Columns polar-embedded

Wilson, N. S., Gilroy, J. J., Dolan, J. W. and Snyder, L. R., Column Selectivity in Reversed-phase Liquid Chromatography VI. Columns with Embedded or End-capped Polar Groups,/. Chromatogr. A, 1026 91—100,2004. 

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)). 

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.

N. Wilson, J. Gilroy, J. Dolan, and L. Snyder, Column selectivity in reversed-phase liquid chromatography VI. Columns with embedded or end-capping polar groups, /. Chromatogr. A 1026 (2004), 91-100. 

Perhaps the biggest limitation to RP-LC is the difficulty with adequate retention for very polar analytes. Three undesirable issues are associated with poor retention (1) poor chromatographic separation, (2) greater ionization suppression, and (3) reduced sensitivity due to poor analyte desolvation in highly aqueous mobile phases. Column manufacturers have used several formats to improve retention of polar solutes that include extended alkyl phases, polar-endcapped alkyl phases, polar-embedded alkyl phases, nonendcapped short alkyl phases, and wide pore-diameter phases [94]. Interested readers are referred to a recent two-part review on this important subject [94,95]. 

RPLC with C8 or C18 columns RPLC with CN, phenyl, or polar-embedded columns... 

Figure 2.24. The concept of orthogonality as shown by retention plots of two sets of columns for a variety of different analytes. (A) Since the log k data of the two columns (C8 and C18) are well correlated for most analytes, these two columns are expected to yield similar elution profiles. (B) The selectivity differences of a C18 and a polar-embedded phase (amide) column lead to very scattered correlation of their respective retention data. Methods using a C18 and a polar-embedded column are therefore termed orthogonal and expected to yield very dissimilar profiles. Diagram courtesy of Supelco, Inc.

Figure 3.15. Comparative diagram showing significant difference in column selectivity of various polar-embedded bonded phases versus that of Luna C18, a more conventional straight-chain C18 chemistry. Diagram courtesy of Dionex Corporation.

FIPLC columns packed with high-purity, silica-based bonded phases continue to dominate the market. Modern columns yield more symmetric peaks for basic analytes (less silanophilic activity) and have better batch-to-batch reproducibility and longer lifetimes. Improved bonding chemistries have widened the usable pFI range from 2-8 to 1.5-10 or more. Although C8- and CIS-bonded phases remain the most common, other phases have become quite popular, including phenyl, cyano, and several polar-embedded phases (e.g., amide, carbamate). 

To better distinguish the contributions of polar interactions to retention, the LEER model was transformed into the so-called hydrophobic subtraction model (HSM) for RPLC, where the hydrophobic contribution to retention is compensated for by relating the solute retention to a standard nonpolar reference compound. This approach was applied to characterize more than 300 stationary phases for RPLC, including silica gel supports with bonded alkyl-, cyanopropyl-, phenylalkyl-, and fluoro-substituted stationary phases and columns with embedded or end-capping polar groups. The QSRR models can be used to characterize and compare the suitabihty of columns not only for reversed-phase, but also for NP and HILIC systems. 

Figure 6.36 Separation of xylene sulfonate on a polar-embedded stationary phase. Separator column Acclaim Surfactant Plus, 3 pm column dimensions 150 mm x 3 mm i.d. column...

Figure 6.45 Gradient elution of various aliphatic quaternary ammonium compounds on a polar-embedded stationary phase. Separator column Acclaim Surfactant Plus, 3 pm column dimensions 150mmx3mm Ld. column temperature 25 °C eluent 5 mmol/L formic acid/ MeCN (95 5 v/v) and (B) 5 mmol/L formic add/ MeCN (60 40 v/v) gradient linear, 100% A to 100% B in 12 min flow rate 0.5 mL/min ...

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