PLANAR CHROMATOGRAPHY
• Planar chromatographic methods include thin-layer chromatography (TLC) and paper
chromatography (PC).
• Each makes use of a flat, relatively thin layer of material that is either self-supporting or is coated on
a glass, plastic, or metal surface.
• The mobile phase moves through the stationary phase by capillary action, sometimes assisted by
gravity or an electrical potential.
• Currently, most planar chromatography is based on the thin-layer technique, which is faster, has better
resolution, and is more sensitive than its paper chromatography equivalent
THIN-LAYER CHROMATOGRAPHY
• Thin-layer chromatography has found widespread use in clinical laboratories and is the backbone of
many biochemical and biological studies.
• Typical thin-layer separations are performed on a glass plate coated with a thin and adherent layer of
finely divided particles; this layer constitutes the stationary phase.
TWO-DIMENSIONAL PLANAR CHROMATOGRAPHY
• the separation of amino acids in a mixture by development in two
dimensions.
• The sample was placed in one corner of a square plate and
development was performed in the ascending direction with
solvent A.
• This solvent was then removed by evaporation, and the plate was
rotated 90°, following which an ascending development with
solvent B was performed.
• After solvent removal, the positions of the amino acids were
determined by spraying with ninhydrin, a reagent that forms a
pink-to-purple product with amino acids.
SIZE-EXCLUSION CHROMATOGRAPHY
• Size-exclusion, or gel, chromatography is a powerful technique that is particularly applicable to high-
molecular-mass species
• Packings for size-exclusion chromatography consist of small (,10 μm) silica or polymer particles
containing a network of uniform pores into which solute and solvent molecules can diffuse
• While in the pores, molecules are effectively trapped and removed from the flow of the mobile phase
• Molecules 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
• Molecules having diameters significantly smaller than the pores can penetrate or permeate throughout
the pore maze and are thus entrapped for the greatest time; these are last to be eluted.
COLUMN PACKINGS
• Two types of packing for size-exclusion chromatography are encountered: polymer beads and silica-
based particles, both of which have diameters of 5–10 μm.
• Silica particles have the advantages of greater rigidity, which leads to easier packing and permits the
use of higher pressures; greater stability, which permits the use of a wider range of solvents, including
water; more rapid equilibration with new solvents; and stability at higher temperatures.
• The disadvantages of silica-based particles include their tendency to retain solutes by adsorption and
their potential for catalyzing the degradation of solute molecules.
• hydrophilic gels are available, making possible the use of aqueous solvents for the separation of large,
water-soluble molecules such as sugars
ION CHROMATOGRAPHY
• Ion chromatography refers to modern, efficient methods for separating and determining ions on
columns with relatively low ion-exchange capacity.
• Ion chromatography was an outgrowth of ion-exchange chromatography,
• Principle:- reversible exchange of ions between the target ions present in the sample solution to the
ions present in the ion exchange
• The most common active sites for cation-exchange resins are the sulfonic acid group SO3-H+ a strong
acid, and the carboxylic acid group COO-H+, a weak acid.
• Anionic exchangers contain strongly basic tertiary amine groups N(CH3)3+OH- or weakly basic
primary amine groups NH3+OH-
Cation exchange:- for the separation of cations
• When a sulfonic acid ion-exchanger is brought in contact with an aqueous solvent containing a cation
Mx+, an exchange equilibrium is set up that can be described by
• where RSO3-H+ represents one of many sulfonic acid groups attached to a large polymer molecule.
Anion exchange:- for the separation of anions
• Similarly, a strong-base exchanger interacts with the anion Ax- as shown by the reaction
INSTRUMENTATION
ELUENT PUMB INJECTOR COLUM SUPPRESSOR DETECTOR
INJECTOR
• 6 pot injector
• Two positions ; load position and Inject position
SUPRESSOR
• The high conductance of eluents was solved by the introduction of an eluent suppressor column
immediately following the ion-exchange column
• The suppressor column is packed with a second ion-exchange resin that effectively converts the ions
of the eluting solvent to a molecular species of limited ionization without affecting the conductivity
due to analyte ions
• When cations are being separated and determined, hydrochloric acid is chosen as the eluting reagent,
and the suppressor column is an anion-exchange resin in the hydroxide form. The product of the
reaction in the suppressor is water. That is
• For anion separations, the suppressor packing is the acid form of a cation-exchange resin and sodium
bicarbonate or carbonate is the eluting agent. The reaction in the suppressor is
CATRIDGE BASED SUPRESSOR
• It consist of Three catridges
1. Analyse mode
2. Regeneration Mode
3. Washing mode
• It can be rotate internally
