Simultaneous determination of Brilliant Green and Crystal Violet dyes in fish and water samples with dispersive liquid-liquid micro-extraction using ionic liquid followed by zero crossing first derivative spectrophotometric analysis method

Article abstract of DOI:10.1016/j.saa.2018.04.061

In this study, dispersive liquid-liquid micro-extraction using ionic liquid (IL-DLLME) combined with zero crossing first derivative spectrophotometric method was applied to quantitative determination of triphenylmethane dyes in binary mixtures. The 1-methyl-3-octylimidazolium hexafluorophosphate [OMIM][PF₆] ionic liquid was used to extract Brilliant Green (BG) and Crystal Violet(CV) dyes from aqueous solutions. The amplitude of the zero crossing first derivative spectra at 670 nm and 532 nm were selected for the determination of BG and CV, respectively. Significant factors influencing the extraction of BG and CV such as sample pH, kind of extraction solvent, amount of extractant, extraction and centrifuging times and ionic strength were investigated. Under the optimal conditions, the calibration curves for the simultaneous determination of both dyes were found to be linear in the range of 10–500 μg L^?1 with detection limits (LODs) of 2.7 μg L^?1 and 1.4 μg L^?1 for BG and CV, respectively. The relative standard deviation (RSD%) for five replicate simultaneous determinations of BG and CV were 4.7% and 1.7%, respectively. Extraction efficiencies of the BG and CV dyes in the presence of interfering ions were also investigated. Sample preparation based on the quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction combined with the IL-DLLME method and zero crossing first derivative spectrophotometric detection was applied for the simultaneous analysis of BG and CV in fish and water samples with quantitative recoveries.

Full text of DOI:10.1016/j.saa.2018.04.061

Accepted Manuscript
Simultaneous determination of Brilliant Green and Crystal Violet
dyes in fish and water samples with dispersive liquid-liquid
micro-extraction using ionic liquid followed by zero crossing first
derivative spectrophotometric analysis method
Susan Sadeghi, Zohreh Nasehi
PII:
DOI:
Reference:
S1386-1425(18)30376-7
doi:10.1016/j.saa.2018.04.061
SAA 16018
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 10 November 2017
Revised date:
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date:
11 April 2018
29 April 2018
Please cite this article as: Susan Sadeghi, Zohreh Nasehi , Simultaneous determination
of Brilliant Green and Crystal Violet dyes in fish and water samples with dispersive
liquid-liquid micro-extraction using ionic liquid followed by zero crossing first derivative
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captured as affiliation for all authors. Please check if appropriate. Saa(2017), doi:10.1016/
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ACCEPTED MANUSCRIPT
Simultaneous determination of Brilliant green and Crystal Violet dyes in fish
and water samples with dispersive liquid-liquid micro-extraction using ionic
liquid followed by zero crossing first derivative spectrophotometric analysis
method
Susan Sadeghi*,1, Zohreh Nasehi1
Department of Chemistry, Faculty of Science, University of Birjand, Birjand, Iran
Abstract
In this study, dispersive liquid-liquid micro-extraction using ionic liquid (IL-DLLME) combined
with zero crossing first derivative spectrophotometric method was applied to quantitative
determination of triphenylmethane dyes in binary mixtures. The 1-methyl-3-octylimidazolium
hexafluorophosphate [OMIM][PF6] ionic liquid was used to extract Brilliant Green (BG) and
Crystal Violet(CV) dyes from aqueous solutions. The amplitude of the zero crossing first derivative
spectra at 670 nm and 532 nm were selected for the determination of BG and CV, respectively.
Significant factors influencing the extraction of BG and CV such as sample pH, kind of extraction
solvent, amount of extractant, extraction and centrifuging times and ionic strength were
investigated. Under the optimal conditions, the calibration curves for the simultaneous
determination of both dyes were found to be linear in the range of 10-500 μg L-1 with detection
limits (LODs) of 2.7 μg L-1 and 1.4 μg L-1 for BG and CV, respectively. The relative standard
deviation (RSD%) for five replicate simultaneous determinations of BG and CV were 4.7% and
1.7%, respectively. Extraction efficiencies of the BG and CV dyes in the presence of interfering
ions were also investigated. Sample preparation based on the quick, easy, cheap, effective, rugged
and safe (QuEChERS) extraction combined with the IL-DLLME method and zero crossing first
derivative spectrophotometric detection was applied for the simultaneous analysis of BG and CV in
fish and water samples with quantitative recoveries.
Keywords: Crystal Violet; Brilliant Green; Dispersive liquid-liquid microextraction; Derivative
spectrophotometry; Food analysis
 Corresponding author. Tel/Fax: +98 5632202009
E-mail address:ssadeghi@birjand.ac.ir (S. Sadeghi)
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1. Introduction
The triphenylmethane dyes such as Brilliant Green (BG) and Crystal Violet (CV) (Fig.1) are kinds
of synthetic colorants which are widely used for coloring in the textile industry and biological
stains ]1]. BG is a member of the triphenylmethane dyes family that has antifungal properties ]2[. In
the paper production industry, about 0.8-1.0 Kg of BG per ton of paper is consumed. It is water
soluble and toxic so that it has mutagenic and carcinogenic effects on aquatic biota and humans. It
may also cause irritation with redness and pain when it comes in contact with the skin]3[. On the
other hand, this dye has been used in the aquaculture industry for many decades due to its anti-
microbial, anti-parasitic and anti-fungal properties for the treatment and prevention of certain fish
diseases [4, 5]. Crystal violet (CV) is a well-known dye that is also applied as a dermatological
agent, veterinary medicine, biological stain, additive to poultry feed to inhibit propagation of mold,
fungus and intestinal parasites, paper printing, textile dying, and etc.
BG, CV and their metabolites may cause human carcinogenesis and mutagenesis, so that these dyes
are classified as the third class of dyes (strong toxins) ]6[. It is proven that such dyes can induce
reproductive abnormalities in fish [7-9[, and renal and hepatic tumors in mice ]10[. The fish tissues
can readily absorb CV from water and reduce it metabolically to the leuco moiety (leucocrystal
violet, LCV). According to the European Union (EU), the developed analytical methods must meet
the European minimum required performance limit (MRPL) at 2 ng/g for the sum of the parent
drugs and their leuco forms. Meanwhile, in the US Food and Drug Administration (FDA), a
minimum sensitivity of 1 ng/g is required for the regulatory testing ]11[.
Although, the EU and the FDA do not authorize BG and CV but BG and CV are often illegally used
in the fish farming industry and treatment of certain fish disease owing to low cost, easy
availability, high efficiency against fungus, bacteria and parasite. However, the use of BG and CV
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causes environmental contamination and affects human health ]12[. Therefore, monitoring of these
residues in aquatic products is very important. The development of simple and sensitive methods for
the determination of BG and CV is of great importance and interest among the chemical analysts
[13[.
Various techniques such as spectrophotometry [14], electrochemistry [15[, electro-
chemiluminescence (ECL) ]16-18], enzyme linked immunosorbent assay (ELISA)]19[, capillary
electrophoresis [20[, liquid chromatography with visible and/or fluorescence detection [21-24[,
liquid chromatography–mass spectrometry ]25[, and liquid chromatography-diode array detection
[26[ have been applied for quantitative analysis of triphenylmethane dyes and their leuco-
metabolites in fish tissue. It is proven that the chromatography and electrophoresis techniques have
high sensitivity for the determination of dyes in complex matrices but they have several potential
drawbacks such as the need for time consuming sample pre-treatment, the usage of toxic solvents,
and the resulting waste products[27, 28[. UV-vis. absorption spectrophotometry is more economical
and simpler for analysis of dyes than chromatography and electrophoresis techniques, however,
employment of this technique for the simultaneous analysis of dyes in multi-component dye
systems often encountered problem owing to overlapping their zero-order absorption spectra. For
resolving the spectral overlaps as well as removing the background of signals originated from the
other components in samples, the conventional first-order derivative spectrophotometry has been
used[28[. Derivative spectrophotometry has widely found applications in different fields of analysis,
clinical and biochemical, pharmaceutical, as well as in inorganic or organic analysis ]29, 30[. If the
derivative peak height (amplitude) of analyte is measured at wavelength in which spectra of the
other components pass through zero, the amplitude of the derivative spectrum is proportional only
to the concentration of analyte. This approach of quantitative determination is called zero-crossing
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technique. It allows quantitative determination of one analyte in the presence of matrix or the
simultaneous determination of two analytes in their binary mixtures. In addition, due to dependence
of derivatization result on the shape of zero-order absorption spectra, broad signals led to flatting
and finally zeroing, so that signals of analyte are amplified. Thus, the influence of the background is
eliminated and compare to the classical determination, selectivity is increased [30].
Due to low concentrations of analytes and presence of matrix effects, often a suitable
pretreatment step is required prior to their determinations. In the recent decade, dispersive liquid-
liquid microextraction (DLLME) is becoming popular extraction treatment due to the simplicity of
operation, rapidity of extraction, low cost, environmental friendliness, and high enrichment factor
[31, 32[. In this method, a ternary solvent system consisting of an extraction solvent (water
immiscible), a dispersing solvent (water and organic solvent miscible) and the aqueous sample
solution is used, so that a cloudy solution consisting of fine droplets of the extractant is formed by
the fast injection of the mixture of extraction and dispersing solvents to the aqueous sample. In this
way, the contacting area between the extraction solvent and the aqueous sample is high so that the
equilibrium state is achieved rapidly, and the analytes are enriched into the microdroplets of the
extracting phase. The small volume of the used extracting phase can be separated by centrifugation,
and collected easily by a microsyringe for the subsequent instrumental analysis [33, 34[. Some
dispersive solvents such as ethanol, acetone, and acetonitrile have been used in DLLME. Ionic
liquids (ILs) have unique characteristics such as lower vapor pressure, miscibility in aqueous
phases, higher viscosity and are generally more environmentally friendly than some organic
solvents. Incorporation of functional groups into cations or anions of ILs producing extractants
with selective binding sites that improves selectivity of the ILs based microextraction methods [35[.
In order to facilitate dispersion of the hydrophobic IL into the aqueous solution and ensure the
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adequate formation of microdroplets, ultrasonic energy, microwaves energy, high temperature or
vortex have also been used [36, 37[.
In this study, we integrate the advantages of ionic liquid based DLLME (IL-DLLME) and first
derivative spectrophotometric method by zero crossing technique for simultaneous determination of
trace amount of BG and CV in their binary mixtures. Various experimental parameters that affect
the extraction process have been carefully studied. To the best of our knowledge, there is no report
on the use of IL-DLLME/zero crossing first derivative spectrophotometry for extraction and
determination of the BG and CV dyes in aqueous solutions. The QuEChERS extraction approach
combined with the developed method was designed for the simultaneous analysis of BG and CV in
real samples.
2. Experimental
2.1 Instrumentation
A UV/Vis spectrophotometer Specord 210 Plus (Analytik Jena, Germany) equipped with 1 cm
quartz microcells (0.7 mL of capacity) with a fixed slit width of 2 nm and a scan speed of 1200 nm
min-1 was used for recording the zero order and first derivative absorption spectra. The
WinASPECT software package was used for data acquisition and manipulation.
An analytical electrical balance model H30 (Gallenkamp, Germany) was employed to weigh the
materials. The pH measurements were carried out by using BP3001 benchtop pH meter equipped
with a combined glass electrode (Trans instruments, Singapour). In order to separate the phases, a
centrifuge model EBA20 (Hettich, Germany) was used. An automatic blender (Fan azmagostar,
Iran) was used to prepare the real samples. A Magnetic stirrer (Gallenkamp, Germany) and a R2062
rotary evaporator (Heidolph, Germany) were used in the synthesis of ionic liquid. Also, deionized
water was prepared by using Aqua max system (Young – Lin, South Korea).
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2.2 Chemicals and reagents
1-Methylimidazole, 1-bromobuane, 1-bromohexane, 1-bromooctane, and potassium
hexafluorophosphate (KPF6) were used to synthesize the 1-methyl-3-butylimidazolium
hexafluorophosphate
([BMIM][PF6]),
1-methyl-3-hexylimidazolium
hexafluorophosphate
([HMIM][PF6]) and 1-methyl-3-octylimidazolium hexafluorophosphate ([OMIM][PF6]) ionic
liquids and provided from Merck (Darmstadt, Germany). The purity of Brilliant green dye (BG,
C27H33N2-HO4S, 482.64 g mol-1) supplied by Merck was 95%. Crystal violet (CV, C25N3H30Cl,
407.97 g mol-1) was provided from Fluka (Neu-Ulm, Germany) with a purity exceeding 90% and
used as received. Deionized water was used for preparing all solutions. The stock solutions of BG
and CV (1000 µg L-1) were prepared by dissolving appropriate amounts of BG or CV in deionized
water. Working standard solutions were freshly prepared by successive dilutions of the stock
solutions with the water. A 0.1 M phosphate buffer solutions were prepared with phosphoric acid
and sodium hydroxide solutions. The pH of the solutions was adjusted by 0.1 M NaOH or
phosphoric acid solutions using pH meter. Ethanol was used as a diluting solvent in
microextraction.
2. 3 Synthesis of ionic liquids
The synthesis of [OMIM][PF6] was performed according to the method previously reported by
Obliosca et al. [38] and purified according to the literature [39, 40]. Briefly, appropriate amounts of
1-methylimidazole and an excess of 1-bromooctane was reacted in a two-necked round-bottom
flask with refluxing at 80 ◦C for 48h to obtain1-methyl-3-octylimidazolium bromide ([OMIM][Br]).
The resulting viscous liquid was cooled to room temperature and then was washed several times
with ethyl acetate (3×20 mL) to remove unreacted starting materials. Finally the product was dried
under vacuum at 80◦C. Afterwards, appropriate amount of [OMIM][Br] and KPF6 aqueous solution
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was stirred for 24h at room temperature. The upper phase was removed and the [OMIM][PF6] ionic
liquid was dissolved in dichloromethane. Then, the mixture was washed several times (3×20 mL)
with deionized water and was dried at 80◦C. Other ionic liquids [BMIM][PF6] and [HMIM][PF6]
were synthesized in the same way except for using 1-bromobutane or 1-bromohexane for the
synthesis of [BMIM][PF6] and [HMIM][PF6] ionic liquids, respectively.
2. 4 Sample preparation
The preparation procedure of a trout fish tissue was carried out as follow: A trout fish was
caught from a local fishing pond (Abiz, Ghaen, Iran). The skin and bones were removed, and the
muscles were minced homogenized in the blender and frozen before being analyzed ]41[. Five
grams of tissue samples were weighed in a 50-mL polypropylene tube and spiked with appropriate
concentrations of BG and CV, mixed thoroughly and kept at room temperature for 30 min. The
QuEChERS sample preparation method was used for the extraction of dyes residue from fish
sample. The QuEChERS method was carried out by adding 5 mL of deionized water and 10 mL of
acetonitrile solution containing formic acid (1%; v/v) to the spiked tissues and shaken for 1 h.
Afterward, 1 g of anhydrous sodium acetate and 4 g of anhydrous sodium sulfate were added to the
tube and the mixture was vigorously shaken for 1 min and then centrifuged at 5,000 rpm for 5 min.
The upper phase was completely separated, filtered to remove impurities and heated to near dryness
in a hot water bath. After cooling, the sample was prepared in a phosphate buffer and the pH was
adjusted to 4.0. Finally, the prepared sample solution was treated with the recommended DLLME
before the simultaneous determination of target dyes by zero-crossing first derivative
spectrophotometry.
The river water samples were obtained from the Band-e-Dareh River (Birjand, Iran). Prior to
analysis, the aqueous sample was passed through 0.45 μm membrane filter and kept in glass bottle
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at 4ºC in a refrigerator. The extraction of the target dyes from unspiked and spiked water samples
was carried out according to the recommended IL-DLLME procedures.
2.5 DLLME procedure
An aliquot of standards or sample solution containing 100 μg L-1 of BG and/or 100 μg L-1 of
CV was pipetted into 15 mL scaled-glass test tubes with conical bottom and diluted to the mark by
addition of phosphate buffer solution (pH = 4). Then, 500 μL sodium chloride (10%, w/v) and 150
μL [OMIM][PF6] were transferred to the tube and the mixture was shaken for 3 min. The turbid
solutions were centrifuged at 4,000 rpm for 10 min. The upper aqueous phase was removed with a
syringe, and the volume of the settled phase was diluted to 0.65 mL with ethanol and then entirely
transferred into a quartz microcell and determined spectrophotometrically. The zero order UV-vis.
spectra were recorded in the wavelength region of 500-700 nm with a resolution of 2 nm against the
reagent blank which was treated on dye free solution by the IL-DLLME. For the simultaneous
determination of BG and CV, the binary mixture solutions containing BG and CV dyes were
prepared and after extraction of the target dyes by the recommended IL-DLLME method, the first
order derivative absorption spectra were recorded for the evaluation of dye contents.
3. Results and discussion
3. 1 Optimization of DLLME procedure
To achieve maximum efficiency in the DLLME procedure, several individual experimental
parameters including the sample pH, the type of buffer, the type and volume of ionic liquid
extractant, the extraction time, the centrifuging time and salt concentration were optimized for each
individual dye solution. In each experiment, zero order absorption spectra of the dyes were recorded
in the wavelength region of 500-700 nm. BG and CV showed maximum absorbencies at 628 nm
and 590 nm, respectively, in their single solutions. The first order derivative spectra were obtained
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using the software of instrument with wavelength interval of 2 nm (Δλ=2 nm). To evaluate various
effective parameters in the IL-DLLME, the amplitudes of zero crossing first order derivative spectra
at 670 nm and 532 nm were selected for the determination of BG and CV, respectively.
3. 1. 1 Effect of sample pH
The pH is an important analytical parameter and plays a special role in the extraction of
triphenylmethane dyes. These dyes can exist in the form of colored cationic form or as
undissociated basic form as the carbinol [42]. The carbinol form is relatively insoluble in water and
forms at high pH value. The formation of carbinol form is also depends on the concentration of the
dye. In this study, the effect of pH on the extraction performance of BG and CV was investigated in
the pH range of 2-7. BG has two pKa values of 2.62 and 4.93 while pKa value for CV is 0.8;
consequently at pH<7, BG and CV are mostly dissociated into the cationic form. Thus, the target
dyes would be well extracted at low pH values. The results are shown in Fig. 2A, indicating that the
maximum extraction efficiency was occurred at pH = 5.0 for BG and pH = 3.0 for CV. At higher
pH value than 7.0, OH- as a nucleophile could be contribute to the structural changes occurred in the
dye molecules, producing carbinol form that became easy to remove dyes from aqueous solutions
[43]. Therefore, the pH of the sample solution was adjusted to 5.0 and 3.0 for the extraction of BG
and CV, respectively, in the following experiments.
The effect of type of buffer on the extraction efficiency of the dyes was also investigated. In this
study, acetate, phosphate and Robinson buffers (0.1 M) were prepared at the optimal pH values of
5.0 and 3.0 for microextraction of BG and CV, respectively. As seen in Fig. 2B, the highest
extraction efficiency was achieved by using phosphate buffer for both dyes. Therefore, phosphate
buffer was used in the preparation of dye samples.
3.1.2 Selection of the microextraction solvent
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The extraction solvent is a very important parameter for DLLME processing. Initial conditions
necessary for the extraction solvent such as larger density than water, low solubility in water, and
high extraction efficiency for the target analytes were considered. In comparison with the organic
solvents that used in DLLME methods, ILs are efficient alternatives with properties of less toxicity,
less volatility, less contaminating effect and higher enrichment factor. Based on these criteria, three
ionic liquids [BMIM][PF6], [HMIM][PF6], and [OMIM][PF6] were examined as extraction solvent.
The results are shown in Fig. 3A revealed that [OMIM][PF6] provided the best extraction
efficiencies among three investigated solvents most probably due to higher hydrophobicity.
Therefore, [OMIM][PF6] was selected as an appropriate extraction solvent for BG and CV dyes.
The ionic liquid based DLLME process was performed without any dispersing solvent.
3.1.3 Effect of the volume of ionic liquid
To investigating the effect of extraction solvent volume, different volumes of [OMIM][PF6] IL
from 25 to 200 μL were tested. The results in Fig. 3B indicated that the extraction efficiency
increased with increase the [OMIM][PF6 ] IL volume from 25 μL to 150 μL, and then it remained
constant over the volume range of 150-200 μL. When the IL volume was too large, the amount of
sedimented IL increased, so that the concentration of target dyes in the IL phase decreased, resulted
in decreasing of the extraction efficiencies. Consequently, 150 L of [OMIM][PF6 ] IL was used for
the microextraction of target dyes.
3.1.4 Effect of extraction time
In the DLLME, the extraction time is defined as the time between injecting the extraction solvent
and centrifugation [44]. In this experiment, the effect of extraction time was examined in the range
of 0.5-10 min at room temperature. It is expected that increasing the extraction time facilitates the
mass transfer of target dyes to the IL phase which lead to enhance the extraction efficiencies. It was
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found that more than 95% of BG ad CV were extracted within 3 min at 100 g L-1 and then
remained relatively constant over the time range of 3-10 min. It seems the extraction of analytes
into the extractant was achieved almost instantaneous. Therefore, the optimum extraction time for
BG and CV was chosen 3 min (Fig. 4A).
3.1.5 Effect of centrifuging time
Centrifugation allows the quick and easy collection of the extractant phase at proper time.
Centrifuging time affects the volume of the settled phase by extraction. Therefore, the right choice
of centrifuging time has a great effect on extraction efficiency [45]. In this regard, the effect of this
parameter on the extraction efficiency of dyes was studied in the range from 5 to 25 min at a
centrifugation speed of 4,000 rpm. The results illustrated in Fig. 4B, showing that the maximum
extraction efficiencies were obtained in 10 min. In short centrifuging times, the IL phase didn’t
completely separate and collect at the bottom of the test tube. At longer centrifuging times,
extraction efficiency decreased could be attributed to combine the IL microdroplets again which led
to more dissolving IL in the aqueous phase. Based on these observations, centrifuging the cloudy
mixture for 10 min at 4,000 rpm was used for further experiments.
3.1.6 Effect of ionic strength
The ionic strength of the sample solution is an important analytical parameter that affects the
extraction efficiency. Generally, ionic strength is affected by two processes: electrostatic
interactions and salting out [11]. The salting out effect increases the extraction efficiency, while
electrostatic interactions between salt ions and the target analyte decrease the extraction efficiency.
In this work, the effect of ionic strength was studied with the addition of NaCl salt to the aqueous
dye solutions ranged from 0.1 to 30% (w/v). It is evident from Fig.5 that the extraction efficiency of
target analytes increased with increasing NaCl concentration from 0.1 to 0.5% (w/v) and then was
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remained constant up to 20% (w/v) and after that eventually decreased. The solubility of IL in the
water phase increased with the increase of high ionic strength above 20% (w/v), because of
increasing the density and viscosity of the aqueous phase. Hence, the IL phase couldn’t separate
from aqueous phase and the sediment phase volume decreased. These results revealed that the
recommended method has potential for analysis of the target dyes from saline solutions up to 20%
(w/v).
3.2 The determination of BG and CV in their single solutions
Under the optimal conditions, the zero order absorption spectra of each dye were recorded in the
wavelength range of 500-700 nm. The maximum absorption at wavelengths of 628 and 590 nm
were used for the analysis of BG and CV at different concentrations in their single dye solutions,
respectively. The calibration curves were linear in the range of 1–500 g L-1 for BG and CV dyes,
however, the correlation deviated at very high mixture concentrations.
3. 3 The simultaneous analysis of BG and CV in binary mixtures
According to the results obtained from the optimization process for target dyes, except for the
pH, other optimal parameters were similar for the microextractions of BG and CV. Therefore, the
simultaneous determination of BG and CV in binary mixtures was carried out under the optimal
conditions at pH 4 where both dyes could extract with high extraction efficiencies. Although, a
large amount of CV extracted at pH= 3, but significant amount of BG remained in the aqueous
phase. At pH=5, the extraction efficiency of BG was higher than CV, however, both dyes could be
extracted at pH=4 with high extraction efficiencies. Accordingly, for the simultaneous
determination of BG and CV in binary solutions, pH= 4 was selected as the optimal pH value.
For the simultaneous determination of BG and CV, a binary mixture containing 100 µg L-1 of each
dye was prepared and after microextraction of target dyes by the proposed method, the zero order
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absorption spectra was recorded (Fig. 6A). Although BG and CV in single solution showed well
defined zero order absorption spectra in the wavelength range of 500-700 nm with maximum
absorbance at 628 nm and 590 nm, respectively, but, these spectra were overlapping in the binary
mixture. Therefore, simultaneous determination of the two dyes was not possible by direct
measurement of absorbance using zero-order absorption spectra. The treatment of derivative
absorption spectra has been the basis of simultaneous analysis of BG and CV dyes in binary
mixtures. Fig. 6B depicts the first order derivative absorption spectra of BG and CV in single and
binary mixture solutions in the wavelength range of 500-700 nm. Simultaneous determination was
performed by first derivative absorption spectra of dyes using zero-crossing method based on
measurement of first derivative absorbance value of BG dye at 670 nm (1D670) in the presence of
CV, where the derived absorbance of CV was zero (zero crossing point), and at 532 nm (1D532) for
CV dye in the presence of BG, where the derived absorbance of BG dye was zero or near to zero
value (zero crossing point). Thus, the both dyes were assayed in the concentration range of 10-500
µg L-1 by measurements of the peak amplitude of the first derivative spectra at 670 nm for BG and
532 nm for CV (Fig. 7).
The accuracy of the zero-crossing first order derivative spectrophotometry method for analyzing the
BG and CV dyes concentrations in binary solutions was checked with the recovery studies ]46[.
Two set standard solutions containing BG and CV dyes at different concentrations were prepared.
In the first set, the CV concentration was fixed at 200 µg L-1 while the BG concentration was
changed from 10 to 500 µg L-1(Fig. 7A). In the second set, the similar trend was used to get
standard solutions of CV having concentrations from 10 to 500 μg L-1 in the presence of 200 µg L-1
BG (Fig. 7B). Finally, to prove the validity of the proposed method, synthetic mixtures of different
concentrations of both dyes changing from 10 to 500 µg L-1 were assayed (Fig. 7C). The results
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were computed against the previously constructed standard curves. The recoveries (R, %), errors (E,
%) and average percentage errors (C, %) were calculated using Eqs. from (1) to (3), respectively,
where Ct is the theoretical concentration and Cm is the measured concentration in µg L−1. In Eq. (3),
N is the number of measurements.
−1
C
(µgL
R% =
퐸% =
) × 100
)
(1)
(2)
m
−1
C (µgL
t
−1
−1
퐶
푚
(µgL )−퐶 (µgL
) × 100
푡
−1
퐶 (µgL
푡
)
C
−C
t
N
i=1
m
∑
|
⁄
|
C
t
C% =
× 100
(3)
N
The results of analysis of the synthetic mixtures are summarized in Table 1. As could be seen from
Table 1, the recovery values for BG and CV were in the range of 96 - 105% and the average
percentage errors (E,%) were 3.2 and 3.5% for BG and CV, respectively. It was shown that the
obtained concentrations agreed reasonably well with the theoretical concentrations of both dyes for
reliable measurements. According to the recovery studies, the simultaneous determination of BG
and CV contents in binary mixtures can be accurately performed by using the zero crossing first
derivative spectrophotometric method.
3.4. Analytical performance
From the measurements made under the optimum conditions, calibration graphs for the
determination of each dye in their single solutions and the simultaneous determination of BG and
CV dyes in the binary mixtures were constructed based on the first derivative spectra of the BG and
CV dye solutions and their mixtures. The linearity was obeyed in the concentration ranges of 1-500
μg L-1 and 10-500 μg L-1 for single and binary dye solutions, respectively. The limit of detection
based on three times of standard deviation of the blank (Sb) divided by the slope of calibration curve
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(m) in simultaneous determinations of BG and CV were 2.7 μg L-1 and 1.4 μg L-1, respectively.
Also, the relative standard deviations (RSD%) for five replicate simultaneous determinations of BG
and CV at 50 μg L-1 of each dye were 4.7% and 1.7%, respectively. Because the amount of BG and
CV in 10 mL sample solution are measured after preconcentration by DLLME in a final volume of
150 μL, the analytes were concentrated by a factor of 67 (Table 2).
3. 5 Interference study
The effect of different ions on the simultaneous determination of BG and CV by the IL-DLLME
method was investigated by the measurement of the absorbencies of dyes using zero crossing point
first derivative spectrophotometry. An ion was considered as interferent when it caused a variation
in the absorbencies of the analytes greater than ± 5% [47]. For this study, various amounts of ionic
species were added to the binary mixtures of dyes each at 50 μg L-1 and processed by the IL-
DLLME. In the first test, a 5000-fold weight ratio of the interferent to the dyes was investigated. If
interference occurred, this ratio was gradually reduced until the interference stopped. As the results
show in Table 3, a large excess amount of common cations and anions did not interfere in the
simultaneous determination of BG and CV.
3.6 Application to real samples
In order to evaluate the analytical applicability of the recommended method, it was applied to the
simultaneous determination of BG and CV in river water and trout fish samples. Real samples were
prepared according to section 2.4. In both samples, concentrations of BG and CV by standard
addition method at 670 nm (1D670) and 532 nm (1D532), respectively, were calculated. The results of
analysis are summarized in Table 4. The presented method was compared with the other liquid
phase microextraction methods based on UV-Vis. spectrophotometric detection for determination of
trace levels of BG and CV dyes [3, 5, 13, 48, 49] and the results are shown in Table 5. The
15

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established IL- DLLME combined with zero crossing first derivative spectrophotometry showed
advantages like relatively low detection limits, wide linear concentration range, cost-effectiveness
and easily sample handling properties that makes the method a reliable tool in simultaneous
monitoring BG and CV in aqueous phase.
4. Conclusion
The IL-DLLME system is an environmentally friendly and simple pre-concentration way prior to
spectrophotometric determination of trace amounts of BG and CV. Although BG and CV in their
single solutions showed well defined zero order spectra in the wavelength range of 500-700 nm
with maximum absorption at 628 nm and 590 nm, respectively, but in the binary mixture, the zero
order absorption spectra of BG and CV dyes overlapped. By the first derivation of zero order
spectra, the broad shape of zero-order spectra could flat, so that signals of analytes were amplified.
Using the first derivative absorption spectra and zero-crossing method, simultaneous determination
of BG ad CV dyes was feasible. The present method showed notable points like simplicity,
efficiently, rapidly and low cost which is a good option for determination of the BG and CV dyes
even in low sample volumes. The obtained results demonstrated the capability of the method for
simultaneous detection of BG and CV at trace level in fish and water samples.
Acknowledgments
Authors are grateful to the Research Conical of the Birjand University for funding this work.
16

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Table 1. Results of percentage recovery, error and average error values for BG and CV in binary
solutions by first order derivative spectra method
Theoretical (µg L-1)
Measurement (µg L-1)
Recovery (%)
Error (%)
CBG
CCV
CBG
CCV
BG
CV
BG
CV
200
200
200
200
200
200
200
200
10
10
25
50
194.1
207.0
207.7
203.7
197.7
208.0
191.0
208.3
9.7
10.4
24.0
52.2
97.0
103.5
103.8
101.8
98.8
104.0
95.5
104.2
96.7
104.4
95.9
104.4
103.2
105.0
96.9
100.4
104.5
95.0
-2.9
3.5
3.8
1.8
-1.2
4.0
-4.5
4.2
-3.3
-2.7
-3.3
4.7
4.7
3. 5
4.9
4.4
-4.1
4.4
3.2
5.0
-3.1
0.4
4.5
-5.0
-4.7
-2.2
2.4
-3.5
3.7
5.0
4.3
-2.0
-2.9
-4.8
4.5
5.0
4.5
0.1
0.4
100
200
300
400
500
200
200
200
200
200
200
200
200
10
103.2
210.0
290.7
401.7
522.7
190.0
190.7
195.5
204.9
193.0
206.9
210.0
208.6
9.8
25
50
24.3
48.3
97.3
96.7
95.3
97.8
100
200
300
400
500
10
104.7
209.3
310.7
419.7
518.0
10.3
24.7
47.7
98.0
197.7
288.7
384.7
488.3
104.7
104.7
103.5
104.9
103.6
103.3
98.7
95.3
98.0
98.8
96.2
102.4
96.5
103.5
105.0
104.3
98.0
3.5
3.3
25
50
25
50
24.3
47.6
97.1
95.2
-1.3
-4.7
-2.0
-1.2
-3.8
-3.8
-2.3
100
200
300
400
500
100
200
300
400
500
104.5
210.0
313.6
400.5
501.9
104.5
105.0
104.5
100.1
100.4
96.2
97.7
%C
3.3
3.5
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Table 2. Figures of merit of the proposed method
Analytical feature
Parameters
BG
CV
Linear range in single solution
1-500
1-500
Correlation coefficient in single solution (R2)
Linear range in binary solution
0.9997
10-500
0.9996
10-500
Correlation coefficient in binary solution (R2)
Limit of detection in binary solution(µg L-1)
%RSD (n = 5) in binary solution
Enrichment factor
0.9974
2.7
0.9955
1.4
4.7
1.7
67
67
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Table 3. Effect of coexisting ions on the absorbance of 50 µg L-1 BG or CV in binary solutions
Tolerance ratioa
Foreign ions
BG
CV
2-
Li+, K+,Mg2+, Ca2+, Sr2+, Ba2+,SO4 ,SCN-
5000
5000
-
H2PO4
4000
3000
2000
1000
500
4000
3000
2000
1000
500
F-, I-
3-
PO4
CH3COO- ,Cu2+
-
NO2
a Amount of foreign ion/amount of BG or CV
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Table 4. Analytical results for BG and CV determination in real samples by the recommended
procedure
Added concentration
Found concentration
Recovery (%)
Sample
(μg L-1)
(μg L-1) ± SDa
BG
0
CV
0
BG
0.7 ± 0.5
CV
0.7 ± 0.4
BG
CV
-
-
10.8 ± 0.4
41.3 ± 0.3
90.3 ± 1.7
9.6 ± 0.4
48.8 ± 1.0
97.1 ± 2.4
100.6±3.4 90.0 ± 3.7
10
50
10
50
Fish
81.5±0.6
89.7±1.7
96.3 ± 2.1
96.4 ± 2.4
100
100
0
0
0.9 ± 0.4
10.9 ± 0.2
41.8 ± 0.8
91.7 ± 1.2
0.8 ± 0.7
9.1 ± 0.6
48.4 ± 0.8
94.8 ± 2.3
-
-
100.0 ±1.7 83.9 ± 4.5
82.1 ± 1.6 95.1 ± 1.6
90.9 ± 1.2 94.0 ± 2.3
10
50
10
50
Water
100
100
a
Triplicate analyses(n=3)
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Table 5. Comparison of the reported liquid phase extraction methods with the recommended
method based on UV-Vis. detection
LOD
RSD
(%)
Linear range
Analyte
Method
EF
Ref.
(μg L-1)
(μg L-1)
BG
a CPE
15
2.7
50-2000
-
3
b TC-IL-DLLME
/HPLC
CV
0.03
7.6
0.25-20
276
5
CV
CV
c IL-CIAME
a CPE
1.16
4.8
2.3
-
3.6-15
152
-
13
48
16-1000
CV
d IL-MAE/HPLC
0.08
5.9
0.1-25
10-500
-
49
BG; 2.7
CV; 1.4
BG; 4.7
CV; 1.7
BG&CV IL-DLLME- ZCDSP
67
This work
a Cloud point extraction
bTemperature-controlled ionic liquid dispersive liquid–liquid microextraction/High performance liquid chromatography
cIonic liquid cold-induced aggregation microextraction
d Ionic liquid-based microwave-assisted extraction/High performance liquid chromatography
e Ionic liquid based dispersive liquid-liquid microextraction followed by Zero-crossing first derivative
spectrophotometric method (ZCDSP)
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Figure captions
Fig. 1 Chemical structures of (A) brilliant green (BG) and (B) crystal violet(CV)
Fig. 2 The effect of (A) pH and (B) type of buffer on the extraction efficiency of BG and CV after
IL-DLLME(Conditions: Sample volume, 10 mL; dye concentration, 100 g L-1; acetate buffer,
0.1M; [OMIM][PF6] IL, 100 L; extraction time, 1 min; centrifugation time, 20 min).
Fig. 3 The effect of (A) type of ionic liquid and (B) volume of ionic liquid on the extraction
efficiency of BG and CV after IL-DLLME (Conditions: Sample volume, 10 mL; pH=5 for BG,
pH=3 for BG; dye concentration, 100 g L-1; phosphate buffer, 0.1M; [OMIM][PF6] IL, 100 L;
extraction time, 1 min; centrifugation time, 20 min)
Fig. 4 The effect of (A) extraction time and (B) centrifuge time on the extraction efficiency of BG
and CV after IL-DLLME(Conditions: Sample volume, 10 mL; pH=5 for BG, pH=3 for BG; dye
concentration, 100 g L-1; phosphate buffer, 0.1M; [OMIM][PF6] IL, 150 L
Fig. 5 The effect of salt concentration on the extraction efficiency of BG and CV after IL-DLLME
(Conditions: sample volume 10 ml, dye concentration 100 μg L-1 pH=5 for BG, pH=3 for BG; dye
concentration,100 g L-1; phosphate buffer, 0.1M; [OMIM][PF6] IL, 150 L; extraction time, 3
min; centrifugation time, 20 min)
Fig. 6 (A) Zero order absorption spectra and (B) first order derivative absorption spectra of BG
and CV in single and binary solutions of each dyes at 100 μg L-1
Fig. 7 First order derivative spectra of BG and CV in binary solutions (A) at 200 μg L-1 constant
concentration of CV with varying concentration of BG in the range of 10-500 g L-1, (B) at 200 μg
L-1 constant concentration of BG with varying concentration of CV in the range of 10-500 g L-1,
and (C) with varying concentrations of BG and CV in the range of 10-500 g L-1
26

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Highlights
► Simultaneous determination of CV and BG dyes that zero order spectrophotometric sepectra are
overlapped
► The use of environmental friendliness ionic liquid extractant [OMIM][PF6]
► Simple, efficient, rapid and cost effectiveness method for determination of the BG and CV dyes
► Successful applicable in determination of BG and CV dyes in fish and water samples
27

Graphics Abstract

Figure 1