3.2 The comparison of three flow rate calibration methods
The comparation results of the using standard flow meter, gravimetric method, and volumetric method at the flow rate of 0.5 and 5 mL/min are showed in Table 4. At the flow rate of 0.5 mL/min, the mean measured flow rates of the using standard flow meter, gravimetric method, and volumetric method were 0.5062, 0.4992, and 0.5012 mL/min, respectively. These measured flow rates from the three calibration methods showed similar results as confirmed with the En ratio results (En ≤ 1). For the En ratio, the using standard flow meter was used as the reference method because it is a direct flow rate measurement method whereas the gravimetric and volumetric method are a realization measurement method. The En ratios of the gravimetric method and volumetric method were 0.6 and 0.5, respectively. These results indicated that the flow rate calibration by the gravimetric and volumetric method were consistent with the using standard flow meter.
At the flow rate of 5 mL/min, the mean measured flow rates of the using standard flow meter, gravimetric method, and volumetric method were 4.9172, 4.8272, and 4.8394 mL/min, respectively. The En ratios of the gravimetric and volumetric method were 0.9 and 0.7, respectively. These results represented that the flow rate calibration by the gravimetric and volumetric method correlated with the using of standard flow meter.
In all methods were defined the CMC to be 1.5% for both flow rate calibration points. The results showed that all methods had the uncertainty below 1.5% which were accordingly to the defined CMC.
Table 4
The calibration results of flow rate by using standard flow meter, gravimetric method, and volumetric method
Calibration method
|
Mean ± SD (mL/min)
|
Uncertainty (%)
|
En ratio
|
Flow rate 0.5 mL/min (n = 3)
|
Standard flow meter
|
0.5062 ± 0.0004
|
1.5
|
0
|
Gravimetric method
|
0.4992 ± 0.0003
|
1.5
|
0.6
|
Volumetric method
|
0.5012 ± 0.0004
|
1.5
|
0.5
|
Flow rate 5 mL/min (n = 3)
|
|
|
Standard flow meter
|
4.9172 ± 0.0011
|
1.5
|
0
|
Gravimetric method
|
4.8272 ± 0.0014
|
1.5
|
0.9
|
Volumetric method
|
4.8394 ± 0.0006
|
1.5
|
0.7
|
The standard flow meter was easy and rapid measurement for the flow rate calibration. In this study, the calibration time by using standard flow meter was spent no more than 30 min in both flow rate calibration points which was not included the waiting time for flow rate and pressure stable. Previous report also suggested to use a calibrated flow meter for HPLC flow rate calibration at the upper and lower flow rate values which used in the laboratory [11].
The gravimetric method was a good flow rate calibration method. The necessary equipment included stopwatch, thermometer, calibrated balance, and water container. For the onsite calibration with no calibrated balance and no temperature control might not be proper for this method. The temperature of water needed to be measured which used to define the water density. The type of mobile phase was also important to liquid density which used to convert the weight to volume and used to calculate in the uncertainty budget. Many studies used DI water to be the mobile phase in the flow rate calibration [6, 8, 9]. However, some studies used a mixing of acetonitrile and water to be the mobile phase [7, 10], that might be hard to calculate the liquid density. This study recommended to use DI water for the mobile phase because the DI water had low cost, easy to provide, and knowing the density when the temperature was changed. Another important point of the gravimetric method was the water weight. At the defined time (20 min), the water container should be stopped collecting the DI water immediately to reduce the excess weight. However, the calibration time by the gravimetric method in each flow rate was taken long time around 1 hour (h) with 3 measurements.
Previous studied, the volumetric method were mostly used to calibrate flow rates of the HPLC pump [3, 8–10]. Various sizes of volumetric flasks such as 5, 10, and 25 mL were contained the delivered mobile phase. The flow rate calibration points were set as 0.3, 0.5, 1, 2, 2.5, 5 mL/min. Therefore, elapsed times could be at least 1 to 20 min. These times had to be concerned because the few minutes of elapsed time could make more error than the long-elapsed time. This study, we recommended that the defined or elapsed time should be at least 20 min for reducing time errors. The calibration time by this method in each flow rate was also around 1 h for 3 measurements. Moreover, the error might be occurred at the high flow rate. The fast flow rate led to hard reading of the meniscus and leading to inaccurate timing [11].
3.3 The verification of each flow rate calibration method
The deviation, uncertainty, precision, and verification of the flow rate results are shown in Table 5. The flow rate deviation values by the using standard flow meter, gravimetric method, and volumetric method at 0.5 mL/min were 1.24, -0.17, and 0.24%, respectively and at 5 mL/min were − 1.66, -3.46, and − 3.21%, respectively. The flow rate deviation by all methods showed close results between group. The acceptance criteria of the flow rate accuracy were interpreted from a combined value of the flow rate deviation and uncertainty which should be ≤ 5%, according to the GEON [6]. At 0.5 and 5 mL/min, the results showed that the flow rate accuracy of all methods passed the acceptance criteria, indicated that the HPLC pump had a good accuracy of flow rates.
The flow rate precision values (%RSD) by the using standard flow meter, gravimetric method, and volumetric method at 0.5 mL/min were 0.07, 0.05, and 0.08%, respectively and at 5 mL/min were 0.02, 0.03, and 0.01, respectively. The acceptance criteria of flow rate precision were defined as ≤ 0.5% RSD, according to the GEON [6]. The precision values (%RSD) from all methods were less than 0.5% indicating that the precision from all methods met the precision criteria.
The results of the three calibration methods indicated that the HPLC pump had a good performance of the flow rate accuracy and precision.
Table 5
The verification results of flow rate by using standard flow meter, gravimetric method, and volumetric method
Calibration method
|
Deviation (%)
|
Uncertainty (%)
|
Precision (%RSD)
|
Acceptance criteria of accuracy
( % )
|
Verification of accuracy
|
Acceptance criteria of precision
( % )
|
Verification of precision
|
|
Flow rate 0.5 mL/min (n = 3)
|
Flow meter
|
1.24
|
1.5
|
0.07
|
5
|
Pass
|
0.5
|
Pass
|
Gravimetric method
|
-0.17
|
1.5
|
0.05
|
5
|
Pass
|
0.5
|
Pass
|
Volumetric method
|
0.24
|
1.5
|
0.08
|
5
|
Pass
|
0.5
|
Pass
|
Flow rate 5 mL/min (n = 3)
|
|
Flow meter
|
-1.66
|
1.5
|
0.02
|
5
|
Pass
|
0.5
|
Pass
|
Gravimetric method
|
-3.46
|
1.5
|
0.03
|
5
|
Pass
|
0.5
|
Pass
|
Volumetric method
|
-3.21
|
1.5
|
0.01
|
5
|
Pass
|
0.5
|
Pass
|