Flashcards in Analytic Methods Deck (60)
An organic molecule is excited and emits electromagnetic radiation as it returns to ground state.
Usually involves an oxidation reaction to excite the molecule...
Two inactive fragments of beta-galactosidase are produced; the "enzyme donor" fragment is attached to a target antigen while the "enzyme acceptor" fragment is attached to an antibody against that antigen.
Presence of antigen results in chromogen formation as the beta-galactosidase is re-formed.
The difference in wavelength between a molecule's excited and ground state. This should be sufficiently large as to not overlap and cause increased background noise in the detector.
A method that binds a lanthanide ion to an organic ligand that is activated by radiation. This confers a longer fluorescence lifetime, improving sensitivity, and also increasing the stokes shift.
What are heterophile antibodies?
Usually human anti-mouse antibodies (HAMAs) that can cause false elevation of many lab tests that utilize mouse-derived capture antibodies. Also applies to antibodies seen in autoimmune conditions, EBV infection, and drug-induced lupus.
Enzyme-labeled drug competes with free unbound drug for a drug-specific antibody. Enzyme that becomes antibody-bound cannot react with substrate; therefore more free unbound drug increases signal.
Fluorophore-labeled drug competes with free unbound drug for a drug-specific antibody. Fluorophore that is antibody-bound is sterically hindered and produces polarized light. Free fluorophore rotates rapidly and produces depolarized light.
Optical detection method that examines light reflectance from a test strip as a chromogenic reaction. As the reaction progresses, light reflectance relative to an all-white standard decreases in a nonlinear fashion.
How are point-of-care glucometers affected by hematocrit levels?
High hematocrit levels result in UNDERestimation of glucose.
Low hematocrit levels result in OVERestimation of glucose.
How do conductivity-based detection methods compare to optical-based methods in terms of performance?
Conductivity-based detection is relatively insensitive to variations of hematocrit but is affected by serum protein and electrolyte concentration.
A = ebc
e (molar absorptivity)
b (path length)
Spectrophotometric assay for detection of creatinine. Combine with picric acid to form the "Jarnovsky chromogen".
Glucose hexokinase method
Add hexokinase to sample, to generate NADPH via G6P intermediate.
What ranges of absorption should always be avoided in spectrophotometric assays?
280nm (aromatic amino acids)
Calcium spectrophotometric detection method
Use EDTA/BAPTA or Arsenazo II to form an optically absorbent chelated complex.
What are the components of a spectrophotometer?
Light source, monochromator, cuvette, detector
What is "double-beam" or "split-beam" spectrophotometry?
Spectrophotometry in which the monochromatic beam is split so that a control background "zero" absorbance can be established.
What is "band pass" in spectrophotometry?
The range of wavelengths detected as signal. The selection of the band pass has significant implications for sensitivity, specificity, and accuracy.
Why should absorption in spectrophotometry be limited to <1.0 OD range?
Above this range, background light significantly interferes with detection
How are light transmittance and absorption related in spectrophotometry?
Absorption = log(1/transmittance)
Or, absorption = -log(transmittance)
How do hemolysis, icterus, and lipemia affect spectrophotometry?
Hemolysis absorbs at 405nm and 575nm.
Icterus absorbs at around 480nm.
Lipemia absorbs progressively more at shorter wavelengths (<340nm).
Contrast between turbidimetry and nephelometry.
Turbidimetry measures the degree of light scatter through a sample.
Nephelometry detects light scattering by usually antibody complexes at specific angles
Heating of vaporized, isolated atoms to an excited state, with measurement of emission spectra upon return to ground state. Appropriate for analysis of metals.
Spectrophotometry using vaporized, isolated atoms to identify them based on absorption spectra.
What is the general structure of a voltaic cell?
An anode furnishes electrons through oxidation which travel to a cathode which receives them via reduction.
E = E' - (RT/nF)ln(aRed/aOx)
What is ion activity (a)?
A measure of the relative concentration of charged particles in solution. a = g * n, where g = activity coefficient (related to ion pairing) and n = molality.
What two methods can an electrode use to detect potential differences?
Redox (electrode itself participates in a redox reaction)
Phase-boundary (look at potential of ion transfer across a gradient, usually a membrane)
What are some examples of redox electrodes?
Standard hydrogen (platinum) electrode (default E = 0)
Calomel (mercuric chloride) electrode
Silver chloride electrode