J. Biochem. Biophys.

Methods 70 (2007) 253 – 261

www.elsevier.com/locate/jbbm

Review
Techniques of preparing plant material for chromatographic
separation and analysis
G. Romanik a,⁎, E. Gilgenast a,1 , A. Przyjazny b , M. Kamiński a,1
a
Gdańsk University of Technology, Chemistry Department, Analytical Chemistry Division, 80 – 952 Gdańsk, ul. Narutowicza 11/12, Poland
b
Science and Mathematics Department, Kettering University, 1700 West Third Avenue, Flint MI 48504, USA
Received 28 June 2006; accepted 29 September 2006

Abstract

This paper discusses preparation techniques of samples of plant material for chromatographic analysis. Individual steps of the procedures used
in sample preparation, including sample collection from the environment or from tissue cultures, drying, comminution, homogenization, leaching,
extraction, distillation and condensation, analyte enrichment, and obtaining the final extracts for chromatographic analysis are discussed. The
techniques most often used for isolation of analytes from homogenized plant material, i.e., Soxhlet extraction, ultrasonic solvent extraction
(sonication), accelerated solvent extraction, microwave-assisted extraction, supercritical-fluid extraction, steam distillation, as well as membrane
processes are emphasized. Sorptive methods of sample enrichment and removal of interferences, i.e., solid-phase extraction, and solid-phase
micro-extraction are also discussed.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Plant material; Sample preparation; Chromatographic analysis

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
2. Preliminary sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
2.1. Collection of plant material and preparation of sample for analysis — general principles . . . . . . . . . . . . . . . . . . 254
2.2. Drying, auxiliary techniques and procedures for volatile compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
2.3. Comminution and homogenization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
3. Techniques of isolation of analytes from plant material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
3.1. Extraction/leaching — general principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
3.2. Soxhlet extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
3.3. Ultrasonic extraction (sonication) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
3.4. Accelerated solvent extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
3.5. Microwave-assisted extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
3.6. Steam distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
3.7. Membrane processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
3.8. Supercritical fluid extraction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
3.9. Solid-phase micro-extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
3.10. Sample disruption method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
3.11. Comparison of extraction techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

⁎ Corresponding author. Tel.: +48 58 347 25 58; fax: +48 58 347 26 94.
E-mail address: anyzar@wp.pl (G. Romanik).
1
Tel.: +48 58 347 25 58; fax: +48 58 347 26 94.

0165-022X/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbbm.2006.09.012
254 G. Romanik et al. / J. Biochem. Biophys. Methods 70 (2007) 253–261
4. Solid-phase extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
1. Introduction
Plant metabolites often occur as complex mixtures of many
substances of a wide range of polarity and hydrophobicity. The
most important groups of substances in plant material are: low-
polar (waxes, terpenoids), semi-polar (lipids, phenolic com-
pounds, low-polar alkaloids), and high-polar (polar glycosides,
polar alkaloids, saccharides, peptides, proteins).
The sample preparation procedure for the investigation of the
composition of plant material includes at least three steps, the
first two being always used, while the third one is common,
although not required:
1. Preliminary sample preparation, preceded by comminution
or homogenization of the examined material. The techniques
commonly employed at this stage involve drying, lyophili-
zation, or steam distillation.
2. Extraction/leaching of soluble components of the examined
material with suitable solvents or their mixtures, or a
supercritical fluid, including desorption, hydrolysis, sapon-
ification, etc. When planning extraction/leaching of the
metabolites, it should be realized that a fraction of the total
amount of the components can be adsorbed or otherwise
bound to the cellular wall or organelles. Oftentimes, the
metabolites can form adducts with peptides or phospholi-
pids. Alternatively, the metabolites can be present in the form
of glycosides. Each step of a sample preparation procedure
can result in loss of a fraction of analyte, and this is
especially important when the amounts of isolated sub-
stances are very small. Consequently, in order to relate the
content of metabolites in the final extract to their content in
the tissue or organism, it is necessary to determine the so-
called recovery, which in turn can be inaccurate for the
metabolites adsorbed or otherwise bound to the cellular wall
or high-molecular-weight components of the cell. The range
of extracted components depends on the kind of extrahent
and conditions of the extraction process. First, solvents and
conditions that enable isolation of a wide range of
metabolites are used. The extracts will contain metabolites
and other substances which can interfere with their analysis
belonging to such groups as lipids, phospholipids, glyco-
sides, saccharide, peptides, and other products of plant
metabolism. To reduce the error in determining the recovery,
the standard addition method is used for homogenized plant
material. However, even this approach may not eliminate the
analytical error, particularly when the standard added does
not undergo natural physiological processes in dried or
homogenized material.
3. Analyte enrichment with the simultaneous removal of
interferences by techniques such as liquid/liquid extraction,
solid-phase extraction (SPE), selective adsorption, prepara-
tive liquid chromatography (PLC) with normal (NP-PLC) or
reversed (RP-PLC) stationary phases, ion chromatography
(IC), size-exclusion chromatography (SEC), as well as solid-
phase micro-extraction (SPME), or solvent micro-extraction.
The sample, prepared by the procedures described above, is
subjected to separation and final determination, primarily by
liquid chromatography, electrophoresis, or gas chromatography.
Qualitative analysis, i.e., identification of selected or (less
commonly) all components, is performed first. Next, quantita-
tive analysis is carried out, following calibration of the
instrument. Simultaneous performance of both qualitative and
quantitative analysis is preferred in order to save time.
2. Preliminary sample preparation
2.1. Collection of plant material and preparation of sample for
analysis — general principles
The determination of the composition of plants, fungi, or
bacteria is based on consecutive (or, very rarely, simultaneous)
application of several techniques for preliminary preparation of
the examined material. First, the material is dried or lyophilized,
followed by comminution or homogenization. Next, the material
is extracted (leached) with a specific solvent or either a series or
mixture of solvents. Each extract (leachate) is purified by
removing solids via filtration, ultrafiltration, or centrifugation.
The majority of these sample preparation techniques have
been used for a long time. However, recently some of the
“classical” extraction techniques carried out at elevated
temperature, such as several-day maceration, long-term leaching
with stirring, agitation in water or a buffer solution, and Soxhlet
extraction are being replaced with more modern techniques,
which are more effective, require less solvent, and permit more
readily automation of the apparatus and procedures [1]. The
main objective of sample preparation procedures is the selective
isolation of analytes with the simultaneous matrix simplifica-
tion. Distillation and purification of the extract via removal of
solids is followed by the removal of interferences with a
concurrent enrichment of the extract in the analytes. The
concentration of interferences can substantially exceed that of
the analytes [2]. Sample enrichment aims at increasing the
concentration of analytes above the detection limit of the
instrument used for their determination [3].
An important requirement in analytical chemistry is that the
sample analyzed be representative. This means that samples
must be collected, treated and stored in such way that their
chemical composition is similar to the average composition of
the total material. In the analysis of plant material the
collection of a representative sample is difficult due to
variability of individual plants among a species or variety.
 G. Romanik et al. / J. Biochem. Biophys. Methods 70 (2007) 253–261 255

Consequently, a specific program of sample collection is often
required. For example, at a selected site a dozen or so plants
are collected from a small area (ca. 5 cm2). Soil and
contaminants are removed from the plant material collected.
Each plant is rinsed with deionized water, removing particles
loosely bound to the plant. In addition, the plant material is
rinsed with other liquids or solutions, containing complexing
agents such as EDTA, with dilute HCl solutions or, sometimes,
with organic solvents [4].
2.2. Drying, auxiliary techniques and procedures for volatile
compounds
The analysis of natural samples is often carried out on dried
materials. This allows the determination of specific components
on a dry mass basis and largely reduces the problems associated
with high water content in crude samples (living organisms, their
fragments, or tissues). It should be realized, however, that drying
plant material does not remove water completely and the term

Table 1
Compilation of various operations and steps of the procedure associated with preparation of biological material for chromatographic analysis
No. Operation, step of procedure
1 Comminution, homogenization (breaking,
cutting, grinding, disintegration or lysis of
cellular wall)
2 Division of sample
3 Decomposition of glycosides, adsorbates,
aggregates, complexation of metal ions,
acidic, basic or enzymatic hydrolysis
4 Leaching, extraction of metabolites
Soxhlet extraction
Ultrasonic extraction (sonication)
Microwave-assisted extraction (MAE)
Accelerated solvent extraction (ASE)
Supercritical fluid extraction (SFE)
Steam distillation
5 Decantation/filtration or centrifugation/
ultracentrifugation
6 Drying extracts
7 Derivatization of analytes
8 Complete or partial solvent evaporation,
usually with solvent exchange
9 Extract enrichment in analytes and removal of
interferences using: SPE, liquid–liquid
extraction, steam distillation, supercritical
fluid extraction
10 Storage of extracts
11 Calibration
12 Method validation
Objective Remarks References
Increasing interfacial surface area; obtaining a
homogeneous sample
Sample storage, saving a spare sample
Obtaining free forms of analytes Used only infrequently
Analyte solubilization [6–8]
Separation of solvent from plant material particles, Especially important when grinding a sample [9]
separation of extract and raffinate, colloid peptization in the presence of a solvent
Removal of water, which can interfere with the Drying can be accomplished by: [8]
analysis due to:
Change in surface activity of the stationary phase Passing the extract through a column
packed with a drying agent
Shortening chromatographic column life Adding a drying agent (e.g. anhydrous
Na2SO4) to the extract
Stopping flow of carrier gas as a result of
“flooding” capillary column in case of gas
chromatography of volatile analytes
Conversion of analytes into derivatives with [11]
properties enabling:
Simple determination
Higher stability of analytes
Increased volatility
Higher sensitivity of determination
Increasing analyte concentration in the extract; This step makes use of: [6,7]
replacing solvent (through redissolving dry Evaporation in a rotary evaporator (often at
residue) with the one more suitable for subsequent a reduced pressure)
steps of the analytical procedure Evaporation in a stream of inert gas
Increasing concentration of analytes, removal of Can be accomplished by using: [6,7]
interferences 1. Gel chromatography
2. Column chromatography with:
silica gel
alumina
Florisil
Proper planning of work in the analytical laboratory Extracts should be stored in tightly capped
vials at lowered temperature (below 0 °C)
Preparation for the final determination Most common calibration methods are:
calibration curve
internal standard
standard addition
Investigation whether the procedure is valid for Reference materials and certified reference
specific applications material are required; typically carried out
together with validation of the entire
analytical procedure
256 G. Romanik et al. / J. Biochem. Biophys. Methods 70 (2007) 253–261
“dry mass” means that the material contains from several to a
dozen or so percent water. The drying of natural materials is
frequently performed at 70 °C in ventilated ovens or in ventilated
ovens with a flow of warm air or, sometimes, nitrogen. Con-
vective ovens are often used in small laboratories. Laboratory
vacuum ovens with water absorption, adsorption, or freezing-out
systems, placed before a vacuum pump, have been found effective
for the isolation of nonvolatile and nonsubliming substances.
Recent literature reports indicate that effective drying can be
accomplished at temperatures as low as 40–50 °C [4,5]. Low
drying temperature is especially important when the analytes are
relatively volatile or subliming. In this case, however, vacuum
ovens cannot be used. In case of highly volatile substances, drying
should be replaced with distillation in a stream of warm gas
(typically nitrogen) and freezing out the distillate or, in case of
nonpolar compounds of medium volatility, with steam distillation.
This refers to substances with boiling points below ca. 250 °C as
well as the substances subliming under these conditions.
Any preparation of natural material for analysis also requires
the determination of water content in the primary sample, while
realizing that it may be highly variable and depend on the
developmental stage of the plant, on the kind of species, cul-
tivation conditions, storage of the material, etc. Following
preliminary preparation of a plant sample, various techniques of
isolation of specific groups of analytes are used. The operations
used to prepare biological material for chromatographic analysis
are compiled in Table 1.
2.3. Comminution and homogenization
The next operation in preparing raw plant material for
analysis proper is comminution and homogenization [10]. The
choice of comminution technique depends on the consistency of
the material and its hardness. In case of raw materials containing
essential oils, any increase in temperature should be avoided.
The material should be comminuted in small batches, which
prevents the loss of essential oils [11]. Roots, hard stems, fruits
Fig. 1. Separation steps used for isolation of plant metabolites [14].
and seeds are first cut mechanically or manually and then
ground in any of a number of mechanical mills [11].
Comminution by manual cutting is simple and does not require
sophisticated tools. It results in fragments of different sizes;
thus, sieving of the cut material is recommended. These
techniques have been used, for example, for comminution of
onion and garlic, which were subsequently extracted without
further homogenization of the sample material [12]. Serine was
isolated from yam by dicing the roots, followed by homoge-
nization in a medium of three extracting agents [13].
Comminution usually precedes the next stage of sample
preparation, i.e., homogenization of the material. In the
laboratory, homogenization is often carried out with ceramic
or agate mortar-and-pestle sets. A variety of mechanical
homogenizers are also employed, although their use can result
in local overheating of the material and thermal degradation of
thermolabile substances despite cooling. High-energy ultrason-
ic vibrations, freezing under conditions resulting in the rupture
of cellular wall or membrane, enzymatic lysis (usually
hydrolysis), and other nonmechanical physicochemical pro-
cesses can also be employed for sample homogenization [5].
3. Techniques of isolation of analytes from plant material
3.1. Extraction/leaching — general principles
In order to isolate the analytes from plant material,
extraction/leaching with various solvents is used, as a rule, in
order of increasing polarity of the extracting agent [11,14]. The
analytical procedure is shown in Fig. 1. Making use of various
solvents, extracts containing different analytes can be obtained
(Extracts A, B, C, D). The procedure should be carried out in
several steps so that particular analytes are present in one extract
only, while others are present in different extracts— A in Fig. 1.
Application of additional operations, for example extract
purification, results in obtaining further fractions (Fractions I,
II) — B in Fig. 1. Each of the fractions can then be
 G. Romanik et al. / J. Biochem. Biophys. Methods 70 (2007) 253–261 257

chromatographically separated into individual components — Table 2

C in Fig. 1. Substances isolated using ultrasonic extraction
Analyte Sample Recovery Time Reference
3.2. Soxhlet extraction [%] [min]
Cobalamins Biological samples 94.8–101.1 1 [21]
Soxhlet extraction is one of the oldest techniques for Tartaric and malic Grapes 30 [22]
acid
isolating metabolites from natural material. The technique is
Isoflavones Soybeans 100 20 [23]
used for the isolation and enrichment of analytes of medium and Isoflavonoids Root 83 60 [24]
low volatility and thermal stability. It allows a high recovery, Flavonoids Plant extract 91.2–95.6 60 [25]
but has a number of shortcomings, including long extraction Polysaccharides Buckwheat hulls 147 70 [26]
time and large consumption of solvents, cooling water and Volatile Citrus flowers and 10 [27]
compounds honey
electric energy. Another disadvantage of Soxhlet extraction is
Aroma compounds Aged brandies 45–113 30 [28]
lowered extraction efficiency due to the fact that the temperature Steroids and Stems, leaves and 30 [29]
of condensed solvent flowing into the thimble is lower than its triterpenoids flowers
boiling point [15,16]. These disadvantages are partially Antioxidants Herbs 60/45 [30]
eliminated by the automated Soxhlet extractor recently
introduced to the market. As a result, the extraction time is
shortened considerably, while reproducibility of the results is carried out by Melecchi et al. [20] have demonstrated that
comparable with the classical Soxhlet extraction [15]. This solvent polarity and extraction time have the greatest effect on
extraction is now in common use, being applied to the the recovery. Examples of substances isolated by ultrasonic
determination of, among others, lipids and polycyclic aromatic extraction along with extraction time and recoveries are
hydrocarbons in natural products (e.g., coffee, soybean and compiled in Table 2. The advantage of this technique is the
coconut oil, mushrooms, fruits, and vegetables) [15,17]. possibility of extraction of many samples at once in an ultrasonic
Soxhlet extraction was also used in investigations of anti- bath. The extraction is carried out at room temperature, which
inflammatory and antibacterial properties of plant metabolites. makes it suitable for the extraction of thermally labile analytes.
Nine African plants were examined and their therapeutical The need for separation of the extract from the sample following
properties determined on the basis of pharmacological proper- the extraction is a disadvantage of this technique.
ties of the extracts [18].
3.4. Accelerated solvent extraction
3.3. Ultrasonic extraction (sonication)
Accelerated solvent extraction (ASE) makes use of the same
Ultrasounds are waves with frequencies ranging from 16 kHz solvents as do other extraction techniques, but at an increased
to 1 GHz, inaudible to humans. Ultrasonic vibrations are the pressure (ca. 100–140 atm) and at an elevated temperature (50–
source of energy facilitating the release of some analytes from 200 °C). The design of the extractor, capable of withstanding
the sample matrix. The improvement in extraction efficiency due high pressures, allows the extraction temperature to be raised
to ultrasound appears at certain values of so-called acoustic above the boiling point of the solvent used. The high pressure
pressure [19]. Among the most important phenomena taking allows maintaining the solvent in a liquid state at a high
place in the acoustic field are: cavitation (generation and temperature. Under these conditions, the solvent has properties
collapse of mostly empty cavities), friction at the boundary and favoring the extraction process, such as low viscosity, high
interfacial surfaces, and increase in the diffusion rate. diffusion coefficients, and high solvent strength. This results in
Cavitation is the most significant phenomenon, because it good kinetics of dissolution processes and favors desorption of
has a direct effect on a number of phenomena occurring in a analytes from the cellular wall or organelles.
liquid subjected to ultrasound. Cavitation involves the forma- The sample is placed in an extraction cell, made of stainless
tion of pulsating bubbles as a result of strong stretching forces, steel. Following addition of the solvent, the cell is pressurized,
originating from abrupt local pressure drops [19]. At constant heated to the desired temperature, and the sample is extracted
ultrasound intensity, dynamic equilibrium is established statically for a specific period of time. Next, the extract is
between the forming and collapsing bubbles. The process of removed from the cell and the cell is flushed with fresh solvent.
generation and collapse of the cavities actively interacts with the The cycle can be repeated. When the extraction is complete,
liquid/solid boundary surface, thus enhancing the erosion compressed nitrogen moves all of the solvent from the cell to
processes of solids [19]. the vial for analysis. The total extraction time typically ranges
The average time of ultrasonic extraction typically ranges from 5 to 15 min, and the volume of the solvent used is about
from a few to 30 min, although it can be as long as 70 min (Table 150% of the volume of the extraction cell. The extract is filtered
2). The recoveries obtained during this time are comparable to prior to being collected in the receiver, thus eliminating the need
those obtained after a dozen or so hours of Soxhlet extraction, for a separate filtration step. ASE has been used successfully for
carried out at the same temperature. The extraction conditions the extraction of analytes from natural plant products, food,
can be optimized with respect to time, polarity and amount of pharmaceuticals, etc. The disadvantage of ASE is the high cost
solvent, and the mass and kind of sample. The investigations of the equipment.
258 G. Romanik et al. / J. Biochem. Biophys. Methods 70 (2007) 253–261
3.5. Microwave-assisted extraction
Microwave-assisted extraction (MAE) is based on absorption
of microwave energy by molecules of polar chemical compounds.
The energy absorbed is proportional to the dielectric constant of
the medium, resulting in rotation of dipoles in an electric field
(usually 2.45 GHz). The extraction is carried out at a temperature
from 150 to 190 °C. The hot solvent allows rapid isolation of
thermally stable analytes. The efficiency of microwave-assisted
extraction depends on solvent properties, sample material, the
components being extracted, and, specifically, on the respective
dielectric constants. The higher the dielectric constant, the more
energy is absorbed by the molecules and the faster the solvent
reaches the boiling point. In most cases, the extracting solvent has
a high dielectric constant and strongly absorbs microwave
radiation. The extraction is then carried out in closed containers
made of materials resistant to high temperatures (e.g., PTFE).
Solvents with a low dielectric constant can also be used. In this
case, the sample matrix is heated and the analytes are released into
a cooler solvent. This approach is employed for the extraction of
thermally labile analytes of low polarity.
Major advantages of microwave-assisted extraction include:
shortened extraction time, reduced size of extraction apparatus,
ease of control of sample heating, reduced amount of solvent
used. The limitation of this technique, when applied to
extraction of nonpolar analytes from nonpolar materials, is the
need for using solvents with dipole moments greater than zero
(n-hexane or iso-octane can be replaced by dichloromethane or
a mixture of acetone and n-hexane) [31–33].
3.6. Steam distillation
Steam distillation is a valuable technique, allowing isolation
from plants of volatile components, such as the essential oils,
some amines and organic acids, as well as other, relatively
volatile compounds, insoluble in water. Among others, steam
distillation has been used to isolate the essential-oil fraction
from either plant material or a previously prepared extract in a
low-boiling solvent (petroleum ether or diethyl ether) [11].
Volatile amines of relatively low polarity can be isolated by
steam distilling them from a medium alkalized with calcium
carbonate, while volatile acids can be steam-distilled from a
medium acidified with orthophosphoric acid [11]. Vitzthym et
al. [34] used steam distillation to isolate the flavor components
of black tea. Steam distillation was also used for the isolation of
antioxidants from herbs and the essential oils having anti-
oxidative properties from caraway, clove, rosemary, sage, and
thyme [34]. However, the technique is not free from short-
comings. It involves substantial energy consumption. An
elevated temperature (ca. 100 °C) may cause thermal decom-
position of substances. This can also affect the essential-oil
components, resulting in flavor changes [35].
3.7. Membrane processes
Membrane techniques are finding ever-increasing applica-
tion in the isolation of groups of components from the plant
extracts. A membrane is a selective barrier between two phases.
The phase from which mass transport takes place is called the
donor phase while the receiving phase is called the acceptor
(permeation) phase. The general principle of separation of
liquid mixture components is depicted in Fig. 2. The main
disadvantages of membrane techniques include their slowness,
low efficiency, and susceptibility to membrane fouling by solid
impurities in the donor phase with sizes comparable to the
membrane pores. On the other hand, the method is characterized
by low solvent consumption, simplicity, and high selectivity.
Membrane separation has been applied, for example, in the
isolation and enrichment of polyphenols from grapes [36],
where a membrane with 0.22-μm pore-size and ethanol as the
extracting agent (acceptor phase) were used. The amount of
polyphenols extracted was 11.4% of the total mass of grape
seeds. The authors [36] suggested that the food and pharma-
ceutical industries could use this technique for the isolation of
polyphenols which are anti-oxidants. Ultrafiltration, and
especially nanofiltration, could also be used for the isolation
of protein fractions of a specific range of molecular masses.
When the concentration of salts is high, proteins and peptides
can also be isolated by dialysis [37].
3.8. Supercritical fluid extraction
Supercritical fluids penetrate samples of plant material
almost as well as gases, and this results from their high
diffusion coefficients and low viscosity. At the same time, their
dissolving power is similar to liquids. The most commonly
used extracting agent is carbon dioxide, because of its low cost,
low toxicity, and favorable critical parameters (Tc = 31.1 °C,
Pc = 74.8 atm). CO2 as a nonpolar substance is capable of
dissolving nonpolar or moderately polar compounds. A mixture
of CO2 with modifiers (polar organic solvents) is used for the
extraction of polar substances. The modifiers increase the
solubility of analytes, preventing them from adsorption on the
active sites of sample matrix. The most important advantages of
supercritical fluid extraction include: considerable reduction in
the volume of solvent used, shortened extraction time, ease of
automation, small sample size needed, possibility of on-line
Fig. 2. Separation of mixtures using membrane techniques.
 G. Romanik et al. / J. Biochem. Biophys. Methods 70 (2007) 253–261
coupling with the separation and determination techniques
(SFE/GC, SFE/HPLC), high purity and small volume of the
extract, and high selectivity.
Supercritical fluid extraction (SFE) is relatively efficient
even for materials with compact and hardly accessible structure.
It is especially well suited for the isolation of substances of low
and medium polarity and high volatility. As a rule, carbon
dioxide or carbon dioxide with a volatile polar modifier, such as
methyl acetate, diethyl ether, methanol, formic acid, or
ammonia, are used as supercritical fluids. A review of recent
literature reveals an increasing number of papers on the
application of SFE to the extraction of tocopherols, terpenes,
fatty acids, steroids, and triglycerides from plant and animal
material and from oils [38].
3.9. Solid-phase micro-extraction
Solid-phase micro-extraction (SPME) is a sample prepara-
tion technique best suited for gas chromatography, Although
SPME has been successfully combined with HPLC, this
requires relatively complicated procedures and additional
devices. Therefore, it can be anticipated that the technique
will be used mostly with gas chromatography. Analyte
enrichment by SPME involves two steps. In the first step, a
fiber, coated with an adsorbent or stationary liquid, is exposed
to a liquid sample or the headspace above a sample and the
analytes are sorbed on the fiber. In the second step, the fiber is
introduced into the injection chamber of a gas chromatograph,
where it is subjected to a high temperature, or it is introduced
into the injector of a liquid chromatograph. The released
analytes are swept into the chromatographic column [39]. The
advantages of SPME include: speed (equilibrium between the
sample and the fiber is reached in 2 to 30 min, so the technique
is suitable for rapid monitoring), sensitivity (detection limits
down to ppt can be achieved), low cost (SPME is solvent-free,
a fiber can be used ca. 100 times), general applicability (SPME
can be used with any gas chromatograph or liquid chromato-
graph having a SPME/HPLC sampling accessory), possibility
of extraction from a variety of matrices, and ease of
automation. Volatile components of medicinal plants and
herbs can be determined by SPME/GC/MS. For example,
terpenoids can be adsorbed on fibers coated with polydi-
methylsiloxane (PDMS) [40]. SPME/GC has also been used for
the determination of tobacco alkaloids. The equilibrium
between the plant extract components and a 100-μm PDMS
fiber was reached in 12 min [41].
3.10. Sample disruption method
In the last few years, matrix solid-phase dispersion (MSPD)
has become an extraction method for naturally occurring
compounds. MSPD is primarily used because of its flexibility,
selectivity, and the possibility of performing extraction and
clean-up in one step (saving analysis time). This results in rapid
pre-treatment and low solvent consumption. This technique is
based on blending of a viscous, solid, or semi-solid sample with
an abrasive solid support material.
259
The process requires only simple devices and it comprises
steps such as:
1. a liquid, viscous, semi-solid, or solid sample is placed in
glass mortar and blended together with a solid support, using
a glass pestle to obtain complete disruption and dispersion of
the sample on the solid support;
2. the sample is packed into an empty column or on top of a
solid-phase extraction (SPE) sorbent, the main difference
between MSPD and SPE being that the sample is dispersed
throughout the column and not retained in just the first few
millimeters;
3. elution can be in two ways: either the target analytes are
retained on the column and interfering compounds are eluted
in the washing step while, the target analytes are subse-
quently eluted by a different solvent, or interfering matrix
components are selectively retained on the column and the
target analytes are directly eluted;
4. additional clean-up is performed or the sample is directly
analyzed [42].
The selectivity of a MSPD procedure depends on the
sorbent/solvent combination used. Often reversed-phase sor-
bents, like C8- and C18-bonded silica, are used as the solid
support [43,44].
3.11. Comparison of extraction techniques
Selection of an appropriate extraction technique entails
consideration of not only the recovery but also the cost, time of
extraction, and the volume of solvent used. A comparison of the
previously described extraction techniques for the isolation of
groups of components from plant material is shown in Table 3
[4,45]. Raised temperature and pressure during the extraction
profitably influence the process efficiency, because the solvent
properties are changed and mass-transfer efficiency is
enhanced. Ong and Len [46] performed the extraction of
baicalein in a Soxhlet extractor and compared the results with
pressurized-liquid extraction. Pressurized-liquid extraction
(with 20–25 ml methanol at a pressure 10–30 atm and at
100 °C) over a period of 20 min gave results comparable to
Soxhlet extraction (with 100–120 ml of methanol/water 70:30)
carried out for 3–4 h.
Grigonis et al. [47] compared different extraction techni-
ques: Soxhlet extraction, MAE, and SFE, carried out as one-
step and two-step extractions. MAE and SFE were found to be
Table 3
Comparison of various liquid–solid extraction techniques used in the analysis of
plant metabolites [45]
Extraction Soxhlet USE ASE MAE SFE
Cost Low Low High Medium High
Extraction time 6–48 h b30 min b30 min b30 min b60 min
Solvent use [mL] 200–600 b50 b100 b40 b10
Designation: USE – ultrasonic extraction; ASE – accelerated solvent extraction;
MAE – microwave-assisted extraction; SFE – supercritical fluid extraction.
260 G. Romanik et al. / J. Biochem. Biophys. Methods 70 (2007) 253–261
suitable for extraction of antioxidants from sweet grass. The
extraction times were 6 h, 0.25 h, and 2/h for Soxhlet
extraction, MAE, and SFE, respectively. A two-step extraction
was found to be more efficient than one-step extraction. For
two-step SFE, the extraction yields of anti-oxidants were
0.46% and 0.058% of the total sample mass for the first and
second step, respectively. Pan et al. [48] compared MAE with
other techniques, including classical liquid/liquid extraction,
ultrasonic extraction, and heat reflux extraction for the
isolation of polyphenols and caffeine from green tea leaves.
MAE was found to be superior in terms of the extraction
efficiency, yielding 7.1% more polyphenols and 11% more
caffeine than the other techniques. D. M. Teixeira et al. [49]
compared between disruption methods and solid-liquid
extraction (SLE) to extract phenolic compounds (phenolic
acid, flavonols and cumarins) from Ficus carica levels. More
compounds and higher yields were obtained by these method,
using smaller amounts of solvents, and less sample preparation
time. Higher extraction yields and smaller RSD values were
obtained with SSDM when compared with MSPD.
4. Solid-phase extraction
Sample preparation often includes an extract enrichment
step, wherein the analyte concentration is increased above the
determination limit of the final determination technique. Several
enrichment techniques are common, including gas, liquid and
solid-phase extraction. One of the primary considerations is the
need for reduction of the amount of organic solvent used. This
has resulted in extensive use of solid-phase extraction (SPE).
SPE involves adsorption of sample components on the surface
of a solid sorbent (aminopropyl or octadecyl stationary phases,
bonded to silica gel, etc.), followed by elution with a selected
solvent. SPE is carried out in glass or polypropylene columns or
on extraction disks. SPE has a number of advantages, including
the ability to isolate and enrich both volatile and nonvolatile
analytes, long storage time of adsorbed analytes, elimination of
emulsion formation (common in liquid/liquid extraction) or
foaming (common in gas/liquid extraction). A wide selection of
sorbents enables substantial selectivity of the enrichment
process. An important advantage of SPE compared to liquid/
liquid extraction is a significant reduction in volume of solvent
used [50]. A variety of sorbents available on the market allows
not only the isolation of analytes but also the removal of
interferences. Dry and wet modes of extraction were compared
and found to be equivalent. Due to its simplicity, SPE is finding
ever-increasing fields of application, including the isolation of
proteins and peptides [51,52]. Nonpolar stationary phases are
used [52] and the technique has been automated and coupled to
HPLC or electrophoresis [53,54]. SPE is not free from
shortcomings, including incomplete recoveries. SPE extracts
can be introduced into the HPLC system if methanol,
acetonitrile, water, or even acetone or 2-propanol are used as
elution solvents. However, sometimes the extract cannot be
introduced into the HPLC column, because it is insoluble in the
mobile phase, requiring solvent replacement. During this
operation the analytes could decompose or precipitate.
5. Summary
The majority of sample preparation procedures for the
determination of plant metabolites are developed in such a way
that the final extract introduced into the GC and HPLC columns
or CE capillary contains only the analytes with all the
interferences removed, although full implementation of this
goal may not always be possible or economically justified.
Modern extraction/leaching techniques, i.e., MAE, SFE, or
ASE are not always available in the average laboratory, due to
the high cost of equipment. However, the use of these
techniques results in shortened sample preparation time and a
reduction in the volume or elimination of organic solvents by
employing, e.g., SPME or SFE. This review shows that even
relatively simple and inexpensive laboratory equipment can be
effective in preparing samples of natural materials for
chromatographic analysis.
In every sample preparation procedure, especially for
complex samples containing a large number of components,
analyte enrichment and interference removal are essential.
These two steps are often combined into one. The most
common analyte isolation/enrichment techniques include SPE,
SPME, and, recently, also solvent micro-extraction (the micro-
drop technique). Development of novel stationary phases for
this step is anticipated. Current research effort is focused on
automation of analytical procedures. Miniaturization is another
recent trend in analytical chemistry. This will result in further
reduction in sample size, analysis time, and the amount of
solvents used. Miniaturization and novel sample preparation
techniques will also be used more often in the control of
industrial processes. The techniques discussed in this review
can be used not only in the preparation of plant material for
analysis but also in practical organic chemistry or in the
synthesis of plant or animal metabolites.
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