Exam 5 Spectrometry & Luminescence – Analytical Techniques

Define the following:
Spectrometry
Measurement of elecromagnetic radiation in waves
What are the 4 analytical Spectrometry Techniques?
Photometry
Spectrophotometry
Atomic Absorption
Mass Spectrometry
What are the 3 analytical Luminescence Techniques?
Fluorescence
Chemiluminescence
Nephelometry
With Photometry what does the instrument measure?
light intensity without consideration of wavelength
What is a common example of photometry?
Urine Dipstick Readers
(Reflectance photometry)
Define Beer’s Law
mathematical relationship between Absorbance and concentration.
The concentration of a substance is directly proportional to the amount of light absorbed
Give the mathematical formula for Beer’s Law
(Concentration Unknown/Concentration Standard) = (Absorbance Unknown/Absorbance Standard)
What is the most widely used light source for visible and near-infrared analysis?
Tungsten lamp
Which lamps are used for UV?
Deuterium and mercury-arc
What does the monochromator do?
Isolates 1 particular wavelength of light
Where does stray light come from? (bad!)
Extraneous, unwanted light caused by scratches,dust particles, open compartments
Define the following:
Photodetector
Converts transmitted light into electrical energy
What does the readout device do?
Converts the electrical signal into a useable/readable unit
What is the use of blanks?
Correct for any absorbance due to the color of the reagent or the sample (ex. hemolysis, lipemia)
Define the following:
Wavelength Accuracy Check
Use standard absorbing solutions or filters with known wavelengths to confirm
Define the following:
Linearity Check
Commercially available sets of solutions with expected Abs readings can be run to verify the
calibration curve
What are the 3 parts of Spectrophotometry Quality Assurance?
Use of Blanks
Wavelength Accuracy Check
Linearity Check
Atomic Absorption Spectrophotometry
Measures absorption of light energy by atoms rather than by molecules
What is Atomic Absorption Spectrophotometry mainly used for?
Quantitation of trace metals (copper, zinc, etc)and toxins
What is the light source for Atomic Absorption Spectrophotometry?
Hollow-cathode lamp
What does the Chopper/Modulator do for Atomic Absorption Spectrophotometry?
Breaks cathode lamp light into pulses per second so that the detector can
differentiate between cathode lamp
light and sample emitted light
What does the Burner do for Atomic Absorption Spectrophotometry?
-Breaks chemical bonds in the sample into free atoms (vaporized).
-When atoms absorb light from the lamp
they become excited. As they return to ground state they emit light of the same wavelength as what they absorbed
What are the 2 light signals that will emitted from the Atomic Absorption Spectrophotometry burner?
Pulsed light from the cathode lamp and a steady signal from the patient sample
What does the Monochromator do in Atomic Absorption Spectrophotometry?
Isolates desired wavelength
What are the Advantages of AA?
Accurate, precise, specific.
Which type of Spectrophotometry is capable of measuring substances that
are not easily excited (metals)?
AA Spectrophotometry
What is used primarily in conjunction with chromatography techniques?
Mass Spectrophotometry
Luminescence is the production of light without the production of what?
Heat
Define Stokes Shift
The difference between the Absorbed energy wavelength and the Emitted energy wavelength
What is the light source for a basic fluorometer?
High intensity mercury-vapor or xenon arc lamp
What does the Primary monochromator select for?
Excitation (Absorbed) wavelength
What does the Secondary monochromator select for?
Emission (Fluorescence) wavelength
What are the sensitivity advantages of Fluorometry?
1000 x more than most specs
What are the specificity advantages of Fluorometry?
Since 2 wavelengths are selected
What are the Disadvantages of Fluorometry?
Sensitive to pH, temperature, viscosity (quenching)
What is the use of Fluorometry?
Measuring substances in low concentrations in plasma or serum
(drugs, hormones, vitamins,
cardiac markers)
With Chemiluminescence, the light emissions are proportional to what?
Analyte Concentration
Chemiluminescence labels include (3)
Luminol, acridinium esters and dioxetanes
With Chemiluminescence, light measures can be made in how many seconds?
10-30 seconds
The following are characteristic of what type of luminescence?
-Cost of reagents and instrumentation are low; easy to perform
-Extremely low detection limits (lower than fluorometry)
Chemiluminescence
Chemiluminescence:
Low or High Sensitivity?
High
Chemiluminescence:
Low or High Specificity?
High
What is Chemiluminescence used to detect?
hormones, drugs, vitamins and cardiac markers
Nephelometry:
Light will scatter in direct proportion to what?
Amount of AGN present in the patient’s sample
What is Nephelometry primary used for?
Quantitate serum proteins
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