Classification of Chromatographic Method
Chromatographic methods can be categorized in two ways.
1.The first classification is based upon the physical means by which the stationary and mobile phases are brought into contact.
❖ In column chromatography, the stationary phase is held in a narrow tube through which the mobile phase is forced under pressure. Mobile phase moves by pressure or gravity.
fig. 1 : column chromatography
❖ In planar chromatography, the stationary phase is supported on a flat plate or in the interstices of a paper; here, the mobile phase moves through the stationary phase by capillary action or under the influence of gravity.
2.A second and more fundamental classification of chromatographic methods is one based upon the types of mobile and stationary phases and the kinds of equilibria involved in the transfer of solutes between phases.
Three general categories of chromatography:
(2) gas chromatography, and
The mobile phases in the three techniques are liquids, gases, and supercritical fluids respectively
Mobile Phase:
The Phase that travels through the column (gas or liquid) – transport sample through the column.
Stationary Phase:
Immiscible solid or liquid phase that fixed in place in the column or on a solid support – retain analytes within the column.
Band or Zone:
-Area across which analyte is distributed on column
-Zones of different analytes gradually separate as bands progress down column.
-Zones of different analytes gradually separate as bands progress down column.
LIQUID CHROMATOGRAPHY
There are several liquid chromatography techniques applied in food analysis, namely paper chromatography, thin layer chromatography (TLC) (both of these techniques may be referred to as planar chromatography), and column liquid chromatography, all of which involve a liquid mobile phase and either a solid or a liquid stationary phase. However, the physical form of the stationary phase is quite different in each case.
There are several liquid chromatography techniques applied in food analysis, namely paper chromatography, thin layer chromatography (TLC) (both of these techniques may be referred to as planar chromatography), and column liquid chromatography, all of which involve a liquid mobile phase and either a solid or a liquid stationary phase. However, the physical form of the stationary phase is quite different in each case.
COLUMN CHROMATOGRAPHY
In column liquid chromatography, the mobile phase is liquid and the stationary phase can be either solid or liquid supported by an inert solid. Having selected a stationary and mobile phase suitable for the separation problem at hand, the analyst must first prepare the stationary phase e (resin, gel, or packing material) for use according to the supplier’s instructions. The prepared stationary phase then is packed into a column (usually glass), the length and diameter of which are determined by the amount of sample to be loaded, the separation mode to be used, and the degree of resolution required. Longer and narrower columns usually enhance resolution and separation. Adsorption columns may be either dry or wet packed; other types of columns are wet packed. The most common technique for wet packing involves making a slurry of the adsorbent with the solvent and pouring this into the column. As the sorbent settles, excess solvent is drained off and additional slurry is added.
This process is repeated until the desired bed height is obtained. (There is a certain art to pouring uniform columns and no attempt is made to give details here.) If the packing solvent is different from the initial eluting solvent, the column must be thoroughly washed (equilibrated) with the starting mobile phase.
This process is repeated until the desired bed height is obtained. (There is a certain art to pouring uniform columns and no attempt is made to give details here.) If the packing solvent is different from the initial eluting solvent, the column must be thoroughly washed (equilibrated) with the starting mobile phase.
PAPER CHROMATOGRAPHY
Paper chromatography was introduced in 1944. In paper chromatography the stationary phase and the mobile phase are both liquid (partition chromatography). Paper generally serves as a support for the liquid stationary phase. The dissolved sample is applied as a small spot or streak one half inch or more from the edge of a strip or square of filter paper (usually cellulose), which is then allowed to dry. The dry strip is suspended ina closed container in which the atmosphere is saturated with the developing solvent (mobile phase), and the paper chromatogram is developed. The end closer to the sample is placed in contact with the solvent, which then travels up or down the paper by capillary action (depending on whether ascending or descending development is used), separating the sample components in the process. When the solvent front has traveled the length of the paper, the strip is removed from the developing chamber and the separated zones are detected by an appropriate method.
THIN LAYER CHROMATOGRAPHY
Thin-layer chromatography (TLC), first described in 1938, has largely replaced paper chromatography because it is faster, more sensitive, and more reproducible. The resolution in TLC is greater than in paper chromatography because the particles on the plate are smaller and more regular than paper fibers. Experimental conditions can be easily varied to achieve separation and can be scaled up for use in column chromatography, although thin-layer and column procedures are not necessarily interchangeable, due to differences such as the use of binders with TLC plates, vapor-phase equilibria in a TLC tank, etc. There are several distinct advantages to TLC: high sample throughput, low cost, the possibility to analyze several samples and standards simultaneously, minimal sample preparation, and that a plate may be stored for later identification and quantification.
GAS CHROMATOGRAPHY
Gas chromatography is a column chromatography technique, in which the mobile phase is gas and the stationary phase is either an immobilized liquid or a solid packed in a closed tube. GC is used to separate thermally stable volatile components of a mixture. Gas chromatography, specifically gas–liquid chromatography, involves vaporizing a sample and injecting it onto head of the column. Under a controlled temperature gradient, the sample is transported through the column by the flow of an inert, gaseous mobile phase. Volatiles are then separated based on several properties, including boiling point, molecular size, and polarity.
SUPERCRITICAL FLUID CHROMATOGRAPHY
SFC refers to chromatography performed above the critical pressure (Pc) and critical temperature (Tc) of the mobile phase. A supercritical fluid (or compressed gas) is neither a liquid nor a typical gas. The combination of Pc and Tc is known as the critical point. A supercritical fluid can be formed from a conventional gas by increasing the pressure, or from a conventional liquid by raising the temperature. Carbon dioxide frequently is used as a mobile phase for SFC; however, it is not a good solvent for polar and high molecular-weight compounds. A small amount of a polar, organic solvent such as methanol can be added to an on polar supercritical fluid to enhance solute solubility, improve peak shape, and alter selectivity. Other
supercritical fluids that have been used in food applications include nitrous oxide, trifluoromethane, sulfur hexafluoride, pentane, and ammonia. Supercritical fluids confer chromatographic properties intermediate to LC and GC. The high diffusivity and low viscosity of supercritical fluids mean decreased analysis times and improved resolution compared to LC.
supercritical fluids that have been used in food applications include nitrous oxide, trifluoromethane, sulfur hexafluoride, pentane, and ammonia. Supercritical fluids confer chromatographic properties intermediate to LC and GC. The high diffusivity and low viscosity of supercritical fluids mean decreased analysis times and improved resolution compared to LC.
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