© SSER Ltd.

General principles

• Substances are separated according to their differential solubility between the stationary phase, represented by the water bonded to the cellulose molecules of paper, and the moving phase, the solvent.

• As the solvent front advances it carried the components at different rates.

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

The tank should be saturated with solvent vapour

Investigating the composition of a protein.

• The peptide bonds can be readily hydrolysed by heating with 6M HCl at 100oC for 10-24 hours in an evacuated tube.

• The amino acids released can then be analysed by chromatography or electrophoresis.

Choice of solvent

• The general principle is “like dissolves like”.

• If a non-polar solvent is used the hydrophobic amino acids will move much further up the paper, as they are more soluble in non-polar solvents.

• In contrast if a polar solvent is used the hydrophilic amino acids will advance further up the paper as they are now more soluble.

origin

Solventfront

solvent

Amino acid spots

Spray the drypaper with locatingagent (ninhydrin)to make the colourless spotsvisible

Dry the paperwith gentle heatto developthe amino acidspots

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

Y

Y

XR f

Y

XR f

The Rf value isalways a value less

than one as thesolvent front alwaysmoves further thanthe solute molecules

X1

X5

X4

X3

X2

Rf values can be compared with

published values to identify the amino

acids.

The mixture ofunknown aminoacids is seen to

contain four different amino acids

Of these fouramino acids,

two can be positivelyidentified

The mixture containsfour amino acids; two

unknown together with arginine & leucine

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

• Electrophoresis, like chromatography, is a technique for the separation of molecules

• Both chromatography and electrophoresis may be used for the separation of small molecules

• Electrophoresis, unlike chromatography, can be used for the separation of relatively large molecules

• Electrophoresis is used mainly for separating mixtures of proteins, peptides and DNA fragments

• Electrophoresis separates molecules from a mixture according to their charge and size

• The technique involves passing a direct current through either filter paper or a gel

- +

Electrophoresis gelsaturated with buffersolution (to keep the

pHconstant)Negative

electrodePositiveelectrode

CATHODE ANODE

GEL

Different moleculesshow as differentbands after staining

Sample slotsin the gel

BuffersolutionBuffertank

Contact wickmaking electricalconnection between buffer and gel

Sample ofmixture placedin a slot cut in

the gel

Preparation of samples

• Filter paper wicks are soaked in the test solution.

• They are then inserted in fine cuts made in the thin layer of silica gel.

• NB Further separation can be achieved using filter paper in combination with chromatography in another direction.

Separation of Serum ProteinsBlood serum containsmany different blood proteins

A serum sample isplaced in a slot in the gel

The pH of the buffer isadjusted to make all theproteins negatively charged

When the direct currentis passed through the gel,all the proteins will moveto the anode

After staining, the proteinswill show up as different bands in different positionsfrom the origin slot

Directionof

migration

albumin

alpha 1globulinalpha 2globulinbetaglobulingamma

globulin(mainly

antibodies)

Amino acids are AMPHOTERIC

The amino group of the amino acid is a basic group

The carboxylic group is an acid group

Acids behave as proton donors

Bases behave as proton acceptors

Protons are hydrogen ions (H+)

A proton is the nucleus of a hydrogen atom

H+

H+

Amphotericmolecules behave as both acids and

bases

Amino acids possess a carboxylicacid end and an amino end: Theamino group behaves as a base

The R group may also possess a carboxylic acid

OR an amino group

In solution the acid group behavesas a proton donor and the amino group (basic) as a proton acceptor

In solution, the acid groupbecomes negatively charged and

the amino group becomespositively charged

Neutral amino acids with no acid or amino groups in the R group

have one negative and one positivecharge. Overall they are electrically

neutral moleculesH+H+

The hydrogen atom contains a singlepositively charged particle in its nucleus

This positively charged particle is a proton

Spinning around the nucleus in orbit isa single negatively charged electron

In solution, the hydrogen atom of the carboxylic acid group of the amino acid releases a proton (H+)

The electron remains with thecarboxylic acid group which becomesnegatively charged

+++

This is an ACIDIC amino acid as itpossesses TWO carboxylic acid groups

In solution the acid groups donate a proton and the amino group accepts a protonThe negative electrons remainwith the carboxylic acid groups

The amino acid now has TWOnegatively charged carboxylic acidgroups and ONE positively chargedamino group

Overall, this acidic amino acid isnegatively charged

H+

H+

H+

This is a BASIC amino acid as itpossesses TWO amino groupsIn solution the acid groupdonates a proton and the amino groups accept a proton

The negative electron remainswith the carboxylic acid group

The amino acid now has TWOpositively charged amino acidgroups and ONE negatively chargedcarboxylic acid group

Overall, this basic amino acid ispositively charged

H+

H+

H+

ANODE(+)

CATHODE(-)

mixture of thethree aminoacids placed

in well

movestowards

movestowards

stays atthe origin

GEL SOAKED IN BUFFER SOLUTION

acidic amino acid basic amino acidneutral amino acid

direct currentpassed throughthe gel and later

sprayed withninhydrin

The gel is examined under uv