CHE20006/CHE60001 Analytical Chemistry Chromatography Assignment. Assignment due Monday 18th May, 2020.Use of a computer-based HPLC simulator: Optimization of the mobile phase composition for a...

CHE20006/CHE60001 Analytical Chemistry Chromatography Assignment. Assignment due Monday 18th May, 2020.Use of a computer-based HPLC simulator: Optimization of the mobile phase composition for a chromatographic separation based on a systematic approach.In this assignment you will use a computer based HPLC simulator to develop an optimized separation for a set of standard solutes using your knowledge of chromatographic separations. The HPLC simulator is a free, open-source software that is available in the Chromatography Assignment Module on Canvas. The program was sourced from the internet at the following link: http://www.hplcsimulator.org/simulator.phpThe Scenario.In a commercial chemical analysis laboratory it is usual to develop reference methods for the analysis of typical samples that may be routinely required. So when a sample is suspected of containing certain compounds that correspond to a standard reference method, an analysis can be readily implemented without the time consuming need to design an analysis for each specific sample. In this assignment, the sample mixture contains the following components: acetophenone ethylparaben propiophenone benzophenone propylparaben 3-nitrophenol ketoprofen butylparaben 4-nitrophenolTheory.A systematic approach to optimization involves varying the experimental conditions in order to separate the components with the highest resolution in the minimum run time. The retention factor k for a solute is defined ask=tr −t0 t0where tr is the retention time for a particular solute and t0 is the void time determined as the time for an unretained solute to pass through the chromatograph. The selectivity factor α is the ratio of two adjacent retention factors where k1 for the first solute elutes before the second solute with k2.=k kUnder isocratic conditions where the mobile phase composition remains constant during the chromatographic run, the relationship between the retention factor and the mobile phase composition Φ has the following formlogk =S+cwhere S and c are the slope and intercept when plotting a graph of log k as a function of the mobile phase composition. Each component has unique S and c values, which means that a graphical plot can be employed when finding the optimum mobile phase composition. The best separation for a set of solutes will be obtained when the differences between log k and Φ are maximized. The spreadsheet Optimisor.xlsx will help you identify these favorable mobile phase compositions but you will need to verify the best overall conditions.The Simulator.In practice, the process of optimizing a separation is a time consuming task because a single chromatographic run could be between 5 and 20 minutes. If the optimization involves varying the mobile phase composition then an equilibration time of about 15 minutes is required between runs to ensure that the mobile phase composition is uniform throughout the system. It is obvious that many hours may be required to identify the optimum conditions. This assignment uses21
software to simulate the chromatographic instrument and simple slide controls replace labor intensive and time consuming tasks. The table under the chromatogram contains the retention time in minutes and the retention factor for each component. The void time is displayed under the Column Properties and is given in seconds. The mobile phase composition is varied either by moving the slider or typing the percentage of Solvent B in the box. The chromatogram will instantly appear and the table updates. You are free to explore how the various parameters influence the chromatogram but this
assignment is based on the default parameters and you should restart the program to reset all parameters.The Assignment.Optimization of the mobile phase composition will occur when the smallest selectivity factor for two adjacent peaks for a set of solutes obtains its maximum value. This means that the two least resolved components will become separated by the greatest margin possible but this has to be considered in the context of the overall separation. As the goal of any separation is to separate all components in a minimum amount of time, it follows that maximizing the retention time difference for the two least resolved solutes has to be balanced against the overall run time.You should know that the optimum range for retention factors is between 2 and 5 which can be extended to between 1 and 10 for particularly complex mixtures. This is because there is only a slight improvement in resolution once the retention factor is larger than 10 but the total run time continuously increases and this must be considered against the overall separation goal.You are required to vary the mobile phase composition and record the retention times in the yellow area of the spreadsheet. You will also need to input the void time in minutes. The spreadsheet then generates two graphs with one being the selectivity factors versus the mobile phase composition and the second one being a plot of the minimum selectivity factor.The Report.You should include the graph of the minimum selectivity factor and chromatograms that correspond to the mobile phase composition for the four highest values of the selectivity factor (i.e. α > 1.08). The chromatograms can be obtained using the Copy button below the chromatogram in the simulator. For each chromatogram you need to identify if all the components are fully resolved and discuss the ‘quality’ of the separation based on the overall separation goal. You should then include a conclusion that recommends a particular mobile phase composition based on your evaluation of the four different chromatograms.References.R. A. Shalliker, S. Kayillo and G. R. Dennis, “Optimizing Chromatographic Separation: An Experiment Using an HPLC Simulator”, J. Chem. Ed. 85 (2008) 1265-1268.HPLC Simulator, http://www.hplcsimulator.org/simulator.php