Accepted Manuscript

A novel and simple deep eutectic solvent based liquid phase

microextraction method for rhodamine B in cosmetic products
and water samples prior to its spectrophotometric determination

Erkan Yilmaz, Mustafa Soylak

PII: S1386-1425(18)30370-6
DOI: doi:10.1016/j.saa.2018.04.073
Reference: SAA 16058
Spectrochimica Acta Part A: Molecular and Biomolecular
To appear in:
Spectroscopy
Received date: 23 July 2017
Revised date: 10 April 2018
Accepted
29 April 2018
date:

Please cite this article as: Erkan Yilmaz, Mustafa Soylak , A novel and simple deep
eutectic solvent based liquid phase microextraction method for rhodamine B in cosmetic
products and water samples prior to its spectrophotometric determination. The address
for the corresponding author was captured as affiliation for all authors. Please check if
appropriate. Saa(2017), doi:10.1016/j.saa.2018.04.073

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 ACCEPTED MANUSCRIPT

A Novel and Simple Deep Eutectic Solvent Based Liquid Phase

Microextraction Method for Rhodamine B in Cosmetic Products

and Water Samples Prior to Its Spectrophotometric

Determination

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Erkan Yilmaz, Mustafa Soylak **

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Erciyes University, Faculty of Sciences, Department of Chemistry, 38039 Kayseri-TURKEY

ABSTRACT
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A novel and green deep eutectic solvent based liquid phase microextraction (DES-

LPME) methodology has been proposed for the assessment of rhodamine B from cosmetic
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products and water samples. A deep eutectic solvent (DES) consist of tetrabutyl ammonium

chloride-decanoic acid (1:2) as extraction solvent and tetrahydrofuran as emulsification agent

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were used for the microextraction of rhodamine B. The quantitative recoveries were achieved
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at pH 3 by using 0.3 mL of DES and 0.3 mL of THF. The rhodamine B concentration in last

volume was analyzed by mirco-cuvette UV-VIS spectrophotometer at 550 nm. The limit of
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detection (LOD), limit of quantification (LOQ), preconcentration factor (PF) and relative

standard deviation (RSD %) were found as 2.2 μg L−1, 7.3 μg L−1, 25 and 2.3 %, respectively.
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Accuracy and validity of the developed method was verified by addition-recovery studies for

water and cosmetic samples.

Key-words: Deep eutectic solvents, Liquid phase microextraction, Rhodamine B, cosmetic

products, UV-VIS spectrophotometer.

*This study is a part of PhD thesis of Erkan Yilmaz

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**corresponding author, fax number: +90 352 4374933, E-mail: soylak@erciyes.edu.tr

*** Dedicated to Prof. Dr. Mehmet Dogan on the occasion of his 76th birthday.

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1. Introduction

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Rhodamin B (Rh-B), also known as rhodamine 610 and basic violet 10, is a

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fluorescent and water-soluble dye. Rh-B, a basic and cationic molecule, is used in textile,

food and pharmaceutical industries, as a fluorescence tracer in microbiology studies and in

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wavelength tuning of electromagnetic bombardment in dye lasers [1]. If Rh-B is inhaled or in

contact with the skin and eye by cosmetic products, it may causes deep irritating on skin and
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eyes, and respiratory tract in humans and animals. Moreover, Rh-B has carcinogenic effect

and their chronic and neuro-toxicities for living cells have been experimentally proven. Rh-B
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as coloring agent in cosmetics and drugs was announced as carcinogens by FDA in 1983 [2].
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Therefore, its use as a colorant in foodstuffs, drugs and cosmetic products is forbidden [3].
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However, due to its low price and bright color, some countries continue to be used as coloring
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additives [4]. Hence, accurate and sensitive determination of Rhodamine B in environmental

samples is an important task for scientists [4, 5].

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Various analytical techniques such as high-performance liquid chromatography

(HPLC) [5], UV-VIS spectrophotometry [3], fluorescence spectrophotometry [6], capillary

chromatography [7] have been used for determination of Rh-B. Among of them, UV-VIS

spectrophotometry provides important advantages such as easier operation, lower-cost

analysis and better accuracy [8]. But, direct determination of Rh-B without any pretreatment

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procedure by using UV-VIS spectrophotometry is difficult or not possible due to the matrix

effect of foreign species and its low concentration in real samples [4, 8].

For the last two decade, the use of conventional pretreatment procedure such as

solvent extraction, solid phase extraction, cloud point extraction and coprecipitation has

dropped because of the necessity of high-cost operation apparatus, high consumption of

reagents and toxic organic solvents which cause secondary waste. To eliminate these

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disadvantages, scientists have made considerable effort to find the efficient, economical and

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miniaturized alternative methods to fulfill the requirements of green chemistry and they have

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found microextraction methods involving liquid phase microextraction (LPME) [9] and solid

phase microextraction methods (SPME) [10]. LPME methods are very popular,
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environmentally friendly, inexpensive and quick techniques. In recent years, number of

research in the area of LPME has increased by development of new solvents (organic
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solvents, ionic liquids, supramolecular solvents, switchable solvents and deep eutectic

solvents) and apparatus (ultrasonic irritation apparatus, vortex mixer and microwave
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apparatus) [11-13].
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The most important step in new generation liquid phase microextraction methods is
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the selection of the right, cheap and green solvents, which to eliminate the use of traditional
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toxicological solvents as well as to provide low cost and improve health and safety [12]. Deep

eutectic solvents (DESs) are called new generation green solvent systems and some of them
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are drinkable. DESs are typically prepared by mixing the two safe salts, which are generally

ammonium salts and hydrogen bond donors (HBD). Choline chloride (ChCl) (Vitamin B4) is

the most used ammonium salt and can be acquired from biomass or fossil reserves with a

simple process. Urea, carboxylic acids or polyols have been used as hydrogen bond donors

(HBD) [13, 14]. Deep eutectic solvents have found many chemical application areas such as

extraction-digestion of inorganic and organic compounds, synthesis of organic, inorganic

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compounds and nanomaterials, dissolution of drugs and metal oxides and purification of

valuable organic compounds such as oil and biodiesel [15, 16].

DESs have been widely used as new generation solvents especially in microextraction

studies. Some important applications of them are summarized as follows; Zounr et al. (2018)

developed a ultrasonic assisted deep eutectic solvent liquid-phase microextraction method for

preconcentration of cadmium (Cd) in water and food samples prior to electrothermal atomic

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absorption spectrometry (ETAAS). They used choline chloride and phenol at molar ratios of 1

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and 4 to prepare the DES as extraction solvent [17].

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Ferrone et al. synthesized a DES as eco-friendly solvent by using phenylacetic acid

and betaine, and used it as extraction solvent in the DLLME method for preconcentration and
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separation of oxyprenylated phenylpropanoids in some vegetable oils prior to ultra high

performance liquid chromatography detection. In this study, eco-friendly solvents were used
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as the first time to extract the oxyprenylated phenylpropanoids [18]. A new deep eutectic

solvent based temperature-controlled liquid phase microextraction method for the extraction
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and preconcentration of some pesticides from different samples prior to gas chromatography-
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flame ionization detection has been developed by Farajzadeh et al.. They prepared the DESs
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as extraction solvent by mixing the p–chlorophenol as hydrogen bonding donor and choline
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chloride as hydrogen bonding acceptor. In this study, dispersion of the extraction solvent into

the aqueous phase was performed by temperature changing [19]. Zeng et al. have been
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developed a DLLME method based on the solidification of DES for the extraction of five

benzoylureas. They applied this DLLME-HPLC procedure for determination of the

benzoylureas in real water samples [20]. Yilmaz and Soylak have been suggested a green

ultrasound assisted-deep eutectic solvent (DES) extraction method for directly extraction of

iron from liver samples of animals prior to flame atomic absorption spectrometric

determination [21].

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In this study, we developed a simple, cheap and safe deep eutectic solvent based liquid

phase microextraction application for extraction and preconcentration of the Rh-B prior to its

mirco-cuvette UV-VIS spectrophotometer determination. Important analytical parameters

such as the pH of aqueous phase, type and volume of DES, sample volume, volume of THF

and matrix effect were optimized to improve the extraction efficiency and sensitivity of the

developed DES-LPME method. We think that the DES-LPME developed can be used as

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routine and widespread analysis method for most of analytical laboratory.

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2. Experimental
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2.1. Instrumental

pH adjustment of all the sample solutions was conducted by A pH-meter with Nel pH
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900 (Ankara-Turkey) Model glass-electrode. The Rhodamine B determinations in extraction

solvent phase was carried out by a Hitachi UH 5300 spectrophotometer with quartz micro–
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cell, which has 700 µL of total volume. The production of nano-sized and/or micro-sized
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emulsions was accomplished in a ultrasonic bath, which has maximum 35 kHz ultrasound
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frequencies (Sonorex, Model No. DT-255). A Hettich Rotofix 32 centrifuge

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(Buckinghamshire, England) was used.

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2.2. Chemicals and reagents

All reagents used were analytical grade and used without purification. Rhodamine B

was obtained from Merck (Darmstadt, Germany). Choline chloride (ChCl) ( Purity ≥ 99 %),

tetrabutylaamonium chloride (TBACl) (Purity ≥ 99 %) and methyltrioctylammonium chloride

(MTOACl) (Purity ≥ 99 %) obtained from Sigma-Aldrich, USA were used as quaternary

ammonium salts, while phenol (Ph) ( Purity ≥ 99 %, Merck) and decanoic acid (DA) (Puritt:

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98 %, SAFC, USA) were used as hydrogen bond donors. Ultra-pure water was obtained using

a Milli-Q system (18.2 MΩ cm resistivity) from Millipore Corporation, USA.

2.3. Synthesis of deep eutectic solvents

Deep eutectic solvents used in the presented work were synthesized by using the same

method, which used in our previous experiments [22, 23]; DES1: Mixing 13.96 g of ChCl

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with 9.41 g of Ph at molar ratios 1:2, DES2: Mixing 40.4 g of MTOACl with 17.3 g of DA at

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molar ratios 1:2, DES3, DES4 and DES5: Mixing 10.96 of TBACl with DA at different

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quantities 17.3 g, 34.6 g and 51.9 g for 1:2, 1:3 and 1:4 molar ratios. The components of

DESs were mixed in a beaker and then stirred at 25 °C until a clear liquid was formed about 5
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min.
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2.4. DES-LPME Procedure

The DES-LPME method were performed in 10 mL of aqueous sample solutions with pH 3,

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containing 1x10-5 M Rhodamine B. 300 µL of DES3 and 300 µL of THF was added in the
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aqueous sample solution and then the obtained mixture was sonicated in a ultrasonic bath for
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2 min. At this stage, a nano-sized and micro-sized cloudy DES droplet was formed because of
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the transient cavitation near the interface of DES droplets. At this stage, rhodamine B in

aqueous phase was extracted into hundreds of DES droplets. The formation of Nano-sized and
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micro-sized cloudy DES droplets by transient cavitation near the interface of DES droplets

cause to obtain homogeneously dispersed extraction solvent with higher surface area in

aqueous phase. So, higher extraction efficiency for rhodamine B was obtained. Separation of

the water phase and DES rich phase was achieved by centrifugation process at 4000 rpm for

10 min. The DES phase containing Rhodamine B was taken by using micropipette and the

volume of DES phase was completed to 600 µL with methanol. Then, the rhodamine B

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concentration in last volume was measured as soon as possible to avoid evaporation of

methanol at 550 nm by Uv-Visible spectrophotometer. The total analyze time is

approximately 15 min. The same procedure was also applied for blank solutions. The

experimental steps of the developed method are represented in Figure 1.

2.5. Applications

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The developed DES-LPME method was applied for determination of the Rhodamine

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B concentration of the different water and cosmetic products collected from different region

of Turkey. Sea water and river water samples were taken from İstanbul and Kayseri, Turkey.

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Two types of lipsticks, cologne and acetone cleaning solution of nail polish samples were
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collected from cosmetic markets served in Kayseri, Turkey. The collected water samples were

used after filtration through 0.45 μm cellulose membrane filter. To prepare the lipstick
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samples, 5 grams of lipstick samples were taken and 10 mL of ethyl alcohol was added to the

lipstick samples. The resulting mixture was shaken in the shaking bath for 1 hour. In this case,
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rhodamine B in the lipstick samples passed into the ethyl alcohol phase. When this mixture
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was centrifuged, a solid portion insoluble in ethyl alcohol collected at the bottom of the
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centrifuge tube and Rhodamine B solution in the upper part were obtained. 0.25 mL of ethyl
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alcohol phase was taken and the volume of the phase was completed to the 10 mL with ultra-

pure water. Then, the developed procedure given in “section 2.4.” was applied to these
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samples.

10 mL of cologne and nail polish remover solutions were taken and acetone in these

solutions was removed in a hot water bath (~ 50 ° C). The volume of the obtained residue was

completed with 10 mL of water, and the method given above was applied to the 1.0 mL

portions obtained from these samples.

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2.6. Quality assurance/Quality control

Besides the development of the analytical method, quality assurance (QA) and quality

control (QC) are considered of extraordinary significance. In this study, QA and QC criteria is

consist of analyses of blank solutions, surrogate standard recoveries and laboratory control

samples. The developed method was applied to three replicate of blank solutions to check

contaminations from the preparative steps and to check laboratory background levels. In our

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method, background levels of laboratory blanks were below the limit of detection. The

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recovery assay was studied by using laboratory control samples. The rhodamine B

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concentration in blank samples was subtracted. Surrogate standard recoveries were checked

by addition-recovery analysis for real samples mentioned section 2.6.

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3. Results and discussion

3.1. Effect of pH
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The pH of the working media is one of the critical parameter for the quantitative
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recoveries of the analytes from extraction studies [24-28]. The effect of pH of working media
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on the extraction efficiency of rhodamine B was investigated in the pH range of 2.0-6.0. The
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results are represented in Figure 2. The quantitative recoveries for rhodamine B were obtained

at all working pH range. There is only a ionizable carboxyl group in rhodamine B molecule
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and in the acidic pH’ s, the carboxyl group of rhodamine B is not ionizing and the rhodamine

B molecule is counter-neutral. Due to its neutrality in the decanoic acid phase, Rhodamine B

easily pass from the water phase to the extraction phase at acidic pH’ s. Moreover, the

separation of the extraction phase from the water phase becomes more difficult as the pH

value of the aqueous solution increases due to ionisation of decanoic acid. Hence, for further

experimental works, pH 3.0 was selected as optimal pH.

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3.2. Type of DES

The influences of different type of deep eutectic solvents given in Table 1 as

extraction solvent on the recoveries of rhodamine B were investigated. The quantitative

recoveries for rhodamine B were obtained with all DES solutions given in Table 1. Tetrabutyl

ammonium chloride-decanoic acid (1:2) (DES3) was selected as extraction solvent due to easy

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phase separation with this combination.

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3.3. Effect of DES volume

The effect of the volume of tetrabutyl ammonium chloride-decanoic acid (1:2) (DES3)
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solution on the recoveries of rhodamine B on the presented extraction system was examined

in the volume range of 0.1-0.5 mL. The results are depicted in Figure 3. In the volume of 0.1
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mL and 0.2 mL, the phase separations were not performed completely. Complete phase

separations were obtained after 0.3 mL. Also quantitative recoveries for rhodamine B were
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obtained in the DES3 volume range of 0.3-0.5 mL. Hence, 0.3 mL of DES3 was selected as
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optimal volume for further studies.

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3.4. Effect of THF volume on the recoveries of Rhodamine B

Self-aggregation and separation of DESs molecules in aqueous phase were occurred

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by adding THF as an emulsifier agent and aggregates become insoluble in the water/THF. The

influences of the volume of tetra hydrofuran (THF) were investigated. The results are

depicted in Figure 4. The recoveries of rhodamine B were quantitative THF volume in range

of 0.3-0.5 mL. 0.3 mL of THF was preferred as optimal value for next experiments. The

optimized parameters on the presented deep eutectic solvent based liquid phase

microextraction are represented in Table 2.

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3.5. Matrix effects

Due to the interferic efffecs of the alkaline ions, earth alkaline ions, cations, anions

and organic [29-33] should be investigated in the extraction studies, the influences of some

anions, cations and dyes on the recoveries of rhodamine B on the presented microextraction

procedure were investigated. The results are represented in Table 3. The tolerance limit of

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foreign ions and dyes were taken as that value which caused an error of not more than ±5 % in

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the absorbance. This results show that, the proposed microextraction method could be used

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for various samples.
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3.6. Analytical performance

The analytical performance including preconcentration factor (PF), limit of detection

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(LOD), limit of quantification (LOQ) etc. were summarized in Table 4. The preconcentration

factor (PF), defined as the ratio of the highest model solution volume (15 mL) to the final
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volume (0.6 mL), was 25. The LOD was calculated with three standard deviation of blank by
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the slope of calibration curve followed by the multiplication of preconcentration factor.

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While, the LOQ was calculated by the ten standard deviation of blank by the slope of
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calibration curve followed by the multiplication of preconcentration factor. The relative

standard deviation (RSD) was calculated from seven replicate measurements of known
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concentration of rhodamine B.

3.7. Method validation

The method validation of the developed DES-LPME method was carried out by

addition-recovery experiments for the spiked real samples. For this purpose, known

concentration of Rhodamine B were added in the multiple type of samples (River water, sea

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water, cologne nail polish cleaner and lipstick samples) and recovery experiments with the

developed DES-LPME method were carried out. The obtained recovery results are

represented in Table 5. The obtained results for real samples showed that the recoveries of the

DES-LPME method were from 97 to 110 % with low standard deviations. The data obtained

for validation of the DES-LPME proved that the method was not affected samples matrix and

applicable for preconcentration and accurate determination of Rhodamine B in different

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samples.

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Conclusions

A combination of DES-LPME and UV-VIS spectrophotometry detection was

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exhibited as environmental friendly, very simple and low cost procedure for the

preconcentration and determination of Rhodamine B in cosmetic products and water samples

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has been presented.

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The developed DES-LPME -UV-VIS spectrophotometer combination method has the

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following advantages and disadvantages;

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 The developed DES-LPME method can be used as an alternative to expensive and

time consuming techniques for routine analysis of Rhodamine B in cosmetic products

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and water samples.

 It is possible to analysis by use of micro-sampling UV-VIS spectrophotometer, which

is cheap and easy to use instead of expensive instruments such as HPLC, LC-MS,

which are used in the determination of rodamin B.

 The DES-LPME method obviously provides a better or comparable limit of detection

and preconcentration factor, when compared with other literature studies (Table 6).

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 This method provides accurate analysis, high reproducibility and high extraction

efficiency by using a low amount of samples

 Even though the use of organic solvents cannot be completely eliminated, it is

minimal compared to other enrichment methods.

Acknowledgement

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The authors are grateful for the financial support of the Unit of the Scientific Research

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Projects of Erciyes University (FBA-2015-5783).

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preconcentration and determination of chromium (VI), Talanta,184 (2018) 15-22.

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by electrothermal atomic absorption spectrometry; Response surface methodology-central

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[32] M. Ghaedi, J. Tashkhourian, M. Montazerozohori, M.N. Biyareh, B. Sadeghian, Highly

selective and sensitive determination of copper ion by two novel optical sensors, Arab. J.

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of Heavy Metals in Water and Food Samples Using 2-(2'-Benzothiazolylazo)-6-

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for preconcentration and determination of rhodamine B, Sep. Pur. Tech. 97 (2012) 216-

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[35] M. Soylak, Y. E. Unsal, E. Yilmaz, M. Tuzen, Determination of rhodamine B in soft

drink, waste water and lipstick samples after solid phase extraction, Food Chem. Toxicol.
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[36] J.S. Xu, J. Zhang, X.H. Sun, Y. Cui, F. Zhang, D.C. Yuan, Determination of rhodamine b
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in beverage by a solid phase extraction-high performance liquid chromatography,

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Chemical Research Application 26 (2014) 741–743.

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[37] Y. Yu, Z. Fan, Determination of Rhodamine B in Beverages Using a Polystyrene-Coated

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Magnetite Nanocomposite for Magnetic Solid Phase Extraction, Anal. Lett. 46 (2016)

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[38] E. Ranjbari, M. R. Hadjmohammadi, Optimization of magnetic stirring assisted

dispersive liquid–liquid microextraction of rhodamine B and rhodamine 6G by response

surface methodology: Application in water samples, soft drink, and cosmetic products,

Talanta 139 (2015) 216-225.

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Figure captions

Figure 1. Presentation of the microextraction procedure for Rhodamine B

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Figure 2. Effect of pH on the recovery of Rhodamine B (N=3).

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Figure 3. The effects of tetrabutyl ammonium chloride-decanoic acid (1:2) (DES3) volume on

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the recoveries of Rhodamine B (N= 3).

Figure 4. Effect of THF volume on the recovery values of Rodamine B (N= 3)

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Table 1. Effect of DES type on the recovery of Rhodamine B (N=3).

DES Component Molar

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Salt Recovery, %
type for H-bonding ratio

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DES1 Chlonine chloride Phenol 1:2 100±1

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DES2 Methyl Trioctylammonium chloride Decanoic acid 1:2 95±2

DES3 Tetrabuthylamonium chloride Decanoic acid 1:2 98±1

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DES4 Tetrabuthylamonium chloride Decanoic acid 1:3 99±3

DES4 Tetrabuthylamonium chloride Decanoic acid 1:4 95±4

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Table 2. Optimal experimental parameters obtained for preconcentration of the

rhodamine B by deep eutectic solvent based liquid phase microextraction

method.

Parameter Optimum value

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pH 3.0

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The molar ratio of TBACl:DA 1:2

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DES volume, µL 300

THF volume, µL 300

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Ultrasonic sonication time, min. 2

Centrifugation time, dk. 10

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Sample volume, mL 15

Last volume, µL 600

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Table 3. Effects of some foreign ions and dyes on the recoveries of rhodamine B in the

presented microextraction procedure (N=3).

Ion or molecules Concentration, mg L-1 Recovery, %

Ca2+ 1000 106±2

Mg2+ 500 103±0

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K+ 2500 99±1

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Na+ 2500 96±2

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Zn2+ 5 99±2

Cu2+ 5 106±3
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SO42- 2500 105±1

Cl- 2500 96±2

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Brillant Black BN 10 93±4

Sunset Yellow 10 106±0

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Quercetin 10 101±5
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Curcumin 10 104±3

Tartrazine 10 102±5
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Table 4. Analytical characteristics of the developed DES-LPME method

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Parameter Value

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Preconcentration Factor 25

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Limit of detection, 3s/m, µg L-1 (N=10) 2.2

Limit of quantification, 10s/m, µg L-1 (N=10) 7.3

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Relative standard deviation, % 2.3

Calibration equation y = 0.0037x + 6.3x10-4

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Regression coefficient , R2 0.998

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Table 5. Addition and recovery test for DES-LPME of Rhodamine B in different samples

(N= 3).

Added Found Recovery Added Found Recovery

Sample Sample
µg mL-1 µg mL-1 % µg mL-1 µg mL-1 %

0.0 UDL a - 0.0 UDL -

River
3.0 3.1±0.1b 103 Sea water 4.5 4.4±0.1 98

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water
6.0 6.1±0.2 102 9.0 9.0±0.2 100

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0.0 1.25±0.40 - 0.0 0.3±0.02 -
Cologne Nail polish

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4.5 5.9±0.1 102 4.5 4.9±0.02 102
cleaner
9.0 10.5±0.3 102 9.0 10.2±0.03 110
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Added Found Recovery Added Found Recovery
Sample Sample
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µg g-1 µg g-1 % µg g-1 µg g-1 %

0.0 9.3±0.40 - 0.0 11.6±1.0 -

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Lipstick-I 6.0 16.1±0.1 105 Lipstick-II 6.0 17.3±0.3 98

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9.0 17.8±0.3 97 9.0 20.5±1.1 100

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a UDL: Under Of The Detection Limit.

b Mean ± standard deviation.
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Table 6. Comparison of the DES-LPME method with other methods for the determination of

Rhodamine B in real samples.

Method PF LOD, µg L-1 Sample Ref.

Dispersive liquid-liquid
65.5 1.05 Water, lipstick, dye and soap [34]
microextraction

Solid phase extraction 40 3.14 Water and lipstick [35]

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Solid phase extraction - 15 - [36]

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Magnetic solid phase
100 2.1 Fruit juice [37]

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extraction

Dispersive liquid-liquid Water, fruit juice and

48 1.15 [38]
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microextraction cosmetic products

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DES-LPME 25 2.2 water and cosmetic products

study

PF: Preconcentration factor

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LOD: Limit of detection

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Figure 1.
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100

80
Recovery, %

60

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40

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2 3 4 5 6
pH
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Figure 2.
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Figure 3.
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100

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Recovery, %

80

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60

40
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0.1 0.2 0.3 0.4 0.5 0.6
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THF volume, mL

Figure 4.
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Graphical abstract

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Highlights
 A novel green deep eutectic solvent based liquid phase microextraction (DES-LPME)

method has been developed for rhodamine B.

 The effects of analytical parameters were systematically investigated and optimized.

 The method has been applied to the analysis of rhodamine B in cosmetic products and

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water samples.

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