Anal. Chem. 1988, 58, 2226-2233
2228
PRP-1 column (0.1-1.0 FgmL-') and on a C18reversed phase (18-58 pgmL-1) were linear with a negative Yaxis intercept equivalent to 0.05 and 0.04 pgmL-' U(V1) respectively. Silica-based supports used for this analysis must have a minimum of free silanol sites. Both U(V1) and Th(1V) interact strongly with bare silica surfaces and this interaction can cause peak tailing. The excellent sensitivity and selectivity of this interaction with HIBA make this approach attractive for the determination of trace concentrations of U(V1) and Th(1V) after trace enrichment, and this approach is being studied. This technique is also being studied for the selective determination of U(V1) in solutions of irradiated fuel. ACKNOWLEDGMENT The authors wish to thank J. L. Dalton (CANMET) for the X-ray fluorescence results and helpful discussions and F. M. B. Pontes (University of Waterloo, Ontario) for technical assistance during parts of this work. Registry No. La, 7439-91-0;Ce, 740-45-1; Nd, 7440-00-8;Sm, 7440-19-9; Eu, 7440-53-1; Gd, 7440-54-2; Tb, 7440-27-9; Dy,
7429-91-6; Ho, 7440-60-0; Er, 7440-52-0; U, 7440-61-1; Tm, 7440-30-4; Th, 7440-29-1; Yb, 7440-64-4; Lu, 7439-94-3; Y, 7440-65-5; Pr, 7440-10-0;Arsenazo 111, 1668-00-4. LITERATURE CITED Ritcey, G. M. Sep. Sci. Technol. 1983, 18, 1617-1646. Barkley, D. J. Tech. Bull. 7B125;Energy Mines and Resources Canada: Ottawa, July 1970. Takata, Y.; Arikawa, Y. Bungeski Kagaku 1975, 2 4 , 762-767. Dubuquoy, C.; Metzer, G. Analysis 1977, 5 , 314-320. Larsen, N. R. J. Radioanal. Chem. 1070, 52, 85-91. Elchuk, S.; Cassidy, R. M. Anal. Chem. 1070, 51, 1434-1438. Cassidy, R. M.; Elchuk, S . Anal. Chem. 1082, 5 4 , 1558-1563. Knight, C. H.; Cassidy, R. M.; Recoskie, B. M.; Green, L. W. Anal. Chem. 1984, 5 6 , 474-478. Cassidy, R. M.; Elchuk, S.;Elliot, N. L.; Green, L. W.; Knight, C. H.; Recoskie, 6 . M. Anal. Chem. 1988, 58, 1161-1186. Lalonde, C. R.; Dalton, J. L. Can. J . Spectrosc. 1982, 2 7 , 163-170. Hunt, G. A.; Guest, R. J.; Ingles, J. C.; Hltchen, A,; Barkley, D. J. A Manual of Analytical Methods Used by the Canadian Mining Industry; Energy, Mines and Resources Canada: Ottawa, 1974; p 196. Cassidy, R. M.; Fraser, M. Chromatographia 1984. 18, 369-373.
RECEIVED for review January 28, 1986. Accepted April 30, 1986.
Liquid Chromatographic Separation and Indirect Detection of Inorganic Anions Using Iron(I I) 1, IO-Phenanthroline as a Mobile Phase Additive Pantelis G. Rigas a n d Donald J. Pietrzyk*
Chemistry Department, T h e University of Iowa, Iowa City, Iowa 52242
Iron(I1) 1,lO-phenanthrdine [Fe(phen):+] salts are used as a m W e phase additlve for the liquid chromatographic separation of inorgank analyte anions on a reverse stationary phase. The retention appears to be consistent with an Ion Interaction type retention. Major variables evaluated are the retention and type of reverse statlonary phase, Fe(phen):' mobile phase concentration, organic modifier effects, pH, buffer components, type and concentration of counteranion, ionic strength, and anion selectivity. Indirect detection Is used to detect analyte anions by monitoring the effluent at 510 nm where Fe(phen),?+ absorbs. Several separations of multkomponent mixtures illustrating excellent resolution and efficiency are shown. A detection limit under favorable conditions approaches about 1 ng. Linear detector response with analyte anion (Ci-, Br-) concentration was found from 10 ng to over 10000 ng of Injected analyte.
A successful strategy used in the liquid chromatographic (LC) separation of ionic analytes is to use a hydrophobic ion as a mobile phase additive and take advantage of the interactions that occur between the analyte ion, hydrophobic ion, and the stationary phase. In general, the result is increased retention, improved selectivity, more favorable resolution, and more mobilestationary phase parameters that can be adjusted for optimization of the factors. This technique, which was first applied to the separation of organic analyte ions, is most often referred to as "ion pair chromatography" (IPC) and the hydrophobic ion is called the pairing reagent. IPC and its 0003-2700/86/0358-2226$0 1.50/0
applications in organic separations have been reviewed extensively (1-4). This same strategy has been shown to be applicable to the separation of inorganic analyte ions (5-12); for example, to separate inorganic anions, a tetraalkylammonium (R,N+) salt can be used as the mobile phase additive (5-9) while for cations an alkylsulfonate (RS03-) salt can be used (9-12). Several studies have focused on the nature of the interactions in IPC; these are reviewed elsewhere in detail ( 1 4 , l l - 1 6 , and references within). Clearly, no single interpretation of the interactions accounts for all observations and it is conclusive that the experimental conditions must be carefully accounted for. For example, appreciable association (ion pair formation) is indicated when both the analyte ion and the pairing reagent have extensive hydrophobic centers, when mobile phase organic modifier concentration is high, or when the stationary phase is modified by the presence of a immiscible, liquid stationary phase. In contrast, no or little association is indicated when using hydrophobic ions with modest hydrophobic centers, inorganic analyte ions, organic analyte ions that contain weak hydrophobic centers, and predominately aqueous mobile phases. Because of the lack of ion pair formation under the latter conditions, this type of retention and separation is often referred to as "ion interaction chromatography" (IIC) and involves a double layer type interaction a t the stationary phase surface (5, 12, 13). Several types of hydrophobic ions have been employed in IPC and IIC. In this report the use of iron(I1) 1,lO-phenathroline [Fe(~hen),~+] salts as the hydrophobic cation mobile phase additive for the separation of inorganic analyte anions tZ 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 11, SEPTEMBER I986
is discussed. Fe(phen)t+ offers several advantages. (1)Fehen)^^+ is highly retained on a reverse stationary phase because of the contribution of the three phenanthroline rings in the complex to its hydrophobicity; thus, the mobile phase
concentration of Fe(phen),2+needed to establish an equilibrium amount of Fe(~hen),~+ retained on the stationary phase is smaU (2) Fe(phen):+ is a divalent cation; thus, its apparent anion exchange capacity is twice the amount retained on the stationary phase. (3) Fe(phen)z+ in the mobile p h pmvidea a way to indirectly detect d y t e anions in the column effluent by monitoring absorbance a t the wavelength a t which Fehen),^+ absorbs. (4) Several mobile phase variables can be manipulated to alter inorganic analyte anion retention, selectivity, and resolution. (5) This approach coupled with indirect detection by absorbance change provides a useful alternative to double and single column ion exchange chromatography (ion chromatography) which are widely used for the separation and determination of inorganic anions.
(In,
EXPERIMENTAL SECTION Reagents. Sulfate and C104- salts of Fe(phen);+ and 1,lOphenanthroline were purchased from GFS Chemical Co. Initially, the Cl- salt was prepared by reaction of FeC13with NH20H.HC1 and l,l0-phenanthroline or by reaction of FeCl, with the ligand (18). Subsequently, the F and C1- salts of Fe(phen),,+ were prepared by passing a solution of the 502-salt through a strong base anion exchanger charged in the F or Cl- form, respectively. Inorganic and organic salts used for buffers, ionic strength adjustment, or anal- were analytical reagent grade when possible. Organic solvents were LC quality. LC quality water was obtained by passing distilled water through a Sybron/Barnstead purification unit. Prepacked columns were obtained from Hamilton Co., PRP-1, and Du Pont Co., Zorbax CU. PRP-1 is a polystyrene divinylbenzene, 10 pm, spherical, reverse phase adsorbent in a 150 mm X 4.1 mm i.d. column. Zorbax CI8 is a reverse phase, 6 pm, spherical, octadecylsilica in a 150 mm X 4.6 mm i.d. column. Instrumentation. The LC instrumentation consisted of a Waters 6OOOA pump and U6K injector and a Spectra Physics 770 or Kratos 773 variable wavelength detector. Column temperature was maintained at 30 OC with a Bioanalytical Instrument temperature controller. A Spectra Physics M4100 computing integrator was used for peak area measurements. Procedures. Analyte anions were prepared as aqueous solutions of 1-2 mg/5 mL of Na or K salts. Sample aliquots of 1-10 r L were introduced by syringe. Mixed mobile phase solvents are percent by volume. Weighed amounts of ionic strength salts, NaClO,, NaCl, or Na$04, and buffer salts, which were prepared by adding weighed amounts of the acid, usually acetic, citric, or succinic acid and adjustment to the desired pH with NaOH solution, were added to the mobile phase prior to dilution to volume. Buffer concentrations were usually 1.0 X lo-" M, when necessary ionic strength accounts for buffer concentration. Column breakthrough volumes were determined by passing a Fe(phen),,+ mobile phase of known concentration through a at a constant flow rate until column that is free of Fe(~hen),~+ the Fe(phen),,+ appeared in the column effluent. This was determined by monitoring the effluent at 510 nm where Fe(phen):+ absorbs. The breakthrough profile appears as a very sharp rise in detector response which reaches a maximum value. The breakthrough volume was taken as the inflection point of the profile. The amount of retained Fe(~hen),~+ is calculated from the breakthrough volume (the volume was always >20 mL; thus column void volume is negligible) flow rate, and Fe(pher&,+ mobile phase concentration. Columns were conditioned prior to use with the desired mobile phase by passing at least 100 mL (1mL/min) of mobile phase beyond that required to reach the breakthrough. The retained Fe(phen)32+salt was rapidly removed from the PRP-1 column with 91 CH3CN:H,0, usually 500 mL was sufficient, while for the Zorbax C18 several liters were required for removal of the Fe(phen)32+salt from the column. Flow rate was usually 1.0 mL/min and inlet pressure depending on the mobile phase was 800-1500 psi. Analyte detection was at 510 nm. Capacity factors,
2227
k', were calculatedin the usual way; column void volume, V, which was determined by using analytes that were not retained at the conditions being studied, were 1.0-1.3 mL. RESULTS AND DISCUSSION Studies (5,9,11-13, 19) with R4N+ and RS03- salts as mobile phase additives and inorganic analyte anions and cations, respectively, indicate that in IIC two major simultaneously occurring equilibria are responsible for analyte ion retention on a reverse stationary phase. One describes retention of the hydrophobic ion and its counterion as a surface double layer on the stationary phase. The primary layer is due to hydrophobic ion retention via its hydrophobic center producing a surface charge. The secondary diffuse layer is composed of inert ions of opposite charge. The second equilibrium defines an ion exchange like selectivity between ions in the diffuse secondary layer and analyte ions of the same charge. Thus,analyte ion retention, selectivity, and resolution are influenced by how the hydrophobic ion used and its properties and the stationary-mobile phase parameters and their optimization affect these two equilibria. Fe(phen)z+ is a dication that also has a significant hydrophobic center. Since it is a water soluble, inexpensive, stable complex, like a R4N+salt, it should be useful as a mobile phase additive for the separation of analyte anions. Thus, for the system composed of a reverse stationary phase, A, and an aqueous mobile phase containing a F e ( ~ h e n ) , ~salt, + a buffer within the pH range of Fe(phen),2+ stability, a known counteranion, C-, provided by the buffer, inert electrolyte which also provides a known counteranion as well as a fixed ionic strength, perhaps a small amount of organic modifier, and the analyte anion, X-,the two major equilibria, if IIC is followed, are
+ Fe(phen)32++ 2C- s A-.Fe(phen)32+2C- (1) A . - F e ( ~ h e n ) ~ ~ + 2+ C -2X- G A-.Fe(phen)32+2X- + 2CA
(2) The parameters are, therefore, the reverse stationary phase (influences the equilibrium in eq 1and column performance), the mobile phase Fe(phen):+ concentration (determines the number of apparent anion exchange sites by controlling the direction of eq 1and determines the counteranion and analyte selectivity defied in eq 2), mobile phase solvent composition (since retention of Fe(phen)z+ is consistent with reverse phase retention, mobile phase solvent composition determines the direction of eq 1; a lesser effect is on selectivity in eq 2 assuming the solvent change is a modest one), type and concentration of counteranion in the mobile phase (the type influences eq 1while its concentration through mass action shifts eq 2), mobile phase pH and buffer (provides a counteranion, influences stability of Fe(phen)z+, and affects the charge of the analyte anion if it is derived from a weak acid). To follow IIC, it is assumed that Fe(phen);+ salts are appreciably dissociated in an aqueous medium. Extraction data suggest that this might not be the case for salts of certain simple counteranions. For example, the distribution ratio values for nitrobenzene/H,O extraction of Fe(phen),2+(C-), salts is highly dependent on the counteranion; for C- = C 1 0 ~ , SCN-, I-, Br-, and C1- the values are 13.5,1.7,0.8,0.03,0.003, respectively (20). The favorable nonpolar solvent solubility relative to the other anions suggests a partial dissociation of the C104- salt and because of this C10,- should be a strong eluent counteranion. Stationary Phase. Two reverse stationary phases, PRP-1 and Zorbax C18, were evaluated. The studies reported here deal primarily with PRP-1 as the stationary phase. Several reasons favored this decision. (1) Retention of Fe(phen),,+ appeared to be more reversible on PRP-1. That is, the column could be repeatedly equilibrated with a Fe(phen)32+mobile
2228
, , -Y
ANALYTICAL CHEMISTRY, VOL. 58, NO. 11, SEPTEMBER 1986
O -
L&.(Ph*n)PJJ,
M
2
-2 1
Lc.dsc4ll. Y
F@re 1. Retention of Fe(phen):+ salts on PRP-1 as a function of the mobile phase variables: (A) a 4.1 X 150 mm, 10 pm PRP-1 column and an aqueous Fe(phen),SO,, 5.0 X loJ M acetate, pH 6.2, mobile phase at 1.0 mL/min and 30 O C ; (6)conditions are the same as A except an aqueous 1.0 X loe4M Fe(phen),'+ perchlorate or sulfate salt, NaCIO, or Na'SO, mobile phase or a MeOH:H,O, 1.O X lo-' M
Fe(phen),SO, mobile phase. phase; the Fe(phen)?+ salt removed from the stationary phase and then recharged in the Fe(phen)?+ salt form. Each time the F e ( ~ h e n ) ~loaded ~ + column still provided reproducible chromatography. In contrast large volumes of CH3CN:H20 were required to remove the Fe(phen),2+salt from the Zorbax Cis. Repeated cycling leads to nonreproducible results. Also, inspection of the C,, particles indicated that not all the Fe(phen)?+ salt was removed. Other studies (9)have indicated that free silanol sites within the Zorbax CIScan participate in cation exchange and it is possible that part of the Fe(phen)?+ is retained as a cation, thus eliminating it as a source for anion exchange. (2) PRP-1 being stable throughout the pH range, unlike the C18, allows chromatography at a basic pH. (3) PRP-1gave favorable column efficiencies even though a 10-pm particle was used. (4) PRP-1 column life was significantly longer. (5) Retention of a Fe(phen)32+salt at a given condition was greater on PRP-1; this contributes to better analyte anion selectivity and resolution. All PRP-1 columns used were of similar hydrophobicity according to a phenol (3.2 min) and benzene (5.4 min) test sample using a 9:l CH3CN:H20mobile phase at 0.5 mL/min. Significant departure from this will alter retention times because of a subsequent effect of F e ( ~ h e n ) , ~retention. + Retention of F e ( ~ h e n ) , ~F+e.( ~ h e n ) , ~salt + retention on PRP-1 and Cla is high and counteranion-dependent. A mobile phase rich in organic modifier is required to elute the Fehen),^+ salts in a reasonable time period. The equilibrium amount of Fe(phen)?+ salt retained on PRP-1 for a given mobile phase F e ( ~ h e n ) concentration ~~+ was calculated from column breakthrough volumes. These experiments are summarized in Figure 1A. As the mobile phase F e ( ~ h e n ) salt ~~+ concentration increases, the amount retained on the PRP-1 column, which is also a measure of the apparent anion exchange capacity, appears to increase over the concentration range studied. Since the F e ( ~ h e n ) , ~is+hydrophobic due to the three phenanthroline rings, it is highly retained. Thus, a significant surface anion exchange capacity is produced even at low mobile phase Fe(phen)32+concentrations. For example, at a mobile phase concentration of 1.0 X M Fe(phen)p, which was used in most subsequent studies, there are 17.2 pmol of Fe(phen)?+ distributed throughout and in equilibrium with the stationary phase surface provided within the 15-cm PRP-1 column. Since Fe(phen)?+ is divalent this represents 34.4 pequiv/column of anion exchange sites. This is typical of the apparent anion and cation exchange capacities generated when using R4N+salts (5, 9) and RS03- salts (9, 11) as mobile phase additives for inorganic analyte anion and cation separations, respectively, and the lower end of exchange ca-
pacities used in single column ion chromatography (17). If the mobile phase organic modifier concentration for a given F e ( ~ h e n ) , ~concentration + is increased, the equilibrium amount of the F e ( ~ h e n ) , ~salt + retained on the PRP-1, and subsequently the apparent anion exchange capacity, decreases. In contrast increasing the mobile phase ionic strength for a given F e ( ~ h e n ) , ~mobile + phase concentration causes the equilibrium amount of the Fe(phen)?+ salt retained on the PRP-1 and the apparent anion exchange capacity to increase. As shown in Figure 1B a modest change in either of these variables significantly alters the amount of retained Fe(phen)?+ salt per column. The amount of retained Fe(phen)?+ salt and the subsequent number of anion exchange sites are also counteranion-dependent. In Figure 1B not only the amount of F e ( ~ h e n ) , ~salt + retained on PRP-1 from an aqueous NaC104 medium is significantly higher but the rate at which the number of sites increases with increased salt concentration is greater than that from the Na2S04medium. Over the NaC104 and Na2S04 salt concentration studied (Figure 1B) the number of sites in the S042-mobile phases even at its most favorable condition is still below the least favorable Clod- condition. This is consistent with eluent strength and inorganic analyte anion elution order which became apparent in subsequent studies. That is the C 1 0 ~ salt provides greater surface coverage and is a stronger eluent counteranion than the SO$- salt. Therefore, for a given column the major variables that determine the number of apparent anion exchange sites due to retention of the Fehen),^+ salt are the F e ( ~ h e n ) salt ~ ~ +mobile phase concentration, the organic modifier and its concentration (at a given percent the amount of Fe(phen)?+ salt retained decreases in the order MeOH < EtOH CH3CN), and the mobile phase ionic strength. The surface coverage for the Fe(phen)? sorption isotherm shown in Figure 1A was calculated by using a PRP-1 surface area of 415 m2/g and column weight of 1g. Thus, from 5 x to 2.86 X loy4M Fe(phen),S04 the equilibrium coverage due to retained Fe(phen),S04 changes from 0.040 to 0.077 pmol/m2, respectively. The isotherm in Figure 1A follows C, = kC,l/X, where C, and C, are the amount of F e ( ~ h e n ) ~ S O ~ in the stationary and mobile phase, respectively, and fits the equation log [pmol of Fe(phen),2+/m2]= 0.38 log [Fe(~hen),~'] + 0.26 with a correlation of 0.9992 and a l / x value of 0.38. When compared to C7S03-Li+retention on PRP-1 (9) the coverage of the Fe(phen),SO, for a given mobile phase concentration is greater but the rate at which it increases in surface coverage with increased mobile phase concentration is much less. For example, at 0.0003 M Fe(phen)$04, 27 pmol of Fe(phen),S04 is retained per column while the amount of C7S03-Li+retained per column is estimated (9) to be about 2.5 Mmol. From Figure 1A a 10-fold increase in Fe(phen),S04 concentration increases the amount retained by about 15 pmol while for C7SO>61 61
5.38 5.41 7.36 9.49 9.61 10.8 14.5 20 37 >>70 68
5.46 6.05 10.9 11.3 12.5 18.1 28 65 9.37 85
8.23 8.39 11.3 11.7 12.8 18.3 29 56 6.64 84
4.1 mm, 10 pm, PRP-1 column and an aqueous 1.0
M Fe(phen)?+X (where X = SO-:, ClOc, el-, or F-), 1.0 X M sodium succinate buffer, pH 6.10 mobile phase at 1.0
mL/min. *System peak.
using F e ( ~ h e n ) ~ ( C land O ~ )NaC104 ~ with the difference being that retention is less in a NaC104 solution of equal ionic strength. Even though the number of anion exchange sites in the column increases with ionic strength due to increased F e ( ~ h e n ) salt ~ ~ +retention (see Figure 1B for Fe(phed3S04 retention from a SO:- solution), the mass action effect of the counteranion to reduce retention is much greater. As shown in Figure 4 the analyte anion retention order stays constant over the ionic strength range studied. A comparison of four common eluent counteranions at 1.0 X 10" M Fe(phen)?+ salt mobile phase concentration is shown in Table I. Counteranion eluent strength follows the order SO4,- 2 C104- > C1- > F-, which is similar to the order observed when using typical quaternary ammonium type anion exchangers (26). The mobile phase used in Table I contains two counteranions, one from the Fe(phen);+ salt and succinate from the buffer. Even though succinate is a divalent anion at the mobile phase pH of 6.1 and is a strong eluent counteranion, the effect of the second counteranion on analyte anion retention is still observed. If NaOAc is used as a buffer, its concentration would have to be about five times the succinate concentration used in Table I to obtain similar k'values. Thus, the mass action effect of an increased concentration of the monovalent acetate anion is used to compensate for the greater eluent strength of the divalent succinate anion. Both mobile phase counteranions produce system peaks. The
ANALYTICAL CHEMISTRY, VOL. 58, NO. 11, SEPTEMBER 1986
Table 11. Effects of Different Mobile Phases on Analyte Anion Retention analyte
A
F-
5.38 5.41 7.36 9.49 9.61 10.8 14.5 20.3
103-
ClNOL BrO;
BrNO< C103I-
BF,SCN-
36.6 44.8 >120
k' for mobile phases A to Fa B C D E 3.06
0.80
4.59
3.50 4.16
1.24 2.07
6.15 7.26
4.38 5.23
2.62 4.62
10.7
10.3 14.8
1.31 1.18 1.93 2.68 2.64 3.25 4.88 7.28 8.92 15.0 23.4 63
I
2231
F
I--,Ll
Bi
F 1.10 1.61 2.29 2.82 4.23 6.46 6.44 14.9 21.4
OA 150 mm X 4.1 mm, 10 pm, PRP-1column and (A)aqueous 1.0 X lo-, M Fe(~hen)~(ClO,)~, 1.0 X lo-' sodium succinate, pH 6.1; (B) 1:9 MeOH:H20, 1.0 X lo4 M Fe(phen)$304, pH 5.30; (C) aqueous 1.0 x lo-" M Fe(phen),SO,, 5.0 x lo-, M Na2S04,pH 5.50; (D) aqueous 1.0 X lo-, M Fe(phen),SO,, 5.0 X lo-, M NaOAc, pH 6.2;(E) aqueous 1.0 X lo-, M Fe(phen),(ClO,),, 1.0 X lo4 M sodium citrate, pH 7.25;(F)aqueous 1.0 x lo-, M Fe(phen),(ClO,),, 1.0 X lo-, M sodium 1,3,5-benzenetricarboxylate,pH 5.8 mobile Dhases at 1.0 mL/min.
stronger the eluent counteranion is, the higher the retention volume for its system peak. Thus, in Table I the stronger eluent counteranions, which are also highly retained analyte anions, such as succinate, C104-,and S O:-, yield system peaks that do not interfere in the chromatogram. The weaker ones both in eluent strength and retention as analytes, such as Cland P, yield system peaks early in the chromatogram and may interfere depending on the analyte anion. Comparison of the data for the retention of different anions as analytes establishes the analyte selectivity order. For PRP-1and F e ( ~ h e n ) salt ~ ~ +as the additive a t a pH 6.1 the retention of inorganic monovalent anions follows the order
C104- > I- > C103- > NO3- > propionate >CNO- > IO4- > IO3- > Br- > Br0,- > NO2- > C1- > C102- > formate > acetate > F- (3) Since several analytes are weak acid anions retention order will be altered depending on pK, by using a mobile phase pH that suppresses ionization. At these conditions elution will be earlier, since the equilibrium shown in eq 2 is shifted to the left. Also, since indirect detection is used, the more ionization is suppressed the smaller the analyte peak area becomes (25). The elution order in eq 3 is similar to the order observed when using quaternary ammonium type anion exchangers (26). A quantitative comparison of retention data for monovalent anions for several mobile phase conditions is shown in Table 11. Adding MeOH or other organic modifier (see Figure 3 and compare eluent A to B in Table 11) and increasing ionic strength (see Figure 4 and compare eluent A to C) decreases analyte anion retention. Changing F e ( ~ h e n ) concentration ~~+ will also affect analyte retention; however, a large change is required (see Figure 2) before a noticeable effect on analyte anion retention is obtained. The variable offering the most versatility in influencing analyte anion retention is the counteranion that can be supplied by the Fe(phen),2+ salt, the buffer, and/or ionic strength salt. Table I illustrates the effect of counteranion due to an ionic strength salt while mobile phases A, D, E, and F in Table I1 focus on the buffer as the counteranion source. At the mobile phase pH used the buffer anions, acetate, succinate, citrate, and 1,3,5-benzenetricarboxylate are predominately -1, -2, -3, and -3, respectively. As the charge or the concentration (note that acetate
Figure 5. Effect of mobile phase variables on the separation of the halides: a 4.1 X 150 mm, 10 pm, PRP-1 column and an aqueous (A) M succinate, pH 6.1; (6)same 1.0 X M Fe(phen),SO,, 1.0 X as (A) except 1:95 MeOH:H,O, pH 6.1; (C) same as (A) plus 4.0 X lo4 M Na,SO,, pH 6.1, mobile phase at 1.0 mL/min, 30 OC,and indirect detection at 510 nm.
Flgure 6. Separation of highly retained inorganic monovalent anions. CondRons are the same as in Figure 5A except in (A) 5:95 MeOH:H,O and in (6) no buffer (pH 5.5) and 1:5 MeOH:H,O.
in mobile phase D is 5 times as concentrated as the others) of these buffer anions increases, analyte anion retention decreases. Table I1 also lists retention data for Clearly, in order to separate multivalent anion mixtures and/or the more highly retained monovalent anions, the strong eluent strength offered by multivalent buffer anions (mobile phases A, E, and F in Table 11) are required for more favorable separation times. Separations. Several mobile phase parameters can be manipulated to optimize retention time, selectivity, and resolution when attempting to separate mixtures of inorganic analyte anions. If indirect absorption detection is used, a high Fe(phen):+ salt mobile phase concentration, which increases the apparent anion exchange capacity on the PRP-1surface due to retention of the F e ( ~ h e n ) salt, ~ ~ +is not recommended to increase analyte retention time because of the need to offset background absorbance with the detector. If conductivity is used, more flexibility is available with F e ( ~ h e n )salt ~~+ concentration providing the mobile phase conductivity is not excessive. Altering mobile phase ionic strength, organic modifier concentration, and type and concentration of counteranion used for the inert ionic strength salt and/or buffer can be readily done to optimize separations. Mobile phase pH is a useful variable to alter elution order only if the analytes are anions of weak acids or to determine buffer anion charge. Retention data from the previous figures and tables indicate that selectivity and resolution are favorable for the separation of complex mixtures of inorganic anions. Several
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 11, SEPTEMBER 1986
I
0
l
/
20
I
I
40
I
l
-
60 m i
/
/
SO
I
I
100
I
I
I
I20
Figure 7. Separation of a multicomponent mixture of inorganic monovalent anions. Conditions are the same as Figure 5A except Fe(phen),(CIO,), is used.
examples illustrating this are shown in Figures 5-7. Also, these separations focus on how the mobile phase parameters can be adjusted to change retention time, selectivity, and resolution. Figure 5 shows the separation of a halide mixture on PRP-1 using several different mobile phase conditions and indirect detection at 510 nm. Each halide was injected a t about 1 pg calculated as the anion. For all examples in Figure 5 the F e ( ~ h e n ) mobile ~ ~ + phase concentration is 1.0 X M, a divalent counteranion is used, and the pH is 6.1; thus, the change in retention is the result of altering other variables. In Figure 5A, an aqueous F e ( ~ h e n ) ~ S succinate O~, buffer mobile phase is used. Although not shown, a succinate system peak occurs at about 67 mL and a broad Sod2system peak appears a t about 90 mL. All chromatographic peaks are well-defined and indicate favorable column efficiency. For example, the C1- peak in Figure 5A corresponds to 21000 plates/m using a width at half-height calculation. If a C1OL salt is used with the succinate buffer, a base line resolution is still obtained even though halide retention decreases; for example, I- retention drops to about 38 mL. Figure 5B shows how adding MeOH to the mobile phase can be used to effectively decrease retention; in this case the column equilibrium anion exchange capacity is decreased. System peaks 1 and 2, which are also shifted to a slower retention time, are due to succinate and S042-, respectively. Increasing the counteranion concentration will reduce halide analyte retention. This can be done in two ways. As shown in Figure 5C, adding Na2S04(4.0 X M), which provides a strong divalent counteranion, reduces halide and the system peaks (only succinate system peak is shown) retention. When only the buffer was increased to 5.0 X lo4 M, which provides the strong divalent succinate counteranion, a similar decrease in retention was obtained but was not as large as shown in Figure 5C; for example, I- retention appeared at 42 mL. The amount of F e ( ~ h e n )salt ~ ~ retained + in Figure 5A corresponds to about 30 pmol/column or 60 pequiv of exchange sites column. Separations of highly retained monovalent anions are shown in Figure 6. In Figure 6A a small MeOH concentration and a strong counteranion, ClO;, is used to reduce retention where the halide oxygenated anions follow a reversed elution order when compared to the halide anions. In Figure 6B a large MeOH concentration is used to reduce analyte anion retention time. Figure 7 illustrates a separation of a nine-component mixture of monovalent anions covering a wide range of analyte retention. The ClO,--succinate combination provides a stronger eluent than the S0,2--succinate combination used in Figure 5; its major effect on reducing retention occurs with the more highly retained anions. Two system peaks are observed, one shown in Figure 7 at 71 mL due to succinate and one not shown due to C104- a t >>120 mL. A discussion of how to adjust mobile phase components so that system peaks are a t a more favorable retention time will be reported later (25). Calibration-Detection Limits. Calibration curves were
prepared over the range of 10-10000ng of C1- and 7-15000 ng of Br- calculated as the anion and injected by a 20-pL fixed loop. Peaks shapes were Gaussian and well-defined over the entire weight range and k'increased by about 10% from the lower to the upper end. If the mobile phase ionic strength is modestly increased, this change is reduced; however, the detection limit is less favorable. A 1.0 X M Fe(phen),(C104)2,1.0 X lo4 M succinate, pH 6.1 mobile phase (aqueous for C1- and 1:95 MeOH:H20 for Br-) and a PRP-1 column was used with a Micrometrics M725 autoinjector, Kratos SF773 or Spectra Physics 770 detector (510 nm), and a Spectra Physics M4100 data analysis center. Linear curves for indirect detection of peak area vs. weight injected were obtained for both anal@ anions indicating a very favorable dynamic range. No attempt was made to determine the upper limit for linearity; however, for the mobile phase conditions used, 10000 ng is approaching about 0.5% of the available anion exchange capacity provided by the retained F e ( ~ h e n ) salt ~ ~ +on the PRP-1stationary phase surface. For Cl- the equation defining the calibration curve using the Spectra Physics 770 detector is given by (peak area X = 0.866 (ng of C1-) + 7.035 with a correlation factor of 0.9999 while for Br- using the Kratos SF773 detector it is (peak area X = 2.406 (ng of Br-) 230.9 with a correlation factor of 0.9997. At 1ng of injected analyte, anion peak height signal was still about 6 times background signal indicating that additional optimization of the injector, detector, and data analysis would permit even a lower detection limit. Because indirect detection is used, analyte anion calibration is very dependent on mobile phase ionic strength (20,23). Both peak area and location are affected by ionic strength. A discussion of the effects of ionic strength and other parameters on indirect detection will be reported later (25). Conductivity detection can also be used since the Fehen)^^+ mobile phases, in general, do not provide excessive background conductance. No attempt was made to determine the limits for conductivity detection. However, for an aqueous 1.0 X M Fe(~hen),(ClO,)~, 6.0 X M NaC104, 5.0 X lo-, M NaOAc, pH 5.29 mobile phase, 1pg of NO2- was easily detected. Reducing and/or eliminating the ionic strength and the buffer salt will improve the detection. Registry No. F-, 16984-48-8;C1-, 16887-00-6;Br-, 24959-67-9; NOZ-, 14797-65-0; NO
