Gas-chromatography/mass spectrometry (GC-MS)

Interpretation of EI spectra

Jeremy Keirsey
CCIC MSP
Mass Spec Summer Workshop
August 17, 2015
Gas Chromatography
Important goals in chromatography
- Achieve the best separation
- Little band-broadening – narrow chromatogram peaks – efficient column
- “Dynamic range”
- Separate and detect the “small” in the close vicinity of the “big”
- Reproducibility
- Stable peak positions, retention times

Even with the best column diffusion always plays a role!

Parameters to control diffusion

Particle size (Column type)

Flow rate
The Van Deemter Equation
HETP = A + (B/u) + (Cs + Cm) × u
HETP = height equivalent to a theoretical plate
-a measure of the resolving power of the column

H = L/N
 The Van Deemter Equation
HETP = A + (B/u) + (Cs + Cm) × u
•HETP = height equivalent to a theoretical plate, a measure of the resolving power of the column [m]
(Height = Length/number of plates=L/N)

•A = Eddy-diffusion parameter, related to channeling through a non-ideal packing [m]

•B = diffusion coefficient of the eluting particles in the longitudinal direction, resulting in dispersion [m2 s−1]

•C = Resistance to mass transfer coefficient of the analyte between mobile [m] and stationary phase [s]

•u = Linear Velocity [m s−1]

Original paper: Van Deemter JJ, Zuiderweg FJ and Klinkenberg A (1956). "Longitudinal diffusion and resistance to mass transfer as causes of non ideality in
chromatography". Chem. Eng. Sc. 5: 271–289

Youtube: https://www.youtube.com/watch?v=8i_4-OMCANE
The Van Deemter Curve
 Resolution

Solute moving through a column spreads into a Gaussian shape with standard deviation σ. Common Measures of
breadth are:
1) The width w½ measured at half-height
2) The width w at the baseline between tangents drawn to the steepest parts of the peak (inflection points).
Derive Van Deemter for Resolution
HETP = L/N

N = L/N substitute H = σ2/L

N = L2/σ2 convert length to time (utility)

N = (tr)2/(σ)2 relate σ to w1/2 (2.35σ) and w (4σ)

N = 16tr2/w2

N = 5.55tr2/w1/22
GC coupled to Mass Spectrometry (MS)
Autosampler

MS Analyzer: GC oven GC controller

-Quadrupole -GC Column
-Ion trap
-Time of flight (TOF)
-Orbitrap
GC Columns
• Two Common Formats
• Packed columns (most common with bonded liquid coating)
• Open tubular (typically long columns with small diameters)

Packed Columns Open Tubular (end on, cross section view)

Polyimide Coating
Column Wall (fused silica)

Mobile phase
Stationary phase (wall coating)
GC Columns
• Advantages of Open Tubular Columns
• Best resolution (negligible A term, small C term in Van Deemter Equation)
• More robust
• Better sensitivity with many detectors (due to less band broadening vs. lower
mass through column)
• Column Selection
• High resolution (thin film, 0.25 mm diameter, 60 m) vs. higher capacity (thick
film, 0.53 mm diameter)
• Stationary phase based on polarity
 GC Stationary Phase

Retention factor (k’) describes the migration of the analyte on the column
k’= (tR – tM)/tM

Selectivity factor (α) describes the separation of 2 species, A and B, on the column
α = k’B/k’A

Resolution (R) = ¼ √N (α-1/α) (k’/k’+1)

efficiency selectivity retention
 GC Adjustments
• k is adjusted by changing temperature (higher T means smaller k)
• Selection of stationary phase affects k and α values
• The α values are adjusted by changing column (will work if there is a difference in solute polarity)
• example: separation of saturated and unsaturated fatty acid methyl esters (FAMEs).

O
CH3 C18:0 FAME bp = 369°C
H3C
O

bp = 352°C
CH3
O
C18:1 FAME
H3C
O

• Retention of C18:0 and C18:1 FAMEs on RTX-5MS columns is very similar (due to similar boiling points)
• Retention on more polar columns (RTX-50MS) is greater for the more polar unsaturated FAMEs

• Main concerns of stationary phase are: polarity, functional groups, maximum operating temperature, and
column bleed (loss of stationary phase)
• More polar columns suffer from lower maximum temperatures and greater column bleed-derivatize?
• Changing carrier gas has no effect on retention
GC Column Options
Type Functional Groups Polarity

RTX-1MS 100% dimethyl Non-polar

RTX-5MS 5% diphenyl/95% dimethyl Low polarity

RTX-50MS Phenyl methyl More polar

Stabliwax-MS PEG High polarity

Chirality Columns

β-cyclodextrin
GC Column Options

RTX-1MS = NonPolar RTX-5MS = Low Polarity

Saturated Hydrocarbons Ethers

Olefinic Hydrocarbons Ketones
Aromatic Hydrocarbons Aldehydes
Halocarbons Esters
Mercaptans Tertiary amines
Sulfides Nitro compounds without α-H atoms
CS2 Nitrile compounds without α-H atoms

Long lifetime and very low bleed at high operating temperatures. Column specifically tested for low-bleed performance.
Temperature range: -60 °C to 350 °C Temperature range: -60 °C to 350 °C.
GC Column Options

RTX-50MS = Medium Polarity stabliwax-5MS = High Polarity

Alcohols Polyhydroxyalcohols
Carboxylic acids Amino alcohols
Phenols Hydroxy acids
Primary and secondary amines Polyprotic acids
Oximes Polyphenols
Nitro compounds without α-H atoms
Nitrile compounds without α-H
atoms
Low bleed Low bleed but rugged enough to with stand repeated cycles without
Temperature range: 40 °C to 320 °C. retention time shifting.
Sensitive to water and oxidation.
Temperature range: 40 °C to 250/260 °C.
 GC-MS Applications
- Environmental monitoring
- Food, beverage, flavor, and fragrance analysis
- Forensic and criminal cases confirmation test
- Biological and pesticides detection
- Fermentation control
- Security and chemical warfare agent detection
- Astro chemistry and Geo chemical research
- Medicine and Pharmaceutical Applications
- Petrochemical and hydrocarbon analysis
- Clinical toxicology
- Energy and fuel applications
- Industrial use
- Academics

http://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdf
GC-MS Projects in the MS&P

https://www.youtube.com/watch?v=7yxWBOPNkJ8
GC-Chromatogram
EI Spectrum
 The Nitrogen Rule
• Compounds* that contain even number of N atoms have even
number of nominal molecular weight
• Compounds* that contain odd number of N atoms have odd number
of nominal molecular weight

But what about singly protonated molecules and accurate molecular weights??

* Common organic compounds

Ion Stabilities
• Even electron ions are more stable than odd electron (radical) ions

How about protonated molecules: even electron or not?

And how about ions formed by electron impact (EI) ionization?

 Selected Isotope Ratios and 13C Contributions

Accurate elemental masses:

C: 12.000000
H: 1.007825
O: 15.9949
N: 14.003
Cl: 34.9688
Br: 78.9183
S: 31.9720
Use of Isotope Ratios to Distinguish Structures
Increasing 13C contribution in polyalanines
 1 0 0

Increasing 13C contribution 375.2118 (exact mass)

R e la t iv e I n t e n s it y
8 0

in polyalanines 6 0

4 0
375.4263 (average mass)

(Ala)5
2 0 C15H29O6N5
0
3 7 4 3 7 6 3 7 8 3 8 0 3 8 2
m /z

1 0 0
1975.9541 (exact mass)

R e la t iv e I n t e n s it y
8 0

6 0 1977.1722 (average mass)

4 0
(Ala)25 C90H129O26N25
2 0
3568 (nominal mass)
0
1 9 7 6 1 9 7 8 1 9 8 0 1 9 8 2 1 9 8 4
m /z

1 0 0
3569.8663 (exact mass)

R e la t iv e I n t e n s it y
8 0
3572.0062 (average mass)
6 0

4 0 (Ala)50 C150H252O51N50
2 0
3568 (nominal mass)
0
3 5 7 4 3 5 7 6 3 5 7 8 3 5 8 0 3 5 8 2
m /z
Increasing 13C 100 a 8 c

fr o m n o m in a l m a s s
374.4183 (average mass)
contribution in
7
average mass

R e la tiv e In te n s ity
80

D e v ia n c e
6

( d a lto n )
[Ala5+H]+
polyalanines 60 5
4
40
374.2040 (exact mass) 3
2
20
1 exact mass
0 0
374 376 378 380 382 0 2000 4000 6000 8000
m /z m /z

100 b 3572.9742 (average mass)

R e la tiv e In te n s ity

80
[Ala50+H]+
60

40 3570.8741 (exact mass)

20

0
3570 3572 3574 3576 3578
m /z

[Ala120+H]
GC Chromatogram- an example for you
EI Spectrum- give the top and bottom a try
Characteristic Isotope Distribution of Selected Transition Metal Elements
190 Origin of m/z 190?
255
Metal
element?
Transition Metal?
Isotope Ratios to Identify Chemical Compositions…No Accurate Mass!
Isotope Ratios to Identify Chemical Compositions…No Accurate Mass!
Isotope Ratios to Identify Chemical Compositions…No Accurate Mass!

10B: 25%
11B: 100%

What Happened?
Renormalized Isotope Distribution in the M+. Region
 43

15
86

Try these!!

105
77 182
…and these!! 102
129

117

Halogen?
…and this!! Use Isotope
Distribution in
the M+. Region
…and these!!
GC-MS Projects in the MS&P

https://www.youtube.com/watch?v=7yxWBOPNkJ8
Untargeted GC-MS Analysis of Spider Silk Droppings
-the hunt for hormones
Untargeted GC-MS Analysis of Plants Exposed to Crude Oil
Targeted GC-MS Analysis of Caffeine in Urine-Full Scan
Targeted GC-MS Analysis of Caffeine in Urine-SIM
Targeted GC-MS Analysis of SCFAs (Short Chain Fatty Acids) in Mouse Feces

acetic acid
propanoic acid
butyric acid
4-methyl valeric acid
Quantitative GC-MS Analysis with Xcalibur
Xcalibur Method Setup-DSQ II
Xcalibur Method Setup-Trace GC Ultra
Xcalibur Method Setup-Autosampler
Xcalibur Processing Method Setup
Xcalibur Processing Method Setup-Identification
Xcalibur Processing Method Setup-Detection
Xcalibur Processing Data
Xcalibur Processing Data
Xcalibur Exported Results
Make Discoveries
700
140000000

600
120000000

500
100000000

400
80000000

60000000 300

40000000 200
y = 118779x - 3E+06
R² = 0.9991
20000000 100

0
0
0 200 400 600 800 1000 1200