Research Article, Issue 1
Analytical Methods in Environmental Chemistry Journal
Journal home page: www.amecj.com/ir
AMECJ
Ehsan Zolfonoun a,*
a Material and Nuclear Fuel Research school, Nuclear Science and Technology Research Institute, Tehran, Iran
to food products and pharmaceutical formulas to
correct possible dietary deficiencies [3]. Therefore,
reliable analytical methods for the determination of
Trp are of great interest.
A variety of analytical methods have been
described for the determination of Trp, including
high performance liquid chromatography
(HPLC) [4], capillary electrophoresis [5],
electroanalytical methods [6], spectrophotometry
and spectrofluorimetry [7, 8]. Compares with the
Chromatographic methods, spectrofluorimetric
determination is a simple, fast and inexpensive
method. However, the direct determination of
Trp at low concentrations by spectrofluorimetry
Spectrouorometric determination of L-tryptophan after
preconcentration using multi-walled carbon nanotubes
1. Introduction
Analysis of amino acids is important in several
fields of research, particularly in food,
soil, biotechnology and pharmaceutical
industries [1, 2]. Tryptophan (2-Amino-3-
(1H-indol-3-yl) propanoic acid) (Trp) is an
essential amino acid for humans and is required
for the biosynthesis of proteins and also is
important in nitrogen balance in adults. This
amino acid cannot be synthesized in the human
body and must be obtained from food or
supplements. Tryptophan is sometimes added
* Corresponding author: Ehsan Zolfonoun
Email: ezolfonoun@aeoi.org.ir
https://doi.org/10.24200/amecj.v2.i01.43
A R T I C L E I N F O:
Received 10 Jan 2019
Revised form 3 Feb 2019
Accepted 23 Feb 2019
Available online 19 Mar 2019
------------------------
Keywords:
L-tryptophan
Solid-phase extraction
Multi-walled carbon nanotubes
Bioanalysis
Spectrofluorometry
A B S T R A C T
A solid-phase extraction method based on multi-walled carbon
nanotubes (MWCNTs) was applied for the preconcentration of
L-tryptophan (α-amino acid) prior to its spectrofluorometric
determination. Due to the high surface area of MWCNTs, satisfactory
concentration factor and extraction recovery can be achieved with
only 10 mg MWCNTs in 5 min. The effects of pH, sorbent amount,
eluent type and time on the recovery of the analyte were investigated.
Under the optimum conditions, the detection limit for L-tryptophan
was 8.9 ng mL−1. The precision of the method, evaluated as the relative
standard deviation obtained by analyzing of 10 replicates, was 2.6%.
The practical applicability of the developed method was examined
using wheat and barley samples.
Bio-Extraction of L-tryptophan by carbon nanotubes; Ehsan Zolfonoun
Analytical Methods in Environmental Chemistry Journal Vol 2 (2019) 43-48
44 Analytical Methods in Environmental Chemistry Journal; Vol. 2 (2019)
is difficult because of insufficient sensitivity of
this technique as well as the matrix interferences
occurring in real samples, and an initial sample
pretreatment, such as preconcentration of the
analyte, is often necessary [9, 10].
Solid phase extraction (SPE) is a routine extraction
method for preconcentration of organic and
inorganic analytes. This technique reduces solvent
usage and exposure, disposal costs, and extraction
time. Various adsorbents, such as octadecyl
functional groups bonded on silica gel, C18 [11],
silica gel [12], chelating adsorbents [13], Amberlite
XAD resins [14, 15], activated carbon [16] and
other sorbents [17] have been used for adsorption
of analytes in SPE methods.
Multi-walled carbon nanotubes (MWCNTs) have
received great attention due to their exceptional
electronic, mechanical, thermal, chemical
properties and significant potential applications in
many fields [18]. Owing to their large surface area
and high reactivity, MWCNTs based adsorbents
have been used for solid phase extraction and
preconcentration of organic compounds and metal
ions [19, 20]. Compared with traditional SPE
sorbents, MWCNTs offer a significantly higher
surface area-to-volume ratio and a short diffusion
route, resulting in high extraction capacity, low
extraction time and high extraction efficiencies
[21].
In this paper, a magnetic solid phase extraction
method based on multi-walled carbon nanotubes is
developed for the extraction and preconcentration
of L-tryptophan(α-amino acid), prior to its
spectrofluorometric determination.
2. Experimental
2.1. Reagents
All reagents used were of analytical grade and
were used as supplied. NaOH, ammonia solution,
were purchased from Merck (Germany). MWCNTs
(purity> 95%) were obtained from Sigma-Aldrich.
Standard stock solution (1000 μg mL–1) of
L-tryptophan was prepared by dissolving the pure
solid (Sigma-Aldrich) in deionized water. Working
solutions were prepared daily by adequate dilution
with deionized water.
2.2. Instrumentation
The fluorescence measurements were performed
using a Perkin-Elmer LS50 spectrofluorometer,
equipped with a xenon discharge lamp. A Metrohm
model 744 digital pH meter, equipped with a
combined glass-calomel electrode, was employed
for the pH adjustments.
2.3. solid-phase extraction procedure
A 40 mL sample or standard solution containing
L-Trp (pH 6) was transferred in a polypropylene
centrifuge tube. Then 10 mg of MWCNTs was
added into the sample solution. The mixture was
shaken for 5 min. The solution was centrifuged
for 5 min at 5,000 rpm, and the aqueous phase
was removed. The preconcentrated target analyte
was eluted using 1.0 mL of a 2 mol L−1 solution of
NaOH. The pH of this solution was adjusted at 10
by addition of 2 mol L−1 hydrochloric acid and then
solution made up to 2.0 ml with deionized water.
Finally, the concentration of L-tryptophan was
determined spectrofluorometrically at λem = 360
nm after excitation at 274 nm.
2.4. Sample preparation
For digestion of wheat and barley samples, 20.0 mL
KOH (10 % m/v) were added to 0.20 g of sample
powder in a 100.0 mL conical flask to hydrolyze
in an oven at 40 °C for 16–18 h. Then, the mixture
was filtered through a filter paper and adjusted
to pH 6 by the addition of 6 M HCI. Finally the
solution made up to 50.0 ml with deionized water.
3. Results and discussion
3.1. Optimization of extraction conditions
3.1.1. Effect of pH
The effect of pH on the extraction of L-Trp was
studied in the range of 3.0–11.0 using nitric acid or
ammonium acetate. The resulting percent recovery-
pH plot is shown in Fig. 1, which indicates that
sorption is maximum and quantitative in the pH
range 3.0–9.0. Therefore, pH 6.0 was selected for
further study.
45
Bio-Extraction of L-tryptophan by carbon nanotubes; Ehsan Zolfonoun
3.1.2. Effect of the sorbent amount
In comparison with the traditional sorbents,
MWCNTs offer a significantly higher surface area-
to-volume ratio. Therefore, satisfactory results can
be achieved with fewer amounts of MWCNTs. In
order to study the effect of the sorbent, 2 to 20 mg
of MWCNTs was added to 40 mL of the sample
solution (Fig. 2). The obtained results showed that
by increasing the sorbent amounts from 2 up to 10
mg due to increasing accessible sites, extraction
recovery increased and after that remained
constant. A 10 mg of the MWCNTs was selected
for subsequent experiments.
3.1.3. Effect of eluent type
In order to find the best eluent, different eluting
solutions such as HCl, HNO3, acetic acid, NaOH,
were tested. Obtained results showed that among
the tested eluent, NaOH was found to be the superior
solvent in comparison with other solvents for
desorption of analytes from surface of the sorbent.
Therefore, NaOH solution was selected and used as
an eluent. The effect of NaOH concentration on the
recovery of the adsorbed L-Trp was examined in
the range of 0.1 to 5 mol L−1 (Fig. 3). Based on the
obtained results, 2.0 mol L−1 NaOH was sufficient
for complete elution of the adsorbed Trp on the
sorbent surface. To achieve the highest recovery
Fig. 1. Effect of pH on the extraction efficiency of L-Trp. Conditions: sample volume, 40 mL; MWCNTs amount,
10 mg; Concentration of L-Trp, 0.10 µg mL–1.
Fig. 2. Effect of the MWCNTs amount on the recovery of L-Trp. Conditions: pH, 6, sample volume, 40
mL; Concentration of L-Trp: 10 µg mL–1.
46 Analytical Methods in Environmental Chemistry Journal; Vol. 2 (2019)
of the adsorbed L-Trp, the effect of the volume of
the eluent was also tested. The minimum volume
of NaOH required for quantitative elution of the
retained analyte was 1.0 mL.
3.1.4. Effect of extraction time
The effect of extraction time on the extraction of
L-Trp was studied in the range of 1–15 min. The
experimental results indicated that there was no
significant effect on the extraction efficiency when
the extraction time increased from 5 to 15 min.
Based on the above considerations, 5 min was
selected for further studies.
3.1.5. Sorption capacity
In order to determine the maximum capacity for
L-Trp, 20 mg of the adsorbent was added to 40
mL of an aqueous solution containing 20 mg L–1
L-Trp and shaken it for 30 min under optimized
conditions. After decantation of the sorbent, the
concentration of retained L-Trp in the supernatant
solution was determined. The maximum capacity
was found to be 36.3 mg g–1 for L-Trp.
3.2. Analytical gures of merit
Table 1 summarizes the analytical characteristics of
the proposed method, including linear range, limit
of detection, reproducibility, and enhancement
factor. In the optimum conditions, a calibration
graph was constructed by preconcentrating a series
of the solutions according to the procedure under
experimental. The linear regression equation for
the calibration graph for the concentration range
of 0.04−3.40 µg mL−1 was I=92.69C+74.43
(r2=0.9984, n=8), where I is the fluorescence
intensity and C is Trp concentration in the sample
solution in μg mL−1.
The limit of detection (LOD) of the proposed
method for the determination of Trp was studied
under the optimal experimental conditions. The
LOD, defined as three times the standard deviation
of 10 measurements of the blank solution divided
by the slope of the calibration curve, was 8.9 ng
mL−1. The reproducibility of the proposed method
for extraction and determination of 0.10 µg mL−1
Trp (n= 10) was also studied. The relative standard
deviation (R.S.D.) of these determinations was 2.6
%.
3.3. Application
The proposed method was applied to the
determination of L-Trp in wheat and barley samples
Table 1. Analytical parameters of the proposed method.
Parameter Analytical feature
Linear range (µg mL−1)0.04−3.40
Calibration equation I=92.69C+74.43
r20.9984
LOD (ng mL−1) 8.9
R.S.D. % (n = 10) 2.6
Fig. 3. Effect of NaOH concentration on the extraction efficiency of L-Trp. Conditions: pH, 6, sample volume, 40
mL; MWCNTs-Fe3O4 amount, 10 mg; Concentration of L-Trp, 0.10 µg mL–1.
47
Bio-Extraction of L-tryptophan by carbon nanotubes; Ehsan Zolfonoun
and the obtained results by proposed method were
compared with HPLC method. The results obtained
are shown in Tables 2. The results demonstrated
that the proposed method was suitable for the
determination of L-Trp in real samples.
4. Conclusions
A simple and fast SPE method based on MWCNTs
was developed for the preconcentration and
spectrofluorimetric determination of L-Trp. The use
of NPs endued the SPE method with high extraction
capacity and preconcentration factors. Also using
spectrofluorimetry as a detection system exhibits
a low primary and operational cost in comparison
with other methods such as HPLC. The method
can be successfully applied to the separation and
determination of tryptophan in real samples.
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