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Anal. Chem. 1981, 53, 1620-1627
(29) Schwelghardt, F. K.; White, C. M.; Friedman, S.; Schukz, J. L. “Organic Chemistry of Coal”; American Chemical Society: Washington, DC, 1978 pp 240-257. (30) Uden, P. C.; Carpenter, A. P.;Hackett, H. M.; Henderson, D. E.; Siggla, S. Anal. Chem. 1970. 51. 38-43. (31) Tesarlk, K.; Ghyczy, S. J.’Chmmatogr. 1874, 91, 723-731. (32) Paudler, W. W.; Cheplen, M. Fuel 1979, 58, 775-778. (33) Brown, D.; Earnshaw, D. G.; McDonald. F. R.; Jensen, H. E. Anal. Chem. 1970, 42, 146-151. (34) Lee, M. L.; Novotny, M. V.; Bartle, K. D. “AnalyticalChemistry of Polycyclic Aromatic Compounds”; Academic Press: New York, 1981; A p aendlx 5-.
(35) Later, D. W.; Lee, M. L., Brigham Young University, Chemistry Depart-
ment, Provo, UT, 1981, unpublished work.
(36) Schabron, J. F.; Hurtubise, R. J.; Silver, H. F. Anal. Chem, 1978, 51, 1426- 1433.
RECEIVED for review April 1, 1981. Accepted June 1, 1981. This work was supported by the Department of Energy, Contract No. B-B4843AV,with Pacific Northwest Laboratory, and the Department of Energy, Division of Biomedical and EnvironmentalResearch, Contract No. DE-AC02-79EV10237, with Brigham Young University.
Distribution Processes of Inorganic Solutes in Gel Chromatography Masami Shlbukawa’ and Naoichl Ohta *
’
Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Fukaza wa, Setagaya-ku, Tokyo 158, Japan
Rokuro Kuroda LBboratory for Analytical Chemistry, Faculty of Engineering, University of Chlba, Yayoi-cho, Chlba 260, Japan
A novel concept regarding the separation mechanism has been presented In Inorganic gel chromatography In aqueous media. The distribution of Ionic species Is taken to be a combined effect of size exciuslon and partltlon, the latter Involving ail of the operative factors other than the exclusion effect. The partition depends strongly on the type of the counterion and the cdon in the eluant. Theoretical resuns are In good agreement wlth experimental data, and R , values of ionic species can be estimated semlempirlcally on the basis of the theoretical approach presented.
Gel chromatography is a form of liquid chromatographyfor separating solute compounds primarily according to their size differences, and this technique has been applied extensively in biochemistry and organic polymer chemistry, In the past decade, gel chromatography of inorganic compounds has also been investigated (1). There are many reports on the gel chromatographicbehavior of metal ions and simple anions in aqueous media mainly on highly cross-linked gels with small pore sizes. Most investigators have attempted to explain the gel chromatographic behavior of these small ions in terms of steric exclusion although other secondary effects, such as adsorption, are assumed to be involved in the separation process. The distribution coefficients and ionic sizes have been found well correlated in some cases, in agreement with predicted values based on the size exclusion effect (2-4). However, the gel chromatographic behavior of ionic substances has not been found to be explained exclusively with the exclusion mechanism. There are few papers, however, which succeeded in explaining the separation mechanism in a consistent manner in inorganic gel chromatography. Ogata et al. (5) have shown that the elution volume of magnesium ion on Sephadex G-15 column depends strongly on the type of counteranion in the eluant, the elution volume increasing in the order sulfate < chloride < nitrate < perPresent address: St. Marianna University School of Medicine, Sugao, Takatsu-ku, Kawasaki 213, Japan. 0003-2700/61/0353-1620$01.25/0
chlorate. They also reported (5,6)that this order corresponds to the order of the elution volumes of these counteranions when eluted with 0.1 M sodium chloride solution. Tarutani et al. (7) have observed a similar anion effect on the chromatographic behavior of some divalent metal ions. On the other hand, the effect of the type of coion, Le., the ion with the same charge as the sample ion, has so far scarcely been investigated. We felt that such counterion and coion effecta should reflect the separation mechanism inherent in gel chromatography of ionic substances and attempted to solve the separation mechanism in this paper by assuming the distribution of the solute to be effected as a consequence of combined size exclusion and partition which involves all factors other than exclusion effect. The counterion and coion effects in gel chromatography of ionic solutes will be understood on the basis of the mechanism proposed.
THEORETICAL SECTION If a solute compound is introduced into a system consisting of liquid and gel phases, and then the system is allowed to come to equilibrium, the distribution coefficient can be expressed by eq 1, provided only the size exclusion effect is
K, = V,,/Vi responsible for the distribution, where K. is the distribution coefficient relating to the size exclusion mechanism, Vi is the volume of the internal solvent of the gel phase, and V, is the nonexcluded volume in gel phase. In such a case, the system can be regarded as a volume controlled partitioning with concentration in the available part of the gel phase identical with that in the external solution phase (8). In the exclusion mechanism, the gel acts as an inert matrix, holding the solvent in its pores. However, it seems likely that the properties of the solvent in gel differ from those of bulk of the solvent by the possible interaction between the solvent molecule and the gel matrix. For instance, water molecules i i ~ macromolecular gels generally exhibit physical properties distinct from those of ordinary free water by the interaction with hydrophilic groups or hydro@ 1981 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 53, NO. 11, SEPTEMBER 1981
phobic part of the gel1 matrix (9-12). Consequently, it is reasonable to assume that the concentration of the solute in the available part of .the gel phase, C,, is not necessarily identical with that in the external solution phase, CW This leads to the partition of solute between these two phases with the partition coefficient, Kp = CP/C+ Heitz (13,14)has also considered that the interaction between the gel phase and a solute substance entering it must not be the same as that between pure solvent and the substance and concluded that gel chromatography is regarded as a network-limitedpartition because the polymer network limits the gel phase for the individual solvated substances according to molecular size. The overall distributioln coefficient Kd i s thus expressed as
Kd = K,Kp
(2)
When a sample ion goes into the gel phase, it must be accompanied by the counterion so that both the gel phase and the external solution phase may be electrically neutral in order to satisfy the principle of electrical neutrality of solution. If ionic groups are absent in the gel phase, the partition equilibrium of a sample ion Sm+ (hereafter sample ion is represented by cation, but, af course, the expressions followed are valid for anion) is expressed as
+ mXn-(l) F= Sm+(g)t- :X"(g) n
Sm+(l)
1621
negative peaks; the positive peak is a pseudopeak caused by exclusion of the eluting agent (chloride anion) from the sample zone, and the negative peak corresponds to the sample ion (fluoride anion) (15). This effect does not concern the partition of a neutral species; the "ion exchange" is considered to take place essentially based on the electrostatic interaction. Therefore, if the sample ion forms an ion pair with the counterion and the resulting neutral species becomes partitioned between the external solution phase and the gel phase, process B can be ruled out. Here, the completely dissociated ion system will be considered. Consequently, there are two processes of sample ion partition, (A) and (B). However, these two processes are not independent, but invariably take place simultaneously. The overall partition equilibrium of the sample ion Sm+in the YX eluant system is thus written as 2P+(l)
+ mX"-(l) + ?YP+(g) n P 2sm+(g)
F=
+ EXn-(g) + FYP+(l) (C) n
and correspondingly we have the thermodynamic partition coefficient K p , F expressed by
(A)
where Xn- is the counterion, and g and 1 denote the available part of the gel phase and the external liquid phase, respectively. This process cam be regarded as the partition of an electrolyte SXm/, (ion aissociation is not necessarily assumed in this partition equilibrium). In equilibrium, the chemical potential psx,,. (=ps (rn/n)Mx)of the hypothetical electrolyte SX,,, is the same in both phases
+
where the quantities in the gel phase are given a superscript bar notation. The cheimical potential of species i is pi =: pio R T In ai (4)
(6) In other words, the partition coefficient of sample ion with YX as the eluant electrolyte is just the equilibrium constant of the equilibrium (C). KP$' is the equilibrium constant in the special case, Y = S. By combining eq 2 and 6, we obtain ultimately the overall thermodynamic distribution coefficient Kd,F of the sample ion in gel chromatography when eluting with YX.
+
where fi0 = the chemical potential of species i in the standard state, ai = the activity of species i, R = the gas constant, and T = the absolute temperature. The standard state is taken to be an external solution or gel phase of infinite dilution. By combining eq 3 and 4, the thermodynamic partition coefficient KpZ of samlple ion Sm+ accompanying the counterion X" is thus represented
We see that the overall distribution coefficient of sample ion depends on the type of counterion and the coion in the eluant, even i f K , , y is constant regardless of the type of eluant electrolyte.
EXPERIMENTAL SECTION Materials. Tris(1,lO-phenanthroline)iron(II)(Fe(phen)82+) and tris(2,2'-bipyridine)iron(II) (Fe(bpy):+) salts were prepared according to literature and tris(glycinato)cobalt(m)(Co(g1y)J was prepared as described previously (18). The purity of each product was checked by elemental analysis and UV-VIS spectrometry. The eluants used were aqueous solutions of the various metal salts with the ionic strength of 0.1, the effect of fixed ionic groups of the matrix of Sephadex G gel turning out negligible in these media (18, 19). Test solutions (0.001-0.03M)were prepared by dissolving the salts of sample ion in the eluant used. Each sample salt used consisted of sample ion and the counterion which is the same as the constituent of eluant electrolyte, unless otherwise stated. For the determinations of Rf values of Br- and I- in alkaline earth nitrate media, and of M F in KI media, NaBr, NaI, and Mg(NOJ2 were used as the sample salts,respectively. There is no problem in these replacements because these salts are strong electrolytes and the sample concentrationsare considerably lower than the eluant concentration. Blue Dextran 2000 (Pharmacia Fine Chemicals, Uppsala, Sweden) was employed as an internal standard to calculate the Rf values of samples.
(In,
where Apia = pio - pio. It should be noted that K$ is the same as the partition coefficient KPgXof the sample ion when SX (subscript numbers indicating the chemical formula omitted for simplicity) in used as the eluant electrolyte in the chromatographic system. In general, a background eluant coion, Y@, differs from the sample ion. Thus, we have to take into account the following "ion exchange" equilibrium
+ EYPt(g) P
Sm+(l)
+ FYP+(l)
Sm+(g)
(B)
It has been observed that the eluant electrolyte is excluded from the sample zones (6,15,16).For instance, when sodium fluoride is eluted with sodium chloride solution, the elution curve obtained by argentmetry shows a pair of positive and
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 11, SEPTEMBER 1981
Cross-linked dextran gels, SephadexG-10 and G-25 (dryparticle size, 40-120 and 10-40 Km, respectively,Pharmacia Fine Chemicals) were used after the pretreatment as described previously (18).
Development. A gel suspension was prepared by thoroughly mixing 15.0 g of Sephadex G-10 or 7.0 g of G-25 with 35 mL of distilled water and allowing it to swell overnight. The swollen gel slurry was spread on two glass plates (20 X 20 cm) at an applied thickness of 500 bm. The gel layer was air-dried for about 1 h. The development was carried out at 25.0 f 1.0 "C by the descending technique (20),after preliminarydevelopment for at least 14 h. One-microliter portions of samples were usually applied. When the spot of Blue Dextran 2000 migrated ca. 100 mm down, the gel plate was taken out of the chamber, and Toyo filter paper no. 51 A (Toyo Roshi, Tokyo, Japan) wetted with distilled water was immediately placed onto the gel layer and gently dried with a warm air stream to get a replica. Detection. Fe(phen)?+ and Fe(bpy),2+were detected on the paper replica by their own characteristic red color, and Co(gly), was detected as dark spots under W light. The spots of the other sample ions were revealed on the paper replica in daylight or under UV light by spraying with the following visualizing reagents: Na': 10 g of uranyl acetate and 30 g of zinc acetate dissolved in 9 g of 30% acetic acid and diluted to 100 mL with water. K+: 20 g of sodium hexa(nitrito)cobaltate(III) dissolved in solution consisting of 56 g of sodium acetate, 75 mL of water, and 25 mL of glacial acetic acid. Mg2+: 0.5% 8-hydroxyquinoline solution in ethanol. Sr2+and Ba2+: 1% aqueous sodium rhodizonate. F-: 11.2 mg of AlC13.6H20and 11.8 mg of morin dissolved in 20 mL of 30% acetic acid and then mixed with 40 mL of 99.5% ethanol. C1-, Br-, and I-: 5 % aqueous AgNOSsolution. Clod-: saturated sodium acetate solution, followed by 0.5% aqueous methylene blue. NO3-: 10% acetic acid, 1% sulfanic acid in 30% acetic acid, 0.03% a-naphthylamine in 30% acetic acid, and zinc powder sprayed, in this order. Sod2-:0.2% aqueous BaClzsolution, followed by 1% aqueous sodium rhodizonate.
RESULTS AND DISCUSSION Counterion Effect. First of all, consider the effect of the type of counterion in the eluant. The overall thermodynamic distribution coefficient Kd,zZof the ion X"-, which is the counterion in eq 7, when eluted with the solution of the salt consisting of Wq+ and'Z is given by the following equation.
where
See the Appendix I for the discussion on the constancy of $/yk In thin-layer chromatography, R, value is defined as
-1)
R , = log(
The relationship between R, and Kd can be given by the Martin and Synge eq (21)
where A, and A, are the cross-sectional areas of the external solution and the gel phases, respectively. The validity of eq 15 in thin-layer gel chromatography was confirmed in the previous work (22). Because A , / A , ratio can be regarded as being constant regardless of the kind of eluant electrolyte, the following relationship is obtained by combining eq 12 and 15
m R,JX = - R,JZ 2n
+ Fl(K,) + kl'
(16)
where
Porath (23) and Ackers (8) have already shown that Ks,iis a function of the size of the species i (e.g., hydrated radius, RJ, but to be more exact, K, of the ionic species should depend not only on the size of the sample ion (RS) but also on that of the counterion (Rx) because of the requirement of the electrical neutrality (5). Hence, the general expression for K , , F can be written
log Kd,yZ =
KSJX= f(RsPx)
(18)
If Rs >> Rx, the contribution of Rx to K;,'
can be neglected
Ka,P = f(Rs)
(19)
On the other hand, when RS > R X
Rs (11) Equation 9 holds not only for a given single ion, X", but rather for all ions of the same valency, n-. The similar relationship should also hold for the molar distribution coefficient, Kd, provided the molar activity coefficient ratio of single ion i, yi/yi, can be regarded as being constant independent of its own concentration and the types and concentrations of coexisting ions.
> Rxv);Rrn,iX6)= log f(Rs)- log --
4
(independent of the type of
= constant
X,),that is, Rm,T:)
-0 5
(24)
1 ~
where Rw
