operate and maintain the Shimadzu LC 2010 series gradient HPLC system

.    PURPOSE :
      To provide a procedure to operate and maintain the Shimadzu LC-2010 series,  gradient HPLC system...

 II.   SCOPE :

        This procedure is applicable for the HPLC 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 :
  
11.0      PRELIMINARY CHECK :

1.1      Check that the instrument and surroundings are clean, if not, clean with a soft cloth duster.

             THE LC-2010A SERIES HPLC WORK STATION CONSISTS OF :

              LC-2010A Integrated system
              Computer with class VP software version 6.10
 
2.0    STARTING UP AND CONNECTING LC 2010 AND CLASS VP :

2.1    Place  the tubes A, B, C and D leading to the pump in suitable solvents or mobile phase.

2.2    Turn ON the LC-2010A main unit’s power.

3.0     STARTING UP AND CONNECTING LC 2010 AND CLASS VP :

3.1    Place  the tubes A, B, C and D leading to the pump in suitable solvents or mobile phase.

3.2     Turn ON the LC-2010A main unit’s power.

3.3    After confirming that the LC-2010A is started up , turn the PC power ON,and select  
 
3.4    Start>Programs> Chromatography > Class-VP from the windows task bar to open the Class VP menu window.

 3.5   In the main menu window, double click on the Instrument icon for the LC-2010A.

 3.6   Input user name and a password to Login to class VP.

3.7    After the LC-2010A beeps indicating connection to the PC has been established the 
        Instrument window opens.

4.0    PURGING MOBILE PHASE AND RINSE SOLUTION 

4.1 Place the tube leading to the purge/rinse degasser in 50:50 (methanol : water)

4.2  Click on the purge icon on the direct control toolbar. Select  the flow lines   to be purged and set purge time for each.

4.3     Click on the purge button displays a window showing the progress of purgeand auto purge starts.      

5.0    SETTING LC PARAMETERS 

5.1 Select the New method from the list opened by clicking on the new method button. Default method parameters loaded and ‘untitled method ‘is displayed on the LC setup window.

5.2    Configure the LC parameters by clicking on the pump ,oven and detector icons in the LC setup.

5.3    LC component icons are highlighted by placing the mouse pointer on them .Click the icon opens the window to set and confirm that components parameters.

  5.4  Select  File> Method>Save as and input a name for the method

  6.0   Down loading methods to LC-2010A

  6.1  After creating method, down load it to the LC-2010A main unit by clicking on   down load button .When the down load is complete , a message window appears ClickOK
       
 7.0    STARTING LC-2010A CONTROL 

7.1      Click on the ON/OFF button on the direct control toolbar.

7.2    The LC-2010A controller is activated , and liquid flow and temperature of the column  oven  adjust to the down load parameters.

7.3    The LC-2010A’s current pump pressure and oven temperature of the column oven are displayed above the icons. When all the components are stable ,a Ready indication appears and the status bar turns green.

8.0    SETTING SEQUENCE FOR SEQUENCE RUN

 8.1 Click on the sequence wizard button in LC setup . 

8.2  Select ‘ Create new file ‘ and click on the next button.

8.3    In the run information dialog box, specify a method file to be used and folder to save data files.

8.4     Indicate the start vial number and Input an Injection volume 

8.5    Click on the Next button after finishing settings.

8.6  Next , specify  a sequence acquisition pattern, enter sample ID’s and data file names Finally set sequence report parameters, after the setting is complete Click on the finish button to save the sequence file created .

 9.0    STARTING A SINGLE RUN

 9.1   Click  on the single run button in the LC setup window or the single run button on the  toolbar.

 9.2  Specify items required , and click on the start button.

10.0    STARTING A SEQUENCE RUN

10.1  Click on the sequence run button in the LC setup or the sequence run button on the  toolbar.

10.2      When created  a sequence via Sequence Wizard in the LC setup , select the sequence and  click on the start button to start acquisition.

11.0 TERMINATING ACQUISTION 

Select control> Extend run  and enter  a value for time added to the end of the run. To shorten the acquisition time , specify a negative value .  

12.0     SETTING INTEGRATION EVENTS

12.1     Click file >data > open to open the data file .
 
12.2    The default values can be used for integration , however it is recommended  to optimize slope in accordance with the base line in advance to prevent undesired  peaks such as noise from being integrated.

12.3       The default values : Width 5 , Slope 200, Drift 0, T.DBL 1000,  Minimum area 0.
                                            
10.     CREATING REPORT TEMPLATES 

10.1    Select File > print setup , and setup the printer connected .

10.2    Click on the edit custom report to open the custom report window.
 
VI.  CALIBRATION :     Calibration frequency : Once in three months

1.0.  AUTO-VALIDATION

1.1     Check the various components meet the performance and functionality  criteria .      
        by carrying Auto validation and the system checks for the following parameters  during auto validartion.

12.4     In the Auto validation screen select GE mode, Press F1 to start auto validation.

12.5     In GE mode it starts purging port B.

12.6     Wait for the system to finish purging . After the system finishes purging , the next   
12.7     screen is displayed and the wavelength accuracy test begins.
12.8     Tests followed one after the other and takes 3hrs to complete the Auto-validation.
12.9     After the  gradient concentration  accuracy , the results are  displayed from all tests in  
              GE mode.
12.10 To save the results press F4 (SAVE)
12.11 Down load all the results to the Class VP  software  by Pressing the Auto validation   button and save the  file , Click print to print the report.

ACCEPTANCE CRITERIA :

Note : Flow rate calibration is part of Auto validation in which system checks for gradient and flow accuracy .

13.0    CARRY OVER TEST 

13.1    Perform this test after 100ul of standard injection from linearity test.

13.2    Purge the system with water for few minutes before starting the carryover   test.

13.3   Inject 100ul of blank to check for any carry over due to caffeine from the     previous 100ul standard caffeine injections.

Note : Flow rate calibration is part of Auto validation in which system checks for gradient and flow accuracy .

13.0    CARRY OVER TEST 

13.1    Perform this test after 100ul of standard injection from linearity test.

13.2    Purge the system with water for few minutes before starting the carryover  test.

13.3   Inject 100ul of blank to check for any carry over due to caffeine from the previous 100ul standard caffeine injections.

Acceptance criterion:    Carry over of caffeine not to exceed 0.01% of Mean area of 100ul standard.

Gradient hplc system, mobile phase, suitable solvents, purge button, purge icon