Validation of gc instrument

PRESENTATIONON
QUALIFICATIONOFGASCHROMATOGRAPHY
KARNATAKA COLLEGE OF
PHARMACY, BANGALORE-64
{Approved by AICTE, PCI }
{Affiliated to RGUHS University}
1
SUBMITTED BY
SANTHOSH KUMAR T S
M. Pharmacy 1ST SEM
Ph ANALYSIS
FACILITATED TO
Dr. C. SRIDHAR SIR
HEAD OF THE
DEPARTMENT
PHARMACEUTICAL
ANALYSIS

Contents :
• Introduction
• Instrumentation of GC
• Qualification of GC
• GC applications in
pharmaceutical analysis

CHROMATOGRAPHY
•Is a technique used to separate and identify the components of a mixture.
•Chroma -"color" and graphein - "to write”
PRINCIPLE
•Physical method of separation that distributes components to separate between
two phases moves in a definite direction.
• Substances are separated based on their differential distribution between two
phases.
•Substances will move with the mobile phase at different rate depending upon their
Partition or Distribution co- efficients.
CHROMATOGRAPHY TERMS
• Chromatograph - equipment that enables a sophisticated separation
• EX. Gas chromatography or Liquid chromatography
•Eluent - Fluid entering column/ solvent that carries the analyte.
• Eluate - Mobile phase leaving the column.

GAS CHROMATOGRAPHY
•Gas chromatography (GC) is a widely used technique for separation & analysis
of gaseous & volatile substances which are difficult to separate & analyze.
•In performing gas chromatographic separation, the sample is vaporized &
injected onto the head of a chromatographic column.
•Elution is brought about by the flow of an inert gaseous mobile phase.
•In GC gas as a moving phase is passed through a column containing solid
adsorbent or liquid adsorbent. Thus adsorption or partition is possible.
Based on stationary phase used in column, G.C is of 2 types :
a. Gas solid chromatography (GSC)
b. Gas liquid chromatography (GLC).
a. GSC : Mobile phase – gas
Stationary phase – solid
b. GLC : Mobile phase – gas
Stationary phase – liquid

Criteria for compounds to be analyzed by GC :
 Volatility
 Thermo stability
 The analyte should have a measurable vapor pressure at the
temperature employed.
Instrumentation :
It consists of:
1. Carrier gas tank
2. Flow regulators
3. Sample injection system
4. Column & Stationary phase
5. Detectors

CARRIER GAS
• The carrier gas must be chemically inert. Commonly used gases include
nitrogen, helium, argon, oxygen, air .
• The choice of carrier gas is often dependant upon the type of detector which
is used.
• The carrier gas system also contains a molecular sieve to remove water and
other impurities.
FLOW REGULATORS:
• Used to maintain the uniform pressure and flow rate.
• Rotameter
• Soap Bubble Flow Meter
SAMPLE INJECTION PORT
• For optimum column efficiency, the sample should not be too large.
• The most common injection method is where a micro syringe is used to
inject sample through a rubber septum into a flash vaporizer port at the
head of the column.
• The temperature of the sample port is usually about 50°C higher than
the boiling point of the least volatile component of the sample.

INJECTION DEVICES:
Gas: Valve Devices
Liquid: Loop Devices
Solid: Dissolve the substance in suitable solvent and then inject.
TEMPERATURE CONTROLLING DEVICES:
The devices are very important role playing.
• Pre heaters (for vapour formation)
• Thermostatically controlled oven .
COLUMNS : Two general types of columns are encountered in GC
A. Packed columns
Have inside diameter of about 2 to 4mms.
B. Capillary columns or open tubular columns.
Inside diameter --- 1mm.
Capillary columns are of 2 basic types :
a) Wall-coated open tubular columns (WCOT)
b) Support-coated open tubular columns (SCOT).
DETECTOR
• Flame ionization (FID)
• Thermal conductivity detector (TCD)
• Electron capture (ECD)

Rotameter Soap bubble flow meter

QUALIFICATION
DEFINATION
Action of proving and documenting that equipment or ancillary systems are properly
installed, work correctly, and actually lead to the expected results.
• Qualification is part of validation,
but the individual qualification steps alone do not constitute process validation.
TypesOf Qualification:
1. Designqualification
2. Installational qualification
3. Operational qualification
4. Performance qualification
1. DESIGN QUALIFICATION (DQ)
• It describe the user requirements and defines the functional and operational
specifications of the instrument.

2. INSTALLATIONQUALIFICATION(I.Q):
• The purpose of I.Q is to check the installation site/ environment, confirms
equipment specifications and verifies the condition of installed equipment.
3. OPERATIONALQUALIFICATION(O.Q):
• O.Q includes procedures and documentation of O.Qof analytical instrument.
• When all procedures are executed and all items pass the inspection, it is verified
that the system operates to satisfy the intended purpose.
4.PERFORMANCEQUALIFICATION(PQ):
• The objective is to ensure that the instrument isperforming within specified limits.
• Hence documented verification that the equipment and ancillary systems, as
connected together, can perform effectively and reproducibly based on the
approved process method and specifications.

Design qualification Installation qualificationOperational qualification Performance qualification

CALIBRATION
•It is set of operation which is established under specified conditions.
•It is necessary to ensure the accuracy of the data produced during the process
•Calibration is the processby which ensure that an instrument readings are accurate the
reference to establish standard. Calibration is performed by using primary standard. It is
done to check the zero error deflection by using standard reference.

NEED FOR CALIBRATION:
• With a new instruments
• When a specified time period is elapsed
• When a specified usage (operating hours) has elapsed
• When an instrument has had a shock or vibration which potentially may have put it out
of calibration
• Sudden change in weather
• Whenever observation appears questionable

QUALIFICATIONOFGC EQUIPMENT
Introduction
• The present document is the core document of “Qualification of Equipment”, and
it should be used in combination with it when planning, performing and
documenting the GCequipment qualification process.
• The core document contains the general introduction and the LevelIand IIof
qualification, common to all type of instruments, and the present annex contains
GC instrument-related recommendations on parameters to be checked and the
corresponding typical acceptance limits, as well as practical examples on the
methodology that can be used to carry out thesechecks.
• The tests proposed in the LevelIIIand IVof qualification are based on an overall
approach, in which several parameters are checked at the same time in a
combined test procedure, to obtain information on the overall system
performance (e.g. peak area precision, retention time precision, temperature
programme reproducibility, etc).
• Nevertheless, it should be noted that it is also acceptable to check these
parameters individually by using other well- defined procedures

Level I.Selection Of Instruments &
Suppliers
• Atlevel Iof the qualification of a gc equipment(selection of
instruments and suppliers)
• It is recommended to select a manufacture of gc that can satisfy the
needs of the laboratory and worksunder ISO9001 certification

• It is recommended to check all requirements set during the selection
of the instrument, and calibration should be performed before
putting into service by an accredited external service supplier, or
• Internally by appropriately qualified personnel, using certified reference
buffers according to an approved procedure.
• It establishes that instrument is properly installed in the selected
environment and that environment should be suitable for operation of the
instrument.
• Run of test samples verifies correct installation of all modules, electrical
and fluid connections
Level IIof Equipment Qualification:
Installation and release for use

COLUMN OVEN
Temperature Range (Ambient + 10°C) ~ 400°C (using liquid CO2 gas: -50°C - 400°C)
Dimensions 250 (W) x 360 (H) x 175 (D) mm
Oven Capacity 15.8L
Temperature Accuracy Set value (K) ±1% (calibration at 0.01°C increments)
Temperature Deviation 2°C max. (on 200mm dia. circumfrence 30 mm from rear)
Temperature Variation Coefficient 0.01°C/°C
Temperature Program Steps Up to 20 (cooling program possible)
Programmed rate setting range -250°C ~ 250°C/min
Total time for all steps 9999.99 minutes max.
Linear Heating Range
30°C/min up to 150°C
7°C/min up to 400°C (at 25°C ambient temperature)
Cooling Rate 300°C ~ 50°C in 6 min max. (at 25°C ambient temperature)
Columns Accepted
Capillary Columns: 2
Packed columns for GC14B: 4 (Glass columns:2)

SAMPLE INJECTION UNIT
Temperature Range Up to 400°C
Heating Settings 1°C steps
No. of units installed simultaneously Up to 3 units
Sample injection unit types Dual packed, single packed, split/splitless
CARRIER GAS FLOW CONTROLLER
For Packed/Dual
Flow rate setting range 0 ~ 100mL/min
Programmable Steps 7
Programmed rate setting range -400 ~ 400mL/min
Correction Function
Maintains column flow rate during column
oven heating

For Capillary Split / Split less
(Split /Split less injection mode)
Pressure Setting Range 0 ~ 970kPa
Programmable Steps 7 (pressure-decreasing program possible)
Programmed rate setting range -400 ~ 400kPa/min
Total flow rate setting range 0 ~ 200 mL/min
Correction Function
Maintains column average linear velocity during
column oven heating (for capillary only)
(Pressure mode direct injection)
Pressure Setting Range 0 ~ 970kPa/min
Programmed rate setting range -400 ~ 400kPa/min
(Flow-rate mode direct injection)
Flow rate setting range 0 ~ 1200mL/min
Programmed rate setting range -400 ~ 400mL/min
Correction Function
Maintains column average linear velocity during
column oven heating (for capillary only)

Instrument module Parameter to be
checked
Typical tolerance limit
1. Inlet system 1.1 Injector leak test
2.Pressure/flow accuracy
and stability
3.Repeatability of injection
(overall test 1) - In split mode
-In split less mode
4.Injector temperature
accuracy and stability
5. Carry-over (overall
test 3)
Pressure drop ≤ 15 kPa within
5 minutes
Covered by overall test 1
RSD ≤ 3.0%
RSD ≤ 3.0%
Covered by overall test 2
≤ 0.2%
2.1. Repeatability of oven
temperature characteristics
Covered by overall test
2
Level III.Periodic and motivatedinstrument
Checks Examples of requirements for GCinstruments with FID
2. Oven

Instrument module Parameter to be checked Typical tolerance
limit
3. FID detector 1.Linearity (overall test 3)
2.Constant detector
response
3. Noise
3.3. Drift
r 2 ≥ 0.999
Covered by overall
test 1 or 2
See Annex I
See Annex I

• Practical examples of tests and their associated tolerance limits for several
parameters related to the performance of the different modules of a GCare
presented below.
• These examples can be considered by the OMCLsas possible approaches to perform
the LevelIIIof the equipment qualification process: “Periodic and motivated
instrument checks”.
• Several tests are proposed to check various parameters at the same time
(overall tests).
• In order to runthe tests in a more economical way, other suitable solutions can be
used, as for example, the “Grob Test”mixture, available from different suppliers
(e.g. Alltech, Sigma, Thames Restek).
• This commercial solution should be appropriate to the column
material used.
• It is recommended to run the overall tests by using always the same test column,
exclusively dedicated to qualification purposes, to guarantee reproducible
conditions.

1.1. Injector leak test method
If it is not specified by the instrument manufacturer, the leak test is carried
out according to the procedure laid down in the instrument manual or by
the built in automatic leak check procedure of the instrument.
Otherwise use the test described below:
• Disconnect the column from the injector and closethe injector outlet
with a sealed cap.
•Close the septum purge and the bypass.
• Adjust the flow and pressure controller to the maximal
•possible value of the pressure gauge.
•Adjust the flow controller to zero.
•Read the pressure after 1 minute and record thevalue.
•Record the pressure after 5 minutes.
• Limits:
•Pressure drop ≤ 15 kPa within 5 minutes.
1. Inletsystem The following tests are
proposed for the
periodic and motivated check of the GCInlet System.

1.2. Inlet pressure / flow accuracy and stability
• Adirect measurement of these parameters was not deemed practical or
necessary, but the optimal conditions of flow/pressure can be verified by the
overall test1.
Limits: Refer to overall test 1.
1.3. Repeatability of injection
• The verification of this parameter is covered by the overall test 1.
• This test is to be performed in both split and split lessmode.
1.4. Injector temperature accuracy and stablity
•Due to the fact that the temperature cannot be reliably measured without
opening and modifying the systemand due to the difficulties of introducing a
probe inside this module, the verification of this parameter is considered to be
covered by the overall test 2.

1.5 Injector carry over
• After having injected the solutions for the linearity test of the FIDdetector, in
increasing order, inject the blank and measure the peaks that correspond
to the major peaks (= analytes ) in the linearity solutions.
• The verification of this parameter is covered by the overall
test 3.
2. OVEN
Repeatability of the oven temperature
characteristics
• Due to the fact that the temperature cannot be reliably measured without
opening and modifying the system conditions and that even when
introducing a probe inside the oven, its location would not reflect the real
temperature conditions at all points, the verification of this parameter is
covered by the overall tests 2Aand 2B.
Limits:Refer to overall test 2.

3. FIDdetector
The following tests are proposed for the periodic and motivated check of the
GCFID detector.
3.1.FIDdetectorlinearity
• Increasing amounts of analyte are injected and a linear response
should be obtained.
• The verification of this parameter is covered by the overall test 3.
Limits:Refer to overall test 3.
3.2. Constant FIDdetectorresponse
• The proper and reproducible functioning of the FIDcan be demonstrated by
checking the peak areas obtained from a pre- defined standard solution.
• The verification of this parameter is covered by the overall test 1 or 2.
Limits: Refer to overall test 1 or 2.

3.3. FID detector noise and drift
• if the instrument has a built-in automatic system for the verification of the
noise and drift, follow the manufacturer’s instructions and apply the defined
acceptance criteria.
• Otherwise, use the test described below:
Settings:
• Column installed
• Suitable flow, depending on column length/diameter
• No injection
• Oven temperature: 40°C
• Detector on and heated at working temperature (270- 300°C)
Method:
• After stabilisation of the system, record the signal for 15 minutes.
• Noise: evaluate 10 periods of 1 minute and calculatethe mean value.
• Drift: Evaluate the slope of the baseline over the 15 minutes.

Limits:
• The acceptance criteria for these parameters have to be chosen in
accordance with the instrument vendor’s instructions and the
intended use of the instrument.
• If no instructions are given, the user has to pre-define these acceptance
criteria by taking into account the previous experience and the intended
use of the instrument.
• No fixed values can be pre-defined in this guideline due to the high variety
of integration systems used and consequently the acceptance criteria may be
expressed in different units (voltage, current, arbitrary units per time).

Level IV.In-use instrument checks Examples of
requirements for GCinstruments with FID
Parameterto be checked Typical tolerance limit
1. System suitability check for the
method
According to Ph. Eur. or MAH
dossier or validatedin-house
method
2. Peak area precision RSD≤ 3.0% unless otherwise
prescribed*
3. Retention time repeatability RSD≤2.0%
4. Sensitivity (where relevant, e.g.
for related substances tests)
According to Ph. Eur. or MAH
dossier or validated in-house
method
*This is to be defined in conjunction with the target
concentration of the analyte

Do you think that
the instrument is Qualified
?

OVERALLTEST 1
• The overall test 1 covers the followingparameters:
- Pressure/flow accuracy and stability in the inlet system: Retention time repeatability
- Repeatability of injection: peak area precision
a) In split mode
b) In split less mode
The test may be combined with overall test 3.
a) Split mode:
• Test solution: 1-octanol in n-hexane 1%(v/v).
Settings:
• Column: SPB-1 (30m x 0.32mm ID x 0.25µm film)
• Carrier gas: He
• Velocity: 25cm/sec
• Split: 1:100
• Injection: 1µl
• Injector temperature: 220°C
• Oven temperature: 100°C isotherm
• Detector temperature: 300°C

• Runtime: 8 min
• Retention time of 1-octanol: about 5 min
b) Split less mode:
• Stock solution: 1-octanol in n-hexane 1%(v/v)
• Test solution: Dilute 10 ml of the stock solution with n- hexane to 100 ml
(corresponds to 1µl/ml of 1-octanol in n- hexane)
Settings:
• Column: SPB-1, 30m, 0.32mm ID, 0.25µm film
• Carrier: He
• Velocity: 30cm/sec
• Split less injection: purge valve closed during 2 min
• Injection: 0.2µl of the test solution
• Injector Temperature: 220°C
• Oven Temperature: Initial 60°C for 4 min, 15°C/min. up to 135°C, final
time 1min
• Detector temperature: 300°C
• Runtime: 9.5 min
• Retention time of 1-octanol: about 8 min

Method:
• Carry out 6 consecutive injections of the test solution
and calculate the RSD of the different peak areas and
retention times.
Limits:
• Retention time repeatability: the RSDof the retention
times should be ≤ 2.0%
• Peak area precision (split and split less mode): the RSDof
the peak areas should be ≤ 3.0%

OVERALL TEST 2
The overall test 2 covers the following parameters:
Injector, oven and detector temperature accuracy and stability: retention time
repeatability
Test solution:
• 0.035 ml 1-octanol
• 0.035 ml 2-octanone
• 0.035 ml 2,6-dimethylanilin
• 0.035 ml n-tridecane
• 0.035 ml n-tetradecane
• 35 mg n-eicosane
• Dissolved in 50 ml Dichloromethane
Settings:
• Column: SPB-1 (30m x 0.32mm ID x 0.25µm film)
• Carrier gas: Helium
• Velocity: 25 cm/s
• Split: 1:100

Injection volume: 1 µl
Injector temperature: 220°C
Detector: FID
Detector temperature: 300°C
Gradient program : 60°C (4 min), 5°C/min, 270°C (3 min)
Method:
 Inject the solution twice and calculate the relative retention
times in relation to n-eicosane (RRT=1)
 The following table shows the approximately expected
relative retention times.
Limits:
The RSDof each RRTfrom two consecutive injections should be ≤ 1.0%

OVERALLTEST3
• This test is a modified version of the overall test 1 to be used for the verification
of:
- Detector linearity: linearity of the areas recorded
- Injector carry-over: area recorded in the blank run
• It is described for both split and split less mode and may be combined with
overall test 1.
Split mode:
Prepare further reference solutions by diluting the test solution as described
below.
Settings:
see overall test 1
• Injection sequence:
• 5.0 ml of the test solution diluted to 25.0 ml with n-hexane(2 µl/ml): 2
injections
• Test solution: 1-octanol in n-hexane 1%(v/v)
• 10.0 ml of the test solution diluted to 25.0 ml with n-hexane (4 µl/ml): 2
injections
• 15.0 ml of the test solution diluted to 25.0 ml with n-hexane (6 µl/ml): 2

Split less mode:
•Stock solution: 1-octanol in n-hexane 1%(v/v)
•Test solution: Dilute 10 ml of the stock solution with n- hexane to 100 ml
(corresponds to 1µl/ml of 1-octanol in n- hexane).
• Prepare further reference solutions by diluting the test solution
with n-hexane.
• Settings: see overall test 1
Injection sequence:
• 5.0 ml of the test solution diluted to 25.0 ml with n-hexane (0.2 µl/ml):
2injections
2 injections
2 injections
2 injections
• If combined with overall test 1 for repeatability: test solution (1µl/ml): 6
injections

Limits:
• Linearity: coefficient of correlation of the calibration line obtained
with the reference solutions and the test solution: r 2 ≥ 0.999.
• Carry-over: the percentage of the peak area corresponding to the
analyte in the blank solution should be ≤ 0.2% of the peak area of this
analyte in the chromatogram obtained with the solution with the highest
concentration within the sequence.

GC applications in pharmaceutical
analysis

GC applications in pharmaceutical analysis
a. It is a simple & inexpensive method, generally efficient with regard to
separation.
b. The technique has a very high resolution power.
c. Small sample is needed – μLs.
d. Sensitivity of detection is very high (PPB or Picograms).
e. The speed of analysis is fast.
e. Qualitative & quantitative analysis at a time is possible. The area produced
under each peak is proportional to that concentration.
f. Gas chromatograms are widely used to establish the purity of organic
compounds.
Contaminants, if present, are revealed by the appearance of additional peaks.
g. The areas under these peaks provide rough estimate of the extent of
contamination.
E.g. : Gas chromatography is used to determine the identity & composition of
propellants that are widely used in aerosols.
h. The purity & acceptability of the propellants is tested with respect to moisture,
halogen & non volatile residue determination by using GC.

Precautions:
•Do not heat the G.C. column without passing carrier gas through the
column.
• Check the leakage of Hydrogen gas before putting the flame on.
•Ensure that no air bubble is entrapped in syringe while injecting the sample.
• If found any difficulty in operating the instrument or G.C. Solution software,
and then refer the instrument manual.
• Inform to the Department Head, If any abnormality is observed while
working with Gas chromatograph.

REFERENCES
• Ph.Eur.2.2.35 chromatography; Gas chromatography
• Guidance on equipment qualification of instrumental analysis
• Journal of Perkin Elmer life &analytical science
• Validation and Calibration of Analytical Instruments, D. Gowrisankar et al / J
Biomed Sci and Res., Vol 2 (2), 2010,89-99 ( page no 94 to 96) .

Software installation
• Connect the serial or USB cable to your computer and the GC. The serial
port connection is on the left-hand side of the GC, and the USB connection
is on the right-hand side.
• Locate your copy of the Peak Simple software just inside the front cover of
your SRI manual. Insert the CD or floppy disks into your computer’s
appropriate drive.
• Double click on “My Computer,” then on the appropriate drive to open it.
Double click on the “setup.exe” icon.
• For USB, refer to “Installing the USB Drivers for Model 302 USB Peak
Simple Data System”
• Double-click on the Peak Simple icon to launch the program.
• Open the Edit menu and choose Overall. In the dialog box that pops up,
enter the number of the COM port to which you have connected the GC.

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