New Ascentis™ C8: Enhanced Hydrophobic Retention and Unique Hydrophilic Selectivity Make it Ideal for Method Development and LC/MS Applications

Reporter EU Volume 19

Introduction

Supelco is pleased to introduce the newest member of our Ascentis line of premiere HPLC columns for small molecule separations: Ascentis C8. This new product expands the user’s choice of stationary phase selectivity to optimise chromatographic resolution, and complements the well established C18 and the orthogonally-selective RP-Amide phases. Ascentis C8 is highly reproducible, highly retentive and exhibits enhanced selectivity towards polar compounds. It excels in both highly aqueous and highly organic mobile phases. Like all members of the Ascentis family, Ascentis C8 is available from microbore (1.0 mm I.D.) to preparative (up to 50 mm I. D. on request) dimensions and is especially suited for LC/MS applications.

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The Ascentis family

The Ascentis family of HPLC columns continues the tradition of innovative yet practical HPLC column technology from Supelco. Ascentis was developed at Supelco’s R&D laboratories in Bellefonte, Pennsylvania, U.S.A. using a powerful three-way combination of ultra pure silica, proprietary “Surface-Optimised Technology” and a unique approach to endcapping. The “Surface-Optimised Technology” gives high, yet stable bonded phase coverage. The result is one of the most inert, low-bleed and reproducible HPLC materials available today. Ascentis is ideal for LC/MS applications because it is extremely low bleed and has high reversed-phase retentivity that allows the use of high organic content mobile phases.

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Leveraging stationary phase selectivity to improve chromatographic resolution

Resolution in HPLC is governed by column efficiency (N), retention (k or k’) and chromatographic selectivity (α). Of these three parameters selectivity has the greatest overall effect on resolution (1). The relationship between selectivity and resolution is nearly linear and does not have a limit as do the relationships between resolution and efficiency or retention (Figure 1).


Figure 1. Effect of selectivity (α) on resolution in HPLC. (from reference 1)

Selectivity is influenced by both the composition of the mobile phase and the stationary phase. With the Ascentis HPLC column product line now comprising three different bonded phase chemistries, optimal resolution can be achieved by using Ascentis C18, the standard in RP-HPLC, the unique polar-embedded Ascentis RP-Amide, which gives orthogonal selectivity to C18, or the new Ascentis C8 phase.

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Materials and methods

The comparison of retention of pentylbenzene between Ascentis C8 and competitive C8 phases was carried out as described by Euerby, et al (2). The instrument was a Waters 2690 HPLC system equipped with a 2996 photodiode array detector. Experiments to demonstrate the differences between the three Ascentis phases were performed using 150 x 4.6mm I.D. columns packed with 5μm particles. Solvents were of high purity LC-MS grade and standards were from Sigma-Aldrich (see Product Listing for details). Separation was performed on an Agilent 1100 HP HPLC system with an 1100 multiple wavelength detector. Details are given in the chromatograms.

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Enhanced hydrophobic retention of AscentisTM C8

The properties and specifications of Ascentis C8 are given in Table 1. The comparison of Ascentis C8 with competitive C8 phases according to their hydrophobic retention is shown in Figure 2. Capacity factor (k or k') of pentylbenzene was determined as described by Euerby (2) and compared with published data. The results in Figure 2 show that the Ascentis C8 column has the largest capacity factor of the columns tested, indicating its superior hydrophobic retentivity. High capacity is important to HPLC for three reasons. First, within a certain range, increasing k increases resolution (see Figure 1). Second, increased hydrophobic retention permits use of higher organic LC/MS mobile phases that desolvate more rapidly. Third, high capacity means higher sample loads in preparative separations.


Table 1. Properties of and specifications of Ascentis C8


Figure 2. Comparison of hydrophobic retention on C8 HPLC columns.

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Unique selectivity and retention of polar compounds on Ascentis C8

Besides being unique among C8 phases, Ascentis C8 is also different from the other Ascentis phases. This is demonstrated in the separation of low molecular weight organic acids shown in Figure 3. Three observations are noteworthy. First, all the Ascentis phases gave excellent resolution and peak shape of these often difficult analytes. Second, the different elution patterns show that the Ascentis C8 has different selectivity than the C18 and RP-Amide. The order of elution of the organic acids is the same for Ascentis C8 and C18. However, on the Ascentis RP-Amide column elution order of acrylic acid and fumaric acid is reversed, demonstrating the orthogonal selectivity of the polar embedded RP-Amide phase. Third, the Ascentis C8 is more retentive toward these polar compounds than the C18.


Figure 3. Organic acids demonstrate selectivity differences between Ascentis C18, C8 and RP-Amide phase columns.

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Conclusions

Ascentis C8 is the latest addition to the Ascentis HPLC column product line. Like all Ascentis columns it provides:

  • Excellent peak shape for difficult compounds
  • Low bleed and high retentivity for LC/MS
  • Rugged, reproducible separations

However, Ascentis C8 also provides:

  • Increased hydrophobic retention compared to competitive C8 phase
  • Different selectivity than Ascentis C18 and RP-Amide phases
  • Enhanced retention of hydrophilic compounds compared to C18

The Ascentis family of HPLC columns, which includes the new Ascentis C8, leverages all three variables of the resolution equation, efficiency, retention, but most importantly, selectivity, to give users maximum flexibility in optimizing HPLC separations.

For more information on Ascentis, please call or consult our website: www.sigmaaldrich.com/ascentis

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References

  1. Zhao, J. H.; Carr, P. W.; Analytical Chemistry, 1999, 71(14), 2623-2632.
  2. Euerby, M. R.; Peterson, P.; J. Chromatogr. A, 2003, 994, 13-36.

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