This is a tool to separate compounds according to its hydrophobicity or hydrophilicity. A gradient is given from hydrophilic to hydrophobic range of solvent moving through a solid matrix. Here are some valuable tips collected through experience and handed over so that the reader can find it useful when the chromatograms and the instruments prove so cross with you. The instrumentation and working for both the systems HPLC and FPLC are almost same.
HPLC is High Performance/Pressure Liquid Chromatography and FPLC is Fast Protein Liquid Chromtography.
Table of Contents
- HPLC and FPLC
- Beer Lambert law
- Theoretical plates
- Calculation of theoretical plates
- Resolution
- Phases
- Calculation of retention time and absorbance
- Column capacity
- Components
- Pumps
- Flow path
- Tubings
- Leak
- System wash
- Injector
- UV detector
- Guard column wash
- Column
- Standards for column
- Normal phase column
- Reverse phase column
- Column wash
- Solvent
- Buffer selection for reverse phase HPLC
- Preliminary purification
- Sample preparation
- HPLC sample
- Gradient
- Elution
- Chromatogram
- Fraction
- Chromatogram patterns
- Elution profiles
- FPLC
- Elution volume
- Diffusion of molecules with matrix
- Resolution
- FPLC system
- FPLC column
- Column types
- FPLC system
- Column packing
- Sample preparation
- Buffer
- Washing
- Gradient
- Chromatogram in FPLC
- Chromatogram profiles troubleshooting
Objectives and Key Themes
The objective of this text is to provide practical troubleshooting and standardization tips for HPLC and FPLC chromatogram purification profiles. It aims to equip readers with the knowledge and techniques to overcome common challenges in these chromatographic techniques.
- Troubleshooting HPLC and FPLC systems
- Optimizing chromatogram purification profiles
- Understanding chromatographic principles (e.g., resolution, retention time)
- Sample preparation and handling techniques
- Effective use of solvents and buffers
Chapter Summaries
HPLC and FPLC: This introductory chapter establishes the context of High-Performance Liquid Chromatography (HPLC) and Fast Protein Liquid Chromatography (FPLC) as tools for separating compounds based on hydrophobicity/hydrophilicity. It emphasizes the similarities in instrumentation and operation between the two techniques and sets the stage for the practical troubleshooting advice provided in subsequent chapters. The chapter highlights the use of a solvent gradient moving through a solid matrix to achieve separation.
Beer Lambert law: This section delves into the fundamental Beer-Lambert law, explaining its relevance to quantifying analyte concentration in HPLC and FPLC analyses. The principle and its application in determining concentration from absorbance measurements are explained, linking it to subsequent chapters discussing quantitation of separated components.
Theoretical plates: This chapter covers the concept of theoretical plates in chromatography. It explains the relationship between plate number, column efficiency, and peak broadening. The discussion likely includes equations related to the calculation of theoretical plates and the importance of optimizing column performance. This provides a foundation for understanding resolution and efficiency in later chapters.
Calculation of theoretical plates: This chapter provides practical steps and examples for calculating the number of theoretical plates in a chromatographic column using the chromatogram data. The calculations are essential for evaluating column performance and comparing different chromatographic conditions. This builds upon the previous chapter explaining the significance of theoretical plates.
Resolution: This chapter focuses on the definition and importance of resolution in chromatography, which describes the ability to separate two closely eluting peaks. Factors affecting resolution, such as column efficiency, selectivity, and retention time, are discussed. The chapter likely includes equations related to resolution calculations and methods for improving the resolution of difficult separations. It further develops the themes introduced in previous chapters on theoretical plates and column efficiency.
Phases: This chapter explains the different stationary and mobile phases used in HPLC and FPLC. It explores normal phase and reverse phase chromatography, discussing the characteristics and applications of each. The choice of phase significantly impacts separation efficiency, and this section is critical for understanding the method development and optimization in later chapters.
Calculation of retention time and absorbance: This section details the practical calculation of retention times and the determination of peak areas from HPLC and FPLC chromatograms. It links these measurements directly to quantitative analysis, building upon the earlier chapters discussing the Beer-Lambert law and the significance of peak area in determining the amount of a given component.
Column capacity: This chapter explains the concept of column capacity, its relation to sample loading and separation efficiency. It explores methods for determining column capacity and strategies for optimizing it to avoid overloading. The impact on separation efficiency and peak shape is discussed.
HPLC and FPLC Chromatogram Purification Profiles: Frequently Asked Questions
What is the purpose of this text?
This text aims to provide practical troubleshooting and standardization tips for HPLC and FPLC chromatogram purification profiles. It equips readers with the knowledge and techniques to overcome common challenges in these chromatographic techniques.
What are the key themes covered?
Key themes include troubleshooting HPLC and FPLC systems, optimizing chromatogram purification profiles, understanding chromatographic principles (resolution, retention time), sample preparation and handling, and effective solvent and buffer use.
What chromatographic techniques are discussed?
The text focuses on High-Performance Liquid Chromatography (HPLC) and Fast Protein Liquid Chromatography (FPLC), comparing and contrasting their applications and operational aspects.
What fundamental principles are explained?
The text covers fundamental chromatographic principles such as the Beer-Lambert law (for analyte quantification), theoretical plates (for column efficiency), resolution (for peak separation), and the roles of stationary and mobile phases (normal and reverse phase chromatography).
How are calculations used in the text?
The text includes practical calculations for determining theoretical plates, retention times, absorbance, and resolution. These calculations are essential for evaluating column performance and optimizing separation.
What aspects of HPLC and FPLC instrumentation are addressed?
The text discusses various components of HPLC and FPLC systems, including pumps, flow paths, tubings, injectors, UV detectors, columns, and guard columns. It also addresses troubleshooting issues related to these components (e.g., leaks, system washes).
What is covered regarding sample preparation and handling?
The text details sample preparation techniques and the importance of proper sample handling for optimal results in HPLC and FPLC analysis.
What is the role of solvents and buffers?
The text emphasizes the importance of solvent and buffer selection, particularly buffer selection for reverse-phase HPLC. It discusses how these choices impact separation efficiency.
How are chromatograms interpreted?
The text covers the interpretation of chromatograms, including the analysis of peak patterns, elution profiles, and troubleshooting of problematic chromatograms in both HPLC and FPLC.
What topics are covered in the chapter summaries?
Chapter summaries cover HPLC and FPLC introduction, the Beer-Lambert law, theoretical plates, their calculation, resolution, stationary and mobile phases, calculation of retention time and absorbance, column capacity, and more.
What are the objectives of this text?
The primary objective is to provide practical, hands-on guidance for troubleshooting and optimizing HPLC and FPLC techniques for chromatographic purification. The text aims to enhance the reader’s ability to interpret chromatograms and solve common problems encountered in these analytical methods.
- Quote paper
- T.S. Amar Anand Rao (Author), 2012, HPLC and FPLC: Troubleshooting and Standardizing Chromatogram Purification Profiles, Munich, GRIN Verlag, https://www.grin.com/document/186925
