S. Wang et al., Eur. J. Mass Spectrom.

22, 127–132 (2016) 127

Received: 13 July 2016 ■ Revised: 8 August 2016 ■ Accepted: 8 August 2016 ■ Publication: 10 August 2016

EUROPEAN
JOURNAL
OF
MASS
SPECTROMETRY

Isomeric differentiation of chloroanilines by

gas chromatography-mass spectrometry in
combination with tosylation
Shanshan Wang,a Guohua Zhu,b Mengmeng Chen,a Jinsong Liub and Kezhi Jianga*
a
Key Laboratory of Organosilicon Chemistry and Material Technology, Hangzhou Normal University, Hangzhou, 311121, China.
E-mail: jiangkezhi@hznu.edu.cn
b
Zhejiang Province Environmental Monitoring Center, Hangzhou, 310015, China

p-Chloroaniline is one of the banned aromatic amines in azo dyes, but it is very difficult to distinguish it from its isomers due to their
identical­retention time in chromatography and similar mass spectra. In this work, derivatization of the isomeric chloroanilines was
carried­out to yield the corresponding N-tosyl chloroanilines, which were completely separated by gas chromatography and also
possessed­clearly different electron ionization mass spectra. Thus, the three isomers could be differentiated and determined at the
same time. Density functional theory calculation results indicated that the effect of the substituent pattern in electron ionization mass
spectrometry is mainly due to the difference in the stability of the product ion (P2) at m/z 126, originating from the loss of tosyl radical
from the precursor ion.

Keywords: chloroanilines, GC-MS, tosylation, isomeric differentiation, DFT

Introduction
Chloroanilines have been used extensively as raw materials
for manufacturing azo dyes, pharmaceuticals and many other
industrial chemicals.1 Unfortunately, they are toxic compounds
with nephrotoxic potential2 and suspected human carcino-
gens.3,4 According to EU Directive 2002/72/EC5 and Directive
2002/61/EC,6 p-chloroaniline, rather than its ortho and meta
isomers, has been ascribed as one of the 23 prohibited
primary aromatic amines related to polyurethane products or
azo colors. For now, determination of these primary aromatic
amines is carried out predominantly via chromatography in
combination with mass spectrometry.7–9 However, the three
isomeric chloroanilines cannot easily be separated by common
gas chromatography (GC) or high-performance liquid chroma-
tography (HPLC) columns, especially for p-chloroaniline­ and
its meta isomer.10–13 What is worse, they show nearly iden-
tical electron ionization (EI) mass spectra (see supplementary
Figure S1), and only the ortho isomer can be differeniated from
the meta and the para ones by tandem mass spectrometry
(MS/MS) analysis (see supplementary Figure S2). Thus, it is
very challenging for analysts to quantitatively determine all of
the isomers at the same time.
Derivatization has been verified as an effective pre-
treatment­method in analytical chemistry, which provides
a promising strategy for solution of many analytical prob-
lems.7,14–17 Amidation of the amino group has been reported
for differentiation of the ortho-haloanilines from the meta and
para ones by mass spectrometry (MS) analysis, but it failed to
differentiate between meta and para isomers.18 Interestingly,
we showed that three isomeric N-tosyl methylanilines could
be simultaneously differentiated one from another by electro-
spray (ESI)-MS/MS analysis in previous work, indicating the
promising potential of tosylation in isomer differentiation.19 To

ISSN: 1469-0667 © IM Publications LLP 2016

doi: 10.1255/ejms.1426 All rights reserved
 128 Isomeric Differentiation of Chloroanilines by GC-MS in Combination with Tosylation
advance our work, we try to develop a useful method to differ-
entiate isomeric chloroanilines simultaneously.
Experimental
Reagents and sample preparation
The analytical reagents of chloroanilines, p-tosyl chloride,
pyridine and dichloromethane were purchased from Aladdin
(Shanghai, China). Methanol (HPLC grade) was purchased
from Sigma-Aldrich (St Louis, MO, USA) and water (HPLC
grade) was generated using a Milli-Q system (Millipore,
Bedford, MA, USA).
Derivatization
Tosylation of chloroanilines was carried out by a classical
method (Scheme 1).20 Each chloroaniline was mixed with
p-tosyl chloride in dichloromethane with the presence of pyri-
dine, and the resulting mixture was kept at 40°C for 60 min.
The solvent was removed in vacuum, and the residue was
purified by silica gel chromatography. Their structures were
further confirmed by MS analysis.
GC-MS analysis
The GC-MS experiments were performed using a Trace 2000
GC/DSQ MS instrument (Thermo Fisher Scientific, Waltham,
MA, USA) equipped with an HP-5ms capillary column (30 m
long, 0.25 mm id, 0.25 μm film thickness; Agilent Technologies,
Santa Clara, CA, USA). Helium (>99.999%) was used as carrier
gas at a constant flow of 1.0 mL min–1. The column tempera-
ture was maintained at 50°C for 2 min, and then programmed
Scheme 1. Derivatization of chloroaniline.
Figure 1. TIC of the three isomeric chloroanilines (a) and their tosylation derivatives (b).
at 15°C min–1 to 300°C, which was maintained for 5 min. The
gas chromatograph inlet temperature was 300°C and the
transfer line temperature was 260°C. Mass spectra were
acquired by EI-MS under normal conditions: the ion source
temperature was 200°C, the electron energy was 70 eV, the
scan rate was 2 scans s–1 and the mass range 33–600 amu.
Xcalibur software (Version 1.4) was used to control the GC-MS
instrument and to acquire and process the data.
Theoretical calculations
Theoretical calculations were carried out on the Gaussian 03
program by using the density functional theory (DFT) method
at the B3LYP/6-311+G(d,p) level.21 The optimized structures for
the precursor ions, intermediates and products were identi-
fied as a true minimum in energy by the absence of imagi-
nary frequencies. The optimized structures were shown by
Gauss View (Version 3.09) software. The energies discussed
here were the sum of electronic and thermal free energy. The
optimized­geometry data are available in the supplementary
data.
Results and discussion
GC separation
Figure 1(a) shows the total ion current (TIC) chromatogram
of the three isomeric chloroanilines, but only two peaks are
observed. The component at tR 7.69 min in the TIC is ascribed
as o-chloroaniline by comparison with the retention time of
the standard, while m- and p-chloroanilines co-elute at tR
7.69 min in the chromatography. Thus, it fails to quantitatively­
 S. Wang et al., Eur. J. Mass Spectrom. 22, 127–132 (2016) 129

Scheme 2. The fragmentation pathways of N-tosyl chloroaniline radical ion.

determine­ the prohibited p-chloroaniline at conventional cation (P2s, m/z 99) via the loss of isocyanide. The proposed
conditions, which might give rise to a false-positive result in fragmentation pathways are given in Scheme 2.
analysis of the real samples.10–13 Fortunately, the tosylation To our interest, the tosylation derivatives of the three isomers
products of the three isomers are completely separated under can be easily recognized in EI-MS analysis, and the corre-
the same condition [Figure 1(b)]. The three components at tR sponding EI-MS data are summarized in Table 1. As displayed
16.85 min, tR 17.94 min and tR 18.19 min in the TIC correspond in Figure 2, the relative abundance of P2, which contains the
to the tosylation derivatives of o-, m- and p-chloroanilines,
Table 1. Electron ionization mass spectrometry data of the three
respectively. A complete chromatographic separation offers
isomeric N-tosylchloroanilines.
a good prospect for the accurate quantification of the three
isomers. Ion Compound/relative abundance (%)
o-M m-M p-M
EI-MS analysis M+. (m/z 281) 60.1 43.8 90.9
Further EI-MS analysis of the three derivatives has been
P1 (m/z 155) 100 82.7 98.5
carried out for isomeric differentiation. As shown in Figure 2,
P1s (m/z 91) 91.4 100 100
fragmentation of the N-tosyl chloroaniline radical ion (M+., m/z
281) in path-1 mainly produces the tosyl cation (P1, m/z 155), P2 (m/z 126) 30.0 7.5 98.5
which undergoes further dissociation to yield the tropilium P2s (m/z 99) 18.7 10.3 25.5
cation (P1s, m/z 91) through the loss of SO2.22 In another frag- P3 (m/z 217) 1.3 15.2 1.3
mentation channel (path-2), dissociation of the precursor ion IM /IP2 2.0 5.8 0.92
gives rise to the fragment ion at m/z 126 (P2), which also
IP2s /IP2 0.62 1.37 0.26
undergoes further dissociation to generate a chloronium

Figure 2. EI-MS of the isomeric N-tosyl chloroanilines.

 130 Isomeric Differentiation of Chloroanilines by GC-MS in Combination with Tosylation

Figure 3. The optimized structures of the isomeric P2.

chloro group in its structure, varies distinctively among the
three isomers, p-P2 (98.5%) > o-P2 (30.0%) > m-P2 (7.5%).
The distinctive intensity of p-P2 in the EI-MS can be attributed
to its stable chloronium structure rather than the carbonium
one, which delocalizes the positive charge to the heteroatom
(Cl) due to the para effect.23,24 This is further supported by
the significantly shorter C–Cl bond length (1.688 Å) in p-P2,
compared with that in o-P2 (1.6961 Å) and m-P2 (1.723 Å),
according to the calculation results (Figure 3). Also, the
C=N bond length follows the order of p-P2 (1.267 Å)  <  o-P2
(1.281 Å)  <  m-P2 (1.290 Å). Thus, p-P2 is much more stable
than o-P2 and m-P2, and it is lower in free energy than o-P2
and m-P2 by 14.90 kJ mol–1 and 37.32 kJ mol–1, respectively.
The different stability of the isomeric P2 also affects the
relative abundance of their subsequent fragment ion P2s.
The most stable structure of p-P2 accounts for the least
intensity ratio (0.26) of IP2s/IP2, because it is most unlikely
for p-P2 to undergo further dissociation to afford p-P2s.
On the contrary, the positive charge cannot be delocalized
to the Cl atom in the relative unstable m-P2 (carbonium);
thereby, it shows the least abundant signal in EI-MS and the
most intensity ratio (1.37) of IP2s/IP2 among the three isomers.
In dissociation of the isomeric P2 (Table 2), the change of
free energy (ΔG P2s = G P2s + GCNH  –  GP2) obeys the order of
ΔGp-P2s (128.67 kJ mol–1) > ΔGo-P2s (113.77 kJ mol–1) > ΔGm-P2s
(91.34 kJ mol–1), and thus the corresponding IP2s/IP2 follows the
reverse order: para (0.26) < ortho (0.62) < meta (1.37). In short,
due to the different stability of the three isomeric P2, there is
a significant difference in the relative abundance of P2 and the
intensity ratio of IP2s/IP2, which can be used for differentiation
of the three isomers.
Moreover, different to o-M+. and p-M+., the meta isomer
undergoes SO2 elimination to give a characteristic product ion
at m/z 217 (m-P3, mainly 2-p-methylphenyl-5-chrolo-aniliene
radical ion).25 According to calculation results (supplementary
Table S1), the C13 atom (at the ortho-site of the aniline) in
m-M+. carries much higher spin densities (0.2604) than that in
o-M+. (0.2179) and p-M+. (0.1603), indicating that m-M+. is more
likely to undergo the C–C coupling reaction in the process of
SO2 elimination (supplementary Scheme S1). Thus, the frag-
ment ion m-P3 (15.2%) is distinctly more abundant than o-P3
(1.3%) or p-P3 (1.3%) in EI-MS (Table 1).
In addition, p-M+. possesses a more stable structure, which is
lower in free energy than o-M+. and m-M+. by 14.05 kJ mol–1 and
14.79 kJ mol–1 (Table 2), respectively. Thus, the relative abun-
dance of M+. obeys the following order, p-M+. (90.9%) > o-M+.
(60.1%) > m-M+. (43.8%). There is also a remarkable difference
in the intensity ratio of (IM/IP2) obtained in EI-MS (Table 1).

Table 2. Free energy (Hartree) and relative free energy (kJ mol–1) of the key species in fragmentation of the N-tosyl chloroaniline radical
ions.
Structure ortho- meta- para-
G (hartree) Rel G (kJ mol–1) G (hartree) Rel G (kJ mol–1) G (hartree) Rel G (kJ mol–1)
+.
M –1565.966683 14.05 –1565.966403 14.79 –1565.972036 0.0*
P1 –819.317455 — –819.317455 — –819.317455 —
N1 –746.604248 — –746.603386 — –746.604940 —
P1 + N1 – M+. –1565.921703 118.10 –1565.920841 119.62 –1565.922395 130.33
P2 –746.308409 — –746.299869 — –746.314085 —
N2 –819.605977 — –819.605977 — –819.605977 —
+.
P2 + N2 – M –1565.913613 137.31 –1565.905846 158.99 –1565.920062 136.46
P1s –270.658530 — –270.658530 — –270.658530 —
SO2 –548.710819 — –548.710819 — –548.710819 —
P1s + SO2 – P1 –0.051894 –136.25 –0.051894 –136.25 –0.051894 –136.25
P2s –652.805693 — –652.805693 — –652.805693 —
CNH –93.459383 — –93.459383 — –93.459383 —
P2s + CNH – P2 0.043333 113.77 0.034793 91.34 0.049009 128.67
 S. Wang et al., Eur. J. Mass Spectrom. 22, 127–132 (2016) 131
Conclusion
In this work, three isomeric chloroanilines can be differenti-
ated from each other by tosylation in combination with GC-MS
analysis. The three isomeric chloroanilines were transformed
to the corresponding N-tosyl chloroanilines. The isomeric
derivatives were completely separated under common GC
conditions. In EI-MS of the para isomer, the fragment ion at
m/z 126 (p-P2, 98.5%) is distinctly abundant, and the intensity
ratio (0.26) of IP2s/IP2 is clearly low. Meanwhile, the meta isomer
possesses a much high intensity ratio (1.37) of IP2s/IP2 and a
characteristic fragment ion at m/z 217 in EI-MS. Thus, the
three isomers can be easily differentiated by EI-MS analysis.
Theoretical calculations showed that the different stability of
the fragment ion of P2 is a key factor influencing the relative
intensity of the product ions in EI-MS. These results provided
a promising solution for simultaneous quantitation of isomeric
chloroanilines, and further work in this field will be carried out.
Acknowledgments
The authors gratefully acknowledge financial support from
the National Science Foundation of Zhejiang Province
(LY15B050007), Analysis and Detection Foundation of
Science and Technology Department in Zhejiang Province
(2016C37017), Science and Technology Plan Projects in
Zhejiang Province (2014C03026) and Environmental Protection
Project in Zhejiang Province (2014A011). Computer time was
made available on the SGI Aktix 450 sever at Computational
Center for Molecular Design of Organosilicon Compounds,
Hangzhou Normal University.
References
1. R. Shoji and M. Kawakami, “Prediction of genotoxicity
of various environmental pollutants by artificial neural
network simulation”, Mol. Div. 10, 101 (2006). doi: http://
dx.doi.org/10.1007/s11030-005-9005-1
2. H.H. Lo, I.P. Brown and O.G. Rankin, “Acute nephro-
toxicity induced by isomeric dichloroanilines in fischer
344 rats”, Toxicol. 63, 215 (1990). doi: http://dx.doi.
org/10.1016/0300-483X(90)90044-H
3. K. Riedel, G. Scherer, J. Engl, H.-W. Hagedorn and A.R.
Tricker, “Determination of three carcinogenic aromatic
amines in urine of smokers and nonsmokers”, J. Anal.
Toxicol. 30, 187 (2006). doi: http://dx.doi.org/10.1093/
jat/30.3.187
4. A. Gottlieb, C. Shaw, A. Smith, A Wheatley and S
Forsythe, “The toxicity of textile reactive azo dyes
after hydrolysis and decolourisation”, J. Biotechnol.
101, 49 (2003). doi: http://dx.doi.org/10.1016/S0168-
1656(02)00302-4
5. EU Commission, “Directive 2002/72/EC of 6 August 2002
relating to plastic materials and articles intended to
come into contact with foodstuffs”, Off. J. Eur. Commun. L
220, 18 (2002).
6. EU Commission, “Directive, 2002/61/EC of 19 July 2002
amending for the nineteenth time Council Directive
76/769/EEC relating to restrictions on the marketing
and use of certain dangerous substances and prepara-
tions (azocolourants)”, Off. J. Eur. Commun. L 243, 15
(2002).
7. T. Zimmermann, W.J. Ensinger and T.C. Schmidt, “In situ
derivatization/solid-phase microextraction: determina-
tion of polar aromatic amines”, Anal. Chem. 76, 1028
(2004). doi: http://dx.doi.org/10.1021/ac035098p
8. M. Aznar, E. Canellas and C. Nerín, “Quantitative
determination of 22 primary aromatic amines by
cation-exchange solid-phase extraction and liquid
chromatography-mass spectrometry”, J. Chromatog.
A 1216, 5176 (2009). doi: http://dx.doi.org/10.1016/j.
chroma.2009.04.096
9. A.a Ramkumar, V.K. Ponnusamy and J.-F. Jen, “Rapid
analysis of chlorinated anilines in environmental water
samples using ultrasound assisted emulsification
microextraction with solidification of floating organic
droplet followed by HPLC-UV detection”, Talanta 97, 279
(2012). doi: http://dx.doi.org/10.1016/j.talanta.2012.04.031
10. G. Skarping, M. Dalene and P. Lind, “Determination of
toluenediamine isomers by capillary gas chromatog-
raphy and chemical ionization mass spectrometry with
special reference to the biological monitoring of 2,4- and
2,6-toluene diisocyanate”, J. Chromator. A 663, 199 (1994).
doi: http://dx.doi.org/10.1016/0021-9673(94)85246-4
11. G.B. Wang, J.P. Santerre and R.S. Labow, “High-
performance liquid chromatographic separation and
tandem mass spectrometric identification of breakdown
products associated with the biological hydrolysis of a
biomedical polyurethane”, J. Chromator. B 698, 69 (1997).
doi: http://dx.doi.org/10.1016/S0378-4347(97)00282-X
12. M.S. Narvekar and A.K. Srivastava, “Separation of iso-
mers of aromatic amines on HPTLC plates impregnated
with 18-crown-6”, J. Planar Chromatogr. 15, 120 (2002).
doi: http://dx.doi.org/10.1556/JPC.15.2002.2.6
13. X.W. Ye, Y. Peng, Z.Y. Niu, Y.G. Gao, X. Luo, L. Zou and
M.H. Zhou, “Identification of banned aromatic amines
and their isomers in textiles by ultra-performance liquid
chromatography-linear ion trap/orbitrap high resolution
mass spectrometry”, Chin. J. Chromatogr. 32, 1005 (2014).
doi: http://dx.doi.org/10.3724/SP.J.1123.2014.04041
14. V.G. Zaikin and J. M. Halket, “Derivatization in mass
spectrometry-2. Acylation”, Eur. J. Mass Spectrom. 9, 421
(2003). doi: http://dx.doi.org/10.1255/ejms.576
15. C.F. Poole, “Derivatization reactions for use with
the electron-capture detector”, J. Chromatogr. A
1296, 15 (2013). doi: http://dx.doi.org/10.1016/j.
chroma.2013.01.108
16. C.R. Sun, H.Z. Sun, Y.Q. Lai, J.J. Zhang and Z.W. Cai,
“Liquid chromatography/mass spectrometry method for
determination of perfluorooctane sulfonyl fluoride upon
 132 Isomeric Differentiation of Chloroanilines by GC-MS in Combination with Tosylation
derivatization with benzylamine”, Anal. Chem. 83, 5822
(2011). doi: http://dx.doi.org/10.1021/ac201117k
17. J.M. Plotka-Wasylka, C. Morrison, M. Biziuk and
J. Namiesnik, “Chemical derivatization processes
applied to amine determination in samples of different
matrix composition”, Chem. Rev. 115, 4693 (2015). doi:
http://dx.doi.org/10.1021/cr4006999
18. B.F. Jariwala, M. Figus and B.A. Attygalle, “Ortho
effect in electron ionization mass spectrometry of
N-acylanilines bearing a proximal halo substituent­”,
J. Am. Soc. Mass Spectrom. 19, 1114 (2008). doi:
http://dx.doi.org/10.1016/j.jasms.2008.05.004
19. S.S. Wang, C. Guo, N.W. Zhang, H.R. Zhang and K.Z.
Jiang, “Tosyl oxygen transfer and ion-neutral complex
mediated electron transfer in the gas-phase fragmenta-
tion of the protonated N-phenyl p-toluenesulfonamides”,
Int. J. Mass Spectrom. 376, 6 (2015). doi: http://dx.doi.
org/10.1016/j.ijms.2014.11.003
20. J.G. Kang, J.H. Hur, S.J. Choi, G.J. Choi, K.Y. Cho, L.N.
Ten, K.H. Park and K.Y. Kang, “Antifungal activities of
N-arylbenzenesulfonamides against phytopathogensand
control efficacy on wheat leaf rust and cabbage club root
diseases”, Biosci. Biotechnol. Biochem. 66, 2677 (2002).
doi: http://dx.doi.org/10.1271/bbb.66.2677
21. M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria,
M.A. Robb, J.R. Cheeseman, V.G. Zakrzewski, J.A.
Montgomery, Jr, R.E. Stratmann, J.C. Burant, S.
Dapprich, J.M. Millam, A.D. Daniels, K.N. Kudin, M.C.
Strain, O. Farkas, J. Tomasi, V. Barone, M. Cossi, R.
Cammi, B. Mennucci, C. Pomelli, C. Adamo, S. Clifford,
J. Ochterski, G.A. Petersson, P.Y. Ayala, Q. Cui, K.
Morokuma, D.K. Malick, A.D. Rabuck, K. Raghavachari,
J.B. Foresman, J Cioslowski, J.V. Ortiz, B.B. Stefanov, G.
Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. GomPerts,
R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng,
A. Nanayakkara, C. Gonzalez, M. Challacombe, P.M.W.
Gill, B. Johnson, W. Chen, M.W. Wong, J.L. Andres, C.
Gonzalez, M.E. Head-Gordon, S. Replogle and J.A. Pople.
Gaussian 03. Gaussian Inc., Pittsburgh, PA (2003).
22. C. Lifshitz, “Tropylium ion formation from toluene:
solution of an old problem in organic mass spectrom-
etry”, Acc. Chem. Res. 27, 138 (1994). doi: http://dx.doi.
org/10.1002/chin.199438315
23. K.V. Tretyakov, N.G. Todua, R.S. Borisov, V.G. Zaikin, S.E.
Stein and A.I. Mikaia, “Unique para-effect in electron
ionization mass spectra of bis(perfluoroacyl) derivatives
of bifunctional aminobenzenes”, Rapid Commun. Mass
Spectrom. 24, 2529 (2010). doi: http://dx.doi.org/10.1002/
rcm.466
24. N.G. Todua and A.I. Mikaia, “Mass spectrometry of
analytical­derivatives. 2. ortho and para effects in elec-
tron ionization mass spectra of derivatives of hydroxyl,
mercapto and amino benzoic acids”, Mass-Spectrometria
13, 83 (2016).
25. M.F. Grostic, R.J. Wunk and F.A. MacKellar, “The mass
spectrometry of sulfonylureas: mechanisms for the loss
of sulfur dioxide”, J. Am. Chem. Soc. 88, 4664 (1966). doi:
http://dx.doi.org/10.1021/ja00972a026