CCHM 321: Clinical Chemistry 1│Lecture

2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

 Osmometry
Analytical Techniques and
Instrumentation  Electrochemistry Techniques
(Preliminary Term, 5th Topic)
Trans Outline: COLORIMETRY
Topic 1: Four Basic Topic 7: Chromatography
Disciplines Topic 8: Fluorometry PHOTOELECTRIC COLORIMETRY:
Topic 2: Colorimetry Topic 9: Chemilumine-  Spectrometry The primary goal is the
Topic 3: Volumetric scence
isolation of discreet portions of the spectrum
(Titrimetric) Topic 10: Osmometry
Topic 4: Turbidimetry Topic 11: Electrochemistry for purposes of measurement
Topic 5: Nephelometry Techniques
Topic 6: Electrophoresis  Used to measure analytes
o Spectrophotometric Measurement
• Measurement of light
FOUR BASIC DISCIPLINES intensity in a narrower
wavelength
1. Spectrometry o Photometric Measurement
a. Spectrophotometry • Measurement of light
b. Atomic absorption (AAS) intensity without
c. Mass Spectrometry (MS) consideration of the
2. Luminescence wavelength
a. Fluorescence
b. Chemiluminescence
3. Electroanalytic Methods
a. Electrophoresis
b. Potentiometry / Amperometry
4. Chromatography
a. Gas
b. Liquid
c. Thin-Layer

 Pre-analytical Colorimetry
 Volumetric (Titrimetric)
 Turbidimetry
 Nephelometry
 Electrophoresis
 Chromatography
 Fluorometry
 Chemiluminescence

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

SPECTROPHOTOMETRY: a. Uses 2 photo detectors for the

 Involves measurement of the light sample and the reference beam
transmitted by a solution to determine the b. 2 beams are always present in
concentration of the light-absorbing space
substance in the solution B. Double-beam in time
o Single Beam Spectrophotometer a. Uses 1 photo detector only and
• Simplest type of alternately passes the
monochromatic light through the
spectrophotometer
sample cuvet and then reference
• designed to make 1 cuvet using a chopper or rotating
measurement at a time at 1 sector minor
specified wavelength b. beam appears in two places over
• absorption maximum of the one cycle in time
analyte must be known in
advance when single beam
instrument is used
o Double Beam Spectrophotometer
• Instrument that splits the
monochromatic light into two
components
• 1 beam passes throughout the
sample and the other passes
through a reference solution
or reagent blank
• there’s additional beam that
corrects for variation in light
source intensity
• absorbance of the sample can
be recorded directly as the
electrical output of the sample
beam

 6 Basic Components of Single or Double-

Beam Configuration Spectrophotometer:
o Stable source of radiant energy
o Filter
• isolates specific region of
EMS
o Sample Holder
2 Types of Double Beam Spectrophotometer
• where cuvette is placed
A. Double-beam in space o Radiation Detector

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

• called photo multiplier  any given beam of light has specific values of
o Signal Processor frequency, wavelength, and energy
o Readout Device associated with it
 visible light travels at a speed of 300,000
km/s and can be broken down into seven
colors from longest to shortest wavelength
• red, orange, yellow, green, blue,
indigo, violet
 visible wavelengths or spectrum of white
light: 350 to 700 nm
PARTS OF SPECTROPHOTOMETER

 Colors and the Complementary Colors of the

Visible Spectrum
Wavelength Color Complementary
 Light / Radiant Source
Absorbed Color
350 - 430 Violet Yellow - Blue o Provides polychromatic light and
431 - 475 Blue Yellow must generate sufficient energy or
476 - 495 Green - Blue Orange power to measure the analyte of
496 - 505 Blue - Green Red interest
506 - 555 Green Purple o response to change in light intensity
Yellow - must be linear to give accurate
556 - 575 Violet
Green absorbance measurement throughout
576 - 600 Yellow Blue its absorbance range
601 - 650 Orange Green – Blue o 2 Types
651 - 700 Red Blue - Green • Continuum Source
Note: Need to memorize • emits radiation that
changes in intensity
VISIBLE LIGHT SPECTRUM
• widely used in
 Newton demonstrated that color is a quality laboratory
of light • Tungsten – Visible
 as a form of electromagnetic radiation, light and Infrafed Region
has properties of both • Deuterium – UV
Light
• waves • Xenon – UV and
Visible Range
• particles • Line Source
 can be thought of as a stream of minute • emits limited radiation
energy pockets radiated at varying and wavelength
frequencies in a wave motion

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

• Mercury and Sodium

Vapor Lamps – UV
and Visible Regions
• Hollow cathode tube
– (AAS)
• Laser – also used as
light sources for
spectrophotometer
 Entrance Slit • Diffraction Gratings
o Minimizes unwanted or stray light • Most commonly used;
and prevents the entrance of scattered has better resolution
light into the monochromator system than prism
o Stray Light • Made by cutting
• Any wavelength outside the grooves or slits into an
band transmitted by the a luminated surface of
monochromator a flat piece of crown
• Limits the maximum glass - wavelengths
absorbance that a are bent as they pass a
spectrophotometer can sharp corners
achieve • grooves are 15,000-
• Most common cause of 30,000 per inch
linearity at high analyte
concentration
 Monochromator
o Isolates specific or individual
wavelength of light
o Kinds of Monochromator:
• Prisms
• Wedge shaped pieces
of glass, quartz or
sodium chloride
• can be rotated,
allowing only the
desired wavelength to
• Filters
pass through an exit
• Simple, less expensive
slit
• not precise but useful
• A narrow light
• Made by placing semi-
focused on a prism is
transparent silver
refracted as it enters
films on both sides
more dense glass
• It produces
monochromatic light
based on the principle
of constructive
interference of waves

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

o Cuvet with scratches on their optical

surface will scatter the light and
should be discarded.
o Silica cuvettes transmit light
effectively at wavelength greater than
or equal to 220 nm
o Alkaline solution left standing in
cuvet could dissolve glass producing
etching.
o The path length of cuvet is 1 cm
o To increase sensitivity, some cuvets
are designed to have path length of10
cm, increasing the absorbance for a
given solution by a factor of 10
• Holographic Gratings o arrow should be placed facing
 Exit Slit towards light source
o Controls the width (bandpass) of light o do not overfill cuvet, usually
beam- allows only a narrow fraction maximum volume is 1 ml
of the spectrum to reach the cuvette.
o Bandpass
• total range of wavelength
transmitted
o Special purity of the
spectrophotometer is reflected by the
band, that is, the narrower the
bandpass, the greater the resolution
 Cuvet
o Also called absorption cell/ analytical
cell/ sample cell
o Holds the solution whose
concentration is to be measured
o where sample or analyte is placed
o Kinds of Cuvet
• Alumina Silica Glass  Photodetector
• most commonly used o Detects and converts transmitted
especially from 350- light into photoelectric energy
2,000 nm o Kinds
• Quartz/Plastic • Barrier Layer Cells/
• for measurement of Photocell/ Photovoltaic Cell
solution requiring • simplest detector, least
visible and ultraviolet expensive,
(UV) spectra temperature sensitive
• Borosilicate Glass • Used in filter
• Soft Glass photometers with a
wide bandpass

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

• no external voltage • Measures light at a

but we can rely on multitude of
internal electron wavelength- detects
transfer less amount of light
• non-linear • Most useful as a
• Basic phototransducer simultaneous
that is used for multichannel detector
detecting and
measuring radiation in
the visible region
• Phototube
• Contains cathode and
anode enclosed in a
glass case
• Has a photosensitive
material that gives off
electron when light
energy strikes it
• Requires external
voltage  Meter or Read-Out Device
• Photomultiplier Tube (PMT) o It displays output of the detection
• Most commonly used system
detector – measure o E.g. Galvanometer, ammeter, LED
visible and UV o Beer’s Law (computation for this will
regions not be included)
• Has excellent • States that the concentration
sensitivity and has of the unknown substance is
rapid response- directly proportional to the
detects very low level absorbed light and inversely
of light related to the transmitted
• Should never be used light
to room light because • Absorbance
it will burn out • Amount of light
• detects and amplifies absorbed
radiant energy • Mathematical derived
• contains dynodes – from %T
series of anodes with • cannot be measured
high positive voltage directly by a
• 200x more sensitive spectrophotometer
than phototube • mathematically
• Photodiode derived from %
• Not as sensitive as transmittance
PMT but with • flipside of
excellent linearity transmittance

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

• states how much of the emerge on the other

light the sample side
absorbed
• referred as optical
density
• formula is logarithmic Blanking Technique
function of
transmittance  Blank contains serum but without the reagent
to complete the assay
 Reagent blank corrects absorbance caused by
the color of the reagent- the absorbance of
reagents is automatically subtracted from
each unknown reading.
• unknown solution  Measures absorbance of the sample and
• Au – absorbance of reagent in the absence of end product
unknown  May not be effective in in some cases of
• As – absorbance of turbidity, ultracentrifugation may be
necessary
sample
 To correct for artifactual absorbance reading,
• Cs – concentration of blanking procedures or dual wavelength
solution method may be used
 done when using spectrophotometer
 reagent blank is included when measuring
analytes with the sample together with the
• Percent Transmittance blank and unknown
• fraction of incident Spectrophotometer Quality Assurance
light which is
transmitted  Wavelength Accuracy
• amount of light that o means that the wavelength indicated
successfully passes on the control dial is the actual
through the substance wavelength of light passed by the
and comes out the monochromator
other side  Stray light
o any wavelengths outside the band
• l (letter L) –
transmitted by the monochromator
transmitted
 Linearity
light/output
o demonstrated when a change in
• I0 (letter I) – incident
concentration results in a straight line
light/input
calibration curve
• eg: a percent
transmittance of 25% FLAME EMISSION PHOTOMETRY
indicate that 25% of
 Measures the light emitted by a single atom
the light pass through burned in a flame
the sample and

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

 Principle: Excitation of electrons from lower

to higher state energy
 Light Source: flame
 Method: indirect internal standard method
 Internal Standard: lithium/cesium (corrects
variations in flame and atomizer
characteristics)
 Used for the measurement of excited atoms
(Na and K)
 Flickering of light indicates changes in the
fuel reading of the instrument
ATOMIC ABSORPTION SPECTROPHOTO-
METRY
VOLUMETRIC (TITRIMETRIC)
 Measures the light absorbed by atoms
dissociated by heat  Principle:
 Principle: Element is not excited but merely o The unknown sample is made to
dissociated from its chemical bond and place react with a known solution in the
in an unionized, unexcited, ground state presence of an indicator
 Light source: hollow cathode tube  Examples:
 Interferences: chemical, matrix, and o Schales and Schales Method
ionization (Chloride Test)
 Used for measurement of unexcited trace o EDTA Titration Method (Calcium
metals Test)
 more sensitive than FEP
 An atomizer (nebulizer/graphite furnace) is TURBIDIMETRY
used to convert ions to atoms.
 A chopper is used to modulate the light  For measuring abundant large particles
source. (proteins) and bacterial suspension.
 Lathanum or Strontium Chloride is added to  Principle:
samples to form stable complexed with o Determines the amount of light
phosphate blocked
 accurate, precise, and very specific  Depends on specimen concentration and
particle size
 Solutions are measured using
visible photometers or
spectrophotometers
 Uses: protein measurements (CSF and urine)
to detect bacterial growth in broth cultures;
measuring antibiotic sensitivities, detecting
clot formation
 measurement of intensity of the transmitted
light is a function of the concentration of the
suspended particles
 yields more reliable results than
nephelometry

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

NEPHELOMETRY ELECTROPHORESIS

 More sensitive for protein measurement than  Migration of charged particles in an electric
turbidimetry field
 For measuring the amount of antigen-  Separates proteins on the basis of their
antibody complexes (proteins) electric charge densities
 Principle: determines the amount of scattered  The acidic and basic amino acids determine
light by a particulate matter suspended in a the net charge on a protein
turbid solution  During electrophoresis, proteins are
 Light scattering depends on the wavelength negatively charged (anions) and they move
and particle size towards the anode
 Light scattered is measured at an angle, 15-  has 5 bands
90 degrees  proteins are always negative
 Components:  Iontophoresis: migration of small charged
o Light Source (mercury-arc lamp, ions
tungsten-filament lamp, LED and  Zone electrophoresis: migrations of charged
laser) molecules
o Collimator  Amphoteric: net charge can be either (+) or
o Monochromator (-)
o Sample Cuvet  Electroendosmosis or Endosmosis:
o Stray Light Trap movement of buffer ions and solvent relative
o Photodetector to fixed support
 The deterctor (PM Tube) output is  Components
proportional to concentration o Electrical power, support medium,
 more sensitive because a small scattered buffer, sample, detecting system.
intensity against black background is easier  Factors affecting Rate of Migration
to measure than small change in intensity of o Net electrical charge
the intense transmitted radiation o Size anmd shape of the molecule
 basic principle involved is measurement of o Electric Field Strength
intensity of the scattered light as a function of o Nature of the supporting medium
the concentration of the dispersed phase o Temperature of operation
 Supporting Media:
 light scattering is expensive
o Paper electrophoresis
 preferred when measuring low
• Electrophoresis where there’s
concentrations
movement of charged
particles in a solution under

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

the influence of an external o Coomassie Blue

electrical field • CSF proteins
• employs a filter paper strips o Gold/Silver Stain
soaked in a buffer solution • very sensitive
usually diethylbarbituric acid DENSITOMETRY:
or barbituric acid dissolved in  Electrophoretic machine
alkaline  Measures the absorbance of the stain
• pH is usually 8.6  Scan and quantify electrophoretic pattern
o Starch Gel  Reads gel and cellulose acetate membrane
• separates by surface charge  Electrophoretic Mobility is directly
and molecular size proportional to net charge and inversely
o Cellulose Acetate proportional to molecular size and viscosity
• separates by molecular size  The ionic strength of the buffer determines
o Agarose Gel the amount of current and the movement of
• neutral proteins for a fixed voltage
• separates by electrical charge  At pH 8.6, the gamma globulins move toward
• it does not bind protein the cathode, despite the fact that they are
o Polyacrylamide Gel negatively charged (endosmosis)
• neutral  After electrophoresis, the gel is treated with a
• separates on the basis of mild fixative, such as acetic acid, that
charge and molecular size, precipitates the proteins at the positions to
separates proteins into 20 which they have migrated. They are stained,
fractions gel is dried and cleared of excess stain.
• used to study isoenzymes  If the electrodes are not properly aligned, the
 Stains for Visualization of Fractions current may be denser on one side of the gel
o separated proteins are being stained than the other.
to reveal their locations  If electrophoresis process too long, the
o different stains come with different proteins may migrate off the gel into the
plates from different manufacturers buffer.
o simplest way to accomplish detection  If there is a break in the electric circuit and no
is visualization under UV light or current passes, the proteins will not move.
densitometer can be used  Frequently, gel shows "smile artifact'’
o densitometer
• one of the most common and
reliable way for quantitation
o the more the pH of the buffer differs
from the proteins, the faster the
movement
o smile artifact – the samples at the
center of the gel migrate further than
those in the edges
o Amido black
o Ponceau S
o Oil Red O
o Sudan Black
o Fat Red 7B

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

ISOELECTRIC FOCUSING:
 Separating molecules migrate through pH
gradient; uses a constant gradient
 It is ideal for separating proteins of identical
sizes but with different net charges
 Proteins move in the electric field until they
reach a pH equal to their isoelectric point
 pH gradient is created by adding acid in the
anodic area of electrolyte cell and adding
base to cathode area
 Supporting Media: Agarose gel,
Polyacrylamide Gel, Cellulose Acetate
 Advantages:
o The ability to resolve mixtures of CHROMATOGRAPHY
proteins
o Detects isoenzymes  Involves the separation of soluble
o Identify genetic variants components in a solution by specific
o Detects CSF oligoclonal band differences in physical-chemical
characteristics of the different constituents
 group of techniques used to separate complex
mixtures on the basis of different physical
interaction between the individual
compounds and stationary phase of the
system
 2 Forms of Chromatography
o Planar Chromatography
• Paper Chromatography
• Fraction of the sugar
and amino acid
• Sorbent – Whatman
paper
• Thin layer Chromatography
CAPILLARY ELECTROPHORESIS: • Used for drug
 Sample molecules are separated by electro- screening
osmotic flow (semiquantitative
 (+) charged ions move faster screening test)
 (-) charged ions move slower • Each drug has a
 It uses nanoliter quantities of specimen characteristic Rf value
 Uses: and it must match the
o Separation, quantitation and Rf value with the
determination of MW of proteins. standard
o Analysis of PCR • Rf – retention
o Analysis of organic and inorganic factor; relative
substances and drugs distance of
migration from

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

point of the o Column Chromatography

application • Gas Chromatography
• Extraction of the drug • Gas Solid Chromato-
is pH dependent graphy
• pH must be • Gas Liquid Chromato-
adjusted to graphy
reduce the • Mass Spectrometry
solubility of • GC - MS
the drug in • MS / MS
• Liquid Chromatography
aqueous phase
• High Performance
• Sorbent: thin plastic
plates impregnated Liquid
with a layer of silica Chromatography
gel or alumna (HPLC)
• e.g: blood, urine,
GAS CHORMATOGRAPHY:
gastric fluid
 Used for the separation of steroids,
barbiturates, blood, alcohol and lipids
 Useful for compounds that are naturally
volatile or can be easily converted into a
volatile form
 Specimens are vaporized and swept onto the
columns
 Flame ionization is used as detector
 Elution order of volatile is based on their
boiling point
 Mobile phase: Nitrogen, Helium. Hydrogen
and Argon

A. Gas Solid Chromatography

a. Differences in absorption at the
solid phase surfaces
B. Gas Liquid Chromatography
a. Separation occurs by differences
in solute partitioning between
gaeseous mobile phase and the
liquid stationary phase

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

LIQUID CHROMATOGRAPHY:
 Is based on the distribution of solutes
between a liquid mobile phase and a
stationary phase
 HPLC is the most widely used liquid
chromatography
 High Performance Liquid Chromatography
(HPLC)
o Uses pressure for fast separations,
controlled temperature, in-line
MASS SPECTROMETRY (MS): detectors and gradient elution
 Based on fragmentation and ionization of technique
molecules using suitable source of energy o Uses: Fraction of drugs, hormones,
 Substance first be first separated by gas lipids, carbohydrates, and proteins;
chromatography separation and quantitation of various
 Can also detect structural information and hemoglobins associated with specific
determination of molecular weight diseases, rapid HbA1c
 molecular fragmentation  Reverse Phase HPLC
 allows identification of substance by o mobile phase is more polar than
comparison with a computer library of stationary phase
unknown fragmentation pattern  5 Separation Mechanisms Used in Liquid
Chromatography
GC-MS: o Gel/ Gel Permeation/ Gel Filtration/
 Gold standard for drug testing Size Exclusion/ Molecular Sieve
 Uses an electron beam to split the drug Chromatography
 Used for xenobiotics, anabolic steroids, • Separates molecules based on
pesticides differences in their size and
shape
TANDEM MASS SPECTROSCOPY • As solute travel through the
(MS/MS): gel, large molecules remain in
 Can detect 20 inborn errors of metabolism the mobile phase are eluted
from a single blood spot rapidly in the column
 used for newborn screening • Hydrophilic Gel (Gel
Filtration)
• For separation of
enzymes, antibodies,
and proteins
• supporting medium:
usually Dextran and
Agarose
• Hydrophobic Gel (Gel
Permeation)
• For separation of
triglycerides and fatty
acids

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

•
supporting medium: • For separation of LPP, CHO
Cephadex and glycated Hgb
o Ion Exchange Chromatography • LPP – lipoproteins
• The exchange of sample ions • CHO – carbohydrates
and mobile phase ions with • glycated Hgb
charged group of the (hemoglobin)
stationary phase • Used to separate and prepare
• For separation of amino acids, larger quantities of proteins
proteins and nucleic acids and antibodies for study
• Separation of nucleic acids o Adsorption Chromatography
and proteins depends • Liquid-Solid
primarily on the size and ionic Chromatography
charge density • Separation is based on the
differences (competition)
between adsorption and
desorption at the surface of a
solid particles
• The compounds are adsorbed
to a solid support such as
silica or alumina

FLUOROMETRY/ MOLECULAR
LUMINESCENCE
SPECTROPHOTOMETRY
 Measures the amount of light intensity
present over a zero background
o Partition Chromatography  Principle:
• Liquid to liquid o It determines the amount of light
chromatography emitted by a molecule after excitation
• Separation compounds are by electromagnetic radiation
based on their partition  Light Source: Mercury ARC or Xenon lamp
between liquid mobile phase 365-366 nm
and a liquid stationary phase  Light Detectors: Photomultiplier tube or
coated on a solid support phototube
• For separation of therapeutic o It uses 2 monochromators (filters,
drugs and their metabolites prisms or gratings)
o Affinity Chromatography  Primary Filter: select wavelength that is best
• Uses immobilized absorbed by the solution to be measured
biochemical ligands as the  Secondary Filter: prevents the incident from
stationary phase to separate a striking the photodetector
few solutes from other o About 1,000x more sensitive than
unretained solutes spectrophotometer - emitted radiation
• Uses the so-called lock and is measured directly
key binding

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

 It is affected by quenching – pH and  Involves the oxidation of an organic

temperature changes, chemical contaminants, compound (dioxetane, luminol, acridinium
UV light changes ester) by an oxidant (H2O2, hypochlorite or
 Uses: measurement of phorphyrins, O2). These oxidation reactions may occur in
magnesium, calcium and cathecolamines the presence of a catalyst, such as enzymes,
 last of the 4 basic discipline metal ions and hemin
 Use: immunoassays
 Photodetector: photomultiplier tube
(Luminator)
 advantages
o can include different assays like sub-
picomolar detection limits
o speed
o has flash type reactions
o light is measured for only 10 seconds
o ease of use, usually 1 step procedure
o simple instrumentation

OSMOMETRY
CHEMILUMINESCENCE  The measurement of the osmolality of an
 Differs from fluorescence and aqueous solution such as serum, plasma, or
phosphorescence in that (-) the emission of urine
light is created from a chemical or  Principle:
electrochemical reaction and not from the o It is based on measuring changes in
absorption of electrochemical energy the colligative properties of solutions
 no excitation is required and no and that occurs owning to variations
monochromators are needed in particle concentration
 More sensitive than fluorescence o colligative properties
 Principle: • physical changes that result
o The chemical reaction yields an from adding solute to a
electronically excited compound that solvent
emits light as it returns it to its ground • includes freezing point
state, or that transfers its energy to depression, boiling point
another compound, which then elevation, vapor pressure
produces emission lowering, and osmotic
pressures

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

 Osmotic Particles
o Glucose, urea nitrogen and sodium
 Freezing Point Depression Osmometry
o Most commonly used method for
measuring the changes in colligative
properties

ELECTROCHEMISTRY TECHNIQUES
 Measurement of current or voltage generated
by the activity of specific ions
 Measurement of blood gas, blood pH,
electrolytes, glucose, urea, ionized Calcium,
lead and Chloride
POTENTIOMETRY:
 Measurement of electrical potential due to the
activity of free ions
o change of voltage indicates activity of
each analyte
 Measurement of differences in voltage
(potential) at a constant current
 Follows the Nernst Equation
o equation that enables the
determination of cell potential under
non-standard conditions
 Concentration of ions in a solution can be
calculated from measured potential
difference between two electrodes
 Reference Electrodes: Saturated Calomel and
Silver-Silver Chloride
 Uses: pH and pCO2
o pCO2 – partial pressure of carbon
dioxide
 Ion Selective Electrode (mmol/L)
o Measures the activity of one ion much
more than the other ions present in the
sample
o Ion selectivity depends on the
membrane or barrier composition
used
o Interference: excess protein
o very sensitive and selective for the
ions it measure

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.
2022-2023 3RD YEAR, 1ST SEMESTER Prof. Kimberly Ann Pulga, RMT, MPH

o 2 types of ISE:  Membrane Electrodes

• Direct ISE: without sample
COULOMETRY:
dilution
• Indirect ISE: with sample  Measurement of the amount of electricity,
dilution reported in Coulomb at a fixed potential
o ISE Membrane can have the  An electrochemical titration in which the
following: titrant is electrochemically generated
and the endpoint is detected
• Glass aluminum silicate for
by amperometry
Sodium (Na)
 Follows the Faraday’s Law
• Valinomycin gel (K) o states that the induced voltage in a
• Organic liquid membrane ion circuit is proportional to the rate of
exchanger (Ca and Li) change overtime of the magnetic flux
• Gas and enzyme electrode through a circuit
pH ELECTRODES  Use: Chloride Test (CEF, Serum and sweat)
 Interference: Bromide, cyanide and cysteine
 highly selective and sensitive to hydrogen
ions AMPEROMETRY:
 generate the stable electrical potential  The measurement of the current flow
 Indicator electrode produced by oxidation-reaction
o silver wire coated with AgCl,  Examples: pO2, glucose, chloride and
immersed into an internal solution of peroxidase determination
0.1 mmol/L HCl, and placed into a  Polarography
tube containing a special glass o Measurement of differences in
membrane tip current at a constant voltage
 Reference electrode o Follows the Ilkovic Equation
o Calomel, a paste of predominantly VOLTAMMETRY:
mercurous chloride, is in direct
contact with metallic mercury  Measurement of the current after which a
in an electrolyte potential is applied to an electrochemical cell
solution of potassium chloride  Allows sample to be preconcentrated, thus
 Other Parts utilizing minimal analyte
o Liquid Junction  Anodic Stripping Voltammetry: for lead and
• Electrical connection between iron testing
the indicator and reference
electrodes is achieved by
allowing a slow flow of
electrolyte from the tip of the
reference electrode.
o Read-out Meter
THREE MAJOR ISE TYPES
 Inert Metal Electrodes in contact with a redox
couple (standard hydrogen electrode)
 Metal Electrodes that participate in a redox
reaction (Ag/AgCl electrode)

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CCHM321 | BSMLS 2024 CLINICAL CHEMISTRY 1 TRANSCRIBER: CRUZ, A. M. A.