The solution to be injected is usually called a sample , and the individually separated components are called analytes. It is often used in protein purification, water analysis, and quality control.
Ion methods have been in use since , when H. Thompson and J. Way, researchers in England, treated various clays with ammonium sulfate or carbonate in solution to extract the ammonia and release calcium.
In , the first zeolite mineral column was used to remove interfering calcium and magnesium ions from solution to determine the sulfate content of water. A technique was required to separate and concentrate the radioactive elements needed to make the atom bomb. Researchers chose adsorbents that would latch onto charged transuranium elements, which could then be differentially eluted. Ultimately, once declassified, these techniques would use new IE resins to develop the systems that are often used today for specific purification of biologicals and inorganics.
In the early s, ion chromatography was developed by Hamish Small and co-workers at Dow Chemical Company as a novel method of IEC usable in automated analysis. Detection: Pulsed amperometric detection Analyte s : myo -inositol, galactinol, mannitol, galactose, glucose, fructose, sucrose, raffinose and stachyose [20]. Sample 3: Source: Soybean Extraction procedure: Soybeans were defatted with petroleum ether for 30 min and centrifuged repeating the procedure twice.
Then proteins were extracted with 0. The supernatant was adjusted to pH 6. The precipitate was dissolved in Tris-HCl buffer and the process was repeated in order to obtain purified precipitated fraction containing the 11S globulin. The supernatant obtained after the first precipitation of the 11S fraction was adjusted to pH 4.
The supernatant was stored at low temperature and the precipitate was dissolved in Tris-HCl buffer pH 8. The process was repeated to obtain a purified precipitated fraction containing the 7S globulin. In all cases, the buffer concentration was 20 mM. For every buffer, different gradients were tried. Sample 4: Source: Cochlospermum tinctorium A.
Extraction procedure: The powdered roots of C. Extraction procedures continue until no color could be observed in the ethanol. The acidic fractions were obtained by elution of linear NaCl gradient The carbohydrate elution profile was determined using the phenol-sulphiric acid method. Finally two column volumes of a 2 M sodium chloride solution in water were eluted to obtain the most acidic polysaccharide fraction.
The relevant fractions based on the carbohydrate profile were collected, dialysed and lyophilized. Detection: UV detector, nm Analyte s : Glucose, galactose, arabinose in neutral fraction Uronic acids Both galacturonic and glucuronic acid , rhamnose, galactose, arabinose and glucose in acidic fraction [22].
Sample 5: Source: Hen egg Extraction procedure: Fresh eggs were collected and the same day extract was obtained. Ovomucin was obtained using isoelectric precipitation of egg white in the presence of mM NaCl solution. After centrifugation at The supernatants obtained during the first step with mM NaCl solution and the second step with mM NaCl solution was further used for ion exchange chromatography to separate other egg white proteins. Separation proteins from mM supernatant were allowed to pass through an anion exchange chromatographic column to separate different fractions.
The unbound fractions were then passed through a cation exchange chromatographic column to separate further. Finally the bound fraction was eluted using gradient elution 0. The unbound fraction was collected and used as starting material for cation exchange chromatography.
The column was equilibrated with 10 mM citrate buffer, which was used as the starting buffer. After sample injection the column was eluted by isocratic elution using 0. The fractions were collected and freeze dried-Cation Exchange Chromatography. The precipitate was removed by centrifugation and the supernatant was extensively dialysed against distiled water.
The dialysed protein extract was freeze dried and used for chromatographic separation. Elution of the bound fraction was carried out by using 1 M NaCl in the equilibration buffer. Sample 7: Source: Sweet dairy whey Extraction procedure: After the cheese making process the sweet whey is produced, it is further processed by reverse osmosis to increase the solids content from approximately 5. Stationary phase: Pharmacia's Q- and S-Sepharose anion- and cation-exchange resins Eluent 1: For the anion-exchange process; it was found that two step changes, simultaneous in pH and salt concentration were necessary to carry out the anion-exchange separation.
After the whey feed was loaded onto the column, one column volume of this buffer was passed through to wash out any material that did not bind to the resin, including the IgG. Next, two column volumes of 0. This was then followed by two column volumes of 0. After this second step change, the cleaning cycle was then implemented to prepare the column for the next run.
Eluent 2: For the cation-exchange process, it was found that one step change in pH was appropriate to carry out the cation-exchange separation. The buffer used was 0. One column volume loading of the anion-exchange breakthrough curve fraction was optimum for loading onto the cation-exchange column. After the anion-exchange breakthrough curve fraction was loaded onto the column, one column volume of the initial buffer was passed through to wash out any material that did not bind to the resin.
Next a step change in pH was implemented to elute the bound IgG. This was accomplished by passing two column volumes of the buffer, 0. As the pH wave of this buffer passed through the cation bed it initiated the elution of the IgG because the upper value of its p I range is 8. After this pH step change the cleaning cycle was then implemented. The buffer used was 3 ml g -1 of the fresh leaves.
An aliquot of the dialysed ammonium sulfate fraction containing protein was applied to the affinity chromatography on the N -acetylgalactosamine-agarose column. And then further separation was performed on Sephacryl S column followed by anion exchange chromatography. Extraction: Fruits of the plant extracted with hot water yielded a crude polysaccharide sample, CLRP. The carbohydrate of CLRP was CLRP was a black Polysaccharide sample in which the pigment could not be removed by colum chromatography.
After decoloration, the carbohydrate content of decolored CLRP was Crude polysaccharide material was dissolved in mL 0. Sample Source: Physalisalkekengi var. The precipitate was dissolved in distilled water and the solution was then washed with sevag reagent isoamyl alcohol and chloroform in ratio , which were centrifuged at rpm for 15 min and the protein was removed.
Total sugars were determined by the phenol—sulfuric acid assay using glucose as standard. Stationary Phase: DEAE anion-exchange column Eluent: The column was eluted first with distilled water, and then with gradient solutions 0.
The column was eluted with 0. The major fraction was collected and then freeze dried. All of these fractions were assayed for sugar content by the phenol—sulfuric acid method using glucose as standard Detection: UV Detector, nm Analyte s : Polysaccharides [29]. Sample Source: Ornithogalum caudatum Ait. The polysaccharide pellets were obtained by centrifugation at rpm for 15 min, and completely dissolved in appropriate volume of distilled water followed by intensive dialysis for 2 days against distilled water cut-off M w Da.
The retentate portion was then concentrated, and centrifuged to remove insoluble material. Finally the supernatant was lyophilized to give crude extract. The crude extract was dissolved in 0. The solution was passed through an anion-exchange chromatography column.
After ion exchange chromatography other chromatographic methods was used for further separations. Detection: UV Detector, nm phenol—sulfuric acid method Analyte s : Water soluble polysaccharides [30].
The separated proteins were then re-suspended in a minimum amount of distilled water and the solution dialyzed using cellulose dialysis tubing for 24 hrs against distilled water and concentrated by freeze-drying. The partially purified enzyme was dissolved in acetate buffer 20 mM - pH 6. The solution was passed through the column at a flow rate of 1 mL. The eluted fractions were collected in an automated fraction collector Pharmacia Biotech and the absorbance of the fractions was measured at nm.
The major peak fractions were then assayed for tannase activity, and only the fractions possessing tannase activity were pooled. More Print chapter. How to cite and reference Link to this chapter Copy to clipboard. Martin and Barbara B. Available from:. Over 21, IntechOpen readers like this topic Help us write another book on this subject and reach those readers Suggest a book topic Books open for submissions.
More statistics for editors and authors Login to your personal dashboard for more detailed statistics on your publications. Access personal reporting. More About Us. Exchange Type. Ion exchange group. Buffer counter ions. Commercial samples. Strong cation. Sulfonic acid SP.
Weak cation. Carboxylic acid. CM Cellulose. Strong anion. Quaternary amine Q. Weak anion. DEAE Cellulose. Working pH. N - 2-acetamido iminodiacetic acid. N,N-bis 2-hydroxyethyl glycine. Bis-Tris propane.
N -Methyl-diethanolamine. Sample Nigella sativa Linn. Extraction procedure:. Water extract of N. Stationary Phase:. Fractions of each were collected with an increasing concentration of NaCl. UV detector at nm. Analyte s :. Number of protein bands ranging from kDa molecular mass [19].
Olea europea L. Extract was prepared from the leaves and roots of two years old olive plants with water at room temperature.
Pulsed amperometric detection. Soybeans were defatted with petroleum ether for 30 min and centrifuged repeating the procedure twice. Notably, there was the drifting of elution times for certain analyte peaks 5,6 and a few analytes such as nitrite were degraded by interactions with the resin in the suppressor.
Also, there were the interruptions for suppressor regeneration, even though we had made the interruptions to regenerate less obtrusive by arranging for the suppressor to last about an 8-h day and regenerate overnight.
The picture on suppressors changed in when it was shown that ion chromatography could be accomplished using ion exchange and conductometric detection without a suppressor 10— This new development turned the spotlight on the suppressor and the drawback of the interruptions for its regeneration.
Additionally, this new version of IC was often promoted as avoiding the "complexity" that the suppressor added. The interruptions argument was a legitimate one; the complexity argument was much less so.
It is true that for samples sufficiently burdened with analyte, suppressorless IC is an adequate performer, but it has been demonstrated in practice and in theory 13 that the so-called complexity of adding a suppressor is the price of extracting the maximum sensitivity from conductometric detection.
Please note: In the context of this article, the term sensitivity describes a technique's ability to detect low levels of analyte. This new development did, however, ignite efforts to devise better suppressors. When I performed the first anion separation in , I used as suppressor a coil of sulfonated polyethylene tubing immersed in a stirred suspension of a cation-exchange resin Dowex 50 in the hydronium form. While the sodium hydroxide effluent from the separator was passed through the lumen, sodium ions diffused across the wall of the tubing and exchanged with hydronium ions from the resin as its particles made bumping contacts with the exterior wall of the tubular membrane.
The hydronium ions in turn diffused in the opposite direction and united with hydroxide ions to form water. These membrane devices worked quite well as a continuous suppressor but were fragile and prone to bursting, and because the bed suppressors were quite robust and we had more pressing priorities, we shelved the continuous tubular suppressor.
When suppressorless IC emerged, we revived the membrane concept and succeeded in fabricating a number of more rugged devices 14,15 and they became the first in a series of continuous, chemically regenerated eluent suppressors. At about the same time, Ban and others obtained a patent on a similar continuous suppressor Dionex's introduction of a flat membrane continuous suppressor, the MicroMembrane Suppressor MMS , was a landmark event.
In this device, using anion analysis as the example, the effluent from the separator passed through the narrow channel between closely spaced, flat cation-exchange membranes in the hydronium form. The outside of the membranes was bathed by a continuous stream of sulfuric acid that supplied hydronium ions in exchange for the sodium extracted from the inter-membrane channel.
These devices were very rugged, could suppress higher concentrations of eluent than their predecessors, and, with their low-volume intermembrane channels, they did not degrade the efficiency of the chromatography to an appreciable extent. With these developments, the suppressor had evolved from being a conspicuous part of IC, and something of a bother, to being practically invisible to the user.
Although the flat membrane continuous suppressor was a major advance, it still had some limitations. In the first place, it required a continuous supply of a chemical regenerant. Secondly, although the membranes were preferentially permeable to the suppressing ion, they did allow some leakage of its co-ion; in an anion suppressor, for example, some regenerant sulfuric acid leaked into the mainstream, raising the background conductivity and compromising the measurement of analytes.
As early as , Jansen and others had shown that electrochemistry could be used in membrane devices to effect eluent suppression By placing electrodes in the regenerant compartments of a device of MMS-like construction, ion transport across the intermembrane space was assisted by the electric potential applied to the electrodes.
However, because electrolyte was used in the electrode chambers, these devices could be expected to show undesirable electrolyte leakage into the mainstream. Another landmark in the development of suppressors was the electrochemically regenerated suppressor or the Self-Regenerating Suppressor SRS of Dionex While these devices used an arrangement of membranes and electrodes similar to the Jansen device and to the MMS, the electrode compartments of the SRS were flushed with deionized water.
Using anion analysis with sodium hydroxide eluent as the example, when the electrodes were DC-polarized, hydronium ions produced at the anode were driven by the applied field across the cation-exchange membranes, forcing sodium ions into the cathode compartment where they united with the cathodically generated hydroxide ions and the sodium hydroxide was flushed to waste.
By eliminating the chemical regenerant, the SRS eliminated the problem of regenerant leakage. And in an improved embodiment of the SRS, the water effluent from the IC operation was directed to the electrode compartments, thus eliminating the need for an extra water pump Electrochemistry also revived the packed-bed suppressor 20, In the Dionex Atlas suppressor, a small packed bed of ion-exchange resin, embraced by ion-exchange membranes, is continuously regenerated by polarizing the bed.
By the s, analytical chemistry was augmented by a powerful ally, the computer. With the computer came the ability to automate many operations. Initially, we had introduced the "dogma" that a packed bed needed to suppress many samples before regeneration, but we now realized that with automated valve switching, a small suppressor with just single-sample capacity was viable and would require little intervention from the user This basic idea was later implemented by manipulating three small suppressor beds in a clever three-compartment-revolver device 23 and marketed by Metrohm.
Two major advantages of this small-bed approach over the earlier large suppressor beds are the virtual elimination of peak drifting and minimal degradation of chromatographic efficiency by peak spreading in the void space of the small suppressor bed.
Pioneering work by Dasgupta and others 24,25 had demonstrated that while membrane systems could remove eluent in IC they could also be used to introduce eluent in a controlled way, simply by pumping water to a suitable electrically polarized membrane device. They also recognized that the production of "pure" sodium hydroxide by such a system could provide major advantages for anion analysis by IC. In the early years of IC, carbonate eluents were successful and widely used but they had a few significant shortcomings.
One was that carbonate suppressed to carbonic acid, which has low conductivity but orders of magnitude higher conductivity than pure water. Another was that the carbonate background conductivity was lowered by the presence of analyte; this was not a big issue when the analyte was abundant, but caused nonlinear responses at lower analyte levels. A third drawback was that gradient elution, as it became more of a requirement, was complicated by the ramping baseline conductivity of the carbonic acid background.
Sodium hydroxide or potassium hydroxide as eluent had always been a sort of holy grail for IC because it could be suppressed to the ideal background, water, but two issues delayed its adoption: Hydroxide ion was a relatively ineffective displacing ion, thus demanding high suppression capacities, and it was notoriously difficult to prevent its contamination by omnipresent carbon dioxide that altered its eluting power in unpredictable ways and led to unstable backgrounds.
Although the new MMS suppressors had much greater suppression capacity, thus diminishing the first issue, the carbonate-in-the-eluent problem remained. The membrane-based electrochemical generator alleviated the contamination problem to a great extent because the generator could be provided with ultrapure water and the generator was intrinsically a generator of pure carbonate-free base Concurrent with these developments in hydroxide generators, new separation media with much greater affinity for hydroxide see a later article in this issue enhanced the impact of the electrochemical generators in anion analysis.
While the new electrochemically based eluent generators have made a major change to the trajectory of IC, they do exhaust and have to be replaced at a cost to the user. In the late s, we invented a hybrid of suppression and regeneration where the effluent from the suppressor is not discarded to waste but instead is recaptured and used again as eluent. In principle, these systems could work perpetually simply by pumping water to the IC system. In one embodiment, called ion reflux, the three components of an IC operation — eluent generation, separation, and suppression — were performed continuously within a single column, with just water as the pumped phase.
In another embodiment of ion reflux the separator phase was uncoupled from the other two functions, allowing any stationary phase to be used 27, Reference 30 provides a comprehensive and detailed review of these developments in electrochemistry as applied to suppression and eluent generation in IC. Although conductometric detection was the method that launched IC, it became obvious that other detectors could be used with the new separation media. UV detectors were effective when the analytes were UV-absorbing.
And of course, UV detectors did not require a suppressor, although it should be added that conductometric detection was often the preferred method when the analyte mixture contained target ions, some of which were UV-absorbing and others not.
It had been a sort of dogma in detection that UV detectors were usable only if the analytes were UV-absorbing. We showed, however, that using ion-exchange separation coupled to UV detectors could indeed be used to detect and sensitively measure UV-transparent ions We called this combination indirect photometric chromatography IPC and the detection principle indirect photometric detection IPD. Although IPD had eliminated the need for a suppressor and performed well in many applications 32 , it lacked the sensitivity reach of conductometric detection and received little promotion in IC.
However, the principle became widely used in capillary electrophoresis and somewhat in HPLC. Our work was rewarded by recognition as a milestone in analytical chemistry Carbohydrates and alcohols are not usually thought of as ionics, but Rocklin and Pohl discovered how their intrinsic ionicity could be expressed and used to chromatographically separate them Because carbohydrates are extremely weak acids they express their ionicity only at very high pH, but Pohl and coworkers saw the opportunity of exploiting this and separated carbohydrates by ion exchange using strongly basic eluents Figure 5.
The anion-exchange resins of IC, with their great stability in high-pH environments, were important facilitators of this new technique. Of course, suppressed conductometric detection was a nonstarter because the carbohydrate anions would revert to their non-conducting form in the typical suppressor, so amperometric detection methods, particularly pulsed amperometric detection PAD 35,36 , became important adjuncts to IC and enabled IC separation of a wide variety of analytes Adapted from reference 43 with permission.
This application of IC to nonionics was a distinctly new and different trajectory for the method and certainly a landmark event. Although in the early years we made little progress using simply water as the eluent, others have been notably more successful.
A significant landmark in the evolution of IC is the work of Lamb and others using macrocylic species that form selective and reversible complexes with electrolytes in pure water It is impossible, in an article of this length, to do justice to the pioneer users of IC and the many landmark applications that expanded the method's usefulness.
Therefore, I will restrict my choice to two: the first symposium on IC and the series of developments that have produced astounding advances in the detectability limits of IC. References 32 and 37 are recommended for accounts of the myriad applications of IC.
After the introduction of IC in , it took a certain boldness to embrace this brand-new technology; there were many who said it would not prosper, or as one guru expressed it — so I've been told — "IC was fatally flawed" by the suppressor. But many did embrace it, and some of their early creative applications are recorded in the proceedings of that first symposium at Gatlinburg organized by the U.
Of particular note are stories of "reinventing for the first time" practices common to HPLC at that time but for which there was little or no experience with respect to challenges unique to ion chromatography. View Author Information. Dionex Corporation, Sunnyvale, CA Cite this: J. Article Views Altmetric -.
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