Turkish Journal of Chemistry Turk J Chem

(2017) 41: 987 – 994

http://journals.tubitak.gov.tr/chem/
⃝c TÜBİTAK
Research Article doi:10.3906/kim-1702-72

Spectrophotometric detection of rhodamine B in tap water, lipstick, rouge, and

nail polish samples after supramolecular solvent microextraction

Nebiye ÖZKANTAR1 , Mustafa SOYLAK2 , Mustafa TÜZEN1,∗

1
Department of Chemistry, Faculty of Science and Arts, Gaziosmanpaşa University, Tokat, Turkey
2
Department of Chemistry, Faculty of Sciences, Erciyes University, Kayseri, Turkey

Received: 01.03.2017 • Accepted/Published Online: 29.06.2017 • Final Version: 20.12.2017

Abstract: A simple and sensitive supramolecular solvent-based dispersive liquid–liquid microextraction method was
described for the separation/preconcentration and spectrophotometric detection of rhodamine B. The microextraction
method, which was realized at ambient temperature for the detection of rhodamine B, was carried out by using
supramolecular solvents such as tetrahydrofuran and decanoic acid. The method was based on analyses of rhodamine B
by using UV-Vis spectrophotometry at 558 nm. The influences of some parameters such as pH, sample volume, eluent
solutions, centrifugation time, and ultrasonic bath time were optimized. The effects of various matrix ions were also
investigated. Moreover, the limit of detection and limit of quantification were calculated as 0.49 µ g L −1 and 1.47 µ g
L −1 , respectively. The preconcentration factor was 30. The relative standard deviation was determined as 5.8% in 0.5
× 10 −4 M rhodamine B. The procedure was validated by addition/recovery tests. The microextraction method was
applied to determination of rhodamine B in tap water samples and cosmetic samples such as nail polish, rouge, and
lipstick.

Key words: Preconcentration, rhodamine B, spectrophotometry, microextraction, cosmetic, water

1. Introduction
In industrial areas, dyes are used to color textiles, leather, paper, plastics, etc., and water is also consumed
remarkably. 1 These dyes, which originate from industrial processes, enter the environment through waste water. 2
The presence of dyes in waste water is a major source of concern as it has negative effects on many areas of life.
The discharge of dyes in the environment is a matter of concern for both toxicological and esthetical reasons. 3
One of these dyes, rhodamine B, which appears red to violet, has a molecular weight of 479.02 g/mol and
molecular formula of C 28 H 31 ClN 2 O 3 . It is commonly used as a tracer dye in water to determine the rate and
direction of flow and transport. Rhodamine dyes are used extensively in biotechnology applications such as
fluorescence microscopy, flow cytometry, fluorescence correlation spectroscopy, and ELISA. 4
Due to its extensive pink color, rhodamine B has been widely used as a dye in many industrial applications,
such as in the food, textile, drug, and cosmetic industries. Rhodamine B is used for coloring in many fields, such
as in drugs, cosmetics, and textile products. It is also used as a tool for tracing water pollution. Its usage in
industrial fields, which is a menace to human health, brings about toxicity for organisms living in water. It may
cause long-term unhealthy effects in aquatic environments and it is harmful when it comes in contact with skin.
On the other hand, rhodamine B may cause congenital diseases and cancer, which is the worst disease of our
∗ Correspondence: mustafa.tuzen@gop.edu.tr

987
 ÖZKANTAR et al./Turk J Chem

age. Rhodamine B is a triphenylmethane dye. Due to the harmful effects of rhodamine B, many countries have
prohibited it from using in food samples. Because of its harmful effects, rhodamine B, which exists in many
samples such as waste water, is supposed to be removed with separation-enrichment and analysis methods. This
requirement leads to the developing of many analytical methods.
Sample preparation is one of the most important steps in the analysis of rhodamine B with a UV-Vis
spectrophotometer. Due to the very low concentrations of rhodamine B that can be found in environmental
samples, cosmetics, and food samples and on account of its matrix effects, a number of effective separation and
enrichment methods have been improved for the determination of rhodamine B. 5−7 Some of these methods are
adsorption, cloud point extraction, and liquid–liquid extraction. These methods have some disadvantages such
as causing considerable amounts of chemical solvent losses and usage, having a small preconcentration factor, and
requiring many processes. Therefore, microextraction methods such as dispersive liquid–liquid microextraction,
supramolecular solvent-based microextraction, and single drop microextraction have become popular. In this
study, a supramolecular solvent-based microextraction process has been developed for the process of separation
and enrichment of rhodamine B that exists at trace levels in various samples. This method is a well-defined
supramolecular solvent system based on the merging of two or more components. As a result of this combination,
the processing of polar and nonpolar solvent systems consists of two parts, which are nano and molecular in
size. 8−13
Analysis of dyes has been performed by fluorescence spectrometry, mass spectrometry, phosphorescence
spectrometry, and UV-Vis spectrometry. UV-Vis spectrometry is very important and has been used substantially
for determination of dyes due to its simple usage and being cheaper than other approaches. Most of the studies
based on the determination of dye were performed using UV-Vis spectrometry. 14−21
The aim of this study was to develop a simple and rapid supramolecular solvent-based microextraction
method for separation/enrichment and analysis of rhodamine B in real samples by using UV-Vis spectrometry.
The analysis of rhodamine B by UV-Vis spectrometry was carried out at a wavelength of 558 nm. The analytical
parameters for quantitative extraction were optimized.

2. Results and discussion

2.1. Effect of pH
The transformation of rhodamine B from liquid phase to extraction phase depends on the pH value. Model
solutions were prepared at different pH values (1.0–6.0) with the help of buffer solution in order to determine
the optimum pH range for the quantitative extraction of rhodamine B. For this purpose, model solutions were
prepared between the values of 1 and 6. Later, the microextraction method was applied to the specimens
by UV-Vis spectrophotometer. All results obtained at pH 3.0 were quantitative and the highest recovery was
obtained at pH 3.0 (Figure 1). The optimum sample solution pH was chosen as 3.0 and the subsequent work
was continued at pH 3.0.

2.2. Effect of supramolecular solvent type and amount

When microextraction studies from preliminary methods are examined, the types and quantities of the compo-
nents rank among the most important parameters. Different organic solvents, which include tetrahydrofuran
(THF) with 1-decanol, undecanol, and decanoic acid, are tested with the intent of optimization of the method.
While recovering rhodamine B, the effects of nonmolecular, water-immiscible, and micelle-forming organic sol-
vents were identified. For this, THF-decanoic acid, THF-undecanol, and THF-1-decanol solvents were used.

988
 ÖZKANTAR et al./Turk J Chem

The results of organic solvent type parameter selection were 99%, 79%, and 86%, respectively. The supramolec-
ular solvent-based microextraction method with decanoic acid was chosen since recovery values of 95% or more
were obtained. For this parameter, the amount of supramolecular solvents, which were chosen to obtain the
best recovery value, were investigated after the solvent selection.
Extraction solvents containing different amounts of decanoic acid (50–200 mg) were prepared to determine
the optimal decanoic acid amount that should be present in the supramolecular solvent. The optimum amount
of decanoic acid was selected as 150 mg (Figure 2).

110

100

90

Recovery (%)
80

70

60

50
50 75 100 125 150 175 200 225
mg, Decanoic acid

Figure 1. Effects of pH on the recoveries of rhodamine B Figure 2. Effects of decanoic acid amount on the recov-
(N = 3). eries of rhodamine B (N = 3).

In addition, the influence of THF volume, which should be present in the supramolecular solvent, was
studied for the recovery of the rhodamine B. For this, 150 mg of decanoic acid and supramolecular solvents
containing THF with increasing volumes were prepared. Quantitative recovery values were obtained when the
volume of THF was between 550 µL and 600 µ L. Optimum THF volume was selected as 600 µ L. The volume
of THF was kept constant at 0.6 mL (Figure 3).

110

100
Recovery (%)

90

80

70

60

50
200 250 300 350 400 450 500 550 600
µL, THF

Figure 3. Effects of THF volume on the recoveries of rhodamine B (N = 3).

As a result, further experiments were carried out using 150 mg of decanoic acid and 0.6 mL of THF,
followed by separation-enrichment by microextraction with supramolecular solvent.

2.3. Effects of centrifugation time and ultrasonic bath time

After extraction solvent was injected into the model solution platform, mixtures were sonicated between 1 and
5 min in order to provide the formation of the micelle with the help of ultrasonic vibration. As the sonication
time increased, more clouding occurred in the mixtures. This indicates that extraction drops whose dimensions

989
 ÖZKANTAR et al./Turk J Chem

are nano or molecular dissolve more in the solution platform. In addition to this observation, concordantly, the
highest and quantitative recovery values were obtained in 5 min. A centrifuge operation was applied to mixtures
at 4000 rpm between 5 and 20 min to separate the supramolecular extraction phase from the water phase. In the
end, a notable phase separation and quantitative results were observed with 10 min for the centrifuge operation.

2.4. Effect of sample volume

Samples in volumes of 10 to 40 mL were prepared so as to investigate the effects of sample volume of the recovery
of rhodamine B under the optimum conditions. While obtaining quantitative recovery values for the sample of
30 mL, recovery values started decreasing in the samples of over 30 mL. In this manner, the preconcentration
factor was calculated as 30 owing to the fact that the last volume was 1000 µ L.

2.5. Matrix effects

Alkaline metals, earth alkaline metals, dyes, and some anions with a concentration above the tolerance level
bring about the matrix effect in the determination of rhodamine B. That is why, once the developed method
is applied, evaluating the matrix effect of foreign ions is one of the most important parameters. The tolerance
concentrations of each of the foreign ions and dyes mentioned above were investigated and the recovery values
were calculated. The results are shown in Table 1.

Table 1. Influences of some foreign ions on the recoveries of rhodamine B (sample volume: 10 mL, final volume: 1 mL,
N = 3).

Foreign ions Added as Concentration (mg L−1 ) Recovery (%)

K +
KCl 2000 96 ± 4*
Mg 2+
MgCl2 1000 95 ± 3
Ca 2+
CaCl2 1000 96 ± 3
SO2−
4 Na2 SO4 2500 95 ± 3
Na+
NaCl 2000 100 ± 1
Pb 2+
Pb(NO3 )2 100 97 ± 2
Cu 2+
Cu(NO3 )2 50 98 ± 3
Zn 2+
Zn(NO3 )2 50 98 ± 1
Chromotrope FB Chromotrope FB 100 96 ± 5
Sudan I Sudan I 1 100 ± 2
Sudan Orange G Sudan Orange G 1 101 ± 3
Chicago Sky Blue 6B Chicago Sky Blue 6B 100 97 ± 5
Brilliant Black BN Brilliant Black BN 20 97 ± 1
*Mean ± standard deviation.

2.6. Analytical performance

Addition/recovery experiments were carried out to prove the validity of the applied supramolecular solvent-
based microextraction method. With the known concentration, the recovery value of the added concentration
was calculated by adding the sample solution of rhodamine B. The results are shown in Table 2.

990
 ÖZKANTAR et al./Turk J Chem

Table 2. Tests of addition/recovery in the experiments for rhodamine B (sample volume: 10 mL, final volume: 1 mL,
N = 3).
Samples Added (µg / mL) Found (µg / mL) Recovery (%)
0.0 N. D.* -
2.4 2.36 ± 0.02* 98
Tap water
4.8 4.75 ± 0.03 98
7.2 7.23 ± 0.05 100
9.6 9.67 ± 0.02 101
0.0 1.12 ± 0.02 -
Lipstick 2.4 3.50 ± 0.02 99
7.2 8.25 ± 0.10 99
0.0 1.49 ± 0.005 -
Rouge 4.8 6.47 ± 0.10 104
7.2 8.72 ± 0.20 100
0.0 0.03 ± 0.01 -
Nail polish 2.4 2.47 ± 0.04 102
4.8 4.84 ±0.05 100
*Mean ± standard deviation, N. D.: not detected.

Limit of detection, limit of quantification, relative standard deviation, and enrichment factors were
calculated to evaluate the analytical performance of the developed supramolecular solvent-based microextraction
method. The results of analytical performance studies are indicated in Table 3. The regression equation was A
= 0.140C + 0.0012. The linear range was calculated as 1.5–70 µ g L −1 .

Table 3. Validation parameters for rhodamine B analysis.

Limit of detection (LOD) (µg L−1 ) 0.49

−1
Limit of quantification (LOQ) (µg L ) 1.47
Relative standard deviation (RSD) (%) 5.8
2
Correlation coefficient (r ) 0.9997
Preconcentration factor 30
Linear range 1.5–70 µg L−1

2.7. Analysis of real samples

The method developed for addition/recovery was applied to cosmetic materials such as lipstick, rouge, and
nail polish and to water samples. Before applying the developed microextraction method, 0.5 g of the lipstick,
rouge, and nail polish samples was weighed and taken out with a scoop and 10 mL of ethyl alcohol was added
to the shaker for about 2 h to dissolve the dye phase. Five parallel tubes were then prepared to apply the
developed microextraction method and 0.0–9.6 µ g/mL of rhodamine B was added to these sample solutions
and the developed microextraction method was applied. Finally, analyte concentrations in the final volume
were analyzed with a UV-Vis spectrophotometer.

991
 ÖZKANTAR et al./Turk J Chem

In addition, the method developed for this study was applied to tap water samples. Blank samples were
also analyzed. The validation of the method was checked by the addition-recovery tests. The results are shown
in Table 2.

2.8. Comparison with existing methods

The proposed supramolecular solvent-based microextraction procedure for rhodamine B was compared with
other methods from a literature survey. According to the literature reviews, this study has good efficiency
compared to other studies related to rhodamine B because of the large surface area between the analyte and
microextraction solvent. In comparison with other techniques for rhodamine B removal, the present study takes
place in a short extraction time and at room temperature. Higher recovery values were obtained in this study
compared to other studies and these values were attained within a shorter period and in lower concentrations
by using rhodamine B and organic solvents. 22 A low detection limit was found in this study compared to some
literature values, as shown in Table 4.

Table 4. Comparison between the proposed method and other procedures from the reported literature for rhodamine
B determination.

RSD*
Preconcentration method Analysis method LOD PF* Samples Ref.
(%)
Ionic liquid aqueous
UV-Vis spectrophotometer 3.8 3.2 ng L−1 Soft drink 4
two-phase systems coupled
Soft drink, waste water,
Solid phase extraction UV-Vis spectrophotometer 5 3.14 µg L−1 40 5
and lipstick samples
Dispersive liquid–liquid Environmental and food,
UV-Vis spectrophotometer 4 2.1 µg L−1 20
microextraction cosmetics, and water samples
High-performance liquid Red wine and river water
Solid phase extraction 3.6 µg L−1 21
chromatography samples
Wine, grape juice, blueberry
Ultrasound-assisted DSPE HPLC-DAD < 6.8 0.28 µg L−1 91 22
juice, and chili oil
microextraction UV-Vis spectrophotometer 5.8 0.49 µg L−1 30 Cosmetics and water samples This work
*RSD: Relative standard deviation; PF: preconcentration factor.

2.9. Conclusions
A supramolecular solvent-based dispersive liquid–liquid microextraction method for determination of trace
amounts of rhodamine B was established using a UV-Vis spectrophotometer. In our study, in low quantities
organic solvents such as 600 µ L of THF and 150 mg of decanoic acid were used to form the supramoleculer
solvent phase. Therefore, it can be said that this study is environmentally friendly and cost-efficient and has
almost no negative effects on health. This method was applied in a simple manner without spending much time
and very efficient results were obtained by using only one extraction method. Moreover, the selectivity of the
method is good; there is no interaction effect in the presence of matrix ions. Owing to such advantages, the
method is practical and reliable for determining rhodamine B in environmental water samples and cosmetics. At
the same time, the developed supramolecular microextraction method can be implemented not only for detection
of organic types but also for separation and enrichment studies of inorganic types such as trace elements.

992
 ÖZKANTAR et al./Turk J Chem

3. Experimental
3.1. Apparatus
A Hitachi 150-20 UV-Vis spectrometer was used in order to measure absorbance values of rhodamine B. A
PHS-3C pH meter (Nel pH-900, Ankara, Turkey) was utilized with a combined glass electrode to determine pH
values. The distilled water was obtained with the help of the Millipore Milli-Q system (18 M Ω cm −1 resistivity,
Millipore, Bedford, MA, USA). An ALC PK 120 model centrifuge (Buckinghamshire, UK) was used.

3.2. Chemicals and reagents

All reagents and chemicals were utilized in analytical purity. A standard rhodamine B 1 × 10 −3 M stock
solution was prepared in ethanol (Sigma-Aldrich Co., Milwaukee, WI, USA). The solution was diluted to 1 ×
10 −4 M in order to form a calibration curve. Moreover, standard solutions with increasing concentrations were
prepared from this solution, which was diluted before. Values of pH were adjusted by adding buffer solutions
within the range of 1 to 6 pH. All chemicals and analytical grade solutions were prepared in deionized water.

3.3. Supramolecular solvent-based microextraction procedure

The sample solution of rhodamine B was taken in a 50-mL centrifuge tube, and then 2 mL of acetate buffer
solution was added to adjust the sample pH to 3 by using diluted NaOH and HCl solutions. After pH regulation,
600 µ L of THF and 150 mg of decanoic acid were added to the rhodamine B sample solution to form the
supramolecular solvent. After these steps, the sample solution of rhodamine B was sonicated for 5 min and
a cloudy solution was obtained. This solution was centrifuged at 4000 rpm for 10 min. At the end of the
centrifugation, the solvent phases were completely separated from the aqueous phase and formed a solvent
solution in the upper phase. The lower water phase was eliminated with an injector. After the liquid phase was
taken from the solution, the volume of the remaining phase was between 200 and 300 µ L. The supramolecular
solvent (about 200–300 µ L) was completed to 1000 µ L with ethanol. Finally, the developed microextraction
method as applied to the final solution was measured by UV-Vis spectrophotometer at 558 nm (Figure 4).

0.7

0.6

0.5
Absorbance

0.4

0.3

0.2

0.1

0
450 500 550 600 650 700
Wavelength nm

Figure 4. UV-Vis spectrum of rhodamine B.

Acknowledgments
The authors are grateful for the financial support of the Scientific Research Projects Unit of Gaziosmanpaşa
University (project number: 2015/124). Nebiye Özkantar thanks Dr Erkan Yılmaz for his contributions.

993
 ÖZKANTAR et al./Turk J Chem

References

1. Ravi, K.; Deebika, B.; Balu, K. J. Hazard. Mater. 2005, 122, 75-83.
2. Hema, M.; Arivoli, S. Arab. J. Chem. 2007, 2, 43-51.
3. Metivier-Pignon, H.; Faur-Brasquet, C.; Cloirec, P. L. Sep. Purif. Technol. 2003, 31, 3-11.
4. Wu, J.; Fu, H.; Zhu, X. Sci. Res. 2014, 2, 17-25.
5. Soylak, M.; Unsal, Y. E.; Yilmaz, E.; Tuzen, M. Food Chem. Toxicol. 2011, 49, 1796-1799.
6. Pourreza, N.; Rastegarzadeh, S.; Larki, A. Talanta 2008, 77, 733-736.
7. Sleiman, M.; Vildozo, D. L.; Ferronato, C.; Chovelon, J. M. Appl. Catal. B Environ. 2007, 77, 1-11.
8. Wang, S.; Xu, Z.; Fang, G.; Duan, Z.; Zhang, Y.; Chen, S. J. Agric. Food Chem. 2007, 55, 3869-3876.
9. Jaina, R.; Mathura, M.; Sikarwara, S.; Mittal, A. J. Environ. Manage. 2007, 85, 956-964.
10. Soylak, M.; Unsal, Y. E.; Tuzen, M. Food Chem. Toxicol. 2011, 49, 1183-1187.
11. Ping, Q.; Tao, Z.; Zejun, W.; Xiaoyan, L.; Xuewu, Z. Food Chem. 2011, 125, 1462-1467.
12. Dinc, E.; Baydan, E.; Kanbur M.; Onur, F. Talanta 2002, 58, 579-594.
13. An, L.; Deng, J.; Zhou, L.; Li, H.; Chen, F.; Wang, H. U; Liu, Y. J. Hazard. Mater. 2010, 175, 883-888.
14. Sohrabi, M. R.; Ghavami, M. J. Hazard. Mater. 2008, 153, 1235-1239.
15. Watanabe, T.; Takizawa, T.; Honda, K. J. Phys. Chem. 1977, 81, 1845-1851.
16. Qu, P.; Zhao, J.; Shen, T.; Hidaka, H. J. Mol. Catal. 1997, 120, 235-245.
17. Alothman, A. Z.; Habila, M. A.; Yilmaz, E.; Al-Harbi, N. M.; Soylak, M. Int. J. Environ. An. Ch. 2015, 95,
1311-1320.
18. Aydin, F.; Yilmaz, E.; Soylak, M. RSC Adv. 2015, 5, 94879-94886.
19. Yigit, S.; Tuzen, M.; Soylak, M.; Dogan, M. Int. J. Environ. An. Ch. 2016, 96, 568-575.
20. Unsal, Y. E.; Soylak, M.; Tuzen, M. Desalin. Water Treat. 2015, 55, 2103-2108.
21. Wu, Z. L.; Liu, Q.; Chen, X. Q.; Yu, J. G. J. Separ. Sci. 2015, 38, 3404-3411.
22. Xu, X.; Zhang, M.; Wang, L.; Zhang, S.; Liu, M.; Long, N.; Qi, X.; Cui, Z.; Zhang, L. Food Anal. Methods 2016,
9, 1696-1705.

994