20210528111518D6369 - Food Analysis 10&11.spectros

FOOD ANALYSIS
SESSION 10-11
SPECTROSCOPY
DEPARTMENT OF FOOD TECHNOLOGY

FACULTY OF ENGINEERING
2021
LEARNING OUTCOME
• LO1 : Demonstrate practical proficiency in a food

analysis laboratory
• LO2 : Explain the principles behind analytical
techniques associated with food
• LO3 : Evaluate the appropriate analytical technique
when presented with a practical problem
• LO4 : Design an appropriate analytical approach to
solve a practical problem
OUTLINE
• Basic Principles of Spectroscopy

• Ultraviolet, Visible, and Fluorescence Spectroscopy
• Infrared Spectroscopy
• Atomic Absorption
• Nuclear Magnetic Resonance
• Mass Spectrometry
BASIC PRINCIPLES OF SPECTROSCOPY
• The science that deals with light and its absorption and emission
by solutions and other material substances is called spectroscopy
or Spectrometry
• The broad term for the instrument used is spectrometer , while a slightly
more specific term (when a light sensor known as a phototube is used) is
spectrophotometer
• The length of an electromagnetic wave is called its wavelength, and its

symbol is the lowercase Greek letter lambda, λ
• The speed with which electromagnetic waves move is called the speed of
light and is given the symbol c. The speed of light in a vacuum is
approximately 3.00 ×10 10cm/sec
• The number of the moving electromagnetic waves that pass a fixed point in
1 sec is called the frequency of the light. Its symbol is the lowercase Greek
letter nu, ν. It is expressed in waves (most often called cycles) per second,
or hertz (Hz)
• Mathematically, wavelength, speed, and frequency are related by the
formula
• The movement of an
electron between electron
energy levels is called an
electronic energy ransition
• Atoms in which no
electrons are in the higher
levels are said to be in the
ground state (E0)
• Atoms in which there is an

electron in the higher level
are said to be in an excited
state (E1, E2, E3, etc)
• An absorption spectrum is a plot of the amount of light absorbed

by a sample vs. the wavelength of the light.
• The amount of light absorbed is called the absorbance
• The absorption vs. wavelength information for molecules and

complex ions may also be displayed as a transmission spectrum,
rather than an absorption spectrum, by plotting the amount of
light transmitted by a sample rather than the light absorbed.
• In this case, the parameter that is plotted on the y axis is the

transmittance, or percent transmittance, rather than the
absorbance.
• Transmittance is symbolized as T.
• Under certain conditions, molecular and ionic analytes present in

samples of matter will emit light, and this light can be useful for
qualitative and quantitative analysis
• Similarly, molecules and complex ions may also emit light under certain
conditions, a phenomenon known as fluorescence
The equation of the straight-line A vs. C standard curve is known as the the
Beer-Lambert law, or simply as Beer’s law.
A statement of Beer’s law is
in which A : Absorbance;
a : Absorptivity, or extinction coefficient: the inherent ability
of a chemical species to absorb light and is constant at a given
wavelength, pathlength, and concentration
b : Pathlength : is the distance the light travels through the
measured solution. It is the inside diameter of the sample
container placed in the light path
c : Concentration.
Under these specific conditions, the absorptivity is called the molar

absorptivity, or the molar extinction coefficient, and is given a
special symbol, the Greek letter epsilon (ε)
• However, that the pattern of the absorption—the

characteristic of the spectrum that makes it useful for
qualitative analysis—does not change.
• The complete absorption spectrum for a given absorbing

species for a series of different concentration levels shows
that the absorption pattern is the same for all concentrations,
but the level of the absorption is different.
ULTRAVIOLET–VISIBLE (UV-VIS)
• Ultraviolet–visible (UV-VIS) molecular spectrochemical methods

utilize light in the ultraviolet and visible regions of the
electromagnetic spectrum to analyze laboratory samples for
molecular compounds and complex ions
• Qualitative analysis (identification of unknowns and detection of

impurities in knowns) is accomplished by comparing absorption
or transmission spectra (molecular fingerprints) with known
spectra.
• Quantitative analysis is accomplished with the use of Beer’s law

• If an instrument is meant strictly for visible light studies, then this

lamp is the only one present in the instrument. Such an
instrument is often referred to as a colorimeter
• A light source used frequently for visible light absorption studies
is the tungsten filament source
• If an instrument is meant strictly for ultraviolet work, then the
deuterium lamp is the only light source present and the
instrument is called a UV spectrophotometer
• A light source used frequently for ultraviolet absorption studies is
the deuterium lamp.
• A light source that can be used for both ultraviolet and visible
studies, the xenon arc lamp.
• In these latter two cases, the instrument is called a UV-VIS
spectrophotometer
A monochromator is made up of three parts:

1. An entrance slit : The entrance slit is where light enters the
monochromator from the source. Its purpose is to create a
unidirectional beam of light of appropriate intensity from the
multidirectional light emanating from the source
2. A dispersing element : disperses the light into its component

wavelengths. For visible light, for example, this would mean that
a beam of white light is dispersed into a spray of rainbow colors
3. An exit slit : The narrow band of the spectrum is then selected by

the exit slit
• The sample compartment is an enclosure with a lid that can be

opened and closed in order to insert and remove the cuvette.
• When the lid is closed, the compartment should be relatively free

of stray light , although this is not a requirement if a xenon arc
lamp is used as the source.
• This is because of the high intensity of the xenon arc lamp. The
cuvette is held snugly in a spring-loaded holder.
1. Single-beam instruments. In a single-beam spectrophotometer, the

monochromatic light beam created by the monochromator passes directly through
the sample solution held in the cuvette and then proceeds to the detector
2. Double-beam instruments. In a double-beam instrument the light beam emerging
from the monochromator is split into two beams at some point between the
monochromator and the detector.
Photomultiplier tubes or photodiodes (light sensors) are used as

detectors in UV-VIS spectrophotometers
1. Photomultiplier Tube
• A photomultiplier tube is a
light sensor combined with
a signal amplifier. The light
emerging from the sample
compartment strikes the
photosensitive surface and
the resulting electrical signal
is amplified.
• The photomultiplier tube
consists of a photocathode,
an anode, and a series of
Dynodes for multiplying the
signal
2. Photodiodes
• A photodiode is a p–n jun ction constructed with the top p layer so thin
that it is transparent to light.
• Light shining through the p layer creates additional free electrons in the n
layer that can diffuse to the p layer, thus creating an electrical current that
depends on the intensity of the light.
Diode array : A diode array

is a series of several hundred
photodiodes arranged in a
linear array. Single-beam
spectrophotometers have
been invented that utilize a
diode array as the detector
FLUORESCENCE SPECTROSCOPY
• Fluorometry is an analytical technique that utilizes the ability of

some substances to exhibit luminescence.
• Luminescence is a phenomenon in which a substance appears to

glow when a light shines on it. In other words, light of a wavelength
different from that of the irradiating light is released or emitted
following the absorption of this light.
• Luminescence caused by such a direct jump is called fluorescence.
• Luminescence resulting from a jump back to the ground state after

routing through other electronic states is called phosphorescence
• Fluorometry and absorption spectrophotometry are competing

techniques in the sense that both analyze for molecular species
and complex ions.
• As stated above, the number of chemical species that exhibit

fluorescence is very limited. However, for those species that do
fluoresce, the fluorescence is generally very intense.
• Thus we can say that while absorption spectrophotometry is

much more universally applicable, fluorometry suffers less from
interferences and is usually much more sensitive
IR SPECTROMETRY
IR spectrometry differs from UV-VIS spectrophotometry in the

following ways:
1. Absorption of IR light results in vibrational energy transitions
rather than electronic transitions.
2. While liquid solutions are often analyzed in IR work, pure liquids
and undissolved solids, including polymer films, are also often
analyzed, as are gases.
3. In IR work, the containers that hold liquids or liquid solutions in
the path of the light are not called cuvettes.
4. The liquid sampling cells have extremely short pathlengths (often
fractions of a millimeter), defined by the thickness of a thin
polymer film spacer
IR SPECTROMETRY
IR spectrometry differs from UV-VIS spectrophotometry in the

following ways:
5. Liquid sampling cells utilize large polished inorganic salt crystals as windows
(cell walls) for the IR light. Water, in which these windows are soluble, must
be scrupulously avoided, meaning that analyte solvents must be water-free
organic liquids that do not dissolve the inorganic salt crystals. Glass and
plastic have significant disadvantages and are not usually used.
6. IR spectra are usually transmission spectra rather than absorption spectra,

and wavenumber, rather than wavelength, is plotted on the x-axis.
7. IR spectra are characterized by rather sharp absorption bands and each such
band is characteristic of a particular covalent bond in the sample molecule.
8. Modern IR spectrometers do not use light dispersion to acquire spectra.

IR instrumentation
IR SPECTROMETRY
The advantages of FTIR over the dispersive technique are:
1) It is faster, making it possible to be incorporated into chromatography

schemes
2) The energy reaching the detector is much greater, thus increasing the
sensitivity.
IR SPECTROMETRY
Sampling
• Sampling, in the context of infrared spectrometry, refers to the method by

which a sample is held in the path of the IR light
• Glass and plastic are undesirable materials for the cells because glass and
plastic are molecular (covalent) materials and would absorb IR light and
interfere with reading the sample.
• Inorganic salts, such as NaCl and KBr, are ionic materials and do not absorb
IR light because ionic bonds cannot undergo vibrational energy transitions.
Covalent bonds can vibrate while ionic bonds cannot.
IR SPECTROMETRY
Liquid Sampling
Pure liquids (often referred to as neat liquids) and liquid solutions, sandwiching a
thin layer of liquid between two large NaCl or KBr crystals (the windows) is the
classic procedure for mounting the sample in the path of the light
Sampling
IR SPECTROMETRY
Solid Sampling
The most straightforward method for analyzing a solid material by infrared
spectrometry is to dissolve it in a suitable solvent and then to measure this
solution using a liquid sampling cell
IR SPECTROMETRY
• The region of the

electromagnetic spectrum
involved here is the infrared
region. This spans the
wavelength region from
about 2.5 μm to about 17
μm, or, in terms of
wavenumber, from about
4000 cm–1 to about 600 cm–
1.
• Infrared spectra are usually

displayed as transmission
spectra in which the x axis is
the wavenumber and 100%
T baseline as the maximum
y-axis
IR SPECTROMETRY
IR SPECTROMETRY
• Quantitative analysis procedures using infrared spectrometry utilize Beer’s

law. Thus only sampling cells with a constant pathlength can be used.
• Once the percent transmittance or absorbance measurements are made

INTRODUCTION
• Atomic spectroscopy refers to the absorption and emission of

ultraviolet–visible (UV-VIS) light by atoms and monoatomic ions
and is conceptually similar to the absorption and emission of UV-
VIS light by molecules
• absorption spectra for atoms are due to atoms in the gas phase
absorbing UV-VIS light from a light source and are characterized
by very narrow wavelength absorption bands called spectral
lines.
INTRODUCTION
Besides these differences in electronic energy levels and spectra, atomic

spectroscopy differs from UVVIS molecular spectroscopy in the following ways:
1. Analysis for atoms means that atomic spectroscopy is limited to the
elements. In fact, the key word for atomic spectroscopy is metals
2. Sample preparation schemes for atomic spectroscopy usually place the
metals in water solution. Atomic spectroscopy methods must have a means
for converting metal ions into free gas phase ground state atoms (a process
called atomization). Most of these methods involve a large amount of
thermal energy.
3. The sample container used for atomic spectroscopy is the source of the
thermal energy needed for the conversion of ions in solution to atoms in
the gas phase (and hence is called an atomizer) and in no way resembles a
simple cuvette.
4. The need for and use of thermal energy as outlined above has resulted in
the invention of a number of separate and distinctly different atomizer and
instrument designs, albeit based on the same theory, under the heading of
atomic spectroscopy.
INTRODUCTION
Besides these differences in electronic energy levels and spectra,

atomic spectroscopy differs from UVVIS molecular spectroscopy in
the following ways:
5. Spectral line sources are used as light sources in atomic absorption

instruments rather than the continuum sources used for UV-VIS molecular
absorption instruments, and several atomic emission techniques require no
light source at all apart from the thermal energy source.
6. Since the analytes for atomic spectroscopy are severely limited (elements
only), compared to the large number of molecular and complex ion
analytes for UV-VIS molecular absorption spectrometry the wavelengths
used for quantitation are well known and do not require the analyst to ever
first measure the absorption or emission spectra
INTRODUCTION
The most important of the techniques and instrument designs

requiring thermal energy (item 4 above) are :
1. Flame atomic absorption (flame AA)
2. Graphite furnace atomic absorption (graphite furnace AA)
3. Inductively coupled plasma atomic emission (ICP).
4. Flame emission and atomic fluorescence
INTRODUCTION
Flame Atomic Absorption
Flames and flame Process

1. Flames and flame Process
• For those atoms that are easily excited under these conditions, the emitted
wavelengths are in the visible region of the spectrum with characteristic
color because each element has its own characteristic line spectrum, the
atomic fingerprint, resulting from the particular energy transitions that
element has.
• Easily excited elements include sodium (yellow), calcium (orange), lithium

(red), potassium (violet), and strontium (red).
• All flames require both a fuel and an oxidant in order to exist. Bunsen
burners and Meker (Fisher) burners utilize natural gas for the fuel and air
for the oxidant.
2. Spectral Line Sources
• The light sources used in atomic absorption instruments are sources that
emit spectral lines. Specifically, the spectral lines used are the lines in the
line spectrum of the analyte being measured.
• Spectral line sources emit these wavelengths because they themselves

contain the analyte to be measured, and when the lamp is on, these
internal atoms are raised to the excited state and emit their line spectrum
when they return to the ground state. It is this emitted light, the same light
that will be absorbed in the flame, that is directed at the flame.
• 1. Hollow Cathode Lamp

2. Electrodeless Discharge Lamp
1. Hollow Cathode Lamp
The internal excitation and emission process occurs inside this cup when the
lamp is on and the anode (positive electrode) and cathode are connected
to a high voltage
• The lamp itself is a sealed glass

1. Hollow Cathode Lamp tube and is filled with an inert
gas, such as neon or argon, at a
low pressure. This inert gas plays
a role in the emission process
• The hollow cathode lamp must
contain the element being
measured. A typical atomic
absorption laboratory has a
number of different lamps in
stock that can be interchanged
in the instrument.
• Some lamps are multielement,
which means that several
different specified kinds of
atoms are present in the lamp
and are all raised to the excited
state when the lamp is on.
2. Electrodeless Discharge Lamp (EDL)
• In this lamp, there is no anode or cathode. Rather, a small, sealed

quartz tube containing the metal or metal salt and some argon at
low pressure is wrapped with a coil for the purpose of creating a
radio frequency (RF) field.
• The tube is thus inductively coupled to an RF field, and the

coupled energy ionizes the argon. The generated electrons collide
with the metal atoms, raising them to the excited state.
• The characteristic line spectrum of the metal is thus generated

and is directed at the flame just as with the hollow cathode tube.
3. Premix Burner
• The burner used for flame AA is a premix burner because all the
components of the flame (fuel, oxidant, and sample solution) are
premixed, as they take a common path to the flame
• The fuel and oxidant originate from pressurized sources, such as

compressed gas cylinders, and their flow to the burner is controlled at an
optimum rate by flow control mechanisms that are part of the overall
instrument unit.
• The sample solution is aspirated (drawn by vacuum) from its original

container through a small tube and converted to an aerosol, or fine mist,
prior to the mixing use of a nebulizer
3. Premix Burner
4. Optical Path
The optical path for flame AA is arranged in this order:
1. light source
2. Flame (sample container)
3. Monochromator
4. Detector
The optical path for flame AA can be either single-beam or

double-beam.
4. Optical Path
Graphite Furnace Atomic
Absorption
• The atomizer for

graphite furnace AA
is a small hollow
graphite cylinder
that can be quickly
electrically heated to
a very high
temperature
• Instruments that
utilize a flame for
the atomizer can
also utilize the
graphite furnace.
• The furnace module
is much more than a
small graphite
cylinder,
Absorption
Absorption
Absorption
• Advantage and disadvantage

Flame Atomic Graphite Furnace
Absorption Atomic Absorption
The number of ground Comparatively small A much larger

state atoms
Detection limit Improved over flame AA
Sample size Milliliters of sample Microliters
Absorbance readings Reproducible Less reproducible
Interferences Common More common and more
severe
Inductively Coupled Plasma
• Unlike flame AA and graphite furnace AA, the ICP technique measures the
emissions from an atomization/ionization/excitation source rather than the
absorption of a light beam passing through an atomizer
• ICP is the most important technique because the excitation source is much
hotter, resulting not only in atomizaton, but also in ionization and many more
emissions, and more intense emissions, than from the flame.
• ICP does not use a flame. This atomization/ionization/excitation source is a

high-temperature plasma. A plasma is a gaseous mixture of atoms, cations,
and electrons that is directed though an induced magnetic field, causing
coupling of the ions in the mixture with the magnetic field, hence the name
inductively coupled plasma.
• The ICP torch resembles a flame as the plasma emerges from the magnetic
field. The temperature of the ICP torch is in excess of 6000 K.
• Unlike a flame, in which only a very limited number of metals emit

light because of the low temperature, virtually all metals present in
a sample emit their line spectrum from the ICP torch.
• Not only does this make for a very broad application for ICP, but it
also means that a given sample may undergo very rapid and
simultaneous multielement analysis.
• One is the sequential optical path in which a monochromator is

used and the emissions are measured one wavelength after the
other sequentially by rotating the dispersing element while the
individual lines emerge from the exit slit. In this case, one
phototube measures all emission lines
• The advantages are:

1) all wavelengths of all elements can be utilized
2) spectral interferences can be avoided by careful wavelength selection.
• The disadvantage is that it is slower than the alternative

• The other option is comparable to the diode array design used with UV-VIS
molecular absorption instruments. This design is known as the simultaneous
direct reading polychromator design, in which there are a number of exit slits
and phototubes for measuring a number of lines at once
• The advantages of this design are:

1. It is fast, since all the desired wavelengths are measured simultaneously
2. It is good for samples with known matrices because it can be preset
to the optimum wavelengths.
• The disadvantages are:

1. The wavelength choices are changeable, but expensive
2. Spectral interferences can be a problem
• The advantage of ICP is

1. That the emissions are of such intensity that it is usually
more sensitive than flame AA (but less sensitive than graphite
furnace AA).
2. In addition, the concentration range over which the
emission intensity is linear is broader.
• These two advantages, coupled with the possibility of simultaneous

multielement analysis offered by the direct reader polychromator
design, make ICP a very powerful technique.
• The only real disadvantage is that the instruments are more

expensive.
Miscellaneous Atomic Techniques
• Flame Photometry
• Cold Vapor Mercury
• Hydride Generation
• Spark Emission
• Atomic Fluorescence
Summary
BASIC PRINCIPLE
• Mass Spectrometry is one of spectroscopic technique
• Unlike all the others, this technique does not use light at
all
• mass spectrometer utilizes a high energy electron beam

to cause total destruction and fragmentation of the
molecules of a gaseous sample
• This fragmentation results in small, positively charged

pieces or fragments of the molecules that are detected
individually on the basis of their mass-to-charge ratios
BASIC PRINCIPLE
BASIC PRINCIPLE
The four main components of a

molecular mass spectrometer
SAMPLE INLET SYSTEM
• Purpose is to introduce a very small amount of sample into

the mass spectrometer so that its components may be
converted into gas ions
• Batch Inlet Systems

• Direct Probe Inlet
• Chromatography and Capillary Electrophoretic Inlet System
SAMPLE INLET SYSTEM
Batch Inlet Systems:

• These systems are the simplest and simply involve the
volatilization of the sample externally and then the gradual
leakage of the volatilized sample into the evacuated ionization
chamber.
• For gases, the sample is introduced into the metering volume
container and then expanded into the reservoir flask where it is
then leaked into the ionization chamber.
• For liquids, a small quantity of sample is introduced into the
reservoir and the pressure of the system is reduced to about
10-5 torr.
• The inlet system is lined with glass to avoid losses of polar
analytes by adsorption.
SAMPLE INLET SYSTEM
The Direct Probe Inlet:

• These systems are used for solids and non-volatile liquids
• in these systems the sample is introduced into the ionization
region by means of a sample holder, or probe, which is inserted
through a vacuum lock.
• Probes are also used
when the amount of the
sample to be analyzed is
small.
• With a probe, the
sample is generally held
on the surface of a glass
or aluminum capillary
tube and positioned
within a few meters of
the ionization source.
SAMPLE INLET SYSTEM
Chromatography and Capillary Electrophoretic Inlet System
• Mass spectrometers are often coupled with gas or high –

performance liquid chromatographic systems or capillary
electrophoresis columns to permit the separation and
determination of the components of complex mixtures.
• Linking a chromatographic or electrophoretic column to a mass

spectrometer requires the use of specialized inlet systems
ION SOURCES
• The starting point for a mass spectrometric analysis is the

formation of gaseous analyte ions, and the scope and the utility
of a mass spectrometric method is dictated by the ionization
process.
• The appearance of mass spectra for a given molecular species is

highly dependent upon the method used for ion formation.
ION SOURCES
• Type of ion sources

the sample in solid or liquid state is converted directly in to
gaseous ions.
1. Matrix-assisted Laser desorption/ionization (MALDI)
2. ElectroSpray Ionization (ESI)
3. Fast atom bombardment (FAB)
4. Secondary ion mass spectrometry (SIMS)
5. Thermospray ionization (TI)
6. Field desorption (FD)
the sample is first vaporized and then ionized.

7. Electron Impact (EI)
8. Chemical Ionization (CI)
9. Field Ionization (FI)
ION SOURCES
Ion Sources make ions from sample molecules

(Ions are easier to detect than neutral molecules.)
Electrospray ionization:
Partial
Sample Inlet Nozzle
Pressure = 1 atm vacuum
(Lower Voltage)
Inner tube diam. = 100 um
N2 MH+
++
+
+ + + + + +
++ + +
+ ++ +
MH2+
++ +
++ + + +
++
Sample in solution ++
+ + + +
+
++ ++ ++ +
+ +
N2 gas ++ +
MH3+
High voltage applied Charged droplets

to metal sheath (~4 kV)
ION SOURCES
MALDI: Matrix Assisted Laser Desorption Ionization
• In this technique the sample is mixed with an equal volume of a

saturated solution of matrix (excess of a radiation-absorbing matrix
material ) prepared in a solvent such as water, acetonitrile, acetone
or tetrahydrofuran .
• The matrix is a small, highly conjugated organic molecule that
strongly absorbs energy in the UV region.
• The resulting solution was evaporated on the surface of metallic
probe that was used for introduction of the sample into the mass
spectrometer.
• The solid mixture was then exposed to a pulsed laser beam ,which
resulting the ions.
ION SOURCES
MALDI: Matrix Assisted Laser Desorption Ionization
Sample plate
Laser
hn
1. Sample is mixed with matrix (X)

and dried on plate.
MH+
2. Laser flash ionizes matrix
molecules.
3. Sample molecules (M) are ionized
by proton transfer:
XH+ + M  MH+ + X.
+/- 20 kV Grid (0 V)
ION SOURCES
Ionization by particle Bombardment

Theory
The analyte is in liquid matrix (with low volatility) and is bombarded with
high current of bombarding particles. The primary partile beam is the
bombarding particle beam, while the secondary ions are the ions produced
from bormbardment of the liquid target surface
This is a soft ionization technique and
suited for the analysis of low volatility
species, typically producing large peaks
for the pseudo-moluiceler ion species
[M+H]+ and [M-H]-, along with
structurally significant fragment ions and
some higher mass cluster ions and dimer
ION SOURCES
Ionization by particle Bombardment

Fast Atom Bombardment (FAB) : Natural
inert gas (typically Ar or Xe at energies of 4-
10 KeV) are accelerated by electric potential
to give high-velocity beam of ions.
Ionization of a neutral molecule occurs by
close encounter with a fast moving atom or
ion
Secondary Ion Mass Spectroscopy (SIMS) :
Ions (Typically Cs+ at enegies of 2-30 KeV)
are focused and accelerated to higher KE’S
than is possible in FAB, so sensitivity is
improved for higher masses
ION SOURCES
The Electron-Impact Source (EI)
• The sample is brought to a temperature high enough to produce a molecular

vapor, which is then ionized by bombarding the resulting molecules with
abeam of energetic electrons.
• Electrons are emitted from a heated tungsten or rhenium filament and
accelerated by a potential of approximately 70 V that is impressed between
the filament and the anode.
• M + e- M+ + 2 e-
• The kinetic energy (KE) given by the equation :
KE = 1/2 mv2 V= velocity
• Thus if all ions acquire the same amount of Kinetic Energy, those ions with
largest mass must have the smallest velocity
ION SOURCES
The Electron-Impact Source
• Advantages :
1. high ion current ,thus giving good sensitivities.
2. Fragmentation unambiguous identification of analyte possible.
• Disadvantages :
3. Disappearance of molecular ion peak (sometime)
4. The need to volatize the sample (degradation before ionization can
occur )
5. Limited to analyte having MW smaller than 103 Daltons.
ION SOURCES
ION SOURCES
Chemical Ionization Source (CI)
• Most modern MS are designed so that electron-impact ionization and

chemical ionization can be carried out interchangeably.
• In Chemical ionization ,gaseous atoms of the sample are ionized by collision

with ions produced by electron bombardment of an excess of a reagent gas.
• One of the most common reagent is methane which react with high-energy
electrons to give several ions such as CH4+,CH3+and CH2+ .The first two
predominate and represent about 90%.This ions react rapidly with additional
methane molecules .
ION SOURCES
ION SOURCES
Field Ionization Source (FI)
• In field ionization sources, ions are formed under the influence of a

large electric field (108V/cm) .
• Such fields are produced by applying high voltages (10-20kV) to
specially formed emitters consisting of numerous fine tips having
diameters of less than 1 μm.
• The emitter often take the form of fine tungsten wire)≈ 10 μm
diameter )
• on which microscopic carbon dendrites, or whiskers ,have been
grown by the pyrolysis of benzonitrile in a high electric filed.
ION SOURCES
Field Ionization Source (FI)
• The result of this treatment is a growth of many hundreds of

carbon microtips projecting from the surface of the wire.
• Field ionization emitters are mounted 0.5 to 2 mm from the

cathode, which often also serves as a slit.
• The gaseous sample from a batch inlet system is allowed to diffuse

into the high-field area around the microtips of the anode.
• The electrons from the analyte are extracted by the microtips of

the anode.
ION SOURCES
Field Ionization Source (FI) VS Field Desorption (FD)

The Theory
A high electric voltage (potential) that gives rise to lines of equipotential result in
an electric field crowd around the needle tip. The electric field is the most
intense at the surface (point) of the tip and is where ionization occurs.
FI : sample is heated in a vacum so as FD : the sample is placed directly onto

to volatilize it onto an ionization the surface (dipping emitter in an
surface. FI is suited for use with analyte solution) before ionization but
volatile, thermally stable compounds. FD is needed for non volatile and/or
FI sources are arranged to function thermally labile substances
also as FD sources
ION SOURCES
ION SOURCES
MASS ANALYZER
• Ideally , the mass analyzer should be capable of distinguishing between

minute mass differences.
• In addition ,the analyzer should allow passage of a sufficient number of ions

to yield readily measurable ion currents.
• As with optical monochromator ,to which the analyzer is analogous.
• Type of mass Analyzer :

1. Time of flight (TOF)
2. Quadrupole
3. Ion Trap
4. Magnetic Sector
5. Fourier Transform Ion Cyclotron Resonance (FT-ICR)
MASS ANALYZER
Resolution of Mass Spectrometers :
• The capability of a mass spectrometer to differentiate between masses is

usually stated in terms of its resolution ,R, which is defined as :
R = m/Δm
Δm : the mass difference between two adjacent peaks that are just resolved,
m : the nominal mass of the first peak.
• The resolution needed in a mass spectrometer depends greatly upon its

application.
• Commercial spectrometers are available with resolution raining from about

500 to 500,000.
MASS ANALYZER
1. Time of Flight (TOF)

• The time-of-flight mass spectrometer is different from the
others in that it does not use a magnetic field.
• Rather, it separates the charged fragments on the basis of
how fast they move through a drift tube in which a strong
electric field is present due to a high-voltage metal grid
positioned in the path.
• The lighter fragments travel through the tube faster than the
heavier fragments, and the different masses are detected at
different times, depending on their time of flight through the
tube.
• The signal is thus measured vs. time, but can be translated
into the traditional mass spectrum.
MASS ANALYZER

MASS ANALYZER
Source Drift region (flight tube)
detector
+
+ +
V
• Ions are formed in pulses.
• The drift region is field free.
• Measures the time for ions to reach the detector.
• Small ions reach the detector before large ones.
MASS ANALYZER
2. Quadrupole
Uses a combination of RF and

DC voltages to operate as a
mass filter.
• Has four parallel metal

rods.
• Lets one mass pass through

at a time.
• Can scan through all

masses or sit at one fixed
mass.
MASS ANALYZER
2. Quadrupole
• The most common mass analyzers in use today.
• More compact , less expensive, and more rugged than most other type of
MS.
• It offers the advantage of low scan times, which is particularly useful for real-
time scanning of chromatographic peaks.
• The heart of a quadrupole instrument is the four parallel cylindrical rods that
serve as electrodes.
• Opposite rods are connected electrically, one pair being attached to the
positive side of a variable dc source and the other pair to the negative
terminal.
• At any given moment ,all of the ions except those having a certain m/z
values strike the rods and are converted to neutral molecules .Thus only ions
having limited range of m/z value reach the transducer.
MASS ANALYZER
2. Quadrupole
MASS ANALYZER
2. Quadrupole
Quadrupoles have variable ion transmission modes
m2 m1
m4 m3 m2 m1
m4 m3
mass scanning mode
m2 m1
m2 m2 m2 m2
m4 m3
single mass transmission mode

MASS ANALYZER
3. Ion Trap
• An ion trap is a device in which gaseous anions or cations can be
formed and confined for extended periods by electric and/or
magnetic fields.
• Several types of ion traps have been developed, and two are
currently used in commercial MS.
1- The ion cyclotron resonance trap.
2- The Simple ion trap.
MASS ANALYZER
4. Magnetic Sector
• In this instrument, the magnet is a powerful, variable-field
electromagnet, the poles of which are shaped to cause a bending
of the path of the fragments through a specific angle, such as 90 °
• It is possible to vary the field strength of the magnet
• in such a way as to scan the magnetic field and to focus the ion
fragments of variable mass-to-charge ratio onto the detector slit
one at a time.
• In this way, specific fragments created at the electron beam can be
separated from other fragments and detected individually.
MASS ANALYZER
4. Magnetic Sector
MASS ANALYZER
4. Magnetic Sector
• It is employ a permanent magnet or an electromagnet to cause the
beam from the ion source to travel in a circular path, most
commonly of 180,90 or 60 deg.
• Will KE= z e V=1/2mv2 ( V= voltage between A and B , and e=
electronic charge =1.60 x 10-19) all ions have the same number of
charges z are assumed to have the same kinetic energy after
acceleration regardless of their mass.
• The heavier ions must travel through the magnetic sector at lower
velocities.
MASS ANALYZER

• Utility
Most complex method of mass analysis but most sensitive of the techniques in
common use today. Almost unlimited mass resolution > 106 is routinely
observable with resolutions in the 104 to 105 range
• Ion Trajectery
Ions drift into a spatially uniform static magnetic field of strength (B) that causes the
motion to become circular in a plane perpendicular to the direction of the magnetic
field. Within this ion-trap, the angular frequency (ωc) is inversly proportional to
the m/z value
MASS ANALYZER

• Ion Trajectery
The presence of ions between a pair of detector electrode (in the trapping
cell) will not actually produce any measurable signal. It is necessary to excite
the ions of a given m/z as a coherent package to a larger orbital radius, by
applying an RF sweep of a few milliseconds across the cell. One frequency
will excite one particular (Fourier transformation allows for all frequencies to
measure simultaneously)
Measurement of the angular frequency leads to values for m/z and thus to the
mass spectrum. Because frequency can be measured more accurately than
any other physical property, the technique has a very high mass resolution
MASS ANALYZER

DETECTOR
1. Electron Multiplier
• We have a discrete-dynode electron multiplier and a continuous – dynode

electron multiplier.
• Ion beam exiting the analyzer is accelerated to several thousand electron volts
prior to striking the first stage.
• An incident ion beam causes two electron to be emitted from the first dynode.
These electrons are accelerated to the second dynode where each causes two
more electrons (full in all) to be ejected.
• These in turn are accelerated to a third dynode and so on, eventually reaching,
say, a tenth dynode by which time the initial two electrons have become a
shower of 29 electrons.
DETECTOR
2. Faraday Cup
• Ion travelling at high speed strike the inside of the metal (faraday) cup and cause
secondary electron to be ejected. This production of electrons constitutes a
temporary flow of electric current until the electrons have been captured. The
faraday cup detector is simple and robust and is used in situations in which high
sensitivity is not required
• The transducer is aligned so that ions exiting the analyzer strike the collector
electrode.
• This electrode is surrounded by a cage that prevents the escape of reflected ions
and ejected secondary electrons.
• The collector electrode is inclined with respect to the path of the entering ions
so that particles striking or leaving the electrode are reflected a way from the
entrance to the cup.
DETECTOR
2. Faraday Cup
DETECTOR
3. Scintillator detector
• A fast ion causes electrons to be emitted and these are accelerated towards a
second dynode. In this case, the dynode consist of a substance (a scintillator)
wich emits photons (light). The emitted light is detected by a photomultiplier
and is converted into an electric current. Since photon multipliers are very
sensitive, high gain amplification of the arrival of a single ion is achieved. This
detectors are also important in studies on metastable ions.
MASS SPECTRA
The data collected by the data system is displayed as a Mass

spectrum , which is a plot of signal intensity vs. mass-to-charge ratio
MASS SPECTRA
• The key is that a given molecule always disintegrates into the

same fragments and therefore always displays the same mass
spectrum, a molecular fingerprint
• Because the fragmentation pattern produced by a mass

spectrometer can be used as a fingerprint of molecule, the mass
spectrum reveals, for example, whether the correct compound
has been synthesized and whether contaminants are present.
• Besides the obviously significant applicability to molecular

compound identification, mass spectrometry also finds
application in elemental analysis, such as to determine what
isotopes of an element might be present in a sample or,
combined with inductively coupled plasma, what the
concentration is of an element in a sample
ICP-MS
• The ICP source provides the mass spectrometer with a source of

charged monatomic ions such that the electron beam is not
needed. The “exhaust” of the ICP source is fed into the mass
spectrometer for analysis by the mass analyzer portion of the
mass spectrometer
• The intensity of the signal due to the analyte ion is proportional

to the concentration of the analyte in the sample.
• In most cases there will be more than one analyte ion for a given
element (due to the presence of more than one naturally
occurring isotope) and therefore more than one signal for that
element.
ICP-MS
• It may happen that more than one element in a sample will

produce a signal with a particular mass–charge ratio.
• There would therefore be a spectral interference if that signal

were to be measured.
• Nearly all elements, however, have at least one isotope mass that
no other element has.
• Thus, a signal with this spectral interference is ignored in favor of

one that is due to just one element.
ICP-MS
• The detection limits for elements analyzed by ICP–mass

spectrometry (ICP-MS) are significantly lower in most cases than
the detection limits for other atomic techniques
ICP-MS
GC-MS and LC-MS
• Chromatography instruments are instruments that separate and

quantitate samples of complex mixtures with surprising ease and
accuracy in a very short time.
• The separation occurs in a column, which is a tube that contains a

stationary material phase, either a liquid or solid, that selectively
impedes the progress of the mixture components as they move
through it.
• A mass spectrometer can be used as the detector system.
• Mass spectrometry offers a special advantage over other

detection schemes for instrumental chromatography because the
mass spectra of the mixture components are acquired, providing
a very powerful qualitative analysis tool for these components.
GC-MS and LC-MS
• When the mobile phase is a gas, the technique is called gas

chromatography–mass spectrometry (GCMS).
• In this case, the mixture is in the gas phase as it moves through

the column, and the individual separated mixture components
emerge in the gas phase and mix with the gaseous mobile phase,
whichn is usually helium.
• When the mobile phase is a liquid, or a mixture of liquids, the

technique is called liquid chromatography–mass spectrometry
(LC-MS).
• In this case, the mixture is in the liquid phase as it moves through

the column and individual separated mixture components
emerge dissolved in this liquid phase.
GC-MS and LC-MS
Reference
Nielsen S. Suzanne. (2017). Food Analysis. 05. Springer. New York. ISBN

978-3-319-45776-5
Nielsen S. Suzanne. (2017). Food Analysis Laboratory manual.
03. Springer. New York. ISBN 978-3-319-44127-6

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FOOD ANALYSIS

DEPARTMENT OF FOOD TECHNOLOGY

• LO1 : Demonstrate practical proficiency in a food

• Basic Principles of Spectroscopy

• The length of an electromagnetic wave is called its wavelength, and its

• Atoms in which there is an

• An absorption spectrum is a plot of the amount of light absorbed

• The amount of light absorbed is called the absorbance

• The absorption vs. wavelength information for molecules and

• In this case, the parameter that is plotted on the y axis is the

• Under certain conditions, molecular and ionic analytes present in

Under these specific conditions, the absorptivity is called the molar

• However, that the pattern of the absorption—the

• The complete absorption spectrum for a given absorbing

• Ultraviolet–visible (UV-VIS) molecular spectrochemical methods

• Qualitative analysis (identification of unknowns and detection of

• Quantitative analysis is accomplished with the use of Beer’s law

• If an instrument is meant strictly for visible light studies, then this

A monochromator is made up of three parts:

2. A dispersing element : disperses the light into its component

3. An exit slit : The narrow band of the spectrum is then selected by

• The sample compartment is an enclosure with a lid that can be

• When the lid is closed, the compartment should be relatively free

1. Single-beam instruments. In a single-beam spectrophotometer, the

Photomultiplier tubes or photodiodes (light sensors) are used as

Diode array : A diode array

• Fluorometry is an analytical technique that utilizes the ability of

• Luminescence is a phenomenon in which a substance appears to

• Luminescence caused by such a direct jump is called fluorescence.

• Luminescence resulting from a jump back to the ground state after

• Fluorometry and absorption spectrophotometry are competing

• As stated above, the number of chemical species that exhibit

• Thus we can say that while absorption spectrophotometry is

IR spectrometry differs from UV-VIS spectrophotometry in the

IR spectrometry differs from UV-VIS spectrophotometry in the

6. IR spectra are usually transmission spectra rather than absorption spectra,

8. Modern IR spectrometers do not use light dispersion to acquire spectra.

The advantages of FTIR over the dispersive technique are:

1) It is faster, making it possible to be incorporated into chromatography

• Sampling, in the context of infrared spectrometry, refers to the method by

• The region of the

• Infrared spectra are usually

• Quantitative analysis procedures using infrared spectrometry utilize Beer’s

• Once the percent transmittance or absorbance measurements are made

• Atomic spectroscopy refers to the absorption and emission of

Besides these differences in electronic energy levels and spectra, atomic

Besides these differences in electronic energy levels and spectra,

5. Spectral line sources are used as light sources in atomic absorption

The most important of the techniques and instrument designs

Flames and flame Process

1. Flames and flame Process

• Easily excited elements include sodium (yellow), calcium (orange), lithium

2. Spectral Line Sources

• Spectral line sources emit these wavelengths because they themselves

• 1. Hollow Cathode Lamp

1. Hollow Cathode Lamp

• The lamp itself is a sealed glass

2. Electrodeless Discharge Lamp (EDL)

• In this lamp, there is no anode or cathode. Rather, a small, sealed

• The tube is thus inductively coupled to an RF field, and the

• The characteristic line spectrum of the metal is thus generated

• The fuel and oxidant originate from pressurized sources, such as