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Ascentis Express Fused-Core® HPLC Columns

Applications for Peptide ES-C18 Columns

A Breakthrough in Bioseparations Performance

Applications - Peptides, Peptide Mapping, Small Proteins
Ordering Information

Ascentis Express Peptide ES-C18 is a high-speed, high-performance liquid chromatography column based on a new 160Å 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 for high MW solutes (up to 20 kDa) due to the shallow diffusion paths in the 0.5-µm thick porous shell and the small overall particle size of 2.7-µm. The non-end-capped, sterically protected C18 bonded phase of Ascentis Express Peptide ES-C18 provides a stable, reversed phase packing with a pore structure and pore size that is optimized for reversed-phase HPLC separations of peptides and polypeptides, using typical acidic mobile phases favored for protein structure-function and proteomic applications.

Column Characteristics
The fused-core particle has a surface area of ~ 80 m2/g and an average pore size of 160Å. The fused core particles are 30% to 50% heavier than commercially available totally porous particles due to the density of the solid cores. Therefore, the effective surface area per column is similar to columns packed with totally porous particles having surface areas of 150 - 200 m2/g. Ascentis Express Peptide ES-C18 has a pH range from 1 to 9 and is capable of operation up to 100 °Celsius.

Column Specifications
Silica: High Purity Type B
Phase: Sterically protected C18
pH range: 1 – 9
Temperature: 100 °C
Average Pore Size: 160 Å
Surface Area, nitrogen: 80 m2/g
Pore Volume: 0.30 mL/g

Applications - Peptides, Peptide Mapping, Small Proteins back to top

The Ascentis Express Peptide ES-C18 columns are best utilized 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 ºC) 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 often can 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 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. 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 Peptides
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 Peptides
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.

Figure 1. Basic Peptides
Column: Ascentis Express Peptide ES-C18, 10 cm x 2.1 mm I.D. (53306-U)
Mobile Phase: A: 0.1% additive in water
B: 25:75, (0.4 % additive):acetonitrile
Additive: formic acid, pH 3.5 (adjusted with ammonium hydroxide)
Gradient: initial = 15% B, slope = 2% MeCN / column volume
Flow rate: 0.3 mL/min
Temperature: 35 °C
Det.: ESI(+)-TOF
Injection: 1 µL
Sample: 5 mg/L peptide 1 & 3, 1 mg/L peptide 2, 15 mg/L peptide 4
Peptide probes: (listed in order of elution):
1. ac-GGGLGGAGGLKG monoisotopic mass: 941.5
2. ac-KYGLGGAGGLKG monoisotopic mass: 1118.6
3. ac-GGAVKALKGLKG monoisotopic mass: 1139.7
4. ac-KYALKALKGLKG monoisotopic mass: 1330.8
Figure 2. Peptide Test Mix
Column: Ascentis Express Peptide ES-C18, 10 cm x 4.6 mm I.D. (53324-U)
Mobile Phase: A: 90:10, (0.1 % TFA):acetonitrile
B: 25:75, (0.1% TFA):acetonitrile
Gradient: initial = 0% B to 50% B in 15 min.
Flow rate: 1.5 mL/min
Temperature: 30 °C
Det.: UV at 220 nm
Injection: 5 µL
Sample: The test mix employed contains the following peptides
1. Gly-Tyr MW = 252
2. Val-Tyr-Val MW = 379
3. Met Enkephalin MW = 574
4. Angiotensin II MW = 1032
5. Leu-Enkephalin MW = 555
6. Ribonuclease MW = 13,700
7. Bovine Insulin MW = 5733
Figure 3. Carbonic Anhydrase Tryptic Digest
Column: Ascentis Express Peptide ES-C18, 10 cm x 4.6 mm I.D. (53324-U)
Mobile Phase: A: 0.1 % TFA in water
B: 40:60, (0.1% TFA):acetonitrile
Gradient: initial = 3% B to 100% B in 53 min.
Flow rate: 1.0 mL/min
Temperature: 30 °C
Det.: UV at 215 nm
Injection: 20 µL
Figure 4. Small Proteins
Column: Ascentis Express Peptide ES-C18, 10 cm x 4.6 mm I.D. (53324-U)
Mobile Phase: A: 90:10, (0.1 % TFA):acetonitrile
B: 25:75, (0.1% TFA):acetonitrile
Gradient: initial = 25% B to 40% B in 15 min.; then to 60% B at 20 min.
Flow rate: 1.5 mL/min
Temperature: 30 °C
Det.: UV at 220 nm
Injection: 4µL
Sample: 1. Ribonuclease MW = 13,700
2. Porcine Insulin MW = 5,780
3. Bovine Insulin MW = 5,730
4. Human Insulin MW = 5,800
5. Cytochrom C MW = 12,327
6. Lysozyme MW = 14,700

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