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Final HPLC

This document establishes standard procedures for operating HPLC to analyze finished products and raw materials. It describes startup, washing, column connection, saturation with mobile phase, starting computer programs, sample analysis, washing after analysis, and shutdown procedures. Responsibilities of managers and engineers are also outlined.

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kanwalgul
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120 views

Final HPLC

This document establishes standard procedures for operating HPLC to analyze finished products and raw materials. It describes startup, washing, column connection, saturation with mobile phase, starting computer programs, sample analysis, washing after analysis, and shutdown procedures. Responsibilities of managers and engineers are also outlined.

Uploaded by

kanwalgul
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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HPLC

1. OBJECTIVE:
To establish a standard procedure for the operation of HPLC for analysis of Finished Product
and Raw Materials.
2. SCOPE:
This Operating procedure covers the procedure of proper handling of all HPLC (Shimadzu 2030
Prominence i plus) in order to analysis of samples of Raw materials and finished products.
3. RESPONSIBILITIES:
i. Lab Manager for implementation of SOPs.
ii. Biomedical Engineer for calibration, maintenance of instrument and log sheet.

4. GENERAL REQUIREMENTS:
i. The chromatography software is Lab Solution.
ii. Mobile Phase: Solvents which are required in specific ration to elute the
active ingredients.
iii. Column Type: Column packing material, particle size, length, diameter and
manufacturer are all important. More than one column type may work, but an HPLC
method must specify a column.

5. A) PROCEDURE:
i. Operational steps.
ii. Start- up of system
iii. Washing of line.
iv. Connection of Column.
v. Saturation of Column with Mobile Phase.
vi. Start- up of Lab Solution program
vii. Sample analysis.
viii. Washing after sample analysis and system shut down

6. B) Start up of System:
i. Check the arrangement of system according to Flow Diagram.
ii. Turn “ON” the main supply.
iii.
7. C) Washing of Lines:
i. Rinse the Suction Filter with the mixture of Purified Water and Methanol.
ii. Then rinse the suction filter with Purified water.
iii. Connect suction filters to all lines.
iv. Wash all the lines without connecting the column.
8. D) Connection of Column:
i. After rinsing with purified water, Connect the column.
ii. Rinse the column with Mixture of Methanol and Purified Water (50:50) for at least 30
minutes.
iii. Then wash the column with Filtered and Degassed Purified Water for at least 45
Minutes.
iv. Place the suction filter in their mobile phase so that water should not remain in tubing
and Column.
9. E) Saturation of Column with Mobile Phase (UV Detector):
i. Start the pump
ii. Pass the Mobile Phase for saturation of column.
10. F) Startup of Computer Based Program (Lab-Solution):
i. Double click the Lab-Solution Program icon on the desktop.
ii. When the system beeps twice, double click the instrument icon. If the system beeps
once, it means that the system is not connected to the instrument.
iii. Set LC Time Program.
iv. Load the required method.
11. G) Sample Analysis:
i. Prepare the standard solution according to the weight of ingredients given in the
product and raw material SOP.
ii. Saturate of column with mobile phase until base line is achieved.
iii. Put the Standard and sample vials in sample tray.
iv. Click the “BATCH START” in main tool bar of LC SOLUTION.
v. Click “BATCH PROCESSING” in main tool bar of Lab SOLUTION software.
Then entering the following parameters.
 Sample name
 Sample ID
 Sample type
 Vial No.
 Tray name
 Method file
 Data file
 Level No.
 Analysis type.

12. H) Washing after sample analysis and system shut down:


i. After the analysis of sample OFF the pump by clicking the Pumps Off / On button on
the main tool bar of Lab Solution software.
ii. Put the suction filter in purified water.
iii. Rinse the tube and Column with degassed and filtered purified water for at least 30
minutes.
iv. Switch OFF the column heater so that it can reach the room temperature before
turning the oven OFF.
v. Turn OFF the pumps by clicking the Pumps Off / On button on the main tool bar of
LC Solution software.
vi. Close the LC Solution Software.
vii. Shut down the the system by pressing the power button adjacent to screen.
viii. Turn “OFF” the system/ Computer.
13. J) Precautions:
i. Water used for any purpose i.e. sample preparation, washing of tubing should be
with purified water.
ii. The entire sample must be filtered through 0.45µm membrane filter before injection.
iii. Water for Plunger washing must be replaced daily.

14. REFERENCES:
i. User’s manual.
15. Record:
i. Logbook
ii. Verification Sheet/ Calibration result sheet.

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