48 CHROMATOGRAPHY


Fig. 3. compares the performance of a regular silica C18 column with the new chemically and mechanically stable phase presented in this work by cleaning them with NaOH. Te materials were washed in various concentrations of NaOH for 10 column volumes and the mobile phase eluent was collected. After that, silica content was analysed using ICP-AES. As shown in Fig. 3, the new phase withstands NaOH concentration 10 times higher than regular silica. Tis new class of material leaked 88ppm silica at 1M NaOH compared to 520ppm for the regular silica at 0.1M NaOH.


Te work shown here illustrates that this new class of chemically and mechanically stable columns can operate under a wide range of pH and be exposed to high concentrations of NaOH, facilitating the chromatographic work in the laboratory as well as in scale-up.


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Fig. 2. Selectivity change on a 50 x 2.1mm EternityXT 2.5-C18 using 20 mM sodium phosphate at pH 2.1, 7.2 and 11.3. Conditions: mobile phase A 20 mM sodium phosphate, mobile phase B acetonitrile, gradient 0 - 0.5 min 10% B, 5.5 min 50% B, flow rate 1.5mL/min, detection UV @ 254nm.


n Angiotensin III Arg-Val-Tyr- Ile-His-Pro-Phe


Fig. 2. shows selectivity changes due to three distinct pH conditions. As seen in the figure, by using a sample mixture of neutral (fenuron), acidic (nitrobenzoic acid, pKA 3.7) and basic (procaine, pKA 9.0) compounds, the user can control retention time and achieve selectivity reversal with pH.


Basic drugs are in ionised form when the pH in the mobile phase is lower than the pKA, therefore basic compounds will exhibit low retention times at low pH in reversed phase chromatography. On the other hand, basic compounds are neutralised at two units higher


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than the pKA, exhibiting longer retention times.


Medicinal chemists and scientists dealing with dirty samples – such as those with incomplete reactions or strongly retained impurities – find it challenging to maintain UHPLC and HPLC column performance when exposing them to harsh conditions.


Until now, chromatographic phases presented poor performance when exposed to caustic conditions.


However, by using the new organic/inorganic reinforced silica, it is possible to have columns that can resist tough conditions.


Fredrik Limé, Robert Fredriksson and Cecilia Mazza are with AkzoNobel, producer of the Kromasil range of products, in Sweden. www.kromasil.com


“Until now, chromatographic phases presented poor performance when exposed to caustic conditions.”


Fig. 3. Comparison of regular C18 and mechanically/chemically stable C18 silica leakage when the stationary phase is exposed to cleaning in place (CIP) using a given NaOH solution/ethanol (50/50).


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