ANALYTICAL METHODS
AND
INSTRUMENTATION
FORENSIC INSTRUMENTATION 1
} Most determinations made in the
laboratory are based upon
measurements of radiant energy:
●Emitted
●Transmitted
●Absorbed
●Reflected under controlled
conditions
RADIANT ENERGY:
➢Energy travelling as electromagnetic
wave
➢Photons of energy traveling in a
wavelike manner
➢The shorter the wavelength, the
higher the electromagnetic energy.
TYPES OF ELECTROMAGNETIC
ENERGIES:
➢Cosmic rays –high penetrating power
➢Gamma rays & X-rays : shorter than
190 nm
➢ Visible
➢ Ultra-violet (UV)
➢ Infrared (IR)
➢Radio, TV, microwave
COLORIMETRY
➢Chemical reaction produces a colored substance
whose concentration is proportional to the analyte
◾Quality of Color
◾Intensity of Color
KINDS OF COLORIMETRY
1. Visual Colorimetry
2. Photoelectric Colorimetry
Photoelectric Colorimetry:
} Spectrophotometry – use prisms
or gratings to disperse the
source of light into a continuous
spectrum
} Filterphotometry – use filters to
isolate a narrow wavelength range
of the spectrum
Nature of Light
Light (white light):
➢form of radiant energy with
wavelength visible to the
naked eye
Wavelength
➢distance between 2 peaks as light
travels in a wavelike pattern
•Units: Ǻ ; mµ ; nm
1 nm = 1 mµ
= 10 Ǻ
= 10 -9 m
•Frequently, a white light source for the visible region or a deuterium source for ultraviolet (UV) light will provide the wavelengths used.
Kinds of wavelength
Wavelength (nm) Range
Below 340 Ultraviolet region
340 – 700 Visible Spectra
Above 700 Infrared region
Visible Light
Wavelength (nm) Color Absorbed Complementary/
Solution Color
340 – 430 Violet Yellow Green
430 – 475 Blue Yellow
475 – 495 Green-blue Orange
495 – 505 Blue-green Red
505 – 555 Green Purple
555 – 575 Yellow-green Violet
575 – 600 Yellow Blue
600 – 620 Orange Green-blue
Light Absorption & Transmittance
% Transmittance: = I x 100
IO
➢ ratio of the radiant energy transmitted
divided by the radiant energy incident on the
sample
Absorbance:
➢ the amount of light absorbed
Light Absorption & Transmittance
A = log 100
%T
A = 2 – log%T
Light Absorption & Transmittance
The amount of light absorbed is
dependent on:
1. The concentration of the
absorbing substance.
}
BEER’S LAW
The concentration of a
substance is directly
proportional to the amount of
light absorbed or inversely
proportional to the logarithm of
the transmitted light.
Light Absorption & Transmittance
2. The thickness or depth of a
solution; the length of light
path or the diameter of a
cuvet
BOUGUER’S OR LAMBERT’S LAW
➢
Absorbance is directly
proportional to length of light
path
Light Absorption & Transmittance
A = abc
Where: A = absorbance
a = molar absorptivity
b = light path of the solution
(cm)
c = concentration of
substance
A = abc c = A
ab
c = A ( 1 ) c = A (K)
ab
K = C *Unknown
A *Standard
Mathematical relationship between
absorption of radiant energy and the
concentration of a solution:
Cu = Au (Cs)
As
SPECTROPHOTOMETRY
❖ PRINCIPLE: Light passes through a monochromator
to provide a selection of desired region of the
spectrum to be used for measurements. Slits are used
to isolate a narrow beam of light and to improve its
chromatic purity. The light next passes through an
absorption cell (cuvet), where a portion of the
radiant energy is absorbed. The amount of energy
absorbed depends upon the nature and concentration
of the solution. Any light not absorbed is transmitted
to a detector (photocell or phototube), which
converts light energy to electrical energy that can be
registered on a meter or a digital read-out.
Internal Components of a
SPECTROPHOTOMETER
Light Entrance Monochro- Exit Cuvet Detector Meter
Source slit mator Slit
Internal Components of a
SPECTROPHOTOMETER
1. Light source
2. Entrance slit
3. Monochromator
4. Exit slit
5. Cuvet
6. Photodetector
7. Meter
Internal Components of a
1. SPECTROPHOTOMETER
Light source
➢ provides electromagnetic
radiation as visible, infrared or
UV light
➢ passes thru the
monochromator to be separated
into discrete wavelengths
Sources of radiant energy
▪Visible and near infra-red ranges:
➢ Tungsten
➢ Halogen quartz
▪Ultraviolet ranges :
➢ Vapor lamps (hydrogen lamp)
➢ Deuterium lamp
➢ Mercury lamp is (less desirable;
uneven emission spectrum)
➢ Tungsten-iodide (high intensity;
lasting)
➢ Xenon lamp - ideal for
applications requiring a narrow
slit
➢ not suited for routine
application owing to problems
resulting from stray light.
➢ Stray light: any wavelength
outside the band transmitted by
Internal Components of a
SPECTROPHOTOMETER
2. Entrance slit
➢ reduces stray light and
prevent scattered light from
entering the monochromator
system
Internal Components of a
SPECTROPHOTOMETER
Light Entrance Monocro- Exit Cuvet Detector Meter
Source slit mator Slit
Internal Components of a
SPECTROPHOTOMETER
3. Wavelength selector or
Monochromator
➢ A device that produces light
of specific wavelengths from a
light source
Internal Components of a
SPECTROPHOTOMETER
•TYPES of Monochromator :
1.Prisms : wedge-shaped pieces of
glass, quartz or sodium chloride.
❖ disperses light to form a
spectrum of colors due to
differing angles of refraction
by different wavelengths
Internal Components of a
SPECTROPHOTOMETER
•TYPES of Monochromator :
2. Diffraction gratings: device w/
small parallel grooves or slits
❖ These grooves act both as a
prism to refract white light and
as slit to diffract it into several
spectra.
Internal Components of a
SPECTROPHOTOMETER
•TYPES of Monochromator :
3. Transmission filters: colored
glass or gelatin sandwiched
between 2 plates of glass.
❖Light outside the transmission
band are absorbed by the
colored material
Internal Components of a
SPECTROPHOTOMETER
•TYPES of Monochromator :
4. Interference filters: semi-
transparent silver films on both
sides of a dielectric such as
MgF.
Internal Components of a
SPECTROPHOTOMETER
Light Entrance Monocro- Exit Cuvet Detector Meter
Source slit mator Slit
Internal Components of a
SPECTROPHOTOMETER
4. Exit slit
5. Analytical cell/Cuvette: holds the
solution in which the absorption is
to be measured.
• Etched to indicate the
position for use
• internal dimension : 1 cm
➢ Square cuvet
• has flat optical surfaces
•presents a flat surface to the
incident light: has less radiant
energy loss from reflection (w/
constant light path)
➢ Round cuvet
Internal Components of a
SPECTROPHOTOMETER
6. Photodetector - device that
measures light intensity by
converting light signal into
electrical signal
➢uses photosensitive materials in
their cathodes that release
electrons when they are exposed to
light energy.
Internal Components of a
SPECTROPHOTOMETER
Light Entrance Monocro- Exit Cuvet Detector Meter
Source slit mator Slit
TYPES of DETECTORS
a. Barrier-layer cells (Photovoltaic tube/
Photocell)
➢ generate their own electrical
output directly from light energy .
•negative electrode : selenium coated w/
Ag
•positive electrode : iron base
•spectral response : 380 to 700 nm
•found in older colorimeters and
spectrophotometers
b. Photomultiplier tube (PMT) – an
electron tube capable of significantly
amplifying current.
Advantages: - rapid response time
- very sensitive
Precautions: - All stray light & daylight
must be carefully shielded from the
photomultiplier to prevent burn out.
PRINCIPLE: (PMT)
•When radiant energy strikes the cathode,
the emitted electrons are attracted to the
adjacent dynode. Each electron then emits
several other electrons. The electrons
emitted from the first dynode are
subsequently attracted to the second dynode,
where the same emission cycle is repeated.
c. Phototube/Photoemissive tube
➢ same as PMT but electrons go
directly to the anode (with 2
electrodes); constructed to respond
to UV radiation
•Limitation : small amount of
photocurrent generated
d. Photoconductive tubes/Photo-
resistive tube
➢ a device whose electrical
resistance decreases as the level of
incident light is raised
➢ sensitive as barrier-layer cell
but do not require an external
power
➢Light sensitive materials (UV):
cadmium sulfide or cadmium selenide
Internal Components of a
SPECTROPHOTOMETER
7. Read-out device/Meter – where the
electrical energy from a detector is
displayed
◦ Direct reading system- the output of the
detector is used to drive a sensitive meter
directly without further amplification
◦ Null point system- the output of the
detector is balanced against that of a
reference circuit
Internal Components of a
SPECTROPHOTOMETER
7. Read-out devices
Digital read-out- numerical display of
absorbance or converted values of
concentrations
Microprocessors
DOUBLE BEAM SPECTROPHOTOMETERS
} DOUBLE-BEAM-IN-SPACE
} DOUBLE-BEAM-IN-TIME
DOUBLE BEAM IN SPACE
} Allcomponents are duplicated EXCEPT
light source
} Thetwo beams pass at the same time
through different components
} Compensate for changes in light
intensity
} Compensate for changes in absorbance
of the reagent blank as the wavelength
is changed in a scanning operation
DOUBLE-BEAM-IN-SPACE
DOUBLE BEAM IN TIME
} Uses the same components as a single-
beam instrument
} Duplicate of cuvet compartments
} Light beam chopper – rotating wheel
w/ alternate silvered sections
(inserted after the exit slit)
} Two beams pass through the same
components but NOT at the same time
DOUBLE-BEAM-IN-TIME
❖Justas a single-beam
instrument is adjusted to zero
absorbance w/ the blank before
& between sample readings, the
double-beam system makes this
adjustments automatically.
FLAME PHOTOMETRY/
FLAME EMISSION PHOTOMETRY
Principle Component Parts Use
Electrons are raised Atomizer, Na+, K+,
to a higher energy flame, air and Li+
level by heat from a gas supply,
flame. As they monochromator, Na: yellow
return to a ground detector, K : violet
state, they emit readout device
Li : red
radiation of a
Rb: red
characteristic
wavelength Mg: blue
FLAME PHOTOMETRY/FLAME EMISSION
PHOTOMETRY/FILTER PHOTOMETRY
ESSENTIAL PARTS OF THE FLAME
PHOTOMETER
1. Gases for Flame Photometry
} mixture of Hydrogen & Oxygen gas
} natural gas
} Acetylene
} Propane – for Na+ and K+ determinations
❖ flame temperature must be held
constant, otherwise sensitivity changes
will result
ESSENTIAL PARTS OF THE
FLAME PHOTOMETER
2. Burner Assembly
} Aspirator
} Atomizer/Nebulizer
} Flame
➢breaks the chemical bonds to produce
atoms
➢Source of energy absorbed by the atoms to
enter an excited state
ESSENTIAL PARTS OF THE
FLAME PHOTOMETER
❖ Internal standard: Lithium or Cesium
Advantages:
▪ high emission intensity
▪ absent from biological fluids/trace element
▪ emits wavelength different from Na & K
▪ Li acts as radiation buffer
Importance: to achieve stability
ESSENTIAL PARTS OF THE
FLAME PHOTOMETER
2 TYPES OF BURNERS OF A FLAME
PHOTOMETER
Total Consumption burner – gases are
passed at high velocity over the end of
a capillary suspended in the solution,
causing liquid to be drawn up through the
capillary into the flame
ESSENTIAL PARTS OF THE
FLAME PHOTOMETER
2 TYPES OF BURNERS OF A FLAME
PHOTOMETER
Laminar flow burner or Pre-mix burner:
gravitational feeding of solution through a
restricting capillary into an area of high-
velocity gas flow where small droplets are
produced & passed into the flame.
ESSENTIAL PARTS OF THE
FLAME PHOTOMETER
Monochromator (Interference filter)
} the narrowest band path
} maximum amount of the line emission
to pass through the detector
} Continuous emission - giving-off of
light of various wavelengths by non-
ionic materials
Atomic Absorption Spectrophotometry
Principle Component Parts Use
Measures light Hollow cathode Calcium,
absorbed by lamp (w/ argon or magnesium,
ground state Neon gas), copper,
atoms. atomizer, burner, zinc, lead
Band path monochromator,
width: 0.001 – detector, readout
0.01 nm device
Atomic Absorption Spectrophotometry
Difference FEP AAS
Light Source Flame Hollow Cathode
Lamp
State of atoms Excited Ground state
Energy Thermal Radiant
measured
Basis of Light Light absorbance
measurement emission
Sensitivity and Lesser greater
specificity
Interferences in AAS
1. Chemical interference
➢ failure of the flame to dissociate the
sample
2. Ionization interference
➢ excitation instead of dissociation
3. Matrix interference
➢ Interference on the Absorption by
evaporation of solvents or formation of solids
FLAMELESS AAS
} Useof carbon rod or graphite
furnace
} Heated until dried/charred
} More sensitive and permits
determination of trace metals in
small samples
Fluorescence Spectrophotometry
Principle Component Parts Use
Atoms absorb Energy source: Porphyrins,
light of a mercury or xenon hormones,
particular arc lamp, slits 1° amino acids,
wavelength and monochromator, vitamins,
emit light of a quartz cuvettes, 2° cathecolamines
longer monochromator,
wavelength detector, readout
(lower energy). device
FLUOROMETRY
} Fluorescence - an energy emission
that occurs when certain compounds
absorb electromagnetic radiation,
become excited and then return to
an energy level that is usually slightly
higher than their original level.
FLUOROMETRY
} Since the energy given off is less than
that absorbed, the wavelength of light
given off is longer than that absorbed
for excitation.
} PHOSPHORESCENCE
◦ Emitted energy is equal to or lower
than the absorbed energy.
Fluorescence Spectrophotometry
TURBIDIMETRY
} measurement of the light blocked
by a suspension of particulate
matter
} measurement of the reduction in
light transmission caused by
particle formation.
} Factors affecting turbidimetry:
◦ A. size and number of the
particles.
◦ B. the depth of the tube
◦ C. cross-sectional area of each
particle
USES of TURBIDIMETRY:
• Microbiology analyzers - measure turbidity
of samples to detect bacterial growth in
broth cultures
•measure the antibiotic sensitivity from
such cultures
•Coagulation analyzers - detect clot
formation in sample cuvets
•Clinical chemistry - quantify protein
concentration in biological fluids such as
urine and CSF
NEPHELOMETRY
} measurement of light scattered by
small particles at an angle to the beam
incident on the cuvette.
} more specific than turbidimetry.
} Immunoglobulins,complement,
immune complexes
NEPHELOMETRY
Refractometry
Principle Refractivity depends:
When light passes from one • wavelength of the
medium into another, the incident light
light beam changes its • Temperature
direction at the boundary • nature of the liquid
surface if its speed in the medium
second medium is different • concentration of the
from the first. solute dissolved in the
medium
Osmometry
Principle Component Parts Use
Determines Cooling bath, Serum and urine
osmolality based thermistor probe, osmolality
on freezing point stirring wire,
depression galvanometer
ELECTROCHEMISTRY
POTENTIOMETRY
➢ Measurement of differences in voltage at a
constant current.
➢The unknown voltage introduced into the
potentiometric circuit opposes the known
reference voltage.
➢Nernst Equation: relationship b/w
measured voltage & the sought for
concentration
POLAROGRAPHY
➢ measurement of differences in current
at a constant voltage
➢used to measure trace metals, oxygen,
vitamin C, and amino acids concentration
➢ILKOVIC Equation: relationship b/w the
difference in current and voltage
COULOMETRY
➢measurement of amount of electricity
(in coulombs) at a fixed potential.
➢Faraday’s Law: relationship of
coulombs consumed with the
concentration
CONDUCTOMETRY
➢measurement of the current flow between
two non-polarizable electrodes between
which a known electrical potential is
established.
AMPEROMETRY
} Measurement of the amount of
current that flows when a constant
voltage is applied to the measuring
electrode
CHROMATOGRAPHY
} Compounds are separated based on
differential distribution between a
mobile phase and a stationary phase.
} Mobile phase: gas or liquid
} Stationary phase: solid support
(coated or uncoated)
GENERAL TYPES OF
CHROMATOGRAPHY
A. ADSORPTION CHROMATOGRAPHY
} Molecules are adsorbed on the
surface of the solid support (non-
mobile)
B. PARTITION CHROMATOGRAPHY
} Solid support coated w/ a film of
water or non-volatile organic liquid
KINDS OF CHROMATOGRAPHY
1. Paper chromatography
} Partition is b/w water & organic solvent
} Immobile phase: cellulose paper/ H2O bound
to paper
} Mobile phase: organic solvent
} Rf = ratio of the distance travelled from
the origin to the distance travelled by the
solvent
} Rf = a/b
Where: a = distance travelled by
substance from origin
b = distance travelled by solvent
2. Thin Layer Chromatography
} Uses flat sheet of chromatographic paper
} Faster
} Compact spots easier to detect
Solid support: H2O bound to silica or
alumina
Mobile phase: organic solvent
3. Ion-Exchange Chromatography
} Separation is based on electrical charges
} Solid Phase: aluminum silicate
polysaccharide
synthetic resins –
polysterene beads
} Mobile Phase: water
4. Gel Filtration / Molecular Sieve
} Gel permeation; size exclusion; molecular
exclusion
} Solid Phase: Polyacrylamide (plastic)
Sephadex (crosslinked
polysaccharide)
Porous beads
} Mobile Phase: flowing water
KINDS OF CHROMATOGRAPHY
5. Gas Chromatography (Drug screening)
Two Phases:
} Stationary Phase: silica coated w/ a non-
volatile organic liquid
} Mobile Phase: Inert carrier gas (He or N)
▪ Gas-Solid: based on adsorption
▪ Gas-Liquid: based on partition
6. High Performance Liquid
Chromatography
▪Selective Adsorption with the
application of pressure
▪For high MW compounds
ELECTROPHORESIS
➢refers to the migration of charged solutes
or particles in a medium under the
influence of an electrical field.
➢Zone electrophoresis
➢Iontophoresis
➢ELECTROPHORETOGRAM – a display of
protein zones
ELECTROPHORETOGRAM
ELECTROPHORETOGRAM
ELECTROPHORETOGRAM
ELECTROPHORESIS
FACTORS AFFECTING MIGRATION:
➢ Net electrical charge of molecule
➢ MW of the particle
➢ Strength of the electrical field
➢ Properties of supporting medium
➢ Temperature of operation
PAPER ELECTROPHORESIS
ELECTROPHORESIS
ELECTROPHORESIS
SUPPORT MEDIA
1. Paper
2. Starch (starch gel)
3. Cellulose acetate
➢ greater resolution, faster &
permanent *
4. Agar (polysaccharide + agarose)
5. Polyacrylamide Gel
ELECTROPHORESIS
General Method:
➢ Place hydrated support material in an
electrophoresis chamber. Remove excess buffer
➢ Place support in contact with electrodes
➢ Apply constant voltage or current
➢ Remove and dry
➢ Add fixative to prevent diffusion
➢ Stain to reveal individual zones
AGAROSE GEL ELECTROPHORESIS
AGAROSE GEL ELECTROPHORESIS
BUFFER FIXATIVE
1. Barbital (pH 8.6) 1. Methanol
2. Cyclohexanol
2. Tris-Boric EDTA
3. Acetic acid
(pH 8.7)
4. Dioxane
ELECTROPHORESIS
Protein stains: - Amido black/Naphthol Blue Black
- Bromphenol blue
- Coomasie Brilliant blue
- Nigrosin
- Ponceau S 5% in TCA
Isoenzymes: - Nitrotetrazoleum Blue
Lipoproteins: - Fat Red 7B (Sudan Red)
- Oil Red O
- Sudan Black B
AUTOMATION
ADVANTAGES:
1. Rapid results
2. Increase in the number of tests performed
3. Saves time and effort
4. Eliminates the needs for personnel increase
5. Economical
6. Errors in calculations & transcriptions are reduced
7. Better precision & accuracy
