Operation of High Performance Liquid Chromatography (HPLC) SOP

Standard Operating Procedure for Shimadzu HPLC | Operating Procedure of Lab Solution Shimadzu HPLC |

• SOP of HPLC Shimadzu covers below points :
  • Precautions before using HPLC
  • Operating procedure steps of HPLC
  • Preparation of Mobile Phase 
  • Start UP of Lab solution software
  • Run Method procedure
  • Run Sequence procedure
  • Integration procedure
  • Create Project procedure
  • Create Method File procedure
  • Create Report Format procedure

1.0 OBJECTIVE :

• To define the Operation procedure for High Performance Liquid Chromatography.

2.0 SCOPE :

• This procedure is applicable to Shimadzu make HPLC, Model No.: LC-2030, LC-2030C, LC-2030C 3D Plus, LC-2030C Plus, located in Quality Control Department

3.0 RESPONSIBILITY :

• Quality Control Officer and above

4.0 ACCOUNTABILITY:

• Quality Control Head

5.0 PROCEDURE :

5.1 PRECAUTIONS :

• Ensure that the plug is connected to the mains.
• Check and ensure that the instrument is clean and calibration is valid.
• Ensure that the pump, column, detector & processors are properly connected.
• Set mobile phase and waste solution bottle in place.
• Ensure that insert the end of solvent tube into solvent bottle.
• Ensure that HPLC is connected with UPS and have battery backup.
• Before you start the analysis of a sample. Ensure that ,
  • Suitable column is installed,
  • System is purged and equilibrated,
  • Solvent bottles are filled by the mobile phase or by solvent,
  • Confirm the wavelength, flow rate, column temperature etc.

5.2 OPERATION :

• Preparation of Mobile Phase :
  • Prepare mobile phase as per IP/BP/USP/In-House specification.
  • Filter through 0.45µ using filtration assembly. Sonicate the mobile phase.
  • Submerge the inlet of line filter in the mobile phase in the reservoir and place it in the provision for the same.

For Adjustment of Chromatographic Conditions as per BP / USP (for HPLC) click below link :

https://pharmablog.in/adjustment-of-chromatographic-conditions/

• Start UP:
  • Switch on the main button.
  • After that start the on/off button on high performance liquid chromatography Instrument.
  • Display will show on screen ;
    • SHIMADZU   Conditioning ….. , Seeking Home ….. , LAMP preheating ….. , Checking Wave ….. , © 2017 Shimadzu Corporation. All rights reserved.       
  • Above all conditions are ok, then put ID Admin & Password then press OK.
  • After that “Ready” display will be shown on screen.
  • Start the computer (Software), Select user and Password, appear on the Monitor.
  • Double click on Lab Solution icon. Login Window will appear.
  • Enter User Name and Password. Click on login tab.
  • Lab Solution Software will open.                                                           

• Run Method:
  • Select the project and click “Ok”.  Realtime analysis window” will appear.
  • To select method, click on “File” then select “Open Method File” and choose method click “Download and Close”.

• Run Sequence:
  • To Create new Sequence, click on Window tab then click on “show window” and select “Realtime Batch.”,
  • Right Click on Sequence and select table style, table style window will appear. Select following options.
    • Vial
      • Enter the number where vial is kept.
    • Tray Name
      • Enter the number where sample is kept.
    • Sample Name
      • Name of Product.
    • Sample ID
      • Identification of sample eg. Blank , Standard solution Etc.
    • Method File
      • Select required method.
    • Data File
      • Give Suitable Data file name.
    • Inj. Volume
      • Put the value which is indicated in specifications.
  • To save the sequence file, click on “File” and select “Save Batch File”. Give suitable batch sequence name.
  • To start a batch sequence , select sequence and Click on “Batch” and click on “Start.” 

• Integration:
  • Click on Window tab then click on “show window” and select “Data Analysis Tools”,
  • Select the project and click Ok. Double click on Post Run Icon.
  • Select the data to integrate and click on “Wizard” icon. Compound wizard table window will appear. Window shows options; width, Slope, Drift, Min. Area / Height. Put value as required.
  • Click Next, Select retention time and click next. Give compound name and click on “Finish” tab.
  • Select “Apply to Method” icon and Save.
  • To integrate the data create on 3D plus HPLC system, Select “Method View -Multi Chromatogram Table”.
  • To integrate the data create on 3D plus HPLC system, Select “Method View -Multi Chromatogram Table”.
  • Click on “Integration” and set the value of  width, Slope, Drift, Min. Area / Height etc. as required.
  • Click on “Compound” and give the compound name and select retention time.
  • After integration select “Apply to Method” icon and Save.
  • To integrate data with multiple wavelength repeat the above steps.
  • For calculating Peak Purity, Click on “Purity” under “Method View” and set the purity parameters.
  • Set the Noise Spectrum, Click in the check box and put the Retention time Range as required.
  • Click on check box to compute noise spectrum from current data for peak purity or Click on “Open Data File” to select alternate data to compute noise spectrum.
  • Results of Peak Purity Such as Impurity, Peak purity index, Single point Threshold and Minimum peak purity Index will appear under “Purity View”.

• Create Project:
  • To Create new project, Click on “Window” tab then click on “show window” and select “administrator tools”.
  • Administration tools will appear then Click on “project administration”.
  • Project Administrator window will appear. Click on “Add” to add new project.
  • Give project name and click “Next”.
  • Select User and then Click “Next”.
  • Select Instrument and then Click “Next”.
  • Verify the details such as project name, Selected User, Selected instrument and click on “FINISH”.

• Create Method File:
  • Select the project and click “Ok”. Realtime analysis window will appear.
  • To Create new Method File, click on “File” then click on “New Method File”, Click “New”.
  • Set the instrument parameters such as LC Time Program, Wavelength, and Temperature etc. as per Standard Test Procedure.
  • For PDA detector set the instrument parameters such as LC Time  Program, Temperature etc. as per Standard Test Procedure. Wavelength range is 190 nm to 800 nm. For single wavelength , set the wavelength near to the wavelength given in Standard Test Procedure. For example for 254 nm set the wavelength range 200 nm to 300 nm.
  • Set the Auto purge time to 3 min., Purge Time (Rinse Port) 20 min., Rinse Deep Time to 5 Sec. Set the maximum Pressure not more than 449 kgf/cm². 
  • Select “Download and Close” to save method file. Give File name and Click “Save”.
  • To save the file in template , click on “File” and select the ” Save Method File As Template”. Saved templates are used for making new method files.

• Create Report Format:
  • Click on Window tab then click on “show window” and select “Data Analysis Tools”.
  • Select the project and click Ok. Double click on Post Run Icon. Post Run Analysis window will appear.
  • Click on Window tab then click on “show window” and select “Report Generator”.
  • Click on “Item” and Select Sample Information, Chromatogram, peak table, Summery (compound) etc. from “common”, “Analysis Common” options.
  • To set the chromatogram parameters such as Peak Top comment, Setting  Scale etc., right click on chromatogram then click on Properties and set the parameters as required.
  • To set the Peak table parameters such as Name, Area, Area %, Retention Time, Relative Retention Time etc. right click on peak table then click on Properties and set the parameters as required.
  • Save the report format and give the name as Assay, RSD etc.

For Calibration of High Performance Liquid Chromatography (HPLC) SOP click below link :

https://pharmablog.in/calibration-of-hplc-sop/

For HPLC Column Management – SOP click below link :

https://pharmablog.in/column-management-sop/

 6.0 ABBREVIATIONS:

Abbreviation	Expanded form
ml	Milliliter
min	Minute
0C	Degree Celsius
nm	Nanometer
%	Percentage
Abs	Absorbance
UPS	Uninterruptible Power Supply
ppm	Parts Per Million
gm	Gram
mm	Millimeter
µl	Microliter
µm	Micrometer
µ	Micron

7.0 ANNEXURES:

NIL

8.0 SOP REFERENCES

•   Manual of High Performance Liquid Chromatography

END OF THE SOP

For HPLC Basics- Principle and Scope click link: HPLC Basics- Principle and Scope

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