Chromatography definition, principle, types, applications. In general, salts that increase surface tension in aqueous solutions also promote hydrophobic interactions. A the basic structure of a hic resin is depicted, and a protein is schematized highli ghting. The first analytical use of chromatography was described by james and martin in. Principles and methods was published describing the. Chapter 4 hydrophobic interaction chromatography of proteins.
If you continue browsing the site, you agree to the use of cookies on this website. When the mobile phase along with the mixture that needs to be separated is introduced from the top of the column, the movement of the individual components of the mixture is at different rates. Pdf on jan 20, 2012, andrea mahn and others published hydrophobic interaction chromatography. In hic, the sample protein molecules are introduced into the column containing a highsalt buffer. Alternatives gel filtration chromatography ion exchange chromatography reverse phase chromatography why hic. In my experience, hic is finding dramatically increased use both in laboratory and production processes. Different basis of separation weaker interactions less structural damage maintain high activity 3. Hydrophobic interaction chromatography hic is a valuable tool used in protein purification applications. For many cases, hydrophobic interaction chromatography hic is an ideal separation method. Sep, 2012 hydrophobic interaction chromatography separates biomolecules, under relatively mild conditions, according to differences in their hydrophobicity.
However, unlike reversedphase chromatography, hic is a nondenaturing technique. Thin layer chromatography tlc calculating retention factors for tlc. Since the molecular mechanism of hic relies on unique. Column chromatography principle, procedure, applications on. 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. Theory and use of hydrophobic interaction chromatography in. Hydrophobic interaction and reversed phase chromatography. Introduction to hydrophobic interaction chromatography. Apr 26, 2014 hydrophobic interaction chromatography,utilizes the reversible interaction of protein and the hydrophobic ligand for the separation of protein mixtures. The sample mixture is allowed to pass through a column of solid stationary phase under the force of a liquid mobile phase. Paper chromatography principle, procedure, applications. Ion exchange chromatography is an interesting type of column chromatography as you know, the chromatography is a process of the separation of molecules from a mixture. Hydrophobicinteraction chromatography polar liquidnonpolar liquid or solid molecular sizepolarity af.
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 may be viewed as a series of equilibrations between the mobile and stationary phase. Gel permeation molecular sieve chromatography the basic principle of this method is to use dextran containing materials to separate macromolecules based on their. Principle of affinity chromatography shodex hplc columns. Hydrophobic interaction chromatography is a separation technique that uses the properties of hydrophobicity to separate proteins from one another. These groups can range from methyl, ethyl, propyl, octyl, or phenyl groups.
It works on the principle of hydrophobic interactions hence the more nonpolar the material is, the longer it will be retained. In reversed phase mode very polar compounds are often not sufficiently retained in low percent organic, or even in 100% aqueous mobile phase. Hydrophobic interaction chromatography hic this is a chromatographic technique in which the sample interacts, at high mobile phase salt concentration, with hilic a hydrophobic stationary phase. Hydrophobic interaction and reversed phase chromatography, principles and methods, 11001269 multimodal chromatography multimodal ligands capture target molecules through multiple types of interaction. A21, with kind permission from elsevier sciencenl, sara burgerhartstraat 25, 1055kv amsterdam. In my experience, hic is finding dramatically increased use both in laboratory and. 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. Hydrophobic interaction chromatography hic newsmedical. Principle of involved in this technique is the separation of components by adsorption. Hydrophobic interaction chromatography cytiva, formerly ge.
Hydrophobic interaction chromatography separates biomolecules, under relatively mild conditions, according to differences in their hydrophobicity. Sep 15, 2016 hydrophobic interaction chromatography is a biological recognition process corresponding to a two. Separation principles in chromatographic purification. The various constituents of the mixture travel at different speeds, causing them to separate. Pdf hydrophobic interaction chromatography researchgate. This separation is done based on the differences in the adsorption coefficient or partition coefficient of the sample with the stationary phase. Samples should be in a high salt concentration to promote hydrophobic interaction. 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 separates molecules based on their hydrophobicity. In hic biomolecules adsorb to a weak hydrophobic surface at high salt concentration. Ionic interactions are commonly involved, but others such as hydrogen bonding and hydrophobic interactions can be significant. Learn the principle, procedure of paper chromatography along with its types and applications. Hic is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices. Pdf hydrophobic interaction chromatography involves the separation of protein.
Hic is used in the purification of proteins over a broad range of scalesin both analytical and preparatory scale applications. Applications of hydrophilic interaction chromatography to. In hydrophobic interaction chromatography the matrix material is lightly substituted with hydrophobic groups. Paper chromatography is an inexpensive method of separating dissolved chemical substances by their different migration rates across the sheets of paper. It provides a complementary approach to ionexchange and sizeexclusion.
Chromatography is a general term applied to a wide variety of separation techniques based on the partitioning or distribution of a sample solute between a moving or mobile phase and a fixed or stationary phase. Sep 25, 2018 chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Principles of chromatography stationary phase article. Hydrophobic interactions between proteins and the chromatographic matrix can be exploited to purify proteins. Hydrophobic interaction chromatography hic is a powerful technique for protein separation. For further details on these two chromatographic modes see also fausnaugh et al. The components with lower adsorption and affinity to stationary phase travel faster when compared to the greater. Therefore, the native form of the proteins is expected. Column chromatography is a technique which is used to separate a single chemical compound from a mixture dissolved in a fluid. The two phases are water held in pores of the filter paper and the other phase is a mobile phase which passes through the paper. Fundamentals and applications in biomedical engineering 605 fig. Reversedphase chromatography also called rpc, reversephase.
Hydrophilic interaction chromatography normal phase and hydrophilic interaction liquid chromatography hilic are primarily used to separate polar and hydrophilic compounds. Hydrophobic interaction chromatography bound with negatively charged groups are known pseudoaffinity chromatography highpressure liquid chromatography hplc. Hydrophobic interaction chromatography introduction hic is a powerful technique for separation and purification of bio molecules. Hic is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions. Theory and use of hydrophobic interaction chromatography. Column chromatography principle, procedure, applications. The opposite of normal phase, or reversed phase chromatography, results from the adsorption of hydrophobic molecules onto a hydrophobic solid support in a. Hydrophobic interaction chromatography hic is a reversedphase separation technique that uses a weakly hydrophobic stationary phase and a negative ionic strength gradient in a buffered aqueous mobile phase for the separation of proteins 5, 209211, 368371. Hydrophobic interaction and reve rsed phase chromatography. Principles of hydrophobic interaction chromatography youtube. The principle involved can be partition chromatography or adsorption chromatography. Introduction to hydrophobic interaction chromatography hic.
Hydrophobic interaction chromatography hic is a technique for separation of proteins, peptides, and other biomolecules based on their relative degree of hydrophobicity. 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 can be used for capture, intermediate purification, or polishing steps. Ionexchange chromatography polar liquidionic solid molecular charge sizeexclusion chromatography liquidsolid molecular size hydrophobicinteraction chromatography polar liquidnonpolar liquid or solid molecular sizepolarity af. Stationary phase in chromatography, is a solid phase or a liquid phase coated on the surface of a solid phase. Hydrophobic interaction chromatography theory and principle. This means hic is wellsuited for capture steps after sample cleanup by ammonium sulfate precipitation or for intermediate steps directly after an ion. Affinity chromatography separates proteins on the basis of a reversible interaction between a protein or group of proteins and a specific ligand coupled to a chromatography matrix. Purification of monoclonal antibodies by hydrophobic. Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins. Fundamentals and applications in biomedical engineering chapter pdf available january 2012 with 1,935 reads how we measure reads.
Based protein separations on waters proteinpak hi res hic. The separation of the components in the sample mixture, with some exceptions, correlates with. Tskgel is available as bulk polymeric resin or in silica or polymericbased prepacked columns. Partition chromatography because the substances are partitioned or distributed between liquid phases. Hydrophobicity hydrophobic interaction chromatography reversed phase chromatography fig. Hydrophobic interaction chromatography,utilizes the reversible interaction of protein and the hydrophobic ligand for the separation of protein mixtures. Hydrophobic interaction chromatography polar liquidnonpolar liquid or solid molecular sizepolarity af. Hydrophobic interaction chromatography hic is a powerful technique used. Agarosegel chromatography is used for the purification of rna, dna particles, and viruses 4. Jennissen introduction and historical aspects hydrophobic interactions have been described as the unusually strong attraction between nonpolar molecules and surfaces in water israelachvili, 1985. Hic is used in the purification of proteins over a broad. Proteomicsprotein separations chromatographyhydrophobic.
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. Sep 25, 20 hydrophobic interaction chromatography slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. Hydrophobic interaction chromatography an overview. Hydrophobic interaction chromatography jennissen major. Principles and methods 80642960 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. This video describes the principles of the technique. 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. Hydrophobic interaction chromatography hic process.
Hydrophobic interaction chromatography hic occupies a unique niche as a polishing step in many monoclonal antibody mab purification processes. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. Apr 12, 2019 the principle of hydrophobic interaction chromatography. Ion exchange chromatography affinity chromatography general principles of liquid chromatography tskgel columns for afc group specific columns. Hydrophobic interaction chromatography hic theory and.
Chapter 4 hydrophobic interaction chromatography of proteins herbert p. Reversed phase chromatography is particularly effective for separating peptides, nucleotides, and restriction fragments. Gas chromatography science class 11 chemistry india organic chemistry some basic principles and techniques methods of purification of organic compounds principles of chromatography. In this type of chromatography, hydrophobic groups such as phenyl, octyl, or butyl, are attached to the stationary column. Hilic uses hydrophilic stationary phases with reversedphase type. In general the conditions under which hydrophobic interaction chromatography is used are relatively mild and protein friendly resulting in. The principle for protein adsorption to hic media is complementary to ion exchange chromatography and gel filtration. Chapter 1 2 3 introduction, chromatography theory, and. Hydrophobic interaction chromatography hic this is a chromatographic technique in which the sample interacts, at high mobile phase salt concentration, with a weakly hydrophobic stationary phase.
Chromatography is a laboratory technique for the separation of a mixture. The power of chromatography 9 comes from its ability to separate a mixture of compounds, or analytes, and. Hydrophobic interactions tend to be strongest at high salt concentrations. Hic is used to remove various impurities that may be present in the solution, including undesirable product.
Hydrophobic interaction chromatography hic is a commonly used technique that exploits these hydrophobic features of proteins as a basis for their separation even in. 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. 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 column affinity chromatography columns chiral separation column orpak cdbs453 chiral separation column orpak crx853 polymerbased column for high temperature analysis etrp1 selection of chiral separation columns principle of affinity chromatography feature of high temperature analysis etrp1. Our mission is to provide a free, worldclass education to anyone, anywhere. The russian botanist mikhail tswett coined the term chromatography in 1906. Paper chromatography principle, procedure, applications on. Based protein separations on waters proteinpak hi res. The principle of hydrophobic interaction chromatography. The components in the mixture get adsorbed to the solid stationary phase during their travel. Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Hydrophobic interaction chromatography cytiva, formerly. Learn the principle, procedure of column chromatography along with its types and applications. Hydrophobic interaction chromatography hic theory and principle.
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