Use of a Spectrophotometer

Question 1

What is the function of a spectrophotometer?

Answer 1

To quantify substances that absorb light. The absorption of light is proportional to concentration at the correct wavelength

Question 2

Which absorbance values are less likely to be accurate?

Answer 2

Values greater than 1.0 or less than 0.05

Question 3

What does wavelength have to do with absorbance?

Answer 3

Each substance has its own absorption pattern and is better at absorbing photons at certain wavelengths. The higher the absorption coefficient, the better the substance is at absorbing light at a specific wavelength

Question 4

What is NADH?

Answer 4

The reduced form of NAD+, the main electron carrier in fuel oxidation. It absorbs strongly at 340nm but NAD+ does not.

Question 5

What is haemoglobin?

Answer 5

The protein in red blood cells that binds haem ligands. It absorbs strongly at 500nm.

Question 6

What is met-haemoglobin?

Answer 6

The iron in the haem group of Hb has been oxidised to Fe3+ due to oxygen in the air. Is unable to bind to oxygen

Question 7

How are enzyme-linked assays used to measure ethanol and why?

Answer 7

Ethanol has very little or no absorbance. Alcohol dehydrogenase is used to catalyse a reaction that produced NADH (with absorbance) in direct proportion to the concentration of ethanol.

Question 8

What does each letter in the beer lambert law mean?
A = εcl
OR
A = log10 (I0/I)

Answer 8

A= absorbance
I0 = light intensity before it passes through the sample
I= light intensity after it passes through
ε = absorption coefficient
C= concentration of the substance
l= path length