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1. Introduction
- Basic concepts in HPLC
- Overview of various kinds of chromatography (Reversed & Normal Phase, Ion Exchange, Gel Filtration/Permeation, Affinity, Hydrophobic Interaction, Chiral, etc.)
- Definition of HPLC "system"
- Isocratic and gradient systems
- Types of pumps, injectors, detectors, data devices
- Columns, construction
- Mechanism of separation on column
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2. Video HPLC Demonstration using Visible Separation
- Reversed phase separation of colored compounds done on glass column to illustrate several separation concepts and anomalies
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3. Separation Theory
- Description of chromatogram
- Resolution equation, good & bad resolution numbers, practice calculating resolution, what the numbers mean
- Introduction to Capacity, Selectivity, and Efficiency
- Definition and use of k', α, and N, parts they play in improving separations
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4. Controlling Resolution
- Concept and multiple examples of adjusting various k', α, and N factors to change a separation
- Above includes role of temp., pH, particle size, column length, column chemistry, surface area, mobile phase strength & chemistry, flow rate, sample load, and so forth
- Examples are given as to how these apply to Ion Exchange, Ion Chromatography, Gel Permeation, Reversed Phase and Normal Phase Chromatographic modes.
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5. Mobile Phase
- Solvent selection according to solvent group
- Solvent strength according to polarity index
- Characteristics of solvents: UV cut-off, RI, viscosity, etc.
- Changing solvents, calculating mobile phase polarity
- Performing "window plots" to predict appropriate solvent mixtures
- Effects of salt concentration, pH
- UV spectra of solvents & buffers
- Checking for impurities, safety concerns
- Methods of filtration and de-gassing
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6. Normal and Reversed Phase Chromatography
- Selection of separation mode
- Comparison of sample, column, mobile phase polarities and how they interplay
- Normal Phase characteristics, columns, how separations occur
- Examples of Normal Phase separations
- Overcoming problems
- Reversed Phase characteristics, columns, how separations occur
- Dealing with ionic samples through ion suppression & ion pairing
- Examples of Reversed Phase separations
- Overcoming problems
- Column selection criteria: column geometry, column type, packing materials, particle size, shape, size distribution, surface area, pore size, volume, distribution, functional groups, carbon load, end capping, etc.
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7. Gradient Chromatography
- Types of gradients and gradient systems
- Dealing with resolution problems by changing gradient shapes
- Examples of gradient changes and their effect on chromatography
- Determination of delay volume and time, accuracy, re-equilibration
- Using gradient to determine isocratic conditions for method development
- Rules for gradient use
- Overcoming gradient problems
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8. Sample Preparation
- Purposes and goals of sample prep.
- Typical sample prep options and routines, when and how to use them
- Examples of sample prep problems and solutions
- Discussion of specific questions from the audience involving sample prep.
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9. Detection and Quantation
- Comparison of detector types
- UV detection, chromophores/wavelength, types of UV/Vis. detectors (including variable, programmable, & PDA), how they work, Beer's Law, molar absorptivity, selectivity, and spectral information
- RI detectors, where used, how they work, examples
- Fluorescence detectors, where used, how they work, examples
- Electochemical detectors, where used, how they work, examples
- Conductivity detectors, where used, how they work, examples
- Light Scattering Detectors, how they work, typical applications, examples
- Concepts of sensitivity and linearity
- Integration, how integrators work, setting parameters
- External and internal standard methods, advantages and disadvantages
- Determining accuracy and precision
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10. Troubleshooting
- Determining system suitability
- Causes of a variety of problems, what baselines tell us
- Divide and conquer approach to system troubleshooting
- Determination of faulty component
- Generic troubleshooting of injectors, pumps, detectors
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