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 autosampler feeds one of two

split/splitless injectors

• Normal methods use “split mode” for

concentrated samples

– Usually aim for <50 ng on column

– 1 uL injection, 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 CO2 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

More EI Mass Spectra

(mainlib) Coca ine10 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

HO

O

H OO

Cocaine

(mainlib) 3,4-Pyrid inedimethanol, 5-hydroxy-6-methyl-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

OH

OH

NVitamin B6

(mainlib) Androst-4-ene-3,17-dione10 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 ’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

04

81

21

62

02

4

01

23

4

pentane

DCM

hexane

pentanal

heptane

Re

ten

tion

Tim

e (m

in)

butanal

hexanal

octane

heptanal

2-pentylfuran

octanal

2-octenal

nonanal

2-nonenal

Octanoic acid

2-decenal

2-undecenal2,4-decenedial

3-methyl-2-

heptanone

Empty vial

Oleic acid heated 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

• 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 to make an appointment