Identification of Components in the
Neutral
Fraction of Green River Shale
by
Brian Dean Andresen
B.S.
Florida State University
(1969)
Submitted in Partial
Fulfillment
of the Requirements for
the
Degree of
Master of Science
at the
fassachusetts Institute of Technology
Feburary 1972
Signature of Author
Certified
Thesis Supervisor, Dept. of Chemistry
Theis Supervisor, Dept. of
Earth and Planetary Sciences
jA
Accepted by
'
I/
\
".
Chai rman, Pepr r tmenta
Committee on
Graduate Students, Department of
Eart
ml
netary Sc iences
WI
M1i
N
197
EIRAR q
page 2
Identification of Components in the
Feutral
Fraction of Green River Shale
by
Brian Dean Andresen
Submitted to the Department of Ear-th
and Planetary Sciences on
V!ovember 2, 1971
in partial
of the requirements for
fulfillment
the degree of
Vaster of Science
Abstract
With
the
aid
of
a
combined gas chromatograph-mass
spectrometer-computer system, the analysis of
fraction
of
the
Aliphatic
and
aromatic
Green
River
compounds
shale
the
was
were
neutral
undertaken.
examined
and
characterized.
A
series
structural
of
An
relatively
mild
components
were
identified
relationship to part of the carbon
8-carotene.
revealed
compounds
skeleton
of
investigation of the products produced upon
thermal
considerable
degradation
similarity
in
of
8-carotene
composition
isolated from Green River shale,
genesis from
which bear
implying
to the
their
8-carotene or closely related polyisoprenoid
page 3
products.
natural
structure
to
Furthermore
Vitamin
E
compounds
were
identified
significance discussed.
Thesis Supervisor:
Title:
Professor of Chemistry
Thesis Supervisor:
Title:
Dr. Klaus Biemann
Dr. John M. Edmond
Assistant Professor of Oceanography
similar
and
in
their
page 4
Acknowledgment
I sincerely wish to thank Dr.
me
to -work in his group.
Riemann
for
allowing
His guidance and generosity has
been most rewarding.
I also wish to thank all the members of
his
research
group for their help and consultation.
Financial
and
Woods
Hole
acknowledged.
support
from
Oceanographic
the Office of Naval Research
Institute- are
gratefully
page 5
Table of Contents
page
10
I,. Introduction
A.
Geochemistry of Sediments
B.
Gas chromatography-Mass Spectrometry-Computer
10
Systems and Their Application To Geochemistry 21
II'.
Analysis of the Neutral Organic Components in
Green River Shale
27
A.
History
27
B.
Analysis and Results
32
C.
Compounds of Biological
Interest in the
Green River Shale
III.
64
Analysis of the Thermal Degradation of
Beta Carotene and Comparison to Green River Shale 75
IV.
V.
VI.
Conclusions
87
Experimental
89
A.
GC-MS-Computer Operating Conditions
89
B.
Green River Shale Extraction and Separation
90
1.
Extraction
2.
Gradient Chromatography
C.
Osmium Tetroxide Oxidation of Olefins
D.
Synthesis of the Green River Shale Components 95
References
95
97
page 6
List of Figures
1.
Schematic of the combined gas chromatographmass spectrometer-computer
system used in
the analysis of Green River shale
2.
Total
22
ionization plot of a typical fraction
in the first few fractions of gradient
33
chromatography
3.
Mass spectrum of an alkylbenzene found
in Green River shale fraction 45
38
4,. Mass spectrum of an alkylnaphthalene found
in Green River shale fraction 45
5.
Mass spectrum of a methyl
substituted
bibenzyl
6.
43
47
Comparison of the authentic and synthetic
mass spectra for first compound of a
homologous series
7.
53
Gas chromatographic retention time comparison
for ionene and the compound in Green
River shale
8.
57
Mass spectral comparison between the Green
River shale component and the ionene
obtained from heated 0-carotene
58
page 7
9.
Mass spectrum of a polycyclic compound found
Green River shale fraction 45
10.
Total
in
61
ionization plot of fraction 45 and
mass chromatograms of m/e=135
65
11.
Mass chromatogram of m/e=149
68
12.
Comparison of mass spectra between
synthetic and authentic chromanes
ionization plot of heated s-carotene
13.
Total
14.
Apparatus used during gradient chromatography
73
77
92
page
List of Tables
page
identified in Ancient sediments
1.
Compounds
2.
Compounds previously identified
in Green
28
River shale
3.
Hydrocarbons in first few fractions obtained
from gradient chromatography
in Green River shale
4.
Alkylbenzenes
5.
Alkylnaphthalenes
6.
Compounds with two aromatic groups separated
in Green River shale
Compounds previously
45
76
Compounds derived from the thermal
degradation of s-carotene
9.
40
identified in heated
B-carotene
8.
35
49
saturated side chains
7.
20
81
Compounds -found both in Green River
shale and
in heated 8-carotene
85
8
page 9
List of Schemes
1.
Extraction of powdered Green
River shale
2.
Gradient chromatography of the
neutral organics
3.
31
Synthetic scheme followed in the
preparation of chromane type compounds
4.
30
71
Fragmentation pattern of the first
compound in the homologous series
84
page 10
1.
A.
of Sediments
Geochemistry
In
years
recent
interest
planet
in
the
and
at
been an ever increasing
has
there
understanding of the origin of life on this
identifying
what
type of life forms were
earliest pe riod s of ti me .
present during the
has
Introduction
Most research
centered arou nd the study of ancient sed iments and
identification of organic compounds which
might
serve
the
as
indicators of living systems.
Many
aspect
problems
of
have
organic
geochemistry
compounds,
isolated
biological
compounds,
compounds
sources.
not
may
arisen using this approach.
have
from
it
long
more
organic
should
resemble
exposures
common
unrelated
to
problem with the
"marker"
these
solely from inorganic
organic
compounds
of
biological
may
This
compounds
to elevated temperature and pressure
Beinng
derived
biologically
so
different
compounds,
biological
isolation
activity.
an d
select ion
from
these
be
dismissed
Thus,
the major
isolated organic chemicals migh t erroneo usly
as
that
resemble biologically derived substances.
while buried deep in sediments.
the
the
possible
produced
may be due to the. metamorphosis
after
is
Conversely, the isolated
at all
that
sediments,
but
been
is
One
of
biological
type compounds can be defined as determining what
page 11
is the true origin of the compounds found in sediments.
The theories concerned
compounds
with
with
origin
of
organic
in sediments can be divided into two categories.
The first is the formation of most
through
the
abiotic processes.
the
formation,
organic
matter,
all
organic
compounds
The second theory is concerned
deposition,
produced
only
and
by
transformation
living
of
systems,
in
sediments.
It has been observed that certain volcanos are rich in
organic
muds(l).
Proponents of the abiotic theory believe
that this organic material
earth
is synthesized deep
within
the
and is forced under pressure through fissures in the
earth's crust.
To support this theory, others
argue
that
the reaction of hot alkali metals with carbon dioxide(2) or
metal carbide reactions
account
for
the
with
larg e
amounts
beneath the surface of the earth.
obtained
from
the
reaction
hydrochloric acid(4) has shown that
variety
of
organic
would
water(3)
material
suffice
to
organic carbon found
of
Analysis of the products
of
iron
an
are
carbide
extremely
with
complex
produced.
chromatographic analysi s of this complex material
shows
Gas
a
smooth Gaussian type di stribution of hydrocarbons, with the
maximum around the C18 region.
The
Fischer-Trops ch(5)
reaction
also
proposes
a
explanation
for the presence of organic material
in ancient sediments.
A Fischer-Tropsch synthesis involves
possible
page 12
the reaction of carbon monoxide with hydrogen on a suitable
hydro carbons.
yield
to
catalyst
Gas
analysis o f this material shows normal
C32,
from C18 t
This
present.
incorporat on
Analy
o rganics
the
less
on mineral
reaction
of
other
hydrocarbons ranging
abundant
surfaces,
is
followed by
another
is
of
possible
many me teorites(6) has shown that a vast
rgani c material is synthesized
amount of
organics
for the presen ce of hydrocarbons in sediments.
explanatio
Many
with many
chromatographic
of
type
deep
in
space.
organic c ompounds, resembling those having
biological origi n, have be en found
in
such
meteorites(7).
t he abiotic theory believe that the presence
Advocates
of
of organic
mater ial
in met erorites is
proof
that
organic
compounds can be syn thesiz ed from inorganic sources.
In
t he
search
for
biological
marker
ancient sed iments, attenti on was focused on
isoprenoid
hydrocarbons
because
structure:
carbons.
characteri stic
The
me thyl
a
characteris tic
two
derived fro m the
molecule,
hydroca rbon S,
phytol
side
the
their
saturated
stability and
branch
every
four
1 ,4-head-to- tail linkage has
been though t to be indicat ive of
particular,
of
compounds in
biologica 1
phytane,
chain
of
systems.
thought
the
to
In
be
chlorophyll
and pristane, the decarboxalated phytanoic acid,
have been considered and detected(8).
page 13
oxidation
-CO 2
Ph tol
reduction
H20
Phytane
Pristane
However, work by Natta(9) using a
Al(Et)
3
-VC1 3 ,
doubt(10)
catalyst,
has been able to polymerize isoprene units
with the above characteristic
casts
suitable
structure.
This
seriously
on earlier findings, for mineral surfaces
may also act in a stereospecific way to exactly
mimic
the
biological- pathway.
In
younger sediments,
rich in organic
compounds, the
dominance of one type of compound over another is observed.
is that many young
of. this fact
6
sediments (less than 10 years old) show the dominance of
A
good
example(11)
page 14
never
which
tends
to
show
this
explained,
be
can
number
waxes
contribution of plant
the
even
rule
out
which
odd
of
numbe red
abiogeinic
synthe tic
of
type
dominance
However, this
specificity.
carbon
fact
This
hydrocarbons.
processes
over
hydrocarbons
numbered
odd
over
by
successfully,
show
also
this
e ven
the
same
characteristic hydrocarbon pattern.
the second theory for explaining the presence of
Thus
organic material
of
in sediments centers around the product ion
The deposition of
complex molecules by living systems.
these compounds is then a
the
Looking for
containing biological markers would
sediments
oldest
record of the past.
then be away to pinpoint the beginning of life.
If
compounds
biological
are
to
be
isolated
they must remain unchanged for long periods of
identified,
This is not generally the case for older
time.
little
Very
preserved
chromatographic
Series(13),
sedimen ts.
of the true biological type material
chromatographic
one-celled
analysis
shows
is found
Electron microscopy has revealed the
in ancient sediments.
oldest
and
of
complex
a
peaks,
very
organisms(12),
this
sediment,
but
gas
Onverwa cht
envelope of overlapping gas
indicative
of
abiogi cal
synthesis.
The
problem
is
that
most
naturally
occurr ing
biochemicals are unstable (for a variety of reasons)
leaving
the protection of the living cell.
af ter
Once releas ed,
page 15
be
or
microbes,
be
either
can
compounds
these
changed
due
by
metabolized
to
a
other
of extreme
variety
conditions.
Different compounds show
a
variety
of
stabilities.
are easily denatured due to elevated temperatures
Proteins
and also can be hydrolyzed to their component peptides
amino
These
acids.
small er
components
lattice
incorporated into the mineral
of
can
the
and
then
be
sediments,
carried far from the site of deposition to then be released
upon pH chang es.
become
The degradation of. the
even more likely if they were bonded to the mineral
surfaces whos e crystal
hea t
elevated
carbon
dioxi de
Carbohydrates
would
microbes and
liv ing
easily
are
only
soon
with
altered
greatly
contribute
and water to the total matter found in the
P igments, usually found in
sediments.
in
is
lattice
pressure.
and
metabolized b y
even
would
compounds
systems,
low
concentrations
easily altered at elevated
are
and can not be considered a major contributor
temperatures
(as unaltered compounds) to the total organics found in the
L ipids,
sediments.
the
on
material
organic
the
to
found in sediment.s and cover a broad
Al bel son et
range of comp ounds.
much
the other hand, contribute
al .(14 )
have
calculated
rate of decomposit ion fo r various n 3tural products and
have found
molecule
that
was
the
best
structural identity.
hydroc arbon
port ion
suited
the
for
of
lipid
the
retention
of
These calculation, based on the
its
time
page 16
required to thermally degrade one half
t he original sample,
establ ishe d a half life
at
calcul atio n
years
S imilar
400 K.
carbohydrates and amin o acids showe d half
o
years
at
400
K.
seem to
These numbers
for
3
lives of <10
indicate
0
of 10
that
most
of
the
material pres ent in
organic
sediments will be high in hydrocarbons
and
Normal
hydrocarbons can be
found
an d
in
attesting
isoprenoid fatty acids and
large
to
concentrations
the
in
ma ny
other
old
1 ipids.
sedi ments,
of hydrocarbon stability if
fact
these
compounds were present in the sediment during deposition.
The trans formation of these
on
dependent
ounds
environmental condi
and pressures, detected with depth
norma 1
under
sedimentat ion)
com plex molecules.
extremely
may
c r ust.
have
sediments.
an
Also
effect
In
a
very
d presumably with age
easily
rrangement,
cause
yielding
The com plexity of crude oils
definitely point toward complex synthesis deep
earth's
thus
High temperatures
s.
ould
polymeriza tion, bond cleavage , and
is
within
the
oxidizing and reducing environments
on
the
organic
material
in
the
reducing environment the organics tend,
wi th depth and age to be transformed into light hydrocarbon
gases,
ult imately
becoming the completly reduced methane.
Whereas oxi dative conditions in the sediments would tend to
eventually
produce
The interac tion of
contribute
to
the
only carbon (in the form of graphite).
all
the
above
sediments,
processes
would
then
through transformations,
a
page 17
complex variety of organic compounds.
oldest
CH 4
Increaseq
lig ht mol. wt. hydrocarbons
paraffins
f
Starting organics
In the sediments
TO C and -depth
pressure
H2
youngest
cycl ic aromatic hydrocarbons
higher mol.
wt.
organics
kerogens
ncrease"
Not only is the stability
structure
oldest
graphite
importan t
for
of
the
consider.
to
of
the
optical
these compounds is anot her important
point to
but
also
Optically active compounds are always considered
be an inherent part of biological
relies on the fact
attached
to
that
four
systems.
diff erent
a single carbon atom.
possible
configurations
The concept
groups
may
If one of
is domi nant, the material
the macroscopic property of rotating plane-polarized
upon irradiation.
be
For a carbon atom with
such asymmetry, two configurations ar e possible.
the
molecular
general
characterization of organic
compounds from biol ogical systems,
activity
the
has
light
Abiogenic processe s would be expected to
produce an equal distribution of the two configurations and
the
material(racemate)
internal
would
show no rotation because of
compensation.
The rotation of the plane polarized light
is
then
a
page 18
good
for
indicator
biologically
produced
compounds and
could be used to signify biological activity
mineral
Again,
surfaces
are
another
in
sediments.
poi nt that must be
considered, for it has been shown that L( +)
be
synthesized(15)
good yiel d solel y b
in
ammonia, carbon dioxide,
water
and
This
seems
to
surface.
mineral
acids can
acid
action of
on
suitable
a
cast some doubt on the
validity of optical activity as a gui depost fo r identifying
biological chemicals.
Because
of
their
concentration in sediments,
in
analyzed
the
Table
and
great
have
1
been
lists
Pre-Cambriain
in
those
sediments.
work has been done
seen from the table, little
be
can
handl in,g
hydrocarbons
detail.
great
hydrocarbons previously found
As
of
ease
with the identification of compoun ds other than the normal,
branched,
and
complex
sediments.
to
due
This has mostly been
highly
hydroca rbons
isoprenoid
of
nature
the
miinor
concentration
components
which
are
structura lly
possibly more significant).
younger
the
under
Thus,
sediments
more
analysis
may
hold
reported in
(and
assump tion
the
of the minor neutral components
River shale (Eocene) was undertaken.
research,
in the
complex
the
understanding the evolutionary pathway of older
the
and
It has now become possi ble to study these minor
components
that
low
the
fatty ac ids.
and
key
for
sediments,
in the Green
It was the aim of the
this thesis, to identify these minor
page 19
components in the shale and to consider the possible
responsible for their formation.
steps
page
20
Table 1
Compounds Identified In Pre-Cambrian Sediments
References
1.
Nonesuch Shale -
a.
normal
1 billion
years old
hydrocarbons - C17 most abundant,
no odd over even preference
b.
(16)
Branched and cyclic - anteiso C16 to C18,
iso alkanes C16 to 018, and cyclohexyl
alkanes C16 to C19
2.
Gunflint Chert - 1.9 billion years old
a.
normal hydrocarbons - even distribution
of the C20 to C30 pa raffins
b.
c.
fatty acids - n-C16 most abundant
Soudan Shale -
a.
4.
5.
(18)
.(19)
2.7 billi on years old
normal hydrocarbons - C17 most abundant,
contains C15 through SC20 only
b.
(18)
branzhed and cyclic - pristane C19,
phytane C20, possibl e steranes
3.
(17)
branched -
presence of C21 isoprenoid
(20)
(20,21)
Fig Tree Shale - 3.1 bil lion years old
a.
normal hydrocarbons - C14 to C25
b.
br-anched -
c.
olefins -
pristane and phytane
22)
23)
23)
C16
Onverwacht series - 3.7 billion years old
a.
normal hydrocarbons - C16 to C31
(24)
b.
branched - pristane and phytane
(25)
c.
fatty acids -
n-C16 most abundant,
also n-C18, n-C15, and n-C14
(25)
page 21
B.
Gas Chromatography-Mass Spectrometry-Computer Systems
and Their Application To Geochemistry
With modern techniques of extraction and analysis, the
elucidation of many organic compounds
possible.
powerful
The
advent
of
gas
chromatography
impact on geochemistry.
previously
unresolved,
components.
can
Compounds with
in sediments
Mlany
now
be
boiling
is
has
now
had a
complex
mixtures,
separated
into their
points
ranging
from
o
below zero to above 400 can now easily be separate.d by this
technique.
Mass
spectrometry
is
another
technique available to the geochemist.
Small
volatile organic materials are bombarded with
beam and the fragment
volatility
quantities of
an
electron
ions thus formed are used to deduce a
structure for the original organic molecule.
sample
important
requirements
The
similar
of gas chromatography and
mass spectrometry have made them exceptionally well
to
be
coupled
separated
by
immediately
together(26,27,28,29).
the
by
gas
mass
chromatograph
can
spectrometry.
The
suited
The
compounds
be
anal yzed
addition
of
a
computer(30).to the system has made gas chromatography-mass
spectrometry
a
powerful
tool,
through
the
continual
organic
materials
acquisition of data.
With this system, the
obtained
from
mixture
of
a particular sediment
the gas chromatography column (see
A
is first separated in
Figure
1)
and
the
components are then carried into the ion source of the mass
Figure 1.
Schematic of the combined gas chromatographmass spectrometer-computer system used in
the analysis of Green River shale.
A.
Gas chromatography column
B.
Source
C.
Analyzer
D.
Electron Multiplier
MASS SPECTROMETER
GAS
CHROMATOGRAPH
VAC.
RMU6- L
CONTROL UNIT
INDICATOR LIGHTS
AND TRIGGERS
.1
i
page 24
spectrometer (B Figure 1).
fragmented,
Here the compound
is
ionized,
and accelerated out of the ion source and into
the analyzer
(C Figure 1).
spectra
the repetitive scanning of the magnetic field
and
The
recording
are controll ed directly by the computer.
400
of
The
the
maximum
spectra (for.this system) allows ample time
ga s
complete
chromatographic
Peak
mixture.
centers
data are de t ermined in
disk
and
real
run
of
a
mass
of
to make a
large
complex
intensities of the digitized
time and stored on
a
magnetic
for s ubsequent conversion to m/e_ vs. intensity data.
These spect ra are immediately plotted and
microfilmed
for
fast easy ac cess and convenient storage(31).
Immedi ately
d ata
following
ionizaton p lot is generated
correlate
directly
Using this
plot,
with
by
the
it is possible
a
acquisi"tion,
the
chromatographi c trace.
gas
during
a
Because
run,
many
are obtained for each GC peak as vwell as along the
baseline where minor
This
components
allovws
of
the
separation .of
mixture
concentration
may
components
partiall y resolved by the gas chromatograph, due
slight
mass
to i mmediately find a
all of the mass spectra are recorded
hidden.
should
and
computer
spectrum fr om any portion of the gas chromatogram.
spectra
total
to
be
only
their
differences on either side of the GC
peak.
In addition to the data and plot described above, Mass
Chromatograms(32)
are
also
generated.
The
Mass
page 25
of
intensity
the
a
particular
type
of
Thus a fragment, characteristic
in
which
the
of
compound or fragmentation pattern,
produces a peak coincident
peak
of
ion as a function of the
one particular
spectrum index number.
plot
generated
Chromatogram is essentially a computer
This
contained.
is
material
chromatographic
gas
the
with
is
especially advantageous when searching for particular types
of
compounds
or
which
available
interpretive
Various
complex mixture.
programs(32)
are
pick
out
to
computer
the
enable
investigator
significant trends and help the
the
compounds in the
of
series
homologous
evaluate
to
vast amounts of information and to direct his energies
most
to areas of the data which are of
direct
value
and
relevance.
High
tool
This
ava ilable.
focusing
magn etic
ion beam.
on
the
double
Placing a n electrostatic field in
focusing
ene rgy
al lows
prior
to
focusing and thu Is allows higher resolution of the
a
In this manner,
mass
resolve not only ions diff ering by
ions with the same nominal mass,
compositi ons.
Fcir examp lie
it
beam at a nominal mass of m/e=135
elemental
relies
technique
principl e(33).
front of the
velocity
is another valuable
resolution mass spectrometry
compositions
C 9 H11 0 (135.08099).
are
spectrometer
can
now
nominal masses, but also
b ut
differing
elemental
i s possible to separate a
i nto two components whose
C 1 0H15+
(135.11737)
and
page 26
Thus with the
low
resolution
aid of the above two systems, high
mass
soectrometry,
neutral components in Green
The
application
of
River
shale
gradient
experimental) permitted separation
shale
the
analysis
was
the
of the
undertaken.
chromatography
of
and
total
(see
neutral
extract into less complex fractions and thus allowed
an optimum use of the GC-HS-Computer system.
page 27
II.
Analysis of the !eutral Organic
Components in Green River Shale
A.
History
The Green River shale formation is located in a
state
area
including
Colorado, Wyoming, and Utah and has
been reported(34). to be the the remains of
water.
lake
(Eocene).
that
existed
those
million years ago
rich
organic
character
in
the
shale)
chromatogram
show
a
has been done on this shale.
in
Table
2.
aith the extraction of the
in
by
followed
spectrometry.
both
this
resembling
characteristic
the
pattern obtained from extracts of the shale.
solvents,
of
Extracts of the present day algae (similar
found
hydrocarbon gas
summarized
fresh
partly due to the deposition of organic material
by these algae.
to
50-60
large
fungi still indigenous to that area(35,36).
It is proposed that the
is
some
a
The lake deposits contain many fossilized algae,
bacteria,. and
shale
three
and
U.S. Bureau of Mines,
Much research
The results of this work
are
Post of this research has dealt
powdered
gas
shale
block form by W.E.
Laramie,
using
chromatography
Samples of Green River shale
powde red
hydrocarbon
Wyoming.
were
organic
and
supplied
Robinson of
This
mass
the
particular
sampl e of shale has come from the U.S. Bureau of Vines Test
[ ine at Rifle, Colorado.
page 28
Table 2
Compounds Identified
In Green River Shale
References
1.
Neutrals
a.
Normal
Hydrocarbons - odd over even
preference, C11 through C33, maxium
(37)
at C17 and C29
b.
Branched Hydrocarbons phytane(C20) 65
of isoprenoids
pristane(C19)
2,6,10-trimethyl pentadecane
2,6,10-trimethyltridecane
c.
2.
2,6,10-trimethyldodecane
(37)
farnesane,squalane
(38)
Steranes ( cholestane, ergostane,
sitostane)
(39)
d.
Triterpanes (gammacerane,lupane)
(39)
e.
Tetraterpanes (perhydrocarotene)
(40)
(41)
Acids
a.
Unbranched Saturated - C10 through C34
w-methyl
keto acids C11, C14
(42)
dicarboxylic acids C13-C19
b.
Branched isoprenoid -
CE,
C9
C14-C17, C18, C19-C21
dicarboxylic alpha methyl
a.
3.
C13,
C15, and C16
(42)
Aromatic acids
(43)
Porphyrins
(44)
page 29
B.
Analysis
A
and
benzene-methanol
the neutrals, acids, and bases were
separated according to Scheme 1.
the
extract
placed
was
column and subjecte
Scheme
2.
separation
Liquid
of
the
on
The
methylene
techn ique
elution
chromato graphy
is
al 1 ows
diagrammed
high
and
methanol
chromatogr aphic ana lysis of every
organics.
high
A fairly
resolu tion
solvent
f ifth
complex mixture
was found between fractions 25-65.
of
system.
275-310
complex
variety
of
of the neutral
organic
compounds
Fractions 70-230 showed
organic
showed
Fraction
compounds.
showed only a very few components.
The individual fracti ons were then
gas
Gas
allowed
only a ver y few components, while fr actions 235-270
another
In all
benzene,
fraction
sep aration
in
capacity
neutra 1 compone nts into types.
chloride ,
capac ity
of
silica gel chromatography
a
fractions were col lected us ing a hexane,
310 5-ml
low
portion
neutral
to gradient column chromatography (see
Th
experiment al).
wi th
extracted
was
shale
powdered
of
sample
chromatographic
mass
(GC-tVS-Computer) system fo r
components in each fractio n.
the
subjected
to
the
spectrometric
computer
the
individual
analysis
page 30
Scheme 1
Extraction of the Powdered
Green River Shale
Powdered Shale (64
g)
1:1 benzene-methanol
extraction
evaporate, residue
taken up in methylene chloride
extract with .1N HC1
methylene chloride
layer
remove bases in
aqueous layer
extract with .1N KOH
1.19 g neutrals
in methylene chloride
remove acids in
aqueous layer
page 31
Scheme 2
Gradient Chromatography
1.,19 g Neutral Organics
column chromatography
hexane
hexane-benzene
benzene
-*fractions 1-142
benzene-methylene chloride
*fractions 143-239
methylene chloride-methanol
I
*fractions 240-310
SGas
chromatography of every fifth fraction.
Those containing many organics were further
analyzed using mass spectrometry.
page 32
Resul ts
Of the solvents to pass through. the silica gel
hexane
was
straight-chain,
saturated
in
hydrocarbons were eluted
Compounds
found
been
previously
in Table 3.
least
and
first
the
in
reported
Consequently,
polar.
isoprenoi d, and cyclic saturated
the firs
fraction
in
fraction
t few fraction.
20
through
have
35
the li terature and are listed
Figure 2 is the total
representative
column,
and
ionization
shows
concentration of the various hydroca rbons.
the
plot
of
a
relative
As can be
seen
from the plot, the n-C27, n-C29, and n-C31 hydrocarbons are
present
previous
in large abundance.
work(37)
on
This
is
in
agreement
the straight chain hydrocarbons and
their high concentrations has been e xplained(45)
the
hydrocarbon
growth.
with
contribution
from
partly
by
plant waxes and algae
Figure 2.
Total
ionization plot of a typical
fraction
in the first few fractions of gradient
chromatography.
GRSN-F25
HI CAL
TOTAL IONIZATION PLOT
l".
2 12 70
NUM- OF SPECTRA = 260
C2 9
Figure 2
C3 1
SPECTRUM INDEX NUMBER
Table 3
Hydrocarbons In First Few Fractions
Obtained From Gradient Chromatography
Spectrum
Index No.
Mol.
Wt.
Formula
6
272
C20H42
Phytane
8
268
C19H40
Pristane
23
226
C16H34
n-Hexadecane
37
296
C21H44
2,6,10,14-Tetramethyl
heptadecane
310
C2 2 H4 6
2,6,10,14-Tetramethyl
octadecane
324
C2 3 H4 8
2,6,10,14-Tetramethyl
nonadecane
75
338
C2 4 H 5
0
Tetracosane
85
352
C2 5 H 5
2
96
366
C2 6 H5
4
102
394
C2 8H5 8
2,6,10,14,18-Pentamethyl
tricosane
107
380
C2 7 H5
6
Heptacosane
121
394
C2 8 H 5
8
135
408
C2 9 H 6
0
151
422
C3 0 H6
2
172
436
C3 1 H6
4
187
400
C29H52
218
464
C33H68
Compound(37)
Pentacosane
Hexacosane
Octacosane
Nonacosane
Tricontane
n-Decylheneicosane
Sitostane(39)
page 36
group
Fractions 45 through 65 held the next large
during gradient chromatography.
eluted
compounds
organic
showed
Gas Chromatographic(GC) analysis of these fractions
many
of
peaks, attesting to the complex nature of
unresolved
the neutral organics.
striking
The first and most
analysis
and
UV
of
spectral
mass
after
was that many
shale.
River
because the Green River shale
unusual
are
results
data)
in the Green
aromatic compounds were present
These
(obtained
result
formation has been condidered to be formed under a reducing
aromatic compounds must then have
These
environment(40).
formed differently than through an oxidative
first
GC-MS-Computer
polysubstituted
compounds
aromatic
of
type
in
analysis)
observed
neutral
the
system.
C H +,
7 7
ions,
upon electron
stable ions as reported in the literature(47).
R1 H
H
-.-c leavag e R2'
R2
R3
m/e=105
m/e=119
P =R 2 =P3 =R
=R =H
:3' 5
1R
P =R =R =R =IHR =CH
1
2
5
3
S= =R =,
12:3
3
R =P =CH
4
5
R4
R
5
yielding
impact(46).
These substituted benzenes are found to yield the
m/e= 91
are
The compounds are characterized
benzenes.
tropylium
(through
extract
by cleavage of the bond be ta to the benzene ring,
stable
The
follwing
page 37
m/e=133
R1 =R 2 =H, R3 =R4=R 5 =CH 3
m/e=147
R1 =H, R2 =R 3 =R4=R 5 =CH
m/e=161
R 1 =R 2 =R 3 =R4=P 5 =CH
Figure
3
3
3
shows the mass spectral fragmentation pattern
(70 ev) of a typical trisubstituted
alkylbenzene
Its spectrum is
for a compound found in Green River shale.
in
agreement
benzene
the
with
compounds(47,48).
is
whose abundance
indicative
of
a
reported
data
concerning
m/e=204,
alkyl
The base peak(largest fragment
nornalized
to
100%)
at
me/=119
dimethyl substituted tropyliur, ion.
side chain, its length interpreted from the
at
obtained
contains
seven
carbon
molecular
atoms.
Thus
is
The
ion
for
alkylbenzenes, containing unbranched saturated side chains,
only the characteristic tropylium and the molecular ion are
needed to suggest the structure of the molecule.
lists
the
Table
4
alkylbenzenes which were found in the first few
fractions of Green River shale.
Figure 3.
Mass spectrum of an alkylbenzene found
in Green River shale fraction 45.
56--122
2 18 70
FIGURE 3. GRSN F45
119-,
M
.1
I
...
............
...q..- -P., P. -- op P-1.
20 I
20
40
.40
I60
60
80
100
120
140
160
180
1
p 1 -4
200
m /e
220
-~
240
260
2e0
300
-
I --
320
~
-
340
---q
----
I
360
'I
320
I" II" ' III'l'"
400
420
440
460
460
page 40
Table 4.
Alkylbenzenes in Green River Shale
suggested-
mol.
wt.
structure
106
Xylene
120
Cumene
134
148
176
I-0
190
204
~w
(31)
agree-
collection
authentic
page 41
the substitutents on the aromatic
of
location
inspection
facts can be obtained by the
fragment
the
of
a few
ring,
of the m/e=120 fragment
abundance
low
The
intensities.
the
exactly
tell
Although mass spectral data cannot
ion, seen in Figure 3, is a good indicator that there is at
benzene.
With
migration
of
type
this
of
at m/e=(base peak + 1) is sterically unfavorable.
ion
chain,
at
of
formation
Consequently, the
have
large
m/e=(base
m/e=92
to
the
+ 1) ions.
The
ortho
peak
an
of
formation
following mechanism(51) demonstrates the
ion
usual
the
substitution,
Conversely, compounds lacking substituents,
side
the alkyl
on
group
hydrogen atom in a VMcLafferty type(49,50)
a
rearrangement is blocked.
the
methyl
ortho-substituted
one
least
for n-butylbenzene with the ortho position
open.
CH
+2
CH
H
H
3
m/e=92
Similar
ions
shale
seen
were
base
intensity differences in the
for
+
1
other aromatic hydrocarb.ons in Green River
noted.
When
of
these
characteristics
peak
the
applicable,
compounds
wi 11
structural
be discussed in
greater detail.
The polycyclic aromatics, previously
literature
unreported in the
concerning Green River shale, are another group
page 42
in the neutral
of compounds found
Notable among
fractions.
These
these componds are the series of alkyl naphthalenes.
stable
their
by
recognized
easily
are
compounds
benzotropylium ions and have been extensively characterized
using mass
spectrometry(52).
generated
ions,
Important
upon electron impact, are listed below.
R1
Hi
H
...
..cleavage
,>__
R
R
2
m/e=141
3
R2
R3
=R 2 =P 3 =H
m/e=155
P =P 2=H,
m/e=169
R1=H,
m/e=183
H
,
_
R3=CH
R2=R =CH3
=R =R 3=CH3
A typical example of the type of spectra produced by a
molecule of the naphthalene type is seen
this
in Figure
length
is
determined
from
m/e=204, contains five carbon atoms.
the
In
spectrum, the base peak at m/e=183 is indicative of a
side
chain,
molecular
ion at
The
trimethyl substituted alkylnaphthalene.
whose
4.
the
Using this
alkylnaphthalene series of hydrocarbons
shale were characterized and are listed
in
approach,
in Green River
Table 5.
Mass spectrum of an alkylnapthalene found
in Green River shale fraction 45.
Figure 4.
-.' '
_r s
i
-J
~1
f
i
2 18 70
FIGURE 4. GR N F45
56--223
183
CH 3
M
II fill
20
40
11
. 11.11111
60
90
100
120
140
160
180
200
220
rn /e
240
260
260
300
320
340
360
390
400
420
440
460
460
r
page 45
Table 5.
Alkylnaphthalenes in Green River Shale
suggested
mol.
142
156
170
184
198
240
wt.
structure
(31) agrees
collection
authentic
page 46
In the la ter portion of the first few fractions,
spectra
were
obtained
for
compounds
characteri stic of t\ wo aromati c
abundant
and molecu lar ion i
the
seen
s
ions
with fragment
revealed
their
by
Usually only the tropylium ions
ions.
tropylium
groups
mass
in each spectrum.
Figure
5
is
mass spectrum of a c ompound found in Green River shale
which seem s to poss
saturated
s tw o aromat ic groups
hydrocartbon
aromatic r ings give:
m/e=119.
The
mol
c hain.
ris e
sC
ular
to
of
background
spect runm.
the
the
between
ions
at
m/e=224,
ion,
characteri zing peak
in
Cleavage
reasonab le
attached
to
a
the two
m/e=105
and
is the only other
intensity
seen
above
These data, along with the
empirical formula(C 1 7 H2 0 ), indicated structure I.
rooC
The base peak at m/e=119 is
this
structure,
for
substituted aromatic
alkyl
the
compounds
substituents(50).
also
in
agreement
appe arance
dec rease
If
with
potentials
with
the
of
increasing
appearance
potential(electron energy required t o produce the
ion)
is
low, then the abundance of a trimeth yl substituted aromatic
ion should be greater than that of a disubstituted aromatic
ion during the ionization under 70 e lectron volts.
This
approach
compounds in
the
was
shale
used
for
possessing
the
two
identification of
aromat ic
groups
Figure 5.
Mass spectrum of a methyl substituted bibenzyl.
2 25 70
FIGIRE 5
CH3
M
;1
I
20
40
I,1
llllllli
1111
II
UIIII
"IIIIIU
Umuu
~U~YYIUII~
80 100
60
120
m/e
140
1~Y17T~r7-~1
160 180 200
220
240
62--190
page 49
connected
saturated hydrocarbon chains.
by
agreeing with the above assumptions were then
the
responsible
compounds
Those spectra
studied
for such spectra are listed in
Table 6.
Table 6
Compounds with two aromatic rings
joined by saturated side chains
(31) agrees
suggested
mol.
wt.
collection authentic
structure
I
T
224
N"~s
NI
252
288
308
330
2(CH2O'
4
(CH2
and
proposed
proposed
page 50
A particular class of polycyclic organic compounds
Gree n
in
noted
shale whose members contai n both an
River
aromati c pa rt and hydroc arbon side
of
of
mo lecu le
the
tropyli um
ion.
in
the
units
chains
eq uivalent unsaturation.
degrees
is
is
e asily
differ ing
with
The aromati c portion
by
recognized
stable
its
These compounds differ usually b y 14 mass
substit uted
aromatic
fragmen tati on
hroughout
rings,
yet
mass
their
po I y-methyl
of
indicative
peak,
base
exhibit
spe ctra.
similar
The
side
chain, char act rized by the molecular ion, usually contains
one
of
equivalent
compounds
unsaturation.
be grouped
can
The
benzylic
type
of
into the following series (based
on mass spectr al data).
99 H 17
mol.wt.=216,base peak=105
-
j
9H 17
mol.wt.=230,base peak=119
-9H17
mol.wt.=244,base peak=133
The
.naphthalene
series
of
hydrocarbons also have a
similar side chains and can be grouped into
the
following
series.
C9H17
mol.wt.=280,base peak=169
page 51
14H27
In
an
mol.wt.=350,base peak=169
to determine the structure of the side
effort
chain, a number of experiments were performed.
fractions
containing
the
gas
chromatographic
(CC)
followed
experimental)
retention
Secondly, osmium tetroxide oxidation
the
mentioned above were
compounds
hydrogenated on an active catalyst (see
the
First,
of
and
times compared.
these
fractions,
by silylation(53,54) and GC retention comparisons
were carried out (see experimental).
In both cases, the GC
for
the
These two experiments had been aimed at detecting
the
time
retention
measurements
did
not
change
compounds of interest.
presence
of
one
bonds
by
either hydrogenation or by
This negative result
oxidation.
the
double
strongly
indicated
that
equivalent of unsaturation can be attributed to a
saturated ring system.
After considering all possible ring systems C9 H1 7 could
form, compound Ila (R =R2 =CH 3 ,R 3 =H)
laboratory(55).
wide
occurence
was synthesized in this
This structure was chosen because
of
its
in nature and, in particular, its presence
in the carotenoid family.
Unfortunately the mass
spectral
data for this synthetic compound did not agree with that of
the naturally occurring compounds.
at
m/e=105,
the
Instead of a base
peak
synthetic material showed a base peak at
m/e=111 (due to stabilized ion formation on the cyclohexane
page 52
Thus compound Ilb (R 1 =R3=CH3,R2=H)
ring).
was synthesized
with the hope of forming a compound whose mass spectrum was
more
like that of the unknown.
The methyl groups in these
positions would decrease the formation of the m/e=111
But
again
the
mass
spectral
fragmentation
ion.
and
gas
chromatographic retention times were different.
H3
R
R2
Ila (RI= R2= CH ,R =H)
3
3
Ilb (R = P3= CH 3 ,R 2 =H)
R3
Figure 6 shows the
between
the
synthetic
differences in
and
the
mass
spectra
naturally-occurring material.
These spectra differ not so much in their ovcrall
pattern,
but in the relative intensities (particularly the molecular
ion) and these variations
difference
ring.
for
in
the
can
methyl
not
be
due
substitution
merely
to
a
of the saturated
A library search(31) using a mass spectrum simulated
a
compound without a methyl group on the benzene ring
showed that the lower homolog (base peak at m/e=91)
mass
spectrum
characteristic
1-phenyl,1-cyclohexylethane.
Later
work
of
(page 80 )
had
a
a
with
a-carotene seemed to indicate that the compound was in fact
1,1,3-trimethyl-2(m-tolyl)-cyclohexane.
Another
fractions
polycyclic-type
compound
found
had an empirical formula C1 3 H1 8
high resolution mass
spectrometry).
The
in
these
(obtained from
low
resolution
Figure 6.
Comparison of the authentic and synthetic
mass spectra for the first compound of
a homologous series.
FIGURE 6.
MASS SECTRiM OF NATRAL PROULCT
i. -.
~~UiL-d1iL.i.J
~
~
.
'. . -. - -. -
20
40
. .
60
.
.
80
.
.
100
.
120
-
.' . -
.--
.
140
160
2 12 70
,1
.
.
180
k..
.
200
I
220
•-- ..
240
260
280
300
320
SYNTHETIC HYEROCARBON
CH3
3H
CH3
Li I
'20 40
20
40
60
6O
80
i L. I ,
100
..
. .
340
360
..
380
3 20 71
CH
'
...
120
140
10
'I
10
2
220
24
260
280
300'I'
90 100 120 140 160 180 200 220 240 260 290 300
56--140
.
.
...
400
j .
420
..
440
406--263
460
480
page
spectrum showed a strong f-15
mass
this
led
five
ion along with the
ion .
The stability of
unsaturation
of
equivalents
to the tentative st ructure IllI.
IV
III
The
exact
structure
III
o f the methyl
locations
considered next.
The close s tructural
and
ionene
(IV),
thermal degradation(56,57,58) of
substituents were
similarity
between
a major product from the
-carotene
was
obvious.
Therefore
ionene, obtained from the thermal
degradation
carotene,
was coinjected with
of
River
shal e.
As
can
be
158
and
grew
in
the
seen
tentatively assigned as ionene,
at
55
sample
Green
f rom Figure 7, the peak,
elut ed from the
size.
the
Thi s
GC
co Iumn
fact, along wi th the
extremely s imilar mass spectra (Figu re 8),
thus
con firmed
the presenc e of ionene in Green Rive r shale.
Compou nds
whose
mass
spectra showed a base pe ak s at
m/e=173 wer e also' noted.
These
concentrati on,
but
spectra seemed to indicat e that
one more me thyl
group was
the
ionene molecule.
and m/e=202,
their
c ompounds
were
in
attached t o the aromatic ring
low
of
Molecular ions were noted at m/e=188
leading to the structures seen below.
Figure 7.
Gas chromatographic retention time comparison
for ionene and the compound in Green
River shale.
Figure 8.
Mass spectral comparison between the Green
River shale component and the ionene
obtained from heated beta carotene.
FIGURE 7
FRACTION 45
10o 140
150 160 10 180 190o 200
IONENE
1
130
I
140
I
150
o 160
COINJECTED
Io
170
o 190
1a
o
180
260
200
ETRM T NATERAL PRODIT
60
;Uk
80
100
120
140
160 - 180
200
220
CH3 CH3
GO
80
-"~~'~~''""''~Y"~-~'
.
--
mII
120
160
100
140
6 70
1G4-274
6
CH
"'_--
56--114
240
FEATED CAROTEE
- -~-~-- '
2 l8 70
.
190
300
20
4
cr 3
page 59
Other mass spectra were obtained for compounds
ion
Besides this ion, only the molecular
to
seen
is
was
m/e=238
at
of reasonable intensity (larger than 201).
be
compound which yields only one or two
impact
the
ion at m/e=223.
an abundant
showed
Green River shale that
in
ions
A
electron
upon
Confirmed by high resolution
usually aromatic.
mass spectrometry, the elemental
composition of the ion
at
+
C1 7 H2 2 .
was
m/e=223
are
increase by 64 mass units
peak
with a methyl
considered.
by these
in the base peak
over
data.
the
of
location
the
methyl
Due to the fact that the
The
base
is also in agreement
substituted aromatic ring fused to an
The
molecule.
suggested
the spectrum of ionene
in
seen
tetramethyl-substituted
or
tetrahydrophenathrene
tetrahydroanthracenes
Tetramethyl-substituted
ionene
groups was also
thermal
degradation
of B-carotene produced compounds with similar mass spectra,
the structures seen below were eliminated.
If
starting
one
assumes
material,
that
it
B-carotene
is
the
probable
seems reasonable to first consider
only the gem dimethyl substituents.
page 60
Another spectrum was also found with only a base
14
units
mass
lower
and the molecular
(m/e=209)
m/e=214 to distinguish the molecule.
a
homolog
of
the
above
The
ions
ion at
This strongly suggest
tetrahydrophenanthrene
tetrahydroanthracene type molecule
substitution.
peak
with
only
one
or
methyl
which were found to characterize
these compounds are seen below.
or
or
R
m/e=209
R
R=H
m/e=223
R=CH 3
Figure 9 shows a
this
group
methyl.
representative
of compounds (C1 8 H2 2 ).
group
would
lead
to
the
mass
spectrum
from
Benzylic cleavage of a
base
peak.
Similar
fragmentation patterns were noted for the material obtained
from the thermal degradation of
for
the
O-carotene.
The rationale
products obtained from the thermal degradation of
the carotene molecule was used to suggest the
presence
of
these molecules in the shale.
Finally,
mass spectra were obtained from compounds in
the Green River shale similar
concerning
the
published
data(52)
substituted and unsubstituted methyl anthracene
and phenanthrenes.
compounds
to
are
What
is
of
interest
is
that
these
also found among the products obtained from
Figure 9.
Mass spectrum of a polycyclic compound found
in Green River shale fraction 45.
4r
2 10 70
FIGLRE 9. GRSN F45
56--181
M-15
CH3
:H3
LE
4poiii
'I
r
l
20
40
60
80
100
120
1
I' I ' 'I '
I
'20
40I
140
160
180
200
m
220
/e
240
260
280
-l1
300
320
340
360
360
1
400
420
Iiial
I-IWqwII
440
460
480
page 63
the thermal
degradation of
0-carotene.
contain
The latter fractions(237-270) seemed
methyl
esters,
and probably are
P reviously
many
reported in the literature(42)
the result of esterification of acids
in
the chromatograph y column during methanol elut ion.
highly
No
condensed
aromatic
ring
systems
were
detected using th is GC-MS-Computer assisted an alysis in the
neutral
fraction
of
Green River shale.
that if these typ e of compounds were
have
been
found
well
fractions.
This
fact
concerning
the
genesis
It seems obvious
present,
of
would
before the very polar methyl ester
also
of
futher
very
condensed aromatic ring
the Green River shale should
if any of these type of compounds.
i nformat ion
supports
systems only unde r oxidative environments.
environment
they
T
T he
reductive
yield little,
F
page 64
C.
Compounds of Biological
Interest in
Green River Shale
In surveying the data, it was
fractions
compounds
observed
characterized
by
These appeared to
those
by
r.
in
some
an abundant ion at
m/e=135 were present.
investigated
that
be
the
in
Preti(55)
same
a
as
parallel
investigation.
A mass chromatogram of fract ion 45 is shown
in
This plot indicates that at three different
Figure 10.
temperatures, compounds responsible for an i on
were
eluted
from
the
GC
column.
spectrometry showed that this abundant
an
elemental
compounds.
along
composition
The fragmentation
with
a
of
2,2-dimethylchromane(59), led to
m/e=135
High resolution mass
ion a t
Cg H110+
pattern
similar
at
m/e=135
for
of
each
all
had
three
mo lecule,
fragm entati on
tenta tive
assignment
in
of
the following structures for the three peaks seen in Figure
10.
r
H3 ,
CH
Y"r
2
3
Figure 10.
Totalionization plot of fraction 45 and
mass chromatogram of m/e=135.
GREEN RIVER SHALE FRACTIDN 45
TOTAL IONIZATION FLOT
RLN NO- =
56
NUML
OF SPECTRA = 389
SPECTRLM INDEX NLUBER
GREEN RIVER SHALE FRACTION 45
M/E 135
F=
O-O
VII
v
LtvI
"'
l
' '' ~ '
' ~
I
~ 'I-
. . . .
I
^-
I .I
I I. . I
. . I I I
I
46
page 67
aromatic
posibility
the
Because
was
ring
a
of
considered,
trimethyl-substituted
chromatogram was
mass
a
plotted for m/e=149 (m/e=135 plus one methylene group).
can
be
from
seen
Figure 11 the same fractions (F42-F50)
that contained the compounds V, VI,
compound
m/e=149.
fragmentation
whose
A
more
As
detailed
and VII also included a
yielded
study
an abundant ion at
of
ion
the
+
+
m/e=149(C 10 H 1 3 0 ) and of the molecular ion(C
2 9 H5 0 0
compound that formed this stable ion, led to. the
at
) of the
proposed
structure VIII.
VIII
This
structure is analogous to that of Vitamin E, but
lacks the phenolic hydroxyl.
Vitamin E
Figure 11.
Mass chromatogram of m/e=149 for fraction 45.
GREEN RIVER SHALE FRACTION
M/E 149
F=
0-03
VIIl
page 70
Var iou Is isomeric compounds of type VII and
by
synthesized
G.Preti(55)
methyl -subs tituted phenol,
refluxing
solution
using
phytol,
the
VIIII
appropri atel y
and zinc chloride
that
in
a
Coinjection, fol 1owed
of acetic acid.
by GC peak enhancement and very similar mass spectral
indicate
we re
the compounds present
in
data
the shale are best
represented by structures IX and X respectively.
r"Y
IX.
Y~
page 71
In order to confirn the proposed
other
members
of
the
isomers(XI,XII, and XIII)
purpose
the
chromane
structures
series,
of VI were
for
the
the appropriate
prepared.
For
this
alco.hol component (tetrahydrofarnesol) had to
be synthesized.
Scheme 3 outlines
leading to sturctures XI,
XII,
the
synthetic
pathway
and XIII.
Scheme 3
Synthesis of Chromane Type Compounds
H 2 Pd/c
0
(EtO) 2 PCH CO 2 Et
1 atm.
NaH/Diglyme
,H
0
LAH
reflux
+d igl yme
H
0-H
ZnC1
HOAc
reflux
XI
R=H R2=R3=CH 3
XII
R3=H R1 =R 2 = C H 3
XIll
R2=H
R113
=R3 =CH
2
page 72
synthetic and
the
from
obtained
spectra
mass
The
12.
naturally occurring material may be compared in Figure
occurring material
a long
spect ra
mass
Green River shal e.
in
considers
that
chromatographic
to be present in
The confirmation of these chromane type
these
molecules
their
derived
probably
on e of the Vitamin E type molecules, yet were
from
not completely metabolized to
The
microorganisms.
a nd
functionality
the
one
the
only
of
loss
molecules
complex
less
may
unit
isoprene
by
phenolic
indicate
enzym e systems, unable to degrade large aromatic
primitive
may
be
G reen River shale is very suprising when one
compounds
rings.
can
region
gas
times indicate compound XI(=VI)
retention
origin
identical
with
naturally
The extremely similar
residual background.
to
attributed
the
mass
low
the
around
of
spectrum
the
in
differences
Slight
the
The loss of the acidic OH from
refle ct
well
the
aromatic
ring
for oxygen by microorganisms
need
subjected to a reducing environment or a specific reductive
pathway
on
a
mineral
compounds may be the remai ns of
is
well
documen ted that the iso
are easily metab olized
presence
o
complexity
during shal
such
The two shorter-chained
surface.
by
Vitamin
acteri al
f the biochemistry
It
renoid hydrocarbons(60, 61)
bacte ria.
soil
E
metabolism.
type compounds
of
the
formation and might well
Finally,
reflects the
organ isms
serve as
the
present
a biological
marker type compound in the study of other sed iments.
Figure 12.
Comparison of mass spectra between synthetic and
authentic chromanes in Green River shale.
k
4
MASS SPECTRUM OF PEAK IV
7 69
0--15
2,5,7-TRIMETfYL,2 4 -DIIMETHYLNAN
) -CR-IMANE
4 26 71
lw
458--125
@Oc~--m
11111
• ;"'
'
IL"u 1.
A i.
L
m6 iL
200
"' '' 'I100
,,
2,7,9-TRIMETHYL-4
..
1
100
.I
..
300
20
4 26 71
-DIMETHYMNONANE) -CHOdANE
458-- 38
40
60
80
100
120
140 160
40
40
60
60
80
'I
1-00
100 120
I
I
140
10
II
180
200
220
220 240
26
290
I
200
320 340
4 26 71
458--175
Qac"
f"r
dr
160
I
200
2,5,8, -TRIMETHYL-(4' ,8'-IMETHYLNONANE)-DalMANE
~8c-~--~
"20
20
10
240
260
280
300 320
I I
I
100
120
i]
140
10
16
200
22
240
I36 26
2
300
60
90
100
120
140
160
10
200
220
240
290
300
I I
i h -..
I,; -~--....~..-..k.....
. . .~
.~
.~
.~
I
m
rr -ri-I
rrr- -r-r rr rr........-.
~_~ ~~.
~..~~
~~~~_____
,I
340
60
40
260
0
30
340
0
340
360
3e0
400
420
I
440
40
400
420
440
460
480
6
page 75
III.
Identification of the Compounds Obtained
From the Thermal Degradation of Beta Carotene
Procedure
plant
Because it was realized that certain decomposed
pigments would contribute many neutral organic compounds to
the
sediment,
degradation of
compounds
through
formed
the
thermal
B-carotene were analyzed.
8-carotene
The
of
choice
was
8-carotene
First,
obvious.
perhydro- 8 -carotene had previously been isolated(40) from
Green
River shale.
shale
formation,
If then
it
might
8-carotene was present during
well
have
rearranged
to
contribute many of the aromatic type compounds found in the
shale.
Secondly, if one
assumes
structure
this
as
the
starting material, one may more easily deduce the
probable
structure
of many of the compounds in Green River shale if
indeed they where formed through the thermal degradation of
B-carotene or related polyenes.
Initially, powdered
ampule
for
and
four
transformed
under
sealed
days,
into
the
8-carotene was placed in a
nitrogen.
normally
dark
After heating at 140
purple
a light yellow liquid.
run with this sample showed a complex
glass
powder
0O
was
A GC-MS-computer
mixture
of
organic
page 76
The
compounds
in Figure 13.
heating
total
ionization plot of this run is seen
Some of the major constituents
obtained
carotene have been isolated and identified.
previousl y reported are listed
per cent concentration found
by
Those
in Table 7 along with
their
in this particular sample.
Table 7
Compounds Previously Identified In Heated Carotene(56,57)
The
Compounds
4 Total
Benzene
3.4%
Toluene
23.2%
Xylene
35.1%
lonene
14.8%
others
23.5%
formation
of
these types of compounds has been
e xplained(58) as cyclization and elimination
reactions
of
th e polyene.
R
.
R
Toluene
The
formation
of
a
variety
$-carotene is thus dependent
unsaturated
hydrocarbon
on
of
.compounds
the
chain.
ionene(IV) can be similarily explained.
the
reaction
below.
by
orientation
heating
of
the
formation
of
The mechanism
for
The
has been elaborated upon(58) and is outlined
Figure 13.
Total
ionization plot of heated beta-carotene.
0
FIGURE 13. HEATED CAROTENE
TOTAL I~OIZATION PLOT .
NU(.
OF SPECTRA t
RULNO- = 362
339
SPECTRUM INDEX NUMBER
4
page 79
-H 2
Ii
HD
CH 3
$-carotene
H
%%
lonene( IV)
Because the
performed on the
of
this
reducing
hydrogenation
was
products.
The
B-carotene rearrangement
experiment
a
constitute
catalytic
extensive
environment,
aim
would
sediments
was to see whether any compounds
would be formed whose mass spectral
data
similar
was
to
compounds found in Green River shale.
After
material
catalytic
was
reduction, the normally light yellow
transformed
into
a
clear
oil.
A
GC-MS-computer analysis of these compounds was then used to
identify compounds in the Green River
similar
mass
spectra.
shale
which
showed
In addition the structures of the
aromatic and olefinic compounds
present
in
the
original
heated carotene sample could now be more easily deduced.
page 80
Results
In the heated carotene sample, many aromatic compounds
violet
8
nm
nm) as well as from
280
and
in
tropylium i ons seen
characte ristic
Table
(255
absorption
the
lists the compou nds that wlere
mass
spectra.
found in the heated
suggested not only by
Structures were
carotene
ultra
of
basis
the
on
deduced
These were
were found.
their mass
spectra, but also by consi deration o f th e possibl e pathways
by which
cyclize.
B-carotene could
inter-molecular
compounds
detecteId
of
possibility
and in
act,
high mol ecular weight(over
600)
in teraction
would explain some of the
The
when
was
considered,
vapori zing
sample
a
thermally degraded heated carotene directly
into
of
the
the
ion
source of the mass spectrometer.
spectra that closely resembled spectra
Of all the mas
obtained from Green River shale, one spect rum found in
the
catalytic reduction sample was of particul ar intere st.
Its
mass spectrum is ve y similar to the mass spectrum
Green
very
River
minor
0-carotene,
shale
Al though a
thermally
degraded
the
from
for
importance
the
identification of a
homologous series of compounds in Green River shale
be
noted.
is
to
The first compound in the homologous series in
the Green River shale
Previously
the
component seen in Figu re 6.
component
its
of
reported
has
a
molecular
ion
at
m/e=216.
in the literature(58), compound (XIV)
Table 8
Compounds derived from the
thermal degradation of a-carotene
(structures Indicated through mass spectral data)
agree
Peak
Structure
Authentic
Collection
(31)
T
T
A
B
6H13
D
proposed
C
x
E
lonene
F
G
Isomer of
Table 8 (continued)
Peak
agree
Collection Authentic
(31)
Structure
r
cho
T
I
OR
proposed
H3
proposed
C8H17
proposed
C6 H13
proposed
10"21
C1 2 H2 5
proposed
proposed
proposed
page 83
has been
identified
compound was 214.
a
After catalytic
molecular
spectrum similar to
component
With
a
in the side chain, the molecular weight of this
double bond
with
in the heated carotene sample.
seen
in
216
%weight of
and
Figure
6
apeared.
a
compound
possessing a mass
the spectrum of the Green
information, structure XV was
first component
reduction,
Thus
River
shale
with
tentatively assigned
as
this
the
in the Green River shale homologous series.
This fact also gives valuable
concerning
information
the
side chain of the higher homologs.
N
reduction
y
H
The
fragmentation
component (seen
in
pattern
Figure
6)
for the Green River shale
can
according to the following Scheme 4.
now
be
rationalized
page 84
Scheme 4
Fragmentation of Compound in the Neutral Fraction
a
>
m/e=132
M =216
H
b
H
----
m/e=105
-H*
m/e=69(100%)
m/e=145
page 85
Table 9 summarizes the compounds which were found both
in Green River shale and in the heated carotene.
Table 9
Compounds present
shale
in both Green River
and in the heated carotene samples
The high concentrations of toluene and xylene
the
extreme
conditions to which B-carotene was subjected.
Presumably,
compounds.
over
longer
in
polyenes
River shale would only be subjected to
increases
lower-weight
the
of
Others(56) have found lesser amounts
aromatic
reflect
slight
the Green
temperature
Extended, milder
periods of time.
heating periods and possible mineral catalysis of
specific
rearrangements may produce many neutral compounds not found
solely by heating polyenes at high temperatures.
It does
seem
evident
that
some
of
the
compounds
page 86
present
in
Green River shale may have been derived by the
degradation of various polyenes.
ionene
(IV),
obtained
from
The high concentration of
the
thermal
B-carotene, points toward the presence of
shale
formation.
Green
River
conjugated
shale
systems
Still
may
of
degradation of
polyenes
during
more of the compounds present
have
plant
other polyunsaturated systems.
had
their
carotenes,
origin
in
in
the
xanthophylls or
page 87
IV.
Conclusions
the
characterize
in
compounds
organic
to
relationships
the
structural
bear
shale
neutral
the
of
many
that
the
high
A
polyenes.
degraded
thermally
between
correlation
shows
her
The work reported
of Green River shale.
organics
neutral
and
identify
to
The purpose of this thesis was
products obtained froml-carotene
and the constituents of Green River shale was observed with
the
aid of the GC-MS-Computer system.
if
indeed
degradation may
their
sedimentation,
were
polyenes
several
This indicates that
during
present
well have produced
the many of the neutral organic compounds found in
younger
sediments.
fact
The
that
a
sediments subjected to
that
environment
reducing
before reduction.
good
indicated
of
In this work an analogy was used for the
in
compounds
the
heated carotene
reduced
sample and those in the Green River shale neutral
A
present in
thermal rearrangement of the compounds occurred
some
formation
were
compounds
aromatic
correlation
was
obtained,
extract.
but future work could
center around reducing a polyene first, thermally-degrading
newly
these
formed compounds and then analyzing to see
any similarites are produced between
the
neutral
fraction
of
Green
these
River
correlation is found, then it might well
thermal
degradation
was,
even
in
a
compounds
shale.
be
if
and
If little
assumed
that
young sediment, an
page
important part of organic geochemical
A
synthetic
scheme
methyl-substituted
for
chromane
the
88
processes.
identification
was
developed.
of
The
a
new
chromane is structurally related to the Vitamin E molecule,
but
lacks
the
the side chain.
phenolic hydroxyl and one
isoprene unit on
This suggests partial metabolism
microorganisms
of
specific
site
reduction
on
research
into
the
extracts
the
acid
original
Vitamin
mineral
Vitamin E is among these organics.
may
E
molecule
surfaces.
well
by
soil
or
Further
reveal
that
page 89
IV.
Experimental
A.
GC-MS-Computer Operating Conditions
See Table IX.
Table IX
Operating Conditions
Helium flow rate - 26 ml/min through
Column - 6' x 1/8"
(I.D.)
metal,
3% OV-1
Interface - split ratio 50:50, GC:MS
Temperatures -
injector 250-2800
manifold 2800
ion source
Hitachi
2500
RMU-6L coupled to an IBM 1800 computer
Ionizing voltage- 70 ev
page 90
Green River Shale Extraction and Separation.
B.
1.
Extraction
Two Soxhlet extractors
rinsed with distilled
solution,
allowed
Into each extractor
1:1)
was
and
added
At the end of that time,
reflux for two days.
to
thimble
one
extractor
400 ml of solvent (benzene-methanol,
cleaning
and then rinsed with
water,
wad of glass wool were placed.
a
with
washed
Into each
reagent grade methanol.
and
were
fresh solvent was added and the old solvent discarded.
While the blank extraction was
were
tubes
test
washed
in progress, 345
in cleaning solution, rinsed in
distilled water, and finally ultrasonically
reagent grade methanol.
of
bath
tubes and other glassware
10-ml
were
cleaned
in
a
After cleaning, the test
stored
in
a
clean,
dry
All contact with the glassware was done with nylon
place.
gloves.
After the
two
days
of
extraction,
the
blank
was
evaporated to a small volumn and gas chromatographed to see
what impurities were present. Only one small
peak was
seen
at 205 on the gas chromatogram.
Next,
64 g of powdered Green River shale were divided
into two equal
Four
hundred
parts which were placed into the extractors.
ml
of
solvent (benzene-methanol,
1:1) were
added to each extractor and allowed to reflux for two days.
At
the end of that time, the solvent was removed and fresh
solvent added.
Again refluxing was allowed to continue for
page 91
two
which the first and second extracts were
after
days,
evaporated to a small volume and redissolved with methylene
chloride ( 100 ml).
The
total
bases, and neutrals.
remove
the
separate the
removed
by
bases,
A
0.1N
and
a
HCl
0.1N
to
used
KOH solutiorn was use d to
neutral s
were
chlcoride.
methylene
into
extraction
was
solution
remaining
The
acids.
then separated into acids,
were
organics
then
This
solvent was evaporated to yield 1.19g of neutr;al organ i CS.
2.
Gradient Chromatography (62)
chromatog raphed
then
was
The neutral fraction
in
a
manner similar that reported by G.Preti(55), b y placing the
column
fraction atop a silica gel
eluting
outlines
with
solvents
of
the
solvents
used
(1"
x
4'
glass)
increasing polari ty.
and
Figure
14
and
Scheme 2
shows
the
apparatus used.during elution.
Scheme 4
Gradient method
300 ml
he ane
benzene
methylene chloride
met anol
The
cleaned
test
tubes were placed in the automatic
fraction collector, and the machine was set to take 5 ml of
effluent
per fraction.
The rate was adjusted for 12 ml an
Figure 14.
Apparatus used during gradient chromatography.
ew solvent
mixed solvents
magnetic
st i rrer
col umn
----
mixture of
neutrals
$silica
fl
gel
collection
page 94
hour.
Every fifth 5-ml sample collected was then placed in
a 10 ml pear-shaped flask, most of the solvent removed, and
the residual solution analyzed by gas chromatography.
page 95
C.
Osmiumn tetroxide
10
ml
to 8.0
Oxidation of
Tetroxide
Osmium
of
to
(51.5 mg, 0.0002 moles) was added
of thi s yellow solution was added
of di oxane. Two ml
mg
Olefins
neutral
organics.
Catalytic
amounts
of
pyr idine were added following procedures outlined by Capell
After 2 hours, this brown solution was
et al.
(53 ).
to' a
mix ture of sodium bisulfate, water, and py ridine and
After extraction with chlor oform
sti rred fo r 8 hours.
dry ing
ov er
res idue
anhydrous
magnesium
(N, O-Bis-T rimethylsilyl-Acetamide)
pyr idine.
D.
and
sulfate, the evaporated
using
silalyted
was
added
and
1
ml
BSA
20
mic ro
liters
GC-MS analysis followed after 2 hours
55
at
.
Synthesis of Green River Shale components
1.
Benzylcycloalkane (IIb)
6-methyl-2-butylthiomethylenecyclohexanone (0.02 moles) was
reduced with 18 g (0.02 moles) Raney nickel as described by
Ireland et al.
The dimethyl ketone (0.006 moles) was
(63).
added to an excess of
a-bromo
o-xylene
the
was
reagent
nitrogen.
extracted
The
newly
formed
then dehydrated with 50% sulfuric acid/water,
neutralized with a saturated solution of
and
from
formed
(0.02 moles) and magnesium (0.02 moles)
in ether and refluxed under
alcohol
Grignard
with
ether.
sodium
carbonate
Reduction of the olefin with
Pd/c (10%) and hydrogen at one atmosphere
yielded
66%
of
compound lib.
2.
Methyl-substituted
Chromanes - Pseudoionene (5.36
page 96
g, 0.003 moles) was catalytically reduced with 10%
The ketone (3.9
hydrogen at one atmosphere.
was added to a solution (40 ml)
moles)
(0.02
moles)
procedures
by
hydride.
The
Wadsworth
et
continue overnight.
a B-unsaturated
was
reaction,
al.
water
4.8
0.48
in
(64),
g (0.02
accord
was
with
allowed
added
g
and
to
the
extracted with ether and dried over
ester
accomplished
(yield
86%).
Reduction of the
by refluxing in glyme with excess
lithium aluminum hydride for 24 hours.
of
and
Excess water was then
anhydrous magnesium sulfate
ester
g, 0.02 moles)
of glyme containing
triethylphosphonoacetate
sodium
Pd/c and
Cautious
addition
followed by extraction from a saturated solution
of sodium chloride y ielded a mixture of compounds including
the
allylic
alchol .
Vacuum distillation of this mixture
yielded 83% of the a llylic alcohol.
equally
of
into
-three parts, added tp a one molar equivelent
2,3-,2,5-,and
solution
of
3,5-dimethylphenol
acetic
acid
and
procedure by Smith e t al.(65).
base
afforded
XII(27.8%)and
The sample was divided
the
and
refluxed
in
a
zinc chloride according to
Extraction
with
Claisen's
methyl-substituted chromane XI(30.8%),
XIII (31.0%)
chromatography peak areas).
(%
calculated
from
gas
page
97
References
1.
E. Coste, Canadian Mining Inst. J., 6, 73 (1903).
2.
M. Berthelot, Ann. de Chim et des Phys.
9, 481 (1866).
3.
D. Mendelie, The Principles of Chemistry, P.F. Collier
and Son,N.Y. (1902), 1, part 2, Russian Translation.
4.
T.C. Hoering, "Criteria for Suitable Rocks
in
Precambrian Organic Geochemistry", Carnegie Institute
Yearbook, 6_,
5.
365
M. Calvin, Chemical
(1966).
Evolution, Oxford University
Press, N.Y., p. 102, ref. 38.
6.
J.M. Hayes and K. Biemann, Geochim. et Cosmochim. Acta,
32, 239 (1968).
7.
J.M. Hayes, Ph.D. Thesis, MIT, Dept. of Chem. (1966).
8.
B.J. Mair, Geochim. et Cosmochim. Acta, 28, 1303, (1964).
9.
G. Natta et al., Chem. et Ind.
(Milan), 40, 362 (1958).
10.
E.D. McCarty and M. Calvin, Nature, 216, 642 (1957).
11.
A.G. Douglas et al.,
Advances In Organic Geochemistry,
p. 315, G.D. Hobson and G.C. Speers,
Perg. Press (1970).
12.
A.E. Engle et al., Science, 161, 1005 (1968).
13.
W.D. .1*acLeod Jr.,
14.
P.H. Abelson, Fortschs. Chem Org. Nat. Stoffe, 17,
J. Gas Chromatography, 6, 591 (1968).
379 (1959).
15.
E.Degens, !ature, 227,
16.
G. Eglinton, P.M. Scott, T. Belsky, A.L. Burlingame,
492 (1970).
M. Calvin, Science, 145,
263 (1964).
page 98
17.
R.B.
Jons,
T. Belshy, E.D. McCarthy, A.L. Burlingame,
P. Haug, H.K. Schnover, W.J. Rechter, and M. Calvin,
Geochim, et Cosmochim. Acta., 30, 1191 (1966
18.
J.
Oro,
D.S.
19.
D.W.
Nooner,
Barghoorn Science, 148,
W.Van Hoeven, J.R.
S.A. Wikstr om.
A. Zlatk ei,
77 (1961).
Maxwell an d M.Calvin,
Geochim. et Cosmochim. Acta, 33, 877 (1969).
einschein, ibid, 22, 58 (1961).
20.
W.G.
21.
D.D. McCarthy, W.Van Hoeven, M. Calvin,
Tetrahedron Letters No. 45,
22.
(1967).
4 437
J. Oro, D.W . Nooner, A. Zlatkis, A.A. Wikstrom and
E.S. Bargho orn, Science, 148, 77 (1965).
23.
J. Oro, D.W . Nooner, Nature, 213, 1082 (1967).
24.
W.D. MacLeo d, J. Chromatog.,
25.
J. Hans and M. Calvin, Nature, 224, 576 (1969).
26.
R. Ryhage, Anal.
27.
J.T. Watson and K. Biemann, Anal.
28.
P. Llewelly n and D. Littlejohn, presented at the
Chem.,
6,
36, 759
591 (1968).
(1964).
Chem., 36, 1135 (1964).
"Pittsburgh Conference on Analytical Chemistry and
Applied Spe ctroscopy" Feb. 1966.
Chem., 38, 1585 (1966).
29.
S.R. Lipsky et al., Anal.
30.
R.A. Hites, Ph.D. Thesis, MIT Dept. of Chem. (1968
R.A. Hites and K. Biemann, Anal.
31.
Chem., 40, 1217 (1968).
H.S. Hertz, R.A. Hites, and K. Biemann, ibid.,
43, 681 (1971).
32.
R. A. Hites and K. Biemann, ibid.,. -42, 855 (1970)
.
page
33.
K. Biemann, Mass Spectrometry,
34.
W.H. Bradley, U.S. Geol.
99
N.Y. (1962).
McGraw-Hill
Surv. Profess.
87 (1929).
Paper 159-E,
Bradley, Amer. J. Sci.,
35.
W.H.
36.
J. Han, M. Calvin et al.,
262,
4 13
(196
4
).
Pro. Nat. Acad. Sci.,
59, 29
(1968).
37.,
W.E. Robinson, J.J. Cummins and G. U. Dinneen, Geochim.
et Cosmochim. Acta, 29, 249
J. Chrom.
46,
(1965).
112 (1970).
38.
B. Cooper,
39.
A.L. Burlingame, P. Haug, T. Belsky, and M. Calvin,
Pro. Nat. Acad. Sci.,
40.
54, 1406 (1965).
Sister M. T.J. Murphy, A. McCormic, G. Eglinton, Science,
157,
1040 (1967).
41.
P.C. Murphy, Ph.D. Thesis MIT Dept. of Chem. (1970).
42.
R.C. Murphy, Science, 165, 695
43.
P. Haug, H.K. Schnoes, A.L. Burlingame, Geochim. et
(1969).
Cosmochim. Acta, 32, 358 (1968).
44.
J. R. Morandi
11,
and H.C. Jensen, J. Chem. and Eng. Data.,
81 (1966).
45.
G. Eglinton and Sister M.T.J. Murphy, Organic Geochemistry
46.
H. Budzikiewicz, C. Djerassi and D.H. Williams,
Mass Spectrometry of Organic Compounds, p. 76, Holden-Day
Inc., San Francisco (1967).
47.
H.M1.
Grubb and S. Myerson,
in F.W. McLafferty, ed.,
Spectrometry of Organic Ions, Academic Press, N.Y.
48.
A.B. King and M.R. Basila, Anal.
Chem.,
37,
Hass
(1963).
734 (1965).
page 100
49.
Ref. 45, page 155.
50.
F.W%.
tVcLafferty,
in F.W.
VcLafferty ed.,
Mass
Spectrometry of Organic Ions, Academic Press, N.Y. (1963).
45,
page 82.
51.
Ref.
52.
A. Cornu and R. Massot, Compilation of Mass Spectral
Data, Heyden and Son limited, Universitaires De
France (1966).
Chem.,
40, 1458 (1968).
53.
P. Capella and C.M. Zorzat, Anal.
54.
C.J.
55.
G.Preti, PhD. Thesis MIT Dept. of Chem.,
56.
W. Day and J.G. Erdman, Science, 141, 808 (1963).
57.
I. Mader, Science, 144, 533 (1964).
58.
F.S. Erdman and A.W. Johnson, J. Chem. Soc.,
59.
H. Budzikiewicz, C. Djerassi and D. H. Williams,
Argondelis and E.G. Perkins, Lipids, 3,
379 (1968).
(1971).
2892 (1965).
Elucidation of Natural Products by Mass
Spectrometry,
60.
Vol.
2, p. 260, Holden-Day Inc.
(1964).
V.E. Voishvillo, A.A. Axrem, U. A. Titov,
Priklad. Biochim. and fikrobiol.,
6,
491,
(1970) (in Russian).
61.
R. Jones and M.A. Edington, J. Gen. Ficrobio., 52,
381 (1968).
62.
E. Heftmann, Chromatography, p. 80,
Reinhold Pub.
N.Y. (1967).
63.
R.E. Ireland and J. A. Marshall,
J. Organic Chem., 27,
64.
W.S. Wadsworth, Jr.,
1615 (1962).
and W.D. Emmons, J.
Am. Chem. Soc.,
page 101
83,
65.
1733 (1961).
L.R. Smith et al.,
J. Organic Chem., 4,
311 (1939).