Optical Techniques Flashcards by Liz Haywood

The distance traveled by one complete wave cycle

Wavelength

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Wavelength is measured in terms of ____

Nanometers (nm)

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The number of cycles per second, inversely proportional to wavelength

Frequency

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The peak height of a wave

Amplitude

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Wavelength that has a high frequency and high energy (400 nm)

Short wavelength

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Wavelength that has a low frequency and low energy (700nm)

Long wavelength

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Wavelength (in nm) of visible light

380-800 nm

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Wavelength (in nm) of UV light

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The visible colors correspond to the wavelengths that are ____ to our eyes

Transmitted

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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

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These experiments were w/ the interaction of monochromatic (one wavelength) electromagnetic radiation w/ varying thickness of absorbing material

Lambert’s Experiments

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____ is inversely and logarithmically related to %T

Absorbance

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Experiments w/ transmittance of a solution under differing concentrations

Beer’s Experiments

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As concentration increases, absorbance increases linearly and transmittance decreases exponentially

Beer’s Experiments

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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

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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

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Function is to be a source of radiant energy

Light source

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Four types of light sources

Tungsten filament w/ halogen vapor
UV
Hollow cathode lamp
Laser

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Function is to focus light into a narrow beam

Entrance slit

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Function is to isolate the desired wavelength and exclude others

Monochromator

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3 types of monochromators

Glass filters
Prisms
Diffraction gratings (used in lab)

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Used for readings in the visible and near visible range of spectrum

Colored agent of a glass filter

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Augment wavelengths in phase, destroy those out of phase

Interference filters (glass)

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Eliminate transmission of wavelengths above or below a set wavelength

Glass cut-off filters

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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)