Polar-embedded

Liu, X.D., Bordunov, A., Tracy, M., Slingsby, R., Avdalovic, N., and Pohl, C., Development of a polar-embedded stationary-phase with unique properties, J. Chromatogr. A, 1119, 120, 2006. 

The fact that the EP wants to replace old TEC methods with more selective, efficient, and sensitive separation methods provides the chance for the introduction of more CE methods. The continuous development of analytical methods is reflected in the national and international pharmacopoeias. This might be demonstrated for atropine sulfate. Whereas the Deutsches Arzneibuch, 7th Edition (DAB 7) only limits the tropic acid by extraction and titration with NaOH and phenolphthalein indication, the 4th edition of the EP looked for foreign alkaloids and decomposition products by means of TEC with a potassium iodobismuthate for detection. By intensity comparison of the obtained spots, it was possible to limit these impurities to 0.5%. The EP 5 utilizes an ion-pair HPLC method that is able to limit most of the impurities to less than 0.2%. To make the method more robust, an HPLC method using a polar embedded was applied, which might be the next step for the EP. However, recently the same authors have reported on a MEEKC method being as robust and precise as the latter HPLC method (see Eigure 6) but far more sensitive and, therefore, a future perspective for the EP. 

The polar groups are, on the other hand, responsible for an induced polar selectivity. Analytes able to form hydrogen bonds like phenols are retarded more strongly with polar-embedded stationary phases than with the corresponding classical RP of an identical carbon content. This is demonstrated in Figure 2.5 for the separation of polyphenolic compounds present in red wine. The retention time of the polyphenolic compound kaempferol with the shielded phase is more than three times longer than with the corresponding RP column of an identical carbon content. The polar... 

FIGURE 2.8 Hydrophobic retention and selectivity with RP columns. The stationary phases are ordered according to the increasing retention of toluene in methanol-water 50-50 v-v. Dashed line Stationary phases with a silica pore diameter below lOnm. Solid line Stationary phases with a silica pore diameter >12nm. ( ) Stationary phases with polar-embedded functional groups. (( ) Stationary phase based on a wide pore silica (30 nm)). 

Since the 1970s numerous HPLC methods using lEC, RP and ion-pair chromatography have been proposed. In the last years, RP chromatography has become the most used method, thanks to its simplicity, sensitivity, and compatibility with different detection techniques. The stationary phases usually used are C18 or phenyl-bonded silica-based phases. More recently, alternative stationary phases, such as polar-embedded, polar endcapped, and perfluorinated phases, have been successfully tested for folate analysis [577]. The mobile phase is usually a mixture of phosphate or acetate buffer and acetonitrile or methanol. 

With the above reservations we turn back to the interaction based classification of the hybrid QM/MM methods which allows us to distinguish the mechanical embedding, polarization embedding etc. We shall consider them subsequently. 

Carabias-Martinez, R. Rodriguez-Gonzalo, E. Smith, N.W. Ruano-Miguel, L. 2006. Use of polar-embedded stationary phase for the separation of tocopherols by CEC. Electrophoresis 27 4423 430. 

J. Layne, Characterization and comparison of the chromatographic performance of conventional, polar-embedded, and polar-endcapped reversed-phase liquid chromatography stationary phases, /. Chromatogr. A 957, (2002), 149-164. 

The separation of chiral compounds will be discussed in Chapter 22. However, the separation of diastereomers can be accomplished using achiral stationary phases. Another alternative is the use of chiral columns for the separation of diastereomers in either the reversed-phase or normal-phase mode. The use of achiral bonded phases without chiral additives, such as phenyl and alkyl bonded phases for the separation of diastereomeric pharmaceutical compounds, is acceptable. Different selectivities can be obtained by employing stationary phases containing varying functionalities (phenyl, polar embedded moieties). The effect of aqueous mobile-phase pH, temperature, and type of organic eluent (acetonitrile versus methanol) can also play a dramatic role on the separation selectivity of diastereomeric compounds. 

Screening columns from each of the following various column classes should provide for the desired chromatographic selectivity, even for the most challenging separations (1-3) C8 or C18 stable at pH < 2, pH 2-8, and pH > 8-11 (4) phenyl (5) pentafluorphenyl (6) polar embedded and stationary phases that could be run in 100% aqueous. A certain number of columns in each of the six column classes and subclasses could be chosen as standard columns that the chromatographers choose as a first choice for performing method development. These standard columns could be chosen based on some... 

For more hydrophobic compounds, a stationary phase that has a lower surface area should be used. For very polar compounds that cannot be retained on traditional CIS phases, less hydrophobic columns such as C4 and polar embedded stationary phases could be used. However, all this is also dependent on the pH of the analysis since some columns are not stable at low pH (<2) and higher pH (>7) for extended periods of time. This should be taken into careful consideration when defining a column(s) during the development of a method. 

Moreover, the effect of pH on a particular compound s retention needs to be determined first before exploring the retentivity and selectivity of different columns. The strategy and choice of the optimal pH for analysis was discussed in Chapter 4 and is further reinforced in the case studies within this chapter. After the optimal pH is chosen for the separation and the gradient has been optimized on a particular column and the optimal selectivity still has not been achieved between critical pairs, then a column screening can be performed. For method column screening, generally columns with 10-cm or 5-cm X 3.0-mm i.d. could be used that are packed with 3-pm particles. Implementation of a column switcher that can use six different types of stationary phases such as two types of C18 from different vendors, phenyl, two polar embedded, and pentafluorphenyl is suggested. 

In Figure 8-18, a mixture of acids and bases was analyzed on three types of columns phenyl, polar embedded, and C18 column. Significant differences in selectivity were obtained. The separation could be further optimized by modifying the gradient slope and employing off-line method development tools such as Drylab for further optimization and resolution of the critical pairs. 

In this case study, two different Cl 8 columns from different manufacturers were used. Alternatively, other stationary phase types could also be used such as a polar embedded phase and a Cl 8 phase. Some systems come also equipped with a six-column switcher and in that case, two different types of polar embedded phases, phenyl phase, pentafluorophenyl phases, two different Cl 8 phases (of different bonding density) and an alternate C8 phase could be used. 

In Figure 8-66, the same selectivity test mix (MIX 1) and conditions were used for a polar embedded column. Multiple columns from three different lots... 

Figure 8-66. Selectivity lot-to-lot reproducibility test. Polar embedded CIS column. Mobile phase A 0.1% H3PO4 in H2O. Mobile phase B MeCN. Gradient 40-75% B in 1.5 min, hold 75% for 0.5 min. Flow, 0.5 mL/min temperature, 35°C injection, IpL sample concentration, 0.1 mg/mL. Diluent 20/S0, ACN/water.

Example of a stationary phase with polar embedded group , different selectivity for polar analytes than the classical alkyl phases, for eluents with high water content. U.D. Neue et al., Chromatographia, 54. 169 (2001) H. Engelhardt et al., Chromatographia Supp ., 53, S 154 (2001). 

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