Transcript Analyzing Small Molecules by EI and GC-MS

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 [email protected] or
[email protected] AFTER 11/7 to make an
appointment