Ascentis® Express Peptide ES-C18 U/HPLC Columns

Separation of peptides, small proteins and other high molecular weight compounds

The Ascentis® Express Peptide ES-C18 columns are best used with mobile phases that are mixtures of acetonitrile and water or methanol and water. Higher levels of the organic solvent component will typically reduce the retention of the sample compounds.

Using elevated temperatures (e.g., 40 – 100 °C) will reduce the viscosity of the mobile phase and allow the use of faster flow rates and lower column pressure for high sample throughput. Gradient-elution techniques using 5-10% organic component as the initial mobile phase and increasing to 100% organic component as the final mobile phase can typically effect separations of complex sample mixtures in minimal time.

Ionizable compounds, such as acids and bases, are generally best separated with mobile phases buffered at a pH of 2 to 3. The use of 10-50 mM buffers is always recommended for optimum results and long-term stability when separating ionizable compounds.

Ascentis® Express Peptide ES-C18 columns utilize a sterically protected C18 bonded-phase with extremely high resistance to acid-catalyzed hydrolysis of the siloxane bond that attaches the C18 chain to the surface. Thus, the combination of low pH and elevated temperature operation of the column is well tolerated.

Ascentis® Express Peptide
ES-C18 Specifications
Silica: Type B (High purity silica)
Particle Platform: Superficially porous
particles (SPP)
Phase Chemistry: Diisobutyloctadecyl
Particle Size: 2.0 µm / 2.7 µm / 5.0 µm
Pore Size: 160 Å
Carbon Load: 4% / 4.6% / 4%
Surface Area: 65 m²/g / 90 m²/g / 60 m²/g
pH Range: 1 - 8
Max Temperature
at Low pH:
90 °C
Max Temperature
at High pH:
40 °C
Endcapped: No


Peptide separations are efficiently conducted using low pH mobile phase modifiers, often at 0.01-0.1% concentration, most popularly employing trifluoroacetic acid (TFA), and the related perfluorocarboxylic acids, pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA). These acids exhibit desirable low UV transparency, volatility, and peptide ion pairing properties. Additional opportunities for low pH operation include the normal short chain carboxylic acids, formic acid and acetic acid, as well mineral acids, such as phosphoric acid (0.001-0.02 M).

Pharmaceutical Peptide Separation

Many peptides have been investigated as therapeutic pharmaceutical drugs and are active vasodilators, vasoconstrictors, hormones, and neuropeptides. Using reversed-phase HPLC, it is possible to solve the tasks of identification, purity monitoring, and quantitative analysis in may cases, including those where the application of other methods is impossible.

Synthetic Peptide Separation

The difficulty with synthetic peptides involves the production of many “deletion variants”. A deletion may occur at any point in the peptides synthesis and so several versions of the peptide are produced which are absent one, two or three amino acids from the desired product. This makes for an interesting chromatographic problem because the resultant peptide mix contains peptides that are very similar in structure.

Peptide Mapping

Protein analysis and characterization has become more crucial due to many biopharmaceutical advances. Peptide mapping via LC-MS is one such technique that is commonly used today. A typical procedure involves the preparation of a tryptic digest from the protein, with subsequent characterization using reversed-phase, gradient HPLC separation followed by mass spectral analysis and database search.

Featured Applications:

  • Basic Peptides
  • Peptide Test Mix
  • Carbonic Anhydrase Tryptic Digest
  • Small Proteins

C18 Fused-Core® phases designed for peptide and protein UHPLC and HPLC separation

Ascentis® Express Peptide ES-C18 is a high-speed, high-performance liquid chromatography column based on a superficially porous Fused-Core® particle design. The Fused-Core® particle provides a thin porous shell of high-purity silica surrounding a solid silica core. This particle design exhibits very high column efficiency due to the shallow diffusion paths. Three different particle sizes are available for your specific needs:

Fused-Core® particle size 2 µm

2.0 µm particles

Best Fused-Core UHPLC Column

An optimized solution for high-throughput small molecule analysis


Fused-Core® particle size 2.7 µm

2.7 µm particles

Fast HPLC on ANY System

A practical solution that delivers UHPLC performance from any HPLC


Fused-Core® particle size 5 µm

5.0 µm particles

The Lab Work-Horse Column

True plug and play solution for improving existing 3 or 5 µm fully porous particle HPLC columns

HPLC Guard Column


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