HPLC CALIBRATION

1.0 Calibration of Flow Rate 1.1. Prime the pump with degassed HPLC Grade water 1.2. Select a flow rate for channel A referring to Table 1 and enter the flow rate in increasing order on the pump controller, then start the pump. 1.3. Referring to Table 1, obtain Class A volumetric flask. Ensure that the flask is thoroughly dry. 1.4. Use the stopwatch to begin timing. Table 1: Flow Rate Volumetric Flask 0.500 ml/min 10 ml 1.000 ml/min 10 ml 2.000 ml/min 10 ml 1.5. After the flow and pressure have stabilized, insert the outlet tubing into the volumetric flask and start the stopwatch. 1.6. Stop the stopwatch when the bottom of the meniscus reaches the volume-level mark on the flask. 1.7. Record the elapsed time (in second) in the calibration. 1.8. Calculate the flow rate using the following equation; 10 ml X 60 Calculated flow rate = ------------------------------ Measured time in seconds 1.9. Record the calculated flow rate in the calibration format. Verify that the flow rate falls within the acceptable flow rate range of + 1 %. 1.10. Repeat the procedure for the B channel and record the results in the Acceptance criteria : + 1 % Frequency : 6 Month  15 days 2.0 Flow Rate Consistency: 2.1 From the experiment DETECTOR LINEARITY take the retention time of Caffeine from 6 consecutive injections and calculate the % RSD 2.2 Record the result. Acceptance criteria: NMT 1% Frequency of calibration: 6 Month  15 days 3.0 Injector Precision test: 3.1 Mobile Phase: Prepare the mixture of 600 ml Methanol (HPLC grade) and 400ml HPLC grade Water. 3.2 Filter the mobile phase through 0.45  membrane filter paper and sonicate for about 5 minutes to degas it 3.3 Connect the system and detector through the HPLC column mentioned in 5.8.1 3.4 Start 1.50 ml/min flow rate through line A 3.5 Sample preparation: Prepare 250 mg/l solution of Caffeine in mobile phase 3.6 Create instrument method with the following parameters 3.6.1 Column : C18 Symmetry 150 X 4.6 mm, 5  3.6.2 Flow rate : 1.5 ml/min 3.6.3 Wave length : 254 nm 3.6.4 Injection Vol. : 10 l 3.6.5 Run time : 5 minutes 3.6.6 Column temp : 40°C 3.7 Inject six replicate injections of the sample solution into the HPLC System 3.8 Record the area of the peak due to Caffeine. 3.9 Calculate the % RSD of the area of six replicate injections 3.10 Record the observations. Acceptance Criteria: % RSD for replicate injections is not more than 1.0 % Frequency : 6 Month  15 days 4.0 Detector Linearity 4.1 Prepare the Mobile phase and chromatographic condition as described in Injector Precision. 4.2 Prepare the 125 mg/l, 250 mg/l and 500 mg/l solutions of Caffeine in mobile phase 4.3 Inject all the three sample solutions in duplicate 4.4 Record the area of the peak due to Caffeine 4.5 Calculate the correlation coefficient for above concentrations Vs peak area Record the correlation coefficient. Acceptance Criteria: The correlation coefficient obtained is not less than 0.99. Frequency : 6 Month  15 days