United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
P.O. Box 93478
Las Vegas NV 89193-3478
EPA/600/R-94/029
April 1994
feEPA
Research and Development
Environmental Screen-
ing For Azo Dyes By
Chemical Reduction And
Mass Spectrometry
01B2QAD94
-------
ENVIRONMENTAL SCREENING FOR AZO DYES BY CHEMICAL
REDUCTION AND MASS SPECTROMETRY
by
ROBERT D. VOYKSNER, ROLF STRAUB, and JEFFREY T. KEEVER
Analytical and Chemical Sciences
Research Triangle Institute
Research Triangle Park, North Carolina 27709
and
HAROLD S. FREEMAN and WHEI-NEEN HSU
College of Textiles
North Carolina State University
Raleigh, North Carolina 27695
CR819555
Project Officer
L. Donnelly Betowski
Methods Research Branch
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89193
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89193
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NOTICE
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development (ORD), partially funded and managed the extramural research described here. It has
been peer-reviewed by the Agency and approved as an EPA publication.
The research reported in this report was adapted from two manuscripts sent to peer-reviewed
journals. They are reprinted with permission from the following sources. Copyright 1993 American
Chemical Society. The citations are as follows:
1. "Determination of Aromatic Amines Originating from Azo Dyes by Chemical Reduction Combined with
Liquid Chromatography/Mass Spectrometry", Voyksner, R.D.; Straub, R.; Keever, J.T.; Freeman,
H.S.; Hsu, W-N. Environ. Sci. Technol. 1993, 27, 1665-1672.
2. "Determination of Aromatic Amines Originating from Azo Dyes by Hydrogen-Palladium Reduction
Combined with Gas Chromatography/Mass Spectrometry", Straub, R.F.; Voyksner, R.D.; Keever,
J.T. Anal. Chem. 1993, 65, 2131-2136.
-------
ABSTRACT
The U.S. Environmental Protection Agency is interested in azo dyes, which form the largest
class of dyes in use and have the potential to form carcinogenic aromatic amines under reducing condi-
tions. Therefore, a method that both affords reductive cleavage products in-situ and permits their char-
acterization would also permit a better assessment of modern complex and structurally unknown textile
dyes for their potential genotoxicity. A logical approach to such a method would involve the evaluation
of procedures for the reductive cleavage of azo dyes and for the mass spectrometric analysis of the
resulting volatile and nonvolatile aromatic amines.
Two general procedures were evaluated for the reductive cleavage of commercial azo dyes.
The first procedure used solutions of sodium hydrosulfite (dithionite) and tin (II) chloride to reductively
cleave 16 azo dyes. Identifications of the chemical reduction products were mainly based upon mass
spectra obtained by particle beam high-performance liquid chromatography/mass spectrometry
(HPLC/MS). Standards of the reduction products, when available, were used to confirm identities. The
chemical reduction methods resulted in nearly complete reduction of the azo bond to form aromatic
amines. Overall, tin (II) chloride was the more powerful reducing agent and yielded a greater number of
products.
The second procedure was evaluated for reductive cleavage of eight commercial azo dyes using
hydrogen (H2) and palladium (Pd). The reduction was accomplished directly in the heated injector of a
gas chromatograph (GC); the resulting products were separated by capillary gas chromatography (GC)
and characterized by mass spectrometry (MS). This in-situ method resulted in nearly complete reduc-
tion of the azo bond to form aromatic amines. For most of the tested (non-sulfonated) azo dyes, the in-
line H2/Pd reduction/analysis procedure yielded the same or a greater number of reduced cleavage
products as did reduction with tin (II) chloride in solution.
The analysis of reduced, industrial waste sludge extracts indicated the presence of identifiable
aromatic amines, which originated from the reduction of unknown dye components. While the identity of
the parent dyes in these sludges could not be determined, this analytical approach appears to provide
the means to assess the environmental significance of a dye-manufacturing effluent based on the pres-
ence of various amines. Therefore, reductive cleavage followed by HPLC/MS permits the screening of
modern, complex synthetic dyes for potentially genotoxic aromatic amines without prior knowledge of the
parent dye structure.
The second procedure, the in-line reduction process, was not affected by the presence of
wastewater interferences. The GC/MS analysis of reduced waste-sludge extracts indicated the pres-
ence of identifiable aromatic amines originating from the reduction of unknown dye components as well
as from other reducible nitrogen-containing compounds. While the identities of the parent dyes in these
sludges were unknown, this analytical approach appears to provide a means to assess the potential
environmental significance of released effluent based on the detection of genotoxic aromatic amines.
This report was submitted in fulfillment of Cooperative Agreement CR819555 and contract 68-
02-4544 by Research Triangle Institute under the sponsorship of the U.S. Environmental Protection
Agency. This report covers a period from November 1989 to April 1993 and work was completed as of
April 1993.
in
-------
Contents
Page
NOTICE : ii
ABSTRACT iii
FIGURES v
TABLES v
LIST OF ABBREVIATIONS AND SYMBOLS vi
ACKNOWLEDGMENT viii
INTRODUCTION 1
CONCLUSIONS AND RECOMMENDATIONS 3
EXPERIMENTAL PROCEDURES 5
Materials 5
Extraction Procedures 5
Dyes 5
Sludges 5
Chemical Reduction Procedures 5
Tin Chloride (SnCL/HCI) Reduction 5
Sodium Hydrosulfite (Na2S2O4) Reduction 9
Chemical Reduction of Sludge Extracts 9
Tin Chloride (SnCL/HCI) Reduction 9
Sodium Hydrosulfite (Na2S2O4) Reduction 9
Instrumentation 9
Equipment for HPLC/PB-MS 9
Equipment for GC/MS 9
Equipment and Conditions for High Resolution Measurements 9
HPLC/PB-MS Conditions 10
Dye Samples 10
Sludges 10
GC/MS Conditions 10
Reduced-Dye Standards 10
In-Line Hydrogenation Experiment 10
RESULTS AND DISCUSSION 11
Chemical Reduction of Commercial Azo Dyes 11
Detection of Aromatic Amines in Reduced Sludge Extracts 17
Sludge 1 17
Sludge II 17
In-Situ Reduction of Commercial Azo Dyes 17
Detection of Aromatic Amines in Sludge Extracts after In-Situ Reduction 24
Sludge 1 24
Sludge II 24
REFERENCES 27
iv
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Figures
Number Page
1. (A) HPLC/PB-MS TIC chromatogram of identical quantities of Solvent 16
Yellow 3 before reduction, (B) after reduction with Na,,S2O4,
and (C) after reduction with SnCI2. Peak 1 = 2-aminotoluene
(M+- = 107); peak 2 = parent dye (Mv = 225); peak 3 = 2-methyl-
1,4-diaminobenzene (M+' = 122).
2. (A) HPLC/PB-MS TIC chromatograms from 70-pg injections of extract 18
of sludge I after reduction with Na^O,,, (B) after reduction with SnCI2,
and (C) untreated. Table 3 lists the PB-EI and TS mass spectral
data.
3. (A) PB-EI mass spectrum of peak 2 in TIC trace of reduced sludge 21
extract, (B) HPLC/PB-MS TIC chromatogram of SnCI2-reduced sludge II
extract, and (C) HPLC/PB-MS TIC chromatogram for the untreated sludge II
extract. The chromatograms generated from B and C were from the analysis
of 70 /jg of sludge extract. Table 3 lists the PB-EI and TS mass
spectral data.
Tables
Number Page
1. Azo Dyes Used in This Study 6
2. Identification of Chemical Reduction Products of Colorants 1-16 by HPLC/MS 12
3. Identified Components in Reduced Sludge Samples I and II 19
4. Identification of Major Chemical-Reduction Products of Colorants by GC/MS 22
5. Major Components Formed after In-Situ Reduction of Sludge I and II 25
-------
Cl
C.I.
cm
cone.
El
EtOAc
eV
FD&C
9
GC/MS
HPLC/MS
HPLC/TS-MS
i.d.
LC
M
M+'
mM
mg
min
mL
mm
mmol
Mol. wt.
ms
MS/MS
m/z
N.D.
nm
OAc
°C
PB
PB-MS
ppb
ppm
PPt
s
TIC
TLC
tR
TS
TS-MS
UV
V
v/v
w/v
LIST OF ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
chemical ionization
Color Index
centimeter
concentrated
electron impact ionization
ethyl acetate
electron volt
Food, drug, and cosmetic
gram
gas chromatography/mass spectrometry
high performance liquid chromatography/mass spectrometry
high performance liquid chromatography/thermospray mass spectrometry
internal diameter
liquid chromatography
molar
molecular radical cation
millimolar
milligram
minute
milliliter
millimeter
millimole
molecular weight
millisecond
mass spectrometry/mass spectrometry
mass-to-charge ratio
microampere
microliter
micrometer
not detected
nanometer
acetate
degrees centigrade
particle beam
particle beam-mass spectrometry
parts per billion
parts per million
parts per trillion
second
total ion current
thin layer chromatography
retention time
thermospray
thermospray-mass spectrometry
ultraviolet
volt
volume-to-volume ratio
weight-to-volume ratio
VI
-------
SYMBOLS
CH2CI2 methylene chloride
-CH2OH hydroxymethyl
Cl chlorine
-CN cyano group
-CO2CH3 acetate
HCI hydrochloric acid
He helium
H2 hydrogen (molecular)
[M+H]+ protonated molecule
MgSO4 magnesium sulfate
N2 nitrogen
Na2CO3 sodium carbonate
Na2S2O4 sodium hydrosulfite
NaOH sodium hydroxide
-NH2 amino group
-N=N- azo group
-NO2 nitro group
-OH hydroxyl
Pd palladium
pH the negative of the log of the hydrogen ion concentration
SnCL tin (II) chloride
vu
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ACKNOWLEDGMENT
We thank Dr. L. Don Betowski (EPA, EMSL-LV) for helpful discussion concerning the project, and
U.S. dye manufacturers for supplying the commercial azo dyes.
via
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SECTION 1
INTRODUCTION
Azo dyestuffs are widely used as colorants in a
variety of products such as textiles, paper, leather,
gasoline, and foodstuffs. These dyes are of great
environmental concern to the U.S. Environmental
Protection Agency due to their potential to form
carcinogenic aromatic amines under reducing con-
ditions (1). Genotoxicity among azo dyes and their
synthetic intermediates has been well documented
(2,3). For instance, 2-naphthylamine, 4-amino-
biphenyl, 4,4'-diaminobiphenyl (benzidine) (4),
and Acid Red 27 (FD&C Red 2, a food, drug, and
cosmetic colorant) have been identified as actual or
potential carcinogens, although evidence suggests
that metabolites of some of the azo compounds are
the actual genotoxic agents (5). Also, certain aro-
matic amines used in the past as precursors of syn-
thetic dyes pose a potential risk to human health
(6,7). This point was initially thought to be impor-
tant only if workers were exposed to a genotoxic
precursor during dye manufacture or when han-
dling an impure dye containing the unreacted,
genotoxic parent arylamine. It was later found,
however, that even a commercially available and
highly purified dye such as Congo Red (Color
Index (C.I.) No. 22120, also known as Direct Red
28) can generate the known carcinogen benzidine,
in the presence of mammalian enzymes (8). Also,
the literature contains additional papers describing
the genotoxicity of dyes or their reductive cleavage
products (9-12). Therefore, it is recognized that
azo dyes, as well as the amines formed from dye
metabolism/reduction, must be monitored to ade-
quately assess the potential risk to humans and the
envkonment.
Over 140 million pounds of synthetic dyes are
produced annually in the USA (13). Following the
production of industrial dyes, impurities as well as
a portion of the dyes themselves may be discharged
in waste streams. The resulting wastewater is a
complex sample matrix having variable propor-
tions of suspended solid particles and dissolved
solutes, and is often subjected to reductive or
oxidative waste treatment processes to remove
color prior to release. This so-called "reductive-
clear" step creates a wide array of new products,
and current methodology does not always provide
the specificity or ability to identify them.
Progress in the area of environmental monitor-
ing of azo dyes will require the development of
specific, sensitive, accurate, rugged, and cost-
effective methods for the detection of environmen-
tal contaminants in a variety of matrices. The fact
that numerous dyes are not volatile, are thermally
unstable, and are active carcinogens places an
enormous burden on conventional analytical tech-
nology. The classical approach for attempting to
detect low levels of nonvolatile/thermally unstable
pollutants has been based on mutagenicity tests,
liquid chromatography, and gas chromatogra-
phy/mass spectrometry (GC/MS) (14-16).
However, these techniques have disadvantages
stemming from nonspecific detection, lack of sen-
sitivity, or incompatibility with nonvolatile, ther-
mally unstable organics. On-line HPLC/MS using
thermospray has overcome some of these disad-
vantages in the analysis of environmental wastes
(17-20). Thermospray has proven to be a suitable
technique for many azo dyes (15,21-32) with struc-
tural information obtained by MS/MS (15,22) or
through repeller-induced fragmentation (27,30,31).
Furthermore, particle beam (PB) MS has been used
to generate electron impact ionization (El) spectra
from a series of commercial dyes (32-36).
However, detection of azo dyes using these
HPLC/MS techniques poses additional problems,
including chromatographic separation, sensitivity,
or specificity of detection, due to the extreme
ranges in polarity, volatility, and stability of the
dyes. This problem is particularly important when
dealing with the thousands of dyes and their
1
-------
homologs, reduction products, and precursors that
can be found in dye-related wastes. Therefore,
rather than developing several methods for the
multitude of compounds that exhibit extremes in
polarity and volatility in dye-related wastes, alter-
native methodology needs to be developed that can
adequately screen for the potential formation of
genotoxic compounds.
This paper reports the analysis by PB-
HPLC/MS and by GC/MS of common products,
originating from reduction of a variety of azo dyes.
The ability to chemically reduce azo dyes, with
concomitant formation of aromatic amines, fol-
lowed by the direct analysis of these reduction
products, can provide the means to qualitatively
screen azo dye content in complex industrial efflu-
ents. In addition, known mutagenic amines
formed from the reduction can be monitored to bet-
ter assess the environmental risk of the waste.
Nitrogen-containing substituents such as nitro
and diazo groups undergo exceedingly easy reduc-
tion by many reagents (1). The nitro group is read-
ily converted to a series of functional forms
through various degrees of reduction - occasional-
ly to a nitroso group, more often to a hydroxy-
lamino group, and most frequently to the amino
group. By controlling the amount of hydrogen and
the pH of the reaction, hydroxylamino, azoxy, azo,
hydrazo, and amino compounds have been
obtained in good yields by catalytic hydrogenation
over 2% palladium-on-carbon at room temperature
and atmospheric pressure (37). Complete reduc-
tion of nitro compounds to amines is accomplished
by catalytic hydrogenation. Catalysts suitable for
reductive conversion of aromatic nitro compounds
to amines include platinum oxide, palladium,
Raney nickel, copper chromite, and rhenium sul-
fide (1). Popular reducing agents for the conver-
sion of aromatic diazo and nitro compounds to
amines are iron, zinc, tin, tin (II) chloride (SnCl2),
hydrogen sulfide or its salts, and sodium hydrosul-
fite (Na^C^, sodium dithionite) (37). However,
the detection of azo dyes by HPLC/MS poses prob-
lems due to their wide range of polarities and
volatilities, creating difficulties with chromato-
graphic separations and MS responses. Rather
than developing several methods for the multitude
of azo dyes in waste streams, cost effective
methodologies need to be developed that can ade-
quately screen for the formation of potential geno-
toxic compounds in waste streams.
Gas chromatography (GC) employing MS
detection (GC/MS) is relatively routine compared
with HPLC/MS, but has rarely been used to ana-
lyze azo dyes due to their limited volatility.
Therefore, only a few GC/MS methods have been
reported for monitoring volatile dyes (38) or dyes
in waste streams (13,39). The ability to chemical-
ly reduce dyes by hydrogenation in a palladium-
filled GC injection port, followed by direct GC/MS
analysis of these usually more volatile reduction
products can provide the means to qualitatively
screen industrial effluents for potential toxicity.
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SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
The reduction of industrial sludges containing
azo dyes usually forms a nearly colorless effluent.
Depending on the identity of the azo dyes in the
waste, the aromatic reduction products may be
more harmful to the environment than the original,
untreated sludge components. The use of Na2S2O4
or SnClj to cleave the -N=N- group of azo dyes fol-
lowed by HPLC/MS analysis of the reaction prod-
ucts is a possible way to assess the toxicity poten-
tial of complex waste sludges. Using a library con-
taining the mass spectra of aromatic amines repre-
senting common couplers used to synthesize azo
dyes can help in the identification of the parent
dye. Also, components in effluents following the
"reductive-clear" steps can be monitored. This
procedure is less suitable, however, for characteriz-
ing wastes containing sulfonated (hydrophilic) azo
dyes, due in part to the significantly higher number
of standards required to build an effective library,
and the limitations of PB-MS for the sensitive
detection of the sulfonated aromatic amines.
However, the latter problem can be addressed using
electrospray negative ion-MS for the detection of
the sulfonated compounds (32,40).
The reduction of azo dyes by H2 in the presence
of Pd at the elevated temperature of a gas chro-
matographic injector has the advantage that the
entire analysis can be accomplished directly by
GC/MS. In numerous cases, the same reduction
products were observed for this in-line H2/Pd
reduction as were formed by reduction of azo dyes
with SnCl2 in solution. This in-line
reduction/analysis procedure represents a cost-
effective and simple way to screen waste streams
for aromatic amines that can be formed during
reductive waste treatments or in the environment
by anaerobic processes. A knowledge of the
amines that can be generated aids the assessment of
the toxic potential of the particular waste.
The procedures outlined in this report were
originally developed for the screening of waste
materials for azo dyes. Since azo dyes do not have
any uniquely characteristic feature that either mass
spectrometry or infrared spectroscopy could use to
identify as belonging to this particular class, the
reduction-before-analysis approach that has been
proposed is recommended for use to identify the
more common aromatic amines. An aromatic
amine then could be a possible indicator of the par-
ent azo dye. Raman spectroscopy is able to identi-
fy the azo linkage in these compounds, but the
application of Raman to trace analysis is still in the
developmental stages. There are other methods
(e.g., SW-846 Method 8321) to perform a more
complete analysis to detect and quantify the parent
dyes, which may be required for regulatory and
enforcement purposes; however, these methods are
more tedious and require purified dye standards,
which are not readily available.
Besides being an effective screening method for
the parent azo dyes, these procedures have proved
successful for identifying potential environmental
breakdown products of these dyes. The disposal of
azo dyes in a reducing environmental will often
result in the formation of aromatic amines. These
methods, therefore, provide a better assessment of
the potential toxicity of azo dyes in the environ-
ment than the analysis of the parent dye. Real
environmental samples can be screened in this
manner. However, the original non-reduced sam-
ple has to be retained for further examination fol-
lowing the screening procedure.
The direct on-column reduction of wastes using
hydrogen and palladium provides an efficient and
cost-effective procedure to assess the concentration
of aromatic amines formed from these wastes and
hence the potential risks associated with their envi-
ronmental management.
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SECTION 3
EXPERIMENTAL PROCEDURES
Materials
Sixteen commercial dyestuffs, identified by
their C.I. name and number (Table 1), were evalu-
ated in this study. The samples having low dye
content were used after isolation from diluents by
CH2C12 extraction. The dyes were obtained from
the following sources: 1-9 and 16 (Aldrich
Chemical Company Inc., Milwaukee, WI, USA);
10 (Ciba-Geigy, Dyestuffs and Chemicals
Division, Greensboro, NC, USA); 11 (BASF,
Charlotte, NC, USA); 12 (Eastman Chemicals,
Kingsport, TN, USA); 13 (Sandoz Colours and
Chemicals, Charlotte, NC, USA); 14 and 15
(Crompton & Knowles, Charlotte, NC, USA). All
other chemicals were purchased from Aldrich. The
solvents used for extractions, reactions, or liquid
chromatography were "HPLC/GC grade" quality
(Baxter Healthcare Corp., Muskegon, MI, USA).
The water was distilled and passed through a Milli-
Q Water System (Millipore Corp., Bedford, MA,
USA) prior to use. Amine standards such as 2-
amino-toluene, 2-methyl-l,4-diaminobenzene, ani-
line, l-amino-2-naphthol, 2,4-dimethylaniline,
4-nitroaniline, 4-aminophenol, 8-amino-7-hydrox-
ynaphthalene, 1,3-disulfonic acid disodium salt, 2-
bromo-4,6-dinitroaniline, benzidine and 3,3'-
dichlorobenzidine were from Aldrich and from
EPA (Las Vegas, NV, USA). Sludges I and II were
provided by EPA (Environmental Research
Laboratory, Athens, GA, USA).
Thin layer chromatography (TLC) for solvent
dyes was performed on normal-phase Macherey
Nagel SDL G/UV^ plates (Alltech, Avondale, PA,
USA) using toluene:ethyl acetate (EtOAc)/4:l as
the mobile phase. For all other hydrophobic dyes,
TLC analyses used toluene:EtOAc/l:4; the
hydrophilic dye (Acid Orange 10), however, was
eluted with ethanol:EtOAc/4:l.
The palladium spun fibers (lot #20248) used for in-
line hydrogenation experiments were 99.05%
(Johnson Matthey Electronics, Ward Hill, MA,
USA).
Extraction Procedures
Dyes. Dye samples having a high diluent con-
tent were separated from their commercial addi-
tives via Soxhlet extraction using methylene chlo-
ride, and then recrystallized from toluene/petrole-
um ether. The pure commercial dyes thus obtained
were used in the subsequent analyses.
Sludges. A set of organic extracts using 60 g of
sludge I and 75 g of sludge II were obtained from
two 300-mL methylene chloride extractions. The
organic extracts of each sludge were concentrated
to give 0.2 g of purple solid from sludge I and 0.35
g of orange solid from sludge n. TLC analysis
showed that both samples contained a major com-
ponent along with two minor components. Each
solid concentrate was divided into three equal
parts, two of which were used in the reduction pro-
cedures. The third part was dissolved in acetoni-
trile (10 mg/mL) under a nitrogen (N2) atmosphere
and used as the unreduced control sample in the
HPLC/MS and GC/MS analyses. For the blank
sample, the same amount of CH2C12 as used in the
extractions was evaporated to dryness and the
residue dissolved in acetonitrile.
Chemical Reduction Procedures
Tin Chloride (SnCl/HCl) Reduction. The reac-
tion was conducted in boiling methanol and
required approximately 1 to 4 mmol of SnCl2 (40%
in cone. HC1) per mmol of dye to remove the last
traces of the parent dye, as judged by TLC. The
variability in the quantity of reducing agent needed
can be attributed to the number and reactivity of
reducible groups (e.g., N=N, NO2) on the dye.
Once the solution was decolorized (15 to 60 min-
-------
CH3
CH3
\
CH3
3. Solvent Yellow 14
C.I. 12055
5. Solvent Red 24
C.I. 26105
2. Solvent Yellow 3
C.I. 11160
,CH3 HO
CH3 (f V-N=N?
4. Solvent Orange 7
C.I. 12140
CH3
6. Pigment Red 3
C.I. 12120
0182QAD94.RPT-1
TABLE 1. AZO DYES USED IN THIS STUDY
Structure, Name, Color Index (C.I.) and Molecular Weights of Commercial Dyes
-------
02N
= N
7. Disperse Red 1
C.I. 11110
O2N
(314)
/CH2CH3
J
XCH2CH2OH
CH2CH3
=/ XCH2CH2CN
8. Disperse Orange 13
C.I. 26080 (352)
Cl
O2N
N=N
-------
,OCH3
CH2CH2OCCH3
N O
XCH2CH2OCCH3
N02 NHCOCH3
13. Disperse Blue 79
C.I. 11345 (624)
O2N
N=N
CH2CH3
N
=/ NCH2CH2CN
Cl
14 Disperse Orange 37
C.I. none (391)
oo
O2N
CH2CH2OH
/ \CH2CH2OH
1 5. Disperse Brown 1
C.I. 11152 (432)
NO2 NHCOCH3
V_N=N - N(CH2CH3)2
Br
17. Industrial Precursor
for Disperse Blue 165 (478)
16. Acid Orange 10
C.I. 16230
CHa OH
ArHN
V
II
O
HO CH3
c'
II
N=N-C-C- NHAr
II
O
18. Generic Structure of Certain Yellow Azo Pigments
TABLE 1. AZO DYES USED IN THIS STUDY Continued
Structure, Name, Color Index (C.I.) and Molecular Weights of Commercial Dyes
-------
utes), 5 to 10 g of solid Na,CO3 was added to
achieve pH 7 to 8. This treatment normally pro-
duced a milky emulsion, which was concentrated
to remove the methanol, diluted with EtOAc, and
filtered or centrifuged to separate the white, solid
precipitate. The EtOAc layer was dried over
MgSO4 and concentrated. TLC was then used to
determine the number of products and to confirm
that reductive cleavage of the dye was complete. A
portion of the reaction product mixture was then
dissolved in acetonitrile (10 mg/mL) and stored
under N2 for HPLC/MS analysis.
Sodium Hydrosulfite (Na^S2O4) Reduction. A
solution (or suspension) of 1 mmol dye in boiling
methanol, under N2 atmosphere, was treated with
14 to 28 mmol of Na2S2O4 (25% w/v in water)
solution until decoloration was completed. This
treatment required about 5-12 min depending on
the structure of the dye. However, Solvent Red 24
(5; a disazo colorant) and Pigment Red 3 (6; a spar-
ingly soluble colorant) required about 98 mmol of
the reducing agent to effect decoloration. The
workup of this reaction involved the evaporation of
methanol under nitrogen followed by EtOAc
extraction of the remaining mixture. The EtOAc
extract was concentrated, and the mixture obtained
was dissolved in acetonitrile (10 mg/mL) and
stored under N2for HPLC/MS analysis.
Chemical Reduction of Sludge Extracts
Tin Chloride (SnCl/HCl) Reduction. Solutions
of the concentrate from extracts of sludges I and n
were dissolved separately in methanol or methyl-
ene chloride and treated with 1 M SnCl2 (in cone.
HC1). This caused an immediate decolorization of
the solution. The solution was made basic (pH 10
to 12) with NaOH and then extracted with methyl-
ene chloride. The extract was dried over MgSO4,
filtered, and evaporated to dryness. The residue
was dissolved in acetonitrile (10 mg/mL) and
stored under N2 for HPLC/MS analysis.
Sodium Hydrosulfite (Na^flJ Reduction.
Solutions of the concentrate from extracts of
sludge I and II were dissolved separately in
methanohmethylene chloride (1:1) and boiled with
1 M NajS^ solution for 20 minutes. Very little
color change was apparent at the end of this treat-
ment. The resulting solutions were evaporated to
dryness, and the residue dissolved in acetonitrile
(10 mg/mL) and stored under N2 for HPLC/MS
analysis.
Instrumentation
Equipment for HPLC/PB-MS. The chromatog-
raphy was performed using a Waters Series 600
multisolvent delivery system (Waters Assoc., Inc.,
Milford, MA, USA) controlled by a Waters 600-
MS System Controller. The samples were injected
with a Waters model U6K universal liquid chro-
matograph injector and separated on a Spherisorb
ODS II, 5-fjm. particle size, 25-cm x 4.6-mm i.d.
column (Regis Chemical Company, Morton Grove,
IL, USA) for dye samples, and on an Alltech
Solvent Miser, C18,5-/mi particle size, 15-cm x 2.1-
mm i.d. column (Alltech Assoc., Inc., Deerfield,
IL, USA) for sludge extracts. A Waters 484 MS
Tunable Absorbance Detector set at 254 nm was
placed in-line before the model 59980A PB inter-
face (Hewlett Packard, Palo Alto, CA, USA). The
PB interface was connected to a Hewlett Packard
quadrupole mass spectrometer, model 5988A. The
equipment and conditions used for HPLC/thermo-
spray (TS)-MS is described elsewhere (32).
Equipment for GC/MS. For the analysis of
reduced azo dye standards, a GC/MS system con-
sisting of a Finnigan MAT 4500 GC (Finnigan
MAT Co., San Jose, CA, USA) and a Finnigan
MAT 4500 quadrupole MS were used. A 15-m x
0.32-mm i.d., 0.25-jum DB-1 (100% methyl) fused
silica column (J & W Scientific, Folsom, CA,
USA) was used for the separation of the reduction
products.
For the in-line hydrogenation experiments, a
Hewlett-Packard GC, model 5890 (Hewlett-
Packard, Palo Alto, CA, USA), with both open
injection liners and liners packed with palladium
filaments, was connected to a Hewlett-Packard
(model 5988A) single quadrupole MS for detection
and characterization of the reduction products. A
12.5-m x 0.28-mm i.d., 0.33-/«n crosslinked
methyl silicon fused silica column (Hewlett
Packard) was used for separation.
Equipment and Conditions for High Resolution
Measurements. The high resolution mass spec-
trometer (VG ZAB-E; Fisons Instruments,
Manchester, U.K.) scanned from m/z 50 to 450 at a
rate of 10 s per scan with a reset time of 2 s and a
response time of 0.01 ms. The ion source was
operated at 150°C. The trap current was 200 juA,
and electron energy was set to 70 eV. The sample
holder was filled with azo dye and Pd wires in a
ratio of about 1:10, w/w. The probe temperature
was ramped from 50°C to 250°C at a rate of 1°C
per s.
-------
HPLC/PB-MS Conditions
GC/MS Conditions
Dye Samples. The injected volume was 75 pL,
and the chromatography was performed with a
water-methanol linear gradient (40% to 100%
methanol in 20 min with a 15-min hold) at a flow
rate of 0.5 mL/min. The PB desolvation chamber
temperature was 60°C, and a helium inlet pressure
of 2.75 bar was maintained. The ion source was
operated at 200°C. The filament emission current
was 0.3 mA, and the electron energy was 70 eV.
The manifold temperatures were set to 90°C (front)
and 45°C (rear), and the mass range was scanned
from m/z 45 to 650 at a rate of 1 s per scan. The
sensitivity of the system was checked by injection
of a standard solution of caffeine.
Sludges. A 1-^JL volume of extract was inject-
ed and eluted with a water-acetonitrile gradient
(40% to 100% acetonitrile in 20 min, with a 25-
min hold) at a flow rate of 0.2 mL/min. The
changes from the HPLC conditions for the dye
samples provide better separation for the broader
range of components that could be found in the
complex sludges. A lower flow rate was used to
provide better PB sensitivity and stability, especial-
ly when operating with high aqueous-content solu-
tions. The PB interface was operated as described
above for dyes, except the ion source was set at
250°C, and the mass range was scanned from m/z
45 to 550 at a rate of 1 s per scan.
Reduced-Dye Standards. The injection volume
was 2/jLof each standard. A temperature gradient
of 50 to 325°C at a ramp rate of 10°C/min was
applied. The helium (He) carrier gas rate was 1
mL/min and the split ratio 20:1 was implemented
after 0.7 min. The injector and the separator tem-
perature were maintained at a temperature of
290°C. The MS operated in the electron ionization
(El) mode and scanned from m/z 40 to 500 with a
scan time of 1 s. The source temperature was
190°C.
In-Line Hydrogenation Experiment. The injec-
tion volume was 1 juL of a ImM dye solution or
slucige extract in acetonitrile. Both empty injection
liners and similar glass liners packed with 11 mg of
0.08-mm palladium wire were used in the GC sep-
aration. A temperature gradient of 40°C to 300°C
at a ramp rate of 10°C/min with hold at the final
temperature for 4 min was applied. For the in-line
hydrogenation experiments, the carrier gas consist-
ed of He doped with 5% (v/v) hydrogen (H2)
(Linde Specialty Gases, Somerset, N.J.). The carri-
er gas flow rate was 0.8 mL/min and the split ratio
10:1 was implemented after 0.5 min. The injector
was maintained at a temperature of 300°C. The
MS operated in the El mode and scanned from m/z
30 to 600 with a scan time of 1 s. The source tem-
perature was 200°C.
10
-------
SECTION 4
RESULTS AND DISCUSSION
Chemical Reduction of Commercial Azo Dyes
The first step in achieving the principal goal of
this work, to screen unknown industrial waste for
the formation of potentially hazardous products
after a reductive treatment, involved comparing
SnCl2 and Na^C^ as reducing agents for the dyes
shown in Table 1. For each dye, an aqueous medi-
um in a nitrogen atmosphere was used during the
reduction, since some of the anticipated reduction
products might have been air sensitive. It was later
determined, however, that reductive treatment of
the azo dyes in the presence of oxygen yielded the
same reduction products as the reaction under a N2
atmosphere. The reduction time was optimized to
maximize the yield of reduction products detected,
while minimizing the amount of the parent dye
remaining. Disperse dyes containing 2,6-dichloro
groups (such as Disperse Orange 37 (14) and
Disperse Orange 30 (11)) were generally quite
resistant to chemical reduction, requiring longer
reaction times to achieve a complete reductive-
cleavage of the parent dyes.
The PB HPLC/MS analysis of the reduced azo
dyes (Table 2) indicated that the major degradation
products formed were aromatic amines generated
by splitting the azo linkage. These aromatic amines
were not detected in the intact dye prior to reduc-
tion. Standards, when available, were used to con-
firm the identity of the reduction products as well
as provide an estimate for reduction efficiency.
The yield of aromatic amines formed (Table 2) was
based upon UV (254 nm) chromatographic peak
areas. The yield ranged from 61-110% of the peak
signal of the parent dye prior to reduction. These
yields were consistent with the yields measured by
MS when appropriate standards existed. While
estimation of reduction-product yields suffers from
variability due to difference in extinction coeffi-
cients, this method is probably as accurate as an
estimation based on MS response in the absence of
a complete set of standards.
The extraction recovery was verified gravimet-
rically at the 10-mg/mL level for selected, non-
volatile reduction products. Recoveries were
greater than 90% for disperse and solvent dyes and
were about 70% for sulfonated dyes. A more thor-
ough investigation of reduction-product recovery
and yields will be the subject of future investiga-
tion and will be necessary if semi-quantitative
determinations are to be performed.
Structurally simple solvent dyes (1-5) in Table
1 formed the anticipated aromatic amines after
reduction (Scheme 1).
Scheme 1: R1-N=N-R2-
SnCyHCl
+ R.-NH,
or
Na2S2O4 reduction of Solvent Yellow 3 (2) and
Yellow 14 (3) did not go to completion, as
unchanged dye was detected in the extracts. Figure
1 shows the HPLC/PB-MS total ion current (TIC)
chromatograms of Solvent Yellow 3 (2) following
reduction with either Na^O, (B) or SnCl2 (C).
11
-------
TABLE 2. IDENTIFICATION OF CHEMICAL REDUCTION PRODUCTS OF COLORANTS
1-16 BY HPLC/MS
No. Dye Identified Reduction Mol.
Products Wt. tRa
1 Solvent Yellow 2
2 Solvent Yellow 3
3 Solvent Yellow 14
4 Solvent Orange 7
5 Solvent Red 24
6 Pigment Red 3
7 Disperse Red 1
aniline" 93
N,N-dimethyl-1,4-di- 136
aminobenzene
2-aminotoluened 107
2-methyl-1,4-di- 122
amino-benzene"
unchanged dyee 225
aniline" 93
1-amino-2- 159
naphthol"
unchanged dye" 248
2,4-dimethylaniline" 121
1-amino-2-naphthol" 159
2-aminotoluene11 107
2-methyl-1,4- 122
diamino-benzene'
1-amino-2-naphthol" 159
1-amino-2-naphthol" 159
4-nitroaniline" 138
12.9
28.4
18.2
30.0
25.8
13.1 ,
19.3
28.3
22.1
20.2
22.4
40.4
19.1
19.5
13.0
Particle Beam
m/z (Relative Intensity)"
93(1 00); 66(39)
136(100); 120(85);
93(37); 81 (41)
107(74); 106(100);
77(26); 51 (15)
122(100); 94(33);
78(26); 58(1 9)
225(58); 134(17); 106(100);
91 (28); 77(23); 75(1 3);
51(4)
93(100); 66(37)
159(100); 130(89);
103(26); 77(22); 51 (15)
248(90); 21 9(7); 171 (15);
143(1 00); 11 5(97); 89(10);
77(41); 51 (10)
121(100); 106(78); 77(17)
159(100); 130(63);
103(13); 51(11)
107(1 00); 91 (55);
77(49); 51 (25)
122(33); 104(46);
71 (41); 55(1 00)
159(1 00); 130(70);
103(20); 77(20)
159(1 00); 130(62);
103(1 5); 77(1 9)
138(100); 108(83);
92(50); 65(75)
UV
,1
88
90
3
3
7
49
20
30
31
18
16
42
70
70
12
-------
TABLE 2. IDENTIFICATION OF CHEMICAL REDUCTION PRODUCTS OF COLORANTS
1-16 BY HPLC/MS Continued
No. Dye Identified Reduction Mol.
Products Wt. tRa
8 Disperse Orange
13
9 Disperse Orange
25
10 Disperse Orange
44
11 Disperse Orange
30
12 Disperse Black 9
13 Disperse Blue 79
aniline" 93
4-aminophenold'° 109
1,4-diamino- 158
naphthalene
4-nitroaniline" 138
N-(2-cyanoethyl)- 189
N-(ethyl)-1 ,4-diamino-
benzene
N,N-bis(2-cyanoethyl)- 214
1 ,4-diaminobenzene11
1,4-diamino-2,6- 176
dichlorobenzene
N-(2-cyanoethyl)- 205
N-(2-hydroxethyl)-1 ,4-
diaminobenzene1
2,6-dichloro-4-nitro- 206
aniline
N-(2-cyanoethyl)-N- 247
(2-acetoxyethyl)-1 ,4-
diaminobenzene
1,4-diaminobenzene" 108
N,N-bis-(2-hydroxyethyl)- 196
1 ,4-diaminobenzene
2-bromo-1,4,6- 201
triaminobenzene1
3-acetamido-4-[N,N-bis- 367
(2-acetoxyethyl)amino]-
13.1
6.2
10.7
12.3
14.8
8.3
13.8
6.1
25.5
10.0
8.3
21.7
10.4
15.1
87(100)
Particle Beam
m/z (Relative Intensity)"
93(1 00); 66(40)
109(10); 108(100);
80(48); 64(7)
158(100); 109(50); 80(37)
138(1 00); 108(83);
92(50); 65(75)
189(25); 149(1 00);
120(34); 92(4)
214(40); 174(100);
120(37); 106(5)
76(27); 149(73); 124(40);
98(1 00); 81 (56); 78(63)
205(45); 174(85); 165(80);
120(1 00); 92(30); 65(20)
208(40); 206(60); 178(50);
176(84); 162(20); 160(30)
135(15); 133(22); 126(30);
124(1 00); 92(28); 90(31)
247(1); 205(32); 174(91);
165(54); 120(1 00);
92(20); 65(20)
108(1 00); 92(44); 80(66);
67(25); 52(64)
196(25); 165(100);
120(20); 93(14)
203(15); 202(5); 201 (10);
88(23); 70(83); 61 (100)
367(10); 294(1 5); 208(9);
UV
10
78
18
75
70
10
35
20
45
30
60
25
60
1 -amino-5-methoxybenzene
13
-------
TABLE 2. IDENTIFICATION OF CHEMICAL REDUCTION PRODUCTS OF COLORANTS
1-16 BY HPLC/MS Continued
No.
14
Dye Identified Reduction
Products
Disperse Orange-
37
1,4-diamino-2,6-
dichlorobenzene
Mol. Particle Beam
Wt. tRa m/z (Relative Intensity)"
176 23.0 176(27);
98(100);
149(73)
84(56);
; 124(40);
74(63)
UV
(%)c
40
15 Disperse Brown 1
16 Acid Orange 10
N-(2-cyanoethyl)-N-ethyl- 189 26.1
1,4-diaminobenzene
4-amino-2,6-dichloro-4' 333
[N-(2-cyanoethyl)-amino]
azobenzene
1,4-diamino-2,6- 176 22.3
dichlorobenzene
3-chloro-N,N-bis(2-- 230 17.4
hydroxyethyl)-1,4-
diaminobenzene
aniline" 93 13.5
189(19); 149(100); 120(73); 40
106(8); 92(21)
334(15); 333(27); 293(100); 265(21); 10
229(7); 201 (33);149(49);
120(41); 100(15); 92(9)
176(56); 149(28); 134(59); 40
98(100); 84(53)
230(21); 199(100); 155(37); 127(13) 40
93(100); 66(40) 40
atR = retention time in TIC chromatogram (min). bm/z (Relative intensity) reports the major peaks (>5%) of each product
down to m/z 50; a maximum of 12 ions are reported in descending m/z. CUV(%) = peak area of identified reduction prod-
uct/I of the peak areas in the chromatogram of the reduced sample at a wavelength of 254 nm; the identified reduction
products were <0.5% of the total peak area in the unreduced HPLC/UV analysis of the parent dye. dldentity confirmed
with standard, 'observed only after Na,,S2O4 reduction, 'observed only after SnCI2 reduction, flonly identified by
HPLC/PB-MS in sample reduced with SnCI2.
14
-------
The response from the reduction products is lower
than that of the unreduced dye because of losses in
the particle beam momentum separator due to sam-
ple volatility. The peaks for 2 appear at the end of
the HPLC run since a low-pressure solvent mixer
on the HPLC pump and a 4.6-mm i.d. column
operating at 0.5 mL/min resulted in a 7-8 min delay
in solvent changes at the detector. TIC chro-
matograms B and C show peaks attributable to the
formation of the two reduction products (Peaks No.
1 and 3), while the parent dye (Peak No. 2) is near-
ly absent. PB-MS response for volatile amines
(e.g., aniline) is 2-10% of the response by GC/MS
(fused silica column fed directly into the MS
source). Reduction products that are volatile or of
low molecular weight, such as aniline from either
Solvent Yellow 2 (1) or Disperse Orange 13 (8),
were difficult to detect by PB-MS, due to losses in
the momentum separator.
The presence of numerous substituent groups
(e.g. -NO2, -CO2CH3, and -CN) in addition to the
azo linkage in dyes 9-11, 13, and 15, increased the
number of components among the reductive-cleav-
age products (Scheme 2). For example, the reduc-
tion of Disperse Orange 30 (11) with SnCl2/HCl
also resulted in hydrolysis of the acetate to the
hydroxyethyl group, giving a product with a
hydroxyethyl group and a molecular weight of 205.
Also, one of the major products (2,6-dichloro-4-
nitroaniline) formed from cleavage of the azo bond
was further reduced to give the corresponding
diamine (2,6-dichloro-l,4-diaminobenzene).
Scheme 2:
for X = NO,
R1-N=N-R2-X
SnCl2/HCl
or
Na2S204
R.-NH, + H2N-R2-NH2
for X = C2H4OAc
SnCL/HCl
R1-N=N-R2-X ^ Rl-NHj + HOC-.H^-NH,
or
Na2S204
Disperse Blue 79 (13) also showed further
reduction of substituents. For example, the expect-
ed reduction product, 2-bromo-4,6-dinitroaniline,
was not detected. Instead, 2-bromo-l,4,6-tri-
aminobenzene was found.
Clearly, substituent groups, such as -OAc and -NO2,
in azo dyes are often affected by the use of a pow-
erful reducing agent such as SnCl2. Under the
right conditions, NO2 and azo functional groups
yield common diamino or triamino products, there-
by simplifying the analysis. In addition, the for-
mation of multiple reduction products reinforces
the point that this approach is best suited for qual-
itative screening purposes and that the reduction
products must be identified if a particular target
azo dye is to be monitored by this methodology.
Sulfonated azo dyes also pose an environmental
concern. These dyes are usually precluded from
PB MS analysis due to nonvolatility and lack of
MS response. One acid dye (Acid Orange 10 (16))
was included in this evaluation to determine if the
reduction methodology could aid in developing
MS screening procedures for this type of dye. PB-
MS analysis of the reduced dye only detected the
volatile reductive-cleavage product, aniline. A sul-
fonated reduction product (8-amino-7-hydroxy-
naphthalene-l,3-disulfonic acid), was confirmed
by thermospray mass spectrometry based on the
retention time of a standard.
Both chemical reduction methods resulted in
reductive cleavage for most of the commercial azo
colorants studied. However, SnCl2/HCl was a
more powerful reducing agent, yielding a greater
number of products as well as thorough conversion
of the starting dye. Therefore, it is preferable to use
SnCyHCl for further reduction studies of com-
mercial azo dyes and for industrial sludges.
Treatment of the azo dyes listed in Table 2 forms
some common reduction products. For example,
aniline, methylaniline, or dimethylaniline are com-
mon reductive cleavage products for seven dyes
(Nos. 1-5, 8 and 16); aminonaphthol is a common
reductive cleavage product for four dyes (Nos. 3-
6); diaminobenzenes (including monochloro and
dichlorodiaminobenzenes) are common reductive
cleavage products for four dyes (Nos. 11, 12, 14,
and 15); and 4-nitroaniline is a reduction product
for two dyes (Nos. 7 and 9). As a result, the detec-
tion of certain amines can give useful information
regarding the possible structures of the parent azo
dye compounds used in a manufacturing proce-
dure.
15
-------
B
1600000 -J
1400000-
1200000-
1000000-
% 800000
c
- 600000
400000-
200000-
0-
CD
in
c
40000-
35000
30000
25000
20000
15000-
10000-
8000-
0-
28000
24000
20000
16000-
12000-
8000-
4000-
0-
Solvent Yellow 3
Unreduced
12 16 20
Time (min)
24
i
28
1
32
Na2SO4 reduced
4 8 12 16 20 24 28 32
Time (min)
SnCU reduced
12 16 20
Time (min)
i
24
i
28
32
Figure 1
16
-------
Detection of Aromatic Amines in Reduced Sludge
Extracts
Extracts of sludges (I and II) from two different
waste treatment sites were reduced with SnCl2/HCl
and
^ and then analyzed by HPLC/MS to
screen for potentially hazardous reduction prod-
ucts, and for azo dyes based upon the detection of
aromatic amines. The HPLC conditions were
changed for the analysis of the sludges compared
with the dye standards to provide a better and faster
separation for a broader range of components that
might be found in a sludge. This was accom-
plished by using a stronger elution solvent, such as
acetonitrile instead of methanol, and a 150- mm x
2.1-mm column operated at a flow rate of 0.2
mL/min. A 2.1-mm i.d. HPLC column offered two
advantages. First the column's operating flow rate
is closer to the optimal flow rate of the PB-MS
interface (0.2-0.4 mL/min) resulting in better sen-
sitivity (especially for an aqueous mobile phase)
than with the 0.5 mL/min for the 4.6-mm i.d. col-
umn. Secondly, the chromatographic peak width is
narrower compared with the 4.6-mm i.d. column,
resulting in a higher concentration introduced into
the MS and better sensitivity. To ensure the valid-
ity of the method, all amine standards were ana-
lyzed using these new conditions to verify elution
order. While the determination of the dye stan-
dards would have benefited from better resolution,
it was decided no additional information would
have been gained about their reduction, and they
were not reanalyzed using these new conditions.
Sludge I. The HPLC/PB-MS chromatograms
(A and B) of the components in both chemically
reduced extracts of the wastewater sludge are pre-
sented in Figure 2. The Na^C^ treatment formed
four new products, while SnCl2 afforded at least six
new products, based upon the total ion current
(TIC) chromatogram. The retention times and m/z
values of the reduction products are found in Table
3. Identification of products was based mainly on
mass spectral library searches (41), spectral inter-
pretation, and available aromatic amine standards
analyzed under the same HPLC/MS conditions.
Thermospray (TS) ionization was used to confirm
the molecular weights of the reduction products.
The HPLC/TS-MS analysis also indicated that the
principal blue colorant present in sludge I was the
monoazo dye 17 (Table 1), molecular weight 478,
an important precursor in the synthesis of Disperse
Blue 165 (TS mass spectrum not shown). Analysis
of the reduction products indicated that dye 17 was
present at the 3-5 /jg level in the sludge extract
based upon the relative intensity of similar reduc-
tion products generated from Disperse Blue 79
(13). This corresponds to about 143-238-ppm level
in the actual sludge sample. The TIC chro-
matogram C of the untreated sludge sample shows
no fully identifiable mass spectrum.
Sludge II. The analysis of the extract from the
Na.,S2C)4 treatment closely resembled that obtained
from the untreated sample. This indicates that
Na^C^ was not a strong enough reducing agent
for the colorant(s) present. However, SnCl, treat-
ment led to several new products, the principal of
which are shown in Figure 3. Table 3 lists the
retention time and m/z values of the two products
(3,3'-dichlorobenzidine and N,N'-dicyclohexy-
lurea) generated from sludge n by SnCl2 treatment.
Although both compounds were found in the
untreated and treated extract, SnCl2 treatment sig-
nificantly increased the amount of the amine to
near 1 pg (based on the relative response to benzi-
dine) in the sludge extract, verifying that the parent
dye contained a benzidine moiety. This corre-
sponded to about 84 ppm of dichlorobenzidine in
the sludge sample. These results suggest that the
principal components in this sludge were colorants
of type 18 (Table 1), a common skeleton of com-
mercial yellow-pigment azo dyes. They are the pri-
mary type colorants for which dichlorobenzidine is
still used today.
In-Situ Reduction of Commercial Azo Dyes
The ability to reduce azo dyes in a H2-Pd injec-
tion port liner, in-b'ne with GC/MS analysis, was
tested for some of the compounds in Table 1.
Initially, each dye was analyzed with an empty
injection port to demonstrate the absence of aro-
matic amines. The GC/MS analysis for eight dyes
did not detect aromatic amines in die dye formula-
tion. Also, most dyes (except Solvent Yellow 2)
failed to show intact molecular ions. Usually the
ions detected in the mass spectrum are a result of
thermal degradation of the parent dye in the heated
injection port.
In-line reduction using the GC injection port
required the presence of Pd and hydrogen and the
reaction required high temperatures (> 250°C) to
reduce relatively simple azo dyes such as Solvent
Yellow 2. Failure to achieve one of these criteria
significantly affected the reduction yield of the azo
dyes. With 5% hydrogen in helium reduction
yields were similar to those with pure hydrogen;
this removes the risk associated with using pure
hydrogen in the experiment.
17
-------
100-1
50 H
B
5 0
50^
o>
CD
cc
Na2S2O4 reduced
Sludge I extract
SnCI 2/HCI reduced
Sludge I extract
4.5
Unreduced
Sludge I extract
CH2CI2
from
extraction
r i i i i i i i i i i i i i
6 8 10 12 14 16 18 20 22 24 26 28 30 32
Time (min)
Figure 2
18
-------
TABLE 3. IDENTIFIED COMPONENTS IN REDUCED SLUDGE SAMPLES I AND II
Sludge Extract
No. and
(Reducing Peak Identified Reduction Mol Wt.
Agent Applied) No. Products
l(Na.,S204) 2 2-bromo-4,6-dinitro- 263
aniline8
5 4-(2-bromo- 478
4,6-dinitro-phenylazo)-
3-acetamido-N,N-di-
ethyl-aniline
l(SnCI2) 1 N,N-bis-(2- 199
cyanoethyl) aniline
2 2-bromo-4,6- 263
dinitro-aniline"
3 N,N-bis-(2- 277
cyanoethyl)
bromoaniline
4 4-(2-chloro-4-nitro- 382
phenylazo)-N,N-bis-
(cyanoethyl)aniline
5 4-(2-bromo-4,6-dinitro- 478
phenylazo)-3-acetamido-
N,N-di-ethylaniline
6 unknown 416
H(SnCI2) 1 N,N'-dicyclo- 225
hexylurea
2 3,3'-dichloro 252
-benzidine
Thermospray
tRa (m/z Relative Intensity)
Positive" Negative0
18.7 322(<1);
262(100)
28.9 496(1); 537(100);
479(100) 477(40)
11.9 217(100);
200(36)'
19.0 322(1);
262(100)
17.9 295(100);
278(10)
28.9 383(100) 441(22);
381(100)
28.9 537(100);
477(45)
24.7 417(100)
17.6 226(100)
20.0 270(2)
253(100)
Particle Beam UV
(m/z Relative Intensity) %d
N.D.1 3
478(10); 403(52); 70
348(20), 253(10);
221 (19); 205(1 00);
161 (63); 79(49)
199(7); 159(100); 7
106(46); 104(52);
91 (42); 77(70)
N.D. 1
277(1); 239(96); 8
237(1 00); 191 (20);
184(46); 155(37);
125(13); 76(64)
N.D 20
N.D. 10
41 8(32); 41 6(30);
403(92); 401 (100);
333(15); 323(12);
120(25); 105(29);
77(37)
225(8); 143(12); 5
99(21); 61 (23); 56(1 00)
254(68); 252(1 00); 45
223(21); 182(1 3);
154(27);126(31);
102(16); 86(33); 54(19)
atR = Retention time in TIC chromatogram (min); "positive ions detected were (M+l\IH4)*and (M+H)+; Negative ions detected were
(M+CH3C02)- and (M-H)-; "UV % = relative UV peak area in UV2M chromatogram; "positively identified also by HPLC/UV and standard
analysis; 'negative ion detection; °N.D. = not detected or spectrum from more than one component.
19
-------
Injection port temperatures over the region of
50 to 400°C were evaluated. Higher temperatures
were more effective for most of the dyes. The
intact dye or thermal degradation products of the
parent dye were vaporized, then subjected to
reduction in the injection port. A temperature of
300°C showed an acceptable combination of high-
er reduction yields and few thermal decomposition
products.
The in-line H^d reduction, GC/MS approach
was used to study eight dyes listed in Table 1. A
summary of the reduction products identified for
the dyes, based upon the El mass spectra, are pre-
sented in Table 4 and are discussed below.
Likewise, off-line solution-phase reductions of the
same azo dyes using SnCl2/HCl, followed by
GC/MS analysis (no reduction in the GC port) pro-
vided a means to compare the number and identity
of the products formed and their yields.
The H2/Pd reduction of the solvent dyes (1 and
5) primarily resulted in the cleavage of the azo
bond to form aromatic amines. The H2/Pd reduc-
tion of Solvent Yellow 2 yielded aniline and an
unexpected product (^ = 10.21 min, M** = 148)
together with a trace of unreduced dye. High reso-
lution mass measurements indicated the empirical
formula of C8H1QN3 (mass error 0.5 ppm), which is
consistent with the diazonium product, 4-diazoni-
um-N,N-dimethylaniline. The SnCl2 reduction
exhibited the two expected amines, aniline and
N,N-dimethyl-4-aminobenzene. The HJPd reduc-
tion of Solvent Red 24 primarily produced the
expected amines (2-aminotoluene, 2,4-diamino-
toluene, and l-amino-2-naphthol) from cleavage of
the azo bonds . The SnC^ reduction produced the
same amines, except 2,4-diaminotoluene was not
detected. Clearly, there is a similarity in reduction
processes for the in-line H2/Pd and solution SnCl2
reduction of these solvent dyes.
20
-------
Peak 2
Figure 3
8000-
7000-
6000-
5000-
4000-
3000-
2000-
1000-
0
77
63
\
80
126 154
108il39|179/2
' / \. 201
j J.U/J.Y
252
235
120
160 200
240
r-110
-100
-90
-80
-70
-60
-50
-40
-30
-20
-10
0
B
100-1
75-
e
>,
> 50 -
in
0)
Ji
§
1 25 -
0)
cc
0 ^
25 -
SnCI 2 - reduced
sludge II extract
Untreated sludge II extract
i
12
i
16
20 24 28
Time (min)
32 36 40
44
21
-------
TABLE 4. IDENTIFICATION OF MAJOR hL/Pd-REDUCTION PRODUCTS OF COLORANTS BY GC/MS
No.
1
5
7
9
12
14
15
16
Dye Identified Reduction Products Mol. Wt.
Solvent Yellow 2 aniline6
N,N-dimethyl-1 ,4-diaminobenzene
4-diazonium-N,N-dimethylaniline
Solvent Red 24 2-aminotoluenee
2,4-diaminotoluenee
1-amino-2-naphthol"
1-isocyanatonaphthalene
Disperse Red 1 aniline6
1,4-diaminobenzene6
4-nitroaniline
N-ethyl-N-(2-hydroxyethyl)-1 ,4-
diaminobenzene
Disperse Orange 25 »1 ,4-diaminobenzene6
4-nitroaniline6
N-(2-cyanoethyl)-N-ethyl-1 ,4-
diaminobenzene
unknown8
4-diazonium-N-(2-cyanoethyl)-N-
ethylaniline
4-hydrazine-N-2-cyanoethyl-N:
ethylaniline
Disperse Black 9 1 ,4-diaminobenzenee
(precursor) N,N-dimethyl-1,4-diaminobenzene-
N,N-bis-(2-hydroxyethyl)-1 ,4-
diaminobenzene
Disperse Orange 37 1,4-diaminobenzenee
unknown
unknown
2-chloro-4-nitroaniline
1 ,4-diamino-2,6-dichlorobenzene
N-(2-cyanoethyl)-N-ethyl-1 ,4-diamino-benzene
2,6-dichloro-4-nitroanilinee
Disperse Brown 1 aniline6
2,3-dichloroaniline
N,N-diethyl-1 ,4-diaminobenzene
2-chloro-4-nitroanilinee
1 ,4-diamino-2,6-dichlorobenzene
unknown
2,3,4-trichloroaniline
2,6-dichloro-4-nitroaniline6
Acid Orange 10 'aniline6
Acid Orange 10 -2Na-S03
93
136
148
107
122
159
169
93
108
138
180
108
138
189
191
201
204
108
136
196
108
136
142
172
176
189
206
93
161
164
172
176
189
195
206
93
326
Solution-Reduction
tR (area %)>*'
1.26(11)
5.00(88)
N.D.
2.19(93)
N.D.
12.35(1)
7.30(6)
1.39(7)
4.13(42)
8.17(11)
9.38(40)6
4.17(18)
8.18(3)
12.77(76)
N.D.
N.D.
N.D.
4.15(35)
N.D.
13.88(60)
N.D.
N.D.
N.D.
N.D.
8.13(45)
11.23(52)
N.D.
N.D.
N.D
N.D.
N.D.
8.23(100)
N.D.
N.D.
N.D.
1.36(2)
17.56(98)
Pd/H2 Reduction
tR(area %)>M
4.15(30)
N.D.'
10.24(45)
5.43(84)
9.38(3)
14.29(9)
11.94(1)
N.D.
8.03(16)
12.35(16)
13.80(62)
8.02(11)
12.31(3)
14.92(49)
16.45(2)
15.47(17)
15.71(18)
8.28(53)
9.49(13)
16.72(32)
8.14(7)
10.14(9)
10.51(20)
13.59(7)
12.41(17)
15.20(32)
14.11(8)
4.07(2)
9.99(2)
10.98(1)
13.52(3)
12.53(53)
15.49(28)
13.05(1)
14.01(10)
N.D.
N.D.
, = Average retention time in TIC chromatogram (min); "(area %) = percentage of the total peak area in the TIC
chromatogram; cReaction product from aqueous SnCI2 reduction; dReaction product from in-line hyPd reduction;
"identity confirmed with standard; 'N.D. = Not detected in TIC chromatogram; ^unknown = structure of formed reduc-
tion product unknown or only partially resolved.
22
-------
The disperse dyes (Nos. 7-15) tend to exhibit a
greater variety of reduction products due to other
reducible groups on the molecule (e.g., NO2) and
functional groups (e.g., Cl, OH) that can react
under the imposed conditions. The H,/Pd reduc-
tion of Disperse Red 1 resulted in the formation of
near equal amounts of the partially reduced prod-
uct (4-nitroaniline) and the completely reduced
product (1,4-diaminobenzene), together with N-
ethyl-N-(2-hydroxyethyl)-1,4-diaminobenzene.
The SnCl2 reduction produced the same products
as did Hj/Pd reduction, plus a trace of aniline, pos-
sibly from loss of NO2 and reduction of the azo
linkage. The H2/Pd reduction of Disperse Orange
25 produced six products. Three of the products
(comprising 63% of the ion current) could be
accounted for by reduction of the azo and nitro
groups for the dye. The other three products were
not as obvious. High resolution MS aided in deter-
mining the empirical formulas for the ions at m/z
201 and 204 which correspond to the respective
diazonium (CUH13N4) and hydrazine (CUH15N4)
products in Table 4 (mass error of 3.4 and 4 ppm,
respectively). However, the peak of lowest relative
intensity, at m/z 191, was not identified.
The SnClj solution-reduction of this dye exhib-
ited three products that were observed for the
H^/Pd reduction, originating from reduction of the
azo and/or nitro groups. The diazonium or
hydrazine products were not detected for the SnCl2
reduction. The H2/Pd reduction of Disperse Black
9 resulted in the formation of two products that
formed by reduction of the azo linkage (1,4-
diaminobenzene and N,N-bis(2-hydroxyethyl)-1,4-
diaminobenzene). Also, losses of methanol were
observed to form an N,N-dimethyl-1,4-
diaminobenzene. However, this product only
accounted for 13% of the ion current. The SnCl2
solution-reduction only resulted in the reduction of
the azo linkage to form the same two products (1,4-
diaminobenzene and N,N-bis(2-hydroxyethyl)-1,4-
diaminobenzene) detected by H2/Pd reduction.
The H^d reduction of Disperse Orange 37
formed seven products. The major product (32%)
was N-(2-cyanoethyl)-N-ethyl-1,4-diaminoben-
zene formed from reduction of the azo linkage.
Four products were identified to form by combina-
tions of the azo linkage or nitro group reductions or
losses of chlorine. These products included 2,6-
dichloro-4-nitroaniline, 1,4-diamino-2,6-dichloro-
benzene, 2-chloro-4-nitroaniline, and 1,4-diamino-
benzene and accounted for 39% of the ion current.
Two products (Mol. wt. 136 and 142) were not
identified. The SnCl, solution-reduction only
formed two products, "l,4-diamino-2,6-dichloro-
benzene and N-(2-cyanoethyl)-N-ethyl- 1,4-di-
aminobenzene. These two products were the two
main products observed by H2/Pd reduction.
The H2/Pd reduction of Disperse Brown 1 was
similar to that of Disperse Orange 37. There were
eight reduction products detected, and the main
product (53%) was l,4-diamino-2,6-dichloroben-
zene. However, other products were detected that
involved the reduction of a nitro group, as well as
losses of nitro groups and losses or addition of Cl
(e.g., aniline, 2,3-dichloroaniline, 2-chloro-4-
nitroaniline, 2,6-dichloro-4-nitroaniline and 2,3,4-
trichloroaniline). Also, a reduction product, N,N-
diethyl- 1,4-diaminobenzene was detected instead
of the expected product, 3-chloro-N,N-(2-hydrox-
yethyl)- 1,4-diaminobenzene. The SnCl2 reduction
only produced one product (l,4-diamino-2,6-
dichlorobenzene), the same product observed in
H2/Pd reduction. While a variety of products can
be formed by H2/Pd reduction of disperse dyes, the
major products correspond to the reduction prod-
ucts observed from SnCl2 solution reductions.
The most difficult dyes to study by mass spec-
trometry are the acid dyes. The salts of sulfonic
acid dyes cannot be volatilized without thermal
degradation. Even the free acids are extremely
nonvolatile (39). This may explain why Hj/Pd
reduction of Acid Orange 10 failed to produce any
significant peaks that could be correlated with the
reduction of this dye. The SnCl2 solution-reduc-
tion of the acid dye resulted in two products; the
main reduction product (98%) was a partially
desulfonated parent dye (M*~ = 326), and the other
was aniline.
Several general reaction schemes for the H2-Pd
reduction in the GC injection port can be formulat-
ed based on the products identified in Table 4.
Structurally simple dyes (Solvent Yellow 2 and
Solvent Red 24) formed the anticipated aromatic
amines as shown in Scheme 3.
H2/Pd
Scheme 3: R,-N=N-R2
The presence of other functional groups (e.g.,
NO2, OH, CN or halogens) increased the number
of products formed. The presence of a nitro group
in the dye usually resulted in the formation of a
diamine (Scheme 4).
23
-------
Sctene4:
H/Pd
R,N=N-R2-NO2
+ O2NR,NH2 + H2NR,NH2
Reaction products resulting in the loss of a
halogen (e.g., Cl), OH, or CH2OH from the dye
were more difficult to predict, but accounted for
less than 10% of the ion current for the reduced
dye.
The H2/Pd reduction yield for the simple azo
dyes (e.g., Solvent Yellow 2) was above 60%. The
yield was based on the molar response of the amine
standard (e.g., aniline) relative to the molar quanti-
ty of the parent dye. More complex azo dyes that
contained additional functional groups resulted in a
wide variety of reduction products. Lower yields
were often observed since the same azo cleavage
products could be further reduced to form other
products (e.g., Disperse Red 1 can be reduced to
form 4-nitroaniline, which then can be reduced to
form 1,4-diaminobenzene). However, if the molar
response for the two possible reduction products
are summed, the reduction yield is about 70%.
Also, it was not possible to calculate the yields of
the more complex dyes in this manner due to a lack
of reference standards. While the reduction was
not 100%, the relative intensities of the reduction
products for Solvent Yellow 2 and Solvent Red 24
were consistent (standard deviation <25%) for their
daily analysis over the course of the study.
The H^d reduction of azo dyes in Table 1
indicates that several common cleavage products
can arise. Aniline is a common cleavage product
for dyes 1, 7, and 15; 1,4-diaminobenzene for dyes
Nos. 7, 9, and 12; 4-nitroaniline for dyes 7 and 9;
and l,4-diamino-2,6-dichlorobenzene for dyes 14
and 15. As a result, detection of a limited number
of amines can aid in determining dye types used in
manufacturing.
Detection of Aromatic Amines in Sludge Extracts
after In-Situ Reduction
Extracts of sludges (I and n) from two different
waste treatment sites were reduced with H/Pd in
the injector and then analyzed by GC/MS to quali-
tatively screen for azo dyes based upon the detec-
tion of aromatic amines or other related reduction
products. The GC/MS conditions were the same as
for the dye standards.
Sludge I. The in-line reduction treatment of a
wastewater sludge formed seven new products.
The retention times and postulated molecular
weights of these reduction products are listed in
Table 5. Identification of products was based
mainly on mass spectral library searches (41),
manual spectral interpretation, and the analysis of
available reference standards. In both the unre-
duced and the reduced wastewater sludge extract,
numerous fairly simple aromatics have been tenta-
tively identified. The major components present in
the reduced and unreduced extracts were aniline
(^ = 4.10, M+" = 93), 3-aminophenyl propanenitrile
(^ = 10.98 min, M+' = 146), 2,4-dinitrobromoben-
zene (^ = 12.56, M+- = 246), 2-chloro-4-nitroani-
line (tu = 13.52 min, M+- = 172), and (1,1'-
biphenyl)-4-ol (^ = 13.96 min, M*" = 170). Three
of the seven reduction products listed in Table 5
were tentatively identified as 4-nitroaniline, 4-
nitro-l,2-diaminobenzene, and 2-nitro-1,4-
diaminobenzene. These products are consistent
with the principal blue colorant in sludge I that was
identified by thermospray LC/MS to be the monoa-
zo dye 17 (Table 1). However, it is evident that
sludge I contains additional azo dyes or other com-
pounds that can be reduced to form aromatic
amines.
Sludge II. The H2/Pd treatment of the waste-
water sludge produced four new reduction products
listed in Table 5. Most of the components identi-
fied in the unreduced extract were present after the
in-line reduction. Both samples contained poly-
substituted aromatic amines and polychlorinated
aromatics. The major components identified in
both the unreduced and the reduced wastewater
sludge were aniline (^ = 4.10 min, M+ = 93), 1,3-
or 1,4-dichlorobenzene (^ = 4.71 min, M+' = 146),
4-ethoxyaniline (^ = 7.97 min, M+- = 137), methyl-
aniline (^ = 5.48 min, M+- = 107), 2-methoxyani-
line (^ = 7.01 min, M+" = 123), 1,2,4-trichloroben-
zene (^ = 7.97 min, M+" = 180), 2,3-dichloroaniline
(^ = 9.18 min, M+' = 161), 2,3,4-trichloroaniline
(tR = 11.98 min, M+' = 195), 4-chloro-2,5-
dimethoxyaniline (^ = 13.00 min, M*- = 187), 4,4'-
dichlorobiphenyl (^ = 14.62 min, M+' = 222), and
3,3'-dichlorobenzidine (^ = 21.01 min, M+ = 252).
Although substituted benzidine derivatives were
found in the untreated and reduced extract, H^d
reduction produced higher levels of dichlorobenzi-
dine. Comparative results, obtained from thermo-
spray LC/MS, confirmed the occurrence of
dichlorobenzidine in this sample. These results
suggested that the principal component in the
24
-------
sludge was a colorant of type 18 (Table 1). This is These yellow azo pigments are the primary type of
a common skeleton of commercial yellow azo pig- colorants for which dichlorobenzidine is currently
ments, which were present in the sludge II sample. used.
TABLE 5. MAJOR COMPONENTS FORMED AFTER IN-SITU REDUCTION OF SLUDGE I AND H
Sludge Extract
No
I
n
Peak No.
1
2
3
4
5
6
7
1
2
3
4
Identified Products
2-chloro-l,4-diaminobenzeneb
4-nitroanilineb
4-nitro-l,2-diaminobenzeneb
2-nitro- 1 ,4-diaminobenzene
unknown0
unknown
unknown
2-methylphenolb
4-chloroanilineb
2,5-dimethoxyaniline
benzidineb
Mol. Wt.
142
138
153
153
203
247
294
108
127
153
184
V
10.73
12.33
15.27
16.01
17.26
18.52
19.29
5.62
6.26
10.43
17.85
\ = Average retention time in TIC chromatogram (min); bldentity confirmed with standard; 'Unknown
Structure of formed reduction product unknown or only partially resolved.
25
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26
-------
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