Please help me answer question (A-D) after reading the following passage. thank

Question

Please help me answer question (A-D) after reading the following passage. thank you . This a biochemistry question. Spectrophotometry You may be wondering as to why you have been given a document titled spectrophotometry, when the title of the experiment is protein assays. The reason behind this is because the protein assays you will be performing. use spectrophotometry as a tool to measure concentration of proteins. Spectrophotometry is one of the most widely used and versatile of all biochemical analytical tools. The analysis of systems by measuring light absorption has several advantages. It is a nondestructive method unless a photochemical reaction occurs, which is not common. It offers selectivity in that each compound has a characteristic absorbance spectrum, and a particular component in a solution or group in a molecule often can be singled out for observation. It averages the properties of the system over a short time interval, 10s which allows one to follow fast reactions, making kinetic studies possible The absorption of visible light that is detected by the human eye (400-700 nm)-is most commonly noted by the color of objects. A solution of vitamin B12. for example, appears red because it absorbs blue, green, and yellow light, which are present in white light, and therefore transmits red light. In contrast, a solution of vitamin B appears yellow because it transmits yellow light. The absorbance spectra of vitamin B12 and vitamin B are shown in figures 1. Visible light is only a small part of the electromagnetic spectrum. o 2.5 Vitamin B12 Riboflavin 1.5 0.5 200 300 500 Wavelength, nm 600 700 Figure 1: Absorption spectra of vitamin Bi (continuous line) and riboflavin (dashed line) with the arrows showing the ma values in the 300-700 nm wavelength range Another key feature of an absorption spectra are wavelength of maximum absorption mas). In figure 1, in the wavelength range above 300 nm. Vitamin B12 has a characteristic a, at 363 nm. whereas the riboflavin has characteristic a·at 350 nm and 450 nm. The values are characteristic of compounds and come in handy in various biological assays. The absorption spectrum shown in Figure 1 was measured using a device called absorption spectro- photometer. a device capable of measuring absorbance of a sample. A typical schematic of a spectrophotometer is shown in Figure 2

Explanation / Answer

Spectrophotometry :

The concentration of the given sample can be determined with the help of spectrophotometer which is based on the amount of light absorbed by the sample.

When the sample is concentrated or the solute concentration is high, the amount of light absorbed increases

Absorbance is measured using spectrophotometer which is directly proportional to the concentration of the sample

Therefore, absorbance is direclty proportional to concentration of the sample.

Blank sample with no absorber cannot absorb any light, so the transmittance of light is 100 %

Transmittance is the amount of light unabsorbed by the sample.

0 % absorbance = 100 % transmittance

If absorbance is 100 % then transmittance of light is zero.

Transmittance = Iout / I in

Absorbance =  Iin/ I out

Therefore, transmittance and absorbance are ratio of intensities of light

And, when path length is higher, transmittance will be less

According to Beer-Lambert Law,

A = a b c

where A = absorbance (no unit)

a = molar extinction coefficient (1/ Molarity cm)

b = path length in cm,

C = molar concentration (M, molarity)

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