SOP for Calibrating the Gas Chromatography.

1.0     Objective:

                 The objective of this Calibration procedure is to lay down the procedure for Calibrating the Gas Chromatography.

2.0      Scope:

           This procedure covers calibration of Gas Chromatography.

3.0     Responsibility:

            Jr. Research Officer, Research Officer: Responsible for calibration of the instrument as per procedure.

          Head of Department: Responsible for calibration, timely as per schedule.

          QA Officer/QA Manager: Review the records and governing the document

4.0       Procedure

4.1     Calibration with Packed column:

4.1.1     Calibration of Flow rate.

Ø  Open the column oven compartment.

Ø  Install the properly Packed column.

Ø  Connect soap film flow meter to the detector port outlet with the help of Teflon or

             rubber tube.

Ø  Fill the pipette bulb partially with a soap solution and attach to the bottom of the flow

             meter.

Ø  Open the knob of carrier gas (N2) and set up proper pressure (i.e. 500 kpa or 5 Kg/cm2) in

             carrier gas pressure controller.

Ø  Check for leaks at each and every point of attachment using soap solution.

Ø  Open the knob of carrier gas flow controller & allow carrier gas to flow through

             corresponding digital flow control.to set the flow N2 gas.

Ø  Gentle squeeze the bulb to force a soap film up into the gas       stream. Start the stopwatch as

            soon as the film reaches to zero ml mark. Stop the watch                   when the film reaches to 30 ml

            marking. Note down the time require to reach the film from               0 to 30 ml mark. Calculate the

            flow rate using the following formula.

            Flow rate ml/min  = 60 X 30.0/60 (Dist. Between two                      points   3.0 ml)

            Time taken in seconds

Ø  Similarly calibrate the flow rate after the time interval of 20 Sec, 40 Sec, & 60 Sec and

             find out the difference between the readings.

Ø  Acceptance criteria: observed flow rate of the equipment should be within ±2ml/min of set flow rate.

         Operate the instrument as per respective procedure.

4.1.2 Prepare suitable solvent mixture and use specified chromatographic conditions as below :

(a)        Preparation of Solvents Mixture:

              Take each 1.0 gm of Isopropyl Alcohol, Isooctane and                       Chlorobenzene in 100 ml

              volumetric flask and make volume with Methanol.                            (Solution B).

(b)          Chromatographic Status:

  Oven Temperature  1   : 160° C     Time 1 Rate 1 :  10° C /Oven Temperature  2   : 240° C       Time 2   :  9.0 minute         Rate 2 :   0° C / minute

                Injector Temperature   :  260° C                

                Detector Temperature  :  280° C

                Carrier flow                 :  30 ml/minute       

                Injection Volume         :  1.0 µl        

               Column                         :  Packed Column (Porapack Q)  – A porous polymer based on 2, 6 –diphenyl – p - phenylene oxide.

              Length   :  2 meter             Diameter   : 3.2 mm OD      
      

(c)          Linearity of Detector Calibration:   
           

Ø  Prepare different concentration solution of solvent mixture solution (B). Pipette 1 ml, 2 ml, 3 ml, 4 ml and 5 ml of Solution B in different 10 ml volumetric flask and make volume with methanol. They will be indicated 1000 ppm, 2000 ppm, 3000 ppm, 4000 ppm and 5000 ppm solutions. 

Ø  Inject 1.0 µl of all different concentration solution, and note down the response of area. Calculate the correlation coefficient of the response of all solvents should not be less than 0.99. 

Ø  Enter the details of calibration as given in Annexure – 1.

(d)          Precision of Detector Calibration:

Ø  Inject 1.0 µl five replicate injection of 5000 ppm concentration solution of solvent mixture.

Ø  Note down the response of area and retention time, calculate the RSD of the area and retention time should not be more than 3.0 %.

Ø  Enter the details of calibration as given in Annexure – 1

4.2            Calibration Frequency:

                  The instrument will be calibrated once in three months (Quarterly).

4.3            Column Calibration:

Ø  Preparation of Solvent mixture solutions and use chromatography conditions as given in Gas Chromatography system calibration foe respective column.

Ø  Calculate the area/ area ratio and the relative retention time (RRT) of first two peaks by considering the last peak as internal standard.

Ø  Calculate the RSD of ratios, and relative retention times. The obtained RSD value should not be more than 3.0 %.

Ø  Calculate the resolution and number of theoretical plates. The Resolution between two peaks should not be less than 2.0 and theoretical plate should not be less than 35 % of Initial Value.

Ø  Record the details in the column calibration record, as given in Annexure - 2.

Ø  Calibration Frequency:

                  Quarterly

5.0             Documentation:

5.1  Annexure - 1    Gas Chromatography Calibration Record   XXX/FRM/000

5.2   Annexure - 2    Gas Chromatography Column Performance Record      XXX/FRM/000  

6.0             History of Revision:

Revision No.	Effective Date	Revision details	Reason for revision

Annexure-1

Calibration of Gas Chromatography	Page no. : 01 of 03
1.0    Calibration of carrier gas flow rate
Name of Equipment       : Gas Chromatography        Model No: Instrument ID No.    :                                                  Make :
Required Gas flow: 30.0ml/min Sr.No Set Flow Rate Observation-1 Observation-2 Observation-3 1 20 Second. 2 30 Second 3 30 Second Remark                            : Complies / Does not Complies Calibration on                 :                                     Calibration Due Date: Calibrated By                   :                                     Checked  By          :
SOP No.: XXX/SOP/000                                                                      Format No. : XXX/FRM/000

CALIBRATION OF GAS CHROMATOGRAPHY	Page no. : 02 of 03
(1)  Detector Calibration Linearity:
Date of Calibration   :                                                  Due date for next Calibration : Instrument ID No.    :                                                  Model No.  : Make :
(1)                        :                                                    Make          :              (Solvent  a)                                                         Batch No. :        (2)                         :                                                     Make         :              (Solvent  b)                                                         Batch No.  :                   (3)                          :                                                    Make         :              (Solvent  c)                                                         Batch No.  :                   Preparation of Solvents Mixture :               Take (1 gm)______  gm Isopropyl Alcohol, (1 gm)_______ gm Isooctane and (1 gm)_______  gm Chlorobenzene in 100 ml volumetric flask and make volume with Methanol. (Solution B). Pipette 1 ml, 2 ml, 3 ml, 4 ml and 5 ml of Solution A in different 10 ml volumetric flask and make volume with Methanol. They will be observed 1000 ppm, 2000 ppm, 3000 ppm, 4000 ppm and 5000 ppm solutions.           CHROMATOGRAPHIC STATUS  : Oven Temperature  1   : _____ ° C         Time 1   : ______minute        Rate 1 : _____ ° C / minute Oven Temperature  2   :______   ° C        Time 2   :______minute        Rate 2 :_____ ° C / minute Injector Temperature   :______° C         Detector Temperature : ______ ° C Carrier flow       : ______ ml/minute        Injection Volume : ______ Column ID No. : Column             :                                                         Manufacturer  :                                                          Length              :                               Diameter   :                                    Film Thickness  :
Calibrated by :                                                                   Checked by :

CALIBRATION OF GAS CHROMATOGRAPHY										Page no. : 03 of 03
(1)  Detector Calibration Linearity
Date of Calibration   :                                                  Due date for next Calibration :
Sr. No.	Concentration		Area						Remarks
			Isopropyl Alcohol		Isooctane		Chlorobenzene
1	1000 ppm								Satisfactory / Not Satisfactory
2	2000 ppm
3	3000 ppm
4	4000 ppm
5	5000 ppm
Correlation Coefficient (Limit : NLT 0.99)
(2)     Precision of Detector Calibration:
Preparation of Solvents Mixture :      Pipette 5 ml Solution B in a 10 ml volumetric flask and make volume with Methanol (5000 ppm). Inject 5 replicate injections.
Sr. No.	Area					Retention Time
	Isopropyl Alcohol	Isooctane		Chlorobenzene		Isopropyl Alcohol		Isooctane			Chlorobenzene
1
2
3
4
5
Average
RSD Limit
	NMT 3.0 %					NMT 3.0 %
Remarks : Satisfactory / Not Satisfactory
Calibrated by :                                                                                      Checked by :

Annexure – 2

Gas Chromatography Column Performance Record	Page No. : 01 of 02
Date of Calibration   :                                                  Due date for next Calibration : Column No. :  Column        : Manufacturer  :          Length          :                            Diameter   :                                   Film Thickness  :
(1)                        :                                                   Make          :              (Solvent  a)                                                         Batch No. :        (2)                      :                                                       Make         :              (Solvent  b)                                                         Batch No.  :                   (3)                       :                                                      Make         :              (Solvent  c)                                                         Batch No.  :                   Preparation of Solvents Mixture :      Take (1 gm)_____ gm Isopropyl Alcohol, (1 gm)______   gm Isooctane and (1 gm)____gm Chlorobenzene in 100 ml volumetric flask and make volume with Methanol. (Solution B). Pipette 1 ml, 2 ml, 3 ml, 4 ml and 5 ml of Solution A in different 10 ml volumetric flask and make volume with methanol. They will be observed 1000 ppm, 2000 ppm, 3000 ppm, 4000 ppm and 5000 ppm solutions.           CHROMATOGRAPHIC STATUS  : Oven Temperature  1   :               ° C            Time 1   :  -                                   Rate 1 :        ° C / minute Oven Temperature  2   :              ° C            Time 2   :           minute                 Rate 2 :        ° C / minute Injector Temperature   :               ° C            Detector Temperature :              ° C Carrier flow       :           ml/minute        Injection Volume :  Column             :
Calibrated by :                                                                   Checked by :
SOP.NO.: XXX/SOP/000          Format No. : XXX/FRM/000

Gas Chromatography Column Performance Record										Page No. : 02 of 02
Date of Calibration   :          Due date for next Calibration : Column No. :  Length          :                            Diameter   :                                   Film Thickness  :
Sr. No.	Relative Ratio					Relative Retention Time
	Isopropyl Alcohol			Isooctane		Isopropyl Alcohol		Isooctane
1
2
3
4
5
Average
RSD Limit
	NMT 3.0 %					NMT 3.0 %
Sr. No.		Theoretical Plate					Resolution
		Isopropyl Alcohol	Isooctane		Chlorobenzene		Isopropyl Alcohol		Isooctane
1
2
3
4
5
Average
Limits		NLT 350	NLT 770		NLT 770		NLT 2.0		NLT 2.0
Remarks : Satisfactory / Not Satisfactory
Calibrated by :                                              Checked by :