Ion exchange is pr obably the most fr equently used chr omatographic technique for the separation and purification of pr oteins, polypeptides, nucleic acids, polynucle otides, and other char ged biomolecules 1. Thus, the two main controlling factors in ion exchange chromatography are the ionic charge z and the ionic radius r. Cm sepharose fast flow deae sepharose fast flow q sepharose. Ion exchange chromatography of antibody fragments anders ljunglo. Chromatography method chapter 2 jingjing li, wei han and yan yu. Effect of ph on protein binding and elution patterns. Hiprep sp ff 1610, hiprep cm ff 1610, hiprep q ff 1610. Ion exchange chromatography sepharose fast flow ion. Both chemists and biochemists have routinely employed this technique for the purification of. One thing we can consider is whether these ions have different strength interactions with the fixed anion in the resin. Hic can be used for capture, intermediate purification or polishing steps in a purification protocol.
Separation principles in chromatography purification. The columns are prepacked glass columns for high performance ion exchange chromatography of proteins, peptides, polynucleotides and. Ion exchange chromatography using a cation exchanger. Ion exchange chromatography, unlike some other chromatography methods, also permits high flow rates, which in some cases can be crucial to the recovery of active protein. Media from ge healthcare can be used for smallscale separations in the laboratory through to production of kilogram quantities of product. Furthermore, binding and elution conditions are easy to optimize, resulting in fast, highresolution. Ion chromatography includes all chromatographic methods that separate ionic substances and substances that dissociate easily. In ions with high polarizability, additional nonionic adsorption processes contribute to the separation mechanism. Sepharose fast flow ion exchange media and prepacked column. A type of ion exchange chromatography, membrane exchange is a relatively new method of purification designed to overcome limitations of using columns packed with beads. This application describes an easy separation of model proteins and explains how cation exchan ge. Depending on the charge of the sample and the resin cation or anion exchange chromatography is used. The total amount of loaded protein and which binds to the column should not exceed the total binding capacity of the chromatography column.
High capacity, high resolving power, mild separation conditions, versatility and wide speared applicability, tendency to concentrate the sample relatively low cost 3. In this technique, the chromatography resin is derivatized with a groups which will bind to a specific site on a given protein of interest. Prepare the column to the desired start bind the target molecules and wash out all unbound. The figure shows the beads blue with negatively charged groups red attached. Hiprep 1610 columns for ion exchange iex chromatography. Separation can be selectively achieved by adsorption and release of samples from the matrix. Ion exchange iex chromatography can separate molecules. Individually download our three volumes of affinity chromatography handbooks. An understanding of these principles will enable the separation power of ion exchange chromatography iex to be fully appreciated. Basic principles and application of dialysis buffer are recommended i.
Oct 25, 2019 introduction ion exchange chromatography is a type of adsorption chromatography. Ion exchange chromatography the separation and purification of various elements by ion exchange chromatography takes advantage of the variation of the electrostatic bond energies of ions in solution. This technique has the advantage of greatly improving the quality of a complex operation, but it can be a very timeconsuming separation. Lacki, jay mueller,2 chithkala harinarayan,2 robert van reis,2 robert fahrner,2 james m. Flow ge healthcare can be used for the initialstage puri.
Charge ion exchange chromatography iex hydrophobicity hydrophobic i nteraction chromatography hic reversed phase chromatography rpc biorecognition ligand specificity affinity chromatography ac gel filtration hydrophobic interaction ion exchange affinity reversed phase fig. This technique is used to analyze ionic substances. The popularity of ion exchange chromatography has been increased in recent years because this technique allows analysis of wide range of molecules in pharmaceutical, biotechnology, environmental, agricultural and other. Nonvolatile buffers for anion exchange chromatography. Access free ion exchange chromatography handbook ge healthcare ion exchange chromatography handbook ge healthcare as recognized, adventure as without difficulty as experience about lesson, amusement, as competently as promise can be gotten by just checking out a book ion exchange chromatography handbook ge healthcare plus it is not directly done, you could acknowledge even. Below its pi, a protein will bind to a negatively charged cation exchanger. A second subcategory of liquid chromatography is known as ionexchange chromatography. However, ion chromatography must be done in conditions that are one unit away from the isoelectric point of a protein. Ion exchange chromatography handbook ge healthcare nacfe. Ion exchange chromatography, which is also known as adsorption chromatography, is a useful and popular method due to its. Sec size exclusion chromatography iex ion exchange chromatography hic hydrophobic interaction chromatography poor resolutionpurity general possible cause remedy column poorly packed do a column performance test. The separation is based on the reversible interaction between a charged protein and an oppositely charged chromatographic medium. It works on almost any kind of charged moleculeincluding large proteins, small nucleotides, and amino acids.
Repack if needed use prepacked columns large mixing spaces at top of column adjust top adapter to resin surface. These short objective type questions with answers are very important for board exams as well as competitive exams of b. Iex ion exchange chromatography igf1 insulinlike growth factor 1. Ion exchange chromatography principles and methods. In this example, the counter ion is sodium, which is positively charged. Van alstine1 1ge healthcare, biosciences ab, se751 84 uppsala, sweden. Ge healthcare ion exchange columns and media selection guide imagination at work. The separation is based on the reversible interaction between a charged protein and an oppositely charged chromatography resin. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid the mobile phase and a porous solid the stationary phase. History of ie chromatographyhistory of ie chromatography 1940s modern ionexchange chromatography was developed during the wartime manhattan project. Chromatography method chapter 2 jingjing li, wei han and. The sample is introduced then flows through the guard and into the analytical ion exchange columns where the ion exchange separation occurs.
There is a reversible exchange of similar charged ions. Regeneration introduction to ion exchange chromatography fig 1. Affinity chromatography ac ac separates proteins on the basis of a reversible. Vbs0072 separation of proteins with cation exchange. Select the ion exchange medium according to the objective. The binding capacity of weak ion exchangers varies considerably more with ph than that of strong ion exchangers, which might affect selectivity. Practical aspects of performing a separation are covered in chapter 2. Ion exchange chromatography is based on adsorption and reversible. Charged substances are separated by column chromatography with resins that carry charged ionic groups. Principles of ion exchange chromatography anion exchanger 0. Charge property of the common types of ion exchangers and example protein with different ph value. Ion exchange chromatography multiple choice questions and answers for competitive exams.
Data file 29067018 ab ion exchange chromatography capto s impact capto s impact chromatography medium resin is a strong cation exchanger fig 1. Thus, the two main controlling factors in ion exchange chromatography are. All ion exchange chromatography relies on electrostatic interactions between the resin functional groups and proteins of interest. These methods are ion pair chromatography 3, ion exchange chromatography 4 and ion exclusion chromatography 5.
Iex ion exchange chromatography, hic hydrophobic interaction chromatography, ac affinity chromatography. Fast flow ion exchange media in the recommended xk 1620 column. Ion exchange chromatography is the reversible adsorption of charged molecules to immobilized ion groups on a matrix of an opposite charge. Apr 28, 2019 modern ion exchange resin were first used in 1935 by adams and holms. Ion exchange chromatography an overview sciencedirect. Data file 29067018 ab ion exchange chromatography capto s. Adsorption chromatography depends upon interactions of different types. Ion exchange chromatography data file, ka64150119df sepharose fast flow ion exchangers include resins that are called weak cm, deae, and anx or strong sp and q. Ion exchange chromatography iex iex separates proteins with differences in charge.
The technique of ion exchange chromatography is based on this interaction. Surface charge of protein the mobile phase in iexc is aqueous solution with proper ph value and ionic strength. Data file 29067018 ab ion exchange chromatography capto. Msc course of methods in biochemistry find, read and cite. These short solved questions or quizzes are provided by gkseries. Ion exchange chromatography ion exchange chromatography is probably the most frequently used and versatile method for fractionating biological substances, even proteins and peptides with small differences in charge can be separated. Types of ion exchange chromatography media andy masters ge healthcare, life sciences sales development uk and ireland chromatography resins. Ge healthcare akta protein purification design book. Guide to ionexchange chromatography harvard apparatus. Prepare the column to the desired start bind the target molecules. Many drugs and pharmaceutical agents are weakly or strongly acidic or basic in nature. A typical ion chromatography consists of several components as shown in figure 3.
Ion exchange chromatography q sepharose high performance sp. Scx chromatography involves allowing peptides to bind to a negatively charged resin followed by elution on an increasing gradient of salt mobile phase, such that peptides with relatively lower charge states and therefore weaker interaction with the column elute earlier than those with strong interaction villen et al. Pdf ion exchange chromatography iec allows for the separation of ionizable molecules. Finally, a limitation of weak ion exchangers is their ph dependence. Ge healthcare protein purification troubleshooting guide pure protein today. Ion exchange chromatography is probably the most powerful and classic type of liquid chromatography. Nonvolatile buffers for cation exchange chromatography. Mostly similar charged ions like cations and anions can be conveniently separated by this technique. The most important aspect is that the sample must be soluble in the eluent and should ideally be dissolved in the mobile phase itself. Ion chromatography or ionexchange chromatography separates ions and polar molecules based on their affinity to the ion exchanger. The medium is designed for polishing of monoclonal antibodies mabs and a wide range of other biomolecules. Detailed information on the technique of ion exchange can be found in the handbook ion exchange chromatography and chromatofocusing.
Ion exchange chromatography is used t o separate charged molecules, including proteins, from complex biological samples. When working outside of their optimal ph range, these resins rapidly lose capacity, and more importantly. Capto s impact is part of a platform of media based on the capto product line. Handbooks cytiva, formerly ge healthcare life sciences. Pressure and flow rate retention time purity and resolution peaks.
Support protocols describe 1 pilot experiments to determine initial conditions for batch or column chromatography i. Ion exchange chromatography principles and methods 18111421 affinity chromatography principles and methods 18102229 hydrophobic interaction chromatography principles and methods 18102090 gel filtration principles and methods 18102218 reversed phase chromatography principles and methods 181416 expanded bed adsorption principles and. This application describes an easy separation of model proteins and explains how anion exchan ge. Ion chromatography or ion exchange chromatography separates ions and polar molecules based on their affinity to the ion exchanger. Analyte and mobile phase are initially always polar andor ionic.
Ion exchange chromatography instrumentation microbe notes. Access free ion exchange chromatography handbook ge healthcare ion exchange chromatography handbook ge healthcare as recognized, adventure as without difficulty as experience about lesson, amusement, as competently as promise can be gotten by just checking out a book ion exchange chromatography handbook ge healthcare plus it is not directly done, you could acknowledge even more concerning this. In this experiment, a protein homogenate is subjected to anion exchange chromatography on an fplc system ge. Ion exchange starts with the equilibration of the exchanger using ph, and ionic strength.
Ion exchange chromatography technique is independent of sample volume provided, that the ionic strength of the sample is low and the target molecule protein of interest is highly charged. Principle of ion exchange chromatography ion exchange iex chromatography can separate molecules or groups of molecules that have only slight differences in charge. In ion exchange chromatography, the support consists of tiny beads to which are attached chemicals possessing a charge. Ion exchange chromatography iex separates proteins with differences in surface charge to give highresolution separation with high sample loading capacity. Summary ion exchange chromatography is one of the most widely used fplc techniques for protein separation and purification. Ion exchange chromatography resins can be used at high flow. Separation is based on the reversible interaction between a charged molecule and an oppositely charged chromatography medium. Principles of ion exchange this chapter provides a general introduction to the theoretical principles that underlie every ion exchange separation. Where to download ion exchange chromatography handbook ge healthcare. Ion exchange chromatography high performance ion chromatography, hpic this separation method is based on an ion exchange process occurring between the mobile phase and ion exchange groups bonded to the support material. Membrane chromatographic 36 37 devices are cheap to massproduce and disposable unlike other chromatography devices that require maintenance and time to revalidate. Anion exchange chromatography workflow buffer preparation.
To modify these instructions for columns of different dimensions, refer to appendix a. Ion chromatography or ionexchange chromatography is a chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. Ionexchange chromatography is widely used in the analyses of petroleum fractions for the isolation and preliminary separation of acid and basic components speight, 2001. Sepharose fast flow ion exchangers, available in different formats for process development to. Hitrap sp hp, 1 ml and 5 ml hitrap q hp, 1 ml and 5 ml vwr. Separation of proteins with anion exchange chromatography on.
Ge healthcare offers a wide variety of prepacked columns and readytouse chromatogra. Fast protein liquid chromatography fplc, is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. The eluent is delivered to the system using a highpressure pump. The principle of separation is thus by reversible exchange of ions between the target ions present in the sample solution to the ions present on ion exchangers. Protein preparation handbook thermo fisher scientific. Separation on the basis of specific binding sites on the protein affinity chromatography. Feb 04, 2021 ion exchange chromatography or ion chromatography is a process that allows the separation of ions and polar molecules based on their affinity to ion exchangers. Protein chromatography kits will aim to cover some of the chromatography techniques routinely used in protein purification. Vbs0073 separation of proteins with anion exchange. Ion exchange chromatography involves the separation of ionizable molecules based on their total charge.
Size size exclusion chromatography sec, also called gel. Samples should be in a high salt concentration to promote hydrophobic interaction. Ion exchange chromatography sepharose fast flow ion exchange. Hydrophobic interaction and reversed phase chromatography. This means hic is wellsuited for capture steps after sample cleanup by ammonium sulfate precipitation or for intermediate steps directly after an ion exchange separation. This technique enables the separation of similar types of molecules that would be difficult to separate by other techniques because the charge carried by the molecule of interest can be readily manipulated by changing buffer ph. Buffer ph and ionic strength are crucial for all forms of ion exchange. Ion exchange chromatography an overview sciencedirect topics. Ion exchange chromatography is one of the most widely used fplc techniques for protein separation and purification. In both situations, the sample is already in a high salt solution. Proteins bind as they are loaded onto a column at low ionic strength. Ge, ge monogram, amersham, akta, axichrom, biacore, bioprocess, capto, cy, cydye, ecl.
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