SOP For Calibration of GC with FID & ECD | Gas Chromatography Calibration

This document details the procedure for the Operation and Calibration of Gas chromatography with FID & ECD (Perkin Elmer 9 Clarus-500). It is the policy of XYZ Pharmaceuticals Limited that the written procedure shall be followed for the operation and calibration of GC in order to ensure smooth operation and its use monitored to obtain consistent and reproducible results and minimize downtime and meet the regulatory requirements.

This procedure is to be applied at the time of Operation and Calibration of GC.

3.0 RESPONSIBILITY & ACCOUNTABILITY

Persons along with their responsibilities and accountability are given below:

Executive - Corporate Quality Assurance:- To prepare SOP and follow the same

Trainee Analyst/ Jr. Analyst/ Analyst/ Sr. Analyst:- To follow the SOP accordingly

5.0 PROCEDURE

5.1 GENERAL INSTRUCTIONS

5.1.1 Disconnect the instrument from the main power supply at the time of cleaning.

5.1.2 Clean the Instrument externally with a clean dry cloth.

5.1.3 Ensure that the GC, PC, and printer are connected to a stabilized uninterrupted power supply (UPS).

5.1.4 Report any discrepancy observed during operation and calibration of instrument to Section In-charge or his representative for corrective and preventive action (as per XYZ/CQA/SOP-030)

5.1.5 Section In-charge or his representative will take the necessary action and report the same to Manager-Quality Assurance.

5.1.6 Affix “Out of Order” label on the GC.

5.2 PRECAUTIONS

5.2.1 Ensure that there is no vibration forming device near to Instrument.

5.2.2 Check Calibration status tag before starting operation.

5.2.3 Handle the column with extreme care (especially capillary columns). Do not connect or disconnect the column when the oven temperature is above ambient.

5.2.4 The column should be conditioned prior to start the analysis at a temperature of 20°C   above the highest operating temperature of a particular method. However, it should not exceed the maximum temperature limit specified for a particular column. Ensure that the carrier gas flow is ‘ON’ before setting the required temperature.

5.3 GENERAL SET UP

5.3.1 Connect the gas cylinders/generators-Nitrogen, Helium, Zero air, and Hydrogen to the   manifold of gas purification unit (ensure that the silica gel in the purification unit is achieved blue color)

5.3.2 Slowly open the cylinders/outlet of generators (when new cylinders are connected, the pressure of the cylinders is 120 kg/cm2) ensure that there is no leakage in the line using a mixture of isopropyl alcohol and water. (Alternately connect the air compressor unit for zero air using appropriate pre-filters of the compressed air supplied by the compressor).

Colour coding for the gasses is as follows.

Red’ color tubing                    -           Hydrogen

 ‘Green’ color tubing                -           Helium

 ‘Yellow’ color tubing                -           Nitrogen

Blue’ color tubing                   -           Air

Black color tubing                  -           Argon

5.3.3 Set the required outlet pressure from the gas purification unit; ensure the outlet pressure is within the specified limits of the gauge.

5.3.4 Check for the presence of any column in the oven, If any column is connected, remove the column verify the column type dimension against the specification against the connected column, if necessary or continue with the same for the next analysis and connect the column which is required for the analysis.

5.3.5 Check the maximum temperature of the column to be used for analysis.

5.3.6 In the case of capillary columns, Ferrules should be replaced when they are too flat to produce a good seal. In the case of SS columns, do not over tighten seals otherwise column fittings may be damaged.

5.3.7 Fix and tighten the column ends with adaptors, to the open ends inside the oven to the appropriate injector and detector ports.

5.3.8 Check the connections of the appropriate carrier gas i.e. Nitrogen/ Helium, according to the Specification.

5.4 OPERATION PROCEDURE

5.4.1 Put on the Gas Chromatograph, PC, monitor, and printer.

5.4.2 The GC display should illuminate after a few moments and go through an initialization mode.

Display Shows

Clarus 500

Gas Chromatograph

Rev. X.XX

System initializing, please wait





5.4.3 After initializing, the display shows “Log in

5.4.4 Enter the “Log in” then the Gas Chromatograph will go to the normal window and now it is ready to take the signals from the software.

5.4.5 After establishing communication with the connected PC, the main screen will appear.

Start the Total Chrom Workstation by the “TCNav” icon from the monitor.

Total Chrom Navigator – PEGC Starts with the following Display at the left top side of the main menu.

PEGC

No Method

ACQ: No data

I/F: Released

CMD: None







5.4.6 LOADING & CREATING A NEW METHOD

5.4.6.1 Loading of a Method:

5.4.6.2 Loading of the GC method can be performed only if the method is already saved in the computer.

5.4.6.3 Click the method under the build Parameters on the main menu window.

5.4.6.4Click for select/load the method from the directory.

5.4.6.5Select the required method and click ‘open’. The selected method will get displayed.

5.4.7 CREATING A NEW METHOD

5.4.7.1 Click the method under the Build parameter on the main menu window.

5.4.7.2 Select and click ‘create new method”

5.4.7.3 Select PEGC under the instrument selection and select “NEXT”.

5.4.7.4 Fill the description of the method under documentation and select “NEXT”.

5.4.7.5 Fill the Instrument notes and select “NEXT”.:

5.4.7.6 Template: GC or CAP-GC and fill the data as per the Test Procedure  / Specifications.

5.4.7.7 Select the Method Parameters as follows:

5.4.8    DATA ACQUISITION:

5.4.8.1Select data Channel A or B [A for Detector A (ECD) or Channel B for detector B (FID)] 

5.4.8.2Select sampling rate 1.56 to 25 pts per s as per the requirements (6.25 pts pers, is an optimum capillary column and 3.125 ml pts pers, is optimum for packed column)

5.4.8.3Set the Real-time plot for the chromatogram as per the peak height and then select “NEXT”.

5.4.9 INSTRUMENT CONTROL:              

5.4.9.1 Oven  / Inlet :

5.4.9.2 Enter the specified column oven temperature or temperature programming as per the test method.

5.4.9.3 Enter the Injector temperature A or B.

5.3.9.4 Also enter the Maximum temperature range of the Column and equilibrium time. Keep the coolant in off condition.

5.4.9.5 Carrier:

5.4.9.6 Specify the Carrier Gas Flow / Pressure / Velocity as per the test method and also specify the Column Length & Diameter and split ratio/flow if required.

5.4.9.7 Detector:

5.4.10 DETECTOR A (FID)

5.4.10.1Specify the Detector Temperature, Time constant, and Range 1 or 20 (Select 1 for High sensitivity and 20 for less sensitivity). And Keep the auto-zero ON.

5.4.10.2 Specify the Flow Rate of the Hydrogen and Air for the FID Detector. (Optimum 450 or 400ml/min for Air and 45 or 40ml/min for Hydrogen).

 Select the Attenuation from 0 to –6 along with offset.

5.4.11 INSTRUMENT TIME EVENT

5.4.11.1 Select the Instrument time event as per the requirement and then select the “NEXT” key to continue.

5.4.11.2 Process:

5.4.11.3 Specify the integration parameter/baseline time event and report as per the peak response and select “NEXT” to continue.

5.4.11.4 Component Default and Component:

5.4.11.5 Specify the Component Default and Component as per the test procedure and select the “FINISH”  key and save the method as per the specified name.

5.5 Preparation of sequence:-

5.5.1 Select the sequence from the Build sequence option of the main menu.

5.5.2 Select- Create a new sequence and follow the following instruction:

5.5.3 Global Parameter – Select the instrument as PEGC and finally select Build the sequence as per template or as per vial by vial and select the ‘OK” Key.

5.5.4 Sequence template –

5.5.5 Study: - Enter the Name of the analysis

5.5.6 Select the specific method for channel A or B.

5.5.7 Specify the calibration if required.

5.5.8 Specify the sample number pattern and number of samples and finally save the sequence as per desired name.

5.5.9 To start Sequence / Method.

5.5.10 Select the Instrument option from the main menu.

5.5.11 Select sequence (to start sequence) or method (to set GC as per specified parameters) along with raw file and result file path.

5.5.12 Select suppresses process and press OK.

5.5.13 After internal setting and equilibration STATUS with the show.

PEGC: Ready

ACQ: No Data

I/F: INST NRDY

CMD: None

PEGC

Ready

 GC: Ready

 

 

 

 

 5.5.14 Now GC is ready for the analysis

5.6 Operation of Head Space (Turbo matrix)

5.6.1 Switch on the main power supply.

5.6.2 Switch on Head Space from the rear panel; the HS splash screen is displayed.

5.6.3 Preparation of New Method:

5.6.3.1 Press anywhere on the screen.

5.6.3.2 Enter the password in the log-in message and press ok.

5.6.3.3 Keep the instrument idle for a few seconds for the initialization process.

5.6.3.4 After initialization set the following

5.6.3.5 Parameter for Status bar:

5.6.3.6 Select the specified method or change the parameter of the following:

5.6.3.7 Temperature settings:

a)    Oven temperature

b)    Needle temperature

c)    Transfer line temperature

Values can be changed by [-] or [+] keys

5.6.3.7.1Time settings:

a)    GC cycle time

b)    Injection time

c)  Pressurization time

d)    Incubation time (Thermo)

e)    Withdrawal time

Values can be changed by [-] or [+] keys

5.6.3.7.2 Option settings:

a)    Set the injection mode to volume or time (set the volume mode as the default unless otherwise specified)

b)    Set the operating mode to constant progressive or MHE (set the constant mode as the default unless otherwise specified)

Select PPC and set column pressure in psi as per requirement.

5.6.3.8   Preparation of Job Queue / Sequence.

5.3.9.8.1Select run option and enter vial number from _____ to ____ by [-] or [+] keys

5.3.9.8.2Select the specified method and press the start key.

5.3.9.8.3Now headspace is ready for the specified operations

  The auto sample will inject the sample when GC will become in Ready Condition.

5.6.3.9   Maintain Logbook of GC on XYZ/CQA/SOP-051/FR-01 Logbook for GC Appendix I  

Operation and Calibration of GC with FID & ECD

5.7 CALIBRATION PROCEDURE

5.7.1 Calibration frequency: Half-yearly or any Major breakdown and after maintenance.

5.7.1.1 Calibration for gas flow rate using Digital Flowmeter

5.7.1.2 Calibration for gas flow rate using FID Detector

5.7.1.3 Calibration for temperature      

5.7.1.3.1 Column Oven          

5.7.1.3.2 Injection Port          

5.7.1.3.3 Detector (FID)         

5.7.1.3.4 Headspace

5.7.13.5 Linearity and reproducibility of response for the Flame Ionization Detector (For Headspace Autosampler)         

5.7.1.3.6 Detector Sensitivity (to be done along with service engineer).

5.7.1.1 CALIBRATION FOR GAS FLOW RATE  (using Digital Flowmeter)  

5.7.1.1.1 Check the flow rate of the gas using the Digital Flowmeter as follows.

5.7.1.1.2 Connect the required column  & set the  flow rate 5ml/min,10ml/min ,15ml/min.

5.7.1.1.3 With the help of a flow meter check the flow rate of the gas per min at the detector outlet.

5.7.1.1.4 Flow rate is to be checked separately for each detector port.

5.7.1.1.5 Take five readings for each flow rate & report the flow rate.

An acceptance criterion for the set flow is not more than ± 5%.

Acceptance criteria for RSD is not more than 2 %

5.7.1.1.8Record the calibration data on XYZ/CQA/SOP-051/FR-02 as Calibration Datasheet Appendix II

5.7.1.2   CALIBRATION OF CARRIER GAS FLOW (using FID detector)

5.7.1.2.1Capillary Column:

5.7.1.2.2Put on the Carrier gas and carry out the calibration of the Gas flow keeping oven temperature, Injector Temperature, and Detector Temperature at ambient.

5.7.1.2.3Note down the readings at a set flow rate of 5 ml/min, 10 ml/ min, and 15 ml/ min for the   

Injection port 1(Capillary Column) 

Calculate flow accuracy by formula:

Flow Accuracy = Observed flow rate (ml / minute)

                  ------------------------------- X   100

                             Set flow  (ml / minute)

An acceptance criterion for the set flow is not more than ± 5%.

5.7.1.3   Record calibration data on XYZ/CQA/SOP-051/FR-02 Calibration Datasheet Appendix II  

5.7.1.3   CALIBRATION FOR TEMPERATURE:  

5.7.1.3.1 Column Oven:

5.7.1.3.1.1 Check the temperature of column oven using temperature sensor probe at 40, 100, 150, 200 & 240 ° C

Acceptance criteria for temperature variation ± 2° C

5.71.3.1.2 Record the calibration data on XYZ/CQA/SOP-051/FR-02 Calibration Datasheet Appendix II

5.7.1.3.2 Injection Port:

5.7.1.3.2.1Check the temperature of each Injection Port using temperature sensor probe at 140, 200, & 280 ° C

Acceptance criteria for temperature variation ± 10° C.

5.7.1.3.2.2Record the calibration data on XYZ/CQA/SOP-051/FR-02 Calibration data sheet Appendix II

5.7.1.3.3 Detector (FID):

5.7.1.3.3.1Check the temperature of Detector using temperature sensor probe at 180, 250 & 300°C

Acceptance criteria for temperature variation  ± 10° C.

5.7.1.3.4 Headspace Incubator:

5.7.1.3.4.1 Check the temperature of Headspace Incubator using temperature sensor probe at 80, 85  & 90 ° C

Acceptance criteria for temperature variation ± 2 ° C.

5.7.1.3.4.3 Record the calibration data on XYZ/CQA/SOP-051/FR-02   Appendix II

5.7.2 LINEARITY OF DETECTOR

5.7.2.1 Linearity and reproducibility of response for the Flame Ionisation Detector (For Headspace Autosampler)

5.7.2.1.1 Preparationof standard solutions of 100pm, 50ppm, and 25ppm.

5.7.2.1.2 Preparation of Standard stock solution: (1000ppm of each of Ethyl Acetate, Isopropyl   Alcohol, Methanol and Methylene Chloride in purified water.)

5.7.2.1.3   Weigh accurately about 100 mg of Ethyl Acetate, 100 mg of Isopropyl Alcohol, 100 mg of Methanol, and 100 mg of Methylene Chloride in a 100 ml volumetric flask containing 10ml of purified water. Mix and dilute with purified water.

5.7.2.1.4 100ppm solution: Dilute 5 ml of the stock solution to 50 ml with purified water. 5.7.2.1.5 50ppm solution: Dilute 5 ml of the stock solution to 100 ml with purified water.

5.7.2.1.6 25ppm solution: Dilute 5 ml of the stock solution to 200 ml with purified water.

5.7.2.1.7   Chromatographic system:

Column type

:

Elite 624, 30m,0.53mm ID ,.25µM

Carrier gas

:

Helium / Nitrogen

Carrier Gas pressure

:

7 psi

Split flow

:

20ml/min

Detector

:

FID ( flame ionization detector ).

Injection temperature

:

200°C

Detector temperature

 

260°C

Hydrogen Flow

:

45.0 ml/min

Air Flow

:

450.0 ml/min

Oven temperature

 

40°C (Isothermal)

Range

:

20

Attenuation

:

-5

 

Head Space Parameters:

HS Mode

:

4.0          Constant

Oven Temperature

:

80°C

Needle Temperature

:

85°C

Transfer Line Temperature

:

110°C

GC Cycle time

:

35min

Thermostat Time

:

30min

Pressurization Time

:

0.1 min

Injection time

:

0.1min

Withdrawal Time

:

0.5min

Vial venting

:

On

Headspace pressure

:

15psi

 

5.7.2.1.8 Procedure:

Pipette 2 µl each of 25ppm solution in six individual headspace vials. Pipette 2µl of each of 50 and 100 ppm solution in two individual headspace vials and seal the vial using Teflon coated septum and metallic cap by using a crimper. Inject the above sample as per the headspace condition given above and record the chromatogram and peak area response and retention time.

5.7.2.1.9 Establish the linearity curve and find out the correlation coefficient

Acceptance Criteria:

 The % RSD of an area of six replicate injections of 25 ppm solution should be less than   10% and the retention time should be less than 2%.

The correlation coefficient r2 should not be less than 0.98

5.7.2.1.10 Record the calibration data on XYZ/CQA/SOP-051/FR-02 as Calibration Datasheet Appendix II

5.7.3 DETECTOR SENSITIVITY:

5.7.3.1 Check the Detector Sensitivity once a year using GC Standard Test Mix.

[FID detector sensitivity can be done either packed or capillary column]

5.7.3.2 Detector ‘A’ sensitivity (FID packed Column):

5.7.3.3 Chromatographic system:

Column type

:

1/8’’S.S., 10%OV-101is suitable.

Carrier gas

:

Helium

Detector

:

FID (flame ionization detector).

PKD Injector temperature

:

250°C

FID Detector temperature

:

300°C

Oven temperature

:

90°C  

Injection volume

:

0.5µl     inject manually

Column flow

:

20 ml/min

Hydrogen Flow

:

45.0 ml/min

Air Flow

:

450.0 ml/min

Range

:

20

Attenuation

:

64(TC 0)

Run Time

:

About 10 min

           

5.7.3.4    Modify the oven temperature to correct for column differences such that C9 peak elutes no earlier than  4 minutes.

Calculation Formula:

                                           ( H  x  WH )

Sensitivity (In coulombs per gram)  =   ----------------- 

                                                                               V

H     = (Ampere gain range 20) x (Integrator Atten) x (signal ‘ mv’ )

 [Ampere gain range 20 =   7 x 10-12 Amp / mv,

Integrator Atten =    64]

WH  = (Width at half  height in seconds)

(Note: width at half height in Minutes x 60  = WH in  seconds)

V     = sample weight C8 in 0.5µl

V     = ( 2.965 x 10 –6 ) Grams carbon

  Acceptance Criteria:

Detector ‘A’ sensitivity (FID packed Column): should be more than 0.015 c/g

5.7.3.5 Record the calibration data on XYZ/CQA/SOP-051/FR-02 as Calibration Datasheet Appendix II

5.7.3.1 Detector ‘A’ sensitivity (FID Capillary Column):

5.7.3.2 Chromatographic system:

Column type

:

15m 0.25 ID methyl silicone is suitable.

Carrier gas

:

Helium

Detector

:

FID  (flame ionization detector).

CAP Injector temperature

:

250°C

FID Detector temperature

:

300°C

Oven temperature

:

100°C  

Injection volume

:

0.5µl     inject manually

Split flow

:

50 ml/min

Column flow

:

1 ml/min

Hydrogen Flow

:

40.0 ml/min

Air Flow

:

400.0 ml/min

Range

:

1

Attenuation

:

8(TC-3)

Run Time

:

About 10 min

 

5.7.3.3 Modify the oven temperature

5.7.3.4 Modify the oven temperature to correct for column differences such that the C9 peak elutes no earlier than 3 minutes.

Calculation Formula:

                                                                             H  x  WH             1 + SP

Sensitivity (In coulombs per gram)     =  ( -------------- )  x  (   -------  )

                                                                                   V                       C

H     = (Ampere gain range 1) x (Integrator Atten) x (signal ‘ mv’ )

[Ampere gain range 1 =   3.33 x 10-13 Amp / mv ,

 Integrator Atten = 8]

WH = (Width at half height in seconds)

 (Note: width at half height in Minutes x 60  = WH in seconds)

V     = sample weight C8 in 0.5µl

V     =  ( 2.965 x 10 –6 ) Grams carbon

SP   = Split flow in ml / min  = 50 ml / min

C     = Column flow in ml / min  = 1 ml / min

Acceptance Criteria:

Detector ‘A’ sensitivity FID Capillary Column): should be more than 0.013c/g

NOTE: If the instrument does not comply with any of the calibration parameters, then follow the relevant procedure of instrument breakdown given in LCP 018.

5.7.3.5 Record the calibration data on XYZ/CQA/SOP-051/FR-02 Calibration Datasheet Appendix II5.7.3.6 Summarize all the data on XYZ/CQA /SOP-051/FR-03   GC Summary sheet Appendix III    

5.5  Generation of Instrument Calibration Number

Generate Instrument calibration number on the calibration data sheet as INSCALXXYYZZZ

Where INS denotes Instrument, CAL denotes Calibration

XX denotes the year, YY denotes the Month and ZZZ denotes the sequence number. 

5.6  ABBREVIATIONS

INS CAL:  Instrument Calibration

 ml             

Milliliters

°C              

Degree Centigrade

min           

Minutes

g         

Gram

%              

percentage.

µl        

microliter

c/g

Coulombs per gram

ppm         

part per million

FID                 

Flame Ionisation Detector

GC                 

Gas Chromatograph

HS

Headspace

RSD                

Relative Standard Deviation

IPA                  

Isopropyl Alcohol

RT                   

Retention Time

Del

Delay

ECD

Electron capture detector

  Download:- Logbook for GC Appendix

>SOP For Operation and Calibration of HPLC

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