SOP for Operation of Shimadzu HPLC LC-2010 CHT

OBJECTIVE

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

PURPOSE

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

RESPONSIBILITY

HPLC operating chemist / Departmental Head.

PROCEDURE

• Switch on the instrument and after initialization enter the password menu will be displayed.
• Purging the system before the start of the analysis.
• Click on purging on the display.
• Press A, B, C, D & INJ on display.
• Keep the column in the system.
• Then go to the menu by pressing the cursor ▲ or ▼.
• Set the system for column washing to the initial composition of A = Mobile phase 100%, B =Methanol 100 %, C = Acetonitrile 100% & D = Water 100% and put column oven @25 to 60ºC and put flow at 1.0ml/min and press the Pump button.
• Set the flow at 1.0 ml/min leave for 1min and check for leaks. Leave the system to equilibrate for 2 min.
• Instrument ready for analysis.

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Connecting to the LC Solution software.

• Click → LC Solution icon.
• Click →Administration
• Click →User Administration.
• User ID – Admin → ok (by default).
• Method Creation.

1. Go to → Assistant Bar.
2. Click → Top.
3. Click → Data Acquisition.
4. Click → Instrument Parameters.
5. Click → Advanced.

• Data Acquisition→ LC stop time 25 min and then click apply to all Acquisition time.
• LC time prog
• Gradient flow → Total flow at 1.0 ml/min.(Adjust the parameters as per the method requirement)

• Pump → Mode → Isocratic flow and Gradient flow.

1. Isocratic flow → Flow at 1.0 ml/min, Port – A and pressure limits Maximum - 380 kgf/cm2, Minimum - 0 kgf/cm2.
2. Gradient flow → Total flow at 1.0 ml/min. (Adjust the parameters as per the method requirement)

• Detector-A

1. Lamp-D2
2. Polarity-+
3. Response-1.0 sec
4. Cell temperature- Low
5. Wavelength-254 nm. (Adjust the parameters as per the method requirement)

• Column oven → No need to change.
• Controller → No need to change.
• Autosampler

1. Sample Rack-1.5 ml 70 vials- Click-Detect Rack.
2. Rinsing volume -200 ul.
3. Needle stroke-52 mm.
4. Sampling speed-15 ul/sec.
5. Rinse mode – Before and after Aspiration.
6. Rinse Dip time – 0.

• Auto purge → No need to change.
• Go to file → save method file as Batch No. and then click Download.
• Click → Batch processing, click, then go to file new batch file → Vial Right click, Add Row → select → click → number of samples-10 ok.

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Report Format Creation

• Click Postrun → click Report format → click file → new report format file.
• Then go Item → General Text one window will come → Text type the company name → go general Font setting → Times new → Bold → ok → apply → ok.
• Click Item → common → sample information.
• Click Item → LC/PDA common → Chromatogram.
• Click Item → LC/PDA common → Peak table → go to file save report format.

Integrate the Data File

• Click Postrun → click Top → LC Data analysis→ select browse folder → elegant select.
• Select the data file → double click → click wizard → Program → Reject peaks → after integration→ click simulates then ok.

Calculate the % RSD for Area and RT

• Top → click report format → click file → new report format file → Item → common → sample information.
• Item → LC/PDA common → Summary compound.
• Click right properties then go file → add → click table.
• Print the required peak and close.
• Printing of the chromatogram.
• Click on chromatogram → preview and publish → window opens → select individual report → ok → print.
• Column washing after sample sequence completed.
• When the sequence is finished, stop the flow by pressing the “Stop pump” button.

1. Turn off the instrument.
2. Use the Annexure to check the column performance details.

ABBREVIATION

SOP: Standard Operating Procedure

LC: Liquid Chromatography

ANNEXURE

Nil

REVISION HISTORY

Nil

ALSO READ: SOP for Operation of Shimadzu HPLC