L8. Proteins.

Separation of aminoacids by chromatography

Chromatography is a laboratory technique for the separation of a mixture. The

mixture is dissolved in a fluid called the mobile phase, which carries it through a structure
holding another material called the stationary phase. The various constituents of the mixture
travel at different speeds, causing them to separate. The separation is based on differential
partitioning between the mobile and stationary phases. Subtle differences in a
compound's partition coefficient result in differential retention on the stationary phase and
thus affect the separation.
The distribution of the molecules between the two phases is governed by one or more
of four basic processes, namely adsorption, ion-exchange, partitioning, and gel filtration (gel
exclusion, molecular sieving). The operating of these processes, coupled with the movement
of the mobile phase, results in a differential migration (resolution) of the molecules along the
stationary phase.
Essential components of a chromatography system are:
• A stationary phase, either a solid (a gel) or an immobilized liquid, held by a support
matrix.
• A chromatographic bed: the stationary phase may be packed into a glass or a metal
column, spread as a thin layer on a sheet of glass or plastic, or adsorbed on cellulose
fibers (paper).
• A mobile phase, either a liquid or a gas that acts as a solvent, carrying the sample through
the stationary phase and eluting from the chromatographic bed.
• A delivery system to pass the mobile phase through the chromatographic bed.
• A detection system to monitor the test substances
Individual substances interact with the stationary phase to different extend as they are
carried through the system, enabling separation to be achieved.
In a chromatographic system, those substances which interact strongly with the
stationary phase will be retarded to the greatest extend while those which show little
interaction will pass through with minimal delay, leading to differences in distances traveled
or elution times.

Classification criteria

1. Classification according the physical state of the two phases, stationary and
mobile, forming the chromatographic system:
a) Solid stationary phase:
- mobile phase gaseous : gas-solid chromatography (GSC)
- mobile phase liquid: liquid-solid chromatography (LSC)
b) Liquid stationary phase:
- mobile phase gaseous : gas-liquid chromatography (GLC)
- mobile phase liquid: liquid-liquid chromatography (LLC)

2. Classification according the nature of interactions between the components of

the mixture and the phases mobile and/or stationary
The distribution of the molecules between the two phases is governed by one or more
of four basic processes, namely adsorption, ion-exchange, partitioning, and gel filtration (gel
exclusion, molecular sieving). Thus, chromatography can be:
 Adsorption Chromatography
In adsorption chromatography the stationary phase is a solid, generally in the form of
a column (filled with the solid in form of very small pearls, with a controlled diameter and
porosity). The mobile phase is either an aqueous or a non-aqueous solution. As the material
moves down the column, the process of adsorption and desorption is repeated many times.
The rate of movement of the molecules depends on the degree of their adsorption to the solid
support.

Adsorption chromatography (polar stationary phase)

Ion-Exchange Chromatography
In ion-exchange chromatography molecules are separated based on their net charge.
The stationary phase is an ion-exchange resin (a cross-linked polymer with many charged
functional groups) and the mobile phase is an aqueous solution. Ion-exchange
chromatography is usually done on columns. The molecules are retarded in their movement
through the column depending on the sign and magnitude of their charge. Electrostatic
binding of the molecules to the oppositely charged groups on the ion-exchanging resin, and
the disruption of these bonds, occurs repeatedly as the material moves down the column.
Ideally, the sample is applied to the column at a pH at which the molecules have an opposite
charge to that of the groups on the resin, so that the molecules bind to the column. The pH of
the eluting buffer is then changed, thereby changing the net charge of the molecules. When
the molecules and the functional groups of the resin carry a charge of the same sign, the
molecules are repelled by the charged groups of the resin and are eluted from the column.
Molecules can also be eluted from an ion-exchange column by an increase in the ionic
strength. A cation exchange resin is a negatively charged resin that binds cations; an anion
exchange resin is a positively charged resin which binds anions.
 Ion-Exchange Chromatography (cation exchanger)

Partition Chromatography
In partition chromatography the distribution of molecules between a mobile phase and
a stationary liquid phase is based upon the solubility of the molecules in the two phases. A
substance, soluble in two immiscible phases, will distribute (partition) itself between these
two phases according to its solubility. The stationary liquid phase is held in place by a porous
solid such as a sheet of filter paper (paper chromatography). In this case the wet filter paper
(a polar layer of water molecules bound to the hydroxyl groups of the cellulose) represents
the stationary phase, and the layer of liquid above the wet paper (usually a relatively non-
polar organic solvent) represents the mobile phase. The distribution of the molecules between
the two phases occurs repeatedly as the material moves up or down on the paper. Non-polar
molecules will move father in this system than the polar ones.
Movement of substances in partition chromatography is characterized by an Rf value.
The Rf is the ratio of the distance moved by the sample to that moved by the solvent, both
measured from the origin line. That is,

distance moved by sample

Rf = ----------------------------------
distance moved by solvent

The Rf value is generally proportional to the solubility of the sample in the mobile
phase.
 Liquid-liquid partition chromatography
(e.g. reverse phase HPLC)

Gel-Filtration Chromatography
In gel filtration (permeation) chromatography the stationary phase consists of
spherical gel particles of controlled size and porosity (crosslinked polymers), which are
generally used in the form of a column. The mobile phase is the liquid passed through the
column. Molecules are separated on such a column based on their size and shape. These two
parameters determine the rate and extend of diffusion of the molecules. Smaller molecules
diffuse more readily into the gel particles than larger ones of the same general shape. The
more likely the penetration into the gel particles, the more retarded the movement of the
molecules through the column. Movement in and out of the gel particles occurs repeatedly as
the material moves through the column. Molecules that are larger than the pore size of the gel
particles are excluded from the gel particles and move rapidly through the space between the
gel particles. Thus, for molecules of the same general shape, the smaller the molecular
weight, the more retarded the movement through the column and the greater the elution
volume. While the penetration of gel particles, based on molecular weight, is the main factor
in gel filtration, other factors (such as charge interactions and adsorptive effects) must be
considered under some conditions.

Gel permeation chromatography

 Affinity Chromatography
It is the most selective type of column chromatography. It relies on binding
interactions between a protein and a ligand. A ligand is a molecule, group, ion, or atom that
binds usually non-covalently to another molecule or atom. In affinity chromatography, the
ligand is covalently attached to the matrix (the solid support). The ligand may be a reactant or
product to which an enzyme binds in vivo, or it may be an antibody that recognizes the
protein of interest (antigen).

Affinity chromatography

3. Types of chromatography according to the form of the chromatographic bed:

Thin layer chromatography (TLC) and paper chromatography

In that case, one applies the sample as a single spot near one end of the sheet, by
microsyringe or microcapillary. This sheet is allowed to dry fully, and then it is transferred to a
glass tank containing a shallow layer of solvent. The sheet is removed when the solvent front
has traveled across 80-90% of its length. One can express movement of an individual substance
in terms of its relative frontal mobility, or RF value (see partition chromatography).

Column chromatography
In that case, a glass column (or steel) is packaged with the appropriate stationary
phase and the mobile phase is equilibrated by passage through the column, either by gravity
or using a low-pressure peristaltic pump. The sample can be introduced to the top of the
column, to form a discrete band of material. This is then flushed through the column by the
mobile phase. If the individual substances have different rate of migration, they will separate
within the column, eluting at different times as the mobile phase travels through the column.
The migration of a particular substance at a given flow rate can be expressed in terms
of its retention time (t), or elution volume (Ve).

High-Pressure Liquid Chromatography (HPLC)

Column chromatography originally used large “soft” stationary phases that required
low pressure flow of the mobile phase to avoid compression; separations were usually time-
consuming and of low resolution (“low performance”).
The resolution of conventional chromatographic methods is greatly increased by the
use of HPLC. The matrix in HPLC consists of beads that are smaller, more uniform in size,
and more tightly packed than the beads used in conventional chromatographic columns. High
pressure (hundreds of pounds per square inch - up to 10 MPa) is applied to pump a mixture of
substances and solvent through the column, which is usually made of stainless steel. All
components, valves, etc., are manufactured from materials that can withstand the high
pressures involved.
The speed, sensitivity and versatility of HPLC make this the method of choice for the
separating of many small molecules of biological interest, normally using reverse phase
partition chromatography. Separation of macromolecules (especially proteins and nucleic
acids) usually requires “biocompatibles” systems in which stainless steel components are
replaced by titanium, glass or fluoroplastics, using lower pressures to avoid denaturation, e.g.
the Pharmacia FPLC system.

Gas chromatography (GC)

Modern GC uses capillary chromatography columns (internal diameter 0.1 - 0.5 mm)
up to 50 m length. The stationary phase is generally a cross-linked silicone polymer, coated
as a thin film on the inner wall of the capillary: at normal operating temperatures, this
behaves in a similar manner to a liquid film, but is far more robust. The mobile phase
(“carrier gas”) is usually nitrogen or helium. Selective separation is achieved as a result of the
differential partitioning of individual components between the carrier gas and silicone
polymer phases. The separation of most biomolecules is influenced by the temperature of the
column, which may be constant during the analysis (“isothermal” - usually 50 - 2500C) or,
more commonly, may increased in a programmed manner (e.g. from 500C to 2500C per min).
Samples are injected onto the “top” of the column, through a sample injection port containing
a gas-tight septum. The output from the column can be monitored by flame ionization,
electron capture or thermal conductivity.

Experimental part: Amino acid separation by paper chromatography

Principle
Paper chromatography is a type of partition chromatography, with a large use the
years 1940-1960. Today it was practically completely replaced with thin layer
chromatography, which uses a solid support laid as a thin layer on a plastic sheet or a glass.
The sample (amino acid solution) is deposited on a starting point on the paper sheet,
in a very low quantity (1-10µl). The paper is dried and then eluted with a solvent system
(usually up the paper due to its porosity, or capillarity). The amino acids will separate due to
their different hydrophobic or hydrophilic properties. The substances are put in evidence by
special color reagents (or UV light).

Reagents
1. Amino acid solution: 0.3% solution of phenylalanine and histidine.
2. Eluent: a mixture of n-buthanol, acetone, acetic acid and water at a volume ratio of:
3,5:3.5:1.0:2.0.
3. Ninhydrin solution: -it is used to put in evidence the migrated amino acids
4. Watman paper.

Materials and Apparatus

1. chromatographic chamber
2. spray, hair drier, hot plate
3. cylinder, pipettes, micropipette
Procedure
1. Introduce 10 ml of eluent in the chromatographic chamber. Attach the cover. Shake the
chamber slowly and horizontally, to create an atmosphere saturated in eluent vapors.
2. Draw a line, with a pencil, on the chromatographic paper, at 1.5 cm from the base. Place a
drop of the amino acid solution (approx. 5 µl) on the middle of the line, using the
micropipette. Dry immediately the paper with the hair drier.
3. Suspend the dried paper in the chromatographic chamber so that it is immersed in the
eluent for approx. 5 mm.
4. Maintain the paper in the covered chromatographic chamber till the eluent arrives at
approx. 2 cm from the top of the paper. Then take out the paper, and mark the eluent front
with the pencil. Dry again the paper with the hair drier.
5. Spray the paper with the ninhydrin solution between the two marks, and then dry it over
the hot plate till the blue-violet color appears.

Calculation
The Rf values for the two amino acids will be calculated.The migration order,
function of the amino acid hydrophobicity, from the most rapid to the slowest one is:
phenylalanine and histidine.