Gas-chromatography/mass spectrometry (GC-MS) Interpretation of EI spectra

Jeremy Keirsey CCIC MSP

Mass Spec Summer Workshop August 17, 2015

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

Gas Chromatography

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

•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 Equation

HETP = A + (B/u) + (Cs + Cm) × u

http://en.wikipedia.org/wiki/HETPhttp://en.wikipedia.org/wiki/Eddy-diffusionhttp://en.wikipedia.org/wiki/Diffusion_coefficienthttp://en.wikipedia.org/wiki/Dispersion_relationhttp://en.wikipedia.org/wiki/Mass_transfer_coefficienthttp://en.wikipedia.org/w/index.php?title=Linear_Velocity&action=edit&redlink=1

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

MS Analyzer: -Quadrupole -Ion trap -Time of flight (TOF) -Orbitrap

GC oven -GC Column

GC controller

Autosampler

GC coupled to Mass Spectrometry (MS)

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)

Column Wall (fused silica)

Mobile phase

Stationary phase (wall coating)

Polyimide Coating

• 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 Columns

GC Stationary Phase

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

Retention factor (k’) describes the migration of the analyte on the column

k’= (tR – tM)/tM

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).

• 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

O

CH3

O CH3

O

CH3

O CH3

C18:0 FAME

C18:1 FAME

bp = 369°C

bp = 352°C

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

GC Column Options

Chirality Columns

β-cyclodextrin

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

GC Column Options

Saturated Hydrocarbons Olefinic Hydrocarbons Aromatic Hydrocarbons Halocarbons Mercaptans Sulfides CS2

Long lifetime and very low bleed at high operating temperatures. Temperature range: -60 °C to 350 °C

Ethers Ketones Aldehydes Esters Tertiary amines Nitro compounds without α-H atoms Nitrile compounds without α-H atoms

Column specifically tested for low-bleed performance. Temperature range: -60 °C to 350 °C.

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

GC Column Options

Alcohols Carboxylic acids Phenols Primary and secondary amines Oximes Nitro compounds without α-H atoms Nitrile compounds without α-H atoms

Polyhydroxyalcohols Amino alcohols Hydroxy acids Polyprotic acids Polyphenols

Low bleed but rugged enough to with stand repeated cycles without retention time shifting. Sensitive to water and oxidation. Temperature range: 40 °C to 250/260 °C.

Low bleed Temperature range: 40 °C to 320 °C.

http://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdf

- Environmental monitoring - Food, beverage, flavor, and fragrance analysis - Forensic and criminal cases - 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

GC-MS Applications

confirmation test

http://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://www.omicsonline.org/open-access/gc-ms-technique-and-its-analytical-applications-in-science-and-technology-2155-9872.1000222.pdfhttp://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?

Accurate elemental masses: C: 12.000000 H: 1.007825 O: 15.9949 N: 14.003 Cl: 34.9688 Br: 78.9183 S: 31.9720

Selected Isotope Ratios and 13C Contributions

Use of Isotope Ratios to Distinguish Structures

Increasing 13C contribution in polyalanines

3 5 7 4 3 5 7 6 3 5 7 8 3 5 8 0 3 5 8 20

2 0

4 0

6 0

8 0

1 0 0

Rel

ativ

e In

tens

ity

m / z

3 7 4 3 7 6 3 7 8 3 8 0 3 8 20

2 0

4 0

6 0

8 0

1 0 0

Rel

ativ

e In

tens

ity

m / z

(Ala)5 C15H29O6N5

375.2118 (exact mass)

375.4263 (average mass)

3 5 7 4 3 5 7 6 3 5 7 8 3 5 8 0 3 5 8 20

2 0

4 0

6 0

8 0

1 0 0

Rel

ativ

e In

tens

ity

m / z

3 7 4 3 7 6 3 7 8 3 8 0 3 8 20

2 0

4 0

6 0

8 0

1 0 0

Rel

ativ

e In

tens

ity

m / z

1 9 7 6 1 9 7 8 1 9 8 0 1 9 8 2 1 9 8 40

2 0

4 0

6 0

8 0

1 0 0

Rel

ativ

e In

tens

ity

m / z

1975.9541 (exact mass)

1977.1722 (average mass)

3569.8663 (exact mass)

3572.0062 (average mass)

(Ala)50 C150H252O51N50 3568 (nominal mass)

(Ala)25 C90H129O26N25 3568 (nominal mass)

Increasing 13C contribution in polyalanines

3 7 4 3 7 6 3 7 8 3 8 0 3 8 20

2 0

4 0

6 0

8 0

1 0 0

Re

lati

ve

In

ten

sit

y

m / z

3 5 7 0 3 5 7 2 3 5 7 4 3 5 7 6 3 5 7 80

2 0

4 0

6 0

8 0

1 0 0

Re

lati

ve

In

ten

sit

y

m / z

a

b

374.4183 (average mass)

374.2040 (exact mass)

3570.8741 (exact mass)

3572.9742 (average mass)

0 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 00

1

2

3

4

5

6

7

8

De

via

nc

efr

om

no

min

al

ma

ss

(da

lto

n)

m / z

c

[Ala5+H]+

[Ala50+H]+

exact mass

average mass

[Ala120+H]

Increasing 13C contribution in polyalanines

GC Chromatogram- an example for you

EI Spectrum- give the top and bottom a try

Characteristic Isotope Distribution of Selected Transition Metal Elements

255

190 Origin of m/z 190?

Metal element?

Transition Metal?

Isotope Ratios to Identify Chemical Compositions…No Accurate Mass!

Isotope Ratios to Identify Chemical Compositions…No Accurate Mass!

What Happened?

10B: 25% 11B: 100%

Isotope Ratios to Identify Chemical Compositions…No Accurate Mass!

Renormalized Isotope Distribution in the M+. Region

86

43

15

182

105

77

Try these!!

129 102

117

Halogen?

…and these!!

…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

0

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700

y = 118779x - 3E+06 R² = 0.9991

0

20000000

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Gas-chromatography/mass spectrometry (GC-MS)�Interpretation of EI spectraGas ChromatographySlide Number 3The Van Deemter EquationThe Van Deemter CurveResolutionDerive Van Deemter for ResolutionGC coupled to Mass Spectrometry (MS)�GC ColumnsSlide Number 10GC Stationary PhaseGC AdjustmentsGC Column OptionsGC Column OptionsGC Column OptionsGC-MS ApplicationsGC-MS Projects in the MS&PGC-ChromatogramEI SpectrumThe Nitrogen RuleIon StabilitiesSelected Isotope Ratios and 13C ContributionsUse of Isotope Ratios to Distinguish StructuresIncreasing 13C contribution in polyalaninesSlide Number 25Slide Number 26GC Chromatogram- an example for youEI Spectrum- give the top and bottom a tryCharacteristic Isotope Distribution of Selected Transition Metal Elements�Slide Number 30Slide Number 31Slide Number 32Isotope Ratios to Identify Chemical Compositions…No Accurate Mass!�Slide Number 34Slide Number 35Renormalized Isotope Distribution in the M+. RegionSlide Number 37Slide Number 38Slide Number 39Slide Number 40GC-MS Projects in the MS&PUntargeted GC-MS Analysis of Spider Silk Droppings�-the hunt for hormonesUntargeted GC-MS Analysis of Plants Exposed to Crude OilTargeted GC-MS Analysis of Caffeine in Urine-Full ScanTargeted GC-MS Analysis of Caffeine in Urine-SIMTargeted GC-MS Analysis of SCFAs (Short Chain Fatty Acids) in Mouse FecesQuantitative GC-MS Analysis with XcaliburXcalibur Method Setup-DSQ IIXcalibur Method Setup-Trace GC UltraXcalibur Method Setup-AutosamplerXcalibur Processing Method SetupXcalibur Processing Method Setup-IdentificationXcalibur Processing Method Setup-DetectionXcalibur Processing DataXcalibur Processing DataXcalibur Exported ResultsMake Discoveries