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.
- Vial
- 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 below link:
HPLC Basics- Principle and Scope
