Anal. Methods Environ. Chem. J. 4 (3) (2021) 21-32
Research Article, Issue 3
Analytical Methods in Environmental Chemistry Journal
Journal home page: www.amecj.com/ir
AMECJ
Cobalt separation from water and food samples based
on penicillamine ionic liquid and dispersive liquid-
liquid microextraction before determination by AT-FAAS
Yaghoub Pourshojaei
a,*
and Alireza Nasiri
b
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran.
b
Environmental Health Engineering Research Center, Kerman University of Medical Sciences, Postal code:7619813159, Kerman, Iran
ABSTRACT
The cobalt compounds have adverse health effect on human and
caused to damage of the DNA cells, neurological and endocrine
systems. Therefore, the separation and determination of cobalt in
water and food samples must be considered. In this research, the
(2S)-2-amino-3-methyl-3-sulfanylbutanoic acid (penicillamine) as a
chelating agent mixed with ionic liquid (OMIM PF
6
) /acetone and
used for extraction of cobalt from 50 mL of water samples by ultra-
assisted dispersive liquid-liquid microextraction (USA-DLLME) at
pH=6. Based on procedure, the samples were shaked for 5 min (25
o
C)
and after complexation of cobalt ions by thiol and amine group of
penicillamine, the ionic liquid phase separated in the bottom of the
conical tube by centrifuging for 3.0 min. The upper liquid phase
was vacuumed by the auto-sampler and the Co
2+
ions back extracted
from the ionic liquid/ penicillamine in acidic pH. Finally, the cobalt

atomic absorption spectrometry (AT-FAAS). The main parameters
such as the sample volume, the penicillamine amount, the ionic liquid
amount and the shaking time were optimized. The linear range, the

L
-1

-1
and 98.5, respectively (r = 0.9995, RSD%=2.2). The
procedure was validated by ET-AAS analysis.
Keywords:
Cobalt,
Water and food,
Penicillamine,
Ionic liquid,
Ultra-assisted dispersive liquid-
liquid microextraction,

spectrometry
ARTICLE INFO:
Received 11 Jun 2021
Revised form 8 Aug 2021
Accepted 30 Aug 2021
Available online 28 Sep 2021
*Corresponding Author: Yaghoub Pourshojaei
Email: pourshojaei@yahoo.com
https://doi.org/10.24200/amecj.v4.i03.148
------------------------
1. Introduction
Cobalt compounds exist in two valence forms include
cobalt (Co II, cobaltous, Co
2+
and Co III, cobaltic,
Co
3+
), the other forms have not environmentally
available. Also, the other cobalt compounds have
toxic effect in the environment and the human body
by extra exposure [1]. The people is exposed to cobalt
through inhalation of air and food and drinking water.
Cobalt ions enter to environment from numerous
industrial factories such as heavy metals activity
process, the grinding, the mining and paint [2].
Furthermore, it can be used for a medical process for
the medicine Company. The cobalt compounds are
widely dispersed in air with a low concentration less
than 2.0 ng m
-3
[3, 4]. Cobalt has a low concentration
   
-1
in drinking water. The
cobalt concentration in river, the groundwater, the

-1
[5].
Feng et al reported the concentrations of cobalt in
the groundwater had lower than 0.01 mg L
-1
which is
lower than other heavy metals [6]. Lim et al showed
an applied model for the heavy metals such cobalt in
22
Anal. Methods Environ. Chem. J. 4 (3) (2021) 21-32
[7]. United
States Environmental Protection Agency (EPA)
reported that the cobalt levels in sediment and surface
   
-1
  
-1
, , respectively
[2]. Food analysis in dietary cobalt intake such as
       
to control cobalt toxicity in human body which is
    [8]. Besides, the
skin contact is a main way that cobalt was entered to
human body. Cobalt as an essential metal exists in the
human body and the maximum amount of it generally
concentrated in the liver. Cobalt in eggs has biological
role in vitamin B12 and named cyanocobalamin [9].
It uses in structure of vitamin B
12
and produce the red

[9]. Cobalt toxicity cause several health problems
such as cardiomyopathy, nerve/thyroid problems,
hearing and visual impairment, neuropathy,
tinnitus
and glomerulonephritis [10, 11]. Therefore, the
accurate results for determination of cobalt must
be considered by a new technology. The normal
concentration of cobalt is equal to 1.0 ng mL
-1
for
environmental or occupational exposure and more
than this value cause to toxicity. The sources
of cobalt can be entering to human body from
occupational/environmental/food exposures. The
blood Co concentration is 100 µgL
-1
and more than
300 µgL
-1
cause toxicity in human [12, 13]. The
penicillamine a chelating agent, is a trifunctional
compound, containing of a thioalcohol, a carboxylic
acid, and an amine that was used for the treatment
of Wilson’s disease, kidney stones , rheumatoid
arthritis, and removal of heavy metal. Based on
disorder of copper metabolism, copper accumulated
in human body and the penicillamine extracted
extra copper from body but, it can be removed the
other essential metals from body [14, 15]. Many
analytical methods such as electrothermal atomic
absorption spectrometry (ET-AAS) [16],  
atomic absorption spectrometry (F-AAS) [17] and
the inductively coupled plasma optical emission/
mass spectrometry (ICP-OES, ICP-MS) [18] have
previously used for the determination of cobalt in
various water and food samples. Moreover, analytical
techniques based on the above instruments cannot
       
For this purpose, the procedures must be developed
for the separation and preconcentration of cobalt
from samples. There are many methodologies for
matrixes including,
the magnetic solid phase extraction (MSPE) [19],
dispersive micro-solid phase extraction (D-
[20], the liquid-liquid extraction (LLE), the dispersive
liquid-liquid microextraction (DLLME) [21], the
electrochemistry methods (ECM) [22], the cloud
point extraction (CPE) [23] and the precipitation
[24]. Recently, the ultra-assisted dispersive liquid-
liquid microextraction (USA-DLLME) [25] has
been used as one of the most practical methods for
the separation of metal ions. The main advantages
of USA-DLLME to other techniques are simple
separation, high preconcentration, fast analysis, low
time, high recovery and good enrichment factor (EF).
The ionic liquid as green solvent plays critical role for
collection of ligand and metals from samples into two
phases; a IL/ligand phase and liquid phase of water
samples. Metal ions can be extracted from aqueous
solution into the small-volume IL/ligand phase with
hydrophobicity, the more density than water samples
and low solubility in water. In this study, the mixture
of (2S)-2-amino-3-methyl-3-sulfanylbutanoic acid
(penicillamine)/ (OMIM PF
6
) /acetone have been
used for extraction of cobalt from water samples
by USA-DLLME at pH=6. The thiol and amine
groups of penicillamine play an important role in the
coordination of metals and have a strong complex
with the cobalt ions [26]. In this study, this ligand
was used as an ion carrier and as a chelating agent to
cobalt ions accompanied with ionic liquid
2. Experimental
2.1. Instrumental Analysis
The cobalt (Co) value in water and digested food
samples was determined by AT-FAAS (GBC, Aus).
The air-acetylene was used for cobalt measurement by
AT-FAAS. The atom trap accessory as SQT-AT devices
is placed on the burner. In order to improve sensitivity,

SQT which the source beam was passed. SQT-AT
devices cause to increase the sensitivity of absorption
23
Cobalt extraction by penicillamine and ionic liquid Yaghoub Pourshojaei et al
(ABS) per concentration before analysis. The limits
of detection (LOD) were obtained at 0.05 and 0.13
mg L
-1
for the AT-FAAS and FAAS, respectively. The
HCL was adjusted by screws up to maximum energy.
The AT-FAAS for cobalt determination was tuned by
wavelength of 240.7 nm (7 mA). The aspiration of
samples into FAAS was done by the auto-sampler
(0.5-1 mL). The linear range for AT-FAAS was 0.15-
6.0 mg L

for cobalt analysis. The working range for
the AT-FAAS and F-AAS was obtained at 0.15-15
and 0.4-15 mg L

for cobalt, respectively. Graphite
furnace accessory coupled to an atomic absorption
spectrophotometer (GBC) was used for validation of
cobalt in digested food and water samples. The pH of
the samples was adjusted by favorite buffer solutions
(Sigma, Germany) and determined by the Metrohm
pH meter (Swiss). The phosphate buffers (Na
2
HPO
4
and NaH
2
PO
4
) were used to adjust the pH from 6.0
to 8.0.
2.2. Reagents and Materials
The ultra-pure H
2
SO
4
, HCl, NaOH and HNO
3
solutions for cobalt analysis in food and water
samples were prepared from Sigma Aldrich
(Germany). The calibration solutions of Co(II) were
made by dissolving 1.0 g of cobalt nitrate (Co(NO
3
)
2
)
in 1 L of deionized water (DW) solution (2% HNO
3
).
The linear ranges of cobalt were daily prepared by
standard solutions (1g L
-1
, 1000 mg L
-1
) and diluted
by DW (Millipore, USA). All of the laboratory
glassware was cleaned with nitric acid (5% v/v)
and washed with DW for 10 times. ionic liquid of

(HMIM PF
6
, CAS N: 304680-35-1), 1-Methyl-3-
  
[PF
6
], CAS N: 304680-36-2), 1-methyl-3-
  
[PF
6
], [BMIM][PF6], CAS N: 304680-36-2), and
 
( [EMIM][PF
6
], CAS N: 155371-19-0), acetone
(CAS N: 67-64-1) and the penicillamine (CAS N:
52-66-4) were purchased from Sigma, Germany.
The reagents of Na
2
HPO
4
and NaH
2
PO
4
(CAS N:
7558-79-4, 99.95%; CAS N: 7558-80-7, 99%) were
prepared from the Sigma Aldrich, Germany.
2.3. Preparation of water and food samples
All food samples (Rice, Spinach, Broccoli, and
Onion) were pulverized and then ground/ dried/
homogenized before analysis. Finally, the powder
samples are converted to a uniform size and then
place in the oven at 90
C for 3 h. After adding DW
to food samples, the homogenization of sample
was digested with microwave (Antom Paar, multi-
wave) based on book catalog procedure. The food
samples were digested at optimum conditions
(200
C, 500 ps UV radiation). First, 1.0 g of food
powder samples were placed in PTFE tube with
surrounding ceramic tube of microwave and then, 5
mL of HNO
3
with 1 mL of H
2
O
2
solution were added
to samples. The powder samples were digested for
58 min and diluted with DW up to 50 mL before
determination of cobalt by the USA-DLLME at
pH=6. By microwave, the all cobalt forms in foods
(organic foods) convert to Co(II) by induced oxygen
combustion and total cobalt can be determined in
food samples. All water samples prepared based on

3
(2%) by
the ASTM sampling method for water and storage in
PE tube at -4
0
C.
2.4. Procedure of cobalt extraction
The Co (II) ions were separated and preconcentrated
based on the complexation of cobalt-penicillamine
in water and food samples by the USA-DLLME
procedure (Fig.1). Also, the total cobalt in food
samples was determined based on penicillamine
ligand by the AT-AAS. The penicillamine (0.12 g)
dispersed into 180 mg of hydrophobic ionic liquid
([HMIM][PF
6
] and 0.5 mL acetone and then, the
mixture of ligand/([HMIM][PF
6
] /acetone was
injected into 50 mL of water or standard solution
 
-1
) by a syringe at pH=6.
After sonication of samples for 5.0 min, the
Co(II) ions were complexed by the thiol group of
      
(2S)-2-amino-3-methyl-3-sulfanylbutanoic acid].
After the extraction process, the Co-ligand was
trapped in the hydrophobic [HMIM][PF
6
] at the
bottom of a conical PE tube by centrifuging for
5 min (3500 rpm). The upper liquid phase was