Analytica Chimica Acta xxx (2018) 1e11

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Tutorial

Solvent-terminated dispersive liquid-liquid microextraction: a tutorial

Fotouh R. Mansour a, *, Neil D. Danielson b
a
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Tanta University, 31111, Egypt
b
Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA

h i g h l i g h t s g r a p h i c a l a b s t r a c t

A remarkable progress of ST-DLLME

has been observed in the last few
years.

The demulsifier type and volume and
the demulsification time control the
extraction efficiency in ST-DLLME.

The different applications of ST-
DLLME and the green aspects of this
new mode have also been discussed.

a r t i c l e i n f o a b s t r a c t

Article history: Solvent-terminated dispersive liquid-liquid microextraction (ST-DLLME) is a special mode of DLLME in
Received 12 July 2017 which a demulsifying solvent is injected into the cloudy mixture of sample/extractant to break the
Received in revised form emulsion and induce phase separation. The demulsification process starts by flocculation of the
13 January 2018
dispersed microdroplets by Ostwald ripening or coalescence to form larger droplets. Then, the extractant
Accepted 2 February 2018
Available online xxx
either floats or sinks depending on its density as compared with that for the aqueous sample. The
demulsifier should have high surface activity and low surface tension in order to be capable of inducing
phase separation. The extraction efficiency in ST-DLLME is controlled by the same experimental variables
Keywords:
Microextraction
of normal DLLME (n-DLLME) such as the type and volume of the extractant as well as the disperser. Other
Dispersive liquid-liquid microextraction parameters such as pH and the temperature of the sample, the stirring rate, the time of extraction and
Solvent-demulsification the addition of salt are also important to consider. Along with these factors, the demulsifier type and
Green analytical chemistry volume and the demulsification time have to be optimized. By using solvents to terminate the dispersion
step in DLLME, the centrifugation process is not necessary. This in turn improves precision, increases
throughput, decreases the risk of contamination through human intervention and minimizes the overall
analysis time. ST-DLLME has been successfully applied for determination of both inorganic and organic
analytes including pesticides and pharmaceuticals in water and biological fluids. Demulsification via
solvent injection rather than centrifugation saves energy and makes ST-DLLME easier to automate. These
characteristics in addition to the low solvent consumption, the reduced organic waste and the possibility
of using water in demulsification bestow green features on ST-DLLME. This tutorial discusses the prin-
ciple, the practical aspects and the different applications of ST-DLLME.
© 2018 Elsevier B.V. All rights reserved.

* Corresponding author. El-Geish Street, the Medical Campus, Faculty of Pharmacy, Tanta, 31111, Egypt.
E-mail address: fotouhrashed@pharm.tanta.edu.eg (F.R. Mansour).

https://doi.org/10.1016/j.aca.2018.02.005
0003-2670/© 2018 Elsevier B.V. All rights reserved.

Please cite this article in press as: F.R. Mansour, N.D. Danielson, Solvent-terminated dispersive liquid-liquid microextraction: a tutorial,
Analytica Chimica Acta (2018), https://doi.org/10.1016/j.aca.2018.02.005
2 F.R. Mansour, N.D. Danielson / Analytica Chimica Acta xxx (2018) 1e11

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1. Principle of ST-DLLME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2. Requirements of organic solvents used as demulsifiers in ST-DLLME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.3. Factors affecting efficiency in ST-DLLME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.3.1. Effect of demulsifier type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.3.2. Effect of demulsifier volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.3.3. Effect of demulsification time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.4. Tips and tricks in ST-DLLME methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.5. Applications of ST-DLLME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.5.1. Low density solvent ST-DLLME (LDS-ST-DLLME) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.5.2. High density solvent-ST-DLLME (HDS-ST-DLLME) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.5.3. Vortex-assisted ST-DLLME (VA-ST-DLLME) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.5.4. ST-DLLME with solidified floating organic droplet (ST-DLLME-SFOD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.5.5. Ultrasound air-assisted ST-DLLME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.6. ST-DLLME and automation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.7. The green aspects of ST-DLLME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction solidification of floating organic droplets (DLLME-SFOD) [7,8]. After

extraction and phase separation, the sample is placed in an icebox
Sample preparation is an essential step in most analytical pro- to allow the extract to solidify before being transferred by a spatula.
tocols to decrease the matrix effect, pre-concentrate the analytes, Instead of manual shaking, other methods of mechanical agitation
derivatize the sample, or protect the instrument [1,2]. The recent were employed to improve the dispersion step using less or no
trends towards miniaturization of analytical systems inspired sci- dispersers. These methods include vortexing (as in vortex-assisted
entists to develop new approaches of sample preparation that DLLME or VA-DLLME) [9,10], ultrasonic waves (as in ultrasound-
require smaller amounts of samples and consume minimal quan- assisted DLLME or USA-DLLME) [11,12] or repeated aspiration/in-
tities of organic solvents. Liquid-phase microextraction (LPME) is jection as in air-assisted DLLME (AA-DLLME) [13,14]. A number of
one of these recent approaches [3]. In spite of the simplicity and the review articles has been published about DLLME [15,16] and its
cost-effectiveness of LPME, the % recovery is small compared with different modalities such as LDS-DLLME [6], DLLME-SFOD [7,8], VA-
conventional liquid-liquid extraction (LLE). That is because LPME is DLLME [9], and ionic liquid based dispersive liquideliquid micro-
not exhaustive; the main objective of microextraction is to extract a extraction (IL-DLLME) [17]. Their applications in water [18], food
reproducible representative amount of the analyte from the sample [19,20] and biomedical analysis [21] have been characterized.
rather than to retrieve all of the analyte. In order to skip the centrifugation step, Chen et al. introduced
To increase the extraction efficiency of LPME, Rezaee et al. [4] solvent-terminated DLLME (ST-DLLME) in 2010 [22]. In ST-DLLME,
developed dispersive liquid-liquid microextraction (DLLME) in an amount of the dispersing solvent is added to the cloudy
2006. In DLLME, the extractant is mixed with an organic disperser mixture to break the emulsion and induce phase separation. The
and the mixture is injected into the aqueous sample to form a centrifugation step which is time-consuming and difficult to
homogenous cloudy solution by manual or mechanical shaking. automate is replaced with a simple fast step of demulsification by
This induced dispersion leads to a significant increase in the contact solvent injection. This mode has been developed in the last few
surface between the extractant and the sample which markedly years to allow for automatic coupling of DLLME with the analytical
increases the extraction efficiency. However, DLLME suffers from device. To the best of our knowledge, ST-DLLME has not been
three limitations: i) the extractants are heavier than water, which reviewed. In this work, the principle, the demulsifying solvents and
complicates the phase separation step after extraction, ii) two the factors affecting extraction efficiency in ST-DLLME are dis-
organic solvents, at least are required (the extractant and the cussed. The applications of ST-DLLME in pesticides, water, soil and
disperser) to perform the dispersion-assisted microextraction biological analysis have also been summarized in both the text and
process and iii) the dispersion is terminated by centrifugation a table.
which is time consuming and renders the technique difficult to
automate. Automation of the extraction procedures is particularly 2. Discussion
important in LPME and DLLME due to the dynamic nature of these
processes which requires strict control of all the procedures. 2.1. Principle of ST-DLLME
In the last decade, a remarkable effort was exerted to overcome
the limitations of DLLME. To widen the range of extractants used in In conventional DLLME (also known as normal DLLME (n-
DLLME, solvents lighter than water, such as toluene, xylene, and DLLME)) [4], the extractant is mixed with an organic disperser
octanol were tried in low density solvent DLLME (LDS-DLLME) before being injected into the aqueous sample. The selected
[5,6]. To further facilitate the extractant transfer after the micro- disperser has to be miscible with the aqueous sample and the
extraction (ME) process, organic solvents with freezing points in organic extractant [23]. By manual shaking, the extractant is
the range of 10e25  C were employed as extractants in DLLME with dispersed as fine droplets in the form of a cloudy solution. The

Please cite this article in press as: F.R. Mansour, N.D. Danielson, Solvent-terminated dispersive liquid-liquid microextraction: a tutorial,
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 F.R. Mansour, N.D. Danielson / Analytica Chimica Acta xxx (2018) 1e11
disperser forms a thin film around the extractant droplets which
facilitates dispersion formation [22]. Then, an extra amount of the
disperser is added to act as a demulsifier which breaks the emul-
sion and induces phase separation. The demulsification process
starts by flocculation of the dispersed extractant droplets by Ost-
wald ripening or coalescence to form larger droplets [24], as shown
in Fig. 1. The molecules on the surface of the dispersed droplets are
less energetically favorable than molecules in the interior; droplet
flocculation is thus thermodynamically-driven to increase the
droplet size and decrease the number of exposed surface molecules
[25]. If the extractant is lighter than water, these flocculated
droplets will float on the top of the extraction vessel in a step called
creaming. On the other hand, the high density solvent can sink to
the bottom in a step known as sedimentation [26]. After that, phase
separation of the floating or the sedimented layer follows. Although
the demulsifier is usually the same solvent used as a disperser, any
substance that fulfills the requirements of a demulsifying solvent
can be used. These requirements will be discussed in more detail in
the next section.
2.2. Requirements of organic solvents used as demulsifiers in ST-
DLLME
The ideal demulsifying solvent should fulfill the following
requirements:
(i) The demulsifier should be available at a reasonable cost to
make the extraction process cost effective.
(ii) The demulsifier should be safe and ecofriendly to avoid any
health or environmental hazard.
(iii) The demulsifier must have high surface activity and a low
surface tension [22] in order to spread over a large area and
break the emulsion.
(iv) A small amount of the ideal demulsifier can break the
emulsion to decrease solvent consumption.
(v) The demulsidifer should not be totally miscible with the
extractant because the enrichment factor (EF) of the ST-
DLLME method will decrease. The EF is calculated accord-
ing to the equation:
Fig. 1. Steps of sample preparation by ST-DLLME.
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3
Co;eq
EF ¼ (1)
Caq;int
where Co,eq is the extracted concentration of the analyte in the
organic layer at equilibrium and Caq,int is the initial concentration of
the analyte in the aqueous sample. If the demulsifier has prefer-
ential miscibility with the extractant, the volume of the extraction
phase will increase, which decreases the concentration of the
extract by dilution and in turn decreases the EF [27].
The most commonly used demulsifier in ST-DLLME is acetoni-
trile [22,28e35]. Other organic solvents have been tried such as
methanol [36], ethanol [37], 1-butanol [27], and acetone [38e40].
Table 1 summarizes physical properties of the commonly employed
demulsifiers in ST-DLLME.
2.3. Factors affecting efficiency in ST-DLLME
The extraction efficiency in ST-DLLME is controlled by the same
factors of n-DLLME. These factors include the type of extractant, the
volume of extractant, the type of disperser, the volume of disperser,
the pH of the sample, the temperature of the sample, the stirring
rate, the time of extraction and the addition of salt. Along with
these parameters, three other experimental variables have to be
studied in order to attain the optimum microextraction (ME) con-
ditions. These variables include the type of demulsifier, the volume
of demulsifier and the time of demulsification. The factors affecting
efficiency in n-DLLME have already been discussed in previous
reviews [7,8]. In this section, only the parameters that are most
pertinent to ST-DLLME will be described in more detail.
2.3.1. Effect of demulsifier type
Selection of the best demulsifying solvent is crucial to obtain the
highest possible efficiency in ST-DLLME. Yet, this factor has been
overlooked in most of the reported ST-DLLME applications. The
majority of the ST-DLLME methods studied the effect of the type of
disperser on the extraction efficiency, and then assumed that the
best disperser would be the best demulsifier. However, there is not
enough evidence that this assumption is true. In the few
4 F.R. Mansour, N.D. Danielson / Analytica Chimica Acta xxx (2018) 1e11

Table 1
Physicochemical properties of the commonly employed demulsifiers.

Demulsifier Surface tension (dyne/cm) Boiling point ( C) Freezing point ( C) Specific gravity Dielectric constant Ref

Acetonitrile 29.1 81.6 44 0.782 37.5 [33]

Acetone 23.3 56 95 0.790 20.6 [43]
Methanol 22.6 64 98 0.792 32.6 [36]
Ethanol 22.3 78 114 0.789 22.4 [37]
1-Butanol 22.8 108 108 0.802 17.7 [27]
Water 72.8 100 0 0.998 79.7 [41]

applications that did study the effect of the type of demulsifier

independently from the disperser type, unexpected results were
obtained. For example, when the effect of various dispersers on the
extraction efficiency of chlorophenols by ST-DLLME was studied,
acetone gave the highest efficiency followed by acetonitrile and
methanol [33]. Thus, 500 mL of acetone was used to disperse 50 mL
of the chloroform extractant. The authors then tried the same three
solvents as demulsifiers; the efficiency of acetonitrile was two
times higher than the efficiency of acetone and three times higher
than the efficiency of methanol. Similar results were obtained by
Hassanpoor et al. [27] who compared the commonly used demul-
sifiers (acetonitrile, acetone, methanol and ethanol) with those
rarely used (1-octanol, ethylacetate and 1-butanol). Surprisingly,
the highest peaks were obtained with 1-butanol and ethylacetate,
although the disperser was ethanol. These results indicate the
importance of studying the effect of demulsifier type even after
selecting the disperser.

2.3.2. Effect of demulsifier volume

Although the aim of adding a demulsifier to the extraction
mixture is to break the emulsion and induce phase separation, the
optimum demulsifier volume should be selected on the basis of
extraction efficiency rather than demulsification efficiency (DE).
The DE can be calculated from the equation:
Fig. 2. Effect of volume of disperser solvents (A) and volume of demulsifier solvents
(B) on peak area of chlorophenols extracted by the HDS-ST-DLLME (n ¼ 3). Conditions:
extraction solvent, 55 mL chloroform (With permission from Ref. [33]).

Vo; t 2.3.3. Effect of demulsification time

DE ð%volÞ ¼  100
Vo; t0
Where Vo,t is the volume of the organic extractant after time ¼ t,
and Vo,t0 is the initial volume of the organic extractant. However,
this equation could be misleading especially when the disperser or
the demulsifier is miscible with the extractant. In this case, the
calculated demulsification efficiency will be increased not because
of the complete emulsion break-up, but because of the increase in
Vo,t due to the added volume of disperser/demulsifier. For this
reason, proper selection of the right demulsifier should be based on
the extraction efficiency [27].
Liang et al. studied the effect of the demulsifier volume on the
efficiency of high density solvent (HDS) ST-DLLME of chlorophenols
using chloroform as an extractant, acetone as a disperser and
acetonitrile as a demulsifier [33]. The volume of acetonitrile was
changed from 400 mL to 1200 mL, and the peak intensity was
measured using HPLC with UV detection. As shown in Fig. 2A, the
largest peaks were observed using 800 mL of acetonitrile. Generally,
most applications use a volume of the demulsifier equal to the
volume of the disperser. However, this has to be verified experi-
mentally first. In the previous application [33], the optimum
extraction was achieved using 500 mL of acetone as a disperser. If
500 mL of acetonitrile were used as demulsifier, a much lower ef-
ficiency would be obtained (Fig. 2B). The effect of the demulsifier
volume on extraction efficiency is particularly important when the
demulsifier is different than the disperser [37,42].
Guo and Lee defined the demulsification time as “the interval
between the completion of injection of the demulsification solvent
and the start of the collection of the extract” [31]. An increase of
demulsification time up to 3 min improved the extraction efficiency
of phthalate esters using ST-DLLME. A study of the effect of
demulsification time is important not only to increase the extrac-
tion efficiency, but also to improve the precision of the method. The
correlation between the demulsifier type and the volume or time
required for demulsification was never investigated in the reported
applications of ST-DLLME. These demulsifier-related experimental
parameters should be optimized together with the other variables
affecting DLLME.
2.4. Tips and tricks in ST-DLLME methods
Although ST-DLLME is a sample preparation technique,
complicated matrices may require treatment before ME proced-
ures. For example, biological samples of high protein content such
as blood derivatives and breast milk can be treated with a suitable
protein precipitant. Two types of precipitating agents are
commonly employed: water-miscible organic solvents and inor-
ganic acids. In this case, using organic solvents such as acetonitrile
or methanol is preferred because these solvents can serve as dis-
persers, decreasing the number of steps and solvent consumption.
For biological samples of low protein content such as urine and
saliva, simply dilution can be effective. Dilution of the sample

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 F.R. Mansour, N.D. Danielson / Analytica Chimica Acta xxx (2018) 1e11 5

decreases the matrix effect and increases the extraction efficiency.
The diluting solvent could be water, aqueous acid or aqueous base
depending on the nature of the analyte. For acidic analytes, aqueous
acids are preferable to decrease %ionization which improves the
extraction. Similarly, aqueous bases are used for basic drugs. The
optimum pH of the diluent depends on the pKa value of the analyte.
The type of extractant chosen depends on the selected mode of
DLLME. In normal DLLME, extractants heavier than water such as
chloroform are used. These solvents are toxic and of limited num-
ber. Low density solvent DLLME (LDS-DLLME) uses safer extractants
that are lighter than water such as toluene, n-hexane and n-octane.
However, transfer of the extractant after phase separation is diffi-
cult; this problem can be solved by the solidification of floating
organic droplet (SFOD) mode of DLLME. In this approach, extrac-
tants lighter than water with melting points in the range 10e25  C
are used. After phase separation, the sample is cooled and the
floating droplet is transferred.
No matter what DLLME mode is chosen, a smaller extractant
volume is often preferable to increase the sample enrichment.
Extractant volumes less than 10 mL are avoided to guarantee a
sample size sufficient for analysis. The type and volume of disperser
are equally important. In the reported ST-DLLME methods, meth-
anol and acetonitrile are the most common dispersers, used pri-
marily in the 400e800 mL range. These solvents can also be used to
terminate the dispersion and induce phase separation. However,
selection of the optimum terminating solvent should be done
irrespective of the type of dispersion. It should be noted that a
setting time is required before the termination step to attain
equilibrium. This setting time is usually short (seconds to a few
minutes); premature termination will affect the method
repeatability.
2.5. Applications of ST-DLLME
ST-DLLME can be classified according to the density of the
extracting solvent or according to the dispersion technique. Based
on the density of the extractant, ST-DLLME can be categorized into
high density solvent ST-DLLME (HDS-ST-DLLME), low density sol-
vent ST-DLLME (LDS-ST-DLLME) and ST-DLLME with solidified
floating organic droplet (ST-DLLME-SFOD). Depending on the
dispersion technique, ST-DLLME can be classified into conventional
ST-DLLME which employs manual shaking, ultrasound-assisted ST-
DLLME (USA-ST-DLLME) and vortex-assisted ST-DLLME (VA-ST-
DLLME). The applications of these different modes are described
below.
GC/MS method was developed using the same disperser, n-hexane
as an extractant, and acetone as a demulsifier [43]. Comparison of
these methods is not easy due to the different GC detectors
employed. The GC/MS method was slightly modified later and
adapted for automation by the same group [28]. An innovative
method was presented by Zhu et al. [37] for determination of PAHs
by using methylene chloride as an auxiliary solvent and adjusting
the density of the extractant through the ratio of methylene
chloride:n-hexane. The higher sensitivity of this method could be
attributed to the high extraction power of the binary solvent
system.
LDS-ST-DLLME was also used for determination of organochlo-
rine pesticides using m-xylene as an extractant and acetonitrile as
the disperser/demulsifier [29]. In this method, a mixture of m-
xylene/acetonitrile (750/40 mL) was injected into a 10 mL volume of
the water sample and stirred. After two min, a 750 mL volume of
acetonitrile was added to the solution in order to terminate the
dispersion step, and the mixture was left for one min. The method
was then applied for determination of mineral and natural water
from Northern Greece using GC/MS. The matrix effect was studied;
the observed differences in % recoveries were within the accep-
tance limits of the United States Environmental Protection Agency
(EPA). An HPLC/UV method was developed by Behbahani et al. [30]
for determination of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-
methyl-4-chlorophenoxyacetic acid (MCPA) in urine and water. The
authors compared their work with the reported methods for
determination of organochlorine pesticides. Although the ST-
DLLME HPLC/UV method was less sensitive than other GC-MS
methods, it was simple, fast and cost-effective [44]. An HPLC-MS/
MS method was performed by Caldas et al. for determination of
organochlorine and other pesticides in addition to pharmaceuticals
and personal care products in water using water as a demulsifier
[41]. The authors compared water and acetone as demulsifiers. As
shown in Fig. 3, the peak areas of different analytes were higher

2.5.1. Low density solvent ST-DLLME (LDS-ST-DLLME)

LDS-ST-DLLME is the most common type of all ST modes. The
reason for that could be attributed to the advantages of LDS which
include using relatively safe organic solvents in the extraction such
as 1-octanol, and the ease of phase separation of the floating
organic layer. In addition, the first reported method of ST-DLLME
introduced by Chen at al employed an LDS [22]. In this method,
carbamate pesticides were extracted from water samples using
toluene as an extractant and acetonitrile as a disperser/demulsifier
solvent. A 5 mL volume of the water sample was mixed with the
mixture of extractant/disperser (15 mL toluene þ 500 mL acetoni-
trile) using a syringe. The solution was allowed to settle to reach an
equilibrium state; then an additional 500 mL volume of acetonitrile
was added to break the emulsion and terminate the extraction step.
LDS-ST-DLLME has also been used for determination of poly-
cyclic aromatic hydrocarbons (PAHs) in environmental water using
Fig. 3. Peak area of pharmaceuticals and personal care products (PPCPs) and pesticides
toluene as an extractant, acetone as a disperser and acetonitrile as a after extraction by ST-DLLME with different demulsifier solvents (acetone left bar,
demulsifier. The extract was analyzed using GC-FID; the limits of water right bar of pair) (sample volume, 10 mL; sample pH 2 and 8; 1-octanol volume,
detection (LODs) were in the range 0.52e5.11 mg/L. A more sensitive 120 mL; acetone or water volume, 0.75 mL) (With permission from Ref. [41]).

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6 F.R. Mansour, N.D. Danielson / Analytica Chimica Acta xxx (2018) 1e11
when water was the demulsifier.
In addition to carbamates and organochlorine pesticides,
ionizable pesticides were extracted by ST-DLLME [38]. Four s-
triazine herbicides including atrazine, propazine, prometryn, and
terbutryn were determined in environmental water using ST-
DLLME with HPLC/UV. The toluene/acetone (50 þ 600 mL)
mixture was injected into 5 mL of the salty sample (10% w/v NaCl)
and left for ten min to equilibrate. This long extraction time was
compensated by the short demulsification time (5e10 s). The
method was comparable to other modes of DLLME [45e47].
Trace levels of three fungicides (azoxystrobin, diethofencarb
and pyrimethanil) were determined in environmental water using
HPLC for analysis and LDS-ST-DLLME for sample preparation [36].
Instead of using a test tube as an extraction container, the authors
used two syringes; one as an extraction vessel and one for injecting
the disperser and the demulsifier and for collecting the extractant
after the ME process. The achieved EFs were 239 for azoxystrobin,
200 for diethofencarb and 195 for pyrimethanil. As shown in Fig. 4,
no peaks were observed for the three analytes in the spiked water
before ME, which indicates the role of ST-DLLME in improving the
method sensitivity.
Besides organic compounds, inorganic analytes have been
determined using LDS-ST-DLLME for sample treatment [27,32,48].
Cyanide concentrations in natural water and plasma were
measured after pre-concentration using MIBK as an extractant,
ethanol as a disperser and 1-butanol as a demulsifier [27]. To
improve the extraction efficiency of the cyanide ion, zinc(II) tetra-
phenylporphyrine (ZnTPP) was employed to form an extractable
complex with the analyte. Fig. 5 shows the chemical structure of
the complex formed between ZnTPP and CN. This reaction does
not only increase the extraction efficiency, but also enables a UV
detection approach for cyanide. As shown in Fig. 5, the wavelength
of maximum absorbance (lmax) of ZnTPP increased after
complexation with CN. It is important to note that the measured
absorbance must be corrected for the absorbance of the extracted
ZnTPP which should be used as a blank. Water contaminated with
palladium was monitored using electrothermal atomic absorption
spectrometry (ETAAS) after LDS-ST-DLLME [32]. Thio-Michler's
ketone was used as a complexing agent before extraction with 1-
octanol and dispersion with acetonitrile. The same disperser was
used as a demulsifier and the attained EF was 231.
Fig. 4. HPLC-UV Chromatograms obtained for the East Lake water (a) spiked (1 mg/L)
East Lake water before extraction; (b) blank East Lake water extracted by the proposed
STeDLLME; (c) spiked (1 mg/L) East Lake water extracted by the proposed STeDLLME.
Peak identification: (1) azoxystrobin, (2) diethofencarb, (3) pyrimethanil (With
permission from Ref. [36]).
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Analytica Chimica Acta (2018), https://doi.org/10.1016/j.aca.2018.02.005
Fig. 5. (a) Absorption spectrum of ZnTPP after extraction in the absence of cyanide
(MIBK as blank), and (b) absorption spectrum of the cyanide-ZnTPP complex after
extraction (extracted ZnTPP to be used as blank). (With permission from Ref. [27]).
2.5.2. High density solvent-ST-DLLME (HDS-ST-DLLME)
Although conventional DLLME utilizes high density solvents
such as methylene chloride [49], chloroform [50e54] and carbon
tetrachloride [55], these solvents are rarely used in ST-DLLME. The
reason for that is the high toxicity of the halogenated hydrocarbons
and the difficulty in separating the sedimented extractant. How-
ever, these heavy solvents usually show higher extraction efficiency
compared with solvents lighter than water [56]. The high lip-
ophilicity of halogenated solvents makes them more efficient in
extracting hydrophobic and non-polar compounds [57,58]. As an
application for HDS-ST-DLLME, chloroform was used for extraction
of chlorophenols from environmental water using acetone as a
disperser and acetonitrile as a demulsifier [33]. The extracted
sample was diluted with sodium dodecylsulfate (SDS) and field-
enhanced sample injection was applied for 5 s. The analytes were
separated by reverse migrating micellar electrokinetic chroma-
tography (MEKC) before being detected by UV absorbance at
200 nm. Using this HDS-ST-DLLME-based method, impressively
high EFs were achieved (Table 2). Although conventional DLLME
was equally efficient, ST-DLLME was faster and simpler.
2.5.3. Vortex-assisted ST-DLLME (VA-ST-DLLME)
As previously mentioned, ST-DLLME can be assisted using
different types of mechanical agitation such as vortexing, ultra-
sound waves or repeated aspiration/injection. Compared with
other methods of mechanical shaking, the vortex is more efficient
[59]. Using vortexing, DLLME can be performed with no or less
disperser [60]. Vortex-assisted ST-DLLME (VA-ST-DLLME) was
performed for pre-concentration of organophosphorus pesticides
in environmental water [34]. The analytes were extracted using 1-
dodecanol, dispersed and demulsified by acetonitrile. The disper-
sion was accelerated via a vortex mixer running at 1800 rpm for
20 s. The achieved extraction efficiency was between 90 and 99%.
The authors collected the floating layer using a syringe compatible
with the HPLC injection port. However, they could have frozen the
extractant to facilitate the process of sample transfer due to the low
melting point (22e24  C) of 1-dodecanol. The applications of this
mode will be discussed in the next section.
2.5.4. ST-DLLME with solidified floating organic droplet (ST-DLLME-
SFOD)
DLLME with solidified floating organic droplet (DLLME-SFOD) is
 F.R. Mansour, N.D. Danielson / Analytica Chimica Acta xxx (2018) 1e11 7

Table 2
Applications of different modes of ST-DLLME.

Analyte Sample Type of Extractant Disperser Demulsifier Detection Linearity LOD %RSD EF Ref
DLLME Range (mg/L)
(mg/L)

PAHs Environmental LDS-ST- Toluene, 40 mL Acetone, Acetonitrile, GC-FID 20e500 0.52 2.2 NR [42]
water DLLME 500 mL 800 mL e5.11 e13.6
PAHs Water LDS-ST- n-Hexane, 50 mL Acetone, Acetone, GC-MS 0.05e50 0.0037 <11 NR [43]
DLLME 500 mL 500 mL e0.039
PAHs Environmental LDS-ST- Methylene chloride/n-hexane Acetonitrile, Ethanol, GC-MS 0.008 0.0021 5.8 94.9 [37]
water DLLME (1:1, v/v), 200 mL 800 mL 600 mL e5.0 e0.0136 e10.9 e103
PAHs Environmental LDS-ST- n-Hexane, 60 mL Acetonitrile, Acetonitrile, GC/MS 0.1e50 0.023 4.8 NR [28]
water DLLME 1400 mL 1600 mL e0.058 e7.3
Organochlorine pesticides Environmental LDS-ST- m-Xylene, 40 mL Acetonitrile, Acetonitrile, GC/MS 0.05e50 0.002 3.5 25 [29]
water DLLME 750 mL 750 mL e0.05 e10.2e319
2,4-D & MCPA Urine and water ST-DLLME 1-Octanol, 75 mL Acetonitrile, Acetonitrile, HPLC-UV 1e400 0.2e1.6 5.6 123 [30]
750 mL 750 mL e131
Pesticides, Water ST-DLLME 1-Octanol, 120 mL Acetone, Water, LC-MS/ 0.0125 0.0038 1e21 NR [41]
pharmaceuticals and 750 mL 750 mL MS e25 e0.38
personal care products
Carbamate pesticides Water ST-DLLME Toluene, 15 mL Acetonitrile, Acetonitrile, GC-MS/ 0.005e20 0.001 2.3 NR [22]
500 mL 500 mL MS e0.05 e6.8
Ionizable pesticides Environmental LDS-ST- Toluene, 50 mL Acetone, Acetone, HPLC/UV 10e400 0.60 8.8 NR [38]
water DLLME 600 mL 500 mL e2.33
Fungicides Environmental LDS-ST- Toluene, 20 mL Methanol, Methanol, HPLC-MS 0.1e200 0.026 7.9 195 [36]
water DLLME 500 mL 500 mL e0.071 e8.2 e239
Phthalate esters Water LDS-ST- Toluene, 60 mL Acetonitrile, Acetonitrile, GC-MS 0.1e50 0.01 <5.9 178 [31]
DLLME 1300 mL 1400 mL e0.02 e272
Cyanide Natural water ST-AS- MIBK, 100 mL Ethanol, 1-Butanol, UV/Vis 4.0e130 1.0 1.1 50 [27]
and plasma DLLME 500 mL 2500 mL
Copper Water and food LDS-ST- Xylene, 250 mL Acetone, Acetonitrile, FAAS 0.22e140 0.08 5.5 NR [48]
samples DLLME 550 mL 500 mL
Palladium Environmental LDS-ST- 1-Octanol, 75 mL Acetonitrile, Acetonitrile, ETAAS 0.025 0.007 3.68 231 [32]
water DLLME 750 mL 750 mL e0.500
Chlorophenols Environmental HDS-ST- Chloroform, 50 mL Acetone, Acetonitrile, MEKC/ 20e4000 3.5e4.0 8.2 233 [33]
water DLLME 500 mL 800 mL UV e9.3 e797
Cadmium Water SPE-ST- 1-Octanol, 50 mL Acetonitrile, Acetonitrile, FAAS 0.1e50 0.03 5.1 96 [34]
DLLME 500 mL 500 mL e165
Organophosphorus Environmental VA-LDSeST 1-Dodecanol, 100 mL Acetonitrile, Acetonitrile, HPLC/UV 0.5e500 0.25 <5.3 72 [35]
pesticides water eDLLME 500 mL 500 mL e1.00 e79
Organochlorine pesticides Environmental ST-DLLME- n-Hexadecane, 100 mL Acetone, Acetone, GC/MS 0.025 0.012 3.15 96 [39]
water SFOD 500 mL 750 mL e2.00 e0.024 e4.53 e101
Sulfonylurea herbicides Water and Soil ST-DLLME- 1-Dodecanol, 120 mL Methanol, Acetone, HPLC/UV 5e1000 0.24 2.73 NR [40]
SFOD 400 mL 1200 mL e0.81 e10.4

NR: not reported.

a modified mode of LDS-DLLME. In DLLME-SFOD, the extracting
organic solvent such as 1-undecanol or 1-dodecanol has to be
lighter than water, with a melting point in the range between 10
and 25  C [8,61]. The demulsification step in DLLME-SFOD can be
induced by adding a demulsifying solvent to the cloudy solution
and the mode is termed ST-DLLME-SFOD. After this step, the
extraction vessel is placed in an icebox to allow the floating layer of
extractant to solidify before being transferred by a spatula.
Nine sulfonylurea herbicides were measured in water and soil
samples using HPLC/UV after ST-DLLME-SFOD [40]. The water
sample was adjusted to pH 2 using 1 M HCl, and the ionic strength
was increased by adding 7 g of NaCl before making up the volume
to 20 mL. The extracting solvent (1-undecanol, 120 mL) was mixed
with the disperser (methanol, 400 mL) and pipetted into the sample.
The formed cloudy solution was heated at 30  C for 1 min; after that
a 1200 mL volume of acetone was added to break the emulsion. The
solution was then incubated in an icebox for 5 min to solidify the
floating layer, which could then be transferred by an Eppendorf
tube to the HPLC instrument. Similar procedures were applied for
soil samples after extraction with 1 mL of methanol and dilution to
50 mL with 0.1 M of NaHCO3. The method was faster and simpler
than the conventional solid-phase extraction (SPE) and LLE
methods.
2.5.5. Ultrasound air-assisted ST-DLLME
DLLME can be assisted by different agitation techniques such as
ultrasonic waves [62,63] or repeated aspiration/injection steps
[13,14]. Ultrasound helps to facilitate the dispersion step instead of
manual shaking. An alternative approach is to use a syringe to
frequently draw and inject the extractant and the sample until an
emulsion is formed. This alternative approach is known as air-
assisted DLLME [64]. Ezoddin et al. [65] merged the two ap-
proaches to extract three antifungals from water and biological
samples using ultrasound-air assisted ST-DLLME-SFOD. The sam-
ple, placed in a tube with a conical bottom, was mixed with the
extractant (1-dodecanol) after adjusting the pH to 10. The tube was
then put in an ultrasonic bath while the mixed extractant/sample
combination was being rapidly aspirated and reinjected for 30s.
After the extraction time, a 200 mL volume of acetonitrile was added
to break the emulsion and terminate the extraction process. Then,
the tube was transferred into an icebox to solidify the floating
extractant before collection.
2.6. ST-DLLME and automation
The ultimate approach for a ST-DLLME method is development
of a fully automated extractive and analytical technique. Method

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8 F.R. Mansour, N.D. Danielson / Analytica Chimica Acta xxx (2018) 1e11
automation improves precision, increases throughput, decreases
the risk of contamination through human intervention and mini-
mizes the overall analysis time. The effort exerted in automation of
DLLME has been recently reviewed [66]. In this review, Alexovi
c
et al. classified the different approaches used in automation of
DLLME into flow, batch, flow-batch and in-syringe modes. These
modes can be achieved by using an autosampler or by employing
one of the flow techniques such as flow injection analysis,
sequential injection analysis or lab-in-syringe.
The critical step in DLLME automation is the phase separation,
which is ideally performed by centrifugation. However, centrifu-
gation is very difficult to automate. Other alternative ways of phase
separation have been tried such as passive floatation [67], SPE [68]
and solvent demulsification [22]. Demulsification via solvent in-
jection requires fewer tools and is easy to automate.
Initially phthalate esters were used as model analytes for
automation of ST-DLLME [31]. The GC autosampler was used to
perform the ME procedures as well as to inject the sample into the
gas chromatograph. Three different narrow-neck vials were
employed; the first vial contained the aqueous sample of phthalate
esters, the second vial was filled with a mixture of the extrac-
tant:disperser in the optimum volume ratio (toluene:acetonitrile,
6:130 v/v), and the third vial had the demulsifier (acetonitrile). The
ST-DLLME started with the repeated injection of 80 mL of mixed
extractant/disperser into the sample vial 17 times. This step took an
average time of 340s and a total volume of 1.36 mL from the second
vial. The sample vial was then left for 2 min before a 70 mL volume
of acetonitrile was injected into the first vial and repeated for 20
times (400s and 1.4 mL of demulsifier). After 3 min, a 10 mL volume
of the upper layer was withdrawn by the syringe and injected into
the GC/MS system.
Although this approach was successfully applied, this technique
had three limitations: the narrow-neck vials were homemade (not
commercially available), the repeated injection steps prolonged the
procedures, and the required large volume of demulsifier could
affect the equilibrium and the extraction recovery. These problems
were addressed by using a dual-rail autosampler with two different
syringes; a large-volume syringe for solvent transfer in the ST-
DLLME procedures and a microsyringe for extract collection and
injection into the GC/MS instrument [28]. The large volume syringe
allowed each of the dispersion and the demulsification steps to be
performed in one injection which accelerated the whole process.
Fig. 6. The twelve principles of green analytical chemistry. The requirements fulfilled by ST-DLLME are indicated by red checkmarks. (For interpretation of the references to colour
in this figure legend, the reader is referred to the Web version of this article.)
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Analytica Chimica Acta (2018), https://doi.org/10.1016/j.aca.2018.02.005
2.7. The green aspects of ST-DLLME
The main objective of current analytical chemistry is to achieve
high quality results using safe reagents and methodologies. Anastas
and Warner were the first to set rules for green chemistry [69,70].
Shortly after that, several attempts have been reported to apply
similar rules for green analytical chemistry (GAC) [71,72]. In 2013,
Galuska et al. proposed twelve principles of GAC [73]. These prin-
ciples covered the operator, the sample, the reagents, the instru-
ment, the method, and the waste. Safety considerations for the
operator should be applied. Sample number and volume should be
reduced when possible. The reagents should be safe and from
renewable sources. The instrument used should be energy efficient,
automated, and miniaturized to perform in situ analysis. The
method applied should be direct with no need for sample deriva-
tization or treatment, and capable of determining multiple analytes
simultaneously by merging analytical procedures. Finally, the waste
should be minimized. Fig. 6 summarizes the twelve principles of
GAC.
ST-DLLME fulfills most of these principles through use of organic
solvents in demulsification to facilitate automation, which makes in
situ analysis possible. Although the energy consumption of ST-
DLLME has not been studied, it is expected that demulsification
by solvent injection requires less energy than centrifugation.
Moreover, the small volume of an organic extractant serves for
sample extraction and preconcentration at the same time, which
decreases the number of steps. This feature also decreases solvent
consumption and waste disposal. Water can replace a toxic organic
solvent in the termination step [41]. These characteristics conse-
quently increase the safety of the operator.
3. Perspective
Development of ST-DLLME methods is the first step to make
fully automated ME procedures. So far, a few methods of ST-DLLME
have been automated. More effort is expected in the future to
integrate the ST-DLLME procedures with the analytical in-
struments. The number of solvents tested as demulsifiers in ST-
DLLME is still very limited. The consideration of other solvents
such as organic polymers to terminate the dispersion step is
anticipated [74e78]. The use of stronger demulsifying solvents such
as commercially available demulsifiers [79] to decrease the solvent
consumption and achieve fast and efficient termination should be
 F.R. Mansour, N.D. Danielson / Analytica Chimica Acta xxx (2018) 1e11
the focus of future research. The effect of the terminating solvent on
the equilibrium state and the demulsification kinetics should be
studied. That is particularly important when water is used as a
demulsifier. The addition of water to the cloudy mixture of sample/
extractant/disperser does not only break the emulsion, but also
increases the volume of sample which may affect the equilibrium
concentration. Other methods of demulsification such as salt-
addition [80,81], nitrogen floatation [82], or agitation-induced
termination [83] are expected to expand the field. Comparison of
the demulsification efficiency (DE) for these different techniques is
important to select the optimum procedure.
4. Conclusion
The principle and the different applications of ST-DLLME have
been reviewed. Compared with other modes of DLLME, ST-DLLME
is simpler and easier to perform. ST-DLLME does not require a
centrifugation step, making it fast and energy efficient. In addition,
ST-DLLME is easier to automate by using a regular or a dual-rail
autosampler. The green aspects of ST-DLLME make it an attractive
research area especially with the recently introduced safe and
renewable demulsifiers. These characteristics improve analytical
performance as well as the safety of the operator. On the other
hand, ST-DLLME has some drawbacks; collection of the extractant
requires special narrow-neck vials that are not commercially
available. In addition, the added demulsifier could shift the equi-
librium and affect the extraction efficiency. Yet, these limitations do
not refute that ST-DLLME is one of the most promising DLLME
modes, especially when automation is desired.
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List of abbreviations
 F.R. Mansour, N.D. Danielson / Analytica Chimica Acta xxx (2018) 1e11 11

2,4-D: 2, 4-Dichlorophenoxyacetic acid Fotouh R. Mansour is a lecturer at the Faculty of Phar-

AS: Auxiliary solvent macy, Tanta University, Egypt. He got his BS (Pharmaceu-
DE: Demulsification efficiency tical sciences) from the same university with an overall
DLLME: Dispersive liquid-liquid microextraction grade: excellent with honors (the third among one thou-
EF: Enrichment factor sand students). In 2009, Fotouh finished his MS in
ETAAS: Electrothermal atomic absorption spectrometry Electrothermal atomic ab- analytical chemistry from the same university. In 2013, he
sorption spectrometry got his PhD in analytical chemistry from Miami University.
FAAS: Flame atomic absorption spectrophotometry In 2014, he became the vice president of the Strategic
FID: Flame ionization detector Planning Unit in Tanta University. In 2015, he became a
GAC: Green analytical chemistry university associate in the University of Tasmania. Fotouh
GC: Gas chromatography is currently the director of the Pharmaceutical Services
HDS: High-density solvent Center in Tanta University. His research interests are
LDS: Low density solvents pharmaceutical analytical chemistry and separation
LLE: Liquid liquid extraction sciences.
LLME: Liquid-liquid microextraction
LPME: Liquid phase microextraction
MCPA: 2-Methyl-4chlorophenoxyacetic acid Neil D. Danielson has been a professor of chemistry at
ME: Microextraction Miami University in Oxford OH for almost 40 years. He
MEKC: Micellar electrokinetic chromatography received his BS degree at Iowa State University, his MS at the
MIBK: Methyl isobutyl ketone University of Nebraska, and his PhD at the University of
MS: Mass spectrometry n-DLLME Georgia with L. B. Rogers as his research advisor. His
n-DLLME: Normal DLLME research direction has always been primarily in separation
PAHs: Polycyclic aromatic hydrocarbons science, particularly liquid chromatography and capillary
PPCPs: Pharmaceuticals and personal care products electrophoresis, but his work in chemiluminescence is most
SDS: Sodium dodecylsulfate cited. He has also been involved in the development of
SFOD: Solidification of floating organic droplet home-built instruments to facilitate teaching of analytical
SPE: Solid-phase extraction chemistry. Neil has directed 28 PhD students and 18 MS stu-
ST-DLLME: Solvent-terminated DLLME dents, and their research is reflected in most of the 150 pub-
TPP: Tetraphenylporphyrine lications. His major service to the separation science
USA-DLLME: Ultrasound assisted DLLME community is as co-editor of the Journal of Chromato-
VA: Vortex assisted graphic Science.

Please cite this article in press as: F.R. Mansour, N.D. Danielson, Solvent-terminated dispersive liquid-liquid microextraction: a tutorial,
Analytica Chimica Acta (2018), https://doi.org/10.1016/j.aca.2018.02.005