((s) principles and uses of paper and tthin layer chromatograaphy

What is thin layer chromatography TLC used for

Separate individual components of a mixture

Separate and identify. Mixture of amino acids in solution

How are the a.a separated

M.p pick up a.a in the organic solvent and moves through s.p and a.a are separated

What is used. In stationary phase and why

Silica gel (adhesive substance) applied to rigid surface (e.g glass or metal)

A.a. Added to one side of the;

This end submerged in organic solvent

O.s moves through silica gel = mobile phase

What dos the rate at which diff a.a move through silica gel deine on

Interaction (hydrogen bonds) they have with silica in s.p

And solubility in m.p

Results in diff a.a moving different distances = separate out

What should you make sure to do when sampling the protein and how

Need to make it concentrated protein sample

Put it on then let it dry and repeat several times

What are the precaution to take

Don’t contaminate so wear gloves or hold onto edges

Wear safety goggles

lien dran in pencil

Solvent below pencil line or will dissolve into solvent ( no more than 1cm deep)

What will travel further up in protein chromatography

Lighter a.a with smaller r groups because thy have less resistance with silica gel

What happens on mobile phase

Allow sample to run and separate a.a

What do you do to see the a.a

Spray chromatogram with ninhydrin spray

A.a reacts with it an a purple brown colour is made

What do you do at the end when everything is separated

Mark how r the solvent has travelled (solvent front )

Work out rf value - distance travelled by component/ distance traveled by solvent

As long as conditions are kept the same then

Same a.a will always ravel the same distance in the same time

What does chromatography rely on?

Differences in solubility In mobile phase affects how far each component can travel

Why are components with higher solubility travel further

Spend more time in mobile phase and are carried further up paper than less soluble components

What is paper chromatography method

Spot is placed on paper and dried

Paper suspended in solvent (water o ethanol)

Solvent travels up through paper the different components within mixture move up paper at different speeds

Larger molecules move slower than smaller ones

This causes the original mixture to separate out into different spots or bands on the paper

What can paper chromatography be used for

Separate a mixture of monosaccharides

Describe using chromatography to separate a mixture of monosaccharides

Need to be stained because they are colourless

Spot of stained monosaccharides placed on line at bottom of paper

Spots of known standard solutions of different monosaccharides are then placed on line beside sample spot

Suspended

Mono. Separate out

Unknown can be identified by comparing and matching them with chromotgrams of known standard solutions of different monosaccharides.

If a spot is same distance from line as a spot of one of the known standard solutions then it must contain this monosaccharide

How do you use rf values to identify chloroplast pigment

Each pigment will have unique rf value

Specific rf values depended on solvent that is being used

Carotenoids rf value is closer to one

Chlorpphyll B has lower

Chlorophyll a is between carotenoid and chlor.B

What do smaller rf values indicate in terms of chloroplast pigments

Pigment is less soluble and larger in size