MLS 414 (Lec)

3 – ANALYTIC TECHNIQUES AND INSTRUMENTATION

Prof. Melvin Misoles | January 31, 2023

OUTLINE
I. Electrophoresis III. Osmometry
II. Electrochemistry
Techniques

TERMINOLOGIES
Ampholyte A molecule that contains both
acidic and basic groups
Electrophoretic The rate of migration of a
Mobility charged solute in an electric field,
expressed per unit field strength
Endosmosis Preferential movement of water in
one direction through
electrophoresis medium due to
selective binding of one type of
charge on the surface of the
medium
Electrophoretogram The migration of charged
macromolecules • Electrodes:
Iontophoresis The migration of small charge a. Cathode – negatively charged
ions b. Anode – positively charged
Zone The migration of charged • Thus:
electrophoresis macromolecules in a porous o Cations (+) → Cathode (-)
support medium such as paper,
o Anions (-) → Anode
cellulose acetate or agarose gel
film

ELECTROPHORESIS
• A versatile and powerful analytical technique capable
of separating and analyzing a diverse range of ionized
analytes
• Migration of charged solutes and particles in a liquid
medium under the influence of an electric field.
• Principle: the movement of electrically charged
compounds in a medium resulting to their separations
based on their electrical charged when an electric
current is applied.
• Macromolecules of interest (clinical lab):
o Proteins in serum
o Urine
o Cerebrospinal fluid (CSF)
o Erythrocytes and tissue
o Other biologic body fluids

→ Electrophoresis can be used to separate serum

proteins based on their electric charge densities.
o VLDL – Very Low Density Lipoprotein
o IDL – Intermediate Density Lipoprotein
o LDL – Low Density Lipoprotein
o HDL – High Density Lipoprotein
→ The farthest molecule to migrate is the densest.
→ Acidic and basic amino acids also determine the net
charge on a protein; hence, its electrophoretic mobility.

THEORY OF ELECTROPHORESIS
• In an electrophoresis system, chemical species which
take on electrical charge by becoming ionized, move
toward either the cathode which has a negative
electrode or the anode which has positive electrode
depending on the kind of charge they carry.

Trans Maker: Aguinaldo, L.G.

Editor: Blas, S.G. | 1
 MLS 414 (Lec)
3 – ANALYTIC TECHNIQUES AND INSTRUMENTATION
Prof. Melvin Misoles | January 31, 2023

CONVENTIONAL ELECTROPHORESIS (ions and leading to a decrease in resistance and an

increase in current
• Heat evolved during electrophoresis increases the
conductance of the system. With constant voltage
power sources, the resultant rise in current due to
increase in thermal agitation of all dissolved ions
causing an increase on both the migration rate of
protein and the rate of evaporation if water from the
stationary support medium.
Supporting Medium
• Provides the matrix in which separation takes place.
• Various types of support media are used in
electrophoresis and vary from pure buffer solutions in a
capillary to insoluble gels (e.g. sheets slabs or columns
of starch agarose or polyacrylamide or membranes of
cellulose acetate)
Cellulose Acetate
→ The general operations performed in conventional • Separates by molecular size
electrophoresis includes separation, staining, • Cellulose is acetylated to form cellulose acetate by
detection, and quantification (concentration of each treating it with acetic anhydride
fraction). • A dry brittle film composed of about 80% air space
PERFORMANCE OF ELECTROPHORETIC SEPARATION • Homogenous medium with uniform pore size and does
not absorb the protein
1. A hydrated support material is plotted to remove
excess buffer and then placed into the electrophoresis
chamber.
2. The sample is added to the support and it is placed in
contact with buffer previously added to the electrical
chambers.
3. Electrophoresis is conducted for a determined length of
time under conditions of either constant voltage or Agarose Gel
constant current. • Separates by electrical charge
• A highly purified uncharged polysaccharide derived
FACTORS AFFECTING RATE OF MIGRATION
from agar and therefore doesn’t produce endosmosis
1. Net electric charge of the molecule • Requires small amounts of sample (approx. 2 mL)
2. Size and shape of the molecule • It does not bind protein and therefore migration is not
3. Strength of electric field affected
4. Properties of Supporting Medium
5. Temperature of operation
COMPONENTS OF ELECTROPHORESIS
1. Driving force (electrical power)
2. Support medium
3. Buffer
4. Sample
5. Detecting System
Polyacrylamide Gel
• Separation of protein on the basis of charge and
molecular size
• Referred to as PAGE
• Layers of gel with different pore sizes and uses
• Gel is prepared before electrophoresis in a tube-
shaped electrophoresis cell
o Top: large pore gel
o Mid: large pore spacer gel
o Bottom: small pore separation gel
o Each layer of gel is allowed to form a gelatin
before the next gel is poured over it.
• Separated serum proteins into 20 or more fractions
Driving force (Electrical Power) rather than the usual five fractions separated by
cellulose acetate or agarose
• Power supplies operating at either constant current or • Widely used to study isoenzymes
constant voltage are available commercially
• In electrophoresis, heat is produced when current flows
through a medium that has resistance, resulting in an
increase in thermal agitation of the dissolved solute
Trans Maker: Aguinaldo, L.G.
Editor: Blas, S.G. | 2
 MLS 414 (Lec)
3 – ANALYTIC TECHNIQUES AND INSTRUMENTATION
Prof. Melvin Misoles | January 31, 2023

Tris-glycine buffer → Protein separation

(pH more than → High buffering capacity, low
8.0) conductivity
What happens after electrophoresis?
• When an electrophoresis is completed, the support is
removed from the electrophoresis cell and rapidly dried
or placed in a fixative to prevent diffusion of sample
components.
• It is then stained to visualize the individual protein
Starch zones
• Separates proteins on the basis of surface charge and • After washing excess dye, the support is dried
molecular size, as does polyacrylamide gel
• Not widely used because of technical difficulty in STAINS
preparing the gel • Used to visualize protein fractions and differ according
Agarose Polyacrylamide Gel to the type of application.
• Polysaccharide • Cross-linked polymer • The amount of dye taken up by the sample is affected
extracted from sea of acrylamide by many factors such as:
weed • Gel casted vertically o Type of protein
• Gel casted horizontally • Potent neuro-toxic o Degree of denaturation by fixing agents
• Non-toxic • Separate small Proteins • Amido black
• Separate large molecules • Coomassie Brilliant Blue
molecules • Used for DNA or • Bromphenol Blue
• Commonly used for protein separations DNA • Ethidium Bromide
DNA separations • Staining can be done • Sybr Green or Sybr Gold
• Staining can be done after pouring the gel
Lipoproteins • Sudan Black
before or pouring the • Potential carcinogen
gel Hemoglobins • Ponceau Red
• Amido Black
• Coomassie Brilliant Blue
Buffer
• Serves as a multifunctional component in the
electrophoretic process as it carries the current,
establishes pH which electrophoresis is performed and
determines the electrical charge on the solute.
• AMPHOLYTE is a molecule whose net charge can be
either positive or negative
• 2 buffer properties that affect the charge of
ampholytes:
o pH
o Ionic strand
o The ions carry applied electric current and allow
the buffer to maintain a constant pH during
electrophoresis.
• If buffer is more acidic than the pl (isoelectric point)
o Binds with H ions
o Becomes positively charged and migrates toward
the cathode
• If buffer is more basic than the pl
o Losses H ions
o Becomes negatively charged and migrates
toward the anode Suggested Wavelengths for Quantification of Protein
• A particle with no net charge will not migrate and Zones by Direct densitometry
remains at the point of application Separation Stain Nominal
Barbitone buffer → Serum protein separation Type Wavelength
(around 8.0 pH) → Poor resolution, weak buffer (nm)
Phosphate buffer → Enzyme separation Serum proteins *Amido Black B 640
(around 7.0 pH) → Low buffering capacity, high in general (Naphthol Blue Black)
conductivity *Coomassie Brilliant 595
Tris-borate-EDTA → Nucleic acid separation Blue G-250 (Brilliant
buffer (TBE) → Good resolution, high buffering Blue G)
(around 8.0 pH) capacity, low conductivity *Coomassie Brilliant 560
Tris-acetate-EDTA → Nucleic acid separation Blue R-250 (Brilliant
buffer (TAE) → High resolution, high buffering Blue R)
(around 8.0 pH) capacity, low conductivity *Ponceau S 520

Trans Maker: Aguinaldo, L.G.

Editor: Blas, S.G. | 3
 MLS 414 (Lec)
3 – ANALYTIC TECHNIQUES AND INSTRUMENTATION
Prof. Melvin Misoles | January 31, 2023

Isoenzymes *Nitrotetrazolium 570 • Albumin – highest concentration in plasma proteins

NAD(P)H(NBTH) Blue (as the • α-1 Globulins
formazan formazan) o α1-fetoprotein
Lipoprotein *Fat Red 7B (Sudan 540 o α-antitrypsin
zones Red 7B) o HDL
*Oil Red O 520 • α-2 Globulins
*Sudan Black B 600 o haptoglobin
DNA fragments *Ethidium Bromide 254 (Ex) o ceruloplasmin
(fluorescent) 590 (Em) o α2-macroglobulin
CSF proteins *Silver nitrate ------ • β-Globulins
o transferrin
→ Once electrophoretic separation and staining are o C-reactive protein
complete, it is possible to quantify the individual zones
• γ-Globulins
either as percentage of the total or as absolute
o immunoglobulins
concentration by direct densitometry
→ These plasma proteins are separated based on their
DENSITOMETRY
molecular mass, isoelectric point, and
• In densitometer, the gel is moved past a measuring electrophoretic mobility.
optical system and the absorbance of each fraction is
displayed on a recorder chart or an electronic display. REFRACTOMETRY
In most cases, the area under each peak is • Based on refractivity (the ability of the substance to
automatically integrated. bend light)
• It measures the absorbance of stain concentration of • When the light passes from one medium into another,
the dye and protein fraction the path of the light beam changes direction at the
• It scans and quantitate electrophoretic pattern boundary surface if its speed in the second medium is
• BASIC COMPONENT: different from that in the first
1. Light source • Can be used to measure protein concentration,
2. Monochromator specific gravity of urine, and column effluent of
3. Movable carriage – scan medium over the entire HPLC analysis
area • Refractive index
4. Photodetector o The ratio of the two speed of light
5. Read-out device

How it works?
• The signals detected by the photodetector are related
to the absorbance of the sample stain on the support
which is proportional to the specimen concentration.
• The support medium is moved through the light beam
at a fixed rate so that a graph may be constructed that
represents multiple density readings taken at different
points.
• Most densitometers have built-in integrator to find the
area under the curve so that all sample fractions can
be quantified.
Normal Serum Protein Electrophoresis
ELECTROCHEMISTRY
• Involves the measurement of electrical signals
associated with chemical system that are incorporated
into an electrochemical cell
• The magnitude of a voltage or current signal originating
from an electrochemical cell is related to the activity or
concentration of a particular chemical species in the
cell.
• Measurement of current or voltage generated by the
activity of a specific ion
Trans Maker: Aguinaldo, L.G.
Editor: Blas, S.G. | 4
 MLS 414 (Lec)
3 – ANALYTIC TECHNIQUES AND INSTRUMENTATION
Prof. Melvin Misoles | January 31, 2023

ELECTRODES
a) Reference Electrode
▪ Produces constant potential
b) Indicator Electrode
▪ Responds to changes in the activity of solution
▪ Measuring electrode
→ The concentration of ions in a solution can be
calculated from the measured potential difference
ELECTROCHEMICAL CELL
between the two electrodes.
• A Galvanic cell converts chemical energy into
Reference Electrode
electrical energy.
• Here, the redox reaction is spontaneous and is • Calomel/electrode
responsible for the production of electrical energy. o Consist of mercury in contact with a solution that
is saturated with mercurous chloride (calomel)
• The two half-cells are set up in different containers,
and also contain a known concentration of
being connected through the salt bridge or porous
potassium.
partition.
o Mercury ions react with fewer sample
o As the electrodes are connected there is a
components than do silver ions. Such reactions
spontaneous flow of electrons from the
can lead to plugging of the junction between
electrode with the lower electron affinity.
electrode and the analyte solution.
o These electrons pass through the external
• Silver/silver chloride
meter to the cathode where hydroxyl ions are
liberated. o Consist of a silver electrode immersed in a
o This reaction continues until one of the solution of potassium chloride that has been
saturated with silver
chemical components is depleted at which point
the cell is dead and cannot produce electrical o ADVANTANGE: can be used at temperatures
greater than 60°C whereas Calomel electrodes
energy to external meter.
cannot
• Here the anode is negative and cathode is the
o Overall better and faster
positive electrode. The reaction at the anode is
• Normal hydrogen electrode
oxidation and that at the cathode is reduction.
• The electrons are supplied by the species getting Ion-selective Electrode
oxidized. They move from anode to the cathode in • Electrochemical transducer capable of responding to
the external circuit. one specific ion
• Indicator electrode that can respond to individual types
of anions or cations
• Very sensitive and selective for the ion it measures
o It measures the activity of one ion much more
than other ions present in the sample
• 2 types of ISE
1) Direst ISE
2) Indirect ISE
• Consist of a membrane/barrier separating a reference
solution and a reference electrode from the solution to
be analyzed
• Its ionic selectivity depends in the membrane/barrier
composition used
Liquid junction a. Glass aluminum silicate (Sodium)
b. Valinomycin gel (Potassium)
• also known as a salt bridge are required to complete c. Organic liquid membrane ion exchangers
the circuit between the reference and without (calcium and Lithium)
contaminating anything d. Gas and enzyme electrodes
• FUNCTIONS:
o It allows electrical contract between the two pH Electrode
solutions • Selective for the detection of hydrogen ions
o It prevents the mixing of the electrode solutions • Indicator electrode has a glass membrane
o It maintains the electrical neutrality in each half cell • Internal reference electrode: Ag/AgCl
as ions flow into and out of the salt bridge • Glass electrode is the first and still the most common
ELECTROCHEMICAL TECHNIQUES electrode for measuring hydrogen ion activity.
• Consist of small bulb made of layers of hydrated and
Potentiometry non-hydrated glass which contains a chloride ion buffer
• Measurement of a cell potential (voltage) under solution with known hydrogen ion concentration
equilibrium conditions
• Measurement of a potential or voltage difference pCO2 Electrode
between two electrodes immersed in solution under the • pH electrode contained within a plastic jacket
condition of essentially zero current • Filled with sodium bicarbonate buffer and permeable
• The measured potential is related to the molar membrane (Teflon or silicone) across its opening
concentration by the Nernst equation
Trans Maker: Aguinaldo, L.G.
Editor: Blas, S.G. | 5
 MLS 414 (Lec)
3 – ANALYTIC TECHNIQUES AND INSTRUMENTATION
Prof. Melvin Misoles | January 31, 2023

• When whole blood containing dissolved CO2 comes in

contact with a Teflon membrane, the CO2 from the
blood passes through and mixes with the buffer
Voltammetry
• The measurement of current after which a potential is
applied to an electrochemical cell
• Anodic stripping voltammetry – used to measure
heavy metals (e.g. lead and iron testing)
THREE ELECTRODES
Working Electrode
• Makes contact with the analyte OSMOMETRY
• Facilitate the transfer of charge to and from the analyte • Measurement of the osmolality of an aqueous solution
Reference Electrode such as serum, plasma and urine; measurement of
• A half cell with a known reduction potential the concentration of dissolve solute particles in a
Auxiliary Electrode solution
• To sustain electrolysis • As osmotically active particles are added to a solution,
• Process by which electric current is passed through a its osmolality will also increase
substance to effect a chemical change (substance • Osmolality of a solution is related to its colligative
loses or gains an electron) properties such as osmotic pressure, boiling point,
freezing point, and vapor pressure.
Coulometry o Called as such because they can be related to
• Involves the application of a constant current generate each other and the osmolality
a titrating agent • ↑ osmolality of the solution = ↑ osmotic pressure, ↓
• An electrochemical titration in which the titrant is freezing point, ↓ vapor pressure
electrochemically generated and the endpoint is • Based on measuring changes in the colligative
detected by amperometry properties of solution that occur owing to variations in
• The time required to titrate a sample at a constant particle concentration that is related to the osmotic
current is measured and is related to the amount of pressure of the solution
analyte in a sample by Faraday’s equation
• Use: Chloride test (CSF, serum, and sweat) COLLIGATIVE PROPERTIES

Amperometry 1. Osmotic pressure

• The measurement of the current flow produced by an 2. Boiling point
oxidation-reduction reaction at a single applied 3. Freezing point
potential 4. Vapor pressure
• A measure of the cell current when the potential • Colligative properties are those properties of a solution
difference between indicator and reference electrodes that are only a function of the concentration (molality)
is controlled of the particles in solution. Ideally, the size of the
• pO2 gas-sensing electrode – uses an amperometric particles, the mass of the particles, and the type of
/current sensing electrolytic cell as the indicator particles do not affect the colligative properties. Only
electrode the molality of the particles (moles of particles per kg of
• glucose electrode – employed in glucometer that solvent) matters.
determines a random determination of glucose by • The colligative properties are expressed as the affect
glucose oxidase principle that the particles in solution have on the solvent. As the
molality of the particles increases, it has the following
effects on the solvent:
1. The vapor pressure decreases
2. The boiling point increases
3. The freezing point decreases
Osmotic Pressure
• The minimum pressure which needs to be applied to a
solution to prevent inward flow of its pure solvent
across a semi-permeable membrane.
• Osmosis – tendency of a fluid (usually water) to pass
through a semi-permeable membrane into a solution
where the solvent concentration is higher thus,
equalizing the concentrations of materials on either
side of the membrane.

Trans Maker: Aguinaldo, L.G.

Editor: Blas, S.G. | 6
 MLS 414 (Lec)
3 – ANALYTIC TECHNIQUES AND INSTRUMENTATION
Prof. Melvin Misoles | January 31, 2023

producing a voltage pulse. The size of which is

proportional to the cell size.
• The number of pulses is directly related to the cell
count.
• 2-20 fL – platelets
• >36 fL – erythrocytes
LYSES OF RBC: How it works?
• The other blood volume is mixed with diluent and a
cytochemicalytic reagent that lyses only the RBC
• A leukocyte count is performed as the remaining cells
pass through an aperture
• >35 fL – leukocytes

Vapor Pressure
• Measure of the tendency of a material to change into
gaseous or vapor state and it increases with
temperature.

COULTER
MACHINES

BECKMAN-
ELECTRICAL IMPEDANCE COULTER
• Measurement is based on the change in electrical CHEMISTRY
resistance across an aperture when a particle in ANALYZER
conductive liquid passes through this aperture

ABOTT
CELL-DYN

• Used primarily in the hematology laboratory to count

leukocytes, erythrocytes, and platelets

PENTRA
Commonly
used for
CBC testing

CELL BATH: How it works?

• In this method, 1 volume is mixed with diluent and is
delivered to the cell bath where erythrocyte and platelet REFERENCE
I. Tietz, Fundamentals of Clinical chemistry
counts are performed.
• As a cell passes through the aperture partially
occluding it, the electrical impedance increases
Trans Maker: Aguinaldo, L.G.
Editor: Blas, S.G. | 7