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High-performance liquid chromatography

About: High-performance liquid chromatography is a research topic. Over the lifetime, 47312 publications have been published within this topic receiving 1012843 citations. The topic is also known as: HPLC & high-pressure liquid chromatography.


Papers
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Journal ArticleDOI
TL;DR: High-performance liquid chromatography is a promising alternative for determining the G+C content of bacterial deoxyribonucleic acid (DNA) and may also be more accurate than indirect methods, such as the buoyant density and thermal denaturation methods.
Abstract: High-performance liquid chromatography is a promising alternative for determining the G+C content of bacterial deoxyribonucleic acid (DNA). The method which we evaluated involves enzymatic degradation of the DNA to nucleosides by P1 nuclease and bovine intestinal mucosa alkaline phosphatase, separation of the nucleosides by high-performance liquid chromatography, and calculation of the G+C content from the apparent ratios of deoxyguanosine and thymidine. Because the nucleosides are released from the DNA at different rates, incomplete degradation produces large errors in the apparent G+C content. For partially purified DNA, salts are a major source of interference in degradation. However, when the salts are carefully removed, the preparation and degradation of DNA contribute little error to the determination of G+C content. This method also requires careful selection of the chromatographic conditions to ensure separation of the major nucleosides from the nucleosides of modified bases and precise control of the flow rates. Both of these conditions are achievable with standard equipment and C18 reversed-phase columns. Then the method is precise, and the relative standard deviations of replicate measurements are close to 0.1%. It is also rapid, and a single measurement requires about 15 min. It requires small amounts of sample, and the G+C content can be determined from DNA isolated from a single bacterial colony. It is not affected by contamination with ribonucleic acid. Because this method yields a direct measurement, it may also be more accurate than indirect methods, such as the buoyant density and thermal denaturation methods. In addition, for highly purified DNA, the extent of modification can be determined.

4,685 citations

Book
01 Jan 1979
TL;DR: A short history of HPLC can be found in this paper, where the authors present a detailed overview of the current state of the art in HPLC and its application in the literature.
Abstract: PREFACE. GLOSSARY OF SYMBOLS AND ABBREVIATIONS. 1 INTRODUCTION. 1.1 Background Information. 1.2 A Short History of HPLC. 1.3 Some Alternatives to HPLC. 1.4 Other Sources of HPLC Information. References. 2 BASIC CONCEPTS AND THE CONTROL OF SEPARATION. 2.1 Introduction. 2.2 The Chromatographic Process. 2.3 Retention. 2.4 Peak Width and the Column Plate Number N. 2.5 Resolution and Method Development. 2.6 Sample Size Effects. 2.7 RELATED TOPICS. References. 3 EQUIPMENT. 3.1 Introduction. 3.2 Reservoirs and Solvent Filtration. 3.3 Mobile-Phase Degassing. 3.4 Tubing and Fittings. 3.5 Pumping Systems. 3.6 Autosamplers. 3.7 Column Ovens. 3.8 Data Systems. 3.9 Extra-Column Effects. 3.10 Maintenance. References. 4 DETECTION. 4.1 Introduction. 4.2 Detector Characteristics. 4.3 Introduction to Individual Detectors. 4.4 UV-Visible Detectors. 4.5 Fluorescence Detectors. 4.6 Electrochemical (Amperometric) Detectors. 4.7 Radioactivity Detectors. 4.8 Conductivity Detectors. 4.9 Chemiluminescent Nitrogen Detector. 4.10 Chiral Detectors. 4.11 Refractive Index Detectors. 4.12 Light-Scattering Detectors. 4.13 Corona-Discharge Detector (CAD). 4.14 Mass Spectral Detectors (MS). 4.15 Other Hyphenated Detectors. 4.16 Sample Derivatization and Reaction Detectors. References. 5 THE COLUMN. 5.1 Introduction. 5.2 Column Supports. 5.3 Stationary Phases. 5.4 Column Selectivity. 5.5 Column Hardware. 5.6 Column-Packing Methods. 5.7 Column Specifications. 5.8 Column Handling. References. 6 REVERSED-PHASE CHROMATOGRAPHY FOR NEUTRAL SAMPLES. 6.1 Introduction. 6.2 Retention. 6.3 Selectivity. 6.4 Method Development and Strategies for Optimizing Selectivity. 6.5 Nonaqueous Reversed-Phase Chromatography (NARP). 6.6 Special Problems. References. 7 IONIC SAMPLES: REVERSED-PHASE, ION-PAIR, AND IONEXCHANGE CHROMATOGRAPHY. 7.1 Introduction. 7.2 Acid-Base Equilibria and Reversed-Phase Retention. 7.3 Separation of Ionic Samples by Reversed-Phase Chromatography (RPC). 7.4 Ion-Pair Chromatography (IPC). 7.5 Ion-Exchange Chromatography (IEC). References. 8 NORMAL-PHASE CHROMATOGRAPHY. 8.1 Introduction. 8.2 Retention. 8.3 Selectivity. 8.4 Method-Development Summary. 8.5 Problems in the Use of NPC. 8.6 Hydrophilic Interaction Chromatography (HILIC). References. 9 GRADIENT ELUTION. 9.1 Introduction. 9.2 Experimental Conditions and Their Effects on Separation. 9.3 Method Development. 9.4 Large-Molecule Separations. 9.5 Other Separation Modes. 9.6 Problems. References. 10 COMPUTER-ASSISTED METHOD DEVELOPMENT. 10.1 Introduction. 10.2 Computer-Simulation Software. 10.3 Other Method-Development Software. 10.4 Computer Simulation and Method Development. References. 11 QUALITATIVE AND QUANTITATIVE ANALYSIS. 11.1 Introduction. 11.2 Signal Measurement. 11.3 Qualitative Analysis. 11.4 Quantitative Analysis. 11.5 Summary. References. 12 METHOD VALIDATION. 12.1 Introduction. 12.2 Terms and Definitions. 12.3 System Suitability. 12.4 Documentation. 12.5 Validation for Different Pharmaceutical-Method Types. 12.6 Bioanalytical Methods. 12.7 Analytical Method Transfer (AMT). 12.8 Method Adjustment or Method Modification. 12.9 Quality Control and Quality Assurance. 12.10 Summary. References. 13 BIOCHEMICAL AND SYNTHETIC POLYMER SEPARATIONS. 13.1 Biomacromolecules. 13.2 Molecular Structure and Conformation. 13.3 Special Considerations for Biomolecule HPLC. 13.4 Separation of Peptides and Proteins. 13.5 Separation of Nucleic Acids. 13.6 Separation of Carbohydrates. 13.7 Separation of Viruses. 13.8 Size-Exclusion Chromatography (SEC). 13.9 Large-Scale Purification of Large Biomolecules. 13.10 Synthetic Polymers. References. 14 ENANTIOMER SEPARATIONS. 14.1 Introduction. 14.2 Background and Definitions. 14.3 Indirect Method. 14.4 Direct Method. 14.5 Peak Dispersion and Tailing. 14.6 Chiral Stationary Phases and Their Characteristics. 14.7 Thermodynamic Considerations. References. 15 PREPARATIVE SEPARATIONS. 15.1 Introduction. 15.2 Equipment for Prep-LC Separation. 15.3 Isocratic Elution. 15.4 Severely Overloaded Separation. 15.5 Gradient Elution. 15.6 Production-Scale Separation. References. 16 SAMPLE PREPARATION. 16.1 Introduction. 16.2 Types of Samples. 16.3 Preliminary Processing of Solid and Semi-Solid Samples. 16.4 Sample Preparation for Liquid Samples. 16.5 Liquid-Liquid Extraction. 16.6 Solid-Phase Extraction (SPE). 16.7 Membrane Techniques in Sample Preparation. 16.8 Sample Preparation Methods for Solid Samples. 16.9 Column-Switching. 16.10 Sample Preparation for Biochromatography. 16.11 Sample Preparation for LC-MS. 16.12 Derivatization in HPLC. References. 17 TROUBLESHOOTING. Quick Fix. 17.1 Introduction. 17.2 Prevention of Problems. 17.3 Problem-Isolation Strategies. 17.4 Common Symptoms of HPLC Problems. 17.5 Troubleshooting Tables. References. APPENDIX I. PROPERTIES OF HPLC SOLVENTS. I.1 Solvent-Detector Compatibility. I.1.1 UV Detection. I.1.2 RI Detection. I.1.3 MS Detection. I.2 Solvent Polarity and Selectivity. I.3 Solvent Safety. References. APPENDIX II. PREPARING BUFFERED MOBILE PHASES. II.1 Sequence of Operations. II.2 Recipes for Some Commonly Used Buffers. Reference. Index.

2,509 citations

Journal ArticleDOI
TL;DR: The system described here gives a direct and precise method for determining DNA base composition by reversed-phase high-performance liquid chromatography (HPLC).
Abstract: DNA base composition was determined by reversed-phase high-performance liquid chromatography (HPLC). DNA was hydrolysed into nucleosides with nuclease P1 and bacterial alkaline phosphatase. The mixture of nucleosides was applied to HPLC without any further purification. One determination by chromatography needed 2 μg of hydrolysed nucleosides and took only 8 min. The relative standard error of nucleoside analysis was less than 1%. The system described here gives a direct and precise method for determining DNA base composition.

2,468 citations

Journal ArticleDOI
TL;DR: Hydrophilic-interaction chromatography fractionations resemble those obtained through partitioning mechanisms, and the chromatography of DNA, in particular, resembles the partitioning observed with aqueous two-phase systems based on polyethylene glycol and dextran solutions.

1,761 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
20231,224
20222,651
2021518
2020661
2019761
2018949