Anal. Chem. 1988, 60,454-459
454
83-32-9; 1,2,3,4-tetrachlorodibenzo-p-dioxin, 30746-58-8; 1,3,5trichlorobenzene, 108-70-3; phenanthrene,85-01-8;fluoranthene, 206-44-0;fluorenone, 486-25-9;anthraquinone, 84-65-1; l-nitronaphthalene, 86-57-7; 2-nitrofluorene, 607-57-8; 9-nitrophenanthrene, 954-46-1; 1-nitropyrene, 5522-43-0.
LITERATURE CITED Bushway, R. J. J. Chromatogr. 1981, 211, 135. Saner, W. A.; Gilbert, J. J. Llq. Chromatogr. 1080, 3 , 1735. Wolkoff, A. W.; Creed, C. J. Liq. Chromatogr. 1981, 4 , 1459. Andrews, J. S.; mod, T. J. Am. Lab. (Fairfled, Conn.) 1982, 14, 70. West, S. D.; Dorulla, G. K.; Poole, G. M. J. Assoc. Off. Anal. Chem. 1983, 6 6 , 111. (6) Bogus, E. R.; Gallagher, P. A.; Cameron, E. A,; Mumma, R . 0. J . Agric. Food Chem. 1085, 33, 1018. (7) Drevenkar, V. 2. Frobe; Stengl, B.; Tkalcevlc, B. Mlkrochim. Acta 1085, 143. (8) Merz, W.; Neu, H.J. Vom Wasser 1085, 6 5 , 189. (9) Tatar, V.; Popl, M. Fresenlus' 2. Anal. Chem. 1085, 322. 419. (IO) Bardalaye, P. C.; Wheeler, W. B. Int. J. Envlron. Anal. Chem. 1086, 2 5 , 105. (11) Hoke, S. H.; Brueggemann, E. E.; Baxter, L. J.; Trybus, T. J. Chroma togr. 1988, 357, 499. (12) Richard, J. J.; Junk, G. A. Mikrochlm. Acta 1986, I , 387. (1) (2) (3) (4) (5)
-
(13) Puyear. R. L.; Fleckensteln, K. J.; Monk, W. E.; Brarnmer, J. D. Bull. Envlron. Contam. Toxlcol. 1081, 2 7 , 790. (14) Saner, W. A.; Jadarnec, J. R.; Sager, R. W.; Klllen, T. J. Anal. Chem. 1878, 5 1 , 2180. (15) Chladek, E.; Marano, R. S. J. Chromatogr. Sci. 1984, 2 2 , 313. (16) Rostad, C. E.; Perelra, W. E.; Ratcliff, S. M. Anal. Chem. 1964, 56, 2856. (17) Stelnhelrner, T. R.; Ondrus, M. 0. Anal. Chem. 1986, 58, 1639. (18) Nielsen, P. G. Chromatographia 1984, 18, 323. (19) Renberg, L.; Lindstrom, K. J. Chromatogr. 1981, 214, 327. (20) Thome, J. P.; Vandaele, Y. Int. J. Envlron. Anal. Chem. 1987, 2 9 , 95. (21) Thyssen, K. J. Chromatogr. 1085, 379, 99. (22) Junk, G. A.; Richard, J. J. Chemosphere 1087. 16. 61. (23) Junk, G. A.; Richard, J. J.; Grleser, M. D.; Wtlak, D.; Witiak, J. L.; Arguello, M. D.; Vlck, R.; Svec, H. J.; Fritz, J. S.; Calder, G. V. J. Chromatogr. 1974, 9 9 , 745.
RECEIVED for review August 31, 1987. Accepted October 28, 1987. This work was performed in the laboratories of the US. Department of Energy and supported under Contract No. W-7405-Eng-82. The work was supported by the Office of Health and Environmental Research, Office of Energy Research.
Liquid Chromatographic Separation of Analyte Anions with Iron(II) 1,IO-Phenanthroline as a Mobile-Phase Additive: Origin and Parameters in Indirect Detection Pantelis G. Rigas and Donald J. Pietrzyk* Chemistry Department, University of Iowa, Iowa City, Iowa 52242
Analyte anions separated on a Hamitton PRP-1 reversephase column uslng Iron(I1) 1,IO-phenanthrollne [Fe(phen):+] salts as mowlephase add#ives can be detected ImRrectly because absorbance wlthln the analyte band differs from the background absorbance prodded by the Fe(phen)z+ salt containing moblle phase. Three malor factors cause Fe(phen):+ concentratlon to change wlthln the analyte band. These are ionic strength change wlthin the band relative to the background, the type of counteranlon(s) (anion selectivity) in the mobile phase, and the concentratlon (mass action) of the counteranlon(8) and analyte anion(s) in the mobile phase. Dependlng on condltlons, Fe( phen):' concentratlon wlthin the band can elther increase (posltlve analyte peaks) or d e crease (negative analyte peaks). MoMbphase varlables that dlrectly or indirectly affect Ionic strength or counteranion conditions, and consequently Influence indlrect photometrlc detection and detectbn Ilmlts, are Fe(phen):+ concentration, solvent composltlon, type and concentration of counteranIon( s) provided by Fe(phen),*+, buffer, and lonlc strength salts, pH, and amount of InJectedanalyte. Molar absorptlvlty of the metal complex used as the ion interaction reagent also affects detectlon Iknlts. Contlngent on the absorption detector, the analyte anion, and optimlzetion of the parameters, cailbratlon prcvides a broad linear dynamic range and detectlon lhnlts wlthin the 1-10 ng range.
In indirect photometric detection (IPD) the mobile phase contains an additive that absorbs at the detection wavelength. When an analyte enters the column equilibrium is disrupted, the chromophoric additive concentration in the analyte band 0003-2700/88/0360-0454$01.50/0
changes, and the change is deteded as the band passes through the detector. IPD is useful in fundamental and analytical studies since it (1)provides a way of examining secondary equilibria and their effects in ion interaction chromatography (1,2) and (2) provides a way to detect nonchromophoric analytes. Three major analytical separation-IPD strategies have been reported. In ion exchange chromatography (IEC) a chromophoric eluent counterion, such as phthalate, can be used for the detection of nonchromophoric inorganic anions (3-5). The equilibrium responsible for the absorbance change in the analyte band is the result of the analyte-chromophoric counteranion ion exchange that takes place on the ion exchanger stationary phase. A second type of IPD is when a chromophoric ion interaction (or ion pairing) reagent is used as a mobilephase additive for ion interaction chromatography (IIC). Inorganic and organic analyte anions and cations have been separated and detected by using chromophoric quaternary ammonium or sulfonic salts, respectively (4-13). This strategy differs from an IEC approach because the I1 reagent provides both the interaction center for analyte ion retention and the chromophoric property. Thus, the absorbance change in the analyte band is influenced by I1 reagent retention on the stationary phase and the interaction between the 11reagent and the analyte ion (8-11). A variation of the IIC strategy is to use to mobile-phase additive derived from a nonchromophoric hydrophobic ion and a chromophoric counterion. For example, tetralkyl quaternary ammonium p-toluenesulfonate salts can be used for the separation and IPD of inorganic and organic anions (1, 2, 14-17). A third IPD strategy is based on association type equilibria that can occur between a nonionic analyte and chromophoric nonelectrolyte additive in the mobile phase (18-20). This permits non0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988
chromophoric nonelectrolytes to be detected after separation on reverse and normal stationary phases. Calibration and detection sensitivity of IPD-IEC strategies are often comparable to single-column IEC and suppression IEC strategies using conductivity detection (4,5). The major advantages of IPD are its flexibility. For example, (1)mobile-phase optimization can focus on retention and resolution and not suppressionand (2) the IPD strategy is not instrument specific. While favorable sensitivities have been reported for PD-IIC it has also been suggested that calibration in IPD-IIC is not reproducible, has a limited range of utility, and requires very careful control of all parameters (8-11). We have shown that iron(I1) 1,lO-phenanthroline [(Fehen)^^+)] salts, which provide both ion interaction and chromophoric properties, are useful mobile-phase chromophoric additives for the IIC separation and IPD of mixtures of inorganic (21) and organic (22) analyte anions. Because of ease in detection, favorable response to mobile-phase variables, and the scope of application as an IIC additive, F e ( ~ h e n ) salt ~ ~ +additives are also ideal probes for investigating the origin of indirect detection and the parameters that influence IPD in IIC. This report focuses on these directions and establishes the following: (1)why absorption changes when a nonchromophoric analyte is in the presence of the chromophoric I1 reagent; (2) the factors that influence IPD in IIC; (3) that the major parameters are (a) difference in ionic strength within the analyte band and the bulk mobile phase, (b) the couteranion and analyte anion exchange selectivities, and (c) the mass action effeds of the counteranion and analyte anion within the band; (4) that if the parameters, particularly those contributing to ionic strength, are adjusted, detection is sensitive and calibration is reproducible and linear over many orders of magnitude. EXPERIMENTAL SECTION Reagents and Instrumentation. Sulfate and C10, salts of F e ( ~ h e nwere ) ~ ~purchased from GFS Chemical Co. Macroporous poly(styrenediviny1benzene)150 mm X 4.1 mm, 10-pm spherical, prepacked columns (PRP-1) were obtained from Hamilton Co. Buffer salts, ionic strength salts, solvents, and instrumentation used were described previously (21). Procedures. Procedures for analyte and mobile-phase preparation, column conditioning, breakthrough determinations, and k'determination were discussed previously (21). Flow rate was 1.0 mL/min, column temperature was 30 O C , void volume was 1.0-1.3 mL, inlet pressure was usually 800 to 1500 psi, and detection was at 510 nm where Fe(~hen),~+ salts absorb. Analyte solutions for the calibration studies were prepared by dissolving weighed quantitites of Na or K salts in LC water in a volumetric flask. Stock solution concentrations were 500 or lo00 ppm, and these were used to prepare calibration curve standards by successive dilution using class A type pipets (>20.0mL) and volumetric flasks (>lo0 mL). A fixed-volume 20-pL loop was used to inject standard samples onto the column by a Micromeritics 725 automatic injector or Rheodyne 7125 fixed-loop injector. RESULTS AND DISCUSSION Indirect Photometric Detection. A F e ( ~ h e n ) salt ~ ~ +in the mobile phase serves a dual role by providing both an I1 mode of retention and an absorbance change that marks the presence of the analyte anion. As a Fe(phen):+ salt containing mobile phase passes through the PRP-1 column and detector set a t 510 nm, an equilibrium amount of Fe(phen)?+ salt is retained on the PRP-1, depending on the mobile-phase conditions, and provides sites for retention and resolution of analyte anions. The analyte anions compete with other mobile-phase counteranions provided by buffer, ionic strength, and F e ( ~ h e n ) , ~salts + according to an anion-exchange-like selectivity (21). The background absorbance due to Fehen)^^+ is electronically offset in the absorbance detector and a change in Fe(phe1-113~' concentration in the flowing
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Figure 1. Effect of Fe(phen),*+ salt concentration on analyte anion peak area. A 4.1 X 150 mm, 10-pm PRP-1 column and an aqueous 0.50 mM acetate buffer, pH 6.20 mobile phase containing (A) 0.025, (b) 0.050, (C) 0.10, (0)0.25, and (E) 0.50 mM Fe(phen),SO, at 30 OC, 1.0 mL/min with detection at 510 nm.
mobile phase will cause a detector response. If the analyte anions are resolved on the column due to selectivity differences, a Fe(phen)?+ concentration change occurs in each analyte anion band and the detector responds to this change. Positive or negative peaks are obtained depending on whether the F e ( ~ h e n ) ~concentration ~+ increases or decreases, respectively, in the analyte band relative to the offset absorbance. Void volume markers, sample matrix components,and other counteranionsprovided by buffers and/or ionic strength salts will also alter F e ( ~ h e n ) concentration ~~+ and produce either positive or negative peaks (system peaks). The variables the influence F e ( ~ h e n ) , ~salt + retention on PRP-1 and subsequently analyte anion retention and resolution are summarized in the following (21). (1)As the Fe(phen)p salt concentrationincreases, the equilibriumamount of F e ( ~ h e n ) salt ~ ~ +retained on the PRP-1 surface increases. (2) Retention on the F e ( ~ h e n ) ~ salt ' + on PRP-1 is counteranion dependent; in general, the higher the counteranion anion exchange selectivity, the larger the equilibrium amount of F e ( ~ h e n ) , ~retained + on the PRP-1 surface. (3) As ionic strength increases, the equilibrium amount of F e ( ~ h e nsalt )~~ retained increases. (4) As organic solvent modifier concentration increases, the equilibrium amount of F e ( ~ h e n ) salt ~~+ retained decreases; the effect is also solvent type dependent. The following focuses on how these same parameters influence both the origin and the sensitivity of IPD when using Fehen),^+ salts as mobile-phase additives. Fe(phen)?+ Concentration. Figure 1shows how analyte anion peak area changes as a function of mobile-phase Fehen)^^+ concentration over the range of 0.025-0.50 mM F e ( ~ h e n ) ~ S OIn & all cases, eluent composition, except for F e ( ~ h e n ) salt ~ ~ +concentration, amount of analyte, and pump and detector settings are identical. Previous studes (21) demonstrated that 0.10 mM F e ( ~ h e n ) is ~ ~optimum + for analyte anion retention and resolution; this also produces the optimum peak area. At F e ( ~ h e n ) concentration ~~+ below and above about 0.1 mM peak area is significantly reduced even though the concentration of the analytes producing the peaks is constant. Particularly striking is the observed decrease in peak area above 0.1 mM, where detedor response can be made to disappear at even higher F e ( ~ h e n ) concentrations. ~~+ For example, in Figure 1E analyte peaks are barely detected. Two factors contribute to this. (1)An increase in mobile-phase F e ( ~ h e n ) , ~concentrations + affects the background by decreasing the radiation reaching the detector photocell. (2) As subsequent experiments show an increase in ionic strength decreases the Fe(~hen),~+ concentration change in the analyte band. The mobile phase in Figure 1provides two system peaks (SP). One is due to OAc- from the buffer, while the second is due to SO:- provided by the Fe(phen)$+ salt. The location
458
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988 120,
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composition. As analyte anions, X-, pass through the column X- competes with C- in the secondary layer according to an 0
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Figure 2. Effect of mobile-phase ionic strength on analyte anlon peak height. condltlons are the same as in Figure 1 except an aqueous 0.10 mM Fe(phen)&IO,),, 0.10 mM citrate buffer, pH 7.25 mobile phase containing (A) 0.50, (B) 1.0, (C) 3.0, and (D) 6.0 mM NaCiO,.
anion-exchange-like selectivity. The two equilibria describing this are given by eq 1 (sorption of the F e ( ~ h e n ) salt) ~ ~ +and eq 2 (anion exchange according to mass action and anion exchange selectivity between C-and X-1. PRP-1 Fe(phen)32+ 2C- * PRP-l-.Fe(phen)32+2C(1) PRP-l-Fe(phen)32+2C- + 2X- * PRP-l-.Fe(phen)32+2X- + 2C- (2)
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of the system peaks and their areas are also affected by Fehen)^^+ concentration in the same way that analyte peaks are affected. The absorbance change detected in the analyte band when chromophoric sulfonate or quaternary ammonium I1 reagents Salt or Buffer Concentration. Increasing mobile-phase counteranion concentration by the addition of a salt or buffer were used was suggested to be consistent with the IIC model will decrease analyte anion retention time due to mass action (8,9, 11,21,22). Other workers suggested that binding and effects. Figure 2 shows the analyte anion peak area also displacement of the chromophoric I1 reagent in competing ion decreases as mobile-phase counteranion (ionic strength) inpair distribution processes between counterions and analyte creases. This is striking since the amount of analyte injected ions cause the absorbance change (6). Clearly, several mois constant and the peak widths become narrower as analyte bile-phase parameters were recognized as factors in IPD (6, retention decreases with increase in ionic strength. In Figure 11,21, 22), particularly mobile-phase ionic strength, which 2 Fe(phen)3(C101)z and citrate buffer concentrations are influences IPD chromatographic analyte peak area (7,8,21, constant, pump and detector settings are the same, and the 22). only parameter changed is the NaC104Concentration. For the F e ( ~ h e n )as ~ ~a +chromophoric I1 reagent provides a high analyte Cl-, whose k'changes from 1.13to 0.70, the peak height molar absorptivity, is reversibly retained on PRP-1, and unis reduced by 1/2 for a NaC104 increase of 0.50 to 6.0 mM. If dergoes a rapid and reversible interaction with analyte anions. ionic strength is increased further, peak area continues to Thus, a F e ( ~ h e n )salt ~ ~ should + be an ideal probe species for decrease and for the more strongly held anions, their peak studying and establishing unequivocally the parameters inareas will disappear even though the analytes are still retained fluencing IPD and for studying which of these are the most significant in determining peak area in IPD. The effects of on the F e ( ~ h e n ) salt ~ ~ +equilibrated PRP-1. This was demonstrated with other electrolytes, since high concentrations Fe(phedsZ+salt, electrolyte, and buffer concentration on of NaC104 decrease F e ( ~ h e n ) ~ ( C lsolubility. O~)~ The location analyte anion peak area are consistent with the view that and peak area of the C10; and citrate systems peaks in Figure mobile-phase ionic strength is a key parameters, however, 2 are also ionic strength dependent. No attempt was made other factors are also significant. to correctly assign the two system peaks. Two key features are indicated in Figure 3 where the equilibrium amount of four retained [ F e ( ~ h e n ) ~ ] ,salts, C~ When succinate buffer at pH 6.10 was increased from 0.10 to 5.0 mM, analyte peak area and retention time decreased calculated from column breakthrough volumes, on the PRP-1 similar to the added electrolyte effect shown in Figure 2. surface are graphed as a function of mobile-phase salt (Na,C) These changes occur even though analyte anion and Feconcentration, where C" = C104-,S042-,C1-, F.First, as ionic (phenI3SO4concentration and pump and detector settings strength increases, the equilibrium amount of F e ( ~ h e n ) salt ~~+ were constant. System peaks were similarly affected. retained increases. Second, the equilibrium amount retained Origin of Absorbance Change. Retention of analyte ions is dependent on the counteranion and for a given ionic on a reverse stationary phase from a mobile phase containing strength the amount retained follows the counteranion order an I1 reagent of opposite charge follows an IIC model (8,10, C104- > S042- > C1- > F-. The active stationary phase as 11,21,23-25). For a mobile phase that contains a F e ( ~ h e n ) ~ ~ + shown in eq 1 is a "layerlike phase" composed of an equilibsalt with counteranion C- and the reverse stationary phase, rium amount of mobile-phase components that are maintained PRP-1, the PRP-1 surface after equilibration with the mobile dynamically by the mobile-phase flow through the column. phase contains a primary positive-charged layer due to reWhen an analyte anion band enters the column, the stationary tention of Fe(phen)32+and a secondary diffuse layer of C-. phase in contact with the analyte zone experiences several Buffer and/or electrolytes anions will also contribute to Cadjustments, which influence the equilibrium condition and
ac Bulk eluent Secondary layer Cn Prlmary layer a'n
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988
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Flguro 5. Effect of moblie-phase counteranlon on analyte peak directlon. Condltlons are the same as in Figure 1 except an aqueous (A) 0.10 mM Fe(phenbF,, 5.0 mM NaF and (B) 0.10 mM Fe(phen),Ci,, 5.0 mM NaCi mobile phase.
band to yield no IPD peak area since the net change in Fe(phe&+ in the band relative to the background is zero. This depend on the analyte, the mobile-phase components, and the has been observed previously by Levin and Grushka (1). anion selectivities. (1)The analyte causes the ionic strength to be larger in the analyte band. This increases the equilibFurthermore, the equilibration can produce a system peak for rium amount of retained F e ( ~ h e n ) salt ~ ~ +on the stationary each counteranion that is present and F e ( ~ h e n )salts ~ ~ +are phase (see Figure 3) and decreases by an equivalent amount also excellent probe systems for study system peak properties. the F e ( ~ h e n ) concentration ~~+ within the analyte band. (2) Figure 3 indicates that C104-,or other strong counteranions like C104-, will contribute to positive analyte peaks in IPD The analyte anion and the counteranion compete for the when the analyte anions are of lower anion selectivity. This charge site provided by the retained F e ( ~ h e n )according ~~+ to mass action and anion selectivity. Since the counteranion is observed experimentally for most common inorganic analyte anions (21),see also Figures 1and 2, and simple monovalent in the analyte band changes according to these processes, the organic analyte anions (22). If F is the counteranion Figure equilibrium amount of Fe(phen)?+ salt retained on the PRP-1 in thie band must also change. If a counteranion of high anion 5A, negative analyte anion peaks are observed because Fexchange selectivity (see Figure 3) is replaced by a counterexchange selectivity is the lowest among common anions and anion of low selectivity by mass action, the equilibrium amount its effect on Fe(phen):+ salt retention (Figure 3) is the lowest. of retained F e ( ~ h e n ) must ~ ~ + decrease and the F e ( ~ h e n ) ~ ~ +When C1- is used, Figure 5B, analyte anions with lower seconcentration in the analyte band must increase by an lectivity than C1- are positive peaks, for example F-, and equivalent amount. If the reverse occurs or a counteranion analyte anions with higher selectivities are negative peaks, for example Br- and NO
