Short Course on Compound-Specific
Isotope Ratio Mass Spectrometry
O
H
C
N
14th April 2003, School of Chemistry, University of Bristol
Organic
Geochemistry
Unit
SIMSUG Short Course on
Compound-Specific Isotope Analysis
Programme:
8:30 to 8:40
Welcome
Rich Pancost
8:40 to 8:50
Introduction to compound-specific isotope analysis
Richard Evershed
8:50 to 9:10
Laboratory techniques in compound-specific isotope
analysis
Ian D. Bull
9:10 to 9:30
Derivatisation of compounds for compound-specific
isotope analysis
Bart van Dongen
9:30 to 9:55
Gas Chromatography-Isotope Ratio Mass Spectrometry
(GC-IRMS) instrumentation
Rich Pancost
9:55 to 10:15
Discussion and Questions
10:15 to 10:30
Coffee
10:30 to 10:50
Troubleshooting in GC-IRMS
Jim Carter
10:50 to 11:20
Data analysis in GC-IRMS
Hazel Mottram
11:20 to 11:55
CSIA for other isotopes
Andreas Hilkert
11:55 to 12:25
Case studies using compound-specific isotope analysis
Zoë Crossman and
Mark Copley
12:10 to 12:45
Discussion and Questions
12:45 to 1:30
Lunch and Further Discussion
8:40 to 8:50
Introduction to compound-specific isotope analysis
Richard Evershed
An introduction to
compound-specific stable isotope
determinations by gas
chromatography-isotope ratio mass
spectrometry
Richard P. Evershed
Definitions
• Stable isotope ratio mass spectrometry (IRMS)
Used to achieve high precision determinations of the
variations in stable isotope composition:
Abundance ratio R = 13C/12C
δ13C values have units of per mil (o/oo)
Rstandard = 0.0112372 for PDB (but assigned value of 0o/oo)
• Stable isotopically labelled compounds have been
determined for many years using conventional MS but
cannot be used to determine high precision stable
isotope ratios at natural abundance - only capable of
determining variations in isotope compositions at the o/o
level, ca. three orders of magnitude lower precision
The profusion (and confusion) of acronyms!
• Isotope ratio monitoring-GC/MS (IRM-GCMS; Matthews
and Hayes, 1978)
• GC-IRMS
• GC-combustion-IRMS (GC-C-IRMS)
• GC-thermal conversion-IRMS (GC-TC-IRMS)
• Compound-specific isotope analysis (CSIA)
• Etc, etc.
• Beware!
Origin of gas chromatography-isotope ratio
mass spectrometry
• Gas chromatography-IRMS
– Concept of linking GC with IRMS evolved during the 1970s
and 1980s (Matthews and Hayes, 1978)
– GC separates organic compounds
– On-line reactor combusts compounds to CO2 (and N2)
– IRMS determines relative abundance ratio of 13C/12C as CO2
(15N/14N as N2)
– Apparently rather simple!
Research opportunities for
exploiting carbon isotopes
•
•
•
•
Differences in natural abundance due to isotopic
fractionation in nature
- Abiological vs biological processes
- C3 and C4 photosynthesis
- Biochemical pathways
- Environmental influences on organisms
Tracer methodologies; 13C replacing radiotracers
- Enriched substrates, i.e. commercially enriched gases,
chemically synthesised compounds, cultures
Versitility and scope
- Laboratory experiments
- Human subjects
- Field experiments
Improvements in stable isotope MS technologies
- Continuous flow instruments
- Compound-specific approaches
Why compound-specific determinations
rather than bulk?
• Dictated by the nature of the research question not by
fashion!
• Compound-specific and bulk determinations complimentary
• Advantages
- Linking molecular structure-stable isotope compositionsource or process
- Small sample sizes; only a few tens of nanograms of a
single compound required for a determination
- Complex materials or mixtures, e.g. living organisms
composed of biochemical components of widely varying
structures and origins
- Isotopic information accessible at the biochemical
building-block level,e.g. individual amino acids in a protein
Why compound-specific determinations
rather than bulk?
• Disadvantages
- Individual analyses slow to perform (hours rather than
minutes)
- Loss of sample integrity during sample preparation
- Technically more demanding; major manpower commitment
- More expensive initially and higher consumable costs
- Analytical precision lower than bulk approaches; no
compound-specific equivalent to the dual inlet although
improvements will come
Continuous-flow bulk and
compound-specific approaches
z
Bulk isotope ratio instruments
GC
Combustion
IRMS
- Large sample sizes, e.g. 10–3 g
- Minimal sample preparation
z
Compound–specific isotope ratio instruments
GC
Combustion
IRMS
- Very small sample sizes, e.g. 10–8 g
- Complex sample preparation procedures
- Requires knowledge of capillary GC, reactor systems and
low dead-volume gas handling systems
Sample preparation
8:50 to 9:10
Laboratory techniques in compound-specific isotope
analysis
Ian D. Bull
9:10 to 9:30
Derivatisation of compounds for compound-specific
isotope analysis
Bart van Dongen
Laboratory Techniques in Compound Specific
Stable Isotope Analysis
Ian D Bull
Sample
•
Aim: To isolate a complex extract containing hundreds of compounds
and separate it into discrete groupings of compound class amenable to
GC analysis
Raw sample
GC sample
An example analytical protocol
Sample
Total lipid extract
Biopolymer analysis
Chromatography
Acid fraction
•
•
Neutral fraction
Polar fraction
Lipids are extracted from the sample matrix and separated by
chromatography prior to analysis
Biopolymers need to be isolated from the lipid extracted residue
Step 1 – Sample preparation
•
•
•
•
Samples are freeze dried and crushed
Freeze dried to remove water and increase the effectiveness of solvent
penetrating the sample matrix
Crushed in liquid nitrogen to provide a greater surface area and a
homogenous sample
Inorganic complications, e.g. S – remove with activated Cu turnings
•
•
•
Glassware
– needs to be ‘clean’
• furnaced
• solvent extracted
– solvent bottles
Plasticisers
You!
Relative Intensity
Contamination
cholesterol
HO
10
15
20
Time (min)
25
30
• Contamination may be more dominant than the compounds of interest
• Co-elution of contaminants with compounds of interest
• Contaminent may be the same as the compounds of interest
Step 2 - Extraction
•
Aim: To extract lipids from the sample matrix whilst maintaining sample
extract integrity and project viability
•
•
•
Soxhlet
Ultrasonication
Bligh-Dyer
– normal
– acidified
Liquid/liquid extraction
Autoextraction
•
•
Factors to consider before extraction
•
•
•
•
What am I actually interested in?
Stability of compounds of interest
Type of matrix being extracted
Sample size - limiting factor
– small samples need high residue recovery rate
•
•
•
•
•
Soils, sediments - Soxhlet
Small samples - ultrasonication
Bacterial cultures, tissue - Bligh-Dyer
Aqueous solutions - liquid/liquid extraction
Proprietary autoextraction instruments – high sample throughput
Soxhlet extraction
•
•
•
•
Pre-extracted cellulose thimble
Continuous extraction for 16-24h
Enables solvent to be recycled
approximately 100 times during 24 h
cycle
Rigourous extraction but not suitable for
light or heat sensitive compounds, e.g.
ergosterol
HO
•
ergosterol
Large solvent volumes
Ultrasonication
•
•
•
•
•
•
Sample agitated ultrasonically to
assist solvent penetration
Normally performed with centrifuge
tube or vial containing sample and
solvent immersed in ultrasonic bath
Sonication applied for 15 min and
solvent removed and repeated
several times with fresh solvent,
solvent fractions combined
Faster than Soxhlet extraction –
large sample throughput
Less rigorous extraction
Good for very small samples
Bligh-Dyer
•
•
•
•
•
Monophasic solvent system
– buffered water, chloroform, methanol
– acidifed Bligh-Dyer using acidified water
Specifically designed for the extraction of fresh biological tissues
(breaks cell membranes)
Carried out in an ultrasonic bath
Simultaneous, efficient extraction of both hydrophobic (lipids) and other
hydrophilic cell components
High sample throughput
Bligh , E.G. and W.J. Dyer (1959) Canadian Journal of Biochemistry
and Physiology 37: 911-917
Total Lipid Extract
•
Lipid extraction yields the Total Lipid Extract (TLE)
– contains hundreds of observable compounds
High temperature GC
chromatogram of the TLE of
oak leaf litter
Step 3 - Separation of lipid extracts
•
Carried out by chromatography using the principles
– Stationary phase (silica, aluminium oxide) and mobile phase
(solvent)
– Different molecules have different affinities for the two phases
hence move through column or along plate at different rates
– Depends on size and/or functional groups
– Specialised stationary phases can exhibit an ionic affinity for
specific functional groups
Column Chromatography
Solvents added in
sequence
Glass ‘column’
Sample
Fraction 1: hydrocarbons
Sorbent
Fraction 2: TAGs, wax esters
Fraction 3: sterols, triterpenols, alcohols
Glass frit
Stopcock
Fraction 1
Hexane
Least polar
Fraction 2
DCM
Elutropic series
Increasing polarity
Fraction 3
DCM/methanol
Most polar
Step 3 - Separation of lipid extracts
•
Carried out by chromatography using the principles
– Stationary phase (silica, aluminium oxide) and mobile phase
(solvent)
– Different molecules have different affinities for the two phases
hence move through column or along plate at different rates
– Depends on size and/or functional groups
– Specialised stationary phases can exhibit an ionic affinity for
specific functional groups
Solid phase extraction
Treatment of non GC-IRMS amenable lipids
•
•
•
Wax esters, steryl esters,
triacylglycerols,
phospholipids are all
examples of compounds
that are non GC-IRMS
amenable yet are present
in the samples and can
yield important
information
Using phospholipids as an
example
Aliquot of the PLFA
fraction taken and
saponified to generate
GC-IRMS amenable
compounds, i.e. fatty
acids
O
O
O
O
O
O
- OH, 0.01 M
-O
O
R
OR'
R
OR'
OH
- OH
H
+
O
- OR'
R
O-
workup
O
OH
P OO-
Step 5 - Biopolymer analysis
•
Carbohydrates – acid hydrolysis of the lipid extracted residue
HO
O
HO
O
O
O
HO
O
HO
O
OH
OH
CH2OH
CH2OH
CH2OH
CH2OH
O
OH
n = 1-10000
72% H2SO4, RT, 1 h
1 M H2SO4, 100oC, 2.5 h
e.g.
H
OH
H
H
H
HO
OH
O
OH
CH2OH
H
glucose
O
O
OH
Step 5 - Biopolymer analysis
O
•
Proteins
e.g.
Gly
OH
NH2
O
OH
Phe
NH2
O
6 M HCl, 100oC, 24 h
Ser
OH
HO
NH2
O
Pro
OH
NH
S
Met
•
Still not ideal but we are getting there!
O
OH
NH2
Our example analytical protocol revisted
Sample
Total lipid extract
Biopolymer analysis
Chromatography
Proteins
Acid fraction
Neutral fraction
Polar fraction
n-alkanoic acids
Hydrocarbons
wax esters
n-alkanols
sterols, triterpenols
PLFAs
Carbohydrates
Summary
•
•
•
•
•
•
•
•
•
Identify you target compounds before proceeding with any form of
sample preparation
- well constructed hypotheses
Design extraction and separation procedures around the compounds of
interest
Be aware of limitations conferred by the remit of the investigation
Acquire the necessary infrastructure
Maintain an analytical environment
Be paranoid about contamination – blank runs
Use suitable standards to verify your analytical procedure and where
appropriate act as internal references for quantitative work
Never use the whole of your sample – mistakes will be made!
The methods shown are not prescriptive – experiment!
Derivatisation of compounds for
compound-specific isotope analysis
Bart van Dongen
Analytical protocol
Sample
Residue analysis
Total lipid extract
Chromatography
Acid fraction
n-alkanoic acids
Amino acids
Neutral fraction
Polar fraction
Hydrocarbons
wax esters
n-alkanols
sterols
triterpenols
PLFAs
Measurement possible?
Derivatisation
GC-IRMS
Carbohydrates
Talk outline
• Introduction
• Why do we need derivatisation?
• Conditions
• What are the general factors that affect derivatisation reactions?
• Different compound classes:
• Fatty acids
• Alcohols
• Monosaccharides
• Amino acids
n-Alkanes
31
Relative intensity
n-Alkanes obtained from a soil
29
27
33
25
21
23
Retention time
No functional groups
No derivatisation needed
GC-run of mixture of compounds
Relative intensity
n-Alkane
Sterols
Fatty acids
Retention time
Derivatisation
• Compounds that are too involatile, because of functional groups,
to analyse using GC can be chemically modified
• Derivatised (i.e. functional groups blocked by apolar groups)
• Examples of functional groups:
-Carboxylic acids
-Hydroxy
-Amino
• Examples of derivatisation reactions:
-Esterification
-Silylation
-Acetylation
• Many derivatisation reaction possible
• Which to choose?
Requirements
• Isotope effect (KIE= lightK/heavyK)
• Addition of as little carbon as possible
• Relatively fast reaction
• Good separation of compounds possible
• No interference with by-products
• Stable end-products
Isotope effect in derivatisation
Derivatisation reaction
Carbon atoms
target molecule
involved
No carbon atoms
involved
No isotope
effect
Isotope
effect
Conversion
100%
Method usable
Carbon atoms
derivatisation molecule
involved
Yes
Isotope
effect
Conversion
not 100%
Reproducible?
No
Method not usable
Addition of carbon by derivatisation
• Derivatisation results in the addition of carbon atoms, with different
δ13C values (compared to the original carbon atoms)
•Correction need to be made for every added carbon
nderivised compoundδ13Cderivitised compound-nderivative groupδ13Cderivative groupa
δ13Ccompound =
ncompound
n = the number of carbon atoms
a Rieley,
1994
Uncertainty due to added carbona
Uncertainty in δ-value
2.4
n=3
n=5
• Increase in uncertainty with increasing
addition of derivative carbon
1.2
n=10
• Effect larger if number of original carbon
atoms is smaller
n=20
n=30
0
1
10
20
Carbon number added
n = the number of original carbon atoms
a after
Rieley, 1994
Fatty acids
• Diazomethane method
Bond broken
O
R
O
C H2N2
OH
+ N2
R
OMe
• Fast, Irreversible reaction
• Isotope effect is on the carbon of the diazomethane
• Added in excess quantity
• Non-reproducable isotope effect
• Method not usable
Fatty acids
• BF3, MeOH derivatization
-
O
O
R
OH
BF3
MeOH
R
Bond broken
BF3
+
OH2
OMe
O
R
OMe
• Isotope effect is on the carbon of the fatty acid
• Conversion 100%
• No isotope effect
• Method usable
Fatty acids, GC-run standard mixture
Fatty acids; derivatised
Relative intensity
Old situation
Retention time
Alcohols
• Silylation using BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide)
and pyridine
Pyridine
R
OH
CF3
R
NSi(CH3)3
OSi(CH3)3
OSi(CH3)3
• No carbon atoms involved in reaction
• Usable method
• Disadvantage: products are stable but only for a relative short time
Alcohols, chromatogram
Alcohols in a forest soil
Relative intensity
24
28
26
22
30
23
25
27
32
29
31
Retention time
Monosaccharides
O
O
OH
OH
OH
HO
6
OH
OH
OH
OH
5
OH
Glucose
Ribose
• Main problems: Relatively large number of protection groups needed
Relatively small number of original carbon atoms (usually 4 to 6)
• How to minimize the addition of carbon?
(Silylation would add 12 to 18 carbon atoms)
Alditol acetate methoda
O
OH
OH
OH
OH
NaBH4
OH
Bond broken
OAc
O
OAc
O
O
OAc
OH
OH
OH
OH
OAc
OH
OH
OAc
Pyridine
OAc
• Isotope effect on the carbon of the reagent
• Fractionation effect seems constantb
• Method usable but correction factor needed
Disadvantages: Still a large number of carbon atoms added (8-12)
Actually measuring alditols; loss of information
a After
Gunner et al., 1961; b Macko et al 1998; Docherty et al., 2001
Loss of structural information
• Two hexoses, two pentoses etc. may lead to the same alditol
O
OH
HO
HO
HO
HO
OH
OH
OH
OH
OH
OH
OH
OH
Arabinose
O
Arabitol
Lyxose
• One hexose, pentose etc. may lead to two alditols
OH
OH
O
OH
HO
HO
OH
OH
OH
OH
OH
Fructose
OH
Glucitol
OH
HO
+
HO
OH
OH
OH
Mannitol
Methyl boroacetylation of monosaccharidesa
HO
HO
O
OH
OH
+
HO O
HO
OH
Bonds broken
OH
1)Methyl boroacetylation
2)Silylation
OH
Si O
O
O
O
B
Total 5
+
O
O
B
O
B
O
O
• Addition of relatively small number of carbon atoms (2-5)
• Measuring monosaccharides, not alditols
• Isotope effect is on carbon atoms of monosaccharidesb
• Reaction quantitative; No isotope effect
a After Reinhold et al., 1974; b van Dongen et al., 2001
OO B
Total 2
GC-IRMS of a monosaccharide mixture
glucose
arabinose
Relative intensity
xylose
mannose
Relative retention time
Amino acids
COOH
H2N
Main problems:
H
• Two different groups which needs protection
• Small number of own carbon atoms
R
2 (Glycine) to 11 (Tryptophan)
COOH
H2N
H
COOH
H2N
H
H
Glycine
N
H
Tryptophan
Amino acids, derivatisation
OH
O
1) iso-propanol/HCl
Bond broken
H 2N
H
2)
O
O
H
N
O
R
Bond broken
Isotope effect: Step 1;
OCH(CH3)2
O
H
R
F3C
O
CF3
CF3
On carbon atom of amino acida
Conversion 100%; method usable
Step 2;
On carbon atom of reagent
Fractionation effect seems constantb
Method usable but correction factor needed
aRieley
1994; bDocherty et al. 2001
Amino acids, GC-run
Relative retention time
Glu
Phe
Pro
Hyp
Leu
Ile
Val
Thr
Ser
Ala
Gly
Relative intensity
Asp
I.S.
Amino acids in a standard mixture
Amino acids, point to think about
Total 5
OCH(CH3)2
O
H
N
O
H
R
CF3
?
• Relatively large number of carbon atoms added (at least 5)
• A reaction with an isotope effect, although can be corrected for
• HF can be formed, causing problems when measuring isotopes
Summary
• Derivatisation methods are available which make it possible to determine
the δ13C values of the majority of functionalised compounds.
• However always bear in mind that:
1) derivatisation can cause an isotope effect
2) corrections are needed for the added carbon
GC-IRMS instrumentation and analysis
9:30 to 9:55
Gas Chromatography-Isotope Ratio Mass Spectrometry
(GC-IRMS) instrumentation
Rich Pancost
10:30 to 10:50
Troubleshooting in GC-IRMS
Jim Carter
10:50 to 11:20
Data analysis in GC-IRMS
Hazel Mottram
11:20 to 11:55
CSIA for other isotopes
Andreas Hilkert
Gas Chromatography-Isotope Ratio Mass
Spectrometry (GC-IRMS) instrumentation
Rich Pancost
GC-IRMS Instrumentation
Gas Chromatograph
Separates individual compounds, allowing
discrete isotopic compositions to be determined
Combustion/Reduction Interface
Converts eluting compounds into CO2 for
analysis
Continuous Flow Instrument
Allows the delivery of sample CO2 to an isotope
ratio mass spectrometer via a He stream
The GC-IRMS
ThermoFinniganMAT
design
The Gas Chromatograph
For further ref: http://gc.discussing.info/
Injector
Column
Backflush valves
The GC Injector
• Ideally, want on-column
injection
• Makes best use of
limited sample size
• Minimizes problems
associated with
isotopic fractionation
A split/splitless injector
An on-column injector
The GC Column
Polyimide Coating
Fused Silica
Stationary Phase
Expanded view of capillary tubing
The GC Column
The importance of investing in good
separation
Differences amongst columns:
Stationary phase
Length
Diameter
15 m
30 m
60 m
The Backflush System:
Necessary for the removal of solvent
• The small quantities of solvent used during injection
represent a large amount of organic material
introduced to the GC/C/IRMS system
• Excess carbon will:
• Saturate the oxidizing power of the combustion
furnace
• Saturate the NafionTM tubing with water
• Introduce excess carbon to the source, which is
quite bad for the filament
• Use of chlorinated solvents is particularly problematic
• Generate HCl in combustion furnace, could
corrode downstream components
Straight v. Backflush Configuration
The Backflush System
Combustion furnace
O2
He
The Combustion Furnace
• Converts organic compounds into CO2 and H2O
• Contains an oxidizing metal (CuO or ZnO) and
typically a catalyst (Pt)
4 CuO
2 NiO
ThermofinniganMAT
design
2 Cu2O + O2
2 Ni + O2
• Configuration dictates operating temperature
• CuO: 825-850°C (cannot operate at higher temperatures due to
thermal decomposition of CuO)
• NiO: 1150°C (can operate at lower temperatures but need
supplemental O2)
• Operation with supplemental O2 ideal for maximizing combustion
but quickly exhausts downstream reduction furnace
• Hybrid reactors favoured by many
• Key: avoid loss of chromatographic resolution
• must allow laminar flow and/or
• convert organic matter fully to CO2 rapidly
The Combustion Furnace
0
24 m
m
0
32 m
m
(240 mm long)
Note: heated zone is
260 mm, bracketing
wires
Capillary from GC (slides ~1.5 cm
into end of reactor)
He
Furnace must extend
at least 3 cm out of
heated zone
Oxidation of the Combustion Furnace
• To maintain oxidizing capacity of the furnace, it must
periodically be oxidized
• NOTE: CuO thermally degrades at 850°C; thus, the
furnace must be oxidized even if the system is not in use
• Immediately after oxidation, thermal desorption results in
high amounts of O2 being released through system
• This is bad for reduction furnace and filament
• Oxidation schemes
• Briefly (10-20 sec at end of each run)
• Every other day (overnight)
• Weekly (overnight)
Depends on usage,
metals and temperature
Oxidation of the Combustion Furnace
• Oxidation must be done in
backflush mode
• Insures flow through reactor
• Insures no flow into reduction
furnace and MS
The Reduction Reactor
• Purpose:
• Nitrous oxides are converted to N2
• Excess O2 is removed from analyte
stream
• Reactor material and components
largely the same as combustion furnace
• 3 Cu wires
• Operated at 650°C
The Water Trap
• Water generated during combustion is a
problem!
• Can protonate CO2 in MS source
resulting in elevated m/z 45 signals
• Can be removed with a cryogenic trap
OR
• Can be removed by passing analyte
stream through a selectively permeable
membrane (NafionTM) with a dry He
counterflow
The Open Split
• Adaptation for GC-IRMS that is
analogous to adaptations for any
continuous flow interface
He
• Capillary to MS has an inner diameter
of 0.1 mm
• Insures that delivery of He stream to
MS is 0.5 ml/min
• Thus, typically 1/4 of the analyte is
delivered to the MS
Analyte
Reference Gas Inlet
Reference
To MS
He
Allows introduction of reference
gas in a line parallel to the
analyte (i.e. two columns deliver
He and CO2 to MS)
Troubleshooting
(Tricks and Tips)
Jim Carter
with special reference to:
ThermoFinnigan GCC I-III
GC-IRMS interface
Three things to consider
Combustion Reactor
Gas Chromatograph
(organic compound)
Interface
(H2, N2, CO, CO2)
Axiom
There is no substitute for good chromatography
The gas measured must correspond to a single compound
Good Gaussian peak shape improves precision
Don’t assume …
The software will not “separate” your peaks
Minor components will affect your result
GC basics
(avoiding fractionation)
INJECTOR
Splitless, on-column or PTV injection
Constant flow rate = constant split ratio
COLUMN
0.32mm id / thick film
0.32mm id / thin film
0.25mm id
high sample loading
improved chromatography
improved split ratio
Tools of the trade
Hewlett Packard
Flowcalc 2.05
www.chem.agilent.com
/cag/servup/usersoft/main.html
Digital
flow meter
Tube reamers
Maximise your chromatography
•
•
•
•
Eliminate the usual chromatographic problems
Cold spots
Dead volumes
Active sites
Cold Spots
Reduce thermal mass
Remove metal
Dead Volumes
ZDV fitting
GC column
Oxidation reactor
Remove coating
Drill through
Dead Volumes
Bleed capillary
100µl / min
helium
Dead Volumes
1% oxygen
helium
Active Sites
GC column
System Checks
•
•
•
•
•
•
•
Know how your instrument performs when its working!
Don’t wait until it breaks!
Know - Flow rates - BF ON / BF OFF
Know - Chromatographic “dead time” (to)
Know - m/z 40 signal - BF ON / BF OFF (flow and leaks)
Know - m/z 18 signal – BF OFF (water in ion source)
Know - Standard ON/OFF test (standard deviation)
The Argon Test
•
•
•
•
Monitor m/z 40
Inject 1µl of air
RT = column t0 + interface t0 (10-15 sec)
Tailing = dead volume, blockage or leak
The Hexane Test
•
•
•
•
•
Monitor m/z 44
GC oven at ca. 100oC
Inject 1µl of hexane vapour
RT = approx. RT for argon
Tailing = cold spots or poor combustion
NB should resolve hexane isomers
What can possibly go wrong? (1)
Poor chromatography
Did RT change? (argon test)
No
Yes
Check m/z 40
Check Injector
(septum/liner)
OK
Check Ox. Reactor
Up
Check for leak
Check “T” piece
Down
Blockage
(check flows)
Check Ox. reactor
What can possibly go wrong? (2)
It’s the wrong numbers
•
•
•
δ13C values are consistently enriched
δ13C values are consistently depleted
δ13C values are unstable
Is chromatography OK?
No
Yes
As before
Which way did the δ value move?
Up
Down
N compounds?
Check m/z 18
High
Check source heater
OK
Check Nafion
Yes
Check Red. reactor
No
Is m/z 40 OK?
Yes
Check Ox. Reactor
(hexane test)
Isotope values are unstable
As before
No
Is chromatography OK?
Yes
Check for leaks
No
Check BF valve
Up
Is m/z 40 OK?
Yes
Is m/z 18 OK?
Yes
Standard ON/OFF test
Poor
Clean Ion Source
Summary
• Chromatography first and foremost
• Know your system when its working
• Have the right tools available
• Fault find systematically
(check/change one thing at a time)
• If all else fails:
rebuild the interface
start at the water trap – GC column
capillaries “go wrong” as do fittings
change one thing at a time
“Once upon a time in Indiana …..”
Data Analysis and Interpretation
Hazel Mottram
Axiom
You cannot get reliable isotope data
without good chromatography!
What is measured?
•
•
Unlike a conventional organic mass spectrometer, in which a range of
ions are measured, in isotope ratio mass spectrometry we only
measure a few ions
For δ13C analyses, three ions are measured:
– m/z 44
– m/z 45
– m/z 46
•
These correspond to the different isotopomers of CO2:
–
–
–
•
12C16O16O
(m/z 44)
13C16O16O and 12C17O16O (m/z 45)
12C16O18O (m/z 46)
A reference gas is used in the same manner as in routine isotopic
analysis to allow measurement of isotope ratios relative to a standard
What a run looks like!
What a run looks like!
Variation across peak
After Ricci et al (1994)
Peak integration - Automated
After Ricci et al (1994)
Manual integration
•
Must integrate the whole peak in both m/z 44 and 45/44 traces –
otherwise will get incorrect representation of isotope ratio
9
²
²
Manual integration
•
•
Must integrate the whole peak in both m/z 44 and 45/44 traces –
otherwise will get incorrect representation of isotope ratio
Peak area important – instrument is only linear over a certain range
Manual integration
•
•
•
Must integrate the whole peak in both m/z 44 and 45/44 traces –
otherwise will get incorrect representation of isotope ratio
Peak area important – instrument is only linear over a certain range
Peak shape important – unusual peak shapes (particularly in 45/44
trace) can indicate coelutions
Lichtfouse et al (1991)
The Background
•
•
It is crucial to have appropriate background as the software calculates
δ13C values from deviations from this
Can be done automatically
– On the basis of immediately preceding and subsequent data points
as described previously
– Or using dynamic background (develops a background from entire
analysis, smoothing data)
– Caution must be used! Especially for peaks eluting near a sudden
shift in the baseline (i.e. when the instrument shifts out of
backflush)
– Or manually (be sure to inspect both m/z 44 and ratio trace!)
Sample analysis:
Routine analysis with minor derivatisation
(Note: different labs have significantly different protocols)
•
Compounds with minimal co-elution and over 0.3 V amplitude:
– Samples should be run twice
– Samples should be run with co-injected standards which have been
measured off-line
• Approximately same abundance
• Same compound class if possible
– If duplicate runs are reproducible within 0.6 ‰ and standards are within
~0.5 ‰ of known values then no further runs are necessary
•
Compounds with 0.1 - 0.3 V amplitudes and minimal co-elution:
– The analytical precision of the instrument decreases at this range
(Merritt and Hayes, 1994)
– Samples should be run in triplicate
– Values should be reported with errors of ± 1.0 ‰
•
Values from compounds with amplitudes < 0.1 V should never be used!
Sample analysis: Coelutions
•
•
•
Ideally co-elution should be avoided using further clean up steps or a
different column
Where this is not possible, co-eluting peaks can be integrated together
using the integration software
For small overlaps, the co-eluting peaks can be integrated separately:
– Maximum co-elution of 25%
– A minimum estimate of analytical error can be gained by running a sample
in different concentrations
– Newer users should probably avoid trying to interpret co-elutions as this is a
very tricky area
•
For a description of the errors arising from co-elution, see Ricci et al
(1994)
Sample analysis:
Compounds requiring extensive derivatisation
•
•
•
•
Compounds such as amino acids and monosaccharides require more
extensive derivatisation, involving addition of numerous carbons and
often involving a reproducible kinetic isotope effect
These are both sources of error which must be accounted for
Kinetic isotope effect must be calculated for each compound under
each set of conditions
More replicates needed to minimise error
Correction for derivatising groups
•
With no kinetic isotope effect, e.g. methylation of a fatty acid, this is a
simple mass balance equation:
RCO2H
BF3/MeOH
RCO2Me
ncdδ13Ccd = ncδ13Cc + ndδ13Cd
where
n is number of moles of carbon
c refers to compound of interest
d refers to the derivative group
fd refers to the derivatised fatty acid
(Rieley, 1994)
Correction for derivatising groups
•
•
•
Measure value of derivatising compound offline e.g. BF3/methanol
Adjust value obtained for compound of interest accordingly
Example:
A value of –28.14 ‰ is obtained from the GC/C/IRMS analysis of C18:0 FAME
The BF3/MeOH is measured offline and found to have a value of -40.15 ‰
What is the -corrected δ13C value for the fatty acid?
ncdδ13Ccd = ncδ13Cc + ndδ13Cd
δ13Cc =
1
(ncdδ13Ccd - ndδ13Cd)
nc
= 1/18 {(19 x –28.14) – (1 x –40.15)}
= -27.47
Correction for derivatising groups
•
•
Where it is not possible to measure the value of the derivatising carbon
offline (e.g. where reagents are obtained in numerous small batches)
an alternative approach can be taken
The derivatised and underivatised compound are analysed and the
contribution of the derivatising reagent
ncdδ13Ccd = ncδ13Cc + ndδ13Cd
δ13Cd
•
=
1
(ncdδ13Ccd - ncδ13Cc)
nd
This should be repeated for each compound of interest
Derivatisation: BSTFA for alcohols
δ13Cd
•
= 1 (ncdδ13Ccd - ncδ13Cc)
nd
The contribution of the BSTFA to the overall δ13C value is normally
measured using a standard alcohol with known δ13C value
OH
OH
HO
myo-inositol
HO
OH
OH
•
The advantage of using myo-inositol is that it has a large number of
hydroxy groups
– Therefore a large quantity of derivative carbon is added and there is less
error in calculating its contribution
Correction for derivatising groups
•
•
Where there is a kinetic isotope effect, δ13Cd cannot be directly
determined
The kinetic isotope effect for each compound can be quantified
according to Rieley (1994):
KIE = 1 + ∆ncd / 1000x
where
•
ncd is the difference between the measured isotope value and that predicted
from mass balance equations
x is the number of groups available for derivatisation
Where the KIE is constant for a set of conditions, correction factors can
be calculated
− δ13C values of underivatised and derivatised compound are used to
calculate the effective stable isotope composition of the derivatising carbons
Errors where there is no KIE
BF3/MeOH
(IRMS)
± 0.1 ‰
Calculation
errors
propagate
FAME of interest
(GC/C/IRMS)
± 0.3 ‰
δ13C of FAME
of interest
±?
2
n 
2  nc + n d 
 + σ 2d  d 

σ c2 = σ cd
 nc 
 nc 
2
σc2 = 0.32 × (19/18)2 + 0.12 × (1/18)2
σc2 = 0.100
σc = 0.32 ‰
Docherty et al (2001)
Errors where a KIE is present
Underivatised
sugar
(IRMS)
± 0.1 ‰
Correction
factor
δcorr ± ?
Calculation
errors
propagate
Derivatised sugar
(GC/C/IRMS)
± 0.3 ‰
2
2
n 
 n + nd 
2  nc + n d 
 + σ cd


σ = σ  s  + σ 2sd  s
 nc 
 nc 
 nc 
2
c
2
2
s
σc2 = 0.12 × 5 2 + 0.32 × 5+102 + 0.32 × 5+10 2
5
5
5
2
σc = 1.63
σc = 1.3 ‰
Derivatised
sugar analyte
(GC/C/IRMS)
± 0.3 ‰
Calculation
errors
propagate
δ13C of sugar of
interest
±?
Docherty et al (2001)
Analysis of highly labelled compounds
•
•
•
How is the analysis of a sample containing highly labelled compounds
different from analysis at natural abundance?
Are the δ13C values of other compounds in that analysis affected?
To investigate potential carryover:
– FAME mixture containing 5 components at natural abundance analysed x 5
– Earlier eluting labelled compound added to mixture and analysed again
Within run carryover:
16:0* + natural abundance fame mix (1:1)
Within run carryover
16:0* + natural abundance fame mix (1:1)
16:1
17:0
17:1
18:0
-14
-16
-18
13
δ C (‰)
-20
-22
-24
-26
-28
-30
-32
after 16:0*
FAME std
18:1
18:2
Within run carryover
16:0* + natural abundance fame mix (2:1)
16:1
17:0
17:1
18:0
-14
-16
13
δ C (‰)
-18
-20
-22
-24
-26
-28
-30
-32
after 16:0*
FAME std
18:1
18:2
Within run carryover
Summary
•
•
Good isotope data requires good chromatography
Care must be taken during data analysis to ensure
1. Peak amplitudes are within range of instrumental linearity
2. Data is only collected from peaks with minimal coelution
3. Backgrounds are selected with care
•
•
Corrections must be made for atoms added during derivatisation
Errors must be accounted for
– This is particularly important when dealing with kinetic isotope effect
•
When analysing highly enriched compounds:
– Highly enriched components within a chromatographic run may adversely
affect the δ13C values of closely eluting compounds
The Way to N, H, O by irm-GC/MS
Andreas Hilkert
ThermoFinnigan MAT GmbH, Bremen
δ15N by irm-GC/MS
Introduced in 1992
Why 15N analysis ?
-30
Carbon Isotope Ratio, δ 13CPDB [‰]
Drugs
-31
-32
Source:
CO2, Air
Heroin
-33
PlantMetabolism
-34
Cocaine
-35
Source: N2, Soil
Plant Metabolism
-36
-14
-12
-10
-8
-6
-4
15
Nitrogen Isotope Ratio, δ Nair [‰]
3
-2
0
Advantages
+ Nitrogen specific detection
+ All carbon of sample matrix is removed
+ All carbon of column bleed is removed
+ Free choice of derivatives
+ Derivatization groups without Nitrogen
+ No isotope dilution
+ No isotope fractionation
+ No intramolecular 15N tracer dilution
e.g. 1-13C-Leucine vs. 15N-Leucine
4
N-selective irm-GC/MS Trace
Ref. gas
Ref. gas
Ref. gas
Comparison of FID trace and m/z 29 trace
N, O – tBDMS Amino Acid Derivatives
5
Challenges
+ Low abundance of 15N (ion statistics)
+ Low abundance in AA (sample amount)
+ N2 background
(signal/background)
+ leak tightness of GC/C system
+ purity of Helium carrier gas
+ 100 % N2 yield
(ox / red efficiency)
+ Interfering masses (m/z 28, 29, 30)
+ CO
(combustion efficiency)
+ CO+ from CO2+
(CO2 trap efficiency)
6
Comparison of δ15N and δ13C Determination
H
15
N
R
H
Accumulated Effect on
13
C
H
C O
O H
13
Element content
Atoms per gas molecule
15N, 13C abundance
Ionization efficiency rel. to CO2
Intensities of
- in relation to m/z 45 (13C)
- m/z 45 set to 100 %
N
C
<10 %
>60 %
2 (in N2)
0.732 %
ca. 70 %
1 (in CO2)
1.08 %
100 %
m/z 29 (15N)
8.33 %
(e.g. 5% N, 60% C)
4.17 %
2.82 %
1.98 %
Theoretical required sample amount for δ15N
50 x higher than for δ13C
if same precision as for δ13C is required
7
Challenges
The Effect of CO contamination
CO
Ratio
29/28
1.08 / 1
29
N2
0.732/ 1
28
δCON2 = (1.08/0.732 -1)*1000
= + 475 ‰
8
Combustion and Reduction
Organic Compound
100 % Combustion
Reduction
CO2, N2, H2O, NOx
Water Removal
CO2, N2, H2O
CO2, N2
CO2 Removal
100 % N2
9
Combustion and Reduction
100 % N2 at 100 % Combustion
10
Combustion Requirements for δ15N
• Complete Oxidation of C to CO2
• N2 Production optimized
– High Temperature (980 °C)
– Pyrolytic aspects
• NOx Production minimized
– No Excess of O2
– Indicator mass m/z 30 (NO)
-NHx
-CHx
11
Ox
Ox
N2
CO
Ox
Red
Ox
NOx
CO2
Combustion Efficiency
0
12
Sample Cleanup
• Water Removal
• CO2 Removal
13
δ15N Applications
O-iPropyl, N-Pivaloyl Amino Acid Derivatives
Data taken from C. Metges
14
Sample Size
1.5 nmol N2 on col.
15
Boosting the Limits
16
Recipe for δ15N irm-GC/MS
17
Injector
splitless
Retention Gap
3m deactivated fused silica
Capillary Column
50 m, 0.32 mm i.d., 0.5 µm filmthickness, e.g. Ultra 2, DB5, ...
Column Connectors
glass deactivated (e.g. Restek)
or metal / Vespel (e.g. Valco)
Oxidation Reactor
980 °C, restricted re-oxidation
Reduction Reactor
650 °C
CO2-Trap
cryogenic trap
Movable Open Split
release of trapped CO2
Sample
< 1.5 nmol N2 on column,
< 600 ng AA derivative o.c.
δ2H by irm-GC/MS
Introduced in 1998
Why δ2H irm-GC/MS ?
relatively D depleted
clay minerals
musts
wine water
natural gas
marine oils
non marine oils
C3 ethanol
C4 ethanol
C3 plants
GISP
SLAP
-400
19
-300
-200
C4 plants
SMOW
-100
0
100 ‰
Hydrogen Isotope Ratios
Schematic Fractionation in the Atmospheric Water Cycle
-94 ‰
Vapor
-110 ‰
Vapor
-14 ‰
Rain
OCEAN
δD = 0 ‰
20
-126 ‰
Vapor
-30 ‰
Rain
CONTINENT
We know where you eat !
13
An orphan's tail: variations of δ O and δ D in a single elephant hair
-50
δΟ
-55
12
-60
-65
δD
10
-70
-75
9
-80
-85
8
-90
7
TC/EA-DELTA+XL
ANALYST: H. Avak
6/23/2000
samples 200-600 µg
-95
6
-100
0
50
100
150
Length (mm)
21
200
250
δDSMOW (‰)
δ18 OSMOW (‰)
11
Comparison of δ2H and δ13C Determination
H
H
13
C
H
13
H
C
O
H
Accumulated Effect on
Intensities of
- in relation to m/z 45 (13C)
- m/z 45 set to 100 %
H
e.g. Ethanol
Element content
Atoms per gas molecule
2H, 13C abundance
Ionization efficiency rel. to CO2
H
C
m/z 3 (DH)
13 %
50 %
2 (in H2)
0.03 %
ca. 10 %
1 (in CO2)
1.08 %
100 %
300 %
(e.g. 6 H, 2 C)
150 %
4.16 %
0.42 %
Theoretical required sample amount for δD
240 x higher than for δ13C
if same precision as for δ13C is required
22
Low Energy Helium Ions
Under continuous flow conditions
4He is about 107 times more
abundant than HD
Due to collisions He ions with
less energy than 3 kV would
fall into the m/z 3 cup
magnet
m/z 4
m/z 3 (HD)
m/z 2 (H2)
ion source
23
universal triple collector
for N2, CO, O2,CO2, SO2
Contribution of He+ Ions at m/z 3
300
10
He abundance at m/z 3 [V]
without retardation lens
with retardation lens
He abundance at m/z 3 [V
250
200
150
8
6
4
2
0
0
100
0.02
0.04
0.06
0.08
0.1
He flow [ml/min]
50
0
0
0.1
0.2
0.3
He flow [ml/min]
24
0.4
0.5
The production of low energy He
ions is related to the He flow
into the ion source
Energy Filter - Retardation Lens
HD Collector - Rejection of 4He+ ions by a retardation lens
entrance slit
4
Ion Kinetic Energy [kV]
HD
retardation lens
ground ~Vacc
3
HD+
∆E>400 V
4
0
25
+
He+
He+
secondary electron
suppressor
-100 V
Faraday cup
ground
1012 Ω
Energy Filter
Complete absence of 4He
at m/z 3 cup with Energy Filter
26
E 0998 032 PO
H3+ Factor
H3+ Factor (K) = 6.01 ppm / nA
[H33++] = [H22]22 • K
30 V (nA)
227 mV (pA)
61 µV (fA)
0.31 µV H33++ (0.5 %)
27
m/z 3
12 mV (pA)
5.4 mV H33++ (45 %)
H3+ Factor – Linearity
6
y = 0.2x [‰ / V]
at H3+ 6.010621
5
δD [‰]
4
30 V (nA)
3
0.05 ppm
2
1
y = -0.01x [‰ / V]
at H3+ 6.06773
227 mV (pA)
0
-1
-2
0
5000
10000
15000
20000
Intensity [mV]
28
25000
30000
35000
Requirements to δ2H irm-GC/MS
¾ Quantitative Conversion
ƒ Empty reactor tube at ≥ 1400 °C
¾ GC Performance
ƒ Inner diameter of recommended GC columns: 0.25 mm
¾ Sensitivity
ƒ GC flow: 0.8 – 1.0 ml/min
Ì Optimal linear velocity
ƒ High He flow into IRMS: 0.4 ml/min
Ì Optimal open-split ratio
¾ Precision and Stability
ƒ Long-term stability of the H3+ factor
¾ Linearity
29
High Temperature Conversion
y
Cn Hx Oy
GC/TC
x/2
>1400 °C
n-y
30
CO
C
δ2H
H2
irm GC/MS
HD
H2
0.015 %
99.985 %
High Temperature Conversion
High Temperature Conversion Interface
31
High Temperature Conversion
Production of methane and hydrogen
from propane as a function of temperature
10
hydrogen
9
8
Signal (V)
7
6
propane
5
4
1440 °C
3
2
methane
1
0
600
800
1000
1200
1400
Temperature (°C)
Chart according to T. Burgoyne et al., Anal. Chem. 1998, 70, 5136-5141
32
Transfer into the Reactor
• Metal Connector / Ceramic Reactor – “Standard”
F.S.
i.d. = 0.25 mm
i.d. = 0.32 mm
GC oven < 320 °C
Al2O3
Heater
Heater 1450
940 °C°C
critical
volume
nowires
wires
• Metal Connector / Ceramic Reactor – “Optimized”
F.S.
i.d. = 0.32 mm
i.d. = 0.5 mm
GC oven < 320 °C
33
Al2O3
Heater
Heater 1450
940 °C°C
ca. 0.5 cm polyimide
are burnt off
nowires
wires
δD of Free Steroids (underivatized)
O
HO
HO
H
No.
O
Etiocholanolone
1
2
3
4
5
6
7
8
Mean-value:
Std-deviation:
H
Androsterone
H22 Intensity
Intensity (mV)
(mV)
H
6000
6000
5000
5000
Etiocholanolone
Etiocholanolone
4000
4000
Androsterone
Androsterone
3000
3000
mass
mass 22
2000
2000
mass
mass 33
1000
1000
00
820
820
840
840
860
860
880
880
900
900
Time
Time (s)
(s)
34
920
920
940
940
960
960
δ D/HSMOW [‰]
δ D/HSMOW [‰]
-227.08
-225.79
-225.84
-226.00
-228.90
-225.24
-224.98
-225.61
-226.18
1.26
-332.54
-332.27
-332.44
-332.56
-337.86
-332.94
-331.20
-333.47
-333.16
2.00
Metabolic Studies
enriched
0.32
0.30
0.28
0.26
0.24
0.22
0.20
0.18
D/H ratio trace
δ2H of Natural and Enriched
natural
3.50
C16:0 C17:0
3.00
Fatty Acid Methyl Esters
m/z 2 trace [V]
2.50
2.00
1.50
C18:1
1.00
0.50
800
900
Time [sec]
35
1000
1100
1200
FAME
Sample 1
Sample 2
Methyl Palmitate
200 ng
200 ng
C16:0
- 313.6 ‰ ± 5.6 ‰
- 321.8 ‰ ± 3.1 ‰
Methyl Heptadecanoate
200 ng
200 ng
C17:0
- 302.4 ‰ ± 2.7 ‰
- 303.4 ‰ ± 2.7 ‰
Methyl Oleate
40 ng
20 ng
C18:1
21305 ‰ ± 191 ‰
21602 ‰ ± 94 ‰
δ18O by irm-GC/MS
Introduced in 1996
Why δ18O irm-GC/MS?
20
0
Honey
-20
Meteoric Water Line
SMOW
(‰)
-40
δD
Mexico
18
δD = 8δ O + 10
-60
Argentina
Chile
Guatemala
-80
USA
-100
18
δD= 7.35δ O - 254
-120
-140
Canada
H. Avak, MAT Application Lab
TC/EA-ConFlo II-delta+XL
-160
-20
0
20
18
δ O SMO W (‰)
37
40
Comparison of δ18O and δ13C Determination
Accumulated Effect
Intensities of
- in relation to m/z 45 (13C)
- m/z 45 set to 100 %
O
C
Element content
10-50 %
>60 %
Atoms per gas molecule
18O, 13C abundance in M+
Ionization efficiency rel. to CO2
1 (in CO)
0.204 %
ca. 70 %
1 (in CO2)
1.08 %
100 %
m/z 30 (CO)
16.66 %
(10% O, 60% C)
16.66 %
3.15 %
2.20 %
Theoretical required sample amount for δ18O
45 x higher than for δ13C
if same precision as for δ13C is required
38
GC/TC Requirements on δ18O Analysis
¾Capillary Reactor Design
ƒ Keep the GC resolution: No peak broadening
¾No Contact to Al2O3
ƒ Inert reactor design
ƒ Avoiding of any exchange of oxygen
¾Surplus of Carbon in the Reactor
ƒ Conditioning of the reactor
ƒ Catalyst:
• Principally suitable: Ni (mp 1455 °C) , Pt (mp 1769 °C)
• Unsuitable:
Fe, Co
¾Quantitative High Temperature Conversion
ƒ Reactor temperature: > 1250 °C
¾No Memory Effects
39
Challenges
Factors influencing the δ18O Determination
40
-
Backgrounds
- N2, O2, CO2, H2O (leaks, He quality)
- Column Bleed
-
Derivatization
- Isotope Dilution
- O - Exchange
-
Compounds containing N
- N2 contamination on CO masses
High Temperature Conversion Interface
1250 °C
in standby
Capillary reactor design
¾ Pt shielded
¾ No contact to Al2O3
¾ Inert tube
¾ H2/He make-up gas
¾ Surplus of carbon (conditioning)
41
δ18O Analysis of Vanillin
Direct after installation of a new reactor
26
δ1818
OO(‰)
(‰)
24
22
20
18
Conditioning
(6 Runs)
Average:
24.52 ‰
n:
26
Std. Dev:
0.23 ‰
Specification: 0.80 ‰
16
1
4
7
10
13
16
19
22
Number of Injection
Operator: Peter Weigel
42
25
28
31
GC-IRMS Methodology
Short Backflush Time = better GC/TC conditions
43
IRMS:
Interface:
GC:
Column:
Flow:
Injector:
Delta plus XL
GC/C&TC
HP 6890
Ultra 1, 25 m x 0.32 mm, df= 0.52 µm
1.2 ml/min, Constant Flow
220 ˚C, Split/Splitless
Backflush
off
CO Ref. gas
Backflush
on
Solvent Peak
CO Ref. gas
CO Ref. gas
18O
δδ18
O of
of flavor
flavor
compounds
compounds
GC/TC - TC/EA – Cross Calibration
δ 18OSMOW [‰ ]
GC/TC
18O
δδ18
O of
of
flavor
flavor
compounds
compounds
No.
1
2
3
4
5
6
7
8
9
10
Mean-value:
Std-deviation:
Vanillin
9.09
9.11
9.17
8.52
9.21
9.24
9.19
9.34
9.20
8.88
9.10
0.23
β− Ionone
17.49
17.47
17.77
18.03
17.73
17.89
17.61
17.92
17.23
17.97
17.71
0.26
Frambinone
14.02
13.97
14.20
14.15
14.19
14.08
14.12
14.37
14.34
14.28
14.17
0.13
9.30
0.04
15.90
0.07
14.12
0.13
TC/EA
Mean-value:
Std-deviation:
¾
Short Backflush Time
¾
44
¾
Inert reaction tube at 1250 °C
Low and constant He flow
Isotope Fingerprinting of Tequila
-11.0
δ13C:
13
δ CSMOW (‰) Ethanol
GC-C/TC DELTA+XL
Analyst: Dr. D. Juchelka
Headspace sampling
4/2000
-11.5
100 %Tequila
Sugar
Cane
-12.0
Enzymatic
Fractionation
of Isotope
-12.5
Ratios
0
Mixed
5
10
18
δ OSMOW (‰) Ethanol
45
δ18O:
15
Physical Fractionation
of Isotope Ratios
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List of attendees
First Name
Last Name
Saoud
Marie
Laura
Claire
Zoe
Les
Pascal
Paul
Charlotte
Georg
Liz
Liam
Kate
Lisa
Peter
Sean
Simon
Andrea
James
Sarah
Kerry
Andrew
Sam
Elisa
Pete
Corinne
Rona
Callum
Chris
Jason
Tamsin
Gwen
Karen
Rhiannon
Gillian
Len
Sheng
Chaunlun
Al Habsi
Archbold
Beramendi-Orosco
Bickers
Billings
Bluck
Boeckx
Brooks
Bryant
Cadisch
Campbell
Chalmers
Clark
Cole
Ditchfield
Doyle
Eaton
Heinmeyer
Howard
Jackson
Jones
Kelly
Kelly
Lopez-Capel
Maxfield
McCulloch
McGill
Murray
Mussell
Newton
O’Connell
O’Sullivan
Privat
Stevens
Taylor
Wassenar
Xu
Zhang
Institution
tour group
University of Newcastle upon Tyne
Queen’s University Belfast
University of Nottingham
University of Bristol
University of York
MRC Human Nutrition Research
Ghent University
UC Berkeley
NERC Radiocarbon Lab.
Imperial College at Wye
Earth Sciences, Uni. of Glasgow
NERC Radiocarbon Lab.
University of Bristol
University of York
RLAHA, Oxford University
Forensics Explosives Lab.
Institute of Child Health, London
CTCD –York
University of Newcastle upon Tyne
MRC Human Nutrition Research
MRC Human Nutrition Research
SUERC
University of Bristol
University of Newcastle upon Tyne
University of Bristol
SUERC
SUERC
NERC Radiocarbon Lab.
LGC
SUERC
University of Oxford
Queen’s University Belfast
University of Oxford
University of Oxford
University of Newcastle upon Tyne
National Water Research Institute, Canad
SUERC
University of Georgia
5
7
5
3
4
7
3
6
6
2
6
3
2
3
4
3
5
4
4
6
5
1
1
1
6
1
2
7
2
2
5
7
1
7
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