Thin Layer Chromatography Practical

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  Modern Instrumental Analysis

  • Satinder Ahuja, Neil Jespersen(Authors)
  • 2006(Publication Date)
  • Elsevier Science

    (Publisher)

  Chapter 13 Thin-layer chromatography Pamela M. Grillini 13.1 INTRODUCTION Thin-layer chromatography (TLC) is one of the most popular and widely used separation techniques because of its ease of use, cost-effectiveness, high sensitivity, speed of separation, as well as its capa-city to analyze multiple samples simultaneously. It has been applied to many disciplines including biochemistry [1,2] , toxicology [3,4] , pharma-cology [5,6] , environmental science [7] , food science [8,9] , and chemi-stry [10,11] . TLC can be used for separation, isolation, identification, and quantification of components in a mixture. It can also be utilized on the preparative scale to isolate an individual component. A large variety of TLC equipment is available and discussed later in this chapter. High-performance thin-layer chromatography (HPTLC) has become widely used and while it follows the same principles as TLC, it makes use of modern technology including automatic application devices and smaller plates, which allow for better sensitivity. TLC is related to paper chromatography (PC) as both use a station-ary phase and a liquid phase to move the sample [12] . A common ex-ample of PC is the separation of black ink into its individual colors. Because the individual molecules behave differently when exposed to a solvent such as water or isopropyl alcohol, they are retained on the paper at different intervals, creating a visible separation of the indi-vidual components. This helps to identify each component in a mixture. 13.2 THEORY AND BASIC PRINCIPLES In TLC, the sample is applied as a small spot or streak to the marked origin of stationary phase supported on a glass, plastic, or metal plate. The sample solvent is allowed to evaporate from the plate that is then placed in a closed chamber containing a shallow pool of mobile phase at Comprehensive Analytical Chemistry 47 S. Ahuja and N. Jespersen (Eds) Volume 47 ISSN: 0166-526X DOI: 10.1016/S0166-526X(06)47013-6 r 2006 Elsevier B.V.

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  Chromatographic Analysis of Alkaloids

  • Milan Popl, Jan Fahnrich, Vlastimil Tatar(Authors)
  • 2018(Publication Date)
  • CRC Press

    (Publisher)

  6 Thin-Layer Chromatography

  In contrast to paper chromatography, which is used rarely nowadays due to its low speed and efficiency, thin-layer chromatography (TLC) is well established and widely used in various fields of analytical chemistry (1 , 2 , 3 , 4 , 5 , 6 ). Because quantitation with this technique is not straightforward, its long-time domain has been qualitative analysis. Besides giving retention data, the chemical reactions with various reagents performed directly on the sorbent layer can easily be used for the identification of unknown samples. Identification can rely on a large amount of data accumulated in the past. An extensive summary of alkaloid TLC was completed by Baerheim Svendsen and Verpoorte (7 ). For identification purposes the preferred separation systems are those in which retention data for a great number of compounds have been established. Unfortunately, only a few systematic studies cover more than hundreds of compounds. As an example, the important systems for drug identification can be mentioned here. Many TLC data for drugs were accumulated in early studies (8 ). A compilation of TLC data (9 ) enabled completion of a computer-based identification system utilizing TLC, GC, and spectral data (10 ) with 1600 drugs included in this data base. A dissimilarity index was used to match unknown samples with the data file. Another approach utilizes principal component analysis of RF values for 362 drugs in four separation systems using silica gel layers (11 ). Quantitative analysis using TLC, although more difficult, is gaining importance. With suitable equipment the accuracy of determination may be comparable to that of other chromatographic methods.

  I. TECHNIQUE OF TLC

  As far as laboratory equipment is concerned, TLC is the simplest chromatographic technique. Essentially only suitable vessels containing mobile phase and precoated plates are required in order to perform separations. Even in that way TLC possesses numerous advantages over column chromatography. Several samples can be analyzed simultaneously in a single run. Since chromatographic plates are used only once, samples with a relatively complex matrix can be separated, in contrast to column liquid chromatography, in which column contamination is to be avoided. Far simpler purification steps are therefore satisfactory. All components of the sample are detectable on the TLC plate. Strongly retained components eluted as broad peaks, difficult to detect, or not eluted at all in column liquid chromatography are in TLC concentrated in small spots near the start and can be sensitively detected. With certain precautions TLC can be used as a pilot technique for solvent system selection in column chromatography. The possibility of two-dimensional separation multiplies the resolving power of the TLC, making possible the analysis of complex mixtures.

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  Chromatography Today

  • C.F. Poole, S.K. Poole(Authors)
  • 2012(Publication Date)
  • Elsevier Science

    (Publisher)

  649 CHAPTER 7 THIN-LAYER CHROMATOGRAPHY 7 .1 Introduction 649 7 . 2 Theoretical Considerations 653 7.2.1 Solvent Migration Through Porous Layers 655 7.2.2 Band Broadening and the Plate Height Equation 662 7.2.3 Resolution and Separation Capacity 667 7 . 3 Stationary Phases for TLC 671 7.3.1 Inorganic Oxide Adsorbents 672 7.3.2 Chemically Bonded Layers 67 6 7.3.3 Cellulose Layers 681 7.3.4 Chiral Stationary Phases 681 7 . 4 Development Techniques 684 7.4.1 Linear and Radial Development 684 7.4.2 Continuous Development 686 7.4.3 Multiple Development 687 7.4.4 Two-Dimensional Development 69 3 7 . 5 Mobile Phase Optimization 697 7.6 Quantitative Evaluation of Thin-Layer Chromatograms 704 7 .7 Instrumentation for Thin-Layer Chromatography 7 09 7.7.1 Sample Application 710 7.7.2 Development Chambers 713 7.7.3 Scanning Densitometry 720 7 . 8 Preparative Thin-Layer Chromatography 726 7. 9 References 728 7.1 INTRODUCTION Thin-layer chromatography (TLC) is a type of liquid chromatography in which the stationary phase is in the form of a thin layer on a flat surface rather than packed into a tube (column). It is a member of a family of techniques that include some types of electrophoresis and paper chromatography, more generally referred to as planar chromatography. Since we will not discuss electrophoresis in this section, and since TLC has virtually superseded paper chromatography in most analytical 650 laboratories, we will confine ourselves to a discussion of TLC. The introduction of new TLC layers prepared from small diameter particles of a narrow size range in the mid-1970s revolutionized the practice of TLC. The techniques employed with these new layers became known as high performance TLC, modern TLC, or instrumental TLC, to distinguish them from earlier practices, generally referred to as conventional TLC. These differences, Table 7.1, are not of a fundamental nature but rather represent a further optimization of all aspects of the separation process in TLC.

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  Paper and Thin Layer Chromatography

  • Ivor Smith, J. W. T. Seakins, Ivor Smith, J. W. T. Seakins(Authors)
  • 2013(Publication Date)
  • Butterworth-Heinemann

    (Publisher)

  CHAPTER 3 THIN LAYER CHROMATOGRAPHY Ivor Smith and R. S. Ersser ALTHOUGH a number of workers had previously described procedures for thin layer chromatography, TLC, the first practical description of apparatus and technique was that of Stahl. (1) His contribution was not limited to this, however, as it was equally important to make avail-able suitable materials for preparing the layer and his investigation of the factors concerning the manufacture of such materials was of at least equal importance. Indeed the first suitable materials were commercially described as after Stahl and many still carry that label. TLC is, in principle, a variant of paper chromatography, PC. Where-as PC came into general use rather slowly because the apparatus was usually home-made and somewhat temperamental, TLC was adopted rapidly. This was to be expected as the wealth of practical information gained over years of use with paper could be applied almost unchanged to the new technique. The technique of TLC can be divided into two discrete techniques, namely the preparation of suitable TL plates and chromatography on such plates. Once the plates are prepared, the technique of chromatography is identical with that used for paper. PC, i.e. chromatography on a sheet (or layer) of cellulose, was highly successful in the field of ionic and polar molecules. It was highly unsuccessful in the field of non-polar or lipid molecules. TLC on silica gel brought the chromatography of lipids into use as a simple routine technique comparable with the paper technique for aminoacids. Not only were the separations as simple to perform but they occurred in minutes instead of hours, the technique required smaller quantities of materials as the spots were smaller and reagents appeared more sensitive but, also, more corrosive location reagents could be used on the inert silica gel layer.

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  A Century of Separation Science

  • Haleem J. Issaq(Author)
  • 2001(Publication Date)
  • CRC Press

    (Publisher)

  4 Thin Layer Chromatography Joseph Sherma Lafayette College, Easton, Pennsylvania The term thin layer chromatography will be used throughout this chapter rather than planar chromatography, a general classification that has been used recently to denote chromatography modes having a flat stationary phase. Paper chromatography is a type of planar chromatography, but the term has been used incorrectly to include electrochromatography or electrophoresis be-cause this method does not involve the elements necessary for designation as a chromatographic method as defined by Strain in 1942 [1], i.e., substances in a narrow initial zone are caused to undergo differential migration on a porous, sorptive stationary phase having a selective resistive action by the nonselective driving force of a liquid or gaseous mobile phase. I. EARLY HISTORY OF THIN LAYER CHROMATOGRAPHY (1889 TO EARLY 1960s) The history of thin layer chromatography (TLC) has been traced back to experiments performed by the Dutch biologist Beyerinck in 1889 [2]. These predate the early work on column chroma-tography reported by Tswett in 1906 [3] but are predated by the paper chromatography work of Runge, Schoenbein, and Goppelsroeder [4] during the period 1834-1888. Beyerinck allowed a drop of a mixture of hydrochloric and sulfuric acids to diffuse through a thin layer of gelatin on a glass plate. The hydrochloric acid traveled faster than the sulfuric acid and formed a ring around the latter. The hydrochloric acid zone was made visible by reaction with silver nitrate and the sulfuric acid with barium chloride. Tswett did not study TLC but discussed adsorption of compounds on strips of paper during capillary analysis [5]. Consden, Martin, and Gordon reintroduced paper chromatography in 1944 [6], and it grew into a universally used microanalyti-cal method during the next 10 years.

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  Handbook of Isolation and Characterization of Impurities in Pharmaceuticals

  • Satinder Ahuja, Karen Mills Alsante(Authors)
  • 2003(Publication Date)
  • Academic Press

    (Publisher)

  9 ISOLATION METHODS I: THIN-LAYER CHROMATOGRAPHY PAMELA M. GORMAN AND HONG JIANG Pfizer Global Research & Development Division,Groton,CT 06340 I. INTRODUCTION TO THIN-LAYER CHROMATOGRAPHY (TLC) A. History B. Fundamentals of TLC C. Why UseTLC? II. TLC APPLICATIONS IN PHARMACEUTICAL INDUSTRY III. TLC METHOD DEVELOPMENT AND VALIDATION A. Drug Substance Method Development and Validation B. Drug Product Method Development and Validation IV. IMPURITY ISOLATION AND CHARACTERIZATION BY TLC A. TLC-Specified Impurities B. Known Impurities C. Unknown Impurities D. Summary of Impurity Isolation and Characterization byTLC REFERENCES I. INTRODUCTION TO THIN-LAYER CHROMATOGRAPHY (TLC) Thin-layer chromatography (TLC) is one of the most popular and widely used separation techniques because of its ease of use, cost-effectiveness, high sensitivity, speed of separation, as well as its capacity to analyze multiple samples simultaneously. It has been applied in the disciplines of biochemistry, 1,2 toxicology, 3,4 pharmacology, 5,6 environmental science, 7 food science, 8,9 and chemistry. 10,11 TLC can be utilized for separation, isolation, identification, and quantification of components in a mixture. It can also be utilized on a preparative scale to isolate a particular component. A large variety of TLC apparatus is commercially available. 203 A. History Pioneer work in thin-layer chromatography to isolate and analyze medicinal compounds was performed by Izmailov and Shraiber on unbound alumina as early as 1938. 12 However, E. Stahl introduced the term ‘‘thin-layer chromatography’’ in 1956, which was considered the beginning of modern TLC. 13 Since the 1960s, commercialization of precoated TLC plates and automation of sample application and detection have made it accessible to all laboratories. A number of valuable texts have been written about the history of TLC. 14–20 The most recent one is reviewed by C. F. Poole.

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  Applied Thin-Layer Chromatography

  Best Practice and Avoidance of Mistakes

  • Elke Hahn-Deinstrop(Author)
  • 2007(Publication Date)
  • Wiley-VCH

    (Publisher)

  The applied substance mainly ends up at the edge of the spot. Then, on chromatographic development, peaks with an ap- proximate Gaussian distribution are obtained whose width increases as the polarity of the solvent increases. Figure 24a–c, in conjunction with [2], shows the substance distri- bution as a function of the solvent used. Up to this point in the description of TLC, we have not differentiated between the differing experimental requirements associated with qualitative, quantitative or pre- parative results. However, very different attitudes lie behind these three concepts and the consequent demands made on the separation techniques used. Preparative work is not the main topic of this book, so that classification must be in accordance with the other areas of TLC, i.e. identity testing, purity testing and assay- ing. The demands made on these and hence the differences between them begin with the choice of the layers and end only with the documentation. 50 3 Before the TLC Development Process Figure 24. Substance distribution as a function of the solvent system used (from [2, p. 210]) (a) After applying (top view) (b) After applying (profile) (c) After developing (profile) (Note: benzene is carcenogenic! It should be replaced with toluene or a similar solvent) 3.6.1 Manual Application of Samples You have now carried out the necessary steps of sample preparation, and your samples are now in solution and ready to be applied. Before you take the selected plate mate- rial out of the package or the desiccator, please consider how your chromatograms should appear later on, e.g. with respect to the positioning of the samples, migration distance etc. Practical Tip for positioning the samples: Fill out the form “TLC III” (see Section 9.3 “GMP/GLP-Conforming Raw Data Sheets”) and place this or fix it with a clothes peg or similar device at your laboratory work bench.

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  Preparative Liquid Chromatography

  • B.A. Bidlingmeyer(Author)
  • 1987(Publication Date)
  • Elsevier Science

    (Publisher)

  PTLC on l a y e r s 1 mm t h i c k was r e p o r t e d by R i t t e r and Meyer i n 1962 E l l . E a r l i e r p r e p a r a t i v e work [ e . g . , 21, a l t h o u g h termed TLC, was a c t u a l l y performed on adsorbent b a r s used as columns or o n a n a l y t i c a l l a y e r s a f t e r column chromatography [ 3 , 4 1 . PTLC has a l s o been c a r r i e d o u t on c o n i c a l [ 5 1 and c y l i n d r i c a l [61 l a y e r s , on layer-coated b e l t s [71, i n l a y e r s supported o n a s t a i n l e s s s t e e l framework [81, and w i t h o t h e r s p e c i a l apparatuses and l a y e r forms. However, p r e p a r a t i v e a p p l i c a t i o n s of TLC a r e most o f t e n performed on r e g u l a r , f l a t t h i n l a y e r s with increased t h i c k n e s s , and i t i s t h i s v a r i a t i o n of TLC t h a t w i l l be discussed m a i n l y i n t h i s c h a p t e r . subsections w i 11 focus on t h e experimental techniques used t o accommodate l a r g e r samples i n TLC and w i l l a l s o i n c l u d e t h e p r i n c i p l e s i n v o l v e d i n t h e scale-up of a n a l y t i c a l TLC, where a p p r o p r i a t e . The f o l l o w i n g 2.2 LAYERS FOR PTLC P r e p a r a t i v e l a y e r s a r e made i n t h e l a b o r a t o r y or a r e purchased commercially precoated. For homemade l a y e r s , s p e c i a l sorbents t h a t a r e s u p p l i e d by some f i r m s for PTLC are u s u a l l y used. t h i c k n e s s for PTLC i s 0.5-2.0 mm (500-2000 pm), and t h e s i z e of t h e p l a t e s i s The most popular l a y e r 107 usually 5 x 20, 10 x 20, 20 x 20. 20 x 40, or even 20 x 100 cm. The conditions for successful separations on preparative plates include a homogeneous layer, regularly applied sample, and a well-saturated developing chamber. The latter is important because a substance moves in the layer with changing velocity according to the rate of evaporation of the solvent.

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  Quality Control and Evaluation of Herbal Drugs

  Evaluating Natural Products and Traditional Medicine

  • Pulok K. Mukherjee(Author)
  • 2019(Publication Date)
  • Elsevier

    (Publisher)

  Sorbents with large surface areas adsorb not only water but also other vapors that may be present in the laboratory air. Layers that have been exposed to the laboratory atmosphere for a prolonged period afford pronounced dirt zones, generally in the area of the solvent front or of β-fronts and fail to give reproducible results. Because the plastic packaging of thin-layer plates is not impermeable to gases, it will not completely protect the plates from environmental contaminants. Storage in plate cassettes protects them from the laboratory air and thus from contamination. Owing to their adsorptive activity, sorbents always contain small quantities of contaminants originating from the environment (e.g., pesticides, plasticizers), which they acquire during the production process. It may therefore be necessary in some cases to preclean the plates. As a rule, the TLC layer can be cleaned by predevelopment with chloroform–methanol (1:1) or with the solvent used for subsequent chromatography; such development should be performed over a longer distance than is planned for a position of the solvent in the subsequent chromatogram. The following points need to be considered:

  • ●  No grease should be used for sealing on storage of plates in glass vessels (desiccators).
  • ●  Storage over drying agents is generally unnecessary.

  8.5 Application of Samples

  In some cases, for example, in trace analysis or for samples with a complex matrix, samples cannot be applied directly to TLC plates but first have to be subjected to a cleaning and/or enrichment step (clean-up). In this way, a crude sample is prepared from the material to be studied, for example, by extraction, distillation, sublimation, or precipitation (to mention just the most important separation processes). The crude sample is subjected to further clean-up steps before chromatographic separation. Such clean-up steps should ideally lead to group or substance-specific separation of the analyte(s) (Mildau et al., 1984 ). For the preparation of the samples, see Table 8.2 . The following points should be kept in mind.

  • ●  Many clean-up steps have to be discovered and optimized empirically.
  • ●  Any error in clean-up will persist all the way through to the evaluation of the finished chromatogram.
  • ●  The equipment and solvents used during sample preparation must be absolutely clean and pure.
  • ●  Sufficient substance should be cleaned-up in order to permit controls (duplicate and replicate determinations).
  • ●  
    After clean-up, the samples should be chromatographed as quickly as possible, particularly in the case of labile substances. Overnight storage should be in a refrigerator and longer storage in a freezer.

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  Thin-Layer Chromatography

  A Modern Practical Approach

  • Peter E Wall, Roger M Smith(Authors)
  • 2007(Publication Date)
  • Royal Society of Chemistry

    (Publisher)

  quality of separation one desires. For quick identification purposes, the spotting technique with basic equipment is quite adequate. But where precision is required with a high degree of accuracy, automated band application is recommended. This is particularly important where the separated zones are very close together. Using concentration zone plates is not the answer to simple band application, except at a purely qualitative level as during sample band focusing most of the solute concentrates in the centre of the band. Hence sample distribution is uneven along the length of the band. 7 References 1. G. Guiochon and A.M. Siouffi, J. Chromatogr. , 1982, 245 , 1–20. 2. D.C. Fenimore in Instrumental HPTLC , W. Bertsch and R.E. Kaiser (eds), A. Hu ¨thig Verlag, Heidelberg, Germany, 1980, 81–95. 3. R.E. Kaiser in HPTLC High Performance Thin-Layer Chromatography , A. Zlatkis and R.E. Kaiser (eds), Elsevier, Oxford, UK, 1977, 21–32. 4. R.E. Kaiser in HPTLC High Performance Thin-Layer Chromatography , A. Zlatkis and R.E. Kaiser (eds), Elsevier, Oxford, UK, 1977, 88–90. 5. D.C. Fenimore and C.J. Meyer, J. Chromatogr. , 1979, 186 , 555–561. 6. D.C. Abbott and J. Thompson, Chem. Ind. , 1965, 310. 7. A. Musgrave, J. Chromatogr. , 1969, 41 , 470. 8. H. Halpaap and K.F. Krebs, J. Chromatogr. , 1977, 142 , 823–853. 85 Sample Application CHAPTER 5 Development Techniques 1 Introduction Thin-layer chromatography can be defined as a differential migration process where sample components are retained to differing degrees in a thin layer of sorbent as a solvent or solvent mixture moves by capillary action through the layer. The retention of analytes will depend on the interactions that occur with the liquid phase on the surface and contained within the porous physical structure of the sorbent, or there may be a direct interaction with the molecular structure of the sorbent. Also retention will be affected by the nature of the moving solvent and any additives contained in it.

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  Separation Methods in Drug Synthesis and Purification

  • Klara Valko(Author)
  • 2020(Publication Date)
  • Elsevier Science

    (Publisher)

  For preparative purposes, plates may have larger thickness, such as 500, 1000 and 2000 μm. TLC plates are supplied generally with binder; however, some manufacturers supply plates without binder. The TLC plates for analytical purposes may be purchased with a UV indicator for 254 nm. Some specialities of TLC plates are well-known. One of them is the plate with a concentrating zone. The concentrating zone is situated at a narrow strip having an extremely large pore diameter (over 1000 nm) and an extremely small surface area (less than 1 m 2 /g). The length and thickness of the concentrating zone is about 25 mm and 150 μm, while the active layer has dimensions such as 75–175 mm and 200–250 μm. The samples are loaded in the concentrating zone. The components will migrate with the front of the developing solvent (the concentrating zone shows practically no sorption) and reach the very sharp interface to the concentrating zone pass it, and in the chromatographic layer, the sample components are subjected to separation. HPTLC plates have finer particle sizes and higher separation power; details are given in books and booklets (for example, by Merck [ 39 ]). The major differences between TLC and HPTLC plates are given in Table 10.8. Precoated layers are available with chemically bonded phases. The most interesting group is silica gel in the reversed-phase (both TLC and HPTLC), silanized and with long-chain hydrocarbons. The silanization degree may be between 50% and 100%. In addition, these layers can also be purchased in a water-compatible form

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