Basic Principles Of Chromatography

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27chapterBasic Principlesof ChromatographyBaraem Ismail Department of Food Science and Nutrition, University of Minnesota,St. Paul, MN 55108-6099, USAbismailm@umn.eduandS. Suzanne NielsenDepartment of Food Science, Purdue University,West Lafayette, IN 47907-2009, USAnielsens@purdue.edu27.1 Introduction 47527.2 Extraction 47527.2.1 Batch Extraction 47527.2.2 Continuous Extraction 47527.2.3 Countercurrent Extraction 47527.3 Chromatography 47527.3.1 Historical Perspective 47527.3.2 General Terminology 476S.S. Nielsen, Food Analysis, Food Science Texts Series, DOI 10.1007/978-1-4419-1478-1 27,c Springer Science Business Media, LLC 2010 473

47427.3.3 Gas Chromatography 47627.3.4 Liquid Chromatography 47727.3.4.1 Paper Chromatography 47727.3.4.2 Thin-LayerChromatography 47827. GeneralProcedures 47827. Factors AffectingThin-LayerSeparations 47827.3.4.3 Column LiquidChromatography 47927.3.5 Supercritical Fluid Chromatography 48027.4 Physicochemical Principles of ChromatographicSeparation 48127.4.1 Adsorption (Liquid–Solid)Chromatography 48127.4.2 Partition (Liquid–Liquid)Chromatography 48227.4.2.1 Introduction 48227.4.2.2 Coated Supports 48327.4.2.3 Bonded Supports 483Part V 27.527.627.727.827.9Chromatography27.4.3 Ion-Exchange Chromatography 48327.4.4 Size-Exclusion Chromatography 48527.4.5 Affinity Chromatography 488Analysis of Chromatographic Peaks 48927.5.1 Separation and Resolution 49027.5.1.1 Developing a Separation 49027.5.1.2 ChromatographicResolution 49127. Introduction 49127. ColumnEfficiency 49227. ColumnSelectivity 49427. Column CapacityFactor 49427.5.2 Qualitative Analysis 49527.5.3 Quantitative Analysis 495Summary 496Study Questions 497Acknowledgments 498References 498

Chapter 27 475Basic Principles of Chromatography27.1 INTRODUCTION27.2.2 Continuous ExtractionChromatography has a great impact on all areas ofanalysis and, therefore, on the progress of sciencein general. Chromatography differs from other methods of separation in that a wide variety of materials,equipment, and techniques can be used. [Readers arereferred to references (1–19) for general and specificinformation on chromatography.]. This chapter willfocus on the principles of chromatography, mainlyliquid chromatography (LC). Detailed principles andapplications of gas chromatography (GC) will bediscussed in Chap. 29. In view of its widespreaduse and applications, high-performance liquid chromatography (HPLC) will be discussed in a separatechapter (Chap. 28). The general principles of extraction are first described as a basis for understandingchromatography.27.2 EXTRACTIONIn its simplest form, extraction refers to the transfer of a solute from one liquid phase to another.Extraction in myriad forms is integral to food analysis – whether used for preliminary sample cleanup,concentration of the component of interest, or as theactual means of analysis. Extractions may be categorized as batch, continuous, or countercurrent processes. (Various extraction procedures are discussed indetail in other chapters: traditional solvent extractionin Chaps. 8, 18, and 29; accelerated solvent extractionin Chap. 18; solid-phase extraction in Chaps. 18 and29; and solid-phase microextraction and microwaveassisted solvent extraction in Chap. 18).27.2.1 Batch ExtractionIn batch extraction the solute is extracted from one solvent by shaking it with a second, immiscible solvent.The solute partitions, or distributes, itself between thetwo phases and, when equilibrium has been reached,the partition coefficient, K, is a constant.K Concentration of solute in phase lConcentration of solute in phase 2[1]After shaking, the phases are allowed to separate,and the layer containing the desired constituent isremoved, for example, in a separatory funnel. Inbatch extraction, it is often difficult to obtain a cleanseparation of phases, owing to emulsion formation.Moreover, partition implies that a single extraction isusually incomplete.Continuous liquid–liquid extraction requires specialapparatus, but is more efficient than batch separation. One example is the use of a Soxhlet extractor forextracting materials from solids. Solvent is recycled sothat the solid is repeatedly extracted with fresh solvent. Other pieces of equipment have been designedfor the continuous extraction of substances from liquids, and different extractors are used for solvents thatare heavier or lighter than water.27.2.3 Countercurrent ExtractionCountercurrent distribution refers to a serial extraction process. It separates two or more solutes with different partition coefficients from each other by a seriesof partitions between two immiscible liquid phases.Liquid–liquid partition chromatography (Sect. 27.4.2),also known as countercurrent chromatography, is adirect extension of countercurrent extraction. Yearsago the countercurrent extraction was done with a“Craig apparatus” consisting of a series of glass tubesdesigned such that the lighter liquid phase (mobilephase) was transferred from one tube to the next,while the heavy phase (stationary phase) remainedin the first tube (4). The liquid–liquid extractions tookplace simultaneously in all tubes of the apparatus,which was usually driven electromechanically. Eachtube in which a complete equilibration took placecorresponded to one theoretical plate of the chromatographic column (refer to Sect. The greaterthe difference in the partition coefficients of varioussubstances, the better was the separation. A muchlarger number of tubes was required to separate mixtures of substances with close partition coefficients,which made this type of countercurrent extractionvery tedious. Modern liquid–liquid partition chromatography (Sect. 27.4.2) that developed from thisconcept is much more efficient and convenient.27.3 CHROMATOGRAPHY27.3.1 Historical PerspectiveModern chromatography originated in the late nineteenth and early twentieth centuries from independentwork by David T. Day, a distinguished Americangeologist and mining engineer, and Mikhail Tsvet,a Russian botanist. Day developed procedures forfractionating crude petroleum by passing it throughFuller’s earth, and Tsvet used a column packed withchalk to separate leaf pigments into colored bands.

476Part V Because Tsvet recognized and correctly interpretedthe chromatographic processes and named the phenomenon chromatography, he is generally creditedwith its discovery.After languishing in oblivion for years, chromatography began to evolve in the 1940s due to thedevelopment of column partition chromatography byMartin and Synge and the invention of paper chromatography. The first publication on GC appeared in1952. By the late 1960s, GC, because of its importance to the petroleum industry, had developed intoa sophisticated instrumental technique, which wasthe first instrumental chromatography to be availablecommercially. Since early applications in the mid1960s, HPLC, profiting from the theoretical and instrumental advances of GC, has extended the area ofliquid chromatography into an equally sophisticatedand useful method. SFC, first demonstrated in 1962,is finally gaining popularity. Modern chromatographictechniques, including automated systems, are widelyutilized in the characterization and quality control offood raw materials and food products.27.3.2 General TerminologyChromatography is a general term applied to a widevariety of separation techniques based on the partitioning or distribution of a sample (solute) betweena moving or mobile phase and a fixed or stationaryphase. Chromatography may be viewed as a series27-1figureChromatographyof equilibrations between the mobile and stationaryphase. The relative interaction of a solute with thesetwo phases is described by the partition (K) or distribution (D) coefficient (ratio of concentration of solutein stationary phase to concentration of solute in mobilephase). The mobile phase may be either a gas (GC) orliquid (LC) or a supercritical fluid (SFC). The stationary phase may be a liquid or, more usually, a solid. Thefield of chromatography can be subdivided accordingto the various techniques applied (Fig 27-1), or according to the physicochemical principles involved in theseparation. Table 27-1 summarizes some of the chromatographic procedures or methods that have beendeveloped on the basis of different mobile–stationaryphase combinations. Inasmuch as the nature of interactions between solute molecules and the mobile orstationary phases differ, these methods have the abilityto separate different kinds of molecules. (The reader isurged to review Table 27-1 again after having read thischapter.)27.3.3 Gas ChromatographyGas chromatography is a column chromatographytechnique, in which the mobile phase is gas and thestationary phase is either an immobilized liquid or asolid packed in a closed tube. GC is used to separatethermally stable volatile components of a mixture. Gaschromatography, specifically gas–liquid chromatography, involves vaporizing a sample and injecting it ontoA scheme for subdividing the field of chromatography, according to various applied techniques.

Chapter 27 477Basic Principles of Chromatography27-1tableCharacteristics of Different Chromatographic MethodsMethodGas–liquid chromatographyGas–solid chromatographySupercritical mal-phase chromatographyIon-exchange chromatographySize-exclusion yAffinity chromatographyMobile/Stationary PhaseRetention Varies withGas/liquidGas/solidSupercritical fluid/solidMolecular size/polarityMolecular size/polarityMolecular size/polarityPolar liquid/nonpolar liquid or solidMolecular size/polarityLess polar liquid/more polar liquidor solidPolar liquid/ionic solidLiquid/solidPolar liquid/nonpolar liquid or solidMolecular size/polarityMolecular chargeMolecular sizeMolecular size/polarityWater/binding sitesSpecific structureReprinted from (8), p. A21, with kind permission from Elsevier Science-NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam,The Netherlands.the head of the column. Under a controlled temperature gradient, the sample is transported through thecolumn by the flow of an inert, gaseous mobile phase.Volatiles are then separated based on several properties, including boiling point, molecular size, andpolarity. Physiochemical principles of separation arecovered in Sect. 27.4. However, details of the chromatographic theory of separation as it applies specifically to GC, as well as detection and instrumentationof GC, are detailed in Chap. Liquid ChromatographyThere are several liquid chromatography techniquesapplied in food analysis, namely paper chromatography, thin layer chromatography (TLC) (both of thesetechniques may be referred to as planar chromatography), and column liquid chromatography, all of whichinvolve a liquid mobile phase and either a solid or aliquid stationary phase. However, the physical form ofthe stationary phase is quite different in each case. Separation of the solutes is based on their physicochemicalinteractions with the two phases, which is discussed inSect. Paper ChromatographyPaper chromatography was introduced in 1944. Inpaper chromatography the stationary phase and themobile phase are both liquid (partition chromatography, see Sect. 27.4.2). Paper generally serves asa support for the liquid stationary phase. The dissolved sample is applied as a small spot or streakone half inch or more from the edge of a strip orsquare of filter paper (usually cellulose), which isthen allowed to dry. The dry strip is suspended ina closed container in which the atmosphere is saturated with the developing solvent (mobile phase), andthe paper chromatogram is developed. The end closerto the sample is placed in contact with the solvent,which then travels up or down the paper by capillaryaction (depending on whether ascending or descending development is used), separating the sample components in the process. When the solvent front hastraveled the length of the paper, the strip is removedfrom the developing chamber and the separated zonesare detected by an appropriate method.The stationary phase in paper partition chromatography is usually water. However, the supportmay be impregnated with a nonpolar organic solventand developed with water or other polar solvents orwater (reversed-phase paper chromatography). In thecase of complex sample mixtures, a two-dimensionaltechnique may be used. The sample is spotted in onecorner of a square sheet of paper, and one solvent isused to develop the paper in one direction. The chromatogram is then dried, turned 90 , and developedagain, using a second solvent of different polarity.Another means of improving resolution is the useof ion-exchange (Sect. 27.4.3) papers, that is, paperthat has been impregnated with ion-exchange resinor paper, with derivatized cellulose hydroxyl groups(with acidic or basic moieties).In paper and thin-layer chromatography, components of a mixture are characterized by their relativemobility (Rf ) value, where:Rf Distance moved by componentDistance moved by solvent[2]Unfortunately, Rf values are not always constant fora given solute/sorbent/solvent, but depend on many

478factors, such as the quality of the stationary phase,layer thickness, humidity, development distance, andtemperature. Thin-Layer ChromatographyThin-layer chromatography (TLC), first described in1938, has largely replaced paper chromatographybecause it is faster, more sensitive, and more reproducible. The resolution in TLC is greater than in paperchromatography because the particles on the plate aresmaller and more regular than paper fibers. Experimental conditions can be easily varied to achieveseparation and can be scaled up for use in columnchromatography, although thin-layer and column procedures are not necessarily interchangeable, due todifferences such as the use of binders with TLC plates,vapor-phase equilibria in a TLC tank, etc. Thereare several distinct advantages to TLC: high samplethroughput, low cost, the possibility to analyze several samples and standards simultaneously, minimalsample preparation, and that a plate may be stored forlater identification and quantification.TLC is applied in many fields, including environmental, clinical, forensic, pharmaceutical, food,flavors, and cosmetics. Within the food industry, TLCmay be used for quality control. For example, cornand peanuts are tested for aflatoxins/mycotoxins priorto their processing into corn meal and peanut butter, respectively. Applications of TLC to the analysisof a variety of compounds, including lipids, carbohydrates, vitamins, amino acids, and natural pigments,are discussed in reference (5). General Procedures TLC utilizes a thin(ca. 250 µm thick) layer of sorbent or stationary phasebound to an inert support in a planar configuration. The support is often a glass plate (traditionally,20 cm 20 cm), but plastic sheets and aluminum foilalso are used. Precoated plates, of different layerthicknesses, are commercially available in a wide variety of sorbents, including chemically modified silicas. Four frequently used TLC sorbents are silica gel,alumina, diatomaceous earth, and cellulose. Modified silicas for TLC may contain polar or nonpolar groups, so both normal and reversed-phase (seeSect. thin-layer separations may be carriedout. High-performance thin-layer chromatography(HPTLC) simply refers to TLC performed using platescoated with smaller, more uniform particles. Thispermits better separations in shorter times.If adsorption TLC is to be performed, the sorbent is first activated by drying for a specified timeand temperature. Sample (in carrier solvent) is appliedas a spot or streak 1–2 cm from one end of the plate.Part V ChromatographyAfter evaporation of the carrier solvent, the TLCplate is placed in a closed developing chamber withthe end of the plate nearest the spot in the solventat the bottom of the chamber. Traditionally, solventmigrates up the plate (ascending development) bycapillary action and sample components are separated. After the TLC plate has been removed from thechamber and solvent allowed to evaporate, the separated bands are made visible or detected by othermeans. Specific chemical reactions (derivatization),which may be carried out either before or after chromatography, often are used for this purpose. Twoexamples are reaction with sulfuric acid to producea dark charred area (a destructive chemical method)and the use of iodine vapor to form a colored complex (a nondestructive method inasmuch as the colored complex is usually not permanent). Commonphysical detection methods include the measurement of absorbed or emitted electromagnetic radiation(e.g., fluorescence) by means of autoradiography andthe measurement of β-radiation from radioactivelylabeled compounds. Biological methods or biochemical inhibition tests can be used to detect toxicologicallyactive substances. An example is measuring the inhibition of cholinesterase activity by organophosphatepesticides.Quantitative evaluation of thin-layer chromatograms may be performed (1) in situ (directly onthe layer) by using a densitometer or (2) after scraping a zone off the plate, eluting compound from thesorbent, and analyzing the resultant solution (e.g., byliquid scintillation counting). Factors Affecting Thin-Layer SeparationsIn both planar and column liquid chromatography,the nature of the compounds to be separated determines what type of stationary phase is used. Separation can occur by adsorption, partition, ion-exchange,size-exclusion, or multiple mechanisms (Sect. 27.4).Table 27-2 lists the separation mechanisms involved insome typical applications on common TLC sorbents.Solvents for TLC separations are selected for specific chemical characteristics and solvent strength(a measure of interaction between solvent and sorbent; see Sect. 27.4.1). In simple adsorption TLC, thehigher the solvent strength, the greater the Rf valueof the solute. An Rf value of 0.3–0.7 is typical. Mobilephases have been developed for the separation of various compound classes on the different sorbents [seeTable 7.1 in reference (15)].In addition to the sorbent and solvent, severalother factors must be considered when performing planar chromatography. These include the typeof developing chamber used, vapor phase conditions (saturated vs. unsaturated), development mode

Chapter 27 479Basic Principles of Chromatography27-2tableThin-Layer Chromatography Sorbents and Mode of SeparationSorbentChromatographic MechanismSilica gelAdsorptionSilica gel RPReversed phaseCellulose, kieselguhrPartitionAluminum oxideAdsorptionPEI celluloseaIon exchangeMagnesium silicateAdsorptionTypical ApplicationSteroids, amino acids, alcohols,hydrocarbons, lipids, aflatoxins, bileacids, vitamins, alkaloidsFatty acids, vitamins, steroids, hormones,carotenoidsCarbohydrates, sugars, alcohols, aminoacids, carboxylic acids, fatty acidsAmines, alcohols, steroids, lipids,aflatoxins, bile acids, vitamins, alkaloidsNucleic acids, nucleotides, nucleosides,purines, pyrimidinesSteroids, pesticides, lipids, alkaloidsReprinted from (15) by permission of Wiley, New York.a PEI cellulose refers to cellulose derivatized with polyethyleneimine (PEI).(ascending, descending, horizontal, radial, etc.), anddevelopment distance. For additional reading refer toreferences (5), (7), and (16). Column Liquid ChromatographyColumn chromatography is the most useful methodof separating compounds in a mixture. Fractionationof solutes occurs as a result of differential migrationthrough a closed tube of stationary phase, and analytescan be monitored while the separation is in progress.In column liquid chromatography, the mobile phaseis liquid and the stationary phase can be either solidor liquid supported by an inert solid. A system forlow-pressure (i.e., performed at or near atmosphericpressure) column liquid chromatography is illustratedin Fig. 27-2.Having selected a stationary and mobile phasesuitable for the separation problem at hand, the analyst must first prepare the stationary phase (resin, gel,or packing material) for use according to the supplier’s instructions. (For example, the stationary phaseoften must be hydrated or preswelled in the mobilephase). The prepared stationary phase then is packedinto a column (usually glass), the length and diameterof which are determined by the amount of sample tobe loaded, the separation mode to be used, and thedegree of resolution required. Longer and narrowercolumns usually enhance resolution and separation.Adsorption columns may be either dry or wet packed;other types of columns are wet packed. The most common technique for wet packing involves making aslurry of the adsorbent with the solvent and pouringthis into the column. As the sorbent settles, excesssolvent is drained off and additional slurry is added.This process is repeated until the desired bed heightis obtained. (There is a certain art to pouring uniformcolumns and no attempt is made to give details here.)If the packing solvent is different from the initial eluting solvent, the column must be thoroughly washed(equilibrated) with the starting mobile phase.The sample to be fractionated, dissolved in a minimum volume of mobile phase, is applied in a layerat the top (or head) of the column. Classical or lowpressure chromatography utilizes only gravity flow ora peristaltic pump to maintain a flow of mobile phase(eluent or eluting solvent) through the column. In thecase of a gravity-fed system, eluent is simply siphonedfrom a reservoir into the column. The flow rate is governed by the hydrostatic pressure, measured as thedistance between the level of liquid in the reservoirand the level of the column outlet. If eluent is fed to thecolumn by a peristaltic pump (see Fig. 27-2), then theflow rate is determined by the pump speed and, thus,regulation of hydrostatic pressure is not necessary.The process of passing the mobile phase throughthe column is called elution, and the portion thatemerges from the outlet end of the column is sometimes called the eluate (or effluent). Elution may beisocratic (constant mobile-phase composition) or agradient (changing the mobile phase, e.g., increasing solvent strength or pH) during elution in orderto enhance resolution and decrease analysis time (seealso Sect. 27.5.1). As elution proceeds, components ofthe sample are selectively retarded by the stationaryphase based on the strength of interaction with thestationary phase, and thus they are eluted at different times.The column eluate may be directed through adetector and then into tubes, changed at intervals by

48027-2figurePart V ChromatographyA system for low-pressure column liquid chromatography. In this diagram, the column effluent is being splitbetween two detectors in order to monitor both enzyme activity (at Right) and UV absorption (at Left). The twotracings can be recorded simultaneously by using a dual-pen recorder. [Adapted from (12), with permission.]a fraction collector. The detector response, in theform of an electrical signal, may be recorded (thechromatogram), using either a chart recorder or a computerized software, and used for qualitative or quantitative analysis, as discussed in more detail later. Thefraction collector may be set to collect eluate at specified time intervals or after a certain volume or numberof drops has been collected. Components of the sample that have been chromatographically separated andcollected then can be further analyzed as needed.27.3.5 Supercritical Fluid ChromatographySFC refers to chromatography performed above thecritical pressure (Pc ) and critical temperature (Tc ) ofthe mobile phase. A supercritical fluid (or compressedgas) is neither a liquid nor a typical gas. The combination of Pc and Tc is known as the critical point.A supercritical fluid can be formed from a conventional gas by increasing the pressure, or from a conventional liquid by raising the temperature. Carbondioxide frequently is used as a mobile phase for SFC;however, it is not a good solvent for polar and highmolecular-weight compounds. A small amount of apolar, organic solvent such as methanol can be addedto a nonpolar supercritical fluid to enhance solute solubility, improve peak shape, and alter selectivity. Othersupercritical fluids that have been used in food applications include nitrous oxide, trifluoromethane, sulfurhexafluoride, pentane, and ammonia.Supercritical fluids confer chromatographic properties intermediate to LC and GC. The high diffusivity and low viscosity of supercritical fluids meandecreased analysis times and improved resolutioncompared to LC. SFC offers a wide range of selectivity (Sect. 27.5.2) adjustment, by changes in pressureand temperature as well as changes in mobile phasecomposition and the stationary phase. In addition,SFC makes possible the separation of nonvolatile,thermally labile compounds that are not amenableto GC.SFC can be performed using either packedcolumns or capillaries. Packed column materials aresimilar to those used for HPLC. Small particle, porous,high surface area, hydrated silica may serve as thestationary phase itself, or simply as a support for abonded stationary phase (Chap. 28). Polymer-basedpacking has been used, but is less satisfactory owingto long solute retention times. Capillaries are generallycoated with a polysiloxane ( Si O Si) film, whichis then cross-linked to form a polymeric stationaryphase that cannot be washed off by the mobile phase.Polysiloxanes containing different functional groups,such as methyl, phenyl, or cyano, may be used to

Chapter 27 481Basic Principles of Chromatographyvary the polarity of this stationary phase. Instrumentation for packed column SFC is similar to that usedfor HPLC with one major difference: A back pressure regulator is used to control the outlet pressureof the system. Without this device, the fluid wouldexpand to a low-pressure, low-density gas. Besides theadvantages of decreased analysis time and improvedresolution, SFC offers the possibility to use a widevariety of detectors, including those designed for GC.SFC has been used primarily for nonpolar compounds. Fats, oils, and other lipids are compounds towhich SFC is increasingly applied. For example, theRnoncaloric fat substitute, Olestra , was characterizedby SFC-MS (mass spectroscopy). Other researchershave used SFC to detect pesticide residues, study thermally labile compounds from members of the Alliumgenus, fractionate citrus essential oils, and characterize compounds extracted from microwave packaging(3). Borch-Jensen and Mollerup (1) highlighted the useof packed column and capillary SFC for the analysis offood and natural products, especially fatty acids andtheir derivatives, glycerides, waxes, sterols, fat-solublevitamins, carotenoids, and phospholipids.27.4 PHYSICOCHEMICAL PRINCIPLESOF CHROMATOGRAPHIC SEPARATIONSeveral physicochemical principles (illustrated inFig. 27-3) are involved in chromatography mechanisms employed to separate or fractionate variouscompounds of interest, regardless of the specific techniques applied (discussed in Sect. 27.3). The mechanisms described below apply mainly to liquid chromatography; GC mechanisms will be detailed inChap. 29. Although it is more convenient to describeeach of these phenomena separately, it must be emphasized that more than one mechanism may be involvedin a given fractionation. For example, many cases ofpartition chromatography also involve adsorption.27.4.1 Adsorption (Liquid–Solid)ChromatographyAdsorption chromatography is the oldest form ofchromatography, originated with Tsvet in 1903 in theexperiments that spawned modern chromatography.In this chromatographic mode, the stationary phaseis a finely divided solid to maximize the surface area.The stationary phase (adsorbent) is chosen to permit differential interaction with the components ofthe sample to be resolved. The intermolecular forcesthought to be primarily responsible for chromatographic adsorption include the following: Van der Waals forcesElectrostatic forcesHydrogen bondsHydrophobic interactionsSites available for interaction with any given substanceare heterogeneous. Binding sites with greater affinities,the most active sites, tend to be populated first, so thatadditional solutes are less firmly bound. The net resultis that adsorption is a concentration-dependent process, and the adsorption coefficient is not a constant(in contrast to the partition coefficient). Sample loadsexceeding the adsorptive capacity of the stationaryphase will result in relatively poor separation.Classic adsorption chromatography utilizes silica (slightly acidic), alumina (slightly basic), charcoal(nonpolar), or a few other materials as the stationary phase. Both silica and alumina possess surfacehydroxyl groups, and Lewis acid-type interactionsdetermine their adsorption characteristics. The elutionorder of compounds from these adsorptive stationaryphases can often be predicted on the basis of their relative polarities (Table 27-3). Compounds with the mostpolar functional groups are retained most stronglyon polar adsorbents and, therefore, are eluted last.Nonpolar solutes are eluted first.One model proposed to explain the mechanismof liquid–solid chromatography is that solute andsolvent molecules are competing for active sites onthe adsorbent. Thus, as relative adsorption of themobile phase increases, adsorption of the solute mustdecrease. Solvents can be rated in order of theirstrength of adsorption on a particular adsorbent, suchas silica. Such a solvent strength (or polarity) scaleis called a eluotropic series. A eluotropic series foralumina is listed in Table 27-4. Silica has a similarrank ordering. Once an adsorbent has been chosen,solvents can be selected from the eluotropic seriesfor that adsorbent. Mobile phase polarity can b

Chromatography 481 27.4.2 Partition (Liquid–Liquid) Chromatography 482 Introduction 482 Coated Supports 483 Bonded Supports 483 27.4.3 Ion-Exchange Chromatography 483 27.4.4 Size-Exclusion Chromatography 485 27.4.5 Affinity Chromatography 488 27.5 Analys

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