CHROMATOGRAPHY
Part 2
Column Chromatography
** It is a common chromatographic technique.
** It is a type of adsorption chromatography that is widely used for the separation of individual
components of interest present in mixture.
** It is a separation technique in which the stationary bed is within a tube.
** Adsorbents (stationary phase) are packed into a column in a glass tube leaving an open
unrestricted path for the mobile phase in the middle of the tube.
** Adsorbents such as silica gel, alumina, charcoal powder & calcium hydroxyapatite are used
**The sample mixture in a solvent is loaded on this column.
**The individual compounds get differentially adsorbed on to the adsorbent.
** The individual compounds come out of the column at different rates which may
be collected separately & identified.
Advantages
1) It is simple and easy technique. •
2) Mixture of any type or quantity can be easily separated •
using this technique.
3) Many types of solvent/mobile phase can be used in this •
technique.
4) It is an inexpensive technique. •
5) This technique can be used both on small and on large •
scale.
Disadvantages
1. It is a time consuming process, especially when
components show colorless bands.
2. A large amount of the solvent is required for the
proper 21 elusion.
Partition Chromatography
• This form of chromatography is based on a thin film formed on the
surface of a solid support by a liquid stationary phase.
• Separation of components of a sample mixture occurs due to
differences in partition coefficients.
• Partition is the distribution of a dissolved substance (solute) between
two immiscible fluids.
• Molecules will partition into the stationary phase based on their
affinity towards the stationary phase and eventually partition into the
mobile phase again.
Ion Exchange Chromatography
** It involves the separation of molecules on the basis of
their electrical charges. • Resins-cation exchangers &
anion exchangers are used.
** Ion-exchange chromatography is used for the
separation of either cations or anions.
** It is used for the separation of almost any kind of
charged molecule including large amino acids, small
nucleotides, and proteins.
Size-Exclusion Chromatography
** It is also referred to as molecular sieving or molecular exclusion
chromatography.
** The phenomenon of separation of the components of interest present
in the sample mixture is based on the size of individual component .
**Smaller molecules are able to enter the pores of the media and,
therefore, molecules are trapped and removed from the flow of the
mobile phase.
**However, molecules that are larger than the average pore size of the
packing are excluded and thus suffer essentially no retention; such
species are the first to be eluted.
Paper chromatography
** It is one of the oldest and simplest methods of
chromatography.
** Chromatography paper is a kind of filter paper which acts as
stationary phase in the chromatographic system.
** It is involves placing a small dot or line of sample solution onto
a strip of chromatography paper.
** The paper is placed in a jar containing a shallow layer of solvent
and sealed.
** As the solvent rises through the paper, it meets the sample
mixture, which starts to travel up the paper with the solvent.
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 glass plate .
** For spotting the sample and standard solution on TLC plate,
capillary tube or micro syringe is used.
** The analyte can be visually detected easily If the analyte is
colored.
• But for the detection of colorless analyte on TLC plate, two types of techniques
are used:
• Destructive Technique Specific reagent, e.g., ninhydrin and stannus chloride is
used in the form of spray on TLC slide.
• Non-destructive Technique Different methods are used, the detection of
radioactive materials, Geiger Muller counter is used, whereas for the detection
of fluorescent compounds, UV chamber is used ..
Advantages
1. TLC is a cheaper chromatographic technique.
2. The separation process is faster.
3. TLC is a simple process because of its short
development time.
4. TLC helps to identify the individual components
easily.
Disadvantages
1. By TLC, only qualitative analysis is possible.
2. Stationary phase in TLC is no longer stable.
3. It cannot differentiate between some isomers
Gas-liquid Chromatography
** It is a process of separation of the components of the given
sample by using a gaseous mobile phase.
** It is the method of choice for the separation of volatile
substances .
** Stationary phase is an inert solid material impregnated with a
non-volatile liquid.
** In gas chromatography, a sample is rapidly heated and
vaporized at the injection port. The sample is transported through
the column by a mobile phase consisting of an inert gas.
** Sample components are separated based on their
boiling points and relative affinity for the stationary
phase, which is most often a viscous liquid (wax) within
the column.
** The higher a component's affinity for the stationary
phase, the slower it comes off the column. The
components are then detected and represented as peaks
on a chromatogram
