PRINCIPLES OF CHROMATOGRAPHY

1. AFFINITY CHROMATOGRAPHY
The principle of affinity chromatography is that the stationary phase consists of a
support medium (e.g. cellulose beads) on which the substrate (or sometimes a
coenzyme) has been bound covalently, in such a way that the reactive groups that are
essential for enzyme binding are exposed. As the mixture of proteins is passed
through the chromatography column, those proteins that have a binding site for the
immobilised substrate will bind to the stationary phase, while all otter proteins will be
eluted in the void volume of the column.
2. ISOELECTRIC FOCUSING
The use of isoelectric focusing (IEF) is limited to molecules which can be either
positively or negatively charged. Proteins, enzymes and peptides are such amphoteric
molecules. The net charge of a protein is the sum of all negative and positive charges
of the amino acid side chains, but the three-dimensional configuration of the protein
also plays a role.
Isoelectric focusing takes place in a pH gradient. The charged molecules move
towards the anode or the cathode until they reach a position in the pH gradient where
their net charges are zero. This pH value is the "isoelectric point" (pI) of the
substance. Since it is no longer charged, the electric field does not have any influence
on it. Should the substance diffuse away, it will gain a net charge again, and the
applied electric field will cause it to migrate back to its pI. This concentrating effect
leads to the name focusing. Thus with IEF there is no problem with diffusion.

3. ION-EXCHANGE CHROMATOGRAPHY
Ion exchange chromatography, proteins are separated on the basis of their overall
(net) charge. If a protein has a net negative charge at PH 7, it will bind to a column
containing positively charged beads, whereas a protein with no charge or a net
positive charge will not bind. The negatively charged proteins bound to a such column
can then be eluted by washing the column with an increasing gradient(increasing
concentration ) of a solution of sodium chloride (Na+ Cl-ions) at the appropriate PH.
The chloride ions compete with the protein for negative charge elute first, followed by
those with a higher density of negative (DEAE) groups (such as DEAE-cellulose or
DEAE-Sphadex) are used for separation of negatively charged proteins (Anionic
proteins). Thia is called anion exchange chromatography. Columns containing
negatively charged carboxymethyl (CM) groups (Such as CM-cellulose or CM-
Sephadex) are used for the separation of positively charged proteins (cationic
proteins). This is called cation exchange chromatography.
4. GEL FILTRATION CHROMATOGRAPHY (GF) OR MOLECULAR
EXCLUSION CHROMATOGRAPHY
GF relies on a stationary phase consisting of spherical gel particles whose
size and porosity are carefully controlled during manufacture.
When the mobile phase is passed through a column of this material, molecules are
fractionated on the basis of their sizes and shapes. Small molecules (shown in blue
in the diagram on the left) are able to diffuse into the pores, while larger molecules
(shown in red) are excluded. This occurs repeatedly as the mobile phase moves
down the column, and as a result, during the elution process, the small molecules
are retarded with respect to the larger molecules. This causes the smaller molecules
to appear in the later fractions of the elute.
Size is not the only factor that influences the rate at which solutes are retarded. For
a given pore size, rod-like molecules will tend to be excluded with respect to
spherical molecules of the same size. In addition, certain molecules may have some
affinity for the stationary phase as a result of non-covalent interactions.
5. THIN LAYER CHROMATOGRAPHY (TLC)
Similar to other chromatographic methods TLC is also based on the principle of
separation. The separation depends on the relative affinity of compounds towards
stationary and mobile phase. The compounds under the influence of mobile phase (driven
by capillary action) travel over the surface of stationary phase. During this movement the
compounds with higher affinity to stationary phase travel slowly while the others travel
faster. Thus separation of components in the mixture is achieved.
Once separation occurs individual components are visualized as spots at respective level
of travel on the plate. Their nature or character are identified by means of suitable
detection techniques.
6. HPLC THEORY & PRINCIPLE
The principle involved in HPLC testing is separation of compounds in a mixture more
efficiently and also quickly than that of traditional column chromatography.
The separation of compounds is due to their relative differences in travel through the
column on application of pressure exerted through mobile phase or carrying liquid. The
compounds of the mixture travel with different rates due to their relative affinities with
the solvent and stationary phase. Compound with higher affinity towards stationary phase
of the column travels slowly and vice-versa.
The above principle is similar to that of column chromatography but in HPLC, The
separation is more effective due to greater surface area achieved due to very small
particle size of stationary phase in comparison to that used in column chromatography.
This decrease in particle size increases has disadvantage that it proportionately enhances
the flow time and run time due to increased surface area. To minimize this obstacle the
high pressure is applied to the flow of mobile phase through the column by use of pumps.
7. PAPER CHROMATOGRAPHY
Paper chromatography is a type of planar chromatography systems wherein a
cellulose filter paper acts as a stationary phase on which separation of compounds takes
place.
Principle of paper chromatography: The principle involved is partition
chromatography where in the substances are distributed or partitioned between to liquid
phases. One phase is the water which is held in pores of filter paper used and other phase
is that of mobile phase which moves over the paper. The compounds in the mixture get
separated due to differences in their affinity towards water (in stationary phase) and
mobile phase solvents during the movement of mobile phase under the capillary action of
pores in the paper


8. COLUMN CHROMATOGRAPHY
Column chromatography is the actual or the basic procedure of chromatography. From it
the principle of chromatography was described. Other types of chromatographies were
developed with column chromatography as a module. Column chromatography is a type
of adsorption chromatography techniques. Here stationary phase is a solid material
packed in a vertical column.
Principle:
When a mixture of mobile phase and sample to be separated are introduced from top of
the column, the individual components of mixture move with different rates. Those with
lower affinity to stationary phase move faster and eluted out first while those with greater
affinity move or travel slower and get eluted out last. The solute molecules adsorb to the
column in a reversible manner. The rate of the components is given as follows
R= Rate of movement of a component / Rate of movement of mobile phase. i.e. it is the
ratio of distance moved by solute to the distance moved by solvent.

9. PRINCIPLES OF NUCLEAR MAGNETIC RESONANCE (NMR)
The nuclei of all elements carry a charge. When the spins of the protons and neutrons
comprising these nuclei are not paired, the overall spin of the charged nucleus generates a
magnetic dipole along the spin axis, and the intrinsic magnitude of this dipole is a
fundamental nuclear property called the nuclear magnetic moment, . The symmetry of
the charge distribution in the nucleus is a function of its internal structure and if this is
spherical (ie analogous to the symmetry of a 1s hydrogen orbital), it is said to have a
corresponding spin angular momentum number of I=1/2, of which examples are
1
H,
13
C,
15
N,
19
F,
31
P etc. Nuclei which have a non-spherical charge distribution (analogous to e.g.
a hydrogen 3d orbital) have higher spin numbers (eg
10
B,
14
N etc) outside the scope of
this particular lecture course.
In quantum mechanical terms, the nuclear magnetic moment of a nucleus can align with
an externally applied magnetic field of strength Bo in only 2I+1 ways, either re-inforcing
or opposing Bo. The energetically preferred orientation has the magnetic moment aligned
parallel with the applied field (spin +1/2) and is often given the notation a, whereas the
higher energy anti-parallel orientation (spin -1/2) is referred to as b. The rotational axis of
the spinning nucleus cannot be orientated exactly parallel (or anti-parallel) with the
direction of the applied field Bo (defined in our coordinate system as about the z axis) but
must precess about this field at an angle (for protons about 54) with an angular velocity
given by the expression



10. CHROMATOGRAPHY
a technique for analysis of chemical substances. The term chromatography literally
means color writing, and denotes a method by which the substance to be analyzed is
poured into a vertical glass tube containing an adsorbent, the various components of the
substance moving through the adsorbent at different rates, according to their degree of
attraction to it, and producing bands of color at different levels of the adsorption column.
The term has been extended to include other methods utilizing the same principle,
although no colors are produced in the column.
The mobile phase of chromatography refers to the fluid that carries the mixture of
substances in the sample through the adsorptive material. The stationary phase (or
adsorbent) refers to the solid material that takes up the particles of the substance passing
through it. Kaolin, alumina, silica and activated charcoal have been used as adsorbing
substances or stationary phases.
Classification of chromatographic techniques tends to be confusing because it may be
based on the type of stationary phase, the nature of the adsorptive force, the nature of the
mobile phase, or the method by which the mobile phase is introduced.
The technique is a valuable tool for the research biochemist and is readily adaptable to
investigations conducted in the clinical laboratory. For example, chromatography is used
to detect and identify in body fluids certain sugars and amino acids associated with
inborn errors of metabolism.

(i). adsorption chromatography
that in which the stationary phase is an adsorbent.

(ii) affinity chromatography
a method of chromatography that utilizes the biologically important binding interactions
that occur on protein surfaces. For example, an enzyme substrate is covalently coupled to
an inert matrix such as a polysaccharide bead. The enzyme can be bound to the bead and
thereby separated when present in very low concentration in a very complex mixture of
other macromolecules.

(iii) column chromatography
the technique in which the various solutes of a solution are allowed to travel down a
column, the individual components being adsorbed by the stationary phase. The most
strongly adsorbed component will remain near the top of the column; the other
components will pass to positions farther and farther down the column according to their
affinity for the adsorbent. If the individual components are naturally colored, they will
form a series of colored bands or zones.
Column chromatography has been employed to separate vitamins, steroids, hormones and
alkaloids and to determine the amount of these substances in samples of body fluids.
(iv) exclusion chromatography
that in which the stationary phase is a gel having a closely controlled pore size.
Molecules are separated based on molecular size and shape, smaller molecules being
temporarily retained in the pores.
(v) gas chromatography
a type of chromatography in which the mobile phase is an inert gas. Volatile components
of the sample are separated in the column and measured by a detector. The method has
been applied in the clinical laboratory to separate and quantify steroids, barbiturates and
lipids.
(vi) gas-liquid chromatography
gas chromatography in which the substances to be separated are moved by an inert gas
along a tube filled with a finely divided inert solid coated with a nonvolatile substance;
each component migrates at a rate determined by its solubility in the stationary phase and
its vapor pressure.
(vii) gel-filtration chromatography, gel-permeation chromatography
exclusion chromatography.

(viii) high performance liquid chromatography (HPLC)
a miniaturized method in which the solution to be analyzed is passed, under high
pressure, through a long, thin column packed with tiny beads such that analyses are
completed in minutes rather than hours and with improved resolution.

(ix) ion-exchange chromatography
that utilizing resins to which are coupled either cations or anions that will exchange with
other cations or anions in the material passed through their meshwork.

(x) molecular sieve chromatography
exclusion chromatography.

(xi) paper chromatography
a form of chromatography in which a sheet of special paper is substituted for the
adsorption column. After separation of the components as a consequence of their
differential migratory velocities, they are stained to make the chromatogram visible. In
the clinical laboratory paper chromatography is employed to detect and identify sugars
and amino acids.

(xii) partition chromatography
a form of separation of solutes utilizing the partition of the solutes between two liquid
phases, namely the original solvent and the film of solvent on the adsorption column.


(xiii) thin-layer chromatography
that in which the stationary phase is a thin layer of an adsorbent such as silica gel coated
on a flat plate. It is otherwise similar to paper chromatography.

TLC CHROMATOGRAPHY OR THIN LAYER CHROMATOGRAPHY
Is a type of planar chromatography. TLC is routinely used by researcher in the field of
phyto-chemicals, biochemistry etc. to identify the components in a compound mixture like
alkaloids, phospholipids, amino acids etc..
It is a semi quantitative method of analysis and its sophisticated version or highly precise
quantitative version is High performance thin layer chromatography (HPTLC).
Thin layer chromatography principle:
Similar to other chromatographic methods TLC is also based on the principle of separation.
The separation depends on the relative affinity of compounds towards stationary and mobile
phase. The compounds under the influence of mobile phase (driven by capillary action)
travel over the surface of stationary phase. During this movement the compounds with
higher affinity to stationary phase travel slowly while the others travel faster. Thus
separation of components in the mixture is achieved.
Once separation occurs individual components are visualized as spots at respective level of
travel on the plate. Their nature or character are identified by means of suitable detection
techniques.
TLC chromatography System components:
TLC System consists of
a) TLC plates preferably ready made with stationary phase: These are stable and chemically
inert plates on to whose surface a thin layer of stationary phase is applied. The stationary
phase on the plates is of uniform thickness and consists of fine particle size.
b) TLC chamber: This is used for the development of TLC plate. The chamber maintains
uniform environment inside for proper development of spots. It also prevents the
evaporation of solvents and keep the process dust free.
c) Mobile phase: This comprises of a solvent or solvent mixture recommended for the
purpose. The mobile phase used should be particulate free and of highest purity for proper
development of TLC spots. The solvents recommended are chemically inert with the
sample, stationary phase.
d) A filter paper moistened in the mobile phase, to be placed inside the chamber. This helps
uniform rise in mobile phase over the length stationary phase.
TLC chromatography

Thin layer chromatography procedure:
The stationary phase is applied onto the plate uniformly and then allowed to dry and
stabilize. But now a days ready made plates are preferred.

A thin mark is made at the bottom of the plate with a pencil to apply the sample spots.
Then samples solutions are applied on the spots marked on the line at equal distances.
The mobile phase is poured into the TLC chamber to a level few centimeters above the
chamber bottom. A filter paper moistened in mobile phase is placed on the inner wall of the
chamber to maintain equal humidity in the entire chamber and there by avoid edge effect.
Then the plate prepared with sample spotting is placed in TLC chamber such that the side of
the plate with sample line is towards the mobile phase. Then the chamber is closed with a
lid.
The plate is immersed such that sample spots are well above the level of mobile phase but
not immersed in the solvent as shown in the picture for development.
Allow sufficient time for development of spots. Then the plates are removed and allowed to
dry. The sample spots are visualized in suitable UV light chamber or any other methods as
recommended for the said sample.
TLC chromatography video demo
Advantages of TLC
The Thin layer chromatography advantages include:
It is simple process with short development time.
It helps in visualization of separated compound spots easily.
The method helps to identify the individual compounds.
It helps in isolation of most of the compounds.
The separation process is faster and the selectivity for compounds is higher (even small
differences in chemistry is enough for clear separation.
The purity standards of the given sample can be assessed easily.
It is a cheaper chromatographic technique.
Applications of Thin layer chromatography
1. To check purity of given samples.
2. Identification of compounds like acids, alcohols, proteins, alkaloids, amines, antibiotics
etc.
3. To evaluate reaction process by assessment of intermediates, reaction course etc.
4. To purify samples i.e for purification process.
5. To keep a check on the performance of other separation processes.
Being a semi quantitative technique, tlc chromatography is used for rapid qualitative
measurements than for quantitative purposes. But due its rapidity of results, easy handling
and inexpensive procedure, it finds its application as one of the most widely used
chromatography techniques