chromatography Flashcards
what is chromatography used for
used to separate a mixture into individual components
what type of mixtures can it be used to separate
mixture of proteins,carbohydrates, nucleic acids
what does chromatography rely on
the difference in solubilities of solutes in the solvent in order to separate the mixture into individual components
what two phases are involved in chromatography
-mobile phase
-stationary phase
when do components in a mixture separate during chromatography
they separate when the mobile phase travels over the stationary phase
-the mobile phase dissolves the mixture and carries it through the stationary phase where it is separated into individual components
what affects how far each component can travel
the solubility of the component in the mobile phase,the more soluble the more time it spend in the mobile phase and so the further its carried through the stationary phase travels further than those that are less soluble as they spend less time in the solvent
what is the mobile phase in paper chromatography
-solvent ,some form of a liquid,water or ethanol
state two forms of chromatography
-paper chromatography
-thin layer chromatography
what the stationary phase in paper chromatography
the chromatography paper
state the method of paper chromatography
-draw a line with a pencil about 2 cm away from the bottom of the chromatography paper-this is our origin line
- a spot of the unknown mixture is placed on the origin line and left to dry
-suspend the chromatography paper in a solvent,make sure the solvent is below the origin line
-place a lid on top of the beaker containing the solvent so solvent doesn’t evaporate
- as the solvent travels up the chromatography paper ,the different components of the mixture move up the paper at different speeds and travel different distances .
-This depends on whether the molecules are large or small, larger molecules travel slower than small ones, and it depends on the solubility of the components in the solvent,the more soluble the more time they spend in the solvent so the further they travel
-the mixture ends up separating into different spots on the chromatography paper -this is known as a chromatogram
-to find the identity of the spots calculate their rf value and compare it to the database value of different components
-if the rf value is the same then that component must be present in the mixture
on what depends at what rate the amino acids in the organic solvent move through the silica gel
-the hydrogen bonds the amino acids form with the silica gel
-depends on the solubility of amino acids in the organic solvent
describe how you would use paper chromatography to find the identity of an unknown monosaccharide mixture
paper chromatography can be used to separate a mixture of monosaccharides
-first stain the mixture of monosaccharides because it’s colourless
-a spot of the mixture is placed on the origin line at the bottom of the chromatography paper
-then spots of known standard solutions of different monosaccharides are placed next to the mixture spot
-suspend the chromatography paper in the solvent,make sure the solvent is not touching the origin line
-place a lid on the beaker so the solvent doesn’t evaporate
- as the solvent travels up the chromatography paper the different monosaccharides within the mixture separate out at different distances from the origin line
-take the chromatography paper out and draw a line along the solvent front
-then unknown monosaccharides can then be identified by calculating the distance travelled by the spots from the origin line
-calculate the rf value of each spot by dividing the distance travelled by the spot by the distance travelled by the solvent
-compare to the rf values of known monosaccharides, match and compare the distances if they are the same then that monosaccharide is present within the mixture
what is the stationary phase in thin layer chromatography
the silica gel on the glass/plate.The amino acids are added to the end of the gel.This end is then submerged under water
what is the mobile phase in thin layer chromatography
a organic solvent .It picks up the amino acids and moves them through the stationary phase
what is thin-layer chromatography used for
to separate a mixture of amino acids into individual components
what does a larger rf value indicate
describe the method of using thin-layer chromatography
-wear gloves, handle the plate by the edges draw a line using a pencil 2 cm away from the bottom-its our origin line
-mark 4 marks equally spaced out on the origin line
-spot the first mark with the unknown amino acid mixture.Wait for it to dry and spot it again.
-spot the 3 remaining marks with 3 different known amino acid solutions
-submurge the plate into a jar containing the organic solvent ,and place a lid on top
-make sure the solvent is not touching the origin line
-the solvent will dissolve the mixtures of amino acids and move them through the silica gel on the plate,separating the mixtures into separate amino acids spots
-when the solvent has travelled 2 cm to the top of the plate ,remove the plate and draw a pencil line along the solvent front
Then wait for it to dry
-Finally spray the plate in a fume cupboard with ninhydrin spray which will turn the amino acids purple making it easier to see the separate sports on the plate,making it easier to compare and match them
what is the name of the spray that turns amino acids purple
ninhydrin spray,and is sprayed ina fume cupboard,wear gloves and goggles while spraying as well as a lab coat,wash your hands afterwards
describe the method of separating a mixture of amino acids using paper chromatography
-draw a line using a pencil 2 cm away from the bottom of the chromatography paper-its our origin line
- a spot of the unknown mixture of amino acids is placed on the origin line of the chromatography paper
-next to it spot known solutions of different amino acids
-submurge the chromatography paper in solvent contained ina breaker and close the beaker with lid so no solvent evaporates
-make sure the solvent isn’t touching the origin line
-the mixture of amino cods will dissolve in the solvent and the solvent will carry it through the chromatography paper where it will separate into separate amino acids ,forming spots at different distances from the origin line depending on the amino acids solubility in the solvent,if more soluble ,spend more time in the solvent so travel further
-then identify the unknown amino acids by comparing and matching them with the chromatograms of the known standard solutions of amino acids
-if they have the same rf value then that amino acid must be present in the mixture
-spray the chromatography paper in a fume cupboard with ninhydrin spray to make the amino acids purple,making them more easily visible and easier to compare
does the rf have units
no
is rf values always ammer or bigger than 1
smaller
what is meant by the solvent front
its the distance trave;lled by the solvent
what is meant by the origin line
it’s the line we draw at the bottom of the chromatography paper/plate
how do you calculate an rf value
distance travelled by solute(distance from the origin line to the centre of the spot)/ distance travelled by solvent(distance to the solvent front)
what does an RF value determine
determines how far a molecule travels on the stationary phase
how can you use rf values to identify unknown molecules
calculate the rf value of the unknown molecule and compare it to the rf value of known standard solution of molecules?search their rf value in the database
what does a smaller rf value indicate
molecule has travelled a shorter distance as its less soluble in the solvent and is larger-
spend less time in the solvent thus carried a shorter distance in the stationary phase
what does a larger rf value indicate
molecule has travelled further up the stationary phase eas it is more soluble in the solvent so spends more time in it thus carried further up and is also likely a smaller molecule
