CALIBRATION OF GAS CHROMATOGRAPHY WITH EMPOWER SOFTWARE

• PROCEDURE:
• CALIBRATION OF FLOW ACCURACY:
• Select detector flow and set it to the specified value listed in the result Table (Annexure-I)
• Ensure that all other detector flows are “OFF”.After completing appropriate preparation steps for the calibration mass flow meter being used ( i.e. Gas selection, purge, and zero).
• Connect the flow meter to the detector exit and measure flow.
• Allow time for flow meters” reading to stabilize.
• Record the flow value displayed on GC front panel in the results table.
• Record the measured flow value displayed on flow meter in results table (Annexure-I).
• Calculate the flow difference between displayed value and measured value and record the results table (Annexure-I).
• Turn flow OFF.Repeat the steps above for each remaining detector flow.After all flows have been measured.
• Remove the flow Cap from the detectors base.
• Acceptance criteria: the difference between measured flow and set flow should not be more than ± 10%.
• Record the details in annexure-I.
• Calibration schedule: Once in six months and after any major maintenance Job.

• CALIBRATION OF HEADSPACE SAMPLER WITH FID DETECTOR:
• Fix the column from capillary inlet to FID.
• Chromatographic parameters

• Column: Fused silica column 30 m long 0.32 m internal diameter with 1.8 micrometers film of 6% Cynopropyl Phenyl. 94% dimethyl polysiloxane.

• Inlet temperature 120°C
• Carrier gas: Helium
• Oven temperature: 60°C (Iso thermal)
• Flow mode: Constant Flow
• Detector Temperature: 200°C
• Column Flow: 0.8 ml/min
• Hydrogen Flow: 45 ml/min
• Air flow :450ml/min
• Make up flow :30ml/min

• Mode: Split
• Split ratio: 1:50
• Stop time: 15 min.

Head space parameters

• Incubation temperature: 80°C
• Agitator Speed: 500 rpm
• Incubation Time:20 min
• Agitator on time: 10 sec
• Agitator off time: 10 sec
• Flush Time: 2.0 min
• Syringe temperature: 90 °C
• Pressurization Time: 0.08 min
• Injection Volume: 1000 µL
• Fill speed: 500 µl /sec
• Fill stroke: 1
• Injection speed: 500 µL/sec
• GC Cycle Time :20 min

• Sample preparation: prepare 0.4 % v/v of Ethanol in water.
• Prepare six vials each contains 2 ml test solution. Seal the vial with PTFE septa and cap, place in the headspace analyzer. Run the experiment.
• Retention time for ethanol shall be about 4.0 min.
• Acceptance criteria:

• RSD for peak area should not be more than 5.0 %
• RSD for retention time should not be more than 0.5%
• Record the details in Annexure-II.

• Calibration Schedule: Once in six months and after any major maintenance Job.
• CALIBRATION OF CAPILLARY INLET WITH FID:
• Fix the column for capillary inlet to FID.
• Chromatographic parameters

• Column: HP –5, 30 mx0.32 mm ID x 0.25µ
• Inlet temperature: 250°C
• Carrier gas: Helium/Nitrogen
• Flow mode: Constant pressure
• Inlet pressure column 25 psi
• Detector Temperature: 300°C
• Injection Volume: 1 µL
• Column Flow: 0.8 ml/min
• Hydrogen Flow: 45 ml/min
• Mode: Split less
• Air Flow: 450 ml/min
• Makeup Flow: 7 ml/min
• Run time: 8.5 min
• Oven temperature Program: Initially held at 60°C then raised to 90°C at 25°C/min. Then again increased to 170°C at 15°C /min. Hold at 170° C for 2 min.

• Procedure: Inject the sample 6 times as per the procedure and calculate the RSD for retention times and peak areas of individual peaks.
• Acceptance criteria:

• RSD for peak area should not be more than 5.0 %
• RSD for retention time should not be more than 0.5%
• The average area of individual peaks should be greater than the value mentioned in the annexure-III.
• Record the details in calibration record in Annexure-3

• Calibration Schedule: Once in six months and after any major maintenance Job.
• CALIBRATION OF DETECTOR LINEARITY FOR FID:
• Chromatographic parameters:

• Column: Fused silica column 30 m long 0.32 m internal diameter with 1.8µ
• Injection temperature 190°C
• Carrier gas: Helium/Nitrogen
• Flow mode: Constant Flow
• Detector Temperature: 250°C
• Hydrogen Flow: 45 ml/min
• Air Flow: 450ml/min.
• Make flow :30ml/min
• Column Flow: 1mL/min
• Mode: Split
• Split ratio: 1:50
• Injection Volume: 1000 µL
• Run time 20 min.
• Oven temperature Program: Initially held at 70 °C for 10 min. Then raised to 260°C at 70°C/min and Hold at 260° C for 5 min.

Head space parameters

• Incubation temperature: 120°C
• Agitator Speed : 500 rpm
• Incubation Time:30 min
• Agitator on time : 10 sec
• Agitator off time: 10 sec
• Flush Time : 2.0 min
• Syringe temperature : 130 °C
• Pressurization Time: 0.08 min
• Injection Volume :1000 µL
• Fill speed : 500 µl /sec . Fill stroke: 1
• Injection speed: 500 µL/sec .
• GC Cycle Time: 25 min

Preparation IPA stock solution :

• Take 500µl of IPA into a 25ml flask having about 20 ml of NN-DMF and then dilute it to volume with NN-DMF and mix well.

Preparation of solutions for Linearity.

S. No	Volume of STD stock (ml)		Dilute to 10 ml of NN- DMF
01	1		10
02		2	10
03		3	10
04		4	10
05		5	10
06		6	10

• Pipette 5 ml of each solution into individual vials. Fit them with a septum and crimp cap and then seal it and inject.
• Calculate the coefficient of correlation.

• Acceptance criteria: Coefficient of correlation NLT 0.99.
• Calibration: Once in six months and after any major maintenance Job.
• Record the Details in annexure-IV.

CALIBRATION OF COLUMN OVEN TEMPERATURE TEST:

• Set the oven temperature to 35°C and wait for at least 5 min to stabilize the oven, take the temperature reading from the data logger and record the temperature.
• Set the oven temperature to 100 °C and wait for at least 5 min, after the oven gets Stabilized record the data logger reading in the annexure.
• Set the oven temperature to 230 °C and wait for at least 5 min, after the oven gets Stabilized record the data logger reading in the annexure.

• Acceptance criteria:
• The difference at 35°C should not be more than 2°C from the set value.
• The difference at 100°C should not be more than 4°C from the set value.
• The difference at 230° C should not be more than 5°C from the set value.

• Record the details in Annexure-V.

• Calibration schedule: Once in six months and after any major maintenance Job

• VERIFICATION OF DETECTOR AND INJECTOR TEMPERATURE:
  • Detector:
• Set the detector temperature to 100°C and wait for at least 15 min and record the Temperature in Annexure -VI.
• Repeat the above step for remaining temperatures 200°C and 300°C.
  • Injector:
• Set the injector temperature to 100°C and wait for at least 15 min and record the temperature in Annexure-VI.
• Repeat the above step for remaining temperatures 200°C and 300°C.
  • Acceptance criteria:
• The difference at 100°C should not be more than 2°C from the set value.
• The difference at 200°C should not be more than 2°C from the set value.
• The difference at 300°C should not be more than 2°C from the set value.
  • Calibration schedule: Once in six months and after any major maintenance Job.

• ANNEXURES: 

ANNEXURE NO.	TITLE OF ANNEXURE
Annexure-I	Calibration record of Flow accuracy
Annexure-II	Calibration record for Headspace sampler With FID
Annexure-III	Calibration record for capillary inlet with FID
Annexure-IV	Calibration record of Detector Linearity for FID
Annexure-V	Calibration record of Column oven temperature Test
Annexure-VI	Verification of Detector and Injector temperature

Annexure-I
CALIBRATION RECORD OF FLOW ACCURACY

Annexure-II
CALIBRATION RECORD FOR HEADSPACE SAMPLER WITH FID

Annexure-III
CALIBRATION RECORD FOR CAPILLARY INLET WITH FID

Annexure-IV
CALIBRATION RECORD OF DETECTOR LINEARITY FOR FID

Annexure-V
CALIBRATION RECORD OF COLUMN OVEN TEMPERATURE

Annexure-VI
VERIFICATION OF DETECTOR AND INJECTOR TEMPERATURE