4.0. PROCEDURE:
4.1 PROCEDURE FOR GENERAL CLEANING:
4.1.1 Ensure that the power supply to the instrument is switched off and main cord is removed from supply.
4.1.2 Clean the instrument with a clean dry cloth everyday. A wet cloth dipped in dilute soap solution may be used occasionally.
4.1.3 Precaution must be taken to clean the instrument immediately with dry cloth.
4.2 OPERATING INSTRUCTIONS:
4.2.1 Ensure that the instrument is properly connected to the power supply.
4.2.2 Fix the column and prepare the mobile phase.
4.3 PREPARATION OF MOBILE PHASE:
4.3.1 Use HPLC grade solvent/ HPLC grade water / HPLC grade reagent only.
4.3.2 Filter the mobile phase through 0.45 membrane filter after mix it in required proportion and degas on ultrasonic bath for about 5 minutes.
4.3.3 Affix the label on the container of Mobile Phase as shown below.
MOBILE PHASE Material : Prepared on : Valid up to : Prepared By : |
4.4 Operating Procedure:
4.4.1 Turn On the mains of LC-2010AHT unit.
4.4.2 Turn on the computer and double click on the icon “class VP “ from the menu window.
4.4.3 In the main menu window, double click on the instrument icon.
4.4.4 After the LC2010 AHT beeps indicating connection to the PC has been established the instrument window opens.
4.4.5 Place the tube leading to the purge/rinse degasser in 50:50 volume of (Methanol or Acetonitrile: water)
4.4.6 Click on the purge icon on the direct control toolbar. Select the tubing to be purged and set purging time for each.
4.4.7 Click on the purge button; display a window showing the progress of purge and auto purging shell start.
4.4.8 Select the new method from the list opened by clicking on the new method button. ‘untitled method’ shell display with Default method parameter on the LC setup window.
4.4.9 Configure the LC parameters by clicking on the pump, oven and detector icons in the LC setup.
4.4.10 Placing the mouse pointer on them highlights LC component icons. Clicking the icon, opens the window to set and confirm that components parameters.
4.4.11 Select file>Method>Save as and give a name for the method.
4.4.12 After creating method, down load it to the LC-2010AHT main unit by clicking on down load button. When the down load is complete, a message window appears and then Click OK
4.4.13 The LC-2010AHT controller shell be activated, and check that the correct parameter has been downloaded successfully.
4.4.14 To create a new sequence:
4.4.15 Click on the sequence wizard button in LC setup
4.4.16 Select ‘Create new file’ and click on the next button.
4.4.17 For making single run, Click on the single run button in the LC setup window or the single run button on the toolbar, specify a method file to be used in a respective dialog box and specify the path to save the data files. And click on the start command.
4.4.18 To make a series of a sequence, Click on the sequence run button in the LC setup or the sequence run button on the toolbar, specify the start vial number, injection volume, method file, data path, sample ID’s, data file names and after the setting is complete Click on the finish button to save the sequence file created in a respective folder by giving a desired name.
4.4.19 After creating a sequence, view Sequence Wizard in the LC setup, select the sequence and click on the start button to start acquisition.
4.4.20 Before injecting system suitability solution ensure that proper base line is obtained .For system suitability test, inject six continuous injections of same standard and RSD should not be more than 2.0 %, otherwise it is specified in standard test procedure.
4.4.21 After every ten injection of sample (5 samples in duplicate), the standard solution shall be injected in triplicate and the RSD shall be calculated and ensure that it is within the limit.
4.4.22 For terminating acquisition, select control>Extend run and enter a value for time added to the end of the run. To shorten the acquisition time. Specify a negative value.
4.4.23 Setting integration event, Click file> open to open the data file.
4.4.24 The default values can be used for integration, however it is recommended to optimize slope in accordance with the base line in advance to prevent undesired peaks such as noise from being integrated.
4.4.25 The default values: Width 5, Slope 200, Drift 0 T. DBL 1000, MINIMUM AREA 0.
4.4.26 For Creating report templates Select file> print setup, and setup the printer connected.
4.4.27 Click on the edit custom report to open the custom report window.
4.5 SHUT DOWN PROCEDURE:
4.5.1 Switch-off the computer.
4.5.2 Switch-off the LC-2010AHT main unit power.
4.6 CALIBRATION PROCEDURE:
4.6.1 AUTO-VALIDATION
4.6.1.1 Check the various components meet the performance and functionality criteria by carrying Auto validation and the system checks for the following parameters during auto validation
Wavelength accuracy (nm) |
Lamp energy |
Pulsation (Mpa) |
Temperature (°C) |
Absorbance accuracy (%) |
Drift (Mau) |
Noise (mAU) |
Gradient A% B% |
Gradient A% C% |
Gradient A% D% |
4.6.1.2 Press VP to display the VP/TOP screen, press F3 (VALID) to open the validation menu, and then 3WXYZ (Enter) to display Auto validation screen.
4.6.1.3 Connect the resistor tubing to the pump outlet, and till the mobile phase reservoirs.
4.6.1.4 When performing in the gradient mode, use 0.05% aqueous acetone in the Port A and HPLC grade water in ports B, C & D.
4.6.1.5 In the auto validation screen select Gradient (GE) mode and select solvent combinations A/B, A/C and A/D. Press F1 to start auto validation
4.6.1.6 In GE mode it starts purging port B, C AND D.
4.6.1.7 Wait for the system to finish purging. After the system finishes purging, the next screen is displayed and the wavelength accuracy test beings.
4.6.1.8 Tests follow one after the other and it takes about 3hrs to complete the Auto-validation.
4.6.1.9 After the gradient concentration accuracy, the results are displayed from all tests in Gradient (GE) mode.
4.6.1.10 To save the results press F4 (SAVE)
4.6.1.11 Down load all the results to the class VP software by pressing the Control> Validation run on the Menu bar, Select complete and enter a name for the file and save the file. Click print to print the report.
4.6.1.12 ACCEPTANCE CRITERIA
PARAMETERS | CRITERIA |
Wavelength accuracy (nm) | ±1 |
Lamp energy | >800 |
Pulsation (Mpa) | <0.50 |
Temperature (°c) | <0.20 |
Absorbance accuracy (%) | ±3.0 |
Drift (mAU/h) | <0.50 |
Noise (mAU) | <0.02 |
Gradient A% B% | 2.0 |
Gradient A% C% | 2.0 |
Gradient A% D% | 2.0 |
Note: Flow rate calibration is part of Auto validation in which system checks for gradient and flow accuracy
4.6.2 FOR PUMP:
4.6.2.1 Disconnect the column and connect the inlet and outlet tubing’s with a union.
4.6.2.2 Prime all the lines at 5 ml/min flow rate with water and ensure that flow line is free from air bubbles.
4.6.2.3 Set the flow rate at 1ml / min and collect the mobile phase (water) in a dry pre-weighed beaker and collect the mobile phase for 10 min. Weigh the beaker to get the weight of mobile phase. Calculate the flow rate by dividing the weight obtained with 10 (run time) and calculate the volume by dividing with weight per ml.
4.6.2.4 Calculate the corresponding flow rate. Carry out the experiment in duplicate. Record the observations in Annexure – 1.
4.6.2.5 Acceptance criteria: Flow rate should be in between 0.99 to 1.01 ml / min.
4.6.3 FOR GRADIENT VALVE:
4.6.3.1 Install union in place of column and flush solvent lines (A, B, C and D) with respective solvents at flow rate of 2ml/min.
4.6.3.2 Prepare 56 mg / litre of Propyl Paraben in Methanol.
4.6.3.3 Fill reservoir A and B with methanol and reservoir C and D with 56 mg / lit of Propyl Paraben in Methanol as mobile phase.
4.6.3.4 INSTRUMENT SET UP:
4.6.3.5 Enter the following time program:
Time | Flow (ml) | % A | % B | % C | % D |
Initial | 2.0 | 50 | 50 | 0 | 0 |
2 | 2.0 | 90 | 0 | 10 | 0 |
4 | 2.0 | 50 | 50 | 0 | 00 |
6 | 2.0 | 90 | 0 | 0 | 10 |
8 | 2.0 | 50 | 50 | 0 | 0 |
10 | 2.0 | 0 | 90 | 10 | 0 |
12 | 2.0 | 50 | 50 | 0 | 0 |
14 | 2.0 | 0 | 90 | 0 | 10 |
16 | 2.0 | 50 | 50 | 0 | 0 |
18 | 2.0 | 50 | 50 | 0 | 0 |
4.6.3.6 Use detector at wavelength of 254nm.
4.6.3.7 Record the printout of gradient valve test as per Annexure – 2.
4.6.3.8 The gradient valve test shall be accepted if percentage RSD of peak heights is not more than 5.0%.
4.6.4 CALIBRATION OF INJECTOR:
4.6.4.1 CHECK FOR PRECISION:
4.6.4.1.1 Purge the injector system with 100% water to ensure the complete washing of injector.
4.6.5 STANDARD PREPARATION:
4.6.5.1 Transfer about 50 mg of Uracil to a 250ml volumetric flask. Add 100ml of Methanol, sonicate to dissolve and make up the volume with Methanol to obtain a solution containing about 0.02 mg/ml of Uracil.
4.6.5.2 Filter the solution through 0.45m membrance filter.
4.6.5.3 Use HPLC grade Methanol as the mobile phase.
4.6.5.4 Instrumental Set Up
Column : Hypersil ODS or equivalent 250 x 4.6 mm, 5.0m
Flow rate : 1.0 ml/min
Injector Volume : 20m litre
Detector : 254 nm
4.6.5.5 Inject the standard preparation six times in the system. The peak areas observed shall be consistent.
4.6.5.6 The relative standard deviation for area counts calculated shall not be more than 1.0%.
4.6.5.7 Record the observation in the format as mentioned in Annexure – 3.
4.6.6 CHECK FOR LINEARITY:
4.6.6.1 Inject 10, 20, 30, 40 and 50m litre of standard preparation in duplicate. Calculate the average area counts corresponding to each set of injection. Tabulate the average area against each injection. Plot a graph for area counts vs. m liter the resulting graph shall be linear. The correlation co- efficient calculated shall be not less than 0.99.
4.6.6.2 Record the observations in Annexure - 3.
4.6.7 CALIBRATION OF DETECTOR:
4.6.7.1 Standard preparation. Mobile phase, Instrument set- up same as mentioned in calibration of Injector.
4.6.7.2 Run the chromatograph at different wavelength (252 nm – 264 nm with 2 nm increment)
4.6.7.3 The largest peak response shall be at 258 nm ± 2nm.
4.6.7.4 Record the results in the detection calibration record as per the Annexure - 4.
4.6.7.5 Affix a calibration status label on the instrument containing “Calibrated On”, “Due On” and “Signature”.
4.6.7.6 Report to Head – QC, if any discrepancy observed during calibration or operating the instrument and affix ‘Under Maintenance’ label on the instrument.
UNDER MAINTENANCE EQUIPMENT : SINCE :SIGNATURE: |
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ReplyDeleteMobile Flow Calibration System