INSTRUMENTATION – II

By: Dwi Priyadi Djatmiko

Moderator: dr. I Putu Adi S.,Sp.PK
INTRODUCTION
Instrumentation & Analytic techniques

Spectrophotometry Chromatography

Electrophoresis

Foundation of all measurements made in

modern clinical chemistry laboratory
SPECTROPHOTOMETRY
DEFINITIONS
PHOTOMETRY
The measurement of light

SPECTROPHOTOMETRY
The measurement of the intensity of light at selected
wavelengths
BASIC CONCEPTS
BEER’S LAW
The concentration of a substance is directly
proportional to the amount of the light absorbed or
inversely proportional to the logarithm of the
transmitted light

Percent transmittance (%T)

Ratio of the radiant energy transmitted (T) divided by
the radiant energy incident on the sample (I)
BASIC CONCEPTS
Percent transmittance (%T)
BASIC CONCEPTS
Absorbance (A)
The amount of the light absorbed
Cannot be measured directly by a spectrophotometer,
but is mathematically derived from %T

%T = I/I0 x 100
I0 = Incident light

A = -log(I/I0) = log (100%) – log %T =

2 – log %T
BASIC CONCEPTS
According to Beer’s law, absorbance is directly
proportional to concentration

A= εxbxc
ε = molar absorptivity
b = the length of light path through the solution
c = the concentration of absorbing molecule
TYPES & COMPONENTS
BASIC TYPES
1. Single Beam Spectrophotometer
2. Double Beam Spectrophotometer

COMPONENTS
1. Light source 5. Photodetector
2. Monochromator 6. Readout device
3. Fiber optics 7. Recorder
4. Cuvets 8. Microprocessor
SINGLE BEAM SPECTROPHOTOMETER
DOUBLE BEAM SPECTROPHOTOMETER
LIGHT SOURCE
1. Incadescent Lamps
• Tungsten light bulb
– acceptable for moderately dilute solutions
– doesn’t supply sufficient radiant energy for
measurements below 320 nm
• Hydrogen lamp
• Deuterium lamp
• Hollow cathode lamp
LIGHT SOURCE
2. Laser
A device that controls the way that energized atoms
release photons
– have spatial coherence
– produce monochromatic light
– have pulse width that vary from microseconds to
picoseconds or less
Example: Argon, Helium-cadmium, Laser diode, etc
MONOCHROMATOR
A system for isolating radiant energy of a desired
wavelength and excluding that of other wavelengths
 Selection depends on the analytical purpose
• Filters
– a thin layer of colored glass
– narrow-bandpass types
– sharp-cutoff types
MONOCHROMATOR
• Prisms
Separates white light into a continuous spectrum by
refraction with shorter wavelengths being bent/refracted
more than longer wavelengths as they pass
• Diffraction gratings
Prepared by depositing a thin layer of alumunium-copper
alloy on the surface of a flat glass plate  ruling many
small parallel grooves into the metal coating
FIBER OPTICS
Bundles of thin, transparent fibers of glass, quartz, or
plastic that are enclosed in material of a lower index of
refraction and transmit light throughout their lengths by
internal refraction

 Advantage
Better directional control of the beam of light within
the geometrical confines of an instrument
FIBER OPTICS
 Disadvantages
– greater amounts of stray light
– refractive index changes in the glass, quartz, or
plastic rods
– the loss of transmitted energy after continued use in
the UV region of the spectrum (solarization)
CUVETS
A small vessel used to hold a liquid sample to be analyzed
in the light path of a spectrometer
 May be round, square, or rectangular
 Constructed from glass, silica, or plastic
PHOTODETECTOR
Device that convert light into an electric signal that is
proportional to the number of photons striking its
photosensitive surface
• Photomultiplier Tubes
– contains a cathode, a light-sensitive metal, and a
series of dynodes all of which enclosed in an
evacuated glass enclosure
– excellent sensitivity, rapid response, slow to fatigue
PHOTODETECTOR
• Photodiodes
– solid-state photodetectors that are fabricated from
photosensitive semiconductor materials  absorb
light over a characteristic wavelength
– capable of measuring light at a multitude of
wavelengths
READOUT DEVICES
Displaying electrical energy from a detector
 Operate on the principle of selective illumination og
portions of a bank of light-emitting diodes (LEDs),
controlled by the voltage signal generated
• Analog devices
• Digital readout devices
RECORDER
May be equipped in a spectrophotometer in addition to or
instead of a digital display
 Synchronized to provide line traces of transmittance or
absorbance as a function of either time or wavelength
MICROPROCESSORS
Along with software:
– Digitally store output from a calibrator
– Substracted digital signals from blanks from calibrators
& unknowns
– Automatically calculate the concentration of unknowns
– Convert kinetic data into concentration or enzymatic
activity
VARIATIONS
• Reflectance Photometry
– Measured diffuse reflected light
– The intensity of the reflected light from reagent carrier
is compared with the intensity of light reflected from a
reference surface
• Flame Emission Spectrophotometry
Based on the characteristic emission of light by atoms of
many metallic elements when given sufficient energy,
such as that supplied by a hot flame
VARIATIONS
• Atomic Absorption Spectrophotometry
An emission technique in which an element in the
sample is excited and the radiant energy given off
measured as the element returns to its lower energy
level
ELECTROPHORESIS
DEFINITIONS & BASIC CONCEPTS
Electrophoresis
The migration of charged solutes or a particle in a liquid
medium influenced by electrical field
• Positive ion (cation)  cathode (negative electrode)
• Negative ion (anion)  anode (positive electrode)

Electropherogram
A densitometric display of protein zones
DEFINITIONS
Zone Electrophoresis
The migration of a charged molecules, usually in a
porous supporting medium like agarose gel film, such
that each protein zone is sharply separated from
neighboring zones by a protein free area
BASIC CONCEPTS
Electrophoretic mobility (μ)
• the rate of migration (cm/s) per unit field strength (V/cm)
• Depend on:
– molecule’s net electrical charge; size & shape
– electrical field strength
– properties of the supporting medium
– temperature of operation
– Endosmotic flow & wick flow
BASIC CONCEPTS
Electrophoretic mobility (μ)
μ = Q/K ↔ r ↔ n
Q = net charge of particle r = ionic radius of the particle
K = constant n = viscosity of the buffer

The rate of migration:

directly proportional to the net charge of the particle
inversely proportional to particle’s size & buffer’s viscosity
COMPONENTS
1. Power Supplies
2. Buffers
3. Support Media
– Starch gel – Polyacrylamide gel
– Cellulose acetate – Agarose
ELECTROPHORESIS APPARATUS
POWER SUPPLIES
Drive the movement of ionic species in the medium and
allow adjustment and control of either the current or the
voltage
 In more sophisticated units, the power may be
controlled as well and conditions may be programmed
to change during electrophoresis
BUFFERS
Carry the applied currents and fix the pH at which
electrophoresis is carried out
 determine the kind of electrical charge on the solute
 determine the electrode toward which the solute will
migrate
SUPPORT MEDIA
Provides the matrix in which protein separation takes place
• Starch gel
– prepared from partially hydrolyzed native starch
– to separate macromolecular ions on the basis of both
charge-to-mass ration & molecular size
– preparation is relatively difficult & requires
considerable skill
SUPPORT MEDIA
• Cellulose acetate
– thermoplastic resin made by treating cellulose with
acetic anhydride to acetylate the hydroxyl groups
– need to presoaked before use & the strips need to be
cleared before densitometry
• Polyacrylamide gel
– Thermostable, transparent, strong, relatively
chemically inert gel that can be made in a wide range
of pore sizes
– Need caution  potential carcinogenicity
SUPPORT MEDIA
• Agarose
– a purified, esentially neutral fraction of agar obtained
by separating agarose from agaropectin
– to separate serum, urine, or CSF proteins, Hb
variants, isoenzymes, lipoproteins, etc
– Advantages: lower affinity for proteins, native clarity
after drying, exhibits little endosmosis
GENERAL OPERATIONS
• Electrophoretic Separation
• Detection
• Quantification
• Blotting Techniques
ELECTROPHORETIC SEPARATION
1. The excess buffer is first removed from the support
surface by blotting
2. 5 – 7 μL of sample is applied, allowed to diffuse into the
gel, then blotted to remove the excess
3. The gel is placed into the electrode chamber
4. Electrophoresis is performed at specified
current/voltage/power
5. The gel is rinsed, fixed, then dried
6. The gel is stained, then redried
7. The gel is scanned in a densitometer
DETECTION
• Staining
– Amido Black – Oil Red
– Coomasie Brilliant Blue – Sudan Black
– Nitrotetrazolium Blue – Ethidium Bromide
– Fat Red – Silver Nitrate
• Combination of a stain molecule & an antiglobulin
QUANTIFICATION
Densitometer
An instrument that measures the absorbance of each
fraction as the medium is moved past a photometric
optical system and displays an electropherogram on a
recorder chart or computer display

Reliable densitometric quantitation requires:

– Light of an appropriate wavelength
– A linear response from the instrument
– A transparent background in the medium being scanned
DENSITOMETER
BLOTTING TECHNIQUES
• Southern Blotting
to separate and detect DNAs or fragment of DNA
• Northern Blotting
to separate and detect RNAs
• Western Blotting
to separate and detect proteins
TYPES OF ELECTROPHORESIS
• Slab Gel Electrophoresis
– using a rectangular gel regardless of thickness
– Able to simultaneously separate several samples in
one run
• Disc Electrophoresis
– discontinuities in the electrophoretic matrix caused by
layers of gel that differ in composition and pore sizes
– was developed to improve the condition of which
protein electrophoresis using agarose gel yields only 5
zones
TYPES OF ELECTROPHORESIS
• Isoelectric Focusing Electrophoresis (IEF)
– Separates amphoteric compounds with increased
resolution in a medium possesing a stable pH
• Isotachophoresis (ITP)
– Completely separates smaller ionic substances into
adjacent zones that contact one another with no
overlap, and all migrate at the same rate
TYPES OF ELECTROPHORESIS
• Pulsed-Field Electrophoresis
– Power is alternately applied to different pairs of
electrodes or electrode arrays, so the electrophoretic
field is cycled between two directions
• Two-Dimensional (2-D) Electrophoresis
– Combining charge-dependent IEF in the first
dimension with molecular weight-dependent
electrophoresis in the second dimension
TYPES OF ELECTROPHORESIS
• Capillary Electrophoresis
– The classic techniques of zone electrophoresis, ITP, IEF,
and gel electrophoresis are carried out in a small-bore
fused silica capillary tube of 20 – 200 cm in length
– Advantages: able to apply much higher voltages than
in traditional electrophoresis and the ease of
automation
CAPILLARY ELECTROPHORESIS
CHROMATOGRAPHY
DEFINITIONS
CHROMATOGRAPHY
A physical process whereby components (solutes) of a
sample mixture are separated by their differential
distribution between stationary and mobile phases

The mobile phase

Carries the complex mixture (sample)
The stationary phase
Through which the mobile phase flows
BASIC CONCEPTS
The mobile phase carries the sample through a
bed/layer/column containing the stationary phase

↓↓↓
The solutes may:
1. Reside only on the stationary phase (no migration)
2. Reside only in the mobile phase (migration with the
mobile phase)
3. Distribute between the two phases (differential
migration)
FORMS OF CHROMATOGRAPHY

Chromatography

Planar Column

Paper Thin Layer Gas Liquid

PAPER CHROMATOGRAPHY
The stationary phase: a layer of water or a polar solvent
coated onto the paper fibers
THIN LAYER CHROMATOGRAPHY
The stationary phase: a thin layer of sorbent is spread
uniformly on a glass plate, plastic sheet, or alumunium foil
GAS CHROMATOGRAPHY
The stationary phase: methyl silicone polymers,
substituted silione polymers, or silicone polyesters

The mobile phase: a gas (an inert gas such as nitrogen,

helium, hydrogen, or argon)
LIQUID CHROMATOGRAPHY
The stationary phase: polar or non-polar substances
(particles of a small diameter  High Performance Liquid
Chromatography)

The mobile phase: a liquid

SEPARATION MECHANISMS
Classified by the chemical or physical mechanisms used to
separate the solutes
• Ion-Exchange
• Partition
• Adsorption
• Affinity
• Size-Exclusion
ION-EXCHANGE CHROMATOGRAPHY
Basis: an exchange of ions between a charged stationary
surface and mobile phase of the opposite charge

The Stationary phase: the particle surfaces of a plastic

resin or silica
PARTITION CHROMATOGRAPHY
Basis: the differential distribution of solutes between two
immiscible liquids

The Stationary phase: one of the immiscible liquids

ADSORPTION CHROMATOGRAPHY
Basis: the differences between the adsorption and
desorption of solutes at the surface of the solid particle

The Stationary phase: adsorbent (nonpolar, acid polar,

basic polar)
AFFINITY CHROMATOGRAPHY
Basis: the unique and specific biological interaction of the
analyte and ligand

The Stationary phase: a ligand immobilized on a particles

of a support either directly or via a spacer
SIZE-EXCLUSION CHROMATOGRAPHY
Basis: the molecular sizes of the solutes

The Stationary phase: a variety of materials (crossed-

linked dextran, agarose, polyacrylamide, porous glass,
polystyrene-divinylbenzene, or combinations of the above)
REFERENCE
1. Burtis CA, Ashwood ER, Bruns DE, editors. Tietz
Textbook of Clinical Chemistry and Molecular
Diagnostics, 4th ed. Missouri: Elsevier. 2006.
2. Bishop ML, Fody EP, Schoeff LE, editors. Clinical
Chemistry Techniques, Principles, Correlations, 6th ed.
Philadelphia: Lippincott Williams & Wilkins. 2010.
THANK YOU