Operation and Calibration of ( GRADIENT HPLC Shimadzu Class VP series )

I.    PURPOSE :
To provide a procedure to operate and maintain the Shimadzu make Class VP series Gradient HPLC system.

 II.    SCOPE :

This procedure is applicable for the GRADIENT HPLC, Shimadzu -Class VP series used in the Quality Control department 
III.    RESPONSIBILITY :
    It is the responsibility of all the personnel involved in the analysis using of the system
   
IV.    DOCUMENT REFERENCE :
    SOPs    :    Core procedure: Calibration of Equipment
    Forms    :    Calibration of HPLC

V.    OPERATION :
       1.0    PRELIMINARY CHECK :

1.1    Check that the instrument is clean and free from dust, if not, clean with a soft cloth duster.

2.0  THE LC-10 AVP SERIES HPLC WORK STATION CONSISTS OF :

2.1.    LC-10AT VP Pump
2.2.    SPD-10 AVP UV vis detector
2.3.    SIL-HT- A System controller and auto sampler
2.4.    Computer with Class VP software version 6.10

3.0    BASIC OPERATION :

3.1.   Switch ‘ON’ the main for LC-10 pump

3.2.   Switch ‘ON’ the main for auto injector

3.3.   Switch ‘ON’ the main for UV vis detector

3.4.    Switch ‘ON’ the system controller and the computer.

3.5.    After switching ‘ON’ all the components, finally switch ‘ON’ the computer.

3.6.    Default screen appears on the respective screen modules. 

3.7.    Double click on the (Shimadzu class VP) icon in the screen.

3.8.    Double click on Instrument 1 icon.  Here for the beep sound, which indicates the communication between the system and the software.

3.9.     OPERATING PROCEDURE FOR LC-10AVP PUMP

3.10.    Open the drain valve of the pump.  Press the purge key.  Ensure that there are no air bubbles in the flow line and press purge/pump key to stop purging.  Close the drain valve of the pump.

          SYSTEM SOFTWARE

3.11     Check the method name displayed on the top of the screen.

3.12     Click on the method option, click the instrument set up.  Click on the pump, set the A. flow   

             in ml/min.  P.max in  kgf/cm2.

3.13     Click on  the detector SPD-10 AVP, icon.  Set the wavelength in nm and run time in min. and save the method.

3.14     After setting the above parameters, click on download at the bottom of the screen.

3.15     Observe for the message displayed on the screen.  Click the OK icon and close the screen. This indicates the software successfully downloaded the set information to the system.

3.16     Click on sequence.  Click on edit, edit table will appear.  Set the vial number.  Repetitions   from the vial (no. of injections) sample ID, File name and finally Description.

       AUTOZERO/PURGE INJECTOR / RINSE INJECTOR/INSTRUMENT ‘ON/OFF’

3.17  Go to control option, click the direct control .  Direct control message will appear on the top  left corner of the screen.  Click on the instrument on/off icon.  It activates the whole system  ACT LED will glow on the system controller.

3.18.    Before injecting a sample observe for the base line by pressing preview run in control option.

3.19.    To start a run, press the single run into the control option or blue arrow icon on the main screen.  If the injections are in sequence mode, press the double green arrow button on the main screen.
3.20.    To stop the single run,  press the stop run  column in the control option.

       RESULTS & PRINTOUTS

3.21       Click on the Shimadzu class-vp button.

3.22      Double click on the instrument off line processing instrument 1 will appear.  Double click instrument 1.

3.23      On double click, instrument 1 (off line).  The method used and the data file namewill appear on the top of the screen.

3.24      To open, select data file, click file, under file option, click data, press open, data files   will appear.  Select the data and press open, chromatogram will appear on the screen.

3.25      To check the results, press view and then method custom report.

3.26    Class-vp custom report will appear on the screen.

3.27    To print click the right button of the mouse and press print.

VI. CALIBRATION                        Calibration frequency : Once in three months

1.0    Calibration of the pump
1.1    Check the flow rate of the filtered and degassed Purified water as follows:
1.2    Disconnect any column if connected to the system and connect restriction capillary
1.3    Set the flow of the pump at 1.0 mL per minute. Keep the system at this flow rate for   about 5 minutes to equilibrate the system.
1.4    Collect the volume of water delivered for 5 minutes and determine the weight of the water .
1.5    Convert the weight in to volume by the following table:

1.6   Repeat the above for 2 and 3 mL flow rates.
1.7 Acceptance criteria: 
   Flow rate:  + 0.1mL 
         
2.0     Column compartment thermostat / oven calibration.
2.1     Disconnect any column if connected to the system.
2.2     Purge the system with Purified water to remove any solvents and previous buffer salts.
2.3    Connect a restricted capillary of dimension 2m x 0.12 mm ID in place of column.
2.4    Keep a flow rate of 1.0 mL/minute.
2.5     Keep the sensor wire of the traceable digital thermometer in the column thermostat/ oven.
2.6     Set the temperature at 20.0°C.Wait till the set temperature is attained and the temperature display on the instrument is stable.
2.7    Wait for 5 minutes before readings are taken so that the temperature on the Instrument and that  displayed on the thermometer are stable.
2.8    Note down the temperature displayed by the instrument and the thermometer.
2.9    Repeat the steps 2.6 to 2.8 for temperature settings at 30.0°C, 40.0°C, 50.0°C,60.0°C & 70.0°C.  

2.10    Acceptance Criteria:
The difference between the set temperature and the displayed temperature on  the thermometer  should not be more than ± 2.0°C.       
      3.0       BASE LINE CHECK (NOISE & DRIFT)

3.1     Connect the restriction capillary and check for any leakages at all possible points.
3.2      Allow the system to stabilize for few minutes by passing purified water through the restriction capillary and the detector. 
3.3    Check monitor base line before starting the Noise & drift test .Once  the base line is  stabilized  proceed to the Noise & drift test as follows.
3.4     Click the base line button in the LC set up or Select Control >Baseline Check   dialog to  display a base line check dialog box.
3.5     Enter the time period for start, end time and Threshold values for the Noise and Drift test Use ASTM method to evaluate the above test. 
3.6     The Threshold values for Noise and drift are : 50uV for Noise 5000uV/h for Drift.
3.7     Click the start button to start the test and Click cancel to  stop the test.
3.8      If the Noise & Drift values are within the specified threshold range the result will be displayed as Pass. If the measured value is larger than the threshold check the system for other maintenance in the pump and detector.
3.9         Export the result file by specifying the data path and file name and print the file  from the folder. Alternatively use print screen to print the result window. 
    Acceptance Criteria: The Noise & drift values obtained from the test should be within the set 
                                      Threshold values.

4.0     DETECTOR LAMP INTENSITY CHECK:

4.1    Click on the control icon in Instrument I, then click instrument status.

4.2    Instrument status will appear on the screen.

4.3    Click on the SPD-10 AVP (Det A) icon in instrument 1.

4.4    Reference energy and sample energy will be displayed on the screen.

 Acceptance Criteria:   Reference energy displayed should not be less than 800 mV at 220 nm.

5.0      WAVELENGTH ACCURACY:

5.1    Preparation of standard solution : Weigh accurately about 25 mg of caffeine in a 100ml volumetric flask, dissolve and dilute to volume with water. Further dilute 10ml of this solution to 100ml with water.
5.2      In the HPLC, connect the outlet of the pump into the inlet of detector.

5.3      Flush the flow cell with water.

5.4      In the ON LINE, go to instrument set up and enter the start and end wavelengths for spectrum
           scan.

5.5     Go to ‘Control’’ press on ‘Spectrum scan’ spectrum scan window appears.

5.6     Press background and acquire a background and spectrum.

5.7     Fill the flow cell with Caffeine  solution and press ‘spectrum’ to acquire a spectrum.

5.8    Right click on the spectrum and select  the mode to calculate  ? max where the maximum absorbance value  is observed.

5.9         Again right click on spectrum  move the cursor to ‘Utility’ and save the spectrum.

5.10      Go to method, custom report, delete all the graphs, right click, insert graph, spectrum control.   The template appears and another window of spectrum control appears.  In that source select     ‘file’ and open the spectrum data and say apply, click ‘OK’. Right click on the graph, select axes  set up and set the ‘x’ axis scale to 200 to 300 nm 
 
5.11    Click on ‘report’ ‘print’ and ‘method custom report’.

5.12   Right click on the graph, select axes set up and set the ‘x’ axis scale to 200 to 300 nm and then  take printout as explained in 8.4.10.

5.13  In the expanded spectrum printout, draw perpendicular from the maximum absorbance to the ‘x’ axis.  Note down the intercept of the perpendicular on the ‘x’ axis.

          ACCEPTANCE CRITERIA: The wavelengths at which maximum absorbance 
           observed (the intercept of the perpendicular on the ‘x’ axis) should be between 
            
                   Standard absorbance maxima                            Limit 
                                  205nm                                               203nm to 207nm
                                  245nm                                               243nm to 247nm
                                  273nm                                               271nm to 275nm

6.0     Precession and Linearity of auto injector and detector response  :      
            Chromatographic conditions:
      Column : ODS C18 15cm x 4.6mm , 5microns 
      Mobile phase  :     Acetonitrile : Purified  water (15 : 85 v/v).Filter and degas
      Flow rate : 1.0mL/min
      Wave length: 273nm
      Temperature : 360C
      Run time     :  10 minutes
6.1       Preparation of standard solution: 
            Weigh accurately 25 mg of Caffeine into a 100 mL volumetric flask, dissolve and dilute to volume with mobile phase. Further dilute 10 ml to 100 ml with mobile phase. (25 mcg/mL)
6.2      Inject the  10uL , 20uL ,50uL  &100uL   of the standard solution in five replicates and record the chromatograms . 
6.3    Theoretical plates of Caffeine peak should not be less than 3000 and tailing  factor should not be  more than 2.0 for 20uL injection (As per USP).
6.4     Calculate the percentage RSD of the peak areas of caffeine from the five replicate injections at   each level.

6.5     Calculate the average peak area of caffeine from the five replicate injections at each level.
6.6    Calculate the percentage RSD of retention times of caffeine at each level.
6.7    Plot the  linearity graph of  injection volume on the x- axis against  average area of Caffeine at  each level on the y-axis.Acceptance Criteria: 

•    The % RSD for the retention time of caffeine from the five replicate injections at each level should be less than 1.5.
•    The % RSD of peak areas of Caffeine from the 5 replicate injections at each level should be less than 2.
•    The linearity coefficient ‘r’ obtained from the linearity graph of caffeine for different levels should not be less than 0.999

  7.0 Gradient accuracy

          7.1Chromatographic Conditions    :
    Column        :    Hypersil ODS      100 x 4.6 mm ID,  5 ?m or equivalent      
    Flow rate        :    2.0 mL /min.
    Detector wavelength    :    254 nm
    Injection volume    :    20 ?L
    Runtime        :    30 minutes
 Mobile Phase A      :     Water generated by Milli-Q Apparatus 
             Mobile phase B    :      0.3%v/v Acetone in filtered water generated by Milli-Q
Preparation of Mobile Phase B: Take 3 mL of acetone in 1000 mL of water 
                                                    generated by     Milli-Q
.

7.2  Gradient program  :
Time (min)    Mobile phase B percentage
0.01                             0
2.00                             0
2.01                           10
8.00                           10
8.01                           50
13.00                         50
13.01                         90
18.00                         90
18.01                         100
23.00                         100
23.01                          0
30.00                          0

                        Procedure: 
7.2.0.    Open the drain valves and purge the flow lines of both pumps
7.2.1.    Equilibrate the column with initial concentration with above mentioned conditions and wait until the baseline is stable    
7.2.2.    Adjust the baseline level to fit the full Scale of the integrator    
7.2.3.    Inject exactly 20 ?L of mobile phase A and start the time program for gradient accuracy test.

7.2.4.    Determine the signal level at 0%(B Con),10%(B  Con), 50%(B  Con), 90%(B  Con) and 100%(B  Con) 
7.2.5.    Calculate the actual B concentration level at 10%(B Con), 50%(B Con), 90%(B Con) Using 0% (B Con) and 100% (B Con)
        Calculation:    
    

Similarly calculate the actual B concentration level at 50%(B Con) and 90%(B Con)

 7.3  Acceptance Criteria :
        At B concentration 10% level should be between 9.0 and 11.0
        At B concentration 50% level should be between 49.0 and 51.0
    At B concentration 90% level should be between 89.0 and 91.0

7.4    Follow the procedure 7.2.0 to 7.3 exactly using C & D channels instead of A& B   

8.0         CARRY OVER TEST 
                  Chromatographic conditions: 
          Column                    :      ODS C18; 15 cm x 4.6 mm, 5 microns 
                                                (Inertsil ODS-2 column is suitable)
          Mobile phase            :     Acetonitrile : Purified Water (15 : 85 v/v).Filter and degas
          Flow rate                  :     1.0 mL / min.

          Wavelength              :     273 nm
          Run time                  :    10 minutes
          Column oven temp.  :    36°C 
          Injection volume      :    100 µL
         Preparation of standard solution:
8.1.     Weigh accurately 25 mg of caffeine into a 100 mL volumetric flask, dissolve and
           dilute to volume with mobile phase. 
8.2      Inject 100 µL of standard solution and note down the chromatogram.
8.3      After completion of the test solution inject 
8.4     Inject 100 µL of blank 3 times and  100 µL of blank again blank .
8. 5    Calculated any carry over  of caffeine in the last blank.

8.6     Acceptance Criteria:
          Carry over should not be more than 0.03% of 100-µL caffeine std peak area.

 9.0   Software calibration.
       1) Perform the validation of the software once in every year  
        2) And whenever software is updated.

10.0  If the instrument fails in calibration, proceed as per the Core Procedure, “Calibration of Equipment”.    

Gradient hplc, calibration of equipment, equilibrate, thermometer