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Discovery BIO Wide Pore C8 |
HPLC Columns and Capillaries |
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Bonded Phase: |
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Covalently-bonded octylsilane (C8), endcapped |
Silica: |
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Spherical, 3µm, 5µm and 10µm diameters, 300Å pore diameter, |
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high purity (<10ppm metals) |
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Solutions for Protein and Peptide Separation Challenges |
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Proteomics and biotherapeutics: high resolution of synthetic peptides and peptide maps
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LC/MS; no-bleed, capillary dimensions, and pH stability
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Peptide purification: Scalable from analytical to preparative
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Intermediate hydrophobicity makes it a good method development starting point
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RESOLUTION OF ANGIOTENSINS AT NEUTRAL pH |
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Human Angiotensins I, II, & III contain a seven amino acid sequence conserved between the three types. Differences between the three peptides vary only by three amino acid residues. Angiotensin II differs from Angiotensin III by an N-terminal aspartate; and Angiotensin I contains an N-terminal asparate as well as two C-terminal amino acids (histidine and leucine). At low pH conditions (most typical for peptide chromatography), angiotensins II & III are not resolved since the asparate residue contains no charge (pH 2.0). Therefore, resolution of the two angiotensins is typically conducted at alkaline pH. Note however that baseline resolution was achieved at pH 7 using Discovery BIO Wide Pore C8.
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Column:
1. Angiotensin II (1.67g/L)
2. Angiotensin III (1.67g/L)
3. Angiotensin I (1.67g/L)
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D |
R |
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Y |
I |
H |
P |
F |
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R |
V |
Y |
I |
H |
P |
F |
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D |
R |
V |
Y |
I |
H |
P |
F |
H |
L |
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Discovery BIO Wide Pore C8, 15cm x 4.6mm, 5µm; Mobile Phase: (A) 10mM NH4H2PO4/ NH4OH, pH 7; (B) 50:50, (20mM NH4H2PO4/ NH4OH, pH 7):MeCN; Flow Rate: 1mL/min; Temp: 30°C; Detection: 215nm; Injection: 6µL; Gradient: 30-60% B in 15 min
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INTERMEDIATE HYDROPHOBICITY
OF DISCOVERY BIO WIDE PORE C8 |
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Because of its intermediate hydrophobicity (less hydrophobic than C18; more hydrophobic than C5), Discovery BIO Wide Pore C8 is an ideal phase for early method development.
Column: Discovery BIO Wide Pore C18, C8, & C5, 15cm x 4.6mm, 5µm; Mobile Phase: (A) 95:5, (0.1% TFA in H2O):(0.1% TFA in MeCN); (B) 50:50, (0.1% TFA in H2O):(0.1% TFA in MeCN); Flow Rate: 1mL/min; Temp: 30°C; Detection: 215nm; Injection: 50µL; Sample: caboxymethylated apohemoglobin tryptic digest; Gradient: 0-100% B in 65 min
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STABILITY OF DISCOVERY BIO WIDE PORE C8 |
AT ALKALINE AND ACIDIC pH |
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Alkaline pH Stability
Retention times of the sample components remained constant after 21,000 bed volumes were passed through the column.
Column: Discovery BIO Wide Pore C8, 5cm x 4.6mm, 5µm; Mobile Phase: 25mM potassium phosphate, pH 8 : MeOH (95:5); Flow Rate: 2mL/min; Temp: 35°C; Detection: 254nm
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Acidic pH Stability
Retention times of the sample components remained constant after 40,000 bed volumes were passed through the column.
Column: Discovery BIO Wide Pore C8, 5cm x 4.6mm, 5µm; Mobile Phase: (A) 0.1% TFA in 5% MeCN; (B) 0.1% TFA in 75% MeCN; Flow Rate: 2mL/min; Temp: 35°C; Detection: 220nm; Gradient: 0-22% B in 22min
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RESOLUTION OF INSULINS FROM VARIOUS SPECIES |
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Insulin is composed of two peptide chains: a 21 amino acid A chain and a B chain of 30 amino acids. The two chains are linked via two disulfide bonds, and much of the sequence is conserved across species (including the locations of the disulfide bonds) allowing for a very similar three dimensional conformation regardless of the species type. For example, porcine insulin differs from human by only one amino acid, and bovine differs by three amino acids. In the application, Discovery BIO Wide Pore C8 was able to resolve all three types of insulin.
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Column:
1. Bovine insulin (5µg)
2. Human insulin (5µg)
3. Porcine insulin (5µg)
Discovery BIO Wide Pore C8, 15cm x 4.6mm, 5µm; Mobile Phase: (A) 71:29, (0.1% TFA in H2O):(0.1% TFA in MeCN); (B) 68:32, (0.1% TFA in H2O):(0.1% TFA in MeCN); Flow Rate: 1mL/min; Temp: 30°C; Detection: 215nm; Injection: 50µL; Gradient: 0-100% B in 30min |
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