PENGENALAN ASAS GAS

CHROMATOGRAPHY

OLEH:ISMAIL IBRAHIM PPN, PJK

Kenapa sampel yang bercampur boleh diasingkan?

Dasar sungai

Arah aliran

Apa perbezaan nya?

Interaction is difference : gravity

BeratRingan

Bagaimana pengasingan boleh Bagaimana pengasingan boleh dilakukan?dilakukan?

• Pengasingan dapat dilakukan oleh turus/ column.

packing material, 3-5um

Turus/column

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

Suntikan boleh digunakan dalam dua cara:

• split

• splitless.

CARA SUNTIKAN

Split/Splitless Flow System

Split Flow Diagram: Pre-Injection

SEAL

(INLET)

(SEPTUM)

SPLIT VENT

TOTAL FLOW

COLUMN

SEPTUM

= CARRIER GAS

LINER

50 mL/min

2 mL/min

0.6 mL/min

48 mL/min

47.4 mL/min

PURGE VENT

PURGE VALVE

Split Flow Diagram: Sample/Carrier Mixing

(INLET) PURGE VALVE

(SEPTUM) PURGE VENT

SPLIT VENT

TOTAL FLOW

COLUMN

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.

= CARRIER GAS

= SAMPLE MOLECULES

= SOLVENT MOLECULES

Split Flow Diagram: Liner Overload

(INLET) PURGE VALVE

(SEPTUM) PURGE VENT

SPLIT VENT

TOTAL FLOW

COLUMN

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.

= CARRIER GAS

= SAMPLE MOLECULES

= SOLVENT MOLECULES

choosing the right liner for the job

LINERS

• 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 bawahLiner tirus bahagian bawah

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.

Liner tirus bahagian atasLiner tirus bahagian atas

SPLIT LINERS

Straight Tube

Straight

Tube with glass wool

Fixed glass wool

Inverted Cup

Baffle

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.

KEGUNAAN GLASS WOOLKEGUNAAN GLASS WOOL

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 SeparationChromatographic SeparationOther considerationsOther 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.

A

B

C

D

Flow

Model of the Chromatographic Process

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.

How To Improve Column Efficiency

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)

1

23

4

5

6

7

89

1 2

3

4

6

9

87

1. C11

2. 4-chlorophenol3. 1-decylamine4. C13

5. methyl caprate6. C14

7. acenaphthylene8. 1-dodecanol9. C15

Liquid Phase Selectivity

5% phenyl methyl silicone

50 % phenyl methyl silicone

Detectors

• Different detectors will give different types of selectivity.

Exit End of Column

Jet

Air

H2 Inlet+

Make-Up

FID DetectorAssembly

Inlet

Capillary ColumnEnd-Position

(1-2 mm from Top of Jet)

Flame Ionization Detector Schematic

FIDFID• 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.

The FID is a destructive, mass sensing detector.Cations generated in the flame are counted and produce the detector signal. Analytes that have the greatest number of low oxidation state carbons produce the largest signal.

H2

H2

H2

H2

H2

H2

CH4

CH4

CH4

CH 4

CH 4

CH 4

CHO+

CHO+

CHO+

CHO+ CHO+

CO2

CO2

CO2

H 02

H 02

H 02

H 02

H2

H2

H2

H2

H2

H2

Column

Jet

Flame Ionization Detector

Rare gasesNitrogen oxidesSilicon halides

H2OPerhalogenated cpds

NH H COCO HCOH

2

2

3 CS COSO N HCOOH

2

2

2

Compounds with Little or No FID Response

Recommended Flow Rates

Gas Type Flow Range Suggested Flow

Recommended Detector Temperature

If < 150º C, flame will not light Detector temp should be 20º C > higher than oven temp.

Carrier Gas(hydrogen, helium, nitrogen)

Packed Columns 10 - 60 ml/min Capillary Columns 1 - 5 ml/min Detector GasesHydrogen 24 to 60 ml/min 40 ml/min.Air 200 to 600 ml/min 450 ml/minColumn plus capillary makeup

10 to 60 ml/min 50 ml/min

Operating the FID

Detector Type Support gases

Selectivity Detectability Dynamic range

Flame ionization (FID)

Mass flow Hydrogen and air

Most organic cpds.

100 pg 107

Electron capture (ECD)

Concentration Make-up Halides, nitrates, nitriles, peroxides, anhydrides, organometallics

50 fg 105

Flame photometric (FPD)

Mass flow Hydrogen and air possibly oxygen

Sulphur, phosphorus, tin, boron, arsenic, germanium, selenium, chromium

100 pg 103

Nitrogen-phosphorus

Mass flow Hydrogen and air

Nitrogen, phosphorus

10 pg 106

Thermal conductivity (TCD)

Concentration Reference Universal 1 ng 107

Flame photometric (FPD)

Mass flow Hydrogen and air possibly oxygen

Sulphur, phosphorus, tin, boron, arsenic, germanium, selenium, chromium

100 pg 103

Photo-ionization (PID)

Concentration Make-up Aliphatics, aromatics, ketones, esters, aldehydes, amines, heterocyclics, organosulphurs, some organometallics

2 pg 107

ELECTRON CAPTURE DETECTOR

• Theory of operation

ECD berdasarkan kepada keelektronegatifan yang bertindak ECD berdasarkan kepada keelektronegatifan yang bertindak balas dengan elektron terma yang membentuk ion bercas balas dengan elektron terma yang membentuk ion bercas negatif. Kehilangan electron bergantung kepada kuantiti analit negatif. Kehilangan electron bergantung kepada kuantiti analit di dalam sampel. di dalam sampel. Dalam menghasilkan elektron bertenaga rendah, gas Dalam menghasilkan elektron bertenaga rendah, gas pembawa diionkan oleh zarah beta melalui sumber radioaktif pembawa diionkan oleh zarah beta melalui sumber radioaktif di dalam sel. Elektron yang mengalir menghasilkan arus yang di dalam sel. Elektron yang mengalir menghasilkan arus yang mana dikumpul dan diukur. Apabila molekul sampel berada di mana dikumpul dan diukur. Apabila molekul sampel berada di dalam sel, elektron ditarik pada elektrod melalui sampel, dalam sel, elektron ditarik pada elektrod melalui sampel, menyebabkan arus berkurangan. Perubahan ini dicatat dan menyebabkan arus berkurangan. Perubahan ini dicatat dan diukur oleh kromatogram.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 richflame, where some species are reduced and excited. The gas flow movesthe excited species to a cooler emission zone above the flame where theydecay and emit light. A narrow bandpass filter selects light unique to onespecies, while a shield prevents intense carbon emission from reaching thephotomultiplier tube (PMT).

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

The current from the PMT is amplified and digitized by the FPD electronicsboard. The signal is available either as a digital signal on the communicationsoutput 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 AcquisitionData Acquisition

1. Chart recorder

2. Integrator

3. Chemstation - Computer & chromatographic

software

Qualitative Qualitative AnalysisAnalysis

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 Qualitative AnalysisAnalysis

1. Identification by Log Plotting of Homologous SeriesA. Semi-log plotting, one columnLog R t vs C n or B p or (CH2)n

B. Log-log plotting, two columnsLog R t(non polar) vs Log R t (polar)

Retention timeRetention time

Number of Carbon AtomsNumber of Carbon Atoms

n-paraffinsn-paraffinsn-paraffinsn-paraffins

EstersEstersEstersEsters

100100

1010

.. 1.01.0

0.10.1

Retention Time Retention Time (non-polar)(non-polar)

Retention Time Retention Time

(polar)(polar)

AlkeneAlkeness100100

1010

1.01.0

0.10.10.10.1 1.01.0 1010 100100

Method of Calibration

• Area %

• Normalisation %

• External Standard

• Internal Standard

Quantitative Quantitative AnalysisAnalysis

For accurate quantitative results:-

• Peaks should be well resolved

• Peak should be undistorted

• Signal to be large

• Baseline should be flat

  ADVANTAGES DISADVANTAGES

AREA % No calibration required Injection size not critical

Must have uniform detector responseAll components must eluteAll components must be detectedAll area must be correct

ESTD Correct for detector responseCalibrate peaks of interestNot all peaks need eluteNot all peaks need detectResults reported in units of choice

Injection size is criticalInstrument stability requiredFrequent recalibrations

NORM % Injection size not critical All peaks must eluteAll peaks must be measuredMust calibrate all peaks

ISTD Known component added to both sample and standardInjection size not critical Calibrate peaks of interestCorrect for detector responseResults reported in units of choice

Must add a component to the sampleMore complex sample and standard preparation steps

Summary of Methods

Quantitative AnalysisQuantitative Analysis

1. External Standard Method- Concentration of an unknown A

CA = C1/A1 x A A

C =concentration , A = peak area

2. Internal Standard Method - Concentration of an unknown X

Cx = (CA /CS)/(AA /AS) x AX /AIS x WiS

IS, S=internal standards

ConcentrationConcentration

Peak AreaPeak Area

concentration ratioconcentration ratio

peak area ratiopeak area ratio

Internal Standard Method

Area ratio

( As/AIS)

Concentration

INTERNAL STANDARD

Amount of y = (Corrected Response) y

(Corrected Response) ISTD

X ISTD Amount X MF

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.

TargetTarget TargetTarget

Disadvantage of external standard calibration method

• Injection error will directly influence the quantitative result.

1.0 uL injection 1.1 uL injection

100 ppm100 ppm 110 ppm110 ppm

Advantage of internal standard calibration method

• Injection error can be eliminated.

2000 / 1000 = 22000 / 1000 = 2 2200 / 1100 = 22200 / 1100 = 2

1.0 uL injection 1.1 uL injection

11001100 22002200

IS10001000 20002000

IS TT

Disadvantage of internal standard calibration method

• Separation is slightly difficult.

ISISTT

TTISIS TTISIS

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.