Analyzing Small Molecules by EI and GC-MS July 2014
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 reduce 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 autosamplerfeeds one of two split/splitless injectors Normal methods use split mode for concentrated samples Usually aim for <50 ng on column 1 ulinjection, 50:1 split is default on most methods 1 mg/ml solution gives 20 ng on column at 50:1 Splitless mode is available for dilute samples Injector kept very hot (280 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 front injector is an Rxi-5Sil MS 95% dimethylsiloxane, 5% phenylsiloxane Very non-polar stationary phase from Restek 0.25 mm i.d., 30 m long, 0.25 μm film thickness Temperatures can range from -40-350 C <30 is available with liquid CO 2 tank Up to 20 C per minute ramp possible Default gradients A slow gradient from 40-300 C over 30 minutes A fast gradient from 70-280 C in 18 minutes
Other Column Chemistries There are entire catalogs full of GC columns with different chemistries Any user-supplied column MUST be compatible with GC- MS (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 (special dimethylsiloxane for volatile compounds) DB-WAX (polyethylene glycol) Column change takes about 4 hours Schedule this DAYS in advance with Dr. Karty
Detection of Compounds As compound come off the end of the column, they need to be detected in some way A 5973 inert mass spectrometer is coupled to the outlet of the 6890 GC Provides both retention time AND electron ionization mass spectrometric information
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
100 50 Cocaine 82 More EI Mass 182 Spectra 77 94 105 42 303 51 15 27 59 68 122 198 140 152 166 272 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 (ma inlib) Coca ine N H O O H O O 100 Vitamin B6 N 94 106 151 169 50 HO OH 27 31 39 53 OH 67 81 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 (ma inlib) 3,4-Pyrid ined imetha nol, 5-hyd roxy-6-methyl- 100 50 O 109 124 148 O 79 91 97 201 41 55 67 162 131 173 187 229 18 29 86 216 258 271 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 (ma inlib) And rost-4-ene-3,17-d ione 122 136 Androstenedione 244 286
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 11 library has over 240,000 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 11 only has 213,000 unique compounds Interpreting EI spectra is an art Suggest reading Interpreting Mass Spectra by McLafferty and Turecek as a reference
Empty vial 3-methyl-2- heptanone Retention Time (min) 0 4 8 12 16 20 24 hexanal heptanal 2-octenal 2-pentylfuran octanal nonanal 2-nonenal Octanoic acid 2,4-decenedial Oleic acid heated to 150 C 0 1 2 3 4 octane 2-decenal butanal pentanal DCM 2-undecenal hexane heptane pentane
Abundance 100000 Scan 5576 (17.680 min): BRON-OLEIC3.D 57 95000 90000 85000 80000 Mass Spectrum from 17.68 minutes 75000 70000 65000 60000 55000 41 98 50000 45000 40000 70 82 35000 30000 25000 20000 15000 10000 5000 0 m/z--> 109 114 124 51 65 77 87 93 142 40 50 60 70 80 90 100 110 120 130 140
Abundance 8000 Scan 5576 (17.680 min): BRON-OLEIC3.D 57 Mass Spectrum from 17.68 minutes 6000 41 70 82 98 4000 2000 0 m/z--> Abundance 114 124 51 91 142 10 20 30 40 50 60 70 80 90 100 110 120 130 140 8000 41 57 #19126: Nonanal Nonanal Library Mass Spectrum 6000 4000 29 70 82 98 m/z--> 2000 0 114 124 13 51 63 91 107 131 142 10 20 30 40 50 60 70 80 90 100 110 120 130 140
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 analyteat 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-BF 4 or TBAF) Any time an error shows up on the computer
Training Times 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.eduor asorg@indiana.edu to make an appointment