Calibration of Gas Chromatography SOP

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SOP for Calibration of Gas Chromatography in Pharmaceutical Industry | SOP of Gas Chromatography | GC Calibration |

  • SOP covers below points :
    • Calibration of Gas Chromatography
    • Safety And Precautions
    • Gas Flow Rate Calibration
    • Precision Of Injector And Linearity Of Detector Response
    • Calibration Of Head Space
    • Calibration For Syringe
1.0 OBJECTIVE :
  • To lay down a procedure for calibration of Gas Chromatography (Head Space GC-HS-10).
2.0 SCOPE :
  • This SOP is applicable for operation and calibration of Gas Chromatography in the Quality Control Laboratory
3.0 RESPONSIBILITY :
  • The QC Staff shall be responsible for:
    • Following the procedure for calibration of Gas Chromatography.
  • The Manager-QC shall be responsible for:
    • Ensuring compliance of the SOP
4.0 ACCOUNTABILITY:
  • Quality Control Head 
5.0 PROCEDURE :

5.1 SAFETY AND PRECAUTIONS:

  • Ensure that the carrier gas flow is ON before starting the  gas chromatograph.
  • Switch on the instrument and ensure that carrier gas is flowing in the column (can be checked in capillary, in the display screen of the instrument).
  • Raise the injector and detector temperature to 200 to 250 respectively.
  • Maintain the column at 30°C for at least 10 minutes 10 minutes, then at the rate of 5°C /min. increase the column oven temperature to 108C below its maximum temperature and hold the column at this temperature for 30 minutes.
  • Handle the column with extreme care (especially capillary columns). Do not connect or disconnect column when oven temperature are above ambient.
  • Solvents and reagents used in the preparation of solution for examined should be of spectroscopic grade/ GC grade/ HPLC grade/ AR grade.
  • Rinse the syringes well in between the injections to avoid carry over.
  • After use replace the column in the designated place with the column ends.

5.2 PRELIMINARY CHECK BEFORE STARTING THE INSTRUMENT:

  • Ensure that the system is clean and free from dust. If not, clean with a soft duster.
  • Connected to a stabilized power supply.
  • Ensure that the  gas flow is on and Sufficient gas is available for complete analysis.
  • Adjust the required outlet pressure by using a knob provided on the filtration unit.
  • Turn on the toggle valves near the instrument.

5.3 GENERAL SETUP:

  • Ensure that the carrier gas flow is ON before starting the gas chromatograph.
  • Switch on the instrument and ensure that carrier gas is flowing in the column (Can be checked in Capillary, in the display screen of the instrument).
  • Raise the injector and detector temperatures to 200 to 250 respectively.
  • Maintain the column at 30°C for at least 10 minutes, then at the rate of 5°C/min. increase the column oven temperature to 10°C below its maximum temperature and hold the column at this temperature for 30 minutes.
  • Handle the column with extreme care (especially capillary columns). Do not connect or disconnect column when oven temperature are above ambient.
  • Solvents and reagent used in the preparation of solution for examination should be of spectroscopic grade / GC grade / HPLC grade
  • Rinse the syringes well in between the injections to avoid carry over.
  • After use replace the column in the designated place with the column ends.
  • In case of capillary columns, Ferrules should be replaced when they are too flat to produce a good seal. Fix and tighten the column ends with adaptors, to the open ends inside the oven to the appropriate injection and detector ports.
  • Check for any leakage at the joints with the help of IPA: water (1:1)
  • Put on the gas cylinders for nitrogen, hydrogen and air and turn ‘ON’ the knobs provided at the back-side of the instrument
    • Red’ colour tubing         – Hydrogen
    • Yellow’ colour tubing     – Nitrogen
    • Blue’ colour tubing        – Air
  • Check the connections of the appropriate carrier gas i.e. Nitrogen, according to the specification

5.4 CALIBRATION:

  • REAGENT/ EQUIPMENT REQUIRED
    • Methanol
    • Isopropyl Alcohol
    • Methylene Chloride.
    • Ethyl Acetate
    • Dimethyl Formamide
  • FREQUENCY OF CALIBRATION : Half yearly ±5 days
    • The recalibration is to be carried out when a major maintenance or change of major part is done or with respective parameter of calibration which is replaced.

5.4.1 GAS FLOW RATE CALIBRATION:

  • GAS FLOW RATE CALIBRATION OF CARRIER GAS FLOW:
    • Check the fittings to confirm absence of any leakage.
    • Set the required flow rate 2.0 ml/min
    • Connect the flow meter tube to Injector  outlet of the coloumn.
    • Keep the system at this flow rate for 5 minutes to equilibrate.
    • Observe the flow on Digital flow meter.
    • Repeat the above operations for 5.0 ml & 10.0 ml.
  • GAS FLOW RATE CALIBRATION OF AIR GAS FLOW:
    • Put off the Hydrogen gas flow.
    • Set the airflow at 400 ml/min.
    • Check the fittings to confirm absence of any leakage.
    • Keep the system at this flow rate for 5 minutes to equilibrate.
    • Observe the flow on Digital flow meter
    • Repeat the above operations for 450 ml/min.
  • GAS FLOW RATE CALIBRATION OF HYDROGEN GAS FLOW:
    • Put off the air flow.
    • Set the Hydrogen gas flow at 40 ml/min.
    • Check the fittings to confirm absence of any leakage.
    • Keep the system at this flow rate for 5 minutes to equilibrate.
    • Observe the flow on Digital flow meter.
    • Repeat the above operations for 45 ml/min.
    • Note down the result in Calibration TDS.
  • ACCEPTANCES CRITERIA :
ParameterSet Flow RateTolerance (± 5% of set flow)
Hydrogen gas40 ml/min.                38 ml to 42 ml.
45 ml/min.43.75 ml to 47.25 ml.
Zero air gas400 ml/min.380 ml to 420 ml.
450 ml/min.427.50 ml to 472.5 ml.
Nitrogen Gas2.0 ml/min.1.90 ml to 2.10 ml.
ml/min.4.75 ml to 5.25 ml.
10.0 ml/min.9.5 ml to 10.5 ml.

5.4.2 PRECISION OF INJECTOR AND LINEARITY OF DETECTOR RESPONSE:

  •  CHROMATOGRAPHIC CONDITIONS:
Equipment:Gas chromatograph GC-2014 with GC-HS-10
Column:DB-624, 30 m x 0.537 mm x 3.0 μm or equivalent
Carries gas:Nitrogen
Column pressure / Flow rate:3 psi  /  5ml / min
Detector:FID
Injector temperature:150°C
Injection mode    
Split ratio		:
:		Split
1 : 5
Detector temperature    
Signal acquire    
H2 flow    
O2 flow		:
:
:
:		240°C
40 msec
40 ml / min.
400 ml / min.
Oven temperature:Initial temperature 40°C for 10 min.
Ramp rate 40°C / min.
Final Temperature 240°C for 10 min.
Equilibration time:1.0 min.

  • Preparation of standard stock solution:
    • Weigh accurately about 0.1 g each of methanol, isopropyl alcohol, methylene chloride and ethyl acetate into a 100 ml volumetric flask containing about 30 ml of Dimethylformamide, shake well and dilute to volume with Dimethylformamide.
      • Level 5 (200 ppm): Dilute 20 ml of standard stock solution to 100 ml with Dimethylformamide.
      • Level 4 (150 ppm): Dilute 15 ml of standard stock solution to 100 ml with Dimethylformamide.
      • Level 3 (100 ppm): Dilute 10 ml of standard stock solution to 100 ml with Dimethylformamide.
      • Level 2 (50 ppm): Dilute 5 ml of standard stock solution to 100 ml with Dimethylformamide.
      • Level 1 (25 ppm): Dilute 2.5 ml of standard stock solution to 100 ml with Dimethylformamide.
  • Preparation for precision:
    • Transfer 100 ppm standard solution in the clean, dry vial. Inject 1µl of this 100 ppm standard solution in six replicates.
  • ACCEPTANCE CRITERIA FOR PRECISION:
    • % RSD of area of six replicate injections should not more than 15.0%.
    • % RSD of retention time should be less than 2.0%.
  • Preparation for Linearity:
    • Inject 1.0, 2.0, 3.0, 4.0 and 5.0 level of standard solution in three replicates respectively. After completion of sample solution  integrate and calibration the peak and take report print out of sample set integrate.
  • ACCEPTANCE CRITERIA
    • % RSD of Retention time of three replicate injections should not more than 2.00%.
    • % RSD of area of three replicate injections should not more than 15.00%.
    • The correlation coefficient ‘r’ obtained from the linearity level for each solvent should be not less than 0.99.

5.4.3 CALIBRATION OF HEAD SPACE :

PRECISION AND  LINEARITY OF DETECTOR RESPONSE: CHROMATOGRAPHIC CONDITIONS:

Equipment:Gas chromatograph GC-2010 PLUS & Headspace HT3 or equivalent
Column:DB-624, 30 m x 0.53 mm x 3.0 μm or equivalent
Carries gas:Nitrogen
Column pressure / Flow rate:3.4 psi  /  5ml / min
Detector:FID
Injector temperature:150°C
Injection mode     
Split ratio		:
:		Split
1 : 5
Detector temperature     
Signal acquire     
H2 flow     
Zero air flow		:
:
:
:		240°C
40 msec
40 ml / min.
400 ml / min.
Oven temperature:Initial temperature 40°C for 10 min.
Ramp rate 40°C / min.
Final Temperature 240°C for 10 min.
Equilibration time:1.0 min.

HEADSPACE CONDITIONS:

Constant heat time:On
GC cycle time:35.0 min.
Valve oven temperature:105°C
Transfer line temperature:110°C
Platen / sample temperature:110°C
Platen temperature equil. time:30 min.
Mixer:On
Mixing level:Level 5
Mixing time:1 min.
Mixer stabilize time:0.5 min.
Pressurize:10 psi.
Pressurize time:1.0 min.
Pressurize equil. time:0.5 min.
Loop fill pressure:5 psi.
Loop fill time:1 min.
Inject time:1 min.

  • Preparation of standard stock solution for Head sapce:
    • Weigh accurately about 0.1 g each of methanol, isopropyl alcohol, methylene chloride and ethyl acetate into a 100 ml volumetric flask containing about 30 ml of Dimethyl sulphoxide, shake well and dilute to volume with Dimethyl sulphoxide.
      • Level 1 (50 ppm)  : Dilute 5 ml of standard stock solution to 100 ml with Dimethyl sulphoxide.
      • Level 2 (100 ppm): Dilute 10 ml of standard stock solution to 100 ml with Dimethyl sulphoxide.
      • Level 3 (150 ppm): Dilute 15 ml of standard stock solution to 100 ml with Dimethyl sulphoxide.
      • Level 4 (200 ppm): Dilute 20 ml of standard stock solution to 100 ml with Dimethyl sulphoxide.
      • Level 5 (250 ppm): Dilute 25 ml of standard stock solution to 100 ml with Dimethyl sulphoxide.
  • Preparation for Precision:
    • Pipette out 2 ml of 100 ppm in 6 replicate into individual vials and seal the vial using Teflon coated septum and metallic cap using crimper.
  • Preparation for Detector Linearity:
    • Pipette out 2 ml of each 50 ppm, 100 ppm, 150 ppm, 200 ppm and 250 ppm in three replicate into individual head space vials and seal the vial using Teflon coated septum and metallic cap using crimper. Inject blank (use same DMSO – 1 injection) and the above solutions as per the head space condition given above and record the chromatogram and peak area response and retention time. Plot a linearity graph between concentration of each solvent (ppm) on the x-axis and average area of each solvent at each level on the y-axis.
  • ACCEPTANCE CRITERIA:
    • % RSD of area of six replicate injections should not more than 15.0%.
    • % RSD of retention time should be less than 2.0%.
    • Theoretical plate Not less than 2,000 of 1st replicate.
    • Resolution Not less than 1.0 of 1st replicate.
    • Tailing factor Not more than 2.00 of 1st replicate.
    • The correlation coefficient ‘r’ should not be less than 0.99.
  • STATIC CARRYOVER ANALYSIS TEST:
    • Chromatography condition and head space condition as per Precision And  Linearity Of Detector Response:
    • Use 250ppm standard solution for carry over test.
    • Sequence for carryover test analysis is three blank, one standard solution 250ppm and two blank.
    • Calculation for carryover analysis test:
  % of carryover =  Avg. area of last 2 blank (4 & 5)  X 100
Area of 250 ppm standard solution

ACCEPTANCE CRITERIA : Not more than 1.0%

5.4.4 CALIBRATION FOR SYRINGE

  • Take methanol & sonicate the syringe. 
  • Wash the syringe with purified water, then rinse with methanol
  • Dry the syringe in oven.
  • Take clean, dried and pre-weighed empty 5 ml volumetric flask.
  • Fill the syringe with purified water and ensure that the air is not created.
  • Dispense 10 µl volumes into the volumetric flask and weigh.
  • Repeat the same procedure for two times and calculate the net volume.
  • Calculate net volume in micro liter by using the formula;
=Weight of collected volume (mg)
Weight of 1.0 g. of water (0.99602) at 25°C

ACCEPTANCE CRITERIA: The net volume should be ± 1.0 % of actual volume.

5.5 MAINTENANCE PROGRAMME:

  • When instrument is under break down put the tag stating “OUT OF ORDER” Inform to Head QC/Designee.
  • Head QC/Designee will inform to instrument manufacturer/service person.
 6.0 ABBREVIATIONS:
AbbreviationExpanded form
SOPStandard Operating Procedure
No.Number
Q.C.Quality Control
QAQuality Assurance
DeptDepartment
No.Number
°CDegree celcius
secSecond
mlMilliliter
minminute
ppmPart per million

7.0 ANNEXURES:
  Annex. No.Title
01Calibration Protocol for Gas Chromatography

8.0 SOP REFERENCES
  • In-House

END OF THE SOP

ANNEXURES :

Annex. No. 01 Calibration Protocol for Gas Chromatography