analyzing small molecules by ei and gc-ms. samples appropriate for gc-ms volatile and semi-volatile...
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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 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 (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 ApplicationsE. De Hoffmann, J. Charette, V. Strooband, eds., ©1996
More EI Mass Spectra
(m a in lib ) C o c a in e10 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
0
50
100
15 27
42
5159 68
77
82
94105
122140 152 166
182
198 272
303
N
H O
O
H OO
Cocaine
(m a in lib ) 3,4-Pyrid in e d im e th a n o l, 5-h yd ro xy-6-m e th yl-10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
0
50
100
27 31
3953
6781
94106
122
136
151
169
HO
O H
O H
NVitamin B6
(m a in lib ) A n d ro st-4-e n e -3,17-d io n e10 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
0
50
100
18 2941 55 67
79
86
91 97
109
124
131
148
162173 187
201
216 229
244
258 271
286
O
O Androstenedione
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
0 4 8 12 16 20 24
0 1 2 3 4
pent
ane
DCM
hexa
ne
pent
anal
hept
ane
Retention Time (min)
buta
nal
hexa
nal
octa
ne
hept
anal
2-pe
ntyl
fura
n
octa
nal
2-oc
tena
l nona
nal
2-no
nena
lO
ctan
oic
acid 2-
dece
nal
2-un
dece
nal
2,4-
dece
nedi
al
3-methyl-2-
heptanone
Empty vial
Ole
ic a
cid
heat
ed to
150
C
40 50 60 70 80 90 100 110 120 130 1400
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
65000
70000
75000
80000
85000
90000
95000
100000
m/z-->
Abundance
Scan 5576 (17.680 min): BRON-OLEIC3.D57
98
41
7082
114
124109
65 7751 9387 142
Mass Spectrum from 17.68 minutes
10 20 30 40 50 60 70 80 90 100 110 120 130 1400
2000
4000
6000
8000
m/z-->
Abundance
Scan 5576 (17.680 min): BRON-OLEIC3.D57
9841
70 82
114
12451 91 142
10 20 30 40 50 60 70 80 90 100 110 120 130 1400
2000
4000
6000
8000
m/z-->
Abundance
#19126: Nonanal57
41
987029 82
114 1245113 91 14263 131107
Mass Spectrum from 17.68 minutes
Nonanal Library Mass Spectrum
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 [email protected] or
[email protected] AFTER 11/7 to make an appointment