Spectrophotometric determination of phenol and chlorophenols by salting out assisted liquid-liquid extraction combined with dispersive liquid-liquid microextraction

Article abstract of DOI:10.1016/j.saa.2019.02.060

In this work, salting out liquid-liquid extraction (SALLE) combined with dispersive liquid-liquid microextraction (DLLME) was developed as a novel extraction method for extraction and preconcentration of phenol and chlorophenols (CPs) in environmental wat

Full text of DOI:10.1016/j.saa.2019.02.060

Accepted Manuscript
Spectrophotometric determination of phenol and chlorophenols
by salting out assisted liquid-liquid extraction combined with
dispersive liquid-liquid microextraction
Reza Tabaraki, Elham Heidarizadi
PII:
S1386-1425(19)30178-7
https://doi.org/10.1016/j.saa.2019.02.060
SAA 16836
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 29 October 2017
Revised date:
Accepted
date:
29 January 2019
1
6 February 2019
Please cite this article as: R. Tabaraki and E. Heidarizadi, Spectrophotometric
determination of phenol and chlorophenols by salting out assisted liquid-liquid extraction
combined with dispersive liquid-liquid microextraction, Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.02.060
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ACCEPTED MANUSCRIPT
Spectrophotometric determination of phenol and chlorophenols by salting
out assisted liquid-liquid extraction combined with dispersive liquid-liquid
microextraction
*
Reza Tabaraki , Elham Heidarizadi
Department of Chemistry, Faculty of Science, Ilam University, Ilam, Iran
ABSTRACT
In this work, salting out liquid-liquid extraction (SALLE) combined with dispersive liquid-
liquid microextraction (DLLME) was developed as a novel extraction method for extraction
and preconcentration of phenol and chlorophenols (CPs) in environmental water samples.
The analytes were derivatized with 4-aminoantipyrineand determined by spectrophotometry.
Experimental parameters such as type and volume of the organic solvent, type and amount of
salt, pH and vortex time were optimized. Under the optimum conditions, calibration curves
were linear in the range of 1-300 µg/L and limit of detections (LODs) were in the range of
0
1
.15–0.22 µg/L. The extraction recoveries and enrichment factors ranged from 94.80% to
06.1% and 78.12 to 82.53, respectively. Repeatability of method based on five replicate
measurements of phenols was in the range of 4.8–7.2%. The results obtained in this study
showed that the proposed method is simple, rapid and environmentally friendly with high
extraction efficiency for preconcentration and determination of phenol and CPs in real
samples. The proposed method was also compared with the reference method.
Keywords: Phenols, Salting out liquid-liquid extraction, Dispersive liquid-liquid
microextraction, Spectrophotometry, Water samples.
---------------------------------------------------------------------------
Corresponding author: 98 841 2227022; fax: 98 841 2227022.
E-mail address: rezatabaraki@ yahoo.com; r.tabaraki@ilam.ac.ir
1

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1
. Introduction
Phenol and substituted phenols are widely distributed in natural waters because of their
wide use in many industrial processes such as the manufacture of plastics, dyes, drugs and
pesticides [1]. Chlorophenols can be found in water, especially in drinking water due to the
chlorination process [2]. Chlorophenols and several other phenolic compounds, considered as
the most toxic and carcinogenic pollutants have been issued by the US Environmental
Protection Agency in a list of highly polluting materials. The European Union decision
(2455/2001/EC) sets a maximum allowable phenol concentration of 0.5 g/L in tap water and
a permissible limit of 0.1 g/L for individual phenol [3].
Derivatization of phenols with 4-aminoantipyrine (4-AAP) for spectrophotometric
determination is still commonly used due to its speed, cost-effectiveness and absence of
laborious steps. Phenols react with 4-AAP in the presence of an oxidant to form an antipyrine
dye. In order to achieve higher selectivity and sensitivity, this complex is extracted from
aqueous solution to chloroform. The standard method for determination of phenol requires a
large volume of chloroform that is the main disadvantage of this method regarding green
analytical chemistry [4, 5].
Many analytical methods for phenol and chlorophenols are based on chromatographic
techniques such as high performance liquid chromatography [6-8], gas chromatography-mass
spectrophotometry [9-11], capillary electrophoresis [12, 13] and spectrophotometry [14, 15].
Salting-out assisted liquid-liquid extraction (SALLE) can be applied successfully for
extraction of polar chlorophenols such as 4-chlorophenol and 2,4dichlorophenol from
aqueous samples. This method is based on salt-induced phase separation phenomena. The
organic phase is separated from a mixture of an aqueous medium containing the analytes and
a water-miscible organic solvent, upon addition of a salt to the mixture. This method can be
applied to inorganic elements, very polar drugs or hydrophobic drugs dissolving in organic
2

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solvent such as acetonitrile that can be homogenized with water [16]. It is noticeable that the
enrichment factor obtained by SALLE is often low and still cannot be satisfied for ultra-trace
analysis.
Recently, studies focus on the miniaturization of the liquid-liquid extraction method to
reduce organic solvent used in sample preparation. Therefore, some microextraction
techniques such as solid phase microextraction (SPME) [17, 18] and liquid phase
microextraction (LPME) [19, 20] have been proposed for the enrichment of phenol and
chlorophenols. Dispersive liquid–liquid microextraction (DLLME) was introduced by Assadi
and co-workers in 2006 as a novel liquid phase microextraction techniques. This method uses
a ternary solvent system, in which a solution of an extraction solvent in a dispersed solvent is
rapidly injected into an aqueous sample containing the analyte. The disperser solvent is
miscible in both aqueous and organic phases [21]. The extraction solvent must be immiscible
in water and should form tiny fine droplets in it. This method has been used widely for
analysis of chlorophenols in environmental and food samples [8, 23, 24].
Combination of DLLME and SPE is a way to increase the enrichment factor of DLLME. By use of
SPE, analytes are pre-concentrated from
a
large volume of sample matrix to
a
small volume of organic solvent, which can act as disperser solvent in DLLME. However, a SPE
procedure requires column conditioning, sample elution and evaporation of solvents, being labor
intensive, time and solvent-consuming. Given that DLLME itself is a green analytical technique, SPE
seems not a perfect pre-concentration technique [8].
The aim of this work was to develop a new extraction method based on the combination of
SALLE, as a simple and environmentally friendly technique, and DLLME for preconcentration of
phenol and chlorophenols. To the best of our knowledge, for the first time, the SALLE-
DLLME is applied to the spectrophotometric determination of phenol and chlorophenols in
water samples.
3

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2
. Experimental
2
.1. Instrumentation
The spectrophotometric determination of phenols in organic phase was carried out by UV–
visible spectrophotometer (Varian 300 Bio, Australia) equipped with a quartz microcell.
Absorption measurements were carried out at 470-480 nm. A PE Co centrifuge was utilized
to accelerate phase separation. A100 µL Hamilton micro syringe (Hamilton Company) was
used to collect the organic phase. A Metrohm pH meter (model-780, Switzerland), water bath
(model CO-W 40, Pars Azma Co, Iran) and a vortex mixer (model VM-10,Wise Mix, Korea)
was also employed.
2
.2. Chemicals
A stock standard solution of phenol (pK =9.99), 4-chlorophenol (4-CP, pK = 9.41) and 2,
a
a
4
-dichlorophenol (2,4-DCP, pK = 7.89) were obtained by dissolving phenols reagent in
a
water. Working standard solutions were prepared daily by diluting the stock standard solution
with ultra-pure water to the required concentrations. 4-aminoantipyrine (4-AAP), potassium
peroxodisulfate (PPD), potassium hexacyanoferrate (III), hydrogen peroxide and ammonium
hydroxide were used with derivatization purposes. Methanol, ethanol, isopropyl alcohol,
acetone and acetonitrile (ACN) were tested as disperser solvents. Dichloromethane and
chloroform were used as potential pre-concentration solvents. All of chemical and reagents
were obtained from Merck (Darmstadt, Germany).
2
.3. Derivatization and extraction method
First, 5 mL of sample (phenol species individually) was buffered at pH=9.5 with 150 ꢀof
.5 M hydrogen phosphate/ammonia solution and mixed with 50 ꢀof 4-AAP (1% w/v) and
0 ꢀof potassium peroxodisulfate (1.5% w/v) in a 15 mL glass test tube and left for 7 min
0
5
4

ACCEPTED MANUSCRIPT
in a water bath (30ꢁꢂ) until purplish product was allowed to develop. Afterward, 1.25 mL of
ACN and then 1.5 g of NaCl (i.e., 30% w/v) were added to the mixture and vortexed at a high
frequency for 1 minute. During this step, two phases were formed and the phenol and CPs
(
quinon complexes) were extracted into the top phase (ACN phase). In the next step
DLLME), the ACN phase was then withdrawn using a 1 mL syringe with a sharp needle tip
(
and mixed with 125µL chloroform (pre-concentration solvent). Then the mixture rapidly
injected into 5 mL deionized water in a 10 mL glass tube with conical bottom. A cloudy
solution, resulting from the dispersion of chloroform droplet in the aqueous solution, was
formed and the analytes were extracted into chloroform. The mixture was then centrifuged
for 5 min at 5000 rpm, which led to settle down the droplets of the extractant in the bottom of
the tube. Finally the settled organic phase (V≈ 110 µL) was removed using a Hamilton
syringe and transferred to a microcell for absorbance measurement of each phenol species at
the respective absorption maxima by UV-Vis spectrophotometry. Blanks were treated in the
same way.
2
.4. Reference procedure
The referent extraction method was performed according to 5530 APHA standard method
for determination of phenols in water and wastewater [24]. Briefly, 500 ml of sample are
transferred to a separatory funnel and the pH is adjusted to the desired value with the aid of
the appropriate buffer solution. After the successive addition of the necessary amounts of
amine and potassium ferrocyanide, there is vigorous agitation at least 10 times. The mixture
is left to stand for 15 min and the purplish-red product developed is extracted with 2×25 ml
of chloroform. The quinone complexes are measured at the optimum wavelength, against a
blank of double distilled water treated in the same manner.
5

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3
. Results and discussion
3
.1. Derivatization reaction
The reaction scheme of 4-AAP with phenol and CPs was shown in Fig. 1. The phenol-4-
aminopyrazolone derivative complexes had good stability with time and no absorbance
change was observed even 1 h after the completion of the analytical procedure. The produced
color was often purplish-red or red, indicating maximum absorbance in the vicinity of 480
nm [25].
3
.2. Selectivity of derivatization
This method is useful to determine phenol, orto and meta-substituted phenols. Para-phenols
where the substitution is nitro, nitrosobenzoyl, alkyl, aryl or aldehyde groups do not react
with 4-AAP. Para substitution prevents the reaction except for the halogens; therefore 4-nitro
phenol did not react while 4-CP reacted. The presence of nitro group in the ortho position
prevents the reaction (2-nitro phenol and 2, 4-dinitro phenol). During the reaction of p-
substituted phenols, 4-AAP expelled the p-substituent or produce the meta or ortho transfer
products [5, 26].
3
.3. Optimization of variable in derivatization and SALLE step
The pH of the sample solution is a very important factor in both derivatization and
extraction processes. Based on mechanism of reaction of 4-AAP with phenols [25], this
reaction occurs in basic medium. Thus, the effect of pH was investigated in the range of 8.5–
1
1.5. The results showed that the maximum absorbance signal and maximum stability of
color occurs in the pH= 9.5 (Fig. 2) and therefore, pH of 9.5 was selected for next
experiments.
6

ACCEPTED MANUSCRIPT
The effect of 4-AAP concentration was also studied. As shown in Fig. 3, an increase in the
absorbance was obtained when the concentration of 4-AAP in the sample was increased.
Concentration of 0.015 % w/v was selected as optimum 4-AAP concentration.
The effect of oxidizing agents such as potassium hexacyanoferrate (III), PPD and hydrogen
peroxide were investigated. The experimental results proved that the applicability of PPD
was superior. Therefore, the effect of PPD concentration was studied. Optimum concentration
of oxidizing agent was 0.015% w/v.
Choice of the extraction solvent is a critical parameter in a SALLE method. The main
requirements for selecting the extraction solvent are high polarity, miscibility in water and in
the pre-concentration solvent and inducing phase separation when adding the appropriate salt
[
27].
Methanol, ethanol, isopropyl alcohol, acetone and acetonitrile (with polarity index of 5.1,
.3, 3.9, 5.1 and 5.8, respectively) were selected as extraction solvent. The results showed
4
that just acetonitrile and isopropyl alcohol formed a two-phase system by adding NaCl. Phase
separation could not be obtained with other solvents. Based on the results, ACN gave the
highest extraction efficiency for antipyrine dyes. This can be explained by its closer polarity
to that of water [28] or antipyrine dyes. Therefore, ACN was selected as an extraction
(disperser) solvent.
To study the effect of ACN volume in SALLE method, different volumes of ACN (0.75-
2
mL) were tested. The aqueous solution volume was kept constant in all the experiments (5
mL). Absorbance increased when the volume of ACN was increased from 0.5 to 1.25 mL and
then decreased with further increase in the volume of the ACN due to dilution effect. Lower
volumes of ACN was insufficient for collection and determination by spectrophotometric.
One of the most important variables that have to be optimized in SALLE is the type of salt.
Different salts were tested as salting out agent such as NaCl, KCl, MgSO and Na CO
4
2
3
7

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(40%w/v). It was found that all salts were capable of promoting the formation of two phases,
but the best signal and reproducible results were obtained using NaCl. Therefore, NaCl was
used for subsequent experiments.
Afterwards, the effect of NaCl concentration was investigated in the range of 0.5-2.5 g (or
1
0-50%, w/v) in the aqueous sample solution. Results show that the absorbances of phenols
were improved when the amount of NaCl increased from 0.5-1.5 g and remain constant at
higher amounts of NaCl. Thus, 30%, w/v (1.5 g) was chosen for the subsequent steps.
3
.4. Optimization of variables in DLLME step
The selection of a suitable organic solvent plays an important role in DLLME. It should
able to form a cloudy solution system with water and disperser solvent. Also it should have
highly immiscibility with aqueous phase (sample solution) and higher density than water
(preferably). In our study, dichloromethane, heptane, toluene, isooctane, xylene and
chloroform as pre-concentration solvents were investigated. Extraction of antipyrine dye was
occurred only when chloroform was used in pre-concentration step.
To examine the effect of the pre-concentration solvent volume in DLLME step, different
volumes of chloroform (75- 200 µL) were investigated. At volume greater than 125 µL, the
volume of sedimented phase at the bottom of the test tube increased and absorbance
decreased. So, 125 µL was chosen as optimum volume for the pre-concentration solvent.
Centrifugation time and rate were evaluated in the range of 2-5 min and 2000-6000 rpm
respectively. The results (not shown) indicated that centrifugation time and rate did not have
significant effects. Therefore, centrifugation is used only for complete settling of chloroform
to the bottom of the centrifuge tube.
3
.5. Analytical figure of merit
8

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The analytical characteristics of the optimized proposed method were investigated respect
to linear dynamic range, limit of detection (LOD), precision (expressed as relative standard
deviation) and enrichment factor. The resulting calibration graphs were linear over the
−
1
2
−1
concentration ranges of 1-200µg L for phenol (R =0.993), 5–300 µg L for 4-CP
2
−1
2
(
R =0.995) and 2–220 µg L for 2, 4-DCP (R =0.996), respectively. The limits of detection
−
1
−1
−1
were 0.15 µg L for phenol, 0.22 µg L for 4-CP and 0.16 µg L for 2, 4-DCP,
ꢃꢄꢅ
respectively. LOD was calculated based on LOD = ꢆ ꢁwhere s is the standard deviation five
b
replicate blank measurements and m is the slope of the calibration curve after
preconcentration. The relative standard deviations (RSDs) were in the range of 4.8–7.2% for
five repeated experiments. The enrichment factors (78.12-82.53) was evaluated by comparing
the calibration graphs before and after the extraction process. As it can be seen from Table 3,
the analytical results agreed well with the results obtained with reference method.
3
.6. Real sample analysis
Applicability of the proposed method was tested by extraction and determination of phenols
in water samples (tap water, river water, seawater and waste water). Each sample (5 mL) was
analyzed according to the proposed method at optimum conditions by spiking the individual
phenol separately. The extraction recoveries of analytes were between 94.8 and 106.1 %, at
−
1
concentration of 50 and 100µg L for phenol and chlorophenols, respectively. The relative
recovery (RR) calculates as following equation:
ꢏꢐꢑꢒꢓ
RR=ꢁꢇꢈꢉꢊꢋꢌꢍ ꢎꢏꢒꢔꢔꢑꢔꢕꢖꢗꢘꢘ
where Cfound, Creal , and Cadded are the concentrations of analyte after addition of known
amount of standard in the real sample, the concentration of analyte in real sample and
the concentration of known amount of standard which was spiked to the real sample,
respectively. The concentration of phenols was below the detection limit in all samples under
9

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study, except in the wastewater. Results of original and spiked solutions are shown in Table
1
. As shown, the presented method can be reliably used for determination of phenols in
environmental water samples.
3
.7. Comparison with other methods
Analytical performance of the proposed method was compared with different published
methods for phenol and CPs. As shown Table 2, proposed procedure has the comparable or
better limits of detection and linear ranges than those reported previously. Combination of
SALLE with DLLME had lower LOD than DLLME alone [4, 5]. The proposed procedure
combines two simple and inexpensive extraction methods to increase the enrichment factor in
comparison with DMSPE–HS-SPME [29]. The procedure is simple and easy to operate
because application of HPLC and GC for analysis is sometime complicated and difficult to
reproduce. The extraction time in this work is remarkably short comparing to the reported
LLLME method [3]. Relative standard deviations of the method are the same with other
reported methods. These results indicate that the SALLE-DLLME-UV is a sensitive,
repeatable and simple method that can successfully be used for preconcentration and
determination of phenols in water samples. Comparison of the proposed method and the 5530
American Public Health Association (APHA) standard method showed that the proposed
method is fast, simple, requires low sample volume and solvent consumption, and provides
lower LOD than APHA standard method [24].
4
. Conclusion
In the present study, for the first time, salting out liquid-liquid extraction combined with
dispersive liquid-liquid microextraction as a new extraction method was developed for the
extraction and preconcentration of phenol and chlorophenols (CPs) in environmental water
1
0

ACCEPTED MANUSCRIPT
samples. High sensitivity and selectivity by derivatization of phenols were achieved for
spectrophotometric analysis. Miniaturization of sample reagent and sample volumes using
SALLE-DLLME makes it suitable alternative for 5530 APHA standard method for
determination of phenols in water and wastewater samples. This combination not only
resulted in a high enrichment factor, but also it could be used in complex matrices. In
addition, the proposed method offers advantages such as simplicity, inexpensive, fast phase
separation, lower consumption of organic solvents and high efficiency. On the other hand, the
main disadvantage of this method from the point of view of green analytical chemistry is the
use of chloroform. Various parameters in this technique were optimized. Under optimum
conditions, low limit of detection, good linearity range, adequate repeatability and good
enrichment factor were obtained.
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1
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Table 1
Real samples analysis.
-1
a
-1
Samples
Tap water
Analyte
phenol
Added(µg L ) Found (SD ) (µg L ) Recovery (%)
b
0
n.d.
-
(
Karaj,Iran)
50
48.5 (2.5)
96.4 (4.7)
97
96.4
100
4
-CP
0
n.d.
-
5
1
0
00
0
52.3 (3.5)
94.8 (5.6)
n.d.
104.6
94.8
-
2
2
2
,4- DCP
phenol
5
0
52.5 (3.2)
103 (6.7)
105
103
100
River water
0
50
n.d.
-
(
Tehran river)
51.4 (3.1)
95.6 (5.2)
n.d.
50.1 (2.8)
98.5 (6.0)
n.d.
102.8
95.6
-
100.2
98.5
-
100
4
-CP
0
5
0
00
0
1
1
,4- DCP
phenol
50
47.5 (2.8)
102.4 (5.7)
95
102.4
00
Seawater
Caspian sea, Iran)
0
50
n.d.
-
98.2
103.2
-
95.8
104.6
-
106.1
96.7
-
(
49.1 (2.7)
103.2 (5.8)
n.d.
47.9 (3.1)
104.6 (6.2)
n.d.
53.05 (3.4)
96.7 (6.5)
52.3( 2.8)
99.8 (5.2)
149.1 (8.4)
100
4
-CP
0
5
0
00
0
1
1
1
,4- DCP
5
0
00
0
c
d
Wastewater
Phenols
50
95
97
00
a
SD, standard deviation (n=3).
b
n.d., not detected.
c
Collected from a municipal wastewater treatment plant.
d
as total content.
1
4

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Table 2
Comparison of the proposed method with other methods.
Analyte
Extraction technique
Linear range
LOD
(µg L )
0.1
EF
RSD (%)
Ref.
-1
-1
(µg L )
a
4
-CP
SA-DLLME
0.5-200
285
6.2
[1]
[3]
b
4
-CP, 2,4-DCP
Phenol
New LLLME
0.5-250
0.056-0.061 67-239
2.8-7.6
DLLME
DLLME
1–900
2.5-150
1-500
0.18
0.8
920
700
954
-
6
[4]
[5]
Phenol
5.2
7.1
7.24
c
4
-CP
,4-DCP
Phenol
LPME
0.3
[19]
[29]
d
2
DMSPE–HS-SPME
2-100
0.6
1-200
5-300
2-220
0.15
0.22
0.16
80.86
78.12
82.53
4.8
7.2
6.5
4
-CP
SALLE-DLLME
This method
2
,4-DCP
a
Surfactant assisted dispersive liquid–liquid microextraction
b
Liquid–liquid–liquid microextraction
liquid phase microextraction
c
d
Dispersive micro solid phase extraction- headspace solid phase microextraction
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ACCEPTED MANUSCRIPT
Table 3
Determination of phenol and chlorophenols in spiked samples.
Analyte
Added
Found by SALLE-
DLLME
Recovery
(%)
Found by
reference method
20.8(1.4)
Recovery
(%)
-1
(
µg L )
Phenol
20
19.3 (1.2)
96.5
104
5
0
51.6 (3.5)
103.5 (6.8)
21.2 (1.5)
103.2
103.5
106
50.6 (3.2)
101.6 (6.6)
19.8 (1.2)
101.2
101.6
99
1
00
4-CP
20
5
0
48.2 (3.4)
96.7 (6.1)
96.4
96.7
50.3 (3.6)
95.8 (5.8)
100.6
95.8
1
00
2,4-DCP
20
20.8 (1.5)
104
18.8 (1.3)
94
5
0
47.4 (3.1)
105.1(6.8)
94.8
105.1
52.2 (2.7)
102.5 (6.5)
104.4
102.5
1
00
a
(
SD): standard deviation (n=3).
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Highlights
>
>
>
A preconcentration technique was developed by combination of SALLE with DLLME.
Lower consumption of solvents and LODs of this method than APHA standard method.
Application of the developed method in real samples.
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Figure 1

Figure 2

Figure 3