SOP FOR HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (JASCO)

1.       PURPOSE

        The purpose of this SOP is

1.       To defined the operating procedure.

2.       To define the precautions to be taken during handling.

3.       Cleaning procedure and maintenance.

2.       SCOPE

        This SOP is applicable for operation, handling cleaning and maintenance of the high performance liquid chromatography. Make: Jasco Model: UV-975, PU-980

3.       RESPONSIBILITY

        To organize the job training program for implementation of SOP.

        To monitor overall activities in adherence to the compliance of approved SOP.

        To arrange necessary servicing and maintenance.

        To check the recorded documents for its correctness, accuracy and completeness.

        Section in charge

        To operate the instrument as per SOP.

        To record the observations as per SOP.

        To maintain the documents as per SOP.

4.       SAFETY WARES

        Wear apron, cap and company foot ware.

5.       START UP PROCEDURE

5.1   Clean the high pressure liquid chromatography (HPLC) system and surroundings.

5.2   Ensure that the system consists of following equipment

5.3   Select the prescribed column and connect in the specified direction i.e. in the direction of flow arrow / sign marked on the column, column outlet end is connected to the inlet of the detector, which in turn is connected to ‘Drain’ through tubing.

5.4   Ensure that the mobile phase flow is in following direction.

5.5   Ensure that UPS is working.

5.6   Connect the power supply and switch ON the mains.

5.7   Instrument is ready for calibration or use.

6.        OPERATIONAL PROCEDURE

6.1   PUMP

6.1.1          Ensure that, the instrument is calibrated and start up procedure is followed.

6.1.2          Turn the power switch, located at the lower left of the front panel, to ‘ON’ position.

6.1.3          The LED display glows on and it follows diagnostic tests.

6.1.4          Wait till initialization is completed.

6.1.5          Press “ EDIT / ENTER” or ‘2’ key. The flow value on display starts blinking then press EDIT / ENTER key.

6.1.6          Set the flow at 0.2ml /min, using the numerical key on the panel, and again press “EDIT / ENTER” key.

6.1.7          Press ‘5’ key. The value of P.MAX will start blinking. Set the value at 500; press “EDIT / ENTER” key.

6.1.8          Press ‘6’ key. The value of P.MIN will start blinking. Set the value at 0; press “EDIT / ENTER “ Key.

6.1.9          Ensure that end of inlet tubing of pump is in the reservoir containing at least 50 ml filtered and degassed methanol (HPLC Grade).

6.1.10      Open the purge valve by rotating the knob in anticlockwise direction.

6.1.11      Press ‘PUMP’ key. The orange pilot lamp indicates that the pump is on.

6.1.12      The methanol flows through the pump and is drained out from the drain tube connected to the outlet of purge valve.

6.1.13      Allow to run methanol for 5 minutes or till any air bubble produced in the tube is drained out.

6.1.14      Press ‘PUMP’ key. The pilot lamp is off.

6.1.15      Close the purge valve by rotating it in clockwise direction.

6.1.16      Press ‘2’ key. And set the flow at 0.2 ml/min. Press “EDIT /ENTER” key. Press ‘PUMP’ key.

6.1.17      The valve on the monitor screen indicates the actual pressure.

6.1.18      Gradually increase the flow with the interval of 0.2 ml / min to 1.0 ml / min.

6.1.19      Allow to run for 10 minutes.

6.1.20      Gradually decrease the flow with the interval of 0.2 ml/ min; to 0.0 ml / min.

6.1.21      Press ‘ PUMP’ key. Remove the tubing from methanol and place it in the reservoir containing filtered and degassed (sonicated) water for HPLC.

6.1.22      Follow the instructions 11 to 18.

6.1.23      Allow to run for at least 20 minutes. Then decreases the flow. And press ‘PUMP’ key.

6.1.24      Replace the water with reservoir containing mobile phase.

6.1.25      Follow the instructions 11 to 18 and increase the flow to the specified flow rate.

6.1.26      Allow the run at the specified flow rate for at least 15 minutes or as specified in the individual monograph so as equilibrate the system with the mobile phase.

6.1.27      The system is now ready for injection.

6.1.28      Inject the samples as per individual specification.

6.1.29      After completing all the injections, switch off the detector, make entry in usage log.

6.1.30      Gradually decrease the flow with the interval of 0.2 ml/min to 0.0 ml/min.

6.1.31      Press ‘PUMP’ key. Remove the tubing from mobile phase and place it in the reservoir containing filtered and (degassed) sonicated water for HPLC.

6.1.32      Follow the instructions 6.1.11 to 6.1.18.

6.1.33      Allow to run for at least 240 minutes. Then decrease the flow and press ‘PUMP’ key.

6.1.34      Replace the water with reservoir containing methanol.

6.1.35      Follow the instructions 6.1.11 to 6.1.18.

6.1.36      Allow to run for at least 20 minutes. Then decrease the flow and press ‘PUMP’ key.

6.1.37      Switch off the pump.

6.1.38      Switch off the mains.

6.2   DETECTOR

6.2.1          Turn the power switch, located at the lower left of the front panel, to ‘ON’ position.

6.2.2          The LED display glows on and it follows diagnostic tests.

6.2.3          Wait till initialization is completed.

6.2.4          Press “EDIT /ENTER” or ‘1’ key. The value of the wavelength on display starts blinking.

6.2.5          Enter the specified wavelength using the numerical keys.

6.2.6          Press “EDIT /ENTER” key for 2 times or press ‘2’ key. The value of the range on display starts blinking.

6.2.7          Enter the specified range using or keys. Again press EDIT / ENTER key.

6.2.8          The absorbance value will be displayed on the screen.

6.2.9          The system is now ready for analysis.

6.3   INTEGRATOR (NT BASED SOFTWARE)

6.3.1          Switch on the computer in which the software is installed.

6.3.2          Wait till the initialization is complete.

6.3.3          Activate the software undergoes start up procedures.

6.3.4          Wait till the software undergoes start up procedures.

6.3.5          Enter the specified ‘OPERATOR’ Name, ‘USER’ name and then click ‘OK’.

6.3.6          The screen now displays the software window with the selected ‘USER’>

6.3.7          Click on ‘RUN’ key, then monitor Base Line, select the specified system.

6.3.8          Enter the appropriate information in the displayed chart. i.e. Run Name, No., Run Length and run information.

6.3.9          Click ‘RUN’ key, the screen will display, chromatogram showing ‘Waiting’ status. The system is now ready for analysis.

7.       ANALYSIS PROCEDURE

7.1   Use only HPLC Grade solvents for analysis.

7.2    Use only calibrated, clean, dry glass apparatus.

7.3   All the solutions which are entering the HPLC System i.e. water, mobile phase, etc. must be filtered through 0.4 µ membrane filter paper, and properly degassed.

7.4   In case of mobile phase containing buffer, it is essential to give water washing to the column prior and after running the mobile phase.

7.5   Prepare the sample and standard solutions as mentioned in the specifications.

7.6    Ensure that the HPLC system is equilibrated, and ready to start.

7.7    Take a clean, dry syringe. Rinse it with water in case of mobile phase containing buffers, or with the mobile phase.

7.8   Remove red cap of Rheodyne injector port.

7.9   Ensure that the injector is on ‘Load’ position.

7.10  Suck water / mobile phase in the syringe, ensure that no air bubble is trapped in the syringe.

7.11   Inject water / mobile phase in the loop. Rinse the port with water for 5 times.

7.12   Inject mobile phase or the solvent system used for sample preparation and turn the injector downward i.e. in ‘Inject’ position,  the channel on the screen gets activated and shows ‘Run in Progress’ signal.

7.13  Turn the injector port on ‘Load’ position.

7.14  After completing the injections again rinse the loop with water and then with Methanol.

7.15   Preserve the column in Methanol.