Optical Techniques Flashcards
wavelength is measured in terms of _______
nanometers (nm)
the distance traveled by one complete wave cycle
wavelength
the number of cycles per second, inversely proportional to wavelength
frequency
the peak height of a wave
amplitude
wavelength that has a high frequency and high energy (400 nm)
short wavelength
wavelength that has a low frequency and low energy (700 nm)
Long wavelength
wavelength (in nm) of visible light
380-800 nm
wavelength (in nm) of ultraviolet light
<380 nm
The visible colors correspond to the wavelenghts that are _______ to our eyes
transmitted
Only colors we can see are transmitted to the eyes, others are ______ but not seen. This color is the complementary color of the transmitted color.
absorbed
these experiements were with the interaction of monochromatic (one wavelength) electromagnetic radiation with varying thickness of absorbing material
Lambert’s Experiments
______ is inversely and logarithmically related to %T
absorbance
experiments with transmittance of a solution under differing concentrations
Beer’s Experiments
As concentration increases, absorbance increases linearly and transmittance decreases exponentially
Beer’s Experiments
A=abC , state the law and what each letter represents
Beer’s Law; A=absorbance, a= molar absorptivity constant in g/L, b=path length of cuvette, C=concentration
Beer’s Law is not valid if…(5 limitations)
1 very elevated concentrations are measured
2 incident light is not monochromatic
3 solvent absorption is significant compared to solute absorption (HIL)
4 radiant energy is transmitted by other mechanisms (stray light)
5 sides of the cuvette are not parallel
function is to be a source of radiant energy
light source
Four types of light sources
tungsten filament with halogen vapor, ultraviolet, hollow cathode lamp, and laser
function is to focus light into a narrow beam
entrance slit
function is to isolate the desired wavelength and exclude others
monochromator
3 types of monochromators
glass filters, prisms, and diffraction gratings (used in lab)
used for readings in the visible and near visible range of spectrum
colored agent of a glass filter
augment wavelengths in phase, destroy those out of phase
interference filters (glass)
eliminate transmission of wavelengths above or below a set wavelength
glass cut-off filters
made of glass or quartz, these do not give a linear dispersion of light, creates bends in light
prisms
a diamond point used to cut tiny furrows into aluminized face of perfect, flat piece of glass; precise angle and distance; will bounce off at different angles to determine which light will go through
diffraction grating
function is to determine the bandpass (bandwidth) or range of wavelengths that will reach the sample, last line of defense before light hits the sample, defined as the bandwidth of the absorbance curve at a point equal to one-half of the max absorbance.
exit slit
function is to act as a receptacle for the sample
cuvette
______ cuvettes are used for visible measurements
glass
_______ cuvettes are used for UV measurements
quartz
function is to convert transmitted light energy to electrical energy
detector
thin layer of semiconductor (selenium) deposited on metal base (iron), selenium coated with thin silver lacquer and covered by protective glass, encased in plastic
barrier layer cell (detector)
semiconductors which change their charged voltage upon being struck by light; voltage change is converted to current (electron flow) and measured
Photodiode
photomultiplier tube components
cathode, anode, photosensitive electrodes, encased in evacuated glass envelope
Light energy strikes cathode, electrons are emitted and focus on first dynode; dynode emits an increased number of electrons, and each successive dynode does the same; final amplification may be 10^6 times more than initial energy
Photomultiplier Tube (PM)
why are PM tubes preferred detectors for clinical laboratory spectrophotometers?
use a small amount of transmitted light to multiply the signal and expand the range of absorption vs concentration. This lessens the need to dilute a sample.
function is to display the electrical signal from the detector in an understandable form; may be an LED, meter, recorder, printer, digital display, etc.
readout device
3 types of spectrophotometers
single beam, double beam (in time/in space/dual), and recording
Most common spectrophotometer
single beam
advantage of double beam spectrophotometers
Useful when you need 100% accuracy
These spectrophotometers run with a reference and are twice as big as a single
double beam and split (dual) beam (see picutres page 14 in notes)
Quality control 1 purpose and 2 method for wavelength accuracy
1 to ensure wavelength is really going through the sample
2 replacement of source lamp, holmium oxide (rare earth) glass filter, didymium filter
Quality control 1 purpose and 2 method for band width
1 monitor range of wavelengths
2 use mercury vapor lamp to verify
Quality control 1 purpose and 2 method for stray light
1 no ambient room light
2 use glass cut-off filters to verify
Quality control 1 purpose and 2 method for linearity
1 make sure absorbance vs concentration
2 didymium filter
Quality control 1 purpose and 2 method for photometric accuracy
1 checking performance of instrument/spectrophotometer
2 National Bureau of Standards (NBS) glass filters or potassium dichromate solutions
failure to get reading in spect
usually electrical
erratic readings in spec
improper warm-up
incorrect wavelength in spec
we want 450 nm but can be a misaligned monochromator
scattered light in spec
scratched or improper cuvette
unsteady baseline in spec
grounding problem; when you zero instrument get crazy results
dark current in spec
residual current when instrument is turned off
emission of light by an atom or molecule after absorption of an excitation photon
fluorescence
Emitted light has a _______ wavelength and _______ energy than the excitation light
longer, less
______ detects emitted light and follows Beer’s law
fluorometry
Why do fluorometers have a right angle design?
To minimize the amount of excitation light that may reach the detector, improving sensitivity and specificity of fluorometers
Technique where a chemical reaction (oxidation) of an organic compound causes immediate light flash
Chemiluminescence
Measures the decrease in percent transmittance through a solution; increased conc of particles=less light transmitted through solution; follows Beer’s Law and assays may be performed on a spectrophotometer
Turbidimetry
Measures the amount of light that is scattered toward a detector at a variety of angles (not 0 or 180)
Nephelometer
Two common chemiluminescent materials used
Luminol and acridinium ester (AE)
