The separation is based on the reversible interaction between the protein and the hydrophobic surface of a chromatographic medium. Shortcomings and benefits of hydrophobic interaction chromatography are discussed in. Samples are adsorbed to the resin at relatively high salt concentrations and eluted by applying. Hydrophilic interaction chromatography or hydrophilic interaction liquid chromatography, hilic is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. Hydrophobic interaction chromatography hic is a widely used technique for separation and purification of proteins and peptides. In theory, hic and reversephase chromatography rpc are closely related lc techniques. Purification of monoclonal antibodies by hydrophobic. Automated hydrophobic interaction chromatography column. Normal phase and hydrophilic interaction liquid chromatography hilic are primarily used to separate polar and hydrophilic compounds. There are a number of definitions in the literature but the best is probably that given by glasstone many years ago but is still directly applicable today. Dec 28, 1984 comparison of hydrophobic interaction and reversedphase chromatography of proteins. Hydrophobic interaction chromatography hic is a widely used chromatographic technique that finds application at different stages of downstream processing. A quantitative method is demonstrated with the characteristics of 3.
From a kinetic plot analysis, it was expected that the monolithic column could provide three times faster separation than particlepacked hilic columns. Hydrophobic interaction chromatography hic is a commonly used technique that exploits these hydrophobic features of proteins as a basis for their separation even in complex biological mixtures 1 2. Hydrophobic interaction chromatography introduction hic is a powerful technique for separation and purification of bio molecules. Hydrophilic interaction chromatography hilic is described as a useful alternative to reversedphase chromatography for applications involving polar compounds. In the hilic mode, an aqueousorganic mobile phase is used with a polar stationary phase. Illustration of hydrophobic interaction chromatography and how it works on a particle level. Hydrophobic interaction chromatography is a separation technique that uses the properties of hydrophobicity to separate proteins from one another. Chromatographic methods for protein purification ispybio. Hydrophobic interaction chromatography toyopearl hydrophobic interaction chromatography hic is a widelyused technique for separation and purification of proteins and peptides. Prediction of protein retention in hydrophobic interaction. Pdf main interactions and influences of the chromatographic. In the early 1990s new phases started to emerge, and the practice was given a name, hydrophilic interaction chromatography hilic.
Hilic uses hydrophilic stationary phases with reversedphase type. Hydrophobic interaction chromatography is used to separate proteins with difference in hydrophobicity. Hydrophobicity hydrophobic interaction chromatography hic reversed phase chromatography rpc charge ion exchange chromatography iex, chromatofocusing cf size gel filtration gf, also called size exclusion chromatography biorecognition ligand specificity affinity chromatography ac fig 1. For many cases, hydrophobic interaction chromatography hic is an ideal separation method. Sep 15, 2016 hydrophobic interaction chromatography is a biological recognition process corresponding to a two. Hydrophobic interaction chromatography hic is commonly used as a polishing step in monoclonal antibody purification processes. Macroprep tbutyl and methyl hydrophobic interaction. The first hplc applications were published more than 30 years ago, and were for a long time mostly confined to carbohydrate analysis.
Ms for metabonomic analysis of zucker rat urine helen g. Hydrophilic interaction chromatography hilic waters corporation. Hydrophobic interaction chromatography media amsbio. Hydrophobic interaction chromatography add a small piece of paper to collection tube where column seats to insure column flow rest pipette tip on side of column to avoid column bed disturbance when adding solutions drain until the meniscus is just. Jan 01, 2005 read protein interactions in hydrophobic charge induction chromatography hcic, biotechnology progress on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Hydrophobic interaction chromatography hic separates biomolecules, under relatively mild conditions, according to differences in their hydrophobicity. Pdf hydrophilic interaction liquid chromatography hilic is a popular. Hydrophobic interaction chromatography hic is one of the basic purification procedures inthebiosciences. Sep, 2012 hydrophobic interaction chromatography separates biomolecules, under relatively mild conditions, according to differences in their hydrophobicity. For historical reasons, it has been reported that hilic is a variant of normal phase liquid chromatography, but the separation mechanism used in hilic is more complicated than that in nplc.
A weakly nonpolar stationary phase is used with an aqueous mobile phase containing a high concentration. Hydrophobic interaction chromatography tosoh bioscience. Hilic is increasingly becoming a method of choice for the analysis of polar compounds 15. Hydrophobic interaction chromatography hic is a powerful technique for protein separation. Reversed phase, hydrophilic interaction and normal phase chromatography columns polymerbased columns for reversed phase chromatography odp2 hp polymerbased columns for reversed phase chromatography asahipak polymerbased columns for reversed phase chromatography rspak. Ge healthcare hic hydrophobic interaction chromatography. Hydrophilic interaction liquid chromatography hilic is a popular technique for the. The use of hydrophilic interaction chromatography with.
Hydrophobic interaction chromatography sciencedirect. The technique utilizes the accessible hydrophobic regions located on protein surfaces and their interactions with a weakly hydrophobic stationary phase. Temperature effects in hydrophobic interaction chromatography. Here, we report a strategy of a dualgradient hydrophobic interaction chromatography hic mode to refold recombinant human granulocyte colonystimulating factor from its inclusion bodies at high protein concentration. Abstract applications of hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs, i.
The hic procedure is a type of affinity chromatography. Hydrophobic interaction chromatography brian mccauley. Highly aqueous systems sometimes lead to problems such. Semiautomated identification of nglycopeptides by hydrophilic interaction chromatography, nanoreversephase lcmsms, and glycan database search. Hydrophilic interaction chromatography hilic separation. However, the retention of polar analytes often requires a highly aqueous mobile phase to achieve retention. Jennissen introduction and historical aspects hydrophobic interactions have been described as the unusually strong attraction between nonpolar molecules and surfaces in water israelachvili, 1985. Gc ngas chromatography hcw nhealthcare worker hd nhazardous drug hepa nhighe % iciency particulate air hilic nhydrophilic interaction chromatography hipec nhyperthermic intraperitoneal chemotherapy hplc nhighperformance liquid chromatography iarc ninternational agency for research on cancer if nifosfamide im nintramuscular ip. An automated method for identifying suitable hydrophobic interaction chromatography hic media to be used in the process of protein purification is presented. Reversed phase, hydrophilic interaction and normal phase. Hydrophilic interaction chromatography hilic can be described as a reversed reversedphase chromatography performed using a polar stationary phase for example, unmodified silica, amino, or diol bonded phases.
The variable hydrophobic nature of proteins allows their separation through differential hydrophobic surface interactions. Comparison of hydrophobicinteraction and reversedphase. Salt tolerant interaction chromatography y combination of unique cellulosebased membrane structure and covalently attached salt tolerant primary amine pa ion exchange ligand primary amine sartobind stic pa february 15, 2012. Large scale process development for hydrophobic interaction chromatography, part 1. Hydrophobic interaction chromatography of various streptavidin samples is compared with electrophoretic results. Possibilities and limitations of hydrophobic interaction. The methodologies discussed consider threedimensional structure data of the protein and its surface hydrophobicity. Expanded bed adsorption chromatographic separation special techniques reversedphase chromatography hydrophobic interaction chromatography twodimensional chromatography simulated movingbed chromatography pyrolysis gas chromatography fast. Hydrophobic interaction and reversed phase chromatography. Hydrophobic interaction chromatography hydrophobic interaction chromatography hic is a powerful technique for both analytical and preparative separations of biomolecules. The various constituents of the mixture travel at different speeds, causing them to separate. Hydrophobic interaction chromatography hic is based on nonpolar interactions that are induced by high salt mobile phases.
Hydrophobic interactions tend to be strongest at high salt concentrations. Macroprep tbutyl and methyl hydrophobic interaction chromatography media instruction manual please read these instructions prior to using macroprep hydrophobic interaction chromatography media. Aiming to improve the attractiveness and the learning methods provided by this experiment, a novel experiment was. Hydrophilic interaction chromatography hilic is a variant of normal phase chromatography in which biomolecules are separated on the basis of polar and hydrophilic interactions with polar neutral and ionizable constituents of stationary phases in hydrophobic mobile phases. Ionic interactions are the basis for purification of proteins by ion exchange chromatography. Commonly, it is the amount of salt in the buffer which is varied. Bottoli in the early 1990s, hydrophilic interaction liquid chromatography hilic emerged as a new separation method. Protein interactions in hydrophobic charge induction. The role of hydrophobic interaction chromatography in process purification hydrophobic interaction chromatography hic is a powerful tool for the process purification of biomolecules. Hic is widely used in protein purification as a complement to other techniques that separate according to charge, size or biospecific recognition. Hydrophobic interaction chromatography monmouth college.
Dispersive interactions are the most difficult to describe probably because they are still not clearly understood. Shortcomings and benefits of hydrophobic interaction chromatography are discussed in respect to the evaluated results. Since the molecular mechanism of hic relies on unique. Hic sorts biomolecules by degree of their surface hydrophobicity. Alternatives gel filtration chromatography ion exchange chromatography reverse phase chromatography why hic. Hydrophobic interaction chromatography springerlink. Chromatographic characterization of stationary phases for. The use of hydrophilic interaction chromatography with mass spectrometry detection for the separation of basic pharmaceuticals and food toxins r. Salt tolerant interaction chromatography y combination of unique cellulosebased membrane structure and covalently attached salt tolerant primary amine pa ion exchange ligand primary amine sartobind stic pa. Theses on chromatography international library for. Hydrophobic interaction chromatography an overview.
Different basis of separation weaker interactions less structural damage maintain high activity 3. Hydrophobic interaction chromatography hic theory and. If you have any questions or comments regarding these instructions, please contact your local biorad laboratories representative. Hydrophobic interaction chromatography hic is commonly used for separating proteins. Interaction chromatography add a small piece of paper to collection tube where column seats to insure column flow rest pipet tip on side of column to avoid column bed disturbance when adding solutions drain until the meniscus is just above the matrix for best separation. Hic offers an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction. Hydrophobic interaction chromatography resins is an alternative for the size exclusion chromatography and ion exchange chromatography. The column showed high separation efficiency, with a theoretical plate height h720. Both are based upon interactions between solventaccessible nonpolar groups hydrophobic patches on the surface of biomolecules and the hydrophobic ligands alkyl or aryl groups covalently attached to the gel matrix.
Read possibilities and limitations of hydrophobic interaction chromatography evaluation with strepavidin, chromatographia on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. Hydrophilic interaction chromatography hemstrom 2006. Kumagai, agilent technologies, hachiojishi, tokyo, japan. The proteins containing both hydrophilic and hydrophobic regions are applied to a hic column under specified salt buffer conditions, which promotes the binding of the biomolecule to the hic resin but also has a. The technique takes advantage of the hydrophobic areas located on the surface of proteins. Hydrophilic interaction liquid chromatography hilic is an alternative highperformance liquid chromatography hplc mode for separating polar compounds. Hydrophilic interaction chromatography request pdf. Sep 25, 20 hydrophobic interaction chromatography 1 1. Illustration of hydrophilic interaction chromatography and how it works on a particle level. Highly efficient analysis of underivatized carbohydrates. In my experience, hic is finding dramatically increased use both in laboratory and production processes. Separation, purification and identification of the components of a mixture. Hydrophobic interaction chromatography resins market.
Property technique hydrophobicity hydrophobic interaction chromatography hic reversed phase chromatography rpc charge ion exchange chromatography iex, chromatofocusing cf. Interactions and influences of the chromatographic parameters in. Hydrophobic interaction chromatography hic today a key method in the purification of monoclonal antibodies involves the separation of protein molecules in their native and biologically active state owing to a differential interaction of these molecules with hydrophobic sites on the surface of a solid support. Hydrophilic interaction chromatography hilic separation of basic drugs using msms detection application note organic portions of typical analytes. In general the conditions under which hydrophobic interaction chromatography is used are relatively mild and protein friendly resulting in. Proteomicsprotein separations chromatographyhydrophobic. Sep 29, 20 illustration of hydrophilic interaction chromatography and how it works on a particle level. Hydrophobic interaction chromatography hic a protein. This technique is ideally used as intermediate step in a purification process or to capture specific protein. Hydrophobic interaction chromatography hic is a powerful tool for the process purification of biomolecules. Hydrophobic interaction chromatography hic theory and principle. Hilic uses hydrophilic stationary phases with reversedphase type eluents. Hydrophilic interaction chromatography for the analysis of.
The method utilizes a mediumpressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multiwavelength. Hydrophobic interaction chromatography resins is defined as a technique used for the process purification of biomolecules. Mar 19, 1996 the effect of temperature from 5 degrees c to 50 degrees c on the retention of dansyl derivatives of amino acids in hydrophobic interaction chromatography hic was investigated by hplc on three stationary phases. Pdf semiautomated identification of nglycopeptides by. Chapter 4 hydrophobic interaction chromatography of proteins. Hydrophilic interaction chromatography, hilic, chromatographic characterization, structural selectivity, ion exchange interactions abstract the work presented herein summarizes the results of a chromatographic characterization study of hilic stationary phases involving ten silica. Hydrophobic interaction chromatography jennissen major. Hydrophobic interaction chromatography hic is a commonly used technique. Hydrophobic interaction chromatography thermo fisher. However, because of its complexity it has not gained the same foothold in the methodological repertoire of protein chemistry as has affinity chromatography or ion exchange chromatography. It was first described by shepard and tiselius in 1949 using the term salting out chromatography.
The mobile phase employed is highly organic in nature 70%. In this type of chromatography, hydrophobic groups such as phenyl, octyl, or butyl, are attached to the stationary column. Hydrophilic interaction chromatography mariana roberto gama, raquel gomes da costa silva, carol h. Efficient refolding of recombinant proteins in the forms of inclusion bodies at higher concentration remains challenging. Hydrophilic interaction chromatography an overview. In general, hydrophobic interaction chromatography hic is advantageous if the sample is sensitive to ph change or harsh solvents typically used in other types of chromatography but not high salt concentrations. Glycerolassisted hydrophobic interaction chromatography. In reversed phase mode very polar compounds are often not sufficiently retained in low percent organic, or even in 100% aqueous mobile phase. Hic is an excellent complement to size exclusion and ion exchange chromatography.
Chapter 4 hydrophobic interaction chromatography of proteins herbert p. Hilic is ideally suited for analysis of polar and hydrophilic compounds. Principles of hydrophobic interaction chromatography youtube. Although hydrophobic groups tend to be located in the interior of a protein molecule and thus would be inaccessible, early investigations 4 showed that hydrophobic effects are important in the interaction of certain enzymes with substrates and modifiers, indicating the acces sibility of hydrophobic sites. Separation, purification and identification of the. Stationary phases are similar to reversed phase chromatography rpc but the density of functional groups is lower. Hydrophobic interaction chromatography hic is a versatile method for the purification and separation of biomolecules by using the function of hydrophobicity. The proteins containing both hydrophilic and hydrophobic regions are applied to a hic column under specified salt buffer conditions, which promotes the binding of the biomolecule to the hic resin but also has a stabilizing influence on. Chromatography is a laboratory technique for the separation of a mixture. Hydrophilic interaction liquid chromatography hilic.
Topics include the molecular mechanism of separation by hic in contrast to reversed phase chromatography, a helpful section on strategies for. The purified gfp was obtained by use of the hic hydrophobic interaction chromatography procedure. Ge healthcare hic hydrophobic interaction chromatography media features high chemical stability, properties to increase productivity and epoxy coupling manufacturer. Hydrophilic interaction liquid chromatography has established itself as the separation mode of choice for uncharged highly hydrophilic and amphiphilic compounds that are too polar to be well retained in rplc but have insufficient charge to allow effective electrostatic retention in ionexchange chromatography. Pdf hydrophilic interaction chromatography using amino. Hydrophobic interaction and reversed phase chromatography principles and methods 11001269 percoll methodology and applications 18111569 ficollpaque plus for in vitro isolation of lymphocytes 18115269 gst gene fusion system handbook 18115758 antibody purification handbook 18103746 the recombinant protein handbook protein expression. The term hydrophilic interaction chromatography was introduced by alpert over 25 years ago to describe a liquid chromatography technique where polar or ionised solutes can be separated on a.
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