Q: A spectrophotometer measures the absorbance of a solution at a specific wavelength. If the path length is 1 cm and the absorptivity ($$\epsilon) is$$ $$0.016\ \mu M$$^{-1}$$ cm$$^{-1}$$, what is the concentration of a solution with an absorbance of 0.32$?

$$c = \frac{0.32}{0.016 \times 1} = 20\ \mu M$$

Q: A student calibrates a 200 μL micropipette using water and a balance. If the average mass delivered is 0.152 g, what is the percent deviation from the set volume of 150 μL? (Assume water density is $$1\ g\ mL$$^{-1}$$)

Percent deviation $$= \frac{152 - 150}{150} \times 100\% = 1.33\%$$

Q: A student prepares a standard curve for bromophenol blue and measures an unknown sample's absorbance as 0.347. If the absorptivity is $$0.016\ \mu M$$^{-1}$$ cm$$^{-1}$$ and path length is 1 cm, what is the concentration of the unknown?

$$c = \frac{0.347}{0.016 \times 1} = 21.7\ \mu M$$

Question 1

Which of the following best describes the relationship between absorbance and concentration according to the Beer-Lambert Law?

Accepted Answer

Absorbance is directly proportional to concentration.

Question 2

A spectrophotometer measures the absorbance of a solution at a specific wavelength. If the path length is 1 cm and the absorptivity ($$\epsilon) is$$ $$0.016\ \mu M$$^{-1}$$ cm$$^{-1}$$, what is the concentration of a solution with an absorbance of 0.32$?

Accepted Answer

$$c = \frac{0.32}{0.016 	imes 1} = 20\ \mu M$$

Question 3

What is the purpose of using a solvent blank in spectrophotometric measurements?

Accepted Answer

To define zero absorbance and account for background absorption by reagents.

Question 4

Which equation correctly represents the Beer-Lambert Law?

Accepted Answer

$$A = \epsilon l c$$

Question 5

If a micropipette is set outside its specified volume range, what is the most likely consequence?

Accepted Answer

The pipette may be damaged and lose calibration.

Question 6

A student calibrates a 200 μL micropipette using water and a balance. If the average mass delivered is 0.152 g, what is the percent deviation from the set volume of 150 μL? (Assume water density is $$1\ g\ mL$$^{-1}$$)

Accepted Answer

Percent deviation $$= \frac{152 - 150}{150} 	imes 100\% = 1.33\%$$

Question 7

Why is absorbance preferred over transmittance for quantitative analysis in spectrophotometry?

Accepted Answer

Absorbance is linearly related to concentration, making calculations easier.

Question 8

In a biuret assay, why must standards and unknowns be treated identically (same volume, time, temperature, etc.)?

Accepted Answer

To ensure the color yield is consistent and results are comparable.

Question 9

A protein solution is diluted by a factor of 10 before analysis. If the measured concentration in the diluted sample is $$0.8\ mg\ mL$$^{-1}$$, what is the concentration in the original sample?

Accepted Answer

$$8.0\ mg\ mL$$^{-1}$$

Question 10

During the determination of a protein's isoelectric point (pI), what observation indicates the pI has been reached?

Accepted Answer

Maximum precipitation or turbidity occurs.

Question 11

If a spectrophotometer is set to a wavelength where the analyte does NOT absorb, what will be the result?

Accepted Answer

The absorbance will be close to zero.

Question 12

A student prepares a standard curve for bromophenol blue and measures an unknown sample's absorbance as 0.347. If the absorptivity is $$0.016\ \mu M$$^{-1}$$ cm$$^{-1}$$ and path length is 1 cm, what is the concentration of the unknown?

Accepted Answer

$$c = \frac{0.347}{0.016 	imes 1} = 21.7\ \mu M$$

Question 13

Which of the following is NOT a correct step in using a micropipette?

Accepted Answer

Depress the operating button to the second stop before aspirating liquid.

Question 14

In spectrophotometry, what is the effect of using a cuvette with a path length different from 1 cm when calculating concentration using the Beer-Lambert Law?

Accepted Answer

The calculated concentration will be incorrect unless the path length is accounted for.

Question 15

A student finds that the absorbance of their unknown sample is higher than the highest standard on their curve. What should they do?

Accepted Answer

Dilute the sample and repeat the measurement, then account for the dilution in calculations.

Question 16

Which of the following best explains why the Beer-Lambert Law may not hold at high concentrations?

Accepted Answer

At high concentrations, deviations occur due to molecular interactions and scattering.

Question 17

In a colourigenic assay, what is measured in the spectrophotometer?

Accepted Answer

The absorbance of the new chromophore formed by the reaction.

Question 18

Which protein from the following list is most likely to precipitate at pH 4.7?

Accepted Answer

Casein

Question 19

If the gradient of a standard curve for absorbance vs. concentration is $$0.016\ \mu M$$^{-1}$$ cm$$^{-1}$$, what does this value represent?

Accepted Answer

The absorptivity ($$\epsilon) of $$the substance at the measured wavelength.

Volumetric Micropipette Use and Spectrophotometry in Biochemical Analysis

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