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CHROMATOGRAPHIC MECHANISMS
The process whereby a solute is transferred from a mobile phase to a stationary phase. Four sorption mechanisms
Adsorption Partition Ion-Exchange Size-Exclusion
SURFACE ADSORPTION
Based on the relative polarities of solute, stationary phase and mobile phase. Components get distributed according to their relative affinity. Components having high affinity towards the stationary phase- Travel slower Components having less affinity towards the stationary phase- Travel faster No two components have the same affinity for a combination of SP, MP, and other conditions.
PARTITION
Based on the relative solubility of the solute in the two phases. (Volatility if the mobile phase is gas). Solutes will be distributed according to their partition coefficients. Components which are more soluble in the stationary phase- Travel slower Components which are less soluble in the stationary phase- Travel faster No two components have the same partition coefficient for a particular combination of Stationary phase, Mobile phase, and other conditions.
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ION EXCHANGE
Based on the relative ion exchange capacity of the solute in the two phases. Stationary Phase contain fixed charged groups and mobile counter ions. Counter ions exchange with ions of the solute. Reversible exchange of ions takes place between similar charged ions of solute in the mobile phase and that of an ion exchange resin. Cation exchange resin used for separation of Cations. Anion exchange resin for anion separation.
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Choice of methods
The choice of a particular mechanism depends on following factors
The complexity of sample Solubility and volatility of sample Chemical and physical properties of the sample Resolution required Required separation efficiency, concentration of analyte Detection limit Number of samples under analysis
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Preparative Chromatography
The Chromatogram
Wh
Wb
tR = retention time: is the time required for the analyte to reach the detector, after the sample injection. tM = void time: is the time required for a non retained species to reach the detector. It is also known as Dead time Wb = baseline width of the peak in time units Wh = half-height width of the peak in time units
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Peak shapes
A tailing peak will have a TF greater than one. The opposite symmetry, fronting will yield a TF less than one. When the asymmetry factor lies outside the range of 0.95-1.15, it reduces the column efficiency and resolution. For example, an AF of 1.3 reduces efficiency by 69% and resolution by 30%.
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Theory
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The relationship governing the column efficiency is N=L/H . Efficiency of a column can be explained with the help of plate theory.
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Plate numbers can be from a few hundreds to several hundred thousands. Plate heights can be from a few tenths to 1/1000th of a cm or even lesser.
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Wh
Wb
Band broadening
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Peak Broadening
The magnitude of kinetic effects on column efficiency depends upon the length of time the mobile phase is in contact with the stationary phase, which in turn depends upon the flow rate of the mobile phase .
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CS and CM are the mass transfer coefficients for the stationary and mobile phases respectively.
u is the mobile phase flow velocity.
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The degree of band-broadening due to stationary phase mass transfer depends on: 1) the retention and diffusion of the solute 2) the flow-rate of the solute through the column 3) the kinetics of interaction between the solute and the stationary phase
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The degree of band-broadening due to mobile phase mass transfer depends mainly on: 1) the size of the packing material 2) the diffusion rate of the solute
It is proportional to the square of the particle diameter dp2 and inversely proportional to the diffusion coefficient DM of the solute in the mobile phase. Decreasing the size of the stationary phase particles is always helpful in decreasing the plate height.
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How can band broadening be reduced? (and thus column efficiency be enhanced)
Minimum value for H is achieved by: Decreasing the particle diameter Decreasing the column width Minimizing thickness of liquid stationary phase Lowering the temperature (reduces diffusion coefficient) Using mobile phases having low viscosity and high diffusion coefficient Using stationary phase having low viscosity and high diffusion coefficient in GC and partition.
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3. Temperature.
High temperature enhances rate of elution with less resolution. Low temperature affords higher resolution Separations carried out at 202C
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4. Pore diameter.
Pore diameter of 20A affords effective polar adsorption.