Application Note 360

Rapid Determination of Azo Dyes in

Textiles Using ASE and HPLC with MS
and UV Detection
Introduction Equipment
Azo dyes are used widely in the manufacture ASE 200 System
of various consumer goods such as leather, textiles, Glass fiber filters (P/N 047017)
plastics, paper, hair care products, and cosmetics. UltiMate® 3000 HPLC system consisting of:
On September 11, 2003, the European Union enacted
SRD 3600 Solvent Rack
European Parliament Directive 2006/61/EC, prohibiting
the manufacture and sale of consumer goods containing LPG 3600 Pump
certain aromatic amines originating from specific azo WPS-3000T Autosampler
dyes.1 The dyes named in this directive can, under certain TCC-3000 Column Compartment
conditions, be reduced to aromatic amines. Twenty two UVD340U Detector
aromatic amines are currently classified as carcinogenic or MSQ™ Plus Mass Spectrometer
potentially carcinogenic to humans and are prohibited by AXP-MS Pump
the directive.
Chromeleon® 6.80 Chromatography Workstation
Current sample preparation and analysis methods for
these compounds are time consuming and labor intensive.
Using accelerated solvent extraction (ASE®) with Reagents and Standards
UltiMate 3000 HPLC/MS/UV analysis has proven to be a Acetonitrile, HPLC grade (Fisher Scientific)
more advantageous alternative compared to other products Methanol, HPLC grade (Fisher Scientific)
currently available. Ammonium acetate, powder (Fisher Scientific)
ASE is a product for extracting organic compounds Acetic acid, glacial (Fisher Scientific)
from solid or semisolid samples using conventional Azo Dyes Mix-6 (Ehrenstorfer Laboratories part
solvents at elevated temperatures and pressures. This 18000376)
application note describes methods for extraction of
Individual azo dye standards (Sigma Aldrich)
textile samples using ASE, along with the analytical
methods for determination of 22 aromatic amines, some
of which are specified in the Parliament Directive.
ASE Conditions Sample Prep
Preheat: 20 min (purge closed) Soak 1 g of material in 3 mL citrate buffer
(pH 6.0) and 3 mL sodium dithionite (200 mg/mL) for
Pressure: 1500 psi
2 min. Transfer the mixture and solution to an ASE 11 mL
Static Time: 5 min
cell containing a glass fiber filter.
Temperature: 100 °C
Cycles: 2 Extraction
Purge Time: 100 s Place the extraction cells into the ASE 200 carousel,
Flush Volume: 30% and extract using the ASE conditions listed above. Record
Solvent: 90% MeOH with 10% 1 M KOH total volume of the extract, and inject 10 µL onto the
Extraction Time UltiMate 3000 HPLC/MS/UV system.
per Sample: 40 min
LC Conditions
Solvent Amount Used
per Sample: 17 mL Time Flow 30 mM Acetonitrile Methanol Curve
Rate Ammonium
Analytical Conditions (µL/min) Acetate, pH 4

Column: Acclaim® 120 C18 3 µm 120 Å 0.00 250 91 5 4

2.1 x 150 mm 25.00 250 69 27 4 5

Eluent: H2O/5%, CH3CN/4%, CH3OH 40.00 250 52 40 8 5

H2O/40%, CH3CN/45% CH3OH 45.00 250 15 40 45 5

48.00 250 15 40 45 5
Column Temperature: 30 °C
48.10 250 91 5 4 5
Flow Rate: 250 μL/min
Injection Volume: 10 μL 30 mM Ammonium Acetate, pH 4.0
UV: 240 nm, 278 nm Weigh 2.31g NH4OAc (FW 77.0825 g/mol). Add 1000
Ionization Mode: APCI, positive polarity mL H2O. Adjust pH to 4.0 with acetic acid.
Nitrogen: 3 bar
Corona Current: 10 μA
Source Temperature: 400 °C
Cone Voltage: Varies by analyte (See Table 1.)
SIM Mode: m/z by analyte (See Table 1.)
Dwell Time: 0.3 s
Span: 0.3 m/z

2 Rapid Determination of Azo Dyes in Textiles Using Dionex ASE and UltiMate 3000 HPLC Systems
Mass Spectrometer Conditions
Table 1. Optimized Detection Conditions of Azo Dye
Individual standards for the azo dye compounds were Standards for Mass Spectrometric Analysis
obtained from Sigma Aldrich. An AXP-MS auxilliary Compound Name Molecular SIM Cone
pump was used to infuse these samples for optimization Weight m/z Voltage (V)
in the mass spectrometer at a flow rate of 100 μL/ 4-Aminoazobenzene 197.09 198 70
min. During the infusions several scan funcions were 2,4-Diaminotoluene 122.08 123 55
performed to determine the optimum ionization technique,
4-Aminobiphenyl 169.09 170 30
cone voltage, and and mass-to-charge ratio (m/z) for each
4,4’-Oxydianiline 200.09 201 60
analyte. These tuned values for cone voltage and m/z were
2-Anisidine 123.07 124 80
used to define the SIM scans used in the method (Table 1).
4,4’-Methylenebis-(2-chloroaniline) 266.04 267 50
The preferred mode of ionization for all analytes
was Atmospheric Pressure Chemical Ionizaion (APCI); 4,4’-Benzidine 184.10 185 50
positive mode. APCI is a gas-phase technique typically o-Toluidine 107.07 108 40
used to analyze small molecules. APCI is a robust 4-Chloro-2-methylaniline 141.03 142 60
technique which is not affected by minor changes in 2-Methoxy-5-methylaniline 137.08 138 55
buffer strength. In positive ionization mode, protonated Bis-4-aminophenylmethane 198.27 199 50
analyte ions are formed by gas-phase ion molecule 4-Chloroaniline 127.02 128 70
reactions, resulting in the formation of [M+H]+ ions. o-Dianisidine 244.12 245 55
In some cases the SIM scans were staggered across o-Tolidine 212.13 213 55
the run time during analysis; focusing on a two minute
2-Aminonaphthalene 143.07 144 60
region before and after the analyte eluted. Sequencing
2,6-Dimethylaniline 121.09 122 55
the scan functions in this fashion minimized the total
2,4-Dimethylaniline 121.09 122 50
analytical cycle time and allowed the mass spectrometer
4,4’-Methylenebis-(2-methylaniline) 226.04 227 55
to collect more data for the peaks of interest, while
excluding data collection where no signal was present. 3,3’-Dichlorobenzidine 252.02 253 55
2-Methyl-5-nitroaniline 152.06 153 50
4-Methoxy-1,3-phenylenediamine 138.17 139 55
4,4’-Thiodianiline 216.30 217 50

Application Note 360 3

 Column: Acclaim 120 C18 3 µm, Flow Rate: 250 µL/min Column: Acclaim 120 C18 3 µm, Flow Rate: 250 µL/min
120 Å (2.1 x 150 mm) Inj. Volume: 10 µL 120 Å (2.1 x 150 mm) Inj. Volume: 10 µL
Eluent: H2O/5% CH3CN/4% CH3OH Detection: UV, 278 nm Eluent: H2O/5% CH3CN/4% MeOH Detection: UV: 240 nm, 278 nm
H2O/40% CH3CN/45% CH3OH MSQ, APCI positive ionization H2O/40% CH3CN/45% MeOH MSQ, APCI positive ionization
Temperature: 30 °C Temperature: 30 °C
Peaks: Peaks:
1. 2,4-Diaminotoluene 12. o-Dianisidine 1. 2,4-Diaminotoluene 15. 4,4’-Methylenebis-(2-methylaniline)
2. 4,4’-Oxydianiline 13. o-Tolidine 2. 4,4’-Oxydianiline 16. 2-Aminonapthaline
3. 2-Anisidine 14. 2-Methyl-5-nitroaniline 3. 2-Anisidine 17. 4,4’-Thiodianiline
4. 4,4’-Benzidine 15. 4,4’-Methylenebis-(2-methylaniline) 4. 4,4’-Benzidine 18. 4-Chloro-2-methylaniline
5. 4-Methoxy-1,3-Phenylenediamine 16. 2-Aminonapthaline 5. 4-Methoxy-1,3-Phenylenediamine 19. 4-Aminoazobenzene
6. o-Toluidine 17. 4,4’-Thiodianiline 6. o-Toluidine 20. 3,3’-Dichlorobenzidine
7. Bis-4-aminophenylmethane 18. 4-Chloro-2-methylaniline 7. Bis-4-aminophenylmethane 21. 4-Aminobiphenyl
8. 2,4-Dimethylaniline 19. 4-Aminoazobenzene 8. 2,4-Dimethylaniline 22. 4,4’-Methylenebis-(2-chloroaniline)
9. 2-Methoxy-5-methylaniline 20. 3,3’-Dichlorobenzidine 9. 2-Methoxy-5-methylaniline
10. 4-Cholroaniline 21. 4-Aminobiphenyl 13
10. 4-Cholroaniline
11. 2,6-Dimethylaniline 22. 4,4’-Methylenebis-(2-chloroaniline) 17
11. 2,6-Dimethylaniline
12. o-Dianisidine 18
4 5 2 20
12 13 13. o-Tolidine 15
4 7 14
19 20 14. 2-Methyl-5-nitroaniline 21
3 16

9 19
1 10 22
17 6 8 11
12

14 16 21
3
1 2 56 7
9
10 15 22
8 18
11 12 13
4
19 20
0 10 20 30 40 45
Minutes 25331
3 17
16
21
Figure 1. UV separation of azo dye standard mixture detected at 6 11 14
1 2 5 7 8 9 10 15 18 22
278 nm.
25330

Results and Discussion

Figure 2. Comparison of azo dye standards detected by UV and
A tertiary gradient was used because acetonitrile was mass spectrometric detection. The UV trace appears at the bottom
found to improve the resolution of the peaks eluting at and is expanded to show the detection of all compounds using
SIM (selected ion monitoring) channels in the mass spectrometer.
the beginning of the analysis, while methanol improved
separation of the peaks at the end of the run. Ammonium
acetate was used to maintain a pH of 4, ensuring analytes
remained in the the acid form; this resulted in improved
detection by the mass spectrometer.
Using the photodiode array detector, two
wavelengths, 240 and 278nm, demonstrated optimum
response for azo dye detection. Figures 1 and 2 show the
detection and separation of the 22 aromatic amines, each
at a concentration of 5 mg/L.

4 Rapid Determination of Azo Dyes in Textiles Using Dionex ASE and UltiMate 3000 HPLC Systems
 Figures 3 and 4 show LC/MS analysis of two
Column: Acclaim 120 C18 3 µm, Flow Rate: 250 µL/min
different leather samples extracted using ASE and 120 Å (2.1 x 150 mm) Inj. Volume: 10 µL
Eluent: H2O/5% CH3CN/4% CH3OH Detection: UV, 278 nm
detected by UV/MS. As demostrated in these figures, H2O/40% CH3CN/45% CH3OH
complete resolution between peaks using UV detection Temperature: 30 °C Peaks: 1. 2-anisidine
2. o-toluidine
is difficult in complex matrices. The specificity provided 18
by mass spectrometry is critical for detection and
positive identification of low level concentrations of azo
mAU
compounds in these solutions

Conclusions
ASE combined with the UltiMate 3000 HPLC system 0
4,500
and MSQ Plus/UV detectors create a powerful tool for 2

extraction and analysis of azo dyes. The ASE system is

ideally suited for the extraction of the complex matrices Counts 1
analyzed here, providing significant savings in time and
solvent use compared to other techniques.
The complexity of the sample extracts makes 0
0.2 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25 27.5 30 32.5 35 37.5 40 42.5 45
identification and quantitation of analytes difficult using Minutes 25329
UV detection alone. Coupling the HPLC system to the
Figure 3. The top trace shows UV separation of an unknown red
MSQ Plus detector provides improved specificity and leather extract sample. Poor resolution between the peaks makes
selectivity for identification of compounds prohibited by identification of azo dyes difficult. The bottom trace shows the
the European Parliament Directive. advantage offered by mass spectrometry; clear, positive identifica-
tion of 2-anisidine and o-toluidine.

Column: Acclaim 120 C18 3 µm, Flow Rate: 250 µL/min

120 Å (2.1 x 150 mm) Inj. Volume: 10 µL
Eluent: H2O/5% CH3CN/4% MeOH Detection: UV: 240 nm, 278 nm
H2O/40% CH3CN/45% MeOH
Temperature: 30 °C Peaks: 1. o-toluidine
2. 4-aminobiphenyl
18

mAU

0
2,000
1
2

Counts

400
0.2 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25 27.5 30 32.5 35 37.5 40 42.5 45
Minutes 25328

Figure 4. Using a mass spectrometer provides positive identifica-

tion of o-toluidine and 4-aminobiphenyl extracted from a brown
leather sample.

Application Note 360 5

 List of Manufacturers References
Ehrenstorfer EQ Laboratories 1. Puntener, A., Page, C. European Ban on Certain
530 Means Street Suite 120, Atlanta, GA 30318 Azo Dyes. TFL Leather and Technology. 2004. 2:1–5
Tel: (404) 586-6828; www.ehrenstorfer.com

Thermo Scientific Corp., a division of

ThermoFischer Scientific, Inc.
81 Wyman St., Waltham, MA, 02545,
Tel: (781) 622-1000; www.thermo.com

Sigma-Aldrich
3050 Spruce St., St. Louis, MO 63178;
Tel: (800) 325-3010; www.sigmaaldrich.com

Chromeleon, ASE, Ultimate, and Acclaim are registered trademarks of Dionex Corporation.
MSQ Plus is a trademark of Thermo Fischer Scientific.

Passion. Power. Productivity.

Dionex Corporation North America Europe Asia Pacific
1228 Titan Way U.S. (847) 295-7500 Austria (43) 1 616 51 25 Benelux (31) 20 683 9768 (32) 3 353 4294 Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 2764 2735
P.O. Box 3603 Canada (905) 844-9650 Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0 Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190
Sunnyvale, CA Ireland (353) 1 644 0064 Italy (39) 02 51 62 1267 Switzerland (41) 62 205 9966 Taiwan (886) 2 8751 6655
South America LPN 2007-01 PDF 06/08
94088-3603 United Kingdom (44) 1276 691722

(408) 737-0700
Brazil (55) 11 3731 5140 www.dionex.com ©2008 Dionex Corporation