The ability to use the full pH range and elevated temperatures are distinct advantages compared to silica-based supports. Anion Exchange Capacity Anion exchange capacity is a measurement of the number of negative charges anions that the exchange resin can bind to and is reported in singly charged ion equivalents per 1 gram of resin. Exchange capacity is dependent upon the pH of the mobile phase and in anion exchange chromatography; as mobile phase acidity decreases pH increases , the exchange capacity decreases.
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.
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Previous Video Next Video. Overview Source: Laboratory of Dr. Jill Venton - University of Virginia Ion-exchange chromatography is a type of chromatography that separates analytes based on charge.
Log in or Start trial to access full content. Preparing the Sample and the Column In this demonstration, a mixture of 2 proteins will be separated on a cation-exchange column: hemoglobin and cytochrome C.
Add 0. Centrifuge for 2 min to remove any froth. Place the cation-exchange column in a test tube for 5 min to allow resin to settle. Clamp the test tube with the column onto a ring stand to make sure it is upright. Open the top cap of the column, and then the bottom cap.
Allow the buffer in the column to drip out under gravity into the test tube. The strong anion exchange matrix is quaternary ammonium, and is designated Q.
The weak anion exchange matrix is diethylaminoethyl, or DEAE. An intermediate strength resin, which contains primarily tertiary with some quaternary amines, is also available. The pH of the buffer in the column determines the charge of each of the constituents in a sample. The charge of strong anion exchange resins is minimally altered by a change in pH and can operate over a broad pH range.
The availability of a wider working pH range with strong anion exchange columns provides the ability to use pH to change the net charge of the target molecules to make them bind more strongly, more weakly, or not at all. Chromatography may be carried out in positive mode, where the target s of interest bind strongly to the column and many other molecules do not bind as strongly.
Login to your personal dashboard for more detailed statistics on your publications. Edited by Dean Martin. Edited by Amal Ali Elkordy. We are IntechOpen, the world's leading publisher of Open Access books. Built by scientists, for scientists. Our readership spans scientists, professors, researchers, librarians, and students, as well as business professionals.
Downloaded: 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. 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.
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