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Buy eBook - 6. Bernard Fried , Joseph Sherma. CRC Press , M01 4 - pages. The fourth edition of this work emphasizes the general practices and instrumentation involving TLC and HPTLC, as well as their applications based on compound types, while providing an understanding of the underlying theory necessary for optimizing these techniques.

The book details up-to-date qualitative and quantitative densitometric experiments on organic dyes, lipids, antibiotics, pharmaceuticals, organic acids, insecticides, and more. Selected pages Title Page. Table of Contents. Contents III. Common terms and phrases acetate adsorbent adsorption amino acids Anal analytical anthocyanins application aqueous Biomphalaria Camag carbohydrates carotenoids cellulose chamber Chapter Chem chemical Chro Chromatogr column compounds CRC Press densitometry described detection reagents determination drugs elution Experiment extraction Figure flavonoids fluorescence fluorescence quenching Fried Eds gradient hemolymph high-performance HPLC HPTLC plates identification impregnated Initial Zones lipids lipophilic Marcel Dekker metabolites methanol method mixture mobile phase mobile-phase multiple development neutral lipids nucleotides Nyiredy OPLC optimization pesticides pharmaceutical phospholipids pigments Planar Chromatogr.

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Quantitative analysis A TLC analysis that determines the weight or concentration of a constituent of a sample. Rf The ratio of the distance of migration of the center of a zone divided by the distance of migration of the solvent front, both measured from the origin.

Rx The same as Rf except that the distance of solvent front migration is replaced by the distance of migration of some reference compound x. Radial circular development Development of a layer in such a manner as to form circular or arc-shaped zones. Some workers differentiate circular and radial TLC by the use of "circular" for the case where one initial zone is developed into circular zones and "radial" for development of a series of initial zones, spotted in a circular pattern, into arcs.

Raman spectroscopy A technique in which a beam of intense monochromatographic radiation, such as from a laser, is focused on a sample, and scattering produces radiation that is shifted slightly in frequency by an increment of energy corresponding to a natural transition of the molecule Raman lines. The method complements IR absorption spectroscopy for measurement of molecular vibrations. Relative standard deviation RSD The standard deviation of a series of replicate analyses is divided by the mean, and the resulting quotient is multipled by Also called coefficient of variation. Resolution The ability to separate two zones.

Migration distances can be used instead of Rf values in this equation. Resolution is the result of the combined contributions of efficiency zone compactness , selectivity separation of zone centers , and capacity average Rf value of the pair of substances to be separated. Retention factor Another name for capacity factor. Re versed-phase chromatography Liquid-liquid partition TLC in which the stationary phase is nonpolar compared to the mobile phase. The layer can be impregnated or bonded.

Sandwich chamber S-chamber A developing chamber formed from the plate itself, a spacer, and a layered or nonlayered cover plate that stands in a trough containing the mobile phase, or some other type of small-volume chamber in which vapor-phase saturation occurs quickly. Saturated The condition of a chamber that is lined with paper and equilibrated with mobile phase vapors before chromatographic development is begun.

Secondary front An additional solvent or mobile phase front below the primary front, that occurs because the mobile phase components demix. Selectivity The ability of a chromatographic system to produce different Rf values for the components of a mixture, i. Sensitivity The ability to detect or measure a small mass of analyte. Silanol An SiOH group on the surface of silica gel. It has an amorphous, porous structure with siloxane and silanol groups on the surface. Siloxane SiOSi groups on the surface of silica gel.

Soft layer A sorbent layer prepared without binder or with gypsum binder see Hard layer. Solid-phase extraction SPE An alternative to traditional separatory funnel extraction in which an analyte is extracted from a liquid sample by use of a solid packed in a small column, cartridge, or disk. The sample is forced through the solid, which is an adsorbent or bonded phase, with the aid of vacuum or pressure; the analyte is retained on the solid, and it is subsequently eluted with a small volume of a strong solvent, usually resulting in significant enrichment.

Solute A general term for the compounds or ions being chromatographed.

Thin-Layer Chromatography (TLC)

Solvent The liquid used to dissolve the sample for application to the layer. Sometimes used to refer to the mobile phase or to the liquid used to elute chromatographed zones from scraped layer material. Solvent front The farthest point of movement of the mobile phase during development. Solvent strength A measure of the polarity of a solvent for liquid-solid adsorption chromatography. It is based on the free energy of adsorption onto a standard surface. Values for common solvents range from 0.

Sorbent The layer material used in TLC. Sorption A general term for the attraction between a layer and a solute, without specification of the type of physical mechanism i. Sorbent is a related general term referring to the layer itself. Spectroscopy An analytical technique based on the interaction of electromagnetic radiation with matter. Also called spectrometry. Spot Used synonymously with zone, but usually meant to indicate a round or elliptical shape.

Spot capacity Same as separation number. Stationary phase The solid sorbent layer, with or without any impregnation agent, preloaded vapor molecules, or immobilized mobile phase component s. Stepwise development Development using a mobile phase whose composition is changed using discontinuous, stepped gradients, in contrast to continuously variable gradient elution. Straight-phase chromatography Another name for normal-phase chromatography. Streak An initial zone in the shape of a narrow horizontal line at the origin. Streaking See Tailing.

Supercritical fluid extraction SFE A technique for extracting analytes from sample matrices by using a dense gas. Carbon dioxide, which becomes a supercritical fluid when used above its critical pressure psi and temperature 31C , is the most widely applied extraction medium for SFE because it is nontoxic and nonflammable and facilitates extractions at low temperatures in a nonoxidizing environment.

Tailing Formation of a zone with an elongated rear portion, often leading to incomplete resolution. Throughput A term used mostly in the context of "sample throughput," which indicates the number of samples that can be analyzed by a particular method in a given period of time. TLC has high sample throughput because multiple samples can be applied to a single plate. For example, there is higher solvent throughput with an unsaturated N-chamber than with a saturated N-chamber because of the greater amount of solvent that passes through the layer to replace the solvent that has evaporated from it.

TLC Thin-layer chromatography. TLG Thin-layer gel chromatography, in which separations are based mainly on solute sizes. Trailing See Tailing. Two-dimensional development Successive development of a chromatogram with the same solvent or different solvents in directions at a 90 angle to each other.

Unsaturated The condition of a chamber that has the mobile phase and plate added together so that equilibration with the vapors is occurring during chromatographic development. UV Ultraviolet. Validation The process of determining the suitability of a given TLC method for an intended application, such as qualitative identification, assays, semiquantitative limit tests, or quantitative determination of impurities in pharmaceutical analysis.

The characteristics tested can include accuracy, precision, specificity, detection limit, quantification limit, linearity, and robustness. Visualization Detection of the zones on a chromatogram. Zone The area of distribution on the layer containing the individual solutes or mixture before, during, or after chromatography. The initial zone is the applied sample prior to development.

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Band, zone, and spot are often used more or less interchangeably, but spot usually denotes a round zone, and band a flat, horizontally elongated zone. The purpose of this chapter is to present an overview of all important aspects of thin-layer chromatography TLC. It briefly reviews information and provides updated references on topics covered in the remaining chapters in Part I and refers readers to the specific chapters. It treats topics that are not covered in separate chapters, such as sampling and sample preparation and the more classical procedures of TLC, in more detail.

A suggested source of additional information, both basic and advanced, on the practice and applications of TLC is the primer written by Fried and Sherma 1. Introduction to TLC. Thin-layer chromatography and paper chromatography comprise "planar chromatography. A suitable closed vessel containing solvent and a coated plate are all that are required to carry out separations and qualitative and semiquantitative analysis. With optimization of techniques and materials and the use of available commercial instruments, highly efficient separations and accurate and precise quantification can be achieved.

Planar chromatography can also be used for preparative-scale separations by employing specialized layers, apparatus, and techniques. Basic TLC is carried out as follows. A small aliquot of sample is placed near one end of the stationary phase, a thin layer of sorbent, to form the initial zone.

The sample is then dried. The end of the stationary phase with the initial zone is placed into the mobile phase, usually a mixture of two to four pure solvents, inside a closed chamber. If the layer and mobile phase were chosen correctly, the components of the mixture migrate at different rates during movement of the mobile phase through the stationary phase. This is termed development of the chromatogram. When the mobile phase has moved an appropriate distance, the stationary phase is removed, the mobile phase is rapidly dried, and the zones are detected in daylight or under ultraviolet UV light with or without the application of a suitable visualization reagent.

Differential migration is the result of varying degrees of affinity of the mixture components for the stationary and mobile phases.

Thin-Layer Chromatography, Revised And Expanded

Various separation mechanisms are involved, the predominant forces depending upon the exact properties of the two phases and the solutes. Among the latter are dipole-dipole Keesom , dipole-induced dipole Debye , and instantaneous dipole-induced dipole London interactions. Sample collection, preservation, and purification are problems common to TLC and all other chromatographic methods.

For complex samples, the TLC development will usually not completely resolve the analyte from interferences unless a prior purification cleanup is carried out. This is most often done by selective extraction and column chromatography. TLC can cope with highly contaminated samples, and the entire chromatogram can be evaluated, reducing the degree of cleanup required and saving time and expense. The presence of strongly adsorbed impurities or even particles is of no concern, because the plate is used only once 2.

Detection is simplest when the compounds of interest are naturally colored or fluorescent or absorb UV light. However, application of a detection reagent by spraying or dipping is required to produce color or fluorescence for most compounds. Absorption of UV light is common for most aromatic and conjugated compounds and some unsaturated compounds.

These compounds can be detected simply by inspection under nm UV light on layers impregnated with a fluorescence indicator fluorescence quench detection. Compound identification in TLC is based initially on a comparison of Rf values to authentic reference standards. Rf values are generally not exactly reproducible from laboratory to laboratory or even in different runs in the same laboratory, so they should be considered mainly as guides to relative migration distances and sequences.

Factors causing Rf values to vary include dimensions and type of chamber, nature and size of the layer, direction of the mobile-phase flow, volume and composition of the mobile phase, equilibration conditions, humidity, and sample preparation methods preceding TLC.

See Chapter 11 in Ret. Confirmation of identification can be obtained by scraping the layer and eluting the analyte followed by infrared IR spectrometry, nuclear magnetic resonance NMR spectrometry, mass spectrometry MS , or other spectrometric methods if sufficient compound is available. These methods can also be used to characterize zones directly on the layer in situ. The history of liquid chromatography, which dates back to the first description of chromatography by Michael Tswett 3 in the early s, was reviewed by Sherma 4.

TLC is a relatively new discipline, and chromatography historians usually date the advent of modern TLC from A review by Pelick et al. In , Izmailov and Schraiber separated certain medicinal compounds on unbound alumina or other adsorbents spread on glass plates. Because they applied drops of solvent to the plate containing the sample and sorbent layer, the procedure was termed drop chromatography.

Meinhard and Hall in used binder to adhere alumina to microscope slides, and these layers were used in the separation of certain inorganic ions with the use of drop chromatography; this method was called surface chromatography. In the s, Kirchner and colleagues at the U.

Department of Agriculture performed TLC as we know it today.

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They used silica gel held on glass plates with the aid of a binder, and plates were developed with the conventional ascending procedures used in paper chromatography. Kirchner coined the term "chromatostrips" for his layers, which also contained fluorescence indicator for the first time. Stahl introduced the term "thin-layer chromatography" in the late s. His major contributions were the standardization of materials, procedures, and nomenclature and the description of selective solvent systems for resolution of important compound classes.

His first laboratory manual 10 popularized TLC, and he obtained the support of commercial companies Merck, Desaga in offering standardized materials and apparatus for TLC. Quantitative TLC was introduced by Kirchner et al. Densitometry in TLC was initially reported in the mids using commercial densitometers such as the Photovolt and Joyce Loebl Chromascan.

Plates with uniform, fine-particle layers were produced commercially in the mids and provided impetus for the improvements in theoretical understanding, practice, and instrumentation that occurred in the late s and s and led to the methods termed high-performance. Centrifugally accelerated preparative layer chromatography PLC and overpressured layer chromatography OPLC , which are the major forced-flow planar chromatographic techniques, were introduced in the late s.

These and other high-performance and quantitative methods caused a renaissance in the field of TLC that is reflected in this Handbook. Although the major use of TLC will probably continue to be as a general low-cost and low-technology qualitative and screening method in laboratories worldwide, there is no doubt that TLC will continue to evolve and grow in the new millennium as a highly selective, sensitive, quantitative, rapid, and automated technique for analysis of all varieties of samples and analytes and for preparative separations.

To keep abreast of this inevitable progress in TLC, the biennial reviews of advances in theory, practice, and applications by Sherma, the most recent of which was published in 11 , are indispensable. TLC involves the concurrent processing of multiple samples and standards on an open layer developed by a mobile phase. Development is performed, usually without pressure, in a variety of modes, including simple one-dimensional, usually in ascending or horizontal mode; multiple; circular rarely ; and multidimensional.

Zones are detected statically, with diverse possibilities. Paper chromatography, which was invented by Consden, Gordon, and Martin in , is fundamentally very similar to TLC, differing mainly in the nature of the stationary phase. Paper chromatography has lost favor compared to TLC because the latter is faster and more efficient, allows more versatility in the choice of stationary and mobile phases, and is more suitable for quantitative analysis. High-performance TLC layers are smaller; contain sorbent with smaller, more uniform particle size; are thinner; and are developed for a shorter distance compared to TLC layers.

These factors lead to faster separations, reduced zone diffusion, better separation efficiency, lower detection limits, less solvent consumption, and the ability to apply more samples per plate. However, smaller samples, more exact spotting techniques, and more reproducible development techniques are required to obtain optimal results. High-performance liquid chromatography involves the elution under pressure of sequential samples in a closed on-line system, with dynamic detection of solutes, usually by UV absorption.

This makes the two methods complementary for compound separation and identification. TLC is the most versatile and flexible chromatographic method for separation of all types of organic and inorganic molecules that can be dissolved and are not volatile. It is rapid because precoated layers are usually used without preparation. Even though it is not fully automated as is possible for HPLC, TLC has the highest sample throughput because up to 30 individual samples and standards can be applied to a single plate and separated at the same time.

The ability to separate samples simultaneously in parallel lanes is important in applications that require high sample throughput, e. Modern computer-controlled scanning instruments and automated sample application and development instruments allow accuracy and precision in quantification that are in many cases equivalent to those obtained with HPLC and gas chromatography GC.

There is a wide choice of layers and developing solvents acidic, basic, completely aqueous, aqueous-organic. Every sample is separated on a fresh layer, so that problems involved with carryover and cross-contamination of samples and sorbent regeneration procedures are avoided. Mobile-phase consumption is low, minimizing the costs of solvent purchase and disposal. Because layers are normally not reused, sample preparation methods are less demanding, and complex, impure samples can be applied to the layer without concern for the extra ghost peaks and noneluting compounds that shorten the life of HPLC columns.

Simultaneous sample cleanup and separation of target compounds are often achieved with TLC The wide choice of development methods and pre- or postchromatographic detection reagents leads to unsurpassed specificity in TLC, and all components in every sample, including irreversibly sorbed substances, can be detected.

There is no need to rely on peaks drawn by a recorder or to worry about sample components possibly remaining uneluted on a column. Because it is an off-line method, the various steps of the procedure are carried out independently. Examples of the advantages of this approach include the ability to apply compatible detection methods in sequence and to scan zones repeatedly with a densitometer using different parameters that are optimum for individual sample components. The pyramidal screening approach, in which TLC is used as a screening step followed by HPLC confirmation and quantification of only positive samples, can result in less analytical time and lower cost than when all samples are analyzed by HPLC Abjean 14 showed that meat samples could be analyzed for sulfonamide drugs by a single analyst in 12 days using TLC screening and HPLC analysis of positive samples compared to 50 days for HPLC multiresidue analysis alone.

The simultaneous identification of chloramphenicol, nitrofurans, and sulfonamides in pork or beef is an example of TLC multiclass screening The drugs were identified by homogenization and extraction from 1 g of tissue with ethyl acetate, cleanup of the extract on a silica gel solid-phase extraction SPE cartridge, and separation by TLC. Spraying with pyridine detected nitrofurans, and subsequently fluorescamine detected chloramphenicol and sulfonamides.

Twenty samples could be analyzed per day per analyst for three residue classes by a single method. The determination of antibiotics in milk 16 and of poly cyclic aromatic hydrocarbons PAHs in soil 17 are other TLC screening methods that have demonstrated advantages in terms of simplicity, time, and cost compared to HPLC. Books that have appeared since the publication of the second edition of this Handbook are those by Kaiser et al.

Book chapters 21,22 and an encyclopedia article 23 covering TLC, several general review articles 13,24,25 , and a guide to method development 26 were published within the last seven years. The capacity factor, k', is the ratio of the quantities of solute distributed between the mobile and stationary phases, or the ratio of the respective times the substance spends in the two phases, , ts tm retention time in stationary phrase retention time in mobile phase.

The classic Van Deemter equation and its modifications have been used to describe zone spreading in GC and HPLC in terms of eddy diffusion, molecular diffusion, and mass transfer. In contrast to column chromatography, in which all solutes move the same distance, separated components migrate different distances in TLC, and their zones are broadened to varying degrees. Separation efficiency and capacity in TLC were discussed by Poole Efficiency is limited by less than optimal velocity of the mobile phase driven by capillary forces, leading to zone broadening that is largely dominated by molecular diffusion.

Mobile-phase velocity decreases approximately quadratically with migration distance, resulting in the migration of zones through regions of varying efficiency and the need to specify plate height for the layer as an average value. For sorbents with narrow particle size range, solvent front velocity is greater for coarseparticle layers than for layers with fine particles It has also been shown that for RP layers with bonded long-chain alkyl groups, mobile phases with larger percentages of water will ascend very slowly, requiring plates to be prepared from particles with a larger diameter pm than those used for the usual HP layers 5 fjim or from sorbents with a lower degree of surface modification.

Polar-bonded sorbents, such as cyano or amino, are wetted by aqueous solvents Guiochon and coworkers showed that for capillary flow TLC on fine-particle HP layers, zone broadening is controlled by the size of the sorbent particles for short migration distances and molecular diffusion for long migration distances. For large-particle sorbent layers, the packing and slow mass transfer processes can both contribute to broadened, irregularly shaped zones.

High plate numbers can be generated on layers with relatively large particles only with long migration distances, especially for solutes with large diffusion coefficients. HPTLC layers produce the highest efficiency for short migration distances of mm, and efficiency eventually is poorer than for TLC as the migration distance increases and molecular diffusion overtakes zone center separation to become the limiting factor.

Longer solvent front migration distances require layers with a larger particle size to obtain a reasonable range of mobile-phase velocities and total number of theoretical plates 13, The results of these studies indicate that HPTLC plates can produce more compact zones in a shorter development distance, increasing the speed and detection limits of the zones. About theoretical plates can be obtained for a cm development on HPTLC plates, whereas a development distance of approximately 15 cm is needed to obtain this.

The experimental zone capacity for baseline separated peaks in a chromatogram resulting from capillary controlled flow is about , and this is not strongly dependent on the average particle size of the layer Zone capacity for forced-flow development is ; for capillary controlled flow automated multiple development AMD , ; and for two-dimensional 2-D capillary flow, approximately The subscript 2 refers to the zone with the higher Rf value.

As in the analogous resolution equation for HPLC, this equation includes terms related to the efficiency of the layer, the selectivity of the TLC system, and the capacity of the system the zone positions on the layer. Resolution increases with the square root of the layer efficiency TV , which depends linearly on the Rf value.

In terms of zone position, studies have shown that maximum resolution is obtained in the R, range of 0. The most effective means for increasing resolution on a TLC or HPTLC layer with the usual capillary flow, one-dimensional single development is to improve selectivity by variation of the mobile phase, the choice of which is aided by systematic optimization methods such as simplex, PRISMA, and others that have been developed 37 see Chap. Other approaches for increasing resolution include the use of capillary flow with multiple or two-dimensional development or forced-flow development.

The foregoing discussion applies to capillary flow TLC, in which the migration velocity of the mobile phase through the layer is controlled by capillary forces and decreases as development distance increases The optimum velocity necessary for maximum efficiency is not realized in capillary flow TLC. In forced-flow planar chromatography, the mobile phase is driven by centrifugal force [rotation planar chromatography RPC ] or by a pump OPLC see Chap.

RPC never reaches an overall mobile-phase velocity that would give the highest separation efficiency, because the radial velocity of solvent migration diminishes from the center to the circumference of the plate In OPLC, mobile-phase velocity can be controlled at a predetermined constant close to optimal value so that solvent front migration is a linear function of time As a result, average plate height is approximately independent of migration distance and is most favorable for HPTLC plates, zone broadening by diffusion is minor even over long migration distances, plate number increases linearly with migration distance, and resolution continues to increase as migration distances increases 30, The time required for the mobile phase to cover the same distance in OPLC is typically five- to tenfold shorter than in TLC, depending on the surface tension, viscosity, and the ability to wet the layer.

Separation time is further reduced because the number of theoretical plates needed to achieve a separation is generated in a shorter time because of the near-optimal mobile-phase flow rate Poole 13 showed that for a development distance of 18 cm, forced-flow development can produce theoretical plates in 9 min. Increased efficiency is obtained by use of longer bed lengths e. Electro-osmotic flow caused by applying an electric field across a wet layer containing both ionized silanol groups and mobile ions is an additional mechanism for moving the mobile phase through the layer.

Nurok 39 reported that separation of six pyrimidines on silica gel with acetonitrile mobile phase was 12 times faster than with conventional TLC and that separation in the RP mode is two to three times faster depending on the mobile phase. Only preliminary studies of this approach have been carried out to date, and Poole 13 reports that the mobile-phase velocity declined with migration distance and showed only moderate increase compared to capillary flow, and that the demonstrated improved performance with electro-osmotic flow has been below that predicted by theory.

The classic book by Geiss 40 is recommended as an excellent source of information on the fundamentals of TLC. Although the book is highly theoretical and mathematical, numerous practical summaries and suggestions can be found throughout its chapters to guide anyone working with TLC. Especially useful in better understanding TLC is Chapter 6 in Geiss 40 , on the role of the vapor phase. It explains and distinguishes chamber saturation saturation of the chamber atmosphere , sorptive saturation preloading of the layer from the atmosphere , and capillary saturation saturation of the layer through the rising mobile phase and the results caused by different chamber types and solvent mixtures.

It is safe to say that few practitioners of TLC clearly understand these complicated effects that occur during development. The Geiss book also contains a discussion and a decision flow chart for optimization of separations of two closely related substances or a wide polarity range multicomponent mixture with the use of different mobile phases, development approaches, chamber types, and layers.

Readers are directed to Chapter 2 of this Handbook and to Ref. Reference 42 covers studies of quantitative structure-retention relationships, one of the more important theoretical fields of TLC. One of the most important steps in analysis is that of obtaining an appropriate sample of the material to be analyzed. If a nonrepresentative sample is taken, the analytical result will be unreliable no matter how excellent the procedure and laboratory work.

As an example, the purity of a bottle of analgesic tablets should not be determined by analyzing one tablet, which might be nonrepresentative of the average tablet. A better plan is to grind together 10 tablets to form a homogeneous powder and take a sample weight equivalent to the average weight of one tablet for the analysis. In this way, the composition of the laboratory sample has a much higher probability of accurately representing the average composition of the entire contents of the bottle. The sample should not change or be lost as a result of storage prior to TLC analysis.

The integrity of most samples can be maintained by storage in a freezer. However, with some samples, freezing and thawing or the introduction of the common fixatives formalin or ethanol can affect the results of subsequent analyses The storage container should be airtight to prevent volatilization of the sample or introduction of air, water, or other vapors. The container should be constructed from a material chosen such that impurities are not leached into the sample from the inside surface and analyte cannot be lost by adsorption on the inside surface.

Plastic is a common choice for storage of samples to be analyzed for metals, and glass for samples with organic analytes. A detailed discussion of sampling procedures for different types of gas, liquid, solid, and bulk samples is beyond the scope of this chapter. Chapter 4 in Ref. Most college textbooks on quantitative analysis and instrumental analysis contain sections or chapters on the theory and practice of sampling e.

Sample Preparation. The only chapter on sample preparation specifically for TLC was written by Sherma 45 , but because of its date it does not contain modern methods. A review paper on sample preparation for chromatographic analysis of plant material 46 and two reports on instruments for sample preparation 47,48 contain information on the newest methods. Sections on sample preparation related to specific compound types will be found in most of the applications chapters in Part II of this Handbook.

If the analyte is present in low concentration in a complex sample such as biological or plant material, then extraction, isolation, and concentration procedures must usually precede TLC. On the other hand, any impurities that would comigrate with the analyte and adversely affect its detection or cause a distorted or trailing analyte zone must be removed prior to TLC. Purification of extracts is accomplished by methods such as solvent partitioning, column chromatography, desalting, and deproteinization.

Direct Spotting of Samples Certain samples can be successfully analyzed by direct spotting without extraction or cleanup. The applied volume must give a detectable zone with a scan area that can be bracketed by the scan areas of a series of standard concentrations if densitometric quantification is desired.

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Impurities must not retain the compound at the origin, distort its shape cause tailing , or alter the Rf value of the zone. The quantification of benzoic and sorbic acid preservatives in beverages directly applied onto a plate with a preadsorbent spotting strip is an example The preadsorbent facilitated the analysis because samples could be quickly and easily applied over a large area, the initial zone was automatically concentrated at the layer interface upon development, and the kieselguhr strip retained sample impurities.

Unpurified urine and serum samples have also been applied successfully to preadsorbent layers for determination of amino acids, drugs, and lipids. Direct Application of Sample Solutions or Extracts For determination of macro constituents in relatively pure matrices, samples can be dissolved in an appropriate volume of pure solvent followed by spotting of an aliquot of solution on the layer.

This approach has been used for HPTLC assay of active ingredients of many pharmaceutical dosage forms, e. Fillers and other inert ingredients in samples such as foods and pharmaceuticals often remain undissolved. This will cause no problem if the analyte is dissolved completely and the insoluble material is filtered or centrifuged into a pellet or allowed to settle to the bottom of the sample container prior to spotting clear test solution.

Extracts of trace constituents in some types of adequately pure samples can also be spotted directly after concentration of an extract to a suitable volume. Any coextracted impurities must be resolved from the analyte by the TLC separation step or not detected by the visualization method used. To minimize the amount of coextractives, the least polar analyte that will quantitatively extract the analyte should be used, leaving as many polar impurities as possible unextracted.

Direct spotting of extracts was used to determine hydrocarbons in wastewater extracted with heptane by means of a microseparator 53 and the pesticide dichlorvos in minced visceral tissue extracted with ethyl acetate Cleanup of Extracts by Solvent Partitioning Extracts that are too impure for direct spotting can be cleaned up by partitioning with immiscible solvents. The principle of differential partitioning is to leave impurities behind in one solvent layer while extracting the analyte into the other layer.

Acids are converted into salts that are soluble in aqueous solutions at high pH but are un-ionized and extractable into organic solvents at low pH. Basic compounds are extracted into organic solvents at high pH and into water in their salt forms at low pH. In practice, the pH should be at least two units below the pKa of an acid and two units above the pKa of a base in order to have a large enough fraction of uncharged molecules to allow efficient extraction into organic solvents.

As an example, the mycotoxin patulin was determined in apples, apple concentrate, and apple juice by extraction with ethyl acetate, cleanup by partition with 1. Other uses of liquid-liquid extraction in sample preparation are to remove oils, fats, and lipids from samples if these compounds will interfere with subsequent TLC and to concentrate sample solutions prior to spotting. Cleanup of Extracts by Column Chromatography Chromatography on gel permeation, silica gel, alumina, Florisil, and carbon columns, among others, has been very widely used for cleanup of samples, often after preliminary purification by solvent partitioning.

Examples are the TLC determination of uracil herbicides in roots of Echinacea angustifolia Moench Asteraceae after acetone extraction, partitioning with cyclohexane and then chloroform, and purification on a Florisil R column eluted with dichloromethane-acetone 56 and 12 dyes in food extracts after elution from an XAD-2 column with acetone, methanol, and water Column chromatographic cleanup, which usually employs large volumes of solvents to elute fractions of the sample, has been largely replaced by SPE in order to speed up and simplify extraction and cleanup and save on the cost of purchasing and disposing of solvents.

Modern Sample Preparation Systems The field of sample preparation has moved increasingly toward the use of disposable microcolumns and cartridges in order to speed up and simplify extraction and cleanup. These sample preparation systems are of two basic types. Columns packed with diatomaceous earth and designed for efficient liquid-liquid extractions in place of separatory funnels are available with capacities ranging from 0. The packing is either unbuffered or buffered at pH 4. The aqueous sample is poured into the column, and after a 5 min wait, organic extracting solvent is poured into the column.

The eluent containing the analyte is collected, evaporated to dryness under nitrogen flow, reconstituted in an appropriate solvent, and spotted for TLC analysis. Extraction columns of this type are used for screening drugs of abuse in urine e. The second method, SPE, uses sorbent phases with a variety of mechanisms and formats. The most common formats are microcolumns or cartridges with mg of sorbent packed in mL syringe barrels. Other SPE formats include pipet tips, disks, fixed well plates, flexible well plates, well plates, and large-volume cartridges and flash Chromatography columns The well plates are compatible with the use of TLC for drug discovery combinatorial chemistry high-throughput applications The sorbents available from Varian in their Bond Elute columns are illustrative of the products of other SPE product manufacturers.

These include the following. Bond Elute sorbents are supplied in 50 mg to 10 g weights in cartridges up to 60 mL in volume. Figure 1 shows a Speedisk J. Totally automated SPE systems are also available commercially SPE is used to concentrate solutes from dilute solution, e. The analytes are recovered by elution from the column with a few milliliters of an appropriate solvent and spotted for TLC. The concentration factor obtained for this method, which has been termed "trace enrichment," is the ratio of the sample volume to the elution volume.

SPE can also be used to purify concentrated solvent extracts in place of classical large columns that require up to hundreds of milliliters of elution solvents. Photograph supplied by Mallinckrodt Baker Inc. The basic steps of SPE, illustrated for the most commonly used reversed-phase C18 cartridge, can be summarized as follows: Conditioning.

The cartridge is prepared for receiving the sample by passing a volume of an appropriate solvent followed by a volume of liquid similar to the sample matrix. For the C,s cartridge, methanol is passed through followed by water for extraction of an aqueous sample. The sample is applied, and the analyte and other components with attraction for the sorbent are retained. Non- or weakly attracted components will pass through, providing the first stage of cleanup. With the C1K cartridge, the most polar interferences will elute first, and retention increases as polarity decreases.

One or more solvents with decreasing polarity are passed through to elute interferences that are more polar than the analyte but keep the analyte on the column. A sufficiently nonpolar eluent is passed to remove the analyte. Interferences more nonpolar than the analyte will have a greater attraction for the C,8 sorbent and remain uneluted. The following is an abbreviated guide to the SPE of different classes of sample analytes: Nonpolar extraction.

A polar solution water, buffers containing a nonpolar analyte is applied to a C l s , Cs, C2, CNE, CH, PH, or 2OH column that was preconditioned with methanol followed by water or buffer see listing above for abbreviations. The sample must be buffered, if necessary, to suppress analyte ionization. Polar interferences are removed. The analyte is eluted with a nonpolar solvent such as methanol, acetonitrile, tetrahydrofuran THF , hexane, or methylene chloride. Polar extraction. A nonpolar solution containing a polar analyte is applied to an SI, CN, 2OH, or NH2 column that was preconditioned with the nonpolar solvent in which the analyte is dissolved, such as hexane or chloroform.

Viscous samples are diluted in a nonpolar solvent, and water is removed from the sample, e. Nonpolar interferences are removed by washing with a nonpolar solvent or a polar-nonpolar mixture that is not strong polar enough to elute the analyte. The analyte is recovered by elution with a polar solvent such as methanol or isopropanol. Anion-exchange extraction.

Both the chosen column and the analyte must be ionic for exchange to occur. The sample pH is adjusted as above for conditioning and applied to the column. Interferences are removed by washing with the sample buffer and with an organic solvent such as acetonitrile or methanol, if necessary. The eluents can be totally aqueous or aqueous-organic mixtures; addition of an organic modifier such as methanol may improve analyte recovery. Cation-exchange extraction. The sample pH is adjusted in the same manner. Interferences are eliminated by elution with the sample buffer and with an organic solvent, if necessary.

Addition of an organic modifier such as methanol may improve analyte recovery. Examples of applications of SPE prior to TLC analysis include analysis for pesticides in fruits and vegetables according to the official German multimethod S19 using SPE on silica gel and amino cartridges prior to HPTLC with gradient elution AMD 60 ; oxygenated cholesterol derivatives in plasma using silica gel SPE 61 ; quinoline and quinuclidine alkaloids in pharmaceutical preparations using cation-exchange SPE 62 ; rutin in glycerinic plant extracts using Envi Supelco cartridges 63 ; and aflatoxins in a variety of foods using phenyl, silica, C18, and FlorisilC18 cartridges A strategy for choosing SPE cartridge elution solvents based on the PRISMA TLC mobile-phase optimization procedure was demonstrated for extraction of furocoumarin isomers and flavonoid glycosides from medicinal and aromatic plants The use of immunoaffinity columns for sample cleanup is among the newest sample preparation procedures.

Immunoaffinity cleanup was used after methanol extraction for determination of aflatoxins B-l, B-2, G-l, and G-2 in various food matrices by TLC-densitometry Automated Soxhlet extraction, microwave-assisted extraction MAE , and accelerated solvent extraction ASE have good potential for preparing solid samples for TLC analysis, but published methods have not yet appeared. Stahl first interfaced SFE with TLC in , and there has been increasing interest in developing new methods in recent years. Examples of SFE-TLC analyses reported include cyanizine herbicide in soil 67 ; flavonoids in Scutellariae radix 68 ; aloin and aloe-emodin in consumable aloe products 69 ; semi volatile compounds in cassia and cinnamon 70 ; and residues of 20 pesticides of multiple classes in soil Hydroperoxides in combustion products were separated from solid matrices using SFE with on-line transfer to TLC plates Desalting is often required for samples such as urine, serum, and tissue culture media in order to eliminate streaking and the formation of unresolved zones in the TLC of amino acids, carbohydrates, and other hydrophilic compounds.

Salts are removed from samples by performing ion exchange, using a desalting column, dialysis, and passage through a nonpolar sorbent. A simple desalting procedure suitable for 0. The sample is dried under air at 45C and then extracted with 1 mL of 0. Deproteinization When proteins may interfere with TLC analysis, they must be removed by deproteinization procedures. The technique has been used to deproteinize biological fluids prior to their analysis for drugs Proteins in samples such as serum, urine, tissue, and milk can be precipitated by addition of trichloroacetic acid 75 , perchloric acid, or sulfosalicylic acid followed by centrifugation and removal of the supernatant, which may or may not require further cleanup prior to TLC.

Protein removal from various types of samples has also been carried out by pH modification, denaturation with chaotropic agents or organic solvents, addition of a compound that competes for binding sites, and the use of restricted-access media. The preparation of derivatives in TLC was reviewed by Edwards 76 , who documented the application of derivatization techniques to a wide range of compounds including amino acids, steroids, drugs, and environmental pollutants.

Fluorescent derivatives for TLC were reviewed by Wintersteiger One of the major advantages of TLC is the use of derivatization postchromatography for the purpose of zone detection. This is normally achieved by spraying the layer with or dipping it into a solution of an appropriate reagent or reagents and then drying or heating to complete the reaction.

Hundreds of such reagents have been described to cause zones to absorb visible or ultraviolet radiation or to become fluorescent for organic species in general or to react selectively with particular compound classes see Sec. Postchromatographic derivatization allows the reaction of all standards and samples simultaneously under the same conditions, and the separation properties of the solutes are not changed by the reaction. Prechromatographic derivatization is advantageous when the parent compound is too volatile for TLC but the derivative is less volatile, the derivative is easier to separate from other sample constituents, the derivative has greater stability e.

A disadvantage of prederivatization is that the introduction of usually high molecular weight functional groups into the derivative may equalize the chromatographic properties of similar substances and make separation more difficult. In addition, prederivatization of each sample prior to its application can be tedious and time-consuming, by-products of the reaction may interfere with the TLC separation, or the presence of excess reagent may cause a background that interferes with quantification by scanning.

It is possible in some cases to derivatize in situ prior to chromatography. This is usually done by applying a spot or band of excess reagent to the origin and overspotting the sample while the reagent zone is still moist, followed by application of heat to accelerate the reaction, if necessary. Zones of sample and. Many different kinds of in situ prechromatographic derivatization have been reviewed In many cases, enantiomers have been resolved by TLC after the formation of derivatives, e.

The latest methodology involves separation of enantiomers of compounds such as chiral drugs by TLC without their prior derivatization Evaporation of Solutions Most sample preparation procedures require concentration or evaporation to dryness of sample extracts, combined partition solvent batches, or column effluents. It is important that evaporations be carried out without loss or degradation of the analyte, and studies may be required to determine which of the available methods is best to use in each particular situation.

A common method of concentration uses a rotary evaporator with an attached round-bottomed flask. A helpful variation is to place the solution in a Kuderna-Danish evaporative concentrator flask with attached lower calibrated tube Kontes , so that the concentrate ends up in the tube and can be applied to the layer without transfer. Nitrogen blowdown is the recommended method for concentration of small volumes of volatile organic solvents. Gas is supplied to the sample, held in a tube or vial, through Tygon tubing connected to a glass capillary. The sample is warmed in a C water bath to speed evaporation.

Various commercial devices that allow simultaneous blowdown of multiple samples are available.

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Reconstitution of Evaporated Residues It is common practice to evaporate solutions just to dryness and then dissolve the residue in an exact volume of the same or a different solvent, from which a known aliquot or the total sample is applied to the layer. The best initial zones on silica gel are obtained if the solvent is highly volatile and as nonpolar as possible, consistent with complete solubility and stability of the analyte s.

By use of a nonpolar solvent, purification can be achieved if some polar impurities in the residue are left undissolved selective solvation. Solvents with a high boiling point or polarity are difficult to remove from the sorbent during application. If a small amount of solvent is retained after application, it can adversely affect the separation by causing zone spreading or deformation or a different Rf value. Care must be taken, however, because hot air used to dry solvent at the origin can decompose labile substances on the surface of an active sorbent.

A volatile sample solvent promotes the production of small, regular initial zones, but containers must be kept tightly sealed except when filling the sample application device. Sorbent materials and layers are described in Chapter 4 of this Handbook and Chapter 3 of Ref. A great variety of commercial precoated layers are available for TLC on glass, plastic, or aluminum foil supports in 20 X 20 cm size. For mechanical stability, 0.

Plates with gypsum binder, which are known as "soft layers" and are designated with a G, must be used with greater care than "hard" organic polymer-bound layers to avoid abrasive conditions. Binder-free silica gel plates containing a small amount of colloidal silica to aid layer adherence are also available.

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For detection of zones by fluorescence quenching, plates are impregnated with indicator compounds e. Glass is the most inert support material, and its planarity is advantageous when the layer will be scanned for quantitative analysis. Procedures and devices for preparing homemade plates are described in Chapter 3 of the third edition of Fried and Sherma 1. Homemade plates, the quality of which is almost never equivalent to that of commercial plates, are rarely made except when a needed layer is not available or cost is a major consideration.

To remove extraneous materials that may be present due to manufacture, shipping, or storage conditions, it is advisable to preclean plates before use. This has often been done by predevelopment to the top with dichloromethane-methanol or the mobile phase to be used for the analysis. The following two-step HPTLC plate cleaning method has been proposed 90 for surface residue removal in critical applications when optimum sensitivity is required for detection and quantification: Develop the plate to the top with methanol, air dry for 5 min, totally immerse the plate in a tank filled with methanol, air dry for 5 min, oven dry for 15 min at 80C, and cool in a desiccator before use.

The routine activation of adsorbents at C for 30 min, or at a higher temperature, is often proposed in the literature, but this treatment is not usually necessary for commercial plates unless they have been exposed to high humidity. RP plates do not require activation prior to use. Suggestions for initial treatment, prewashing, activation, and conditioning of different types of glass- and foil-backed layers have been published Silica gel is by far the most frequently used layer material for adsorption TLC.

Some characteristic properties, including porosity, flow resistance, particle size, optimum velocity, and plate height, have been tabulated for three popular brands of silica gel TLC and HPTLC plates Separations take place primarily by hydrogen bonding or dipole interaction with surface silanol groups by using lipophilic mobile phases, and analytes are separated into groups according to their polarity. Specific differences in the types and distributions of silanol groups for individual sorbents may result in selectivity differences, and separations will not be exactly reproducible on different brands of silica gel layers Other TLC adsorbents include aluminum oxide alumina , magnesium oxide [used mostly for carotenoid pigment separations 92 ], magnesium silicate Florisil 93 , polyamide, and kieselguhr Alumina 95 is a polar adsorbent that is similar to silica gel in its general chromatographic properties, but it has an especially high adsorption affinity for carbon-carbon double bonds and better selectivity toward aromatic hydrocarbons and their derivatives.

The alumina surface is more complex than silica gel, containing hydroxyl groups, aluminum cations, and oxide anions, and pH and hydration level alter separation properties It is available in basic pH , neutral , and acid Polyamides 6 Nylon 6; polymeric caprolactam and 11 polymeric undecanamide have surface CONH groups and show high affinity and selectivity for polar compounds that can form hydrogen bonds with the exposed carbonyl groups.

However, depending on the type of analyte and mobile phase, three separation mechanisms can operate with polyamide: adsorption, partition normal- and re versed-phase , and ion exchange. This has led to separations of compounds from a wide array of chemical classes such as amino acids, phenols, phenolic compounds, carboxylic acids, cyclodextrins 96 , coumarins, and flavonoids Polyamide has been impregnated with various metal salts to improve the separation of sulfonamides Homemade mixed sorbent layers have been used by various workers to increase the resolution of certain samples compared to that obtained on the separate phases.

Binary layers that have been reported include silica gel-alumina , kieselguhr-alumina, alumina-calcium sulfate, magnesia-kieselguhr, cellulose-silica gel, poly amide-silica gel, polyamide-kieselguhr, polyamide cellulose, polyamide-glass powder similar to silica gel , silica gel-kieselguhr , and alumina-cellulose The properties of these mixed layers are usually somewhere between those of the two separate phases but are impossible to predict or explain with certainty.

Information on and applications of mixed layers are mostly contained in older standard TLC texts and reviews. Partition, Preadsorbent, and Impregnated Layers. Compounds that have the same polarity and functional group and migrate together on silica gel can often be resolved by partition TLC.

Crystalline cellulose AVICEL or high-purity fibrous cellulose serves primarily as a support material for the NP liquid-liquid partition TLC of polar substances, such as amino acids , and water-soluble biopolymers, although adsorption effects cannot be excluded in many cases. The stationary phase is either water or an impregnated polar liquid such as dimethylformamide. Cellulose used to prepare thin layers differs from that in chromatography paper mainly by having shorter fiber length yum , resulting in the same migration sequence for a series of compounds developed with a given mobile phase but less diffusion and higher efficiency than in paper chromatography.

Kieselguhr diatomaceous earth and synthetically prepared silicon dioxide Merck silica 50, are small surface area, weak adsorbents that are used as the lower cm inactive sample application and concentrating zone in the manufacture of silica gel and C18 preadsorbent plates. Layers have been impregnated with buffers, chelating agents, metal ions, or other compounds to aid in the resolution or detection of certain compounds see Ref.

If plates are prepared in the laboratory, the reagent is usually added to the stationary-phase slurry. Reagents are applied to precoated plates by spraying, brushing, horizontal or vertical dipping, development, or overdevelopment Analtech precoated plates are available already impregnated with potassium oxalate to facilitate resolution of polyphosphoinositides, magnesium acetate for phospholipids, 0. Other reagents that have been added to thin layers to improve separations include ion-pairing reagents , molybdic acid for separation of carbohydrates , boric acid carbohydrates and lipids , polycyclic aromatic hydrocarbons PAHs , formation of charge transfer complexes with numerous organic compounds , surfactants sulfa drugs and substituted pyrazoles , EDTA reduces tailing of drugs , urea wax esters and hydroxybenzenes , ferric ion carboxy- and hydroxybenzenes , cupric ion glucose and sorbitol , caffeine PAHs , and ammonium sulfate surfactants.

High-Performance Layers. High-performance layers are more efficient, leading to tighter zones, better resolution, and more sensitive detection. Flow resistance is higher migration time per centimeter is slower , but overall development time is shorter because smaller migration distances are used for HPTLC than for TLC typically cm versus cm.

Sample sizes are generally 0. Silica gel is the most widely used type of HP plate, but other HP layers, including bonded phases, are also commercially available. Layers with spherical particles offer better efficiency, spot capacity, and detection limits than those with nonspherical particles. The silica gel matrix on the sheets is designed to have the least possible spectral interference for direct coupling of TLC with Raman spectrometry see Sec. Bonded Layers. Reversed-phase TLC, in which the stationary phase is less polar than the mobile phase, was originally carried out on silica gel or kieselguhr layers impregnated with a solution of paraffin, squalane, silicone oil, octanol, or oleyl alcohol.

Analtech sells RP plates with hydrocarbon liquid phase physically adsorbed onto the surface of a silica gel layer. Impregnated plates of this kind require the use of aqueous and polar organic mobile phases saturated with the stationary liquid, and they cannot tolerate the use of nonpolar organic solvents, which will strip the coating from the support. Bonded phases with functional groups chemically bonded to silica gel eliminate stripping of the stationary liquid from the support by incompatible mobile phases.

Alkylsiloxane-bonded silica gel with CH3, C2H5, C 8 H 17 , and C18H37 functional groups are most widely used for RP-TLC of organic compounds polar and nonpolar homologous compounds and aromatics , weak acids and bases after ion suppression with buffered mobile phases, and strong acids and bases using ion-pair reagents.

Layers from different companies but with the same bonded group can have different percentages of carbon loading and give different results. The hydrophobic nature of the layer increases with both the chain length and the degree of loading of the groups. Alkylsiloxanebonded layers with a high level of surface modification are incompatible with highly aqueous mobile phases and are used mainly for normal-phase separations of low-polarity compounds The latter layers with a low degree of surface coverage and more residual silanol groups exhibit partially hydrophilic as well as hydrophobic character and can be used for RP-TLC and NP-TLC.

Chemically bonded phenyl layers are also classified as reversed-phase, but their use has only seldom been reported in the literature. Hydrophilic bonded silica gel containing cyano , amino , or diol groups bonded to silica gel through a trimethylene chain [ CH2 3] are compatible with aqueous mobile phases and exhibit multimodal mechanisms. Cyano layers can act as a normal or reversed phase, depending on the characteristics of the mobile phase, with properties similar to a low-capacity silica gel and a short-chain alkylsiloxane bonded layer, respectively Amino layers are used in NP and weak anion-exchange modes.

In NP-TLC, compounds are retained on amino layers by hydrogen bonding as with silica gel, but the selectivity is different. Charged substances such as nucleotides or sulfonic acids can be separated by ion exchange using. Although there is limited retention in RP-TLC, the separation of oligonucleotides on amino layers based on differences in hydrophobic properties of the compounds has been reported.