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Advantage of chemically and mechanically stable reversed chromatography phases

30th May 2014

Posted By Paul Boughton


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), mobile phase 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. Conditions (high pH): mobile phase A 0.1% ammonium hydroxide in water (pH 11.0), mobile phase B acetonitrile, gradient 0 min 5% B, 10 min 40% B, flow rate 0.7 mL/min, detection UV @ 225 nm
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, mobile phase B acetonitrile, gradient 0 - 0.5 min 10% B, 5.5 min 50% B, flow rate 1.5 mL/min, detection UV @ 254 nm
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)

Fredrik Limé, Robert Fredriksson and Cecilia Mazza discuss a new class of chemically and mechanically stable columns that can operate under a wide range of pH.

Reversed phase ultra-high performance liquid chromatography (UHPLC) and high-performance liquid chromatography (HPLC) are the most common techniques for screening samples in discovery laboratories, method development and quality control. Traditional silica-based materials are the most popular materials in HPLC and UHPLC, however they are limited to a pH range between 2 and 8. Working outside this range can 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 active pharmaceutical ingredients (APIs), dealing with tough sample mixtures, require stationary phases with high mechanical stability and chemical resistance at a higher pH than the traditional silica.

The work here illustrates a new class of columns that are both chemically and mechanically stable, can be exposed to a wider range of pH and high concentrations of NaOH for eluting tough compounds and impurities, and their relevant benefits for medicinal chemists as well as bio-chromatographers.

Fast peptide screening

Quick screening of peptide is a critical success factor for the biotechnology industry. Fig. 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 below, there is selectivity reversal between Antiotensin I and III. These results illustrate elution order changes with pH that 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

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 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.

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. 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 analysed using ICP-AES. As shown in Fig. 3, the new phase withstands NaOH concentration 10 times higher than regular silica. This new class of material leaked 88ppm silica at 1M NaOH compared to 520ppm for the regular silica at 0.1M NaOH.

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.

For more information at www.scientistlive.com/eurolab

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





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