R.P 12 (TLC) Flashcards
Outline the stages in TLC chromatography for amino acids
Wear plastic gloves to hold onto the TLC plate to prevent contamination of hands to plate
Draw pencil line 1.5cm from bottom of plate
Use capillary tube to apply small drop of amino acid to mid point of pencil line
Allow spot to dry
Add solvent of depth 1cm (must be below pencil line or will dissolve mixture from plate)
Place TLC plate in developing tank
Allow developing solvent to rise up the plate (not reaching the very top)
Remove plate and quickly mark position of solvent front with pencil mallow to dry in fume cupboard (solvent may be toxic)
Spray with developing agent Ninhydrin
Measure distance from initial pencil line to spots (A)
Measure distance from initial pencil line to solvent front (B)
Calculate RF values (A/B)
Why do we only use a tiny drop of each solution when using TLC to separate species
Too big a drop will cause different
spots to merge
Why does the solvent have to be below the pencil line
If the solvent is too deep it will
dissolve the sample spots from the plate
Why must we use a lid
To prevent evaporation of toxic solvent
Why must we dry in a fume cupboard
Dry in a fume cupboard as the solvent is toxic
How can we get more accurate results in TLC
Will get more accurate results if the solvent is
allowed to rise to near the top of the plate but the
Rf value can be calculated if the solvent front does
not reach the top of the plate
What is the equation for Rf value
Rf value = distance moved by amino acid/
distance moved by the solvent
Why do different species move up further on the TLC plate
Separation by chromatography depends on the
balance between solubility in the moving phase and
retention in the stationary phase
How does a solid stationary phase separate
A solid stationary phase separates by adsorption
How does a liquid stationary phase separate
A liquid stationary phase separates by relative solubility
If the stationary phase was polar and the mobile
phase was non- polar, which would travel further up the plate, a polar or non polar compound?
Then non-polar compounds would pass through the plate more quickly than polar compounds as they would have a greater solubility in the non-polar mobile phase.
