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4. (25 marks) a) (9 marks) Discuss the following statements about HPLC (3 marks each): 3) Explain why at least 60 µL should be loaded onto a 20 μL loop for injecting a sample onto an HPLC column and what will occur if only 20 µL is loaded. ii) Discuss why HPLC pumps have two pistons that work 180° out of phase. ii) Explain why a monolithic column can be used at higher flow rates than particle packed columns and what implications this has on the chromatography. b) (10 marks) A sample containing compounds A, B, C and D (each containing a UV chromophore) requires separation by chromatography. Currently the HPLC method is a 30 minute isocratic elution with a flow of 0.7 mL/min using 65% water, 35% acetonitrile as the solvent. A C18 column (4.6 x 250 mm) with 3.5 um particles is used for separation and UV is used for detection. The column temperature is 40 °C and injection volume is 10 µL. Compound A elutes at 4.63 minutes; compounds B and C co-elute at 11.2 minutes and compound D elutes at 29.06 minutes. The chromatogram can be seen below.
240- 200- 180- 100- 140- 120- 100- -08 -09 For 20 10 12 15 18 20 24 sme (min)
3) (4 marks) Explain how the following changes to the method will affect the peaks in the chromatogram (retention time, peak shape, resolution): A. The flow rate is increased to 2 mL/min B. The injection volume (loop size) is increased C. A weaker solvent is used with isocratic elution. State what the solvent composition may be D. A column with larger particle size (10 μm) is used ii) (6 marks) Discuss how you would change the method (keeping the same column) to achieve better separation and a shorter analysis time. For each change, explain the effect the change will have on the chromatogram. Your answer should include, but is not limited to: elution type and profile, mobile phases (solvent type, strength and polarity), flow rate. You may wish to draw figures to supplement your answer. c) (6 marks) A biologist has approached you to measure the concentration of proteins in their samples using ion chromatography. 1) (2 marks) Explain which stationary phase you would choose for your column. ii) (4 marks) Based on the chosen column, explain in which direction the pH gradient needs to change in order to increase the solvent strength and how does the elution of the compounds relate to the isoelectric point of the protein.