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Anal. Chem. 1988,60,1390-1393
(7) Malmstadt, H. V.; Enke, C. 0.: Crouch, S. R. Electronics and Instrumentatbn for SdenWs: BenjamlnlCummings: Menlo Park, CA, 1981. (8) H a m m t s u PhOtvCell Cats@; Hamamatsu Corp.: BrMgewater, NJ. (9) Bauer, W. E.; Wade, A. P.; Crouch, S. R. Anal. Chem. 1988, 8 0 , 267-288. (10) Sargeant. K.; Sheridan, A,: O’Kelly, J. Nature (London) 1961, 792, 1096. (1 1) Nesbltt, B. F.: O’Kelly, J.: Sargeant, K.: Sheridan, A. Nature (London)
1962, 195, 1062.
RECEIVED for review September 22, 1987. Accepted March 5,1988. This work was funded by a grant from Neogen Corp., Lansing, MI.
Computer-Controlled Flow Injection Analysis System for On-Line Determination of Distribution Ratios Howard L. Nekimken,* Barbara F. Smith,* Gordon D. Jarvinen, E. J. Peterson, and Marianne M. Jones Chemical a n d Laser Sciences Division, Anal.ytica1 Chemistry Group, M S G740, Los Alamos National Laboratory,
Los Alamos, New Mexico 87545
An automated flow lnJectionanalysls (FIA) system has been developed for the rapid acqulsHlon of iiquid/llquld, metal ion distrlbutlon ratlos ( D ). The system features automatic swltchlng between aqueous metal sample and wash s d u t b , on-line solvent extraction, phase separation, and the simuitaneous detection of the separated phases by diode-array spectrophotometry. A comparative study of manual, singiestage iiquid/iiquid extractions wlth the flow injection system was completed by uslng a new extraction system UOt+/ benzene/TOPO (trloctylphosphine oxide)/HBMPPT (4benzoyC2,4-dlhydrob-methyC2-phenyi-3H-pyra~oC3-thlone). The batch and F I A methods yielded results generally withln 5 % of each other. The major dlfferences between the two systems are that the FIA system is at least twice as fast, Is less labor intensive, is more reproducible, and yields better statlstlcr (a result of the FIA’s speed and automation features). Slope analysls of the plotted data from the uranyl extractlon studles indicates that the extraction complex is UO,(BMPPT),(TOPO).
The use of liquidlliquid extraction for separation of metal ions (on an analytical or process scale) requires determination of metal ion distribution ratios (D) under a variety of conditions. Information about the composition of the extracted species is commonly obtained from slope analysis ( I ) , Le., determining the dependency of D on such factors as pH, ligand, and synergist concentrations. Obtaining the large amount of distribution ratio data necessary to fully evaluate a new ligand system is labor intensive. For rapid acquisition of D values, an automated flow injection analysis (FIA) system was developed. The FIA technique has become very useful in analytical chemistry (2,3). Recently, some work has been reported that uses FIA in analyses that required an extraction procedure (4-15).Most often, flow injection methods rely on controlled or reproducible dilution (generally referred to as dispersion in FIA literature (2))of a sample injected into a carrier stream, but for the direct, equilibrium measurement of D values (not requiring any extrapolation), virtually no sample dispersion (of the center of the sample plug) throughout the entire flow system is desirable. The FIA system described in this paper features no significant sample dispersion (of the center of the sample plug) along with on-line extraction; phase separation; automated, simultaneous solution-phase detection; and quantitation. The use of this FIA system to perform an
extraction study and a comparison of the FIA results with batch mode extractions are discussed.
EXPERIMENTAL SECTION Reagents. All organic solutions were made in benzene (Baker), with the exception of a solvent dependency study, where chloroform (EM), toluene (Baker),and xylene (Baker)were used. The appropriate amounts of crystalline solid HBMPPT (whose synthesis is described in ref 16) and trioctylphosphineoxide (TOPO) (Eastman) were weighed into volumetric flasks (followed by the addition of the organic solvent)to provide the appropriate solution concentrations of ligand and synergist (TOPO), respectively. The U02-HBMPPT complex was synthesized (to assure the proper stoichiometry) by combining uranyl nitrate and HBMPPT in a 1:2 ratio in ethanol and then adding HzO to precipitate the complex out of solution. To all aqueous solutions, LiC104(Aldrich) was added to adjust the ionic strength and chloroacetic acid (Eastman) to buffer the solution. Any pH adjustment was carried out by adding HCIOl (Baker) or LiOH (Aldrich) to the solution. All distilled water used was purified with a Barnstead water purification unit. Arsenazo III (Baker) was used as a color reagent to quantitate uranyl in the aqueous phase solution. Three studies were completed for the uranyl-HBMPPT extraction system: HBMPPT concentration, TOPO concentration, and pH dependencies. For all experiments, 0.1 M LiCIOI and 0.01 M chloroacetic acid were present in the aqueous phase (with the exception of a comparison between the buffered and unbuffered solutions). For all uranyl extractions,the aqueous phase solution contained 5 X lo4 M UOz(N03)2.6H20 (Strem). During the pH dependency study, the ligand and synergist (TOPO) concentrations (in the organic solution phase) were held at 0.01 and 0.005 M, respectively, while the pH of the aqueous solution phase was varied between 0.9 and 2.4. For the ligand dependency study, the TOPO concentration was held constant at 0.005 M and the pH at 1.5, while the HBMPPT concentration was varied from 0.015 to 0.075 M. Finally, during the synergist dependency study, the HBMPPT concentration was held at 0.02 M and the pH at 1.5, while the TOPO concentration was varied from 0.00075 to 0.005 M. For the aqueous phase quantitation of lJ02*+, 1.5 X lod M arsenazo I11 was used. Apparatus and Instrumentation. The batch extraction experiments were carried out in &mL Teflon tubes with snap caps. The solutions were shaken on a Kraft Apparatus, Inc., linear shaker, and the phases were separated in an International Equipment Co. centrifuge. Aqueous and organic samples (1-2 mL) were drawn with Pasteur pipets and were transferred to separate Teflon tubes for analysis. Detection was carried out via absorbance measurements with a HP-8451 diode-array spectrophotometer (with a Hellma 8O-wL flow cell). Figure 1shows a block diagram of the flow injection extraction system. The flow injection extractions were carried out with a custom flow injection analyzer. The organic, wash, and sample aqueous solutions were
0003-2700/88/0360-1390$01.50/0 0 1988 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 14, JULY 15, 1988
I
1
Figure 1. Block diagram of flow injection system: E, extraction coil: T,, T, mixing tees; V, two-way valve; V, three-way valve; M,, phase separator; P, peristaltic pump.
all pumped by Control Equipment Corp. gas displacement pumps (with He gas) as part of the flow injection analyzer. The aqueous color reagent was pumped by a Rainin peristaltic pump (P). Most of the Teflon tubing in this system was 0.5-mm i.d., and the extraction coil (E,) was approximately 3-m long. An HP-85 computer was used to control the two on-off (V,) and the three-way (V,) valves (from Control Equipment Corp.) via an RS-232C serial interface. The phase separator (MI) (also from Control Equipment Corp.) consisted of a Teflon membrane sandwiched between two Teflon blocks. Spectrophotometric studies indicate that the efficiency of the phase separator is a t least 99%. No benzene could be detected in the aqueous phase spectrophotometrically, and because this was the method of detection used in this extraction study, no problems were encountered. The two separated phases were detected simultaneously with two separate detectors. The organic phase was detected with a HP-8451 diode-array spectrophotometer (controlled by a HP-85 computer) and the aqueous phase with a HP-8452 diode-array spectrophotometer (controlled by an IBM PC-XT computer). Flow cells (80 pL) were utilized with both of these detectors. Procedure. The extraction consisted of contacting the organic phase solution (containing HBMPPT and TOPO) with the aqueous phase solution (containing LiClO,, UO,", and chloroacetate) long enough for the system to reach equilibrium (5 min for the batch method and 2 min for the FIA method). It was determined that preequilibration of both solution phases did not significantly change the resulting D values and therefore was not carried out for this particular extraction system. For all batch extractions, 3 mL of each solution phase was dispensed into a Teflon tube. Each solution was then shaken for 5 min, followed by centrifugation for 1-2 min. The phases were then distributed in separate tubes for analysis. The organic phase was detected directly by injecting the solution into the detector flow cell, followed by the acquisition of an analytical measurement. For the aqueous phase, the arsenazo color reagent was added to the sample solution and mixed in a Branson ultrasonic bath, and then the resulting mixture was injected into the flow cell before the acquisition of an analytical measurement. The color reagent to sample volume ratio utilized was 6:l. In the flow injection system (shown in Figure l),the aqueous wash stream and the organic stream were configured to flow a t equal flow rates (approximately 0.3 mL/min). The two phases were merged a t a mixing tee (T,) so that small, alternating segments (approximately 1 p L in volume) of each phase were produced. After the system had been sufficiently rinsed with wash solution, a three-way valve (V,) that controls the aqueous stream was automatically actuated and the sample solution was allowed to flow in place of the wash solution at the same flow rate (0.3 mL/min). Sample switching between different samples was not executed under computer control for this extraction study; however, the FIA system now utilizes an autosampler to do this automatically. At flow rates of 0.3 mL/min and with an extraction coil length of 3 m, the two-phase contact time was nearly 3 min. Analytical measurements were automatically taken for the organic phase directly after the phase separator (M,) by the spectrophotometer. For the aqueous phase, color reagent was added to the stream (at T2)a t a flow rate of approximately 1.8 mL/min (addition a t a 6:l flow rate ratio) after the phase separator. The
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solution was then directed through a mixing coil and then to the spectrophotometer,where analytical measurements were obtained. Quantitation. The same standards were employed for the organic phase for both batch and flow injection modes. The standard solutions all contained the initial HBMPPT and TOPO concentrations most commonly used in the extraction experiments (10 mM HBMPPT and 5 mM TOPO) plus a varying concentration of the U02-HBMPPT complex (from the crystalline solid). The spectral reference was a solution that contained only the ligand and synergist. Standard spectra were run between 470 and 570 nm (the charge-transfer absorbance band of the complex). Precontact of the organic standards with the aqueous phase (with no metal ion present) did not change the resulting absorption spectra. These standard spectra were then stored on a flexible disk for future use in sample quantitation. To obtain the batch aqueous phase standards, a 6:l volume ratio of color reagent to aqueous metal solution (of known concentration) was mixed. The spectral reference consisted of a solution containing every component normally in the aqueous phase with the exception of uranyl ion. Spectral measurements were obtained from 620 to 720 nm and were stored on a flexible disk. The flow injection standard aqueous phase spectra were obtained in exactly the same manner as for the batch method, except the aqueous phase was precontacted with benzene. Again precontact did not change the results significantly. These standard spectra were stored on the IBM computer's hard disk. A BASIC computer program (ACONTROL4) was written to automate the flow injection system. Before program execution, the wash and organic streams are allowed to flow through the system to establish good analytical base lines. When the program is first executed, the user is asked to enter the appropriate experimental parameters, including the analytical wavelengths of interest and information about data storage and timing. After these parameters are entered, a starting run time is printed; the aqueous stream three-way valve is actuated, allowing the sample rather than the wash stream to flow; and a reference measurement is obtained on the HP-8451 spectrometer. Approximately 2 min after the run is initiated, a refarence measurement is obtained on the HP-8452 spectrometer. The program checks the organic phase absorbance a t 500 nm (or some other selected wavelength) every 18 s. If an absorbance value of less than 4.005 is obtained, a new reference measurement is automatically executed. Every fifth measurement on the HP-8451 is printed out. When the absorbance at 500 nm exceeds the threshold value (e.g., A = 0.01 or some other preprogrammed value), the time is printed, and the HP-8451 obtains a spectral measurement every 9 s between 470 and 570 nm and stores the spectra in the temporary memory of the HP-85 computer. This spectral measurement procedure is carried out for a total of 335 s. Approximately 10 s before the end of this spectral measurement cycle, an analytical spectral measurement is obtained on the HP-8452 and is stored on the IBM PC-XT. Of the spectra stored in the temporary memory of the HP-85, only those spectra, where the absorbance at 500 nm is 99% of the maximum value recorded at that wavelength during a given measurement cycle, are stored on a flexible disk for later quantitation. After the spectral measurement cycle has ended, the run time is again printed, the aqueous phase three-way valve is turned off, and wash stream flows in place of the sample. This wash cycle lasts for 250 s (long enough for spectra to be transferred to a flexible disk and to prevent carry over between samples). After this cycle is completed, the above procedure is repeated. Usually four separate sample runs are obtained for each experimental condition. Beer's law plots are constructed every 2 nm in the spectral region of interest (e.g., 47C-570 nm) based on the standard spectra stored on the computer. The sample is then quantitated at each of these wavelengths by the computer. A plot of metal ion concentration vs wavelength for each sample is then constructed for inspection by the user. Alternatively, an average concentration value is calculated and reported as the actual metal ion concentration.
RESULTS AND DISCUSSION Preliminary Work. Some initial studies were necessary before the extraction studies could be completed. These initial measurements were obtained with the same care as later
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 14, JULY 15, 1988
._
_ ~
-7-
--_
Figure 2. Plots of log D vs pH: W, batch data: HBMPPT, 5 mM TOPO).
+, F I A data (10 rnM (5 rnM TOPO, pH 1.5). _
measurements. Before any of the D data could be critically evaluated, the metal ion accountabilities for both the batch and FIA methods were determined. Once the proper quantitation methods were developed, accountabilities were usually between 90 and 110%. Some preliminary work was performed to determine if the chloroacetate buffer severely effected the D values of the U0,-HBMPPT extraction system. Extractions run with and without chloroacetate present yielded slightly different but readily corrected results, and therefore the buffer was included in all subsequent experiments for pH control. The presence of the buffer was responsible for preventing any significant pH changes during the extraction. D values for extractions with prior preequilibration of both the aqueous and organic phase solutions and extractions with no prior preequilibration were compared. No significant difference was found between extractions carried out with or without prior preequilibration. By varying the solution flow rates, which changed the two-phase contact time, a contact time study was completed for the U0,-HBMPPT extraction (on the FIA). Changing either the flow rate or coil length alters the phase contact time, and therefore, either method may be used to determine the necessary time to reach equilibrium. However, changing the flow rate is accomplished more easily. Only about 2 min of contact time between the two phases was required to reach equilibrium because contact times greater than 2 min produced no change in the resulting absorbance values. A brief study (on the FIA) was also undertaken for a comparison of the D values obtained in benzene with the values obtained in toluene, xylene, and chloroform. The extraction behavior was similar in the three aromatic solvents (benzene, toluene, and xylene), whereas chloroform suppressed the distribution ratio by more than an order of magnitude. Extractions of uranyl by H B M P P T and TOPO separately were compared with extractions containing both of these species. D values of 0.45 and 0.3 were obtained for 0.1 M HBMPPT and 0.005 M TOPO, respectively, while the combination of 0.1 M H B M P P T and 0.005 M TOPO produced a D value of 63 (an obvious extraction synergism for the extraction of uranyl). Critical Comparison of the FIA and Batch Systems. For comparison of the two systems, D measurements (for the U0,-HBMPPT extraction system) were acquired as a function of solution pH, HBMPPT, and TOPO concentration for both the batch and FIA methods. The mean of three data points (for the batch method) or four data points (for the FIA method) at each p H or concentration is reported for each of the D measurement methods. The relative standard deviations for the replicate D values were usually within 10%. A data point at every 0.3 p H unit from pH 0.9 to pH 2.4 was obtained for the pH dependency study. Five data points were
-
&
9[
”
-Ll
Flgure 4. Plots of log D vs log [TOPO]: W, batch data: (20 mM HBMPPT, pH 1.5).
+, FIA data
Table I. Statistical Data for Plots of log D vs log [HBMPPT), log [TOPO], and pH (for Both FIA and Batch Data)
log D vs (plot)
data acquisitn
correlatn coeff
PH PH log [HBMPPT] log [HBMPPT] log [TOPO] log [TOPO]
batch FIA batch FIA batch FIA
0.995 0.994 0.998 0.998 0.997 0.997
slope f std dev 1.96 2.11 1.94 1.85 1.10 1.14
std error (18) of Y est
f 0.05
0.11
f 0.05 f 0.03 f 0.03 f 0 02 0.02
0.11 0.03 0.03
*
0.03 0.03
obtained in the concentration range of 15-75 mM HBMPPT for the ligand dependency study. Finally, six data points in the concentration range from 0.75 to 5 mM of TOPO for the synergist dependency study were acquired. All D values were corrected for the competitive complexation of uranyl by chloroacetate, where D = Dexptl[1 + &A,(ClAc)] (17). Dexptl is the experimentally measured D value, PCMcis the uranylchloroacetate binding constant, and (ClAc) is the concentration of chloroacetate. The correction factor ranged from 1.03 (at pH 0.9) to 1.66 (at pH 2.4). At pH 1.5 (the pH used for the extractions in the HBMPPT and TOPO concentration studies), the correction factor was 1.12 (17). Plots of log D vs pH, log D vs log [HBMPPT], and log D vs log [TOPO] are shown in Figures 2, 3, and 4, respectively. Each of these figures has data plotted from both the batch and FIA measurement methods. Least-squares statistical data for these plots are listed in Table I. From all three of these plots, good agreement between the FIA and the batch data is evident. When the precision of the data is taken into consideration (in ad-
Anal. Chem. 1988, 60, 1393-1397
dition to the least squares standard deviation), these results indicate that the differences between the FIA and batch data points are within statistical error. The FIA can produce data significantly faster than a manual procedure (a D value is obtained in 20 min or less), and because the method is almost totally automated, it can be run with minimal supervision and carries out operations in a more reproducible manner. These factors enabIe multiple D values to be acquired with the FIA easier, faster, and with better analysis statistics than with the batch system. It can also be advantageous to have a system such as the FIA that features on-line extraction, phase separation, and detection. This FIA should also be useful for the study of extraction systems that feature either unstable ligands or extracting complexes because there is much less time between the extraction and detection. The solution flow rates and even the extraction coil length on the FIA can be readily adjusted. These simple adjustments should facilitate the acquisition of kinetic measurements for extraction systems. The major limitation of this type of measurement on the FIA would be encountered when very slow flow rates or a very long extraction coil is required because of slow extraction kinetics (i.e., 5 min or more of contact time is required). Solvent Extraction Chemistry. Least-squares fits were employed for the pH, ligand, and synergist dependency plots (shown in Figures 2,3, and 4, respectively) for both the batch and FIA data. These data are summarized in Table I. The least-squares slopes were 2.04, 1.90, and 1.12 (obtained by averaging the FIA and batch data), respectively, for the pH, ligand, and synergist dependencies. The correlation coefficients of these plots were 0.994 or better. With use of the equation log D = log C + npH + n log [HL].,, + n log [SI,,, (derived elsewhere (1)) and the data obtained, the resulting extracting complex is readily derived as U02(BMPPT)2(TOPO),-whereC is a constant and [HL] and [SI are the ligand and synergist concentrations respectively. For this study, [HL],,, and [S],,, are equal to the original concentra-
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tions of ligand and synergist because their solubilities in the aqueous phase are very small and [L-I,, is also very small (1). Deviations from the whole number slopes can be attributed to variations in activity coefficients (as described in ref 16). A similar solid-state structure has been reported with dimethyl sulfoxide as the synergist (16).
ACKNOWLEDGMENT We thank the Los Alamos National Laboratory for funding for this project. We also thank Raul Morales and Bob Ryan for helpful discussions on topics relevant to this paper. Registry No. TOPO, 78-50-2; HBMPPT, 62574-32-7; U, 7440-61-1.
LITERATURE CITED Lo, T. C.; Baird, M. H. I.; Hanson, C. Handbook of Solvent Extraction; Wiley: New York, 1983; Chapter 2. Ruzicka, J.; Hansen, E. Now Injection Analysis; Wiley: New York, 1981. Ruzicka, J. Anal. Chem. 1983, 55, 1040A-1053A. Kariberg, B.; Thelander, S. Anal. Chim. Acta 1978, 9 8 , 1-7. Bergamin, H.; Medeiros, J. X.; Reis, 8 . F.; Zagatto, E. A. G. Anal. Chim. Acta 1978, 101, 9-16. Johansson, P. A,; Karlberg, B.; Thelander, S.Anal, Chim. Acta 1980, 114, 215-226. Deratani, A,; Sebille, B. Anal. Chem. 1981, 5 3 , 1742-1746. Nord, L.; Karlberg, B. Anal. Chim. Acta 1981, 125, 199-202. Ooms, P. C. A,; Leendertse. G. P.;Das, H. A,; Brinkman. U. A. T. J . Radioanal. Chem. 1981, 67, 5-14. Nord, L.; Karlberg, B. Anal. Chim. Acta 1983, 145, 151-158. Fossey, L.; Cantwell, F. Anal. Chem. 1985, 5 7 , 922-926. Sahiestrom, Y.; Karlberg, 8.Anal. Chim. Acta 1986, 185, 259-269. Castro, L. J . Autom. Chem. 1986, 8 , 56-62. Lucy, C. A.; Cantwell, F. F. Anal. Chem. 1986, 58, 2727-2731. Valcarcel, M.; Castro, L. J . Chromatogr. 1987, 393, 3-23. JaNinen, G. D.; Smith, 8. F.; Ritchey, J. M. Inorg. Chim. Acta 1987, 129, 139-148. Kotrly, S.; Sucha, L. Handbook of Chemical Equilibria in Analytical Chemistry; Ellis Horwood: New York, 1985; Chapters 2, 7. Draper, N.; Smith, H. Applied Regression Analysis, 2nd ed.; Wiiey: New York, 1981; Chapter 1.
RECEIVED for review September 14,1987. Accepted February 29, 1988.
Investigation of Poly(L-amino acids) by X-ray Photoelectron Spectroscopy Kenneth D. Bomben* T h e Perkin-Elmer Corporation, Physical Electronics Laboratory, 1161-C S a n Antonio Road, Mountain View, California 94043 Sukhendu B. Dev' Battelle Columbus Diuision, 505 King Auenue, Columbus, Ohio 43201 A systematlc Investigation of 11 homopolymeric amino acids by X-ray photoelectron spectroscopy wlll be dlscussed. The chemlcal shifts In the core levels and the relatlve Intensities of the atomlc environments, obtalned from least-squares curve fitting, can be shown to correspond to partlcuiar structural features. I n addltlon, the atomic concentratlons of the varlous elements have been obtalned and compared to those expected from the structures. Shake-up satellite intensltles from those compounds with aromatlc rlngs are also reported. Human serum albumin was examined, and the results are compared to the expected structure and to prevlously reported results. 1Present address: D e p a r t m e n t of A p p l i e d Biological Sciences, Massachusetts I n s t i t u t e of Technology, Cambridge, M A 02139.
Since the first reports of X-ray photoelectron spectroscopy (XPS, also known as electron spectroscopy for chemical analysis, ESCA) by Siegbahn et al. (1,2), the technique has been applied to numerous fields. The study of biological materials, which are chemically complex, presents special problems in the interpretation of the chemical shift data available from XPS. Nonetheless, over the last 2 decades there has been much work done on biological systems. For example, Siegbahn et al. reported the investigation of cystine hydrochloride (3) and insulin ( 4 ) ,and Millard (5-9) undertook a series of studies that have made use of XPS for the characterization of the surfaces of cells and microorganisms. Ratner et al. (10-12) have used the techniaue to investigate thin protein films' adsorbed on metal support surfaces.- Meisenheimer et al. (13) investigated blood, Klein and Kramer (14)
0003-2700/88/0360-1393$01.50/00 1988 American Chemical Society
