High Performance Liquid Chromatography in Plant Sciences

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.