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Leveraging the Unexpected Behavior of HS F5 at High Temperatures for Enhanced Retention and Selectivity of Basic Compounds

By: David S. Bell, Carmen T. Santasania, Reporter EU Volume 22

David S. Bell, Applications and Technical Service Manager and Carmen T. Santasania, Senior Applications Chemist

Discovery HS F5 offers mixed-mode separations, which gives it distinct selectivity advantages. The intriguing influence of temperature on ion exchange retention and selectivity of Discovery HS F5 is explored in this short article.

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Challenges of HPLC of basic compounds

The HPLC analysis of low molecular weight, basic compounds is challenging from both peak shape and retention standpoints. Modern HPLC phases have addressed the peak shape issue for the most part, but adequate retention of bases, and indeed polar compound in general, has not been resolved. Polar-embedded stationary phases and unmodified silica particles (1) have been put forth as solutions to the retention problem, but each has its limitations especially when dealing with ionized compounds and LC-MS.

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The Discovery HS F5 solution to basic compound retention

We sought to develop an HPLC phase that provides excellent peak shape and retention of basic compounds under conditions compatible with conventional UV and LC-MS analysis. Discovery HS F5 has these attributes. Discovery HS F5 comprises a pentafluorophenylpropyl phase bonded to high purity, spherical, porous silica. In addition to the dispersive interactions of traditional alkyl phases and ion exchange interactions at high organic like silica, the Discovery HS F5 phase also can interact via dipole-dipole, π-π and charge transfer interactions. Although its main feature is its orthogonal selectivity relative to C18 (2), an interesting characteristic of Discovery HS F5 is that it can exhibit both reversed-phase and ion exchange retention depending on the concentration of organic modifier in the mobile phase (3, 4). The ion exchange retention is also influenced by the temperature of the analysis, a fact that can be leveraged to improve chromatographic selectivity.

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Temperature influences on basic compound retention on silica and HS F5 columns

Temperature is known to influence reversed-phase retention, but the unexpected observation that retention and selectivity of quaternary amines is increased on a cation exchange column by increasing the temperature (5) prompted our investigation of the influence of temperature on ion exchange retention on both unmodified silica and Discovery HS F5 columns. The van’t Hoff plots for a mixture of amphetamines with pKa ranging from 8.47 to 10.38 on silica and HS F5 columns are shown in Figures 1a and 1b (note x-axis is from high to low temperature). Results obtained in the 25 – 55°C range in acetonitrile-0.1% ammonium acetate are compared and contrasted in Table 1.

Figure 1a. Ion exchange retention of amphetamines as a function of temperature

Methamphetamine (), ephedrine (), norephedrine (+), MDMA (3,4-methylenedioxymethamphetamine (ectasy) (♦), amphetamine (X), MDA (3,4-methylenedioxyamphetamine ) () and phentermine (). Column: silica, 5 cm x 2.1 mm, 5 μm particles. Mobile phase: 10:60:30, v/v, 0.1% ammonium acetate in water:acetonitrile:0.1% ammonium acetate in acetonitrile. Flow rate: 200 μL/min. Temperature: 25 – 55 oC. Detection: MS, ESI(+), SIR mode.


Figure 1b. Ion exchange retention of amphetamines as a function of temperature:

Methamphetamine (), ephedrine (°), norephedrine (+), MDMA (3,4-methylenedioxymethamphetamine (ectasy) (♦), amphetamine (X), MDA (3,4-methylenedioxyamphetamine ) () and phentermine (). Column: Discovery HS F5, 5 cm x 2.1 mm, 5 μm particles. Mobile phase: 10:60:30, v/v, 0.1% ammonium acetate in water:acetonitrile:0.1% ammonium acetate in acetonitrile. Flow rate: 200 μL/min. Temperature: 25 – 55 oC. Detection: MS, ESI(+), SIR mode.


Table 1. Comparison of effect of temperature on chromatography of basic compounds on silica and HS F5 columns


For basic compounds, the HS F5 exhibits increasing retention with increasing temperature at high percentages of organic modifier, which is in contrast to behavior observed in purely reversed-phase separations. Also, the slopes of the van’t Hoff plots on the HS F5 are less than those for the silica column indicating a weaker influence of temperature on retention on the HS F5. This is a benefit of the HS F5 because retention will not be susceptible to small fluctuations in ambient conditions. An example of the optimized MS analysis of amphetamines on Discovery HS F5 is shown in Figure 2. Short columns provided both necessary efficiency and rapid analysis. The mobile phase, which contained 90% acetonitrile and 0.1% of the volatile salt, ammonium acetate, was conducive to high-sensitivity MS detection.

Figure 2. LC-MS of amphetamines at 35 °C on Discovery HS F5

Column: Discovery HS F5, 5 cm x 2.1 mm, 5 μm particles. Mobile phase: 10:60:30, v/v, 0.1% ammonium acetate in water:acetonitrile:0.1% ammonium acetate in acetonitrile. Flow rate: 200 μL/min. Temperature: 35 oC. Detection: MS, ESI(+), SIR mode.


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Solvation: possible source of temperature influence in ion exchange

Why does ion exchange retention increase with increasing temperature? One hypothesis is a change in solvation: the wetting of the stationary phase by the mobile phase. As solvation power of the mobile phase decreases, decreased solvation of both mobile phase and ionized surface groups of the stationary phase leads to stronger ionic interactions between analyte and stationary phase. Decreased solvation power at higher temperatures results in an increase in ionic interactions between cationic solutes and the anionic support. Stronger solvent–solute interactions at low temperatures are more effective at shielding the ions from interacting. As the temperature increases, weaker solvent–solute interactions render the ions more interactive, resulting in an increase in ion exchange interactions and increased retention. This is in contrast with purely dispersive interactions, like those provided by alkyl groups and neutral analytes, where increasing temperature increases solvent–solute interactions with resulting decrease in retention, a very familiar relationship in RP-HPLC.

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Conclusions

Because it offers mixed mode reversed-phase (at lower temperature) and ion exchange (at higher temperatures), Discovery HS F5 gives longer retention of basic analytes and the power to alter selectivity and peak spacing using both ionic strength and temperature. Discovery HS F5 has distinct advantages over silica in terms of more separation modes, wider applicability and lessened influence of minor ionic strength and temperature fluctuations. The temperature effect on ion exchange is believed to be a result of changes in the solvation which drives the basic analytes onto the acidic cation exchange sites on the HS F5 phase. Discovery HS F5 offers the flexibility of standard reversed-phase mobile phase modifications, but for basic compounds, Discovery HS F5 also offers the power of ionic strength and temperature, while using LC-MS compatible mobile phases, to optimize retention and resolution.

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References

  1. Naidong, W.; J. Chromatogr. B 2003, 796, 209-224.
  2. Neue, U. D.; Van Tran, K.; Iraneta, P.C.; Alden, B.A.; J. Sep. Sci. 2003, 26, 174-186.
  3. Bell, D. S.; Jones, A. D.; J. Chromatogr. A 2005, 1073, 99-109.
  4. Bell, D. S.; Cramer, H. M.; Jones, A. D.; J. Chromatogr. A 2005, 1095, 113-118.
  5. “Discovery Zr: High pH and High Temperature HPLC” Supelco Bulletin 931 (T102931)

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