12
Anal. Chem. 1983, 5 5 , 12-16
Science and Landscape Architecture a t the University of Minnesota, St. Paul, for providing the plant tissue homogenates, and Linda Baker for her expert assistance typing the manuscript. Registry NO. GSSG, 27025-41-8; N-acetylcysteine, 616-91-1. LITERATURE C I T E D (1) Eiiman, G. H. Arch. Biochem. Biophys. 1959, 82, 70-77. (2) Fowler, B.;Robins, A. G. J. Chromatogr. 1972, 72, 105. (3) Beaies, D.; Finch, R.; McLean, A. E. M. J. Chromatogr. 1981, 226, 498-503. (4) Studebaker, J. F.; Siocum, S.A,; Lewis, E. L. Anal. Chem. 1978, 50, 1500-1503. (5) Rabensteln, D. L.; Saetre, R. Anal. Chem. 1977, 49, 1036-1039. (6) Bergstrom, R. F.; Kay, D. R.; Wagner, J. G. J. Chromatogr. 1981, 222, 445-452. (7) Saetre, R.; Rabenstein, D. L. Anal. Chem. 1978, 5 0 , 278-280.
Eggii, R.; Asper, R. Anal. Chim. Acta 1978, 101, 253-259. Allison, L. A.; Keddington, J., unpublished results. Shoup, R. E.; Mayer, G. S.Anal. Chem. 1982, 54, 1164-1189. Mayer, G. S.;Shoup, R. E., accepted for publication in J. Chromatogr. MacCrehan, W . A.; Durst, R. A. Anal. Chem. 1981, 5 3 , 1700-1704. LC-4B Manual, Bioanalyticai Systems Inc., 1982, Sections 5, 6. Kissinger, P. T. Anal. Chem. 1977, 49, 447A-456A. Mefford, I.; Adams, R. N. Life Sci. 1978, 2 3 , 1167-1174. Sakane, Y.; Matsumoto, K.; Ohtsuka, R.; Osajima, Y. Nippon Kagaku Kaishi 1982, (I), 81-86. Chem. Abstr. 1982, 9 6 , 7 6 3 7 4 ~ . (17) Stankovlch, M. T.; Bard, A. J. J. Nectroanal. Chem. 1977, 7 5 , 487. (18) Maresse-Ducarmois, C. A.; Patriarche, G. J.; Vandenbaick, J. L. Anal. Chim. Acta 1974, 7 1 , 165. (19) Guy, C. L.; Carter, J. V. “Plant Cold Hardiness and Freezing Stress”, Li, P. H., Sakai, A., Eds.; Academic Press: New York, 1982. (8) (9) (10) (11) (12) (13) (14) (15) (16)
RECEIVED for review August 2,1982. Accepted September 28, 1982.
Addition of Complexing Agents in Ion Chromatography for Separation of Polyvalent Metal Ions Gregory J. Sevenlch” and James S. Fritz Ames Laboratory and Department of Chemistry, Iowa State University, Ames, Iowa 5001 1
The scope of Ion chromatography wlth a conductlvlty detector has been expanded to Include several addltlonal divalent metal Ions and the trivalent lanthanide cations. A complexing anlon Is Incorporated In the eluent whlch Increases the number of metal Ions that can be separated and Improves the sharpness of the eluted peaks. Further selectivity is obtalned by adding a complexing reagent to the sample and then eluting wlth an eluent containing ethylenediammonium tartrate. Thls technique provldes a rapid and highly selective method for separating and determing magneslum, calcium, and strontium in various samples.
Chromatographic methods for the separation of inorganic cations have tended to be rather time-consuming and often have not employed automatic detection of eluted peaks. In 1975 Small, Stevens, and Bauman (1) invented a clever dual-column method for the separation of inorganic cations that uses a conductivity detector. This system is excellent for separation of the alkali metal ions and the alkaline earth cations, but its scope is limited because the hydroxide-form suppressor column would precipitate most polyvalent metal cations. Fritz, Gjerde, and Becker (2) developed a singlecolumn method for cation chromatography that uses a conductivity detector and permits the separation of additional inorganic cations. They established the principle that a decrease in conductance, as well as an increase in conductance, can be used for detection and quantitative estimation of eluted ions. Other modern methods of inorganic ion-exchange chromatography have been reviewed in a recent book (3). Complexing eluents have been used in many published methods to achieve more selective chromatographic separations of metal ions, but the presence of a complexing agent often makes detection of the metal ions difficult. However, Elchuk and Cassidy have obtained excellent chromatographic separation of the lanthanides with a-hydroxyisobutyric acid using postcolumn derivatization and spectrophotometric detection (4). Conductivity detectors have been improved greatly
in recent years and have the advantage of providing sensitive and “universal” detection of ions in solution. However, the conductivity detector has not been previously used in ion chromatography in conjunction with a complexing eluent. In the present work eluents containing the ethylenediammonium cation and either tartrate or hydroxyisobutyrate as the complexing anion have been used for separation of polyvalent metal cations. The use of a complexing agent in the eluent improves the sharpness of separations and broadens the scope of cation chromatography with a conductivity detector. The eluted metal ions are only partly complexed and are mostly in solution as cations. As in our previous work (2), the eluted ions have a lower equivalent conductance than the eluent cation and thus appear as peaks of lower conductance. In some cases an additional complexing reagent, such as EDTA, is added to the sample (but not to the eluent) to increase the selectivity of the chromatographic separations. EXPERIMENTAL SECTION Apparatus. The instrument used was described previously (2) and consists of a Model 396 Milton Roy minipump, Rheodyne Model 7010 sample injection valve equipped with a 100-pLsample loop, a low-capacity cation-exchange column, a Model 213 conductivity detector from Wescan Instruments (Santa Clara, CA), and a strip chart recorder. All fittings in contact with the eluent were either Teflon, Kel-F, or stainless steel. The column, detector, and eluent were each located in a Styrofoam-lined cabinet to minimize temperature effects. All chromatograms were obtained at room temperature. Flow rates were 0.85 mL/min, flow rates much higher than this gave pressures approaching the limits of the fittings. Cation Exchange Column. The column is of thick-walled glass and measured 350 mm in length with a 2 mm i.d. The column was packed with a gel-type resin with a particle size of 20 pm. The resin was lightly sulfonated t o give a low cationexchange capacity. This material was obtained by special order from Benson Co. (Reno,NE) and was packed by use of a balanced density slurry method. Eluents. Reagent grade ethylenediamine was used after redistillation. Reagent grade tartaric acid and a-hydroxyisobutyric acid were used as received. All eluents were prepared in distilled-deionized water and filtered through a 0.45-pm membrane
0003-2700/83/0355-0012$01.50/0 0 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 1, JANUARY 1983
filter before use. The pH of each eluent was adjusted with either perchloric acid or sodium hydroxide, depending on the pH desired. Sample Solutions, .A11 samples were prepared from reagent grade salts and dissolved in distilled-deionizedwater. Solutions for quantitative work were standardized by EDTA titration. Lanthanide samples were obtained courtesy of J. E. Powell, Ames Laboratory. All lanthanides were received as the oxides and were at least 99.95% pure. Samples Masked with EDTA. Samples were prepared by mixing appropriate amounts of Mg(II), Ca(II), Sr(II), and interfering-ion solutions. Ihough EDTA was added to completely mask the excess metal ioins. The pH of each solution was monitored and adjusted to be about 3.6 while adding the EDTA. The usable pH range was about 3.5-4.0. At higher pH values Mg(II), Ca(II), and Sr(I1) are significantly complexed with complete loss of the peaks at a pH around 5. At lower pH values the EDTA precipitates. The solution can be made basic to redissolve the precipitated EDTA and then reacidified without affecting the results. In samples containing Fe(III), hydrolysis occurred at pH values above 2.0. However the large formation constant of the iron(111)-EDTA complex permitted a pH of 1.7 without any precipitation. The solutions were diluted to give final coiicentrationsof Mg(II), The EDTA concentration was Ca(II), and Sr(I1) of 1.0 X 0.01 M. Interfering metal ions tested included Al(III), Cu(II), Fe(III), Ni(II), Pb(II), and Zn(I1). Amounts in 1-,lo-, and 100-fold molar excess were examined. At 1-fold excess no real interference existed in most cases so this concentration was not examined.
RESULTS AND DISCUSSION The system used was similar to that previously described (2) for the separation of divalent metal cations on a low-capacity cation-exchange resin with an ethylenediammonium salt as the eluent, except that a complexing anion was incorporated in the eluent to modify the elution behavior of sample ions. Of several complexing reagents tried, tartrate was the most succes&.~l, although a-hydroxyisobutyrate (HIBA) was also useful for some separations. With complexing eluents it was found tlhat better results were obtained with a resin of somewhat higher exchange capacity (0.059 mequiv/g) than that used in the earlier work (0.017 mequiv/g). With eluents containing approximately equal molar concentrations of ethylenediammonium cation and tartrate anion, well-formed peaks were obtained for each of the following metal cations: Ba(II), Ca(II), Cd(II), Ce(III), Co(I1) , Dy(III), Er(III), Eu(III), Fe(II), Gd(III), Ho(III), La(III), Lu(III), Mg(II), Mn(II), Nd(III), Ni(IIj, Pb(II), Pr(III), Sm(III), Sr(II), Tb(III), Tm(III), Yb(llI), and Zn(I1). The retention times varied sufficiently to suggest that good separation of mixtures would be possible. The retention times decreased with increasing pH owing to the greater complexing ability of tartrate at higher pH. A practical upper limit of approximately p H 5 was established because the protonation of ethylenediamine is incomplete at higher pH values. The lower end of the practical pH range was3 found to be approximately pH 3. At more acidic pH values much of the complexing ability of tartrate is lost and the detection sensitivity for metal cations is significantly lower because of the higher background conductance. Eluents containing ammonium tartrate and no ethylenediammonium salt were ineffective for elution of the metal cations studied. It apioears that the elution mechanism is a combination of the mass action "pushing" effect of the ethylenediammonium cation and the weakly complexing or "pulling" effect of the tartrate anion. The effect of various ratios of tartrate to ethylenediammonium ion molar concentration was studied. The retention times, shown in Table I decrease as more tartrate is used in the eluent. The most dramatic changes are in the elution of lead(II), which is very strongly retained when no
13
Table I. Adjusted Retention Times (&') (in Minutes) of Metal Ions for Constant Ethylenediamine Concentration M), Constant pH (4.50), and Varied (2.0 X Tartrate Concentration metal ion MgZt
Zn2+ Ni2' Mnzt CdZ+ Sr2+ Pb2+
tartrate concentration, M x l o 3 1.0 2.0 3.0 4.0
0.0
2.8 3.2 3.4 4.3 5.2 11.2 41.2
2.9 2.6 3.0 4.0
2.8 2.0 2.4 3.8 4.5 10.3 7.8
5.0 11.0
12.7
2.8 1.6 2.0 3.7 4.0 9.7 5.7
2.9 1.5 1.8 3.6 4.0 9.6 4.9
5.0 2.8 1.4 1.6 3.5 3.7 9.1 4.1
I: iZn
I1 I
0
3
6
9
1
2
1
5
TIME ,min
Figure 1. Separation of Zn(I1) (10.3 pprn), Co(1I)(9.1 ppm), Mn(I1) (16.0 ppm), Cd(I1)(16.1 ppm), Ca(1I)(17.1ppm) (16.0 ppm), and Sr(I1) (20.3 ppm): eluent, 1.5 X M ethylenediammine, 2.0 X M tartrate, pH 4.0.
tartrate is present but elutes much more rapidly when tartrate is added to the eluent. Separations with EthylenediammoniumTartrate. By use of conditions suggested by these preliminary experiments, several nice separations of metal ion mixtures were obtained with eluents containing ethylenediammonium tartrate. Figure 1shows the separation of several divalent cations in less than 15 min. The rather difficult separation of cadmium(I1) and manganese(I1) is demonstrated in Figure 2. It is even possible to separate several of the individual rare earth cations, as demonstrated in Figure 3. Theoretical Considerations. The effect of elution parameters can be shown more precisely than was done in the earlier experiments. The cation-exchange equilibrium is represented by
+
+ 2MY+
2MY+RY yE2+ + yE2+Rz
(1)
where E'' represents the eluent cation (ethylenediammonium), MY+ represents the sample metal ion, and the subscript on R represents the number of exchange sites on the resin used by the ion. The selectivity coefficient, KME,for this reaction is
At low loading of sample ion, the resin capacity/2 is ap-
ANALYTICAL CHEMISTRY, VOL. 55, NO. 1, JANUARY 1983
14
lp
ICOt
8
0
~
6 3-
w
Y
L
4c-
i;i
r 20-
1
Figure 4. Plot of the logarithm of the adjusted retention time vs. the logarithm of the eluent concentration for several representative lanthanides. Both the pH and the tartrate concentration are held constant.
L--
0 3 6 9 TIME, min.
300:
Flgure 2. Separation of 20 ppm Mn(1I) and 20 ppm Cd(I I). Conditions are the same as those given In Figure 1.
’
2ool
’
I
l
l
ZrnM etnyleneaiammne
1
pY.45
I
- 80 - 6 5
1
150 t35
Icg
I20
C5 O X
Q m u
Flgure 5. Plot of the logarithm of the adjusted retention time for several representative lanthanides vs. the logarithm of the fraction of that same
metal as free metal cations. Both the ethylenedlammonium ion concentration and the pH are heid constant. in eq 2, where [M’] is the sum of free and complexed metal in solution and aMis the fraction of the metal in solution that exists as the free cation. The capacity factor, k , is now the ratio of [My+R,] to [M’]. Continuing the derivation as before, we obtain an equation that is identical with eq 4, but with an additional term containing aM. ELUTION T I M E , MINUTES
Figure 3. Separation of Lu(III), Tm(III), Ho(III), Gd(III), Nd(III), Pr(1 I I), Ce(I I I), and La(111). All concentrations are 20 ppm except M ethylenediammine,2 X M Pr(II1)(30 ppm): eluent, 2 X tartrate, pH 4.5.
proximately given by [E2+Rz].The capacity factor, k , is equal to the ratio [MY+EtJ/[MY+]. Thus the equation can be written
(3) The adjusted retention time for an eluted peak ( t ? is equal to t&, where tois the retention time of a nonsorbed substance. Substituting t’/t, for k and taking the log of each term gives
’
log t’ = 2
log
(y )+
Y
log t o - - log [E2+]- 1 / 2 log KME (4) 2
In eluents containing a complexing anion such as tartrate, some of the metal cation will be in solution as a neutral or anionic complex. The effect of this complexing on the exchange equilibrium can be calculated by methods worked out primarily by Ringbom (5). We substitute [ M ’ l a ~for [MY+]
’
2 log [E”] - 1 / 2 log KME ( 5 )
The validity of eq 4 was tested by measuring the adjusted retention times of a number of cations at constant tartrate and pH but at varying concentrations of ethylenediammonium cation in the eluent. As shown in Figure 4,linear plots were obtained. The theoretical slope for a 3+ cation is -1.5, but the experimental values for the rare earth ions in the figure were slightly below -1.0. However, the experimental slopes for divalent metal ions such as cadmium, magnesium, calcium, manganese, strontium, and zinc averaged about -0.9 or slightly lower. This is in fairly good agreement with the theoretical slope of -1.0. Linear plots were also obtained in every case when the concentration of tartrate in the eluent was varied and the log of adjusted retention time was plotted against log aM(see eq 5 and Figure 5). The slopes of the rare earth cations varied slightly but most were around 1.2 compared with a theoretical slope of 1.5. From the values of aMin Figure 5, it can be seen that about 90% or more of the lanthanide sample ions are complexed. However, 50% or less of divalent sample ions are
ANALYTICAL CHEMISTRY, VOL. 55, NO. 1, JANUARY 1983
15
Lu
7I I I I 3 6 9 I2 15
0
I
I
18
21
TIME, min
Flgure 6, Separation of the seven heaviest lanthanldes using a-hydroxyisobutyrate eluent. All metal ion concentrations are 10 ppm. M a-hyM ethylenediammine and 3.0 X Eluent is 4.0 X droxyisobutyrate, pH 4.5.
complexed in the mobile phase. These complexing conditions are mild compared to older methods, but complexing is sufficient to sharpen most of the peaks considerably. Eluents Containing a-Hydroxyisabutyrate. Success with tartrate as a complexing eluent anion suggested the use of a-hydroxyisobutyrate, especially for separation of the rare earths. The use of such eluents showed no particular advantage over tartrate for chromatography of divalent metal ions, but separation of' several individual rare earth cations was successful. Figure 6 shows a separation of the seven heaviest rare earths using an eluent of ethylenediammonium a-hydroxyisobutyrate. The tailing of the later peaks suggests that the complexing-dlecomplexing equilibrium probably is slow. Quantitative Measurements and Detection Limits. Plots of either peak height or peak area with ethylenediammonium tartrate eluents gave linear calibration curves for magnesium(I1) over a concentration range of 1.0-15.0 ppm, for calcium(I1) over a range of 2.0-30.0 ppm, and for zinc(I1) over a range of 2.0-14.0 ppm. Other metal cations behave similarly with both tartrate and a-hydroxyisobutyrate eluents. Figure 7 shows the excellent reproducibility obtained for a rapid separation of magnesium, calcium, and strontium. Quantitative measurements are not limited to the concentration ranges mentioned above. The detector sensitivity can be adjusted to work in different concentration ranges. The practical detection limit of this system is 49 ppb of magnesium and 80 ppb of calcium, $whichrepresents only 200 pmol of each metal ion. Use of Masking Reagents. In work with eluents containing a complexing anion it became apparent that the complexed metal moves rapidly through the column under conditions where strong complexing occurs. If a selective complexing reagent could be employed, it should be possible to elute the strongly complexed metal ions quite rapidly and then to separate the remaining cations with the ethylenediammonium tartrate eluent described above. A further requirement would be that the auxiliary complexing agent must work in the 3 to 5 p H range needed for the eluent. Simple calculations showed that EDTA does not complex metal ions significantly such as magnesium(I1)and calcium(l1)
e
10
2e
TIME, min
Flgure 7. Rapld separation of Mg(II), Ca(II),and Sr(I1)for successive injections of the same solution. Each metal is 5.0 X M. Eluent M tartrate, pH 4.5. M ethylenediamine and 3.0 X is 4.0 X
n
-4 0
5
10
TIME, min
Figure 8. Injectlon of Mg(II), Ca(II),and Sr(I1)(1.0 X M each) in lOOX excess of Fe(II1). A sufficient amount of EDTA (0.01 M) was
added to complex the Fe(II1) present. Chromatographic conditions 2.0 X M ethylenediamine and 2.0 X M tartrate, pH 4.5; sample pH is 1.7. The Fe peak present is an Fe(I1) impurity.
at pH 4 but it does complex many metal cations that form more stable EDTA complexes. Therefore, experiments were performed in which EDTA is added to the metal ion sample and the column was eluted with ethylenediammonium tartrate as before. The amount of EDTA used was more than enough to complex the metal ions present, but an unduly high concentration of EDTA was avoided. The results obtained show that conditions can easily be established whereby magnesium and the alkaline earth cation peaks are hardly affected but metal ions that form stable EDTA complexes at about pH 4
16
ANALYTICAL CHEMISTRY, VOL. 55, NO. 1, JANUARY 1983
I
I
I
1
I
J
0
5
IO
15
20
25
is used. This excess of iron(II1) in a sample of magnesium, calcium, and strontium will totally obscure the magnesium peak while calcium and strontium appear on the tail of the “pseudopeak”. Figure 8 shows a chromatogram of the same sample in which EDTA is added to complex the iron(II1). The additional peak is from an iron(I1) impurity in the iron(I1) solution used. Work thus far indicated that any metal ion that has an EDTA formation constant of about l O I 5 or higher should be masked effectively by adding EDTA to the sample. Figure 9 shows a nice separation of magnesium and calcium in samples containing a large excess of aluminum(III), copper(II), or iron(II1). Quantitative data are presented in Table I1 for recovery of magnesium, calcium, and strontium from a much larger concentration of selected metal ions using masking with EDTA. Although a 100-fold molar excess causes slightly high results in several cases, the results are definitely good enough to show that this is a very useful and selective method for quantitative determination of magnesium, calcium, and strontium.
T I M E , min
ACKNOWLEDGMENT
Flgure 9. Determination of Mg(I1) and Ca(I1)(2.0 X M each) in a 50-fold excess of Fe(III), Cu(II), and AI(II1). Each sample had EDTA added to mask the interfering metal. Sample pH was as follows: Fe(III), 1.7; Cu(II), 3.7;AI(III), 3.7. Eluent was 2.0 X M ethylenediamine and 2.0 X M tartrate, pH 4.5.
Table 11. Chromatographic Separation of 20 @molof MgZ+,Ca2+,and Srz+from a Large Excess of Foreign Metal Ion Using EDTA Masking foreign metal ion A13+
coz+
cuz+ Fe3+
molar excess
lox lOOX
1ox
1oox lox 1oox lox lOOX
Nil+
lox
Pb”
lOOX 1ox lOOX
Zn2+
lox
lOOX
recovery, % Mg2+
Caz+
Sr2+
102.5 107.4 100.1 107.3 99.4 104.0 99.8 107.0 102.5 105.2 99.5 107.8 100.9 106.0
101.7 108.1 99.0 97.9 98.2 103.1 96.7 96.5 102.8 104.2 98.3 104.9 98.1 104.9
99.5 102.0 97.7 95.8 96.4 92.3 96.0 96.6 102.1 98.8 97.3 97.5 96.4 102.3
are rapidly eluted. the EDTA, being added only to the sample and not to the eluent, also moves rapidly through the column and appears as part of the initial peak, or pseudopeak. Samples containing a large excess of iron(II1) give extremely wide “pseudopeaks”, when the ethylenediammonium tartrate
The authors with to thank D. T. Gjerde for his consultation and advice and J. Benson for supplying the cation exchange resins used in this work. Registry No. Ca, 7440-70-2;Mg, 7439-95-4;St, 7440-24-6; Pr, 7440-10-0; Ba, 7440-39-3; Cd, 7440-43-9; Ce, 7440-45-1; Co, 7440-48-4; Dy, 7429-91-6; Er, 7440-52-0; Eu, 7440-53-1; Fe, 7439-89-6; Gd, 7440-54-2; Ho, 7440-60-0; La, 7439-91-0; Lu, 7439-94-3; Mn, 7439-96-5; Nd, 7440-00-8; Ni, 7440-02-0; Pb, 7439-92-1; Sm, 7440-19-9; Tb, 7440-27-9; Tm, 7440-30-4; Yb, 7440-64-4; Zn, 7440-66-6; ethyl, 107-15-3; tartaric acid, 87-69-4; a-hydroxyisobutyric acid, 79-31-2.
LITERATURE CITED (1) Small, H.; Stevens, T. S.; Bauman, W. C. Anal. Chem. 1975, 4 7 , 1801. (2) Fritz, J. S.; Gjerde, D. T.;Becker, R. M. Anal. Chem. 1980, 52, 1519. (3) Fritz, J. S.; Gjerde, D. T.; Pohlandt, C. “Ion Chromatography”; Alfred Huthig: Heidelberg, 1982. (4) Elchuk, S.;Cassidy, R. M. Anal. Chem. 1979, 51, 1434. (5) Ringbom, A. “Complexation in Analytical Chemistry”; Wiley-Intersclence: New York, 1963.
RECEIVED for review July 30, 1982. Accepted October 1, 1982. Operated for the U.S. Department of Energy by Iowa State University under Contract No. W-7405-ENG-82. This work was supported by the director of Energy Research, Office of Basic Energy Sciences. This work was presented at the 24th Annual Rocky Mountain Conference, Aug 1-5,1982, Denver,
co.
