10. Theory of Spectrophotometry

Question 1

Describe Spectrophotometry (1pt)

Answer 1

Spectrophotometry is a quantitive method of studying materials by measuring the amount of light transmitted/ absorbed.

Question 2

Why do different solutions have distinctive colours (1pt)

Answer 2

Many solutions have distinctive colours because they absorb and transmit different wavelengths of visible light.

Question 3

What happens if we see all wavelengths at the same time? (1pt)

Answer 3

If we see all wavelengths at the same time the light appears white

Question 4

What colour is transmitted (1pt)

Answer 4

The colour that is not absorbed is transmitted. For example, chlorophyll absorbs red and blue light therefore transmits green.

Question 5

How do we measure the concentration of a solution? (2pt)

Answer 5

1. The intensity of a colour is an indication of the solutions concentration. The more concentrated the solution the darker it is.
2. If we can engineer a situation where a substance is detected by a colour change reaction we can then determine the concentration of that substance by measuring the absorbance of light.

Question 6

Describe the Spectrophotometer (2pts)

Answer 6

The Spectrophotometer measures absorbance or transmission of light at a particular wavelength

1. The sample is placed into a cuvette and light of selected wavelength is passed through the sample.
2. The instrument measures the amount of light that is absorbed by the sample.

Question 7

A= E x C x L

Answer 7

1. E= Tendency for the solution to absorb a particular wavelength. (known as Molar extinction Coefficient)
2. C= Concentration of the solution. The more concentrated the solution, the darker the colour is and the greater the absorbance.
3. L= Path length. The longer the path length the more solution the beam travels through and so the higher the absorbance. In practice all the cuvettes are 1cm wide.

Question 8

Describe the Beer-Lambert Law (3pts)

Answer 8

1. A= E x c x l
   Absorbance= constant x concentration x path length
2. The Beer-Lambert law describes the relationship between the absorbance of a solution and its concentration.
3. The amount of light absorbed is proportional to the concentration of the solution. This means that if we plot a graph off absorbance versus concentration we get a straight line.

Question 9

What is the molar extinction coefficient? (3pts)

Answer 9

1. The molar extinction Coefficient= Is the theoretical absorbance of a one molar solution of the substance being studied per centimetre path length.
2. c= absorbance / concentration
3. limitations:
4. We might not know the molar extinction coefficient of our substance
5. If we have a reaction mixture then the molar extinction coefficient cant be used as it only applies to a single substance.

Question 10

Describe Transmission (1pt)

Answer 10

Transmission= Light out/ light in

Question 11

What happens if the substance you want to measure is not coloured (3pt)

Answer 11

1. The substance might be conjugated to a coloured compound or specifically bound a coloured dye.
2. Many substances can be made to undergo a chemical reaction that give rise a coloured result. Absorbance is then measured.
3. Some substances appear colourless because they absorb only in the Ultra-violet or infra-red regions of the spectrum. For example a solution of DNA.