Calibration of Gas Chromatography (GC)

Standard Operating Procedure (SOP) for Calibration of Gas Chromatography (GC), parameters are Carrier Gas flow Accuracy,  Calibration of Flame Ionization Detector (FID) by linearity Measurement, Calibration of Auto-injector by linearity measurement, Calibration for Column Oven temperature measurement, etc.

SOP / Protocol for GC Calibration

GC Calibration Procedure:

    • At the time of calibration enter the details in the respective calibration template and compare the obtained results to the specified limits given in the respective template.
    • After completion of calibration submit the report for checking.
    • Update the calibration status on the instrument label and fill the respective logs after completion of calibration.
    • Check the leakage at the Joints with 50% v/v solution of soap solution in water prior to use (Near septum nut, around injection port nut, at column connection).
    • Calibration procedure and acceptance criteria are given in respected calibration templates.

Calibration Frequency:

    • The following GC calibration parameter shall be performed monthly. (± 3 days)GC Calibration
      • (A)- Calibration of Carrier gas flow accuracy.
    • The following GC calibration parameter shall be performed six monthly. (± 15 days)
      • (A)-GC Calibration of Flame Ionization Detector by linearity measurement.
      • (B)-GC Calibration of the injector by Linearity measurement ( Liquid injection)
      • (C)-GC Calibration of Auto-injector by linearity measurement.
      • (D)-GC Calibration of Column oven for temperature accuracy.
      • (E)-GC Calibration of Vial temperature accuracy.
    • Calibration requirement in case of maintenance shall be evaluated case-by-case ( Instrument manual can be referred).

CALIBRATION TEMPLATE FOR CARRIER GAS FLOW

Instrument Gas Chromatography
Make Instrument code
Model
  • Calibration requirements:
    • Flow meter Code No.____________ Calibration Valid up to :_____________________
    • Stopwatch Code No.____________ Calibration Valid up to :______________________
  • System requirements:
Testing conditions for flow Rate

Applied conditions

For capillary column mode :

1.0, 2.0, 5.0 ml/min.

 
For packed column mode :

10,20,30,40,50 ml/min.

 

 

  • Sample/ standard preparation: N/A
  • Calibration procedure:

    • Switch on the instrument as per the instrument SOP.
    • The analyst shall open the carrier gas like Nitrogen or Helium from the cylinders or use a gas generator for respected carrier gas.
    • Ensure that Hydrogen and zero air shall be off, whenever checking carrier gas flow.
    • Set up the instrument to check the flow rate and attach the silicon tube inlet of the flow meter.
    • Adjust the pressure between 5.5 and 6.5 kg/cm2 by opening and closing of the valve located at the cylinder. (If applicable)
    • Attach another end of the silicon tube to the injector end.
    • Set the flow 1 ml/min and observe the flow reading in the flow meter. Calculate the reciprocal flow rate with the help of an inbuilt stopwatch and record the observations in the observation table.
    • Follow the same procedure to calibrate the flow for other flow as per table and record the results in the observation table.
    • Verify the results against the acceptance criteria and in case of any non- compliance, inform Head-QC or designee.
  • Observation table:

Sr. No Set Flow Observed Flow Acceptance criteria: (Difference between set flow and observed flow should meet the criteria given in the table below.
For capillary column mode :
1. 1 ml/min 0.90-1.10 ml/min
2. 2 ml/min 1.80-2.20 ml/min
3. 5 ml/min 4.50-5.50 ml/min
For packed column mode :
1. 10 ml/min 9.0-11.0 ml/min
2. 20ml / min 18.0-22.0 ml/min
3 30ml / min 17.0-33.0 ml/min
4 40ml / min 36.0-44.0 ml/min
5. 50ml / min 45.0-55.0 ml/min
  • Acceptance criteria:
    • The difference between the set flow and observed flow should meet the criteria given in the table.
  • Result: ………………………………(Satisfactory/Dose not satisfactory)
Date of calibration:
Next Due Date of calibration (Frequency monthly):
Calibration type: Scheduled / Not scheduled (Reason: _________________________)

 

Calibrated by:

Date:

Checked by:

Date:

Approved by:

Date:

 

GC CALIBRATION – FLAME IONIZATION DETECTOR BY LINEARITY MEASUREMENT.

(BY USING PACKED COLUMN)

Instrument Gas Chromatography
Make Instrument code
Model
  • Calibration requirements:

    • DMF (AR grade) Code no : _______________ Valid up to: _______________
    • Isopropyl alcohol Code no : _______________ Valid up to: _______________
    • Ethyl alcohol Code no : _______________ Valid up to: _______________
  • System requirements:

Parameter Test Conditions Applied Conditions
Column Porapak Q – 80 / 100#, 2M,

S.S. Column.

Carrier gas Nitrogen
Flow rate 30 ml/min
Detector FID
Column temperature 180°C
Injector temperature 250°C
Detector temperature 250°C
Injection volume 1 ml
Run Time 20.0 min
  • Sample/ standard preparation:
    • Take _______(5.0 ml) of ethyl alcohol in ______(50ml) volumetric flask, dilute up to mark with ________DMF. (Stock solution-1).
    • Take _______(5.0 ml) of Isopropyl alcohol in ______(50ml) volumetric flask, dilute up to mark with ________DMF. (Stock solution-2).
    • Prepare the final concentration of ethyl alcohol as per the following procedure.
      • 2000 ppm: Take _______(1.0 ml of stock solution 1) and _______(5.0 ml of stock solution 2) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
      • 4000 ppm: Take _______(2.0 ml of stock solution 1) and _______(5.0 ml of stock solution 2) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
      • 6000 ppm: Take _______(3.0 ml of stock solution 1) and _______(5.0 ml of stock solution 2) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
      • 8000 ppm: Take _______(4.0 ml of stock solution 1) and _______(5.0 ml of stock solution 2) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
      • 10000 ppm : Take _______(5.0 ml of stock solution 1) and _______(5.0 ml of stock solution 2) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
  • Calibration procedure:

    • Set up the instrument as per the conditions.
    • Inject the samples three times from each solution and calculate the ratio of IPA and Ethyl alcohol. Record the observation in table-1
    • Calculate the %RSD of the mean ratio. It should not be more than 5.0 %.
    • Plot a linearity curve of concentrations Vs corresponding mean area, using the least square method. Calculate the correlation coefficient (r) and record the observations in the given table.
  • Observation table:

Table-1

Sr. No. Ethyl Alcohol Conc. Area of IPA

(X)

Area of Ethyl alcohol

(Y)

Area Ratio of Ethyl alcohol to IPA
1 2000 ppm
Mean
RSD%

 

Sr. No. Ethyl Alcohol

Conc.

Area of IPA

(X)

Area of Ethyl alcohol

(Y)

Area Ratio of Ethyl alcohol to IPA
2 4000 ppm
Mean
RSD%
3 6000 ppm
Mean
RSD%
4 8000 ppm
Mean
RSD%
5 10000 ppm
Mean
RSD%

 

Table-2

Sr. No Ethyl Alcohol std

Conc.

Mean Area ratio Acceptance Criteria
1 2000 ppm Correlation coefficient should be NLT 0.990
2 4000 ppm
3 6000 ppm
4 8000 ppm
5 10000 ppm

 

  • Acceptance criteria:

    • Correlation coefficient should be NLT 0.990
  • Result: ………………………………(Satisfactory/Dose not satisfactory)

GC CALIBRATION – FLAME IONIZATION DETECTOR BY LINEARITY MEASUREMENT.

(BY USING CAPILLARY COLUMN – LIQUID INJECTION MODE)

Instrument Gas Chromatography
Make Instrument code
Model
  • Calibration requirements:

    • DMF (AR grade) Code no : _______________ Valid up to: _______________
    • Isopropyl alcohol Code no : _______________ Valid up to: _______________
    • Tetrahydrofuran (THF) Code no : _______________ Valid up to: _______________
  • System requirements:

Parameter

Test Conditions

Applied Conditions

Column AT-5 (30m X 0.53mm X 5.0µ)
Carrier gas: Nitrogen or Helium
Oven temperature 60°C – 3 min. – @ 30°C/min.-210°-2min.
Detector temperature 220°C – Flame Ionization detector (FID)
Head pressure 25 kpa
Injection volume 1µl
Split ratio 40:1
Injector temperature 180°C
  • Sample/ standard preparation:

    • Take _______(5.0 ml) of Isopropyl alcohol in ______(50 ml) volumetric flask,dilute up to mark with ________DMF. (Stock solution).
    • Internal Standard Preparation: Take ______(5 ml of THF) to _______(50ml) of Volumetric flask and dilute upto the mark with ____________(DMF).
    • Prepare the final concentration of Isopropyl alcohol as per the following procedure.

    • 5000 ppm : Take _______(2.5 ml of stock solution ) and _______(5.0 ml ) of THF (IS) in ______(50 ml) volumetric flask, dilute up to with ________DMF.
    • 8000 ppm : Take _______(4 ml of stock solution ) and _______(5.0 ml ) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
    • 10000 ppm : Take _______(5 ml of stock solution ) and _______(5.0 ml ) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
    • 12000 ppm : Take _______(6 ml of stock solution ) and _______(5.0 ml ) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
    • 15000 ppm : Take _______(7.5 ml of stock solution ) and _______(5.0 ml ) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with________DMF.
  • Calibration procedure:

    • Set up the instrument as per the conditions.
    • Inject the samples three times from each solution and calculate the ratio of IPA and THF. Record the observation in table-1
    • Calculate the %RSD of the mean ratio. It should not be more than 5.0 %.
    • Plot a linearity curve of concentrations Vs corresponding mean area, using the least square method. Calculate the correlation coefficient (r) and record the observations in given table-2.
  • Observation table:

 Table-1

Sr. No. IPA Conc. Area of IPA

(X)

Area of THF

(Y)

Ratio
1 5000 ppm
Mean
RSD%

 

Sr. No. Ethyl Alcohol

Conc.

Area of IPA

(X)

Area of THF

(Y)

Ratio
2 8000 ppm
Mean
RSD%

 

3 10000 ppm
Mean
RSD%

 

4 12000 ppm
Mean
RSD%

 

5 15000 ppm
Mean
RSD%

 

Table-2

Sr. No IPA std

Conc.

Mean Area ratio Acceptance Criteria
1 5000 ppm Correlation coefficient should be NLT 0.990
2 8000 ppm
3 10000 ppm
4 12000 ppm
5 15000 ppm

 

  • Acceptance criteria:
    • Correlation coefficient should be NLT 0.990
  • Result: ………………………………(Satisfactory/Dose not satisfactory)

 

GC CALIBRATION -FLAME IONIZATION DETECTOR BY LINEARITY MEASUREMENT.

(BY USING CAPILLARY COLUMN – HEADSPACE MODE)

Instrument Gas Chromatography
Make Instrument code
Model
  • Calibration requirements:

    • DMF (AR grade) Code no : _______________ Valid up to: _______________
    • Isopropyl alcohol Code no : _______________ Valid up to: _______________
    • Tetrahydrofuran (THF) Code no : _______________ Valid up to: ___________
  • System requirements:

Table: 1

Parameter Test Conditions Applied Conditions
Column AT-5 (30m X 0.53mm X 5.0µ)
Carrier gas Nitrogen or Helium
Oven temperature 60°C – 3 min. – @ 30°C/min.-210°-2min.
Detector temperature 220°C – Flame Ionization detector (FID)
Head pressure 25 k pa (3.5 psi) ( can vary as per instrument make)
Injection volume 1 ml ( can vary as per instrument make)
Split ratio 40:1
Detector range 1
Attenuation -3

 

Table: 2

HSS Conditions:
Vial temperature 85°C
Syringe temperature 100°C
Equilibration time 20 min.
Injector temperature 200°C
Transfer line temperature 90°C
Withdrawn time 0.2 min
Pressurize time 3 min
GC Cycle time 20 min
Carrier gas pressure 14 psi
  • Sample/ standard preparation:

    • Internal standard solution preparation: Take _______(0.3 ml) of THF in a ______(100 ml) volumetric flask, dilute up to mark with ________Water.
    • Stock solution: Take _______( 5ml) of Isopropyl alcohol in a ______(100 ml) volumetric flask, dilute up to mark with ________Water.
    • Prepare the final concentration of Isopropyl alcohol as per the following procedure.
    • 2500 ppm: Take _______(2.5 ml of stock solution ) and _______(5.0 ml ) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
    • 4000 ppm: Take _______(4 ml of stock solution ) and _______(5.0 ml ) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF. 5000 ppm: Take _______(5 ml of stock solution ) and _______(5.0 ml ) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
    • 6000 ppm: Take _______(6 ml of stock solution ) and _______(5.0 ml ) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
    • 7500 ppm: Take _______(7.5 ml of stock solution ) and _______(5.0 ml ) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
    • Pipette _____ (5 ml) of each linearity solution in three HSS vial respectively. Seal the vial with Septa followed by the crimp.
  • Calibration procedure:

    • Set up the instrument as per the conditions.
    • Inject the samples three times from each solution and calculate the ratio of IPA and THF. Record the observation in table-1
    • Calculate the %RSD of the mean ratio. It should not be more than 5.0 %.
    • Plot a linearity curve of concentrations Vs corresponding mean area, using the least square method. Calculate the correlation coefficient (r) and record the observations in given table-2.
  • Observation table:

Table-1

Sr. No. IPA Conc. Area of IPA

(X)

Area of THF

(Y)

Ratio
1 2500 ppm
Mean
RSD%

 

Sr. No. Ethyl Alcohol

Conc.

Area of IPA

(X)

Area of THF

(Y)

Ratio
2 4000 ppm
Mean
RSD%

 

3 5000 ppm
Mean
RSD%

 

4 6000 ppm
Mean
RSD%

 

5 7500 ppm
Mean
RSD%

 

Table-2

Sr. No IPA std

Conc.

Mean Area ratio Acceptance Criteria
1 2500 ppm Correlation coefficient should be NLT 0.990
2 4000 ppm
3 5000 ppm
4 6000 ppm
5 7500 ppm

 

  • Acceptance criteria:
    • Correlation coefficient should be NLT 0.990
  • Result: ………………………………(Satisfactory/Dose not satisfactory)

 

CALIBRATION OF AUTO INJECTOR BY LINEARITY  MEASUREMENT

( BY USING CAPILLARY COLUMN)

Instrument Gas Chromatography
Make Instrument code
Model
  • Calibration requirements:

    • DMF (AR grade) Code no : _______________ Valid up to: _______________
    • Ethyl Acetate             Code no : _______________ Valid up to: _____________
  • System requirements:

Table: 1

Parameter Test Conditions Applied Conditions
Column : DB-624

(30m X 0.53 mm X FT 3.0 mm)

Carrier gas : Nitrogen
Flow rate : 4.50 ml/ min
Column Temperature : 45 oC – 4 min Rate : 15 oC/min

230 °C – 7 min

 

Injector Temperature : 180 oC
Detector Temperature : 230 oC
Hydrogen gas flow : 40 ml/min.
Air-gas flow : 400 ml/min.
Makeup : 30 ml/min with nitrogen
Split ration: 1:50 1:50
Run time : 23 min.

 

Table: 2

HSS Conditions:
Injector Temperature : 85 oC
GC Cycle time : 30 min
Transfer line temperature: 90 oC

 

Oven temperature: 35 oC
Carrier pressure: 14 psi
Pressurize time: 3.0 min
Withdrawn time: 0.2 min
Thermostat time: 20 min
  • Sample/ standard preparation:

    • Standard stock solution :Take _______(2.0 ml) of Ethyl acetate in ______(100ml) volumetric flask, dilute up to mark with ________DMF.
    • Standard solution Preparation :Take _______(1.0 ml) of stock solution in ______(100ml) volumetric flask, dilute up to mark with ________DMF.
    • Pipette out 5.0ml of this solution into HSS vial and seal the vial with septa followed by a magnetic cap.
  • Calibration procedure:

    • Set up the instrument as per the conditions.
    • Inject the sample 0.5ml , 1 ml , 1.5 ml , 2.0ml , and 2.5 ml.
    • Inject each volume three times (triplicate) and record the observation in the table.
    • Plot the graph volume Vs. mean area of each volume, and calculate the correlation coefficient (r). It should not be less than 0.990.

 

CALIBRATION OF AUTO INJECTOR BY LINEARITY MEASUREMENT

( BY USING CAPILLARY COLUMN)

  • Calibration requirements:
    • DMF (AR grade) Code no : _______________ Valid up to: _______________
    • Ethyl Acetate             Code no : _______________ Valid up to: _______________
  • System requirements:

Table: 1

Parameter Test Conditions Applied Conditions
Column Porapak Q – 80 / 100#, 2M,

S.S. Column.

Carrier gas Nitrogen
Flow rate 30 ml/min
Injector temperature 250°C
Detector FID
Column temperature 180°C
Detector temperature 250°C
Injection volume 0.5ml, 1ml, 1.5ml, 2.0ml and 2.5ml
Run time : 20 min.
  • Sample/ standard preparation:
    • Take _______(5.0 ml) of ethyl alcohol in ______(50ml) volumetric flask, dilute up to mark with ________DMF. (Stock solution-1)
    • Take _______(5.0 ml) of Isopropyl alcohol in ______(50ml) volumetric flask, dilute up to mark with ________DMF. (Stock solution-2)
    • Take _______(3.0 ml of stock solution 1) and _______(5.0 ml of stock solution 2) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF. (6.0 ppm ).
  • Calibration procedure:
    • Set up the instrument as per the conditions.

Related: HPLC Calibration

    • Inject the sample 0.5m l, 1m l, 1.5m l, 2.0m l, and 2.5m l every three times. (calculate the ratio of IPA and Ethyl alcohol. Record the observation in table-1.
    • Calculate the %RSD of the mean ratio for each injection volume. It should not be more than 5.0 %.
    • Plot a linearity curve of Injection volume Vs corresponding mean area, using the least square method. Calculate the correlation coefficient (r) and record the observations in the table.

 

CALIBRATION TEMPLATE FOR COLUMN OVEN TEMPERATURE MEASUREMENT

Instrument Gas Chromatography
Make Instrument code
Model
  • Calibration requirements:
    • Temperature Indicator Device Code no : ___________Valid up to: _____________
  • System requirements for GC Calibration:

Table: 1

Parameter Test Conditions Applied Conditions
Column Oven Temperature Temperature:

A). 35°C

B)  100°C

C)  150°C

D)  200°C

E)   250°C

F)   300°C

Temperature:

A). ………..°C

B)  ………..°C

C)  ………..°C

D)  ………..°C

E)   ………..°C

F)   ………..°C

 

  • Sample/ standard preparation: NA
  • GC Calibration procedure:

    • Insert the probe of the temperature indicator device in the column oven and close it.
    • Set the column oven temperature 35°C and keep idle to stabilize the temperature and take the reading
    • Repeat the same procedure for 35°C, 100°C, 150°C, 200°C, 250°C and 300°C record the observations in the table.
  • Observation table:

Table-1

Sr. No Set Temperature Observed Temperature Acceptance criteria:
1. 35°C  

 

Difference between the set temperature and observed temperature should be ±2°C

2. 100°C
3. 150°C
4. 200°C
5. 250°C
6. 300°C
  • Acceptance criteria:
    • Difference between the set temperature and observed temperature should be ± 2°C
  • Result: ………………………………(Satisfactory/Dose not satisfactory)

 

GC CALIBRATION TEMPLATE FOR VIAL TEMPERATURE MEASUREMENT

Instrument Gas Chromatography
Make Instrument code
Model
  • Calibration requirements:
    • Temperature Indicator Device Code no : ___________Valid up to: _____________
  • System requirements: NA

 

  • Sample/ standard preparation: NA
  • Calibration procedure:
    • Set the vial temp at 35°C and keep idle to stabilize the temperature and take the reading
    • Repeat the same procedure for 90°C and 130°C. Record the observations in the table.
  • Observation table:

Table-1

 

Sr. No.

 

Set Temperature

Observed Temperature Acceptance criteria:
Temperature indicative device G.C
1. 50°C Difference between observed temperature from Temperature indicative device and G.C should be ±2°C
2. 90°C
3. 130°C
  • Acceptance criteria:
    • Difference between the set temperature and observed temperature should be ± 2°C
  • Result: ………………………………(Satisfactory/Dose not satisfactory)

 

RelatedCalibration of UV Spectrophotometer

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Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an ultimate pharmaceutical blogging platform. Email: [email protected]

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