PENGENALAN ASAS

GAS
CHROMATOGRAPHY
OLEH:
ISMAIL IBRAHIM PPN, PJK
Kenapa sampel yang bercampur
boleh diasingkan?
Arah aliran

Dasar sungai
 Apa perbezaan nya?

Ringan
Berat
Interaction is
difference : gravity
 Bagaimana pengasingan boleh
dilakukan?
• Pengasingan dapat dilakukan oleh turus/ column.

Turus/
column
packing material, 3-5um
 BAGAIMANA PEMISAHAN
TERJADI DALAM KROMATOGRAFI

• Pemisahan berlaku hasil dari interaksi

sebatian sasaran (“target compound”
dengan fasa pegun (“stationary phase”)
yang berbeza di antara satu dengan lain.
Proses kromatografi melibatkan
dua fasa

• Fasa Pegun (“Stationary Phase”)

Fasa Bergerak (“Mobile Phase”)

 Kenapa sampel yang bercampur
dapat dipisahkan ?

• Kerana sifat tiap sampel (compound)

adalah berbeza.
 PEMISAHAN
• Pemisahan terhasil dari
persaingan daya molekul-
molekul fasa gerak dan fasa
pegun menarik atau menolak
molekul sampel
KEGUNAAN KROMATOGRAM
1.Kualitatif
• Masa Penahanan (“Retention Time”) (Rt) puncak
selalunya tetap/konstan di dalam kromatografik yang
sama dan dengan itu boleh digunakan untuk
mengenalpasti sesuatu komponen.
2.Kuatitatif
• Luas Puncak (“Peak Area”) adalah setanding
“proportional” dengan kompenen yang disuntik dan
dengan itu boleh digunakan dalam kiraan kandungan
kompenon di dalam sampel.
 pengenalan

Gas Chromatography (GC) melibatkan

sampel yang meruap dan disuntik pada
kepala turus kromatografi (Injector).
Sampel ini alirkan melalui turus dengan
fasa geraknya adalah gas. Pada turus
terdapat fasa pegun.Pada peringkat
akhirnya adalah pengesan.
schematic diagram of a gas chromatograph
 Apa yang terjadi di GC?
• Penyuntik (injector)- sampel atau larutan
akan meruap.
• Ketuhar (oven)- tempat pemanasan pada
suhu tertentu
• Turus (column)- pemisahan melalui takat
didih dan sebatian berkutub.
• Pengesan (detector)- mengesan sebatian
 komponen alatan

Gas pembawa (carrier gas)

- Gas pembawa terdiri daripada gas nitrogen,
helium, argon dan karbon dioksida.
- Pemilihan gas pembawa bergantung kepada jenis
pengesan yang digunakan. gas pembawa mesti
melalui molecular seieve bagi menapis bahan tidak
tulen dan juga untuk membuang kandungan wap
air.
 Bekalan gas

• Tiub Bekalan gas

– Tiub yang digunakan terdiri daripada tiub kuperam
atau tiub besi tahan karat dan yang sudah
dibersihkan.
– Tiub plastik tidak boleh digunakan kerana
tiub plastik yang boleh ditembusi oleh gas, oksigen
atau gas lain yang mudah meruap yang boleh
diserap ke dalamnya.
 Ketulenan dan kadar
pengaliran gas pembawa
1. Gas pembawa yang mempunyai ketulenan yang tinggi dapat
menambahkan masa jangka hayat turus dan meningkatkan
kesensitifan pengesan
• Penapis bahan tak tulen hendaklah dipasang pada laluan gas.
• Hidrogen > 99.99%
• Helium > 99.995%
• Nitrogen > 99.999%
• Kadar pengaliran gas yang dicadangkan;
– Turus pack 40~60 ml/min
– Turus kapilari 0.5~20 ml/min
 Bahagian suntikan
(Sample injection port)

• Bagi mendapatkan kecekapan turus yang

optima, sampel hendaklah tidak terlalu
banyak.
• Suntikan yang perlahan bagi sampel yang
banyak menyebabkan jalur melebar dan
resolusi berkurangan
 CARA SUNTIKAN

Suntikan boleh digunakan dalam dua cara:

• split
• splitless.
Split/Splitless Flow System
Split Flow Diagram: Pre-Injection
SEPTUM

(SEPTUM)
2 mL/min PURGE VENT
TOTAL FLOW

50 mL/min

SPLIT VENT

48 mL/min (INLET)
PURGE VALVE
47.4 mL/min

= CARRIER GAS

LINER

0.6 mL/min
SEAL
COLUMN
Split Flow Diagram: Sample/Carrier
Mixing
(SEPTUM)
PURGE VENT
TOTAL FLOW

= CARRIER GAS SPLIT VENT

= SAMPLE MOLECULES
= SOLVENT MOLECULES (INLET) PURGE VALVE

For the concept of SPLIT RATIO to

be valid, the sample (solvent +
analyte) must be mixed with the
carrier gas to give a homogeneous
mixture.

At the bottom of the injection port a

small part of this mixture will transfer
to the column, while the bulk of the
mixture will leave the chromatograph
via the SPLIT VENT.
COLUMN
Split Flow Diagram: Liner Overload
(SEPTUM)
PURGE VENT

TOTAL FLOW

= CARRIER GAS SPLIT VENT

= SAMPLE MOLECULES
= SOLVENT MOLECULES (INLET)
PURGE
VALVE

A large injection of a solvent with a

large expansion volume can cause
an overload of the injection port
liner.

This can result in loss of sample out

the PURGE VENT as well as
contamination of the in-coming
carrier gas line.

COLUMN
 LINERS

choosing the right liner for

the job
 Liner tirus bahagian bawah
• Liner yang tirus pada bahagian bawah
dapat mengurangkan cecair yang disuntik
daripada terkena bahagian bawah
penyuntik.
• Jika Glass wool yang diletakan longgar,
kemudian pemanasan dilakukan,ia nya
dapat menghalang daripada “Glass Wool”
tersebut bergerak kebawah liner yang
mana boleh menyebabkan tekanan tinggi
didalam “Injector Port”
 Liner tirus bahagian atas
Liner yang tirus pada bahagian atas
dapat mengurangkan kesan Flashback.
Ini terjadi apabila jumlah cecair
berlebihan yang disuntik ke dalam
liner menyebabkan isipadu gas yang
meruap lebih besar daripada isipadu
cecair yang disuntik.
 SPLIT LINERS

Straight Straight Fixed Inverted Baffle

Tube Tube with glass wool Cup
glass wool
 KEGUNAAN GLASS WOOL

Bertindak sebagai penapis dan

mengurangkan peluang bagi sebarang
bahan yang tak meruap daripada sampai
ke turus (Column).
• Ia dapat menangkap cecair yang disuntik
daripada picagari dan meningkatkan
pemeruapan
• Mengelakkan cecair terkena pada
bahagian bawah penyuntik.
 GLASS WOOL
• ALWAYS USE DEACTIVATED
(SILYLATED) WOOL.

• BOROSILICATE OR QUARTZ MATERIAL

NO!
 GLASS WOOL
Liner Packaging Recommendations

• Amount size and placement must be

consistent for consistent result

• Liner deactivation glass wool plug in place

is ideal
 GLASS WOOL
PLACEMENT IN LINER
NEAR TOP OF LINER
• Wipe syringe needle of sample
• Can improve injector precision
• Helps to prevent backflash

NEAR BOTTOM OF LINER

• Helps in volatilization of high MW
component
• Increases mixing
SPLITLESS LINERS
 Chromatographic Separation
Other considerations

Column materials
• Tubing
1. Metal - Stainless steel, nickel, copper,
aluminum
2. Glass - Pyrex, fused silica
3. Polymer - Teflon
 Turus (Columns)

Pada umumnya turus (column) terdiri

daripada dua jenis, ia itu:

• Pack Column

• Capillary Column.
Model of the Chromatographic
Process
Flow A

D
 How To Improve Column
Efficiency
1. Use smaller diameter column
2. Use a lower % or thinner film of stationary phase
3. Use smaller sample size
4. Use longer column.
5. Use temperature programming for sharper later
eluting peaks.

Effective column efficiency is dependent upon good sample introduction technique.

Samples should be introduced in a tight, rapid plug to avoid band broadening.
 COLUMN DIAMETER
(Capillary Columns)
I.D. (mm) Common Name

• 0.53 Megabore
• 0.45 High Speed Megabore
• 0.32 Wide
• 0.20-0.25 Narrow
• 0.18 Minibore
• 0.10 Microbore
• 0.05 “Nanobore”
 INLET HEAD PREASSURES
• I.D(mm)Pressure(psig)
• 0.10 225-250
• 0.20 25-35
• 0.25 15-25
• 0.32 10.20
• 0.53 2-4

30meter Column (Helium)

 Liquid Phase Selectivity
5% phenyl methyl silicone
3
2
1
6
1. C11
4 7
9 2. 4-chlorophenol
5 8
3. 1-decylamine
4. C13
5. methyl caprate
6. C14
36 50 % phenyl methyl silicone 7. acenaphthylene
14 2 8. 1-dodecanol
9
9. C15

8
7
 Detectors

• Different detectors will give different types

of selectivity.
 Flame Ionization Detector
Schematic
FID Detector
Assembly
Air
Inlet

Capillary Column
End-Position
(1-2 mm from Top of Jet) Jet
H2 Inlet
+
Make-Up

Exit End of Column

 FID
• Nyalaan FID terujud daripada pembakaran
Hidrogen dan udara. Apabila sampel
dilalukan, pembakaran akan terjadi
menyebabkan berlakunya pengionan dan
melepaskan elektron.
• Pemungut yang mempunyai voltan berkutub,
menarik elekton yang terbebas mewujudkan
arus letrik yang seimbang dengan jumlah
hidrokarban didalam sampel. Isyarat dari
pengesan ini akan dibesarkan dan diproses.
Flame Ionization Detector
CO H0
2
+ 2 CHO+
CHO
H0 + CO
2 CHO 2
+
CHO CHO
+ The FID is a destructive, mass sensing
CO
2 detector.
H0
2 Cations generated in the flame are
H0
2 counted and produce the detector
signal.
Analytes that have the greatest
H H number of low oxidation state carbons
2 CH 2
4 produce the largest signal.
H H
2 CH 2
4
H H
2 CH 2
4
H CH H
2 4 2

CH
Column
H 4 H
2 2
CH Jet
H 4 H
2 2
Compounds with Little or No
FID Response

Rare gases NH 3 CS 2
Nitrogen oxides H2 COS
Silicon halides CO O2
H 2O CO 2 N2
Perhalogenated cpds HCOH HCOOH
 Operating the FID
Recommended Flow Rates
Gas Type Flow Range Suggested Flow
Carrier Gas
(hydrogen, helium,
nitrogen)
Packed Columns 10 - 60 ml/min
Capillary Columns 1 - 5 ml/min
Detector Gases
Hydrogen 24 to 60 ml/min 40 ml/min.
Air 200 to 600 ml/min 450 ml/min
Column plus capillary 10 to 60 ml/min 50 ml/min
makeup
Recommended Detector Temperature

If < 150º C, flame will not light

Detector temp should be 20º C > higher than oven temp.
Detector Type Support Selectivity Detectability Dynamic
gases range
Flame Mass flow Hydrogen Most organic 100 pg 107
ionization and air cpds.
(FID)

Electron Concentration Make-up Halides, nitrates, 50 fg 105

capture nitriles,
(ECD) peroxides,
anhydrides,
organometallics

Flame Mass flow Hydrogen Sulphur, 100 pg 103

photometri and air phosphorus, tin,
c (FPD) possibly boron, arsenic,
oxygen germanium,
selenium,
chromium
Nitrogen- Mass flow Hydrogen Nitrogen, 10 pg 106
phosphorus and air phosphorus

Thermal Concentration Reference Universal 1 ng 107

conductivity
(TCD)
Flame Mass flow Hydrogen Sulphur, 100 pg 103
photometric and air phosphorus, tin,
(FPD) possibly boron, arsenic,
oxygen germanium,
selenium,
chromium
Photo- Concentration Make-up Aliphatics, 2 pg 107
ionization aromatics, ketones,
(PID) esters, aldehydes,
amines,
heterocyclics,
organosulphurs,
some
organometallics
 ELECTRON CAPTURE DETECTOR

• Theory of operation
ECD berdasarkan kepada keelektronegatifan yang bertindak
balas dengan elektron terma yang membentuk ion bercas
negatif. Kehilangan electron bergantung kepada kuantiti analit
di dalam sampel.
Dalam menghasilkan elektron bertenaga rendah, gas
pembawa diionkan oleh zarah beta melalui sumber radioaktif
di dalam sel. Elektron yang mengalir menghasilkan arus yang
mana dikumpul dan diukur. Apabila molekul sampel berada di
dalam sel, elektron ditarik pada elektrod melalui sampel,
menyebabkan arus berkurangan. Perubahan ini dicatat dan
diukur oleh kromatogram.
 NITROGEN PHOSPHORUS DETECTOR
• Theory of operation
The NPD (also called a thermionic detector) uses a jet
and collector similar in appearance to a Flame Ionization
Detector. In an NPD, however, ions of alkali metal are
introduced into a flame where hydrogen and air flows are
less than those for an FID, minimizing the normal
hydrocarbon ionizations, and increasing the ionization of
nitrogen or phosphorous compounds. This causes the
NPD to be both sensitive and selective for organic
compounds containing nitrogen and/or phosphorous.
This thermionic source efficiently ionizes nitrogen and
phosphorous containing organic molecules. Ions are
collected and the resulting current measured for the
chromatogram.
 NITROGEN PHOSPHORUS
DETECTOR
• NPD juga dikenali sebagai pengesan thermoionic
menggunakan jet dan Collector adalah lebih kurang
sama dengan FID. Walau bagaimanapun, didalam NPD
ion logam alkali diajukan(introduced) kepada nyalaan api
hidrogen dan udara yang kurang berbanding FID,
pengionan hidrokarbon yang kurang dan meningkatkan
kadar pengionan sebatian nitrogen atau fosforus.
• Ini menyebabkan NPD lebih sensitif dan berupaya untuk
memilih sebatian organik yang mengandungi sebatian
nitrogen dan/atau fosforus.
• Ion yang dikumpul dan arus yang terhasil diukur untuk
kromatogram.
 Flame Photometric Detector (FPD)
Theory of operation

In the Flame Photometric Detector (FPD), the sample burns in a hydrogen rich
flame, where some species are reduced and excited. The gas flow moves
the excited species to a cooler emission zone above the flame where they
decay and emit light. A narrow bandpass filter selects light unique to one
species, while a shield prevents intense carbon emission from reaching the
photomultiplier tube (PMT).

The light strikes a photosensitive surface in the PMT where a light photon
knocks loose an electron. The electron is amplified inside the PMT for an
overall gain of up to a million.

The current from the PMT is amplified and digitized by the FPD electronics
board. The signal is available either as a digital signal on the communications
output or as a voltage signal on the analog output.
FLAME PHOTOMETRIC DETECTOR
 How a photoionization detector works

•
A photoionization detector uses an ultraviolet (UV) light
source to ionize components of the incoming sample.
When a compound enters the detector, it passes by the
UV lamp and is bombarded by high energy photons.
Components that have a low enough ionization potential
are ionized, producing free electrons that are directed to
a polarizing electrode within the detector. The
photoionization detector senses the electron stream as a
current that is proportional to the amount of ionizable
gases in the sample. The signal from the photoionization
detector is then amplified and output to a display or
external device.
 THERMAL CONDUCTIVITY DETECTOR
• Theory of operation
The TCD responds to any compounds whose thermal conductivity is
different from the thermal conductivity of the carrier gas alone. The
TCD cell is a dual channel device, with an empty flow path and a
path containing a detector filament. A switching valve alternates
between sending the column effluent (containing analytes) through
the empty and the active flow paths. When the column effluent flows
through the empty channel, a pure stream of reference gas
maintains an equilibrium through the filament path. The reference
gas is used to compare thermal conductivity changes caused by the
column effluent.

A gas with high thermal conductivity, such as helium, is used as the

carrier /makeup/reference gas. When the analyte is present in the
gas stream, the thermal conductivity drops, and less heat is lost to
the cavity wall. Under constant applied voltage, a silicon nitride
coated filament in the TCD cell will heat up and its resistance will
increase. This change is what is recorded and measured for the
chromatogram.
DATA ANALYSIS

QUALITATIVE AND
QUANTITATIVE
 Data Acquisition

1. Chart recorder
2. Integrator
3. Chemstation
- Computer & chromatographic
software
 Qualitative
Analysis
1. Direct Comparison of Retention Time
The simplest procedure in the identification of
unknown. A known standard is first analysed under
the specific GC conditions, followed by the
unknown also under the similar conditions.
The difference in the retention times should not be
more than +/-0.1 min for proper identification
 Qualitative
Analysis Retention time

1. Identification by Log 100

n-paraffins
n-paraffins
Plotting of Homologous
Series 10

A. Semi-log plotting, one . 1.0

column
Esters
Esters
Log R t vs C n or B p or (CH2)n 0.1

Number of Carbon Atoms

B. Log-log plotting, two Retention Time
columns (non-polar)
Alkene
Log R t(non polar) vs Log R t 100
s
(polar) 10
Retention Time
1.0
(polar)
0.1
0.1 1.0 10 100
 Method of Calibration
• Area %
• Normalisation %
• External Standard
• Internal Standard
 Quantitative
Analysis
For accurate quantitative
results:-
• Peaks should be well resolved
• Peak should be undistorted
• Signal to be large
• Baseline should be flat
 Summary of Methods
  ADVANTAGES
AREA % No calibration required
Injection size not critical
ESTD Correct for detector response
Calibrate peaks of interest
Not all peaks need elute
Not all peaks need detect
Results reported in units of choice
NORM % Injection size not critical
ISTD Known component added to both sample
and standard
Injection size not critical
Calibrate peaks of interest
Correct for detector response
Results reported in units of choice
DISADVANTAGES
Must have uniform detector response
All components must elute
All components must be detected
All area must be correct
Injection size is critical
Instrument stability required
Frequent recalibrations
All peaks must elute
All peaks must be measured
Must calibrate all peaks
Must add a component to the sample
More complex sample and standard
preparation steps
 Quantitative Analysis
Concentration
1. External Standard
Method
- Concentration of an unknown A
CA = C1/A1 x A A
C =concentration , A = peak area Peak Area

2. Internal Standard
Method concentration ratio

- Concentration of an unknown X
Cx = (CA /CS)/(AA /AS) x AX /AIS x WiS
IS, S=internal standards

peak area ratio

 Internal Standard Method

Area ratio
( As/AIS)

Concentration
 INTERNAL STANDARD

(Corrected Response) y
Amount of y = X ISTD Amount X MF
(Corrected Response) ISTD
 EXTERNAL STANDARD

Amount of y = (Corrected Response) y X MF

 Criteria for Choosing Internal
Standard
• Not present in the sample
• Readily available
• Chemically similar to sample
• Same concentration range
• Does not react with sample or other matrix
• Elutes near components of interests
• Isolated, clean peak
• Chromatographically stable
 Advantage of external
standard calibration method
• Only the target compound separation can
be focused.

Target Target
 Disadvantage of external
standard calibration method
• Injection error will directly influence the
quantitative result.

1.0 uL injection 1.1 uL injection

100 ppm 110 ppm

 Advantage of internal
standard calibration method
• Injection error can be eliminated.

1.0 uL injection 1.1 uL injection

2200
2000 1100
1000 T
T IS
IS

2000 / 1000 = 2 2200 / 1100 = 2

 Disadvantage of internal
standard calibration method
• Separation is slightly difficult.

IS T T
IS

T
IS
 Disadvantage of internal
standard calibration method

• It is difficult to look for the IS

compound.
– The chemical structure of IS compound
is similar with one of target compound.
– IS sample is not existent in the actual
sample.