1) Why do molecules absorb light? Why are only certain wavelengths absorbed? 2)

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 ?max rather than a wavelength nearby such as 580 nm?

3) Identify the n --> ?* and ? -->?* transitions in the spectrum below. Support your conclusion with two reasons.

Explanation / Answer

Molecules absorb light because they have internal energy levels,so, when light of that particular frequency matches with energy difference between these levels, the electron gets exited to higher energy states, and while returning back to their ground state, they emit radiation in the form of colour. This is the reason why only certain wavelength of light will get absorbed.

In organic molecules the energgy levels are named

as '' sigma, pi, n , pi* , sigma* ''.

All the transitions are not observed, because only

some of them fall under visible spectrum, The

transitions that fall under visible spectrum are
n -----> pi*
pi ----> pi*

The maximum wavelength is always considered

because this will be exactly equal to the energy

difference between the two energy levels. Hence

this taken into consideration will result in

accurate calculations of absorbance.

3) In n --> * and -->* transitions, the energy

difference between n --> * is less than -->*

transitions, hence wavelength of 1st will be

greater than 2nd transition. Therefore
In the figure,

wavelength 395 ---> n --> *
                    255 ---> -->*

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