INSTRUMENTS AND
PRINCIPLES USE IN
BIOCHEMICAL ASSAYS
DR. APPIAH MICHAEL
SPECTROPHOTOMETRY
• Assay of a substance in solution by spectrophotometry is done by
measuring the amount of light absorbed by that solution after appropriate
treatment.
• The more light absorbed the higher the concentration of the analyte in
consideration.
• Colorimetry is an associated procedure used to describe the same technique,
the only difference being the sophistication of the instrument used.
• A colorimeter is a simpler instrument usually with less variable wavelengths
and of limited variability.
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Spectrophotometry instrumentation
• A light source provides the incident ray for the system.
• The wavelength of the incident ray is usually within the visible range
(400-800) and can use a tungsten lamp.
• The light required is to be of a single wavelength but the light source
emits light of several wavelengths.
• The proper wavelength is selected by a prism or a grating system (a
monochromator).
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• The incident light passes through the cuvette holding the analyte with
minimal absorption.
• The transmitted light falls on a detector, which is a phototube that
responds to light striking it.
• This transmitted light falling on the detector generates an electric
signal to a read out device to indicate the amount of light passing
through the sample.
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Three Types of Spectrophotometric Methods
• Three examples of common types of chemical reactions that are measured by the
spectrophotometer are endpoint colorimetric, endpoint enzymatic, and kinetic
reactions.
• The Jaffe reaction for creatinine is an example of an endpoint colorimetric
reaction.
• The hexokinase reaction with glucose is an example of an endpoint enzymatic
reaction in which enzymes catalyze the reaction to measure the analyte.
• An example of a kinetic method, which employs substrates and coenzymes to
measure the activity of the enzyme, is that of measurement of alanine
transaminase (ALT) using alanine, alpha-ketoglutarate, and pyridoxyl-5’phosphate.
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Common Types of Chemical Reaction Measured With
Spectrophotometry
Chemical Reaction Type
What Is Measured
How Absorbance Is
Measured*
Endpoint colorimetric
Chromogen
One reading
Endpoint enzymatic
Colorless coenzyme
One reading
Kinetic
Colorless coenzyme
Multiple absorbance
readings
*Spectrophotometer is first set to 100% T/zero absorbance with a reference solution.
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Atomic Absorption Spectrophotometry
• Measures light to determine the concentration of an analyte. However, this
is a more complex type of spectrophotometry.
• Only metals such as divalent or some trivalent cations can be atomized
easily, so this is a common method of analysis for metals such as lead.
• The element of interest is separated from the molecular state and placed in
its unexcited, ground state.
• In the ground state, the element absorbs radiation at a narrow bandwidth of
spectrum that is specific to the element.
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• The excitation source emits the same wavelength of light that the
atoms can easily absorb.
• Absorption of radiant energy follows Beer’s law, such that
concentration of the number of atoms present in the sample is
proportional to the amount of absorption of the specific wavelength of
radiation.
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• AAS uses a high-energy hollow cathode lamp (HCL) containing a filament
of the same metal to be tested and a rare gas.
•
• Electrical discharge produced by the power source ionizes the rare gas
atoms, causing collisions of the gas atoms with the metal atoms.
• The metal atoms emit radiant energy when they return to ground state.
• A rotating beam splitter, also known as a rotary chopper, generates a
reference beam that bypasses the sample in the flame and, alternatively, a
sample beam.
• The ratio between both paths is determined.
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• Essentially, the decrease in radiant energy coming from the sample beam as
compared to the reference beam is detected by the photodetector.
• The difference in energy between the two beams is due to the amount of energy
absorbed.
• The placement of the monochromator is after the cuvette, unlike in simple
spectrophotometry.
• A monochromator between the flame and the photodetector eliminates extra
wavelengths of light emitted by the flame before it reaches the photodetector.
• This helps to ensure that only light emitted by the lamp and not absorbed by the
sample is detected.
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• AAS is highly sensitive for quantification of trace and toxic metals.
• It is not suitable for metals found in larger concentrations in plasma,
such as sodium or potassium.
• Interferences in AAS include chemical,ionization, and matrix
interference.
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BEER-LAMBERT LAW
• Beer-Lambert law states that the concentration of the sample
and path length is directly proportional to the absorbance of the
light.
• By comparing the amount of incident light entering a solution with the
amount of transmitted light, we can calculate the concentration of
analyte in solution.
• The difference between the amounts of incident light and transmitted
light can be expressed mathematically and is referred to as absorbance
of the solution.
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• A simple expression of Beer-Lambert law is as follows: A = c e l
• Where
➢A is the measured absorbance,
➢c is the concentration of the analyte in solution,
➢e is the coefficient of absorptivity (and is specific for each compound), and
➢l is the length of the light path i.e the length of the cuvette
• A simple interpretation of the law is that within certain limits concentration
of analyte is directly proportional to the absorbance
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Why does Beer-Lambert law fails at higher concentrations?
❑Beer-Lambert law fails at higher concentrations because;
✓The linearity of the law is limited to chemical and instrumental
factors. When the solution has higher concentrations, the proximity
between the molecules of the solution is so close that there are
deviations in the absorptivity.
✓Also, when the concentration is high, the refractive index changes.
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FLUOROMETRY
• The Nature of Fluorescence: There are several types of luminescence
phenomena including ;
➢Fluorescence
➢phosphorescence and
➢chemiluminescence.
• These 3 phenomena occur when radiant energy is absorbed by a
molecule.
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• Fluorescence occurs when excited electrons give off light as they drop from
the excited state back to their ground state.
• It has the property that the emitted fluorescence light is of a less energy or
has a longer wavelength than the excitation light.
• Phosphorescence, shows a larger shift in emitted light wavelength than does
fluorescence.
• Chemiluminescence and bioluminescence differ from the other
luminescence phenomena (fluorescence and phosphorescence) in that the
excitation event is caused by a chemical reaction and not by
photolumination.
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• The physical event of the light emission in chemiluminescence and
bioluminescence is similar to fluorescence in that it occurs from an excited
singlet state and the light is emitted when the electron returns to the ground
state.
• For all practical purposes, fluorescence and phosphorescence differ only in
the exact route the electron takes in returning to the ground state.
• An increasingly useful tool in the analytical field is the use of fluorescence
in chemical assays because in many instances it is more sensitive than
spectrophotometry in that it allows smaller samples and fewer reagents to
be used in a shorter time.
• Other advantages include elimination of interference seen in
spectrophotometry
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Basic Concepts of Fluorescence:
• As with spectrophotometry, fluorescence measurements involve the
interaction of light with a chemical compound.
• The difference, however, in fluorescence is that the compound emits
light, usually of a longer wavelength, in response to the light striking
it.
• This emitted light is detected by a phototube that determines the
intensity.
• The amount of light the compound emits is propotional to
concentration of the compound.
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Fluorescence Instrumentation
• In a fluorometer, light passes through an entrance slit and interacts with a
wavelength selector (filter or grating) before striking a sample in cuvette.
• The emitted light radiates out of the cuvette in all directions.
• A wavelength selector and detector systems are located at 90 degree to the
path of the incident ray to avoid detection of light coming into the sample.
• Emitted light from the sample passes through the wavelength selector and
strikes the phototube of the detector.
• An electronic signal is sent to the read out system to indicate the intensity of
the fluorescence detected.
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Nephelometry
• Nephelometry is defined as the detection of light energy scattered or
reflected toward a detector that is not in the direct path of the transmitted
light.
• Common nephelometers measure scattered light at right angles to the
incident light.
• The design principle of a nephelometer is similar to the design principle
applied in fluorescence measurements.
• The major operational difference between the fluorometer and the
nephelometer is that the excitation and detection wavelength will be set to
the same value in the nephelometer.
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FLAME EMISSION SPECTROPHOTOMETRY (FES)
• Energy for excitation electrons comes from a flame. The energy
produces electron transitions within the atom/ion.
• As the electrons drop back to the ground state, light energy of specific
wavelength is emitted.
• This light is detected and measured by specific optical systems.
• Only Na+ & K+ are run routinely as electrolytes whilst Lithium levels
in serum are determined by flame photometry for manic-depressive
patients on lithium therapy.
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Principle of Instrumentation
• FES requires a sample handling system, a burner and a light detecting
system.
• The sample is burned by diluting it and presenting it to the flame in the
form of spray.
• The sample enters the aspirator by suction and passes through the atomizer,
which disperses the sample in a fine mist.
• The mist then interacts with the flame for excitation of the ions in solution.
For most commonly used flame photometers, a mixture of propane and
compressed air is used for the flame.
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• As the excited electrons drop back to a lower energy level, the light
generated passes through a mechanism to eliminate stray radiation and
collimate the beam to some extent.
• Interaction with a monochromator determines the wavelengths that
impinge on the phototube in the detector system.
• The signal generated by the phototube is proportional to the
concentration of the ion of interest in the sample.
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Precautions
• Haemolysed blood should be avoided.
• Avoid using anti-coagulant tubes and plain tubes should be used.
Heparin is the only anti-coagulant that may be used if necessary.
• High lipid or protein concentrations produce lower values of Na+ K+
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ION SELECTIVE ELECTRODES (ISE)
• This method uses a different principle for the determination of
electrolytes.
• Most chemical analysers are fitted with ISEs, which usually contain
Na+ with glass membranes and K+ electrodes with liquid ionexchange membranes.
• High selectivity is one its advantages.
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Principle
• The ion-selective electrode works based on the principle of a galvanic cell. It
consists of a reference electrode, ion-selective membrane, and voltmeter.
• The transport of ions from an area of high concentration to low concentration,
through the selective binding of ions with the specific sites of the membrane,
creates a potential difference.
• This potential is measured with respect to a stable reference electrode having a
constant potential, and a net charge is determined. The difference in potential
between the electrode and the membrane depends on the activity of the specific
ion in solution.
• The strength of the net charge measured is directly proportional to the
concentration of the selected ion.
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AUTOMATION
• Automation is the process of using a machine to perform steps in
laboratory testing with only minimal involvement by the analyst.
• Steps needed to produce laboratory results from a given sample
includes: specimen identification, specimen volume measuring,
sample pretreatment, reagent volume measuring, sample and reagent
mixing, incubation, reaction timing and reaction analysis and
calculations, and result presentation on a visual screen and/or in print.
• Automation can handle all or some of these steps.
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Terms to describe automations
• these terms are used in operating manuals and manufacturer’s literature.
✓single-channel or multichannel
✓discrete analysis
✓continuous flow analysis
✓random access
✓batch analysis
✓sequential analysis and
✓centrifugal analysis.
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Centrifugal Analyzers
• Centrifugal analyzers were developed as a result of space-aged technology.
• Samples and reagents were mixed together, reacted, and flowed by
centrifugal force into separate cuvettes in which spectrophotometric
analysis could occur.
• These analyzers were capable of performing a batch of one type of test to
completion.
• Then, after a washing out phase and setting up phase, a new batch of a
different test could be performed.
• All patient test results were reported out at once for a particular batch.
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Continuous Flow Analyzers
• The process is a rapid way of introducing samples to the ion-selective
electrodes.
• Carryover is minimized since the products of electrochemistry are ions of
low concentrations rather than a large amount of chromagen.
• However, air bubbles and wash fluids are used to remove remnants of
sample before the next one flows in.
• Continuous flow is also used in some spectrophotometric instruments in
which the chemical reaction occurs in one reaction channel and then is
rinsed out and reused for the next sample, which may be an entirely
different chemical reaction.
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• Since disposable reaction cells are not used and liquid reagents are
used, cost is low per test.
• Sample carryover is minimized by the use of air bubbles and an inert
coating substance that forms a reaction capsule.
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Discrete Analyzers
• Sample reactions are kept discrete through the use of separate reaction
cuvettes, cells, slides, or wells that are disposed of following chemical
analysis.
• This keeps sample and reaction carryover to a minimum but increases the
cost per test due to disposable products.
• One system uses multiple layers of dry reagent embedded into gel layers
compiled onto a plastic slide the size of a postage stamp.
• This dry slide system only introduces liquid from the patient sample and,
although is more costly due to the disposable components, generates only
dry waste.
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• Some discrete systems reuse the reaction cuvette by thoroughly
washing, rinsing with pure water, and drying it between patient
samples.
• This technique helps to minimize cost by limiting disposable
components as well as minimizing sample and test carryover.
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Specimen Identification
• The sample container, whether it is a cup or the original tube, are
properly labeled to trace back to the original patient sample and
matched to the patient identification information and test requisition
form.
• Barcode labels are applied to sample tubes that fit onto analyzers to
make identification and pairing of the sample with the results easier.
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Sample Pretreatment
• Proteins are present in large concentrations in serum (e.g., albumin at
35 g/L) and may interfere with some analyses. The effect of protein
interference can be lessened by sample pretreatment.
• This is achieved usually by adding a large amount of diluent to dilute
out interfering substances such as protein.
• Instead of diluting the sample, one analyzer system has a topspreading layer of film that traps proteins and prevents them from
reaching the chemical reaction layer.
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Reagent Handling and Pipetting
• Reagents are stored by automated analyzers in room-temperature and
refrigerated compartments.
• Small refrigeration compartments with computerized sensors monitoring the
temperature and inventory of reagents are used to store labile reagents.
• Reagent containers often contain barcode labels for ease of identification by
the instrument and help in maintaining inventory.
• Specimen volume and reagent measuring is usually done by positive
displacement pipettes and tubing, with pumps generating suction to aspirate
the sample and reagent volumes and deliver them to a reaction cell.
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The Chemical Reaction
• Sample and reagent mixing is performed by a variety of techniques,
including centrifugal force, sound waves, vibration, stirring paddles,
and a physical beater.
• Incubating in a carefully controlled heating block is necessary for
enzyme-catalyzed reactions and is required by many testing methods.
• Reaction timing is carefully controlled by computerized timing
devices that may control pumps or chains or other physical steps to
move the reaction cell through the system.
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Reaction Analysis
• Reaction analysis is achieved at the end of the incubation phase by
endpoint or continuous monitoring of light transmittance using
spectrophotometry for most analytes.
• Ions are commonly measured by changes in potentiometry using ionselective electrodes.
• A microprocessor performs calculations to convert absorbance or other
measurements into units of concentration based on a standard curve.
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Result Reporting
• Patient and quality control results are presented on a visual screen and printed on
paper.
• Results can be sent to the electronic patient chart that is stored in the database of
the hospital computer system.
• Sophisticated sensors send signals to the computer system to warn the operator of
errors or problems of analysis.
• Warning sounds alert the operator to indicate technical problems; error flags on the
laboratory report indicate results that must be verified.
• These cautionary activities provide continuous quality assurance of the technical
process.
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Thank you
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