Chapter 19 - Spectrophotometers Flashcards
What is the difference between single-beam and double-beam Spectrophotometers?
Single-beam Spectrophotometers can only analyze one sample at a time whereas a double beam can simultaneously analyze a sample and a reference
How does a double-beam spectrophotometer work?
- light is passed through a monochromator where it is split into different wavelengths
- the light passes through a sample and hits a detector where it measures the irradiance “P”
- a chopper diverts the beam through a reference cuvet and the detector measures “P0”
- the beam is chopped several times per second and the circuitry compares “P” and “P0” to obtain transmittance and absorbance
Why do UV-Vis spectrometers have both a deuterium and a quartz halogen lamp?
To provide a source of ultraviolet radiation as well as a source of visible light
What does a monochromator do?
It splits light into its component wavelengths and selects a specific wavelength to pass onto the sample or detector
What happens when you decrease the monochromator slit width?
- increase resolution
- decrease bandwidth
- decrease sensitivity
- decrease signal to noise
Which is a more sensitive detector?
Photomultiplier tube, phototube, or a monochromator
Photomultiplier tube
How does a phototube detector work?
Visible light or ultraviolet radiation strikes a cathode emitting electrons. These electrons flow through a vacuum to a collector whose current is proportional to the radiation intensity.
(For every photon that comes in, approximately one electron comes out)
What is a photomultiplier tube and how does it work?
It is a detector.
Photons enter the photomultiplier tube and strike a cathode in which electrons are released and these electrons strike a dynode which is positive. When the electrons strike the dynode they are accelerated towards it with more kinetic energy than originally. Each electron knocks more electrons from the dynode and they’re accelerated towards another dynode. This process is repeated several times.
What is a photodiode array and how does it work?
It is a detector.
A reverse bias creates a depletion region; when radiation strikes each diode, free electrons and “holes” migrate to regions of opposite charge and partially discharge the array. Following a certain time, the current needed to recharge each diode is measured and proportional to the amount of irradiation received.
What is a polychromator?
Opposite to a monochromator, a polychromator disperses light into its component wavelengths and directs the light at a photodiode array.
How can UV-Vis spectroscopy aid in the prevention of kidney stones?
It helps to determine the level of phosphates.
Which assay can be used to determine protein concentration?
The Bradford protein assay as well as the Bicinchoninic acid (BCA) protein assay
What is a Soret band?
A Soret band is an absorbance due to heme.
What is Fourier analysis?
Fourier analysis is a procedure in which a curve is decomposed into a sum of sine and cosine terms, called a Fourier series.
How does an interferometer work?
Radiation from the source strikes a beam splitter which transmits some light and reflects some light. When light strikes the beam splitter at point 0, some is reflected to a stationary mirror at a distance 0S and some is transmitted to a movable mirror at a distance 0M. Rays reflected by the mirrors travel back to the beam splitter where half of each ray is transmitted and half is reflected. One recombined ray travels in the direction of the detector and another heads back to the source.
