CLINICAL

CHEMISTRY 1
Prepared by: Shayne Eyre Dichos,
RMT, MSMT (CAR)
ANALYTICAL METHOD
 Light energy, wavelength and radiant
energy spectrum:
•  Energy - is transmi?ed via electromagneCc waves that are characterized
by their frequency and wavelength.
•  Wavelength - is the distance between two successive peaks and it is
expressed in terms of nanometer (nm).
400-700nm - visible spectrum
<400nm - ultraviolet region(UV)
>700nm — infrared region (IR)
•  The relaConship between wavelength and energy (E) is described by
Planck's formula: E = hv , where:
E - is the energy of a photon in Joules or eV
h - constant (6.626 x 10-34 erg sec)
v - frequency
•  Frequency - is the number of vibraCons of wave moCon per second.
–  The lower the wave frequency, the longer the wavelength.
–  The wavelength is inversely related to frequency and energy; the shorter
the wavelength, the higher the frequency and energy and vice versa.
 Light energy, wavelength and radiant
energy spectrum:
Notes to Remember:
–  Nominal wavelength represents the wavelength in
nanometers at peak transmi?ance.
–  A slight error in wavelength adjustments can introduce a
significant error in absorbance readings.
–  Wavelength accuracy—the wavelength indicated on the
control dial is the actual wavelength of light passed by the
monochromator.
–  Didymium or holmium oxide filter—used to check
wavelength accuracy
–  Neutral density filters and dichromate soluMon - verify
absorbance accuracy on linearity
I. COLOROMETRY
•  Photoelectric colorimetry
–  The primary analyCcal uClity of
spectrophotometry or filter photometry is the
isolaCon of discreet porCons of the spectrum for
purposes of measurement.
a.  Spectrophotometric Measurement - of light
intensity in a narrower wavelength
b.  Photometric measurement - measurement of
light intensity without consideraCon of
wavelength
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A. Spectrophotometry
•  It involves measurement of the light
transmi?ed by a soluCon to determine the
concentraCon of the light-absorbing
substances in the soluCon.
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A. Spectrophotometry

Single beam
spectrophotometer
•  It is the simplest types of
absorpCon spectrometer.
•  It is designed to make
one measurement at a
Cme at one specified
wavelength.
•  The absorpCon maximum
of the analyte must be
known in advance when a
single-beam instrument is
used.

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A. Spectrophotometry
Double-beam spectrophotometer
•  Is an instrument that splits the monochromaCc light into two
components.
•  One beam passes through the sample, and the other through a
reference soluCon or , blank.
•  The addiConal beam corrects for variaCon in light source intensity.
•  The absorbance of the sample can be recorded directly as the
electrical output of the sample beam.

2 Types of Double-beam Spectrophotometer:

a.  double-beam in space - uses 2 photodetectors for the sample
beam and reference beam.
b.  double-beam in Mme – uses one photodetector and alternately
passes the monochromaCc light through the sample cuvet and
then reference cuvet using a chopper (rotaCng sector mirror).
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A. Spectrophotometry
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A. Spectrophotometry

6 basic components of single or double-beam

configuraCon spectrophotometer:
•  stable source of radiant energy
•  filter that isolates a specific region of the
electromagneCc spectrum
•  sample-holder
•  radiaCon detector
•  signal processor
•  readout device.
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A. Spectrophotometry
Parts of the Spectrophotometer
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A. Spectrophotometry
Parts of the Spectrophotometer
1.  Light/Radiant source
•  It provides polychromaCc light and must
generate sufficient radiant energy or power
to measure the analyte of interest.
•  An intense beam of light is directed through
the monochromator and the sample.
•  To give accurate absorbance measurements
throughout its absorbance, its responsible to
change in light intensity must be linear
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A. Spectrophotometry
Parts of the Spectrophotometer
1. Light/Radiant source
2 Types:
a.  ConMnuum source — emits radiaCon that changes
in intensity; widely used in the lab.
examples: tungsten, deuterium and xenon lamps
•  Tungsten light bulb - is the commonly used light
source in the visible and near infrared region
•  Deuterium lamp – is rouCnely used to provide UV
radiaCon in analyCc spectrometers.
•  Xenon discharge lamp - produces a conCnuous
source of radiaCon, which covers both the UV and
the visible range.
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A. Spectrophotometry
Parts of the Spectrophotometer
1. Light/Radiant source
2 Types:
b.  Line source — emits limited radiaCon and
wavelength.
examples: Mercury and sodium vapor lamps in
spectrophotometers (UV and visible regions), and the
hollow cathode lamp (AAS
•  Line sources that emit a few discrete lines find wide
use in atomic absorpCon, molecular, and fluorescent
spectroscopy.
•  light AmplificaCon by sCmulated Emission of
radiaCon (LASER) is also used as light sources for
spectrophotometry.
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A. Spectrophotometry
Parts of the Spectrophotometer
Factors for choosing a light source:
•  Range
•  Spectral distribuCon within the range
•  The source of radiant producCon
•  Stability of the radiant energy and temperature
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A. Spectrophotometry
Parts of the Spectrophotometer
1. Light/Radiant source
AlternaCves:
1.  Mercury arc (visible and UV)
2.  Deuterium lamp (165nm) – UV
3.  Hydrogen lamp – UV
4.  Xenon lamp – UV
5.  Merst glower - IR
6.  Globar (silicone carbide) - IR
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A. Spectrophotometry
Parts of the Spectrophotometer
2. Entrance Slit
•  Minimizes unwanted or stray light and
prevents the entrance of sca?ered light into
the monochromator system.
•  Stray light - refers to any wayelength outside
the band transmi?ed by the monochromator;
it does not originate from the polychromaCc
light source; it causes absorbance error.
•  Stray light limits the maximum absorbance
that a spectrophotometer can achieve.
•  Straylight is the most common cause of loss
of linearity at high-analyte concentraCon.
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A. Spectrophotometry
Parts of the Spectrophotometer

3. Monochromator -
Isolates specific or
individual wavelength
of light.
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A. Spectrophotometry
Parts of the Spectrophotometer
Kinds of Monochromators:
a.  Prisms
•  are wedge-shaped pieces of glass, quartz or sodium chloride.
•  a narrow of light focused on a prism is refracted as it enters the
more dense glass.
•  can be rotated, allowing only the desired wavelength to pass
through an exit slit.
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A. Spectrophotometry
Parts of the Spectrophotometer
Kinds of Monochromators:
b. DiffracMon graMngs
•  are the most commonly used
•  be?er resoluCon than prism.
•  made by cuing grooves (parallel grooves) or slits into an
aluminized surface of a flat piece of crown glass - wavelengths
are bent as they pass a sharp corner.
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A. Spectrophotometry
Parts of the Spectrophotometer
Kinds of Monochromators:
c. Filters
•  are simple, least expensive, not precise but useful.
•  made by placing semi-transparent silver films on both
magnesium fluoride
•  Produce monochromaCc light based on the principle
of construcCve interefrence of waves - light waves
enter one side of the filter and are reflected on the
second surface.
•  usually pass a wide band of radiant energy and have
selected wavelength.
d. Holographic GraMng
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A. Spectrophotometry
Parts of the Spectrophotometer
4. Exit Slits
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A. Spectrophotometry
Parts of the Spectrophotometer
4. Exit Slits
•  It controls the width of light beam (bandpass) allows only a
narrow fracCon of the spectrum to reach the sample
cuve?e.
•  Spectral purity of the spectrophotometer is reflected by the
bandpass — the narrower the bandpass, the greater the
resoluCon.
•  Accurate absorbance measurement requires a bandpass
< 1/5 the natural bandpass of the spectrophotometer.
•  The degree of wavelength isolaCon is a funcCon of the type
of device used and the width of entrance and exit slit.
•  Bandpass - is the total range of wavelengths transmi?ed.

I. COLOROMETRY
A. Spectrophotometry
Parts of the Spectrophotometer
[Link]
•  Also called absorpCon cell/analyCcal cell/
sample cell
•  It holds the soluCon whose concentraCon
is to be measured.
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A. Spectrophotometry
Parts of the Spectrophotometer
Kinds of Cuvets:
1.  Alumina silica glass- most
commonly used (can be used
in 350-2000nm)
2.  Quartz/plas9c - used for
measurement of soluCon
requiring visible and
ultraviolet spectra
3.  Borosilicate glass – used for
strong alkaline soluCon
4.  So? glass – used for strong
acidic soluCon
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A. Spectrophotometry
Parts of the Spectrophotometer
Notes to remember on Cuvets:
•  Cuvets with scratches, on their opCcal surface sca?er light
and should be discarded.
•  Silica cuve?es transmit light effecCvely at wavelengths
>220nm
•  Alkaline soluCons should not be lem standing in cuvets for
prolonged periods because alkali slowly dissolves glass
producing etching
•  The path length of cuvets is 1 cm, although much smaller
path lengths are used in automated systems.
•  To increase sensiCvity, some cuvets are designed to have
path lengths of 10 cm, increasing the absorbance for a
given soluCon by a factor of 10.
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A. Spectrophotometry
Parts of the Spectrophotometer
6. Photodetector
•  It detects and converts traps.
•  It detects the amount of light that passes
through the sample in the cuvet.
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A. Spectrophotometry
Parts of the Spectrophotometer
Kinds of Detectors:
a.  Barrier layer cell/Photo cell/Photovoltaic cell
•  It is the simpliest detector;Ieast expensive; temperature-
sensiCve
•  It is a basic phototransducer that is used for detecCng and
measuring radiaCon in the visible region.
•  It is composed of selenium on a plate of iron covered with
transparent layer of silver.
•  It require no external volts but uClized internal electron
transfer for current producCon — low internal resistance
•  This cell typically has a maximum sensiCvity at about 550
nm and the response falls off to about 10% of the
maximum at 350 and 750 nm
•  It is used in filter photometers with a wide bandpass.
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A. Spectrophotometry
Parts of the Spectrophotometer
Kinds of Detectors:
b. Phototube
•  It contains cathode and
anode enclosed in a glass
case
•  It has a photosensiCve
material that gives off
electron when light energy
strikes it
•  It requires external voltage
for operaCon
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A. Spectrophotometry
Parts of the Spectrophotometer
Kinds of Detectors:
c.  PhotomulCplier (PM) tube
•  It is the most common type — measures
visible and UV regions.
•  It has excellent sensiCvity and has a rapid
response - detects very low levels of light.
•  The response of the PMT begins when
incoming photons strike a photocathode.
•  These tubes are limited to measuring low
power radiaCon because intense light
causes irreversible damage to the
photoelectric surface.
•  It should never be exposed to light room
because it will burn out

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A. Spectrophotometry
Parts of the Spectrophotometer
Kinds of Detectors:
d.  Photodiode
•  It is not as sensiCve as PM but with excellent linearity.
•  It measures light at a mulCtude of wavelength
•  It detects less amount of light.
•  It has a lower dynamic range and higher noise compared
to PM tube.
•  It is most useful as a simultaneous mulCchannel detector.

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A. Spectrophotometry
Parts of the Spectrophotometer
7. Meter or read-out device
•  It displays output of the detecCon system.
Examples: galvanometer/ammeter/light-emiing diode
(LED) display
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A. Spectrophotometry

Beer's Law
•  It states that the concentraCon of the
unknown substance is directly proporConal to
the absorbed light (absorbance or opCcal
density) and inversely proporConal to the
amount of transmi?ed light (%Transmi?ance).
•  It mathemaCcally establishes the relaConship
between concentraCon and absorbance.
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A. Spectrophotometry
Absorbance (A)
•  is the amount of light absorbed.
•  is proporConal to the inverse log of transmi?ance.
•  it is mathemaCcally derived from % T.
A = abc = 2-log%T
where:
A = Absorbance
a = molar absorpCvity; absorpCvity of the compound under
standard condiCons
b = length of light through the soluCon
c = concentraCon of absorbing molecules/soluCon

Unknown soluMon: Au x C5
As
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A. Spectrophotometry
Percent Transmicance
•  is the raCo of the radiant energy transmi?ed (T) divided by the radiant
energy incident on the sample (I).
%T = It x 100
I0
where: It = transmi?ed light thru the sample
to = intensity of light striking the sample

Ø  the %T measured by commercial spectrophotometers is the raCo of the

sample transmi?ed beam divided by the blank transmi?ed beam.
Ø  in actual pracCce, the light transmi?ed by a blank is subsCtuted for I0

%T = sample beam signal x 100
blank beam signal
 COLORS AND COMPLIMENTARY COLORS
OF THE VISIBLE SPECTRUM
Wavelength Color Absorbed Complementary Color
350 - 430 Violet Yellow - blue

430 - 475 blue Yellow

475 - 495 Green blue Orange

495 - 505 Blue green Red

505 - 555 Green Purple

555 - 575 Yellow-green Violet

575 - 600 Yellow blue

600 - 650 Orange Green-blue

650 - 700 Red Blue-green

Note: If a soluCon absorbs light of a certain color (2nd column), the observed color of
the soluCon is the complementary color.
I. COLOROMETRY
A. Spectrophotometry
Notes to Remember:
•  The amount of light absorbed at a parCcular wavelength depends on molecular
and ion types present and may vary with concentraCon, pH or temperature.
•  The light path must be kept constant to have absorbance proporConal to
concentraCon.
•  DeviaCons from Beer's law may be caused by changes in instrument funcCons or
chemical reacCons.
•  Instrument deviaCon is commonly a result of the finite band pass of the filter or
monochromator.
•  Turbidity readings on a spectrophotometer are greater in the blue region than in
the red region of the spectrum.
•  An absorbance check is performed using glass filters or soluCons that have known
absorbance values for a specific wavelength - the operator simply measures the
absorbance of each soluCon at a specified wavelength and compares the results
with the stated values.
•  The linearity of a spectrophotometer can be determined using opCcal filters or
soluCons that have known absorbance values for a given wavelength.

I. COLOROMETRY
A. Spectrophotometry
Blanking Technique
•  the blank contains serum bur without the reagent to complete the assay
or vice versa
•  Reagent blank corrects absorbance caused by the color of the reagents -
the absorbance of reagents is automaCcally subtracted from each of
unknown reading.
•  Sample blank measures absorbance of the sample and reagent in the
absence of the end product, and corrects the measurement for opCcal
interference (like hemoglobin) absorbing the wavelength of
measurement.
•  A blanking process may not be effecCve in some cases of turbidity and
ultracentrifugaCon may be necessary to clear the serum or plasma of
chylomicrons.
•  Lipids interfere mainly by increasing light sca?er (turbidity))
•  To correct for arCfactual absorbance readings, "blanking" procedures or
dual-wavelength methods may be used.
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B. Flame Emission Photometry (FEP)
•  It measures the light emi?ed by a single atom
burned in a flame
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B. Flame Emission Photometery
Principle: ExcitaCon of electrons from lower to higher
energy state.
Light source: Flame (also serves as the cuve?e)
Method: Indirect internal standard method
Internal standard: Lithium/Cesium — to correct for
variaCons in flame and atomizer characterisCcs
•  It is used for the measurement of excited ions
(sodium and potassium).
•  Flickering light indicates changes in the fuel reading
of the instrument.
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C. Atomic AbsorpMon Spectrophotometry
•  It measures the light absorbed by atoms dissociated
by heat
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C. Atomic AbsorpMon Spectrophotometry
Principle: Element is not excited by merely dissociated from its
chemical bond and place in an unionized, unexcited , ground state
Light source: Hollow-cathode lamp
Interferences: chemical, matrix (differences in viscosity), ionizaCon
•  It is used for the measurement of unexcited trace metals (calcium
and magnesium)
•  It is more sensiCve than FEP; it is accurate, precise and very
specific.
•  Internal standard is not needed — changes in aspiraCon have li?le
effect on the number of ground state atoms.
•  An atomizer (nebulizer/graphite furnace) is used to convert ions to
atoms; a chopper is used to modulate the light source.
•  Lanthanum or stronCum chloride is added to samples to form
stable complexes with phosphate—to avoid calcium interference.
II. VOLUMETRIC (Titrimetric)
Principle: The unknown
sample is made to react with
a known soluCon in the
presence of an indicator

Examples:
•  Schales and Schales
method (chloride test)
•  EDTA TitraCon method
(Calcium test)
III. TURBIDIMETRY
For measuring abundant large parCcles (proteins) and
bacterial suspensions.
III. TURBIDIMETRY
Principle: Determines the amount of light blocked
(reducCon of light) by a parCculate ma?er in a turbid
soluCon.
•  It depends on specimen concentraCon and parCcle size.
•  The measurement of reducCon of light is due to parCcle
formaCon.
•  SoluCons requiring quanCtaCon by turbidimetry are
measured using visible photometers or visible
spectrophotometers.
•  Uses: protein measurements (CSF and urine); to detect
bacterial growth in broth cultures; to measure the
anCbioCc sensiCviCes; to detect clot formaCon
IV. NEPHELOMETRY
ü  For measuring the amount of anCgen-anCbody
complexes (proteins).
Principle: Determines the amount of sca?ered light by a
parCculate ma?er suspended in a turbid soluCon.
•  Light sca?ering depends on the wavelength and parCcle
size.
•  Light sca?ered by parCcles is measured at an angle,
typically 15-90 degrees to the beam incident on the
cuvet.
•  Most anCgen—anCbody complexes have a diameter of
250-1500 nm, and the wavelengths used are 320-650
nm, thus lighCs sca?ered forward (Rayleigh—Debye
type)
IV. NEPHELOMETRY
ü  Components of nephelometer: light source ( mercury-arc
lamp, a tungsten—filament lamp, a light emiing diode, and a
laser), collimator, monochromator, sample cuvet, stray light
trap, and photodetector
ü  The detector (PM tube) output is proporConal to
concentraCon
V. ELECTROPHORESIS
ü  MigraCon of charged parCcles in an electric field.
ü  Separates proteins on the basis of their electric charge densiCes.
ü  The acidic and basic amino acids determine the net charge on a
protein, hence its electrophoreCc mobility.
ü  During electrophoresis, proteins are negaCvely charge (anion) and
they move towards the anode.
V. ELECTROPHORESIS
Terminologies to remember:
•  Iontophoresis - migraCo of small charged ions
•  Zone electrophoresis — migraCon of charged
macromolecules
•  Amphoteric — is a molecule whose net charge can be either
posiCve or negaCve depending on pH condiCons.
•  Electroendosmosis/Endosmosis - movement of buffer ions
and solvent relaCve to the fixed support. Components of
electrophoresis: electrical power, support medium, buffer,
sample and detecCng system
V. ELECTROPHORESIS
Factors affecMng rate of
migraMon:
1.  Net electric charge of the
molecule
2.  Size and shape of the
molecule
3.  Electric field strength
4.  Nature of the supporCng
medium
5.  Temperature of operaCon
V. ELECTROPHORESIS
SupporMng Media:
1.  Paper electrophoresis
2.  Starch Gel — separates surface
charge and molecular size
3.  Cellulose acetate — separates
by molecular size
4.  Agarose gel — neutral;
separates by electrical charge it
does not bind protein
5.  Polyacrylamide Gel - neutral;
separates on the basis of charge
and molecular size; separates
proteins into 20 fracCons; used
to study isoenzymes.
V. ELECTROPHORESIS
Stains for VisualizaMon of FracMons
((Bands):
1.  Amido black
2.  Ponceau S
3.  Oil Red 0
4.  Sudan Black
5.  Fat Red 7B
6.  Coomassie Blue
7.  Gold/Silver stain - very sensiCve down
to nanogram quanCCes of proteins
Densitometry
•  It measures the absorbance of stain -
concentraCon of the dye and protein
fracCon.
•  Scan and quanCtate ectrophoreCc
pa?ern
V. ELECTROPHORESIS
Notes to Remember:
•  ElectrophoreCc mobility is directly proporConal charge and inversely
proporConal molecular size and viscosity of the supporCng medium.
•  The ions carry the applied electric current and allow the buffer to maintain
a constant pH during electrophoresis.
•  The ionic strength of the buffer determines the amount of current and the
movement of the proteins for a fixed voltage — if ionic strength is low,
more current is carried by the charge proteins which move a longer
distance (fast mobility); if ionic strength is high, less current is carried by
the proteins which move a shorter distance (slow mobility).
•  Increasing the strength of the field (increase current) also increases
migraCon.
•  The more the pH of the buffer differs from the isoelectric point (p1), the
greater is the magnitude of the net charge of that protein and the faster it
will move in the electric field.
V. ELECTROPHORESIS
PrecauMons:
•  If the electrodes are not properly aligned, the current may be denser on
one side of the gel than on the other; proteins will migrate farther on the
side with more current.
•  If electrophoresis proceeds too long, the proteins may migrate off the gel
into the buffer.
•  If there is a break in the electrical circuit and no current passes, the
proteins will not move from the point of applicaCon.
•  Frequently, gels show the "smile arCfact," in which samples at the center
of the gel migrate farther than those at the edges.
V. ELECTROPHORESIS
Kinds of Electrophoresis
1. Isoelectric Focusing
•  SeparaCng molecules migrate through a pH gradient using constant power
•  pH gradient; is created by adding acid to the anodic area of the
electrolyte cell and adding base to the cathode area.
•  It is ideal for separaCng proteins of Proteins move in the electric field unCl
they reach a pH equal to their isoelectric point.
•  SupporCng media: agarose gel, polyacrylamide gel and cellulose acetate

Advantages:
1.  The ability to resolve mixture of proteins.
2.  To detect isoenzymes of ACP, CK and ALP in serum.
3.  To idenCfy geneCc variants of proteins such as alpha-l-anCtyrpsin.
4.  To detect CSF oligoclonal banding.
V. ELECTROPHORESIS
Kinds of Electrophoresis
Isoelectric Focusing
V. ELECTROPHORESIS
Kinds of Electrophoresis
2. Capillary Electrophoresis
•  Sample molecules are separated by electro-osmoCc flow (EOF).
•  PosiCvely (+) charged ions in the specimen emerge early at the capillary
outlet because the EOF and the ion movement are in the same direcCon.
•  NegaCvely (-) charged ions in the specimen move towards the capillary
outlet but at a slower rate.
•  It used nanoliter quanCCes of specimens.
•  Uses: separaCon, quanCtaCon and determinaCon of moleciular weights of
proteins and pepCdes; analysis of PCR products; analysis of organic and
inorganic substances and drugs.
V. ELECTROPHORESIS
Kinds of Electrophoresis
Capillary Electrophoresis
V. ELECTROPHORESIS
Kinds of
Electrophoresis
3. Western Bloing
•  Is a method used to
separate, detect and
idenCfy one or more
proteins in a complex
mixture.
VI. CHROMATOGRAPHY
•  Involves the separaCon of soluble components in a
soluCon by specific differences in physical-chemical
characterisCcs of the different consCtuents.
VI. CHROMATOGRAPHY
2 Forms of Chromatography
A. Planar
1. Paper Chromatography
–  FracConaCon of sugar and amino-add.
–  Sorbent -Whatman paper
2. Thin Layer Chromatography (TLC)
–  It is used for drug screening (semiquanCtaCve screening test).
–  Sample components are idenCfied by comparison with standards on the same plate.
–  Each drug has a characterisCc Rf value and it must match the Rf value of the drug
standard.
–  When all drug spots including the standards have migrated with the solvent front, it is
cause by incorrect aqueous to nonaqueous solvent mixture.
–  ExtracCon of the drug is pH dependent — the pH must be adjusted to reduce the
solubility of the drug an aqueous phase
–  Biological samples such as blood, urine and gastric fluid can be used for the test.
–  Sorbent - thin plasCc plates impregnated with a layer of silica gel or alumina.

RetenMon factor (Rf) Value – relaMve distance of migraMon from the point
applicaMon
Rf = distance leading edge of component moves
Total distance solvent front moves
VI. CHROMATOGRAPHY
B.  Column
1. Gas Chromatography (GC)
•  It is used for the separaCon of steroids, barbiturates, blood, alcohol
and lipids
•  It is useful for compounds that are naturally volaCle or can be easily
converted into a volaCle form
•  If the molecule of interest is not volaCle enough for direct injecCon,
it is necessary to derivaCze it into a more volaCle form.
•  Samples (urine or blood) are introduced into the GC column using a
hypodermic syringe or an automated sampler.
•  The specimens are vaporized and swept onto the column.
•  Flame ionizaCon is used as a detector for GLC.
•  EluCon order of volaCles is based on their boiling point.
•  tR - the retenCon Cme of the solute in GC or HPLC
•  Mobile phase: nitrogen, helium, hydrogen and argon (inert gases)
VI. CHROMATOGRAPHY
B. Column
1. Gas Chromatography (GC)
2 Types:
a.  Gas Solid Chromatography (GSC)
•  differences in absorpCon at thesolid phase surfaces
b.  Gas Liquid Chromatography (GLC)
•  separaCon occurs by differences in solute parCConing
between the gaseous mobile phase and the liquid
staConary phase.
VI. CHROMATOGRAPHY
B. Column
Mass Spectrometry (MS)
•  Based on the fragmentaCon and ionizaCon of molecules using a suitable source of
energy.
•  Before a compound can be detected and quanCfied by MS, it must be separated
by GC.
•  It can also detect structural informaCon and determinaCon of molecular weight.

GC-MS - is the gold standard for drug tesCng. It uses an electron beam to split the
drug emerging from the column into its component ions. QuanCtaCve measurement
of drug can be performed by selecCve ion monitoring. It is also used for xenobioCcs,
anabolic steroids and pesCcides.

Tandem mass Spectroscopy (MS/MS)- can detect 20 inborn errors of metabolism
from a single blood spot
VI. CHROMATOGRAPHY
B. Column
1. Gas Chromatography (GC)
2 Types:
a.  Gas Solid Chromatography (GSC)
•  differences in absorpCon at thesolid phase surfaces
b.  Gas Liquid Chromatography (GLC)
•  separaCon occurs by differences in solute parCConing
between the gaseous mobile phase and the liquid
staConary phase.
VI. CHROMATOGRAPHY
B. Column
2. Liquid Chromatography
•  Is based on the distribuCon of solutes
between a liquid mobile phase and staConary
phase.
•  HPLC is the most widely used liquid
chromatography.
VI. CHROMATOGRAPHY
B. Column
2. Liquid Chromatography
High Performance Liquid Chromatography (HPLC)
•  It uses pressure for fast separaCon, controlled
temparature, in-line detectors and gradient eluCon
technique.
•  Uses: fracConaCon of drugs, hormones, lipids,
carbohydrates and proteins; separaCon and quanCtaCon of
various hemoglobins associated with specific diseases (e.g.,
thalassemia); rapid HbA1c test (within 5 minutes)

Reverse phase HPLC - mobile phase, it is more polar than
staConary phase.

Note: An internal standard is used in HPLC and GC methods to
compensate for variaCon in extracCon and injecCon.
VI. CHROMATOGRAPHY
B. Column
5 SeparaMon Mechanisms Used in Liquid
Chromatography:
1.  Gel/Gel PermeaMon/Gel FiltraMon/Size Exclusion /
Molecular Sieve Chromatography.
•  Separates molecules based on differences in their size and
shape
•  As solutes travel through the gel, large molecules remain in
the mobile phase are eluted rapidly from the column
A.  Hydrophilic gel (Gel FiltraCon)
–  For separaCon of enzymes of enzymes anCbodies and proteins
–  Example: dextran and agarose
C.  Hydrophobic gel (Gel PermeaCon)
–  For separaCon of triglyceride and fa?y acid
–  Example: sephadex
VI. CHROMATOGRAPHY
B. Column
5 SeparaMon Mechanisms Used in Liquid
Chromatorgraohy:
2. Ion Exchange Chromatography
•  The mechanism in this type of chromatography is
the exchange of sample ions and mobile phase
ions with the charged group of the staConary
phase
•  For separaCon of amino acids, proteins and
nucleic acids
•  SeparaCon of nucleic acids and proteins depends
primarily on eb primarily on the sign and ionic
charge density
VI. CHROMATOGRAPHY
B. Column
5 SeparaMon Mechanisms Used in Liquid
Chromatorgraohy:
3. ParMMon Chromatography (Liquid-Liquid
Chromatography)
•  SeparaCon compounds is based on thier
parCCon between a liquid mobile phase and a
liquid staConary phase coated on a solid
support.
•  For separaCon of therapeuic drug and their
metabolites.
VI. CHROMATOGRAPHY
B. Column
5 SeparaMon Mechanisms Used in Liquid
Chromatorgraohy:
4. Affinity Chromatography
•  It uses immobilized biochemical ligands as the
staConary phase to separate a few solutes from other
unretained solutes.
•  This type of separaCon uses the so-called lock-and-key
binding that is widely present in biologic systems.
•  For separaCon of lipoproteins, carbohydrates and
glycated hemoglobins
•  it is also used to separate and prepare larger quanCCes
of proteins and anCbodies for study.
VI. CHROMATOGRAPHY
B. Column
5 SeparaMon Mechanisms Used in Liquid
Chromatorgraohy:
5. AdsorpMon Chromatography (Liquid-Solid
Chromatography)
•  SeparaCon is based on the differences
(compeCCon) between the adsorpCon and
desorpCon of solutes at the surface of a solid
parCcle.
•  The compounds are adsorbed to a solid
support such as silica or alumina
VI. CHROMATOGRAPHY
B. Column
Bases of SeparaMon:
1.  Rate of Diffusion
2.  Solubility of the solute
3.  Nature of the solvent
4.  Sample volaClity/solubility
5.  DistribuCon between
6.  Molecular Size (molecular sieving)
7.  Hydrophobicity of the molecule
8.  Ionic a?racCon
9.  DifferenCal distribuCon between two immiscible liquids
10.  SelecCve separaCon of substances
11.  Differences in adsorpCon and desorpCon of solutes
VII. FLUOROMETRY/MOLECULAR
LUMINESCENCE SPECTROPHOTOMETRY
•  It measures the amount of right intensity
present over a zero background
VII. FLUOROMETRY/MOLECULAR
LUMINESCENCE SPECTROPHOTOMETRY
•  Principle: It determines the amount of light emi?ed by a
molecule amer excitaCon by electromagneCc radiaCon.
•  Light sources: mercury arc or xenon lamp
•  Light detectors: PhotomulCplier tube or phototube
•  It uses 2 monochromators (either filters , prisms or
graCngs) - the wavelength that is best absorbed by the
soluCon to be measured is selected by the primary filter;
the incident light is prevented from striking the
photodetector by the secondary filter.
•  It is about 1000x more sensiCve than spectrophotometer -
emi?ed radiaCon is measured directly.
•  It is affected by quenching - pH andtemperature changes,
chemical contaminants, UV light changes.
•  Uses: measurement of porphyrins, magnesium, calcium
and chatecolamines
VIII. CHEMILUMINESCENCE
•  It differs from fluorescence and phosphorescence
in that the emission of light is created from a
chemical or electrochemical reacCon, and not
from absorpCon of electromagneCc energy.
•  In this method, no excitaCon radiaCon is required
and no monochromators are needed because the
chemiluminescence arises from one species.
•  It is more sensiCve than fluorescence.
•  Principle: The chemical reacCon yields an
electronically excited compound that emits light
as it returns to its ground state, or that transfers
its energy to another compound, which then
produces emission.
VIII. CHEMILUMINESCENCE
•  It involves the oxidaCon of an organic compound (e.g.,
dioxetane, luminol, acridinium ester) by an oxidant
(hydrogen peroxide, hypochlorite, or oxygen). These
oxidaCon reacCons may occur in the presence of catalysts,
such as enzymes (alkaline phosphatase, horseradish
peroxidase, or microperoxidase), metal ions (Cu2+ or Fe3+
phthalocyanine complex), and hemin.
•  The excited products formed in the oxidaCon reacCon
produce chemiluminescence on return to the singlet state.
•  A typical signal from a chemiluminescent compound rises
rapidly with Cme and reaches a maximum when reagent
and analyte are completely mixed.
•  Use: Immunoassays
•  Photodetector: PhotomulCplier tube (luminometer)
IX. OSMOMETRY
•  Is the measurement of the osmolality of an aqueous
soluCon such as serum plasma, or urine.
•  Principle: It is based on measuring changes in the
colligaCve properCes that occur owing to variaCons in
parCcle concentraCon.
•  OsmoMc parMcles: glucose, urea nitrogen and sodium
•  ColligaMve properMes of the soluMon: osmoCc pressure,
boiling point, freezing point, and vapor pressure
•  When acCve osmoCc parCcles are added to a soluCon,
osmolality increases and four other properCes of the
soluCon are also affected.
•  As the osmolality of a soluMon increases the following
reacMons occur: osmoCc pressure increases; boiling point is
elevated; freezing point is depressed; and the vapor
pressure is also depressed
IX. OSMOMETRY
•  Is the most commonly used method for
measuring the changes in a colligaCve properCes
of a soluCon.
•  It is based on the principle that addiCon of solute
molecules lowers the temperature at which a
soluCon freezes at which a soluCon freezes
•  A 1.0 mOsm/kg soluCon has a freezing point
depression of 0.00186°C when compared with
pure solvent (usually water).
•  Blood plasma, with an osmolality of about 285
mOsm/kg, has a freezing point of about -0.53° C.
X. ELECTROCHEMISTRY TECHNIQUES
The measurement of current or voltage generated by the acCvity of a specific ion.

1.  PotenMometry
•  Is the measurement of electrical potenCal due to the
acCvity of free ions - change in voltage indicates
acCvity of each analyte.
•  It is also the measurement of differences i-voltage
(potenCal) at a constant current.
•  It follows the Nernst equaCon.
•  ConcentraCon of ions in a soluCon can be calculated
from the measured potenCal difference between the
two electrodes.
•  Reference electrodes: calomel and silver-silver chloride
(pH)
•  Uses: pH and pCO2 tests Ion
X. ELECTROCHEMISTRY TECHNIQUES
1.  PotenMometry cont…
Ion SelecMve Electrode (ISE)
•  An electrochemical transducer capable of responding to one given
ion
•  It is very sensiCve and selecCve for the ion it measures - it
measures the acCvity of one ion much more than other ions
present in the sample \
•  Its ionic selecCvity depends on the membrane/barrier
composiCon used.
•  ISE analyzers measure the electrolyte dissolved in the fluid phase
of the sample in mmol/L of plasma water.
•  ISE analyzers using undiluted samples are not subject to
pseudohyponatremia caused by hyperlipimec samples.
•  Protein coaCng the ISE membrane would cause a response error.
•  If interference is due to excess protein, an alternaCve mode of
analysis such ISE in undiluted specimen can be employed to yield
correct electrolyte acCvity (i.e., equivalent of osmolality)
X. ELECTROCHEMISTRY TECHNIQUES
1.  PotenMometry
Ion SelecMve Electrode (ISE) cont…
2 types of ISE:
–  direct ISE ( without sample diluCon)
–  indirect ISE (with sample diluCon)
•  ISE membrane: glass aluminum silicate
(sodium), valinomycin gel (potassium) organic
liquid membrane ion exchangers (calcium and
lithium), gas and enzyme electrodes
X. ELECTROCHEMISTRY TECHNIQUES
2. Coulometry
•  Is the measurement of the amount of
electricity (in coulombs) at fixed potenCal
•  Is an electrochemical CtraCon in which the
Ctrant is electrochemically generated and the
endpoint is detected by amperometry.
•  It follows the Faraday's law.
•  Use: Chloride test (CSF, sefum and sweats)
•  Interferences: bromide, cyanide and cysteine
X. ELECTROCHEMISTRY TECHNIQUES
3. Amperometry
•  Is the measurement of current flow produced
by an oxidaCon-reacCon
•  Examples: p02, glucose, chloride and
peroxidase determinaCons
Polarography
•  Is the measurement of differences in current
at a cnnstant voltage
•  It follows the Ilkovic equaCon.
X. ELECTROCHEMISTRY TECHNIQUES
4. Voltammetry
•  The measurement of current amer which a
potenCal is applied to an electrochemical cell.
•  It allows sample to be preconcentrated, thus
uClizing minimal analyte.
•  Anodic stripping voltametry- led and iron
tesCng