CHP 5312

CHEMICAL PATHOLOGY I
Principles of analytical techniques in clinical
chemistry
Introduction
Analytic techniques and instrumentation provide the
foundation for all measurements made in a modern clinical
chemistry laboratory. The majority of techniques fall into one
of four basic disciplines within the field of analytic
chemistry:
Spectrometry (including spectrophotometry, atomic
absorption, and mass spectrometry
Luminescence (including fluorescence, chemiluminescence,
and nephelometry);
 electroanalytic methods (including electrophoresis,
potentiometry, and amperometry); and chromatography
(including gas, liquid, and thin-layer).
SPECTROPHOTOMETRY AND PHOTOMETRY
 The instruments that measure electromagnetic radiation have several
concepts and components in common. Photometric instruments
measure light intensity without consideration of wavelength.
 Most instruments today use filters (photometers), prisms, or gratings
(spectrometers) to select (isolate) a narrow range of the incident
wavelength. Radiant energy that passes through an object will be
partially reflected, absorbed, and transmitted.
 Electromagnetic radiation is described as photons of energy traveling in
waves. The relationship between wavelength and energy E is described
by Planck’s formula:
E =hv
 where h is a constant (6.62 ×10_27 erg sec), known as Planck’s constant,
and v is frequency.
 Because the frequency of a wave is inversely proportional to the
wavelength, it follows that the energy of electromagnetic radiation is
inversely proportional to wavelength.
 Electromagnetic radiation includes a spectrum of energy from short-
wavelength, highly energetic gamma rays and x-rays on the left to long-
wavelength radiofrequencies on the right. Visible light falls in between,
with the color violet at 400-nm and red at 700- nm wavelengths being the
approximate limits of the visible spectrum.
 Photometric instruments measure either absorption or emission of radiant
energy to determine concentration of atoms or molecules. The two
phenomena, absorption and emission, are closely related. For a ray of
electromagnetic radiation to be absorbed, it must have the same frequency
as a rotational or vibrational frequency in the atom or molecule that it
strikes. Levels of energy that are absorbed move in discrete steps, and any
particular type of molecule or atom will absorb only certain energies and
not others.
 When energy is absorbed, valence electrons move to an orbital with a
higher energy level. Following energy absorption, the excited electron will
fall back to the ground state by emitting a discrete amount of
 energy in the form of a characteristic wavelength of radiant energy.
 Absorption or emission of energy by atoms results in a line spectrum.
Because of the relative complexity of molecules, they absorb or emit a bank
of energy over a large region. Light emitted by incandescent solids
(tungsten or deuterium) is in a continuum.
Beer’s Law
 The relationship between absorption of light by a solution and the
concentration of that solution has been described by Beer and others.
Beer’s law states that the concentration of a substance is directly
proportional to the amount of light absorbed or inversely proportional to
the logarithm of the transmitted light. Percent transmittance (% T) and
absorbance (A) are related photometric terms.
 Percent transmittance is the ratio of the radiant energy transmitted (T)
divided by the radiant energy incident on the sample (I). All light absorbed
or blocked results in 0% T. A level of 100% T is obtained if no light is
absorbed. In practice, the solvent without the constituent of interest is
placed in the light path. Most of the light is transmitted, but a small
amount is absorbed by the solvent and cuvet or is reflected away from the
detector. The electrical readout of the instrument is set arbitrarily at 100%
T, while the light is passing through a “blank” or reference.
 The sample containing absorbing molecules to be measured is placed in the
light path. The difference in amount of light transmitted by the blank and
that transmitted by the sample is due only to the presence of the compound
being measured. The %T measured by commercial spectrophotometers is
the ratio of the sample transmitted beam divided by the blank transmitted
beam.
 Equal thicknesses of an absorbing material will absorb a constant fraction of
the energy incident upon the layers. Absorbance A is the amount of light
absorbed. It cannot be measured directly by a spectrophotometer but rather
is mathematically derived from % T as follows:
%T= I / I0 ×100
 where I0 is incident light and I is transmitted light. Absorbance is defined as
follows:
A = -log (I/I0) = log100 - log %T
A= 2 - log %T
 According to Beer’s law, absorbance is directly proportional to
concentration:
A =ɛ× b× c
 Where; ɛ =molar absorptivity, the fraction of a specific wavelength of light
absorbed by a given type of molecule; b is the length of light path through
the solution; and c is the concentration of absorbing molecules.
 Absorptivity depends on molecular structure and the way in which the
absorbing molecules react with different energies. For any particular
molecular type, absorptivity changes as wavelength of radiation
changes. The amount of light absorbed at a particular wavelength
depends on the molecular and ion types present and may vary with
concentration, pH, or temperature.
 Because the path length and molar absorptivity are constant for a given
wavelength,
A ̴ C
 Unknown concentrations are determined from a calibration curve that
plots absorbance at a specific wavelength versus concentration for
standards of known concentration. For calibration curves that are
linear and have a zero y-intercept, unknown concentrations can be
determined from a single calibrator. Not all calibration curves result in
straight lines. Deviations from linearity are typically observed at high
absorbances. The stray light within an instrument will ultimately limit
the maximum absorbance that a spectrophotometer can achieve,
typically 2.0 absorbance units.
 Turbidity and Nephelometry
 Turbidimetric measurements are made with a spectrophotometer to
determine concentration of particulate matter in a sample. The amount
of light blocked by a suspension of particles depends not only on
concentration but also on size. Because particles tend to aggregate and
settle out of suspension, sample handling becomes critical. Instrument
operation is the same as for any spectrophotometer.
 Nephelometry is similar, except that light scattered by the small
particles is measured at an angle to the beam incident on the cuvet.
Light scattering depends on wavelength and particle size. For
macromolecules with a size close to or larger than the wavelength of
incident light, sensitivity is increased by measuring forward light
scatter. Instruments are available with detectors placed at various
forward angles, as well as at 90 degrees to the incident light.
Monochromatic light obtains uniform scatter and minimizes sample
heating. Certain instruments use lasers as a source of monochromatic
light; however, any monochromator may be used.
Measuring light scatter at an angle other than at 180 degrees in
turbidimetry minimizes error from colored solutions and increases
sensitivity. Because both methods depend on particle size, some
instruments quantitate initial change in light scatter rather than total
scatter. Reagents must be free of any particles, and cuvets must be free of
any scratches.
ELECTROCHEMISTRY
Many types of electrochemical analyses are used in the
clinical laboratory, including potentiometry,
amperometry, coulometry, and polarography. The two
basic electrochemical cells involved in these analyses
are galvanic and electrolytic cells.
Galvanic and Electrolytic Cells
 It consists of two half-cells and a salt bridge, which can be a piece
of filter paper saturated with electrolytes. Instead of two as
shown, the electrodes can be immersed in a single, large beaker
containing a salt solution. In such a setup, the solution serves as
the salt bridge.
 In a galvanic cell, as the electrodes are connected, there is
spontaneous flow of electrons from the electrode with the lower
electron affinity (oxidation; e.g., silver). These electrons pass
through the external meter to the cathode (reduction), where OH -
ions are liberated. This reaction continues until one of the
chemical components is depleted, at which point, the cell is
“dead” and cannot produce electrical energy to the external meter.
 Current may be forced to flow through the dead cell only by
applying an external electromotive force E. This is called an
electrolytic cell. In short, a galvanic cell can be built from an
electrolytic cell. When the external E is turned off, accumulated
products at the electrodes will spontaneously produce current in
the opposite direction of the electrolytic cell.
Half-Cells
 It is impossible to measure the electrochemical activity of one half-cell;
two reactions must be coupled and one reaction compared with the
other. To rate half-cell reactions, a specific electrode reaction is
arbitrarily assigned 0.00 V. Every other reaction coupled with this
arbitrary zero reaction is either positive or negative, depending on the
relative affinity for electrons. The electrode defined as 0.00 V is the
standard hydrogen electrode: H2 gas at 1 atmosphere (atm). The
hydrogen gas in contact with H+ in solution develops a potential. The
hydrogen electrode coupled with a zinc half-cell is cathodic, with the
reaction:
2H+ +2e- → H2
 Because H2 has a greater affinity than Zn for electrons. Cu, however, has
a greater affinity than H2 for electrons, and thus the anodic reaction:
H2 → 2H+ +2e-
 Occurs when coupled to the Cu-electrode half-cell. The potential
generated by the hydrogen-gas electrode is used to rate the electrode
potential of metals in 1 mol/L solution. A hydrogen electrode is used to
determine the accuracy of reference and indicator electrodes, the
ELECTROPHORESIS
 Electrophoresis is the migration of charged solutes or particles in an
electrical field. Iontophoresis refers to the migration of small ions,
whereas zone electrophoresis is the migration of charged
macromolecules in a porous support medium such as paper, cellulose
acetate, or agarose gel film. An electrophoretogram is the result of zone
electrophoresis and consists of sharply separated zones of a
macromolecule.
 In a clinical laboratory, the macromolecules of interest are proteins in
serum, urine, cerebrospinal fluid (CSF), and other biologic body fluids
and erythrocytes and tissue.
 Electrophoresis consists of five components: the driving force
(electrical power), the support medium, the buffer, the sample, and
the detecting system. A typical electrophoretic apparatus is illustrated
in
Charged particles migrate toward the opposite charged electrode. The
velocity of migration is controlled by the net charge of the particle, the
size and shape of the particle, the strength of the electric field,
chemical and physical properties of the supporting medium, and the
electrophoretic temperature. The rate of migration is directly
proportional to the net charge of the particle and inversely proportional
to its size and the viscosity of the buffer.
Detection and Quantitation
 Separated protein fractions are stained to reveal their locations.
Different stains come with different plates from different manufacturers.
The simplest way to accomplish detection is visualization under UV
light, whereas densitometry is the most common and reliable way for
quantitation. Most densitometers will automatically integrate the area
under a peak, and the result is printed as percentage of the total.
 The movement of buffer ions and solvent relative to the fixed support is
called endosmosis or electroendosmosis. Support media, such as paper,
cellulose acetate, and agar gel, take on a negative charge from adsorption
of hydroxyl ions. When current is applied to the electrophoresis system,
the hydroxyl ions remain fixed while the free positive ions move toward
the cathode. The ions are highly hydrated, resulting in net cathodic
movement of solvent. Molecules that are nearly neutral are swept toward
the cathode with the solvent. Support media such as agarose and
acrylamide gel are essentially neutral, eliminating electroendosmosis.
The position of proteins in any electrophoresis separation depends not
only on the nature of the protein but also on all other technical variables.
 CHROMATOGRAPHY
Chromatography refers to the group of techniques used to separate complex
mixtures on the basis of different physical interactions between the
individual compounds and the stationary phase of the system. The basic
components in any chromatographic technique are the mobile phase (gas or
liquid), which carries the complex mixture (sample); the stationary phase
(solid or liquid), through which the mobile phase flows; the column holding
the stationary phase; and the separated components (eluate).
 Modes of Separation-Adsorption
Adsorption chromatography, also known as liquid-solid chromatography,
is based on the competition between the sample and the mobile phase for
adsorptive sites on the solid stationary phase. There is an equilibrium of
solute molecules being adsorbed to the solid surface and desorbed and
dissolved in the mobile phase. The molecules that are most the soluble in
the mobile phase, move fastest; the least soluble, move slowest. Thus, a
mixture is typically separated into classes according to polar functional
groups.
The stationary phase can be either
acidic polar (e.g., silica gel), basic
polar (e.g., alumina), or nonpolar
(e.g., charcoal). The mobile phase
can be a single solvent or a mixture
of two or more solvents, depending
on the analytes to be desorbed.
Partition Chromatography
 Partition chromatography is also referred to as liquid-liquid
chromatography. Separation of solute is based on relative solubility in
an organic (nonpolar) solvent and an aqueous (polar) solvent. In its
simplest form, partition (extraction) is performed in a separatory
funnel. Molecules containing polar and nonpolar groups in an
aqueous solution are added to an immiscible organic solvent. After
vigorous shaking, the two phases are allowed to separate. Polar
molecules remain in the aqueous solvent; nonpolar molecules are
extracted in the organic solvent.
 This results in the partitioning of the solute molecules into two
separate phases. The ratio of the concentration of the solute in the two
liquids is known as the partition coefficient:

K=
Modern partition chromatography uses pseudo liquid
stationary phases that are chemically bonded to the support or
high-molecular-weight polymers that are insoluble in the
mobile phase.
Partition systems are considered normal phase when the
mobile solvent is less polar than the stationary solvent and
reverse phase when the mobile solvent is more polar.
Partition chromatography is applicable to any substance that
may be distributed between two liquid phases.
Because ionic compounds are generally soluble only in water,
partition chromatography works best with nonionic
compounds.
Steric Exclusion
 Steric exclusion, a variation of liquid-solid chromatography, is
used to separate solute molecules on the basis of size and shape.
The chromatographic column is packed with porous material. A
sample containing different-sized molecules moves down the
column dissolved in the mobile solvent. Small molecules enter
the pores in the packing and are momentarily trapped. Large
molecules are excluded from the small pores and so move
quickly between the particles.
 Intermediate-sized molecules are partially restricted from
entering the pores and, therefore, move through the column at
an intermediate rate that is between those of the large and small
molecules. Early methods used hydrophilic beads of cross-linked
dextran, polyacrylamide, or agarose, which formed a gel when
soaked in water.
 This method was termed gel filtration. A similar separation process
using hydrophobic gel beads of polystyrene with a nonaqueous
mobile phase was called gel permeation chromatography. Current
porous packing uses rigid inorganic materials such as silica or glass.
The term steric exclusion includes all these variations. Pore size is
controlled by the manufacturer, and packing materials can be
purchased with different pore sizes, depending on the size of the
molecules being separated.
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