Separation Techniques

Question 1

Size exclusion SEC - How does it separate, what elutes first

Answer 1

Separates via size, large molecules elute first

Question 2

TLC (What does it separate by, what elutes last?)

Answer 2

Separates small amounts via differences in polarity

Polar compounds elute last (low R_f) because they have a high affinity for the polar silica gel

Question 3

What are nonpolar compounds in TLC and what is their R_f value?

Answer 3

Alkenes and aromatics

High R_f

Question 4

What are intermediate polar compounds in TLC and what is their R_f value?

Answer 4

Ketones, esters, alkyl halides

Intermediate R_f

Question 5

What are highly polar compounds in TLC and what is their R_f value?

Answer 5

Alcohols, amines, carboxylic acids

Low R_f

Question 6

Column chromatography

What does it separate, by what, and what elutes last?

Answer 6

Separates large amounts of solids or high boiling point liquids (oils)

Separates via differences in polarity

Polar compounds elute last

Question 7

High performance liquid chromatography (HPLC)

What does it separate by, and how is it different than column chromatography?

What are the two types?

Answer 7

Separates via differences in polarity

More efficient and effective than column chromatography

Normal phase HPLC - polar substances elute last

Reverse phase HPLC - nonpolar substances elute last because the stationary phase is nonpolar

Question 8

Ion Exchange Chromatography

What does it separate, what is retained, what is the mobile/stationary phase?

Answer 8

Separates amino acids or peptides based on charges states

Cation-exchange - positive ions are retained

Anion-exchange - negative ions are retained

Stationary phase = resin

Mobile phase = buffered solution

Question 9

In ion-exchange chromatography, what allows for control of the charges and how can you isolate the ions?

Answer 9

The buffered solution allows for control of the charges

Altering the pH of the solvent or you can flush with a counterion (Na+)

Question 10

Affinity Chromatography

What does is separate by, what does is separate, and how do you elute it?

Answer 10

Separates via highly specific lock-and-key interactions

Separates proteins from blood serum or a cell lysate

To elute the target protein, add a competitive antibody binding protein or alter the pH

Question 11

Gas chromatography

What does it separate, how does it separate, and what elute first?

Answer 11

Separates via differences in volatility or boiling point

Separates small amounts of low BP compounds

Low boiling point compound elutes first

Question 12

How does volatility relate to boiling point?

Answer 12

Higher boiling point = less volatile

Question 13

In gas chromatography what do the number of peaks represent and how do you find the quantity of the compound?

What does the intensity indicate?

Answer 13

peaks = # compounds

Area under the curve = relative quantity of that compound

Increased intensity = lower boiling point = more volatile

Question 14

Simple distillation

What does it separate, why is it used, what is the criteria?

What is an example?

Answer 14

Separates large amounts of low BP compounds

The difference in BP is > 30 degrees C

Used to remove impurities

The purified, low BP compound is separated

Ex: Separating fresh water from seawater

Question 15

Fractional distillation

What does is separate, what is the criteria, how does the % change as you go up?

What is an example?

Answer 15

Separates diastereomers

Difference in BP < 30 C

As you go up the tube, the relative % of the lower BP compound increases. You want 100% of the lower BP compound at the end

Ex: separating components of crude oil

Question 16

What is boiling point determined by?

What two things can alter boiling point?

Answer 16

The strength of the Intermolecular forces of the compound

Increased molecular weight = increased BP

Increased branching = decreased boiling point

Question 17

Solvent extraction

What does it separate by?

Answer 17

Separates via solubility differences

Question 18

What are the solubility rules?

Answer 18

Like dissolves like

Less than 5 carbon atoms and a polar group is water soluble

Charged functional groups are more soluble in water than nonpolar (organic) solvents

Question 19

What compounds aren’t useful in solvent extractions? Why?

Answer 19

Alkyl alcohols because they are not acidic enough

Question 20

What is the relative strength of NaHCO3 and what does it deprotonate?

Answer 20

It is a weak base

Only deprotonates carboxylic acids

Question 21

What is the relative strength of NaOH and what does it deprotonate?

Answer 21

It is a strong base

Deprotonates phenols and carboxylic acids

Question 22

What does HCl protonate?

Answer 22

Amines

Question 23

What compromises the aqueous and organic layers?

Answer 23

Aqueous layer = small, polar molecules

Organic layer = large, nonpolar molecules

Question 24

Which species are preferentially soluble in the aqueous layer?

Answer 24

Charged species, regardless of size

Question 25

How do the chemical and physical properties of enantiomers relate

Answer 25

Enantiomers share chemical and physical properties, so they cannot be separated by any physical process

Question 26

How do you separate enantiomers? (3 steps)

Answer 26

Convert enantiomers to diastereomeric salts via a chiral resolving agent

Separate the salts (Recrystallization)

Revert salts back to enantiomers

Question 27

What are the requirements of the chiral resolving agent when separating enantiomers?

Answer 27

Acids and base must be complementary (Acids go w basic diastereomeric salts)

It must be enantiomerically pure (R,R or S,S)

Question 28

What is an example of separating enantiomers (process)

Answer 28

R/S bases are treated with an R,R acid resolving agent (could be S,S acid)

This forms R,R,R and S,S,R salts

Separate via recrystallization

Revert the salts back to enantiomers by reacting with a base (NaOH)