PURIFICATION AND ANALYSIS OF

BIOMOLECULES CHROMATOGRAPHY
 The primary goal of biochemical research
is to understand the molecular nature of life
processes.

• The molecular details of a biological

process cannot be fully elucidated until the
interacting molecules have been isolated and
characterized.
Chromatography
INTRODUCTION TO CHROMATOGRAPHY
 Chromatography;

 has developed into the ultimate tool for not only the isolation
and purification, but also for the characterization, of
biomolecules.
 has now been expanded into multiple forms, continues to be
the most effective technique for separating and purifying all
types of biomolecules.
 widely used as an analytical tool to measure biophysical and
other quantitative properties of molecules.
 Chromatography is a technique for separating
mixtures into their components in order to analyze,
identify, purify, and/or quantify the mixture or
components.

• Analyze
Separate • Identify
• Purify
• Quantify
Mixture Components
 Uses for Chromatography
Chromatography is used by scientists to:
Analyze – examine a mixture, its components, and their
relations to one another
Identify – determine the identity of a mixture or
components based on known components
Purify – separate components in order to isolate one of
interest for further study
Quantify – determine the amount of the a mixture and/or
the components present in the sample
Uses for Chromatography
Real-life examples of uses for chromatography:
Pharmaceutical Company – determine amount of each
chemical found in new product
Hospital – detect blood or alcohol levels in a patient’s blood
stream
Law Enforcement – to compare a sample found at a crime scene
to samples from suspects
Environmental Agency – determine the level of pollutants in
the water supply
Manufacturing Plant – to purify a chemical needed to make a
product
 All types of chromatography are based on a very simple
concept:
 The sample to be examined is allowed to interact with two
physically distinct entities
1. mobile phase
2. stationary phase
 Chromatographic methods
(according to how analytes bind to or interact with the stationary phase)

Partition chromatography Adsorption chromatography

(distribution of an analyte between two phases is

based primarily on solubility differences) relies on relatively specific interactions
between the analytes and binding sites on the
•Paper chromatography surface of the sorbent.
•Thin-layer chromatography
•Gas-liquid chromatography •Coloum chromatography
•Ion-exchange chromatography
Partition chromatography
 The distribution of analytes between the two phases is based
primarily on solubility differences.

 The distribution may be quantified by using the partition

coefficient, KD

 most effective for the separation of small molecules, especially for

separation and identification of amino acids, carbohydrates,
and fatty acids.
 Adsorption chromatography

 Adsorption chromatography relies on relatively specific

interactions between the analytes and binding sites on the
surface of the sorbent.
 The attractive forces between analyte and support may be
ionic, hydrogen bonding, or hydrophobic interactions.
 Binding of analyte is, of course, reversible.
 Most effective when applied to the separation of
macromolecules including proteins and nucleic acids.
 Chromatographic methods
(according to the type of mobile phase used in the system)

Gas chromatography Liquid chromatography

Mobil phase = gas Mobil phase = liquid

 Chromatographic methods

(according to the on the type of support material used in the system)

Packed bed (column) Open tubular (capillary)

Open bed (planar)

Paper Chromatography
Principles of Paper Chromatography
The principle of separation in paper
chromatography is partition chromatography.
 Capillary Action – the movement of liquid within the spaces of
a porous material due to the forces of adhesion, cohesion, and
surface tension. The liquid is able to move up the filter paper
because its attraction to itself is stronger than the force of
gravity.

 Solubility – the degree to which a material (solute) dissolves

into a solvent. Solutes dissolve into solvents that have similar
properties. (Like dissolves like) This allows different solutes
to be separated by different combinations of solvents.
Separation of components depends on both their
solubility in the mobile phase and their differential
affinity to the mobile phase and the stationary phase.

The main difference of paper chromatography is that a

sheet of paper is used for the inert phase.
The degree of retention of a component is called the
retardation factor (Rf)
= distance migrated by an analyte (Da)
distance migrated by the solvent (Ds)
Good resolution is resulted when Rf is about 0.4 to 0.8.
 Ascending
Technique

In a chromatographic chamber, the

solvent rises up the paper by
capillary action and allows a
separation of the components as it
ascends. Very simple equipments are
required. However, the solvent will
rise only 20 to 25 cm and separations
of the analytes are limited.
 Descending
Technique
In a chromatographic chamber,
a paper strip is hanged
vertically with the spotted end
up. The solvent rises up the
wick and descends across the
spot and down the paper. This
technique permits a separation
over a longer distance and
allows an increase in
resolution.
Thin Layer Chromatography
Thin Layer Chromatography
Here the mobile phase is a liquid

Flowing past a thin layer of powder on a solid

support (silica gel, alumina).
Substances that are less attracted to the solid or
are more soluble in the liquid move faster.

And so move further up the plate by the time that

the process has been stopped by taking the plate
out of the liqiud. - larger Rf
Rf = distance moved by substance
distance moved by solvent front

For substances that are very soluble in the

liquid Rf will be close to ....

1
For substances that are rather insoluble in
the liquid Rf will be close to ....

0
Two-Dimensional Technique
A solution of interested is subjected to two separation processes in
two-dimensional technique. A sample solution is spotted in one
corner of a square or rectangular sheet of paper or Thin Layer
plate.

One solvent is applied to

separate the analytes in
one direction. After the
paper/TL is dried and
turned 90o, a second
solvent is used. High
degree of separation of
the analytes can be
achieved.
Column Chromatography
Column chromatography,

 in which the stationary phase is confined to a glass, metal

column or plastic tube and the mobile phase (a solvent or
buffer) is allowed to flow through the solid adsorbent either
by gravity feed or pressure.
 A small amount of the sample to be analyzed is layered on top
of the column.
 The sample mixture enters the column of adsorbing material
and the molecules present are distributed between the mobile
phase and the stationary phase.
 The various components in the sample have different affinities
for two phases and move through the column at different rates.
 Collection of the liquid phase emerging from the column yields
separate fractions containing the individual components in the
sample.
 Chromatographic Equipment

Pump: to give an even flow of liquid

Column: in which separation occurs
Dedector: to provide a continual measurement of a physical
parameter of the eluent
Recorder: to give a continious visual read ot of the dedector
out-put
Fraction collector: to separate the column eluent into
samples
Operation of a Chromatographic Column
The heart of the system is, of course, the column of adsorbent

The most suitable forms are dry powders or a slurry form of the material in an
aqueous buffer or organic solvent…
 Adsorbing materials come in various forms and sizes.
For most biochemical applications, 100 to 200 mesh size is suitable.

*The size of particles in an adsorbing material is defined by mesh size. This refers to a standard sieve
through which the particles can pass.
 In general, the longer the column, the better the resolution
of components.
 Flow rate decreases with increasing column length.
 The actual size of a column depends on the nature of
the adsorbing material and the amount of chemical
sample to be separated.
 For preparative purposes, column heights of 20 to 50 cm are
usually sufficient to achieve acceptable resolution.
Packing the column
 Once the adsorbing material and column size have been
selected, the column is poured.
 Most columns are packed by pouring a slurry of the sorbent
into the tube and allowing it to settle by gravity into a tight
bed. The slurry is prepared with the solvent or buffer that
will be used as the initial developing solvent.
 Sometimes it is necessary to pack a column under pressure
Loading the column
 The sample to be analyzed by chromatography should be
applied to the top of the column in a concentrated form.

 If the sample is solid, it is dissolved in a minimum amount of

solvent;

 If already in solution, it may be concentrated by

ultrafiltration
Eluting the column
 Adsorption columns are eluted in one of three ways:
1. Continual elution (isocratic elution): All components may
be eluted by a single solvent or buffer.
2. Stepwise elution: the column is first eluted with a volume
of one solvent (polar) and then second solvent (greater
polarity or ionic strenght).
3. Gradient elution: the concentration of a component in
thesolvent can be gradually increased (by addition of a salt
(KCl, NaCl, etc.)
Collecting the eluent

 The separated components emerging from the column in the

eluent are usually collected as discrete fractions.
 If many fractions are to be collected, a mechanical fraction
collector is convenient
Detection of Eluting Components
 The detection method used will depend on the nature of the
analytes.
 Smaller molecules such as lipids, amino acids and
carbohydrates can be detected by TLC or PC.
 Proteins and nucleic acids are conveniently detected by
spectroscopic absorption measurements at 280 nm and 260
nm.
 Enzymes can be detected by measurement of catalytic
activity.
 Most often the eluent is directed through a flow cell where
absorbance or fluorescence characteristics can be measured.

 The detector is connected to a recorder or computer for a

permanent record of spectroscopic changes.
Column
Chromatography

Chromatogram
Typical response obtained by chromatography
chromatogram - concentration versus elution time

Wh

Wb

Inject
Where:
tR = retention time, tM = void time
Wb = baseline width of the peak in time units
Wh = half-height width of the peak in time units
 Measures of Solute Separation:
separation factor () – parameter used to describe how well two
solutes are separated by a chromatographic system:

= k’2/k’1
k’ = (tR –tM)/tM

k’1 = the capacity factor of the first solute

k’2 = the capacity factor of the second solute

A value of a ~ 1.1 is usually indicative of a good separation

resolution (RS) – resolution between two peaks is a
second measure of how well two peaks are separated:

RS = tr2 – tr1
(Wb2 + Wb1)/2
where:

tr1, Wb1 = retention time and baseline width for the first
eluting peak

tr2, Wb2 = retention time and baseline width for the second
eluting peak
 Rs is preferred over
a since both
retention (tr) and
column efficiency
(Wb) are considered
in defining peak
separation.

Rs ~ 1.5 represents baseline resolution, or complete separation of two neighboring

solutes  ideal case.
Rs ~ 1.0 considered adequate for most separations.
 Factors affecting separation:
Factors due to Stationary Phase:

1- Particle size of the stationary phase: Reducing the particle size

increases the surface area and improve separation. However,
reduction of the particle size will decrease the flow rate of the
mobile phase.
2- Adsorbent activity: The choice of the suitable adsorbent is
very important.

3- Uniformity if packing of the column: If the stationary phase is

not packed uniformly then the bands will be irregular and
less uniform resulting in poor separation.

4- Concentration of the mixture: the proper ratio between

sample to be separated and the amount of stationary phase
is very important too much samples resulted in bad
separation.
 Factors due to Mobile Phase:
1- Selection of the proper mobile phase: Very polar mobile phase will wash out
all components without any separation. On the other hand very non polar
mobile phase will result in broad band and poor separation.

2- Rate of flow: Slower flow rate usually resulted in a better separation and
narrower bands.

3- Consistency of flow: The continuous flow of the mobile phase during the
whole experiment gives better separation than interrupting the flow then
continue it later.
 Factors due to Columns:
 Column dimensions: Increasing the length of the column
improve separation. However, that usually leads to slower
flow rate. Also increasing the column length some times is
impractical.

 Column temperature: Increasing the temperature usually

reduces the adsorption power of the stationary phase and
increase elution speed. This may leads to decrease in the
efficiency separation.
 GENERAL FACTORS INCREASING RESOLUTION

1. Increase column length

2. Decrease column diameter
3. Decrease flow-rate
4. Pack column uniformly
5. Use uniform stationary phase (packing material)
6. Decrease sample size
7. Select proper stationary phase
8. Select proper mobile phase
9. Use proper pressure
10. Use gradient elution