Techniques of preparing plant material for chromatographic separation and analysis

Techniques of preparing plant material for chromatographic separation and analysis

Techniques of preparing plant material for chromatographic

separation and analysis

G Romanik a,⁎ , E Gilgenasta,1, A Przyjazny b, M Kamińskia,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

www.elsevier.com/locate/jbbm

⁎ 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

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-low-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

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

1 Comminution, homogenization (breaking,

cutting, grinding, disintegration or lysis of

cellular wall)

Increasing interfacial surface area; obtaining a homogeneous sample

2 Division of sample Sample storage, saving a spare sample

3 Decomposition of glycosides, adsorbates,

aggregates, complexation of metal ions,

acidic, basic or enzymatic hydrolysis

Obtaining free forms of analytes Used only infrequently

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

Separation of solvent from plant material particles, separation of extract and raffinate, colloid peptization

Especially important when grinding a sample

in the presence of a solvent

[9]

6 Drying extracts Removal of water, which can interfere with the

analysis due to:

Drying can be accomplished by: [8]

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

Na 2 SO 4 ) to the extract Stopping flow of carrier gas as a result of

“flooding” capillary column in case of gas chromatography of volatile analytes

7 Derivatization of analytes Conversion of analytes into derivatives with

properties enabling:

[11]

Simple determination Higher stability of analytes Increased volatility Higher sensitivity of determination

8 Complete or partial solvent evaporation,

usually with solvent exchange

Increasing analyte concentration in the extract;

replacing solvent (through redissolving dry residue) with the one more suitable for subsequent steps of the analytical procedure

Evaporation in a rotary evaporator (often at

a reduced pressure) Evaporation in a stream of inert gas

9 Extract enrichment in analytes and removal of

interferences using: SPE, liquid–liquid

extraction, steam distillation, supercritical

fluid extraction

Increasing concentration of analytes, removal of interferences

Can be accomplished by using: [6,7]

1 Gel chromatography

2 Column chromatography with:

silica gel alumina Florisil

10 Storage of extracts Proper planning of work in the analytical laboratory Extracts should be stored in tightly capped

vials at lowered temperature (below 0 °C)

11 Calibration Preparation for the final determination Most common calibration methods are:

calibration curve internal standard standard addition

12 Method validation Investigation whether the procedure is valid for

specific applications

Reference materials and certified reference material are required; typically carried out together with validation of the entire analytical procedure

“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 inTable 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

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 inFig 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 inFig 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

Fig 1 Separation steps used for isolation of plant metabolites [14]

chromatographically separated into individual components —

C inFig 1

3.2 Soxhlet extraction

Soxhlet extraction is one of the oldest techniques for

isolating metabolites from natural material The technique is

used for the isolation and enrichment of analytes of medium and

low volatility and thermal stability It allows a high recovery,

but has a number of shortcomings, including long extraction

time and large consumption of solvents, cooling water and

electric energy Another disadvantage of Soxhlet extraction is

lowered extraction efficiency due to the fact that the temperature

of condensed solvent flowing into the thimble is lower than its

boiling point [15,16] These disadvantages are partially

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

comparable with the classical Soxhlet extraction [15] This

extraction is now in common use, being applied to the

determination of, among others, lipids and polycyclic aromatic

hydrocarbons in natural products (e.g., coffee, soybean and

coconut oil, mushrooms, fruits, and vegetables) [15,17]

Soxhlet extraction was also used in investigations of

anti-inflammatory and antibacterial properties of plant metabolites

Nine African plants were examined and their therapeutical

properties determined on the basis of pharmacological

proper-ties of the extracts[18]

3.3 Ultrasonic extraction (sonication)

Ultrasounds are waves with frequencies ranging from 16 kHz

to 1 GHz, inaudible to humans Ultrasonic vibrations are the

source of energy facilitating the release of some analytes from

the sample matrix The improvement in extraction efficiency due

to ultrasound appears at certain values of so-called acoustic

pressure [19] Among the most important phenomena taking

place in the acoustic field are: cavitation (generation and

collapse of mostly empty cavities), friction at the boundary and

interfacial surfaces, and increase in the diffusion rate

Cavitation is the most significant phenomenon, because it

has a direct effect on a number of phenomena occurring in a

liquid subjected to ultrasound Cavitation involves the

forma-tion of pulsating bubbles as a result of strong stretching forces,

originating from abrupt local pressure drops[19] At constant

ultrasound intensity, dynamic equilibrium is established

between the forming and collapsing bubbles The process of

generation and collapse of the cavities actively interacts with the

liquid/solid boundary surface, thus enhancing the erosion

processes of solids[19]

The average time of ultrasonic extraction typically ranges

from a few to 30 min, although it can be as long as 70 min (Table

2) The recoveries obtained during this time are comparable to

those obtained after a dozen or so hours of Soxhlet extraction,

carried out at the same temperature The extraction conditions

can be optimized with respect to time, polarity and amount of

solvent, and the mass and kind of sample The investigations

carried out by Melecchi et al [20] have demonstrated that solvent polarity and extraction time have the greatest effect on the recovery Examples of substances isolated by ultrasonic extraction along with extraction time and recoveries are compiled in Table 2 The advantage of this technique is the possibility of extraction of many samples at once in an ultrasonic bath The extraction is carried out at room temperature, which makes it suitable for the extraction of thermally labile analytes The need for separation of the extract from the sample following the extraction is a disadvantage of this technique

3.4 Accelerated solvent extraction

Accelerated solvent extraction (ASE) makes use of the same solvents as do other extraction techniques, but at an increased pressure (ca 100–140 atm) and at an elevated temperature (50–

200 °C) The design of the extractor, capable of withstanding high pressures, allows the extraction temperature to be raised above the boiling point of the solvent used The high pressure allows maintaining the solvent in a liquid state at a high temperature Under these conditions, the solvent has properties favoring the extraction process, such as low viscosity, high diffusion coefficients, and high solvent strength This results in good kinetics of dissolution processes and favors desorption of analytes from the cellular wall or organelles

The sample is placed in an extraction cell, made of stainless steel Following addition of the solvent, the cell is pressurized, heated to the desired temperature, and the sample is extracted statically for a specific period of time Next, the extract is removed from the cell and the cell is flushed with fresh solvent The cycle can be repeated When the extraction is complete, compressed nitrogen moves all of the solvent from the cell to the vial for analysis The total extraction time typically ranges from 5 to 15 min, and the volume of the solvent used is about 150% of the volume of the extraction cell The extract is filtered prior to being collected in the receiver, thus eliminating the need for a separate filtration step ASE has been used successfully for the extraction of analytes from natural plant products, food, pharmaceuticals, etc The disadvantage of ASE is the high cost

of the equipment

Table 2 Substances isolated using ultrasonic extraction

[%]

Time [min]

Reference

Cobalamins Biological samples 94.8–101.1 1 [21]

Tartaric and malic acid

Volatile compounds

Citrus flowers and honey

Steroids and triterpenoids

Stems, leaves and flowers

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 isolaapplica-tion 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 CO2with 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.

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

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 inTable 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-one-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 time 6–48 h b 30 min b 30 min b 30 min b 60 min Solvent use [mL] 200–600 b 50 b 100 b 40 b 10 Designation: USE – ultrasonic extraction; ASE – accelerated solvent extraction; MAE – microwave-assisted extraction; SFE – supercritical fluid extraction.

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 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

References [1] Giergielewicz - Możajska H, Dąbrowski Ł, Namieśnik J Ekol Tech 2000; VIII(6).

[2] Huie CW A review of modern sample-preparation techniques for the extraction and analysis of medicinal plants Anal Bioanal Chem 2002; 373:1–2.

[3] Benthin B, Danz H, Hamburger M Pressurized liquid extraction of medicinal plants J Chromatogr A 1999;837:211–9.

[4] Namieśnik, J., Przygotowanie próbek materiału roślinnego do analizy chromatograficznej, Chemia i inzynieria ekologiczna, T.7,Nr 8/9 (2000), 805–821.

[5] Market B Sample preparation (cleaning, drying, homogenization) for trace element analysis in plant matrices Sci Total Environ 1995;176:45–6 [6] Kipopoulou AM, Manoli E, Samara C Bioconcentration of polycyclic aromatic hydrocarbons in vegetables grown in an industrial area Environ Pollut 1999;106:369.

[7] Schultz H, Papp P, Huhn G, Stark HJ, Schuurmann G Biomonitoring of airborne inorganic and organic pollutants by means of pine tree barks I Temporal and spatial variations Sci Total Environ 1999;232:49.

[8] Tikhomiroff C, Joliocoeur M Screening of Catharanthus roseus

secondary metabolites by high-performance liquid chromatography.

J Chromatogr A 2002;955:87.

[9] Loponen J, Ossipov V, Lempa K, Haukioja E, Pihlaja K Concentrations and among-compound correlations of individual phenolics in white birch leaves under air pollution stress Chemosphere 1998;37:1445–56.

[10] Quevauviller Ph Conclusions of the workshop-improvements of trace

element determinations in plant matrices Sci Total Environ

1995;176:141–8.

[11] Jerzmanowska Z Substancje roślinne Metody wyodrębniania Warszawa:

PWN; 1970.

[12] Jaillais B, Cadoux F, Auger J SPME-HPLC analysis of Allium

lacrymatory factor and thiosulfinates Talanta 1999;50:423–31.

[13] Chen T-E, Huang D-J, Lin Y-H Isolation and characterization of a serine

protease from the storage roots of sweet potato (Ipomoea batatas [L.]

Lam) Plant Sci 2004;166:1019–26.

[14] Nyiredy Sz Separation strategies of plant constituents-current status.

J Chromatogr B 2004;812:35.

[15] Hofler F, Richter B, Felix D Accelerated Solvent Extraction Prospekt

reklamowy firmy U.S.A.: DIONEX; 1995.

[16] Lao RC, Shu YY, Holmes J, Chiu C Environmental sample cleaning and

extraction procedures by microwave-assisted process (MAP) technology.

Microchem J 1996;53:99–108.

[17] Prange G, Liepelt G, Kayser G, Korhammer S, Market B GIT

Labor-Fachzeitschrift 1999;3(218).

[18] Lin J, Opoku AR, Geheeb-Keller M, Hutchings AD, Terblanche SE, Jager

AK, et al Preliminary screening of some traditional zulu medicinal plants

for anti-inflammatory and antimicrobial activities J Ethnopharmacol

1999;68:267–74.

[19] Śliwiński A Ultradźwięki i ich zastosowanie Warszawa: WNT; 1993.

[20] Melecchi MIS, Peres VF, Dariva C, Zini CA, Abad FC, Martinez MM, et al.

Optimization of the sonication extraction method of Hibiscus tiliaceus L.

Flowers Ultrason Sonochem 2006;13:242–50.

[21] Visimnas P, Lopez-Erroz C, Balsalobre N, Hernandez-Cordoba Speciation

of cobalamins in biological samples using liquid chromatography with

diode-array detection Chromatographia 2003;58:5 [M.].

[22] Palma M, Barroso CG Ultrasound-assisted extraction and determination

of tartaric and malic acids from grapes and winemaking by-products Anal

Chim Acta 2002;458:119.

[23] Rostagno MA, Palma M, Barroso CG Ultrasound-assisted extraction of

soy isoflavones J Chromatogr A 2003;1012:119.

[24] Xiao HB, Krucker M, Albert K, Liang XM Determination and

identification of isoflavonoids in Radix astragali by matrix solid-phase

dispersion extraction J Chromatogr A 2004;1032:117.

[25] Sladkovsky R, Urbanek M, Solich P Off-line coupling of isotachophoresis

and high performance liquid chromatography for determination of

flavonoids in plant extract Chromatographia 2003;58:187.

[26] Hromadkov Z, Ebringerov A Ultrasonic extraction of plant materials —

investigation of hemicellulose release from buckwheat hulls Ultrason

Sonochem 2003;10:127.

[27] Alissandrakis E, Daferera D, Tarantilis PA, Polissiou M, Harizanis PC.

Ultrasound-assisted extraction of volatile compounds from citrus flowers

and citrus honey Food Chem 2003;82:575.

[28] Caldeira I, Pereira R, Clýmaco MC, Belchior AP, Bruno de Sousa R.

Improved method extraction of aroma compounds in aged brandies and

aqueous alcoholic wood extracts using ultrasound Anal Chem Acta

2004;513:125.

[29] Schinor EC, Salvador MJ, Turatti ICC, Zucchi OL, Dias DA.

Comparisson of classical and ultrasound – assisted extractions of steroids

and triterpenoids from three Chresta spp Ultrason Sonochem

2004;11:415.

[30] Albu S, Joyce E, Pomiwnyk L, Lorimer JP, Mason TJ Potential for the use

of ultrasound in the extraction of antioxidants from Rosmarinus officinalis

for the food and pharmaceutical industry Ultrason Sonochem

2004;11:261.

[31] Pan X, Niu G, Liu H Microwave-assisted extraction of tanshiones from

Salvia miltiarrhiza bunge with analysis by high-performance liquid

chromatography J Chromatogr A 2001;992:425.

[32] Pan X, Niu G, Liu H Microwave-assisted extraction of tea polyphenols and tea caffeine from green tea leaves Chem Eng Process 2003;42:2 [33] Fulzele PD, Satdive RK Distribution of anticancer drug comptothecin in

Nothapodytes foetida J Chromatogr 2005;1063:537.

[34] Farag RS, Badei AZMA, Hewedi FM, El-Baroty GSA Antioxidant activity of spice essential oils on linolenic acid oxidation in aqueous media.

J Am Oil Chem Soc 1989;66:792–9.

[35] Ammann A, Hinz DC, Addleman RS, Wai CM, Wenclawiak BW J Anal Chem 1999;364:650–3.

[36] Nawaz H, Shi J, Mittal GS, Kakudac Y Extraction of polyphenols from grape seeds and concentration by ultrafiltration Sep Purif Technol 2006;48:176–81.

[37] Valcarcel M, Arce L, Rǀos A Coupling continuous separation techniques

to capillary electrophoresis J Chromatogr A 2001;924:3–30.

[38] Lang Q, Wai Ch M Supercritical fluid extraction in herbal and natural product studies — a practical review Talanta 2001;53:4.

[39] Camel V, Caude M Trace enrichment methods for the determination of organic pollutants in ambient air J Chromatography A 1995;710:3–19 [40] Czerwiński J, Zygmunt B, Namieśnik J, Fresenius J Head-space solid phase microextraction for the GC–MS analysis of terpenoids in herb based formulations Anal Chem 1996;356:80.

[41] Yang SS, Smetena I Determination of tobacco alkaloids using solid phase microextraction and GC–NPD Chromatographia 1998;47:443 [42] Kristenson EM, Ramos L, Brinkman UA Th Recent advances in matrix solid-phase dispersion Trends Anal Chem 2006;25:96.

[43] Teixeira DM, Teixiera da Costa C Novel methods to extract flavonones

and xanthones from the root bark of Maclura pomifera J Chromatography

A 2005;1062:175–81.

[44] Manhita AC, Teixiera DM, da Costa CT Application of sample disruption methods in the extraction of anthocyanins from semi-solid vegetables samples J Chromatography A 2006;1129:14–20.

[45] Leach MJ Complement Ther Nurs 2004;10.

[46] Ong ES, Len SM Pressurized hot water extraction of berberine, baicalein and glycyrrhizin in medicinal plants Anal Chim Acta 2003;482:81–9 [47] Grigonis D, Venskutonis PR, Sivik B, Sandahl M, Eskilsson CS Comparison of different extraction techniques for isolation of antioxidants

from sweet grass ( Hierochlo¨e odorata) J Supercrit Fluids

2005;33:223–33.

[48] Pan X, Niu G, Liu H Microwave-assisted extraction of tea polyphenols and tea caffeine from green tea leaves Chem Eng Process 2003;42:129–33.

[49] Teixiera DM, Patao RF, Coelho AV, Teixeira da Costa C Comparison between sample disruption methods and solid–liquid extraction (SLE) to

extract phenolic compounds from Ficus carica leaves J Chromatogr A

2006;1103:22–8.

[50] Camel V Extraction techniques Anal Bioanal Chem 2002;372:39–40 [51] Visser NFC, Lingeman H, Irth H Sample preparation for peptides and proteins in biological matrices prior to liquid chromatography and capillary zone electrophoresis Anal Bioanal Chem 2005;382:535–58.

[52] Schmerr MJ, Cutlip RC, Jenny A Capillary isoelectric focusing of the scrapie prion protein J Chromatogr A 1998;802:135–41.

[53] Naylor S, Tomlinson AJ Membrane preconcentration-capillary electro-phoresis tandem mass spectrometry (mPC-CE-MS/MS) in the sequence analysis of biologically derived peptides Talanta 1998;45:603–12 [54] Tomlinson AJ, Benson LM, Jameson S, Johnson DH, Naylor S Utility of membrane preconcentration-capillary electrophoresis-mass spectrometry

in overcoming limited sample loading for analysis of biologically derived drug metabolites, peptides, and proteins Am Soc Mass Spectrom 1997;8:15–24.