Evolve.

Adapt.
Overcome.
CEFI is now ready.

MODULE 3 / INSTRUMENTATION
MLS 109: Clinical Chemistry 1

WILLIAM CHRISTOPHER C. SALAZAR, RMT

College of Medical Technology
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

Light
Electromagnetic radiation with a range of wavelength
between 390 (violet) and 770 (red) nanometers (nm),
capable of stimulating the subjective sensation of light.
Visible range: approximately 400nm – 700nm
<400nm – ultraviolet region (UV)
>700nm – infrared region (IR)
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Parts of a wave
a) Amplitude
The distance from the midpoint of the crest or trough to the
midpoint of vibration
b) Crest
High points of the wave
c) Through
Low points of the wave
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d) Wavelength
The distance between the top of one crest and the top of the
next one
Distance from any point of a wave to another identical point to a
next wave
e) Frequency
How often a vibration occurs
The lower the wave frequency, the longer the wavelength
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COLORIMETRIC
A. Photometry
Measurement of the intensity of light
Light from a light bulb contains the entire visible
spectrum. Materials absorb light at one wavelength
and reflect the other parts of the spectrum
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Reflectance photometry
• Measure light reflected from solid surfaces
• Principle
Light is directed on unpolished solid surface of sample
Some light is absorbed, the rest is reflected
Light reflected is measured with a detector and filter
The more light absorbed, the less light reflected
Reflectance is nonlinear; therefore, a microprocessor is used
to transform the data to a linear response.
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Used in
Dipstick reagent pads
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B. Spectrophotometry
Involves measurement of the light transmitted by a
solution to determine the concentration of the light
absorbing substances in the solution.
It measures the light intensity in a narrower
wavelength.
Measures the array of lights or radiant energy
absorbed or transmitted
Measures the light transmitted by the analyte in
order to determine the concentration of light-
absorbing analyte
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Types of Spectrophotometer
1. Single-beam spectrophotometer
• Simplest type of absorption spectrophotometer
• Designed to make one measurement at a time at one
specified wavelength
• The absorption maximum of the analyte must be known
in advance when a single-beam instrument is used.
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Types of Spectrophotometer
2. Double-Beam Spectrophotometer
• An instrument that splits the monochromatic light into
two components – one beam passes throughout the
sample, and the other through a reference solution or
blank.
• The additional beam corrects for variation in light source
intensity
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2. Double-Beam Spectrophotometer
Types
a) Double-beam in space
• Uses 2 photodetectors, one each for the sample beam and the
reference beam
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b) Double-beam in time
• Uses only one photodetector and alternately passes the
monochromatic light through the sample cuvet and then
reference cuvet using a chopper or rotating sector mirror.
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Parts of the Spectrophotometer

1. Light source
• Provides polychromatic light
• Must generate sufficient and consistent 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 range, its response to change in light
intensity must be linear.
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• 2 types
• Continuum source
• Emits radiation that change in intensity
• Most widely used in the laboratory
• Examples
• Tungsten (halogen / iodide) lamp
• Commonly used for visible – near infrared region
• Deuterium lamp
• Provides UV radiation
• Xenon lamp
• Produces a continuous source of radiation which covers both
the UV and visible range
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• 2 types
• Line source
• Emits limited radiation and wavelength
• Used in atomic absorption, molecular and f lu orescent
spectroscopy
• Examples
• Mercury and sodium vapor lamps
• LASER – Light Amplif ic ation by Stimulated Emission of
Radiation
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2. Entrance Slit
• Minimizes unwanted or stray light and prevents the
entrance of scattered light into the monochromator
system.
• Stray light
• Any wavelengths outside the band transmitted by the
monochromator
• Does not originate from the polychromatic light source
• Causes absorbance error
• Limits the maximum absorbance that a spectrophotometer
can achieve
• Most common cause of loss of linearity at high analyte
concentration
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3. Monochromator
• It isolates specific wavelength of light
• Types of monochromator
a) Prism
• Wedge-shaped piece of glass, quartz or sodium chloride
• Can be rotated, allowing only the desired wavelength to pass
through an exit slit
• A narrow light focused on a prism is refracted as it enters the
more dense glass
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Prism
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b) Diffraction gratings
• Most commonly used
• Has better resolution than prism
• Made by cutting grooves or slits into an aluminized surface
of a flat piece of crown glass
• Wavelength are bent as they pass a sharp corner
• Made up of
• Grooves = for refraction
• Slits = for defraction
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Diffraction gratings
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Diffraction gratings
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c) Filters
• Simplest, least expensive, least precise
• Made by placing semi-transparent silver films on both sides
of a dielectric such as magnesium fluoride
• Produce monochromatic light based on the principle of
constructive interference of waves – light waves enter one
side of the filter and are reflected at the second surface
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Filters
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4. Exit slit
• Controls the width of light beam
• Bandpass
• The total range of wavelengths transmitted
• Accurate absorbance measurement requires a bandpass less
than 1/5 of the natural bandpass of the spectrophotometer
• Spectral purity of the spectrophotometer is reflected by the
bandpass o The narrower the bandpass, the greater the
resolution
• Allows only a narrow fraction of the spectrum to reach
the sample cuvette
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5. Cuvet
• Holds the solution to whose concentration is to be
measured
• Also called absorption / analytical / sample cell
• Types
a) Alumina silicate glass
• Most commonly used
• Can be used in 350 -2000 nm
• Transmit light effectively at wavelengths >200nm
b) Quartz and Plastic
• Used for measurement of a solution requiring visible and
ultraviolet spectra
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c) Borosilicate glass
• For alkaline solutions
• Alkaline solutions should not be left standing in cuvets for
long periods because alkali slowly dissolves glass,
producing etching.
d) Soft glass
• For acidic solutions
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6. Photodetector
• Detects the amount of light that passes through the
sample in the cuvet
• Detects and converts transmitted light into photoelectric
energy
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Types of photodetectors
a) Barrier layer cell/Photocell/photovoltaic cell
• Simplest detector, least expensive, temperature sensitive
• Used in filter photometers with a wide bandpass
• Basic phototransducer that is used for detecting and
measuring radiation in the visible region
• Composed of selenium on a plate of iron covered with
transparent layer of silver
• Has a maximum sensitivity at about 550nm and the
response falls off to about 10% of the maximum at 350nm
to 750nm
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b) phototube
• Contains cathode and anode enclosed in a glass case
• It has a photosensitive material that gives off electron
when light energy strikes it
• Requires external voltage for operation
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c) Photomultiplier tube
• Most commonly used detector – measures visible and UV
region
• Excellent sensitivity and rapid response (detects very low
level of light)
• Limited to measuring low power radiation because intense
light causes irreversible damage to the photoelectric
surface.
• May burn-out if exposed to room light
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d) Photodiode
• Not as sensitive as PMT but with excellent linearity
• Measures the light at a multitude of wavelengths but
detects less amounts of light
• Has lower dynamic range and higher noise compared to
PMT
• Most useful as a simultaneous mechanical detector.
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7. Read-out Device/Meter
• Displays the output of the detection system
• Example
• Galvanometer
• Ammeter
• LED display
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Things to consider
✔ Light shield
used to protect cuvette from unwanted light that may
cause measurement errors
✔ Holmium oxide / didymium filters
used to check wavelength calibration of
spectrophotometer
✔ Nickel II sulfate
solution with sharp cutoff wavelength used for detection
of stray light
Evolve.
Adapt.
Overcome.
CEFI is now ready.

MODULE 3 / INSTRUMENTATION
MLS 109: Clinical Chemistry 1

WILLIAM CHRISTOPHER C. SALAZAR, RMT

College of Medical Technology
 COLLEGE OF MEDICAL TECHNOLOGY
Calayan Educational Foundation, Inc.

Beer’s Law
States that the concentration of the unknown
substance is directly proportional to the absorbed
light (absorbance or optical density) and inversely
proportional to the amount of transmitted light (%
transmittance).
Mathematically establishes the relationship
between concentration and absorbance.
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Percent transmittance
The ratio of the radiant energy transmitted (T)
divided by the radiant incident on the sample (I)
Measured by spectrophotometers to derive the
concentration of the unknown
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Percent transmittance
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Absorbance
Amount of light absorbed
Inverse logarithm of transmittance
Cannot be directly measured by the
spectrophotometer
Mathematically derived from % transmittance
Absorbance = abc = 2 – log % transmittance
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Blanking Techniques
Used to correct for artifactual absorbance readings
Reagent blank
Corrects the absorbance caused by caused by the color
of the reagents
The absorbance of reagents is automatically subtracted
from each of unknown reading.
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Blanking Techniques
Sample blank
Measures absorbance of the sample and reagent in the
absence of the end product.
Corrects the measurement for optical interference (like
hemoglobin) abs orbing at the wavelength of
measurement.
Ineffective in cases of turbidity (due to lipemia)
Ultracentrifugation may be necessary to clear the serum or
plasma of chylomicrons
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C. Flame/Atomic Emission Photometry

It measures the light emitted by a single atom
burned in a flame
Used for measurement of excited ions (sodium and
potassium)
Internal standard: Lithium/Cesium – corrects
variation in flame and atomizer characteristics
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Principle :
Substances being exposed to high temperature results
in excited atoms which almost immediately return to
ground state giving off energy of a specific wavelength
Excitation of electrons from lower to higher energy state.
Concentration of the substance is directly proportional
to the amount of light produced
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Components
1. Gas
source of flame
mixture of hydrogen and oxygen, natural gas, acetylene
and propane in conjunction with air and O2
flame temperature should be held constant
2. Atomizer / Burner
introduces heat to atoms
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3. Light source
Each element produces an individually characteristic
wavelength
Sodium – yellow
Potassium – violet
Magnesium – blue
Lithium and Rubidium - red
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4. Entrance slit
5. Monochromator
Isolates the specific wavelength of interest
6. Exit slits
7. Detector
Requires water and standards to establish thermal
equilibrium before measurements
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Internal standards
Provides a reference point in sample, control and
standards
Corrects variations in flame and atomizer characteristics
Minimizes the effects of changes in f lame temperature
and aspiration rate.
Acts as a radiation buffer to reduce the effects of mutual
excitation
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1. for sodium and potassium measurement

Lithium
Cesium
2. for lithium measurement
Potassium
Cesium
Flickering light indicates changes in the fuel reading
of the instrument.
Not routinely used
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D. Atomic Absorption Spectrophotometry

It measures the light absorbed by atoms
dissociated by heat.
Used for measurement of unexcited trace minerals
(calcium and magnesium)
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Principle
Measures the amount of light absorbed by the element
sample in an unexcited state, unionized state, neutral
atom ground state and dissociated from its chemical
bond
Ground state atoms absorb light at defined wavelengths
Measures light absorbed by ground state atoms
Inverse of flame photometry
After the f lame dissociates the element from the chemical
bonds, the atom remain in the ground state and the amount if
light absorbed is measured rather than emitted.
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More sensitive than FEP

Used for detecting small amounts of metals (heavy
metals, trace elements)
Reference method for calcium
Phosphate would interfere so lanthanum or strontium are used
as diluents to bind the phosphorus.
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Operation
sample is atomized in a flame
atoms of the metals to be quantif ied are maintained at
ground state
beam of light from Hollow Cathode Lamp (HCL) is
passed through a chopper to the flame
ground state atoms in the f la me absorb the same
wavelength of the from HCL
light not absorbed by the atoms is measured as a
decrease in light intensity by the detector
difference of light leaving the HCL and the amount of
light measured by the detector is indirectly proportional
to the concentration of the analyte
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Components
1. Hollow Cathode Lamp
source of radiation energy
f ilter gas such as neon or argon gas is placed inside the
lamp
2. Nebulizer / Atomizer
sprays the sample into the flame
used to convert ions to atoms
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3. Chopper
Used to modulate the light source
4. Monochromator
isolates light by f iltering out extraneous energy from the
flame
5. Slits
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6. Detector
measures the intensity of light energy produced from the
monochromator
usually a photomultiplier
internal standard is not needed – changes in aspiration
have little effect on the number of ground state atoms
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Interferences
❑Chemical
Occurs when flame cannot dissociate the sample
❑Ionization
Occurs when sample in flame become excited
❑Matrix
Occurs when light absorption is enhanced by organic
substance
Occurs when sample is evaporated in the f lame and produces
a solid particle
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VOLUMETRIC/ TITRIMETRIC
The unknown sample is made to react with a known
solution in the presence of an indicator
Examples
Schales and Schales method = for chlride
EDTA titration method = for calcium
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TURBIDIMETRY
Measures the amount of light reduced or blocked by
particle formation
Determines the amount of light blocked (reduction
of light) by a particulate matter in a turbid solution.
The measurement of reduction of light is due to
particle formation.
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Specimen concentration is directly proportional to

the amount of light blocked which depends on the
specimen concentration and particle size.
Solutions requiring quantitation by turbidimetry are
measured using visible photometers or visible
spectrophotometers.
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Uses
Detection of bacterial growth in broth cultures
Antibiotic sensitivity tests
Coagulation studies
Protein measurements in CSF and urine
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NEPHELOMETRY
Measures the amount of light scattered by a
particular solution
Amount of light scatter is directly proportional to
the number and size of particles in the solution
Light scattered is measured at angles 15-90˚ of the
cuvette
Forward scatter - for large molecules
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Used for measurement of large particles like Ag-Ab

complexes
Components
1. Light source
mercury arc lamp
tungsten filament lamp
LED
laser
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2. Collimator
A device that produces a beam of parallel rays
3. Monochromator
4. Cuvette
5. Stray light trap
6. photodetectors
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Note
Sensitivity depends on the absence of background
scatter from scratched cuvettes and particulates in
reagents
Uses
Measurement of antigen-antibody complexes (proteins)
Evolve.
Adapt.
Overcome.
CEFI is now ready.

MODULE 3 / INSTRUMENTATION
MLS 109: Clinical Chemistry 1

WILLIAM CHRISTOPHER C. SALAZAR, RMT

College of Medical Technology
 COLLEGE OF MEDICAL TECHNOLOGY
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ELECTROPHORESIS
Migration of charged particles in an electric field.
Principle
The ionized ampotheric molecules will move either to
the cathode (negative electrode) or the anode (positive
electrode) depending on the net charge on the molecule.
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It is the movement of electrically charged

compounds in a medium resulting to their
separation based on their electrical charges when
an electric current is applied
Cation moves toward the cathode
Anion moves toward the anode
A charged colloid particle or ion will migrate toward
either anode or cathode under the influence of an
externally applied electrical field
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Migration depends on
i. Electric charge of a molecule at a given pH
a) Acid pH buffer = molecules are positive (cations) and
migrate toward the negatively charged electrode (cathode)
b) Alkaline pH buffer = molecules are negative (anions) and
migrate toward the positively charged electrode (anode)
c) Electrophoretic mobility is proportional to the net charge
d) A particle without a net charge will not migrate and remains
in the point of application
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ii. Particle size and shape

Electrophoretic mobility is inversely proportional to the
molecular size and viscosity of the supporting medium
iii. Electric field strength
Higher voltage = faster migration
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iv. Nature of the supporting medium

a) Cellulose acetate and agarose gel
Most commonly used for protein electrophoresis
Affected by electro-osmosis or endosmosis o Endosmosis
flow of ions toward the negative electrode
b) Polyacrylamide gel
not affected by endosmosis
v. Temperature of operation
Higher temperature = faster migration
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vi. Buffer ionic strength

Lower ionic strength = better separation = less resolution =
faster migration = longer pattern
Higher ionic strength = better resolution = less separation =
slower migration = shorter pattern.
The ions carry the applied electric current and allow the buffer
to maintain a constant pH during electrophoresis.
The more the pH of the buffer differs from the isoelectric
point, the greater is the magnitude of the net charge of that
protein and the faster it will move in the electric field.
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Components
1. Electrical Power
Voltage : 100-200 volts for about 25 minutes.
2. Support Medium
a) Paper
Oldest support medium
No longer used
b) Cellulose acetate
Replacement for paper
Separates based on molecular size
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Cellulose acetate
Advantages
substances clears with alcohol and controlled heat or acetic
acid+alcohol
excess dye extracted rapidly from the background
migration occurs in short time (20-60')
can be used for isoenzymes and hemoglobin fractions
tough when wet
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c) Agarose
Separates by electrical charge
It does not bind protein
Advantages
Reproducibility
7-8 distinct protein bands o improved resolution
no clearing
increased sensitivity
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d) Polyacrylamide gel
Separates on the basis of charge and molecular size
Not affected by endosmosis
Separates proteins into 20 fractions, used to study
isoenzymes
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3. Buffer
Carries out the applied current.
The pH of the buffer determines the charge on the
protein molecule and therefore its direction of migration.
At pH 8.6, all proteins are negatively charged and
migrate toward the anode.
Sodium barbital
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4. Sampling and Detecting System

After electrophoresis, the gel is treated with a mild
fixative, such as acetic acid, that precipitates the
proteins at the positions to which they have migrated.
They are then stained, and the gel is dried and cleared of
excess stain.
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Stains for visualization of fractions

Amido black
Ponceau S
Oil Red O
Sudan Black
Fat Red 7B
Coomassie Blue
Gold / Silver Stain
Most sensitive
Can detect nanogram quantities of proteins
*****stains must bind all fractions equally
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Densitometry
Measures the absorbance of stain – concentration of the dye
and protein fraction
It scans and quantitates the electrophoretic pattern.
Measurements are reported in g/dL
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Precautions
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
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Precautions
If there is a break in the electrical circuit and no current passes,
the proteins will not move from the point of application.
“Smile” artifact
Samples at the center of the gel migrate farther than those at
the edges
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Uses
Screening for protein abnormalities
Fractionation of isoenzymes
Identification of abnormal hemoglobins
Phenotyping of lipoproteins
Identifying the type and class of an abnormal protein by
reacting it with specific antibodies
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Isoelectric Focusing
Separation of molecules by migration through a pH
gradient
Similar to electrophoresis
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Principle
pH gradient is created by adding acid to the anodic area of the
electrolyte cell and adding base to the cathode area.
The separating molecules migrate into an area where pH is
equal to their isoelectric point
Proteins move in the electrical field until they reach a pH equal
to their isoelectric point.
Movement of molecules are based on pH
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Ideal for separating proteins of identical sizes but with

different net charges
Advantages
Able to resolve mixture of proteins
Detects isoenzymes
Identify genetic variants of proteins such as alpha-1-
antitrypsin
Detect CSF oligoclonal banding
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Capillary Electrophoresis
Sample molecules are separated by electro-osmotic flow
Positively charged ions in the specimen emerge early at the
capillary outlet because the EOF and the ion movement are in
the same direction.
Negatively charged ions in the specimen move towards the
capillary outlet but at a slower rate.
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Advantage : uses nanoliter quantities of specime

Uses
Separation, quantitation and determination of molecular
weights of proteins and peptides
Analysis of PCR products
Analysis of organic and inorganic substances and drugs
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CHROMATOGRAPY
A technique where solutes in a sample are
separated for identification based on physical
differences that allow their differential distribution
between a mobile phase and a stationary phase
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Mobile phase
Inert gas or a liquid
Stationary phase
may be silica gel bound to the surface of a glass plate or
plastic sheet
may be silica or a polymer that is coated or bonded within a
column
Involves separation of soluble components in a solution
by specific differences in physical and chemical
characteristics of the different constituents.
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Principle
Mixture can be separated into individual components on the
basis of specific differences of their physical characteristics
Separation of chemical mixture into different components
based on their physical characteristics and the interactions of
molecules with the mobile and stationary phase through a
support medium
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Principle
Technique used to separate complex mixtures on the basis of
different physical interactions between the individual
compounds and the stationary phase of the system (a solid or
a liquid - coated solid).
The goal of this technique is to produce “fractions” for
quantitation
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Bases of separation
Rate of diffusion
Solubility of the solute
Nature of the solvent
Sample volatility / solubility
Distribution between 2 liquid phases
Molecular size
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Bases of separation
Hydrophobicity of the molecule
Ionic attraction
Differential distribution between two immiscible liquids
Selective separation of substances
Differences in adsorption and desorption of solutes
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Forms of Chromatography
A. Planar
1. Paper Chromatography
Used for fractionation of sugar and amino acid
separation is determined by the rate of diffusion, solubility of
the solute, and nature of the solvent
Stationary phase : Whatman paper
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2. Thin layer Chromatography

The mobile phase moves through the stationary phase by
absorption and capillary action
Principle
Stationary phase is a thin layer of fine particles on a sheet of
glass or plastic
Mobile phase is a solvent or a mixture of solvents
Sample is applied on lower edge and is carried upward with the
solvent separated by differences between the solvent and the
sorbent
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Basic steps
sample extraction using a liquid-liquid or column technique
concentration of the extracted sample
sample application by spotting onto the silica gel plate
development of the solute in the sample using stationary and
mobile phases
solute detection using chromogenic sprays, UV light,
fluorescence and heat
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Basic steps
interpretation of chromatographic results utilizing Rf values
of solute in comparison to aqueous standards
Rf value
Retention factor value
Relative distance of migration from the point of
application
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Used for semiquantitative drug screening test

Each drug has a characteristic Rf value and it must match the Rf
value of the drug standard
Sample components are identified by comparison with
standards on the same plate
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Forms of Chromatography
B. Column
1. Gas Chromatography
Used for separation of compounds that are naturally volatile or
can be converted to gas.
Has high resolution, accuracy, sensitivity and short TAT
Components
1. Gas Tank
contains the source of gases for mobile phase (inert gases)
nitrogen, helium, argon, hydrogen, CO2, NH3, NO
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2. Injector
introduces the sample to the machine
samples are heated and vaporized then introduced
if volatilization cannot be carried out, derivation of a
substance into a more volatile form is done by
silylation - most common
alkylation
acylation
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3. Column
it is where interaction between molecules take place
Types
1. Packed Column
large sample capacity
2. Capillary Column
has higher efficiency and better detection limits
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4. Detectors
flame ionization detector
thermal conductivity detector
nitrogen-phosphorus detector
electron capture detector
flame photometric detector
mass spectrophotometric detector
5. Data system
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Types
a) Gas Solid Chromatography
Separation occurs based on differences in absorption at the
solid phase surfaces.
b) Gas Liquid Chromatography
Separation occurs by differences in solute partitioning
between the gaseous mobile phase and the liquid stationary
phase.
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Mass spectroscopy
Based on the fragmentation and ionization of molecules using a
suitable source of energy
It can also detect structural information and determination of
molecular weight.
Before a compound can be detected and quantified by MS, it
must be first separated by GC
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Mass spectroscopy
Gas Chromatography – Mass Spectroscopy
Gold standard for drug testing
In this method, quantitative
Tandem Mass Spectroscopy
Can detect 20 inborn errors of metabolism from a single blood
spot
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Forms of Chromatography
B. Column
2. Liquid Chromatography
It is based on the distribution of solutes between a liquid
mobile phase and a stationary phase.
Analyzes substances that are not readily volatile
Uses low temperature, suitable for thermolabile compounds
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Separation Mechanisms Used in Liquid Chromatography

1. Gel Filtration / Molecular Sieve Chromatography
Separates based on size and shape
As solutes travel through the gel, large molecules that remain
in the mobile phase are eluted rapidly from the column.
Hydrophilic Gel (Gel Filtration)
For separation of enzymes, antibodies and proteins
Ex : dextran and agarose
Hydrophobic Gel (Gel Permeation)
For separation of TG and fatty acids
Ex : sephadex
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2. Ion Exchange Chromatography

Separation depends on the molecular weight, size and
charge of the ions of the molecules
Involves exchange of sample ions and mobile phase
ions with the charged group of stationary phase
Stationary phase
polymers with covalently bound ions
Ions with the greatest densities are held most strongly
on ion exchange material
Used in separation of
Amino acids
Proteins
Nucleic acid
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3. Partition Chromatography (Liquid – Liquid

Chromatography)
Separation is based on differences in solubility between
2 liquid phases - one aqueous, one organic
Types
a) normal phase LC
uses polar stationary phase
b) reverse phase
uses non-polar stationary phase
Used for separation of therapeutic drugs and their
metabolites.
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4. Affinity Chromatography
Separates solutes by using immobilized biochemical
ligands as a stationary phase.
This type of separation uses the so-called lock-and-key
binding
Uses in separation of
Lipoproteins
Carbohydrates
Glycated hemoglobins
Antibodies
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5. Adsorption Chromatography (Liquid-Solid

Chromatography)
Separates based on the differences (competition)
between the adsorption and desorption of solutes at the
surface of a solid particle.
The compounds are adsorbed to a solid support such as
silica or alumina.
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Types of Liquid Chromatography

High Performance Liquid Chromatography
Uses pressure for fast separations, controlled
temperature, in-line detectors and elution technique.
Components
1. solvent reservoir
2. pumps
pushes / propels the solvent
3. injector
for sample introduction
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4. chromatographic column
functions as stationary phase
operates at room temperature
5. detector
it is where the effluent from the column passes
solutes are introduced in the order that each was
eluted
6. computer
process data and control the operation of the
system
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Uses
Fractionation of drugs, hormones, lipids,
carbohydrates and proteins
Separation and quantitation of various hemoglobins
associated with specific diseases (HB A1c)
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Liquid Chromatography – Mass Spectroscopy

It requires interface methods to convert nonvolatile to
volatile compounds and is used for detecting
nonvolatile substances in body fluids.
Utilized to confirm positive results from screening of
illicit drugs (complementary for GC-MS)
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Evolve.
Adapt.
Overcome.
CEFI is now ready.

MODULE 3 / INSTRUMENTATION
MLS 109: Clinical Chemistry 1

WILLIAM CHRISTOPHER C. SALAZAR, RMT

College of Medical Technology
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FLUOROMETRY
It measures the amount of light intensity present over
a zero background.
Determines the amount of light emitted by a
molecule after excitation by electromagnetic radiation.
Measures the amount of light emitted by a substance
due to its excitation from a source rendering a higher
or equal energy from its original state
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Spectrophotometric measurement of light emitted by a

substance that has been previously excited by a source
of UV light
After it is excited and driven into a higher energy state, a
molecule loses energy by fluorescing
The amount of light emitted is proportional to the
concentration of the substance in solution.
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Components
Light Source
Excitation/Primary Monochromator
Selects the wavelength that is best absorbed by the solution
Cuvette
Emission/Secondary Monochromator
Prevents the incident light from striking the photodetector
Photodetector
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Factors Affecting Fluorometry

Light scattering
occurs when the detector is positioned at the right angle to the
incidence light
Self quenching
as the absorbance in the sample with a high fluorophore
concentration increases, more of the excitation light is
absorbed before it reaches the molecules near the center of
the cuvette
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Self absorption
occurs when excited light is absorbed before it gets out to the
cuvette
Temperature
pH
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1000x more sensitive than spectrophotometer

Used in measurement of
Porphyrins
Magnesium
Calcium
Cathecolamines
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CHEMILUMINESCENCE
Emission of light that occurs from excitation of
chemical or electrochemical compounds
Differs from fluorescence in that the emission of
light is created from a chemical or electrochemical
reaction, and not from absorption of
electromagnetic energy.
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The chemical reaction yields an electronically

excited compound that emits light as it return to its
ground state, or that transfers its energy to another
compound, which then produces emission
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Involves oxidation of an organic compound to

produce light – the excited products formed in the
oxidation reaction produce chemiluminescence on
return to unexcited state.
Organic compounds
Dioxetane
Luminol
Acridinium ester
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Oxidant
Hydrogen peroide
Hypochlorite
Oxygen
Catalysts
Enzymes (ALP, HP, microperoxidase)
Metal ions ( Cu2+, Fe3+)
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Does not require light source and monochromators

More sensitive than fluorometer
Used in immunoassays.
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OSMOMERTY
Measures the amount of dissolved substances in a
solution
Based in measuring changes in the colligative
properties of solutions that occur due to variations
in particle concentration.
Colligative properties
osmotic pressure
increases with osmolarity
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boiling point
increases with osmolarity o freezing point decreases with
osmolarity
vapor pressure
decreases with osmolarity
Freezing point depression osmometry o Most
commonly used method for measuring the changes
in colligative properties of a solution.
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ELECTROCHEMISTRY TECHNIQUES
A. Potentiometry
Principle
measurement of potential between 2 electrodes in a solution
to measure analyte concentration
potentials are produced by interaction of metals and its ions
when ions of different concentration are separated by a semi-
permeable membrane
technique used to determine the concentration of a substance
using an electrochemical cell that consists of 2 half cells,
where the potential difference between an indicator electrode
and a reference electrode is measured
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technique used to determine the concentration of a substance

using an electrochemical cell that consists of 2 half cells,
where the potential difference between an indicator electrode
and a reference electrode is measured
Components that measures electrode potentials
1. Reference Electrode
constant voltage source
Includes the ff
a) Standard Hydrogen Electrode
international standard, seldom used in routine work
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b) Saturated Calomel Electrode

consists of mercury covered layer by a layer of mercurous
chloride in contact with saturated solution of KCl
widely used as reference electroode
unstable at 80˚C
c) Silver-Silver Chloride Electrode
more stable in high temperature than saturated calomel
electrode
most common
2. Indicator Electrode
measuring electrode
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Ion Selective Electrode

An electrochemical transducer capable of responding to one
given ion
Measures the activity of one ion much more than other ions
present in the sample.
Consists of a membrane separating a reference solution and a
reference electrode
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Its selectivity depends on the membrane / barrier composition

used.
Glass aluminum silicate – sodium
Valinomycin gel – potassium
Organic liquid membrane ion exchangers – calcium and lithium
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Types of ISE
Direct ISE
Without sample dilution
Less prone to interference
Indirect ISE
With sample dilution
Prone to interference due to lipemia
Interferences
Protein coating
Defective ISE membrane
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B. Coulometry
An electrochemical titration in which the titrant is
electrochemically generated and the endpoint is
detected by amperometry.
The number of equivalent weights of a reactant
oxidized/reduced is directly proportional to the quantity
of electricity used in the reaction o Quantity is measured
in coulomb
Used in chloride test
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C. Amperometry
Electrochemical technique that measures the amount of
current produced through the oxidation or reduction of
the substance to be measured at an electrode held at a
fixed potential
Measurement of the electrical current when a constant
potential is applied
Used in measurement of
pO2
glucose
chloride
peroxidase
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D. Voltammetry
Measures solution composition based on the current-
potential relationship in an electrochemical cell when the
potential is varied.
The measurement of current after which a potential is
applied to an electrochemical cell
It has the capacity for multi-element measurements.
Allows sample to be preconcentrated, thus utilizing
minimal analyte
Used for lead and iron testing
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D. Voltammetry
Measures solution composition based on the current-
potential relationship in an electrochemical cell when the
potential is varied.
The measurement of current after which a potential is
applied to an electrochemical cell
It has the capacity for multi-element measurements.
Allows sample to be preconcentrated, thus utilizing
minimal analyte
Used for lead and iron testing