chromatography Flashcards
what is the aim of chromatography
to separate a mixture into its constituent biological molecules
what are the components of chromatography
stationary and mobile phase
what is the stationary phase
either the chromatography paper or a thin layer chromatography plate. the paper is made of cellulose. the TLC plate is a sheet of plastic, coated with a thin layer of silica gel or aluminium hydroxide.
in each case, there are free -OH groups pointing outwards in contact with the mobile phase
what is the mobile phase
the solvent for the biological molecules. at a simple level, we use water (for polar molecules) or ethanol (for non polar molecules) the mobile phase flows through and across the stationary phase, carrying the biological molecules within with
what is the procedure of chromatography
- wear eye protection
- draw the line in pencil and put a tiny dot on the line to show you where to place the solution mixture (if you draw it in ink, the ink pigments will separate)
- spot the solution mixture onto the pencil dot several times using capillary tubing. wait for the spot to dry before putting on the next spot and try to make it as thin as possible. when its completely dry, lower it in the solvent. ensure the level of the solvent at the start is below the pencil line
- cover the beaker with a watch glass
- let the apparatus run until the solvent has reached a point just underneath the top of the paper/TLC plate
- remove it from the solvent and lay it on a white tile to dry
what happens as the solvent travels up the paper
the components of the solution mixture travel with it. the pigments travel at different speeds and so are different positions on the plate
how to calculate Rf value
the distance travelled by the spot/distance travelled by the solvent
when should you calculate the Rf value
before the solvent dries so you can see it
what happens if you repeat the investigation under the same conditions
each pigment will have the same Rf value
what can you do if you know the values of particular pigments
allows you to identify them
what can you use to see colourless solutions in thin layer chromatography
solutions such as UV light, ninhydrin and iodine
how can you use ultraviolet light
TLC plates have a chemical which fluoresces under UV light. if you look at the plate under UV light, most of it will glow except the places the spots have travelled to. they mask the plate from UV light
how can you use ninhydrin
to see amino acids, allow the plate to dry and spray it with ninhydrin. this binds to the amino acids which are then visible as brown or purple spots
how can you use iodine
allow the plate to dry and place in an enclosed containter with a few iodine crystals. the iodine forms gas which binds to the molecules in each of the spots
what does the speed at which the molecules move along the paper of TLC plate depend on
depends on their solubility in the solvent and their polarity and may also depend on their size
how does chromatography work
exposed OH groups make the surface of the paper/plate very polar and allow it to form hydrogen bonds with the molecules alongside other dipole interactions.
which will move slowly
a highly polar solute will tend to stick to the surface and hence move up slowly
what will travel quickly
a non polar solvent
what will happen if two molecules travel at exactly the same speed
it will be difficult to separate them
how can you separate two molecules that move at exactly the same speed
you could use a different solvent or change the pH
how is chromatagraphy used
- TLC is used to monitor the progress of reactuons because it works relatively quickly.
- it is also used for urine testing of athletes for illegal drugs
- used for analysing drugs for purity of components
- analysis of foods to determine the presence of contaminants
