COLLEGE OF MEDICAL LABORATORY SCIENCE

Clinical Chemistry 1
Introduction to Clinical Chemistry

 Drugs of Abuse
o (e.g. Cocaine, Barbiturates,
 A quantitative science that is concerned
Acetaminophen)
with measurement of amounts of
c. Large Molecules
biologically important substances (called
 Transport Proteins
analytes) in body fluids.
o (e.g. Albumin, Transferrin,
 A science, a service and an industry.
Haptoglobin)
o As a science, it links the
 Enzymes
knowledge of general chemistry,
o (e.g. Lipase, Amylase, Creatinine
organic chemistry, and bio-
Kinase)
chemistry with an understanding
 Specific Proteins
of human physiology,
o (e.g. Immunoglobulins, C-
o As a service, it produces
reactive proteins, Complement)
objective evidence from which
 Diabetes Marker
medical decisions may be made,
o (e.g. Hemoglobin A1c or HbA1c)
and
o As an industry, clinical
laboratories are businesses,
which operate under the
1. Blood
regulations and practices that
2. Urine
guide commerce in the United
3. Cerebrospinal Fluid (CSF)
States.
4. Amniotic Fluid
 Also a separate section or department in
5. Saliva
the hospital laboratory. The clinical
6. Synovial Fluid
chemistry services is the most utilized in
7. Pleural Fluid
the hospital laboratory.
8. Pericardial Fluid
9. Peritoneal Fluid

a. Ions, Salts, and Minerals

 (e.g. Sodium, Potassium, Calcium,
Chloride CO2, Lead Iron)
b. Small Organic Molecules
 Aim is to ensure an accurate, reliable,
 Metabolites
o (e.g. Glucose, Cholesterol, Uric and timely test results performed in a
labotory.
acid)
 Therapeutic Drugs  Quality standards for all clinical
o (e.g. Vancomycin, Theophylline, laboratories to ensure accuracy,
Digoxin) reliability and timeliness of patient test
results regardless of where the test was
 Toxicology
performed.
o (e.g. Alcohol, Salicylate,
 Defines clinical laboratories broadly.
Acetaminophen)

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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Introduction to Clinical Chemistry
a. Waived Tests
 Simple laboratory examinations and
procedures that are cleared by the U.S.
Food and Drug Administration (FDA)
for home use.
b. Nonwaived Tests
 Moderately and highly complex tests as
defined by the requirements for operator
skill, reagent preparation, and
automation and the difficulty of
interpretation of results.
 These are regulated under guidelines
that cover quality standards for
proficiency testing (PT), patient test
management, quality control, personnel
qualifications, and quality assurance.
o (This will be further discussed in
the topic of Quality Assessment
and Quality Control)
 Provide guidelines for safe operation of
testing processes.
 Regulations include guidelines for
operating safety equipment and
identifying, handling and storing
chemical hazards.
o (This will be further elaborated in
the laboratory discussions)
 Perform analytical procedures that
yields accurate and precise information,
aiding in patient diagnosis and
treatment.
 Correctly use basic supplies and
equipment.
 Possesses an understanding of
fundamental concepts critical to any
analytic procedure.
1. Assuring reliable test results that
contribute to the prevention, diagnosis,
prognosis, and treatment of physiological
and pathological conditions. This
assurance requires:
a) Producing accurate test results.
b) Correlating and interpreting test data.
c) Assessing and improving existing
laboratory test methods.
d) Designing, evaluating, and implementing
new methods.
2. Designing and implementing cost-effective
administrative procedures for
laboratories, including their services and
personnel.
3. Designing, implementing, and evaluating
processes for education and continued
education of laboratory personnel.
4. Developing and monitoring a quality
assurance system to include:
a) Quality control of services.
b) Competence assurance of personnel.
5. Promoting an awareness and
understanding of the services they render
to the consumer, the public, and other
health-care professionals.
 A method of monitoring accurate
outcome; in which test samples from an
external source are analyzed and results
compared to those reference
laboratories and scored for accuracy.
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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Laboratory Supplies

 Used for the manufacture of weighing

bottles because it develops less static
A. High Thermal Resistant Glass
surface changes.
 Usually a borosilicate glass with low
 Composed of a mixture of the oxides of
alkali content.
silicon, calcium and sodium.
 Resistant to heat, corrosion and thermal
shock.
 Used when heating or sterilization is
A. Colored and Opal Glasses
required.
 Used in light fitters, lamp bulbs and
 Most common Resistant Borosilicate:
lightning lenses.
o Beakers
B. Coated Glasses
o Flasks
 Have thin metallic oxide permanently
o Pipettes
fine-bonded to the surface of the glass.
B. High Silica Glass
 Have electronic applications as heat
 Made by removing all elements from
shield to protect against infrared light.
borosilicate glass.
C. Optical Glasses
 Has good optical qualities, temperature
 Mostly soda-lime, lead and borosilicate
capabilities and is radiation-resistant.
of high optical purity.
 Used for high precision analytical work
 Used in making prisms, lenses and
and for optical reflectors and mirrors.
optical mirrors.
 Not used for the type of glassware
D. Glass Ceramics (Pyroceram)
generally used in the laboratory.
 Have high thermal resistance, chemical
C. High Alkali-Resistant Glass
stability and corrosion resistance like
 Partially used for strong alkaline
borosilicate glasses.
solutions.
 Useful in hot plates, table tops and heat
 Often referred to as “soft glass” as its
exchanges.
thermal resistance is much less than of
E. Radiation-Absorbing Glasses
borosilicate glass; used primarily
 Made of soda-lime and lead.
whenever digestion with strong alkali is
 Useful in preventing transmission of huge
made.
energy radiation as gamma rays and X-
D. Low Actinic Glass
rays.
 Has materials that usually impart or red
color to the glass that reduce the
amount of light passing through
substance inside the glassware.  Beginning to replace glassware in the
 Provides protection to reagents highly laboratory setting.
sensitive to light ranging from 3,000-  Its unique high resistance to corrosion
5,000 Angstrom (A). and breakage as well as its varying
 Used for substances that are particularly flexibility, had made it most appealing.
sensitive to light such as bilirubin or  It is relatively inexpensive, allowing for
Vitamin A. most items to be completely disposable
E. Standard Flint Glass or Soda-Lime Glass after each use.
A. Polyolefins

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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Laboratory Supplies
 Unique group of resins with relatively
inert properties.
 Unaffected by acids, alkalis, salt
solutions and aqueous solutions.
 Can be autoclaved.
D. Teflon-Fluorocarbon Resin
a. Polypropylene
o More vulnerable to attack by
oxidizing agents.
o Can withstand higher
temperatures.
b. Polyethylene
o Both polypropylene are used
primarily to fabricate bottles,
beakers, jars jugs, funnels pipette
jars, pipette baskets, tanks,
burette covers, check valves,
disconnect valves, twistcock
connectors, needle valves, hollow
stoppers, dropping pipettes,
hydrometer jars, stirring rods,
tubings and reagent dispensers.
B. Polycarbonate resin
 Twice as strong as polypropylene and
may be used at temperatures ranging
100˚C to 160˚C.
 Unsuitable for use with bases such as
amines, ammonia, alkalies and oxidizing
agents.
 Dissolved by chlorinated aliphatic and
aromatic hydrocarbon.
 Insoluble in aliphatic hydrocarbons,
some alcohols and dilute aqueous
solutions and salts.
 Used extensively in centrifuge tubes and
graduated cylinder.
C. Tygon
 Non-toxic, clear plastic of modified
plasticized polyvinyl chloride.
 Can be used to handle most chemicals
but should not be subjected to prolonged
immersion in aliphatic or aromatic
hydrocarbons, ketones and esters.
 Flexible at 30˚C, brittle at 45˚C and
resists dry heat to 95˚C.
 Can be steamed, autoclaved or
chemically sterilized.
 Used for the manufacture of tubing (i.e.,
tubing used in Auto-Analyzers).
 Pure translucent white and inert to
corrosive reagents boiling agua regia,
nitric and sulfuric acids, boiling
hydrocarbons, ketones, esters and
alcohols.
 Can resist extreme temperatures
ranging from -270*C to +255˚C, used in
cryogenic experiments or work at
temperatures over extended periods.
 Used for self-lubricating stopcocks,
stirring bars, and bottle cap liners and
tubing because of its anti-adhesive
properties.
a. To contain/TC Pipette
o Holds a particular volume but
does not dispense that exact
volume.
o Calibrated by introducing the
exact weight of mercury required
to give the desired volume at a
specific temperature.
o Mercury does not wet glass and
pipettes calibrated this way will
contain but not deliver the stated
volume.
b. To deliver/TD Pipette
o Dispense the indicated volume.
o Calibrated by weighing the
volume of water that will flow
from them by gravity.
o Rate of delivery must never be
hastened by blowing.
c. “To Blow-Out” Pipette
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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Laboratory Supplies
o Same as TD pipette but drops o No graduations to the tip.
remaining at the tip after delivery o Self-draining pipette.
is blown out to receiving vessel. d. Micropipettes
o An etched ring is seen near the o “To contain” pipette which is
mouthpiece. calibrated with mercury.
o Entire content of the pipette must
a. Volumetric/Transfer Pipette be emptied.
o Has the greatest degree of o Used when small amount of
accuracy and precision. blood or specimen is needed (< 1
o Designed to dispense one volume ml).
c/o further subdivisions.
o Calibrated to deliver a fixed
volume of liquid.
o Has a bulb between mouthpiece  By far the most routinely used pipette.
and tip that decreases surface  Advantages:
area/unit volume and diminished o Time savings
error from water film. o Safety
o Self-draining. o Stability
b. Ostwald-Folin Pipette o Ease of use
o Used in measuring viscous fluids o Increase in precision
such as whole blood. o Lack of required cleaning
o Measures smaller volume (2.0  Tips (contaminated) are often
mm or less). disposable.
o Has a bulb near the tip.  Types:
o Etch mark, ring near mouthpiece. o Air Displacement
o Used with biologic fluids having a o Positive Displacement
viscosity greater than that of o Dispenser/ Dilutor
water. e. Pasteur Pipette
o Blow-out pipette. o No calibration.
c. Graduated/Measuring Pipette o For biologic fluid w/o specific
o Used to deliver an amount of volume.
liquid contained between two
calibration marks.

a) Serologic Pipette
o The rate of fall of liquid is much
too fast.
o Has an etched band on the
suction piece.
o Has calibration marks to the tip.
o Blow-out pipette.
b) Mohr Pipette
o Calibration lies between two
marks on the stem.

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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Laboratory Supplies
6. Activated alumina (AL2O3)
 Most common desiccants.
 Most commonly used in Volume
Measurements and THERMALLY
RESISTANT.

 Temperature Monitoring Devices: 6 or

12 months interval.

 Should be made of glass that is resistant
to many chemicals used and resistant to
heat.
 Used for general mixing and reagent
preparation.
 Wide, straight-sided cylindrical vessels
and are available in many sizes in
several forms.
a. Total Immersion
o Used for refrigerators and
freezers.
b. Partial Immersion
o Used for heating blocks and
water baths.
c. Surface Thermometer
o Used for incubators and heating
oven.
A. Analytical Balance
 Precision is up to 1/1000 of a gram.
B. Rough or Platform Balance
 Precision is up to 0.1 gram.

Example:
 Used to measure volumes of liquids
when high degree of accuracy is not o Torsion Balance - for weighing
essential. chemicals.
o Triple Beam Balance - three
beams are present in the
 Pear-shaped flasks. balance.
 Have one calibration mark on narrow
part of the neck.
 Used to contain a specific amount or a. Relative Centrifugal Force (RCF)
volume of liquid. o Force acting on sample being
centrifuged.
o Obtained by using NOMOGRAM.
1. Anhydrous calcium chloride
𝑅𝐹𝐶 = 1.118 × 10−5 × 𝑟 × (𝑟𝑝𝑚)2
2. Magnesium perchlorate
3. Magnesium sulfate b. Revolution per minute (rpm)
4. Sodium sulfate
5. Calcium sulfate

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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Laboratory Supplies
o Speed of centrifugation;
determined by TACHOMETER or
i. Class M Weights
STROBE LIGHT.
 Are primary standard quality.
c. Radius (r)
 Used only to calibrate other weights.
o Distance in cm from center of
ii. Class S Weights
rotation to bottom of the tube
 Are used for calibrating balances.
when rotating.
iii. Class S-1 Weights
d. CAP recommends
 Have greater tolerance than Class S
o Daily: cleaning of any spills or
weights.
debris such as blood, and glass.
 Are used for routine analytic work.
o Every three months: timer
iv. Class P Weights
brushes and speed be checked.
e. Horizontal-head (Swinging Bucket)  Have greater tolerance than Class S-1.
o Tubes are in horizontal position v. Class J Weights
when rotating.  Are intended for micro analytical work
o Recommended for serum and range from 50 to 0.05 mg.
separator tubes.
o Produces a tightly packed, flat
sediment surface.
f. Angle-head
o Tubes are at fixed angle (25-40˚)
when rotating.
o Capable of higher speeds.
o Produces a slanted sediment
surface that isn't tightly packed.
o Decantation is not
recommended.
g. Ultra-centrifuge
o High-speed, capable of 100,000
rpm.
o Refrigerated to reduce heat.

1. Fixed-angle head
2. Swinging-bucket type
3. Microfuge
4. Tachometer
5. Strobe light

 Analytical Weights:
o Used to verify the performance of
analytical balance.

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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Analytical Method and Instrumentation

 Invisible spectra – below 340 nm

(ultraviolet region (UV) above 700
 Radiant energy from short wavelength
(infrared region [IR])
gamma rays to long wavelength radio
waves.
 They are photons of energy traveling in a
 Number of vibrations of wave motion per
wavelike manner. The shorter the
second.
wavelength, the lighter the
electromagnetic energy.

1. Cosmic rays
2. Gamma rays
3. X-rays
4. Visible
5. Ultra-violet (UV)
6. Infrared (IR)
7. Radio, TV, microwave, etc.  There are two primary considerations in
every colorimetric analysis:
o Quality of the color
 Transmitted via electromegnetic waves o Intensity of the color
characterized by frequency and
wavelength.
a. Visual Colorimetry
 Uses the eyes in determining end point.
 Shows the relationship between b. Photoelectric Colorimetry
wavelength and energy.
𝐸 = ℎ𝑣
a. Spectrophotometry
Wherein;  Spectrophotometric measurements.
 h -> constant (6.62 x 10-27 erg sec) b. Filter photometry
 v -> frequency  Photometric measurements.

 Distance between peaks as light is

envisioned to travel in wavelike manner.
 Visible spectra – 340 nm – 700 nm
 Measurement of light intensity in a much
narrower wavelength. It uses a device
(prisms and/or gratings) to disperse the
source of light into a continuous
spectrum.

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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Analytical Method and Instrumentation
 Measurement of light intensity of
multiple wavelengths. It uses filter to
isolate part of the spectrum.
 States that the concentration of a
substance is directly proportional to the
amount of the light absorbed or inversely
proportional to the logarithm of
transmitted light.
 Ratio of the radiant energy transmitted,
divided by the radiant energy incident on
the sample.
%𝑇 = 𝐼𝑡 /𝐼𝑜 × 100
 The amount of light absorbed.
 Proportional to the inverse log of
transmittance.
 Mathematically derived from %T.
𝐴 = 2 − 𝑙𝑜𝑔%𝑇
𝐴 = −𝑙𝑜𝑔%𝑇 = 1⁄𝑙𝑜𝑔 %𝑇
𝐴 = 𝑎𝑏𝑐
Where:
 A= absorbance
 a= molar absorptivity (absorptivity of the
compound under standard conditions)
 b= length of light through the solution
(light path)
 c= concentration of absorbing
molecules/solution
 Provides radiant energy in the form of
visible or non-visible light that may pass
through the monochromator. The light of
proper wavelength will be made incident
on the analytical cell.
 Continuum source
o Emits radiation that changes in
intensity.
 Line source
o Emits limited radiation and
wavelength; uses an intense
beam of light directed through
the monochromator and the
sample.
 Tungsten Light bulb
o Commonly used light source in
the visible and near infrared
region (up to 1,200 nm)
 LASER (Light Amplification by
Stimulated Emission of Radiation)
1. Range
2. Spectral distribution
3. Source
4. Stability
a. Tungsten Iodine lamp
 Produces energy wavelength from 340 to
700 nm (visible region).
b. Quartz Halide lamp
 Contains small amounts of halogen such
as iodine to prevent the decomposition
of the vaporized tungsten from the very
hot filament.
c. Deuterium Discharge lamp
 Provides energy source with high output
in the UV range (down to 165 nm).
d. Infrared Energy source
 used above 800 nm.
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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Analytical Method and Instrumentation
Examples:  Has small grooves cut at such as angle
that each groove behave like a very
 Merst glower
small prism.
o An electrically heated rod of rare
 Separates white light into various color
earth element oxides.
component.
 Globar
 Based on the principle that wavelengths
o Uses silicon carbide.
are bent as they pass a sharp corner.
 Mercury Vapor lamp
c. Colored filters
o Exits narrow bands of energy at
 Made of a glass that absorbs some
well-defined places in the
portion of the electromagnetic spectrum
spectrum (UV and visible).
and transmit others.
 Hollow Cathode lamp
 Light energy is absorb by dye
o Consists of a gas-tight chamber
compounds on the glass and is
containing anode, a cylindrical
dissipated as heat.
cathode, and insert gas such as
 Band pass in 35 to 50 nm or more.
helium of argon.
d. Interference filter
 Utilizes the wave character of light to
 Minimizes unwanted or stray light and
enhance the intensity of the desired
prevents the entrance of scattered light
wavelength by constructive interference
into the monochromator system.
and eliminates others by destructive
 Stray light causes systematic error;
interference and reflections.
causes loss of linearity.
 Band pass is 10 to 20 nm.

 Isolate specific wavelength of light.

 Monochromatic light
 Produces linear spectrum and therefore
o Light radiation of a single
maintaining a constant and pass which
wavelength.
is simple.
 Can be used in the regions of spectrum
where light energy is absorbed by glass
prism.

 Controls the width of light beam

a. Prisms
 Wedge-shaped pieces of glass, quartz,
NaCl, or some other material that allows
transmission of light.
 Disperses white light into a continuous
spectrum of colors based on variation of
refractive index for different wavelength.
b. Gratings
(bandpass.
 Accurate -> <1/5 the natural bandpass of
the spectrophotometer).
 Used to hold the solution in the
instrument whose concentration is to be
measured.
 It is made of glass, quartz or plastic.

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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Analytical Method and Instrumentation
a. Borosilicate glass cuvette
 For solutions that do not etch glass.
b. Quartz or plastic
 Does not absorb UV radiation at
wavelength below 320.
c. Alumina silica glass
 Good for 340 nm and above (visible).
 Electron tube amplifying a current that
can convert transmitted energy into an
equivalent amount of electrical or
photoelectric energy.
a. Barrier layer cells (Photocell or
Photovoltaic cell)
 Composed of a film of light sensitive
material like selenium on iron plate with
transparent layer of silver.
 Requires no external voltage sources.
b. Photoemissive tube or Phototube
 Has photosensitive material that gives off
electron when light energy strikes it.
 Consists of 2 electrodes (cathode and
anode) sealed in an evacuated glass to
prevent scaterring of photoelectrons by
collision with gas molecules.
 Require an outside voltage for operation.
c. Photomultiplier tube
 Uses a series of electrodes to internally
amplify the photosignal before leaving
the tube.
o Most common type -> measures
visible and UV regions.
o Excellent sensitivity and rapid
response.
o Must never be exposed to room
light.
 Simplest method of displaying output of
the detection system. Also called read-
out device.
 Galvanometer/ammeter
 Didymium and holmium oxide filter
o Used to check wavelength
accuracy.
 Neutral density filters and dichromate
solution
o Verify absorbance accuracy on
linearity.
 Splits monochromatic light into two
components.
a. Double-beam in space
 Uses 2 photodetectors, for the sample
beam and reference beam.
b. Double-beam in time
 Uses one photodetector and alternately
passes the monochromatic light through
the sample cuvet and then reference
cuvet using a chopper.
Principle;
 The measurement of emitted light when
an electron in an atom becomes excited
by heat energy produced by the flame.
 Excited atoms return to ground state by
emitting light energy that is
characteristic of that atomic species.
 It is used primarily to determine
concentration of sodium, potassium or
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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Analytical Method and Instrumentation
lithium since these alkali metals are easy
to excite.
Purposes of the Flame in EFP:
 Breaks the chemical bond to produce
atoms.
 Source of energy absorbed by the atoms
to enter an excited state.
 Using a mixture of hydrogen and oxygen
gas, (acetylene, propane or natural gas).
 Breaks up the solution into finer droplets
so that the atom will absorb heat energy
from the flame and get excited.
a. Total consumption burner
 Aspirate sample directly into the flame,
the gases are passed at high velocity
over the end of the capillary suspended
in the solution.
b. Premix burner
 Involves the gravitational feeding of
solution through a restricting capillary
into an area of high velocity gas flow
where small droplets are produced and
passed into the flame.
r detecting absorption of electromagnetic
 Na filter
o Transmit yellow light (589 nm).
 K filter
o Transmit violet light (767 nm).
 Lithium
o Transmit red light (761 nm).
 Uses photocell as detector.
 Lithium
o Referred internal standard; also
acts as a radiation buffer.
 Reasons why lithium is preferred
o Its emission characteristics are
similar to those of Na+ and K+
o Normally present as a trace
element in human tissues and
does not present interferences in
the determination.
 Purposes
o To achieve stability where there is
fluctuations caused by changes in
fuel of air pressure which affects
flame temperature and rate of
sample aspiration.
 Its concentration must be precisely the
same in all samples and standards.
 Energy required of the internal standard
must be close to that required to excite
the element being measured.
 Must not be normally found in ion being
analyzed.
Note:
 In cases where lithium is the analyte
cesium is used as internal standard.
Principle:
 Measures concentration of element by
radiation by atoms, rather than by
molecules.
 The element is not excited but they are
dissociated from their chemical bonds
and placed in the unionized, unexcited
ground state.
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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Analytical Method and Instrumentation
 It is ideal for alkali metals that are not
easily excited (e.g. calcium, magnesium).
 Hollow cathode lamp, which produces a
wavelength of light, specific for the kind
of metal in the cathode.
 Modulates light beam coming from the
hollow cathode lamp.
 Uses flame to dissociate the chemical
bonds and form free, unexcited atoms.
a. Total consumption burner
 Flame is more concentrated and can be
made hotter, thus lessening chemical
interferences.
 Produces large droplets in the flame and
produces a high acoustical noise.
b. Premix burner
 Gases are mixed and the sample is
Example:
atomized before entering the flame and
the large droplets go to waste and not in
the flame.
 It has less noisy signals with longer
pathlength and greater absorption and
sensitivity.
 Flame is less hot and therefore cannot
dissociate metal complexes.
 Selects the desired wavelength from a
spectrum of wavelength which could
either be a prism or diffraction gratings.
 Uses photomultiplier tubes to measure
the intensity of the light signal.
 Chemical
o Situation at which the flame
could not dissociate the sample
into neutral atoms.
 Ionization
o Situation at which atoms in the
flame become excited and emit
energy instead of staying in the
ground state.
 Matrix
o Formation of solids from sample
droplets due to enhancement of
light absorption by organic
solvents.
 Lanthanum or strontium chloride –
forms stable complexes with
phosphate -> to avoid calcium
interference
 The unknown sample is made to react
with a known solution (titrating agent) in
the presence of an indicator.
 Chloride determination
o (Schales and Schales)
 Calcium determination
o (EDTA Titration)
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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Analytical Method and Instrumentation

 It is the solution of the pure form of the  It is the measurement of the light blocked
sample and its derivatives and the by a suspension of particulate matter as
determination of its dry weight. light passes through the cuvette.

Example:
 Total Lipid determination

 Determines the amount of light emitted

by a molecule after excitation by
electromagnetic radiation.
 Measures the amount of light intensity
present over a zero background.
 Light source: mercury arc and xenon
lamp.
 Light detector: photomultiplier tubes.
 It is affected by quenching – pH and
temparature changes, chemical
contaminants, UV light changes.
 Uses:
o Measurement of porphyrins
o Magnesium
o Calcium
o Catecholamines
Fluorescence concentration measurement is
related to:
 Molar absorptivity of the compound.
 Intensity of incident radiation.
 Quantum efficiency of every emitted per
quantum absorbed.
 Length of light path.
Factors affecting Turbidimetry:
 Size and number of the particles.
 The depth of the tube.
 Cross-sectional area of each particle.
Disadvantage
 Variable absorption due to aggregation
particles which tend to settle out of the
solution.
 It is the measurement of light scattered
by small particles an at angle to the
beam incident on the cuvette.
 Factors affecting turbidimetric
measurements are the same factors
affecting nephelometric measurements.
 More specific than turbidimetry.

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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Analytical Method and Instrumentation
o Substance being analyzed.
 Radiolabeled antigen Ag)
 It is used to measure the disintegration
o Acts as label.
per minute of time of a radiosotope.
 Antibody
o Provide binding site for the two
antigens.

 The measurement of differences in

voltage at a constant current.
a. Alpha  Follows the Nernst equation.
 Positively charged particles, resembles  Reference electrode: calomel and silver-
the nucleus of a helium atom with mass silver chloride.
of 4.  Ion selective Electrode (ISE).
 Have very little of energy.  Nicolsky-Eisenmann – conceptualized
b. Beta ISE selectivity.
 Resembles an electron with both
negative (B-) and positive (B+) charges
but essentially no mass.
 Exists in two forms: soft and hard beta.
c. Gamma
 A form of electromagnetic energy with
no mass, only energy.
 Exists in two forms: soft and hard
gamma.

 The measurement of differences in

current at a constant voltage.
a. Solid Scintillation Counter
Polarography is used to measure trace
 Measure gamma radiation using
metals, oxygen, Vitamin C and amino
thallium activated NaI crystal as
acids concentration.
scintillator and PM tube as detector with
 Follows the Ilkovic equation.
preamplifier circuit.
b. Liquid Scintillation Counter
 Measures beta radiation using liquid
flour as scintillator.

 An immunologic procedure involving the

use of radioisotope.

Substances involved in RIA:  The measurement of amount of

 Unlabeled antigen (Ag) electricity (in terms of coulombs) at a
fixed potential.

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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Analytical Method and Instrumentation
 Follows Faraday’s law.
 Example: Chloride test (serum and
sweat, CSF)
 Pilocarpine – sweat inducer
 Interferences: bromide cyanide and
cysteine.
 The measurement of the amount of
current that flows when a constant
voltage is applied to the measuring
electrode.
 The measurement of the current flow
between two non-polarizable electrodes
between which a known electrical
potential is established.
 It involves the separation of a mixture on
the basis of specific differences of the
physical-chemical characteristics of the
different components on a supporting
medium called absorbent or sorbent.
 The amount of the mixture are
separated by a continuous
redistribution between two (2) phases:
o Stationary phase
o Mobile phase (also called eluent
or carrier fluid)
 A spot of the substance to be
fractionated is placed on the paper just
above the solvent level.
 Organic solvent (mobile phase) moves
up through the paper by capillary action
and various fractions in the sample
move at different rates.
Sorbent
 Special grades of filter paper.
Example: Whatman Phase Separating Paper
Basis of Separation
 Rate of diffusion
 Solubility of the solute
 Nature of the solvent
Clinical Use
 Fractionation of sugars, amino acids and
barbiturates.
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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Analytical Method and Instrumentation
 For the separation of unwanted
 When a mixture of small and large substances present in a solution mixture.
molecules is allowed to pass over small  Concentration of solute of interest
particles in a column, the smaller suspended in highly diluted samples can
molecules diffuse into the gel, whereas be determined.
larger molecules tend to pass rapidly in
the column and appear in the eluate
first.

Basis of Separation

 Molecular weight and size

 Charge of the ions
 Hydrophobicity of the molecules

Clinical Use

 Fractionation of polysaccharides, nucleic

acids, proteins including enzymes and
isoenzymes.
Sorbent

 Thin plastic plates impregnated to a

layer of silica gel, alumina,
polyacrylamide gel or starch gel.

Basis of separation

 Rate of diffusion
 Substances to be separated are passed  Solubility of the solute
on the ion- exchange column and  Nature of the solvent
depending on the net charge and pH of
the solution; the substance is absorbed
from solution in the ion- change resin.
 Ions with greatest charge densities will
be held most strongly on an ion-
exchange material.

Sorbent

 Anion or cation resin with functional

groups.
 A highly polar substance tends to be
Basis of separation more soluble in a highly polar solvent
 Differences in sign and ionic charge (water), while the less polar substance
densities. tends to be more soluble in a less polar
solvent, (organic solvent).
Clinical Use  Follows the “Like dissolves like” principle.

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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Analytical Method and Instrumentation
separation of high molecular weight
components and many labile biologic
compounds such as peptides, drugs,
hormones, barbiturates, lipids, steroids
and antibiotics.

Basis of separation

 Differences in pH
 Polarity of solvent

Clinical use  It refers to the migration or movement of

 Fraction of sugars
 It is capable separating and measuring
nanogram and picogram amounts of
volatile substances.
 It follows the concept of selective
adsorption.
 It applies 4,000- 10,000-lbs/square inch
pressure for the rapid identification and
charged particles in an electric field.
 Charged particle or ion will migrate
towards the anode or cathode
depending on the isoelectric pH (pI) of
the solution under the influence of an
externally applied electric field.
 Iontophoresis
o Migration of small charged ions.
 Zone electrophoresis
o Migration of charged
macromolecules.
 Amphoteric
o Eitherr positive or negative net
charge.
 Electroendosmosis/endosmosis
o Movement of buffer ions and
solvent relative to the fixed
support.
 Isoelectric point
o Zero net charge.
 CHON are negatively charged during
electrophoresis due to:
o Barbital = pH 8.6
o Tris-boric EDTA = pH 8.7
Factors affecting rate of Migration:
 Net electric charge
 Size and shape of the molecule
 Electric fluid strength

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COLLEGE OF MEDICAL LABORATORY SCIENCE
Clinical Chemistry 1
Analytical Method and Instrumentation
 Nature of supporting media

 Paper
 Starch Gel
o Separates by surface charge and
molecular size.
 Cellulose acetate
o Separates by molecular size.
 Agarose gel
o Neutral; separates by electrical
charge; does not bind protein
(lipoprotein).
 Polyacrylamide gel
o Neutral; separates on the basis of
charge and molecular size; very
sensitive.

a. Serum Protein Electrophoresis

o Amido black
o Ponceau S
b. Lipoprotein electrophoresis
o Oil red O
o Sudan Black
o Fat red 7B
c. Coomasie Blue
o CSF CHON electrophoresis ->
multiple sclerosis
d. Gold/Silver

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