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Alternative Selectivity of Amide-based Embedded Polar Group HPLC Stationary Phases – Role of Hydrogen Bonding

By: Dave Bell, Reporter EU Volume 24

Dave Bell


High-performance liquid chromatographic (HPLC) method development often commences using alkyl (C8, C18) stationary phases. The method development scientist will commonly adjust the mobile phase organic modifi er percentage and type as well as the solvent pH in an attempt to obtain the desired retention and resolution for a given analysis. When these common practices fail to yield the desired result, utilization of alternative stationary phase chemistries may be considered. What stationary phase, however, is most suitable for a given set of analytes?

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Among the more common commercially-available chemistries, embedded polar group (EPG) phases are becoming a preferred alternative stationary phase. EPG stationary phases generally consist of an alkyl chain with polar groups such as an amide, urea, sulfonamide or carbamate embedded in the bonded ligand. EPG phases have been observed to provide improved peak shape for basic compounds, differential selectivity, decreased retention of hydrophobic analytes and suitability for 100% aqueous applications. The types of analytes that show differential selectivity and the retention mechanisms that govern such differences, however, remain unclear.

In these investigations, the fundamental interactions that contribute to alternative retention on EPG phases as compared to traditional C18 chemistries is briefl y reported. The studies include a comparison between Ascentis C18 and Ascentis RP-Amide using a column classifi cation protocol as reported by Euerby et. al.(1,2), and a comprehensive linear solvation energy relationship (LSER) investigation (3-5). Contributions from hydrogen bonding interactions are determined to be significant in governing alternative selectivity using the EPG phases relative to alkyl stationary phases. Representative chromatograms for the separation of resorcinols and catechols on both a C18 and EPG phase demonstrate alternative selectivity.

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Column Classification Study

The column classification study was conducted according to the protocol utilized by Euerby, et. al.(2). Ascentis C18 and Ascentis RP-Amide (amide-based EPG phase) columns (15 cm x 4.6 mm, 5 μm) along with a Discovery C18 (15 cm x 4.6 mm, 5 μm, as a control) were employed.

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Linear Solvation Energy Relationship (LSER) Study

Retention data for 25 neutral analytes with known molecular descriptors were acquired in triplicate according to the following conditions:

column: Ascentis RP-Amide (565323-U) or Ascentis C18 (581323-U) , 5 cm x 4.6 mm I.D., 5 μm particles
mobile phase: 50:50 (v/v), water:methanol
flow rate: 1 mL/min.
temp.: 35 °C
det.: UV at 220 nm
injection: 10 μL

Multiple linear regression analysis on the resultant data was performed using Minitab ver.13 (State College, PA USA)

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Separation of Catechols and Resorcinols

column: Ascentis RP-Amide (565324-U) or Ascentis C18 (581324-U) , 15 cm x 4.6 mm I.D., 5 μm particles
mobile phase: 75:25, 20 mM phosphoric acid (pH 2.0 unadjusted):acetonitrile
temp.: 30 °C
flow rate: 1.5 mL/min.
det.: UV, at 270 nm
injection: 25 μL
sample: 50 μg/mL in 20 mM phosphoric acid, pH 2.0

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Results and Discussion

The goal of a column classification protocol is to provide performance comparisons between the myriad of stationary phases that are commercially available. The HPLC practitioner can use this information to determine the brands of columns likely to provide similar performance for an existing method or what columns might provide different performance for method development purposes. The studies attempt to break down the important interactions that contribute to retention and selectivity using test probes that attempt to ascertain interaction presence and dominance on a given stationary phase. The column classifi cation protocol reported by Euerby includes tests for hydrophobic retentivity (k’ pentylbenzene, k’PB), hydrophobic selectivity (k’/k’ amylbenzene/butylbenzene, αCH2), shape selectivity (k’ triphenylene/k’ o-terphenyl, αT/O), hydrogen bond selectivity (k’ caffeine/ k’ phenol, αC/P) and ion-exchange capacity at pH 2.7 and pH 7.6 (k’ benzylamine/k’ phenol, αB/P, 2.7 and αB/P, 7.6, respectively). By comparing the values obtained for different stationary phases, specifi c interactions can be identifi ed as having relatively greater or lesser contributions to overall retention.

A C18 stationary phase, Ascentis C18 and an EPG phase, Ascentis RP-Amide were compared using the Euerby protocol and the results are presented in Table 1. In terms of selectivity differences between the phases, the hydrogen bonding capacity is observed to be dominant. Caffeine is a hydrogen bond acceptor whereas phenol is a hydrogen bond donor.

The lower value obtained on the EPG phase signifies that the phase preferentially retains the hydrogen bond donating phenol (denominator) compound. Based on these data, it is concluded that the amide stationary phase acts as a hydrogen bond acceptor.

Table 1. Column Classification Results

Linear solvation energy relationship studies attempt to relate fundamental molecular solute descriptors such as molecular volume (V), polarizability (S), electron lone pair interactions (E) and hydrogen bonding interactions (donating/acidity, A and accepting/basicity, B) to the free energy related to a phase transfer process (differential interaction with the mobile phase and stationary phase) through equations such as Eq. 1.

Eq 1: log k’ = c + eE + sS + aA + bB + vV

Where k is the capacity factor for a given probe analyte, c is a constant and e, s, a, b and v are stationary phase characteristics that complement the analyte interaction descriptors. Retention data for a set of probe analytes with known interactions properties may be used to elucidate the dominant interactions contributing to retention on a given stationary phase using multiple linear regression analyses (for further information on LSER studies see references 3-5).

In the present study, Ascentis C18 and Ascentis RP-Amide were once again compared using the LSER approach. The data presented in Table 2 indicate that the polarization and hydrogen bonding terms are statistically different between the two stationary phase chemistries and thus contribute to the differences in selectivity often observed when employing the phases. Figure 1 shows the preferential retention of phenolic and aniline compounds used in the study on the EPG phase. Again, the preferential retention of compounds capable of donating toward a hydrogen bond is observed. The LSER study also implicates polar interactions (such as dipole-dipole) as a potentially differentiating interaction between EPG and C18 phases.

Table 2. LSER Multiple Linear Regression Analysis Results

Figure 1. Preferential Retention of Hydrogen Bond Donors on EPG Phases

With hydrogen bonding being implicated as a dominant contributor to differential retention and selectivity on EPG phases, the separation of a group of catechols and resorcinols was compared on the amide and C18 phases. Figure 2 demonstrates both enhanced retention and alternative selectivity for the hydrogen bond donors using the EPG phase.

Figure 2. Separation of Catechols and Resorcinols on Ascentis C18 and Ascentis RP-Amide

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Hydrogen bonding plays a key role in the alternative retention and selectivity observed for EPG phases when compared to traditional C18 chemistries. The role of hydrogen bonding is supported by column classifi cation studies, fundamental LSER investigations and in specific application. When stationary phase chemistries other than traditional alkyl phases are required for the separation of hydrogen bond donating analytes, EPG phases such as the Ascentis RP-Amide should be explored as a viable alternative.

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  1. M.R. Euerby, McGeown, A. P., Petersson, P., J. Sep. Sci. 26 (2003) 295.
  2. M.R. Euerby, P. Petersson, J. Chromatogr. A 994 (2003) 13.
  3. L.C. Tan, Carr, P. W., Abraham M. H., J. Chromatogr. A 752 (1996) 1.
  4. A. Wang, P.W. Carr, J. Chromatogr. A 965 (2002) 3.
  5. J. Zhao, P.W. Carr, Anal. Chem. 70 (1998) 3619.

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