Interface Engineering Co., Ltd. Gas Chromatography -이론과 실제- 2004. Kim, Wang-Yu Ph.D. Interface Engineering Co., Ltd.

GC의 정의 두개의 상(고정상, 이동상) 사이에서 혼합물 각각의 성분이 분배(혹은 흡착)되는 정도가 다른 성질을 이용하여 각각의 성분을 분리하는 크로마토그래피 기술이다. 이동상으로는 혼합물과 반응하지 않는 기체를 사용하는 것이 HPLC와 크게 다른 점이다.

GC vs. LC Mobile phase Gas Liquid Sample state Gas Liquid Stationary phase Liquid or solid Liquid

Outside GC

Inside GC Injecting and vaporizing Detecting Separating

Injection Port Septum Graphite ferrule Inlet liner Nut

Septum

Ferrule

Capillary columns 1. Stationary phase 2. Inner Diameter(mm) 3. Length(m) 4. Film Thickness(um)

FID Flame Ionization Detector

GC Column 종류 (단면) Open (Capillary) Packed Regular Wall Coated Open Tube (WCOT) Conventional Porous Bead Porous Layer Packed and Layer Column Open Tube (PLOT) Micropacked Bead

Capillary Column Types orous L ayer O pen T ubular (PLOT) Carrier Gas W all C oated O pen T ubular (WCOT) Liquid Phase

GC column의 특성 1. Stationary phase 2. Inner Diameter(mm) 3. Length(m) 4. Film Thickness(um)

Polarity

POLARITY Polarity Solubility Phase = Solute High Phase ¹ Solute Low High solubility = High retention and capacity

DISPERSION INTERACTION Kcal/mole 2 4 6 8 10 Benzene Phenol C10 C12 8.1 11.9 10.9 DB-1 (non-polar)

DIPOLE INTERACTION Compounds With Dipole Differences Cl H C=C C=C Cl Cl H Cl 1,2-dichloroethylene 1,1-dichloroethylene Smaller differences require a stronger dipole phase

HYDROGEN BONDING INTERACTION Example Compounds Strong: alcohols, carboxylic acids, 1° and 2° amines Moderate: aldehydes, esters, ketones Weak: hydrocarbons, halocarbons, ethers

SELECTIVITY Interaction Strengths Phase Dipersion Dipole H Bonding Methyl Strong None Phenyl Weak Cyanopropyl Moderate Trifluoropropyl PEG

COMPOUNDS Properties Compounds Polar Aromatic Hydrogen Bonding Dipole Toluene no yes induced Hexanol Phenol Decane Naphthalene Dodecane

50% CYANOPROPYL 1. Toluene 2. Hexanol 3. Phenol 4. Decane (C10) 5. Naphthalene 6. Dodecane (C12) 2 4 6 8 10 12 14 16 100% Methyl 1 3 5 50% Cyanopropyl Strong Dispersion Strong Dipole Moderate H Bonding

Polysiloxanes (Methyl Substituted) % = # of sites on silicon atoms occupied 100 % methyl (HP-1, DB-1 etc.) Nonpolar

Polysiloxanes (Phenyl methyl Substituted) 5% phenyl (HP-5, DB-5, etc.) 35% phenyl (HP-35, DB-35, etc.) 50% phenyl (HP-50+, DB-17, etc.) Nonpolar Mid-polar Mid-polar

Polysiloxanes (Cyanopropylphenyl methyl Substituted) 6% cyanopropylphenyl (HP-1301, DB-1301, etc.) 14% cyanopropylphenyl (HP-1701, DB-1701etc.) 50% cyanopropylphenyl (HP-225, DB-225, etc.) Mid-polar Mid-polar Polar

Two columns are different !! HP-17: 50% phenyl and 50% methyl siloxane vs HP-50+ : (50%)-Diphenyl (50%)-Dimethylpolysiloxane 3 CH m n Si O CH 3 n Si O Two columns are different !!

Poly(ethylene) Glycol HO - C-C-O- -H n 100% PEG (HP-WAX) Less stable than polysiloxanes Unique separation characteristics Polar

POLARITY Solubility And Retention C12 C10 Hexanol 100% Methyl (non-polar) 2 4 6 8 Same GC conditions Same column dimensions Only differ in stationary phase C10 C12 Hexanol 100% PEG (polar) 2 4 6 8

1. Stationary phase 3. Length(m) 4. Film Thickness(um) 2. Inner Diameter(mm)

COLUMN DIAMETER Retention 80°C isothermal 0.25 mm 5 10 15 20 21.81 0.32 mm 2 4 6 8 12 14 16 16.06 Isothermal: Retention is inversely proportional to column diameter Temperature program: 1/3-1/2 of isothermal values

COLUMN DIAMETER(0.05~0.53mm) 0.53 mm 0.32 mm n =58,700 n =107,250 Sample capacity : < 2 ug < 500 ng Instrumental condition

Phase Ratio The combined effect of the column diameter and film thickness is described by , the phase ratio. = r/2d f where r = column radius ( m) d f = film thickness ( m)

Differing Column Inner Diameter, Equal Betas Time (min) 5 10 15 20 Carrier: Oven: 65° C Injection: Split Detector: FID Column A: HP-624 B: HP part no. 19091V-413 30 m, 0.32 mm, 1.8 m Helium, 40 (m/sec) 19095V-423 0.53 3

1. Stationary phase 2. Inner Diameter(mm) 4. Film Thickness(um) 3. Length(m)

COLUMN LENGTH Theoretical Efficiency Length (m) N 15 71,430 30 142,860 60 285,720 0.25 mm ID N/m = 4762 (for k = 5)

COLUMN LENGTH(10~120m) Resolution and Retention: Isothermal 2.29 min R=1.16 4.82 min R=1.68 8.73 min 15 m 30 m 60 m Double the plates, double the time but not double the the resolution

1. Stationary phase 2. Inner Diameter(mm) 3. Length(m) 4. Film Thickness(um)

FILM THICKNESS(0.1~5um) Retention: Isothermal 7.00 0.25 µm 2 4 6 8 Thermal stability Sample b.p. 25.00 1.00 µm 5 10 15 20 25

More stationary phase = More degradation products FILM THICKNESS Bleed More stationary phase = More degradation products

“Back biting” Mechanism of Bleed Formation Si HO H3C CH3 CH3 Si O OH CH3 OH Si O CH3 + Si O CH3 H3C Again

Four Types Of Low Bleed Phases Phases tailored to “mimic” currently existing polymers -Examples: DB-5ms, DB-35ms, DB-17ms New phases unrelated to any previously existing polymers -Example: DB-XLB Optimized manufacturing processes -Example: DB-1ms

Column Installation Choosing ferrule Cutting the capillary column Inlet installation Leak-checking Outlet installation Establishing flow Conditioning

Ferrule Graphite(100%) - 450℃ - general purpose and reused Choosing ferrule Cutting the capillary column Inlet installation Leak-checking Outlet installation Establishing flow Conditioning Ferrule Graphite(100%) - 450℃ - general purpose and reused - FID, NPD, ECD - Not good for Mass Vespel(100%) - 280℃ - reused - for only isothermal operation

Choosing ferrule Cutting the capillary column Inlet installation Leak-checking Outlet installation Establishing flow Conditioning Cutting Jagged silica edges or exposed polyimide cause adsorption and tailing peaks, so it is very important that the column ends are cut uniformly.

Inlet installation 먼저 column nut를 capillary column에 끼우고 ferrule을 끼운다. Choosing ferrule Cutting the capillary column Inlet installation Leak-checking Outlet installation Establishing flow Conditioning Inlet installation 먼저 column nut를 capillary column에 끼우고 ferrule을 끼운다. 3cm 이상 capillary가 나오게 한 후 먼저 inlet에 조여 capillary가 ferrule에 적당히 물리게 한다. 일단 푼다. capillary를 5mm정도 남겨놓고 자른다. 다시 inlet에 연결한다. 손으로 꽉 조인 상태에서 ¼~반바퀴 더 wrench로 돌린다. 이동상을 흘렸을 때 이 부분에 Leak가 있을 경우 injector 압력 gauge가 올라가지 않는다.

Leak-checking Use acetone in vial Choosing ferrule Cutting the capillary column Inlet installation Leak-checking Outlet installation Establishing flow Conditioning Leak-checking Use acetone in vial

Choosing ferrule Cutting the capillary column Inlet installation Leak-checking Outlet installation Establishing flow Conditioning Outlet installation 보통 detector jet orifice보다 1-3mm 아래쪽에 column end가 있게 한다(보통 capillary를 detector안쪽으로 끝까지 밀어 넣어 넣은 후 2mm정도 뒤로 뺀다). column nut와 ferrule을 끼우는 요령은 inlet과 같다.

Carrier gas Flow Choosing ferrule Cutting the capillary column Inlet installation Leak-checking Outlet installation Establishing flow Conditioning Carrier gas Flow

Conditioning 40℃ start and 5~10℃/min below 20~25℃ Choosing ferrule Cutting the capillary column Inlet installation Leak-checking Outlet installation Establishing flow Conditioning Conditioning 40℃ start and 5~10℃/min below 20~25℃

Storage septum on each end of the column Especially for PEG type column

Split/Splitless Injection

Splitless Injection

And Now Let’s do Some TROUBLESHOOTING

DB-624 COLUMN QC Test Mix Column: DB-624 1. 1,2-Dichloropropane 2.71 Column: DB-624 30m x 53mm I.D., 3.0µm Carrier: Helium at 40 cm/sec measured at 35°C Injector: Mega Direct, 260°C Detector: FID, 300°C Oven: 35°C for 1.50 min 30°/min to 65° for 10 min 1. 1,2-Dichloropropane 2. Octane 3. Tetrachloroethylene 4. Chlorobenzene 5. Nonane 8.0e4 7.0e4 6.0e4 7.43 10.92 5.0e4 4.0e4 17.42 12.49 3.0e4 20.78 2.0e4 1.0e4 5 10 15 20 25 Time (min.)

Example of Column Contamination 2.21 1.5e4 1.4e4 DB-624 QC Test Mix* After 75 Injections of Oily Sample 1.3e4 3.30 1.2e4 1.1e4 1.0e4 6.03 9000 9.26 8000 10.46 7000 14.40 17.86 6000 5 10 15 20 Time (min.)

Column and Liner Contamination Inlet coil of column

Example of Column Contamination 2.80 1.2e4 1.1e4 Removed 1 1/2 m from injector end * 1.0e4 7.34 9000 10.79 8000 17.19 12.33 7000 20.56 6000 5000 5 10 Time (min.) 15 20 25

Backflush Column Rinse with 10ml each: Methanol, Methylene Chloride, Hexane Capillary column Special connector and ferrule Flexible teflon tubing 1/16" flexible teflon line to regulated pressure source Special adapter Cap Vial Beaker for solvent collection Capillary column

Remember Complete system = Carrier Gas + Injector + Column + Detector + Data System Multiple cause and effect Do not change too many variables at once