Physical and Chemical Methods

of Food Analysis
Assoc. Prof. Dr. PHAN TẠI HUÂN
Faculty of Chemical Engineering and Food Technology
Nong Lam University - HCMC

Lecture 4:
Spectroscopy

• Reading : Chapter 22, 23 and 24 in Book “Introduction to

the Chemical Analysis of Foods”, Nielsen, S. Suzanne,
1994, Jones and Bartlett Publishers, London.

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 Introduction
• Spectroscopy deals with the production,
measurement, and interpretation of spectra arising
from the interaction of electromagnetic radiation with
matter.
• Spectroscopic methods can be used for both
quantitative and qualitative analyses.
– based on the absorption or emission of radiation in the
ultraviolet (UV), visible (VIS), infrared (IR) frequency
ranges

Electromagnetic radiation
• The wave properties of electromagnetic radiation are described in
terms of the wave's frequency, wavelength, and amplitude.

Representation of plane-polarized electromagnetic radiation propagating along the x-

axis. The electric and magnetic fields are in phase, perpendicular to each other,4 and to
the direction of propagation.

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Properties of light

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 Electromagnetic radiation
• The wave is plane polarized in that the oscillating
electric and magnetic fields making up the wave are
each limited to a single plane.
• The frequency (ν) of a wave is defined as the number
of oscillations the wave will make at a given point
per second.
• The wavelength (λ) represents the distance between
successive maxima on any given wave.
• The wavenumbers (σ or ֿν ) are reciprocal
wavelengths in units of cm-1.

Electromagnetic radiation

E= hν = hc/λ
where:
– E = energy of a photon
– h = Planck's constant (6,6262.10-34 J s)
– ν =frequency of associated wave
– λ = wavelength of associated wave
– c = speed of light

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 Energy State of Matter
• The internal energy of an atom is described in terms
of its electronic energy levels.
Eatom = Eelectronic
• The internal energy of a molecule is dependent on its
electronic, vibrational, and rotational energies.
Emolecule = Eelectronic + Evibrational + Erotational

Eelectronic => UV-Vis

Evibrational => Infrared
Erotational => Microwave
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 Energy Level Transitions in Spectroscopy
Absorption of Radiation
• The absorption of radiation by an atom or molecule is
that process in which energy from a photon of
electromagnetic radiation is transferred to the
absorbing species.
• When an atom or molecule absorbs a photon of light,
its internal energy increases by an amount equivalent
to the amount of energy in that particular photon.

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Energy Level Transitions in Spectroscopy

Emission of Radiation
• Emission is essentially the reverse of the absorption
process, occurring when energy from an atom or
molecule is released in the form of a photon of
radiation.
• A molecule raised to an excited state will typically
remain in the excited state for a very short period of
time lime before relaxing back to the ground state.

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Energy Level Transitions in Spectroscopy

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UV-Vis Spectroscopy

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 UV-Vis Spectroscopy
• Spectroscopy in the ultraviolet-visible (UV-Vis)
range is one of the most commonly encountered
laboratory techniques in food analysis.
– The UV range: 200 - 380 nm
– The Vis range: 380 to 780 nm
• Spectroscopy utilizing radiation in the UV-VIS range
may be divided into two general categories
– Absorbance spectroscopy (more common)
– Fluorescence spectroscopy

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Transmittance (T)
T = I/Io = P/Po
%T = (P/Po) x 100
where:
• T = transmittance
• Po = radiant power of beam incident on absorption cell
• P = radiant power of beam exiting the absorption cell
• Io = radiant Intensity of beam incident on absorption cell
• I =radiant intensity beam exiting the absorption cell
• %T= percent transmittance

Io (Po) I (P)

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 Absorbance (A)
A = log(Po/P) = -logT= 2 - log%T
where:
– A =absorbance
– T = transmittance

• In quantitative spectroscopy, the fraction of the

incident beam that is not transmitted does not equal
the solution's absorbance.

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Beer's law
A = a.b.c
where:
– A =absorbance
– c = concentration of absorbing species
– b = path length through solution (cm)
– a = absorptivity

• Absorbance is directly proportional to the

concentration of the absorbing species in the solution.
(T and %T are not directly proportional to the
concentration)
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 Beer's law
• The absorptivity, a, of a given species is a
proportionaliry constant dependent on the molecular
properties of the species. The absorptivity is
wavelength dependent and may vary depending on
the chemical environment (pH, ionic strength,
solvent, etc.).
A = ɛ.b.c
• In case where the concentration of the analyte is
reported in units of molarity, the absorptivity term
has units of (cm)-1(M)-1 , designated by the symbol ɛ
which is referred to as the molar absorptivity.
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Example
• Caffeine in coffee and tea can be determine by its
absorption at 276 nm. The caffeine is first extracted
with dilute NH4OH and then saparated from other
materials on a Celite column eluted with CHCl3. If a
standard sample containing 0.5 mg caffeine/50 ml of
CHCl3 had a reading of 80%T, calculate the
concentration of caffeine in an unknown sample
solution with a %T of 60. 10-mm cells were used.

• Ans: 0,023 mg caffeine/ml CHCl3

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Reflection and scattering

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 Reflection and scattering

A= log(Psolvent / Panalyte solution) ≈ log(Po/P)

where:
– Psolvent = radiant power of beam exiting cell containing
solvent (blank)
– Panalyte solution = radiant power of beam exiting cell
containing analyte solution

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Deviations From Beer's Law

• Beer‘s law is applicable only to dilute solutions, up to
approximately 10 mM for most analytes.
– Increase analyte concentrations => decrease the
intermolecular distances => affect the charge
distribution of the other => affect the ability of the
analyte to capture photons of a given wavelength.

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 Procedural Considerations
• Sample preparation
• Homogenization and clarification => measured
directly
• Selection of reference solution for samples: sample
solvent, the solvent being water or an aqueous buffer
in many cases.
• In more complex situations, the analyte to be
quantified may need to be chemically modified prior
to making absorbance measurements.

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Equipment

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http://www.chemicool.com/img1/graphics/spectrometer1.gif

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Arrangement of component in a simple single-
beam, UV-Vis absorption

Radiation Wavelength Po P Detector

Source Isolator Sample

Readout
Device
Power Supply

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Schematic illustrating the property of

diffraction from a reflection grating

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Schematic illustrating the property of
diffraction from a reflection grating

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Schematic illustrating the property of

diffraction from a prism

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 Instrumentation

• Cuvette:
Squartz and
fused silica

Size:
1cm2 x 4,5cm

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Instrumentation
Detector

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 Instrumentation
Spectronic 20D

1. Wavelength Selection (C), 2. 0%T adjustment(A), 3. Cell compartment (E),

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4. 100%T adjustment (B), 5. Mode selection (D).
http://www.biology.lsu.edu/INTROBIO/tutorial/webtutorial/images/spec/spectrophotometer.jpg

Instrumentation

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Optimum wavelength

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'Complementary hue refers to the color observed for a solution
that shows maximum absorbance at the designated wavelength
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Calibration Curves

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(Skoog, 2004)

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(Skoog, 2004)

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 Relative Error

36,7%

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Relative Error

• Minimum relative error occurs 37%T.

• Satisfactory results can be obtained over the range of
15–65%T. This range corresponds to an absorbance
range of 0.82–0.19.
• Samples that are too concentrated (A > 0.82) should
be diluted to bring their absorbance values below 0.8.
• Samples that are too dilute (A < 0.19) should be
concentrated if possible, by evaporation or solvent
extraction.

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 A standard addition protocol

• In some cases, representative calibration standards

cannot be prepared due to the complexity of the
unknown sample.
– Interfering compounds absorb radiation in the same
spectral region as the analyte.
– Interfering compounds influence the absorbance of the
analyte.
– Interfering compounds react with modifying reagents that
are supposedly specific for the analyte.
• Calibration curves are potentially in error if the
unknown and the standards differ with respect to pH,
ionic strength, viscosity, types of impurities 51

A standard addition protocol

A=k[(VsCs+VuCu)/(Vt)]
where:
– Vs = volume of standard
– Vu= volume of unknown
– Vt=total volume
– Cs= concentration of standard
– Cu= concentration of unknown
– k = proportionality constant
(path length x absorptivity)

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 A standard addition protocol

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Spectrophotometric analysis of a mixture

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 Spectrophotometric analysis of a mixture

120.CA + 15.CB = 1,10

30.CA + 40.CB = 1,65

=> CA = 0,0044 (mol/l)

CB = 0,038 (mol/l) 55

Application of UV-Vis Spectroscopy

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 Application of UV-Vis Spectroscopy

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Application of UV-Vis Spectroscopy

Figure 3.18. Determination of protein concentration using a standard curve – the Bradford assay.
Coomassie brilliant blue has a λmax at 596 nm when it complexes with protein. A standard curve 58
is constructed with various known concentrations of bovine serum albumin.

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Application of UV-Vis Spectroscopy

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 Application of UV-Vis Spectroscopy

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Application of UV-Vis Spectroscopy

• Fig. UV–visible absorption spectra of the pigment, carminic acid,

flavokermesic acid and kermesic acid (A). Spectra for flavokermesic and
kermesic acid, showing the wavelengths at which the two spectra are most
different (B).
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Application of UV-Vis Spectroscopy

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Fluorescence Spectroscopy

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Arrangement of component in a
spectrofluorometer

Wavelength Po
Radiation P
Sample
Source Isolator
PF
Wavelength
Isolator
Power supply
Detector

Readout
Device 68

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 Fluorescence spectroscopy

PF = Φ(Po – P)
where:
• PF = radiant power of beam emitted from fluorescent
cell
• Φ = constant of proportionality
• Po = radiant power of beam incident on absorption
cell
• P = radiant power of beam exiting the absorption cell

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Fluorescence spectroscopy

A = log(Po/P) = ɛ.b.c
=> P = Po.10- ɛ.b.c

=> PF = Φ(Po - Po.10- ɛ.b.c)

=> PF = ΦPo (1 - 10- ɛ.b.c)
=> PF = Φ.Po.2.303 ɛ.b.c
=> PF = k.Po.c

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 Fluorescence spectroscopy

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Application of Fluorescence Spectroscopy

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 Application of Fluorescence Spectroscopy
EEMs of a virgin olive oil between λex = 300 and 400 nm, λem = 400 and 695
nm

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Application of Fluorescence Spectroscopy

EEMs of a pure olive oil between λex = 300 and 400 nm, λem = 400 and 695 nm

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 Application of Fluorescence Spectroscopy
Total component spectra of a virgin olive oil

λem = 600–
695 nm:
chlorophylls

λem = 400–
600 nm:
oxidation
products and
Vitamin E

(a) Fluorescence emission spectra between 400 and 695 nm; (b) between 400 75
and 600 nm. Continuous line: before oxidation; dotted line: after 3 h heating.

Infrared Spectroscopy

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 The IR Region of the Electromagnetic Spectrum

• Infrared radiation is electromagnetic energy with

wavelengths longer than visible light but shorter than
microwaves.
– Near-IR : 0.8 – 2.5 µm
– Mid-IR: 2.5 – 15 µm
– Far-IR: 15 – 100 µm
• The near- and mid-IR regions of the spectrum are
most useful for quantitative and qualitative analysis
of foods.

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Molecular Vibrations
• A molecule can absorb IR radiation if it vibrates in
such a way that its charge distribution, and therefore
its electric dipole moment, changes during the
vibration.
• The most important vibrations that produce a change
in dipole moment are stretching and bending
(scissoring) motions.

3652 cm-1 3756 cm-1 1596 cm-1

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 Factors Affecting the Frequency of Vibration
The energy level for any molecular vibration is given
by the following equation:

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Factors Affecting the Frequency of Vibration

• Hooke´s law:

• Where: ν is frequency and f is force constant (bond

strength)

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Factors Affecting the Frequency of Vibration

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Molecular Vibrations

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(Mid-infrared spectroscopy, Mid-IR)

• Mid-IR: 2,5 – 15 µm (Fundamental absorptions are

primarily observed ).
• Two types of spectrometers are routinely used for
mid-IR spectroscopy:
– Dispersive IR instruments ) (≈5% instruments).
– Fourier transform IR (FTIR) instruments.

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 Dispersive IR

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Fourier transform IR (FTIR)

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Fourier transform IR (FTIR)

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Fourier transform IR (FTIR)

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 Fourier transform IR (FTIR)

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Sample Handling Techniques

• Liquids are most commonly measured by
transmission IR spectroscopy.
• Cell windows with a path length of 0.01-1.0 mm
of halide or sulfide salts are most commonly used.
• Care must be taken when selecting cells for use
with aqueous samples or those with high moisture
content.
• Solvents commonly used for IR spectroscopy
include carbon tetrachloride, carbon disulfide,
methylene chloride, and some alkanes such as
hexane.
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 Sample Handling Techniques
Solid sample:
• Mixing about 1 mg of a finely ground (< 2µ mm
diameter) solid sample with about 100 mg
powdered dry potassium bromide. The mixture is
compressed under very high pressure (> 50,000
psi) in a vacuum to form a small disk about 1 cm
in diameter.
• Alternatively, a small amount of the powder, 2–4
mg, can be made into a thick slurry, or mull, by
grinding it with a few drops of a greasy, viscous
liquid, such as Nujol (a paraffin oil) or
chlorofluorocarbon greases.

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 Sample Handling Techniques
Gas samples:
• Transmission spectra can be obtained from gas
samples using a sealed 2-10 cm KBr cell with IR
transparent windows.
• For trace analysis, multiple-pass cells are used.
• FTIR instruments also can be interfaced to a gas
chromatograph. to obtain spectra of compounds
eluting from the chromatography column.

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Background Correction in Transmission Measurements

Solvent Absorption
• The solvent absorption spectrum is measured by
putting pure solvent in the liquid sample cell and
recording its spectrum.
Air Absorption
• Gaseous CO2 and H2O vapor are both strong IR
absorbers. Both occur in air in the optical light
path and contribute to any IR absorption signal
measured.
– The air spectrum may be recorded by running a
spectrum with no sample present (more common)
– The optical system can be purged with dry N2 or
argon, removing CO2 and H2O in the process.
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 IR Emission
• If the sample molecules are heated, many will occupy
excited vibrational states and will emit radiation upon
returning to the ground state.
• Advantage:
– sample can be remote (gases, flames and smokestacks)
– emission measurements are nondestructive
– do not suffer from atmospheric background problems
• Disadvantage:
– thick samples cannot be measured due to reabsorption
of the emitted radiation by cool parts of the sample.

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Applications of Mid-IR Spectroscopy

Qualitative Applications
• Identify specific functional groups present in an
unknown substance.
• A set of standard spectra are available from the
Sadtler Standard Spectra (Sadtler Division of Bio-
Rad, Inc., Philadelphia, PA).
• Common food applications include the
identification of flavor and aroma compounds,
particularly when FTIR measurements are coupled
with gas chromatography.

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Mid-IR Absorption Frequencies of Various
Organic Functional Groups

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 Applications of Mid-IR Spectroscopy
Quantitative Applications
• IR spectroscopic measurements obey Beer's law,
although deviations may be greater than in UV-
Vis spectroscopy due to the low intensities of IR
sources, the low sensitivities of IR detectors, and
the relative narrowness of mid-IR absorption
bands.
• Infrared Milk Analyzers, which have the ability to
analyze hundreds of samples per hour
– ester carbonyl groups of lipid: 5,73 µm
– amide groups of protein: 6,46 µm
– hydroxyl groups of lactose: 9,6 µm
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Near-Infrared, NIR
• NIR (0.8-2.5µm) are more widely used for
quantitative analysis of foods than are mid-IR
measurements.
• A major advantage of NIR spectroscopy is its
ability to measure directly the composition of
solid food products by use of diffuse reflectance
techniques.

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 Diffuse Reflectance (DR)

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Attenuated Total Reflectance

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 Reflectance (R)
R=I/Io
where:
– I= the intensity of radiation reflected from the
sample at a given wavelength.
– Io= the intensity of radiation reflected from the
reference at the same wavelength.
• Reflectance measurements are expressed as
differences, or derivatives, of the reflectance
values obtained from adjacent wavelengths
– (1/R)
– (logR2 – logR1)
– (2logR2 – logR1 – logR3) 107

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Quantitative Methods Using NIR Spectroscopy

• Because of the overlapping nature of the NlR

absorption bands, it is usually necessary to take
measurements at two or more wavelengths to
quantitate a food component reliably.

% constituent= z + a.log(1/R1) + b.log(1/R2) + c.log(1/R3)+ …

• Absorbance or derivatized reflectance data also can

be used in lieu of the log (1/ R) format.

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 Quantitative Methods Using NIR Spectroscopy

• Protein determination of flour :

• log(1/R2180) (absorbance at 2180 nm) is the key

absorption band for the protein.
• log(1/R2100) is absorbance at the characteristic
absorption wavelength of starch and works as an
adjusting term that eliminates any influence caused by
starch.
• Log(1/R1680 ) is the reference which eliminates any
effects caused by the particle size of the sample. 111

The IR spectra of a polyglycol with no water present and with 4%

water. The peak marked with an X is the O22H bend at 1640 cm -1
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that will be used for quantitation

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Standards of the polyglycol containing 0.5–8% water were scanned and the
spectra saved. The spectra of the standard solutions were collected using a
horizontal attenuated total reflectance (HATR) liquid sample cell using a ZnSe
crystal. All spectra were collected on a PerkinElmer Instruments Paragon1131000
FTIR spectrometer at 8 cm-1 resolution.

The data from (b) were input into the Spectrum ® Beer’s Law
quantitative analysis software. The calibration curve is shown.
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 Applications of NIR Spectroscopy to Food Analysis

• Measuring protein in wheat flour, and protein and oil in

soybeans.
• Measuring moisture, protein, and fat in red meats and
processed meat products, poultry, and fish.
• Measuring moisture and fat in butter.
• Measuring moisture, fat, and protein in cheese and egg.
• Measuring lactose, protein, and moisture in milk and
whey powders.
• Measuring total sugars and soluble solids in fruits and
vegetables.
• …
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FTIR spectra of codliver oil adulterated with lard at concentrations
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ranging from 0.5 to 50%

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Fig. 1 FTIR spectra of codliver oil, lard, mutton, beef, and chicken fats
at wavenumber region of 4,000 – 650 cm-1 119

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