Packed - As suggested by the term, it is filled with a coated inert solid support such as fire brick, alumina, and graphite with a specific mesh size. The coatings are called phases and for best results are chemically bonded to the support. Chemical bonding provides for longer column life and less bleeding (major source of background noise) contributing to lower sensitivity. Column dimensions 1/8” - 1/4” ID x up to about 6’ using glass or stainless steel. Advantages - higher capacity (higher conc). Disadvantages: low resolution and low S/N. Capillary - Here the phase (film) is coated on the inside diameter of the capillary wall with film thickness range of 0.1 to 5μ where the ticker film provides for better resolution but also allows for more bleed. Typical dimensions .25mm - .53mm ID x up to 60m made of fused silica coated with polyamide. Advantages: high resolution and better S/N. Disadvantages: low capacity and cost.
Note peaks 15, 16 17 & 18 on the DB-5 column and note the same peaks on the DB-1701 column. This shows the need for confirmatory columns (columns with different phases) so that separation of the compounds can be verified.
The adsorbent is a sheet of paper of suitable texture and thickness
Development may be ascending in which case the solvent is carried up the paper by capillary forces, or descending , in which case the solvent flow is also assisted by gravitational force.
C- Thin-layer chromatography Separation is based on migration of the sample spotted on a coated (stationary phase) plate with one edge dipped in a mixture of solvents (mobile phase). However, it is not usually as accurate or sensitive as liquid chromatography.
LLC: the column consists of an inert support usually silica gel on which the stationary partitioning phase is adsorbed .
In the normal phase mode , the stationary phase is polar (e.g. methanol, acetonitrile or water) while the mobile phase is less polar (e.g. iso-octane, chloroform or n-hexane). This mode is usually used for the separation of polar components.
In the reverse phase LLC , the stationary phase is less polar and the mobile phase is polar. It is usually used for the separation of non-polar components.
LSC : The packing may be silica (polar packing) or octadecylsilica, ODS (C 18 -silica, non-polar packing). Adsorption mechanism is involved here.
In the normal phase LSC, the packing is polar (silica) and the mobile phase is less polar (e.g. n-hexane).
In the reverse phase LSC , the packing is non-polar (eg. ODS) and the mobile phase is polar (e.g. acetonitrile-water or methanol-water).
Again, as under LLC, normal phase LSC is used for polar solutes while reverse phase LSC is used for separation of non-polar compounds.
F) SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) A supercritical fluid: is a substance above its critical temperature and pressure. Critical temperature (T c ): is that above which it is impossible to liquefy a gas, no matter how great a pressure is applied. Critical pressure (P c ): is the minimum pressure necessary to bring about liquefaction at T c . Critical volume (V c ): is the volume occupied by one mole of gas or liquid at the critical temperature and pressure. SFC , is a column chromatographic technique in which a supercritical fluid is used as a mobile phase .The used mobile phase is frequently cooled to be maintained in a liquid state for easier pumping to the column. Carbon dioxide is the most frequently used mobile phase .Other mobile phases include ammonia, nitrous oxide , and xenon .SFC ,is an intermediate between GC and HPLC and offers the advantages of both .
Compound T c , °C P c , atm. Carbon dioxide 31.05 72.9 Nitrous oxide 36.4 71.5 Ammonia 132.4 111.3 2-Propanol 235.1 47.6 Methanol 239.4 79.9 Acetonitrile 274.8 47.0 Water 374.1 217.6
Advantage of supercritical fluids as mobile phases in chromatography compared with liquid chromatography is that
solutes generally have much higher diffusion coefficient in them than in liquids. This leads to enhanced speed of separation and possibly greater resolution with complex mixtures, especially for large molecules.
SFC possesses also advantages over GC in that solutes do not have to be volatile or thermally stable .
C) ELECTROPHORESIS (Electrochromatography) Introduction : Electrophoresis is a technique in which solutes are separated by their different rates of travel through an electric field. - commonly used in biological analysis, particularly in the separations of proteins, peptides and nucleic acids The rate of migration (electrophoretic mobility) of each species is a function of its charge, shape and size .
– another type of zone electrophoresis – It involves high voltage electrophoresis in narrow bore fused-silica capillary tubes and on-line detectors similar to those used in HPLC. - On passing through the detector, they produce response profiles that are sharper than chromatographic peaks. High Performance Capillary Electrophoresis (HPCE):