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December 2022
eternityxt_armadillo

Improving alkaloid selectivity and resolution by adjusting the pH of the mobile phase

When dealing with UHPLC/ HPLC analysis of drug samples, selectivity and resolution are key parameters. Reversed phase analysis based on silica stationary phases is the most common technique but it is limited to acidic and neutral pH conditions. Kromasil® EternityXT UHPLC/HPLC columns can operate within a wider pH window; therefore, we illustrate the benefit of using these columns to exploit selectivity and resolution at high pH to analyse a mixture of alkaloids.

Nowadays an extensive number of alkaloid drugs are available and administered for numerous ailments. Presently, scientists use both natural sources, such as plants and fungi, as well as apply synthetic chemistry to obtain new alkaloids drugs. Reproducible alkaloid analysis is, therefore, critical for scientists from the initial screening stages in drug discovery to method development and quality control.

When dealing with UHPLC/ HPLC analysis of drug samples, selectivity and resolution are key parameters. Reversed phase analysis based on silica stationary phases is the most common technique but it is limited to acidic and neutral pH conditions. Kromasil® EternityXT UHPLC/HPLC columns can operate within a wider pH window; therefore, we illustrate the benefit of using these columns to exploit selectivity and resolution at high pH to analyse a mixture of alkaloids.

2 chromatograms of separation at low and high pH
  • Conditions
  • Column: Kromasil EternityXT-5-C18, 4.6 × 150 mm
  • Eluent low pH: acetonitrile / water / formic acid (30/70/0.1), pH ~2.7
  • Eluent high pH: acetonitrile / 10 mM ammonium carbonate in water (50/50), pH ~10.5
  • Flow rate: 1 ml/min
  • Temperature: 30 °C
  • Detection: UV @ 220 nm
  • Substances: 1 = lidocaine
  •   2 = papaverine,
  •   3 = noscapine,
  •   4 = Diphenhydramine
  •   s.p. = solvent peak
Figure 1 - Separation of alkaloids: improved resolution at high pH

Figure 1 shows two chromatographic results for an alkaloid mixture. As seen in the figure, at low pH, the compounds tend to elute earlier since they are in ionised form and resolution is limited. On the other hand, at high pH there is complete separation. The most significant changes in retention times between the two pH conditions are given by lidocaine, noscapine and diphenhydramine which, at pH 10.5, are in their neutral state. Also, diphenhydramine’s peak shape improved under basic conditions.

Due to the outstanding mechanical and chemical stability under a wide pH range, Kromasil® EternityXT UHPLC/HPLC columns exhibit consistent results over time. Figure 2 illustrates lifetime studies carried out at low and high pH using Kromasil® EternityXT C18. As seen in the figure, the efficiency of the columns as well as retention times of the compounds remain unchanged after 1000 injections.

plots and chromatograms showing maintained efficiency over 1000 injections
  • Conditions
  • Column: Kromasil EternityXT-5-C18, 4.6 × 150 mm
  • Eluent low pH: acetonitrile / 20 mM potassium phosphate in water, pH 2.5 (50/50)
  • Eluent high pH: acetonitrile / 10 mM ammonium carbonate in water, pH 10.5 (70/30)
  • Flow rate: 1 ml/min
  • Temperature: ambient
  • Detection: low pH: UV @ 220 nm, high pH: UV @ 210 nm
  • Substances: acetophenone and toluene
Figure 2 - Lifetime test at low and high pH: efficiency with toluene over 1000 injections

This excellent performance of Kromasil® EternityXT UHPLC/HPLC columns confirms their utility for discovery and development as well as for routine analysis in the laboratory.