CIULLA_INSTRUMENTATION & ANALYTICAL PRINCIPLES Flashcards
referred to as the distance traveled by one complete wave cycle (distance between two successive crests)
Wavelength
Wavelength is measured in _____
nanometers (nm)
characterized as a spectrum from short wavelength to long wavelength
Radiant energy
order of wavelengths from shortest to longest
Gamma ray X-ray UV Visible Infrared Microwave Radio
Two properties of electromagnetic radiation
wave-like
particle-like
T/F:
The shorter the wavelength, the greater the energy
TRUE
The shorter the wavelength, the greater the energy (also the greater number of photons
Visible light wavelength
400-700 nm
What is detected when all visible wavelengths of light are combined?
White light
Visible color
wavelength of light transmitted (not absorbed) by an object
the particles of light
photons
three ways by which an atom becomes excited upon absorbing a photon
- electron is moved to a higher energy level
- the mode of the covalent bond vibration is changed
- rotation around covalent bonds is changed
When energy is absorbed as a photon, an electron is moved to a higher energy level where it is _________.
unstable
T/F:
An excited electron is unstable and will return to ground state
TRUE
An excited electron emits energy in the form of _____.
Light
Absorption or emission of energy forms a ________ that is characteristic of a molecule and can help identify a molecule.
line spectrum
an instrument used to determine the concentration of a light-absorbing analyte in solution.
spectrophotometer
T/F:
An analyte may absorb, transmit and reflect light to varying degrees but always of a characteristic nature for the analyte
TRUE
Components of a Spectrophotometer
- power supply
- light source
- entrance slit
- monochromator
- exit slit
- cuvet/sample cell
- photodetector
- readout device
component of a spectrophotometer which produces an intense, reproducible, constant beam of light
light source (exciter lamp)
Types of incandescent lamps
tungsten
deuterium
type of incandescent lamp regarded as most common and is used in visible and infrared regions
tungsten
type of incandescent lamp used in UV region
deuterium
T/F:
Changing the light source in a spectrophotometer changes the angle of the light striking the monochromator
TRUE
Recalibration is a must upon changing light source.
a component of the spectrophotometer which converts the electromagnetic radiation transmitted by a solution into an electric signal
Photodetector
the electromagnetic radiation detected in a spectrophotometer is converted into a ______.
electric signal
T/F:
The more light transmitted, the more energy, and greater electric signal is measured.
TRUE
a component of the spectrophotometer where the electrical energy from a detector is displayed on some type of digital display
Readout device
Readout system may be a ________.
chart recorder
computer printout
the range of wavelengths in nanometers that is transmitted by the monochromator and exit slit between two points of a spectral scan where the light transmitted is one-half of the peak (maximum) resistance
band pass or spectral bandwidth
refers to the wavelengths at which an atom absorbs light; each metal exhibits a specific line spectrum
line spectrum
an instrumentation technique wherein the ground-state atoms absorb light at defined wavelengths
Atomic Absorption Spectrophotometry
an instrumentation technique where the sample is atomized in a flame and the atoms are maintained at a ground state. Light from HCL is passed through the chopper to the flame and the ground-state atoms absorb the wavelengths of light from HCL
Atomic Absorption Spectrophotometry
The difference in the amount of light leaving the HCL and the amount of light measured by the detector is (inversely/directly) proportional to the concentration of the metal analyte in the sample
inversely proportional.
The less light measured by the detector, the greater the concentration of metal analyte in the sample
Components of AAS
Hollow-cathode lamp Chopper Burner head for flame Monochromator Detector Readout device
inert gas present in an HCL
helium or argon
a component of AAS that contains an anode, a cylindrical cathode made of metal being analyzed and an inert gas
HCL
the measurement of light scattered by a particulate solution
Nephelometry
In nephelometry, the amount of scatter is (directly/inversely) proportional to the number and size of particles present in the solution
directly proportional
In nephelometry, when small particles are involved, the light is measured through
a. at an angle to the incident light
b. via forward light scatter
a
In nephelometry, when large particles are involved, the light is measured through
a. at an angle to the incident light
b. via forward light scatter
b
The ____________ of nephelometry depends on the absence of background scatter from scratched cuvets and particulate matter in reagents
sensitivity
measures light blocked as a decrease in the light transmitted through the solution
Turbidimetry
Turbidimetry mainly depends on
particle size and concentration
What instrument does turbidimetry use for measurement?
Spectrophotometer
A process where atoms absorb energy at a particular wavelength, electrons are raised to higher-energy orbitals, and the electrons release energy as they return to ground state by emitting light energy of a longer wavelength and lower energy than the exciting wavelength.
Fluorescence
The emitted light in fluorescence has a very (short/long) lifetime.
short
Referred to as the emission of light produced by certain substances after they absorb energy.
Phosphorescence
The time delay between absorption of radiant energy and release of energy as photons of light in phosphorescence
> 10^-4 sec
The process where the chemical energy of a reaction produced excited atoms, and upon electron return to ground state, photons of light are emitted
Chemiluminescence
The process where an enzyme-catalyzed chemical reaction produces light emission.
Bioluminescence
generic term for the type of instrument that is used to measure chemiluminescence and bioluminescence
Luminometer
a technique where solutes in a sample are separated for identification based on physical differences that allow their differential distribution between a mobile phase and a stationary phase
Chromatography
the phase in chromatography which may be an inert gas or liquid
Mobile phase
the phase in chromatography which may be silica gel bound to the surface of a glass plate or plastic sheet; may be silica or polymer that is coated or bonded within a column
Stationary phase
Thin-Layer Chromatography is a type of __________ chromatography.
planar
technique used clinically for urine drug screening
TLC
In chromatography, the mobile phase moves through the stationary phase by means of what principles
Absorption and capillary action
T/F:
In chromatography, the solute components move at different rates because of solubility in the mobile phase and electrostatic forces of the stationary phase that retard solute movement
TRUE.
Mobile phase and stationary phase work together to provide solute resolution and separation
In chromatography, solute will stay at (solvent front/origin) if solvent is too polar for the solute
solvent front
In chromatography, solute will stay at (solvent front/origin) if solvent is insufficiently polar
origin
types of luminescence where excitation requires absorption of radiant energy
fluorescence
phosphorescence
types of luminescence where excitation does not require absorption of radiant energy
chemiluminescence
bioluminescence
Factors that affect rf values in chromatography
chamber saturation
temperature
humidity
composition of the solvent
a technique used to separate volatile solutes
Gas-liquid chromatography
This component in gas-liquid chromatography carries the vaporized sample into the
column
inert carrier gas (mobile phase)
This component in gas-liquid chromatography produces a signal for identification and quantification of the
solutes
detector
commonly used detectors in gas-liquid chromatography
flame ionization
thermal conductivity
electron capture
mass spectrometer
components of gas-liquid chromatography
carrier gas (w/ flow control device) heated injector chromatographic column heated column oven detector computer
in gas-liquid chromatography, the injector is maintained approx. at ___________ higher than the column temperature
50degC
The carrier gas in GLC must have a __________ to regulate gas flow
flow-control device
This component in GLC is used to separate solutes
chromatographic column
This component in GLC is used to process data and control the operation of the system
computer
In GLC, the sample is injected into the injector where the sample is _______.
vaporized
carrier gases commonly used in GLC
hydrogen
helium
nitrogen
argon
T/F:
The carrier gas flow rate is critical to maintaining column efficiency and reproducibility of elution times
TRUE
in GLC, the types of columns (stationary phase) used are designated as what form?
packed
capillary
When the volatile solutes carried by the gas over the stationary phase
of the column are eluted, the column effluent is introduced to what component of the GLC?
detector
in chromatography, this is the portion of the mobile phase which carries the sample components with it
Eluent
in chromatography, this is the combination of the mobile phase and the analytes
Eluate
in chromatography, the mobile phase that exits the column
Effluent
in chromatography, the solute-mobile phase mixture which exits the column
Eluate
the process of passing of mobile phase through the chromatographic bed to transport solutes
Elution
In GLC, the identification of a solute is based on its ________.
retention time
In GLC, the quantification of a solute is based on ________.
peak size
In peak size (GLC), the amount of solute present is ___________ to the size of the peak.
proportional
T/F:
In GLC, the more volatile a solute, the faster it will elute from the column
TRUE
T/F:
In GLC, the less interaction of the solute with the column, the faster it will elute
TRUE
HPLC stands for
High-Performance Liquid Chromatography
Components of HPLC
solvent reservoir(s) one or more pumps injector chromatographic column detector computer
In HPLC, this component is used to propel the solvents
pump
In HPLC, this component is used to process data and control the operation of the system
computer
a type of liquid chromatography where the mobile phase is a liquid
that is passed over the stationary phase of the column. The separation of
solutes in a sample is governed by the selective distribution of the solutes
between the mobile and stationary phases.
HPLC
solvents commonly used in HPLC
acetonitrile methanol ethanol isopropanol water
a technique where the strength of solvent remains constant during separation
Isocratic elution
a technique where the strength of solvent continually increases (%/min)
during separation
Gradient elution
In HPLC, this is an organic material covalently bonded to silica that
may be polar or nonpolar in composition.
Stationary phase
composed of polar stationary phase and
nonpolar mobile phase
normal-phase liquid chromatography
composed of nonpolar stationary phase
and polar mobile phase
reversed-phase liquid chromatography
in HPLC, the column which functions as the stationary phase, generally operates at what temperature
room temperature
In HPLC, this produces a signal for identification and quantification of the
solutes
detector
commonly used detectors in HPLC
spectrophotometer photodiode array fluorometer electrochemical mass spectrometer
is an instrument that uses the principle of charged
particles moving through a magnetic or electric field, with ions being
separated from other charged particles according to their mass-to-charge
ratios.
mass spectrometer
In mass spectrometry, the ions are separated from other charged particles according to _________.
mass-to-charge ratio
In mass spectrometry, the mass spectrum produced is for (identification/quantification).
identification
In mass spectrometry, the number of ions produced relates proportionally to concentration and is for (identification/quantification).
quantification
a high-quality technique for identifying drugs or drug
metabolites, amino acid composition of proteins, and steroids.
mass spectrometry
an instrumentation technique used in the field of proteomics
mass spectrometry
The eluate gas from a gas chromatograph may be introduced into a mass spectrometer that functions as _________.
detector system
Components of mass spectrometer
ion source vacuum system analyzer detector computer
component of mass spectrometer which prevents the collision of ions with other molecules when electronic or magnetic separation is occurring
vacuum system
Types of analyzer that may be used in mass spectrometer
beam-type
trapping-type
Analyzer in mass spectrometer which involves a destructive process, where ions pass through the
analyzer one time and then strike the detector.
Beam-type
a beam-type analyzer (in mass spectrometer), where mass-to-charge ratios are scanned during a prescribed time period to form a mass spectrum.
Quadrupole
In mass spectrometry, the detector detects ions using _________.
electron multipliers
Electron multiplier types (detector in mass spectrometer)
discrete dynode
continuous dynode
In mass spectrometry, this converts the detector’s signal to a digital form.
computer and software
In mass spectrometry, sample identification is achieved because each compound produces a __________, which is analyzed by a database for matching to a computerized reference library
unique spectrum
a system where a gas chromatograph or a high-performance liquid chromatograph is connected to two mass spectrometers
(GC/MS/MS) or (HPLC/MS/MS).
tandem mass spectrometer
an instrumentation technique which employs an electrochemical cell
Polarography
a technique used to determine the concentration of a substance in solution employing an electrochemical cell that consists of two half-cells
Potentiometry
In potentiometry, what is being measured?
potential difference (between indicator electrode and reference electrode)
common type of reference electrode (potentiometry)
silver-silver chloride (Ag/AgCl)
common type of reference electrode, consisting of mercury covered by a layer of mercurous chloride in contact with saturated solution of potassium chloride
(potentiometry)
calomel
In potentiometry, this employs a pH-sensitive glass electrode for measuring blood pH, and it employs PCO2 and PO2 electrodes for measuring gases in blood
ph/blood gas analyzer
In potentiometry, the pH electrode is what type of electrode?
glass electrode
Electrochemical technique that measures the amount of current produced through the oxidation or reduction of the substance to be measured at an electrode held at a fixed potential
Amperometry
Coulometry is based on what law?
Faraday’s law
states that in an electrochemical system, the number of equivalent weights of a reactant oxidized or reduced is directly proportional to the
quantity of electricity used in the reaction
Faraday’s Law
Used clinically to separate and identify proteins, including serum, urine and cerebrospinal fluid (CSF) proteins, lipoproteins, isoenzymes, and so on.
electrophoresis
defined as the movement of charged molecules in a liquid medium when an electric field is applied
electrophoresis
defined as the movement of charged molecules in a
porous supporting medium where the molecules separate as distinct zones
zone elctrophoresis
In electrophoresis, this provides a matrix that allows molecules to separate (e.g.,
agarose gel, starch gel, polyacrylamide gel, and cellulose acetate membranes).
support medium
In electrophoresis, the pH at which negative and positive charges are equal on a protein is the _____.
isoelectric point
In serum electrophoresis, buffer solutions are of what pH?
8.6
In electrophoresis, detection and quantification of the separated protein is accomplished using a _____.
densitometer
In electrophoresis, this indicates that the analyte present in too high a concentration
Holes in staining pattern
In electrophoresis, this indicates that the voltage is too low
very slow migration
In electrophoresis, this indicates that the pH is too high or low or there may be excessive heat production
sample precipitates in support
commonly encountered problems in electrophoresis
holes in staining pattern
very slow migration
sample precipitates in support
a type of zone electrophoresis in which protein separation is based on the isoelectric point (pI) of the proteins.
Isoelectric focusing
Capillary electrophoresis is based on what principle
Electroosmotic flow (EOF)
AUTOMATION PARAMETERS/TERMINOLOGY:
Centrifugal force moves samples and reagents into cuvet areas for simultaneous analysis.
Centrifugal analysis
AUTOMATION PARAMETERS/TERMINOLOGY:
Each sample reaction is compartmentalized. This may
relate to an analyzer designed to assay only one analyte (e.g., glucose) or an
analyzer capable of performing multiple tests where the sample and reagents are in a separate cuvet/reaction vessel for each test.
Discrete analysis
AUTOMATION PARAMETERS/TERMINOLOGY:
Able to perform individual tests or panels, and allows for stat samples to be added to the run ahead of other specimens
Random access
AUTOMATION PARAMETERS/TERMINOLOGY:
Samples processed as a group
Batch analysis
AUTOMATION PARAMETERS/TERMINOLOGY:
Instrument from a single discipline with automated capability
Stand-alone
AUTOMATION PARAMETERS/TERMINOLOGY: Instrument from a single discipline with additional
internal automated capability (e.g., auto-repeat and auto-dilute)
Automated stand-alone
AUTOMATION PARAMETERS/TERMINOLOGY:
At least two instruments from a single discipline with one controller
Modular workcell
AUTOMATION PARAMETERS/TERMINOLOGY:
Instrument able to perform tests from at least two disciplines
Multiple platform
AUTOMATION PARAMETERS/TERMINOLOGY:
At least two analytical modules supported by
one sample and reagent processing and delivery system
Integrated modular system
AUTOMATION PARAMETERS/TERMINOLOGY:
Transports specimens quickly from one location
to another
Pneumatic tube system
Maximum number of tests generated per hour
throughput
Amount of time to generate one result
turnaround
Mechanism for patient/sample identification; used for reagent
identification by an instrument
Bar coding
Amount of serum that cannot be aspirated
Dead volume
The contamination of a sample by a previously aspirated sample
Carry-over
Use of preliminary test results to determine if additional tests
should be ordered or cancelled on a particular specimen; performed manually
or automated
Reflex testing
Automated systems exist for laboratories
where samples are received, centrifuged, distributed to particular instruments
using a conveyor system, and loaded into the analyzer without operator
assistance. This kind of automation is seen in large medical center laboratories
and commercial laboratories where the volume of testing is high.
Total laboratory automation
Dry reagents are packaged as ________.
lyophilized powder
tablet form
In automation, some instruments have _____ that detect the amount of serum or plasma in the tube.
level-sensing probes
a vessel where the mixing of sample and reagents occur
cuvet
the permanent, non-disposable cuvets in some automated instruments are made of ____.
quartz glass
disposable cuvets are made of _____.
plastic
most common reaction temperatures
37degC and 30degC
Determination of sample concentration is based on change in absorbance over time
kinetic assays
composition of hemoglobin
tetramer
4 globin chains
4 heme groups
4 iron atoms
hemoglobin type composed of two alpha chains and two beta chains
H A1
hemoglobin type composed of two alpha chains and two delta chains
H A1
hemoglobin type composed of two alpha chains and two gamma chains
H F
hemoglobinopathy involving substitution of valine for glutamic acid in position 6 of
the beta chain
Hemoglobin S
hemoglobinopathy involving substitution of lysine for glutamic acid in position 6 of
the beta chain
Hemoglobin C
Hemoglobinopathies occur when one amino acid is substituted on what chain types?
alpha
beta
hemoglobin differentiation is best achieved by use of
electrophoresis
How is hemoglobin F differentiated from the majority of human hemoglobins?
alkali resistance
at what pH do hemoglobins have a net negative charge and migrate from the point of application toward the anode?
8.6
at what pH do hemoglobins exhibit different electrophoretic mobilities on agar gel
6.2
order of migration of hemoglobins on agar gel (anodal to cathodal)
C&S
A1, A2, D, E, G
F
T/F:
Reaction temperatures and times vary for each analyte.
TRUE
Order of migration of hemoglobins using cellulose acetate
A1
F
S, G, D
A2, C, O, E
Performing diagnostic tests outside the main laboratory and at or
near patient care areas
Point-of-care testing
Point-of-care instruments must be evaluated in accordance with ______?
Clinical Laboratory Improvement Amendments of 1988 (CLIA ‘88)
simple tests that carry a low risk for an incorrect result.
Waived testing
the only type of tests that can be performed using POCT instruments
Waived laboratory tests
immunochemical techniques used to
detect an extremely small amount of analyte (functions as antigen) by reacting
it with an antibody (functions as reagent) to form an antigen-antibody complex
Immunoassay
In immunoassays, the small amount of analyte functions as _______?
antigen
In immunoassays, the antibody functions as _____?
reagent
T/F:
In immunoassays, the label may be attached to either a reagent antigen or a reagent antibody
TRUE
What can increase the specificity of immunochemical procedures?
use of monoclonal antibodies
Antibodies produced in an animal from many cell clones in response to an immunogen
Polyclonal antiserum
heterogenous mixture of antibodies
polyclonal antiserum
Antibodies produced from a single clone or
plasma cell line
monoclonal antiserum
homogenous antibodies
monoclonal antiserum
type of immunoassay based on the competition
between an unlabeled antigen (sample analyte) and a labeled antigen
for an antibody
Competitive-binding immunoassay
In competitive-binding immunoassay, which acts as the sample analyte?
unlabeled antigen
original labels used for immunoassays
radioactive isotopes (I125)
Nonradioactive labels used today in immunoassays
enzyme
fluorophore
chemiluminiscent labels
assays that require that free labeled antigen be physically removed from the labeled antigen bound to antibody
Heterogenous
assays that do not require physical removal of free
labeled antigen from bound-labeled antigen
Homogenous
Examples of heterogenous assays
RIA
ELISA
IRMA
a homogeneous
immunoassay where the sample analyte (functions as unlabeled antigen)
competes with the enzyme-labeled antigen for the binding sites on the antibody
Enzyme multiplied immunoassay technique
T/F:
In EMIT, the more analyte (unlabeled antigen) present in the mixture,
the less binding of enzyme-labeled antigen to the antibody.
TRUE
immunochemical technique based on the amount of
polarized fluorescent light detected when the fluorophore label is excited with polarized light
Fluorescent polarization immunoassay (FPIA)
immunochemical technique based on measuring
the degree to which fluorescence intensity is greater in one plane than in another (polarized versus depolarized).
FPIA
a homogeneous technique where the sample analyte
(functions as unlabeled antigen) competes with the fluorophore-labeled antigen for the binding sites on the antibody.
FPIA
T/F:
The more analyte
(unlabeled antigen) present in the mixture, the more binding of
fluorophore-labeled antigen to the antibody
FALSE
a technique between antigen and antibody that employs a chemiluminescent indicator molecule such as isoluminol and acridinium ester as labels for antibodies and haptens.
Chemiluminescent immunoassay
chemiluminescent indicator molecules used in chemiluminescent immunoassay
isoluminol
acridinium ester
a homogeneous technique that is an adaptation of the chemiluminescent
immunoassay.
Luminescent oxygen channeling immunoassay (LOCI)
indicator label used in electrochemiluminescence immunoassay
ruthenium
