CHROMATOGRAPHY 47
Advantage of chemically and mechanically stable reversed chromatography phases
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.
R
eversed 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.
Fig. 1. Separation of Angiotensin I, II, and III using a 50 x 2.1mm 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 @ 220nm. 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 0.7mL/min, detection UV @ 225nm.
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.
Te work here illustrates a new class of columns that are both
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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.
Te 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. Tese results illustrate elution order changes with pH that can benefit scientists:
n Angiotensin I Asp-Arg-Val-
Tyr-Ile-His-Pro-Phe-His-Leu n Angiotensin II Asp-Arg-Val- Tyr-Ile-His-Pro-Phe
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