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Basic Principles of Chromatography 27 chapter Basic Principles of Chromatography Baraem Ismail ∗ Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN 55108-6099, USA [email protected] and S. Suzanne Nielsen Department of Food Science, Purdue University, West Lafayette, IN 47907-2009, USA [email protected] 27.1 Introduction 475 27.2.3 Countercurrent Extraction 475 27.2 Extraction 475 27.3 Chromatography 475 27.2.1 Batch Extraction 475 27.3.1 Historical Perspective 475 27.2.2 Continuous Extraction 475 27.3.2 General Terminology 476 S.S. Nielsen, Food Analysis, Food Science Texts Series, DOI 10.1007/978-1-4419-1478-1_27, 473 °c Springer Science+Business Media, LLC 2010 474 Part V • Chromatography 27.3.3 Gas Chromatography 476 27.4.3 Ion-Exchange Chromatography 483 27.3.4 Liquid Chromatography 477 27.4.4 Size-Exclusion Chromatography 485 27.3.4.1 Paper Chromatography 477 27.4.5 Affinity Chromatography 488 27.3.4.2 Thin-Layer 27.5 Analysis of Chromatographic Peaks 489 Chromatography 478 27.5.1 Separation and Resolution 490 27.3.4.2.1 General 27.5.1.1 Developing a Separation 490 Procedures 478 27.5.1.2 Chromatographic 27.3.4.2.2 Factors Affecting Resolution 491 Thin-Layer 27.5.1.2.1 Introduction 491 Separations 478 27.5.1.2.2 Column 27.3.4.3 Column Liquid Efficiency 492 Chromatography 479 27.5.1.2.3 Column 27.3.5 Supercritical Fluid Chromatography 480 Selectivity 494 27.4 Physicochemical Principles of Chromatographic 27.5.1.2.4 Column Capacity Separation 481 Factor 494 27.4.1 Adsorption (Liquid–Solid) 27.5.2 Qualitative Analysis 495 Chromatography 481 27.5.3 Quantitative Analysis 495 27.4.2 Partition (Liquid–Liquid) 27.6 Summary 496 Chromatography 482 27.7 Study Questions 497 27.4.2.1 Introduction 482 27.8 Acknowledgments 498 27.4.2.2 Coated Supports 483 27.9 References 498 27.4.2.3 Bonded Supports 483 Chapter 27 • Basic Principles of Chromatography 475 27.1 INTRODUCTION 27.2.2 Continuous Extraction Continuous liquid–liquid extraction requires special Chromatography has a great impact on all areas of apparatus, but is more efficient than batch separa- analysis and, therefore, on the progress of science tion. One example is the use of a Soxhlet extractor for in general. Chromatography differs from other meth- extracting materials from solids. Solvent is recycled so ods of separation in that a wide variety of materials, that the solid is repeatedly extracted with fresh sol- equipment, and techniques can be used. [Readers are vent. Other pieces of equipment have been designed referred to references ( 1–19 ) for general and specific for the continuous extraction of substances from liq- information on chromatography.]. This chapter will uids, and different extractors are used for solvents that focus on the principles of chromatography, mainly are heavier or lighter than water. liquid chromatography (LC). Detailed principles and applications of gas chromatography (GC) will be discussed in Chap. 29. In view of its widespread 27.2.3 Countercurrent Extraction use and applications, high-performance liquid chro- matography (HPLC) will be discussed in a separate Countercurrent distribution refers to a serial extrac- chapter (Chap. 28). The general principles of extrac- tion process. It separates two or more solutes with dif- tion are first described as a basis for understanding ferent partition coefficients from each other by a series chromatography. of partitions between two immiscible liquid phases. Liquid–liquid partition chromatography (Sect. 27.4.2 ), also known as countercurrent chromatography, is a 27.2 EXTRACTION direct extension of countercurrent extraction. Years ago the countercurrent extraction was done with a In its simplest form, extraction refers to the trans- “Craig apparatus” consisting of a series of glass tubes fer of a solute from one liquid phase to another. designed such that the lighter liquid phase ( mobile Extraction in myriad forms is integral to food anal- phase) was transferred from one tube to the next, ysis – whether used for preliminary sample cleanup, while the heavy phase ( stationary phase) remained concentration of the component of interest, or as the in the first tube ( 4). The liquid–liquid extractions took actual means of analysis. Extractions may be catego- place simultaneously in all tubes of the apparatus, rized as batch , continuous , or countercurrent pro- which was usually driven electromechanically. Each cesses. (Various extraction procedures are discussed in tube in which a complete equilibration took place detail in other chapters: traditional solvent extraction corresponded to one theoretical plate of the chromato- in Chaps. 8, 18 , and 29; accelerated solvent extraction graphic column (refer to Sect. 27.5.2.2.1). The greater in Chap. 18 ; solid-phase extraction in Chaps. 18 and the difference in the partition coefficients of various 29 ; and solid-phase microextraction and microwave- substances, the better was the separation. A much assisted solvent extraction in Chap. 18 ). larger number of tubes was required to separate mix- tures of substances with close partition coefficients, 27.2.1 Batch Extraction which made this type of countercurrent extraction very tedious. Modern liquid–liquid partition chro- In batch extraction the solute is extracted from one sol- matography (Sect. 27.4.2 ) that developed from this vent by shaking it with a second, immiscible solvent. concept is much more efficient and convenient. The solute partitions , or distributes, itself between the two phases and, when equilibrium has been reached, the partition coefficient , K, is a constant. 27.3 CHROMATOGRAPHY Concentration of solute in phase l K = [1] 27.3.1 Historical Perspective Concentration of solute in phase 2 Modern chromatography originated in the late nine- After shaking, the phases are allowed to separate, teenth and early twentieth centuries from independent and the layer containing the desired constituent is work by David T. Day, a distinguished American removed, for example, in a separatory funnel. In geologist and mining engineer, and Mikhail Tsvet, batch extraction, it is often difficult to obtain a clean a Russian botanist. Day developed procedures for separation of phases, owing to emulsion formation. fractionating crude petroleum by passing it through Moreover, partition implies that a single extraction is Fuller’s earth, and Tsvet used a column packed with usually incomplete. chalk to separate leaf pigments into colored bands. 476 Part V • Chromatography Because Tsvet recognized and correctly interpreted of equilibrations between the mobile and stationary the chromatographic processes and named the phe- phase. The relative interaction of a solute with these nomenon chromatography , he is generally credited two phases is described by the partition (K) or distri- with its discovery. bution (D) coefficient (ratio of concentration of solute After languishing in oblivion for years, chro- in stationary phase to concentration of solute in mobile matography began to evolve in the 1940s due to the phase). The mobile phase may be either a gas (GC) or development of column partition chromatography by liquid (LC) or a supercritical fluid (SFC). The station- Martin and Synge and the invention of paper chro- ary phase may be a liquid or, more usually, a solid. The matography. The first publication on GC appeared in field of chromatography can be subdivided according 1952. By the late 1960s, GC, because of its impor- to the various techniques applied (Fig 27-1 ), or accord- tance to the petroleum industry, had developed into ing to the physicochemical principles involved in the a sophisticated instrumental technique, which was separation. Table 27-1 summarizes some of the chro- the first instrumental chromatography to be available matographic procedures or methods that have been commercially. Since early applications in the mid- developed on the basis of different mobile–stationary 1960s, HPLC, profiting from the theoretical and instru- phase combinations. Inasmuch as the nature of inter- mental advances of GC, has extended the area of actions between solute molecules and the mobile or liquid chromatography into an equally sophisticated stationary phases differ, these methods have the ability and useful method. SFC, first demonstrated in 1962, to separate different kinds of molecules. (The reader is is finally gaining popularity. Modern chromatographic urged to review Table 27-1 again after having read this techniques, including automated systems, are widely chapter.) utilized in the characterization and quality control of food raw materials and food products. 27.3.3 Gas Chromatography 27.3.2 General Terminology Gas chromatography is a column chromatography technique, in which the mobile phase is gas and the Chromatography is a general term applied to a wide stationary phase is either an immobilized liquid or a variety of separation techniques based on the parti- solid packed in a closed tube. GC is used to separate tioning or distribution of a sample ( solute) between thermally stable volatile components of a mixture. Gas a moving or mobile phase and a fixed or stationary chromatography, specifically gas–liquid chromatogra- phase. Chromatography may be viewed as a series phy, involves vaporizing a sample and injecting it onto 27-1 A scheme for subdividing the field of chromatography, according to various applied techniques. figure Chapter 27 • Basic Principles of Chromatography 477 27-1 table Characteristics of Different Chromatographic Methods Method Mobile/StationaryPhase RetentionVarieswith Gas–liquid chromatography Gas/liquid Molecular size/polarity Gas–solid chromatography Gas/solid Molecular size/polarity Supercritical fluid Supercritical fluid/solid Molecular size/polarity chromatography Reversed-phase Polar liquid/nonpolar liquid or solid Molecular size/polarity chromatography Normal-phase chromatography Less polar liquid/more polar liquid Molecular size/polarity or solid Ion-exchange chromatography Polar liquid/ionic solid Molecular charge Size-exclusion chromatography Liquid/solid Molecular size Hydrophobic-interaction Polar liquid/nonpolar liquid or solid Molecular size/polarity chromatography Affinity chromatography Water/binding sites Specific structure Reprinted from ( 8), p.
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