CLINICAL CHEMISTRY 4: Lecture
Instrumentation
 Chemical analysis of blood samples
 Differ in their principle and application
 The most common used in clinical chemistry involves the
principle of light absorption
Colorimetry
 Measured is colored, it absorbs light within the visible
spectrum (400-700 nm Light)
 Relies on the human eye for interpretation
 Beer’s law states that the concentration of an analyte in a
solution is directly proportional to the amount of light it
absorbs
 The more light absorbed the more analyte present
Two types Parts of the spectrophotometer
 Visual colorimetry, the color intensity of the solution is
marked against a standard solution. e.g. Dobowski
(Duboscq) colorimetry
 Photoelectric colorimetry, measurement of light intensity
(independent of wavelength) involves the use of a
photoelectric device or detector
*insert exit slit
Light source
Instrumentation
Spectrophotometry
 The quantitative measurement of the reflection or
transmittance properties of a solution as a function of
wavelength
 Allows measurement of light intensity in a much narrow
wavelength range thus it is suitable for both colored and
colorless solutions
 Both visible and UV (<400 nm) spectrophotometer
depending on the absorbance peak of the analyte
Two types
Single Beam (light source, diffraction grating
apparatus, sample detector)
Double Beam (light source, monochromator,
standard + sample, sample detector
and standard detector)
 provide radiant energy
 Visible
o Tungsten bulb (400-700 nm)
JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS
 CLINICAL CHEMISTRY 4: Lecture
 Non-visible UV light
o Hg arc lamp, deuterium lamp,
o H vapor lamp,
o Tungsten iodide lamp (high intensity and long
lasting visible),
o UV light
 Non- visible infrared (IR) light
o Used for both analysis of the composition of renal
and gallstones, purified drugs, and toxicologic
substances
Entrance slit
 Minimizes stray lights emitted by the lamp and prevents
scattered light from entering the monochromator
Prevents absorbance error
Monochromator
 Replaces the filter of colorimeter
 Has the ability to isolate sharply specific wavelength of
light or to select the amount of light that will pass
through the cuvette
 May either be prism (triangular or wedge piece of
transparent material capable of resolving specific
wavelengths) or diffracting grating (grooved, may
contain about 3000 or more grooves per nm
Glass (visible spectrophotometry)
Quartz (for UV spectrophotometry)
Sodium chloride or Potassium bromide (infrared
spectrophotometry)
Exit slit
 this controls the amount of emergent light the passes
through the cuvette
Instrumentation
Cuvette
 A.K.A. analytical cell, absorption cell
 Holds the solution
 Aluminosilicate for strong acidic solutions, resistant to
etching
 Borosilicate for strong alkaline solutions
 Glass are not suitable for UV spectrophotometry since
they could block the passage of UV light
 Quartz or plastic for UV
Common errors in handling cuvette
1. Failure to position the analytical cell properly in the
photometer
2. Failure to match the absence absorbance reading of the cell
Detector, Photocell or Photomultiplier tube
 Measures the intensity of the emergent light
 Some detectors are made of barrier layer cells
 When the light falls on certain metals, electron flows in
proportion to the intensity of light
 Photomultiplier tube for Visible and UV sensitive
Meter or Readout Device
 This numerically present absorbance or percent
transmittance
JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS
 CLINICAL CHEMISTRY 4: Lecture

Beer’s Law (Beer’s-lambert’s Law)  The light intensity of the characteristic wavelength produced
by the atom is directly proportional to the number of atoms
 States that the concentration of the solution is directly
emitting the energy, which in turn directly proportional to
proportional to the amount of light absorbed and inversely
the concentration of the substance of interest in the sample.
proportional to the light transmitted.  Excitation of the analyte
 Beer’s Law can be expressed in equation form as shown  Visible spectrum
below:
 Qualitative (color)
 Quantitative (photons of light)
100
𝐴 = 𝑎𝑏𝑐 = log = − log %𝑇  Different electropositive elements when excited by heat
%𝑇 given off by a hot flame emit in the form of a colored light
A=Absorbance
characteristic of that element, when the atoms return to the
a=Is the absorptivity value
ground state
b= Is the light path of the solution in cm
c= Concentration of the substances of interest
Analyte Wavelength (nm) Color
%T= Percent transmittance
Lithium 670 nm Red
Blank Correction Sodium 589 nm Yellow
Magnesium Blue
 ensures the accuracy of the results by eliminating interfering Potassium 466 nm Violet
substances Calcium 682 nm Red orange
Rubidium Red
types of blanks Barium 559 nm Lime green
1. Reagent Blank
- Contains only the reagent (no sample)
- Used to correct for the absorbance reading
2. Sample Blank
- Contains only the sample (no reagent)
- Used to correct non-specific absorbance of
other substances in the sample (hemoglobin,
bilirubin, lipids)
Flame Emission Spectroscopy / FES
 Quantitative analysis of serum and plasma electrolytes

Instrumentation JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS

 CLINICAL CHEMISTRY 4: Lecture

Parts of Flame Emission Spectroscopy Atomic Absorption Spectrophotometry/ AAS

 The inverse of flame emission spectroscopy
 NO EXCITATION of electrons but merely dissociation of
their chemical bonds as free atoms in the neutral ground
state.
 Use hollow cathode lamp as the energy source
Parts of the Atomic Absorption Spectroscopy

Main components
1. Burner assemble
2. Optical system
3. Photodetector
Two methods of doing flame spectroscopy
Potential interference
Direct method 1. Chemical interference
- Standards of sodium or potassium is - Fails to dissociate the sample into free atoms
aspirated directly into the flame to provide a e.g. Phosphate interference determination of
series of meter readings against which is as calcium caused by the formation of calcium
unknown solution is compared phosphate.
Indirect or internal - Lanthanum and strontium is added to
- Lithium is used as the internal standard displace the Calcium phosphate or a higher
- It acts as a radiation buffer to prevent mutual temperature may be used instead
co-excitation 2. Ionization interference
- Atoms are in the flame becomes excited
instead dissociated only and the atoms emit

Instrumentation JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS

 CLINICAL CHEMISTRY 4: Lecture

energy of the same wavelength as that being Nephelometry

measured.
- Solve this by lowering the flame  Measured the amount light scattered by suspending particles
 Greater precision than turbidimetry
Two advances that improved the quality of metal analysis by AAS:  Measured at right angle 90 
 The amount of light scattered is related to the number and
1. Zeeman Background Correction Method size in the light beam
- Employ very powerful electromagnet at the
sample site interacting with one plane of the
Difference between Nephelometry and Turbidimetry
polarized light
2. Smith-Hieftje Method
- Involves pulsing the hollow cathode lamp at
high current until it undergoes self–reversal
Turbidimetry
 Determines the amount light blocked (180) by the
suspending particles in the sample as light passes through the
cuvette of a spectrophotometer or a colorimeter
 Ideal for determination of proteins in cerebrospinal fluid and
urine.
 Determinants:
1. Size and number of particles
2. Cross-sectional area of each particles
3. Depth of the tubes (cuvette) Fluorometry
Interferences:  Measures analyte which have the ability to absorb light of
- Difference in the particle size between lower wavelength and transmit it at a higher (visible)
standard and the test solutions wavelength.
- Settling of the heavy particles in solution  The usual source of exciting radiation is the UV light
(remedied with the use of stabilizing produced by a mercury arc lamp or a xenon lamp
colloids like Gum Arabic and Gelatin to  Extremely sensitive and specific
provide a viscous medium that will retard  Determination of porphyrins, magnesium, calcium, and
the rate of precipitation. catecholamines

Instrumentation JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS

 CLINICAL CHEMISTRY 4: Lecture

Fluorometry measures two different fluorescence spectra: Parts of the Refractometer

1. Excitation spectrum
- Wavelength of light used to excite the
electrons
2. Emission spectrum
- Wavelength where fluorescence is at its
highest
Parts Fluorometry

Types of Refractometer
 Traditional handheld refractometer
 Digital handheld refractometer (LED light)
 Inline process refractometer (LED light, monitor
concentration more accurate)
 Abbé refractometer (highly precise)
Refractometry
Osmometry
 Determination of the concentration of the solution by
measuring its refractive index  Use to measure the concentration of solutes particles
(molecular weight) in a solution.
Interferences:
Types
 Wavelength of the incident light
 Nature of the liquid medium  Freezing point osmometer
 Concentration  Vapor Pressure osmometer
 Temperature  Membrane osmometer

Instrumentation JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS

 CLINICAL CHEMISTRY 4: Lecture

Part of freezing osmometer Membrane osmometer

Note:
 1 mOsm/kg of solution has a Freezing depression of
.00186C when compared to solvent
 Blood plasma with osmolality (ℓ) 285 mOsm/kg has freezing
point of -.53C

Colligative properties
Freezing point
- Depressed by 1.86C
Boiling point
- Elevated by .52C
Osmotic pressure
- Increased .3 mm/Hg at 25C
 more analytes the lower the freezing point Vapor pressure
- Decreased
Vapor of osmometer
Interferences:
Lipemic samples-freezing point
Volatile solutions- vapor pressure

Instrumentation JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS

 CLINICAL CHEMISTRY 4: Lecture
Densitometry
 Use to measure the absorbance of the stain on a supporting
medium (paper or Gel)
Components:
Light sourceFilterMonochromatorElectrophoretic
stripDetectorX-Y strip chart record
Types of Densitometer
Transmission Densitometer (transparent film) Reflected and
transmitted light
Reflection Densitometer (components: light sourcelensFilter
printedlens (the same with spectrophotometer)
Combination Densitometer
Spot- single spot
Line- Successive spot
Bi-dimensional- 2D value light absorbance
Electrochemistry
 Measurement of current or voltage generated by activity of
specific ions.
 Applied for the measurement of blood gas and pH.
Potentiometry
 Measures the potential between two electrodes in a solution.
 Concentration of ions in solutions is calculated from the
measured potential difference between the two electrodes
 Deference is related to the molar concentration of the
solution as expressed by the Nernst equation.
Instrumentation
Potentiometry uses two types of electrodes:
Indicator Electrode
 AKA measuring electrode (platinum wire and carbon rod)
 Respond proportionately to the concentration of the analyte
Reference Electrode
 Maintain a constant voltage source
 Under controlled condition for the significant length of time
 E.g. saturated calomel electrode, Ag-AgCl electrode and
normal hydrogen electrode
Reference Electrodes
 Normal Hydrogen Electrode (NHE) -Consists of a
platinized platinum electrode in a 1.228 N HCl solution with
hydrogen at atmospheric pressure bubbled over the platinum
surface
 Saturated calomel electrode –most reference electrode
 Standard redox potential is -0.242 v
 Unstable at >80C use Silver-silver electrode constructed
low-voltage (-6 v) battery, a platinum wire and a silver wire
pH
 Difference in the potential between the internal and external
electrode determines the hydrogen ion activity
 First and still the most common pH electrodes are glass
electrode operating the principle of potentiometry.
 Hydrogen ion activity is measured
 Electrode consist small bulb made layers of hydrated and
non-hydrated glass contains chloride ion buffer solution
 Buffer has a known hydrogen ion activity
JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS
 CLINICAL CHEMISTRY 4: Lecture
 Internal electrode: Ag-AgCl- reference electrode
 External electrode
pCO2 Gas electrode
 pH electrode contained within a plastic jacket filled with
sodium bicarbonate buffer and has a gas permeable
membrane (Teflon or Silicon) across its opening
 Blood CO2 diffuses through the membrane and mixes with
the buffer
 Chemical reaction:
CO2 + H2 O HCO3 - + H+
.: the production of hydrogen ion decreased the pH which is
then measured by the potentiometric pH electrode
 CO2 measured by the Severinghaus
Ion-Selective electrode
 Very sensitive and selective for the ion it is measuring
 Used measuring serum and urine electrolytes
 Used to measure Na, K, Cl, Ca, Mg, and NH 3 in serum
 It consists of a membrane separating a reference solution
from the solution being analyzed
There are three basic ISE classes:
1. Ion-selective glass (for H+, Na+, NH4 )
2. Solid-state electrolytes (Ag-AgCl membrane for chloride
determination in sweat)
3. Liquid ion-exchange membrane (for pH determination)
Instrumentation
Coulometry
 It is an electrical titration where the titrant is
electrochemically generated and the endpoint is detected
by amperometry.
 based on Faraday Law
Amperometry
 measures the electrical current at a single applied
potential
 coulometry and amperometry are combined in the
measurement of Chloride in the body fluids such as sweat
(cystic fibrosis), urine, and CSF.
Coulometry and Amperometry
- demonstrate in the determination of chloride
by a chloridometer or Cotlove titrator.
pO2 Gas electrode
 O2 measured by Clark electrode
 These gas sensing electrodes use amperometric or current-
sensing electrolytic cell as indicator
 Consist of gas-permeable membrane (polypropylene) which
allows only dissolved oxygen to pass through. The oxygen
mixes with phosphate buffer solution and reacts with
polarized platinum cathode via the following reaction:
O2 + 2H2 O + 4e-  4 OH-
 Changes is directly proportional to the partial pressure of
oxygen in the sample
 Silver anode provides the oxidizing electrode to complete
the circuit
JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS
 CLINICAL CHEMISTRY 4: Lecture

Electrophoresis Electric field

 method of separation and purification involves the migration  an increase in potential gradient increase the rate of
of charged particles in an electric field migration
 Components should be CHARGED
 Suitable for the separation and the quantitation of protein in Medium
the body  inert medium exert adsorption and molecular sieving effects
 A charged particles or iron will migrate towards the anode or on the particles, influencing its rate of migration
the cathode depending on the isoelectric pH (pI) –no excess  Sephadex- molecules larger than the pores are excluded
net charge, of the solution under the influence of an from entering the gel bead and those molecules migrate
externally applied electric field.
faster
Mainly used for separation of complex mixtures of biological  Polyacrylamide, starch and agarose- the larger the
substances possess charged functional group molecules are made to squeeze through the pores. The
smaller molecules pass through the pores easily but the
1. Proteins larger molecules are retarded.
2. Polysaccharides
3. Nucleic acids Buffers
4. Peptides
 Choice of buffer depends upon the type of sample being
5. Amino acids
electrophoresed
6. Oligosaccharides
Composition:
The migration of the particles is influenced
Commonly used are formate acetate, citrate, phosphate,
1. Net electric charge of the molecules EDTA
2. Size and shape of the molecules Ion strength:
3. Electric field strength Usually between 0.05 to .1M
4. Nature of the supporting medium, or type electrophoretic pH
medium and; Determines the degree of ionization of organic
5. Temperature of operation components

Sample
 the higher the charge, greater is the electrophoretic mobility

Instrumentation JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS

 CLINICAL CHEMISTRY 4: Lecture

Common buffers used: Stains

 May used to visualize the protein bands
1. Barbital (Veronal) buffer
 Commonly used stains:
- Provides pH 8.6
- Proteins in normal serum assume a net 1. CSF proteins- Coommasie blue
negative charge 2. Lipoproteins- Oil Red O, Sudan black, Fat Red, 7B
3. Protein and enzyme electrophoresis- Amido Black,
- Migrates towards the anode
Ponceau S
- Pattern of separation –electrophoretogram
4. DNA- Ethydium bromide
- Albumin (farthest from the origin),  1 -
5. Nigrosin
Globulin,  2 -Globulin, -Globulin, (origin)
6. Eosin
and -Globulin (towards the cathode)
2. Tris-boric acid EDTA buffer Clearing agents
- Provides the pH 8.7
Components  may added to prevent diffusion of separate fractions to each
1. Power supply- operates either constant current or voltage other
2. Buffer  examples: methanol, cyclohexane acetic acid, and dioxane
3. Support media Types of Electrophoresis
4. Sample
5. Detecting system 1. Moving boundary or frontal electrophoresis
Serum and Plasma Electrophoretogram - Separation of molecules using homogenous
solution
- No distinct zones are formed
- Fraction involved: albumin, , , -
Globulins
2. Zonal electrophoresis
- Use of support medium
- Fraction involved: albumin,  1 ,  2 , , -
Globulins
Examples of zonal electrophoresis:
 Paper electrophoresis –carried out on filter paper
 Cellulose acetate electrophoresis – widely used because of
it uniformity and ease of preparation

Instrumentation JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS

 CLINICAL CHEMISTRY 4: Lecture
 Gel electrophoresis- method for preparation and analysis of
macromolecules and their fragments, based on their size and
charge
2 types of gel:
Agarose –highly purified uncharged polysaccharides derived
from agar; use to separate macromolecules such as nucleic
acids, large proteins and protein complexes
Polyacrylamide- used to separate most proteins and small
oligonucleotides because of the presence of small pores
 Agarose Gel electrophoresis- used as a purified fraction
and therefore, does not produce electroendosmosis; based on
electric charge
 Starch electrophoresis- separates proteins on the basis of
surface charge and molecular size; now widely used because
of technical difficulty in preparing the gel
 Polyacrylamide Gel electrophoresis- involves separation of
proteins on the basis of charge and molecular serum
Polyacrylamide gel electrophoresis (PAGE)
 Inert support whose porosity can be adjusted by changing
the composition of acrylamide prior to polymerization
 Done for standard separation of negative protein
 Sodium dodecyl sulfate (SDS)-PAGE involves the use of
SDS to denature the protein
Two-dimensional electrophoresis 2-DE
 Standard electrophoretic separation in one direction is
followed by SDS-PAGE in the perpendicular direction
 Hundreds of identifiable protein peaks
Instrumentation
Isoelectric Focusing
 Separation of proteins based on its isoelectric points
 Involves the migration of proteins in a pH gradient created
by addition of an acid to the anodic area of the electrolytic
cell and a base to the cathode area
 They stop migrating when they reach their isoelectric points
 Isoelectric point is the pH where the net charge of the protein
molecule is zero
 Isoelectric focusing has been applied in the assay of ACP
isoenzymes
Application: detection isoenzymes of CK and ALP in serum
Flow Cytometry
 Flow cytometry is a technique used for counting and
examining microscopic particles by suspending them in a
stream of fluid and passing them by an electronic detector
apparatus.
Principle:
 Diagram of the optics of a flow cytometer
 a source of monochromatic light, usually a laser diode (A)
sends a beam that is collimated (LT) and focused (L2) by
JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS
 CLINICAL CHEMISTRY 4: Lecture
lenses on the laminar flow chamber (C), after passing
through the camera, the direct light beam is arrested by a
screen (s), while the scattered light is focused by another lens
(L3) on a photodiode (D1), this constitutes the Forward
Scatter detector (FSC).
 The side scattered light and the fluorescence are focused by a
lens onto a dichroic mirror (that reflects most of the light of
wavelength equal to that produced by the Source (A), goes
through a filter (Fl) and impinges on a photodiode
detector(D2). This constitutes the Side Scatter detector
(SSC).
Instrument Overview
Flow Cytometers
-are able to analyze several thousand particles every second, in "real
time, and can actively separate and isolate particles having specified
properties.
 A flow cytometer is similar to a microscope, except that,
instead of producing an image of the cell, flow cytometry
offers "high throughput for a large number of cells)
automated quantification of set parameters.
 The flow cytometer performs this analysis through a laser
beam and capturing the light that emerges from each cell as
it passes through.
Instrumentation
 The data gathered can be analyzed statistically by flow
cytometry software to report cellular characteristics such as
size, complexity, phenotype, and health.
 The fluidic system - which presents samples to the
interrogation point and takes away the waste.
 The lasers - which are the light source for scatter and
fluorescence.
 The optics - which gather and direct the light.
 The detectors - which receive the light the electronics
 The peripheral computer system – which convert the
signals from the detectors into digital data and perform the
necessary analyses
Factors Affecting instrumentation:
 Sample pressure
 Proper operation of fluidic components
 Alignment of the laser beam with the stream
 The wavelength of excitation
 Sample preparation
Electrical Impedance
 Electrical Impedance - is the measure of the opposition that a
circuit presents to the passage of a current when a voltage is
applied.
JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS
 CLINICAL CHEMISTRY 4: Lecture
Principles of Electrical Impedance
 The impedance principle of cell counting is based on
the detection and measurement of changes in electrical
impedance (resistance) produced by a particle in
conductive fluid as it passes through a small aperture
 Coulter Principle or Impedance method by Wallace
Coultier.
 Number of pulses = blood cell count
 Amplitude (height) of the pulse= volume of the cell
 A Backwash or sweep flow mechanism has been use
to prevent recirculation of cell in the aperture
Components:
 Aperture - the orifice where the particles or blood cells will
pass through
 Aperture diluent - a low concentration electrolyte solution
where the blood is diluted.
 Aperture tube - the glass tube that contains the aperture.
 External electrode – the electrode immersed in a low
concentration electrolyte solution outside the aperture tube.
Instrumentation
 Internal electrode - an electrode inside the aperture tube.
 Histogram – graphical presentation of blood cell
distribution; provide information about erythrocyte,
leukocyte and platelet frequency and distribution as well as
depict the presence of subpopulations,
Counting chambers
Common chambers that uses impedance
 RBC/ Platelet chamber
 WBC chamber
RBC chamber
 Aspirated blood is divided into two separate volumes for
measurements.
 One volume is mixed with diluents and delivered to the cell
bath, where erythrocyte and platelet counts are performed.
 As cell passes through the aperture, partially occluding it, the
electrical impedance increases, producing a voltage pulse,
the size of which is proportional to the cell size.
 The number of pulses is the number of cell counted.
WBC chamber
 The other blood volume is mixed with diluent and a
cytochemicalytic reagent that lyses only the red blood cells.
 A leukocyte count is performed as the remaining cells pass
through an aperture.
Factors Affecting Instrumentations
 Size of platelets
 Cells into 2-20 femtoliter range are counted as platelets;
 Those counter above 35 femtoliter as counted as red blood
cells. This presents a problem when attempting to obtain
platelet counts in individuals that possess small red blood
JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS
 CLINICAL CHEMISTRY 4: Lecture

cells, as counting area between platelets and cells may Types of Chromatography
overlap.
INTERFERENCES Paper chromatography
- Based on the nature of solvent, solubility of
 Aperture diameter - is crucial, with the red blood cell solute and rate of diffusion.
(RBC)/platelet aperture smaller that the WBC aperture to Gel permeable (size-exclusion) chromatography
increase platelet counting sensitivity - Size or molecular weight of the molecules
 Protein buildup - It decreases the diameter of the orifice, Ion-exchange chromatography
slowing the flow of cells and increasing their relative - Net charge of the molecule
electrical resistance. It results to lower cell counts, which
Liquid-liquid chromatography
result to falsely elevated cell volumes,
- Solubility of the solutes (partition-
 Count reduction or coincident passage loss - Coincident
passage of more than once cell at a time through the orifice coefficient)
Causes artificially large pulses, resulting in falsely increased Gas-liquid chromatography
cell volumes and falsely decreased cell counts. - Basis of sample volatility
 Recirculation of cells back into the sensing zones - It Thin-layer chromatography (TLC)
creates erroneous pulses and falsely elevates cell counts. - Similar to paper chromatography
 When the WBCs are present in extremely high numbers, - Uses glass or paper plates with thins silica,
they become a source of interference. polyacrylamide, or starch gel as matrix
High performance liquid chromatography (HPLC)
Chromatography
- Uses pressure (500-5000 lbs psi) for
pumping of aqueous or organic solution
 Method of separation based on the specific difference in the
through a column
physico-chemical chemicals e.g. column and the mobile
Gas chromatography
phase e.g. eluent
- Most powerful
 Introduced by Russian botanist Mikhail Tswett
- Can separate nanograms or picograms of
 Compounds interacting more strongly with the stationary
volatile substances
phase retained longer in the medium than those that favor
the mobile phase.
 The smaller the affinity a molecule have for the stationary
phase the shorter the time spent during reaction, and the
longer it travels.

Instrumentation JE CABANA ; MD VILLA ; NJ HABLADO ; S CAHIS