Spectral Basics
This type of light consists primarily of a single wavelength.
What is monochromatic light?
This law states that absorbance is directly proportional to concentration and path length.
What is the Beer-Lambert law?
This technique measures the absorption of light by ground-state atoms.
What is atomic absorption spectroscopy (AAS)?
This technique separates charged molecules by applying an electrical field.
What is electrophoresis?
What is EOF
Electro-osmotic flow—the bulk movement of liquid through a capillary caused by an applied electric field acting on charged surfaces.
This technique measures light scattered by particles, usually using a detector positioned at an angle to the incident light.
What is nephelometry?
This phenomenon occurs when white light separates into its component wavelengths.
What is dispersion?
If a sample has a transmittance of 100%, its absorbance is:
What is 0?
This technique measures radiation emitted by excited atoms.
What is atomic emission spectroscopy (AES)?
This serum protein normally migrates the fastest during serum protein electrophoresis because of its high negative charge and relatively small size.
What is albumin?
The pH at which a protein has no net electrical charge is called its:
What is the isoelectric point (pI)?
This technique measures the reduction in transmitted light caused by particles in a suspension.
What is turbidimetry?
This phenomenon occurs when light changes direction as it passes from one medium into another.
What is refraction?
A sample has a % transmittance of 25%. Calculate its absorbance.
A = 2 − log(%T)
A = 2 − log(25)
A = 2 − 1.397 = 0.603
Before absorption can occur, the sample must be converted into individual free atoms. This process is called:
What is atomization?
A negatively charged molecule placed in an electrophoresis system will migrate toward which electrode?
What is the anode (positive electrode)?
What is the major difference between IEF and IFE?
IEF separates proteins according to their pI. IFE uses antibodies to identify specific proteins after electrophoretic separation
A small fluorescent molecule rotates more rapidly than a large fluorescent molecule. Which molecule generally produces greater fluorescence polarization?
What is the larger molecule?
Name the three major interactions of electromagnetic radiation with matter that are important in spectral techniques.
What are absorption, emission, and scattering?
A standard has a concentration of 20 mg/dL and an absorbance of 0.50. An unknown has an absorbance of 0.75. Assuming Beer-Lambert behavior, what is the concentration of the unknown?
What is 30 mg/dL?
(20 / 0.50 = X / 0.75
X = 30 mg/dL )
This type of light source is commonly associated with atomic absorption and produces element-specific wavelengths.
What is a hollow cathode lamp?
Name the five major serum protein electrophoresis fractions in order from fastest to slowest migration.
What are:
Albumin → α₁ → α₂ → β → γ
This detergent binds to proteins and gives them a relatively uniform negative charge-to-mass ratio, allowing separation primarily by molecular size.
What is sodium dodecyl sulfate (SDS)?
Match the technique with what it detects:
Southern blot
Northern blot
Western blot
Southern = DNA
Northern = RNA
Western = Protein
Name four major components found in a typical spectrophotometer and state the function of each.
What are light source, wavelength selector/monochromator, sample holder/cuvette, and detector?
A spectrophotometer gives inaccurate results because the instrument has not been properly adjusted using a blank, standards, or reference materials. What analytical process is needed to establish accurate measurement?
What is calibration?
Compare atomic absorption and atomic emission spectroscopy. Include what happens to the atoms and what is actually measured.
Atomic absorption: ground-state atoms absorb element-specific radiation.
Atomic emission: excited atoms return toward lower energy states and emit radiation.
A densitometry tracing of an SPEP pattern shows a large, narrow peak in the gamma region. What type of protein abnormality should be suspected, and what technique could be used to further characterize it?
What is a monoclonal immunoglobulin/gammopathy, and immunofixation electrophoresis (IFE) can be used for further characterization?
A protein sample is treated with SDS and β-mercaptoethanol before electrophoresis. What does each reagent do?
SDS: denatures proteins and gives them a relatively uniform negative charge.
β-mercaptoethanol: reduces disulfide bonds, separating protein subunits.
Differentiate these three forms of luminescence: bioluminescence, chemiluminescence, and electrochemiluminescence.
Bioluminescence: light generated through a biological process.
Chemiluminescence: light produced by a chemical reaction.
Electrochemiluminescence: light generated through an electrochemical process.