Practical Instrumentation for Automation and Process Control-IDC
Instrumentation and Automation
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Transcript of Instrumentation and Automation
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The primary objective of instrumentation is to provide the clinician with the
best possible data to be of value to the patient. Thorough understanding of the
principles associated with the machines used is quite necessary for the operators in
order to have an easier time in performing maintenance procedures, calibration,
and in troubleshooting problems that may arise.
Automation in the clinical laboratory has been driven by the need to create
automated systems which are capable of reducing or eliminating manual tasks in
perform analytical procedures, and thus enhancing the reduction of errors.
AUTOMATION
3 stages in automation:
Pre-Analytical Stage
The pre-analytical stage is concerned with the handling of the sample or
specimen before processing. The two goals involved in this stage are to minimize
non-value added steps in the laboratory process and to increase available time for
value-added steps in order to produce better results.
Automated pre-analytical system attempts to provide the user with some of
the tasks necessary to prepare samples for testing, namely: pre-sorting,
centrifugation, volume checks, clot detection, decapping, secondary tube labeling,
aliquoting, and destination sorting in analyzer racks.
Analytical Stage
The analytical stage is primarily concerned with the processing of the sample
or specimen.
Tasks:
1. Introduction of the sample may be accomplished through the use of
peristaltic pumps and positive liquid displacement.
2. Addition of reagent reagent used must be handled, prepared and
stored properly. Automated analyzer can be classified based upon reagent.
Automated analyzers classified based upon the reagent used:
o Open reagent system - a system in which the reagents other than the
instrument manufacturers reagent can be used.
o Close reagent system - the operator can only used the manufacturers
reagent.
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3. Mixing
Mixing devices and techniques used:
o Magnetic stirring
o Rotating paddles
o Forceful dispensing
o Use of ultrasonic energy
o Use of vigorous lateral displacement
4. Incubation - uses an elongated cuvet path length and fluorocarbon oil
incubation bath to maximize result accuracy by enhancing absorbance value,
while using microvolume technology for samples and reagent (Bayer
Diagnostics) or uses a thermal electric module in the shape of a ring tomaintain a constant temperature for analysis (Beckman Coulter).
5. Detection
o Absorption spectroscopy - principal means of measurement in
automated analyzer.
o Reflectance photometry adapted to dry slide analysis and used in
chemistry laboratories.
o Fluorescent compounds - used for measurement of drugs, hormones,
and vitamins in several immunoassay analyzers.
POST ANALYTICAL STAGE
The post analytical stage plays its role after processing of the sample or
specimen. The instrument computer plays a major role in this stage as it represents
a means to accomplish several tasks, which include signal processing, data handling
and process control.
The processing of data by computers has allowed automation of nonisotopicimmunoassays, reflectance photometry and other nonlinear assays because
computer algorithms can transform nonlinear standard input signal into linear
calibration plots. Computers can also perform data correction, subtract blank
response, and monitor patients result against reference values.
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INSTRUMENTATION
PRINCIPLES USED:
SPECTROPHOTOMETRY
Spectrophotometry involves the measurement of the amount of light
absorbed by a solution and relating it to the concentration of the solution. It uses
Beer-Lambert Law (often referred to as the Beers law), which 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, as its
fundamental principle.
8 basic components:
1. Light source - light source provides light and must provide enough energy orpower to measure the analyte of interest.
Tungsten lamp or tungsten-halogen lamp used for wavelengths in the
visible region
Deuterium lamp used for wavelengths in the ultraviolet region
Silicon carbide - used for wavelengths in the infrared region
2. Entrance slit - excludes unwanted or stray light and prevents scattered light
from entering the system.
3. Monochromator - produces the light of specific wavelength from the light
source. It arranges the wavelength of light in such a way that
results to the wavelength required in the process.
Quartz prism separates white light into a continuous spectrum and uses
the principle of refraction.
Diffraction gratings bends light and forms wave front for ultraviolet and
near infrared spectrum. It uses the principle of diffraction.
Interference filter based on constructive interference of waves, utilizes
several mirrors, and uses the principle of reflection.
4. Exit slit - also known as bandpass act as the passage way of the filtered light
from the monochromator to the sample cell.
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5. Sample holder - also known as cuvet or analytical cell, maybe square or
round in shape and is used to hold samples and must be scratch-free so as
not to obtain erroneous results.
Soft glass cuvet for acidic solutions
Borosilicate cuvet for strong basic solution
Quartz or plastic cuvet for ultraviolet measurements
6. Photodetector - convert transmitted energy into an equivalent amount of
electrical energy.
7. Signal processor - a device that alters the signal and filters it to remove
unwanted components.
8. Readout device
Monitor
Ammeter
Galvanometer
Recorder
REFLECTOMETRY
Reflectometry makes use of a filter photometer (reflectometer), which
measures the quantity of light reflected by a liquid samples that has beendispensed onto a coarse solid support. Reflectometry is used in urine dipstick
analysis and dry slide chemical analysis.
2 types of reflectance:
Specular reflectance occurs of polished surface
Diffuse reflectance occurs on nonpolished surface
MOLECULAR LUMINESCENCE SPECTROSCOPY (FLUOROMETRY)
Fluorometry is based on the principle of luminescence in wherein an
exchange of energy occurs when compounds absorb electromagnetic radiation,
become excited and return to an energy level lower or equal to their original level.
Chemiluminescence is an example.
Basic components:
Light source
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Excitation (primary) monochromator
Cuvet
Emission (secondary) monochromator
Photodetector
NEPHELOMERTY AND TURBIDIMETRY
Both nephelometry and turbidimetry are based on the principle of scattering
of radiation by particles in suspension. Nephelometry, which is often used in the
measurement of antigen-antibody reaction, is the measurement of the light
scattered by a particulate solution whereas turbidimetry, commonly used in
coagulation analyzers, measurement of antibiotic sensitivities, and quantification of
protein concentration in body fluids, is the measurement of the reduction in light
transmission caused by particle formation.
REFRACTOMETRY
Refractometry is based on the principle of light refraction, which is the
bending of light. Refractometry is used in measuring protein concentration, specific
gravity of urine, and column effluent of high-performance liquid chromatography
analysis.
OSMOMETRY
Osmometry involves the measurement of the osmolality of an aqueous
solution and is based on the measuring changes in the colligative properties ofsolutions owing to variations in particle concentration.
FLOW CYTOMETRY
Flow cytometry is the measurement of multiple properties of cells suspended
in a moving fluid medium. It is used to count and sort cells, is the core component
of hematology cell counters, and is the technology used to differentiate white blood
cells.
ELECTROCHEMISTRY
Electrochemistry involves the measurement of the current or voltagegenerated by the activity of specific ions.
Potentiometry the measurement of voltage between two electrodes in a
solution
Coulometry the measurement of the quantity of electricity needed to
convert an analyte to a different oxidation state
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Voltametry - a method in which a potential is applied to an electrochemical
cell and the resulting current is measure
aniodic strippling voltametry - an electrochemical technique used in
measuring heavy metals
Amperometry - the measurement of the current flow produced by an
oxidation-reduction reaction.
CONDUCTANCE
Conductance is a measurement of the ability of a solution to carry an electric
current. This principle is used monitoring water purity, measuring analytes in blood.
It is also the principle used in the components of detectors used in high
performance liquid chromatography (HPLC) and gas chromatography (GC), cell
counters, and capillary electrophoresis.
IMPEDANCE
Electrical impedance, a measurement based on the change in electrical
resistance across an aperture when a particle in the liquid passes through this
aperture, is primarily used in enumerating leukocytes, erythrocytes, and platelets.
ELECTROPHORESIS AND DENSITOMETRY
Electrophoresis is the separation of charged compounds based on their
electrical charge, and in order to obtain a quantitative profile of the separated
fractions, the principle of densitometry is used, which is performed on the stained
support medium.
ISOELECTRIC FOCUSING
Isoelectric focusing is a technique which is performed similar to other
electrophoresis methods, the difference lies on the medium through which the
separating molecules migrate through. In isoelectric focusing, the separating
molecules migrate through a pH gradient. This principle is useful in measuring
serum acid phosphatase isoenzymes and in detecting oligoclonal immunoglobulin
bands in CSF and isoenzymes of creatine kinase and alkaline phosphatase in serum.
CHROMATOGRAPHY
Chromatography is a method of separation which is based on the different
interactions of the specimen compounds with the mobile phase and with the
stationary phase as the compounds travel through a support medium.
Gas chromatography - useful for volatile compounds
2 types of stationary phases commonly used in gas chromatography:
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Gas-solid chromatography- solid absorbent and uses the same
material for both the stationary and the support phase
Gas-liquid chromatography - uses liquid phases to coat the solid
support material
Liquid chromatography - for better separation of thermolabile compounds
High performance liquid chromatography - better method of liquid
separation over other forms due to a superior resolution, shorter analysis
time, and a greatly improves reproducibility
5 commonly used separation techniques in liquid chromatography:
Adsorption (liquid-solid) chromatography - compounds are adsorbed to a solid
support
Partition (liquid-liquid) chromatography - separates compounds based on
their partition between a liquid mobile phase and a liquid stationary phase
coated on a solid support
o Normal-phase liquid chromatography - uses a polar liquid stationary
phase
o Reverse-phase liquid chromatography - uses a nonpolar liquid
stationary phase
Ion-exchange chromatography - uses column packings that have charge-
bearing functional groups attached to a polymer matrix and uses the
mechanism of the exchange of sample ions and mobile phase ions with the
charged group of the stationary phase
Affinity chromatography - uses immobilized biochemical ligands as the
stationary phase to separate a few solutes from other unretained solutes
Size-exclusion chromatography - separates molecules according to the
difference in their sizes
MASS SPECTROMETRY
Mass spectrometry is based on fragmentation and ionization of molecules
using a suitable source of energy. The resulting fragment masses and their relative
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abundance yield a characteristic mass spectrum of the parent molecule. Mass
spectrometry typically involves the following major steps which include, conversion
of parent molecule into a stream of ion; separating the ions by mass-to-charge
ratio; counting the number of ions of each type or measuring current produced
when the ions strike a transducer.
SCINTILLATION COUNTER
A scintillation counter is an instrument that detects scintillations (flashes of
light that occur when gamma rays or charged particles interact with matter) using a
photomultiplier tube and counts the electrical impulses produced by the
scintillations. Scintillation counting is usually applied in radio immunoassays.
2 types of scintillation method:
Crystal scintillation used to detect gamma radiation
Liquid scintillation used to count radionuclides that emit beta particles
CAPILLARY ELECTROPHORESIS
Capillary electrophoresis is a separation technique that is said to be better
than conventional electrophoresis and high performance liquid chromatography due
to its short analytical time, resolving power, and microsample volumes. Capillary
electrophoresis is applied in the separation of serum proteins and haemoglobin.
NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
Nuclear magnetic resonance spectroscopy is a technique for determining the
surface of organic compounds. It is non-destructive, but requires a larger volume of
the sample compares to mass spectroscopy. Nuclear magnetic resonance is a
phenomenon that occurs when the nuclei of certain atoms are immersed in a static
magnetic field and exposed to a second oscillating magnetic field. Lipoprotein
particle measurement is the most popular application of nuclear magnetic
resonance spectroscopy.