Spectrometry

Question 1

Definition: Spectroscopy

Answer 1

The study of the interaction of matter with light (i.e electromagnectic radiation)

Question 2

Definition: Spectrometry

Answer 2

The method used to acquire a spectrum

Question 3

Definition: Spectrophotometry

Answer 3

Spectrometry in the UV-visible-near infrared range

Used most in biochemistry labs

Question 4

Applications of Spectrophotometry

Answer 4

• analysis of materials
• quantitation of components, including in complex mixtures
• time-resolved (monitoring of rates of changes of reactants and products)

Question 5

What is the Planck-Einstein relation

Answer 5

E= hv
Energy = Plank constant x frequency
J=J.s s^-1

Question 6

What is the Planck constant

Answer 6

~6.626x10^-34 J.s

Question 7

Which has the higher energy X-rays or Radio waves

Answer 7

Xrays

Question 8

Which has the higher frequency X-rays or Radio waves

Answer 8

Xrays

Question 9

Which has a longer wavelength X-rays or radio waves

Answer 9

Radio waves

Question 10

Energy and frequency are___________

Energy and wavelength are _______ proportional

Answer 10

1. proportional

2. inversely

Question 11

What equation relates wavelength, speed of light and frequency

Answer 11

λ= c/v
m = ms-1 / s-1

Question 12

What is the range of the visible spectrum

Answer 12

~700nm - ~400 nm

Question 13

What is an electronic transiton

Answer 13

When photons in the UV/visible/near-IR range cause electrons to move from one orbital to another of higher energy

Question 14

When does electronic transition occur

Answer 14

When the E of the absorbed photons is equal to the energy required to move the electrons between the orbitals

Question 15

What happens when you place a substance in a light path

Answer 15

It results in a band of absorption at specific wavelengths by a substance

Question 16

_________ ,___________, __________ of the bands of absorption are characteristic of the absorbing substance

Answer 16

Wavelength
Shapes
Intensities

Question 17

Haem groups have strong bands in the ____ and ____

Wavelengths depends on the _____and on the _____ state e.g _____, _____ and _____

Answer 17

400-450 nm and 550-610 nm
haem type
chemical
oxidizes, reduced, ligand-bound

Question 18

What colour is oxidised Haemoglobin (Fe3+)

Answer 18

Brown

Question 19

What colour is reduced Haemoglobin (Fe2+)

Answer 19

Purple

Question 20

What colour is oxygen-bound, reduced Haemoglobin (Fe2+ … O2)

Answer 20

Red

Question 21

Wavelengths:
Tyrosine
Tryptophan
Nucleic acid bases

Answer 21

280nm
280nm
260nm
Proteins with known number of Try and Trp can be quantitated from their absorbance at 280nm

Question 22

What is used for quantitative analysis of absorption

Answer 22

Spectrophotometer

Question 23

What are the basic components of a typical spectrophotometer

Answer 23

Light source: Halogen lamp/ tungsten filament λ>300nm
Deuterium arc λ ~ 350-200nm (for Tyr, Trp, DNA)
Dispersion device: prism/ diffraction grating to separate the different wavelengths
Exit slit: Select specific wavelengths with a narrow slit
Sample: Within a cuvette (below visible light quartz cuvette)
Detector: T measure the intensity of light. Converts a stream of photos into a stream of electrons.

Question 24

What can absorbance spectroscopy be used for

Answer 24

Absorbance bands- Qualitative.

Absorbance spectrum- Measure the absorbance at each wavelength, plot absorbance vs wavelength- Quantitive