28
Anal. Chem. 1991, 63,28-33
microelectrodes for equilibrium measurement of absolute O2 concentrations (2). The use of fast-scan cyclic voltammetry gives a method with high spatial resolution and time resolution on the order of 100 ms, while having a minimal effect on the surrounding chemical environment (29).The technique described here has many of the characteristics required of an in vivo amperometric detector (2): it is flow rate independent, it has a restricted diffusion layer and is stable, and it is essentially nonperturbational to the chemical environment.
ACKNOWLEDGMENT The helpful comments on this manuscript by Robert Kennedy are greatly appreciated. Registry No. 02, 7782-44-7; dopamine, 51-61-6.
LITERATURE CITED (1) Hitchman, M. L. Measurement of Dissolved Oxygen. I n Chemical Analysls; Elving, P. J., Winefordner, J. D., Kolthoff, I.M., Eds.; Wiley: New York, 1978. (2) Davies, P. W.; Brink, F. Rev. Sci. Instrum. 1942, 73,524-533. (3) Silver, I. A. M a l . Electron. Blol. Eng. 1965, 3 , 377-387. (4) Whalen, W. J.; Riley, J.; Nair, P. J. Appl. Physlo/. 1967, 23, 798-801. (5) Crawford, D. W.; Cole, M. A. J. Appl. Physiol. 1985, 58, 1400-1405. (6) Kelly, R.; Wightman, R. M. Anal. Chim. Acta 1986, 787, 79-87. (7) Kristensen, E. W.; Kuhr, W. G.; Wightman, R. M. Anal. Chem. 1987, 59, 1752-1757. (8) Wiedemann, D. J.; Tomusk, A. B.; Wilson, R. L.; Rebec, G. V.; Wightman, R. M. J. Neurosci. Methods, in press. (9) Baur, J. E.; Kristensen, E. W.; May, L. J.; Wiedemann, D. J.; Wightman, R. M. Anal. Chem. 1988, 6 0 , 1268-1272. (IO) Freeman, G. B.; Gibson, G. E. J. Neurochem. 1986, 47, 1924-1931. (11) Wise, K. D.; Smart, R. B.; Mancy, K. H. Anal. Chim. Acta 1960, 776, 297-305.
(12) Winlove, C. P.; Parker, K. H.; Oxenham, R. K. C. J. Electroanal. Chem. 1984, 770, 293-304. (13) Lawson, D.; Whltely, L. D.; Martin, C. R.; Szentirmay, M. N.; Song, J. I. J. Electrochem. SOC. 1988, 735, 2247-2252. (14) Taylor, R. J.; Humffray, A. A. J. Electroanal. Chem. InterfacialElectrochem. 1975, 6 4 , 85-94. (15) Engstrom, R. C.; Strasser, V. A. Anal. Chem. 1984, 5 6 , 136-140. (16) Wightman, R. M.; Amatore, C.; Engstrom, R. C.; Hale, P. D.; Kristensen, E. W.; Kuhr, W. G.; May, L. J. Neuroscience 1988, 25, 513-523. (17) Paxinos, G.; Watson, C. The Rat Braln In Stereotaxic Coordinates, 2nd ed.;Academic Press: New York, 1986. (18) Hawley, M. D.; Tatawawadi, S.V.; Piekarski, S.;Adams, R. N. J. Am. Chem. SOC. 1967, 89, 447-450. (19) Deakin, M. R.; Kovach, P. M.; Stutts, K. J.; Wightman, R. M. Anal. Chem. 1986, 58, 1474-1480. (20) Nagaoka, T.; Yoshino, T. Anal. Chem. 1986, 58. 1037-1042. (21) Nagy, G.; Gerhardt, G. A.; Oke, A. F.; Rice, M. E.; Adams, R. N.; Moore, R. B., 111; Srentirmay, M. N.; Martin, C. R. J. Nectroanal. Chem Interfacial Electrochem . 1965, 788, 85-94. (22) Rice, M. E.; Nicholson, C. Anal. Chem. 1989, 6 7 , 1805-1810. (23) Wightman, R . M.; May, L. J.; Michael, A. C. Anal. Chem. 1988, 60, 769A-779A. (24) Siesjo, B. K. Braln Energy Metabollsm; Wiley: New York, 1978; p 408. (25) Feng, 2.-C.; Roberts, E. L.; Sick, T. J.; Rosenthal, M. Brain Res. 1988, 445, 280-288. (26) Lubbers, D. W.; Leniger-Foliert, E. I n Brah Work: The Coupling of Function, Metabolism and Blood Flow in the Brain; Ingvar, D. H., Lassen, N. A., Eds.; Academic Press: New York, 1975; pp 85-100. (27) May, L. J.; Wightman, R. M. Brain Res. 1989, 487, 311-320. (28) Leniger-Follert, E.; Lubbers, D. W. fflugers Arch. 1976, 366, 39-44. (29) Stamford, J. A. J. Neurosci. Methods 1986, 77, 1-29.
.
RECEIVED for review June 25,1990. Accepted September 28, 1990. This research was supported by the NIH (Grant PHS R01 NS15841).
Separation and Simultaneous Determination of Aluminum, Iron, and Manganese in Natural Water Samples by Using High-Performance Liquid Chromatography with Spectrophotometric and Electrochemical Detection Yukio Nagaosa* and Hiroki Kawabe Faculty of Engineering, Fukui University, Bunkyo 3-9-1, Fukui 910, Japan
Alan M. Bond Division of Chemical and Physical Sciences, Deakin University, Geelong 321 7, Victoria, Australia
Reversed-phase llquld chromatography has been used to determine slmultaneously trace levels of AI, Fe, and Mn In natural water samples afler direct Injectlon of thelr 8qulndlnd complexes onto a Bondasphere ODS column. Chromatographic separatlon can be made with the mobile phase of 2 3 acetonltrlle/20 mM acetate buffer solutlon containing 5 mM 8-qulnollnol reagent. Excellent sensltlvlty Is obtained by spectrophotometrlc detection at 390 nm. The spectrometric detection llmits of these metals are at the part per bllllon levels of test solutlon. The tolerance llmlts of numerous other metal Ions are reported. Only Cr(II1) and NI(I1) Interfere with the determination of AI, and Mo(V1) Interferes with the determination of Mn at concentrations less than a 100-fold excess. Analytical data obtained on river and sea water samples are In agreement wlth expected values. Amperometric detection of Mn and Fe wlth a thin-layer flow cell and a glassy-carbon worklng electrode Is also described. This method Is less sensitive but more speclflc than spectrophotometric detection.
In the past decade, a number of reports have appeared on the reversed-phase high-performance liquid chromatographic (RPHPLC) separation and determination of trace metals after precolumn derivatizing with the use of bonded stationary phase (1-5). The most frequently used derivatizing agents include dithiocarbamic acids (6-18) and related compounds (19-21), 8-quinolinol (22-24), P-diketones (25), and 4-(2pyridy1azo)resorcinol (26-33). Many of the reagents used to date form stable but nonselective complexes with many "hard" metal ions, thereby enabling multielement determination to be undertaken. However, on account of having nitrogen and oxygen coordinating atoms, 8-quinolinol has the advantage of selectively complexing somewhat harder multivalent metal ions such as Al(III), Fe(III), Mn(III), and Co(II1) (34). Furthermore, 8quinolinol is relatively stable under RPHPLC conditions and may therefore be included directly in the mobile phase. Consequently, 8-quinolinol is useful as an in situ precolumn derivatizing agent for RPHPLC separation and determination
0003-2700/91/0363-0028$02.50/00 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 1, JANUARY 1, 1991 8-Qlhln
of these metals. Previously, we reported the determination of Cu, Fe, and Mn by RPHPLC techniques as their 8-quinolinol complexes with reductive thin-layer amperometric detection (21). UV spectrophotometric detection also has been employed in the determination of A1 (24). In the present paper, we have attempted to develop a simple and sensitive RPHPLC method for the simultaneous determination of Fe, Mn, and A1 using spectrophotometric detection. The method can be applied to the simultaneous determination of the three metal ions at the parts per billion level in natural water samples. We also compare results by spectrophotometric detection with those of the highly selective determination of Fe and Mn by amperometric detection with a thin layer electrochemical cell. EXPERIMENTAL S E C T I O N Apparatus. The liquid chromatograph used consisted of a Tosoh (Tokyo,Japan) Model CCPD pump and a Rheodyne Model 7125 injector with a 100-pL sample loop. The chromatograph was equipped with a Shimadzu (Kyoto, Japan) Model SPD-6AV UV-VIS spectrophotometric detector (8-pL flow cell) and a Model R-101 laboratory recorder. Cyclic voltammetric experiments were performed with a Princeton Applied Research (Princeton, NJ) Model 174A polarographic analyzer and a Rika Denki (Tokyo, Japan) Model RW-11 x-y recorder. A Yanako (Kyoto, Japan) Model P-010 glassy-carbon minielectrode was used for stationary cell work. The polarographic analyzer was connected with a BAS (Bioanalytical System; West Lafayette, IN) TL-5 thin-layer flow cell consisting of a glassy-carbon working electrode and a silver/silver chloride reference electrode (BAS RE-l) and with a Toa Denpa (Tokyo, Japan) Model EPR-2OOA recorder for amperometric detection. A Toa Denpa Model HM-5A pH meter was used to measure the pH of the solution. The columns used for this study were the following: p-Bondasphere ODs-loo& 5 pm, 150 mm X 3.9 mm (Waters); Ultrasphere, ODS, 5 pm, 150 mm X 4.6 mm (Beckman); TSKgel ODS-EOTM, 5 pm, 150 mm X 4.6 mm (Tosoh); Lichrosorb RP-18, ODS, 5 pm, 150 mm X 4.6 mm (Merck). Reagents. Acetonitrile was of HPLC grade from Tokyo Kasei Chemicals (Tokyo, Japan). The other reagent grade chemicals were purchased from Wako Junyaku Kogyo (Tokyo, Japan) and used without further purification. Metal ion standards of a desired concentration were prepared by dilution of stock 1000 pg/mL standard solutions for atomic absorption spectrophotometry (Wako Junyaku Kogyo). Deionized water from a Millipore Milli-Q water purification system was used for all solutions and dilutions. General Procedure. An aliquot of metal ion solution and 5.0 mL of 20 mM 8-quinolinol solution in acetonitrile were placed into a 10-mL volumetric flask, to which 0.6 mL of 2.0 M acetate buffer (pH 5.9) was added. Finally, the volume was increased to 10.0 mL with deionized water. A part of this solution was transferred to a 10-mL Teflon test tube, and it was heated at 50 "C for 10 min to complete the complex formation. After cooling, a 100-pL aliquot of the resulting solution was injected onto an ODS column. The mobile phase (chromatographic eluent) was 2 3 acetonitrile/20 mM acetate buffer (pH 5.9) solution containing 5 mM 8-quinolinol. All eluents were degassed with nitrogen during the HPLC runs. The other chromatographic conditions were as follows: flow rate, 1.0 mL/min; column temperature, 20 "C; wavelength for spectrophotometric detection, 390 nm; applied potential for amperometric detection, -0.40 V vs Ag/AgCl reference electrode (3 M NaC1) in dc mode; low-pass filter (time constant), 3 s. Sample Pretreatments for River Water Determinations. Asuwa and Kuzuryu River samples taken were pretreated by procedures A and B. (A) After collection, the samples were filtered immediately through a 0.45-pm filter (Millipore filter). Hydrochloric acid was then added to the filtered solution to adjust the pH t o about 1.5. For the complex formation, 5.0 mL of the pretreated sample solution, 2.0 mL of 2.0 M acetate buffer (pH 6.5), and 10 mL of 20 mM 8-quinolinol solution in acetonitrile were transferred into a 20.0-mL volumetric flask and the solution was diluted to the mark with deionized water. The other procedures for complex formation were the same as that described in the previous section. (B) The pH of the samples was initially
29
rzrJ n n
0
4
8
12
16
18
Retention time, min Figure 1. Chromtograms of metal Bquinolinol complexes with different ODS columns: (a) p-Bondasphere C18, Waters; (b) Ultrasphere ODs, Beckman; (c) Lichrosorb RP-18, Merck; (d) TSKgel ODSIOTM, Tosoh. Chromatographic conditions: concentration of each metal ion, 20 pg/L; sensitivity, 0.005 AUFS (absorbance units full scale); wavelength, 390 nm; eluent, 2:3 acetonitrile/acetate buffer containing 5 mM 8quinolinol;
Other conditions are reported in the text. adjusted to about 1.5 by adding an appropriate amount of hydrochloric acid. Filtration with a 0.45-pm filter was made prior to RPHPLC analysis. Other procedures were the same as described in procedure A. Sample Treatment for Sea Water Determinations. Coastal sea water was collected at a point off the Echizen coast of Fukui prefecture in Japan. Nitric acid was then added to the sample in order to adjust the pH of solution to about 2.0. The sea water sample was filtered through a 0.45-pm filter and stored in polyethylene bottles. Twenty-five milliliters of the sample was placed in a 100-mL beaker, and 10 mg of sodium actate and a known amount of sodium hydroxide were added to adjust the pH of solution to between 6.0 and 6.5. The volume of this solution was increased to 50.0 mL with water. Equal volumes of the pretreated sample and 20 mM 8-quinolinol solution in acetonitrilewere mixed in a 20-mL Teflon test tube. The other procedures were the same as the general procedure described above. R E S U L T S A N D DISCUSSION Changes in Chromatographic Behavior of Metal 8Quinolinol Complexes f o r Different Reversed-Phase Columns. Four ODS columns were tested for the RPHPLC determination of metal ions after formation of those complexes with 8-quinolinol under a variety of eluting conditions. Typical chromatograms obtained with spectrophotometric detection are shown in Figure 1. In the case of the p-Bondasphere (Figure l a ) and Ultrasphere (Figure l b ) columns, the Cu(II), Al(III), Fe(III), and Mn(II1) complexes could be well resolved chromatographically with a 2:3 acetonitrile/ aqueous mobile phase mixture, provided 5 mM 8-quinolinol was also present in the mobile phase. Under the conditions
ANALYTICAL CHEMISTRY, VOL. 63, NO. 1, JANUARY 1, 1991
30
Table I. Tolerance Limits of Foreign Ions for the Determination of AI, Fe, and Mn by Chromatography with Spectrophotometric Detectiona tolerance limit lo6
105 lo4 lo2
10 1
ion or salt added
Fe
A1
NaBr, NH4Cl,NaCl, KNO,
Mn
NaBr, NH,Cl, NaC1, KNO,, Na3C6H6o7
NaBr, NH4Cl, NaCl, KNO, Na4P207
Na4P207
Na3C8H507, Na4P207 a3 6 5 7 MnZ+,Cu2+,Fe3+,CdZt, Hg2+, Mn2+,Cuzt, Fe3+,Cd2+,Hg2+,MOO:-, As02-, MnZt, Cuzt, Fe3+,Cd2+,Hg2+,AsOr, MOO^^-, As02-,Se4+,Ti4+, Sb4+,Ti4+,Pb2+,Zn2+,CaZt, Co2+,Mg2+,Bis+, Se4+,Ti4+,Pb2+,Zn2+,Ca2+,Co2+, Pb2+,Zn2+, Ca2+,Co2+,Mg2+, Sb3+,Sn2+,W042-, V02+, Ni2+,Cr3+,A13+ Mg2+,BiP+, Sb3+,Sn2+,WO:-, V02+, Bi3+,Sb3+,Sn2+,WO:-, V02+ Ni2+,Cr3+, A13+ Ni2+ MOO:Cr3+
10 rg/L of each of Al(III), Fe(III), and Mn(I1) were used in these experiments. Experimental conditions and parameters are given in the
text.
of the experiments, any Fe(I1) or Mn(I1) present is converted to the Fe(II1) or Mn(II1) complexes and the method therefore detects the sum of the two oxidation states of the metal. In the remainder of this paper the formalisms of Fe(II1) and Mn(II1) are therefore used since 3+ is the oxidation state of the complexes formed. The retention times of each complex were less than 15 min, and responses were observed in the same order of Cu(I1) < Al(II1) < Fe(II1) < Mn(II1) for both columns. The retention times decrease with increasing content of acetonitrile in the mobile phase. However, if the acetonitrile content is raised above 50% in the mobile phase, coelution of the Cu(I1) or Al(II1) complex with the unreacted 8quinolinol is observed on the chromatogram, as reported previously with an Ultrasphere ODS column (21). In the present study, a 1-Bondasphere column is recommended as the analytical column because it gave the highest signal to noise ratio for each complex among the columns investigated. All subsequent data reported refer to this column. Since the RP-18 column is composed of an unspherical and nonendcapped ODS supporting material, the peak heights obtained were low and insufficient for peak resolution of Cu(I1) and Al(II1) complexes (Figure IC). The finding of small peak heights and tailing suggests on-column degradation or hydrolysis of the complexes during the elution on the R P column (31, 35, 36). Distinct peaks were obtained for the Al(III), Fe(III), and Mn(III), but not Cu(II), complexes with the TSKgel column, although they were somewhat broader and smaller than those obtained with the r-Bondasphere and Ultrasphere columns (Figure Id). A low peak height for the Co(II1) complex was seen between the unreacted 8-quinolinol and the Al(II1) peaks with all columns used. However, this response was less useful for the RPHPLC determination of the metal ion. The Ni(I1) and Cr(II1) peaks were overlapped partly with the Al(II1) peak under the present experimental conditions, and the Mo(V1) complex gave an ill-defined chromatogram at retention times that could overlap with the Mn(II1) peak if present a t relatively large concentrations. Table I summarizes the tolerance of the method for determining Al(III), Fe(III), and Mn(II1) with numerous other metals. A high degree of specificity is achieved apart from the problems with Ni(II), Cr(III), and Mo(V1) mentioned above. Further discussion of interferences is given later. RPHPLC Determination of Aluminum, Iron, a n d Manganese by Spectrophotometric Detection. Simultaneous calibration curves of peak height versus concentrations were prepared for Al(III), Fe(III), Cu(II), and Mn(II1) according to the general procedure described in the Experimental Section. Aqueous metal solution were prepared with concentrations over the range 0.10-100 ng/mL and injected onto the w-Bondasphere column after complexation. Parts a and b of Figure 2 show the chromatographic separation of the four complexes a t different concentrations. The Cu(I1)
I
0
4
8
12
Retention time, min Figure 2. Chromatograms of metal 8-quinolinol complexes obtained with spectrophotometric detection (390 nm, 0.005 AUFS): (a) 20 pg/mL of each metal ion; (b) 10 hg/mL of each metal ion; (c) the blank solution.
peak is not suitable for RPHPLC determination, because it appears as a shoulder on the front of the unreacted reagent peak and the two peaks are insufficiently resolved. The detection of the metals was carried out bymonitoring the absorbance of analytes in the eluate a t 390 nm. At this wavelength these 8-quinolinol complexes have absorption maxima and a molar absorptivity of (3-8) X L cm-I mol-' (34,37). Although the 8-quinolinol complexes have much larger molar absorptivities in the UV region, the mobile phase, containing 5 mM of ligand, has a very strong UV absorption that could not be suppressed electronically with the apparatus used. Linear regression analyses of the calibration curves are listed in Table 11. Good linearity of the curves was obtained over approximately 3 orders of magnitude for the three metals. The calibration curves obtained for Al(II1) and Fe(II1) has a
ANALYTICAL CHEMISTRY, VOL. 63,
NO. 1, JANUARY
1, 1991
31
T a b l e 11. L i n e a r Regression Analysis o f Calibration Curves for the Determination of Metals
metal ion
regression line"
A1
Fe
y = 1 . 4 0 ~ 3.50 (0.005 AUFS) y = 0.9990 y = 0.490~- 0.538 (0.005
Mn
y = 0.9995 y = 4 . 4 3 ~+ 0.495 (0.001
+
re1 std
detection limit,*
dev, 5%
pg/L
6.9
2.0
1.8
4.0
3.8
0.35
AUFS) AUFS)
y = 0.9999 " x : Metal ion concentration, pg/L. y: peak height, arbitrary units (2 mm = 1 arbitrary unit). y: relative standard deviation obtained at a 20 pg/L metal ion concentration. *Detectionlimit was determined as a signal to noise ratio of 3:l.
positive and negative y intercept value at the added metal ion concentration of zero, respectively. The blank test on the chromatogram (Figure 2c) showed that there are absorption peaks at the same retention times as for the two complexes. The positive peak for Al(II1) in the blank test presumably indicates the presence of this metal ion a t the trace level in the mobile phase. The appearance of the negative peak at the retention time for iron is presumably due to a decreased concentration of the ligand in the eluate, but the exact reason for this is unclear. Linear correlation coefficients greater than 0.9990 were obtained for all three metal complexes. Included in Table 11are the relative standard deviations for 10 replicate determinations of each 20 ng of metal ion/mL, and the limits of detection are expressed as a signal to the baseline noise ratio of 3:l under the cited chromatographic conditions. The present RPHPLC system enables the simultaneous determination of the three metals a t the nanogram per milliliter concentration level in aqueous samples. Interestingly, the limit of detection for Mn(II1) is 0.35 ng/mL (35 pg) with respect to the concentration of the injected sample solution. This sensitivity is so high that the present RPHPLC method is suited for the direct determination of Mn(I1) in natural water samples. The oxidation of Mn(I1) to Mn(II1) due to dissolved oxygen and the complexation with 8-quinolinol can be completed simultaneously by heating the aqueous sample solution to 50 "C for 5 min. Interferences from the foreign metal ions and salts were investigated for the RPHPLC determination of each 1.0 ng of Al(III), Fe(III), and Mn(III)/100 pL (injection volume) by the chromatographic method. The maximum amount added was 1.0 mg for salts and 0.1 pg for metal ions, which corresponded to a IO6- and 102-foldexcess over the concentration of each metal ion, respectively. The tolerance limit for the RPHPLC analysis was expressed as the maximum amount to be determined within an error of h5%. The results are shown in Table I. As expected, foreign ions that inhibit complexation with 8-quinolinol and/or coelute with the metal ion of interest interfered with the RPHPLC determination. For example, the presence of 1.0 pg of Cr(II1) and 10 ng of Ni(I1) was permissible for the Al(II1) determination. There was no interference from most of the common metal ions with the determination of Fe(II1). For the manganese determination, more than 10 ng of Mo(V1) caused serious interference because this is a coeluting metal ion. Being masking agents, sodium citrate and sodium pyrophosphate must not be over the tolerance limits listed in Table I. RPHPLC D e t e r m i n a t i o n of I r o n and M a n g a n e s e by E l e c t r o c h e m i c a l D e t e c t i o n . In general, the electrochemical detection method is known to be more selective than the spectrophotometric one (38, 39). In the present work, amperometric detection using a thin-layer cell and a glassy-carbon
0 E,
-0,E
-0.3 V
VS.
AUAgC1
Figure 3. Cyclic voltammograms (scan rate 200 mV s-') for reduction of Fe(II1) and Mn(II1) 8-quinolinol complexes in the chromatographic eluent. Concentration of metals: 1.0 pg/L Fe(II1) and 2.0 pg/L Mn(I1). For other conditions, see Experimental Section (general procedure).
c
50
40
10
0
0.2
0.4
0.3 -E,
V
0.5
0.6
V S . Ag/AgC1
Figure 4. Amperometric hydrodynamic voltammograms for reduction of Fe(II1) and Mn(II1) 8-quinolinol complexes. Concentration of each metal ion: 100 pg/L. Experimental conditions are reported in the text.
working electrode has been exploited to monitor the current (response) of reductive species after RPHPLC separation with the K-Bondasphereanalytical column. Initially, the technique of cyclic voltammetry was used to investigate for the reduction of Fe(II1) and Mn(II1) complexes with 8-quinolinol in the eluent of interest, which included 8-quinolinol a t a stationary glassy-carbon electrode. The cyclic voltammograms shown in Figure 3 indicate that for both metals, the reduction and subsequent oxidation took place at the electrode with a scan rate of 200 mV s-l. The reductive peaks obtained with the cathodic scan were -0.10 and -0.45 V vs Ag/AgCl for the Mn(II1) and Fe(II1) complex, respectively. A probable electrode process at a glassy-carbon electrode is M(8-q~inolinol)~ + e- M(8-q~inolinol)~(40, 41). The ligand 8-quinolinol is not electroactive over this potential range. The applied potential of the working electorde was varied from -0.20 to
-
32
ANALYTICAL CHEMISTRY, VOL. 63, NO. 1, JANUARY 1, 1991
Table 111. Determination of Al, Fe, and Mn in River Water Samples procedure
A A A A
B B B B B B
A1
sample
Kuzuryu River Kuzuryu River
+ 20 pg/L Al(II1) + 20 pg/L Fe(II1) + 12 wg/L Mn(I1) Asuwa River Asuwa River + 40 pg/L Al(II1) + 40 pg/L Fe(II1) + 8 @g/LMn(W Kuzuryu River Kuzuryu River + 40 pg/L Al(II1) + 80 pg/L Fe(II1) + 10 wg/L Mn(II) Asuwa River Asuwa River + 400 pg/L Al(II1) + 800 pg/L Fe(II1) + 40 pg/L Mn(I1) Asuwa River" Asuwa River" + 400 pg/L Fe(II1) + 80 pg/L Mn(I1)
concentration, gg/L Fe
17 f 1.7 34 f 3.9 30 f 3.1 68 f 1.9 61 f 0.7 107 f 2.2 580 f 9.6 984 f 14
24 f 3.1 44 f 2.2 10 f 0.7 60 f 1.2 63 f 3.4 143 f 2.5 648 f 14 1456 f 18 764 f 15 1256 f 94
Mn
3.0 f 0.5 15 f 0.2 6.0 f 0.3 14 f 0.3 6.0 f 0.2 16 f 0.3 44 f 0.7 84 f 0.8 50 f 3.0 133 f 5.0
Amuerometric detection method. Other samules determined bv suectrouhotometric method.
-0.60 V vs Ag/AgCl, in order to find the optimum applied potential for the RPHPLC determination. Figure 4 shows the peak current vs applied potential curve for 100 ng/mL Fe(II1) or Mn(II1). The Fe(II1) peak height for the reductive process increased as the potential gradually decreased in the range from -0.20 to -0.60 V, being approximately constant between -0.50 and -0.60 V vs Ag/AgCl. On the other hand, the Mn(II1) peak height was a maximum a t around -0.20 V. At more negative potentials, the peak height changes slowly with the working electrode potential. The Fe(II1) complex gave a larger peak height than the Mn(II1) complex a t any potentials presumably because it has a much shorter retention time. An applied potential of -0.40 V vs Ag/AgCl for detection was chosen as being suitable for the simultaneous determination of the two metals by the RPHPLC method. At the applied potential, the response is moderately free from the background current due to the reduction of traces of dissolved oxygen present in the eluate. At -0.40 V vs Ag/AgCl, no response was observed for Al(III), Cr(III), Co(III), M O W ) , or 8-quinolinol. Accordingly, these ions caused no interference on the simultaneous RPHPLC determination of iron and manganese even at large concentrations when electrochemical detection was used. The present amperometric detection method, while not offering as high a sensitivity as the spectrophotometric one, has the advantage of minimizing interferences from other species that cause problems with spectrophotometric detection. Simultaneous calibration plots for the determination of Fe(II1) and Mn(II1) with electrochemical detection were prepared under the experimental conditions of the general procedure. The peak heights were directly proportional to the metal concentration in the range 0-300 ng/mL. From the regression analyses, the following relationships have been obtained: y(nA) = 0.174x(ng/mL) + 4.58 and y (correlation coefficient) = 0.9990 for Fe(II1); y = 0 . 2 9 1 ~ + 0.629 and y = 0.9989 for Mn(II1). The lower limit of determination was 10 ng/mL for both metals. Analyses of River and Sea Water Samples. River and sea water samples were examined according to the above procedures. Figure 5 shows typical chromatograms obtained for Asuwa River water when spectrophotometric (a) and electrochemical detection methods (b) were used. This water sample was pretreated by using procedure B, followed by addition of 0.01 M 8-quinolinol solution and heating for 5 min at 50 OC to convert Mn(I1) to Mn(II1). Two different detection modes produced an identical result within experimental error with respect to Fe(II1) and Mn(II1) concentration, as shown in Table 111. For other river water samples, the spectrophotometric detection was used because the manganese concentration was below the limit of detection using amperometric detection. Table I11 summarizes the results obtained for various river water samples, together with those of the samples spiked with the metal standards to ascertain the validity of analytical data. The spiked metal ion concentration corre-
A1
I
Mn
2 nA
n
0
Fe
"1 4
8
1 nA
'wn
1
2
Retention time, min Figure 5. Chromatograms for the simultaneous determination of Fe( H I ) and Mn(I1) in Asuwa River water with (a) spectrophotometric and (b) electrochemical detection. Sensitivity: 0.02 AUFS for AI(II1); 0.01 AUFS for Fe(II1); 0.002 AUFS for Mn(I1).
Table IV. Results for the Determination of Al, Fe, and Mn in Sea Water sample
concentration, pg/L Fe Mn
A1
sea water 30 f 4.3 14 f 3.9 1.4 f 0.4 sea water + 20 kg/L Al(II1) + 20 55 f 2.2 32 f 1.9 9.2 f 0.5 ue/L Fe(II1) + 8.0 ue/L Mn(I1) sponds to the difference between the two analytical values, as required if the procedure is valid. The analytical results depended upon the pretreatment method. The concentrations obtained with procedure B were larger than those found procedure A. Since no filtration of the sample was made in procedure A, it can be concluded that the metals are also incorporated in particulates or substances of a size larger than 0.45 pm. Asuwa River samples showed a larger difference between the two analytical values obtained after the pretreatments A and B than the Kazuryu River samples did. The determination of metals in coastal surface water that was collected from the Echizen coast a t Fukui prefecture in
Anal. Chem. 1991, 63, 33-44
Japan also was made by the present RPHPLC method using spectrophotometric detection a t 390 nm. Table IV lists the analytical results for Al(III), Fe(III), and Mn(III), together with those obtained on sample solutions spiked with metal ion standards. Comparison of both sets of data revealed that the sea water sample could be determined successfully for the three metal ions and that there was no interference from matrices such as alkali and alkaline-earth metals. The sensitivity of this method could be lowered by using a preconcentration technique, i.e., a liquid-liquid extraction or a liquid-solid extraction using a (&-bonded silica cartridge column.
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RECEIVED for review October 2, 1990. Accepted October 8, 1990.
Sequential Multimodal Elution for Pseudomultidimensional Liquid Chromatography on a Single Column Edward L. Little, Mark S. Jeansonne, and Joe P. Foley* Department of Chemistry, Louisiana State University; Baton Rouge, Louisiana 70803-1804
A method of elutlon is described that ylelds stgnlflcantly higher (2-3 tlmes) peak capacltles and facllltates the separatlon of compounds by class (e.g., aclds versus neutrals), thereby lncreaslng the lnformlng power and the selectlvlty. The method Is based on the sequential appllcatlon of two or more elutlon modes, all of whlch, except the last one, are selective, Le., deslgned to elute only a certain class of compounds. The total peak capaclty Is glven approximately by rcj ,, where r Is the number of elutlon modes and 6, Is the peak capaclty of an lndlvldual elution mode, roughly equal to that obtained In a conventlonal onedlmenslonal separatlon. The Increase In lnformatlon content Is proportlonal to log r but Is also dependent on the relative proportlon of sample components eluted durlng lndlvldual separatlon modes. Several reversed-phase examples utlllzlng sequential pH and solvent gradients are presented, Including separatlons of benzoic aclds from neutral aromatic compounds, peptides from neutral specles, and phenols from polyaromatlc hydrocarbons. Solute bandwidths, retention time, and area reproduclblllty are comparable to that of conventlonal reversed-phase separations. Although the resoivlng power of this multlmodal, singlecolumn elutlon method is somewhat less than that of a true two-dlmenslonai (multiple column) method, it can be an effective and simple alternative for the analysts of moderately complex samples. 0003-2700/9 1/0363-0033$02.50/0
INTRODUCTION Liquid chromatography has emerged as the separation method of choice for numerous complex solute mixtures. The introduction of high-performance liquid chromatography (HPLC) has further increased its usefulness due to greater column efficiency and overall reproducibility. Reversed-phase high-performance liquid chromatography (RPLC) has received more attention than any other separation mode due to its broad applicability. Other advantages of RPLC include greater column stability than in conventional liquid-liquid chromatography (LLC); the variety, economy, and low toxicity of common RPLC solvents; and the ease of gradient elution. As samples become more complex, the ability of a particular separation method to resolve all components decreases. Several factors may lead to insufficient resolution for complex samples, including inadequate column efficiency and/or gradient optimization. Even in cases where these factors are optimized, however, the separation may still be unsuitable due to limitations of selectivity and/or peak capacity. With regard to the latter, a statistical study of component overlap has shown that "a chromatogram must be approximately 95% vacant to provide a 90% probability that a given compound of interest will appear as an isolated peak" (1). In instances where this condition is not met, the additional resolution of components within a complex sample would typically require supplemental separation steps, thereby reducing the speed 0 1990 American Chemical Society
