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The importance of overloading studies in method development
– a case study

Preparative HPLC differs from analytical HPLC since it needs to be performed in the nonlinear part of the adsorption isotherm in order to maximize important parameters such as column loading and productivity. In preparative HPLC, method development screening is commonly performed with analytical injections. This makes it possible to establish and compare selectivity in different chromatographic systems. From the screening, one or more candidate phases are selected for a closer preparative study with overloaded injections. This case study illustrates the risk of relying only on selectivity during preparative method development.
The scope of the study was to separate an API (Mw ≈ 900 g/mol) from its impurities and achieve 99.0% purity with as high productivity as possible.

Analytical screening

Several stationary phases and mobile phase systems were screened to find which conditions would give the best selectivity between the target API and the impurities. Below is a table showing the selectivity between the target molecule and its closest front impurity using different stationary phases. A representative chromatogram from the screening process is also shown.

Selectivity between target molecule and its closest front impurity for various stationary phases
Stationary phaseSelectivity (α)
Kromasil 100-3.5-C181.08
Kromasil 100-3.5-C81.07
Kromasil 100-3.5-C41.09
Kromasil 100-5-Phenyl1.09
Kromasil 60-5-CN1.12
analytical injection
Figure 1 Analytical separation on Kromasil 100-3.5-C18
  • Column: Kromasil 100-3.5-C18 4.6 × 150 mm
  • Mobile phase: acetonitrile/water (55/45)
  • Flow rate: 1.5 ml/min
  • Temperature: 30°C
  • Detection: UV @ 210 nm

The initial screening process showed no significant difference in separation between the product and the key impurity when comparing various stationary phases. Furthermore, since the selectivity is low, preparative purification could prove to be problematic with respect to throughput and yield at the desired purity level.

Overloading studies

Since no stationary phase showed any significant advantage for this particular separation, several stationary phases were used during the overloading studies. While many conventional reversed-phase resins showed similar adsorption behavior, and cross contamination of the front impurity, one resin definitely stood out. Below are the conditions, and reconstructed elution profiles of two preparative purifications:

analytical injection
Figure 2a Overloaded injection on Kromasil 100-10-C18
prep injection
Figure 2b Overloaded injection on Kromasil 60-10-CN
  • Overloading conditions

  • Columns: A: Kromasil 100-10-C18 4.6 × 250 mm
  •   B: Kromasil 60-10-CN 4.6 × 250 mm
  • Mobile phase: A: acetonitrile/water (55/45)
  •   B: acetonitrile/water (35/65)
  • Load: 1.0 g/kg stationary phase
  • Flow rate 1 ml/min
  • Temperature 25°C
  • Detection UV @ 254 nm

The reconstructed elution profiles clearly show that using Kromasil 60-10-CN alters the adsorption behavior of the target API resulting in an anti-langmurian (type III) isotherm. In this case, the product peak rises away from the closest impurity resulting in much higher yield at the same purity level. Furthermore, it is evident that the elution behavior of the impurities is also altered. The front impurity shows an elongated elution profile (the so called tag-along effect), while the tail impurity is pushed together (compression effect). This is due to the competitive isotherm which is a result of two different molecules competing for the same site on the resin surface.

In conclusion, this study shows the importance of incorporating overloading studies, along with analytical screening, in order to find the most suitable preparative chromatographic method.

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