Anal. Chem. 1990, 62, 2059-2062
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Minimal Dispersion Flow Injection Analysis Systems for Automated Sample Introduction Stephen H. Brooks* and Gregory Rullo I C I Pharmaceuticals Group, Concord Pike and Murphy Road, Wilmington, Delaware 19897
INTRODUCTION The dispersion coefficient, D, is the most common descriptor of dilution in a flow injection analysis (FIA) system and is most generally defined as the ratio of the analyte concentration introduced into the system to the concentration at peak maximum after transport through the defined manifold. Limited dispersion systems (D= 1-3) in FIA are used when the analyst desires to measure the composition of a nearly undiluted sample (I). These systems have received minimal application for the analysis of real samples. The determination of metal ions by atomic absorption spectrophotometry (2) and the potentiometric determination of pH in soil extracts (3)and calcium activity in serum ( 4 ) are examples of limited dispersion FIA systems which have appeared in the literature. In these applications, the limited dispersion system provides for the accurate and precise transport of the sample to the detector. The minimal dilution of the sample also allows the analyst to enhance sensitivity (due to limited dilution) while taking advantage of the speed and reproducibility of the flow injection technique. The measurement of equilibrium constants (i.e. measurement of ionization constants, metal-ligand coordination complexes, ion pair formation, and dimerization and investigation of charge transfer complexes) provides specific examples where dilution of the analyte is deleterious to the accuracy of the determination. The accurate measurement of critical micelle concentrations of surfactant solutions affords another example of a physicochemical measurement which is highly dependent upon maintenance of the integrity of the sample solution prior to the measurement itself. In all of these applications, dilution of the sample within the flow system would result in inaccurate determinations of solution properties. The present work introduces the practical application of flow injection systems employing a large ratio of injection to manifold volume for automated sample introduction. In this study, FIA and spectrophotometric detection are utilized for the determination of proton-transfer equilibrium constants. The advantages of the speed and reproducibility of automated sample introduction are realized and the steady-state nature of the process ensures the maximum accuracy of the determination. Other presented applications using minimal dispersion flow injection sample introduction which are dependent upon the establishment of a steady-state signal include the conductometric determination of critical micelle concentrations and the accurate spectrophotometric measurement of samples which possess viscosities which are significantly different from each other. The developed technique is considered to be a hybrid between air-segmented continuous flow analysis (CFA) and classical FIA, with an analytical signal reminiscent of the former while employing the instrumentation of the latter. It is not affected by the many drawbacks of air segmentation, yet provides, to a first approximation, a steady-state signal. The use of traditional FIA instrumentation allows the technique to be utilized as an extremely reproducible, automated sample introduction method in conjunction with all available high-performance liquid chromatography (HPLC) flow-through detectors. The technique exploits certain advantages of both air-segmented CFA and FIA and allows the analyst to perform measurements that would be extremely more difficult, if not impossible, using either system alone.
EXPERIMENTAL SECTION Apparatus. A Waters (Milford, MA) Model 6000A HPLC pump was used to propel the carrier streams. Samples were introduced into the system by a Rheodyne (Cotati, CA) Model 7126 sample injection valve in conjunction with a Varian (Walnut Creek, CA) Model 8055 autosampler. Sample volume used in all experiments was 500 pL. The injector was connected to the spectrophotometric detectors using a 20 cm length (1.6 mm 0.d. x 0.25 mm i.d.) of Alltech Associates (Deerfield,IL)stainless steel tubing and to the conductance detector flow cell by a 5 cm length (1.6 mm 0.d. X 0.25 mm id.) of Alltech Teflon tubing. The manifold used for the viscosity experiments was a 100-cm coil (1.6 mm 0.d. x 0.50 mm id.) of Alltech Teflon tubing with a coiling diameter of 23 mm. The FIA spectrophotometric measurements were made with a Kratos (Ramsey, NJ) Spectroflow 783 (0.8 cm path length, 0.1-s rise time) programmable absorbance detector. Ionic surfactants were detected by a Dionex (Sunnyvale, CA) Model CDM-1 conductivity detector. The total volumes of the FIA flow manifolds (including injector and preflow cell detector tubing) were determined by measuring the time from sample injection to initial baseline disturbance for duplicate injections at 0.1 mL/min and were found to be 67 and 8 pL for the Kratos and Dionex detectors, respectively. Detector output signals were acquired by a VG Lab Systems, Ltd. (Manchester, England), Vax Multichrom Data Aquisition System and were simultaneously monitored on a Houston Instrument (Austin, TX) Omniscribe Series D5000 recorder. Static UV measurements were obtained with a Perkin-Elmer (Norwalk, CT) Lambda 5 UV/VIS spectrophotometer with a 1.00-cm path length. Measurements of solution pH were made with a Corning (Medfield, MA) Ion Analyzer 150 pH meter with a Corning general purpose combination electrode. All experiments were performed at ambient conditions (21 f 1 "C)without temperature control. Reagents. All water used in the preparation of solutions and carrier streams was doubly deionized and passed through a Barnstead (Boston, MA) Nanopure I1 activated carbon system to remove organic impurities. Reagent grade hydrochloric, nitric, acetic, succinic, and phosphoric acids and HPLC grade methanol were from J. T. Baker (Phillipsburg, NJ). Certified ACS grade sodium hydroxide and potassium hydroxide pellets as well as Certified Primary Standard grade benzoic acid were from Fisher (Fair Lawn, NJ). Alldrich Chemical (Milwaukee, WI) was the supplier of the 2-nitrophenol and 4-nitrophenol while thiamine hydrochloride was from Kodak (Rochester, NY). Microselect grade sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB) were from Fluka (Hauppauge, NY). All chemicals were used as received without further purification. Procedure. The procedure used for the spectrophotometric determination of ionization constants followed the method described by Albert and Serjeant (5). In summary, an aqueous stock solution of the analyte was prepared, from which two solutions of equal analyte concentration were diluted with the appropriate buffer to obtain the absorbance spectra of the fully ionized and molecular (un-ionized)forms of the anal*. The fully protonated and deprotonated forms of the analyte were achieved by adjusting solution pH to be greater than 2 pH units, in each direction, from the pKa (ensuringthat >99% of the analyte is in the desired form). The spectra of these solutions were compared and the analytical wavelength was chosen in a spectral region where a difference between the absorbance of the two species existed. Additional aliquots of stock solution were used to prepare a total of nine solutions of equal analyte concentration. These solutions and corresponding blanks were diluted with a series of non-UV absorbing buffers, of constant ( I = 0.01) ionic strength whose pH values had been adjusted to achieve solutions at the estimated pKa, and f0.2, f0.4, f0.6, and *2 pH units from the estimated pKa. For each of these solutions, the pH and UV absorbance at
0003-2700/90/0362-2059$02.50/00 1990 American Chemical Society
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Table I. Experimental Conditions for Determination of pK, Values at 21 f 1 "C, Z = 0.01 compound
concn,O mg/mL
detection wavelength, nm
buffer* (0.01 M)
carrier composition
benzoic acid (static UV) (limited dispersion) (minimal dispersion)
0.081 0.082 0.082
274 274 274
sodium acetate sodium acetate sodium acetate
0.01 M acetic acid
0.037 0.037
250 250 400 400
succinic acid succinic acid
thiamine HC1
not applicable 0.01
M acetic acid
0.01 M succinic acid
aqueous phosphoric acid aqueous 0.0075 phosphoric acid aqueous 'Benzoic acid solutions contained 0.1% (v/v) ethanol, all others were totally aqueous. *Bufferswere adjusted to the desired pH with 1 M KOH.
2-nitrophenol 4-nitrophenol
0.010
Table 11. Comparison of Methods for the Spectrophotometric pK, Determination of Benzoic Acid flow injection*
staticn
classical
(D< 3)
UV-vis
LL, . 0.4 ,
1
,
,
-. ,
i
w \
0.8
1.2
Time ( m i n i
Flgure 1. Response curves resulting from the injection of 500 pL of (a) 0.149, (b) 0.124, (c) 0.0872, (d) 0.0498, and (e)0.0249 mglmL of a methanolic solution of benzoic acM into the minimal dispersion FIA system: detection wavelength, 259 nm; methanol carrier stream: flow rate, 1.0 mL/min.
the analytical wavelength were accurately measured. For the static UV measurement, samples were read against the corresponding blank. When the low volume FIA manifolds were used, both sample and blank absorbances were independently measured and the blank absorbance was subtracted from the sample reading. Table I summarizes the experimental conditions (buffers,analyte concentrations,and analytical wavelengths) used for determination of the proton-transfer equilibrium constants in the present work.
RESULTS AND DISCUSSION The integrity of the resulting response curve was assessed by using an injection of 500 pL of a methanolic 0.0872 mg/mL benzoic acid test solution. This large injection volume (when compared to the total manifold volume) ensures that the signal achieved is in excess of 99.99% of the steady-state condition, maintaining the integrity of the injected sample. At this injection volume, it was shown that the resulting response height was independent of flow rate and exhibited excellent within and between injection precision and that the spectrophotometric detector exhibited linearity of response using either peak area or peak height response as a basis for calibration. Figure 1 shows the FIA response curves resulting from a series of standards into the defined system. Spectrophotometric Determination of Proton-Transfer Equilibrium Constants. The pK, of benzoic acid was first determined by the classical static spectrophotometric approach. The results of this study are presented in Table 11. The reported standard deviation of 0.013 in conjunction with excellent agreement with literature values indicates that an accurate determination of the pK, of benzoic acid has been obtained. An attempt was then made to use a traditional limited dispersion FIA system as a sample introduction technique to both accurately and precisely determine a value for the benzoic acid ionization constant. Injections of 1O-pL volumes of the prepared solutions resulted in dispersion coefficients which range from 2.02 (pH = 4.773) to 2.36 (pH = 3.558), verifying classification as a limited dispersion system. This experiment underscores the inability of a classical FIA
minimal dispersion
PH
PK,
PH
PK,
PK,
3.585 3.773 3.943 4.168 4.344 4.553 4.735
4.199 4.181 4.197 4.180 4.178 4.182 4.160
3.558 3.740 3.950 4.159 4.363 4.526 4.773
4.207 4.262 4.300 4.383 4.451 4.534 4.602
4.197 4.187 4.179 4.187 4.185 4.187 4.173
mean 4.182 4.391 4.185 0.146 0.007 std dev (*) 0.013 Completely ionized and deionized forms were achieved by solutions of pH = 1.08 and 11.92, respectively. bCompletelyionized and deionized forms were achieved by solutions of pH = 1.61 and 11.97, respectively. See text for description of classical FIA manifold. Injection volume is 500 pL. system to be utilized as the sample introduction method for pK, determinations. The developed low-volume FIA manifold was then utilized in conjunction with absorbance detection for the determination of the pK, of benzoic acid. The results of this experiment are also given in Table I1 and indicate excellent agreement between the manual static UV determination and the automated sample introduction procedure for the determination of pK, values. In the FIA system, all blanks and samples were injected in duplicate with the exception of those corresponding to the fully ionized and deionized forms of the acid, which were injected in quadruplicate. The standard deviation of measurement is an indication of the enhanced reproducibility of automated sample introduction. With the validity of the minimal dispersion system for the spectrophotomeric determination of ionization constants demonstrated, all subsequent pK, values were determined by using the FIA system. The technique was then applied to the determination of the pK, of the weaker base of thiamine hydrochloride (vitamin Bl). The resulting average pK, value from the seven determinations was 4.617 f 0.018. This value was highly precise but was in poor agreement with available literature references, which indicated the pK, to be 4.8 (6-9). Thiamine hydrochloride is known to decompose in aqueous solutions a t pH values greater than 5.5 (9). All solutions used in this study had pH values less than 5.5, except for a pH = 6.554 solution, which was used to form the singly deprotonated form of the compound. In an attempt to ensure that decomposition had not occurred, solutions were reprepared and the pK, was redetermined and calculated to be 4.629 f 0.027. A more thorough search of the literature revealed that refs 7-9 had
ANALYTICAL CHEMISTRY, VOL. 62, NO. 18, SEPTEMBER 15, 1990
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Table 111. Spectrophotometric Determination of pK, Values by FIA compound value FIA" (fstd dev)
1it.b
ref 5, p 29 5, p 141 10, p 1858 13, p 5-51 5, p 145 13, p 5-51 5, p 145
benzoic acid
4.185 f 0.007
4.16 (20 "C) 4.204
thiamine HCl' 2-nitrophenol
4.632 f 0.026 7.166 f 0.020
4.5d 7.222 7.23
4-nitrophenol
7.087 f 0.003
7.150
I
.' F'
O A l l experimentally determined values are at 21 f 1 "C, I = 0.01, injection volume = 500 rL. bAll reported literature values 25
unless otherwise indicated. 'The standard deviation reported is calculated from the mean of 21 determinations (14 using a succinic acid carrier and 7 using an aqueous carrier) of the pK,. dSee text for further discussion.
*
O C
0
7' ,,'
4
8
12
16
SDS Concentration (mM) obtained the value of 4.8 from Windheuser and Higuchi (6). In an apparent transcription error, these authors (6) incorrectly reported the pK,, value determined by Williams and Ruehle (IO). The pioneering work of Williams and Ruehle in 1935 determined pKnl of the weaker base of thiamine hydrochloride to be 4.5 by the half-neutralization technique for pKa determination. The errors associated with acceptance of the half-neutralization point as an approximation of pKa (activity considerations and the autoprotolysis of water are ignored) are well documented ( I I , 1 2 ) . Significant errors result when using the half-neutralization method for determination of pKa values outside the range of 7 f 2.5 (11). The present work, therefore, represents the first known spectrophotometric determination of pK,, for thiamine hydrochloride and is believed to be more accurate than previously reported values determined by the half-neutralization method. A basic assumption of the low-volume FIA systems described here is that there is no penetration to the center of the sample zone by the carrier stream. Therefore, the use of a buffered carrier stream should not be necessary for this work. This assumption was shown to be valid by the determination of pK,, for thiamine hydrochloride using a 100% aqueous carrier stream. The resulting pKa of 4.653 f 0.021 is in agreement with the two previous determinations of this pK, value. A major drawback of a CFA technique which requires establishment of a steady-state signal is the increased consumption of carrier stream. This work had shown that FIA is able to utilize a 100% aqueous carrier for experimentation and all subsequent work uses water as the vehicle for transporting the sample through the system. The large decrease in throughput observed in the low-volume FIA systems is of little consequence when employing an aqueous carrier. The pK, values for 2-nitrophenol and 4-nitrophenol were also determined via the described system utilizing a water carrier. Table I11 summarizes the pK, values determined by this approach as well as a comparison to literature values. In the case of the nitrophenol isomers, the precision of FIA is utilized in conjunction with the spectrophotometric method to differentiate between pKa values separated by only 0.08 pK, unit. The slight difference between the literature (25 "C) and experimental (21 f 1 OC) values can be explained by the effect of temperature on ionization constants. For phenol, it is known that the pKa decreases by 0.012 unit for every 1 "C decrease in temperature (5). When this fact is taken into consideration, there is excellent agreement with the literature values. Conductance Determination of Critical Micelle Concentrations. The critical micelle concentration (crnc) is the concentration range in which surfactant monomers aggregate to form micelles. Experimentally, the cmc is obtained by monitoring the change in slope of a response vs concentration
Figure 2. Plot of response (pS) vs concentration (mM) for a 500-pL volume of SDS obtained by using the FIA sample introduction technique with conductance detection.
curve of a physicochemical property (surface tension, conductance, refractive index, etc.) of the solution. Linear extrapolation of the response curves obtained at surfactant concentrations below and above the cmc yields an intersection concentration equivalent to the cmc. For any method which relies upon measurement of a physicochemical property of a series of surfactant solutions, it is crucial that the technique utilized to introduce surfactant samples to the detection system does not result in dilution of the sample. The cmc is usually determined by a series of these manual measurements. Here, FIA is utilized as the sample introduction technique in conjunction with conductance detection for the determination of cmc's of two common ionic surfactants, SDS and CTAB. Figure 2 is the resulting response vs concentration curve obtained for SDS. This work determined the cmcs of SDS and CTAB to be 8.0 and 0.91 mM, respectively. These values are in good agreement with literature value ranges (14) of 8.1-8.5 and 0.92-0.99 mM for these ionic surfactants. Accurate CFA Quantification of Viscous Solutions. Traditionally, it has been necessary to match the viscosity of the sample and standard in order to perform accurate quantification by FIA. This precaution is necessary due to the influence of viscosity on sample zone dispersion (15). A 1.0 mg/mL solution of benzoic acid was prepared in water and in 8.56 X M CTAB. This concentration of CTAB is roughly 10 times the cmc for the surfactant and results in a significant increase in viscosity when compared to a purely aqueous solution. Dissolution of the benzoic acid in the micellar media resulted in a slight spectral shift, and when compared to the aqueous solution, an isosbestic point is evidenced at 284.7 nm by static UV measurement. In this spectral region, the rate of change of absorbance with wavelength is approximately 0.1 AU/nm. Preliminary investigation using the flow-through UV spectrophotometer indicated that the isosbestic lies between 285 and 286 nm for the detector used. The resolution of the HPLC detector is 1 nm and the wavelength of detection was chosen to be 286 nm. At this wavelength, the benzoic acid dissolved in CTAB exhibited an absorbance of approximately 0.004 AU greater than that of the aqueous solution. Monitoring the absorbance of these solutions a t the isosbestic wavelength ensures that any differences in response between injected samples in an aqueous or micellar matrix are a direct result of transport phenomena. The results of an injection of these two solutions into a traditional flow manifold are shown in Figure 3. The dispersion coefficient, D, is 1.76 for the aqueous sample and 2.29 for the high viscosity sample. Similar peak profiles have been
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 18, SEPTEMBER 15, 1990
aqueous sample (0.1170relative standard deviation), ensuring that the viscosity of the solution did not interfere with the reproducible establishment of the steady-state signal.
b
Time ( m i n )
Figure 3. Response curves for the injection of a 1.O mglmL solution of benzoic acid showing the effects of sample viscosity upon sample zone dispersion in (A) a traditional ( 5 0 - ~ Linjection, 100 cm coil, 1.6 m m 0.d. X 0.25 i.d.) and (6) minimal dispersion F I A system (500-pL injection). Injections a and b correspond to samples in an aqueous and 8.56 X lo3 M CTAB matrix, respectively. (Detection wavelength, 286 nm; aqueous carrier stream; flow rate, 1.0 mL/min.)
observed by previous workers (16, 17) and this observed difference in peak height responses accentuates the need for a careful matching of sample matrices (viscosity, particulates) of injected solutions in order to achieve reliable results for measurement of analyte concentrations (vs standards) by FIA. In contrast with the response curve resulting from the traditional FIA system (Figure 3A), Figure 3B shows that sample introduction via the minimal dispersion FIA system results in peak height responses which are truly representative of the composition of the injected samples. The shape of the response curve is clearly effected on the leading and tailing portions of the peak, and for the viscous sample, the time of the steady state condition is noticeably decreased. These alterations in the response curve, however, do not interfere with the accuracy or precision of the steady-state measurement. The precision ( n 24)of injection of the viscous solution (0.21% relative standard deviation) was similar to that of the
CONCLUSION This work has introduced the practical application of lowvolume FIA manifolds and has demonstrated its utility as a mode of sample introduction for making accurate and precise measurements of solution parameters while not disturbing the integrity of the injected sample. Proton-transfer equilibria represent a specific example of an association complex. The concept can be applied more generally in conjunction with a wide variety of HPLC flow-through detectors to allow the analyst to more easily investigate association phenomena (ligands and metal ions, ion pair formation, and dimerization association). Utilization of the technique in conjunction with conductance, refractive index, and spectral probes should allow a more rapid and efficient method for sample introduction to aid in the determination of cmc’s for a wide variety of ionic and nonionic surfactant systems. Use of low manifold volume FIA and associated detection for the analysis of viscous samples and samples containing particulates (in systems where no reaction is occurring, i.e. tablet dissolution samples) eliminates the need for matrix matching while still providing the advantages of automated sample introduction to the analyst. In all of these applications, both presented and proposed, maintenance of solution composition is paramount in obtaining accurate results. The developed technique has demonstrated its utility to provide rapid and reproducible delivery of an undisturbed sample bolus which allows the analyst to perform measurements that would not be possible with a classical limited dispersion system. LITERATURE CITED (1) Ruzicka, J.; Hansen, E. H. Now Injection Analysis, 1st ed.; Wiley: New York, 1981. (2) Olsen, S.; Pessenda, L. C. R.; Ruzicka, J.; Hansen, E. H. Analyst 1983, 108, 905. (3) Hongbo, C.; Hansen, E. H.; Ruzicka, J. Anal. Chim. Acta 1985, 769, 209. (4) Hansen, E. H.; Ruzicka, J.; Ghose, A. K. Anal. Chim. Acta 1978, 700, 151. (5) Albert, A.; Serjeant. E. P. The Determination of Ionization Constants, 3rd ed.; Chapman and Hall: New York, 1984. (6) Windheuser, J. J.; Higuchi, T. J . Pharm. Sci. 1962, 57, 354. (7) Carlin, H. S.;Perkins, A. J. Am. J . Hosp. fharm. 1968, 25, 271. (8) Newton, D. W.: Kluza, R. B. Drug. Intell. Clin. pharm. 1978, 72, 546. (9) CRC Handbook of Hormones, Vitamins, and Radiopaques; CRC Press: Boca Raton, FL. 1986; p 248. (10) Williams, R. R.; Ruehle, A. E. J. Am. Chem. SOC. 1935, 5 7 , 1856. (11) Cookson, R. F. Chem. Rev. 1974, 7 4 , 5 . (12) Benet, L. 2.;Goyan, J. E. J. fharm. Sci. 1987, 56, 665. (13) Lange’s Handbook of Chemistry; McGraw-Hill: New York, 1985. (14) Brooks, S. H.; Berthod. A.: Kirsch, B. A,; Dorsev. J. G. Anal. Chim. Acta 1988, 209, 111. (15) Ruzicka, J.; Hansen, E. H. Flow Injection Analysis, 2nd ed.; Wiley: New York, 1988. (16) Loscascio-Brown. L.; Plant, A. L.; Durst, R. A. Anal. Chem. 1988. 6 0 , 792. (17) Brooks, S . H.: Leff, D. V.; Hernandez Torres, M. A,; Dorsey, J. G. Anal. Chem. 1988. 6 0 , 2737.
RECEIVED for review January 25,1990. Accepted June 6,1990.
