EPA 660/2-74 076
August 1974
Environmental Protection Technology Series
Automated Analysis of Individual
Refractory Organics in Water
Polluted
Office of Research and Development
U.S. Environmental Protection Agency
Washinqton, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
U. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL
PROTECTION TECHNOLOGY ; series. This series
describes research performed to develop and
demonstrate instrumentation, equipment and
methodology to repair or prevent environmental
degradation from point and non-point sources of
pollution. This work provides the new or improved
technology required for the control and treatment
of pollution sources to meet environmental quality
standards.
EPA REVIEW NOTICE
This report has "been reviewed "by the Office of Research and
Development, EPA, and approved for publication. Approval
does not signify that the contents necessarily reflect the
views and policies of the Environmental Protection Agency, nor
does mention of trade names or commercial products constitute
endorsement or recommendation for use.
For sale by the Superintendent or Documents, U.S. Government Printing Office, Washington, B.C. 20402 - Price $1.78
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EPA-660/2-7^-076
August
AUTOMATED ANALYSIS OF INDIVIDUAL REFRACTORY ORGANICS
IN POLLUTED WATER
by
W. Wilson Pitt, Robert L. Jolley and Sidney Katz
Contract No. 14-12-833
Project 16 ACG 03
Program Element 1BA027
Project Officer
A. Wayne Garrison
Southeast Environmental Research Laboratory
Athens, Georgia 30601
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
Washington, D.G. 20460
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ABSTRACT
High-resolution anion-exchange chromatography has been applied to
the problem of analyzing for the residual organic compounds present in
municipal sewage plant effluents at microgram-per-liter levels. Two
different chromatographic systems have been used: one capable of
analyzing for compounds which are uv-absorbing and/or oxidizable
with sulfatoceric acid, and the other for carbohydrate analysis.
It was necessary to concentrate the samples of effluent 50~ to
3000-fold prior to their analysis on the chromatographs. A two-step
procedure, consisting of 10- to ^0-foid concentration by vacuum
evaporation, followed by freeze-drying to the desired final volume,
was developed. Loss of noncarbonate carbon with this procedure was
generally less than 15$.
Samples of sewage plant effluents were concentrated and analyzed.
Techniques of positively identifying compounds present in sewage were
established. Using these techniques, 56 organic compounds have been
identified in samples of effluent from a primary treatment plant and
13 organic compounds have been identified in samples of effluent from
a secondary treatment plant.
The concentration, chromatographic, and identification procedures
were also applied to the analysis of chlorinated effluent from primary
and secondary sewage plants. More than 60 chromatographic peaks con-
taining chlorine have been found, and specific chlorinated compounds
were tentatively identified by cochromatography and quantified at the
0.5- to 4-ug/liter level.
A detector system for liquid chromatography based on cerate
oxidimetry was adapted as a rapid, sensitive continuous monitor for
measuring the COD of water. Analysis of COD levels as low as 100
micrograms-per-liter can be obtained in a few minutes by using per-
chloratoceric acid as the oxidant and measuring the resulting Ce(lll)
fluorometrieally.
ii
-------
An experimental study of the effects of column geometry and operating
parameters on chromatographic resolution was made to permit optimization
of the ion exchange resin systems.
One high-resolution, ion exchange chromatograph (UV-Analyzer) was
constructed for the Advanced Waste Treatment Research Laboratory and
another for the Southeast Environmental Research Laboratory. These
instruments are being used in the analysis of treated sewage effluents
and other polluted waters.
This report was submitted in fulfillment of Project Number 16ACG 03,
Contract Number 14-12-833, by the Oak Ridge National Laboratory under
the sponsorship of the Environmental Protection Agency. Work was completed
as of May 1974.
iii
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CONTENTS
Abstract
List of Figures
List of Tables
Acknowledgement s
Sections
I
II
III
IV^
V
VI
VII
VIII
IX
X
Conclusions
Recommendations
Introduction
Methods and Instrumental Development
Identification of Stable Organic Pollutants
Effects of Chlorination of Sewage Plant Effluents
Cooperative Efforts with EPA Laboratories
Discussion
References
Publications
Page
ii
v
vii
ix
1
2
3
5
U2
69
89
93
9^
97
iv
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FIGURES
No. Page
1 Procedure for Concentrating Sewage Plant
Effluent Samples 7
2 Schematic Diagram of Vacuum Distillation System 8
3 High-Resolution Anion Exchange Chromatograph for UV-
Absorbing Compounds(UV-Analyzer) 10
k High-Resolution Anion Exchange Chromatograph for
Carbohydrates (Carbohydrate Analyzer) 11
5 Reference UV-Analyzer Chromatogram of 1000X
Concentrate of Primary Sewage Treatment
Plant Effluent 12
6 Reference UV-Analyzer Chromatogram of 2000X Concentrate
of Secondary Sewage Treatment Plant Effluent 13
7 Carbohydrate Analyzer Chromatograms of Primary and
Secondary Sewage Treatment Plant Effluents, Human
Urine, and Sugar Standards 15
8 Schematic Diagram of Dual-Column, High Resolution,
Liquid Chromatograph for Analyzing Two Polluted
Water Samples Simultaneously 16
9 Dual-Column Sample Injection Valve 17
10 Electrical Heating System for Chromatographic Columns 19
11 Dual-Column Chromatograms of Two Identical Samples 20
12 Dependence of Chromatographic Resolution on Column
Length and Linear Velocity of Eluent 22
13 Schematic of Cerate Oxidative Monitor 26
1^ Schematic of Flow Fluorometer 27
15 Exploded View of Flow Fluorometer Body 28
16 Calibration Curve for* Flow Fluorometer 30
17 Chromatogram of Primary Sewage Plant Effluent
Analyzed by the UV-Analyzer with a Cerate
Oxidative Monitor 3!
18 Chromatogram of Secondary Sewage Plant Effluent
Analyzed by the UV-Analyzer with a Cerate
Oxidative Monitor 32
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No- page
19 Cerium Fluorescence Oxidimetry 35
20 Schematic of Continuous Chemical Oxygen
Demand Analyzer 37
21 Recorder Trace from Continuous COD Analyzer 39
22 Ultraviolet Absorption Spectra of Unknown
Sample Taken at Different pH Values ^9
23 Ultraviolet Absorption Spectra of Guanosine
Taken at Different pH Values 50
2k Gas Chromatograms of TMS Derivatives of Reference
Compound (Guanosine) and Liquid Chromatographic
Fraction from Primary Sewage Treatment Plant
Effluent 52
25 Mass Spectra of TMS Derivatives of the Reference
Compound (Guanosine) and Liquid Chromatographic
Fraction from Primary Sewage Treatment Plant
Effluent 53
26 UV-Analyzer Chromatograms of Primary Sewage Treat-
ment Plant Effluent Chlorinated with Different
Amounts of Calcium Hypochlorite 71
27 Schematic of Chlorine Generator and Sample
Chlorinator 73
28 Schematic of Apparatus Used for Concentration of
Radioactive Samples 7^
29 Dual-Column UV-Analyzer Chromatograms of Chlorinated
Primary Sewage Treatment Plant Effluent 76
30 Dual-Column UV-Analyzer Chromatograms of Chlorinated
Effluent from a Secondary Sewage Treatment Plant 79
31 Chromatograms of Secondary Sewage Treatment Plant
Effluent Chlorinated with Hypochlorite Solution
for 15-, 45-, and 90-min Reaction Times 80
32 Chromatograms of UV-Absorbing Constituents Developed
on the Southeast Environmental Research
Laboratory UV-Analyzer
a) 0.25 ml of Reference Urine (URS-IV)
b) 0.25 ml of Primary Effluent (lOOOX) 91
vi
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TABLES
No.
1 Resolution of Coupled Anion-Cation Columns
During Chromatography of ORESP Secondary
Sewage Effluent Samples 25
2 Oxidation Potential Available with Various Oxidants
Used in COD Analysis 34
3 Oxidation of Organic Compounds and Polluted Waters
by Perchloratoceric Acid COD Method 40
4 Physical Characteristics and Operating Parameters
for the Analytical and Preparative Anion
Exchange Columns 44
5 Guanosine Identification Data 51
4'
6 Description of Samples 54
7 Status of Sewage Plant Effluent Samples (5/1/74) 55
8 Identification of Molecular Constituents in 1000-
to ^000-Fold Concentrates of Primary Domestic
Sewage 57
9 Data on Unknown Compounds Found in Unchlorinated
Primary Effluent (SPJ-l) From the Oak Ridge
East Sewage Plant 60
10 Data on Unknown Compounds Found in Chlorinated
Sewage Plant Effluent (SPJ-3) From the Oak
Ridge West Sewage Plant 6l
11 Data on Unknown Compounds Found in Chlorinated
Effluent (SPJ-8) from the Oak Ridge West
Sewage Plant 62
12 Data on Unknown Compounds Found in SPJ-11,
Composite of SPJ-3 and SPJ-8 66
13 Identification of Molecular Constituents in
1000- and 2000-fold Concentrates of Secondary
Domestic Sewage Effluent 67
14 Data on Unknown Compounds Found in Secondary
Effluent (SPJ-9 and SPL-l) From a Domestic Sewage
Treatment Plant 68
vii
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Ncr. Page
15 Stable Chlorine-Containing Organic Constituents
in Chlorinated Effluents from Domestic
Sanitary Sewage Treatment Plants.(Concentrations
of the Constituents, ng Cl/liter of original
effluent) 82
16 Tentative Identifications and Concentrations of
Chlorine-Containing Constituents in Chlorinated
Effluents 86
17 Percentage Chlorination Yield of Chlorine-Containing
Constituents with Respect to Reaction Time for
the Chlorination of Effluents 88
viii
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ACKNOWLEDGMENTS
The suggestion that high-resolution chromatography might be useful
in water pollution studies emerged from a conversation between Dr. A. A.
Rosen of the Division of Field Investigation, Environmental Protection
Agency, Cincinnati, Ohio, and Dr. C. D. Scott of the Oak Ridge National
Laboratory. Dr. Rosen served as the original Project Officer and was
intimately involved with the maturation of the project. Dr. Scott, as
Chief of the Experimental Engineering Section of the Chemical Technology
Division at ORNL, has been in close contact with the developmental effort.
The responsibility for the principal development effort and report prepara-
tion has been shared by Dr. R. L. Jolley, Dr. W. W. Pitt, Jr., and
Dr. S. Katz.
The assistance and encouragement of Mr. W. T. Donaldson, and
Dr. A. W. Garrison, Southeast Environmental Research Laboratory, Environ-
mental Protection Agency, as Project Officers have been particularly
gratifying.
The cooperation and continued interest of Mr. Charles Mashni,
Advanced Waste Treatment Research Laboratory, Environmental Protection
Agency, throughout the program, particularly during the time he served
as interim Project Officer, have been most welcome.
Samples from the East and West Sewage Treatment Plants, Oak Ridge,
Tennessee, were collected in cooperation with Mr. T. C. Stephens, Mr. J.
Robinson, Jr., and Mr. Henry Sturgill operating under the supervision
of Mr. 0. K. Rickman, Director of Public Works, City of Oak Ridge.
The four-place vacuum distillation equipment was made available
by Dr. W. Davis, Jr.
The numerous chromatographic analyses on the UV-Analyzers and
the carbohydrate analyzer were made by Mr. G. Jones, Mr. R. C.
Lovelace, and Mr. J. E. Thompson.
The support of the Chemical Identification Group working under
Dr. J. E. Mrochek, and including Mr. S. R. Dinsmore and Dr. W. T.
Rainey, contributed greatly to the success of the program.
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SECTION I
CONCLUSIONS
1. Over 100 refractory organic compounds can be present in effluents
from municipal sewage treatment plants at microgram-per-liter levels;
some of these refractory compounds are chlorinated under conditions
existing when effluents from sewage treatment plants are chlorinated.
2. High-resolution anion exchange chromatography provides a reliable
and useful tool for determining refractory organic compounds present
at low levels in sewage effluents and various other polluted waters.
3« In. addition to uv-absorbing compounds, numerous other compounds
can be detected by sulfatoceric acid oxidimetry.
4. The detection of sources of pollution, the testing of the effective-
ness of sewage treatment steps, including possible tertiary steps, and
the determination of the ultimate disposition of pollutants are obvious
end uses of the high-resolution anion exchange chromatographic systems.
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SECTION II
RECOMMENDATIONS
This program has been limited to the adaptation of existing
analytical systems for use in the determination of refractory organic
compounds in polluted waters with a minimum of instrumental develop-
ment. The scope of the program did not include exploiting the
capabilities of the analyzers for detecting sources of pollution,
testing the effectiveness of sewage treatment plants, or determining
the ultimate fate of pollutants; nor did it include positive identifi=
cation of all the separated organic constituents.
It is recommended that the use of high-resolution liquid chromato-
graphs with uv photometers and cerate oxidative monitors for determining
refractory organic compounds in industrial waters and other polluted
waters be significantly expanded. This can be accomplished either by
fabricating additional UV-Analyzers as described herein or by modifying
commercially available high-pressure chromatographs• A vigorous effort
should be continued to determine the identities of as many as possible
of the residual stable organic compounds being discharged to surface
waters. Also, potential hazards of these compounds, particularly those
which are chlorinated, should be evaluated. High-resolution analyzers
have been developed to the point that they could be used by appropriate
agencies to determine sources of pollution, the effectiveness of
sewage treatments, and the fate of organic pollutants — all on a
molecular level. This effort should be closely coordinated with the
analytical development program to take advantage of improvements as
they are made and to "feed back" information relating to problem areas
that would lead to necessary modifications of the instruments.
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SECTION III
INTRODUCTION
The continuing discharge of stable organic compounds in sewage
effluents to surface waters and the buildup of organic constituents
1 2
by recycle constitute a serious threat to our water quality. '
Furthermore, as such water contamination becomes excessive, additional
processing steps may become necessary to permit reuse of the water.
Because present analytical techniques for sewage effluents are non-
specific and often indirect, they are not adequate for use in
evaluating new developments of advanced treatment processes. These
analytical techniques do not provide sufficient information to
determine the chemical forms or concentrations of the stable organic
compounds that resist degradation. Thus, we do not know the effective-
ness of various treatment steps for specific refractory compounds;
nor do we know what harmful effects might result if these refractory
compounds continue to build up in the water supply.
In an effort to better define the pollution problem, this program
to determine the specific organic compounds present in sewage plant
effluents was undertaken. An important step in providing the analytical
information to answer pertinent questions and permit rational develop-
ment of additional sewage treatment steps has been the demonstration
that the high-resolution anion exchange analytical system can provide
reliable measurement of refractory organic compounds at microgram-
per-liter levels in sewage effluents and other polluted waters. In
3 4
the first year under this contract, we demonstrated that at least
50 to 100 specific refractory organic compounds are present in
effluents from municipal sewage treatment plants at microgram-per-
liter levels and that many more refractory compounds are present
at higher concentrations in effluents from industrial sewage treat-
ment plants.
It was demonstrated that previously developed automated, high-
resolution liquid chromatographs would be useful in the (a) detection
-------
of sources of pollution, (b ) testing of the effectiveness of sewage
treatment steps including possible tertiary steps, and (c) determination
of the ultimate disposition of pollutants. Subsequently, emphasis of
this program has been directed toward the exploitation of this type
of instrumentation, primarily by identifying specific compounds found
in the plant effluents but also by improving the chromatographic
separation system.
A major effort has been to define more clearly the magnitude of
the pollution threat by identifying and quantifying the specific stable
organic compounds that are difficult to degrade during common sewage
treatment steps. This has resulted in establishing the identities
of 56 of the organic compounds detected in primary sewage plant
effluents and 13 of the separated organic compounds in secondary
sewage plant effluents. An effort has also been made to determine
whether the chlorination of sewage effluents produces undesirable
levels of stable chlorinated organic compounds. Additionally, several
detection systems for increasing the sensitivity and detection capability
of the existing high-resolution chromatographs were evaluated. One
detector was adapted for use as a continuous oxygen demand (COD)
analyzer that is several times more sensitive than any presently
available.
In the interest of more closely coordinating the direction and
emphasis of the program with other activities in EPA Laboratories,
several informal meetings of the contributing personnel were held.
The participants at the meetings included personnel from the Southeast
Environmental Research Laboratory and the Advanced Waste Treatment
Research Laboratory.
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SECTION IV
METHODS AND INSTRUMENTAL DEVELOPMENT
The primary developmental effort of this program has been directed
toward adaptation of existing high-resolution ion exchange chromato-
graphs to the analysis of individual refractory organic compounds in
effluents from sewage treatment plants. To achieve this objective,
it was necessary to develop a technique to easily and reliably con-
centrate sewage plant effluent samples by as much as several thousand-
fold with little loss of noncarbonate carbon and to modify and improve
the existing UV-Analyzer and carbohydrate analyzer. In addition, an
oxidative detector which was developed as a liquid chromatograph
monitor was modified for use as a very sensitive continuous COD
monitor.
CONCENTRATION OF SEWAGE SAMPLES PRIOR TO ANALYSIS
Since the lower limit of detection for the UV-Analyzer is
100 ug/liter to 100 mg/liter, "depending on the ultraviolet absorp-
tivity of the individual compound, and the concentrations of specific
contaminants in effluent samples may be 10 ug/liter or less, con-
centration of sewage effluents by factors up to ^000 may l>e necessary
prior to analysis. As shown in later sections, the concentration
factor depends on the history of the effluent. Concentration methods '
such as freezing, extraction, adsorption, and low-temperature distil-
lation were considered. Freezing is inadequately understood for the
large number of compounds of interest and would require multiple
stages to achieve the concentration required in most cases. Extraction
^
and adsorption may not quantitatively concentrate all compounds of
interest. Therefore, the method of low-temperature distillation
appears to be the most convenient and should provide adequate recovery
of stable, nonvolatile organic compounds.
Low-temperature distillation may be carried out by freeze-drying,
rotary evaporation, or vacuum distillation. Considerations relative
-------
to the choice of equipment are: the desirability of maintaining the
sample at a low temperature to avoid decomposition, the necessity of
collecting the solids that separate during the concentration step in
order to redissolve coprecipitated organic compounds, and the need to
reduce the volume of sample from several liters to a few milliliters
in a reasonable time. Although freeze-drying provides maximum sample
integrity and simplified collection of separated solids, it is not
well suited to the reduction of volumes in excess of 200 ml. Rotary
evaporation is also unwieldy for large volumes; in addition, it
exposes the sample to temperatures higher than ambient and leaves
the precipitate distributed over a large surface. Vacuum distil-
lation appears to be the best method in that it is more rapid than
either of the other distillation methods, the temperature of the
sample is maintained at or below ambient, and the precipitated solids
are well concentrated; however, reduction to a final volume less than
50 ml is difficult in existing equipment. Based on these considerations
7
and the availability of a vacuum still, a two-step concentration
(Fig. 1) procedure was adopted. First, a concentration of 10- to
30-fold is effected in the vacuum still; then the resulting volume
(150 ml) of concentrate is further reduced by freeze-drying.
Normally, a volume of several liters of waste effluent is reduced
to a few milliliters to provide for a working sample and a spare sample,
and to allow for minor losses. In the first step, the effluent is
filtered through a 0.h-^-[aa. membrane to remove suspended matter; only
negligible uv-absorbing material is lost in this separation. The
volume of the filtrate is then reduced in the vacuum still (Fig. 2)
to about 150 ml of liquid, plus some separated solids. The concentrated
liquid, the solids, and a rinse solution are transferred to the freeze-
dryer, where the final reduction in volume is made. Water and either
acetic acid (for the UV-Analyzer) or borate buffer solution (for the
carbohydrate analyzer) are added to attain the desired liquid volume
and to adjust the pH to ~4-5 for the UV-Analyzer or to 8-5 for the
carbohydrate analyzer. Finally, the resulting sample is well mixed,
and the solids are separated by centrifugation.
6
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ORNL DWG. 74-1971 Rl
SAMPLE FOR
DOC+
SEWAGE TREATMENT PLANT
EFFLUENT SAMPLE
10-100 LITERS
1
| 045 jt FILTER]
DOC= DISSOLVED
ORGANIC CARBON
10-100 LITERS
LOW-TEMP
EVAPORATOR
< 50/j.
PRESSURE
200 mfl
FREEZE
AT
-60°C
AND
DRY
< I O/i.
DISSOLVE IN ACETIC ACID
FREEZE
AT •
- 60°C
AND
DRY
DISSOLVE IN DILUTE ACETATE
BUFFER AND CENTRIFUGE
SAMPLE FOR
DOC
TO CHROMATOGRAPH
Fig. 1. Procedure for Concentrating Sewage Plant Effluent Samples.
7
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ORNL-DWG 70-12480 RI
MMbtt'w.VE
DOM* IT.ASK-
e xo
icoouxr surra
OMPCKStTE
RCSEHVOR
TtWOUTUIIC
Fig. 2. Schematic Diagram of Vacuum Distillation System.
8
-------
The recovery of the noncarbonate organic compounds in the final
concentrate appears to be satisfactory (greater than £&%), as determined
by carbon analyses of the effluent and the separated phases of the
final concentrates.
HIGH-RESOLUTION ANALYZERS
Because of their sensitivity and capability for detecting and
quantifying many individual organic compounds in complex aqueous
8-12
samples, two high-resolution anion exchange chromatographs have
been adapted for use in analyzing for the molecular components in
sewage plant effluents and other water supplies. Each of these
systems, the UV-Analyzer for ultraviolet-absorbing compounds (Fig. 3)
and the carbohydrate analyzer (Fig. 4), consists primarily of a heated,
high-pressure, anion exchange column; a sample injection valve; a
concentration-gradient generating and pumping system; a two-wavelength
dual-beam photometer; and a strip-chart recorder. The ion-exchange
column for each system is a 150-cm length of type 316 seamless stainless-
steel tubing (0.22 to 0.62 cm ID) packed with strongly basic anion
exchange resin. A 0.05- to 2.5-ml sample (the volume depending on the
inside diameter of the ion exchange column and the nature of the sample)
is applied to the column by a 6-port injection valve mounted as near
to the top of the column as possible in order to minimize peak
broadening.
On the UV-Analyzer the chromatograms are developed by eluting the
sample constituents with an ammonium acetate--acetic acid buffer solu-
tion (pH 4.4) whose acetate concentration gradually increases from
0.015 to 6.0 M. The eluent is pumped through the ion exchange column
-2-1
at the rate of about 250 ml cm - hr with a pressure drop of 100 to
200 atm. The absorbances of the column effluent at 254 and 280 nm,
referred (at the same wavelengths) to the stream entering the column,
are monitored by a two-wavelength, dual-beam flow photometer and
recorded on a strip chart. Typical UV-Analyzer chromatograms of
municipal sewage treatment plant effluent samples are shown in Fig. 5
(primary effluent) and Fig. 6 (secondary effluent). The. compounds
-------
ORNL DWG 69-1243 R3
RECORDING
POTENTIOMETER
9 CHAMBER GRADIENT BOX
•CHROMATOGRAM
TO FRACTION COLLECTOR
"OR TO DRAIN
PRESSURE
RELIEF VALVE
6-PORT SAMPLE
INJECTION VALVE
HIGH-PRESSURE
CHROMATOGRAPHIC
COLUMN
HIGH-PRESSURE
PUMP
CONSTANT-
TEMPERATURE*
CIRCULATOR
Fig. 3. High-Resolution Anion Exchange Chromatograph for TJV-Absorblng
Compounds (UV-Analyzer).
10
-------
ORNL DWG 70-9001
SAMPLE
IN
9-CHAMBER
GRADIENT BOX
SIX PORT SAMPLE
INJECTION VALVE
TO
WASTE
SAMPLE
LOOP
PRESSURE
GAUGE
in
FOLDED, HIGH PRESSURE
CHROMATOGRAPHIC
COLUMN AT 55°C
PHENOL SOLUTION
H2S04
ORNL
LORIMETER
REACTION ZONE
OVERFLOW
WASTE
HIGH
PRESSURE
PUMP
Fig. If. High-Resolution Anion Exchange Chromatograph for Carbohydrates
(Carbohydrate Analyzer).
11
-------
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ELUTION TIMC.ftr, O I
M
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CUmOH VOL..M 0
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NOTE:
» (LUTION VOLUME) FOR ANALYTICAL CHROMATOOIUPHS
WITH 0,45-c. 1.0. t 190-M COLUMN
LO
.«
t
' .4
t
» _
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— i1 i- • i — i i i i i i i i 'i i ii r • i - i • • i • • i n 'i •• T • -\ —i "-"i ~T - i T 1 —
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0,»4-»«-IO > Otttm STAINLESS STEIL KITH «-ll f OIAU. AMINEX A-tT REIIN i
TEMPERATURE PftOaRAU, AMBIENT TO BC'C AT 11.6 kr i ELUENT tRAOIEMT INCREAtW*
LINEARLY IN CONCENTRATION FROM 0.015 M TO • 0 M AMMONIUM ACETATE, »H 4.4i
tLUENT FLOW RATE tlnl/kr , COLUMN PREWUKE IWO M*-
I i i 1 1 I 1 1 1 i i 1 I 1 I 1 I I 1 i 1 I I I i i i i |
-
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1
ILUTION TlME.kr
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SrMJ>404l4S43
4S4*
-------
shown on the chromatograms are those that have been identified by
the techniques described in Section V-
The carbohydrate analyzer utilizes a sodium borate--boric acid
buffer (pH 8-9) as the eluent. This eluent, whose boron concentration
increases from 0.085 to 0.8^5 M during an analysis, is pumped through
-2 -1
the column at the rate of about 350 ml cm hr , also with a pressure
drop of 100 to 200 atm. The separated carbohydrate compounds are
monitored with a phenol — sulfuric acid color development system which
mixes the column effluent with 5$ phenol and concentrated sulfuric
acid and subsequently measures the absorbance, at if 80 and ^90 nm,
of the reaction mixture after reaction for 3 min at 100° C« The more-
13
sensitive Mark III Carbohydrate Analyzer, which was originally
developed for physiologic fluids, has been applied to municipal primary
and secondary effluents concentrated 500-fold by the technique described
earlier. The resulting chromatograms contained 38 and 19 peaks, respec-
tively, and are compared to those obtained for urine and reference
sugar standard samples in Fig. 7- &1 the first 1-1/2 hr of elution,
the primary and secondary effluent chromatograms contain several large
peaks, similar to those in the urine chromatogram, that are probably
due to complex sugars. Among the simple sugars appearing in the
primary and secondary effluent samples are sucrose, raffinosej
allulose, mannose, fructose, xylose or sorbose, and glucose. These
are tentative identifications based on elution positions, but several
of these sugars have been definitely identified in UV-Analyzer fractions
DUAL-COLUMN UV-ANALYZER
A dual -column UV-Analyzer, which has two columns operating in
parallel, has been demonstrated to be a powerful tool for comparing
the chromatograms of related samples . The system, although primarily
12
developed for biomedical applications, has been adapted for use in
the water pollution effort. The final design (shown schematically
in Fig. 8) was based on the Mark II -A UV-Analyzer design but incor-
porates two recent improvements and a dual -column sample injection
valve (Fig. 9)- This valve, which essentially consists of two,
-------
ORNL DWG 71-3828
OJ043
OjOU
w
w
3 O.OM
o
••
9 0.013
OJDOS
SAMPLE: tee OF MUNICIPAL PRIMARY EFFLUENT CONCENTRATED 500-FOLD
SECONDARY EFFLUENT
4SO >•
4*0 ••
t
HOURS
10 II It IS
SAMPLE: Ice OF MUNICIPAL SECONDARY EFFLUENT CONCENTRATED 300-FOLD
19 I* IT l>
SAMPLE: lee OF NORMAL URINE
• » 10 II B II M n 16
HOURS
SAMPLE: lee CONTAINING O.05 MICROMOLES OF EACH SUGAR
Fig. 7. Carbohydrate Analyzer Chromatograms. of Primary and Secondary
Sewage Treatment Plant. Effluents, Human Urine, and Sugar Standards.
-------
ORNL DWG 71-3797 R 3
SAMPLES IN
CONCENTRATED
BUFFER
SAMPLE
LOOPS
HIGH- PRESSURE
CHROMATOGRAPH
COLUMNS
DILUTE Y
BUFFER
12 PORT DUAL COLUMN
SAMPLE INJECTION VALVE
-COLUMN
JACKET
TEMPERATURE
INDICATOR
CONTROLLER
MIXING
CHAMBER "»/-h
H
CTS
PRESSURE
GAGE
PRESSURE
RELIEF
VALVE
-RECORDING
POTENTIOMETER
-CHROMATOGRAMS
HIGH
PRESSURE
PUMP
Fig. 8. Schematic Diagram of Dual-Column, High Resolution, Liquid Chromatograph for Analyzing
Two Polluted Water Samples Simultaneously.
-------
ORNL-DWG 71-9486A
MOVABLE
METAL WASHER
NUT FOR TIGHTENING
PLASTIC SLEEVE
INNER PLASTIC
SLEEVE
TAPERED
METAL SHAFT
METAL
RESTRAINT
HANDLE FOR
ROTATING SHAFT
Fig. 9- Dual-Column Sample Injection Valve.
17
-------
ganged 6-port sample injection valves, provides simultaneous injection
of two different samples onto two columns; the same design principle
could be used to construct multicolumn injection valves. The improve-
ments consisted of: (a) replacement of the constant-temperature
circulator with a more reliable thermistor-controlled, resistance
heater wound around the outside of the column jacket (Fig. 10); and
(b) replacement of the metal gradient generator with a clear-plastic
device which produces a more gentle transition into the gradient.
To convert the Mark II Analyzer to a dual-column system, the
single 0.62 cm x 150 cm ion exchange column in the Mark II unit
was replaced initially with two 0.^5 cm x 150 cm columns, thep. later
with two 0.30 cm x 150 cm columns. Each of the smaller pair of columns
contained one-fourth the volume of the single large column; thus the
amounts of resin and eluent required for the two columns are only one-
half of the amounts needed for the single column in the Mark II
prototype. The effluent from each column was monitored with a two-
wavelength, dual-beam photometer, and the absorbances at 25^ and
280 nm were recorded on a single strip chart.
The operating procedure for the dual-column UV-Analyzer is
identical with that used for the Mark II UV-Analyzer except that, in
the dual-column system, two samples are loaded into the 12-port sample
injection valve and are simultaneously injected, one onto each chromato-
graphic column.
The dual-column Analyzer yields chromatograms that are almost
superimposable when identical samples are injected onto each column
(Fig. 11); however, the patterns strikingly show even small dissimi-
larities when different samples are injected. The dual-column analyzer
is particularly useful for comparing sewage samples before and after
treatment. The results obtained from routine use of the dual-column
UV-Analyzer have demonstrated the usefulness of the multicolumn mode
of operation. In addition to the higher sample capacity, the capability
for comparing samples simultaneously represents a significant improve-
ment over single-column operation. With relatively minor modification,
the existing Mark II UV-Analyzers can be adapted to dual-column operation.
18
-------
ORNL DWG 72-I4I8RI
JACKET—*
FILLING
AND
CONVECTION
LEG
-~»
<
<
<
<
<
<
<
<
^
^--""
•^•^
^^ — -
^5
..
^**— ^^
^,
,
ii
i
•i
f
^
«
^
^COLUMNS
CHEATING JACKET (FILLED WITH
S ETHYLENE GLYCOL)
,»~500 W RESISTANCE
UCHTCO _
MtATER .^
xCHROMEL-ALUMEL ^^
j THERMOCOUPLE ^^^
TO- Pl '^S'
^_j-|_> -^
1 .
IT D
TJ-LT-
^THERMISTOR
rro ^-^5
RESET
T!
HIGH
115
Vac
ac POWER
CONTROL-
LER
;
1
115 Vac
POWER
TEMPERATURE
INDICATOR
a
HIGH-TEMPERATURE
CUTOFF
TEMPERATURE
ADJUST
POTENTIOMETER
u u
Fig. 10. Electrical Heating System for Chromatographic Columns.
-------
CO
o
80
TlME(hre)
6 7
OftNL DWG 71-2891 Rl
II 12 _. B
O 12
24 36 48 60 72 64 96 106 120 132 CM B6 168 180 192 204 216 22B 246 264 282 300 318 336 354 372 390
ELUTION VOLUME (ml)
TIME(hre)
I? 20
COLUMNS. 0.45.ISOem/UilHEX*-Zr«C5"«
TOFCMTIfflEi AHKENT— CO-CAT>ln.
«Mn.ri
-------
COLUMN GEOMETRY AND OPERATING PARAMETER STUDIES
1 An experimental study of the effects of column geometry and
•
operating parameters on chromatographic resolution has been made in
cooperation with the Body Fluids Analyses Program with the goal of
->
determining the best combination of column dimensions and operating
parameters. According to theoretical considerations, faster analyses
and higher resolution are conflicting requirements for a given chromato-
graphic system. One can define the resolution, R , of peaks 1 and 2
s
by:
Rs S &2 ~
where v^ and v = elution volumes of peaks 1 and 2,
o = average standard deviation of the peaks in volume units
Ik
Van Deemter's differential model for chromatography results in:
, ce -L ft
R* -n •
but, on the other hand, analysis time, t , will have the following
cl
proport iona lity :
r"
*.-? ' •,
o
where L = column length,
U = linear velocity of eluent.
The experimental results confirmed the first proportionality (Fig. 12).
One fact should be borne in mind here; that is, although higher values
for resolution (as defined above) are generally desired, each system
has an upper limit above which any increase would be superfluous and,
in fact, would only increase separation time. For example, two adjacent
peaks are almost completely separated if their R is greater than about
S
-.0. Of course, in the separation of a very complex mixture such as
sewage plant effluent, it is always possible that additional compounds
21
-------
ORNLDWG 7I-3869RI
to
ro
a
CT
1.
O
s?
§
§
8.0
7JO
6.0
5.0
3 4-0
O
(O
U
QL
3.0-
2.0
1.0
10
30
40
50
6O
[LENGTH/LINEAR VELOCITY]
7O
1/2
80
90
100
-------
might be eluted between any two peaks. (indeed, it is known that other
compounds elute between the two pairs shown in Fig. 12). However, one
can reach a point of diminishing returns when trying to improve res-
olution. It was the purpose of this investigation to determine where
that point is. From plots such as the one in Fig. 12, it is possible
to establish the minimum time required to separate two given compounds.
This is done by multiplying the square of the abscissa at R = 1.0 by
s
the ratio of the elution volume to the column geometric volume; that
is,
t
min
'L_\ 1/2
2
x
AL
Rs=1.0
where A = cross -sectional area of the column. Thus, the separation of
pseudouridine and uracil (Fig. 12) would require a minimum time of
(10)2 x = 167 sec.
j • (
Considering the above data and typical sewage effluent chroma tograms, it
became apparent that a shorter column operated at a higher linear velocity
could be used effectively. Hence, a O.V?-cm-diam by 50-cm-long column
was fabricated and tested at a linear velocity of 0.1 cm/sec. The
results showed that sewage effluent samples would be analyzed in 6 to
8 hr with little, if any, loss of resolution. This column was then
sent to the AWTR Laboratory for incorporation in their UV -Analyzer.
CATION-ANION COLUMNS IN SERIES
A coupled anion-cation exchange column system was devised which
vastly improves the resolution of the UV -Analyzer. The improved resolution
results from a greater separation between the compounds that elute early
from the standard ani on -exchange column. In order to optimize this
coup led -column system for analysis of sewage samples, a sample of ORESP
secondary effluent was analyzed using three different combinations of
23
-------
0.^5-cm-ID columns whose lengths totaled 100 cm. The results shown
in Table 1 indicate that equal lengths of cation and anion exchange
columns provide the best resolution.
OXIDATIVE DETECTOR
An oxidative system, which has been under development in the Body
Fluids Analyses Program for use in monitoring the eluates from liquid
chromatographs (Fig. 13 ).> appeared to have potential as a monitor for
dissolved aqueous pollutants. This system relies on the reduction of
tetravalent cerium reagent to fluorescent, trivalent cerium by compounds
in the column effluent and is thus capable of monitoring many non-uv-
absorbing compounds. Although its primary application has been in
the analysis of samples of human body fluids, it has also been used
to analyze several sewage plant effluent samples for oxidizable compounds.
The resulting chromatograms showed that this detector has a high potential
for ion-exchange chromatography of polluted waters. One of these detectors
was also installed on a small (~ 1$) side stream of the preparative col-
umn effluents, where it acted as a monitor for those fractions which
contained oxidizable compounds.
When used as a column monitor in conjunction with a uv photometer,
the cerate fluorescence monitor is placed immediately downstream from
the photometer. The column effluent, after passing through the photo-
meter, is continuously combined with an equal volumetric flow of the
reagent [2.5 x 10 N Ce(iv) in h N sulfuric acid]-in a small glass
jet mixer. The reaction mixture then flows, with a 10-min residence
time, through a coiled tube reactor immersed in boiling water. This
17
stream passes through a flow fluorometer, ' shown schematically in
Fig. 14. It consists of a fluorometer body, an electronic chassis,
and a high-voltage power supply. The fluorometer body (Fig. 15) is
a machined aluminum block which contains a low-pressure mercury lamp,
a 254-nm interference primary filter, quartz tube flow-cell, two
Corning J-60 secondary filters, photomultiplier tube and housing, and
a photoconductor for compensation of changes in lamp intensity. In
2k
-------
Table 1. RESOLUTION OF COUPLED ANION-CATION COLUMNS
DURING CHROMATOGRAPHY OF ORESP SECONDARY
SEWAGE EFFLUENT SAMPLES
Column
Anion
75
50
25
length, cm
Cation
25
50
75
Resolution
(No. of chromatographic peaks)
1*0
^5
35
25
-------
ORNL DWG 72 * I38O R-2
GAS
PRESSURE tSOmm Hg)
L-a
COLUMN
ON
REAGENT
RESERVOIR
ELUATE FLOW
7cc/hr.
PHOTOMETER
CAPILLARY
FLOW
CONTROL
7cc/hr.
MIXER
RECORDER
o
o
o
TO
WASTE
!4-24cc/hr
FLUOROMETER
BOILING
WATER
REACTION
= SECTION
HEATER
Fig. 13. Schematic of Cerate Oxidation Monitor.
-------
OWL DWG. NO. 72-4213
N
Fig. l^i. Schematic of Flow Fluorometer.
-------
ORNL DWG. NO. 72-4212RI
SAMPLE OUTLET
CO
CO
FLUOROMETER BODY
EXCITATION SLIT
EXCITATION FILTER
FLUORESCENCE VIEWING SLIT
BLOCKING FILTER
COMPENSATION
APERTURE
ADJUSTMENT
1— UV CONVERSION FILTER
• LAMP COMPENSATION PHOTOCONDUCTOR
-INSULATING MOUNT
PLUG
PHOTOMULTIPLIER HOUSING
PHOTOMULTIPLIER TUBE
VOLTAGE DIVIDER CIRCUIT
-HIGH VOLTAGE CONNECTOR
•SIGNAL CONN
Fig. 15. Exploded View of Flow Fluorometer Body.
-------
this situation, nature was exceptionally benevolent because the
excitation maximum for trivalent cerium is very close to the most
intense emission line of the low-pressure lamp, 25^- nm, and the
emission spectrum of Ce(lll) corresponds almost precisely with the
band-pass of the Corning f-60 filters. These characteristics plus
the photo response characteristics of the S-ll photomultiplier pro-
vided an exceedingly low background due to reflected light and allowed
the construction of a very sensitive instrument from relatively inex-
pensive components. The calibration curve for this fluorometer with
Ce solutions is shown in Fig. 16. As can be seen, the fluorometer
•4 3+
has a linear signal output up to about 2 x 10 M Ce and a slight
decrease in a slope up to 10 M. Although the sensitivity of this
detector depends on the oxidizability of the substance being determined,
moderately oxidizable compounds can be detected at levels of about
100 ng/ml in column effluents. Chromatograms of primary and secondary
sewage effluents analyzed on the Mark III UV-Analyzer with an oxidative
monitor are shown in Figs. IT and 18•
OXYGEN DEMAND MONITOR
The cerate-fluorescence monitor, which was developed as an
alternative detector system for liquid chromatography, has been adapted
as a rapid, sensitive cerate oxidative monitor for measuring the COD
of waters. The pollutants are oxidized with perchloratocerate reagent,
and the resulting Ce(lll) is determined fluorometrically. Analysis
requires only a few minutes for determinations at levels as low as
100 ug oxygen demand per liter.
Cerate oxidimetry has been in general use in volumetric oxidimetry
18
for several decades, and its applicability to the analysis of COD
in polluted waters was suggested as early as 19^1. For valid reasons,
this method was not adopted as the standard in the United States; how-
ever, the intrinsic fluorescence of trivalent cerium coupled with
recent advances in fluorescence analysis and cerate oxidimetry made
reconsideration of cerate oxidation methods for COD analyses seem
warranted.
29
-------
ORNL DWG 72-8766 Rl
O
P.
a
3
o 0.1
ENLARGED SECTION
SEE ENLARGED
SECTION
0 O.I 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
TRIVALENT CERIUM CONCENTRATION (millimoles/liter)
FLUOROMETER CALIBRATION CURVE
-------
ORNL DWG 72-11725
MANY AROMATIC ACIDS FOUND IN HUMAN
WASTES ELUTE IN THIS REGION
IX
i-o-
>!-.:
-i5 = «A"i"'Li'">
270 Ml SAMPLE OF
1000 x CONCENTRATE
PRIMARY EFFLUENT
Fig. 17- Chromatogram of Primary Sewage Plant Effluent Analyzed by
the UV-Analyzer with a Cerate Oxidative Monitor.
31
-------
ORNL DWG. 72-13503
FLUORESCENCE
—UV ABSORBANCE
SAAiS
IUU
90
80
CO
t 70
Z
3 60
ui
s«
u
2 40
IT
§30
_i
^20
10
1
1
I ...
-
j
22
illlllll'Illllll1!!!
97CI u 1 <>AMP( F OF FLUORESCENCE
fooV; CONCENTRATE ORESP -uv ABSORBANCE
SECONDARY EFFLUENT
-
-
-
A"
. ^J V . ^ J u ^
*
J J ^ 1 l__j-_ I, L 1 1 J 1 1 _- - | 1 _'j- ... [ ' ' -J-i_-.-,.-'- ' -'
84
86
88 in
o
90 ^
^
92 |
94 1
I"*
96 >
98 1
100 8
ir>9
24 26 28 30 32 34 36 38 40 42 44
"HOURS
Fig. 18. Chromatograni of Secondary Sewage Plant Effluent Analyzed
"by the UV-Analyzer with a Cerate Oxidative Monitor.
32
-------
Many of the organic compounds normally found in sewage and
industrial wastes are not oxidized as efficiently with eerie sulfate
(sulfatoceric acid) as with dichromic acid, particularly when silver
is used as a catalyst with the dichromate. This is understandable
when one compares the available oxidation potentials as shown in
Table 2; as can also be seen from the table, perchloratoceric acid
provides an oxidation potential approaching that of silver-catalyzed
chromic acid and thus should also oxidize many of the same organic
compounds•
An attractive feature of a cerate oxidation method is that the
trivalent cerium which results from any redox reaction with compounds
in the water sample is strongly fluorescent (Fig. 19)- Concentrations
_O _|i
of Ce(lll) from 10 to 10 M can be readily determined fluorometrically
by excitation with ultraviolet light (254 nm) and measuring the 90°
20
light emission at 350 nm. With this information in mind, a method
which requires only a few minutes for measuring the COD of waters
at levels as low as 100 pg per liter was developed. This method is
quite amenable to automation and can be used for continuous, remote
stream analysis.
Several organic compounds representative of the various constituents
of waste and natural waters were analyzed by this method as a means of
testing its applicability. The classes of such compounds included
carbohydrates, alcohols, amino acids, surfactants, straight-chain
acids, volatiles, and others. In addition, samples of sewage plant
effluents and a river water sample were analyzed.
The "perchloratoceric acid" method involves the mixing of the
-3 -2
waste sample with 10 to 10 M perchloratoceric acid reagent and
heating at 100°C for 5 min. The reagent concentration and the ratio
of reagent to sample are selected so that about 10 to 50$ of the
Ce(iv) will be utilized. After this, the mixture is diluted to a
total cerium concentration of about 10 M with cold 0.2 N sulfuric
acid, which halts the reactions. The fluorescence of the mixture
resulting from trivalent cerium is then measured.
Preliminary tests of the perchloratoceric acid method were carried
out with conventional laboratory glassware and a Perkin-Elmer Model 203
33
-------
Table 2. OXIDATION POTENTIALS AVAILABLE WITH VARIOUS
OXIDANTS USED -IN COD ANALYSIS
18 M H*
3+
Ag+ «•
2+
H2S<\
Ce
3+
-1.98 v
-lA2 V
-1.70-to -1.87 V
-------
ORNL DWG 72-8759
U)
VJl
CERIUM(IV) + ELUTED COMPOUND
(NONFLUORESCENT)
CERIUM(III) , 4- OXIDIZED PRODUCTS
(FLUORESCENT)
FLUORESCENCE EXCITATION MAXIMUM: 260 nm
'FLUORESCENCE EMISSION MAXIMUM: 350 nm
Fig. 19. Cerium Fluorescence Oxidimetry.
-------
fluorescence spectrophotometer. However, once the general procedure
was worked out, a continuous-flow apparatus of two pressurized reagent
reservoirs, a sample puoip (peristaltic), two jet mixers, a boiling water
bath, a flow fluorometer, and a strip-chart recorder (Fig. 20) was
assembled. This apparatus can be used as a continuous stream monitor
or, as described here, as a discrete sample analyzer. In the discrete
mode of operation, it is capable of analyzing about ten samples per
hour.
Pressurizing the reagent reservoirs is a very simple but reliable
method for metering a low flow of the reagents. Using a simple gas
pressure regulator and a length of capillary tubing provides a constant
21
flow in the range of a few milliliters per hour. The sample stream
and the perchloratoceric acid stream are mixed in the first jet mixer,
the sample entering through the jet and the reagent entering through ,
the annulus. The cross sections of the jet and annulus are designed
so that the linear velocities of the two fluids at the jet exit are
nearly equal, thus minimizing back-mixing. From this mixer the stream
flows through a coil of thin-walled Teflon tubing immersed in a boiling-
water bath. The residence time in the bath is 5 rain. This mixture
is then diluted tenfold in the second jet mixer with 0.2 N sulfuric
acid, which effectively stops the reaction. The concentration of
trivalent cerium in this diluted stream is continuously measured by
the flow fluorometer described in the previous sections and recorded
on a strip-chart. -£
Three or more samples of nine organic compounds dissolved in
triply distilled water and three samples of polluted waters were run
using .this apparatus in the discrete sample mode. A blank of triply
distilled water was run between each sample. Periodically, a cali-
bration standard containing a known amount of trivalent cerium in
distilled water was also analyzed.
A quantity of each organic compound which would require about 10 mg
of oxygen for complete combustion to carbon dioxide and water is weighed
out and dissolved in distilled water. At approximately 5-min intervals,
the sample tube is placed into one of these solutions and a 1-min sample
36
-------
ORNL DWG 72-7924
RECORDER
PRESSURE(a2-°-5atm)
DILUENT
RESERVOIR
^CAPILLARY
[FLOW
.DNTROL
> 180 cc/hr
MIXER
REAGENT
RESERVOIR
SAMPLE
6 cc/hr
CAPILLARY
FLOW
91 CONTROL
12 cc/hr
TO
WASTE
FLUOROMETER
Fig. 20. Schematic of Continuous Chemical Oxygen Demand Analyzer.
37
-------
is withdrawn. During the remainder of the cycle, the sample tube is
inserted into distilled water. The COD of the sample is indicated
by the height of a peak on the strip-chart recorder 10 min after
sampling. Once each day, usually at the start of the operation, the
system is calibrated with three samples of a Ce(lll) solution of
known concentration (1 to 2 x 10 M or 8 to 16 mg oxygen demand per
liter). An example of the recorder trace is shown in Fig. 21. The
results of several series of runs using the selected organic compounds
and pollution samples are shown in Table 3.
Although some compounds were not as completely oxidized as with
the catalyzed chromic acid, all of the samples with the exception of
acetone, benzene, and pyridine were oxidized to a sufficient level
after 5 min at 100° C. Note that, under these conditions, pyridine
and benzene were oxidized to a greater extent than by the Standard
22
Methods procedure. If greater levels of oxidation are required,
longer reaction times or a higher temperature can be used. Tempera-
tures above 125° C should not be used because rapid degradation of
the cerate occurs .
The results obtained from analyses of polluted water samples show
that the perchloratoceric method is indeed rapid and sensitive, and
should be a useful complement to the standard method. A proportionality
factor relating this method to the standard method may be determined
by running several samples by each procedure. Such a factor would
probably be necessary in cases where samples contain significant
quantities of compounds which are oxidized to a lesser extent than
with the standard method. This factor can then be applied to later
samples run by the perchloratoceric acid method, if its accuracy is
ensured by an occasional check by the standard method.
The major advantages of this method are rapidity, sensitivity,
and little need for manual attention. Even though no effort has
been made to optimize conditions for maximum sample throughput, 10
to 12 samples per hour can be analyzed on the apparatus described
here. With the addition of an automatic sampler, operator attention
would be reduced to about 1 hr per day for charging the reagent
38
-------
VO
...
I I x 10 M Ce — 8 ppm 0.0.
2 33mg/j£ BENZENE
3 10 mg/j£ ACETIC ACID - II ppm O.D.
4 14.5 mg/Jt GLUCOSE- 15 ppm 0.0.
5 5 mg/JL SODIUM LAURYL SULFATE - - 10 ppm O.D.
6 lOOmg/jePYRIDINE- 220 ppm O.D.
7 5 mq/JL LINEAR ALKYL SULFONATE ( M.W. 315 )
8 0.75mg/jeETHANOL— 16 ppm O.D.
9 I0mg/j2 GLYCINE— 7 ppm O.D.
10 10 mq/Ji ACETONE— 18 ppm O.D.
10
20 30 40
TIME(min)
50
60
70
Fig. 21. Recorder Trace from Continuous COD Analyzer.
-------
Table 3. OXIDATION OF ORGANIC COMPOUNDS AND POLLUTED WATERS
BY PERCHLORATOCERIC ACID COD METHOD
Sample
Acetic acid
Acetone -
Benzene
Ethanol
Glucose
Glycine
Linear aXkyl sulfonate
Pyridine
Sodium lauryl sulfate
Sewage plant effluent
(industrial plant)
Secondary sewage plant
effluent (Oak Ridge, Tenn. )
,— -
Clinch River water
Concentration,
mg/liter
10
8
3-3
7.5
Hk 5
10
5
10
5
19 COD
6 BOD5
8 COD
Number of
det erminat ions
4.
5
k
3
k
k
5
6
5
5
5
3
Oxygen consumed,
Average
6.1
3.2
2.8
11.0
17- k
3-0
6.9
3-5
13-^
16.5
25.9
7.8
mg/liter
Range
5.2
3.0
2. If
10.8
15-9
2.8
6.3
3-2
12.8
Ilk 8
2U.2
7-5
- 7.U
- 3-3
- 3-5
- 11.1
- 17-9
- 3-2
- 7A
- 3.6
- lif.6
- 18.1
- 27. k
- 8.0
% of
theoretical
COD
57
18
26
69
112
1*6
105
16
135
i
-------
reservoirs and samples and for reading the strip chart. The disadvantages
of the method are its relative low dynamic range (less than two decades)
for a given set of conditions and the necessity for running a standard.
-------
SECTION V
IDENTIFICATION OF STABLE ORGANIC POLLUTANTS
Thirteen samples of "domestic" sewage plant effluents, five from
the primary stage and eight from the secondary stage, were obtained
and concentrated during this investigation. Fifty-six compounds were
identified in the primary effluent, while thirteen compounds were
identified in secondary effluent.
IDENTIFICATION TECHNIQUES
The preparation of samples for analysis, the separation of con-
stituents, and the application of analytical methods to separate
fractions involve an integrated and complex series of manipulations
and investigate
is given below.
23
and investigative techniques. A brief description of this process
Preparation of Sewage Effluent Samples for Analysis
Concentration of sewage effluents by factors up to 3000 is necessary
prior to analysis. Of the several concentration techniques considered,
low-temperature distillation described in Section IV was selected as
the most desirable. One sample was concentrated on XAD-4 macroreticular
resin; however, since this technique gave disappointingly poor results,
it was not investigated further.
Anion Exchange Chromatography
The major effort during the program was concerned with the identifi-
cation of uv-absorbing molecular components of samples of sewage plant
"effluents. The UV-Analyzer used for the analysis of these samples
employs a 0.30 cm x 150 cm column and is restricted to sample volumes
of less than 1 ml. Thus, repeated 40-hr chromatographic runs would
be required to obtain sufficient quantities of many of the molecular
o
constituents for identification purposes. A preparatory system with
several times the capacity of the analytical system has been used to
42
-------
increase the quantities of compounds collected per run. This system,
which is coupled to a fraction collector, is capable of chromato-
graphing 5 ml °f sample with a resolution approaching that of the
smaller analytical column. By combining several adjacent 5" or 10-
min fractions from a single chromatographic run, sufficient quantities
of many chromatographed compounds were collected to allow identifi-
cation by suitable analytical techniques. Comparative data on column
sizes, resin, and operating parameters for the analytical and preparative
systems are listed in Table 4-
Rechromatography Using a Cation Exchange Resin
For most separations, chromatographic peaks that eluted earlier
than uric acid were further purified using a 0.62 cm x 150 cm jacketed
column packed with cation exchange resin (Aminex A-7., Bio-Rad Labora-
tories, Richmond, California). The fractions corresponding to individual
chromatographic peaks from the preparative anion-exchange system were
lyophilized and then redissolved in 0-3 M ammonium acetate—acetic acid
buffer (pH, 4.65)« Each fraction was loaded on the column and chromato-
graphed at 50°C using the same buffer that was used for dissolution.
The effluent was monitored at 2$k nm with an ultraviolet photometer.
In addition to purification, the use of cation exchange established
a second characteristic elution position for individual organic
compounds, providing an additional means of identification.
Preparation of Fractions for Analyses
Adjacent 5~ or 10-min eluate 'fractions corresponding to individual
chromatographic peaks from either the anion or cation exchange separations
were combined, frozen at -70° C, and then lyophilized in a freeze-dryer
at -60°C and a pressure of 50 microns for removal of the ammonium
acetate—acetic acid buffer. The samples were then dissolved in 2 to
3 ml of spectrescopic-grade methanol and subjected to uv spectral, gas
chromatographic, and mass spectral analyses.
-------
Table k. PHYSICAL CHARACTERISTICS AND OPERATING PARAMETERS
FOR THE ANALYTICAL AND PREPARATIVE ANION EXCHANGE COLUMNS
Columns
a
Parameters
Analytical
Preparative
Column dimensions, cm
Resin
Resin particle size, p.
Sample size, ml
Run time, hr
Temperature program
Operating pressure, psig
Flow rate, ml/hr
Eluent
Concentration gradient
Gradient generator
0. 30 x 150
Bio-Rad A- 2?
8-12
0.28
Ambient, in-
creasing to 60 °C
after 11.0 hr
1900
10
Ammonium acetate —
acetic acid buffer,
pH JJ-.44
0.015 M - 6.0 M
2 -chamber
0.9k- x 150
Bio-Rad A- 2?
12-15
5
Ambient, in-
creasing to 60 °C
after 11. -5 hr
1600
Ammonium acetate—
acetic acid buffer,
pH k.kk
0.015 M - 6.0 M
Q
9-chamber
o
Columns were constructed of stainless steel and jacketed for
temperature control.
^One chamber was filled with 0.015 M buffer, and the other was filled
with 6.0 M buffer.
°Two chambers contained 87^ ml of 0.015 M, two chambers contained 851 ml
of 1.0 M, two chambers contained 828 ml of k. 0 M, and three chambers
contained 1208 ml of 6.0 M buffer.
-------
Ultraviolet Spectrometry
Ultraviolet spectra were obtained for each of the collected
fractions in methanol solution from 320 to 210 nm on a Beckman DB-G
recording spectrophotometer, and compared with uv spectra of reference
compounds obtained in the same manner.
Gas Chromatography
A 1-ml aliquot of the methanol solution of the fraction to be
identified was evaporated to dryness under a stream of dry nitrogen.
Trimethylsilyl (TMS) derivatives were then formed by adding 50 to
100 ul of dry pyridine (Pierce Chemical Company) and 50 to 100 ul
of bis(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1%
trimethylchlorosilane (Regis Chemical Company) and heating the
reaction mixture overnight at 50°C. Separate aliquots (k ul) of
the reaction mixture were injected directly onto the two columns
of a MicroTek MT-220 gas chromatograph equipped with dual flame
ionization detectors and dual electrometers. The two columns
(6-ft x 0.25-in.-O.D. Pyrex tubing packed with 3$ OV-1 or OV-1T on
80/100 mesh Chromosorb W-HP) were placed in the same oven, which
was programmed for a temperature increase from 100 to 325°C at
the rate of 10°C/min. The helium carrier gas flow rate was 85 cc/min
for each column.
Mass Spectrometry
Mass Spectrometry was performed on 2-ul aliquots of the TMS-
derivatized samples described above or on nonderivatized samples.
In each case, 1-ml aliquots of the methanol solution of the samples
were reduced in volume, transferred to the glass insert of the mass
spectrometer probe, and evaporated to dryness in a vacuum desiccator.
In the case of samples containing compounds that eluted later than
uric acid in the anion exchange chromatogram, a drop of 6 N HCl
was added prior to evaporation to ensure that the compounds remained
in the acidic form.
-------
Three mass spectrometers were available for use. The TMS-
derivatized sample was run routinely on a Finnigan model 3000 gas
chromatograph—mass spectrometer using a J-ft x 0.25-in. glass
column packed with 2ff0 Dexsil on 80/100 mesh Chromosorb W-HP for
additional resolution. This usually provided data concerning the
molecular weights of the constituents and the number of active
hydrogen atoms per molecule. Comparison of the fragmentation pattern
with that for reference standards was necessary for absolute
identification.
A second mass spectrometer, constructed at Oak Ridge National
Laboratory, was used for low-resolution spectra (N/Am ~ 800). This
instrument was a single-focusing mass spectrometer with a 12-in.
*
radius, 90°-sector magnet, operating at an accelerating voltage of
^ kV. Solid samples were introduced via a vacuum-lock direct
insertion probe operating at the lowest temperature required to
produce a suitable spectrum, generally between 150 and 250°C.and
the spectra were recorded with electron energies of 70 and 15 eV-
These spectra were then manually transferred from the oscillograph
output to punched cards for subsequent computer calculation of
metastable data, relative intensities, and plotting.
A Varian Aerograph model 1200 gas chromatograph was coupled
with this instrument for gas chromatographic--mass spectrometric
(GC-MS) studies. The gas chromatograph column consisted of a J-ft x
0.25-in. glass tube packed with 2$ Dexsil on 80/100 Chromosorb W-HP.
Effluent from the column was divided, with approximately 10$ being
directed to the flame ionization (Fl) detector. The remaining
effluent was fed through a heated stainless steel capillary to the
helium separator, contained in a separate oven. The separator was
a Biemann-Watson type, constructed of porous stainless steel tubing
2k-
as described by Krueger and McCloskey. A micro-needle valve
located between the chromatograph and separator was adjusted to allow
operation of the FT detector at atmospheric pressure. The enriched
sample passed from the separator through a cutoff valve and heated
stainless steel capillary into the ionizing region of the mass
-------
spectrometer. At a helium carrier flow rate of 30 ml/min, the separator
operated at approximately 0.5 torr, and the source can at < 1 x 10 torr
(uncorrected). For normal operation, the column temperature was programmed
such that the GC resolution was adequate but retention time for the peak
of interest was minimized. The separator and interconnecting tubing were
maintained at 250°C. The mass spectra for the GC-MS system were obtained
using an ionizing electron energy of 30 eV and manually transferred from
the recording of the oscillograph to punched cards.
High-resolution spectra were obtained using an instrument which
was constructed at the Oak Ridge National Laboratory. This instrument
had a Nier Johnson configuration, with a IJ-in. radius and 90°-sector
magnetic stage. A l4-stage electron multiplier served as the collector,
and its output was obtained in the form of an oscillogram of the integra-
ted ion current. An ion accelerating voltage of 5000 V and an ionizing
electron energy of JO eV were used in routine operation. Samples were
introduced by means of a direct inlet probe, while the mass standard
perfluorokerosene was introduced from a reservoir through an adjustable
leak. An IBM 1130 computer was used to acquire data from the spectro-
meter, to process the data, and to print the exact masses and possible
empirical formulas of selected peaks.
Fluorescence Spectrometry
The use of fluorescence spectrometry, which might prove advantageous
as an identification aid, was explored with several reference compounds
and used in the identification of indican. Although it did not con-
clusively prove the existence of indican (this was primarily established
by anion exchange position and gas chromatographic MQ values), it did
show that several other fluorescent compounds were present in the eluate
fraction, and that indican was a minor constituent of this fraction.
The extreme sensitivity of fluorescence spectrometry should prove
useful in detecting constituents present at less than microgram-
per-liter concentrations.
Example of Typical Identification Data
The information which resulted in the identification of guano-
sine is presented as an example of the identification data and
-------
procedure. Eluate fraction 95 from the chromatographic separation
of SPJ-1 concentrate of primary effluent was further chromatographed
on the cation exchange system. The elution positions determined
for both the original anion exchange separation and the cation
exchange separation suggested that the unknown might be guanosine.
The fraction collected from the cation separation was then lyophilized,
and uv absorbance scans from 220 to 320 nm of the methanol solution
were obtained for the residue dissolved in methanol (Fig. 22).
Comparison of these scans with spectra of a reference standard
(Fig. 23) further supported the identification of guanosine. The
spectra of the unknown determined at different pH values (Fig. 22)
showed a spectral shift at alkaline pH similar to that of guanosine.
The methanol solution was evaporated under nitrogen gas and derivatized
in preparation for gas chromatography. Retention values determined
for the unknown compared very well with those for the guanosine
reference standard (Table 5). The gas chromatograms for the OV-1T
column are shown in Fig- 2^. Absolute confirmation as guanosine
was obtained from the mass spectra as determined by the low-resolution
mass spectrograph. Figure 25 shows the mass spectra of the unknown
offset 0-5 mass unit and superimposed on the mass spectra of the
reference standard.
SAMPLE STATUS
Thirteen effluent samples were taken from a domestic sewage plant,
concentrated, and separated on a preparative-scale anion exchange
system. Five of these were of primary effluents, some of which were
chlorinated; the remaining eight were of secondary effluents, also
both unchlorinated and chlorinated (Table 6). The status of analytical
work on these samples at the close of the report period is given in
Table T. One sample of primary effluent and one sample of secondary
effluent were involved in the Cl tracer chlorination study described
in Section VI. Two primary samples were taken for identification of
carbohydrates, while the others were obtained-for identification of
uv-absorbing and other organic compounds.
48
-------
ORNL DWG 72-5307
Ul
o
z
<
CD
OC
O
(/)
CD
METHANOL-WATER, pH 7.0
METHANOL-WATER, pH 11.8
220
240
260 280
WAVE LENGTH, nm
300
320
Fig. 22. Ultraviolet Absorption Spectra of Unknown Sample Taken at Different pH Values.
-------
ORNL DWG 72-11698
GUANOSINE
0.0207 mg/ml
0.0
220
240
260
280
300
320
WAVELENGTH, nm
Fig. 23. Ultraviolet Absorption Spectra of Guanosine Taken at Different pH Values.
(Data from Circular OR-10 Sixth Printing, 1969, Pabst Laboratories, Division of Pabst Brewing Co.)
-------
Table 5. GUANOS IKE IDENTIFICATION DATA
Guano sine,
SPJ 1-95-2 unknown
Guanosine,
reference standard
VJI
H
Anion exchange elution position, hr
Cation exchange elution position, ml
Ultraviolet spectra
Gas chromatographic retention value, M.U.
Mass spectra
Molecular weight, TMS derivative
Molecular weight, minus TMS groups
Major fragments, m/e
8.3 (prep.)
13-8
255 (max), 275 (sh)
28.00, 29.5^
283
2^5, 32^, 368, ij-10,
556, 571, 628
7.5 (urine, analyt.)
13-8
25^ (max), 275 (sh)
28.00, 29. te
283
2*f5, 32if, 268,
556, 571, 628
-------
ORNL DWG 72-6001 R I
REFERENCE
COMPOUND
CI6
C22
'20
wvj
-24
in
o>
UJ
z
CO
o
z
<
o
"281
l
I
J I I
PRIMARY EFFLUENT
COMPOUND
6' x 1/4" 3% OV-17
80/100 CHROMOSORB W(HP)
100° —» 325°C AT I0°/min
UJ
FCM
C9
8
10
12
14
16
18
20
TIME (mm)
Fig. 214-. Gas Chromatograms of IMS Derivatives of Reference Compound
(Guanosine) and Liquid Chromatographic Fraction from Primary Sewage Treatment
Plant Effluent. ;
-------
ORN1_ DWG T2-53O9R2
100
LD
GO-
0
h B
i I r
30 110 130 150 170 190 E10 E30 E50 E70 E30 310 330 350 370
M/E
VJ1
LO
100
BO
60 \
d
GUANOSINE REFERENCE STANDARD, IMS DERIVATIVE
P-l, F 95 UNKNOWN, TMS DERIVATIVE (OFFSET 0.5 M/E)
0
390
1 r—i 1 1 1 1—1 1
T 1 1 1 1—T r
410 430 450 470 490 510 530 550 570 590 610 G30 G50
M/E
Fig. 25- Mass Spectra of TMS Derivatives of the Reference Compound (Guanosine) and Liquid
Chromatographic Fraction from Primary Sewage Treatment Plant Effluent.
-------
Table 6. DESCRIPTION OF SAMPLES
Sample
Description
SPJ-1 ORESP primary effluent (10-5-71).
SPJ-2 ORESP primary effluent (10-5-71) chlorinated in the laboratory.
SPJ-3 ORWSP chlorinated primary effluent (10-28-71); 2 ppm residual
chlorine (orthotolidine).
SPJ-li- ORESP secondary effluent (U-22-71).
SPJ-5 ORESP chlorinated secondary effluent (11-22-71); 0.5 ppm residual
chlorine (orthotolidine).
SPJ-6 ORESP secondary effluent (1-18-72).
SPJ-7 ORESP chlorinated secondary effluent (1-18-72); 0.75 ppm
residual chlorine.
SPJ-8 ORWSP chlorinated primary effluent (2-23-72); 0.5 ppm residual
chlorine (orthotolidine).
SPJ-9 ORESP secondary effluent (lf-19-72).
SPJ-10 ORESP chlorinated secondary effluent (l|~19~72); 1 ppm residual
chlorine (orthotolidine).
SPJ-11 Composite of SPJ-3 and SPJ-8.
SPK-1 ORESP primary effluent (3-1-72) for carbohydrate determination.
SPK-2 ORESP secondary effluent (li-2^-72) for carbohydrate determination.
SPL-1 ORESP secondary effluent (12-12-73).
H-l ORWSP primary effluent (2-23-72) chlorinated in the laboratory
with 3oci tracer as Cl2 gas to approximately 2 ppm residual
chlorine (orthotolidine).
H-2 SPJ-9 chlorinated in the laboratory with ^ Cl tracer as Clg gas to
1.0 ppm residual chlorine (orthotolidine).
H-5 SPJ-9 spiked with ^ Cl tracer as chloride.
H-6 SPJ-9 chlorinated in the laboratory with ^ Cl tracer as Cl, gas
to 1.0 ppm residual chlorine (orthotolidine). Chlorine residual
destroyed with potassium thiosulfate after 90 min.
H-8 SPJ-9 chlorinated in the laboratory with ^ Cl tracer as hypochlorite
to 1.0 ppm residual chlorine (orthotolidine). Chlorine residual
destroyed with thiosulfate after 15 min.
H-9 Same as H-8. Chlorine residual destroyed after ^5 min.
H-10 Same as H-8. Chlorine residual destroyed after 90 min.
-------
Table 7- STATUS OF SEWAGE PLANT EFFLUENT SAMPLES (5/1/7*0
CO
SPJ-1
SPJ-2
SPJ-3
SPJ-1+
SPJ-5
spj-6
SPJ-7
SPJ-8
H-l
SPK-1
spj-9
SPJ-10
SPK-2
SPJ-11
SPL-1
H-2
H-5
H-6
H-8
H-9
H-10
o
•p
Concentration f
1000
1000
1000
1000
1000
1000*
1000
1000
570
1000
2000
2000
2000
3000
31+00
IQl+O
880
1110
6ifO
580
k 90
§g
H
Chromatographed
analytical coluj
X
X
X
X
X
X
X
X
X
b
X
X
X
X
X
X
X
X
X
M
3
Chromatographed
preparative coli
X
X
X
X
X
X
X
X
X
X
X
*
fl) 43
fo
-------
IDENTIFICATION RESULTS
The primary effluent samples in which 56 compounds were identified
included both unchlorinated and chlorinated samples; several of the
constituents were also quantified. The identities and concentrations
of these molecular pollutants are given in Table 8. Thus far, the
sugars galactose, glucose, and maltose have been found in fractions
collected from the carbohydrate analyzer. The remaining compounds
were found in fractions collected from the UV-Analyzer. The copper
(II) acetate (binuclear) identified using mass spectrometry in a
cooperative effort with the Southeast Environmental Research Laboratory
at Athens, Georgia, may have resulted from the reaction of copper ion
in the sewage plant effluent with the acetate ion of the chromatographic
column eluent. It is worth noting that the fractions containing the
copper (ll) acetate (binuclear) were blue, probably because of the
presence of Cu(ll), and that this blue color has appeared only in
chlorinated sewage effluents. The copper may have been present as the
result of corrosion in the concentration apparatus. A standard reference
uv chromatogram of primary sewage plant effluent has been prepared (shown
with identified compounds in Fig. 5)- It will be used for reporting
additional identifications and elution positions. The elution positions
for identified compounds are given in Table 8-
In addition to the 56 identified compounds, other chromatographic
fractions have been partially characterized. Tables 9 an^ 10 list
the molecular weights obtained by mass spectrometry and gas chromato-
graphic retention data for 15 unknown compounds found in five chromato-
graphic fractions of unchlorinated domestic sewage plant effluent, and 8
unknown compounds found in four chromatographic fractions of chlorinated
sewage plant effluent. Thirty-eight unknown compounds were found on
more extensive characterization of a chlorinated sewage effluent which had
been concentrated 2000-fold. These compounds are listed in Table 11
according to increasing molecular weight. It is obvious that, as more
interpretative information regarding TMS-mass spectra becomes available,
numerous additional compounds will be identified. Thirty unknown
compounds were characterized in the SPJ-11 separation. SPJ-11 was a
56
-------
Table 8. IDENTIFICATION OF MOLECULAR CONSTITUENTS IN 1OOO- TO 3OOO-FOIJD CONCENTRATES
OF PRIMARY DOMESTIC SEWAGE
Compound
•L.
Ethylene Glycol
Maltose
Galactose
Glucose
Glycerine
Galacitol
Erythritol
Urea
!T~-Methyl-lf-pyridone-3-carboxamide
Phenylalanine
Uracil
5 -Ace tylamino -6 - amino - 3 -methyl ur ac 11
IN -Methyl - 2 -pyr idone - 5 -carboxamide
Tyro sine
Thymine
Theobromine
7 -Methylxanthine
Inosine
Hypoxanthine
Xanthine
Copper (II) acetate (binuclear)
Adenosine
1, 7-Dimethylxanthine
Q
Identification method
AC, GC, MS
AC, GC
AC, GC
AC, GC
AC, GC, MS
AC, GC, MS
AC, GC, MS
AC, GC, MS
AC, UV, GC
AC, GC, MS
AC, CC, UV, GC, MS
AC, CC, UV, GC
AC, CC, UV, GC
AC, GC, MS
AC, CC, GC, MS
AC, CC
AC, CC
AC, CC, UV, GC, MS
AC, GC, MS
AC, CC, UV, GC, MS
MS
AC, CC, UV, GC, MS
AC, CC
Concentration,
lag/liter
10
90
ifO
1^0
20
~7
~90
50
25
70
Anion exchange elution
position, ml
8
8
8
8
10
10
10
10
ill
16
18
20
21
23
29
29
29
29
30
55
60
6k
6k
V/l
-------
Table 8 (continued). IDENTIFICATION OF MOLECULAR CONSTITUENTS IN 1000- TO 3000-FOLD CONCENTRATES
OF PRIMARY DOMESTIC SEWAGE
Compound
3 -Methylxant hine
Caffeine
:\ Guano sine
2-Deoxyglyceric acid
3-Jfcrdroxybutyric acid
3-Deoxyarabinohexonic acid
Quinic acid '
1-Methylxanthine
2-Deoxytetronic acid
Glyceric acid
b
4-Deoxytetronic acid
3-Deoxyerythropentonic acid
2, 5-dideoxypentonic acid '
3) 4- Dideoxypentonic acid
Ribonic acid , ' ' '
Oxalic acid
2-Hydroxyisobutyric acid
Uric acid
Orotic acid
Succinic acid
Q
Identification method
AC, CC
AC, CC, UV, MS
AC, CC, UV, GC, MS
MS
GC, MS
MS
MS
AC, CC, UV
MS
MS
MS
MS
MS
MS ?
MS
AC, GC, MS
GC, MS
AC, GC, MS
AC, UV, GC, MS
AC, GC, MIS
Concentr at ion,
Hg/liter
-10
50
70
20
5
Anion exchange elution
position, ml
6k
67
76
80
80
80
80
82
85
90
90
90
90
90
95
95
95
106
145
205
\J1
00
-------
Table 8 (continued). IDENTIFICATION OF MOLECULAR CONSTITUENTS IN 1000- TO 30OO-FOLD CONCENTRATES
OF PRIMARY DOMESTIC SEWAGE
Compound
Phenol
3 -Hydroxyphenylhydr acrylic acid
Phenylacetic acid
4- Hydroxyphenylacetic acid
Benzoic acid
2-Hydroxybenzoic acid
^-Hydroxybenzoic acid
3-Hydroxybenzoic acid
3-^droxyphenylpropionic acid
Indican
3-Hydroxyindole
o-Phthalic acid6
£-Cresol
Q
Identification method
AC, GC, MS
AC, UV, GO, MS
AC, 'GC
AC, UV, GC, MS
AC, GC, MS
AC, GC, MS
AC, GC
AC, GC, MS
AC, GC, MS
AC, GC, F
MS
AC, UV, MS
AC, GC, MS
Concent rat ion,
lig/liter
6
10
~10
190
7
~lfO
~20
~2
200
20
Anion exchange elution
position, ml
205
205
230
235
260
290
295
295
295
325
3^0
14-00
koo
\J\
VO
AC - anion exchange chromatography; CC - cation exchange chromatography; UV - ultraviolet spectrum;
GC - gas chromatography on two columns; MS - mass spectroscopy; F - fluorescent spectrum.
^From chlorinated effluent.
cldentified by A. W. Garrison, Southeast Environmental Research Laboratory.
dNo reference spectra available. Structure deduced from mass spectra analysis.
eIdentified in Mill Creek sewage effluent.
-------
Table 9. DATA ON UNKNOWN COMPOUNDS FOUND
US UNCHLORINATED PRIMARY EFFLUENT (SPJ-l)
FROM THE OAK RIDGE EAST SEWAGE PLANT
Elation
time,
hr
0.6
0.6
0.6
0.6
0.9
0.9
0.9
0.9
1.8
1.8
1.8
15.8
15.8
15.8
1*6.5
Molecular
•weight
135
130
202
10?
100
130
li*6
158
111*
130
136
118
131
211
150
GC retention data,
OV-1
1^.99
15.0
17-5
20.0
15-1*2
18.00
17.58
13.13
13-88
15.0
15.0
13.50
16.92
21.68
13.51
OV-17
15.90
-
-
-
-
17. 91*
18.17
13.89
15.36
-
-
ll*. 96
19.05
2l*.7l*
15.00
M.U.
A
+0.91
-0.06
+0.59
+0.76
+1.1*8
+1.1*6
+2.13
+2.88
+1.1*9
60
-------
Table 10. DATA ON UNKNOWN COMPOUNDS FOUND IN CHLORINATED SEWAGE
PLANT EFFLUENT (SPJ-3) FROM THE OAK RIDGE WEST SEWAGE PLANT
Elution
time
hr
1.05
lA
lA
lA
ll*.0
ll*.0
58
58
Molecular
weight
221
98
127
113
110
138
138
236
GC retention data,
OV-1
17-58
13.1
1^.79
15 ^5
ll*.10
15-0
15-01*
25-61*
07-17
17-86
-
15.^9
16. Ol*
ll*.88
-
16.21J-
27.10
M.U.
A
+0.28
+0.70
+0.59
+0.78
+1.20
+1.1*6
2 ppm chlorine residual.
61
-------
Table 11. DATA ON UNKNOWN COMPOUNDS FOUND
IN CHLORINATEDa EFFLUENT (SPJ-8)
FROM THE OAK RIDGE WEST SEWAGE PLANT
Molecular
weight
61
74
95
95
97
105
107
108
112
113
113
116
124
Elution
time,
hr
0.8
3.6
3.2
9-8
16.5
10.5
40.5
67
1.4
0.8
0.8
2.0
16
GC
retention data,
OV-1 OV-17
11.
14.
11.
10.
14
13-
18.
16
15.
17.
11.
15.
15.
0
5 15-3
4 12.8
8
15.3
6 15.7
9 20.0
-
4 16.0
7
2
2 16.0
0
M.U.
A
-
0.8
1.4
-
1.3
2.1
1.1
-
0.6
-
-
0.8
-
62
-------
Table 11 (continued). DATA ON UNKNOWN COMPOUNDS FOUND
IN CHLORINATEDa EFFLUENT (SPJ-8)
FROM THE OAK RIDGE WEST SEWAGE PLANT
Molecular
weight
125
12?
128
129
130
130
132b
132
1^0
11*6
ll+7
1U8
151
152
15*
159
160
Elution
time,
hr
3-0
l.lf
12.7
3-0
10.0
l.V
10.0
16.0
1*0.5
3-2
10.5
18
16
16
1*0
10
18
GC
retention, data
OV-1 OV-17
15- *
lit-. 7
12.2
16.0
17.8
1*.5
20.2
16.1
15
1^.5
20
15.8
17.7
13.5
17.5
22. k
16.9
-
-
12.8
17-5
-
-
20.5
16.4
16.2
15.2
20.8
16.5
20.6
lU.9
18.6
-
17.6
M.U.
A
-
-
0.6
1.5
-
-
0.3
0.3
1.2
0.7
0.8
0.7
2.9
1.1
1.1
-
0.7
63
-------
Table 11 (continued). DATA ON UNKNOWN COMPOUNDS FOUND
IN CHLORINATED8" EFFLUENT (SPJ-8)
FROM THE OAK RIDGE WEST SEWAGE PLANT
Molecular
weight
166
177
IB*
218
219
233
28^
JO*
Elution
time,
hr
to. 5
2.0
16
10.5
3.0
12.7
to. 5
10.0
GC
retention data,
OV-1 OV-1?
16.
18.
20.
16.
lfc.
12.
23
*.
8 18 A
7
k 20.6
k 17.1
8 15.3
2 12. 8
-
8 26.1
M.U.
A
1.6
-
0.2
0.7
0.5
0.6
-
1.3
0.5 ppm residual.
^Aglycon molecular weight.
glucuronide.
Probably present as a
-------
ERRATA SHEET
AUTOMATED ANALYSIS OF INDIVIDUAL REFRACTORY
ORGANICS IN WATER POLLUTED
EPA-660/2-7^-076
Should read
AUTOMATED ANALYSIS OF INDIVIDUAL REFRACTORY
ORGANICS IN POLLUTED WATER
-------
composite of SPJ-3 and -8 samples concentrated 3000-fold. These compounds,
along with pertinent identification data, are listed in Table 12.
Thirteen organic compounds were identified in samples of unchlo-
rinated secondary effluent from a domestic sewage plant. Most of
these constituents have been quantified, and their concentrations
and anion exchange elution positions are given in Table 13. A
standard reference chromatogram of secondary sewage plant effluent
for reporting elution positions and identifications is given in
Fig. 6. In addition, 20 unknown compounds were chararacterized in
2000- and 3^00-fold concentrates of secondary effluent. These
compounds, along with the characterization data, are listed in
Table 14.
-------
Table 12. DATA ON UNKNOWN COMPOUNDS FOUND
IN SPJ-11, COMPOSITE OF SPJ-3 AND SPJ-8
Molecular
weight
57
76
82
9^
101
106
117
117
128
129
132
ll*7
ll*9
152
15^
157
161
162
163
168
170
173
177
192
208
2ll*
21*6
266
285
281*
Elution
time, hr
53
53
0.8 -
0.8
1.0
1.0
385
1.5
32
38
1.5
0.8
0.8
0.8
0.8
68
38
1*1*
0.8
1.5
53
32
68
25
32
68
68
68
53 /,
53 ^'<
M
201
205a
370
295a
. 21*5
235a
189
295a
3^
3^5
333a
276a
365
28la
355a
373a
290a
29la
292a
369a
299a
37l*a
l*50a
2l*9a
280
502
591a
1*82
573a
356
TMS
Base (1)
73
73
73
75
ll*7
11*7
73
73
259
3^5
73
11*6
262
73
73
73
203
73
189
217
209
271*
73
2l*9
75
297
73
73
73
73
peaks
(2)
116
75
117
116
116
73
70
75
73
330
201*
73
350
<• 11*7
188
75
73
11*7
73
73
221*
273
75
75
73
73
75
161*
75
75
(3)
158
ll*Y
ll*7
ii*9
73
117
171*
17l*
75
73
217
75
73
117
*i)[)i
17!*
ll*7
129
ll*7
116
117
73
129
ll*l*
265
75
93
21*5
117
117
GC retention data, M. U.
for OV-1 column
12
13.5
12.1*
10.1*
11.1*
12.1*
11.8
11.1*
ll*.6
16.0
17.6
16
16.1*
16
ll*
15.2
ll*.7
ll*.5
19
17-7
15.1*
17-1*
16.2
21 -
18.9
18.5
20
22
22
22.5
M-15 peak.
66
-------
Table 13. IDENTIFICATION OF MOLECULAR CONSTITUENTS IN 1000- AND 2000-FOLD CONCENTRATES
OF SECONDARY DOMESTIC SEWAGE EFFLUENT
Compound
Glycerine
Uracil
5-Acetylamino-6-amino-3-methyl uracil
1 -Met hylino s ine
Inosine
7 -Methylxanthine
1 -Methylxanthine
1, 7-Dimethylxanthine
Succinic acid
Catechol
Indole-3-acetic acid
3 - Hy droxy indole
p_-Cresol
Q
Identification method
AC, GC, MS
AC, CC, UV, MS
AC, CC, UV
AC, CC, UV
AC, CC, UV
AC, CC, UV
AC, GC
AC, CC, UV
AC, MS
MS
MS
MS
AC, GC, MS
Concentration,
M.g/liter
30
30
80
20
5
6
~6
90
Approximate anion-
exchange elution
position, ml
10
18
20
29
29
" 29
82
6k
135
235
235
320
~300
AC - anion exchange chromatography; CC - cation exchange chromatography; UV - ultraviolet spectrum;
MS - mass spectrescopy.
-------
Table 14. DATA ON UNKNOWN COMPOUNDS FOUND IN SECONDARY EFFLUENT (SPJ-9
AND SPL-1) FROM A DOMESTIC SEWAGE TREATMENT PLANT
Molecular
weight
95
111*
115
124
131
133
157
161
163
166
177
190
202
203
208
270
274
307
321
345
Elution
time, hr
0.8
12.9
12.9
52.5'
57-5
2.0
2.0
0.8
2.0
0.8
2.0
2.0
29-5
5-5
5-5
5-5
4o.o
52.5
0.8
5-5
M
167
315a
331
196
203
349
373
305
379
310
393
406
346
347
352
486
346
379
320*' c
705
TMS
Base (l)
98
143
147
166
73
280
147
171
310
24l
73
-
277
73
73
73
331
75
99
131
peaks
(2)
44
147
73
181
188
284
2£2
73
163
225
147
-
75
146
40
79
315
79
44
117
(3)
167
73
262
196
174
147
304
166
379
73
324
-
73
52
52
357
215
73
69
73
GC retention data, M. U.
for OV-1 column
10.4
15
t
12.9
12.9
11.5
12.9
12.0
10.2
16.0
14.3
16.0
16.0
17.0
19.0
22 '
29-5
18.9
15
11.8
27-4
peak.
TMS derivative.
M-1 peak.
68
-------
SECTION VI
EFFECTS OF CHLORINATION OF SEWAGE PLANT EFFLUENTS
Because little definitive information is available concerning
the potential hazards resulting from the chlorination of sewage plant
effluents and other waters, a study was undertaken to determine
whether chlorinated organic compounds are produced during chlorina-
tion of various waters. Indeed, effects on compounds present in
both primary and secondary effluents were indicated by preliminary
studies on three sewage effluent samples, SPJ-2, -3, and -5, each
chlorinated to different residual levels prior to concentration.
Comparison of chromatograms before and after chlorination showed
that some chromatographic peaks disappeared, while others appeared,
apparently as the result of chlorination (see Fig. 5)-
It is a matter of some significance whether these effects of
chlorination are a consequence of oxidation or result from chlorine
addition and/or substitution products. The latter is, of course,
of great significance in terms of possible ecological effects, both
in the short and long terms. Perhaps the most direct and sensitive
technique available to determine whether any of the chromatographic
changes are due to chlorine addition or substitution products is the
use of a radioactive isotope of chlorine as a tracer. Therefore,
the radioactive isotope Cl was added as a tracer in the chlorine
/
gas used to chlorinate some of the sewage effluent samples. Chlorine-
36 is a 0-Tl-Mev beta particle emitter with a 3.0 x 10^ year half-
per •z/T
life. The presence of Cl in chromatographic fractions other
than those containing chloride ion would confirm that some compounds
in the sewage had been chlorinated.
Anion exchange chromatography of chlorinated primary sewage
effluent tagged with Cl and concentrated 500- to 1000-fold revealed
as many as 60 radioactive peaks, several of which coeluted with uv-
absorbing peaks. These peaks were shown to be mostly chlorine-
containing organic compounds. Under the conditions of chlorination
69
-------
used at the Oak Ridge Sewage Treatment Plants, about 1% of the chlorine
dosage was associated with these stable chlorinated organics. Seven-
teen of the compounds were tentatively identified.
A detailed study of the chlorination effects on organic constituents
26
has been published as a doctoral thesis.
CHLORINATION OF SAMPLES
In the initial chlorination experiments with nonradioactive chlorine,
chlorination was effected by addition of dry calcium hypochlorite to
3-liter aliquots of primary sewage plant effluent at ambient temperature.
After being stirred with a Teflon-coated stirring bar for 1 hr, each
aliquot was then concentrated by vacuum distillation. The dry calcium
hypochlorite used in the chlorination experiments contained 6l$. available
27
chlorine as determined by the iodometric method for chlorine residual.
In these scouting experiments, the amount of hypochlorite added to
the effluents provided 8-5* 17, and 85 ppm total chlorine. An orthotolidine
determination of the combined chlorine residual of the 8-5 PPm experi-
ment indicated an approximate combined chlorine residual of k ppm.
The effluents were then concentrated 1000-fold and chromatographed
(Fig. 26). Comparison of these chromatograms indicates considerable
differences, with most of the chromatographic peaks disappearing in
the concentrate of the effluent chlorinated at the highest concentration
(SPJ-2).
CHLORINATION OF SAMPLES WITH 5 Cl
Gaseous chlorine generated by the reaction of KCl in dilute
H2SO^ with KMnO^ powder, 10 KC1+ 2KMnO^ + 8 HgSO^—>2 MnSO^ + SKgSO^ +
8 H 0 + 5 ClQ, was used for the radioactive chlorination experiments.
2 36
A potassium chloride solution tagged with Cl and containing an
excess of sulfuric acid to convert the chloride ion to HCl was dropped
onto potassium permanganate powder, and the chlorine gas was sparged
from the reaction tube with nitrogen gas. The nitrogen gas containing
TO
-------
.0*
at
J07
M
W as
I •»•
lfl3
*.o«
01
ttifLt n«uinnTuicinp CHOMC M9n>UAL.o*THOTouD»icicONCiNTRATcD TJO >
ANIOH [ICHAMf CHRONATOMAPH RUN CONDITION! I
AISORfANCC MEAMRIO AT IMM, 0 3-M-IO • I9O%» ITAINLCM mtL COLUMN WITH ••» * DIA*. AMINC» A-IT ««1N,
TEMPERATURE PRMRAM, AMMNT TO U*C AT II to | IIUINT tRADICHT MCMAIIIN LIMIAIILT IN CONCCNTMTION FKOH O.OI9 M
TO t.O M AMMONIUM ACtTATE. »H 4.4 , CLUINT FLOW RATt IO.I/W ; COLUKK NtMUIIC IBM Mif TOTAL
uo fig maun em.unT(iwnAi.ocomc ooHamnuTioN iro «»«•) CONCCHTRATCD »K) «
ANION E«CHAIMI CHROMATOWAFH HUH CONCITIOHI' I[C AMVI
ITS
CUmONVOUWC.M
.n
.OB
.07
M
Vat
tat
6.03
»o /ig mum crnjuCNTiMmAi. CM.CHMC oaMCCMnunoN 69 nt/iimieoNciNTiuTeo 1000 «
ANION CXCHANU CHROMATOORAPH RUN CONDITION* ' l« AMVC
Fig. 26. UV-Analyzer Chromatograms of Primary Sewage Treatment Plant Effluent Chlorinated with
Different Amounts of Calcium Hypochlorite.
-------
the chlorine was then bubbled through 2 liters of sewage effluent in
a reaction flask at room temperature. A gas trap containing potassium
iodide downstream from the reaction flask collected the unreacted
chlorine and allowed the efficiency of chlorination to be determined.
Milligram quantities of chlorine could be generated at an efficiency
of up to 95$ using this experimental setup (Fig. 27). To ensure zero
leakage of cl to the atmosphere, the experiments were performed in
a hood equipped for radioactivity experiments; in addition, the system
was operated at a negative pressure (-1 in. HpO) with a controlled air
leak into the "hot" off-gas system. During chlorination, the reaction
mixture of sewage plant effluent was sparged with a slow flow of
nitrogen gas from the chlorine generator while being stirred
with a Teflon-coated magnetic stirring bar. After 1 hr, the combined
chlorine residual was determined with orthotolidine.
The remaining chlorinated effluent was taken to dryness using
the vacuum distillation apparatus shown in Fig. 28. This equipment
was also operated so as to ensure that the radioactivity remained
confined within the concentration apparatus. Subsequently, the
residue was dissolved in acetic acid to eliminate the carbonates
and reevaporated to dryness. The final dissolution was made with
dilute ammonium acetate buffer (0.015 M> pH 4.4) in preparation for
chromatographic analysis. The pH of the Cl tagged concentrate was
determined using a pH meter (usually 4-5), and the pH of the untagged
sample to be compared with it on the dual-column chromatograph was
adjusted to correspond.
A fraction collector was used to serially collect the eluate
fractions from the column on which the tagged concentrate had been
chromatographed. Half-milliliter aliquots of each of the eluate
fractions collected during the chromatographic run, plus samples
resulting from various steps of the chlorination, were analyzed for
Cl by liquid scintillation counting.
-------
POTASSIUM
CHLORIDE-
SULFURIC ACID
POTASSIUM
PERMANGANATE
CO
REACTION
MIXTURE
H — 3
/KW^^i^
, .--;:•.• -.-^•_:•;•;.;:."«.
POTASSIUM
IODIDE
SOLUTION
9\tf&
fi«?l¥'
efiH0t
fc
&%-.*
|lX,A4V.u<
ll
i
>'^iV«1
&tf«?
W/«
I^A
ORNL DWG. 72-11702 R 2
I
j
*?
i
ao
?s
4
r
L
OFF-GAS
MANOMETER
PACKED
SODA-LIME
BED
Fig. 27. Schematic of Chlorine Generator and Sample Chlorinator.
-------
ORNL DWG. 72-11703 R
ROTARY
EVAPORATOR
NO. 2
WATER TRAP
(DRY ICE)
NO. 3
WATER TRAP
tORY ICE-TRICHLORO-
ETHYLENE)
MERCURY
MANOMETER
NO. 1
WATER TRAP
(DRY ICE-TRICHLOROETHYLENE)
PACKED
SODA-LIME
BED
. WATER
BATH
VACUUM
PUMP
Fig. 28. Schematic of Apparatus Used for Concentration of Radioactive Samples.
-------
EXPERIMENTAL RESULTS
Five samples of effluent from the secondary stage and one
from the primary stage of Oak Ridge sewage treatment plants were
chlorinated in the laboratory with Cl-tagged chlorination agents
(see Table 6). The primary effluent sample was chlorinated to 2 ppm
chlorine residual (orthotolidine method) using chlorine gas (H-l),
and the secondary effluent samples were chlorinated to 1 ppm residual
using chlorine gas in two cases (H-2 and -6) and hypochlorite solu-
tion in the others (H-8, -9, and -10). Essentially the same results
were obtained with each reagent with one major exception. One of
the chlorine-containing constituents subsequently separated by anion
exchange chromatography was considerably higher in concentration
after chlorination with hypochlorite solution than when chlorine
gas was used.
In the initial experiment, a 2-liter volume of unchlorinated
primary effluent (sample H-l) from the Oak Ridge West Sewage Plant
was chlorinated in the laboratory with 18-7 mg of chlorine gas
containing approximately 0.12 mCi of Cl. The chlorinated mixture
was then reduced to a volume of 3-5 ml in a rotary evaporator. Of
the initial radioactivity, h&fo was found in the concentrate, 10$
in the condensate, and the remainder in the chlorine generator, the
potassium iodide trap following the chlorination reactor, and the
solid residue from the concentration step.
A 15.8-liter sample of primary effluent, chlorinated at the sewage
plant,- was taken with a slight time delay so as to be representative
of the unchlorinated sample used for the tracer experiment. This
sample (SPJ-8) was concentrated 1000-fold using the routine concen-
tration techniques previously reported. For comparison purposes,
the Cl-tagged concentrate and the untagged concentrate were
chromatographed simultaneously on the dual-column chromatograph.
The chromatograms of the two concentrates are shown in Fig. 29 •
75
-------
ClUTIONTIMC.tr 0
CLUTION VOLVIK,*! o
I
It
r
100
its
n i i i
i i i r r^i \ * v r
WHOM EXCHAME CHROMA! O0RAW RUN CONDITIONS-
DUAL. 0.>*»-IB i lBO-t» STAINLESS STf CL WITH «"« * (MAM, AHlKEX A-IT RCtlNi
TCUPCRATUIIE PR09MU, AMBIENT TO «B*C ATUtWi CLUKHT 8 RAO IE NT IKCRCASIK9
UWAMLV IN CONCCNTRATION FROM 0.019 M TO «.0M AHHOMUH ACETATE. P» 4.41
CLUE NT FLO* RATC IO«1/M CACH COLUMN t COUHW PRCMURt IftOO H*f TOTAL.
® @ ®
i I I I
i i i.i iii i
i i i i
1 '
CLUTim VOUIMt.Bl OO
MO H Or CHLOIHNATIDtFrUllllTIU^nkf COHIIWO CHLOKHI KHIOUiL. OKTHOTOLIDINCI CONUNTRATU IOOOX, ttO»
no f\ or irrLUUT CHLOHINATIO IN LAKMATORT OVMOK i •»*« COUMCO CHUWI« RUIDUAL, ORTHOTOUOIM! I CONCINTMTU) «TO x ,
Fig. 29. Dual-Col\nnn UV-Analyzer Cliromatograms of Chlorinated Erimaiy Sewage Treatment
Plant Effluent.
-------
Analysis of the eluate fractions for ^ Cl showed 37 radioactive
peaks in the chromatogram of the tagged concentrate. Four of the
large peaks (peak maximum > 400 cpm/ml) and two of the smaller peaks
(peak maximum ~ 50-400 cpm/ml) were associated with uv-absorbing
constituents. The one extremely large peak (peak maximum = 900*000
cpm/ml) eluting at 200 ml was found to be chloride ion. Four moderately
large and two small peaks were not associated with uv-absorbing constit-
uents. The elution positions of the remaining peaks were such that no
positive conclusion could be made concerning association with uv-
absorbing constituents. A material balance of 112$ of Cl was
determined for the chromatographic separation. The •* Cl activity
level of this 280-^1 sample, which was chromatographed, analyzed
7
1.11 x 10 cpm. Ninety-nine percent of the activity was found in
the chloride peak, 0.6 was found in the remaining eluate fractions,
and 0.4 was found on the sacrificial resin cartridge which precedes
the ion exchange column and removes constituents that tend to
irreversibly adsorb on the resin. The main resin column, although
not analyzed, should have little activity on it.
Comparison of the two chromatograms in the dual-column analysis
(Fig. 29) revealed that 13 chromatographic peaks in the untagged
concentrate (0-5 ppm combined chlorine residual) were apparently
common with radioactive peaks in the tagged concentrate (~ 2 ppm
combined chlorine residual). Five milliliters of this untagged
concentrate was chromatographed on the preparative system, and those
fractions which correspond to the radioactive peaks are being
processed according to our routine identification procedure in an
effort to establish the identities of the chlorinated compounds.
Using similar radioactive tracer experiments with secondary
effluent, it was also demonstrated that chlorinated organic residues
are produced during chlorination at sewage treatment plants. Several
chromatographic experiments were necessary to rigorously prove that:
(l) chlorine-containing compounds are found when a concentrate of
secondary sewage effluent is chromatographed; (2) these chlorinated
11
-------
residues are not artifacts of the concentration procedure; (3) the
chlorinated residues result from chlorination and not from chloride
ion interaction and are, therefore, reasonably stable in aqueous
media; and (4) the chlorinated residues are organic in nature.
Several dual -column chromatographic runs were made in which Cl-
tagged samples were compared with similar samples not tagged. The
radioactive sample, H-2, was a l(AO-fold concentrate of secondary
effluent which had been chlorinated to a chlorine residual of 1 ppm
using Cl -tagged chlorine gas to determine whether organic constit-
uents in secondary effluent were indeed chlorinated as in primary
effluent. This proved to be the case, as is shown in Fig. JO,
because many peaks of radioactivity were found in addition to the
chloride peak. The radioactive sample, H-6, was a 1110-fold con-
centrate of secondary effluent chlorinated in the same manner as
H-2; however, in this case, the chlorine residual was destroyed with
potassium thiosulfate prior to concent rat ion» Because essentially
the same results were obtained from this experiment as for H-2, it
was concluded that the chlorine -containing peaks were not artifacts
of the concentration procedure. It was also concluded that all the
chromatographic peaks represent stable compounds and are probably
not chloramines, since thiosulfate would probably destroy most
chloramines. Radioactive samples H-8, H-9, and H-10 were 640-, 580-,
and lj-90-fold concentrates of secondary effluent chlorinated with
Cl -tagged hypochlorite to a 1 ppm chlorine residual which was
destroyed after 15> ^-5* ai*d 90 min °f reaction time, respectively.
Chromatograms of the chlorine -containing constituents in these
experiments are shown in Fig. Jl. It was concluded from this
experimental series that the yield of chlorine -containing constit-
uents increases with increasing reaction time and that chlorination
with either hypochlorite or chlorine gas gives essentially equivalent
results. A comparison of radioactive chromatographic peaks in
secondary sewage effluents with those found in primary sewage effluent
78
-------
VO
10
25
50
100
ELUTION VOLUME.ml
225
300
325
10'
09
.08
SfT
I H0«
»i- 3.O5
10
10
.02
.01
o
1 1 1 1 I
f MTt ••!/»* I
nn,M(44l
P4 TOTAL.
^7^
M
_l_
40
42 44
425
46
i ' i J - --•-——,.—
EUIT10N TIME. hr
54
56
350
ISO ELUTION VOLUME.ml 525
I nm
{—^ M^i Af*Tn/tTY
w At i mil
•" ~ ~~ ^ Z5A ft(n
SSO 373 4OO 423 45O 'ELUTION VOLUME.ml 525
OF CHLORINATED EFFLUENT (1.0 mg/llllr COMBINED CHLORINE RESIDUAL .ORTHOTOLIDINE I CONCENTRATED I68OX 254 nm
62
375
68
6OO
Fig. 30. Dual-Column UV-Analyzer Chromatograms of Chlorinated Effluent from a Secondary Sewage
Treatment Plant.
-------
0*HL 0*« T3-24M »«
oo
o
3! (8) (9) (II) (13) (15) (18
A^IJ, ^,V . V.--,-^
10
2*5
90
100
ELUTION VOLUME.nl
200
250
325
280/a OF EFFLUENT CHLORINATED IN LABORATORYll.Omg/lltor COMBINED CHLORINE RESIDUAL. ORTHOTOLIDINE }
IS mln REACTION TIME. CONCENTRATED 644X. "Cl ACTIVITY
45 mln REACTION TIME. CONCENTRATED 582 X. MCI ACTIVITY
90 mln REACTION TIME. CONCENTRATED 490 X. "CI ACTIVITY
Fig. 31. Chromatograms of Secondary Sewage Treatment Plant Effluent Chlorinated with Ifypochlorite
Solution for 15-, k5~, and 90-min leaction Times.
-------
(see Figs. 29-31) indicated that most of the chlorine-containing
peaks in the secondary effluent are also found in the primary effluent.
As noted in these figures, the chromatographic peaks were correlated
and, for purposes of identification, assigned number designations.
Several experiments were required to determine the nature of the
chlorine-containing peaks. In two runs (samples H-3 and H-^), only
Cl-tagged chloride ion was chromatographed, proving that the
extremely large chlorine-containing peak (peak 3!) was indeed chloride.
In one run, H-5, the Cl was introduced to the secondary effluent
sample as chloride ion and concentrated. The results obtained by
chromatographing the 880-fold concentrate conclusively showed by
absence of radioactive peaks other than the chloride peak that the
chlorine-containing constituents had indeed been created by the
chlorination of the secondary effluent, and did not result from
chloride-complex formation with inorganic material and/or from isotopic
exchange with chlorine-containing constituents which may have been
in the effluent prior to chlorination. In one run, H-7, a synthetic
effluent containing only inorganic constituents at approximately the
same concentrations as that in the secondary effluent, as determined
by spark-source mass spectrometry, was chlorinated to a 1 ppm chlorine
residual using chlorine gas and concentrated. After chromato-
graphing the 850-fold concentrate, no significant peaks
other than chloride were found, thus proving that none of the
chromatographic peaks of radioactivity resulted from complexes of
hypochlorous acid, hypochlorite ion, or chloramines with inorganic
constituents in the secondary effluent. Thus it was concluded from
this series of experiments that the large majority of chlorine-
containing peaks found in the chlorinated effluents were stable
organic compounds.
Analyses of all the chromatograms (Figs. 29-3!) showed that at
least 62 chlorine-containing constituents which possibly were stable
chloro-organic compounds were separated on the chromatograph. The
concentrations of these constituents, in nanograms of Cl per liter
of original effluent, are given in Table 15.
81
-------
TalDle 15. STABLE CHLORINE-CONTAINING ORGANIC CONSTITUENTS HT CHLORINATED EFFLUENTS
FROM DOMESTIC SANITARY SEWAGE TREATMENT PLANTS
(Concentration of the constituents, ng Cl per liter of original effluent)
Constituent
1
2
3
k
5
6
7
8
9
10
11
12
13
ib
15
16
17
18
19
20
21
22
23
Experiment
H-ia
3120
221*0
30lK>
lj-520
7320
630
1070
1^20
9260
980
2130
6200
3950
2860
1090
19^0
3150
3550
H-2*
12^0
890
760
630
2k60
560
900
W>
520
2760
6ko
700
I&IO
1000
770
1210
2610
1730
H-6C
1000
560
ij-60
3^0
1550
260
130
310
600
190
13^0
^30
290
870
530
580
hko
610
1060
1^20
82 -
H-8d
U60
150
150
150
120
1^0
100
110
670
1^0
170
2i|-0
k6o
210
190
^30
230
330
510
H-9e
i^O
2^0
350
lOlf-0
250
120
170
210
1120
200
10l)-0
220
270
2lfO
370
170
320
H-10f
610
260
290
2^0
1070
260
360
230
1230
' 260
230
1530
ij-70
210
2^0
6ko
680
560
-------
Table 15 (continued). STABLE CHLORINE-CONTAINING ORGANIC CONSTITUENTS
IN CHLORINATED EFFLUENTS FROM DOMESTIC SANITARY SEWAGE TREATMENT PLANTS
(Concentration of the constituents, ng Cl per liter of original effluent)
Constituent
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Experiment
H-ia
4810
3330
2170
4030
3160
4230
[21.3 mg]g
840
1500
1520
910
350
800
220
14400
180
530
90
160
430
380
503
190
350
H-2*
1780
n4o
1210 -
1280
1810
; [20.9mg]g
240
140
190
150
5610
120
490
120
150
90
790
H-6C
590
910
1010
930
850
[17.2 mg]g
390
190
90
2450
160
90
50
120
160
80
70
430
H-8d
550
560
770
910
[24.0 mg]§
340
170
230
i4o
50
1670
270
4320
80
60
120
160
230
130
H-9e
370
330
610
990
[21.6 mg]g
290
190
70
170
220
180
20
1750
60
3260
60
50
100
210
130
140
170
H-10f
490
510
910
1690
[17.3 mg]S
270
120
150
no
130
50
100
2600
i4o
338o
190
50
170
190
180
150
290
83
-------
Table 15 (continued). STABLE CHLORINE-CONTAINING ORGANIC CONSTITUENTS
IN CHLORINATED EFFLUENTS FROM DOMESTIC SANITARY SEWAGE TREATMENT PLANTS
(Concentration of the constituents, ng Cl per liter of original effluent)
Constituent
1*9
50
51
52
53
51*
55
56
57
58
59
60
61
62
Total
Number of
constituents
Experiment
H-ia
170
210
120
1700
21*30
108,183
1*7
H-2b
5**0
220 /
650
'
100
130
260
ll*0
210
120
38,910
1*1*
H-6°
20
120
70
70
170
200
30
90
60
30
60
60
80
180
22,780
52
H-8d
1*0
30
730
130
150
110
60
170
17,800
,6
H-9e
250
100
80
1*70
80
170
250
190
ll*0
220
300
260
270
18,550
52
H-10f
220
90
100
1*80
200
80
80
220
90
180
120
220
3^0
300
23,990
*
aPrimary effluent; 2 ppm chlorine residual; estimated chlorination contact time,
2l*0 min; chlorinating agent, 36ci-tagged chlorine gas.
^Secondary effluent; Ijppm residual; estimated chlorination contact time, 2l*0 min;
chlorinating agent, 36ci-tagged chlorine gas.
°Secondary effluent; 1,-Ppm chlorine residual; chlorination contact time, 90 min;
chlorinating aeent. 3°ci-taeeed chlorine gas.
^Secondary effluent; 1 ppm chlorine residual; chlorination time, 15 min; chlorinating
agent, 3oci-tagged hypochlorite solution.
Secondary effluent; 1 ppm chlorine residual; chlorination time, 1*5 min; chlorinating
agent, 3°ci-tagged hypochlorite solution.
fSecondary effluent; 1 ppm chlorine residual; chlorination time, 90 min; chlorinating
agent, 36d-tagged hypochlorite solution.
^Theoretical values are 2l*.0, 2l*. 8, 25.0, 25.6, 25.6, and 25.6 mg, respectively.
81*
-------
Tentative identifications of If of the chlorine-containing constit-
uents were established by comparison of their anion exchange elution
volumes with those determined for reference standards. Reasonable
agreement of elution volumes is presumptive evidence that the unknown
may indeed be the same compound as the reference standard. Table 16
gives tentative identifications of these organic compounds, along
with their concentrations as calculated from data for Experiment H-9
in Table 15. Because the extent of chlorination of this sample is
approximately equivalent to that expected at the Oak Ridge East Sewage
Treatment Plant, the concentrations of the tentatively identified
compounds would approximate those expected at that sewage treatment
plant. The concentrations were calculated from data which were
based on the reasonable assumption that complete isotopic dilution
of the Cl occurred rapidly during the chlorination process. There-
fore, our values may be high if the chlorination reaction by which
the compounds are formed proceeds at a rate equivalent to or faster
than the chlorination rate for the formation of chloramines.
The magnitude of the chlorination effect is dependent on the
chlorine dosage and the total reaction time. In Experiment H-9>
with a chlorination reaction time (^-5 min) and conditions (chlorine
residual, 1 mg/liter) approximately equivalent to those at ORESP,
0.6$ of the chlorine dose is associated with stable chlorine-containing
organic constituents separated chromatographically from the chlorinated
effluent; an additional Q.k% is associated with chlorine-containing
constituents which did not elute from the resin. Therefore, the
total chlorination yield was about 1$ of the chlorine dose. The
chlorination yields are calculated based on the assumption that
during the chlorination, complete isotopic dilution of the Cl
in the chlorinating agent occurs with the "pool" of nonradioactive
chlorine in the effluents. The remainder of the available chlorine
was apparently utilized in oxidation reactions.
Since the experimental conditions are similar to the field
conditions at ORESP, chlorination at the sewage treatment plant
85
-------
Table 16. TENTATIVE IDENTIFICATIONS AND CONCENTRATIONS OF CHORINE-CONTAINING CONSTITUENTS
IN CHLORINATED EFFLUENTS
Constituent
16
••^8
** "19
22
32
42
43
45
52
53
55
56
57
59
61
62
Peak elution
volume,
ml
72.4 + 2.7
80.2+ 4.0
86. 2 + 4. 2
102 + 3. 3
218
302+ 5-9
312+ 5-9
334 + 6.4
403 ±1.7
415 ± 3.8
436+ 5.4
444+ 4.4
464+ 8.7
496 +8.7
527 + 15.6
547 + 20. 2
Tentative
identification
5-Chlorouracil
5-Chlorouridine
8-Chlorocaffeine
6-Chloro-2-aminopurine
8-Chloroxanthine
2-Chlorobenzoic acid
5 -Chloro salicylic acid
4-Chloromandelic acid
2 -Chlorophenol
4-Chlorophenylacetic acid
4-Chlorobenzoic acid
4-Chlorophenol
3-Chlorobenzoic acid
and/or. 3 -Chlorophenol
4-Chlororesorcinol
3 -Chloro -4-hydroxy-
benzoic acid
4-Chloro - 3 -methylphenol
Reference standard
elution volume,
ml
72a
81
86
109
218
307 ± 9
310
338
400
4ll
434 + 11°
446
455 + 10°
456
495
540
550
Concent rat ion
of organic
compound,
lag/liter
4.3
1.7
1.7
0.9
1.5
0.26
0.24
1.1
1.7
0.38
1.1
0.69
0.62 /
o.51d
1.2
1.3
1.5e
a
'Average of two determinations.
"Average of seven determinations + standard deviation.
°Average of four determinations + standard deviation. ,
^Values based on the assumption that chlorine is present as the pure compound of either, and
not a mixture of both.
Concentration in sample H-2.
-------
should produce similar chlorination yields; i.e., about 1% of the
chlorine dose should be associated with stable chlorine-containing
organic constituents. Table IT summarizes the chlorination yields
obtained in the experimental series.
87
-------
Table 17. PERCENTAGE CHLORINATION YIELD OF CHLORINE -CONTAINING
CONSTITUENTS WITH RESPECT TO REACTION TIME
FOR THE CHLORINATION OF EFFLUENTS
Reaction time,
min
15
^5
90
2kO
Percentage chlorination
yield of chromatograph-
able chlorine-containing
constituents
0.55
0.58
0-75-0.87
1.6-1.8
Percentage chlorination
yield of total chlorine-
containing constituents
0.93
0.98
1.27
Q
The yield is expressed as percent of chlorine dose which, after chlorina-
, tion, is associated with stable chlorine-containing constituents.
Percentage chlorination yield of chromatographable chlorine-containing
constituents separated as peaks in the radioactive tracer experiments.
Percentage chlorination yields of the total chlorine-containing con-
stituents separated from the chlorinated effluent. The total is defined
as the sum of both chromatographable constituents and the constituents
that did not elute from the resin during the chromatographic separation,
prorating the noneluting constituents between H-8, -9, and -10 based
on the proportionate amount of chromatographable constituents determined
for the respective experiments.
-------
SECTION VII
COOPERATIVE EFFORTS WITH ENVIRONMENTAL PROTECTION AGENCY LABORATORIES
In accordance with the specific aims stated in the program proposal,
two high-resolution chromatographs for the analysis of stable organic
compounds were designed, constructed, and tested. One was delivered
to the Advanced Waste Treatment Research Laboratory (AWTRL), the
other to the Southeast Environmental Research Laboratory (SERL).
Supportive efforts in applying the instrument to field problems included
assistance in the areas of operation, maintenance, and minor modifications
Other efforts under this contract made in cooperation with personnel
from two EPA laboratories included: (1 ) identification of constituents
in samples furnished by C- I. Mashni of AWTRL; and (2) lyophilization
and preparative mode chromatography of sewage plant effluent samples
for subsequent analysis by A. W. Garrison of SERL.
CONSTRUCTION OF TWO UV-ANALYZERS FOR ENVIRONMENTAL PROTECTION AGENCY
LABORATORIES
A significant part of this program has been concerned with the
construction of two UV-Analyzers for use at EPA Laboratories. The
design of the Mark II UV-Analyzer (from the Body Fluids Analyses
Program) was selected for the instrument that was constructed for
the AWTRL. This instrument was subsequently converted to a Mark IIA;
the analyzer constructed for SERL was an updated Mark IIA version.
The use of this analyzer design simplified the problem of peak
identification since the identifications that are made and the techniques
that are used in the parallel work of the Body Fluids Analyses Program
are directly applicable.
Fabrication of the first analyzer was completed in November 1970
at a cost of $13,200, and the second was completed in November 1973
at a cost of $14,500. Both analyzers were tested at ORNL to ensure
satisfactory operation, and excellent results were obtained. The
89
-------
resolution in each case was equal to or better than that obtained with
the UV-Analyzers (see Fig. 32). During the shakedown period of each
instrument, a scientist from the EPA Laboratory was given three days
of training in operation and maintainance of the instrument. After
shakedown, the instruments were delivered to the respective EPA
Laboratories, where they continue to be operated by EPA personnel.
IDENTIFICATION OF ORGANIC CONSTITUENTS IN AWTRL SAMPLES
A 20-liter sample of raw sewage was taken on January 5> 1971 > at
the Mill Creek Plant in Cincinnati by Mr. Charles Mashni of the AWTRL.
He concentrated the sample to 30 ml by vacuum diatillation and freeze -
drying. A 5-ml portion of concentrate was then shipped to ORNL and
chromatographed on our preparative-scale UV-Analyzer. Chromatographic
eluent fractions associated with the major peaks were isolated and
investigated using gas chromatography and mass spectrometry. One
organic compound which eluted from the anion exchange column at kO hr
was identified by mass spectrometry as o_-phthalic acid. The molar
extinction coefficient of a reference sample of o_-phthalic acid was
o
determined to be 1120 absorbance-cm /g-mole at a wavelength of
280 nm. The chromatographic peak representing this compound was then
quantitated to give an p_-phthalic acid concentration of 0.2 ug/ml
in the original sewage. Although gas chromatographic and mass spectral
data on some of the other peaks were obtained, no identifications could
be assigned and further characterization was not considered to be
profitable.
A sample of raw sewage was chlorinated at AWTRL, and portions of
this material were chromatographed, before and after chlorination, on
their UV-Analyzer. Fractions containing uv-absorbing peaks were
collected, lyophilized, and sent to ORNL for possible identification.
One fraction of the raw sewage sample contained xanthine; another
contained guanosine. Two others coeluted with adenosine and
1-methylxanthine, but these identifications were discounted by other
90
-------
OKNL OWt 74 - TS«e
400
Huron VOLUHC,at
(a)
(b)
Fig. 32. Chromatograms of UV-Absorbing Constituents Developed on the Southeast Environmental
Research LaTx>ratory UV-Analyzer. a) 0.25 ml of Reference Urine (URS-IV); b) 0.25 ml of Primary
Effluent (1000X).
-------
tests. One fraction of the chlorinated sample contained uracil,
5-acetylamino-6-amino-3-methyl uracil, and N-methyl-2-pyridone-5-
carboxamide. The remainder of the fractions from both samples did
not contain a sufficient quantity of material to obtain gas chromato-
graphic and/or mass spectral data for identification.
ASSISTANCE TO THE SOUTHEAST WATER LABORATORY IN IDENTIFICATION
OF COMPOUNDS IN SEWAGE PLANT EFFLUENTS
Concentrates of three samples taken at AWTRL were lyophilized and
chromatographed on the preparative-scale UV-Analyzers. Large volumes
(20 liters ) of raw sewage influent and effluents from the Activated
Sludge Sewage Treatment and Physical-Chemical Pilot Plants at the
Robert A. Taft Water Research Center were collected and concentrated
to less than 200 ml by Wayne Garrison and Frank Allen of SERL. These
concentrates were shipped to ORNL, where they were lyophilized to
dryness and then redissolved in a minimum volume of dilute ammonium
acetate buffer solution. Five-milliliter aliquots of each sample were
chromatographed on the preparative-scale UV-Analyzer (0«9 x 150 cm
column), and J-min fractions of the column eluate were collected.
The individual fractions that contained uv-absorbing constituents
were sent to SERL for subsequent gas chromatographic and mass spectral
analyses.
92
-------
SECTION VIII
DISCUSSION
The value of high-resolution liquid chromatography for analyzing
sewage plant effluents has been demonstrated; and, with the identification
of specific organic compounds present in effluents, the potential use of
the UV-Analyzer, particularly in the dual-column configuration, to
monitor and improve sewage plant operations becomes obvious. As
additional compounds are identified and their presence or absence noted
before and after various treatments, the efficacies of these treatments
can be further evaluated. The combination of radioactive tracer techniques
using Cl with the UV-Analyzer has demonstrated that chlorinated organic
compounds result from chlorination of water containing dissolved sewage
residues, and this combination can be used to study the effects of
chlorination on other waters. The use of the cerate-oxidative system
as an additional monitor has further broadened the capability of the
UV-Analyzer by adding to the list of detectable compounds and increasing
its sensitivity so that its applicability would extend to many other
compounds. Perchloratoceric acid is an extremely useful reagent for
rapidly determining the chemical oxygen demand at less than lOOjig/liter
levels.
Some of the more important contributions that the results of this
project could make to an understanding of the problem of water pollution
are: (1) identification of most of the uv-absorbing refractory organics
and many of the carbohydrates and other oxidizable components found
in "domestic" sewage plant effluents; (2) determination of the fate of
such compounds, particularly when the effluents are chlorinated, and
identification of the stable chlorinated compounds which result; and
(3) development of techniques for evaluation of the various methods
of removing these pollutants. It is expected that a significant
effort will continue to be directed toward the identification of
stable organic compounds separated on the UV-Analyzers at AWTRL
and SERL.
93
-------
SECTION IX
REFERENCES
1. Bunch, R. L«, E. F. Earth, and M. B. Ettinger. Organic Materials
in Secondary Effluents. J Water Poll Control Fed 3^; 122, 1961.
2. Middleton, F. M«, and A. A. Rosen. Organic Contaminants Affecting
the Quality of Water. Public Health Rept (U.S.) £1: 1125,
3. Katz, S., and W. W. Pitt, Jr. Automated Analysis of Polluted Water.
Oak Ridge National Laboratory, Oak Ridge, Term., USAEC Report
ORNL-TM-3862. August 1972. 53 p.
4. Katz, S., W. W. Pitt, Jr., C- D. Scott, and A. A. Rosen. The
Determination of Stable Organic Compounds in Waste Effluents at
Microgram per Liter Levels by Automatic High -Re solution Ion
Exchange Chroma tography. Water Res 6: 1029, 1972-
5« Ryckman, D. W., J. W. Irwin, and R. H. F. Young. Trace Organics
in Surface Waters. J Water Poll Control Fed 39; 661, 1967.
6. Baker, R. A. Trace Organics Contaminant Concentration by
Freezing, II. Inorganic Aqueous Solutions. Water Res 1^: 97;
1967.
7« Stevens, R. H. A Vacuum Distillation System for Concentrating
Heat Labile Solutions. Oak Ridge National Laboratory, Oak Ridge,
Tenn., USAEC Report K-1594. May 8, 1964. 25 p.
8. Scott, C- D., J. E. Attrill, and N. G. Anderson. Automatic, High-
Resolution Analysis of Urine for Its Ultraviolet -Absorb ing Constit-
uents. Proc Soc Exptl Biol Med 125; 181, 1967.
9- Scott, C- D. Analysis of Urine for Its Ultraviolet -Absorbing
Constituents by High-Pressure Anion Exchange Chroma tography.
Clin Chem 14; 521, 1968.
10. Scott, C- D., R. L. Jolley, W. F. Johnson, and W. W. Pitt, Jr.
Prototype Systems for the Automated, High -Resolution Analyses of
UV-Absorbing Constituents and Carbohydrates in Body Fluids. Clin
Pathol 535: 701, 1970.
11. Pitt, W. W., Jr., C. D. Scott, W. F. Johnson, and G. Jones, Jr.
A Bench-Top, Automated, High Resolution Analyzer for Ultraviolet-
Absorbing Constituents of Body Fluids. Clin Chem 16: 657, 1970.
12. Pitt, W. W-, Jr., C. D. Scott, and G. Jones, Jr. Simultaneous
Multi-column Operation of the UV-Analyzer for Body Fluids. Clin
Chem 18; 7^7, 1972.
-------
13- Katz, S., S. R. Dinsmore, and W. W. Pitt. A Small Automated
High -Resolution Analyzer for Carbohydrate Constituents of Body
Fluids. Clin Chem 17_: 731, 1971-
Ik- Van Deemter, J. J., F. J. Zuiderweg, and A. Klinkenberg. Chem
Eng Sci £: 271, 1956.
15. Scott, C. D., D. D. Chilcote, and N. E. Lee. Coupled Anion
and Cation Exchange Chromatography of Complex Biochemical
Mixtures. Anal Chem 44: 85, 1972.
16. Katz, S., and W. W. Pitt, Jr. A New Versatile and Sensitive
Monitoring System for Liquid Chromatography: Cerate Oxidation
and Fluorescence Measurement. Anal Letters £.(3): 177, 1972-
17. Thacker, L. H. A Miniature Flow Fluorometer for Liquid
Chromatography. J Chromatog. In Press.
18. Smith, G. F. Cerate Oxidimetry. 2nd Ed. Columbus, Ohio, The
G. Frederick Smith Chemical Co., 1964. 81 p.
19. Klein, L. J. In: Proc Inst Sewage Purif (England). 1941.
p. 174-191.
20. Armstrong, W. A., D. W. Grant, and W. F. Humphreys. Anal Chem
25.: 1300, 1963.
21. Jolley, R. L., W. W. Pitt, Jr., and C. D. Scott. Nonpulsing
Reagent Metering for Continuous Colorimetric Detection Systems.
Anal Biochem 2£: 300, 1969.
22. Standard Methods for the Examination of Water and Waste Water.
13th Ed. APHA, AWWA, WPCF, p. 495-499-
23. Mrochek, J. E., W. R. Butts, W. T. Rainey, Jr., and C. A. Burtis.
Separation and Identification of Urinary Constituents by Use of
a Multiple -Analytical Technique. Clin Chem 17: 72, 1971.
24. Krueger, P. M., and J. M. McCloskey. Porous Stainless Steel as
a Carrier Gas Separator Interface Material for Gas Chromatography-
Mass Spectrometry. Anal Chem 4l; 1930, 1969.
25" McKinney, F. E., S. A. Reynolds, and P. S. Baker. Isotopes
User's Guide. Oak Ridge National Laboratory, Oak Ridge, Tenn.,
USAEC Report ORNL-IIC-19. Sept. 29, 1969. 96 p.
26. Jolley, R. .L. Chlorination Effects on Organic Constituents in
Effluents from Domestic Sanitary Sewage Treatment Plants. Ph.D.
dissertation. University of Tennessee, Knoxville, Tenn., 1973;
Oak Ridge National Laboratory, Oak Ridge, Tenn., USAEC Report
ORNL-TM-4290. October 1973. 342 p.
95
-------
27- Standard Methods for the Examination of Water and Waste Water.
13th Ed. APHA, AWWA, WPCF, p. 385-386. 1971-
96
-------
SECTION X
PUBLICATIONS
1. Gehrs, C- W., L. D. Eyman, R. L. Jolley, and J. T. Thompson.
Effects of Stable Chlorine -Containing Organics on Aquatic
Biota. Nature. Accepted for Publication.
2. Jolley, R. L. Chlorination Effects on Organic Constituents in
Effluents from Domestic Sanitary Sewage Treatment Plants. Ph.D.
dissertation, University of Tennessee, Knoxville, Tenn., 1973;
Oak Ridge National Laboratory, Oak Ridge, Tenn., USAEC Report
ORNL-TM-4290, October 1973. $k2. p.
Jolley, R. L. Determination of Chlorination Effects on Organic
Constituents in Sewage Treatment Plant Effluents : A Coupled
36Cl Tracer — High -Resolution Chroma tographic Technique. Presented
at the l67th American Chemical Society. Los Angeles, Calif.
Mar. 31 - Apr. 5,
4. Jolley, R. L. Chlorine -Containing Organic Constituents in
Chlorinated Effluents from Sewage Treatment Plants. Journal
Water Pollution Control Federation. Accepted for Publication.
5. Jolley, R. L., S. Katz, J. E. Mrochek, W. W. Pitt, Jr., and
W. T. Rainey. A Multicomponent Analytical Procedure for Organics
in Complex, Dilute Aqueous Solutions. Chemical Technology.
Accepted for Publication.
6. Jolley, R. L., W. W. Pitt, Jr., and C. D. Scott. High -Resolution
Analyses of Refractory Organic Constituents in Aqueous Waste
Effluents. In: Realism in Environmental Testing and Control.
Proceedings of the 19th Annual Technical Meeting, The Institute
of Environmental Sciences, Anaheim, California, 1973- p. 2Vf-252.
7- Jolley, R. L., C. D. Scott, W. W. Pitt, Jr., and M. D. McBride.
Determination of Trace Organic Contaminants in Natural Waters
by High -Resolution Liquid Chromatography. Proceedings of the
First NSF Trace Contaminants Conference, Oak Ridge, Tenn.,
August 8-10, 1973. In Press.
8. Katz, S., and W. W. Pitt, Jr. A New Versatile and Sensitive
Monitoring System for Liquid Chromatography: Cerate Oxidation
and Fluorescence Measurement. Anal Letters £_: 177, 1972.
9. Katz, S., W. W. Pitt, Jr., and G. Jones, Jr. Sensitive Fluorescence
Monitoring of Aromatic Acids after Ani on -Exchange Chromatography
of Body Fluids. Clin Chem 1£(8): 817, 1973-
97
-------
10. Katz, S., W. W- Pitt, Jr., J. E. Mrochek, and S. Dinsmore. Sensitive
Fluorescence Monitoring of Carbohydrates Eluted by a Borate Mobile
Phase from an Anion Exchange Column. Journal of Chromatography.
Submitted for Publication.
11. Katz, S., W. W- Pitt, Jr., C. D. Scott, and A. A. Rosen. The
Determination of Stable Organic Compounds Present in Water at
ppb Levels by Automatic High-Resolution Ion Exchange Chromatography.
Presented at the l6lst American Chemical Society National Meeting,
Los Angeles, Calif., Mar. 29 to Apr. 2 1971.
12. Katz, S., W. W..Pitt, Jr., C. D. Scott, and A. A. Rosen. The
Determination of Stable Organic Compounds in Water Effluents
at ug/liter Levels by Automatic High-Resolution Ion Exchange
Chromatography. Water Res 6: 1029, 1972.
13- Pitt, W. W., Jr. Analysis for Trace Organic Contaminants in
Polluted Water by High-Resolution Ion Exchange Chromatography.
Presented at the Gordon Conference on Ion Exchange, Wolfeboro,
N. H., Aug. 1, 1973.
14- Pitt, W. W., Jr., and R. L. Jolley. High-Pressure Ion Exchange
Chromatography for Analysis of Water Pollutants. Presented at the
Symposium on Identification and Transformation of Aquatic
Pollutants, Athens, Ga, Apr. 8-10, 1974.
15. Pitt, W. W., Jr., R. L. Jolley, and C. D. Scott. Determination
of Trace Organic Contaminants by High-Resolution Ion Exchange
Chromatography. Environmental Science and Technology. Submitted
for Publication.
16. Pitt, W. W., Jr., S. Katz, and L. H. Thacker. A Rapid Sensitive
Method for the Determination of the Chemical Oxygen Demand of
Polluted Waters. Presented at the 73rd National Meeting of
the American Institute of Chemical Engineers, Minneapolis,
Minn., Aug. 30, 1972; Water 1972. A-I.Ch.E. Symposium Ser.
129, Vol. 69, 1973-
17. Pitt, W. W., Jr., C- D. Scott, and M. D. McBride. Determination
of Trace Organic Contaminants by High-Resolution Liquid Chromato-
graphy. Presented at the Seventh Annual Conference on Trace
Substances in Environmental Health, University of Missouri,
Columbia, Mo., June 12-14, 1973-
98
-------
SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
W
T:tls
Automated Analysis of Individual Refractory Organics In
Polluted Water'
/ Auihor(s)
W. Wilson Pitt, Robert L. Jolley and Sidney Katz
> Or%an 3: n
Oak Ridge National Laboratory (operated by Union Carbide
Corporation for the U.S. Atomic Energy Commission)
Oak Ridge, Tennessee 3?830
5. .Report D
6.
8. Pe-formir Organization
Rt^ortNo.
16ACG 03
lU-12-833
13. Type c ' Repor and
Period Covered
12. sponsoring Organiza- on Environmental Protection Agency
.' ", Slip iTT.r . - '
Environmental Protection Agency report number, EPA-660/2-7U-076, August
(6. Abstract
Residual organic compounds present in municipal sewage treatment plant effluents at
microgram-per-liter levels were analyzed using high-resolution anion-exchange chromato-
graphy. Effluents were concentrated 50- to 3000-fold by vacuum evaporation and freeze-
drying and then analyzed by liquid chromatographs capable of detecting uv-absorbing,
oxidizable (with sulfatoceric acid), or carbohydrate constituents. Using techniques
such as uv spectroscopy, gas chromatography, and mass spectrometry, 56 organic compounds
were identified in primary effluent and 13 organic compounds in secondary effluent.
Some of these constituents were quantified.
Chromatographic procedures, coupled with radioactive tracer chlorination, were
applied to the analysis of chlorinated primary and secondary effluents. More than 60
peaks containing chlorine were found, and specific chlorinated compounds were tenta-
tively identified by cochromatography and quantified at the 0. 5- to k-ug/liter level.
A detector system for liquid chromatography based on cerate oxidimetry was adapted
as a rapid, sensitive continuous monitor for measuring the COD of waters. The effects
of column geometry and operating parameters on Chromatographic resolution were studied.
Two high-resolution, ion exchange chromatographs (UV-Analyzers) were constructed for
U.S. Environmental Protection Agency research laboratories, and are being used in the
analysis of treated sewage effluents and other polluted waters. (Jolley — Oak Ridge
National Laboratory)
n a. Descriptors ^Pollutant identification, *Sewage effluents, ^Chromatography, ^Organic
compounds, *Chlorination, Water analysis, Water pollution, Pollutants, Municipal wastes,
Carbohydrates, Analytical techniques, Separation techniques, Instrumentation, Chemical
oxygen demand, Gas chromatography, Spectrometry, Photometry, Fluorometry, Ion exchange,
Radioactive tracers, Chlorine, Sewage treatment, Waste water treatment.
176. identifiers *High-resolution liquid chromatography, -^Primary effluent, ^Secondary
effluent, ^Ultraviolet-absorbing constituents, *Chlorine-containing organics,
*0xidisable constituents, Unknown organics characterization, Concentration technique,
Column geometry studies, Operating parameter studies, Cerate oxidimetry, Continuous COD
monitor, flow fluorometer, chlorination effects, chlorinated effluent, chlorination
reaction yield, chlorine-36.
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Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C. 2O24O
institution Oak Ridge National Laboratory
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