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C h r n o o I
Retros hy
g o t a rap m
ctive pe
A seminal 1975 paper and strong commercial development launched ion
Paul R. Haddad University of Tasmania (Australia) 266 A
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n the 1960s and early 1970s, various chromatographic methods were used to separate low molecular weight inorganic anions and cations; however, the separations were only moderately efficient, and detecting those analytes that did not absorb UV radiation was rather difficult. It was more common for researchers to determine these analytes by wet-chemical methods (especially titrations) or electroanalysis (usually with an ion-selective electrode). The real impetus for the development of ion chromatography (IC) came in a paper by Small, Stevens, and Bauman in 1975 (1). This paper presented two very significant developments that laid the foundation for the emergence of IC as a powerful analytical technique—a new method for preparing highly efficient stationary phases to separate inorganic ions and a new approach to sensitive conductivity detection. It is rare for two such profound developments to appear within a single publication, but these developments together created a form of IC that still dominates the field today. IC should not be regarded as a single, specific analytical technique but rather as a collection of LC techniques used to separate inorganic anions and cations and low molecular weight water-soluble organic acids and bases. These techniques include ion-exchange, ion-exclusion, and ion-interaction (ion-pair) chromatographies. We cannot cover all of these techniques, so we will deal only with ion-exchange systems. These are the most common and comprise about 75% of published IC applications. Furthermore, we will emphasize determining inorganic anions because this is the most common IC application. Readers interested in comprehensive coverage of the development of IC should check elsewhere (2–6). About the same time as the publication of the 1975 paper, Dow Chemical (where Small, Stevens, and Bauman worked) patented and subsequently licensed the technology, which became known as “suppressed IC”, to Durrum Instruments, which later became Dionex, for commercial development. This form of IC proved to be commercially successful and became widely used. The development of IC by Dionex was swift, sustained, and scientifically elegant. The patents protecting the technology had the rather unexpected effect of stimulating several alternative approaches to ion analysis, many of which are still used today.
Principles of suppressed IC The IC methodology reported in 1975 is illustrated in Figure 1a. The apparatus consisted of some conventional LC instrumentation in the form of a pump, injector, and conductivity detector, but it differed from convention in that two columns were used. The innovation of the system lay in the nature and purpose of these two columns. The first column consisted of a specialty ion-exchange resin. This particular type of column was used because conventional, high-capacity ion-exchangers had two major limitations. First, as the capacity of the ion-exchanger increased, the concentration
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of mobile phase required to elute analyte ions also increased; acting with the eluent ions to produced a weakly conducting this created problems for detection by conductivity because the product; therefore, the second column became known as the background conductance of the mobile phase became too high suppressor. In the case of anion separations, the suppressor column was to achieve sensitive detection. Second, conventional highcapacity ion-exchangers were generally macroporous in nature, a strong cation-exchanger in the H+ form, and an eluent of so analyte ions were required to diffuse in and out of the par- NaOH was used. The reactions occurring between the eluent ticles, which were often relatively large in diameter, resulting (NaOH) and the suppressor column are in long diffusion paths, band broadening, and low separation (2) Suppressor-H+ + Na+OH– ⌱ Suppressor-Na+ + H2O efficiencies. These two problems were overcome by physically confining the active ion-exchange groups to the outer surface of the sta- and the reactions between the analyte (NaCl) and the suppressor tionary-phase particles. The procedure can be illustrated for column are anion-exchangers by taking microporous core particles, which (3) have been sulfonated on the surface to produce a negative charge, Suppressor-H+ + Na+ + Cl– ⌱ Suppressor-Na+ + H+ + Cl– and allowing them to interact with a suspension of very The conductance G (microSiemens, µS) of an small particles, which have been aminated to proelectrolyte solution is duce a positive charge. Electrostatic attraction between the two types of particles results G = (+ + –)C/10– 3K (4) in an agglomerated material in which the aminated particles coat the core (a) Eluent in which + and – are the limitparticles (Figure 2a). The resulting Regenerant reservoir ing equivalent ionic conducstationary-phase particles have tances (S·cm2 equiv–1) of the short diffusion paths; anionic sulColumn Suppressor Pump Injector Detector cationic and anionic compofonate groups on the surfaces of nents of the electrolyte, rethe particles repel anionic anaRecorder or integrator spectively; C is the molar conlytes. In addition, the ion-excentration; and K is the cell change capacity is low because (b) constant (cm–1). Equation 4 the functional groups exist only Eluent reservoir shows that, for the reacon the limited area comprising tions in Equations 2 and 3, the outer surface of the core Column Pump Injector Detector the conductance of a 5-mM particles. NaOH eluent decreases from The original microparticles Recorder or 124 µS (C = 0.005 M; + for used for coating were obtained integrator Na+ = 50 S·cm2 equiv–1; – for by grinding larger anion-exOH– = 198 S·cm2 equiv–1; K change resins but were later = 10 cm–1) to 0.05 µS (the replaced with monodisperse conductance of water). At the latex particles in the 20- to 100same time, the conductance of nm range. Agglomerated ionFIGURE 1. Block diagram showing the instrumental compoa sample band comprising 1exchangers therefore offered mM NaCl increases from 12.6 nents used in (a) suppressed and (b) nonsuppressed IC. the possibility of efficient sepµS to 42.6 µS as a result of rearations using dilute eluents placing Na+ (+ = 50 S·cm2 that are the the perfect combination for IC. A typical ionequiv –1) with H+ (+ = 380 exchange reaction for anions S·cm2 equiv–1). The suppresin IC is sion reactions have therefore greatly enhanced the detecResin+A– + E– ⌱ Resin+(1) E– tion signal by simultaneously + A– reducing the background conductance and increasing the in which an analyte A– is dissample conductance. placed from the positively Nonsuppressed IC and charged ion-exchange resin by indirect detection the eluent E–. The second column in Figure 1a enhanced Four years after the original the sensitivity of the conducdescription of suppressed IC, tivity detection system by reGjerde, Fritz, and Schmuck-
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ler (7, 8) reported an alternative approach—nonsuppressed IC. Just like Small’s group, they used a low-capacity ion-exchange material in which the functional groups were confined to the surface of the particles. However, Fritz’s group reacted inert polymer beads with sulfuric acid or aminating reagents for a very short time so that only the surface of the particles became functionalized with either sulfonate groups (for cation-exchangers) or quaternary ammonium groups (for anion-exchangers). This led to ion-exchangers with capacities as low as 10–30 µequiv/g. These particles were described as “surfacefunctionalized materials” (Figure 2b) and were used in the experimental setup in Figure 1b. Although the stationary phases used in suppressed and nonsuppressed IC were similar in concept, the nonsuppressed method did not use a suppressor column. Sensitive detection was achieved by carefully choosing the eluent composition. The change in conductance ⌬G produced when an anionic analyte S– is eluted by an anionic eluent E– from an anionexchange column is (a) ⌬G = (S- – E-)CSIS /10–3K
(5)
in which CS is the concentration of the analyte; S- and E- are the limiting equivalent ionic conductances of the sample ion and eluent ion, respectively; and IS is the fraction of the analyte present in the ionic form. Equation 5 arises because of the stoichiometric replacement of eluent ions by analyte ions at elution, and the equation shows that the detector response depends on analyte concentration, the difference in the limiting equivalent ionic conductances of the eluent and analyte anions, and the degree of analyte ionization. Sensitive conductivity detection in nonsuppressed IC could therefore occur as long as there was a considerable difference in the limiting equivalent ionic conductances of the analyte and eluent ions. This difference could be positive or negative, depending on whether the eluent ion was strongly or weakly conducting. If the limiting equivalent ionic conductance of the eluent ion was low, then an increase in con-
Aminated latex particles
+ + + + + + + +
+
+ + + + + +
+
+ + + + + +
Core particle
+
+ + + + +
+
+
ductance occurred when the analyte entered the detection cell. This mode was described as direct detection because the analyte had a higher conductance than the eluent ion. Alternatively, an eluent ion with a high limiting equivalent ionic conductance could be used, and a decrease in conductance would occur when the analyte entered the detection cell. This type was described as indirect detection because the analyte had a lower value of conductance than the eluent ion. Typical eluents used for the nonsuppressed IC of anions with conductivity detection were aromatic carboxylates, such as benzoate and phthalate, whereas aromatic bases could be used for cation separations (9). All of these species were bulky ions with low limiting ionic conductances (< 35 S·cm2 equiv–1), so direct detection of more conductive analyte ions, such as chloride and sulfate, was possible. Alternatively, species can be detected by indirect conductivity using competing eluent ions with very high values of limiting ionic conductance, such as H3O+ for cation separations and OH– for anion separations. (b) A UV-absorbance detector (that was often borrowed from Quaternary ammonium functional groups the lab’s HPLC setup) was frequently used instead of a con+ + + + ductivity detector. When an + anionic analyte S– is eluted + from an ion-exchanger by an + + eluent containing a compet+ + ing anion E–, the absorbance change ⌬A is + +
+
+ +
+
+
+
FIGURE 2. Schematic representation of (a) an agglomerated anion-exchange particle and (b) a surface-sulfonated anionexchange resin. Note that the interior of the bead is a macroporous polymer and is not functionalized.
⌬A = (S- – E-)CSm
(6)
in which S- and E- are the molar absorptivities at the detection wavelength of the analyte and eluent anions, respectively; CS is the molar concentration of the analyte; and m is the detector pathlength (cm). The absorbance change measured by the detector when an analyte is eluted is proportional to the analyte concentration, cell pathlength, and difference in molar absorptivities between the analyte and eluent anions. Equation 6 shows that the analyte could be detected directly if its absorptivity exceeded that of the eluent (leading to a positive value for ⌬A) or indirectly if its absorptivity was less than that of the eluent anion (leading to a negative value for ⌬A).
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Indirect spectrophotometric detection (also called indirect photometric chromatography and vacancy detection) was widely used in the early days of IC, particularly in academia (10, 11). Although absorbance decreases when analytes are eluted, the polarity of the detector output was often reversed to make positive the peaks on the recorder. If the eluent behaved according to Beer’s law, a linear calibration plot was obtained, permitting sample quantification. It was highly fortuitous that the aromatic carboxylate eluents used for direct conductivity detection were also ideal for indirect spectrophotometric detection. Implementing this method required only changing the detector, thus avoiding the need to reoptimize the eluent composition or other separation conditions. Nonsuppressed IC was developed commercially by a number of companies, including Waters, Wescan, Metrohm, Vydac, and Hamilton, and promoted actively as an alternative to suppressed IC. Technically, a suppressor removed from a suppressed IC system creates a nonsuppressed system. However, the distinction between the two techniques persisted because the unique characteristics of the agglomerated stationary phases used in suppressed systems readily identified a separation as being from that type of stationary phase regardless of whether a suppressor was used.
such concentrated eluents). In addition, pure NaOH eluents were difficult to prepare. Many other eluent ions, which were the conjugate bases of weak acids (e.g., borate), were amenable to suppression and used for specific separations. Using similar technology and making some changes to the detection system, researchers developed suppressed IC into a technique that successfully separates inorganic cations (often with color-forming postcolumn reactions as the basis of detection [12]), organic acids and bases (13), carbohydrates (14), and amino acids (15). Stationary phases for nonsuppressed IC were also developed; but once again, the original surface-functionalization approach remained the most popular, although sometimes manifested in new ways. Because the eluent did not have to be compatible with a suppressor, many new varieties appeared. Eluents for anion separations included aromatic sulfonates (e.g., toluenesulfonate), complex ions (such as the anionic complex formed between gluconate and borate), metal ions, and metal complexes. Conductivity detection was used frequently, but many reports appeared with spectrophotometric and electrochemical (amperometry, coulometry, potentiometry) detection, operated in both the direct and indirect modes. Given the chemical similarities of the separation processes in suppressed and nonsuppressed IC, and the strong commercial interests in each, it is not surprising that many of the developments in one technique appeared quickly in the other. It is fair to say that this rich, productive environment was a substantial factor that accelerated the development of IC at a spectacular rate.
Suppressed methods -im proved primarily - be cause of new station ary phases and better
New stationary phases and eluents The intensely competitive IC market has seen proponents of both the suppressed and nonsuppressed methods striving for continuous improvement. For suppressed methods, these improvements were primarily new stationary phases and better suppressors. For nonsuppressed methods, the development of new stationary phases was accompanied by a much greater selection of eluents and detection methods. Stationary phases for suppressed IC continued to use the agglomeration approach, but better separation selectivity was achieved by using various core and latex particles with different compositions and functionalities. A wide range of columns was developed, which were optimized for general use or a specific application or separation. The columns had high separation efficiencies and gave fast separations. NaHCO3–Na2CO3 buffer replaced NaOH as the standard eluent, chiefly because OH– is a very weak ion-exchangedisplacing ion, and concentrated eluents were therefore needed (and hence, high-capacity suppressors capable of dealing with
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Suppressors get better and better Special mention must be made of the striking improvements in suppressors. The original suppressor had been in the form of a column and had functioned well. A testament to the power of this approach is that, some 25 years after they were first introduced and after the expiration of the patent, commercially available, modern column suppressors, with most of their original problems eliminated, have reappeared as the suppression technology of choice for some manufacturers. There can be few examples of such a rebirth of a technology. These second-generation packed-column suppressors differ from the original in two important ways. First, they are much smaller and last for
only a single chromatographic run. Second, they are regenerated automatically between runs, for example, by using electrolysis to produce H+ and OH– from the waste electrolyte that converts the suppressor back to H+ or OH– (16). This leads to a practical system that can be left unattended. Because column suppressors needed to be regenerated periodically and caused some problems with particular analytes, continuously regenerated suppressors based on ionexchange membranes were used. In these devices, the replacement of eluent ions by regenerant ions, which lies at the heart of any suppression reaction, was accomplished by separating the eluent and the regenerant by an ion-exchange membrane that selectively transfers ions. For example, a cation-exchange membrane could be used for continuous transfer of H+ from a H2SO4 regenerant to replace Na+ from a NaHCO3 eluent, resulting in a reaction similar to that shown in Equation 1. The membrane could be configured as a hollow fiber (with the eluent passing through the lumen of the fiber and the regenerant flowing over the outer surface of the fiber) or as flat sheets arranged in a sandwich. In a typical flat-sheet (or micromembrane) suppressor, the eluent was passed through a central chamber that had ion-exchange membrane sheets as the upper and lower surfaces. Regenerant flowed in a countercurrent direction over the outer surfaces of both membranes. Analyte ions were prevented from penetrating the membrane by the repulsion effect of the membrane’s functional groups and therefore remained in the eluent stream. Mesh screens constructed from a polymeric ion-exchange material were inserted into the eluent cavity and also into the cavities that housed the flowing regenerant solution. The entire device was constructed in a layer configuration, and gaskets were used to define the desired flow paths. The volume of the eluent chamber was very small (
