Discovery BIO Wide Pore C5

HPLC Columns and Capillaries

Bonded Phase:		Covalently-bonded pentylsilane (C5), endcapped
Silica:		Spherical, 3 µM, 5 µM and 10 µM diameters, 300Å pore diameter,
		high purity (<10ppm metals)

Solutions for Protein and Peptide Separation Challenges

• Proteomics and biotherapeutics: high efficiency protein and hydrophobic separations
• LC/MS; no-bleed, capillary dimensions, and pH stability
• Protein purification: Scalable from analytical to preparative
• Enhanced lifetime and pH stability over C4 phases

Here's the Proof

Excellent Performance for Large Molecules

Column: Discovery BIO Wide Pore C5, 15cm x 4.6mm, 5µm; Mobile Phase: (A) 0.1% (v/v) TFA in H₂O: MeCN (75:25), (B) 0.1% (v/v) TFA in H₂O: MeCN (25:75); Flow Rate: 1mL/min; Temp: 30°C; Detection: 220nm; Injection: 10µL; Sample: Protein Test Mix; Gradient: 0-100% B in 25 min.

1. RNase (1mg/mL) (13.7kDa)
2. Cytochrome C (1mg/mL) (12.4 kDa)
3. Lysozyme (1mg/mL) (14.3 kDa)
4. Bovine Serum Albumin (2.5mg/mL) (67.0 kDa)
5. Myoglobin (1mg/mL) (18.8 kDa)
6. Ovalbumin (3.5mg/mL) (45.3 kDa)

Figure 1. Excellent Performance for large molecules

Increased Stability of Discovery Bio Wide Pore C5 Over Conventional C4

After 25,000 column volumes (222 gradient cycles) were passed through the column, the Discovery BIO Wide Pore C5 showed very little change in efficiency. The competitive C4 column showed a significant loss of efficiency, as much as 50%.

Column: Discovery BIO Wide Pore C5, 5cm x 4.6mm, 5 µM; Mobile Phase: (A) 0.1% TFA in H₂O : MeCN (95:5), (B) 0.1% TFA in H₂O : MeCN (25:75); B 2 mL/min; Temp: 35 °C; Detection: 220nm; Injection: 5 µL; Sample: Protein/Peptide Test Mix; Gradient: 0-20% B in 20 min

Figure 2. Increased stability of discovery bio wide pore

Stability of Discovery Bio Wide Pore C5 at pH 8.0

Retention times of the sample components remained constant after 14,000 bed volumes were passed through the column.

Column: Discovery BIO Wide Pore C5, 5cm x 4.6mm, 5 µM; Mobile Phase: 25mM potassium phosphate, pH 8 : MeOH (95:5); Flow rate: 2 mL/min; Temp: 35 °C; Detection: 254nm

Figure 3. Stability of discover

Undetectable LC/MS Bleed

LC/MS Bleed Analysis on Discovery BIO Wide Pore C5
Column: Discovery BIO Wide Pore C5, 15cm x 4.6mm, 3 µM; Mobile Phase: (A) 0.1% TFA in H₂O, (B) 0.1% TFA in MeOH; Flow Rate: 1 mL/min; Temp: 30 °C; Gradient: 0-100% B in 15 min, hold for 5 min, then back to 0% B for 10 min

No TIC baseline rise relative to blank run (gradient run w/o column)

Figure 4. M/S Spectrum (shaded area above)

Figure 5. Essentially no m/z peaks generated from column when compared to blank run. ("X" indicates peaks also found in blank run)

Reproducibility of Discovery Bio Wide Pore C5

Column: Discovery BIO Wide Pore C5, 15cm x 4.6mm, 5 µM; Mobile Phase:(A) 0.1% PFPA (pentaflouropropionic acid) in H₂O : MeCN (81:19), (B) 0.1% PFPA in H₂O : MeCN (62:38); Flow Rate: 1 mL/min; Temp: 30 °C; Detection: 215nm; Injection: 10 µL; Sample: Peptide Mix (Sigma Cat. No. P2693); Gradient: 0-100% B in 19 min.

Peak Identity:	Amino Acid Sequence:
1. Arg8-vassopressin 2. Brandykinin, fragment 1-5 3. Oxytocin 4. Met-enkaphalin 5. Luteinizing Hormone Releasing Hormone 6. Leu-enkephalin 7. Brandykinin 8. Bombesin 9. Substance P	CFQNCPRG-amide; disulfide RPPGF CYIQNCPLG-amide; disulfide PEHWSYGLRPG-amide YGGFM YGGFL RPPGFSPFR PEQRLGNQWAVGHLM-amide RPKPQQFFGLM-amide

Figure 6. Lot to lot reproducible of discovery

Ordering Information