Spectrophotometry

Question 1

What is electromagnetic radiation?

Answer 1

Radiant energy of any wavelength.

Photons of energy travelling in waves.

Question 2

What is a wavelength?

Answer 2

The distance between to successive crests of troughs in a wave.

Question 3

What is frequency?

Answer 3

The number of wavelengths passing through a certain point in a given time.

Question 4

What is the wavelength range of visible light and give the associated colours?

Answer 4

400 nm (violet) to 700 nm (red)

Question 5

What is polychromatic light?

Answer 5

Light consisting of all or a portion of the wavelengths of the spectrum.

Question 6

What is monochromatic light?

Answer 6

Light of one wavelength.

Question 7

In general what do we see as an object absorbs light?

Answer 7

We see the complementary colours to those that are absorbed.

Question 8

Does violet which has a shorter wavelength than red, have more or less energy level than red light?

Answer 8

Violet has a higher energy level than red light does.

Question 9

What are different ways that light can interact with objects and describe them?

Answer 9

Transmission
Reflection
Refraction
Diffraction
Absorption
Scattering
(See slide 9 for descriptions and illustrative figure)

Question 10

What does the light source do and name some various sources?

Answer 10

Light Source: Provides radiant energy of constant intensity
Several sources:
Visible light: Tungsten 
UV light: Deuterium 
Visible and UV: Mercury

Question 11

What does the monochromator do in the spectrophotometer?

Answer 11

Monochromator:

a. Allows for the isolation of radiant energy of a specific λ using filters, prisms, diffraction gratings, entrance and exit slits, mirrors and collimating lenses
b. Used to minimize stray light, focus and direct a beam of light onto the dispersing device
c. Prisms or diffraction gratings are commonly used

Question 12

What do prisms do, how can they be controlled, and what kinds are used?

Answer 12

Prisms:

1. Split white light into a continuous spectrum via refraction. Amount of refraction depends on wavelength.
2. Control - Can be rotated so only the desired wavelength will pass through the exit slit.
3. Types:
   a. Visible light: glass
   b. UV light: quartz or silica

Question 13

What do diffraction gratings do?

Answer 13

Diffraction Gratings:
Spectrum resulting from light striking a surface engraved with a large number of parallel lines (20-6000 lines/mm).
Think hologram/CD

See slide 34 for illustration.

Question 14

What do the entrance slit, collimator, exit slit, and filter do in a spectrophotometer?

Answer 14

Entrance slit: Restricts/isolates angle of light striking prism or diffraction grating
Collimator: Collect as much light as possible on the entrance slit
Exit slit: Controls bandpass of light
Selects specific wavelength from spectrum. Example: if the bandpass of spectrophotometer is set for 10 nm and the instrument is set at 540 nm, the wavelengths that would hit the sample would be from 535 to 545 nm.
Filter: Isolates individual wavelengths of light

Question 15

What do the sample cells do? Size, material and interfering factors that are related to them?

Answer 15

1. Sample Cell: Holds solution to be tested
   (aka cuvettes).
2. Materials
   a) Visible light: borosilicate or plastic
   b) UV light: quartz
3. Size: 0.1-1.0 cm light path
4. Interfering factors: bubbles, dust, fingerprints, scratches … anything that interferes with the light path could interfere with reading

Question 16

What is the processor, display, readout devicer?

Answer 16

Processor converts electrical signal into a value of absorbance or transmittance.
Can be in the form of a terminal, chart recorder, printer, digital display or meter.

Question 17

What are the different types of photodetectors?

Answer 17

1. Photocell and Phototube: Current is produced proportional to incident radiation. Excited electrons travel from selenium coated iron to silver layer
   Photocell = No external voltage source is required
   Phototube = Need for external voltage source
2. Photomultiplier Tube: Detects and amplifies radiant energy
3. Photodiode Array: Polychromatic light measures many λ’s at once (multichannel)

Question 18

What is a double beam spectrometer? Compare to a single beam spectrometer.

Answer 18

Similarities:
1. Both have one light source
2. Measurement of absorbance or transmittance of monochromatic light
Differences:
1. Double beam spectrometer - Splits light beam using mirrors and choppers.
2. Double beam overcomes the intensity fluctuations and drift problems of single beam by splitting the beam between a reference and sample cuvette
Two types:
Double beam in space – uses two photodetectors
Double beam in time – uses one photodetector

Question 19

What is reflectance spectrophotometry?

Answer 19

Reflectance Spectrophotometry

1. Light reflected from solid surface and measured.
2. Beam of light is directed at 45° towards surface.
3. Some light is absorbed and some if reflected. Reflected light (λ not absorbed by analyte) is measured by detector.
4. Inversely proportional to concentration of analyte
5. Maximum reflectance = no absorbing material/analyte in the optical path

Question 20

What do turbidimetry and nephelometry make measurements based on?

Answer 20

Turbidimetry and nephelometry make measurements based on light scatter.

Question 21

What is Turbidimetry?

Answer 21

Measured at 180˚

Amount of light reaching photodetector is inversely proportional to concentration

Question 22

What is Nephelometry?

Answer 22

Nephelometry

1. Various forward angles such as 30○ and 90○ used to measure (Different than incident light).
2. Amount of light reaching photodetector is directly proportional to concentration

Question 23

What are the similarities between turbidimetry and nephelometry? Example uses. Shared sources of error?

Answer 23

1. Measures the concentration of particles in suspension through the use of light scatter. E.g. Bacteria in suspension, immune complexes, coagulation, cell counting, etc. Particles must be properly suspended prior to measurement
2. Sources of error: variation in size, sample matrix (lipemia) may scatter light, particles in reagent, scratches in cuvette, and dust

Question 24

What is fluorometry?

Answer 24

Substance exposed to light of a certain λ → Light of a different λ is emitted → Longer than incident
Fluorescent molecules are referred to as flurophores
Light source is usually mercury or xenon
Emits shorter λ (high energy)
Light source at right angles to photodetector
Prevents source light from striking photodetector
Measures fluorescent light
Detector at 90○
2 filters – one to isolate excitation λ and one to isolate emission λ
Used in immunoassays due to its sensitivity and specificity
Consideration:
Quenching can occur due to energy transfer between molecules in sample resulting in a decrease in fluorescence

Question 25

What is chemiluminescence?

Answer 25

No light source required
Chemical reaction supplies light to be measured
Oxidization reactions of chemicals such as luminol, acridinium esters, and dioxetanes
Emit light then decay
Intensity of light measured followed by gradual decay
Like a glow stick

Question 26

What is an atomic absorption spectrophotometer?

Answer 26

Measures concentration of elements via the use of wavelengths of light specifically absorbed by elements
Flame atomizes sample causing excited atoms to emit light energy specific to the element on return to ground state.
Hollow cathode lamp is used as a light source
Rotating chopper used to distinguish between lamp light and emitted light
Used in clinical laboratory to test for trace metals such as lead