Training in Stable Isotope

Methods, Mass Spectrometery &

Isotopology
IB 400
Todd Dawson (2-6090) tdawson@berkeley.edu

Paul Brooks (3-1748) isotopes@berkeley.edu

Kevin Simonin (2-1054) ksimonin@berkeley.edu

& Elizabeth Wenk for all her powerpoint magic

9-28-05 Version
 Training in Stable Isotope Methods, Mass Spectrometery
& Isotopology

Goal: provide some of the fundamentals needed to understand isotopes and

their use in the biological sciences

Six sessions:
1. Fundamentals of isotope physics & chemistry
2. Case studies of how stable isotopes have been applied in natural systems
3. Sampling issues: what, where, how and help! of isotope sample collection
and preparation
4. The isotope ratio mass spectrometer: how it works and how you make it
work for your research
5. Correcting your data - tour de Excel!
6. Discussion about isotope applications & your work - the questions
you face and the challenges you need to resolve

» Introductions: who you are, what you do, why isotopes?

TODAY: Introduction to Stable Isotopes
1. Introduction to isotopes

2. Isotopes used in ecological studies

3. Stable isotope notation

4. Correct usage of stable isotope expressions

5. Causes of variation in stable isotope abundances

6. Fractionation factors

7. Rayleigh distillation: using fractionation factors

Why use stable isotopes?
» They are non-radioactive TRACERS of resource
origin, fate and flux including:
1. organismal movements
2. energy or resource flow across levels of ecological
organization

» They are non-destructive and non-disruptive

INTEGRATORS of ‘system’ processes including:
1. organismal function/tradeoffs
2. spatial and temporal responses to environment
What are
isotopes?
History of Isotopy:

• 1914 Fredrick Soddy proposed that any place on

periodic table can be occupied by more than one
kind of atom

He proposed isotope ---- meaning “same place”

Isotopes  Nuclides of single element having
different atomic weights

•Presently more than 2500 nuclides are known from

~110 elements
To be or not to be? - A stable isotope that is!
Nuclear Entities

These entities are of greatest interest to us

Atoms First:
First
An atom is composed of three types of particles:

PROTONS,
PROTONS NEUTRONS,
NEUTRONS and ELECTRONS

Each element has a unique number of protons, its atomic

number. The number of protons (Z) determines many of
the chemical and physical properties associated with an
atom.

The atomicThe atomic

mass is thenumber = Z (Protons)
sum of protons and neutrons
(N), particles with nearly identical weights. (Electrons,
negatively charged particles, have insignificant weight –
The
to atomic mass = Z + N (Protons + Neutrons)
ecologists!)
 Partial chart of the elements
Each square represents a nuclide, an isotope specific atom
Atomic number = Z (Protons): bottom left of each atom
Atomic mass = Z + N (Protons + Neutrons): top left of each atom
13 14 15 16 17 18 19 20
8 8 O 8 O 8 O 8 O 8 O 8 O 8 O 8 O
12 13 14 15 16 17 18
7 N N N N N N N
Proton Number (Z)

7 7 7 7 7 7 7

9 10 11 12 13 14 15 16
6 6 C 6 C 6 C 6 C 6 C 6 C 6 C 6 C
8 9 10 11 12 13
5 5 B 5 B 5 B 5 B 5 B 4 B
6 7 8 9 10 11 12
4 4 Be 4 Be 4 Be 4 Be 4 Be 4 Be 4 Be
5 6 7 8 9
3 3 Li 3 Li 3 Li 3 Li 3 Li
3 4 5 6 8
2 2 He 2 He 2 He 2 He 2 He
1 2 3
1 1 H 1 H 1 H

0 1 2 3 4 5 6 7 8 9 10 11 12 13
Neutron Number (N)
 Partial chart of the elements
Each green row represents nuclides that are isotopes:
isotopes they
share a common number of protons (Z) but differ in their
number of neutrons (N).
13 14 15 16 17 18 19 20
8 8 O 8 O 8 O 8 O 8 O 8 O 8 O 8 O
12 13 14 15 16 17 18
7 N N N N N N N
Proton Number (Z)

7 7 7 7 7 7 7

9 10 11 12 13 14 15 16
6 6 C 6 C 6 C 6 C 6 C 6 C 6 C 6 C
8 9 10 11 12 13
5 5 B 5 B 5 B 5 B 5 B 4 B
6 7 8 9 10 11 12
4 4 Be 4 Be 4 Be 4 Be 4 Be 4 Be 4 Be
5 6 7 8 9
3 3 Li 3 Li 3 Li 3 Li 3 Li isotopes
3 4 5 6 8
2 2 He 2 He 2 He 2 He 2 He
1 2 3
1 1 H 1 H 1 H

0 1 2 3 4 5 6 7 8 9 10 11 12 13
Neutron Number (N)
 Partial chart of the elements
Each green row represents nuclides that are isobars:
isobars they share
a common atomic weight (N + Z).

13 14 15 16 17 18 19 20
8 8 O 8 O 8 O 8 O 8 O 8 O 8 O 8 O
12 13 14 15 16 17 18
7 N N N N N N N
Proton Number (Z)

7 7 7 7 7 7 7

9 10 11 12 13 14 15 16
6 6 C 6 C 6 C 6 C 6 C 6 C 6 C 6 C
8 9 10 11 12 13
5 5 B 5 B 5 B 5 B 5 B 4 B
6 7 8 9 10 11 12
4 4 Be 4 Be 4 Be 4 Be 4 Be 4 Be 4 Be
5 6 7 8 9
3 3 Li 3 Li 3 Li 3 Li 3 Li
3 4 5 6 8
2 2 He 2 He 2 He 2 He 2 He
1 2 3
isobars
1 1 H 1 H 1 H

0 1 2 3 4 5 6 7 8 9 10 11 12 13
Neutron Number (N)
 Partial chart of the elements
Each green row represents nuclides that are isotones:
isotones they
share a common number of neutrons (N).

13 14 15 16 17 18 19 20
8 8 O 8 O 8 O 8 O 8 O 8 O 8 O 8 O
12 13 14 15 16 17 18
7 N N N N N N N
Proton Number (Z)

7 7 7 7 7 7 7

9 10 11 12 13 14 15 16
6 6 C 6 C 6 C 6 C 6 C 6 C 6 C 6 C
8 9 10 11 12 13
5 5 B 5 B 5 B 5 B 5 B 4 B
6 7 8 9 10 11 12
4 4 Be 4 Be 4 Be 4 Be 4 Be 4 Be 4 Be
5 6 7 8 9
3 3 Li 3 Li 3 Li 3 Li 3 Li
isotones
3 4 5 6 8
2 2 He 2 He 2 He 2 He 2 He
1 2 3
1 1 H 1 H 1 H

0 1 2 3 4 5 6 7 8 9 10 11 12 13
Neutron Number (N)
 Partial chart of the elements
The shaded squares are stable and the un-shaded squares are
unstable or radioactive nuclides.

13 14 15 16 17 18 19 20
8 8 O 8 O 8 O 8 O 8 O 8 O 8 O 8 O
12 13 14 15 16 17 18
7 N N N N N N N
Proton Number (Z)

7 7 7 7 7 7 7

9 10 11 12 13 14 15 16
6 6 C 6 C 6 C 6 C 6 C 6 C 6 C 6 C
8 9 10 11 12 13
5 5 B 5 B 5 B 5 B 5 B 4 B
6
Be
7
Be
8
Be
9
Be
10
Be
11
Be
12
Be -decay occurs along
4 4 4 4 4 4 4 4
the line of isobars
5 6 7 8 9
3 3 Li 3 Li 3 Li 3 Li 3 Li
3 4 5 6 8
2 2 He 2 He 2 He 2 He 2 He
1 2 3
1 1 H 1 H 1 H

0 1 2 3 4 5 6 7 8 9 10 11 12 13
Neutron Number (N)
 Stable isotope trends
3 important points:

1. Stable isotopes tend to

have an N/Z near 1 for
masses less than 20

2. Stable isotopes tend to

N
have an even Z-number for N/Z = 1
masses greater than 20

3. Most biologically O
important elements have
masses less than 20
Z
H
N
C
What isotopes are
used in ecological
studies?
Average terrestrial abundances of the stable isotopes
of elements used commonly (), occasionally (), and
rarely () in ecological studies
Element Isotope Abundance Element Isotope Abundance Element Isotope Abundance

Hydrogen 1
H 99.985 Sulfur 32
S 95.00 Iron 54
Fe 5.82
2
H 0.015 33
S 0.76 56
Fe 91.66
Carbon 12
C 98.89 34
S 4.22 57
Fe 2.19
13
C 1.11 36
S 0.014 58
Fe 0.33
Nitrogen 14
N 99.63 Chlorine 35
Cl 75.53 Copper 63
Cu 69.09
15
N 0.37 37
Cl 24.47 65
Cu 30.91
Oxygen 16
O 99.759 Potassium 39
K 93.10 Zinc 64
Zn 48.89
17
O 0.037 40
K 0.0118 66
Zn 27.81
18
O 0.204 41
K 6.88 67
Zn 4.11
Magnesium 24
Mg 78.70 Calcium 40
Ca 96.97 68
Zn 18.57
25
Mg 10.13 42
Ca 0.64 70
Zn 0.62
26
Mg 11.17 43
Ca 0.145 Strontium 84
Sr 0.56
Silicon 28
Si 92.21 44
Ca 2.06 86
Sr 9.86
29
Si 4.70 46
Ca 0.0033 87
Sr 7.02
30
Si 3.09 48
Ca 0.18 88
Sr 82.56
 Common isotopes
When we see this list of isotopes used in ecological
studies, note that it includes many of the most common
isotopes in the solar system:

The 10 most common isotopes in the solar system are:

H >> 4He >> O >

16
C >>
12 20
Ne > N >
14 24
Mg > 28
Si > 56
Fe > S
32

The isotopes we study occur

throughout the solar system and
are important in lots of processes!
 Atomic Elemental Molecular Relative Mass Terrestrial Technical
Element Relative

Isotope
weight Relative Difference
range Precision
Mass
Abundance Difference

(%) ‰ ppm ‰ ppm

1.0078 D/H HD / 1H1H
1
700 109 0.25 0.17
Hydrogen 1H
1
2.0141 99.984 HEAVIER ISOTOPES
100% (3/2)
(Deuterium) 0.0156 50%
1H2 ARE RARE!
(D)
12.0000 13
C / 12C C 16O 16O / 12C 16O 16O
13
100 1123 0.05 0.56
Carbon 6C
12
13.0034 98.892 8.3% (45/44)

6C
13 1.108 2.3%

14.0031 15
N/ 14N N 14N / 14N 14N
15
50 181 0.1 0.72
Nitrogen 7N
14
15.0001 99.635 7.1% (29/28)

7N
15 0.365 3.6%

15.9949 O / 16O
18
C 16O 18O / 12C 16O 16O
12
100 200 0.1 0.20
Oxygen 8O
16
16.9991 99.759 12.5% (46/44)
17.9992
8O
17 0.037 4.5%

0.204
8O
18

31.9721 34
S / 32S 34
S 16O 16O / 32S 16O 16O 100 4580 0.2 9.16
Sulphur 16S
32
32.9714 95.02 6.3% (66/64)
33.9679
16S
33 35.9671 0.76 3.1%

4.22
16S
34

0.014
16S
36
 Atomic Relative Molecular Relative Mass Terrestrial Technical
Element Elemental

Isotope
weight Abundance Difference
range Precision
(%)
Relative Mass
Difference ‰ ppm ‰ ppm
1.0078 99.984 HD / 1H1H
1
700 109 0.25 0.17
Hydrogen 1H
1
2.0141 0.0156 D/H (3/2)
(Deuterium) 50%
1H2 100%
(D)
12.0000 98.892 C 16O 16O / 12C 16O 16O
13
100 1123 0.05 0.56
Carbon 6C
12
13.0034 1.108
13
C / 12C (45/44)
2.3%
6C
13
8.3%
14.0031 99.635
HYDROGEN
N N/ N N
HAS50
THE
181 0.1 0.72
15 14 14 14

Nitrogen 7N
14
15.0001 0.365
15
N/ 14N (29/28)

7N
15
7.1% LARGEST 3.6%
MASS DIFF
BETWEEN ISTOPES
15.9949 99.759 C 16O 18O / 12C 16O 16O
12
100 200 0.1 0.20
Oxygen 8O
16
16.9991 0.037 O / 16O
18
(46/44)
17.9992 0.204 4.5%
8O
17
12.5%
8O
18

31.9721 95.02 34
S 16O 16O / 32S 16O 16O 100 4580 0.2 9.16
Sulphur 16S
32
32.9714 0.76
34
S / 32S (66/64)
33.9679 4.22 3.1%
16S
33 35.9671 0.014 6.3%
16S
34

16S
36
 Atomic Relative Elemental Terrestrial Technical
Element Molecular Relative

e
Isotop
weight Abundance Relative
range Precision
(%) Mass
Difference Mass Difference
‰ ppm ‰ ppm
1.0078 99.984 D/H 700 109 0.25 0.17
Hydrogen 1H
1
2.0141 0.0156 100% HD / 1H1H
1

(Deuterium)
1H2 (3/2)
(D) 50%
12.0000 98.892 13
C / 12C 100 1123 0.05 0.56
Carbon 6C
12
13.0034 1.108 8.3%
13
C 16O 16O / 12C 16O 16O
6C
13 (45/44)
2.3%
We analyze gases that
14.0031 99.635 15
N/ 14N 50 181 0.1 0.72
Nitrogencontain
7N the
14
isotopes
15.0001 0.365 7.1% N 14N / 14N 14N
15

N15interest!
7of (29/28)
3.6%
15.9949 99.759 O / 16O
18
100 200 0.1 0.20
Oxygen 8O
16
16.9991 0.037 12.5%
12
C 16O 18O / 12C 16O 16O
17.9992 0.204
8O
17 (46/44)
8O
18 4.5%
31.9721 95.02 34
S / 32S 100 4580 0.2 9.16
Sulphur 16S
32
32.9714 0.76 6.3%
34
S 16O 16O / 32S 16O 16O
33.9679 4.22
16S
33 35.9671 0.014 (66/64)
16S
34 3.1%
16S
36
 Atomic Relative Elemental Molecular Relative Mass
Element Terrestrial Technical

e
Isotop
weight Abundance Relative Difference
(%) Mass range Precision
HYDROGEN HAS A LARGE Difference
‰ ‰
TERRESTRIAL RANGE,
ppm ppm

Hydrogen 1H1 1.0078 99.984 D/H HD / 1H1H 109 0.17

1

700 0.25+
BUT ALSO RELATIVELY
(Deuterium)
2.0141 0.0156 100% (3/2)
50%
LOW
1H PRECISION
2

(D)
12.0000 98.892 13
C / 12C 13
C 16O 16O / 12C 16O 16O 1123 0.56
Carbon 6C
12
13.0034 1.108 8.3% (45/44) 100 0.05
2.3%
6C
13

14.0031 99.635 15
N/ 14N N 14N / 14N 14N
15
181 0.72
Nitrogen 7N
14
15.0001 0.365 7.1% (29/28) 50 0.1
3.6%
7N
15

15.9949 99.759 O/ O C O O/ C O
18 16 12 16 18 12 16
O
16
200 0.20
Oxygen 8 O 16
NITROGEN16.9991
HAS A SMALLER
0.037 12.5% (46/44) 100 0.1
17.9992 0.204 4.5%
O 17
TERRESTRIAL
8 RANGE, BUT
8O BETTER TECHNICAL
18

PRECISION
31.972195.02 S/ S S 16O 16O / 32S 16O 16O 4580 9.16
34 32 34

Sulphur 16S
32
32.9714 0.76 6.3% (66/64) 100 0.2
33.9679 4.22 3.1%
16S
33 35.9671 0.014

16S
34

16S
36
Expressing differences
in stable isotope
abundance
Element Relative

Isotope
Absolute isotope abundances
Abundance
are found at the third decimal
(% atoms) leading to small relative differences
Hydrogen H1
1 99.984
(Deuterium) 0.0156
1H2
(D) Are these relevant?

Carbon 6C12 98.892

6C
13 1.108

Nitrogen 7N14 99.635

7N
15 0.365

Oxygen 8O16 99.759

8O
17 0.037
1.0860% 13C 1.0805% 13C
0.204
8O
18
Stable isotope composition is expressed in  (delta)
notation:

R in ‰ = Rsample
–1 x 1000
Rstandard
Stable isotope composition is expressed in  (delta)
notation:

R in ‰ = Rsample
–1 x 1000
Rstandard

R is the isotope ratio of the HEAVY / LIGHT isotopes in

either your sample or a standard
i.e. D/H, C/12C ,
13
N/14N ,
15
O/16O
18

and is a very small number

Stable isotope composition is expressed in  (delta)
notation:

R in ‰ = Rsample
–1 x 1000
Rstandard

Delta notation indicates the isotope ratio in your

sample relative to a standard.
If the isotope ratio in your sample equals the
standard, Rsample/Rstandard = 1 and R = 0‰

The International Atomic Energy Association (IAEA)

maintains a set of standards used for stable isotope
measurements.
Stable isotope composition is expressed in  (delta)
notation:

R in ‰ = Rsample
–1 x 1000
Rstandard

Because Rsample never deviates much

from Rstandard (natural variation in isotope ratios is
limited), [(Rsample / Rstandard)-1] is a small number.
In order to make the variation more apparent, one
multiplies the value by 1000, thereby expressing the
value in per mil (parts per thousand ; ‰) notation
The isotope abundance ratios measured and their
internationally accepted reference standards
Isotope Ratio Standard Abundance Ratio
of reference
Measured
standard

2
H (D) 2
H/1H (D/H) V-SMOW: “Vienna-Standard Mean 1.5575 x 10-4
Ocean Water”
13
C 13
C/12C V-PDB: Vienna-PeeDee Belemnite” 1.1237 x 10-2

15
N 15
N/14N N2-atm: atmospheric gas 3.677 x 10-3

18
O 18
O/16O V-SMOW 2.0052 x 10-3
V-PDB 2.0672 x 10-3

34
S 34
S/32S CDT: a troilite (FeS) from the 4.5005 x 10-2
“Canyon Diablo” meteorite
The isotope abundance ratios measured and their
internationally accepted reference standards
Isotope Ratio Abundance Ratio
Measured
Standard (R) of reference
standard

H (D) H/1H (D/H) 1.5575 x 10-4

V-SMOW: “Vienna-Standard
2 2

Mean Ocean Water”

C C/12C 1.1237 x 10-2
V-PDB: “Vienna-PeeDee
13 13

Belemnite” [a fossil]
N N/14N 3.677 x 10-3
N2-atm: atmospheric gas
15 15

O O/16O 2.0052 x 10-3

V-SMOW
18 18

2.0672 x 10-3
V-PDB
S S/32S 4.5005 x 10-2
CDT: a triolite (FeS) from the
34 34

“Canyon Diablo” meteorite

The isotope abundance ratios measured and their
internationally accepted reference standards
Isotope Ratio Measured Standard
Abundance Ratio of
reference standard These values
(w/ 95% CI) are the
H (D) H/1H (D/H) V-SMOW ratios of
1.5575 x 10-4 ± .001
2 2

atoms in the
C C/12C V-PDB standards
1.1237 x 10-2 ± .0009
13 13
and reflect
N N/14N N2-atm the very low
3.677 x 10-3 ± .00081
15 15

abundance
of the
O O/16O V-SMOW heavier
2.0052 x 10-3 ± .00043
18 18

V-PDB isotope
2.0672 x 10-3 ± .0021
O O/16O V-SMOW
373 x 10-6 ± 15
17 17

V-PDB
379 x 10-6 ± 15
S S/32S CDT
4.5005 x 10-2
34 34
 Standards

Some other international standards of known  value:

Standard Light Antarctic Precipitation (SLAP) with values:

D = -428‰ 18O = -55.5‰

Greenland Icesheet Precipitation (GISP) with values:

D = -189.7‰ 18O = -24.8‰

 Working standards
The internationally accepted reference standards are
obviously in limited supply, expensive, and cannot be used
as the daily reference standard in labs around the world.

Instead isotope labs employ WORKING STANDARDS.

Working standards are:
 used on a regular (daily) basis
 homogeneous
 well matched to your analyses
 easily obtained or made
 easily corrected back to the international standards
 delta notation
Those same two leaves have more interpretable isotope
“values” in delta notation.

1.0860% 13C = 13C -23‰

1.0805% 13C = 13C -28‰

 delta notation
A quick note, to be elaborated on in lecture 5:

 You can’t do chemical calculations with  units.

 They are just for comparative purposes.

 That said, for small ranges of units “you are

allowed” to be sloppy.
 Atom % notation
Xheavy Xheavy
Atom % = •100 = •100
Xheavy + Xlight Xtotal

Where X is the FRACTION of the heavy or light isotope

in a mixture.

 Unlike delta notation, atom % notation does not accentuate

small changes in isotope abundance.

 You will NOT see this notation used in the NATURAL

ABUNDANCE stable isotope literature

 You WILL see this notation used if you are working with
ENRICHED stable isotope methods
 Using and
referring to the
delta values
Some comparative terms:
LIGHT vs. HEAVY SAMPLES
a LIGHTER sample contains more of the lighter isotope,
relative to another sample
a HEAVIER samples contains more of the heavier isotope,
relative to another sample

DEPLETED vs. ENRICHED SAMPLES

a sample “DEPLETED” IN THE HEAVY ISOTOPE contains
less of the heavy isotope and more of the light isotope,
relative to another sample
a sample “ENRICHED” IN THE LIGHT ISOTOPE contains
more of the light isotope and less of the heavy isotope,
relative to another sample
Using the D signature in H2O as an example:

0
 isotopically heavier such values are found in
warm climates, at low
 enriched in D (2H)
D elevation and low
 depleted in H latitudes, evaporated
(‰)
water

-200

 isotopically lighter such values are found in

cold climates, at high
 depleted in D (2H)
elevation and high
 enriched in H latitudes

-400
 Some causes of
variation in stable
isotope values
 Isotope Effects

Urey’s Axiom: “The heavy isotopes concentrate in the

compound in which the element is most strongly held”

If Urey is correct then this implies that issues such as:

 Bond-strength,
 Mass of an element, isotope or atom,
 Rates of a chemical reaction (chemical behavior),
 System properties (open vs. closed),
 Etc. …….

Could all have effects on isotope distributions in the materials

we measure and help explain the variation in stable isotope
composition (e.g., variation in ).
 Key points about isotopes

1. Chemical properties of any element are largely

determined by the number and configuration of
electrons (e-)

 Since isotopes have the same number and

configuration of electrons . . . . .

isotopes have the same chemical properties

13
CO2 is chemically identical to CO2
12
 Key points about isotopes

1. isotopes have the same chemical properties

2. However, isotopes differ in then number of neutrons,

N they possess, and therefore in mass

How do mass differences lead to

variation is isotope abundance?

Mass differences
influence chemical BEHAVIOR
in reactions or mixtures
 Isotope mass effects
Differences in mass influence:
1. The RATES at which the isotopes react
Lighter isotopes react faster. Therefore different
isotopes involved in a chemical reaction display
differential representation in different phases of the
reaction
2. The PHYSIO-CHEMICAL properties of molecules
composed of different isotopes
That is, factors including vapor pressure, boiling
temperature, freezing point, and melting point are
affected by the isotope composition of a molecule.

 As we’ll see next, for water composed of different

isotopes this has a large, measurable, and significant
influence.
 Physio-chemical differences

Characteristic physical properties of H216O, D216O, H218O

(from Hoefs 1973, 1997)

Property H216O D216O H218O

Density (20ºC, in g cm-2) 0.997 1.1051 1.1106
Temperature of greatest density (ºC) 3.98 11.24 4.30
Melting point (@760 Torr, in ºC) 0.00 3.81 .028
Boiling point (@760 Torr, in ºC) 100.00 101.42 100.14
Vapor pressure (@100ºC, in Torr) 760.00 721.60 758.07
Viscosity (@20ºC, in centipoise) 1.002 1.247 1.056
Molar volume (@20ºC, in cm3/mole) 18.049 18.124 18.079
 Physio-chemical differences

Characteristic physical properties of H216O, D216O, H218O

(from Hoefs 1973, 1997)

Property H216O D216O H218O

Density (20ºC, in g cm-2) 0.997 1.1051 1.1106
Temperature of greatest density (ºC) 3.98 11.24 4.30
Melting point (@760 Torr, in ºC) 0.00 3.81 .028
Boiling point (@760 Torr, in ºC) 100.00 101.42 100.14
Vapor pressure (@100ºC, in Torr) 760.00 721.60 758.07
Viscosity (@20ºC, in centipoise) 1.002 1.247 1.056
Molar volume (@20ºC, in cm3/mole) 18.049 18.124 18.079
 Physio-chemical differences
Isotope effect associated with zero-point energy

DISSOCIATED ATOMS

{Morse Potential Curve}

Potential energy

Differences in ZPEs are the

fundamental cause of equilibrium
isotope fractionation

H-H ZERO POINT ENERGY

D-D LEVELS (ZPEs)
ABSOLUTE ZERO

Interatomic distance
 Physio-chemical differences
Isotope effect associated with zero-point energy

DISSOCIATED ATOMS

THESE VALUES
ARE THE AMOUNT
OF ENERGY
Potential energy

REQUIRED TO
BREAK THE BOND;
EL = 103.2 MORE ENERGY IS
NEEDED TO
EH = 105.3 BREAK THE D-D
BOND THAN THE
H-H BOND,
H-H LEADING TO
D-D ISOTOPE EFFECTS

Interatomic distance
 Physio-chemical differences
In summary:
 Higher vibrational frequency, the stretching and
compressing of chemical bonds between atoms, leads to
a higher zero point energy and lower stability

 Bond strengths are proportional to isotope mass, so an

isotope with a higher mass has a higher bond strength

 Molecules with heavier isotopes will be more stable than

light isotopes but diffuse more slowly

Therefore, partial vaporization of a liquid pool will lead

to increased concentration of the lighter isotope in the
vapor phase
 Reaction rate differences

 Differences in mass also influence the RATES

at which the isotopes react

 The lighter isotope reacts at a faster

rate,leading to a heavier  value in the remaining
substrate relative to the product.

-- Therefore, differences in MASS influence

RATES and lead to ISOTOPE FRACTIONATION
 Fractionation

 Both the differences in physiochemical

properties and reaction rates lead to the
REDISTRIBUTION of ISOTOPES

 This process is known as FRACTIONATION

 Fractionation can be caused by either

BI-DIRECTIONAL or UNIDIRECTIONAL
reactions
 Types of fractionation
1. Exchange/equilibrium [BI-directional]
• Complete back-reaction
• Product/reactant offset by constant
fractionation factor
2. Kinetic [UNI-directional] = “biological”
• Incomplete back-reaction (extreme case-
Rayleigh distillation)
• Product and reactant can evolve in concert
(closed system), or product composition
determines reactant composition (open
system)
3. Transport/Diffusion [UNI-directional]
• Subset of kinetic fractionation reactions
involving flux along a concentration gradient
 Fractionation
BI-DIRECTIONAL REACTIONS:
 Known as EQUILIBRIUM FRACTIONATION

 Differences in physio-chemical properties

and sometimes reaction rates (very fast) allow
bi-directional exchange of isotopes

 In such a reaction the difference in  value

between the two pools REMAINS CONSTANT
when there is CONTINUOUS EXCHANGE
between the substrate and the product
 Fractionation
An example on an EQUILIBRIUM REACTION:

CO2 + H2O  H2CO3

Or alternatively: CO2  H2O

since we are interested in the exchange of oxygen atoms
between CO2 and H2O

Initially the H2O and CO2 have

different isotope compositions, but
as they exchange 18O and 16O’s back
and forth they reach an equilibrium. If
fractionation occurs that the concentration
of each isotope species will not be the same in
both the H2O and CO2 pools [in this case,if the
initial 18O of H2O is –12.95‰
–12.95 the CO2’s 18O
will be 28.83‰
28.83 - we’ll see this next]
 Fractionation
UNI-DIRECTIONAL REACTIONS:

 Known as KINETIC FRACTIONATION

 Differences in physio-chemical properties and

reaction rates never result in uni-directional
exchange of isotopes (there is no back-reaction)

 Referred to as DISCRIMINATION if it is
biologically (enzyme mediated) fractionation
Attaching a number
to “fractionation”
 Fractionation Factors

The  values of the substrate and the product

are related to one another through a:

FRACTIONATION FACTOR, 

defines the relationship between the

substrate (A) and product (B) in either an
equilibrium or kinetic reaction such that,

AB= RA / RB [R is the isotope ratio]

 Fractionation Factors

If  = 1, no fractionation is occurring

If  > 1, there is more of the heavier isotope in

the substrate than before the reaction
began
If  < 1, there is more of the lighter isotope in
the substrate than before the reaction
began
 Fractionation Factors
How to relate  and  values:
Actual value
We know: AB = RA/RB

In addition: AB = (1000 + A) / (1000 + B)

And since: A = ((RA/RS) –1)•1000

Derivation: AB = [((RA/RS) –1)•1000] + 1000

[((RB/RS) –1)•1000] + 1000

= [(1000•RA / 1000•RS) – 1000] + 1000

[(1000•RB / 1000•RS) – 1000] + 1000

= (1000•RA / 1000•RS)
(1000•RB / 1000•RS)

= RA / RB = (1000 + A) / (1000 + B)

 Fractionation Factors

In other words you can calculate  using:

AB = RA/RB

-or-

AB = (1000 + A) / (1000 + B)

 Fractionation Factors
With an equilibrium equation,  is really an
EQUILIBRIUM CONSTANT - at equilibrium, it will tell
you the distribution of isotopes between two species.

 With kinetic fractionation it is the same idea, except

AB = k1 / k2, where k1 and k2 are the RATE
CONSTANTS for the two isotopic species

 In a multi-step process, equilibrium fractionations are

ADDITIVE

 In a multi-step process, kinetic fractionations are NOT

ADDITIVE
 Fractionation
Adding isotope stoichiometry to our equilibrium reaction example, we get:

(CO16O)gas + (H218O)liquid <=> (CO18O)gas + (H216O)liquid

 this is the equilibrium equation for each atom

 you need to empirically determine the quantities of each species
 once you have these numbers, you can calculate the fractionation factor,
because:

K = ((CO18O)g•(H216O)l) / ((CO16O)g•(H218O)l)

= (18O/16O)g/(18O/16O)l

= R A / RB or
 = RA / RB
 Fractionation Factors…
What is the -constant in our equilibrium example?

H2O  CO2
where: 18OH2O = -12.95‰
-12.95
18OCO2 = 28.83‰

therefore: RH2O = 0.001979

RCO2 = 0.002063

(18O/16O)water = RA = 0.001979 = 0.95939

(18O/16O)carbon dioxide RB 0.002063

 Switching between delta units and atom %
Converting delta units to atom%, of the heavier isotope:

100
1
atom% = + 1

+ 1 * RR
1000

On a spreadsheet: atom% = 100/((1/(((/1000)+1)*RR))+1)

For 13
C: RR = 0.0112372
15
N: RR = 0.0036765 (Europe Sci. Handout)
SMOW 2HRR = 0.00015575
18
O PDB RR = 0.002067 (Finnigan MAT Isodat Manual)
18
O SMOW RR = 0.0020052 (Colorado Plateau Stable
34
S RR = 0.045005 Isotope Lab Web Site)
 Calculating RA from atom%

You’ve now calculated Atom% O

18

Atom% O = 1 – Atom%
16
O
18

RA = Atom% O / Atom%
18 16
O
 Fractionation Factors
In our kinetic example: CO2 + H2O  carbohydrate

What is the -constant of the enzyme?

If the CO2 being used has a value of –12.4‰
–12.4 and
the carbohydrates have a value of -28‰, we can
calculate that they have atomic ratios of 0.01110
and 0.01092 respectively.
Next:
(13C/12C)carbon dioxide R=A =0.01110 = 1.01605

(13C/12C)carbohydrate RB 0.01092
 More fractionation terminology
There are many other terms which tell you the ‘per mil’
difference between compound A and compound B.

 Some are used in the biological literature and others in

the geological literature.

 Although the numbers they yield are not identical, they

are close approximations of one another.

 When you work up numbers it is very important that you

indicate which calculation you are using.
 More fractionation terminology

By definition: AB = A - B

 and you should therefore use the

previous equations to obtain A and B
from to calculate AB
 More fractionation terminology
By definition: AB = A - B
 and you should therefore, use the previous equations to
obtain A and B to calculate AB

But you will also see a jumble of other calculations, like:

Isotopic enrichment
a. In the geological literature: AB = (AB – 1)•1000
b. In the biological literature: AB = 103lnAB

Isotopic separation (big Delta, )

a. In the geological literature: AB = 103lnAB  a – b

Isotope discrimination
a. Used in the biological literature and refers specifically to enzyme-
mediated fractionation where A is the source and B is the product
b. AB = (AB – 1)•1000

It is much better to use the (AB – 1)•1000 calculation. There is no mathematical reason to use an
 More fractionation terminology

Just to compare some  numbers:

AB (AB–1)•1000 1000*lnAB A - B

Equilibrium 0.95939 - 42.33 - 40.61 - 41.77

example
Kinetic 1.01605 16.05 15.92 16.4
example

It is very important to know what

terminology is being used!
TEMPERATURE:
A key cause for
variation in 
 Temperature dependence of 

This graph of 
values for 18O
and D in water
shows that
fractionation
increases as the
temperature
decreases!
 Temperature dependence of 

At 20º:
1.0092
18O = [18O / 16O]Liquid

[18O / 16O]Vapor

1.0092 = 9.2‰
D = [D / H]Liquid
1.074
[D / H]Vapor

1.0740 = 74‰
 Temperature dependence of 

At 80º:
1.0092
18O = [18O / 16O]Liquid

[18O / 16O]Vapor

1.0055 = 5.5‰ 1.0055

D = [D / H]Liquid
1.074
[D / H]Vapor
1.038

1.038 = 38‰
 Tying together
isotope fractionation
concepts:
Rayleigh distillation
 Rayleigh distillation
• Rayleigh fractionation occurs when a parent mass is
depleted by equilibrium fractionation to a phase
continually removed. The  values of all elements that
show mass dependent fractionation are affected; the
process occurs in all natural systems.
• The equation describing Rayleigh processes is:
R=
R0f(1-)

R and R0 are the ratios at t and at t=0 f is

the fraction remaining at t  is
the fractionation factor

“Condensation example”
 WATER & Rayleigh distillation
Rayleigh distillation describes
the observed patterns of
progressive ISOTOPE
FRACTIONATION such as when This is a classic Rayleigh
a liquid pool evaporates (e.g. Plot; A, B and C are for
during cloud formation) an OPEN system; D and E
for a CLOSED system

Rayleigh distillation is an
EQUILIBRIUM
FRACTIONATION process
which creates differences in 
values

This fractionation is due to

the different PHYSIO-
CHEMICAL BEHAVIORS of the
isotopes
 WATER & Rayleigh distillation
“Evaporation example”
As Rayleigh distillation
proceeds, the isotope
values of both the
accumulated vapor mass
and the remaining water
change. The pattern is
dependent on whether
the system is “OPEN” or
“CLOSED”

In this example ∆18O

is -9.8‰ ( = 1.0098)
 WATER & Rayleigh distillation
“Evaporation example”

In a closed system,
the vapor pool is in
continuous contact
with the liquid pool

D = 18O of water in a
CLOSED system D
E = 18O of vapor in a
CLOSED system” E
 WATER & Rayleigh distillation

In a CLOSED system the

two pools never differ by
more than because as
distillation proceeds, the
isotopes in the two pools
will always equilibrate
with one another

D 

 E
 WATER & Rayleigh distillation
“Evaporation example”

In an open system, the

vapor is removed as soon
as it forms.

A = remaining water in
OPEN system (liquid)
The initial  is -9.8

B = instantaneous vapor
in OPEN system A
C = accumulated vapor

}
fraction being removed B
from the OPEN system
C
 WATER & Rayleigh distillation
In either an OPEN or a “Evaporation example”

CLOSED system, the

remaining liquid pool (A) and
instantaneous vapor (B) must
be related to one another by
the fractionation factor, 

However, in an OPEN

system, since the
accumulated vapor (C) is not  
in contact with (A), these A
two pools are related to one
another by  only at the B
start of the distillation
 C

process.
 WATER & Rayleigh distillation
“Evaporation example”

For both systems, if

distillation is complete,
the accumulated vapor
mass (C & E) must have a
 value equal to the initial
water mass

However OPEN vs.

CLOSED systems display
different instantaneous 
offsets betweens the two
pools
 Rayleigh fractionation from rainfall
“Condensation example”
 in cloud vapor and
condensate plotted as
a function of the total
fraction of remaining rain

vapor in the clouds

follows the same rain

Rayleigh process as
evaporation cloud
Change in cloud temperature as condensate forms
18O in a cloud vapor and
condensate plotted as a
function of the fraction of
remaining vapor in the clouds (liquid H2O)
for a Rayleigh process.

A twist: as evaporation
proceeds, the temperature
of the remaining cloud
decreases.

The increase in
fractionation with the
decreasing temperature
is taken into account
 Uni-directional reaction patterns
Rayleigh distillation can also
INSTANTANEOUS
be applied (similar rules) to PRODUCT
UNI-DIRECTIONAL (Kinetic)
REACTIONS (but they act the
opposite from what we just saw)
SUBSTRATE

ONCE AGAIN

With uni-directional
reactions the important
distinction is between
FINITE and INFINITE CUMULATIVE
amounts of substrate PRODUCT
 Uni-directional reaction patterns
If INFINITE amounts of
substrate exist, the
conversion of substrate to
product does not
noticeably change the 
value of the remaining
substrate (now ‘open’)

Therefore the  values of

the substrate and product
remain constant over time
and are always related by   
the fractionation factor 
 Uni-directional reaction patterns
If FINITE amounts of
substrate exist, the
creation of product will
change the  value of the
remaining substrate (now
‘closed’) 

Therefore, the value of

both the substrate and the 
instantaneous product will
change over time, although
they will always be related 
to one another by the
fractionation factor 
 How can Stable
Isotopes be used
as a tool?

On to some case studies in isotope ecology

Mistake
Referring to the symbol  as
del
13C composition
Isotopically depleted water
Heavy (light) 18O values
Depleted 13C value
Oxygen isotopes in chert;
inferred from carbon isotopes;
isotopes of soil water
The isotopic composition of
water was 18O = - 4.3‰
The isotopic value changed;
the carbon value changed
Enriched (depleted) carbonates
Depleted carbon source
The isotopic signature of the
rock was 18O = 5.7 ‰
15, 18 , 13 , etc.
15
, 18 , 13 , etc.
-15, -13 , -18 etc.
The mineral equilibrated with
the fluid
Sulfur was measured
Recommended Expressions
Since the time of the early Greeks, the name
of this symbol has been and remains delta
13C value; or carbon isotope composition
18
O (or D) depleted water
High (low) 18O values
low13C value (relative to another value)
Oxygen isotope ratio (composition) of chert;
inferred from carbon isotope measurements;
isotopic composition of soil water
The 18O value of the water was - 4.3‰
The isotopic composition changed; the 13C
value changed
Isotopically heavy (light) carbonates;
(relatively) 18O-rich carbonates;
(relatively) 13C-poor carbonates
Low 13C source; source with a low 13C value
The 18O value of the rock was 5.7 ‰. Thus
this rock has the oxygen isotope signature of
the mantle
15N, 18O , 13C, etc.
The mineral exchanged with the fluid
The sulfur isotope composition was measured
Explanation
The word del describes either of two things in mathematics: an
operator () of the partial derivative sign ()
A composition of values is not possible
Water is not depleted (or enriched) in isotopes
As number,  values are either high or low
13C values are numbers and cannot be depleted
Such mistakes are a carryover from loose oral communication
A matter of redundancy
The phrase “isotopic value” is ambiguous. Does it mean a
ratio? A delta value?
These phrases culled from the literature make no sense. More
importantly, the words enrich and deplete are overused and
much abused. These words should be reserved for describing a
process that changes the content of the heavy isotope of some
element in the substance being considered
The word signature should apply to the isotopic composition
of a significant reservoir like the mantle, the ocean, or a major
part of the system being studied, not to the isotopic
composition of ordinary samples
Introduction of new symbolism that saves one character of
space is unnecessary at best and confusing at worst.
Isotopic equilibrium, may not have been attained during the
process being described
Confusing because the sulfur content of a rock or mineral may
be understood
Mistake Recommended Expressions Explanation

Referring to the Since the time of the early The word del describes either of
symbol  as del Greeks, the name of this two things in mathematics: an
symbol has been and operator () of the partial
remains delta derivative sign ()
Mistake Recommended Expressions Explanation
Referring to the symbol  as Since the time of the early Greeks, the name The word del describes either of two things in mathematics: an
del of this symbol has been and remains delta operator () of the partial derivative sign ()

13C composition 13C value; or carbon A composition of values is not

isotope composition possible
Mistake Recommended Expressions Explanation
Referring to the symbol  as Since the time of the early Greeks, the name The word del describes either of two things in mathematics: an
del of this symbol has been and remains delta operator () of the partial derivative sign ()
13C composition 13C value; or carbon isotope composition A composition of values is not possible

Isotopically 18
O (or D) depleted water Water is not depleted (or enriched)
depleted water in isotopes
Mistake
Referring to the symbol  as
del
13C composition
Isotopically depleted water
Recommended Expressions
Since the time of the early Greeks, the name
of this symbol has been and remains delta
13C value; or carbon isotope composition
18
O (or D) depleted water
Explanation
The word del describes either of two things in mathematics: an
operator () of the partial derivative sign ()
A composition of values is not possible
Water is not depleted (or enriched) in isotopes

Heavy (light) High (low) 18O values As number,  values are either
18O values high or low
Mistake
Referring to the symbol  as
del
13C composition
Isotopically depleted water
Heavy (light) 18O values
Recommended Expressions Explanation
Since the time of the early Greeks, the name The word del describes either of two things in mathematics: an
of this symbol has been and remains delta operator () of the partial derivative sign ()
13C value; or carbon isotope composition A composition of values is not possible
18
O (or D) depleted water Water is not depleted (or enriched) in isotopes
High (low) 18O values As number,  values are either high or low

Depleted 13C low13C value (relative to 13C values are numbers and
value another value) cannot be depleted
Mistake
Referring to the symbol  as
del
13C composition
Isotopically depleted water
Heavy (light) 18O values
Depleted 13C value
Recommended Expressions
Since the time of the early Greeks, the name
of this symbol has been and remains delta
13C value; or carbon isotope composition
18
O (or D) depleted water
High (low) 18O values
low13C value (relative to another value)
Explanation
The word del describes either of two things in mathematics: an
operator () of the partial derivative sign ()
A composition of values is not possible
Water is not depleted (or enriched) in isotopes
As number,  values are either high or low
13C values are numbers and cannot be depleted

Oxygen isotopes Oxygen isotope ratio Such mistakes are a carryover

in chert; inferred
from carbon
isotopes; isotopes
of soil water
(composition) of chert; from loose oral communication
inferred from carbon
isotope measurements;
isotopic composition of
soil water
Mistake
Referring to the symbol  as
del
13C composition
Isotopically depleted water
Heavy (light) 18O values
Depleted 13C value
Oxygen isotopes in chert;
inferred from carbon isotopes;
isotopes of soil water
Recommended Expressions
Since the time of the early Greeks, the name
of this symbol has been and remains delta
13C value; or carbon isotope composition
18
O (or D) depleted water
High (low) 18O values
low13C value (relative to another value)
Oxygen isotope ratio (composition) of chert;
inferred from carbon isotope measurements;
isotopic composition of soil water
Explanation
The word del describes either of two things in mathematics: an
operator () of the partial derivative sign ()
A composition of values is not possible
Water is not depleted (or enriched) in isotopes
As number,  values are either high or low
13C values are numbers and cannot be depleted
Such mistakes are a carryover from loose oral communication

The isotopic The 18O value of the A matter of redundancy

composition of water was - 4.3‰
water was 18O =
- 4.3‰
Mistake
Referring to the symbol  as
del
13C composition
Isotopically depleted water
Heavy (light) 18O values
Depleted 13C value
Oxygen isotopes in chert;
inferred from carbon isotopes;
isotopes of soil water
The isotopic composition of
water was 18O = - 4.3‰
The isotopic
value changed;
the carbon value
changed
Recommended Expressions
Since the time of the early Greeks, the name
of this symbol has been and remains delta
13C value; or carbon isotope composition
18
O (or D) depleted water
High (low) 18O values
low13C value (relative to another value)
Oxygen isotope ratio (composition) of chert;
inferred from carbon isotope measurements;
isotopic composition of soil water
The 18O value of the water was - 4.3‰
The isotopic composition
changed; the 13C value
changed
Explanation
The word del describes either of two things in mathematics: an
operator () of the partial derivative sign ()
A composition of values is not possible
Water is not depleted (or enriched) in isotopes
As number,  values are either high or low
13C values are numbers and cannot be depleted
Such mistakes are a carryover from loose oral communication
A matter of redundancy
The phrase “isotopic value” is
ambiguous. Does it mean a ratio?
A delta value?
Mistake
Referring to the symbol  as
del
13C composition
Isotopically depleted water
Heavy (light) 18O values
Depleted 13C value
Oxygen isotopes in chert;
inferred from carbon isotopes;
isotopes of soil water
The isotopic composition of
water was 18O = - 4.3‰
Recommended Expressions
Since the time of the early Greeks, the name
of this symbol has been and remains delta
13C value; or carbon isotope composition
18
O (or D) depleted water
High (low) 18O values
low13C value (relative to another value)
Oxygen isotope ratio (composition) of chert;
inferred from carbon isotope measurements;
isotopic composition of soil water
The 18O value of the water was - 4.3‰
Explanation
The word del describes either of two things in mathematics: an
operator () of the partial derivative sign ()
A composition of values is not possible
Water is not depleted (or enriched) in isotopes
As number,  values are either high or low
13C values are numbers and cannot be depleted
Such mistakes are a carryover from loose oral communication
A matter of redundancy

The isotopic value changed; The isotopic composition changed; the 13C The phrase “isotopic value” is ambiguous. Does it mean a
the carbon value changed value changed ratio? A delta value?

Enriched Isotopically heavy (light) These phrases culled from the

(depleted) carbonates; literature make no sense. More
carbonates (relatively) 18O-rich importantly, the words enrich and
carbonates; deplete are overused and much
Depleted carbon (relatively) 13C-poor abused. These words should be
source carbonates reserved for describing a process
that changes the content of the
Low 13C source; source heavy isotope of some element in
with a low 13C value the substance being considered
Mistake
Referring to the symbol  as
del
13C composition
Isotopically depleted water
Heavy (light) 18O values
Depleted 13C value
Oxygen isotopes in chert;
inferred from carbon isotopes;
isotopes of soil water
The isotopic composition of
water was 18O = - 4.3‰
The isotopic value changed;
the carbon value changed
Enriched (depleted) carbonates
Depleted carbon source
The isotopic
signature of the
rock was 18O =
5.7 ‰
Recommended Expressions
Since the time of the early Greeks, the name
of this symbol has been and remains delta
13C value; or carbon isotope composition
18
O (or D) depleted water
High (low) 18O values
low13C value (relative to another value)
Oxygen isotope ratio (composition) of chert;
inferred from carbon isotope measurements;
isotopic composition of soil water
The 18O value of the water was - 4.3‰
The isotopic composition changed; the 13C
value changed
Isotopically heavy (light) carbonates;
(relatively) 18O-rich carbonates;
(relatively) 13C-poor carbonates
Low 13C source; source with a low 13C value
The 18O value of the rock
was 5.7 ‰. Thus this rock
has the oxygen isotope
signature of the mantle
Explanation
The word del describes either of two things in mathematics: an
operator () of the partial derivative sign ()
A composition of values is not possible
Water is not depleted (or enriched) in isotopes
As number,  values are either high or low
13C values are numbers and cannot be depleted
Such mistakes are a carryover from loose oral communication
A matter of redundancy
The phrase “isotopic value” is ambiguous. Does it mean a
ratio? A delta value?
These phrases culled from the literature make no sense. More
importantly, the words enrich and deplete are overused and
much abused. These words should be reserved for describing a
process that changes the content of the heavy isotope of some
element in the substance being considered
The word signature should apply
to the isotopic composition of a
significant reservoir like the
mantle, the ocean, or a major part
of the system being studied, not to
the isotopic composition of
ordinary samples