19 6 0.003
0.197 6 0.001 0.101 6 0.001 0.477 6 0.001 0.248 6 0.001 0.982 6 0.002 0.502 6 0.001
0.100 2
0.5
3
1.0
0.48 6 0.005 0.250 0.98 6 0.008 0.500
a b
Recovery (%)
101.0 99.2 100.4
Injections supplied by three manufactures. Average of three determinations 6 standard deviation.
Interference The influence of foreign compounds that commonly accompany SPB in pharmaceutical preparations was studied by preparing solutions containing 0.200 mg ml 21 SPB and increasing concentrations of the potential interferents up to 1.00 mg ml 21 or by adding an amount to give an error of 3%. The errors were determined by comparison with the peak heights given by a solution of analyte containing no foreign substances. Up to 1.00 mg ml 21 of glucose, lactose, sucrose, asparate, benzoate, citrate, oxalate, salicylate, and tartrate 0.200 mg ml 21 of tetraphenyl borate and benzenesulfonate, and 0.020 mg ml 21 of dodecyl sulfate and dodecyl benzenesulfonate were tolerated in the determination of 0.200 mg ml 21 SPB. Analysis of Pharmaceutical Preparations The proposed method was satisfactorily applied to the determination of SPB in pharmaceutical preparations. Commercially available formulations were analyzed and the results obtained are summarized in Table 1. As can be seen, for all formulations the assay results were in good agreement with values obtained by the nonaqueous titrimetric method (16). The recoveries obtained by adding SPB to each pharmaceutical formulation are also given in Table 1. CONCLUSIONS Ion-pairing flow injection with piezoelectric detection has been based on ion association between a cationic surfactant and a anionic drug. In comparison with conventional approaches using ion pairing, an important aspect of this method is the elimination of separation between the free ion-pairing reagent and the ion pair. The sensitivity, accuracy, and precision of this method have been improved by using reference and working detectors with higher frequencies, a buffer, and a short connecting tube. Moreover, developments would be an advance in gradient ion-pairing flow injection with piezoelectric detection used in multicomponent analysis in pharmaceuticals.
258
MO ET AL.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
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