Applications

Reversed phases are the most commonly used stationary phases in contemporary LC and are employed in over 65% of all analytical applications. They are largely used with water as a component of the mobile

Figure 5 Separation of a sample of explosives. Column: 25x4.6 mm i.d. packed with C18 reversed phase. Particle diameter 5 im. Solvent methanol-water, 50/50 at a flow rate of 1.5 mL min-1. Sample volume 100 |L. 1, HMX (1 igmL-1); 2, RDX (1 ig mL-1); 3, 1,3,5-trinitrobenzene (2 igmL-1); 4, 1,3-dinitrobenzene(10 ig mL-1); 5, 2,4,6-trinitrotoluene (1 ig mL-1); 6, tetryl (1 ig mL-1); 7, nitrobenzene (1 ig mL-1); 8, 2,6-di-nitrotoluene (2 igmL-1); 9, 2,4-dinitrotoluene (1 ig mL-1); 10, 2-nitrotoluene (0.1 igmL-1); 11, 4-nitrotoluene (0.5 igmL-1); 12, 3-nitrotoluene (0.1 ig mL-1).

Figure 5 Separation of a sample of explosives. Column: 25x4.6 mm i.d. packed with C18 reversed phase. Particle diameter 5 im. Solvent methanol-water, 50/50 at a flow rate of 1.5 mL min-1. Sample volume 100 |L. 1, HMX (1 igmL-1); 2, RDX (1 ig mL-1); 3, 1,3,5-trinitrobenzene (2 igmL-1); 4, 1,3-dinitrobenzene(10 ig mL-1); 5, 2,4,6-trinitrotoluene (1 ig mL-1); 6, tetryl (1 ig mL-1); 7, nitrobenzene (1 ig mL-1); 8, 2,6-di-nitrotoluene (2 igmL-1); 9, 2,4-dinitrotoluene (1 ig mL-1); 10, 2-nitrotoluene (0.1 igmL-1); 11, 4-nitrotoluene (0.5 igmL-1); 12, 3-nitrotoluene (0.1 ig mL-1).

Figure 6 Separation of some urea pesticides on a C8 reversed-phase column. Column LC 8 reversed phase, 15 cm long, 4.6 mm i.d., 5 |im particles used with a mobile-phase gradient of 18 : 82 acetonitrile-waterto 65 : 35 to acetonitrile-water in 9 min. 1, Methomyl; 2, oxamyl; 3, fenuron; 4, manuron; 5, carbofuran; 6, propoxur; 7, carbaryl; 8, fluometuron; 9, duron, 10, propham; 11, siduron; 12, linuron; 13, chioprophan; 14, barban; 15, neburon.

Figure 6 Separation of some urea pesticides on a C8 reversed-phase column. Column LC 8 reversed phase, 15 cm long, 4.6 mm i.d., 5 |im particles used with a mobile-phase gradient of 18 : 82 acetonitrile-waterto 65 : 35 to acetonitrile-water in 9 min. 1, Methomyl; 2, oxamyl; 3, fenuron; 4, manuron; 5, carbofuran; 6, propoxur; 7, carbaryl; 8, fluometuron; 9, duron, 10, propham; 11, siduron; 12, linuron; 13, chioprophan; 14, barban; 15, neburon.

phase, in binary, ternary and occasionally quaternary mixtures with aliphatic alcohols, nitriles and ethers, e.g. methanol, acetonitrile and tetrahydrofuran. They are also used with ion-pairing reagents in ion chromatography where the reagent is adsorbed as an interactive layer on the surface of the stationary phase. The mechanism of ion-pairing reagents is complex and will be discussed elsewhere.

Reversed phases are used in industrial product quality control, environmental testing, clinical analyses, biotechnological assays, forensic examination and many research programmes. Numerous examples of the use of reversed phases are included elsewhere in this book: some will be given here to illustrate the types of separation possible using predominantly dispersive interactions with the stationary phase. An example of an environmental or forensic type of application is given in Figure 5. The separation was carried out on an octadecyl bonded phase using a 50% v/v methanol-water mixture as the mobile phase. The selectivity of the dispersive stationary phase is clearly demonstrated, the more polar materials having strong polar interactions, with the strongly polar mobile phase being eluted first. In contrast, the relatively strong dispersive interactions of the monosubstituted toluenes with the reversed phase cause them to be eluted last. It is also seen that the explosive components are well resolved and detected quantitatively (mostly at the sub-microgram level) with no difficulty. Some samples can be separated by weaker dispersive interactions using reversed phases with shorter chains. An example of the use of a C8 reversed phase for the separation of a number of carbamate and urea pesticides is shown in Figure 6. The separation is achieved using a gradient that starts with a highly polar binary aqueous mixture containing only 18% v/v acetonitrile to a moderately polar solvent mixture containing 65% v/v acetonitrile. It is seen that excellent separation is obtained in only 12 min.

As discussed earlier, the use of dispersive interactions to separate biologically labile compounds demands the use of short chain length reversed phases to prevent irreversible adsorption and/or solute de-naturation. An alternative approach is to use one of the polar bonded phases in a manner where the dispersive interactions dominate but not sufficiently to cause strong adsorption. The amino or diol polar phases have been used in this manner to separate protein and protein-like materials and an example is given in Figure 7. It is seen that water containing no solvent is used as the mobile phase, which virtually completely negates the polar interactive capability of the polar stationary phase. The only significant interactions remaining are those with the dispersive moieties of the bonded material. This results in only weak interactions between the solutes and the stationary phase and the separation is attained without significant tailing (a symptom of strong adsorption) or denaturation.

1. Ferritin

2. Ovalbumin

3. Myoglobin

4. Insulin

5. Glycyl-Tyrosin

Time (min)

An LC-Diol column, 25 cm long and 6.2 mm I.D. and a Mobile Phase of 0.1 M Potassium Phosphate Buffer at pH 6.8

Figure 7 The separation of some proteins on a polar phase used in the reversed phase mode.

Due to limited pressure available in liquid chromatographs, which is not constrained by pump design but rather by other parts of the chromatograph (in particular the sample valve), the reduction in particle diameter of a column packing is limited in practice. Small particles can only be used with shorter columns, which results in reduced separation times but not an increase in the attainable column efficiency. The shorter columns packed with very small particles also produce extremely small peak volumes that place serious constraints on the sample system and in particular the detector sensor cell. Although in the research laboratory, particles having diameters less than 1 |im may well be developed and their efficacy demonstrated, their general use in analytical laboratories will be very limited for some years to come. Due to the versatility and wide range of operating variables available, difficult separations are more easily achieved by using procedures other than by reducing the particle size to a level where practical difficulties become paramount.

Reversed phases are probably the most effective and popular stationary phases in use today. However, stationary phases employing bonded silica may well not be the best form of reversed stationary phase to use in the future. The great disadvantage to silica-based stationary phases is their instability in aqueous solvents, particularly at extreme pH. The future stationary phases that will be used in LC are more likely to be some form of macro-reticulated polymeric materials that have been developed and are already quite widely used. These polymeric particles can be prepared with significant surface area and porosity and are extremely inert and stable in aqueous solvent mixtures at the extremes of pH. They can also be prepared with a wide range of polarities linking different chemical groups to their surface and by using different polymer bases.

Seealso: II/Chromatography: Liquid: Column Technology. III/Pharmaceuticals: Basic Drugs: Liquid Chromatography. Porous Polymers: Liquid Chromatography.

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