Analytical Techniques and Measureme Flashcards
Regions in Electromagnetic Spectrum: Ultraviolet light, Visible light, Infrared
absorbance wavelengths
Ultraviolet light < 400 nm
Visible light 400-700 nm
Infrared > 700 nm
Planck’s formula for energy: E = hν
E = energy, h = Planck’s constant, ν = frequency
Relationship between wavelength (λ) and energy (E)
Inversely related
E = hc/λ, where h is Planck’s constant, c is the speed of light
Beer-Lambert’s Law relationship
Directly proportional
A = abc
ɛ = molar absorptivity; b = path length; c = concentration
To compute the absorbance value given the % transmittance:
A =2 -log %T
To determine the concentration of an unknown analyte:
c = A / (ɛ × b)
Abs of sample / (A STD x STD known)
Blank used in Spectrophotometry
Distilled water, reagent, or sample to subtract absorbances not due to analyte
Corrects absorbance caused by reagent color
Reagent blank
Subtracts absorbance from hemolysis, icterus, turbidity, or drug interference
Sample blank
Substance of known purity and concentration used to determine unknown analyte concentration
Standard solution
Contains various analyte concentrations to monitor analytical performance
Control solution
Characteristics of Control solution
Commutable, stable, no matrix effects, spanning clinically important range
Values provided by manufacturer
Assayed control
Values determined by the laboratory
Unassayed control
Checked using didymium glass or holmium oxide, directly proportional to Beer’s Law.
Wavelength accuracy
Done using glass filters and solutions that have known absorbance values.
Absorbance check
Change in concentration resulting in a straight-line calibration curve, related to Beer’s Law.
Linearity
Any wavelength outside the band of interest, detected using sharp cut-off filters.
Stray light
Sources include extraneous room light, light dispersed by a darkened lamp envelope, deteriorated optics, scratches on optical surfaces, dust particles in the light path, higher order spectra produced by diffraction gratings.
Sources of stray light
Provides polychromatic light which the sample will modify or attenuate by absorption.
Light source
UV light source; commonly used in the UV region.
Deuterium/Hydrogen
UV-visible light source.
Xenon/Mercury
Visible to near infrared light source.
Tungsten; LASER
Prevents stray light from entering the monochromator system.
Entrance slit
Effects of stray light includes
absorbance error and loss of linearity
Isolates a portion of the spectrum emitted by the source and focuses it on the sample.
Monochromator
Continuous, non-linear spectrum; better separation of high-frequency light.
Prism
Continuous, linear spectrum; uniform separation of wavelengths; most common.
Diffraction gratings
Controls the bandpass; allows only a narrow fraction of the spectrum to reach the cuvette.
Exit slit
Range of wavelengths transmitted, calculated as width at more than half the maximum transmittance.
Bandpass
Sample cell; may be round or square.
Cuvette
Material used for UV and IR measurement.
Quartz/Fused silica
Material used for UV to visible measurements.
Plastic
Material used for visible measurements only.
Glass
Converts the transmitted light energy into an equivalent amount of electrical energy.
Photodetector
Simple photodetector type.
Barrier layer cell
Requires an external voltage source for operation (photodetector)
Phototube
Photodetector with excellent linearity.
Photodiode
Most commonly used and most sensitive photodetector; amplifies light signal.
Photomultiplier tube
Processes the electrical signal, performs mathematical operations, and displays the output.
Readout device
Formula used to process and display absorbance:
Absorbance readout formula:
A = 2 - log(% transmittance)
Designed to compensate for variations in intensity of the light source by splitting the light beam.
Double-beam spectrophotometry
Type of double-beam spectrophotometry where a beam splitter directs one portion of light to the sample cuvette and the other to the reference cuvette.
Double-beam-in-space
Type of double-beam spectrophotometry where a chopper rotates continuously and strikes one cuvette at a time.
Double-beam-in-time
Measurement of light emission caused by a chemical, biochemical, or electrochemical reaction, not by photo illumination.
Luminometry
Emission of light caused by oxidation of organic compounds catalyzed by an enzyme, a metal, or hemin.
Chemiluminescence
A special form of chemiluminescence where an enzyme-catalyzed chemical reaction produces light emission and involves the use of natural substrates.
Bioluminescence
Emission of light caused by a reaction generated electrochemically on the surface of an electrode.
Electrochemiluminescence
Detection of scintillations (flashes of light) using a PM tube and counting electrical impulses.
Scintillation Counting
Type of scintillation counting used for detecting gamma radiation (I125 and I131).
Crystal scintillation (gamma counter)
Type of scintillation counting used for detecting beta radiation (H3 and C14).
Liquid scintillation (Beta counter)
Involves fragmentation and ionization of molecules, followed by separation of ions by mass-to-charge ratio.
Mass Spectrometry
Mass spectrometry technique that uses MALDI for analyzing proteins and other large molecules.
MALDI TOF MS
Mass spectrometry technique that combines gas chromatography with mass spectrometry for separating and analyzing compounds.
GCMS or HPLC-MS
Mass spectrometry technique that uses multiple stages of mass spectrometry for detailed analysis.
Tandem MS (MS/MS)
A quantitative method for determining the amount of an analyte based on isotope dilution.
IDMS
Non-destructive method for determining the structure of organic compounds, used in lipoprotein particle measurements.
Nuclear Magnetic Resonance (NMR) Spectroscopy
Approach where specimens are pumped through a continuous tubing system at the same rate and subjected to the same analytical reactions; can result in carry-over problems.
Continuous flow
Automation method using centrifugal force to transfer liquids into separate cuvets for measurement, capable of batch analysis.
Centrifugal analysis
Automation method that places each sample and accompanying reagents in separate containers, allowing for batch analysis, random access, or stat capabilities.
Discrete analysis
Analyzer configuration where specimens enter the analytical process one after another, and results are produced in the same order as specimens.
Sequential analysis
Analyzer configuration where all specimens are subjected to a series of analytical processes simultaneously, in parallel.
Parallel analysis
Analyzer configuration that groups many specimens in the same analytical session for analysis, handling a large number of specimens in one run.
Batch analysis
Analyzer configuration that allows each specimen to be analyzed for a different set of tests and can analyze stat specimens out of sequence as needed.
Random-access analysis
Type of analyzer that requires reagents to be provided in a unique container or format by the manufacturer.
Closed-system analyzer
Type of analyzer that allows operators to change parameters and use reagents from various suppliers.
Open-system analyzer
Testing device with advantages of reduced turnaround time and electronic documentation of testing.
Point of Care Testing (POCT) Devices
Most commonly used POCT device that uses enzymatic methods coupled with photometric or electrochemical detection but should not be used to diagnose diabetes mellitus.
Blood glucose monitors (glucometers)
SMA, Technicon is an example of
Continuous flow
Cobas-Bio (Roche) is an example of
Centrifugal analysis
OCD Vitros 350, Beckman Unicel DXC, Dupont ACA, Abbott Architect, Cobas 6000, Siemens Vista are examples of
Discrete analysis
Point of Care Testing (POCT) Devices
Blood glucose monitors (glucometers)
