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
1. HPLC and FPLC
2. Beer Lambert law
3. Theoretical plates
4. Calculation of theoretical plates
5. Resolution
6. Phases
7. Calculation of retention time and absorbance
8. Column capacity
9. Components
10. Pumps
11. Flow path
12. Tubings
13. Leak
14. System wash
15. Injector
16. UV detector
17. Gaurd column wash
18. Column
19. Standards for column
20. Normal phase column
21. Reverse phase column
22. Column wash
23. Solvent
24. Buffer selection for reverse phase HPLC
25. Preliminary purificatino
26. Sample preparation
27. HPLC sample
28. Gradient
29. Elution
30. Chromatogram
31. Fraction
32. Chromatogram patterns
33. Elution profiles
34. FPLC
35. Elution volume
36. Diffusion of molecules with matrix
37. Resolution
38. FPLC system
39. FPLC column
40. Column types
41. FPLC system
42. Column packing
43. Sample preparation
44. Buffer
45. Washing
46. Gradient
47. Chromatogram in FPLC page
48. Chromatogram profiles troubleshooting
Objectives and Core Topics
This work aims to provide a comprehensive guide on the operational principles, maintenance, and standardization of High Performance Liquid Chromatography (HPLC) and Fast Protein Liquid Chromatography (FPLC) systems. It addresses the practical challenges of purification by detailing troubleshooting strategies and optimized protocols for varying laboratory conditions.
- Theoretical foundations of chromatography including resolution, theoretical plates, and Beer-Lambert law applications.
- System components and maintenance, including pump operation, column packing, and leak prevention.
- Optimization of solvent gradients and buffer selection for specific purification requirements.
- Troubleshooting chromatogram profiles and interpreting common artifacts or elution patterns.
Excerpt from the Book
Theoretical Plates
Separation of the solute happens based on the number of theoretical plates of matrix.
The number of theoretical plates can be calculated from the bead size of the column and from the length of the column.
Alternatively it can also be calculated from the peak width and the height.
Theoretical plates are the imaginary plates of size the thickness of one bead size.
So one theroretical plate is a plate consisting of a single layer of beads of silica particles. From this, the number of theoretical plates running throughout the column can be calculated from the length of the column.
The use of calculating this is to directly determine the efficiency of the column.
The more the number of theoretical plates, the more the efficiency.
This is because the sample gets more distance to get separated out by flowing for a longer distance and it diffuses more laterally depending on the flow rate.
Summary of Chapters
HPLC and FPLC: Introduces the basic tools and instruments used for separation based on hydrophobicity or hydrophilicity.
Beer Lambert law: Details the mathematical relationship between absorbance and analyte concentration within the flow cell.
Theoretical plates: Explains the concept of theoretical plates as a metric for column efficiency and separation quality.
Calculation of theoretical plates: Outlines practical methods for calculating column performance indices by hand using peak dimensions.
Resolution: Discusses the capacity of the system to separate closely eluting compounds through gradient management.
Keywords
HPLC, FPLC, Chromatography, Theoretical Plates, Elution, Gradient, Resolution, Solvent, Buffer, Protein Purification, Absorption, Troubleshooting, Chromatogram, Matrix, Silica Beads
Frequently Asked Questions
What is the primary purpose of this document?
This document serves as a technical manual and troubleshooting guide for operating HPLC and FPLC systems in a laboratory environment, focusing on protein and compound purification.
What are the central themes covered in the text?
The text focuses on system maintenance, column handling, buffer optimization, and the interpretation of chromatography results through chromatogram analysis.
What is the primary research goal addressed?
The goal is to standardize purification profiles and assist researchers in resolving common technical difficulties encountered during liquid chromatography experiments.
Which scientific methods are primarily discussed?
The manual covers gradient elution, gel filtration chromatography, and reverse-phase separation techniques.
What is covered in the main section of the document?
The main sections provide detailed instructions on system components, maintenance, sample preparation, gradient programming, and detailed troubleshooting charts.
What keywords characterize the work?
The work is defined by terms such as liquid chromatography, protein purification, system maintenance, and chromatogram optimization.
How should air bubbles be handled in the pump?
Air bubbles are removed by purging the system, using a syringe needle to extract air from the pump head, or by loosening the purge valve while the system is running.
Why is column storage critical in this context?
Proper storage in solvents like 20% ethanol is essential to prevent microbial growth, drying of the matrix, and degradation of the column material.
What is the importance of the Beer-Lambert law here?
It is used to calculate the concentration of proteins or other analytes based on their absorbance at specific wavelengths, usually 280nm.
- 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
