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Using EternityXT stationary phase for analysis and purification under reversed-phase chromatography

Silica based chromatography materials are typically considered stable in the range of pH 2 to pH 8 and separations and purifications are usually carried out under these conditions. However, there are applications where higher pH could be used to achieve better purifications to attain higher purity and/or yield. EternityXT stationary phase materials are ideal for production facilities that need to work with an extended range of pH because it is stable under low and high pH conditions.

An extension of the pH to more alkaline conditions can improve the selectivity and resolution between the main compound of interest and its impurities, increase loading or the productivity of, for example, amine containing substances where pH > 8 could be beneficial.

The chemical stability of EternityXT material was tested for an extended period of time and compared with classic silica materials available in the market today by monitoring the change in k' with time for the phenanthrene at elevated temperature using 10 mM carbonate buffer at pH 10.5. The results are presented in Figure 1. As seen in the figure, changes of k’ are reported against time for: Kromasil EternityXT 10 μm C18, C8 and C4 as well as Kromasil 10 μm C18 and a competitor C18 material. Eternity XT materials show very high chemical stability throughout the study independent of the derivatization. Still, Kromasil EternityXT C18 and C8 remain unchanged throughout the studies, C4 experiences a slight decrease in k’ with time, but the %k’ varies only by 5% within 120 hours under harsh conditions.

As expected traditional silica materials have lower chemical stability at pH 10.5. Still Kromasil 10 μm C18 has been shown to have superior chemical stability beyond its traditional pH range of operation, which is reflected in the results presented in figure 1.

plot of evolution of retention times for phenanthrene for various C18 materials
Figure 1: Chemical stability of 10 μm C18 materials at high pH and high temperatures
  • Conditions
  • Substance:Phenanthrene
  • Hydrolysis conditions
  • Mobile phase: acetonitrile / 10 mM ammonium carbonate, pH 10.5 (10/90)
  • Flow rate: 0.2 ml/min
  • Temperature: 60 °C
  • Analytical conditions
  • Mobile phase: acetonitrile / water (70/30)
  • Flow rate: 1 ml/min
  • Temperature: 25 °C

To further depict the chemical stability of the Kromasil EternityXT materials, analytical chromatograms for monitoring the hydrolysis study for one of the phases are shown in Figure 2. These are chromatograms from analytical injections for Kromasil EternityXT 10 μm C18 with 12 hour hydrolysis between each injection. The top chromatogram corresponds to the beginning of the study and the bottom chromatogram is the last result from the test. The results shown here indicate that the k’ remains constant for all the compounds in the sample mixture. It is to note that the same sample vial was used for this study. As it is known, toluene is a volatile compound, and while toluene's retention time is maintained during this study, its peak height and peak area decrease with time, due to the evaporation of the compound in the sample vial. Finally, these results show that Kromasil EternityXT 10 μm C18 remains unchanged during the hydrolysis confirming the chemical stability of the material.

chromatograms from the hydrolysis of a C18 material
Figure 2: Analytical chromatographic results of the hydrolysis study of EternityXT 10 μm C18 at high pH and high temperatures
  • Conditions
  • Substances: 1 = sodium nitrate, 2 = dimethyl phthalate, 3 = toluene, 4 = biphenyl, 5 = phenanthrene
  • Analytical conditions
  • Mobile phase: acetonitrile / water (70/30)
  • Flow rate: 1 ml/min
  • Temperature: 25 °C

Similarly to small organic bases that have been shown to have improved separation, resolution and loading at high pH, certain peptide separations and isolations can benefit from being carried out under basic pH. Figure 3 illustrates the purification of a 3.5 kDa peptide at pH 8 where classic silica material is normally used and compared with pH 9.5 using EternityXT. As shown in Figure 3, the purity and yield are superior when carrying out the purification at higher pH.

This study has shown that Kromasil EternityXT organosilane enforced silica functionalized with C4, C8 and C18 have high chemical resistance under harsh alkaline conditions while maintaining the high performance. This stability at high pH expands the working range for users when they need to separate and purify compounds such as the peptide example depicted here.

chromatogram for peptide purification at high pH
Figure 3: Peptide purification at high pH
  • Preparative conditions
  • Column: Kromasil EternityXT-C18 4.6 × 250 mm
  • Mobile phases
  • @ pH 8: n-propanol / 100 mM TRIS buffer, pH 8.0
  • @ pH 9.5: n-propanol / 100 mM ammonium carbonate pH 9.5
  • Gradient: 0-30 min: 5%, 35 min: 10%, 40 min: 23%, 100 min: 31%, 110 min: 45% n-propanol
  • Flow rate: 0.55 ml/min
  • Fraction analytsis
  • Column: Kromasil 100-3.5-C4 4.6 x 100 mm
  • Mobile phase: acetonitrile / 25 mM phosphate buffer, pH 2.5
  • Gradient: 0 min: 43%, 40 min: 46.5% acetonitrile
  • Flow rate: 1 ml/min
  • Temperature: 25 °C
  • Detection: UV @ 214 nm

Parts of this note were previously published as an article on Chimica oggi - Chemistry Today