Basic

Principles of
Spectroscopy
DE ALA, ERICK NOAH S.

MSFS
Spectroscopy
• A rapid, non-destructive test which focuses on the interaction of light and matter
(Tkachenko, 2006).

• It can be used for qualitative and quantitative analysis (Baeten, 2002).

• Provides a high-sensitivity measurements while does not require complex sample

preparation and calibration.

• Deals with the production, measurement, and interpretation of spectra arising

from the interaction of electromagnetic radiation with matter.
Light
• Refers to electromagnetic radiation of any
wavelength, whether visible or not.

• Possesses both: particulate and wavelike

properties.

• Phenomena associated with light propagation, such

as interference, diffraction, and refraction, are most
easily explained using the wave theory of
electromagnetic radiation.

• Whilst interaction of light with matter, which is the

basis of absorption and emission spectroscopy, may
be best understood in terms of the particulate nature
of light.
Light
• Wave properties of electromagnetic radiation, or
light are described in terms of the wave’s frequency,
wavelength, and amplitude.

• Frequency (v) of a wave refers to the number of

oscillations the wave will make at a given point per
second.
• Wavelength (λ) refers to the distance between
successive maxima on any given wave.
• Amplitude (A) represents the magnitude of the
electric vector at the wave maxima.
Light
• The term brightness is not commonly used in spectroscopy, instead the use of
Radiant power (P) or Radiant Intensity (I).

• Radiant power and Radiant intensity often are used synonymously when referring
to the amount of radiant energy striking a given area per unit time.
Light
Photons are particles of energy that move through space with wave-like properties.

The energy a photon possess is defined in terms of the frequency of a wave as

shown in the equation;

E = energy of a photon
h = Planck’s Constant
v = frequency of associated wave
The Electromagnetic Spectrum
Energy States
Atoms and molecules, under normal conditions, exist predominantly in the ground
state, which is the state of lowest energy.

Ground state atoms and molecules can gain energy, in which case they will be
elevated to one of their higher energy states, referred to as excited states.
Energy States
Atoms and molecules, under normal conditions, exist predominantly in the ground
state, which is the state of lowest energy.

Ground state atoms and molecules can gain energy, in which case they will be
elevated to one of their higher energy states, referred to as excited states.

Consequently, there will be specific “allowed” internal energy levels for each
atomic or molecular species.
Energy States
The set of available energy levels for any given atom or molecule will be distinct for that
species. Likewise, the potential
energy spacing's between allowed internal energy levels will be
characteristic of a species.

The potential energy spacing's between allowed internal energy levels will be characteristic of a
species.

Qualitative absorption and emission spectroscopy make use of this phenomenon in that these
techniques attempt to determine an unknown compound’s relative energy spacing by measuring
transitions between allowed energy levels.
Energy Level Transitions in Spectroscopy
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 matter absorbs a photon, its internal energy

increases by an amount
equivalent to the amount of energy in that particular
photon.

Thus, in the process of absorption, the species goes

from a lower energy state to a more excited state.
Energy Level Transitions in Spectroscopy
The emission of radiation is essentially the reverse of
absorption, 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 before relaxing back
to the ground state

There are several relaxation processes through which an excited

molecule may dissipate energy. The most common relaxation
process is for the excited molecule to dissipate its energy
through a series of small steps brought on by collisions with
other molecules.
Energy Level Transitions in Spectroscopy
However in some cases, molecules excited by the
absorption of UV or Vis light will lose a portion of
their excess energy through the emission of a photon.
This emission process is referred to as either
fluorescence or phosphorescence, depending on the
nature of the excited state.
Ultraviolet & Visible Light
(UV-Vis) Spectroscopy
UV-Vis Spectroscopy
• Most commonly encountered laboratory techniques in food analysis.

• Allows vast forms of analysis such as quantification of macronutrients,

micronutrients, estimation of rancidity, surveillance testing (enzyme-
linked immunoassays).
UV-Vis Spectroscopy
Ultraviolet
• Colorless to a human observer.
Visible Light
• Distinct color is observed at different
wavelengths (ranging from violet at the
short wavelength to red at the longer
wavelength of the spectrum).
UV-Vis Spectroscopy
• Electromagnetic radiation in the
Ultraviolet range (UV) portion of the
spectrum ranges in wavelength from
approximately 200 to 700 nm.

• Electromagnetic radiation on Visible

Light Range (Vis) portion of the
spectrum ranges in wavelength from
350 to 700 nm.
Basis of Quantitative Absorption Spectroscopy
• Determine the concentration of analyte in a given sample solution.

• Measurement is based on the amount of light absorbed from a reference beam as

it passes through the sample solution.

• The presence of analyte in the solution will affect the amount of radiation
transmitted through the solution thus, the relative transmittance or absorbance of
the solution may be used as an index of analyte concentration.
Basis of Quantitative Absorption Spectroscopy

• The solution to be analyzed is contained in an absorption cell

and placed in the path of radiation of a selected wavelength(s).

• The amount of radiation passing through the sample is then

measured relative to a reference sample.

• The relative amount of light passing through the sample is then

used to estimate the analyte concentration.
Po P
Basis of Quantitative Absorption Spectroscopy

The transmittance (T) of a solution is defined as the ratio of

P to Po, and often is expressed as percentage as shown;

Where;
%T – percentage Transmittance
Po – radiant power of beam incident
Po P
P – Radiant power exiting the absorption cell
Basis of Quantitative Absorption Spectroscopy
• It is important to note that Transmittance is not necessarily directly proportionate
to the concentration of the absorbing analyte in the sample solution.

• Thus, a second tem is used describe the relationship between P and Po, the
absorbance (A).
Basis of Quantitative Absorption Spectroscopy
Absorbance is defined with respect to Transmittance (T) as shown in the equation;

Absorbance is a convenient expression in that, under appropriate conditions, it is

directly proportional to the concentration of the absorbing species in the solution.
Basis of Quantitative Absorption Spectroscopy
The relationship between the absorbance (A) and the concentration of the absorbing
species is defined in Beer’s Law.

The law states that the absorptive capacity of a dissolved substance is directly
proportional to its concentration in a solution, thus shown in the equation;

A = Absorbance
a = absorptivity
Ɛ = molar absorptivity
b= path length through solution
c = concentration of absorbing species
Basis of Quantitative Absorption Spectroscopy
• Absorptivity (a) of a given species is a proportionality constant dependent on the
molecular properties of the species.

• It is wavelength dependent and may vary depending on the chemical

environment (pH, ionic strength, solvent, etc.) the absorbing species is exposed
to.

• In the special case where the concentration of the analyte is reported in units of
molarity. In these conditions, the designation of the symbol Ɛ, referring to the
molar absorptivity is used.
Basis of Quantitative Absorption Spectroscopy
• Scattering of Radiation Beam as it passes through an
absorbing solution is a major factor to the reduction of
beam radiation.

• In practice, a reference cell is used to correct for these

processes. A reference cell is one that, in theory, exactly
matches the sample absorption cell with the exception
that it contains no analyte.

• A reference cell often is prepared by adding deionized

distilled water to an absorption cell (cuvette).
Basis of Quantitative Absorption Spectroscopy
• This procedure assumes that all processes except the selective absorption of
radiation by the analyte are equivalent for the sample and reference cells.

• The absorbance measured in the laboratory is approximated by the equation;

P solvent – Radiant power exiting cell containing blank solvent

P analyte solution – Radiant power exiting cell containing analyte
solution.
Procedural Considerations to Minimize Experimental
Errors
Calibration Curves

• Calibration curves are used in Food Analysis

to establish the relationship between analyte
concentration and absorbance.
• They are established experimentally through
the analysis of a series of samples of known
analyte concentration.
Calibration Curves

• Linear Calibration Curves are expected and

preferred for systems obeying Beer’s Law.

• In the case of Non-Linear Calibration

Curves, it is caused by slight inconsistencies
to the change in absorbance per unit change in
analyte concentration. This is often pointed
due to concentration-dependent changes, or
some instrument limitations.
Calibration Curves

• Interfering Compounds, are substances that

absorb radiation, in the same spectral region
as the analyte.

• Influences the absorbance of the analyte.

• Substances that reacts to the modifying

reagents that are supposedly for the analyte.
Calibration Curves

• Differences of standards and unknown with

respect to pH, ionic strength, viscosity, types
of impurities, may cause errors to the
calibration curve.

• To avoid these errors, the assay system is

calibrated using the standard addition
protocol.
Calibration Curves
Steps to Standard Addition Protocol

Step 1: To a series of flasks, add a constant

volume of the unknown (Vu), for which you are
trying to determine the analyte concentration
(Cu).

Step 2: to each individual flask add a known

volume (Vs) of a standard analyte solution of
concentration Cs, such that each flask receives a
unique
volume of standard.

Step 3: Dilute all flasks to the same total volume,

(Vt)
Calibration Curves
Steps to Standard Addition Protocol

Step 4: Each of the Flask is then assayed, and is

treated identically.

• Referring to the Beer’s law, it is then

concluded that the measured absorbance of
each of the flask will be proportional to the
total analyte concentration.
Calibration Curves
Adjusted Absorbance is then calculated using the equation;

Where;
Vs – Volume of standard
Vu – Volume of the Unknown
Vt – Total Volume
Cs – Concentration of Standard
Cu – Concentration of Unknown
k – Proportionality constant (path length x absorptivity)
Calibration Curves
• The result from the assay is then plotted with the
volume of standard added to each of the flask (Vs)
as the independent variable and the resulting
Absorbance (A) as the dependent Variable.

• Assuming Beer’s law, the line describing the

relationship will be expressed in the equation;
 Calibration Curves
Calculation of Slope, intercept and is done by using the equations provided;
 UV-Vis Spectrophotometer
There are many variations of spectrophotometers available for UV–Vis
spectrophotometry.

A basic spectrophotometer is composed of five essential components: the light

source, the monochromatic, the sample/reference holder, the radiation detector,
and a readout device.
Basic Components of a Spectrometer
 UV-Vis Spectrophotometer
1. Light Source
• Must consistently emit a strong band of radiation encompassing the entire
wavelength range for which the instrument is designed.
• Tungsten Filament Lamp is the most common radiation source for Visible
Light spectrophotometers, capable of emitting radiation from 350nm to
2500nm.
• For Ultraviolet radiation, the deuterium-discharge lamps are used,
providing continuous radiation spectrum from 160nm to 375nm.
 UV-Vis Spectrophotometer
2. Monochromator
• The component that functions to isolate the specific, narrow, continuous
group of wavelengths to be used in the spectroscopic assay.
• The monochromator is so named because light of a single wavelength is
termed monochromatic, however visible radiation are polychromatic in
nature.
• Polychromatic radiation from the source enters the monochromator and is
dispersed according to wavelength, and monochromatic radiation of a
selected wavelength exits the monochromator.
 UV-Vis Spectrophotometer
2. Monochromator
 UV-Vis Spectrophotometer
3. Detector
• It is where the light transmitted through the reference or sample cell is
quantified.

• An ideal detector would;

 give a signal directly proportional to the radiant power of the beam
striking it;
 have a relatively consistent response to light of different wavelengths,
such that it was applicable to a wide range of the radiation spectrum.
 UV-Vis Spectrophotometer
3. Detector
 UV-Vis Spectrophotometer
4. Readout Device/Display

• The signal from the detector is generally amplified and then displayed in a
usable form to the analyst.
• In the simplest case, the analog signal from the detector is displayed on an
analog meter.
• Analog readouts are adequate for most routine analytical purposes; however,
analog meters are somewhat more difficult to read and, hence, the resulting
data are expected to have somewhat lower precision.
 UV-Vis Spectrophotometer
4. Readout Device/Display

• Digital readouts express the signal as numbers on the face of a meter. In

these cases, there is an obvious requirement for signal processing between the
analog output of the detector and the final digital display.
• Many of the newer instruments include microprocessors capable of more
extensive data manipulations on the digitized signal.
Fluorescence Spectroscopy
Fluorescence Spectroscopy
• In fluorescence spectroscopy, the signal being measured is the electromagnetic radiation
that is emitted from the analyte as it relaxes from an excited electronic energy level to its
corresponding ground state.

• This technique is to be far more sensitive than Absorption Spectroscopy methods.

Fluorescence Spectroscopy

• In fluorometers and spectrofluorometers, there is a

need for two wavelength selectors, one for the
excitation beam and one for the emission beam.

• The photon detector of fluorescence

instrumentation is generally arranged such that the
emitted radiation that strikes the detector is
traveling at an angle of 90◦ relative to the axis of
the excitation beam
Fluorescence Spectroscopy
• This detector placement minimizes signal
interference due to transmitted source radiation and
radiation scattered from the sample.
Fluorescence Spectroscopy
The Radiant Power (I) of fluorescence beam emited from a fluorescent sample is proportional
to the change in radiant power of the source beam as it passes through the sample cell as shown
in the equation;

Where;
Pf = radiant power of beam emitted from
fluorescent cell
Φ = constant of proportionality
Po = radiant power entering the sample cell
P = radiant power exiting the cell
Fluorescence Spectroscopy
The Radiant Power (I) of fluorescence beam emited from a fluorescent sample is proportional
to the change in radiant power of the source beam as it passes through the sample cell as shown
in the equation;

Where;
Pf = radiant power of beam emitted from
fluorescent cell
Φ = constant of proportionality
Po = radiant power entering the sample cell
P = radiant power exiting the cell
Fluorescence Spectroscopy
The constant of proportionality used is termed the quantum efficiency (Φ). The quantum
efficiency equals the ratio of the total number of photons emitted to the total number of photons
absorbed.

the combining of transmittance and the Beer’s Law equations, we are able to define P (as
analyte concentration) and Po as shown in the equation;

Substituting this equation to the previous Pf equation results to;

Fluorescence Spectroscopy
Such derived equation is very valid given that the
analyte concentrations are kept very low. The
equation also emphasizes two important points:

• First, the fluorescent signal will be directly

proportional to the analyte concentration,
assuming other parameters are kept constant.

• Second, the sensitivity of a fluorescent assay is

proportional to Po, the power of the incident
beam, the implication being that the sensitivity of
a fluorescent assay may be modified by adjusting
the source output.
Fluorescence Spectroscopy

• This equation will eventually decay if

analyte concentrations are increased to
relatively high values.

• Thus, the linear concentration range for each

of the analysis must be done experimentally.
Fluorescence Spectroscopy

• This equation will eventually decay if

analyte concentrations are increased to
relatively high values.

• Thus, the linear concentration range for each

of the analysis must be done experimentally.
Fluorescence Spectroscopy

• For the Calibration curve of a fluorescence

assay; The nonlinear portion of the curve at
relatively high analyte concentrations results
from decreases in the fluorescence yield per
unit concentration.

• Temperature, solvent, impurities, and pH

may influence the fluorescence yield per
unit of concentration.
Fluorescence Spectroscopy

• Thus, it is vital that these environmental

parameters be accounted for in the
experimental design of fluorescence assays.
End of Report Presentation
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