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

chromatographytank

lid

chromatographypaper

origin

solvent

solventfront

spots

x

y

Rf = x/y

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.

first run second run

original spot

original spot

rotatethrough

90°

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