Advantage of Chemically and Mechanically Stable Reversed Chromatography Phases from Kromasil

By: Frank Limé, Robert Fredriksson, and Cecilia Mazza, AkzoNobel

Reversed-phase UHPLC and HPLC are the most common techniques for screening samples in discovery laboratories, method development and quality control. Traditional silica-based materials are also the most popular materials in HPLC and UHPLC, however they are limited to a pH range between 2 and 8. Working outside this range could lead to retention time changes, loss of performance and consequently, higher laboratory costs.

Stationary phases that can work beyond pH 8 have increased flexibility in analytical and discovery laboratories since these materials allow for free choice of buffers, wider pH window for screening potential drug candidates and biopharmaceuticals. Pharmaceutical industries and producers of peptide and oligonucleotide APIs, dealing with tough sample mixtures, require stationary phases with high mechanical stability and chemical resistance at higher pH than the traditional silica materials offer. This work illustrates a new class of columns that are both chemically and mechanically stable, can be exposed to wider range of pH and high concentrations of NaOH for eluting tough compounds and impurities, relevant benefits for medicinal chemists as well as bio-chromatographers.

Quick screening of peptide is a critical success factor for the biotechnology industry. Figure 1 illustrates the benefit of working at low and high pH to explore selectivity and resolution power in analytical chromatography and subsequent, effective scale-up. The mixture used in this study contains three peptides, but the low pH conditions result in a co-elution of two out of three compounds at pH 1.9. By increasing the pH of the mobile phase to 11 complete resolution of the three peaks is achieved. As seen in the figure, there is selectivity reversal between Antiotensin I and III. These results illustrate elution order changes with pH which can benefit scientists.

Angiotensin I Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu
Angiotensin II Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
Angiotensin III Arg-Val-Tyr-Ile-His-Pro-Phe

Figure 1. Separation of Angiotensin I, II, and III using a 50 x 2.1 mm EternityXT 1.8-C18 column.

Figure 1. Separation of Angiotensin I, II, and III using a 50 x 2.1 mm EternityXT 1.8-C18 column.
CONDITIONS (LOW pH); mobile phase: A 0.1% TFA in water (pH 1.9), B 0.1% TFA in acetonitrile; gradient: 0 min 9% B, 10 min 36% B; flow rate: 0.7 mL/min.; detection: UV @ 220 nm.
(High pH); mobile phase: A 0.1% ammonium hydroxide in water (pH 11.0), B acetonitrile; gradient: 0 min 5% B, 10 min 40% B; flow rate: 0.7 mL/min; detection: UV @ 225 nm

 

Figure 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 ionized 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 neutralized at 2 units higher than the pKA, exhibiting longer retention times.

<b>Figure 2.</b> Selectivity change on a 50 x 2.1 mm EternityXT 2.5-C18 using 20 mM sodium phosphate at pH 2.1, 7.2 and 11.3.

Figure 2. Selectivity change on a 50 x 2.1 mm 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, B acetonitrile; gradient: 0 - 0.5 min 10% B, 5.5 min 50% B; flow rate: 1.5 mL/min; detection: UV @ 254 nm

 

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. Figure 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. The materials were washed in various concentrations of NaOH for 10 column volumes and the mobile phase eluent was collected. After that, silica content was analyzed using ICP-AES. As shown in the figure, the new phase withstands NaOH concentration 10 times higher than regular silica. This new class of material leaked 88 ppm silica at 1M NaOH compared to 520 ppm for the regular silica at 0.1M NaOH.

Figure 3. Comparison of regular C18 and mechanically/chemically stable C18 silica leakage

Figure 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).

 

The 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.