Application NoteMaterials Testing &Research

Determination of Aromatic Amines
Derived from Azo Colorants byGC/MS Using Supported Liquid
Extraction Chem Elut S Cartridges
AuthorDerick Lucas
Agilent Technologies, Inc.

Abstract
Agilent Chem Elut S is a sample preparation product that uses synthetic
media for supported liquid extraction (SLE). This study uses the Chem Elut S
cartridges for the quantitative analysis of aromatic amines derived from azo
dyes according to European method ISO 14362-1. A sodium citrate/sodiumhydroxide solution containing aromatic amines was loaded onto the cartridgeand held for 15 minutes, then eluted with methyl tert-butyl ether (MTBE) to givean extract for GC/MS analysis. The method measured amines at 30 µg/mL forthree different SLE cartridge formats, all delivering high recovery (87 to 119%) andexcellent reproducibility (<9% RSD). Results demonstrate that Chem Elut S is ahigh‑performance SLE product for the determination of aromatic amines.

Introduction
Azo dyes are the mostly widely used
dyes in industry, accounting fortwo‑thirds of all synthetic dyes.1 Many
common products, including textilefibers, foods, drugs, and cosmetics, usethem as coloring additives. Certain azo
dyes are banned in textile manufacturing
due to environmental hazards caused by
reductive degradation into carcinogenic
aromatic amines.2
The quantification of aromatic aminesfrom their corresponding azo dyes intextiles is outlined in European method
ISO 14362‑1.3 The azo dye is extracted
from the material using refluxing
chlorobenzene, which is then dried
down. The extraction residue is treatedwith a sodium citrate/sodium hydroxide
buffer and sodium dithionate to reduceazo dyes to aromatic amines. This work
evaluated the Chem Elut S extraction
recovery and reproducibility by spiking
the appropriate aromatic amines into a
blank sodium citrate/sodium hydroxide
solution.
In traditional SLE, an aqueous sample isloaded onto the sorbent bed containing
diatomaceous earth (DE) where theaqueous sample coats as a thin film on
the material. A water-immiscible solvent
is then passed through the SLE bed,
which extracts target analytes from thesample with high efficiency, and elutes
them into a collection tube for analysis
with or without post-treatment. Thisfeature provides significant time andlabor savings over conventional LLE, anduser-to-user reproducibility is improved.

2The Chem Elut S products use asynthetic medium optimized toovercome the issues of traditional
DE sorbent. Unlike irregularly shaped
DE particles that have considerable
variability and fines, Chem Elut S
particles have a narrow particle size
distribution and are free of fines.
These features promote ideal flow
characteristics and higher reproducibility.
Also, Chem Elut S has higher sample
holding capacity than DE sorbents,
delivering efficient sample adsorption
and reducing the chance of sample
breakthrough. The three large format
Chem Elut S cartridges, 5 mL, 20 cc;
10 mL, 60 cc; and 20 mL, 60 cc, operatewith gravity loading and elution,
simplifying the workflow.
This work demonstrates high recovery
and reproducibility of aromatic amines
using Chem Elut S cartridges and GC/MS(SIM) detection.

Experimental
All reagents and solvents were HPLCor analytical grade. MTBE was from
VWR-BDH Chemicals (Radnor, PA, USA).
Sodium citrate dihydrate (Sigma‑Aldrich,St. Louis, MO, USA) was used to prepare
citrate buffer at 0.06 M in water. Sodium
dithionite was from Sigma‑Aldrich andprepared as a 200 mg/mL solution
daily. Sodium hydroxide was fromSigma‑Aldrich as a 50% solution inwater. The aromatic amines andinternal standards were purchasedfrom Sigma‑Aldrich and AccuStandard(New Haven, CT, USA) as neat solids
and liquids for preparation of stock
standards. Table 1 lists retention times,
CAS numbers, and GC/MS ions for target
amines.

Standards and solutions
Individual stock solutions were preparedfor each amine at 10 mg/mL in wateror DMSO, as required. These standards
were then combined into working
standards for calibrants and sample
spiking.

Sample preparation equipment
and supplies•

Agilent Chem Elut S 5 mL, 20 cc(p/n 5610-2009);•Agilent Chem Elut S 10 mL, 60 cc(p/n 5610-2010)•Agilent Chem Elut S 20 mL, 60 cc(p/n 5610-2011)•

40 and 150 mL glass collectionbottles•

Eppendorf pipettes

Instrument conditions•

Agilent 7890 GC•

Agilent 5977 GC/MSD
Agilent 5977 GC/MSD Parameters
GC Column

Agilent J&W DB-35ms, 30 m × 250 µm × 0.250 µm (p/n 122-3832)

Liner

Ultra Inert single taper, splitless, with wool (p/n 5190-2293)

Injection Volume

1 µL

Inlet Temperature

280 °C

Flow Rate

2 mL/min, constant flow

Oven Temperature

100 °C then 10 °C/min to 320 °C

Aux Temperature

320 °CMS Source

250 °C

Quad Temperature

180 °CMS ModeSIM (see Table 1 for ions)

Table 1. Target amines and internal standards, retention time, SIM parameters, and recovery specification.3

CAS No.

Retention Time(min)Quantifier Ion(m/z)

Qualifier Ion 1(m/z)

Qualifier Ion 2(m/z)%RecSpec.3

o-Toluidine

95-53-4

2.783

106

4-Chloroaniline

106-47-8

4.511

127

107

89>50

129

100

2,4,5-Trimethylaniline

137-17-7

4.872

>70

120

135

134

p-Cresidine>70

120-71-8

3-Chloro-o-toluidine

87-60-5

5.224

122

137

94

No spec

5.585

141

106

140

4-Chloro-o-toluidine>70

95-69-2

5.700

141

106

140

>70

2,4-Diaminotoluene

95-80-7

7.271

121

122

94

>50

3-Nitro-p-toluidine

119-32-4

9.268

152

107

135

>70

2-Naphthylamine

91-59-8

9.326

143

115

116

>702-Aminobiphenyl

90-41-5

9.427

169

168

167

>70

4-Aminobiphenyl

92-67-1

11.591

169

168

170

>70

Anthracene-d10

1719-06-8

11.784

188

184

189

I.S.

p-Aminoazobenzene

60-09-3

15.200

197

92

120

>70

4,4’-Oxydianiline

101-80-4

15.836

200

171

108

No spec

4,4’-Diaminophenylmethane

101-77-9

15.937

198

197

106

>70Benzidine

92-87-5

16.012

184

185

92

>70

3,3’-Dimethyl-4,4’-diaminodiphenylmethane

838-88-0

17.363

226

211

120

No spec

3,3’-Dimethylbenzidine

119-93-7

17.623

212

213

106

No spec

4,4’-Thiodianiline

139-65-1

18.520

216

184

215

>70

4,4’-Methylenebis(2-chloroaniline)

101-14-4

18.856231266

195

No spec

3,3’-Dimethoxybenzidine

119-90-4

18.973

244

201

229

No spec

Analyte3Sample pretreatment procedure: TheE.4.6. check protocol was followedin ISO 14362-1 to determine the SLE
extraction efficiency of aromatic
amines.3 A solution was prepared with
0.06 M sodium citrate adjusted to pH
6 with sodium hydroxide. The sample
was then spiked at 30 µg/mL witharomatic amines and mixed well prior
to SLE extraction. After the additionof the amines, the solution was brightorange/yellow.
SLE procedure1.

Set up Chem Elut S cartridges withcollection tubes/bottles.

2.

Transfer the sample onto a cartridgeand load under gravity (see Table 2for volumes).

3.

Hold the sample in media for
15 minutes.

4.

Gravity elute with methyl tert-butylether (see Table 2 for volumes).

5.

Mix eluent and aliquot into
autosampler vials for GC/MS SIManalysis.

Recovery and reproducibility
evaluation
The Chem Elut S protocol for aromatic
amines was tested for recovery andreproducibility. Each cartridge wasrun in three replicates at 30 µg/mL, asindicated in the EN method.3 Isotopicallylabeled internal standard (IS)anthracene-d10 was spiked at 30 µg/mLto ensure accurate volume correction.
Calibrants were prepared at 7.5 µg/mL
in MTBE to account for a 4x dilution, asindicated in the SLE procedure.

4×101
6.0

122 3

34

4 55 66 7

7 8

8 9

1718

5.5
5.0
4.5
Counts (%)

Sample preparation

4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
345

678910 11 12 13 14
Acquisition time (min)1516192021Figure 1. GC/MS SIM chromatogram overlay of 20 aromatic amines and IS at 7.5 µg/mL.

Aqueousphase
Porousmesh

1. Load

3. Add solvent

4. EluteGravity

Gravity

2. Hold for 15 minutes

Figure 2. Diagram of the general Agilent Chem Elut S workflow. The steps include: 1) loading sample withgravity; 2) holding the sample in the SLE media for 15 minutes; 3) adding a water-immiscible solvent toextract the analytes; and 4) eluting the organic solvent with gravity.

Table 2. Cartridge load and elution volumes for aromatic amine SLE extraction.
Part Number

Load Volume (mL)Elution Volume (mL)

5 mL, 20 ccCartridge

5610-2009

52010 mL, 60 cc

5610-2010

104020 mL, 60 cc

5610-20112080

Results and discussion

Table 3. Recovery and reproducibility of carcinogenic aromatic amines by Agilent Chem Elut S.
Chem Elut S Format

Recovery and reproducibility results
The study produced excellent results,
as shown in Table 3. Recovery
was between 87 and 119% for all
three Chem Elut S formats tested,with %RSD <9. Extending the SLE
hold time to 15 minutes increased
recoveries for p-aminoazobenzene,4,4'-methylenebis(2-chloroaniline), and3,3'-dimethoxybenzidine by 29 to 38%(Figure 3). These recovery improvements
were demonstrated on both synthetic
and DE-based SLE tubes. All large-format
cartridges performed exceptionally well,particularly the 10 mL, 60 cc tube, which
demonstrated excellent reproducibility.

5 mL, 20 cc
Analyte

10 mL, 60 cc

20 mL, 60 cc%Rec.

%RSD%Rec%RSD%Rec%RSD

o-Toluidine

96.8

5.7

91.9

2.4

91.3

4.5

4-Chloroaniline

98.6

3.7

91.3

0.7

92.2

4.7

2,4,5-Trimethylaniline

98.8

3.9

92.2

0.8

93.8

4.3

p-Cresidine

96.9

4.3

90.60.8

91.3

3.9

3-Chloro-o-toluidine

97.8

3.7

91.0

1.0

92.3

4.4

4-Chloro-o-toluidine

96.4

3.9

89.8

0.6

90.7

4.7

2,4-Diaminotoluene

96.7

4.4

90.7

0.7

90.6

4.2

3-Nitro-p-toluidine

99.8

4.7

95.0

0.8

94.9

5.0

2-Naphthylamine

97.8

4.3

91.7

0.8

93.0

5.1

2-Aminobiphenyl

96.4

3.4

90.7

0.6

92.8

4.4

4-Aminobiphenyl

97.9

4.3

93.5

0.6

94.1

4.5

p-Aminoazobenzene

98.9

6.2

96.2

0.5

95.3

4.1

4,4'-Oxydianiline

112.9

6.5

109.50.8

107.7

5.8

4,4'-Diaminophenylmethane

99.5

6.1

95.3

0.6

95.0

5.5

Sample preparation with Chem Elut SBenzidine

116.4

5.6

110.4

1.3

111.7

6.1

The Chem Elut S tubes were simple
to use, fast, and gave high analyte
recovery and excellent precision foraromatic amines. The synthetic medium
is carefully manufactured to provide
high sample holding capacity, uniform
packing, consistent batch‑to‑batch
reproducibility, and optimal flow
characteristics. Chem Elut S 5 mL,20 cc, 10 mL, 60 cc, and 20 mL, 60 cc
cartridges are designed to allow
gravity sample loading and elution
for simple operation. This application
benefits from a 15-minute hold timeto ensure exceptional recovery for all
analytes. Performance characteristics
were consistent with DE-basedsorbents in direct comparisons. Thesefeatures provide excellent data quality,ease‑of‑use, and matrix removal (that is,salts).

3,3'-Dimethyl-4,4'-diaminodiphenylmethane

119.6

2.4

114.0

0.5

113.1

5.3

3,3'-Dimethylbenzidine

103.4

5.4

96.8

0.4

96.8

5.7

4,4'-Thiodianiline

107.4

5.9

101.9

1.0

99.7

5.9

4,4'-Methylenebis(2-chloroaniline)

107.2

5.8

101.6

1.0

99.6

5.7

3,3’-Dimethoxybenzidine

91.6

6.1

91.5

8.2

87.1

5.8

Impact of hold time on recovery for three amines
120

5 min hold
15 min hold

100%Rec

80
60
40
20

3,3'-D
im
et
ho
xy
benz
idin
e

4,4'-Methylenp-eb

is(Am2-chinoazoloro

benz

anili
neene)

0

Figure 3. Experiment demonstrating the impact of holding time on recovery of three aromatic amines.
Samples containing 30 µg/mL amines were extracted with Agilent Chem Elut S 10 mL, 60 cc tubes (n = 3).

5Conclusion

References

Agilent Chem Elut S uses a synthetic
medium designed for consistency andhigh aqueous sample holding capacity.
In this study, Chem Elut S deliveredhigh recovery and reproducibility foraromatic amines derived from azo dyes
as outlined in ISO 14362-1. Excellent
performance was demonstrated for
all three large Chem Elut S cartridges
tested, giving 87 to 119% recovery and<9% RSD for all 20 aromatic amines.
Many other sample and analyte types
benefit from Chem Elut S samplepreparation and are featured in other
Agilent applications.

1.

Freeman, H. S. Aromatic Amines:
Use in Azo Dye Chemistry. Front.
Biosci. (Landmark Ed.) 2013, 18,145–164.

2.

Pielesz, A.; et al. Detection andDetermination of Aromatic Amines
as Products of Reductive Splittingfrom Selected Azo Dyes. Ecotoxicol.
Environ. Saf. 2002, 53(1), 42–47.

3.

Textiles - Methods for Determinationof Certain Aromatic Amines Derived
from Azo Colorants - Part 1:
Detection of the Use of Certain
Azo Colorants Accessible withand Without Extracting the Fibres.
EN 14362-1:2012, Feb. 2012.

www.agilent.com/chem
This information is subject to change without notice.
© Agilent Technologies, Inc. 2019
Printed in the USA, June 24, 20195994-0951EN