Standard operating procedure for HPLC (High performance of Liquid Chromatography)

 
1.       Title:
Standard operating procedure for HPLC
2.          Purpose:         
To lay down the procedure for the operation of HPLC used in the raw material, intermediates &
finished product analysis in FBL lab.
3.         Scope:
This procedure shall be applicable for operation of HPLC ( Shimadzu ) in FBL Lab.
4.         Responsibility: Analyst.
5.         Procedure:
5.1       Setting instrument parameters:
5.1.1     Switch on the instrument.          .
5.1.2    Click on the instrument from the LC-solution Launcher with ‘Admin “as user
             name & password.
            LC real time analysis window will open
5.1.3     From the assist bar (left side) choose the data acquisition icon.
Cursor will appear in the instrument parameter view.
5.1.4    Set the method as per analysis and save the method file form file menu.
5.1.5    From the assist bar select report format go to file menu and choose the open report format,
           <c:\ Labsolution\Template\Quantination\Detector A>
5.1.6    Click < Download parameter> to the instrument. Activate the instrument on/off icon
             From the icon bar.
5.1.7          Wait till the instrument is stabilized.
5.2      Making a single – run analysis
  5.2.1  Prior to analysis, confirm that ‘Ready ‘is displayed in the < chromatogram> view and that the
            Baseline is stable enough.In the < data acquisition > window, click on the (single start) icon on                                  the Assistant bar.
   5.2.2   < Single run > window will appear.
   5.2.3    In this window, open the method file, enter the name of data file, the vial no, injection volume
            specify the report format and its path required automatic printout.
   5.2.4   After entering the above data, click on the (ok) button. A single analysis run will start.
 
5.3       Creating Report format:
5.3.1    Click on the (report format) icon located on the top page of assistant bar in the < LC Postrun>
            Program. The report window will be displayed.
5.3.2    From the tool bar, select the chromatogram and specify the area to be pasted to
            the work space.
5.3.3    Click on the drag mouse diagonally where a report item will be pasted.
5.3.4    When releasing the mouse button, chromatogram properties window appears.
5.3.5    Alternatively, you may want to  click  on  the (file) tab of the  properties  window  and
            Specify the desired data file using (Browse) the  specified  file  will be  loaded   to  the
            report.
5.3.6    The report format can be checked in the workspace in the < preview> window in the properties                            window customize the report item specifying a report display format and whether the report         item is to be displayed or hidden. The <properties> window can be displayed by double –       clicking on the report.
5.3.7    Item or selecting  (properties) from  the  menu displayed  by clicking with the right mouse                                button.
5.3.8     Next, using the same procedure as in positioning   the chromatogram position the (peak table)
            in the work space.            
5.3.9     Now the layout of the report is completed. Click on the (preview) icon on the (report)
             assistant   bar to check  the  contents  of report   click  on the (print)   button  located  at 
             the   top of   the   preview   to print  the  report. Other data. Click on the tool bar and name
             the file.
 
5.4            Creating  Batch  table:
5.4.1          Assist bar < click on the batch processing> batch window will open.
5.4.2          Click  new  batch  file   from  the   file  menu  right  click  on  the  table  and  choose  the  table
style
5.4.3     Choose   the desired item from the left side list added.
5.4.4     If  you want  add one row,  just  right  click  on the   table  and  choose  the  add row
5.4.5     Enter  the  vial,  injection  volume  method  file  report  file  and  details  for  row  one  by  one
5.4.6     Then save the batch file from file menu.
5.4.7     The same batch creation can be followed   using < WIZARD> icon from the  file
              assistant bar   and  then  <click  next  with  necessary    details  >
 
5.4.8     To  run  the   batch  file  just  click  on  the  batch  start   button  from  assist  bar.
 
5.5      Carrying   out batch analysis:
5.5.1    Display the desired batch table (batch file) selecting   <LC analysis>- < Batch table>
            Click  on  the  (Batch  start)  icon  on  the  ( batch ) assistant  bar.   The batch analysis 
            will be carried  out  starting  with  first  row.
5.5.2    To  pause/ resume   or  stop  batch  analysis,  click  on  the  (pause/restart)  icon  or  the
            ( Stop  )   icon  the  (batch)  page  of  assistant  bar  .when  batch  is  paused.  Now progressing
            analysis   is  not  stopped  and  scheduled  rows  before  execution  can   be edited  such  as 
            addition and  deletion.
5.5.3          The  progress  of  batch  analysis  can  be  checked  on  the  < output  window>  after  the  batch  analysis   has  been  completed.  Check  the  log  message  to  ensure  that  no error  occurred  during  analysis .
 
5.6         Setting Peak Integration   parameter:
5.6.1   Click  on  the (data  analysis)  icon  on  the  (acquisition) page  of  the  assistant  bar.  the 
< Lc  data  analysis  > window  in the  < lc postrun >  program  will  be  displayed  with the
Previously obtained data file loaded.
5.6.2    In the  <lc  data  analysis  >  window   open  the  data file  crated  from  the  analysis  using  the  (file) menu  or  < Data  Explorer>
5.6.3    When the data file is opened the chromatogram.
5.6.4    Display  the  integration  parameter window  select  (data analysis  parameters)  menu   or
            click on the (integration)  tab
5.6.5   In this  window adjust  the integration  parameters to  determine  the  optimal 
            integration parameters  such  as  drift  width , lock  on / off etc.
 5.6.6     After  entering  the  appropriate  values  for  the  integration  parameters  click  on the  (apply) 
            Button. The resulting effect can be checked in the <chromatogram> click   on the ok.
 5.6.7     To  save  the  integration   parameters a  part  of  the  method  file  <click  apply to
              method  in the assistant   bar>  Make  sure  he  method  file  is  not  used  for  analysis 
              in the  real-  time  and  run  is  going.
5.6.8    Set  the  drift  value higher  around  5000 for  valley calculation.
5.6.9    Adjust  the time  program  for  integration  based  on  the  needs similar  to  LC-10
5.6.10   The  same  integration  can  be  carried  out  by  wizard  menu  also.
5.6.11   Right  click  in  the  chromatogram  window-   click  wizard-  set  the  integration 
             parameters and  follow  the  display- click  ok.
 
5.7        Applying setting to original method:
5.7.1          Save the analytical parameters set in the < lc data Analysis >   window as a method file.
5.8.2   Click  on  the (apply  method  ) icon  on  the (lc / PDA  data ) page  of  the  assistant  bar.
            The  dialog  box  will  be  opened  promoting  you to enter  the  name  of    the  method  file                                                        to be  saved   the  original   method  file  name  is  entered  as  the  default  settings.  To apply       the  settings  in  the  < lc  data  analysis  >  window  to  the  original  method   file  click the     (ok)   button  to  overwrite  it  the dialog  box  will  be  opened  prompting  you  to  select  whether        or  not  to apply  the  parameters   for  the  method  . Click on the (ok) button.  The above operation  will  overwrite the  original  method  file with  the  changed  data  analysis             parameters.
6.0  Abbreviations:
                    1) HPLC: High Performance Liquid Chromatograph
   
7.0  Records: Nil
 
8.0  Reference : Shimadzu HPLC Manual