Anal. Chem. 1994,66,793-797
Kinetics and Equilibria of the Zn-EDTA-PAR Postcolumn Reaction Detection System for the Determination of Alkaline Earth Metals Charles A. Lucy’ and Huyen N. Dlnh Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N IN4
Zn-EDTA-PAR is the archetypical “displacement” reagent for the postcolumndetection of metals in ion chromatography. However, only a superficial understanding of the mechanism of this system exists. The displacement reaction of the ZnEDTA-PAR reagent was investigated using stopped and continuous flow methods. The kinetics and sensitivity of the Zn-EDTA-PAR reagent are strongly dependent upon the nature and the concentrationof the auxiliary complexingagent. Ethylenediamine was more effective than ammonia for the determination of alkalineearth metals, both in terms of reaction kinetics ( t l l 2 of 0.7 s for ethylenediaminevs 3.6 s for NH3 for Mg2+)and sensitivity (2-9-fold improvement). Furthermore, auxiliary agents suppress the direct reaction of PAR with metal ions such as Zn2+and so should be used sparinglyin postcolumn reagents. Postcolumn reaction is a sensitive and convenient means of detecting metal ions separated by liquid chromatography. In this technique, the effluent from the chromatographic column merges with a reagent stream containing a metallochromic reagent, and the resultant colored metal complex is monitored. Ideally, the reagent should form a stableintensely colored complex with the metals of interest and not form any complex with other metals. A wide variety of reagents fulfill these requirements.’q2 Examples include Arsenazo I for alkaline earth metal^;^ 4-(2-pyridylazo)resocinol (PAR) for t r a n ~ i t i o n ~and . ~ lanthanide metals;6 Arsenazo I11 for l a n t h a ~ ~ i d and e ~ - ~actinide metals;l0-I2 Tiron for aluminum and its nonlabile complexes;13 and diphenylcarbazide for hexavalent ~ h r 0 m i u m . l ~
Of these reagents, PAR is applicable to the widest range of metals,’5 although only Mn, Fe(II/III), Co, Ni, Cu, Zn, Cd, Pb, and the lanthanide metals are typically monitored with PAR as a postcolumn reagent.16 Addition of a solution containing equimolar concentrations of Zn2+ and EDTA to PAR extends the applicability of the reagent to include the alkaline earth metal^.^ This Zn-EDTA-PAR postcolumn reagent is widely quoted in the literature and has become the prototype for other displacement postcolumn reagents for spectros~opic,~~ fluorescent,I8 chemilumine~cent,~~ and coulometric20 analysis. Despite the significance of the Zn-EDTA-PAR system in postcolumn detection of metals, the kinetic and equilibrium behavior of this archetypical displacement detector is not well understood. This is best demonstrated by the trend in the required reaction conditions: initial studies found that the reaction was rapid and did not require a delay l00p;3 later studies used 2 m of reaction coil;21and most recently, 320 cm of reaction coil and 60 O C were necessary.22 The objective of this paper is to determine the chemical factors affecting the kinetics and equilibria of Zn-EDTA-PAR, thus enabling optimization of the Zn-EDTA-PAR and other displacement postcolumn reagents on a chemically sound basis. BACKGROUND Equilibria. Zinc forms an intensely colored, stable (log 02 = 17.1)23complex with PAR. However, in the presence of ED,TA,formation of the Zn-EDTAchelate (log& = 16.44)24 will predominate:
Zn(PAR),
+ EDTAb + 2PAR- + Zn(EDTA)2-
(1)
(1) Cassidy, R. M.; Karcher, B. D.In Reaction Detection in Liquid ChromaaZn2+aYaZn(EDTA) K>n(EDTA) tography; Krull, I. s.,Ed.; Chromatographic Science Series, Vol. 34; Marcel Dekker: New York, 1986; Chapter 3. kackground aZn2+(aPAR-) 202,Zn(PAR), rB12*Zn(PAR)t (2) (2) Dasgupta, P. K. J . Chromatogr. Sci. 1989, 27, 422-448. (3) Arguello, M. D.;Fritz, J. S. Anal. Chem. 1977, 49, 1595-1598. (4) Cassidy, R. M.; Elchuk, S.Anal. Chem. 1982, 54, 1558-1563. Thus, essentially no Zn(PAR)2 exists in a mixture of PAR (5) Siriraks, A.; Kingston, H. M.; Riviello, J. M. Anal. Chem. 1990, 62, 11851193. (6) Heberling, S.S.;Riviello, J. M.; Mou, S.;Ip, A. W. Res. Dev. 1987, Sept, (15) Shibata, S. In Chelates in Analytical Chemistry; Flaschka, H. A., Barnard, 74-17. Marcel Dekker: New York, 1972; Vol. 4, Chapter 1. A. J., Jr., Us.; (7) Knight, C. H.; Cassidy, R. M.; Recoskie, B. M.; Green, L. W. Anal. Chem. (16) Heberling, S.S.;Riviello, J. M. Presentation at the 27th Rocky Mountain 1984, 56, 474-478. Conference, Denver, CO, July 1985. (8) Cassidy,R.M.;Elchuk,S.;Elliot,N.L.;Green,L.W.;Knight,C.H.;Rccoskie, (17) Bowles, C. J.; Bader, L. W.; Jackson, K. W. Talanra 1990, 37, 835-840. B. M. Anal. Chem. 1986, 58, 1181-1186. (18) Williams, T.;Barnett, N. W. Anal. Chim. Acta 1992, 264, 297-301. (9) Elchuk, S.;Lucy, C. A.; Burns, K. I. Anal. Chem. 1992, 64, 2339-2343. (19) Jones, P.; Williams, T.; Ebdon, L.Anal. Chim. Acta 1990, 237, 291-298. (10) Barkley, D.J.; Blanchette, M.; Cassidy, R. M.; Elchuk, S . Anal. Chem. 1986, (20) Takata, Y.; Fujita, K. J . Chromatogr. 1975, 108, 255-263. 58, 2222-2226. (21) Jezorek, J. R.; Freiser, H. Anal. Chem. 1979, 51, 373-376. (11) Elchuk, S.; Burns, K. I.; Cassidy, R. M.; Lucy, C. A. J . Chromaroar. 1991, (22) Yan, D.-R.; Schwedt, G. Fresenius Z . Anal. Chem. 1987, 327, 503-508. 558, 197-207. (23) Smith, R. M.; Martell, A. E. Critical Stability Constants, Amines; Plenum (12) Harrold, M. P.; Siriraks, A.; Riviello, J. J . Chromatogr. 1992,602, 119-125. Press: New York, 1975; Vol. 2. (13) Bertsch, P. M.; Anderson, M. A. Anal. Chem. 1989, 61,535-539. (24) Martell, A. E.; Smith, R. M. Critical Stability Constants,First Supplement; (14) Arar, E. J.; Pfaff, J. D. J. Chromatogr. 1991, 546, 335-340. Plenum Press: New York, 1982; Vol. 5.
-
0003-2700/94/0366-0793$04.50/0 0 1994 Amerlcan Chemical Society
Analytical Chem&try, Voi. 66,No. 6,March 15, 1994 703
and equimolar Zn2+and EDTA. Introduction of an alkaline earth metal ion, M2+, to this reagent does not result in the formation of an alkaline earth-PAR complex. Rather, the alkaline earth metal displaces zinc from the EDTA, which then reacts with PAR to form the colored Zn(PAR)2 chelate. The overall detection reaction is
+ M2++ Zn(EDTA)2-
2PAR-
Zn(PAR),
+
M(EDTA)~- (3)
-- P1Z,Zn(PAR)2K;,M(EDTA)
''detection
';,Zn(EDTA)
Z
= ~ M ( ~ P A R - Kdetection ) (4)
Thus despite Zn2+ forming a more stable EDTA complex than Mgz+,the displacement reaction 3 proceeds to the righthand side because of the stability of the Zn(PAR)2 chelate. Kinetics. The displacement reaction involves a two-step process: M2++ Zn(EDTA)'-
+
--
+ Zn2+
M(EDTA)'-
Zn2+ 2PAR-
(5)
Zn(PAR),
(6) The complexation of zinc with PAR is rapid, as will be shown below. Therefore, the metal displacement from EDTA must be rate determining. A general mechanism for the exchange of metal ions, M', with M-EDTA complexes has been proposed.25 This mechanism proceeds via several steps involving dinuclear transition states in which each metal coordinates with an iminodiacetate segment of EDTA: M(EDTA)'-
+ M'
kl
k-1
0
I \
0
/
M-N-N \
0
\
0
+ M'
__ k2
k-2
0
/ \
0
/ \
M-N-NT/M'
0
As predicted by this mechanism, experimental exchange rate constants correlate well with the stability of the metaliminodiacetate complexes.28Martell suggested that the fourth step is fast, and so the location of the rate-determining step depends on the relative dissociation rates of M (k3)to M' (k-2) from the M-EDTA-M' reaction intermediate. The relative rates of the forward (k3) and back (k-2) reactions are proportional to the relative instabilities of the metal-iminfor the initial metal ion, odiacetate linkages (~M.H*o/KM-IDA) zinc in the case of Zn-EDTA-PAR reagent, and the exchanging metal ion. Estimated values of the M-IDA linkage instabilty are26
Thus in the case of the Zn-EDTA-PAR reagent, the alkaline earth metal will dissociate from the intermediate reaction Martell, A. E. Coordination Chemistry, 2nd ed.;ACS Monograph 174; American Chemical Society: Washington, D.C., 1978; pp 165-168. The value for Zn'+ is from ref 25, p 167. Values for the alkaline earth metals were determined using kM.ns values from Table 1.1 and Figure I. 1 of ref 25 and iminodiacetate stability wnstants (8,)from Martell, A. E.; Smith, R. M. Critical Stability Constants, Amino Acids; Plenum Press: New York, 1974; VOl. 1.
794
Analytcal Chem&try, Vol. 66,No. 6, March 15, 1994
species (k-2) much more rapidly than will the Zn2+ (k3). Therefore, k3 will be the rate-determining step for the reaction of Zn-EDTA-PAR with alkaline earth metals. EXPERIMENTAL SECTION Apparatus. Stopped-flow kinetic studies were performed at 25.0 f 0.1 O C on a HPSF-56 high-pressure stopped-flow spectrophotometer (Hi-Tech Scientific, Salisbury, Great Britain) monitored at 540 nm by an Hi-Tech MG-10 monochromator interfaced with an Apple IIe microcomputer. Kinetic measurements were conducted under pseudo-firstorder conditions. Typically 4-8 runs were recorded and averaged for each set of experimental conditions. The HiTech data acquisition and analysis software (ADS-1) was modified to convert the photomultiplier output to absolute absorbance units and then calculate the pseudo-first-order rate constant, koa,in an iterative procedure using the infinite time absorbance as the adjustable parameter. The HPLC system used for the postcolumn reaction studies consisted of a Waters metal-free solvent delivery system (Model 625, Waters Associates, Milford, MA), a Rheodyne sampling valve (Model 9125, Rheodyne, Berkeley, CA) fit with a 50-pL loop, a fixed-wavelength (546 nm) detector (Model 440, Waters), and a recording integrator (Model 745, Waters). In most experiments, no column was present in the HPLC, Le., the system was run in a flow injection analysis mode. The postcolumn reagent was delivered by constant pressure pumping supplied by application of nitrogen pressure (50 psi) to a multireagent cylinder2'~28fit with a six-port low-pressure switching valve (Model R603 1V6, Rheodyne) to allow selection between up to six reagents. Low-pressure tubing and fittings (Alltech Associates) connected the postcolumn reagent delivery system to the column effluent stream. The carrier stream from the HPLC pump (0.5 mL/min) merged with the postcolumn reagent (0.4-0.5 mL/min, measured for each experiment) at the mixing tee (316 ss, 90° ports) and then flowed through either 50 cm of 0.010-in. i.d. polyether ether (PEEK) tubing or a tightly spiraled reaction coil (RXN 1000 Coil, Waters; 0.50-mm i.d. knitted Teflon tubing; 1000 pL volume). Studies of the stability of the PAR reagent solutions were performed on a Lambda array 3840 UV/Vis spectrophotometer (Perkin-Elmer) interfaced with a professional computer (Model 7300 PECUV Version, Perkin-Elmer). Reagents. All reagent solutions were prepared using deinoized water (Milli-Q Ultra Pure Water System, Millipore). Analytical-grade reagents were used throughout. Postcolumn reagent solutions were prepared by dissolving PAR (Merck) in the appropriate buffer, passing the solution through a 3 cm X 1.5 cm i.d. column of Chelex 100 (50-100 mesh sodium M Znform, Bio-Rad), and adding an aliquot of 5 X EDTA stock solution. The Zn-EDTA stock solution was prepared by combining stock Znz+and EDTA solutions using the ratio determined by prior titration of the 0.01 M Zn2+ (ZnCl2, Fisher Scientific) in 1 M hexamine with 0.01 M EDTA ( N ~ ~ H ~ E D T A - ~ BDH) H z O , using 0.2% xylenol orange as the indicator.29 All PAR solutions were stored in plasticz1 and in the dark, unless otherwise indicated. (27) Fossey, L.; Cantwell, F. F. Anal. Chem. 1983, 55, 1882-1885. (28) Fossey, L.; Cantwell, F. F. Anal. Chem. 1985.57.922-926,
Magnesium and zinc metal standard solutions were prepared from successive dilutions of certified atomic absorption standards (1000 ppm Reference Solution, Fisher Scientific). Calcium, barium, and strontium metal standard solutions were prepared by dissolving CaCl2-2H20, BaC126Hz0, and S r C l ~ 6 H 2 0respectively, , in deionized water. The desired metal concentrations were obtained from appropriate dilutions. The HPLC eluent was 0.050 M tartaric acid (Fisher) and 0.0012 M octanesulfonic acid (Janssen Chimica), adjusted to pH 3.4 with KOH. The eluent was passed through a cationic exchange resin (AG 50W-X8,50-100 mesh hydrogen form, Bio-Rad) and vacuum filtered through 0.45-pm nylon filters prior to use. The column was a 3.9 mm i.d. X 15.0 cm analytical column packed with 5 pm of c18 reversed-phase particles (Delta Pak C18 300 A, Waters). Unless otherwise indicated the concentrations in the tables and figures refer to the concentration in the final reaction solution and not those in the initial reagent solutions. RESULTS AND DISCUSSION PAR Stability. PAR solutions are unstable when either exposed to light or stored in glass bottles2' but are stabilized by ammonia buffers with a pH above 9.21v22Similar results were observed herein for ethylenediamine buffers. Azobenzene and its derivatives undergo a wide variety of photochemical r e a c t i o n ~ .Not ~ ~ surprisingly, Huckel calculations using the simple Huckel molecular orbital (SHMO) program31 reveal that the valence electrons in PAR concentrate at the lowest unoccupied molecular orbital (LUMO), the N-N portion of the molecule. In other words, promotion of an electron by light to the LUMO to form a radical is fairly easy and, consequently, so is degradation of PAR. Using plastic bottles and storing the reagent in the dark reduce the degree of photodegradation simply by reducing the irradiation. However, the presence of either ammonia or ethylenediamine was more important in ensuring the stability of PAR. PAR in pH 10 ammonia buffer was stable for greater than 2 weeks whether stored in the dark or continuously exposed to laboratory light. Purging the PAR solutions with nitrogen yielded a further small improvement in stability. Thus, all subsequent PAR and Zn-EDTA-PAR solutions were buffered to about pH 10 with ammonia or ethylenediamine, purged with nitrogen, stored in the dark, and used within 2 weeks of preparation. Stopped-FlowKineticStudiesof PAR and Zn-EDTA-PAR. Stopped-flow kinetic studies investigated the effect of experimental variables on the rate of the reactions involved in the Zn-EDTA-PAR postcolumn detection of metals. The direct reaction between Zn2+and PAR was rapid. A pseudo-firstorder rate constant of 85 s-l was observed for the reaction of 7.6 X 106 M Zn2+with 1 X 10-4 M PAR in 1 M ammonia at pH 10. Addition of 1 X 10-4 M Zn-EDTA to the solution did not affect the rate of reaction between Zn2+ and PAR (kob,60 f 24 s-l). However, for the same conditions the reaction rate of 2.1 X M Mg2+ with 1 X 10-4 M Zn(29) Almonte, W.; Hinman, A. S.; Malck, L.; Yeager, H. L. Chemistry 410 Laboratory Manual; University of Calgary; Calgary, 1989; pp 81-88. (30) Griffiths, J. Chem. Soc. Reu. 1972, I , 481-493. (31) Rauk, A. Orbital Interaction Theory of Organic Chemistry; Wiley-Interscience: New York, 1994.
EDTA-PAR (kob, 0.2 s-I) was much slower than that of Zn2+. This indicates that the displacement step (reaction 5 ) must be rate determining. Thereactionrate(kob,0.14f0.02s-1)between2.1 X 10-5 M Mg2+and 1 X lo" M Zn-EDTA-PAR was independent of pH for solutions buffered between pH 9.25 and 10.00 under a constant 1.0 M unprotonated ammonia (NH3). Likewise, variationof the concentration of Zn-EDTA between 1 X lo" and 4 X 10-4M at constant pH (1O.O), ammonia concentration (1.0 M), and PAR concentration (1 X 10-4 M) yielded no significant change in the reaction rate (kob,0.20 f 0.03 s-l), Nevertheless, these variables were held constant within each subsequent experiment. Ammonia is commonly added to Zn-EDTA-PAR postcolumn reagents to buffer the solution and to prevent metal hydroxide formation. Studies were performed on the effect of increasing the ammonia concentration on the displacement rate. The reaction rate varied between 0.029 ( t l p = 24.3 s) and 0.19 s-l (t1/2 = 3.6 s) and displayed a second-order dependence on the concentration of unprotonated ammonia ([NH3]) at low ammonia concentrations (10.5 M, slope 2.09; r = 0.9994) and a first-order dependence on [NH3] at higher concentrations (10.5 M, slope = 0.89; r = 0.9994). Such variable order behavior was reported previously for the decomplexation kinetics of nickel aminopolycarboxylate complexes by cyanide.32 These results indicate that at least two ammonia are incorporated within the transition state for the displacement of Zn2+ from EDTA. Metal hydroxide formation prohibited investigations at the low ammonia concentrations required to determine the absolute number of NH3 involved in the transition state. Regardless, the key conclusion drawn from this study is that the maximum displacement rate observed in ammonia buffer is slower (tll2 = 3.6 s) than is desirable in a postcolumn detector. Also, further increases in the ammonia concentration would yield only minor improvements in the reaction rate. Thus, another means of enhancing the reaction rate is required. A second auxiliary reagent that has been used with ZnEDTA-PAR is ethylenediamine (en). However, previously ethylenediamine was added to the eluent solely to optimize the separation of the metal ions3J3-36 and not for enhancing the postcolumn detection. In the presence of ethylenediamine, the displacement reaction proceeds much more rapidly than observed previously for ammonical solutions, with half-lives in the range 4.3-0.7 s observed for 0.019-0.225 M ethylenediamine. The dependence of the reaction rate on ethylenediamine concentration is approximately first order (slope0.84; r = 0.92). This agrees with previous studies of the effect of ethylenediamine on the dissociation of metal-aminocarboxylate complexes.37 Most importantly, the reaction rate ( t l p = 0.7 s) is now appropriate for postcolumn reaction detection. The presence of reagents in addition to ethylenediamine can have a marked effect on the reaction between 1 X lo-+ (32) Coombs, L. C.; Vasiliadcs, J.; Margerum, D.W. Anal. Chem. 1972,4423252331. (33) Yan, D.-R.; Schwcdt, G. Anal. Chim. Acta 1985, 178, 347-353. (34) Yan, D.-R.; Schwcdt, G. Fresenius 2.Anal. Chem. 1985, 320, 325-329. (35) Yan, D.-R.; Schwedt, G. Fresenius 2.Anal. Chem. 1985. 320, 252-257. (36) Yan, D.; Zhang, J.; Schwcdt, G. Fresenius Z . Anal. Chem. 1989,335,687691. (37) Carr, J. D.; Libby, R. A.; Margerum, D. W. Inorg. Chem. 1%7,6,1083-1088.
AnatyticalChemWy, Vol. 66,No. 6,Mer& 15, 1994
795
70 I
Figure 1 also shows the response of Zn-EDTA-PAR to Mg2+. Ethylenediamineformsonly a weakcomplex with Mg2+ ( a M 8 ranged from 0.994 to 0.77 for the ethylenediamine concentrations studied). Each Mg2+is then free to displace one Zn2+ from its EDTA complex. Once free however, the Zn2+will be subject to the competitive complexation between PAR and ethylenediaminediscussed above. The net result is that the sensitivity of the Zn-EDTA-PAR reagent in ethylenediamine buffer is essentially the same for Mg2+ as Zn2+. 2 0 I , , . , I Table 1 shows the response of the Zn-EDTA-PAR reagent 0.00 0.05 0.10 0.15 0.20 0.25 for Mg2+in ammonia buffers. The sensitivity of the reagent for Mg2+ is much less than that of Zn2+, and the relative [ethylenediamine], mol/L sensitivity (MgZ+/ZnZ+) decreases as the ammonia concentration is increased. The decrease in Mg2+sensitivity results Fbun 1. Effect of ethylenediamine on the detector senrltlvlty for Zn” and Mg2+ under equlllbrlum postcolumn reaction detection from the formation of M ~ ( N H s ) , ~ +The . fraction of free condltlons. Symbob: ( 0 )Zn2+ for t,,,,, = 80 8; (B)Zn2+ for t,,,,, = 1.8 ( a M S z + ) decreases from 0.66 to 0.22 as [NH,] was magnesium s;and (A)Mop+ for t,,,,, = 80 s. Experhentaiconditions: mode, flowincreased from 0.37 to 1.5 M. Thus, the presence of ammonia Injection analysb; carder eolutlon, 0.5 mL/mln water; reagent solution, 0.5 mL/mln of 2.0 X lo-‘ M Zn-EDTA-PAR; pH, 10.0. decreases the sensitivity of the Zn-EDTA-PAR reagent to alkaline earth metals by (i) decreasing the amount of Zn2+ displaced from the EDTA complex and (ii) decreasing the proportion of the displaced Zn2+ which complexes with PAR. In conclusion,while the addition of an auxiliary agent such as ammonia or ethylenediamineto a Zn-EDTA-PARreagent 0.373 59 40 is desirable to stabilize the PAR reagent and to enhance the 46 23 0.671 1.49 32 15 reaction rate, auxiliaryagents will also suppressthe sensitivity. Ethylenediamineis superior to ammonia as an auxiliary agent Ekperimental conditions: mode,flow i ‘ectionanal is.carrier solution 0.5 mL/min.water; re enteolutionY.0 x ~ W M ~ ~ E D T A - within the Zn-EDTA-PAR reagent as it results in faster PAR;ph, 9.Mreaction time., 8. Defied as the peak area divided reaction kinetics and less inhibition of Zn(PAR)* formation. by the concentration of metal injected. Postcolumn Detection Sensitivity. In the design of a postcolumn reactor, the reaction time can either be long so as to allow equilibrium to be achieved or short. While the M Zn-EDTA and 2.1 X M Mg2+. In 0.075 M latter approach is less sensitive,its simplicity (directlyconnect ethylenediamine(pH 10.0), the reaction rate was 0.77 f 0.13 the mixing tee to the detector) and minimal peak broadening s-l. Addition of 0.5 M tris(hydroxymethy1)aminomethane make it attractive. Under such preequilibrium conditions, (Tris), a noncomplexing buffer, reduced the rate to 0.43s-l, the detector sensitivity will be a function of both the reaction presumably due to the increased ionic strength. Alternatively, kinetics and equilibrium effects, as discussed above. the addition of 0.375 M ammonia (pH 10.0) to the 0.075 M Figure 2 shows the effect of increasing ethylenediamine ethylenediaminecaused a more severe reduction of the reaction concentration on the detector sensitivity under preequilibrium rate to 0.20 s-I. Such retardation of the ethylenediaminereaction conditions for Mg2+, Sr2+, and Ba2+ and equilibrium assisted dissociation of a metal by ammonia has been reported conditions for Zn2+. As discussed above, the Zn2+ sensitivity p r e v i ~ u s l y .Thus, ~ ~ in order to optimize the reaction rate, decreases due to ethylenediamine complexation of the metal ethylenediamine should be used as an auxiliary complexing ion. The sensitivity behavior of Zn2+ is approximated as an agent in a noncomplexing buffer of minimal ionic strength. (r = 0,991) in Figure 2. exponential function Equilibrium Studies. Figure 1 and Table 1 display the detection sensitivity for Zn2+ in the presence of ethylenediThe detection sensitivity of the alkaline earth metals increases with increasing ethylenediamine to a maximum at amine and ammonia, respectively, under conditions in which approximately 0.04 M (in the reaction mixture) and then the reaction has achieved equilibrium (m, tlxn = 1.8 s; 0 and decreases as the ethylenediamine concentration increases A, tlm = 60 s). In both cases the response factor, defined as the peak area per unit concentration, decreases as the further. At low ethylenediamineconcentrations,increases in the auxiliary complexing agent concentration result in more concentration of auxiliary agent increases. This behavior results from the auxiliary agent competing with PAR for rapid displacement kinetics, and thus the reaction proceeds coordination with Zn2+. The curve in Figure 1 is the response much further toward equilibrium during the 1.8-s reaction factor predicted using literature stability constants for the time. However by 0.04M ethylenediamine, the reaction halfformation of Zn(en),2+.23 The general behavior predicted is life is on the order of the reaction time, and so further increases in the reaction rate result in smaller gains in sensitivity. Under in agreement with the observed behavior for Zn2+. The these conditions, the decreasing sensitivity due to the Zn2+ discrepancies between the predicted and observed behavior at high auxiliary agent concentrationsare believed to result from complexation with the auxiliary agent overwhelms the small uncertainties associated with the stability constants and the increases in sensitivity from the faster reaction rate. Thus, varying ionic strength. the overall sensitivity decreases. To confirm that the dee
i
8
796 A N M h I - b y ,
Vd. 66, No. 6, h&M 15, lfifi4
0:
2
70
X
8 u
56
4
28
42 *i W
\
5 ce
24
e
2
14
o 0.00
0.05
0.10
0.15
0.20
0.25
[ethylenediamine], mol/L Flgwr 2. Effect of ethylenedlamlne on the detector senstthrity for Zn2+ and alkaline earth metals under preequlllbrlum (&, = 1.8 8 ) postcolumn reactlon detectlon condltions. Symbols: ( 0 )Zn2+; (U) Mg2+; (0) Sr2+; and (A) Ba*+. Curves, nonlinear ftt to eq 8. Experbnentalcondltbns: mode, flow Injectbnanalysls;carrler solutlon, 0.5 mL/mln water: reagent solution, 0.5 mL/mln of 2.0 X lo-‘ M Zn-EDTA-PAR; pH, 10.0.
creasing sensitivity to alkaline earth metals is due to the Zn2+ complexation by ethylenediamine,the alkaline earth data were fit to the expression sensitivity = ~e-B[en]- ~/e-C[enl (8) This expressiondescribes the increase in sensitivity due to the first-order increase in the reaction rate with ethylenediamine concentration, tempered by an exponential decrease in sensitivity due to the Zn2+ complexation. The lines in Figure 2 are the best fits to eq 8 (InPlot, GraphPAD Software, San Diego). The values for constant B, the exponential decrease in sensitivity, for the alkaline earth metals were statistically equivalent to that observed for Zn2+. This confirms that the decreasing alkaline earth metal sensitivity at high ethylenediamine concentration results from ethylenediamine complexation of Zn2+, which prevents formation of Zn(PAR)2. Dynamic ion exchange chromatography was used to verify the effectiveness of the ethylenediamine-based Zn-EDTAPAR reagent relative to a conventional ammonia-buffered reagent. Table 2 presents the sensitivities observed under preequilibrium and equilibrium conditions for Zn2+, Ca2+, and Mg2+ using each auxiliary reagent. The sensitivities in Table 2 are half those shown in Figures 1 and 2 and Table 1 due to the higher flow rate in this study. The reaction time of 0.9 s is sufficient for Zn2+ to fully react with PAR, as indicated by the statistical equivalence of the sensitivities with those achieved for a reaction time of 30 s. Thus, as discussed above, the sensitivity is solely a function of the competitivecomplexation of Zn2+by the auxiliaryagent. Calculation of the fraction of Zn2+ which would be present as the Zn(PAR)* complex correctly predicts that the ethylenediamine buffer results in greater sensitivity than the ammonia solution. However, the calculations exaggerate the
tm = 0.9 8 Tris/end
metal ion
NHaC
Zn2+ Ca2+ Mg2+
11.7 0.2 1 0 . 6 i 0.2 1.34 i 0.02
*
21.9 i 0.7 19.7 i 0 . 3 12.2 i 0.3
t,=30e NHsC Wi/end 10.9 f 0.6 1 1 . 6 i 0.3 6.0 i 0.1
22.6 i 0.2 21.0i 0.2 18.7 i 0.2
0 Experimental conditions: column, 3.9 mm i.d. X 16.0 cm Delta Pak C18; eluent, 0.06 M acid (pH 3,4) with 0.0012 M C&Oa-; eluent flow rate, 1.0 mL/mm; reagent flow rate, 1.0 mL/min. All other conditions ae in the Experimental Section. b Determined ae the slope of the calibrationcurve fori ’ectionof (1-6) X 1Od M Znz+, (2-12) X lOdMCa2+and(3-16)X 1V%Mg2+. Poetcolumnr solution: 2 x IO-’ M Z~-EDTA-PAR in 2 M ~ $ 1 CH&OO- ( H 10.0). d Poetcolumn reagent solution: 2 X lO-’M ZnEDTA-PA.8 in 1.0 M Tris buffer (pH 10.0) containing 0.16 M ethylenediamine.
MI@
difference in sensitivity expected ((CYZn(PAR)2 (en)) / ((YZn(PAR)r (NH3)) = (0.77/0.15) = 5.1) vs that observed (en/NHj = 2.0 in Table 2). This bias is believed to be due to uncertainties in the stability constants for the Zn(NHs)n2+complex and/or variation in the ionic strength. The 0.9-s reaction time is not sufficient for Ca2+ or Mg2+ to achieve equilibrium. Thus, the sensitivitywill be a function of both the displacement reaction kinetics and the auxiliary agent complexation. For Ca2+,the sensitivity is 90 3% of that of Zn2+ in both the ammonia and the ethylenediamine buffers, indicating that the displacement reaction is rapid in both solutions and has almost achieved equilibrium. Magnesium, on the other hand, reacts much slower and is far short of equilibrium. As predicted by the studies above, the use of ethylenediamineresults in much faster Mg2+displacement of Zn2+ and so much greater sensitivity under these preequilibrium conditions. After 30-sreaction, the sensitivity for Ca2+in both buffers is equivalent to the theoretical maximum, the sensitivity for Zn2+. Magnesium sensitivities however are below those of Zn2+, as a result of competitivecomplexation of Mg2+ by the auxiliary complexing agents.
*
ACKNOWLEDGMENT This work was supported by the Natural Sciences and Engineering Research Council and the University of Calgary. Special thanks are extended to Dr. Tom Swaddle for the use of the stopped-flow apparatus and to Dr. Michael Grace and Peter Metelski for their assistance in its operation. Dr. Arvi Rauk is thanked for the use of the SHMO program for the Hiickel orbital calculations. The Undergraduate Travel Assistance Grant provided to H.N.D. by the Analytical Division of the Canadian Society of Chemistry is gratefully acknowledged. Presented at the 76th Canadian Chemistry Conference in Sherbrooke, Quebec, June 1993. Recelved for revlew August 24, 1003. Accepted December 7, 1993.’ Abstract published in Advance ACS Abstracts, January IS, 1994.
A M W I
Vd. 66, No. 6, krbrd, 15, la04
707
