**3.3. Columns**

The principle of ion exchange chromatography is that, charged molecules bind electrostatically to oppositely charged groups that have been bound covalently on the matrix. Ion exchange chromatography is a type of adsorption chromatography so that, charged molecules adsorb to ion exchangers reversibly so, the molecules can be bounded or eluted by changing the ionic environment. Ion exchangers can be used in column chromatography to separate molecules according to charge; actually other features of the molecule are usually important so that the chromatographic behavior is sensitive to the charge density, charge distribution, and the size of the molecule. An ion exchanger is usually a three-dimensional network or matrix that contains covalently liked charged groups. If a group is negatively charged, it will exchange positive ions and is a cation exchanger. An example of a group used in cation exchanger is the carboxy-methyl group. However, if a group is positively charged, it will exchange negative ions and is an anion exchanger. An example of a group used in anion exchanger is the di-ethylamino-ethyl group (DEAE). The matrix (stationary phase) can be made of various materials, commonly used materials are dextran, cellulose, and agarose.

The separation on an ion exchanger is usually accomplished in two stages: first, the substances to be separated are bound to the exchanger using conditions that give stable and tight binding; then the column is eluted with buffers of different pH, ionic strength or composition and the components of the buffer compete with the bound material for the binding sites. To choice whether the ion exchanger is to be anionic or cationic depend on the material to be separated. If the materials to be bound to the column have a single charge (i.e., either plus or minus), the choice is clear. However, many substances (e.g., proteins), carry both negative and positive charges and the net charge depends on the pH. In such cases, the primary factor is the stability of the substance at various pH values. Most proteins have a pH range of stability (i.e., in which they don't denature) in which they are either positively or negatively charged. So, if a protein is stable at pH value above the isoelectric point, an anion exchanger should be used; but if stable at values below the isoelectric point, a cation exchanger is required. Ion exchange columns vary widely in size, packing material and material of construction. Depending on its ultimate use and area of application, the column material may be stainless steel, titanium, glass or an inert plastic such as PEEK. The column can vary in diameter from about 2mm to 5 cm and in length from 3 cm to 50 cm depending on whether it is to be used for normal analytical purposes, microanalysis, high speed analyses or preparative work. The life of a column will depend largely on the type of samples it is used to separate but the conditions under which the separations are carried out will also place limits on it useful lifetime.

**Guard column** is placed anterior to the separating column. This serves as a protective factor that prolongs the life and usefulness of the separation column. They are dependable columns designed to filter or remove particles that clog the separation column and compounds and ions that could ultimately cause "baseline drift", decreased resolution, decreased sensitivity or create false peaks

### **3.4. Suppressor**

**3.2. Injector**

8 Column Chromatography

Sample introduction can be accomplished in various ways. The simplest method is to use an injection valve. In more sophisticated LC, automatic sampling devices are incorporated where

In liquid chromatography, liquid samples may be injected directly and solid samples need only to be dissolved in an appropriate solvent. The solvent need not to be the mobile phase, but frequently it is judiciously chosen to avoid detector interference, column/component interference or loss in efficiency. It is always best to remove particles from the sample by filtering, or centrifuging since continuous injection of particulate materials will eventually

Injectors should provide the possibility of injecting the liquid sample within the range of 0.1 to 100 ml of volume with high reproducibility and under high pressure (up to the 4000 psi). They should also produce minimum band broadening and minimize possible flow disturban‐ ces. The most useful and widely used sampling device for modern LC is the **microsampling injector valve**. With these sampling valves, samples can be introduced reproducibly into pressurized columns without significant interruption of flow, even at elevated temperatures. With commercially available automatic sampling devices, large numbers of samples can be routinely analyzed by LC without operator intervention. Such equipment is popular for the analysis of routine samples (e.g., quality control of drugs), particularly when coupled with automatic data-handling systems. Automatic injectors are indispensable in unattended searching (e.g., overnight) for chromatographic parameters such as solvent selectivity, flow

Most of the autosamplers have a piston metering syringe type pump to suck the preestablished sample volume into a line and then transfer it to the relatively large loop (~100 ml) in a standard six-port valve. The simplest autosamplers utilize the special vials with pressuarization caps. A special plunger with a needle, push the cap down to the vial and displace the sample through the needle into the valve loop. Most of the autosamplers are microprocessor controlled and

The principle of ion exchange chromatography is that, charged molecules bind electrostatically to oppositely charged groups that have been bound covalently on the matrix. Ion exchange chromatography is a type of adsorption chromatography so that, charged molecules adsorb to ion exchangers reversibly so, the molecules can be bounded or eluted by changing the ionic environment. Ion exchangers can be used in column chromatography to separate molecules according to charge; actually other features of the molecule are usually important so that the chromatographic behavior is sensitive to the charge density, charge distribution, and the size of the molecule. An ion exchanger is usually a three-dimensional network or matrix that contains covalently liked charged groups. If a group is negatively charged, it will exchange positive ions and is a cation exchanger. An example of a group used in cation exchanger is the carboxy-methyl group. However, if a group is positively charged, it will exchange negative

sample introduction is done with the help of auto-samplers and microprocessors.

cause blockage of injection devices or columns.

rate, and temperature optimization.

**3.3. Columns**

can serve as a master controller for the whole instrument

The suppressor reduces the background conductivity of the chemicals used to elute samples from the ion-exchange column which improves the conductivity measurement of the ions being tested. IC suppressors are membrane-based devices which are designed to convert the ionic eluent to water as a means of enhancing the sensitivity. It can be used with universal detectors to act as a desalting device, thereby removing the interference resulting from the presence of ionic salts in the eluent. Suppressors are normally used with purely aqueous eluents, so there is a need to establish whether these suppressors can be used with the aqueous/ organic eluents needed to elute organic analytes which are retained on the stationary phase during their interaction. Eluents using ionic gradients and containing organic solvents can be suppressed satisfactorily using either chemical suppression with a micromembrane suppres‐ sor or electrolytic suppression using a self-regenerating suppressor. For utilization in industry, the electrolytic suppressor is usually more appropriate since it can employ water as the suppressor regenerant and is fully automated in terms of response to changing eluent conditions. Care needed to be taken with controlling the suppressor current in order to avoid damage to the suppressor and also the generation of ionic components from oxidation of the organic solvents (especially methanol) present in the eluent. Further potential problems, arising when using suppressors as de-salting devices with organic analytes, are the possibility of analytes loss in the suppressor as a result of adsorption or precipitation effects and disper‐ sion of the analyte band in the suppressor.

concentration in the cell. Thus, the electronic circuit modifies the response of the detector to

Ion Exchange Chromatography - An Overview

http://dx.doi.org/10.5772/55652

11

The amplifier output is then either digitized, and the binary number sent to a computer for storage and processing, or the output is passed directly to a potentiometric recorder. This would result in a false change in impedance due to the generation of gases at the electrode surfaces. The frequency of the AC potential that is applied across the electrodes is normally about 10 kHz. In its simplest form, it can consist of short lengths of stainless steel tube insulated

**Amperometric detection** is a very sensitive technique. In principle, voltammetric detectors can be used for all compounds which have functional groups which are easily reduced or oxidized. Apart from a few cations (Fe3+, Co2+), it is chiefly anions such as cyanide, sulfide and nitrite which can be determined in the ion analysis sector. The most important applications lie however in the analysis of sugars by anion chromatography and in clinical analysis using a

**Mass Spectrometry**: Mass to charge ratio (m/z) allows specific compound ID determination. Several types of ionization techniques: electrospray, atmospheric pressure chemical ionization, electron impact. The detector usually contains low volume cell through which the mobile phase

**Detector sensitivity** is one of the most important properties of the detector. The problem is to distinguish between the actual component and artifact caused by the pressure fluctuation, bubble, compositional fluctuation, etc. If the peaks are fairly large, one has no problem in distinguishing them however, the smaller the peaks, the more important that the baseline be smooth, free of noise and drift. Baseline noise is the short time variation of the baseline from a straight line. Noise is normally measured "peak-to-peak": i.e., the distance from the top of one such small peak to the bottom of the next. Noise is the factor which limits detector sensitivity. In trace analysis, the operator must be able to distinguish between noise spikes and component peaks. For qualitative purposes, signal/noise ratio is limited by 3. For quantitative purposes, signal/noise ratio should be at least 10. This ensures correct quantification of the trace amounts with less than 2% variance. The baseline should deviate as little as possible from a horizontal line. It is usually measured for a specified time, e.g., 1/2 hour or one hour and called drift. Drift usually associated to the detector heat-up in the first hour after power-on.

Sensitivity can be associated with the slope of the calibration curve. It is also dependent on the standard deviation of the measurements. The higher the slope of your calibration curve the higher the sensitivity of your detector for that particular component, but high fluctuations of your measurements will decrease the sensitivity. The more selective the detection, the lower is signal/noise and the higher the sensitivity. The detector response is linear if the difference in response for two concentrations of a given compound is proportional to the difference in

form of amperometric detection know as Pulsed Amperometric Detection (PAD).

provide an output that is linearly related to the ion concentration.

from each other by PTFE connecting sleeves.

passes carrying the sample components.

concentration of the two samples.

Weakly acidic analytes are anionic in the presence of the high pH eluents used with anionexchange IC, but become protonated in the suppressor and are therefore prone to hydrophobic adsorption or precipitation. Similarly, weakly basic analytes are separated as cations with low pH eluents but are deprotonated in the suppressor to form neutral species. The micromembrane suppressor consists of layered ion-exchange membranes and fibrous chamber screens with the regenerant chamber screen modified to possess a high ion-exchange capacity which serves as a reservoir for regenerant ions. There is also a possibility of losses of analytes resulting from penetration of the analyte through the suppressor membrane into the regener‐ ant chamber. Theoretically, anionic analytes are not able to penetrate the cation-exchange membranes of the anion suppressor due to the effects of Donnan exclusion.

Introduction of a suppression device between the column and the detector can be expected to cause some degree of peak broadening due to diffusional effects. The shape of the analyte band will also be influenced by hydrophobic adsorption effects, especially when the adsorption and desorption processes are slow. Examination of peak shapes and analyte losses can therefore provide important insight into the use of suppressors with organic analytes which are weakly acidic or weakly basic. It can be expected that peak area recovery rates after suppression are governed by a combination of hydrophobic interactions with the suppressor and permeation through the membranes with the balance between these mechanisms being determined by eluent composition, suppression conditions and analyte properties.
