High Performance Liquid Chromatography in Plant Sciences (Modern Methods of Plant Analysis, New Series, Vol 5)

Modern Methods of Plant Analysis When the handbook Modern Methods of Plant Analysis was first introduced in 1954 the considerations were: 1. the dependence of scientific progress in biology on the improvement of existing and the introduction of new methods; 2. the difficulty in finding many new analytical methods in specialized journals which are normally not accessible to experimental plant biologists; 3. the fact that in the methods sections of papers the description of methods is frequently so compact, or even sometimes so incomplete that it is difficult to reproduce experiments. These considerations still stand today. The series was highly successful, seven volumes appearing between 1956 and 1964. Since there is still today a demand for the old series, the publisher has decided to resume publication of Modern Methods of Plant Analysis. It is hoped that the New Series will be just as acceptable to those working in plant sciences and related fields as the early volumes undoubtedly were. It is difficult to single out the major reasons for success of any publication, but we believe that the methods published in the first series were up-to-date at the time and presented in a way that made description, as applied to plant material, complete in itselfwith little need to consult other publications. Contributing authors have attempted to follow these guidelines in this New Series of volumes.

High Performance Liquid Chromatography of Gibberellins.- 1 Introduction.- 2 Extraction and Purification.- 3 Group Separation Procedures.- 3.1 PVP Adsorption Chromatography.- 3.2 Gel Permeation or Steric Exclusion Chromatography.- 3.3 Anion Exchange Chromatography.- 3.4 Charcoal Adsorption Chromatography.- 3.5 Sephadex G-10 Chromatography.- 3.6 Countercurrent Distribution.- 3.7 Silica Gel Coated Disposable Extraction Columns.- 4 High Performance Liquid Chromatography (HPLC).- 4.1 Gel Permeation or Steric Exclusion HPLC.- 4.2 Normal Phase HPLC.- 4.3 Reversed Phase HPLC.- 4.4 Detection of Gibberellins After HPLC.- 5 Recent Developments and Prospects.- 5.1 Diode Array UV Detection.- 5.2 Electrochemical Detection.- 5.3 Combined HPLC Immunoassay.- References.- Characterization of Cytokinins and Related Compounds by HPLC.- 1 Introduction.- 1.1 Background.- 1.2 Structure and Physicochemical Properties of Cytokinins and Related Compounds.- 2 Cytokinin Isolation and Sample Preparation.- 2.1 Cytokinin Isolation.- 2.2 Sample Preparation.- 3 Preparation of Mobile Phase.- 3.1 Water.- 3.2 Organic Solvents.- 4 Chromatography.- 4.1 Column Selection.- 4.2 Separation Examples.- 4.2.1 Ion Exchange HPLC.- 4.2.2 Normal Phase HPLC.- 4.2.3 Reverse Phase HPLC.- 4.2.4 Ion-Pair Reverse Phase HPLC.- 5 Concluding Remarks.- References.- Separation and Purification of Abscisic Acid and Its Catabolites by High Performance Liquid Chromatography.- 1 Introduction.- 2 Abscisic Acid and Its Catabolites.- 3 Use of High Performance Liquid Chromatography to Characterize Abscisic Acid and Its Catabolites.- 3.1 Synthesis of (±)-Abscisic Acid and Catabolites.- 3.2 Resolution of (±)-Abscisic Acid.- 3.3 Methods for the Purification of Abscisic Acid and Catabolites by High Performance Liquid Chromatography.- 4 Concluding Remarks.- References.- The Determination of Abscisic Acid by High Performance Liquid Chromatography.- 1 Introduction.- 1.1 HPLC and ABA.- 1.1.1 Why Have Many Different Methods Been Used?.- 1.1.2 Which Method is Correct? How to Choose.- 1.1.3 General Approach to HPLC of ABA.- 1.2 HPLC General.- 1.2.1 Theoretical Considerations.- 1.2.1.1 Retention.- 1.2.1.2 Band Spreading.- 1.2.1.3 Resolution.- 1.2.1.4 Controlling RS by N, k’, and ?.- 1.2.2 Preparative Versus Analytical Operation.- 1.2.3 Elution.- 1.2.4 The Different Modes of HPLC.- 1.2.4.1 Partition or Liquid-Liquid Chromatography (LLC).- 1.2.4.2 Adsorption or Liquid-Solid Chromatography (LSC).- 1.2.4.3 Size Exclusion Chromatography (SEC).- 1.2.4.4 Ion Exchange Chromatography (IEC).- 1.2.4.5 Bonded-Phase Chromatography (BPC).- 1.2.5 Detection.- 1.3 ABA General.- 1.3.1 The ABA Molecule.- 1.3.2 Extraction and Preparation.- 1.3.3 Determination of ABA.- 1.4 Generalized Scheme for the Separation of ABA.- 1.4.1 Choosing the Columns.- 1.4.2 Typing the System.- 1.4.3 Extraction and Preparation for RPLC.- 1.4.4 The Second Column.- 1.4.5 Chromatography of ABA in Conjunction with Other Compounds.- 1.4.6 Summary of General Separation Scheme.- References.- High Performance Liquid Chromatography and the Analysis of Indole-3-Acetic Acid, and Some of Its Decarboxylated Catabolites in Scots Pine (Pinus sylvestris L.).- 1 Introduction.- 2 Internal Standardization.- 3 Extraction and Purification.- 4 High Performance Liquid Chromatography.- 5 Examples.- 5.1 Indole-3-Acetic Acid.- 5.1.1 Extraction.- 5.1.2 Purification.- 5.1.2.1 PVP, XAD-7 and Sep-Pak.- 5.1.2.2 Immunoaffinity Chromatography.- 5.1.3 HPLC Analysis.- 5.2 Indole-3-Carboxylic Acid.- 5.2.1 Extraction and Purification.- 5.2.2 HPLC Analysis.- 5.2.3 Internal Standards.- 5.3 Indole-3-Methanol.- 5.3.1 Extraction and Purification.- 5.3.2 HPLC Analysis.- 5.3.3 Internal Standards.- 6 Conclusions.- References.- HPLC for the Separation and Determination of Phenolic Compounds in Plant Cell Walls.- 1 Introduction.- 2 General Considerations.- 3 The Recommended Procedure — Isolation of Cell Walls.- 3.1 Reagents and Equipment.- 3.1.1 Reagents (All Analytical Grade).- 3.1.2 Equipment.- 3.2 Isolation of Cell Walls.- 3.3 Isolation of Dietary Fibre.- 4 The Recommended Procedure — Release of Phenolic Acids and Aldehydes from Cell Walls and Their Separation and Determination by HPLC (C18 Reverse Phase) (Methods 1 and 2).- 4.1 Reagents and Equipment.- 4.1.1 Reagents (All Analytical Grade).- 4.1.2 Equipment.- 4.2 Release of Phenolic Acids and Aldehydes from Cell Walls and Preparation of Solutions for Analytical HPLC (C18 Reverse Phase).- 4.3 Analytical HPLC (C18 Reverse Phase) of Phenolic Acids and Aldehydes (Methods 1 and 2).- 5 Alternative Procedure — Release of Phenolic Acids and Aldehydes from Cell Walls and Their Separation and Determination by HPLC (PRP-1 Resin) (Methods 3 and 4).- 5.1 Reagents and Equipment.- 5.2 Release of Phenolic Acids and Aldehydes from Cell Walls and Preparation of Solutions for HPLC (PRP-1 Resin).- 5.2.1 Method for Analytical HPLC (PRP-1 Resin).- 5.2.2 Method for Preparative HPLC (PRP-1 Resin).- 5.3 Analytical HPLC (PRP-1 Resin) of the Phenolic Acids and Aldehydes.- 5.3.1 Phenolic Aldehydes and trans,trans-Diferulic Acid (Method 3).- 5.3.2 p-Coumaric and Ferulic Acids (Method 4).- 5.4 Preparative HPLC (PRP-1 Resin) of the Phenolic Acids and Aldehydes.- 5.4.1 Preparation of the Aldehydes and trans,trans-Diferulic Acid.- 5.4.2 Preparation of p-Coumaric and Ferulic Acids.- 6 Amount of Phenolic Acids and Aldehydes Released from the Cell Walls of Graminaceous Plants.- References.- HPLC of Thiophenes for Phytochemical and Biochemical Research.- 1 Introduction.- 2 Material and Methods.- 3 Use of HPLC for Phytochemical Research on Thiophenes.- 3.1 Preliminary Experiments: HPLC of Commercially Available Reference Substances.- 3.2 HPLC of Naturally Occurring Thiophenes.- 3.2.1 Thiophenes from Tagetes patula Seedlings.- 3.2.2 Thiophenes from Other Plant Sources.- 4 Use of HPLC for Enzymatic Research on Thiophenes.- 5 Conclusions.- References.- High Performance Liquid Chromatography of Ascorbic Acid.- 1 Introduction.- 2 Sample Preparation.- 3 Stationary and Mobile Phases.- 3.1 Separation of Ascorbic Acid from Dehydroascorbic Acid and from Reducing Agents.- 3.2 Separation of Ascorbic Acid and Metabolically Related Compounds.- 3.3 Separation of Isomeric Forms of Ascorbic Acid.- 4 Detection Techniques for Ascorbic Acid.- References.- High Performance Liquid Chromatography of Phytoalexins.- 1 Introduction.- 2 Instrumentation.- 3 Preparative HPLC of Phytoalexins.- 3.1 Principles.- 3.1.1 Elicitation.- 3.1.2 Extraction and Clean Up.- 3.1.3 Chromatography.- 3.2 Practice.- 3.2.1 Isolation of Stilbene Phytoalexins from Kernels.- 3.2.2 Isolation of Hydroxyflavan Phytoalexins from Narcissus Bulbs.- 3.2.3 Isolation of Pterocarpan Phytoalexins (Glyceollins) from Soybean Cotyledons.- 3.2.4 Isolation of Isoflavone and Isoflavanone Phytoalexins from Pigeonpea Seeds.- 3.2.5 Isolation of Chalcone and Stilbene Phytoalexins from Pigeonpea Leaves.- 4 Analytical HPLC of Phytoalexins.- 4.1 Principles.- 4.1.1 Elicitation.- 4.1.2 Sampling.- 4.1.3 Extraction and Clean Up.- 4.1.4 Chromatography.- 4.2 Practice.- 4.2.1 Analytical HPLC of Furanoacetylenic Phytoalexins from Broad Bean.- 4.2.2 Analytical HPLC of Pterocarpan Phytoalexins (Glyceollins) from Soybean.- 4.2.3 Analytical HPLC of Phytoalexins from Potato.- 4.2.4 Analytical HPLC of Phytoalexins from French Bean.- 4.2.5 Analytical HPLC of Phytoalexins from Cotton.- 5 Application of HPLC to the Solution of Outstanding Problems in Phytoalexin Research.- 5.1 Assessment of the Role of Phytoalexins in Resistance to Microbial Attack.- 5.2 Measurement of the Phytoalexin Potential of the Plant.- 5.3 The Elicitation, Biosynthesis and Degradation of Phytoalexins.- 6 Concluding Remarks.- References.- Analysis of Lipids by High Performance Liquid Chromatography.- 1 Introduction.- 2 Instrumentation.- 2.1 Pumps, Injectors, and Columns.- 2.2 Detectors.- 2.3 Mobile Phase Selection.- 3 Lipid HPLC Applications.- 3.1 Initial Lipid Extraction and Purification.- 3.1.1 Lipid Extraction.- 3.1.1.1 Comments.- 3.1.1.2 Procedure.- 3.2 Separation of Lipid Classes.- 3.2.1 HPLC Separation of Lipid Classes.- 3.2.2 Separation of Lipid Classes by Other Techniques.- 3.2.2.1 Column Chromatography.- 3.2.2.2 Thin Layer Chromatography.- 3.3 HPLC Analysis of Individual Molecular Species of Lipid Classes.- 3.3.1 Separation of Phospholipid Molecular Species by HPLC of Diglyceride Derivatives.- 3.3.1.1 Procedure.- 3.3.1.2 Comments.- 3.3.2 HPLC Separation of Phosphatidylglycerol Molecular Species.- 3.3.2.1 Sample Preparation.- 3.3.2.2 Instrumentation and Mobile Phases.- 3.3.3 HPLC Separation of Glycolipid Molecular Species.- 3.3.3.1 Sample Preparation.- 3.3.3.2 Instrumentation and Mobile Phase.- 3.3.4 Fatty Acid Analysis.- 3.3.4.1 Production of Free Fatty Acids from Lipid Classes.- 3.3.4.2 Preparation of Fatty Acid Derivatives.- 3.3.4.3 HPLC Analysis of Nitrophenacyl Fatty Acid Derivatives.- 3.3.4.4 Trace Analysis of Fatty Acid by HPLC.- 3.3.5 HPLC of Other Lipid Classes.- References.- Practical High Performance Liquid Chromatography of Proteins and Peptides.- 1 Introduction.- 2 Size Exclusion HPLC.- 2.1 Columns.- 2.1.1 Packings and Surface Interactions.- 2.1.2 Fractionation Limits.- 2.2 Operational Parameters and Resolution.- 2.2.1 Mobile Phase.- 2.2.2 Sample.- 2.2.3 Mobile Phase Velocity.- 2.3 Practical Applications.- 2.3.1 Molecular Weight Determination.- 2.3.2 Preservation of Biological Activity.- 2.3.3 Selected Applications.- 2.4 Large-Scale Separations.- 2.5 Maintenance.- 3 Ion Exchange HPLC.- 3.1 Columns.- 3.2 Operational Parameters and Resolution.- 3.2.1 Mobile Phase.- 3.2.2 Mobile Phase Velocity.- 3.2.3 Gradient Slope.- 3.2.4 Sample and Load.- 3.3 Selected Applications.- 4 Reversed Phase HPLC.- 4.1 Columns.- 4.2 Operational Parameters and Resolution.- 4.2.1 Mobile Phase.- 4.2.1.1 Organic Solvent.- 4.2.1.2 Mobile Phase pH.- 4.2.1.3 Ion-Pairing Agents.- 4.2.1.4 Other Aqueous Solvent Compositions.- 4.2.1.5 Acid Concentration.- 4.2.2 Mobile Phase Velocity and Temperature.- 4.2.3 Gradient Slope.- 4.2.4 Sample Composition and Load.- 4.2.5 Prediction of Retention Times.- 4.3 High Sensitivity RP-HPLC.- 4.4 Applications.- 4.4.1 Two-Dimensional RP-HPLC.- 4.4.2 Hydrophobic Polypeptides.- 4.4.3 Others.- 4.5 Maintenance.- 5 Multidimensional HPLC.- References.- Monitoring Polyamines in Plant Tissues by High Performance Liquid Chromatography.- 1 Introduction.- 2 High Performance Liquid Chromatography of Dansylamines.- 2.1 Dansyl Derivatives.- 2.2 Reagents and Stock Solutions.- 2.3 Extraction of Polyamines.- 2.4 Purification of Extracts.- 2.5 Dansylation of Amines in a Sample.- 2.6 Chromatographic Separation of Dansylamines.- 2.7 Quantification.- 3 High Performance Liquid Chromatography of Benzoylamines.- 3.1 Benzoylation of Amines in a Sample.- 3.2 Chromatographic Separation of Benzoylamines.- 4 Ion-Pair Reverse Phase Chromatography of Underivatized Polyamines.- 4.1 o-Phthalaldehyde Derivatives.- 4.2 Sample Preparation.- 4.3 Chromatographic Separation of Underivatized Polyamines.- 4.4 Post-Column Derivatization with OPA.- 4.5 Detection of OPA Derivatives.- 5 Ion-Exchange Chromatography of Underivatized Polyamines.- 5.1 Chromatographic Separation of Underivatized Amines and Related Compounds.- 6 Conclusion.- References.- Analysis of Alkaloids in Tobacco Callus by HPLC.- 1 Introduction.- 1.1 Background.- 1.2 Analytical Methods.- 2 Experimental.- 2.1 Plant Material.- 2.1.1 Expiant Origin and Callus Induction.- 2.1.2 Callus Culture.- 2.2 Alkaloid Extraction.- 2.2.1 In Dry Tissue.- 2.2.2 In Fresh Tissue.- 2.3 Analysis by HPLC.- 2.3.1 Sample Preparation and Purification of Standards.- 2.3.2 Instrumentation and Separation of Alkaloids.- 2.3.3 Alkaloid Quantitation. Calibration Curves.- 3 Results and Discussion.- 3.1 Callus Culture Method.- 3.2 Alkaloid Extraction.- 3.3 Analysis by HPLC.- 3.4 Applications to the Study of Alkaloid Production and Metabolism In Vitro.- 3.4.1 In Vitro Biosynthesis of Nornicotine.- 3.4.2 Effect of Organic Acids on Alkaloid Content.- 3.4.3 Effect of Putrescine Biosynthetic Inhibitors on Alkaloid Content.- 4 Conclusions.- References.