Instrument Calibration Protocol for Gas Chromatography – Shimadzu
| INSTRUMENT | Gas chromatography (Shimadzu) |
| Instrument code no. | |
| Ref. SOP No. | |
| Make / Model No. | |
| Calibration Frequency | Half Yearly “-15 days”. |
| Date of Calibration | |
| Next Due Date |
1. Flow rate calibration:
Flow Meter ID: ___________________]
| Set flow rate (mL / min) | Observed flow rate (mL /min) – 1 | Observed flow rate (mL /min) – 2 | Observed flow rate (mL /min) – 3 | Mean flow rate (mL / min) | Acceptance criteria (mL / min) |
| 5 mL | 4.75 to 5.25 | ||||
| 20 mL | 19.0 to 21.0 | ||||
| 30 mL | 28.5 to 31.5 | ||||
| 40 mL | 38.0 to 42.0 | ||||
| 50 mL | 47.5 to 52.5 |
Done By: Checked By:
2. Verification of temperature:
a. Column oven:
Instrument used for Calibration:
Instrument No. / Code No.
Calibration Date
Next Due Date of Calibration
| Sr. No. | Set Temperature | Temperature observed on display | Temperature observed on Calibrator | Limits |
| 30 °C | 30 °C ± 2 °C | |||
| 50 °C | 50 °C ± 2 °C | |||
| 100 °C | 100 °C ± 2 °C | |||
| 150 °C | 150 °C ± 2 °C | |||
| 200 °C | 200 °C ± 2 °C | |||
| 250 °C | 250 °C ± 2 °C | |||
| 350° C | 350 °C ± 2 °C |
b. Injector:
| Sr. No. | Set Temperature Injector A | Set Temperature Injector B | Temperature observed on display Injector A | Temperature observed on display Injector B | Limits |
| 1. | 50 °C | 50 °C | 50 °C ± 2 °C | ||
| 2. | 100 °C | 100 °C | 100 °C ± 2 °C | ||
| 3. | 150 °C | 150 °C | 150 °C ± 2 °C | ||
| 4. | 200 °C | 200 °C | 200 °C ± 2 °C | ||
| 5. | 250 °C | 250 °C | 250 °C ± 2 °C | ||
| 6. | 300 °C | 300 °C | 300 °C ± 2 °C | ||
| 7. | 350° C | 350° C | 350 °C ± 2 °C |
c. Detector:
| Sr. No. | Set Temperature FID A | Set Temperature TCD B | Temperature observed on display FID A | Temperature observed on display TCD B | Limits |
| 1. | 50 °C | 50 °C | 50 °C ± 2 °C | ||
| 2. | 100 °C | 100 °C | 100 °C ± 2 °C | ||
| 3. | 150 °C | 150 °C | 150 °C ± 2 °C | ||
| 4. | 200 °C | 200 °C | 200 °C ± 2 °C | ||
| 5. | 250 °C | 250 °C | 250 °C ± 2 °C | ||
| 6. | 300 °C | 300 °C | 300 °C ± 2 °C | ||
| 7. | 350 °C | 350 °C | 350 °C ± 2 °C |
Done By: Checked By:
3. Auto Injector and Detector Calibration :
a. Calibration of FID by Linearity Measurement. (By Using Capillary column):
| Solvent Name | Batch No. | Calibration Standard No. | Purity in % | Use Before |
| Tetrahydrofuran | ||||
| Isopropyl alcohol | ||||
| Dimethylformamide |
(a) THF Internal standard (IS) solution preparation: Take ______ (1.0 gm) of ____________________ Tetrahydrofuran in a ________ (50ml) volumetric flask containing 10ml of DMF and dilute up to mark with __________DMF.
(b) Sample preparation
Stock solution: Weigh accurately _______( 2.0 g) of Isopropyl alcohol standard in a ______(50 ml) volumetric flask containing about 10 ml of DMF, dilute up to mark with ________DMF.
Further dilute this to prepare the different concentration as below,
- 800 ppm: Take _______(1.0 ml of stock solution) and _______(2.0 ml) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
- 1600 ppm: Take _______(2.0 ml of stock solution) and _______(2.0 ml) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
- 2400 ppm: Take _______(3.0 ml of stock solution) and _______(2.0 ml) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
- 3200 ppm: Take _______(4.0 ml of stock solution) and _______(2.0 ml) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
- 4000 ppm: Take _______(5.0 ml of stock solution) and _______(2.0 ml) of THF (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________DMF.
(c) Chromatographic condition :
| Parameter | Test Conditions | Applied Conditions |
| Column No. | ||
| Column | AT-5 (30m x 0.53mm x 5.0µ) or Equivalent | |
| Oven temperature | 100°C-2 min.- @ 30°C/min. 250°-1min. | |
| Detector | Flame Ionization detector (FID) | |
| Detector Temperature | 270°C | |
| Head pressure | 30 kpa | |
| Injection volume | 1.0 µl | |
| Split ratio | 1:40 | |
| Injector temperature | 200°C | |
| Run time | 8 min. |
(d) 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 below.
- Calculate the %RSD of mean ratio. It should not be more than 10.0%.
- Plot a linearity curve of concentrations Vs corresponding mean area ratio, using least square method. Calculate the correlation coefficient (r) and record the observations in a table below.
(e) Observation Table
| Concentration of IPA | Area of IPA X | Area of THF Y | Area Ratio X/Y | Mean Area Ratio and % RSD |
| 800 ppm | ||||
| — | ||||
| — | ||||
| 1600 ppm | ||||
| — | ||||
| — | ||||
| 2400 ppm | ||||
| — | ||||
| — | ||||
| 3200 ppm | ||||
| — | ||||
| — | ||||
| 4000 ppm | ||||
| — | ||||
| — |
Correlation Coefficient _____________ (Limit: NLT 0.990)
(f) Result: Complies / does not comply
Done By: Checked By:
b. Calibration of FID by Linearity Measurement. (By Using Pack column):
| Solvent Name | Batch No. | Calibration Standard No. | Purity in % | Use Before |
| Ethyl alcohol | ||||
| Isopropyl alcohol |
| Water | Ultra Purified (Milli-Q) |
(a) IPA Internal standard (IS) solution preparation: Weigh accurately _____________ (5.0 g) of Isopropyl alcohol standard in _______ (50 ml) volumetric flask containing 10 ml of water, dilute up to mark with ___________ water.
(b) Sample preparation
Stock solution: Weigh accurately _____________ (5.0 g) of ethyl alcohol standard in _______ (50 ml) volumetric flask containing 10 ml of water, dilute up to mark with ___________ water.
Further dilute this to prepare the different concentration as below,
- 2000 ppm: Take _______(1.0 ml) of stock solution and _______(5.0 ml) of IPA (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________water.
- 4000 ppm: Take _______(2.0 ml) of stock solution and _______(5.0 ml) of IPA (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________water.
- 6000 ppm: Take _______(3.0 ml) of stock solution and _______(5.0 ml) of IPA (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________water.
- 8000 ppm: Take _______(4.0 ml) of stock solution and _______(5.0 ml) of IPA (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________water.
- 10000 ppm: Take _______(5.0 ml) of stock solution and _______(5.0 ml) of IPA (IS) in ______(50 ml) volumetric flask, dilute up to mark with ________water.
(c) Chromatographic condition :
| Parameter | Test Conditions | Applied Conditions |
| Column No. | ||
| Column | Porapak Q – 80 / 100#, 2M, S.S. Column. | |
| Carrier gas | Nitrogen | |
| Flow rate | 50 ml/min | |
| Detector | FID | |
| Column temperature | 180°C | |
| Injector temperature | 250°C | |
| Detector temperature | 250°C | |
| Injection volume | 1.0 µl | |
| Run Time | 15.0 min |
(d) Calibration procedure:
- Set up the instrument as per the conditions.
- Inject the samples three times from each solution and calculate the ratio of
Ethyl alcohol and IPA. Record the observation in table below.
- Calculate the %RSD of area ratio at each level. It should not be more than 10.0 %.
- Plot a linearity curve of concentrations Vs corresponding mean area ratio, using least square method. Calculate the correlation coefficient ® and record the observations in given table.
(e) Observation Table:
| Concentration of Ethyl alcohol | Area of Ethyl alcohol X | Area of IPA Y | Area Ratio X/Y | Mean Area Ratio and % RSD |
| 2000 ppm | ||||
| — | — | |||
| — | — | |||
| 4000 ppm | ||||
| — | — | |||
| — | — | |||
| 6000 ppm | ||||
| — | — | |||
| — | — | |||
| 8000 ppm | ||||
| — | ||||
| — | ||||
| 10000 ppm | ||||
| — | — | |||
| — | — |
Correlation Coefficient: _____________ (Limit: NLT 0.990)
(f) Result : Complies / Does not comply
Done By: Checked By:
c. Calibration of Auto Injector by Linearity Measurement. (By Using FID with Capillary column):
| Solvent Name | Batch No. | Calibration Standard No. | Purity in % | Use Before |
| Tetrahydrofuran | ||||
| Dimethylformamide |
(a) Standard stock solution: Weigh accurately _______(1.0 g) of Tetrahydrofuran (THF) standard in ______(50ml) volumetric flask containing 10 ml of DMF, dilute up to mark with ________DMF.
Standard / Sample solution Preparation: Take _______(2.0 ml) of stock solution in ______(50ml) volumetric flask containing 10 ml of DMF, dilute up to mark with ________DMF.
(b) Chromatographic condition :
| Parameter | Test Conditions | Applied Conditions |
| Column No. | ||
| Column | AT-5 (30m X 0.53mm X 5.0µ) or Equivalent | |
| Oven temperature | 100°C – 2 min. – @ 30°C/min.-250°-1min. | |
| Detector temperature | 270°C – Flame Ionization detector (FID) | |
| Carrier gas | Nitrogen | |
| Head pressure | 30 kpa | |
| Injection volume | 0.5µl, 1.0µl, 2.0µl, 5.0µl | |
| Split ratio | 1:40 | |
| Injector temperature | 200°C | |
| Run time | 8 min. |
(c) Calibration procedure:
- Inject the sample with injection volume 0.5µl, 1.0µl, 2.0µl, 5.0µl.
- Inject each volume three times and record the observation in table.
- Calculate the %RSD of area at each volume. It should not be more than 10.0 %
- Plot the graph of injection volume vs. mean area of each volume, and calculate the correlation coefficient (r). It should not be less than 0.990.
(d) Observation Table
| Injection Volume (µl) | Area of Tetrahydrofuran | Mean Area | % RSD (NMT 10.0 %) |
| 0.5 µl | |||
| — | |||
| — | |||
| 1.0 µl | |||
| — | |||
| — | |||
| 2.0 µl | |||
| — | |||
| — | |||
| 5.0 µl | |||
| — | |||
| — |
Correlation Coefficient _________________ (Limit: NLT 0.990)
(e) Result: Complies / Does not comply
Done By: Checked By:
d. Calibration of Auto Injector by Linearity Measurement. (By Using FID with Pack column):
| Solvent Name | Batch No. | Calibration Standard No. | Purity in % | Use Before |
| Isopropyl alcohol |
| Water | Ultra Purified (Milli-Q) |
(a) Sample / Standard stock solution:
Weigh accurately _______(5.0 g) of Isopropyl alcohol standard in ______(50ml) volumetric flask containing 10 ml of water, dilute up to mark with ________water.
Take _______(3.0 ml of stock solution) in ______(50 ml) volumetric flask, dilute up to mark with ________water.
(b) Chromatographic condition:
| Parameter | Test Conditions | Applied Conditions |
| Column No. | ||
| Column | Porapak Q – 80 / 100#, 2M, S.S. Column. | |
| Carrier gas | Nitrogen | |
| Flow rate | 50 ml /min | |
| Detector | FID | |
| Column temperature | 180°C | |
| Injector temperature | 250°C | |
| Detector temperature | 250°C | |
| Injection volume | 0.5µl, 1.0µl, 2.0µl, 5.0µl | |
| Run time | 15.0 min. |
(c) Calibration procedure:
- Set up the instrument as per the conditions.
- Inject the sample 0.5µl, 1.0µl, 2.0µl, 5.0µl each three times. (Calculate the area of IPA. Record the observation in below table.
- Calculate the %RSD of area for each injection volume. It should not be more than 10.0 %.
- Plot a linearity curve of Injection volume Vs corresponding mean area, using least square method. Calculate the correlation coefficient ® and record the observations in given below table.
(d) Observation Table:
| Injection Volume (µl) | Area of Isopropyl alcohol | Mean Area | % RSD (NMT 10.0 %) |
| 0.5 µl | |||
| — | |||
| — | |||
| — | |||
| 1.0 µl | |||
| — | |||
| — | |||
| — | |||
| 2.0 µl | |||
| — | |||
| — | |||
| — | |||
| 5.0 µl | |||
| — | |||
| — | |||
| — |
Correlation Coefficient _______________ (Limit: NLT 0.990)
(e) Result: Complies / Does not comply
Done By: Checked By:
e. Calibration of TCD by Linearity Measurement. (By Using Pack column):
| Solvent Name | Batch No. | Calibration Standard No. | Purity in % | Use Before |
| Isopropyl alcohol | ||||
| Acetone |
(a) Standard preparation:
Prepare the different Linearity preparations by mixing of IPA and Acetone (internal standard) as specified in the below table.
| Linearity Preparation | Volume of IPA (ml) | Volume of Acetone (ml) IS |
| 1 | ___________(1 ml) | ___________(2 ml) |
| 2 | ___________(2 ml) | ___________(2 ml) |
| 3 | ___________(3 ml) | ___________(2 ml) |
| 4 | ___________(4 ml) | ___________(2 ml) |
| 5 | ___________(5 ml) | ___________(2 ml) |
(b) Chromatographic condition :
| Parameter | Test Conditions | Applied Conditions |
| Column No. | ||
| Column | 10% PEG, 2 meter x 1/8” diameter, mesh rang 80-100 | |
| Carrier gas | Helium / Hydrogen | |
| Flow rate | 45 ml /min | |
| Column temperature | 55°C | |
| Injector temperature | 100°C | |
| Detector temperature | 160°C | |
| Current | 80 mA | |
| Injection Volume | 1 µl | |
| Run time | 10 min. |
(c) Calibration procedure:
- Set up the instrument as per the conditions.
- Inject each Linearity preparation in triplicate; calculate the mean area ratio of IPA to Internal standard Acetone.
- Calculate the %RSD of triplicate area ratio of IPA to Internal standard Acetone. It should not be more than 10.0%.
- Plot the graph of Concentration in ml Vs Corresponding mean area ratio of IPA /Acetone (IS) and calculate the Correlation coefficient (r) and it should not be less than 0.990.
- Record the all observations in the following table.
(d) Observation Table:
| Volume of IPA (ml) | Area of IPA X | Area of Acetone Y | Area Ratio X/Y | Mean Area Ratio and % RSD |
| 1.0 ml | ||||
| — | — | |||
| — | — | |||
| 2.0 ml | ||||
| — | — | |||
| — | — | |||
| 3.0 ml | ||||
| — | — | |||
| — | — | |||
| 4.0 ml | ||||
| — | — | |||
| — | — | |||
| 5.0 ml | ||||
| — | — | |||
| — | — |
Correlation Coefficient ____________________ (Limit: NLT 0.990)
(e) Result: Complies / Does not comply
Done By: Checked By:
Remarks: The instrument is calibrated & qualified / Out of calibration & not qualified for use.
| — | Calibrated By | Checked By | Approved By |
| Signature | |||
| Date | |||
| Name | |||
| Department | Quality Control | Quality Control | Quality Control |