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

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Flashcards in Lab 1:Separation and Identification of AA's via Paper chromatography Deck (17):

define the term mobile phase!

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


Stationary phase?

- solid or liquid coated support that is immobile


Whats the third component of chromatography?

- solutes that are separated from the mixture.


Explain the characteristics of the chromatograph paper used in lab!

- 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.


interaction of mobile and stationary?

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


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

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


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

k = solubility in mobile phase/ solubility in stationary phase


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

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


What is the formula for the retention or retardation factor?

Rf= distance solute migrates/ distance mobile phase travelled


What term best describes paper chromatography?

partition chromatography


Was ascending or descending chromatography used in this experiment?



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.

- purple
- proline
- hydroxyproline


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

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?


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!

3ul x 1mL/1000ul = 3x10^-3mL
3x10^-3mL x 1L/1000mL = 3x10^-6L
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


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

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


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



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

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