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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 47827.3.4.2.1 GeneralProcedures 47827.3.4.2.2 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 49127.5.1.2.1 Introduction 49127.5.1.2.2 ColumnEfficiency 49227.5.1.2.3 ColumnSelectivity 49427.5.1.2.4 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. 27.5.2.2.1). 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.