Analyzing Small Molecules by EI and GC-MS Samples Appropriate for GC-MS • Volatile and semi-volatile organic compounds – Rule of thumb, <250 u will likely work by GC-MS – Highly polar or ionic compounds may not be sufficiently volatile for GC-MS – PNP ligand @ 429 u has been analyzed by GC-MS • Mixtures of semi-volatiles – Crude reaction mixtures – Stable isotope labeled samples • Solvent should be in volatile matrix – Please remove TBAF, NaCl, urea, or other involatiles if at all possible Gas Chromatography Basics • Vapor-phase molecules separate by their interactions with the inner wall of the column and the He stream • Oven temperature is raised to weaken interaction with stationary phase • Temperature profile can be altered to improve resolution of similar compounds or speed up analysis time Basic GC Diagram Image source: http://www.cubic.uni-koeln.de/research/horstmann/gas_chromatography2.jpg Agilent 6890/5973 GC-MS GC-MS Injection • A 100-position autosampler feeds a split/splitless injector • Normal methods use “split mode” for concentrated samples – Usually aim for sub-ug on column – 2 uL injection, 30:1 split is default on most methods • 1 mg/mL solution gives 67 ng on column • Splitless mode is available for dilute samples • Injector kept very hot (250 °C) to ensure instant vaporization of sample Split Injection Mode Diagram Image source: http://www.sge.com/uploads/yB/sx/yBsxaFxQS4C3FWzlvBJ6Uw/split_injection_2.gif Default 6890 Settings • Default column on 6890 is an RTX-5silMS – 95% dimethylsiloxane, 5% phenylsiloxane • Very non-polar stationary phase – 0.25 mm i.d., 30 m long, 0.25 μm film thickness • Temperatures can range from 30-320 °C – Sub-30° is available with liquid CO2 tank – Up to 20 °C per minute ramp possible • Default gradients – A “slow” gradient from 40-280 °C over 37 minutes – A “fast” gradient from 70-280 °C in 19 minutes Other Column Chemistries • There are entire catalogs full of GC columns with different chemistries – Any user-supplied column MUST be compatible with GCMS (bonded phase, no particles, limited bleed, etc) • MSF has the following columns available – – – – DB-1 (100% dimethylsiloxane) DB-17 (50% phenyl, 50% dimethyl siloxane) DB-VRX (proprietary DB-1 for volatile compounds) DB-WAX (polyethylene glycol) • Column change takes about 4 hours – Schedule this DAYS in advance with Dr. Karty Electron Ionization (EI) • Gas phase molecules are irradiated by beam of electrons • Interaction between molecule and beam results in electron ejection – M + e- M+• + 2e- • EI is a very energetic process – Molecules often fragment right after ionization EI Diagram Image from http://www.noble.org/Plantbio/MS/iontech.ei.html EI Mass Spectrum Figure from Mass Spectrometry Principles and Applications E. De Hoffmann, J. Charette, V. Strooband, eds., ©1996 82 100 Cocaine More EI Mass Spectra H O O 50 94 77 42 51 0 O N 182 27 15 10 20 30 40 (mainlib) Cocaine 50 H O 105 303 122 59 68 60 70 80 140 152 198 166 272 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 151 100 94 Vitamin B6 106 N 169 HO OH 50 122 OH 39 81 53 27 31 136 67 0 10 20 30 40 50 60 70 (mainlib) 3,4-Pyridinedimethanol, 5-hydroxy-6-methyl- 80 90 100 110 120 130 140 150 160 170 180 286 100 Androstenedione 124 O 244 148 50 109 O 79 41 0 18 55 91 97 67 29 10 20 30 40 50 60 70 (mainlib) Androst-4-ene-3,17-dione 86 80 131 201 162 173 187 216 229 258 271 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 EI Advantages • Simplest source design of all • EI mass spectrometers even go to other planets! • Robust ionization mechanism – Even noble gases are ionized by EI • Fragmentation patterns can be used to identify molecules – NIST ’08 library has over 220,000 spectra – Structures of novel compounds can be deduced EI Disadvantages • Fragmentation makes intact molecular ion difficult to observe • Samples must be in the gas phase • Databases are very limited – NIST’08 only has 190,000 unique compounds • Interpreting EI spectra is an art Empty vial 0 3-methyl-2heptanone 4 8 octane 2 3 Retention Time (min) 4 16 20 2,4-decenedial 2-decenal nonanal 2-nonenal Octanoic acid 12 2-octenal 1 octanal 2-pentylfuran heptanal hexanal 2-undecenal pentanal butanal DCM heptane hexane pentane Oleic acid heated to 150 C 0 24 Abundance m /z--> Scan5576(17.680m in): BR O N-O LEIC3.D 57 100000 95000 90000 Mass Spectrum from 17.68 minutes 85000 80000 75000 70000 65000 98 60000 41 55000 70 50000 82 45000 40000 35000 30000 25000 20000 114 15000 10000 124 109 5000 65 51 77 93 87 142 0 40 50 60 70 80 90 100 110 120 130 140 Abundance Scan5576(17.680min): BRON-OLEIC3.D 57 Mass Spectrum from 17.68 minutes 8000 6000 98 41 70 82 4000 2000 114 124 51 91 142 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 m/z--> Abundance m/z--> #19126: Nonanal 57 Nonanal Library Mass Spectrum 8000 41 6000 98 4000 29 70 82 2000 114 51 13 0 10 20 30 40 50 91 63 60 70 80 90 124 107 100 110 131 120 130 142 140 A Word About Quantification • GC-MS is a quantitative technique • Use only 1 m/z when quantifying a compound • Intensity is proportional to concentration – I α [X] – α is unique to each compound – The more two compounds differ chemically, the more careful one must be when comparing their intensities • Ideally a calibration curve is constructed using multiple solutions of pure analyte at varying concentrations Let MSF Staff Know • If peaks look broad or non-Gaussian – Implies time to change liner or septum • If the same unexplainable mass is seen in multiple injections – 207, 281, 355, 429 are septum bleed – 185 is tributylamine (from TBA-BF4 or TBAF) • Any time an error shows up on the computer Training Times • We will start training new walk-up users AFTER November 7 • Angie and I usually train groups of 2 or 3 • Training takes about 1 hour • Read through online training manual prior to your session to speed the process • Email jkarty@indiana.edu or asorg@indiana.edu AFTER 11/7 to make an appointment