Chromatographic separation stationary phases

Fio. 10. Effect of pressure programming on chromatographic separation. Stationary phase, nitrobenzyl-silica, SI 200 d, 35 /tm column, 50 x 2 mm eluent, n-hepune, temp., 23 C. Sample components 1. unretained 2, bromobenzene 3, toluene 4, naphthalene 5, anthracene 6, brasan 7, o,A-[Pg.50]

In a chromatographic separation procedure the parameters of the chromatographic system (stationary phase, flow, temperature, etc.) have to be selected respectively optimized with respect to some criterion (resolution, time, etc.). In gas chromatography retention data series are published and used for the sttidy of solvent/solute interaction, prediction of the retention behaviour, activity coefficients, and other relevant information usable for optimization and classification. Several clKmometrk techniques of data anal s have been employed, e.g. PCA, numerical taxonomic methods, information theory, and j ttern recognition. 

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. 

Chiral crown ethers based on IB-crown-6 I Fig. 4> can form inclusion complexes with ammonium ions and proionated primary amines. Immobilization of these chiral crown ethers on a chromatographic support provides a chiral stationary phase which can resolve most primary amino acids, amines and amino alcohols. However, the stereogenic center must be in fairly close proximity in the primary aininc lor successful chiral separalion. Significantly, ihe chiral crown ether phase is unique in that ii is one of the few liquid chromatographic chiral stationary phases that does not require the presence of an aromatic ring to achieve chiral separations. 

H. Y. Aboul-Enein and M. R. Islam, Direct separation and optimization of timolol enantiomers on a cellulose tris-3,5-dimethyl-phenylcarbamate high-performance liquid chromatographic chiral stationary phase, J. Chromatogr., 55 109 (1990). 

E. D. Lee, J. D. Henion, C. A. Brunner, I. W. Wainer, T. D. Doyle, and J. Gal, High-performance liquid chromatographic chiral stationary phase separation with filament on thermospray mass spec-trometric identification of the enantiomer contaminant (S)-( + )meth-amphetamine, Anal. Chem., 55 1349 (1986). 

While the mechanisms of retention for various types of chromatography differ, they are aU based on establishment of an equilibrium between a stationary phase and a mobile phase. Figure 19.1 illustrates the separation of these components on a chromatographic column. A small volume of sample is placed at the top of the column, which is filled with the chromatographic particles (stationary phase) and solvent. 

Gas-Liquid Chromatography. Oxidation products were analyzed using either a Pye Argon, Perkin-Elmer Fll or Pye F104 gas chromatograph. The stationary phases and operating conditions have been reported previously 21). An improved method for separating cresol isomers (28) was introduced in this research in place of the previous method which used tris(2,4-xylenyl) phosphate as the liquid phase. 

Fig. 11.2.2, Separation of native types I and III collagen. Chromatographic conditions stationary phase, CIS Bakerbond wide-pore column (250x4.6 mm I.D.) mobile phase, 0.05 M ammonium bicarbonate adjusted to pH 3.2 with trifluoroacetic acid (Buffer A), tetrahydrofuran (Buffer B), stepwise gradient of 20% Buffer B for 10 min, a linear gradient of 20-30% Buffer B over 30 min and 30% B for 15 min flow rate, 1 ml/min temperature, 21°C. Peaks 1, artifact 2, type III collagen 3, type 1 collagen. Reproduced from Smolensk et al. (1983), with permission.

Fig. 11.2.8. Normal phase HPLC separation of six isomeric hexapeptides. Chromatographic conditions stationary phase, pPorasU silica column (300 x 3.9 mm I.D.) mobile phase, cyclohexane-isopropanol-methanol (92 6 2) flow rate, 1.0 ml/min temperature, ambient. Peaks 1, Boc-Met3 Gly-Met2-OMe 2, Boc-Met2-Gly-Met3-OMe 3, Boc-Mets-Gly-OMe 4, Boc-Gly-Mets-OMe 5, Boc-Met4 Gly-Met-OMe. Reproduced from Naider et al. (1983), with permission.

Fig. 11.8.1. Reversed phase separation of vitamin A alcohol and esters. Chromatographic conditions stationary phase, Zorbax C-8 (250 x 4.6 mm I.D.) mobile phase, 99.8% water-0.2% perchloric acid (60%), and 99.8% methanol-0.2% perchloric acid flow rate, 1.6 ml/min detection, UV at 254 nm. Peaks 1, vitamin A alcohol 2, impurity 3, vitamin A acetate 4, vitamin A palmitate. Reproduced from Dupont Analytical Systems with permission.

For the stereochemical determination, it seemed theoretically that the most efficient method of separation for the three pristane isomers A, B and C would be direct gas chromatography on an optically active stationary phase. Such a method was successfully employed for amino acids and amine enantiomers . However, this method was found ineffective for pristane, and hence various gas-chromatographic inactive stationary phases were employed for the separation of the diastereoisomers. The relative stereochemistry could be determined, provided standards with known respective stereochemistry are available. 

The principle of displacement chromatography for separation is based on the Langmuir isotherm. Only a finite number of sites are on the chromatographic support (stationary phase) for the binding of sample components, and if a site is occupied by one molecule, it is not available to the other sample components. Because the number of binding sites is limited, they are saturated when the concentration of the molecules in the sample is large in comparison to the dissociation constant for the sites. 

Figure 8-12. Examples of las eluted enantiomers In a chromatographic separation on chiral HPLC with teicoplanin stationary-phase.

Chromatographic separations are accomplished by continuously passing one sample-free phase, called a mobile phase, over a second sample-free phase that remains fixed, or stationary. The sample is injected, or placed, into the mobile phase. As it moves with the mobile phase, the sample s components partition themselves between the mobile and stationary phases. Those components whose distribution ratio favors the stationary phase require a longer time to pass through the system. Given sufficient time, and sufficient stationary and mobile phase, solutes with similar distribution ratios can be separated. 

Analytical separations may be classified in three ways by the physical state of the mobile phase and stationary phase by the method of contact between the mobile phase and stationary phase or by the chemical or physical mechanism responsible for separating the sample s constituents. The mobile phase is usually a liquid or a gas, and the stationary phase, when present, is a solid or a liquid film coated on a solid surface. Chromatographic techniques are often named by listing the type of mobile phase, followed by the type of stationary phase. Thus, in gas-liquid chromatography the mobile phase is a gas and the stationary phase is a liquid. If only one phase is indicated, as in gas chromatography, it is assumed to be the mobile phase. 

In gas chromatography (GC) the sample, which may be a gas or liquid, is injected into a stream of an inert gaseous mobile phase (often called the carrier gas). The sample is carried through a packed or capillary column where the sample s components separate based on their ability to distribute themselves between the mobile and stationary phases. A schematic diagram of a typical gas chromatograph is shown in Figure 12.16. 

A chromatographic column provides a location for physically retaining the stationary phase. The column s construction also influences the amount of sample that can be handled, the efficiency of the separation, the number of analytes that can be easily separated, and the amount of time required for the separation. Both packed and capillary columns are used in gas chromatography. 

Another important characteristic of a gas chromatographic column is the thickness of the stationary phase. As shown in equation 12.25, separation efficiency improves with thinner films. The most common film thickness is 0.25 pm. Thicker films are used for highly volatile solutes, such as gases, because they have a greater capacity for retaining such solutes. Thinner films are used when separating solutes of low volatility, such as steroids. 

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