Normal-phase columns, polar

Mode of operation for the chiral column. NP normal phase PO polar organic RP reverse phase. Discussed in the Section 14.4.1. 

Most of the mentioned troubleshooting tools will work with other silica-based columns. With normal-phase columns, you obviously need not worry about bonded-phase removal, but silica still dissolved at high pH and high salt concentrations. Polar materials like some proteins adhere very tightly and require... 

A MicroPak MCH-10 reverse phase column was chosen for separation of the hexane and ether extracts. The monomolecular bonded phase provides efficient separation of both polar and non-polar substances and rapid equilibration to initial activity after gradient elution programs. The reverse phase column provides symmetrical, narrow peaks for the cannabinoic acids, which tend to tail on polar, normal phase columns (e.g. silica). 

Endogenous substances in the extracts are more polar than the cannabinoids and elute before them on the reverse phase column. On polar, normal phase columns, strong adsorption of endogenous species requires periodic column clean-up. This problem was not encountered with the reverse phase gradient system. 

A. W. Salotto, E. L. Weiser, K. P. Caffey, R. L. Carty, S. C. Racine, and R. L. Snyder, Relative Retention and column selectivity for the common polar bonded-phase columns The diol-silica column in normal-phase high-performance liquid chromatography,/. Chromatogr. 498 (1990), 55-65. 

In comparing normal phase to reversed phase, several general attributes can be noted. Reversed-phase HPLC provides better separation between compounds varying in alkyl carbon number and elution is in order of decreasing polarity. Normal-phase HPLC provides better resolution of compounds differing in polar substituents and for achiral isomers and elution is in order of increasing polarity. However, more substantial a priori prediction of which specific column will be most suitable for a given API and related substances has remained elusive. 

In order for the separation to take place, a more polar solvent (than the original sample matrix) will be used to effect the desorption of the analyte molecules from the packing material. Examples of packing material include silica bonded with cyano, amine, and diol groups, as with the stationary phase of columns in normal phase chromatography (see Chapter 4 for further explanation). 

In our laboratory we make our own columns for normal-phase chromatography (diol-modified silica). We have noticed that by packing the columns under a high pressure, that is, 950-1000 bar, instead of at the recommended 500-600 bar, we obtained far better separation of the phospholipid classes. Accordingly, this way of packing columns may be a way to achieve better separation of intact polar lipids, if choice of column packing, solvent mixture gradient and sample clean-up have already been optimized. 

Similar to the new polar organic mode, the retention of analytes in normal phase is not difficult to predict. For all the compounds, the average of the retention on individual columns is fairly close to the retention on the coupled columns. The selectivity of most compounds on coupled columns is an average of the selectivities of individual columns (Fig. 2-9). However, it was found that the elution order for some compounds was reversed on ristocetin A and teieoplanin or vancomycin. As a result. 

Another important issue that must be considered in the development of CSPs for preparative separations is the solubility of enantiomers in the mobile phase. For example, the mixtures of hexane and polar solvents such as tetrahydrofuran, ethyl acetate, and 2-propanol typically used for normal-phase HPLC may not dissolve enough compound to overload the column. Since the selectivity of chiral recognition is strongly mobile phase-dependent, the development and optimization of the selector must be carried out in such a solvent that is well suited for the analytes. In contrast to analytical separations, separations on process scale do not require selectivity for a broad variety of racemates, since the unit often separates only a unique mixture of enantiomers. Therefore, a very high key-and-lock type selectivity, well known in the recognition of biosystems, would be most advantageous for the separation of a specific pair of enantiomers in large-scale production. 

In NPLC, which refers to the use of adsorption, i.e. liquid-solid chromatography (LSC), the surface of microparticulate silica (or other adsorbent) constitutes the most commonly used polar stationary phase normal bonded-phase chromatography (N-BPC) is typified by nitrile- or amino-bonded stationary phases. Silica columns with a broad range of properties are commercially available (with standard particle sizes of 3, 5 and 10 im, and pore sizes of about 6-15nm). A typical HPLC column is packed with a stationary phase of a pore size of 10 nm and contains a surface area of between 100 and 150m2 mL-1 of mobile phase volume. 

Table 7.89 lists the main characteristics of MDHPLC (see also Table 7.86). In MDHPLC the mobile-phase polarity can be adjusted in order to obtain adequate resolution, and a wide range of selectivity differences can be employed when using the various available separation modes [906]. Some LC modes have incompatible mobile phases, e.g. normal-phase and ion-exchange separations. Potential problems arise with liquid-phase immiscibility precipitation of buffer salts and incompatibilities between the mobile phase from one column and the stationary phase of another (e.g. swelling of some polymeric stationary-phase supports by changes in solvents or deactivation of silica by small amounts of water).

Construction of the cyclopentane ring was accomplished by utilization of Trosf s Pd-mediated diastereoselective [3+2] trimethylenemethane (TMM) cycloaddition [4] on the cinnamate 5 having an Evans type chiral auxiliary [4b], The resulting diastereomeric mixture (3 1 at best) of 7a and 7b was separated by careful silica gel column chromatography (7a is less polar than 7b under normal phase). Puri-... 

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