lab 2

Question 1

What does the Biuret assay measure?

Answer 1

Protein concentration based on peptide bonds reacting with alkaline copper to form a purple color.

Question 2

Why do proteins form color in the Biuret reaction?

Answer 2

Peptide bonds react with alkaline copper salts to produce a blue-purple complex proportional to protein amount.

Question 3

Why must a blank be used in photometry?

Answer 3

To set incident light (Io) to zero absorbance so only sample absorbance is measured.

Question 4

What is absorbance (A)?

Answer 4

A measure of light absorbed by the sample; defined as A = log10(Io/I).

Question 5

What is transmittance (T)?

Answer 5

The fraction of incident light that passes through the sample (T = I/Io).

Question 6

How do you convert transmittance to absorbance?

Answer 6

A = -log10(T).

Question 7

What does Beer’s Law state?

Answer 7

Absorbance is proportional to concentration: A = Ecl.

Question 8

Why do we create a standard curve?

Answer 8

To determine the concentration of an unknown by comparing its absorbance to known standards.

Question 9

Why is the linear/valid range important?

Answer 9

Only absorbances within the linear range give accurate protein concentration estimates.

Question 10

Why is the 1/2-dilution usually the best estimate for unknown protein?

Answer 10

Its absorbance falls within the valid range and avoids pipetting errors from over-dilution.

Question 11

Why is the undiluted sample’s estimate unreliable?

Answer 11

Its absorbance is too high and lies outside the linear range, requiring extrapolation.

Question 12

What happens if you use too much unknown in a colorimetric assay?

Answer 12

Color reagents become limiting, causing non-linear data and inaccurate readings.

Question 13

Why must cuvettes be matched?

Answer 13

Different path lengths or scratches change absorbance readings.

Question 14

What is the purpose of the riboflavin dilution experiment?

Answer 14

To test whether riboflavin obeys Beer’s Law by comparing measured vs expected absorbance.

Question 15

How do you calculate expected absorbance after dilution?

Answer 15

Multiply the undiluted absorbance by the dilution factor.

Question 16

What does it mean when measured absorbance differs from calculated?

Answer 16

Possible pipetting error, instrument variability, or non-linearity.

Question 17

What are riboflavin’s four absorption peaks?

Answer 17

215 nm, 265 nm, 370 nm, 445 nm.

Question 18

How do you calculate molar absorption coefficient (E)?

Answer 18

E = A / (c × l).

Question 19

Does concentration change E for a given wavelength?

Answer 19

No, E remains roughly constant if Beer’s Law holds.

Question 20

Why are some compounds non-linear at high concentration?

Answer 20

Aggregation or reagent limitation can cause deviations from Beer’s Law.