Anal. Chem. 1983, 55, 493-497
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Cadmium- 'I 13 and Carbon- 13 Nuclear Magnetic Resonance Spectrometry of Cadmium-Amino Acid Complexes S. M. Wang' and R. K. Gilpin"' Department of Chemistry, Kent State University, Kent, Ohio 44242
I n the present Investiqatlon,the solution chemistry of several cadmlum complexes has been studied by multinuclear NMR technlques (Ii3Cd anti I3C). Ligands which have been employed include a serles of amlno aclds (glycine, P-alanine, y-aminobutyrlc acid, and c-amlnocaproic acid) as weYl as ammonia, methylamine, and other substltuted slmllar compounds, such as glyclne methyl ester and N,N-dimethylglyclne. Of particular interest have been changes in the overall profile of chemlcal shift vs. pH obtained between glyclne and slmilar amino acids which contain greater numbers of methylene unlta separatlng the termlnal functlonalities. On the basis of the observed trends, the formatlon of fivemembered chelate rings between the cadmlum( 11) ion and molecules slmilar to glycine seems plausible. I n the case of glycine, an octahedral complex formed from three five-membered chelate rlngs Is suggested.
Multinuclear NMR has been employed for the study of a wide variety of chemical systems. The heavy metal nuclides such as lead, mercury, and cadmium have been of particular interest because of their biotoxicity. Additionally, for these systems the advantage of large changes in chemical shift as a function of often subtle alterations in chemical environment and structure make multinuclear NMR techniques especially attractive. In the case of the cadmium-113 nucleus, a chemical shift range of over 800 ppm has been reported ( I ) . Since the initial cadmium NMR work of Maciel(2) and Ellis (3) a variety of investigations have been carried out, a number of which have been concerned with the nature of metal binding sites in metalloenzymes and other metalloproteins. Although considerable amounts of data have been collected on various cadmium systems, often wide variations in results have been reported. Significantly different values for the chemical shift of identical cadmium compounds have been obtained between laboratories employing very similar experimental parameters. In the case of cadmium-substituted human carbonic anhydrase B, Armitage and co-workers ( 4 ) reported a rather sharp resonance (25 Hz) centered at 146 ppm a t p H 9.6. However, for nearly identical procedures, Sudmeier and Bell (5)observed a broad (3100 Hz) line centered a t 228 ppm with a p H 9.7 solution. For carboxypeptidase A, chemical shifts of 240 and 217 ppm were obtained in p H 8 perchlorate and chloride buffers, respectively (6). In these and more recent investigations, the chemical shift of cadmium as well as other metal complexes has been found to change dramatically as a function of often small modifications in solution parameters. For example, when Jonsson et al. (7) examined the effect of pH on the 'I3Cd carbonic anhydrases for both bicarbonate and cyanide solution, the chemical shift data obtained were extremely sensitive to changes in both the solutions p H and the Present address: Analytical Section, Laboratory of Carcinogen Metabolism, Division of Cancer Cause & Prevention, National Cancer Institute. NIH. Bethesda. MD. Present address: IBM Instruments, Inc., Orchard Park, Danbury, CT 06810.
counterions employed. In addition to these studies, when Kostelnik and Bothner-by (8)investigated the effect of more than 20 diverse ligands, they also found significant changes in chemical shift as a function of counterion. In view of these as well as other similar studies, the need for fundamental investigations is clearly evident before the nature of more complex systems such as protein-metal binding can be interpreted fully. In the current study, several cadmium complexes have been examined by combined I3C and Il3Cd multinuclear NMR techniques. Ligands which have been employed include a homologous series of amino acids and sterically hindered similar compounds. Of particular interest have been changes in the overall profiles of chemical shift vs. pH curves obtained between shorter (e.g., glycine) and longer amino acids. The observed trends seem to support arguments (9) for the formation of five-membered chelate rings between the cadmium(I1) ion and molecules similar to glycine.
EXPERIMENTAL SECTION Materials. Fisher activity buffers, pH 4.00, 7.00, and 10.00, were used as calibration standards. All additional chemicals unless otherwise specified were reagent grade. Glycine, &alanine (6ALA), t-aminocaproic acid (c-ACA),and y-aminobutyric acid (yABA) (Sigma) were used as received. Methylamine (25%)was purified by distillation and standardized against HCl by a potentiometric titration method. Free glycine methyl ester was prepared from its hydrochloride salt (Sigma) by using a modification of that reported by Frankel (10). In the modified procedure, 50 g of glycine methyl ester hydrochloride was suspended in 300 mL of pure, dried diethyl ether and a stream of dry (over KOH) ammonia was passed through the suspension with constant stirring at 0 "C. After 2-3 h, the solution containing the ester was separated from the suspended ammonium chloride by filtration. The ether was removed with a Rotavapor (Buchii)at room temperature. The free glycine methyl ester was distilled at 20 mm pressure and 49 "C. The purity of the ester was satisfactory by chromatographic and spectroscopic procedures. NJV-Dimethylglycinewas obtained from its hydrochloride salt by slowly eluting through an Amberlite CG-400 (Mallinckrodt) anion exchange column (hydroxyl form), with a 70:30 methanol-water mixture. The resulting solution was evaporated to dryness and the purified N,N-dimethylglycine obtained by recrystallization from an ethanol-ether mixture. The product obtained melted at 181-182 "C. The purity and the identity of the compound were verified by TLC and spectrometry. Solutions. Solid chemicals were weighed directly into volumetric flasks and diluted to volume with boiled degassed triply distilled water. These samples were stored under nitrogen. An EDTA solution was prepared and standardized against primary standard calcium carbonate by a complexometric titration method. The cadmium perchlorate solutions were then standardized with the EDTA solution. To minimize dilution effects of all metalligand solutions, we adjusted the pH of each by adding either concentrated perchloric acid (70%)or sodium hydroxide (25%). Measurements. All pH measurements were made with an Orion Model 701.4 Digital Ionanlyzer (pH AO.001, MV k 0.1) against a saturated calomel reference electrode. The temperature of each solution was maintained at 25.0 i 0.1 "C by thermostating the titration cell with water circulating from a Hotpack refrigerated bath. A calibration curve of potential vs. pH for the Fisher activity buffers 4.00, 7.00, and 10.00 was generated from four
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
Table I. '13Cd Chemical Shifts, pH+lPpm and pKa2 - pH+lppm Values for Cadmium Complexes ligand CH,COOH CH,NH, NH,(CH,)COOH NH,(CH,),COOH NH,(CH,),COOH NH,(CH,),COOH NH,CH,COOCH, N( CH,)2CH,COOH
PKal"
pKaza
4.75 2.35 3.60 4.23 4.43 2.08
10.74 9.78 10.19 10.43 10.75 7.66 9.80
First and seconci ionization constants of ligands ( I 7). positive change of 1 ppm in chemical shift.
RESULTS AND DISCUSSION
-28.33 -0.75 -5.83 -19.95 -23.58 -25.39 -2.46 -8.50
6.1 3.9 5.5 6.0 6.4 3.9 4.3
Chemical shift for curve minimum.
potential readings per solution. pH values of the samples were determined by linear regression analysis. All NMR measurements were made at a magnetic field of 1.88 T on a Varian Model FT-80 NMR spectrometer at ambient temperature (26-29 "C). Changes in chemical shift resulting from variations in probe temperature were considered to be negligible (11). The spectrometer was locked internally at 12.2 MHz on the 'H resonance of DzO. Since dioxane was added as an internal standard for the 13CNMR studies, a 4-mm coaxial insert was used to hold the DzO. For the l13Cdmeasurements, the 1.0 M Cd(C104)2 was contained in a 5-mm tube as an external reference. A 2-mm coaxial insert with D20was mounted within the 5-mm tube. Both of these were mounted coaxially within the 10-mm tube used to hold the sample solution. 13C spectra were obtained at 20.000 MHz by using either a 43" flip angle or 90" flip angle with a 1.20-1.68-s acquisition time and no pulse delay. Although dioxane was added as a secondary standard, all 13C chemical shifts are reported vs. Measi. The resonance of the I3C nuclei of dioxane was 67.39 ppm downfield from that of Me4Si. lI3Cd spectra were obtained at 17.6 MHz with a Varian Model FT-80 broad-band VT probe and a Harris PRD 7838 frequency synthesizer. The pulse width and acquisition time used were different for different ligands. Due to the negative gyromagnetic ratio of lL3Cdnucleus, all spectra were obtained with the decoupler off, or with N.O.E. suppressed decoupling. Aqueous solutions of either 1.0 M Cd(C104)2or neat Cd(CH& were used as external standards. However all chemical shifts reported are referenced to 0.1 M Cd(C104)2. This was done in accordance with literature values ( I ) where 1.0 M Cd(C104)2and neat Cd(CH3), are at -1.85 ppm and 642.9 ppm from 0.1 M Cd(C104)2,respectively. Chemical shifts at higher and lower frequencies than the reference are reported as positive and negative values, respectively. In these studies no bulk susceptibility correction was applied since it was negligible. Plots of chemical shift vs. pH for solutions of cadmium (0.3 M) and a series of monoaminomonocarboxylic acids (0.6 M) are shown in Figure 1. These solutions as well as all others were prepared from the perchlorate salts of the particular compounds studies. The perchlorate salts were chosen to minimize anion effects on chemical shift. At lower solution pH, shifts to negative values (reported in parts per million) were observed as the result of cadmium complexing initially through the acidic end of the ligand followed by shifts to positive values as the solution p H was increased. Positive chemical shifts are consistent with cadmium binding with the amino site. These observations are in agreement with earlier investigations of cadmium nitrate binding with acetic acid and ammonia (12, 19) as well as with the diverse ligand effects reported by Kostelnik (8). In both studies binding by oxygen donor ligands resulted in shielding of the cadmium nucleus and shifts to lower frequency whereas binding by nitrogen donor ligands resulted in cadmium deshielding and shifts to higher frequency. Of interest from Figure 1 are changes in the overall profiles of the curves obtained between glycine and the other amino acids studied. On an absolute basis as the length of the central
shift,b P P ~ PH+lppmC P K , ~- PH+lppm
I
-30'
2
4.6 5.9 4.7 4.4 4.4 3.8 5.5 Determined for a
I
I
I
1
I
10
6 PH
Figure 1. Ii3Cd chemical shift of 0.3 M Cd(CIO,), with 0.6 M NH,(CH,),COOH: n = 1, glycine (0);n = 2,@-alanine(a);n = 3, y-ABA (A);and n = 5, t-ACA (+).
carbon chain was increased, the more pronounced the chemical shift minimum. However, on a relative basis, with each carbon addition smaller and smaller changes were observed. Values of chemical shift for each curve minimum are tabulated in Table I. Except for glycine, the onset of the upward trends are consistent with the pK, values of the amino acids investigated. These positive changes in chemical shift are indicative of the complexation of Cd(I1) with the amino groups. The pH value (defined as pH+,,,,) corresponding to a positive change of 1ppm in the chemical shift from the curve minimum has been tabulated in Table I. This point was chosen to represent the beginning of the upward chemical shift. Also reported in Table I is the calculated value for the different between the pK, and pHflppmfor each acid. Differences (pK, - pH+,,,,) of 4.4 log units were observed for e-aminocaproic acid and y-aminobutyric acid and 4.7 log units for @-alanine. This difference increased to 5.9 log units for glycine. Additionally, changes in chemical shift for glycine at lower solution pH were relatively small compared to the other amino acids as well as acetic acid. Plotted in Figure 2 are 13Cdata from the carbonyl carbon of the free amino acids and their corresponding cadmium complexes. These are shown as opened and darkened points, respectively. The 13Cdata also show glycine binding weakly with cadmium through the carboxyl group a t values of solution p H below 4 and binding more strongly with the amino group a t higher pH.
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
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t
901 a E 60VI .c
” 0
s
.c
301751
0-
2
6
10
6
2
Figure 2. 13C chemical shift of carbonyl carbon in 0.6 M NH,(CH,),,COOH and its cadmium complex of 0.3 M Cd(CiO,),: glycine (A), Cd-glycine (A);@-alanine(0), Cd-@-alanine (+); y A B A (0),Cd-yABA ( 0 ) ;+ACA ( O ) , Cd-e-ACA (W).
The 13C trends as in the case of the l13Cd trends are explainable only partially by electronic contributions resulting from extension of the central alkyl chain. However, the data are consistent with the idea of the promoted deprotonation of the glycine amino group by cadmium(I1) ion through the formation of a more stable chelate ring. This is seen in Figure 2 as a shift in the bound vs. the unbound curve of approximately 4 p H units for glycine. The shift difference (bound vs. unbound) decreases for the longer amino acids. The formation of five-membered cyclic compounds has been suggested by others (8,23). After Rabenstein and co-workers (8)examined by proton NMR heavy metal complexes of polyglycine peptides, they ,proposed that certain divalent metal ions form five-membered ring chelates by simultaneously coordinating through the N-terminal nitrogen and carbon:yl oxygen atoms. Additionally, when Krishnan and Plane (13) investigated glycine complexes in aqueous solutions by Raman spectrometry, they found a weak line a t 150 cm-l for zinc solutions. The line was suggested to be the result of a chelate-ring bending vibration. Although they did not report similar results for the cadmium-glycine system, it is not unreasonable to assume like behavior for other chemically similar ions such as cadmium. When the chelate ring size increases beyond five, the stability of the complex which is formed decreases dramatically clue to the increased strain (14). I n the case of the data shown in Figure 1,the formations of cyclic compounds are unlikely for y-ABA and 6-ACA ligands since this would result in seven- and nine-membered chelates, respectively. Additional supporting evidences for the formation of a cadmiumslycine five-membered chelate are the data reported by Low (15). Although it is recognized that a complex formed in solution may be different than that found in the crystalline form, it is interesting that for a crystal with the formula of Cd(NH2CH2C0&-H20,the complex is octahedral with two glycine ligands chelating with cadmium in the transplanar array while the other two coordination positions are occupied1 by carbonyl oxygens of neighboring glycine ligands. To investigate the above trends and arguments further, we examined cadmium complexes of compounds similar to but more sterically hindered than glycine. Plots of chemical shift,
10
PH
PH
Figure 3. ‘I3Cd chemical shifi of 0.3 M Cd(Ci02)2complexing with 0.6 M methylamlne (+), glycine (O),glycine methyl ester (A),and N , N dimethylglycine I.(
B PH 4.47
rI
C pH 5.99
I
D pH 6.19
I
I
Figure 4. I3C spectra of Cd-glycine methyl ester: (A) pH 1.49, (a) 41.30, (b) 54.39, (c) 169.75 ppm; ( 6 )pH 4.47, (a) 41.34, (b) 54.37, (c) 169.75, (d) 42.63 ppm; (C) pH 5.99, (a) 41.34, (b) 54.34, (c) 170.6, (d) 42.62, (e) 50.10 ppm; (D) pH 6.19, (d) 42.76, (e) 50.10, (c) 175.73 ppm. Peak r is dioxane, reference, 67.39 ppm from Me&.
vs. pH for the data obtained appear in Figure 3. The pK, values, pH+lppmvalues, and the differences between pK, and pH+lppm(pK,, - pH+lPpm)for each compound are tabulated in Table I. The chemical shift of methylamine remained relatively unchanged over a wide pH range. Although no chemical shift data were measurable for solutions of methylamine with a pH above 6.4 because of solubility problems, the chemical shift profile might be expected to be similar to that of the Cd-NH3 complex (12, 19).
496
ANALYTICAL CHEMISTRY, VOL. 55, NO. 3, MARCH 1983
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~
I
$. 1t 200
j 5
l
4 100
u
t
2
10
6 PH
Flgure 5. '%d chemical shift vs. pH for mole ratio studies of cadmium-glycine complexes, 0.3 M Cd(C104)2complexing with glycine of various concentrations: 0.3 M (B), 0.6 M (e), 0.9 M (A),and 1.2 M (0).
Significant negative chemical shifts were not observed for the cadmium-glycine methyl ester system. This is reasonable since the possibility of cadmium binding through the deprotonated oxygen is removed. The I13Cd chemical shift beyond pH 4.5 was found to rise more sharply than expected based on the other observed data. This is explainable in terms of a catalytic hydrolysis of the ester by the metal ion at higher solution pH via a five-membered cyclic intermediate. This argument is supported by other published data (16) as well as by the 13C results obtained from the Cd-glycine methyl ester at different values of solution pH. Spectra from these experiments are shown in Figure 4. At a pH value of 1.49, only three peaks corresponding to the methyl (a), methylene (b), and carbonyl (c) carbons of glycine methyl ester were observed as shown in Figure 4A. However, at higher pH values, partial hydrolysis of the ester produced two additional peaks (Figure 4B,C). Peaks d and e are the methoxylate and the methylene carbons, respectively, of glycinate, the hydrolyzed product of the ester. At a solution pH of 6.19, as shown in Figure 4D, the ester was hydrolyzed completely into glycinate and methanol. For solutions with pH values higher than 6.19, the l13Cd chemical shift curve rises sharply which is likely due to the cyclic cadmium-glycinate complex. Based only on electronic considerations, the nitrogen in N,N-dimethylglycine is more basic than in glycine. This increased basicity should have resulted in increased cadmium binding. However, because the steric availability of the lone pair electrons of the nitrogen are hindered partially by the two attached methyl groups, the formation of the chelate ring was found to be reduced. This is shown in Figure 3 both by a small negative shift in the curve minimum and by a slight delay in the onset of the upward chemical shift at the higher pH range. For N,N-dimethylglycine, the difference, pKa2pH+lppm,was 5.5 log units which was similar to the glycine complex (5.9) and also was likely the result of the formation of the cyclic complex. The small difference in the pKa2 pHflppmvalue which was observed is reasonable in view of value for the mesteric considerations. The pKa2- pH+lppm thylamine complex was similar t~ values for yABA and eACA where ring formation is unfavored.
Following the above investigations, mole ratio studies for glycine were carried out. Shifts in the lI3Cd resonance as a function of pH for various metal to ligand ratios are shown in Figure 5 . Because of solubility problems encountered in the 1:l and 1:2 (cadmium:glycine) systems, measurements on solutions with pH values above 7 were not possible. Nevertheless, a slight leveling in the chemical shift curve at approximately 70 ppm was found at pH 7 for the 1:1 system. Additionally based on the low temperature studies of Ackerman (18) a plateau in the chemical shift curve at a value of about 150 ppm seems reasonable for the 1:2 system. The 1:3 and 1:4 solutions resulted in similar plateaus between 240 and 250 ppm. Ackerman et al. (18) using W d NMR have studied cadmium-glycine complexation at subzero temperatures by emulsifying solutions with a mixture of sorbitan tristearate, heptane, and water. At pH values of 7 , 8 , and 9, and a temperature of -55 'C, four peaks were detected at chemical shift values of -77, +55, +153, and +263 ppm. Although it was suggested by Ackerman that these peaks were the result of the 1:1, 1:2, 1:3, and 1:4 complexes, other studies (12) have shown that cadmium ions, in the presence of nitrates, form complexes in aqueous solution with the most negative value for the chemical shift at approximately -80 ppm. It therefore seems reasonable to suggest that the peak at -77 ppm reported by Ackerman and co-worker was the cadmium-nitrate complex and that the other peaks at 55,153, and 263 ppm were from the 1:1, 1:2, and 1:3 species, respectively. These results thus are consistent with the data obtained in the current studies. Although large changes in chemical shift for the 1:1, 1:2, and 1:3 systems were observed a t higher solution pH, only a small difference was noted between 1:3 and 1:4 systems. These results suggest that the highest order complex formed for the cadmium-glycine system is octahedral, with three glycinates forming three five-membered chelate rings. These rings occupy the six coordination sites of the cadmium ion. During the review process of the current paper, Jakobsen and Ellis (20) have reported the solution structure of cadmium-glycine complexes by Il3Cd NMR in supercooled aqueous solutions. By using lSN-enrichedglycine and carrying out studies at -40 "C, they have found a singlet at -35.9 ppm, a 1:l doublet at 53.6, a 1:2:1triplet at 153.9 ppm, and a poorly resolved multiplet at 262.8 ppm. These chemical shifts and I13Cd-15N multiplicities are consistent with complexes of Cd-Gly, Cd-Gly,, and Cd-Gly3. Likewise these results strongly support the conclusions set forth in this publication. Registry No. '13Cd, 14336-66-4; 13C, 14762-74-4.
LITERATURE CITED (1) Cardin, A. D.; Ellis, P. D.; Odom, J. D.; Howard, J. W.. Jr. J. A m . Chem. SOC. 1975, 97, 1672-1679. (2) Maciel, G. E.; Borzo, M. J. Chem. SOC.,Chem. Commun. 1973, 394. (3) Ellis, P. D.; Waish, H. C.; Peters, C. S.;Codrington, R. J. Magn. Reson. 1973, 1 1 , 431-436. (4) Armitage, M.; Pajer, R. T.; Miterkamp, A. J. M. S.; Chlebowski, J. F.; Coleman, J. J. Am. Chem. SOC. 1976, 98,5710-5712. (5) Sudmeier. J. L.; Bell, S. J. J. Am. Chem. SOC.1977, 99, 4499-4500. (6) Bailey, D. B.; Ellis, P. D.; Fee, J. A. Biochemistry 1980, 19, 591-596. (7) Jonsson, N. B. H.; Tibell, L. A. E.; Evelhoch, J. L.; Bell, S. J.: Sudmeier, J. L. R o c . Nati. Acad. Sci. U . S . A . 1980, 77,3269-3272. (8) Kostelnik, R. J.; Bothner-By, A. A. J. Magn. Reson. 1974, 14, 141-151. (9) Rabenstein, D. L.; Liblch, S. Inorg. Chem. 1972, 1 1 , 2960-2967. (IO) Frankel, M.; Katchaiski, E. J. A m . Chem. S O C . 1942, 64, 2264-2268. (11) Haberkorn, R. A.; Que, L., Jr.; Gillum, W. 0.; Holm, R. H.; Liu, C. S.; Lord, R. C. Inorg. Chem. 1976, 15, 2408-2414. (12) Wang, S. M.; Greenberg, M. S. Presented at the 12th Regional Meeting of the American Chemical Society, Pittsburgh, PA, Nov 1980. (13) Krishnan, K.; Plane, R. A. Inorg. Chem. 1967, 6, 55-60. (14) Irving, H.; Williams, R. J. P.; Ferrett, D. J.; Williams, A. E. J. Chem. Soc. 1954, 3494-3504. (15) Low, B. W.; Hirshfeld, F. L.; Richards J. A m . Chem. SOC.1959, 81, 44 12-441 6.
Anal. Chem. 1983, 55, 497-501 (18) Bender, M. L.; Turnguest, 8. W. J . A m . Chem. SOC. 1957, 7 9 , 1889- 1893. (17) Sllien, L. G.: Martell, A. E. "Stability Constants of Metal-Ion Complexes"; The Chismlcal Society: London 1964. (18) Ackerman, M. J. 6.; Ackerman, J. J. H. J . fhys. Chem. 1980, 8 4 , 3151-3157.
497
(19) Wang, S.M. Ph.D. Dissertation, Kent State University,Kent, OH, 1981. (20) Jakobsen. H. J.; Ellis, P. D. J . fhys. Chem. 1981, 8 5 , 3367-3369.
for review September 309 lg8'. Resubmitted 23, 1982. Accepted November 8, 1982.
Nonsegmented Rapid-Flow Analysis with UltravioIetNisible Spectrophotometric Determination for Short Sampling Times Peter W. Alexander" and Amlius Thallb Department of Analytical Chemistry, University of New South Wales, P.O. Box 7, Kensington, New South Wales, Australia 2033
The development of a nonsegmented, rapid-flow system wlth short sampling times In shown to allow hlgh-speed sampling with a UV-vislble spectrophotometric detector. For methyl orange as a model color indlcator, a sampling rate of 360 samples h-' is obtained experimentally with a 3-s sampllng time followed by a 7 a flush In a narrow bore (0.5 mm), short pathlength (800 mm) flow. The sampllng is performed manually with a precision of 0.85% relatlve standard devlatlon (RSD) and negligible carry-over, wlth a dwell time of 8 s. Determination of chloride by a fast chemical reactlon Is used as an example to compare rapld-flow analysis wlth flow-injectlon and bubble-gating techniques.
Studies of theoretical factors (1)and sample peak formation ( 2 ) in air-segmented continuous flow analyzers have been reported. It was concluded (2) that operation with short sampling times with sharp sample peak formation is inferior t o steady-state operation, particularly when computer acquisition of the sample data is required. It is clear, however, from the work of Evenson e t al. (2),that increased sampling speed can be achieved bj7 operation of continuous-flow systems under non-steady-state conditions. Increased sampling sipeeds have instead been obtained by bubble-gating techniques (3-5) and by the development of various flow-injection analysis (FIA) systems (6-10). FIA operates under non-steady-state conditions and gives sharp sample peak readout, a ~reviewed i by several authors (6-10). A comparison of the segmented-flow and FIA systems has been described recently ( 5 ) . In this study, we report the results obtained by using a nonsegmented flow analysis system designed to maintain rapid-flow velocity through the system. With the improved hydraulic control and more sensitive UV-vis spectrometric detectors now available, we show that a rapid-flow analysis system can be operated a t very short sampling times to give precise data with no carry-over between samples, without the need for either air-segmented flow or flow injection of the sample. With the short sampling times, sampling rates as high as 360 samples h-' are demonstrated with a peak readout equivalent t o the flow-injection technique b u t without the need for a loop injector. In previous rapid-flow studies (11-14) we showed that both air-segmented and nonsegmented flow can be used to give very rapid sample analysis with ion selective electrodes (12),polarography (11, 13),and plasma emission spectrometry (14) as detectors in flow systems. In this work, however, we have miniaturized the complete flow system with a UV-vis spec-
trometric detector and show that nonsegmented flow can be used for fast sample-throughput with manual sampling provided rapid-flow velocity and short pathlength flow systems are used with fast chemical reactions.
EXPERIMENTAL SECTION Reagents and Solutions. Methyl orange (May & Baker) was used as an acid-base indicator for flow-analysis experiments. A stock solution of methyl orange (50 mg L-') was prepared in distilled water. Sample solutions in the concentration range 1-6 mg L-l were then prepared by serial dilution of the stock solution. The acid reagent was AnalaR hydrochloric acid (Ajax Ltd.) prepared in 0.1 M concentration in aqueous solution. The reagent for chloride determination was prepared according to a published procedure (9) by dissolving and mixing mercuric thiocyanate (0.626g), ferric nitrate (30.3 g), and concentrated nitric acid (4.72g) in water, adding methanol (150 mL), and diluting to a final volume of 1 L. The standard chloride solutions were prepared from a stock solution of sodium chloride containing 1.648 g in 1 L of distilled water. Chloride standards in the range 0.1-10.0 mg L-' were prepared by serial dilution in distilled water. Instrumentation. A Desaga Type 131-900 peristaltic pump with six channels equipped with a speed controller was used with a Pye-Unicam (SP-400)spectrophotometer, fitted with a flowthrough cuvette of 10 mm pathlength and 20 pL volume (Zeiss). The spectrophotometer was coupled to a Linear strip chart recorder. All pump tubing and transmission tubing was of PVC (Elkay). Procedure. Sample solutions of methyl orange were pumped into the acid reagent stream, as shown in Figure 1A. The tubing for the sample line was 0.5 mm internal diameter and the mixing point was a zero dead-volume T-piece constructed from stainless steel with 0.5 mm bore. After mixing of the sample and reagent, the solution was pumped through uncoiled tubing (0.5 mm bore) to the optical flow cuvette in the spectrophotometer set at 510 nm and then to waste. A debubbler constructed of glass with 1.0 mm bore was inserted in the flow path just prior to the cuvette, in order to remove intersample bubbles. The line lengths of each flow segment are shown in Figure lA, giving a total flow path of 80 cm for the sample solution. The experimental procedure consisted of initially pumping the flush solution (water) through the system continuously until a steady base line absorbance reading was established on the chart recorder. Samples were then aspirated into the system by manual sampling with timing measured using a stopclock, followed by a flush solution also timed accurately between each sample. The absorbance readout was continuously recorded at the fixed wavelength of 510 nm. The only air bubbles introduced into the system occurred between each sample and the following flush solution. The bubbles were removed by use of the debubbler. Flow rates were controlled by use of the pump speed controller, using fixed-bore pump tubes of 0.63 mm for the sample line and 1.02 mm for the reagent for all experiments. The maximum flow rates possible using the pump tubes in this system are shown in
0003-2700/83/0355-0497$01.50/00 1983 American Chemical Societv
