Joed T.

Ticse, MD
1st Yr Resident, Dr. PJGMRMC
 Photometry is defined as the measurement of light.
 Spectrophotometry is defined as the measurement of the
intensity of light at selected wavelengths.
 Qualitative and quantitative analysis.
Beer’s Law—Relationship Between Transmittance, Absorbance,
and Concentration
 Concentration of a substance is directly proportional to the amount
of light absorbed
A=abc
where
A = Absorbance
a = Proportionality constant defined as absorptivity
b = Light path in centimeters
c = Concentration of the absorbing compound,
usually expressed in moles per liter
1. Radiant energy source (light source)
2. Wavelength selectors (monochromator)
3. Sample holder (cuvet)
4. Photodetector
5. Signal processor
6. Read out (display unit)
1. Radiant energy source 3. Sample holder (cuvet)
(light source) a. Silica, quartz
a. Tungsten light bulb
4. Photodetector
b. Xenon lamp
a. PMT
c. Laser
b. CTD
2. Wavelength selectors
(monochromator) 5. Signal processor
a. Filters (pre- and 6. Read out (display unit)
post-sampling) a. LED
b. Prisms b. LCD
c. Graters
 Atomic absorption (AA) spectrophotometry is used widely in
clinical laboratories to measure trace elements.
 measure concentration by detecting the absorption of electro-
magnetic radiation by atom rather than by molecules
 The flame emission photometer, which measures light emitted
by excited atoms, was widely used to determine concentration
of Na+, K+, or Li+.
 In reflectance photometry, diffuse reflected light is
measured. The reaction mixture in a carrier is illuminated with
diffused light, and the intensity of the reflected light from the
chromogen is compared with the intensity of light reflected
from a reference surface.
 Reflectance photometry is used as the measurement method
with urine dipstick analysis and dry-film chemistry
systems.

 Fluorescence refers to the condition when a molecule absorbs
light at one wavelength and reemits light at a longer
wavelength. An atom or molecule that fluoresces is termed a
fluorophore.
 Fluorometry is defined as the measurement of emitted
fluorescent light.
 Fluorometric analysis is a widely used method of quantitative
analysis in the chemical and biological sciences; it is a very
accurate and sensitive technique.
Instrumentation
 Operationally, a fluorometer uses interference filters, glass
filters, gratings, or prisms to produce monochromatic light for
sample excitation and for isolation of fluorescence emission.
Components
1. excitation source
2. excitation monochromator
3. cuvet
4. emission monochromator, and
5. detector
 Chemiluminescence, bioluminescence, and electrochemi-
luminescence are types of luminescence in which the
excitation event is caused by a chemical, biochemical, or
electrochemical reaction, and not by photoillumination .
 In bioluminescence, an enzyme or a photoprotein increases the
efficiency of the luminescence reaction.
 Luciferase and aequorin

 Chemiluminescence assays are ultrasensitive (attomole to

zeptomole detection limits).
 Electrochemiluminescence differs from chemiluminescence
in that the reactive species that produce the chemiluminescent
reaction are electrochemically generated from stable
precursors at the surface of an electrode.
 Used in both immunoassays and nucleic acid assays.
 Advantages of this process include (1) improved reagent
stability, (2) simple reagent preparation, and (3) enhanced
sensitivity with detection limits of 200 fmol/L.
 Light scattering is a physical phenomenon that results from the
interaction of light with particles in solution.
 Nephelometry and turbidimetry are analytical techniques used
to measure scattered light. Light scattering measurements have
been applied to immunoassays of specific proteins and
haptens.
 Nephelometry and turbidimetry are used to measure the
concentrations of large particles (such as antigen–antibody
complexes, prealbumin, and other serum proteins) that
because of their size cannot be measured by absorption
spectroscopy.
 Nephelometry detects light that is scattered at various angles;
scattered light yields a small signal that must be amplified. In
contrast,
 turbidimetry measures a reduction in light transmission due to
particle formation; thus it detects a small decrease in a large
signal.
 Refractometry is based on light refraction.
 The ability of a substance to bend light is called refractivity, and
it is measured as a difference between the angle of incidence
and the angle of refraction.
 Refractometry can be used to measure protein concentration,
specific gravity of urine, and column effluent of high
performance liquid chromatography analysis.
 Osmometry is the measurement of the osmolality of an
aqueous solution such as serum, plasma, or urine.
 These properties are osmotic pressure, boiling point, freezing
point, and vapor pressure.
 As the osmolality of a solution increases, (1) the osmotic
pressure increases, (2) the boiling point is elevated, (3) the
freezing point is depressed, and (4) the vapor pressure is
depressed.
 Flow cytometry is used to count and sort cells, as well as viral
particles, DNA fragments, bacteria, and latex beads. It is a core
component of hematology cell counters and the technology
used to differentiate white blood cells.
 A flow cytometer measures multiple properties of cells
suspended in a moving fluid medium. As each particle passes
single file through a laser light source, it produces a
characteristic light pattern that is measured by multiple
detectors for scattered light (forward and 90 degrees) and
fluorescent light (if the cell is stained with a fluorochrome).
 In flow cytometry, the term particle describes any object
flowing through the instrument. An event is anything that is
interpreted by the instrument to be a single particle.
 Size restrictions also apply; cells or particles must be from 1 to
30 µm in diameter. Specialized flow cytometers are designed to
handle smaller particles such as DNA fragments or bacteria.
 Flow cytometers are able to measure multiple parameters,
including
 cell size (forward scatter),
 granularity (90° scatter),
 DNA content,
 RNA content,
 DNA (A+T)/(G+C) nucleotide ratios,
 chromatin structure,
 antigens,
 total protein content,
 cell receptors,
 membrane potential,
 calcium ion concentration as a function of pH.
 The measurement of potential (voltage) between two
electrodes in a solution forms the basis for a variety of
procedures for measuring analyte concentration.
 Potentiometry is widely used clinically for the measurement of
pH, PCO2, and electrolytes (Na+, K+, Cl-, Ca2+, Mg2+, Li+) in
whole blood, serum, plasma, and urine, and as the basis for
some biosensors for metabolites of clinical interest .
 Many different types of electrodes are used for potentiometric
applications. They include redox, ion-selective membrane
(glass and polymer), and PCO2 electrodes.
 Coulometry measures the electrical charge passing between
two electrodes in an electrochemical cell.
 Given the volumetric amount of serum or plasma sample
originally used, it is possible to calculate the concentration of
Cl- in the sample.
 Coulometric titration is one of the most accurate electro-
chemical techniques because the method measures the
absolute quantity of electroactive substance in the sample.
 Coulometry is considered the gold standard for
determination of chloride in serum or plasma.

 Amperometry is the measurement of the current flow produced
by a redox reaction.
 Several immobilized enzyme electrodes use this principle, as
do pO2 electrodes
 Voltammetry is a method in which a potential is applied to an
electrochemical cell and the resulting current is measured.
 The most important advantages of voltammetry are sensitivity
and the capability for multielement measurements.
 Conductometry is an electrochemical technique used to
determine the quantity of an analyte present in a mixture by
measurement of its effects on the electrical conductivity of the
mixture. It is the measure of the ability of ions in solution to
carry current under the influence of a potential difference.
 Blood analytes such as urea
 components of detectors used HPLC, GC, cell counters, and
capillary electrophoresis.
 Electrical impedance measurement is based on the change in
electrical resistance across an aperture when a particle in
conductive liquid passes through this aperture.
 Electrical impedance is used primarily in the hematology
laboratory to enumerate leukocytes, erythrocytes, and
platelets.
 Electrophoresis is a comprehensive term that refers to the
migration of charged solutes or particles of any size in a liquid
medium under the influence of an electrical field.
 Depending on the charge they carry, ionized solutes move
toward either the cathode (negative electrode) or the anode
(positive electrode) in an electrophoresis system.
 The net charge of a compound, in turn, depends on the solution
pH. Electrophoresis separations often require high voltages
(50-200 V DC)
 To obtain a quantitative profile of the separated fractions,
densitometry is performed on the stained support medium.

 A densitometer measures the absorbance of the stain on a

support medium.
 The basic components of a densitometer include a light source,
a monochromator and a movable carriage to scan the medium
over the entire area, an optical system, and a photodetector.
 Electrophoresis are carried out in a small-bore (10 to 100 µm)
fused silica capillary tub
 Applications of CE include separation of
 Uncharged molecules (MEKC) mode
 inorganic ions,
 amino acids,
 organic acids,
 drugs,
 vitamins,
 porphyrins,
 carbohydrates,
 oligonucleotides,
 proteins,
 DNA fragments.
 At a specifc pH, a protein will have a net charge of zero when
the positive charge and the negative charge of its amino acids
cancel each other out. At this pH value, known as the protein’s
isoelectric point (pI), the protein is isoelectric.
 Isoelectric focusing (IEF) techniques are performed similarly to
other electrophoresis methods, except that the separating
molecules migrate through a pH gradient.
 Chromatography is a separation method based on different
interactions of the specimen compounds with the mobile phase
and with the stationary phase as the compounds travel through
a support medium.
 Compounds interacting more strongly with the stationary
phase are retained longer in the medium than those that favor
the mobile phase.
 Gas chromatography (GC) is useful for compounds that are
naturally volatile or can be easily converted into a volatile
form.
 GC has been a widely used method for decades owing to its
high resolution, low detection limits, accuracy, and short
analytic time.
 Applications involve various organic molecules, including
many drugs
 Its basic design consists of five components: a gas cylinder as a
mobile phase source, a sample injector, a column, a detector,
and a computer for data acquisition.
 Many organic compounds are too unstable or are insufficiently
volatile to be assayed by GC without prior chemical
derivatization.
 LC techniques use lower temperatures for separation, thereby
achieving better separation of thermolabile compounds.
 Finally, it is easier to recover a sample in LC than in gas
chromatography. The mobile phase can be removed, and the
sample can be processed further or reanalyzed under different
conditions.
 When the stationary phase in LC consists of small-diameter
particles, the technique is known as high performance liquid
chromatography (HPLC).
 High-performance liquid chromatography (HPLC) emerged in
the late 1960s as a viable form of liquid chromatography that
provided advantages over other forms of liquid chroma-
tography and gas chromatography.
 HPLC columns can be used many times without regeneration.
The resolution achieved with HPLC columns is superior to that
of other forms of liquid chromatography, analysis times are
usually much shorter, and reproducibility is greatly improved.
 A widely used clinical application of HPLC is the separation
and quantitation of various hemoglobins associated with
specific diseases (e.g., thalassemia).
 Whole blood measurement of glycosylated hemoglobin
(HbA1c) can be accomplished using HPLC with complete
analysis within 5 minutes.
 Five separation techniques are commonly used in liquid
chromatography. They include adsorption, partition, ion-
exchange, affnity, and size exclusion.

 MS is a very powerful qualitative and quantitative analytical
technique that is used to measure a wide range of clinically
relevant analytes.
 Mass spectrometry (MS) is based on fragmentation and
ionization of molecules using a suitable source of energy.
 The resulting fragment masses and their relative abundance
yield a characteristic mass spectrum of the parent molecule.
 Mass analysis is the process by which one or more ionic
species are identified according to the mass-to-charge ratio
(m/z).
 Before a compound can be detected and quantified by mass
spectrometry, it must be isolated by another method: GC or
HPLC.

Basic Components
 ion source,
 mass analyzer, and
 ion detector.
 Nuclear magnetic resonance spectroscopy (NMR) is a
technique for determining the structure of organic compounds.
 Unlike mass spectroscopy (MS), NMR is nondestructive,
although it does require a larger sample volume than MS.
 Although NMR is widely used as a diagnostic imaging
technique, it has been adapted for only a limited number of
clinical laboratory analyses, the most popular being
lipoprotein particle measurements.