INTRODUCTION TO GAS CHROMATOGRAPHY

GAS CHROMATOGRAPHY (GC) GC is a technique used to separate , identify and quantify compounds in a sample mixture. Compounds are separated primarily by differences in their volatilities and structures. Compounds must vaporize into the gas phase below 400 0C and thermally stable.

How the GC Operating? Recorder/Data System Flow Controller Injection Port Chromatogram Detector Column Oven Carrier Gas Column Injection Migration Elution Peak Signal Column Operating

GC ? ● GC : Gas Chromatography ● Is an Analytical Instruments Technique used for Separating Chemical Mixtures ● Column coated with non-Volatile is a Stationary Phase ● Solutes are Carried through GC dissolved in Carrier gas is a Mobile Phase ● Solutes must be in vapor state at GC Operating Temperature

GAS CHROMATOGRAPHY เหมาะกับตัวอย่างที่กลายเป็นไอได้ง่าย (volatility) และมีความเสถียรทางความร้อน (thermal stability) สารผสมถูกแยกออกจากกันเนื่องจากความแตกต่างทางคุณสมบัติทางฟิสิกส์หรือทางเคมี ใช้ก๊าซเฉื่อย (inert gas) เป็นก๊าซพา (carrier gas)

หลักการของ Chromatography เป็นเทคนิคการวิเคราะห์ที่ใช้สำหรับการแยกสารผสม การแบ่งส่วน (partitioning) ระหว่าง mobile phase และ stationary phase นำไปสู่การแยก GC ใช้ก๊าซเฉื่อยเป็น mobile phase และใช้ของเหลวซึ่งกลายเป็นไอได้ยาก มีน้ำหนักโมเลกุลสูง เป็น stationary phase Chromatogram ถูกนำไปใช้เพื่อช่วยในการพิสูจน์เอกลักษณ์ของสารและการวิเคราะห์หาปริมาณองค์ประกอบต่างๆในตัวอย่าง

SEPARATION OF SAMPLE IN THE COLUMN time (A) (B) (D) (C)

HOW SEPARATION OCCURS Chromatography is a separation method achieved by the distribution of substances between two phases: a mobile phase and a stationary phase. Mobile Phase Stationary Phase Gas Solid Chromatography Gas Solid (GSC) Gas Liquid Chromatography Gas Liquid (GLC)

DETECTOR DATA SYSTEM GAS SUPPLY SAMPLE INTRODUCTION COLUMN OVEN GAS CHROMATOGRAPH SAMPLE INTRODUCTION COLUMN DETECTOR OVEN TEMPERATURE CONTROL DATA SYSTEM GAS SUPPLY DATA OUTPUT CHROMATOGRAM

CARRIER AND DETECTOR GASES chosen with the consideration of the type of detector used Carrier Gas: He, N2, H2 Inert Dry Pure

Gas for GC 1.Carrier 2.Fuel & 3.Oxidation Non Flammable Detector ECD,TCD Flammable Detector FID,FPD,TSD Carrier Gas is He,N2,H2,Argon purity UHP > 99.995 Fuel = H2 purity UHP > 99.995 Oxidation = Air purity UHP > 99.995

การเลือกก๊าซพา (Carrier Gas) มีคุณสมบัติเฉื่อย (inert) ปราศจากความชื้น และบริสุทธิ์ (มีสิ่งปนเปื้อน <1 ppm) H2 เร็วที่สุดมีความหนืดต่ำที่สุด แต่ไม่เหมาะกับ GC/MS เนื่องจาก ยากต่อกับขับออกด้วย vacuum pumping system N2 ดีกว่า H2 และHe เล็กน้อยที่ optimum carrier gas flow rate สำหรับ fast analysis: H2 ดีกว่า He และ He ดีกว่า N2 คำนึงถึงเครื่องตรวจวัดที่ใช้ ใช้ He เป็นก๊าซพาใน GC/MS

Gas Filters & Accessories

Gas-Clean Filters Using high-grade gases in analytical labs is considered critical for modern high-resolution gas chromatography Leaks in the gas lines connecting the gas cylinders to the GC are sometimes inevitable. Oxygen, water moistures, hydrocarbons may enter into your GC system may result in damage to column loss of sensitivity damage to detector High sensitivity indicators Fast and simple replacement No instrument down time No tools required Will fit the filter base of Agilent, SGE and Alltech

Sample Introduction Purpose: To introduce the sample on to the column in the vapor state. Syringe Injection Manual injection Auto-sampler injection Valve Injection Gas sampling valves Liquid sampling valves Auxiliary Sampling Devices Purge and Trap Headspace

TYPES OF INLET SYSTEMS Packed column inlet Septum - purged packed column inlet Split/splitless capillary inlet Split - only capillary inlet Dedicated on - column capillary inlet

BASIC VAPORIZATION INJECTOR SPLIT INJECTOR carrier gas line septum nut flow controller or needle valve septum liner o-ring 100 mL/min 2 mL/min 98 mL/min flow controller or pressure regulator liner 97 mL/min split vent 1 mL/min pressure regulator or needle valve column column nut and ferrule BASIC VAPORIZATION INJECTOR SPLIT INJECTOR

flow controller or pressure regulator flow controller or needle valve septum purge vent 3 mL/min 2 mL/min 30 mL/min 2 mL/min 1 mL/min 28 mL/min pressure regulator or needle valve mL/min 27 mL/min split vent solenoil-closed solenoil-open 1 mL/min 1 mL/min PURGE ON PURGE OFF

Ratio of amount of sample in the column and total sample split ratio = Fcolumn+ Fsplit vent / Fcolumn Ratio of amount of sample in the column and the amount leaving via the split line split ratio = Fsplit vent / Fcolumn Fsplit vent = Flow from split vent (mL/min) Fcolumn = Column flow (mL/min)

Packed 0pen (Capillary) Regular Conventional Packed Wall Coated COLUMN TYPES Regular Conventional Packed Wall Coated Open Tube and Micro Packed Bead Column Porous Layer Open Tube Porous Layer Bead

Pack Column

Capillary Column GC Column

Efficiency, plates/m 2000-4000 1000-4000 600-1200 500-1000 Properties and Characteristics of Typical GC Columns Type of Column FSOT WCOT SCOT Packed Length, m 10-100 10-100 10-100 1-6 Inside diameter,mm 0.1-0.53 0.25-0.75 0.5 2-4 Efficiency, plates/m 2000-4000 1000-4000 600-1200 500-1000 Sample size,ng. 10-75 10-1000 10-1000 10-106 Relative pressure Low Low Low High Relative speed Fast Fast Fast Slow Chemical inertness Best Poorest Flexible Yes No No No FSOT : Fused-silica, open tubular column. WCOT : Wall-coated, open tubular column. SCOT : Support-coated, open tubular column.

COLUMN SEPARATION CHARACTERISTICS Efficiency : Ability of the column to produce sharp peaks. Resolution : Ability of the column to separate two peaks from each other. Selectivity : Ability of the column to determine chemical and/ or physical difference in two peaks.

COLUMN SEPARATION CHARACTERISTICS Efficiency --------- Resolution --------- Selectivity --------- Efficiency -------- Resolution -------- Selectivity --------

COLUMN SEPARATION CHARACTERISTICS ISOTHERMAL TEMPERATURE PROGRAMMED

COLUMN TEMPERATURE OPERATION ISOTHERMAL TEMPERATURE PROGRAMMED

GC DETECTOR Definition : A GC Detector is a device which senses the presence of a component different from the carrier gas, and converts that information to an electrical signal.

DETECTOR RESPONSE CHARACTERISTICS Sensitivity: The response per amount of sample, that is, the slope of the response / amount curve. The minimum amount on the curve is defined as the minimum detectable level (MDL). Selectivity: A measure of which categories of compounds will give a detector response. Dynamic Range : The range of sample concentrations for which the detector can provide accurate quantitation.

GC DETECTOR Thermal Conductivity Detector (TCD) Flame lonization Detector (FID) Electron Capture Detector (ECD) Nitrogen Phosphorus Detector (NPD) Flame Photometric Detector (FPD) Photo-ionization Detector (PID) Electrolytic Conductivity Detector (ELCD,HALL) Mass Selective Detector (MSD) Infrared Detector (IRD)

Detectors Types of Detectors Brief Description TCD Filament temperature increases as analytes present in the carrier gas pass over it . Causing the resistance to increase. FID Components burn in a flame producing ions which are collected and converted into a current ECD As electronegative species pass through the detector , they capture low energy thermal electrons causing a decrease in cell current.

Detectors Types of Detectors Brief Description NPD Nitrogen and phosphorous compounds produce increased currents in a flame enriched with vaporized alkali metal salt. FPD Sulfur and phosphorous compounds burn in a flame producing chemiluminescent species which are monitored at selective wave lengths. .

Detectors Types of Detectors Brief Description ELCD Halogens , sulfur , or nitrogen compounds are mixed with a reaction gas in a reaction tube . The products are mixed with a suitable liquid which produces a conductive solution PID Molecule are ionized by excitation with photons from a UV lamp . The charged particles are then collected , producing a current.

Detectors Types of Detectors Brief Description MSD Molecules are bombarded with electrons producing ion fragments which pass into the spectrometers mass filter . The ions are filtered based on their mass / charge ratio. IRD Molecules absorb infrared energy , the frequencies of which are characteristic of the bonds within that molecule.

Detectors Types of Detectors Brief Description AED Molecules are energized by a plasma source and separated into excited atoms. As electrons return to their stable state , they emit light,which is element specific.

DETECTORS COMMONLY USED IN CAPILLARY GC Type Typical Sensitivity Carrier+ Samples Range Makeup H2 Air FID Hydrocarbons 1 - 5 pgC/s. 20 - 60 30-40 200-500 10 ppb - 99 % TCD General 5 - 100 ng 15 - 30 n.a. n.a. 10ppm - 100% ECD Organohalogens 0.05 - 1 pg. 30 - 60 n.a. n.a. Chlorinated 50ppt-1ppm solvents & pesticides

DETECTORS COMMONLY USED IN CAPILLARY GC Type Typical Sensitivity Carrier+ Samples Range Makeup H2 Air NPD Organonitrogen 0.1-10 pg. 20-40 1-5 70-100 &organo 100ppt-0.1% phosphorus compounds FPD Sulfur 10 - 100pg 20-40 50-70 60-80 (393 nm) compounds 10ppb-00ppm FPD Phosphorus 1-10 pg. 20-40 120-170 100-150 (526nm ) compounds 1ppb-0.1%

DETECTORS COMMONLY USED IN CAPILLARY GC Type Typical Sensitivity Carrier+ Samples Range Makeup H2 Air PID Compounds 2pg C/sec 30-40 * n.a. ionized by UV ELCD Halogens,N,S 0.5pg CI/sec 20-40 80 n.a. 2pg S/sec 4pg N/ sec FTIR Molecular 1000pg of 3-10 n.a. n.a. vibrations Strong absorber

DETECTORS COMMONLY USED IN CAPILLARY GC Type Typical Sensitivity Carrier+ Samples Range Makeup H2 Air MSD Tunable for any 10pg to 10 ng 0.5-30** n.a. n.a. species (depending on SIM vs.Scan) AED Tunable for any 0.1-20pg/sec 60-70 PRESET PRESET Element (depending on element) * Refer to Detector Manual ** Dependent upon the type of interface

APPLICATION Qualitative Analysis Quantitative Analysis

Qualitative Analysis relative retention retention indices Comparision of retention data relative retention retention indices

Quantitative Analysis Area % Normolization % External standard Internal standard

AREA % Area % of A = Area(A)/S Area Area % of A = 280/1070 = 26.17

NORMALIZATION % RF = amount/area NORM%= (RF*area)*100/S(RF* area)

External standard Calibration

External standard Calibration

Calibration curve : compound A area Concentration(mg/ml) area 1 488 2 980 4 1850 5 2450 concentration

Calibration curve : compound B area Concentration(mg/ml) area 1 598 2 1210 4 2350 5 3000 concentration

Calibration curve : compound C area Concentration(mg/ml) area 1 400 2 780 4 1590 5 3995 concentration

External standard Sample

Internal standard Calibration

Internal standard Calibration

Internal standard Calibration curve Area ratio Concentration Concentration Area ratio mg/ml A B C 1 0.208 1 0.170 2 0.417 1 0.332 4 0.829 1 0.677 5 1.042 1 0.851 COMPOUND A Concentration

Internal standard Calibration curve Area ratio Concentration Concentration Area ratio mg/ml A B C 1 0.208 1 0.170 2 0.417 1 0.332 4 0.829 1 0.677 5 1.042 1 0.851 Area ratio COMPOUND C Concentration

Internal standard Sample

Summary Of Methods Injection size not critical detector response ADVANTAGES DISADVANTAGES AREA % No calibration required Must have uniform Injection size not critical detector response All components must elute All components must be detected

Summary Of Methods ADVANTAGES DISADVANTAGES All peaks must be measured NORM % Injection size not critical All peaks must elute All peaks must be measured Must calibrate all peaks

Summary Of Methods ADVANTAGES DISADVANTAGES ESTD Correct for detector response Injection size is critical Calibrate peaks of interest Instrument stability required Not all peaks need elute Frequent recalibrations Not all peaks need detect Results reported in units of choice

Summary Of Methods to both sample and standard to the sample ADVANTAGES DISADVANTAGES ISTD Known component addaded Must add a component to both sample and standard to the sample Injection size not critical More complex sample Calibrate peaks of interest and standard preparation Correct for detector response steps Results reported in units of choice

CHROMATOGRAM Column type , stationary phase Condition: temperature , flow rate Carrier gas Detector Injection volume Sample