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Optimization of High-Performance Liquid Chromatography methods
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Advanced Spectroscopic and Analytical Techniques
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Academic year 2023/2024
- Teacher
- Claudio Minero
- Teaching period
- Second semester
- Type
- Basic
- Credits/Recognition
- 4
- Credits 24 CFU plan
- 4 CFU (16 h)
- Course disciplinary sector (SSD)
- CHIM/01 - analytical chemistry
- Delivery
- Formal authority
- Language
- English
- Attendance
- Obligatory
- Type of examination
- Oral
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Sommario del corso
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Program
The main parameters affecting separation and resolution in HPLC are examined, starting from basic chromatographic theory. The main separation methods are examined: RPC for non-polar and ionic analytes, direct phase and HILIC, with focus on relevant parameters for method development (solvent strength, temperature, type of solvents, type of columns).
The detailed Program involves the following topics.
Method development: Column parameters, the peak width and resolution. Band broadening and factor affecting it. Knox equation and the conceptual basis for the optimization of HPLC conditions.
REVERSED PHASE CHROMATOGRAPHY (RPC). RPC theories, Solvent-Strength Selectivity, regular and irregular analytes. Optimization by changes in %B, change of solvent, fine-tuning the B-solvent, Temperature. Solvent selectivity triangle. Stationary phases. Optimization of particle size, thickness of stationary phase, flow rates, mobile phases viscosities. Method development (isocratic), peak shape, tailing, column overload in Volume and Mass. Gradient Elution. Gradient Shape, linear-solvent-strength (LSS) gradients, Band Migration in Gradient Elution, Gradient peak shape, gradient retention, Band compression, Resolution α*. Optimization of gradient elution. Segmented gradients. Steps for Method development (gradient). Gradient problems. Optimization by RPC column selection. RPC column selectivity, and the interactions that can affect column selectivity. Hydrophobic subtraction model. Method development by column change. RPC for ionizable samples. The effect of pH change on retention, buffer type and concentration. Method development for ionic samples. ION-PAIR chromatography (IPC). Basic of retention and mechanism. Ion-Pair Reagents, effect of Concentration and Type on retention. Method development and choice of Initial Conditions. IPC problems.
ION-EXCHANGE CHROMATOGRAPHY (IEC). Basis of Retention and Retention factor for IEC, Role of the Counter-Ion. Applications for Separations of Carbohydrates by AEC.
NORMAL PHASE CHROMATOGRAPHY (NPC). The background and actual uses. Synthetic (unbonded) silica, HILIC phases (polar-bonded-phase packings): cyano, diol, amino. NPC retention mechanism, eluent strength. Non localizing and basic or nonbasic localizing solvents: effect on selectivity. Solvent blends. Isomer Separations. NPC method development.
Hydrophilic Interaction Chrom. (HILIC). Type of columns and B solvent composition. HILIC mechanism, columns and method development. HILIC Problems.
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Course delivery
lessons in the presence of teacher, remote connections are allowed
Suggested readings and bibliography
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- Book
- Title:
- Introduction to Modern Liquid Chromatography
- Year of publication:
- 2010
- Publisher:
- Wiley
- Author:
- L.R. Snyder, J.J. Kirkland, J.W Dolan
- Required:
- Yes
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- R. Meyer, Practical High-Performance Liquid Chromatography, 5th ed., Wiley, 2010
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Notes
Lectures are scheduled on:
08/07 - 14.00-16.00 - AULETTA I
09/07 - 14.00-16.00 - AULETTA I
10/07 - 14.00-16.00 - AULETTA I
12/07 - 14.00-16.00 - AULETTA I
16/07 - 09.00-11.00 - AULA AG3
17/07 - 09.00-11.00 - AULA VALLAURI
18/07 - 09.00-11.00 - AULETTA II
19/07 - 09.00-11.00 - AULETTA II- Enroll
- Open
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