Chromatography Flashcards
How does it work
Separates mixtures of molecules based on density
what is chromatography used for
- separating mixtures of monosaccharides and identifying their components
- separating mixtures of amino acids and identifying their components
Rf calculation
Distance moved by spot /
distance moved by solvent front
What information about density of the molecule does the position on the chromatogram tell you
Less dense molecules move further and more dense molecules move less distance
Is it easy to measure the distance to the solvent front
No, in reality, the solvent doesn’t move up to give a completely horizontal straight line
Problems with technique
- paper touches the side - doesn’t run straight
- solvent continues to move
- solvent front not marked - no Rf values possible
- pen used to draw origin line, ink dissolves in solvent and moves up the paper
- molecules similar densities, can’t be separated by one-way chromatography
Two-way chromatography
Fixes problem of separating out substances which have very similar Rf values
A chromatogram is made starting from a single spot of mixture placed towards one end of the base line.
It is stood in a solvent as before and left until the solvent front gets close to the top of the paper
Wait for the paper to dry out completely, then rotate it through 90 and develop the chromatogram again in a different solvent
Electrophoresis
Technique that uses the net charge of peptides (amino acids) as a basis for separation
A potential difference is applied across a solid material (e.g. paper for amino acid analysis) permeated by an electrolyte
Anions migrate to the anode and cations to the cathode. The rate of diffusion is related to the size and net charge. Small highly charged proteins migrate more quickly.
Why can electrophoresis be used to separate a mixture of amino acids
As amino acids can ionise they can be separated using electrophoresis and their R groups contain different groupings to give both anions and cations
