HPLC

HPLC DiscoveryBIO Wide Pore C8

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Ordering Information

 
Discovery BIO Wide Pore C8
HPLC Columns and Capillaries
 
Bonded Phase:   Covalently-bonded octylsilane (C8), endcapped
Silica:   Spherical, 3µm, 5µm and 10µm diameters, 300Å pore diameter,
    high purity (<10ppm metals)
 
Solutions for Protein and Peptide Separation Challenges
 

Proteomics and biotherapeutics: high resolution of synthetic peptides and peptide maps

LC/MS; no-bleed, capillary dimensions, and pH stability

Peptide purification: Scalable from analytical to preparative

Intermediate hydrophobicity makes it a good method development starting point


 
Here's the
Proof
 
RESOLUTION OF ANGIOTENSINS AT NEUTRAL pH
 

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.


Column:

1. Angiotensin II (1.67g/L)
2. Angiotensin III (1.67g/L)
3. Angiotensin I (1.67g/L)

D R V Y I H P F    
  R V Y I H P F    
D R V Y I H P F H L
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

g001503

 
INTERMEDIATE HYDROPHOBICITY
OF DISCOVERY BIO WIDE PORE C8
 

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

g001504_06


 
STABILITY OF DISCOVERY BIO WIDE PORE C8
AT ALKALINE AND ACIDIC pH
 

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

g001508

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

g001509


 
RESOLUTION OF INSULINS FROM VARIOUS SPECIES
 

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.


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
g001507


Ordering Information
5µm Discovery BIO Wide Pore C8 HPLC Columns
ID(mm) Length(cm) Cat. No.
4.6 25 568323-U
10µm Discovery BIO Wide Pore C8 HPLC Columns
ID(mm) Length(cm) Cat. No.
21.2 25 567225-U