Lecture 13

CHROMATOGRAPHY

Chromatography (from Greek chroma, colour) is the collective term

for a family of laboratory techniques for the separation of mixtures. It
involves passing a mixture which contains the analyte through a
stationary phase, which separates it from other molecules in the
mixture and allows it to be isolated.

History
It was the Russian botanist Mikhail Semyonovich Tsvet who invented
the first chromatography technique in 1901 during his research on
chlorophyll. He used a liquid-adsorption column containing calcium
carbonate to separate plant pigments. The Russian word "tsvet"
means "color," so literally "color writing". In 1952 Archer John Porter
Martin and Richard Laurence Millington Synge were awarded the
Chemistry Nobel Prize for their invention of partition chromatography.
Since then, the technology has advanced rapidly. Researchers found
that the principles underlying Tsvet's chromatography could be
applied in many different ways, giving rise to the different varieties of
chromatography.

Chromatography Terms
The analyte is the substance which is to be purified or isolated during
chromatography.
Analytical chromatography is used to determine the identity and
concentration of molecules in a mixture.
A chromatogram is the visual output of the chromatograph. Different
peaks or patterns on the chromatogram correspond to different
components of the separated mixture.
A chromatograph takes a chemical mixture carried by liquid or gas
and separates it into its component parts as a result of differential
distributions of the solutes as they flow around or over the stationary
phase.
The mobile phase is the analyte and solvent mixture which travels
through the stationary phase.
Preparative chromatography is used to purify larger quantities of a
substance.
The retention time is the characteristic time it takes for a particular
molecule to pass through the system.
The stationary phase is the substance which is fixed in place for the
chromatography procedure and is the the phase to which solvents and
the analyte travel through or bind to. Examples include the silica plate
in thin layer chromatography.

Chromatography Defined
Chromatography is classically defined as “a separation process that
is achieved by distributing the substances to be separated between
two phases, a moving phase and a stationary phase”.

Those substances distributed preferentially in the moving phase

will pass through the chromatographic system faster than those that
are distributed preferentially in the stationary phase. As a
consequence, the substances will be eluted from the chromatographic
system in the inverse order of the magnitude of their distribution
coefficients with respect to the stationary phase’. (In chromatography,
by convention, distribution coefficients are always given with
reference to the stationary phase). In general, the moving phase will
be either a gas or a liquid which gives rise to two basic types of
chromatography; gas chromatography (GC) where the moving phase
is a gas and liquid chromatography (LC) where the moving phase is a
liquid. In the same way the stationary phase will normally be either a
liquid or a solid which gives rise to four sub groups of
chromatography, gas-liquid chromatography (GLC), gas-solid
chromatography (GSC), liquid-liquid chromatography (LLC) and
liquid-solid chromatography (LSC). There are some anomalies to these
generalizations, the mobile phase, a gas or a liquid, may be operated
under super critical conditions. Under super critical conditions, the
moving phase can possess the attributes of both a liquid and a gas
and exhibit the chromatographic advantages of both physical states.
This type of chromatography is termed super critical fluid
chromatography (SCFC). In addition, a continuous form of preparative
chromatography has been described where the stationary phase
packing falls continuously down a column and a gaseous mobile phase
simultaneously passes up the column. By adjusting the temperature of
different portions of the column, a continuous feed of relatively pure
individual substances can be obtained from different parts of the
column. This type of chromatography has been termed moving bed
chromatography (MBC). The moving bed has been simulated using an
ingenious mobile phase valve system which has been called simulated
moving bed chromatography (SMBC).
Chromatographic Mechanisms
During a chromatographic separation solute molecules are
continuously moving back and forth between stationary and mobile
phases. The process whereby a solute is transferred from a mobile to
a stationary phase is called, “sorption”. Chromatographic techniques
are based on four different sorption mechanisms, viz., surface
adsorption, partition, ion-exchange and exclusion.

The rise in the concentration of a solute on the solid stationary phase

is called, “surface adsorption”. If a liquid is coated onto the surface of
an inert solid support, the sorption process is one of partition and the
movement of the solute is determined solely by its relative solubility in
the two phases or by its volatility if the mobile phase is a gas. In ion-
exchange, the stationary phase is a permeable polymeric solid
containing fixed charged groups and mobile counter-ions which can
exchange with the ions of a solute as the mobile phase carries them
through the structure. Inert, porous, stationary phase (a gel structure)
which has a small pore size into which small molecules up to a certain
critical size can diffuse. Molecules larger than the critical size are
excluded from the gel and move unhindered through the column,
whilst smaller ones are retarded to an extent dependent on molecular
size. In each chromatographic technique, one of the four mechanisms
predominates, but two or more may be involved simultaneously.
Partition and adsorption frequently occur together and in paper
chromatography, ion-exchange and exclusion certainly play minor
roles.
Chromatography Theory
Chromatography is a separation method that exploits the differences
in partitioning behavior between a mobile phase and a stationary
phase to separate the components in a mixture. Components of a
mixture may be interacting with the stationary phase based on
charge, relative solubility or adsorption. There are two theories on
chromatography, the retention theory and the plate theory.

Retention Theory
The retention is a measure of the speed at which a substance moves in
a chromatographic system. In continuous development systems like
HPLC or GC, where the compounds are eluted with the eluent, the
retention is usually measured as the retention time Rt or tR, the time
between injection and detection. In interrupted development systems
like TLC the retention is measured as the retention factor Rf, the run
length of the compound divided by the run length of the eluent front:
 Distance moved by the compound
Rf = -----------------------------------------------------
Distance moved by the solvent

The retention of a compound often differs considerably between

experiments and laboratories due to variations of the eluent, the
stationary phase, temperature, and the setup. It is therefore
important to compare the retention of the test compound to that of
one or more standard compounds under absolutely identical
conditions.

Plate Theory
The plate theory of chromatography was developed by Archer John
Porter Martin and Richard Laurence Millington Synge. The plate
theory describes the chromotography system, the mobile and
stationary phases, as being in equilibrium. The partition coefficient K
is based on this equilibrium, and is defined by the following equation:

Concentration of the solute in stationary phase

K = ------------------------------------------------------------------
Concentration of the solute in mobile phase

K is assumed to be independent of concentration, and can change if

experimental conditions are changed, for example temperature is
increased or decreased. As K increases, it takes longer for solutes to
separate. For a column of fixed length and flow, the retention time (tR)
and retention volume (Vr) can be measured and used to calculate K.

Paper Chromatography
This is an older technique which involves placing a small spot of
sample solution onto a strip of chromatography paper. The paper is
dipped into a suitable solvent and placed in a sealed container. As the
solvent rises through the paper it meets the sample mixture which
starts to travel up the paper with the solvent. Different compounds in
the sample mixture travel different distances according to how
strongly they interact with the paper. This allows the calculation of an
Rf value and can be compared to standard compounds to identify an
unknown substance.

Thin Layer Chromatography

Thin layer chromatography (TLC) is a widely-employed laboratory
technique and is similar to paper chromatography. However, instead
of using a stationary phase of paper, it involves a stationary phase of a
thin layer of adsorbent like silica gel, alumina, or cellulose on a flat,
inert carrier. Compared to paper, it has the advantage of faster runs,
better separations, and the choice between different adsorbents.
Different compounds in the sample mixture travel different distances
according to how strongly they interact with the adsorbent. This
allows the calculation of an R f value and can be compared to standard
compounds to identify an unknown substance.

Column Chromatography
Fig. 6.1. A standard column chromatography and a flash column
chromatography setup

Column chromatography encompasses a number of techniques based

around utilizing a vertical glass column filled with some form of solid
support, with the sample to be separated placed on top of this
support. The rest of the column is filled with a solvent which, under
the influence of gravity, moves the sample through the column.
Similarly to other forms of chromatography, differences in rates of
movement through the solid medium are translated to different exit
times from the bottom of the column for the various elements of the
original sample.

In 1978, W. C. Still introduced a modified version of column

chromatography called flash column chromatography. The technique
is very similar to the traditional column chromatography, except for
that the solvent is driven through the column by applying positive
pressure. This allowed most separations to be performed in less than
20 minutes, with improved separations compared to the old method.
Modern flash chromatography systems are sold as pre-packed plastic
cartridges, and the solvent is pumped through the cartridge, with
automation.

Fast Protein Liquid Chromatography

Fast protein liquid chromatography (FPLC) is a term applied to
several chromatography techniques which are used to purify proteins.
Many of these techniques are identical to those carried out under high
performance liquid chromatography.

High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) is a form of column
chromatography used frequently in biochemistry and analytical
chemistry. The analyte is forced through a column (stationary phase)
by a liquid (mobile phase) at high pressure, which decreases the time
the separated components remain on the stationary phase and thus
the time they have to diffuse within the column. Specific techniques
which come under this broad heading are listed below. It should also
be noted that the following techniques can also be considered fast
protein liquid chromatography if no pressure is used to drive the
mobile phase through the stationary phase.

Ion Exchange Chromatography

Ion exchange chromatography is a column chromatography that uses
a charged stationary phase. It is used to separate charged compounds
including amino acids, peptides, and proteins. The stationary phase is
usually an ion exchange resin that carries charged functional groups
which interact with oppositely charged groups of the compound to be
retained. Ion exchange chromatography is commonly used to purify
proteins using FPLC.

Size Exclusion Chromatography

Size exclusion chromatography (SEC) is also known as gel permeation
chromatography or gel filtration chromatography and separates
particles on the basis of size. Smaller molecules enter a porous media
and take longer to exit the column, whereas larger particles leave the
column ealier. It is generally a low resolution chromatography and
thus it is often reserved for the final, "polishing" step of a purification.
It is also useful for determining the tertiary structure and quaternary
structure of purified proteins, especially since it can be carried out
under native solution conditions.

Affinity Chromatography
Affinity chromatography is based on selective non-covalent interaction
between an analyte and specific molecules. It is very specific, but not
very robust. It is often used in biochemistry in the purification of
proteins bound to tags. These fusion proteins are labelled with
compounds such as His-tags, biotin or antigens, which bind to the
stationary phase specifically. After purification, some of these tags are
usually removed and the pure protein is obtained.

Gas-Liquid Chromatography
Gas chromatography (GC) is based on a partition equilibrium of
analyte between a liquid stationary phase and a mobile gas. The
stationary phase is adhered to the inside of a small-diameter glass
tube (a capillary column) or a solid matrix inside a larger metal tube
(a packed column). It is widely used in analytical chemistry; though
the high temperatures used in GC make it unsuitable for biochemistry,
it is well suited for use in the petrochemical, environmental
monitoring, and industrial chemical fields. It is also used extensively
in chemistry research.
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