Instrumentation and Analytical Principles Flashcards
How is light classified? what is visible light?
according to wavelength: UV light has very short wv, infrared has very long; Visible light is that between 400 and 700 nm, which when combined produces white light
What does a spectrophotometer measure?
the light transmitted by an analyte in solution, in order to determine the concentration. Analytes may absorb, transmit, and reflect light to varying degrees but always characteristic to the analyte
what types of lamps are most common in spectrophotometers, and for which regions?
Tungsten: for visible and IR
Deuterium: for UV
what must be done when the lamp, is changed in a spectrophotometer?
recalibration bc changing the light source changes the angle of the light striking the monochromator
what is the monochromator?
a device that allows selection of a particular set of wavelengths at the input: allows polychromatic light to be separated into narrow bands of monochromatic light
what types of monochromators are used in photometers vs spectrophotometers?
glass and interference filters vs diffraction gratings and prisms
How does the monochromator allow wavelength selection?
the exit slit is used to select the bandpass which allows the light of the selected wavelength to pass through the cuvette onto the detector
what is atomic absorption spectrophotometry based on, principle-wise?
what is it used to do?
the fact that ground-state ATOMS (not molecules) absorb light at defined wavelengths/ each metal has a specific line spectrum
put another way: measure concentrations by detecting absorption of electromagnetic radiation by atoms
what are the basic steps of atomic absorption spectrophotometry?
- sample in sample cell is atomized in a flame or by electrothermal method; the atoms to be measured are kept at ground-state
- beam from hollow cathode lamp is passed through a chopper to the flame
- the ground-state analyte atoms in the flame absorb the same wavelengths of light from the lamp as they would emit when excited
- the light NOT absorbed by the atoms is measured as a decrease in light by the detector (photomultiplier tube)
what is measured in nephelometry?
light scattered by particulates in solution; amount of scatter is directly proportional to number and size of particles present in solution
what does turbidimetry measure? what instrument does it use?
the light blocked by particles in solution, as a decrease in transmittance; uses a spectrophotometer
briefly describe fluorescence
a type of luminescence in which atoms absorb energy at a particular wavelength, their electrons are raised to higher-energy orbitals and then when return to ground state they emit light of longer wavelength and lower energy than the exciting wavelength
what is chemiluminescence?
the process where the chemical energy of a reaction produces excited atoms and when electrons return to ground state, emit light
chromatography is a technique in which solutes in a sample are separated for ID based on:
physical differences that allow their differential distribution between a mobile phase and a staionary phase
describe some mobile phase and stationary phase types in chromatography
mobile: liquid or gas
stationary: can be silica gel bound to the surface of a glass plate or plastic sheet, or silica/polymer bound to a column
IN Gas-Liquid chromatography, will a more volatile solute elute faster or slower? what other solute attribute causes faster elution?
faster; a solute that has less interaction for the solid phase (the column)
When a sample is injected into a gas-liquid chromatographer, what happens to it? How does it move through the column?
It is vaporized bc the interior is much higher temp; then an inert carrier gas carries it into the column
what are some inert carrier gases used in chromatography?
helium, hydrogen, nitrogen, argon
what determines separation of solutes in GLC?
both the differences between vapor pressures of solutes and amount of interaction with the column
What is HPLC and what governs separation of solutes?
High Performance liquid chromatography; separation of solutes is governed by selective distribution of solutes between mobile and solid phases (using polarity and nonpolarity)
what is the diff btw isocratic and gradient elution in HPLC?
In isocratic, the solvent strength remains constant; In gradient it continually increases during separation
What is the diff btw normal-phase and reversed-phase liquid chromatography?
IN normal phase, the stationary phase is polar and the mobile phase is nonpolar
In reverse……obviously they are the opposite
If a solute is introduced to the detector for HPLC before all the other solutes, and appear first, were they faster or slower to be eluted? What does this mean regarding their properties?
they are faster to elute if detected first; this means they were more volatile and/or less attracted to the solid phase.
what is the principle of mass spectrometry?
electrons bombard a sample and ionizes it into fragment ions that are separated by their mass-to-charge ratios; a spectrum is produced that is unique for a compound so it can be id’d, as well as the number of ions produced relates to quantity
What are the most important applications of Mas spectrometry in clinical labs?
identification of drugs or drug metabolites, steroids, as well as the amino acid composition of proteins
what can be used in conjunction with mass spec to identify the eluate?
both gas-liquid and HPL chromatography
what is anodic stripping voltammetry used to assay and what is it based on (a technique using an electrochemical cell and the voltage at which sharp rise in current id’s the analyte electrochemically and the wave height reflects its concentration)
it is used to assay heavy metals, such as lead in blood; The description of this method is polarography
what method of assay common to electrolytes in serum is based on potentiometry? what is the principle
Ion-selective electrodes; the concentration of a substance in solution is determined using an electrochemical cell made of two half-cells and the potential diff btw an indicator electrode and reference electrode is measured
what antibiotics are used as neutral carriers for Iion exchange (selective) electrodes for potassium, and for ammonium?
K: valinomycin
NH4+: nonactin and monactin
what determines the charge of proteins in a solution?
the pH of the solution, bc they are amphoteric
In buffer solutions of pH 8.6 for electrophoresis, what net charge do serum proteins take on?
a net negative charge, and they migrate toward the POS ANODE
what are some stains that are used to visualize separated serum proteins in agarose or cellulose gel?
coomassie blue, amido black B, Ponceau S
what stains are used for visualizing proteins separated by electrophoresis from CSF, for lipoproteins, and LD isoenzymes?
CSF: silver nitrate
lipoproteins: 7B and oil red O
LD: nitrotetrazolium blue
