EPA/600/A-95/072
LC/MS TECHNIQUES FOR THE ANALYSIS OF DYES
bv
J. Ymon1. L, D. Betowskr and R. D Voyksner
Weizmann Institute ot" Science. Deportment ol" Environmental Sciences and Energy Research.
Rehovot 76100. Israel.
U.S. Environmental Protection Agency. Environmental Monitoring Svstems Laboratory. Las Vegas.
NV 89119. USA
Research Triangle Institute. Analytical and Chemical Sciences. Research Triangle Park. NC 27709.
USA.
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CONTENTS
1. Introduction 1
2. Thermospray (TS) - LC/MS 3
2.1. Principles of Operation 3
2.2. Applications to Dye Analysis 4
3. Particle Beam(PB) - LC/MS 10
3.1. Principle of Operation 10
3.2. Applications to Dye Analysis 12
4. Ion Spray and Electrospray (ES) - LC/MS 15
4.1. Principles of Operation 15
4.2 Applications to Dye Analysis 16
4.2.1. API-MS of Sulfonated Dyes 16
4.2.2. LC/MS of Dyes 17
5. Summary 22
Notice 23
References 24
Figure Captions 30
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LC/MS TECHNIQUES FOR THE ANALYSIS OF DYES
1 Introduction
Dvesmffs ore of major environmental interest because oi* their widespread use as colorants in a
variety of products, such as textiles, paper, leather, gasoline, and foodstuffs. S\ nthettc intermediates, by-
products. and degradation products of these dyes could be potential health hazards because of their toxicuy or
carcinogenicity
The analysis of ekes poses special problems for the chemist. The d\es Jo not belong to one group of
chemical compounds, but encompass main chemical functionalities, ranging from mostly ionic to ptirclv
covalenl. The anahsis of such a large \nrietv of compounds poses difficulties because ol large differences in
solubility, volatility, ionization efficiency, etc. A semantic problem often leading to confusion in the anahsis
of dyes is the difference between dye classification and dye use. D\ c classification is based on the major
functionality of the dye: azo. amhraquinone. poKmethine. phthaloc\ anine. sulfur, an Imethane. sulbcne. and
coumarin being the main classes. The use of a dye generally refers to the manner in which the dye is applied
Some of the more common applications are in acidic or basic media, as mordants, lakes, pigments, solvents,
or dispersants.
As an additional complication, some of the manufacturing precursors to dyes are carried over to. and
are not removed from, the final dye product. The result is a complex mixture characterized not only by the
dye itself, but also by several other compounds. Most dyes, including sulfonated azo dyes, are nonvolatile or
thermally unstable, and therefore are not amenable to gas chromatography (CC) or gas phase ionization
processes. Therefore. GC/MS techniques cannot be used
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Several desorption ionization methods have been used for the analysis of dyes:
a. Field Desorption (FD) Mass Spcctrometiy 11-3|
b. Secondary Ion Mass Spectrometry (SIMS) 14-71
c. Califomium-252 Plasma Desorption Mass Spectrometry (PDMS) |X.9|.
d. Fast Atom Bombardment (FAB) Mass Spectrometry ) 10-141.
e Laser Desorption Mass Spectrometry 1151
Because of low sample purity, modern complex dyes cannot be annly/ed using the above mentioned
direct probe techniques. However, the combination of liquid chromatography with mass spectrometry
(LC/.V1S) enables the separation of nonvolatile, thermallv unstable, and polar d\es tor introduction into the
mass spectrometer lor identification. Significant advances in combining LC and MS have occurred in recent
years and have been extensively reviewed 11 (i- 19).
Most of the earlier work on LC/MS foenssed on ihc incompatibility of LC mobile phase tlows arid
the vacuum requirements of the mass spectrometer An aqueous reversed-phasc LC mobile phase at a flow
rate of 1 mL/irun can generate I-4 liters of gas when introduced into a mass spectrometer at 10"" ton. This
exceeds the operational requirements of most MS systems. In addition, the thermal lability or low volatility
of the analytes may impede their transformation into the vapor state and subsequent ionization by electron
ionization (EI) or chemical ionization (CI). As a result of research in interfacing LC with MS. three major
types of interfaces and LC/MS techniques have been developed
a. Thermospray (TS),
b. Particle Beam (PB).
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c. Ion spray and Electrosprnv (ES).
These LC/MS melhods have been aupiied in n inruc variety of anahucal problems This chapter will
describe the application of these LC/MS techniques in die analysis ol\ives,
2 Thermosprny (TS) - LC/MS
2.1. Principles ol Operation
Thermospray is a widely accepted technique because it can handle most conventional LC solvents ana
flow rates, as well as provide a means to gently ionize most nonvolatile or thermalh unstable samples. It has
good sensitivity, within a factor of 10 to that of GC/MS. The interface is commercially available for most
mass spectrometers and is simple to use
The techniques and mechanisms of ihcrinospnn have been reviewed j 191. The aqueous solution of
the sample contains a volatile electrolyte (typically, ammonium acetate) at concentrations near 10"' \1 The
TS interface consists of a vaporizer, where the mobile phase is heated to form a high-velocity spray As a
result of the statistical distribution of ions in this spray, some of the micrometcr-si/c droplets of the spray are
electrically charged The high electric Held induces desorption of performed ions from the liquid solution into
the gas phase. These ions could result from the solute molecule by protonation or addition of solvent cluster
ions. The primary ions produced m the TS process arc identical w ith those produced in solution; in
ammonium acetate solution, the ions are NH," and CH-,CO:'. and clusters of these ions with water, ammonia,
and acetic acid. Equal amounts of positive and negative ions are produced The droplets enter the source,
where the ions are extracted through the ion exit cone, while neutral molecules go to a cold trap connected to
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a mechanical vacuum pump. This extraction process allows the introduction of a total of 1-2 iiiL/nim of LC
effluent, while maintaining a pressure of 10" torr m the mass spectrometer
The TS interface can accept tlow rates as low as 0.1 mL/min through the addition of solvent
post-column to result in a total [low rate of I mL/min. It can accommodate most solvents used in normal or
reversed-phase LC and any volatile buffer This is the only interface that operates optimally under highly
aqueous conditions, with best sample ion currents at 100% water 120 ( The interface can be operated
smoothly through a solvent gradient LC analysis if the vapori/cr temperature is adjusted to compensate for
changes in the heat of vaporization of the changing LC solvent Buffers necessary for thermospray ion
formation do not have to interfere with the LC chromatographic separation because they can be added
post-column [21 ]. resulting in optimal LC and MS operation.
Thermospray is both an ionization and enrichment technique. Ions may be produced by CI. initiated
by a filament or discharge, or through ion evaporation. The major disadvantage of thermospray is that the
ionization occurs in the solvent at a relatively high source pressure of at least I torr As a result, electron
ionization (EI) cannot be used. The spectra, therefore, cannot be compared with those of the readily
accessible, commercially available El libraries
2.2. Applications to Dye Analysis
Betowski and Ballard (221 used tandem mass spectrometry in conjunction with a TS interface to
elucidate the structure of Basic Red 14. The instrument was a Fmnigan MAT Triple Stage Quadrupole
modified for thermospray ionization. The HPLC consisted of a Rheodyne Model 7125 injector valve and a
Waters 6000A solvent delivery system The column was a Brownlee RP-2. 10cm x 4 6mm I D .
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analytical cartridge column. Aqueous 0 I M ammonium ncctate/melhanol (93 3 wv) ;u a How rate of
I 3 inL/min was used as TS buffer
The TS positive ion mass specinim of Basic Red I 4 generated a base peak of nvz 344 that was
construed to be the parent ion. However, m addition to this ion. there were peaks of significant abundances at
m/z 174 and 189. There was also a peak fen. 35-40 percent relative abundance) at nvz 34fi. Since TS
ionization is a soft technique, tiiese ions wore attributed to impurities in the Basic Red 14 This assumption
was strengthened by the MS/MS collision,il induced dissociation (GDI spectrum of ni/'z 344 The ions at
m/z 174 and 189 were not present in this spectrum. The CID spectra of the ions at m/z 174 and 189
suggested these were the " ions of ail indoiinc and a ben/aldch\de. rcspcctn cl\. and the structures were
confirmed w ith the authentic standards. An important class of canonic d\cs. the mcthincs. is preparea bv the
condensation of an aldehvdc w ith an indohne. When the condensation reaction of the identified mdoline and
benzaldehyde w as performed, the mixture turned red. and the thermospray spectrum of the product show ed a
m/z 344. which, under CID conditions generated a spectrum identical to the initial spccinim. The nvz 346
ion proved to have originated from a compound formed as a result of a reduction of the parent dye
Ballard and Betowski [231 continued their work on TS of dyes, using the same instrument, with a
study of 16 dyes belonging to six different classes They reported detections limits from 15 to 200 ng for a
variety of dyes. Thcmiosprav ionization worked well for the representative dyes of the az.o, methine.
arylmethane. anthraquinone. coumarin. and xanihene classes Howev er, this technique worked less well for
the sulfonated dyes. For example. Acid Orange (> (Fig 1) is n sodium salt of a monosullonated. monoazo
dye having a molecular weight of 3 16 daltons. In the positive ion TS spectrum, the (M + H) and the (VI +
Na)+ ions are observed at m/z 3 17 and 339. respectively The base peak in this spectrum, however, is the ion
at m/z 295. which corresponds to the protonated sulfonic acid form of this dye.
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Tlie negative ion TS spectrum shows an ion at m/x 293. which corresponds 10 the anion of tins cive.
(M - NaV VVTien the auxiliary- filament was turned on. m/z 294 appeared. This ion was attributed to the
electron capture product of the free sulfonic acid, which may be present as an impurity-. The negative ion TS
mode was found to be less sensitive than the positive ion TS mode b\ aL least a factor of ten
Covey and Henion [241 used a dual purpose DLI/TS LO'MS interface, which was introduced into a
Hewlett-Packard 5985B GC7MS via the standard, direct insertion probe inlet. A \ aricty of compounds were
analyzed with this instrument, including an industrial d\c. sulforhodamine B. The TS mass spectrum
included an MH* ion at m/z 559 and a fragment ion at m/z 4f>7
Voyksner [25 j demonstrated the use of TS-LC/MS to characterize azo. diazo. and anthraquinone dyes
in wastewater, soil, and gasoline. TS mass spectra of the analyzed d\es produced mainly MH* ions with few
fragments. Switching the filament "on", produced additional fragment ions which helped in the structural
elucidation of these dyes The commercial diazo and aiuhraquinonc dyes proved to be very complex
mixtures of nearly 40 alkyi-substiluied dye components, making monitoring and identification of a particular
dyestuff difficult The detection limits were found to be 10 ppl in wastew ater. 100 ppb in soil, and I ppm in
gasoline. Fig. 2 shows the mass chromatograms of a commercial red dye spiked into gasoline.
Thermosprav LC/MS/MS was found to be effective in the analysis of wastew ater for disperse azo
dyes [26], In this study. Disperse Red I was used to test the effectiveness of an activated sludge process.
Primary effluent from a municipal wastewater treatment plant M as used as the feed for the system. The
system was spiked with two concentrations of the dye. The samples were analysed by a combination of
HPLC/UV-visible. TS-LC/MS. and TS-LC/MS/MS. The results from the mass spectrometric methods for
various samples agreed with the HPLC/UV-usible results within 5 to IX percent The average precision for
f,
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the mass spectrometry methods was 12 percent. The IS- LC7.MS and TS-LGM.S/MS s\stems were
important for not only monitoring the disappearance ol' Disperse Red 1. but also lor identifying breakdown
products from the aetivatcd waste process A ir.ajor degradation product had the same nominal mass as a
major interference ion. Tandem mass spectroniei\ was required to differentiate between the background ton
and the degradation product ion. Another complication in identifying tins product was thai the initial mobile
phase gradient did not cause this compound to elute. !l look the addition of 0.1 M ammonium acetate to the
mobile phase to help elute this breakdown product.
Flor\ ei ai. 1271 investigated various factors that affected the ihermosprav response for nine
sulfonated azo dves. They used a mouilled Hew leu-Packard ?^XKA mass spectrometer, connected to a
Scientific S\stems Model GS400 HPLf gradient .sv-uem \ ia a Vcstcc TS interlace Their major llndtng was
that too high a concentration of ammonium acetate buffer suppresses the ionization of these anionic uyes.
They suggested that the major ionization processes in these dyes is anion evaporation directly from the
droplet. If too high a concentration of ammonium acetate is added, ejection of the more volatile acetate ion
will compete with the evaporation of the dye anion
Yinon el nl. [28. 291 worked on increasing the sensitivity of the TS technique by use of a w ire repeller.
A series of dyes belonging to different chemical classes ( Fig 3) were analyzed by TS-LC/MS using a
modified source containing a wire repeller. The instrument used was a Fumigan MAT Iriple-stage
quadnipole (TSQ) equipped with a Vcstcc ion source and thcrmospray interface This ion source was
originally not provided with a repeller A hole was drilled exactly opposite the ion-extraction funnel and a
0,025 in. diameter copper wire was introduced, insulated with a ceramic tube The wire faces the funnel but
does not protrude into the ion chamber volume The repeller was operated at a voltage range of 220-250
Volts. The HPLC consisted of a Rheodyne Model 7125 injector valve and a Specira-Phvsics SP8700XR
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solvent delivery- system. A syringe pump (ISCO LC-5000) was connected to the system lo deliver the buffer
(0.1 M ammonium acetate) postcolwnn-via the TSP interlace into the source Mcthanol-water was used as
mobile phase. An increase of about two orders of magnitude'was obtained with the wirc-repellcr. as well as
an increase in the relative intensity of the molecular ion \ersus the fragment ions Figure 4 shows the mass
spectra of Disperse Blue 79 at repeller 0 and 250 Volts, respecmch
The TS positive ion mass spectra of several sulfonated dyes were recorded for the first time because
of the increased sensitivity. For example, the mass spectrum of Acid Blue 40 has a base peak at m/z 372.
probably due to loss of NaSO, from the protonnted molecule and replacement of this N'nSO, group h\ a
hydrogen atom. Lower-abundancc ions in the mass spectrum included the MH ion at in// 474. an | Vl-j-Nn|~
ton at m/z 496. (M+Na-HCOCH,]" ai mi/. 452. |MH-H:COCH«|' at m/V 429. |MH-NH:-H,COCH,|' at m/z
412. IMH-SO,]* at m/z 394. and f,VIH-NaSO-+H-CH;CO| at m// 330
Losses of SO.Na and 2SO.Na as well as losses of Na and 2Na. from the protonatcd molecule, were
observed in the sulfonated dyes The loss of each one of these groups involved the replacement by a
hydrogen atom. Acid Blue 40. which has only one SO,Na croup, and Acid Red I 14. w hich has two SO,Na
groups attached to the same ring, lose one and two SO.Na groups, respectively. Direct Red 81 and Acid Blue
113, each having two SO,Na groups on different rings, lose both one and two Na atoms, but do not lose SO,
groups, which seem to be more strongly attached to the molecule
The same instrument was used to acquire rcpeller-activated collisionally induced dissociation
(repeller-CID) mass spectra of dyes |30|, These were obtained by applying a voltage of 400 V on die
wire-repeller. The mass spectra contained a large number of fragment ions that were useful for structural
elucidation. Some of these fragment ions were found lo be similar lo those obtained by thermospray tandem
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mass spectrometry with C'lD. and some of them were similar in those obtained by El. using a particle benm-
LC/MS. Fig. 5 show s a compnrison of three mass spectra ol'Disperse Yellow 5. TS-rcpeller-CID.
TS-MS/'MS-CID a no EL
A TS-LC/MS system was modified for the analysis of ihcs bv restricting the TS vaporizer exit orifice
and adding a nccdlc-up repcller to the ion'source |3 1 j. An increase in signal response for disuifonatcd azo
dyes was observed in the negative-ion nuxie. TS mass spectra contained inainiv
fM-Na|'. |M-2Na|:. and |.Vl-2N'a^H| ions.
The red dyes of woven fabrics from the Greco-Roman period, excavated at Akons in middle Eg}pt.
were investigated bv a series of analytical techniques, including TS-LC/MS with selected ion monitoring
[32], The instrument used w as a Shimad/u QP-1000 LCV.VIS The HPLC consisted of a LC 6-A pump and a
Shim-pack CLC-ODS(M) 15cm \ 4.6 mm 1 D column The solvent was methanol. I) IM ammonium acetate:
acetic acid (100:40:7) at a llow rate of 1 niL/min Results show ed the presence of Alizarin (MH~ at m/z. 240).
suggesting that the fabrics were dyed w ith madder
TS-LC/MS together with GC/.VIS and particle bcam-LCVMS were used to study photodcgradation
products of Disperse Red 167 1331 This dye is a widcl> used dvc cspcciallv for automobile cloth interiors.
The instrument used was a Finnigan MAT 4500 qtiadrupole mass spectrometer equipped with a Vestec TS
interface. The chromatographic separation was performed on a Waters 600 MS HPLC pmnp with a
Supelcosil LC-1 25cm x 4 6 mm I D. column The flow rate w as 1 mL/mm through the column with
post-column addition of 0 3 M ammonium acetate at 0 2 mL/min. The TS data provided insight into a great
number of photodegradation products but w as less helpful in structure elucidation
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TS-LC/MS was used to analvze a series of disperse dyes extracted from polyester and cellulose
acetate fibers, a basic dye from orlon fiber, and n vat dye from denim (Table i) [341. Molecular
characterization of each dye was obtained from the extract of a single fiber. 5* 10 mm long. The instrument
used was a 450IB Finnigan MAT quadrupole mass spectrometer with a TS interlace and ion source. The
HPLC system consisted of CM4000 LDC Milton Roy solvent-delivery system with a Rhcodyne 7125 injector
valve and a Merck Cl:<. 15 cm x 4mm I D. column The mobile phase, at a lion rate of 0 9 mL/min. was
inethanol-water. starting at 50:50 and changing w ilhm 5 min. to 100% methanol. The buffer. 0.1 M
ammonium acetate, was delivered post-column via the TS interface into the ion source by a Constamemc Bio
3000 Milton Roy delivery pump. The ion source repeller was operated at a voltage of 100 V Figure 6 shows
the reconstructed ion chromatogram and TS mass spectrum of Indigo (Vat Blue 11.
Thermospray. panicle beam, and elcctrosprav LG'MS were used for the analysis of a series of 14
commercial azo and diazo dyes (Fig. 7) [351 The HPLC consisted of Waters Series 600 multi-solvent
delivery system, a Waters U6K injector and a Spherisorb ODS II. 25cm x 4.6 mm I D.. 5-;'.m particle size,
column. Posteolumn ammonium acetate buffer addition was done with a Waters Model 6000 pump. The
HPLC was connected to a Finnigan MAT 4500 quadrupole mass spectrometer via a Vestec Model 701C TS
interface. TS analyses of the inv estigated dyes produced mass spectra consisting primarily of MH~ ions with
very few fragments.
3 Particle Beam(PB) - LOWS
3.1. Principle of Operation
Particle beam (PB) LC/MS provides a means of obtaining both El and CI mass spectra for
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compounds that can be brought into the gas phase but mas not be amenable to GC due lo thermal Inbilu\ or
lack of volatility. EI mass spccLra arc more casih referenced so mass spectral data bases, which can assist in
the identification of unknown components in \ annus matrices In addition. El fragmentation patterns offer
valuable information in structure elucidation The nbilitv to obtain CI spectra wuh the same interface makes
it possible to also obtain molecular weight information on the analyzed compound
In the PB interface, the transfer of the neutral analvte from the column to the ion source of the mass
spectrometer is accomplished only hv aerodynamic means The PB interlace |36-3,S| has three basic
functions: (1) aerosol formation. (2) desolvation. and (?) momentum separation of analvte from solvent.
Following LC chromatographic separation, the column eliiatc is passed to a nebulizer. where the citient
(solveni + analvte) is dispersed into a fine mist of droplets. Aerosol formation is done bv a coaxial (low of
helium, which nebulizes the LC eilluent The resulting aerosol is then desolv ated in a heated desolvauon
chamber, where the volatile sohent evaporates, while the dissolv ed analvte condenses to form solid
micromeier sized panicles The resulting mixture of particles. sol\ em molecules and helium atoms is drawn
through a small nozzle into a pumped chamber, causing a rapid expansion to occur. The relatively high-mass
solute panicles will gam high momentum from the expansion, enabling tlicm to traverse the separator as a
linear beam. Separation is achieved by sampling the panicle beam through a small onfice (skimmer), leaving -
the gaseous solvent molecules and helium atoms to be pumped away. The process is repeated in the second
stage of the separator, with a resulting enrichment of 10"' - 10 s particles relative to solvent Finally, the
panicles pass through a short transfer line into the ion source The heated walls of the source Hash-volatilize
the particles into the gas phase for EI or CI ionization. The PB interface can handle common solvents for
normal or reversed phase separations, v olatile mobile phase additives, and How rates up to 1 mL/min.
The use of El or CI is limited lo compounds that can be brought into the gas phase This is a severe
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limitation when dealing with the nonvolatile and thermally unstable d\cs that are oltcn found in LC
separations.
3.2. Applications to Dye Analysis
Vlarbury et al. |39] used PB-LC/Y1S lor the analysis of a series of d\es. including Solvent Red 1.
Disperse Red I 1 and Disperse Blue 3 The instrument was an Extrel 400-1 quadnipole mass spectrometer
fitted with a PB interface and standard EI/CI source Methane was used as CI reagent HPLC separations
were done with a Waters gradient system with either a C,„ RCM or C: SS column Figure 8 shows the EI and
CI spectra of Solvent Red I. In addition to the molecular ion at mi v. 278. structurally significant fragment
ions are obtained.
Yinon et al. [401 interfaced an HPLC to a Finmgan-MAT Triple Stage Quadnipole equipped with a
4510 EI ion source by means of an Extrel ThermaBcam PB-LC/MS interface and used the system for the
analysis of a series of commercial dyes The HPLC consisted of a Spectra-Physics SP8700XR
solvent-delivery system with a Rheodync 712? injector valve and a Varian MicroPak MCH-5-N-CAP Cls 15
cm x 4 mm I D column Methanol-watcr. in a gradient mode, was used as mobile phase at a How rate of 0.9
mL/min. Table 2 shows the EI mass spectra of the analyzed dyes. Characteristic EI fragmentations in azo
dyes included cleavages of the N - C and C - N bonds on either side of the azo linkage and cleavage of the N
= N double bond, with transfer of two hydrogen atoms to fonn an amine The mass spectra of most azo dyes
contained a small molecular ion or none at all. No fragmentation resulting from ring cleavage was observed
in the azo dyes.
The detection limits of the analyzed dyes ranged from 50 ng. lor Disperse Orange 3. to 2 7 ng for
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Disperse Broun I. As a comparison, the LO'.VIS system's sensitivity was checked with caffeine, for which n
detection limit off ng was found.
Pliotodegradation products of Disperse Red I 67 were determined by PB-LC/'MS (together with
TS-LC/MS-sce section 2.2) with a Hewlett-Packard 5')XXA quudrupole mass spectrometer 1331. The
chromatographic system used was a Waters 600 ,VIS HPLC pump and a Supelcosil 5-^m LC-I. 25 cm \ 4 6
mm ID. column. The mobile phase was mcihanot-waier. m a gradient mode, at a llow rate of 0.5 mL/min.
The major photodegradation pathways uerc found to be reduction of the nuro group, free radical loss of N2
followed by coupling to form a substituted biphcml. hvdrohsis of the ester, and N-dealkyiation.
The EI mass spectra of a series of 14 commercial a/o and din/o d\cs I Fig 7) uerc studied using
PB-LC/MS [351. The HPLC was a Waters Series (>()() multi-solvent dcli\ cry s\ stem controlled by a Waters
600-MS system controller. The dyes, dissolved in methanol or acetonitrile. were injected through a Waters
U6K injector and separated on a Spherisorb ODS II. 25cm x 4 6 mm 1 D . 5 .-.m particle size, column. The
HPLC was connected through a PB interface to a Hewlett-Packard Model 59XXA quadrupole mass
spectrometer.
From most azo dyes it was possible to obtain molecular ion information and characteristic
fragmentation patterns for structural elucidation Fig l) shows the RIC chromatogram of Disperse Brown 1
and the corresponding El mass spectra of two resolved components. The major peak (number 1). at a
retention time of 29.1 mm shows a mass spectrum with a relative!} small molecular ion at m/z 432 and an
intense p cleavage fragment |M - CH:OH|~ at m/z 401. as well as an intense C-N cleavage product at m/z
183. The shoulder peak (number 2) is due to a decomposition product of the dye. The two smaller peaks
(numbers 3 and 4) are probably due to plastieizer impurities. The detection limits were in the range of 500 ng
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to 5 ;tg. with n signal-to-noise ratio of 3:1. ;it lull scan
The chemical reduction of n/.o dyes containing industrial .sludges usuaiK forms a neariy colorless
effluent. Depending on the identity of azo dves in the waste, the aromatic reduction products are more
harmful to the environment than the untreated sludge components
The use of Na-S-0., or SnCk to cleav e the N=N- moietv of a/.o dy es followed by LC/MS analy sis of
the reduction products is a possible way to assess toxicity potential of complex w aste sludges Voyksner et
al. [411 identified the reduction products from the reciucti\ e cleavage of i (> commercial a/.o dyes by
PB-LC/MS. The instrumentation used w as the same as in the previous application 135 | Standards of the
formed reduction products were used to coniirm identities The chemical reduction methods resulted in nearly
complete reduction of the a/.o bond to form aromatic amines Overall, tin chloride was the more powerful
reducing agent, yielding a greater number of products Table 3 shows the chemical reduction products of the
investigated dyes.
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Measurements ofdves with T.S-LC'MS were lound to be In two to three orders of magnitude more
sensitive than with PB-LC/MS Pace and Robv 142) anaiv/ed several d\e wastes lor synthetic precursors,
transformation products, and selected aromatic amine target unaiues associated with the manufacture of azo
dyes. They used PB-LC/MS and GC/MS The LC7MS >\sicm umsisted of a Hewlett-Packard K1W HPLC
with photometric detector, coupled bv a PB unci lace to a Hew leti-Paekard 5MXXA quadnipole mass
spectrometer Both El and NCI loni/.aiion modes were used in both PB-LC/MS and GC7MS s>stems Tabic
4 shows the comparison of ten tat i\civ identiiieci compounds m an a/.o d\c sample by PB-LC/MS ana
GC/MS, Sev eral compounds were detected In GC/M.S. but not in PB-LC'/MS. because of the higher
sensitivity of GC/MS. in contrast, several a/.o compounds were detected in PB-LC/MS but not by GC/MS
because the azo compounds decomposed m the heated GC
4 Ion Spray and Electrospray (ES) - LC/MS
4.1. Principles of Operation
In a topical ES mass spectrometer, a solvent How of I to iO . ,L/min is introduced at atmospheric
pressure into an ion source chamber through a stainless steel hvpodcmuc needle, which is at ground potential
[43], The solvent How forms a fine sprav of charged droplets in response to an applied electric field of 2 - 4
kV. As the droplets evaporate, the annlvte ions, whose poiaritv is opposite to the applied potential, migrate to
the surface of the droplets, where Coulomb repulsion causes the droplets to break up into \ei smaller
droplets, thus enhancing ev aporation. At some minimum droplet diameter, the analvtc ions are believed to
desorb from the droplets into Lhc gas phase This process is known as ion ev aporation. and is Lhe primary
mechanism for gas phase ion formation in e!eclrospra\
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A compound will yield an observable ion if it is able to ionize in solution Thus, basic compounds are
dissolved in acidic solutions and acidic compounds in basic solutions The resulting cations and anions are
analyzed in positive and negative ion modes, rcspcctivelv
Ion spray (pneumatically assisted electrospnn I uses pneumatics 10 assist nebuli/ation and desoKation
of liquids in electrosprav Nitrogen is added coaxially lo the samples to assist in ilie nebulization of the
liquid. A potential of 3 - (> kV on the needle assembly charges the droplets for subsequent ion evaporation.
Ion spray is effective in handling mosi reverse-phase solvents and mobile-phase addimes. and n expands the
upper flow rate (e.g.. 50 .:L/nnni. compared with unassisted nelmii/ation in clcctrosprax
4.2 Applications to Dye Analysis
4.2.1. API-MS of Sulfonated Dyes
Atmospheric pressure ionization (API)-MS techniques of pneumatic assisted electrospra\ (ion spray)
and electrosprav [17. IS. 441 have been demonstrated for the detection of dyes. Electrosprav achieves the
best sensitivity for compounds that are precharged in solution (c g . ionic species or compounds that can be
protonated or deprotonated by the adjustment of pH) |43|. For this reason, the initial work with electrosprav
MS focused on ionic d\cs such as sulfonated azo dves. which have eluted analysis by panicle beam or
thermospray LC/MS. The ion spray or electrosprav of monosullbnatcd azo dyes consists of an |M-Na| anion
with little fragmentation. Monsulfonatcd dyes including Acid Orange 7. Acid Red 337. Acid Red 151. Acid
Red 88. Acid Yellow 151. and Acid Yellow 49 have been analyzed by electrosprav or by ion spray [35. 45.
46],
Disulfonated azo dyes including Acid Red I. Acid Black I. and Acid Blue I 13 could be analyzed by
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API-MS [45-47], The mass spectra of these d\cs consist of |M-2Ka|"- and i Vl-Nn |* ions. Howexcr. Acid
Blue I 13 does not exhibit an |M-Na|- anion ThermnlK assisted clcctrospra\ was demonstrated bv Hemon et
ai. to detect a he.xasullbnaied uyc i Direct Red XOi ol' M \V I 240 US|. The mass spectrum shoncd only
multiply charged ions ranging from |M-6H|" to |M-2H|": 14s |
API-MS techniques could also pro\ tdc structuralh rele\ ant fragir.entation information on the
sulfonated azo dye b\ collision induced decomposition (CID) in the transport region of the interface
Typically this is accomplished by increasing a capillary or orifice potential resulting in the CID of molecular
species generated by elecirospray or ion spray. The fragment ion ustialK results from breakage of the azo
linkage with the charge slaying with the moiety containing SO.. Also. |SO,| ion at m/z. SO is a common CID
fragment for these sulfonated azo dves. Figure in shows the CID spectrum of Acid Orange 10 using
elecirospray transport CID (200V capillary voltage) to obtain structural information for this riisulfonated azo
dye. Similarly. CID infomiation could be obtained by tandem MS on the |M-Na|' anion.
The API technique also proved to be sen. sensitive for the detection of the sulfonated azo dyes in
negative ion detection. Full scan detection w as demonstrated on I -10 ng quantities of these sulfonated azo
dyes.
4.2.2. LC/MSofDyes
The flexibility of API-MS permits the coupling of separation using high How rates or very low flow
rates for online LC/MS. The use of a heated pneumatic nebulizer with corona discharge (atmospheric
pressure chemical ionization - APC1) permitted 1-2 mL/min operation, while electrospray or ion spray
permitted operation at the 1-40 .;L/nnn range. In cither case, chromatographic conditions for the separation
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of the sulfonated azo d\cs must employ \ ointiic buffers to minimize suppression oi"the signal. .Sulfonated
azo dyes were separated on a C,„. column using a gradient of aceioniinlc til water containing U, I VI
ammonium acetate at 2 niL/'min using the APC'I approach |45|
Wliile APCI could tolerate higher le\cl> of buffer, response lor the poK sulfonated a/.o dyes were
usually poorer compared with the monositlfonaied n/,o dyes. This difference m response may be attributed to
the lower volatility of the pol\ sulfonated d\cs. resulting in less material in the gas phase for ionization by
APCI, LC/MS using ion spray successfully detected the pol\.sulfonated a/.o d\cs 1451. To achie\c the best
sensitivity the LC conditions were changed to a I 0 mm i D C v column to reduce the How rate to 41). :L mm
and the ammonium acetate concentration was reduced to u uu I M Higher llow rates or higher lc\cls of
buffer would result in signal suppression
Also, capillary electrophoresis (CE) coupled to eiecirosprav-MS was demonstrated for the separation
and detection of sulfonated a/.o d\es |-U>| CE separations using aceioniinlc ilow rates of 1.8 .;L/min
permitted rapid separation of the sulfonated a/.o dyes. While the high resolution separation capabilities of CE
resulted in high peak concentrations, permitting detection of low pmole quantities of dyes, the low injection
volume of CE (2-30 nL) limited the concentration detection limits Low ppm detection limits were reported
for the sulfonated azo dyes by CE/MS [46|,
Cationic dyes have been analyzed using positive ion detection electrospray-MS with great success
These pre-charged cationic dyes arc well suited for ion evaporation ionization in clcctrospray MS. analogous
to the negative ion formation for the sulfonated a/.o dyes previously discussed For this reason, cationic dyes
such as Basic Yellow 11 (methine class of dyes). Basic Violet i and Basic Green 4 (arylmethane class of
dyes), nnd Baste Violet 10 and Solvent Red 4(J (xnnthcne class of dyes) could be detected at sub-ng quantities
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under full scan conditions using elecirosprav-VIS |4lJ| The mass spectra oi'lhese canonic dves exhibited onlv
[Ml* ions at condil.tons thai do not cause CID (low capillar or urilxe potentials) ;is shown in Figure I Li for
Basic Violet 10 Several ways were evaluated m obtaining siruiuual information on these elves including'
(1) Elecirosprav transport CID.
(2) Triple quadmpole MS/MS.
(3) Ion trap mass spectroniem ilTMSi MS.'VIS
The three CID techniques showed structural information but ihcrc were major (.inferences 111 energies
available for activation. The use of elecirosprav transport CID made use of the voltage at tiie capillary exit
(Anahtica of Brantord elecirosprav source) to generate the CID fragment ions (Figure 12).
At a low capillar, exil \oltage (50-X0V). only molecular ions are delected. At a high capillary exit
voltage (160-240 V) significant fragmentation is observed Of the three techniques, the elecirosprav transport
CID has the capability lo place ihe largest quantities of internal energy iM6 eV) into the ion to generate the
most fragments |50|, This has been demonstrated lor Basic Violet 10 (Figure I lb) .Sensitivity of this
approach was superior due to the minimal ion losses at the various capillary exit voltages (the total ion
current was'nearly constant over the voltage range evaluated in Figure 12) evaluated for CID. However, since
there is no mass selection prior lo C'lD. the signal/noise and specilkily of the approach may be limned in
complex samples or from coekition of components under LC/VIS conditions.
The elecirosprav ion trnp-MS (ITMS) |5 1-5.11 makes use of initial mass selection prior to CID of the
selected ion tn the trap Molecular ions generated by elecirosprav . gated into the trap, are mass selected by a
combination of rfand dc fields. A tickle voltage of I-2V applied to the cndcap for 30 ms increases the
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kinetic energy of the selected ion for (.'ID vvnh helium in the trap (4 \ 10 : lorn. The tickle voltage, tickle
time, and rf trapping well depth tq/) can ne v nncd so ctjnirol the extent of energy transfer to the selected ion
resulting m different amounts of fragmentation Figure 13 shows the optimization of tickle v oltage for the
CID of the f M |' Ion of Basic Yellow I 1 For the canonic eves. tickle v oltages of
2-2.8V were optimal (qz 0.3. tickle time 30 ms. pressure 4 \ 10"' torn lor the generation of structurally
relevant product ions. Higher tickle voltages eiecied the molecular :ons into the trap walls, resulting in loss of
sensitivitv Lower voltages aid not provide sufficient kinetic energy lor CID of the selected molecular 1011
The product ions were ejected from the trap to the multiplier using the mass selection instamhiv mode of
operation
While the ion trap imparts lower energies compared with CID ,n me elcctrosprav transport, significant
structural information couid be obtained for these dves such as Basic Violet 10 f Figure ; Ics. Furthermore,
increasing the q/. value enables more cnergv to he imported to the ions resulting in near equivalent
fragmentation to elcctrosprav- transport CID Also. MS1' capabilities of the ITMS could generate additional
structural information 1531 Sensitivity of the ITMS approach was comparable to elcctrosprav transport CID
by the ITMS approach, offering superior signal/noise and specificiiv due 10 mass selection prior to CID of the
ions.
Triple quadmpole |54. 55] was also evaluated to obtain CID information on a mass selected ion In
this instance, the molecular ion of interest was selected bv quadmpole I. the CID of the ion occurred in
quadnipole 2 (30 eV lab energy with Argon target gas at I niiorn. and the third quadrupole was scanned to
detect the product ions formed. Although the triple quadrupole offered good specificity due to mass selection
prior to CID. the extent of fragmentation was limited due to a maximum of 30 cV collision energy
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available on [lie instrument. Figure i Id displays the triple quaurtipoie (.'I0 mass spectrum of Basic
Violet 10 for comparison 10 the other C1D techniques discussed
Electrospra\ has been also suecesslui mi numerous a/;) d\es liiai are nui iodic sails A/o oves
consisting of both disperse and solvent classes of dyes have been analyzed by eleclrospra> MS |4N. 4s>. 521.
Specifically, disperse dvcs including Blue 7lJ. Yellow 5. Bron/c 2. Orange 13. Orange 3. Red I. Red 13.
Brown I. Solvent Red 3. Solvent Red 23 and 24. Pigment Reel 3. and Methyl Red ha\e been analyzed b\
electrospray or ion spray Optimal sensitiv its u as usually observ ed at low pH conditions te g.. 1 % acetic
acid) that promote the protonation of a basic sue on the dve to form a cation in solution, under positiv e ion
detection. The scnsiti\ itv for negative ion detection thigh pH using ii ;"'l, ammonium hydroxide) did not
compare with positive ion detection, possibh due to the lack of sites lor deprotonauon lo form anions in
solutions. All these d\es exhibited |M—H| ions and fragmentation under CID conditions in the electrospray
transport. Examples of electrosprav MS spectra of two a/o d\cs are presented m Figures 14 and 15. Figure
14 shows the clectrospra> mass spectrum of Solvent Red 24 The fragmentation was generated b\ CID m the
electrospray transport tcapillary voitage of I Ci()V i Figure 15 shows the electrospray transport CID spectrum
of Bronze 2 obtained on a Vcstec electrospray source.
The electrospray MS analysis of the azodye was usually more sensitive compared to panicle beam or
thermospray. However, the response did not compare to the signals generated by electrospray for the cation
or sulfonated dy es.
Several anthraquinonc dyes have been analyzed by electrospray MS 1521 These dyes include
Disperse Blue 1 and 3 Analogous elcclrospray-MS conditions mentioned for the az.o dyes (low pH, positive
ion detection) were optimal for the detection of these nnthraquinone dyes The basic nitrogens on the
anthraquine nng serv e as the site of protonation to generate a cation to obtain optimal electrospray- MS
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sensitivity. The clectrospray .spectra ol' Shc.sc dyes consisted ol' only |M-rH I ion under non-CID conditions.
Structural information could be obtained bv CID in the electrospray transport as demonstrated for Disperse
Blue 1 (Figure K>)
Research in new methodologies such as e!eetrospra\ LC. MS have greatly enhanced tr.e ability io
characterize increasingly complex, polar and nonvolatile d vest tiffs The sensitiv it\ and specificity (In CID in
the electrospray transport or Dy MS/MS) achieved uuh clectrospray LCVMS cnanles ihe monitoring lor trace
levels of dyes in mixtures, a necessity for environmental monitoring or production process control
5. Summary
Table 5 attempts to compare the various LC/MS techniques, according to class of dye io be analy zed.
in terms of sensitn itv and specificity. Techniques like particle hcam-LlV.VJS.uhich is based on gas phase
ionization process, are not suitable for nonvolatile components such as sulfonated a/.o dyes.
Thermospray-LC/MS on a single quadrupole system usually results m singic ion spectra, lacking structural
information for compound confirmauon. Elccirosprav LC/MS probably offers the best combination of
sensitivity and specificity' (CID in electrospray transport rcgioni However, electrospray sensitivity is often
reduced for non-polar dyes that do not have sues of protonation or deprotonation to form cations or anions
for their respective positive or negative ion detection
7?
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Notice
This review lias been funded, in pan. bv ihc U S Environmental Proiection Agencv through us
Office of Research and Development (ORD). and conducted through a collaboration of lite
Environmental Monitoring Systems Laboratory in Las Vegas with the Research Triangle Institute and th
Wcizmann institute of Science
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References
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11. J, J. Monnghan. M Barber. R. S Bordoli. R. D Sedgwick, and A N. Tvlcr, ()r<; \-1oss Snvarom.. 18
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12. R. M. Brown. C S. Creascr. and H .1. Wriglu. ()rx Moss Spectrum . 19 119S4> 311
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25.
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20. R. D. Voyksncr and C A Hnncv. .4w/. r/
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34 J Yinon and J Saar../. f 'hrtunoioi^r . 5Kf> (1 W!) 73
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42 C Pace and M. Rohy. Clinracicri/aiion and AimKsis of liic 3n.sLv\'cutial Fraction From A/.o Dve
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1 52, H.-Y Lin and R, D Vo\ksncr. Anal. ('hem . «>5 l l%W» 45
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2()
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Figure Captions
Figure I. Chemical Strucutre of Acid Orange 6
Figure 2. Mass ehromatograms of i ppm of a commercial red d\e spiked into gasoline The major red
eomponeiil (A) al mi/. 3XI and llie major orange component (B) ai mi/ 249 arc displayed.
Figure 3. Chemical structures of dyes analyzed by TS-LC/MS
Figure 4 Comparison of three mass spectra of Disperse Yellow 5, obtained b\ (a) TS-repellcr-CID. (b)
TS-MS/MS-CID. and (c) EL
Figure 5. TS-LC/MS (with u ire-repeller) mass spectra of Disperse Blue 79 at repel ler 0 and 250 Volts,
respectively.
Figure 6. Reconstructed ion chromatogram (RIC) to TS mass spectrum of Indigo ('Vni Blue 1)
Figure 7, Chemical structures of commercial dyes analyzed b\ TS-. PB-. and ES-LC7MS.
Figure 8 EI- and Cl-PB-LC/MS mass spectra of Solvent Red 1.
Figure 9. RIC chromatogram of Disperse Brown I and the corresponding PB-EI mass spectra of two
resolved components, peaks I and 2.
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Figure 10
Electrosprav mass spectrum of 100 ng/,;L .solution (2-propanoi water. 11) of Acid Orange 10 at
a capillars voltage of-200V
Figure 1 f Positiv e ion mass spectra of Basic Violet 10
a} Eleclrosprav-MS non-CID conditions
b) Electrosprav-MS ('ID conditions (35
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1 Figure 15. Elcctrosprnv mnss spectrum of Bronze 2 at a repeller \oltayc of 40V (V'cstcc electrospray
2 source) lo yenerme structurnIK rele\ant CID ions. Conditions: 20 nu', L solution in 80%
3 ¦ tVleOH. 20% H-0 with |% acetic acid in fused at 5 . L.'min
4
5 Figure 16. Electrospray mass spectrum of Disperse Blue I ni a capillars \oltage ol' 160V to generate
6 structurally rcle\ ant CID frnyment ions Conditions: 50 ng/,il_ solution (75/25 McOH/H:0. 1%
7 acetic acid) infused at 4.',L/min.
-------
Na03S-
n=n—<( : OH
HO
Acid Orange 6
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9:50
r
13:40
t A _
nri*J
20;M
Tlm« (mJn)
27-20
14:10
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-¦ AZO CUSS
OH
l^jA,N=N
NO,
I
CM,
1, Disp«r»a Yellow 5
C.l. 12790
OH
P
2. Oispors« Oranga 13
C.L 26080
= N-O-0"
O •
3. Solvent Red 3
C.l. 12010
o,n^0^"n=n^>"nh'
4, Disperse Orange 3
C.l. 11005
5. Disperse Red 1 3
C.l. 11115
HO
" - ©
6. Solvent Red 23
C.l. 26100
a a
NIC.H
CI
7. Disperse Brown 1
C.l. 1 1152
.OH),
0,'N~"©~N"N'<@M
C.H.OH
8. Disperse Red 1
C.l. 11110
9. Disperse Orange 25
C.I. 11227
.Br
QCH,
¦C2H4OCCH3
OjN-^ V* N = N—# 'V-Nx
'NO, NW0 ^C2H4OCCH3
/ \
H CCH3
10. Disperse 8lue 79
C.l. None
0
ANTHRAQUINONE CLASS
O NHCH,
isy^
o NHCjH.OH
18 Disperse Blue 3
C.l. 61505
COUMARIN CLASS
iS)
19 Fluorescent Brighionar 236
C.l. None
XANTHENE CLASS
NtC,H,),
20 Solvent Rsd 49
C.l. 45170.1
SULFONATED AZO CLASS
Q-...-Q-C
M
HO H,C CH, HO
11. Acid Oranga 6 m.c-(5)-S0,-o-^^n = = ""0
cx 14270 «•<>.*-o
SO,Mm
Q MH.
or to
IS. Acid Had 114
C.l. 23836
METHINE CLASS
12. Acid Blua 40
M,C r-id
C.l. 62126 *
I 9 OCH,
OH CH,
1 6. Ba»ic Yellow 11
C.l. 48055
13. Oiract Red 81
C.l. 28160 ARYLMETHANE CLASS
(O) (0>
o.
^ ki/
14. Acid Blua 113 1 7,'Ba.ic Graan 4
C»l.,"26360 C.l. 42000
-------
180,8
EE.5
»/i
SPEC:
180.8
56.
i/l
SPEC:
Repelter at 0 V
486 5jq
S13 . 451
Repeiier at 250 V
2892
28000
T ' I ' I ' I
550 - 611
-------
-------
100.S1
S31
:53
841.
262,079
i 0.500
594
533 . . 593 S40 670 7p 7gs 603 S33 ,
A I'i ... A t >1 -Aii i Iil< 1 I li,U 1-- . L J -, lli J-AV-.yWftill
r'lUUw/.k
390 4?2, 4?4
1332S.
sao
2:«?
-00
11; 40
30Q
13:26
200
15:66
'."CAN
tine
130.0-
INDIGO (VAT BLUE 1)
0 H
H o'
7712
5-3.9-
-------
1. SoK^n V»Morw Z
c.i tio?a
2. 5eh*m Y#Ho* 3
CI ntso
,CHj
•NH,
CH, /CHJ *0^
CH, HO^
CH,—y—MsN (( \
3. Sokwf* Yiikm U
C.L \2GS5
4, Sefc»nt Onuigp 7
C.L 12140
fw/1
0,N
5. OspM^fUdt
C.t 11110
CH.CH.
CH,C KjOM
rs-^Q^-r\
O
6. 0iao«a« Qranq« 13
C.L 2&oaa
ZH^ CM,
CHjCHjCN
7. 0«p«nMi Ofang« 25
C.L H227
CI
„-( ^ H=H
I. DeScnmui Or«flg«
C.I ?ern
\ /
\
CMjCHjCN
CH,CH-CN
OjN'
V-N=H-/
CI
CH^CMjOCCK,
o
9. CXse«m» OrmnQt 30
C.L 11114
J /CHiCH?0H
VCH,CH,OH
19. 0isp«r»i Slack 9 {pr»ar»|
C.L mrw
OOij
OjH
CHjCHjOCCH,
/ h=m—/ \ f/
M H
NO, NHCOCH, O
11, Doom** 8fc« 79
C.I nws
O-jH-
\=/ CHjCHjO*
12, Oopm-m CSriflg* 3?
C.L MOna
O,N
13. QapamaBrawnl
C.L 11I5Z
\ H=M—^ \ H
CKjCHjOM
\=/ ^CHjCHjOH
HO,
N»0yS
14, Aod Ontng* 10
C.L 1S230
SO
-------
-------
150000
2 100000
>
J2
tr 50000
(/)
c
o
CD
>
CD
cr
n:
aN
If)
c
CD
IS
>
Jo
0
cc
Peak 1
B IOO-i 29.1 min
t 1 r
4 8 12 16 20 24 28 32 36 40 44
min
401v
\
^ ^r>vs,loH
y-fH - N--f V-M
\ J \
CH; SMjOH
50-
357
I
313
242 285 \ 328
M+-
432
\
80 120 160 200 240 280 320 360 400
_ , „ m/z
Peak2 139
100-1 30.3 min
CjHjOH
CjHsOH
80 120- 160" "200 - 240 280 320 360 400
— . m/z
-------
100 -1
&
>
jo
©
CO.
[SO3I-
80
113
145
141
118
0 - 1 I > ! | 1 ! I | r I I | I ! I I I
03 Na
[M-2Na-S03-H]"
325
[OHC10H4(SO3)2]-
301
221
[M-Na]*
429
349
401
t j 1 \ f \ r 1 1 ¦[ 1 j f
1 1 n 1 11 11
50 100 150 200 250 300 350
m/z
r
400 450 500
-------
CD
>
Relative Intensity (%)
Relative Intensity (%)
u
CO
CD
Ul
u
u
Ul
CTJ
-------
D
£ 50
-------
100
0"
>»
%
jj 50
0)
>
• HI
IS
0)
DC
© [M+H]+
Sum of Product Ions
d Total Ion Current
50 100 150
Capillary Exit Voltage
200
250
-------
100
80
U)
I 60
CD
I 40
d)
cr
20
0
M+H +
E Product Ions
I i I I I II I f I
0.6 0.9 1 1.21.4 1.6 1.8 2.0 2.2 2.4 2.6
Tickle Voltage (V)
-------
OH
CH
CH
MW 380
158
276
381
100
'35
c
c
50
149
224
109
JS
0>
XL
276
158
209!
lit—
400
100
500
200
300
-------
w
c
03
CD
•>
J2
03
oc
209 (-C2H5)
N = N
100 200 300 400 500 600
m/z
-------
Relative Intensity
-------
Table 1. Investigated Samples
C.I. = Color Index.
Commercial
Name of Dye
C.I. .
Name
C.I.
Numbe
r
M.W.
Type of
Fiber
Manufacturer
1.5% Serisol Fast Yellow GD
Disperse Yellow 3
r,M
11855
269
Diacetate
Yorkshire
2.0% Serisol Fast Yellow PL 150
Disperse Yellow 9
10375
274
Diacetate
Yorkshire
1.2% Resolin Yellow 5GS
Disperse Yellow 5
12790
324
Polyester
Bayer
0.72% Dlspersol Orange B-A Grains
Disperse Orange
1
11080
318
Polyester
IC)
0.6% Serilene Orange 5R300
Disperse Orange
1
11080
318
Polyester
Yorkshire
0.6% Serilene Orange 2RL200
Disperse Orange
25
11227
323
Polyester
Yorkshire
0.72% Dispersol Orange B-2R 200
Grains
Disperse Orange
25
11227
323
Polyester
ICI
1% Resolin Orange F3R 200%
Disperse Orange
25
11227
323
Polyester
Bayer
2.2% Resolin Orange RL
Disperse Orange
13
26080
352
Polyester
Bayer
0.6% Serilene Yellow Brown 2RL 150
Disperse Orange
37
-
391
Polyester
Yorkshire
1.5% Serisol Brilliant Red X3B 200
Disperse Red 11
62015
268
Diacetate
Yorkshire
0.6% Serisol Fast Scarlet BD 200
Disperse Red 1
11110
314
Diacetate
Yorkshire
0.6% Serisol Fast Crimson BD 150
Disperse Red 13
11115
348
Diacetate
Yorkhire
0.6% Serilene Red Brown R-FS 150
Disperse Brown 1
11152
432
Diacetate
Yorkshire
1.5% Serisol Brilliant Blue BGN 300
Disperse Blue 3
61505
296
Diacetate
Yorkshire
1.0% Resolin Blue BBLS
Disperse Blue 165
-
405
Polyester
Bayer
1.0% Yoraeryl Yellow RL
Basic Yellow 28
-
309
Orion
Yorkshire
Indigo
Vat Blue 1
73000
262
Denim
Levi Strauss
-------
Table 2. Particle Beam El Mass Spectra of Dyes
Dye
Mol.wt.
m/z of ions observed (% relative abundance)
Disperse Yellow 5
324
324(1); 295(1.5); 202(3); 174(7); 138(9); 108(100); 92(17)
Disperse Orange 13
352
352(2); 247(10); 142(26); 115(22); 109(22); 93(100)
Solvent Red 3
292
292(17): 263(3): 235(4); 171(6): 149(9); 143(100); 121(48); 115(36);
108(18)
Disperse Orange 3
242
242(3); 213(4); 212(10); 120(55); 92(100)
Disperse Red 13
348
317(22); 287(20); 154(17); 144(25); 142(28); 134(25); 133(100);
126(40): 120(30): 105(50); 104(50); 99(20); 92(32); 90(40)
Solvent Red 23
352
352(4); 267(1.5); 197(20); 143(30); 120(46); 115(32): 108(11);
93(40); 92(100)
Disperse Brown 1
432
432(1.5): 403(15); 402(5); 401(17); 359(5.5); 357(7); 313(5); 214(15);
208(17); 206(36); 185(40); 183(78); 176(39); 167(32); 149(77);
139(100); 124(49); 104(82). 90(48)
Disperse Red 1
314
314(4); 297(2); 283(34); 267(1 1); 253(19); 237(8); 207(9); 180(15);
168(18); 149(15); 147(18); 133(100); 120(49); 108(63); 105(55);
103(47)
Disperse Orange 25
323
323(1); 293(12), 283(7); 253(26); 240(9); 224(3); 189(3); 173(10);
149(20); 133(35); 120(62); 108(31); 105(19); 104(20); 93(18);
92(100)
Disperse Blue 79
624
87(100)
Basic Green 4
329
330(38); 329(13); 287(8); 255(10); 254(21); 253(100); 237(13),
223(12); 210(35); 209(20): 208(32); 194(29); 181(15): 165(82),
135(22): 126(78); 120(32); 118(37); 103(45); 95(22)
Disperse Blue 3
296
267(12); 266(100); 249(49); 234(28); 220(22); 204(11); 194(10);
181(8); 180(9); 165(17), 164(13); 152(22); 139(12); 124(15);
110(13); 104(19)
Fluorescent
Brightener 236
389
390(29); 389(100); 361(19); 333(11); 304(19); 207(75); 206(28);
195(38); 181(26); 180(18); 179(43); 178(82); 165(18); 152(78);
151(47); 139(35); 127(35); 114(21): 105(30); 102(57)
Solvent Red 49
442
399(18); 398(34); 397(26); 327(18); 326(100); 282(18); 199(20);
184(40); 177(23); 170(23); 163(20); 162(18); 156(32); 149(48);
142(20); 105(16); 91(19)
162-tbl.2
-------
Table 3. identification of Chemical Reduction Products of Colorants 1-16 by HPLC/MS3
No.
Dye
Identified Reduction Products
Mol
wl.
t.
Particle Beam (mfr, relative intensity)
uv
(%)
• 1
Solvent Yellow 2
aniline"
93
12.9
93(100); 66(39)
>1
N,/V-dimethyl-1,4-diaminobenzene
136
28.4
136(100); 120(85); 93(37); 81(41)
88
2-aminotoluene 5
107
18.2
107(74); 106(100); 77(26); 51(15)
90
2
Sotvenl Yellow 3
2-m#thyt-1,4-diaminoben2«ne"
122
30.0
122(100); 94(33); 78(26); 58(19)
I 3
unchanged dye *
225
25.8
225(58); 134(17); 106(100); 91(28); 77(23); 75(13); 51(4)
3
aniline"
93
13.1
93(100); 66(37)
7
3
Solvent Yellow 14
1-amino-2-naphthole
159
19.3
159 (100); 130(89); 103(26); 77(22); 51(15)
49
unchanged dye'
248
283
248(90); 219(7); 171(15); 143(100); 115(97); 89(10); 77(41); 51(10)
20
Solvent Orange 7
2,4-dimethytaniline "
151
22.1
151(11); 121(100); 106(78); 77(15)
I
30 |
4
1-amino-2-naphthol5
159
20.2
159(100); 130(63); 103(13); 77(17); 51(11)
31
i
2-aminotoluene6
107
22.4
107(100); 91(55); 77(49); 51(25)
18
5
Solvent Red 24
2-methyl-1,4-diaminobenzene"
122
40.4
122(33); 104(46); 71(41); 55(100)
16
1-amino-2-naphthol"
159
19.1
159(100); 130(70); 103(20); 77(20)
42
6
Pigment Red 3
1 -amino-2-naphtbol1'
159
19.5
159(100); 130(62); 103(15); 77(19)
70 i
7
Disperse Red 1
4-nitroaniline!
138
13.0
138(100); 108(83); 92(50); 65(75)
70
anilines
93
13.1
93(100); 66(40)
>1
8
Disperse Orange 13
4-aminophenol "
109
6.2
109(10); 108(100); 80(48); 64(7)
10
1,4-diamino-naphthalene
158
10.7
158(100); 109(50); 80(37)
78
I
Disperse Orange 25
4-nttroaniline11
138
12.3
138(100); 108(83); 92(50); 65(75)
18
9
N-(2cyanoethy1)-W-(e1hy1)-1,4-
diaminobenzene
189
14.8
189(25); 149(100); 120(34); 92(4)
75
10
Disperse Orange 44
/V,/V-bis(2-cyanoelhy<)-1,4-
diaminobenzene °
214
8.3
214(40); 174(100); 120(37); 106©
70
1,4-diamino-2 ,6-dichlorobenzene
176
13.8
176(27); 149(73); 124(40); 98(100); 81(56); 78(63)
35 i
W-(2-cyanoethyt)-W-(2-
[iydroxyethyf)-1,4-diaminobenzene
205
6.1
205(45); 174(85); 165(80); 120(100); 92(30); 65(20)
35 |
11
Disperse Orange 30
2,6-dichloro-4-nitroamline
206
25.5
208(40); 206(60); 178(50); 176(84); 162(20); 160(30); 135(15);
133(22); 126(30); 124(100); 92(28); 90(31)
20 .
«-(2-cyanoethyl)-AH2-
acetoxyethy!)-1,4-diaminobenzene
247
10.0
247(1); 205(32); 174(91); 165(54); 120(100); 92(20); 65(20)
45 ;
1,4-diaminobenzene •
108
8.3
108(100); 92(44); 80(66); 67(25); 52(64)
30 ;
12
Disperse Black 9
/V,/V-bis(2-hydroxyethyt)-1,4-
diaminobenzene
196
21.7
196(25); 165(100); 120(20); 93(14)
60 •
2-bromo-1,4,6-triaminobenzene"
202
10.4
203(15); 202(5); 201(10); 88(23); 70(83); 61(100)
25 :
13
Disperse Blue 79 j
3-acetamido-4-{W,W-bis(2-
acetaxyethyf)-amino]-1 -amino5-
methoxybenzene
367
15.1
367(10); 294(15); 208(9); 87(100)
60 ;
j
-------
No
Dye
Identified Reduction Products
Mol
wt.
t.
Particle Beam (nVt, relative intensity)
UV
(%)
1.4-diamino-2,6-diChlofobenzene
176
23.0
176(27); 149(73); 124(40); 90(100); 64(S6); 74(63)
40
14
Disperse Orange 37
/W-cyanoethyt-M-ethyM ,4-
diaminobenzene
189
28.1
189(19); 149(100); 120(73); 106(8); 92(21)
40
4-amino-2,6-dichloro-4'[[M-(2-
cyanoethyt)-amino|azobnn2ene
333
334(15); 333(27); 293(100); 265(21); 229(7); 201(33); 149(49);
120(41),100(15); 92(9)
10
1,4-diamino-2,6-diehlOfc benzene
176
22.3
176(56); 149(28); 134(59); SB(100), 84(53)
40
15
Disperse Brown 1
3-chloro-A/l/V-bi5(2-hydro*yelhyl)-
1,4-diaminobenzone
230
17.4
230(21); 199(100); 155(37); 127(13)
40
aniline"
93
13.5
93(100); 66(40)
40
16
Acid Orange 10
8-amino-7-hydro*ynaphthaten®-
1,3-disul?onic acid, disodium salt"
363
16,6
Not detected1
48
* t „ = retention time in TiC chromstogram (min); rryir {relative intensity) reports the major peaks (>5%) of each product down to rrVx SO.
A maximum of 18 ions are reported in descending nVz ; UV (%) = [peak area of identified reduction product |/V [of the peak areas in the ehromatogram of the
reduced sample at a wavelength of 254 nm] The identified reduction products were «0.5% ol the total peak area in the unreduced HPLCAJV analysis of the
parent dye.
5 Identify confirmed with standard.
1 Observed only after NajSjO, reduction.
3 Observed only after SnCI, reduction.
' Only identified by HPLC/PB-MS in sample reduced with SnCI,.
' Identity confirmed by thefmospray-MS; ions detected include 371(100), 364(5), 319(4), and 274(9).
Reprinted with permission from R.O. Voytaner el a! . Environ. Sci. Technol., 27 (1993) 1665. Copyright 1993 by the American Chemical Society.
-------
Table 4. Comparison Of Particle Beam LC/MS an a GC/MS In Tentatively Identifying Compounds In An Azo
Dye Sample
PB LC/MS
GC/MS
MW
El
NCI
El
NCI
1.
Aniline
S3
X
2.
N-phenylformarnide
121
X
3.
4-nitroanilme
138
X
4.
N-cyanoethylaniline
146
X
5,
4-phenylphenol
170
X
X
X
6.
2,4,6-trimethoxy-l ,3,5-triazine
171
X
7.
2-chloro-4-nitroaniline
172
X
X
X
8,
2,6-dimethoxy-4-(N.N-dimethyl amino)-1 3.5-
triazine
184
X
9.
N.N-bis(cyanoethyl)anilme
199
X
X
X
10.
2-bromo-4-nitroaniline
216
X
X
11,
Sulfur (S8)
256
X
X
X
12.
2-bromo-4.6-dinitroaniline
261 !
1
X
X
X
13.
4-bromo-N.N-bis(cyanoethyl)-aniline
277
X
X
X
14.
Hexachlorobenzene
282
X
X
15.
Tribromoanisole
342
X
X
X
16.
4-(2'-chloro-4'-nitrophenylazo)-N.N-
bis(cyanoethyl)aniline
382
X
X
17.
4-(2'-bromo-4',6'-dinitropheny! azo)-3-acetamido-
N.N-diethyl aniline
478
X
X
18.
4-(2'-chloro-4'-nitrophenylazo)-3-acetamido-N.N-
bis(ethyl ethonate)aniline
505
X
19.
4-(2'-bromo-4',6'-dinitrophenyl azc)-5-acetam:do-
2-methoxy-N.N- diethylaniline
508
X
X
20.
Disperse blue 79
624
X
X
MW = molecular weight
PB = particle beam
El = electron ionization
NCI = negative chemical ionization (carbon dioxide)
-------
Table 5. Predicted Success Of Varous LC/MS Techniques for The Characterization Of Selected
Dye Classes
MS Techniques
Dye Classes
Particle Beam-LC/MS
(EI/CI)
Thermospray
LC/MS
Electrospray LC/MS
Sulfonated Azo
O
H
•
Cationic
o
•
Azo (disperse)
9
•
Azo (solvent)
H
9
•
Anthraquinone
H
• R
O H 9
> >
Increasing success for MS analysis in terms of sensitivity and specificity.
-------
EMSL-LV 95-090 TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing;
-
1. REPORT NO. 2.
EPA 600/A-95/07?
3. RECt
4, TITLE AND SUBTITLE
LC/MS Techniques for the Analysis of Azo Dyes
S. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7 authorisi Yinon, J. (1); Betowski. L.D (2);
Vcyksner, R.D. (3)
a, PERFORMING organization REPORT NO,
9. PERFORMING ORGANIZATION NAME ANO AODHESS
(1) Weizmann Institute of Sci., Dpt of Env. Sci,
Rehovot, 76100 ISREAL; (2) U.S. EPA; (3)
Research Triangle Institute, RTP, NC
10. PROGRAM ELEMENT NO.
11, CONTRACT/GRANT NO.
CR 819555
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. EPA/ORD/NERL
13, TYPE OF REPORT AND PERIOD COVERED
Book Chapter
Characterization Research Division
POB 93478, Las Vegas, NV 89193
14 SPONSORING AGENCY CODE
EPA 600/07
15. SUPPLEMENTARY NOTES
Yinon, J Betowski, L.D.; Voyksner,,R.D. J LC/MS Techniques, for the Analysis of Azo.Dyes, " In Applications..of,LC/MS.in..Environmental.
Chemistry; D. Barcelo,- ed. Elsevier (publisher) igW..'-*"- y'f.' '¦/" —
16. ABSTRACT
^••Dyestuffe are of major environmental interest because of their widespread use as colorants for e.g., textiles,
paper, leather, and foodstuffs. Synthetic intermediates, by-products and degradation products could be
potential health hazards because of their toxicity or carcinogenicity ¦- The dyes do not belong to one group of
chemical compounds. The analysis of such a large variety of compounds poses difficulties because of
differences in solubility, volatility, ionization efficiency, etc. Furthermore,, some of the manufacturing precursors
to dyes are carried over to and are not removed from the final dye product. The result is a complex mixture
characterized not only by the dye itself, but also by several other compounds./ Most dyes, including sulfonated
azo dyes'are nonvolatile or thermally unstable, and therefore, are not amenable to GC'or gas phase ionization .
processes. Thus, GC/MS techniques cannot be used. However, the combination of 1C with MS enables the
separation of nonvolatile, thermally unstable, and polar dyes for introduction into the MS for identification. As a
result of interfacing LC with MS, three major types of interfaces and LC/MS techniques have been developed:
(1) Thermospray, (2) Particle Beam, and (3) Ion Spray and Electrospray. This chapter describes the application
of-these LC/MS techniques in the analysis of dyes.- - ...
17. KEY WORDS AND DOCUMENT ANALYSIS
». DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Croup
Analytical Methods, Dye Analysis,
Sulfonated Dyes, Thermospray
18, DISTRIBUTION STATEMENT
Release to the Public
19. SECURITY C
21. NO. OR5P(*a§8S
atc/
<3 a
20. SECURITY CllMSlSSSffWKfc/
22. PRICE
EPA Form 2220-1 («•». 4-77) previous edition is obsolete
------- |