Chapter 19 - Spectrophotometers

Question 1

UV-Visible spectrophotometers:

1. Uses a reference cell to monitor differences in transmittance
   - sample and reference must be placed alternately in the beam.
   - One wavelength at a time.
2. 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.

Answer 1

1. Single beam

2. Double beam

Question 2

Light Sources:

1. Common Light Sources used (3)

For smaller handheld colorimetric probes, ____ are used

4. 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)

Answer 2

1. Deuterium
   Xenon arc lamps
   Tungsten
2. LEDs
3. Lasers

Question 3

Wavelength Selection:

What are two ways select wavelength of light

Answer 3

monochromators

optical filters

Question 4

selects a narrow range of wavelength of light and allows only that narrow range to pass through the device

Answer 4

monochromators

Question 5

what are two types of monochromators

Answer 5

prism monochromators

grating monochromators

Question 6

Type of monochromators:

1. 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.

Answer 6

1. Prism monochromators

Question 7

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).

Answer 7

dispenses

narrow, wavelength

rotation of the monochromator

Grating

Question 8

Remove unwanted bands of radiation

Bandpass filters are

Answer 8

Filters

simplest filters and interfernece and holographic filters

Question 9

__________ 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.

Answer 9

Simplest filter

interference and holographic filter

Question 10

Basic function is to convert electromagnetic energy (photons) into electrical energy

Answer 10

Detector

Question 11

Most common detector and why?

Answer 11

Photomultupler tube (PMT)

• useful for light detection of very weak signal
• sensitive device

Question 12

Detectors - PMTs works:

1. Incoming photon strikes a surface inside the tube called a _____
2. Electrons are _____ and strike a second _____ with more energy.
3. Each electron _____ more electrons from the _____.
4. Rinse and repeat.
5. Scans through ______.

Answer 12

1. dynode
2. accelerated, dynode
3. knocks, dynode
4. Rinse and repeat.
5. wavelengths

Question 13

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.

Answer 13

photodiode array

polychromator

diode, wavelenth

Question 14

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.

Answer 14

Flourescence

Question 15

What two board classes divide fluorophore?

Answer 15

intrinsic (occur naturally) and extrinsic (syntheitc)

Question 16

aromatic amino acids
neurotransmitter
porphyrins green fluorscent proteins (GFP)

synthetic dye
modified biochemicals

Answer 16

Intrinsic - occur naturally

Extrinsic - syntheitic

Question 17

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.

Answer 17

Enzyme-linked immunosorbent assay (ELISA)

Question 18

Lamp Power Supply:

Emission from \_\_\_ \_\_\_\_ \_\_\_ emits radiation throughout: 
a.
b. 
c. 
- 200 to over 1400nm

Answer 18

xenon arc lamp

a. ultraviolet
b. visible
c. infrared

Question 19

In fluoroesence, how many monochromators exist and what are they?

Answer 19

two monochromators:
exciter (emission)
analyzer (absobance)

Question 20

what does the analyzer monochromator does?

Answer 20

filters out stray light

Question 21

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.

Answer 21

Sample chamber

Question 22

Three basic filters in fluoroescence

Answer 22

excitation
dichroic beamsplitter or mirror
emission

Question 23

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.

Answer 23

excitation filter

dichroic beamsplitter or mirror filter

emission filter

Question 24

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

Answer 24

signal averaging

signal-to-noise ratio

Question 25

Applications to fluorescence
K
T
LB
PA

Answer 25

kinetics
titrations
ligand binding
protein analysis