CHEMISTRY & CHEMICAL TECHNOLOGY

Vol. 9, No. 4, 2015 Chemistry

Maria Smolinska1,2, Оlga Коrkunа2, Тeodozia Vrublevska2 and Grigory Tеslyar1

ERIOCHROME BLACK T – A NEW ANALYTICAL REAGENT

FOR SPECTROPHOTOMETRIC DETERMINATION
OF SULPHANILAMIDES
1State Scientific-Research Control Institute of Veterinary Medicinal Products and Feed Additives,
11, Donetska str., 79019 Lviv, Ukraine
2Ivan Franko National University of Lviv, Faculty of Chemistry, Department of Analytical Chemistry,

6, Kyryla and Mefodiya str., 79005 Lviv, Ukraine; boiko_maria@ukr.net

Received: August 29, 2014 / Revised: September 22, 2014 / Accepted: April 20, 2015

 Boiko M., Korkuna O., Vrublevska T., Teslyar G., 2015

Abstract. Optimal conditions of the sulphanilamides thiazolylazo) resorcinol [19], which are characterized by a
diazonium salts interaction with o,o'-dihydroxysubstituted high sensitivity and selectivity at the presence of other
azo dye eriochrom black T have been defined. Therefrom classes of biologically active substances. The goal of our
the method of spectrophotometric determination of ten investigation was the search of effective analytical
sulphanilamides in finished medicinal forms has been reagents for SA determination by the examining of azo
developed. This method is based on the azocoupling dyes; one of them is o,o'-dihydroxysubstituted azo dye
reaction of the sulphanilamide diazo salt with one of the eriochrom black T (EBT). Structural formula and some
products of the redox destruction of azo dye with physico-chemical properties of this azo reagent are
formation of a colored azo compound. This method is presented in the Table 1.
characterized by the wide linearity range (from 3 to
64 µg/ml depending on the detectable sulphanilamide), Table 1
simplicity, rapidity (duration of analysis is under 30 min),
Structural formula and some characteristics of azo dye
and reproducibility (Sr ~ 0.040).
EBT (С.І. 14645, CAS No. 1787-61-7) [20-23]
Keywords: sulphanilamides, eriochrome black T, spectro- Structural formula Characteristics
photometry, finished dosage forms. dark blue powder
OH
HO
рКH3Ind = 3.9; red

1. Introduction N
N SO3Na
рКH2Ind- = 6.4; red; рН < 6
рКHInd2-= 11.5; ε615 = 2.35∙104;
Numerous methods of spectrophotometric NO2
рН = 10;
determination of sulphanilamides (SA) content in finished -
Ind3 orange; рН > 12
dosage forms have been elaborated. They are based on the
interaction of both SA [1-5] and SA diazonium salts
Within the pH range from 6–7 to 12–13 the EBT
[6-13] with organic reagents leading to formation of the
colored analytical forms. Even though dyes are common molecules in solution are present in the form of
analytical reagents in pharmaceutical analysis, they are dissociated ion (with one dissociated hydroxyl group),
rarely used for SA determination. In the literature the use while azo form of reagent dominates at lower values of
of alizarine and its derivatives [14], which form charge this pH range at the acid and neutral media (Scheme 1)
transfer complexes with SA, is described. However these [20-23].
reactions are characterized by a low contrast. Previously In analytic chemistry EBT is more often used in the
we developed techniques of spectrophotometric SA inorganic analysis. For the analysis of organic substances
determination with application of azo reagents, such as its application is limited. According to literature data it is
acid mono azo dye tropaeolin O [15, 16], heterocyclic azo known that EBT interacts with organic molecules, resulting
reagents 4-(2-pyridylazo) resorcinol [17, 18], and 4-(2- in formation of ion associates, which are extracted by
402 Maria Smolinska et al.

organic solvents. In this way the following substances:

2. Experimental
diphenhydramine in pharmaceutical and biological objects
[24], lansoprazole in finished dosage forms, such as
capsules [25], levofloxacin in pills [26], trace amounts of 2.1. Reagents and Instruments
nifedipine [27], amlodipine besylate [28], and promethazine We studied the interaction of ten most common in
[29] in pharmaceutical forms are determined. Using the medical practice SA (Table 2).
EBT the following proteins such as a human and ox serum Sulphanilamides were purchased from Sigma
albumin [30], globulin, albumin, and lysozyme [31] are (USA). Solutions of sulphamethoxazole, sulphamethazine,
determined by the method of flow-injection analysis in the sulphamerazine, sulphadimetoxine, sulphathiazole, sulpha-
combination with Rayleigh scattering. diazine, sulphametoxypyridazine, and sulphamono-
metoxine were prepared by dissolving appropriate
amounts of the reagents of pharmacopoeia grade in 0.1 M
sodium hydroxide solution. Solutions of sulphanilamide
were prepared by dissolving appropriate amounts of the
reagents of pharmacopoeia grade in 0.1 M hydrochloric
acid. Solutions of sulphaguanidine were prepared by
dissolving appropriate amounts of the reagents of
pharmacopoeia grade in the mixture of equal amounts of
0.1 M hydrochloric acid and 96 % ethanol.
Solutions of eriohrom black T (Merck, Germany)
were prepared by dissolving appropriate amounts of the
reagent of analytical grade ≥ 98 % purity in the 30 %
mixture of ethanol (96 %) and water.
The solutions of hydrochloric acid, sodium
hydroxide, sodium nitrite, urea, sulphamic acid, and
universal buffer mixture (UBM) were prepared from the
Sheme 1. Forms of existence of the EBT in aqueous solutions chemicals of the analytical grade.
Table 2
Physical and chemical properties of SA
Structural formula Name Some characteristics [32-34]
1 2 3
O
М = 172.2 g/mol;
H2N S NH2 Sulphanilamide (SAM),
рК1 = ―
O p-aminobenzenesulphanilamide
рК2 = 10.1
CAS No. 63-74-1
N O
М = 250.3 g/mol;
N S NH2 Sulphadiazine (SDA),
рК1 = 2.49
N H O 2-(p-aminobenzenesulfonamido)-pyrimidine
рК2 = 6.48
CAS No. 68-35-9
H3C
O
N Sulphamerazine (SMR), М = 264.3 g/mol;
N S NH2 2-(p-aminobenzenesulfonamido)-4- рК1 = ―
N H O methylpyrimidine рК2 = 7.00
CAS No. 127-79-7
O
N
N S NH2
Sulfathiazole (STZ), М = 255.3 g/mol;
S H O
2-(p-aminobenzenesulfonamido)- рК1 = 2.62
1,3-thiazol рК2 = 7.37
CAS 72-14-0
H3C
N O
N S NH2 Sulphamethazine (SMZ), М = 278.3 g/mol;
N H O
2-(p-aminobenzenesulfonamido)- рК1 = 2.65
H3C 4,6-dimethylpyrimidine рК2 = 7.58
CAS No. 57-68-1
 Eriochrome Black T – a New Analytical Reagent for Spectrophotometric Determination of… 403

Table 2 (Continued)
1 2 3
HN O
N S NH2
Sulphaguanidine (SGN), М = 214.2 g/mol;
H2N N-(p-aminobenzenesulfonamido)- рК1 = 2.72
H O
aminoiminomethyl рК2 = 11.82
CAS 57-67-0
H3C O
N O
N S NH2 Sulphadimethoxine (SDM), М = 310.3 g/mol;
N 6-(p-aminobenzenesulfonamido)-2,4- рК1 = 2.65
H O
H3C O dimethoxypyrimidine рК2 = 6.82
CAS No. 122-11-2
H3C O
O Sulphamonomethoxine (SMM), М = 280.3 g/mol;
N N S NH2 4-(p-aminobenzenesulfonamido)-2- рК1 = 2.51
N H O methoxypyrimidine рК2 = 7.28
CAS 1220-83-3
H3C O
М = 253.3 g/mol;
N S NH2 Sulphamethoxazole (SMX),
O N рК1 = 1.74
H O 3-(p-aminobenzenesulfonamido)-5-methyloxazole
рК2 = 5.70
CAS No. 723-46-6
O
H3 C O N S NH2
Sulphamethoxypyridazine (SMP), М = 280.3 g/mol;
N N H O
3-(p-aminobenzenesulfonamido)-6- рК1 = ―
methoxypyridazine рК2 = 7.20
CAS No. 000080-35-3

UV-Vis measurements were performed with UV- 2.2. General Procedure of SA

Vis scanning spectrophotometers CARY.WIN–UV-VIS-
50 (Varian, USA) and SPECORD M-40 (Carl Zeiss Jena, Determination with EBT
Germany) using l cm cuvettes. All absorbance 5.0 ml of 1.0 M hydrochloric acid solution was
measurements were performed at 293–298 K. placed into a 25 ml volumetric flask. Then a sample of
The pH value was measured by pH-meter RV 11 solution containing 4–64 μg ml–1 of SA in final volume
(Sartorius, Germany) as well as pH-meter model was added. Next 0.5 ml of 5.0·10-2 M sodium nitrite
pH 150M (Gomelsky Plant of Measuring Devices, Bela- solution was added into the flask. After stirring the
rus), equipped with a combination electrode, which com- mixture was held for 20 min at a room temperature, then
bines both the glass and the reference silver chloride elect- 0.5 ml of 2.5 M urea solution was added, the mixture was
rodes into one body. The required pH of each solution was stirred and was held for 10 min at a room temperature for
adjusted using diluted HCl and NaOH solutions. destruction of nitrite-ions excess. Then 1.5 ml of
Potentiometric titration was performed using a potentio- 6.0 · 10-3 M EBT solution as well as 2.5 ml of 0.04 M
meter pH 150M with a platinum indicator electrode EPL- solution of UBM were added into the flask. Obtained
02 and a silver chloride reference electrode EVL-1M4 mixture was neutralized by adding of sodium hydroxide
(Gomelsky Plant of Measuring Devices, Belarus). solution so that the pH value was adjusted to pH = 8.0.
Next, distilled water was added to the full volume of
Polarographic researches were performed using an
25 ml. Then the solution was mixed thoroughly and the
oscillopolarograph PO-5122 model 03 (Russia) with
absorbance measurements (at room temperature ~ 293 K)
additional hardware and a three-electrode thermostated were carried out for all blank solutions of corresponding
cell with an indicator mercury dropping electrode, reagents at 485 nm in 1.0 сm cuvettes. SA concentration
auxiliary platinum electrode, and the reference saturated was calculated using the methods of calibration curve and
calomel electrode (linear potential range from -0.2 to single-point standardization.
-1.75 V by the defined conditions: potential sweep rate –
2.0 mV/s, the delay imposition voltage – 4.0 s). 2.3. Procedure of Tablets Preparation for
Polarograms were recorded at a room temperature SA Determination
(293–298 K). Dissolved oxygen was eliminated from the
test solutions by means of bubbling with purified argon Twenty tablets were weighed and finely powdered
for 15 min. in a porcelain mortar. The accurate amount of powder,
404 Maria Smolinska et al.

equal to ~ 100 mg of SA, was placed into a 100 ml λmax = 485 nm, while the absorbance maximum of azo dye
volumetric flask and dissolved in 50 ml of 0.1 M NaOH. EBT is at λmax = 600 nm under the reaction conditions, and
Then the solution was mixed for 10 min and 0.1 M NaOH it decreases with a slight bathochromic shift for all
was added to complete the volume to 100 ml. The products of interaction.
obtained solution was mixed again and filtered through
the fold filter of medium porosity. The filtrate was used
for analysis.

2.4. Sample Preparation of Suppository

for SA Determination
Sample of suppository containing 100 mg of SA in
accordance with the content of the product, was placed
into a chemical glass and 50 ml of 0.1 M sodium
hydroxide was added. The mixture was heated on a water
bath at the temperature of ~ 353 K for at least 10 min to
complete melting of the ointment. Received mixture was
filtered through a folded paper filter (white ribbon) in a
volumetric flask 100 ml (glass with mixture was kept in a
hot water bath to prevent solidification of the preparation
till filtering was complete). The filter was washed several
times with hot 0.1 M sodium hydroxide solution. The Fig. 1. Absorption spectra of solution EBT (1) and products of
filtrate was cooled. Next, the same solvent was added to its interaction with SA diazonium salts aqueous solution:
the mark of the flask. The obtained filtrate was used for SAM (2), SMX (3), SMZ (4), SDM (5), SMR (6), STZ (7),
analysis. SGN (8), SMP (9), SMM (10), SDA (11). СHCl = 1.0 M,
СSА = 6.0·10-5 М, CNaNO2 = 8.0·10-4 М, CUrea = 4.0·10-2 М,
СEBT = 3.6·10-4 М, СUBM = 4.0·10-3 М, pH = 8.0, λ = 485 nm
3. Results and Discussion Obtained colored products of this reaction are most
probably formed because of the redox process between
3.1. Spectrophotometry of SA with EBT diazotized SA and azo dye. Diazonium salt can oxidize
azo dye and destroy it with the release of N2 and colored
3.1.1. Electronic spectrums of product of SA
degradation products, which in its turn can interact
diazonium salts interaction with EBT between themselves or with SA diazonium salts [35].
During the preliminary research of SA interaction it
3.1.2. Conditions for SA diazotization
was discovered that only diazotized SA form colored
compounds with EBT. On the UV-Vis absorbance spectra The conditions of SA diazotization and destruction
of products of interaction the diminishing of absorbance of excess amount of nitrite ions for receiving maximum
maximum of azo dye itself, and the appearance and efficiency of products interaction of SA with azo dye have
increase of the new absorbance maximum proportional to been tested. As follows from the research results, the
SA concentrations were observed. It was established that maximum efficiency of the products of SA interaction
the excess amount of nitrite ions, which have been used with EBT is observed at SA diazotization in 0.5−1.0 M
for obtaining of diazonium salts, interacts with azo dye hydrochloric or sulphuric acids, whereas the use of
because the absorbance at EBT λmax on spectra of both phosphate and acetic acids does not allow achieving the
proper dye and products of SA diazonium salts interaction same result. Therefore we used 1 M HCl for SA
with EBT decrease. Following from this, the action of diazotization in all following experiments.
nitrite-ions on EBT is the same as the action of diazotized To achieve maximum analytical signal it is
SA. Therefore, nitrite ions remaining unreacted with SA necessary to use excess of nitrite ions compared to SA
should be removed from the reaction mixture by means of content, and after diazotization reaction to remove
urea. remaining unreacted nitrite ions by urea or sulfamic acid.
As can be seen from absorption spectra (Fig. 1), The optimal conditions of SA diazotization for obtaining
maximum absorbance of compounds, which were formed of maximum analytical signal at the SA interaction with
after the SA diazonium salts interaction with EBT is at EBT are shown in Table 3.
 Eriochrome Black T – a New Analytical Reagent for Spectrophotometric Determination of… 405

Table 3
Optimal conditions of SA diazonium salts obtaining for maximum analytical signal
at the SA interaction with EBT
Concentration of HCl 1.0 mol·l-1
Amount of NaNO2 > 3-fold excess to the concentration of SA
Diazotation time 20 min at 293 K
Amount of urea > 50-fold excess to the concentration of NaNO2
Reaction time 10 min at 293 K

3.2. Conditions of Interaction of

Diazotized SA with EBT
3.2.1. Effect of medium acidity on the
interaction of diazotized SA with EBT
In order to establish maximum efficiency of SA
diazonium salts interaction with EBT, the investigations
of pH effect on the analytical signal value were carried
out. As follows from Fig. 2, the maximal yield of products
for reaction of the most SA diazonium salts with EBT is
observed in the slightly alkaline medium within the range
of рН = 7.5–9.5.
According to literature data in alkaline aqueous
solutions EBT has dissociated one of the OH-groups
(Scheme 1), and in condition of interaction with the SA
diazonium salts its azo-form dominates over hydrazo- Fig. 2. Effect of pH value on EBT interaction with SA
form. The interaction between diazotized SA and azo dyes diazonium salts: SAM (1), SMX (2), SMZ (3), SDM (4),
SMR (5), STZ (6), SGN (7), SMP (8), SMM (9), SDA (10).
in the acidic medium does not take place as phenol and
СHCl = 1.0 M, СSА = 6.0·10-5 М, CNaNO2 = 8.0·10-4 М,
naphthol compounds are easier oxidized in alkaline
medium [36, 37]. CUrea = 4.0·10-2 М, СEBT = 3.6·10-4 М, СUBM = 4.0·10-3 М, λ = 485 nm

3.2.2. Effect of concentration of azo dye

on interaction of SA diazonium salts with EBT
It is known that certain excess amount of reagent
promotes the shift of reaction equilibrium toward the
formation of reaction products that makes it possible to
get the maximum yield of the colored products. For these
reasons, the dependence of absorbance of the products of
SA diazonium salts interaction with EBT on the excess of
the reagent amount has been investigated. According to
the experimental results (Fig. 3), maximal value of
absorbance of the interaction products of diazotized SA
with EBT is observed at 6-fold reagent excess.
At the azo group destruction in EBT molecule the
formation of two particles: α-naphthol and β-naphthol
containing substituents (sulfonate and nitro groups) is Fig. 3. Dependence of the azo reagent concentration on the
possible (Scheme 2). absorbance of product interaction of the EBT with SA
From literature data it is known that SA diazonium diazonium salts: SAM (1), SMX (2), SMZ (3), SDM (4),
salts azocouple with β-naphthol and the absorption spectra SMR (5), STZ (6), SGN (7), SMP (8), SMM (9), SDA (10) with
of formed products are characterized by the maximum EBT for concentration azo reagent. СHCl = 1.0 M,
absorbance at 477 nm [6]. The presence of the nitro group СSА = 6.0·10-5 М, CNaNO2 = 8.0·10-4 М, CUrea = 4.0·10-2 М,
in the products of EBT destruction deactivates them as azo СUBM = 4.0·10-3 М, pH = 8.0, λ = 485 nm
406 Maria Smolinska et al.

Sheme 2. The scheme of EBТ destruction

component, thus only unsubstituted α-naphthol can

interact with electrophilic particles [36, 37]. We verified
whether SMT diazonium salt can react with α-naphthol
under the same reaction conditions as with the
o,o'-hydroxysubstituted azo dye.
It was established that the α-naphthol reacts with
SMT diazonium salt, and the product of the SMT
interaction with α-naphthol is characterized by maximum
absorbance at 475 nm (Fig. 4). Thus, the maximum
absorbane which is observed on absorption spectrum of Fig. 4. Absorption spectra of the aqueous solution of the EBT,
α-naphthol and products of their interaction with diazotized
the products of SMT diazonium salt interaction with EBT
SMZ: EBT (1), NO2-+EBT (2), (NO2-+Urea)+ EBT (3),
at 470–485 nm is the maximum absorbance of compound (SMZ+NO2-+Urea)+ EBT (4),
of the SMT diazonium salt interaction with destruction (NO2 +Urea)+α-naphthol (5), (SMZ+NO2-+Urea)+
-

product of this dye, namely with α-naphthol. This leads to +α-naphthol (6). СHCl = 1.0 M, СSMZ = 6·10-5 М,
an assumption about a slow oxidation of azo dye and fast CNaNO2 = 8.0·10-4 М, CUrea = 4.0·10-2 М, СEBT = 3.6·10-4 М,
azocoupling of unreacted SMT diazonium salt with Сα-naphtole = 1.2·10-4 М, СUBM = 4.0·10-3 М, pH = 8.0
products of EBT oxidation, namely, α-naphthol, to form
new azo compound. Therefore a part of SA diazonium salt 3.3. Potentiometry of SA with EBT
is used for oxidation of the dye’s azo group while the rest
is azocoupled with products of redox reaction. However, To confirm the redox interaction between SA and
under the same conditions of reaction their parts in both EBT a potentiometric redox titration of diazotized SMZ
processes are constant, which allowed developing the with EBT solution has been carried out (Fig. 5) in
efficient methods of the SA determination by means of optimum conditions of their spectrophotometric deter-
EBT. mination.

Fig. 5. Curves of the redox titration of SMZ diazonium salt (1) and blank solution (2) by the EBT solution. СHCl = 1.0 M,
СSMZ = 1.75·10-3 М, CNaNO2 = 1.75·10-2 М, CUrea = 0.875 М, СЕBТ = 1.75·10-3 М, СUBM = 4.0·10-3 М
 Eriochrome Black T – a New Analytical Reagent for Spectrophotometric Determination of… 407

Fig. 6. Polarograms of the reduction processes: Urea (1), NO2- (2), SMZ (3), SMZ+NO2- (4), EBT (5), NO2-+EBT (6),
(SMZ+NO2-+Urea)+EBT (7), α-naphthol (8), (SMZ+NO2-+Urea)+α-naphthol (9). СHCl = 1.0 M,
СSMZ = 6.0·10-5 М, CNaNO2 = 8.0·10-4 М, CUrea = 4.0·10-2 М, СEBT = 3.6·10-4 М, СUBR = 4.0·10-3 М, pH = 8.0

3.4. Polarography of SA with EBT The polarogram curve of recovery of the

diazonium salt SMT interaction product with EBT has a
Previously the polarographic activity of all the similar character as the polarogram of the product of the
reagents that take part in the interaction of SA with EBT interaction of the dye with nitrite, namely the height of
(Fig. 6 (1)) has been established. reduction peaks at -0.60 V is decreased and the peak
It was established that in the conditions of a at -0.75 V turns into a broad shoulder. This indicates the
maximum efficiency of reaction products such substances identical effects of both nitrite ions and SMZ diazonium
as urea and nitrite ions are not polarographically active, as salt on azo dye, namely the destruction of dye azo group
evidenced by the absence of peaks at the polarograms of under the action of these both reagents. Potential of the
their reduction process. With regard to the SMZ and its reduction peak of azo group of the product of SMZ
diazonium salt two peaks at -0.50 and -1.41 V are diazonium salt interaction with α-naphthol is -0.55 V,
observed on the polarograms. In weak alkaline medium however the current value of its reduction is very small,
(pH = 8.0), where the interaction of SA with EBT takes that is why the reduction peak of the EBT excess on the
place, the compounds, which contain a primary aromatic polarogram is much higher and masks the reduction peak
amino group (SA), or their diazonium salts can interact of a newly formed azo compound.
with each other to form azo compounds [36-40]. Peaks on The results of spectrophotometric research of the
polarogram correspond to reduction of formed azo groups. products interaction of the EBT with SA diazonium salts
The results of polarographic research of EBT and correspond to the results of polarographic and
products of its interaction with SA diazonium salts are potentiometric research and allow to suggest the following
shown in Fig. 6 (2). scheme of SA interaction with EBT: oxidation of azo dyes
Two peaks of azo group at -0.59 and -0.75 V on under the action of SA diazonium salt with the following
polarogram of azo dye EBT redution are observed. The azocoupling of unreacted SA diazonium salts with
presence of urea does not influence the polarographic α-naphthol, which is formed as a result of the azo dye
characteristics of azo dye. However, under the action of destruction (Scheme 3).
nitrite ions the peak height of azo dye reduction at -0.59 V
is decreased and the peak at -0.75 V is converted into a 3.5. Spectrophotometric and Metrologic
broad shoulder, which indicates the destruction of the dye
azo group. It is most probable that azo group is oxidized Characteristics of SA Determination
to azoxygroup or destroyed with the release of N2, as with EBT
follows from the literature on the oxidative properties of
nitrite ions toward azo compounds [36-40]. As follows We have investigated that the absorbance of
from the polarogram of azo dye reduction the excess of colored products at the SA determination with EBT
nitrite ions is completely destroyed under the action of linearly depends on SA concentration in the solution.
urea, since polarograms at the presence as well as at the Validation results of spectrophotometric determination of
absence of nitrite ions are identical. ten SA by means of EBT are presented in Table 4.
408 Maria Smolinska et al.

Sheme 3. Scheme of SA diazonium salts interaction with EBT

Table 4
Spectroscopic characteristics of the products of SA interaction with EBT and validation results of SA
spectrophotometric determination. n = 3, P = 0.95
ε·10-3, Optimum photometric Calibration equation, СLOD, СLOQ,
SA R2
M-1 cm-1 linear range, µg ml-1 СSА, µg ml-1 µg ml-1 µg ml-1
SAM 2.2 7– 8 ΔА = 0.009 + 0.015∙C 2.41 7.23 0.9997
SMO 4.1 3–48 ΔА = 0.010 + 0.019∙C 1.05 3.15 0.9997
SMZ 4.2 3–48 ΔА = 0.017 + 0.018∙C 1.14 3.42 0.9995
SDM 4.8 3–48 ΔА = 0.014 + 0.017∙C 1.26 3.78 0.9995
SMR 4.2 3–48 ΔА = 0.012 + 0.017∙C 1.09 3.27 0.9994
STZ 3.3 3–48 ΔА = 0.013 + 0.013∙C 1.03 3.10 0.9992
SGN 1.6 14–64 ΔА = 0.013 + 0.014∙C 4.95 14.9 0.9998
SMP 2.8 7–48 ΔА = 0.011 + 0.016∙C 2.46 7.38 0.9998
SMM 2.7 7–48 ΔА = 0.012 + 0.015∙C 2.43 7.29 0.9997
SDA 3.4 3–48 ΔА = 0.011 + 0.019∙C 1.17 3.51 0.9997

Sheme 4. Series of sensitivity decrease for ten SA determinations at redox reaction with EBT
Table 5
The results of spectrophotometric SA determination with EBT in one-component medicines and veterinary
drugs. СHCl = 1.0 M, СSА = 6.0·10-5 М, CNaNO2 = 8.0·10-4 М, CUrea = 4.0·10-2 М, СEBT = 3.6·10-4 М, СUBM = 4.0·10-3 М,
pH = 8.0, λ = 485 nm, n = 5, Р = 0.95
Amount of drug found x ± ∆x
Determined SA and relative standard deviation (Sr)
(regulated content in preparation)
Pharmaceutical method (nitritometry) Spectrophotometric method with EBT
"Intrauterine candle with sulphadimezine" suppository of the LLC "Basalt", Kyiv
(excipients – polyethylene glycol, propylene glycol)
Sulphamethazine 298 ± 11 301 ± 8
(300 ± 30 mg/g) (0.032) (0.023)
"Streptocide" tablets of the PTC "Darnytsya" Kyiv
(excipients – starch, gelatin, aerosil, calcium stearate)
Sulphanilamide 504 ± 16 501 ± 18
(500 ± 50 mg/tabl) (0.027) (0.031)
 Eriochrome Black T – a New Analytical Reagent for Spectrophotometric Determination of… 409

There is a correlation between the structure of the 5. The method is characterized by a wide linearity range
SA and the sensitivity of their determination with EBT. (from 3 to 64 µg/ml depending on the detectable
As one can see from the Scheme 4, the most sensitive are sulphanilamide), simplicity, rapidity, and reproducibility
SDM and SMZ determinations, which contain six- (Sr ~ 0.040).
membered heterocycles with two hetero atoms of nitrogen
and two substituents of methoxy- or methyl-radicals that References
have +M-effect [35-37]. Less sensitive is the [1] Vaid F., Aminuddin M. and Mehmood K.: Pakist. J. Pharm. Sci.,
determination of SMR containing six-membered 2004, 17, 77.
heterocycle with one substituent (methyl-radical), or [2] Klokova E. and Dmitrienko S.: Vestn. Mosk. Univ. Serіya 2.
unsubstituted SDA. STZ and SMO, whose molecules Khimiya, 2008, 49, 339.
[3] Evgeniev M., Garmonov S., Shakirova L. et al.:
contain five-membered heterocycles with two different Zh. Anal. Khimii, 2000, 55, 888.
heteroatoms, nitrogen and Sulphur and nitrogen and [4] Ogoda Onah J. and Eromi Odeiani J.: J. Pharmaceut. Biomed,
oxygen, respectively, are determined with less sensitivity. 2002, 30, 851.
The determinations of SA, which contain six-membered [5] Vijaya Raja G., Bala Sekaran C., Siva Kumari P. et al.: Orient. J.
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[17] Boiko M., Vrublevska T., Korkuna O. et al.: Voprosy Khimii i
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technical documentation of investigated medicines. The [18] Boiko M., Vrublevska T., Korkuna O. et al.: Zavodskaya
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[21] Zhou H., Wu X., Meng F. et al.: Spectrochim. Acta A, 2011,
interaction with о,о′-dihydroxysubstituted azo dye EBT 78, 681.
have been established. [22] Ghosh S.: Chem. Phys. Lett, 2010, 500, 295.
2. It was shown that the interaction of SA diazonium salts [23] Zhu M., Huang X. and Shen H.: Talanta, 2001, 53, 927.
with EBT is based on the azocoupling reaction of SA [24] El-Didamony A. and Moustafa M.: Arabian J. Chem., 2010, 3,
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[25] Aydogmus Z.: Acta. Pharm. Sci., 2006, 48, 45.
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3. The correlation between the SA structure and Indian J. Pharm. Sci., 2004, 66, 799.
sensitivity of their determination with EBT has been [27] Rahman N., Khan N.A. and Azmi S.: Farmaco, 2004, 59, 47.
detected. The determination sensitivity series of ten SA [28] Narayana Reddy M., Tulaja Rani G., Prasada Rao K. et al.:
Indian J. Pharm. Sci., 1997, 59, 188.
with azo dye has been established.
[29] Patil D. and Chafle D.: Asian J. Chem., 2007, 19, 3253.
4. On the basis of carried out researches the method of [30] Li Y., Dong L., Wang W. et al.: Anal. Biochem, 2006, 354, 64.
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