Analyzing Phenyl Ureas and Carbamates with HPLC/API-MS Presented by Chen-Kai Meng, Ph.D. Applications Chemist
Outline Overview of ESI, APPI, and APCI sources Comparing ESI, APPI, and APCI results Identifying compounds with the same MW Quad CID (Collision Induced Dissociation) Trap MS/MS (AutoMS^2 and MS^3) Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 2
Outline Overview of ESI, APPI, and APCI sources Comparing ESI, APPI, and APCI results Identifying compounds with the same MW Quad CID (Collision Induced Dissociation) Trap MS/MS (AutoMS^2 and MS^3) Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 3
Relative Applicability of Atmospheric Pressure Ionization Techniques Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 4
ESI Source HPLC inlet Nebulizer gas inlet Nebulizer Capillary Nebulizer tip aimed at 90 to inlet of MS Waste heated N 2 Countercurrent gas for efficient evaporation and desolvation Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 5
ESI Process Charged Droplets Analyte Ion - - - - - Evaporation - - de/dr = 10 9 V/cm Rayleigh Limit Reached - - - - - - - Coulombic Explosions - - Solvent Ion Cluster Analyte Ion Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 6
Dr. John Fenn Nobel Lecture Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 7
Nebulizing the Sample Nebulizing nitrogen Polymeric Damping Mechanism Rigid stainless steel eluent delivery capillary Micromachined tip Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 8
ESI Droplets Distribution Agilent LC/MSD Our sprayer design leads to smaller and more uniform droplets Amount Some competitive designs Spray Droplet Size Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 9
ESI Source Design Ion capture zone Heated N 2 Cone design has small sweet spot for ion capture Flat field presented to ions in Agilent source gives a large sweet spot for ion capture plus heated N 2 repels uncharged species Slide 10 Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio
Agilent 1100 LC/MSD, ESI Source Enhanced Sensitivity, Optimized Geometry Unique nebulizer and electrical field design require no X,Y, Z position adjustments - even when flow rate changes!! Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 11
APCI Source HPLC inlet Nebulizer gas inlet Heater Capillary Corona needle heated N 2 Waste Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 12
APCI Process Analyte containing aerosol Evaporation DISCHARGE Charge transferred to analyte Vapor Charged reagent gas formed Analyte ions Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 13
Agilent 1100 LC/MSD APCI Source Corona Needle Ionize the gas with a discharge Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 14
APPI Source Nebulizer Heater Capillary Ionizes gas phase analytes with light instead of a corona discharge Similar to PIDs for GC hν UV Lamp heated N 2 Waste Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 15
APPI Process Direct ionization of analyte to radical cation Analyte containing aerosol Evaporation Vaporization of LC eluent hυ hυ Dopant is photoionized and acts as reagent gas Ionization of solvent or dopant with charge exchange to analyte Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 16
Energetics for Photoionization lamp Krypton 10.0 and 10.6 ev Ionization Potentials Solvent Ionization Potentials Anthracene 7.4 ev Toluene 8.82 ev Fluoranthene 7.8 ev Acetone 9.70 ev Caffeine 8.0 ev 4-Nitrotoluene 9.5 ev 2,4,6-Trinitrotoluene 10.59 ev Methanol Acetonitrile Water 10.85 ev 12.19 ev 12.61 ev Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 17
Agilent 1100 LC/MSD PPI Source Lamp Source (Krypton), instead of Discharge Needle Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 18
Flow Rate Performance for APPI and APCI APCI versus APPI Response for Cortisol by Area Area 1400000 1200000 1000000 800000 600000 400000 200000 0 1.0/47 2.1/208 3.0/425 4.6/1000 Column I.D./Flow Rate (µl/min) APCI APPI Zorbax SB-C18 150 mm columns in 1.0, 2.1, 3.0 or 4.6 mm i.d. Isocratic elution with 80% methanol in water at 47, 208, 425 or 1000 µl/min depending on column i.d. Data were collected on the LC/MSD in SIM mode. Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 19
Positive Ion FIA in THF:H2O or IPA:H2O Compounds: MW ESI APCI APPI Analgesics Aspirin 180.04 Ibuprofen 206.13 Acetaminophen 151.06 Naproxen 230.09 Caffeine 194.08 PAH s anthracene 178.08 benzo(a)anthracene 228.09 benzo(ghi)perylene 276.09 benzo(a)pyrene 252.09 naphthalene 128.06 Vitamins A 286.21 D 2 396.34 E 430.38 K 450.35 C 176.03 Conditions: 0.4 ml/min, 100 picomoles injected Key: not seen weak good signal Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 20
Break Number 1 Please type your question into the Chat Box at any time during the presentation. Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 21
Outline Overview of ESI, APPI, and APCI sources Comparing ESI, APPI, and APCI results Identifying compounds with the same MW Quad CID (Collision Induced Dissociation) Trap MS/MS (AutoMS^2 and MS^3) Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 22
Carbamates and Phenyl Ureas Methomyl 162 Fenuron 164 Propham 179 Monuron 198 Carbaryl 201 Diuron 232 Aminocarb 208 Fluometuron 232 Propoxur 209 Siduron 232 Chlopropham 213 Linuron 248 Oxamyl 219 Neburon 274 Swep Carbofuran 219 221 Mexacarbate 222 Methiocarb 225 Barban 257 Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 23
LC/MSD Method Parameters ESI APCI APPI Solvent A H 2 O, 0.1% acetic acid H 2 O H 2 O Solvent B ACN, 0.1% acetic acid ACN MeOH Post column n/a n/a 40 µl/min Acetone Solvent gradient B: 10% @0, 30% @4, B: 30% @0, 40% @4, 70% @13, 80% @16 80% @10, (hold 6min) Inj. Volume 2 µl 2 µl 2 µl Column (30 o C) Zobrax Eclipse XDB-C8 4.6 x 50 mm, 3.5 µm (p/n 935967-906) Col. Flow 1 ml/min 1 ml/min 1 ml/min Frag 60 V 110 V 110 V Vcap 3500 V 4500 V 4500 V Dry gas 12 L/min 11 L/min 11 L/min nebulizer 60 psi 35 psi 35 psi Vaporizer n/a 225 C 225 C dry gas temp 350 C 275 C 275 C Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 24
Don t use ACN as Mobile Phase for APPI 1200000 1000000 800000 600000 H 2 O/ACN (noisy baseline) 400000 200000 2 4 6 8 10 min 1000000 800000 H 2 O/MeOH 600000 400000 200000 0 2 4 6 8 10 min Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 25
Acetone (dopant) needed in APPI 120000 100000 80000 60000 40000 Without post-column Acetone (poor signal) 20000 0 2 4 6 8 10 12 14 min 2000000 1500000 1000000 With 40 µl/min post-column Acetone 500000 0 2 4 6 8 10 12 14 min Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 26
API Results (20 ng on column) 2250000 2000000 1750000 1500000 1250000 1000000 750000 500000 250000 0 3000000 2500000 2000000 1500000 1000000 500000 0 25000000 20000000 15000000 MSD1 TIC, MS File (CAS\020304A\UREA0042.D) APPI, Pos, Scan, Frag: 110 APPI 2 4 6 8 10 12 14 MSD1 TIC, MS File (CAS\020304A\UREA0050.D) APCI, Pos, Scan, Frag: 110 APCI 2 4 6 8 10 12 14 MSD1 TIC, MS File (F:\UREA0225\M6230001.D) API-ES, Pos, Scan, Frag: 60, "pos scn" ESI min min 10000000 5000000 0 2 4 6 8 10 12 14 min Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 27
Baseline noise 1000 800 600 400 200 MSD1 SPC, time=14.991:15.488 of CAS\020304A\UREA0042.D Max: 1091 0 3000 2500 150 200 250 300 350 MSD1 SPC, time=14.975:15.488 of CAS\020304A\UREA0050.D Max: 3155 m/z 2000 1500 1000 500 0 20000 17500 15000 12500 10000 7500 5000 2500 0 150 200 250 300 350 MSD1 SPC, time=14.940:15.463 of F:\UREA0225\M6230001.D Max: 20907 m/z 200 400 600 m/z APPI APCI ESI Slide 28 158.9 183.0 210.0 233.1 256.2 279.1 299.2 338.1 363.0 385.1 426.1 485.0 522.5 550.5 633.1 794.6 298.3 337.1 136.1 149.1 171.2 205.1 233.2 250.1 259.2 280.2 288.3 313.2 391.3 279.2 116.1 128.1 138.2 158.1 170.2 188.1 201.1 220.2 260.2 288.3 179.2 219.2 233.2 259.2 275.1 117.1 Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio
Signal-to-Noise Ratio (Scan mode) ESI is better for Carbamate analysis APPI is better for Phenyl urea analysis ESI APPI APCI Carbofuran Mexacarbate Diuron Monuron (Carbamate) (Phenyl urea) Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 29
Break Number 2 Please type your question into the Chat Box at any time during the presentation. Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 30
Outline Overview of ESI, APPI, and APCI sources Comparing ESI, APPI, and APCI results Identifying compounds with the same MW Quad CID (Collision Induced Dissociation) Trap MS/MS (AutoMS^2 and MS^3) Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 31
AP Electrospray LC/MSD HPLC inlet Nebulizer gas inlet Nebulizer Skimmers Lenses HED detector Capillary Octopole Quadrupole Waste heated N 2 Fragmentation zone (CID) Molecular fragmentation by ion collisions with nitrogen molecules Controlled via a single ion optics parameter -- "fragmentor voltage" Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 32
Example of CID Fluometuron, MW=232 F F F 213 NH 188 O N CH 3 CH 3 80 60 40 20 0 12 10 8 6 4 2 0 Frag = 75 V Max: 4.63411e006 233.1 234.1 140 160 180 200 220 240 Frag = 225 V 168.0 Max: 610944 213.0 233.1 160.0 188.0 191.0 214.0 234.0 140 160 180 200 220 240 m/z m/z Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 33
Example of CID Frag V. 175 V Fluometuron, MW=232 200 V 225 V 250 V 275 V Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 34
Example of CID H 3 C CH 3 N Cl Cl Diuron, MW=232 188 O NH 160 197 80 60 Frag = 75 V Max: 1.49094e006 233.0 235.0 40 20 234.0 237.0 0 140 160 180 200 220 m/z 8 6 4 2 Frag = 200 V 159.9 Max: 178432 187.9 197.1 233.0 235.0 234.0 237.1 0 140 160 180 200 220 m/z Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 35
Example of CID Diuron, MW=232 Frag V. 175 V 200 V 225 V 250 V 275 V Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 36
Outline Overview of ESI, APPI, and APCI sources Comparing ESI, APPI, and APCI results Identifying compounds with the same MW Quad CID (Collision Induced Dissociation) Trap MS/MS (AutoMS^2 and MS^3) Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 37
AutoMS^2, the first 233 peak O Cl F F F NH N CH 3 CH 3 72 72 H 3 C CH 3 N O NH Cl This mass 72 fragment is common to both Fluometuron and Diuron Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 38
AutoMS^2, the second 233 peak Cl 72 H 3 C CH 3 N O NH Cl All three precursors (233, 235, and 237) gave the same mass 72 fragment. Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 39
AutoMS^2, the third 233 peak 114 O Siduron, MW=232 NH NH CH 3 137 94 Can we confirm the mass 94 and 137 fragments? Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 40
AutoMS^3, the third 233 peak 121 94 is part of peak 137. Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 41
Fragments from Siduron 114 O Siduron, MW=232 MS2 NH NH 94 114 137 CH 3 137 121 94 94 121 MS3 Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio Slide 42
Summary In APPI, don t use Acetonitrile (use Methanol) and add Acetone (or Toluene) post-column as dopant Phenyl Ureas respond better in APPI, Carbamates respond better in ESI AutoMS^n (Trap) and CID (Quad) help to identify compounds with the same masses Reference: C. Kai Meng, Analyzing Phenyl Ureas and Carbamate Pesticides Using ESI-, APPI-, and APCI-LC/MSD, Agilent Application Note, Publication number 5988-6635EN. Slide 43 Dial 44 207 162 0125, Chairperson John Vis for e-seminar Audio