Solution chromatography

FIGURE 5.4 Effect of the gradient dwell volume, V7>. the elution volume, Vj, in reversed-phase chromatography. Solute neburon, retention equation (Equation 5.7) with parameters a=A, m = 4. Linear gradients 2.125% methanol/min (a) from 57.5% to 100% methanol in water in 20min ( i = 50) (b) from 75% to 100% methanol in water in 11.75 min (k = 10). Vg uncorrected calculated from Equation 5.8, Vg + Vg, Vg, added to Vg uncorrected, Vg corrected calculated from Equation 5.21. (A) A conventional analytical C18 column, hold-up volume y ,= ImL flowrate l.OmL/min. (B) A microbore analytical C18 column, hold-up volume y = 0.1mL flow rate 0.1 mL/min. 

Adsorption chromatography. This chromatographic technique is best known because of its use in the last century as a preparative method of separation. Stationary phases have made a lot of progress since Tswett. who used calcium carbonate or sugar. The separation of organic compounds on a thin layer of silica gel or alumina with solvent as a mobile phase are examples of this type of chromatography. Solutes bond to the stationary phase because of physisorption or chemisorption interactions. The physico-chemical parameter involved is the coefficient of adsorption. 

Figure 23-5 The idea behind chromatography Solute A. with a greater affinity than solute B for the stationary phase, remains on the column longer.

Partition chromatography. A liquid stationary phase is bonded to a solid surface, which is typically the inside of the silica (Si02) chromatography column in gas chromatography. Solute equilibrates between the stationary liquid and the mobile phase, which is a flowing gas in gas chromatography. 

Ion-exchange chromatography. Anions such as —S03 or cations such as —NfCH I are covalently attached to the stationary solid phase, usually a resin, in this type of chromatography. Solute ions of the opposite charge are attracted to the stationary phase by electrostatic force. The mobile phase is a liquid. 

In high-pressure adsorption chromatography, solutes adsorb with different affinities to binding sites in the solid stationary phase. Separation of solutes in a sample mixture occurs because polar solutes adsorb more strongly than nonpolar solutes. Therefore, the various components in a sample are eluted with different retention times from the column. This form of HPLC is usually called normal phase (polar stationary phase and a nonpolar mobile phase). 

Zafiron and Ohver [494] have developed a method for characterising environmental hydrocarbons using gas chromatography. Solutions of samples containing oil were separated on an open tubular column (15.2m x().()5cm) coated with OV-101 and temperature programmed from 75 to 275°C at 6°C per minute helium (50mL min-1) was used as carrier gas and detection was by flame ionisation. 

Adsorption chromatography The process can be considered as a competition between the solute and solvent molecules for adsorption sites on the solid surface of adsorbent to effect separation. In normal phase or liquid-solid chromatography, relatively nonpolar organic eluents are used with the polar adsorbent to separate solutes in order of increasing polarity. In reverse-phase chromatography, solute retention is mainly due to hydrophobic interactions between the solutes and the hydrophobic surface of adsorbent. Polar mobile phase is used to elute solutes in order of decreasing polarity. 

GEL CHROMATOGRAPHY Solute-matrix interactions and their application Gel chromatography in organic solvents New media for gel chromatography New column designs... 

Gas chromatography Solution and emulsion polymerization systems (VAc/BA, all acrylics, BA/St, St/AN,.,.) [87-90] Ring-opening polymerization [91] and polyolefin gas-phase polymerization [92] Direct measurement of concentrations/Invasive, non-robust in industrial environment, requires sampling and dilutions loops or head-space (equilibrium parameters required) All polymerization techniques... 

CPC/SEC (gel permeation chromatography/size exclusion chromatography) Solution polymerization [95, 96, 102, 108, 109] The whole MWD obtained/ Invasive, non-robust, sampling loop required, large delay times (30 min to 1 h) Homogenous polymerization systems (free-radical / step-growth)... 

MLC is a good example of the use of secondary equilibria in liquid chromatography. Solutes are partitioned between the mobile and stationary phases,... 

Figure 6 Detection of thermal aggregation of jS-lactoglobulin by gel-permeation chromatography. Solutions of 8-lactoglobulin (3.8 mg ml" ) in 50 mmol I" phosphate, pH 6.8, were heated for 15 min at the temperatures given in the figure (°C). Aliquots of the clear solutions of the heated protein were run on a Superdex column in 50 mmol I" phosphate, 0.1 moll" NaCI, pH 7.0.

Immobilized metal-ion affinity chromatography solutions Buffer A 0.1 M acetic acid. To make 10 ml, add 57.5 1 glacial acetic acid to high-purity water and adjust to 10 ml. 

For planar chromatography, solute spot areas or densities can be measured in situ, or the solute sjxjts can be removed, dissolved and measurements made by another analytical technique such as UV spectrometry (Topic E9). 

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