Anal. Chem. 1987,59, 363-366
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Gradient Membrane-Suppressed Anion Chromatography with N-Substituted Aminoalkylsulfonic Acid Salts as Eluents Knut Irgum
Department of Analytical Chemistry, University of UmeP, S-901 87 UmeA, Sweden
Gradlents of sodium 2-amlnoethanesuifonate (I), sodium hydroxlde, and sodlum 3-( N-cyclohexylamlno)-1-propanesulfonate (11) were used as eluents for separating elght mono- and divalent anions with a polyacrylate-basedstrong anion exchange separator column, a catlon exchange membrane suppressor, and conductlvlty detectlon without postsuppression. Fluoride, acetate, formate, and chiorlde could be separated, and thlosuifate still eluted in less than 15 mln on a 50-mm column, uslng a gradient changlng from 2 mM I to 16 mM I1 over 10 min.
Although ion chromatography (IC) (1)has been established as the method of choice for many anion analyses, the development of gradient elution for this important technique has been slow. So far, only a few publications (2-6) have shown the potential of eluent gradients for separating anions with widely different partition coefficients in a single run. This languor is due to the use of the conductivity measurement, which is extremely sensitive to changes in eluent composition, as a principal detection method. The limited choice of useful eluent substances has been a major obstacle for continued development of suppressed anion chromatography. Carbonate was suggested in one of the first papers describing the concept of IC (7) and has remained the most widely used eluent ever since. It has also, until recently, been considered the only common eluent substance with sufficient eluting power for gradient use. The residual conductivity of carbonic acid is, however, relatively high by todays standards, and gradient use of these eluents is as a consequence problematic ( 4 ) . Gradients changing between equal concentrations of hydrogen carbonate and carbonate ion should intuitively, as stated by Small (8), give a constant conductivity after the suppressor step, but experience from our laboratories shows that this is not the case (2). The reason for this, as explained by Sunden et al., is that a carbonate ion displaces twice the number of hydrogen carbonate ions in the separator column, thus creating a temporary increase in total carbon concentration a t the detector during the gradient. When steep gradients are used, this causes a significant hump in the base line. Additionally, the difference in eluent strength between hydrogen carbonate and carbonate ions is not adequate to provide sufficiently strong gradients for many applications by compound variation only; the total carbon content of the eluent must also be changed to achieve the desired increase in eluent power during the run ( 2 ) . Postsuppressor devices have been employed to overcome the problem of fluctuations in residual conductivity caused by varying total carbon concentration with carbonate eluents. These reduce the carbonic acid concentration in the suppressor effluent by diffusion of C02(g)through porous PTFE tubing ( 4 ) or silicone tubing (9). Carbon dioxide free air or a hot potassium hydroxide solution are used as COPsinks, respectively, and the reduction in background is significant. These add-ons contribute, however, to the complexity of the system 0003-2700/87/0359-0363$0 1.50/0
and cause an additional band broadening. Monovalent anions will be the best eluents in gradient applications, since the change in eluent strength with concentration is higher with these than with polyvalent anions (10). Stillian (6) has also shown that sodium hydroxide can be successfully employed in gradient elution with a new high-capacity membrane suppressor, but the pH of eluents approaching 0.1 M NaOH makes them incompatible with alkaline-instable samples. The documented detrimental effects of hydroxide ions on strongly basic anion exchangers (11) must also be considered before NaOH is chosen as eluent. Aminoalkylsulfonates were introduced as IC eluents by Ivey (12)and were recently further evaluated in our laboratories (13). The promising results from these tests indicated that such eluents could be useful for gradient IC. This paper reports the use of two such substances in a gradient application.
EXPERIMENTAL SECTION Reagents and Solutions. Taurine (2-aminoethanesulfonic acid) and CAPS (3-(N-cyclohexylamino)-l-propanesulfonicacid) were purchased from Sigma and recrystallized from water/ methanol, as described by Good et al. (14). Taurine was additionally purified for some experiments by the procedure outlined by Ivey (12). It comprised pumping a 0.5 M solution of taurine through a 6 mm i.d. X 150 mm column containing Dowex 1x8 (20-50 mesh, pract.; Serva, Heidelberg, FRG). This column had been extensively cleaned in methanol and water, and converted to the C1- and the OH- forms in the usual way. It was then converted to the taurinate form by 20 bed volumes of 0.5 M taurine, whereafter it was cleaned with water. Sodium hydroxide was prepared as a 1.00 M solution from Titrisol ampules (Merck, Darmstadt, FRG). Eluents, except those containing sodium hydroxide only, were prepared as buffers by dissolving the purified free acid in a small volume of water, adding NaOH, and diluting to volume. The total eluent flow rate was 1.0 mL/min. H2S04(12.5 mM) was used as suppressor regenerant at 4 mL/min. Water was purified by Milli-Q (Millipore, Bedford, MA) equipment and had a conductivity less than 60 nS cm-'. Three different gradient systems were tested: one concentration gradient from 1.25 mM CAPS/1.0 mM NaOH to 20.0 mM CAPS/lG.O mM NaOH, and two compound/concentration gradients, from 2.0 mM NaOH to 20.0 mM CAPS/16.O mM NaOH and from 2.5 mM taurine/2.0 mM NaOH to 20.0 mM CAPS/16.0 mM NaOH. The concentrationswere programmed to vary linearly over 10 min, with s t a r t at the injection. Owing to tube and mixing chamber dead volume, the onset of the gradient at the column head was delayed 150 s. Equipment. The instrumentation was identical with that described in ref 13, except that two LDC Constametric I11 pumps (Laboratory Data Control, Riviera Beach, FL), controlled by an LDC Gradient Master gradient programmer, were substituted for the Altex pump. The active mixing chamber included in the LDC system was used. RESULTS AND DISCUSSION Gradient Test Sample Composition. The short Waters column performed surprisingly well with the Good buffer eluents; the separation of halides that has earlier called for column switching and multiple detectors (15), or gradient elution (5) for separation within reasonable time, was com0 1987 American Chemical Society
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pleted in less than 15 min by using isocratic elution with CAPS (13). A less facile sample was therefore sought. Fluoride, acetate, and formate were not separated in the isocratic mode using 2 mM NaOH, an eluent that did not elute sulfate or thiosulfate within 45 min. If 12.5 mM CAPS/10.0 mM NaOH was used, the retention time for thiosulfate, the last eluted anion, was 12 min, but the four earliest peaks coeluted (see Figure 5 in ref 13). The separation of fluoride, acetate, formate, and chloride with thiosulfate eluting within reasonable time would be a hard match for a 50-mm column. A sample with these (and some additional ions to fill the gap) was accordingly chosen as test sample. Concentration vs. Compound Gradients. The choice of eluents for gradient ion chromatography can follow two different strategies: changing the concentration of a single eluent compound or using two or more eluent substances with different affinities for the ion exchange groups on the analytical column. The substances used in this work were chosen for their low residual conductivities and for their different affinities for the ion exchange groups on the separator (13). This allowed both these gradient techniques to be used. Chromatograms using a CAPS concentration gradient, as well as taurine/CAPS and NaOH/CAPS compound/concentration gradients are provided to illustrate this. The taurine/CAPS concentration gradient achieves near base line separation of the four earliest eluted ions, at the same time as it elutes thiosulfate in less than 15 min (see Figure 1). Considering the length of the column, and that the system was not optimized for resolution, the result is remarkable. (Peak width at half height for 0.1 mM NaCl injected directly into the suppressor, with a low dead volume straight connector substituted for the separator column, was 210 pL.) The same separation could not be achieved with sodium hydroxide as the weaker eluent, even if used at a concentration as low as 0.5 mM. The CAPS/CAPS gradient was also incapable of separating the early eluting ions. It is not as much the initial concentration of the weak eluent as its displacing potential that determines resolution of early eluting peaks (see Figures 1,2, and 3, and confer with retention data in ref 13). When the column is in equilibrium with a very weak eluent, the onset of a gradient with a stronger eluent ion will cause exchange of practically all the attached weak eluent before breakthrough of the strong eluent occurs. This means that the earliest eluted ions will actually be eluted by an increasing concentration of the weaker eluent. The effective eluting power of the gradient will thus not be a simple function of the eluent composition at the column head, but a gradual slow increase followed by a step and further slow increase for a programmed linear gradient. This was not verified but could certainly be measured by monitoring a property unique to the stronger eluent after the separator column. An effective eluent profile like this can explain the sharp peak seen a t the retention time of chloride in the taurine/CAPS chromatogram. This will actually occur where the CAPS breaks through. Another interesting feature of this phenomenon is that, even if the resolution of the early eluting ions is influenced by the type of the weaker eluent, the retention times of these ions will be essentially unaffected by variations in the concentration of a given substance as the weaker eluent. This holds as long as the initial concentration is not so high that the ions are eluted to a significant degree before the onset of the gradient. This was clearly seen in the NaOH/CAPS system. A chromatogram like the one in Figure 2, but with 0.5 mM used instead of 2 mM NaOH as the weak eluent, was practically identical with the one shown. With 5 mM NaOH as the weaker eluent, the earliest eluted peaks were slightly compressed. (These chromatograms are not shown.)
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Figure 1. (a) Gradient chromatogram using 2.5 mM taurine/2.0 mM NaOH as the weaker, and 20 mM CAPS16 mM NaOH as the stronger eluent. Peak identification was as follows: 1, F-; 2, CH,COO-; 3, HCOO-; 4, Cl-; 5, NO,; 6, NO3-; 7, S O:-; 8, S,032-. Fluoride concentration was 50 pM; all others were 100 pM. Dotted line indicates background without injection, but with column in place; dashed line indicates background for gradient pumped directly into the membrane suppressor. The sample was injected 10 min after programmed gradient had returned to 100% of the weaker eluent. (b) As in Figure la, except that the sample was injected 1 h after programmedgradient had returned to 100% of the weaker eluent.
In some experiments, a delay was introduced between injection and start of the gradient. The effect was a shift in the retention times for all peaks, accompanied by a slight band broadening of the earliest peaks. This procedure cannot be recommended for increasing resolution. Base Line Disturbances during Gradient. Base line fluctuations and ghost peaks from the suppression step alone, indicated by the dashed lines in Figures 1, 2 , and 3, are virtually absent for all gradients tested. The residual conduc-
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Figure 2. &adient chromatogram using 2.0 mM NaOH as the weaker and 20 mM CAPS/16 mM NaOH as the stronger eluent. Sample composition and legend are given in Figure 1. The sample was injected 10 min after programmed gradient had returned to 100% of the weaker eluent.
tivities of both concentration and compound gradients, compensated by 3.8 p S cm-' for co-ion penetration of the suppressor membrane (16), were less than 1 p S cm-' after the suppressor. Spurious peaks and a base line increase did, however, appear with the separator column in place; the taurine/CAPS gradient was worst in this respect. The most probable cause of these artifacts is contamination of the weaker eluent by foreign anions or carbon dioxide (3). Most foreign anions, including carbonate, will be enriched on the separator column due to the poor elution capability of the weak eluent. At the onset of the gradient, they elute as peaks or bands creating the effects seen. When comparing these results with other gradient methods, note that the gradients used here are drastic and that the problems just described are a function of the difference in relative eluent strength of the weak and strong eluent of the pair, as well as the steepness of the gradient. A peak is seen in all blank chromatograms at the retention time of sulfate. When excess time is allowed for column reequilibration between consecutive runs with the taurine/ CAPS gradient, the magnitude of this peak increases, whereas with the other eluent combinations tested, both having a more powerful weak eluent, its shape is distorted toward an earlier retention time. The appearance of sulfate is not unexpected, a t least when using aminoalkyl sulfonic acids as the weaker eluent. Apart from these distinct peaks, a broad hump, which is attributed to carbonate, is evident. It could be argued that carbonic acid is incapable of producing a signal of this magnitude, but the hump increased when the flask containing the weak eluent was left open overnight. Sunden et al. (2) and Tarter ( 5 ) ,who used carbonate as eluents, also saw a varying carbonic acid concentration at the detector as a base line disturbance. Dasgupta has even issued a warning against carbonate contamination (3). Between-run residual conductivity, when excess time is allowed between consecutive taurine/CAPS gradient runs, is shown in Figure 4. After returning to its minimum value after a run, the base line dwells for some 15 min and then increases
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t, (min) Figure 3. (a) Gradient chromatogram using 1.25 mM CAPS/1.O mM NaOH as the weaker, and 20 mM CAPS/16 mM NaOH as the stronger eluent. Sample composition and legend are given in Figure 1. The sample was injected 10 min after programmed gradient had returned to 100% of the weaker eluent. (b) As in Figure 3a, except that the sample was injected 1 h after programmed gradient had returned to 100% of the weaker eluent.
in a step, whereafter it continues to increase slowly, and has not reached its final level even after 3 h. This general behavior is seen for all tested eluent combinations, but the weaker the eluent strength of the first eluent, the more pronounced becomes the effect. Note also from Figures l, 2, and 3, that the conductivity after the gradient run is below that of the same gradient pumped directly into the suppressor. These observations further strengthen the contamination theory. The steps in Figure 4 can be explained by the breakthrough of contaminant (monovalent?) anions that have relatively weak interaction with the separator, while the continued increase after the steps is ascribed to the more strongly retained carbonate and sulfate ions. Carbon Dioxide Contamination. It must seem a paradox to users of the technique that a warning has to be issued
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Figure 4. Long-term base line drift between two blank gradient runs (no injection), uslng the same eluents as in Figure 1. Arrows indicate start of gradients. The first gradient was started 10 min after the pump had returned to 100% of the weaker eluent.
against carbon dioxide contamination in suppressed anion chromatography. During these experiments it was found that base line noise during the gradient was significantly decreased by substituting stainless steel tubing for the P T F E tubing commonly used in ion chromatography. These findings are in accordance with those of Dasgupta (3). Care should be taken when preparing the eluents, by using C02-free water and by preventing prolonged exposure to air. Carbonate-free sodium hydroxide for converting eluent acids to salts can be prepared as described in ref 17. Eluent Purification by Ion Exchange. Ivey (12) described a method for purifying zwitterionic eluent substances before use. The method involves passing a solution of the zwitterion through a strong anion exchange resin in the OHform. Attempts to purify taurine by this technique were not successful. This may have been due to the long time that was used; the solution was pumped by a peristaltic pump through a silicon tube at a flow of 0.2 mL/min. As silicon rubber is permeable to carbon dioxide (9), a considerable uptake of CO,(g) might have taken place. If the lack of success was due to carbon dioxide permeation, an in-line modification of this method, as proposed by Dasgupta (3),would be worth testing. Between-Run Equilibration Time. Gradient anion chromatography is fundamentally different from, e.g., gradient reversed-phase HPLC (18)in one important respect. The ion exchange separator column contains a finite number of active groups, each of which must have an anion attached to it at all times to avoid violation of the electroneutrality principle. In principle, the only anions present in the eluent are the anion of the eluent compound and hydroxide ions. Hydroxide concentration is generally much lower than the eluent anion, so the changes in the ratio of e1uent:hydroxide ions attached to the active groups of the column should be modest when a concentration gradient is used. With a compound gradient, the stronger eluent anion is attached to the active groups at the end of the run. These have to be replaced by the weaker eluent and the column equilibrated with the latter, before the
next run commences. Longer effective analysis times might thus be expected. This was tested for all eluent pairs. The difference in eluent strength is largest in the taurine/CAPS gradient, and for this eluent pair, a difference in retention times for the first eluted ions was indeed seen when 10 min and 1 h reequilibration times were used between runs (Figure 1). If equal time intervals are maintained between the injections, this should not represent any problems. The other eluents showed no such time dependence and the separator column is ready for the next sample as soon as the base line has reached its minimum, approximately 10 min after returning to the weaker eluent. Consecutive runs should, in fact, be performed in as close succession as possible to avoid accumulation of eluent contaminants on the column, as discussed above. To summarize, gradient suppressed IC with aminoalkylsulfonate eluents compares favorably with other gradients described (2-6). Artifact peaks and base line drift caused by impurities in the weaker eluent is, and will most probably remain to be, a problem in gradient IC. Extremely pure substances must therefore, as is the case for other gradient techniques (18,19) as well, be used as the weaker eluent of a gradient pair, regardless of compound type.
ACKNOWLEDGMENT The author thanks Michael Sharp for linguistic revision of the manuscript, Anders Cedergren for valuable discussions, and Karin Olsson for tracing the chromatograms. Registry No. I, 7347-25-3;11, 105140-23-6;F-, 16984-48-8; CH,COO-, 71-50-1; HCOO-, 71-47-6; C1-, 16887-00-6; NO;, 14797-65-0;NO
