Question

1. Why do molecules absorb light? Why are only certain wavelengths absorbed?

2. Shown below is the absorbance spectrum of CBBG in the presence of a large excess of BSA. The x-axis shows wavelength (in nm) and the y-axis is absorbance – the y-axis is arbitrary since the absorbance depends on the concentration. You’ll notice that the maximum of this absorbance spectrum occurs at 595 nm, which is where you measured the absorbance of the complex.

a. Why must we use a dye such as CBBG to measure protein concentration by visible spectrophotometry?

b. What assumptions must be made in the Bradford assay to determine the amount of protein in a sample?

c. What is an advantage of using the λmax rather than a wavelength nearby such as 580 nm?

Identify the nπ* and π π* transitions in the spectrum below. Support your conclusion with two reasons.

Answer 1

1.

They can absorb light since they absorb it int he ways of "energy". The electrons get excited and they increase their energetic levels from low "n" levels to high "n" levels.

Some certain wavelengths are absorbed depending on the type of molecule, bonding/antibonding, hybridization, orbitals involved, etc...

2a.

The dye will help us identify the excitation level of the dye-protein complex, therefore we can measure it /i.e. quantitativ emeasurment)

2b.

Assume this is initially has no concentration of protein/enzymes. You also must be informed that htis is a pretty qualitative test

2c

WL max is always a unique point since it is the maximum value, that is, there is no repetition, there is only this unique point. If we use near values, we might get at least 2 values and that will mess up the calculation (since the curve is 2-tailed)