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ChromatographyChromatography
Learning objective:Learning objective:To be able to describe the To be able to describe the
method of method of chromatography and its chromatography and its
applicationsapplications
ChromatographyChromatography• Chromatography is used to Chromatography is used to
separate pure substances from a separate pure substances from a mixture of substances, such as a mixture of substances, such as a cell extract. cell extract.
• It is based on different substances It is based on different substances having different solubilities in having different solubilities in different solvents. different solvents.
• A simple and common form of A simple and common form of chromatography uses filter paperchromatography uses filter paper
Substances to be identifiedare ‘spotted’ near one endof the filter paper
As the solvent moves up the paper, different molecules move at different rates with the smallest molecules moving the fastest
The technique is used for small molecules suchas amino acids, small peptides and sugars
Paper chromatography isa technique used for theseparation & identificationof relatively small chemicalsubstances by a moving solvent on sheets or stripsof filter paper
Filter papercylinder
Clip
Solvent
Concentratedspot of
chemicals tobe separatedand identified
Direction ofsolvent
movement
origin
Solventfront
solvent
Amino acid spots
Purple spotsdevelop located atdifferent distancesfrom the origin line
Mark the solventfront & allowpaper to dry
amino acid spotson the origin line
Chromatography separates small molecules in a mixture on the basis of size
As the solvent moves up the paper, molecules move at different rates
When the spots are colourless (most amino acids), a locating agent is needed to visualise their positions on the chromatography paper
• The chromatogram can be analysed by The chromatogram can be analysed by measuring the distance travelled by the measuring the distance travelled by the solvent front, and the distance from the solvent front, and the distance from the origin to the centre of each spot. origin to the centre of each spot.
• This is used to calculate the This is used to calculate the RfRf (relative (relative front) value for each spot:front) value for each spot:
• An An RfRf value is characteristic of a value is characteristic of a particular solute in a particular solvent. particular solute in a particular solvent. It can be used to identify components of It can be used to identify components of a mixture by comparing to tables of a mixture by comparing to tables of known known RfRf values. values.
R f distance moved by spot
distance moved by solvent
YY
XR f
Y
XR f
The Rf value isalways a value less
than one as thesolvent front alwaysmoves further than
the solute molecules
X1
X5
X4
X3
X2
The mixture ofunknown aminoacids is seen to
contain four different amino
acids
Of these fouramino acids,
two can be positivelyidentified
The mixture contains
four amino acids; two
unknown together with arginine &
leucine
• Sometimes chromatography with Sometimes chromatography with a single solvent is not enough to a single solvent is not enough to separate all the constituents of a separate all the constituents of a mixture. mixture.
• In this case the separation can be In this case the separation can be improved by improved by two-dimensional two-dimensional chromatographychromatography, where the , where the chromatography paper is turned chromatography paper is turned through 90° and run a second through 90° and run a second time in a second solvent. time in a second solvent.
• Solutes that didn't separate in Solutes that didn't separate in one solvent will separate in one solvent will separate in another because they have another because they have different solubilities.different solubilities.
Mixture ofamino acidson origin line
Paper dried and rotated clockwise
through 90o
Solventfront
First solventSecond solvent
Two-way chromatography provides better separation of substances that behave in a similar fashion in the first
solvent.A second run in a different solvent resolves two very close
spots more clearly
Solventfront
Different types of Different types of chromatographychromatography
• Paper chromatographyPaper chromatography is the simplest, but is the simplest, but does not always give very clean does not always give very clean separation.separation.
Different types of Different types of chromatographychromatography
• Thin layer chromatographyThin layer chromatography (tlc) uses a thin layer of (tlc) uses a thin layer of cellulose or silica coated cellulose or silica coated onto a plastic or glass sheet. onto a plastic or glass sheet. This is more expensive, but This is more expensive, but gives much better and more gives much better and more reliable separation.reliable separation.
Different types of Different types of chromatographychromatography
• Column chromatographyColumn chromatography uses a uses a glass column filled with a cellulose glass column filled with a cellulose slurry. Large samples can be slurry. Large samples can be pumped through the column and pumped through the column and the separated fractions can be the separated fractions can be collected for further experiments, collected for further experiments, so this is so this is preparative preparative chromatographychromatography as opposed to as opposed to analytical chromatographyanalytical chromatography..
Different types of Different types of chromatographychromatography
•High performance liquid High performance liquid chromatographychromatography (HPLC) is (HPLC) is an improved form of an improved form of column chromatography column chromatography that delivers excellent that delivers excellent separation very quickly.separation very quickly.
Different types of Different types of chromatographychromatography
• ElectrophoresisElectrophoresis uses an uses an electric current to separate electric current to separate molecules on the basis of molecules on the basis of charge. It can also be used charge. It can also be used to separate on the basis of to separate on the basis of molecular size, and as such molecular size, and as such is used in DNA sequencingis used in DNA sequencing