Reproducibility chromatography

In HPLC, the mobile phase is a liquid delivered under high pressure (up to 400 bar (4 x 10 Pa)) to ensure a constant flow rate, and thus reproducible chromatography, while the stationary phase is packed into a column capable of withstanding the high pressures which are necessary. 

Although the method is simple and straightforward, there are a few important points to consider. First, since 200 pL are being injected on to a 2-mm diameter HPLC column, compatible solvents must be injected on to the column and the amount of acetonitrile used in the prepared samples should be exactly as directed. Second, the temperature of the column and the use of a degassing system for the mobile phase are critical components required to guarantee reproducible chromatography. In addition, the standards should be stored in a refrigerator when not in use. 

Figure 3.10 exemplifies the application of packed-column SFC for the rapid determination of the enantiomeric purity of pharmaceutical materials. The method employed a CHIRALCEL OD CSP with a carbon dioxide/methanol/ isopropylamine mobile phase under isocratic conditions. A flow rate of 4 mFmin was used to generate a high linear velocity whilst maintaining an outlet pressure of 200 bar by use of a back-pressure regulator to ensure reproducible chromatography. Baseline resolution of the enantiomers of propanolol was achieved in approximately 3 min with an overall cycle time of 4 min. Detection was by UV... 

Determinations of the adsorption isotherms for a number of organic solvent-water systems in contact with hydrocarbonaceous stationary phases have shown that a layer of solvent molecules forms on the bonded-phase surface and that the extent of the layer increases with the concentration of the solvent in the mobile phase. For example, methanol shows a Langmuir-type isotherm when distributed between water and Partisil ODS (56). This effect can be exploited to enhance the resolution and the recoveries of hydrophobic peptides by the use of low concentrations, i.e., <5% v/v, of medium-chain alkyl alcohols such as tm-butanol or tert-pentanol or other polar, but nonionic solvents added to aquo-methanol or acetonitrile eluents. It also highlights the cautionary requirement that adequate equilibration of a reversed-phase system is mandatory if reproducible chromatography is to be obtained with surface-active components in the mobile phase. 

Even so variations in these properties can be encountered with the same type of paper and thus procedures for the storage, pre-treatment and handling of the paper should be standardised in order to ensure reproducible chromatography. In order to appreciate more fully the processes occurring during development, it is necessary to look closely at the structure of paper itself. Paper is a random pile of cellulose fibres, each fibre comprises a number of chains of 2000 anhydroglucose units. The structure of the cellulose chain is shown in Figure 3.4. 

In order to obtain reproducible chromatography whether in analytical or preparative scale work the column must be conditioned prior to use. As a guide the amount of solvent required for equilibration is 15-20 column volumes. If equilibration is incomplete this can lead to poor reproducibility and separation. 

Buffer is required to keep the pH constant for reproducible chromatography and to minimize nonspecific, adsorptive interactions with the silica... 

The chromatographic system should comprise a precolumn located between the pump and the injector, packed with silica gel, in order to saturate the mobile phase with silicic acid, thus increasing the lifetime of the analytical column. Caution must be taken to insure that the steady-state condition required for reproducible chromatography is reached. Before the sample is injected into the chromatograph, the system must be equilibrated with the micellar mobile phase. Special care is required to work with micellar mobile phases such as those given in Chapter 4 (Section IV). 

The. selection of a solvent for application of the sample can be a critical factor in achieving reproducible chromatography with distortion-free zones. In general, the application solvent should be a good solvent for the sample and should be as volatile and weak as possible. For silica gel TLC, a weak solvent is nonpolar for reversed-phase TLC, it is polar. High volatility promotes solvent... 

If we consider Fig. 1 in more detail, it is clear that the largest changes in retention time occur when the pH of the mobile phase is close to the pfQ of the analyte. In addition, the secondary equilibria that result in peak broadening can also be emphasized at this pH. There is a fairly simple solution to this problem where analyte pfCj values are known, the chromatographer can simply retrieve these values and be sure to work at least 2 pH imits above or below the pK, . Chromatographic mechanisms should be relatively free from secondary equilibria effects in this area i.e., the analytes will exist in either fully ionized or fully unionized form. This should result in better peak shape and more reproducible chromatography. 

---