Set the pump at a nominal mobile phase flow rate (for example, 2 milliliters per minute). Place the solvent outlet line into a graduated cylinder. Start the stopwatch and allow the pump to function for a set period of time. Read the volume of solvent in the graduated cylinder. The volume collected should equal the flow rate multiplied by the time period to demonstrate the pump flow rate programming is working.
Set the pump to produce a given ratio of the two mobile phase solvents. Place the solvent outlet line into a pre-weighed graduated cylinder and allow the pump to function until 50 to 100 milliliters of solvent collect. Read the exact volume of solvent in the cylinder and then reweigh the cylinder with the solvent. Calculate the weight of solvent dispensed. From the weight and the respective densities of the two pure solvents, calculate the ratio of solvents dispensed into the cylinder. This ratio should equal the ratio programmed into the HPLC pump.
Load a standard check sample into the autosampler and set it to perform at least ten repeated injections of the standard. The check sample should contain a specific chemical that the HPLC detector responds to and the concentration of the chemical in the sample should be known. For each injection, record the detector output including the retention time of the analyte and the detector response (peak height or area). Calculate the average and standard deviation for all ten retention times and responses. Check that the standard deviation in each case is small compared to the absolute value of the average retention time and detector response. This will allow you to validate the precision of the autosampler, column and detector.