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Traditional silica-based reversed phase materials very often have an upper limit for use at neutral to slightly basic pH. At higher pH levels, the silica matrix starts to dissolve. With Kromasil® Classic RP phases this limit has been moved up to pH 9.5, and in some cases, even higher. With the Eternity platform, the boundaries are moved beyond what could be expected from the strongest silica matrix.

Up to pH 12

The first generation of Eternity C18 set a new standard for column lifetime expectations for hybrid materials. With EternityXT C18, users get the flexibility to develop methods for quick UHPLC analysis as well as isolation and large-scale purification between pH 1-12, for long-term use.

Long-term chemical stability

In theses figures the long-term chemical stability at low and high pH is shown. Low pH conditions simulate a very long-term use by applying an elevated temperature and a highly aqueous mobile phase. The hybrid materials still show excellent stability, with very low shift in k’ over time. High pH conditions also include highly aqueous buffer and elevated temperature. It has been shown that carbonate buffer is especially aggressive when used with silica-based packing materials, but it has little effect on the retention factor for EternityXT, due to the very dense C18 derivatization and the EternityXT gradient, protecting the silica matrix.
  • Conditions
  • Column size: 4.6 x 250 mm
  • Acidic hydrolysis
  • Mobile phase: methanol / water / trifluoroacetic acid (5/95/0.1), pH ≈ 1.9
  • Flow rate: 0.2 ml/min
  • Temperature: 80 °C
  • Basic hydrolysis
  • Mobile phase: acetonitrile / 10 mm ammonium carbonate, pH 10.5 (10/90)
  • Flow rate: 0.2 ml/min
  • Temperature: 60 °C
  • Chromatographic test conditions
  • Mobile phase: acetonitrile / water (70/30)
  • Test substance: phenanthrene
  • Flow rate: 1 ml/min
  • Detection: UV @ 254 nm

Flexibility at your fingertips

The main proportion of all synthetic pharmaceutical APIs are basic in nature, and will exhibit an increased loadability, and hence productivity, at a high pH. Basic peptides, oligos and PNAs will also benefit from high pH separation methods. in addition, it is possible to sanitize or regenerate Kromasil® EternityXT in-column (CIP) even using 1 M NaOH when necessary. 1 m NaOH is a standard in biochromatography for polymeric resins.

With Kromasil® EternityXT, users have the flexibility to develop analytical and separation methods for virtually the entire pH range, and to sanitize or regenerate the column using conditions previously reserved only for polymeric resins. This gives scientists the best of both worlds: highest performance and excellent stability at high pH.

Chemical stability – CIP conditions

Effect of CIP on silica based materials
In purification of polypeptides and proteins it is common to use high pH CIP processes to remove irreversibly adsorbed depositions on the packing material. The figures show retention time change after a number of CIP cycles, and the leakage of silicon during the process. For 0.5 M NaOH it can be seen that the leading hybrid C18 competitor exhibits a much lower stability compared to EternityXT, both in terms of retention time change and leakage of silicon. At 1.0 M NaOH, i.e. standard cleaning conditions for polymeric materials, EternityXT still shows very high chemical stability, while a regular C18 competitor is quickly impaired already at ten times lower hydroxide concentration, i.e. 0.1 M NaOH.
  • Conditions
  • Column size: 4.6 x 250 mm
  • Mobile phase: 10 column volumes of NaOH solution / ethanol (50/50)
  • Flow rate: 1 ml/min for 10 column volumes (contact time 41.5 min)
  • Test substance: nortriptyline at pH 7.0
  • Temperature: ambient

Chromatographic performance
– EternityXT vs polymeric packing

Stability that matches a polymeric material
It is well known that PS/DVB-based packing materials exhibit very high chemical stability at high pH, allowing cleaning steps involving for example 1 M NaOH. However, the material can unfortunately not compete with silica-based packing materials in terms of chromatographic performance. The graph shows a typical comparison between a silica- and a polymer-based packing material: Kromasil® EternityXT and the market leader for PS/DVB-based packings, where identical conditions have been used. The chromatogram shows a preparative separation of insulin, where it can be seen that the silica-based material, EternityXT, has markedly sharper peaks, with roughly only 50% of the band broadening seen on the PS/DVB-based material. Both analytical efficiency and loading capacity is significantly better for EternityXT.
With Kromasil® EternityXT it is possible to obtain the high separation power associated with silica-based materials, and at the same time experience very high chemical stability at high pH, as can be seen in the figures.
  • Conditions
  • Column size: 4.6 x 250 mm
  • Mobile phase: ethanol / ammonium acetate, 0.2 mM
  • Gradient: for EternityXT; 0 min: 30%, 60 min: 38% ethanol
  •   for PS/DVB; 0 min: 34%, 60 min: 42% ethanol
  • Flow rate: 0.7 ml/min
  • Temperature: 25 °C
  • Detection: UV @ 280 nm