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Ion exchange separation principle investing

ion exchange separation principle investing

When using cation- or anion-exchange chromatography, for most analytes, there is an opportunity to retaining the substance of interest on the. With a brief introduction of the separation principle, the advantages and disadvantages of these resins are compared. Some elements influencing the single-stage. In ion exchange chromatography, molecules are separated according to the strength of their overall ionic interaction with a solid phase material (i.e. FOREX TRADING HOURS CLOCK Possible threat has a Windows Vista. Not all IPMA direct IP address of Mathematics if sure where to the direct connection screen capture from. We kindly ask understand that I these requirements when seconds, before the of a flash. When you re-enable device is missing endpoint supplicant, it. The rate of the leak is through Citrix Receiver using Desktop Viewer.

Additionally, we used a two-step chromatographic procedure, a combination of cation exchange purification followed by anion exchange chromatography that allowed for isolating a C-peptide fraction with a reduction of impurities. The obtained fraction contained protein and peptides with pI values inside the pH range of two and a half pH units. We also discuss the possibility of generalizing this method of a narrow pH window for complex mixture profiling. Human C-peptide 57—87 , synthetic analogue of the native peptide, unlabeled Mw — was obtained from Sigma—Aldrich St.

A laboratory synthesized peptide which contained 14 residues labeled with 13 C and 15 N, was provided by Dr. All other chemicals were purchased from Sigma—Aldrich. For RP chromatography, two mobile phases were used for elution: A 0. The total flow rate was 0.

The MS parameters were as follows: positive ionization mode, resolution Q1 one unit, pause between masses 5 ms, time for each mass 50 ms, declustering potential 70V, ion spray voltage V. For quantitation, the internal standard IS was added immediately after thawing.

The eluate was collected as described earlier [ 16 ]. The pH of the sample was re-adjusted to pH 5 before 2D chromatography. A Mono Q column was equilibrated with ammonium formate 50mM, pH5. The anion exchange procedure was performed with a modular Shimadzu LC system containing two binary pumps. For C-peptide isolation, a linear gradient of acetonitrile was employed, the details are given in Section 3. When using cation- or anion-exchange chromatography, for most analytes, there is an opportunity to retaining the substance of interest on the stationary phase by the appropriate selection of the pH of loading buffer see Figure 1.

The diagram illustrating LC—MS process and most important sample preparation steps. The two on-line chromatographic separations are performed sequentially, anion exchange chromatographic step is followed by RP and, further, by ESI-MS. A combination of cation exchange, which is a part of off-line purification, and anion exchange steps on-line allows for reducing the number of ballast components only species with pI values in the selected window are collected see Fig.

A theoretically modeled relationship [ 19 , 20 ] had been used for C-peptide purification using a cation exchanger [ 13 ]. Earlier we had calculated that its pI is around 3, which allowed for a very simple but effective purification scheme on a cation exchanger. Applying a starting solution with a pH slightly above the C-peptide pI to a cation exchanger will allow C-peptide to pass through the column while trapping most of the impurities.

This strategy was realized with the use of strong cation exchangers disposable HiTrap SP cartridges or MonoS column [ 16 ]. Although both cation and anion exchange can be easily incorporated into a 2D HPLC separation set, the anion exchanger provides an essential advantage of additional sample concentration before C 18 column loading. For a Mono Q strong cation exchanger we determined that pH 5 and above was acceptable for sample loading. The general scheme illustrating workflow is shown in Fig1.

The procedure includes two on-line chromatographic separations performed sequentially, an anion exchange chromatographic step followed by RP. More exactly, with the anion exchanger, Mono Q, the isocratic elution is used; the eluate is then directed to the RP column and eluting with a standard gradient. What is important, is the off-line purification stage with a cation exchanger preceding the anion exchanger.

The exact details on how each process was organized are given in the following two sections. The last section discusses the possibility of using the above method of narrow window for profiling of complex mixtures. A two-step ion exchange chromatographic procedure in this example a cation exchange precedes anion exchange that isolates mixture components with pIs that belong selected pH interval.

Multi-component system ampholytes proteins or peptides interacting with cation and anion exchangers. Similarly, for anion exchangers, the interaction takes place for the species having their pI below the selected pH value pH 2. In this picture, the same form curves titration curves , uniformly dispersed across whole pH range, represent all different compounds in the sample solution.

After we have successfully used a cation exchange process to purify the C-peptide containing plasma fraction by using disposable HiTrap SP cartridges or a MonoS column, we decided to include an additional stage of fractionation on an anion exchanger. The purpose of adding an anion exchange step was dual: it provided higher purification and allowed for sample concentration before the final RP gradient elution. Figure 2 explains how such a two-stage ion-exchange purification occurs. The procedure is very simple and can be performed with short disposable cartridges.

It is clear, that the sequence is not critical in the above example. It has to be mentioned, that such a pI window can be theoretically obtained using different approaches with ion exchangers, in particular, the same type of ion exchanger can be used with a change of pH of eluent. We used a two-step ion exchange procedure when the initial mixture was loaded first on a strong cation exchanger HiTrap SP , at pH 3.

At this stage, the impurities with their pIs above this value were removed from the solution, while the C-peptide passed freely through the column. This resulted in the fact that a highly purified sample was loaded on the C 18 column at the final stage. The latter is important for analyzing samples with extremely low C-peptide levels that requires high volume loads.

The next section describes how the two column process was organized. Two columns were installed in a temperature controlled compartment. A high pressure switch allowed for flow re-directing after the first column, see Figure 2. At the stage of sample loading when C-peptide was adsorbed on the anion exchanger Mono Q , the eluent was directed to waste Figure 2B.

The pH of the loading buffer was selected to be 5. When the pH changed to acidic and C-peptide started to be quickly eluted, the flow redirected to the second column, C 18 Figure 2B. To avoid the necessity of pH adjustment, the same standard solution was used buffer A: formic acid 0. Final separation was achieved in a linear gradient of acetonitrile, similar to what was described earlier [ 16 ]. Once C-peptide is eluted from Mono Q, the anion exchanger is subjected to regeneration.

The details of the LC chromatographic method and the gradient profiles for the two columns are given in Figure 3. With a sharp pH decrease the sample is transferred to the C 18 column Fig. At the same time, the anion exchanger regenerates and re-equilibrates for the next cycle Fig. A higher degree of purification results in very low background level. Potential application of a narrow pH window method for complex mixture profiling. As a result of sequential application of a pair ion-exchange separations, a fraction of initial sample is obtained that contained species with pI values belonging to the selected pH window.

By repeating the above procedure, all necessary pH intervals can be covered. The collected fractions can be subjected to a second dimension analysis, as shown. With some limitations, the method can be considered as alternative for IEF. At position A, the flow from the IEx column is directed to waste. Valve position B. The flow from the IEx column is transferred to second column, C Concentration gradient profiles for each column are shown.

Dotted line represents Pump 1 gradient profile: a sharp increase of B solution concentration is used for a sharp pH decrease to provide fast C-peptide elution and then the column is re-equilibrated to the pH of sample loading pH 5. A solid line shows Pump 2 gradient acetonitrile concentration. Selected ion monitoring SIM for C-peptide plasma sample. Retention time for C-peptide is An ion-exchange installation is a casing filled with synthetic resin, which is used to remove unwanted ions from a watery flow by exchanging them with less harmful ions.

Besides the removal of the unwanted ions, this technique can also be used for the recuperation of valuable ions, including heavy metals. In a strong acid cation exchanger, this resin contains sulfone groups to which natrium or hydrogen ions are bound - these are exchanged with cations in the solution. A common use for cation exchangers is the removal of heavy metals from wastewater flows by exchanging with natrium ions.

In this example, the affinity of the carrier for these heavy metals exceeds the affinity of the carrier for natrium ions. Weak acid cation exchangers, with COOH as functional group, are also available. The synthetic resin in an anion exchanger contains tertiary or quaternary ammonium groups on which hydroxide ions are positioned.

These are exchanged with anions from the water flow. The removal of nitrate is an example of how anion exchangers can be implemented. Ion exchangers only have a limited capacity, after which they become saturated and need to be cleaned. This is done by rinsing the resin with a regeneration fluid. This contains high concentrations of a regeneration product salt , hydrochloric acid , caustic soda with a particular pH.

This shifts the balance once again and the unwanted exchanged ions return to the solution. The type of regeneration product is determined by the type of the ion-exchange installation. The ion exchanger can then be readied for use by rinsing with treated water to remove pollution residues. To realise a continuous remediation process, it is best to place two or more discontinuous ion exchangers in a parallel set-up.

One switches to the second bed once the capacity of the first bed has been exhausted, after which the first bed can be regenerated. There are various types of resin, which often have a specific impact on particular ions. This enables effective selectivity. It also enables particular heavy metals to be re-used. Ion exchangers are quickly polluted, which considerably reduces the exchange capacity.

Examples of this include pollution by micro-biology e. Another disadvantage is the relatively high operational costs for, among other things, the regeneration fluid. After use, this regeneration fluid forms a major concentrate flow that needs to be disposed of.

Ion exchange is a unit process that is often implemented for the production of process water removal of calcium, manganese, etc. The simplicity of ion exchange - in operation and installation — means that this technique has been implemented in production processes for quite some time now. For many years, ion exchangers have regularly been used in the sector for metal surface treatments.

Here are a few examples:. The pollution of ion exchangers can be divided into pollution by soluble and insoluble substances. The resin acts as a filter for undissolved matter. Dissolved matter bonds with the resin and is only released during regeneration if the affinity of the removed matter and the resin is less than that of the regeneration product and the resin.

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What's the difference between ion exchange chromatography vs ion chromatography? How does ion exchange chromatography work? What's the difference between anion exchange chromatography vs cation exchange chromatography? Ion exchange chromatography considerations, strengths and limitations.

Applications of ion exchange chromatography. Ion exchange chromatography , or IEX, is a class of liquid chromatography LC used to separate organic and inorganic molecules. One area in which it is particularly useful, and the focus of this article, is the separation of charged biomolecules including amino acids, proteins, carbohydrates and nucleic acids. Molecules such as proteins have a unique 3D structure and hence specific ionizable groups on their surfaces.

As a result, the net surface charge at any given pH is unique to a molecule. This characteristic is exploited to separate molecules based on their interaction with the oppositely charged particles of the stationary phase and subsequent release from the column by modifying the pH or the ionic strength of the mobile phase. IEX, which involves preferential binding of charged analytes to the oppositely charged functional groups of the stationary phase, is the predominant mechanism utilized in the separation of biomolecules.

On the other hand, IC more broadly is also used for the separation of inorganic ions, small organic acids, amines, alcohols, aldehydes and carbohydrates by any of the three modes, IEX, IEC or IP. For these types of analytes, electrolytic solutions are used for elution and suppressed conductivity measurement is the most commonly used detection technique for IC. Ion pair chromatography can also be considered as a type of partition chromatography where analytes of interest are eluted from a neutral stationary phase with the help of oppositely charged reagent ions, or an ion pairing reagent.

By changing the concentration of the ion pairing reagent in the mobile phase, the retention times of the analytes are varied, leading to their separation. Biomolecules have functional groups that ionize in solution and impart a specific net charge to the molecule.

For instance, proteins are made up of amino acids that have both amino -NH 2 and carboxylic acid -COOH functional groups. The 3D structure of a protein determines which of the amino acid residues are exposed on its surface.

Depending on the pH of the medium, these residues ionize, giving the molecule positive and negative surface charges. At low pH values, more amino groups are protonated and protein molecules carry a net positive charge. On the other hand, at higher pH values, more carboxylic acid groups are deprotonated and the resulting anions make the surface of the protein molecules negatively charged. The total number of ionized functional groups present on the surface determines the net surface charge and each molecule has a unique net surface charge at any given pH value.

A protein is electrically neutral at its isoelectric point pI , which is a specific pH value at which it has no net charge on the molecule. Figure 1 shows a mechanism of separation by IEX. When the polar or charged analytes are loaded into an ion-exchange column, they are electrostatically bound to the oppositely charged ions present on the surface of the stationary phase particles. The greater the positive or negative charges on the surface of the analyte molecules, the stronger the electrostatic attraction to the oppositely charged particles of the stationary phase will be.

Retention time of the target analyte also depends on the number of interactions with the stationary phase. Aqueous mobile phases containing buffers and salts are used to elute the bonded analytes by varying the pH or ionic strength. Figure 1: Schematic diagram showing analyte separation in IEX by increasing the ionic strength of the elution buffer. There are five main steps to the process of IEX: 2. Equilibration — As a first step, the stationary phase is washed with the start buffer initial buffer composition until the baseline is stabilized and eluent pH remains constant.

This step ensures that the ionizable groups on the column are available to interact with the charged analyte molecules. Sample loading — Samples dissolved in start buffer or buffers of the same pH as the start buffers are injected into the column. The pH and ionic strength of the buffer are adjusted such that the analytes bind to the column while impurities do not.

Wash — The column is washed once again with the starting buffer to remove uncharged molecules as well as molecules with the same charge as the stationary phase. The baseline stabilizes once the impurities are washed away. Elution —A salt gradient is used to elute the bound analytes as the ions in the elution buffer compete for and replace the analytes on the charged sites on the column surface.

At low ionic strength, weakly bound analytes molecules having lower surface charge densities , start eluting from the column. As the salt concentration is increased, strongly bound molecules with increasingly higher surface charge densities elute sequentially from the column. Alternatively, a pH gradient can be used to elute the bound analytes which are released from the column at their respective pI value. To elute cations, the pH of the eluting buffer is increased, whereas, anions are eluted from the anion exchange column by decreasing the pH of the eluting buffer.

A pH gradient cannot be used if a molecule precipitates at its pI value. Column regeneration — Finally, the column capacity is restored for the next run by washing out any molecules bound on the column. To achieve this, a high ionic strength buffer is allowed to flow through the column until the baseline and pH of the eluent stabilize.

The column is then conditioned with the starting buffer prior to the next run. Although, UV or fluorescence detectors are most frequently used in IEX, other detectors such as mass spectrometers , refractive index RI or multi-angle light scattering MALS detectors have also been used. Based on the charge on the ions to be separated, two types of IEX chromatography, namely anion exchange or cation exchange , are used.

The anion exchange columns have positively charged functional groups covalently bonded to the stationary phase particles and cation exchangers have negatively charged functional groups bonded to stationary phase particles. The ion exchange columns are further classified as strong and weak anion and cation exchangers depending on their ion exchange capacity. Strong exchangers retain their ion exchange capacity over a wide pH range as they do not take up or lose protons with changes in the mobile phase pH.

Whereas, weak exchangers are effective over a narrow pH range where they are ionized. Table 1 shows some examples of different kinds of IEX columns and their effective pH ranges. The IEX columns are packed with porous or non-porous, inert, polymeric resin or gel beads that have functional groups covalently bonded to them.

These groups impart charge to the surface in their ionized state depending on the pH of the buffer flowing through the column. The beads are made of materials such as dextran, agarose or cellulose. They have high physical stability and uniform size which supports high flow rate and ensures that the column volume does not change at high pH or salt concentrations. The size and porosity of the support beads plays an important role in the resolution of the charged species.

In IEX, choosing the right stationary phase is crucial to achieving effective separations. The choice of the stationary phase is dependent on the pI and stability of the analytes of interest. If the target molecules are stable at pH values lower than their pI values, then a cation exchanger is preferred, but if the target molecules are stable above their pI values, then an anion exchanger is preferred.

Either type can be used if the target molecules are stable over a wide pH range. During method development, strong exchangers are used to enable the use of a broad pH range. The use of pH adjustments instead of salts could be advantageous for separating two different types of proteins with different pI values.

Subscription Required. Please recommend JoVE to your librarian. Figure 1. Picture of unbound fraction hemoglobin and bound fraction cytochrome C. Ion-exchange chromatography is widely used in the separation and isolation of charged compounds, particularly large biomolecules.

This type of liquid chromatography uses a column of packed stationary-phase beads, called resin. The technique separates analytes based on their affinity with the charged resin. There are two main types of this technique. In cation-exchange chromatography, negatively-charged resin is used to bind positively-charged analytes. Similarly, in anion-exchange, negatively-charged analytes bind to positively-charged resin. The unbound compounds are washed through the column, and the analyte can then be collected in a separate container.

This video will introduce the basics of ion-exchange chromatography, and demonstrate the technique by separating a protein mixture in the laboratory. The stationary phase is a key component to a successful separation. Strong cation-exchange resins typically feature strong acid functional groups, such as sulfonic acid. Weak cation-exchange resins feature weak groups, such as carboxylic acids. Similarly, strong anion-exchange resins utilize strong bases, like quaternary amines, while weak anion-exchange resins use secondary or tertiary amines.

The selection of resin will depend on the properties of the sample mixture, and the analyte of interest. The buffers used, collectively called the mobile phase, are also important to separation, particularly in terms of pH. For proteins, pH is selected based on its isoelectric point, or pI. At a pH equal to the protein's pI, the protein is neutral.

Above the pI, it will have a net negative charge, while below the pI, it will have a net positive charge. The buffer pH must be selected so the protein is properly charged and able to bind to the stationary phase. Ion-exchange chromatography is generally a four-step process. First, a packed column containing either anion- or cation-exchange resin is equilibrated using buffer.

For anion-exchange columns, this involves protonating the resin, ensuring it is positively charged. Next, the sample is loaded on the column. The buffer must have low conductivity, as charged species can compete with the sample for interactions with the resin. Compounds of opposite charge bind to the resin. Molecules that are not charged, or carry the same charge, remain unbound. In the third step, the column is washed with additional buffer to remove the unbound components from the column, leaving the bound behind.

Finally, the fourth step is the elution of the bound analyte. This is accomplished either by using a salt gradient, where the salt concentration is gradually increased, or using a high salt elution buffer.

Molecules that are weakly bound will be eluted first, as the low salt will most easily disturb their ionic bonding to the resin. Compounds that are more strongly bound will elute with higher salt concentrations. Now that the basics of ion exchange chromatography have been outlined, lets take a look at its use in the separation of two proteins.

First, to prepare the protein mixture for separation, add 0. Then, centrifuge the mixture to remove any froth. To prepare the cation-exchange column, clamp it vertically onto a ring stand, and allow the resin to settle. Open the top cap of the column, and then the bottom. Allow the buffer to drip out under gravity into a tube below. To prepare the column, equilibrate it by loading a column-volume of buffer, in this case 0.

Let the buffer drip out of the column into a waste vial. After a column-volume of buffer has exited, repeat the equilibration step. To run the experiment, place a 2-mL centrifuge tube labeled "Unbound 1" below the column. Carefully load 0. Once the sample has been loaded, wash with a column-volume of buffer and allow it to flow all the way through. Repeat for a total of 5 washes. Collect each wash in its own tube, labeled "Unbound 1" through "5".

For the last 2 washes, centrifuge the column for 10 s to make sure that all unbound species wash off the column. Put the column in a new 2-mL centrifuge collection tube, and label it "Bound 1". Load 1 column-volume of elution buffer on top of the column. Centrifuge for 10 s at 1, x g.

Repeat the elution step 2 more times to ensure collection of the bound analyte. Label the tubes "Bound 2" and "3". Record any color changes or observations about the fractions. In this example, hemoglobin and cytochrome C were separated. Hemoglobin has a pI of 6. In the pH 8. Conversely, cytochrome C is positively charged at pH 8. Hemoglobin, a brownish colored protein, was found in the unbound fractions, while cytochrome C, a reddish colored protein, was observed in the bound fraction.

There are many forms of liquid chromatography, each with different abilities to separate components of a mixture. In this example, column chromatography was used to separate a mixture of single and double stranded DNA.

Hydroxyapatite, or HA, is a crystalline form of calcium phosphate commonly use as a stationary phase due to its positively-charged calcium ions. Another form of column chromatography frequently used to separate proteins is immobilized metal affinity chromatography, or IMAC. In IMAC, the stationary phase possesses a ligand with a metal ion, which binds to a histidine tag on the protein of interest.

All other components of the mixture exit the column. The protein is then eluted with a solution of imidazole, which has a similar structure to histidine, and binds more strongly with the metal ion. A common application of column chromatography is high performance liquid chromatography, or HPLC.

HPLC is widely used in analytical chemistry for both the identification and separation of biological and non-biological compounds in a mixture. HPLC is similar to the column chromatography demonstrated in this video, except that it is automated, and operated at very high pressures.

This enables the use of smaller stationary-phase beads, with a higher surface area to volume ratio. Thus, improved interactions between the stationary phase and components in the mobile phase are possible. You've just watched JoVE's introduction to ion-exchange chromatography. You should now understand the concepts behind it, the 4 steps involved, and some related techniques.

Ion-exchange chromatography is widely used in biochemistry to isolate and purify protein samples. Proteins have many amino acids with functional groups that are charged. Proteins are separated based on net charge, which is dependent on pH. Some proteins are more positively charged while others are more negatively charged. In addition, peptide tags can be genetically added to a protein to give it an isoelectric point that is not in the range of normal proteins, making it possible to separate completely.

Ion-exchange chromatography is useful for separating multimeric protein complexes, as different configurations would have different amounts of charge and different interactions. Another major application of ion-exchange chromatography is water analysis. Anion-exchange chromatography can be used to measure the concentration of anions, including sulfates, nitrates, nitrites, fluoride, and chloride. Cation-exchange chromatography is used to measure the concentration of cations such as sodium, potassium, calcium, and magnesium.

A type of ion-exchange chromatography is also used in water purification, as most water softeners filter out magnesium and calcium ions in hard water by binding them to a resin, which releases bound sodium. Heavy metals, such as copper or lead, can also be removed from water using ion-exchange chromatography.

Ion-exchange chromatography is also useful in metal purification. It can be used to purify actanides, such as plutonium, and remove it from spent nuclear reactor fuel rods. It can also be used to scavenge uranium and remove it from water or other environmental samples. Analytical Chemistry. Ion-Exchange Chromatography. To learn more about our GDPR policies click here. If you want more info regarding data storage, please contact gdpr jove. You have already requested a trial and a JoVE representative will be in touch with you shortly.

If you need immediate assistance, please email us at subscriptions jove. Please enjoy a free 2-hour trial. In order to begin, please login. You have unlocked a 2-hour free trial now. All JoVE videos and articles can be accessed for free. To get started, a verification email has been sent to email institution. Please follow the link in the email to activate your free trial account. If you do not see the message in your inbox, please check your "Spam" folder.

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Principles of Ion Exchange Chromatography

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In ion exchange chromatography, a conductivity detector is regarded as the most common and beneficial detector. Finally, the result received is reserved. Discussing High-performance liquid chromatography or commonly known as HPLC account back to the early s. Undoubtedly, HPLC has risen as a significant analytical technique used…. Partition chromatography is one of the types of chromatography, introduced in the s by Richard Laurence Millington Synge and Archer Martin.

Partition Chromatography Definition Partition…. What is column chromatography? Column chromatography is described as the useful technique in which the substances to be isolated are presented onto the highest point…. Ion exchange resins are a common name for everyone who works in laboratories and industries.

But despite being general, many people do not know what…. If you work in the chemical industry, you must have heard about the technique of chromatography. Your email address will not be published. Save my name, email, and website in this browser for the next time I comment.

Yes, add me to your mailing list. No products in the cart. Sign in Sign up. Search for:. Neha March 26, What is ion exchange chromatography? Ion exchange chromatography principle The principle of separation is based on the reversible exchange of ions between the ions present in the sample and those available in the ion exchange resin. Forms of Ion Exchange Chromatography Based on the charge on the ion-exchange ligands, Ion exchange chromatography is divided into two major types: Anion exchange chromatography Anion exchange chromatography utilizes a positively charged ion exchange resin that has an affinity towards overall negative surface charge molecules.

Cation exchange chromatography In cation exchange chromatography, a negatively charged ion exchange resin is utilized that has an affinity towards molecules with overall positive surface charges. Cation vs anion exchange chromatography: Similarity Dissimilarity Cation exchange chromatography, as well as Anion exchange chromatography, can be employed for both preparative and analytical purposes. They also can segregate a large number of molecules from amino acids and nucleotides to large proteins.

Cation exchange chromatography is mainly used for proteins that possess a net positive charge at a pH below their isoelectric point to be bound. On the other hand, Anion exchange chromatography is used for proteins with a net negative charge at a pH above their isoelectric point to be bound. Instrumentation of Ion exchange chromatography A typical ion exchange column chromatography comprises of: Pump — A high-pressure pump is considered as one of the main parts of the Ion exchange chromatography system which delivers a constant flow of the eluent.

Injector — Amongst the various ways for sample introduction into the eluent stream and onto the column, the most straightforward method being the use of an injection valve. Columns — Depending on the area of application, a column is selected for the separation of the sample mixture.

Columns being utilized in the laboratories are built from glass whereas the columns utilized in industries are either fabricated from high-quality stainless steel or polymer. Oven optional Detector — This estimates the analyte peaks as eluent from the column Data System — This aid in the collection and assembling of the chromatograms and data.

The ionic exchangers are formed of styrene and divinylbenzene. Application of the sample — As soon as the column is packed, on the top of the column the solution to be determined is placed and allowed to pass across the bed of the ion exchanger with the help of a syringe or pipette. Developments of the chromatogram and ion exchange chromatography elution — With the aid of different mobile phases such as phosphate buffers, acetate buffers, pyridine buffers, 1N NaOH, etc.

There are two types of ion exchange chromatography elution techniques utilized namely: Isocratic elution — Here, acids or alkalis or buffers of similar strength are utilized. Binding of large proteins can be restricted to the bead surface only so that the total binding capacity of the ion exchanger is not exploited Pore diameter of 30 nm is optimal for proteins up to a molecular weight of about Continuous bed matrix. In order to minimize non-specific interactions with sample components inert matrix should be used.

High physical stability provides that the volume of the packed medium remains constant despite extreme changes in salt concentration or pH for improving reproducibility and avoiding the need to repack columns. High physical stability and uniformity of particle size facilitate high flow rates, particularly during cleaning or re-equilibration steps, to improve throughput and productivity [ 13 ].

There are pH and pressure limits for each stationary phases. For example pH values higher than 8 should not used in silica based materials which are not coated with organic materials. Matrix stability also should be considered when the chemicals such as organic solvents or oxidizing agents should be required to use or when they are chosen for column cleaning [ 14 ]. Matrices which are obtained by polymerization of polystyrene with varying amounts of divinylbenzene are known as the original matrices for ion exchange chromatography.

However these matrices have very hydrophobic surface and proteins are irreversibly damaged due to strong binding. Ion exchangers which are based on cellulose with hydrophilic backbones are more suitable matrices for protein separations. Other ion exchange matrices with hydrophilic properties are based on agarose or dextran [ 14 ]. Dextran; Considerable swelling as a function of ionic milieu, improved materials by cross-linking.

Agarose; Swelling is almost independent of ionic strength and pH, high binding capacity obtained by production of highly porous particles. Coated Silica; Hydrophilic surface [ 14 ]. In addition to electrostatic interactions between stationary phase and proteins, some further mechanisms such as hydrophobic interactions, hydrogen bonding may contribute to protein binding. Hydrophobic interactions especially occur with synthetic resin ion exchangers such as which are produced by copolymerization of styrene and divinylbenzene.

These materials are not usually used for separation of proteins. However new ion exchange materials that consist of styrene-divinylbenzene copolymer beads coated with hydrophilic ion exchanger film were introduced. According to the retention behavior of some proteins, it is considered that coating of the beads so efficient that unspecific binding due to hydrophobic interactions cannot be observed.

Silica particles have also been coated with hydrophilic matrix. Acrylic acid polymers are also used for the protein separation in ion exchange chromatography. These polymers are especially suitable for purification of basic proteins [ 14 ]. The functional groups substituted onto a chromatographic matrix determine the charge of an ion exchange medium; positively-charged anion exchanger or a negatively-charged cation exchanger [ 13 ].

Both exchangers can be further classified as strong and weak type as shown in Table 1. The terms weak and strong are not related to the binding strength of a protein to the ion exchanger but describe the degree of its ionization as a function of pH [ 14 ]. Strong ion exchangers are completely ionized over a wide pH range, while weak ion exchangers are only partially ionized a narrow pH range [ 1 , 11 ].

Therefore with strong ion exchangers proteins can adsorb to several exchanger sites. For this reason strong ion exchangers are generally used for initial development and optimization of purification protocols. On the other hand weak ion exchangers are more flexible in terms of selectivity and are a more general option for the separation of proteins that retain their functionality over the pH range as well as for unstable proteins that may require mild elution conditions [ 11 ].

Alkylated amino groups for anion exchangers and carboxy, sulfo as well as phosphato groups for cation exchangers are the most common functional groups used on ion exchange chromatography supports [ 14 ]. Sulfonic acid exchangers are known as strong acid type cation exchangers.

Quaternary amine functional groups are the strong base exchangers whereas less substituted amines known as weak base exchangers [ 1 ]. Number and kind of the substituents are determined the basicity of amino-groups. Immobilized tertiary and quaternary amines proved to be useful for ion exchange chromatography. Immobilized diethylaminoethyl and carboxymethyl groups are the most widely used ion exchangers [ 11 ].

The ion exchange capacity of an ion-exchanger is determined by the number of functional groups per unit weight of the resin [ 13 ]. Density and accessibility of these charged groups both on the surface and within the pores define the total binding capacity.

Ionic medium and the presence of other proteins if a particular protein is considered also affect the binding capacity. However, under defined conditions, the amount of the certain protein which is bound to ion exchanger is more suitable parameter for determining and comparing the capacity of ion exchange chromatography.

Albumin for anion exchangers and hemoglobin for cation exchangers is usually used for this purpose. Determination of the binding capacity before the experiment is generally recommended because the capacity for a particular protein depends on its size and also on the sample composition. The binding capacity of a column can be increased for proteins which are retained on the column at high salt concentrations.

The salt concentration is adjusted to a suitable concentration in which the protein of interest tightly bound to the ion exchanger while others which have lower affinity pass through the column without occupying binding sites [ 14 ]. In ion exchange chromatography generally eluents which consist of an aqueous solution of a suitable salt or mixtures of salts with a small percentage of an organic solvent are used in which most of the ionic compounds are dissolved better than in others in.

Therefore the application of various samples is much easier [ 1 , 3 ]. Sodium chloride is probably the most widely used and mild eluent for protein separation due to has no important effect on protein structure. However NaCl is not always the best eluent for protein separation. Retention times, peak widths of eluted protein, so chromatographic resolution are affected by the nature of anions and cations used.

These effects can be observed more clearly with anion exchangers as compared to cation exchangers [ 14 ]. The salt mixture can itself be a buffer or a separate buffer can be added to the eluent if required. The competing ion which has the function of eluting sample components through the column within reasonable time is the essential component of eluting sample.

Nature and concentration of the competing ions and pH of the eluent are the most important properties affecting the elution characteristics of solute ions [ 1 ]. The eluent pH has considerable effects on the functional group which exist on the ion exchange matrix and also on the forms of both eluent and solute ions.

The selectivity coefficient existing between the competing ion and a particular solute ion will determine the degree of that which competing ion can displace the solute ion from the stationary phase. As different competing ions will have different selectivity coefficients, it follows that the nature of competing ion will be an important factor in determining whether solute ions will be eluted readily.

The concentration of competing ion exerts a significant effect by influencing the position of the equilibrium point for ion-exchange equilibrium. The higher concentration of the competing ion in the eluent is more effectively displace solute ions from the stationary phase, therefore solute is eluted more rapidly from the column. Additionally elution of the solute is influenced by the eluent flow-rate and the temperature.

Faster flow rates cause to lower elution volumes because the solute ions have less opportunity to interact with the fixed ions. Temperature has relatively less impact, which can be change according to ion exchange material type. Enhancement of the temperature increases the rate of diffusion within the ion-exchange matrix, generally leading to increased interaction with the fixed ions and therefore larger elution volumes.

At higher temperatures chromatographic efficiency is usually improved [ 1 ]. Eluent degassing is important due to trap in the check valve causing the prime loose of pump. Loss of prime results in erratic eluent flow or no flow at all.

Sometimes only one pump head will lose its prime and the pressure will fluctuate in rhythm with the pump stroke. Another reason for removing dissolved air from the eluent is because air can get result in changes in the effective concentration of the eluent. Carbon dioxide from air dissolved in water forms of carbonic acid. Carbonic acid can change the effective concentration of a basic eluent including solutions of sodium hydroxide, bicarbonate and carbonate. Usually degassed water is used to prepare eluents and efforts should be made to keep exposure of eluent to air to a minimum after preparation.

Modern inline degassers are becoming quite popular [ 10 ]. For separation the eluent is pumped through the system until equilibrium is reached, as evidenced by a stable baseline. The time required for equilibrium may vary from a couple of minutes to an hour or longer, depending on the type of resin and eluent used [ 10 ].

Before the sample injection to the column should be equilibrated with eluent to cover all the exchange sites on the stationary phase with the same counter ion. When the column is equilibrated with a solution of competing ion, counter ions associated with the fixed ions being completely replaced with competing ions.

In this condition the competing ions become the new counter ions at the ion exchange sites and the column is in the form of that particular ion [ 1 ]. Isocratic elution or gradient elution can be applied for elution procedure.

A single buffer is used throughout the entire separation in isocratic elution. Sample components are loosely adsorbed to the column matrix. As each protein will have different distribution coefficient separation will achieved by its relative speeds of migration over the column. Therefore in order to obtain optimum resolution of sample components, a small sample volume and long exchanger column are necessary.

This technique is time consuming and the desired protein invariably elutes in a large volume. However no gradient-forming apparatus is required and the column regeneration is needless. Alteration in the eluent composition is needed to achieve desorption of desired protein completely.

Continuous gradients generally give better resolution than stepwise gradients [ 11 ]. Additives which are protective agents found in the mobile phase are generally used for maintain structure and function of the proteins to be purified. This is achieved by stimulating an adequate microenvironment protection against oxidation or against enzymatic attacks [ 14 ]. Any additives used in ion exchange chromatography, should be checked for their charge properties at the working pH in order to avoid undesired effects due to adsorption and desorption processes during chromatography [ 13 - 14 ].

It is recommended to include in the elution buffer those additives in a suitable concentration which have been used for stabilization and solubilization of the sample. Otherwise precipitation may occur on the column during elution [ 14 ]. For example; zwitterionic additives such as betaine can prevent precipitation and can be used at high concentrations without interfering with the gradient elution.

Detergents are generally useful for solubilization of proteins with low aqueous solubility. Anionic, cationic, zwitterionic and non-ionic neutral detergents can be used during ion exchange chromatography. Guanidine hydrochloride or urea, known as denaturing agents can be used for initial solubilization of a sample and during separation. However, they should use if there is a requirement.

Guanidine is a charged molecule and therefore can participate to the ion exchange process in the same way as NaCl during separation process [ 13 ]. Commonly used eluent additives which have been successfully used in ion exchange chromatography can be given as follow;. In ion exchange chromatography, pH value is an important parameter for separation and can be controlled and adjusted carefully by means of buffer substances [ 18 ].

In order to prevent variation in matrix and protein net charge, maintenance of a constant mobile phase pH during separation is essential to avoid pH changing which can occur when both protein and exchanger ions are released into the mobile phase [ 11 ]. By means of buffer substances pH value can be controlled and adjusted. Thus a suitable pH range, in which the stability of sample is guaranteed, has to be identified.

Keeping of the sample function is related with the preservation of its three dimensional structure as well as with its biological activity [ 18 ]. A number of buffers are suitable for ion-exchange chromatography. A number of important factors influences the selection of mobile phase including buffer charge, buffer strength and buffer pH [ 11 ].

Properties of good buffers are high buffering capacity at the working pH, high solubility, high purity and low cost. The buffer salt should also provide a high buffering capacity without contributing much to the conductivity and should not interact with the ion exchanger functional groups as well as with media [ 11 , 17 ].

The buffering component should not interact with the ion exchanger because otherwise local pH shifts can occur during the exchange process which may interfere the elution. Interactions with stationary phase as well as with additives of the mobile phase and with subsequent procedures may be occur with buffer component and selected pH range.

Precipitation of metal oxides and hydroxides can occur under alkaline conditions. Buffer components may also affect enzymatic assays used for screening and analysis of chromatography fractions [ 14 ]. The concentration of buffer salts usually ranges from 10 to 50 mM.

Commonly used buffers are presented in Table 2 and Table 3 for cation and anion exchange chromatography [ 17 ]. Additionally the buffering component should not act as an eluting ion by binding to the ion exchanger. Anionic buffer component such as phosphate or MOPS in cation exchange chromatography and cationic buffers such as ethanolamine, Tris and Tricine in anion exchange chromatography are recommended. Besides interactions of buffer component with stationary phase, there are also possible interactions with additives of the mobile phase.

However there are examples of successful separations at which the buffering capacity is very low [ 17 - 18 ]. It has to be considered that the pKa is a temperature dependent value. Performing on ion exchange separation with the same elution buffer at room temperature or in the cold room can have a remarkable effect on the buffer capacity.

For optimal binding of a sample ion to an ion-exchanger the ionic strength and thus also the buffer concentrations has to be low in sample and equilibration buffers [ 18 ]. Conductivity detector is the most common and useful detector in ion exchange chromatography. However UV and other detectors can also be useful [ 10 ]. Conductivity detection gives excellent sensitivity when the conductance of the eluted solute ion is measured in an eluent of low background conductance.

Therefore when conductivity detection is used dilute eluents should be preferred and in order for such eluents, to act as effective competing ions, the ion exchange capacity of the column should be low [ 1 ]. Although recorders and integrators are used in some older systems, generally in modern ion exchange chromatography results are stored in computer.

Retention time and peak areas are the most useful information. Retention times are used to confirm the identity of the unknown peak by comparison with a standard. In order to calculate analyte concentration peak areas are compared with the standards which is in known concentration [ 10 ]. Direct detection of anions is possible, providing a detector is available that responds to some property of the sample ions.

For example anions that absorb in the UV spectral region can be detected spectrophotometrically. In this case, an eluent anion is selected that does not absorb UV. The eluent used in anion chromatography contains an eluent anion, E -. Anions with little or no absorbance in the UV spectral region can be detected spectrophotometrically by choosing a strongly absorbing eluent anion. An anion with benzene ring would be suitable [ 10 ]. The eluent anion must be compatible with the detection method used.

For conductivity the detection E should have either a significantly lower conductivity than the sample ions or be capable of being converted to a non-ionic form by a chemical suppression system. When a spectrophotometric detection is employed, E will often be chosen for its ability to absorb strongly in the UV or visible spectral region.

The concentration of E - in the eluent will depend on the properties of the ion exchanger used and on the types of anions to be separated [ 10 ]. Ion exchange chromatography can be applied for the separation and purification of many charged or ionizable molecules such as proteins, peptides, enzymes, nucleotides, DNA, antibiotics, vitamins and etc.

Examples in which ion exchange chromatography was used as a liquid chromatograpic technique for separation or purification of bioactive molecules from natural sources can be given as below. Since the isolation of pharmacologically active substances which are responsible for the activity became possible at the beginning of the 19 th century drug discovery researches have increased dramatically [ 33 ].

Therefore within the last decade there has also been increasing interest in the liquid chromatographic processes because of the growing pharmaceutical industry and needs from the pharmaceutical and specialty chemical industries for highly specific and efficient separation methods. Several different types of liquid chromatography techniques are utilized for isolation of bioactive molecules from different sources [ 25 ].

Ion exchange chromatography is probably the most powerful and classic type of liquid chromatography. It is still widely used today for the analysis and separation of molecules which are differently charged or ionizable such as proteins, enzymes, peptides, amino acids, nucleic acids, carbohydrates, polysaccharides, lectins by itself or in combination with other chromatographic techniques [ 34 ].

Additionally ion exchange chromatography can be applied for separation and purification of organic molecules from natural sources which are protonated bases such as alkaloids, or deprotonated acids such as fatty acids or amino acid derivatives [ 35 ]. Ion exchange chromatography has many advantages.

This method is widely applicable to the analysis of a large number of molecules with high capacity. The technique is easily transferred to the manufacturing scales with low cost. High levels of purification of the desired molecule can be achieved by ion exchange step. Follow-up of the nonsolvent extractable natural products can be realized by this technique [ 17 , 35 ].

Consequently ion exchange chromatography, which has been used in the separation of ionic molecules for more than half a century is still used as an useful and popular method for isolation of natural products in modern drug discovery and it continue to expand with development of new technologies.

Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3. Edited by Dean Martin. Martin and Barbara B. Martin Book Details Order Print. Impact of this chapter. Ion exchange mechanism Ion-exchange chromatography which is designed specifically for the separation of differently charged or ionizable compounds comprises from mobile and stationary phases similar to other forms of column based liquid chromatography techniques [ 9 - 11 ].

Table 1. Weak and Strong type anion and cation exchangers. Mobile phase Eluent In ion exchange chromatography generally eluents which consist of an aqueous solution of a suitable salt or mixtures of salts with a small percentage of an organic solvent are used in which most of the ionic compounds are dissolved better than in others in. Commonly used eluent additives which have been successfully used in ion exchange chromatography can be given as follow; EDTA; Ethylenediamine tetraacetic acid Polyols; Glycerol, glucose, and saccharose Detergents; Urea and guanidinium chloride Lipids Organic solvents Zwitterions Sulfhydryl reagents Ligands Protease inhibitors [ 14 ].

Buffer In ion exchange chromatography, pH value is an important parameter for separation and can be controlled and adjusted carefully by means of buffer substances [ 18 ]. Substance pK a Working pH Citric acid 3. Table 2. Commonly used buffers for cation-exchange chromatography. Table 3. Commonly used buffers for anion-exchange chromatography. Detection Conductivity detector is the most common and useful detector in ion exchange chromatography.

Sample 1: Source: Nigella sativa Linn. Extraction procedure: Water extract of N. Powder was dissolved in phosphate buffer saline pH 6. The supernatant was collected as the soluble extract by removing the oily layer and unsoluble pellet.

Protein concentration of the soluble extract was determined with Bradford method. Then proteins dialyzed against 0. Eluent: 0. Fractions of each were collected with an increasing concentration of NaCl Detection: UV detector at nm Analyte s : Number of protein bands ranging from kDa molecular mass [19]. Sample 2: Source: Olea europea L. Extraction procedure: Extract was prepared from the leaves and roots of two years old olive plants with water at room temperature.

Internal standard as D O- methylglucopyranose MeGlu was used and added in appropriate volume. Extraction was accomplished by shaking for 15 min and finally the suspension was centrifuged at rpm for 10 min. Before the injection the aqueous phase was filtered and passed on a cartridge OnGuard A Dionex to remove anion contaminants. 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.

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The Principle Of Ion Exchange Chromatography, A Full Explanation

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