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 enters 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 Annexure to check the column performance details.

ABBREVIATION

SOP: Standard Operating Procedure

LC: Liquid Chromatography

ANNEXURE

Nil

REVISION HISTORY

Nil

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