Other Designs in Automation
Task
To introduce basic designs and overview procedures of clinical chemistry automation.
Objectives
Upon completion of this exercise, the student will:
1.
Discuss common clinical applications of non-photometric instruments.
2.
Discuss the principles and applications of the following:
a. flame photometer
b. thermal cycler
c. scintillation counter
c. thin-layer chromatography
d. gas chromatography
e. high-performance liquid chromatography
3. Describe the principle of measuring electromagnetic radiation using fluorometry.
4. Discuss the advantages and disadvantages of using fluorometry.
Discussion:
Equipment /
Procedure
Flame Photometer
Equipment /
Procedure
Thermal cycler
(aka: thermocycler /
PCR machine / DNA
amplifier)
Molecular Diagnostic
Testing
Clinical
Applications
Na+, K+, Li+
Clinical
Applications
Molecular
Diagnostics coming quickly to
the clinical
laboratory.
Applications:
hepatitis
cystic fibrosis
pathogenic
microorgs
CT / NG
HIV
Principle
1. Outer shell electrons of Na, K, Li, & Cs expand their orbits when
heated.
2. As they cool, returning to ground state, the energy is given off in
the form of light.
3. The wavelength of light is characteristic for the particular element.
4. The intensity of the light is proportional to the amount of the
element being measured.
5. An internal standard is added to the diluent to compensate for
variation in flame temperature, flow rate, etc.
Principle
Under very strict conditions of environmental cleanliness, the cycler
raises and lowers the temperature creating conditions whereby the
target DNA is denatured, (where the double strands become single
stands). The action of reagent-like primers, nucleotides, and
enzymes result in the formation of newly synthesized double
stranded DNA. This process is repeated many times resulting in
amplification of the target DNA which can then be analyzed by
electrophoresis.
There are also RNA applications.
Equipment /
Procedure
Clinical
Applications
Fluorometer
protoporphyrins,
some
therapeutic
drugs, a few
coagulation
applications
Principle
1.
2.
3.
4.
Basic
fluorometer
filter
5.
6.
Fluorometers measure substances that absorb short  and
release energy of longer wavelength; for those few molecules
are capable of absorbing light of one wavelength, then emitting
light at a different, longer wavelength.
Mercury arc or xenon arc lamp produces short wavelength
(UV) light which is passed through a monochromator.
Monochromatic light of an appropriate short wavelength
passes through a quartz / fused silicon cuvet holding the
specimen.
The light energy is absorbed by the molecules which then
release some of the energy as a longer wavelength.
A second monochromator at a 90 angle to the light source
filters out wavelength other than the long wavelength being
emitted.
The amount of light being released is proportional to the
fluorescing molecules.
Advantages
Specific and sensitive
Disadvantages Few molecules fluoresce, susceptible to pH and temperature
changes
Equipment /
Procedure
Scintillation Counter
Clinical
Applications
trace levels of
hormones and
drugs
Principle
A particle tagged with a radio nuclide emits gamma rays which strike
a detector of a scintillation counter producing an electrical pulse of a
size proportional to the energy of the gamma ray striking it.
The scintillation counter is another laboratory instrument that measures electromagnetic radiation
emission. The scintillation counter measures gamma rays, rather than visible light.
Gamma rays come from an unstable nucleus that is rearranging to become more stable. During this
process they emit anenergy in the form of gamma rays and/or particles. Gamma rays have an extremely
short wavelength and extremely high energy (high frequency). Unlike visible light, they are usually
discussed in terms of their energy rather than wavelength.
Because of their tremendous energy, instruments for measuring gamma rays must have a few different
components than the instruments which measure lower energy visible light. Gamma rays are measured in
a scintillation counter. (Beta emissions counters are also available.)
Clinical chemistry procedures that use gamma / Beta emitters were very popular at one time due to their
high sensitivity in measuring very low levels of constituents such as drugs and hormones. Today
scintillation counters are rarely found outside of research institutions for several reasons:
Costs - Usually a special area of the laboratory was set up and isolated from rest of lab. Equipment was
expensive. Reagents were expensive and had short shelf. If the test was not ordered frequently and only
a few tests were performed out of the kit, the laboratory would lose money by offering the test. Additional
costs were incurred during the disposal of the nuclear wastes.
Regulations, regulations, regulations.... The hassle of nuclear waste disposal with federal, state and local
disposal laws meant there was plenty of extra paper work!
Development of enzyme and other non-radioactive immunoassay tags replaced gamma - tags.
Chromatography
Equipment /
Procedure
Clinical
Applications
Chromatography is the collective term for
a family of techniques for the separation
of mixtures involving the passing of the
mixture containing the analyte through a
‘stationary phase’ to separate it from other
molecules in the mixture and allowing it to
be isolated.
Thin-Layer
Chromatography
Equipment /
Procedure
Principle
Chromatography is a separation method that exploits the differences
in partitioning behavior between a mobile phase and a stationary
phase to separate the components in a mixture. Components of a
mixture may be interacting with the stationary phase based on
charge, relative solubility or adsorption.
Under specific conditions, separation of mixtures depends upon the
relative amount of time the specific compound is in a moving state
(liquid or gas) as opposed to a stationary state of crystal/solid.
TLC technique is
1. The sample is placed on a ‘thin layer’ stationary phase, usually
most useful in
applied to glass or plastic plate.
separating organic 2. The prepared plate is then placed into a chamber containing a
compounds; ID
small amount of solvent (referred to as the ‘mobile phase’).
drugs, sugars, and 3. The solvent/mobile phase travels up the stationary phase
amino acids
separating the mixture into its various components.
Clinical
Applications
Gas Chromatography Though gas
chromatography
(GC) is one of the
most widely used
techniques in
modern analytical
research
chemistry labs, it
has limited
applications in the
clinical chemistry
lab, though it could
be used to ID
various volatile
organic molecules,
alcohols, etc.
Principle
1. In its basic form, GC is used to separate complex mixtures of
different molecules based on their physical properties, such as
polarity and boiling point. It is an ideal tool to analyze gas and
liquid samples containing many hundreds or even thousands of
different molecules, allowing the analyst to identify both the
types of molecular species present and their concentrations.
2. Gas-liquid chromatography (GLC), or simply gas chromatography
(GC), is a type of chromatography in which the mobile phase is
a carrier gas, usually an inert gas such as helium or nitrogen,
and the stationary phase is a microscopic layer of liquid on an
inert solid support, inside glass or metal tubing, called a column.
3. As the separated compounds exit the end of the column they are
‘sensed’ by detectors.
4. Detectors rely on a change in refractive index, UV-VIS
absorption, or fluorescence after excitation with a suitable
wavelength.
5. Modern GC s work in tandem with mass spectrometers (MS) to
further assist in the identification of the unknown compound.
Equipment /
Procedure
Clinical
Applications
High-Performance
Liquid
Chromatography
(HPLC)
Mass Spectrometry
Principle
1. High-performance liquid chromatography (HPLC) is a form of
liquid chromatography to separate compounds that are
dissolved in a solution. HPLC instruments consist of a reservoir
of mobile phase, a pump, an injector, a separation column, and
a detector. The separation of the compounds begins by injecting
a plug of the sample mixture into the column. The different
components in the mixture pass through the column at different
rates due to differences in their partitioning behavior between
the mobile liquid phase and the stationary phase.
2. The pumps provide a steady high pressure with no pulsating, and
can be programmed to vary the composition of the solvent
during the course of the separation.
3. As the separated compounds exit the end of the column they are
‘sensed’ by detectors.
4. Detectors rely on a change in refractive index, UV-VIS
absorption, or fluorescence after excitation with a suitable
wavelength.
5. Like GC, HPLC instruments can have MS detectors.
detector for GLC
& HPLC
1. When used as the detector for gas-liquid and high-pressure
chromatographs, the MS will aid in the identification and
quantitation of compounds providing structural information and
molecular weight determination.
2. Mixtures of compounds are separated by the gas chromatogram.
3. The substances /molecules enter the mass spec where they are
bombarded by electrons to form charged molecular ions and
fragments
4. The molecules breakdown into characteristic fragments
depending on their molecular structure.
5. The fragments are then forced through a filtering section where
they are sorted according to their mass-to-charge ratio and are
counted by an electron multiplier.
6. The fragmentation pattern formed will be characteristic for the
molecule. There are extensive computerized libraries and
matching algorithms to compare the mass spec patterns.
7. Tandem mass spectrometers - the addition of a second mass
spectrometer to the system will allow further breakdown and ID
resulting in increased sensitivity and lower detection limits.
Laboratory: Other Designs in Automation
Study Questions
Points=5
Name:______________
Date: _____________
Instructions: Each question worth one point unless indicated.
1. List the type of chemistry equipment that is commonly used for measuring the following (0.5
each)
a. Lithium:________________
b. Protoporphyrins:________________
c. Amino acids:_________________
d. Volatile organic acids:_____________
2. State the instrument that is capable of reproducing copies of nucleic acid fragments.
3. What is the source of gamma rays?
4. If gamma/beta emitting procedures offered such high specificity and sensitivity, why are they no
longer widely used in the clinical laboratory?