SOP on Operation And Calibration of HPLC

Purpose

This SOP describes the procedure for operation and calibration of High Performance Liquid Chromatography (Model Shimadzu LC-2010 CHT).

Procedure:

Operation

• Ensure the calibration of HPLC before Use.
• Refer the manual for detail operation.
• Switch “ON” mains of instrument and PC. Switch “ON” instrument by switch provided on front side.  Enter the login ID No. “00000” to operate the instrument from display of front side and press OK. Select the column / Mobile phase as per the specification and fix the column in place provided.
• PC display will show LC solution. Select LC SOLUTION and proceed further as steps given below.

System configuration:

• Connect RS-232 set the SCL settings Login LC-Solution.
• Click <LC Analysis1 > icon on the <LC Solution Launcher>.
• In the Login window User Id: “Admin” Password “–” Program will start up.
• Go to <View> Select <Assistant bar> from the left side Assistant bar <Click System   configuration> Icon.
• Initial Configuration, <Instrument> Property <set the name or ID> appears. Then <System Configuration> window is subsequently displayed.
• Select [Instrument Type: VP or 2010] select the Communication < SCSI or RS232> Enter port numbers.  Click on OK button.
• Modules are configured for Analysis list. Set the parameters of each module by Double click on the individual module. ---Finally Click <OK>.

Setting Instrument Parameters

• Click on the Instrument from the LC -Solution Launcher with above user name and password.  LC Real Time Analysis Window will open.
• From the Assist bar (Left side) choose the Data Acquisition icon.
• Click on the file Menu and choose the new method file.
• Cursor will appear in the instrument Parameter View.
• Choose the Advanced button in Instrument parameter View.
• Set the Method as per analyses and save the method file form file menu.
• From the Assist bar Select Report format. Go to file Menu and choose the open Report format <C:\Labsolution\LCsolution\Template\Quantitation\Detecor A>.
• Click <Download parameters> to the instrument.  Activate the Instrument on/off icon from graphical menu.
• Wait till the instrument is stabilized.

Making a Single – run analysis

• Prior to analysis, confirm that “Ready” is displayed in the <Chromatogram> View and that the baseline is stable enough.
• In the <Data Acquisition> window, click on the [Single Start] icon on the Assistant Bar (or). The <Single Run> window will appear.
• In this window, open the method file, enter the name of data file, the vial number and injection volume, specify the Report format and its path if required automatic printout.
• After entering the above data, click on the [OK] button. A single analysis run will start.

Creating Report format

• Click on the [Report Format] icon located on the top page of the Assistant Bar in the <LC Post run > program. The <Report> window will be displayed.
• From the Toolbar, select the [LC/ Chromatogram] item and specify the area to be pasted to the workspace.
• Click and drag mouse diagonally where a report item will be pasted. 
• When releasing the mouse button, <LC/ Chromatogram Properties> window appears.
• Alternatively, you may want to click on the [File] tab of the <Properties> window and specify the desired data file using [Browse]. The specified file will be loaded to the report.
• The report format can be checked in the workspace (or more precisely in the <Preview> window that will be described later). In the <Properties> window, customize the report item by specifying a report display format and whether the report item is to be displayed or hidden. The <Properties> window can be displayed by double-clicking on the report.
• Item or selecting [Properties] from the menu displayed by clicking with the right mouse button.
• Next, using the same procedure as in positioning the [Chromatogram] report item, position the [Peak Table] report item in the workspace.
• Now the layout of the report is completed. Click on the [Preview] icon on the [Report] Assistant Bar to check the contents of the report. Click on the [Print] button located at the top of the preview to print the report.
• If the created report format is saved, a report in the same format can be created with any other data. Click on the tool bar and name the file.

Creating Batch Table

• Assist bar < Click on the Batch Processing> Batch window will open
• Click New Batch file from the file menu right click on the table and choose the Table Style (that is necessary columns).
• Choose the desired item from the left side list and add.
• If you want add one row, just right click on that table and choose the add row.
• Enter the Vial, injection volume, method file, report file and details for row one by one.
• Then save the batch file from file menu
• The same Batch creation can be followed using < WIZARD> icon from the Assistant Bar and then <Click Next with necessary details>
• To run the batch file just click on the Batch Start button from the assist bar.

Carrying out Batch analysis

• Display the desired batch table (batch file) by selecting <LC Analysis> - <Batch Table>. Click on the [Batch Start] icon on the [Batch] Assistant Bar. The batch analysis will be carried out starting with the first row.
• To pause/resume or stop batch analysis, click on the [Pause/Restart] icon or the [Stop] icon on the [Batch] page of the Assistant Bar. When a batch is paused, now progressing analysis is not stopped, and scheduled rows before execution can be edited such as addition and deletion. The same buttons are also available on the Toolbar.
• The progress of the batch analysis can be checked on the <Output Window>. After the batch analysis has been completed, check the log messages to ensure that no error has occurred during analysis. If the batch analysis was not successful, also refer to this log.
• <From the Next chapter is explained as example in the LC-Solution Post run>.   

Setting Peak Integration Parameters

• Note:  This can be set in Real time only Prior to Analysis as the part of the method file).
• Note 2:  This is saved for each data in the post run individually).
• Note 3:  Please note the File structure of LC-Solution.  With each data file the copy of the Method; batch and report file will be saved >.
• Note 4:  Any changes made in a particular data will not reflect in the other data’s
• Click on the [Data Analysis] icon on the [Acquisition] page of the Assistant Bar. The <LC Data Analysis> window in the <LC Post run> program will be displayed with the previously obtained data file loaded.
• In the <LC Data Analysis> window, open the data file created from the analysis using the [File] menu or <Data Explorer>.
• When the data file is opened, the chromatogram will be displayed.
• Display the Integration Parameters window. Select [Data Analysis Parameters] menu (displayed by right-clicking on the chromatogram), or click and click on the [Integration] tab.
• In this window, adjust the integration parameters to determine the optimal integration parameters such as Drift Width, Lock on/off etc.,
• After entering the appropriate values for the integration parameters, click on the [Apply] button. The resulting effect can be checked in the <Chromatogram> View. When the appropriate integration result is obtained, click on the [OK] button.
• To save the integration parameters as a part of the method file < click Apply to Method in the Assistant bar>.  Make sure the method file is not used for analyses in the real-time and run is going.
• Set the Drift Value higher around 5000 for Valley to valley calculation.
• Adjust the Time program for Integration based on the needs. Similar to LC-10
• The same integration can be carried out by Wizard menu also.
• Right click in chromatogram window ---Click Wizard--- set the integration    parameters and follow the display---click OK.

Setting identification and quantitative analysis parameters

• Note1:  This can be set in Real time only Prior to Analysis as the part of the method file)
• (Note2: If you do the Id table directly makes sure it is in edit mode)
• With the data file opened in the <LC Data Analysis> window, [Wizard] (on the Assistant Bar) can be selected. Click on the [Wizard] icon. The <Compound Table Wizard> window will be displayed.
• <Compound Table Wizard 1/5> Check that the integration parameters are properly set, and then click on the [Next] button. (The integration parameters may be changed. For information on the integration parameter settings, refer to the previous section.)
• <Compound Table Wizard 2/5> Check the peak (retention time) to be registered in the “Compound Table”, and then click on the [Next] button.
• <Compound Table Wizard 3/5> Specify a quantitative calculation method and a calibration curve type. Set the necessary parameters such as Quantitative Method, Calculated by, Curve Fit Type, and Units
• <Compound Table Wizard 4/5> Specify a method for identifying peaks. After completing the settings for the identification method, click on the [Next] button.
• <Compound Table Wizard 5/5>Finally, Enter the component name of the peak, Type parameters to “Target”. For the internal standard method, specify only the Type of the internal standard substance as “ISTD” and enter the same number in the [ISTD group] of the internal standard peak and the peaks that are calculated using that peak. For Conc. 1, 2, and 3, enter the level-1 concentration, level-2 concentration, and level-3 concentration of each compound included in the standard sample. (The “Level” of the sample to be analyzed will be specified for each sample in the Batch Table.
• After finishing the settings in the <Compound Table Wizard>, click on the [View] button located in the upper right corner of the “Compound Table” to establish the edited “Compound Table”. If you want to cancel the edited “Compound Table”, select [Table Edit] - [Cancel Edit] from the menu before clicking on the [View] button.

Applying settings to original method

• Save the analytical parameters set in the <LC Data Analysis> window as a method file.
• Click on the [Apply to Method] icon on the [LC/ Data] page of the Assistant Bar.
• The dialog box will be opened prompting you to enter the name of the method file to be saved. The original method file name is entered as the default setting. To apply the settings in the <LC Data Analysis> window to the original method file, click the [OK] button to overwrite it. The dialog box will be opened prompting you to select whether or not to apply the parameters for the method (see the following window). Click on the [OK] button. The above operations will overwrite the original method file with the changed data analysis parameters.

QA\QC Parameters

(Note: only with Batch this should be done not for single run.)

• Click on <<LC Post run>> from the LC Solution Launcher.
• << LC Post run>> Window will open. Then Choose Method file from <<Data explore window>>.
• From the menu Choose Method àQA/QC Parameter.
• QA/QC Parameters window will open in that window, please select the sample type, report type and select the option box “Calculate sample type statistics per batch table”.
• For Ex: To Check the “Noise/Drift Check”, then Tick the “Noise/Drift Check” check box, Right side Detail Grid will Enabled.
• In that Detail grid, select “Report” Check box and “Check” Check Box for the desired item.
• Then click on Criteria Tab.
• In that, Selected report type will appear with Compound name (Peak Name), If compound Name is not showing, Add the Peak name to the compound window from <<LC Post run>> Analysis.
• Set the limit value in criteria tab. Then click on OK button
•  Then click “Apply to Method” from the Assist Bar and save the method file.
•  Then select the particular batch table, go to the User prog column, if it is not showing the column in that batch table, just right click on the batch table and select <<Table Setting>> and select the user prog item. It will come in the batch table.
•  In the first row of the batch file select “BeginQCTool.exe” file. That file is stored in the Lab solution\program folder.
•  In the last row of the batch file select “EndQCTool.exe” file. This file is stored in the Lab solution\Program folder.
•  In the between rows of the batch file select “RunQCTool.exe” file. This file is stored in the Lab solution\Program folder.
•  Then select the Setting icon through Assist bar under batch heading.
•  In that select QA/QC tab select, Check box “Execute QA/QC” select and select the check box “Output HTML Style File. Click on ok.
•  Then Save the Batch file. And Execute the Batch file.
•  After executing the batch file, HTML file will open. If not, you can select that file from Data explore window, by selecting the all files tab.
•  The HTML file with the name QA /QC Data is stored in the Batch file folder. Just double click on that html file; it gives the report based on the QA/QC Parameters.

Calibration

Frequency of calibration:  Quarterly

Calibration of Pump: 
Check the Flow Rate by collecting Milli-Q water at the detector outlet over a specified time period. 
Procedure: Put the HPLC grade water in the channel A and the connect the 2 meter capillary column or union between auto injector & detector. Set the required flow rate and Collect the water in 10 ml volumetric flask. Note the time required in second and calculate the corresponding flow rate.
Repeat the procedure for 2.0 ml/min & 2.5 ml/min and record in same in format.

Acceptance criteria ± 1.0 % of Flow rate.

Retention Time Reproducibility:

Set the HPLC parameters and prepare the sample solution for caffeine as given below:

Mobile phase                        :  Methanol:  Milli-Q water (70 ml: 30 ml)

Column                                  :  ODS Hypersil 4.6 x 150 mm 5 µm

Flow rate                               : 1.0 ml/min

Column temperature            : Ambient

Diluent                                   : Mobile Phase

Detector wavelength           :  272 nm

Rum time                              : 7.0 minutes

Injection volume                   : 20µl

Standard solution Preparations:

Stock Solution:

• Weigh accurately about 100.0 mg caffeine in 100 ml volumetric flask and make up Volume with mobile phase.
• Prepared 40 ppm solution from stock solution for Retention time reproducibility and note down the retention time of 6 replicate injections in the calibration format.

40 ppm solution: Dilute 2 ml of Caffeine standard stock solution in clean and dry 50 ml of volumetric flask, mix and make up the volume up to 50 ml with diluent.

Calculate the RSD of the retention time .It should not be more than 1.0 %.

Gradient Composition:

Purge channel A & B with Purified Water and channel C & D with 0.3 % Acetone in purified water to remove the air bubble from the line .Set the Chromatographic condition as mentioned below:

HPLC condition          :           Use union for test

Detector                     :           265 nm

Injection volume        :           0.1µl.

Mobile phase  :                      Solvent A/B-water HPLC Grade, Solvent C/D-0.3 % Acetone.

Temperature             :           40°C

Gradient Program:
No. of step	Time	Solvent A/B	Solvent C/D
1	0.01	100	0
2	5.00	100	0
3	5.01	90	10
4	10.0	90	10
5	10.01	50	50
6	15.00	50	50
7	15.01	10	90
8	20.00	10	90
9	20.01	0	100
10	25.00	0	100
11	25.01	100	0
12	30.00	100	0

After setting all HPLC Conditions, start pump and record the chromatogram and check the M.V (Height) from the chromatogram and calculate the % of different composition by using formula given below.

Calculation Formula: -For channel A and C        

Actual concentration of 10.0 % (c) =     Height at 10.0% x100

                                                                               Height at 100 %   

Actual concentration of 50.0 % (c) =        Height at 50.0% x100

                                                                                  Height at 100 %

Actual concentration of 90.0 % (c) = Height at 90.0% x100

                                                                           Height at 100 %

Note:  Repeat the above calculation for channel B & D.

Acceptance Criteria: ±1 of set composition.

For record of observation refer the format no. TML/F/RDC/016-02.

Calibration of Injector

Injection precision (area reproducibility): (use solution 40 mcg/ml of caffeine solution)

Exiting system Record the area of 6 replicate injections in calibration format.

Calculate the RSD of the area and it should not be more than 2.0 %.

Injector linearity:

 Inject the solution 40 mcg/ml 5 µl, 10 µl, 20 µl, 30 µl, 40 µl, 50 µl & 100 µl (one injection each).  Record the peak area. The peak area and injection volume is represented in a graph and determine the regression line. Calculate the correlation coefficient for linearity. It should not less than 0.99.

Detector Linearity Test:

Stock Solution:

Weigh accurately about 100.0 mg caffeine in 100 ml volumetric flask and make up

Volume with mobile phase.

Prepare five different concentration of caffeine solution (Concentration 10 mcg/ml,

20 mcg/ml, 40 mcg/ml, 80 mcg/ml and 100 mcg/ml).

Chromatographic conditions are same as wavelength accuracy test instead of Detector wavelength.

Detector wavelength           : 272 nm

Preparation of five different concentration of  caffeine solution:

10mcg/ml of caffeine solution: Take 1ml of Caffeine standard stock solution in clean and dry  100 ml volumetric flask, mix and dilute up to 100 ml with same diluent.

 20 mcg/ml of caffeine solution: Take 1ml of Caffeine standard stock solution in clean and dry.

50 ml volumetric flask, mix and dilute up to 50 ml with same diluent.

40 mcg/ml of caffeine solution: Take 2ml of Caffeine standard stock solution in clean and dry  50 ml volumetric flask, mix and dilute up to 50 ml with same diluent.

 80 mcg/ml of caffeine solution: Take 2ml of Caffeine standard stock solution in clean and dry 25 ml volumetric flask, mix and dilute up to 25 ml with same diluent.

100 mcg/ml of caffeine solution: Take 5ml of Caffeine standard stock solution in clean and dry 50 ml volumetric flask, mix and dilute up to 50 ml with same diluent.

Inject each different concentration of caffeine solution.

Calculate the Coefficient of correlation (r2).

Acceptance criteria: Coefficient of correlation (r2) NLT 0.99
Carry over test: (Use solution 40 mcg/ ml and Blank m.p)
The peak area is measured for 20 µ injection of mobile phase as a blank , followed  by six 20 µl. injections of 40 mcg/ml of caffeine standard solution .The Injection carry over  is calculated for area as the peak response from the mobile phase injection with the comparison  to average area of six standard caffeine injection. The value should not more than 0.2 %.

Injector accuracy:

To confirm the auto injector has withdraw the proper sample amount from vial

Fill a sample vial, full with degassed & filtered water and cap the vial with septum. Carefully keep the vial on the analytical balance by using the forceps. Record the weight W1 (in mgs). Record the same in the format.

Placed the vial in sample position 1 and generate the sequence for 6 injections by putting the sample volume 20 µl. and run the system.

After all 6 injections, weigh the vial, record the weight W2 in format.

Using the formula calculate the average amount injected per injection.

For record of observation refer the format No.TML/F/RDC/016-02.

Water withdraw per injection= (W3=W1-W2)/0.9972x6

Where 6= total number of injection.

Acceptance criteria: ± 2.0% of set injection volume (i.e. 19.6 to 20.4 µl).
Detector Performance

Wavelength accuracy
Caffeine standard stock solution (1000mcg/ml): Take 100 mg of caffeine standard in clean and dry 100 ml volumetric flask, add 60 ml of diluent and sonicate to dissolve. Make up the volume up to 100 ml with diluent.

40 mcg/ml caffeine solutions: Dilute 2 ml of Caffeine standard stock solution in clean and dry 50 ml of volumetric flask, mix and make up the volume up to 50 ml with diluent.   Inject the 40 mcg/ml caffeine standard solution in each individual wavelength.

To verify the wavelength accuracy using standard caffeine solution (40 mcg /ml caffeine solution in mobile phase.) Wavelength verification from 265 to 275 nm.

Chromatographic condition: 

Mobile phase                        :  Methanol:  MilliQ water (70 ml: 30 ml)

Column                                  :  ODS Hypersil 4.6 x 150 mm 5 µm

Flow rate                               : 1.0 ml/min

Column temperature            : Ambient

Diluent                                   : Mobile Phase

Detector wavelength           : 265 nm to 275 nm

Rum time                              : 7.0 minutes

Injection volume                   : 20µl

Record the max & min Absorbance value of caffeine in calibration format

Acceptance criteria:

Maxima at 272 nm ± 2 nm.
Temperature Accuracy:

Procedure: Set the oven temperature at 40°C and 25°C from system .Check the oven temperature from display and note down temperature in the format. Also verify the oven temperature by keeping the external pre calibrated thermometer in the oven and verify the same against the displayed temperature by the system. Difference should be ± 1.0°C from set temperature.

Acceptance criteria:

± 1.0 °C of set temperature. 

Sample cooler temperature accuracy:

Procedure:   Set the sample cooler temperature at 10°C and 40°C from system .Check the sample cooler temperature from display and note down temperature in the format. Also verify the sample cooler temperature by keeping the external pre -calibrated thermometer in the oven and verify the same against the displayed temperature by the system. Difference should be ± 1.0°C from set temperature.

Acceptance criteria:

± 1.0 °C of set temperature

Action to be taken in case unsatisfactory results:

Attach Not to be used / under maintenance label to the instrument.

Inform the maintenance & Engineering department or inform the authorised service agent of the Manufacturer. After rectification recalibrate the instrument before use.

Precaution

Proper handling of the equipment.

Do not keep the door of the oven open for a long period when the temperature is ‘ON’.

Applying and releasing of the vacuum should be done slowly so as to avoid the substance from propelling out of the bottle.