Lab 1:Separation and Identification of AA's via Paper chromatography

Question 1

define the term mobile phase!

Answer 1

something that carries the solutes through the stationary phase.
L> usually a gas or liquid

Question 2

Stationary phase?

Answer 2

• solid or liquid coated support that is immobile

Question 3

Whats the third component of chromatography?

Answer 3

• solutes that are separated from the mixture.

Question 4

Explain the characteristics of the chromatograph paper used in lab!

Answer 4

• paper is made of cellulose which is a polymer made of sugar…has a lot of hydroxy groups which are polar and strongly bind water molecules through hydrogen bonding.
• paper resembles a liquid coated solid.

Question 5

interaction of mobile and stationary?

Answer 5

• comprising of polar organic solvents will be passed through the stationary paper either ascending by capillary action or descending due to influence of gravity .

Question 6

What happens when a solute is exposed to two immiscible liquids?

Answer 6

• it will distribute into liquids according to its solubility in each.

Question 7

The ratio of solubility in each liquid is called the distribution coefficient, K also called the partition coefficient. What is the formula?

Answer 7

k = solubility in mobile phase/ solubility in stationary phase

Question 8

Polar solutes will interact stronger with ____ and less polar solutes will interact stronger with___.

Answer 8

• stationary phase
• mobile phase
  L> polar solutes will be retained in the paper earlier on and the less polar ones will travel further.

Question 9

What is the formula for the retention or retardation factor?

Answer 9

Rf= distance solute migrates/ distance mobile phase travelled

Question 10

What term best describes paper chromatography?

Answer 10

partition chromatography

Question 11

Was ascending or descending chromatography used in this experiment?

Answer 11

ascending

Question 12

Most aa’s will produce a ___ colour when reacting with ninhydrin. __ and ___ have the amino group as part of a ring and give a yellow colour.

Answer 12

• purple
• proline
• hydroxyproline

Question 13

What is the purpose of using two different solvent systems (pH’s) to develop the chromatogram?

Answer 13

to separate co-elating solutes in a system to identify if two solutes can be the same if the relationship sustains during development.
ie: show up same on one test
will they on a different one?

Question 14

If 3 ul of 0.01M alanine (MW 89g/mol) is applied to a spot on paper how many micrograms of solute are present? How many milligrams? Show work!

Answer 14

3ul x 1mL/1000ul = 3x10^-3mL
3x10^-3mL x 1L/1000mL = 3x10^-6L 
M=moles/v 
moles= 0.01M x 3x10^-6L = 3x10^-8 moles 
3x10^-8 moles  x 89g/mol = 2.67x10^-6 g 
2.67x10^-6 g x 1x10^6 ug/1g= 2.67ug of alanine 
2.67ug x 1x10^-3mg/ug= 2.67x10^-3 mg

Question 15

Why do aa’s structure change in response to pH’s of varying environments ?

Answer 15

they have both a basic amino group and an acidic carboxylic acid group…….they also have side chains that are basic, acidic, polar or non polar….protonation can lead to development of a net ionic charge which changes the polar nature of something

Question 16

More ionic charge = the more nonpolar/polar therefore the material will bind /stronger/weaker to the polar stationary phase

Answer 16

polar

Question 17

Will an aa have a higher Rf value in a mobile phase that causes it to be more polar or non polar?

Answer 17

non polar because it will not be retained as easily by the polar stationary phase.