Discovery Bio C8 HPLC Columns

Analytical / Chromatography

Analytical Reagents

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)
Column IDs:		0.32mm to 21.2mm

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: 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

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

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

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

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

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: 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

1. Bovine insulin (5µg)
2. Human insulin (5µg)
3. Porcine insulin (5µg)