A comprehensive review of the practice of modern liquid chromatography covering a wide range of techniques, such as Reversed Phase LC, Ion Chromatography, Size Exclusion Chromatography and Chiral Chromatography, is provided. This review provides the student with the knowledge required to select an appropriate separation technique for a given analysis. In addition, new approaches to separation science, including capillary electrophoresis and green analytical chemistry tools such as UHPLC is given. A systematic approach to method development for LC techniques is given and an overview of the latest advances in columns for LC and impacts on chemical waste production is discussed.
The module is designed to provide a comprehensive understanding of modern hyphenated techniques (GC-MS, LC-MS, GC-MSMS, LC-MSMS and ICP-MS) for separation, identification and quantification and their examples of their uses in analytical chemistry are shown.
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Separation Science
HPLC: Mode of separation (Normal and Reversed phase LC), instrumentation used, and UHPLC. Chiral separation.Ion Chromatography (IC): Mode of separation, suitable stationary and mobile phase used, anionic and cationic columns, suppressor columns and conductivity detectors. Size Exclusion Chromatography (SEC): Separation mode, stationary phase and mobiles phases used for Gel permeation chromatography (GPC) and gel filtration chromatography. Capillary electrophoresis: Electroosmosis, electrophoresis, flow dynamics, efficiency and resolution, capillary gel electrophoresis, micellar electrokinetic capillary chromatography,Resolution Equation: Understanding how the degree of separation is dependent on both thermodynamic factors (k, α) and kinetic factors (peak width, w, and N). Method Development for LC: Optimising the solvent strength for mobile phase, choice of buffers, selecting the appropriate pH and using ion-pair reagents and choosing green organic solvents where possible. Selecting an appropriate column, mobile phase and detector for the analysis while considering chemical waste, energy use and overall carbon emissions. Assessing if isocratic or gradient methods are more appropriate.Column design: effect of length, diameter, particle size, UHPLC columns, monolithic columns. Chiral separations through the addition of modifiers to the mobile phase, case studies, cyclodextrins, derivatised surfactants. Column coupling and column switching, solid phase extraction.Detectors: discuss the range of detectors used for LC including photodiode array, fluorescence, refractive index, electrochemical methods of detection. Conductivity detectors for Ion Chromatography. Applications: Pharmaceutical (USP/BP monographs), Forensic and Environmental.
Hyphenated Techniques
Introduction to hyphenation (GC/MS, HPLC/MS, MS/MS)Mass spectrometers – quadrupole, ion trap, time of flight (ToF). Mass Spectrometer Resolution.Selected Ion Monitoring (SIM) and Multiple reaction Monitoring (MRM); multi-analyte methods.Tandem spectrometer MS/MS detection in GC and LC. Helium, a finite resource: alternative gases for GCMS instruments.GC Interfaces: Electron Impaction Ionization (EI) and Chemical Ionization (CI). HPLC Interfaces: Electrospray ionization (ESI), Atmospheric Chemical Ionization (APCI) and Atmospheric Pressure Ionization (API). Applications: Drug discovery, environmental, combinatorial chemistry, biochemistry (proteins/peptides).MS as a detector, interpreting the mass spectra for qualitative and quantitative analysis. The use of deuterated internal standards in MS. Overview of Inductively Coupled Plasma MS (ICP-MS). Applications of MS and MSMS.
Laboratory Work
Laboratory work which complements the lecture material is sourced from the School of Chemical and Pharmaceutical Sciences Laboratory Manual. Includes ethical practical to assess quality of medicines from lower and middle-income countries.
Delivery by means of lectures (20 hours), tutorials (4 hours), laboratory practicals (8 hours) and self-study (68 hours) to include computational problems and report writing.
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| Assessment Breakdown | % |
| Formal Examination | 75 |
| Other Assessment(s) | 25 |