AN 360: Rapid Determination of Azo Dyes in Textiles Using ... - Dionex

Rapid Determination of Azo Dyes in 

Textiles Using ASE and HPLC with MS 

and UV Detection 

IntroductIon 

Azo dyes are used widely in the manufacture 

of various consumer goods such as leather, textiles, 

plastics, paper, hair care products, and cosmetics. 

On September 11, 2003, the European Union enacted 

European Parliament Directive 2006/61/EC, prohibiting 

the manufacture and sale of consumer goods containing 

certain aromatic amines originating from specific azo 

dyes. 1 The dyes named in this directive can, under certain 

conditions, be reduced to aromatic amines. Twenty two 

aromatic amines are currently classified as carcinogenic or 

potentially carcinogenic to humans and are prohibited by 

the directive. 

Current sample preparation and analysis methods for 

these compounds are time consuming and labor intensive. 

Using accelerated solvent extraction (ASE ® ) with 

UltiMate 3000 HPLC/MS/UV analysis has proven to be a 

more advantageous alternative compared to other products 

currently available. 

ASE is a product for extracting organic compounds 

from solid or semisolid samples using conventional 

solvents at elevated temperatures and pressures. This 

application note describes methods for extraction of 

textile samples using ASE, along with the analytical 

methods for determination of 22 aromatic amines, some 

of which are specified in the Parliament Directive. 

Application Note 360 

EquIpmEnt 

ASE 200 System 

Glass fiber filters (P/N 047017) 

UltiMate ® 3000 HPLC system consisting of: 

SRD 3600 Solvent Rack 

LPG 3600 Pump 

WPS-3000T Autosampler 

TCC-3000 Column Compartment 

UVD340U Detector 

MSQ Plus Mass Spectrometer 

AXP-MS Pump 

Chromeleon ® 6.80 Chromatography Workstation 

rEagEnts and standards 

Acetonitrile, HPLC grade (Fisher Scientific) 

Methanol, HPLC grade (Fisher Scientific) 

Ammonium acetate, powder (Fisher Scientific) 

Acetic acid, glacial (Fisher Scientific) 

Azo Dyes Mix-6 (Ehrenstorfer Laboratories part 

18000376) 

Individual azo dye standards (Sigma Aldrich)

asE condItIons 

Preheat: 20 min (purge closed) 

Pressure: 1500 psi 

Static Time: 5 min 

Temperature: 100 °C 

Cycles: 2 

Purge Time: 100 s 

Flush Volume: 30% 

Solvent: 90% MeOH with 10% 1 M KOH 

Extraction Time 

per Sample: 40 min 

Solvent Amount Used 

per Sample: 17 mL 

analytIcal condItIons 

Column: Acclaim ® 120 C18 3 µm 120 Å 

2.1 x 150 mm 

Eluent: H O/5%, CH CN/4%, CH OH 

2 3 3 

H O/40%, CH CN/45% CH OH 

2 3 3 

Column Temperature: 30 °C 

Flow Rate: 250 µL/min 

Injection Volume: 10 µL 

UV: 240 nm, 278 nm 

Ionization Mode: APCI, positive polarity 

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 

Sample Prep 

Soak 1 g of material in 3 mL citrate buffer 

(pH 6.0) and 3 mL sodium dithionite (200 mg/mL) for 

2 min. Transfer the mixture and solution to an ASE 11 mL 

cell containing a glass fiber filter. 

Extraction 

Place the extraction cells into the ASE 200 carousel, 

and extract using the ASE conditions listed above. Record 

total volume of the extract, and inject 10 µL onto the 

UltiMate 3000 HPLC/MS/UV system. 

LC Conditions 

Time Flow 

Rate 

(µL/min) 

30 mM 

Ammonium 

Acetate, pH 4 

Acetonitrile Methanol Curve 

0.00 250 91 5 4 

25.00 250 69 27 4 5 

40.00 250 52 40 8 5 

45.00 250 15 40 45 5 

48.00 250 15 40 45 5 

48.10 250 91 5 4 5 

30 mM Ammonium Acetate, pH 4.0 

Weigh 2.31g NH OAc (FW 77.0825 g/mol). Add 1000 

4 

mL H O. Adjust pH to 4.0 with acetic acid. 

2 

2 Rapid Determination of Azo Dyes in Textiles Using Dionex ASE and UltiMate 3000 HPLC Systems

Mass Spectrometer Conditions 

Individual standards for the azo dye compounds were 

obtained from Sigma Aldrich. An AXP-MS auxilliary 

pump was used to infuse these samples for optimization 

in the mass spectrometer at a flow rate of 100 µL/ 

min. During the infusions several scan funcions were 

performed to determine the optimum ionization technique, 

cone voltage, and and mass-to-charge ratio (m/z) for each 

analyte. These tuned values for cone voltage and m/z were 

used to define the SIM scans used in the method (Table 1). 

The preferred mode of ionization for all analytes 

was Atmospheric Pressure Chemical Ionizaion (APCI); 

positive mode. APCI is a gas-phase technique typically 

used to analyze small molecules. APCI is a robust 

technique which is not affected by minor changes in 

buffer strength. In positive ionization mode, protonated 

analyte ions are formed by gas-phase ion molecule 

reactions, resulting in the formation of [M+H] + ions. 

In some cases the SIM scans were staggered across 

the run time during analysis; focusing on a two minute 

region before and after the analyte eluted. Sequencing 

the scan functions in this fashion minimized the total 

analytical cycle time and allowed the mass spectrometer 

to collect more data for the peaks of interest, while 

excluding data collection where no signal was present. 

Table 1. Optimized Detection Conditions of Azo Dye 

Standards for Mass Spectrometric Analysis 

Compound Name Molecular 

Weight 

SIM 

m/z 

Cone 

Voltage (V) 

4-Aminoazobenzene 197.09 198 70 

2,4-Diaminotoluene 122.08 123 55 

4-Aminobiphenyl 169.09 170 30 

4,4’-Oxydianiline 200.09 201 60 

2-Anisidine 123.07 124 80 

4,4’-Methylenebis-(2-chloroaniline) 266.04 267 50 

4,4’-Benzidine 184.10 185 50 

o-Toluidine 107.07 108 40 

4-Chloro-2-methylaniline 141.03 142 60 

2-Methoxy-5-methylaniline 137.08 138 55 

Bis-4-aminophenylmethane 198.27 199 50 

4-Chloroaniline 127.02 128 70 

o-Dianisidine 244.12 245 55 

o-Tolidine 212.13 213 55 

2-Aminonaphthalene 143.07 144 60 

2,6-Dimethylaniline 121.09 122 55 

2,4-Dimethylaniline 121.09 122 50 

4,4’-Methylenebis-(2-methylaniline) 226.04 227 55 

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, 

120 Å (2.1 x 150 mm) 

Eluent: H2O/5% CH3CN/4% CH3OH 

H2O/40% CH3CN/45% CH3OH 


Peaks: 

1. 2,4-Diaminotoluene 

2. 4,4’-Oxydianiline 

3. 2-Anisidine 

4. 4,4’-Benzidine 

5. 4-Methoxy-1,3-Phenylenediamine 

6. o-Toluidine 

7. Bis-4-aminophenylmethane 

8. 2,4-Dimethylaniline 

9. 2-Methoxy-5-methylaniline 

10. 4-Cholroaniline 


1 2 3 

4 

5 6 7 8 

9 

10 

11 

12 13 


Inj. Volume: 10 µL 

Detection: UV, 278 nm 

MSQ, APCI positive ionization 

12. o-Dianisidine 

13. o-Tolidine 

14. 2-Methyl-5-nitroaniline 

15. 4,4’-Methylenebis-(2-methylaniline) 

16. 2-Aminonapthaline 

17. 4,4’-Thiodianiline 

18. 4-Chloro-2-methylaniline 

19. 4-Aminoazobenzene 

20. 3,3’-Dichlorobenzidine 

21. 4-Aminobiphenyl 

22. 4,4’-Methylenebis-(2-chloroaniline) 

0 10 20 30 40 45 

Minutes 

25331 

Figure 1. UV separation of azo dye standard mixture detected at 

278 nm. 

rEsults and dIscussIon 

A tertiary gradient was used because acetonitrile was 

found to improve the resolution of the peaks eluting at 

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. 

14 

16 

15 

17 

18 

19 20 

21 

22 


120 Å (2.1 x 150 mm) 

Eluent: H2O/5% CH3CN/4% MeOH 

H2O/40% CH3CN/45% MeOH 


Peaks: 

1. 2,4-Diaminotoluene 

2. 4,4’-Oxydianiline 

3. 2-Anisidine 

4. 4,4’-Benzidine 

5. 4-Methoxy-1,3-Phenylenediamine 

6. o-Toluidine 

7. Bis-4-aminophenylmethane 


9. 2-Methoxy-5-methylaniline 

10. 4-Cholroaniline 


12. o-Dianisidine 

13. o-Tolidine 

14. 2-Methyl-5-nitroaniline 

3 

3 

1 2 5 6 

16 

7 10 

17 

11 

14 

8 15 18 

9 



Detection: UV: 240 nm, 278 nm 

MSQ, APCI positive ionization 

15. 4,4’-Methylenebis-(2-methylaniline) 

16. 2-Aminonapthaline 

17. 4,4’-Thiodianiline 

18. 4-Chloro-2-methylaniline 

19. 4-Aminoazobenzene 

20. 3,3’-Dichlorobenzidine 

21. 4-Aminobiphenyl 

22. 4,4’-Methylenebis-(2-chloroaniline) 

Figure 2. Comparison of azo dye standards detected by UV and 

mass spectrometric detection. The UV trace appears at the bottom 

and is expanded to show the detection of all compounds using 

SIM (selected ion monitoring) channels in the mass spectrometer. 

4 Rapid Determination of Azo Dyes in Textiles Using Dionex ASE and UltiMate 3000 HPLC Systems 

4 

12 13 

1 

6 

19 

8 

2 

4 14 

5 

15 

7 

16 

9 

10 22 

11 

12 

20 

21 

22 

19 

13 

17 

21 

18 

20 

25330

Figures 3 and 4 show LC/MS analysis of two 

different leather samples extracted using ASE and 

detected by UV/MS. As demostrated in these figures, 

complete resolution between peaks using UV detection 

is difficult in complex matrices. The specificity provided 

by mass spectrometry is critical for detection and 

positive identification of low level concentrations of azo 

compounds in these solutions 

conclusIons 

ASE combined with the UltiMate 3000 HPLC system 

and MSQ Plus/UV detectors create a powerful tool for 

extraction and analysis of azo dyes. The ASE system is 

ideally suited for the extraction of the complex matrices 

analyzed here, providing significant savings in time and 

solvent use compared to other techniques. 

The complexity of the sample extracts makes 

identification and quantitation of analytes difficult using 

UV detection alone. Coupling the HPLC system to the 

MSQ Plus detector provides improved specificity and 

selectivity for identification of compounds prohibited by 

the European Parliament Directive. 


120 Å (2.1 x 150 mm) 

Eluent: H2O/5% CH3CN/4% CH3OH 

H2O/40% CH3CN/45% CH3OH 


18 

mAU 

0 

4,500 

Counts 

1 

2 




Detection: UV, 278 nm 

Peaks: 1. 2-anisidine 

2. o-toluidine 

0 

0.2 2.5 5 7.5 10 12.5 1517.5 2022.5 2527.5 3032.5 3537.5 4042.5 45 

Figure 3. The top trace shows UV separation of an unknown red 

leather extract sample. Poor resolution between the peaks makes 

identification of azo dyes difficult. The bottom trace shows the 

advantage offered by mass spectrometry; clear, positive identification 

of 2-anisidine and o-toluidine. 


120 Å (2.1 x 150 mm) 

Eluent: H2O/5% CH3CN/4% MeOH 

H2O/40% CH3CN/45% MeOH 


18 

mAU 

0 

2,000 

Counts 

1 




Detection: UV: 240 nm, 278 nm 

Peaks: 1. o-toluidine 

2. 4-aminobiphenyl 

400 

0.2 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25 27.5 3032.5 35 37.5 40 42.5 45 

Figure 4. Using a mass spectrometer provides positive identification 

of o-toluidine and 4-aminobiphenyl extracted from a brown 

leather sample. 

25329 

25328 

Application Note 360 5 

2

lIst of manufacturErs 

Ehrenstorfer EQ Laboratories 

530 Means Street Suite 120, Atlanta, GA 30318 

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 

Passion. Power. Productivity. 

Dionex Corporation 

1228 Titan Way 

P.O. Box 3603 

Sunnyvale, CA 

94088-3603 

(408) 737-0700 

North America 

U.S. (847) 295-7500 

Canada (905) 844-9650 

South America 

Brazil (55) 11 3731 5140 

Europe 

Austria (43) 1 616 51 25 Benelux (31) 20 683 9768 (32) 3 353 4294 

Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0 

Ireland (353) 1 644 0064 Italy (39) 02 51 62 1267 Switzerland (41) 62 205 9966 

United Kingdom (44) 1276 691722 

rEfErEncEs 

1. Puntener, A., Page, C. European Ban on Certain 

Azo Dyes. TFL Leather and Technology. 2004. 2:1–5 

Chromeleon, ASE, Ultimate, and Acclaim are registered trademarks of Dionex Corporation. 

MSQ Plus is a trademark of Thermo Fischer Scientific. 

Asia Pacific 

Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 2764 2735 

Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190 

Taiwan (886) 2 8751 6655 

LPN 2007-01 PDF 06/08 

©2008 Dionex Corporation 

www.dionex.com

AN 360: Rapid Determination of Azo Dyes in Textiles Using ... - Dionex

Create successful ePaper yourself

Delete template?

Save as template?