Analysis of commercial Acid Black 194 and related dyes by micellar electrokinetic chromatography

Article abstract of DOI:10.1016/j.dyepig.2011.12.014

The commercial dye C.I. Acid Black 194 was analyzed by reversed-phase HPLC, Capillary Zone Electrophoresis and Micellar ElectroKinetic Chromatography under different operative conditions, with the scope to detect the impurity distribution typical of any production processes and synthetic batch. The three chrome(III) complexes deriving from the industrial synthesis of C.I. Acid Black 194, and one of the main impurities were isolated by silica flash chromatography and identified by mass spectrometry and NMR. More than twelve compounds present in the commercial mixture, but undetectable by the analytical protocols known in literature, were fully separated by the MECK mode capillary electrophoresis with low % Relative Standard Deviation of the main electrophoretic parameters. Two of them were identified by isolation from the commercial mixture. As additional examples two other commercial metal-based dyes, C.I. Acid Brown 432 and C.I. Acid Brown 434, were analyzed by the same protocol with very good results.

Full text of DOI:10.1016/j.dyepig.2011.12.014

Dyes and Pigments 94 (2012) 258e265
Contents lists available at SciVerse ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Analysis of commercial Acid Black 194 and related dyes by micellar electrokinetic
chromatography
*
Roberto Sebastiano , Nunzia Contiello, Stanislao Senatore, Pier Giorgio Righetti, Attilio Citterio
Department of Chemistry, Material and Chemical Engineering “Giulio Natta”, Polytechnic of Milan, Via Mancinelli, 7 e 20131 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The commercial dye C.I. Acid Black 194 was analyzed by reversed-phase HPLC, Capillary Zone Electro-
phoresis and Micellar ElectroKinetic Chromatography under different operative conditions, with the
scope to detect the impurity distribution typical of any production processes and synthetic batch. The
three chrome(III) complexes deriving from the industrial synthesis of C.I. Acid Black 194, and one of the
main impurities were isolated by silica flash chromatography and identified by mass spectrometry and
NMR. More than twelve compounds present in the commercial mixture, but undetectable by the
analytical protocols known in literature, were fully separated by the MECK mode capillary electropho-
resis with low % Relative Standard Deviation of the main electrophoretic parameters. Two of them were
identified by isolation from the commercial mixture. As additional examples two other commercial
metal-based dyes, C.I. Acid Brown 432 and C.I. Acid Brown 434, were analyzed by the same protocol with
very good results.
Received 12 September 2011
Received in revised form
23 December 2011
Accepted 28 December 2011
Available online 20 January 2012
Keywords:
Textile dyes
Metal complexes
Micellar capillary electrophoresis
REACH legislation
Acid Black analysis
Acid Brown analysis
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
with b-cyclodextrin. Subsequently, Borrós et al. [7] adopted CZE for
studyingtheformationofcarcinogenic aryl amines in azodyes. There
has been a significant increase in the use of CZE for analyzing dyes,
especially artificial colourants added to foodstuff, such as brilliant
blue and azorubine in red wines [8] and a variety of azo dyes in
alcoholic beverages [9], not to mention synthetic dyes in ice creams
[10] and milk beverages [11]. The list could continue with reports on
CZE analyses of a variety of food colourants [12], including red food
colourants [13]. The work done up to the year 2000 has been nicely
reviewed by Boyce [14] and not only includes colourants in foodstuff,
but also a variety of additives as well, such as preservatives, antiox-
idants, sweeteners and other modifiers.
Among the commercial dyes, Acid Black 194 is one of the most
popular in both leather and wool, polyamide, silk and wool blended
fabric dyeing [1], direct printing in wool, silk fabric fibre and nylon
non-woven microfabric dyeing [15,16]. In the last case, hydrophobic
interactions between dye and fibre and ioneion electrostatic
interactions between the dye anion and the protonated amino
groups of the fibre are thought to contribute to the dye-substrate
strong affinity [17]. ORISAN Black M-RL is a 1:2 symmetrical
metal complex dye constituted by a mixture of three geometrical
isomers [18], where the chrome(III) central atom is coordinated to
two molecules of a tridentate organic ligand, according to an
octahedral mer-distorting geometry. The chemical structure of one
The total worldwide consumption of dyes in several industries
such as textile, leather, paper, pulp, plastics is estimated will grow to
2.3 million metric tons in 2013 [1]. Approximately 10,000 different
dyes and pigments are produced annually worldwide and used
extensively in the dye and printing industries [2]. Unfortunately, it is
estimated to grow to 10e15% of unexhausted dyes, after the col-
ouring process, are discharged into the waste streams irrespective of
the substrate involved (e.g., leather, plastic) [3]. The complex
chemical formulae of dyes, along with the presence of heavy-metal
ions, has the potential of inducing chronic toxicity, for instance
through mutagenic and carcinogenic effects. This is particularly true
with azo dyes, which alone constitute about 50% of all industrial
colourants produced worldwide [4]. These can be transformed into
carcinogenic compounds under anaerobic conditions [5].
The main analytical methods applied to the characterization
of metal dyes are HPLC and capillary zone electrophoresis (CZE).
After a timid approach [6,7], the CZE analysis, has gained momentum
and has been applied to a variety of cases. Perhaps the first report
appeared in 1998, when Pérez-Ruiz et al. [6] described the CZE
separationoffluoresceindyes byexploitinghost-guestcomplexation
of the three N-
a,b isomers [19] of the unsymmetrical azo ligands, is
shown in Fig. 1A. The organic ligands are multi functional azo dyes
* Corresponding author. Tel.: þ39 (0)2 2399 3014.
E-mail address: roberto.sebastiano@polimi.it (R. Sebastiano).
0143-7208/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2011.12.014

R. Sebastiano et al. / Dyes and Pigments 94 (2012) 258e265
259
A
3 -
NO2
B
N
OH
N
SO3
N
N
O
O
O
3Na+
Cr
N
HO
O
SO3
NO2
N
SO3Na
O2N
C
A
N
N
B
A
N
B
A
N
N
B
N
O
O
O
O
O
O
O
O
O
O
O
Cr
N
Cr
N
Cr
N
O
A
A
B
N
N
B
B
N
A
α α−
,
α β−
,
form
β β−
, form
form
Fig. 1. Acid Black 194: A) one of the chemical structure of the three metal complex positional isomers. B) Chemical structure of the organic ligand in the mono sodic form. C) The
three forms of the positional isomers.
of the family called Mordant Black, characterized by the presence of
one azo group, one strong and two middle acidic functions in
appropriated positions that allow multiple coordination to the
metal ion and give to the complexes a multiple negative charge (at
least 3ꢀ) necessary to ensure a good solubility of the dye in water. In
Fig. 1B a chemical structure of the organic ligand, in the mono
the EU marketplace without the requisite data. For this area the
REACH compliance activities are complex and challenging owing to
the serious amount of work to provide deep toxicological, ecotox-
icological, chemical and physicalechemical properties which are
not often available. There are fears that some of these products
could be categorized as being very persistent and very bio-
accumulative and therefore registration can be more expensive
than expected without suitable safer replacements available. To
harmonize and provide consistent registration dossiers, in 2010
seven consortia for phase 1 substances and five for phase 2
substances were formed. The complex and sometimes huge mix of
ingredients within a given product exacerbates the difficulty of
providing information but development of scientific data to
increase understanding of the possible health and environmental
effects of dyes, contaminants, impurities, by products and residues
remain unavoidable. The starting point for this information flow is
a quantitative and accurate fingerprint distribution of organic
compounds present in the dye mixtures provided by organic
analysis and of the various techniques available, capillary electro-
phoresis can play a relevant role for its versatility, selectivity and
accuracy.
So, with the purpose of identifying a more practical analytical
method able to detect the largest possible number of compounds,
with high efficiency and reproducibility, the analysis of the dye
mixtures were handled by chromatographic and electromigration
approaches in accordance with the analytical protocols applied in
literature. After testing two methods HPLC and CE, under different
analytical conditions identified in the scientific data [20e22]
available and focussing on protocols, at the beginning, compatible
with MS detectors, no satisfactory results were achieved.
sodium salt form, is shown, whereas in Fig. 1C the three
a,a-, a,b-
and - forms of the three N- isomers ring A and B being
b
,b
a,b
differently substituted, are reported [19].
Considering the toxicity towards the environment of the metal
complex dyes and in particular that of C.I. Acid Black 194, that is at
least one order of magnitude higher than in the case of other dyes
(DL50 for fish of the order of 10 mg/L, as compared with Acid
Violet 90 with >100 mg/L), analytical protocols based on CE-MS
able to screen its presence in waste waters were proposed [20].
The same analytical method were applied with good results in
forensic analysis too [21] however, the protocols suggested were
focused on the separation of the complexes of the metal isomers
only.
In relation to the European legislation known as REACH
(Registration, Evaluation, Authorization of Chemical), the problem
has occurred to develop simple, fast and stable analytical methods
for the analysis of the metal complex dyes able to highlight the
main impurity present in the commercial products so to give, at
least, a “finger print” to the batches produced and marketed.
Within the European Union, 2000e2500 different dyes and organic
pigments are produced and marketed and an even wider number of
substances must be considered when their synthetic intermediates
are taken into account. These substances are subjected to REACH
registration requirements and can not be produced or sold within

260
R. Sebastiano et al. / Dyes and Pigments 94 (2012) 258e265
Then we directed our attention to Micellar Electrokinetic
An AcOEt: MeOH: AcOH ¼ 8:1:1 mixture was utilized as eluent. The
isolation of the three metal complex dyes and of a yellow impurity
present in the commercial sample, was performed by flash chro-
Chromatography [23e25] from which we obtained optimal results
since it was possible to separate more than twelve compounds with
high repeatability in about 8 min of analysis. The MECK mode was
also the best applied for the analysis of other two metal dyes of the
same family: the Acid Brown 432 and Acid Brown 434.
matography using direct-phase silica gel 35e70
mm, purchased
from Merck, and the same eluent described above. For 0.5 g of dye
mixture, a 5 cm diameter chromatographic column filled up to
20 cm with silica gel was utilized. The mobile phase flow was set to
2.5 cm/min and 20 mL fractions of eluent were collected.
2. Materials and methods
2.1. Chemicals and materials
2.3. HPLC analysis
All the aqueous solutions were prepared with SigmaeAldrich
(St. Louis e MO e USA) HPLC grade water (Chromasolv Plus). Iso-
propyl alcohol, hexafluoroisopropanol (HFIP), formic acid, acetic
acid, sodium tetraborate, sodium dodecyl sulphate (SDS), were
from SigmaeAldrich (St. Louis e MO e USA). Sodium hydroxide
1.0 M, CE commercial buffer solution at pH 7.4 (89 mM Tris, 332 mM
boric acid) and pH 8.3 (89 mM Tris, 89 mM Boric Acid) were from
Hewlett Packard GmbH (Waldbronn, Germany).
The commercial acid dyes Acid Brown 432, Acid Brown 434, Acid
Black 194 and its organic ligand, 3-hydroxy-4-[(E)e(2-
hydroxynaphthalen-1-yl)diazenyl]-7-nitronaphtalene-1-sulfonate
sodium salt, were supplied from Origo Sas, Milano, Italy.
The HPLC analyses were performed by a Merck-Hitachi instru-
ment, equipped with two pumps (L-6200A and L-6000 intelligent
Pumps), auto sampler AS 200A and UVeVIS detector L-4250. A
Hypersil BDS-C18, 5
m
m, 125 ꢁ 4 mm column by Merck was utilized
as column. The best analytical results were obtained with the
following conditions: column temperature 30 ꢂC, eluent flow
0.5 mL/min, wavelength 254 nm, injection 10
mL, programmed
gradient mode eluting as reported: time,%MeCN,%H2O ¼ 0,10,0;
5,10,0; 10,8,2; 15,8,2; 20,7,3.
2.4. Capillary electrophoresis
The main pigments, isolated by flash chromatography from
the dye Black 194, are the Cr(III) complexes Trisodium,bis[(E)-7-
nitro-3-oxido-4-((2-oxidonaphthalen-1-yl)diazenyl)naphtha-lene-
1-sulfonate] chromate(3-), in the forms reported in Fig. 1(A and C).
The main pigment present in the Acid Brown 432 dye is the Tri-
The analyses were performed with a HewlettePackard 3DCE
instrument, equipped with a UVeVis diode array detector. Fused
silica capillaries (I.D. 50 mm) were purchased from Composite Metal
Services LTD (UK). Typical capillary pre-treatment: a new silica
capillary (50 cm total length) is washed for 10 min with a 1 M
sodium hydroxide solution then, for 5 min water and 5 min with
the background electrolyte (BGE) solution. After which it is ready
for use. Typical preconditioning step (between analyses): flushing
for 1e2 min with the BGE solution. All the capillary flushings were
performed at high pressure (4.5 bar). Typical electrophoretic
conditions: voltage 25 or 29 kV, cassette temperature 20 ꢂC,
working wavelength 233 nm. A 1 mg/mL of aqueous mother solu-
tion of any sample was diluted 2e5 times before injecting.
Injection: diluted sample 30e50 mbar ꢁ 3 s followed by BGE
10 mbar ꢁ 5 s (specific conditions are reported in the legends).
sodium,
bis
[2-((E)-(4-hydroxy-2-oxido-3-((E)-(5-
sulfonatonaphthalen-1-yl)diazenyl)-phenyl)diazenyl)benzoate]
chromate(3-). The ma-inpigment present in theAcid Brown434 dye is
the (Trisodium bis [5-((E)-(4-((E)-(3,5-dinitro-2-oxidophenyl)dia-
zenyl)-2-hydroxy-3-oxidophenyl)diazenyl)-naphthalene-2-
sulfonate]ferrate(ꢀ) (see Fig. 7A and B).
2.2. Thin layer chromatography and flash chromatography
Thin layer chromatography (TLC) was performed by TLC pre-
coated glass plates of Silica Gel 60 F254, purchased from Merck.
-
SO3
-
SO3
N
N
O2N
O2N
X
O
HO
OH
OH
OH
OH
OH
O
H2O
N
N
H
O
+
-HX
N
N
NO2
- ArNHNH2
-
SO3
organic ligand
B
A
O
O
2
O
O
O
3
O
O
ox
- H+
4
1
5
H
6
9
12
O
O
7
11
8
10
Yellow impurity
Fig. 2. Possible synthetic pathway of the yellow impurity isolated from a commercial sample of the Acid Black 194 dye. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

R. Sebastiano et al. / Dyes and Pigments 94 (2012) 258e265
261
D)
mAU
mAU
8
mAU
12
A
C
B
10
10
8
6
8
6
4
6
4
2
4
2
2
0
0
0
-2
-2
6
8
2
3
6
7
8
min
min
min
Fig. 3. CZE of commercial Acid Black 194 dye at different BGE compositions and pH. A: BGE 89 mM Tris, 89 mM boric acid, pH 8.3; 33.5-cm long capillary, voltage ꢀ25 kV. B: BGE
25 mM sodium tetraborate, pH 9.3; 50-cm long capillary, voltage þ29 kV. C: BGE BGE 5 mM ammonium acetate pH 9e40% MeCN [20]; 33.5 cm long capillary, voltage þ20 kV.
Fig. 4. A) Full scale electropherogram of commercial Acid Black 194 dye by MECK mode. Peak n.1 ¼ yellow impurity; peaks n. 2, 3 and 5 ¼ metal complex positional isomers, peak
n.6 ¼ free organic ligand. BGE: 10 mM SDS in 25 mM sodium tetraborate, pH9.3. Operative conditions: 50-cm long capillary, voltage þ29 kV. B) Electropherogram magnification of
commercial Acid Black 194 dye. To note the large number of impurities separable and detectable by UVeVis CE MECK mode. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

262
R. Sebastiano et al. / Dyes and Pigments 94 (2012) 258e265
0.025
0.02
Peak n.
0.015
0.01
1
2
3
4
5
6
0.005
ΔMt/aMt
0
-0.005
-0.01
-0.015
Mt
Mt
Mt
(11-10)/aMt
Δ
Δ
Δ
(1-10)/aMt
(1-11)/aMt
Fig. 5. Analysis of the commercial Acid Black 194 dye by the MECK mode. Trend of the migration time differences among several injections with and without replacing the working
BGE: Mt(1e10)/aMt, Mt(1e11)/aMt, Mt) on the average migration time (aMt) among, respectively, the first and the tenth
Mt(11e10)/aMt ¼ migration time difference (
injections without working BGE replacement, the first and the eleventh injection (this last with BGE replacement), the eleventh and tenth injection.
D
D
D
D
2.5. 4-(naphthalen-2-yloxy)naphthalene-1,2-dione): 1H NMR and
MS-ESI characterization
3. Results and discussion
Normal phase TLC analysis of the dye Black 194, allowed the
separation of the three positional isomers as black spots with
Rf ¼ 0.17, 0.13, 0.09, respectively. Due to the strong polarity of the
metal complexes, the three compounds can be not separated as
clean spots but as lightly streaked stains. The same problem was
encountered during the flash chromatography separation of the
complexes but, due to an over-dimensioned chromatographic
column, it was possible to isolate the three isomers with very small
1H NMR spectra were recorded in DMSO-D6 at 300 K on
a Brucker Avance 400 MHz:
d
ppm 8.18(dd, J ¼ 7.39, 1.39, 1H, H9),
7.84(dd, 1H, H6), 7.56(d, J ¼ 7.3, 1H, H10), 7.74(dt, J ¼ 7.47, 1.23, 1H,
H4), 7.36 (M, 4H, H3,5,7,8), 7.30 (s, 1H, H12), 7.28 (d,d, J ¼ 7.14, 1.05, 1H,
H2), 6.88(dd, 7.74, 1.15, H11) 6.53 (s, 1H, H1).
MS-ESI spectra were recorded bya Bruker Esquire 3000 Plus: 301
(Mþ1, 23.1), 283(15.4), 273(100), 255(38.5), 254(92.3), 231(30.8).
Fig. 6. Analysis of the commercial Acid Black 194 dye by the MECK mode. Importance of the working BGE solutions replacement (inlet and outlet) between the runs on the
migration time reproducibility and resolution. A: first injection with fresh working BGE solutions; B: tenth injection with the same working BGE solutions; C: eleventh injection with
fresh working BGE solutions. BGE: 10 mM SDS in 25 mM sodium tetraborate, pH 9.3. Operative conditions: 50-cm long capillary, voltage þ29 kV.

R. Sebastiano et al. / Dyes and Pigments 94 (2012) 258e265
263
A
mAU
25
3-
O3S
N
N
OH
O
N
O
O
20
Na+
N
3
N
N
Cr
O
O
O
HO
N
15
10
5
N
SO3
EOF
0
1
2
3
4
6
min
5
7
8
9
B
mAU
5
3-
O2N
N
O3S
4
3
N
OH
N
O2N
O
O
O
N
Na+
3
N
Cr
N
N
O
NO2
HO
SO3
N
2
1
NO2
EOF
0
-1
2
4
6
8
10
12
14
16
min
Fig. 7. A: analysis of the commercial Acid Brown 432. B: analysis of Acid Brown 434 BGE: 10 mM SDS in 25 mM sodium tetraborate, pH 9.3. Operative conditions: 50-cm long
capillary, voltage þ29 kV.
reciprocal contamination, and confirm them by mass spectrometry
(MS-ESI) (data not shown). The TLC analysis of the mixture also
showed the presence of a yellow, low-polarity compound with
Rf ¼ 0.75, which was also isolated by flash chromatography and
identified by 1H NMR and MS as the 4-(naphthalen-2-yloxy)
naphthalene-1,2-dione (see Section 2.5). The presence of this
compound in the dye is connected to the typical synthesis of
mordant azo dyes performed at alkaline pH. In this case the
coupling process is between the6-nitro-1,2,4-acid and 2-naphthol,
as reported in Fig. 2. The tautomeric form B of the ligand [26], can
be hydrolyzed to the 1,2 naphthoquinone which can react with the
2-naphthol to give the yellow impurity isolated.
The classical chromatographic approach to the analysis of
commercial samples of the Acid Black 194 dye appeared quite
difficult. Direct and reversed-phase HPLC experiments were per-
formed with several eluents, in all cases, however, with poor
resolution among the three main compounds and a low sensitivity
on the detection of other impurities. The ability of these metal
derivatives to establish multiple interactions with the silica surface
was clearly confirmed also by preliminary experiments performed
via CZE: the classical, MS-compatible, background electrolyteat
acidic pH, based on HCOOH/HCOOꢀ or AcOH/AcOꢀ pairings, as
the typical neutral or basic running buffers resulted thoroughly
inappropriate. Also the use of hydro-organic buffers did not give
satisfactory results in particular in the detections of the dye
contaminants: in Fig. 3 some examples of electropherograms ob-
tained with these types of BGE are reported.
Excellent results were instead obtained by the Micellar Elec-
troKinetic Chromatography (MECK), based on the use of SDS in
tetraborate buffer. Under these conditions, optimal separations of
more than twelve compounds, present in the commercial sample,
were achieved in about 8 min of analysis. The three positional metal
complex isomers were fully separated, together with the impurities
described above, and the presence of free ligand was also detected.
Fig. 4 displays the electropherogram obtained by MECK mode in
a BGE composed by 10 mM sodium dodecyl sulphate (SDS) in
25 mM sodium tetraborate. For evaluating the robustness of the
method, three sets of eleven analyses of the same sample were
performed. The quantitative data obtained are reported in Table 1:
as evidenced, the % Relative Standard Deviation (%RDS) of the main
parameters is very low, which confirms the validity of the tech-
nique, applicable also for developing validated methods for
analyzing this type of mixtures.
As a last observation, it is interesting to note the relative
migration time sensitivity of the different compounds due to the
chemical composition modification of the working BGE (inlet and

264
R. Sebastiano et al. / Dyes and Pigments 94 (2012) 258e265
Table 1
%RDS of the main electrochromatographic parameters collected during the analysis of the commercial Acid Black 194 dye by MECK mode.
Peak n.
% RSD day by day (three days)
Mt (min.)
% RSD daily
Corrected
Plates
Resolution
Mt (min.)
Corrected
Plates
Resolution
area (mAU)
area (mAU)
1
2
3
4
5
6
Average
St. Dev.a
%RSD
Average
St. Dev.a
%RSD
Average
St. Dev.a
%RSD
Average
St. Dev.a
%RSD
Average
St. Dev.a
%RSD
Average
St. Dev.a
%RSD
4.965
0.030
0.61
5.539
0.037
0.68
6.135
0.029
0.47
6.434
0.048
0.74
6.646
0.028
0.42
7.965
0.044
0.55
0.086
0.002
2.10
0.283
0.006
2.02
0.410
0.008
2.02
0.032
0.001
3.24
0.177
0.004
2.28
0.027
0.001
3.06
242,731.342
16,643.019
6.86
9806.367
445.068
4.54
8784.241
212.658
2.42
242,362.926
26,757.382
11.04
33,585.407
2851.317
8.49
190,952.714
5702.605
2.99
3.632
0.099
2.73
4.351
0.091
2.08
2.444
0.063
2.57
1.907
0.141
7.42
2.094
0.299
14.28
4.702
0.033
0.69
Average
St. Dev.
%RSD
Average
St. Dev.
%RSD
Average
St. Dev.
%RSD
Average
St. Dev.
%RSD
Average
St. Dev.
%RSD
Average
St. Dev.
%RSD
4.917
0.031
0.63
5.471
0.038
0.69
6.078
0.031
0.51
6.354
0.047
0.73
6.588
0.026
0.39
7.880
0.046
0.58
0.083
0.002
2.14
0.278
0.006
2.02
0.394
0.009
2.16
0.031
0.001
3.42
0.171
0.004
2.35
0.027
0.001
3.37
249,452
15,912
6.38
10,070
445
4.42
9689
216
2.23
280,547
27,596
9.84
34,624
2213
6.39
196,719
6129
3.600
0.116
3.22
4.300
0.090
2.09
2.600
0.063
2.41
1.870
0.143
7.64
2.470
0.301
12.20
4.820
0.038
0.79
3.12
a
Average of the St. Dev. calculated on the three days analysis.
outlet vials) subjected to protracted electrolysis. In Fig. 5 the trend
of the migration time differences between several injections with
and without replacing of the working BGE is reported. The peaks n.
2 and n. 6 follow respectively the almost perfect V and L shape
expected; also the peaks 3 and 5 follow the expected trend but less
rigorously, while the peaks 1 and 4 move clearly away from it.
The analytical result of this behaviour is particularly relevant for
the peaks n. 4 and 5 (see Fig. 6), where the relative position of these
two peaks change significantly during ten analyses performed
without replenishing the working BGE, thus compromising their
resolution. The appropriated relative positions are promptly re-
established just by replacement of the two working solutions
with fresh running buffer.
Shigeru Terabe in 1987 [25], is still alive and well in Anno Domini
2011. Infact, as known, the main limitation that have reduced the
use of MECK in CE is its incompatibility with the MS detectors. But,
as shown in a very recent work of Barbula et al. [27], this disad-
vantage will be hopefully removed in the near future, so that the
immense potential of this technique in combination with a mass
spectrometer detector will be fully expressed.
Acknowledgements
The authors thank Itaca Nova S.r.l. and Origo Sas for support.
PGR is supported by a grant from PRIN-2008 (Rome, Italy).
Just for evaluating the possible generality of this analytical
method for the analysis of metal complex dyes, other two metal-
based commercial dyes, Acid Brown 432 and Acid Brown 434, were
analysed by MECK. The results are reported in the Fig. 7A and B.
Also here, the Acid Brown 432 displays a complex mixture of at
least eleven compounds, clearly visible by this technique. On the
contrary, the MECK analysis of the Acid Brown 434 exhibited
a single-peak electropherogram (Fig. 7B).
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