1650
Anal. Chem. 1988, 60, 1650-1654
Ruthenium(I I) Complexes as Ion Interaction Reagents for the Liquid Chromatographic Separation and Indirect Fluorometric Detection of Analyte Anions Pantelis G . Rigas and Donald J. Pietrzyk*
Chemistry Department, T h e University of Iowa, Iowa City, Iowa 52242
Ruthenium( I I ) 1,lO-phenanthrotlne and 2,2'-blpyridlne complexes are used as mobile phase additives for the liquid chromatographic separatlon of inorganic and organic analyte anlons on Hamilton PRP-1. The Ru( II ) complexes act as ion interactlon reagents for analyte anion separations and, because the complexes are fluorescent, anions can be detected by Indirect fluorescence detectlon (IFD). Indirect photometric detectlon (IPD) is also posslble. Parameters affecting analyte anion retention are Ru( II ) complex concentratlon, type of ligand in the complex, analyte anion selectivity, type and concentration of counteranlon, pH, and type and concentration of buffer components. Complex mixtures of inorganlc and/or organic analyte anlons can be separated and detected. For IFD a linear detector response was found for an analyte anion (F-) concentratlon from 20 to 500 ng; the detection limit at a 2 1 slgnai:ndse ratlo was 10 ng of F-. IPD detection limlt for F- using a Ru(bpy)t+ or a Ru(phen)t+ complex was about 0.8 and 0.4 ng, respectively.
Indirect detection (ID) in high-performance liquid chromatography is growing in application because it offers a way to monitor analytes that are not easily detected by other strategies. This is particularly true when separating simple inorganic and organic analyte ions. Conductivity and refactive index detection are most often used in these cases. While detection limits are often favorable for conductivity, particularly if postcolumn suppression is possible (1,2 ) , this is not usually the case with refractive index. ID also offers the advantage in that it is not instrument specific and mobile phase conditions can be optimized on column chromatographic principles rather than from a suppression viewpoint. ID, however, often has the disadvantage that secondary equilibria are involved and optimization of the parameters requires understanding of these equilibria if analyte retention, k', and peak area-concentration relationships are too be reproducible. Different equilibria can be involved in ID and parameters affecting ID are therefore not uniform. For example, in ion exchange separations ID is based on exchange between a detector active counterion and the analyte ion according to ion exchange selectivities ( 1 , 3, 4 ) . Association equilibria between a nonionic anal@ and a chromophoric nonelectrolyte can be the basis for separation and ID (5-7). A third approach that is versatile in scope is to use a detector active ion interaction (11) reagent (ion pairing reagent) as a mobile phase additive. Most studies have focused on an additive and/or its counterion that is chromophoric (8-13). In addition to its involvement in ID the I1 additive is also responsible for the separation (11, 13). Indirect fluorescence detection (IFD) has not been widely studied; however, IFD has been shown to be feasible in anion exchange separations using salicylate as a fluorescent counteranion (14)and in I1 chromatographic separation of amines
using 2-amino-5-methylbenzenesulfonicacid as the I1 fluorescent reagent (15). We have recently shown that iron(I1) 1,lO-phenanthioline salts are excellent I1 reagents for the separation and indirect photometric detection (IPD) of inorganic and organic analyte anions (11-13). The studies discussed here focus on the use of highly fluorescent ruthenium(I1) 1,lO-phenanthroline, Ru(phen)?+, and ruthenium(I1) 2,2'-bipyridine, Ru(bpy)?+, salts as mobile phase I1 reagents for the separation and IFD of inorganic and organic analyte anions. Since the Ru(I1) complexes are chromophoric, IPD can also be used.
EXPERIMENTAL SECTION Reagents and Instrumentation. 1,lO-Phenanthroline, 2,2bipyridine, and Ru(bpy),C12were purchased from GFS Chemical Co. Salts of Ru(phen)?+ and Ru(bpy)?+ were prepared by reduction of RuC13.3H20(Aldrich Co.) and reaction of Ru(I1) with the ligand (16). Macroporous poly(styrenediviny1benzene) 150 mm X 4.1 mm, 10 wm spherical, prepacked columns (PRP-1) were obtained from Hamilton Co. Buffer salts, ionic strength salts, and solvents were analytical reagent or HPLC grade. The instrumentation used was described previously (11) except that a Kratos FSA 950 fluoresecent detector equipped with a FSA 115 excitation lamp, FSA 404 blue band excitation filter, and a FSA 433 580 nm cutoff emission filter was used. In several separations fluorescence and absorbance detectors were used in series. Procedures. Procedures for analyte and mobile phase preparation, column conditioning, breakthrough determinations, and k'determination are described elsewhere (11). Flow rate was 1.0 mL/min, column temperature was 30.0 "C, void volume was 1.0-1.3 mL, and inlet pressures were 800-1500 psi. Analyte stock solutions were prepared by dissolving quantities of the Na or K salts (some organic acids were neutralized) in LC water and were usually 500 to 1000 ppm. Carefully weighed standard solutions were prepared for calibration studies and these solutions were used for other standards by successive dilution (class A >20-mL pipets and 1100-mL volumetric flasks). A fixed-volume 20-fiL loop was used in combination with a Micromeritics 725 automatic injector or a Rheodyne 7125 injector. RESULTS AND DISCUSSION The Ru(I1) complexes of 2,2'-bipyridine (bpy) and 1 , l O phenanthroline (phen) can be used as mobile phase I1 additives for the separation of inorganic and organic analyte anions like the corresponding Fe(I1) complexes (11-13). The two key equilibria involved in the separation are (1)retention of the Ru(1I) complex as a primary layer and its counteranion as a diffuse secondary layer onto the PRP-1 stationary phase and (2) competition between the counteranion and the analyte anion according to mass action and anion selectivity. The Ru(I1) complex in the mobile phase provides a continuous fluorescent background as it passes through the column and fluorescent detector. Adjustment of the detector electronic offset minimizes the fluorescent background. As the analyte band passes through the column, the Ru(I1) complex concentration in the band changes relative t o the background concentration due to ionic strength, mass action, and counteranion selectivity effects (13). The concentration
0003-2700/88/0360-1650$01.50/062 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
1651
Table I. Effect of Ligand on the Retention of Inorganic Anions on PRP-1
Ionic Strength ~ ~ C I MI , ( x ~ o - ~ )
analyte
F-
capacity factor: k ’ Ru(bpy),(ClO4)z Ru(~hen)~(ClO~),
BF,
3.45 4.55 5.34 5.76 7.31 10.1 14.7 17.8
SCNSP-lb SP-2b SP-3b
11.6 31.2 70
c1-
NO;
BrNO3-
c1031-
5.85 8.43 10.9 12.6 17.5 25.0 47 60 170 11.7 68
I 3 0
0.5
1
1.5
2
2.1
Ru(bipy)JCIZ, M (X10-4)
Figure 1. Effect of mobtie phase R~(bpy)~Ci, concentration (A) and NaCl concentration (B) on the equilibrium amount of Ru(bpy),CI, retained on PRP-1. conditions for (A) are 150 mm X 4.1 mm, 10 km, PRP-1 column and an aqueous, R~(bpy)~Cl,, 0.10 mM succinate buffer (pH 6.1) mobile phase at 1.0 mL/min, 30 O C , and visible detection (445 nm); for (E) conditions are the same as (A) except for an aqueous 0.10 mM Ru(bpy),CI,, NaCI, 0.10 mM succinate buffer (pH 6.1) mobile phase.
difference is sensed by the fluoresence detector and marks the presence of the analyte anion. If the Ru(I1) complex sorption on the stationary phase increases within the equilibrium region of the analyte band because of the combined effects of the aforementioned factors, fluorescence response within the band must decrease and a negative analyte peak is observed. If it decreases, fluorescence must increase and a positive peak is obtained. In the separations reported here positive peaks were obtained because strong eluent counteranions, such as succinate or C104-, were used. For weaker eluent counteranions, such as C1- or OAc-, negative analyte peaks depending on the analyte were obtained in agreement with the origin of ID (13). System peaks (SP) due to the presence of counteranions and their contribution to the equilibria will also be detected. Ru(I1) Complexes. Several advantages are realized when using R ~ ( p h e n ) ~or~ R + ~ ( b p y ) , ~salts + as mobile phase I1 additives. (1) The complex’s fluorescent or absorption property can be used for IFD or IPD, respectively. (2) The complexes are divalent cations and provide anion exchange sites when an equilibrium amount of Ru(I1) complex is maintained on the stationary phase surface. (3)The Ru(I1) complex retention is greater than the corresponding Fe(I1) complexes for a given set of conditions and thus higher exchange capacities are obtained. (4) The Ru(I1) complexes are stable from pH 1 to 13, thus permitting a mobile phase pH to be used that will ionize weak organic acids. (5) The Ru(I1) complexes we inert complexes, stable at elevated temperature, and thud permit elevated elution temperatures. (6) Variables such as complex, counteranion, buffer, and organic modifier concentration can be adjusted to alter analyte anion retention and resolution. Ru(I1) Complex Retention. The equilibrium retention of the Ru(I1) complexes on PRP-1 from a mobile phase containing the complex is dependent on the complex concentration, the counteranion concentration, the type of counteranion, and the type and amount of organic modifier. The amount retained was calculated from the determination of breakthrough volumes as each of these variables was systematically changed. Figure 1 summarizes the results for Ru(bpy)32+as the additive. If the Ru(phen):+ complex is used, the equilibrium micromoles of complex retained is significantly higher because this complex is more highly retained on PRP-1
Conditions are 150 mm X 4.1 mm, 10 wm PRP-1 column and an aqueous, 0.10 mM Ru(I1) complex, 0.10 mM succinate buffer (pH 6.10) mobile phase at 1.0 mL/min, 30 O C , and IFD [A,, 460 nm, &, 580 nm for R ~ ( b p y ) ~ and l + A,, 465 nm, A, 600 nm for Ruhen)^*']. bSystem peak (SP)1, 2, and 3 are due to fluorescence quenching, succinate anion, and perchlorate anion, respectively. (I
compared to the bpy complex. As the R ~ ( b p y ) , ~concen+ tration increases, the equilibrium micromoles of complex retained on the PRP-1 increases. Similarly, for a given Ru(bpy)32+concentration, the amount retained increases as the counteranion concentration (ionic strength), see Figure 1, increases. As the equilibrium micromoles of retained complex increases, the exchange capacity and subsequently analyte retention will increase. If organic modifier is present, the amount retained decreases as the modifier concentration increases; also the rate of change is dependent on the type of organic modifier. The type of counteranion affects t h e equilibrium retention of the complex and the affect follows the anion elution order; the greater the counteranion is retained as an analyte anion, the stronger it is as an eluent counteranion, and the greater the retention of the complex. All of these trends are consistent with using Fe(I1) phen and bpy complexes as the additive, the differences being in the magnitude of the effect. Analyte Anion Retention. The retention of a series of inorganic analyte anions was determined as a function of R ~ ( b p y ) , ( C l Oand ~ ) ~ R ~ ( p h e n ) , ( C l O ~concentration )~ from an aqueous pH 6.10 (succinate buffer) mobile phase. As the Ru(I1) complex concentration in the mobile phase increases, analyte anion retention increases; however, the rate of change is small above 0.10 mM Ru(I1) complex. Subsequent experiments on the effects of the Ru(I1) complex concentration on IFD and IPD analyte peak area/height also favored 0.10 mM since this concentration provided a maximum peak area/ height. This is consistent with previous results found when using F e ( ~ h e n ) , ~salts + as I1 mobile phase additives. Table I, which compares analyte anion retention for the two Ru(I1) complexes at 0.1 mM concentration, illustrates two major points. (1)Analyte anion retention differs significantly and Table I establishes the elution order. (2) Retention is greater from the R ~ ( p h e n ) ~ ( C l mobile O ~ ) ~ phase because this condition yields a larger equilibrium amount of the Ru(phen)3(C10,)z on the PRP-1 surface and a larger anion exchange capacity (37.5 micromoles/column for R ~ ( p h e n ) ~ (C104)zat the conditions in Table I). A similar comparison using organic analyte anions was consistent with these two observations; the typical elution order is indicated in the separation shown later in Figure 4. Table I lists three system peaks. SP-1 at k’ = 11.7 is due to an apparent general fluorescence quenching that is characteristic of IFD. SP-2 and SP-3 in Table I are due to succinate and C104-, respectively. If IPD is used, only the latter
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988 d-
II F-I
D
F-
e
a
8 a-
I mL 0-1
,
0
I
2
4
6
8
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Percent CH3CN
Figure 2. Effect of CH,CN:H,O on the retention of inorganic analyte anions on PRP-1 using Ru(bpy),CI, as a I 1 mobile phase additive. Conditions are the same as in Table I except for CH,CN/H,O, 0.080 mM Ru(bpy),CI, in the mobile phase. Table 11. Effect o f Counteranion on Inorganic Analyte Retention on PRP-1U s i n g a Ru(phen)& I1 Mobile Phase Additive
analyte FBrNOSc103SP-l* SP-2b
capacity factor,nk’ c1c10,6.05 14.2 22.4 36.1 10.7 12.0
5.85 12.6 17.5 25.0 >>25 11.7
a Conditions are the same as in Table I except for 0.10 mM Ru(phen),C2 where C = C1- or Clod- in the mobile phase. *System peak (SP) 1 and 2 are due to counteranion and fluorescence quenching, respectively.
two system peaks are observed indicating that SP-1 in IFD is not counteranion specific. Since its position is also independent of mobile phase Ru(I1) complex concentration and its counteranion (for example, if Ru(bpy),C12 and Ru(phen),Cl, are used in Table I, the fluorescence quenching system peak for the two is still at k ’ = 12), it appears that the fluorescence quenching peak is due to a quenching of the complex and is affected by the sample injection. M o b i l e P h a s e S o l v e n t - C o u n t e r a n i o n E f f e c t s . When organic modifier is increased analyte anion retention decreases. Figure 2 shows the change when Ru(bpy),Clz is used as the additive. A similar trend, but a t higher retention, was observed with Ru(phen):+. As organic modifier concentration increases, the equilibrium amount of Ru(I1) complex retained on PRP-1 decreases, reducing the anion exchange capacity and subsequently anion retention. The type of counteranion, introduced by the Ru(1I) complex, an ionic strength salt, and the buffer, and its concentration affects analyte retention in several competing ways. Table I1 compares the affect of C1- (a weaker eluant counteranion) and Clod- (a stronger eluant counteranion) on anion retention. As counteranion affinity for the exchange sites increases (the more highly retained an analyte anion is as an analyte, the higher is its affinity as a counteranion) or its concentration increases, the more effective the counteranion is in reducing analyte anion retention. Opposing these two effects, however, is that these two changes also cause an increase in the equilibrium amount of Ru(I1) complex retained
~
1
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1
20
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50
1
60
,
70
,
80
,
90
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as I I mobile phase additives for the separation of inorganic analyte anions on PRP-1. Conditions are the same as in Table I except for (A) 0.10 mM Ru(phen)(CIO,), and (B) 0.10 mM R~(bpy),(ClO,)~ in the mobile phase. Flgure 3. Comparison of Ru(bpy),(CI04), and Ru(phen),(CIO,),
on the PRP-1. This increases anion exchange capacity which contributes to an increase in analyte anion retention. These same trends were observed when using Ru(phen)?+ salts. The location of system peaks, if several counteranions are present in the mobile phase, is also dependent on the type and concentration of the counteranion. Furthermore, the analyte peak direction is also counteranion dependent (13). IFD P a r a m e t e r s . The parameters that cause the Ru(I1) complex concentration to change in the analyte band relative to the mobile phase background which then produces a change in fluorescence and absorbance are the same ones that affect absorbance change when using F e ( ~ h e n ) , ~salts + as mobile phase I1 additives (13). These are (1)the Ru(I1) complex concentration and fluorescence yield or absorptivity, (2) the change in ionic strength between the analyte band and the background, (3) the type of counteranion(s) present and differences in anion selectivity relative to the analyte anion, and (4) the concentration of the counteranion(s). To obtain an optimum analyte peak area or height and a reproducible calibration curve, the mobile phase parameters that affect these four factors must be carefully adjusted and controlled. Since analyte retention time and resolution are also affected, a compromise is frequently required to satisfy separation and IFD analyte peak area/height goals. A discussion of this optimization is available elsewhere (11, 13). Separations. Figures 3 to 6 illustrate the feasibility and scope of inorganic and organic analyte anion separations obtained by using R ~ ( b p y ) , ~and + R ~ ( p h e n ) , ~salts + as mobile phase I1 additives and their role in IFD. In Figure 3 a comparison is made of the ability of the two Ru(I1) complexes as the C104- salts to act as I1 mobile phase additives. Mobile phases in parts A and B of Figure 3 are identical except for the ligand difference. The system peak due to fluroescence quenching is shown as SP-1 while SP-2 and SP-3 a t long retention times (not shown) are due to succinate and Clod-, respectively. Analyte retention is significantly higher from the Ru(phen),,+ mobile phase (Figure 3A) than from the Ru(bpy)gP+mobile phase (Figure 3B) because the equilibrium amount retained from the former is larger. Thus, this results in a greater exchange capacity. As electrolyte was added to
ANALYTICAL CHEMISTRY, VOL. 60, NO. 17, SEPTEMBER 1, 1988
0
IO
' 1 1 '
mL
20
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3?
1653
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40 I
lb
0
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20
30
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Flgure 4. Separation of organic acids as anions: (a) glycolic; (b) acetic; (c) lactic; (d) sp; (e) propionic; (f) acetoacetic; (9) a-hydroxybutyric; (h) chioroacetic; (I) isobutryic; (i) butyric. Conditions are the same as in Table I except for 0.10 mM Ru(phen),(CIO,), in the mobile phase and IPD at 448 nm.
the mobile phase in Figure 3, retention times and analyte peak areas decrease consistent with counteranion and ionic strength effects. Also, if a OAc- buffer is used and the increasing electrolyte is NaCl positive analyte peaks shift to negative ones (except F-) as NaCl increases because C1- is a weak eluent counteranion relative to most of the analyte anions. Resolution of organic acid anions is shown in Figure 4. Figure 4 also shows that both IPD and IFD are effective detection strategies. The differences in retention times for corresponding organic acid anion peaks are due to the connection between the fluorescence and absorbance detectors when hooked in series. The two chromatograms are offset and the IPD chromatogram is inverted for convenience; both were recorded as positive peak deflections. Peak d in Figure 4,the fluorescence quench peak, appears in the IFD chromatogram but not in the IPD chromatograms indicating its uniqueness to IFD. Although not shown in every figure, all other separations reported here were also carried out using IFD and IPD. For analyte anions that are more highly retained either of two options are feasible in order to reduce separation time and maintain resolution. One is to use a Ru(bpy)z+ salt mobile phase. As shown in Figure 3B anion retention is reduced because the retention and the equilibrium amount of the Ru(bpy)z+ salt on the PRP-1 are lower. A second option is to use a strong eluent counteranion in the mobile phase. This is illustrated in Figure 5 where citrate, a stronger counteranion compared to the one used in Figure 3, is used. Detection by both IFD and IPD is possible. Only IFD is shown in Figure 5. The Ru2+ complexes are stable up to pH 13 unlike the corresponding Fe2+ complexes, which are stable only up to p H 9. Thus, by use of a basic mobile phase pH it should be possible to separate and detect very weak acids as anions. This is illustrated in Figure 6 where a pH 10.2 carbonate buffer is used in a R ~ ( b p y ) ~ ( C l mobile O ~ ) ~ phase. At this high pH even CN- is separated and detected. Carbonate and the Ru(bpy)?+ salt also contribute to the reduced retention. The fluorescence quench peak and the I- analyte peak overlap in Figure 6 when IFD is used. This was confirmed from the IPD chromatogram (not shown in Figure 6) since the quench peak is absent in this chromatogram. The disadvantage of the basic mobile phase is that a OH- system peak, which depends on
Figure 5. Separation of more highly retained inorganic anawe anions: (a) F-; (b) CI-; (c) NOT; (d) HP04-'; (e) H,AS04-; (f) NO3-; (g) C103-; (h) SP; (i) SO4-,; (j)CrO,-'; (k) I-; (I) BF4-. Conditions are the same as in Table I except for 0.10 mM Ru(phen),(CIO,), 0.10 mM citrate buffer (pH 7.10) in the mobile phase.
m
s
P e
2
1'0
20 mL
30
40
Figure 6. Separation of inorganic analyte anions in a basic mobile phase. Conditions are the same as in Table I except for 0.080 mM R~(bpy)~(ClO~)~, 0.20 mM C03'- buffer (pH 10.2) in the mobile phase.
the pH, appears in the chromatogram. Calibration and Detection Limits. For an aqueous 0.10 mM R ~ ( p h e n ) ~ ( C l O0.10 ~ ) ~mM , succinate, pH 6.10 mobile phase a linear calibration curve for F- was obtained from 20 to 2000 ng (calculated as F-) of injected F- by using a fixed volume 20-pL sample loop. No attempt was made to determine the upper limit of linearity. The equation defining the calibration curve is given by (peak area x lo3) = 0.547 (ng F-) - 3.10, with a correlation coefficient of 0.9999. When 10 ng of F- was injected, the signal to noise ratio was 2:l and this represents the IFD detection limit for the instrumentation available in this study. A IFD detection limit using Ru(bpy)$+, although not determined accurately, probably also approaches 10 ng of F-. When an aqueous, 0.10 mM R u ( b ~ y ) ~ ( C l O0.10 ~ ) ~mM , succinate pH 6.20 mobile phase and IPD were used, the detection limit for F- using a 2:l signa1:noise ratio was reduced to 0.8 ng of F using 0.01 AUFS. Over the range of 1-5000 ng, an IPD linear calibration curve was obtained and followed the equation (peak area X = 6.528 (ng F-) + 138, with a correlation coefficient of 0.999; the detection limit was 0.8 ng of F-. For R ~ ( p h e n ) and ~ ~ +IPD it is 0.4 ng of F-. The more favorable detection limit for IPD obtained when using the bpy complex results from two major factors. First, the molar absorptivity is a contributing factor to sensitivity
1654
Anal. Chem. 1988, 60, 1654-1659
in IPD. As the molar absorptivity for the additive increases, detection limit becomes more favorable (13) providing the chromophoric I1 additive concentration in the mobile phase is adjusted so that its absorbance is
