STANDARD OPERATING PROCEDURE OPERATION OF THE HPLC SYSTEM SHIMADZU LC-2010CHT

STANDARD OPERATING PROCEDURE

SUBJECT:                      OPERATION OF THE HPLC SYSTEM SHIMADZU  LC-2010CHT

DEPARTMENT           : QA SOP NO.                      : Page No                       : Effective Date             : Next Review                : SOP REVISION           :

1.0       OBJECTIVE: 

To provide guidelines for the operation of the HPLC SHIMADZU LC-2010CHT system.

2.0       POLICY: 

To lay down a procedure for the operation of the HPLC SHIMADZU LC-2010CHT system.

3.0       PROCEDURE:

3.1       Switch on the instrument and after initialization enter the password menu will be displayed.

3.2       Purging the system before start of analysis.

3.3       Click on purging on the display.

3.4       Press A, B, C, D & INJ on display.

3.5       Keep the column into the system.

3.6       Then go to menu by pressing the cursor ▲ or ▼.

3.7       Set the system for column washing to the initial composition of A = Mobile phase 100%,

B =Methanol 100 %, C = Acetonitrile 100% & % D =Water100% and put column oven @25 to 60ºC and put flow at 1.0ml/min and press the Pump button.

3.8       Set the flow at 1.0 ml/min leave for 1min and check for leaks. Leave the system to equilibrate for 2 min.

3.9       Instrument ready for analysis.

3.10     Connecting to the LC Solution software.

3.11     Click → LC Solution icon.

3.12     Click →Administration

3.13     Click →User Administration.

3.14     User ID – Admin → ok (by default).

3.15     METHOD CREATION.

3.15.1  Go to → Assistant Bar.

3.15.2  Click → Top.

3.15.3  Click → Data Acquisition.

3.15.4  Click → Instrument Parameters.

3.15.5  Click → Advanced.

3.15.6  Data Acquisition→ LC stop time 25 min and then click apply to all

Acquisition time.

3.15.7  LC time prog

·         Gradient flow → Total flow at 1.0 ml/min.(Adjust the parameters as per the method requirement)

Time	Module	Action	value
0.01	Pump	Pump B conc.	50
5.00	Pump	Pump B conc.	60
10.00	Pump	Pump	80
20.00	controller	stop	.

3.15.8  Pump → Mode → Isocratic flow and Gradient flow.

·         Isocratic flow → Flow at 1.0 ml/min, Port –A and pressure limits Maximum-380 kgf/cm^2, Minimum-0 kgf/cm^2.

·         Gradient flow → Total flow at 1.0 ml/min.(Adjust the parameters as per the method requirement)

3.15.9  Detector-A

·         Lamp-D2

·         Polarity-+

·         Response-1.0 sec

·         Cell temperature- Low

·         Wavelength-254 nm .(Adjust the parameters as per the method requirement)

3.15.10            Column oven → No need to change.

3.15.11            Controller → No need to change.

3.15.12            Autosampler

·         Sample Rack-1.5 ml 70 vials- Click-Detect Rack.

·         Rinsing volume -200 ul.

·         Needle stroke-52 mm.

·         Sampling speed-15 ul/sec.

·         Rinse mode – Before and after Aspiration.

·         Rinse Dip time – 0.

3.15.13 Autopurge → No need to change.

3.15.14 Go to file → save method file as Batch No. and then click Download.

3.15.15 Click→Batch processing, click, then go to file new batch file → Vial Right click, Add Row → select → click → number of samples-10 ok.

 

3.16     REPORT FORMAT CREATION

3.16.1  Click Postrun→click Report format→ click file → new report format file.

3.16.2  Then go Item → General Text one window will come → Text type the company name → go general Font setting → Times new → Bold → ok → apply → ok.

3.16.3  Click Item → common → sample information.

3.16.4 Click Item → LC/PDA common → Chromatogram.

3.16.5  Click Item → LC/PDA common → Peak table → go to file save report format.

                                                                                                           

3.17     INTEGRATE THE DATA FILE

3.17.1  Click Postrun → click Top → LC Data analysis→ select browse folder→ elegant select.

3.17.2  Select the data file → double click → click wizard → Program → Reject peaks → after integration→ click simulate then ok.

3.18     CALCULATE THE % RSD OF AREA AND RT

3.18.1  Top → click report format → click file → new report format file → Item → common → sample information.

3.18.2 Item → LC/PDA common → Summary compound.

3.18.3  Click right properties then go file → add → click table.         

3.18.4  Print the required peak and close.

3.18.5  Printing of the chromatogram.

3.18.6  Click on chromatogram → preview and publish → window opens → select individual report → ok → print.

3.18.7  Column washing after sample sequence completed.

3.18.8  When the sequence is finished, stop the flow by pressing “Stop pump” button.

              i.      Turn off the instrument.

            ii.      Use Annexure-1 to check the column performance details.

3.19     Calibration of HPLC:

3.19.1  Calibration is done once in a quarterly internally and once annually by the service engineer.

3.19.2  Flow rate check of pump at the below mentioned flow rate and solvents and run for 30 mins.

3.19.3  Flow rate          solvent            

            1 ml                 methanol                     

            1 ml                 IPA                             

            1 ml                 water               

            1.5 ml              methanol         

            2 ml                 methanol

           

3.19.4  Record the results in the calibration record.

4.0       FREQUENCY: As and when analysis is planned.

5.0       RESPONSIBILITY: HPLC operating chemist /Departmental Head.

6.0       REVIEW GUIDE:  HPLC log book.

7.0       ANNEXURE: 1. Column performance data.

8.0       SOPs DISTRIBUTION:

            This SOP shall be distributed to following Departments:

Name of the Department	Master Copy	Circulatory Copy
QA/QC	√	√
Production	x	x
Warehouse	x	x
General	x	x
Maintenance	x	x