SPECTROPHOTOMETRIC

METHODS
Maverick V. Sustiguer, RMT, MSMT
 LEARNING OUTCOMES
At the end of the session, the students must be able to:
a. Explain the basic terminologies about spectrophotometry;
b. List and discuss the basic components of a
spectrophotometer, and
c. Elucidate the working principle of different
spectrophotometric methods.
 DEFINITION OF TERMS
Transmitted via electromagnetic waves that are
ENERGY
characterized by their frequency and wavelength
Distance between two successive peaks
WAVELENGTH
A widely used unit in the visible spectrum is the
(λ) nanometer
FREQUENCY Is the number of vibrations of wave motion per second
(v) Number of oscillations of the waveform in a second
AMPLITUDE Length of the electronic vector at maximum peak height
Unit of frequency
HERTZ (Hz)
Corresponds to one cycle per second

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 ENERGY
FREQUENCY
WAVELENGTH

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 IR Light >700 nm
Visible light 300 to 700 nm
UV light <300 nm

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 Wavelength Light absorbed Complementary color
350 nm to 430 nm Violet Yellow-blue
431 nm to 475 nm Blue Yellow
476 nm to 495 nm Green-blue Orange
496 nm to 505 nm Blue-green Red
506 nm to 555 nm Green Purple
556 nm to 575 nm Yellow-green Violet
576 nm to 600 nm Yellow Blue
601 nm to 650 nm Orange Green-blue
651 nm to 700 nm Red Blue-green
 Increasing wavelength

Increasing frequency and energy

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 Increasing frequency and energy

Increasing wavelength
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 BEER–LAMBERT LAW
• Describes the relationship between absorption of light by a solution and the
concentration of the solution
• Concentration is directly proportional to the amount of light absorbed or
inversely proportional to the logarithm of the transmitted light
• Absorbance and percent transmittance are inversely related as given by the
following:

A = 2 - log%T
SAMPLE PROBLEM: A solution that has a transmittance of 2.3 %T
would have an absorbance of _______.

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 A=axbxc
Where:
A: absorbance (dependent on pH, concentration, temperature and
molecules and ions present)
a: molar absorptivity (function of specific wavelength of light absorbed
by a given type of molecule
b: length of light path
c: concentration of absorbing molecules

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 ABSORBANCE
CONCENTRATION
TRANSMITTANCE

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 SPECTROPHOTOMETER
• An instrument used to measure the light transmitted by a solution to determine the concentration
of the light-absorbing substance in the solution.
• Uses prisms or gratings to isolate a narrow range of the incident wavelength

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 LIGHT SOURCE
• AKA exciter lamp or radiant source
• Produces an intense, reproducible, constant beam of light
• Provide polychromatic light and must generate sufficient radiant
energy or power to measure the analyte of interest.

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TUNGSTEN OR TUNGSTEN-IODIDE Most common
LAMP Visible and near infrared regions
DEUTERIUM-DISCHARGE LAMP
DEUTERIUM LAMP Ultraviolet region
HYDROGEN LAMP
XENON LAMP
MERCURY ARC LAMP Visible and UV regions
SODIUM VAPOR LAMPS
NERNST GLOWER
Infrared region
GLOBAR (SILICON CARBIDE)

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 ENTRANCE SLIT
• Allows lamp light to enter
• Slit is fixed in position and size
• Minimizes unwanted or stray light and prevents the entrance of scattered
light into the monochromator

Stray light
• Wavelength outside the band (does not originate from the polychromatic light
source)
• Limits the maximum absorbance
• Most common cause of loss of linearity (high analyte concentration)
• Most common caused by second-order spectra, deteriorated optics, light
dispersed by a darkened lamp envelope, and extraneous room light.

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 MONOCHROMATOR
• Isolates monochromatic light, or at least as narrow a range of wavelengths
of light as possible, and direct it to the sample or to the photodetector
• Photometers: Glass filters and interference filters
• Spectrophotometer: Diffraction gratings and prisms

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 EXIT SLIT
• Controls the width of the light beam
• Allow only a narrow fraction of the spectrum to reach the
sample cuvet
• Selects the bandpass of the monochromator that allows
light of the selected wavelength to pass through the cuvet
onto the detector

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 BANDPASS
• AKA spectral bandwidth or half power point
or full width at half peak maximum
• Refers to the range of wavelengths passing
through the sample
• This means if wavelengths 550 and 560 nm
pass 50% of the maximum transmitted light,
the range of wavelengths between 550 and 560
nm represents a 10-nm bandpass
• Can be made smaller by reducing the width of
the exit slit
• Spectral purity is reflected by the bandpass
wherein the narrower the bandpass the
greater the resolution.

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 SAMPLE CONTAINERS (CUVET)
• AKA analytical cell, absorption cell, cuvette, sample cell
• Used to hold samples and must be made of material that is
transparent to radiation in the spectral region of interest
• May be round or square

Quartz/plastic cuvets Visible and UV radiation

Alumina silica glass Most common

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 PHOTODETECTORS
• Detects the amount of light that passes through the sample in the
cuvette
• Converts the transmitted radiant energy into an equivalent amount of
electrical energy
• The more light that is transmitted, the more energy, and the greater
the electrical signal that is measured.

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 • AKA photocell or photovoltaic cell
• Basic phototransducer that is used for detecting
BARRIER LAYER and measuring radiation in the visible region
CELL • Used mainly in photometers
• Composed of selenium on a plate of iron covered
with transparent layer of silver
• Contain a negatively charged cathode and
positively charged anode enclosed in a glass
case
PHOTOTUBE
• Has a photosensitive material that gives off
electrons when light energy strikes it
• Requires an external voltage for operation
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 Most common
Detects and amplifies radiant energy
PHOTOMULTIPLIER
200 times more sensitive than the
TUBE
phototube
10,000 times more sensitive than the BLC
Not as sensitive as PM tubes
Has excellent linearity, speed and smaller
size
PHOTODIODE
Has a lower dynamic range and higher
noise and most useful as a simultaneous
multichannel detector
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 READOUT DEVICE
• Electrical energy from a detector is displayed on some type of digital
display or readout system.
• The readout system may be a chart recorder or a computer printout.
• Displays output of the detection system
• Ex: Digital meters, d’Arsonval meters, recorders, light-emitting diodes
(LEDs), cathode-ray tubes (CRTs), and liquid crystal displays (LCDs).

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 Simplest type
SINGLE BEAM
Designed to make one
SPECTROPHOTO
measurement at a time at
METER
one specified wavelength
Splits the monochromatic
DOUBLE BEAM
light into two components
SPECTROPHOTO
(sample and to the reference
METER
solution)
1. Double beam in space: 2 photodetectors
2. Double beam in time: 1 photodetector with
chopper or rotating sector mirror

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 FLAME EMISSION PHOTOMETRY (FEP)
• Principle: Excitation of electrons from
lower to a higher energy state
• Measures the light emitted by a single
atom burned in a flame
• Light source: Flame
• Internal standard: Lithium or Cesium
(corrects variations in flame)
• Used for the measurement of excited
ions such as sodium and potassium

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 ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS)
• Principle: Element is not excited by merely
dissociated from its chemical bond and place in
a unionized, unexcited ground state
• Measures the light absorbed by atoms
dissociated by heat
• Used for the measurement of trace metals such as
calcium and magnesium
• It is more sensitive and specific than FEP
• Hollow-cathode lamp: light source
• Atomizer/nebulizer/graphite furnace: convert ions to
atoms; convert to fine aerosol
• Flame: remove water; breaks the ionic bond between
salts (ground state ions)
• Chopper: modulate light source

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 REFLECTOMETRY
• Used for the measurement of analytes in biological fluids
• Employed in urine dipstick analysis and dry slide chemical analysis

• Reflectometer: a filter photometer that measures the quantity of light

reflected by a liquid sample that has been dispensed onto a grainy or
fibrous solid support

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 MOLECULAR EMISSION SPECTROSCOPY

Types of luminescence where excitation Fluorometry

requires absorption of radiant energy Phosphorescence

Types of luminescence where excitation does Chemiluminescence

not require absorption of radiant energy Bioluminescence

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 FLUOROMETRY
(MOLECULAR LUMINESCENCE SPECTROSCOPY)
• Principle: Atoms absorb energy at a particular
wavelength wherein electrons are raised to
higher-energy orbitals and the electrons release
energy as they return to ground state by emitting
light energy of a longer wavelength and lower
energy than the exciting wavelength
• 1000 times more sensitive than absorption
techniques and has increased specificity

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 PHOSPHORESCENCE
• Emission of light produced by certain substances after they absorb
energy.
• Similar to fluorescence except that the time delay is longer (greater
than 10~4 sec) between absorption of radiant energy and release of
energy as photons of light.

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CHEMILUMINESCENCE
• A process where the chemical energy of a reaction produces excited
atoms, and upon electron return to ground state, photons of light are
emitted

BIOLUMINESCENCE
• A process where an enzyme-catalyzed chemical reaction produces light
emission.

LUMINOMETER: type of instrument that is used to measure

chemiluminescence and bioluminescence

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 REFRACTOMETRY
• Principle: When light passes from one medium into another, the light
beam changes its direction at the boundary surface if its speed in the
second medium is different from that in the first.
• The angle created by the bending of the light is called the critical
angle
• The ability of a substance to bend light is called refractivity
• The refractivity of a solution is an indirect measurement of total
solute concentration.
• Applications: measurement of protein concentration, specific gravity
of urine, and column effluent of HPLC

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NEPHELOMETRY
• Detects light that is scattered at various angles by a particulate solution
• Scattered light yields a small signal that must be amplified

TURBIDIMETRY
• Measures a reduction in light transmission due to particle formation
• It detects a small decrease in a large signal

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 SHORT QUIZ
All answers must be in capital letters.
Acronyms and abbreviations are not allowed.
Any form of alterations, superimpositions, and
erasures will invalidate your answer.
 IDENTIFICATION. Give what is asked.
1.This component of spectrophotometer prevents the entry of stray light.
2.This is the most common monochromator used in spectrophotometer.
3.This is the most common light source used in spectrophotometer.
4.This is the most common cuvet used in spectrophotometer.
5.This is the light source used in fluorometry.
6.This is the light source used in flame emission photometery.
7.This is the most common photodetector used in spectrophotometer.
8.This technique measures the reflected light.
9.This technique measures the reduction in light.
10.This equipment is used to measure bioluminescence.
 ELECTROPHORESIS
• The separation of charged compounds based on their electrical charge.
• When a voltage is applied to a salt solution (usually sodium chloride), an electrical
current is produced by the flow of ions: cations toward the cathode, and anions
toward the anode.
• Common support media: cellulose acetate, agarose, and polyacrylamide gels
• Once the electrophoresis is completed, the support medium is treated with a dye to
identify the separated fractions and the most common dyes include Amido Black,
Ponceau S, Fat Red 7B, and Sudan Black B.
• Densitometry: performed to obtain a quantitative profile of the separated
fractions, is performed on the stained support medium.
• Densitometer measures the absorbance of the stain on a support medium.

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ASSESSMENT OF NUCLEIC ACID CONCENTRATION,
YIELD, AND PURITY
Method Principle Interpretation
dsDNA: A260 X 50𝜇g/mL X DF*
ssDNA: A260 X 33𝜇g/mL X DF*
Nucleic acids absorb RNA: A260 X 40𝜇g/mL X DF*
UV light at 260 nm
Absorbance is directly proportional to the
Spectrophotometry concentration of the nucleic acid.
Proteins absorb light DNA A260/A280 ratio:
at 280 nm • <1.6: protein contamination
• 1.6-2.0: good quality of DNA
• >2.0: contamination with RNA

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