Book - Microwave Green Extraction

R.C. Richter - C.
Pirola
MICROWAVE
GREEN EXTRACTION
Modernizing trace organic analysis
Milestone Press
R.C. Richter - C. Pirola
MICROWAVE
GREEN EXTRACTION
Microwave Green Extraction Layout:
Modernizing trace organic analysis A. Fenili
Authors: Cover and Graphics:

R.C. Richter A. Fenili
C. Pirola S. Lorenzi
G. Pedrini
ISBN 978-88-96006-32-0
Printed in Italy by:
© 2017 Milestone Srl Ikonos Srl
All rights reserved under international copyright
conventions. No part of this book may be reproduced November 2017
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Milestone Press
CONTENTS
INTRODUCTION 9
PART I - SAMPLE PREPARATION

CHAPTER 1
Fundamentals of extraction 13
CHAPTER 2
Modern extraction techniques 25
Soxhlet Extraction 27
Automated Soxhlet (“Randall modification”of the Soxhlet
method) 28
Ultrasonic Extraction (Acoustic Cavitation) 29
Supercritical Fluid Extraction (SCFE) 32
Pressurized Liquid Extraction (PLE) 34
Microwave Assisted Extraction (MAE) 36
CHAPTER 3
Post extraction processing 39
Filtration 41
Water Removal 42
Concentration (Solvent Removal) 42
Interference Removal 46
Derivatization 49
PART II - TRACE ORGANIC ANALYSIS
CHAPTER 4
Chromatography (Compound Separation) 53
CHAPTER 5
Gas Chromatography (GC) 57
Carrier Gas (mobile phase) 59
Sample Introduction (Inlet) 60
Columns for GC 62
GC Oven 67
Detectors 68
CHAPTER 6
Liquid Chromatograph (LC) 75
Mobile Phase and Pump 77
Sample Introduction 79
Columns for LC 81
Column Thermostat 87
CHAPTER 7
Mass spectometer based hyphenated techniques 95
Mass Spectrometry 97
Ion source 99
Mass Analyzer 106
Time of Flight (TOF) 108
Vacuum System and Detectors 111
PART III - THE MICROWAVE ADVANTAGE
CHAPTER 8
Technique comparison 115
Microwave-Assisted Extraction vs Soxhlet 117
Microwave-Assisted Extraction vs Automated Soxhlet 118
Microwave-Assisted Extraction vs Ultrasonic Extraction 118
Microwave-Assisted Extraction vs Supercritical Fluid
Extraction (SCFE) 119
Microwave vs Pressurized Liquid Extraction (PLE) 120
CHAPTER 9
Microwave instrumentation 121
Milestone ETHOS X 123
Consistency and Reliability 125
Productivity 125
Low running cost 126
Green Technology 126
Compliance 126
CHAPTER 10
Applications 129
Environmental Analysis 131
Polymers/Plastics 133
Food and Drugs 134
Future Applications 136
APPENDIX A
References 139
AUTHORS
About the Authors 146
INTRODUCTION
7
Introduction
The purpose of organic trace analysis is to obtain information about

the organic molecules present in the sample matrix of interest.
So why is organic trace analysis so hard?
There are millions of organic compounds that have been characterized

in the chemical literature, with still millions more that are waiting to
be discovered. The biggest problem with organic trace analysis is
isolating the single compound of interest from the rest of the organic
soup. For example hexane has the chemical formula of C6H14, there
are also a least four other compounds that have the same chemical
formula (Table 1), so simple elemental analysis will not tell us if hexane
is present in the sample.
Compound CAS No. Structure
n-Hexane 110-54-3
2-Methylpentane 107-83-5
3-Methylpentane 96-14-0
2,2-Dimethylbutane 75-83-2
2,3-Dimethylbutane 79-29-8
Table 1. Compounds with the chemical formula of C6H14
9
Introduction
Analytical instrumentation has evolved to the point where is capable

of simultaneously detecting the hexane molecule, and all of the
other C6H14, compounds in a matter of minutes at levels less than
one part per billion (ppb). Despite the sophisticated arsenal of
analytical instrumental techniques available, current instrumentation
has not evolved to the point where we put some dirt into a chamber
and the answer appears. Samples must still undergo some type of
pretreatment because the sample matrices often are not compatible
with the modern instrumentation. In addition, the sample homogeneity
must be reduced to a molecular level because today’s analytical
instruments compare molecular relationships on a molecular level
with time integration. This additional pretreatment component for
modern analysis makes the determination of trace organic analytes
challenging for analytical chemists. The various operations performed
on the sample during its preparation for instrumental analysis account
for 60% of the analyst’s time and are responsible for 30 to 50% of
error in performing the analysis. The top problems reported with
sample preparation are: 1) labor intensity; 2) cost; 3) poor recoveries;
4) reproducibility; and 5) contamination. In addition to these problems
the trace organic chemists on average uses three or more sample
preparation techniques per sample just to produce an analytical
result. This book focuses on helping today’s trace organic analysists
understand and overcome the difficulties of sample preparation by
learning how to THINK GREEN.
10 MICROWAVE GREEN EXTRACTION - Modernizing trace organic analys

PART I
SAMPLE PREPARATION
11
CHAPTER 1
FUNDAMENTALS OF EXTRACTION
13
Fundamentals of extraction
The fundamental thermodynamic relationship related to the extraction

of an organic analyte from a solid matrix involves the distribution of
analyte between the sample matrix and the extraction solvent as
shown in Equation 1.
Kd = Ca / Ce
Equation 1
Kd is the distribution coefficient, Ca is the concentration of the analyte in

the sample and Ce is the concentration of the analyte in the extraction
solvent. The distribution coefficient is an equilibrium relationship
and the magnitude of Kd is determined by the relative affinity of the
analyte for the two phases (extraction solvent and sample matrix). The
distribution constant is dependent on various parameters including
temperature, pressure and sample matrix conditions such pH and
organic component concentration as well as kinetic factors defined by
the nature of Kd. All of these factors need to be optimized in order to
achieve maximum extraction efficiency.
The first critical parameter involving extraction is the extraction
solvent. Maximum extraction is achieved when the extraction solvent
has a higher affinity for the analyte that the sample matrix. The affinity
of molecules for one another is determined by their intermolecular
forces of attraction. In general, when predicting which analytes will
be extracted by which solvents we can say “like dissolves like”.
15
Part I - Chapter 1 • Fundamentals of extraction
This means that if similar intermolecular forces of attraction occur

in the analyte and solvent, they can replace each other. The main
intermolecular forces of attraction experienced between the analyte/
matix and analyte/solvent are: dipole-dipole, ion-dipole, ion induced-
dipole, dipole-induced dipole and dispersion. Dipole-Dipole forces
result from the interactions of polar molecules. Polar molecules are
molecules that possess a permanent dipole moment, a separation of
positive (electron deficient region) and negative (electron rich region)
charges. The differences in electronegativity, the ability of an atom to
attract electrons, between atoms in a chemical bond results in the
shared electrons being pulled closer to the more electronegative
element. This results in a concentrating of the negative charge on one
end of the bond, creating negative partial charge (δ-) and a partial
positive charge (δ+) at the other end of the bond where the electrons
were displaced (Figure 1). When individual polar molecules approach
each other, an electrostatic force of attraction (a “glue”) forms between
the positive and negative partial charges on the adjacent molecules
holding them together. A special case of dipole-dipole interactions is
hydrogen bonding. Hydrogen bonding is considered the “super glue”
of dipole-dipole interactions. This “super glue” effect results from large
partial positive and negative charges that form when hydrogen is
bonded to a very electronegative element such as F, O, or N. The large
partial positive and negative charges result in very strong interactions
of these molecules, making them hard to separate (Figure 2).
Ion-dipole forces occur when a polar molecule and an ion interact. The
positively charged end of a polar molecule interacts with negative ions
such as Cl-, NO3-, SiO4-4 and the negatively charged end is attracted
to positive ions such as Na+, Ca2+, Fe3+. Ion-induced dipole forces
result when an ion interacts with a non-polar molecule. If a negatively
charged ion approaches a non-polar molecule, the electrons in the
16 MICROWAVE GREEN EXTRACTION - Modernizing trace organic analysis

H Cl N
H H
Polar Polar
Cl Cl
F
C C
B
Cl Cl H H
F F
Cl H
Nonpolar Nonpolar Polar
Figure 1. Examples simple of a polar and non-polar molecules. The arrow

points to the more electronegative molecule
H O H O H
H H H N H O
H H H H
H N H N H
H H H O H N
H H
Figure 2. Examples of hydrogen bonding
17
non-polar molecule are repelled by the negative charge, causing

them to become unevenly distributed within the non-polar molecules
electron cloud. This results in a partial positive charge (δ+) forming in
the area evacuated by the repelled electrons and creating negative
partial charge (δ-) in the electron rich area. The negative ion is now
attracted to the newly created positive charge. The opposite occurs
when a positively charged ion or molecule approaches a non-polar
molecule. Polar molecules can also induce this phenomena in the
same manner as ions, but the interactions are just not as strong. This
is called dipole-induced dipole forces.
The last intermolecular force of attraction is dispersion. This type of
force occurs between all molecules, and is the only force of attraction
that can occur between non-polar molecules. Non-polar molecules,
by their nature, do not possess a permanent dipole moment, but at
any one instant the electrons in their electron cloud may be unevenly
distributed. This uneven distribution results from the random motion
of the electrons in the electron cloud or through the influence of the
electrons in a nearby atom i.e. the negative electrons repel each other.
These situations result in the formation of temporary dipole moments
within non-polar molecules causing them to be attracted to each other.
The strength of the molecular interactions due to dispersion forces
increases with the molecular weight of the molecules involved. This
is due to the number of electrons available for an instantaneous shift,
which results in larger partial positive and negative charges. Table 2
summarizes the various intermolecular forces of attraction and there
relative strengths.
These forces create a ‘tug of war’ for the analyte molecules between
the extraction solvent and the matrix. For example a soil matrix is
composed of four distinct materials: humus, clay, silt and sand. Humus
is the top layer and consists mainly of organic matter produced from

Force Type Example Strength (kJ/mol)
Dispersion Hexane-Hexane 0.05-30
Dipole Induced Dipole Hexane-Acetone 2-10
Ion Induced Dipole Na+ - Hexane 3-15
Dipole-Dipole Acetone-Water 2 to 25
Hydrogen bonding Methanol-Water 10 to 40
Ion-dipole (Na+) - Water 30-600
Table 2. Intermolecular forces of attraction and their relative strength
the decay of plants and animals. It is typically composed of organic

acids (humic acid), sugars, phenolic compounds, amino acids and
non-humic acid biopolymers, proteins, polysaccharides, lipids and can
interact with the analyte through dipole-dipole, dipole-induced dipole
and dispersion forces (Figure 3). In addition, microorganisms present
in the humus, break down the organic materials into ions (NO3-, NH4+,
etc.) that can interact with the analyte through ion-dipole interactions.
HOOC
A CHO HOOC B O
O OH HC OH COOH
HO
HO CH
OH
COOH
HC OH
O
HO
CH3(CH2)13CH2 OH
HC OH O
O N
HO OH R C O O
O O O
C
O
N O O O O
HN
R O
O
HO OH
NH
NH2
Figure 3. Examples of organic molecules found in soil. Humic Acid (A) Fatty acid
- Hexadecanoic acid (B) Amino acid – L-Glutamic acid (C)
19
Clay is mainly composed of silicate minerals containing the SiO4-4

ion group. These negatively charged oxides of silicon (Al2Si6O18-6,
Si2O5-3, etc.) can form strong ion-dipole interactions with water and
polar or large non-polar analyte molecules. Silt and sand consists of
small neutral molecules (SiO2, Quartz, etc.) that can interact through
dispersion forces.
So how can we tip the “tug of war” in the favor of the extraction
solvent? The equilibrium nature of the distribution coefficient (Equation
1) means that the analyte has a natural tendency to be removed from
the matrix by an extraction solvent with similar intermolecular forces
of extraction, but the amount of analyte removed in the extraction
process is limited, in part, by the rate at which equilibrium is achieved.
Equilibrium can only be achieved when the analyte and the soil matrix
are physically interacting with the extraction solvent. For a liberated
analyte to be fully extracted from the sample matrix, the following steps
must take place: 1) Penetration and diffusion of the solvent into the
solid matrix, 2) Solubilization of the analyte of interest by the solvent,
3) Transport of the solvated analyte to the surface of the solid matrix,
and 4) Migration of the extracted analyte from the surface of the solid
matrix to the bulk solution. Understanding and optimizing these four
processes is the key to achieving maximum extraction efficiency for
liquid-solid extractions.
The mass transfer rate of an analyte particle from the interior of the
matrix to the bulk solvent is driven by the concentration difference
between Ca and Ce. The rate at which the analyte is transferred to the
extraction solvent over time is expressed mathematically using Fick’s
law (Equation 2).

dCe/dt = (ADVe /d) (Ca - Ce )
Equation 2
Ce is the concentration of the analyte in the extraction solvent, t is time,

A is the surface area of the solid sample, D is the diffusion coefficient
(a measure of how easily the analyte can move through the matrix
permeated with solvent), Ve is the volume of the extracting solution, d
is the depth of the analyte in the sample, and Ca is the concentration of
the analyte in the matrix. Examining the terms of this equation we can
determine that maximizing the A,D,Ve and Ce terms and minimizing
the d and Ca terms leads to the fastest extraction rate and efficiency.
Continuing with our example a matrix soil, the morphology of the
soil will have a dramatic impact on the magnitude of the d term. The
distance the analyte has to move within the soil matrix affects the time
is takes to reach the bulk solution to increase the Ce term.
Soils are composed of a variety of particles of varying sizes. These
particles compact and form channels within the matrix where the
analyte molecule can become trapped requiring the solvent to move
a greater distance to reach the analyte (Figure 4). In addition, the
individual soil particles contain micropores, long channels, where the
analyte can become trapped deep inside limiting its accessibility to the
solvent (Figure 5).
The average distance the solvent molecules have to travel in order to
reach the analyte can be minimized by grinding or sieving. Grinding
eliminates the large channels within the bulk material and disrupts
the particle lattice structure providing better access to the analyte for
the extraction solvent. Sieving out large particles can also improve
average recovery of the analyte by eliminating particles that have a
21
Large pores between

large particles.
Intermediate sized
Tiny pores pores between
between clay middle sized
particles. particles.
Figure 4. Schematic representation of available pores in a soil matrix
Figure 5. Scanning electron microscope image of a clay soil particle
high probability of containing deep channels where analytes can be

trapped. These two processes are routine laboratory operations,
which can be employed for soil as well as other matrices. The net
effect increases the overall surface area (A term) reducing the overall
time it takes to reach equilibrium.
Maximizing the Ve and D terms in Fick’s law is easily accomplished by

using larger volumes and extracting at elevated temperatures. Larger
solvent volumes increase the numerical value Ve as well as increase
the number molecules available to interact with the analyte molecules.
Elevated extraction temperatures increase diffusion coefficient, D,
according to the following equation.
D = Doe-Ea/RT
The final diffusion coefficient, D, follows Arrhenius type behavior which

is expressed mathematically by the following equation.
kT
D=
6πηR
D0 is temperature independent pre-exponential (constant), Ea is the

activation energy for desorption of the analyte from the matrix, T is
the absolute temperature and R, the ideal gas constant. The diffusion
coefficient, D, is also related to the viscosity of the extraction solvent
and is expressed mathematically by the Einstein-Stokes equation.
The Einstein-Stokes equation shows that the solvent viscosity, n
term, and diffusion coefficient are inversely related. This means that
large diffusion coefficient’s are achieved with low viscosity extraction
solvents. Viscosity also follows Arrhenius type behavior so performing
the extraction at higher temperatures will result in exponentially lower
viscosity for the extraction solvent.
In summary, there are a variety of factors that affect the extractability
of an analyte from a solid matrix. The best results are often achieved
23
by optimizing a combination of these factors, mainly solvent selection,

solvent volume, and extraction temperature. For solvent selection,
keep in mind that there is no single universal solvent that works for all
analytes and all matrices. The best extracting solvent will also extract
a large amount of the matrix components, which may not be desirable
for further steps in the analysis, requiring significant cleanup of the
extract. In some situations, a solvent that extracts most of the analytes
but does not dissolve (or extract) much of the matrix is preferable
to a solvent able to achieve a more thorough extraction, which also
dissolves much of the matrix. Sometimes, a mixture of water-miscible
solvents (polar solvents like acetone) with nonmiscible ones (non-
polar solvents like hexane) are used. The water-miscible solvents can
penetrate the layer of water molecules bound to the surface of the
solid particles, blocking openings to pores where the analyte might
be trapped. Once liberated, the analyte can then be solvated by
either solvent in the mixture. The next chapter focuses on the modern
extraction techniques developed in order to take advantage of the
fundamental extraction principles discussed in this chapter.

CHAPTER 2
MODERN EXTRACTION TECHNIQUES
25
Modern extraction techniques
Today’s organic trace analyst has a variety of extraction methodologies

from which to choose. This chapter outlines the fundamental theory
and operating principles of the six major extraction techniques (Soxhlet,
Automated Soxhlet, Ultrasonic, Supercritical Fluid, Pressurized Fluid
and Microwave Assisted) that are in use in today’s analytical labs for
the extraction of analytes from solid matrices.
Soxhlet Extraction
This methodology relies on optimizing the concentration difference, the
(Ca – Ce) term in Fick’s law, to shift the extraction equilibrium in favor
of the extraction solvent. This is achieved by providing the sample
matrix with a consistent supply of analyte free solvent, ensuring the
(Ca – Ce) term is as large as possible, promoting diffusion of the analyte
of interest into the extraction solvent.
The Soxhlet extraction apparatus consists of three main pieces, a vapor
condenser, extraction chamber, and a solvent reservoir (Figure 6). The
process begins by weighing the sample to be extracted into a cellulose
thimble, which is permeable to the extraction solvent, and placing it
into the extraction chamber. The extraction chamber is placed on the
solvent reservoir containing the extraction solvent and a few boiling
chips to prevent bumping. A typical extraction requires 10 to 15 grams
of sample and 300 to 400 mL of solvent. The solvent vapor condenser
is placed on top of the extraction chamber and cooling water is turned
on. As the extraction solvent is heated, it evaporates from the solvent
reservoir, passes through the side arm of the extraction chamber and
onto the solvent vapor condenser. The condensed solvent then drips
onto the sample contained in the cellulose thimble.
27
Part I - Chapter 2 • Modern extraction techniques
water water water

out out out
Water
cooled water water water
condensed
in in in
Sample
thimble
Upper
reservoir
Return
tube
Solvent Lower
reservoir
Figure 6. Schematic of Soxhlet extraction apparatus
This process continues until the condensed solvent reaches the top
the siphon tube where gravity action empties the solvent, containing
the extracted analytes, from the extraction chamber back into the
solvent reservoir. The extracted analytes typically have higher boiling
points than the extraction solvent and remain trapped in the solvent
reservoir, while the solvent continuously recirculates. This allows the
sample to be extracted multiple times with fresh solvent. The number
of extraction cycles depends on the solubility of the analytes and the
capacity of the solvent to penetrate the sample. Typically Soxhlet
extractions take 6 to 24 hours to complete.
Automated Soxhlet (“Randall modiﬁcation” of the Soxhlet

method)
The shortfall of the Soxhlet methodology is that it only relies on
optimizing one of the terms in Fick’s law. Faster, more efficient

extractions can be achieved when multiple terms of Fick’s law are
improved simultaneously. In 1974, Edward Randall developed an
extraction apparatus that cut the time of Soxhlet extractions to less
than an hour by improving both concentration difference, the (Ca – Ce)
term and diffusion coefficient, D, term during the extraction process.
Randall achieved a dramatic reduction in extraction time by immersing
the sample in boiling solvent then applying the Soxhlet methodology.
The immersion step increases the mass transfer rate, lowers the
viscosity of the solvent and provides energy to overcome molecular
forces of attraction between the analyte.
The Randall modification of the Soxhlet extraction has been
commercialized by several companies and is now a fully automated
process involving three stages: boiling, rinsing, and drying. In the
first stage, the thimble containing the sample is immersed in boiling
extraction solvent. In the second stage, the thimble is lifted out of the
boiling solvent and subjected to the normal Soxhlet extraction process
to remove any residual analyte from the matrix. Finally, the drying step
removes the solvent from the solvent flask and concentrates the
analyte for further processing (Figure 7).
Ultrasonic Extraction (Acoustic Cavitation)

Ultrasonic extraction uses sound waves (acoustical energy) imparted
into traditional solvents to extract the analytes of interest from a
solid matrix. The acoustical energy interacts with the solvent on a
mechanical level transmitting it through the solvent and sample. The
interaction induces vibrational motion within the solvent molecules,
creating regions of compression and stretching. This results in the
production of acoustic pressure, alternating high pressure and low
pressure cycles at the frequency of the wave. The acoustic pressure
causes a gas bubble to form in the solvent.
29
1 2 3 4
Boiling
1 Rapid solubilisation in boiling solvent.
3 Recovery
Automatic collection of distilled
solvent for re-use.
Rinsing Auto-shut down

2 Efficient removal of remaining soluble 4 The system closes down and the cups
matter. are lifted from the hot plate.
Figure 7. Automated Soxhlet Extraction
The gas bubble oscillates and grows for several cycles until it reaches
an unstable size. The unstable bubble undergoes implosive collapse
releasing large amounts of localized energy, “hot spots” within the
solvent and sample matrix. This energy is high enough to overcome
the molecular interactions of the analyte and the matrix, allowing
it to be extracted by the solvent. The collapse of bubbles is also
accompanied by the generation of shock waves within the extraction
environment. The shock waves lead to increased turbulence and shear
forces within the sample matrix, eliminating the large channels within
the bulk material and disrupting the particle lattice structure. This, in
turn, provides better access to the analyte for the extraction solvent
i.e. improved surface area, A term in Fick’s law.
There are two types of ultrasonic extractors available for the trace
organic analyst. Bath-type ultrasonic devices employ a large stainless
steel container with the ultrasonic transducers located under the

bottom of the container. The stainless steel container is filled with
water to transfer the acoustical energy through the walls of the
sample container into the solvent and sample. This type of system is
generally low power (1-5 W/cm2) and is somewhat are less effective
for extraction of analytes strongly bound to the matrix components.
Probe-type ultrasonic devices delivery the acoustic energy directly to
the sample and extraction solvent. This results in 50 to 100 times
more energy being delivered to the extraction system (Figure 8).
Stainless steel container Extraction Ultrasonic Bubble
Trasducer Trasducer
Ultrasonic Generator
Figure 8. Bath type ultrasonic extractor
The choice of which ultrasonic system depends on the matrix and

specific analytes. The advantages of using an ultrasonic bath are:
1. The ultrasonic bath is the most widely available laboratory source

of ultrasonic radiation.
2. Small cleaning baths are inexpensive.
3. The acoustic field is evenly distributed throughout the bath liquid.
4. Conventional glassware is used.
31
The advantages of using an ultrasonic probe are:
1. Ultrasonic probes provide better bulk mixing when inserted directly

into the extraction system since energy losses during the transfer
of ultrasound through the bath media and reaction vessel walls
are eliminated.
2. The probe can be tuned to give optimum performance.
Supercritical Fluid Extraction (SCFE)

When a gas is compressed and heated above its critical temperature
and pressure (critical point), its physical properties change and it is
referred to as a supercritical fluid (Figure 9). As a supercritical fluid, it has
the solvating power of a liquid and the diffusivity of a gas. Extractions
performed with supercritical fluids increase the diffusion coefficient by
extracting a higher temperature and lower solvent viscosity. In addition,
the high pressure promotes diffusion of the solvent into the matrix to
expedite the equilibrium process.
The most common gas used to perform supercritical fluid extraction
is carbon dioxide. Carbon dioxide is favored for the following reasons:
1. Highly stable and can be obtained in high purity

2. It is nontoxic and nonflammable.
3. It is an environmental friendly solvent.
4. It is highly selective acting as a pure non-polar solvent.
5. It is easily removed, allowing simple product isolation by
evaporation to 100% dryness.
6. It is a tunable solvent whose density can be varied by pressure
changes to control product solubility.

10000
solid
1000 supercritical
pressuere (bar) fluid
liquid
100
10
gas
1
200 250 300 350 400
temperature (K)
Figure 9. Phase diagram for CO2
SCFE is performed with dedicated instrumentation necessary for

generating a supercritical fluid and also for controlling the fluid
movement. The sample to be extracted is loaded into a high pressure
extraction cell, usually made of stainless steel, PEEK (polyether
ether ketone), or any other suitable material that can withstand high
pressure (up to 10,000 psi). The loaded cell is inserted into the heating
oven and connected to the fluid delivery system, fitted with finger-
tight fittings, which eliminate use of a wrench to connect the vessel
to the system. This also reduces the wear and tear that can result
from over-tightening of connectors. The SCFE process begins with
carbon dioxide from the cylinder being cooled to a liquid. The liquid
CO2 is then compressed to a pressure above it’s critical pressure. The
pressurized liquid CO2 is pumped into the heating zone where it is
heated above it’s critical temperature to form a supercritical fluid. The
supercritical carbon dioxide flows into the extraction vessel, where it
rapidly diffuses into the solid matrix and extracts the soluble analytes.
After a variable static extraction period, the supercritical fluid and the
extract leave the extraction vessel from the top, through a pressure
33
reduction valve. Depressurizing the fluid reverts the CO2 back to a

gaseous state. The extracted material is collected as a solid precipitate,
dissolved in a suitable solvent as the gas bubbles through or on a
sorbent trap allowing the analyte to be selectively eluted (cleaned-up)
before analysis. The gaseous CO2 exits the separator vessel where it is
vented or condensed then recycled back into the system. (Figure 10)
700 PSI
400
1200
PSI
PSI
Compressor Pump
1200 PSI
CO2
Figure 10. Schematic of SCFE system
Pressurized Liquid Extraction (PLE)

Pressurized liquid extraction evolved from the supercritical fluid
extraction methodology and was developed in an attempt overcome
the limitations SCFE. The PLE methodology employs the use of typical
extraction solvents instead of a supercritical fluid. PLE, like SCFE,
improves multiple terms of Fick’s law simultaneously. PLE is performed
at temperatures from 75°C to 150°C and pressures of 1500 to 2000
psi. The higher temperature increases the mass transfer rate, lowers
the viscosity of the solvent and provides energy to overcome molecular

forces of attraction between the analyte. The high pressure promotes
diffusion of the solvent into the matrix to expedite the equilibrium
process and the use of organic solvents promotes a small Kd by using
an extraction solvent that has a high affinity for the analyte of interest.
PLE can be performed with essentially the same instrumental setup
as SCFE just replacing the carbon dioxide with a suitable extraction
solvent. The PLE system consists of extraction cell(s), solvent tanks, a
solvent pump, a heating oven, collection vessel(s), and inert gas tank
(Figure 11). The process starts when a stainless steel extraction cell is
loaded with the sample to be extracted. The loaded extraction cell is
transferred to a thermal oven where the extraction solvent is pumped
into the vessel, then pressurized with an inert gas. The elevated
pressure is used to maintain the solvent as liquid when the extraction
temperature is above its normal atmospheric boiling point. The oven
is 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
Solvent Solvent Pump

????
????
mp
Oven
Valve
Reserf
valve Vent
Extraction
Oven
cell
Extraction Cell
Vent
Nitrogen
Static valve Solvent
cylinder
Collection
vial
Collection Bottle
Figure 11. Schematic of PLE extraction system
35
fresh solvent. The cycle can be repeated to improve the extraction

efficiency. When the extraction is complete, compressed nitrogen
moves all of the solvent from the cell to the vial for analysis. The extract
is filtered, via an inline filter in the bottom of the extraction vessel prior
to being collected in the receiver, thus eliminating the need for a
separate filtration step.
Microwave Assisted Extraction (MAE)

The ability of microwaves to act as a heating source was discovered in
1946 and commercially available microwave heating devices became
available in 1967. Microwave radiation has the ability to rapidly heat
the sample matrix and extraction solvents when coupled with closed
extraction vessels. Closed-vessel microwave heating allows organic
solvents to be heated to 2 to 3 times their atmospheric boiling points.
The elevated temperature lowers solvent viscosity and the increased
solvent pressure, allowing the extraction solvent to penetrate the
sample matrix more efficiently. These factors significantly increase the
diffusion coefficient leading to faster more efficient extractions.
Heating by microwave energy is a “cold” in situ process, producing
heat only when there is absorption or coupling of the microwave energy
to the solution or microwave absorbing objects. The two primary
mechanisms for the absorption of microwave energy by a solution
are dipole rotation and ionic conductance. In the dipole rotation
mechanism, molecular dipoles align with the applied electric field.
Oscillation of the electric field results in forced molecular movement
of the dipole molecules with the resulting friction heating the solution.
At 2.45 GHz, the frequency of most laboratory microwave ovens,
the dipoles align, then randomize 2450 million times a second. In the
ionic conduction mechanism, the ionic species present in solution
migrate in one direction or the other according to the polarity of the

electromagnetic field. The accelerated ions meet resistance to their
flow and heating is a natural consequence. These two unique heating
mechanisms result in rapid heating of solutions in comparison to
conduction and convection (Figure 12).
MAE is performed with dedicated laboratory microwave systems.
These systems consist of two components, the microwave oven
and extraction rotor (Figure 13). The microwave oven is specially
constructed to withstand the harsh laboratory environment. The
microwave power input is computer controlled to ensure precise
temperature control during the temperature ramp and hold at the
desired extraction temperature. The extraction rotor is a carousel
design than can hold 8 to 24 samples. The MAE process begins by
loading the sample into the extraction vessel along with the extraction
solvent. The extraction vessel is then placed into a secondary vessel
designed to accommodate the high pressure generated when a
solvent is heated above it’s atmospheric boiling point.
The vessel is sealed and placed into the rotor with the other previously
+ Relaxation Relaxation
Alignment with Field
Dielectric Polarization
O
H H
time
H H
O

-
+
Ionic Conductance
CI- H
+
NO - +2
3 Cu
+ CI-
time
H
+2 NO -
Cu 3
Figure 12. Mechanism’s of microwave heating
37
prepared samples. The loaded rotor is placed into the microwave

system and the heating profile is entered. Microwave extractions
typically use a 10 to 20 minute ramp to temperature and a 10 to 20
minute hold at temperature. During the heating process, the rotor is
rotated 360° to ensure even heating of all the samples. When the
extraction run is complete, the samples are allowed to cool before
opening for analysis.
Figure 13. Microwave extraction system

CHAPTER 3
POST EXTRACTION PROCESSING
39
Post extraction processing
Under the most ideal conditions extracts would be ready for immediate
analysis, but this is rarely the case. Even the most advanced sample
preparation methods often require some form(s) of post extraction
processing to produce an extract that meets the instrumental and
reporting specifications.
Filtration
Several of the previously described extraction methodologies
include a method for separating the extraction solution from the
matrix. This separation process must be completed in a quantitative
manner in order to achieve the most accurate analysis results. The
most common method of filtration is simple filter paper (Figure 14a).
Fluted filter paper allows for faster flow since the wet paper is not
pressed against the walls of a filter funnel. Filter paper filtration is most
commonly used as the initial filtration step for samples that must be
concentrated. Disposable syringe filters are also used. These types
of filters come in various pore sizes and materials, with 0.45 micron
nylon being the most commonly used. The filter is simply attached
to a syringe with a Luer fitting and the extract then introduced into
the syringe. Constant pressure is applied to move the extract through
the filter. The first few drops are discarded, with the remaining portion
being deposited directly into an autosampler vial (Figure 14b). Syringe
filtering is necessary step for all samples, even ones that have been
paper filtered, since particles (filter paper fibers, drying agent particles.
etc), can adversely affect analytical instrumentation performance and
lifetimes.
41
Part I - Chapter 3 • Post extraction processing
a b
Figure 14. Filtration techniques. Fluted filter paper (a) Syringe Filter (b)
Water Removal
Since polar solvents are often necessary for achieving complete
extraction, water contained within the sample matrix can be
coextracted along with the analytes. This coextracted water has to
be removed from extracts when it will cause problems the analysis, or
interference removal (discussed below). The most common method for
removing coextracted water is to use a drying agent. A drying agent is
an anhydrous inorganic salt which acquires waters of hydration (water
scavenger) when exposed to the moistness of a wet solution. The
drying agent dries the sample without removing any of the analyte.
The drying agent can be incorporated as part of the filtration process,
by adding it to the filter paper or post drying by adding it to the filtered
solution then decanting. The most common drying agents are listed
in Table 3.
Concentration (Solvent Removal)

This technique is used to increase the intensity of the analytical
signal, lowering the limit of detection or to exchange the solvent from

Force Type Capacity1 Completeness2 Rate3 Use
Magnesium Sulfate High Medium Rapid General
Sodium Sulfate High Low Medium General
Calcium Chloride Low High Rapid Halides
Calcium Sulfate Low High Rapid General
Molecular Sieves High Extremely High Medium General
Table 3. Common Drying Agents

(1Amount of water removed per given wt of drying agent. 2Amount of water still
in solution at equilibrium with drying agent. 3Rate of drying action.)
the extraction solvent to one that is more compatible with analysis

or interference removal technique (discussed below). This task is
performed by evaporating the solvent to a 1–2 mL volume and then
making it up to a 5-mL volume in a volumetric flask. Evaporation
occurs when some of the molecules at the liquids surface move fast
enough to escape the liquid never to return. This is accomplished by
heating the extract to vaporize the solvent and remove it as a gas
leaving only the analyte behind. Applying vacuum during the process
decreases a solvent’s boiling point so that vaporization occurs at lower
temperatures reducing the risk of analyte decomposition i.e. water boils
at 7.5°C at a pressure of 10 mbar. The three most commonly used
concentration techniques are nitrogen blow down, vortex evaporation
and centrifugal concentration.
- Nitrogen Blow Down

In this technique nitrogen is blown down through needles onto the
sample vials to create a flow over the liquid surface. This flow of
nitrogen continually force off the layer of air that has become saturated
with vapor. This increases the evaporation rate by forcing the liquid to
43
release solvent molecules in an attempt to reestablish equilibrium with

the vapor phase. The gas flow rate must be precisely controlled to
prevent ‘splashing’ of the sample and lose of analyte. The evaporation
rate can be further enhanced by heating from below with a water bath,
but the temperature needs to be controlled to prevent the liquid from
becoming too hot during the process leading to analyte decomposition
or loss. Nitrogen blow techniques sometimes suffer from poor batch
to batch reproducibility (Figure 15a).
- Vortex Evaporation
In this technique the extract is placed under vacuum, while
simultaneously swirling to create a vortex inside the container. The
vortex increases the evaporative surface area of the sample speeding
up the solvent removal process. The downside of this technique is that
the sample can spread over the vessel walls and dry during the final
stages. It is a prone to solvent bumping if the vacuum pressure and
vortex rate are not precisely controlled. The evaporation rate is further
accelerated by heating, but may result in overheating of the analyte
when all or part dries on the container walls (Figure 15b).
- Centrifugal Concentration
With this technique the extract is placed into a rotor and spun at high
speed under vacuum. The solvent below the surface experiences a
higher pressure due to the extra weight of solvent multiplied by the
gravitational force exerted by the high speed spinning. The denser
material is captive in the bottom of the vessel allowing vaporization
of the less dense solvent occupying the upper level of the extract. A
flow of steam or infrared lamps are used to heat the spinning samples
to further increase evaporation rate. Centrifugal concentration is the
most reproducible of the three techniques (Figure 15c).

Blow down Hot gas
Heat
Figure 15a. Nitrogen blow down schematics
Vortexing Pump
Heat
Figure 15b. Vortex evaporation schematics
Centrifugal evaporation
IR
Lamps
Vacuum
pump
Figure 15c. Centrifugal evaporation schematics
45
Interference Removal
The aim of interference removal is to isolate the analyte(s) from
coextracted elements of the matrix that prevent accurate identification
and quantification. This is the most commonly technique to accomplish
this task is solid phase extraction (SPE). SPE uses a solid sorbent,
contained in a cartridge or a tube, to either retain the interferences
allowing the analytes to pass freely or retain the analytes passing the
interferences as waste then eluting the concentrated analytes. The
two main type of SPE sorbents used for trace organic analysis are
polar and non-polar. Polar SPE sorbents are used for the extraction of
polar analytes from non-polar matrices. The sorbent surface has polar
functional groups such as diol, aminopropyl, cyanopropyl, unbonded
silica, alumina, and Florisil. Analytes containing polar functional groups
can interact and are retained at the sorbent surface via dipole–dipole or
hydrogen bonding interactions. To maximize interactions, the sample
should be in a non-polar solvent, so interactions with the sorbent are
preferred. If the polar analyte is already in a polar medium, a simple
solvent exchange, as described above, into a non-polar solvent can
be performed before the SPE extraction. Polar SPE phases are usually
used to retain interferences while letting the analyte pass through and
is typically used for pesticide and PCB analysis.
Non-polar SPE phases contain non-polar functional groups such as
Octadecly (C18, CH3(CH2)17), Octyl (C8, CH3(CH2)7), Phenyl ((CH2)3C6H5)
and Cyano (CN, (CH2)3CN). Interaction between the analyte and
sorbent surface is done via dispersive forces (discussed in Chapter 1).
These sorbent types are used for the extraction of molecules that
contain non-polar functional groups from predominantly polar matrices
(for example, water). The interaction between the analyte and the
sorbent surface group is facilitated by polar solvents, which repel the
analyte from the solution phase and more strongly onto the sorbent

surface. Non-polar SPE phases are usually used to retain the analyte
while washing away the interferences, then eluting the analyte.
The SPE process consists of four main steps: conditioning, retention,
rinsing, elution.
- Conditioning
Before molecules can be absorbed by the sorbent, it must be made
compatible with the sample solvent to promote close contact between
the sorbent and the sample molecules. Without conditioning a polar
sample will flow through the channels and pores of a non-polar
sorbent without making any contact. Conditioning involves the use
of a mediating solvent to promote better contact between the sample
solvent and the sorbent. For example for a non-polar C18 sorbent it
is first washed with hexane to uncoil the long C18 chains, then with
methanol to displace the hexane and fill all the pores followed by a
water wash. This procedure promotes good surface contact with the
sorbent and causes the C18 chains to stick out like thorns into the
solution to grab the analytes.
- Retention
The sample is passed through the sorbent bed with the aid of a gentle
vacuum or applied pressure. The interference or analyte is retained in
the bed while the non-retained molecules pass through the column.
The flow rate will depend on the amount of sorbent used, column
dimensions, and particle size. If the flow rate is too fast the interferences
or analytes will pass through without being retained.
- Rinsing
Washing selectively removes interferences. The solvent used for
washing steps has a higher elution strength than the sample solvent,
47
but is weaker than the final elution solvent to ensure that analytes are
not coeluted with the interferences. Water with 5-10% organic solvent
is typically used since it will remove ionic as well as weakly bound
organic molecules.
- Elution
In the elution step the absorbed analyte(s) are removed from the
sorbent and returned to the liquid phase for analysis. The elution solvent
must be capable of disrupting all of the retentive interactions between
the analyte and sorbent, but not too strong to remove tightly bound
contaminants. In addition the eluting solvent must be compatible with
the analytical measurement technique, should be contaminate free,
and environmentally friendly.
The SPE process is illustrated pictorially in Figure 16.
Figure 16. Demonstration of non-polar sorbent SPE. The mixture containing

the analyte (Blue) and the interference (Yellow) is loaded, then retained on the
column. The interference is washed off and discarded, then the analyte is eluted,
interference free, for analysis

Derivatization
Derivatization involves a chemical reaction between an analyte and a
reagent to change the chemical and physical properties of the analyte
for improved analysis. For analysis by gas chromatography (GC) the
goal is to improve the analyte(s) thermal stability and improve it’s
volatility. This involves masking the polar functional groups (i.e. OH,
COOH) with a less-polar substituent to improve the compounds thermal
stability and improve it’s volatility. The most common derivitization is
to replace the hydrogen of OH of COOH a group with a trimethylsilyl
((CH3)3Si ) group (Equation 3). This effectively removes the hydrogen
bonding capabilities lowering the compound’s boiling point.
For analysis by liquid chromatography the goal of derivatization is to
introduce functional groups to increase the sensitivity and selectivity of
the detection technique. The most common derivatization for LC is to
add fluorescence or UV functionality, by adding a group that contains
an aromatic group.
Based on this brief overview of post extraction processing you can see
why trace organic analysis is a time consuming and error prone. Even
small improvements or elimination of steps can have dramatic effects on
the overall accuracy and precision of the analysis. The accompanying
list of references provides a more in depth look at these techniques
and provides best practices for their implementation. In addition, one
should regularly consult supplier’s and manufacture’s websites for
technical updates and helpful advice with these techniques.
(CH3)3Si-Cl + RCOOH (CH3)3Si-COOR + HCl
Equation 3
49
PART II
TRACE ORGANIC ANALYSIS
CHAPTER 4
CHROMATOGRAPHY
(COMPOUND SEPARATION)
53
Chromatography
(Compound Separation)
Part I of this book described methodologies for the isolation of analytes
from the matrix and preparing them for analysis. Part II will describe
the instrumental techniques used for the subsequent identification and
quantification of these analytes of interest. This section is not indented
to be a comprehensive guide to all analytical aspects associated with
trace organic analysis, but to be a companion to first section providing
the background knowledge to understand the synergy between all
steps involved in the trace organic analysis of solid samples. A list of
comprehensive theoretical sources on the presented subjects will be
provided at the end of each major section for those wishing to further
enhance their knowledge and understand of these techniques.
There only a few analytical methods that are specific for a single
chemical compound. Most methods are geared for a particular class
of compounds or chemical species, so separation of the compounds
of interest from the other matrix elements is a necessary step in
the analytical process. These separations are achieved through the
chromatographic process.
The chromatographic process involves partitioning the extract
components between two phases based on their physical properties.
The extract components are initially dissolved in the mobile phase
and then forced through a stationary phase, typically attached to the
inner walls of a column. As the mobile phase moves over a stationary
phase, the extract components interact with the stationary phase via
their intermolecular forces of attraction as described in Chapter 1.
Compounds that interact strongly with the stationary phase will be
slowed, while compounds with weak interactions will travel more
rapidly. The differences in travel rates cause the individual molecules, of
55
Part I| - Chapter 4 • Chromatography (Compound Separation)
the same compound, to gather together and move as discrete group

as they traverses the column. As these molecular groups traverse
the length of the column to the exit they ultimately separate from
each. Upon exiting the column each group of compound molecules
is then subjected to qualitative and quantitative analysis. The various
chromatographic processes are named according to the physical
state of the mobile phase. In gas chromatography (GC), the mobile
phase is a gas, and in liquid chromatography (LC) the mobile phase
is a liquid. Each technique has its advantages and disadvantages and
are summarized as follows:
1. LC is applied to the separation of any compound that is soluble in

a liquid phase such as drugs and pharmaceuticals, nucleic acids,
amino acids, lipids, carbohydrates, antioxidants, natural and
synthetic polymers and inorganic compounds. GC is applied only
to volatile compounds.
2. LC analysis is usually performed at room temperature and therefore
compounds that are thermally labile can be analyzed
3. Retention of analytes in LC depends on their interaction with both
the mobile and the stationary phase making it a more flexible in
optimizing separations.
4. LC is subject to greater peak or band-broadening which results in
lower resolution compared to GC.

CHAPTER 5
GAS CHROMATOGRAPHY (GC)
57
Gas Chromatography (GC)
The key parts of a gas chromatograph include: 1) a source of gas

as the mobile phase, 2) an inlet to deliver sample to a column, 3)
the column where separations occur, 4) an oven to control column
temperature, 5) a detector to register the presence of a chemical in
the column eluent, and 6) a data system to record and display the
chromatogram (Figure 17).
Figure 17. Modern GC System with autosampler
Carrier Gas (mobile phase)

The mobile phase in GC is called the carrier gas and serves as the
means to move constituents of the extract through the column. Most
columns do not tolerate moisture and oxygen well when operated at
temperatures over 100 °C so for the best results, column longevity
59
Part I| - Chapter 5 • Gas Chromatography (GC)
and chromatographic reproducibility the carrier gas needs to be

cleaned over molecular sieve beds to remove moisture, oxygen,
and hydrocarbon impurities. The three most commonly used carrier
gases are Helium, Hydrogen, and Nitrogen. Helium is currently the
carrier gas of choice because it inert, non-toxic and is a compromise
between the speed of hydrogen and the safety cost of nitrogen.
Recent spikes in the cost of helium have lead many trace organic
chemists to use hydrogen or nitrogen as alternatives in spite of some
safety concerns with the use of hydrogen and perceived theoretical
limitations of nitrogen. The advent of hydrogen generators eliminates
the safety concerns associated with the use and storage of hydrogen
gas cylinders and while there are real differences among the carries
gases, there are not dramatic changes in the final results once the
operational parameters are optimized.
Sample Introduction (Inlet)

The chromatographic process begins when sample is introduced
into the column, without disrupting the flow through the column.
The internal diameter for capillary columns used in modern GC
instrumentation ranges from 0.10mm to 0.53mm, making direct
injection of the sample on the column very difficult. To overcome this
problem a microsyringe is used to inject the sample through a rubber
septum into a flash vaporization chamber (i.e. inlet or injection port)
at the head of the column. The sample vaporizes to form a mixture
of carrier gas, vaporized solvent and vaporized analytes which is then
carried into the column by the carrier gas flow (Figure 18).
The inlet needs to be held at a temperature hot enough to vaporize
the sample rapidly, to ensure the entire sample is deposited on the
column, but low enough to avoid sample decomposition. Most GC
methods use injection port temperature ranges from 250°C to 290°C.

Figure 18. Cross Section of GC inlet
The small diameter of capillary GC columns limits the amount of sample

that can be loaded into a GC column so in order to accommodate
both high and low concentration extracts the inlet can be operated
in one of two modes, split or splitless. When analyzing a neat or high
concentration extract very small sample sizes, usually much less than
a microliter, must be used to avoid overloading the column. It is very
difficult to handle such small sample sizes with a microsyringe, but
split mode provides a way to inject a normal-size sample, vaporize
it, and then transfer only part of it to the column for analysis. This
is achieved through the use of a split valve. When the split value is
open the vaporized sample divides itself between entering the column
and exiting the split vent. This results in only a fraction of the sample
entering the column. The relative ratio of sample passing through the
split vent and on to the column can be controlled by adjusting the split
purge valve in the vent line (Figure 19a). When sample concentrations
61
are low (ppb range) it is desirable for all the extract to be transferred
onto the column, so splitless mode is used. In splitless mode, the
split valve remains closed during the injection process allowing the
majority extract components to enter the column and be trapped. For
efficient trapping the column must be held at a lower temperature than
the injection port, typically 5°C to 10°C below the boiling point of the
extract solvent. After the extract components have been trapped on
the column, the split valve is opened and the residual vapor in the inlet,
now mostly solvent, is swept out the vent (Figure 19b). Splitless mode
has some disadvantages. You must start with a cold column requiring
more time between runs; 2) optimization of both the initial column
temperature and the time of opening the split valve.
Inlet Septum Inlet Septum

flow Septum nut purge flow Septum nut purge
control with septum control control with septum control
Liner Split Split Liner Split Split

valve vent valve vent
(open) control (closed) control
(a) (b)
Figure 19. Split (a) and Splitless (b) injection modes
Columns for GC
Most common GC columns used for trace organic analysis are capillary
columns. Capillary GC columns are manufactured from fused silica
and coated with polyamide, a high temperature plastic, for support
and flexibility (Figure 20). They are generally 30 meters in length and

have an internal diameters ranging from 0.10mm to 0.53mm. The
stationary phase is an immobile, high molecular weight liquid which
is chemically bonded to the inner walls of the silica capillary tubing
(Figure 21).
Polymide coating
Fused silica
Stationary phase
Figure 20. Cross-section diagram of a GC capillary column
R R
Si OH + Cl Si Cl Si O Si Cl + HCI
R R
Silica Phace Bonded
Support Backbone Stationary
Phase
R R
Si O Si Cl + CH3OH Si O Si OCH3
R R
Bonded and capped
Stationary Phase
Figure 21. Overview of the stationary phase bonding process. The R groups are
customizable and are chosen based on the chemical properties of the analyte.
Bonding minimized phase and analyte degradation
63
Choosing a capillary column for GC analysis the often the most difficult
and ambiguous decision since one must balance the internal diameter
(I.D.), column length, stationary phase thickness and stationary phase
composition to achieve the best separation of the target analytes in
the shortest time possible.
- Internal Diameter
Smaller I.D. columns produce better separations compared to larger
I.D. columns. Smaller I.D columns mean that less analyte is traveling
down the center of the column increasing interactions with the
stationary phase increasing separation efficiency. Smaller I.D. columns
require higher operating pressure and have smaller analyte capacities.
The most popular I.D. for capillary GC columns is 0.25mm because
it is the best compromise between separation efficiency and sample
capacity. Capacity increases as column I.D. increases. Exceeding
the sample capacity of a column will result in skewed peaks and
decreased resolution. Therefore, if the extract to be analyzed contains
analytes at high concentrations, or a wide range of concentrations,
then a column with 0.32mm or larger I.D should be considered. If
the proper I.D. is chosen, the column should allow the system to
provide sufficient sensitivity for the minor components without being
overloaded with the major components. The analyst must decide
if the loss in efficiency resulting from using a larger I.D. column is
problematic for their application. As a general rule non-polar phases
have higher capacities for non-polar analytes, and polar phases have
higher capacities for polar analytes.
- Column Length
Capillary GC columns are made in various lengths, typically 10, 15, 30,
60, and 105 meters. Longer columns provide more resolving power

than shorter columns of the same inner diameter, but they also increase
analysis time and should be used only for applications demanding the
utmost in separation power. Doubling the column length (e.g., 30 m
to 60 m) increases resolution by approximately 40%, while analysis
time can be twice as long. In addition, longer columns cost more.
Conversely, if a separation can be performed on a shorter column
(e.g., 15 m versus 30 m), then both analysis time and column cost will
be less.
- Stationary Phase Thickness

Stationary phase thickness has a direct effect on both the retention of
each sample component and the maximum operating temperature of
the column.
The benefits of using a thin stationary phase:

1. Sharper peaks which lead to less overlap.
2. Reduced stationary phase breakdown i.e. column bleed.
3. Higher maximum operating temperature (350 to 400°C)
4. Faster elution times.
5. Lower elution temperatures.
The main drawback of using thin stationary phases is a decreased

analyte capacity and the analyte might interact with the column wall if
it penetrates to deep. Thin films are mainly used for analytes with high
boiling points (>300 °C) such as pesticides, PCBs, FAMEs, phthalate
esters, and other semivolatile compounds.
The benefits of using a thicker stationary phase:

1. Increased sample capacity
2. Increases analyte retention sometimes leading to better separation
65
The main drawbacks of using a thicker stationary phase include:

increased peak widths, increased column bleed, and a reduced
maximum operating temperature for the column. Thicker films are
mainly used for analytes with low boiling points or high concentration
samples to reduce the risk of overload.
- Stationary Phase Composition

Choosing the best stationary phase is the most important decision
when selecting a capillary column because it determines the types
of intermolecular forces of attraction that are available to separate
the analytes of interest. As with the extraction process described in
Chapter 1, stationary phases are selected using the “like dissolves
like” principle. A non-polar stationary phase is best for the analyses
of non-polar compounds. Polar stationary phases are most effective
for separating polar compounds. The default non-polar stationary
phase is polydimethylsiloxane (Figure 22) which will exhibit only
dispersion forces which increase with the size of the molecule.
Thus, larger compounds with higher boiling points will have stronger
interactions with the stationary phase, resulting in the elution order
of the analytes generally following the boiling points of the analytes.
Replacing some of the methyl groups in the stationary phase with
phenyl groups increases the polarity of the column. This is due to the
ability of the free π electrons in the phenyl to have weak dipole and
hydrogen bonding interactions with the analytes. The replacement of
5% or 50% of the methyl with phenyl groups are the most common
substitutions. Replacing the methyl group with more polar groups
such as cyanopropal (C3H6CN) or trifluoropropyl (C3H6CF3) increases
the polarity even more. There are numerous possible combinations
of substitution groups available to make stationary phases. The
best method for selecting a stationary phase is to consult the large

collection of example applications provided by column manufacturers
and suppliers, GC manufacturers and in published literature. While an
exact example application may not be available, enough information
can usually be obtained to simplify the decision or reduce the number
of potential columns. The most difficult situation is when no previous
information is available. Stationary phase selection is much easier
even if only one chromatogram is available for all or most of the analyte
compounds.
CH3 CH3 CH3
Si O Si O Si
CH3 CH3 CH3

n
Figure 22. Polydimethylsiloxane stationary phase
GC Oven
After injection the analytes must be maintained as a vapor throughout
their passage through the GC column. Therefore, most gas
chromatographs are equipped with ovens to precisely and reproducibly
control the column temperatures from 30 to 350°C within a few tenths
of a degree (Figure 23). The optimal column temperature depends
on the boiling points of the analytes and the degree of separation
required. Setting a temperature equal to or slightly above the average
boiling point of the analytes is sufficient for separation in a reasonable
time. This is called isothermal since the temperature is held constant
during the run. For a complex extract with many components covering
67
a wide range of boiling points and polarities; temperature programming

can be employed. In temperature programming mode the column
temperature increases at a set rate as the separation proceeds.
Figure 24 illustrates the improvements in the separation efficiency and
analysis time when temperature programming is employed.
Column
Oven
Figure 23. Schematic of GC oven with column
Detectors
The separated extract components exiting the column enter a detector
where the composition of the carrier gas stream is characterized
through one of several possible chemical or physical properties of the
molecules. There have been dozens of detectors developed for GC
analysis, but the most commonly used GC specific detectors are the
thermal conductivity detector (TCD) flame ionization detector (FID),
and the electron capture detector (ECD).

250K
A) Isothermal 70˚C
200K
150K
100K
50K
0
0 5 10 15 20 25 30
Time (min)
350K B) Isothermal 130˚C
300K
250K
200K
150K
100K
50K
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Time (min)
250K
C) Temperature Programming
200K
150K
100K
50K
Time (min)
Figure 24. Effect of temperature programming on separation efficiency
- Thermal Conductivity Detector (TCD)

All gases conduct heat (thermal conductivity) this property can be
used to detect molecules in the effluent exiting the column. A thermal
conductivity detector is simply an enclosed wire filament placed at the
exit of the column which allows the gas mixture to flow over it before
69
it is vented to the atmosphere. When a constant voltage is applied to

the filament it heats up. The resistance of the filament and the rate at
which the filament loses heat depends on the thermal conductivity
of the gas mixture passing over it. When the gas mixture consists
of only helium carrier gas, which has a thermal conductivity of 196
mW/mK, the filament is cooled to a constant temperature producing
a baseline current. When the gas mixture passing over the filament
contains a mixture of carrier gas and analyte, say hexane which has a
thermal conductivity 23.4 mW/mK, the filament temperature increases
due to the lower thermal conductivity of the hexane. This causes the
measured current to deviate from the baseline producing a peak in
the chromatograph. Modern detectors use a rapid switching valve
to alternate between carrier effluent and a mobile phase gas. When
an analyte is present in the gas stream, the filament temperature
drops and then recovers when the reference gas is switched in. The
electronics sense this change and adjusts the power to the filament
to keep the temperature constant. The recorded power differential,
in volts, is plotted versus time to produce the chromatograph. This
configuration minimizes the effects of variations in temperature,
pressure and electrical power noise associated with traditional design
(Figure 25).
- Flame Ionization Detector (FID)

Almost all organic compounds, except CO, CO2,and HCN, will burn
when exposed to a high temperature flame. When pyrolyzed organic
compounds produce ions and electrons proportional to the number of
carbon atoms present in the molecule. Analyte molecules are detected
by monitoring the current produced by collecting the charged particles.
The FID functions by first igniting hydrogen and air flame in a chamber
near the exit of the column. The burner tip is charged with a positive

Vent
Filament
Reference gas
Switching
valve
Makeup gas
Column
Figure 25. Modern TCD. Agilent Technologies design
voltage relative to the upstream collection electrode located above the

flame (Figure 26). The resulting current is then measured and plotted
as mA versus time to produce a chromatograph.
- Electron Capture Detector (ECD)

For environmental trace analysis of organochloride pesticides,
herbicides and polychlorinated biphenyls the electron capture detector
is the detector of choice due to it’s selective response to these types
of compounds. An electron capture detector capitalizes on the ability
electronegative and polarizible compounds to interact with free
electrons. The ECD functions by first creating an ionic environment
at the exit of the column. A radioactive isotope, usually Ni, in the
63
detector cell emits beta particles. The beta particles collide with
carrier gas molecules to create a shower of low-energy free electrons.
These free electrons create a constant standing current between the
detector electrodes (Figure 27). When an electronegative or polarizible
71
molecule enters the detector cell some of the free electrons are
captured, causing a significant reduction in the measured current
producing a peak in the chromatograph. Halogenated compounds
containing I, Br, and Cl along with peroxides, quinones, and nitrogen
groups are detected with high sensitivity. The detector is insensitive to
hydrocarbons, amines, alcohols.
Vent
Electrode
Air
Flame
Hydrogen
Makeup gas
Figure 26. Schematic of a FID detector

Anode gas
Vent
63Ni plating
Makeup gas
Figure 27. Schematic of ECD detector
Detector Signal Signal Sensitivity

Advantages Disadvantages
Type Generator Produced LOD
Universal
Thermal Change Low sensitivity.
detector.
Thermal conductivity electrical Often can’t use 1 to
Simple.
Conductivity of analyte resistance of with capillary 10 ppm
Linear range
gas the wire columns
(105)
General purpose.
Ionization of Insensitive to
Flame Total ion Linear range 1 to
analyte in H2O, CO2, SO2,
Ionization current (107). 100 ppb
H2/air flame and NOx
Low Noise
Decreases in Selective toward
Reduction
ion current in electronegative
in the Insensitive
the presence functional groups
ionization other organic
Electron of organic (e.g. halogens, 0.01 to
of a carrier molecules.
Capture molecules peroxides, 0.1 ppb
gas by a Small linear
which can quinines, and
ß- emitter range (~100)
capture nitro groups).
usually 63Ni
electrons Very sensitive
Table 4. Summary of GC detectors properties and capabilities
73
CHAPTER 6
LIQUID CHROMATOGRAPH (LC)
75
Liquid Chromatograph (LC)
The key parts of a liquid chromatograph include: 1) mobile phase,

degasser and pump, 2) sample introduction system (injector) to
deliver sample to a column, 3) the column where separations occur,
4) column thermostat to control temperature, 5) a detector to register
the presence of a chemical in the column eluent, and 6) a data system
to record and display the chromatogram (Figure 28).
Mobile Phase and Pump

Liquid chromatography systems contain one or more solvent
reservoirs, typically 1L in size, to hold high purity solvents to be used
as the mobile phase. Water, Methanol, Acetonitrile, and buffered water
Figure 28. Modern HPLC System (Agilent technologies)
77
Part I| - Chapter 6 • Liquid Chromatograph (LC)
are the most common mobile phase solvents. The solvents have to be
filtered and degassed, in or offline, before use. Dust particles, dissolved
gases, and other particulate matter cause irreproducible flows and
interfere with the performance many of the detectors. A LC analysis
can be performed with a single pure solvent as the mobile phase or
a solvent mixture with constant proportions. Separations performed
in this manner are called isocratic elutions which are analogous in
function to isothermal temperature programming for GC discussed
previously. Isocratic elution’s are best used for simple separations
where the polarity of the analytes does not very over a wide range.
For complex mixtures of widely varying polarities a gradient elution is
often employed to separate and elute more rapidly. In gradient mode
one of the solvents in the mix is held constant and the other solvent
increases in concentration with time during the run. This is analogous
to temperature programming in GC analysis. Figure 29 illustrates the
Isocratic 40% Organic
0 35
Gradient 35% to 70% Organic
0 40
Figure 29. Comparison of isocratic vs gradient separation of eight compounds

improvements in the separation efficiency when gradient elution is
employed. The only drawback of gradient elution is that it takes some
time to re-equilibrate back to the starting mobile phase composition,
so the improvements in analysis time may only be marginal.
Pumps for LC deliver mobile phase through the column that contains
the stationary phase. The pump must deliver the mobile phase at high
pressures between 50 and 1300 bar to overcome the resistance of
the stationary phase in the column. A fluctuating flow rate can create
detector noise that can obscure weak analyte signals. The most
common type of pump in use is a dual reciprocating piston pump.
Two small ball check valves open and in an alternating fashion to
control the flow into and out of the pump cylinder. The fill stroke pulls
the mobile phase in from the solvent side, then on the exit stroke
it is pushed through the injector onto the column. The dual piston
design creates a more uniform pressure and flow because one piston
while one piston is in the exhaust stoke the other is in the fill stoke.
A pulse damper is used downstream of the first piston to control the
flow surges and to produce a constant flow. Gradients made up from
the source solvents are produced by proportioning the liquids through
a multichannel gradient valve (MCGV) at low pressure then pumping
them through the column. The gradient is electronically controlled and
programmable in 0.1% increments (Figure 30).
Sample Introduction
Optimum separation efficiency is achieved by introducing the sample
as a thin uniform band at the top of the column. To introduce the
sample with a syringe through a septum, as in GC, into a high pressure
system without leaking is practically impossible. Instead, LC systems
a sampling loop system is used to seamlessly introduce the sample
into the mobile phase. This system uses a rotating valve which allows
79
degasser
solvents
MCGV as isocratic
pump
Figure 30. Schematic of MCGV and HPLC pump system
the sample to be loaded into a sampling loop outside the flow of

the mobile phase, then rotated to become part of the mobile phase
flow (Figure 31). If the valve is in the load position, a syringe can be
used to fill the sample loop, typically 1 to 50 µL, through the needle
port in the center of the valve. Any excess sample leaves the valve
through the waste port 6. While the valve is in load position, the mobile
phase enters the valve through position 2 and leaves it at position 3.
When the valve is switched to the inject position, the solvent flows
through the sample loop carrying the sample to the column. As soon
as the valve is switched, a start signal is sent to the LC system to
start recording the detector output. Most LC systems today are sold
with an autosampler that is capable of loading samples from small
glass vials, contained in a numbered carousel, on to the LC column
unattended (Figure 32).

pump
1 2 1 2
6 3 column 6 3
5 4
5 4
load position inject position
Figure 31. Schematic of HPLC Injection Valve
Figure 32. HPLC autosampler (Agilent technologies)
Columns for LC
Columns are typically made out of stainless steel tubing ranging from
5 to 25 cm in length (Figure 33). Stainless steel frits with pore sizes of
0.2 to 0.5 µm allows the mobile phase to pass through while the larger
stationary phase particles are retained within the column.
Choosing column packing material for LC analysis the often most
difficult and ambiguous decision since one must balance the internal
81
diameter (I.D.), column length, packing material size, and stationary

phase composition to achieve the best separation of the target
analytes in the shortest time possible.
Figure 33. HPLC columns
- Column Internal Diameter (I.D.)

HPLC columns have variable internal diameters ranging from 1.8 mm
to 4.6 mm. Large diameter HPLC columns require higher flow rates and
therefore larger volumes of mobile phase will be used. Changing from
a 4.6 mm to a 2.1 mm ID column can reduce the flow rate and solvent
volume required to reach the same optimum linear velocity without
increasing the run time. This results in improved separation efficiency,
reduced solvent consumption, and higher sensitivity because the
analyte is diluted by less solvent. These effects are shown in Figure 34.
- Column Length
LC columns are made in various lengths ranging from 15 to 250 mm.
Longer columns provide more resolving power than shorter columns
of the same inner diameter, but they also increase analysis time
and should be used only for applications demanding the utmost in
separation power (Figure 35). Doubling the column length increases
resolution by approximately 40%, while analysis time can be twice

as long. As a result, the column length needs to be chosen such
that resolution is good enough but retention/analysis time does not
become too large. Typical column lengths are 50 mm to 150 mm.
4.6 mm ID 2.1 mm ID
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Retention Time, min. Retention Time, min.
Figure 34. Effect of column diameter on separation and sensitivity
15 mm
50 mm
150 mm
0 1 2 3 4 5 6 7 8 min
Figure 35. Effect of column length on separation effectiveness
83
- Packing Particle (Structure and Size)

Columns used for LC are not coated with an immobilized liquid like
capillary GC columns but are instead packed with small diameter silica
particles bonded with the stationary phase in the same manner as
described for capillary GC columns in the previous chapter. Figure 36
shows the two most common particles types used in LC columns and
Figure 37 illustrates a particle coated with the stationary phase.
The particle size effects the efficiency separation in the same way as the
film thickness in a capillary GC column. Smaller particles have a higher
separation efficiency producing sharper peaks while larger particles
have a higher sample capacity (Figure 38). The main drawback of
using smaller particles is that they require pressures greater than 400
bar, which require the capabilities of the newer pumps that have upper
limits of 1300 bar.
Figure 36. Common materials of these particles are silica sols or gels. Sols are
aggregates of many small non-porous particles. Gels consist of large rigid and
porous particles

CH3
Silica
particle Si O Si R
CH3
Bonded stationary
phase
Figure 37. The R group is changed based on the desired properties of the
column. The stationary phase coats the particles analogous to fuzz on the skin
of a peach
1.8 um
3.5 um
5 um
Figure 38. Effective of particle size on the separation of coeluting compounds
85
- Particle Stationary Phase

In Chapter 3 the SPE technique was described. LC uses essentially
the same materials to achieve the analytical separation of the target
compounds. In contrast to polar phases used for primarily for SPE
clean-up, reversed phased packings are the most popular packing
material, with over 90% of trace analytical chemist’s using this mode
of seperation. Reversed phase packings are popular because they can
be used for non-polar, polar, ionizable, and ionic molecules making is
the most versatile phase available. The versatility of reversed phase
arises from the fact that in GC there are only two polarities involved in
the separation process, those of the analyte and those of the stationary
phase. In LC we have a third polarity, the mobile phase, which can be
adjusted to optimize the separation process. This allows the chemist
to use a stationary phase that is the opposite polarity of the analyte,
then elute the compounds by adjusting the mobile phase polarity
to ‘strip’ the analyte off the particles. The mobile phase polarity is
adjusted by using a mixture of water and a water soluble organic such
as methanol or acetonitrile. The most common non-polar compounds
used in stationary phases are: Octadecly (C18, CH3(CH2)17), Octyl (C8,
CH3(CH2)7), Phenyl ((CH2)3C6H5) and Cyano (CN, (CH2)3CN). C18 offers
the most retention, maybe too much for some samples. C8 is less
retentive than C18, but with similar selectivity in most cases. Phenyl is
significantly less retentive for non-polar compounds but has a strong
affinity for polar compounds, so reduces analysis time for mixture
if polar/non polar analytes. CN is the affinity for non-polar analytes,
and is good for reducing analysis time of late eluting compounds
and avoiding the use of gradient separations. As with GC capillary
column selection one should consult the large collection of example
applications provided by HPLC column manufacturers and suppliers.
While an exact example application may not be available, enough

information can usually be obtained to simplify the decision or reduce
the number of potential columns. The most difficult situation is when
no previous information is available. Stationary phase selection is
much easier even if only one chromatogram is available for all or most
of the analytes of interest.
Column Thermostat
A thermostatted column compartment (TCC) is used for controlling
the mobile phase and column temperature. A constant temperature
is required for stable reproducible separation conditions. Temperature
variations in the laboratory will cause peaks to shift making it difficult to
assign “peaks” to specific compounds in the chromatogram.
Certain chemical compounds may have low solubility in the HPLC
mobile phase and may precipitate. Performing the separations
at elevated temperature improves the solubility of these analytes
preventing precipitation. Elevated temperatures also lower the mobile
phase viscosity leading to lower operating pressures and improved
safety.
- Detectors
The eluent exiting the column enters a detector where the composition
of the mobile is characterized through one of several possible chemical
or physical properties of the molecules. There have been dozens of
detectors developed for LC analysis, but the most commonly used
LC specific detectors are the absorbance (VWD or DAD), fluorescence
(FLD), refractive index (RID), and the evaporative light scattering
(ELSD).
- Absorbance (VWD or DAD)

Absorbance detectors are the most common detectors used for LC.
87
Absorbance detectors operate on the principle that as a beam of light

passes through a liquid and is partially absorbed by the components
of the liquid Figure 39. The amount of light absorbed is directly
proportional concentration according to Beer’s law (Equation 4).
A variable wavelength detector (VWD) operates on a single wavelength
of light set by the operator. A beam splitter splits the incident beam
from a deuterium lamp on to a grating to isolate a specific wavelength.
l0 l
Figure 39. Ilustration of the absorption process. I0 is the initial light intensity and I
is the intensity after a portion is absorbed by the sample
I0
A = -logT = log = ε·c·d
I
A Absorption
T Transmission T = I ⁄ I0
I0 Intensity of the light that has passed the flow cell
I Intensity of the light going to the flow cell
ε Extinction coefficient: how strong a specific substance absorbs light
c Concentration of the substance
d Length of the beam path through the cell, cell length
Equation 4

The beam of the isolated wavelength is then split into two. Half of the
intensity goes to the reference diode, which measures the intensity
without absorption (I0). The other half passes through the flow cell,
where it is partially absorbed by the sample. The sample diode
measures the light intensity after absorption, which is I (Figure 40).
VWD’s can be programmed to change wavelengths during a
chromatographic run to compensate a low response of analyte. This
leads improvements the detection limits for each individual analyte. At
low UV wavelengths (<210 nm), just about every organic compounds
absorbs light, making UV detectors somewhat universal.
A dioade array detector (DAD) operates on the same fundamental
absorption principles as a VWD except all wavelengths are measures
simultaneously. Each dioade in the array responds to a narrow
wavelength ranges of the dispersed light, so that all wavelengths can
be monitored simultaneously. This allows an absorption spectra to be
produced (Figure 41) that can be used to identify sample compounds
through library matching. While the VWD uses a reference diode, for the
Figure 40. Schematic of a Variable Wavelength Detector
89
DAD there is no direct measurement of a signal without absorption (I0).

Instead a detector balance is used, which can be done automatically
when switching on the detector or when starting a measurement.
During a detector balance, absorption values for all wavelengths are
set to zero. All intensities measured during an experiment are now
relative to this zero absorption intensity (Figure 42).
- Fluorescence (FLD)
When a molecule absorbs a photon of light, the molecule becomes
more energetic for a short time then releases the excess energy by
omitting a photon of light i.e. when a black light is shown on certain
substances they glow in the dark. This system is very specific to
molecules that contain conjugated π electron systems such as PAH’s
or molecules that can be made to fluoresce through derivatization. A
fluorescence detector operates on the same basic principle as a VWD
except a high energy Xenon lamp is used in place of Deuterium and the
molecular fluorescence is observed by a photoelectric transducer that
is a right angle to excitation beam and sample cell (Figure 43). FLD’s
are about 100 times more sensitive than VWD and DAD detectors, but
only for molecules than can produce the fluorescence effect.
- Refractive Index Detector (RID)

Refraction is the bending of a wave when it enters a medium where
it’s speed is different. The refraction of light when it passes from a fast
medium to a slow medium bends the light ray away from it’s travel
path. This detector operates by passing light through a flow cell that
is separated by a glass plate. One half of the flow cell allows pure
mobile to pass through, while the other half allows the eluent from the
column to pass. When the column eluent contains analyte molecules a
displacement of the light beam centered on the flow cell will occur. This

0.6
absorbance 0.3
0
200 300 400 500 600
wavelength (nm)
Figure 41. An absorbance spectrum is a graph that shows how absorbance

varies with wavelength and is unique characteristic of a compound
Figure 42. Schematic of a Diode Array Detector
displacement causes a change in the light intensity which is registered

by the detector and a peak in the chromotograph is produced (Figure
44). RID is considered a universal detector because every organic
molecule can refract light when present in the mobile phase. RID is
especially useful for substances that do not absorb visible light as
carbohydrates, lipids and polymers. The RID is sensitive to changes in
91
the mobile phase composition and ambient temperature. This limits its
applicability to isocratic elution and temperature control of the mobile
phase must be precisely controlled.
- Evaporative Light Scattering (ELSD)

The ELSD measures the intensity of light scattered by molecules in the
sample. When the mobile phase containing analyte molecules enters
the detector, a nebulizer generates fine mist of droplets by a flow of
nitrogen. The mobile phase is evaporated while the sample itself is
retained in a microparticle form. The cloud of microparticles passes
through a laser beam where the microparticles cause the beam to
scatter. The scattered radiation is detected at right angles to the flow
by a photodiode (Figure 45). The degree of light scattering is related to
the concentration of the compound of interest in the sample. ELSD is
a universal mass-responsive detector, that can detect all compounds
which are less volatile than the solvent. Since the solvent is evaporated
and it the mobile phase composition has no effect on detection so
ELSD can be used with gradient elution. It is especially useful for
natural substances like carbohydrates, lipids and amino acids that do
not absorb light in the UV-Vis range.
Emission
monochromator
Lens
Mirror Lens
Xenon
flash lamp
Photomultiplier
Excitation Photodiode
monochromator
Sample
Figure 43. Schematic of a fluorescence detector

Tungsten lamp
Condenser lens
Light-receiving elements
“Zero” glass
Heat exchangers
Collimator lens
Flow cells
Mirror
Figure 44. Schematic of a refractive index detector (RID)
Figure 45. Schematic diagram of ELSD process
93
Signal Gradient LOD 10 µL

Detector Advantages Disadvantages
Generator Elution injection
Universal low wavelengths.
UV/VIS Selective higher wavelengths.
DAD allows increased Compounds must
VWD/ ID Yes compound Identification. absorb light
100 ppb
DAD Usable with buffered mobile
phase. Liner range 105
Selective. Usable with buf- Requires derivatiza-
1 to
FLD ID Yes fered mobile phase. Linear tion for non-fluore-
range 104 scent compounds 10 ppb
Universal. Can detect com-
Temperature
pounds that do not absorb or
RID ID Yes fluoresce.
sensitive. 100 ppm
Linear range 103
Requires Auxiliary
Universal. Can detect com- gas. Not usable with
1 to
ELSD ID Yes pounds that do not absorb or buffered mobile
fluoresce. phase. 10 ppm
Linear range 103
Table 5. Summary of LC detectors properties and capabilities

CHAPTER 7
MASS SPECTROMETER BASED

HYPHENATED TECHNIQUES
95
Mass spectometer based hyphenated
techniques
With classic GC and LC analysis the instrumentation components and
techniques developed in tandem to achieve the best analytical results.
Hyphenated techniques developed independently then converged
to provide some of the most powerful analytical techniques for trace
organic analysis. This section will describe the techniques of GC and
LC coupled with mass spectrometry.
Mass Spectrometry
Mass Spectrometry is an analytical technique that provides
information about an analyte’s molecular mass and structure. A mass
spectrometer separates ions generated from the analyte based on
their mass and charge, and generates an individual electronic signal
for ion. The electronic signals are then displayed as a series of sharp
peaks, one for each ion, to produce what is commonly called a mass
spectrum (Figure 46).
Each mass spectrum is unique to an individual molecule making
qualitative analysis possible. In addition the magnitude of each peak
is proportional to the number of ions produced allowing quantitative
determinations to be performed. How do we go about extracting
meaningful information from a mass spectrum and identify the
compounds we have separated? A number of libraries of printed
and computerized spectral databases are available to us. We can
use these spectra to compare both masses of fragments and their
intensities. Once a likely match is found, we can obtain and run the
same compound on our instrument to confirm the identity both by
GC retention time and mass spectra. This ability to simultaneously
obtain qualitative and quantitative information about a molecule is
97
Part I| - Chapter 7 • Mass spectometer based hyphenated techniques
???
Sample
124.1
m/z156
CH
3
m/z186
O N
O S
186.0
80000 NH N NH
3
279.1
m/z108
+
Ionization + m/z213
+
156.1
+
(positive or negative) + + 60000 [M+H]+
H N
2
m/z124
108.2
40000
+
+
3010
+ + + [M+Na]+
Manipulate according
323.0
+
213.2
to mass to charge ratio
107.1
20000
280.1
125.1
(or size to charge)
187.0
157.1
0
100 200 m/z
Detection
Figure 46. Illustration of the mass spectrometer process

and sample mass spectrum
what makes using a mass spectrometer as a detector a powerful tool

to trace organic analysis.
Another unique feature of using a mass spectrometer as a
chromatographic detector is the ability to resolve chromatographic
peaks by mass. Bis(2-chloroisopropyl)ether and 2-methylphenol
coelute when analyzed by GC as shown in Figure 47. Analyzing the
individual mass spectrums for these compounds we can see that
2-methylphenol has a unique ion at m/z 108 and bis(2-chloroisopropyl)
ether has a unique ion at m/z 45 (Figure 48). If us analyze only these
ions the individual chromatograms will produce a single peaks that
can be used to accurately quantify each compound. (Figure 49)
A mass spectrometer detection system consists of the ion source, the
mass analyzer, vacuum system and detector.

6.7 6.8
m/z
Figure 47. Total Ion Chromatograph (TIC) of bis(2-chloroisopropyl)ether

and 2-methylphenol both coelute at 6.7 minutes
100 100
108
45
80 80
60 60
40 40
20 20
0.0 0.0
0.0 20 40 60 80 100 120 0.0 40 80 120 160
m/z m/z
a b
Figure 48. Mass Spectrums of 2methylphenol (a)

and bis(2-chloroisopropyl)ether (b)
Ion Source
The mass analyzer requires gaseous ions this means that methods
for producing ions are very different for GC eluents when compared
to LC elutents. For GC the analyte molecules are already in the
gaseous phase so they can be directly ionized, while LC eluents
require volatilization prior to ionization. Electron Impact Ionization
(EI) and Chemical Ionization (CI) are applicable to GC eluents, while
Electrospray (ESI), Atmospheric Pressure Chemical Ionization (APCI),
and Atmospheric Pressure Photo Ionization (APPI).
99
bis(2-chloroisopropyl)ether
m/z45
6.7 6.8
2-methylphenol
m/z108
6.7 6.8
m/z
Figure 49. Individual target ion TIC’s for bis(2-chloroisopropyl)ether

and 2-methylphenol
- Electron Impact Ionization (EI)

The sample from the GC is exposed to a stream of 70ev electrons from
filaments. The emitted electrons collide with the sample molecules
where an electron knocked off, leaving behind a molecular ion with
a positive charge. In most cases ions with a single positive charge
are formed. The positive ion is forced from the ionization chamber by
a positively charged repeller into the mass spectrometer (Figure 50).
On average, one ion is produced for every 1000 molecules entering
the source under the usual spectrometer conditions, at 70 eV.
Furthermore, between 10 and 20 eV is transferred to the molecules
during the ionization process. Since approximately 10 eV is enough to
ionize most organic molecules, the excess energy leads to extensive
fragmentation. Every time a molecule of the same compound is ionized

under the same conditions, it forms the same quantity and pattern of
ions. This fragment pattern becomes a fingerprint that can be used
to identify and quantitate the molecule being analyzed. The limitation
of the technique is that under the voltage used, many molecular ions
first formed do not survive fragmentation. Since this molecular ion
gives us the molecular weight of the compound this information is
often missing and requires a secondary technique to molecular weight
identification/confirmation.
~70 Volts
Electron Collector (Trap)
Positive
Ions
+
Repeller Neutral Inlet
Molecoles
+ +
+
+
+ to
+ Analyzer
Electrons
Filament Extraction
Plate
Figure 50. Schematic of Electron Ionization
- Chemical Ionization (CI)

EI is a direct energy transfer process with electron kinetic energy
deposited directly into an analyte molecule. CI is an indirect process
involving an intermediate chemical agent. Methane, isobutene and
ammonia are most commonly used. When these agents are introduced
into the ionization chamber in large concentrations the electron beam
reacts exclusively with the reagent molecules. The ionization process
when methane is used as the chemical reagent is as follows.
101
- + -
CH4 + e CH4* + 2e
The main ion can undergo further reactions
+ +
CH4* CH3 + H*
+ +
CH4 + CH4 CH5 + CH3*
+ +
CH4 + CH3 C2H5 + H2
The sample molecules will interact with the ions via one of the following
reactions.
+ +
CH5 + M MH + CH4 + H2 (proton transfer)
+ +
CH5 + M M + CH4 + H2 (charge transfer)
The chemical ionization of the sample molecule occurs at much lower

energy than in electron impact ionization. The sample ion formed is
more stable and usually retains the molecular ion structure without
fragmentation or rearrangements with the molecular peak either M+1
or M-1.

- Electrospray Ionization (ESI)
The eluent from the LC pass through a small stainless steel capillary.
A converging nozzle mechanism accelerates a stream of nitrogen
nebulizing gas flowing parallel to the stainless steel capillary. The
resulting high velocity, uniform flow of nitrogen produces smallm
uniform droplets. Smaller, more uniform droplets desolvate in a more
consistent manner, ultimately delivering more ions into the instrument
increasing the signal strength.
The ESI nebulizer is oriented 90° in relation to the mass spectrometer
sampling capillary. This greatly reduces the number of neutral
particles entering the mass spectrometer, eliminating noise that would
otherwise be generated by those particles. A charge, either positive
or negative, is attached to each droplet by passing them through
an electric field that is created by applying a potential difference of
several kilovolts between the metal capillary and a counter electrode
(discharge needle). A stream of heated nitrogen drying gas effectively
desolvates the ions formed in the ESI spray. As the solvent evaporates
the droplets shrink until the surface tension can no longer support the
charge, resulting in an explosion of the droplet into smaller droplets.
The process continues until bare ions are ejected free of the solvent.
These ions are attracted, then pass through the sampling capillary
sampling orifice into the mass analyzer (Figure 51).
- Atmospheric Pressure Chemical Ionization (APCI)

APCI is an ionization technique which uses gas-phase ion–molecule
reactions at atmospheric pressure analogous to CI described above.
The evaporated mobile phase acts as the ionizing gas and reactant
ions are produced from a corona discharge, electrical discharge
brought on by the ionization of a fluid surrounding a conductor, on
the nebulized solvent. The solvent ions, (H2O+, CH3OH+, CH3CN+),
103
Nebulizing gas Ions Heated nitrogen drying gas
Eluent spray
+ ++
+ ++
+ + + + + + + + + + +
Entrance to
dielectric capillary Dielectric capillary
Figure 51. Schematic of ESI source
collide with the analyte and undergo cascade reactions which ionize
the analyte via proton or charge transfer like methane CI described
previously. A schematic of an APCI source is shown in Figure 52. APCI
is mainly applied to polar and relatively non-polar compounds with
moderate molecular weight up to about 1500 amu.
- Atmospheric Pressure Photo Ionization (APPI)

The ionization efficiency of non-polar analytes and aromatic is usually
poor in APCI. By substituting a discharge lamp emitting photons
rather than the corona discharge needle emitting electrons non-polar
analytes can be ionized (Figure 53).
The analytes molecules can be ionized directly through direct
interaction with the light photons;
+ -
M + hv M* - e
+ +
M* + Solvent MH + (Solvent - H)

HPLC inlet
Nebulizer (sprayer)
Nebulizing gas
Vaporizer (heater)
Drying gas
+ +
+ +
+ + + + + + + + + + +
Corona
discharge
needle Capillary
Figure 52. APCI source
HPLC inlet
Nebulizer (sprayer)
Nebulizing gas
Vaporizer (heater)
UV lamp Drying gas
hv +
+
+ +
+ + + + + + + + + + +
Capillary
Figure 53. Schematic of APPI source
105
or by using a large amount of a readily ionizable substance (dopant,

D, (acetone, toluene).
+
D + hv D*
+ +
D* + Solvent (D - H)* + (Solvent + H)
+ + +
M + (Solvent + H) MH + Solvent
+ +
D* + M M*
*+ +
M + (Solvent) MH + (Solvent - H)
+ +
The molecular ion can appear as either the M* or the MH molecule.
+ +
The formation of either the radical cation M* , the MH protonated
molecule, or both, will depend on the relative ionization energies or
proton affinities of the analyte molecules and the extract components.
Mass Analyzer
All mass analyzers use static or dynamic electric and magnetic fields
alone or combination. Most of the basic differences between the
various common types of mass analyzers lie in the manner in which
such fields are used to achieve separation. The most commonly used
mass analyzers for trace organic hyphenated GC and LC techniques
are quadrupole, time of flight (TOF), and tandem MS/MS.
- Quadrupole
Quadrupole mass analyzer separates ions in an electric field that is
varied with time. This design consists of a square array of four parallel
metal rods. Opposite pairs of rods are connected to opposite ends

of a DC source, then each pair is connected to an oscillating radio-
frequency (RF) source. By controlling RF and DC voltages to these
rods, an oscillating electric field is generated. A positive ion entering
the space between the rods will be drawn towards a negative rod. If
the potential changes sign before the ion discharges itself on the rod,
the ion will change direction. Ions that can maintain a stable trajectory
will exit the quadrupole spectrometer and strike the detector (Figures
54 and 55). Quadrupole mass analyzers are small, cheap, and have
good reproducibility, but suffer from a limited mass range, up to
1000 amu, and low resolution. A quadrupole mass analyzer can be
operated in one of two modes; full scan or selected ion monitoring
(SIM). In full scan analyzer control software steps the quadrupole
through increasing DC and RF voltages, which sequentially filters
the corresponding ions across the entire mass spectrum. A typical
mass scan will cover the range of 35-500 m/z four times per second.
Scanning mode provides a complete picture of all the ionized
compounds that occur in the eluent above the detection limit in the
chosen mass range. Laboratories have extensive computer libraries
containing mass-spectra of many different compounds to identify
the unknown analyte through spectra matching. Full scan mode is
quite useful when identifying unknown compounds in an extract and
provides a starting point for development of SIM methods.
To detector
From ion
source
Figure 54. Quadrupole separation process
107
Figure 55. Quadrupole Mass Analyzer
In SIM mode the analyzer gathers data for only the masses of interest
rather than all masses over a wide range. Typically two to four ions
are monitored per analyte (Figure 56). Since the quadrupole spends
more time sampling each of the chosen masses instead of a whole
mass range sensitivity of SIM is 10 to 100 times greater than full
scan mode. The down side of SIM is a lower degree of confidence
in the qualitative identification with SIM, but this can be overcome
by using several ions for SIM and comparing their ratios, which are
unique to each analyte, as well as by matching retention times to that
of standards. Examples of analyses carried out using SIM are PAH,
PCB and pesticide determinations.
Time of Flight (TOF)

In contrast to the quadrupole mass analyzer, TOF does not use an
externally applied force to separate the ions. In TOF separation of ions
by mass occurs during flight to the detector. If all ions are produced

1 scan
scan mass range
abundance
m/z
time m/z
1 scan discrete masses

SIM
abundance
m/z
time m/z
Figure 56. Mass Spectrometer Scanning vs SIM
with the same kinetic energy, then their velocity depends on its mass.
Lighter ions will arrive at the detector before the heaver ones. TOF
spectrometer operates by applying a high voltage pulse which imparts
the same kinetic energy to all the ions accelerating them down the
flight tube, ~1 meter in length, where they separate before arriving
at the detector (Figure 57). TOF spectrometers are very fast, have a
mass range up to 10,000 amu and have superior resolution compared
to quadruple mass analyzer (Figure 58).
- Tandem MS/MS
Tandem MS/MS consists of operating two mass analyzers in tandem
with a collision cell separating the two analyzers. Tandem MS/MS is
109
Accelerating
energy (E) Flight path distance (d)
Ion
pulser
Ion
Flight tube
optics
Detector
Ion source
Figure 57. Schematic of Time of Flight separation process
used to obtain more structural information on a particular ionic species.

This is the case when the ionization method yields very few diagnostic
fragments, fragmentation is obscured by column bleed/impurities, or
is obscured by other ions generated from matrix elements. Tandem
MS/MS instruments can be operated in one of three modes. In the first
mode, the first spectrometer is set to scan over the chosen mass range
and the second mass spectrometer is set to a pre-selected mass to
charge ratio. In this configuration only the collision fragments that match
the preselected mass to reach the detector and are recorded. This
method is selective for ions containing particular functional groups i.e.
m/z 77 corresponds to the phenyl group (C6H5+). In the second mode,
ions of a given mass are selected by the first mass spectrometer.
These selected ions then pass into a collision cell, filled with nitrogen,
where they collide and further fragment. The new fragments analyzed
by a second spectrometer using full scan mode. The third mode is
identical to the second mode except the second spectrometer is
also set to a predetermined mass so only the collision fragments that
match the preselected mass reach the detector and are recorded.

a Low resolving power
Target mass
Abundance
Interference
b High resolving power
Target mass
Abundance
Interference
Mass
Figure 58. Resolution comparison Quadrupole (a) vs TOF (b) mass

spectrometer
This double filtering results in a very sensitive method and is mainly

used for quantitation. Tandem quadrupole/quadrupole and tandem
quadrupole/TOF systems are the most common configurations used
for trace organic analysis and are shown in Figures 59 and 60.
Vacuum System and Detectors

All mass spectrometers must function under high vacuum (low
pressure). This is necessary to allow ions to reach the detector without
undergoing collisions with other gaseous molecules. Collisions with
111
could produce a deviation of the trajectory of the ion causing it to miss

the detector, cause it lose its charge making it inseparable or produce
unwanted reactions increasing the complexity of the spectrum. The
detectors are simple electron multipliers where the ions exiting the
mass analyzer collide with a dynode, which emits electrons when it is
struck by fast moving ions. The emitted electrons create a cascade
effect along the length of the dynode amplifying the signal.
Figure 59. Schematic of a tandem quadrupole/quadrupole system
Figure 60. Tandem quadrupole/TOF analysis system

PART III
THE MICROWAVE ADVANTAGE
CHAPTER 8
TECHNIQUE COMPARISON
115
Technique comparison
In 2013, Majors reported in his LC/GC survey that microwave-assisted

extraction, accelerated solvent extraction and supercritical fluid
extraction were expected to see increased use, but as of 2015 only
microwave-assisted extraction actually saw increased use in trace
organic analysis laboratories. So what makes microwave assisted
extraction so attractive for organic trace analysis?
Microwave-Assisted Extraction vs Soxhlet

Soxhlet can be applied universally to almost any sample matrix. The
Soxhlet methodology is very simple and the initial investment equipment
for a basic level system is low compared to MAE. In addition, the Soxhlet
methodology eliminates the need for post extraction filtration, which is
an added step for the MAE methodology. The disadvantages of using
the Soxhlet methodology are long extraction times, typically 8 to 24
hours; a large amount of solvent is required for each extraction, typically
300 mL; poor selectivity, mandatory concentration of the extract, and
artifact formation. For example, it has been shown that PAH’s maintain
a higher affinity for organic soil material than the extraction solvent
when using the Soxhlet methodology. MAE, in contrast, uses 10 to
20 mL of solvent per extraction, can be completed in as little as 30
minutes, and does not suffer from artifact formation. In addition, MAE
instrumentation can process 24 samples simultaneously and occupies
only 4 square feet of bench space (the system does not need to be in
a hood to operate). In contrast, Soxhlet extractions must be carried
out in a hood, occupies 5 square feet of space for each group of six
setups (30 square feet for 24 samples simultaneously), and needs a
supply of cooling water for the condensers.
117
Part III - Chapter 8 • Technique comparison
Microwave-Assisted Extraction vs Automated Soxhlet

The automation of the Soxhlet methodology improves on traditional
Soxhlet with respect to extraction time, solvent usage, and post
extraction processing. The commercial systems process six samples
simultaneously, use 70-90 mL of solvent per extraction, and can
process 24 samples for environmental analytes in a typical work
day. The startup cost for a six position automated Soxhlet system
is comparable to a MAE system, with the main advantage being the
integrated filtration and concentration. The automated systems must
be operated in a fume hood, occupy 2.5 square feet of bench space,
and require a supply of cooling water for the condensers. Overall, MAE
still outperforms automated Soxhlet in terms of sample throughput,
ease of use, solvent use and operating costs.
Microwave-Assisted Extraction vs Ultrasonic Extraction

Ultrasonic extractions are the simplest of all the extraction techniques
to perform requiring no specialized training. They require the least
time to execute, typically 2-3 minutes. Six to eight samples can be
processed simultaneously with a bath system and the technique is
relatively inexpensive, even if multiple probe type units are purchased.
When compared to MAE, ultrasonic extraction has the following
disadvantages: 1) the samples routinely need to be extracted 2-3
times, using fresh solvent each time, to achieve acceptable results;
2) the ultrasonic probes wear with time resulting in metallic particle
contamination of the extracts and loss of extraction efficiency; 3)
ultrasonic waves have been shown to produce radicals from water by
the following reaction:
H2O H* + OH*

The hydroxy and hydroxyl radicals formed in this reaction are highly
reactive and can produce interferences or artifacts of the analytes;
4) the probe type devices have to be thoroughly cleaned between
samples to avoid cross contamination; 5) large solvent consumption,
typically 200-300 mL; 6) acoustical isolation is needed to protect
operator. In spite of the advantages of fast extraction times and ease
of use, MAE still outperforms ultrasonic extraction in terms of sample
throughput, solvent use, extraction efficiency, labor involved, and
operating costs.
Microwave-Assisted Extraction vs Supercritical Fluid Extraction

(SCFE)
Supercritical fluid extraction is the most environmentally friendly
extraction method available for the trace organic analyst due to its
ability to easily obtain isolated analyte without further steps and solvent
can be recycled with a high level of purity. It is fast, 10-50 minutes
per sample and requires minimal amount of solvent, typically 5 to
10mL. The disadvantages of using the SCFE methodology are: 1) it’s
a complicated technique requiring precise control of the operational
parameters to obtain reproducible results; 2) matrix conditions effect
the penetration of supercritical fluid; 3) samples must be dried prior to
extraction because inter-matrix water could prevent the formation of
the supercritical fluid; 4) even at high densities, CO2 has a limited ability
to dissolve highly polar compounds. In addition, SCFE systems range
from $35,000 for a manual single extraction cell system to $125,000
for a fully automated system, and require extensive oversight to prevent
operational problems. MAE is the clear winner when compared to
SCFE extraction in terms of price, overall ease of use, flexibility and
sample throughput.
119
Part III - Chapter 8 • Technique comparison
Microwave vs Pressurized Liquid Extraction (PLE)

Pressurized liquid extraction is the most comparable technique to
MAE. It is fast, typically 15 minutes per sample, uses a small amount
of solvent, 15 to 45 mL per sample, and operates at elevated
temperatures and pressures. In addition, PLE offers in-line filtration and
the whole process is fully automated for unattended operation. The
disadvantages using the PLE methodology are: 1) the instrumentation
requires sources of air and nitrogen in order to function; 2) all system
seals, o-rings, require inspection before each run; 3) the system
needs to be rinsed between sample runs to prevent sample cross
contamination; 4) transfer lines can become clogged if analyte or
matrix elements precipitate when cooled, samples that contain very
fine particles can become compacted under high pressure making
extraction and filtration difficult or impossible; 5) any system or
computer errors associated with the automated process will stop the
sample queue making fully unattended operation questionable. In
addition, PLE systems cost twice as much as a microwave-assisted
extraction systems and takes 6 hours to process 24 samples. MAE
in contrast can process 72 samples, including filtration, in 6 hours,
easy to use instrumentation, uses disposable glass inserts to minimize
cross contamination, and has no matrix restrictions.

CHAPTER 9
MICROWAVE INSTRUMENTATION
121
Microwave instrumentation
Milestone ETHOS X
Figure 61. Milestone ETHOS X with fastEX-24 rotor
The ETHOS X (Figure 61) microwave cavity has a volume in excess

of 70 liters and, with a power of 1900 Watt, it’s the most powerful
microwave platform system available for solvent extraction. Equipped
with the most advanced contact-less temperature sensor, the ETHOS
X is controlled via a compact terminal with an easy-to-read, bright,
full-color, touchscreen display. The terminal runs an icon-driven, multi-
language software (Figure 62) with pre-loaded extraction methods. An
entirely new instrument setup has been specifically developed to fulfill
and exceed the US EPA method 3546 requirements.
123
Part III - Chapter 9 • Microwave instrumentation
US EPA 3546
T1:
°C T2: 120 °C
(set: 120 °C)
200 P1:
P2:
MW:
150
1
12 2
24 13
11 23 14 3
22 15
10 4 1
21 16
9 20 17 5
19 18
100
8 6
7
50
5 10 15 20 min
T1 T2 P1 P2 MW
Figure 62. Milestone ETHOS X operating software
The fastEX-24 rotor consists of a 24-position carousel, which holds

large pressure vessels made of the Milestone unique WeflonTM, ensuring
fast and homogeneous heating and providing high reproducibility
and complete recovery of the target compounds. At the core of the
vessel there is a disposable and inexpensive 100 mL glass vial, for
an unsurpassed ease of use and the lowest running cost (Figure 63).
When determining organic pollutants, most environmental labs aim
to achieve lower and lower detection limits. The sample preparation
technique play a very important role in this challenge. Although new
extraction technologies have been developed in the recent years, no
one is entirely matching the needs of the environmental labs. The new
ETHOS X with fastEX-24 rotor extracts up to 30 gram sample with
a minimal solvent volume, thus helping the analysts to accomplish
their tasks. The Milestone fastEX-24 rotor uses disposable glass vials,
that completely eliminate the need for cleaning and the possibility of
memory effect between different runs. The vials have a volume of 100
mL, ensuring the extraction of large sample amount. The very easy

handling and the affordable cost of the vials lead to high productivity
at very low running cost.
Load the sample Add the solvent
Insert the glass vial Close the vessel
Figure 63. Milestone ETHOS X working sequence
Consistency and Reliability

Data quality and reliability are key for environmental labs.
The ETHOS X with fastEX-24 provides fast, accurate and precise
analysis.
The unique design of the fastEX-24 rotor with its large volume,
disposable glass vials fully eliminates cross contamination and memory
effect thus contributing to achieve great analytical results.
Productivity
The ETHOS X fulfill the productivity required by the modern
environmental lab-indeed a factory for analyses.
125
Part III - Chapter 9 • Microwave instrumentation
While other technologies process sequentially, one sample at the

time, increasing the sample preparation time, the ETHOS X process
simultaneously 24 samples in 40 minutes.
Low running cost

Low solvent consumption along with the high productivity of the
ETHOS X strongly reduce the running cost of the environmental
pollutants analysis. The environmental labs often o er metal analysis
as well. The ETHOS X also match these requirements providing the
flexibility and all the additional accessories suitable to perform sample
preparation for AAS, ICP, ICP-MS. Two different applications with a
single microwave platform.
Green Technology
Microwave solvent extraction is inherently a green process, because of
the low solvent usage and the high sample amount vs solvent volume
ratio. This prevents waste to treat, clean-up or dispose. All process
takes place in a closed environment, without having the user exposed
to solvent vapors, and with reduced energy consumption.
Compliance
The US EPA method 3546 is a procedure (Table 6) for extracting
water insoluble or slightly water soluble organic compounds from
soils, clays, sediments, sludges, and solid wastes. This method
is applicable to the extraction of semi-volatile organic compounds,
organophosphorus pesticides, organochlorine pesticides, chlorinated
herbicides, phenoxyacid herbicides, substituted phenols, PCBs, and
PCDDs/PCDFs (Table 7).

US EPA 3546 method outline
Sample amount 2-20 gram
Solvents type Hexane and Acetone (1:1)
Solvents volume 25 mL
Temperature 100-115°C
Time at temperature 10-20 minutes
Table 6. US EPA 3546 method outline
Compound Analysis
PCBs EPA 8082
PAHs EPA 8270, 8100
Semivolatile organics EPA 8270
Phenols EPA 8151
Chlorinated pesticides EPA 8081
Organophosphorus pesticides EPA 8141
Chlorinated herbicides EPA 8141
Table 7. The US EPA 3546 method is suitable for the

above standard analytical procedure
127
CHAPTER 10
APPLICATIONS
129
Applications
Environmental Analysis
Determination of priority organic pollutants is one of the fundamental
aspects of many trace organic analysts jobs. Microwave extraction
has been used for these applications for over 20 years. There are three
standard microwave extraction methods for environmental analysis
1. USEPA SW846-3546 Microwave extraction. This method

extraction of semivolatile compounds, organophosphours
pesticides, organochlorine pesticides, chlorinated herbicides,
phenoxyacid herbicides, substituted phenols, and PCB’s from
soils sediments and sludges.
2. American Society of Testing Materials ASTM D5765-05(2010)
Total petroleum hydrocarbons.
3. ASTM D6010-12 is the Standard Practice for Closed Vessel
Microwave Solvent Extraction of Organic Compounds from Solid
Matrices. This covers the same parameters as EPA method.
The following tables (8, 9 and 10) contain examples of the use of
microwave extraction for environmental analysis. The microwave
results are comparable and offer better precision than Soxhlet.
131
Part III - Chapter 10 • Applications
Sample 1 Sample 2 Sample 3

Soxhlet Microwave Soxhlet Microwave Soxhlet Microwave
Phenol 2.49 ± 0.43 2.63 ± 0.41 2.13 ± 0.59 2.32 ± 1.11 2.34 ± 0.29 2.87 ± 0.35
2-chlorophenol 1.69 ± 0.37 1.72 ± 0.64 0.87 ± 0.54 0.99 ± 0.71 1.63 ± 0.40 1.96 ± 0.33
Isophorone 2.45 ± 1.93 2.67 ± 0.58 2.63 ± 2.14 3.25 ± 0.73 2.25 ± 1.44 3.03 ± 0.72
2-chloronaphthalene 1.43 ± 0.30 1.74 ± 0.14 1.61 ± 0.35 2.02 ± 0.58 1.41 ± 0.24 1.83 ± 0.32
Anthracene 0.83 ± 0.19 1.82 ± 0.20 0.39 ± 0.12 0.78 ± 0.18 1.18 ± 0.48 1.89 ± 0.33
Benzo(b)fluoranthene 0.98 ± 0.78 1.38 ± 0.31 1.23 ± 0.81 1.57 ± 0.27 0.86 ± 0.69 1.32 ± 0.39
Benzo(k)fluoranthene 0.88 ± 0.65 1.21 ± 0.23 1.07 ± 0.69 1.35 ± 0.20 0.78 ± 0.54 1.21 ± 0.34
Benzo(a)pyrene 0.96 ± 0.37 2.18 ± 0.25 0.59 ± 0.12 1.51 ± 0.21 1.18 ± 0.62 2.22 ± 0.54
Table 8. Extraction of Phenol and PAH compounds from soil. Concentrations

expressed as ug/g and error expressed as standard deviation (n=6)
Force Type Certified Value Microwave

Lidane 10.6 ± 4.8 10.6 ± 4.5
p,p’ DDE 18.7 ± 1.8 18.7 ± 1.8
Endosulfan I 6.9 ± 3.8 8.9 ± 1.2
Endrin 12.97 7.5 14.3 ± 2.0
Methoxychlor 16.4 ± 7.9 17.3 ± 2.2
Table 9. Extraction of Chlorinated Pesticides from soil. Concentrations

expressed as ug/g and error expressed as 95% CI (n=8).
Sample was certified using Soxhle
Force Type Certified Value Microwave

Aroclor 1016 279 ± 10 263 ± 5
Aroclor 1254 45.3 ± 5.8 44.0 ± 3.1
Table 10. Extraction of Polychlorinated Biphenyls (PCB) from soil and sediment.
Concentrations expressed a ug/g and error expressed as 95% CI (n=8).
Sample was certified using Soxhlet

Polymers/Plastics
Synthetic polymer materials are becoming the materials of choice for
many industrial and commercial applications. Development of new
polymers to meet a specific need is a costly endeavor. A cost effective
alternative is to blend two compatible polymer materials to achieve
the desired effect. For example PTFE (Polytetrafluroethylene is added
to thermoplastics to increase impact strength, weather and chemical
resistance. The blending process has to be strictly monitored. Changes
in the reaction stoichiometry or processing will alter the properties of
the polymer resulting in a compromised or undesired product. The
traditional batch quality control method for PTFE polymer blends is
to dissolve the PTFE with trifluoroacetic acid and determine percent
incorporation gravimetrically. The time needed to process 24 samples
using the traditional Soxhlet method is 18 hours, while it only takes
3 hours using microwave technology to achieve comparable results
(Table 11).
Traditional Method Microwave Method
Sample One 1.43 to 1.83 1.44 ± 0.08
Sample Two 2.68 to 3.46 3.03 ± 0.17
Sample Three 2.00 to 2.16 2.10 ± 0.09
Table 11. Comparison of Soxhlet with Microwave for PTFE extraction.

Concentrations expressed as percent PTFE in sample and error expressed as
standard deviation (n=6)
In addition to blends every polymer or plastic in commercial use

contains additives. Additives are compounds are added to either alter
the properties of the polymer or enhance its processability. Flame
retardants, ultraviolet stabilizers, biocides, and antioxidants (Irganox
1076, Irganox 1010) are common chemical property modifiers. The
levels of the additives incorporated into the polymer matrix need
133
to be monitored because changes in the reaction stoichiometry or

processing will alter the properties of the polymer and result in a
compromised or undesired product. The time needed to process
24 samples using the traditional Soxhlet method is 18 hours, while
it only takes 3 hours using microwave technology. For this study the
concentration of Irganox 1076 and 1010 in polypropylene 303 ± 5
μg/g and 79.2 ± 4 µg/g respectively with MAE which compares well
with the Soxhlet results of 294 ± 14 μg/g and 80.4 ± 6 μg/g.
There are two standard method available for the extraction of polymers:
ASTM D7210-13 is the Standard Practice for Extraction of Additives
in Polyolefin Plastics. This provides guidelines for extracting phenolic
antioxidants, phosphite antioxidants, UV stabilizers, antistatic agents,
and slip additives from milled polyolefin plastics. The Consumer
Product Safety Commission (CPSC) issued Test Method: CPSC-
CH-C1001-09.3. This test method uses a microwave extraction
technique determine the phthalate content analysis in children’s toys
and childcare articles.
Food and Drugs

Food products need to be analyzed for a variety analytes for labeling
and regulations including fat and amino acid content. Traditional fat
methods rely on the Soxhlet methodology and takes 24 hours to
process 24 samples, while it takes only 3.5 hours using microwave
technology. The results for a comparative study are shown in Table
12. The microwave methodology provides comparable results with
improved precision.
Amino acid content is also an important parameter for many food
products. The standard AOAC method for cysteine is a six hour
performic acid oxidation and evaporation followed by a 24 hr extraction
with 6M HCL. Using microwave technology the performic acid

Soxhlet Microwave
Soy Beans 43.4 ± 2.1 43.5 ± 0.8
Peanuts 48.5 ± 2.6 47.9 ± 0.5
Cookies 26.9 ± 1.1 26.2 ± 0.9
Rape Seeds 19.0 ± 1.3 19.3 ± 0.7
Chocolate 30.2 ± 6.8 34.1 ± 3.4
Table 12. Extraction of Fat from Food Products. Comparison of Soxhlet with
Microwave extraction. Concentrations expressed as percent fat in sample and
error expressed as standard deviation (n=6)
oxidation/evaporation step can be reduced to 45 minutes followed by

a 1 hr extraction with 6M HCl. The percent recovery of cysteine from a
standard reference material with using the new MAE was 98 ± 3 while
the AOAC method was 96 ± 6.
Color additives used in foods, drugs, cosmetics, and medical
devices must comply with regulations issued by FDA. The use of an
unlisted color additive, the improper use of a listed color additive, or
the use of a color additive that does not conform to the purity and
identity specifications of the regulation may cause a product to be
adulterated according to the provisions of the FD&C Act. The FDA
may take enforcement action against such products. D&C RED 30 is
a synthetic dye produced from petroleum or coal tar sources and is
FDA approved for use in pharmaceuticals and cosmetics. The Code
of Federal Regulations, Title 21 states D&C RED 30 must not contain
more than 5% acetone extractable material. The standard AOAC
method for determining acetone extractable material in D&C RED
30 requires a 20 hour Soxhlet extraction with 300 mL of acetone,
followed by filtration and evaporation to dryness. The time needed to
process 24 samples using the traditional Soxhlet method is 18 hours.
135
This same determination can be performed in only 2 hours using

microwave technology. The average extractable material for the D&C
RED 30 sample used in this study using microwave technology was
1.68% ± 0.06 which compares well with the AOAC method result of
1.70% ± 0.5.
Testing blood samples for drugs of abuse is an important part of
a forensic analytical chemists job. The extraction method for the
determination of free morphine in human serum involved the following
steps:
-- Addition of organic solvent for extraction and protein precipitation.

-- pH modification
-- Derivatization reaction
-- Extraction into organic solvent
-- pH modification
Using microwave technology one can reduce the process from

6 steps to three. This cuts the overall analysis time for 35 samples
from 1.5 days to 3.5 hours. The additional benefit of using microwave
technology is that microwave radiation significantly aids protein
denaturation resulting in better recoveries of morphine from standards
(94% ± 2) when compared to the traditional methodology (85% ± 4).
Future Applications
The simplicity and diversity of microwave extraction makes it amendable
to any extraction application. Future applications are expected to
include essential oil production, “Restriction of Hazardous Substances
(RoHS) and Waste from Electrical and Electronic Equipment (WEEE)
Directives, Food Safety and speciation.

APPENDIX A
137
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IMAGES
© Agilent Technologies <2017>
Reproduced with Permission, Courtesy of Agilent Technologies

AUTHORS
145
Authors
About the Authors
Robert C. Richter received his B.S in chemistry

from Valparaiso University in 1992 and his
Ph.D. from the University of Missouri in 1995
under the direction of S. Roy Koirtyohann, who
authored the first paper on microwave-assisted
acid digestions. He completed his postdoctoral
training at the New York State Department
of Health, and then worked at Duquesne University with Prof. H.M.
‘Skip” Kingston and participated in the method validation for EPA
microwave extraction method 3546. In 2000, he joined Milestoneand
served as the product specialist for the digestion, clean chemistry
and extraction product lines. In his six year tenure with Milestone he
developed over 100 unique microwave application for companies
such as Boeing, Dow, Dupont, Pfizer, Genetech, NASA, Abbot,
and MEMC. He is currently a professor of chemistry at Chicago
State University and director of the Center for Microwave Enhanced
Chemistry (CMEC). CMEC is engaged in variety of microwave
research including nanoparticle synthesis, optimization of organic
synthetic methodologies, environmental analysis, and integrating
microwave enhanced chemistry to Aquaponics system development.
He was the lead author on the 2001 Analytical Chemistry A-Page
article, “Microwave Enhanced Chemistry”, and the author of the first
microwave clean chemistry book, “Clean Chemistry for the Modern
Laboratory”.

Camillo Pirola is the Marketing and Business
Development Manager at Milestone. After
receiving his degree in Chemistry, in May 1999,
Camillo joined Milestone as an Application
Chemist. His professional career interlaces with
Milestone’s overall growth. He then became
Application Manager, providing application
support to all Milestone distributors around the world. In the late 90’s,
he became Area Manager for several countries including Japan, Italy,
The United Kingdom, Spain, Russia, India, and Brazil. While retaining
his position of Area Manager, in the mid-2000’s, Camillo became
responsible for the Milestone marketing at a global level. Since
2012, he is also the Business Development Manager at Milestone.
Camillo has contributed to several scientific papers and lectures, and
is often invited to give presentations, seminars, and training courses
worldwide.
147
MICROWAVE
GREEN EXTRACTION
The role of the analytical chemist has not changed since the inception
of the discipline. However, the questions for which society wants
answers have become more challenging. Instrumental analysis has
continually evolved to keep up with the analysis demands, but sample
preparation has failed to keep up with the evolutions of modern trace
organic analysis instrumentation.
In this book we discuss the importance of sample preparation for trace

organic analysis. Part I focuses on the fundamental theory of extracting
an analyte from a sample matrix, modern extraction techniques, and
post extraction processing. Part II reviews modern instrumental
analysis techniques as they relate to the sample preparation process
and Part III discusses how advances in microwave technology bring
sample preparation to the same standards as instrumental techniques.
The goal of the authors was to produce a text that helps today’s trace
organic analyst understand and overcome the difficulties of sample
preparation by learning how to THINK GREEN. We hope you enjoy it.

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Authors: Cover and Graphics:

Edited by: Milestone Srl and Ikonos Srl

PART I - SAMPLE PREPARATION

The purpose of organic trace analysis is to obtain information about

So why is organic trace analysis so hard?

There are millions of organic compounds that have been characterized

Compound CAS No. Structure

Table 1. Compounds with the chemical formula of C6H14

Analytical instrumentation has evolved to the point where is capable

10 MICROWAVE GREEN EXTRACTION - Modernizing trace organic analys

The fundamental thermodynamic relationship related to the extraction

Kd is the distribution coefficient, Ca is the concentration of the analyte in

This means that if similar intermolecular forces of attraction occur

16 MICROWAVE GREEN EXTRACTION - Modernizing trace organic analysis

Nonpolar Nonpolar Polar

Figure 1. Examples simple of a polar and non-polar molecules. The arrow

non-polar molecule are repelled by the negative charge, causing

18 MICROWAVE GREEN EXTRACTION - Modernizing trace organic analysis

Dipole Induced Dipole Hexane-Acetone 2-10

Ion Induced Dipole Na+ - Hexane 3-15

Hydrogen bonding Methanol-Water 10 to 40

Ion-dipole (Na+) - Water 30-600

Table 2. Intermolecular forces of attraction and their relative strength

the decay of plants and animals. It is typically composed of organic

Clay is mainly composed of silicate minerals containing the SiO4-4

20 MICROWAVE GREEN EXTRACTION - Modernizing trace organic analysis

Ce is the concentration of the analyte in the extraction solvent, t is time,

Large pores between

Figure 4. Schematic representation of available pores in a soil matrix

Figure 5. Scanning electron microscope image of a clay soil particle

high probability of containing deep channels where analytes can be

22 MICROWAVE GREEN EXTRACTION - Modernizing trace organic analysis

The final diffusion coefficient, D, follows Arrhenius type behavior which

D0 is temperature independent pre-exponential (constant), Ea is the

by optimizing a combination of these factors, mainly solvent selection,

24 MICROWAVE GREEN EXTRACTION - Modernizing trace organic analysis

MODERN EXTRACTION TECHNIQUES

Today’s organic trace analyst has a variety of extraction methodologies

water water water

Figure 6. Schematic of Soxhlet extraction apparatus

Automated Soxhlet (“Randall modiﬁcation” of the Soxhlet

28 MICROWAVE GREEN EXTRACTION - Modernizing trace organic analys

Ultrasonic Extraction (Acoustic Cavitation)

Rinsing Auto-shut down

Figure 7. Automated Soxhlet Extraction

30 MICROWAVE GREEN EXTRACTION - Modernizing trace organic analys

Stainless steel container Extraction Ultrasonic Bubble

Figure 8. Bath type ultrasonic extractor

The choice of which ultrasonic system depends on the matrix and

1. The ultrasonic bath is the most widely available laboratory source

The advantages of using an ultrasonic probe are:

1. Ultrasonic probes provide better bulk mixing when inserted directly

Supercritical Fluid Extraction (SCFE)

1. Highly stable and can be obtained in high purity

32 MICROWAVE GREEN EXTRACTION - Modernizing trace organic analys

Figure 9. Phase diagram for CO2

SCFE is performed with dedicated instrumentation necessary for

reduction valve. Depressurizing the fluid reverts the CO2 back to a

Figure 10. Schematic of SCFE system

Pressurized Liquid Extraction (PLE)

34 MICROWAVE GREEN EXTRACTION - Modernizing trace organic analys

Solvent Solvent Pump

Figure 11. Schematic of PLE extraction system