Atomic Absorption Spectrophotometer Calibration, Purpose :
Atomic Absorption Spectrophotometer Calibration, The purpose of this SOP is to describe the operation and cleaning procedure of Atomic Absorption Spectrophotometer Model No. – A Analyst 400 with Graphite Furness HGA 900 used for analysis of the finished product & raw materials in the Quality Control Laboratory at XX Pharmaceuticals Ltd.
Atomic Absorption Spectrophotometer Calibration, Scope :
This procedure is applicable for the Atomic Absorption Spectrophotometer, installed in the Quality Control Laboratory of XX Pharmaceuticals Limited.
Definitions / Abbreviation:
Standard Operating Procedure (SOP): A written authorized procedure, which gives instructions for performing operations.
QC: Quality Control.
Responsibilities:
The roles and responsibility is as follows:
Sr. Executive/Executive, QC
[][]To ensure that this procedure is followed.
[][]To maintain the records properly as per SOP.
Manager, Quality Control
[][]To ensure that this procedure is kept up to date.
[][]To confirm that the SOP is technically sound and reflects the required working practices.
[][]Arrange training on the SOP to all concerned personnel.
[][]To ensure implementation of the SOP after training.
[][]Schedule calibration of the instrument at the defined intervals.
Head of Quality Assurance
[][]Approval of the SOP.
[][]To ensure overall implementation of the SOP.
Procedure:
Precaution(s):
[][]Care must be taken in handling the instrument opening, putting sample, and closing especially.
[][]Ensure optimum fluid level, otherwise flame will not ignite.
[][]All the solutions must be prepared carefully.
[][]The detection chamber must be clean and free from any dust of foreign particles.
[][]The selection of correct hollow cathode lamp of a particular atom must be present on a correct position of the lamp holder.
[][]The Air conditioning system and air exhauster must be open during operation of the Instrument.
Operation
[][]Procedure for getting concentration of a particular atom in Flame Method
[][]Ensure proper cleaning of the machine before operation.
[][]Turn on the computer and printer.
[][]Turn on the switch of the instrument.
[][]Turn on the switch of air compressor.
[][]Let open the exhaust switch for a few minutes of the compressor to expel moisture from it.
[][]Close the exhaust switch to retain huge amount of compressed air in it.
[][]Set the accessories apparatus of the flame mode.
[][]Turn on the switch of the Flame mode.
[][]Turn on the operating software on the computer.
[][]Align and optimize the instrument.
[][]Turn the screw of the acetylene gas cylinder and ensure its proper flow to the ignition chamber.
[][]Method setting: A method set by following way-
[][]At first click to file manager.
[][]Then go to new and click to method.
[][]Select the element (test element) from starting condition and click OK.
[][]Click on spectrometer at define element & write the method name in method description.
[][]Then select AA/AA-BG (as per method) from signal option.
[][]Click on setting and select the time, delay time & replicates.
[][]Click on sampler and select Air from Oxidant.
[][]Click on calibration and select Linear Through zero then select results unit (ppb, ppm etc) from equation & units.
[][]Click on standard concentration & select Blank and standard (1, 2, 3 etc).
[][]Then check sequence setting: set the number of standard and samples sequentially and save it.
[][]Then check sequence setting: set the number of standard and samples sequentially and save it.
[][]After completion the program, arrange & displayed the results window, the calibration window, the manual analysis control window and the flame control window from monitor window bar.
[][]Click “ON” button on the right side of the Flame option.
[][]Then follow the instruction of the software and input information accordingly.
[][]After completion of the calibration curve input sample according to software instructions.
[][]After completing analysis print the output or it may be saved.
[][]Turn off the software.
[][]Turn off the gas flow.
[][]Turn off the main instrument and expel air from compressor.
[][]Turn off the Switch of the computer.
Cleaning
[][]Clean all parts of the machine with cotton cloth using methanol after use.
[][]Procedure for getting concentration of a particular atom in Graphite Method
[][]Ensure proper cleaning of the machine before operation.
[][]Turn on the computer and printer
[][]Turn on the switch of the instrument
[][]Turn on the switch of air compressor
[][]Let open the exhaust switch for a few minutes of the compressor to expel moisture from it
[][]Close the exhaust switch to retain huge amount of compressed air in it.
[][]Set the accessories apparatus of the Graphite mode.
[][]Check the cooling system, the water level retain between maximum and minimum level.
[][]Turn on the switch of the Graphite mode.
[][]Turn on the operating software on the computer.
[][]Align and optimize the instrument.
[][]Turn the screw of the Argon gas cylinder and ensure its proper flow to the ignition chamber.
[][]Method setting: A method set by following way-
[][]At first Click to file manager.
[][]Then go to new and click to method.
[][]Then go to new and click to method.
[][]Then select the element (test element) from starting condition and click OK.
[][]Then go to spectrometer at define element & write the method name in method description and select AA/AA-BG from signal option. (as per method)
[][]Click on setting and select the time, delay time, BOC time & replicates.
[][]Click on sampler and go to Furness program & select Temperature, Ramp time, Hold time, internal flow & gas type.
[][]Click on Autosampler and select sample volume, Diluent location, matrix modifiers volume & location.
[][]Click on calibration and select Linear Through zero from calibration equation.
[][]Then select result unit (ppm, ppb etc) from equation and unit.
[][]Click on standard concentration & select Blank and standard (1, 2, 3 etc).
[][]Click on calculate standard volume & select stock standards, location & concentrations. Select the location of blank & reagent blank. Then click to OK and save it.
[][]Then click on Displayed the sample information editor. Write sample location and sample ID then save as in sample information file.
[][]Click on Auto (Automated analysis control)
[][]Click on Open from results data set name (for Data save).
[][]Click on analyze.
[][]After completion the program, arrange & displayed the results window, the calibration window, the automated analysis control window and the Furness control window from monitor window bar.
[][]Then click on analyze all
[][]Then follow the instruction of the software and input information accordingly
[][]After completion of the calibration curve automatically input sample according to software instructions.
[][]After completing analysis print the output or it may be saved.
[][]Turn off the software.
[][]Turn off the gas flow.
[][]Turn off the main instrument and expel air from compressor.
[][]Turn off the Switch of the computer.
Cleaning
[][]Clean all parts of the machine with cotton cloth using methanol after use.
[][]Procedure for getting concentration of a particular atom in MHS Method
[][]Ensure proper cleaning of the machine before operation.
[][]Ensure proper cleaning of the machine before operation.
[][]Set the MHS part with AAS properly.
[][]Set the sample tube on the flame chamber properly.
[][]Turn on the computer and printer.
[][]Turn on the switch of the instrument.
[][]Turn on the switch of air compressor.
[][]Let open the exhaust switch for a few minutes of the compressor to expel moisture from it.
[][]Close the exhaust switch to retain huge amount of compressed air in it.
[][]Turn on the switch of apparatus of the Graphite mode.
[][]Check the cooling system, the water level retain between maximum and minimum.
[][]Turn on the switch of the Flame mode.
[][]Turn on the operating software on the computer.
[][]Align and optimize the instrument.
[][]Turn the screw of the Argon & acetylene gas cylinder and ensure its proper flow to the ignition chamber.
[][]Method setting: A method set by following way-
[][]At first Click to file manager.
[][]Then go to new and click to method.
[][]Select the element (test element) from starting condition and click OK.
[][]Then go to spectrometer at define element & write the method name in method description.
[][]Click on setting and select the time, delay time, BOC time & replicates.
[][]Then click on sampler and go to Furness program & select Temperature, Ramp time,
[][]Hold time, internal flow & gas type.
[][]Click on calibration and select Linear Through zero from calibration equation.
[][]Then select result unit (ppm, ppb etc) from equation and unit.
[][]Click on standard concentration & select Blank and standard (1, 2, 3 etc).
[][]Then check sequence setting: set the number of standard and samples sequentially and save it.
[][]After completion the program, arrange & displayed the results window, the calibration
[][]window, the manual analysis control window and the flam control window from monitor window bar
[][]Then click “ON” button on the right side of the Flam option.
[][]Then follow the instruction of the software and input information accordingly
[][]After completion of the calibration curve input sample according to software instructions.
[][]After completing analysis print the output or it may be saved.
[][]Turn off the software.
[][]Turn off the gas flow.
[][]Turn off the main instrument and expel air from compressor.
[][]Turn off the Switch of the computer.
Cleaning
Clean all parts of the machine with cotton cloth using methanol after use.
Calibration:
Calibration for Flame System:
Wavelength Accuracy Using Ni:
Test Conditions:
[][]Open the default Nickel method (File-New-Method-Ni)
[][]Open the continuous Graphics window so the system will set up for Nickel.
[][]Ensure the lamp has been on for 15 minutes before measuring g absorbance with the Nickel standard.
Test prerequisites:
[][]Burner position Optimized and Nebulizer adjusted using Copper.
Test Steps:
[][]Under the tool bar, double click on the instrument icon in the ‘Spectrometer status panel’ to open the diagnostic/spectrometer window.
[][]Select the ‘Optical Position’ button at the button of the diagnostics window.
[][]Select the ‘Optical Position’ button at the button of the diagnostics window.
[][]A graphic plot of the peaked positions for the prism and Grating will be displayed
The Prism Tolerance is ±190 motor steps
The Grating Tolerance is +380, -260 motor steps
Sensitivity and Precision Using Ni:
Apparatus:
Volumetric flask with stopper, 1000 ml
Glass pipette 3 ml
Beaker 10 to 200 ml
Reagents:
1000 ppm Ni AAS standard solution.
Nitric acid
Preparation for 3ppm Ni standard solution:
[][]Pour a small quantity of 1000 ppm Ni AAS standard solution in the beaker. Use it to rinse the 3 ml pipette and the beaker, then discard it.
[][]Pour another 5ml (minimum) 1000ppm Ni AAS standard solution into the beaker.
[][]Pipette 3 ml standard from the beaker into the 1000 ml volumetric flask.
[][]Half fill the volumetric flask with de-ionized water.
[][]Pour 10 ml (approximately) of nitric acid into the volumetric flask.
[][]Fill the volumetric flask with de-=ionized water exactly to the mark.
Note: 3ppm Ni standard should be produced before the day of test.
Test Steps:
Edit the Nickel default method and enter/verify the following parameter:
Signal type: A
Read time:10.0
Replicates: 10
[][]While in the method editor, go to the calibration/ standard concentrations page. Enter blank for calibration blank ID and Nickel standard for standard ID.
[][]In the ‘Flame Control’ window, select the ‘Flam On/off’ icon to light the flame.
[][]In the ‘Manual Analysis Control’ window, aspirate blank and select ‘Analysis Blank’. 10 Replicates will be measured and autozero will occur.
[][]Aspirate a 3ppm Ni standard and select ‘Analysis Sample’. 10 Replicates will be measured.
[][]The results will be displayed in the result window. Record the values for mean absorbance and relative standard deviation (RSD).The value should meet following requirements.
Mean Absorbance ≥ 0.200
RSD ≤ 0.3%
[][]Return to the ‘Flame Control’ window and extinguish the flame by pressing Flame On/Off.
Wavelength Accuracy Using As:
Test Conditions:
[][]Open the default Arsenic method (File-New-Method-Ni)
[][]Open the continuous Graphics window so the system will set up for Arsenic.
[][]Ensure the lamp has been on for 15 minutes before measuring absorbance.
Test prerequisites:
[][]Burner position Optimized and Nebulizer adjusted using Copper.
Test Steps:
[][]Under the tool bar, double click on the instrument icon in the ‘Spectrometer status panel’ to open the diagnostic/spectrometer window.
[][]Select the ‘Optical Position’ button at the button of the diagnostics window.
[][]A graphic plot of the peaked positions for the prism and Grating will be displayed.
The Prism Tolerance is ±200 motor steps
The Grating Tolerance is ±380 motor steps
[][]Select the ‘X’ in the upper right hand corner to exit the ‘Optical position’ window. Select the X in the Upper right corner to close the window.
AA-BG Baseline Noise at 1 Abs. Using As:
Test Steps:
Edit the Arsenic default method and enter/verify the following parameter:
Signal type:AA-BG
Read time: 2
Replicates: 99
[][]In the Manual Analysis Control window, select the ‘Analyze blank’ button. The system will take 99 readings and perform an autozero.
[][]Insert the 1.0 A neutral density filter into the filter holder.
[][]In the Manual Analysis Control window, select the ‘Analyze Sample’ button. When the reading is complete, record the SD value. It should be ≤ 0.005.
AA Baseline Noise and Drift using Cu:
Test prerequisites:
[][]Burner position Optimized and Nebulizer adjusted using Copper.
[][]Copper lamp should be warmed up at least 15 minutes.
Test Steps:
[][]Open the default Copper method (File-New-Method-Ni)
[][]Open the continuous Graphics window so the system will set up for Copper.
[][]Edit the Copper default method and enter/verify the following parameter:
Signal type: AA
Read time: 0.5
Replicates: 99
[][]In the Manual Analysis Control window, select the ‘Analyze blank’ button. The system will take 99 readings and perform an autozero.
[][]In the Manual Analysis Control window, select the ‘Analyze Sample’ button. Data collection for 99 replicates will begin.
[][]At the end of 99 replicates, record the standard deviation (SD) value in the table below. It should be ≤0.001. Record the mean absorbance value as well.
[][]Wait 15 minutes and repeat the measurement. Record the mean absorbance value for the second measurement.
[][]Calculate the difference between the absorbance measurements. It should be ≤ 0.002 Abs.
Wavelength Accuracy Using Cu:
Test Steps:
[][]Under the tool bar, double click on the instrument icon in the ‘Spectrometer status panel’ to open the diagnostic/spectrometer window.
[][]Select the ‘Optical Position’ button at the button of the diagnostics window.
[][]A graphic plot of the peaked positions for the prism and Grating will be displayed.
The Prism Tolerance is ±120 motor steps
The Grating Tolerance is ±380 motor steps
[][]Select the ‘X’ in the upper right hand corner to exit the ‘Optical position’ window. Select the X in the Upper right corner to close the window.
Copper Capacitance:
The Optical position box also displays two capacitances values. Cap: in the upper left corner. The first value is the cap: used for peaking and the last value is the Final Cap: Record the Final Cap. It should be ≥1.0 pF
Flame Sensitivity and Precision using Cu:
Apparatus:
Volumetric flask with stopper, 1000 ml
Glass pipette 2 ml
Beaker 10 to 200 ml
Reagents:
1000 ppm Cu AAS standard solution.
Nitric acid.
Preparation for 2ppm Cu standard solution:
[][]Pour a small quantity of 1000 ppm Cu AAS standard solution in the beaker. Use it to rinse the 2 ml Pipette and the beaker, then discard it.
[][][][]Pour another 5ml (minimum) 1000ppm Cu AAS standard solution into the beaker
Pipette 2 ml standard from the beaker into the 1000 ml volumetric flask.
[][]Half fill the volumetric flask with de-ionized water.
[][]Pour 10 ml (approximately) of nitric acid into the volumetric flask.
[][]Fill the volumetric flask with de-=ionized water exactly to the mark.
Note: 2ppm Ni standard should be produced before the day of test.
Test Steps:
[][]Edit the Copper default method and enter/verify the following parameter:
Signal type: AA
Read time:10.0
Replicates: 10
[][]In the ‘Flame Control’ window, select the ‘Flam On/off’ icon to light the flam.
[][]In the ‘Manual Analysis Control’ window, aspirate blank and select ‘Analysis Blank’. 10 Replicates will be measured and autozero will occur.
[][]Aspirate a 2ppm Cu standard and select ‘Analysis Sample’. 10 Replicates will be measured.
[][]The results will be displayed in the result window. Record the values for mean absorbance and relative standard deviation (RSD).The value should meet following requirements.
Mean Absorbance ≥ 0.250
RSD ≤ 0.30%
[][]Select the Spectrometer- Setting tab and enter/verify the following parameter:
[][]Signal type: AA
Read time:0.1
Replicates: 10
[][]In the ‘Flame Control’ window, select the ‘Flam On/off’ icon to light the flam.
[][]In the ‘Manual Analysis Control’ window, aspirate blank and select ‘Analysis Blank’. 10 Replicates will be measured and autozero will occur.
[][]Aspirate a 2ppm Cu standard and select ‘Analysis Sample’. 10 Replicates will be measured.
[][]The results will be displayed in the result window. Record the value for relative standard deviation (RSD).The value should meet following requirements.
RSD ≤ 3.0%
[][]Return to the ‘Flame Control’ window and extinguish the flame by pressing Flame On/Off.
Calibration for Flame System:
Chromium Baseline Noise For Furness:
Test Steps:
[][]Click on the File pull down menu, then on Open. Select Method.
[][]Click on Browse. Go up 2 levels and double-click on Service. Then double click on Method.
[][]Double click on HGA Cr Test method. Select the Method Ed icon to open the Method Editor window.
[][]In the spectrometer section, select set. Select 2.7 Slit Width and 0.8 Slit Height. Select OK. Click on the file pull down menu, then on Save. Select Method. Close the Method Editor window.
[][]Click on the Tools pull down menu and then select Continuous Graphics. This will set up the Lamp. Close the Continuous Graphics window.
[][]Open the Align lamps window. Look at the Bar Graph status window and verify that the Cr HCL Lamp current is 15 mA and the Slit is se4t to 2.7/0.8. Also verify that the Background corrector is On. Allow the lamp to warm up for 30 minutes.
[][]Close the Align Lamps window.
[][]From the Tools pull down menu open the windows Results, Peaks and Automated Analysis. Arrange and align the windows as desired.
[][]Open the Furness control window and select the Furness ON/Off button to run at least one dry firing (without any sample) to make sure that there is no residual signal (Peak area less than 0.005) from any previous injections or tube contamination.
[][]Make sure position 3 of the Autosampler is empty. Type in 3 as the sample lactation in the setup page of the Automated Analysis window, click on the Analyze tab and then on the Analyze Samples button to measure 5 Furness dry firings (without any sample).
[][]Ensure that the AA and BG signals do not diverge from the baseline. Realign the Furness into the optical beam if necessary.
[][]The standard deviation of the mean dry-firing result in integrated absorbance (Peak area) for the Cr wavelength must not exceed a maximum value of 0.002. Record the result.
Chromium Characteristic Mass and Precession:
Test Steps:
[][]Use the same method as in the previous test. Demonized water should be in position 1 and Cr standard should be in position 2 of the Autosampler.
[][]Type in 2 as the sample lactation in the setup page of the Automated Analysis window, click on the analyze tab and then on the Analyze Samples button to measure 5 furnace firings using 20 µl sample injections.
[][]Calculate the characteristic mass using the Calculate characteristic mass tool from the Analyses pull down menu.
Characteristic Mass Result:
The characteristic mass (m0) results in pg and calculated from the mean integrated absorbance (Peak area) values should lie within the following ranges:
Element: Cr
Lower Limit (pg): 2.3
Target Value (pg): 3.0
Upper Limit (pg): 3.8
Note: If the characteristic mass exceeds the lower limit, check for possible contamination of the water used for sample preparation. Prepare a new solution if required.
Precision:
[][]The relative standard deviation (%RSD) of the mean sample solution readings calculated from the integrated absorbance (peak area) values for Cr must not exceed the maximum value of 2.0%.
[][]Record the results in the OQ Test Certificate on page 40.
[][]AS-800 Autosampler Linearity
[][]Click on the File pull down menu, then on Open. Select Method.
[][]Verify that you are in the Service\Methods directory. If not, click on Browse. Go up 2 levels and double-click on Service. Then double click on Method.
[][]Double click on HGA AS-800 Lin method.
[][]Select the Method icon to open the Method Editor window.
[][]In the spectrometer section, select Set. Select 2.7 Slit Width and 0.8 Slit Hight. Select OK.
[][]On the right side of the Method window select the Settings tab.
[][]In the Lamp Current section, select Use current (mA) and then type 15 in the Lamp Current box.
[][]Click on the File pull down menu, then on Save. Select Method.
[][]Close the Method Editor window.
[][]Clock on the Tools pull down menu and then select Continuous Graphics. This will set up the Lamp. Close the Continuous Graphics windows.
[][]Open the Align lamps windows. Look at the Bar Graph status window and verify that the Cr HCL Lamp current is 15mA and Slit is set to 2.7/0.8. Also verify that the Background Correction is ON. Allow the lamp to warm up for 30 minutes (if not already warmed up).
[][]Close the Align Lamps window.
[][]From the Tools pull down menu open the windows Results, Peaks, Examine Calibration and Automated Analysis. Arrange and align the windows as desired.
[][]Click on the Analyze tab of the Automated Analysis window and then on the Calibration button top start the calibration.
[][]When the calibration is finished check the Correlation Coefficient Result in the Examine Calibration window.
[][]The Correlation Coefficient Result Cr must exceed the minimum value of 0.999.
Annexure: Atomic Absorption Spectrophotometer Calibration
Annexure-I: Calibration Information Sheet for Wavelength Accuracy, Sensitivity and Precision using Ni
Annexure-II: Calibration Information Sheet for Wavelength Accuracy and AA-BG Baseline Noise at 1 Abs. using As
Annexure-III: Calibration Information Sheet for Baseline Noise, Wavelength Accuracy, Copper Capacitance, Flame Sensitivity and Precision using Cu
Annexure-IV: Calibration Information Sheet for Baseline Noise, Characteristic Mass and Precession, Autosampler Linearity using Cr
Annexure-V: Operation logbook for Atomic Absorption Spectrophotometer