United States
Environmental Protection
Agency
EPA-600/2-80-031
July 1980
Research and Development
Formation and
Significance of
N-Chloro Compounds
in Water Supplies
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EPA-600/2-80-031
July 1980
FORMATION AND SIGNIFICANCE OF N-CHLORO COMPOUNDS IN WATER SUPPLIES
by
J. Carrell Morris
Neil Ram
Barbara Baum
Edmund Wajon
Harvard University
Division of Applied Sciences
Cambridge, Massachusetts 02138
Grant No. R803631
Project Officer
Edward L. Katz
Drinking Water Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 45268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, Office of Research and Development, U.S. Environmental Protection
Agency, and approved for publication. Approval does not signify that the
contents necessarily reflect the views and policies of the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
ii
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FOREWORD
The Environmental Protection Agency was created because of increasing
public and government concern about the dangers of pollution to the health
and welfare of the American people. The complexity of that environment
and the interplay between its components require a concentrated and inte-
grated attack on the problem.
Research and development is that necessary first step in problem
solution and it involves defining the problem, measuring its impact,
and searching for solutions. The Municipal Environmental Research Labor-
atory develops new and improved technology and systems for the prevention,
treatment, and management of wastewater and solid and hazardous waste
pollutant discharges from municipal and community sources, for the pre-
servation and treatment of public drinking water supplies, and to minimize
the adverse economic, social, health, and aesthetic effects of pollution.
This publication is one of the products of that research; a most vital
communications link between the researcher and the user community.
This report addresses the hygienic and aesthetic importance of nitro-
genous organic compounds in natural waters and the formation and properties
of their chlorinated derivatives. The areas studied include: (1) an
investigation of the reactivity toward aqueous chlorine of selected nitro-
genous compounds analogous to those anticipated as likely constitutents
of raw drinking water supplies; (2) an attempt to isolate, concentrate
and identify nitrogenous compounds actually present in such supplies as
an aid to understanding reactions occurring with nitrogen-containing
materials during water chlorination; (3) an evaluation of several analy-
tical methods now in use for distinguishing between free available
chlorine and combined chlorine; and (4) the determination of nitrogenous
precursors to haloform formation.
Francis T. Mayo, Director
Municipal Environmental
Research Laboratory
iii
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ABSTRACT
.secure hygienic quality in water supplies is dependent upon the main-
tenance of a free chlorine residual./ The available analytical methods used to
differentiate between free and combined chlorine, however, are subject to
interference from organic chloramines, though it appears some differentiation
is achieved using the amperometric method of analysis.
A^arge number of naturally occurring nitrogenous organic compounds \
readily react with aqueous chlorine, exerting significant chlorine demands.]
Several of these compounds also produce chloroform upon reaction with chlorine
with maximum formation occurring between pH 8.5 and pH 10.5. The correlation
between chloroform formation and chlorine demand, however, is tenuous. It also
appears that intermediates may be formed under neutral or slightly acidic con-
ditions which produce chloroform upon exposure to more alkaline conditions.
analytical scheme was developed to identify trace quantities of N-
organic contaminants in dilute aqueous solution. \ This involved: (1) selective
removal of non-nitrogenous compounds using XAJ5-8 and Tenax GC macroreticular
resins; (2) concentration of water samples from 1,000 to 2,000 fold by flash
evaporation and lyophilization; and (3) separation and identification by high
performance liquid chromatography (HPLC). Seven N-organic compounds were
identified in municipal water supplies (adenine, 5-chlorouracil, cytosine,
guanine, purine, thymine, and uracil) at concentrations ranging from 20ug/L
to 860ug/L. All of the samples exhibited a large unresolved group of compounds
rapidly eluting from the chroma tographic column. Exhibition of a corresponding
fluorescamine responsive fluorescent peak, determination of the organic nitrogen
content of this group of materials, and retention positions of reference com-
pounds suggested that this group was composed of primary amine compounds.
The demonstration of a parallel increase in organic nitrogen content with
population density in two laboratory grown blue-green algal cultures, and the
finding of elevated organic nitrogen values in a water supply sample collected
during the occurrence of a blue-green algal bloom, suggested that summer algal
bloom occurrences can add considerably to the organic nitrogen content of a
water supply.
The levels of chloroform which might be formed at pH 7 were calculated
by assuming the N-organic compounds identified in the water supplies were not
removed prior to chlorination and yielded CHC13 according to the results of
Baum (10). The calculated CHC13 levels were well below the maximum contaminant
level (MCL) of 0.1 mg/L proposed by EPA for total trihalogenated methanes. The
calculated CHC13 formed under more alkaline conditions, however, was more than
10% of the MCL and was therefore significant. The calculated levels of com-
bined forms of chlorine yielding falsely positive tests for free chlorine in
iv
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some samples were slightly less or exceeded the 0.5 mg/L free chlorine
residual generally taken as an acceptable level of disinfection. If a major
portion of the free chlorine residuals determined in finished drinking water
consists of the less germicidal combined forms, additional chlorine should be
added to assure protection of public health.
This investigation of the formation and significance of organic N-chloro
compounds in chlorination of water supplies funded under research grant
R803631 involves the identification and determination of nitrogenous organic
materials in natural waters and the elucidation of their behavior with aqueous
chlorine. The areas studied include: (1) an investigation of the reactivity
toward aqueous chlorine of selected nitrogenous compounds analogous to those
anticipated as likely constituents of raw drinking water supplies; (2) an
attempt to isolate, concentrate and identify nitrogenous compounds actually
present in such supplies as an aid to understanding reactions occurring with
nitrogen-containing materials during water chlorination; (3) an evaluation of
several analytical methods now in use for distinguishing between free avail-
able chlorine and combined chlorine; and (4) the determination of nitrogenous
precursors to haloform formation.
This report was submitted in fulfillment of Grant # R-803631 by Harvard
University under the sponsorship of the U.S. Environmental Protection Agency.
This report covers the period April 1, 1975 to June 30, 1979, and work was
completed on June 30, 1979.
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CONTENTS
Foreword
Abstract v
Acknowledgment viii
1. Introduc tion i
2. Summary 5
3. Differentiation Between Free and Combined Chlorine 7
Experimental methods 9
Chlorinated cyanuric acid 10
Nitrogenous organic compounds 12
Conclusions 20
4. Identification of Nitrogenous Organics in Water Supplies 21
Analytical methods 21
Results 35
5. Reactions Between Nitrogenous Organic Compounds and Aqueous
Chlorine 187
Analytical methods 187
Chloroform standards 189
Results and discussion 191
Conclusions 232
References 235
Appendices
A. Appended Tables from Section 4 243
B. Appended Figures from Section 4 277
C. Literature Review 287
The hazards of consuming chemically contaminated
drinking water 287
Organic impurities in natural waters 289
Nitrogenous organic compounds 294
Algal production of N-organic extracellular metabolites.. 306
Urochromes and humic substances 306
Macroreticular resins 308
Isolation of trace organic compounds from dilute aqueous
solution 312
Kjeldahl and ammonia determinations 332
Appendices References 335
vii
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ACKNOWLEDGMENTS
This work was supported by Grant No. R803631 from the U.S. Environmental
Protection Agency.
The investigators studying the research topic during the period of this
grant included: Dr. Barbara Baum, Dr. M.M. Varma, Dr. Joseph Gould, Mr.
Edmund Wajon and Dr. Neil Ram. Ms. Agnes Straud also assisted the study.
The investigators wish to thank the personnel at the Lawrence Experiment
Station, Lawrence, Massachusetts for the use of their gas chromatograph and
for their help in the chloroform analyses, and Mrs. Elaine Zagarella for
gathering reference material.
viii
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SECTION 1
INTRODUCTION
Chlorine was first used in America to disinfect a municipal water supply
in 1908 in Jersey City, New Jersey (1). Since then it has become the primary
defense against the transmission and spread of waterborne disease. It has
been known for the past 60 years or more that free aqueous chlorine (HOC1 and
OC1~) reacts readily with ammonia or other nitrogenous organics to form com-
bined forms of chlorine generically termed chloramines. In general, these
compounds are much less germicidal than free aqueous chlorine. A secure
hygienic quality in water supplies is therefore, dependent upon the mainte-
nance of a free chlorine residual. The combined forms, however, retain an
oxidizing capacity and tend to react similarly with many analytical reagents
for active chlorine. When these N-chloro compounds are formed, tests for free
chlorine may be falsely positive and may indicate a nonexistent germicidal or
virucidal behavior.
The ability of varying analytical methods to differentiate between free
and combined chlorine is therefore of great importance and was investigated in
this study.
It has not always been recognized that there are two types of problems
concerned in chemical selectivity of the sort that is required to differenti-
ate between free and combined chlorine. The first type, the one commonly
recognized, is that the reagent must not react directly with the combined form
of chlorine as it does with the residual free chlorine. But, it must also be
kept in mind that even though the reagent reacts only with free residual
chlorine, it may nonetheless give a false positive response when hydrolysis of
the type reaction (1) occurs.
RNHC1 + H20 * RNH2 + HOC1 . (1)
-Reaction (1) is rapid compared with the time required for the chemical deter-
mination.
Differentiation of combined and free residual chlorine is relatively easy
when the only combined forms of active chlorine are the ammonia chloramines,
for then the hydrolysis equilibria lie sufficiently to the left to make selec-
tivity secure on a thermodynamic basis. The situation is more complex, how-
ever, when organic chloramines are concerned, for then hydrolysis may be rapid
enough and great enough for response as if there were much free residual
chlorine present. But only the instantaneously present free residual chlorine
is effective virucidally, not that generated by hydrolysis in response to
chemical reaction.
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A recent investigation of particular significance in this regard is the
study by Guter, Cooper and Sorber (2). Their evaluation of field methods for
determining free active chlorine, which included variations on most of the
standard techniques, showed that only one, a technique based on the use of
syringaldazine, was free of false positive tests as compared with ampero-
metric investigations. So, most methods for determining free active chlorine
may give misleading information about the bactericidal or virucidal effective-
ness associated with specified chlorination procedures.
An even more intriguing aspect of their work, however, was the finding
that the false positives occurred only with organically polluted water and
not for synthetic waters containing ammonia. The implication is that organic
N-chloro compounds are formed when polluted waters are chlorinated and that
some of these react more readily with supposedly selective reagents for free
active chlorine than do the ammonia-chloramines.
Even this study had its ambiguities, however, for it was necessary to
assume that the amperomctric method gives reliable results for free residual
chlorine, an assumption that may not always be correct. Only purely physical
methods that can measure the free active chlorine instantaneously present in
the aqueous solution without disturbance of the' labile equilibria can provide
thoroughly reliable standards against which proposed chemical determinations
can be checked.
The problem has been solved, in part, by the recent work of O'Brien (3),
in which all the hydrolysis and ionization constants were evaluated for the
chlorinated isocyanurate system. As a result, it is now possible to compute,
on purely thermodynamic grounds, the exact concentration of free active
chlorine instantaneously present in any chlorinated isocyanurate solution.
Indeed, it is possible by adjustment of chlorine to isocyanurate ratio and
pH to prepare solutions with desired known concentrations of free and com-
bined residual chlorine. Unfortunately, conclusions reached on the basis of
this organic-chloramine system may not be valid for all organic chloramines.
There have been numerous investigations of the reactions of free aqueous
chlorine with ammonia, epitomized by the papers of Palin (4) and of Wei and
Morris (5) . Studies like these have provided reasonably accurate information
about the conditions of formation and properties of the ammonia-chloramines
that their behavior and germicidal effectiveness relative to free aqueous
chlorine can be considered satisfactorily known. Moreover, it has been the
custom to check the performance of new analytical methods supposedly specific
for free chlorine against prepared solutions of ammonia-chloramines in the
assessment of interferences.
In contrast, knowledge about the organic nitrogenous compounds likely to
be found in raw waters or about the reactions of these compounds with aqueous
chlorine and the properties of the products is almost nonexistent. This is
despite the fact that the concentration of organic amine-type nitrogen is
likely to be considerably greater than that of free ammonia in natural
surface waters not recently contaminated with municipal sewage. A review of
older books on water chemistry published in the times when nitrogen analyses
were still used as prime indicators of water quality, such as Thresh and
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Suckling, Examination of Waters and Water Supplies (6), or Mason and Buswell's
Chemistry of Water Supply and Treatment (7), shows that expected concentra-
tions of free ammonia in upland surface waters are less than 0.06 mg/1 N,
whereas total organic amino-N measured by the Kjeldahl methods may be ex-
pected to be several tenths of a milligram per liter.
In addition to the disinfecting problems caused by the less germicidal
N-chloro compounds discussed previously, nitrogenous organics have been shown
to produce complex stable mutagenic chlorinated products when reacted with
chlorine (8,9). They have also been suggested as possible precursors in
haloform formation (10).
Although the composition and extent of hydrocarbon contamination in
natural waters has been under extensive examination, the identification of
nitrogenous organics in water supplies has not been significantly pursued.
This is because many nitrogenous organics are relatively non-volatile and
consequently their identification and analysis by gas chromatography is not
possible without the prior formation of volatile derivatives. Amino acids
are known to be present in the nitrogenous organic fraction found in natural
waters, and some attempts have been made to determine them (11-13), but the
quantities found—some yg per 1 of N in toto—seem small compared with total
organic-N expected. One would also expect considerable concentrations of
pyrrolic-N from the breakdown of chlorophyll and similar plant compounds, of
purine derivatives and of pyrimidine-related compounds such as uracil and
other nucleic acid components, but there appears to be little specific in-
formation available.
Recent advances in the field of high pressure liquid chromatography
(HFCL), have made the detection of non-volatile nitrogenous compounds in-
creasingly feasible. Amino acids commonly found in protein hydrolysates,
physiological fluids (14) and standard amino acid mixtures (15-25) have been
resolved using HPLC with fluorescence detection at the picomole level. Dr.
R. Jolley and Dr. W. Pitt have made significant progress in the separation
and identification of trace organic compounds in urine (26), primary and
secondary stages of municipal sewage treatment plants, and natural waters
(27-29) using a strongly basic anion exchange resin (Bio-Rad aminex A-27)
with ammonium acetate acid buffer eluent. Jolley (30,31) also found that a
number of chlorinated pyrimidines and purines were formed during the chlori-
nation of sewage effluent. Among these were 5-chlorouracil, 5-chlorouridine,
8-chlorocaffeine, 6-chloroguanine and 8-chloroxanthine.
There have been some studies on the reactions of amino acids with
aqueous chlorine, but very little on reactions of other probable aqueous
nitrogenous compounds. The survey by Tarras (32), although widely quoted,
provides little positive information. Palin (4) extended his work with
ammonia to include some observations of breakpoint phenomena. Culver (33),
made a detailed study of reactions in the chlorination of glycine, finding,
among other things, that cyanogen chloride could be formed as an intermediate
product. Friend (34), showed that many amino-acids gave N-chloro derivatives
rapidly, more rapidly than NHa at neutral pH. Conditions of formation and
properties of many other N-chloro compounds are known, of course, but not of
those that are likely to be of interest in connection with water chlorination.
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One category of reactions between chlorine and aqueous nitrogenous
compounds of particular concern is that which produces haloforms. The dis-
coveries by Rook (35) and by Bellar et a.1. (36) that the chlorination of
water supplies containing organic matter produces chloroform and other tri-
halome thanes, have evoked concern because of the potential carcinogenic and
toxic properties of these compounds. Morris (37) calculated the intake of a
50 kg individual consuming one liter per day of water containing 0.1 mg/1 of
chloroform to be .002 mg/kg, about one two-hundredth of the minimum observed
chronic toxicity level. It was suggested (38) that compounds which ionize
rapidly to give carbanions account for the formation of chloroform within the
contact time of most water treatment plants and distribution systems. One
structure in which active carbanion formation occurs is the nitrogen con-
taining pyrrole ring. The hydrogens ortho to the nitrogen are activated like
those in phenol and provide sites for chlorination and subsequent haloform
formation. The pyrrole ring is naturally important because of its occurrence
in chlorophylls, xanthophylls and heme. Indole and derivatives like trypto-
phan also contain pyrrolic units. An investigation into the haloform
producing potential of selected nitrogenous organics was therefore pursued in
this study.
This report addresses the hygienic and aesthetic importance of nitro-
genous organic compounds in natural waters and the formation and properties
of their chlorinated derivatives. Such information is important hygienically
because assured disinfection of all forms of pathogens can be maintained
reliably only if specific determinations for free aqueous chlorine are not
subject to interference by organic N-chloro compounds that may be formed. It
is also important aesthetically because it appears that some of the noxious
tastes and odors commonly attributed to aqueous chlorine may actually be due
either to organic N-chloro compounds or to NCI 3 formed from such compounds
under conditions where it is not normally formed from
This investigation of the formation and significance of organic N-chloro
compounds in chlorination of water supplies funded under research grant
R8036301 involves the identification and determination of nitrogenous organic
materials in natural waters and the elucidation of their behavior with
aqueous chlorine. The areas studied include: (1) an investigation of the
reactivity toward aqueous chlorine of selected nitrogenous compounds analo-
gous to those anticipated as likely constituents of raw drinking water
supplies; (2) an attempt to isolate, concentrate, and identify nitrogenous
compounds actually present in such supplies as an aid to understanding
reactions occurring with nitrogen-containing materials during water chlori-
nation; (3) an evaluation of several analytical methods now in use for
distinguishing between free available chlorine and combined chlorine; and
(4) the determination of nitrogenous precursors to haloform formation.
A detailed literature review on the occurrence and environmental
significance of nitrogenous organic contaminants in water, and analytical
methods for their determination is included in Appendix C.
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SECTION 2
SUMMARY
1. All of the methods used to measure free chlorine (DPD, orthotolidine-
arsenite (OTA), LCV, SNORT, SYRING, and amperometric titration) are subject
to interference from organic chloramines.
2. The amperometric method displays some differentiation between free
and total chlorine.
3. If only amino acids are present along with ammonia, the DPD or
SYRING methods should be used since these appear to be the most specific for
free chlorine under these conditions.
4. XAD and Tenax resins selectively remove non-nitrogenous organic
contaminants from water although not with 100% efficiency.
5. A large number of naturally occurring nitrogenous organic compounds
readily react with aqueous chlorine, exerting significant chlorine demands.
6. Several nitrogenous organic compounds produce chloroform upon
reaction with chlorine.
7. Chlorine demand does not appear to be a very good indication of the
chloroform producing potential of an individual compound.
8. For chloroform products, increasing the pH has a considerable in-
fluence on the quantity of chloroform formed. Maximum chloroform formation
occurs between pH 8.5 and pH 10.5.
9. Compounds that show significant chloroform production only under
alkaline conditions may form other chlorinated derivatives under neutral or
slightly acidic conditions. These intermediates produce chloroform upon
exposure to more alkaline conditions.
10. Chlorination may have the beneficial effect on chemical water
quality in reacting to eliminate amines which are natural precursors of car-
cinogenic nitrosamines formed by reaction with salivary or gastric nitrite.
11. Chromatograms of resin-filtered samples showed no improvement in
either U.V. or fluorescence background traces over chromatograms of unfil-
tered samples. The total number of compounds identified at a given collec-
tion site was maximized, however, by analyzing both raw and resin-filtered
samples. This relates to item //4.
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12. The average values for compounds identified in the water supply
samples were 367, 60, 20, 167, 200, 110, and 160 yg/L, for adenine, 5-chloro-
uracil, cytosine, guanine, purine, thymine and uracil, respectively. Uracil,
adenine, and guanine were found most frequently in the water supply samples
while 5-chlorouracil, cytosine and purine were only encountered once in these
sources.
13. Summer algal blooms may contribute significantly to the organic
nitrogen content of water supplies.
14. The levels of chloroform which might be formed at pH 7 were calcu-
lated by assuming N-organic compounds identified In the water supplies were
not removed prior to chlorination and yielded CHCla according to the results
of Baum (10). The calculated CHC13 levels were well below the maximum con-
taminant level (MCL) of 0.1 mg/L proposed by EPA for total trihalogenated
methanes. The calculated levels of CHC13 formed under more alkaline condi-
tions, however, were more than 10% of the MCL and were therefore significant.
15. The calculated levels of combined forms of chlorine yielding
falsely positive tests for free aqueous chlorine in some samples were
slightly less or exceeded the 0.5 mg/L free chlorine residual generally taken
as an acceptable level of disinfection." If a major portion of the free
chlorine residuals determined in finished drinking water consists of the less
germicidal combined forms, additional chlorine should be added to assure
protection of public health.
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SECTION 3
DIFFERENTIATION BETWEEN FREE AND COMBINED CHLORINE
As discussed previously combined available chlorine is a much less active
disinfectant than free chlorine. Analytical methods capable of differenti-
ating between free available chlorine and the various forms of combined chlo-
rine and the various forms of combined chlorine are, therefore, needed to
ensure that water supplies are adequately disinfected. However, interconver-
sion of these species occurs in solution through the hydrolysis of combined
chlorine to free chlorine (equation (2)), thus presenting difficulties in
developing suitable analytical methods.
R2NC1 + H20 -*• R2NH + HOC1 (2)
The methods currently most used for differentiating free chlorine from
the combined forms are DPD (39-43), orthotolidine-arsenite (OTA) (40,44,45),
LCV (39,46,47), SNORT (39,48), SYRING (39,48,51), and amperometric titration
(39,40,52-58). These methods rely upon the more rapid rate of reaction
between the given reagent with HOC1 as compared to the N-chloro compound
(kinetic selectivity), and on the slowness of hydrolysis of the N-chloro
compound.
The errors involved in the kinetic selectivity of the analytical reagents
are illustrated by their reaction with NH2C1 and NHC12. DPD and SNORT, for
example, react with these chloramines at a rate of 3.6% and 1.1% per minute,
respectively. The reaction with SYRING is slower, however, with interference
only being observed at higher NH2C1 concentrations (48,49). The selective
differentiating ability of the analytical reagents decreases (40,59,60) at
lower pH values. This may be partially attributable to the acid catalysis of
the hydrolysis reaction (equation (2)) (61,62) as illustrated by the inability
of the acid orthotolidine method to distinguish between free and combined
chlorine without rapid addition of arsenite.
Several of the analytical reagents have been tested for their response to
N-chloro compounds. Palin (4) found that DPD could distinguish between free
chlorine and several types of chlorinated amino acids. The OTA procedure was
found to distinguish between free and combined oxidant when seawater con-
taining several amino acids, diphenylamine and uracil was chlorinated (65).
In studying the SYRING procedure, it was found that although it worked
well in simple solutions of known composition, there were several reports of
false positive chlorine in natural waters (49,50). It was also found that N,
NJ-2, 2!-4, 4^6, e^octachloro-N, N^diphenylurea reacted with SYRING (66).
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Recently, a membrane electrode has been developed by Johnson (67,68)
which shows promise as a discriminatory method. Selectivity is based on
species volatility and the application of an appropriate positive potential
to the electrode.
The experimental work on this topic examined the selective differenti-
ating ability of the six analytical reagents for free aqueous chlorine in the
presence of a variety of N-chloro compounds. Several types of nitrogenous
organic compounds were investigated and are shown in Table 1.
TABLE 1. NITROGENOUS ORGANIC COMPOUNDS STUDIED
AM I NO ACIDS
KETEROCYCLIC BASES
GLYCYLGLYCINE (GLY) NHjCHjCNHCHjCootf SUCCINIMIDE
(Su)
SARCOSINE
($A)
CREATINUIE
GLUT AM ic ACID (GLuHiooccK,cHjCnccioH INDOLE
NK
PHENYLALANINE (PA)
TYROSiNE
PROLINE (PR)
GREATINE
HISTIDINE
M-AM1NOPHENOL (AP)
TRYPTOPHAH
PYRROLE
URACIL
CYTOSINE
THYMINE
BARBITURIC ACID
ADEN INE
GUANINE
PURINE
URIC ACID
CAFFEINE
4-AM1NO-
ANTIPYRINE
(TRY) rl
H
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EXPERIMENTAL METHODS
Chlorine-Demand Free Water (CDFW)
All solutions used In this study were prepared using chlorine-demand
free water, prepared by dosing distilled water with 1-2 mg/1 chlorine and and
destroying the chlorine remaining after 6-8 hours by UV radiation. All glass-
ware was dosed with 1-2 mg/1 chlorine for several hours and rinsed with CDFW.
Chlorine Solutions
Concentrated chlorine solution was prepared by bubbling chlorine gas
into distilled water, adjusting the pH to 10 with KOH and then distilling
this solution at 90°C under suction. This produced a 10~2 M solution of
chloride-free HOC1. A 1x io~3 M stock solution of HOC1 was prepared by
dilution and stored in a low actinic glass bottle at 5°C. Total available
chlorine in this solution was determined periodically using the thiosulphate-
iodide titration method.
Buffer Solutions
A 10"3 M sodium bicarbonate buffer solution in CDFW was used in which
the pH was adjusted to pH 7.0 using COa so that the chloride concentration
could be kept as low as possible.
10 3 M Stock Soj-utions of Nitrogenous Compounds
The samples of compounds investigated were of the highest quality
commercially available, and were not purified further before use. The com-
pounds were dissolved in 10"3 M pH 7.0 bicarbonate buffer.
Analytical Methods
The methods employed were DPD, ORTHO (and later OTA), LCV, SNORT, SYRING
and amperometric titration. The reagents were prepared according to Standard
Methods (39). Color development was measured on a Beckmann DU Spectrophoto-
meter. A Fisher Model 393 Chlorine Titrimeter was used for amperometric
titrations. Reagents were discarded if they became discolored or were found
to have lost sensitivity to the stock chlorine solution. The limit of
detection of the colorimetric methods varied between batches of reagents from
about 0.02 mg/1 to 0.15 mg/1; the limit of detection for the amperometric
method was about 0.05 mg/1.
Analytical Procedures
10 ml of a 10~3 molar concentration of each compound tested was dosed
with sufficient 1x 10"3 M stock chlorine solution to give an initial molar
chlorine to compound ratio of 1:1, 2:1 or 3:1. Reaction was allowed to pro-
ceed for half an hour in the dark, at which time the mixture was diluted to
1000 ml with 10"3 M pH 7 buffer and transferred to an amber bottle where it
was mixed thoroughly for another 15 minutes. A portion of the solution was
then added to the analytical reagent and analysed as rapidly as possible.
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Analysis using DPD, ORTHO, LCV, SNORT, SYRING and amperometric titration was
carried out, in that order, a process which generally took 1 to 1-1/2 hours.
Very little change in residual chlorine was found to occur during this time.
In the later experiments with cyanuric acid, the reaction procedure was
altered so that the reactions could be carried out at 10~5 M concentrations
instead of 10~3 M concentrations. 500 ml solutions containing the appropriate
amount of the compound and chlorine were mixed in less than 30 seconds from
individual separatory funnels leading directly into an amber bottle, where
the solution was thoroughly mixed for 45 minutes before analysis.
CHLORINATED CYANURIC ACID
Considerable emphasis in the investigation of the analytical methods was
focused on the cyanuric acid/aqueous chlorine system because of the ability
to predict the equilibrium composition of the solution from the equilibrium
constants measured by O'Brien et al. (69), or by Pinsky and Hu (70). At
equilibrium in aqueous solution, mono, di and trichloro derivatives coexist
along with substantial quantities of free chlorine (F).
Aqueous chlorine was mixed with cyanuric acid and solutions of the
chlorinated cyanuric acids were also prepared. The results of the analysis
of free (F) and total (T) chlorine under different conditions at each stated
chlorine: cyanuric acid molar ratio are presented in Tables 2 to 4. This is
compared with the theoretical concentrations of free chlorine (F), the mono-
chlorinated species (M) , the dichlorinated species (D) and total chlorine (T)
at pH 7.0, derived from O'Brien (3).
There is considerable scatter in the concentration measured by each
analytical method with a standard deviation of about 10-15%. There is also
some variation between the categories. This may be due to (i) differences
in mixing and thus demand, or (ii) low reproducibility in the analytical
method.
The results indicate that, with the exception of the amperometric
method:
(i) none of the methods for free chlorine can distinguish between free
and combined chlorine;
(ii) all methods measure the total available chlorine.
The amperometric method displayed some differentiation between free and
total chlorine, indicating that with at least some of the combined
species, hydrolysis is slow. The interference observed in the other methods
for free chlorine is, therefore, caused by the reaction of the analytical
reagent with the N-chloro compound. In the amperometric method, this
reaction appears to be slow, while in the colorimetric methods it is rapid.
10
-------
TABLE 2. CHLORINATED CYANURIC ACID
MOLAR RATIO 1:1 0.71 MG/L CL2 pH 7.0
RESIDUAL AVAILABLE CHLORINE (MG/L)a
Reaction
Condition
10~3M
Reaction
10~5M
Reaction
Average
Theory
Species
Measured
F
T
F
T
F
T
F
M
D
T
METHOD
DPD
0.55
0.50
0.40
0.50
0.50
0.50
ORTHO
0.55
0.60
0.40
0.40
0.45
0.50
LCV
0.45
0.55
0.60
0.45
0.55
0.50
0.33
0.31
0.07
0.71
SNORT
0.50
0.55
0.50
0.50
0.50
0.50
SYRING AMP
0.55 0.40
0.50
0.45 0.40
0.50
0.50 0.40
0.50
Averages in MG/L CLa, rounded to nearest 0.05 MG/L.
TABLE 3. CHLORINATED CYANURIC ACID
MOLAR RATIO 2:1 1.42 MG/L CL2 pH 7.0
RESIDUAL AVAILABLE CHLORINE (MG/L)a
Reaction
Condition
10~3M
Reaction
10~5M
Reaction
Dichloro-
Cyanurate
Average
Theory
Species
Measured
F
T
F
T
F
T
F
T
F
M
D
T
METHOD
DPD
1.20
1.10
1.00
1.05
0.80
0.90
1.00
1.00
• ORTHO
1.25
1.30
1.00
1.00
0.70
1.00
1.10
LCV
1.45
1.20
1.10
1.15
1.00
0.90
1.25
1.10
0.79
0.41
0.22
1.42
SNORT
1.45
1.25
1.05
1.10
0.75
0.80
1.10
1.05
SYRING AMP
1.30 0.40
0.95
1.00 0.90
1.05
0.80 0.80
0.95
1.00 0.75
0.95
"
Averages in MG/L CLa, rounded to nearest 0.05 MG/L.
11
-------
TABLE 4. CHLORINATED CYANURIC ACID
MOLAR RATIO 3:1 2.13 MG/L CL2 pH 7.0
RESIDUAL AVAILABLE CHLORINE (MG/L)a
Reaction
Condition
10~3M
Reaction
10~5M
Reaction
Trichloro-
Cyanurate
Average
Theory
Species
Measured
F
T
F
T
F
T
F
T
F
M
D
T
METHOD
DPD
1.85
1.65
1.60
1.60
1.60
1.70
1.65
1.65
ORTHO
1.95
2.10
1.60
1.55
1.70
1.75
1.80
LCV
2.10
2.05
1.90
1.80
2.15
2.05
2.05
2.00
1.34
0.41
0.38
2.13
SNORT
2.05
1.90
1.75
1.80
1.90
1.90
1.95
1.85
SYRING AMP ELEC
2.15 1.10
1.55
1.75 1.40
1.70
1.75 1.60
1.80
1.85 1.50
1.75
Averages in MG/L CLa, rounded to nearest 0.05 MG/L.
NITROGENOUS ORGANIC COMPOUNDS
Amino Acids
The results of free chlorine measurement in the presence of amino acids
with a 1:1 ratio of aqueous chlorine are presented in Fig. 1. Several
patterns are evident in this graph. Zero free chlorine is reported by SYRING
in the presence of 6 amino acids (glycylglycine, glutamic acid, sarcosine,
phenylalanine, tyrosine and histidine), in contrast to ORTHO, LCV and SNORT.
Similarly, DPD reports zero free chlorine in solutions containing 5 of these
amino acids (i.e., with the exception of sarcosine). Because DPD, LCV and
SNORT report a high total chlorine concentration (see Fig. 2) in these
solutions, it seems likely that DPD and SYRING can differentiate effectively
between free and combined chlorine in these systems.
At the 2:1 ratio, DPD again appears to be differentiating effectively.
However, the trend observed in free chlorine measurements at the 1:1 ratio
with the SYRING and SNORT methods are reversed (see Fig. 3). The SYRING
method seems to also be measuring combined chlorine especially in those
solutions containing glycylglycine, sarcosine and tyrosine.
12
-------
1:1 CI2:AMltfO /VC-J PS
FREE CHLORINE
GLY
_._,_*_. GLl/
L_, SA
AP 0_0_,,_«,
H
0-4-
0-3 .
0-2
0-1 .
o-o.
Figure 1. 1:1 Chlorine:amino acids. Free chlorine measured in
solution initially containing 0.7 mg/1 Cl«.
13
-------
i;l CI2:AM!WO ACIDS
TOTAL CHLORINE
- OL Y
SA
T Y
OOODDDa
AP
PP
CR
H
CM
O
08-
0-7
0-6
05
0-4
0-3
02
D P D
LCV SNORT ORTHO
Figure 2. 1:1 Chlorine:amino acids. Total chlorine measured in
solution initially containing 0.7 mg/1 Cl-
-------
; I C|2 r A M INO AGIOS
FREE CHLORINE
cur
0-7 i
0-6-
0-5-J
c\j
o
en
E
0-1
03
02
0-1
0-0
DPD
LC V
SNORT ORTH
Figure 3. 2:1 Chlorine:amino acids. Free chlorine measured in
solution initially containing 1.4 mg/1 C12.
15
-------
This study confirmed Palin's finding (4) that DPD was able to differen-
tiate between free chlorine and chlorinated amino acids, although we also
found several amino acids (creatine and sarcosine) where such discrimination
was not found. Some doubts exist about the capabilities of SYRING and SNORT.
It seems SYRING may be able to distinguish between free chlorine and chlor-
inated amino acids (i.e., with the exception of creatine and sarcosine),
while SNORT does not. Moreover, SNORT seemd to suffer from a bleaching of
the blue color, formed by free chlorine, when total chlorine was measured
(see Fig. 4).
2:1 C12:AMINO ACIDS
TOTAL CHLORINE
6 LY
• ^ f^ ••«
*_, i OLD
SA
oooaoo TY
0_ Q _ O _Q
1-2-
00
o
x 08
ov
0-6
02
0-0
p R >.
CR
LJ M.. m_-
DPD
LGV
Figure 4. 2:1 Chlorine:amino acids. Total chlorine measured in
solution initially containing 1.4 mg/1 Cl™.
16
-------
ORTHO and LCV measure some combined chlorine in the free chlorine deter-
mination. It is therefore surprising that Duursma and Parsi (65) were able
to differentiate between free and combined oxidant formed from glycine,
alanine, cysteine, leucine and tyrosine using OTA. However, this may in-
dicate the behavior of N-bromo compounds towards the analytical reagents is
different from the N-chloro compounds.
Differentiation between free chlorine and chlorinated amino acids thus
seems to be a result of the kinetics of hydrolysis. Methods employing a
neutral pH can differentiate, but decreasing the pH even to pH 4 increases
the rate of hydrolysis sufficiently to cause artificially high determinations
of free chlorine.
Heterocyclic Bases
For several of the heterocyclic compounds, chlorine demand was so high
that no available chlorine remained in solutions mixed with equal molar
ratios of aqueous chlorine. There is not as striking a pattern in the
measurement of free chlorine in the presence of these compounds as with the
amino acids (see Fig. 6). SNORT does tend to report higher concentrations of
free chlorine than the other methods and in most cases, the total and free
concentrations reported by SNORT are the same (see Figs. 5 and 6) suggesting
it does not differentiate between free and combined chlorine. All the ana-
lytical methods are subject to some false positive readings for free chlorine.
Since at least one method measured a zero free chlorine concentration in
each solution examined, with the exception of those containing purine or
succinimide, hydrolysis of these N-chloro compounds must be slow and inter-
ference is due to direct reaction with the analytical reagent.
Selected Amino Acids and Bases
Glycylglycine—
DPD can differentiate between free chlorine and the mono- and di-chloro
derivatives, the combined chlorine being measured in the monochloramine
fraction in both solutions. SYRING can also differentiate between free
chlorine and the monochloro derivative.
Sarcosine—
Since only a mono-chloro derivative is possible, the results shown in
Figs. 4 and 5 for the 2:1 ratio indicate this derivative is relatively stable
in the presence of excess chlorine. Complete differentiation between this
species and free chlorine is only achieved by SYRING.
Creatine—
Lomas (71) carried out some chlorination studies on this compound and
found that, using an orthotolidine-ferrous titration procedure (similar to
the SNORT procedure at pH 7), he could differentiate between free and com-
bined chlorine. Our results using SNORT show there is some interference of
combined chlorine with the free chlorine measurement.
17
-------
4_,_,_
. ----- . c
^ ____________ u
T
B
P
CA
o
O-5-i
0-4
r 0-3
O>
E
0'2-i
0-1
0-0
DPD
LCV
Figure 5. 1:1 Chlorine:heterocyclic bases. Free chlorine measured
in solution originally containing 0.7 mg/1.
18
-------
: i CJg: HEJE^OCXCUC BASES
TOTAL
0-8-
07
049
•O
w O • o
o
- 0-4
0*
e
0-3
o-z
• 1
n-n
•jv 0
T CA
/ 'x«
N*
"'-
\ /
""«.•».! t_ • • , I , \
/ / V '\
/ \ x
^'^ y •'"' .-••"' "v\"
*•«. ^X ,/ X X
*"."'" / / XNX
.„.-•"' ">x--..^ ^-"V\
DPI>
LCV SHORT
Figure 6. 1:1 Chlorine:heterocyclic bases. Total chlorine
measured in solution originally containing 0.7 mg/1 Cl,
19
-------
Succinimide—
About 4% of the total available chlorine can exist as free chlorine at
equilibrium with N-chloro succinimide. All methods suffer from some inter-
ference of N-chloro succinimide. However, ORTHO measures a lower free and
total chlorine concentration than the other methods, suggesting the hydro-
lysis of N-chlorosuccinimide and its reaction with orthotolidine are both
slow at pH 1.3.
Creatinine—
SNORT is the only method which is capable of differentiating between
free chlorine and the chlorinated creatinines, either the mono- or the di-
chloro derivative. In the methods were distinction between monochloramine
and dichloramine can be made (DPD and SNORT), concentrations of combined
chlorine were reported in both fractions. This may be evidence that either
derivative reacts readily in the total method, but may slowly interfere in
the monochloramine fraction, and even with the measurement of free chlorine.
Lomas (71) obtained similar results, both with his "SNORT" procedure and the
DPD method. However, he interpreted his results as being evidence for the
formation of a mono-chloro derivative only.
Cytosine—
The SNORT method measured combined chlorine species in the dichloramine
and monochloramine fractions at high cytosine concentrations using chlorine
to cytosine molar ratios of 1 and 2, respectively. This can be explained by
the greater reactivity of the dichlorocytosine. At lower cytosine concen-
trations, however, no differentiation was observed. DPD does differentiate
between free and monochlorocytosine, the later being observed as dichloramine.
Some dichlorocytosine does interfere in the measurement of free chlorine in
the DPD method while the remainder is observed as monochloramine. LCV, how-
ever experiences no interference from either of these compounds in the
measurement of free chlorine.
CONCLUSIONS
All of the methods currently used to measure free chlorine are subject
to interference from organic chloramines. Reliable measurements of the free
chlorine present in water supplies or wastewaters containing organic nitrogen
can thus not be made unless the types of organic nitrogen compounds present
are known. If only amino acids are present along with ammonia, the DPD or
SYRING methods should be used since these seem to be the most specific for
free chlorine under these conditions.
20
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SECTION 4
IDENTIFICATION OF NITROGENOUS ORGANICS IN WATER SUPPLIES
Until recently the organic content of water was generally evaluated using
gross organic analytical determinations such as Total Organic Carbon (TOC),
Chemical Oxygen Demand (COD), various extracting methods (CAE and CCE) or
Biological Oxygen Demand (BOD). Quantitative determination of individual
molecular species present in the microgram per liter range represented a
formidable analytical task. The need to understand the specific nature of
the array of contaminants present in our water supplies, however, has lead to
significant progress in the development of methods and instrumentation re-
quired for the identification and quantification of such contaminants.
Resins, capable of removing specific categories of trace organics, and the
development of high pressure liquid chromatographic techniques have made
possible the detection of non-volatile nitrogenous organic compounds from
dilute sources.
Quantitative determination of organic constituents involves concentration,
separation, and identification techniques. Because of the broad types of
organic compounds likely to be present in natural waters the analytical pro-
cedure must also include a pretreatment step in which "interfering" non-
nitrogenous organic compounds are selectively removed. The remaining organics,
present at only microgram per liter concentrations must then be concentrated
by factors of a thousand fold prior to chromatographic separation.
The procedure used in this study to identify trace nitrogenous organics
involved: (1) the selective removal of interfering, non-nitrogenous organic
material using macroreticular resins [(XAD-2,4 and 8) (72), (Tenax) (73),
(XE-340, 347, 348) (74)], concentration of water samples by flash evaporative
technique followed by freeze drying, and separation and identification of
specific compounds using high pressure liquid chromatography.
ANALYTICAL METHODS
Sample Sites and Collection
Field samples were collected from several water supplies of northeastern
Massachusetts as well as one from Bethesda, Ohio. The Massachusetts locations
were chosen because hydrologic records indicated that summer blue-green algal
blooms were likely to occur in these sources and because of their proximity
to the research laboratory of this investigator. The municipal water supplies
selected for the study were: Billerica, MA (Concord River), Lawrence, MA
21
-------
(Merrimack River), and Danvers, MA (Merrimack River), and Danvers, MA
(Middleton Pond). Other Massachusetts water supplies were considered but the
absence of observed algal blooms in the hydrologic records of these sources
precluded their inclusion in this study. It was supposed that sampling
during a blue-green algal bloom would increase the likelihood of detecting
individual N-organic compounds, because organic nitrogen compounds are known
to be released as extracellular metabolites by this group of algae. Arrange-
ments were also made for the sampling and shipment of water samples from
Fairfax County, VA during bloom occurrences. Plant superintendents at all
locations cooperated by reporting the occurrence of any increased algae
growth in the water supplies under investigation. Water samples were
collected immediately after notification of a bloom occurrence and prior to
addition of algicides to the water supply. Samples were also taken from the
Marlboro East and West sewage treatment plants to confirm the ability to the
analytical procedure to separate and identify organic components from pre-
sumably more concentrated sources. Samples from Spy Pond, Arlington, MA, and
the Charles River, Cambridge, MA, were also collected.
Non-bloom water samples were collected by grab sampling from approxi-
mately the upper six inches of the river or pond using a plastic bucket. The
samples were then transferred into several acid-washed (10% HC1) one-gallon
polyethylene jugs for transport to the laboratory. The Danvers 'bloom1
sample was collected by skimming the upper inch of water from regions of
dense algal growth. The sample was collected prior to treatment of the pond
with copper sulfate. Water samples taken during the second sampling of the
Concord River (Billerica, 7/14/78) were obtained by selectively collecting
water from shallow regions containing algal mats or dense aquatic weeds. All
river sites with the exception of the second Billerica sampling were located
at the intake channels of the water treatment facilities. Pond samples were
taken at convenient spots along the shore. Samples collected during the
second sampling of the Concord River were taken just downstream of the water
treatment plant near Bridge Street and River Street (Figures 7 through 10).
Approximately 15 liters of water were collected at each site. Samples
were returned to the laboratory within one hour of sampling where they were
either analyzed immediately for ammonia and Kjeldahl nitrogen, or stored at
5 degrees centigrade for subsequent analysis. Filtration and concentrations
were carried out within one to five days of the sample collection. Ammonia
and Kjeldahl-nitrogen contents were determined no more than 24 hours after
collection of the sample.
In addition to the field sites, samples were also taken from laboratory
grown cultures of two blue green algae, Anabaena flos aquae and Osci,llatoria
tenuis (obtained from the Texas culture collection of algae, Dr. Richard
Starr, University of Texas, Austin, TX). These two species were chosen
because they are known to liberate large quantities of nitrogenous organic
compounds during growth (75,76,77).
The Anabaena flos aquae culture was incubated for 42 days while the
Oscillatoria tenuis culture was grown for 35 days. The cultures were
sacrificed, filtered to 0.7 mu with Whatman GF/F filter paper and concentrated
by rotary evaporation followed by lyophilization. Concentrated samples were
22
-------
MASSACHUSETTS
BAY
Chorlcs
WATERTOWN Driver site
Figure 7. Massachusetts sampling sites.
-------
LAWRENCE
.TRAIN
PLANT
Figure 8. Sampling site: Lawrence, MA.
24
-------
FILTRATION
PLANT
Figure 9. Sampling site: Danvers, MA.
25
-------
NORTH
BILLERICA
FILTRATION
PLANT v
Figure 10. Sampling sites: Billerica, MA.
redissolved in chromatographic eluant, centrifuged, and frozen at -25°C for
several weeks until chromatographic analysis was performed.
Filtration of Collected Samples
In the laboratory, the water samples were first filtered through Whatman
#1 filter paper to remove suspended material greater than 10 ym in diameter.
The filter paper was replaced whenever the filtration rate decreased appre-
ciably because of clogging. Whatman filter-papers GF/D (2.7 ym) and GF/F
(0.7 ym) were also used for the second Danvers sample and with the filtration
of the laboratory-grown algae cultures to remove suspended algal cells. Katz
et at. (78) reported that only negligible U.V. absorbing material was lost by
filtration through a 10 ym frit to remove suspended material.
26
-------
Ammonia-N Determination
Aramonia-N was initially determined using the indophenol colorimetric
reaction described by Dora Scheiner (79). A twenty-five milliliter filtered
sample was allowed to react in a 50-ml flask with 10 ml of pH 12 buffer
solution containing phenol and nitroprusside followed by dilution to 50-ml
with hypochlorite reagent.
The buffer solution (pH 12) consisted of 30 g trisodium phosphate
(NasPOif -12H20), and 3g disodium ethylenediaminetetraacetate (EDTA) diluted to
one liter with distilled ammonia-free water. The ammonia-free water was pre-
pared by dosing distilled water with 1-2 mg/L chlorine and decomposing the
active chlorine remaining after 6-8 hours with U.V. radiation. The phenol
nitroprusside solution was made by dissolving 30g of phenol in a part of the
pH 12 buffer solution followed by addition of 0.1 g sodium nitroprusside
(Na2Fe(CN)5NO-2H20) and dilution to 500 ml with buffer solution. It was
important to add the nitroprusside only after the phenol had been dissolved
in the buffer solution to ensure a low reagent blank.
The alkaline hypochlorite reagent consisted of 15 ml of commercial
bleach (Chlorox, 3.5% available chlorine) and 200 ml of sodium hydroxide
solution diluted to 500 ml with distilled ammonia free water. Sodium
hydroxide solution was made by dissolving 40g NaOH in distilled ammonia free
water and diluting to one liter. Reagent grade sodium hypochlorite solution
(Fisher Scientific Co., 4-6% NaOCl) was later used in place of the Chlorox
to obtain lower reagent-blank values.
The sodium nitroprusside solution was prepared freshly every three weeks
and stored at 4 degrees centigrade. Alkaline hypochlorite reagent was pre-
pared daily when needed. All reagents were brought to room temperature prior
to use.
After addition of the reagents to the water sample the flask was mixed
well by inversion. Color development was considered complete after 45 minutes
at room temperature. Absorbance was read at 635 nm against a reagent blank
carried through the procedure along with each set of samples. A one cm cell
was used to measure the absorbance of solutions having ammonia concentrations
less than about 1 mg/L
All glassware was acid washed in 10% HC1 prior to use to remove trace
ammoniacal impurities. A calibration curve was prepared by submitting samples
containing known ammonia concentrations to the standard procedure. Stock
ammonia solution (1,000 mg/L N; stable at least 6 months) was prepared by
dissolving 3.810 g NHi»Cl, previously dried one hour at 100°C, in distilled
ammonia-free water and diluting to one liter. Intermediate ammonia solutions
(20 mg/L; stable 4 days) were made by diluting 2 ml stock ammonia solution to
100 ml with distilled, ammonia-free water. Standard ammonia solutions con-
taining 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, and 1.0 mg/L were prepared by
dilution of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 ml of intermediate
ammonia solution to 100 ml with distilled ammonia-free water. A Beckman
double-beam spectrophotometer was used for the absorbance readings.
27
-------
Because of difficulties in the reproducibility and color development in
determining NH3-N content after Kjeldahl digestion using Scheiner's method,
ammonia-N was later determined using the scaled-down indophenol method of
Strickland and Parsons (80). For this method phenol solution was prepared by
dissolving 20 g crystalline analytical grade phenol in 200 ml of 95% ethyl
alcohol. Sodium nitroprusside solution consisted of 1.0 g sodium nitro-
prusside in 200 ml ammonia-free water. The solution was stable for at least
one month. Alkaline reagent was made by dissolving 100 g sodium citrate and
5 g analytical grade sodium hydroxide in 500 ml ammonia-free water. This
solution was stable indefinitely. The oxidizing solution was prepared daily
as needed and consisted of 10 ml reagent grade sodium hypochlorite diluted to
50 ml with alkaline stock solution.
Acid-washed 20-ml Pyrex test tubes were filled with ammonia-free water
and capped with aluminum foil prior to use to lessen contamination by atmo-
spheric ammonia. This water was discarded immediately prior to use for a
determination. Five ml of sample were then pipetted into each test tube.
The following solutions were sequentially added with automatic pipets and the
samples were vortex mixed after each addition:
0.2 ml phenol solution;
0.2 ml nitroprusside solution;
0.5 ml oxidizing solution.
The test tubes were then capped with aluminum foil and allowed to stand at
room temperature for one hour for complete color development. The color
produced is said to be stable for at least 24 hours (80). Absorbance readings
and preparation of calibration curves were done according to the previously
described method.
Kjeldahl-Nitrogen Determination
Total Kjeldahl nitrogen was measured by ammonia determination after
decomposition of the organically bound nitrogen in the minus three oxidation
state in the water sample to ammonia by acid digestion. This method fails to
account for the nitrogen in azides, azines, azo, hydrazones, nitrate, nitrite,
nitrile, nitroso, oximes, and semicarbazones (44). Several digestion methods
were used during the study. The digestion solution of Scheiner (79) which
was employed initially, consisted of 134 g of potassium sulphate (K2SOi,)
dissolved in 650 ml of ammonia-free water and 200 ml of concentrated HzSOi,.
Five ml selenyl chloride (SeOCla) were added and the combined solution was
diluted to one liter, after cooling, with ammonia-free water. Five ml of
this digestion solution was added to a 25-ml water sample in a 100-ml Kjeldahl
flask, and heated under a hood until all the water was removed. The residue
was digested for a further 30 minutes and then cooled to room temperature.
Small glass funnels were inserted into the mouths of the Kjeldahl flasks to
help the digestion and prevent loss. After the sample had cooled, it was
transferred to a 100-ml beaker by sequential rinsing with ammonia free water
and neutralized to pH 7 or greater with 1 M NaOH. The neutralized sampled
was then transferred to a 100-ml volumetric flask and filled to the mark with
ammonia-free water. Twenty-five ml of the diluted neutralized sample was
then analyzed according to Scheiner's ammonia method described previously.
28
-------
Other digestion mixtures were employed with essentially the same pro-
cedure. These included:
(1) Standard Methods digestion reagent (44):
This is the same as Scheiner's reagent with the exception that 2 g of
HgO is used as the catalyst per liter instead of 5 ml selenyl chloride.
(2) Mague and Mague digestion reagent (81):
0.2 g SeOa and 20 g K2SOi, were dissolved in 600 ml ammonia-free distilled
water. 110 ml concentrated reagent grade H2SOi» was then added. After the
mixture had cooled it was diluted to one liter with ammonia-free water.
(3) Strickland and Parsons' digestion reagent (80):
0.1 g of analytical, reagent-grade selenium dioxide (SeOa) was dissolved
in 500 ml ammonia-free distilled water. 500 ml concentrated, reagent-grade
HzSOi, was then added. It was diluted to one liter with ammonia-free water
after it had cooled.
Only 2 ml of the Strickland and Parsons' digestion solution was added to each
water sample because of its stronger sulfuric acid content.
The most satisfactory results were obtained using a modified digestion
solution of Strickland and Parsons' (80) followed by ammonia determination
using the scaled-down indophenol method of Strickland and Parsons described
previously. Twenty grams of KaSOu was added to each liter of Strickland and
Parsons digestion solution to raise the boiling point of the acid and help
keep it refluxing close to the sample. Two ml of digestion solution was
added to each 25-ml water sample in a Kjeldahl flask and digested for two
hours following evaporation of the water. After they had cooled, the acid
residues were each diluted with approximately 10 ml of ammonia-free water
and transferred to 100-ml volumetric flasks. Sequential rinsing with
additional ammonia-free water assured complete transfer. The diluted samples
were then neutralized with 1 M NaOH to pH 6.0-7.6 after addition of one drop
of bromothymol blue indicator. The solution was titrated to the bromothymol
blue endpoint (yellow to faint blue) and then diluted to the mark with
ammonia-free water. Five-mi aliquots were then analyzed for ammonia by the
scaled-down indophenol method. The indophenol-blue color intensity was read
against a reagent blank carried through the entire procedure because of the
potential adsorption of atmospheric ammonia by the sulphuric acid during
digestion.
Great care was required to prevent the contamination of reagents and
samples by atmospheric ammonia or particulate ammonium salts. Solutions were
kept in tightly stoppered bottles. All glassware was cleaned copiously with
10% HC1 and rinsed thoroughly with ammonia-free water immediately prior to
use. Kjeldahl flasks were initially cleaned by steeping them in near-boiling
sulphuric acid for several hours. Ultra-pure sulfuric acid ('Ultrex,' J.T.
Baker Co.) having a lot analysis of no more than 0.5 ppm ammonia was used in
the digestion solution to ensure a low blank value.
Calibration curves were obtained with the standard ammonia solution
described in Section entitled "Ammonia-N Determination". The calibration
29
-------
curve was stable, so that recalibration was not required for each batch of
determinations, or even for new reagents carefully prepared. Organic nitrogen
was calculated as the difference between the nitrogen found for digested
(Kjeldahl) and undigested (NHs) samples.
Resin Adsorption
After the water samples had been filtered through Whatman filter paper
to remove suspended material, macroreticular resins were used to remove
selectively potentially interfering hydrophobic carbonaceous compounds prior
to concentration and chromatographic analysis of the remaining organic
materials. The XAD macroreticular resins (Rohm and Haas Co., Philadelphia,
PA) were thoroughly purified by sequential solvent extractions, of about 10
grams per batch, with methanol, acetonitrile and diethylether in a Soxhlet
extractor for 8 hours per solvent. The purified resins were stored in glass
stoppered bottles under methanol to maintain their high purity. Tenax
(Applied Science Laboratories Inc., State College, PA) was purified with
methanol and acetonitrile extractions while Ambersorb XE-340 was supplied
already purified and hydrated with acetic acid solution from the manufacturer
(Rohm and Haas Co.).
The purified resins were added as methanol slurries into 1 cm I.D. glass
columns and were used either individually or as mixed resin beds. Single
resin beds were prepared by addition of an individual resin to a depth of
about 6 cm (1.5-2.0 grams dry resin). A glass wool plug was inserted near
the stopcock of the column and above the resin bed. Mixed resin beds,
consisted of equal volume combinations of either XAD-2 and XAD-4 or XAD-8 and
Tenax-GC macroreticular resins, and were prepared by sequential addition of
each resin to a depth of about 6 cm. Glass wool plus were inserted near the
stopcock, between the two resin beds, and above the final resin layer.
The procedure for eluting compounds through the prepared column was the
same for both the single and mixed resin beds. The methanol was drained to
the top of the uppermost resin bed and then flushed with approximately 200 ml
of distilled water. The distilled water was drained to the top of the resin
bed prior to passage of a sample. A flow rate of about 10 ml/rain was main-
tained by application of one psi pressure supplied from a regulated, filtered-
air line. The first 20-ml of eluted sample was discarded because of the
corresponding dead volume in the column containing the residual distilled
water. The remaining eluted sample was collected for analysis. The resin
was regenerated after elution of each liter of sample. Regeneration was
initially achieved by equilibration with about 50 ml of methanol and diethyl-
ether for the XAD resins and 100 ml of methanol of the Tenax and XE-340
resins. The regenerating solvents were discarded. Later regeneration for
the XAD-8 and Tenax resins was achieved by sequential equilibration with
50 ml of 10~2 M NaOH, 10~2 M HC1, and methanol followed by rinsing with
300 ml distilled water.
Field samples initially were passed through the resins without pH ad-
justment. Later samples were acidified to pH 2.0 with concentrated
prior to passage through the resins.
30
-------
Adsorption of commercially available humic acid on a combination of
XAD-8 and Tenax resins at acid and basic pH values was investigated. The
humic acid was dissolved in 1 liter of .03 M NaOH. Acidic humic acid solution
was prepared by titration of about 980 ml of the basic humic acid solution to
pH 2.0 with concentrated HsPOu followed by dilution to one liter with ammonia-
free water. Humic acid concentration was determined spectrophotometrically
at 330 nm.
The ability of each macroreticular resin to adsorb selected carbonaceous
substances or nitrogenous organic compounds was investigated by passage of
known concentrations of reference compounds through individual resin beds, or
a combination of them, at several pH values. Breakthrough curves for indi-
vidual test compounds were determined by measuring their concentrations in
the column effluents after passage of increasing volumes of sample through
the resin. The filtered aliquots were then analyzed spectrophotometrically.
U.V. absorbance was converted into concentration on the basis of standard
curves of absorbance readings vs. known concentrations of the reference
compound.
Breakthrough curves for mixtures of test compounds were also determined
by measuring their concentrations in column effluents after passage of in-
creasing volumes of sample through the resin. The filtered aliquots were
first chromatographed to separate the mixtures into their constituent com-
ponents. Concentrations of the separated compounds were obtained by
integration of the areas of the resolved chromatographic peaks with a disc
integrator.
Concentration
One to four liters of water previously filtered through Whatman filter
paper and macroreticular resins was evaporated to approximately 200 ml using
a Buchi, model R, flash evaporator. Evaporation was carried out below 30°C
to avoid reaction and decomposition. The partially concentrated sample was
then lyophilized using a VirTis automatic Freeze-Dryer (Model No. 10-010) to
remove all remaining water. The residue was redissolved in the chromato-
graphic eluant, and the resulting mixture centrifuged. The supernatent was
then ready for chromatographic analysis. Concentrated samples were frozen at
-25°C and stored for several weeks prior to analysis. Thawed samples were
further centrifuged to remove suspended material immediately prior to chroma-
tographic analysis.
Recovery values of total organic nitrogen were determined on samples
concentrated by low temperature distillation and lyophilization using the
scaled-down indophenol method of Strickland and Parson (80) described in
Section 2. These values ranged from 11% for the non-XAD filtered Danvers
bloom sample to 100% for the Concord River (6/8-78) and Oscillator-La tenuis
resin filtered samples. A mean organic nitrogen recovery value of 51.7%
(standard deviation = 29.2%) was observed for the field samples concentrated
by this method.
31
-------
Chromatographic Methods
Description of Chromatographic System—
The Chromatographic system consisted of a DuPont 848 high-pressure pump
with a DuPont 838 programmable gradient accessory, a Schoeffel (Westwood, NJ)
SF 770 Spectroflow U.V. monitor with a SFA 339 wavelength drive assembly, a
FS 970 spectrofluoro fluorescence monitor with a wavelength drive, a Duplex
minipump (Laboratory Data Control, Riviera Beach, FL) and a Schoeffel MM 700
module.
The gradient accessory was used to increase gradually the strength of
the mobile phase during a Chromatographic separation. Gradient elution
provided better resolution for a wider range of sample polarities than could
be attained by conventional isocratic elution. The variable wavelength
feature of the U.V. detector provided analytical flexibility to monitor
compounds having maximum absorbance anywhere in the U.V. spectrum. Both the
U.V. and fluorescence monitors were fitted with wavelength drive mechanisms
for use in identifying unknown peaks by stopped-flow spectrum scanning. The
memory module provided baseline correction during spectrum scanning and
gradient programming. The minipump was used to deliver buffer and reagents
for producing fluorescent derivatives to the column effluent before it
entered the fluorometer.
Figure 11 is a schematic presentation of the Chromatographic system.
The fluorescence detector could be used without post-column derivatization to
monitor naturally fluorescing compounds, or with post-column derivatization
by introducing fluorescamine (4-phenylspiro-[furan-2(3H),l'-phthalan]-3,3'-
dione) (Hoffman-LaRoche Inc., Nutley, NJ) and borate buffer prior to the
fluorometer. These reagents react with primary amines to form fluorescent
derivatives. All Teflon tubing, connectors and tees were purchased from
Rainin Instrument Co. (Boston, MA). 250-ml reservoir bottles were placed
approximately 3 feet above the minipump and connected to it by 1.5 mm I.D. x
3.0 mm O.D. teflon tubing. Air displacing the fluorescamine and borate buffer
was first passed through 50% sulphuric acid in a gas trap to remove traces of
primary amines. The exit valves of the pump were connected to 0.8 mm
I.D. x 1.5 mm O.D. tubing. Coils A, B, and C were approximately 0.40 m,
1.5 m, and 9.0 m lengths of 0.3 mm I.D. * 1.5 mm O.D. tubing, respectively.
The coiled pieces of tubing, A and B, were inserted to provide sufficient
mixing of the reagents. Coil C was included to induce sufficient backpressure
to prevent gas bubble formation when acetone was mixed with the aqueous buffer
at the second tee. A back-pressure of about 40 psi was maintained and was
monitored by two liquid pressure gauges. These gauges also helped to dampen
the pulsating flow of reagents produced by the positive displacement recipro-
cating piston mechanism of the minipurap.
Borate buffer was prepared by titrating 0.1 M boric acid to pH 9.3 with
4.5 M LiOH-H20 and was introduced at a flow rate of about 15 ml/hour. The
f luorescamine solution consisted of 15 mg/L in acetone and was used at a flow-
rate calculated to be less than 25-30% of the total flow (equal to 15 ml/
hour). The use of more concentrated fluorescamine solution has been reported
in the literature (82). However, reagent precipitation in the lines tends to
occur at such higher concentrations (83). Fluorescamine is stable at room
32
-------
x-
Wastes-
Secondary
Eluant
Reservoir
ond
Gradient
Device
High
Pressure
Pump
1°
Eluant
H?S04 Borote
Buffer
Fluorescomine H2S04
Reagent
Memory Module
Wavelength
Drive
Variable
Wavelength
U.V.
Detector
Liquid
pressure
gauges
Wavelength
Drive
Variable
Wavelength
Fluorescence
Detector
COIL C
Figure 11. Schematic diagram of chormatographic system,
-------
temperature in both solution and powdered form (83,84). The acetone solution
and borate buffer solution were made up only when the reservoirs were de-
pleted. Before shut-down of the chromatographic system the tubing was
flused with column eluant and borate buffer to avoid precipitation of fluo-
rescamine in the lines. All glassware was acid washed in dilute HC1 solution.
Caution was taken to prevent contamination by traces of primary amine.
Early work was done using only the isocratic pump and U.V. detector
without wavelength drive. It was found then difficult to achieve sharp
resolution of constituent compounds and the analytical ability to identify
unknown substances was limited severely. Later funding permitted the
purchase of the additional chromatographic equipment described earlier in
this section. Before purchase of the fluorometric equipment a preliminary
study was undertaken to evaluate the ability of fluorescamine derivatization
and fluorescence detection to monitor primary amines not detectable by
conventional U.V. spectroscopy. Separate one to five ml portions were
collected from the effluent of a chromatographic column after passage of a
concentrated field sample. Two ml of eluate or diluted eluate was then
allowed to react with 1 ml 0.2 M boric acid buffer (pH 9.5), 1 ml acetone and
0.2 ml fluorescamine solution with vortex mixing after addition of each
reagent. The buffer was prepared, as suggested by the manufacturer of fluo-
rescamine (Hoffman LaRoche, Inc.), by titrating 0.2 M boric acid with NaOH.
Stein later recommended that LiOH be used in place of the NaOH when using the
boric acid buffer in post column derivatization procedures, to help avoid
precipitation of fluorescamine in the lines containing the mixed reagents.
(Stein, S., personal communication, Roche Institute of Molecular Biology,
Nutley, NJ 07110.) The fluorescamine solution was comprised of 100 mg
fluorescamine per 100 ml acetone. The fluorescence intensity of derivatized
sample was read immediately on a Turner, model 11 fluorescence meter using a
Corning filter 5158 and a combination of filters 4-65 and 3-71 to set the
excitation and emission wavelengths, respectively. Final fluorescence values
were calculated from the differences between sample eluate and chromato-
graphic eluant reagent blank.
Resolution of Injected Samples—
f
Injection volumes—Injection volumes from 5 to 50 yl of concentrated
sample or reference compounds were introduced into the chromatographic system
through a Rheodyne (Berkeley, CA) model 7120 syringe-loading injector. The
reference standards, which were of the highest quality commercially available,
were not purified further before use. The reference compounds were dissolved
either in ammonia-free distilled water or in the chromatographic eluant.
Guanine was dissolved in 0.14 M LiOH. The criterion used in determining the
injection quantity was the volume required to produce maximum on-scale peak
displacement and resolution on the U.V. and fluorescence chart recorders.
Other investigators have used larger injection volumes of 100 ]j1 to
165 ml to achieve improved detection of trace contaminants (85,86). The
maximum injection volume used in this study was limited to the capacity of
the injection loop, 50 yl. Large-bore columns would also have been required
to handle the increased load of a large injection volume.
'34
-------
Mobile phases and column supports — The different modes of chromato-
graphy studied to attain resolution of the largest number of nitrogenous
compounds included: (1) cation exchange chromatography (Zipax SCX, DuPont
Co.); (2) anion exchange chromatography (Aminex A-27, Bio-Rad Laboratories);
(3) paired ion chromatography (Zorbax CN, DuPont Co.); (4) reversed phase
chromatography (Zorbax C-8, Zorbax CN) and (5) normal phase chromatography
(Corasil II, Waters Associates, or Zorbax CN) . Concentrations of reagents
used in making up sodium acetate-acetic acid; sodium citrate-citric acid;
and ammonium acetate-acetic acid buffers at desired pH values and salt
concentrations were determined from the equation
PH = PKa + log - . (3)
0.05 M phosphate buffer (pH = 6.9) was made from equal molar concentrations of
KHaPO and Na2HPOi*. Borax buffer was composed of 0.05 M sodium borate
(Na2Bi»07-5H20) adjusted with phosphoric acid.
Detection, Identification and Quantification of Resolved Constituents —
Eluted compounds were detected by U.V. and fluorescence spectroscopy
with or without f luorescamine derivatization. Identification of unknown
resolved chromatographic peaks was achieved by comparison of retention
position and ultraviolet data with those of reference compounds. U.V. data
was obtained by stopped-flow spectral scanning of individual chromatographic
peaks. The memory model was used to store initially the background spectrum
of the mobile phase, flow cell, and photomultiplier, and later subtract this
background spectrum from that of the eluant to obtain the spectrum for the
compound of interest. The resulting corrected spectra of reference compounds
were then compared with corrected spectra of unknown chromatographic peaks.
Positions of maximum and minimum UV absorbance and relative absorbance at
several wavelengths were used as criteria for compound identification. In-
ternal standards and fluorescence peaks resulting from f luorescamine respon-
sive compounds were also used to identify unknown constituents. The internal
standard consisted of a known quantity of reference compound which was
thought to be present in the concentrated field sample. Sometimes the in-
ternal standard was added to the unknown solution before the chromatographic
separation, while other times it was injected separately immediately before
or after injection of the sample. When injected with the concentrated field
sample, the suspect peak increased in proportion to the quantity of known
reference compound injected. Either method compensated for errors made in
the preparation of the eluting solution. The occurrence of a fluorescent
peak indicated either the presence of a f luorescamine-response primary amine
or a non-derivatized compound also fluorescing at the excitation and emission
wavelengths 'used to detect f luorescamine-responsive compounds (390 nra
excitation, 470 run emission). The probability of identifying the unknown
peak correctly increased with corroborative evidence from different identi-
fication methods.
Determination of peak area for some reference compounds was achieved
with a series 200 disc integrator (Disc Instruments, Inc., Santa Anna, CA)
attached to a potentiometric recorder. The integrator automatically computed
peak area and displayed this computation as a second trace directly under
35
-------
each recorder peak. The number of integrator 'counts' was converted to
concentration units by determination of the number of 'counts' of known
quantities of reference compounds. Because of difficulties in maintaining
the required baseline for proper use of the disc integrator amounts were
later estimated by measurement of peak heights.
RESULTS
Introduction
A number of different analytical methods, isolation and concentration
techniques, and chromatographic systems were used to obtain the results
described in this section. Although the results of some methods proved to
be unsatisfactory, the data are presented to give a complete picture for
future investigators. The chromatographic results, both prior to and after
the acquisition of the gradient pump and fluorescence equipment, are pre-
sented to illustrate the enhanced analytical ability resulting from the use
of the more sophisticated equipment.
Retention of Organic Materials on Macroreticular Resins
Test Compounds—
The concentration and fractional recoveries of individual test compounds
after passage of increasing sample volume through macroreticular resins are
shown in Appendix A (Tables A-l to A-23). Concentration values were calcu-
lated from least-square-fit equations determined from absorbance measurements
of reference compounds at several concentrations (Appendix A, Table A-24).
Recovery values were calculated according to the equation:
% recovery = effluent concentration of test compound (..
influent concentration of test compound '
The percentage recoveries of constituents in mixtures of nitrogenous com-
pounds after passage of increasing sample volumes through a combination of
XAD-8 and Tenax resins are shown in Appendix A (Tables A-25 and A-26).
Recovery values were calculated from concentration measurements determined
either by peak height (H) or peak area (A) on chromatograms of the resolved
mixture. The approximate volume of sample filtered at 80% and 90% recovery
and the ultimate percentage recoveries for the individual compounds and
mixtures of test compounds are summarized in Tables 5 and 6, respectively.
100% recovery of constituent compounds in a mixture containing: 5-chloro-
uracil, barbituric acid, thymine, guanine, creatinine, purine, pyrrole,
adenine and tryptophan was observed after passage of only 70 ml through
XAD-8 resin alone. Recovery values of constituent compounds in a mixture
containing these nine nitrogenous materials after equilibrium for 15 minutes
in various resin slurries are shown in Table 7.
Recovery values greater than 90% after passage of less than 100 ml of
sample or after equilibration for 15 minutes in resin slurries occurred for
most nitrogenous compounds using the XAD and/or Tenax resins under the in-
dicated conditions. One noteworthy exception was the poor recovery of indole
36
-------
TABLE 5. RECOVERY OF INDIVIDUALLY TESTED NITROGENOUS COMPOUNDS AFTER PASSAGE THROUGH
MACRORETICULAR RESINS
Compound
Adenlne
Adenlne
5-Chlorouracll
5-Chlorouracll
Creatlne
Creatlnlne
Cytoalne
Cytoalne
Cytoalne
Humlc Acid
Humlc Acid
Indole
Influent
Concentra-
tion (rag/L)
9.54
9.64
9.69
10.43
10.0
3.19
8.56
8.35
10.0
13.48
18.34
8.74
Reeln(a)
XAD-8 &
Tenax
XAD-8 &
Tenax
XAD-8 6
Tenax
XAD-8 &
Tenax
Tenax &
XAD-8
XAD-2 &
XAD-4
XAD-2 6
XAD-4
XAD-2 &
XAD-4
Tenax 6
XAD-8
XAD-8 &
Tenax
XAD-8 &
Tenax
XAD-2 &
XAD-4
Condi tlona
pH 2.5
pH 7.1
pH 2.0
pH 7.01
pH
unadjuated
pH 2
with HC1
pH
unadjusted
pH 3
with HNOj
PH
unadjusted
pH 2
pH 11.7
10-3
NaOH
•la to mis to
801 break- 90S break-
through through
50 80
60 80
60 80
45 65
80 95
65
75
40 165
71 91
-
120
— _
Ultimate
recovery
(Z) a
98.95
100
100
100
99.17
81
88
90
96
J6.0
87.9
10.3
•la filtered
at ultimate
recovery
300-350
300-350
175-200
350-400
266-286
90-190
175-200
165-190
206-287
475-545
175-200
125-215
Adaorptlve
capacity of
realn(a) for ,
teat compound
0.16
0.19
0.19
0.16
0.27
0.07
0.21
0.11
0.24
_
-
-
(continued)
-------
TABLE 5 (continued)
Cnpound
Indole
Purlne
Purine
Pyrlnldlne
w Pyrlmldlne
GO
Succlnimlde
Succlnimlde
Trypcophan
Trypeophan
Uracil
Influent
Concentra-
tion (ng/L)
6.57
10.00
10.24
10.0
9.75
30.00
30.00
9.66
10.0
10.0
Realo(s)
XAD-4
XAD-8 &
Tenax
XAD-8 &
Tenax
XAD-8 &
Tenax
XAD-8 6
Tenax
XAD-8 &
Tenax
XAD-8 &
Tenax
XAD-8 6
Tenax
XAD-8 6
Tenax
XAD-8
Conditions
lO'3
HNOj
pH 2.0
pH 7.0
pH 2.0
pH 7.0
pH 2.0
pH 7.05
pH 2.0
pH 6.9
PH
unadjusted
•la to
BOX break-
through
-
35
30
70
50
175
60
150
35
56
•la to
90Z break-
through
-
60
40
175
65
250
125
300
45
72
Ultlaate
recovery
«£
4.8
98.18
100
94.62
99.32
93.00
93.15
92.52
100.00
98
•la filtered
at ultimate
recovery
2^0-370
150-175
175-200
250-300
350-400
350-400
175-200
350-400
350-400
147-322
Adaorptlve
capacity of
realn(a) for
test coopound "
mg/g
0.12
0.10
0.23
0.16
1.75
0.60
0.48
0.12
0.37
t Recovery • (maxlnua effluent concentration/Influent concentration), x 100
Adaorptlve capacity calculated at SOX recovery(for 3 g combined resin)
-------
TABLE 6. RECOVERY OF MIXTURE OF NITROGENOUS COMPOUNDS AFTER PASSAGE THROUGH XAD-8 AND TENAX RESINS
(3 GRAMS TOTAL RESIN)
(.0
compound
uracil
Indole
tyrosine
purine
guanine
cytosine
adenine
creatinine
tryptophan
uracil
indole
tyrosine
purine
guanine
cytosine
adenine
creatinine
tryptophan
condition
pH of
mixture
unadjusted
11 £
pH of
tn-f vf 11T*P
•iij. A 1»UL C
adjusted to
pH 2.0 with
HP1
n\j j.
concen-
tration
(mg/L)
5
5
10
10
15
40
20
50
20
5.0
5.0
10.0
10.0
15.0
40.0
20.0
50.0
20.0
mis to
80%
break-
through
45
-
100
100
200
100
150
100
100
100
_
100
75
50
100
75
200
mis to
90%
break-
through
100
-
200
100
300
150
200
200
100
150
_
150
150
50
100
75
200
ultimate
recovery
(%;a
100
0
100
100
91.4
100
100
100
100
100
_
100
100
100
100
100
100
mis
filtered
at ultimate
recovery
200
400
300
200
300
300
300
300
300
300
500
150
300
300
300
300
200
adsorptlve
capacity of
resins for test
compounds (mg/g)
.08
-
.34
.34
1.00
1.33
1.00
1.67
0.67
.17
_
.33
.25
.37
0.67
0.67
1.25
1.33
% recovery « (maximum effluent concentration/influent concentration) x 100
adsorptlve capacity calculated at 80% recovery.
-------
TABLE 7. RECOVERY OF MIXTURE OF NITROGENOUS COMPOUNDS AFTER EQUILIBRATION
FOR 15 MINUTES IN RESIN SLURRY (pH UNADJUSTED)3
Compound
5-chlorouracil
barbituric acid
thymine
guanine
creatinine
purine
pyrrole
adenine
tryptophan
recovery
from XE
slurry
35
75
39
0
25
0
0
13
42
recovery
from Tenax
slurry'3
88-100
100
98-100
100
100
93-100
95-100
95-100
79-100
recovery
from XAD-8
slurry
100
100
84-100
100
85-100
63- 89
-
49- 68
72- 95
•a
Upper percentage numbers are maximum recovery values computed from the
highest observed concentration of compound after equilibration with resin
slurry divided by the lowest observed concentration value of compound deter-
mined before equilibration with resin slurry.
/concentration of compound after \
% recovery = [ equilibration with resin slurry \ x
I concentration of compound before I
\equilibration with resin slurry/
Single or initial numbers are mean values.
after passage through all the resins tested. Most nitrogenous compounds
were strongly adsorbed onto XE resin and, therefore, poor recovery values
were observed. No significant differences were observed for the recovery
values of nitrogenous compounds through different resins with or without pH
adjustment. Humic acid, however, was more strongly adsorbed onto a combi-
nation of XAD-8 and Tenax resins at pH 2.0 than at pH 11.7.
The variability in recovery values obtained from different chromatograms
of resolved mixtures of nitrogenous compounds (Appendix A, Tables A-25 and
A-26) is partially attributable to the difficulty in maintaining the baseline
on the potentiometric chart recorder at zero, as is required for proper
operation of the disc integrator. Baseline fluctuation in excess of the 3%
recommended for maximum drift correction was frequently encountered, making
integration difficult and inexact. This problem may also have contributed
to the somewhat greater recovery values observed in these mixtures in
40
-------
comparison with the recovery values obtained with individually tested
compounds.
The adsorptive capacities of the resins for the several tested compounds
are shown in Tables 5 and 6. These values were calculated from the equation:
influent liters filtered to
Adsorptive capacity = concentration reach 80% recovery
(mg compound/gram resin) grans resin used
and represent the maximum quantity of compound adsorbed per weight of resin.
Quantities of compounds exceeding the adsorptive capacities will break
through into the column effluent. The adsorptive capacity of the resins
(with the exception of indole) for the N-organic compounds ranged from only
0.08 to 1.75 mg per gram of resin as compared to the greater adsorption
(5-25 mg carbon per gram resin) of non-nitrogenous compounds reported in the
literature (87,88). In addition, the similar values obtained from filtered
mixtures of N-organic compounds and individually tested materials indicate a
non-competitive adsorption phenomenon.
NH3-N and Kjeldahl-N—
Adsorption of NH3-N and total Kjeldahl-N onto the macroreticular resins
was also examined. Table 8 summarizes the percentages of NH3-N, Kjeldahl-N,
and organic-N found to pass through the macroreticular resins under study.
The mean percentage recoveries for NH3-N, Kjeldahl-N, and organic-N, for the
different filtration conditions and their standard deviation are shown in
Table 9. Statistical tests (Table 9) showed that the observed differences in
recoveries were not attributable to the different resins and conditions used
within the indicated confidence intervals. Over 93% of the NH -N and 88% of
the organic-N contained in the field and laboratory samples were not adsorbed
onto the individual resins or combinations of resins tested.
Kjeldahl-N and NHa-N Determination
Concentration vs. Absorbance Data—
Concentration vs. absorbance readings at 635 nm using Scheiner's (79)
method and the scaled down procedure of Strickland and Parsons (80) are
shown in Tables 10 and 11, respectively. The following least squares linear
equations were derived from these data and were used to calculate unknown
Kjeldahl-N and NH3-N concentrations in field and laboratory samples from U.V.
absorbance readings. The equations were constrained to pass through the
origin.
NH3-N[mg/L] = 1.265[(mg/L)/AU] x (absorbance at 635 nm)[AU] (6)
(Scheiner's method)
Kjeldahl-N[mg/L] = 1.475[(mg/L)/AU] x (absorbance at 635 nm)[AU] (7)
(Scheiner's method)
NH.-N and Kjeldahl-N[mg/L] =
1.070[(mg/L)/AU] x (absorbance at 635 nm)[AU] (9)
(Strickland and Parsons' method)
41
-------
TABLE 8. PERCENTAGES OF NITROGENOUS MATERIAL PASSING THROUGH MACRORETICULAR RESINS*
to
—
date
11/15/77
12/1/77
12/16/77
1/30/78
3/2/78
3/2/78
3/2/78
3/2/78
3/9/78
3/9/78
5/3/78
method of
NH3-N deter-
mination &
pH conditions
N-l
N-l
N-l
N-l
N-l
N-l
N-l
N-l
N-l
N-l
N-l
source0
Charles River
Spy Pond
Charles River
Charles River
Billerica WTP influent
Billerica WTP effluent
Lawrence WTP influent
Lawrence WTP effluent
Marlboro West WW
pre C12
Marlboro West WW
post Cl2
Marlboro West WW
pre Cl2
resin
XAD-4 &
XAD-2
XAD-4 &
XAD-2
XAD-8
XAD-8
XAD-8
XAD-8
XAD-8
XAD-8
XAD-8
XAD-8
XAD-8 &
Tenax
%
NH3-N
99
100
98
100
100
100
81
96
98
99
89
recoveries
Kjeldahl-N
87
100
95
100
100
100
100
52
93
100
75
organic-N
78
100
94
100
100
100
100
48
89
100
62
(continued)
-------
TABLE 8 (continued)
u>
date
6/9/78
6/14/78
6/21/78
6/21/78
6/23/78
incubation
day 44
incubation
43
method of
NH3-N deter-
mination & ,
pH conditions
N-2
N-2
N-2
A-2
A- 2
A-2
day A-2
c
source
Billerlca water supply
Lawrence water supply
(first 100 ml)
(100-1,000 ml)
Danvers water supply
(first 100 ml)
(100-1,000 ml)
Danvers water supply
(first 100 ml)
(100-1,000 ml)
Ohio supply
(first 100 ml)
(100-1,000 ml)
flask //2 Oscillatoria
tenuis
(first 100 ml)
(100-1,000 ml)
flask #2 Anabaena
fjjps aquae
resin
XAD-8 &
Tenax
XAD-8 &
Tenax
XAD-8 &
Tenax
XAD-8 &
Tenax
XAD-8 &
Tenax
XAD-8 &
Tenax
XAD-8 &
Tenax
NH3-N
92
81
93
0
100
91
-
100
35
100
68
100
% recoveries
Kjeldahl-N
100
84
97
66
100
76
83
84
87
37
73
72
organlc-N
100
86
98
67
98
78
83
82
91
35
73
71
(continued)
-------
TABLE 8 (continued)
date
method of
NH3-N deter-
mination &
pH conditions
% recoveries
source
resin
Kjeldahl-N organic-N
7/4/78
7/11/78
A-2
A-2
Billerlca:
Bridge Street
Danvers bloom
XAD-8 &
Tenax
XAD-8 &
76
94
81
98
82
98
(filtered to 0.62 ym) Tenax
Samples eluted through approximately 3 g (dry weight) resin or combination of resins.
% recoveries were determined after elution of 1 liter of sample through the individual resin or
resin combination.
N = Influent pH value unadjusted
A = influent pH adjusted to 2.0 with
1 = NH3 determined using Sheiner's method
2 » NH3 determined using Strickland and Parsons' method
WTP = water treatment plant
WW = waste water
pre Cl2 = pre chlorinated
post Cl2 - post chlorinated
-------
TABLE 9. MEAN PERCENTAGE RECOVERIES OF Nl^-N, KJELDAHL-N, AND ORGANIC-N THROUGH MACRORETICULAR RESINS
Influent
resins pH value
XAD-8 & pH 2
Tenax
XAD-8 & unadjusted
Tenax (neutral)
XAD-2 6 unadjusted
XAD-4 or (neutral)
XAD-B
alone
overall -
average
for all
resins &
conditions
tested
mean percent recovery
(standard deviation) t-statlstlc
NH3-N
86
(13)
94
(4)
97
(6)
94
(9)
KJeldahl-N
82
(10)
93
(12)
93
(15)
90
(13)
organ Ic-N N N recovery
83 6 -0.65
(10) (8)
90 4 -0.12
(18) (12)
91 10 0.42
(17)
88 20 -
(15)
F accept or
P(|t|>computed statistic reject
t value) (two for organic null
tailed test)c N recovery*1 hypothesis
53Z 3.25 accept
(6.23)
91Z 1.23 sccept
(4.47)
68Z 1.23 accept
(2.77)
• • —
*H • sample site
t • (Xj - X2)/(S1/n1 + S2/n2) ; where degrees of freedoa are indicated in parentheses (• HJ + i»2 - 2); t test
*
performed on recovery values for conditions shown in successive line of table; S • variance.
0 P(|t|>conputed C value): expected frequency of observing given difference In means assuming both sample means taken
from same population.
S./$2 • —$- F(N1 - 1, NZ - 1); number In parenthesis is the SZ level (two tailed) of the distribution of P
°1
• 22
Null hypothesis: a\ • 02, i.e., sample variance taken from same population
-------
TABLE 10. CONCENTRATION vs. ABSORBANCE READINGS8 AT 635 run
(1 cm PATHLENGTH)•
determination
NH3-N
Kjeldahl-N
concentration of
standard (mg/L)
0.10
0.10
0.20
0.40
0.60
0.80
0.80
0.10
0.10
0.20
0.20
O.AO
0.40
0.60
0.60
absorbance
at 635 run
.095
.101
.147
.307
.463
.635
.639
0.026
0.026
0.105
0.103
0.260
0.265
0.422
0.423
Determined 'by Scheiner's method (79).
46
-------
TABLE 11.
NH3-N CONCENTRATION vs. ABSORBANCE AT 635 run DETERMINED BY STRICKLAND AND PARSONS' (80)
INDOPHENOL-HYPOCHLORITE METHOD
concentration
(mg/L)
0.10
0.30
0.50
replicates of
a
0.104
0.294
0.450
b
0.099
0.277
0.472
absorbance at 635 nm a
c
0.100
0.275
0.485
d
0.094
0.287
0.500
e
0.080
0.287
0.440
f
0.086
0.245
0.458
mean
0.094
0.278
0.468
standard deviation
.009
.017
.023
a
1 cm cell
-------
where
AU = absorbance units = -log — .
o
Strickland and Parsons' scaled down method was used in later work of
this research because of its excellent reproducibility (9.5%, 6.1%, and 4.9%
standard deviations at 0.1, 0.3, and 0.5 mg/L NH3-N, respectively), and ease
in analyzing replicate samples. Reagent blanks were carried throughout all
procedures because of increased absorbance attributable to ambient NH3-N
contamination. Typical NH3-N reagent blank absorbance readings (635 run),
after Kjeldahl digestion using different digestion solutions and durations
are shown in Appendix A (Table A-28). All the digestion conditions yielded
similarly low reagent blank values with good reproducibility. A mean of
0.149 AU and standard deviation equal to 0.014 AU (9.5%) were observed for
replicate reagent blank absorbance values of digestion solution D read against
a dstilled water reference. Mean NH3-N reagent blank absorbance readings
(635 nm) without digestion were 0.050 mg/L and 0.04 mg/L, using reagent grade
sodium hypochlorite solution and commercial Chlorox bleach, respectively.
The calculated mean reagent blank absorbance value for digestion solution D
with an undigested NH3-N reagent blank reference was, therefore, 0.105 AU, or
0.11 mg/L NH3-N. The blank values were most likely attributable to absorption
of atmospheric ammonia by sulfuric acid.
Kjeldahl Digestion Solutions—
Table A-29 in Appendix A shows Kjeldahl-N concentration values for
several field samples obtained after digestion with sulfuric acid solutions
containing different catalysts for several periods of time. SeOCla and SeOa
as catalysts yielded similar, consistent, results after 1, 2 or 3 hours of
digestion. One very poor recovery of Kjeldahl-N was observed using HgO as
catalyst.
Field and Laboratory Samples—
Table 12 shows Kjeldahl-N, NH3-N and organic-N concentrations of field
and laboratory samples. Organic nitrogen was computed as the difference
between nitrogen values after digestion (Kjeldahl-N) and before digestion.
Organic nitrogen comprised 65-99% of the total Kjeldahl-N of the water supply
samples (mean =84%, standard deviation = 14%) , and ranged from 0.30-21.7 mg/L.
The unusually high organic-N content in the second Danvers water supply
sample was attributable to the extracellular products of a blue-green algal
bloom that was occurring at the time of sampling. The possibility that algal
cell materials rather than extracellular products were contributing to this
high organic-N value was investigated by measuring the organic-N content
after further filtration to 0.7 ym. Although the bulk of residual green
color was removed by the ultrafiltration, no decrease in organic-N or NH3-N
was observed. The mean 'non-bloom' organic-N content and standard deviation
for the water supplies were 0.76 mg/L and 0.24 mg/L, respectively.
Concentration Efficiencies
By concentration efficiency is meant the percentage of original organic-
nitrogen found still present after concentration. Concentration efficiencies
for field samples and test compounds were calculated from Kjeldahl-N and U.V.
48
-------
TABLE 12. NH3-N AND KJELDAHL-N DETERMINATIONS
date
11/15/77
12/1/77
12/16/77
1/30/78
3/2/78
3/2/78
3/2/78
3/2/78
3/9/78
3/9/78
3/9/78
,
6/9/78
6/14/78
6/21/78
pR valiu
6 NHj-N.
method a
Nl
Nl
Nl
Nl
Nl
Nl
Nl
Nl
Nl
Nl
Nl
N2
N2
N2
non-resin
filtered
, Kjeld-N
source (mg/L)
Charles River
Spy Pond
Charles River
Charles River
Blllerlca WTP Inf.
Blllerlca WTP eff.
Lawrence WTP inf.
Lawrence WTP eff.
Marlboro E. WW
pre Cl.
Marlboro W. WW
pre C\2
Marlboro W. WW
post Cl2
Blllerlca water
supply
Lawrence water
supply
(first 100 ml)
(100-1.000 ml)
Danvers water supply
(first 100 ml)
(100-1.000 ml)
1.9
0.8
7.3
5.1
0.9
0.7
1.5
1.3
0.1
4.4
4.3
0.4
0.8
0.7
-
NU-j-N Z
(mg/L)
0.8
0.3
1.4
1.8
0.3
0.1
0.5
0.1
0.1
1.8
2.1
0.1
0.2
0.1
-
non-resin
filtered
organ Ic-
N
60
66
81
66
65
91
68
92
0
60
52
69
75
99
-
Z NII3-N
40
34
19
34
35
9
32
8
100
40
48
31
25
1
-
resin
filtered
KJeld-N
(mg/L)
1.7
0.8
6.9
5.3
2.7
1.1
1.8
0.7
0.1
4.0
4.4
0.5
0.6
0.7
0.5
0.7
NH3-N
(mg/L)
O.B
0.3
1.4
1.8
0.5
0.1
0.4
0.1
0.1
1.7
2.1
0.1
0.2
0.2
0
0.1
organic
nitrogen
non-resin
filtered
me/L
1.2
0.5
5.9
3.4
0.6
0.6
1.1
1.2
0
2.6
2.2
0.3
0.6
0.6
0.7
0.7
res In
filtered
mg/L
0.9
0.6
5.5
3.5
2.2
1.1
1.4
0.6 *
0
2.3
2.2
0.3
0.5
0.6
0.5
0.7
(continued)
-------
TABLE 12 (continued)
pH value
•
& HHj-Na .
date method source
6/21/78 A2
_ _
_ „
6/23/78 A2
Incubation A2
day: 44
incubation A2
day: 43
7/4/78 A2
7/4/78 A2
Danvers water
(first 100 ml)
(100-1,000 ml)
huinlc acid
(19.9 mg/L)
humlc acid
(19.9 mg/L
replicate
Ohio supply
(first 100 ml)
(100-1.000 ml)
flask n
non-resin
filtered
Kjeld-N
(mg/L)
supply
0.7
-
0.5
0.4
0.6
-
•
NH3-N
(mg/L)
0.1
-
0.1
0.1
0.1
-
non-resin resin
filtered filtered
X organlc-
N
98
-
91
82
92
-
X NH3-N Kjeld-N
(mg/L)
2 0.5
06
9
19
R 0.5
0.5
•
NH3-N
(mg/L)
0.1
0.1
_
.
0.1
0.1
organic nitrogen
non-resin
filtered
rur/L
0.7
0.7
0.4
0.3
0.5
0.5
resin
filtered
mg/L '
0.5
0.6
_
-
0.5
0.3
Osclllatoria tenula
(first 100 ml)
(100-1,000 ml)
flask 92
Anahaena floa
Blllerlca
(River Street)
Blllerlca
1.8
-
aquae 2.3
1.6
2.8
0.1
-
0.1
0.1
1.0
97
-
98
92
65
3 0.7
1.3
2 1.7
8
35 2.2
0.1
0.1
0.4
_
0.7
1.8
1.8
2.2
1.5
1.8
0.6
1.3
1.6
.
1.5
(Bridge Street)
7/11/78 A2
Danvers bloom
filtered to 0.
22.0
62 ion)
0.3
99
1 21.5
0.3
22.0
21.2
,
N • influent pH value unadjusted UTP • vater treatment'plant
A i influent pH adjusted to 2.0 vlth 113704 WW • waste water
1 • NHj determined using Shelner's method (79) pre Cl2 - pre chlorinated
2 - NH3 determined using Strickland and Parsons' method (80) post C12 • post chlorinated
Inf. • influent
eff. • effluent
-------
absorbance measurements, respectively. Table 13 summarizes the organic
nitrogen recovery values of field samples subjected to low temperature
rotary evaporation from a pear shaped flask followed by lyophilization and
dissolution in chromatographic mobile phase solvent. Recovery values of field
samples subjected to this method were variable, raging from 11% to 100% and
having a mean of 51% and a standard deviation of 29%. Incomplete recovery of
organic nitrogen in these samples was attributed to: incomplete transfer of
lyophilized materials, and inability to redissolve all the lyophilized
material in the small volume of solvent required to achieve a 1,000 to 2,000
fold concentration factor. Recovery values greater than 100% for some samples
was attributed to the presence of small quantities of particulate or colloidal
materials in the concentrated sample. It is thought that the presence of
these undissolved materials during acid digestion resulted in increased or-
ganic nitrogen values in the digested solution.
The lowest recovery values were observed in the raw and resin-filtered
Middleton Pond sample (11% and 13%, respectively), which had been collected
during the occurrence of a blue-green algae bloom. The amount of lyophilized
material, in the raw and filtered sample was substantially greater than for
other sites. This was partially attributed to the comparatively greater
level or organic nitrogen (equal to about 21 mg/L) in the sample. It was
therefore hypothesized that the inability to completely dissolve this larger
amount of lyophilized material in the required small volume of solvent re-
sulted in the lower recovery values.
The mean recovery value for the field and laboratory samples, excluding
the Middleton Pond site just discussed, was equal to 60% (standard deviation =
24%). This represented a somewhat greater value for the recovery of nitro-
genous organic material than obtained using the low temperature evaporation
procedure previously described.
Chromatographic Resolution of Reference Compounds
Initial Studies: Isocratic Elution—
A number of liquid chromatographic conditions were tested for their
ability to resolve mixtures of known nitrogenous compounds. The results are
shown in Appendix A (Tables A-32 to A-36). Attempts to separate nitrogenous
mixtures on Aminex A-27 using 0.325 M ammonium acetate eluant, on Zipax SCX
using perchloric, phosphoric, acetic and nitric acid mobile phases of varying
strength, and on Zorbax CN using sodium-acetate-acetic-acid buffer with
varying acetonitrile or methanol composition proved unsuccessful. Separation
was later achieved on the Zipax column using mobile phases comprised of either
KHaPOi,, KaHPOi,, NaH2POit, or NH^HaPOi, at different molar concentrations and
adjusted to pH values of 2.5, 3.5, or 4.5 (Tables 14 to 16). Resolution of
the larges number of compounds on this column was achieved using NaH2POi» or
NHi,H2POi, adjusted to pH 2.5 with HsPOi,. Compounds were eluted more rapidly
using eluants having greater salt concentrations, and at more elevated temper-
atures. Acetonitrile modifier did not improve resolution. Additional column
length increased retention time but did not improve resolution of rapidly
eluting compounds. Elution with a weaker mobile phase (distilled water ad-
justed to pH 2.5 with H3POi,) did not improve the resolution of this group of
51
-------
TABLE 13. CONCENTRATION EFFICIENCY: ROTARY EVAPORATION IN PEAR-SHAPED FLASK FOLLOWED BY
LYOPHILIZATION AND DISSOLUTION IN CHROMATOGRAPHIC MOBILE PHASE SOLVENT
Cn
10
sample
Concord River,
Billerica, MA
6/8/78c
Merrimack River,
Lawrence, MA,
6/lA/78c
Middle ton Pond,
Danvers, MA
6/21/78£
O&CA^lcutofUa. tejnwii
flask //2c
flask //3d
bnabae.no. ftto& aquae.
flask //2C
flask //3d
surface impoundment
Bethesda. Ohioc
organlc-N
before
concentration
(mg/L)
0.3
0.6
0.6
0.6
1.1
2.2
2.8
0.5
concentration
factor
2,000
2,000
2,000
1,630
1,800
1,615
1,750
2,000
calculated
organ! c-N
value after
concentration3
(mg/L)
671
1,182
1,130
1,006
2,030
3,621
4,818
986
observed
organic-N
value after
concentration
(mg/L)
965
755
582
1,157
983
1,236
1,925
610
concentration
efficiency^
(%)
MOO
64
52
>100
48.
35
40
62
(continued)
-------
TABLE 13 (continued)
sample
organic-N
before
concentration
(mg/L)
concentration
factor
calculated
organlc-N
value after
concentration
(mg/L)
observed
organlc-N
value after
concentration
(mg/L)
concentration
efficiency
U)
Concord River, 1.8
(Bridge Street)
Billerica, MA
7/4/78c
Middleton Pond, 21.3
Danvers, MA
(filtered to 0.1 pm)
7/ll/78c
Middleton Pond, 21.7
Danvers, MA
(filtered to 0.1 ym)
7/ll/78)d
1,000
1,000
1,000
1,840
21,280
21,681
797
2,840
2,448
43
13
11
a
Calculated organic-N value after concentration » organic-N value before concentration x concentration
factor (assumes 100% recovery)
Concentration efficiency = (observed organic-N value after concentration)/(calculated organic-N
value after concentration)
c Filtered through XAD-8 and Tenax GC macroreticular resins
Unfiltered
-------
a
TABLE 14. RETENTION VALUES (ML) OF A MIXTURE CONTAINING 11 N-ORGANIC COMPOUNDS ON ZIPAX SCX
.05 N KH2P04 .05 M K2HPOft
adjusted to adjusted to
•lution pH 2.5 with pH 2.5 with
position H3P04 H3P04
1 1.4 1.5
2 2.0 2.0
3 3.3 2.9
4 4.7 3.4
5 8.5 4.9
6 10.8 6.3
7 17.5 12.8
8
9
10
11
12
.05 M NaH2P04 .05 M Na2HPO«
adjusted to adjusted to •
pH 2.5 with pH 2.5 with
H3P04 H3P04
1.4
1.9
2.7
3.4
4.9
5.6
6.7
8.6
12.0
20.1
28.2
1.4
2.0
2.5
3.2
4.2
5.3
6.7
11.6
15.9
no other peaks
observed after
elution of addi-
tional 100 Bl
.01 M NaH2P04
adjusted to
.05 N NaH2P04 pH 2.5 with
pH 4.5 H3P04
1.6
3.3
12.6
18.7
23.1
no other peaks
observed after
elution of addi-
tional 127 al
1.9
2.1
6.9
9.1
14.1
20.0
26.0
31.8
41.7
72.0
95.0
104.3
1 uracll. indole, alanlne, tyrosioe. purine. guanlne. creatine, cytosine. adenlne, crcatinine, tryptophan;
elution order of these coapounds was not determined.
-------
TABLE 15. RETENTION VALUES (ML) OF NITROGENOUS ORGANIC COMPOUNDS ON ZIPAX SCX
(ONE METER COLUMN UNLESS OTHERWISE INDICATED)
Ul
.05 M .01 M .05 N H20 .005 N .05 H .05 M .05 M .05 M .05 H NH&H2P04
NH^PO^ NH^PO^ NH41I2P04 adjusted Nh$H2P04 NII^I^PO^ NaH2P04 sodium NH6H2P04 adjunted to pH
adjusted adjusted adjusted to pH adjusted adjusted adjusted perchlorate adjusted 2.5 with I^POft
to pH 2.5 to pH 2.5 to pH 2.5 2.5 with to pH 2.5 to pH 2.5 to pH 2.5 adjusted to to pH 2.5 10Z acetonltrlla
with H3P04 with H3P04 with H3P04 h^PO* with h*3P04 with h^PO* with HsPOft pll 2.5 with H3P04 3 meter column
Compound temperature temperature with per- 3 meter
• 50°C - 10°C chloric acid column
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
IS.
16.
17.
18.
19.
20.
d-l serlne
uric acid
barbituric
acid
succlnlnlde
t-histldlne
t-aspartlc
acid
taurlne
uracll
pyrrole
2,6,8, trl-
chloropurlne
5-clilorouracil
thymlne
l-hydroxy-
prollne
Imtole
caffeine
alanlne
glyclne
glycylglyclne
m- ami no phenol
pyrlmldlne
1
1
1
1
1
1
1
1
2
1
1
1
1
2
2
2
2
2
2
3
.7
.5
.5
.9
.7
.6
.7
.5
.0
.9
.3
.5
.9
.2 2.0
.4
.7 1.8
.1
.4
.3 & 4.3
.3
1.6
1.4 2
1
1.6 1
1.5
1.6
1.0 1.4 1.3 1.5 1
1
1.6 1.6 1
1.4 1.4 2.1 2.1 2.0 1
1.8 1.4 1.4 ft 7.3 2.8 3.5 9
1.6
1.4 3.3 4
.0
.2
.7 4.3
.3
.6
.4 4.6 4.4
.7
.5
.2
(continued)
-------
TABLE 15 (continued)
Ul
.05 N
NH4H2P04
adjusted
to pH 2.5
with H3P04
Conpound
21. tyros Ine 4.6
22. purlne 5.4
23. guanlne 6.0
24. creatlna 8.6
25. cytoslne 10.5
26. adenlne 12.5
27. creatlnine 15.0
28. pyrrole 2.0
29. tryptophan 25.3
30. urea ND
31. humic acid 13.5
32. phenyl-
a Ian Ine
33. t-prollne 20.3
34. mixture: 8, all re-
14, 16, 29, solved
21-27
35. Marlboro W.
concentrated
effluent: 2.3
spiked wlthi
guanine 8.0
cytoslne 9.8
creatlnine 13.7
tryptophan 17.9
.01 M .05 M
NH4H2P04 NH4H2P04
adjusted adjusted
to pH 2.5 to pH 2.5
with H3P04 with H3P04
temperature
. 50°C
2.1
2.7
5.8 3.9
5.5
5.9
7.0 7.6
7.8 8.6
ND
all
resolved
H20 .005 M .05 M
adjusted NH4H2P04 NH^POi
to pH adjusted adjusted
2.5 with to pH 2.5 to pH 2.5
H3P04 with H3P04 with H3P04
.05 M
NaH2P04
adjusted
to pH 2.5
with H3P04
temperature
. 10°C
3.3
6.5
7.7
10.0
12.2
17.2
18.9
ND
only two
initial
peaka
observed
6.0
8.3
10.5
20.2
22.8
45.0
ND
29.4
3.0 & 12.4
11.5
19.6
26.2
42.0
.05 M .05 M
sodium NH^Il2P04
perchlorate adjusted
ad lusted to to pH 2.5
pH 2.5 with H3P04
with per- 3 meter
chloric column
acid
9.4
29.9
38.8
20.4
29.2 37.6
31.9 45.8
26.5 53.1
4.3
91.5
_
.05 M NH4H2P04
adjusted to pH
2.5 with H3P04
10Z acetonltrlle
3 meter column
4.2
(continued)
-------
TABLE 15 (continued)
Compound
.OS H
NH4H2P04
adjuatad
to pH 2.S
with H3P04
.01 M
HH«H2P04
adjuated
to pH 2.5
with H3P04
.05 M
NH4H2P04
adjuated
to pH 2.5
with H3P04
temperature
- 50"C
H20
adjuated
to pH
2.5 with
H3P04
.005 M
NH4H2P04
adjuated
to pH 2.5
with H3P04
.05 H
NH4H2P04
adjusted
to pH 2.5
with H3P04
temperature
- 10«C
.05 H
NaH2P04
adjuated
to pH 2.5
with H3P04
.05 H
sodium
perchlorata
adjuated to
pH 2.5 with
perchloric
acid
.05 M
NH4H2P04
adjuated
to pH 2.5
with H3P04
3 meter
column
.05 M NH
adjuated
2.5 with
«H2P04
to pH
«3POA
10Z acctonltrlla
3 meter
column
36. SHPAC 1.4-10.5
derlvatlzed
urine
37. SNPA blank 1.7-13.0
Ol
Hyphenated valuea represent one large peak beginning and ending at the Indicated valuea.
bND - not detected
CSSPA • N-aucclnimldyl P-nltrophenylacetate (Regis Chemical Co.)
dOnly 6 peaks observed In chromatogram of mixture of 11 compounda (8. 14, 16. 29. and 21-27) using 0.2 M NH4H2F04 (pH - 3.3) eluant.
-------
TABLE 16. REPRODUCIBILITY OF RETENTION POSITIONS FOR SEVERAL NITROGENOUS COMPOUNDS ON ZIPAX SCX
USING AN ELUANT OF .05 M NHPO ADJUSTED TO pH 2.5 with HO
Ul
00
replicate retention positions (ml)
compound
uracil
indole
tyroslne •
purine
guanlne
cytosine
adenlne
creatinine
tryptophan
a b c d e
1.9 - 1.9 -
5.3 5.1 5.4 5.6 5.5
5.9 5.6 6.0 6.2 6.0
10.3 10.2 10.6 10.9 10.7
12.2 11.9 12.7 13.2 12.8
15.0 15.0 15.1 15.7 15.1
29.2 27.3 29.7 31.3 29.7
f g h
5.3 5.4 5.4
5.9 5.9 5.8
10.5 10.3 10.7
- 12.4 12.3
15.3 14.8 14.7
- 29.2 28.2
mean
(ml)
1.9
5.4
5.9
10.5
12.5
15.1
29.2
absolute
standard
deviation
(ml)
- 0.04
0.12
0.15
0.17
0.24
0.43
0.31
1.26
percent
standard
deviation
3.08
2.73
2.78
2.88
2.29
3.44
2.05
4.32
-------
compounds. Increased retention and subsequent resolution of some compounds
in this group was achieved, however, on the Zorbax C8 column.
The reproducibilities of retention positions for several compounds are
shown in Table 16. Although the absolute standard deviations were somewhat
greater for slowly eluting compounds, the percentage standard deviations for
these substances were not appreciably larger than for rapidly eluting
materials. Absolute standard deviations ranged from 0.04 for a compound
eluting at a mean position of 1.3 ml, to 1.26 for a compound eluting at a
mean position of 29.2 ml. The corresponding percentage standard deviations
for these two substances, however, were 3.08 and 4.32 respectively. Thus,
while the absolute standard deviations varied by about 32-fold, the range in
percentage standard deviation varied by a factor of about 1.4.
Injection of a mixture containing eleven compounds using a Zorbax CN
column and .005 M paired ion exchange reagent mobile phase with or without
20% methanol (Table 17) produced only nine and six chromatographic peaks,
respectively, suggesting that resolution was favored on the Zipax SCX column.
The small number of peaks observed using the mobile phase without methanol was
attributable to incomplete elution of the compounds by this weaker eluant.
Elution and perhaps resolution of all the injected compounds might have been
achieved by incremently increasing the methanol content of the mobile phase
using a gradient making device. The large volume of mobile phase required to
elute the most retentive compound from the column, illustrated the lack of
rapid and clear resolution of constituent compounds using isocratic elution methods.
TABLE 17. RETENTION VALUES (ML) OF A MIXTURE CONTAINING 11 N-ORGANIC
COMPOUNDSa ON ZORBAX CN USING PAIRED ION CHROMATOGRAPHY (PIC)
elution position
*7 * ****** -005 1 B l hePtane
1
2
3
4
5
6
7
8
9
2.7
3.1
4.0
5.0
6.8
16.3
32.0
157.0
161.1
2.9
3.2
4.1
6.2
8.3
26.6
no further peaks observed
after elution with addi-
tional 242 ml mobile phase
no further peaks observed
after elution with additional
108 ml mobile phase
uracil, indole, alanine, tyrosine, purine, guanine, creatinine, cytosine,
adenine, creatine, tryptophan; elution order of these compounds was not
determined.
59
-------
Tables 18 to 20 indicate the operating conditions for the resolution of
18 different nitrogenous compounds by isocratic elution technique. Resolu-
tion of all these compounds is not feasible using any one column and mobile
phase but can be obtained from the chromatographic conditions shown in Table
20. Zorbax C8 proved to be very useful in separating a number of compounds
which were unresolvable on the Zipax SCX column. Different retention
characteristics were achieved by varying the pH of the buffered eluant. In-
creasing methanol content decreased the retentivity of compounds on the
Zorbax C8 column. The strong retentivity observed for indole, using a small
percentage of methanol in the eluant, suggested that gradient elution would
facilitate resolution of mixtures of compounds within more reasonable periods
of time.
0
Several chromatograms are included in Appendix B (Figures B-l to B-4) to
illustrate the varying adsorptivities of 11 compounds at three different wave-
lengths (220 nm, 233 nm, and 254 nm). The chromatograms also illustrate the
phenomenon of peak spreading characteristic of isocratic elution techniques.
Maximum sensitivity was achieved on the U.V. monitor at an attenuation
setting of 0.01 A.U. At this sensitivity setting, a full scale peak on the
potentiometric chart recorder was equal to 1x10~2 absorbance units. The
minimum detectable concentration of a trace compound was defined as the value
which resulted in a peak height equal to 5% of a full scale deflection at the
0.01 A.U. sensitivity setting. Minimum detectable concentrations of nitro-
genous compounds were then calculated according to the equation:
Minimum detectable concentration ._ x -,Q-^\
at 0.01 A.U. attenuation setting = (9)
(moles/L)
where E is equal to the molear extinction value of a particular compound and
the path length of the U.V. detector is equal to one cm. Minimum detectable
concentrations for some nitrogenous organic compounds are shown in Table 21.
Sensitivity settings of 2.0 or 1.0 A.U. were used when concentrated field and
laboratory samples were analyzed, to minimize the number of response peaks
going off the scale of the potentiometric recorder. Minimum detectable con-
centrations of nitrogenous materials monitored at these two settings were
2 x io2 and ixio2, respectively greater than those reported in Table 21.
Table A-37 (in Appendix A) illustrates the changing elution position of
creatine with increasing concentration, attributable to column overloading.
It is interesting to note that the creatine overloading did not effect the
retention characteristics of other compounds introduced simultaneously in-
dicating that saturation was occurring only at selected sites on the resin.
Later Studies: Gradient Elution—
A programmable gradient device was acquired later in this study to
improve the resolution of concentrated field samples and to permit the
separation of compounds having a wide range of polarities. Mixtures of
reference compounds were not satisfactorily separated on either a Durrum DC
1-A resin using a pH 3, 0.2 M citrate buffer to 0.1 M citrate buffer (adjusted
to pH 9.0 with NaOH) 20 minute, x3 gradient, or an an Aminex A-27 column
60
-------
TABLE 18. RETENTION VALUES (ML) OF NITROGENOUS COMPOUNDS ON ZORBAX C8 WITH CITRIC ACID BUFFER ELUANT
Mobile phase
100Z.OS M 201 MeOH; 501 MeOH; 301 ace to- 100Z 0.1 M 75Z HeOH; 100Z SOZ MeOH; 100Z .OS
pH 2.5 BOX .05 H SOZ .05 M nltrlle 70Z pH 2.5 25Z .05 N .05 M SOZ .05 M pH 3.9
buffer pH 2.S pH 2.S 0.1 M pH 2.5 buffer pH 2.5 pH 2.0 pH 2.0 buffer
Compound buffer buffer buffer buffer buffer buffer
•. taurlne
NDb
b. t-hydroxyprollne ND ND
c. Indole
d. creatlnlne
e. t-nspartic acid
f. l-histldine
g. dl-serine
h. glyclne
1. barb 1 curie acid
J. guanlne
k. cytoslne
1. uracll
n. uric acid
n. succlnlmlde
o. tyrosine
p. adenlne
q. 5-chlorouracll
r. alanlne
a. purlne
t. Chymine
u. pyrrole
v. tryptophan
ND 47 & 52 13 & 14 34
ND ND
ND ND
ND
ND
ND ND
3.1 2.2
3.8
3.3
3.5
3.8
4.2 3.0
5.8
5.3
6.4
7.1
9.6 3.6
11.8 6.0 8.1
35.0
ND ND ND ND
ND ND ND ND
4.9 & 5.1 12.9
2.7
ND ND ND ND
ND ND ND ND
ND ND ND ND
ND ND ND ND
4.5
4.7
5.8 7.3
11.4
12.0
8.8 7.8 3.4 8.4
12.8 11.3 4.8 8.9
M SOZ HeOH;
SOZ 1.0 M
pH 3.9
buffer
ND
ND
ND
ND
ND
ND
3.15
3.0
5.0
(continued)
-------
TABLE 18 (continued)
Compound
100X .05 M
pH 2.5
buffer
20Z MeOH;
80Z .05 M
pH 2.5
buffer
50Z MeOH;
SOX .05 M
pH 2.5
buffer
Mobile phase
30Z aceto- 100Z 0.1 M
nltrile 70Z pH 2.5
0.1 M pH 2.5 buffer
buffer
75Z MeOH;
25Z .05 M
pH 2.5
buffer
1001 •
.05 M
pH 2.0
buffer
50Z MeOH;
50Z .05 M
pH 2.0
buffer
100Z .05 M
pH 3.9
buffer
50Z McOR;
50Z 1.0 M
pH 3.9
buffer
Mixture
1. m, n, q, t, & u IT
Raw Marlboro 3.0 S 5.3
West pre- NFC 150
chlorinated
secondary
sewage
effluent
Marlboro West 2.8
XAD filtered HP - 100
prechlorinated
secondary
sewage
effluent
?H - all components resolved
CND - none detected
NF - no further peaks observed after volume Indicated
-------
TABLE 19. RETENTION VALUES (ML) OF NITROGENOUS COMPOUNDS ON ZORBAX C8 WITH HIGHER pH BUFFER ELUANTS
Ul
50% MeOH: 50% 100% .05 M 100% .01 M 50% MeOH; 50% 100% PH
0.2 M pH 6.9 PO, pH 6.9 PO, pH 6.9 pH 8.9 borax 8.9 borax
compound buffer buffer PO^ buffer buffer buffer
a.
b.
c.
d.
e.
£.
g.
h.
i.
J.
k.
1.
m.
n.
o.
P.
taurine
£-hydroxyproline
indole
creatinlne
l-aspartic acid
l-histldlne
d£-serine
glycine
barbituric acid
guanine
cytosine
uracil
uric acid
succinimide
tyros ine
adenine
NDb
ND
15.9
2.8
ND
ND
ND
ND
ND
2.8
ND ND
ND ND
265 14.5 & 15.2
4.4 4.9 2.65 4.1
ND ND
2.6 2.6
2.7 2.7
ND ND
3.4 2.4
5.6 2.6 5.3
3.8 4.5 3.5
3.7 2.6 3.8
ND
5.1 3.3 4.1
4.5 4.4
15.4 14.5
(continued)
-------
TABLE 19 (continued)
50% MeOH; 50%
0.2 M pH 6.9 PO,
compound buffer
q. 5-chlorouracil 2.9
r . alanine
s . purine
t . thymine 2 . 9
u. pyrrole 5.1
v. tryptophan 3.5
u, p, v, & t
Marlboro West
pre C\2 2° sewage
effluent XAD filtered;
concentrated x 200
a
R « resolved
b
ND = not detected
100% .05 M 100% .01 M 50% MeOH; 50%
pH 6.9 P04 pH 6.9 pH 8.9 borax
buffer P04 buffer buffer
2.03
2.6 2.6
11.6
8.7 3.2
11.8 5.2
23.1
Ra
One elution
peak 1.5-8.0 ml
100% pH
8.9 borax
buffer
3.8
8.5
8.8
11.7
22.0
-------
TABLE 20. COMPOUNDS RESOLVABLE8 ON ZIPAX SCX OR ZORBAX C-8
Zorbax C8
Zipax SCX pH 2.5 citric
compound .05 NH4H2P04 acid buffer
taurine
i-hydroxyproline
indole
creatinine
fc-aspartic acid
fc-histidine
dfc-serine
glycine
barbituric acid
guanine
cytosine
uracil
succinimide
tyro sine
adenine
5-chlorouracil
alanine
purine
thymine
pyrrole
tryptophan
creatine
1
1
2
10
1
1
1
1
1
6
8
1
1
4
9
1
3
5
1
1
11
7
a Numbers indicate elution order.
resolvable for conditions shown.
b
ND = not detectable
NDb
ND
ND
ND
ND
ND
ND
ND
1
1
1
1
2
3
3
4
4
5
6
7
8
—
Compounds
Zorbax C8 Zorbax C8
pH 6.9 phos- pH 8.9 borax
phate buffer buffer
ND
ND
11
3
ND
1
1
ND
2
5
2
2
5
4
9
1
1
7
6
8
10
-
having same
ND
ND
10
4
ND
1
1
ND
1
5
2
2
4
4
9
3
-
6
6
7
8
-
number are un-
65
-------
TABLE 21. MINIMUM DETECTABLE CONCENTRATIONS OF SOME NITROGENOUS ORGANIC COMPOUNDS'
compound
cytoslne
creatlnlne
Indole
creatine
creatine
creatine
uracil
uracil
alanlne
tryptophan
adenlne
succinimlde
5-chlorouracll
pyrimidine
purlne
wavelength
254
254
254
254
233
210
254
233
254
278
256
215
272
242
260
mean Eb
3.921 x 103
3
1.074 x 10J
3
3.905 x 10J
2.610 x 101
1.454 x 102
3
2.365 x 10
7.779 x 103
3
2.215 x 10 J
2.70 x 10'1
6.559 x 103
*
1.388 x 10
3.999 x 102
•)
8.183 x 10
•»
2.564 x 10J
•>
7.603 x 10 J
standard
deviation
1.423 x 103
319
261
_
_
431
275
26
_
384
279
56
395
129
193
minimum detectable
concentration (MDC)
moles /L mg/L
1.26 x 10~7
_7
4.66 x 10
_7
1.28 x 10 '
1.92 x 10"5
3.44 x 10~6
_7
2.11 x 10 '
6.43 x 10"8
_7
2.27 x 10 '
1.83 x 10~3
7.62 x 10"8
-8
3.60 x 10 °
1.25 x 10"6
-8
6.11 x 10 °
_7
1.95 x 10 '
-R
6.58 x 10
9.57 x 10"3
_2
5.27 x 10
_2
1.50 x 10
2.51
4.51 x 10"1
_2
2.77 x 10
7.21 x 10"3
_2
2.53 x 10
165.0
1.56 x 10~2
_3
4.87 x 10
1.24 x 10"1
-3
8.95 x 10 J
_2
1.56 x 10
_3
7.80 x 10 J
"Peak height of 5 chart units at 0.01 A.U. attenuation setting
llolar extinction values calculated from the mean values of standard
compounds dissolved In ammonia-free water (pH unadjusted; see Table
solutions of nitrogenous
A-24).
-------
using a 0.01 to 1.0 M ammonium-acetate-acetic acid buffer (pH=4.4) gradient.
Use of the Zorbax CN column with a pH 5.0, citrate buffer to 50% methanol,
20 minute gradient, resulted in the separation of eight compounds from a
mixture of eleven materials. All eight resolved peaks, however, eluted
rapidly and prior to the methanol phase of the gradient. Elution of a mix-
ture of ten compounds (uracil, indole, pyrimidine, sarcosine, tyrosine, crea-
tinine, cytosine, adenine, guanine, and tryptophan) on Zipax SCX (1 meter)
using 0.01 M sodium perchlorate adjusted to pH 2.5 with perchloric acid
eluant for 19 minutes followed by initiation of a 10 minute x5 gradient to
1.0 M of the buffer, resulted in resolution of only seven of the compounds.
Better resolution of this mixture was obtained using the Zipax SCX column
with NHnHaPOi, instead of the sodium perchlorate salt.
Successful separations of mixtures of reference compounds were obtained
on either the Zorbax C-8 or the Zipax SCX column using the chromatographic
conditions shown in Tables 22 and 23 and Figures 12 and 13. The Zorbax C-8
column exhibited greater selectivity for these compounds than did the Zipax
SCX column. The C-8 column was therefore used most frequently in analyzing
concentrated field samples.
TABLE 22. ELUTION POSITIONS3 (MINUTES) OF REFERENCE NITROGENOUS COMPOUNDS ON
ZIPAX SCX
0.01-1.0 M sodium 0.01-1.0 M NHi»H2POi,
perchlorate adjusted adjusted to pH 2.5 with
to pH 2.5 with perchloric H3POi»; 20 minute x2
Compound acid; 10 minute, x5 gradient gradient
5-chlorouracil
indole
tyrosine
guanine
adenine
trptophan
uracil
alanine
purine
creatine
cytosine
creatinine
1.1
1.5
7.2
7.3
8.6
25.9
—
-
—
—
—
_
2.3
4.9
7.9
10.0
13.1
1.8
4.0
6.7
8.1
10.2
in.fi
Values represent mean retention times.
After several weeks, the Zorbax C-8 column (column 'a') began exhibiting
peak aberations. This was later attributed to dissolution of the silica
support in the analytical column, and the accumulation of colloidal or part-
iculate material in the mobile phase or injected samples at the inlet of the
chromatographic column. A new C-8 column (column 'b') was therefore obtained
from the manufacturer. Although this replacement initially resulted in
67
-------
00
I mi 1.0. • I.I •»
I. 401 IWM: 10 •!• •' «.,JI.«I
1-MitUlM WO •«,
c,i..l~ 10 ../I
crr.ll.lM 10 .,/L
,»..l~ W .£/(
tkr»C>. M .«/!.
,j • tfttffcri ••rllillMi • 1*0 ••(
!."• • »IO ~. •n.lil.Uit • »0.0« I..... - t.l «A)[J- J|J^"JJ" " J|JJ
>li>rti MO'C II. IM..I, «o ,,/L
l*r U V i.«c.
Figure 12. Chromatogram of a mixture of 12 nitrogenous compounds resolved on Zorbax C8
with a 0.05 M phosphate buffer (pH = 6.9) to 50% MeOH; 10 minute, X5 gradient
-------
,it, - O.I
fl-or..c.«. *.t.f(o,- r.cii.tlu. - MO M; Mta.UM
• 70 —. ..~m.HT - ».01 (,„,. . o.J U)
: I4M p.i
I I I
II » !•
Ei-lloo tlm. (mlnul.. I lo. UV tiM«
Figure 13. Chromatogram of a mixture of 12 nitrogenous compounds resolved on Zorbax C-8 with a 0.05 M
borate buffer (pH-8.9) to 50* MeOH, 20 minute, X5 gradient.
-------
TABLE 23. ELUTION POSITIONS (MINUTES) OF REFERENCE NITROGENOUS COMPOUNDS ON
ZORBAX C8 COLUMN "a", USING .05 M PHOSPHATE BUFFER (pH = 6.9) TO 50% MeOH,
10 MINUTE, x5 GRADIENT
elution position6
compound
elution time (minutes)
1
1
1
1
1
1
2
3
3
3
4
4
5
6
7
8
9
10
10
11
11
12
urea
alanine
barbituric acid
creatine
uric acid
proline
fc-histidine
cytosine
uracil
tyrosine
succinimide
creatinine
guanine
5-chlorouracial
thymine
pyrrole
purine
tryptophan
adenine
pyrimidine
fc-hydroxyproline
indole
1.0
1.7
1.8
2.1
2.3
2.5
2.7
3.5
3.5
3.5
3.8
3.9
5.2
6.2
8.4
9.3
11.7
12.9
13.1
14.2
16.0
23.9
Numbers indicate elution order. Compounds having same number are unresol-
vable for conditions shown. Values represent mean retention time.
separations similar to those attained on the original C-8 column (Table 24),
loss in column retentivity was later observed after passage of about 20 to
50 ml of phosphate buffer through the column. Purine, for example, eluted
more than twice as fast after the column had been in contact with about 30 ml
of phosphate buffer in comparison to when the column was used immediately
after it had been regenerated with metHanoi and reequilibrated with phosphate
buffer. This occurrence was attributed to rapid bonding of trace contaminants
in the phosphate buffer onto adsorption sites in the analytical column.
Loss of retentivity on the Zorbax C-8 column was observed to occur more
slowly using a 0.05 M borate buffered eluant to 50% methanol gradient
(Figure 13). Loss of retentivity was observed under these conditions only
after several hundred milliliters of borate buffer had been eluted through
the analytical column. This occurrence was again attributed to bonding of
trace contaminants in the buffer onto adsorption sites of the chromatographic
resin. Column retentivity was restored by regeneration with methanol.
70
-------
TABLE 24. ELUTION POSITIONS3 (MINUTES) OF REFERENCE NITROGENOUS COMPOUNDS ON
ZORBAX C8, COLUMN "b", USING A .05 M PHOSPHATE BUFFER (pH=6.9) TO 50% MeOH,
10 MINUTE, x5 GRADIENT
elution position compound elution time (minutes)
1
1
2
3
4
5
6
7
8
9
10
11
barbituric acid
£-histidine
cytosine
creatinine
guanine
5-chlorouracil
thymine
pyrrole
purine
adenine
pyrimidine
indole
2.0
2.0
3.2
3.8
6.2
7.9
9.8
12.6
13.0
13.8
15.5
37.4
Values represent mean retention times.
Numbers indicate elution order. Compounds having same numbers are unresol-
vable for conditions shown.
With the exception of 5-chlorouracil, all of the reference compounds
eluted in the same order using the borate buffered eluant (Table 25) as pre-
viously observed using the phosphate buffered mobile phase (Table 24).
Thirteen compounds were resolved under these conditions. Most of the amino
acids, however, were not strongly retained on the chromatographic column.
An unidentified chromatographic peak, eluting from about 27-30 minutes
was observed in chromatograms of the mixture of reference compounds resolved
on Zorbax C-8 using the borate buffered eluant (Figure 13). This was attri-
buted to desorption by the methanol, of trace contaminants in the buffer which
had been retained on the column during the first phase of the gradient.
A chromatogram of the ultraviolet and fluorescamine derivatized fluor-
escence traces obtained without injection of a sample is shown in Figure 14.
The contaminant peak just discussed again appeared in the chromatogram during
the methanol phase of the gradient. The fluorescence baseline remained quite
flat during the buffer phase of the gradient but then increased by about 10%
increasing methanol eluant composition. This was attributed to a correspond-
ing pressure drop of about 56 to 50 p.s.i. in the post column lines. No base-
line change was observed, however, in an underivatized fluorescence trace,
obtained without injection of a sample, at a sensitivity setting two times
greater than that used in monitoring the derivatized fluorescence baseline.
71
-------
TABLE 25. ELUTION POSITIONS3 (MINUTES) OF REFERENCE NITROGENOUS COMPOUNS ON
ZORBAX C8, COLUMN "b", USING A .05 M BORATE BUFFER (pH 8.9) TO 50% MeOH,
20 MINUTE, x5 GRADIENT
elution position
1
1
1
1
1
1
1
2
2
2
2
3
3
3
4
4
4
5
6
7
8
8
9
10
10
11
12
13
compound
aspartic acid
glycine
glutamic acid
serine
barbituric acid
thioruea
glycylglycine
proline
lysine
valine
il-histidine
succinimide
tyrosine
cytosine
leucine
uracil
5-chlorouracil
creatinine
guanine
thymine
pyrrole
phenylalanine
purine
adenine
tryptophan
pyrimidine
indole
skatole
elution time ' (minutes)
1.5
1.8
1.7
1.8
1.9
2.0
2.0
2.5
2.6
2.7
2.8
3.4
3.7
3.7
4.3
4.1
4.3
4.6
6.3
10.5
14.4
14.2
15.5 '
21.1
21.0
23.0
46.3
67.5
3Values represent mean retention times.
Numbers indicate elution order. Compounds having same numbers are unresol-
vable for conditions shown.
72
-------
i •> uaplxl lnrod.,.4 IMC,
Mchltilt: Xorfcai i:«
4la0Ml«M! l««*th • 2) rm: l.n. • 2.1 M
plwMt .OV1 barm. buff.r (p* • •••) to Ut MOI; » •>•»•. »
«Hott
lreter: «av,.|.-««ll. . |n w •miitvlfy . 1.0
vlwtor: vmcicallua • no ••: WIM|M • 410 M:
>lil>lt. . 40.07 (rnur . 0.: Ut)
r: two p»l
l*w ratv) kvfrrr • 1.1 •!/•!•
Ourl •(••irft I r«/«m
.t Fl«nr.*i-<.l».
Mltoc
•
Figure 14.
^^^''^""''""""""""'"""^•""^'"'"'""'"'""""T™"^"1™""""""1™!?"'"™™1""111^""'""^^""""
•HIM. HM 0-lMln) I0> IIMMICMC* UK*
Chromatogram of U.V. and fluorescamine-derivatized fluorescence
traces obtained without injection of a sample.
The Zorbax C-8 'b1 column began exhibiting peak aberation after being
used for only several days. The analytical life of this column, using the
borate buffered eluant, was shorter than that of the Zorbax C-8 'a' column,
using phosphate buffered eluant. This was attributed to the more rapid
dissolution of the silica support in the column using the more alkaline
mobile phase.
Another replacement column (column 'c') was therefore obtained from the
manufacturer and used with a guard column. The guard column prevented fouling
of the analytical column by sample or mobile phase contaminants. In addition,
the silica-containing guard column presaturated the mobile phase with dis-
solved silica, thereby decreasing the rate of silica dissolution in the ana-
lytical column. The guard column consisted of a 4.6 i.d. by 5 cm stainless
steel tube packed with permaphase ODS (DuPont Co.) and fitted with a 20 urn
inlet frit and 2 ym outlet frit. Elution positions for some reference nitro-
genous compounds on the Zorbax C-8 'c' column using a 0.05 M borate buffer
(pH-8.9) to 50% methanol, twenty minute, x5 gradient and the 5 cm permaphase
guard column are shown in Table 26. With the exception of purine all of the
reference compounds exhibited the same retention times using the borate buffer
with the guard column (Table 26) as previously observed without the guard
column (Table 25) . Purine eluted slightly more rapidly using the guard
column and the new Zorbax C-8 column, and was therefore not resolvable from
pyrrole. The guard column did not appear to decrease the overall separation
efficiency to any observable extent.
73
-------
TABLE 26. ELUTION POSITIONS3 (MINUTES) OF REFERENCE NITROGENOUS COMPOUNDS ON
ZORBAX C8 COLUMN "c", USING A .05 M BORATE BUFFER (pH 8.9) TO 50% MeOH, 20
MINUTE x5 GRADIENT (USING A 5 cm PERMAPHASE GUARD COLUMN)
elution position column elution time (minutes)
1
2
3
4
4
5
6
7
8
8
9
10
11
barbituric acid
£-histidine
cytosine
5-chlorouracil
uracil
creatinine
guanine
thymine
pyrrole
purine
ader.ine
tryptophan
pyrimidine
2.3
3.3
3.9
4.2
4.3
4.9
6.5
10.7
14.0
14.0
21.6
22.5
23.8 . .
a
Values represent mean retention times.
Numbers indicate elution order. Compounds having same numbers are unresol-
vable for conditions shown.
Ultraviolet spectra of reference nitrogenous compounds were recorded
after correction for the background spectrum of 50% methanol in water, the
flow cell and photomultiplier, using the memory module. Figures 15 and 16
show the uncorrected and corrected spectra, respectively, of 50% methanol in
water. Figures 17 to 30 and 31 to 56 show the corrected spectra of reference
nitrogenous compounds eluted through Zorbax C-8 using a 0.05 M phosphate
buffer (pH = 6.9) to 50% methanol, 10 minute, x5 gradient and a 0.05 M sodium
borate buffer (pH = 8.9 with H3P04) to 50% methanol, 20 minute, x5 gradient,
respectively. Neither of the two buffers had significant absorbance at wave-
lengths greater than about 225 nm for the signal attenuation used in the
spectral scans (Figures 57 and 58). End absorbance at wavelengths less than
225 nm for compounds eluting in the buffer phase of the gradient program was
partially attributable to absorbance by the buffer. Most of the reference
nitrogenous compounds exhibited the same U.V. spectra in the phosphate buffer
as in the borate buffer. Shifts in the positions of maximum and minimum
absorbancies attributable to the different buffers were noted, however, for
purine, succinimide, tyrosine, and 5-chlorouracil. Spectral data for some of
the reference compounds are summarized in Table 27.
74
-------
^F-
I
I
aso
aoo
Wavelength
200
(nm)
Figure 15. 50% methanol uncorrected signal.
75
-------
I
t
s
I
•so
i
300
280
200
Wtoratength (nm)
Figure 16. 50% methanol corrected signal.
76
-------
t
e
w
360
360
Wavelength (nm)
Figure 17. Acenine (in 0.05 M phosphate buffer)
77
-------
o
^
9
I
t
e
w
4
380
300
(nm
2SO
2OO
Figure 18. 5-chlorouracil (in phosphate buffer).
78
-------
350
300
2 SO
200
(nm)
Figure 19. Creatinine (in 0.05 M phosphate buffer).
79
-------
t
e
>
360
300
WavaUngth (nm)
250
200
Figure 20. Cytosine (in 0.05 M phosphate buffer)
80
-------
V
e
t
P
8
4
300
Wavelength (nm)
1ST
Figure 21. Guanine (in 0.05 M phosphate buffer)
81
-------
w
!
t
p
360
2*0
200
(nm)
Figure 22. Indole (in 0.05 M phosphate buffer).
82
-------
t
p
w
4
300
Wavelength (nm)
2OO
Figure 23. Purine (in 0.05 M phosphate buffer)
83
-------
*
*
t
p
w
4
960
300
Wav«l*ngth (nm)
—T
200
Figure 24. Pyrimidine (in 0.05 M phosphate buffer)
84
-------
I
I
t
e
u
4
3*60
TOO
Wavelength (nm)
200
Figure 25. Pyrrole (in 0.05 M phosphate buffer)
85
-------
o
t
p
e
350
300
Wavelength (nm)
250
200
Figure 26. Succinimide (in 0.05 M phosphate buffer).
86
-------
a
n
t
I
3*0
300
Wavalcngth (nm)
200
Figure 27. Thymine (in 0.05 M phosphate buffer).
87
-------
I
8
p
w
990
300
Wavalcngth (nm)
250
200
Figure 28. Tryptophan (in 0.05 M phosphate buffer).
88
-------
I
A
t
e
c
4
1ST
300
Wavelength (nm)
260
200
Figure 29. Tyrosine (in 0.05 M phosphate buffer)
89
-------
I
e
A
t
35O 300 25O
Wav«l*ngth (nm )
Figure 30. Uracil (in 0.05 M phosphate buffer)
2OO
90
-------
350
300
W»v« length (nm)
250
200
Figure 31. Adenine (in 0.05 M borate buffer)
91
-------
t
e
4
950
300
(nm)
290
200
Figure 32. Barbituric Acid (in 0.05 M borate buffer).
92
-------
^
^
I
t
4
300
Vfewtongth (nm)
200
Figure 33. 5-chlorouracil (in borate buffer)
93
-------
a
A
t
«*
e
I
950
300
W«v«l«ngth (nm)
280
200
Figure 34. Creatinine (in 0.05 M borate buffer)
94
-------
t
e
g
4
350
300
Wavelength (nm)
280
200
Figure 35. Cytoslne (in 0.05 M borate buffer)
95
-------
t
2
9
4
3SO
I "
aoo
i
280
I
200
Wavelength (nm)
Figure 36. D-glutamic acid (in 0.05 M borate buffer).
96
-------
3
n
t
4
350
300 250
WavcUngth (nm)
2OO
Figure 37. DL-phenylalanine (in 0.05 M borate buffer).
97
-------
350
300 250
Wav*l«ngth (nm)
200
Figure 38. DL-serine (in 0.05 M borate buffer).
98
-------
>
•
e
t
p
b
I
300
280
200
W«v«Un0th
-------
I
•
a
A
t
O
5
>
,
aao
3OO
Wavelength (rnn)
280
2OO
Figure 40. Glycine (in 0.05 M borate buffer)
100
-------
>
•
n
t
I
350
300
Wavelength (tint)
•
25O
200
Figure 41. Glycylglycine (in 0.05 M borate buffer).
101
-------
|
380
300
W«v«l*ngth
2SO
200
nm
Figure 42. Guanine (in 0.05 M borate buffer)
102
-------
>
V
o
•I
II
9
n
t
o
4
•
350
3OO
I
250
200
W«v«l«ngth (nm)
Figure 43. L-(+)-Aspartic Acid (in 0.05 M borate buffer)
103
-------
aoo
Wavelength (nm|
280
abo
Figure 44. L-(-)-Histidine (in 0.05 M borate buffer)
104
-------
t
o
b
P
4
350
I I
300 250
Wav«ltngth iron)
200
Figure 45. L-(+)-Lysine (in 0.05 M borate buffer)
105
-------
t
o
S>
4
35O
300
Wav*l«ngth (nm)
2SO
200
Figure 46. L-(-)-Proline (in 0.05 M borate buffer).
106
-------
or
•
^
9
\
\
2
»
>
4
aoo
300 290
Wav« length (nm)
200
Figure 47. 3-methylindole (skatole) (in 0.05 M borate buffer)
107
-------
>
V
•
o
•
t
e
^
gi
360
300
Wavelength (nm)
290
200
Figure 48. Purine (in 0.05 M borate buffer)
108
-------
»
I
£
I
W«**Mngth (MM)
200
Figure 49. Pyrimidine (in 0.05 M borate buffer)
109
-------
or
t
e
8
4
300
I
250
20O
Wavelength (nm)
Figure 50. Pyrrole (in 0.05 M borate buffer)
110
-------
>
cr
a
o
t
o
I
350
300
250
200
Wavelength (nm)
Figure 51. Succinimide (in 0.05 M borate buffer)
111
-------
>
o-
o
a
1
o
g
350
300 250
Wavelength (nm)
ZOO
Figure 52. Thiourea (in 0.05 M borate buffer)
112
-------
9
e
a
A
t
O
•
01
I
350
300
Wavelength (nm)
250
200
Figure 53. Thymine (in 0.05 M borate buffer)
113
-------
I
o
t
p
8
300
Wavelength (nm)
250
"T"
200
Figure 54. Tryptophan (in 0.05 M borate buffer)
114
-------
MO • 280
Wmclmgth (MM}
Figure 55. Tyrosine (in 0.05 M borate buffer)
115
-------
300
Wavelength (nm)
280
200
Figure 56. Uracil (in 0.05 M borate buffer)
116
-------
350
300
Wavelength (nm)
250
2OO
Figure 57. .05 M phosphate buffer, pH = 6.9.
117
-------
8
t
e
*
4
aso
aoo
280
200
Figure 58. .05 M sodium borate adjusted to pH 8.9 with H-PO,.
118
-------
TABLE 27. SPECTRAL DATA FOR SOME REFERENCE NITROGENOUS COMPOUNDS
position of maximum position of minimi
absorbance (nm)a absorbance (nm)
compound
adenine
barbituric acid
5-chlorouracil
creatinine
cytosine
guanine
fc-histidine
purine
pyrimidine
pyrrole
succinlraide
thymine
tryptophan
.05 M
phosphate
buffer
260
-
276
234
267
276 & 250
-
265
242
flat
239
265
279
.05 M
borate
buffer
260
257
293
233
266
272 & 243
293
268
240
flat
211
265
276
.05 M
phosphate
buffer
227
-
240
217
247
259 263
-
223
218
flat
232
233
243
.05 M
borate
buffer
229
230
248
215
248
& 228
249
233
219
flat
205
235
243
absorbance
im
absorbance
.05 M
phosphate
buffer
4.9
-
3.3
1.6
1.4
-
4.9
4.4
-
5.0
3.3
2.7
max
min
.05 M
borate
buffer
4.0
4.8
3.6
1.5
1.4
-
3.2
4.0
2.9
-
1.07
2.9
3.0
absorbance
absorbance
.05 M
phosphate
buffer
1.4
1.2
2.9
0.7
1.3
1.3
1.7
0.8
0.4
5.1
1.9
2.2
6.0
at 220 nm
at 233 nm
.05 M
borate
buffer
1.3
1.7
1.7
0.8
1.3
1.4
1.7
2.4
2.1
-
-
2.3
7.1
(continued)
-------
TABLE 27 (continued)
position of maximum position of minimum absorbance
absorbance (nm)
absorbance (nm)
max
absorbance
.
min
absorbance at 220 nm
absrobance at 233 nm
compound
tyrosine
uracil
indole
.05 M
phosphate
buffer
276 & 222
259
273 & 217
.05 M
borate
buffer
276 & 222
259
'
.05 M
phosphate
buffer
245
228
240
.05 M •
borate
buffer
260
229
"
.05 M
phosphate
buffer
7.2
4.0
3.7
.05 M
borate
buffer
10.3
2.9
"
.05 M
phosphate
buffer
1.6
1.3
8.4
.05 M
borate
buffer
2.3
1.3
^Underlined values were used in computation of absorbance ratio.
-------
Chromatographic Resolution of Concentrated Natural Samples
Isocratic Elution With U.V. Detection Only—
Chromatograms of concentrated Charles River (Appendix B, Figure B-5),
early Concord River (Appendix B, Figure B-6), and Merrimack River (Appendix B,
Figure B-7) water samples all contained only large peak eluting rapidly from
the Zipax SCX and Zorbax C-8 columns, and occasionally smaller secondary
peaks. No distinct peaks characteristic of identifiable nitrogenous compounds
were detected. Both Kjeldahl-N (Table 28) and fluorometric analyses (Appendix
B, Figures B-8 and B-9) of aliquots taken from the chromatographic column
after injection of concentrated field samples, however, indicated the presence
of nitrogenous organic compounds beyond the initial large elution peak. The
approximate mass of Kjeldahl-N in each aliquot was calculated from the product
of the Kjeldahl-N value (mg/L) and the volume analyzed (liters). These data
showed that approximately 82% of the nitrogen content of the eluting nitro-
genous compounds was found in the first 10 ml of the chromatographic effluent.
Some of the higher Kjeldahl-N values occurred in aliquots having corresponding
large U.V. absorption. Kjeldahl-N recovieries in other aliquots, however,
were observed in low U.V. absorbant portions. Kjeldahl-N values of resolved
individual N-organic compounds were probably obscured by the relatively large
volume of aliquot required for analysis.
Resolution of some 15 distinct peaks from a concentrated primary sewage
effluent collected at Marlboro East sewage treatment facility was achieved
using Zorbax C-8 with citric acid, phosphate, and borate buffered eluants of
increasing methanol composition. Peak identification was not pursued because
of the inability reproducibly to regulate increasing mobile phase strength
with the isocratic pump. Separation of this concentrated sample using Zorbax
CN (1-octanesulfonic acid eluant), Zipax SCX (NHi»H2POi»; pH 2.5 eluant) and
Aminex A-27 (sodium acetate-acetic acid buffer; pH 4.4 eluant) resulted in
the resolution of only 5, 5, and 4 peaks, respectively. Gradient elution
would most likely have resulted in improved resolutions.
The ability to resolve several complex mixtures on Zipax SCX using
NHi»H2POi, (pH 2.5) eluant was also investigated. Urine samples were resolved
into six U.V. detectable (233 nm) constituent peaks while urochrome con-
stituents in urine and in secondary sewage effluent were resolved into only
two large peaks in each case.
Gradient Elution with U.V. and Fluorescence Detection—
The gradient device significantly improved the chromatographic analysis
of concentrated field samples over conventional isocratic elution. Chromato-
grams of concentrated samples resolved on the Zorbax C-8 column using the
phosphate to methanol, or borate to methanol gradient elution operating
conditions, displayed from 1, to over 40 distinct and reproducible peaks.
The Zorbax C-8 column exhibited greater selectivity over the Zipax SCX column
and was therefore used exclusively in analyzing the concentrated samples.
Nitrogenous compounds in 8 of the 12 samples concentrated by the low temper-
ature distillation-lyophilization method were characterized (Figures 59 to
82).
121
-------
TABLE 28. NITROGEN VALUES IN ALIQUOTS TAKEN FROM THE CHROMATOGRAPHIC EFFLUENT OF ZIPAX SCX AFTER
INJECTION OF CONCENTRATED (200 FOLD) MARLBORO WEST POST CHLORINATED EFFLUENT
to
IS)
sample
aliquot
(ml)
0-2
2-4
4-8
8-10
10-12
12-14
14-18
0.05 ml
concentrated
sample
volume
analyzed
(ml)
1
1
2
1
1
1
2
.05
b
KJeldahl-N
(tng/L)
7.7
1.2
2.8
4.7
0.9
1.6
0.9
354
approximate mass
of Kjeldahl-N in
volume analyzed0
(mg)
0.77 x 10"2
0.12 x 10~2
0.55 x 10~2
0.47 x 10~2
-2
0.09 x 10
0.16 x 10"2
0.18 x 10"2
1.77 x 10"2
percent Kjeldahl-N
in aliquot
33
5
24
20
4
7
8
-
^Volume analyzed • 1/2 sample aliquot volume. Aliquots were divided equally for separate NH3 and
Kjeldahl-N analysis.
3Kjeldahl-N = organic-N since NH3-N = 0
=Mass Kjeldahl-N = concentration of Kjeldahl-N (mg/L) x volume analyzed (liters)
-------
ho
OJ
flwllen IIm« Iw UV tree* |Mny|«B)
-r-
60
Figure 59. Chromatogram of raw concentrated Middleton Pond sample (7/11/78), phosphate buffered eluant,
with fluorescaraine derivatized fluorescence.
-------
V) »l HlMUto* PM4. BMVW*. NA
-------
co
«-»!.. • .1 MMUU. N*. tan... m (,-, . MM.
C.1— nctl.,, «—»«. C-i
(ringt • 0.1 t*A)
*VO Ml
»l«i i.t«; bu(f., . l.l BI/.U) lMi/w.t«r • 0.
*JO*C
Ch*rl •(>•»<)( 1 CB/Ml«
M Mm. (HlnlMl Iw U« III
Figure 61. Chromatogram of raw concentrated Middleton Pond sample (7/11/78), borate buffered eluant,
with underivatized fluorescence.
-------
. l-II-It)
to
IMlo W .l| »Ut\*tm »••*. •—..... Ill In. I. Mil. ,.4, I
c-l
m i.». • t.i •
Mrtiu pi»Mi .CM »»•!• k»rr« (f» i.t) >• m man N
O.f . 4«tB«r«rl *«v«Un«tfc • 110 M| MMlllVltf - 1-0
- 0.1
*MMT«l 1430 »•!
r.l.l »<•»« - 1.) .1/.1.I WOl/MUr - OH •!/•!•
Figure 62. Chromatogram of resin-filtered, concentrated Middleton Pond sample (7/11/78), borate
buffered eluant, with fluorescamine derivatized fluorescence.
-------
. • (iota Itlun*. • MM, l-u->«)
> I] _i I. >.*>.! •
.OM k.,.t. k.Il.r (f»4.t) t. Ml
t.f . 4*l«cl*n IMMlMftk - 110 »| •MMtllvltf • 2. ft
n»»Mc«K> <« MO BM oKilx • 4M Ml »—ilil.li, . M.M
IB
u V lr*c«
ffhill«> tlM« (M4n
Figure 63. Chromatogram of resin-filtered, concentrated Middleton Pond sample (7/11/78), borate
buffered eluant, with underivatized fluorescence.
-------
to
CC
M .Li CMMN llm. IIIUclu, M ».<>.l.llk -!»•,, uultlvtl, • l.t
""HI'"'! !"""" """""• • "• *' -'••'•• • «» -I .~l.i.lr, - M.M
Pr«»ur»i MX) MI
io. otii kuff.r * 1.1 .!/.!.< IMI/mur - O.TJ «!/.!.
I««f«r«tur«i xlO'C
!•
.r. lliiin.ilii 1.
tlMM«n 11m*
-------
M
l«Wl>: W ill
llmi. Illlrrl... M (. — . • IOM. I-4-II)
CeliM 4lBM*l«Bii iMfttli - 21 r«| l.». • 1.1 w
IMIU f*-".: .OM fcor... W-rr.f .. HI IMMt M «lMt«
V.V. 4«t*ct*ri w«**lra|tli - 1X0 mn MMlilvlty • l.t
(r*n|« O.I iiA)
PTMawrci 14V) Ml
riov >.t.i fc.ff.r - 1.) . I/.1-, H^M/M*« • O.I) .1/.|.
tlMM (MIAWf**) f«f UV !>•<•
llm> IOIKX.. )
M
Figure 65. Chromatogram of raw Concord River sample (7/4/78), borate buffered
eluant, with underivatized fluorescence.
-------
H
OJ
O
Ua»)Ui JO til UD-t/TaoM flltarad Concord ll*ar. Illlarlca. HA
(*-»-7»). •«*>!• cooc.ntr.l.d 2.000 fold
ColuaM packlD|l Zorbax C-8
Coliim dlamnalona: laagth - 2) »t I.D. • 2.1 •»
Hobila phaaat .05* boratc buffar (pH 8.9) to 501 rhOH; JO alnu...
i5 gradlant
D.V. datKtori ««vrlength - 220 n»; acnattt*!ty - 2.0
CoaBjenti: fluoresc&alne and boratc buffar latroducad bafora fl
to Monitor derlvatlzad primary aftlna coapounda
10
Elullon lima (mlmilai) for U.V Iraca
1O 15 20
Elullon Urn* (mlnulaa I for tluotaacanca Iraca
J5
30
Figure 66. Chromatogram of resin-filtered Concord River sample (6/8/78), borate buffered eluant,
with fluorescamine derivatized fluorescence.
-------
"D-"T""" f"""<" Cohort llnr. llll.rlc.. HA
, •*•?!• conccntratad J.OOO fold
Column poking: lort.. C-8
Moilon.: l«,,th - 1J cm; l.D. - 2.1 „
Mobil. ph«.: .Oi« bor.t. buffet (pH S.9) to SOX tMH; W .lou
• Rr»dleat
U.». d.t.ctor: i<>vcl.n«th - JZO t... .m.uivlt; • 1.0
cenu d.tector: nclt.tlon - )90 n.; ral.iloi - 470
•riulclvlty - 37.01 (r.ng. . 0.2 UA)
t«: buff.r - I.J •!/.!„; M.UH/H 0 - 0.73 ml/mix
fur*- "v^O"/* *
P«d: 1 <••/•! n
• : undrt iv.tdrd fluoroetDci
I
to **
glutton tim» (minutai) for U.V. trace
10
30
10 IS 20
Elullon llm* (mlnulci) lor fluor««c.nc« lr«c«
25
Figure 67. Chromatogram of resin-filtered Concord River sample (6/9/78) , borate buffered eluant,
with underivatized fluorescence.
-------
OJ
ro
ta^Ul SO Hi rmi OicllUtorll (twit ••»!«. e«K*»tral>4 l.fOO f°
CO!M« Mckl«t> *«rb«m r-8
&>!••• Jlaawloui Iwmth > 35 oil I.D. - 1.1 **
febtU pk«.«. .0«1 bor.t. buffer (pd - •.*) to 501 IMXIl M •!•«»
i5 Ittiiml
O.V. <«t.rror: «.v.l.r,tth • 110 >•! ».o.Itlvlti - 2.0
Fluor««c«ne« 4*(ccror: •xcitatton - MO M; wtHlMi • 470 ••{
•mcitlvlty - 55.01 (r«n|« - 0.2 gA)
Pr««iurc: 14V) pfl
riou r>»: kufftr - 1.1 .1 .lo; lto<*/H2O - 0.7) ml I ml a
fiver««c*nc«
IS 20
Hut Ion llm« (mlnul.i) for U V tract
If 10
Clvllon llm« (mlnut«t) tor 'l
10
Figure 68. Chromatogram of raw filtrate from a culture of Oscillatoria tenuis, borate buffered
eluant, with fluorescamine derivatized fluorescence.
-------
H
LO
Figure 69. Chromatogram of raw filtrate from a culture of Oscillatoria tenuis, borate buffered
eluant, with underivatized fluorescence.
-------
I— pU. JO 111 UD-I/TXMI filtered O.clll.torl« tenul. u^ple.
concentrated 1.610 fold
ColMn packlnft Zorba* C-8
Colon dlaeulonit lenfth - 2S cm; 1.0. - 2.1 —
Mofcll. ph.M: .OSN boriti buffer (pH - 8.9) to V)I HrOH; 20 .lour.
U.V. 4>
fh.rt
*ctor: wcv*l«ngth - 220 am: •rnfltlvtty • 2.0
ence detector: excitation - 190 am', eaieeloa • 470 na»:
a* altlvlty - 16.01 (ran|e - 0.2 uA)
USO pel
buffer - 1.1 »l/»l»; HeOH/H2O - 0.71 ml/mlm
art: ->.20'C
fluorc«c«aln« and boratc buffer introduced before fluor
10
-------
H
UJ
in
p
\
^
J
t
I
leiepUi 50 ul IAD-1/Tenu filtered O.ctlletorl. temjle mol*.
concentrated 1.6)0 fold
ColiMBn packing: Zorba* C-fl
Calnaa ilmfoMtont: length * 1} CBI I.D. - I.I IB
Mobil _ph..«: .03M bor.t. buff.t (pB - 8.9) to SOI NrfM; 20 .lout.
U.V.
terror: wavelength - 220 «; aaiMttlvlty • 2.0
niltlvltjr - 36.01 (txige - 0.2 uA)
te: butter - l.J .l/.ln. HcOH/HTO - 0.73 •!/•!•
underivettird fluoreecence
W 1*
tlutlon llm« fmlnulei) (or U.V. lnc«
10 IS JO
ElUllon lime (mlnulei) lor 'luoreicence tr«c«
Figure 71.
Chromatogram of resin-filtered filtrate from a culture of OsaillatoTia tenu~is, borate
buffered eluant, with underivatized fluorescence.
-------
cr-
Mil It •!••«• .'
Figure 72. Chromatogram of raw filtrate from a culture of Anabaena flos aquae,
borate buffered eluant, with fluorescamine derivatized fluorescence.
-------
OJ
I.. M
m MrkKl 1..%.. C-t
MM 4IMMIMMI iMflfc • t> Ml I.B. • 1.1 M
M1U fluuc .OW koc«l« ».ff.r | •
Figure 73. Chromatogram of raw filtrate from a culture of Andbaena flos aquae,
borate buffered eluant, with underivatized fluorescence.
-------
OJ
00
tm,\,i it >|| tmtitm OM •«••« ». 0 1«A
, fcimwi nclldlm - HO «.i ~l.il— - . ft a, MMIU.II> • tO.H
- O.i
rnnwrti
flaw !•!•: K«tf.r <• 1.1 •(/•INI MaOM/Mtcf • O.M •!/•!•
Ckiri .,.*, I r./.i
Figure 74. Chromatogram of resin-filtered filtrate from a culture of Andbaena flos
aquae, phosphate buffered eluant, fluorescamine derivatized fluorescence.
-------
i
^
teaplei V) ul IAD-«/T«um filtered »«cheed«, on eeepl.. co»«etr« C-l
Coluen dl.rn.10ni: length > 25 c»; I.D. - 2.1 Mi
Mobil* phe»: .05 M borete buffer (p« - I.*) Co Ml HeOl; 10 Btnu,
gradient
U.V. detector: Mevelen|th • 220 im: ecnelclvlty - 0.4
eence detector: ••cltetloo • MO am; ewleilo* • 470 ••;
neltlylty - «0.OX (r
-------
S«pl«: 50 ul UD-l/T«ftu (llt.rrd B.-.h..d.. OH >«^>lr. roocntr.t
2000 fold
Coluon p.ctlng. Zorb.i C-8
Coluan di«rnilon«: length - 2) <••; 1.0. • 2.1 n
Hob II. ph».r .OW bui.tf buff.r (p« - t.t) to SOI ItKW; JO .lout.
*'* glfdtfnt
U.V. dcicrior: «v«len|th * 220 am; ••n.ltlvllr - 0.4
Fluorv.ccnce d«c*ctor: eiclt.clon • 390 rm; cvli.lon - 470
n.ltlvlty • 40.01 (ranRC - 0.2 uA)
e: UV) P.I
tt: buffer - 1.) -I/.In. (VOH/HJO - 0.7) .l/.la
«: -*.20"C
d: 1 r./.ln
underlv.t lied fluorc.renct
1 10 It
((•lion lim« (minulti) lor U-V. trie*
19 10
Elutlon Urn* (hilnulM ) lor liuor.«c.nc. Inc.
Figure 76. Chromatogram of raw surface impoundment sample, Bethesda, Ohio, borate buffered eluant,
with underivatized fluorescence.
-------
f**?le: JO »1 UD-i/T«nn filtered Mlddletnn Pond enable (p* un.id |u.t.d)
*-21-7a, cuncrntratrd 2000 fold
Colum packing: Zorbai C-8
Coliaan dlienalona: length • 25 cm; I.D. - 2.1 an
Mobile phaae: .05* borate buffer (pH - 1.9) to SOt NaOH( JO ejlnute.
«5 gradient
U.V. detector: vavelrngth • 220 n«- ««n«ltl»lty - 2.0
Fluoreacence detector: eicltatlon - J90 am; eailaalon - «70 na-
aenaltlvlty - 36.01 (range - 0.2 nA)
Preaaure: 1450 pal
Ploii rate: buffer -1.3 .]/.!„• M«OH/H,0 . 0.71 •!/•!>
Teap'ralure: i20*C
Chart apeed: I i«/.ln
Coawnta: riuorearailne and borate buffer Introduced before
f luor
10 •
Clutlon lima
IS X
fltilnulap lor U.V. lrac«
EluHon urn*
IB
mlnul • •
30
lor
Figure 77. Chromatogram of resin-filtered (pH unadjusted) Middleton Pond sample (6/21/78), borate
buffered eluant, with fluorescamine derivatized fluorescence.
-------
•: V> pi UD-*/Teiui» filtered NlddUtcm Pond ..„. 1, r-8
Tnlmn dluniloiu: length - 2S ,-«; I.D. - 2.1 IB
Mobil* ph«.«: .OiH borne buffer (pH - ».») to SOI NeOH; 20 »lnul«,
»' iridlenc
U.V. detector: vnelen|th - 220 n., >en>ltlvlty - 2.0
Pluore.cenc. detector: e,cit.tlon . 1,0 «; »i..lo» - 470 n.;
•entltlvlty • J4.0I (ran(e - 0.2
Preiture: 14V) pal
Plou rite: buffer - 1.1 .l/.ln: KeOH/H20 - 0.73 .l/.In
Tevperature: %20'C
Ctiirt (peed: 1 cm/mln
underlvit lied fluorescence
tlullen II m« (mlnutel) tor U.V. Itece.
10 IS 30
C lulled Urn* (jnlnulaO far fluoxscenc* lr»e«
25
Figure 78. Chromatogram of resin-filtered (pH unadjusted) Middleton Pond sample (6/21/78), borate
buffered eluant, with underivatized fluorescence.
-------
1.0), MlddlatM road .je»la
M Hi lAB-t/raau fllt.rW (at
), c«tc*«trat>d 2000 fold
Calaan F.<-kln,i lorkaa C-l
Co I nan dleameloaal length - IS e*| I.D. - 1.1 ••
Itoblla ahaaa: .OM karat* kaffar (•« - t.t) to 501 NrCM; JO •!•
*5 gradient
U.V. detector: « r
• : buCIrr - 1.3 «l/.lo. HrW/HjO - 0.7] mlfmim
art: ^ZO'C
•cd: 1 cm/min
float fmctmlnt and bor.i. buffer titrod»e«d >«for*
lury aailnc colpounde.
Cklltan urn* (mlnulaa) for U.V. trace
T
<5 .20 »
Eklllon tin* (mlnul«») for lluor«ac«nc« tract
Figure 79. Chromatogram of resin-filtered (at pH= 2), Middleton Pond sample (6/21/78), borate
buffered eluant, with fluorescamine derivatized fluorescence.
-------
•aaail.i 90 ill IAB-l/Teui fllt.r.d (.c pH - 2.0). Nlddleton Pood l
(6-21-76), coac.iit rated 2000 (old
Colueai p*ckln«: Zorbai C-B
Colian dlBenaloa*! length - 25 cm; I.D. • 2.1 an
Nobtle phaae: .05H borttt buff*r 'pH - 6.9) to 50J IMM; 20 limit*;
• gradient
U.V. dcttctot: Mvclrnith - 220 r,.. Multlvltr • 2.0
Pluorcicrncr detector: rx
-------
Figure 81. Chromatogram of resin-filtered Merrimack River sample (6/14/78), borate buffered eluant,
with fluorescamine derivatized fluorescence.
-------
n
ILJ
•«*l.< M nl lAD-ifr— rilurvd fer Match •!»•» .«.*!• (*-l4_7lf
co«r*mtr.tH 2000 t»l4
Col*m 41a.M*loa*i laagik • 19 cmi 1.0. • 1.1 ••
-to*iU_.A»*«; .OM bor.f. k.fUr to Wl IWM| 20 tilMt*.
i * 120 MI MMlclvltr • 2.0
*t*cteri ncltatloa • WO em; *«ia«la* • *ft M«I
t«f U V. H«c«
Figure 82. Chromatogram of resin-filtered Merrimack River sample (6/14/78), borate buffered eluant,
with underivatized fluorescence.
-------
Identification of unknown resolved chromatographic peaks was achieved by
comparison of retention position and U.V. data with those of reference
compounds. U.V. data were obtained by stopped-flow spectral scanning of in-
dividual chromatographic peaks. It was found, however, that the retention
positions for some compounds were effected by the stopped-flow analytical
method. For example, after scanning cytosine using the stopped flow tech-
nique, it was found that thymine eluted only 2.5 minutes after resumption of
mobile phase flow through the Zorbax C-8 column 'a' using the phosphate to
methanol gradient. With continuous eluant flow, thymine eluted 4.5 minutes
after cytosine. The shorter elution time of thymine, observed in the separa-
tion where the mobile phase had been temporarily stopped, indicated that this
compound probably continued to diffuse through the column while the eluant
was static. Pyrrole, on the other hand, was eluted the same amount of time
after thymine, with or without stopping of the mobile phase during the chroma-
tographic run. Positions of unknown chromatographic peaks could therefore be
unambiguously related to those of reference materials only in that portion of
the chromatogram obtained prior to and including the first stopped-flow
spectral scan of an eluting material. Stopped-flow spectral scanning of un-
known compounds eluting at positions of reference materials was therefore
done only one time for each chromatographic run.
The principal information used in comparing U.V. spectra of unknown
materials with those of reference compounds was the positions of maxima and
minima U.V. absorbance, and ratios of maximum to minimum U.V. absorbance and
absorbance at 220 nm to 233 run. U.V. spectra of materials isolated from
concentrated field samples are shown in Figures 83 to 109. A summary of the
information taken from these spectra for use in the identification of these
materials is shown in Table 29.
The sample taken at Middleton Pond, Danvers, MA (7/11/78) was analyzed
using both the borate and phosphate buffer to methanol gradients on the first
Zorbax C-8 column (column 'a'). The probability that the chromatographic
peaks were correctly identified in this sample increased because of the
corroborative retention data and U.V. spectra obtained using the two different
operating conditions. Unfortunately, loss in retentivity on replacement
Zorbax C-8 columns using phosphate buffer as the mobile phase, precluded the
use of this eluant in obtaining corroborative information on other samples.
Absorbance ratio values of sample to reference compound equal to 1 re-
presented a perfect correlation of the unknown compound to the reference
material. Values above or below 1 were attributable to increased or decreased
absorbance in the unknown material arising from the presence of coeluting
compounds.
Table 30 lists the samples which did not result in identifiable chroma-
tographic peaks, In some cases only one large unresolved group of compounds,
rapidly eluting from the column, was observed. Other chromatograms contained
distinct peaks which did not coincide with the retention positions of any of
the reference nitrogenous compounds (Figures 75 to 82).
147
-------
«c
>
V
m
e
a
n
350
300
Wav«l*ngth (nm)
250
Figure 83. Middleton Pond, Danvers, MA (raw; x 1,000; 7-11-78). Phosphate
buffered eluant, suspected compound: uracil.
148
-------
9
e
300
Wav*l«ngth (ran)
250
Figure 84. Middleton Pond, Danvers, MA (raw; x 1,000; 7-11-78). Phosphate
buffered eluant, suspected compound: 5-chlorouracil.
149
-------
E
or
•
o
o
350
3OO
Wavelength (ntn)
250
Figure 85.
Middleton Pond, Danvers, MA (raw; x 1,000; 7-11-78). Phosphate
buffered eluant, suspected compound: thymine.
150
-------
<
II
9
•
e
^
9
n
350
3OO
Wavelength (nm)
I
25O
Figure 86. Middleton Pond, Danvers, MA (raw; x 1,000; 7-11-78). Phosphate
buffered eluant, suspected compound: adenine.
151
-------
c
je
0)
•
e
a
o
300 25O 200
Wavelength (nm )
Middleton Pond, Danvers, MA (raw; x 1,000; 7-11-78). Phosphate
buffered eluant, suspected compound: purine.
350
Figure 87.
152
-------
c
jc
9
•
e
^
er
380
300
250
I
2OO
Wavelength (nm)
Figure 88. Middleton Pond, Danvers, MA (raw; x 1,000; 7-11-78)
buffered eluant, suspected compound: uracil.
Borate
153
-------
V
•
e
•*
o-
350
300
Wavelength (nm)
2 SO
Figure 89. Middleton Pond, Danvers, MA (raw; x 1,000; 7-11-78)
buffered eluant, suspected compound: thymine.
Borate
154
-------
9
e
•4
V
n
350
3OO
250
Wav«l*ngth (nm
Figure 90. Middleton Pond, Danvers, MA (raw; x 1,000; 7-11-78)
buffered eluant, suspected compound: adenine.
Borate
155
-------
c
je
e
^
a
•
9
3 SO
300
250
Wavelength (nm )
Figure 91. Middleton Pond, Danvers, MA (raw; x 1,000; 7-11-78)
buffered eluant, suspected compound: purine.
Borate
156
-------
o-
e
^
o-
a
9
n
300
Wavelength
250
(nm)
Figure 92. Middleton Pond, Danvers, MA (resin filtered; x 1,000; 7-11-78)
Borate buffered eluant, suspected compound: guanine.
157
-------
«<
e
•*
cr
300 250
Wavelength (nm)
200
Figure 93. Middleton Pond, Danvers, MA (resin filtered; x 1,000; 7-11-78)
Borate buffered eluant, suspected compound: thymine.
158
-------
350
3OO
Wavelength (nm)
250
Figure 94. Middleton Pond, Danvers, MA (resin filtered; x 1,000; 7-11-78)
Borate buffered eluant, suspected compound: uracil.
159
-------
e
•*
a
3
O
350
300
Wavelength (nm)
250
200
Figure 95. Concord River, Billerica, MA (resin filtered, x 2,000, 6-9-78)
Borate buffered eluant, suspected compound: guanine.
160
-------
e
•»
v
•
350
300
W«v«l*ngth (nm)
250
Figure 96. Concord River, Billerica, MA (resin filtered; x 1,000; 7-4-78)
Borate buffered eluant, suspected compound: uracil.
161
-------
cr
t>
3
n
350
300 250
Wavelength (nm)
200
Figure 97. Concord River, Billerica, MA (resin filtered; x 1,000; 7-4-78)
Borate buffered eluant, suspected compound: guanine.
162
-------
a-
o>
e
n
0
350
300
Wavelength (nm)
Figure 98. Concord River, Billerica, MA (resin filtered; x 1,000; 7-4-78)
Borate buffered eluant, suspected compound: adenine.
163
-------
a
•
o
^
a
a
r>
350
300
Wavelength
250
(nm )
Figure 99. Anabaena flos aquae (resin filtered; x 1,615). Phosphate
buffered eluant, suspected compound: cytosine.
164
-------
350
Figure 100.
Wavelength (nm )
Anabaena flos aquae (resin filtered; x 1,615)
buffered eluant, suspected compound: uracil.
Phosphate
165
-------
o-
•
o
^
V
01
3
O
360
300
Wavelength (nm)
250
Figure 101. Anabaena flos aquae (resin filtered; x 1,615). Phosphate
buffered eluant, suspected compound: 5-chlorouracil.
166
-------
B
9
O
350
300
Wavelength (nm)
250
Figure 102.
Anabaena flos aquae (resin filtered; x 1,615).
buffered eluant, suspected compound: pyrrole.
167
Phosphate
-------
a»
«
e
3
n
I
350
Figure 103.
300
Wavelength (nm)
I
2SO
Andbaena flos aquae (resin filtered; x 1,615). Phosphate
buffered eluant, suspected compound: tryptophan.
168
-------
9
e
^
v
•
a
n
350
300
Wavelength
250
200
(nm )
Figure 104. Andbaena flos aquae (resin filtered; x 1,615).
buffered eluant, suspected compound: thymine.
Phosphate
169
-------
c
5
|
e
350
300
W«v«l«ngih (nm)
250
200
Figure 105. Anabaena flos aquae (raw; x 1,750).
suspected compound: tyrosine.
170
Borate buffered eluant,
-------
350
3OO
Figure 106.
Wavelength (nm)
Andbaena flos aquae (raw; x 1,750)
suspected compound: uracil.
250
Borate buffered eluant,
171
-------
0-
•
e
.
aao
3OO
Wavelength (nm
250
Figure 107. Osaillatoria tenuis (resin filtered; x 1,630)
eluant, suspected compound: adenine.
Borate buffered
172
-------
c
'<
e
9
A
350
300
Wavelengt h CnitO
250
Figure 108. Osaillatoria tenuis (resin filtered; x 1,630)
eluant, suspected compound: purine.
Borate buffered
173
-------
tt
V
«
o
t»
3
n
3 SO
3OO
2 SO
Wavelength (nm)
Figure 109. Osoillatoria tennis (raw; x 1,860)
suspected compound: uracil.
Borate buffered eluant,
174
-------
TABLE 29. U.V. ADSORBANCE DATA FOR COMPOUNDS RESOLVED ON ZORBAX C8 USING 0.05 M PHOSPHATE
OR .05 M BORATE BUFFERED ELUANT TO MeOH GRADIENTS
I.
la.
II.
III.
suspected compound
similarity similarity . c
to reference to reference peak
Xpeak ""PO"^3 *trough compound Atrough
Mlddleton Pond, Danvera, MA, raw, 7/11/78
uracil
5-chlorouracll
thymlne
adenine
adenine
purlne
Mlddleton Pond, Danvers,
uracll
thymtne
adenine
purlne
Mlddleton Pond, Danvera,
guanlne
thymlne
uracil
Middleton Pond, Danvera,
cytoalne
adenine
260 444b
273 444
261 444
262 444
256 444
267 444
MA. raw. 7/11/78
256 444
261 444
260 444
261 44
(phosphate buffered
231
250
230
232
232
245
eluant)
444
4
444
444
444
0
1.30
1.57
-
2.11
-
1.58
/A eflk v C
* trough'aample
* trough'reference
0.33
0.48
-
0.43
-
0.32
A220C
A233 (
0.87
1.48
1.55
1.86
1.61
-
'A220\C
lA233'aample
'A220\
kA233^reference
0.67
0.52
0.70
1.29
1.12
-
(borate buffered eluant)
234
231
247
230
MA. resin filtered. 7/11/78 (borate
276 & 250 444
260 444
253 44
MA, entralnment
263 444
260 44+
263 & 230
230
227
444
444
0
444
1.60
3.0
—
2.36
0.55
1.03
—
0.56
1.24
1.14
—
1.0
0.95
0.50
-
0.42
buffered eluant)
444
444
444
precipitation method, 7/11/78
247
230
444
444
-
2.87
1.7
(phoaphate
1.07
1.88
-
0.99
0.59
buffered eluant)
0.77
0.58
0.92
1.67
1.3
1.83
1.52
0.66
0.73
0.97
1.36
1.05
(continued)
-------
TABLE 29 (continued)
•uapected compound
W
similarity similarity
to reference to reference
compound * trough COBP°un«'
/Ape.k \ C /A220\ C
^trough 'sample „ ^A233'samDle
Apeak c
trough
IV. Concord River. Blllerlca, MA, resin filtered. 6/9/78 (borate buffered eluant)
guanlne 272 & 241 +++ 264 & 231 -H-+
V. Concord River. Billerlca, MA. resin filtered, 7/4/78 (borate buffered eluant)
uracil 257 -»-H- 243 -H-f 1.15
guanlne 279 & 249 +++ 262 & 227 -H-f
adenine 262 -H-f 252 0
VI.
VII.
VIII.
IX.
(Vak \ A220
^At rougher aference A233
0.36
1.15
1.70
1.92
2.02
fA220\
^A233/referenM
0.82
1.31
1.36
1.56
Anabaena flos aquae, reain filtered (phosphate buffered eluant)
cytoslne
uracil
5-chlorouracll
pyrrole
tryptophan
thymine
Anabaena floa aquae.
tyroslne
uracil
Osclllatoria tenuis.
adenine
purlne
Osclllatoria tenula.
uracil
a see Table 27
b
++* - + 5 ra
268
257
272
flat
273
266
raw (borata
272
260
•H-f 250 ++4-
•H-f 229 4-H-
•H-f 243 -H-f
- .
•H- 256 0
•H+ 239 ++
buffered eluant)
•H-f 260 0
•H-f 233 -ff*
1.13
3.21
1.10
_
1.07
1.36
—
2.33
0.81
0.79
0.33
_
0.40
0.41
—
0.80
2.33
1.52
1.85
2.04
2.12
2.27
2.04
1.73
1.19
0.64
0.33
0.97
0.99
1.57
realn filtered, x 1630 (borate buffered eluant)
259
271
raw, x 1860
- 256
•H-f 245 0
•H-f 242 +
(borate buffered eluant)
•H+ 237 -f
_
1.82
1.29
_
0.46
0.44
2.03
1.79
2.33
1.56
0.74
1.79
++ - ± 7 nm
•f - + 10 nn
0 •
CA - absorbance value at wavelength shown
-------
TABLE 30. SAMPLES RESULTING IN EITHER POORLY RESOLVED
OR UNIDENTIFIABLE CHROMATOGRAPHIC PEAKS
I. Samples concentrated by low temperature distillation followed by lyo-
philization:
1. Surface impoundment, Bethesda, Ohio (resin-filtered)
2. Middleton Pond, Danvers, MA, 6/21/78 (resin-filtered at both pH 2.0
and pH 7.0)
3. Merrimack River, Lawrence, MA, 6/14/78 (resin-filtered)
All of the samples exhibited a large unresolved group of compounds
rapidly eluting from the column. A corresponding fluorescamine-fluorescence
peak was frequently observed. Underivatized fluorescence for this rapidly
eluting group of compounds was substantially less than the corresponding
derivatized fluorescence indicating the presence of primary amine compounds
in the concentrated samples. The total concentration of this group of
compounds was later calculated to be greater than about an equivalent of
40 mg/L of glycine. Retention data of reference amino acid compounds (Table
25) additionally suggested that this rapidly eluting group of compounds may
have consisted of primary amine materials.
The chromatograms display both a U.V. and fluorescence trace for most
samples. The U.V. ordinate is in absorbance units. A full scale reading is
equal to the sensitivity value reported in each chromatogram. The fluor-
escence ordinate is calibrated by both a sensitivity and range setting.
Fluorescence settings equal to a sensitivity of 40.0% and 45.0% and a range
of 0.2 for each, resulted in a full scale reading for 36 mg/L and 12 mg/L
fluorescamine derivatized glycine, respectively. Every 10% increase in the
sensitivity setting approximately doubled the fluorometric sensitivity.
The recording pens for the U.V. and fluorescence traces are displaced
from each other by 1.3 cm as indicated on the abscissa of the chromatograms.
Corresponding U.V. and fluorescence peaks are separated by only 1.2 cm
because of the lag between the U.V. and fluorescence detectors.
Chromatograms of raw and resin-filtered samples are included for most
sites. The same compounds were identified at some sites in both the raw and
resin-filtered samples, while other compounds were observed in only one of
the differently treated samples. No significant improvement in the chroma-
tographic baseline was observed for any of the resin-filtered samples.
Table 31 shows the concentrations of organic nitrogen compounds identi-
fied in the concentrated field and laboratory samples. Concentrations were
calculated from peak heights of known concentrations of reference compounds
according to the equation:
177
-------
TABLE 31. CONCENTRATIONS OF ORGANIC NITROGEN COMPOUNDS IDENTIFIED IN CONCENTRATED FIELD
AND LABORATORY SAMPLES3
oo
Mlddleton
Pondb,
Danvers,
MA (raw)
Compound 7/11/78
adenlne 860 (445)
5-chlorouracll 60 (11)
cytosine
guanlne
purlne 200 (93)
pyrrole
thymlne 240 (S3)
tryptophan
tyros Ine
uracll 250 (62)
organic nitro- .
gen (pg N/L) 21.7 x 1(T
Z of organic 3.1
nitrogen identi-
fied In sample
Mlddleton
PomP,
Danvers,
MA (resin
filtered)
7/11/78
190 (88)
90 (20)
170 (42)
21.3 x 103
0.7
Pond b,
Danvers, MA
(entralnment
precipitation
method
7/11/78
90 (46)
20 (8)
—
Concord
River
Blllerlca,
MA (resin
filtered)
6/9/78
140 (64)
0.34 x 103
18.8
Concord
River,
Blllerlca Anobaena
MA (raw) jloA aqu&e.
7/4/78 (raw)
150 (77)
170(79)
340 (26)
60 (15) 100 (25)
1.84 x 103 2.24 x 103
9.3 2.3
Anobaena OidLUatoiua.
j£o4 aquae OtcAJUatoiua, temuA
(resin leniLU (resin
filtered) (raw) filtered)
40 (21)
10 (2)
40 (16)
40 (19)
160 (33)
40 (9)
30 (4)
80 (20) 20 (5)
1.60 x 103 1.78 x 103 1.31 x 103
6.2 0.2 3.1
Values out of parentheses Indicate compound concentrations as ug/L
Values in parentheses are calculated organic nitrogen concentrations (as pg N/L)
Sampled during the occurrence of blue-green algal bloom
-------
concentration
of suspected
compound
peak height of
suspected com-
pound at 220 run
(in chart units)
absorbance units
chart unit
absorbance units
at 220 nm per
mg/L reference
compound
(10)
Absorbance values (220 nm) per mg/L of reference compound for materials
separated using either the phosphate or borate buffered eluant are shown in
Tables 32 (see page 180) and 33, respectively.
TABLE 33. VALUES FOR CONVERTING PEAK HEIGHTS OF NITROGENOUS COMPOUNDS
RESOLVED ON ZORBAX C8 TO CONCENTRATION IN mg/L (BORATE BUFFERED ELUANT)
Compound
concentration
(mg/L)
absorbance at 220 nm
(absorbance units)
absorbance at 220 nm
per mg/L of compound
(absorbance units)3
barbituric acid
£-histidine
cytosine
5-chlorouracil
uracil
creatinine
guanine
thymine
pyrrole
purine
adeine
tryptophan
pyrimidine
tyrosine
20
610
15
60
10
10
50
75
100
75
100
50
40
20
0.150
0.230
0.100
0.700
0.060
0.140
0.086
0.280
0.090
0.230
0.170
0.220
0.250
0.056
.0075
.0004
.0067
.0117
.0060
.0140
.0017
.0037
.0009
.0031
.0017
, .0044
.0063
.0028
^Absorbance at 220 nm per mg/L of compound
[absorbance at 220 nm/concen-
tration of compound (mg/L)]
Middleton Pond, sampled during the occurrence of a blue-green algal
bloom (Figures 58 to 63) and the laboratory grown culture of Anabaena flos
aquae (Figures 72 to 74) exhibited the greatest number of identifiable nitro-
genous compounds. 5-chlorouracil, thymine, and uracil were identified in
both of these samples. Adenine, guanine, and purine were identified in
Middleton Pond and not in the Anabaena sample, while the reverse was found
for cytosine, pyrrole, and tryptophan.
Comparison of raw and resin-filtered samples at the same collection
sites, showed that both different and some of the same compounds were iden-
tified in the dissimilarly treated samples. One treatment did not appear to
favor the identification of a larger number of compounds. The total number
179
-------
TABLE 32. VALUES FOR CONVERTING PEAK HELGHTS OF NITROGENOUS COMPOUNDS RESOLVED ON ZORBAX C8
TO CONCENTRATION IN rag/L (PHOSPHATE BUFFERED ELUANT)
oo
o
concentration
compound (mg/L)
barbituric acid
/-histidine
cytosine
creatinlne
guanine
5-chlorouracil
thymine
pyrrole
purine
tryptophan
pyrlmidine
indole
20
600
10
in
50
50
75
100
75
50
40
90
Absorbance at 233 nm
(absorbance units)
0.066
0.074
0.072
0.066
0.068
0.058
0.084
0.062
0.108
0.144
0.102
0.086
absorbance at 220 ran3
absorbance at 233 nm
1.22
1.72
1.35
0.66
1.28
2.87
2.23
5.13
0.82
6.09
0.36
8.40
absorbance at
220 nm per mg/L
of compound
(absorbance unit)"
.0040
.0002
.0097
.0044
.0006
.0033
.0025
.0032
.0012
.0175
.0009
.0080
aAbsorbance ratios were determined from U.V. spectral scans of reference compounds. (Figures 17
to 30)
Absorbance at 220 nm per mg/L of compound = [(absorbance at 233 nm) x (absorbance at 220 nm/
absorbance at 233 nm)]/(concentration of compound in
mg/L)
-------
of compounds identified at a given collection site was maximized by analyzing
both raw and resin-filtered samples.
Concentrations of organic nitrogen compounds ranged from 860 yg/L of
adenine in Middleton Pond (7/11/78) to 10 yg/L of 5-chlorouracil in the raw
laboratory grown culture of Anabaena flos aquae. The average values for the
compounds identified in the water supply samples were 367, 60, 20, 167, 200,
110, and 160 yg/L of compounds, for adenine, 5-chlorouracil, cytosine,
guanine, purine, thymine and uracil, respectively. Uracil, adenine and
guanine were found most frequently in the water supply samples while 5-chloro-
uracil, cytosine and purine were only encountered once in these sources. A
total of 7 N-organic compounds were identified in the water supplies while 9
compounds were found in the laboratory grown blue-green algal cultures.
Pyrrole, tryptophan and tyrosine were found in the algal cultures and not in
the water supplies. Guanine was found in the water supplies but not in the
algal cultures.
The percent of organic nitrogen identified in each sample was calculated
according to the equation:
sum of organic nitrogen concentrations
of individual compounds identified in
% of organic nitrogen _ sample (yg N/L) ,,,-
identified in sample total organic nitrogen (yg/L)
determined in sample
The total organic nitrogen was determined by Kjeldahl-N analysis. Organic
nitrogen concentrations of individual compounds identified in the samples
were calculated according to the equation:
organic nitrogen
concentration of „ , ..
. ,. . , , , ,/T yM compound v yM nitrogen
individual compound = yg compound/L * — c -r x -^ °—T
identified in ^ comP°und ^M compound
sample (yg N/L) , .
x 14 ug nitrogen (12)
yM nitrogen
The percent of organic nitrogen identified in the water supplies and blue-
green algal cultures ranged from 0.7% to 18.8% (mean= 8%) and 0.2% to 6.3%
(mean= 3%), respectively. The majority of organic nitrogen in these samples,
therefore, was not characterized.
The types of compounds identified included: amino acids (tyrosine and
tryptophan), nucleic acid bases (adenine, cytosine, guanine, and uracil),
purines (adenine, guanine, purine), and pyrimidines (cytosine, thymine, and
uracil). Three of these substances, uracil, pyrrole, and tryptophan, were
previously shown to be precursors for the formation of trihalomethanes (89,
38). Purines and pyrimidines were shown to be intermediates for compounds
181
-------
causing mutagenic activity in finished waters upon chlorination (90-96, 64).
5-chlorouracil was shown to be mutagenic (30,97).
Significance of Findings
The types and concentrations of compounds identified in municipal water
supplies are environmentally significant because of their ability to yield
interference or false positive tests in determining free chlorine, and to be
precursors of trihalomethanes during chlorination. Several trihalomethane
precursors were found in the municipal water supply samples and in laboratory-
grown blue-green algal cultures: pyrrole, thymine, tryptophan, and uracil.
The molar yield of chloroform of these materials was previously determined in
laboratory studies at pH 7 by Baum (10) and are shown in Table 34. Molar
yield is defined as: (moles of chloroform formed)/(moles of compound used).
TABLE 34. HIGHEST CONCENTRATION OF CHLOROFORM PRODUCED AT pH 7a
compound time (hours) molar yield of CHC1_(%)
pyrrole
thymine
tryptophan
tryptophan (pH=7.5)d
uracil
0.3
3.4
24.0
7.0
0.7
0.7
0.5
7.8
17.9
0.6
a —5
Initial chlorine concentration = 9.0x10 M; HOCl/compound ratio = 9:1
except where noted; after Baum (10).
b% molar yield of CHC1, = ^oles °* chloroform formed) x WQ
3 (moles of compound used)
c —5
Initial chlorine concentration = 10x10 M; HOCl/compound ratio = 10:1
Initial chlorine concentration = 10 x io~ M; HOCl/compound ratio =20:1
Table 35 shows the calculated levels of chloroform produced by chlori-
nation of these compounds in samples studied. It was assumed that the in-
organic compounds yielded chloroform according to the values shown in Table
34, and that they would not be removed prior to chlorination of the water
supplies. The calculated total production of chloroform for all the samples
was well below the proposed maximum contaminant level of 0.1 mg/L (100 ppb)
for total trihalogenated methanes (73).
The molar yield of chloroform, however, is highly pH dependent. The
molar yield of tryptophan, for example, increases from 7.8% to pH 7, to 17.9%
at pH 7.5, 24% at pH 8, and about 31% at pH 9. The molar yield of uracil
182
-------
TABLE 35. CALCULATED LEVELS OF CHC1, PRODUCED IN WATER SAMPLES CONTAINING
N-ORGANIC SUBSTANCES UPON CHLORINATION (AT pH 7)a
site
Middleton Pond
Danvers, MA
7/11/78
compound
thymine
uracil
concentration
(UM/L)
1.90
2.23
chloroform
produced^5
(uM/L)
0.010
0.013
chloroform
produced
(ug/D
1.26
1.46
Concord River
Billerica, MA
7/4/78
uracil
Andbaena flos aquae uracil
culture tryptophan
Osci.'llatoria tenu-is uracil
culture
0.54
0.89
0.15
0.18
0.003
0.005
0.011
0.001
0.36
0.60
2.34
0.12
Proposed maximum contaminant level = 100 Mg/L chloroform.
'Chloroform produced (pM/L) = concentration (yM/L) * molar yield of CHC13 (%)
increases from 0.6% at pH 7 to about 8% at pH 8 and about 40% at pH 9. One
hundred percent molar yield of chloroform is obtained for uracil above about
pH 10. The molar yield for pyrrole increases (after about 24 hours contact
time) from 0.5% at pH 7.5 to 7.6% at pH 9.1. Some of these values were
obtained by allowing mixtures to react at neutral or slightly acidic pH values
for several hours with subsequent increase of pH by addition of NaOH. The
reader is referred to Baum's thesis (10) for a full discussion of these
reactions. The chloroform which could be produced by the N-organic compounds
present in the water supplies and laboratory blue-green algal cultures under
more alkaline conditions are shown in Table 36. The calculated levels of
chloroform formed under the more alkaline conditions in the Middleton Pond
and Concord River samples are more than 10% of the proposed maximum conta-
minant level for total trihalomethanes and are therefore significant.
Additional N-organic compounds not identified in the dissolved N-organic
fraction of these samples might additionally contribute to the level of CHCla
production. The increased molar yield of CHCls in alkaline waters stresses
the need to carefully monitor the pH of water supplies in treatment facilities
and to provide increased organic contaminant removal capabilities in alkaline
waters.
The significance of the levels of N-organic materials identified in the
water supplies can also be evaluated by calculating the amount of combined
chlorine which may be formed during chlorination. As discussed previously,
183
-------
TABLE 36. CALCULATED LEVELS OF CHCl^ PRODUCED IN WATER SUPPLIES CONTAINING
N-ORGANIC SUBSTANCES UPON CHLORINATION (AT HIGHER pH VALUES
chloroform produced
site compound (yg/L)
Q
Middleton Pond thymine NC
Danvers, MA uracil 20.0 (pH 8)
7/11/78 99.9 (pH 9)
Concord River uracil 4.8 (pH 8)
Billerica, MA 24-2
7/4/78
Anabaena flos aquae
culture
Oscillatoria tenu-is
culture
uracil
tryptophan
uracil
8.0 (pH 8)
39.9 (pH 9)
5.5 (pH 7.5)
7.4 (pH 8)
9.5 (pH 9)
1.6 (pH 8)
8.1 (pH 9)
a
Proposed maximum contaminant level = 100 yg/L chloroform
b
Chloroform produced (yg/L)
concentration of compound
(uM/L) J
[molar yield of CHCl-j atl x yg compound
[particular pH value (%)] yM compound
CNC: not calculated because data on % chloroform yield at higher pH values
was unavailable.
combined chlorine is a much less active disinfectant than free chlorine. The
combined forms, however, tend to react similarly with many analytical reagents
for active chlorine. When these N-chloro compounds are formed, tests for free
chlorine may indicate a non-existent bactericidal or virucidal behavior.
Table 37 shows the calculated values of combined chlorine that could be
formed during chlorination. The total conversion of N-organic constituents
to combined forms and a 1:1 molar reaction are assumed.
Chlorine residuals of 0.2 to 1 mg/L after 15 to 30 minues of contact
time generally result in 99.9% destruction of Escheriohia ooli (1) (an in-
dicator species for pathogenic bacteria) in drinking water. A 15 minute free
chlorine residual of 0.5 mg/L is generally taken as an acceptable level of
disinfection (1). If this residual is comprised of the less germicidal
combined forms, however, the water supply may not be hygienically safe. The
calculated levels of combined forms of chlorine yielding falsely positive
184
-------
TABLE 37. CALCULATED VALUES OF COMBINED CHLORINE FORMED UPON CHLORINATION
sample
compound
molar concentration
(yM/L)
Billerica, MA
6/9/78
Concord River
Billerica, MA
7/4/78
adenine
guanine
uracil
1.11
1.12
0.54
active chlorine
concentration3
(mg/L)
Middle ton Pond
Danvers , MA
7/11/78)
Concord River
adenine
5-chlorouracil
purine
thymine
uracil
total ,
guanine
6.36
0.41
1.67
1.90
2.23
0.93
0.452
0.029
0.119
0.135
0.158
0.982
0.066
0.79
0.80
0.38
total 0.197
active chlorine concentration =
[concentration of compound (yM/L)] x
71 pg chlorine determined
as free aqueous chlorine
yM combined chlorine
x 10~ mg/pg
185
-------
tests for free aqueous chlorine in the Middleton Pond sample and in one
sample from the Concord River were calculated to be 0.982 and 0.197 mg/L
free aqueous chlorine, respectively. Assumed free chlorine residuals of
0.5 mg/L in the finished drinking water from these sources, then, might not
provide adequate disinfection, since a significant portion of this value may
be comprised of the less germicidal combined forms.
The finding of trihalomethane (THM) precursors at levels which could
produce significant amounts of chloroform upon chlorination under alkaline
pH conditions, and the demonstration that the N-organic constituents iden-
tified could lead to false positive tests for free chlorine stresses the
environmental significance of the findings of this study. A subcommittee of
the National Research Council recently reported (98) that it was virtually
impossible for researchers to establish a link between THM's in drinking
water and an increase in human cancer because of the inherent complexities
of such epidemiological studies. It did not, however, refute the possible
causal relationship between carcinogenesis in humans and the presence of
THM's and other carcinogens in water supplies. A study by Cantor and McCabe
(99) supported the suspicion that a real link existed between organic con-
taminants in drinking water supplies and cancer rates in the human population.
The study fell short, however, of providing the association. The presence of
N-organic compounds in water supplies, from the standpoint of human health,
exertion of chlorine demand, reaction with chlorine to form less bactericidal
and virucidal chloramines, production of objectionable tastes, and direct
carcinogenic and mutagenic effects is therefore significant.
This study also raises concern about the potability of finished water
during occurrences of summer blue-green algal blooms. Aside from the taste
and odors associated with such occurrences, high levels of N-organic material
released by these algae, could result in increased THM formation as well as
combined N-chloro forms yielding falsely positive tests for free chlorine.
During the occurrence of such algal blooms, superintendents of treatment
facilities should be advised to carefully monitor the pH of the water during
chlorination and to provide increased organic contaminant removal capabilities
in alkaline waters where THM formation is more favored. Removal of organic
contaminants could be achieved by use of resins which selectively remove
organic contaminants or conventional methods such as carbon adsorption, or
chemical coagulation. Because all of the methods currently used to measure
free chlorine are subject to interference from organic chloramines, enumera-
tion of bacteria after chlorination should be examined closely in addition
to maintaining a chlorine residual somewhat greater than what is normally
acceptable, to ensure a sufficiently disinfected water.
186
-------
SECTION 5
REACTIONS BETWEEN NITROGENOUS ORGANIC COMPOUNDS AND AQUEOUS CHLORINE
A broad spectrum of nitrogenous compounds at concentrations in the range
of milligrams per liter were examined for their reactions with aqueous
chlorine in dilute aqueous solution. The major types of reactivity studied
were: (1) chlorine demand, as a measure of the occurrence of either redox
processes or the formation of chlorinated organic compounds; (2) chloramine
formation as shown by alteration in the reactions of the residual available
chlorine with selective analytical reagents; (3) details of reactions as
shown by changes in the ultraviolet spectra of mixtures of the compounds with
aqueous chlorine; and (4) analysis of appropriate reaction mixtures for
formation of halogenated methanes or other volatile chlorinated compounds
resulting from the chlorination of nitrogenous materials.
Selected nitrogenous organic compounds were assessed during a series of
screening experiments using chlorine demand and ultraviolet spectrophoto-
metric scans to detect the formation of chlorinated derivatives. The chlorine
demand exerted by these compounds was most significant as shown in Table 38.
All of these nitrogenous compounds exhibited substantial chlorine demand at
concentrations in the range of 1-10 mg per liter within several hours of
contact, indicating the importance of nitrogenous materials with respect to
the total organic content of natural waters. Although nitrogenous materials
may constitute only 5% of the organic matter in a natural water, for example,
they may account for 25% of the demand.
The potential of selected compounds as precursors to chloroform formation
was also investigated. Initial experiments were carried out near pH 7 with
concentrations of aqueous chlorine typically less than 7 milligrams per liter.
The amount of chloroform produced as a function of time was determined for
selected compounds, at pH values between 6 and 11. Studies were also made to
determine the effect of increasing the pH of reaction mixtures after they had
been allowed to react at an initial pH value for several hours.
ANALYTICAL METHODS
The concentrations of chlorine solutions and organic reagents typically
utilized in the experiments were 10"1* molar or less. Frequently, studies
were carried out with concentrations as small as one micromole per liter.
Because of this, scrupulous care had to be taken to diminish the likelihood
of contamination during the preparation of reagents. Furthermore, additional
precautions had to be taken throughout the analyses to reduce or eliminate
errors due to extraneous substances or specific problems inherent in some of
the analytical techniques. Routine laboratory operations were carried out con-
sistently according to the following procedure.
187
-------
TABLE 38. NITROGENOUS COMPOUNDS UNDER INVESTIGATION
Compound
Adenine
Alanine
m-Aminophenol
Arginine
Aspartic Acid
Barbituric Acid
Caffeine
Creatinine
Cyanuric Acid
Cytosine
Glutamic Acid
Glycylglycine
Histidine
Indole
Phenylalanine
Proline
Purine
Pyrrole
Sarcosine
Succinimide
Thymine
Tryptophan
Uracil
Uric Acid
Tyrosine
N-Chlor
Formation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Chlorine
Demand3
+ 3
+ 1.5
+ 5
+ 2.5
+ 3
+ 4
0
+ 1 (v.s.)
0
+ 2
+ 2
0
+ 2
+ 8
+ 2.5
+ 1.5
0
+ 5.5
0
0
+ 3
+13
+ 3
+ 3
+ 3
U.V. Spectrum
Study
X
X
X
X
X
X
X
X
X
X
X
a
A plus sign indicates demand greater than one mole of chlorine per mole of
compound after several hours of reaction with excess chlorine. Numbers
give the moles of chlorine demand per mole of substrate after 5 to 10 hours
reaction time with excess chlorine present.
Glassware
Prior to initial use, all glassware was thoroughly cleaned using
laboratory detergent, rinsing with dilute HC1, and final rinsing with
distilled, chlorine-demand-free water. Spectrophotometric cells were
periodically soaked in a solution of 50% ethanol and 50% 3N HC1 prior to a
distilled water rinse.
188
-------
Reagent Preparation and Analytical Procedure
Chloride-Free-HOCl Solutions —
In order to minimize the loss of HOC1, concentrated aqueous chlorine
stock solutions were prepared under a hood by bubbling chlorine gas from a
sintered-glass diffuser into a 2 liter Erlenmeyer flask containing distilled
water until a chartreuse color was attained. After neutralization to about
pH 6 with concentrated sodium hydroxide, the chlorine solution was distilled
in a rotary vacuum distiller at 30-35°C. The condensate (chloride-free) was
stored under refrigeration in low-actinic, Pyrex-glass bottles. This con-
centrated stock solution, which remained stable for months, was used to
prepare all aqueous chlorine (HOC1) solutions.
Buffer — To simulate conditions encountered in natural systems, a bicar-
bonate C02 buffer (pH approximately 7.0) was employed. Two methods generally
were used to prepare buffer solutions. In the first dilute HC1 (and, if
necessary, dilute NaOH) was used to adjust the pH of 10~3 M (1.0 mM) NaHC03
solution to 7. In the second method carbon dioxide gas was bubbled through
the 10~3 M (1.0 mM) NaHC03 until pH 7 was obtained. With the latter tech-
nique, a high background concentration of chloride was avoided.
Phosphate and Borax buffers, shown below, were used to achieve pH values
between 5 and 10.
5.91 KHP0 (6-OOx 10~4M); NaHP0 (8.38xlO~M)
6.98 KHP0 (2.67 x 1Q~4M); NaHP0 (5.02xlO~4M)
7.73 KH2P04 (6.69 x 10"5M); Na2HP04 (7.53xlO~4M)
9.50 Na2B40? - 10 H20 (.05M)
10 Carbonate/bicarbonate buffer
All buffers were prepared with chlorine-demand-free distilled water and
stored in glass carbons.
CHLOROFORM STANDARDS
Standard solutions of chloroform for the calibration of the gas chroma-
tographic equipment were prepared by dissolving 1 ml of chloroform in 20 ml
of 9.5% ethanol and diluting to one liter with distilled water. After
mixing, 5 ml of this solution was diluted to 250 ml with distilled water,
followed by further dilution of 5 ml to one liter with distilled water.
This produced a chloroform standard that contained 148 mg per liter of
chloroform. Screw cap vaccine vials (25 ml) were filled to overflowing with
portions of this final solution, capped with Tuf-Bond (making sure that no
air bubbles were formed), and placed in the refrigerator until use. Fresh
standards were prepared in this manner at least once a weak.
189
-------
Equipment
Spectrophotometers—
A Beckman DU spectrophotometer was used for the various colorimetric
determinations of free available chlorine. Analyses were carried out in
the visible wavelength range with matched one cm cells using distilled water
as the reference solution.
A Beckman DK-2 spectrophotometer was used to monitor the ultraviolet
absorption of aqueous solutions of individual compounds investigated prior
to and during contact with aqueous chlorine. Distilled water was used as
the reference solution.
Gas Chromatographs—
Gas chromatographic measurements of concentrations of chloroform pro-
duced during experiments were carried out at the Lawrence Experiment Station
of the Massachusetts Department of Environmental Quality Engineering in
Lawrence, Massachusetts. The instrument most frequently used consisted of
a Chromalytics 1047 Concentrator, Tracer 222 gas chromatograph, and Tracer
310 Hall electrolytic conductivity detector. A second chromatograph, the
programmable Tracer 560 equipped with a Tekmar Liquid Sample Concentrator
(model LSC-1) and a Tracer 700 Hall electrolytic conductivity detector, was
used periodically. Although the response of the Tracer 560 was approximately
an order of magnitude more sensitive than that of the 222, the selective
adsorption principle of operation remained the same. In each case, the
volatile components were purged with nitrogen (Matheson cylinder) from a
five-mi aqueous sample and collected on a site-sampling tube packed with
TENAX resins. The tube was then heated to desorb the adsorbed volatiles and
pass them from the first tube to a concentrator U-tube trap again packed with
TENAX. Then the latter tube was heated in a programmed manner from 130°F to
180°F to transfer the volatile materials to the chromatographic column.
This precolumn technique was needed to provide the slug sample required for
gas chromatographic determinations.
Individual compounds were selectively desorbed during the temperature
program and combined with deionized solvent in a gas-liquid contactor. The
Hall detector continuously monitored the electrical conductivity of the
liquid. Thus, the concentrations of the halogenated materials were shown
potentiometrically as a strip chart recorder print-out. Peak heights were
quantitatively calibrated by comparison with standards which were run at
least once each day.
Chlorine-Demand
Chlorine-demand studies were conducted according to the following
general procedure: 0.50-0.20 millimolar of each organic compound was made
up in a 500 ml volumetric flask using 0.01 M NaHC03 solution. A volume of
stock aqueous chlorine solution sufficient to give a molar ratio for chlorine
to organic compound usually about 4 or 5 to one was placed in a second 500 ml
volumetric flask and diluted to the mark with 0.01 M NaHC03.
190
-------
For initiation of reaction the contents of the two flasks were poured
simultaneously into a two-liter reagent bottle of low actinic glass. Samples
(100 ml) were removed from the reagent bottle at suitable times after mixing
and the concentrations of residual chlorine were determined by thiosulfate
titration following addition of iodide and acetic acid using starch as in-
dicator. Some attempts were also made to determine free chlorine by titration
with methyl orange. The results agreed generally with the total residual
chlorine measurements.
Samples from many of the reaction mixtures were placed in spectrophoto-
metric cells and measurements of ultraviolet spectra were made at a number of
times with a Beckman-DK2 spectrophotometer. The proportion of chlorine
concentration remaining (chlorine at time, t/initial chlorine) was computed
at various times.
RESULTS AND DISCUSSION
Chlorine-Demand of N-Organic Compounds
Chlorine-demand provides a direct measure of the overall extent to which
organic matter has been oxidized, and, when combined with differential
chloride determinations, provides a measure of total chloro-organic compound
produced by means of the equation
Cl-demand = Cl -formed + C-C1 bonds formed (13)
Studies concerning the chlorine demand of selected N-organic compounds are
shown graphically in Figures 110 through 121.
The chlorine-demand ratio (CDR) is given by equation (14) :
(C1Q) - (Clt)
Chlorine-demand ratio (CDR) = - ,„ , ^ - (14)
where
Cl = the added initial molar concentration of chlorine in the
reaction mixture
Cl = the molar concentration of chlorine remaining at any time, t
Cpd = the added initial molar concentration of compound in the
reaction mixture.
Alanine, CH3CH(NH2)COOH—
A demand experiment was made on this typical amino acid at a molar ratio
of aqueous chlorine to alanine equal to 4, the initial concentrations being
5 * 10~5 alanine, and 2x10~"M chlorine. The results obtained were:
time, hr. 2 5 7.5 72
demand ratio 1.4 1.5 1.6 1.7
191
-------
3.0
3.o
1.0
0.5
Glut ami c acid
Chlorine
T/ME.
05
,20
ppm
7.36
molar
ratio
i c» CMC'-"V>e.oo*
L-Alanine
Chlorine
Arginine
Chlorine
.05
.02
.20 llf.15
8.71
Figure 110. Chlorine demand of selected N-organic compounds,
192
-------
3-S
3-o
J 5
1-5
1.0
o-s
° I 2
m-Amino phenol
Chlorine
.10 10.9
.80 56.6
/
/
j>f.f^ffy
35 Jt. S? it
molar
ratio
*
Wri,
Creatinino
Chlorine
.20
p.65
o''
Din: thyl -Tiir.o-
benzaldolrrJe
Chlorine
10
28.3
CHO
Figure 111. Chlorine demand of selected N-organic compounds.
193
-------
o
c
tv
7-0
•
(..o
££>
L-Tryptopha-i
Chlorine
Try pi- 6: I
X
X (•
^uisj
1J
ki
^ 2.0
|.<3
0
Ph. flja ^/:' x
A A • « —-" *"
Cfiff.
77*vr
TOT
.20
PPi"
10.2
14-15
molar
ratio
8:1
Phony lalanine
Chlorine
l.k 231.0
5.6 396.0
Caffeine
Chlorine
.05
.20
9.7
Proline
Chlorine
Figure 112,
.05 5.76
28.30
8:1
Chlorine demand of selected N-organic compounds.
194
-------
Barbituric acid
9 _
Chlorine
Indole
• —
Chlorine
.10
.60
12.8
5.86
.20 11;. 15
Xndotff
molar
ratio
6:1
Figure 113. Chlorine demand of selected N-organic compounds,
195
-------
y-s
3.S
3.0
2.S
X-5
/.o
Jo ff (,0 fS 9o /of /
O
'f
-------
3-0
1-5
l-o
0.5
/ 2.
Creatinine
Chlorine
* 9
.20
St. si 6? -Z'f to
molar
ratio
5.65
.15
Phenylalanine
Chlorine
l.if 231.0
5.6 396.0
Dime thy ia^iino-
benzaldehyde
Chlorine
.10
23.30
Figure 115. Chlorine demand of selected N-organic compounds,
197
-------
o.s
0.3
0.2
o.l
i y * /
Creatinine
Chlorine
Caffeine
•
Chlorine
ft to
.10
.10
10.3
7.08
.05 9.7
.20 llj.,15
molar
ratio
1:1
Figure 116. Chlorine demand of selected N-organic compounds.
198
-------
f.
£o
2-0
l.o
A.
X
Trypt-
t:l
-------
J.o
/•S
/.*
O.S
Pyrrole
• -_
Chlorine
Pyrrole
t
Chlorine
.30 20.1
1.50 106.25
.15 10.1
.60 U2.5
molar
ratio
5:1
Figure 118. Chlorine demand of selected N-organic compounds,
200
-------
c
A
rv» - fln*. -Ph.
m.« /}»»». -ph.
3-o
. "Ph. j:
2.0
1-0
HOURS
Figure 119. Chlorine demand of selected N-organic compounds.
201
-------
_ffM_
.10
.50
.10
.1*0
10.9
35A
10.9
28.3
molar
ratio
5:1
U:1
m-Ar.iinophcnol
1
Chlorine
m-Aminophenol
•
Chlorine
m-Aminophenol .10 10.9
'— 3:1
Chlorine .30 21.2lj
m-Aminophenol .10 10.9
2:1
Chlorine .20 1^.15
m-Aminophenol .10 10,9
-n
Chlorine .10 7.08
Figure 119 (continued)
202
-------
O.i
Phsnylanino
Chlorine
MM
.01
.06
S
1.65
I3Tȣ>
molar
ratio
6:1
-Q-sarie as above, deten^.ined by Hethvl Orange nethod
Creatinine
Chlorine
.01
.05
1.13
3.51;
5:1
o-sane as above, detcrnined by Methyl Orange method
Figure 120. Chlorine demand of selected N-organic compounds.
203
-------
/
°r,-<
Figure 121. Chlorine demand of selected N-organic compounds.
-------
molar
ppn ratio
Pyrrole .0072 .l;b
Chlorine ,06 lj.,25
as ;bove, det^r.-.ined by llethyl Cr-:ji6e rr.ethod
?yrrole ^011^ .97
• 7:1
Chlorine .10 7.08
as above, determined by llethyl Orange method
Tyrrole .OIlUj. .97
• 5.7:1
Chlorine .08 5.66
—O-sane as above, as determined by Methyl Orroige method
Figure 121 (continued)
205
-------
(Demands, here and in the other tables, are expressed as moles of
chlorine reduced per initial mole of organic compound.)
The results are reasonably consistent with the equation;
CH3CH(NH2)COOH + 2HOC& -»• CH COCOOH + NH_C£ + H+ + CH~ + HJ3
provided there is some concurrent or subsequent oxidation of ammonia to
nitrogen.
Arginine, H2NCH2CH2CH(NH2)COOH—
This basic amino acid is of interest because of the alternate opportuni-
ties for N-chlor compound formation. An experiment with a molar ratio of
aqueous chlorine to arginine_equal to 4, the initial concentrations being
5 x io~5M arginine and 2 x 10~2M chlorine, gave the following data:
time, hr. ? 5 7.5 72
demand 1.9 2.3 2.4 2.5
The results suggest oxidative deamination at one amino group followed
by breakpoint oxidation of the liberated ammonia. The fact that the behavior
is similar to that of other natural amino acids suggests that this reaction
is occurring at the a-amino group. The suggested reaction, then, is
H2NCH2CH2CH(NH2)COOH+2.5 HOCfc •* H2NCH2CH2COCOOH + ~ NZ + 2.5 Cfc~
+ 2.5 H+ + 1.5 H0 .
Creatinine,
Creatinine, a urinary excretory product, was considered a likely nitro-
genous constituent of waters polluted by animals or man. It has two pro-
spective sites for N-chlor ination, one a ring N-H, amide-like in structure,
the other an amino group.
Three kinetic runs were performed with mixtures of creatinine and aqueous
chlorine, at molar chlorine to creatinine ratios of 1:1, 4:1 and 5:1.
-4 -4
Mixture 1; 1 x 1Q M chlorine; 1 x IQ ^M creatinine
t ime , hr .
demand
Mixture 2: 2 x
time, hr.
demand
Mixture 3 : 5 x
time, hr.
demand
0
0
.1 2
.5 0
10~4M
0
0
.1
.1
10" 5M
0
0
.1
.5
.65
7
0
chlorine;
0
0
.25
.3
1
0
chlorine;
0
0
.25
.8
1
1
.9 25,
.65 0,
5 x
.5
.4
lx
.2
.0
ID"5
3.
0.
It)'5
1.
1.
M
5
7
M
6
3
.5
.75
creatinine
5
0
.8
.85
9.3 57
1.3 3.1
creatinine
5
1
.8
24
3.0
206
-------
Concurrent spectrophotometric observations on the 1:1 and 4:1 mixtures
were not very informative. The 1:1 mixture showed enhanced absorbance in the
region near 230 nm, the wavelength of maximum absorption for creatine. This
might indicate of an N-chloro derivative or oxidation product of creatinine
in which the fundamental absorbing structure had not been changed. The ab-
sorption at 230 nm continued increasing throughout the run for this chlorine
to creatinine ratio. With the 4:1 mixture the increase was less pronounced,
the wavelength of maximum absorption was shifted toward 220 nm and the over-
all absorption began decreasing after about 9 hours of reaction and continued
to do so subsequently throughout the reaction period.
Creatinine thus exhibits a small immediate demand followed by a continued
slow exertion of demand to at least 3 moles of chlorine for each mole of
creatinine. Presumably some oxidized intermediate is formed which is then
subject to additional oxidation slowly in the presence of excess chlorine.
Other than this, no plausible reaction scheme can be proposed at present.
Glutamic Acid, HOOC2CH?CH CH(NH2)COOH—
A single experiment at 4:1 molar ratio of chlorine to glutamic acid, the
concentrations being 2 x 10~'*M and 5 * 10~5M, gave the following results:
time, hr. 2 5 7.5 72
demand 1.8 2.0 2.0 2.0
Although the molar demand is a bit low for completion of the reaction
HOOCCH2CH2CH(NH2)COOH + 2.5 HOC£ -»• HOOCCH2CH2COCOOH +2.5 Cfc~ + 0.5 NZ
+ 2.5 H+ + 1.5 H) ,
this seems the most reasonable mechanism to propose. A corresponding experi
ment with aspartic acid, HOOCCH2CH(NH2)COOH, gave somewhat greater molar
demands, 1.8 after 2 hours, 2.0 after 5 hours and 3.5 at 72 hours. There is
potential chloroform formation from the initial oxaloacetic acid product,
HOOCCH2COCOOH in this case.
Indole, C8H7N—
Indole is one of the substances studied showing strong chlorine demand.
At chlorine to indole ratios up to 4 the chlorine was essentially completely
reduced in an hour or less. The tabulated data are for molar ratios of 4:1
and 8:1, both with 5 x 10~5M indole, the initial chlorine concentrations
being 2 x 10~4M and 4 x 10~"M, respectively.
4:1 molar ratio
time, hrs. 0.1 0.5 1.5 3.5 6.2 25 32
demand 1.1 2.6 3.7 3.9 4.0 4.0 4.0
8:1 molar ratio
time, hrs. 0.1 0.5 2.6 5 9 26
demand 2.0 2.5 4.9 5.5 6.2 7.4
207
-------
Although the reaction of chlorine in these experiments was extensive,
the maximum chlorine demand shown, 7.4 moles of chlorine per mole of indole,
is still far less than the 19.5 moles needed for complete oxidation to COa,
Na and H20. Accordingly, there is good indication of partially oxidized
organic intermediates, but whether these are chlorinated or not cannot be
judged.
Formation of partial oxidation products was also shown visually by the
appearance of yellow colors in both solutions within 0.5 hr. of the start of
reaction that persisted throughout the runs to at least 55 hours with the 4:1
molar ratio.
Spectrophotometrically, both reaction mixtures exhibited initial intensi-
fication of absorption in the 270-280 nm absorption band and a batho chromic
shift of absorption near 220 nm. After 30 minues these maxima gradually
diminished and were replaced by increasing absorption near 250 nm. This ab-
sorption continued to increase for 24 hours in the solution with the molar
ratio equal to 4 and thea became substantially constant. At the molar ratio
of 8, maximum absorption at 250 nm was reached after 80 minutes; thereafter
the absorption decreased continuously throughout the ultraviolet range,
indicating general oxidation to nonabsorbing products.
m-Aminophenol,
This compound is of interest because of its structural similarity to
resorcinol, which Rook (35) found to yield molar quantities of chloroform
when chlorinated. It also possesses structural similarities to some naturally
occurring aromatic amines. Because ingested amines may react in the stomach
with nitrite from saliva to give nitrosamines, the general destructiveness of
aqueous chlorine toward amines with consequent elimination of carcinogenic
potential is noteworthy as a contrast to the general nonreactivity of chlorine
dioxide and ozone towards amines and amides as a class.
A series of experiments was conducted with aqueous chlorine and m-
aminophenol at molar chlorine to amine ratios ranging from 0.5 to 8. Sub-
stantially complete disappearance of available chlorine occurred within two
minutes for all but the two largest ratios, 5 and 8. Demand data for the
runs at these two ratios, both with 1.0 x lO'^M in-aminophenol, were as follows:
5:1 molar ratio; initial Cl = 5 x
time, hr.
demand
8:1 molar
time, hr.
demand
0.
4.
1
6
ratio;
0.
2.
1
7
0
4
.6
.7
3.
4.
initial
0
2
.5
.9
1.
3.
5
8
Cl
7
0
7
4
=
6
3
.8
Q xx
.3
.4
24
4
.8
10~4M
9.
3.
5
8
57
4.5
The reason for the relatively small molar demand observed with the 8:1
chlorine to amine ratio is unknown. It is inconsistent with the rapidity and
completeness of chlorine consumption observed in all the other experiments.
208
-------
A number of color changes was observed in the different reaction mix-
tures. With the 0.5 molar ratio of chlorine to amine a pale pink color
developed as the reactants were mixed. This color persisted for some time;
the solution changed to a peach hue over several hours and eventually became
pale yellow after about 30 hours. With molar chlorine to amine ratios of 1,
2 and 3 the solutions developed more intense pink colors that persisted for
longer times. In all instances, however, the colors had changed to peach
after about 18 hours and faded to haylike tinges after 25 to 30 hours. At
the molar chlorine to amine ratio of 5 there were flashes of pink color
during mixing, but the fully mixed solution- was initially pale yellow and
became colorless within 3 minutes. Formation of transient intermediates that
are further oxidized in the presence of excess free chlorine is indicated.
Ultraviolet spectral changes during these color shifts were not dramatic.
Generally increased ultraviolet absorbance was observed for all studied ratios
of chlorine to amine. The increase was quite large for the first few minutes
of reaction, but rather minor thereafter. An absorption band developed near
310 nm, most prominently when the chlorine to amine ratio was 3.
Phenylalanine , CaHiiOaN
Three experiments were conducted with this amino acid, at molar chlorine
to amine ratios of 4, 6 and 8. The results obtained were:
4;1 ratio;
time, hr.
demand
6;1 ratio;
time, hr.
demand
8;1 ratio; 4 x 1Q~3M Cl.
5.
0.
2.
6:
0.
1.
6
2
1
x 10
0.
2.
M
8
4
< 10~5M Cl
1
6
0.
1.
2
7
Cl,
5
2.
i
0.
2.
1.4
x
10
M
phenylalanine
60
6
1
7
2
x 10
1
2
3
•
-5
•
•
5
5
8
M
phenylalanine
5
2.5
5 x 1Q~4M phenylalanine
time, hr.
demand
0.1
1.4
2.8
1.9
9.4
2.3
26
3.5
The observed demands, for the first 5 hours or so, corresponded to the
expected value of 2.5 for the reaction.
C,H,.CH0CH(NH-)COOH + 2.5 HOCJ, •* C,H,.CH-COCOOH + ^ N. + 4 H00
O j / i. O J / 2. 2. L 2.
+ | a" + | H+
A pungent odor, characterized as sharp, cabbage-like and "organic", was
produced during the reaction between aqueous chlorine and phenylalanine. It
may be due to phenylpyruvic acid or perhaps to some chlorinated intermediates
Its occurrence suggests a source of tastes and odors developed as a result of
chlorination.
209
-------
Spectrophotometric observations showed a strong increase of overall
absorption in the region of the 257 nm peak of phenylalanine soon after start
of reaction, but no loss of structure of the absorption band occurred, in-
dicating lack of change in the benzene rings itself. At the molar ratio of
chlorine to amino acid equal to 4 the spectrum became quite stable after
about 30 minutes. At the molar ratio of 8 there was a continuing slow in-
crease in absorption at 250-260 nm up to 26 hours.
Pyrrole, C^H^N—
Several quantitative demand runs were conducted with pyrrole at molar
chlorine to amine ratios ranging from 4 to 8.3. Qualitative observations
were made on mixtures with smaller ratios.
When pyrrole was mixed at the 10"** level with an equal molarity of
aqueous chlorine, there was essentially complete reaction of the available
chlorine within 2 minutes. The reaction was accompanied by increased ultra-
violet absorption, indicating that the process was not simple oxidative
destruction of the pyrrole. Little further spectral change occurred for the
next several hours, but overnight a small absorption maximum appeared at
280 nm, indicating continuing reaction even in the absence of available
chlorine .
Data from the quantitative runs were as follows:
ratio 4:1; 6 * 10~4M C&. 1.5 *10~4M pyrrole
time, hr.
demand
0.1
2.6
0.25
3.1
1.0
3.6
3.0
3.7
6.0
3.8
23.5
3.9
ratio 5:1; 1.5 x 1Q~3M Cl, 3 * 10~Sl pyrrole
t ime , hr .
demand
0.1
2.6
0.25
3.6
1.0
3.7
3.0
4.2
6.0
4.4
23
4.5
time, hr.
demand
0.1
3.7
0.2
3.9
0.5
4.4
1.0
4.5
2.0
4.8
3.0
5.0
ratio 7:1;
1x1Q"4M C&.
1.4 x10 5M pyrrole
46
3.9
46
4.6
ratio 5.7:1; 8 x 1Q 5M C&. 1.4 * 1Q 5M pyrrole
23
5.7
time,
demand
ratio
time,
demand
hr.
8.3
hr.
0
3
:1?
0
4
.1
.3
6
.1
.3
0.
3.
x 10
0.
4.
2
7
-5M
3
8
1
4
C
2
5
.8
.9
X* j
.3
.4
5.3
5.1
0.72
5.2
5.8
23
5.
xlO~5
24
6.
7
M
5
pyrrole
As can be seen, the molar demand of pyrrole for aqueous chlorine is
great, increasing to at least 6.5 when there is substantial residual chlorine
in the solution. When the initial molar ratio of pyrrole to chlorine was 4,
the nearly immediate molar demand of about 3 was accompanied by the develop-
ment of an ultraviolet absorption band with a maximum near 250 nm. The
210
-------
intensity of this band decreased gradually over the succeeding several hours
as the remaining available chlorine reacted. Similar results were obtained
with an initial molar ratio of 5 except that the demand seemed to stabilize
at about 4.5 with some residual chlorine left in the solution after about six
hours reaction. However, continuing greater demand was shown with greater
molar ratios of chlorine to pyrrole, showing that additional oxidation was
still possible.
The reactivity of pyrrole with aqueous chlorine was particularly
important because of the relation of pyrrole to porphyrins (such as chloro-
phyll and heme) , the essential amino acid tryptophane, and other decomposition
products of proteins (such as indole and indole derivatives) . Tryptophane
and indole, both of which contain pyrrole rings, exerted significant chlorine
demands. A third nitrogen heterocycle, proline, having a pyrrolidine
structure, also exhibited a high chlorine demand.
The results so far point to considerable destructive oxidation of pyrrole
by aqueous chlorine that. overshadows possible chloramine formation. The
spectra show the formation of absorptive intermediates, some of which may be
chlorinated. It seems likely that pyrrole may be a source of volatile
chlorinated organic compounds like chloroform.
Tryptophane, C
Tryptophane is an amino acid of interest because it contains both a
pyrrolic-N and a primary amino-N. The pyrrole function appears to take
precedence, for the immediate molar chlorine demand of tryptophane solutions
is greater than 3 and significant residual chlorine does not last for much
more than 2 hours even at an initial molar ratio of chlorine to tryptophane
equal to 8. There is some indication that the demand for 10" 5M solutions
over 24 hours may be approximately 12.
Quantitative results for the initial molar ratio equal to 8, with
4 x 10~"*M chlorine and 5 x 10~5M tryptophane, were:
time, hr. 0.1 1.0 2.0 7 7.2
demand 4.2 7.1 7.6 7.8 8.0
Spectrophotometric observations on the reaction mixture with the 8:1
molar ratio indicated a complex reaction picture. Initially the tryptophane
absorption peak near 280 nm was replaced by a stronger absorption band with
maximum near 265 nm. Then this band was superseded over a period of about 30
minutes by another with a maximum near 240 nm. The intensity of this last
absorption continued increasing for at least 5 hours and a small secondary
peak with maximum near 330 nm also developed. A visible yellow color also
appeared in the solution after about 2 hours.
It appears probable that there is extensive reaction with the pyrrolic
ring at least and that volatile chlorinated products may be formed as with
pyrrole itself .
211
-------
Barbituric Acid—
The reaction of aqueous chlorine with barbituric acid was studied with a
6:1 molar ratio of chlorine to barbiturate at lO"1**! concentration.
There was a rapid initial demand of 2.0 moles of chlorine per mole of
babiturate that was accompanied by a substantially complete disappearance of
the barbiturate absorption maximum at 250-260 nm. Substituted for the
absorption maximum was a generally increasing absorption toward shorter wave
lengths. This general absorption then decreased slowly with increasing
reaction time. The results imply a rapid partial reaction with all the
barbiturate rather than a more extensive reaction with some of it.
The molar chlorine demand increased slowly with increasing time of
reaction from 2.8 to 75 minutes to 4.3 at 27 hours.
Caffeine—
This purine differs from the barbiturate in that the pyrimidine nitrogens
are fully methylated and there are no methylene hydrogens alpha to carbonyl
groups as potential chlorination sites. As a result very little reaction
with chlorine was observed. In a mixture of 2 x 10~"*M chlorine with .5x10" M
caffeine the chlorine decreased only to 1.7x10""* over a period of 67 hours.
Correspondingly the absorption spectrum for caffeine with its maximum at
273 nm changed only slightly. There was a slight general decrease in ab-
sorption over the 67 hour period, perhaps corresponding to the slight
reduction observed in the chlorine concentration.
£-Dymethylaminobenzaldehyde—
This substance, like the previous one, is not of direct, primary concern.
It happened to be available as a representative of a para-substituted aromatic
derivative and a structural analog of p-aminobenzoate.
In the one run with this compound, at a molar ratio of 4, there was an
initial molar demand about 0.6 that increased to 1.5 over 10 minutes, to
about 2.0 after 2 hours, to 3.0 after 6 hours and eventually to 4.0 after
55 hours.
Spectrophotometrically there was an initial shift in absorption from a
peak at 340 nm to one at 315 nm and from one at 245 nm to a broad maximum
centered near 233 nm that was complete in 10 minutes. Subsequently, the
absorption remained substantially constant for about 4 hours followed by a
slow uniform decrease in intensity across the spectrum for the duration of
the experiment.
Cytosine—
One series of studies was carried out in which fairly concentrated
solutions of cytosine were mixed with portions of aqueous chlorine, the molar
ratios of chlorine to cytosine ranging from less than one to several fold.
After overnight reaction the solutions were evaporated under vacuum and the
concentrated residues were subjected to thin-layer chromatography. When the
chromatographs were developed by spraying with starch-iodide solution, blue
spots appeared indicating the presence of oxidizing species (presumably
212
-------
N-chloro compounds) of good stability in the treated solutions and evaporated
residues. When the molar chlorine to cytosine ratio was less than one, only
a single spot appeared, at the same distance from the origin for all samples.
When the ratio was greater than one, multiple spots appeared, at different
distances and having different intensities depending on the initial ratio.
The formation of a number of N-chloro derivatives or decomposition products
of cytosine is clearly indicated.
Generally speaking the demands exhibited by the amino acids and related
compounds can be accounted for semi-quantitatively as an oxidative hydrolysis
of N-chlorinated amine groups, followed by breakpoint oxidation of the re-
leased ammoniacal-N to nitrogen gas. The greater part of the demand is
therefore attributable to oxidation of nitrogen rather than oxidation or
substitution on carbon by the aqueous chlorine. Only when the demand exceeds
2.5 moles of chlorine per mole of compound is it likely that any general
oxidative breakdown of the organic compound has occurred.
Three of the five compounds exhibiting molar demands much in excess of
2.5, pyrrole, indole and tryptophane, are structurally related in that all
possess the pyrrole ring. It appears that the presence of this ring, which
is a component of such important natural products as porphyrin, chlorophyll
and hemoglobin, may provide a point of attack for general oxidation by aqueous
chlorine. Even so, the demands shown do not represent complete oxidation to
CO2 and N2 .
The strong demand exhibited by m-aminophenol, is noteworthy because of
its structural resemblance to resorcinol, in-dihydroxybenzene, which is known
to produce substantial chloroform when allowed to react with aqueous chlorine.
Initial Chlorine Demand
Initial chlorine demand ratios (ICDR) defined as the chlorine demand
ratio determined for reaction times of approximately 30 seconds are shown in
Table 39. It is easily seen that many organic compounds exert substantial
chlorine demand within a half a minute after mixing.
Those compounds which exhibited high initial chlorine demands are
typically members of one of the following groups: 1. benzenoids with at
least one attached hydroxyl group (m-aminophenol); or 2. heterocyclic nitro-
genous compounds having a pyrrole or pyrrolidine structure (tryptophane,
indole and proline).
Long Term Chlorine Demand
Several compounds were chosen for aqueous chlorination studies that
lasted a minimum of 20 hours. Table 40 lists these compounds together with
highest chlorine demand observed after many hours of reaction with HOC1.
Compounds that exhibited a high initial chlorine demand, such as trypto-
phane, frequently underwent subsequent slow increase in chlorine demand over
time. In the majority of experiments on compounds of this type, the greater
part of the total chlorine demand was exerted within the first 30 seconds or so of
reaction.
213
-------
TABLE 39.
Compound
alanine
m-aminophenol
arabinose
arginine
aspartic acid
glutamic acid
indole
proline
tryptophane
COMPOUNDS SCREENED FOR INITIAL
Initial chlorine concentration
0.20
0.60
0.05
0.20
0.10
0.10
0.20
0.30
0.40
CHLORINE DEMAND
(mM) HOCl/Cpd3
2:1
6:1
5:1
2:1
3:1
1:1
4:1
3:1
4:1
ICDRb
c
ns
4.50
0.46
ns
ns
ns
1.04
2.93
2.90
Cpd = the added initial molar concentration of compound in the reaction
mixture
ICDR = initial chlorine demand ratio
i
"ns = no significant chlorine demand (<0.25 ICDR)
TABLE 40. HIGHEST CHLOROFORM RATIOS OF SELECTED COMPOUNDS
Compound Initial Chlorine
Concentration (mM) "^-"-'^P0
alanine
m-aminophenol
arginine
aspartic acid
caffeine
creatinine
dime thy laminobenzaldehyde
glutamic acid
indole
phenylalanine
proline
pyrrole
tryptophane
0.20
0.80
0.20
0.20
0.10
0.05
0.04
0.20
0.40
0.20
0.20
0.20
0.40
4:1
8:1
4:1
4:1
2:1
5:1
4:1
4:1
8:1
4:1
4:1
13.9:1
8:1
Time
(hours)
72
41
72
72
67
29
33
72
31
67
72
24
72
CDR
1.73
7.18
2.56
3.50
0.62
3.28
3.80
2.05
7.40
3.87
2.76
12.67
8.00
214
-------
Chloroform Formation
The stoichiometry of chlorine demand as it relates to chloroform form-
ation shows that each molecule of chloroform produced reduces the theoretical
chlorine demand for oxidation to C02 and water by one molecule of HOC1.
Equations can be written for each chloroform-producing substance to give a
maximum expected stoichiometric or theoretical chlorine demand (TCD, noted
by underlined values). These are shown in Table 41. Actual demand figures
can approach these stoichiometric ones only if the rest of the organic
compound is oxidized fully to C02 and H2). (The values for the N-containing
compounds are somewhat uncertain. Breakpoint oxidation to N2 has been
assumed, but if oxidation proceeds all the way to NOT, then the theoretical
figure should be greater by 2.5 per N atom.) When this happens, there is no
organic matter left in the form of additional chloroorganic compounds, except
possibly trichloroacetate or hexachloroacetone. In these circumstances the
supposition that substantial quantities of many chloroorganic substances
besides chloroform have been produced is simply not tenable.
The percent theoretical chlorine demand utilized (%TCD) can be evaluated
using equation (15) from the observed chlorine demand ratio and the theore-
tical chlorine demand ratio.
% Theoretical chlorine demand _ observed chlorine demand ratio (CDR)
(% TCD) theoretical chlorine demand ratio (TCDR) X
(15)
Chlorine demand ratios (CDR), % theoretical chlorine demand values
(%TCD) along with the highest concentration of chloroform produced during
initial studies at pH 7.0 are shown in Table 42. The percent molar yield of
CHC13 is defined by:
% Molar Yield of CHCl^ = "oles of chloroform formed x
-> moles of compound used
It is noteworthy that in all the investigations of chloroform formation the
percent molar yields were not greater than 100%, even with the most productive
compounds under the most favorable conditions.
The relationship between the percent molar yield of chloroform formed
during the 16:1 sample run using tryptophane, and the associated chlorine-
demand ratio over a contact period of about 8 hours is depicted in Figure 122.
The percent molar yields and chlorine demand ratios for indole, a degradation
product of tryptophane, exhibit a similar relationship, but with markedly
reduced molar yield of chloroform as compared with that of tryptophane.
Figure 123 shows the results of chlorination studies at pH 7 on indole and
tryptophane with initial aqueous chlorine concentrations of 1.0xlO~5M and
molar ratio or HOC1 to compound equal to 10.
Several nitrogenous organic compounds, including some with pyrrolic
rings, are effective producers of chloroform. Detailed results with 5 x 10~6M
tryptophane, generally typical of findings with the other nitrogenous compounds
215
-------
TABLE 41. STOICHIOMETRIC EQUATIONS OF COMPOUNDS HAVING
CHLOROFORM FORMING POTENTIAL
Compound +
adenine:
W5
Alanine:
Cof-LNO-
372
4-amino antipvrine:
C11H13N3°
m-aminophenol :
CCH7NO
o /
arginine:
C6H14N4°2
aspartic acid:
C4H?N04
barbituric acid:
C4H4N2°3
caffeine:
81042
creatine:
C4H7N3°
cyanuric acid:
C3H3N3°3
cytosine:
C.Hj-N^O
HOC1 ->
11.5
6.5
26.5
13.5
11
6.5
6
Ii
9.5
3.5
8.5
CHC13 H
1
1
1
1
1
1
1
1
1
1
1
f- C02 H
4
2
10
5
5
3
3
7
3
2
3
h H20
3.5
4.5
7.5
4.5
7
4.5
3
6
4.5
2.5
3.5
+ HC1 +
8.5
3.5
23.5
10.5
12
3.5
3
15
6.5
0.5
5.5
N2
2.5
0.5
1.5
0.5
2
0.5
1.0
2.0
1.5
1.5
1.5
p-dimethyl ami nobenzal dehye :
C9HnNO
glutamic acid:
C5HgN04
21.5
9.5
1
1
8
4
6.5
5.5
18.5
6.5
2.5
0.5
aNumber represents moles required for balancing equations.
216
(continued)
-------
TABLE 41 (continued)
Compound +
glycylaglycine:
C4H8N2°3
histidine:
C6H9N3°2
L-hydroxyproline:
C5H9M03
indole:
CfiH7N
phenylalanine:
C9H11N02
proline:
C5HgN02
purine:'
C5H4N4
pyrrole:
C4H5N
sarcosine:
C3H7N02
thymine:
C5H6N2°2
tryptophane:
C11H12N2°2
tyro sine:
C9H11N03
uracil:
C4H4N2°2
uric acid
R A A Q
HOC1 H
8
13.5
10.5
1G.5
20.5
11.5
11
9.5
6.5
12.
25_
19.5
T_
8
" CHC13 H
1
1
1
1
1
1
1
1
1
1
1
1
1
1
* C02 H
3
5
4
7
8
4
4
3
2
4
10
8
3
4
i- H20
5
5.5
5.5
4.5
6.5
5.5
3
3.5
4.5
4
7
6.5
3
3
+ HC1 +
5.0
10.5
7.5
15.5
17.5
8.5
8
6.5
3.5
7
22
16.5
4
5
M?
1.0
1.5
0.5
0.5
0.5
0.5
2.0
0.5
0.5
1.0
1.0
0.5
1
2
217
-------
TABLE 42. HIGHEST CONCENTRATION OF CHLOROFORM PRODUCED DURING INITIAL
CHLOROFORM PRODUCTION STUDIES AT pH 7a
Compound
adenine
alanine
m-aminophenol
barbituric acid
caffeine
chlorophyll
creatinine
cytosine
L-hydroxyproline
(pH 10.5)
indole
phenylalanine
proline
purine
pyrrole
thymine
tryptophane
(pH 7.5)e
tyrosine
uracil
uric acid
Time
(hours)
4.0
25
3.8
4.4
3.3
4.0
3.7
5.0
98
98
25
24
3.7
2.8
0.3
3.4
24
7.0
4
0.7
2.0
Chlorine Theoretical
Demand Demand Ratio
Ratio (Equation 15)
5.7
3.0
4.6
4.3
1.8
1.2
3.6
8.5
8.5
8.5
2.5
1.6
.7
3.1
5.4
10.0
13.4
9.0
4.3
6.1
50
46
34
72
10
16
42
81
81
46
12
14
6
33
54
40
54
46
61
76
Molar Yield
CHC1, (%)
J
b
ns
1.0
ns
1.4
ns
10 yg/1
0.5
0.5
6.9
37.2
4.9
1.1
0.6
ns
0.7
0.5
7.8
17.9
ns
0.6
ns
Initial chlorine concentration of
except where noted.
9.0xio~5M and HOCl/Cpd ratio of 9:1
ns = less than 0.5% molar yield of CHC13
CInitial chlorine concentration 20x 10~*M; HOCl/Cpd =10:1
Initial chlorine concentration 10 x 10~5M; HOCl/Cpd = 10:1
elnitial chlorine concentration 12 x10~5M; HOCl/Cpd = 12:1
218
-------
ro
•14
hours
Figure 122. Chlorination of tryptophane, pH. 7.5. Solid line, chloroform formed; dashed
line, molar chlorine demand ratio. Initial tryptophane, 0.50xio~5M;
initial aqueous chlorine, 8 x 10~5M (5.7 mg/1).
-------
N5
M
O
200
160
CHCI.
(In ug/D
80
40
pH=7
t
Tryptophane
10-1
Indole
I
hours
•tt
C.D.R.
1.5
Figure 123. Chlorination of tryptophane and indole, pH 7. Solid lines, chloroform formed;
dashed lines, molar chlorine demand ratio. Initial tryptophane, 1.0*10 M;
initial aqueous chlorine, l.Oxlo^M (7.1 mg/1). Initial indole, 1.0xlQ~5M;
initial aqueous chlorine, 1.0xlQ~1*M (7.1 mg/1).
-------
are depicted in Figure 124. An increased chloroform production resulting
from increases in the ratio of the chlorine to tryptophane applied was
observed. Pyrrole and L-hydroxyproline exhibited similar behavior.
Perhaps most interesting of all were the results with chlorophyll.
"Soluble" chlorophyll at a nominal concentration of 1.7 mg per 1 (the
material was an aqueous paste with solids contents unknown) was mixed with
5.7xlO-"M (40 mg per 1) of aqueous chlorine at pH 5.8, 6.6, 7.0, 9.2 and
10.0 and allowed to stand for about 100 hours. The chloroform produced, in
Ug per 1, was found to be 12, 32, 56, 260 and 230, respectively. A plot of
the chloroform produced as a function of pH is shown in Figure 125.
The fact that chlorophyll, even if only at elevated pH, was able to
produce such substantial quantities of chloroform is strongly suggestive
that algae as well as fulvates may be sources of haloforms in the treatment
of water supplies.
Effects of pH Alteration
One of the most significant findings of these studies was the increased
yields of chloroform obtained when reaction mixtures were made alkaline an
hour or two before analyses for chloroform were performed. Since these
increases were observed without significant changes in chlorine consumption
and in some instances even when solutions had been dechlorinated prior to
pH change, they are strongly indicative of the presence of intermediate
chlorinated compounds like trichloroacetate that require alkaline conditions
for hydrolysis.
Examples of the results obtained are shown in Figures 126 through 130
for a number of different nitrogenous compounds.
Results for proline are shown in Figure 126 along with values of the
molar chlorine-demand ratio at the time of the chloroform determinations.
Although increasing the pH produced some increased chlorine demand, the
final value was the same for the region pH 9 to 11, the region in which the
great increase in yield of chloroform occurred. This was indicative that
the greater chloroform production at higher pH was not a result of greater
chlorination.
Detailed results for tryptophane are depicted in Figure 127. Perhaps
the most remarkable feature in this experiment was the occurrence of 100%
molar yield of CHCla near pH 11. This is to be compared with a maximum
observed yield of 18% in the studies at pH 7.5. It becomes clear, once
again, that chlorination to intermediate products occurs during the reaction
period in neutral or mildly acidic solutions, with CHCla being liberated by
hydrolysis when the pH is raised.
Chlorination reactions with parallel samples of tryptophane and indole
conducted over a period of 28 hours at pH 6 yield considerably different
molar percentages of chloroform, about 14% for tryptophane and 3% for indole.
This is shown by Figure 128. After the 28 hours of contact, the pH of each
sample was sequentially increased by addition of increments of sodium
221
-------
100i
TRYPTOPHAN
20:1
80-
ug/l
CHCI
NJ
N>
S3
40
18
12
%
molr.r
yield
CHCL
-6
80
160
minutes
360
440
0
Figure 124. Chlorination of tryptophane, pH 7.5. Initial tryptophane concentration,
0.50x_lO~5M; initial chlorine concentrations, 6.0xiQ~5M (4.3 mg per 1),
8x10 5M (5.7 mg per 1), 1.0 x lO'^M (7.1 mg per 1).
-------
275
200-
ug/i
CHCU
100^
CHLOROPHYLL
1.7
CHLORINE
4 0.0m o/|
-IOO hours
9
11
Figure 125. Chlorination of chlorophyll at varied pH. Initial chlorophyll
concentration (nominal), 1.7 mg per 1; initial chlorine,
5.7x10" M (40 mg per 1). pH values, 5.8, 6.6, 7.0, 9.2, 10.0.
Chlorine demand after 100 hours 36 mg per 1 in all solutions.
223
-------
16
14
Cl
demand
rntio
12
PROLINE
16.5:1
Figure 126. Yields of CHCla from proline with PH change. Solid line, chloroform yield;
dashed line, molar chlorine-demand ratio. Initial proline concentration,
2.0x10 M; initial aqueous chlorine, 3.3x10"^ (23 mg per 1). pH at 6.'?
for 21 hours, then separate samples increased in about 0.5 pH unit steps
with greatest pH at 11. Chloroform and residual chlorine determinations
-------
2.4
1.8
mg/l
0-6
TRYPTOPHANE
25.2:1
100
75
c/
10
0
molar
yield
CHCL
25
,H
11
Figure 127. Yields of chloroform from tryptophane with pH change. Initial
tryptophane concentration, 2.0*10 M; initial aqueous chlorine,
5.04xlO~'tM (36 mg/l). pH at 5.4 for 45 hours, then separate
samples increased in pH in 0.5 unit step with greatest pH near
11. Chloroform determinations at 47 hours.
225
-------
14
12
10
Molar Yield
CHCI3
6
PH-6
Tryptophane
37.5:1
8
hours
20 24
28
Figure 128. Chlorination of tryptophane and indole, pH 6. Initial
tryptophane, 1.0 x 10~5M; initial aqueous chlorine, 3.75x10"^
(26.5 mg/1). Initial indole, 1.0xlCT5M; initial aqueous
chlorine, 2.85x10-^1 (20.2 mg/1).
226
-------
30
Molar Yield
CHCL
20
10
Tryptophane
Indole
S5
PH
9
11
Figure 129. Yields of chloroform from tryptophane and indole with pH
change. Yields of chloroform from tryptophane and indole with
pH change after reduction of residual chlorine with thiosulfate
after 28 hours at pH 6. Initial tryptophane, 1x1(T5M; initial
aqueous chlorine 3.75xi0-*M (26.5 mg/1) . Initial indole,
IxlQ M; initial aqueous chlorine, 2.85xio~'*M (20.2 mg/1).
227
-------
2.4
100
URACIL
8.25:1
Figure 130. Yields of chloroform from uracil with pH change. Initial
uracil concentration, 2.0*10~5M; initial aqueous chlorine,
1.65x10"^ (11.7 mg/1). After reaction at pH 6.6 for 21
hours virtually all chlorine reduced. Separate samples
increased in pH to a maximum of 10.6. Chloroform determinations
at 26 hours.
228
-------
hydroxide after removal of any remaining available chlorine by addition of
sodium thiosulfate. Within minutes of each pH alteration the concentrations
of chloroform were determined. The results, shown in Figure 129, indicate
the amount of chloroform obtained from indole does not significantly increase
when pH is increased after dechlorination whereas that obtained from trypto-
phane increases greatly. Since there was no available chlorine remaining
to provide additional chlorination, it appears that an intermediate was
formed at pH 6 which hydrolyzed or decomposed to give chloroform when
sufficient base was added. This observation is consistent with the inform-
ation derived from the spectrophotometric studies.
A similar pattern was exhibited for the chlorination of uracil with pH
change presented in Figure 130. Uracil had not previously been regarded as
a significant chloroform producer based on studies near pH 7. This experi-
ment showed conclusively that extensive chlorination had occurred during the
reaction, so that when the pH was increased, with virtually no residual
available chlorine present. 100% molar yield of chloroform was obtained.
The study of L-hydroxyproline illustrates the differences resulting from
different pH values. Parallel samples of L-hydroxyproline were prepared in
different buffer systems, one at neutral pH and the other at pH 10.5. Each
was then mixed with an equal volume of chlorine solution diluted with the
same buffer as the L-hydroxyproline sample. Shoftly after mixing, both
solutions exhibited similar chlorine demand and chlorine production. However,
after two hours had past, the alkaline sample showed a significantly greater
chlorine demand in addition to a more than forty-fold increase in chloroform
formation as compared to that of the solution at neutral pH (see Figure 131).
Results of chloroform production by L-hydroxyproline at various pH
values are shown below:
L-hydroxyproline 2.0 x 10 M ( 2.6 ppm)
chlorine 55.5 x 10~5M (39.3 ppm)
Approximate time: 170 hours
£H Molar Yield of Chloroform Observed Demand Ratio
5-6 ( 7.0)a 0.1% ( 1.1%) 25.4 (25.9)
( 8.2) ( 2.4 ) (26.1)
(10-0) (110.0 ) (26.1) LI
6.6 (10.0) 0.6 ( 96.8 ) 25.9 (26.0) f]
. .
7.0(10.0) 1.5 (89.4) 26.1 (26.1)
9-2 37.2 26.0
10.0 41.5 25.9 y - 1
Sodium hydroxide was added to the low pH samples fifteen to twenty hours be-
fore chloroform determination was carried out at Lawrence. In addition, the
sample originally at the lowest pH was separated into four portions: the first
was placed in a screw-capped vial without any alteration while each of the
other three had sodium hydroxide added until pH values of 7.0, 8.2 and 10.0 were
attained at which time they were placed in screw-capped vials for transport to
Lawrence.
229
-------
time, min.
Figure 131. Production of chloroform (o) and chlorine demand ratio (•)
chlorination of 10 x 10~5M L-hydroxyproline with 9 x 10~5
aqueous chlorine at pH 7 and 10.5 (chlorine demand ratio =
moles of chlorine used/mole of L-hydroxyproline added) .
for
230
-------
The results of this study were very interesting. As was the case in
other compounds, the main increase in chloroform production occurred between
pH 7.0 and pH 9.2. However, the subsequent increase, that occurred between
pH 9.2 and pH 10.0, was not substantial. This suggested that the chloroform
production tended to level off at the higher pH values, or perhaps, as in the
case of chlorophyll, the maximum chloroform yield was reached prior to the
highest pH. Again, the observed demand ratios were not significantly
different for any of the original pH values.
The samples which were increased in pH also produced rather interesting
results. The first sample, pH 5.6 exhibited little increase in chloroform
formation when raised to pH 8.2. Yet, when the pH reached 10.0 the molar
yield of the chloroform exceeded 100%. Apparently, a very dramatic increase
occurs at some point between these two pH values. Also, the two other
samples which underwent pH adjustment showed significantly greater quantities
of chloroform formed than the sample originally at pH 10.0. The changes in
demand ratios were negligible. Additional nitrogenous compounds which
produced considerable quantities of chloroform under alkaline conditions
were pyrrole and m-aminophenol.
Pyrrole; pyrrole 2.0xio~^M (1.3 ppm)
chlorine 25.0xio~5M (17.7 ppm)
Approximate time: 24 hours
£H
5.7
6.5
7.5
9.1
10.0
Molar Yield of Chloroform
0.2 %
0.4
0.5
7.6
30.0
Observed Demand Ratio
12.0
11.9
11.9
12.0
12.0
H
A significant increase in chloroform production occurred between pH 7.5
and pH 9.1 and again between pH 9.1 and pH 10.0. However, it is quite
interesting to note that the demand ratios for all pH values were almost
identical. This indicated the formation of an intermediate compound which,
at the lower pH values, did not readily convert to chloroform
m-Aminophenol; in-aminophenol
chlorine
Approximate time: 150 hours
2. Ox 10~i?M
37.0x10 ^M
(2.2 ppm)
(26.2 ppm)
PH Molar Yield of Chloroform
4.4 (10.1) 3.4 % (17.8%)
6.3 (10.1) 7.6 (12.5%)
6.8 (10.1) 13.4 (16.2%)
9.2 17.3
9.9 30.8
Observed Demand Ratio
17.6 (17.6)
17.6 (17.6)
17.6 (17.6)
17.7
17.7
OH
231
-------
In the case of m-aminophenol, the first large increase in chloroform
formation occurred between pH 6.3 and pH 6.8, the next occurring between pH
9.2 and pH 9.9 (Figure 132). As observed previously, the difference among
the demand ratios was not significant. In addition, even after the acidic
samples were raised to pH 10.1 their demand ratios (determined by thiosulfate
titration after gas chromatography analysis) did not change. On the other
hand, the amount of chloroform produced increased markedly. This substanti-
ated the previous hypothesis that an intermediate was formed initially, but
its conversion to chloroform was inhibited due to the low pH of some of the
samples. When the pH values were subsequently increased, the formation of
chloroform was favored. Also, the lowest pH sample produced the greatest
amount of chloroform after the pH was raised. This might mean that formation
of the intermediate (or intermediates) was favored at the lowest pH value.
A possible explanation for the striking chloroform production of the
nitrogenous compounds discussed is that heterocyclic structures often react
in a manner similar to ketones (100). The elevated pH results in rapid
tautomerism to the enol form, thus allowing the reaction to proceed ana-
logously to that of diketo compounds. These diketo compounds have been
known to produce significant amount of chloroform when allowed to react with
aqueous chlorine (35).
Although altering the pH and concentrations can result in different
values of chlorine demand and quantities of chloroform formed, there are
other variables which can also affect these values. Recent studies con-
ducted at the Lawrence Experiment Station on chloroform concentrations in
Massachusetts drinking water supplies indicate that contact time is one such
important parameter. In this investigation all sample runs were carried out
over a period of several hours with demand and concentration of chloroform
being determined periodically for extrapolation necessitated by time con-
straints on the use of the equipment. Other variables, such as temperature,
were not investigated in this study.
CONCLUSIONS
A large number of naturally occurring nitrogenous compounds readily
reacted with aqueous chlorine exerting significant chlorine demands. The
groups of compounds found to be moderately to highly reactive included:
benzoids containing one or more hydroxyl groups (in-aminophenol) , amino acids
(alanine, tryptophane, proline and L-hydroxyproline); structures containing
the pyrrole ring (indole, pyrrole, chlorophyll and some amino acids already
mentioned); pyrimidines (cytosine, uracil, thymine and barbituric acid);
and some purines (adenine and uric acid). Not all organic compounds that
exhibited high chlorine demand produced chloroform as a product suggesting
either simple oxidation or the formation of other chlorinated organic
compounds which have so far remained unidentified. For those compounds which
did produce chloroform the chlorine demand did not appear to be a very good
indication of the chloroform producing potential. For chloroform producers,
increasing the pH had a considerable influence on the quantity of chloroform
formed, with maximum chloroform formation occurring between pH 8.5 and
pH 10.5.
232
-------
60
50
Molar Yield
CHCL
20
10
m-Aminophenol
« 5 6 7 9 I'D 11
pH
Figure 132. Yields of chloroform from m-aminophenol with pH change. Initial
m-aminophenol, 2 x 10~5M; initial aqueous chlorine, 3.3xlo~'*M
(23 mg/1). pH at 4.3 for 41 hours, then pH increased.
form determinations at 67 hours.
Chloro-
233
-------
Compounds that reacted with aqueous chlorine near pH 7 to yield chloroform
did not necessarily exhibit enhanced production of chloroform with increase
in pH. Moreover, several compounds that showed sharp increases in chloro-
form formation under alkaline conditions also began to show decreases in
chloroform production with increasing pH values greater than about 10. Thus,
aqueous chlorine reactions of selected compounds (or raw waters) having pH
values greater than 10 can give misleading indications of the ultimate
capacity for formation of chloroform.
Some compounds that showed significant chloroform production only under
alkaline conditions generally formed other chlorinated intermediates under
neutral or slightly acidic conditions. These intermediates when subjected
to a higher pH, produced chloroform. Samples for haloform should, there-
fore, be made alkaline to pH 11 or greater and held at the pH for an hour
or so before haloform determinations.
It seems possible that a very simple technique for preliminary assess-
ment of the potential for chloroform production in a water supply can be
obtained by determination of the four to six-hour chlorine demand with enough
excess chlorine present to give a free residual chlorine of several milli-
grams per liter.
234
-------
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APPENDIX A
TABLE A-l. RECOVERY OF CREATINE IN WATER AFTER PASSAGE THROUGH TENAX AND
XAD-8 MACRORETICULAR RESINS (pH UNADJUSTED)
aliquot (ml)
influent (= 10 mg/L)
0-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
85-90
90-95
95-100
100-105
105-110
110-116
116-121
121-126
126-146
146-166
166-186
186-206
206-226
226-246
246-266
absorbance at 254 nma
0.600
0.033
0.035
0.064
0.109
0.157
0.204
0.257
0.308
0.360
0.405
0.450
0.468
0.491
0.510
0.535
0.542
0.550
0.553
0.568
0.575
0.568
0.581
0.578
0.585
0.581
0.582
0.581
0.595
% recovery
^
5.5
5.8
10.7
18.2
26.2
34.0
42.8
51.3
60.0
67.5
75.0
78.0
81.8
85.0
89.2
90.3
91.7
92.2
94.7
95.8
94.7
96.8
96.3
97.5
98.5
97.0
98.5
99.2
1 cm pathlength
243
-------
TABLE A-2. RECOVERY OF URACIL IN WATER AFTER PASSAGE THROUGH TENAX AND
XAD-8 MACRORETICULAR RESINS (pH UNADJUSTED)
aliqout (ml)
influent = 10 mg/L
0-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
85-91
91-96
96-101
101-106
106-111
111-116
116-121
121-126
126-152
152-177
177-202
202-227
302-326
absorbance at 254 nma
.715
.009
.026
.053
.147
.250
.364
.472
.510
.508
.629
.645
.659
—
.680
.681
.681
.690
.690
.692
.692
.692
.705
.725
.720
.715
.715
% recovery
—
1.3
3.6
7.4
20.6
35.0
50.9
66.0
71.3
71.1
88.0
90.2
92.2
—
95.1
95.2
95.2
96.5
96.5
96.8
96.8
96.8
98.6
101.4
101.7
100.0
100.0
cm pathlength
244
-------
TABLE A-3. RECOVERY OF URACIL IN WATER AFTER PASSAGE THROUGH XAD-8
MACRORETICULAR RESIN (pH UNADJUSTED)
aliquot (ml)
Influent (= 10 mg/L)
0-20
20-25
25-31
31-36
36-41
41-46
46-51
51-56
56-62
62-67
67-72
72-77
77-82
82-87
87-92
92-97
97-102
102-107
107-122
112-117
117-122
122-147
147-172
297-322
*a
absorbance at 254 nm
.690
.018
.064
.126
.264
.330
.410
.480
.541
.569
.599
.612
.640
.649
.649
.655
.660
.662
.651
.651
.640
.672
.658
.678
.687
% recovery
—
2.6
9.3
18.3
38.3
47.8
59.4
69.6
78.4
82.5
86.8
88.7
92.8
94.1
94.1
94.9
95.7
95.9
94.4
94.4
92.8
97.4
95.4
98.3
98.3
1 cm pathlength
245
-------
TABLE A-4. RECOVERY OF CREATININE IN WATER ADJUSTED TO pH 2.0 WITH HC1 AFTER
PASSAGE THROUGH XAD-2 AND XAD-4 RESINS
aliquot (ml)
influent
0-5
5-10
10-15
15-20
20-25
25-30
30-40
40-65
65-90
90-190
concentration (mg/L)
3.2
1.5
1.1
1.5
1.6
1.9
2.4
2.2
2.5
2.5
2.6
% recovery
47.7
34.6
47.1
49.0
58.2
75.8
68.6
74.5
80.0
81.0
TABLE A-5. RECOVERY OF CYTOSINE IN WATER AFTER PASSAGE THROUGH XAD-2
XAD-4 MACRORETICULAR RESINS (pH UNADJUSTED)
aliquot (ml)
influent
0-5
5-10
10-15
15-20
20-25
25-50
75-100
175-200
concentration (mg/L)
8.6
0.7
0.4
0.9
2.0
5.3
5.4
7.1
7.5
% recovery
^
7.9
4.6
10.6
23.6
62.2
63.6
82.6
88.0
246
-------
TABLE A-6. RECOVERY OF CYTOSINE IN WATER ADJUSTED TO pH 2 WITH NITRIC ACID
AFTER PASSAGE THROUGH XAD-2 AND XAD-4 RESINS
aliquot (ml) concentration (mg/L) % recovery
influent
0-5
5-10
10-15
15-20
20-25
25-30
30-40
40-65
65-90
165-190
8.3
1.1
1.4
2.6
5.3
4.9
5.4
6.5
7.2
7.3
7.6
.
13.4
11.0
31.2
63.0
59.1
64.6
77.4
85.8
87.5
90.8
TABLE A-7. RECOVERY OF INDOLE IN 10~3 NaOH AFTER PASSAGE THROUGH
XAD-2 AND XAD-4 RESINS
aliquot (ml) concentration (mg/L) % recovery
influent
0-5
5-10
10-15
15-20
20-25
25-50
50-75
175-200
8.7
1.8
1.3
1.1
2.3
1.3
1.6
1.5
1.7
20.3
20.0
12.7
26.8
14.7
18.6
17.0
19.3
247
-------
TABLE A-8. RECOVERY OF INDOLE IN 10
-3
M HNO., AFTER PASSAGE
THROUGH XAD-4 RESIN
aliquot (ml)
concentration (mg/L)
% recovery
influent
0-5
5-10
10-15
15-20
20-25
25-30
30-55
55-80
125-150
190-215
6.6
0.4
0
0.3
0.
0.
0.
0,
0
0,
0.
.1
.1
.1
6.1
0
4.4
0.9
0.9
1.7
1.3
0
4.8
4.8
248
-------
TABLE A-9. RECOVERY OF CYTOSINE IN WATER AFTER PASSAGE THROUGH XAD-8 AND
TENAX RESINS (pH UNADJUSTED)
aliquot (ml)
influent (= 10 mg/L)
0-25
25-30
30-35
35-40
40-45
45-50
50-56
56-61
61-66
66-71
71-76
76-81
81-86
86-91
91-96
96-101
101-106
106-111
111-116
116-121
121-126
126-146
146-166
166-186
186-206
206-226
267-287
absorbance at 233 run3
.435
.170
.090
.111
.161
.195
.236
.262
.290
.323
.335
.353
.365
.380
.387
.392
.408
.412
.414
.414
.418
.413
.403
.412
.416
.416
.418
.418
% recovery
39.1
20.7
25.5
37.0
44.8
54.3
60.2
66.6
74.3
77.0
81.2
83.9
87.4
89.0
90.0
93.8
94.7
95.2
95.2
96.1
92.6
92.6
94.7
95.6
95.6
96.1
96.1
al cm pathlength
249
-------
TABLE A-10. RECOVERY OF HUMIC ACID SOLUTION (pH 2.0) AFTER PASSAGE THROUGH
XAD-8 AND TENAX MACRORETICULAR RESINS
volume eluted
(ml)
influent
0-25
50-75
75-100
100-125
125-175
175-275
275-375
375-474
474-545
absorbance
at 330 run
0.322
0.070
0.046
0.050
0.050
0.053
0.077
0.090
0.190
0.116
concentration
(mg/L)
13.5
2.9
1.9
2.1
2.1
2.2
3.2
3.8
4.6
4.9
% recovery
.
21.7
14.3
15.6
15.5
16.5
23.9
28.0
33.9
36.0
1 cm pathlength
250
-------
TABLE A-ll. RECOVERY OF HUMIC ACID SOLUTION ADJUSTED TO pH 11.7
AFTER PASSAGE THROUGH XAD-8 AND TENAX MACRORETICULAR RESINS
volume eluted
(ml)
Influent
0-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
85-90
90-95
95-100
100-110
110-120
120-145
145-170
170-195
195-370
absorbance
at 330 nma
0.438
-
.102
.104
.164
.185
.191
.204
.228
.246
.265
.278
.288
.295
.307
.318
.323
.331
.350
.363
.374
.368
.385
concentration
(mg/L)
18.3
-
4.3
4.4
6.9
7.8
8.0
8.5
9.6
10.3
11.1
11.6
12.1
12.4
12.9
13.3
13.5
13.9
14.7
15.2
15.7
15.4
16.1
% recovery
_
-
23.3
23.7
47.4
42.4
43.6
46.6
52.1
56.2
60.5
63.5
65.8
67.4
70.1
72.6
73.7
75.6
79.8
82.9
85.4
84.0
87.9
1 cm pathlength
251
-------
TABLE A-12. RECOVERY OF TRYPTOPHAN IN WATER ADJUSTED TO pH 2.0 WITH
HYDROCHLORIC ACID AFTER PASSAGE THROUGH XAD-8 AND TENAX GC
MACRORETICULAR RESINS
aliquot (ml)
influent
0-5
5-10
10-15
15-20
20-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
85-90
90-95
95-100
100-125
125-150
150-175
175-200
200-250
250-300
300-350
350-400
concentration (ng/L)
9.7
0.2
0.3
0.5
0.7
1.1
1.5
2.1
2.7
3.2
3.6
3.9
4.2
4.6
4.8
5.1
5.3
5.5
5.9
6.0
6.1
6.5
7.6
7.9
8.2
8.5
8.7
8.8
8.9
% recovery
—
2.4
2.7
5.4
7.1
10.9
16.0
21.8
27.9
32.7
37.4
40.5
43.9
48.0
49.7
53.1
54.4
57.1
60.5
61.6
63.6
67.7
78.9
81.6
84.8
87.8
89.8
91.5
92.5
252
-------
TABLE A-13. RECOVERY OF TRYPTOPHAN IN WATER ADJUSTED TO pH 6.9 WITH
NaOH AND HC1 AFTER PASSAGE THROUGH XAD-8 AND TENAX GC MACRORETICULAR RESINS
aliquot (ml)
influent
0-5
5-10
10-15
15-20
20-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
85-90
90-95
95-100
100-125
125-150
150-175
175-200
200-250
250-300
300-350
350-400
a
absorbance at 278 run (A.U.)
0.365b
0.041
0.130
0.190
0.218
0.241
0.275
0.290
0.310
0.320
0.330
0.335
0.340
0.345
0.348
0.350
0.354
0.353
0.353
0.358
0.360
0.352
0.360
0.361
0.361
0.359
0.362
0.363
0.365
% recovery
—
11.2
35.6
52.1
59.7
66.0
75.3
79.5
84.9
87.7
90.4
91.8
93.2
94.5
95.3
95.9
97.0
96.7
96.7
98.1
98.6
96.4
98.6
98.9
98.9
98.4
99.2
99.5
100
al cm pathlength; H20 reference
b influent concentration = 10 mg/L
253
-------
TABLE A-14. RECOVERY OF ADENINE IN WATER ADJUSTED TO pH 2.5 WITH
HYDROCHLORIC ACID AFTER PASSAGE THROUGH XAD-8 AND TENAX GC
MACRORETICULAR RESINS
aliquot (ml)
influent
0-5
5-10
10-15
15-20
20-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
85-90
90-95
95-100
100-125
125-150
150-175
175-200
200-250
250-300
300-350
concentration (mg/L)
9.54
0.5
2.1
3.6
4.4
5.1
5.7
6.2
6.8
7.2
7.5
7.8
8.1
8.2
8.3
8.4
8.5
8.7
8.7
8.9
8.9
9.0
9.2
9.4
9.3
9.3
9.4
9.4
% recovery
5.0
21.8
37.6
45.8
53.1
59.3
64.6
70.9
75.0
78.1
81.2
84.4
85.4
86.5
88.5
89.5
91.1
91.6
93.7
93.7
94.8
96.9
99.0
97.9
97.9
99.0
99.0
254
-------
TABLE A-15. RECOVERY OF ADENINE IN WATER ADJUSTED TO pH 7.1 WITH NaOH
AND HC1 AFTER PASSAGE THROUGH XAD-8 AND TENAX GC
MACRORETICULAR RESINS
aliquot (ml)
influent
0-5
5-10
10-15
15-20
20-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
85-90
90-95
95-100
100-125
125-150
150-175
175-200
200-250
250-300
300-350
350-400
concentration (mg/L)
9.6
0.0
1.0
2.5
3.7
4.4
5.1
5.9
6.4
6.9
7.4
7.5
7.6
7.8
8.1
8.3
8.5
8.7
8.8
8.9
8.9
8.9
9.3
9.4
9.4
9.3
9.5
9.6
9.6
% recovery
0.3
10.6
25.7
37.9
46.1
53.1
60.8
66.0
71.7
76.3
77.3
78.4
80.4
83.5
86.6
88.7
90.2
91.8
92.8
92.8
72.8
96.9
97.9
97.9
96.9
99.0
100
100
255
-------
TABLE A-16. RECOVERY OF 5-CHLOROURACIL IN WATER ADJUSTED TO pH 2.0 WITH
HYDROCHLORIC ACID AFTER PASSAGE THROUGH CAD-8 AND TENAX GC
MACRORETICULAR RESINS
aliquot (ml)
influent
0-5
5-10
10-15
15-20
20-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
85-90
90-95
95-100
100-125
125-150
150-175
175-200
200-250
250-300
300-350
350-400
concentration (mg/L)
9.7
0.2
1.2
3.1
3.5
3.9
4.4
5.0
5.6
6.3
6.8
7.3
7.7
8.0
8.2
8.4
8.6
8.8
8.9
8.9
9.0
9.2
9.5
9.5
9.7
9.7
9.7
9.7
9.7
% recovery
2.3
16.0
31.9
36.5
40.0
45.2
51.7
58.1
65.0
70.4
75.4
79.2
82.3
84.2
86.5
88.7
90.2
91.5
92.3
93.1
94.4
97.7
98.5
100
99.6
100
100
100
256
-------
TABLE A-17.
RECOVERY OF 5-CHLOROURACIL IN WATER ADJUSTED TO pH 7.01 WITH NaOH
AND HC1 AFTER PASSAGE THROUGH XAD-8 AND TENAX GC
MACRORETICULAR RESINS
aliquot (ml)
influent
0-5
5-10
10-15
15-20
20-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
95-90
90-95
95-100
100-125
125-150
150-175
175-200
200-250
250-300
300-350
350-400
concentration (mg/L)
10.4
0.5
2.0
3.5
4.7
5.5
6.3
7.1
7.7
8.2
8.6
8.9
9.1
9.3
9.5
9.6
9.6
9.7
9.9
9.9
10.0
10.0
10.3
10.3
10.4
10.2
10.3
10.3
10.4
% recovery
—
4.5
18.9
33.2
44.6
52.9
60.7
67.9
74.1
78.7
82.1
89.8
87.5
89.3
91.1
92.0
92.6
92.9
94.6
94.6
95.5
95.5
98.2
99.1
99.6
97.3
99.1
99.1
100
257
-------
TABLE A-18. RECOVERY OF SUCCINIMIDE IN WATER ADJUSTED TO pH 2.0 WITH
HYDROCHLORIC ACID AFTER PASSAGE THROUGH XAD-8 AND TENAX GC
MACRORETICULAR RESINS
aliquot (ml)
influent
0-10
10-20
20-30
30-40
40-50
50-60
60-70
70-80
80-90
90-100
100-125
125-150
150-175
175-200
200-250
250-300
300-350
350-400
absorbance at 215 run (A.U.)a
1.000b
0.127
0.196
0.245
0.318
0.383
0.435
0.470
0.540
0.550
0.590
0.620
0.690
0.780
0.810
0.860
0.920
0.920
0.930
% recovery
12.7
19.6
24.5
31.8
38.3
43.5
47.0
54.0
55.0
59.0
62.0
69.0
78.0
81.0
86.0
92.0
92.0
93.0
4 cm pathlength; H-0 reference
influent concentration = 30 mg/L
258
-------
TABLE A-19. RECOVERY OF SUCCINIMIDE IN WATER ADJUSTED TO pH 7.05 WITH
NaOH AND HC1 AFTER PASSAGE THROUGH XAD-8 AND TENAX GC
MACRORETICULAR RESINS
aliquot (ml)
influent
0-10
10-20
20-30
30-40
40-50
50-60
60-70
70-80
80-90
90-100
100-125
125-150
150-175
175-200
200-250
250-300
300-350
350-400
concentration (mg/L)
29.5
7.0
11.6
15.5
18.8
21.0
22.7
23.8
24.2
25.1
25.9
25.9
26.7
27.3
27.5
27.5
27.5
27.5
27.5
% recovery
—
23.8
39.5
52.6
63.8
71.2
77.0
80.8
82.2
84.9
87.7
87.7
90.4
92.6
93.2
93.2
93.2
93.2
93.2
259
-------
TABLE A-20. RECOVERY OF PURINE IN WATER ADJUSTED TO pH 2.0 WITH
HYDROCHLORIC ACID AFTER PASSAGE THROUGH XAD-8 AND TENAX GC
MACRORETICULAR RESINS
aliquot (ml)
influent
0-5
5-10
10-15
15-20
20-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
85-90
90-95
95-100
100-125
125-150
150-175
175-200
200-250
250-300
'' a
absorbancc at 260 nm (A.U.)
0.550b
0.056
0.185
0.268
0.282
0.330
0.380
0.428
0.444
0.468
0.480
0.485
0.490
0.498
0.500
0.510
0.510
0.510
0.520
0.520
0.520
0.520
0.530
0.540
0.540
0.530
0.540
% recovery
_
10.2
33.6
48.7
51.3
60.0
69.1
77.8
80.7
85.1
87.3
88.2
89.1
90.6
90.9
92.7
92.7
92.7
94.6
94.6
94.6
94.6
96.4
98.2
98.2
96.4
98.2
1 cm pathlength; H-O reference
Influent concentration = 10 mg/L
260
-------
TABLE A-21. RECOVERY OF PURINE IN WATER ADJUSTED TO pH 7.02 WITH NaOH
AND HC1 AFTER PASSAGE THROUGH XAD-8 AND TENAX GC
MACRORETICULAR RESINS
aliquot (ml)
influent
0-5
5-10
10-15
15-20
20-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
85-90
90-95
95-100
100-125
125-150
150-175
175-200
200-250
250-300
300-350
350-400
concentration (mg/L)
10.2
3.9
5.6
6.9
7.4
8.0
8.5
8.8
9.2
9.4
9.5
9.6
9.7
9.5
9.8
9.9
9.9
10.0
10.0
10.0
10.0
10.0
10.2
10.2
10.2
9.8
10.0
10.2
10.2
% recovery
38.4
55.1
67.7
72.6
77.9
82.6
85.8
89.8
91.3
92.9
93.7
94.4
94.4
96.0
96.8
96.8
97.6
97.6
97.6
99.9
97.6
99.2
99.2
100
96.0
99.9
99.9
99.9
261
-------
TABLE A-22. RECOVERY OF PYRIMIDINE IN WATER ADJUSTED TO pH 2.0 WITH
HYDROCHLORIC ACID AFTER PASSAGE THROUGH XAD-8 AND TENAX GC
MACRORETICULAR RESINS
aliquots (ml)
influent
0-5
5-10
10-15
15-20
20-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
85-90
90-95
95-100
100-125
125-150
150-175
175-200
200-250
250-300
300-350
350-400
absorbance at 242 nm (A.U.)3
0.465b
0.002
0.055
0.104
0.137
0.182
0.222
0.257
0.282
0.305
0.320
0.338
0.349
0.359
0.369
0.373
0.381
0.380
0.380
0.390
0.390
0.399
0.410
0.418
0.485
0.480
0.440
0.440
0.440
% recovery
^
0.4
11.8
22.4
29.5
39.1
47.7
55.2
60.7
65.6
68.8
72.7
75.1
77.2
79.4
80.2
81.9
81.7
81.7
83.9
83.9
85.8
88.2
89.9
91.4
90.3
94.6
94.6
94.6
a 1 cm pathlength; H20 reference
k Influent concentration = 10 mg/L
262
-------
TABLE A-23.
RECOVERY OF PYRIMIDINE IN WATER ADJUSTED TO pH 7.00 WITH NaOH
AND HC1 AFTER PASSAGE THROUGH XAD-8 AND TENAX GC
MACRORETICULAR RESINS
aliquot (ml)
influent
0-5
5-10
10-15
15-20
20-25
25-30
30-35
35-40
40-45
45-50
50-55
55-60
60-65
65-70
70-75
75-80
80-85
85-90
90-95
95-100
100-125
125-150
150-175
175-200
200-250
250-300
300-350
350-400
concentration (mg/L)
9.8
0.6
1.8
3.3
4.4
5.4
6.0
6.7
7.4
7.8
8.1
8.4
8.7
8.9
9.0
9.1
9.2
9.3
9.4
9.4
9.4
9.4
9.6
9.5
9.6
9.5
9.6
9.7
9.7
% recovery
—
5.6
18.3
34.1
45.3
55.0
61.5
68.8
74.5
79.8
83.1
86.1
89.0
91.1
92.4
93.7
94.7
95.4
96.0
96.0
96.7
96.7
98.0
97.6
98.4
97.6
98.7
99.0
99.3
263
-------
TABLE A-24.
LEAST SQUARE EQUATIONS AND MOLAR EXTINCTION VALUES FOR SELECTED NITROGENOUS COMPOUNDS
DISSOLVED IN AMMONIA FREE WATER (pH UNADJUSTED)
ro
compound
(X: pathlength
cm)
uracil (233:4)
uracll (254:1)
creatine (210:4)
creatine (233:4)
creatine (254:4)
concentration absorbance
mg/L M/L (A.U.)
1.0
2.5
5.0
10.0
1.0
2.5
5.0
10.0
1.0
2.5
5.0
10.0
10.0
10.0
8.92 x 10~!j
2.23 x 10 5
4.46 x 10~5
8.92 x 10
8.92 x 10~ij
2.23 x 10~5
4.46 x 10~5
8.92 x 10
6.71 x 10~!j
1.68 x 10*5
3.35 x 10~^
6.71 x 10
6.71 x 10~5
6.71 x 10"5
.079
.197
.398
.789
.067
.182
.343
.692
.077
.125
.295
.681
.039
.007
molar ' least square
extinction fit through
valueb origin0
2.214 x 103
2.179 x 10^
2.230 x 10.. concentration (mg/L) =
2.236 x 10 » 125.5 x absorbance
( X = 2.215 x 10 )
(/I2 = 25.6)
7.510 x-103
8.161 x 10-
7.689 x 10^ concentration (mg/L) •
7.757 x 10 144.4 x absorbance
( X = 7.779 x 103)
(^ = 274.9)
2.871 x 103
1.865 x 10
2.200 x 10;? concentration (mg/L) •
2.522 x 10 » 15.2 x absorbance
( X = 2.365 x 10 )
(^2 = 431.2)
1.454 x 102
2.610 x 101
(continued)
-------
TABLE A-24 (continued)
NJ
a*
Oi
compound
(A: pathlength concentration absorbance3
cm) mg/L M/L (A.U.)
cytosine (254:1) 1.0
2.0
4.0
5.0
10.0
20.0
creatinlne (254:1) 1.0
4.0
5.0
8.0
40.0
indole (254:1) 1.0
4.0
5.0
10.0
11.7
indole
9.0 x 10~^
1.8 x 10";?
3.6 x 10~i?
4.5 x 10"^
9.0 x 10"?
1.8 x 10"*
8.84 x 10"^
3.54 x 10~l
4.42 x 10~^
7.07 x 10":
3.54 x 10
8.54 x 10~*
3.42 x 10~|?
4.27 x 10~^
8.54 x 10~^
9.99 x 10
.010
.072
.161
.191
.458
.830
.007
.028
.043
.093
.530
.036
.125
.155
.334
.410
molar least square
extinction fit through
value b origin0
1.111 x 103
4.000 x 10^
4.472 x 103 concentration (mg/L) -
4.244 x 10 23.7 x absorbance
5.089 x 10,
4.611 x 10J
( X = 3.921 x 103)
(/s~2 = 1,424.8)
7.918 x 10*
7.918 x 10,
9.728 x 103
1.315 x 10, concentration (mg/L) «
1.499 x 10 76.2 x absorbance
( X = 1.074 x 10J)
(/s2 = 319.5)
4.217 x 103
3.661 x 10,
j
3.631 x 10 concentration (mg/L) »
3.912 x 10, 29.5 x absorbance
4.105 x 10J
( X = 3.905 x 103)
(Ss2 = 260.8)
(continued)
-------
TABLE A-24 (continued)
Ni
a\
compound
(X: pathlength
cm)
humic acid (330:1)
tryptophan (278:1)
adenine (256:1)
succinimlde (215:4)
succinimide (215:4)
concentration absorbance3
mg/L M/L (A.U.)
1.0
4.0
9.9
19.8
1
2
3
5
10
1
2
3
5
10
1
2
3
5
10
4.896 x 10~5
9.793 x 10';?
1.469 x 10~^
2.448 x 10~^
4.896 x 10
7.400 x 10~\
1.480 x 10"^
2.220 x 10~*
3.700 x 10~^
7.400 x 10
1.009 x 10~^
2.018 x 10
.
3.028 x 10":
5.046 x 10~*
1.009 x 10
.027
.107
.236
.470
.034
.062
.101
.162
.295
.105
.209
.306
.510
1.000
0.013
0.034
0.045
0.058
'0.120
molar least square
extinction fit through
value- origin0
6.944
6.331
6.875
6.618
6.025
( X =
(•/s~2.=
1.419
1.412
1.378
1.378
1.351
( x =
(^2 =
3.220
4.213
concentration (mg/L) =
41.9 x absorbance
xlO3
x 10*
x 10-
x 10^ concentration (mg/L) »
x 10 _ 32.9 x absorbance
• 6.559 x 10J)
: 383.6)
xioj
x 10,
A
x 10, concentration (mg/L) «
x 10. 9.9 x absorbance
A
x 10
1.388 x 10 )
278.6)
x 10^
x 10
?
3.715 x 10"
2.873 ,x 10, concentration (rag/L) »
2.973
( x =
(*S ' —
x 10 7 80.8 x absorbance
3.399 x 10Z)
56.0) ( continued ^
-------
TABLE A-24 (continued)
ISJ
0«
compound
(X: pathlength
cm)
5-chlorouracll (272:
pyrimldine (242:1)
purine (260:1)
purine (260:1)
Q
a
concentration absorbance
mg/L M/L (AtU.)
1) 1
2
3
5
10
1
2
3
5
10
1
2
3
5
10
6.826 x 10~*
1.365 x 1Q~1
2.048 x IQ~1
3.413 x 10~f
6.862 x 10"4
1.249 x 10~*
2.497 x I0~l
3.746 x 10~i?
6.243 x 10"?
1.249 x 10
8.325 x 10~1?
1.665 x 10~^
2.498 x 10~j?
4.163 x 10
8.235 x 10"5
0.059
0.117
0.164
0.269
0.533
.031
.066
.103
0.156
0.305
0.064
0.126
0.197
0.311
0.616
molar least square
extinction fit through
value origin0
8.644 x lo!?
8.570 x 10,
8.009 x 10^
7.882 x 10_ concentration (mg/L) •
7.808 x 10 18.6 x absorbance
( X' = 8.183 x 10J)
(^"2 = 394.9)
2.483 x 103
2.643 x 10,
2.750 x 10^
2.499 x 10 concentration (mg/L) =
2.443 x 10 , 32.3 x absorbance
( X = 2.564 x 10 )
(/s2 = 128.7)
7.688 x 103
7.568 x 10^
7.888 x 10_ concentration (mg/L) -
7.471 x 10 16.1 x absorbance
7.399 x 103 .
( X = 7.6028 x 10 )
(/? = 192.8)
pathlength = 1 cm; absorbance read against water reference
A(CJO where A = absorbance; C = molar concentration; E = molar extinction value;
i = pathlength = 1 cm
2
(least squares equation constrained to pass through origin)
proportionality constant = EAC/EA
-------
TABLE A-25.
PERCENT RECOVERY OF MIXTURE OF NITROGENOUS COMPOUNDS AFTER PASSAGE THROUGH XAD-8
TENAX RESINS (pH OF MIXTURE UNADJUSTED)
AND
10
ON
00
compound
uracll
indole
tyros ine
purlne
guanlne
cytoslne
adenlne
creatlnlne
tryptophan
15-20 ml ,.
Aa
35.5
0
17.1
29.9
20.2
18.4
37. B
33.1
0
HD
63.6
0
16.7
16.7
7.8
19.4
20.8
25.3
0
20-45 ml
A
59.7
0
55.7
44.4
29.6
23.3
14.1
11.1
0
H
81.8
0
60.4
41.7
39.7
25.0
11.5
13.3
0
45-100 ml
A
93.5
0
70.0
99.1
40.5
77.9
58.4
67.8
97.9
H
98.9
0
83.3
70.8
65.5
74.1
57.3
63.9
75.0
100-150 ml
A
88.7
0
67.1
81.2
52.6
88.3
77.0
89.2
65.1
H
99.4
0
85.4
83.3
78.4
93.5
78.1
85.5
75.0
150-200 ml
A
93.5
0
82.9
100
48.6
98.8
86.5
98.5
17.5
H
100
0
93.8
91.7
84.5
97.2
95.8
96.4
58.3
200-300 ml
A
100
0
88.6
100
55.5
100
98.9
100
100
H
100
0
100
100
91.4
100
100
100
69.4
300-400 ml
A
82.3
0
67.1
94.0
53.0
89.9
69.2
56.0
73.2
H
93.2
0
93.8
95.8
86.2
100
93.8
94.0
83.3
A • peak area computed from integrator counts
H - peak height
-------
TABLE A-26. PERCENT RECOVERY OF MIXTURE OF NITROGENOUS COMPOUNDS DISSOLVED IN A SOLUTION CONTAINING
20 mg/L HUMIC ACIDC ACIDIFIED TO pH 2.0 AFTER PASSAGE THROUGH XAD-8 AND TENAX RESINS
Compound
uracll
Indole
tyros ine
purlne and]
guanlne ]
cytosine
adenlne
creatlnlne
tryptophan
ISO
30-40 ml
A a H^
25.7
0
0
12.6
11.0
7.8
-
0
40.0
0
0
12.5
B.O
7.8
12.5
0
40-50 ml
A H
34.3
0
0
39.3
43.7
29.5
-
0
46.7
0
0
38.5
34.0
33.3
29.2
0
50-75
A
77.1
0
60.0
74.8
90.4
61.1
69.2
0
ml
H
74.7
0
50.0
74.0
70.0
68.6
75.0
0
75-100 ml
A H
65.7
0
67.5
80.8
81. 5
70.5
70.9
0
77.3
0
63.3
82.3
72.1
72.5
100
0
100-150 ml
A H
77.1
0
84.2
86.7
92.7
76.3
22.9
84.0
0
47.5
78.1
74.0
74.5
100
9.4
150-200 ml
A H
94.3
0
100
99.1
85.9
96.9
92.3
74.7
93.3
0
100
94.8
90.0
92.2
95.8
62.5
200-300 ml
A H
91.4
0
53.3
85.0
90.4
72.5
65.4
-
82.7
0
87.4
86.5
76.0
78.4
75.0
100
300-400 ml
A H
100
0
100
100
100
100
100
34.9
100
0
100
100
100
100
100
100
400-500 ml
A H
94.3
0
62.9
82.3
79.3
40.4
77.8
100
81.3
0
81.3
BO. 2
74.0
74.5
88.7
62.5
A • peak area as computed from Integrator counts
H • peak height
humlc acid not eluted
-------
TABLE A-27. VALUES OF KJELDAHL-N BLANKS (mg/L)
a
NJ
-»j
o
digestion solution
composition
A.
B.
C.
D.
134 g K2S04
5 ml SeOCl2
200 ml H2S04
diluted to
1 liter
20 g K2S04
0.2 g Se02
110 ml H2S04
diluted to
1 liter
134 g K2S04
2 g HgO
200 ml H2S04
diluted to
1 liter
20 g K2S04
0.1 g Se02
sample
reagent
blank
reagent
blank
reagent
blank
reagent
blank
reference
cell
undigested
NH3-N
blank
undigested
NH3-N
blank
undigested
NH3-N
blank
distilled
water
digestion duration
(minutes)
60
120
180
120
180
60
120
180
120
absorbance
at 635 nm
.075
.082
.068
.086
.072
.081
.052
.044
.140
.136
500 ml H2S04
diluted to
1 liter
determined by Scheiner's method (79)
.147
.152
.172
(mean = 0.149)
(standard deviation
= 0.014)
-------
TABLE A-28. EFFICIENCIES OF DIGESTION COMPOSITION AND DURATION
N>
source
Charles River
(12/19/77)
Charles River
(12/19/77)
Charles River
(12/16/77)
Charles River
(12/16/77)
Charles River
(12/16/77)
Charles River
(12/16/77)
sample
XAD-8
filtered
XAD-8
filtered
filtered to 10 ym
XAD-8
filtered
filtered to 10 pm
XAD-8
filtered
digestion
duration
(minutes)
60
60
120
120
180
180
180
180
70
150
70
180
180
Kjeldahl-Na
(mg/L)
4.2
4.0
4.1
4.0
4.0
3.9
4.0
3.8
7.0
7.0
7.1
7.8
2.4
digestion
solution
composition
134 g K2S04
5 ml SeOCl2
200 ml H2S04
diluted to liter with HJ
z
20 g K2S04; 0.2 g Se02
110 ml H2S04 diluted to
1 liter with water
134 g K2S04; 4 ml SeOCl2
200 ml H2S04 diluted to
1 liter with water
134 g K2S04; 2 g HgO
200 ml H2S04 diluted to
1 liter with water
Determined by Scheiner's method (79).
-------
TABLE A-29. RETENTION TIMES (IN MINUTES) OF NITROGENOUS ORGANIC COMPOUNDS
0-1
pyridlne
pyrrole
creatinine
uric acid
hydroxyproline
aspartic acid
succinimide
alanine
barbituric acid
hydroxyproline
i-(-)histidine
1-3
6-8
12-13
19-23
creatine
thymine
uracil
caffeine
tryptophan
indole
purine
guanine
cytosine
adenine
column: Zipax SCX
mobile phase: 0.01 M HN03
flow rate: 1.2 ml/min
wavelength: 254 nro
temperature: 30°C
TABLE A-30. RETENTION TIMES (IN MINUTES) OF NITROGENOUS ORGANIC COMPOUNDS
1 - 1.5
1.5 - 2
2 - 2.3
column: Aminex A-27
mobile phase: 0.325 M ammonium acetate
flow rate: 0.5 ml/min
wavelength: 254 nm
temperature: 30°C
4-(-)histidine
creatinine
uric acid
succinimide
aspartic acid
alanine
hydroxyproline
tryptophan
indole
2.3 - 2.5
cytosine
pyridine
uracil
proline
pyrrole
thymine
creatine
adenine
purine
guanine
272
-------
TABLE A-31.
AVERAGE ELUTION POSITIONS (ml) OF NITROGENOUS COMPOUNDS USING DIFFERENT
CHROMATOGRAPHIC COLUMNS AND MOBILE PHASES
u>
column
Zlpax SCX
Zlpax SCX
Zlpax SCX
Zlpax SCX
Zlpax SCX
Zlpax SCX
Zipax SCX
Zlpax SCX
Zlpax SCX
Zlpax SCX
Zlpax SCX
Zlpax SCX
Zlpax SCX
Zlpax SCX
Arainex A- 27
Anlnex A- 27
Zlpax SCX
Zlpax SCX
mobile phase
10~ M perchloric
10", M perchloric
10 M H3P04
0.172 M HAc (pN-2.5)
0.05 M H3P04 (pH-2.1)
10"2 HN03 (pH-2.0)
\Q~ UNO 3
10 II NO 3
ID'2 HN03
0.7 x 10-2 HN03
0.3 x 10-2 HN03
0.5 x 10-2 HN03
0.6 x 10~2 HNO;J
ID'2 HN03 with 1Z
acetonltrlle
3 M NH 4 Ac /HAc
0.3 M NH^Ac/HAc
0.5 M NaN03, 4 x 10~3
HN03
ID'2 I1N03
cytoslne
u
N.obs0
c
42 & 55
10.3 & 24
46
39
36
50.5
N.obs
N.obs
72.5
37.8
21.1
25.3
creatlnlne
8.6 & 34
N.obs
29 & 41
27 & 43
30 & 46
N.obs
75 & 115
35 & 58
N.obs
14.5
alanlne
7.55
4.84
6.6
6.6
6.2
3.74
5.09
Indole
2.0
2.0
1.91
1.87
1.96
2.0
1.77
1.8
1.9
2.02
1.88
water
1.37
1.52
N.obs
1.54
1.51
1.59
1 6 1.5
1.5
1.5
1 6 1.4
0.8 & 4.0
1.48
1.55
a
resolution
1/1/1
1/1/1/1
1/1/1
1/1/2
1/1/2
poor
poor
1/1
1/1/1/1
resolution: numbers indicate quantity of compounds each successive chromatographic peak (not
including water peak).
N.obs = no peak observed.
f\
Two values indicate more than one peak observed for the compound.
-------
TABLE A-32. RETENTION VALUES (ml) OF SELECTED NITROGENOUS COMPOUNDS ON ZORBAX CN
mobile phase
.05 M NaAc/HAc buffer
(pH 5.0); 29Z MeOH
.05 M NaAc/HAc buffer
(pH 5.0); 15Z acetonltrlle
.05 M NaAc/HAc buffer
(pH 5.0); 1Z acetonltrlle
.05 M NaAc/HAc buffer
(pH 5.0); 30Z acetonltrlle
.005 M NaAc/HAc buffer
(pH 5.0); 40Z acetonltrlle
.05 M NaAc/HAc buffer
(pH 5.0); 40Z acetonltrlle
.05 M NaAc; .05 N HAc;
(pH 4.74); 25* acetonltrlle
.05 N NaAc; .025 N HAc
(pH 5.0)
.01 M phosphate buffer
(pH 6.9)
cytoslne
3.25
3.42
3.78
16.2
14.7
2.89
2.85
4.07
4.26
creatlnlne
3.29
3.3
3.86
17.4
10.4
2.89
3.01
4.16
4.16
^^^^^•^^^••^^g^^^^^
alanlne Indole water
13.3 46.8 2.5 & 5.0
19.8 29.9 2.2 & 5.0
V
N. obs 87.54 2.8 & 3.7
N. obs 14.6 & 18.8 3.2 & 3.3
20.64 2.6 & 3.4
10.8 2.3 & 3.1
24.6
103.67
96.0
-?^^^^== =====
a
resolution
2/1/1
2/1/1
2/1
1/1/1
2/1
2/1
2/1
2/1
2/1
!
Resolution: numbers indicate quantity of compounds comprising each successive chromatographic peak
(not including water peaks).
N.obs = no peaks observed.
-------
TABLE A-33. RETENTION VALUES (ml) OF SELECTED NITROGENOUS COMPOUNDS ON ZORBAX C8
mobile phase cytoslne
.05 M NaAc/HAc buffer 2.64
(pH 5.0); 30% acetonitrile
(1/26/78)
.01 M phosphate buffer b 2.35
(pH 6.9); 1/27/78
.001 M phosphate buffer 2.22
(pH 6.9)
.01 M phosphate buffer 2.66
(pH 7.0); KH2P04 + NaOH
.05 M phosphate buffer
(pH 6.9); 30% acetonitrile
1/27/78
.05 N NaAc/HAc buffer 3.08
(pH 5.0); 15% acetonitrile
n
creatinlne alanine, indole resolution
* uracil &
guanine
2.67 - 42.5 2/1
2 . 35 - - unresolved
2.23 - - unresolved
2.71 - - unresolved
35.96
3.08 2.6 158.0 3/1
aresolution: numbers indicate quantity of compounds comprising each successive chromatographic peak
(not including water peaks)
column temperature heated to 33°C
-------
TABLE A-34. RETENTION VOLUMES OF CREATINE ON ZIPAX SCX3
concentration (mg/L) retention (ml)
100 10.7
200 9.9
300 10.2
500 9.2
1,000 8.5
2,000 7.9
2,300 7.6
'mobile phase: .05 M NH^PO adjusted to pH 2.5 with H
276
-------
APPENDIX B
a
»
o
o
3
I
Suplf:
1.
2.
1.
4,
5.
6.
ill of Mixture of 11 nltrogenoui coapoundi:
urccll 5.0 ag/L
Indol* i.O «K/L
(linlni 1,000 ag/L
tyroilnr 5.0 ti^/L
purlne 10.0 m«/L
guanlne 15.0 ng/L
Colupn picking: Zlpax SCX
Coluan dlnpnsiona: lenatti • 1
Mobile phase:
U.V. detector
Pressure: 3.000
Flow rat*: 2.2 ml/nln
Temperature: "v-20'C
Chart speed: 1'Vmln
7.
8.
9.
10.
11.
cr*itin»
cyton In*
•den in*
creatinlne
tryptophin
2,300
40.0
20.0 «g/L
50.0 «/L
20.0 «g/l
•; I.D. •
.05 • NH^PO*, adjmted to pH 2.5 with
lenRth - 233 nm: lensUlvlty
.0*
•1
10
is
20
MIIIIIIKrl
Figure B-l. Chromatogram of mixture of 11 nitrogenous compounds resolved on Zipax SCX (233 nm)
-------
ro
t
o
5
c
I
S«»pt«: SO ill of Mixture of 1 nitroft*noufl covpoundv:
ur«c 11
Indolr
alanlnc
tvroilne
pur 1 in-
Kuan tnc
'.0 •«/!,
S.O .K/I.
1.000 -g/1.
5.0 «R/L
10.0 ng/L
15.0 «K/I-
7. rreitlne
8. cytoaine
9. adenlne
10. crejitlnlfie
11. tryptaphin
1,100 .,/!,
40.0 «K/L
20.0 ««/L
SO.O mgfl
20.0 «K/1.
CollKKI picking: Zlp» STV
Cnlmn dln^nrtlons: length • 1 •; l.D. • 2.1 ••
Mobile ph«sr: .0') « NH4l'iP()4 .d)o.i,.d to pH ?.S with
U.V. detrctur: wnv«lrnpih - 254 n«; •rniltlvlty - .04
Prrtsurr: I.OHO p
-------
ro
t
o
fl
a
Compounds:
Sample: mxture of 11 compound*
Colum: Zlpax SCX (1 meter)
Eluent: 0.05 H NH.H.PO. adjusted to pH 2.5
•Hh HjPOj
Wavelength: 270 nm
Attenuation: 0.04
Temperature: ambient
Flow rate: 2.2 ml/mln
Chart speed: 1 Inch per Minute
Uradl 1.0 «K|/1
2. Indole 0.5 mg/1
3. Alantne 500 mg/1
4. Tyrostne 5.0 mg/1
Purine 10.0 mg/1
Guanlne 25.0 mg/1
Creatlne 200 mg/1
Cytoslne 10.0 mg/1
Adenlne 25.0 mg/1
Creatlntne 10.0 ng/1
11. Tryptophan 10.0 ing/1
Elutlon Time 1n Minutes
10
11
I
12
Figure B-3. Chromatogram of mixture of 11 nitrogenous compounds resolved on Zipax SCX (220 nm)
-------
t
i
c
1
. •lllllll...
'igure B-4. Chromatogram of mixture of 11 nitrogenous organic compounds
resolved on Zipax SCX (220 nm).
pl«: RAW Charlvi Klver. t ;i«^ r Id*;,-, MA. 1-2H-7A, cunctntr«E«d 125 fold
(- 22: mi/L ora.-inU-»)
( -H
Column dlB«.-nitonis: Irnxili - .'s on; l.D. • 2.1 M
Mobil* (rfij.c- .C'>« X.,N(M'IL'., hufii-r (pH • S.G)
L'.V. detocor: wjv.Ui^tli • 211 n>: ,inslilvil. - 0.0,
«: 2.00 40 10
•IllllllCrl
Figure B-5 . Chromatogram of Charles River water sample, resolved on Zorbax C-
280
-------
s
£
!•: l*w Coaci
-»)
.. .
I '
-------
t
o
4
Sample: Merrlmack River, Lawrence, MA, concentrated 100 fold, after filtra-
tion through XAD-4 nacroretlcular resin (• 69.5 mg/L organic-N).
Column packing: Zlpax SCX
Column dimensions: length -In; l.D. • 2.1 mm
Mobile phase: .OSM NH4H2P04 adjusted to pH 2.5 with HlPOi,
U.V. detector: wavelength - 233 nm; sensitivity - 0.04
Pressure: 2,500 psl
Flow rate: 2.0 ml/mln
Temperature: •v-20'C
Chart speed: 0.2"/mln
T- —p-
K> 20
Mlllllit*r«
30
4U
50
Figure B-7. Chromatogram of Merrimack River water sample, resolved
Zipax SCX.
on
282
-------
500-1
400 -J
c
o
-1
(0
(A
O
o
<0
90-
2| 70^
00
10
o;
*•»
i
o>
C
3
*-»•
cn
50-
30-
1ug/l
glycine—»•
10-
blank—*>
Figure B-8.
10
26
30
34
38
14 18 22
Millilltars Eluted
Fluorescence of aliquots from the chromatographic column after injection of 50 yl concen-
trated Marlboro West, raw, prechlorinated sewage effluent, column: Zipax SCX. Mobile
phase: .05 M NaHPO. adjusted to pH 2.5 with
^, .
34
-------
110-
to
CO
^ o 90-
•* "^
to
-t (A
< o
«= *
2. o
£ • 70-
1ug/l
glycine—t
50-
blank
30-
10-
I
4
8 10 12
I
14
16 18 20 22
1
24 26
I
28
30 32 34
I I I
36 38 40
Milliiiters Eluted
Figure B-9..
Fluorescence of aliquots from the chromatographic column after injection of 50 yl concen-
trated Marlboro West, raw, post chlorinated sewage effluent. Column: Zipax SCX. Mobile
phase:
0.05 M NaH^PO, adjusted to pH 2.5 with
^,.
-------
Pyrrole
H~
Purlne
'rollru
H
Succinlmlde
H
Pyrlmldine
Guonme
0 u
U
HN^SxV
^J-^
B
Y
NH2
Thymine
H
NH
0
Jrocil
H
N
f Y
L^NH
U
0
Borbituric Acid
r
5-Chlorourocil
Figure B-10. Chemical structure of some nitrogenous organic compounds.
285
-------
p-j Tyrosine
Tryptophon
HO
Creotinlne
CH,
CH2CH(NH2)C02H 0
NH
Phenylolonine
CH2CH(NH2)CC^H
m -ominophenol
OH
NH2
Indole
Histldlne
H
H02CHCH2
I
NH2
N
Figure B-10. (continued)
286
-------
APPENDIX C
LITERATURE REVIEW
THE HAZARDS OF CONSUMING CHEMICALLY CONTAMINATED DRINKING WATER
The proliferation of synthetic chemicals resulting from our expanding
industrialized economy has led inevitably to the entry of organic compounds
into our nation's water resources. Growing awareness of the deleterious
effect of these trace contaminants on human health and the increasing number
of organic chemicals identified in municipal water supplies have resulted in
a nationwide effort to identify and quantify the full spectrum of organic
compounds reaching the water consumer. Hundreds of organic contaminants,
present at nanogram to parts per million concentrations, have now been iden-
tified in municipal water supplies (1). Improved analytical techniques have
increased the number of organic chemicals identified in drinking water from
just 10 in 1970 (2) to 300 in 1976 (3) and more than 700 specific compounds
in 1978 (A). These values represent the total number of compounds identified
for all communities investigated. The actual number of contaminants present
in a single water supply is probably much smaller. The National Cancer
Institute, working collaboratively with the U.S. Environmental Protection
Agency, recently compiled a list of over 1,700 organic compounds found in
various kinds of waters ranging from industrial effluents to drinking water
(5-8).
The direct adverse effects of these compounds on the consumer include:
1) decreased aesthetic quality resulting from taste and odors, color, and
foaming; 2) toxicological hazards, both acute and chronic; and 3) increased
carcinogenic; or 4) mutagenic risks resulting from long-term, low-level
exposure. Additional harmful compounds are produced indirectly by reaction
of organic compounds with inorganic constituents.
Rook (9), and Bellar, Lichtenberg and Kroner (10) found that chloroform
and other halogenated methanes are formed during the chlorination of water
for disinfection. The carcinogenicity of these chlorinated products has
caused concern about their persistence in the environment and the extent of
their formation.
Unfortunately very little information is available about the human health
effects of trace quantities of organic chemicals in water. Epidemiological
studies on the effects of organic compounds identified in drinking water on
animal or bacterial populations have been minimal (11) . Heuper and Payne (12)
demonstrated the formation of spindle cell sarcomas at the site of sub-
cutaneous injection of carbon chloroform extracts (CCEO as well as internal
papilloma and neoplastic reactions in mice. Jolley et al. (13) reported that
287
-------
organic residues isolated from drinking water by reverse osmosis produced
mutagenes?s in Salmonella. A number of unpublished studies reported by the
director of the National Cancer Institute (14) have shown a pattern of
statistical association between elevated cancer risk rates and surrogates for
organic contaminants in drinking water.
Toxicological data on the risks of ingesting chemical pollutants in
drinking water are also scarce. Tardiff (15) reported that 128 slightly to
super toxic compounds and 43 suspect or positive carcinogens were present
among the organic compounds listed as having been found in tap water.
Kopfler (16) cited chloroethers and chlorobenzenes as examples of compounds
identified in drinking water that may be responsible for some forms of
chronic illness. The Environmental Protection Agency recently commissioned
the Medical College of Virginia to undertake a two year study on the impact
of trace organic compounds on human health. Although the leader of the
research team was reported to have asserted that none of the 700 trace
chemicals studied posed any significant threat to human health (17-19), this
was later refuted by both the research chairman (20) and EPA (11). Findings
of a 1977 National Academy of Sciences study on the effects of potential
toxicants (11) listed one compound, vinyl chloride, as a known human carci-
nogen, and two (benzene and benzo (a)-pyrene) as suspected human carcinogens.
Nineteen other compounds were listed as animal or suspected animal carcinogens.
The study also presented acceptable daily intake (ADI) levels for 45 other
organic compounds. ADI values indicate the level at which exposure to a
single chemical is not anticipated to produce an observable toxic response in
humans. The health effects of the majority of the 74 non-pesticides and 309
volatile organic compounds identified in drinking water and selected for
review, could not be assessed because of inadequate or unavailable toxicolo-
gical information.
The effects of chronic exposure to low does of organic micropollutants
remain largely unknown. However, EPA's scientific advisory board (21) agreed
that a currently unquantifiable human health risk exists from consumption of
organically polluted drinking water. The bases for the opinion that there
are hazards from consumption of chemically contaminated drinking water were
summarized by the director of the National Cancer Institute as follows:
1. Chemicals which have been shown to cause cancers in animal
studies are commonly found in drinking water in small amounts.
2. Some known human carcinogens have been found in drinking water.
3. Exposure to even very small amounts of carcinogenic chemicals
poses some risk and repeated exposures amplify the risk.
4. Cancers induced by exposure to small amounts of chemicals may not
be manifested for 20 or more years and thus are difficult to relate
to a single specific cause.
5. Some portion of the population that is exposed is at greater risk
because of other contributed factors such as prior disease states,
exposure to other chemicals, or genetic susceptibility (14)."
288
-------
The Interim Primary Drinking Water Standards promulgated in December,
1974 and effective June 1977 (22) reflected the insufficiency of epidemiolo-
gical and toxicological data on the health effects of organic chemicals by
their exclusion of maximum contaminant levels (MCL) for specific organic
chemicals other than certain pesticides. The original regulations did, how-
ever call for a maximum contaminant level for the total concentration of
organic compounds, as measured by the carbon chloroform extract which was
later withdrawn (21). A recent proposed amendment to the National Interim
Primary Drinking Water Regulations (4) contained a maximum contaminant level
of 0.1 mg/L for total trihalomethanes (TTHM's) including chloroform.
ORGANIC IMPURITIES IN NATURAL WATERS
Organic substances occur in the environment: either as the result of
natural processes or their introduction by man. Natural sources contribute
the majority of organic material in natural waters (4,23), via decay of
vegetation and animal tissues (humic matter), animal excretion, photosynthetic
byproducts and extracellular release of organic matter b> plankton and
aquatic macrophytes. Sources of man-derived organic contaminants include:
domestic, agricultural and industrial wastes, accidental spillages, dispersed
pesticides, rainfall, seepage and non-point-source runoff.
Until recently the organic content of water was generally evaluated
using gross analytical determinations such as Total Organic Carbon (TOC),
Chemical Oxygen Demand (COD), various extracting methods (Carbon Chloroform
Extract, CCE; or Gabon Alcohol Extract, CAE), or Biological Oxygen Demand
(BOD). Quantitative determination of individual molecular species present in
the microgram per liter range represented a formidable task. The need to
understand the specific nature of the array of contaminants present in our
water supplies, however, has led to significant progress in the development
of methods and instrumentation required for the identification and quanti-
fication of such contaminants. Resins, capable of removing specific cate-
gories of trace organic compounds, and the development of gas- and liquid-
chromatographic techniques have made possible the detection and measurement
of many types of both volatile and non-volatile organic compounds from dilute
sources.
Measurements of organic carbon in natural waters were reported as early
as 1926 by Birge and Juday (24) who investigated the organic matter content
of Wisconsin lakes. They found concentrations of 3-13 mg/L organic carbon
and 0.14-0.75 mg/L organic nitrogen. In 1934 during a survey of 529 lakes,
they reported (25) organic carbon concentrations ranging from 1.2-28.5 mg/L
with a mean of 7.7 mg/L. These determinations were based on total solids,
and may therefore have had positive errors, attributable to carbonate
decomposition.
A summary of the extent of organic impurities in natural waters is
presented in Table C-l. One particularly noteworthy study is the Environ-
mental Protection Agency's National Organics Reconnaissance Survey (NORS)
which was undertaken in response to the Safe Drinking Water Act of 1974 (40).
This act directed EPA to, "conduct a comprehensive study of public water
289
-------
TABLE C-l. ORGANIC IMPURITIES IN SURFACE WATERS
VO
o
Water Resource
Lake water
529 lakes
Tap water
Lake Huron
municipal water
(unidentified)
river water
(unidentified)
Municipal waters
Surface waters
Potable water
Constituent
organic carbon
organic nitrogen
organic carbon
organic compounds
eluted from carbon
filter with dlethyl
ether
average
organic carbon
average organic
carbon
carcinogenic
substances
chlorinated
hydrocarbons
Concentration
3-13 mg/L
0.14-0.75 mg/L
1.2-28.5 rag/L
(mean =7.7 mg/L)
113 Mg/L organic-C
3.1 mg/L
1.6 mg/L
11.8 mg/L
0.9 mg/L
3.2 mg/L
recovered
-
Place Year
1926
1934
Cincinnati 1951
1963
12 cities 1968
in AZ, CA,
NM, and OR
1969
1963
1965
lower 1970
Mississippi
Reference
Birge and Juday
Birge and Juday
Braus , H . et al.
Van Hall e£ al.
Nelson and Lysyj
(24)
(25)
(26)
(27)
(28)
Borneff (29)
Borneff e£ al,. (30)
Takemura et al. (31)
Sweet (32)
(continued)
-------
TABLE C-l (continued)
water Resource
Constituent
Concentration
Place
Year
Reference
NJ
\0
Water supplies
Potable water
Surface waters
Raw and finished
water supplies
Surface water
Surface water
Surface water
Surface water
carbon chloroform many exceeded
extractable organics Public Health
Service's recom-
mended limit
(= 200 ug/L)
chlorinated
hydrocarbons
carbon chloroform
extract
carbon alcohol
extract
46 organic
chemicals
0.02-0.57 mg/L
(weighted mean
0.08 mg/L)
0.03-4.6 mg/L
(weighted mean
0.21 mg/L)
trace amounts
di-n-butyl phthalate 1-30 ppb
dibutoxyethoxymethane
di-2-ethylhexyladipate
di-octylphthalate
di-isodecylphthalate
biphenyl, trichloro- 0.1-0.5 ppb
benzene butylbenzoate
dissolved organic
carbon
dissolved organic
carbon
0.1-15'mg/L
(mean =>1.2 mg/L)
< 1 mg/L
1970
U.S. Public Health
Service (33)
lower 1971
Mississippi
129 stations 1957
throughout 1972
United States
Friloux (34)
Committee Report (41)
lower 1972 E.P.A. (35)
Mississippi
Monatlquot 1972 Hites and Blemann (36,
River, MA 37)
Merrimack 1973 Malcolm and Leenheer
River, MA (38)
100 sites 1973 Malcolm and Leenheer
in 21 U.S. (38)
cities
Wilmington, 1973 Malcolm and Leenheer (38)
N.C. ^
(continued)
-------
TABLE C-l (continued)
Water Resources Constituent
Surface water CHC13
CHCl2Br
CHClBr2
CHBr3
Potable water chloroform
bromodichloro-
me thane
dibroraochloro-
me thane
Natural waters 16 organic
compounds
ISJ
VO
10 Raw and finished chloroform
water supplies
non volatile
organic carbon
85 organic compounds
Surface water total organic
carbon
Surface water total organic
carbon
Drinking water 187 compounds
Concentration
6.0-54.0 yg/L
A. 3- 20.0 yg/L
1.7-13.3 yg/L
1.1-10.0 yg/L
37.3-152 yg/L
2.9-20.8 ug/L
0.1-2.0 yg/L
identified
trace-0.9 yg/L
in 62% of the
sampled raw water;
0.1-311 yg/L in all
of the sampled fin-
ished water.
< 0.05-12.7 mg/L
(median = 1.5 mg/L
identified
7-45 mg/L
5 mg/L
identified
Place Year
stored 1974
surface
water
tap water 1974
from var-
ious muni-
cipal
supplies
1975
National 1975
Organics
Reconnaisance
Survey of
80 water
supplies
Minnesota 1975
river basins
Lake Super- 1975
ior
U.S. 1975
Reference
Rook (9)
Bellar, Lichtenberg,
and Kroner (10)
Pitt, Jolley, and
Scott (39)
E.P.A. (21)
Symons et al. (40)
Maier et_ al (23)
Maier e£ al (23)
Mullaney (42)
(continued)
-------
TABLE C-l (continued)
IO
u>
Water Resource
Surface waters
Surface waters
Natural waters
Drinking waters
Drinking waters
Drinking water
Drinking waters
Surface waters
Drinking water
Constituent
33 and 22 trace
organic compounds
140 trace organlcs
chloroform
16 organlcs
8 organics
72 organlcs
117 peaks in carbon
chloroform extract
27 classes of 360
organic solutes
160 acid extractable
compounds
89 base extractable
compounds
81 purgeable organic
compounds
700 trace chemicals
Concentration
identified
found
present in over
80% of the samples
ug/L levels
ug/L levels
identified
found
Identified at
concentrations
< 1 ppb
found
Place
2 Phila-
delphia
water
supplies
around
Industrial
centers
in the U.S.
5 supplies
in U.S.
13 U.S.
cities
England
204 sites
in S.W. U.S.
near heavily
Year
1976
1976
1976
1976
1976
1976
1977
Reference
Suf fet e_t al. (43)
Chian et al. (44)
Pitt, Jolley, and
Katz (45)
Coleman £t al. (46)
Keith e£ al. (47)
Newell (48)
E.P.A. (49)
industrialized
reported from
previous
areas
U.S.
cities
1978
E.P.A. (4)
findings
-------
supplies and drinking water sources, to determine the nature, extent, sources,
and means of control of contamination by chemical or other substances sus-
pected of being carcinogens (50)." The survey confirmed that the problem of
organic compounds in drinking water was widespread and that trihalomethanes
were present in yg/L quantities in most finished drinking waters as a result
of chlorination (51). The most recent comprehensive data on the presence of
organic chemicals in drinking water are found in the National Organics
Monitoring Survey (NOMS) of 1976 and 1977 (4,52). The NOMS was intended to
provide a more comprehensive survey of synthetic organic contamination in
finished drinking water by monitoring 21 specific organic compounds, and
four general parameters of organic content in 113 community water supplies.
The compounds were selected on the basis of possible occurrence, available
toxicological data, and the existence of analytical methodology for their
identification and quantification. The general parameters monitored in-
cluded: 1) total nonpurgeable organic carbon; 2) carbon chloroform extract;
3) ultraviolet absorbance; and 4) emission fluorescence. Initial results
(52) indicated that the occurrences and concentrations of trihalomethanes in
finished water were greater than for any of the other selected compounds
studied in the survey. Total trihalomethane concentrations ranged from 0.02
to 550 yg/L. Many of the selected contaminants were frequently found at low
concentrations in many of the cities. Supplies located on rivers downstream
from large industries were more susceptible to raw-water contamination.
General parameters did not correlate with the occurrences of specific organic
compounds.
Although large strides have been made towards quantifying the vast array
of organic contaminants in water supplies, the identity of only 40% by weight
of the non-humic material present has been established (21,11). Table C-2
shows the percentage of organic compounds identified in different categories
of materials present in natural waters. About 90% of the volatile organic
compounds has been identified, as compared to only 30-60% of the non-humic
non-volatile constituents (53,11). Included in the larger fraction of un-
identified compounds is the category of compounds comprising the topic of
this report: nitrogenous organic materials. The non-volatile property of
most of these substances has made their identification and analysis by gas
chromatography difficult without the prior formation of volatile derivatives.
Recent advances in the field of high performance liquid chromatography (HPLC),
however, have made the detection of non-volatile nitrogenous compounds in-
creasingly feasible.
NITROGENOUS ORGANIC COMPOUNDS
Although previous attention has been focussed on the environmental
hazards of non-polar chlorinated organic contaminants, nitrogenous organic
compounds are also environmentally significant. Many naturally occurring
nitrogenous organic compounds react readily with aqueous chlorine exerting
significant chlorine demand (54,55). Guter, Cooper and Sorber (56)
hypothesized that N-chloro compounds were formed during the chlorination of
polluted waters and that some of these react more readily with supposedly
selective reagents for free active chlorine than do the ammonia chloramines,
Several types of N-chloro organic compounds have been identified that yield
294
-------
TABLE C-2. COMPOSITION OF MATERIALS IDENTIFIED IN NATURAL WATERS
Classification
Volatile
Non Volatile
Humic Non Humic
Approximate % com-
position in natural
waters
Z of organic com-
pounds identified
in classification
10
90
75
15
Z of total com-
pounds identified
in natural waters
Z of non humic
compounds identi-
fied in natural
waters
36
The specific struc- 5-10
tures of the humic (of total non
substances have not volatile com-
been fully established pounds)
33-67
(of non-vola-
tile non humic
compounds)
A.5-9
18-36
aHumic substances comprise the major portion of the organic material
in natural waters. Percent composition of total organic material in
different waters will vary.
295
-------
interference or false positive tests in determining free chlorine (57),
Because these combined forms are generally much less germicidal than free
aqueous chlorine, falsely positive tests for free chlorine may indicate a non-
existent bactericidal or virucidal behavior. Reliable measurements of the
free chlorine present in water supplies or wastewater containing nitrogen can,
therefore, not be made unless the types of organic nitrogen compounds present
are known.
Reactions of a broad range of nitrogenous organic substances representa-
tive of materials thought likely to occur as part of the organic nitrogen of
water sources have been studied (54, 234) and some (m-aminophenol, uracil,
tryptophan, pyrrole, chlorophyll, alanine, proline, Jl-hydroxyproline, and
indole) have been found to be potential precursors of trihalomethanes in the
chlorination of water supplies. Purine and pyrimidine bases such as caffeine,
cytosine, and uracil (commonly found in wastewaters) have been shown to
produce complex stable mutagenic chlorinated products upon chlorination (58-
65) suggesting that N-chloro compounds may be significant intermediates for
the compounds causing mutagenic activity in finished waters. 5-chlorouracil
was also shown to effect the hatchability of carp eggs (66) and to cause ab-
normalities in the larval fish produced (67). In addition certain types of
nitrogenous compounds which may be present in polluted drinking water sources
are themselves potential health hazards or direct precursors. Included among
these compounds are secondary amines capable of forming carcinogenic nitro-
samines (68), nitroaromatic compounds, and some hetero-cyclic materials. Some
trace nitrogen containing polycyclic compounds are known carcinogens (69).
Brown et al. (70) found the formation of local sarcomas in rats following
subcutaneous injection of unspecified oxides of purines. Nitro and amino
derivatives of pyrrole may also be carcinogenic (71). Tryptophan and two
related compounds, indole, and indolacetic acid have been shown to enhance the
incidence of bladder cancer in certain closely defined situations (72-76).
Organic nitrogen enters the environment via the pathways previously
described for organic substances in general. Table C-3 summarizes the sources
and properties of selected nitrogenous organic compounds. The chemical
structures of some of these substances are shown in Appendix B, Figure B-10.
Several studies have begun to characterize the organic nitrogen compounds
present in domestic sewage (39, 81-84) and urine (85). Organic nitrogen
concentrations have been reported to be between 0.064 mg/L - 0.24 mg/L in
several English lakes (86) and from trace amounts to 0.28 mg/L in streams of
southern New Jersey (87). Ram (87) reported that the organic nitrogen fraction
ranged from traces to 15.6% of the total Kjeldahl nitrogen in streams of
southern New Jersey. Some attempts have been made to distinguish and determine
amino acids in natural waters (88-90). The quantities found, some pg/L of
nitrogen -in toto seem small compared with the total organic nitrogen expected.
Briggs (91) estimated that free amino acids were present at concentrations of
approximately 10~9 g/L in water while Semenov and his coworkers (92) reported
levels of free amino acids in surface waters of the Soviet Union ranging from
2-25 yg/L. Peptide organic nitrogen has been reported to comprise between
15-43% of the total organic nitrogen in surface waters (86,93) with concen-
trations in the range of lO""* mg/L (91). A summary of the organic nitrogen
contents found in natural waters is shown in Table C-4.
296
-------
TABLE C-3. SOURCES AND PROPERTIES OF SELECTED NITROGENOUS ORGANIC COMPOUNDS (77-80, 54, 41)
Compound
Natural Sources
Man Made Sources
Properties
NJ
VO
•vj
Adenine
Alanine
m-Aminopheno1
a purine; nucleic acid unit.
widespread throughout animal and
plant tissues; constituent of
nucleic acids and coenzymes
an amino acid.
In soil: sllty loam:
clay soils:
excreted in urine:
6-160 yg/kg 0.55 mg/kg body wt
30-400 ug/kg per day; In domestic
sewage: 5 ug/L
derivative of phenol, coal
tar
Aspartlc acid an amino acid.
occurs in animals and plants
Barbituric acid pyrimidine derivative
exreted in urine:
0.37-3.7 mg/kg
body weight per day
MWa= 135.14
LD5Q orally in rats 745
mg/kgb
W - 256.4 (H20)C
MW = 89.09
Amax = no maxima at pH 8
potential trichloro-
me thane precursor
MW = 109.12
= 0.144 g/kg in white
mice
= 1 g/kg In rats
MW = 133.1
MW - 128.09
*max = 257 (neutral)
(continued)
-------
TABLE C-3. (continued)
Compound
Natural Sources
Man Made Sources
Properties
Caffeine
to
vD
00
Chlorophyll
5-Chlorouracil
Creatine
Creatlnine
a purine;
occurs in tea, coffee, and mat£
leaves, guarana paste and cola
nuts
green pigment of plants
present in muscular tissue of
many vertebrates
occurs in all soils and in grain
seeds and other vegetable matter
Iso cyanurlc acid
undisinfected sewage
effluent:
-------
Table C-3. (continued)
Compound
Natural Source
Man Made Source
Properties
Cytosine
Glutamic acid
Glycine
Glycylglycine
Guanine
4-Histidine
a pyrimidine; nucleic acid unit*
widely distributed In nature,
constituent of yeast and of
wheat embryo
an amlno acid
an amlno acid; gelatin and silk
fibroin are best sources
normal constituent of proteins
simplest of all peptides
a purlne; nucleic acid unit
occurs in animal and vegetable
tissues, in excreta; fish scales
an amino acid .
excreted by man in
urine 1.8-11.5 mg/kg
body wt per day
excreted by man In
urine 2.3-18.0 mg/kg
body wt per day
excreted by man:
in urine: 0.98-6.6
mg/kg body wt/day
in feces: 1.4-2.1
mg/kg body wt/day
in sweat: 6-10 mg/
100 ml
MW = 75.07
xmax " 27° (water)
MW = 147.13
MW - 75.07
*max • 630 (water,
copper chelate)
MW = 132.12; Xmax - no
maximum at pH 7
MW = 151.13
xmax • 248 (cation)
MW = 155.16
*max = 217.5
(pH=7)
(continued)
-------
TABLE C-3. (continued)
Compound
Natural Source
Man Made Sources
Properties
fc-Hydroxyproline an amino acid
Indole
Leuclne
constituent of coal tar
natural pigments
an amino acid
UJ
o
o
i-Proline
an amino acid
Pyridlne
excreted by man in
urine: 0.02 mg/kg
body weight per day
excreted in human
feces, contents of
domestic sewage:
0.25 ug/L
excreted by man:
in urine: 0.2-0.52
mg/kg body wt/day
in feces: 4.3-6.9
mg/kg body wt/day
in sweat: 1.2-4.2
mg/100 ml
excreted in man in
urine: 0.3-0.9 mg/kg
body wt/day
constituent of coal
tar
MW = 131.13
potential trichloro-
methane precursor
MW = 117.14
Amax = 278 (H20)
potential trichloro-
methane precursor
MW = 131.7
no maximum U.V. absorp-
tion at pH 8
MW - 115.13
xmax = 200 (MeOH) poten-
tial trlchloromethane
precursor
MW = 79.1 faint odor at
.0037 mg/L; acute oral
LD50 in rat = 0.8-1.6
g/kg; transient symptoms
in man: 125 ppm, 4 hrs/
day, for 1-2 weeks;
Amax = 250 (H20)
(continued)
-------
TABLE C-3. (continued)
Compound
Natural Source
Man Made Source
Properties
Pyrlraidlne
Pyrrole
Sarcoslne
dfc-Serine
Succlnlmlde
Taurlne
Thymlne
Tryptophan
basic heterocyclic ring
building block for chlorophyll
hemoglobin, hemocyanin, etc.
found in star fishes and
sea urchins
an araino acid
present in bile, lungs and
flesh of oxen, shark blood
muscles, oysters
pyrimidine derivative, isolated
from thymus; nucleic acid unit
an amino acid
constituent of coal
tar and bone oil
excreted by man in
urine 0.35-1.4 nig/kg
body wt/day
MW = 80.09
*max = 200 (MeOH)
potential trichloro-
methane precursor
MW = 67.09; minimum
lethal dose: in mice =
60.5 g/kg; potential
trihalomethane precursor;
Xmax = 205 (H20)
MW = 89.09
MW
105.09
MW - 99.09
MW • 125.14
MW = 126.11
266
excreted in man in MW = 204.22; Xmax a 278
urine: 0.23-1.3 mg/kg (pH 7); potential tri-
body wt/day also found halomethane precursor
in human saliva & blood _ .
(continued)
-------
TABLE C-3. (continued)
Compound
Natural Sources
Man Made Sources
Properties
10
O
TyrosIne
Urea
Uracil
Uric acid
widely distributed amlno acid
In soil:
sllty loam: 0-65 ug/kg soil
clay soils: 10-60 ug/kg soil
product of nitrogen metabolism
in mammals
pyrlmidine derivative
hydrolysis product of nucleic
acids
chief end product of the nitro-
genous metabolism of birds and
scaly reptiles, found in their
excrement; present in urine of
all carnivorous animals
excreted in man in MW » 181.19
urine: 0.35-1.45 mg/kg Amax - 274 (PH 7)
body weight per day
contents of domestic
sewages: 2-6 mg/L;
domestic sewage efflu-
ent: .020 mg/L
in sewage effluent:
0.013 mg/L
contents of domestic
sewages: 0.2-1.0 mg/L
MW = 60.06
Amax = 260 (pH 9.5)
MW - 112.09
^max = 203, 258 (pH :7)
potential trihalomethane
precursor
MW = 168.11
*MW » molecular weight
LD5Q calculated lethal dose expected to kill 50% of an experimental animal population
*
( ): parentheses indicate solvent or conditions used in absorbance measurement
-------
TABLE C-4. CONCENTRATION OF TYPES OF ORGANIC NITROGEN IN SURFACE WATER
Water Resource Constituent
Concentration
Place
Reference
10
o
Natural Waters amino acids
Lake Water
Lake Water
River Water
peptide-N
total organic
nitrogen
total organic
nitrogen
1 ug/L nitrogen
in toto
0.056-0.436 rag/L Lake Mendota
Lake Michigan
0.064-0.24 mg/L England
trace-0.28 mg/L Southern N.J.
Gardner & Lee (88)
Peake, Baker & Hodgson
(89)
Georgiadls & Coffey (90)
Domogalla, «2t jil. (93)
Fogg & Westlake (86)
Ram (87)
Lake, estuary,
and ocean waters
Lake Water
dissolved free
amino acids
particulate
organic nitro-
gen
found
approximately
15% of the
particulate
organic carbon
Grob (94)
N.E. Wisconsin Croll (224)
(continued)
-------
TABLE C-4. (continued)
U)
o
Water Resource
Lake Water
Tap Water
Constituent
free amlno
acids; pep tides
free amino
acids
Concentration
* 10~9 g/L
^ ID'7 g/L
none found
Place
North Island,
New Zealand
London, England
Reference
Briggs (91)
Sidle (95)
Surface Water
Surface Water
River Water
Hydrolyzed tap
water
River Water
free amino acids 2-25 yg/L as N
proteins 20-340 yg/L as N
volatile amines 6-100 yg/L as N
peptides
common amino
acids (serlne
and glycine)
pyrrole
isoleucine +
leucine
valine
glycine
alanine
glutamic acid
aspartic acid
aromatic amines
aliphatic amines
proline
tryptophan
indole
skatole
hydroxybenzamide
226-250 yg/L
36-61 yg/L
6.03 ug/L
15.23 yg/L
6.45 yg/L
3.83 yg/L
5.00 yg/L
3.06 yg/L
Soviet Union
Rhine River
Semenov (92)
Newell (96)
Rhine River Holluta (98)
London; England Sidle (95)
Holluta & Talsky (97)
(continued)
-------
TABLE C-4. (continued)
Water Resource
Constituent
Concentration
Place
Reference
Lake Water
u>
o
Receiving Water
below an Indus-
trial plant
Drinking water,
river water,
and ground water
glyclne, a-alanlne,
aspartlc acid, glu-
tamlc acid, methlonlne
sulphoxide, arginine,
glycine, hydroxypro-
llne, isoleucine,
leucine, lyclne,
norleucine, norva-
llne, ornlthlne,
phenylalanine, prollne,
serlne, threonlne and
tyrosine
N-containing
heterocycles and
other nitrogen
containing com-
pounds
North Island,
New Zealand
Briggs (91)
Jungclaus e_£ al. (99)
Compound;
aspartlc acid
serine
glutamic acid
glycine
alanine
leucine
phenylalanine
Drinking River
Water Water
Ground
Water
.67 ug/L 2.66 ug/L 1.33 ug/L
1.05 ug/L 4.20 yg/L 1.58 ug/L
.74 yg/L 4.41 ug/L .74 ug/L
.75 ug/L 4.50 ug/L 1.50 ug/L
.45 ug/L .89 ug/L .45 ug/L
.33 ug/L .33 ug/L .33 gg/L
.83 ug/L .83 ug/L
Kasiske et al. (231)
-------
ALGAL PRODUCTION OF N-ORGANIC EXTRACELLULAR METABOLITES
Blue-green algae have received considerable attention as major nuisance
algae because they proliferate in waters where conditions are unfavorable for
most other types. They are usually found in neutral to alkaline waters, are
able to tolerate a relatively wide temperature range, and are often associated
with taste and odor problems (100, 101). Although most algae are known to
liberate organic compounds in general into the surrounding aquatic environ-
ment during growth (102), blue green algae are known also to liberate
comparatively large amounts of organic nitrogen (86, 103, 104). Up to 50% of
the carbon fixed by a population can be released (105). The amount of release
per cell is known to be inversely related to population density (102,106,107),
and is favored by high pH (108) and inorganic nutrient deficiency (105,109).
(High oxygen)-(low carbon dioxide) concentrations and low light intensities
typical of conditions in natural waters also favor release (104). Greatest
liberation in laboratory grown cultures of blue-green algae occurs during the
exponential phase of growth (104-106,110) although Thompson (104) observed a
second per cell increase in liberated total organic carbon in late growth.
Thompson (104) also reported a linear increase in secreted TOC with time and
a linear increase of TOC with time except for an initial peak production at
about ten days for laboratory grown cultures of Oscilaatoria tenuis and
Anabaena flow aquae respectively after a two-day lag-period in both. Various
authors have reported release of leucine, serine, alanine, aspartic acid,
valine (103,111), alanine, aspartic acid, arginine, leucine, proline, valine
(112), glycine (103), and other amino acids, polypeptides and proteins (102,
103,113,114).
These reports suggest that isolation and identification of N-organic
compounds in water supplies can be facilitated by sampling during a blue-
green algal bloom.
UROCHROMES AND HUMIC SUBSTANCES
Urochromes are yellow pigments discharged in human urine as the metabolic
product of porphyrins (115). They are found in urine (116-119), sewage (10-
243 mg urochrome per liter), water from drainage canals (3-14 mg urochrome
per liter) and treated surface waters (0.6-1.5 mg urochrome per liter) (120).
Their presence in water sources is a clear 'indication of fecal contamination
and is therefore of extreme concern from the standpoint of public health
(115,121). In addition to the indirect hazards arising from their association
with enteric bacteria (121), their presence in water has been related to the
occurrence of endemic endocrine goiter in humans (122-124).
Elementary analyses of urochromes oxy-A and B have shown them to be
comprised of about 2.44% and 2.61% nitrogen respectively (122,125). Since
they have some of the nitrogenous structure of pyrrole and porphyrin they are
potential haloform precursors. They are therefore, considered in this report.
The enrichment and isolation of urochromes from water samples according
to Hettche (122,126,127) is carried out by adsorption onto aluminum oxide or
entrainment precipitation with alum and ammonia. After elution with formic
306
-------
acid and dilution to a known volume they are determined by absorptiometry at
380 nm. Both humic acids and lignin derivatives interfere with quantitative
evaluation of urochromes (121,115). Humic substances are high-molecular-
weight compounds comprising an unresolved range of complex polymeric aromatic
organic acids. They result from the aqueous extraction of the soluble fraction
of wood tissues, dissolution of decomposition products of decaying wood and/or
leaching of soluble soil components (26,94). Knorr et dl. (128) could not
distinguish humic acids from urochromes A or B infrared or ultraviolet
spectroscopy, electrophoresis, fluorescence, surface tension, or nitrogen
content. Hettche (122) proposed distinguishing between urochromes and humic
acids by computing the difference in the logarithms of the absorbance values
at 380 and 530 nm for urochromes and humic acid respectively, and multiplying
by 1000. A value equal to 0.9 for pure urochrome and 0.537 for humic acids,
was suggested (115). Sattlemacher and Furstenau (129) however, observed that
no specific extinction curve could be plotted for humic substances in the
visible region. They concluded that the method of Hettche for the determi-
nation of urochromes was inexact. They developed a separation scheme
utilizing paper chromatography after esterification with diazooctadecane and
extraction with chloroform.
Humic substances are also considered in this report because they form the
major part of the organic material in naturally high-colored waters (26).
They are derived from vegetative material and have certain similar character-
istics. Humification takes place under a wide range of environmental
conditions. Being resistent to microbial and chemical decomposition, humus
tends to accumulate even under aerobic conditions. Unfortunately, the
chemical structures of aquatic humic substances are not known with any
certainty (233). Oden (130) classified all humic substances into three groups
according to their solubility in strong acid and alcohol:
TABLE C-5. CLASSIFICATION OF HUMIC SUBSTANCES, AFTER ODEN (130)
Classification Strong Acid Alcohol
fulvic acid soluble -
hymatomelanic acids insoluble soluble
humic acids insoluble insoluble
Croll (224) reported the elemental analysis on an ash free basis for the humic
plus hymatomelanic acids and for the fulvic acids to be: Carbon 51.52%,
Hudrogen 4.59%, Nitrogen 2.84% and Carbon 55.61%, Hydrogen 5.91%, Nitrogen
2.13%, respectively.
Because humic substances usually comprise a large percentage of the
organic content of natural waters and because they contribute minimally to the
total organic nitrogen content (224) it was hypothesized that their removal
307
-------
from water samples might facilitate the isolation of individual nitrogenous
compounds. Stuermer and Harvey (131) commented on the hydrophobic character-
istics of humic substances, implying the use of selective macroreticular
resins for their removal. Since the bulk of organic nitrogen is associated
with hydrophilic substances (131,38) it appeared analytically feasible to
remove the potentially interfering humic materials from water samples with
resins which selectively adsorb hydrophobic compounds. Theoretically, a
prefiltration through such a resin bed would not significantly effect the
hydrophilic nitrogenous compounds. Generally speaking, macroreticular resins
of low and intermediate polarity adsorb hydrophobic solutes, while they do not
adsorb hydrophilic solutes (38).
Several studies have been conducted on the adsorption of humic substances
onto XAD macroreticular resins (Rohm and Haas Company, Philadelphia, PA).
Although Oulman (133) reported that XAD-4 had poor capacity for humic sub-
stances, Chen (134), Blunk (135), Stuermer and Harvey (131), and Weber and
Wilson (136) observed strong adsorption of humus onto macroreticular resins of
varying polarity. Cheng (134) found that adsorption of humic acid by XAD
resins was generally favored at neutral conditions (pH 6-7). Blunk (135)
adsorbed 97-99% of humic material from untreated river water as assayed by
absorbance at 450 nm onto XAD-7. Weber and Wilson (136) adsorbed fulvic acid
onto XAD-2 at pH 1 and Stuermer and Harvey (131) reported adsorption
efficiencies of greater than 90% for humic substances in seawater acidified to
pH 2 onto XAD-2. Junk et al. (132) observed an increase in the adsorption of
organic compounds onto XAD by adding 5 ml of hydrochloric acid per liter
(equal to .056 M or pH of about 1.3) of standard water sample tested. Oliver
(233), and Christman (234) acidified water samples to pH 2.0 and pH 2.2,
respectively to adsorb humic material in these samples onto XAD resins.
Cheng (134) found that humic substances at pH 5 were more strongly ad-
sorbed onto XAD 1, 2, 4, 7, and 12 than onto XAD-8. Leenheer and Huffman
(38) reported that XAD-8 was more effective in adsorbing fulvic acid than
were XAD-2 and 4. XAD-8, however, exhibits less irreversible bonding than do
XAD-2 and XAD-4 (Leenheer, J.A., personal communication). Its use in the
pretreatment filtration step of natural waters is therefore favored.
MACRORETICULAR RESINS
Natural and polluted waters contain a numerous variety of natural and
synthetic compounds that seriously interfere with the isolation, identifi-
cation and determination of individual nitrogenous organic compounds. Rohm
and Haas macroreticular resins are known to adsorb selectively a broad range
of hydrophobic organic compounds (Table C-6). These undesired or interfering
carbonaceous organic materials may be removed by filtration through appropriate
XAD resins prior to concentration and chromatographic separation without sig-
nificant reduction in most nitrogenous constituents, with the exception of
heterocyclic aromatic substances.
XAD macroreticular resins are hard insoluble beads, 20-50 mesh, varying
from white to light brown in color. XAD-2 and XAD-4 have nonpolar surfaces,
XAD-7 and XAD-8 have intermediate surfaces and XAD-12 is highly polar (38).
308
-------
TABLE C-6. ORGANIC COMPOUNDS ADSORBED ONTO XAD AND TENAX MACRORETICULAR RESINS
Resin
Compounds adsorbed
% recovered'
Reference
o
VO
XAD 2
XAD 2
XAD 2
XAD 2
XAD 2
XAD 2
polycyc-Hc aromatics, n-alkanes, 36 - 100
phthalates, halogen compounds, phenols average:
fatty acids, fatty acid methyl esters, 83.27%
steroids
pesticides in natural waters
coprostanol (a characteristic sterol
found in the feces of man and higher
animals)
organic contaminants
alcohols, aldehydes, acids, aromatic
halides, alkylbenzenes, phenols, esters,
ethers, ketones, polynuclear aromatics,
herbicides, pesticides and various
compounds containing halogens, nitrogen
or sulfur
Shinohara et al. (137)
97% at flow
rate = 3 ml/
min
Junk £t al (138)
Wun e_t al. (139)
Burnham et al. (14°)
average recov- Junk et al. (132)
ery rate of 110
individual organ-
ic solutes was
78% with a
standard devia-
tion of 6.3%
Nitrogen compounds:
Hexadecylamine 94
Nitrobenzene 91
Indole 89
0-Nitrotolutene 80
N-Methylaniline 84
Benzothlazole 100
Quinoline 84
Isoquinoline 83
Junk et al. (132)
(continued)
-------
TABLE C-6. (continued)
Resin
Compounds adsorbed
% recovered
Reference
XAD 2
XAD 2
XAD 2
Benzonitrile
Benzoxazole
visible color of 6 textile dyes
alcohols
aldehydes and ketones
esters
acids
phenols
ethers
halogen compounds
polynucleic aromatics
alkylbenzenes
nitrogen and sulfur compounds
pesticides and herbicides
weighted average
alcohols
aldehydes and ketones
alkanes
amines
aromatics
benzothizoles
esters and ethers
halogenated compounds
PCB's
phenols
weighted average
98
92
all visible
color removed
94
95
93
101
89
90
87
89
90
89
90
91
100
74
5
14
68
67
74
57
78
46
59
Webb (141)
Junk et al. (142)
Junk et al. (143)
(continued)
-------
TABLE C-6. (continued)
Resin
Compounds adsorbed
% recovered
Reference
XAD 8
XAO 2, 4,
7 and 8 and
mixtures
XAD
General
14 compounds previously identified
in industrial effluents
13 organic pollutants
phenols, alkyl sulfonic acids, dyes,
steroids, vitamin B-12, fulvic acid
XAD-2
XAD4/8
Tenax GC
20 compounds representing aliphatic
hydrocarbons, aliphatic and aromatic
halogenated hydrocarbons, phthalates,
polynuclear aromatic hydrocarbons
average: 63.31 Webb (141)
an equal weight
mixture of XAD
4 and XAD-8 was
most efficient
Van Rossum and Webb (144)
many non-ionic Burnham et al.
organic compounds
extracted from
dilute aqueous
solution with
approximately
100% efficiency
(145)
All of these
resins gave com-
parable results
of 64% average
recovery for
equal volumes and
equal weighto of
resin. Tenax
averaged 80%;
XAD-2, 64%;
and XAD 4/8,
69% for equal
surface areas of
resin
Webb (146)
£
after elution from resin with appropriate solvent
-------
XAD-2 and XAD-4 are styrene-divinyl benzene copolymers while XAD-8 is an
acrylate ester. XAD 2, 4 and 8 have average surface areas of 330 m2/g, 750
m g and 140 m /g respectively and pore sizes of 90 angstroms, 50 angstroms and
250 angstroms, respectively (141). Surface adsorption is the principal con-
tributing factor to retention on the XAD resins (147,141). No ion-exchange
mechanism is involved (141). Leenheer and Huffman (38) reported the absorptive
capacity of XAD to vary from 5-20 mg organic carbon per gram of resin for
different hydrophobic organic compounds.
Liquid-liquid extraction and carbon adsorption were also considered as
alternative methods for removing interfering hydrophobic materials from water
samples. Each of these methods, however, has several significant draw-backs
in comparison with the XAD resins (Table C-7), and were therefore not employed.
In addition to the XAD series other macroreticular resins have been
developed for selective adsorption of organic compounds from water. Ambersorb
XE 340 (Rohm and Haas, Philadelphia, PA) resin was recently designed to remove
nonpolar hydrophobic organic compounds or halogenated organic molecules from
the aqueous or gaseous phase (148). It is comprised of hard, nondusting,
black spheres whose chemical composition is intermediate between that of
activated carbon and polymeric substances. It has a surface area (N2, BET
method) of 400 m2/g and a pore size distribution ranging from 6-300 angstroms
(148). XE 340 has a high capacity for removal of low molecular weight
chlorinated organic compounds such as chloroform (148).
Tenax GC (Applied Sciences Laboratory Inc., State College, PA) is a
porous polymer based on 2,6-diphenyl-p_-phenylene oxide. It is designed as a
gas-chromatographic support for the separation of high-boiling polar compounds
such as alcohols, polyehtylene glycol compounds, diols, phenols, mono- and
diamines, ethanolamines, amides, aldehydes, and ketones (150). It has been
used to concentrate organic compounds from air (151) and has been suggested
as a useful direct accumulator for organic compounds from water in a manner
similar to XAD resins (146). Novotmy (152) proposed that Tenax be used as an
adsorbant for a wide range of both polar and nonpolar compounds. Webb (146)
found significantly better recoveries of phenol (72%), camphor (83%), and
alphaterpineol (87%) on a 13 cm Tenax column in comparison with a comparable
XAD column. Twenty organic compounds were recovered with 80% efficiency on
the Tenax resin.
ISOLATION OF TRACE ORGANIC COMPOUNDS FROM DILUTE AQUEOUS SOLUTION
Determination of trace nitrogenous organic compounds, present at only
nanogram levels, requires several analytical procedures: 1) selective ad-
sorption of extraneous interfering organic compounds with macroreticular
resins; 2) concentration of the water sample to achieve detectable elevels of
dissolved solutes; 3) separation and detection of constituent compounds using
high pressure liquid chromatography; and 4) identification of resolved con-
stituents. Selective adsorption of the hydrophobic portion of the dissolved
organic carbon in natural waters was considered in the previous section.
312
-------
TABLE C-7. COMPARISONS WITH XAD RESINS
Method
Comments
Reference
Carbon Adsorption
LJ
M
U)
difficult to extract large volumes of water
containing small amounts of organic pollutants
unacceptable background contamination values
incomplete recovery of compounds
non-specific adsorption of compounds
Irreversible adsorption of solutes
lack of adsorption/desorption control
bacterial and oxidizing attack on the adsorbed
organic compounds
meticulous purification required
Van Rossum and Webb (144)
Van Rossum and Webb (144)
Webb (141)
Webb (141)
Burnham e± al. (145)
Malcolm and Leenheer (38)
Sproul and Ryckman (149)
Sproul and Ryckman (149)
Burnham e_t al. (145)
Burnham et al. (145)
Solvent Extraction
distribution coefficient for the contaminants
between water and an extracting solvent may be
unfavorable
labor requirement excessive
difficult for highly polar organic solutes
comprising a major fraction of organic solutes
should be convenient to extract hydrophobic
compounds without extracting hydrophilic
substances
Burnham et al. (145)
Van Rossum and Webb (144)
Webb(141) ; Van Rossum and
Webb(144)
Malcolm and Leenheer (38)
(continued)
-------
TABLE C-7. (continued)
Method Comments Reference
Macroreticular selectively adsorb compounds of different polarity
Resins
low energy requirements, not Involving any phase
transition.
low temperature and high vapor pressure at which
adsorption occurs
favorable kinetics Rohm and Hass publication (148)
homogenious surface Malcolm and Leenheer (38)
high surface area and capacity
good stability
only one adsorption mechanism operative,
allowing solutes to be quantitatively sorbed
and desorbed
good hydraulic flow characteristics for column
operation
-------
Numerous methods of concentration have been reported in the literature
including: liquid-liquid extraction, freeze concentration, adsorptive bubble
separation, chromatography, ion exchange, ultra-filtration, adsorption,
distillation, evaporation, sublimation, and reverse osmosis (26,53,16). Con-
siderations relative to the choice of concentration method are: 1) the
desireability of maintaining the sample at a low temperature to avoid decom-
position or reaction of solutes; 2) the necessity of collecting the solids
that separate during concentration in order to redissolve coprecipated organic
compounds; 3) the need to reduce the volume of sample within a reasonable
period of time; and 4) the requirement that the method of concentration not
alter or chemically degrade the constituents. Extraction and adsorption do
not quantitatively concentrate all compounds of interest (81,154). Low-
temperature vacuum-distillation appears to be the most desirable method, since
it provides efficient recovery of stable, nonvolatile organic compounds and
fulfills the requirements described above. This method cannot, however, be
used for concentrating unstable or volatile compounds. Low-temperature
vacuum-distillation followed by removal of residual water by lyophilization
has been used with great success by many workers (39,53,82,83,153,154).
Recent advances in high-performance liquid chromatography (HPLC) have
made the separation and determination of nonvolatile nitrogenous organic
compounds analytically feasible. The ability to grade resins selectively
into narrow and uniform micropartides, advances in design of high-pressure
columns and pumps, and the development of sensitive detection systems have
resulted in an increased range of chromatographic capability.
Chormatography can be characterized as a separation method based on the
differential migration of solutes through a system of two phases, one of which
is mobile. The basis of the chromatographic separation is the distribution
(or partition) of the sample components between two phases, which are
immiscible. Chromatographic methods are classified either according to the
type of mobile and stationary phases utilized or according to the mechanism
of retention. A description of many modes of chromatography is presented in
Table C-8. Ion-exchange chromatography has been used extensively in the
separation of amino acids, and more recently, in the separation of nucleic
acid components (155). It was, therefore, closely examined as background for
this study. Paired-ion chromatography, previously applied to inorganic
compounds, has also been used successfully in the separation of very polar
compounds (156). In this method a large organic counter-ion added to the
mobile phase forms a reversible ion-pair complex with the ionized sample.
This complex behaves as an electrically neutral and non-polar (lipophilic)
compound. The extent to which the ionized sample and the counter-ion form
an ion-pair complex affects the degree of retention obtained (157). Reversed
phase adsorption chromatography has also been shown to be useful (155).
Amino acids commonly found in protein hydrolysates, physiological fluids
(90) and standard amino acid mixtures (90,158) have been resolved at the
picomole level using HPLC with fluorescence detection. Dr. R. Jolley and Dr.
W. Pitt have made significant progress in the separation and identification
of trace compounds in urine (159), primary and secondary stages of municipal
sewage treatment plants, and natural waters (53,79,82), using a strongly
basic anion-exchange resin (Bio Rad Aminex A-27) with (ammonium acetate)
315
-------
TABLE C-8. MODES OF CHROMATOGRAPHY
Stationary Phase
Mobile Phase
Non Polar Liquid
Polar Liquid
polar solid
non-polar solid
solid surface coated with a
to polar liquid
solid surface coated with a
non-polar liquid
ion-exchange resin with either
acidic or basic mobile counter-
ions
non-polar solid
porous packing gel
normal phase liquid-solid
chromatography a
normal phase liquid-liquid
chromatography
reversed phase liquid-solid
chromatography
reverse phase liquid-liquid
chromatography
ion exchange chromatography
paired-ion exchange chromato-
graphy
gel permeation chromatography or exclusion chromatography
also called adsorption chromatography
-------
(acetic acid) buffer as eluant. A summary of the chromatographic supports
used to separate various mixtures of nitrogenous organic compounds is shown
in Table C-9.
Different modes of operation and chromatographic columns were evaluated
in this study, including: cation-exchange chromatography (Zipax SCX, DuPont
Company), anion-exchange chromatography (Aminex A-27, Bio Rad Laboratories),
paired-ion chromatography (Zorbax CN, DuPont Company), reversed-phase chroma-
tography (Zorbax C8, DuPont Company), and normal-phase chromatography (Corasil
II, Waters Associates). Zipax SCX is a strongly acidic, cation exchanger
consisting of a sulfonated fluorocarbon polymer bonded to spherical glass
microbeads nominally 30 microns in diameter (190,186). These beads have a
surface are of 0.8-1.0 m2/g. It can be used only with water solutions having
pH values ranging from 2-9. Ionic strength has the greatest effect on solute
retention.
Zorbax particles are tiny, uniform, silica-sol beads which have been
produced by agglutination in a polymerization process. Zorbax C8 is a re-
versed-phase packing for compounds with moderate to high polarity and can
also be used in paired-ion chromatography. Zorbax CN is a polar-bonded phase
for both normal and reversed phase chromatography (190,157). Aminex A-27 is
a porous, moderately cross-linked, polystyrene based, quarternary ammonium
type strong anion exchange resin. Corasil II is a normal-phase column packing
consisting of a solid glass bead core with either single or double porous
silica layers.
Eluted compounds have been detected by various methods including:
refractive index, ultraviolet and fluorescence absorbance, heat of adsorption,
electrolytic conductivity, flame ionization, polarography, and dielectric
constant monitoring. Because of its relatively high sensitivity to most
solutes and its insensitivity to changes in temperature, flow rate, and mobile
phase composition, ultraviolet adsorption is the most widely used method with
HPLC. When ultraviolet detection is used at very short wavelengths, near
200 nm, it becomes a general non-specific detector, since almost all compounds
exhibit very strong adsorption in the far U.V. (192).
Identification of a resolved chromatographic peak may be accomplished by:
1) elution position; 2) U.V. spectrum; 3) fluorescence spectrum; 4) nmr
spectrum; 5) internal standard; 6) isotopic labeling; 7) enzymatic peak
shift techniques; and 8) derivatization methods. Positive identification
usually requires corroborative evidence from several identification methods.
The probability of correct identification increases with each additional unit
of information that shows correspondence between the unknown and known
reference standards. Katz and Pitt (159) have recently developed a new
liquid chromatography detector which depends upon the fluorescence measure-
ment of cerium III produced from the reaction of cerium IV with eluted
reducible compounds. It was reported to be more sensitive than previous
oxidative detectors by more than a factor of 100. Gomez and others (193) have
begun to try to develop a sensitive method involving dual-beam Fourier trans-
form infrared spectrosocopy for the on-line identification of organic water
pollutants separated by high pressure liquid chromatography.
317
-------
TABLE C-9. HPLC SEPARATION OF NITROGENOUS ORGANIC COMPOUNDS
Compounds
Column
Mobile phase
Reference
u>
M
00
nucleic acid bases
nucleic acid bases
Zorbax CN
paired-ion
chromatography
Zipax SCXa
nucleic acid hydrolysis XAD-4 support
products: purines, coated with triethyl-
pyrimidines, nucleo- ammonium bicarbonate
sides, RNA hydroly-
zates, nucleotides
nucleosides
nucleotides
nucleosides and bases
in serum and plasma
nucleosides and their
bases
Aminex A-28b
Spherisorb 10 if
uBondapak C.g^
liBondapak C10
lo
propionic acid with heptane DuPont Co. (160)
sulfonic acid
0.01 HNO.
Kirkland (161)
linear gradient, 0.1-0.4 M Vematsu and Suhadolnik
triethylammonium bicarbonate (162)
Na Borate at pH 3-9 with
varying molarity
tetra-n-butylammonium
hydrogen sulfate and 10-
camphorsulfonic acid pH 3.9
KH2P04 pH 5.5 60/40 MeOH/
H20
.01 F KH2P04 pH 5.5
MeOH-H20 gradient (80/20)
Schneider and Glazko
(163)
Hoffman and Llao (164)
Strop £t ail. (165)
Hartwick and Brown (166)
(continued)
-------
TABLE C-9. (continued)
Compounds
Column
Mobile phase
Reference
UJ
(-•
VO
amlno acids with DC-AA
fluorescamine detection
Na citrate buffers
Georgladls and Coffey
(90)
dansyl amlno acids
dansyl amino acids
amino acids
urinary constituents
urinary U.V. adsorb-
ing metabolites
urine amlno acids
Micropak MCH-IO*1
Ion pairing technique
dansyl amino acids Micropak-NH4-KT
Particil PAC
Poragel PN*
Vydac polar phase1
resin coated glass
beads Poracil C
Corasil 11°
BioRad A-15
BioRad A-2?b
Zerolit
Aminex A-7
buffered MeOH/H20 0.01 M Ellis and Garcia (174)
(CH3)4 NCI counter ion
source
dichloromethane-acetic Johnson e_t al. (175)
acid (99:l)/acetonitrile-
acetic acid (90:10) gradient
acetonitrile-water-acetic Hsu and Currie (224)
acid
distilled and delonized
water pH = 6
Grushka and Scott (176)
ammonium acetate-acetic Mrochek ej^ al_. (177)
acid buffer pH 4.28 (0.015 -
6 M)
acetic acid-ammonium acetate Geeraerts e± al. (178)
(pH 4.4) varying from .015 -
6 M
sodium acetate-acetic acid Hamilton (179)
buffer, pH 4.4; .015 - 6 M
(continued)
-------
TABLE C-9. (continued)
Compounds
Column
Mobile phase
Reference
u>
IS}
o
purlne nucleotide, uBondapak
nucleoslde and base
metabolites from
biological extracts
adenosine in the pre-
sence of other nucleic
acid components
amino acids and amlno
sugars
fluorescamine
derivatized amino
acids
0-phthalaldehyde
derivatives of amino
acids
amlno acids as dansyl
derivatives with fluoro-
metric detection
ninhydrin chromagens
of amino acids
dansyl amlno acids
uBondapak
Aminex A-6
Durrum DC-1A
Aminex A-6
LiChrosorb SI60'
LiChrosorb RP8
g
Technicon chromo-
beads, type B
Zipax Ra
.05 M ammonium dihydrogen Anderson and Murphy (167)
phosphate buffer
.007 F KH2P04 l^O/MeOH Hartwick and Brovm (168)
gradient
sodium citrate buffers of Hadzija and Keglevic (169)
varying pH
citrate buffers pH 3.28
and 4.25
Stein et al. (170)
citrate buffers: pH 3.2, Roth and Hampal (171)
4.25, and 6.4
benzene-pyridine-acetic Bayer et_ al. (172)
acid mixture
citrate buffers pH 2.88 Ellis and Garcia
and 5.00
methyl ethel ketone with Frei and Lawrence (158)
light petroleum
(continued)
-------
TABLE C-9. (continued)
Compounds
Column
Mobile Phase
Reference
OJ
serum and urine
components
human urine, blood
serum, cerebrospinal
fluid, and amniotic
fluid
U.V. absorbing con-
stituents of human
urine
acidic urinary
constituents
U.V. absorbing
constituents of
human urine
dansyl polyamlne
derivatives
complex biological
mixture
aromatic bases
Aminex A-27
Amlnex A-27
Aminex BRX1
LiChrosorb ODS
BloRad AGI-18
MicroPak CH-10
Aminex A-27
Aminex A-6
sodium acetate-acetic
acid buffer, pH 4.4;
.014 - 6 M
sodium acetate-acetic
acid buffer, pH 4.4;
.014 - 6 M
sodium acetate-acetic
acid buffer, pH 4.4;
.014 - 6 M
increasing acetonitrlle
concentration in dilute
acid solution
sodium acetate-acetic
acid buffer, pH 4.4;
.015 - 6 M
•
water/acetonitrlie
gradient
sodium acetate-acetic
acid buffer, pH 4.4;
.015 - 6 M
Katz et al. (180)
Katz et al. (180)
Burtis (181)
Molnar and Horvath (182)
Scott (183)
Johnson et al. (175)
Scott et al. (155)
Zipax SCX
water with 0.15 M NaN03 DuPont Co (184)
(continued)
-------
TABLE C-9. (continued)
Compounds
Column
Mobile Phase
References
u>
NJ
cyanopyridines
Neuroamines,
phene thylamines,
B-hydroxyphenethyl,
amine and indoleamines
aza arenes
aromatic amlne
carcinogens
Caffeine in coffee
organic constituents
in primary and secon-
dary sewage treat-
ment plant effluents
trace organic compounds
in municipal sewage
effluent
organic halogen pro-
ducts in chlorinated
municipal sewage
effluents
Zipax SCX
Zlpax SCX
yBondapak ,C-18
V Porasild
Zipax SCX
Zlpax SCX
strong cation-
exchange column
Amiriex A-27
Amlnex A-27
water with 0.10 NaNC>3
and 0.1 N
ammonium phosphate pH 7
Talley (185)
McMurtrey (186)
Dong and Locke (187)
0.1 ammonium acetate buffer Mefford et al. (188)
20-80% CH3CN in water
1% propanol in hexane
0.01 M nitric acid
sodium acetate-acetic
acid buffer, pH 4.4;
.015 - 6 M
sodium acetate-acetic
acid buffer, pH 4.4;
.015 - 6 M
Madison et al. (189)
Jolley et al. (82)
Pitt elt al (39)
Jolley et al. (84)
(continued)
-------
TABLE C-9. (continued)
Compounds
Column
Mobile Phase
Reference
CO
N>
LJ
trace organic compounds C.._ pBondapak
organic compounds uPorasil
extracted from drinking
water by adsorption onto uBondapak
XAD macroreticular resins
tryptophan and some of
its metabolites in
biological fluids
benzamide, benzene-
sulfonamlde, or 4-
methoxybenzamide
derivatives or primary
and secondary amine
compounds
dansyl amino acids
amlno acids and
aromatic amlno
acid derivatives
Spherisorb ODS
uBondapak C
18
yBondapak
Spherisorb 50DS
Amberlite CG-120
Type IIIb
20-100% acetonitrile
in water
hexane to chloroform +
hexnne gradient
hexane to ethanol +
hexane gradient
50% MeOH + 50% paired
ion chromatographic
solution
mixtures of water and
MeOH or acetonitrile
linear gradient formed
from acetonitrile and
sodium phosphate
buffers (neutral pH)
gradient from pH = 3.25
to 4.25 to 7.70 using
sodium citrate or
Borax
Hites and Biemann (36,
37)
Thurston (226)
Riley et al. (227)
Clark and Wells (228)
Wilkinson (229)
Ohtsuki and Hatano (230)
(continued)
-------
TABLE C-9. (continued)
Compounds
Column
Mobile phase
Reference
LO
N>
JJ-
amlno acids
mono-, di-f and
trlphosphate
nucleotides of
adenlne, guanine,
hypoxanthine, xanthine,
uracil, thymine and
cytosine
Durrum DC-6A
Partisil 10-SAX1
citrate buffers (pH
3.2, 3.5, and 4.0)
.007 F KH2POA and .007
F KC1 (pH 4.0) to 0.25 F
KH2P04 and 0.50 F KC1
(pll 5.0) gradient
Kasiske et al. (231)
McKeag and Brown (232)
DuPont Company, Wilmington, Delaware
Bio Rad Laboratories, Richmond, California
Spectra Physics, Santa Clara, California
Waters Associates, Milford, Massachusetts
Durrum Chemical Company, Palo Alto, California
Merck, A.G., Darmstadk, Germany
Technicon Industrial Systems, Tarrytown, New York
Varlan Associates, Palo Alto, California
Whatman Inc., Clifton, New Jersey
Separation Group, Hesperia, California
Permutit, London, Great Britain
Jones Chromatography Ltd., Llanbradach, United Kingdom
-------
Fluorescence spectroscopy has long been a valuable method of analysis
because of its enhanced sensitivity and selectivity over conventional ab-
sorbance spectroscopy. Luminescence phenomea include fluorescence, phosphor-
escence, chemiluminescence, triboluminescence, and electroluminescence.
Fluorescence and phosphorescence are the emission of long wave light following
absorption of short excitation wavelength energy. If the energy emission
ceases in about 10~8 seconds after the excitation source is removed the
phenomenon is called fluorescence. If the energy emission persists for a
longer time than this, the term phosphorescence is used (194)
Many organic compounds either display natural fluorescence or can be
made fluorescent by derivatization. Classes of naturally fluorescing compounds
include: catecholamines, polycyclic aromatics, drugs, vitamins, nucleotides,
prophyrins, flavins, purines, pyrimidines, coenzymes, dyes and steroids (195).
Table C-10 shows luminescence data for a number of nitrogenous organic
compounds. The emissions of the purines, pyrimidines, nucleotides, and nucleic
acids themselves are weak, especially at ambient temperatures (196). Most
amino acids do not fluoresce to any appreciable extent (194). Tyrosine,
tryptophan and phenylalanine however, do have fluorescent properties (197).
The fluorescence adsorption and emission spectra of solutes in solution
depend greatly on several parameters including: solvent polarity, pH,
presence of non-aqueous acids or bases in organic solvents, anion presence
(Cl~, Br~, I~, and NOI), and solute concentration (194). Aniline, for example,
fluoresces intensely in neutral form at pH 7 but is non-fluorescent in its
protonated form at acid pH (196).
Sensitivity in response by two or three orders of magnitude greater than
conventional ultraviolet spectroscopy can be expected from fluorescing com-
pounds or fluorescent derivatives (195). In addition, enhanced peak identi-
fication is possible through fluorescence excitation and emission spectra
using stopped flow techniques. Sylvia et at, (200) reported a strong
correlation (mean correlation coefficient equal to 0.92) between carbon chloro-
form extract (CCE) and luminescence values obtained on a fluorescence spectro-
photometer at selected wavelengths. The authors were unable to define the
actual compound or group of compounds responsible for the fluorescent response
at the selected wavelength.
Weigele et al. (201) recently synthesized a novel fluorogenic reagent,
fluorescamine (fluram: 4 phcnylspiro-[furan-2 (3H), l'phthalan]-3,3'dione)
which reacts very rapidly at room temperature with primary amines to yield
highly fluorescent pyrrolinone derivatives which can be measured at picomole
levels. This fluorometric method was further developed by Udenfriend (202)
and has since then been used by many other researchers (170,175,192,203-205).
Fluorescamine reaction with primary amines proceeds at room temperature with
a half time at pH 9 of about 200-500 milliseconds for most amino acids (170).
Excess reagent is hydrolyzed within seconds to water soluble nonfluorescing
furanones (195). Fluorescamine, as well as its hydrolysis products, is not
fluorescent. It reacts directly with amines to form stable fluorophors (390
excitation, 475 emission) whose intensity is linear with the concentration of
reactant amine (170,202). Kroll et al. (206) found enhanced detection of
naturally fluorescent compounds and fluorescent derivatives at excitation
325
-------
TABLE C-10. LUMINESCENCE DATA FOR A NUMBER OF NITROGENOUS ORGANIC COMPOUNDS
t-o
Compound
adenine
adenoslne
alanine
aniline
aspartic acid
l-aspartic acid
J£-aspartic acid
barbituric acid
Fluorescence (F)
Phosphorescence (P)
Solvent Luminescence (L) A A Comments Reference
ex em
glycol-water
water-MeOH
powdered
glycol-water
EtOH
powdered
original state
original state
water at pH 7
water at pH 7
original state
original state
original state
original state
NaOH
NH40H
H2S04
F
P
L
F
P
L
L
L
F
F
L
L
L
L
L
L
L
294 * - 0.06
278 406 DL - 0.02
260-280 355
296-313 355
315 * - 0.003
280 422 LD = 0.001
260-280 375
296-313 375
320-395 white
253 phosphoresces
280 344 fair
291 361 fair
320-395 slate
365 medium purple-
blue
365 purplish white
320-395 whitish-violet
320-295 light green
320-395 violet
320-395 violet
(197)
(196)
(199)
(197)
(196)
(199)
(198)
(198)
(196)
(196)
(198)
(198)
(198)
(198)
(198)
(198)
(198)
(continued)
-------
TABLE C-10. (continued)
Compound
u>
N3
-J
Solvent
Fluoresence (F)
Phosphorescence (P)
Luminescence (L)
em
Comments Reference
caffeine
6-chloropurlne
cytosine
guanine
Indole
EtOH
original state
alkali
water-methanol
glycol-water (1:1)
powdered
original state
glycol-water
powdered
original state
NaOH
NH^OH
sulphuric acid
water pH 7
dime thylsulf oxide
original state
original state
sulphuric acid
P
L
L
P
F
L
L
L
F
L
L
L
L
L
F
F
P
L
L
285 440 DL = 0.2
320-395 bluish white
320-395 green
273 419 DL = 0.002
312 * = 0.06
260-280 400
296-313 400
365 blue-violet
320 * - 0.06
260-280 370
296-313 370
320-395 purple
320-395 light green
320-395 violet
320-395 violet
269 or 315 350
335 » - 0.42
404
365 violet- blue
365 green
(196)
(198)
(198)
(196)
(197)
(199)
(198)
(198)
(199)
(198)
(198)
(198)
(198)
(196)
(196)
(197)
(198)
(198)
(continued)
-------
TABLE C-10. (continued)
Compound
Solvent
Fluorescence (F)
Phosphorescence (P)
Luminescence (L)
ex
em
Comments Reference
00
nucleic acid
nucleotide
phenylalanine
purlnes
pyrimidine
pyrrole
NaOH
NH^OH
HC1
water
EPA
0.5% glucose
in water, original
state
original state
original state
original state
EPA
water-methanol
original state
original state
water
original state
original state
original state
EtOH
L
L
L
L
F
F
F
F
F
F
P
L
L
L
L
F
F
L
L
365 yellow green
365 yellow green
365 yellow green
320-395
250-300
258,205
250-275
272
320-395
320-395
320-395
298
240,207
320-395
320-395
283-380
285
290
282 small quantum
yield
280
357
405 DL - 0.01
yellowish
330-480
310-480
326 low quantum
yield
400-480
310-480
(198)
(198)
(198)
(198)
(197)
(197)
(196)
(197)
(199)
(196)
(197)
(196)
(198)
(198)
(198)
(197)
(197)
(198)
(198)
(continued)
-------
TABLE C-10. (continued)
Compound
Solvent
Fluorescence (F)
Phosphorescence (P)
Luminescence (L)
ex
em
Comments Reference
N3
pyridine
2-thiouracil
thymlne
Cryptophan
EtOH
EtOH
powdered
powdered
glycol-water (1:1)
original state
original state
original state
original state
EPA
original state
original state
original state
water pH 2.0
water
ethanol
0.5% glucose
in water,
0.5% glucose in water
EPA
original state
phosphoric acid
P
P
L
L
F
F
P
F
P
F
F
F
F
F
F
P
P
F
P
L
L
310
296
260-280
296-313
350
350
280
280
280
250-300
280,218
295
320-395
320-395
440 DL = .0001
510 DL = 0.02
310
395
316
450
500
350
500
325
315
348
350 » - 0.20
348
440 DL - 0.002
435
330
490
grey
greenish
(196)
(196)
(199)
(199)
(197)
(197)
(197)
(197)
(197)
(197)
(194)
(196)
(197)
(199)
(199)
(196)
(197)
(197)
(197)
(198)
(198)
(continued)
-------
TABLE C-10. (continued)
Compound
Solvent
Fluorescence (F)
Phosphorescence (P)
Luminescence (L)
ex
X Comments Reference
em
w
U)
o
tyrosine
fc-tyrosine
uracil
urea
original state
original state
water pH 2
water pH 12
aqueous
original state
EPA
0.5% glucose in water
EPA
0.5% glucose in water
EtOH
EtOH
EtOH
original state
original state
powdered
powdered
original state
NaOH
NH^OH
H£S04
glycol-water
original state
F
F
F
F
F
F
F
F
P
P
P
P
P
L
L
L
L
L
L
L
L
L
L
280
250-300
275,222
295,240
270
395
395
253
291
290
320-395
365
260-280
296-313
365
365
365
365
320-395
308
303
300 * - 0.21
305
300
394 DL - 0.02
390 DL - 0.02
389 DL ='0.02
purple
strong rose-white
355
430
violet
light green
blue-violet
blue-violet
315 » - 0.008
grey
(194)
(196)
(199)
(199)
(199)
(197)
(197)
(197)
(197)
(197)
(196)
(196)
(196)
(198)
(198)
(199)
(199)
(198)
(198)
(198)
0198)
(197)
(198)
(continued)
-------
TABLE C-10. (continued)
Compound
Solvent
Fluorescence (F)
Phosphorescence (P)
Luminescence (L)
ex
en
Comments Reference
uric acid
original state
320-295 medium violet
(197)
LO
Solvent abbreviations:
MeOH - methanol
original state - compound In its original and unaltered state
EtOH - ethanol; NaOH - sodium hydroxide; HC1 - hydrochloric acid
EPA - mixture of ethanol, isopentane, and ether usually in the proportion of 2:5:5
NH^OH - ammonium hydroxide
ex
Lem
excitation wavelength; DeMent (199) reported U.V. values only as "U.V.11 but indicated
that this most likely represented values of 320-395 nm.
emission wavelength; DeMent (199) reported color of emitted light. Approximate wavelengths
are: ultraviolet •» 4'M nm violet = 400-450 nm blue = 450-500 nm green » 500-570 nm
yellow = 570-590 nm orange = 590-620 nm red = 520-760 nm infra red =>760 nm
Comments: DL = limit of detection in yg/mL
* = quantum yield (or efficiency) where:
number of fluorescence quanta emitted
number of quanta absorbed to a singlet excited state
(0 < * < 1)
-------
wavelengths below 250 nm but did not investigate fluorescamine derivatives.
Fluorescamine has the added advantage of not being reactive with ammonia or
urea (201,204), although Georidis and Coffey (90) reported a small amount of
fluorescence resulting from ammonia contamination in the buffers used in their
fluorometric system. Because of the extreme sensitivity to fluorescent con-
taminants all sampling equipment and analytical glassware must be washed in
dilute hydrochloric acid and carefully rinsed prior to use (90,204). Primary
amine compounds capable of forming potentially fluorescing derivatives with
fluorescamine include: alanine, aspartic acid, adenine, m-aminophenol,
creatine, cytosine, glycine, glycylglycine, histidine, leucine, serine,
taurine, tryptophan, and tyrosine.
The utility of fluorescamine was more recently expanded by Weigele et ai.
(207), who reported that proline and other secondary amino acids could be
transformed into fluorescamine responsive primary amines via oxidative
decarboxylation with N-chlorosuccinimide. Felix and Terkelson (205) incor-
porated N-chlorosuccinimide and fluorescamine reagents into a fluorometric
analyzer capable of detecting both primary and secondary amine compounds.
The presence of 0.05 M hydrochloric acid in the N-chlorosuccinimide solution
was required to achieve pH 2 for the oxidation of the secondary amines.
Derivatization may be accomplished either before or after the chromato-
graphic column. In post-column derivatization the physical properties of the
underivatized solutes determine their characteristic retention. In pre-
column derivatization the retention values result from the properties of the
derivatized solute. Although it is often assumed that pre-column derivati-
zation tends to make a chromatographic separation more difficult, Johnson et
at. (175) suggested that in many instances it may lead to enhanced selectivity.
KJELDAHL AND AMMONIA DETERMINATIONS
The indophenol-hypochlorite reaction for the measurement of ammonia
utilizes the sensitive method of Berthelot (208) and Solorzano (209) in which
ammonia is converted to a deep-blue colored compound, indophenol, by reaction
with hypochlorite and phenol in alkaline solution (191). The intensity of the
blue colored indophenol, measured at 635 nm, is proportional to the ammonia
concentration (210). The phenol-hypochlorite reaction is extremely sensitive,
so that under optical conditions yg/1 levels of ammonia-nitrogen should be
determinable (191,211). The method has been modified (212) by introduction
of nitroprusside as catalyst. This change accentuates the blue color at room
temperature.
Total Kjeldahl nitrogen includes ammonia and organic nitrogen but does
not include nitrite and nitrate-nitrogen without special modification. It is
measured by determination of ammonia after decomposition of the organic
compounds to ammonia and COz by either ultraviolet light or H2SOi» digestion.
Ultraviolet decomposition, although potentially sensitive, gives variable
recovery of organic nitrogen and, therefore, has not been widely used (191).
Digestion mixtures containing a variety of catalysts and concentrations of
potassium sulfate have been reported in the literature (210,211,191,213).
These are shown in Table C-ll. Addition of potassium sulfate to the digestion
332
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TABLE C-ll. DIGESTION MIXTURES REPORTED IN THE LITERATURE
Composition
Digestion time
Neutralized prior
to ammonia
determination
Reference
0.1 g Se02; 500 ml H
diluted to one liter
with distilled water
H2S04; K2SO;
mercuric sulfate
0.2 g Se02; 20 g K2SO,
110 ml H2S04 diluted to
one liter with distilled
water
134 g K2S04; 200 ml
5 ml delenyl chloride
diluted to 1 liter with
distilled water
approximately
1.5 hours
approximately
1 hours
approximately
3.5 hours
approximately
1 hour
bromothymol blue
end point (pH 6.0-
7.6)
phenolphthalein
end point
(pH 8.3 - 10.0)
phenolphthalein
end point
neutralized to
pH 8 or 'higher'
Strickland and Parsons
C13;
Mann (2ii)
Hague and Hague (191)
Scheiner (214)
-------
mixture raises the boiling point of the H2SOi». The presence of phenol-
phthalein in the reaction mixture does not affect the final color (191).
Strickland and Parsons (213) caution that ultra-pure concentrated acid must
be used to ensure low blank values.
There is considerable discussion in the literature over the effect of
reagent concentration, catalyst composition, temperature, reagent sequence,
pH and development period on the intensity and stability of the colored indo-
phenol (191,211,215). Copper, selenium and manganese catalysts have been
employed (211,216,217). Reliable and reproducible methods for determining
ammonia and Kjeldahl nitrogen in natural waters using the indophenol hypo-
chlorite reaction, however, have been reported (213,214). Excellent repro-
ducibility was observed by Scheiner (214) who found the maximum relative
difference between duplicate determinations to be ±1%. Bolleter (218)
reported standard deviations of 0.03 ppm at a level of 1 ppm of ammonia.
Although Wearne (210) reported that amino acids have an inhibitory
effect on the determination, Strickland and Parsons (213) found that urea and
several amino acids in the concentration of 3 Mg at N/L in filtered sea water
caused negligible interference. Cocking (219) found that creatine and
creatinine at concentrations of 5 mg/L each did not interfere with the
indophenol-blue color formation. Bolleter et al. (218) found that aliphatic
amines, NaCl, KN03, NaSOi,, and BaCl2 did not interfere. Increased absorbance
resulted from the presence of copper, zinc and iron salts. Interference also
resulted from the presence of aromatic amines. Manabe (220) found that at
concentrations up to 5 mg/L none of 24 electrolytes tested gave rise to any
significant interference in the ammonia determination. Of the 20 nitrogenous
compounds tested at 0.2 mg/L amino acid nitrogen, only fc-cystine, £-glutamic
acid, £-histidine, £-methionine, and Jl-phenylalanine showed interference, in
the range of 5-13%. All of these compounds, however, were at concentrations
substantially higher than those normally found in water. Similar results
were reported by other investigators studying interference effects of electro-
lytes, amino acids and urea both in sea water and fresh water (209, 221-223).
334
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1 REPORT NO
EPA-600/2-80-031
3 RECIPIENT'S ACCESSION NO.
4 TITLE AND SUBTITLE
FORMATION AND SIGNIFICANCE OF N-CHLORO COMPOUNDS IN
WATER SUPPLIES
5 REPORT DATE
July 1980 (Issuing Date)
6 PERFORMING ORGANIZATION CODE
7 AUTHOR(S)
Carrell Morris, Neil Ram, Barbara Baum, Edmund Wajon
8 PERFORMING ORGANIZATION REPORT NO
9 PERFORMING ORGANIZATION NAME AND ADDRESS
Harvard University
Division of Applied Sciences
Cambridge, Mass. 02138
10 PROGRAM ELEMENT NO
1CC614 SOS# 2 Task 5
11 CONTRACT7GRANT NOT
R803631
12 SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory - Cin.,OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13 TYPE OF REPORT AND PERIOD COVERED
Final Rpt. April 1,1975-June 30
14 SPONSORING AGENCY CODE
EPA/600/14
15 SUPPLEMENTARY NOTES
Project Officer: Edward L. Katz, (513) 684-7235
16 ABSTRACT
Many naturally occurring nitrogenous organic compounds readily react with aqueous
chlorine, exerting significant chlorine demands. Several N-organic compounds also
produce chloroform upon reaction with chlorine with maximum formation occurring between
pH 8.5 and pH 10.5. The correlation between chloroform formation and chlorine demand,
however, is tenuous. It also appears that intermediates may be formed under neutral or
slightly acidic conditions which produce chloroform upon exposure to more alkaline
conditions.
Available analytical methods used to differentiate between free and combined chlo-
rine are subject to interference from organic chloramines. Some differentiation, how-
ever, may be achieved using amperometric titration.
Seven N-organic compounds were identified in municipal water supplies (adenine,
5-chlorouracil, cytosine, guanine, purine, thymine, and uracil) at concentrations rangi
from 20ug/L to 860ug/L. A large unidentified group of primary amine compounds was ob-
served in all of the samples. Field and laboratory data suggested that summer algal
bloom occurrences add considerably to the organic nitrogen content of a water supply.
Calculated levels of CHCl^ which might have formed in the water supplies under
alkaline conditions were more than 10% of EPA's maximum contaminant level for trihalo-
methanes. Calculated levels of combined forms of chlorine yielding falsely positive
tests for free chlorine in some samples were significant.
17
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b IDENTIFIERS/OPEN ENDED TERMS
C COSATI I Icld/Grtiup
chlorination, nitrogen organic
compounds, organic compounds, chloroform
high performance liquid
chromatography, Northeast
Mass., fluorescence
spectroscopy, nitrogenous
haloforms
13B
18 DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19 SECURITY CLASS (Tint Report)
Unclassified
21 NO OF PAGES
361
20 SECURITY CLASS (This page}
Unclassified
22 PRICE
EPA Form 2220-1 (Rev 4-77)
353
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