Chem 202 Quiz with Complete Solutions
In order to remove highly charged compounds from an ion exchange column, the elution
solution should be high or low ionic strength? ANSWER High ionic strength
What would happen if the highest ionic strength elution solution is used first?
Correct! ANSWER All the charged compounds come off the column
Using UV-Vis Spectroscopy, you will quantify the complex ions that you purified earlier in
the lab.
The Beer-Lambert Law states that the intensity of the light passing through the sample cell is
also measured for that wavelength - given the symbol, I. If � is less than Io (light through a
cuvette), then the sample has absorbed some of the light.
This absorbance can be used to determine the concentration of the sample.
A=ϵcl
A is the absorbanceϵ is the molar absorption constant (a constant that is known for each
possible complex ion)c = concentration of solutionl = length of the cuvette
Example: Guanosine has a maximum absorbance of 275 nm. E275 = 8400M-1 cm-1 and the
path length is 1 cm. Using a spectrophotometer, you find the that A275 = 0.70.
What is the concentration of guanosine? ANSWER 8.33 x10-5 mol/L
You will be purifying a smaller peptide from a mixture of a protein and a peptide using a size
exclusion gel column. Which compound will elute first? ANSWER Protein
In a mixture of the five proteins listed below, which should elute second in size-exclusion
(gel filtration) chromatography?
a. cytochrome c MW = 13,000
b. immunoglobulin G MW = 145,000
, c. ribonuclease A MW = 13,700
d. RNA polymerase MW = 450,000
e. serum albumin MW = 68,500 ANSWER b
The best way to increase the resolution of your chromatography column would be to:
ANSWER Use a longer column
Your peptide is not colored. So you will need to determine which fractions that elute from the
column will contain your purified peptide.
What will be a good method to determine which fractions contain your sample? ANSWER
UV-Vis to detect any molecules that absorb in the UV-vis region
You will analyze the LC/MS of your assigned protein. All MS of proteins can be found here.
Remember for MS of proteins:
Proteins are often multiply charged
Proteins have different protonation states
The spectrum will have multiple molecular ions
The molecular ion the mass of the protein divided by the total charge of the protein.
Example MS of a protein
The largest peak (most abundant form) of this protein had a +18 charge. If the m/z ratio of
that peak is 950 Da, what is the mass of the protein? ANSWER 17,100 Daltons
A column made with 3-aminophenylboronic acid will bind tightly to glycoproteins (proteins
that have sugar molecules attached to it).
In order to remove highly charged compounds from an ion exchange column, the elution
solution should be high or low ionic strength? ANSWER High ionic strength
What would happen if the highest ionic strength elution solution is used first?
Correct! ANSWER All the charged compounds come off the column
Using UV-Vis Spectroscopy, you will quantify the complex ions that you purified earlier in
the lab.
The Beer-Lambert Law states that the intensity of the light passing through the sample cell is
also measured for that wavelength - given the symbol, I. If � is less than Io (light through a
cuvette), then the sample has absorbed some of the light.
This absorbance can be used to determine the concentration of the sample.
A=ϵcl
A is the absorbanceϵ is the molar absorption constant (a constant that is known for each
possible complex ion)c = concentration of solutionl = length of the cuvette
Example: Guanosine has a maximum absorbance of 275 nm. E275 = 8400M-1 cm-1 and the
path length is 1 cm. Using a spectrophotometer, you find the that A275 = 0.70.
What is the concentration of guanosine? ANSWER 8.33 x10-5 mol/L
You will be purifying a smaller peptide from a mixture of a protein and a peptide using a size
exclusion gel column. Which compound will elute first? ANSWER Protein
In a mixture of the five proteins listed below, which should elute second in size-exclusion
(gel filtration) chromatography?
a. cytochrome c MW = 13,000
b. immunoglobulin G MW = 145,000
, c. ribonuclease A MW = 13,700
d. RNA polymerase MW = 450,000
e. serum albumin MW = 68,500 ANSWER b
The best way to increase the resolution of your chromatography column would be to:
ANSWER Use a longer column
Your peptide is not colored. So you will need to determine which fractions that elute from the
column will contain your purified peptide.
What will be a good method to determine which fractions contain your sample? ANSWER
UV-Vis to detect any molecules that absorb in the UV-vis region
You will analyze the LC/MS of your assigned protein. All MS of proteins can be found here.
Remember for MS of proteins:
Proteins are often multiply charged
Proteins have different protonation states
The spectrum will have multiple molecular ions
The molecular ion the mass of the protein divided by the total charge of the protein.
Example MS of a protein
The largest peak (most abundant form) of this protein had a +18 charge. If the m/z ratio of
that peak is 950 Da, what is the mass of the protein? ANSWER 17,100 Daltons
A column made with 3-aminophenylboronic acid will bind tightly to glycoproteins (proteins
that have sugar molecules attached to it).
