Proop
`
`ANALYSIS
`
`THIRD EDITION
`
`©
`
`S. SUZANNE NIELSEN
`
`RIMFROST EXHIBIT 1179 Page 0001
`RIMFROST EXHIBIT 1179 Page 0001
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`

`

`Library of Congress Cataloging-in-Publication Data
`
`Food aralysis/edited by S Suzanne Nielsen. —ird ed,
`F-
`om.
`Includes bibliographical references and index.
`ISBN (}30b47495-4
`1. Food—Analysis.
`TAS4 2003
`oh07dell
`
`[. Nielsen, 5. Suzanne.
`
`ESBN: (+06-47495-6
`
`(62009 Kluwer Academic / Plenum Publishers, New York
`233 Spring Street, New York, New York W013
`http:wwwowkap.com
`Wee 7F 6543 2
`
`ACCLLP record for this book is available from the Library of Congress
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`
`RIMFROST EXHIBIT 1179 Page 0002
`RIMFROST EXHIBIT 1179 Page 0002
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`

`

`Chapter 27
`
`© Basic Principles of Chromatography
`
`449
`
`Ligands for affinity chromatography may be either
`specific or general (iec., group specific). Specific lig-
`ands, such as antibodies, bind only one particular
`solute. General ligands, such as nucleotide analogs and
`lectins, bind to certain classes of solutes. For exam-
`ple, the lectin concanavalin A binds to all molecules
`that contain terminal glucosyl and mannosyl residues.
`Bound solutes then can be separated as a group or indi-
`vidually, depending upon the elution technique used.
`Some of the more common general ligands are listed
`in Table 27-4. Although less selective, general ligands
`provide greater convenience.
`Elution methods for affinity chromatography may
`be divided into nonspecific and (bio)specific methods.
`Nonspecific elution involves disrupting ligand ana-
`lyte binding by changing the mobile-phase pH, ionic
`strength, dielectric constant, or temperature. If addi-
`tional selectivity in elution is desired, for example in
`the case of immobilized general ligands, a biospecific
`elution technique is used. Free ligand, either identical
`to or different from the matrix-bound ligand, is added
`to the mobile phase. This free ligand competes for
`binding sites on the analyte, For example, glycopro-
`teins bound to a concanavalin A (lectin) column can be
`eluted by using buffer containing an excess of lectin.
`In general,
`the eluent ligand should display greater
`affinity for the analyte of interest than the immobilized
`lizard.
`
`In addition to protein purification, affinity chro-
`matography may be used to separate supramolecular
`structures such as cells, organelles, and viruses; concen-
`trate dilute protein solutions; investigate binding mech-
`anisms; and determine equilibrium constants. Affinity
`chromatography has been useful especially in the sep-
`aration and purification of enzymes and glycoproteins.
`Inthe case ofthe latter, carbohydrate-derivatized adsor-
`bents are used to isolate specific lectins, such as con-
`canavalin A, and lentil or wheat-germ lectin. The lectin
`then may be coupled to agarose, such as concanavalin
`A- or lentil lectin-agarose,
`to provide a stationary
`phase for the purification of specific glycoproteins,
`glycolipids, or polysaccharides.
`
`27.3.4 Chromatographic Techniques
`
`The same general principles of chromatography apply,
`regardless of the specific method or technique used.
`Paper, thin-layer, and column liquid chromatography
`all utilize a liquid mobile phase, but the physical form
`of the stationary phase is quite different in each case.
`SPC is an analytical technique similar to LC, except that
`a supercritical fluid, instead of a liquid, is used as the
`mobile phase.
`
`27.3.4.1 Paper Chromatography
`Paper chromatography was introduced in 1944.
`Although adsorption by the paper itself has been uti-
`lized, paper generally serves only as a support for the
`PIE General Affinity Ugands and thelr
`liquid stationary phase (partition chromatography).
`To carry out this technique, the dissolved sample is
`|toble|Specificitias
`applied asa small spot or streak one half inch or more
`from the edge of a strip or square of filter paper (usu-
`ally cellulose) and is allowed to dry. The strip is then
`Certain dashydrogenases via
`Cibacron Blue FSG-A dye,
`suspended in a closed container,
`the atmosphere of
`derivatives of AMP, NADH,—binding at the nucelotide
`which is saturated with the developing solvent (mobile
`and NADPH
`binding site
`Concanavalin A, lentil lectin,
`1
`phase), and the paper chromatogram is developed.
`wheat-genm lectin
`giycoprotains, glycolipids,
`The end closer to the sample is placed in contact with
`and membrane proteins
`solvent, which then travels up or down the paper by
`contaming sugar residues of
`capillary action (depending on whether ascending or
`certain configurations
`descending developmentis used), separating sample
`Various proteases
`components in the process. When the solvent front has
`traveled the length of the paper, the strip is removed
`from the developing chamber and the separated zones
`are detected by an appropriate method.
`The stationary phase in paper partition chromato-
`graphy is usually water. However, the support may
`be impregnated with a nonpolar organic solvent and
`developed with polar solvents or water (reversed-
`phase paper chromatography). In the case of complex
`sample mixtures, a two-dimensional technique may be
`used. The sample is spotted in one corner of a square
`sheet of paper, and one solvent is used to develop
`the paper in one direction. The chromatogram is then
`
`Ligand
`
`Specificity
`
`SoyDean trypsin inhibitor,
`methyl esters of vanous
`aming acids, D-amine
`acids
`Phenyboronic acid
`
`Protein A
`
`DAA, ANA, muckeosides,
`nucleotides
`
`‘Ghycosylated hamoaglobina
`Sugars, nucleic acids, and
`other cis-dial-cantaining
`subsiances
`Many immunoglobulin classes
`and subclasses via binding
`to the F, region
`Nucieases, polymerases,
`nuciest acids
`
`Reprinted wih patron from (73). Copyright 1985 American
`‘Ghemical Society.
`
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`
`
`450 Pan ¥ «® Chromatography
`
`dried, turned 90°, and developed again, using a second
`solvent of different polarity. Another means of improv-
`ing resolution is the use of ion-exchange papers. Both
`paper that has been impregnated with ion-exchange
`resin and paper in which cellulose hydroxyl groups
`have been derivatized (with acidic or basic moieties)
`are available commercially.
`In paper and thin-layer (planar) chromatography,
`components of a mixture are characterized by their
`relative mobility (Ry) value, where:
`
`13]
`
`27.3.4.2.1 General Procedures TLC utilizes a thin
`(ca. 250 jum thick) layer of sorbent or stationary phase
`bound to an inert support in a planar configuration.
`The support is often a glass plate (traditionally, 20 cm x
`20 cm) but plastic sheets and aluminum foil also are
`used, Precoated plates, of different layer thicknesses,
`are commercially available in a wide variety of sor-
`bents,
`including chemically modified silicas. (Plates
`are seldom hand-coated today.) Four frequently used
`TLC sorbents are silica gel, alumina, diatomaceous
`earth, and cellulose, Many separations achieved by
`paper chromatography can be transferred to TLC on
`cellulose, Modified silicas for TLC may contain polar
`or nonpolar groups, analogous to bonded phases for
`column chromatography (see section 27.3.3.2.3,), and
`both normal and reversed-phase thin-layer separa-
`tions may be carried out, High performance thin-layer
`chromatography (HPTLC) simply refers to TLC per-
`formed using plates coated with smaller, more uniform
`particles. This permits better separations in shorter
`times.
`If adsorption TLC is to be performed, the sorbent
`is first activated by drying for 4 specified time and
`temperature, Sample (in carrier solvent) is applied as
`a spot or streak 1-2 cm from one end of the plate,
`After evaporation of carrier solvent, the TLC plate is
`placed in a closed developing chamber with the end
`of the plate nearest the spot in the solvent at the bot-
`tom of the chamber. Traditionally, solvent migrates up
`the plate (ascending development) by capillary action
`and sample components are separated, After the TLC
`plate has been removed from the chamber and solvent
`allowed to evaporate, the separated bands are made
`visible (visualized) or detected by other means. Spe-
`cific chemical reactions (derivatization), which may
`be carried out either before or after chromatography,
`often are used for this purpose. Two examples are
`reaction with sulfuric acid to produce a dark charred
`area (a destructive chemical method), and the use
`of iodine vapor to form a colored complex (a mon-
`destructive method inasmuch as the colorecl complex
`is usually not permanent), Common physical debee-
`tion methodsinclude the measurement of absorbed or
`emitted electromagnetic raciation (e.g., fluorescence)
`and measurement of f-radiation from radioactively
`labeled compounds. Biological methods or biochemi-
`cal inhibition tests can be used to detect toxicologically
`active substances. An example is measuring the inhi-
`bition of cholinesterase activity by organophosphate
`pesticides.
`Quantitative evaluation of thin-layer chromato-
`grams may be performed: (1) in situ (directly on the
`layer) by using a densitometer or (2) after scraping a
`sone off the plate, eluting compound from the sorbent,
`and analyzing the resultant solution (e.g., by liquid
`scintillation counting).
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`Distance moved by component
`sa Distance moved by solvent
`
`Unfortunately, Ry values are not always constant for
`a given solute/sorbent/solvent, but depend on many
`such as the quality of the stationary phase,
`layer thickness, humidity, development distance, and
`temperature,
`
`27.3.4.2 Thin-Layer Chromatography (TLC)
`TLC, first described in 1934, has largely replaced paper
`chromatography because it
`ia faster, more sensitive,
`and more reproducible, (Both of these techniques may
`be referred to as planar chromatography.) The resolu-
`Hon in TLC is greater than in paper chromatography
`becouse the particles on the plate are amaller and more
`regular than paper fibers. Experimental conditions can
`be easily varied to achieve separation and can be scaled
`up for use in column chromatography. (Thin-layer and
`column procedures are not necessarily interchange-
`able, due to differences such as the use of binders with
`TLE plates, vapor-phase equilibria ina TLC tank, ete.)
`Some distinct advantages of TLC include high sam-
`ple throughput and low eost; the possibility to analyze
`several samples and standards simultancously; and
`minimal sample preparation (since the stationary phase
`is disposable). In addition, a plate may be stored for
`later identification and quantitation,
`TLC has been applied to the analysis of lipids (see
`Chapter 14). HPLC of lipids is complicated by the
`lack of chromophores that permit ultraviolet-visible
`(UV-Vis) detection, and most GC analyses require
`prior derivatization; however, many good lipid detec-
`tion reagents are available for TLC. TLC is applied in
`many fields, including environmental, clinical, foren-
`sic, phamaceutical, food, flavors, and commetics. Within
`the food industry, TLC may be used for quality control.
`For example, corn and peanuts are tested for aflatox-
`ingmycotoxins prior to their processing into corn meal
`and peanut butter, respectively. Applications of TLC
`bo the analysis of a variety of compounds, including
`lipids, carbohydrates, vitamins, amino acids, and nat-
`ural pigments, are discussed in the book by Fried and
`Shermia (4).
`
`

`

`27.3.4.2.2 Factors Affecting Thin-Layer Separations
`In both planar and column liquid chromatography,
`the nature of the compounds to be separated deter-
`mines what type of stationary phase is used. Separa-
`Hon can occur by adsorption, partition, ion-exchange,
`size-exclusion, or multiple mechanisms as previously
`discussed in section 27.3.3. Table 27-5 lists the separa-
`tion mechanisms involved in some typical applications
`on common TLC sorbents.
`Although selection of both mobile and stationary
`phases determines the success of a given TLC separa-
`tion, the rationale behind choice of mobile phase for a
`particular fractionation often is not described. Solvents
`for TLC separations should be selected on the basis of
`their chemical characteristics and solvent strength (a
`measure of interaction between solvent and sorbent;
`see section 27.3.3.1). In simple adsorption TLC, the
`higher the solvent strength, the greater the Ry value
`of the solute. One usually tries to use a mobile phase
`such that Ry values of 0.3-0.7 are obtained. (Although
`single solvent mobile phases may provide adequate
`mobility, they often do not give adequate separation.)
`Fortunately for the beginner, mobile phases have been
`developed for the separation of various compound
`classes on specific sorbents; see, for example, table 7.1
`in reference (12).
`In addition to the sorbent and solvent, several other
`factors must be considered when performing thin-layer
`
`Sorbent
`
`Typical Application
`
`Silica gel
`
`Adsorption
`
`Silica gel AP Reversed phase
`
`Cellulose,
`kieseigquhr
`
`Partition
`
`Aluminum
`oxide
`
`Adsorption
`
`Pel
`
`,
`
`lon exchange
`
`Adsorption
`
`silicate
`
`Nucleic acids, nucleotides
`pyrimidines
`Steroids, pesticides, ipids
`i
`
`27.3.4.3.1 General Procedures A system for low-
`pressure (Le., performed at or near atmospheric pres-
`sure) column liquid chromatography is illustrated in
`Fig. 27-9. (While the procedure outlined below is appli-
`cable to column chromatography in general, the reader
`is referred to subsequent chapters for details specific to
`HPLC or GC).
`Having selected a stationary and mobile phase suit-
`able 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 phase often
`must be hydrated or preswelled in the mobile phase.)
`The prepared stationary phase then is packed into a col-
`ii Thin-Layer Chromatography Sorbents
`umn (usually glass), the length and diameter of which
`|table ‘nd Mode of Separation
`are determined by the amount of sample to be loaded,
`the separation mode to be used, and the degree of reso-
`Chromatographic
`lution required. Adsorption columns may be either dry
`Mechanism
`or wet packed; other types of columns are wet packed.
`The most common technique for wet packing involves
`making a slurry of the adsorbent with the solvent and
`pouring this into the column. As the sorbent settles,
`excess solvent is drained off and additional slurry is
`added. This process is repeated until the desired bed
`height is obtained. (There is a certain art to pour-
`ing uniform columns and no attempt is made to give
`details here.) If the packing solvent is different from
`the initial eluting solvent, the column must be thor-
`oughly washed (equilibrated) with the starting mobile
`phase.
`The sample to be fractionated, dissolved in a min-
`imum volume of mobile phase, is applied in a layer
`at the top (or head) of the column. Classical or low-
`pressure chromatography utilizes only gravity flow
`or a peristaltic pump to maintain a flow of mobile
`phase (eluent or eluting solvent) through the column.
`In the case of a gravity-fed system, eluent is simply
`siphoned from a reservoir into the column. The flow
`rate is governed by the hydrostatic pressure, measured
`
`
`
`Chapier 27 © Basic Principles of Chromatography 451
`
`(or paper) chromotography. These include the type of
`developing chamber used, vapor phase conditions
`(saturated Versus unsaturated), development mode
`(ascending, descending, horizontal, radial, etc.), and
`development distance.
`
`27.3.4.3 Column Liquid Chromatography
`Column chromatography is the most useful method
`of separating compounds in a mixture. Fractionation
`of solutes occurs as a result of differential migration
`through a closed tube of stationary phase, and analytes
`can be monitored while the separation is in progress.
`This section of the chapter will cover general proce-
`dures, theory, and the quantitation of data from column
`liquid chromatography.
`
`Reprinted from (12) by enmission of John Wiley & Sons, Mere ‘York,
`PE] cafuiese refers to celluikes derivatized with polyathylenaimine
`(PEN.
`
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`

`Part¥ © Chromatography
`
`A wystem for low-pressure column liquid chromatography. In this diagram, the column effluent is being split
`iid
`between two detectors in order to monitor both enzyme activity (at
`and UV absorption (at left), The two
`[_Nowre|
`tracings can be recorded simultaneously by using a dual-pen recorder,
`[Adapted from (9), with permission,|
`
`that have been chromatographically separated in this
`manner then can be analyzed as needed,
`
`as the distance between the level of liquid in the reser-
`voir and the level of the column outlet, Lf eluent is fed
`to the column by a peristaltic pump (see Fig, 27-9), the
`flow rate is determined by the pump speed and, thus,
`27.3.44.2 Qualitative Analysis The volume of liquid
`regulation of hydrostatic pressure is not necessary,
`required to elute a compound from an LC column is
`The process of passing the mobile phase through
`called the retention volume, Vg. The associated tine
`is the retention the, fg, Comparing Vg or fy to that of
`the column is called elution, and the portion that
`emerges from the outlet end of the column is some-
`standards chromatographed under identical conditions
`often enables one to identify an unknown compound.
`times called the eluate (or effluent), Elution may be
`isocratic (constant mobile-phase composition) or a
`(One should remember that different compounds may
`gradient may be used. Gradient elution refers to chang-
`have identical retention times) A related technique is
`ing the mobile phase (e.g., increasing solvent strength
`toapike the unknown sample with a known compound
`or pH) during elution in order to enhance resolu-
`and compare chromatograms of the original and spiked
`samples to see which peak has increased,
`In most
`Hon and decrease analysis time. The change may be
`continuous or stepwise, Gradients of increasing ionic
`cases, it will be necessary to collect the peak(s) of inter-
`strength are extremely valuable in ion-exchange chro-
`est and establish their identity by another analytical
`rethesel.
`matography. Gradient elution is commonly used for
`Olten it is necessary to compare chromatograms
`desorbing lange molecules, such as proteins, which
`can undergo multiple-site interaction with a station-
`obtained from two different systems or columns. Dil-
`ary phase. As elution proceeds, components of the
`ferences in column dimensions, loading, temperature,
`flow rate, system dead-volume, and detector geome-
`sample are selectively retarded by the stationary
`phase according to one (or more) of the mechanisms
`try may lead to diserepancies for uncorrected retention
`discussed earlier, and thus are eluted at different
`data, By subtracting the time required for the mobile
`times.
`phase or a nonretained solute (fy orto) to travel through
`The column effluent may be directed through a
`the column to the detector, one obtains an adjusted
`retention time,f,, (or volume) as depicted in Fig. 27-10.
`detector and then into tubes, changed at intervals by
`a fraction collector. The detector response, in the form
`The adjusted retention time (or volume) corrects for dif-
`ferences insystem dead-volume;it may be thought ofas
`of an electrical signal, may be recorded (the chro-
`the time the sample spends adsorbed on the stationary
`matogram) and used for qualitative or quantitative
`phase.
`analysis, as discussed in more detail later. The frac-
`tion collector may be set to collect eluate at specified
`A simple, reliable method for the identification of
`time intervals or after a certain volume or number of
`peaks is to use relative retention as expressed by the
`drops has been collected, Components of the sample
`separation factor, a. Values for a (Fig. 27-10) depend
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