United States
Environmental Protection
Agency
Environmental Research
Laboratory
Athens GA 30613
Research and Development
EPA-600/S4-84-072 Sept. 1984
Project Summary
Application of an Analysis
Protocol to Identify Organic
Compounds Not Identified by
Spectrum Matching
Joan T. Bursey
Industrial wastewater survey sam-
ples were analyzed for organic com-
pounds not identified by spectrum
matching. Analysis of the samples pro-
ceeded from an initial packed column
GC/MS analysis for Priority Pollutants,
through computerized spectrum
matching for other organic compounds
to the present capillary column GC/MS
analysis of a chosen set of sample ex-
tracts. Attention was focused on the
spectra seen to occur frequently yet not
tentatively identified by spectrum
matching.
A plan for systematic study of these
sample components was devised that
included, in step-wise fashion, the use
of high resolution gas chromatography
(HRGC), high resolution mass spec-
trometry (HRMS), chemical ionization
mass spectrometry (CIMS) with posi-
tive and negative ion detection (NCI),
and Fourier transform infrared spec-
troscopy (FT-IR). Sample cleanup was
used at all levels to mitigate interfer-
ence.
For 55 extracts in which components
of interest were observed, accurate
mass measurement was successfully
used to generate chemical formulas in
35 cases. Of these, the results for 16
could be narrowed to one or two pos-
sibilities each. Tentative structures
were proposed in six cases. Since the
proposed compounds were not com-
mercially available and the costs of syn-
thesis were prohibitive, no further con-
firmation was made.
Conclusions were: 1) that this type
of compound isolation/identification
effort is very time and labor intensive,
2) that the labor costs are high because
highly trained and experienced person-
nel are required, and 3) that the amount
of definitive information that can be ob-
tained by application of any one of the
analytical techniques discussed above
(or of several of the techniques in suc-
cession) ranges from minimal to very
high, but integration of all the informa-
tion available is often not as simple as
the analyst might wish.
This Project Summary was de-
veloped by EPA's Environmental Re-
search Laboratory, Athens, GA, to an-
nounce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
In June 1976, a Consent Decree was
signed by EPA and several public in-
terest groups that set deadlines for EPA
to promulgate regulations for treating
industrial wastewater. Beginning in Au-
gust 1977, some 4,000 samples of plant
influents and effluents were taken in 21
industrial categories to gather data on
treatment processes. The analytical
work was centered on 114 organic com-
pounds and 15 inorganic substances
called Priority Pollutants.
The sampling and analysis effort con-
tinued through August 1980. In 1979, an
amendment to the Consent Decree di-
rected EPA, among other things, to de-
termine, by mass spectrum matching,
the compounds, in addition to the Prior-
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ity Pollutants, that might be present,
chemically confirm those compounds
of highest concentration and frequency
of occurrence, and attempt to identify
by other means significant components
not identified by spectrum matching.
Computerized spectrum matching of
the raw GO/MS data was performed by
the Athens Environmental Research
Laboratory (ERL). The chemical confir-
mation of matching results was pre-
sented in Analysis of Industrial Waste-
water for Organic Pollutants in Consent
Decree Survey Samples (EPA-600/4-83-
028).
The original computerized analysis
aimed at deconvoluting and identifying
all of the peaks of the chromatogram. A
number of compounds, however, could
not be identified by the computer but
were observed frequently in several in-
dustrial categories. The objective of the
second part of this program was to en-
deavor to identify these compounds or
at least arrive at a point where a struc-
ture could be postulated on the basis of
available information.
There were several steps in the
screening process used to select the ex-
tracts for analysis. Because a primary
criterion was frequency of occurrence
of a characteristic mass spectrum that
could not be identified, a compilation
was made of these mass spectra, or-
dered by frequency of occurrence. The
spectra were screened by the Project
Officer and the Principal Investigator,
both experienced mass spectros-
copists, a few identifications that ap-
peared obvious were made, and some
of the mass spectra were deleted from
consideration for further analytical ef-
fort.
Another major criterion for the selec-
tion of a mass spectrum for further
analytical effort was an obvious indica-
tion of the presence of halogen, that is,
the presence of the isotopic clusters
characteristic of the occurrence of halo-
gen. The ultimate and deciding criterion
was the availability of an extract in
which the unknown compound was
present in a concentration sufficient to
produce a mass spectrum of reasonable
quality. If several industrial effluent ex-
tracts containing the unknown mass
spectrum were available, the highest
priority was given to the extract in
which the component was present at
the highest concentration.
Experimental Procedures
Capillary gas chromatography with
accurate mass measurement was per-
formed under the same conditions as
those shown in Table 1 for the Finnigan
4021 GC/MS system. The accurate mass
measurements were performed on a
VG Micromass 7070HS, at a resolving
power of 2000, with a 5% valley. Ioniz-
ing energy was 70 eV, 200 jtA trap cur-
rent, a source temperature of 210°C,
and a transfer line temperature of
275°C. The scan speed was 700 msec/
decade, from m/z 550-20, with a 500
msec reset time. C2I4 was used as a sec-
ondary reference. Data were processed
on a VG 2035 data system.
Chemical ionization analyses were
performed in the pulsed positive/nega-
tive chemical ionization mode. The in-
strument used was a Finnigan 4500 GC/
MS/DS. Capillary columns and condi-
tions correspond to those used on the
Finnigan 4021 and reported in Table 1.
The reagent gas was methane, at a
source pressure of 0.7 torr and an
analyzer pressure of 2 x 10~5 torr. The
source temperature was 120°C, with an
electron energy of 100 eV and an emis-
sion current of 0.2 mA. The scan cycle
was 1 sec, with a scan range of 90-550
daltons. The multiplier was operated at
1300 V, with the amplifier at 10~7 A/V.
The Unacon 81 OA is a commercial in-
jection system that uses a preconcen-
tration technique to increase injection
volume and thereby decrease analytical
detection limits. In operation, the Una-
con removes solvent before injection of
analytes onto the chromatographic col-
umn. The Unacon 81 OA system was in-
Table 1. Instrumental Parameters
Column
Initial Temperature
Minutes Isothermal
Programming Rate
Maximum Temperature
Column Flow
Injector Temperature
Injection Mode
Column
Initial Temperature
Minutes Isothermal
Programming Rate
Maximum Temperature
Column Flow
Injector Temperature
Injector Mode
Transfer Line/Separator Oven
Ionization Energy
Emission Current
Mass Range
Scan Speed
Finnigan 3300
Gas Chromatography Parameters
Finnigan 4021
60 m OV-101 wide-bore thick film fused silica capillary.
30PC
6 minutes at 75°C
4°C/min
270PC
1.2mUminHe 1.6 mL/min He
250°C 270°C
Splitless for 60 sec, then Split/ess for 24 sec, then
-10:1 split 7.5:1 split
60 m Carbowax 20 M wide-bore thick film fused silica capillary.
3CPC
6 minutes at 75°C
fClmin
220°C
1.2 mL/min He
250PC
Splitless for 60 sec, then
-10:1 split
Mass Spectrometer Parameters
40-500 da/tons
2 sec/cycle
23CPC
Splitless for 24 sec, then
2.5.1 split
27CTC
70 eV
0.5mA
1 sec/cycle
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terfaced to a Varian 3700 gas
chromatograph using a fused silica
capillary column. The capillary column
was attached directly to the Unacon
810A's transfer line, which was wrap-
ped with heating tape and heated to
165°C to prevent condensation of the
less volatile organics during transfer.
The Unacon 81OA system was equipped
with a large-bore trap (6.35 mm O.D,
2.54 mm ID x 20.3 cm in length) contain-
ing Tenax GC« and glass beads and a
small-bore trap (6.35 mm ID, 0.9 mm ID
x 20.3 cm in length) also containing
Tenax and glass beads as sorbent mate-
rial.
A Nicolet 7199 Fourier transform in-
frared spectrometer was used with a
Model 7001A Michelson interferometer,
a high energy cooled glow bar source,
a heated 0.3 mm ID x 42 cm (3 cm3)
gold-plated light pipe, and a 4000 to 800
cm"1 range liquid nitrogen cooled Mer-
cury-Cadmium-Telluride detector, with
related optics. The entire system was
enclosed within a sealed nitrogen-
purged enclosure.
The spectrometer was controlled by
a Nicolet 1180 data system with 40K
solid state memory interfaced to the
spectrometer by a high precision 15 bit
analog-to-digital converter. Twenty-bit
spectral words were acquired and
stored on a Diablo 44B dual density,
dual disk memory system. Long-term
data storage was accomplished using a
Kennedy 9000 9-track, 800 bits-per-inch,
37 inches-per-second tape deck.
Results and Discussion
An analysis protocol developed by
the Athens ERL was adhered to in this
phase of the program. A flow chart of
the protocol is presented in Figure 1.
Progress was evaluated in relation to
the points in the flow chart labeled A, B,
etc.
Point A ("capillary GC/low resolution
EIMS analysis. Pertinent component
still observed?") was the starting point
for each industrial effluent extract.
Three outcomes were possible from
this analysis: 1) the component of in-
terest was observed (progress to points
B and C); 2) the component of interest
was not observed (analysis of the perti-
nent industrial effluent extract was
abandoned and another extract was
substituted, if available); or 3) a mass
spectrum only similar to (but not
exactly corresponding to) the compo-
nent of interest was observed (same
outcome as [2], above). At point B ("suf-
ficient GC separation?"), the GC separa-
tion did not prove crucial to the capabil-
ity of observing a mass spectrum for
the component of interest. Using com-
puterized background subtraction tech-
niques, it was possible to deconvolute
mass spectra even in the face of severe
coelution problems. Several extracts
(see point E) were subjected to cleanup
procedures, however, because of their
extreme complexity.
At point C ("Is the component still un-
identified?"), the answer was almost in-
variably that the component was still
unidentified. At point D ("Manual in-
terpretation of low resolution El mass
spectrum: tentative identification?"),
some structural features could be eluci-
I Tentatively Identified
Compound
M
Std. available at reasonable
cost within 4-6 wks?
No ,
YeS y
for purchase
or synthesis
I No
Anal, known amt. of std.
with cap GC/low res. EIMS.
MS & approx. rel. ret. time
of std match that of
sample component!'
[Stop]
Yes
Anal, appropriate extract
with cap GC/low res EIMS.
Pertinent component still
observed?
1
No
Yes
\Yes
P. O. decision to anal.
another extract
containing same
component.
No
\Sufficient GC separation?
TyTs <
I Unidentified component^
Component becomes
unidentified, P.O.
decision required.
.No
1
Add known amt of std. to
ext. & anal, with GC/MS
Coelution of std, & sample
component observed?
No
I Yes
Use diff. column or
reas. cleanup procedures
t. Yes
Sufficient separation?
Yes
Manual interp. (<4 hr.)
of low res. El spectrum.
Clean MS of appropriate
GC peak observed?
Yes
Estimate concentration;
classify as confirmed.
utner anal.
techniques
justified?
k
No
i No
'0
High
need
tuffici
— J
H
No
I Yes
Figure 1. Flow chart for Ab initio compound identification.
P.O. decision
required
Yes
(Sufficient clues to
tentatively identify?
Has this point been
reached previously?
f/Vo
[MW known?\
["/Vo
Perform CIMS analvsisiYesf
Successful? I
Est cost to this
point & cost to
continue. P.O
decision.
Perform
analysis
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dated but only rarely could a postula-
tion of a complete structure be made.
For all practical purposes, point F ("Add
standard and reanalyze. Coelution of
standard and sample component?")
was never reached. Only once was a
standard obtained commercially corres-
ponding to a postulated structure; in
this instance, elution times of standard
and component of interest did not cor-
respond. At point L ("Standard avail-
able at reasonable cost in 4-6 weeks?"),
the standards to correspond to tentative
structures were found to be unavailable
commercially. At point M ("Project Of-
ficer decision for purchase or synthe-
sis."), synthesis was considered at one
time when the postulated structure
seemed to be narrowed down to one
possible isomer, but the projected cost
of $2,000-3,000 for a single compound
was prohibitive, and no synthesis was
performed.
At point G ("Perform CIMS. Success-
ful?"), chemical ionization mass spec-
trometry (in both positive and negative
mode) was performed for a number of
samples. One of the primary instances
of application of the chemical ionization
technique was the occurrence of com-
ponents of interest that appeared to be
highly halogenated alkanes and al-
kenes. These compounds do not pro-
duce molecular ions (or any species
characteristic of molecular weight)
under electron ionization conditions.
The mass spectrometric literature does
not show instances of successful appli-
cation of chemical ionization tech-
niques to these types of compounds,
but an effort was made (in both positive
and negative mode) to see whether any
information could be obtained.
Point H ("High resolution CIMS
analysis needed?") proved to be crucial
in the decision-making process, and the
high resolution EIMS technique was
applied to most of the extracts. At-
tempts to use high resolution tech-
niques without the benefits of the pre-
ceding capillary separation were uni-
formly unsuccessful.
At point I ("Other analytical tech-
niques justified?"), a major decision
needs to be made. The criterion for de-
ciding whether other analytical tech-
niques are justified is a cost-benefit
analysis: relative to the cost in time and
dollars of performing the proposed
analysis, is the structural information to
be gained from the application of the
technique proportionate?
Results of the most successful at-
tempts at compound identification are
shown in Table 2. Structure postulation
was not made in most cases due to the
lack of functional group information
that might have been obtained from a
more sensitive GC/FT-IR analysis. For-
mulae were not postulated in cases
where no molecular ion could be pro-
duced by CIMS. No standards for any
of the postulated compounds were ob-
tainable commercially, and the expense
of custom synthesis was deemed too
great.
If the objective of the analytical proce-
dures is to confirm a structural postula-
tion by co-injection of a standard, five
conditions must be met:
1) micrograms of sample material
must be available for analytical
methods that can provide the
most definitive structural informa-
tion;
2) extensive resources must be avail-
able from the point of view of ac-
cessibility to major analytical in-
strumentation;
3) extensive financial resources must
be available in order to make the
most effective use of analytical
techniques that require major in-
strumentation. Few people are
sufficiently specialized in inter-
preting data from the wide range
of analytical techniques that must
be used;
4) a major financial commitment
could be required to obtain analyt-
ical standards that corresponds to
the structures postulated. Where it
is impossible to decide between
two or three possible isomers on
the basis of available spectral in-
formation, extensive custom syn-
thesis could be required;
5) an overall success rate is very
likely to be low despite effective
use of all available techniques. It
would be possible, for example, to
Table 2. Summary of Selected Analysis Results
Extract #
(Fraction) Industrial Category
584 (AC II
7367 (B/N)
Pulp and Paper
Amusements and Athletic
Instrumental
Number of Postulated Postulated Techniques Used
Occurrences Formula(s) Structure Successfully
9 CaH7O3CL None
CSH,,SO3CI
10 CgH,aN4O None
HRMS
HRMS
13230 (ACII
19436(ACI)
21258 (B/N)
5855 (ACI)
11338(ACI)
11536 (B/N)
18959 (ACI)
20250 (BN)
Goods
Mechanical Products
Plastics and Synthetics
Aluminum
CgH2oO3
CieHieO2
C1BH1(pN2
None
None
None
HRMS, Cl
HRMS
HRMS
Pesticides
Pesticides
Inorganic Chemicals
Pulp and Paper
Explosives
5
29
5
5
16
CJOH,202
None
None
None
CH3(CH2)2CH-
OH
None
HRMS, FT-IR
HRMS
HRMS, Cl
HRMS
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Table 2. (Continued)
Extract #
(Fraction) Industrial Category
2389 (ACI)
Inorganic Chemicals
Number of Postulated
Occurrences Formula(s)
6 CeH19CI3
Instrumental
Postulated Techniques Used
Structure Successfully
Cl HRMS
Cl
2390 (ACI)
5694 (B/N)
5849 (ACI)
5859 (ACI)
Inorganic Chemicals
Organic Chemicals
Pesticides
Pesticides
54
17
5
7
Series of
halo alky Is
C13HI3S02N
CSHION20S
C
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Joan T. Bursey is with Research Triangle Institute. Research Triangle Park, NC
27709.
Walter M. Shackelford is the EPA Project Officer (see below).
The complete report, entitled "Application of an Analysis Protocol to Identify
Organic Compounds Not Identified by Spectrum Matching" consists of two
parts:
Part 1: Text (Order No. PB 84-229 715; Cost 23.50. subject to change).
Part 2: Appendices (Order No. PB84-229 723; Cost 22.00. subject to change).
The above reports will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Research Laboratory
U.S. Environmental Protection Agency
Athens, GA 30613
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use S300
pi
o
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