Subsidiary colors in D&C Red No. 34 and its lakes: Synthesis, structural characterization, and analysis by ultra-performance liquid chromatography

Article abstract of DOI:10.1016/j.dyepig.2012.05.001

D&C Red No. 34 is the calcium salt of 3-hydroxy-4-[(1-sulfo-2- naphthalenyl)azo]-2-naphthalenecarboxylic acid. Its lakes are insoluble pigments formed by precipitating the dye anion onto an insoluble substratum, typically rosin. Subsidiary colors are colored impurities that are positional isomers of the dye anion or have higher or lower numbers of substituent groups. D&C Red No. 34 and its lakes are color additives listed in the U.S. Code of Federal Regulations (CFR) for use in externally applied drugs and cosmetics. These color additives are required to be batch certified by the U.S. Food and Drug Administration (FDA) to ensure compliance with the CFR requirements, including a limit of not more than four percent subsidiary colors in the straight color. There is no specified limit for subsidiary colors in the lakes, but the impurities may be analyzed for compliance with good manufacturing practices. In this paper, the syntheses of seven D&C Red No. 34 subsidiary colors are reported. Various combinations of impurities in the two intermediate starting materials for the dye, 2-amino-1-naphthalenesulfonic acid and 3-hydroxy-2-naphthalenecarboxylic acid, were used to synthesize the new compounds, which were purified by recrystallization and fully characterized by elemental analysis, ultra-performance liquid chromatography (UPLC), nuclear magnetic resonance spectroscopy, infrared spectroscopy, and visible spectrophotometry. The new compounds were used as reference materials in UPLC analyses of test samples from 33 certified lots of D&C Red No. 34 and its lakes. FDA is currently using these new reference materials for batch certification of the color additives.

Full text of DOI:10.1016/j.dyepig.2012.05.001

Dyes and Pigments 95 (2012) 304e312
Contents lists available at SciVerse ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Subsidiary colors in D&C Red No. 34 and its lakes: Synthesis, structural
characterization, and analysis by ultra-performance liquid chromatography
,
a
b
c
c
Nebebech Belai a , Bhakti Petigara Harp , Eugene P. Mazzola , Yiu-fai Lam , Ehab Abdeldayem ,
*
Aaron Aziz c, Magdi M. Mossoba b, Julie N. Barrows a
a Office of Cosmetics and Colors, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD 20740, USA
b Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD 20740, USA
c Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20740, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
D&C Red No. 34 is the calcium salt of 3-hydroxy-4-[(1-sulfo-2-naphthalenyl)azo]-2-naphthalenecarboxylic
acid. Its lakes are insoluble pigments formed by precipitating the dye anion onto an insoluble
substratum, typically rosin. Subsidiary colors are colored impurities that are positional isomers of the dye
anion or have higher or lower numbers of substituent groups. D&C Red No. 34 and its lakes are color
additives listed in the U.S. Code of Federal Regulations (CFR) for use in externally applied drugs and
cosmetics. These color additives are required to be batch certified by the U.S. Food and Drug Adminis-
tration (FDA) to ensure compliance with the CFR requirements, including a limit of not more than four
percent subsidiary colors in the straight color. There is no specified limit for subsidiary colors in the lakes,
but the impurities may be analyzed for compliance with good manufacturing practices. In this paper, the
syntheses of seven D&C Red No. 34 subsidiary colors are reported. Various combinations of impurities in
the two intermediate starting materials for the dye, 2-amino-1-naphthalenesulfonic acid and 3-hydroxy-
2-naphthalenecarboxylic acid, were used to synthesize the new compounds, which were purified by
recrystallization and fully characterized by elemental analysis, ultra-performance liquid chromatography
(UPLC), nuclear magnetic resonance spectroscopy, infrared spectroscopy, and visible spectrophotometry.
The new compounds were used as reference materials in UPLC analyses of test samples from 33 certified
lots of D&C Red No. 34 and its lakes. FDA is currently using these new reference materials for batch
certification of the color additives.
Received 25 October 2011
Accepted 5 May 2012
Available online 16 May 2012
Keywords:
D&C Red No. 34
Color additive
Lakes
C.I. Pigment Red 63:1
Subsidiary colors
Azo dyes
Published by Elsevier Ltd.
1. Introduction
prepared by synthesizing the dye anion “in situ” during the
laking process [3,4].
D&C Red No. 34 is the calcium salt of 3-hydroxy-4-[(1-sulfo-2-
naphthalenyl)azo]-2-naphthalenecarboxylic acid (R34, Colour
Index (C.I.) 15,880:1, CAS No. 6417-83-0, C.I. Pigment Red 63:1). The
R34 dye anion is prepared by a two-step reaction: diazotization of
2-amino-1-naphthalenesulfonic acid followed by an azo coupling
reaction with 3-hydroxy-2-naphthalenecarboxylic acid, as shown
in Fig. 1 (1) [1,2]. R34 lakes are insoluble pigments formed by
extending the dye (“straight color”) onto an insoluble substratum
R34 and its lakes are color additives listed in the U.S. Code of
Federal Regulations (CFR) for use in externally applied drugs and
cosmetics [5,6]. The U.S. Food and Drug Administration (FDA)
routinely analyzes R34 and its lakes as part of its batch certifi-
cation program. FDA batch certification ensures that these color
additives comply with published specifications and other
requirements, including a limit of not more than four percent
subsidiary colors in the straight color [5]. There is no specified
limit for subsidiary colors in R34 lakes, but the impurities
may be analyzed for compliance with good manufacturing
practices [6].
with
a precipitating cation. Typically, rosin is used as the
substratum and calcium chloride is used as the precipitant. The R34
straight color exhibits limited solubility in water so its lakes are
In commercially prepared R34 straight colors and lakes,
unreactedintermediatestartingmaterials and subsidiarycolors may
be present as impurities [1]. The unreacted intermediates result
from incomplete diazotization of 2-amino-1-naphthalenesulfonic
acid or an excess of 3-hydroxy-2-naphthalenecarboxylic acid. In
* Corresponding author. Tel.: þ1 240 402 1116; fax: þ1 310 436 2961.
E-mail address: nebebech.belai@fda.hhs.gov (N. Belai).
0143-7208/$ e see front matter Published by Elsevier Ltd.
doi:10.1016/j.dyepig.2012.05.001

N. Belai et al. / Dyes and Pigments 95 (2012) 304e312
305
H+, NO2
-
I
Na2CO3, NaOH (for 1, 2, 4, 6-8)
K2CO3, KOH (for 3)
-
1 R1=SO3 , R5=COOH, R6=OH, R2=R3=R4=H
5 R5=COOH, R6=OH, R1=R2=R3=R4=H
-
-
2 R2=SO3 , R5=COOH, R6=OH, R1=R3=R4=H
6 R1=SO3 , R6=OH, R2=R3=R4=R5=H
-
-
3 R3=SO3 , R5=COOH, R6=OH, R1=R2=R4=H
7 R1=SO3 , R4=OH, R2=R3=R5=R6=H
-
4 R1=SO3 , R4=OH, R5=COOH, R2=R3=R6=H
II
-
8 R1=SO3 , R4=OH
Fig. 1. (I) The structures, azo coupling reaction scheme, and 1H and 13C NMR chemical shift numbering for D&C Red No. 34 (1) and seven subsidiary colors (2e7). (II) The structure
and chemical shift numbering for 8.
general, subsidiary colors are positional isomers of the R34 dye
anion or are related compounds containing greater or fewer
substituent groups. Subsidiary colors arise due to side reactions of
reactive impurities that may be present in either starting material.
For example, monosulfonated reactive impurities such as 7-amino-
1-naphthalenesulfonic acid and 6-amino-2-naphthalenesulfonic
acid are commonly present in small amounts in 2-amino-1-
naphthalenesulfonic acid due to sulfonation side reactions [7].
naphthalenecarboxylic acid (4), 3-hydroxy-4-[(2-naphthalenyl)
azo]-2-naphthalenecarboxylic acid (5), 2-[(2-hydroxy-1-
naphthalenyl)azo]-1-naphthalenesulfonic acid (6), 2-[(4-hydroxy-
1-napthalenyl)azo)]-1-naphthalenesulfonic acid (7), and 2-[(1-
hydroxy-2-naphthalenyl)azo]-1-naphthalenesulfonic acid (8).
Subsidiary color 6 (C.I. 15,630, CAS No. 1248-18-6) was formerly
certifiable as D&C Red No. 10 (the sodium salt) and D&C Red No. 11
(the calcium salt). The R34 subsidiary color structures are shown
Fig.1, and Table 1 lists the starting materials used in their syntheses.
In the past, FDA determined the subsidiary colors in R34 straight
colors by thin-layer chromatography (TLC). The TLC method could
take up to 8 h to complete and did not completely separate the
subsidiary colors from the main R34 color band. High-performance
liquid chromatography (HPLC) [9e14] and more recently ultra-
performance liquid chromatography (UPLC) [15] have been
successfully applied to the determination of impurities in color
additives and color additives in products. We recently reported
a UPLC method for the determination of the intermediates and
subsidiary colors in R34 and its lakes [16]. As part of the UPLC
method development, it was essential to obtain purified reference
materials for the subsidiary colors. Herein we report the synthesis,
isolation, and purification of seven R34 subsidiary colors as well as
the structural characterization of each compound by elemental
analysis, nuclear magnetic resonance (NMR) spectroscopy, infrared
Likewise, reactive impurities such as a- or b-naphthol are commonly
present in 3-hydroxy-2-naphthalenecarboxylic acid [8]. Subsidiary
colors in R34 can form by two different pathways involving these
reactive impurities. Either an impurity in 2-amino-1-
naphthalenesulfonic acid can undergo diazotization and coupling
with 3-hydroxy-2-naphthalenecarboxylic acid or an impurity in 3-
hydroxy-2-naphthalenecarboxylic acid can undergo coupling with
diazotized 2-amino-1-naphthalenesulfonic acid. The reactive
impurities in both of the starting materials also can undergo diaz-
otization and coupling reactions with each other. However, those
subsidiary colors are generally not observed in R34 samples and,
therefore, are not considered here.
The subsidiary colors reported in this paper are 3-hydroxy-
4-[(8-sulfo-2-naphthalenyl)azo]-2-naphthalenecarboxylic
acid
(2), 3-hydroxy-4-[(6-sulfo-2-naphthalenyl)azo]-2-naphthalene
carboxylic acid (3), 1-hydroxy-4-[(1-sulfo-2-naphthalenyl)azo]-2-
Table 1
D&C Red No. 34 subsidiary colors and impurities and intermediates used in their syntheses.
Compound
Impurity
Intermediate
3-hydroxy-4-[(8-sulfo-2- naphthalenyl)azo]-2- naphthalenecarboxylic acid (2)
3-hydroxy-4-[(6-sulfo-2- naphthalenyl)azo] -2- naphthalenecarboxylic acid (3)
7-amino-1-naphthalenesulfonic acid
6-amino-2-naphthalenesulfonic acid
3-hydroxy-2-naphthalenecarboxylic acid
3-hydroxy-2-naphthalenecarboxylic acid
1 -hydroxy-4-[(1 -sulfo-2- naphthalenyl)azo]-2- naphthalenecarboxylic acid (4) 1-hydroxy-2-naphthalenecarboxylic acid 2-amino-1-naphthalenesulfonic acid
3-hydroxy-4-[(2- naphthalenyl)azo]-2- naphthalenecarboxylic acid (5)
2-[(2-hydroxy-1- naphthalenyl)azo] -1- naphthalenesulfonic acid (6)
2-[(4-hydroxy-1- naphthalenyl)azo] -1- naphthalenesulfonic acid (7)
2-[(1-hydroxy-2- naphthalenyl)azo] -1- naphthalenesulfonic acid (8)
2-naphthylamine
2-naphthol
1-naphthol
3-hydroxy-2-naphthalenecarboxylic acid
2-amino-1-naphthalenesulfonic acid
2-amino-1-naphthalenesulfonic acid
1-hydroxy-2-naphthalenecarboxylic acid 2-amino-1-naphthalenesulfonic acid

306
N. Belai et al. / Dyes and Pigments 95 (2012) 304e312
(IR) spectroscopy, and visible spectrophotometry. We also report
results from UPLC determinations of subsidiary colors in 33 certi-
fied lots of R34 dyes using the new reference materials.
preparation of compound 7, from the reaction of diazotized 2-
amino-1-naphthalenesulfonic acid with 1-naphthol, similarly
resulted in a trace amount of compound 8 which is also produced as
a result of an ortho coupling. Similar types of side reactions have
been reported by Bourne et al. in the diazo coupling reaction of 1-
naphthol and 4-aminobenzene sulfonic acid (sulfanilic acid) [17].
Bourne reported that the reaction of diazotized sulfanilic acid with
1-naphthol proceeded by two parallel reactions that gave primarily
the monoazo dyes coupled at para- and ortho- positions and then,
by subsequent secondary coupling reactions of the monoazo dyes,
gave bisazo dyes. (Bisazo products were not detected by UPLC in
compounds 4 and 7).
2. Results and discussion
2.1. Synthesis of subsidiary colors
Seven subsidiary colors related to the R34 dye anion were
synthesized from starting materials representative of impurities in
either 2-amino-1-naphthalenesulfonic acid or 3-hydroxy-2-
naphthalenecarboxylic acid. The compounds were prepared as
sodium or potassium salts following the two-step sequence
described in Fig. 1, I. In general, the compounds were obtained via
diazotization of a naphthylamine derivative, and the resulting
Compound 5, was not synthesized due to the toxicity of the
starting material (2-naphthylamine) [18]. Instead, an archived
batch that was a historically synthesized FDA sample was recrys-
tallized and used as the reference material.
diazonium salt was coupled with
a corresponding naphthol
Compounds 1e8 were obtained in 45e97% yield and purified by
recrystallizing from hot water. Compounds 1, 2, 4, and 6e8 were
isolated as sodium salts, while compound 3 was isolated as
potassium salt because the sodium salt of 3 was very soluble and
formed an oily residue, making it difficult to crystallize. Compound
5 was isolated with neutral charge, and the absence of any cations
was confirmed by elemental analysis. The purity and chemical
identity of the compounds were established by elemental analysis
and UPLC. NMR and IR spectroscopy were used to confirm the
structural identity of each compound.
derivative: see Table 1. The sodium salt of R34 (R34-Na), compound
1, was also synthesized for NMR and visible spectrophotometric
characterization since R34 is insoluble in water.
Diazotization of 2-amino-1-naphthalenesulfonic acid followed
by coupling with 3-hydroxy-2-naphthalenecarboxylic acid gave
compound 1. Diazotization of 7-amino-1-naphthalenesulfonic acid
and 6-amino-2-naphthalenesulfonic acid followed by coupling
with 3-hydroxy-2-naphthoic acid gave compounds
respectively. Similarly, diazotization of
2
and 3,
2-amino-1-
naphthalenesulfonic acid followed by coupling with 2-naphthol
gave compound 6. All three subsidiary colors were obtained as
pure compounds.
2.2. NMR spectroscopy
Compound
4 was produced when diazotized 2-amino-1-
naphthalenesulfonic acid was reacted with 1-hydroxy-2-
naphthoic acid. However, during the preparation of compound 4,
a trace amount of compound 8 was also formed (<2% determined
by UPLC). In the 1H NMR spectrum of the crude mixture, only the
characteristic patterns of compound 4 were observed. However,
after isolating compound 8 as a pure compound, its structure was
elucidated by NMR. The results show that compound 8 is appar-
ently produced in a side reaction by ortho coupling (at the alpha
position relative to the hydroxyl group) with 1-hydroxy-2-
Compounds 1e8 were initially characterized by 1H and 13C NMR
spectroscopy. Two-dimensional NMR experiments including
correlation spectroscopy (COSY), nuclear Overhauser effect spec-
troscopy (NOESY), heteronuclear single quantum coherence
(HSQC), and heteronuclear multiple bond correlation (HMBC)
permitted complete assignments of the 1H and 13C NMR chemical
shifts. The results are given in Tables 2 and 3.
For the most part, well-resolved 1H multiplets and signal inte-
grals were observed. COSY spectra were used to identify hydrogen
spin systems, while NOESY spectra were used to establish the
naphthoic acid, resulting in
a decarboxylated product. The
Table 2
1H NMR assignments for compounds 1e8, chemical shifts
d
(ppm) obtained in DMSO-d6, multiplicity, and coupling constants (3JHH and 4JHH in Hz).
Position Chemical shifts, multiplicity (Coupling constants)
1
2
3
4
5
6
7
8
1
2
3
4
5
7.02, d (9.5)
7.12, d (9.5)
8.38, s
7.77, d (10.2)
6.77, d (10.2)
6.63, d (9.7)
7.82, d (9.7)
8.68, s
8.63, s
8.66, s
8.49, s
7.90, dd
(7.5,1.2)
7.48, td
(7.5, 1.0)
7.73, ddd
(7.7, 7.5, 1.2)
8.43, dd
(7.7, 1.0)
7.95, dd (7.5, 1.2) 7.98, dd (7.7, 1.0) 8.33, dd (7.3, 1.3) 7.90, dd (7.9, 1.4) 7.65, dd (7.3, 1.2) 8.53, dd (7.8, 1.0)
8.33, dd (7.4, 1.0)
6
7
8
7.52, td (7.5, 1.0) 7.54, td (7.7, 1.0) 7.47, td (7.3, 1.2) 7.32, ddd
(7.9, 6.9, 1.1)
7.73, td (7.5, 1.2) 7.77, td (7.7, 1.0) 7.63, td (7.3, 1.3) 7.55, ddd
(8.3, 6.9, 1.4)
8.56, dd (7.5, 1.0) 8.61, dd (7.7, 1.0) 8.91, dd (7.3, 1.2) 8.80, dd (8.3, 1.1) 8.42, dd (7.3, 1.0) 8.05, dd (8.0, 1.4)
7.41, td (7.3, 1.0) 7.76, ddd (7.8, 7.5, 1.4) 7.50, td (7.4, 1.0)
7.57, td (7.3, 1.2) 7.56, ddd (8.0, 7.5, 1.0) 7.70, td (7.4, 1.0)
7.64, dd (7.4, 1.0)
9.10, dd (7.3, 1.0)
20
30
8.61, d (1.4)
8.21, d (1.4)
8.17, d (9.0)
8.47, d (1.7)
9.06, dd
9.31, dd (7.4, 1.5) 8.15, dd (6.8, 2.6) 9.10, dd (7.4, 1.0) 8.88, dd (8.6, 1.0)
(8.6, 1.2)
7.57, ddd
(8.6, 6.8, 1.6)
7.51, ddd
(7.7, 6.8, 1.2)
7.88, dd
40
50
60
8.25, dd (7.7, 0.6) 8.16, dd (9.0, 1.8) 7.53, ddd
(7.4, 6.8, 1.6)
7.49, ddd
7.60, ddd
(6.8, 6.6, 2.7)
7.53, ddd
(7.4, 6.8, 1.5)
7.51, ddd (8.6, 7.5, 1.5) 7.54, ddd
(7.4, 7.3, 1.0)
7.39, ddd (7.5, 7.3, 1.0) 7.46, td (7.4, 1.0)
7.71, t (7.7)
7.59, td (6.6, 2.6) 7.46, ddd
(7.0, 6.8, 1.0)
8.00, dd (6.6, 2.7) 7.87, dd (7.0, 1.5) 7.84, dd (7.3, 1.5)
(7, 6.8, 1.5)
7.97, dd (7.7, 0.6) 8.41, d (1.8)
7.89, dd (7, 1.6)
7.86, dd (7.4, 1.0)
(7.7, 1.6)
8.05, d (9.2)
8.40, d (9.2)
70
80
8.05, d (8.5)
8.02, d (8.5)
7.93, d (8.6)
8.06, d (8.8)
8.07, dd (8.8, 1.7) 8.41, d (8.8)
8.03, d (8.8)
7.99, d (9.1)
8.24, d (9.1)
7.99, d (9.2)
8.30, d (9.2)
7.88, dd (8.5, 1.4) 7.79, dd (8.5, 1.4) 7.33, d (8.6)

N. Belai et al. / Dyes and Pigments 95 (2012) 304e312
307
Table 3
functional group. R34 has three functional groups, eOH at C2,
eCOOꢀ at C3, and eSOꢀ3 at C20. Compounds 2e4 contain all three
functional groups in different positions relative to R34 and, there-
fore, are isomers of R34. Compounds 5e8 each contain two of the
three functional groups. All seven subsidiary colors contain an eOH
group, compounds 2, 3, 5, and 6 at C2 and compounds 4, 7, and 8 at
C4. Compounds 2e4 contain a eCOOꢀ group at C3 and an eSOꢀ3
group at C30, C50, and C20, respectively. Compound 5 is missing an
eSOꢀ3 group and contains a eCOOꢀ group at C3. Compounds 6, 7
and 8 are missing the eCOOꢀ group and contain eSOꢀ3 groups at
C20. In compounds 1e7, the azo group connects C1 and C10, whereas
in compound 8 the azo group connects C3 and C10.
13C NMR assignments for compounds 1e8 and their chemical shifts
d
(ppm)
obtained in DMSO-d6.
Position Chemical shifts
1
2
3
4
5
6
7
8
1
2
3
4
4a
5
6
7
8
130.07 130.68 129.57 135.84 131.80 129.90 135.60 121.60
165.80 165.60 165.56 118.62 157.72 177.13 124.96 129.17
123.66 124.85 124.66 109.81 123.37 126.94 128.48 130.90
148.17 145.31 145.34 170.52 134.54 141.53 183.84 176.60
126.23 126.20 126.06 127.05 125.33 128.02 129.57 132.94
131.87 131.01 130.98 124.05 129.58 129.08 122.91 127.04
127.32 126.88 126.62 124.17 123.03 126.37 132.40 126.66
132.73 131.84 131.76 128.55 129.15 129.32 127.80 133.16
122.08 121.67 121.61 122.88 122.44 121.82 125.38 127.76
135.87 134.28 134.50 134.15 130.86 133.94 131.89 137.00
175.58 171.47 171.60 171.58 170.27
136.57 146.97 146.44 149.05 151.53 137.33 136.75 136.59
131.03 116.54 124.30 136.68 125.40 129.67 129.95 131.28
130.50 123.43 131.69 130.67 133.54 130.62 130.67 130.57
128.73 138.56 130.78 128.73 129.21 128.46 127.26 128.36
127.20 114.34 116.93 125.76 126.95 126.46 126.79 126.52
126.54 127.19 141.00 125.28 127.17 125.19 124.18 125.18
128.42 128.11 117.53 127.63 127.96 127.84 128.19 127.86
132.41 133.67 133.07 133.11 133.76 131.35 124.56 129.06
132.16 128.84 127.98 129.90 129.15 131.15 131.67 131.09
115.98 124.97 125.40 118.17 117.17 115.85 114.42 115.40
8a
C]O
10
2.3. IR spectroscopy
20
Infrared spectroscopy is a widely used technique for the deter-
mination of molecular structure, particularly functional groups, and
for the identification of compounds. IR spectra for R34-Na (1) and
the seven subsidiary colors 2e8 were obtained and compared. The
various characteristic frequencies were used to evaluate the extent
of frequency shifts that might have occurred due to changes in the
local environment of a functional group; see Table 4. The phenolic
hydroxyl group gave rise to a broad and prominent band covering
the region 3600e3200 cmꢀ1, which appeared to be hydrogen
bonded. The aromatic ¼ CH stretch vibrations were observed near
3100e3030 cmꢀ1. The IR spectrum in the range from 1700 to
700 cmꢀ1 was relatively complex; the analytically useful group
vibrations were intermixed with those characteristic of aromatic
rings. The C]C stretch vibrations of the aromatic rings were
20a
30
40
50
60
6’a
70
80
spatial proximities of adjacent spin systems. HSQC spectra identi-
fied 1H and 13C spin pairs, and HMBC spectra permitted assignment
of all of the quaternary carbons. In this manner, all of the 1H and 13
C
chemical shifts could be assigned unambiguously, with the sole
exception of the primed-ring carbon and hydrogen atoms of
compound 3. This is due to the pseudo-symmetry of the primed-
ring hydrogen atoms (70, 80, 20 and 30, 40, 60; each 3-spin system
has outer hydrogen atoms arrayed ortho and meta to the central
hydrogen) and because the chemical shifts of the azo- and
sulfonate-bearing carbon atoms are only 5.44 ppm apart. The ACD/
NMR Predictor program (v.12.00 by Advanced Chemistry Devel-
opment, Inc) suggested 13C chemical shifts for the sulfonate- and
azo-carbons of 143.82 and 149.59 ppm, respectively (5.77 apart)
[19]. Thus, the observed shifts of 141.00 and 146.44 ppm were
assigned to C-50 and C-10, respectively.
observed at 1625e1600 cmꢀ1
,
1598e1500 cmꢀ1
,
and
1490e1420 cmꢀ1. The aromatic ring ¼ CeH in-plane bending and
out-of-plane bending vibrations were observed in the regions
1200e1100 cmꢀ1 and 905e870 cmꢀ1, respectively. The antisym-
metric and symmetric stretch bands for the compounds with
carboxylate salts 1, 2, 4 and 5 were located at 1580e1550 cmꢀ1 and
1400e1390 cmꢀ1, respectively. In the IR spectrum observed for
compound 3, the conjugation of C]O in COOH with the aromatic
ring was also observed at 1721 cmꢀ1. For compounds 1e4 and 6e8,
the sulfonic acid salts, SOꢀ3 stretching vibrations were observed in
the range of 1260e1255 cmꢀ1. The naphthalene rings substitution
patterns could also be examined. The vibration bands for four
adjacent hydrogen and two adjacent hydrogen atoms were
observed at 790e700 cmꢀ1 and 860e800 cmꢀ1, respectively. The
observed spectra were consistent with compounds 1, 4, and 5e8
The 1H, 13C, and two-dimensional NMR characterization
confirmed the accuracy of the predicted structures for all of the
compounds. The NMR results show that compounds 2e8 are either
positional isomers of the R34 dye anion or are missing one
Table 4
IR frequencies for compounds 1e8.a
Compounds eOH
Aromatic O¼C-O- Aromatic ring C]C
ring ¼ CH
ReSO3- salt Aromatic ring in-plane Aromatic ring Two adjacent
H bending out-of-plane H in aromatic
H bending ring
Four adjacent
H in aromatic
ring
1
2
3
4
5
6
7
3455m
1568sh 1622m 1603m 1473s 1448s 1422m
1555m
1564s 1660sh 1618m 1598m 1492m 1450m 1258m
1259m
1142m 1056m 1011m 878w
868w 834w 811w 774w 751w
3481m 3058w
3426m
3439m 3055w
1199s 1124m 1050m 883w
1003m
869w 831m
813w
752m 737w
756w 743w
1555s
1721m 1613m 1596m 1495s 1452m 1433m 1255m
1556s
1576s 1622s 1603s 1576s 1528m 1502vs
1490sh 1444m 1426m
1555s 1613m 1593m 1496s 1451sh
1397m 1442m 1422m
1192s 1123m 1035s
1016m
895w 876w
3441m 3066w
3439m 3059w
1258m
1175m 1161m 1072m 876w
1046m 1028m
1162m 1146m 1014m 904w 875w
824w 814w 798w 761w 751sh
862w 814w 762sh 752w
3557m 3097w
3493m 3044w
3439m 3064w
1473s 1448m 1424m
1255m
1260m
1140m 1059m
871w
852w 812w 786w 775w
1620s 1606m 1597s 1579m 1502s
1488m 1477m 1442m 1412m
1186m 1168m 1161m 880w
1126m
852w 838w 812m 757m 748m
1057m 1047m 1018m
8
3447m 3051w
1480s 1464sh 1426m
1249m
1054m
879w
818w 792w
772w 747w
a
IR band intensity: s, strong; m, medium; w, weak; sh, shoulder.

308
N. Belai et al. / Dyes and Pigments 95 (2012) 304e312
1.2
1
0.8
0.6
0.4
0.2
0
400
450
500
550
600
650
700
Wavelenght (nm)
Fig. 2. Visible absorption spectra of observed for compounds 1e8 in DMSO.
having two sets of adjacent hydrogen atoms, in both primed and
unprimed naphthalene rings, and compounds 2 and 3 having only
one set of four adjacent hydrogen atoms in the unprimed naph-
thalene ring. All of these results are consistent with those obtained
by NMR.
that were certified by FDA between 2005 and 2011. Sample 33 was
from a lot produced by a domestic manufacturer that was used for
toxicological testing in support of the color additive listing (the
toxicology test lot). The results of these analyses are summarized in
Table 5. Samples 4, 22, 29, 30, 31, and 33 are straight colors and
were found to contain relatively higher amounts (0.598e2.97%) of
the subsidiary colors compared to the R34 lakes, for which the
subsidiary colors fell in the range 0.0805e1.97%. Compounds 3 and
6 were the most commonly found subsidiary colors and all samples
contained both of those compounds except samples 2, 10, and 31.
The three exceptions contained only one subsidiary color; sample 2
contained compound 3 while samples 2 and 31 each contained
compound 6. In addition, eleven of the samples contained
compound 2. Within these eleven, samples 27 and 32 also con-
tained compounds 7 and 5, respectively. Compound 4 was not
found in any of the 33 samples. Compound 8 is a side reaction
impurity in compounds 4 and 7 and as expected also was not found
in any of the samples. Results for total subsidiary colors determined
by UPLC in the straight colors are consistently lower than the
results previously obtained by TLC (the lakes were not analyzed by
TLC). The higher TLC results are attributed to the incomplete
separation of the individual subsidiary colors from the main R34
color band and, therefore, we consider the UPLC results to be more
accurate.
2.4. Visible spectrophotometry
The visible absorption spectra of compounds 1e8 in DMSO
(0.02 mg/mL) are shown in Fig. 2 and selected spectral data are
summarized in Table 5. The absorption spectra exhibit significant
differences in absorptivity values and absorption maxima (lmax
)
due to the differences in functional group substitution. Compounds
1e3 have a reddish color, compounds 4 and 5 have a yellowish
color, and compounds 6e8 have an orange color. Compounds 1e3
each have a lmax at 520e535 nm, compounds 6e8 each have
a
lmax at 485e510 nm, and compounds 4 and 5 each have a lmax at
420e430 nm. The absorptivity values for compounds 6, 7, and 8 are
relatively highest, followed by those for compounds 1e4.
Compound 5 has the lowest absorptivity value (Table 6).
2.5. UPLC analyses
Survey UPLC analyses were conducted of samples from 33
certified lots of R34 straight colors and lakes using the seven
subsidiary colors as reference materials. Thirty-two samples were
from lots produced by five foreign and domestic manufacturers
3. Experimental
3.1. Materials
Table 5
All chemicals were used as purchased without further purifi-
cation. 1-Hydroxy-2-naphthalenecarboxylic acid, 3-hydroxy-2-
naphthalenecarboxylic acid, and 2-amino-1-naphthalenesulfonic
acid were purchased from Aldrich Chemical Co. 2-
Naphthol, 6-amino-2-naphthalenesufonic acid, and 7-amino-1-
naphthalenesulfonic acid were purchased from Fluka, TCI Amer-
ica, and 3B Scientific Corporation, respectively. 1-Naphthol was
purchased from TCI America. Water was purchased from Thermo-
Fisher Scientific and methanol was purchased from J.T. Baker.
Dimethyl sulfoxide (DMSO) and DMSO-d6 were purchased from
Thermo-Fisher Scientific and Isotec Inc., respectively. All solvents
were HPLC grade. Samples of the D&C Red No. 34 lots analyzed in
Absorption maxima (lmax) and absorptivity values ( 3 ) for compounds 1e8 obtained
in DMSO.
D&C Red No. 34 and
subsidiary colors
lmax (nm)
3
(L/mgꢁcm)
1
2
3
4
5
6
7
8
523
533
533
427
430
493
495
504
0.0319
0.0345
0.0420
0.0355
0.0240
0.0618
0.0957
0.0696

N. Belai et al. / Dyes and Pigments 95 (2012) 304e312
309
Table 6
UPLC determinations of subsidiary colors in certified lots of D&C Red No. 34 straight colors and lakes using compounds 2e8 as reference materials.
Sample number
Manufacturer
Color additivea
Total subsidiary
colors found (%)b
2
3
5
6
7
1
2
3
4
5
6
7
8
A
A
B
C
A
A
B
B
B
B
F
B
B
A
A
A
A
A
A
B
B
C
B
B
B
B
A
B
C
D
E
R34L
R34L
R34L
R34
0.445
1.40
0.437
2.43
1.35
1.47
0.692
0.568
0.343
0.289
1.33
0.345
0.484
0.803
1.58
1.54
1.77
1.77
1.80
0.980
1.53
2.97
0.443
1.40
0.435
2.04
1.28
1.40
0.382
0.255
0.0196
0.00201
0.00198
0.392
0.0681
0.0636
0.310
0.312
0.324
0.289
0.0427
0.318
0.0124
0.0388
0.0271
0.0542
0.0836
0.0898
0.0886
0.902
1.47
0.971
0.437
0.449
R34L
R34L
R34L
R34L
R34L
R34L
R34L
R34L
R34L
R34L
R34L
R34L
R34L
R34L
R34L
R34L
R34L
R34
R34L
R34L
R34L
R34L
R34L
R34L
R34
R34
R34
R34L
R34
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
1.29
0.001710
0.0253
0.360
0.764
1.55
1.48
1.69
1.68
1.71
0.0100
0.0674
0.0552
1.89
0.0582
0.0537
0.0537
0.369
1.26
0.110
0.105
0.119
0.0963
0.600
0.622
0.678
0.687
1.44
0.643
0.598
2.40
0.528
0.318
0.00777
0.531
0.377
0.411
0.0805
0.492
0.143
0.0238
0.0795
0.0514
0.0673
0.0327
0.169
1.92
0.0805
0.796
2.57
G
0.0738
0.0265
1.73
0.204
Tc
0.842
a
Lots certified during 2005e2011, R34 - straight colors and R34L e R34 lakes (67e81% straight color).
All values are averages of duplicate analyses; note that compounds 4 and 8 were not found in any lots.
Toxicology test lot.
b
c
this study were obtained from FDA’s Office of Cosmetics and Colors.
Galbraith Laboratories, Inc., performed the elemental analyses of
compounds 1e8.
bell weighting. Phase-sensitive NOESY NMR spectra were deter-
mined using the same spectral windows and data points as for
COSY. 1H pulse widths of 11.5 s (90ꢂ) and 2.5-s relaxation delay
m
times were used to acquire 256 incremented proton NMR spectra of
28 scans each. Mixing times of 0.6 s were employed. Free-induction
decays were processed as 1024 ꢃ 1024 matrices with appropriate
zero filling in t1 and squared sine bell weighting.
3.2. Physical measurements
NMR solutions were prepared by dissolving 18e20 mg of each
purified compound in 1 mL of DMSO-d6. 1H, 13C, DEPT, COSY, NOESY,
and HSQC spectra were recorded on a Bruker AV-400 spectrometer
and HMBC spectra were recorded on a Bruker AV-600 spectrom-
HSQC NMR spectra were obtained with average spectral widths
of 24,000 and 4000 Hz in the carbon and proton dimensions,
respectively, and with 2048 data points in the F2 dimension; 256
incremented 1H spectra of 24 scans each were acquired by using
eter. Peak positions of the residual CD2H signal (
13CD3 signal (
39.51) were used as secondary references relative to
internal (CH3)4Si at 0 ppm. Chemical shifts ( ) and coupling
d 2.50) and the
d
11.5-m
s (90ꢂ) 1H pulse widths and 2.2-s relaxation delay times. Free-
d
induction decays in both dimensions were processed as
2048 ꢃ 1024 matrices with appropriate zero filling in t1 and
squared sine bell weighting. The value 1J(CH) ¼ 145 Hz was used for
constants (J) are reported in parts per million (ppm) and hertz (Hz),
respectively. Experiments were performed using standard Bruker
software.
calculating the delay D, which is equal to 3.45 ms.
1H NMR spectra were obtained with average spectral widths of
18 ppm; 32,768 experimental points (zero-filled to 65,536); 30ꢂ
pulses; 2.28-s acquisition times; and 1.5-s relaxation delay times.
13C and DEPT NMR spectra were acquired with average spectral
widths of 240 ppm; 65,536 experimental points; 30ꢂ (13C) and 135ꢂ
(final DEPT) pulses; 1.37-s acquisition times; and 2-s relaxation
delay times.
HMBC NMR spectra were recorded with average spectral widths
of 14,000 and 2100 Hz in the carbon and proton dimensions,
respectively, and with 2048 data points in the 1H dimension; 256
incremented 1H spectra of 40 scans each were acquired by using
10.3-m
s (90ꢂ) 1H pulse widths and 2.4-s relaxation delay times.
Free-induction decays in both dimensions were processed as
2048 ꢃ 1024 matrices with appropriate zero filling in t1 and sine
bell weighting. The value nJ(CH) ¼ 9 Hz was used for calculating the
delay DLR, which is equal to ca. 55 ms, and 1J(CH) ¼ 155 Hz was used
COSY NMR spectra were determined with average spectral
widths of 4000 Hz in each domain and with 2048 data points in the
F2 dimension. 1H pulse widths of 11.5
m
s (90ꢂ) and 1.5-s relaxation
for D in the J-filter.
delay times were used to acquire 256 incremented proton NMR
spectra of 8 scans each. Free-induction decays were processed as
1024 ꢃ 1024 matrices with appropriate zero filling in t1 and sine
Fourier transform infrared (FT-IR) spectroscopic measurements
were carried out on an Agilent (formerly Varian) FTS 7000e IR
spectrometer operating with Resolution Pro 4.0 software in the

310
N. Belai et al. / Dyes and Pigments 95 (2012) 304e312
transmission mode. The optical bench included a Michelson inter-
ferometer with an air bearing moving mirror, a potassium bromide
substrate beam splitter, and a linearized mercury cadmium tellu-
ride (MCT) detector operating at liquid nitrogen temperature. The
spectrometer performance met the following criteria: In the
absence of a test sample, a 3 min data collection at 4 cmꢀ1 reso-
lution yielded between 2200 and 2000 cmꢀ1 a peak-to-peak noise
level <0.0005 absorbance unit (AU) for absorption spectra. Test
samples (w2 mg) were measured as potassium bromide pellets
(w200 mg). Test sample single-beam spectra were ratioed against
that of an open beam (air). FTIR spectra were collected over the
wavenumber range of 4000 cmꢀ1e700 cmꢀ1 at a resolution of
4 cmꢀ1. To enhance the signal-to-noise ratio, 256 scans were co-
added and signal averaged.
3.3.2. 3-Hydroxy-4-[(8-sulfo-2-naphthalenyl)azo]-2-
naphthalenecarboxylic acid (2)
7-Amino-1-naphthalenesulfonic acid (0.017 mol) was dissolved
in 50 mL of water by adding 3 mL of 30% NaOH solution.
Concentrated HCl (30 mL) was added to this solution, which was
then cooled to w0 ꢂC. An aqueous solution of NaNO2 (10 mL,
0.019 mol) was added dropwise. The mixture was vigorously
stirred at w0 ꢂC for 30 min and checked for excess HNO2 (I2-starch
test). An aqueous solution of 10% sulfamic acid (3 mL) was added
to eliminate excess HNO2. The light brown diazonium salt was
filtered and repeatedly washed with ice water and the wet
precipitate was immediately used in the following coupling reac-
tion. 3-Hydroxy-2-naphthalenecarboxylic acid (0.017 mol) and
sodium carbonate (0.028 mol) were added to a 150 mL solution of
2:1 water:ethanol (v/v) and cooled to w0 ꢂC. The wet diazonium
salt was then added in small portions with vigorous stirring. A red
solution formed followed by precipitation of the dye anion. The
product was filtered, dried, and recrystallized twice from hot
water.
Visible absorption spectra were obtained with a double beam
PerkinElmer Lambda 25 UVevisible spectrophotometer over
a wavelength range 300e750 nm. The spectra were recorded in
DMSO at room temperature (22 ꢂC). The lmax values are those
selected by the spectrophotometer software.
UPLC analyses were conducted using the method described
previously.[16] The subsidiary color peaks in the chromatograms
were determined at 485 nm and identified from the retention
times and photodiode array spectra. The subsidiary color(s)
present in the samples were quantified by using a calibration
curve generated by analyzing separate standard solutions spiked
with the subsidiary color reference materials (2e8) prepared in
the absence of the R34 matrix. The calibration solutions contained
0e2.01% by weight of each subsidiary color and the instrument
response was linear over this range. The UPLC system consisted
of an Acquity Separation Module, a photodiode array detector
(monitored at 485 nm for the subsidiary colors), an Aquity UPLC
Yield: 6.84 g (85%). Anal Calc. for Na2C21H12O6N2S: C 54.08, H
2.59, N 6.01, S 6.88. Found: 54.01, H 2.45, N 5.98, S 6.88, Na 9.54.
lmax
(
) in DMSO: 533 nm (0.0345 L mgꢀ1 cmꢀ1).
3
3.3.3. 3-Hydroxy-4-[(6-sulfo-2-naphthalenyl)azo]-2-
naphthalenecarboxylic acid (3)
6-Amino-2-naphthalenesulfonic acid (0.025 mol) was dissolved
in 50 mL of water by adding 2.16 g of KOH. Concentrated HCl
(20 mL) was added to this solution, which was then cooled to
w0 ꢂC. An aqueous solution of NaNO2 (10 mL, 0.029 mol) was added
dropwise. The mixture was vigorously stirred at w0 ꢂC for 30 min
and checked for excess HNO2 (I2-starch test). An aqueous solution
of 10% sulfamic acid (6 mL) was added to eliminate excess HNO2.
The light brown diazonium salt was filtered and repeatedly washed
with ice water and the wet precipitate was immediately used in the
following coupling reaction. 3-Hydroxy-2-naphthalenecarboxylic
acid (0.024 mol) and potassium carbonate (0.035 mol) were
added to a 150 mL solution of 2:1 water:ethanol (v/v) and cooled to
w0 ꢂC. The wet diazonium salt was then added in small portions
with vigorous stirring. A red solution formed immediately followed
by precipitation of the dye anion. The product was filtered, dried,
and recrystallized twice from hot water.
BEH C18 column (50 ꢃ 2.1 mm id, 1.7
mm particle size), a guard
column, and a column heater set at 25 ꢂC (all from Waters Corp.,
Milton, MA). Eluant A was 20 mM aqueous ammonium acetate
and eluant B was acetonitrile. The gradient consisted of linear
segments of 0e25% B in 2.5 min, hold at 25% B for 5 min, 25%e
35% B in 1.67 min, and hold at 35% B for 5 min. The column was
re-equilibrated with 0% B for 5 min. The injection volume was
1 mL. The flow rate was 0.25 mL/min. The apparatus was controlled
and the data were collected and analyzed using Empower
2 software.
Yield: 10.0
g
(87%). UV lmax
(
3 )
in DMSO: 533 nm
3.3. Synthesis and characterization of compounds 1e8
(0.0420 L mgꢀ1 cmꢀ1).
Anal Calc for KC21H13O6N2S: C 52.82, H 2.95, N 5.87, S 6.72, K
8.19. Found: C 53.10, H 2.87, N 5.98, S 6.60, K 8.01.
3.3.1. 3-Hydroxy-4-[(1-sulfo-2-naphthalenyl)azo]-2-
naphthalenecarboxylic acid (1)
2-Amino-1-naphthalenesulfonic acid (0.025 mol) was dissolved
in 50 mL of water by adding 3 mL of 30% NaOH solution. Concen-
trated HCl (30 mL) was added to this solution, which was then
cooled to w0 ꢂC. An aqueous solution of NaNO2 (10 mL, 0.033 mol)
was added dropwise. The mixture was vigorously stirred at w0 ꢂC
for 30 min and checked for excess HNO2 (I2-starch test). An aqueous
solution of 10% sulfamic acid (4 mL) was added to eliminate excess
HNO2. The light brown diazonium salt was filtered and repeatedly
washed with ice water and the wet precipitate was immediately
used in the following coupling reaction. 3-Hydroxy-2-
naphthalenecarboxylic acid (0.025 mol) and sodium carbonate
(0.027 mol) were added to a 150 mL solution of 2:1 water:ethanol
(v/v) and cooled to w0 ꢂC. The wet diazonium salt was then added
in small portions with vigorous stirring. A red solution formed
followed by precipitation of the dye. The product was filtered,
dried, and recrystallized twice from hot water.
3.3.4. 1-Hydroxy-4-[(1-sulfo-2-naphthalenyl)azo]-2-
naphthalenecarboxylic acid (4)
2-Amino-1-naphthalenesulfonic acid (0.050) was dissolved in
100 mL of water by adding 6 mL of 30% NaOH solution. Concen-
trated HCl (30 mL) was added to this solution, which was then
cooled to w0 ꢂC. An aqueous solution of NaNO2 (10 mL, 0.061 mol)
was added dropwise. The mixture was vigorously stirred at w0 ꢂC
for 30 min and checked for excess HNO2 (I2-starch test). An
aqueous solution of 10% sulfamic acid (8 mL) was added to elim-
inate excess HNO2. The yellow diazonium salt was filtered and
repeatedly washed with ice water and the wet precipitate was
immediately used in the following coupling reaction. 1-Hydroxy-
2-naphthalenecarboxylic acid (0.050 mol) and sodium carbonate
(0.060 mol) were added to a 500 mL solution of 4:1 water:ethanol
(v/v) and cooled to w0 ꢂC. The wet diazonium salt was then added
in small portions with vigorous stirring. A red solution formed
immediately followed by precipitation of the dye anion. The
product was filtered, dried, and recrystallized twice from hot
water.
Yield: 10.41 g (89%). Anal. Calc. for Na2C21H12O6N2S$H2O: C 52.1,
H 2.91, N 5.78, S 6.62. Found: C 53.5, H 2.81, N 6.02, S 6.73. lmax ( ) in
3
DMSO: 523 nm (0.0319 L mgꢀ1 cmꢀ1).

N. Belai et al. / Dyes and Pigments 95 (2012) 304e312
311
Yield: 21.6
g
(97%). UV lmax
(
3
)
in DMSO: 427 nm
3.3.8. 2-[(1-hydroxy-2-naphthalenyl)azo]-1-naphthalenesulfonic
acid (8)
The compound was serendipitously isolated as
(0.0355 L mgꢀ1 cmꢀ1).
Anal Calc. for Na2C21H12O6N2S: C 54.08, H 2.59, N 6.01, S 6.88.
Found: C 51.37, H 2.76, N 5.78, S 6.85.
a
pure
compound from the synthesis of compound 4. 2-Amino-1-
naphthalenesulfonic acid (0.029 mol) was added in 50 mL of
water. Concentrated HCl (6.5 mL) was added to this mixture, which
was cooled to w0 ꢂC. An aqueous solution of NaNO2 (10 mL,
0.029 mol) was added dropwise. The mixture was vigorously stir-
red at w0 ꢂC for 30 min and checked for excess HNO2 (I2-starch
test). An aqueous solution of 10% sulfamic acid (2 mL) was added to
eliminate excess HNO2. The solution containing the diazonium salt
was kept cold for the subsequent coupling reaction. 1-Hydroxy-2-
naphthoic acid (0.025 mol) and sodium carbonate (0.035 mol)
were added to a cooled solution (w0 ꢂC) of solution of 200 mL of
water and 25 mL of 30% NaOH. The solution containing the yellow
diazonium salt was then added dropwise to the cold 1-hydroxy-2-
naphthalenecarboxylic acid solution with vigorous stirring. A red
solution formed immediately followed by precipitation of the dye
anion. The product was filtered, dried, and recrystallized twice from
hot water.
3.3.5. 3-Hydroxy-4-[(2-naphthalenyl)azo]-2-
naphthalenecarboxylic acid (5)
A
previously synthesized FDA sample (produced from 2-
naphthylamine and 3-hydroxy-2-naphthalenecarboxylic acid as
shown in Fig. 1) was recrystallized from hot water as follows. 2.05 g
was dissolved in 3 L of hot water at pH 11 (adjusted with 30% NaOH
solution). A precipitate formed immediately after the pH was
adjusted to 7 with concentrated HCl. The precipitate was filtered,
washed with water, and allowed to air-dry overnight.
Yield: 0.93
g
(45%). UV lmax
(
3 )
in DMSO: 430 nm
(0.0240 L mgꢀ1 cmꢀ1).
Anal Calc. for C21H14O3N2: C 73.67, H 4.12, N 8.18. Found: C 72.88,
H 3.96, N 8.04.
3.3.6. 2-[(2-hydroxy-1-naphthalenyl)azo]-1-naphthalenesulfonic
acid (6)
Yield: 1.39 (12%). UV lmax
(
3 )
in DMSO: 504 nm
2-Amino-1-naphthalenesulfonic acid (0.025) was dissolved in
50 mL of water by adding 5 mL of 30% NaOH solution. Concentrated
HCl (20 mL) was added to this solution, which was then cooled to
w0 ꢂC. An aqueous solution of NaNO2 (10 mL, 0.030 mol) was added
dropwise. The mixture was vigorously stirred at w0 ꢂC for 30 min
and checked for excess HNO2 (I2-starch test). An aqueous solution
of 10% sulfamic acid (3 mL) was added to eliminate excess HNO2.
The yellow diazonium salt was filtered and repeatedly washed with
ice-cold water and the wet precipitate was immediately used in the
following coupling reaction. 2-naphthol (0.025 mol) and sodium
carbonate (0.032 mol) were added to a 250 mL solution of 4:1
water:ethanol (v/v) and cooled to w0 ꢂC. The wet diazonium salt
was then added in small portions with vigorous stirring. A red
solution formed immediately followed by precipitation of the dye
anion. The product was filtered, dried, and recrystallized twice from
hot water.
(0.0696 L mgꢀ1 cmꢀ1).
Anal Calc. for NaC20H13O4N2S: C 60.00, H 3.27, N 7.00, S 8.01.
Found: C 59.44, H 3.59, N 6.85, S 8.14.
4. Conclusion
Seven subsidiary colors related to the major dye component of
R34 were successfully synthesized and characterized. The
compounds were synthesized from various combinations of start-
ing materials representative of impurities in either of the inter-
mediate starting materials, 2-amino-1-naphthalenesulfonic acid
and 3-hydroxy-2-naphthalenecarboxylic acid, using azo coupling
reactions, and were purified by recrystallization. The compounds
were fully characterized by elemental analysis, UPLC, NMR spec-
troscopy, IR spectroscopy, and visible spectrophotometry. They
were used as reference materials in a UPLC survey of 33 certified
lots of R34 and R34 lakes. FDA is currently using the new
compounds as reference materials in the batch certification of R34
straight colors and lakes.
Yield: 9.34
g
(89%). UV lmax
(
3 )
in DMSO: 493 nm
(0.0618 L mgꢀ1 cmꢀ1).
Anal Calc. for NaC20H15O5N2S$H2O: C 57.41, H 3.61, N 6.70, S 7.66.
Found: C 56.64, H 3.66, N 6.69, S 7.81.
Appendix A. Supplementary data
3.3.7. 2-[(4-hydroxy-1-naphthalenyl)azo]-1-naphthalenesulfonic
acid (7)
Supplementary data related to this article can be found online at
doi:10.1016/j.dyepig.2012.05.001.
2-Amino-1-naphthalenesulfonic acid (0.050) was dissolved in
150 mL of water by adding 6 mL of 30% NaOH solution.
Concentrated HCl (30 mL) was added to this solution, which was
then cooled to w0 ꢂC. An aqueous solution of NaNO2 (10 mL,
0.051 mol) was added dropwise. The mixture was vigorously
stirred at w0 ꢂC for 30 min and checked for excess HNO2 (I2-
starch test). An aqueous solution of 10% sulfamic acid (4 mL) was
added to eliminate excess HNO2. The yellow diazonium salt was
filtered and repeatedly washed with ice-cold water and the wet
precipitate was immediately used in the following coupling
reaction. 1-naphthol (0.049 mol) and sodium carbonate
(0.074 mol) were added to a 250 mL solution of 4:1 water:-
ethanol (v/v) and cooled to w0 ꢂC. The wet diazonium salt was
then added in small portions with vigorous stirring. A red
solution formed immediately followed by precipitation of the
dye anion. The product was filtered, dried, and recrystallized
twice from hot water.
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Yield: 10.1 (50%). UV lmax
(
3 )
in DMSO: 495 nm
(0.0957 L mgꢀ1 cmꢀ1).
Anal Calc. for NaC20H13O4N2S: C 60.00, H 3.27, N 7.00, S 8.01.
Found: C 59.11, H 3.25, N 6.78, S 8.02.

312
N. Belai et al. / Dyes and Pigments 95 (2012) 304e312
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