Thin Layer Chromatography in PhytochemistryHajnos/Thin Layer Chromatography in Phytochemistry 46772_C000 Final Proof page i 28.1.2008 7:41am Compositor Name: PJayarajCHROMATOGRAPHIC SCIENCE SERIESA Series of Textbooks and Reference BooksEditor: JACK CAZES1. Dynamics of Chromatography: Principles and Theory, J. Calvin Giddings2. Gas Chromatographic Analysis of Drugs and Pesticides, Benjamin J. Gudzinowicz3. Principles of Adsorption Chromatography: The Separation of NonionicOrganic Compounds, Lloyd R. Snyder4. Multicomponent Chromatography: Theory of Interference, Friedrich Helfferich and Gerhard Klein5. Quantitative Analysis by Gas Chromatography, Josef Novk6. High-Speed Liquid Chromatography, Peter M. Rajcsanyi and Elisabeth Rajcsanyi7. Fundamentals of Integrated GC-MS (in three parts), Benjamin J. Gudzinowicz, Michael J. Gudzinowicz, and Horace F. Martin8. Liquid Chromatography of Polymers and Related Materials, Jack Cazes9. GLC and HPLC Determination of Therapeutic Agents (in three parts),Part 1 edited by Kiyoshi Tsuji and Walter Morozowich, Parts 2 and 3edited by Kiyoshi Tsuji10. Biological/Biomedical Applications of Liquid Chromatography, edited by Gerald L. Hawk11. Chromatography in Petroleum Analysis, edited by Klaus H. Altgelt and T. H. Gouw12. Biological/Biomedical Applications of Liquid Chromatography II, edited by Gerald L. Hawk13. Liquid Chromatography of Polymers and Related Materials II, edited by Jack Cazes and Xavier Delamare14. Introduction to Analytical Gas Chromatography: History, Principles, and Practice, John A. Perry15. Applications of Glass Capillary Gas Chromatography, edited by Walter G. Jennings16. Steroid Analysis by HPLC: Recent Applications, edited by Marie P. Kautsky17. Thin-Layer Chromatography: Techniques and Applications, Bernard Fried and Joseph Sherma18. Biological/Biomedical Applications of Liquid Chromatography III, edited by Gerald L. Hawk19. Liquid Chromatography of Polymers and Related Materials III, edited by Jack Cazes20. Biological/Biomedical Applications of Liquid Chromatography, edited by Gerald L. Hawk21. Chromatographic Separation and Extraction with Foamed Plastics and Rubbers, G. J. Moody and J. D. R. ThomasHajnos/Thin Layer Chromatography in Phytochemistry 46772_C000 Final Proof page ii 28.1.2008 7:41am Compositor Name: PJayaraj22. Analytical Pyrolysis: A Comprehensive Guide, William J. Irwin23. Liquid Chromatography Detectors, edited by Thomas M. Vickrey24. High-Performance Liquid Chromatography in Forensic Chemistry,edited by Ira S. Lurie and John D. Wittwer, Jr.25. Steric Exclusion Liquid Chromatography of Polymers, edited by Josef Janca26. HPLC Analysis of Biological Compounds: A Laboratory Guide, William S. Hancock and James T. Sparrow27. Affinity Chromatography: Template Chromatography of Nucleic Acidsand Proteins, Herbert Schott28. HPLC in Nucleic Acid Research: Methods and Applications, edited by Phyllis R. Brown29. Pyrolysis and GC in Polymer Analysis, edited by S. A. Liebman and E. J. Levy30. Modern Chromatographic Analysis of the Vitamins, edited by Andr P. De Leenheer, Willy E. Lambert, and Marcel G. M. De Ruyter31. Ion-Pair Chromatography, edited by Milton T. W. Hearn32. Therapeutic Drug Monitoring and Toxicology by LiquidChromatography, edited by Steven H. Y. Wong33. Affinity Chromatography: Practical and Theoretical Aspects, Peter Mohrand Klaus Pommerening34. Reaction Detection in Liquid Chromatography, edited by Ira S. Krull35. Thin-Layer Chromatography: Techniques and Applications, Second Edition, Revised and Expanded, Bernard Fried and Joseph Sherma36. Quantitative Thin-Layer Chromatography and Its IndustrialApplications, edited by Laszlo R. Treiber37. Ion Chromatography, edited by James G. Tarter38. Chromatographic Theory and Basic Principles, edited by Jan ke Jnsson39. Field-Flow Fractionation: Analysis of Macromolecules and Particles,Josef Janca40. Chromatographic Chiral Separations, edited by Morris Zief and Laura J. Crane41. Quantitative Analysis by Gas Chromatography, Second Edition,Revised and Expanded, Josef Novk42. Flow Perturbation Gas Chromatography, N. A. Katsanos43. Ion-Exchange Chromatography of Proteins, Shuichi Yamamoto,Kazuhiro Naka-nishi, and Ryuichi Matsuno44. Countercurrent Chromatography: Theory and Practice, edited by N. Bhushan Man-dava and Yoichiro Ito45. Microbore Column Chromatography: A Unified Approach to Chromatography, edited by Frank J. Yang46. Preparative-Scale Chromatography, edited by Eli Grushka47. Packings and Stationary Phases in Chromatographic Techniques,edited by Klaus K. Unger48. Detection-Oriented Derivatization Techniques in LiquidChromatography, edited by Henk Lingeman and Willy J. M. Underberg49. Chromatographic Analysis of Pharmaceuticals, edited by John A. Adamovics50. Multidimensional Chromatography: Techniques and Applications,edited by Hernan Cortes51. HPLC of Biological Macromolecules: Methods and Applications, edited by Karen M. Gooding and Fred E. RegnierHajnos/Thin Layer Chromatography in Phytochemistry 46772_C000 Final Proof page iii 28.1.2008 7:41am Compositor Name: PJayaraj52. Modern Thin-Layer Chromatography, edited by Nelu Grinberg53. Chromatographic Analysis of Alkaloids, Milan Popl, Jan Fhnrich, and Vlastimil Tatar54. HPLC in Clinical Chemistry, I. N. Papadoyannis55. Handbook of Thin-Layer Chromatography, edited by Joseph Shermaand Bernard Fried56. GasLiquidSolid Chromatography, V. G. Berezkin57. Complexation Chromatography, edited by D. Cagniant58. Liquid ChromatographyMass Spectrometry, W. M. A. Niessen and Jan van der Greef59. Trace Analysis with Microcolumn Liquid Chromatography, Milos KrejcI60. Modern Chromatographic Analysis of Vitamins: Second Edition, edited by Andr P. De Leenheer, Willy E. Lambert, and Hans J. Nelis61. Preparative and Production Scale Chromatography, edited by G. Ganetsos and P. E. Barker62. Diode Array Detection in HPLC, edited by Ludwig Huber and Stephan A. George63. Handbook of Affinity Chromatography, edited by Toni Kline64. Capillary Electrophoresis Technology, edited by Norberto A. Guzman65. Lipid Chromatographic Analysis, edited by Takayuki Shibamoto66. Thin-Layer Chromatography: Techniques and Applications: Third Edition, Revised and Expanded, Bernard Fried and Joseph Sherma67. Liquid Chromatography for the Analyst, Raymond P. W. Scott68. Centrifugal Partition Chromatography, edited by Alain P. Foucault69. Handbook of Size Exclusion Chromatography, edited by Chi-San Wu70. Techniques and Practice of Chromatography, Raymond P. W. Scott71. Handbook of Thin-Layer Chromatography: Second Edition, Revised and Expanded, edited by Joseph Sherma and Bernard Fried72. Liquid Chromatography of Oligomers, Constantin V. Uglea73. Chromatographic Detectors: Design, Function, and Operation,Raymond P. W. Scott74. Chromatographic Analysis of Pharmaceuticals: Second Edition,Revised and Expanded, edited by John A. Adamovics75. Supercritical Fluid Chromatography with Packed Columns: Techniquesand Applications, edited by Klaus Anton and Claire Berger76. Introduction to Analytical Gas Chromatography: Second Edition,Revised and Expanded, Raymond P. W. Scott77. Chromatographic Analysis of Environmental and Food Toxicants,edited by Takayuki Shibamoto78. Handbook of HPLC, edited by Elena Katz, Roy Eksteen, Peter Schoenmakers, and Neil Miller79. Liquid ChromatographyMass Spectrometry: Second Edition, Revised and Expanded, Wilfried Niessen80. Capillary Electrophoresis of Proteins, Tim Wehr, Roberto Rodrguez-Daz, and Mingde Zhu81. Thin-Layer Chromatography: Fourth Edition, Revised and Expanded,Bernard Fried and Joseph Sherma82. Countercurrent Chromatography, edited by Jean-Michel Menet and Didier Thibaut83. Micellar Liquid Chromatography, Alain Berthod and Celia Garca-Alvarez-CoqueHajnos/Thin Layer Chromatography in Phytochemistry 46772_C000 Final Proof page iv 28.1.2008 7:41am Compositor Name: PJayaraj84. Modern Chromatographic Analysis of Vitamins: Third Edition, Revised and Expanded, edited by Andr P. De Leenheer, Willy E. Lambert, and Jan F. Van Bocxlaer85. Quantitative Chromatographic Analysis, Thomas E. Beesley, Benjamin Buglio, and Raymond P. W. Scott86. Current Practice of Gas ChromatographyMass Spectrometry, edited by W. M. A. Niessen87. HPLC of Biological Macromolecules: Second Edition, Revised and Expanded, edited by Karen M. Gooding and Fred E. Regnier88. Scale-Up and Optimization in Preparative Chromatography: Principles and Bio-pharmaceutical Applications, edited by Anurag S. Rathore and Ajoy Velayudhan89. Handbook of Thin-Layer Chromatography: Third Edition, Revised and Expanded, edited by Joseph Sherma and Bernard Fried90. Chiral Separations by Liquid Chromatography and RelatedTechnologies, Hassan Y. Aboul-Enein and Imran Ali91. Handbook of Size Exclusion Chromatography and Related Techniques:Second Edition, edited by Chi-San Wu92. Handbook of Affinity Chromatography: Second Edition, edited by David S. Hage93. Chromatographic Analysis of the Environment: Third Edition, edited by Leo M. L. Nollet94. Microfluidic Lab-on-a-Chip for Chemical and Biological Analysis and Discovery, Paul C.H. Li95. Preparative Layer Chromatography, edited by Teresa Kowalska and Joseph Sherma96. Instrumental Methods in Metal Ion Speciation, Imran Ali and Hassan Y. Aboul-Enein97. Liquid ChromatographyMass Spectrometry: Third Edition, Wilfried M. A. Niessen98. Thin Layer Chromatography in Chiral Separations and Analysis, edited by Teresa Kowalska and Joseph Sherma99. Thin Layer Chromatography in Phytochemistry, edited by Monika Waksmundzka-Hajnos, Joseph Sherma, and Teresa Kowalska Hajnos/Thin Layer Chromatography in Phytochemistry 46772_C000 Final Proof page v 28.1.2008 7:41am Compositor Name: PJayarajHajnos/Thin Layer Chromatography in Phytochemistry 46772_C000 Final Proof page vi 28.1.2008 7:41am Compositor Name: PJayarajMonika Waksmundzka-HajnosMedical University of LublinLublin, PolandJoseph ShermaLafayette CollegeEaston, Pennsylvania, U.S.A.Teresa KowalskaUniversity of SilesiaKatowice, PolandThin Layer Chromatographyin PhytochemistryCRC Press is an imprint of theTaylor & Francis Group, an informa businessBoca Raton London New YorkHajnos/Thin Layer Chromatography in Phytochemistry 46772_C000 Final Proof page vii 28.1.2008 7:41am Compositor Name: PJayarajCRC PressTaylor & Francis Group6000 Broken Sound Parkway NW, Suite 300Boca Raton, FL 33487-2742 2008 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa businessNo claim to original U.S. Government worksPrinted in the United States of America on acid-free paper10 9 8 7 6 5 4 3 2 1International Standard Book Number-13: 978-1-4200-4677-9 (Hardcover)Thisbookcontainsinformationobtainedfromauthenticandhighlyregardedsources.Reprinted materialisquotedwithpermission,andsourcesareindicated.Awidevarietyofreferencesare listed. Reasonable efforts have been made to publish reliable data and information, but the author andthepublishercannotassumeresponsibilityforthevalidityofallmaterialsorfortheconse-quences of their use. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafterinvented,includingphotocopying,microfilming,andrecording,orinanyinformation storage or retrieval system, without written permission from the publishers.Forpermissiontophotocopyorusematerialelectronicallyfromthiswork,pleaseaccesswww.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged.Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe.Library of Congress Cataloging-in-Publication DataThin layer chromatography in phytochemistry / editors, Monika Waksmundzka-Hajnos, Joseph Sherma, Teresa Kowalska.p. cm. -- (Chromatographic science series)Includes bibliographical references and index.ISBN 978-1-4200-4677-9 (hardback : alk. paper) 1. Plants--Analysis. 2. Thin layer chromatography. I. Waksmundzka-Hajnos, Monika. II. Sherma, Joseph. III. Kowalska, Teresa. IV. Title. V. Series.QK865.T45 2008572.362--dc22 2007040781Visit the Taylor & Francis Web site athttp://www.taylorandfrancis.comand the CRC Press Web site athttp://www.crcpress.comHajnos/Thin Layer Chromatography in Phytochemistry 46772_C000 Final Proof page viii 28.1.2008 7:41am Compositor Name: PJayarajContentsPreface.................................................................................................................... xiiiEditors ..................................................................................................................... xvContributors ........................................................................................................... xixPart IChapter 1 Overview of the Field of TLC in Phytochemistryand the Structure of the Book ............................................................. 3Monika Waksmundzka-Hajnos, Joseph Sherma,and Teresa KowalskaChapter 2 Plant Materials in Modern Pharmacy and Methodsof Their Investigations....................................................................... 15Krystyna Skalicka-Wozniak, Jarosaw Widelski, and KazimierzGowniakChapter 3 Medicines and Dietary Supplements Producedfrom Plants......................................................................................... 37Anita Ankli, Valeria Widmer, and Eike ReichChapter 4 Primary and Secondary Metabolitesand Their Biological Activity............................................................ 59Ioanna ChinouChapter 5 Plant Chemosystematics .................................................................... 77Christian ZidornChapter 6 Sorbents and Precoated Layers for the Analysisand Isolation of Primary and Secondary Metabolites ..................... 103Joseph ShermaChapter 7 Chambers, Sample Application, and ChromatogramDevelopment .................................................................................... 119Tadeusz H. Dzido and Tomasz TuzimskiHajnos/Thin Layer Chromatography in Phytochemistry 46772_C000 Final Proof page ix 28.1.2008 7:41am Compositor Name: PJayarajixChapter 8 Derivatization, Detection (Quantication),and Identication of Compounds Online ...................................... 175Bernd SpangenbergChapter 9 Biodetection and Determination of Biological Activityof Natural Compounds .................................................................. 193Erno Tyihk, gnes M. Mricz, and Pter G. OttChapter 10 Forced-Flow Planar Layer Liquid ChromatographicTechniques for the Separation and Isolationof Natural Substances .................................................................... 215Emil MincsovicsPart IIPrimary MetabolitesChapter 11 TLC of Carbohydrates................................................................... 255Guilherme L. Sassaki, Lauro M. de Souza, Thales R. Cipriani,and Marcello IacominiChapter 12 TLC of Lipids................................................................................ 277Svetlana Momchilova and Boryana Nikolova-DamyanovaChapter 13 Amino Acids.................................................................................. 299Ravi BhushanSecondary MetabolitesShickimic Acid DerivativesChapter 14 Sample Preparation and TLC Analysisof Phenolic Acids .......................................................................... 331Magdalena Wjciak-Kosior and Anna OniszczukChapter 15 Application of TLC in the Isolation and Analysisof Coumarins ................................................................................. 365Monika Waksmundzka-Hajnos and Mirosaw A. HawryHajnos/Thin Layer Chromatography in Phytochemistry 46772_C000 Final Proof page x 28.1.2008 7:41am Compositor Name: PJayarajxChapter 16 Application of TLC in the Isolation and Analysisof Flavonoids ................................................................................. 405Marica Medic-aric, Ivona Jasprica, Ana Mornar,and eljan MaleChapter 17 TLC of Lignans ............................................................................. 425Lubomr Opletal and Helena SovovSecondary MetabolitesIsoprenoidsChapter 18 TLC of Mono- and Sesquiterpenes ............................................... 451Angelika Koch, Simla Basar, and Rita RichterChapter 19 TLC of Diterpenes......................................................................... 481Micha . HajnosChapter 20 TLC of Triterpenes (Including Saponins) ..................................... 519Wieslaw Oleszek, Ireneusz Kapusta, and Anna StochmalChapter 21 TLC of Carotenoids....................................................................... 543George BrittonChapter 22 TLC of Sterols, Steroids, and Related Triterpenoids .................... 575Laurie Dinan, Juraj Harmatha, and Rene LafontChapter 23 TLC of Iridoids.............................................................................. 605Gra_ zyna ZgrkaSecondary MetabolitesAmino Acid DerivativesChapter 24 TLC of Indole Alkaloids ............................................................... 623Peter John HoughtonChapter 25 TLC of Isoquinoline Alkaloids...................................................... 641Monika Waksmundzka-Hajnos and Anna PetruczynikHajnos/Thin Layer Chromatography in Phytochemistry 46772_C000 Final Proof page xi 28.1.2008 7:41am Compositor Name: PJayarajxiChapter 26 TLC of Tropane Alkaloids ............................................................ 685Tomasz MroczekChapter 27 TLC of Alkaloids from the Other Biosynthetic Groups ............... 701Jolanta FliegerSecondary MetabolitesCompounds Derivedfrom Acetogenine (Acetylocoenzyme A)Chapter 28 Polyacetylenes: Distribution in Higher Plants,Pharmacological Effects, and Analysis ......................................... 757Lars P. Christensen and Henrik B. JakobsenChapter 29 Quinone Derivatives in Plant Extracts .......................................... 817Gra_ zyna Matysik, Agnieszka Skalska-Kaminska, and AnnaMatysik-WozniakIndex..................................................................................................................... 853Hajnos/Thin Layer Chromatography in Phytochemistry 46772_C000 Final Proof page xii 28.1.2008 7:41am Compositor Name: PJayarajxiiPart IHajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 1 11.1.2008 1:15pm Compositor Name: VAmoudavallyHajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 2 11.1.2008 1:15pm Compositor Name: VAmoudavally1Overview of the Fieldof TLC in Phytochemistryand the Structureof the BookMonika Waksmundzka-Hajnos, Joseph Sherma,and Teresa KowalskaCONTENTS1.1 Survey of Phytochemistry................................................................................ 31.2 Procedures of Thin Layer Chromatography .................................................... 51.3 Organization of the Book ................................................................................ 91.1 SURVEY OF PHYTOCHEMISTRYPhytochemistry is a broad area, generally termedplant chemistry. Investigationsin theeld of phytochemistry are important for numerous research disciplines, suchas plant physiology, plant biochemistry, chemosystematics (which is often referredto as chemotaxonomy), plant biotechnology, and pharmacognosy.Plant physiologyfocusesonthelifeprocessesoccurringinplants. Especiallyimportant are the investigations on the inuence of various external factors, such asultravioletvisible(UVVis)radiation,temperature,thenatureofsoil,theclimate,etc., on the composition of active compounds contained in plants. One part of thisdiscipline is known as allelopathy. Within the framework of allelopathy, theresponses of the plant organisms to external pathological factors (e.g., environmentalpollution, the presence of pathogens, insects, etc.) are investigated.Plant biochemistryfocusesonbiochemical transformationsthat playafunda-mental role in the biosynthesis of active compounds contained in plants, which arereferred to as primary and secondary metabolites.Chemosystematics involves the classicationof plants onthe basis of theirbiochemistryandchemistry.Itprovestobeofspecialimportancewhensearchingfor and collectingoral specimens. Within the framework of chemosystematics, therelations are investigated between the classes of plants and the occurrence ofthe specic substances or substance groups in the plant tissues.Hajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 3 11.1.2008 1:15pm Compositor Name: VAmoudavally3The most important application of phytochemical investigation methods is to theeld of pharmacognosy. Pharmacognosy is a part of the pharmaceutical sciences andis focusedonnatural products (mainlyonplant materials) andthecomponentsthereof that show biological activity and are, therefore, used in therapy.The history of phytotherapy is almost as long as the history of civilization. Thetermpharmacognosy has beeninuse for little more thana century, but itsfoundationswerelaidoutbyearlycivilizations.TheAssyrian,Egyptian,Chinese,and Greek records of great antiquity make reference to the nature and use of herbsand herbal drugs. Knowledge of medicinal plants spread in West Europe and then inthe whole Western World, to a large extent through the monasteries and their schoolsof medicine. In 16th century, early botanists published herbalsusually illustratedwith the woodcut picturesdescribing the nature and use of an increasing number ofplants. In modern science, phytotherapy appeared in the 19th century, when therstbiologicallyactivecompounds (basicallyalkaloids) wereisolatedfromtheplantmaterial (e.g., morphine, strychnine, narcotine, caffeine, etc.) Thegoldenageofphytotherapy lasted until 1935, when therst sulfonamides and then antibiotics weresynthesized and used in therapy. Then the age of chemotherapy began. However, it isa widely recognized fact that numerous synthetic drugs exertalong with a positivetherapeutic effectalso harmful and often irreversible side effects. To the contrary,in the plant world, one very often encounters strongly active substances coexistingwith the other compounds that mitigate their negative side effects. Because of this, inrecent years a return to phytotherapy has been observed. This return has further beenspurred by an appeal of the World Health Organization to screen plant material forthe presence of biologically active compounds contained therein and exerting, e.g., awellpronouncedanticanceractivity. Itis rmlybelievedthatagreat,yetstillnotfully revealed, therapeutic potential exists in plants, because so far only a few percentout of 250,000 plant species have been investigated with regard to their usefulness inmedicine.Nowadays, medicines of natural origin are appreciated for their high effective-ness and low toxicity, and they are the widely used commercial products. The marketvalueofherbal preparationssellinginUnitedStatesaloneisestimatedat severaldozenmilliondollars per year. Plant materials are oftenobtainedfromnaturalsources, althoughmanyof themedicinal plants arealsocultivated. Fromthesefacts, it is clearthat thereis a high and increasing need forefcientpurity controlof plant material, andfurther for the assessment of their identityandchemicalcomposition, in order to obtain the expected therapeutic effect.The paramount goal of pharmacognosy is comprehensive investigations of plantmaterials by use of physical, chemical, and biological methods, and also the searchfor a possibility to use these materials as natural medicines. Modern pharmacognosyfocuses on thechemicalcomponentsof the plantmaterials,includingthe structureand pharmacological properties that are responsible for their use in therapy. Thus, itcanbeconcludedthat themainareaof interest is inthechemistryof biogeniccompounds(i.e., thechemistryof natural compoundsof plant origin). Thisnewapproach to the subject of pharmacognosy is based on the dynamic treatment of thenatural sourcesofdrugsthat takesintoaccount theirbiochemical transformationsand consequently allows synthesis of the new biologically active substances. In thatHajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 4 11.1.2008 1:15pm Compositor Name: VAmoudavally4 Thin Layer Chromatography in Phytochemistryway, links are being established between pharmacognosy and plant biotechnology,which involves breeding tissue cultures as a source of technological amounts of thebiologically active substances.An interest of modern pharmacognosy, in particular compounds that occur in theplant materials, is due to their already recognized signicance in therapy and also tothe importance of a steady search for new natural substances with a curing potential.Inthissense, plant material hastobetreatedasasourceof suitablemedicines.The therapeutic effect can be obtained by direct use of plant materials, by use of theplant confections, or by use of substances or substance groups isolated from the planttissues. The latter case occurs only when a given plant contains highly activesub-stances, e.g., the alkaloids in Secale cornutum, Tuber Aconiti, and Rhizoma Veratri,orcardiacglycosidesinFoliumDigitalispurpureaeandFoliumDigitalislanatae.These materials are an important source of selected alkaloids or cardiac glycosides.Plant materials, galenic preparations, and isolated compounds proposed fortherapy have to meet certain strictly determined standards. With the most importantmaterials,thesestandardssimplyarethepharmacopoeialrequirements,althoughavast number of herbs used in formal and popular medicine are not included in anypharmacopoeia.Standardizationoftheplantmaterialandofherbalpreparationsismeant toguaranteetheirtherapeuticvalue, andit isaresult oftheinvestigationson biologicallyactivecomponents.Thereareawidenumber ofmethodstoinves-tigate plant material, namely macroscopic (focused on botanical identity and purityof the plant material); microscopic (mostly histochemical investigations, whichprovide the basis for identicationof the material); biological (microbiologicaland biomolecular investigations and investigations of biological activity); and chem-ical methods. Chemical investigations of the plant material have a variety of goals,suchas determinationof the substance groups, quantitative analysis of activecompounds, isolation of substances from the plant tissues for their further identi-cation, or physicochemical characterization, and,nally, structural analysis of theisolated unknown compounds.1.2 PROCEDURES OF THIN LAYER CHROMATOGRAPHYAmong the chemical methods of plant examination, chromatographic analysis playsa very important role, and it has been introduced to all the modern pharmacopoeias.Becauseof numerousadvantagesof thechromatographicmethods(suchastheirspecicityandapossibilitytousethemforqualitativeandquantitativeanalysis),they comprise an integral part of the medicinal plant analysis.The following chromatographic methods are most frequently applied in phyto-chemical analysis: one- and two-dimensional paper chromatography, one- and two-dimensionalthin layer chromatography (TLC;alsocalledplanarchromatography),high-performance column liquid chromatography (HPLC), gas chromatography(GC), andcounter current chromatography(CCC). These methods canalsobeusedfortheisolationoftheindividual componentsfromthecomponentmixtureson a preparative and micropreparative scale.TLCis achromatographictechniquewidelyusedfor qualitativeanalysis oforganiccompounds, isolationoftheindividual compoundsfrommulticomopnentHajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 5 11.1.2008 1:15pm Compositor Name: VAmoudavallyOverview of the Field of TLC in Phytochemistry and the Structure of the Book 5mixtures, quantitativeanalysis, andpreparative-scaleisolation. Inmanycases, itoutperformstheotherchromatographictechniques. Firstly, thereisamultitudeofchromatographic systems that can be applied in TLC. Many kinds of TLC and high-performance TLC (HPTLC) precoated plates are commercially available, e.g., thosewith the inorganic adsorbent layers (silica or silica gel and alumina); organic layers(polyamide, cellulose); organic, polar covalently bonded modications of the silicagel matrix(diol, cyanopropyl, andaminopropyl); andorganic, nonpolar bondedstationary phases (RP2, RP8, RP18) with different densities of coverage of the silicamatrix (starting from that denoted as W, for the lowest density of coverage and thuswettable with water). Sorbents applied in TLC have different surface characteristicsand, hence, different physicochemical properties. Moreover, there is a wide choice ofmobilephasesthat canbeusedtoseparatemixturecomponents; thesebelongtovarious selectivitygroups and, thus, havedifferent properties as protondonors,protonacceptors, anddipoles. InTLC, ultraviolet (UV)absorptionofthemobilephase solvents does not play a signicant negative role in detection and quanticationofthe analytes,because the mobile phase is evaporated fromthe plate priorto thedetection. High viscosity of a solvent can be viewed as a sole property limiting itschoice as a mobile phase component. These plate and mobile phase characteristicsallow a choice from among an unparalleled abundance of TLC systems that offer abroadspectrumof separationselectivities, whichis particularlyimportant whencomplex mixtures of the plant extracts have to be separated.Another advantage of TLC is that each plate is used only once, thereby allowingsimplersamplepreparationmethodswhencomparedwithtechniquessuchasGCand HPLC, in which multiple samples and standards must be applied to the columnin sequence. Highly sorbed materials in plant extract samples can be left behind in acolumn and interfere in the analysis of subsequent samples. Multiple samples can beanalyzed at the same time on a single TLC or HPTLC plate, reducing the time andsolvent volumeusedpersample; theprocessingofstandardsandsamplesonthesameplateleadstoadvantagesintheaccuracyandprecisionofquanticationbydensitometry.Last, but notleast,TLC enables usageof numerousspecial developmenttech-niques. Most separationsarecarriedout byacapillaryowdevelopment withasingle mobile phase (isocratic) in the ascending or horizontal conguration. Gradientelutionwithstepwise variations inmobile phase composition, whichis widelyappliedinHPLC, is alsousedinTLC. Besides, therearethefollowingspecialmodes of developing a chromatogram: unidimensional multiple development (UMD),incremental multipledevelopment (IMD), gradient multipledevelopment (GMD),and bivariant multiple development (BMD). Moreover, the circular and anticirculardevelopment methods can also be applied. UMD consists of repeated developmentof the chromatogram over the same development distance, with a given mobile phaseof constant composition and with drying the plate between the individual develop-ment runs. IMD is performed by the stepwise increase in the development distance(the increment in the development distance is kept constant), using a steady mobilephase composition and drying the plate between the development runs. It results innarrowing of the spots or zones and improved resolution. In GMD, each step of therepeated chromatogram development is performed with a mobile phase of differentHajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 6 11.1.2008 1:15pm Compositor Name: VAmoudavally6 Thin Layer Chromatography in Phytochemistrycomposition, thusenablinggradient development. Thedevelopment distanceoftheconsecutivedevelopmentrunsiskeptsteadyanditisonlythemobilephasecompositionthatchanges, thusenablingtheanalysisofcomplexmixturesspan-ning a wide polarity range. When a lowstrength mobile phase is used, theseparationofthelowpolaritycomponentsisachievedonasilicalayer. Whenamedium polarity mobile phase is used, then the medium polarity components areseparated(therstgroupisthenelutedtotheupperedgeoftheplate).Withthehighpolaritymobilephases, separationof thehighpolar componentsof plantextractscanbeobtained.BMDinvolvesastepwisechangebothofthedevelop-mentdistanceandthemobilephasecomposition.Withuseofaspecialchamberand computer program, an improved version, known as Automated Multiple Devel-opment (AMD), can be applied, with the distance of the development increasing andthemobilephasestrengthdecreasingat eachstep. AMDenablestheanalysisofcomplexsamplesover a wide polarity rangeand providesfocusing (tightening)ofthezones. Inthecircular andanticircular development modes, themobilephasemigrates radially from the center to the periphery or from the periphery to the center,respectively. Analytes with lower RF values are better resolved by means of circularchromatographythanbymeans of linear chromatography, andtheadvantageoftheanticircularmodeisthat itallowsbetterresolutionofcompoundswithhigherRF values.TLCis alsothe easiest technique withwhichtoperformmultidimensional(i.e., two-dimensional)separations. Asinglesampleisappliedinthecorner ofaplate, andthelayer isdevelopedinthe rst directionwithmobilephase1. Themobile phase is dried by evaporation, and the plate is then developed with mobilephase2at aright angle(perpendicular or orthogonal direction); mobilephase2has different selectivity characteristics when compared with mobile phase 1. In thisway, completeseparationcanbeachievedofverycomplexmixtures(e.g., ofthecomponents of a plant extract) over the entire layer surface.Particularlyvaluableseparationresults canbe achievedwhenusingvariousmobile phase systems to benet from different separation mechanisms. For example,with cellulose one can apply a nonaqueous mobile phase to achieve the adsorptionmechanismof retentionandanaqueous mobilephasetoachievethepartitionmechanism. In a similar way, with the polar chemically bonded stationary phasesonecanusenonaqueousmobilephasestoachievetheadsorptionmechanismofretention and the aqueous mobile phases to achieve the reversed-phase mechanism.Shifting from the adsorption to the partition mode causes marked differences in theseparation selectivity.After performingtheseparationwiththeoptimumlayer, mobilephase, anddevelopment techniquecombination, thezonesmust bedetected. Ifthezonesarenot naturally colored oruorescent, or do not absorb 254 nm UV light so they can beviewed asuorescence-quenched zones on special F-plates containing auorescentindicator, a detectionreagent must be applied by spraying or dipping, usuallyfollowed by heating. This derivatization is mainly used in the postchromatographicmodeforlocalizationoftheseparatedcomponent zonesonthelayer. Veryoftenuniversal reagents are used, such as iodine vapors or sulfuric acid. These reagents canlocate almost all of the existing organic compound classes. Selective reagents can beHajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 7 11.1.2008 1:15pm Compositor Name: VAmoudavallyOverview of the Field of TLC in Phytochemistry and the Structure of the Book 7usedasderivatizingreagentsforindividualorgroupidenticationoftheanalytes.For example, the Dragendorffs reagent (KBiI4) is used for identication of hetero-cyclic bases (e.g., of alkaloids), ninhydrin for identication of compounds containingan amino group in their structure (e.g., of the amines and amino acids), and2-(diphenylboryloxy)-ethylamine polyethyleneglycol (PEG)foridenticationofpolyphenols.TLC is coupled with densitometry to enable detection of colored, UV-absorbing,oruorescent zones through scanning of the chromatograms with visible or UV lightin transmission or reectance modes. By comparison of a signal obtained with thatforthestandardsprocessedwithcomparablechromatographicconditions,densito-metric measurements can be used for quantitative analysis of the components con-tained in the mixtures. With multiwavelength scanning of the chromatograms,spectral data of the analytes can be directly acquiredfrom the TLCplates and canfurther be compared with the spectra of the analytes from a software library or fromstandardsdevelopedonthesameplate. Thus, adensitometer withadiodearraydetectorenablesdirect(insitu)identicationoftheanalytes.Otherpossibilitiestoidentifyanalytesareofferedbyoff-lineoron-linecouplingofTLCwithFouriertransform infrared spectrometry, mass spectrometry, etc.Further, itisworthnotingthat TLCcoupledwithbioautographicdetectionofmicrobiologicallyactivecompoundscanbesuccessfullyappliedintheanalysisofplant extracts. Especiallysuitablefor thispurposeisdirect bioautography, whichuses microorganisms (e.g., bacteria or fungi) growing directly on a TLC plate withthe previously separated mixtures of the plant extracts. In this procedure, antibacter-ial or antifungal compounds appear as clear spots (i.e., without microorganismsgrowing on them) on an intensely colored background. This approach can be usedas an additional analytical option in screening of biological samples, as a standard-ization method for medicinal plant extracts, and as a selective detection method.Additionally, special instruments enable the use of the forced-ow migration ofthemobilephase. Overpressured-layer chromatography(OPLC), alsocalledopti-mumperformancelaminar chromatography, makesuseof apumpthat feedsthesorbentbedwithmobilephaseataselectedowrate.Rotationplanarchromatog-raphy (RPC) uses centrifugal force in order to obtain an analogous effect. Electro-osmoticallydrivenTLCmakesuseofelectroosmotic owtoforcemobilephaseacross a layer. All of these forced-ow methods provide a constant ow rate of themobilephase; thelinearproleofthe owandeliminationofvapor phasefromthe system may improve system efciency and peak resolution.The advantages of TLC are particularly important with plant extracts, which arevery complex mixtures of the structurally differentiated chemical compounds. Suchextractsveryoftencontainpolar(e.g., tanninsandphenols)andnonpolar(lipids,chlorophylls, and waxes) ballasts, apart from a fraction of active substances that is ofmainimportanceforphytochemistryandpharmacognosy.Thislatterfractioncon-tains closely related compounds of a similar structure and physicochemical proper-ties.Isolationofafractionofinterestfromsuchamixturerequiresacomplicatedprocedure, usuallyliquidliquidorsolid-phaseextraction. TLCenablesseparationofacrudeplant extract without anearlierpurication. Forexample, inanormalphasesystemanonpolarfractionmoveswiththemobilephasefront(oritcanbeHajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 8 11.1.2008 1:15pm Compositor Name: VAmoudavally8 Thin Layer Chromatography in Phytochemistryprewashedwithanonpolarmobilephasepriortothedevelopmentofachromato-gram),and the polarfractionremainsstronglyretainednear tothe origin; thenthefraction of interest is separated in the central part of the chromatogram.Summing up, TLCis a principal separation technique in plant chemistryresearch. It can be used in a search for the optimumextraction solvents, foridenticationof knownandunknowncompounds, andwhat isat least equallyimportantforselectionofbiologicallyactivecompounds. TLCalsoplaysakeyroleinpreparativeisolationofcompounds, puricationofthecrudeextracts, andcontrol of the separation efciency of the different chromatographic techniques andsystems.TLChasmanyadvantagesinplantchemistryresearchanddevelopment.These include single use of stationary phase (no memory effect), wide optimizationpossibilities withthe chromatographic systems, special development modes anddetectionmethods, storagefunctionof chromatographicplates (all zones canbedetected in every chromatogram by multiple methods), low cost in routine analysis,and availability of purication and isolation procedures.1.3 ORGANIZATION OF THE BOOKThe book comprises 29 chapters, divided into two parts. Part I consists of 10 chaptersandprovides general information onthose areas of science that are related tophytochemistryandcanbenet fromtheuseofTLC. Moreover, it alsocontainschaptersdevotedtothetechnicalaspectsofTLC,suchastheinstrumentationandchromatographic systems involved.Followingthischapter, Chapter2focusesonmedicinal plantsasasourceofnaturaldrugsandontheirroleinmodernpharmacy.Italsoprovidesabriefover-viewof themethods usedfor theinvestigationof theplant material andof thetechniquesusedfortheextraction, purication, andnal assessment ofthedrugshaving a plant origin.Chapter 3 is devoted to the medicines and the diet supplements produced fromplants. Firstly, the authors introduce denitions of the plant medicines and plant dietsupplements, andthentheypresent thehistoryof herbal drugsinthetraditionalmedicines of various cultures throughout the world. The botanical supplements arethen discussed, and the chapter ends by discussing the tasks of TLC in theeld ofbotanical drugs and dietary supplements.Chapter 4focuses onthe primaryandthe secondarymetabolites andtheirbiological activity. Because classication of the metabolites as primary and second-ary is not straightforward and can be viewed differently by the different authors, weeditors will explain in the next paragraph our own ideas on this very important issue,which shape the structure of the entire volume.Primary metabolites are those that occur ineach plant andfulll its basicphysiological functions (i.e., appear as the building, energetic, or the reserve mater-ial). Inotherwords, primarymetabolitesareindispensableforthelifeofaplant.Secondary metabolites are the products of metabolism and play no crucial role in theplants life. This classication can be regarded as a rough and provisional only, as itoften happens that the secondary metabolites have a well recognized physiologicalfunction in the plants as well. In practice, all metabolites can be classied in differentHajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 9 11.1.2008 1:15pm Compositor Name: VAmoudavallyOverview of the Field of TLC in Phytochemistry and the Structure of the Book 9ways. Most often classication is based on their chemical structure, which generallyremains ina goodagreement withthe biogenetic origin. Sometimes, however,problemscanarisewithastraightforwardclassicationofcertaingroupsofcom-pounds that belong to the same biogenetic group and yet completely differ in termsof chemical structure. For example, steroidal alkaloids are traditionally included inthealkaloidgroup. However, theirbiogeneticoriginisnot fromaminoacidsbutfromsteroids, andfor this particular reasoninthis booktheyare classiedassteroids. In a similar way, taxoids are sometimes classied as pseudoalkaloidsduetothepresenceof thetertiarynitrogenatominthemolecules of certaintaxoidrepresentatives.Atthesametime,alltaxoidsbiogeneticallybelongtotheclassofditerpenes. It is noteworthy that classication of metabolites based on their biogen-eticoriginissometimesimpractical, andthenit isrecommendedtorefertotheirchemical structureor physicochemical properties. For example, naphthoquinonesand anthraquinones may originate both from shickimic acid and acetogenine. In fact,quinones can have a different biogenetic origin, but are joined in one group based ontheir similar physicochemical properties. Iridoids could formally be included in theclass of monoterpenes, but this is not done because of their differentiated physico-chemical andpharmacological properties(classical monoterpenesarethevolatilecompoundspresent inessential oils, whereasiridoidsusuallyarethenonvolatilespecies). Fortheabovereasons, wedecidedtoclassifythequestionablegroupsof compounds accordingtotheir chemical structure. Consequently, all of themetabolitesbothprimaryandsecondaryareadditionallydividedaccordingtotheir biogenesis.Chapter5focusesonchemosystematics,alsoknownaschemotaxonomy.Thischapter startsfromdenitionof thisparticular branchof phytochemical science,which involves classication of the plant organisms based on the differences at thebiochemical level, especially in the amino acid sequences of common proteins. Thenthe author highlights the areas of the main interest for the chemosystematic studiesanddiscusses applicabilityof the mainchromatographic modes tothis area ofresearch.In Chapter 6, the sorbents and precoated layers that are particularly useful in theanalysisandpreparativeisolationoftheprimaryandsecondarymetabolitesfromplant extracts are described. This chapter covers virtually all of the TLC and HPTLCanalytical andpreparativelayersusedforseparation, determination, andisolationwithintheeldofphytochemistry,includingsilicagel,reversedphaseandhydro-philic bondedphases, nonsilica sorbents (alumina, cellulose, polyamides, modiedcelluloses, and kieselguhr), and miscellaneous layers (resin, impregnated, mixed, anddual layers).Chapter7 starts with description of the chromatographic chambers and mobilephase compositions that can be utilized in phytochemical research. Then the authorsdiscuss the development of the chromatograms in the different thin layer chromato-graphic modes. This chapter covers the methods of sample application to theadsorbent layer as well.Thentwochaptersfollowingdealwiththedetectionoftheanalytesaftertheirthinlayer chromatographicseparation. Themainpart of Chapter 8isdevotedtoderivatizationof theplant extract components byuseof universal andselectiveHajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 10 11.1.2008 1:15pm Compositor Name: VAmoudavally10 Thin Layer Chromatography in Phytochemistryreagents. The specicity of TLCnot shared with any other chromatographicmoderesults fromthe possibility of applying several differentdetection methodsinsequence inorder toidentifygroups or individual analytes. Inthis chapter,physical methods of detection are also discussed (such as UVVis light absorption,uorescence, and mass spectrometry), as well as the methods of quantitative analysiswith use of TLC combined with densitometry.Chapter 9 deals with the methods of biodetection in TLC that enable rapid andselective determination of the biological activity (antibacterial, antifungal, and other)of plant metabolites. In this chapter the mechanisms of bioactivity of the individualcompounds are explained.Part I ends withthe descriptionof the forced-owplanar chromatographicdevelopment techniques(Chapter10), includingtheir inuenceexertedonsepar-ability of the plant metabolites.Part II of thebookis dividedintothechapters that reect thetypes of themetabolites that occur in plants. Chapters 11 through 13 refer to primary metabolites.Chapter 11 deals with the chemistry of carbohydrates, with their occurrence intheplantsasmono-, oligo-, andpolysaccharides, andalsoasglycoconjugates. Itprovidesanoverviewof therecommendedanalytical methods, includingsamplepreparation, derivatization, and the most suitable TLC systems.InChapter 12, different classes of plant lipids are presented, andthe TLCsystems applied to their separation (including normal- and reversed-phase andargentation) are discussed. Class separation of lipids, their isolation, and quantica-tion are taken into the account.Chapter 13 focuses on free amino acids, peptides, and proteins, including theiroccurrence in plants and the use of TLC to separate the individual groups of thesecompounds.The next part of the book deals with the secondary metabolites occurring in planttissues, and it is divided into sections according to the metabolic pathways in whichindividual substances are synthesized.Chapter 14startswiththephenoliccompoundsthat belongtothemetabolicpathway of shickimic acid, i.e., phenols, phenolic acids, and tannins. It describes thestructure and classication of these compounds, their biological importance, samplepreparationmethods,and thevariousTLCsystemsand special techniquesthat areused for their separation and analysis.Chapter15dealswithcoumarinsthat belongtothephenol classandarealsoderived from shickimic acid. Details are provided on sample preparation and isola-tion of coumarins with aid of classical TLC and HPTLC and the forced-ow planarchromatographic techniques. Application of TLC to the measurement of biologicalactivityofcoumarinsisalsodescribed. Thechapterendswithtablesoftheplantfamilies in which coumarins occur.Chapter16isdedicatedtothephenoliccompoundsoriginatingfromasimilarpathwayas coumarins, i.e., avonoids. After ashort introductiononchemistry,biochemistry, and medical signicance ofavonoids, the methods for their analysisusing various TLC systems are presented, including forced-ow development tech-niques. In this chapter, sample preparation methods and quantication ofavonoidsby means of TLC combined with slit-scanning and video densitometry are discussed.Hajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 11 11.1.2008 1:15pm Compositor Name: VAmoudavallyOverview of the Field of TLC in Phytochemistry and the Structure of the Book 11The section of the book on secondary metabolites ends with lignans, alsooriginating from shickimic acid. Chapter 17 is focused on the chemistry, occurrencein plant material, and pharmacological activity of the representatives of this group,followed by the sample preparation techniques and the TLCanalysis of thesecompounds. Detailsabout quanticationoflignansinherbal extractsandprepar-ations are also reported.The next section of the book is focused on isoprenoid derivatives, which includeseveral groupsof compounds. It starts with Chapter 18 on the volatile compounds(mono- and sesquiterpenes), including their denition, classication, occurrence, andimportance. Thenthe followingapplications of planar chromatographyare dis-cussed: identicationofthevolatilefractionsinpharmaceutical drugs, taxonomicinvestigations, tracing of various adulterations, and analysis of cosmetics.Chapter 19coversditerpenoidsandpresentstheir structure, physicochemicalproperties, natural occurrence, and pharmacological activity. The details ofsamplepreparationandtheanalytical andpreparativeTLCseparations of this groupofcompounds with the aid of different chromatographic systems are described, includ-ing derivatization and quantication methods. The chapter ends with a comparison oftheperformanceofTLCwiththat oftheotherchromatographicandrelatedtech-niques used in diterpenoid analysis.The next group of compounds that belong to the isoprenoid methabolic pathwayare triterpenes, and they are described in Chapter 20. After a short introduction onstructure and properties of this group, chromatographic systems and detectionmethodsappliedintheanalysisof triterpenes(saponinsincluded) arepresented.The chapter emphasizes the role of planar chromatography as a technique supportingcolumn chromatography in identication and determination of the biological activityof triterpenes.Chapter 21focusesontetra- andpolyterpenes, andamongthemcarotenoidsrepresent the most important group of compounds. First, structure, occurrence, andproperties arepresented. Thenthespecial aspects of theTLCanalysis (suchasdetectionandinstabilityof carotenoids) are emphasized. The use of silica andalumina, andalsoofthebasicnormal phaseadsorbents, isdiscussed. Usefulnessof TLC in screening of the plant material, in preparative separations, and in isolationof individual carotenoids is also described.The next large groupof compoundsthat belong tothe isoprenoid pathwayaresteroids, andtheyare presentedinChapter 22. Intheintroductorypart of thischapter, thechromatographicsystemsandtechniquesuseful forplanarseparationof steroids are described. Then an overview of the literature is provided, taking intotheaccount theclassesof phytosterols, steroids(brassinosteroids, bufadienolides,cardenolides, ecdysteroids, steroidal saponins, steroidal alkaloids, vertebrate-typesteroids, andwithanolides), andoftherelatedtriterpenoids(cucurbitacins). Struc-tural diversity, the separation systems, and the detection and quantication for eachclass of compounds are presented.Iridoides are the last group of compounds that belong to the isoprenoid pathway,andtheyaredescribedinChapter23. Aftertheintroductorypart onthestructureandphysicochemical propertiesof iridoides, theissuesareemphasizedrelatedtoHajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 12 11.1.2008 1:15pm Compositor Name: VAmoudavally12 Thin Layer Chromatography in Phytochemistryisolation of this group of compounds from the plant material and to sample prepar-ation. Then the TLC systems and techniques applied to the analysis of iridoids aredescribed, taking into account the detection methods and the forced-ow techniques.The preparative layer chromatography of iridoids is also discussed.The next four consecutive chapters deal with alkaloids synthesized in the plantorganisms from amino acids. There are several groups of alkaloids differing in theirstructure, properties, and biological activity.Chapter24focusesonindolealkaloids. Firstly, thechemicalstructure, occur-rence, andpharmacological, ecological, andchemosystematicimportanceof thisgroup are discussed. This preliminary information is followed by a detailed descrip-tion of the TLC separations of indole alkaloids, including chromatographic systems,techniques, and detection methods. Details on the separations of the particular typesof indole alkaloids are also presented.Chapter 25is devotedtothestructure, properties, andbiological activityofisoquinoline alkaloids. Information concerning problems with chromatographic sep-aration of basic compounds is also provided, and the normal phase, reversed phase,and pseudoreversed phase systems are described in detail. The use of TLC plates andgrafted plates for two-dimensional separations and forced-ow techniques applied tothe separation of the isoquinoline alkaloids are presented. Examples of TLC appli-cations to quantitative analysis are shown, along with the preparative separations.Tropane alkaloids are handled in Chapter 26. Chemistry and stereochemistry oftropaneandtherelatedalkaloids, andtheirnaturaloccurrence, arepresentedrst.Various methods of extraction of this entire group of compounds from plant materialare described, followed by the pretreatment of the extracts by liquidliquid partition-ing (LLP), solid assisted liquidliquid partitioning (Extrelut), and solid-phase extrac-tion (SPE). Then the information on TLCof tropane alkaloids including theirquanticationis provided. Thechapter gives detailedinformationontheOPLCanalysis of tropane alkaloids, and a comparison is made with the results originatingfrom the other separation techniques in use.Chapter 27 focuses on the remaining groups of alkaloids, including phenylethy-lamine derivatives, quinoline derivatives (Cinchona alkaloids), and pyrrolidine,pyrrolizidine, piridine, and piperidine derivatives (Tobacco, Lobelia, Pepper, Pelle-tierine, Sedum, Senecioalkaloids), quinolizidine alkaloids (Lupine alkaloids),xanthine, imidazolederivatives, andditerpene alkaloids. Preparationof extracts,themostfrequentlyemployedTLCsystems,andthedetectionmethodsapplicableto each individual group are presented.Thelast twochaptersaredevotedtothesecondarymetabolitesderivedfromacetogenine(acetylocoenzymA). Chapter 28dealswiththedistributionof poly-acetylenes inplants andpharmacological activityof polyacetylenes. Separation,detection, and isolation by means of TLC in various different systems are described.The results are compared with those originating from HPLC.Chapter 29 is focused on quinones (antraquinones and naphthoquinones), theiroccurrence in plants, and pharmacological activity. Applicability of the conventionalTLC techniques applied to the separation of quinines, and also of the special modes(e.g., gradient or two-dimensional TLC), is discussed.Hajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 13 11.1.2008 1:15pm Compositor Name: VAmoudavallyOverview of the Field of TLC in Phytochemistry and the Structure of the Book 13Theauthorswhoagreedtocontributechapterstothebookareall recognizedinternational experts in their respectiveelds. The book will serve as a comprehensivesourceof informationandtrainingonthestate-of-the-art phytochemistrymethodsperformed with aid of TLC. It will help to considerably popularize these methods forpractical separations and analyses in aeld that will undoubtedly grow in importancefor many years to come. A computer assisted search has found no previous book onTLCinphytochemistry. Three editions of the book Phytochemical Methods(1973, 1984, and1998)byJ.B. Harborne(ChapmanandHall, London, UK)hadchapters organizedby compound type, mostof which contained some informationon TLC analysis. A chapter on Thin Layer Chromatography in Plant Sciences byJ. Pothier was contained in the book Practical Thin Layer Chromatography editedby B. Fried and J. Sherma (CRC Press, 1996), a chapter on planar chromatography inmedicinal plant research in Planar Chromatography edited by Sz. Nyiredy(Springer, 2001) andachapter onnatural mixturesbyM. Waksmundzka-Hajnoset al. in Preparative Layer Chromatography edited by T. Kowalska and J. Sherma(CRC=Taylor&Francis,2006)includedinformationonplantextracts.AbookbyE. Reich and A. Schibli (Thieme Medical Publishers, Inc., 2007) covers the theor-etical concepts and practical aspects of modern HPTLC as related to the analysis ofherbal drugs.However, these information sources are not comprehensive, and therst two arenow out of date. Our proposed book will solve this void in information in the criticaleld of phytochemical analysis.Hajnos/Thin Layer Chromatography in Phytochemistry 46772_C001 Final Proof page 14 11.1.2008 1:15pm Compositor Name: VAmoudavally14 Thin Layer Chromatography in Phytochemistry2Plant Materialsin Modern Pharmacyand Methods of TheirInvestigationsKrystyna Skalicka-Wozniak, Jaros/law Widelski,and Kazimierz G/lowniakCONTENTS2.1 Plant Material and Marine Products as Sources of ActiveSecondary Metabolites................................................................................... 162.2 The Distribution and Concentration of Natural Compounds withBiological Activity in Different Organs of Medicinal Plants........................ 172.3 Methods of Investigations of Plant Material ................................................. 182.3.1 Macroscopic Investigations ............................................................... 182.3.2 Microscopic and Microchemical Methods of Investigations ............ 182.3.3 Chemical Methods of Investigations ................................................. 202.3.3.1 Approximate Group Identication...................................... 202.3.3.2 Quantitative Analysis of Active Compoundsin Plant Material by Various Methods(Titration, Spectrophotometric Methods) ........................... 202.3.3.3 Isolation of Active Compounds.......................................... 212.3.4 Biological Methods of Investigations................................................ 212.4 Modern Extraction Methods of Active Compounds from Plant Materialand Marine Products ...................................................................................... 222.4.1 Classic Extraction Methods in Soxhlet Apparatus ............................ 222.4.2 Supercritical Fluid Extraction............................................................ 232.4.3 Pressurized Liquid Extraction............................................................ 252.4.4 Medium-Pressure SolidLiquid Extraction Technique ..................... 272.5 Purication of Crude Extracts and Sample Preparation ................................ 272.5.1 LiquidLiquid Partition ..................................................................... 272.5.2 Solid Phase Extraction....................................................................... 272.5.3 Gel Permeation Chromatography ...................................................... 29Hajnos/Thin Layer Chromatography in Phytochemistry 46772_C002 Final Proof page 15 11.1.2008 1:16pm Compositor Name: VAmoudavally152.6 Chromatographic Methods and Their Role in Investigationsof Plant Material ............................................................................................ 292.6.1 Gas Chromatography......................................................................... 292.6.2 High-Performance Liquid Chromatography...................................... 292.6.3 Electrophoresis and Electrochromatography ..................................... 302.6.4 Coupled Methods (GCMS, LCMS, LCNMR) ............................ 32References ............................................................................................................... 32Pharmacognosyisthesciencewhichtreatsofthehistory, production, commerce,collection, selection, identication, valuation, preservationanduseof drugs andother economic materials of plant and animal origin.The termpharmacognosy derived from the ancient Greek words pharmakon,drugor medicine, andgnosis, knowledge, andliterallymeans the knowledgeof drugs.2.1 PLANT MATERIAL AND MARINE PRODUCTS AS SOURCESOF ACTIVE SECONDARY METABOLITESDrugsarederivedfromthemineral, vegetable, andanimal kingdoms. Theymayoccurinthecrudeorrawform, asdriedorfreshungroundor groundorgansororganisms or natural exudations of these(juiceor gum), whentheyaretermedcrude drugs.Theseareknownas herbal medicinal products (HMPs), herbal remedies, orphytomedicines and include, for example:.Herb of St. Johns wort (Hypericum perforatum), used in the treatment ofmild to moderate depression.Leaves of Gingkobiloba, usedfor cognitive deciencies (oftenintheelderly), includingimpairment of memoryandaffectivesymptomssuchas anxietyThere are also derived substances, such as alkaloids (e.g., caffeine, from the coffeeshrubCoffeaarabicausedasastimulan), glycosides(e.g., digoxinandotherdigitalis glycosides, fromfoxgloveDigitalis spp.usedtotreat heart failure),alcohols, esters, aldehydes, or other constituents or mixtures of constituents isolatedfrom the plant or animal.Finally, also pure chemical entities exist, which are produced synthetically andreferred to as nature identical, but originally discovered fromplant drugs.Examples include:.Morphine, from opium poppy (Papaver somniferum), used as an analgesic.Quinine, fromCinchonabark(Cincoinaspp.), usedinthetreatment ofmalariaHajnos/Thin Layer Chromatography in Phytochemistry 46772_C002 Final Proof page 16 11.1.2008 1:16pm Compositor Name: VAmoudavally16 Thin Layer Chromatography in PhytochemistryAlso many foods are known to have benecial effects on health:.Garlic, ginger, and many other herbs and spices.Anthocyanin- oravonoid-containing plants such as bilberries, cocoa, andred wine.Carotenoid-containingplants suchas tomatoes, carrot, andmanyothervegetables [1]2.2 THE DISTRIBUTION AND CONCENTRATION OF NATURALCOMPOUNDS WITH BIOLOGICAL ACTIVITY IN DIFFERENTORGANS OF MEDICINAL PLANTSIsolated pure natural products such as numerous pharmaceuticals used in pharmacyare thus not botanical drugs, but rather chemically dened from nature. Botanicaldrugs are generally derived from specic plant organs of plant species. The follow-ing plant organs are the most important:.Herba or aerial parts (herba).Leaf ( folium).Flower ( os).Fruit ( fructus).Bark (cortex).Root (radix).Rhizome (rhizoma).Bulb (bulbus)Fruitsandseedshaveyieldedimportant phytotherapeuticproducts, e.g., caraway(Carum carvi), fennel (Foeniculum vulgare), saw palmetto (Serenoa repens), horsechestnut seeds (Aesculus hippocastanum), or ispaghula (Plantago ovata), which areused often in phytotherapy.Numerous drugs contain also leaf material as the main component. Some widelyused ones include balm (Melissa ofcinalis), deadly nightshade (Atropa belladonna),ginkgo (Ginkgo biloba) peppermint (Mentha 3piperita), bearberry (Arctostaphylosuva-ursi), and many others.Althoughtheowersareofgreatbotanicalimportance,theyareonlyaminorsource of drugs used in phytotherapy. One of the most important example ischamomile (Chamomilla recutita (Matricariase os)). Other examples includecalendula (Calendula ofcinalis) and arnica (Arnica montana).Stemmaterial whichisoftenapart ofthosedrugsisderivedfromallabove-groundparts,e.g.,ephedra(Ephedrasinica),hawthorn(CrataegusmonogynaandCrataegus oxyacantha), passion ower (Passiora incarnata), or wormwood(Arthemisia absynthium). Also parts of the stem are used in phytotherapy like barkof Rhamnus frangula (frangula) or bark of Salix alba (willow).Finally,undergroundorgans(rhizomeandroot)ofmanyspecieshaveyieldedpharmaceutically important drugs. Examples include: Devils claw (HarpagophytumHajnos/Thin Layer Chromatography in Phytochemistry 46772_C002 Final Proof page 17 11.1.2008 1:16pm Compositor Name: VAmoudavallyPlant Materials in Modern Pharmacy and Methods of Their Investigations 17procubens), tormentill (Potentillaerecta), rhubarb(Rheumpalmatum), andkava-kava (Piper methysticum) [1].2.3 METHODS OF INVESTIGATIONS OF PLANT MATERIALThe analytical side of pharmacognosy is embraced in the expression the evaluationofthedrug,forthisincludestheidenticationofa druganddeterminationofitsquality and purity.The identication of the drug is ofrst importance, for little consideration can begiven to an unknown drug as regards its quality and purity. The identication of adrug can be established by actual collection of the drug from plant or animal (amongthemmarineorganism) whichcanbepositivelyidentiedfromthebotanical orzoologicalstandpoint.Thismethodisrarelyfollowedexceptby an investigatorofthe drug, who must be absolutely sure of the origin of his samples. For this reasondruggardens are frequentlyestablishedinthe connectionwithinstitutionofpharmacognostical research.Quality of a drug refers to intrinsic value of the drug, that is, to the amount ofmedicinal principles or activeconstituents present inthedrug. Ahighgradeofqualityinthedrugissuchimportancethat effort shouldbemadetoobtainandmaintainthishighquality.Themostimportantfactorstoaccomplishthisinclude:collection of the drug from the correct natural source at proper time and in the propermanner, the preparation of the collected drug by proper cleaning, drying and garblingand proper preservation of the clean, dry, pure drug against contamination with dirt,moisture, fungi, lth, and insects.The evaluation of the drug involves a number of methods, which may be classiedas follows: organoleptic, microscopic, biological, chemical, and physical [2].2.3.1 MACROSCOPIC INVESTIGATIONSOrganoleptic (lit.impression on the organs) refers to evaluation by means of theorgans of sense, and includes the macroscopic appearance of the drug, its odor andtaste, occasionally the sound orsnap of its facture, and thefeel of the drug tothe touch.For convenience of description the macroscopic characteristic of a drug may bedivided into four headings, viz.: shape and size, color and external markings, fractureand internal color, andnally odor and taste.Forexample, descriptionoflinseed(Linumusitatissimum)isasfollows: Theseedis exalbuminous, of compressed ovate or oblonglanceolate outline, pointed atone end, rounded at the other and from 4 to 6 mm in length; externally glabrous andshiny, brown to dusky red with a pale-yellow, linear raphe along one edge; the hilumandmicrophyleinaslightdepressionnearthepointedend;odorslight,becomingvery characteristic in the ground or crushed drug; taste mucilaginous and oily [3].2.3.2 MICROSCOPICAND MICROCHEMICAL METHODSOF INVESTIGATIONSMicroscopical methodsofvaluingdrugsareindispensableintheidenticationofsmall fragments of crude drugs and of the powdered drugs as well as in the detectionHajnos/Thin Layer Chromatography in Phytochemistry 46772_C002 Final Proof page 18 11.1.2008 1:16pm Compositor Name: VAmoudavally18 Thin Layer Chromatography in Phytochemistryoftheiradulterans,forthesepossessfewfeaturesotherthancolor,odor,andtastewhereby clues toward their identitymay be afforded. Moreover, owingto thesimilarityof some plant organs of alliedspecies, denite identicationevenofcertainentire,cellularvegetable drugscannot be madewithouttheexamination ofmounts of thin sections of them under a microscope. Every plant possess a charac-teristic histology in respect to its organs and diagnostic features of these are ascer-tainedthoughthestudyof thetissuesandtheir arrangement, cell wallsandcellcontents, when properly mounted in suitable stains, reagents or mounting media [3].Somecharacteristicfeaturescanbeeasilyusedtoestablishbotanical identityand quality of the drugs. The typical example is the various types of crystals formed bycalciumoxylate. Several species of thefamilySolanaceaeareusedfor obtainingatropine, alkaloid used as spasmolytic in cases of gastrointestinal cramps and asthma.Species containing high amount of atropine like Atropa belladonna (deadly nightshade),Datura stramonium(thorn apple), or Hyoscyamus niger (henbane) are characterized bytypical crystal structures of oxalate: sand, cluster crystals and microspheroidalcrystals, respectively.Thesearesubcellularcrystalstructures, whichcanbeeasilydetected using polarized light and are thus a very useful diagnostic means.Second typical example are the glandular hairs, which are characteristic for twofamilies(LamiaceaeandAsteraceae) containingmanyspecieswithessential oils.Figure 2.1 shows diagnostic features of botanical drugsmicroscopic examinationView from sideFIGURE2.1Diagnostic features of botanical drugs, that are revealeduponmicroscopicexamination include typical glandular hair as found in the Lamiaceae (a) and Asteraceae (b).Top: lateral view; bottom: viewfromabove. (FromHeinrich, M. et al., FundamentalsofPharmacognosy and Phytotherapy, Elsevier Science, Churchill Livingstone, 2004.)Hajnos/Thin Layer Chromatography in Phytochemistry 46772_C002 Final Proof page 19 11.1.2008 1:16pm Compositor Name: VAmoudavallyPlant Materials in Modern Pharmacy and Methods of Their Investigations 19includingtypical glandularhair(LamiaceaeandAsteraceaefamily)lateral viewand view from above.Inmanyinstances,agoodideaofthequalityofadrugcanbeascertainedbyusing microchemical methods. These may consist of examining mounts of sectionsorpowdereddruginvariousreagentswhicheitherformsaltsofcontainedactiveprinciples, that have constant characters (microcrystallization) or show denite colorreactions, or of isolation of constituents of the powdered drug with a suitable solvent,ltering 2 or 3 drops of the extract on to a slide, permitting the solvent to evaporateandexaminingtheresidue. Thereis alsopossibleisolationof aconstituents bymicrosublimation.It is often possible to detrmine whether a powdered drug has been exhausted byexamining the crystals found in its sublimate. These have been found to be charac-teristicformanydrugs. Microsublimationuponaslideisasuperiortechniqueincomparisonwithtest tubesublimation. Thesublimatesmaybedirectlyexaminedunder the microscope without mechanical alteration.2.3.3 CHEMICAL METHODSOF INVESTIGATIONS2.3.3.1 Approximate Group IdenticationIdenticationofthecharacteristicgrouporgroupsofactiveconstituentsisoneofthe basic methods of the evaluationof the drugandthe rst stepinisolationprocedure.For example, Borntragers test is commonly applied to all anthraquinone drugs.As effect of the reaction a deep rose color is produced.Another example is a reactionthat give acids and avonoids withArnovsreagent phenolic (the products of this reactiongive purple color). All of thesereactions arealsousedbothfor qualitativeandquantitativeanalysis(colometricreactions).Characteristic reaction foravonoids, like 1% methanolic solution of AlCl3, 5%methanolicsolutionofKOH, and1%methanolic solutionofNaturstoffreagenzA,are used for derivatization of TLC plates. It enables general evaluation of differentgroups of active compounds, in this caseavonoids.2.3.3.2 Quantitative Analysis of Active Compounds in Plant Materialby Various Methods (Titration, Spectrophotometric Methods)Evaluationof plant drugs uses all of themethods knowninchemical analysis.Amongthemwe cansingle out the titration. Titrationis a commonlaboratorymethod of quantitative chemical analysis which can be used to determine theconcentration of known reactant. Because volume measurements play a key role intitration, it is also known as volumetric analysis. A reagent, called titrant, of knownconcentration(astandardsolution) isusedtoreact withameasuredquantityofreactant(the analyte).Titration is usedin quantitativeanalysisof tropan alkaloids,where KOH is used as a titrant and methyl red as the indicator.Spectrophotometric techniques are used to measure the concentration of solutesinsolutionbymeasuringtheamountoflightthatisabsorbedbythesolutioninaHajnos/Thin Layer Chromatography in Phytochemistry 46772_C002 Final Proof page 20 11.1.2008 1:16pm Compositor Name: VAmoudavally20 Thin Layer Chromatography in Phytochemistrycuvette placed in the spectrophotometer. According the BeerLambert Law there isthe linear realationship between absorbance and concentration of an absorbingspecies. It enablesaquantitativedeterminationofcompoundsinwhichsolutionsabsorb light. For example total concentration of pyrrolizidine alkaloids in Symohy-tum ofcinale root were investigated using UV-VIS spectra of adducts of3,4-dehydroPAs and Erlichs reagent [4].2.3.3.3 Isolation of Active CompoundsWhen a crude extract obtained by a suitable extraction procedure shows interestingactivity (e.g., an antibacterial activity), demonstrated in bioassay, the next and one ofthemost difcult stepsistofractionatetheextract usingadifferent (sometimescombined) separation method(s) so that a puried biologically active component canbe isolated.Figure 2.2 gives a general isolation protocol starting with selection of biomass(e.g., plant, microbe or tissue culture), which is then extracted using differentextraction methods. Hydrophilic (polar) extracts will then usually undergoionexchangechromatographywithbioassayofgeneratedfractions. Afurtherionexchangemethodofbioactivefractionwouldyieldpurecompounds,whichcouldnext be submitted for structure elucidation (MS, NMR).2.3.4 BIOLOGICAL METHODSOF INVESTIGATIONSThe biological evaluation of the plant drugs is one of the most important issues ofpharmacognosy. For drugs obtainedfromnatural sources, all activecompoundspresent in the plant are responsible for therapeutic effect.There are plenty of methods for evaluation of biological properties of plant drug.For example, bacteria, such as Staphylococcus aureus are used to determineantisepticvalueofthedrugs.ForstandardizationofDigitalisspp.(Foxglove)andother heart tonic drugs, pigeons and cats are used. Bioassay is the use of biologicalsystem to detect properties of a mixture or a pure compound.Active fractionsHydrophilic extractLipophilic extractExtraction(soxhlet orhot/coldpercolation)Organism selectionPurified extractGel chromatographyBiotage flashchromatographyTLC Biotage flashchromatographyIon exchangechromatographyPartitioningIon exchangechromatographyHPLCActive fractionsActive compoundsActive compoundsStructure elucidationFIGURE 2.2General isolation strategy for purication of bioactive natural products. (FromHeinrich, M. et al., Fundamentalsof PharmacognosyandPhytotherapy, ElsevierScience,Churchill Livingstone, 2004.)Hajnos/Thin Layer Chromatography in Phytochemistry 46772_C002 Final Proof page 21 11.1.2008 1:16pm Compositor Name: VAmoudavallyPlant Materials in Modern Pharmacy and Methods of Their Investigations 21Bioassays could involve the use of in vivo systems (clinical trials, whole animalexperiments), ex vivo systems (isolated tissues and organs), or in vitro systems (e.g.,cultured cells). Collection of materials for testing in bioassays couldeither berandomcollectionof samples or directedcollection, i.e., fromplants knowntobe used traditionally. Bioassays were often linked with the processes of fractionationand isolation, known as bioassay-guided fractionation.2.4 MODERN EXTRACTION METHODS OF ACTIVE COMPOUNDSFROM PLANT MATERIAL AND MARINE PRODUCTSSample pretreatment is one of the most time-consumingsteps of the analyticalprocessandalsooneof themost important. Plants containmanyof bioactivecompounds and it is important to extract all of them in the best, short and effectiveway with minimal solvent usage. Proper extraction technique should also be cheapand simple [5,6]. Important is high recoveries, reproducibility, lowdetectionlimits, andautomation[7]. Solidliquidextractionis oneof theoldest ways ofsolidsamplepretreatment [5]. Extractionandfractionationof extract is alsoanimportant methodinisolationofcompoundgroupsorindividual substancesfromplant material.2.4.1 CLASSIC EXTRACTION METHODSIN SOXHLET APPARATUSExtractioninSoxhlet apparatushasbeentheleadingtechniquemostlyusedforalong time and still is considered to be a standard technique and the main reference towhich the other new leaching methods are compared [5].The sample is placed in a thimbleholder and during operation is continuouslylled with fresh portion of solvent from distillationask. When the liquid reaches theoverow level, a siphon aspirates the solute of the thimble holder and unloads it backintothedistillationask, carryingtheextractedanalytesintothebulkliquid. Theoperation is repeated until complete extraction is achieved [5,6].Different solvents can be used in extraction process. Addition of co-solvents as amodier toincrease the polarityof liquidphase is possible. Soxhlet extractionmainlydependsoncharacteristicofmatrixandofsizeofparticlesastheinternaldiffusion may be a limiting step during extraction [6].The most important advantage of conventional Soxhlet extraction technique is itscontinuous character. The sample has a contact with the fresh portion of the solvent.Afternishing the process of extraction noltration is required. It is also very cheapand simple methodwhere small experienceis required. The methodhas the possi-bility to extract more sample mass than the other methods [5]. Also wide industrialapplicationandbetter efciencyareadvantagesof Soxhlet extractionover novelextraction methods [6].The most signicant disadvantages compared with other techniques are the longtimeof extraction, poor penetrationof thematrixbythesolvent, andthelargeamount of solvent required, whichis veryexpensive andcauses environmentalproblems. Somesolventshaverecentlybeenquestionedbecauseoftheirtoxicity.Also we cannot forget that extraction occurs for a long time at the boiling point ofHajnos/Thin Layer Chromatography in Phytochemistry 46772_C002 Final Proof page 22 11.1.2008 1:16pm Compositor Name: VAmoudavally22 Thin Layer Chromatography in Phytochemistrythesolvent sothethermal decompositionof compoundsandcreatingartifactsispossible [5,6]. Due to all advantages and disadvantages of Soxhlet extraction methodit is the most popular one and many scientist have tried to improve it.First, thechangesfocusedonthenewdesignof basicunits(thimbleholder,siphons, condenser) what improve application and obtained results [5]. There is alsosomemodicationshorteningthetimeof extractionbyusingauxiliaryenergies.Thus led to create high-pressure and focused microwave-assisted Soxhlet extraction(FMASE). High pressure was achieved by placing the extractor in autoclave or usingsupercritical uid Soxhlet extractor [8].FMASEshows some differences comparing with other microwave-assistedextraction techniques: extraction is under normal pressure, irradiation is focused onthesample, ltrationisnotrequired. Theadvantagesofthattechniqueareshorterextractiontime, capabilityfor automation, andquantitativeextraction. Extractionefcienciesand precisionare better thanin conventionalmethod. Still it keepstheadvantagesofconventionalmethod[8].Alsotherearesomeapplicationsofultra-sound-assisted Soxhlet extraction [9].2.4.2 SUPERCRITICAL FLUID EXTRACTIONSupercriticaluid extraction (SFE) is one of the most successful techniques. Super-critical state is achieved when the temperature and the pressure of a substance is overitscritical value. Undersupercritical conditionsthe uidhasthecharacteristicofboth liquid and gases what makes extraction faster and more effective. Manysupercritical uids have been used, such as freons, ammonia, organic solvents, butthe most common is CO2 because it has lowest toxicity and inammability. The lowsupercritical temperature of carbon dioxide makes it attractive for the extraction ofthermolabilecompounds[5,6]. Applicationofsomeofthemislimitedbecauseoftheir unfavorable properties with respect to safety and environmental consideration.Water in supercritical state has higher extraction ability for polar compounds, but isnot suitable for thermally labile compounds [10].Many compounds such as phenols, alkaloids, glycosidic compounds are poorlysoluble in CO2 because of its low polaritydifculties in extracting polar analytesarethemaindrawbacksofthemethod. Toimproveefciencythepolarityoftheextractantcanbeincreased(byadditionmethanol,ethanol,pentane,acetone).Themostcommonsolventismethanolbecauseitisaneffectivemodierandisupto20%misciblewithCO2. Sometimes ethanol is abetter choiceinnutraceuticalsbecauseofitslowertoxicityThepolarityoftheanalytescanalsobereduced(byformingthecomplexorionpair)[1,1014]. BecauseSFEwithCO2alsoextractslipidsfromthematrix, furthercleanupmaybenecessarytoremovelipidsbeforethe analysis.SFEisfrequentlyusedfor extractionoffreshplant material. Theproblemisinhighlevel of moisture what cancause mechanical difculties. ToretainthemoisturesomechemicalssuchasNa2SO4or silicagel aremixedwiththeplantmaterial [10].Also very important is plant particle size. Large particles can prolong extractionprocess because the process is controlled by internal diffusion andne powder canHajnos/Thin Layer Chromatography in Phytochemistry 46772_C002 Final Proof page 23 11.1.2008 1:16pm Compositor Name: VAmoudavallyPlant Materials in Modern Pharmacy and Methods of Their Investigations 23speedup extraction. However, powdered material may cause difculties in keeping aproperow rate causing decrease in the total yield of the extracted substances [15].Themost signicant advantagesoftheSFEtechniquewhencomparedwithclassical Soxhlet extraction are time reduction, its cleanness and safety, possibilityfor couplingon-linewithdetectors andchromatographs, quantitativedetermin-ation, the preconcentration effect, high mass transference, completeness of extrac-tion(supercritical uidshaveahigher diffusioncoefcient andlower viscositythanliquidsolvent). Veryimportant isthat SFEofferspossibilityfor selectiveextractionandfractionationsubstancesfromtheplant material bymanipulatingwithpressure andtemperature. Choi et al. [16] conrmedit incomparisonofsupercritical carbon dioxide extraction with solvent extraction of squalene andstigmasterol from Spirodela polyrhiza. The SFE of squalene (10 Mpa, 508C608C)was comparable to n-hexane Soxhlet extraction but the stigmasterol was not detectedunder these condition. The method can be easily used in the laboratory for a largescale [17].Extracts with fewer unwanted analytes may be obtained by careful manipulationoftheSFEconditions(pressure,temperature,anduseofmodiers).However,thesmall volume of the extractor, which contains only a few grams of the material, is adisadvantage when a higher sample mass is required.Supercritical carbon dioxide is a promising solvent for the extraction of naturalcompounds, especially thermolabile ones. Prevention of degradation could beachieved by eliminating oxygen from the CO2. Apparently, the addition of antioxi-dants would be a reasonable solution if there were no mechanical items to adsorb theoxygen [18]. Also SFE eliminates time-consuming process of concentration and usesno or minimal organic solvent what makes method environmental friendly[5,6,12,14,19].It is worth to notice that sometimes the efciency is higher than 100% referred toconventional Soxhlet method (some analytes are strongly bound with matrix and notenough energy is involved in the Soxhlet process for their separation) [5].SFEmethodwithmodierwaseffectiveforextractionofcoumarinsfromthepeel of Citrus maxima [12], furanocoumarins and pentacyclic terpenoids in rhizomeofDorsteniabryoniifolia Mart. ex Miq. and barkrootsofBrosimumgaudichaudiiTrcul (Moraceae) [20].MacerationundersonicationgavebetterthanSFEextractionofcoumarinsinMikaniaglomerataleaves. SFextractscontainahighlevel ofchlorophylls. Alsoaddition of polar modier (EtOH) did not present signicant advantages [21].SFE with modier, and pressurizeduid extraction (PFE) with dichloromethaneshows that avanones and xanthones are removed from plant material at similar orslightlyhigher yieldsthanobtainedbysolidliquidextraction, inamuchshorterperiod of time and with decreased amount of solvent [22].Biologically active substances of rose hip seeds like unsaturated fatty acids andcarotenewasextractedbySFEwithcarbondioxideandpropane. Oil yieldwashigher in comparison with traditional Soxhlet extraction [23].Effect of low, medium, and high polarity under very high pressure and with polarmodiers has beeninvestigatedbyHamburger et al. [24]. Calendulaofcinalis,Hajnos/Thin Layer Chromatography in Phytochemistry 46772_C002 Final Proof page 24 11.1.2008 1:16pm Compositor Name: VAmoudavally24 Thin Layer Chromatography in PhytochemistryCrataegus sp, and Matricaria recutita were used as models in this study. Extractionyieldsunder different conditionsdependedtothelargeextent ontheprolesofsecondary metabolites present in the plant material. Extractability of lipophiliccompoundsincreasedsubstantiallyat pressurehigher than300bar, theyieldsofpolyphenolics and glycosides remained low even at pressure about 700 bar with 20%of modier in the extractionuid. A wide range of applications for the extraction ofbiologically active substances are described in review article [6,7,25,26].2.4.3 PRESSURIZED LIQUID EXTRACTIONPressurized liquid extraction (PLE) also known as an accelerated solvent extraction(ASE), high-pressure solvent extraction (HPSE), or pressurized uid extraction(PFE) is a technique which uses small volume of conventional solvents at elevatedtemperatures (>2008C) in a very short time to extract solid samples. The pressure ishigherthan200barinordertokeepthesolventinliquidstate(thesolventisstillbelowitscritical point), increasingtemperatureacceleratestheextractionkinetics(processofthedesorptionofanalytesfromthematrixarefastercomparedtotheconditionwhensolventatroom temperatureareused),whichgivessafeand rapidmethod[6,7,27,28]. ASEallowstheuniversaluseofsolventsorsolventmixtureswith different polarities.InPLE, samplewithsand, sodiumsulfateorHydromatrixasadispersant areplaced in a cell. Extraction cell islled with the solvent and the cell is set to certainvalues, then is heated in an oven to the set values. During the heating cycle, solvent ispumpedinandout ofthecell tomaintainthepressureandtoperformthecyclesindicated.Theuidcomingoutoftheextractioncelliscollectedinthecollectionvial. Before loading the plant materials into the extraction cell, the samples are oftenpretreated. Proper size of sample enables right diffusion of analytes from the sampleto the solvent extract. Drying the sample removes any moisture which may diminishextractionefciency.Thechosenextractionsolventmustbeabletosolubilizetheanalytesof interest, minimizingthecoextractionof other matrixcomponents. Itspolarity should be close to that of the target compound [27,29].PLE can be accomplished in the static (sample and solvent are maintained for adenedtimeat constant pressureandtemperature)ordynamicmode(thesolventows throughthe sample ina continuous manner). Because inmost cases thedynamicmodeuseswaterasextractant, several authorshavepreferredtousethetermpressurizedhotwaterextraction(PHWE)toreferit.Waterisnonammable,nontoxic, readily available, and an environmentally acceptable solvent. PHWEwith and without the addition of a small percentage of organic solvent suchas ethanol is highlysuitedfor the chemical standardizationandqualitycontrolof medicinal plants. At thesametime, it canbeappliedat thepilot scaleas amanufacturingprocessformedicinal plants. Furtherinformationaboutapplicationof PHWEin extraction of active compounds can be found in reviewpapers[27,29,30].Veryimportant is completeautomationof thewholeanalytical process andhighly selective extractions of compounds of different polarities [27].Hajnos/Thin Layer Chromatography in Phytochemistry 46772_C002 Final Proof page 25 11.1.2008 1:16pm Compositor Name: VAmoudavallyPlant Materials in Modern Pharmacy and Methods of Their Investigations 25Compared with classic extraction in Soxhlet apparatus, complete extraction canbe achieved in shorter time with a small volume of organic solvent and much betterpenetration of sample by the solvent in PLE. Keeping the high temperature reducessolventviscosityandhelpsinbreakingdownanalytematrixbonds.Alsonoadd-itional ltration is required [27].The suitability of pressurized liquid extraction (PLE) in medicinal plant analysiswas investigated. PLEextracts fromrepresentative herbs containingstructurallydiverse metabolitesofvaryingpolarity and solubility werecompared withextractsobtained according to Pharmacopoeia monographs with respect to yield of relevantplant constituents, extraction time, and solvent consumption. Experiment shows thatone to three extraction cycles of 5 to 6 min at high temperatures afforded exhaustiveor almost exhaustiveextraction(insteadof manyhoursof Soxhlet extraction). Itmarkedlyreducesnot onlytimebut alsosolvent consumptionandprotect againstartifacts of extracted compounds at high temperatures. Reproducibility of results wasgenerally better [31].AmongextractioninSoxhlet apparatus, ultrasonication, microwave-assistedsolvent extraction in open and close system and pressurized solvent extraction ASEgiveshigheryieldoffuranocoumarins(especiallyforhydrophobic)fromPastinacsativa and Archangelica ofcinalis fruits [32,33]. Dawidowicz et al. have optimizedthe extraction condition for the analysis of rutin and isoquercitrin in Sambucus nigraowers, leaves, and berries [34].OngandLen[35]havedevelopedamethodfortheanalysisofglycosidesinmedicinal plants using PHWE. Th