Chemistry
Column Chromatography
Column chromatography is a laboratory technique used to separate and purify individual components from a mixture. It involves passing a mixture through a column filled with a stationary phase, which interacts differently with each component, causing them to separate as they flow through the column. This technique is widely used in chemistry for isolating and purifying organic compounds.
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10 Key excerpts on "Column Chromatography"
- Prakash Singh Bisen, Anjana Sharma(Authors)
- 2012(Publication Date)
- CRC Press(Publisher)
Today, the technique has developed in all dimensions as perhaps the single most powerful analytical and preparative method available in the laboratory. 4.2 GENERAL PRINCIPLES The basis of all forms of chromatography is the differential partition of a compound between two immiscible phases, one of which is stationary and the other, mobile. The way in which a compound is partitioned or distributed between two immiscible phases is given by the partition coefficient ( K d ): K d Concentration in phase A Concentration in phase B = The value of K d is constant at a given temperature. Depending on the different types of phases involved, there are different forms of chromatography (Table 4.1). Depending on the mode by which separation is achieved, there are three types of chromatography: 1. Column Chromatography, in which the stationary phase is packed into glass or metal col-umns and the mobile phase percolates through the column. 2. PC, in which the stationary phase is supported by cellulose fibers of paper and the mobile phase moves through the interstitial spaces by capillary action. Sometimes, the cellulose fibers of the paper act as the solid stationary phase. 3. Thin-layer chromatography (TLC), in which the stationary phase is thinly coated onto glass plates and the mobile phase moves along the stationary phase. 62 Introduction to Instrumentation in Life Sciences 4.3 Column Chromatography In the Column Chromatography technique, the components of a mixture are separated in a column as distinct zones and each zone is eventually displaced from the column as a series of fractions (Figure 4.1). The apparatus and general techniques used for column adsorption partition, ion exchange, exclusion, and affinity chromatography have much in common and are discussed in Section 4.3.1. Gas–liquid chromatography (GLC) and high-performance liquid chromatography (HPLC) have their own unique apparatus and procedures and are discussed separately.
eBook - PDF- Raja Ghosh(Author)
- 2006(Publication Date)
- WSPC(Publisher)
151 Chapter 9 Chromatography 9.1. Introduction Chromatography is a solute fractionation technique which relies on the dynamic distribution of molecules to be separated between two phases: a stationary (or binding) phase and a mobile (or carrier) phase. In its simplest form, the stationary phase is particulate in nature. The particles are packed within a column in the form of a packed bed. The mobile phase is passed through the column, typically at a fixed velocity. A pulse of sample containing the molecules to be separated is injected into the column along with the mobile phase. The velocities at which these molecules move through the column depend on their respective interactions with the stationary phase. For instance, if a molecule does not interact with the stationary phase its velocity is almost the same as that of the mobile phase. With molecules that do interact with the stationary phase, the greater the extent of interaction, the slower is the velocity. This mode of chromatographic separation is also called pulse chromatography to distinguish it from step chromatography which is operated differently. Chromatography is used for the separation of different substances: proteins, nucleic acids, lipids, antibiotics, hormones, sugars, etc. When used for analysis of complex mixtures, chromatography is referred to as analytical chromatography while when used to separate molecules as part of a manufacturing process, it is referred to as preparative chromatography. Some of the applications of chromatography in biotechnology are listed below. 1. Biopharmaceutical production 2. Biopharmaceutical and biomedical analysis Principles of Bioseparations Engineering 152 3. Environmental analysis 4. Foods and nutraceuticals production 5. Diagnostics 6. Process monitoring 9.2. Chromatography system A chromatographic separation system consists of a column, mobile phase reservoir/s, pump/s, sample injector, detector/s and sometimes a fraction collector.
eBook - ePubFood Protein Chemistry
An Introduction for Food Scientists
- Joe Regenstein(Author)
- 2012(Publication Date)
- Academic Press(Publisher)
Chapter 15Column Chromatography
Publisher Summary
Column Chromatography is used to separate proteins during preparation. It can also be used to analyze or characterize different materials and/or reactions, and it can be used to study the kinetics of an enzyme reaction. This chapter discusses various techniques and types of columns for Column Chromatography. Partition chromatography is used to obtain a continuous series of separatory funnels. Thin-layer chromatography and paper chromatography use the same partition principle to separate materials but on a continuous basis. Paper chromatography can be done in either an ascending or descending mode. Ion exchange chromatography uses the preferential binding of various positive ions to a particular negative ion bound to the column (or vice versa) to yield the separation of material. Adsorption chromatography utilizes the force of attraction of protein to the column support material. It may result from hydrophobic interaction and/or van der Waals interaction. Molecular exclusion chromatography, also called gel filtration or molecular sieve chromatography is often referred to by the trade name of the most commonly used support material, Sephadex. An adaptation of affinity chromatography is the use of immobilized enzymes.Column Chromatography is generally used to separate proteins during a preparation. However, it can also be used to analyze or characterize different materials and/or reactions, and it can even be used to study the kinetics of an enzyme reaction. Clearly, then, this is a broad and important topic of study.Let us start our conceptual introduction with the basic question of how to interpret data presented in the form of peaks. We have already seen this format in the amino acid analysis, and we will have to deal with the problems inherent in this form of presentation in many of the standard techniques. Our greatest specific problem is that in “real life” we rarely get perfect separations on columns.
eBook - ePub- Milan Popl, Jan Fahnrich, Vlastimil Tatar(Authors)
- 2018(Publication Date)
- CRC Press(Publisher)
3 ChromatographyPeople working with chromatography have yet to devise an appropriate and comprehensive definition of this method. Unfortunately, chromatography involves so many specific forms and procedures that no definition comes close. Therefore let us not belabor definition and emphasize instead the ability of chromatography to separate complex mixtures into discrete bands. Chromatography is, before anything else, a separation method, although for the absolute definition of separated components, it must be linked to other analytical methods, mostly spectroscopic.I. PRINCIPLES, CLASSIFICATION, AND NOMENCLATURE A. PrinciplesChromatography in principle is based on the equilibrium distribution of a sample between two phases. The phases are composed of compounds other than the sample components. The volumes of both phases are much larger than that of sample and therefore the distribution coefficients of individual substances remain essentially constant. The separation of a sample by chromatography is achieved by the distribution of substances between mobile and stationary phases. The substances which are held strongly in the mobile phase pass through the system more rapidly than those held strongly in the stationary phase. Thus, the velocity of a component’s movement through the system will depend on intermolecular forces holding the substance in the stationary phase or, more exactly, on the difference of forces between the solute molecules and the molecules of each phase. Because these forces for individual components are different, various substances will travel with different velocity, and in this way, they will be separated.B. ClassificationChromatographic methods may be classified according to (a) type of mobile phase with the subdivision due to stationary phase used, (b) method of sample movement through the bed, and (c) technique. The classification which would be based on the nature of intermolecular forces is not fully accepted because generally two or more kinds of forces are operating simultaneously.
eBook - ePub- John Kenkel(Author)
- 2013(Publication Date)
- CRC Press(Publisher)
12 . In this chapter, we introduce the general concepts of chromatography and give a perspective on its scope. Since there are many different classifications, this will include an organizational scheme covering the different types and configurations that exist.Chromatography is the separation of the components of a mixture based on the different degrees to which they interact with two separate material phases. The nature of the two phases and the kind of interaction can be varied, and this gives rise to the different “types” of chromatography, which will be described in the next section. One of the two phases is a moving phase (the “mobile” phase), whereas the other does not move (the “stationary” phase) (see Figure 10.1 ). The mixture to be separated is usually introduced into the mobile phase, which then is made to move or percolate through the stationary phase either by gravity or some other force. The components of the mixture are attracted to and slowed by the stationary phase to varying degrees, and as a result, they move along with the mobile phase at varying rates, and are thus separated. Figure 10.2 illustrates this concept.The mobile phase can be either a gas or a liquid, whereas the stationary phase can be either a liquid or solid. One classification scheme is based on the nature of the two phases. All techniques that utilize a gas for the mobile phase come under the heading of “gas chromatography” (GC). All techniques that utilize a liquid mobile phase come under the heading of “liquid chromatography” (LC). Additionally, we have gas–liquid chromatography (GLC), gas–solid chromatography (GSC), liquid–liquid chromatography (LLC), and liquid–solid chromatography (LSC) if we wish to stipulate the nature of the stationary phase as well as the mobile phase. It is more useful, however, to classify the techniques according to the nature of the interaction of the mixture components with the two phases. These classifications we refer to in this text as “types” of chromatography.
- Chavan, U D(Authors)
- 2021(Publication Date)
- Daya Publishing House(Publisher)
CHAPTER 8 Chromatography 1. CHROMATOGRAPHY Principles and Applications Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures . The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase . The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound’s partition coefficient result in differential retention on the stationary phase and thus changing the separation. Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for more advanced use (and is thus a form of purification ). Analytical chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive. History Thin layer chromatography is used to separate components of a plant extract, illustrating the experiment with plant pigments that gave chromatography its name. Chromatography was first employed in Russia by the Italian-born scientist Mikhail Tsvet in 1900. He continued to work with chromatography in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls . Since these components have different colours (green, orange, and yellow, respectively) they gave the technique its name. New types of chromatography developed during the 1930s and 1940s made the technique useful for many separation processes. Chromatography technique developed substantially as a result of the work of Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s.
eBook - PDFChromatography
Fundamentals and applications of chromatography and related differential migration methods - Part A: Fundamentals and techniques
- E. Heftmann(Author)
- 2004(Publication Date)
- Elsevier Science(Publisher)
Recommendations for the terminology of chromatography [18,19] and liquid-phase separations [20–22], including chiral chromatography [23], and supercritical-fluid chromatography [24] have been published by IUPAC. The historical development of HPLC has been covered in a number of articles and texts [25,26]. The wide range of applications of HPLC (covered in Part B of this book) derives from its versatility, as usually the only requirement for this method of analysis is that the analyte must be soluble in the mobile phase. HPLC can also handle a wide range of sample sizes, from preparative to trace levels, although this can vary with the detectability of the analyte (Sec. 2.4). 2.1.1 Modes of liquid chromatography Chromatography is defined as “…a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (the stationary phase), while the other (the mobile phase) moves in a definite Column Liquid Chromatography 97 direction” [18]. The dominance of liquid chromatography is due to its great versatility, as it encompasses a variety of separation modes, the principal modes being adsorption, partition, ion exchange and size exclusion. In adsorption chromatography, the analytes interact with a solid stationary surface and are displaced by competition with the eluent for active sites on the surface. The separation in partition chromatography results from a thermodynamic distribution between two liquid (or liquid-like) phases, whereas ion-exchange chromatography is primarily governed by ionic interactions between ionized analytes and an oppositely charged stationary-phase surface. In size exclusion chromatography the resistive force for separation is the physical size of the analytes, which determines their accessibility to differently sized pores in the stationary-phase material.
eBook - PDF- Haleem J. Issaq(Author)
- 2001(Publication Date)
- CRC Press(Publisher)
Classical liquid chromatography based on adsorption-desorption was essentially a nonlinear process where the time of retardation (what we call today the retention time) and the quantitative response depended on the position on the adsorption isotherm. Essentially it was a preparative technique: the aim was to obtain the components present in the sample in pure form, which then may be subject to further chemical or physical manipulations. In addition, as we have already pointed out, the columns were not reusable and the success of separation depended to a great extent on the skill of the operator who prepared the adsorbent and packed the columns. Classical chromatography was also a slow process: the mobile phase flow was mainly caused by gravity, although in some cases slight pressure was added to the head of the column to enhance the flow. However, even with these fairly simple systems, remarkable results were achieved. The next step in the evolution of chromatography further extended its range and even-tually revolutionized the field. This was the introduction of partitioning as the basis of separation. V. PARTITION CHROMATOGRAPHY Liquid partition chromatography was developed by A. J. P. Martin and R. L. M. Synge and first described in 1941 [20]. The thoughts that led to the invention of the technique are well documented [3b-c,2l,22]. They originally tried to separate monoamino monocarboxylic acids present in wool by counter-current extraction but had great practical difficulties with the tech-nique. Eventually Archer Martin had the brilliant idea to fix one of the solvents and move only the other. In their first work, water was used as the liquid stationary phase (the fixed phase), silica gel as the support of it, and chloroform, containing 0.5% alcohol, as the mobile phase.
eBook - PDF- Douglas Skoog, F. Holler, Stanley Crouch, , Douglas Skoog, F. Holler, Stanley Crouch(Authors)
- 2017(Publication Date)
- Cengage Learning EMEA(Publisher)
The varieties include (1) partition , or liquid-liquid, chromatography ; (2) adsorption , or liquid-solid chromatography ; (3) ion-exchange , or ion, chromatography ; (4) size-exclusion chromatography ; (5) affinity chromatography , and (6) chiral chromatography . Most of this chapter deals with column applications of these important types of chromatography. The final section, however, presents a brief description of planar liquid chromatography because this technique provides a simple and inexpensive way of determining likely optimal conditions for column separations. 1 For detailed discussions of HPLC, see L. R. Snyder, J. J. Kirkland, and J. W. Dolan, Introduction to Modern Liquid Chromatography , 3rd ed., Hoboken, NJ: Wiley, 2010; V. Meyer, Practical High-Performance Liquid Chromatography , 5th ed., Chichester, UK: Wiley 2010. 2 S. Fekete et al., Trends Anal. Chem ., 2014 , 63 , 2, DOI : 10.1016/j.trac.2014.08.007. Copyright 2018 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part. WCN 02-300 747 28B Column Efficiency in LC 28A SCOPE OF HPLC LC is the most widely used of all of the analytical separation techniques. The reasons for the popularity of the method are its sensitivity, its ready adaptability to accurate quantitative deter-minations, its ease of automation, its suitability for separating nonvolatile species or thermally fragile ones, and above all, its widespread applicability to substances that are important to industry, to many fields of science, and to the public. Examples of such materials include amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, drugs, terpenoids, pesticides, antibiotics, steroids, metal-organic species, and a variety of inor-ganic substances. Figure 28-1 reveals that the various liquid chromato-graphic procedures are complementary in their application.
eBook - PDF- Erich Heftmann(Author)
- 1991(Publication Date)
- Elsevier Science(Publisher)
Column switching is a very versatile, powerful technique that has found application in both LC and GC. In most cases, the physical system is more complex than shown in Fig. 1.21a. Often two separate chromatographic systems are coupled together, and the action of the valve is usually controlled automatically (by a timer or computer). There are many possible applications of this procedure, some of which are summarized in Table 1.7. (For an additional discussion of column switching, see Refs. 26 and 109-124.) 1.10 LARGE-MOLECULE SEPARATIONS Liquid chromatography has played an important role in the separation and analysis of large-molecule samples, including both synthetic and natural polymers (cf. Chapters 14, 17, and 22). In some respects, the chromatography of these compounds appears to differ from that of small molecules, i.e., compounds with molecular weights < 1000. The special characteristics of large-molecule chromatography therefore merit some discus- sion. References on p. A65 A56 1.10.1 Unique features of large molecules 1.10.1.1 Molecular size The molecular weight range of the compounds under discussion is roughly lo3 to lo6. The diffusion coefficient, Dm, decreases with increasing solute molecular weight, so that two orders of magnitude can separate the diffusion rates of small and large molecules in the mobile phase outside of the particles. This is illustrated in Fig. 1.22 by the solute molecules myoglobin (a protein) and a small, substituted benzene derivative. The arrows adjacent to each molecule represent the relative distance each molecule can diffuse in a given time. As seen in Fig. 1.17, this difference in solute diffusion rates means that the separation of large-molecule samples generally is carried out at much higher reduced velocities than that of small-molecule solutes, with a corresponding increase in h and decrease in the platenumber, N; i.e., poorer separation.
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