Optical Techniques Flashcards
The distance traveled by one complete wave cycle
Wavelength
Wavelength is measured in terms of ____
Nanometers (nm)
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 (700nm)
Long wavelength
Wavelength (in nm) of visible light
380-800 nm
Wavelength (in nm) of UV light
?
The visible colors correspond to the wavelengths 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 experiments were w/ the interaction of monochromatic (one wavelength) electromagnetic radiation w/ varying thickness of absorbing material
Lambert’s Experiments
____ is inversely and logarithmically related to %T
Absorbance
Experiments w/ 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 (g/L), b = path length of cuvette, C = concentration
Beer’s Law is not valid if…. (5 limitations)
- Very elevated concentrations are measured
- Incident light is not monochromatic
- Solvent absorption is significant compared to solute absorption (HIL)
- Radiant energy is transmitted by other mechanisms (stray light)
- Sides of the cuvette aren’t parallel
Function is to be a source of radiant energy
Light source
Four types of light sources
- Tungsten filament w/ halogen vapor
- UV
- Hollow cathode lamp
- 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
- 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, but creates a bend 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 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) deposted on metal base (iron). Selenium is coated w/ 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 10^6x 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 spectorphotometers
Single beam, double beam (in time/in space/dual), and recording
The most common spectrophotometer
Single beam
Advantage of double beam spectrophotometers
Useful when you need 100% accuracy
These spectophotometers run w/ a reference and are twice as big as a single
Double beam and split (dual) beam
Quality control
- Purpose
- Method for wavelength accuracy
- To ensure wavelength is really going through the sample
- Replacement of source lamp, holmium oxide (rare earth) glass filter, didymium filter
Quality control
- Purpose
- Method for bandwidth
- Monitor range of wavelengths
- Use mercury vapor lamp to verify
Quality control
- Purpose
- Method for stray light
- No ambient room light
- Use glass cut-off filters to verify
Quality control
- Purpose
- Method for linearity
- Make sure absorbance vs. concentration
- Didymium filter
Quality control
- Purpose
- Method for photometric accuracy
- Checking performance of instrument/spectorphotometer
- National Bureau of Standards (NBS) glass filters or potassium dichromate solutions
Failure to get reading in spectrophotometer?
Usually electrical
Erratic readings in spectorphotometer?
Improper warm-up
Incorrect wavelength in spectrophotometer
We want 450 nm but can be a misaligned monochromator
Scattered light in spectrophotometer?
Scratched or improper cuvette
Unsteady baseline in spectorphotometer?
Grounding problem; when you zero instrument, get crazy results
Dark current in spectrophotometer?
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 amoutn 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 %T through a solution; increased concentration of particles = less light transmitted through solution; follows Beer’s Law and assays may be performed on a spectorphotometer
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)
