Anal. Methods Environ. Chem. J. 6 (4) (2023) 19-36

Research Article, Issue 4

Analytical Methods in Environmental Chemi s try Journal

Journal home page: www.amecj.com/ir

AMECJ

Rapid extraction and separation of mercury in water and food

samples based on micelles and azo-thiazoles complexation

before determination by UV-Vis Spectrophotometry

Hesham H. El-Fekya,*, Talaat Y. Mohammeda, Alaa S. Amina and Mohammed A. Kassema, b

a Chemis try Department, Faculty of Science, Benha University, Benha 13518, Egypt.

b Chemis try Department, Faculty of Applied Science, Umm Al-Qura University, Makkah, Saudi Arabia

ABS TRACT

A simple and sensitive procedure has been es tablished for analyzing

mercury (II) ions spectrophotometrically in the presence of micellar

medium using three azo-thiazoles complexing reagents: 2-amino-

6-(thiazole-2-yldiazenyl)-3-pyridinol (C8H7N5OS), 8-hydroxy-7-

(thiazole-2-yldiazenyl) quinoline-5-sulfonic acid (C12H8N4O4S2), and

1-hydroxy-4-(thiazole-2-yldiazenyl)-2-naphthoic acid (C14H9N3O3S).

H1 NMR spectra validated the three azo thiazoles synthesized

material. Tween 80 (polysorbate 80) and cetyltrimethylammonium

bromide (C19H42BrN as molecular biology) are micellar mediums

to enhance sensitivity. Absorbances were measured for Hg (II)

complexation with R1, R2, and R3 at λmax of 617, 633, and 554 nm,

respectively. The UV-Vis spectrophotometer showed calibration

curves in the 0.2-15 mg L-1. The molar absorptivity, Sandell’s

sensitivity, detection, and quantication limits (LOD, LOQ) were

determined. The interferences of various ions were inves tigated, and

a s tatis tical assessment of the results was performed. The methods

have been applied for trace determination of mercury (II) in food

and environmental water samples. For food samples, all samples

were diges ted before complexation with the azo-thiazoles material

at optimized pH before determination by UV-Vis spectrophotometry.

Keywords:

Mercury,

UV-Vis spectrophotometry,

Ligand,

Azo-thiazoles,

Complexation,

Water and Food samples

ARTICLE INFO:

Received 15 Aug 2023

Revised form 13 Oct 2023

Accepted 11 Nov 2023

Available online 29 Dec 2023

*Corresponding Author: Hesham H. El-Feky

Email: hesham.elfeky@fsc.bu.edu.eg

https://doi.org/10.24200/amecj.v6.i04.258

1. Introduction

Environmental monitoring is a subject that requires

the development of novel analytical methods. The

speciation of potentially hazardous metal ions is

essential for comprehending their eco-toxicological

and biological properties, which depend on the

chemical species. Extensive research has been

devoted to developing sensitive, relatively simple,

accurate, rapid, and cos t-eective methods for

determining indus trially pertinent metals that may

harm human health [1]. Mercury is a problematic

natural pollutant because it can hurt almos t all living

things [2]. As a result of human environmental

achievements, mercury compounds can exis t in

various settings [3]. They frequently exis t in trace

amounts in natural water types [4]. Mercury

pollution is a signicant problem in the lakes

and rivers near indus trial zones. Therefore, it is

essential to develop new, selective, ecient, and

cos t-eective monitoring procedures for mercury

ions [5]. Low Hg (II) concentrations in the target

species pose a signicant challenge in mercury

determination. In naturally occurring water samples,

the predominant forms of mercury are inorganic

------------------------

mercury (mercurous and mercuric) and organic

mercury (CH3Hg+). Modern records indicate that

the total (Hg2+, MHg+) concentrations range from

0.2-100 ng L-1 and the organic mercury (CH3Hg+)

has a a concentration of 0.05 ng L-1 in water [6].

Many analytical techniques, such as inductively

coupled plasma (ICP) [7], cold vapour atomic

absorption spectrometry (CV-AAS) [8], neutron

activation analysis (NAA) [9], x-ray uorescence

[10], atomic uorescence spectrometry (AFS) [11],

and spectrophotometric technique [12] had been

advanced to monitor mercury ion at a micro level.

Each of the above methods has some advantages;

however, they may have disadvantages, such as

low reproducibility and limited sample exibility.

Without pre-concentration, the inductively coupled

plasma method was suitable for determining trace

amounts of Hg (II). However, this ins trument

is quite expensive to purchase and maintain.

Additionally, this technique has some signicant

interference [11]. Due to its simplicity, atomic

absorption spectrometry was a suitable and widely

utilised technique for accurate Hg(II) determination.

In the meantime, its application is limited due to

its limited linear range and signicant spectral

interference from volatile subs tances [13]. Due to

the low Hg (II) concentration, these practises are not

directly applicable to environmental and biological

models and/or frequently require pre-concentration

s teps to enhance selectivity. Several photometric

reagents have been used for spectrophotometric

mercury ion determination. Dithizone was the mos t

common reagent used for this purpose [14]. Before

photometric analysis, the Hg(II)-dithizone complex

is taken out with either CCl4 or CHCl3 [15]. Based

on the absorbance measurements of the formed

complexes in the presence of surfactants, we report

for the rs t time the direct spectrophotometric

determination of the Hg(II) ion with three novel azo

dyes. Also, many methods based on nanotechnology

were used for mercury extraction/removal from

dierent water, air, human and food (vegetable and

nut) samples. Mousavi et al used Tetraethyl thiuram

disulde [(C2H5)2-NCSS2CSN-(C2H5)2; TET] mixed

with ionic liquids for extraction/speciation mercury

in human samples by dispersive liquid-liquid

microextraction (DLLME) coupled to CV-AAS

[16]. Osanloo et al used silver nanoparticles coating

on micro glassy balls for removal mercury from air

and Rouhollahi et al can be determined mercury

in air and human samples [17-19]. Golbabaei,

Bagheri and hassani showed that mercury can be

determined in biological human samples based on

adsorbent by the CV-AAS. In addition, they used

the nano-palladium functionalized on the silica

nanoparticles for mercury removal from air by the

GFSC method [20-22]. Moreover, some methods

based on adsorbents such as MWCNTs, pyrrolic

and pyridinic nitrogen doped porous graphene

nanos tructure(N-D-PNG) extracted mercury from

water, food, and air samples [23-25].

In this s tudy, we prepared the azo dyes based

on new synthesis include, 2-amino-6-(thiazole-

2-yldiazenyl)-3-pyridinol (C8H7N5OS) [R1],

8-hydroxy-7-(thiazole-2-yldiazenyl) quinoline-5-

sulfonic acid (C12H8N4O4S2) [R2], and 1-hydroxy-

4-(thiazole-2-yldiazenyl)-2-naphthoic acid

(C14H9N3O3S) [R3] (Fig. 1). The H1 NMR spectra

of all synthesized azo dyes. Herein, Hg(II)

was successfully measured at the micro level

in dierent water and food samples using the

proposed methodologies by UV-Vis spectrometry

after complexation with azo dyes. The food

samples were diges ted before the complexation

and determination procedure. The approach has

several advantages, including its low cos t, ability to

be applied to real samples, and broad linear range.

2. Experimental

2.1. Ins trumentation

All absorption measurements are taken with a

Jasco UV-Vis spectrophotometer (model V530,

Jasco, Tokyo, Japan) with a scanning speed of

400 nm/min, a bandwidth of 2.0 nm, and 1.0 cm

pair-matched quartz cells. A pH meter (HI 8014,

HANNA Ins truments, Woonsocket, RI, USA) was

used to adjus t the pH of all solutions. We used a

Fluoromax-4 (Horbiba Scientic, Kyoto, Japan)

for the spectrouorimetric observations. Both the

excitation and emission slit widths were 9 nm.

Anal. Methods Environ. Chem. J. 6 (4) (2023) 19-36

2.2. Reagents and Chemicals

All chemicals and reagents employed in this s tudy

were of analytical grade (Merck, Darms tadt,

Germany), and the solutions were prepared using

bi-dis tilled water. A s tock solution of 1×10-2 M

mercuric chloride was prepared by weighing out

0.679 g HgCl2.2H2O, dissolved in the leas t amount

of bi-dis tilled water and completed in a 100-mL

measuring ask to the mark with bi-dis tilled water.

The s tock solution was then s tandardized by EDTA

[26]. All used solutions were carefully diluted from

a s tock solution. At room temperature, the solution

held up for a whole month. The preceding method

was used to prepare universal buer solutions. As

a result of the low Hg (II) concentration, these

methods are either not directly applicable to

environmental and biological models or necessitate

additional pre-concentration processes to increase

selectivity. Mercury ion concentrations have been

measured spectrophotometrically using a variety

of photometric reagents. The mos t commonly

used reagent for this was dithizone [27]. Tween

80 [0.5% (v/v)], Triton X-100 [0.5% (v/v)],

cetyltrimethylammonium bromide (CTAB) [0.5%

(w/v)], and sodium dodecyl sulphate (SDS)

[0.5% (w/v)] were used as surfactants due to their

commercial availability in a highly puried form,

low toxicity, and low charge. Tween 80 and Triton

X-100 were prepared by adding 0.5 mL of each

surfactant to 50 mL of bi-dis tilled water and then

bringing the volume to 100 mL to achieve a 0.5%

(v/v) solution. In the case of SDS and CTAB, a

0.5% (w/v) solution was intended by dissolving 0.5

g of the surfactant in 50 mL of bi-dis tilled water

and then lling a 100 mL measuring ask with bi-

dis tilled water to the desired volume. Nitric acid

(HNO3), 70 %, and hydrogen peroxide (H2O2),

30 % (w/w) in H2O, were obtained from Aldrich.

Rapid mercury determination by azo-thiazoles and UV-Vis Hesham H. El-Feky et al

Fig.1. Synthesis of azo dyes include, 2-amino-6-(thiazole-2-yldiazenyl)-3-pyridinol (C8H7N5OS)

[R1], 8-hydroxy-7-(thiazole-2-yldiazenyl) quinoline-5-sulfonic acid (C12H8N4O4S2) [R2],

and 1-hydroxy-4-(thiazole-2-yldiazenyl)-2-naphthoic acid (C14H9N3O3S) [R3]

2.3. Synthesis of reagents

A solution of 2-aminothiazole (10.014 g, 0.1 mole)

dissolved in 1:1 (v/v) HCl aqueous solution was

cooled in an ice bath at ca. – 5.0 oC. To this solution,

while s tirring vigorously, a cold aqueous solution of

sodium nitrite (6.903 g, 0.1 mole) was added, and

the reaction mixture was kept in an ice bath at a

temperature range of 0-5.0oC for 30 min. The obtained

cold diazonium salt was used for coupling with an

equivalent quantity of cold solution of 2-amino-3-

hydroxypyridine (11.01 g, 0.1 mole), dissolved in

10 % (w/v) NaOH. The formed azo dye (R1) was

kept for 40 min in an ice bath at ca. -5.0oC, ltered

o, washed with bi-dis tilled water, and dried. The

obtained azo compound was nally re-crys tallized

using absolute C2H5OH. For the preparation of the

other azo compounds, 8-hydroxy-7-(thiazole-2-

glaze) quinoline-5-sulfonic acid [R2] and 1-hydroxy-

4-(thiazole-2-ylazo)-2-naphthoic acid [R3], a typical

procedure was used using 8-hydroxyquinoline-5-

sulphonic acid (26.125 g, 0.1 moles) and 1-hydroxy-

2-naphthoic acid (18.81 g, 0.1 moles), respectively.

The reaction yield was in the range of 75-85 %.

The azo compounds showed a sharp melting

point, indicating high purity. The compounds were

characterized using 1H-NMR spectroscopy (Fig. 2a-

c). The reagent solutions were prepared by dissolving

0.110, 0.033 and 0.148 g of R1, R2 and R3 in 100 mL

ethanol to obtain 5×10-3, 1×10-3 and 5×10-3 mol L-1 of

R1, R2 and R3, respectively.

2.4. General procedure

In a typical procedure, for the reagents R1 and R2,

an appropriate volume of the sample containing 1 ×

10-3 M of Hg(II) was placed in a 10 mL measuring

ask. The universal buer of pH 6.0 (5 mL) or pH

Anal. Methods Environ. Chem. J. 6 (4) (2023) 19-36

Fig. 2a. 1H-NMR spectroscopy of 2-amino-6-(thiazole-2-yldiazenyl)-3-pyridinol (C8H7N5OS) [R1]

Fig. 2b. 1H-NMR spectroscopy of 8-hydroxy-7-(thiazole-2-yldiazenyl) quinoline-5-sulfonic acid (C12H8N4O4S2) [R2]

R1 R2 R3

7.13 (4 mL) was added to the sample for R1 or R2,

respectively. Afterwards, 1.0 mL of (5 × 10-3 M) R1

or 2.0 mL of (1×10-3 M) R2 was added, then 1.0 mL

of tween [0.5% (v/v)] or 1.5 mL of CTAB [0.5%

(w/v)] was added, respectively. A blank solution

with all the chemicals except Hg(II) was equipped

and treated as the sample solution. The procedure

was performed at room temperature (25oC). As

outlined above, the dierence in absorbance

between the sample and its respective blank was

measured at wavelengths of 617 or 633 nm for R1

and R2, respectively. On the other hand, a similar

procedure was used when the R3 reagent was

used. The optimal order of addition was reagent

R1 (1.0 mL, 1 × 10-3 M), metal (1 × 10-3 M),

buer (pH 4.97, 4 mL), and surfactant (0.5 mL of

tween [0.5% (v/v)]). In addition, at a wavelength

of 554 nm, the dierence in absorbance between

the sample and its corresponding negative was

measured (Scheme 1). After sample preparation,

the optimal time was determined for each of the

three reagents by measuring the absorbance at

various intervals(times). Dierent experimental

parameters, such as pH, buer volume, reagent

concentration, surfactant type, concentration,

reaction time, and reaction temperature, have been

inves tigated to determine the optimal conditions

for the proposed spectrophotometric method.

Rapid mercury determination by azo-thiazoles and UV-Vis Hesham H. El-Feky et al

Fig. 2c. 1H-NMR spectroscopy of 1-hydroxy-4-(thiazole-2-yldiazenyl)-2-naphthoic acid (C14H9N3O3S) [R3]

Scheme 1. Procedure for extraction and separation of mercury in water and food samples

2.5. Determination of the molecular s tructures

using the mole ratio method

In the mole ratio procedure illus trated in [28],

the Hg(II) ion concentration was held cons tant

at 1 mL of 1.0 × 10-4 M, while that of the ligand

was sys tematically modulated between 0.2 and

2.4 mL of 1.0 × 10-4 M. The absorbance of the

prepared solutions was measured at the optimal

wavelength under the optimal conditions. The

obtained absorbance was then plotted agains t

the mole ratio of ligand, [ligand/metal]. The

s toichiometric ratio of the formed s table

complexes was extracted from the purified

intersections.

2.6. Mercury ion determination in some real

samples

The food samples (potatoes, beans, rice,

soybeans, and legumes) were dried in an oven

at 75oC until their weights s tabilized and then

ground into a fine powder. In a 100 mL vial,

2.5 g of powder was weighed, and 10 mL

of concentrated nitric acid was added. The

obtained mixture was diges ted for 20 minutes

in a microwave oven. After allowing the flask

to settle, an additional 10 mL of nitric acid and

1 mL of hydrogen peroxide were added, and the

mixture was allowed to s tand for approximately

25 minutes [29, 30]. The mélange was then re-

heated in the microwave for 40 minutes. The

diges t was then permitted to chill. Finally, 1.0

mL of nitric acid was added and allowed to

sit for 10 minutes. The obtained solution was

adjus ted to a pH range of 8.0 to 9.0 by adding

sodium carbonate. The solutions were then

processed by the general procedure. S tandard

addition was used to es timate the recovery

percentage and verify the results’ precision.

2.7. Determination of mercury (II) in water

samples

Water samples were collected around the

settlements of Benha and Qalyub in the Al-

Qalyubia Governorate. After adding (1:2)

(v/v) concentrated H2SO4 and concentrated

HNO3 to each filtered water sample in a fume

chamber [29, 30], each sample was roughly

evaporated to dryness. After chilling, the

remaining solution was re-heated with 10.0

mL of double-dis tilled water. The solution was

cooled and neutralized with diluted ammonium

hydroxide. The solution was then filtered and

transferred precisely into a 25 mL measuring

vial. The solutions were then processed by the

general procedure.

3. Results and Discussion

Under ideal conditions, mercury (II) creates pink

complexes with R1, R2, and R3. Figure 3 shows

the absorption spectra of Hg(II)–R1, Hg(II)–

R2, and Hg(II)–R3 complexes at maximum

values of 617 nm, 633 nm, and 554 nm,

respectively. The mercury complexes exhibited

a bathochromic shift in their absorption

spectra compared to the free reagents, which

absorbed at 557 nm, 563 nm, and 477 nm.

S tudies were conducted to determine the

optimal conditions for obtaining high colour

intensity and maximal colour development

for micro quantification of mercury. Each of

the following parameters’ effects on colour

development was inves tigated.

3.1. Influence of pH of the medium

Specifically, pH is a crucial factor influencing

the chelation of the Hg ion by the proposed

reagent molecules. A universal buffer sys tem

ranging from 2.7 to 12.0 was utilized to

ascertain the optimal pH. As shown in Figure

4, the maximum absorption values for Hg (II)

complexes with R1, R2, and R3 were obtained

at pH 6.0, 7.13, and 4.97, respectively. In

addition, the effect of buffer volume on the

analytical peak was inves tigated between 0.5

and 6 mL (in a total volume of 10 mL). With

a buffer volume of 5.0 mL for R1-Hg (II) and

4.0 mL for both R2-Hg (II) and R3-Hg (II)

complexes, the highes t analytical absorbance

was found (Fig.4).

Anal. Methods Environ. Chem. J. 6 (4) (2023) 19-36

Rapid mercury determination by azo-thiazoles and UV-Vis Hesham H. El-Feky et al

Fig. 3. The absorption spectra of the complex formed between reagents R2 with Hg(II) where A: Spectrum of

pure ligand at pH 7.13 agains t buer solution as a blank, B: Spectrum of a complex solution containing ligand,

metal ion and buer using the same buer as blank. C: Spectrum of solution (B) agains t ligand and buer of

the same pH as blank and D: spectrum of solution (C) in the presence of CTAB.

Fig. 4. Eect of pH on the absorption spectra of complexes formed

between Hg(II) and the reagents under s tudy.

3.2. Influence of the reagent concentration

The eect of R1, R2 and R3 concentrations on

the analytical signal (absorbance) of the formed

complexes with Hg(II) is shown in Figure 5.

It was clear that for R1-Hg(II) and R3-Hg(II)

complexes, the absorbance increased with the

reagent concentration up to 5 × 10-4 M and then

suered from some decrease by increasing the

reagent concentration. Therefore, 5 × 10-4 M of the

reagents R1 and R3 was selected as the optimum

concentration. However, the absorbance enhanced

with R2 concentration and reached its maximum

value at 2 × 10-4 M. Consequently, 2 × 10-4 M was

chosen as the optimal concentration for R2 reagent.

3.3. Effect of surfactant volume

Tween 80 was the mos t appropriate solvent for

R1 and R3, while CTAB was the mos t suitable for

the R2 complex. The optimal volumes of 0.5%

(v/v) Tween-80 were 1 and 0.5 mL for Hg(II)

complexes with R1 and R3, respectively. For the

Hg(II)-R2 complex, however, the optimal volume

for obtaining a high absorbance value was 1.5 mL

of 0.5% (w/v) solution.

3.4. Effect of sequence of addition

The eect of the sequence of addition [reagent

(R), metal (M), buer (B), and surfactant (S)] on

the formation of complexes was inves tigated by

measuring the absorbance of sample solutions

prepared using various addition sequences. The

sequence [M.B.R.S.] was the bes t for Hg(II)-R1

and Hg(II)-R2 complexes, while [R.M.B.S.] was

the bes t for reagent R3. The optimal addition

sequence and other optimal conditions for each

metal complex are lis ted in Table 1.

3.5. Effect of temperature and time

The inuence of temperature and time on the

proposed methodologies was inves tigated over

25 - 60oC and 2 - 50 min, respectively. The optimal

reaction temperature for R1-Hg(II), R2-Hg(II), and

R3-Hg(II) complexes was determined to be 25oC.

The optimization of the reaction time revealed that

all complexes ins tantly attained their maximum

absorbance, except for R1, which achieved its

maximum absorbance 10 minutes after blending.

Table 1 presents the optimal time and temperature

for other complexes.

Anal. Methods Environ. Chem. J. 6 (4) (2023) 19-36

Fig. 5. Eect of reagent concentration on the absorption spectra of complexes

formed between Hg(II) and the reagents under s tudy.

3.6. S toichiometric ratio

Utilizing a molar ratio routine, the s toichiometry of

Hg (II)-R1-3 complexes was determined. The optimal

absorbance wavelengths for each complex were

617 nm, 633 nm, and 554 nm for the R1, R2, and

R3 complexes, respectively. The results indicated

that the absorbance curves for R1 and R2 complexes

attained their maximum value at the same molar

ratio (1:1). This demons trated that a singular

complex compound composed of (Hg-R1) and (Hg-

R2) can be formed from these two sys tems. The

results indicated that the R3 complex’s molar ratio

curve attained two maximum values with molar

ratios of 1:1 and 1:2 (M: L). This indicated that two

complex compounds with the s tructures (Hg-R3)

and (Hg-[R3]2) may be present in the method. The

calculated s tability cons tants for the formed (1:1)

complexes of Hg(II) with the reagents R1, R2, and

R3 were 11.11, 7.62, and 9.50, respectively. Due

to the high chemical s tability of its s tructure, the

Hg(II)-R1 complex has a high value for its s tability

cons tant. This is likely because reagent R1 contains

two electron-donating groups (-OH and -NH2),

which increase the electron cloud on the chelation

centre and subsequently increase the s tability of

this complex relative to the other complexes under

inves tigation.

3.7. S tudy of the interference

Taking into account the high selectivity provided

by the spectrophotometric technique at the

selected wavelengths of 617 nm, 633 nm, and 554

nm for Hg(II) chelated with R1, R2, and R3, the

tolerance limits for a maximum error of 3.0% were

determined. Various interfering ions of varying

concentrations were added to a solution containing

2 mg L-1 of Hg(II) for inves tigation. The results

demons trated that common coexis ting ions had

no appreciable eect on determining analyte

ions. Co(II), Ni(II), and Mn(II) species were

discovered to cause interference at high tolerance

limits between 1 and 20. Therefore, they mus t be

eliminated or disguised prior to mercury analysis.

Sodium chloride, sodium borate, and sodium

tungs tate do not interfere with detecting mercury

(II) ions. In addition, the concentrations of these

ions are typically decient in mos t water and food

samples, so these procedures can be used to detect

Hg(II) ions in actual water and food samples.

3.8. Spectrofluorometric measurements

As revealed in Figures 6-8, an aqueous solution of R2

and R3 with a concentration of 1.0 × 10-5 M displays a

vigorous uorescence intensity at λex/em=507/614 and

λex/em=480/610 nm, respectively. Two phenomena were

Rapid mercury determination by azo-thiazoles and UV-Vis Hesham H. El-Feky et al

Table 1. The optimum condition parameters of Hg(II) with the azo-dye reagents.

Parameters Hg(II)

R1R2R3

Type of buer Universal Universal Universal

Working pH 6.00 7.13 4.97

Volume of buer, mL 5.00 4.00 4.00

λmax, nm 617 633 554

Reagent’s concentration, M 5 x 10-4 2 x 10-4 5 x 10-4

Surfactant used Tween 80 CTAB Tween 80

Volume of surfactant, mL 1.0 [0.5% (v/v)] 1.5 [0.5% (w/v)] 0.5 [0.5% (v/v)]

Sequence of addition M.B.R.S M.B.R.S R.M.B.S

Time, min 10 1 1

S toichiometric ratio (M: L) (1:1) (1:1) (1:1), (1:2)

S tability cons tanta11.11 7.62 9.54

R: Reagent B: Buer M: Metal S: surfactant

aS tability cons tant using the molar ratio method

observed for R2 and R3. The emission uorescence

peak of free reagents R3 has suered from quenching

after adding 1.0 × 10-4 M mercury ion. This quenching

increased seriously by increasing the concentration of

Hg(II). The second observation was shown in the case

of reagent R2, where the absence of a new emission

peak at λem 664 nm as shown in Fig. 6. These two

observations gave evidence that the formation of

nonluminous complexes between R2, R3 and Hg(II)

[31]. Therefore, these observations can accurately

es timate mercury ions using the prepared reagents R2

and R3 in this research. It turns out that mercury ion

acts as a uorescent quencher in the case of R2 and R3.

Experimentally, it was found that the R1 detector is not

given uorescence intensity, which may be attributed

to the s tructure of R1 compared to the other R2 and R3.

3.9. Analytical characteris tics

The calibration graphs for Hg(II) complexes were

linear in the concentration range 0.2-10.0 µg mL-1

with an excellent correlation coecient (r2) of

0.998 for R1 complex while 0.6-13.0 µg mL-1 with

(r2) of 0.998 in case of R2 complex and 0.9-5.0 µg

mL-1 with (r2) of 0.999 in case of R3 complex. The

regression equation may be expressed by

A = 0.064 C-0.024 for Hg(II)-R1

A = 0.020 C+0.001 for Hg(II)-R2

A = 0.028 C-0.013 for Hg(II)-R3

where C is the Hg(II) concentration in a sample

solution in µg mL-1, and A is the absorbance. The

molar absorptivity and Sandell`s sensitivities of the

formed complexes were calculated and exhibited a

high sensitivity of the three reagents.

In addition, Table 2 tabulated the analytical

parameters for the three proposed methods. The

limits of detection and quantication were 51 µg L-1

and 156 µg L-1 for R1-complex, 15 µg L-1 and 45 µg

L-1 for R2 complex, and 26 µg L-1 and 785 µg L-1 for R3

complex. These concentration intervals are suitable

for the measured [Hg(II)] in four real water and ve

food samples. The relative s tandard deviation (RSD)

for six replicate measurements of 2 mg L-1 of Hg(II)

with R1, R2 and R3 was obtained at 2.88%, 3.42% and

3.32%, respectively.

Anal. Methods Environ. Chem. J. 6 (4) (2023) 19-36

Fig. 6. The proposed s tructures of the complexes formed between Hg(II) and reagents where

A) is (1:2)(Hg(II)-2R3) and B) is (1:1)(Hg(II)-R1)

Rapid mercury determination by azo-thiazoles and UV-Vis Hesham H. El-Feky et al

Fig. 8. Fluorescence excitation (a), emission (b) and after the addition of 1.0 × 10-4 M Hg(II)

Fig. 7. Fluorescence excitation (a), emission (b) and after addition of 1.0 × 10-4 M Hg(II)

30 Anal. Methods Environ. Chem. J. 6 (4) (2023) 19-36

3.10. Analytical applications

For the availability and reliability of the proposed

procedures, the methods were applied to four

dierent real water samples and ve dierent

food samples with three azo dyes. The obtained

results are similar based on the s tandard addition

method. The accuracy was tes ted by the S tudent’s

t-tes t. According to this tes t, the calculated

t-values ranging from 0.23 to 2.27 are less than

that of the theoretical value (2.44) at a condence

level of 95 %. In addition, the s tatis tical F-tes t

was also applied to compare the precision of

the s tudied methods with another reference

method for the determination of trace mercury

(UV-visible diuse reectance spectroscopy

after complexation and membrane ltration

enrichment) [32, 33]. The calculated F4,2 tes t

value at a 95% condence level did not exceed

the theoretical value (19.2) with a value ranging

from (1.10 to 17.77), indicating no signicant

dierence between the performance of the two

methods. The results were validated for three dyes

in food and water samples in Tables 3-5. Also,

the proposed spectrophotometric methods are

compared to other published analytical methods

in Table 6 [34-40].

Table 2. Cumulative data of the analytical conditions

for spectrophotometric determination of Hg(II) with dierent reagents

Parameters

Hg(II)

R1R2R3

Color Violet Violet Violet

pH 6.00 7.13 4.97

λmax (nm), A curve 557 563 477

λmax (nm), B curve 567 521 496

λmax (nm), C curve 624 632 572

λmax (nm), D curve 617 633 554

Beer’s law range (µg mL-1) 0.2-10.0 0.6-13.0 0.9-5.0

Ringbom range, (µg mL-1) 1.99-6.30 1.99-5 1.99-3.4

Detection limit, (µg mL-1) 0.051 0.150 0.26

Quantication limit, (ng mL-1) 0.156 0.450 0.785

S tandard deviation (SD) (n=6) 0.022 0.007 0.015

RSD (%) 2.88 3.42 3.32

Variance x 10-4 4.80 0.57 2.20

Error % 0.9% 0.31% 0.61%

Slope (b) 0.064 0.020 0.028

Intercept (a) -0.024 0.001 -0.013

Correlation coecient, r20.998 0.998 0.999

Molar absorptivity (L mol-1 cm-1) 12838 4012 5617

Sandell’s sensitivity (ng cm-2) 0.015 0.050 0.035

Condence limit 5.2 ± 0.023 4.5 ± 0.007 4.8 ± 0.015

aAverage of six consecutive measurements.

Rapid mercury determination by azo-thiazoles and UV-Vis Hesham H. El-Feky et al

Table 3. Determination of mercury ion in environmental samples using reagent R1

Sample Added

(µg mL-1)

Found

(µg mL-1)Recovery (%) RSD (%)

The calculated

s tudent’s t-tes t and

F-values b

Potato

2.92±0.008

5.98±0.026

7.88±0.023

102.0

99.2

0.44

4.42

4.16

0.60, 4.00

Beans

0.70±0.001

3.66 ±0.008

5.90 ±0.001

98.6

104.0

4.87

0.33

0.13

0.41, 2.50

Rice

6.98±0.011

7.90±0.054

9.85±0.011

96.5

95.6

2.36

3.30

0.17

0.44, 1.56

Soybean

3.35±0.009

6.28±0.018

8.4±0.008

97.6

101.0

2.37

4.24

1.36

2.27, 4.93

Lentils

2.89±0.008

5.85±0.038

7.95±0.009

98.6

101.2

0.16

1.42

1.66

0.23, 1.10

Tap water

0.60±0.001

3.57±0.018

5.56±0.002

99.0

99.2

5.60

2.97

0.60

0.57, 4.93

Pump

1.08±0.057

4.15±0.017

6.02±0.001

102.4

98.7

1.11

3.25

0.27

2.11, 5.50

Surface water

0.70±0.001

3.48±0.001

5.59±0.003

98.5

97.8

4.70

0.55

0.90

0.54, 17.77

Well

0.68±0.001

3.72±0.005

5.61±0.005

101.3

98.6

5.50

1.90

1.20

1.80, 10.00

a The average values and their s tandard deviations for ve replicate measurements.

b The tabulated s tudent’s t-and F(4,2) values are 2.44 and 19.2 for a 95% condence level and four degrees of freedom

Table 4. Determination of mercury ion in environmental samples using reagent R2

Sample Added

(µg mL-1)

Found

(µg mL-1)Recovery (%) RSD (%)

The calculated

s tudent’s t-tes t and

F-values b

Potato

2.97±0.002

6.15±0.003

8.12±0.001

106.0

103.0

2.66

1.77

0.12

2.10, 4.20

Beans

0.67±0.001

3.68±0.001

6.05±0.015

100.3

107.6

0.57

0.3

1.09

1.99, 2.20

Rice

6.94±0.018

7.85±0.016

9.70±0.005

97.0

96.0

1.10

2.86

2.05

0.87, 7.20

Soybean

3.33±0.001

6.35±0.006

8.29±0.001

100.6

99.2

0.44

3.90

0.36

1.97, 5.23

Lentils

2.91±0.001

5.96±0.086

7.92±0.006

101.6

100.1

0.67

2.23

3.14

0.54, 4.57

Tap water

0.57±0.001

3.70±0.003

5.06±0.011

104.3

100.6

1.77

4.00

2.67

1.66, 3.47

Pump water

1.10±0.003

4.25±0.002

6.20±0.009

105.0

102.0

1.56

2.25

2.94

0.89, 2.35

Surface water

0.85±0.003

3.65±0.002

5.62±0.001

97.6

95.4

1.66

2.16

0.13

0.68, 8.21

Well water

0.63±0.001

3.40±0.003

5.62±0.003

98.4

99.8

1.84

3.7

0.26

0.74, 7.89

a The average values and their s tandard deviations for ve replicate measurements.

b The tabulated s tudent’s t-and F(4,2) values are 2.44 and 19.2 for 95% condence level and four degrees of freedom

Anal. Methods Environ. Chem. J. 6 (4) (2023) 19-36

Table 6. Comparison of the proposed spectrophotometric methods with other analytical methods

Reagent λmax

(nm)

Range of Beer’s law

(µg mL-1)Remark Reference

TMK 570 5.0-80.0 Acetate buer, Triton X-114 [34]

Iodide 330 10.0-400.0 H2SO4, Triton X-114 [35]

Rhodamine 556 10-100 pH5.0, Triton X-114 [36]

Dithizone 490 50-500 pH1-3, Triton X-100 [37]

PAN 554 10.0-1000.0 pH9.0, BR buer, Triton X-114 [38]

TAR 389 500-2500.0 pH8.0, BR buer, Triton X-114 [38]

Dithizone 490 0.2-4.0 Triton X-100 [39]

617 0.2-10 Tween 80

This Work633 0.6-13 CTAB

554 0.9-5 Tween 80

TMK: Thio-Michler’s Ketone

PAN: 1-(2-pyridyl azo)-2-naphthol

TAR: 4-(2-thiazolylazo) resorcinol

Table 5. Determination of mercury ion in environmental samples using reagent R3

Sample Added

(µg mL-1)

Found

(µg mL-1)Recovery (%) RSD (%)

The calculated

s tudent’s t-tes t and

F-values b

Potato

2.69±0.009

3.62±0.003

4.65±0.004

96.5

98.0

2.65

2.27

2.50

0.69, 14.2

Beans

0.64±0.002

1.66±0.020

2.62±0.030

102.0

99.0

2.85

3.94

3.35

1.05, 7.21

Rice

1.50±0.004

2.54±0.003

3.48±0.001

104.0

99.0

4.20

3.40

1.06

2.11, 6.51

Soybean

2.66±0.004

3.67±0.007

4.60±0.006

101.0

97.0

5.00

3.55

3.47

1.68, 1.98

Lentils

2.84±0.016

3.87±0.002

4.70±0.007

103.0

94.7

1.66

1.05

4.16

1.58, 4.54

Tap water

0.73±0.002

1.67±0.009

2.71±0.004

94.5

99.0

1.43

1.82

2.84

1.58, 8.94

Pump

1.04±0.006

1.96±0.011

2.97±0.009

97.3

96.5

2.50

1.90

2.75

1.58, 4.54

Surface water

0.77±0.001

1.72±0.006

2.75±0.004

96.8

99.0

2.35

1.13

2.78

1.78, 4.69

Well

0.66±0.003

1.68±0.009

2.70±0.006

102.0

3.20

1.76

2.45

1.42, 4.57

a The average values and their s tandard deviations for ve replicate measurements.

b The tabulated s tudent’s t-and F(4,2) values are 2.44 and 19.2 for a 95% condence level and four degrees of freedom

Rapid mercury determination by azo-thiazoles and UV-Vis Hesham H. El-Feky et al

4. Conclusions

The proposed paper es tablished three new approaches

for the micro-determination of mercury ions. Three

prepared azo dyes, R1, R2 and R3, were used as complex

reagents. Absorbances of the formed complexes were

measured at 617 nm, 633 nm and 554 nm, respectively.

The mercury concentrations of 0.2-10.0 µg mL-1 for

R1, 0.6-13.0 µg mL-1 for R2 and 0.9-5.0 µg mL-1 for

R3 were s tudied and determined. The detection limits

are 50 µg L-1 for R1, 150 µg L-1 for R2 and 260 µg

L-1 for R3. The procedures were applied eectively

to determine mercury in water and food matrices.

The results were in excellent agreement relative to

accuracy and precision. The developed methods can

be used for the micro-determination of mercury in real

samples by the UV-Vis-spectrophotometer without

requiring any cos tly tool.

5. References

[1] M. Salgarello, G. Visconti, L.B. Adesi,

Interlocking circumareolar suture with undyed

polyamide thread: a personal experience,

Aes thetic Plas t. Surg., 37 (2013) 1061-1062.

https://doi.org/10.1007/s00266-013-0186-1

[2] R. Gürkan, T. Çepken, H.I. Ulusoy,

Surfactant-sensitized spectrophotometric

determination of Hg (II) in water samples

using 2-(2-thiazolylazo)-p-cresol as ligand and

cetylpyridinium chloride as cationic surfactant,

Turk. J. Chem., 36 (2012) 159-177. https://doi.

org/10.3906/kim-1104-32

[3] A. Ghaari, Performance comparison of neural

network training algorithms in modeling of

bimodal drug delivery, Int. J. Pharm., 327

(2006) 126-138. https://doi.org/10.1016/j.

ijpharm.2006.07.056

[4] M. Saber Tehrani, F. Ras tegar, A.

Parchehbaf, M. Khatamian, Determination of

Pb (II) and Cu (II) by electrothermal atomic

absorption spectrometry after preconcentration

by a Schi base adsorbed on surfactant coated

alumina, Chin. J. Chem., 24 (2006) 765-769.

https://doi.org/10.1002/cjoc.200690145

[5] M.S. Hosseini, H. Hashemi-Moghaddam,

Sensitized extraction spectrophotometric

determination of Hg (II) with dithizone after

its otation as ion-associate using iodide and

ferroin, Talanta, 67 (2005) 555-559. https://doi.

org/10.1016/j.talanta.2005.01.010

[6] R.M. Blanco, M. Tagle Villanueva, J. Enrique

Sánchez Urıa, A. Sanz-Medel, Field sampling,

preconcentration and determination of mercury

species in river waters, Anal. Chim. Acta,

419 (2000) 137-144. https://doi.org/10.1016/

S0003-2670(00)01002-3

[7] A.N. Anthemidis, G. A. Zachariadis, C. E.

Michos, J. A. S tratis, Time-based on-line

preconcentration cold vapour generation

procedure for ultra-trace mercury determination

with inductively coupled plasma atomic

emission spectrometry, Anal. Bioanal. Chem.,

379 (2004) 764-769. https://doi.org/10.1007/

s00216-004-2593-2

[8] S. Rıo-Segade, C. Bendicho, Determination of

total and inorganic mercury in biological and

environmental samples with on-line oxidation

coupled to ow injection-cold vapor atomic

absorption spectrometry, Spectrochim. Acta Part

B: Atom. Spect., 54 (1999)1129-1139. https://

doi.org/10.1016/S0584-8547(99)00052-X

[9] Q. Tu, J. Qvarns tröm, W. Frech, Determination of

mercury species by capillary zone electrophoresis-

inductively coupled plasma mass spectrometry:

a comparison of two spray chamber–nebulizer

combinations, Analys t, 125 (2000) 705-710.

https://doi.org/10.1039/A908880F

[10] M. Bacci, D. Magrini, M. Picollo, M. Vervat,

A s tudy of the blue colors used by Telemaco

Signorini (1835–1901), J. Cult. Herit., 10

(2009) 275-280. https://doi.org/10.1016/j.

culher.2008.05.006

[11] V. Fernández-Pérez, L.E. Garcia-Ayuso, M. L.

de Cas tro, Focused microwave Soxhlet device

for rapid extraction of mercury, arsenic and

selenium from coal prior to atomic uorescence

detection, Analys t, 125 (2000) 317-322. https://

doi.org/10.1039/A905217H

[12] S. Suresha, M. Fawaz Silwadi, A. Ahmed Syed,

Sensitive and selective spectrophotometric

determination of Hg (II), Ni (II), Cu (II) and Co (II)

Anal. Methods Environ. Chem. J. 6 (4) (2023) 19-36

using iminodibenzyl and 3-chloroiminodibenzyl

as new reagents and their applications to

indus trial euents and soil samples, Int. J.

Environ. Anal. Chem., 82 (2002) 275-289.

https://doi.org/10.1080/03067310290024300

[13] C.C. MagalhŃes, F. Krug, A.H. Fos tier, Direct

determimnation of mercury in sediments by

atomic absorption spectrometry, J. Anal. At.

Spectrom., 12 (1997) 1231-1234. https://doi.

org/10.1039/A701870C

[14] D. C. Nambiar, N. N. Patil, V.M. Shinde,

Liquid-liquid extraction of mercury (II) with

triphenylphosphine sulphide: Application

to medicinal and environmental samples,

Fresenius J. Anal. Chem., 360 (1998) 205-207.

https://doi.org/10.1007/s002160050675

[15] K.L. Cheng, K. Ueno, T. Imainura, Handbook

of organic analytical reagents, CRC Press,

1982. https://library.unitech.ac.pg/cgi-bin/koha/

opac-detail.pl?biblionumber=25868

[16] H.Z. Mousavi, M.M. Eskandari, A.A. Miran-

Beigi, Ultra-trace arsenic and mercury

speciation and determination in blood samples

by ionic liquid-based dispersive liquid-liquid

microextraction combined wif ow injection-

hydride generation/cold vapor atomic absorption

spectroscopy, Chem. Papers, 69 (2015) 779-790.

https://doi.org/10.1515/chempap-2015-0086

[17] M. Osanloo, M. Ghazaghi, H. Hassani,

Validation of a new and cos t-eective method

for mercury vapor removal based on silver

nanoparticles coating on micro glassy balls,

Atmos. Pollut. Res., 8 (2017) 359-365. https://

doi.org/10.1016/j.apr.2016.10.004

[18] A. Rouhollahi, Determination of mercury

concentration in the air of dental clinics and the

urines of their personnel with cold vapor atomic

absorption spectrometry, Iran. J. Toxicol., 2

(2009) 287-291 http://ijt.arakmu.ac.ir/article-

1-66-en.html

[19] M. Osanloo, O. Qorban Dadrass, Using silver

nano particles for sampling of toxic mercury

vapors from indus trial air sample, J. Health

Safe. Work, 4 (2014) 21-30. http://jhsw.tums.

ac.ir/article-1-5119-en.html

[20] F. Golbabaei, A. Vahid, A. Faghihi Zarandi,

A novel nano-palladium embedded on the

mesoporous silica nanoparticles for mercury

vapor removal from air by the gas eld

separation consolidation process, Appl.

Nanosci., 12 (2022) 1667-1682. https://doi.

org/10.1007/s13204-022-02366-0

[21] F. Golbabaei, H. Hassani, F. Eftekhar, M.J.

Kian, Occupational exposure to mercury: air

exposure assessment and biological monitoring

based on dispersive ionic liquid-liquid

microextraction, Iran. J. Public Health, 43

(2014) 793-799. http://ijph.tums.ac.ir

[22] M. Bagheri Hosseinabadi, N. Khanjani, M.D.

Mobarake, Neuropsychological eects of long-

term occupational exposure to mercury among

chloralkali workers, Work, 66 (2020) 491-498.

https://doi.org/ 10.3233/WOR-203194

[23] F Golbabaei, A Ebrahimi, A Koohpaei, A

Faghihi-Zarandi, Single-walled carbon

nanotubes (SWCNTs), as a novel sorbent

for determination of mercury in air, Global

J. Health Sci., 8 (2016) 273-280. https://doi.

org/10.5539/gjhs.v8n7p273

[24] M. Habibnia, A. Rashidi, A.F. Zarandi, M.D.

Mobarake, Simultaneously speciation of

mercury in water, human blood and food samples

based on pyrrolic and pyridinic nitrogen doped

porous, graphene nanos tructure, Food Chem.,

403 (2023) 134394. https://doi.org/10.1016/j.

foodchem.2022.134394

[25] F. Golbabai, A. Ebrahimi, Performance

comparison survey of multi-walled and single-

walled carbon nanotubes for adsorption and

desorption of mercury vapors in the air, Iran.

Occup. Health, 10 (2013) 21-31. https://espace.

library.uq.edu.au/view/UQ:9d03f02

[26] A. I. Vogel, Ά textbook of quantitative inorganic

analysis, Longman, London, 1978. https://

archive.org/details/vogels textbookof0000voge

[27] A. B. Fowler, R. K. Zalups, Mercury, Chapter

22, Handbook on the Toxicology of Metals,

Academic press, fth Edition, Volume II (2022)

539-599. https://doi.org/10.1016/B978-0-12-

822946-0.00020-9

Rapid mercury determination by azo-thiazoles and UV-Vis Hesham H. El-Feky et al

[28] J.H. Yoe, A. Jones, Colorimetric determination

of iron with disodium-1, 2-dihydroxybenzene-3,

5-disulfonate, Am. Chem. Soc., 16 (1944)111-

115. https://doi.org/10.1021/i560126a015

[29] A. Keramat, R. Zare-Dorabei, Ultrasound-

assis ted dispersive magnetic solid phase

extraction for preconcentration and

determination of trace amount of Hg (II) ions

from food samples and aqueous solution by

magnetic graphene oxide (Fe3O4@ GO/2-

PTSC): Central composite design optimization,

Ultrason. Sonochem., 38 (2017) 421-429.

https://doi.org/10.1016/j.ultsonch.2017.03.039

[30] M.H. Mashhadizadeh, Solid phase extraction

of trace amounts of silver, cadmium, copper,

mercury, and lead in various food samples based

on ethylene glycol bis-mercaptoacetate modied

3-(trimethoxysilyl)-1-propanethiol coated Fe3O4

nanoparticles, Food Chem.,151 (2014) 300-305.

https://doi.org/10.1016/j.foodchem.2013.11.082

[31] M.A. Kassem, I. I. Althaga, Sensitive

spectrouorimetric s tudy of the interaction

between Europium (III) and 1, 2-Phenylenebis

(azan-1-yl-1-ylidene) bis (methan-1-yl-1-

ylidene) diphenol Schi Base, J. Fluorescence,

26 (2016) 2087-2093. https://doi.org/10.1007/

s10895-016-1903-3

[32] Y.G. Yin, Dithizone-functionalized solid

phase extraction–displacement elution-high

performance liquid chromatography–inductively

coupled plasma mass spectrometry for mercury

speciation in water samples, Talanta, 81

(2010) 1788-1792. https://doi.org/10.1016/j.

talanta.2010.03.039

[33] L. Li, Transformation of cefazolin during

chlorination process: products, mechanism and

genotoxicity assessment, J. Hazard. Mater.,

262 (2013) 48-54. https://doi.org/10.1016/j.

jhazmat.2013.08.029

[34] A. Niazi, T. Momeni-Isfahani, Spectrophotometric

determination of mercury in water samples

after cloud point extraction using nonionic

surfactant Triton X-114, J. Hazard. Mater., 165

(2009) 1200-1203. https://doi.org/10.1016/j.

jhazmat.2008.09.091

[35] A. Afkhami, T. Madrakian, H. Siampour,

Flame atomic absorption spectrometric

determination of trace quantities of cadmium

in water samples after cloud point extraction in

Triton X-114 without added chelating agents,

J. Hazard. Mater., 138 (2006) 269-272. https://

doi.org/10.1016/j.jhazmat.2006.03.073

[36] M. Pandurangappa, K.S. Kumar, Micellar

mediated trace level mercury quantication

through the rhodamine B hydrazide spirolactam

ring opening process, Anal. Methods, 3 (2011)

715-723. https://doi.org/10.1039/C0AY00693A

[37] M. Garrido, M.S. Di Nezio, A.G. Lis ta, M.

Palomeque, B.F. Band, Cloud-point extraction/

preconcentration on-line ow injection method

for mercury determination, Anal. Chim. Acta,

502 (2004) 173-177. https://doi.org/10.1016/j.

aca.2003.09.070

[38] H. I. Ulusoy, R. Gürkan, S. Ulusoy, Cloud point

extraction and spectrophotometric determination

of mercury: species at trace levels in environmental

samples, Talanta, 88 (2012) 516-523. https://doi.

org/10.1016/j.talanta.2011.11.026

[39] M. Arjomandi, A review: analytical methods for

heavy metals determination in environment and

human samples, Anal. Methods Environ. Chem.

J., 2 (2019) 97-126. https://doi.org/10.24200/

amecj.v2.i03.73

[40] H.B. Singh, B. Kumar, R.L. Sharma, M. Katyal,

Direct spectrophotometric determination of

trace amounts of mercury (II) in aqueous media

as its dithizonate complex in the presence of

a neutral surfactant, Analys t, 114 (1989) 853-

855. https://doi.org/10.1039/AN9891400853

Anal. Methods Environ. Chem. J. 6 (4) (2023) 19-36