The comparison of flow rate calibration methods for high-performance liquid chromatography (HPLC) pump

High-performance liquid chromatography (HPLC) pump has a main function to deliver a mobile phase which needs accuracy and stable flow rate. Recently, the reference of General European OMCL Network 2005: Qualification of Equipment Annex 1 qualification of liquid chromatography equipment presents three flow rate calibration methods that have been used to calibrate the HPLC pump performance such as using standard flow meter, gravimetric method, and volumetric method. In this reference, the calibration methods have not been clearly described. Therefore, this study aimed to investigate and compare the flow rate calibration by the using standard flow meter, gravimetric method, and volumetric method. The flow rate was calibrated at 0.5 and 5 mL/min with three replications. The stability between days of each method was observed for three days. The calibration procedure, traceability chart, and uncertainty budget of each method had been described. The flow rate results were analyzed by using the E_n ratio. The results showed that the stability of all methods had no statistically significant difference between days when compared to the first day itself. The average measurement flow rates by using standard flow meter, gravimetric method, and volumetric method at 0.5 mL/min were 0.4908, 0.4992, and 0.5012 mL/min, respectively, and at 5 mL/min, they were 4.8346, 4.8272, and 4.8394 mL/min, respectively. These flow rates showed consistent results between each other as confirmed with the E_n ratio ≤ 1. The precision of the three methods was 0.01 to 0.08 %RSD. This study concluded that the flow rate results of three calibration methods had been consistent with each other. All methods had a good stability and precision for the flow rate calibration of the HPLC pump.