N

CH

O

Short Course on Compound-SpecificIsotope Ratio Mass Spectrometry

Organic GeochemistryUnit

14th April 2003, School of Chemistry, University of Bristol

SIMSUG Short Course onCompound-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 analysisZoë Crossman and Mark Copley

12:10 to 12:45 Discussion and Questions

12:45 to 1:30 Lunch and Further Discussion

8:40 to 8:50Introduction to compound-specific isotope analysis

Richard Evershed

An introduction tocompound-specific stable isotope

determinations by gas chromatography-isotope ratio mass

spectrometry

Richard P. Evershed

• 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

Definitions

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 composition-source 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

Bulk isotope ratio instruments

- Large sample sizes, e.g. 10–3 g- Minimal sample preparation

Compound–specific isotope ratio instruments

- Very small sample sizes, e.g. 10–8 g- Complex sample preparation procedures- Requires knowledge of capillary GC, reactor systems andlow dead-volume gas handling systems

Combustion GC IRMS

CombustionGC IRMS

Sample preparation

8:50 to 9:10Laboratory techniques in compound-specific isotope

analysisIan D. Bull

9:10 to 9:30Derivatisation of compounds for compound-specific

isotope analysisBart 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

Acid fraction Neutral fraction Polar fraction

Chromatography

Total lipid extract Biopolymer analysis

Sample

• 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

Contamination

• Glassware– needs to be ‘clean’

• furnaced• solvent extracted

– solvent bottles• Plasticisers• You!

Rel

ativ

e In

tens

ity

10 15Time (min)

20 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

OH cholesterol

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

OHergosterol

• 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

Glass frit

Sorbent

Glass ‘column’

Sample

Stopcock

Fraction 1 Fraction 2 Fraction 3

Hexane DCM DCM/methanol

Elutropic seriesIncreasing polarityLeast polar Most polar

Solvents added in sequence

Fraction 1: hydrocarbons

Fraction 2: TAGs, wax esters

Fraction 3: sterols, triterpenols, alcohols

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 andsaponified to generate GC-IRMS amenable compounds, i.e. fatty acids

-O

O

OP

OOO

O

O

-

OH

O

R OR'

O

OH-

O

R OHOR'

-

R O

O- OR'-

-

H+

OH, 0.01 M-

workup

Step 5 - Biopolymer analysis

• Carbohydrates – acid hydrolysis of the lipid extracted residue

OOH

OH

OOOOH

OH

OOOH

OH

OOOH

OH

O

CH2OH CH2OH CH2OH CH2OH

n = 1-10000

O

OH

OH CH2OH

H

OH

HH

H H

OH

72% H2SO4, RT, 1 h

1 M H2SO4, 100oC, 2.5 h

e.g.

glucose

Step 5 - Biopolymer analysis

• Proteins NH2

O

OH

NH2

O

OH

NH

O

OH

NH2

OS

OH

NH2

OH

O

OH

Gly

Phe

Ser

Pro

Met

6 M HCl, 100oC, 24 h

• Still not ideal but we are getting there!

e.g.

Our example analytical protocol revisted

n-alkanoic acids

Acid fraction

Hydrocarbonswax estersn-alkanols

sterols, triterpenols

Neutral fraction

PLFAs

Polar fraction

Chromatography

Total lipid extract

Proteins Carbohydrates

Biopolymer analysis

Sample

Summary

• Identify you target compounds before proceeding with any form ofsample 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

n-alkanoic acids

Acid fraction

Hydrocarbonswax estersn-alkanolssterols triterpenols

Neutral fraction

PLFAs

Polar fraction

Chromatography

Total lipid extract

Amino acids Carbohydrates

Residue analysis

Sample

GC-IRMS

DerivatisationMeasurement possible?

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

Rel

ativ

e in

tens

ity

Retention time

n-Alkanes

No functional groups

No derivatisation needed

29

31

3327

2523

21

n-Alkanes obtained from a soil

Rel

ativ

e in

tens

ity

Retention time

Fatty acids

Sterols

n-Alkane

GC-run of mixture of compounds

Derivatisation

• Examples of functional groups: -Carboxylic acids-Hydroxy-Amino

• Examples of derivatisation reactions: -Esterification-Silylation-Acetylation

• Many derivatisation reaction possible

• Which to choose?

• 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)

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

Method usable

No isotope effect

No carbon atoms involved

Isotope effect

Carbon atoms target moleculeinvolved

Conversion 100%

Conversion not 100%

Isotope effect

Carbon atoms derivatisation moleculeinvolved

Reproducible?Yes

Method not usable

No

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

a Rieley, 1994

nderivised compoundδ13Cderivitised compound-nderivative groupδ13Cderivative groupncompound

δ13Ccompound =a

n = the number of carbon atoms

Carbon number added

Unc

erta

inty

in δ

-val

ue

1 10 20

2.4

1.2

0

n=3

n=5

n=10

n=20

n=30

Uncertainty due to added carbona

a after Rieley, 1994

n = the number of original carbon atoms

• Increase in uncertainty with increasing addition of derivative carbon

• Effect larger if number of original carbonatoms is smaller

Fatty acids

• Diazomethane method

R OH

O

R

O

O

N2H2N2 +

C

Me

• Fast, Irreversible reaction

• Isotope effect is on the carbon of the diazomethane

• Added in excess quantity

• Non-reproducable isotope effect

Bond broken

• Method not usable

Fatty acids

• BF3, MeOH derivatization

R OH

O

R

O

OMeR

O

OH2OMe

BF3

+

-

BF3MeOH

• Isotope effect is on the carbon of the fatty acid

• Conversion 100%

• No isotope effect

• Method usable

Bond broken

Fatty acids, GC-run standard mixtureR

elat

ive

inte

nsity

Retention time

Fatty acids; derivatised Old situation

• Silylation using BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) and pyridine

Alcohols

• No carbon atoms involved in reaction

• Usable method

• Disadvantage: products are stable but only for a relative short time

R OH R OSi(CH3)3

CF3 OSi(CH3)3

NSi(CH3)3

Pyridine

Alcohols, chromatogram

Retention time

Rel

ativ

e in

tens

ity

3123 25

22

24

2628

27

30

32

Alcohols in a forest soil

29

Monosaccharides

• Main problems: Relatively large number of protection groups needed

O

OH

OH

OH

OH

OH

O

OH

OH

OH

OH

Glucose Ribose

6 5

• How to minimize the addition of carbon?(Silylation would add 12 to 18 carbon atoms)

Relatively small number of original carbon atoms (usually 4 to 6)

Alditol acetate methoda

O

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OAc

OAc

OAc

OAc

OAc

OAc

O

O O

NaBH4

Pyridine

Disadvantages: Still a large number of carbon atoms added (8-12)Actually measuring alditols; loss of information

Bond broken

• Isotope effect on the carbon of the reagent• Fractionation effect seems constantb• Method usable but correction factor needed

a After Gunner et al., 1961; b Macko et al 1998; Docherty et al., 2001

Loss of structural information

OHOHOHOH

OOH

OHOHOH

OH

OH

OHOH

O

OH

• Two hexoses, two pentoses etc. may lead to the same alditol

Arabinose Arabitol Lyxose

• One hexose, pentose etc. may lead to two alditols

OHOHOHOH

OOH

OHOHOHOH

OHOH

OH

OHOH

OHOH

OH+

Fructose MannitolGlucitol

+OH

OH

OHOHO

HO OHO

OH

HO

OH

a After Reinhold et al., 1974; b van Dongen et al., 2001

1)Methyl boroacetylation

Methyl boroacetylation of monosaccharidesa

• Isotope effect is on carbon atoms of monosaccharidesb

• Reaction quantitative; No isotope effect

2)SilylationO

O

O

O

OB

B

+

OSi

O

OOO

OB

BTotal 5

Total 2

• Addition of relatively small number of carbon atoms (2-5)

• Measuring monosaccharides, not alditols

Bonds broken

arabinosexylose

mannose

glucose

Relative retention time

Rel

ativ

e in

tens

ity

GC-IRMS of a monosaccharide mixture

Amino acids

H

R

NH2

COOH Main problems:

• Two different groups which needs protection

• Small number of own carbon atoms

2 (Glycine) to 11 (Tryptophan)

H

H

NH2

COOH

NH

HNH2

COOH

Glycine

Tryptophan

H

R

NH2

OHO

1) iso-propanol/HClH

R

NH

O OCH(CH3)2

CF3

O

O CF3

O O

F3C

2)

Amino acids, derivatisation

Bond broken

Isotope effect: Step 1; On carbon atom of amino acida

Conversion 100%; method usable

aRieley 1994; bDocherty et al. 2001

Bond broken

Step 2; On carbon atom of reagent

Fractionation effect seems constantb

Method usable but correction factor needed

Amino acids, GC-runR

elat

ive

inte

nsity

Amino acids in a standard mixture

Ala

Gly

Thr Se

r

Val Le

uIle

I.S.

Pro

Hyp

Asp G

luPh

e

Relative retention time

Amino acids, point to think about

• Relatively large number of carbon atoms added (at least 5)

H

R

NH

O OCH(CH3)2

CF3

O

• A reaction with an isotope effect, although can be corrected for

• HF can be formed, causing problems when measuring isotopes

Total 5

?

Summary

• Derivatisation methods are available which make it possible to determinethe 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 9:55Gas Chromatography-Isotope Ratio Mass Spectrometry

(GC-IRMS) instrumentationRich Pancost

10:30 to 10:50Troubleshooting in GC-IRMS

Jim Carter

10:50 to 11:20Data analysis in GC-IRMS

Hazel Mottram

11:20 to 11:55CSIA for other isotopes

Andreas Hilkert

Gas Chromatography-Isotope Ratio Mass Spectrometry (GC-IRMS) instrumentation

Rich Pancost

GC-IRMS Instrumentation

Gas ChromatographSeparates individual compounds, allowing discrete isotopic compositions to be determined

Combustion/Reduction InterfaceConverts eluting compounds into CO2 for analysis

Continuous Flow InstrumentAllows the delivery of sample CO2 to an isotope ratio mass spectrometer via a He stream

The GC-IRMS

ThermoFinniganMATdesign

The Gas ChromatographFor 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

15 m 60 m30 m

The GC ColumnThe importance of investing in good

separation

Differences amongst columns:Stationary phaseLengthDiameter

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

O2 He

Combustion furnace

The Combustion Furnace

• Converts organic compounds into CO2 and H2O

• Contains an oxidizing metal (CuO or ZnO) and typically a catalyst (Pt)

• 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

ThermofinniganMATdesign

4 CuO 2 Cu2O + O22 NiO 2 Ni + O2

The Combustion Furnace

Capillary from GC (slides ~1.5 cm into end of reactor)

(240 mm long)

240mm

320mm

He

Note: heated zone is 260 mm, bracketing

wires

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

• Can be removed by passing analyte stream through a selectively permeable membrane (NafionTM) with a dry He counterflow

OR

The Open Split

• Adaptation for GC-IRMS that is analogous to adaptations for any continuous flow interface

• 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

He

Analyte

Reference Gas Inlet

Allows introduction of reference gas in a line parallel to the

analyte (i.e. two columns deliver He and CO2 to MS)

Reference To MS

He

Troubleshooting(Tricks and Tips)

Jim Carter

with special reference to:ThermoFinnigan GCC I-III

GC-IRMS interface

Three things to consider

Gas Chromatograph(organic compound)

Combustion Reactor

Interface (H2, N2, CO, CO2)

Axiom

There is no substitute for good chromatography

The gas measured must correspond to a single compoundGood 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)

INJECTORSplitless, on-column or PTV injectionConstant flow rate = constant split ratio

COLUMN0.32mm id / thick film high sample loading0.32mm id / thin film improved chromatography0.25mm id improved split ratio

Tools of the trade

Hewlett PackardFlowcalc 2.05

www.chem.agilent.com/cag/servup/usersoft/main.html

Digitalflow 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

Drill throughRemove coating

GC column Oxidation reactorZDV fitting

Dead Volumes

Bleed capillary

100µl / minhelium

Dead Volumes

1% oxygenhelium

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)

Check Ox. Reactor

Check m/z 40

Check for leak Blockage(check flows)

Check “T” piece Check Ox. reactor

No Yes

Up Down

Check Injector(septum/liner)

OK

What can possibly go wrong? (2)

• δ13C values are consistently enriched• δ13C values are consistently depleted• δ13C values are unstable

It’s the wrong numbers

Is chromatography OK?

As before Which way did the δ value move?

Check m/z 18 N compounds?

Check source heater Check Red. reactor

No Yes

Up Down

Check Nafion

Is m/z 40 OK?

Check Ox. Reactor(hexane test)

Yes No

Yes

High

OK

Is chromatography OK?As before

Check for leaks

Standard ON/OFF test

No

Yes

Is m/z 40 OK?

Clean Ion Source

Isotope values are unstable

Up

Check BF valve

No

Is m/z 18 OK?

Yes

Yes

Poor

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 interfacestart at the water trap – GC columncapillaries “go wrong” as do fittingschange one thing at a time

“Once upon a time in Indiana …..”

Data Analysis and Interpretation

Hazel Mottram

AxiomYou 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

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 ofbackflush)

– 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 RCO2Me

ncdδ13Ccd = ncδ13Cc + ndδ13Cd

where n is number of moles of carbonc refers to compound of interestd refers to the derivative groupfd refers to the derivatised fatty acid

(Rieley, 1994)

BF3/MeOH

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 FAMEThe 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 = (ncdδ13Ccd - ndδ13Cd)

= 1/18 (19 x –28.14) – (1 x –40.15)

= -27.47

1nc

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 = (ncdδ13Ccd - ncδ13Cc)

• This should be repeated for each compound of interest

1nd

Derivatisation: BSTFA for alcohols

• The contribution of the BSTFA to the overall δ13C value is normally measured using a standard alcohol with known δ13C value

• 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

OH

OH

OH

OH

OH

OH

myo-inositol

δ13Cd = (ncdδ13Ccd - ncδ13Cc)1nd

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 equationsx 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

2

c

d2d

2

c

dc2cd

2c n

nσn

nnσσ

+

+=

BF3/MeOH(IRMS)± 0.1 ‰

FAME of interest(GC/C/IRMS)

± 0.3 ‰

δ13C of FAME of interest

± ?

σc2 = 0.32 × (19/18)2 + 0.12 × (1/18)2

σc2 = 0.100

σc = 0.32 ‰

Calculation errors propagate

Docherty et al (2001)

Errors where a KIE is present

Underivatised sugar

(IRMS)± 0.1 ‰

Derivatised sugar(GC/C/IRMS)

± 0.3 ‰

Correction factorδcorr ± ? δ13C of sugar of

interest± ?

Derivatised sugar analyte(GC/C/IRMS)

± 0.3 ‰

σc2 = 0.12 × 5 2 + 0.32 × 5+102 + 0.32 × 5+10 2

σc2 = 1.63

σc = 1.3 ‰

2

c

dc2cd

2

c

ds2sd

2

c

s2s

2c n

nnσn

nnσnnσσ

++

++

=

5 55Docherty et al (2001)

Calculation errors propagate

Calculation errors propagate

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 carryover16:0* + natural abundance fame mix (1:1)

-32

-30

-28

-26

-24

-22

-20

-18

-16

-1416:1 17:0 17:1 18:0 18:1 18:2

δ13

C (‰

)

after 16:0*FAME std

Within run carryover16:0* + natural abundance fame mix (2:1)

-32

-30

-28

-26

-24

-22

-20

-18

-16

-1416:1 17:0 17:1 18:0 18:1 18:2

δ13

C (‰

)

after 16:0*FAME std

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 linearity2. Data is only collected from peaks with minimal coelution3. 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 irmThe Way to N, H, O by irm--GC/MSGC/MS

Andreas HilkertThermoFinnigan MAT GmbH, Bremen

δδ1515N by irmN by irm--GC/MSGC/MS

Introduced in 1992

3

Why Why 1515N analysis ?N analysis ?

Source:Source:COCO2, Air2, Air

PlantPlant--MetabolismMetabolism

Source: NSource: N2, Soil2, Soil Plant MetabolismPlant Metabolism-36

-35

-34

-33

-32

-31

-30

-14 -12 -10 -8 -6 -4 -2 0

Nitrogen Isotope Ratio, δ15Nair [‰]

Car

bon

Isot

ope

Rat

io, δ

13C P

DB [‰

]

Drugs

Heroin

Cocaine

4

+ 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

AdvantagesAdvantages

5

NN--selective irmselective irm--GC/MS TraceGC/MS Trace

Comparison of FID trace and m/z 29 trace

N, O – tBDMS Amino Acid Derivatives

Ref

. gas

Ref

. gas

Ref

. gas

6

+ 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)

ChallengesChallenges

7

Accumulated Effect on Intensities of

- in relation to m/z 45 (13C)- m/z 45 set to 100 %

N C m/z 29 (15N)

Element content <10 % >60 % 8.33 %(e.g. 5% N, 60% C)

Atoms per gas molecule 2 (in N2) 1 (in CO2) 4.17 %15N, 13C abundance 0.732 % 1.08 % 2.82 %Ionization efficiency rel. to CO2 ca. 70 % 100 % 1.98 %

H

RH

13C

HH

OO13C

Comparison of Comparison of δδ1155N and N and δδ1133C C DeterminationDetermination

15N

Theoretical required sample amount for δ15N50 x higher than for δ13C

if same precision as for δ13C is required

8

1.08 / 1 0.732/ 1

CO N2Ratio29/28

29 28

δCON2 = (1.08/0.732 -1)*1000

= + 475 ‰

The Effect of CO contamination

ChallengesChallenges

9

Combustion and ReductionCombustion and Reduction

100 % Combustion

Reduction

Water Removal

CO2 Removal

Organic Compound

100 % N2

CO2, N2, H2O, NOx

CO2, N2, H2O

CO2, N2

10

Combustion and ReductionCombustion and Reduction

100 % N2 at 100 % Combustion

11

Combustion Requirements for Combustion Requirements for δδ1515NN

• 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 N2 NOxOx Ox

Red

-CHx CO CO2Ox Ox

12

Combustion EfficiencyCombustion Efficiency

0

13

Sample CleanupSample Cleanup

• Water Removal• CO2 Removal

14

δδ1515N ApplicationsN Applications

O-iPropyl, N-Pivaloyl Amino Acid Derivatives

Data taken from C. Metges

15

Sample SizeSample Size

1.5 nmol N2 on col.

16

Boosting the LimitsBoosting the Limits

17

Recipe for Recipe for δδ1515N irmN irm--GC/MSGC/MS

Injector splitless

Retention Gap 3m deactivated fused silica

Capillary Column 50 m, 0.32 mm i.d., 0.5 µm film-thickness, 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.

δδ22H by irmH by irm--GC/MSGC/MS

Introduced in 1998

19

Why Why δδ22H irmH irm--GC/MS ?GC/MS ?

natural gas

marine oilsnon marine oils

C3 plantsSMOWSLAP GISP

musts

wine water

C4 plantsC3 ethanol C4 ethanol

-400 -300 -200 -100 0 100

clay minerals

relatively D depleted

‰

20

OCEAN CONTINENTδD = 0 ‰

-94 ‰Vapor

-110 ‰Vapor

-126 ‰Vapor

-14 ‰Rain

-30 ‰Rain

Schematic Fractionation in the Atmospheric Water Cycle

HydrogeHydrogenn Isotope RatiosIsotope Ratios

21

We know where you eat !We know where you eat !

An orphan's tail: variations of δO and δD in a single elephant hair

6

7

8

9

10

11

12

13

0 50 100 150 200 250

Length (mm)

δ18O

SMO

W (‰

)

-100

-95

-90

-85

-80

-75

-70

-65

-60

-55

-50δ D

SMO

W (‰)

TC/EA-DELTA+XLANALYST: H. Avak6/23/2000samples 200-600 µg

δΟ

δD

22

Comparison of Comparison of δδ22HH and and δδ1133C C DeterminationDetermination

Accumulated Effect on Intensities of

- in relation to m/z 45 (13C)- m/z 45 set to 100 %

H C m/z 3 (DH)

Element content 13 % 50 % 300 %(e.g. 6 H, 2 C)

Atoms per gas molecule 2 (in H2) 1 (in CO2) 150 %2H, 13C abundance 0.03 % 1.08 % 4.16 %Ionization efficiency rel. to CO2 ca. 10 % 100 % 0.42 %

HH

13CHH

O13C

H

H

Theoretical required sample amount for δD240 x higher than for δ13C

if same precision as for δ13C is required

e.g. Ethanol

23

Low Energy Helium IonsLow Energy Helium Ions

ion source

magnet

m/z 2 (H2)m/z 3 (HD)

universal triple collector for N2, CO, O2,CO2, SO2

m/z 4

Under continuous flow conditions4He is about 107 times more abundant than HD

Due to collisions He ions withless energy than 3 kV would

fall into the m/z 3 cup

24

0

50

100

150

200

250

300

0 0.1 0.2 0.3 0.4 0.5

He flow [ml/min]

He

abun

danc

e at

m/z

3 [V

without retardation lens with retardation lens

Contribution of HeContribution of He++ Ions at m/z 3Ions at m/z 3

The production of low energy He ions is related to the He flow into the ion source

0

2

4

6

8

10

0 0.02 0.04 0.06 0.08 0.1He flow [ml/min]

He

abun

danc

e at

m/z

3 [V

]

25

0

3HD+

4He+

∆E>400 V

Ion

Kin

etic

Ene

rgy

[kV]

ground ~Vacc ground-100 V

HD+4He+

entrance slitretardation lens

secondary electron suppressor

Faraday cup 1012 Ω

HD Collector - Rejection of 4He+ ions by a retardation lens

Energy Filter Energy Filter -- Retardation LensRetardation Lens

26E 0998 032 PO

Complete absence of 4He at m/z 3 cup with Energy Filter

Energy FilterEnergy Filter

27

HH33++ FactorFactor

227 mV (pA)

30 V (nA)

H3+ Factor (K) = 6.01 ppm / nA

[H3+] = [H2]2 • K[H[H33++] = [H] = [H22]]22 •• KK

12 mV (pA)5.4 mV H3

+ (45 %)12 mV (pA)

5.4 mV H3+ (45 %)

61 µV (fA)0.31 µV H3

+ (0.5 %)61 µV (fA)

0.31 µV H3+ (0.5 %) m/z 3

28

y = -0.01x [‰ / V] at H3

+ 6.06773

y = 0.2x [‰ / V] at H3

+ 6.010621

-2

-1

0

1

2

3

4

5

6

0 5000 10000 15000 20000 25000 30000 35000

Intensity [mV]

δD [‰

]

HH33++ Factor Factor –– Linearity Linearity

227 mV (pA)

30 V (nA)

0.05 ppm

29

Requirements to Requirements to δδ22HH irmirm--GC/MSGC/MS

GC Performance

Quantitative Conversion

Sensitivity

Linearity

Precision and Stability

Inner diameter of recommended GC columns: 0.25 mm

Empty reactor tube at ≥ 1400 °C

GC flow: 0.8 – 1.0 ml/minOptimal linear velocity

High He flow into IRMS: 0.4 ml/minOptimal open-split ratio

Long-term stability of the H3+ factor

30

High TemperatureHigh Temperature CConversiononversion

CnGC/TCHx Oy H2x/2

COy

Cn-y

>1400 °C

irm GC/MS 0.015 %

δ2H

99.985 %H2

HD

31

High TemperatureHigh Temperature CConversiononversion

High Temperature Conversion Interface

32

0

1

2

3

4

5

6

7

8

9

10

600 800 1000 1200 1400

Temperature (°C)

Sign

al (V

)

propane

methane

hydrogen

1440 °C

Production of methane and hydrogenfrom propane as a function of temperature

Chart according to T. Burgoyne et al., Anal. Chem. 1998, 70, 5136-5141

High Temperature ConversionHigh Temperature Conversion

33

• Metal Connector / Ceramic Reactor – “Optimized”

GC oven < 320 °C

Al2O3i.d. = 0.5 mmF.S.i.d. = 0.32 mm

ca. 0.5 cm polyimideare burnt off

• Metal Connector / Ceramic Reactor – “Standard”

GC oven < 320 °C

Al2O3F.S.i.d. = 0.32 mm

i.d. = 0.25 mm

criticalvolume

Heater 940 °C

wires

Heater 1450 °C

no wires

wires

Heater 940 °C Heater 1450 °C

no wires

TransferTransfer into the Reactorinto the Reactor

34

0

1000

2000

3000

4000

5000

6000

820 840 860 880 900 920 940 960

Time (s)

H2

Inte

nsity

(mV)

mass 2mass 3

Etiocholanolone Androsterone

0

1000

2000

3000

4000

5000

6000

820 840 860 880 900 920 940 960

Time (s)

H2

Inte

nsity

(mV)

mass 2mass 3

Etiocholanolone Androsterone

No. δ D/HSMOW [‰] δ D/HSMOW [‰]1 -227.08 -332.542 -225.79 -332.273 -225.84 -332.444 -226.00 -332.565 -228.90 -337.866 -225.24 -332.947 -224.98 -331.208 -225.61 -333.47Mean-value: -226.18 -333.16

Std-deviation: 1.26 2.00

H

O

HO

EtiocholanoloneH

O

HO

Androsterone

δδDD of Free Steroids (of Free Steroids (underivatizedunderivatized))

35

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 ‰

Metabolic StudiesMetabolic Studies

δ2H of Natural and EnrichedFatty Acid Methyl Esters

0.50

1.00

1.50

2.00

2.50

3.00

3.50

800 900 1000 1100 1200

m/z 2 trace [V]m/z 2 trace [V]C16:0C16:0 C17:0C17:0

C18:1C18:1

0.180.200.220.240.260.280.300.32 D/H ratio traceD/H ratio trace

Time [sec]Time [sec]

enriched

natural

δδ1818O by irmO by irm--GC/MSGC/MS

Introduced in 1996

37

Why Why δδ1818O irmO irm--GC/MS?GC/MS?

δD = 8δ18O + 10

δD= 7.35δ18O - 254

-160

-140

-120

-100

-80

-60

-40

-20

0

20

-20 0 20 40

δ 18O SMO W (‰)

δD

SMO

W (‰

)

Meteoric Water Line

Canada

Mexico

ChileArgentina

USA

Guatemala

H. Avak, MAT Application LabTC/EA-ConFlo II-delta+XL

Honey

38

Accumulated Effect Intensities of

- in relation to m/z 45 (13C)- m/z 45 set to 100 %

O C m/z 30 (CO)

Element content 10-50 % >60 % 16.66 %(10% O, 60% C)

Atoms per gas molecule 1 (in CO) 1 (in CO2) 16.66 %18O, 13C abundance in M+ 0.204 % 1.08 % 3.15 %Ionization efficiency rel. to CO2 ca. 70 % 100 % 2.20 %

Comparison of Comparison of δδ1188OO and and δδ1133C C DeterminationDetermination

Theoretical required sample amount for δ18O45 x higher than for δ13C

if same precision as for δ13C is required

39

GC/TC Requirements GC/TC Requirements on on δδ1818OO AnalysisAnalysis

Quantitative High Temperature Conversion

No Contact to Al2O3Inert reactor designAvoiding of any exchange of oxygen

Surplus of Carbon in the ReactorConditioning of the reactorCatalyst:

• Principally suitable: Ni (mp 1455 °C) , Pt (mp 1769 °C)• Unsuitable: Fe, Co

Reactor temperature: > 1250 °C

No Memory Effects

Capillary Reactor DesignKeep the GC resolution: No peak broadening

40

ChallengesChallenges

- Backgrounds- N2, O2, CO2, H2O (leaks, He quality)- Column Bleed

- Derivatization- Isotope Dilution- O - Exchange

- Compounds containing N- N2 contamination on CO masses

Factors influencing the δ18O Determination

41

High TemperatureHigh Temperature CConversiononversion InterfaceInterface

Capillary reactor designPt shieldedNo contact to Al2O3

Inert tubeH2/He make-up gasSurplus of carbon (conditioning)

1250 °C

in standby

42

16

18

20

22

24

26

1 4 7 10 13 16 19 22 25 28 31

Number of Injection

18O

(‰) Average: 24.52 ‰

n: 26

Std. Dev: 0.23 ‰

Specification: 0.80 ‰Conditioning

(6 Runs)

δδ1818O Analysis of VanillinO Analysis of Vanillin

Operator: Peter Weigel

Direct after installation of a new reactorδ18

O (‰

)

43

Short Backflush Time = better GC/TC conditions

Back-flushon

Solv

ent P

eak Back-

flushoff

δ18O of flavorcompounds

δ18O of flavorcompounds

IRMS: Delta plus XLInterface: GC/C&TCGC: HP 6890Column: Ultra 1, 25 m x 0.32 mm, df= 0.52 µm Flow: 1.2 ml/min, Constant FlowInjector: 220 ˚C, Split/Splitless

GCGC--IRMS MethodologyIRMS Methodology

CO

Ref

. gas

CO

Ref

. gas

CO

Ref

. gas

44

Short Backflush Time

Low and constant He flowInert reaction tube at 1250 °C

δ18O of

flavor

compounds

δ18O of

flavor

compounds

GC/TC GC/TC -- TC/EA TC/EA –– Cross CalibrationCross Calibration

GC/TC δ18OSMOW [‰] No. Vanillin β− Ionone Frambinone

1 9.09 17.49 14.022 9.11 17.47 13.973 9.17 17.77 14.204 8.52 18.03 14.155 9.21 17.73 14.196 9.24 17.89 14.087 9.19 17.61 14.128 9.34 17.92 14.379 9.20 17.23 14.34

10 8.88 17.97 14.28Mean-value: 9.10 17.71 14.17Std-deviation: 0.23 0.26 0.13

TC/EA Mean-value: 9.30 15.90 14.12Std-deviation: 0.04 0.07 0.13

45

100 %TequilaSugarCane

Isotope Fingerprinting of TequilaIsotope Fingerprinting of Tequila

Mixedδ13C:EnzymaticFractionationof IsotopeRatios

δ18O:Physical Fractionationof Isotope Ratios

-12.5

-12.0

-11.5

-11.0

0 5 10 15

δ18OSMOW (‰) Ethanol

δ13C S

MO

W (‰

) Eth

anol

GC-C/TC DELTA+XLAnalyst: Dr. D. JuchelkaHeadspace sampling 4/2000

References for Compound-Specific Isotope Analysis

Preparatory Chemistry Abidi, S. L. (2001). "Chromatographic analysis of plant sterols in foods and vegetable oils." Journal of Chromatography A

935(1-2): 173-201. Bligh , E.G. and W.J. Dyer (1959) "A Rapid Method of Total Lipid Extraction and Purification." Canadian Journal of

Biochemistry and Physiology 37: 911-917 Christie, W. W. (1976) Lipids Analysis. Pergamon Press, New York. Manirakiza, P. et al (2001) "Comparative Study on Total Lipid Determination using Soxhlet, Roese-Gottlieb, Bligh & Dyer

and Modified Bligh & Dyer Extraction Methods." Journal of Food Composition and Analysis 14, 93-100 Myher, J. J. and A. Kuksis (1995). "General Strategies in Chromatographic Analysis of Lipids." Journal of Chromatography

B-Biomedical Applications 671(1-2): 3-33. Randall, R.C. et al (1991). “Evaluation of Selected Lipid Methods for Normalizing Pollutant Bioaccumulation.”

Environment Toxicology and Chemistry 10: 1431-1436 Ruizgutierrez, V. and L. J. R. Barron (1995). "Methods for the Analysis of Triacylglycerols." Journal of Chromatography B-

Biomedical Applications 671(1-2): 133-168. Smedes, F. (1999). "Determination of total lipid using non-chlorinated solvents." Analyst 124(11): 1711-1718. Smedes, F. and T. K. Askland (1999). "Revisiting the development of the Bligh and Dyer total lipid determination method."

Marine Pollution Bulletin 38(3): 193-201. Touchstone, J. C. (1995). "Thin-Layer Chromatographic Procedures for Lipid Separation." Journal of Chromatography B-

Biomedical Applications 671(1-2): 169-195.

GC-IRMS Instrumentation and Data Analysis

Barrie A., Bricout J., and Koziet J. (1984) Gas-Chromatography - Stable Isotope Ratio Analysis at Natural Abundance Levels. Biomedical Mass Spectrometry 11(11), 583-588.

Becchi M., Aguilera R., Farizon Y., Flament M. M., Casabianca H., and James P. (1994) Gas-Chromatography Combustion Isotope Ratio Mass-Spectrometry Analysis of Urinary Steroids to Detect Misuse of Testosterone in Sport. Rapid Communications in Mass Spectrometry 8(4), 304-308.

Bernreuther A., Koziet J., Brunerie P., Krammer G., Christoph N., and Schreier P. (1990) Chirospecific Capillary Gas-Chromatography (Hrgc) and Online Hrgc-Isotope Ratio Mass-Spectrometry of Gamma-Decalactone from Various Sources. Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung 191(4-5), 299-301.

Brand W. A. (1996) High precision isotope ratio monitoring techniques in mass spectrometry. Journal of Mass Spectrometry 31(3), 225-235.

Brand W. A., Tegtmeyer A. R., and Hilkert A. (1994) Compound-Specific Isotope Analysis - Extending toward N-15 N-14 and O-18 O-16. Organic Geochemistry 21(6-7), 585-594.

Burgoyne T. W. and Hayes J. M. (1998) Quantitative production of H-2 by pyrolysis of gas chromatographic effluents. Analytical Chemistry 70(24), 5136-5141.

Caimi R. J. and Brenna J. T. (1993) High-Precision Liquid Chromatography-Combustion Isotope Ratio Mass-Spectrometry. Analytical Chemistry 65(23), 3497-3500.

Caimi R. J. and Brenna J. T. (1995) High-Sensitivity Liquid-Chromatography Combustion Isotope Ratio Mass-Spectrometry of Fat-Soluble Vitamins. Journal of Mass Spectrometry 30(3), 466-472.

Docherty, G., Jones. V. and Evershed R.P. (2001) Practical and theoretical considerations in the gas chromatography/combustion/isotope ratio mass spectrometry δ13c analysis of small polyfunctional compounds. Rapid Commuications in Mass Spectrometry 15, 730-738.

Ellis L. and Fincannon A. L. (1998) Analytical improvements in irm-GC/MS analyses: Advanced techniques in tube furnace design and sample preparation. Organic Geochemistry 29(5-7), 1101-1117.

Engel M. H., Macko S. A., and Silfer J. A. (1990) Carbon Isotope Composition of Individual Amino-Acids in the Murchison Meteorite. Nature 348(6296), 47-49.

Evershed R. P., Arnot K. I., Collister J., Eglinton G., and Charters S. (1994) Application of Isotope Ratio Monitoring Gas-Chromatography Mass-Spectrometry to the Analysis of Organic Residues of Archaeological Origin. Analyst 119(5), 909-914.

Fantle M.S., Dittel, A.I., Schwalm S.M., Epifanio, C.E. and Fogel, M.L. (1999) A food web analysis of the juvenile blue crab, Callinectus sapidus, using stable isotopes in whole animals and individual amino acids. Oecologia 120, 416-426.

Gaschnitz R., Krooss B. M., Gerling P., Faber E., and Littke R. (2001) On-line pyrolysis-GC-IRMS: isotope fractionation of thermally generated gases from coals. Fuel 80(15), 2139-2153.

Guo Z. K., Luke A. H., Lee W. P., and Schoeller D. (1993) Compound-Specific Carbon-Isotope Ratio Determination of Enriched Cholesterol. Analytical Chemistry 65(15), 1954-1959.

Hayes J. M., Freeman K. H., Popp B. N., and Hoham C. H. (1990) Compound-Specific Isotopic Analyses - a Novel Tool for Reconstruction of Ancient Biogeochemical Processes. Organic Geochemistry 16(4-6), 1115-1128.

Johnson, B.J., Fogel M. and Miller G.H. (1993) Paleoecological reconstructions in southern Egypt based on the stable carbon and nitrogen isotopes in the organic fraction and stable carbon isotopes in individual amino acids of fossil ostrich eggshell. Chemical Geology 107, 493-497.

Leckrone K. J. and Hayes J. M. (1998) Water-induced errors in continuous-flow carbon isotope ratio mass spectrometry. Analytical Chemistry 70(13), 2737-2744.

Lichtfouse E., Freeman, K. A., Collister, J. A. and Hayes, J. M. (1991) Enhanced resolution of organic compounds from sediments by isotopic gas chromatography-combustion-mass spectrometry. Journal of Chromatography 585, 177-180.

Macko, S.A., Ryan, M. and Engel, M.H. (1998) Stable isotopic analysis of individual carbohydrates by gas chromatographic/combustion/isotope ratio mass spectrometry. Chemical Geology 152, 205-210.

Meier-Augenstein W. (1999) Applied gas chromatography coupled to isotope ratio mass spectrometry. Journal of Chromatography A 842(1-2), 351-371.

Meier-Augenstein W. (2002) Stable isotope analysis of fatty acids by gas chromatography- isotope ratio mass spectrometry. Analytica Chimica Acta 465(1-2), 63-79.

Merritt D. A., Brand W. A., and Hayes J. M. (1994) Isotope-Ratio-Monitoring Gas-Chromatography Mass-Spectrometry - Methods for Isotopic Calibration. Organic Geochemistry 21(6-7), 573-583.

Merritt D. A., Freeman K. H., Ricci M. P., Studley S. A., and Hayes J. M. (1995a) Performance and Optimization of a Combustion Interface for Isotope Ratio Monitoring Gas-Chromatography Mass-Spectrometry. Analytical Chemistry 67(14), 2461-2473.

Merritt D. A. and Hayes J. M. (1994a) Factors Controlling Precision and Accuracy in Isotope-Ratio- Monitoring Mass-Spectrometry. Analytical Chemistry 66(14), 2336-2347.

Merritt D. A. and Hayes J. M. (1994b) Nitrogen Isotopic Analyses by Isotope-Ratio-Monitoring Gas- Chromatography Mass-Spectrometry. Journal of the American Society for Mass Spectrometry 5(5), 387-397.

Merritt D. A. and Hayes J. M. (1995) Reactor Selection for Carbon and Nitrogen Isotopic Analyses by Isotope-Ratio-Monitoring Gc/Ms. Abstracts of Papers of the American Chemical Society 210, 40-GEOC.

Merritt D. A., Hayes J. M., and Marias D. J. D. (1995b) Carbon Isotopic Analysis of Atmospheric Methane by Isotope- Ratio-Monitoring Gas-Chromatography Mass-Spectrometry. Journal of Geophysical Research-Atmospheres 100(D1), 1317-1326.

Mosandl A. (1992) Capillary Gas-Chromatography in Quality Assessment of Flavors and Fragrances. Journal of Chromatography 624(1-2), 267-292.

Preston T. and Owens N. J. P. (1985) Preliminary C-13 Measurements Using a Gas-Chromatograph Interfaced to an Isotope Ratio Mass-Spectrometer. Biomedical Mass Spectrometry 12(9), 510-513.

Prosser S. J. and Scrimgeour C. M. (1995) High-Precision Determination of H-2/H-1 in H-2 and H2o by Continuous-Flow Isotope Ratio Mass-Spectrometry. Analytical Chemistry 67(13), 1992-1997.

Reinhold, V.N., Wirtz-Peitz, F. and Biemann, K. (1974) Synthesis, gas-liquid chromatography, and mass spectrometry of per-o-trimethylsilyl carbohydrate boronates. Carbohydrate Research 37, 203-221.

Ricci M. P., Merritt D. A., Freeman K. H., and Hayes J. M. (1994) Acquisition and Processing of Data for Isotope-Ratio-Monitoring Mass-Spectrometry. Organic Geochemistry 21(6-7), 561-571.

Rice A. L., Gotoh A. A., Ajie H. O., and Tyler S. C. (2001) High-precision continuous-flow measurement of delta C-13 and delta D of atmospheric CH4. Analytical Chemistry 73(17), 4104-4110.

Ricci, M. P., Merritt, D. A., Freeman, K. H. and Hayes J. M. (1994) Acquisition and processing of data for isotope-ratio-monitoring mass spectrometry. Organic Geochemistry 21, 561-571.

Rieley, G. (1994) Derivatization of organic compounds prior to gas Chromatographic-Combustion-isotope ratio mass spectrometric analysis: Identification of isotope fractionation processes. Analyst 119, 915-919.

Sauer P. E., Eglinton T. I., Hayes J. M., Schimmelmann A., and Sessions A. L. (2001) Compound-specific D/H ratios of lipid biomarkers from sediments as a proxy for environmental and climatic conditions. Geochimica Et Cosmochimica Acta 65(2), 213-222.

Sephton M. A. and Gilmour I. (2001) Pyrolysis-gas chromatography-isotope ratio mass spectrometry of macromolecular material in meteorites. Planetary and Space Science 49(5), 465-471.

Sessions A. L., Burgoyne T. W., and Hayes J. M. (2001a) Correction of H-3(+) contributions in hydrogen isotope ratio monitoring mass spectrometry. Analytical Chemistry 73(2), 192-199.

Sessions A. L., Burgoyne T. W., and Hayes J. M. (2001b) Determination of the H-3 factor in hydrogen isotope ratio monitoring mass spectrometry. Analytical Chemistry 73(2), 200-207.

Silfer J. A., Engel M. H., Macko S. A., and Jumeau E. J. (1991) Stable Carbon Isotope Analysis of Amino-Acid Enantiomers by Conventional Isotope Ratio Mass-Spectrometry and Combined Gas- Chromatography Isotope Ratio Mass-Spectrometry. Analytical Chemistry 63(4), 370-374.

Sohns E., Gerling P., and Faber E. (1994) Improved Stable Nitrogen Isotope Ratio Measurements of Natural Gases. Analytical Chemistry 66(17), 2614-2620.

Tetens V., Kristensen N. B., and Calder A. G. (1995) Measurement of C-13 Enrichment of Plasma Lactate by Gas Chromatography/Isotope Ratio Mass-Spectrometry. Analytical Chemistry 67(5), 858-862.

Tissot S., Normand S., Guilluy R., Pachiaudi C., Beylot M., Laville M., Cohen R., Mornex R., and Riou J. P. (1990) Use of a New Gas-Chromatograph Isotope Ratio Mass-Spectrometer to Trace Exogenous C-13 Labeled Glucose at a Very Low-Level of Enrichment in Man. Diabetologia 33(8), 449-456.

van Dongen, B.E., Schouten S. and Sinninghe Damsté J.S. (2001) Gas chromatography/combustion/isotope-ratio-monitoring mass spectrometry analysis of methylboronic derivatives of monosaccharides: A new method for determining natural 13C abundances of carbohydrates. Rapid Communications in Mass Spectrometry 15, 496-500.

Woodbury S. E., Evershed R. P., Rossell J. B., Griffith R. E., and Farnell P. (1995) Detection of Vegetable Oil Adulteration Using Gas- Chromatography Combustion Isotope Ratio Mass-Spectrometry. Analytical Chemistry 67(15), 2685-2690.

Yu Z. Q., Sheng G. Y., Piu J. M., and Peng P. A. (2000) Determination of porphyrin carbon isotopic composition using gas chromatography-isotope ratio monitoring mass spectrometry. Journal of Chromatography A 903(1-2), 183-191.

Compound-specific hydrogen isotope analysis

Brand W.A., 2000, Method and apparatus for isotopic hydrogen determination in a sample. UK GB 2314155B, applied 11.06.96, published 02/02/2000 [PATENT assigned to Finnigan MAT]

Angerosa F, Bréas O., Contento S., Guillou C., Reniero F, and E. Sada, 1999, Application of stable isotope ratio analysis to the characterisation of the geographical origin of olive oils. Journal of Agricultural and Food Chemistry, 47, 1013-1017

Begley I.S. and C.M. Scrimgeour, 1997, High-precision δD and δ18O measurement for water and volatile organic compounds by continuous-flow pyrolysis isotope ratio mass spectrometry. Analytical Chemistry, 69, 1530-1535.

Brand W., Tegtmeyer A.R. and A. Hilkert, 1994, Compound-specific isotope analysis: extending towards 15N/14N and 18O/16O. Organic Geochemistry, 21, 585-594.

Brenna J.T., Tobias H.J. and T.N. Corso, 1997, High-precision D/H measurements from organics and position-specific carbon isotope analysis, In Stable Isotopes. H. Griffiths, Ed., BIOS Scientific Publishers Ltd, Oxford, UK. 1997, Chapter 1, pp. 1-12.

Burgoyne T. and J. Hayes, 1998, Quantitative production of H2 by pyrolysis of gas chromatographic effluents. Analytical Chemistry, 70, 5316-5141.

Corso T.N. and J. T. Brenna, 1999, On-line pyrolysis of hydrocarbons coupled to high-precision carbon isotope ratio analysis. Analytica Chimica Acta, 397, 217-224

Corso T.N. and J.T. Brenna, 1997, High precision position-specific isotope analysis. Proceedings of the National Academy of Sciences of the USA, 94, 1049-1053.

Corso T.N., Lewis B.A. and J. T. Brenna, 1998, Reduction of fatty acid methyl esters to fatty alcohols to improve volatility for isotopic analysis without extraneous carbon, Analytical Chemistry 1998, 70(18): 3752-3756. GC-TC=DELTA+XL; first report of 13C and 18O from vanillin by CSIA

Hor K., Ruff C., Weckerle B., Konig Th., and P. Schreier, 2000, On-line HRGC-IRMS determination of δ13C and δ18O values of flavour compounds-on the way to multi-element isotope ratio analysis for authenticity assessment in Frontiers of Flavour Science, Proc. 9th Wurman Symposium, editors P. Schieberle and K.H. Engle, in press.

Hener U., Brand W.A., Hilkert A.W., Juchelka D., Mosandl A., and F. Podebrad, 1998, Simultaneous on-line analysis of 18O/16O and 13C/12C ratios of organic compounds using GC-pyrolysis-IRMS. Z. Lebensm. Unters. Forsch. A., 206, 230-232.

Hener U., Mosandl A., Hilkert A., Bahrs-Windsberger J., Groomann M., and W.R. Sponholz, 1998, (Headspace-) GC-IRMS zur 13C/12C- und 18O/16O-Analyse von Ethanol aus alkoholhaltigen Getränken und Destillaten. (GC-IRMS for the 13C/12C and 18O/16O-analysis of ethanol from alcoholic beverages and distillates). Viticultural and Enological Sciences, 53, 49-53.

Hilkert A.H., Douthitt C.B., Schlueters H.J., and W. Brand, 1999, Isotope ratio monitoring GCMS of D/H by high temperature conversion isotope ratio mass spectrometry. Rapid Communications in Mass Spectrometry, 13, 1226-1230.

Konig T., Hor K., Ruff C., and P. Schreier, 1999, Multielement isotope ratio analysis for detection of authenticity of natural substances. GIT Labor-Fachz., 43, 496-498 (German).

PATENT Brenna, J. T., Tobias H. J., and K.J. Goodman, 1995, Interface system for isotopic analysis of hydrogen. U.S. US 5,661,038 (Cl. 436-173; G01N24/00), 26 Aug 1997, Appl. 442,059, 16 May 1995; 5 pp.

Ruff C., Hor K., Weckerle B., Konig T., and P. Schreier, 2000, Authentizit tskontrolle von Aromastoffen: Gaschomatographie-Isotopenverhaltnis-Massenspektrometrie zur Bestimmung des 2H/1H-Verhaltnisses von Benzaldehyd in Lebensmitteln. Deutsche Lebensmittel-Rundschau, 96, 243-247.

Ruff C., Hor K., Weckerle B., Schreier P., and T. Konig, 2000, 2H/1H ratio analysis of flavor compounds by on-line gas chromatography pyrolysis isotope ratio mass spectrometry (HRGC-P-IRMS): benzaldehyde. Journal of High Resolution Chromatography, 23, 357-359

Ruff C., Hör K., Weckerle B., König T., and P. Schreier, 2000, Authentizitätskontrolle von Aromastoffen: Benzaldehyd in alkoholischen Getränken. Alkohol-industrie, 9, 144-145.

Scrimgeour C.M., Begley I.S. and M.L. Thomason, 1999, Measurement of deuterium incorporation into fatty acids by gas chromatography/isotope ratio mass spectrometry. Rapid Communications in Mass Spectrometry, 13, 271-274.

Session A.L., Burgoyne T.W. and J.M. Hayes, 2000, Correction of the H3+ contribution in hydrogen isotope ratio mass

spectrometry. Analytical Chemistry, Session A.L., Burgoyne T.W. and J.M. Hayes, 2000, Determination of the H3 contribution in hydrogen isotope ratio mass

spectrometry. Analytical Chemistry, Sessions A.L., Burgoyne T.W., Schimmelmann A., and J.M. Hayes, 1999, Fractionation of hydrogen isotopes in lipid

biosynthesis. Organic Geochemistry, 30, 1193-1200. Tobias H.J. and J.T. Brenna, 1997, On-line pyrolysis as a limitless reduction source for high-precision isotopic analysis of

organic-derived hydrogen. Analytical Chemistry, 69, 3148-3152. Tobias H.J., Goodman K.J., Blacken C.E. and J.T. Brenna, 1995, High-precision D/H measurement from hydrogen gas and

water by continuous-flow isotope ratio mass spectrometry. Analytical Chemistry, 67, 2486-2492. Ward J.A.M., Ahad J.M.E., Lacrampe-Couloume G., Slater G.F., Edwards E.A., and B. Sherwood-Lollar, 2000, Hydrogen

isotope fractionation during methanogenic degradation of toluene: potential for direct verification of bioremediation. Environmental Science and Technology, 34, 4577-4581.

Werner R.A., 1998, Entwicklung neuer Verfahren fuer die on-line 18O/16O und 2H/1H Isotopenverhaeltnismessung und ihre Anwendung zur Authentizitaets- und Herkunftsbestimmung bei Naturstoffen. Ph.D. Thesis, Technical University of Munich ISBN 3-933083-73-7.

Xie S., Nott C.J., Avsejs L.A., Volders F., Maddy D., Chambers F.M., Gledhill A., Carter J.F., and R.P. Evershed, 2000, Palaeoclimate records in compound-specific δD values of a lipid biomarker in ombrotrophic peat. Organic Geochemistry, 31, 1053-1057

Biochemistry – Partitioning of isotopes amongst different compound classes Abelson PH, Hoering TC (1961) Carbon isotope fractionation in formation of amino acids by photosynthetic organisms.

Proc NAS 47:623-632 Anderson LA (1995) On the hydrogen and oxygen content of marine phytoplankton. Deep-Sea Research 42:1675-1680 Belyaev SS, Wolkin R, Kenealy WR, DeNiro MJ, Epstein S, Zeikus JG (1983) Methanogenic bacteria from the

Bondyuzhskoe oil field: general characterization and analysis of stable-carbon isotopic fractionation. Appl Environ Microbiol 45:691-697

Benner R, Fogel ML, Sprague EK, Hodson RE (1987) Depletion of 13C in lignin and its implications for stable carbon isotope studies. Nature 329:708-710

Bidigare RR, Hanson KL, Buesseler KO, Wakeham SG, Freeman KH, Pancost RD, Millero FJ, Steinberg P, Popp BN, Latasa M, Landry MR, Laws EA (1999) Ironstimulated changes in 13C fractionation and export by equatorial Pacific phytoplankton: toward a paleogrowth rate proxy. Paleoceanography 14:589-595

Bidigare RR, Popp BN, Kenig F, Hanson K, Laws EA, Wakeham SG (1997) Variations in the stable carbon isotopic composition of algal biomarkers. Abstracts, 18th International Meeting on Organic Geochemistry 22-26 September, Maastricht, The Netherlands p. 119-120.

Bird CW, Lynch JM, Pirt FJ, Reid WW, Brooks CJW, Middleditich BS (1971) Steroids and squalene in Methylococcus capsulatus grown on methane. Nature 230:473-474

Blair N, Leu A, Muñoz E, Olsen J, Des Marais D (1985) Carbon isotopic fractionation in heterotrophic microbial metabolism. Appl Environ Microbiol 50:996-1001

Boucher Y, Doolittle WF (2000) The role of lateral gene transfer in the evolution of isoprenoid biosynthesis pathways. Mol Microbiol 37:703-716

Calvin M, Bassham JA (1962) The photosynthesis of carbon compounds. WA Benjamin, New York Cavalier-Smith T (2000) Membrane heredity and early chloroplast evolution. Trends Plant Sci 5:174-182 Cleland WW, O’Leary MH, Northrop DB (eds) (1977) Isotope Effects on Enzyme-Catalyzed Reactions. Appendix A: A note on the Use of Fractionation Factors versus Isotope

Effects on Rate Constants. University Park Press, Baltimore, Maryland Coffin RB, Velinsky DJ, Devereux R, Price WA, Cifuentes LA (1990) Stable carbon isotope analysis of nucleic acids to

trace sources of dissolved substrates used by estuarine bacteria. Appl Environ Microbiol 56:2012-2020 DeNiro MJ, Epstein S (1977) Mechanism of carbon isotope fractionation associated with lipid synthesis. Science 197:261-

263

Descolas-Gros C, Fontugne M (1990) Stable carbon isotope fractionation by marine phytoplankton during photosynthesis. Plant, Cell and Environment 13:207-218

Disch A, Schwender J, Müller C, Lichtenthaler HK, Rohmer M (1998) Distribution of the mevalonate and glyceraldehydes phosphate/pyruvate pathways for isoprenoid biosynthesis in unicellular algae and the cyanobacterium Synechocystis PCC 6714. Biochem J 333:381-388

van Dongen, B.E., Schouten S. and Sinninghe Damsté J.S. (2002)Carbon isotope variability in aquatic algae and terrestrial plants. Marine Ecology Progress Series 232, 83-92.

Estep MF, Hoering TC (1980) Biogeochemistry of the stable hydrogen isotopes. Geochim Cosmochim Acta 44:1197-1206 Farquhar GD (1983) On the nature of carbon isotope discrimination in C4 species. Aust J Plant Physiol 10:205-226 Farquhar GD, Ehleringer JR, Hubick KT (1989) Carbon isotope discrimination and photosynthesis. Annu Rev Plant Physiol

Plant Mol Biol 40:503-537 Fogel ML, Tuross N (1999) Transformation of plant biochemicals to geological macromolecules during early diagenesis.

Oecologia 120:336-346 Garrett RH, Grisham CM (1999) Biochemistry, second edition. Harcourt College Publishers, Philadelphia, Pennsylvania Gelwicks JT, Risatti JB, Hayes JM (1989) Carbon isotope effects associated with autotrophic acetogenesis. Org Geochem

14:441-446 Guy RD, Fogel ML, Berry JA (1993) Photosynthetic fractionation of the stable isotopes of oxygen and carbon. Plant

Physiol 101:37-47 Hatch MD (1977) C4 pathway of photosynthesis: mechanism and physiological function. Trends in Biochemical Sciences

2:199-202 Hare PE, Fogel ML, Stafford Jr. TW, Mitchell AD, Hoering TC (1991) The isotopic composition of carbon and nitrogen in

individual amino acids isolated from modern and fossil proteins. J Archaeol Sci 18:277-292 Hayes JH (2000) Lipids as a common interest of microorganisms and geochemists. PNAS 97:14033-14034 Ivanovsky RN (1985) Carbon metabolism in phototrophic bacteria under different conditions of growth. In: IS Kulaev, EA

Dawes, DW Tempest (eds) Environmental Regulation of Microbial Metabolism, FEMS Symposium No.23, p 263-272 Academic Press, New York

Jahnke LL, Summons RE, Hope JM, Des Marais DJ (1999) Carbon isotopic fractionation in lipids from methanotrophic bacteria II: The effects of physiology and environmental parameters on the biosynthesis and isotopic signatures of biomarkers. Geochim Cosmochim Acta 63:79-93

Keil RG, Fogel ML (2001) Reworking of amino acid in marine sediments: Stable carbon isotopic composition of amino acids in sediments along the Washington coast. Limnol Oceanogr 46:14-23

Lange BM, Rujan T, Martin W, Croteau R (2000) Isoprenoid biosynthesis: The evolution of two ancient and distinct pathways across genomes. PNAS 97:13172-13177

Laws EA, Popp BN, Bidigare RR, Kennicutt MC, Macko SA (1995) Dependence of phytoplankton carbon isotopic composition on growth rate and [CO2]aq: Theoretical considerations and experimental results. Geochim Cosmochim Acta 59:1131-1138

Lichtenthaler HK, Schwender J, Disch A, Rohmer M (1997) Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway. FEBS Letters 400:271-274

Lichtenthaler HK (1999) The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 50:47-65

Luo Y, Sternberg L (1991) Deuterium heterogeneity in starch and cellulose nitrate of CAM and C3 plants. Phytochemistry 30:1095-1098

Luo Y-H, Sternberg L, Suda S, Kumazawa S, Mitsui A (1991) Extremely low D/H ratios of photoproduced hydrogen by cyanobacteria. Plant Cell Physiol 32:897-900

Macko SA, Fogel ML, Hare PE, Hoering TC (1987) Isotopic fractionation of nitrogen and carbon in the synthesis of amino acids by microorganisms. Chem Geol 65:79-92

Madigan MT, Takigiku R, Lee RG, Gest H, Hayes JM (1989) Carbon isotope fractionation by thermophilic phototrophic sulfur bacteria: Evidence for autotrophic growth in natural populations. Appl Env Microbiol 55:639-644

Madigan MT, Martinko JM, Parker J (2000) Brock Biology of Microorganisms, ninth edition. Prentice Hall, New Jersey Melzer E, Schmidt H-L (1987) Carbon isotope effects on the pyruvate dehydrogenase reaction and their importance for

relative carbon-13 depletion in lipids. J Biol Chem 262:8159-8164 Melzer E, O’Leary MH (1987) Anapleurotic CO2 fixation by phosphoenolpyruvate carboxylase in C3 plants. Plant Physiol

84:58-60 Monson KD, Hayes JM (1980) Biosynthetic control of the natural abundance of carbon 13 at specific positions within fatty

acids in Escherichia coli. J Biol Chem 255:11435-11441 Monson KD, Hayes JM (1982a) Carbon isotopic fractionation in the biosynthesis of bacterial fatty acids. Ozonolysis of

unsaturated fatty acids as a means of determining the intramolecular distribution of carbon isotopes. Geochim Cosmochim Acta 46:139-149

Monson KD, Hayes JM (1982b) Biosynthetic control of the natural abundance of carbon 13 at specific position within fatty acids in Saccharomyces cerevisiae. Isotope fractionations in lipid synthesis as evidence for peroxisomal regulation. J Biol Chem 257: 5568-5575

Ohlrogge JB, Jaworski JG (1997) Regulation of fatty acid synthesis. Annu Rev Plant Physiol Plant Mol Biol 48:109-136 O’Leary MH (1981) Carbon isotope fractionation in plants. Phytochemistry 20:553-567 O’Leary MH, Rife JE, Slater JD (1981) Kinetic and isotope effect studies of maize phosphoenolpyruvate

carboxylase.Biochemistry 20:7308-7314 Popp BN, Kenig F, Wakeham SG, Laws EA, Bidigare RR (1998a) Does growth rate affect ketone unsaturation and

intracellular carbon isotopic variability in Emiliania huxleyi? Paleoceanography 13:35-41 Popp BN, Laws EA, Bidigare RR, Dore JE, Hanson KL, Wakeham SG (1998b) Effect of phytoplankton cell geometry on

carbon isotopic fractionation. Geochim Cosmochim Acta 62:69-77 Popp BN, Trull T, Kenig F, Wakeham SG, Rust TM, Tilbrook B, Griffiths FB, Wright SW, Marchant HJ, Bidigare RR,

Laws EA (1999) Controls on the carbon isotopic composition of Southern Ocean phytoplankton. Global Biogeochem Cycles 13:827-843

Preub A, Schauder R, Fuchs G, Stichler W (1989) Carbon isotope fractionation by autotrophic bacteria with three different CO2 fixation pathways. Z Naturforsch 44:397-402

Riebesell U, Revill AT, Holdsworth DG, Volkman JK (2000) The effects of varying CO2 concentration on lipid composition and carbon isotope fractionation in Emiliania huxleyi. Geochim Cosmochim Acta 64:4179-4192

Roberts RB, Abelson PH, Cowie DB, Bolton ET, Britten RJ (1955) Studies of biosynthesis in Escherichia coli.Carnegie Institution of Washington Publication 607, Washington, D.C.

Robinson JJ, Cavanaugh CM (1995) Expression of form I and form II Rubisco in chemoautotrophic symbioses: Implications for the interpretation of stable carbon isotope values. Limnol Oceanogr 40:1496-1502

Roeske CA, O’Leary MH (1984) Carbon isotope effects on the enzyme-catalyzed carboxylation of ribulose bisphosphate. Biochemistry 23:6275-6284

Roeske CA, O’Leary MH (1985) Carbon isotope effect on carboxylation of ribulose bisphosphate catalyzed by ribulosebisphosphate carboxylase from Rhodospirillum rubum. Biochemistry 24:1603-1607

Rohmer M (1993) The biosynthesis of triterpenoids of the hopane series in the Eubacteria: A mine of new enzyme reactions. Pure and Appl Chem 65:1293-1298

Rohmer M, Knani M, Simonin P, Sutter B, Sahm H (1993) Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem J 295:517-524

Rossmann A, Butzenlechner M, Schmidt H-L (1991) Evidence for a nonstatistical carbon isotope distribution in natural glucose. Plant Physiol 96:609-614

Sakata S, Hayes JH, McTaggart AR, Evans RA, Leckrone KJ, Togasaki RK (1997) Carbon isotopic fractionation associated with lipid biosynthesis by a cyanobacterium:Relevance for interpretation of biomarker records. Geochim Cosmochim Acta 61:5379-5389

Sakata S, Hayes JH, Rohmer M, Hooper AB, Seeman M (2001) Molecular and carbon isotopic compositions of lipids isolated from an ammonia-oxidizing chemoautotroph. Proc Nat Acad Sci USA

Schleucher J, Vanderveer PJ, Sharkey TD (1998) Export of carbon from chloroplasts at night. Plant Physiol 118:1439-1445 Schouten S, Klein Breteler WCM, Blokker P, Schogt N, Rijpstra WIC, Grice K, Baas M, Sinninghe Damasté JS (1998)

Biosynthetic effects on the stable carbon isotopic compositions of algal lipids: Implications for deciphering the carbon isotopic biomarker record. Geochim Cosmochim Acta 62:1397-1406

Schwender J, Gemünden C, Lichtenthaler HK (2001) Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-Cmethylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids. Planta 212:416-423

Scott JH, Nealson KH (1994) A biochemical study of the intermediary carbon metabolism of Shewanella putrefaciens. J Bacteriol 176:3408-3411

Seto H, Watanabe H, Furihata K (1996) Simultaneous operation of the mevalonate and non-mevalonate pathways in the biosynthesis of isopentenyl diphosphate in Streptomyces aeriouvifer. Tetrahedron Lett 37:7979-7982

Sternberg LdSL, DeNiro MJ, Ajie HO (1986) Isotopic relationships between saponifiable lipids and cellulose nitrate prepared from red, brown and green algae. Planta 169:320-324

Sternberg LdSL (1988) D/H ratios of environmental water recorded by D/H ratios of plant lipids. Nature 333:59-61 Strauss G, Fuchs G (1993) Enzymes of a novel autotrophic CO2 fixation pathway in the phototrophic bacterium

Chloroflexus aurantiacus, the 3-hydroxypropionate cycle. Eur J Biochem 275:633-643 Summons RE, Jahnke LL, Roksandic Z (1994) Carbon isotopic fractionation in lipids from methanotrophic bacteria:

Relevance for interpretation of the geochemical record of biiomarkers. Geochim Cosmochim Acta 13:2853-2863 Summons RE, Franzmann PD, Nichols PD (1998) Carbon isotopic fractionation associated with methylotrophic

methanogenesis. Org Geochem 28:465-475 Teece MA, Folge ML, Dollhopf ME, Nealson KH (1999) Isotopic fractionation associated with biosynthesis of fatty acids

by a marine bacterium under oxic and anoxic conditions. Org Geochem 30:1571-1579 van der Meer MTJ, Schouten S, Sinninghe Damasté JS (1998) The effect of the reversed tricarboxylic acid cycle on the 13C

contents of bacterial lipids. Org Geochem 28:527-533

van der Meer MTJ, Schouten S, Rijpstra WIC, Fuchs G, Sinninghe Damasté JS (2001a) Stable carbon isotope fractionations of the hyperthermophilic crenarchaeon Metallosphaera sedula. FEMS Microbiol Lett

van der Meer MTJ, Schouten S, van Dongen W, Rijpstra WIC, Fuchs G, Sinninghe Damasté JS, de Leeuw JW, Ward DM (2001b) Biosynthetic controls on the 13C-contents of organic components in Chloroflexus aurantiacus. J Biol Chem

Vogler EA, Hayes JM (1978) The synthesis of carboxylic acids with carboxyl carbons of precisely known stable isotopic composition. International Journal of Applied Radiation and Isotopes 29:297-300

Vogler EA, Hayes JM (1979) Carbon isotopic fractionation in the Schmidt decarboxylation: evidence for two pathways to products. J Org Chem 44:3682-3686

White DW (1999) The Physiology and Biochemistry of Prokaryotes, 2nd edition. Oxford University Press, New York Winters JK (1971) Variations in the natural abundances of 13C in proteins and amino acids. Ph.D. Thesis, University of

Texas, Austin, Texas, 76 p. Yakir D (1992) Variatons in the natural abundance of oxygen-18 and deuterium in plant carbohydrates. Plant, Cell and

Environment 15:1005-1020 Yakir D, DeNiro MJ (1990) Oxygen and hydrogen isotope fractionation during cellulose metabolism in Lemna gibba L.

Plant Physiol 93:325-332 Zelitch I (1975) Improving the efficiency of photosynthesis. Science 188:626-633 Zubay G (1998) Biochemistry, fourth edition. WCB/McGraw-Hill, New York

CSIA in Archaeological Chemistry

Ambrose, S. A. and L. Norr (1993). "Experimental evidence for the relationship of the carbon iostope ratios of whole diet and dietary protein to those of bone collagen and carbonate", in Prehistoric Human Bone. Archaeology at the molecular level. J. B. Lambert and G. Grupe (eds.). Berlin, Springer-Verlag: 1-37.

Ambrose, S. H., B. M. Butler, et al. (1997). "Stable isotopic analysis of human diet in the Marianas archipelago, Western Pacific." American Journal of Physical Anthropology 104(3): 343-361.

Beerling, D. J., D. P. Mattey, et al. (1993). "Shifts in the d13C composition of Salix herbacea L. leaves in response to spatial and temporal gradients of atmospheric CO2 concentration." Proceedings of the Royal Society London Series B- Biological Sciences 253: 53-60.

Copley, M. S., R. Berstan, et al. (2003). "Direct chemical evidence for widespread dairying in prehistoric Britain." Proceedings of the National Academy of Sciences 100(4): 1524-1529.

Copley, M. S., P. J. Rose, et al. (2001). "Detection of palm fruit lipids in archaeological pottery from Qasr Ibrim, Egyptian Nubia." Proceedings of the Royal Society Biological Section 268: 593-597.

De Niro, M. J. and S. Epstein (1977). "Mechanisms of carbon isotope fractionation." Science(197): 261-263. Docherty, G., V. Jones, et al. (2001). "Practical and Theoretical Considerations in the Gas

Chromatography/Combustion/Isotope Ratio Mass Spectrometry δ13C Analysis of Small Polyfunctional Compounds." Rapid Communications in Mass Spectrometry 15: 730-738.

Evershed, R. P., S. N. Dudd, et al. (1999). "Lipids as carriers of anthropogenic signals from prehistory." Philosophical Transactions of the Royal Society of London Series B-Biological Sciences 354(1379): 19-31.

Evershed, R. P., S. N. Dudd, et al. (2002). "Chemistry of Archaeological Animal Fats." Accounts of Chemical Research 35(8): 660-668.

Friedli, H., H. Lotscher, et al. (1986). "Ice core record of the 13C/12C ratio of atmospheric CO2 in the past two centuries." Nature 324: 237-238.

Macko, S. A., M. H. Engel, et al. (1999). "Documenting the diet in ancient human populations through stable isotope analysis of hair." Philosophical Transactions of the Royal Society of London Series B-Biological Sciences 354(1379): 65-75.

Macko, S. A., M. E. Uhle, et al. (1997). "Stable Nitrogen Isotope Analysis of Amino Acid Ennatiomers by Gas Chromatography/Combustion/Isotope Ratio Mass Spectrometry." Analytical Chemistry 69: 926-929.

Raven, A. M., P. F. van Bergen, et al. (1997). "Formation of long-chain ketones in archaeological pottery vessels by pyrolysis of acyl lipids." Journal of Analytical and Applied Pyrolysis 40-1: 267-285.

Richards, M. P. and R. E. M. Hedges (1999). "Stable Isotope Evidence for Similarities in the Types of Marine Foods Used by Late Mesolithic Humans at Sites Along the Atlantic Coast of Europe." Journal of Archaeological Science 26: 717-722.

Schwarcz, H. P., J. Melbye, et al. (1985). "Stable isotopes in human skeletons of southern Ontario:reconstructing palaeodiet." Journal of Archaeological Science 12: 187-206.

Stott, A. W. and R. P. Evershed (1996). "δ13C analysis of cholesterol preserved in archaeological bones and teeth." Analytical Chemistry 68(24): 4402-4408.

Stuart-Williams, H. L. Q., H. P. Schwarcz, et al. (1996). "The isotopic composition and diagenesis of himan bone from Teotihuacan and Oaxaca, Mexico." Palaeogeography, Palaeoclimatology, Palaeoecology 126: 1-14.

van der Merwe, N. J. and H. Tschauner (1999). C4 plants and the development of human societies. C4 plant biology. R. F. Sage and R. K. Monson. San Diego, Calif., Academic Press: 509-549.

CSIA in Microbiology Abelson, P. H. and Hoering, T. C., 1961. Carbon isotope fractionation in formation of amino acids by photosynthetic

organisms. Proc. Natl. Acad. Sci. 47, 623-632. Aloisi, G., Pierre, C., Rouchy, J.-M., Foucher, J.-P., Woodside, J., and the MEDINAUT Scientific Party, 2001. Methane-

related authigenic carbonates of Eastern Mediterranean Sea mud volcanoes and their possible relation to gas hydrate destabilization. Earth Planet. Sci. Lett. 184, 321-338.

Blair, N., Leu, A., Nunoz, E., Olsen, J., Kwong, E., Des Marais, D., 1985. Carbon isotopic fractionation in heterotrophic microbial metabolism. Appl. Environ. Microbiol. 50, 996-1001.

Boschker, H. T. S., Nold, S. C., Wellsbury, P., Bos, D., de Graaf, W., Pel, R., Parkes, R. J., Cappenberg, T. E., 1999. Direct linking of microbial populations to specific biogeochemical processes by 13C-labelling of biomarkers. Nature 392, 801-804.

Bull, I. D., Parekh, N. R., Hall, G. H., Ineson, P., and Evershed, R. E., 2000. Detection and classification of atmospheric methane oxidizing bacteria in soil. Nature 405, 175-178.

Deines, P., 1980. The Isotopic Composition of Reduced Organic Carbon. In: Fritz, P., Fontes, J. C. (Eds.), Handbook of Environmental Isotope Geochemistry. Elsevier, Amsterdam, The Netherlands.

Elvert, M., Suess E., and Whiticar, M. J., 1999. Anaerobic methane oxidation associated with marine gas hydrates: superlight C-isotopes from saturated and unsaturated C20 and C25 irregular isoprenoids. Naturwissenschaften 86, 295-300.

Elvert, M., Suess, E., Greinert, J. and Whiticar, M. J., 2001. Archaea mediating anaerobic methane oxidation in deep-sea sediments at cold seeps of the eastern Aleutian subduction zone. Org. Geochem. 31, 1175-1187.

Hayes J. M. (1993) Factors controlling 13C contents of sedimentary organic compounds: Principles and evidence. Mar. Geol. l, 111-125.

Hinrichs K. –U., Hayes J. M., Sylva S. P., Brewer P. G., DeLong E. F., 1999. Methane-consuming archaebacteria in marine sediments. Nature 398, 802-805.

Hinrichs, K.-U., Summons, R. E., Orphan, V., Sylva, S. P., Hayes, J. M., 2000. Molecular and isotopic analysis of anaerobic methane-oxidizing communities in marine sediments. Org. Geochem. 31, 1685-1701.

Hoefs M. J. L., Schouten S., de Leeuw J. W., King L. L., Wakeham S. G., Sinninghe Damsté J. S., 1997. Ether lipids of planktonic archaea in the marine water column. Appl. Environ. Microbiol. 63, 3090-3095.

Jahnke L. L., Summons R. E., Dowling L. M., Zahiralis K. D., 1995. Identification of methanotrophic lipid biomarkers in cold-seep mussel gills: chemical and isotopic analysis. Appl. Environ. Microbiol. 61, 576- 582.

Jahnke, L.L., Summons, R.E., Hope, J.M., Des Marais, D.J., 1999. Carbon isotopic fractionation in lipids from methanotrophic bacteria II: The effects of physiology and environmental parameters on the biosynthesis and isotopic signatures of biomarkers. Geochim. Cosmochim. Acta 63, 79-93.

Kuypers M.M.M., Blokker P., Erbacher J., Kinkel H., Pancost R.D., Schouten S. and Sinninghe Damsté J.S., 2001. Massive expansion of marine archaea during a mid-Cretaceous oceanic anoxic event. Science, in press.

van der Meer, M. T. J., Schouten, S., Sinninghe Damsté, J. S., 1998. The effect of the reversed tricarboxylic acid cycle on the 13C contents of bacterial lipids. Org. Geochem. 28, 527-533.

van der Meer, M. T. J., Schouten, S., van Dongen, B., Rijpstra, W. I. C., Fuchs, G., Sinninghe Damsté, J. S., de Leeuw, J., Ward, D. M., 2001a. Biosynthetic controls on the 13C contents of organic components in the photoautotrophic bacterium Chloroflexux aurantiacus. J. Biol. Chem. 276, 10971-10976.

Van der Meer M.T.J., Schouten S., Rijpstra W.I.C., Fuchs G. and Sinninghe Damsté J.S., 2001b. Stable carbon isotope fractionations of the hyperthermophilic crenarchaeon Metallosphaera sedula. FEMS Microbiol. Lett. 196, 67-70.

Monson, K. D., Hayes, J. M., 1982. Carbon isotopic fractionation in the biosynthesis of bacterial fatty acids. Ozonolysis of unsaturated fatty acids as a means of determining the intramolecular distribution of carbon isotopes. Geochim. Cosmochim. Acta 46, 139-149.

Pancost, R. D., Sinninghe Damsté, J. S., de Lint, S., van der Maarel, M. J. E. C., Gottschal, J. C., The Medinaut Shipboard Scientific Party, 2000a. Biomarker evidence for widespread anaerobic methane oxidation in Mediterranean sediments by a consortium of methanogenic archaea and bacteria. Appl. Environ. Microbiol., 66, 1126-1132.

Pancost, R. D., van Geel, B., Baas, M., Sinninghe Damsté, J. S., 2000b. δ13C values and radiocarbon dates of microbial biomarkers as tracers for carbon recycling in peat deposits. Geology 28, 663-666.

Pancost, R. D., Hopmans, E. C., Sinninghe Damsté, J. S., and the MEDINAUT Shipboard Scientific Party, 2001. Archaeal lipids in Mediterranean Cold Seeps: Molecular proxies for anaerobic methane oxidation. Geochim. Cosmochim. Acta 65, 1611-1627.

Preuß, A., Schauder, R., Fuchs, G., Stichler, W., 1989. Carbon isotope fractionation by autotrophic bacteria with 3 different CO2 fixation pathways. Zeitschrift fur Naturforschung C-A Journal of Biosciences 44, 397-402.

Quandt, I., Gottshalk, G., Ziegler, H. and Stichler, W., 1977. Isotope discrimination by photosynthetic bacteria. FEMS Microbiol. Lett. 1, 125-128.

Ruby, E. G., Jannasch, H. W., Deuser, W. G., 1987. Fractionation of stable carbon isotopes during chemoautotrophic growth of sulfur-oxidizing bacteria. Appl. Environ. Microbiol. 53, 1940-1943.

Sakata, S., Hayes, J. M., McTaggart, A. R., Evans, R. A., Leckrone, K. J., Togasaki, R. K., 1997. Carbon isotopic fractionation associated with lipid biosynthesis by a cyanobacterium: Relevance for interpretation of biomarker records. Geochim. Cosmochim. Acta 61, 5379-5389.

Sirevag, R., Buchanan, B. B., Berry, J. A., Throughton, J. H., 1977. Mechanisms of CO2 fixation in bacterial photosynthesis studied by the carbon isotope technique. Arch. Microbiol. 112, 35-38.

Summons, R. E., Jahnke, L. L., Roksandik, Z., 1994. Carbon isotopic fractionation in lipids from methanotrophic bacteria: relevance for interpretation of the geochemical record of biomarkers. Geochim. Cosmochim. Acta 58, 2853-2863.

Summons R. E., Franzmann P. D., Nichols P. D., 1998. Carbon isotopic fractionation associated with methylotrophic methanogenesis. Org. Geochem. 28, 465-476.

Teece, M. A., Fogel, M. L., Dollhopf, M. E., Nealson, K. H., 1999. Isotopic fractionation associated with biosynthesis of fatty acids by a marine bacterium under oxic and anoxic conditions. Org. Geochem. 30, 1571-1579.

Thiel, V., Peckmann, J., Seifert, R., Wehrung, P., Reitner, J., and Michaelis, W., 1999. Highly isotopically depleted isoprenoids: molecular markers for ancient methane venting. Geochim. Cosmochim. Acta 63, 3959-3966.

Thiel, V., Peckmann, J., Richnow, H. H., Luth, U., Reitner, J., Michaelis, W., 2001. Molecular signals for anaerobic methane oxidation in Black Sea seep carbonates and a microbial mat. Mar. Chem. 73, 97-112.

Yamada, K., Ishiwatari, R., Matsumoto, K., and Naraoka, H., 1997. δ13C records of diploptene in the Japan Sea sediments over the past 25 kyr. Geochem. J. 31, 315-321.

Zhang, C. L., Li, Y., Ye, E., Fong, J., Peacock, A. D., Blunt, E., Fang, J. Lovley, D., and White, D. C., in press. Carbon isotope signatures of fatty acids in Geobacter metallireducens and Shewanella algae. This volume.

CSIA in Geological Sciences (just a small sample)

Bidigare R. R., Fluegg A., Freeman K. H., Hanson K. L., Hayes J. M., Hollander D., Jasper J. P., King L. L., Laws E. A.,

Millero F. J., Pancost R. D., Popp B. N., Steinberg P. A., and Wakeham S. G., 1997, Consistent fractionation of 13C in nature and in the laboratory: Growth-rate effects in some haptophyte algae: Global Biogeochemical Cycles 11, 179-192.

Bidigare, R. R., Hanson, K. L., Buessler, K., Wakeham, S. G., Freeman, K. H., Pancost, R. D., Millero, F. J., Steinberg, P., Popp, B. N., Latasa, M., Landry, M. R., and Laws, E. A. (1999) Iron-stimulated changes in carbon isotopic fractionation by phytoplankton in equatorial Pacific waters. Paleoceanography 14, 589-595.

Collister J. W., Summons R. E., Lichtfouse E. L. and Hayes J. M. (1992) An isotopic bio-geochemical study of the Green River oil shale. Org. Geochem. 19, 265-276.

Collister J. W., Rieley G., Stern B., Eglinton G., and Fry B. (1994) Compound-specific δ13C analysis of leaf lipids from plants with different carbon dioxide metabolisms: Organic Geochemistry 21, 619-627.

Collister J. W. and Wavrek D. (1996) δ13C compositions of saturate and aromatic fractions of lacustrine oils and bitumens: Evidence for water column stratification. Org. Geochem., v. 24, p. 913-920.

Deines, P., 1980. The Isotopic Composition of Reduced Organic Carbon. In: Fritz, P., Fontes, J. C. (Eds.), Handbook of Environmental Isotope Geochemistry. Elsevier, Amsterdam, The Netherlands.

Ficken, K.J., Barber, K.E. and Eglinton, G. 1998: Lipid biomarker, δ13C, and plant macrofossil stratigraphy of a Scottish montane peat bog over the last two millennia. Organic Geochemistry 28, 217-237.

Freeman K. H., Hayes J. M., Trendel J. M., and Albrecht P. (1990) Evidence from carbon isotope measurements for diverse origins of sedimentary hydrocarbons. Nature 343, 254-256.

Freeman K. H. and Hayes J. M. (1992) Fractionation of carbon isotopes by phytoplankton and estimates of ancient CO2 levels. Global Biogeochem. Cycles 6, 185-198.

Freeman K. H. and Wakeham S. G. (1992) Variations in the distributions and isotopic compositions of alkenones in the Black Sea. Org. Geochem. 19, 277-285.

Freeman K. H., Wakeham S. G., and Hayes J. M. (1994) Predictive isotopic biogeochemistry: hydrocarbons from anoxic marine basins. Organic Geochemistry 21, 629-644.

Goericke R., Montoya, J. P., Fry, B., 1994. Physiology of isotope fractionation in algae and cyanobacteria. In: Lajtha, K. and Michener, B. (Eds.), Stable Isotopes in Ecology, Blackwell Scientific Publications, Oxford, UK, 187-221.

Grice K., Schaeffer P., Schwark L. and Maxwell J.R. (1997) Changes in palaeoenvironmental conditions during deposition of the Permian Kupferschiefer (Lower Rhine Basin, N.W. Germany) from variations in isotopic compositions of biomarker components. Org Geochem 26 677-690.

Grice, K., Schouten, S., Nissenbaum, A., Charrach, J., Sinninghe Damsté, J. S., 1998. Isotopically heavy carbon in the C21 to C25 regular isoprenoids in halite-rich deposits from the Sdom Formation, Dead Sea Basin, Israel. Org. Geochem. 28, 349-359.

Hartgers W. A., Sinninghe Damste J. S., Requejo A. G., Allan J., Hayes J. M., and de Leeuw J. W. (1994) Evidence for only minor contributions from bacteria to sedimentary organic carbon, Nature 369, 224-227.

Hayes, J.M., Takigiku, R., Ocampo, R., Callot, H.J., Albrecht, P., 1987. Isotopic compositions and probable origins of organic molecules of the Eocene Messel shale. Nature 329, 48-51.

Hayes J. M., Popp B. N., Takigiku R., and Johnson M. W. (1989) An isotopic study of biogeochemical relationships between carbonates and organic carbon in the Greenhorn Formation. Geochimica et Cosmochimica Acta 53, 2961-2972.

Hayes J. M., Freeman K. H., Popp B. N., and Hoham C. (1990) Compound-specific isotopic analyses: A novel tool for the reconstruction of ancient biogeochemical processes, In Durand, B., and Behar, F., eds., Advances in Organic Geochemistry 1989, Organic Geochemistry, p. 1115-1128.

Hayes J. M., Des Marais D. J., Lambert I. B., Strauss H., and Summons R. E. (1992) Proterozoic Biogeochemistry, In The Proterozoic Biosphere: A Multidisciplinary Study (Edited by J. Williams and C. Klein), Schopf, p. 81-134.

Hayes J. M. (1993) Factors controlling 13C contents of sedimentary organic compounds: Principles and evidence. Mar. Geol. 113, 111-125.

Jasper J. P., Mix A. C., Prahl F. G. and Hayes J. M. (1994) Photosynthetic fractionation of 13C and concentrations of dissolved CO2 in the central equatorial Pacific during the last 255,000 years, Paleoceanogr. 9, 781-798.

Jasper J. P. and Hayes J. M. (1990) A carbon isotope record of CO2 levels during the late Quaternary. Nature 347, 462-464. Joachimski, M. M., Pancost, R. D., Freeman, K. H., Ostertag-Henning, C., and Buggisch, W. (2002) Carbon isotope

geochemistry of the Frasnian-Fammenian transition. Palaeogeography, Palaeoclimatology, Palaeoecology 181, 91-109.

Kenig F., Sinninghe Damste J. S., Frewin N. L., Hayes J. M. and de Leeuw J. W. (1995) Molecular indicators for paleoenvironmental change in a Messinian evaporitic sequence (Vena del Gesso, Italy). II: High-resolution variations in abundances and 13C contents of free and sulphur-bound carbon skeletons in a single marl bed. Org. Geochem. 23, 485-526.

Köster, J., Rospondek, M., Schouten, S., Kotarba, M., Zubrzycki, A., Sinninghe Damsté, J.S., 1998. Biomarker geochemistry of a foreland basin: The Oligocene Menilite Formation in the Flysch Carpathians of Southeast Poland. Org. Geochem. 29, 649-669.

Kuypers, M. M. M., Pancost, R. D., and Sinninghe Damsté, J. S. (1999) A large and abrupt fall in atmospheric CO2 concentrations in the Cretaceous. Nature 399, 342-345.

Lichtfouse E., Derenne S., Mariotti A., and Largeau C. (1994) Possible algal origin of long chain odd n-alkanes in immature sediments as revealed by distributions and carbon isotope ratios, Organic Geochemistry 22, 1023-1027.

Logan G. A., Hayes J. M., Hieshima G. B., and Summons R. E. (1995) Terminal Proterozoic reorganization of biogeochemical cycles. Nature 376, 53-56.

Logan G. A., Summons R. E., and Hayes J. M. (1997) An isotopic biogeochemical study of Neoproterozoic and Early Cambrian sediments from the Centralian Superbasin, Australia. Geochimica Cosmochimica Acta 61, 5391 – 5409.

Pancost, R. D., Freeman, K. H., and Wakeham, S. G. (1999) Controls on the carbon-isotope compositions of compounds in Peru surface waters. Organic Geochemistry 30, 319-340.

Pancost, R. D., Freeman, K. H., Wakeham, S. G., and Robinson, C. Y. (1997) Environmental and physiological controls on carbon-isotope fractionation by marine diatoms. Geochimica et Cosmochimica Acta, v. 61, p. 4983-4991.

Pancost, R. D., Freeman, K. H., and Patzkowsky, M. E. (1999) Organic-matter source variations and the expression of a Middle Ordovician carbon isotope excursion. Geology 27, 1015-1018.

Pancost, R. D., van Geel, B., Baas, M., and Sinninghe Damsté, J. S. 2000: δ13C values and radiocarbon dates of microbial biomarkers as tracers for carbon recycling in peat deposits. Geology 28, 663-666.

Pancost, R. D., Telnæs N., Sinninghe Damsté, J. S. (2001) Controls On The Carbon Isotopic Composition Of An Isoprenoid-Rich Oil and Potential Source Rock. Organic Geochemistry 32, 87-103.

Pancost, R.D. and Sinninghe Damsté, J. S. In Press: Carbon Isotopic Compositions of Prokaryotic Lipids as Tracers of Carbon Cycling in Diverse Settings. Chemical Geology.

Schoell M., Hwang R. J., Carlson R. M. K., and Welton J. E. (1994) Carbon isotopic compositions of individual biomarkers in gilsonites (Utah). Org. Geochem. 21, 673-683.

Schoell M., Schouten S., Sinninghe Damste J. S., and de Leeuw J. W., and Summons R. E. (1994b) A molecular organic carbon isotope record of Miocene climate changes. Science 263, 1122-1125.

Schouten, S. van Kaam-Peters, H., Rijpstra, W. I. C., Schoell, M., and Sinninghe Damste, J. S., 2000. Effects of an oceanic anoxic event on the stable carbon isotopic composition of Early Toarcian carbon. American Journal of Science 300, 1-22.

Schouten, S., Rijpstra, W. I. C., Kok, M., Hopmans, E. C., Summons, R. E., Volkman, J. K., Sinninghe Damsté, J. S., 2001. Molecular organic tracers of biogeochemical processes in a saline meromictic lake (Ace Lake). Geochim. Cosmochim. Acta 65, 1629-1640.

van Kaam-Peters, H. M. E., Schouten, S., Koster, J. and Sinninghe Damsté, J. S. 1998: Controls on the molecular and carbon isotopic composition of organic matter deposited in a Kimmeridgian euxinic shelf sea: Evidence for preservation of carbohydrates through sulfurisation. Geochimica Cosmochimica Acta 62, 3259-3283.

List of attendees

First Name Last Name Institution tour group

Saoud Al Habsi University of Newcastle upon Tyne 5Marie Archbold Queen’s University Belfast 7Laura Beramendi-Orosco University of Nottingham 5Claire Bickers University of Bristol -Zoe Billings University of York 3Les Bluck MRC Human Nutrition Research 4Pascal Boeckx Ghent University 7Paul Brooks UC Berkeley 3Charlotte Bryant NERC Radiocarbon Lab. 6Georg Cadisch Imperial College at Wye 6Liz Campbell Earth Sciences, Uni. of Glasgow 2Liam Chalmers NERC Radiocarbon Lab. 6Kate Clark University of Bristol -Lisa Cole University of York 3Peter Ditchfield RLAHA, Oxford University 2Sean Doyle Forensics Explosives Lab. 3Simon Eaton Institute of Child Health, London 4Andrea Heinmeyer CTCD –York 3James Howard University of Newcastle upon Tyne 5Sarah Jackson MRC Human Nutrition Research 4Kerry Jones MRC Human Nutrition Research 4Andrew Kelly SUERC 6Sam Kelly University of Bristol -Elisa Lopez-Capel University of Newcastle upon Tyne 5Pete Maxfield University of Bristol -Corinne McCulloch SUERC 1Rona McGill SUERC 1Callum Murray NERC Radiocarbon Lab. 1Chris Mussell LGC 6Jason Newton SUERC 1Tamsin O’Connell University of Oxford 2Gwen O’Sullivan Queen’s University Belfast 7Karen Privat University of Oxford 2Rhiannon Stevens University of Oxford 2Gillian Taylor University of Newcastle upon Tyne 5Len Wassenar National Water Research Institute, Canad 7Sheng Xu SUERC 1Chaunlun Zhang University of Georgia 7

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