Practical HPLC - Philadelphia University：高效液相色谱-费城大学.pptVIP

Practical HPLC In This Section, We Will Discuss: How to set up an HPLC System for a sample injection including: Solvent Handling Mobile Phase preparation Priming the HPLC Column Handling - Equilibration System Performance Checks Solvent Handling Solvent Characteristics (Specifications): Purity Viscosity Refractive index Boiling Point Toxicity UV Transparency/UV-Cutoff Solubility Solvent Miscibility Solvent UV-Cutoff/Transparency Mobile Phase Preparation Major Steps: Measure appropriate volume of each solvent Mix solvents Add buffers and additives* Filter mobile phase Degas mobile phase Priming HPLC System Priming HPLC System Priming HPLC System Priming HPLC System Priming HPLC System Priming HPLC System Priming HPLC System Priming HPLC System Priming HPLC System Priming HPLC System Priming the HPLC Sample Preparation At a minimum - filter samples: Nylon - hydrophilic nature works with aqueous and solvent based samples, autoclavable to 121oC, pH range 3-12, no concentrated acids. PTFE- a hydrophobic membrane which is highly resistant to solvents, acids, and alkalis. This filter is generally used for non-aqueous samples. pH range 1-14. Cellulose Acetate- good filter for aqueous biological samples with very low protein retention. pH range 4-8. PVDF- highly resistant to most solvents, exhibits low protein binding. pH 2-12. Ultrafilter Membranes- molecular weight cut-off filters for biological samples. Nitrocellulose- exhibits high protein retention. Solid Phase Extraction. Sample Preparation Dissolve the sample in the mobile phase or in a solvent weaker than the mobile phase. The sample volume should be kept as small as possible. Column Storage Column Installation Each column has a defined flow direction! The flow direction is shown by the arrow or direction of writing. Don’t change the flow direction, this will decrease column performance. Column Installation Column Installation cont. Column Equilibration Column Check New columns should be delivered with a