Instrumentation and Analytical Principles Flashcards
Spectrophotometer
Measures light transmitted by that analyte in solution
Tungsten lamp
Most common
Visible and near-infrared region
Heat absorbing filter between lamp and sample to absorb radiation
Not enough energy for UV region
Deuterium lamp
UV region
Continuous emission down to 165 nm
Mercury arc lamp
UV region
Low-pressure emit sharp UV and vis line spectra (not good got absorbance)
Medium and high-pressure lamps emit continuum from UV to mid-vis
Xenon arc lamp
UV region
Continuous spectra
Laser light source
Intense, focused, nearly nondivergent beam of light
Monochromator
Isolate radiant energy of wider wavelengths to mechanically selected narrow band of chosen wavelengths
Wavelength isolator
Bandpass
Width of the band of light at one-half the peak maximum
Ex. Set to read at 550 nm, light from 540-560 nm, so band pass is 10 from 545-555 nm
Absorption spectrophotometry
Ground-state atoms absorb light at defined wavelengths/line spectrum
Sample is atomized in flame, light from hollow-cathode lamp passed through chopper to flame, to detector
Hollow-cathode lamp
Contains anode, cathode is metal being analyzed, and inert gas
Voltage causes ionization of gas, attracted to cathode to collide and knock atoms off, become excited then emit light when return to ground state
Flameless absorption spectrophotometery
Has rod to hold sample in chamber, increase temp to vaporize sample, then absorb light from HCL
More sensitive than flame
Nephlometry
Measure of scattered light
Amount of scatter is directly proportional to number and size of particles present
Sensitivity depends on absence of background scatter
Turbidmetry
Measures light blocked (decrease in transmitted light), dependent on particle size and conc.
Uses spectrophotometer for measurement
Fluorescence
Atoms absorb energy at particular wavelength (excitation), release energy of longer wavelength/lower energy on return to ground state
Fluorometry
UV light used for excitation, pass through primary filter to select excitation wavelength, energy released passed through secondary filter to detector (both at right angle to source to prevent measuring incident light)
Luminescence
Excitation doesn’t require absorption of radiant energy
Chemiluminescence
Chemical energy excites atoms, photons of light emitted
Bioluminescence
Enzyme-catalyze reaction produces light emission
Luminometer
Generic term for instrument to measure chemiluminescence and bioluminescence
Chromatography
Solutes from sample are separated/identified based on physical differences that allow for distribution between mobile and stationary phase
Thin-layer chromatography
Solute resolution and separation by solubility in mobile phase and electrostatic forces in stationary phase
Polarity of solvent important
Gas-liquid chromatography
Separates volatile solutes
Sample is vaporized, inert cattier gas carries sample into column, sample interacts with stationary phase, more volatile/less interaction with stationary is eluted faster
High-performance liquid chromatography
Liquid mobile phase passed over stationary phase of column (usually organic material covalently bonded to silica)
Normal = polar stationary, non-polar mobile
Reverse = non-polar stationary, polar mobile
Mass spectrometry
Charged particles moving through electric/magnetic field, ions separated from other charged particles by mass-to-charge ratio
Sample bombarded by ionization beam, vacuum prevents collision of ions
Polarography
Electrochemical cell, gradually increase voltage between two electrodes in contact with analyte, measure current and voltage change, increase in current proportional to conc. of analyte
Anodized stripping voltammetry
Based on polarography
Negative potential applied to one electrode, metal in solution reduced and plated onto anodic electrode, used as anode in polarographic cell, metal stripped, current during stripping identifies and quantifies analyte
Potentiometry
Determine concentration by using electrochemical cell with 2 half cells, potential difference between indicator and reference measured
Amperometry
Measure current from oxidation/reduction of analyte, measures PO2
Coulometry
Based off idea that amount oxidized/reduced is proportional to electricity used
Measure Cl, Chloride binds with silver, when chloride all used up remaining silver causes increase in current which is measured
Hemoglobin A1
2 alpha and 2 beta chains
Hemoglobin A2
2 alpha and 2 delta chains
Hemoglobin F
2 alpha and 2 gamma chains
Hemoglobin S
Substitution of valine for glutamic acid in position 6 of beta chain
Hemoglobin C
Substitution of lysine for glutamic acid in position 6 of beta chain
Throughput
Max number of tests generated per hour
Dead volume
Amount of serum that cannot be aspirated
