Improved loadability for basic APIs
- run at high pH

The main proportion of all synthetic pharmaceutical APIs are basic in nature, and will in many cases exhibit an increased loadability, and hence productivity, at high pH. Basic peptides, oligos, PNAs, etc, will also benefit from high pH separation methods.

Advantages of running at high pH

There are two main advantages by running at high pH:

  1. The high pH conditions will very often result in an improved selectivity between the target compound and the most-critical impurities, and thereby a higher productivity. A rule of thumb says that if the distance between the substances is increased by a factor 2, the loading can be increased by a factor 4.
  2. If basic APIs are considered, these will become neutral at high pH, and very often exhibit a significantly improved loading capacity compared to the protonated and positively charged form that will be seen at low pH. In many cases loading capacity can be increased 10- or even 100-fold as a result of this.

Both factors above result in a possibility to increase loading, yield and purity for the separation, leading to a higher productivity of target substance.

A new generation silica-based packing materials

A new generation of silica-based hybrid type packing materials introduced recently has led to the possibility to vary pH and other conditions widely, to maximize selectivity and loading capacity, and hence productivity, in industrial scale HPLC. Kromasil EternityXT, with a pH user window from 1 to 12, is a good example, allowing superior flexibility to optimize conditions.

Some practical examples

Increased loading by improved selectivity

Selectivity increase at high pH opens for high loadability
Figure 1 Separation of alprenolol/oxprenolol
  • Conditions

  • ColumnKromasil EternityXT-10-C18 4.6 × 250 mm
  • Mobile phaseslow pH: acetonitrile/water, 0.1% TFA pH 1.9 (30/70)
  •  high pH: acetonitrile/water, 50 mM ammonium bicarbonate pH 10.5 (40/60)
  • Flow rate1 ml / min
  • Temperatureambient
  • DetectionUV @ 280 nm

The chromatograms above illustrate well the selectivity increase that can be obtained at higher pH in many cases. Alprenolol and oxprenolol are run at pH 1.9 and 10.5, respectively, and selectivity can be seen to increase from 1.2 to 2.3. In the high pH case the loading was increased to 21 mg crude on the analytical column (≈ 7 mg crude/g packing), while band broadening can be seen to still be quite moderate. It can be estimated that in a real production purification case the loading could probably be increased to > 100 mg crude on the column (> 35 mg crude/g packing) before the peaks would start to merge. The increased loading could be attributed to both factors described above: the increased selectivity, and the loadability increase obtained by the formation of neutral substances at high pH.

Loadability increase at high pH

Loadability of diphenhydramine at low and high pH
Figure 2 Loadability of diphenhydramine
  • Conditions

  • ColumnKromasil EternityXT-10-C18 4.6 × 250 mm
  • Mobile phasesacetonitrile/25 mM ammonium format, pH 3.7 (35/65)
  •  acetonitrile/25 mM ammonium bicarbonate, pH 10.5 (70/30)
  • Flow rate1 ml / min
  • DetectionUV @ 254 nm

The loadability increase that can be obtained at high pH for basic compounds is illustrated in the chromatograms in figure 2, where diphenhydramine is run at pH 3.7 and 10.5, respectively. At low pH the molecule is ionized, leading to a large band broadening even at very low loadings. The same loading at high pH (upper right chromatogram) produces a sharp peak without any tendency to broaden as a function of concentration overload. If the same band broadening should be obtained at high pH the loading has to be increased more than 160 times. Hence, loading capacity is increased by a factor >160!


The invention of new hybrid phases like Kromasil EternityXT, being available in particle sizes 10 µm and above in large volumes, means that the flexibility to optimize conditions, to maximize productivity in large scale, is greater than ever before. Continuous use at pH values far beyond the normal limits for a silica-based material is now a reality.

In addition, it is possible to sanitize or regenerate in-column (cleaning in place, or CIP) using conditions previously reserved only for polymeric resins. This gives you the best of both worlds: highest performance and excellent stability at high pH values.


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