Proteinpak hi res hic columns contain nonporous, polymethacrylatebased particles 2. 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. Separation of mabs molecular variants by analytical. Hydrophobic interaction chromatography theory and principle. The principle for protein adsorption to hic media is complementary to ion exchange and size exclusion chromatography. Hic offers an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction. High pressure liquid chromatographyhplc is a type of column chromatography generally used in biochemistry and analysis of active compounds to identify, quantify and. Chromatography hic this is a chromatographic technique in which the sample interacts, at high mobile phase salt concentration, with hilic a hydrophobic stationary phase. Property technique hydrophobicity hydrophobic interaction chromatography hic reversed phase chromatography rpc charge ion exchange chromatography iex, chromatofocusing cf. Protein a and protein g affinity chromatography are widely used for antibody purification despite their grossly disproportionate expense.
The basis of separation in hydrophobic interaction chromatography hic is the same as for rpc. A chromatographic matrix containing hydrophobic groups, binds proteins from aqueous solutions to different extents depending on the protein structures and a range of controllable factors including concentrations of salts, ph, temperature and organic solvents fig 2. Currently offered reagents are an array of hydrophobic interaction chromatography hic tools for the separation and purification of proteins and other biomolecules based on their. However, unlike reversedphase chromatography, hic is a. Hydrophobic interaction chromatography hic is a technique for separation of proteins, peptides, and other biomolecules based on their relative degree of hydrophobicity.
Highperformance liquid chromatography is an effective type of column chromatography which is widely used in pharmaceuticals. Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase stable phase is separating from each other while moving with the aid of a mobile phase. Since the molecular mechanism of hic relies on unique. In a hic separation, the hydrophobic ligands on the stationary phase interact with. Introduction to hydrophobic interaction chromatography.
Hic technique is based on hydrophobic interactions between side chains bound to chromatography matrix 22, 23. Basis of chromatography what is the chromatography. Hydrophobic interaction chromatography hic process. Principles and methods was published describing the. Manual media screening, method development and optimization. Hydrophobic interaction chromatography hic in this method the adsorbents prepared as column material for the ligand binding in affinity. 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. Chromatography introduction to chromatography chromatography is a nondestructive procedure for resolving a multicomponent mixture of trace, minor, or major constituents into its individual fractions. It is very useful to determine the assay and related substances in drug. Schematic of affinity purification using protein a, g, or l. Based protein separations on waters proteinpak hi res. Ion chromatography introduction ion chromatography ic was introduced in 1975 by small, stevens and baumann as a new analytical method. Separation principles in chromatographic purification.
Hic is an excellent complement to size exclusion and ion exchange chromatography in difficult sepa. If youre seeing this message, it means were having trouble loading external resources on our website. Hydrophobic interaction chromatography hic is a technique for separation of proteins. Hydrophobic interaction chromatography hic is commonly used as a polishing step in monoclonal antibody purification processes. Hic is carried out with an aqueous solution of higher salt concentration at neutral conditions and uses a weak hydrophobic stationary phase. The principle of hydrophobic interaction chromatography. Hic is widely used in protein purification as a complement to other techniques that. Within a short period of time, ion chromatography developed from a new detection scheme for a few selected inorganic anions and cations to a versatile analytical technique for ionic species in general. Highperformance liquid chromatography hplc has been widely used for years as an analytical method and is a key tool for the separation and analysis of. Explain the principle of affinity chromatography, why a spacer arm. Hydrophobic interaction chromatography hic theory and. In hic biomolecules adsorb to a weak hydrophobic surface at high salt concentration. Hydrophobic interaction chromatography hic is a versatile method for the purification and separation of biomolecules by using the function of hydrophobicity.
The factors effective on this separation process include molecular characteristics related to adsorption liquid. Hydrophobic interaction chromatography hic separates biomolecules, under relatively mild conditions, according to differences in their hydrophobicity. Hydrophobic interaction chromatography hic resins and prepacked columns for the purification of proteins and biomolecules based on their surface hydrophobicity. The stationary phase may be a solid or a liquid supported on a solid, or a gel and cant be gas. Method development for hydrophobic interaction chromatography. Department of food science and nutrition, university of minnesota, st. In hic, the sample protein molecules are introduced into the column containing a highsalt buffer. Interaction principle according to hydrophobic interaction and reversed phase chromatography principles and methods ge healthcare 4 close to the surface of the hydrophobic ligand and solute, the water molecules are more highly ordered than in the. Hic sorts biomolecules by degree of their surface hydrophobicity. The principle of column chromatography is to separate a large pool of proteins into many smaller pools, some of which are enriched in the protein of interest.
Hydrophobic interaction chromatography hic has gained popularity over recent years for the purification of compounds on a preparative and semianalytical scale. Hydrophobic interaction chromatography hic is a widelyused technique for separation and purification of proteins and peptides. In general the conditions under which hydrophobic interaction chromatography is used are relatively mild and protein friendly resulting in. Hydrophobic interaction chromatography thermo fisher. Provides students and practitioners with a solid grounding in the theory of chromatography, important considerations in its application, and modern instrumentation. Principles of hydrophobic interaction chromatography youtube. Pdf applications of chromatography in separation of. Principles of hydrophobic interaction chromatography 9. Phenyl sepharose high performance and butyl sepharose high performance, available in a variety of formats, allow highresolution, highcapacity purification of biomolecules. In my experience, hic is finding dramatically increased use both in laboratory and production processes. Reversed phase chromatography principles and methods 11001269 percoll methodology and applications 18111569 ficollpaque plus for in vitro isolation of lymphocytes 18115269.
Hydrophobic interaction chromatography is widely used for the, separation and purification of proteins, in their native state, as well as for isolating, protein complexes and studying protein. The various constituents of the mixture travel at different speeds, causing them to separate. If youre behind a web filter, please make sure that the domains. Chromatography is based on the principle of separation of compounds into different bands color graphs and the identification of those bands. Ionic ionexchange with bonded phase or ionized surface silanol groups. Use of hydrophobic interaction chromatography with a non. While expensive and specialized equipment is available for column chromatography, only basic equipment is required. The main difference between reverse phase and hydrophobic interaction chromatography is that the reverse phase chromatography rpc uses a more hydrophobic medium, which leads to more stronger interactions whereas the hydrophobic interaction chromatography hic uses a less hydrophobic medium when compared to the medium in the. Hydrophobic interaction chromatography an overview. Hydrophobic interaction chromatographyhigh performance liquid chromatography hichplc is a powerful analytical method used for the separation of molecular variants of therapeutic proteins.
Protein hydrophobic interaction chromatography hic columns hydrophobicbased separations. Purification of monoclonal antibodies by hydrophobic. Partition phase transfer of analytes to and from a polar stationary solvent aqueous and relatively nonpolar organic mobile phase. Fast protein liquid chromatography fplc, is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. Pdf on jan 20, 2012, andrea mahn and others published hydrophobic interaction chromatography. The salt in the buffer reduces the solvation of sample solutes. Chapter 1 2 3 introduction, chromatography theory, and. Stationary phase substance that stays fixed inside the column. 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. For many cases, hydrophobic interaction chromatography hic is an ideal separation method. The method has been employed for monitoring various posttranslational.
Different variations may be applied to solids, liquids, and gases. In theory, hydrophobic interaction chromatography hic and rplc are closely related, as in both techniques separation is based on hydrophobic interactions between the surface of an analyte and the stationary phase. Theory and use of hydrophobic interaction chromatography in. Hydrophobic interaction chromatography hic is a powerful technique for both analytical and preparative separations of biomolecules. Hic reversed phase chromatography rpc charge ion exchange chromatography iex, chromatofocusing cf. However, in application the techniques are very different. Polar interactions dipole hydrogen bonding, induced dipole, etc. Hydrophobic interaction chromatography hic newsmedical. Learn how high performance liquid chromatography works hplc principle and how chromatographic column separates the compounds from a mixture. Hydrophobic interaction chromatography hic is a commonly used technique that exploits these hydrophobic features of proteins as a basis for their separation. Gel permeation molecular sieve chromatography the basic principle of this method is to use dextran containing materials to separate macromolecules based on their differences in molecular sizes. Sample molecules containing hydrophobic and hydrophilic regions are applied to an hic column in a highsalt buffer. Introduction to hydrophobic interaction chromatography hic. The preferential separation is done due to differential affinities of compounds towards stationary and mobile phase.
This is largely due to the absence of more economical generic alternatives. Hic can be applied to characterize mab variants resulting from posttranslational modifications ptms. Read and learn for free about the following article. Hic separations are often designed using the opposite conditions of those used in ion exchange chromatography. Phenyl sepharose high performance butyl sepharose high performance fig 1. Chromatography is a laboratory technique for the separation of a mixture. Chromatography column eluent in eluate out mobile phase solvent moving through the column. Hydrophobic interaction chromatography hic is a valuable tool used in protein purification applications. These methods differ in the properties of the mobile and stationary phases. Chromatography is a physical method of separation in which the components to be separated are distributed between two phases one is the stationary phase and the other is the mobile phase. More detailed packing instructions are to be found in the instruction manual. Introduction to hydrophobic interaction chromatography hic lsr. After separation of the compounds, they are identified by suitable detection methods.
Hic is used in the purification of proteins over a broad. Pdf phenyl sepharose high performance butyl sepharose. 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. The power of chromatography 9 comes from its ability to separate a mixture of compounds, or analytes, and.
The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. This leads to stronger interactions that, for successful elution, must be reversed using nonpolar, organic solvents such as acetonitrile or methanol. Principles of chromatography process by which one separate compounds from one another by passing a mixture through a column that retains some compounds longer than others. Hydrophobic interaction chromatography hic products. Chromatography definition, principle, types, applications. Hydrophobic interaction and reversed phase chromatography. In general, salts that increase surface tension in aqueous solutions also promote hydrophobic interactions.
1249 412 1601 1146 427 684 1128 1192 13 489 315 1270 519 1058 1263 1055 913 73 1531 1058 1495 857 1094 295 101 971 1049 629 622 239 1310 452 929