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1. Introduction to HPLC of Proteins & Peptides
- Structure of proteins and peptides which provide means for separation
- Overview of various kinds of chromatography used to separate proteins and peptides (Reverse Phase, Size Exclusion, Ion Exchange, Affinity Chromatography and Hydrophobic Interaction)
- Overview of types of instrument systems and components available, isocratic and gradient, etc.
- Column construction
- General 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 chromatographic retention mechanisms, source of separation selectivity, and role of efficiency
- 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
- 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
- Peak tailing and fronting determination
- General concepts of achieving good HPLC separations
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4. Mobile Phases
- Properties of solvents and their role in HPLC
- Solvent strengths, polarity index values
- Changing solvents to optimize a method and determining optimal proportions
- Optimizing solvent mixtures using "window plots"
- Common buffers, salts and acids used in protein/peptide separations
- Optimizing salt concentration, pH, modifiers
- UV spectra of solvents & buffers, appropriate buffer ranges
- Various surfactants & detergents, additives, and denaturants for proteins
- Checking for impurities, discussion of safety concerns
- Methods of filtration and de-gassing, effect on auto samplers and final results
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5. Ion Exchange Chromatography of Proteins
- Review of Ion Exchange characteristics
- Discussion of types of HPLC columns available
- Mechanisms of anion and cation separations
- Role of pH in enhancing or suppressing charge
- Side chain amino acids which propagate molecular charge, amphoteric property of proteins
- Examples of working with anion and cation exchange separations, how pH, flow rate, gradient, temperature, and buffer choice enhance separations
- Steps to optimize ion exchange separations
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6. Size Exclusion Chromatography
- Review of SEC characteristics
- A look at some common columns for SEC, how they are constructed, how they work
- The permeation process, molecular weight vs. pore size, calibration curves
- Examples of adding columns of same and different pore sizes and their effect on the separation
- The role of pH, buffers, salts, modifiers on the separation
- Steps to optimize SEC separations
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7. Reversed Phase Chromatography
- Reversed phase characteristics, mechanism of separations, how separations of peptides differ from proteins
- Examples of columns, how they are made, cautions regarding pH
- Polarity contributions of each amino acid to overall polarity of a protein or peptide, and how pH affects polarity
- Effects of solvent choice, flow rate, gradient rate, ion pair reagents
- A look at column size, narrow/micro-bore, preparative scale
- Reducing tailing, use of modifiers, end-capped packings
- Role of carbon loading
- Optimizing reversed phase separations
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8. Hydrophobic Interaction Chromatography
- Characteristics of HIC, how the separation is achieved
- Maintaining protein activity
- HIC columns available, characteristics, properties
- Role of column chemistry, selecting binding salts, effect of pH, gradient, mobile phase modifiers, temperature
- Scaling up to prep., improving recovery
- Adjusting conditions, achieving optimal separations
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9. Gradient Chromatography
- Types of gradients and gradient systems
- Dealing with resolution problems
- Examples of gradient changes and their effect on chromatography
- Dealing with baseline problems, TFA gradient issues
- Using gradient to determine isocratic conditions for method development
- Rules for gradient use
- Optimizing gradient separations
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10. Detection & Quantitation
- Comparison of detector types, properties
- UV detection, chromophores/wavelength, spectrum of proteins
- Types of UV/Vis. detectors (including variable, programmable, & PDA), how they work, Beer's Law, molar absorptivity, selectivity, and spectral information
- Fluorescence detectors, forming protein, peptide, amino acid derivatives
- Radioactivity detectors, their use for radiolabeled proteins/peptides
- Mass spectrometer detectors, various interfaces for HPLC
- Light scattering detectors, their use in determining molecular weight of proteins
- Electrochemical detectors, use in high sensitivity detection of proteins
- Conductivity detectors, use in monitoring salt gradients
- Concepts and definitions of sensitivity and linearity
- Integration, how integrators work, setting parameters
- External and Internal Standard modes of quantitation
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10. Sample Preparation for Protein/Peptide Analysis
- Purposes and goals of sample preparation, desired results
- Typical sample prep options and routines for proteins, when and how to use them
- Examples of: extraction of proteins from complex samples, precipitation of proteins, centrifugation/ultra-centrifugation, filtration/ultra-filtration, molecular sieving, solid phase extraction, derivatization to enhance sensitivity
- De-salting options for proteins,
- Scheme for handling crude protein samples
- Removing lipids from protein samples.
- Discussion of specific questions from the audience involving sample prep.
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10. Troubleshooting
- Maintenance of columns, pumps, injectors, and detectors
- Determining system suitability
- Causes of a variety of problems (retention time shifts, broad/tailing peaks, leaks, short column life, baseline problems, detection sensitivity issues, etc.) how to avoid them, and how to quickly identify their cause.
- Divide and conquer approach to system troubleshooting
- Determination of faulty component
- Troubleshooting of injectors, pumps, detectors
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