Chapter 19 - Spectrophotometers Flashcards
UV-Visible spectrophotometers:
- Uses a reference cell to monitor differences in transmittance
- sample and reference must be placed alternately in the beam.
- One wavelength at a time. - Light alternately passes through the sample and reference directed by a rotating mirror (chopper) into and out of the light path
- Measures both simultaneously.
- Scans wavelengths.
- Single beam
2. Double beam
Light Sources:
- Common Light Sources used (3)
For smaller handheld colorimetric probes, ____ are used
- proved isolated lines of a single wavelength for many applications
- has a bandwidth (range of wavelengths) for wich it works
- all light is coherent (light waves oscillate in phase with each other)
- Deuterium
Xenon arc lamps
Tungsten - LEDs
- Lasers
Wavelength Selection:
What are two ways select wavelength of light
monochromators
optical filters
selects a narrow range of wavelength of light and allows only that narrow range to pass through the device
monochromators
what are two types of monochromators
prism monochromators
grating monochromators
Type of monochromators:
- make use of the wavelength dependence of index of refraction.
- By rotating the prism, you can select the bandwidth of light that reaches the exit slit.
- Prism monochromators
Monochromators:
_____ light into its component wavelengths.
Selects a _____ band of ______ to pass on to the sample or detector.
Wavelength is selected by ______ __ __ ______
are reflective or transmissive optical components with a series of closely ruled lines.
Different wavelengths are reflected by different angles from the grating (diffraction).
dispenses
narrow, wavelength
rotation of the monochromator
Grating
Remove unwanted bands of radiation
Bandpass filters are
Filters
simplest filters and interfernece and holographic filters
__________ is colored glass which absorbs a broad portion of the spectrum and transmits others.
_______ and ______ filters are constructed to pass radiation in the region of interest and reflect others.
Simplest filter
interference and holographic filter
Basic function is to convert electromagnetic energy (photons) into electrical energy
Detector
Most common detector and why?
Photomultupler tube (PMT)
- useful for light detection of very weak signal
- sensitive device
Detectors - PMTs works:
- Incoming photon strikes a surface inside the tube called a _____
- Electrons are _____ and strike a second _____ with more energy.
- Each electron _____ more electrons from the _____.
- Rinse and repeat.
- Scans through ______.
- dynode
- accelerated, dynode
- knocks, dynode
- Rinse and repeat.
- wavelengths
all wavelengths are recorded simultaneously
- faster
Light enters a ________ which disperses the light into its component wavelengths and directs the light at the diode array.
Each ____ receives a different ______ and all are measures simultaneously.
photodiode array
polychromator
diode, wavelenth
Excitation of the molecule by an incoming photon.
Vibrational relaxation of excited state electrons to the lowest energy level.
Emission at a longer wavelength photon and return of the molecule to the ground state.
Flourescence
What two board classes divide fluorophore?
intrinsic (occur naturally) and extrinsic (syntheitc)
aromatic amino acids
neurotransmitter
porphyrins green fluorscent proteins (GFP)
synthetic dye
modified biochemicals
Intrinsic - occur naturally
Extrinsic - syntheitic
Immunoassays are an important application in biochemistry.
An antibody is a protein produced by the immune system of an animal in response to a foreign molecule (antigen)
_____________ is done to recognize this process.
Enzyme-linked immunosorbent assay (ELISA)
Lamp Power Supply:
Emission from \_\_\_ \_\_\_\_ \_\_\_ emits radiation throughout: a. b. c. - 200 to over 1400nm
xenon arc lamp
a. ultraviolet
b. visible
c. infrared
In fluoroesence, how many monochromators exist and what are they?
two monochromators:
exciter (emission)
analyzer (absobance)
what does the analyzer monochromator does?
filters out stray light
Can get adaptors to fit all sizes of cuvettes.
Incident light is absorbed by the sample.
- The fluorescent light is emitted in all directions.
- Some of the light passes through a second filter or monochromator and reaches a detector.
- Usually placed at 90° to the incident light beam.
- Minimize the risk of transmitted or reflected incident light reaching the detector.
Sample chamber
Three basic filters in fluoroescence
excitation
dichroic beamsplitter or mirror
emission
A bandpass filter that passes only the wavelengths absorbed by the fluorophore.
An edge filter used at an angle of incidence (45°) to efficiently reflect light in the excitation band and to transmit light in the emission band.
A bandpass filter that passes only the wavelengths emitted by the fluorophore and blocks all undesired light outside this band – especially the excitation light.
excitation filter
dichroic beamsplitter or mirror filter
emission filter
improves the quality of data
Literally your signal output divided by your noise. Lots of types of noise: White noise Shot noise Johnson noise Drift Interference Chemical Instrumental Flicker Environmental
signal averaging
signal-to-noise ratio
Applications to fluorescence K T LB PA
kinetics
titrations
ligand binding
protein analysis
