1.0
Title: Standard operating procedure for
operation and calibration of Gas Chromatography.
2.0 Purpose: To describe
the procedure for the operation and calibration of Gas Chromatography
3.0
Scope: This procedure shall be applicable for operation and calibration of Gas Chromatography at Fermenta Biotech Limited.
4.0 Responsibility: Research
Associate/ Research Scientist.
1.1
OPERATING PROCEDURE:
1.1.1
Open the valve of Hydrogen, Oxygen and Nitrogen
cylinder and check the pressure.
1.1.2
Switch on instrument and system. Click the ‘Empower’
icon on desktop of computer. Enter user name and password.
1.1.3
‘Empower pro’ displays. Click on ‘Browse project’
and select desire project. Browse project window will open.
1.1.4
New instrument method preparation. In Browse
project window click on ‘File’ ⟶new method⟶ click
on ‘instrument method’ and make changes as per your specification and save the instrument
method.
1.1.5
New method set preparation. In Browse project
window click on ‘File’ ⟶new method⟶ click
on ‘Method set’ and add update above instrument method in method set.
1.1.6
Click on ‘Run sample’ icon in browse project and
select system FBL_GC_04 System. New window will appear. For conditioning the
column go on INSTRUMENT METHOD window select desire method then click on setup.
1.1.7
New sample set preparation Click on the File ⟶new
sample set method ⟶Empty prepare sequence as per
specification and save it.
1.1.8
Start sample set by click on green Run icon.
After
completion of sample set, process the chromatogram and take print in suitable
report template.
1.2
CALIBRATION PROCEDURE :
1.2.1
CARRIER GAS FLOW FOR CAPILLARY COLUMN:
1.2.1.1 PROCEDURE:
5.3.1.1.1
Connect the digital flow Meter to the Detector
outlet port.
5.3.1.1.2
Set the Carrier Gas (Nitrogen) flow and wait till
it reaches the set flow.
5.3.1.1.3
Note the observed flow.
5.3.1.1.4
Record the Results in protocol.
1.2.1.2 FLOW
CALIBRATION:
1.
1.0 ml/min
2.
3.0 ml/min
3.
5.0
ml/min
1.2.1.3 ACCEPTANCE
CRITERIA: (Set
Flow ± 10.0 %)
1.2.2
TEMPERATURE CALIBRATION FOR OVEN:
1.2.2.1 PROCEDURE:
5.3.2.1.1
Put the Probe in Oven Close the Door attached
with calibrated digital Thermometer.
5.3.2.1.2 Set
the Oven Temperature and wait till it Stabilize.
5.3.2.1.3 Note
the observed Temperature.
5.3.2.1.4 Record
the Results in protocol.
1.2.2.2
TEMPERATURE CALIBRATION:
1.
35.0°C
2.
100.0°C
3.
150.0°C
4.
200.0°C
5.
250.0°C
1.2.2.3 ACCEPTANCE
CRITERIA: (Set
temperature ± 2.0°C)
1.2.3
TEMPERATURE CALIBRATION FOR INJECTOR
1.2.3.1 PROCEDURE :
5.3.3.1.1
Insert the Probe in Injector from Top Side
piercing Glass wool attached with calibrated digital Thermometer.
5.3.3.1.2
Set the Injector Temperature and wait till it Stabilize.
5.3.3.1.3
Note the observed Temperature.
5.3.3.1.4
Record the Results in protocol.
1.2.3.2
TEMPERATURE CALIBRATION:
1)
100.0°C
2)
150.0°C
3)
200.0°C
1.2.3.3
ACCEPTANCE CRITERIA: (Set temperature ± 2.0°C)
1.2.4
TEMPERATURE CALIBRATION FOR DETECTOR
1.2.4.1
PROCEDURE :
5.3.4.1.1 Attached
the Probe in Detector end Side with calibrated digital Thermometer.
5.3.4.1.2 Set
the Detector Temperature and wait till it Stabilize.
5.3.4.1.3 Note
the observed Temperature.
5.3.4.1.4 Record
the Results in protocol.
1.2.4.2 TEMPERATURE
CALIBRATION:
1)
200.0°C
2)
250.0°C
3)
300.0°C
1.2.4.3 ACCEPTANCE
CRITERIA:( Set temperature ± 10.0°C)
1.2.5
SYSTEM PRECISION :
1.2.5.1
GC CONDITIONS :
|
Detector |
: |
FID |
|
Column |
: |
DB-624
Capillary column, 30M, 0.53 mm. id, 3.0mm
film thickness or HP-5 Capillary column, 30M, 0.32mm id, 0.25mm or CP SIL
5 WCOT Capillary column, 30M,032mm,ID3.0mM |
|
Injector
temp |
: |
200°C |
|
Detector
temp |
: |
250°C |
|
Oven
Temp |
: |
40°C
hold for 2 minutes, first ramp at 5°C/min to 60 °C hold for 0 Minutes, second
ramp at 50°C/min to 220 °C hold for 1 Minutes. |
|
Run
time |
: |
10
min |
|
Carrier
gas |
: |
N2 |
|
Inject
volume |
: |
1.0
ml |
|
Split |
: |
1:50 |
|
Flow
rate |
: |
Nitrogen 4.5Kg/cm2 Hydrogen 4.0Kg/cm2 Zero air 4.0Kg/cm2
|
1.2.5.2 REAGENT REQUIRED:
1)
Ethanol
2)
Methanol
1.2.5.3 STANDARD
PREPARATION :
4.0%
w/v Methanol in ethanol (100.0 %)
1.2.5.4 PROCEDURE :
5.3.5.4.1
Inject 1.0 ml of
Ethanol as blank.
5.3.5.4.2 Inject
1.0 ml of Standard
preparation.
5.3.5.4.3 Repeat
Standard injection 6 times.
5.3.5.4.4 Determine
the RSD of retention time and area of peak due to Methanol.
1.2.5.5 ACCEPTANCE
CRITERIA:
RSD of area should not be more
than 15.0% & RSD of retention time should not be more than 1.0%.
1.2.6
DETECTOR LINEARITY:
1.2.6.1 PREPARE THE SOLUTION IN FOLLOWING CONCENTRATION:
5.3.6.1.1
50% Solution Take 2.0 g Methanol in 100 ml.
5.3.6.1.2
75% Solution Take 3.0 g Methanol in 100 ml.
5.3.6.1.3
100% Solution Take 4.0 g Methanol in 100 ml.
5.3.6.1.4
125% Solution Take 5.0 g Methanol in 100 ml.
5.3.6.1.5
150% Solution Take 6.0 g Methanol in 100 ml.
5.3.6.1.6
Inject 1.0 ml Ethanol
as blank.
5.3.6.1.7
Inject 1.0 ml of
Standard preparation of in triplicate 50% 75%, 100%, 125% and 150%
concentration solution.
5.3.6.1.8
Calculate the average area of each concentration
and record the correlation coefficient (r2).
1.2.6.2 ACCEPTANCE
CRITERIA:
1.2.6.2.1 RSD
for Each Standard area should not be more than 15.0% & RSD of Retention
time should not be more than 1.0%.
1.2.6.2.2
Correlation coefficient (r2) should be
more than 0.99.
1.2.7
INJECTOR LINEARITY:
1.2.7.1 PROCEDURE:
5.3.7.1.1 Inject
1 ml Ethanol as blank.
5.3.7.1.2 Inject
Above 100 % Prepare Solution Each
0.5 ml, 1.0 ml,
1.5ml, 2.0 ml,
2.5ml, Standard preparation
in triplicate.
5.3.7.1.3 Calculate
the average area of each Injected Standard and record the correlation Co-efficient
(r2).
1.2.7.2 ACCEPTANCE
CRITERIA:
1.2.7.2.1 RSD
for Each Standard area should not be more than 15.0% & RSD of Retention
time should not be more than 1.0%.
1.2.7.2.2 Correlation
coefficient (r2) should be more than 0.99.
Note- After calibration, record the
results in calibration record of Gas Chromatography as per Format No.:
FBL/F/QC/082 and after analysis fill
the entries in logbook for instrument usage as per Format No. FBL/F/QC/078
(Refer SOP/QC/023).
1.2.8
CALIBRATION
STATUS DISPLAY: After completion of calibration display the calibration
status as per current version format: FBL/F/QC/453
(Refer SOP/QC/064).
1.2.9
BREAKDOWN ACTION:
1.2.9.1 Inform to the QC in charge and to the
Service Engineer.
1.2.9.2 Affix the label “OUT OF ORDER”.
1.2.10 SERVICING & MAINTENANCE:
1.2.10.1 The
instrument is carried under annual maintenance contract.
6.0
ABBREVIATIONS:
|
CCIF |
: |
Change control initiation form |
|
cm |
: |
centimeter |
|
°C |
: |
degree Celsius |
|
g |
: |
gram |
|
GC |
: |
Gas chromatography |
|
ml |
: |
milli liter |
|
min |
: |
minutes |
|
mm |
: |
milli meter |
|
mm |
: |
Micrometer |
|
N2 |
: |
Nitrogen |
|
QA |
: |
Quality Assurance |
|
QC |
: |
Quality Control |
|
SOP |
: |
Standard operating procedure |
|
w/v |
: |
weight / volume |
7.1
REFERENCES:
GC
Operation Manual
8.0 RECORDS : Calibration format.
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