Liquid phase

microextraction
LPME
Liquid Phase Microextraction (LPME)

• Use either 2 immiscible liquids (droplet technique)

or 2 liquid phases separated by membrane
(membrane microextraction technique)

• Strategy is to reduce volume ratio of acceptor-to-

donor phase (enrichment) by transferring analyte
from high volume to micro extract (5-50 µL)

• Consumption of organic solvent is greatly

minimized.
2
Donor, acceptor and intermediate
phases
• Donor phase: the sample that contains and donates the
analyte. It is usually aqueous phase.

• Acceptor phase: the solution that contains the analyte in

its final stage prior the instrumental analysis, it can be
aqueous or organic.

• Intermediate phase: the phase use to transfer the analyte

(bridge) between donor and acceptor phases, it has
immiscible with the acceptor and donor phase.

3
• EF is calculated according to the formula:

EF = Ca/Cd

Ca:is the concentration of analyte in the acceptor phase

(extracting phase).
Cd: is the concentration of analyte in the donor phase
(sample).

4
Single-drop microextraction (SDME)
• SDME is one of the first extraction
techniques were proposed to replace
LLE technique.
• Immiscible single drop (1–10 μL) of
organic solvent was suspended at the
end of a micro-syringe needle in an
aqueous solution with continuous
stirring.
• The analytes partition between the
bulk aqueous phase and the organic
solvent microdrop.
• The hanging drop is withdrawn
Once equilibrium was achieved and
injected directly into a GC, HPLC,
or CE. 5
 SDME
• Microextraction performed by suspending a
µL drop of organic solvent on the needle tip of
microsyringe.
• Immersed in a stirred aqueous solution.
• The analytes partition between the bulk
aqueous phase and the organic solvent
microdrop.
• The micro-droplet was withdrawn into the
syringe and injected into CG.

6
Advantages of SDME
• high enrichment factor (EF) due to the high-volume
ratio of the phases (mass transfer).
• Inexpensive.
• Simple.
• Efficient.
• Less organic solvents is used.
• Wide range of pH.

7
Disadvantages of SDME
• Instability of the hanging drop.

• High stirring speed and the slight solubility of the

organic solvent in the aqueous phase may cause the
drop to be dislodged from the syringe needle.

• Analysis of biological samples such as plasma can

emulsify considerable amounts of organic solvent.

• Works best with clean matrix – particles or bubbles in

the sample affect the extraction by making the drop
unstable. 8
 Hollow Fiber-Liquid phase
microextraction (HF-LPME)
The HF-LPME technique
developed in 2000 to overcomes
the problem of drop stability by
protecting the drop within the
lumen of a porous hydrophobic
polypropylene hollow fiber.
SEM images of the
inner surface of the
hollow fiber

9
HF-LPME……
• The polypropylene hollow fiber (1.5-10 cm length, 600 µm
ID, wall thickness 200 µm and 0.2 µm pore size) is used as
extraction device.
• Organic solvent, intermediate phase, (15-20 µL) is
immobilized in the pores of the hollow fiber.
• Analytes are extracted from sample (0.1-10 mL) into the
organic solvent, and then into an acceptor phase filled inside
the hollow fiber.

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 HF-LPME
Two phase Three phase
HF-LPME HF-LPME

11
 Cross section of the hollow fiber inside the aqueous sample
during (i) two-phase and (ii) three-phase LPME.

12
 Two phase Three phase
HF-LPME HF-LPME
• Effeciency increases as the • Effeciency increases as the
log P* increases log P increases and
(The analytes should have decreases at higher values
high affinity to organic) (The analytes should have
• Functional group is not high affinity to organic)
requiered • Functional group is
• The analytes are deionized requiered.
in the donor and acceptor • The analytes are deionized
phases in the donor phase and
• The analytes are extracted ionized in the acceptor phase
to organic solvent • The analytes are extracted to
• Compatable with GC aqouse solution
* P: partitioning coefficient, partitioning • Compatable with HPLC and
constant of the analyte between octanol/water CE 13
 Two-Phase System
• Donor phase – aqueous containing analyte (A); acceptor
phase – organic
• ASample↔AOrganic acceptor
• Partition coefficient, Ka/d=[A]a/[A]d
• Useful for separating hydrophobic molecules with
neutral, acidic or basic groups
• pH of sample solution adjusted for analytes to be
deionized form.
• Directly compatible with CG.

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 Tow phase HF-LPME

pH: 2-3
pH: 2-3
units lower
Acidic Basic units higher
than pKa Donor Donor than pKa
value
value phase phase

ACIDIC BASIC
ANALYTE ANALYTE

15
Three-Phase System
• ASample↔AOrganic phase ↔AAqueous acceptor
• Korg/d= [A]org/[A]d
• Ka/org=[A]a/[A]org
• Overall, Ka/d=[A]a/[A]d=Korg/d*Ka/org
• Useful for basic or acidic analytes with ionizable
functionalities.
• For basic compounds, pH of sample adjusted to
become alkaline, acceptor should be acidic.
• Final extract is aqueous – directly compatible with
CE, HPLC
16
 Three phase HF-LPME

pH: 2-3 Acidic Basic pH: 2-3

units lower Donor Donor units higher
than pKa phase phase than pKa
value
value

ACIDIC BASIC
ANALYTE ANALYTE

pH: 2-3 Basic Acidic pH: 2-3

units higher Acceptor Acceptor units lower
than pKa phase phase than pKa
value value
17
 Ion pair-three phase system
• Suitable for hydrophilic
analytes with poor
partition coefficients.
• A carrier (hydrophobic
ion-pair reagent) is
added to the sample
solution.
• Forms an ion-pair with
the analyte, extraction
of ion-pair complex into
the organic phase in the
pores of the hollow
fibre. 18
Technical set-up for LPME based on U-shaped fiber
(left) and rod-like fiber (right)

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 Cheap
Simple
Wide range of pH
Less waste generated
Less time consuming
Provide clean extracts
The fiber is disposable
No carry-over problems
Less organic solvents is used
High analyte enrichments which allows trace determinations
20
• Low selectivity compared to SPME.
• Small surface area.
• Evaporation or floating of the organic solvent.
• The presence of air-bubbles could influence the
extraction efficiency.
• Twisting of the fiber is possible at high stirring speed
which affects the extraction efficiency (low diffusion
rate) and leaching the organic solvent.
• Extraction time is long.

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 HP-LPME
Developed to
use only one
single drop

SD-LPME

LLE

HF-LPME

22 22
 Solvent Bar Microextraction (SBME)

• The organic extractant was confined

within a short length of a hollow fiber
(sealed at both ends) that was placed in a
stirred aqueous sample solution.
• Free movement of the device within the
sample solution facilitated extraction.
• Organic phase either analysed using GC
or normal phase LC

• Ref: X. Jiang and H.K. Lee, Anal. Chem. 76

(2004), p. 5591.

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Dispersive Liquid-Liquid
Microextraction (DLLME)
• Considered the most acceptable microextraction
technique.
• Simple, rapid, high enrichments, low cost, minimum
requirements for sample and organic solvents…..Green
methods
• Many innovations, e.g.
• Modes of dispersion (ultrasound, vortex, air-assisted)
• Density of extracting solvent (high and low density)
• Types of extracting solvents (organic, ionic liquid, deep
eutectic solvents)
• Combinations with other techniques (SPE, SFE, magnetic-
24
nano materials, etc)
 DLLME
• The fundamental of the technique is based on the mixing of an
aqueous sample containing analytes with a low amount (normally
in the order of microliters) of an extractant solvent, which is non-
miscible with water, with the aid of a dispersive solvent (normally
0.5– 1 mL), which is miscible in both water and the extractant
solvent.
• Thus, only the mixing of the three components forms multiple
microdroplets in solution in which partition of the analytes takes
place. Analytes experience enrichment in the low volume of
extraction solvent, which was dispersed into the bulk aqueous
solution, and are then commonly separated by centrifugation (in the
case of high-dense extracting solvent).
DLLME
 DLLME
• DLLME is a successful extraction technique due to the
high contact surface of fine droplets of extractant solvent
and analytes, which speeds up the mass-transfer processes
of analytes.

• The application of vortex or ultrasound in DLLME if a

solvent less dense than water is used as extraction solvent.
Type of organic solvents use in
DLLME
• The solvent should have very low solubility in water to
form immiscible layers.
• Different solvents is used such as:
• Chlorinated solvents (high density).
• Low density solvents such as: long chain alkanes, long chain
alcoholic solvents, ethers and acetate (with large Carbone
number): vortex or ultrasound are used as agitation methods.
• Ionic liquid solvents.
• Deep eutectic solvents.
DLLME modes
DLLME modes-other classification
A) Conventional DLLME: The mixture can be manually
stirred, and the preconcentrated analytes in the IL
microdroplet are then separated by centrifugation.
B) Temperature assisted- DLLME or Temperature elevated
DLLME
• Heating of mixture (50–90 °C) to ensure adequate formation of
microdroplets.
• The solubility of solvent evidently increases with temperature,
so the heating favors dispersion of the solvent into the aqueous
solution.
• The method further requires cooling of the solution to facilitate
settling of the microdroplet containing extracted analytes.
C) Ultrasound-assisted, microwave-assisted, or vortex-
assisted DLLME
• Ultrasound, microwaves, vortex, or any additional strong
mixing requirement to facilitate dispersion of the
hydrophobic solvent into the aqueous solution and so to
ensure the adequate formation of microdroplets.
• In some cases, a dispersive solvent is needed to improve
the kinetics, and it can be an organic solvent, a surfactant,
or a hydrophilic IL.
D) In-situ
derivatization
DLLME
E) In-situ-IL-DLLME
F) Air-agitated dispersive liquid-
liquid microextraction
Ionic liquids
• Ionic liquids are ionic materials (salt-like compounds)
that are liquid at low temperatures.
• Currently, it’s “official” definition uses the boiling point
of water as a point of reference: “These are ionic
compounds which are liquid below 100 °C.”
• More commonly, These have melting points below room
temperature; some of them even have melting points
below 0 °C.
• These materials are liquid over a wide temperature range
(300–400 °C) from the melting point to the
decomposition temperature of these compounds.
 ILs properties
• Low melting point.
• non-flammable substance.
• Low vapor pressure (unless decomposition occurs):
depends on the strong ionic (Coulomb-) interaction
within these substances
• High thermally stable.
• High mechanically and electrochemically stability.
• Immiscibility with water or organic solvents that result in
biphasic systems.
ILs materials

• ILs are composed of cations interact with anions.

• The properties and stability of the ILs are determined by
the choice of cation. (In general)
• The chemistry and functionality is controlled by the
choice of the anion. (In general)
E) In-situ-IL-DLLME

• In-situ IL-DLLME method, is based on utilizing a hydrophilic IL as

extractant solvent of the analytes contained in the aqueous solution.

• An anion-exchange reagent is then added to promote a metathesis

reaction, and the hydrophilic IL is transformed into a hydrophobic IL,
which settles to contain the preconcentrated analytes.
Ionic liquids used as stationary phases
Ionic liquids used as stationary phases
Deep Eutectic solvents (DES)
• DES is firstly proposed in 1919.
• DES was defined as a mixture of a quaternary ammonium salt
with a metal salt of hydrogen bond donor.
• DES is recently defined as a mixture of hydrogen bond donor
and hydrogen bond acceptor substances which are physically
bonded by hydrogen bonding.
• The resulted mixture will have different properties than the
mixing substances in terms of melting point.
Deep Eutectic solvents (DES)
• Properties:
• Highly polar
• Low freezing point (less than 100 °C)

• Advantages:
• Low vapor pressure (non-flammable)
• Biodegradable and biocompatible
• Non-toxic
• Low cost
• Easy to prepare.
• Easy to scale-up
Deep Eutectic solvents (DES)

• The mixture is prepared in

mole ratios of 1:1, 1:2, 2:1,
1:3 or 3:1.
• Higher ratios have not been
reported.
• The use of more than two
solvents has been reported.
Natural Deep Eutectic solvent (NADES)

• NADES is solvent prepared from natural substances.

• One of the solvent can be a component in the matrix.

• DES or NADES are used as extracting solvent for he

microextraction technique.
Solidification of floating organic
droplet (SFOD-DLLME)
Solidification of floating organic
droplet (SFOD-DLLME)
• The organic solvent should have density lower than
water and the melting point of water should be lower
than extracting medium. Some organic solvent used:
Factors affecting extraction efficiency
in DLLME-SFOD
• Type and volume of extractant.
• Type and volume disperser
• Addition of salt
• Sample pH
• Sample temperature
• Stirring rate
• Extraction time
Vortex-assisted liquid-liquid microextraction
(VALLME) and vortex-assisted liquid-
liquid-liquid microextraction (VALLLME)
• VALLME and VALLLME are sometimes considered as
a modes of DLLME. However, in these techniques
dispersive solvent is not used.
• VALLME is a two phase microextraction technique in
which the final extract is in the organic phase.
• VALLLME is a three phase microextraction technique in
which the final extract is aqueous phase.
Vortex-assisted liquid-liquid microextraction
(VALLME) and vortex-assisted liquid-
liquid-liquid microextraction (VALLLME)
• VALLLME involves two major steps:
(i) VALLME to extract the analyte from aqueous (DP) to
organic phase (intermediate phase)
(ii) a micro-vortex assisted liquid–liquid extraction (μ-
VALLE) procedure to extract the analyte in the intermediate
phase back to an aqueous phase (AP).
• A major hallmark of the technique is that the extracts are
aqueous-base and thus can be directly analyzed using reversed
phase HPLC and CE.
Comparison
The efficiency of the extraction is
reported as:
• Enrichment factor (preconcentration factor):

EF = Ca/Cd

• % Extraction recovery:

% recovery = (Ca * Va /Cd * Vd) * 100%