ORNL DWG 72-600! RI

Coq

REFERENCE
COMPOUND

GUANOSINE (29.54)

| | | | 1 I i i
PRIMARY EFFLUENT

COMPOUND
wi
ae
6' x 1/4" 3% OV-I7 se
80/100 CHROMOSORB W(HP) 38
100° —> 325°C AT 10°/min =
Lg
! L L ! ! L ! ! ! L
2 4 6 6 0 @ = (6G 8 20

TIME (min)

Fig. 2h. Gas Chromatograms of TMS Derivatives of Reference Compound
(Guanosine) and Liquid Chromatographic Fraction from Primary Sewage Treatment
Plant Effluent. :

52


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 0.45 cm x 150 cm columns, then 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


RESEARCH REPORTING SERIES

Research reports of the Office of Research and
Monitoring, Envixonmental 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

4. 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 of Documents, U.S. Government Priating Office, Washington, D.C. 20402 - Price $1.75


a
Table 10. DATA ON UNKNOWN COMPOUNDS FOUND IN CHLORINATED SEWAGE
PLANT EFFLUENT (SPJ-3) FROM THE OAK RIDGE WEST SEWAGE PLANT

Elution
time Molecular GC retention data, M.U.
hr weight OV-1 OV-17 A
1.05 221 17-58 17-86 +0.28
LA 98 13.1 -
14 127 14.79 15.49 +0.70
Ly 113 15.45 16.04 40.59
14.0 110 U.10 14.88 +0.78
14.0 138 15.0 -
58 138 15.04 16.24 +1.20
58 236 25.64 27.10 +1046

ay ppm chlorine residual.

61


TL

ARsonDANCE
oP RERRRRSE

ohm owe ra 3210 #3

{290/28 PRMARY EFFLUDT (AS mytie BATIA. concen 40 meter Comme JORTHOTOLIOMEXCONCENTRATED 730 x -|
ANION EXCHANGE CHROMATOORAPH RUK CONDITIONS! 4
ABSORBANCE MEASURED AY 280m; 0 Sym «t20cm ATAINLERS STEEL COLUMN WITH S-18 ; DIAM. AMINEX 4-27 RESIN,

‘TEMPERATURE PROGRAM, AWBIENT TO BS'C AY Ibe, ELUENT GRADIENT WCREASING LINEARLY 16 CONCENTRATION FROW OID a
70 6.0 AMMONIUM ACETATE, 9H 4.4, ELUENT FLOW RATE 1Om1/be; COLUM PREESUME 1900 pug TOTAL 4

Fig. 26. UV-Analyzer Chromatograms of Primary Sewage Treatment Plant Effluent Chlorinated with

Different Amounts of Calcium Hypochlorite.

A ” . A 1. 1 1 1 A 1
° Fy = 7 c = 7 700 ) m0 ) x0 =
arin vane at
on + : r : 7 - 4
‘a :
at 290 19 PRMART EFFLUENT (HTL CLOWRE CONCENTRATE 17.0 mpi) CONCENTRATED 90 X -
a AMON EXCHANGE CHROMATOORAPH RUM CONDITIONS: BEE ABOVE a
aa :
od
it {
sr
al
a :
1 mn . 1 fi . '
° = % = 7 a 7 = 5 7s os as
euyron vous mt
or a
cr 290 JA PRMLARY EFFLUENT LDETIAL, CHLORD CONCENTRATION 88 mq/iter) CONCENTRATED 1000 3
id ANION EXCHANGE CHROMATOGRAPH RUM CONDITIONS: SEE ABOVE: 4
a
bal ?
sl AI
a


ORNL-OWG 70-12480 RI

Fig. 2. Schematic Diagram of Vacuum Distillation System.

8


Table 12.

DATA ON UNKNOWN COMPOUNDS FOUND

IN SPJ-11, COMPOSITE OF SPJ-3 AND SPJ-8
Molecular Elution TMS peaks GC retention data, M.U.
weight time, hr M Base (1) (2) (3) for OV-1 column
57 53 201 73 né = -158 12 ~
76 53 205" 73 7 = 7 13.5
82 0.8 ~ | 370 73 117.147 12.4
on 0.8 295% 5 6 149 10.4
101 1.0 2h5 147 116 73 11.4
106 1.0 235° 1s7 73 «17 12.4
117 385 189 73 70 174 11.8
117 5 295° 3 75 17h 14
128 32 344 259 73 ve) 14.6
129 38 345 3u5 330 73 16.0
132 1.5 333” 73 20h = 217 17.6
147 0.8 276" 146 73 5 16
149 0.8 365 262 350 73 16.4
152 0.8 281° 73 we 7 16
15h 0.8 355 3 18814 wy
157 68 373" BB 7 174 15.2
161 38 290” 203 73 («1K 14.7
162 Wy 291 B wi7 129 1.5
163 0.8 292" 189 73 «147 19
168 1.5 369 217 73 116 17.7
170 53 299% 209 22h 7 15.4
173 32 374 aa 27373 17.4
1IT 68 450” 73 75 «129 16.2
192 25 2h 2h9 7 yy al -
208 32 280 5 73 (265 18.9
214 68 502 297 73 io) 18.5
246 68 591° 3 % 93 20
266 68 482 73 164 ahs 22
285 53... | 573° 73 7 uy 22
28h, 53 | 356 73 > 7 22.5
"v-15 peak.

66


Table 1. RESOLUTION OF COUPLED ANION-CATION COLUMNS
DURING CHROMATOGRAPHY OF ORESP SECONDARY
SEWAGE EFFLUENT SAMPLES

Colum length, cm

Resolution

Anion Cation (No. of chromatographic peaks)
15 25 ho
50 50 45
25 15 35

25


13.

14.

15.

16.

17-

18.

20.

al.

22.

23.

oh.

25-

26.

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-

van Deemter, J. J., F- J. Zuiderweg, and A. Klinkenberg. Chem
Eng Sci 5: 271, 1956.

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.

Katz, S., and W. W. Pitt, Jr. A New Versatile and Sensitive
Monitoring System for Liquid Chromatography: Cerate Oxidation
and Fluorescence Measurement. Anal Letters 5(3): 177, 1972.

Thacker, L. H. A Miniature Flow Fluorometer for Liquid
Chromatography. J Chromatog. In Press.

Smith, G. F. Cerate Oxidimetry. 2nd Ed. Columbus, Ohio, The
G. Frederick Smith Chemical Co., 1964. 81 p.

Klein, L. J. In: Proc Inst Sewage Purif (England). 1941.
p- 174-191.

Armstrong, W. A., D. W. Grant, and W. F. Humphreys. Anal Chem
35: 1300, 1963.

Jolley, R- L., W. W. Pitt, Jr., and C. D. Scott. Nonpulsing
Reagent Metering for Continuous Colorimetric Detection Systems.
Anal Biochem 35: 300, 1969.

Standard Methods for the Examination of Water and Waste Water.
13th Ed. APHA, AWWA, WPCF, p- 495-99.

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.

Krueger, P. M., and J. M. McCloskey. Porous Stainless Steel as
a Carrier Gas Separator Interface Material for Gas Chromatography-
Mass Spectrometry. Anal Chem 41: 1930, 1969.

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.

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


ORNL DWG. 72- 13502

YOO a er
90- —— FLUORESCENCE 484
—"~UV ABSORBANCE
80r 486
E 8
60F go
g 5
ig 50r jez 2
8 a
us 40r 494
C4
3 3
330 96
ain! g
20) ee, ee OT iad ¢
tor Na canes donee “leo “
fs
Oo fey 4 6 8 10 12 14 16 is 20 22
2 HOURS
ay g
200 =
940 E
2 5
sie we
abr rye
Sup bei
eQesos
She LZe
100) T T T —_—T 7 T T T T T T T T T T IT
90F 270 »| SAMPLE OF —— FLUORESCENCE
sol 1000 x CONCENTRATE ORESP 77 WW ABSORBANCE
o SECONDARY EFFLUENT
© To} uw
z 2
60F =
u E
= SO Fe
8 2
8 ad 2
5 A is
30 it ES
Z f + :
20; naw \ yi £
ie — a eed LA. =
10F nu
o! a 1 4. 4 4 i 4. L 4 1 4
22 24 26 28 30 32 34 36 38 40
“HOURS
Fig. 18. Chromatogram of Secondary Sewage Plant Effluent Analyzed

by the UV-Analyzer with a Cerate Oxidative Monitor.

32


LS

Table 8. IDENTIFICATION OF MOLECULAR CONSTITUENTS IN 1000- TO 3000-FOLD CONCENTRATES
OF PRIMARY DOMESTIC SEWAGE

a Concentration, Anion exchange elution
Compound Identification method ug/liter position, ml
Ethylene Glyco1” AC, GC, MS 8
Maltose AC, GC 8
Galactose AC, GC 8
Glucose AC, GC 8
Glycerine AC, GC, MS 10
Galacito1” AC, GC, MS 10
Erythritol” AC, GC, MS 10
urea? AC, GC, MS 10
w-Methy1--pyridone-3-carboxamide AC, UV, GC 10 14
Phenylalanine” AC, GC, MS 390 16
Uracil AC, CC, UV, GC, MS 4o 18
5-Acetylamino-6-amino~3-methyl uracil AC, CC, UV, GC 140 20
N*-Methy1-2-pyridone-5-carboxamide AC, CC, UV, GC 20 21
Tyrosine” AC, GC, MS 23
Thymine AC, CC, GC, MS ~T 29
Theobromine AC, CC 2
7-Methylxanthine AC, cc ~90 29
Inosine AC, CC, UV, GC, MS 50 29
Hypoxenthine” AC, GC, MS 25 30
Xanthine AC, CC, UV, GC, MS 70 55
Copper (II) acetate (binuclear)*° MS 60
Adenosine AC, CC, UV, GC, MS 64
1,7-Dimethylxanthine AC, CC 64



92

SIGNAL CONN

Fig. 15.

SAMPLE OUTLET
FLUOROMETER BODY

QUARTZ FLOW CELL
COMPENSATION
APERTURE
ADJUSTMENT.
FLUORESCENCE VIEWING SLIT:

BLOCKING FILTER

UV CONVERSION FILTER

INSULATING MOUNT
PHOTOMULTIPLIER HOUSING

PHOTOMULTIPLIER TUBE

VOLTAGE DIVIDER CIRCUIT

HIGH VOLTAGE CONNECTOR

Exploded View of Flow Fluorometer Body.

ORNL DWG. NO. 72-4212 Ri

EXCITATION SLIT
EXCITATION FILTER

LAMP COMPENSATION PHOTOCONDUCTOR PLUG


Table 9. DATA ON UNKNOWN COMPOUNDS FOUND
IN UNCHLORINATED PRIMARY EFFLUENT (SPJ-1)
FROM THE OAK RIDGE EAST SEWAGE PLANT

Elution
time, Molecular GC retention data, M.U.
br weight OV-1 OV-17 A
0.6 135 14.99 15.90 +0.91
0.6 130 15.0 -
0.6 202 17.5 -
0.6 107 20.0 -
0.9 100 15.42 -
0.9 130 18.00 17. 94 -0.06
0.9 146 17.58 18.17 +0.59
0.9 158 13.13 13.89 +0.76
1.8 ny 13.88 15.36 +148
1.8 130 15.0 -
1.8 136 15.0 -
15.8 118 13.50 14.96 +146
15.8 131 16.92 19.05 +2.13
15.8 ran 21.68 2k. 7h +2.88
46.5 150 13.51 15.00 41.49



27. Standard Methods for the Examination of Water and Waste Water.
13th Ed. APHA, AWWA, WPCF, p. 385-386. 1971.

96


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(IIL) solution of
known concentration (1 to 2x 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
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


6L

Lomas owe 75-ncarey

or
$
10°
9 1-8
\ \,
‘\
0 14 ELUTION TIME, hr 20
TT ELUTION VOLUME, mi 225 250 275, 300 ry i
a) —— r

10 cy
s 7
£ 08
§ 10%} 395)
= i 04
Fae

2

rotl

a a
ob
a 1 eer ennaer ees
0 4 % 38 46 az ae ie ELUTION TIME. Rr
380 ats “400 “488 ELUTION VOLUME. mi bs 280 8 600 mi
280m2 OF CHLORINATED EFFLUENT (1,0 mg/liter COMBINED CHLORINE RESIOUAL ORTHOTOLIDINE } CONCENTRATED 1680X,—.._ 254.0m

— er activity

280ut OF EFFLUENT CHLORINATED IN LABORATORY (1.0 mg/Iller COMBINED CHLORINE RESIDUAL. ORTHOTOLIDINE ) CONCENTRATED 1040x,{——__ pact AC

Fig. 30. Dual-Colum UV-Analyzer Chromatograms of Chlorinated Effluent from a Secondary Sewage
Treatment Plant.

ACTIVITY (epm /mi)


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.


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


ce

ORNL DWG 72-8759

CERIUM(IV) + ELUTED COMPOUND —»
(NONFLUORESCENT)

CERIUM(III) | + OXIDIZED PRODUCTS
(FLUORESCENT)

FLUORESCENCE EXCITATION MAXIMUM: 260 nm

‘FLUORESCENCE EMISSION MAXIMUM: 350 nm

Fig. 19. Cerium Fluorescence Oxidimetry.


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 3400-fold concentrates of secondary effluent. These

compounds, along with the characterization data, are listed in

Table 14.

65


4

ORNL DWG. 72-11703 Ri

ROTARY
EVAPORATOR

-}------+-

No.3 MERCURY

WATER TRAP MANOMETER
NO. 2 {ORY ICE-TRICHLORO- Hag
WATER TRAP ETHYLENE) Areas
(ORY ICE)
SODA- LIME
WATER TRAP BED

(DRY ICE -TRICHLOROETHYLENE)

35°C WATER
BATH

Fig. 28. Schematic of Apparatus Used for Concentration of Radioactive Samples.


EPA 660/2-74-076
August 1974

Environmental Protection Technology Series



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 30-fold 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(III)
fluorometrically.

Li


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

the ratio of the elution volume to the column geometric volume; that

is,
af (U2 ]* x
min Uy AL”

R, = 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° x 28 = 167 sec. -

Considering the above data and typical sewage effluent chromatograms, it
became apparent that a shorter column operated at a higher linear velocity
could be used effectively. Hence, a 0.45-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 AWIR 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 anion-exchange colum. In order to optimize this
coupled-column system for analysis of sewage samples, a sample of ORESP
secondary effluent was analyzed using three different combinations of

Cs)


(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-4), only
3 Co1-tagged chloride ion was chromatographed, proving that the
extremely large chlorine-containing peak (peak 31) was indeed chloride.
In one run, H-5, the 36g, 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. 29-31) 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


9L

ACTIVITY (opm fat?

T T T T T T T tT T —T T T T T —1—T T T T
wt AMION EXCHANGE CHROMATOGRAPH RUN CONDITIONS!
jm-iD 4 180-am ATAINLESS STEEL, WITH S-12 p DIAM. AMIMEX AZT REBIK,
GAT Sb, ELUENT ORAGIEWT WERE
ii comer TO S.O1 AimOMOUM ACETATE, ph 4.4
‘COLUMN PRESSURE 1600 poig TOTAL

‘200 pt OF CHLORINATED E7FLUEKT 28 mg/tier COMBINED CHLORDIE AESIOUAL, ORTHOTOLIOINE} CONCENTRATED (009%, ——-—— 280.00

Lommerounie) eovcaraares o70 x, { ———— Melati

{290 pt OF EFFLUENT CHLORINATED [M LABORATORY (APPROC 2 myth coon

Fig. 29. Dual-Column UV-Anelyzer Chromatograms of Chlorinated Primary Sewage T
Plant Effluent. imery age Treatment

ACWTY opm sa?


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 g/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 pg/liter or less, con-
centration of sewage effluents by factors up to 3000 may be 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


spectrometer. At a helium carrier flow rate of 50 ml/min, the separator

operated at approximately 0.5 torr, and the source can at < 1x 107 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 17-in. radius and 90° ~sector
magnetic stage. A lh-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 70 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 MU 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

47


Table 17.

PERCENTAGE CHLORINATION YIELD* oF CHLORINE-CONTAINING
CONSTITUENTS WITH RESPECT TO REACTION TIME
FOR THE CHLORINATION OF EFFLUENTS

Reaction time,

Percentage chlorination
yield of chromatograph-
able chlorine-contgining

Percentage chlorination
yield of total chlorine-

min constituents containing constituents
15 0.55 0.95
45 0.58 0.98
90 0.75-0.87 1.27
2ho 1.6-1.8

"the yield is expressed as percent of chlorine dose which, after chlorina-
ption, 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.

88


19

Table 13. IDENTIFICATION OF MOLECULAR CONSTITUENTS IN 1000- AND 2000-FOLD CONCENTRATES
OF SECONDARY DOMESTIC SEWAGE EFFLUENT

Approximate anion-

; ; a Concentration, exchange elution

Compound Identification method pe/liter position, ml
Glycerine AC, GC, MS 10
Uracil AC, CC, UV, MS 30 18
5-Acetylamino-6-amino-3-methyl uracil AC, CC, UW 30 20
1-Methylinosine AC, CC, UV 80 29
Inosine AC, CC, UV 20 29
- '7-Methyl xanthine Ac, cc, UW 5 “29
1-Methylxanthine AC, GC 6 82
1, 7-Dimethylxanthine Ac, CC, UV ~6 64,
Succinic acid AC, MS 135
Catechol MS 235
Indole-3-acetic acid MS 235
3-Hydroxyindole MS 320
p-Cresol AC, GC, MS 90 ~300

Bac - anion exchange chromatography; CC ~- cation exchange chromatography; UV - ultraviolet spectrum;

MS - mass spectroscopy.


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, 254 nm, and the
emission spectrum of Ce(III) corresponds almost precisely with the
band-pass of the Corning 7-60 filters. These characteristics plus

the photo response characteristics of the S-11 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

has a linear signal output up to about 2 x 107 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. 17 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(III) is determined fluorometrically. Analysis
requires only a few minutes for determinations at levels as low as
100 pg oxygen demand per liter.

Cerate oxidimetry has been in general use in volumetric oxidimetry
for several decades, 18 and its applicability to the analysis of COD
19 For valid reasons,
this method was not adopted as the standard in the United States; how-

in polluted waters was suggested as early as 1941.

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


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.

AL


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 3-ft x 0.25-in. glass
colum packed with 2% 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 3-ft x
0.25-in. glass tube packed with 24 Dexsil on 80/100 Chromosorb W-HP.
Effluent from the column was divided, with approximately 10% being
directed to the flame ionization (FI) 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
as described by Krueger and McCloskey .2* A micro-needle valve
located between the chromatograph and separator was adjusted to allow
operation of the FI 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

46


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 investigative techniques.~° A brief description of this process

is given below.

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
constituents for faentification purposes. A preparatory system with

several times the capacity of the analytical system has been used to

4a


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
has been published as a doctoral thesis .7©

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 61%. available
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 4 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).

36

CHLORINATION OF SAMPLES WITH ~ Cl

Gaseous chlorine generated by the reaction of KCl in dilute
H,S0), with Kno), powder, 10 KC1+ 2RMn0), +8 4,80, —>2 MnSO,, + 6K,80,, +
8 #0 +5 cls, was used for the radioactive chlorination experiments.
A potassium chloride solution tagged with 36cy 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

70


0g

ACTIVITY (cpm/mi)
3
T

a or ao
12 ELUTION TIME, hr 18

10
é a 30 7s ~ 160 ELUTION VOLUME.mi | 2b0 225° abo abs 30d
Tt tt a a a
woth von ume canounoanan ave comcron:
Seniesa ae NES CR
z Eitan ruse sare wat Caen Gall, eal pea wpa tic
g
5
ra
36 32 34 36 38 40 a2 ELUTION TIME hr 48 50 82 34 36 38 60
<—
nm 325 350 378 460 ELUTION VOLUME. mi ats 500 825 350 ,

280 pi OF EFFLUENT CHLORINATED IN LABORATORY (1Omg/titer COMBINED CHLORINE RESIDUAL .ORTHOTOLIOINE )
15 min REACTION TIME ,CONCENTRATED 644X,---—"#Ci ACTIVITY
45 min REACTION TIME, CONCENTRATED $82 X,——-—-S#CI ACTIVITY
90min REACTION TIME, CONCENTRATED 490 X.———*#CI ACTIVITY

ACTIVITY (epm/mi)

Fig. 31. Chromatograms of Secondary Sewage Treatment Plant Effluent Chlorinated with Hypochlorite

Solution for 15-, 45-, and 90-min Reaction Times.


fluorescence spectrophotometer. However, once the general procedure
was worked out, a continuous-flow apparatus of two pressurized reagent
reservoirs, a sample pump (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
oy 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
flow in the range of a few milliliters per hour. 2! 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 min. 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. bs

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


EPA-660/2-74-076
August 1974

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.C. 20460


10.

11.

le.

13.

1h.

15.

16.

17-

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.

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 16lst American Chemical Society National Meeting,
Los Angeles, Calif., Mar. 29 to Apr. 2 1971.

Katz, S., W. W..Pitt, Jr., C. D. Scott, and A. A. Rosen. The
Determination of Stable Organic Compounds in Water Effluents
at pg/liter Levels by Automatic High-Resolution Ion Exchange
Chromatography. Water Res 6: 1029, 1972.

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. 7, 1973-

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.

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.

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.1I.Ch.E. Symposium Ser.

129, Vol. 69, 1973.

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


66

Table 8 (continued). IDENTIFICATION OF MOLECULAR CONSTITUENTS IN 1000- TO 3000-FOLD CONCENTRATES
OF PRIMARY DOMESTIC SEWAGE

a Concentration, Anion exchange elution
Compound Identification method pe/liter position, ml
Pheno1” . AC, GC, MS 6 205
3-Hydroxyphenylhydracrylic acid? AC, UV, GC, MS 10 205
Phenylacetic acid? AC, “GC ~10 230
4-Hydroxyphenylacetic acid AC, UV, GC, MS 190 235
Benzoic acid AC, GC, MS 260
2-Hydroxybenzoic acid AC, GC, MS 7 290
4.-Hydroxybenzoic acid AC, GC 295
3-Hydroxybenzoic acid AC, GC, MS ~hO 295
3-Hydroxyphenylpropionic acid AC, GC, MS ~20 295
Indican” AC, GC, F ~2 325
3-Hydroxyindole” MS 3h0
o-Phthalic acid® AC, UV, MS 200 400
p-Creso1” AC, GC, MS 20 400

®n0 ~ anion exchange chromatography; CC - cation exchange chromatography; UV - ultraviolet spectrum;
GC - gas chromatography on two colums; MS - mass spectroscopy; F - fluorescent spectrum.

brrom chlorinated effluent.

CIdentified by A. W. Garrison, Southeast Environmental Research Laboratory.

@No reference spectra available.

Identified in Mill Creek sewage effluent.

Structure deduced from mass spectra analysis.


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 l-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 pl of dry pyridine (Pierce Chemical Company ) and 50 to 100 yl
of bis (trimethylsilyl )-trifluoroacetamide (BSTFA) containing 1%
trimethylchlorosilane (Regis Chemical Company) and heating the
reaction mixture overnight at 50°C. Separate aliquots (4 pl) 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.-0.D. Pyrex tubing packed with 3% OV-1 or OV-17 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-p1 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.

45


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 36g, 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 100 pg/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


61

ORNL OWG 72-1418RI

power
COLUMNS
HEATING JACKET (FILLED WITH
v7 ETHYLENE GLYCOL) TEMPERATURE
Oy,
~~" ]}--500 W RESISTANCE oe INDIEATOR
JACKET] J _| HEATER HIGH- TEMPERATURE
FIND +7 || -cHROMEL-ALUMEL RESET CUTOFF
CONVECTION Pat |/ THERMOCOUPLE
<
5 -—~ us

a Vac

PE!

TM~ —_

ae-7" ac POWER

. {CONTROL- 50K
4-7 THERMISTOR LER TEMP ERATURE

4 ADJUST

qo" POTENTIOMETER

~~ 115 Vac

Fig. 10. Electrical Heating System for Chromatographic Columns.


ORNL DwG 71-3828

ABSORBANCE

PRIMARY EFFLUENT

.
mourns

SAMPLE: icc OF MUNICIPAL PRIMARY EFFLUENT CONCENTRATED 500- FOLD

SECONDARY EFFLUENT

MELROSE

Uinternel stendersa)

mouRS
SAMPLE: icc OF MUNICIPAL SECONDARY EFFLUENT CONCENTRATED 500-FOLD

Fooss

§ 00s
3

0.008

MORWAL URINE:

2
a

SAMPLE: ice OF NORMAL URINE

Fig. 7.

‘SUGAR STANDARDS:

ee

ABSORBANCE 4800)

g
8

2
S

8

ours.
SAMPLE: Ice CONTAINING 0.05 MICROMOLES OF EACH SUGAR

Carbohydrate Analyzer Chromatograms. of Primary and Secondary

Jewage Treatment Plant. Effluents, Human Urine, and Sugar Standards.

Be)


SECTION X

PUBLICATIONS

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.

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.

Jolley, R. L. Determination of Chlorination Effects on Organic
Constituents in Sewage Treatment Plant Effluents: A Coupled

38¢] Tracer--High-Resolution Chromatographic Technique. Presented
at the 167th American Chemical Society. Los Angeles, Calif.

Mar. 31 - Apr. 5, LOT.

Jolley, R. L. Chlorine-Containing Organic Constituents in
Chlorinated Effluents from Sewage Treatment Plants. Journal
Water Pollution Control Federation. Accepted for Publication.
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.

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. 247-252.

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.

Katz, S., and W. W. Pitt, Jr. A New Versatile and Sensitive
Monitoring System for Liquid Chromatography: Cerate Oxidation
and Fluorescence Measurement. Anal Letters 5: 177, 1972.

Katz, S., W. wW. Pitt, Jr., and G. Jones, Jr. Sensitive Fluorescence

Monitoring of Aromatic Acids after Anion-Exchange Chromatography
of Body Fluids. Clin Chem 19(8): 817, 1973-

oT


Lz

ORNL DWG. NO. 72-4213

a ----—
LAMP I 7
COMPESSATION | sv i
100 aC gue uisicered h H 20080 !
BRIE WSULRTED FROM PECEPIMCLE oe '
FLUORIMETEM B00Y c-c- -q P-r an) for{TM dory. wh 1
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By 65154,WVE Se) | Siok {
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CVS-68 ' tf H Soe |
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1 stam L wy —l | fRECERIACLE
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Fig. 14. Schematic of Flow Fluorometer.


TS

Table 5.

GUANOSINE IDENTIFICATION DATA

Guanosine,
SPJ 1-95-2 unknown

Guanosine,
reference standard

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, 29.54

643

283
245, 324, 368, 410,
556, 571, 628

7.5 (urine, analyt. )
13.8

254 (max), 275 (sh)
28.00, 29.42

643

283

245, 324, 268, 410,
556, 571, 628



Table 14.

DATA ON UNKNOWN COMPOUNDS FOUND IN SECONDARY EFFLUENT (SPJ-9

AND SPL-1) FROM A DOMESTIC SEWAGE TREATMENT PLANT

GC retention data, M.U.

Molecular Elution TMS peaks
weight time, hr M Base (1) (2) (3) for OV-1 column
9 0.8 | 167 98 uh 167 10.4
ny 12.9 | 3157 143 uy 15
115 12.9 | 331 147 73 © 262 12. 9
124 52.5 | 196 166 181 196 12.9
131 57.5 203 73 188174 11.5
133 2.0 | 349 280 284 «1h7 12.9
157 2.0 | 373 147 262 304 12.0
161 0.8 | 305 171 73 «166 10.2
163 2.0 | 379 310 163 (379 16.0
166 0.8 | 310 aul 225 73 14.3
177 2.0 | 393 73 whk7 32h 16.0
190 2.0 | 406 - - - 16.0
202 29.5 | 3h6 277 75 73 17.0
203 ore) 347 73 146 52 19.0
208 5.5 352 73 ho 52 22°
270 5.5 486 73 19 357 29.5
274 40.0 3h6 331 315 215 18.9
307 52.5 379 15 79 73 15
321 0.8 | 320° 99 ue 69 1.8
345 5.5 | 705 131 u7 73 27.4
°v-15 peak. Os

DNot TMS derivative.

CM-1 peak.

68


COLUMN GEOMETRY AND OPERATING PARAMETER STUDIES

‘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, Ry» of peaks 1 and 2

by:
R, = (vy - vy, )/ho,

where V, and ¥, = elution volumes of peaks 1 and 2,

@ = average standard deviation of the peaks in volume units.

Van Deemter' s!+ differential model for chromatography results in:

1 L \l/2
Re Go

but, on the other hand, analysis time, t. will have the following

proportionality:
L
t.* US a4
where L = column length,
U_ = linear velocity of eluent.

Q

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

+-0. Of course, in the separation of a very complex mixture such as

sewage plant effluent, it is always possible that additional compounds

al


Table 16. TENTATIVE IDENTIFICATIONS AND CONCENTRATIONS OF CHLORINE-CONTAINING CONSTITUENTS
IN CHLORINATED EFFLUENTS

Concentration
Peak elution Reference standard of organic
volume, Tentative elution volume, compound,
Constituent mL identification mL pg/liter
16 724+ 2.7 | 5-Chlorouracil 72" 4.3
_— wi8 80.2 + h.o 5-Chlorouridine 81 1.7
19 86.2+ hee 8-Chlorocaffeine 86 1.7
22 102+ 3.3 6-Chloro-2-aminopurine 109 0.9
32 218 8-Chloroxanthine 218 L5
ke 302 + 5.9 2-Chlorobenzoic acid 307 + g? 0. 26
43 312 + 5.9 5-Chlorosalicylic acid 310 0. 2h
5 334+ 6.4 k-Chloromandelic acid 338 La.
52 4o3 + 1.7 2-Chlorophenol 400 L7
53 WI5 + 3.8 4-Chlorophenylacetic acid 411 0. 38
55 436 ‘+ 5.4 4-Chlorobenzoic acid 43h + 11° Li
56 Wht WL 4.-Chlorophenol YANG 0.69
57 46h + 8.7 | 3-Chlorobenzoic acid 455 + 10° 0.6255
and/or, 3-Chlorophenol 456 0.521
59 496 + 8.7 4Chlororesorcinol 495 1.2
61 527 + 15.6 3-Chloro-l\-hydroxy- 540 1.3
benzoic acid
62 5u7 + 20.2 4-Chloro-3-methylphenol 550 15°

* average of two determinations.

Daverage of seven determinations + standard deviation.
Caverage of four determinations + standard deviation.

Gvalues based on the assumption that chlorine is present as

not a mixture of both.

©Concentration in sample H-2.

the pure dompound of either, and


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.


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,

36

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 0.71-Mev beta particle emitter with a 3.0 x 10° year half-
1ife.-? The presence of 36g, 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 36q, 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


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 3601 -tagged chlorination agents
(see Table 6). The primary effluent sample was chlorinated to 2 ppm
chlorine residual (orthotolidine method) using chlorine gas (H-1),
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-1) 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 3 Cl. The chlorinated mixture
was then reduced to a volume of 3.5 ml in a rotary evaporator. Of
the initial radioactivity, 48% 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 3 Sol -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.

16)


ont nw

10

11

12

13

wy

TABLES

Page

Resolution of Coupled Anion-Cation Columns

During Chromatography of ORESP Secondary

Sewage Effluent Samples 25
Oxidation Potential Available with Various Oxidants

Used in COD Analysis 3h
Oxidation of Organic Compounds and Polluted Waters

by Perchloratoceric Acid COD Method ho
Physical Characteristics and Operating Parameters

for the Analytical and Preparative Anion

Exchange Columns Ay
Guanosine Identification Data 51

y

Description of Samples 54
Status of Sewage Plant Effluent Samples (5/1/74) 55
Identification of Molecular Constituents in 1000-

to 3000-Fold Concentrates of Primary Domestic

Sewage 5T
Data on Unknown Compounds Found in Unchlorinated

Primary Effluent (SPJ-1) From the Oak Ridge

East Sewage Plant 60
Data on Unknown Compounds Found in Chlorinated

Sewage Plant Effluent (SPJ-3) From the Oak

Ridge West Sewage Plant 61
Data on Unknown Compounds Found in Chlorinated

Effluent (SPJ-8) from the Oak Ridge West

Sewage Plant 62
Data on Unknown Compounds Found in SPJ-11,

Composite of SPJ-3 and SPJ-8 66
Identification of Molecular Constituents in

1000- and 2000-fold Concentrates of Secondary

Domestic Sewage Effluent 67
Data on Unknown Compounds Found in Secondary

Effluent (SPJ-9 and SPL-1) From a Domestic Sewage

Treatment Plant 68

vii


€L

ORNL DWG. 72-1702 R2

POTASSIUM
CHLORIDE-
SULFURIC ACID

© OFF-GAS

POTASSIUM
PERMANGANATE

MANOMETER

REACTION
MIXTURE

POTASSIUM
PACKED
SOWUTION BOR LIME

Fig. 27. Schematic of Chlorine Generator and Sample Chlorinator.


SELECTED WATER 1 Rept No.
RESOURCES ABSTRACTS W
INPUT TRANSACTION FORM
4. Title 5. ResortD
Automated Analysis of Individual Refractory Organics In 6.
Polluted Water’ 8B Be formir Organization
eyort No.

Author(s)

W. Wilson Pitt, Robert L. Jolley and Sidney Katz Bhs ‘
16ACG 03

+ Organcas n

Oak Ridge National Laboratory (operated by Union Carbide ‘ Y
Corporation for the U.S. Atamic Energy Commission) 14-12-833

Oak Ridge, Tennessee 37830 13. Typec‘ Repor and

Period Covered
12, Sponsoring Organiza'on Environmental Protection Agency

Sup. cme

Environmental Protection Agency report number, EPA~660/2-74-076, August 1974

15, 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.
Same 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 cochramatography and quantified at the 0.5- to 4-yg/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)

17a, 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.

17b. Identifiers *High-resolution liquid chromatography, *Primary effluent, *Secondary
effluent, *Ultraviolet-absorbing constituents,-*Chlorine-containing organics,
*Oxidizable constituents, Unknown organics characterization, Concentration technique,
Colum geometry studies, Operating parameter studies, Cerate oxidimetry, Continuous COD
monitor, flow fluorometer, chlorination effects, chlorinated effluent, chlorination
reaction yield, chlorine-36.

I7e. COWRR Field & Group O5A, O7B, O5D

18. Availability 19. Se-urity CRE [31. No.of | send To:
(Report) ‘J. Pages ‘end To
20. Seourny Cis. | 22. Price | US"SRARsouRCES seieMTuTIGisronMariON cenren
.. (Page). - gl | WASHINGTON, D. c. 20240
Abs::,-:or Robert L. Jolley Institution Oak Ridge National Laboratory

WRSIC 102 (REV SUE aT


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)

Experiment
Constituent H-1 H-2> H-6° H-8¢ H-9° H-10°
kg 170 20 ho 250 220
50 210 120 30 100 90
51 120 70 80 100
52 1700 70 730 4:70 480
53 2430 540 170 130 80 200
5u 220 + 200 170 80
55 650 30 150 250 80
56 ‘ 90 110 190 220
57 100 60 140 90
58 130 30 220 180
59 260 60 60 300 120
60 140 60 170 260 220
61 210 80 270 340
62 120 180 300
Total 108,183 38,910 22,780 17,800 18,550 23,990
Number of
constituents 47 yy 52 6 52 Sh
eDrimary effluent; 2 ppm chlor ie residual; estimated chlorination contact time,
pet? min; chlorinating agent, 3°cl-tagged chlorine gas.
bgecondary effluent; 1 ppm residual; estimated chlorination contact time, 240 min;
ochlorinating agent, 36c1-tagged chlorine gas.
“Secondary effluent; ppm chlorine residual; chlorination contact time, 90 min;
aghlor inating agent, 'Cl-tagged chlorine gas.
“Secondary effluent; 1 ppm chlorine residual; chlorination time, 15 min; chlorinating
agent, Cl-tagged hypochlorite solution.
“Secondary effluent; 1 ppm chlorine residual; chlorination time, 45 min; chlorinating
agent, 3 Cl-tagged hypochlorite solution.
fgecondary effluent; 1 ppm chlorine residual; chlorination time, 90 min; chlorinating

agent,

36ci-tagged hypochlorite solution.

8Theoretical values are 24.0, 24.8, 25.0, 25.6, 25.6, and 25.6 mg, respectively.

8h


€¢

ORNL DWG 72-5309R2

100

80

60

40

RELATIVE INTENSITY

20

fpr =n nnnnnn anne nnnnennneeneeecereennncenne|

|
90 110 130

150° 170 190 210 230 250 270 2480 310 330 350 370
M/E ——>

100
—— GUANOSINE REFERENCE STANDARD, TMS DERIVATIVE
= go} ---- P-1, F.95 UNKNOWN, TMS DERIVATIVE (OFFSET 0.5 M/E)
te
a
5 60
me
S 40
fa
s
20
Fe ee Se oe ie
390 410 430 450 470 490 S40 S30 S50 570 S90 610 630 650
M/E ———>

Fig. 25. Mass Spectra of TMS Derivatives of the Reference Compound (Guanosine) and Liquid
Chromatographic Fraction from Primary Sewage Treatment Plant Effluent.


6h

ORNL DOWG 72-5307

T T T T T
_————  METHANOL-WATER, pH 7.0
METHANOL -WATER, pH I1.8
wW
cs)
2
<q
o -
© METHANOL
a
a
<
I _ i | { { i a L
220 240 260 280 300 320

WAVE LENGTH, nm

Fig. 22. Ultraviolet Absorption Spectra of Unknown Sample Taken at Different pH Values.


ORNL DWG 70-9001

SAMPLE
IN

9- CHAMBER SIX PORT SAMPLE
GRADIENT BOX INJECTION VALVE

FOLDED, HIGH PRESSURE
CHROMATOGRAPHIC
COLUMN AT 55°C

}——{ PHENOL SOLUTION

LX t280,

ORNL
PRESSURE
GAUGE
® [FLREACTION ZONE
OVERFLOW
_ WASTE
HIGH
LH) pREssuRE
My pump

Fig. 4. High-Resolution Anion Exchange Chromatograph for Carbohydrates
(Carbohydrate Analyzer).

i


Table 3. OXIDATION OF ORGANIC COMPOUNDS AND POLLUTED WATERS
BY PERCHLORATOCERIC ACID COD METHOD
Concentration, Number of Oxygen consumed, mg/liter h of
Sample mg/liter determinations Average Range theoretical

Acetic acid 10 y. 6.1 5.2 - 74 57
Acetone - 8 5 3.2 3.0 - 3.3 18
Benzene 3.3 4 2.8 24 - 3.5 26
Ethanol 7.5 3 11.0 10.8 - 11.1 69
Glucose 14.5 4 17.4 15.9 - 17.9 112
Glycine 10 4 3.0 2.8 - 3.2 46
Linear alkyl sulfonate 5 5 6.9 6.3 - 74 105
Pyridine ~ 10 6 3.5 3.2 - 3.6 16
Sodium lauryl sulfate 5 5 13.4 12.8 - 14.6 135
Sewage plant effluent 19 COD 5 16.5 14.8 - 18.1

(industrial plant)
Secondary sewage plant 6 BODs 5 25.9 24.2 - 27.4

effluent (Oais Ridge, Tenn. )
Clinch River water 8 coD 3 7.8 7.5 - 8.0 ’



Table 4. PHYSICAL CHARACTERISTICS AND OPERATING PARAMETERS
FOR THE ANALYTICAL AND PREPARATIVE ANION EXCHANGE COLUMNS

Columns”

Parameters Analytical Preparative
Column dimensions, cm 0.30 x 150 0.94 x 150
Resin Bio-Rad A-27 Bio-Rad A-27
Resin particle size, up 8-12 12-15
Sample size, ml 0. 28 5
Run time, hr ho ke
Temperature program Ambient, in- Ambient, in-

creasing to 60°C creasing to 60°C
after 11.0 hr after 11.5 hr
Operating pressure, psig 1900 1600
Flow rate, ml/br 10 88

Eluent

Concentration gradient

Gradient generator

Ammonium acetate--
acetic acid buffer,
pH 4. 4h

0.015 M- 6.0M

2-chanber®

Ammonium acetate--
acetic acid buffer,
pH 4. uy

0.015 M- 6.0 M

9-chamber®

®colums 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 874 ml of 0.015 M, two chambers contained 851 ml
of 1.0 M, two chambers contained 828 ml of 4.0 M, and three chambers
contained 1208 ml of 6.0 M buffer.


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 3 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. 4,0) 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 3 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-colum 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

36 by liquid scintillation counting.

72


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
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 0.45-pm 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


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-17
column are shown in Fig. 24. 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 7. One sample of primary effluent and one sample of secondary
3

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


Abstract
List of Figures
List of Tables

Acknowledgements

Sections
I
II
TIL

Iv.

VII

VIII

CONTENTS

Conclusions
Recommendations

Introduction

Methods and Instrumental Development
Identification of Stable Organic Pollutants
Effects of Chlorination of Sewage Plant Effluents

Cooperative Efforts with EPA Laboratoriés

Discussion
References

Publications

iv

Page
di

vii

ix


ERRATA SHEET

AUTOMATED ANALYSIS OF INDIVIDUAL REFRACTORY
ORGANICS IN WATER POLLUTED

EPA-660/2-74~076

Should read

AUTOMATED ANALYSIS OF INDIVIDUAL REFRACTORY
ORGANICS IN POLLUTED WATER


‘% TRANSMIT TANCE,

02

% TRANSMIT TANCE,

ORNL OWG 71-2891 RI

202 1 2 3 4 5 eet), 8 9 1 ta &
— saa La 1
@| 4

4
ea q

q
as 4
8s q
20) J
a 4
4 J
96 4
98) we
100) porting |

O 2 24 36 48 60 72 84 96 106 120 132 44 6 GO 100 192 204 AS 228 246 264 282 300 BS 336 354 372 390
ELUTION VOLUME (mt)
TIME (tre)
a a a ro
eat i couse, 0.45 s1s0em AMEN A-27 RESIN 1
Pow Rates 4S'mifw TOTAL
i ATURE; AMGIENT-- 60°C ATS brs. 71
eal H SAMPLE! 0.30 I URINE COMPORITE EACH COLUMN
est q 1
atk 1
eat Sit 1
90- 2 J
t : & A
ser i = K
C 4 h 4
oat 4
96 4
98 WKF
100)

908 26 44a 462 400438 “Sea ~'546- “570584 la 648 G8 880 ia
ELUTION. VOLUME (rat

Fig. 11. Dual-Column Chromatograms of Two Identical Samples.


Table 11 (continued). DATA ON UNKNOWN COMPOUNDS FOUND
IN CHLORINATED? EFFLUENT (SPJ-8)
FROM THE OAK RIDGE WEST SEWAGE PLANT

Elution
Molecular time, GC retention data M.U.

weight hr OV-L OV-17 A
125 3.0 15.4 - -
127 14 14.7 - -
128 12.7 12.2 12.8 0.6
129 3.0 16.0 17.5 1.5
130 10.0 17.8 - -
130 14 14.5 - -
132° 10.0 20.2 20.5 0.3
132 16.0 16.1 16.4 0.3
140 40.5 15 16.2 1.2
146 3.2 U5 15.2 0.7
17 10.5 20 20.8 0.8
148 18 15.8 16.5 0.7
151 16 17.7 20.6 2.9
152 16 13.5 1.9 La
154 ko 17.5 18.6 LL
159 10 22.4 - -
1o =| 38 16.9 17.6 0.7

63


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 (II) acetate (binuclear) were blue, probably because of the
presence of Cu(IZ), 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 and 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-ll was a

56


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. LINEARLY IN CONCENTRATION FROM 0.018 M TO 6.0 it AMON!
3 al ELUENT FLOW RATE O89 mi/br , COLUMN PRERSURE 1800 pig. 4
H
:
3
3.
:

“_ELUTION TIME,

Reference UV-Analyzer Chromatogram of 2000X Concentrate of Secondary Sewage Treatment
Plant Effluent.

Fig. 6.


0€

‘OT *BTa

*J@YSWOLONT MOTZ TOF oaIng uopyzyezqtrep

FLUORESCENCE — (arbitrary units)

ORNL DWG 72-8766 RI

80 r “T T T T T T T T T
: b
7OFr 4
60F 4
OF 4
5 10
r e
40F . rT 4
C ‘a
30-F r Va 4
20+ 7A 4
(0) Var se ee ee ee Ce
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TRIVALENT CERIUM CONCENTRATION (millimoles /liter )

FLUOROMETER CALIBRATION CURVE


os”

ORNL DWG 72-11698

GUANOSINE
0.0207 mg/mi

ABSORBANCE

0.9
220 240 260 280 300 320

WAVELENGTH, nm

Fig. 23. Ultraviolet Absorption Spectra of Guanosine Taken at Different pH Values.

(Date, from Circular OR-10 Sixth Printing, 1969, Pabst Laboratories, Division of Pabst Brewing Co.)


oT

SAMPLE
LOOPS

HIGH-PRESSURE

CHROMATOGRAPH
COLUMNS
COLUMN
CONCENTRATED OILUTE JACKET
BUFFER BUFFER
TEMPERATURE
INDICATOR
CONTROLLER
VOLUMETRIC VOLUMETRIC
SYPHON SYPHON
MIXING
CHAMBER A

ORNL DWG 71-3797R 3

(2 PORT DUAL COLUMN
SAMPLE INJECTION VALVE

— RECORDING
POTENTIOMETER

CHROMATOGRAMS

| aenenieieneiieeenennennte |

Vee

HIGH
PRESSURE
PUMP

Fig. 8. Schematic Diagram of Dual-Column, High Resolution,
Two Polluted Water Samples Simultaneously.

Liquid Chromatograph for Analyzing


Table 15. 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 )

Experiment.

Constituent H-1* H-2° H-6° u-6t H-9° Helo"
1 3120 1240 1000 460 Lo 610
2 220 890 560 150 260
3 30h0 760 460 150 2ho 290
4 4520 630 340 150 350 2ho
5 7320 2460 1550 120 1040 1070
6 630 560 260 140 250 260
7 130
8 310
9 1070 900 600 100 120 360

10 1420 Who 110 170 230
aa 520 190 210

12 9260 2760 1340 670 1120 1230
13 980 6h0 430 140 ” 260
14 2130 700 290 170 200 230
15 2ho

16 6200 1410 870 460 1040 1530
17 3950 1000 330

18 2860 770 580 210 220 470
19 1090 4ho 190 270 210
20 1940 1210 610 430 2h0 240
a1 3150 2610 1060 230 370 6h0
22 330 170 680
23 3550 1730 1420 510 320 560

82. =e


ORNL DWG 72-7924

TO
WASTE

FLUOROMETER

GAS
PRESSURE (0-2- 0-Satm)

Li

REAGENT
RESERVOIR

SAMPLE. MIXER } HEATER

Fig. 20. Schematic of Continuous Chemical Oxygen Demand Analyzer.

37


36

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 36o1 was
determined for the chromatographic separation. The 36g) activity

level of this 280-1 sample, which was chromatographed, analyzed

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:
(1) chlorine-containing compounds are found when a concentrate of

secondary sewage effluent is chromatographed; (2) these chlorinated

TT


Table 7.

Tdgg 103
sasfteue yo Zequmy

310

310

310

310

310

310

peufeqgo etqoeds sseu
suopQoVry Jo zaqumN

27

14

25

15

yderZoqeuoryo se8 uumtoo
-Tenp uo paeydesr307;euo0ryo
suoTqoerz Jo requaN

35

35
43

17

59

29

13
ke

peuteqqo erqoads yaToTA
~B24TN SUOCTZOBIZ JO TEquMN

4L

17

16

39

59
Wn

paqoeTToo suoTqowry
uumTOO uoTZeo JO ZequMN

hg

10

uIMTOO aBweyoxe uotzeo uo

peyderZozemoIyo suoyzoVry
uumToo uote Jo zequmN

14
wy

17

paqosTToo suoyzowry uMTO
eBueyoxe uotue Jo zequMN

10

55

a1

59
250

59
” 35

290
290

290

ummToo saTyeredead
uo peyder307em0I49

STATUS OF SEWAGE PLANT EFFLUENT SAMPLES (5/1/74)

uumToo TeoTyATeure
uo peydez09eu0r1y9

x

x

x

x

x

Aoyoey UoTZeIZUaOUOD

1000

1000

1000

1000

1000

1000”

1000

1000

370

1000

2000

2000

2000

3000
3400

880

10

490

aTdures

SPJ-1

SPJ-2

SPJ-3

SPs-k

SPJ-5

SPJ-6

SPJ-7

SPJ-8

Hel

SPK-1

SPJ-9

SPJ-1O

SPK-2

SPJ-11

SPL-1

H-5

H-6

H-8

#9

H-10

Concentrated using XAD-4 macroreticular resin.

Analyzed on cerate oxidative monitor.

a,
b

92


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.

36.

Several dual-column chromatographic runs were made in which
tagged samples were compared with similar samples not tagged. ‘The
radioactive sample, H-2, was a 1040-fold concentrate of secondary
effluent which had been chlorinated to a chlorine residual of 1 ppm

3661 tagged chlorine gas to determine whether organic constit-

using
uents in secondary effluent were indeed chlorinated as in primary
effluent. This proved to be the case, as is shown in Fig. 30,
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 concentration. 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 490-fold concentrates of secondary effluent chlorinated with

3 61 -tagged hypochlorite to a 1 ppm chlorine residual which was
destroyed after 15, 45, and 90 min of reaction time, respectively.
Chromatograms of the chlorine-containing constituents in these
experiments are shown in Fig. 31. 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


0.45-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 107+ N Ce(IV) in 4 N sulfuric acidJ-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
stream passes through a flow fluorometer, 1T 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 7-60 secondary filters, photomultiplier tube and housing, and

a photoconductor for compensation of changes in lamp intensity. In

2k


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. O. 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.

ix


6€

ARBITRARY FLUORESCENCE UNITS

- nr wa Oo x
s6h6c8lhC<C SKlULCUDULUCUCBhLCUUCUC

°

| x10°M cet** —8 ppm 0.D.

1
L 2 33mg/L BENZENE
4 3 1O0mg/2 ACETIC ACID — Il ppmo.D.
6 4 145mg/Z GLUCOSE — 15 ppm 0.0.
[ 5 5mg/Q£ SODIUM LAURYL SULFATE — ~ IO ppmo.D.
6 100mg/Z PYRIDINE ~ 220 ppm 0.D.
7 Smg/2 LINEAR ALKYL SULFONATE (M.W. 315)
8 8 0.75mg/Z ETHANOL— !6 ppm 0.D.
9 9 10mg/X GLYCINE — 7ppm.D.
10 10mg/2 ACETONE — 18 ppm 0.0.
7
fe)
f l i
° 10 20 30 40 50 60 70

TIME (min)

Fig. 21. Recorder Trace from Continuous COD Analyzer.



No.

15

16

TT

Stable Chlorine~Containing Organic Constituents
in Chlorinated Effluents from Domestic
Sanitary Sewage Treatment Plants. (Concentrations

of the Constituents, ng Cl/liter of original
effluent )

Tentative Identifications and Concentrations of
Chlorine-Containing Constituents in Chlorinated
Effluents

Percentage Chlorination Yield of Chlorine-Containing
Constituents with Respect to Reaction Time for
the Chlorination of Effluents

viii

E

82

86


Many of the organic compounds normally found in sewage and
industrial wastes are not oxidized as efficiently with ceric 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
of Ce(III) from 108 to 10° M can be readily determined fluorometrically
by excitation with ultraviolet light (254 nm) and measuring the 90°
light emission at 350 nm.7° 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
waste sample with 1077 to 1072 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


eT

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‘as 902 4v aonvevosey ———.
one, BOA MOLLOTA 94

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Fig. 5. Reference UV-Analyzer Chromatogram of 1000X Concentrate of Primary Sewage Treatment
Plant Effluent.


ORNL DWG. 74-I97!1RI

SEWAGE TREATMENT PLANT
EFFLUENT SAMPLE

10-100 LITERS

O45 p FILTER
SAMPLE FOR

+
boc ——{ 10-100 LITERS

LOW-TEMP
EVAPORATOR

(50 pn
PRESSURE

| 200 ma

FREEZE
AT
- 60°C
AND

. DRY

- (10pn

DISSOLVE IN ACETIC ACID

. FREEZE
AT +
~ 60°C
AND
DRY
(10p

|

DISSOLVE IN DILUTE ACETATE
BUFFER AND CENTRIFUGE

SAMPLE FOR
boc

TO CHROMATOGRAPH

+
0OC= DISSOLVED
ORGANIC CARBON

Fig. 1. Procedure for Concentrating Sewage Plant Effluent Samples.

7


10
ll

12

13
1h

15
16
17

18

FIGURES

Procedure for Concentrating Sewage Plant
Effluent Samples

Schematic Diagram of Vacuum Distillation System

High-Resolution Anion Exchange Chromatograph for UV-
Absorbing Compounds (UV-Analyzer )

High-Resolution Anion Exchange Chromatograph for
Carbohydrates (Carbohydrate Analyzer )

Reference UV-Analyzer Chromatogram of 1000X
Concentrate of Primary Sewage Treatment
Plant Effluent

Reference UV-Analyzer Chromatogram of 2000X Concentrate
of Secondary Sewage Treatment Plant Effluent

Carbohydrate Analyzer Chromatograms of Primary and
Secondary Sewage Treatment Plant Effluents, Human
Urine, and Sugar Standards

Schematic Diagram of Dual-Column, High Resolution,
Liquid Chromatograph for Analyzing Two Polluted
Water Samples Simultaneously

Dual-Column Sample Injection Valve

Electrical Heating System for Chromatographic Columns

Dual-Column Chromatograms of Two Identical Samples

Dependence of Chromatographic Resolution on Column
Length and Linear Velocity of Eluent

Schematic of Cerate Oxidative Monitor

Schematic of Flow Fluorometer

Exploded View of Flow Fluorometer Body

Calibration Curve for Flow Fluorometer

Chromatogram of Primary Sewage Plant Effluent
Analyzed by the UV-Analyzer with a Cerate
Oxidative Monitor

Chromatogram of Secondary Sewage Plant Effluent

Analyzed by the UV-Analyzer with a Cerate
Oxidative Monitor

Page

10

1l

12

13

5

16
17

20

22
26

28
30

31

32


Table 11.
IN CHLORINATED®

DATA ON UNKNOWN COMPOUNDS FOUND
EFFLUENT (SPJ-8)
FROM THE OAK RIDGE WEST SEWAGE PLANT

Elution

Molecular time, GC retention data, M.U.

weight hr OVv-1 OV-17 A
61 0.8 11.0 - -
Th 3.6 14.5 15.3 0.8
95 3.2 1.4 12.8 14
95 9.8 10.8 - -
97 16.5 14 15.3 1.3
105 10.5 13.6 15.7 2.1
107 40.5 18.9 20.0 L1
108 67 16 - -
112 1.4 15.4 16.0 0.6
113 0.8 17.7 - -
113 0.8 11.2 - -
116 2.0 15.2 16.0 0.8
124 16 15.0 - -

62


ORNL~ DWG 71-94864

MOVABLE
METAL. WASHER

NUT FOR TIGHTENING
METAL CYLINDER

TAPERED
METAL SHAFT-

Jf AANDLE FOR
ROTATING SHAFT

Fig. 9. Dual-Column Sample Injection Valve.

17


SECTION III

INTRODUCTION

The continuing discharge of stable organic compounds in sewage
effluents to surface waters and the buildup of organic constituents
by recycle constitute a serious threat to our water quality. 17°
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

3oh we demonstrated that at least

the first year under this contract,
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


100

ORNL DWG 71-3869 RI

[LENGTH/LINEAR VELociTy] “*

oe ee |
333

8.0

70 ;-
6.0r-
20.

NOILN10S3y

Fig. 12. Dependence of Chromatographic Resolution on Colum Length
and Linear Velocity of Eluent.

22


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 AWIRL 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 AWIRL.
He concentrated the sample to 30 ml by vacuum distillation 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 0 hr
was identified by mass spectrometry as o-phthalic acid. The molar
extinction coefficient of a reference sample of o-phthalic acid was
determined to be 1120 absorbance-en“/g-mole at a wavelength of
280 nm. The chromatographic peak representing this compound was then
quantitated to give an o-phthalic acid concentration of 0.2 pe/ml1
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 AWIRL, 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

l-methylxanthine, but these identifications were discounted by other

90


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)

Experiment

constituent H-1* He2? H-6° H-8¢ H-9° H-10"
ay 4810 1780 590 550 370 4.90
25 3330 iho 910 560 330 510
26 2170 1210 - 1010 TT0 610 g10
27 4030 1280
28 3160
29 4230 1810 930 910 990 1690
30 850
31 {21.3 mg]® [20.9 mgJ® [17.2 mg]® [2h.0 mgJ® [21.6 mg]® [17.3 mg]®
32 840 390 290 270
33 1500 340 190 120
34 1520 170 70 150
35 910 2ho 190 230 170 110
36 350 1ho 90 220 130
37 800 190 140 180 50
38 220 150 50 20 100
39 14400 5610 2450 1670 1750 2600
40 180 120 270 60 140
WL 530 490 160 4320 3260. 3380
he 90 120 90 80 60 190
43 160 50 60 50 50
dh 430 150 120 120 100 170
45 380 90 160 160 210 190
46 503 80 130 180
vg 190 70 230 140 150
48 350 790 430 130 170 290

83


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.


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 (AWIRL), 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 AWIRL; 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 AWIRL. This instrument was subsequently converted to a Mark IIA;
the analyzer constructed for SERL was an updated Mark LIA 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


10.

11.

12.

SECTION IX

REFERENCES

Bunch, R. L., E. F. Barth, and M. B. Ettinger. Organic Materials
in Secondary Effluents. J Water Poll Control Fed 33: 122, 1961.

Middleton, F. M., and A. A. Rosen. Organic Contaminants Affecting
the Quality of Water. Public Health Rept (U-S.) Tl: 1125, 1956.

Katz, S., and W. W. Pitt, Jr. Automated Analysis of Polluted Water.
Qak Ridge National Laboratory, Oak Ridge, Tenn., USAEC Report
ORNL-TM-3862. August 1972. 53 p-

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-Resolution Ion
Exchange Chromatography. Water Res 6: 1029, 1972.

Ryckman, D. W., J. W. Irwin, and R. H. F. Young. Trace Organics
in Surface Waters. J Water Poll Control Fed 39: 661, 1967-

Baker, R- A. Trace Organics Contaminant Concentration by
Freezing, II. Inorganic Aqueous Solutions. Water Res 1: 97,
1967.

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.

Scott, C. D-, J. E. Attrill, and N. G. Anderson. Automatic, High-
Resolution Analysis of Urine for Its Ultraviolet-Absorbing Constit-
uents. -Proc Soc Exptl Biol Med 125: 181, 1967.

Scott, C. D. Analysis of Urine for Its Ultraviolet -Absorbing
Constituents by High-Pressure Anion Exchange Chromatography.
Clin Chem 14: 521, 1968.

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.

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.

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: 767, 1972.

ok


gs

Table 8 (continued).

OF PRIMARY DOMESTIC SEWAGE

IDENTIFICATION OF MOLECULAR CONSTITUENTS IN 1000- TO 3000-FOLD CONCENTRATES

Suceinic acid

Concentration, Anion exchange elution
Compound. Identification method® peg/liter position, ml

3-Methylxanthine Ac, cc 6
Caffeine AC, CC, UV, MS ~10 67
oy guanosine AC, CC, UV, GC, MS 50 76
"B-Deoxyglyceric acid” MS . 80
3-Hydroxybutyric acid? GC, MS 80
3-Deoxyarabinohexonic acid? Ms 80
Quinic acid??? Ms 80
1-Methylxanthine AC, CC, UW 70 82
2-Deoxytetronic acid? MS 85
Glyceric acia? MS 90
4.-Deoxytetronic acid? MS 90
3-Deoxyerythropentonic acid” Ms 90
2,5-dideoxypentonic acia”?t Ms 90
3, 4-Dideoxypentonic acid? ws ? 90
Ribonic acid? MS 95
oxalic acid? AC, GC, MS 95
2-Hydroxyisobutyric acid” GC, MS 95
Uric acid AC, GC, MS 20 106
orotic acid? AC, UV, GC, MS 5 145
P AC, GC, MS 205


Table 11 (continued).

IN CHLORINATED® EFFLUENT (SPJ-8)
FROM THE OAK RIDGE WEST SEWAGE PLANT

DATA ON UNKNOWN COMPOUNDS FOUND

Elution
Molecular time, GC retention data, M.U.
weight hr OV-1 OV-17 ry
166 4O.5 16.8 18.4 1.6
177 2.0 18.7 - -
184 16 20.4 20.6 0.2
218 10.5 16.4 17.1 0.7
219 3.0 14.8 15.3 0.5
233 12.7 12.2 12.8 0.6
284 40.5 23 - -
4g 10.0 24.8 26.1 1.3

30, 5 ppm residual.

Pagiycon molecular weight.

glucuronide.

Probably present as a


The recovery of the noncarbonate organic compounds in the final
concentrate appears to be satisfactory (greater than 85%), 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
samples, two high-resolution anion exchange chromatographs -1e 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
at the rate of about 250 ml en”= nr 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

9


T6

ORNL DWe 74— 7532

to 7 vee
LUTION Time, wr

400
Elbriom vowuwe, mt

(a)

\

Fig. 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 (1000X).


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 0.845 M during an analysis, is pumped through
the column at the rate of about 350 ml en”? 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 480 and 490 nm,
of the reaction mixture after reaction for 3 min at 100°C. The more-
sensitive Mark III Carbohydrate Analyzer, u 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- In 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, raffinose;
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
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,

1h


increase the quantities of compounds collected per run. This system,
which is coupled to a fraction collector, is capable of chromato-
graphing 5 ml of 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 254 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 spectroscopic-grade methanol and subjected to uv spectral, gas

chromatographic, and mass spectral analyses.

43


should produce similar chlorination yields; i.e., about 1% of the
chlorine dose should be associated with stable chlorine-containing
organic constituents. Table 17 summarizes the chlorination yields

obtained in the experimental series.

87


92

ORNL DWG 72-1380 R-2

GAS
pressure (30mm Hg)

RECORDER

f |

TO
3 WASTE

CAME LARY ,

LOW
CONTROL.
T7oc/hr.

COLUMN REAGENT
RESERVOIR

ELUATE FLOW

Teoc/hr. :
14-24cc/hr FLUOROMETER
PHOTOMETER == ]:] REACTION
BOILING E= Hera
WATER = S:

HEATER

Fig. 13. Schematic of Cerate Oxidation Monitor.


ORNL DWG 69-1243 R3

RECORDING

POTENTIOMETER
ccc
|
i} CHROMATOGRAM
!
|
|

9 CHAMBER GRADIENT BOX

| TO FRACTION COLLECTOR

OR TO DRAIN

-+—————

HIGH-PRESSURE
y Y CHROMATOGRAPHIC
rrrt COLUMN

ISPECTROPHOTOME TER
PRESSURE
RELIEF VALVE
SAMPLE
PRESSURE P
GAGE @ 7 on
4
IN OUT]
6-PORT SAMPLE
INJECTION VALVE -
HIGH-PRESSURE CONSTANT- 7
PUMP TEMPERATURE.
CIRCULATOR

Fig. 3. High-Resolution Anion Exchange Chromatograph for UV-Absorbing
Compounds (UV-Analyzer).

10


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-) ORESP secondary effluent (11-22-71).

SPJ-5 ORESP chlorinated secondary effluent (11-22-71); 0.5 ppm residual
chlorine (orthotolidine).

SPI-6 ORESP secondary effluent (1-18-72).

SPJ-7 ORESP chlorinated secondary effluent (1-18-72); 0.75 ppm
residual chlorine.

sPg-8 ORWSP chlorinated primary effluent (2-23-72); 0.5 ppm residual
chlorine (orthotolidine).

SPJ-9 ORESP secondary effluent (4-19-72).

SPJ-10 ORESP chlorinated secondary effluent (4-19-72); 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 (4-24-72) for carbohydrate determination.

SPL-1 ORESP secondary effluent (12-12-73).

H-1 ORWSP pEjmary effluent (2-23-72) chlorinated in the laboratory
with 3°cl tracer as Clg gas to approximately 2 ppm residual
chlorine (orthotolidine).

H-2 SPJ-9 chlorinated in the laboratory with oy tracer as Clo gas to
1.0 ppm residual chlorine (orthotolidine).

H-5 SPJ-9 spiked with 6c, tracer as chloride.

H-6 SPJ-9 chlorinated in the laboratory with 36) tracer as Cl, gas
to 1.0 ppm residual chlorine (orthotolidine). Chlorine fesidual
destroyed with potassium thiosulfate after 90 min.

H-8 SPJ-9 chlorinated in the laboratory with 36 oy 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 45 min.

H-10

Same as H-8. Chlorine residual destroyed after 90 min.

5h


No.
19 Cerium Fluorescence Oxidimetry

20 Schematic of Continuous Chemical Oxygen
Demand Analyzer

al Recorder Trace from Continuous COD Analyzer

22 Ultraviolet Absorption Spectra of Unknown
Sample Taken at Different pH Values

23 Ultraviolet Absorption Spectra of Guanosine
Taken at Different pH Values

2h Gas Chromatograms of TMS Derivatives of Reference
Compound (Guanosine ) and Liquid Chromatographic
Fraction from Primary Sewage Treatment Plant
Effluent

25 Mass Spectra of TMS Derivatives of the Reference
Compound (Guanosine ) and Liquid Chromatographic
Fraction from Primary Sewage Treatment Plant
Effluent

26 Uv-Analyzer Chromatograms of Primary Sewage Treat -
ment Plant Effluent Chlorinated with Different
Amounts of Calcium Hypochlorite

eT Schematic of Chlorine Generator and Sample
Chlorinator
28 Schematic of Apparatus Used for Concentration of

Radioactive Samples

29 Dual-Column UV-Analyzer Chromatograms of Chlorinated
Primary Sewage Treatment Plant Effluent

30 Dual-Column UV-Analyzer Chromatograms of Chlorinated
Effluent from a Secondary Sewage Treatment Plant

31 Chromatograms of Secondary Sewage Treatment Plant
Effluent Chlorinated with Hypochlorite Solution
for 15-, 45-, and 90-min Reaction Times

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 (1000X)

vi

Page
35

3T
39

49

50

52

D3

71

16)

Th

76

19

80

g1


Tentative identifications of 17 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 36

fore, our values may be high if the chlorination reaction by which

Cl occurred rapidly during the chlorination process. There-

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 (45 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 0.44% 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
3 Ser
in the chlorinating agent occurs with the “pool” of nonradioactive

during the chlorination, complete isotopic dilution of the

chlorine in the effluents. The remainder of the available chlorine
(99%) 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


ORNL OWG 72-11725

100 ‘inal fr ial
—— FLUORESCENCE
90] —--uv ABSORBANCE 784
280) ih 186
2 70) i es
wy 60} i 90
2 i
Z 50 ti 92
@ 40) (| | il i 94
« | Moh
$ 30) hii | ty A f rh. 198
3 Md \ Aa Nw
ea \ ll all j WJ | iw i Np \ arb wal n 98
ae t ik f Ane.
19 | 100
: he. m4 _ ‘a os n n
% al \\4 6 8 io 2 4 16 202224
6 a HOURS
wu] {| 2\ 2
3 a\8 g MANY AROMATIC ACIDS FOUND IN HUMAN
z\{ oelz WASTES ELUTE IN THIS REGION
ay] 35 aa 3
3s | S53 4u2 2
eal Sse Za 8
FS arEROE ES
wm OB wa w & S Q
FSESFISTSS 5
25 5am
100-F
— FLUorEscence
90} 270 pl SAMPLE OF —-— uv assorsance 184
oe 1000 x CONCENTRATE OF ORESP les 8
—E PRIMARY EFFLUENT 2
2 70 ea
> E
y so =
& 50} 2
u 3
9 40 oa
6 30 jes >
8 E
i 20} 98 bd
10} 100 S
loz

40 42 48

4446
HOURS

FeifyoRoxveenzorc

ee
ew
8

Fig. 17.
the UV-Analyzer with a Cerate Oxidative Monitor.

31

254 nm UV TRANSMITTANCE.

Chromatogram of Primary Sewage Plant Effluent Analyzed by


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 AWIRL 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 5-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


Table 2.

OXIDATION POTENTIALS AVAILABLE WITH VARIOUS
OXIDANTS USED -IN COD ANALYSIS

Cry eee, =" or eV
+ > nett “1.98 V
at ; 1,80), , celtt -L.42 Vv
3 ; HC1O, ; cel -1.70 to ~1.87 V

3h