Download Vydac 214TP™ C4 Reversed-Phase Columns for Polypeptides

Vydac 214TP™ C4 Reversed-Phase Columns
for Polypeptides
• Polymeric bonding results in exceptionally long column lifetime and
negligible phase leaching.
• 300 Å silica allows full access to the
reversed-phase surface by even
large polypeptides.
• Applications-focused quality control
assures highest level of performance
and reproducibility.
• Now available in 1.0-mm Microbore,
2.1-mm Narrowbore, and 3.2-mm
Solvent Saver.
Description
Applications
Vydac C4 (214TP) reversed-phase material consists
of butyl aliphatic groups bonded to the surface of
300 Å pore diameter silica. The large pores of the
300 Å TP silica allow polypeptide molecules complete
access to the interior of the silica pores. The unique
process by which we manufacture Vydac TP silica
results in a very high purity synthetic silica with
carefully controlled characteristics. Vydac TP silica is
the standard that has defined large pore HPLC silica
for polypeptide separations for nearly two decades
(see Ref. 1 on page 11).
Vydac C4 reversed-phase materials are typically
used and recommended for the separation of:
The hydrophobic (reversed) phase is attached to the
silica using polyfunctional chlorobutylsilanes, resulting
in cross-linking, or polymerization, of the hydrophobic
phase. The practical benefits of the polymeric phase
are:
■ very
■ no
long column lifetime
measurable phase leaching
Carbon loading and end-capping are closely monitored
by applications-focused quality control tests of the
separation of a selected set of peptides.
Vydac 214ATP54 is a special column for which the
adsorbent chemistry is optimized for use in stability
analysis of human growth hormone. (See chromatogram
and Ref. 4 on page 23.)
“214TP” is a trademark of The Separations Group, Inc.
• Glycoproteins (Ref. 1)
• Hemoglobin variants (Ref. 2)
• Histones (Ref. 3)
• Human Growth hormone (Ref. 4)
• Insulin variants (Ref. 5)
• Membrane proteins (Ref. 6)
(All references above refer to listing on page 23.)
Ordering Information
Cat. No.
Description
214ATP54
214TP54
214TP5415
214TP5405
Popular Analytical Sizes
Column, C4, 300 Å, 5 µm, 4.6 mm i.d. x 250 mm
Column, C4, 300 Å, 5 µm, 4.6 mm i.d. x 250 mm
Column, C4, 300 Å, 5 µm, 4.6 mm i.d. x 150 mm
Column, C4, 300 Å, 5 µm, 4.6 mm i.d. x 50 mm
214TP53
214TP5315
Solvent-Saver Analytical
Column, C4, 300 Å, 5 µm, 3.2 mm i.d. x 250 mm
Column, C4, 300 Å, 5 µm, 3.2 mm i.d. x 150 mm
214TP510
Semipreparative
Column, C4, 300 Å, 5 µm, 10 mm i.d. x 250 mm
Other column sizes and particle diameters are available for
both analytical and preparative applications. Please consult
the selection guide on page 32 for additional listings.
Vydac C4 Reversed-Phase Columns for Polypeptides
Ribosomal proteins
Large, hydrophobic proteins are best separated on C 4
reversed-phase columns. While suitable for peptides as small
as 10-20 residues, the short, mildly hydrophobic C 4 phase
gives especially good recovery and resolution for large or
hydrophobic proteins.
Conditions
Column: Vydac 214TP54 (C4, 5 µm, 4.6 mm i.d. x 250 mm).
Eluent: 10-38% isopropanol with 0.1% TFA over 355 min.
From R.M. Kamp, A Rossenthoff, D. Kamp, and B. Wittman-Liebold,
J. Chrom 317, 181-192 (1984).
360 min
0
Human Growth Hormone
Human growth hormone is routinely assayed for stability by measuring
degradation products. Both deamidation and oxidation products are
resolved by isocratic reversed-phase HPLC.
214ATP54
human growth hormone
(HGH)
mono-desamido
HGH
Conditions
Column: Vydac 214ATP54 (C4, 5 µm, 4.6 mm i.d. x 250 mm L).
Eluent: 29% isopropanol/71% 0.01 M Tris buffer, pH 7.5
di-desamido
HGH
Data from R.M. Riggin, G.K. Dorulla, and D.J. Miner, Anal. Biochem. 167,
199-209 (1987).
(Note: The use of an eluent at pH 7.5 will decrease the column lifetime and is not
generally recommended.)
0
42 min
References
1. Microscale Structure Analysis of a High Molecular Weight
Hydrophobic Membrane Glycoprotein Fraction with PlateletDerived Growth Factor-Dependent Kinase Activity, P. Tempst,
D. Woo, D. Teplow, R. Aebersold, L. Hood, and S. Kent, J. Chrom.
359, 403-412 (1986).
2. High Performance Liquid Chromatographic Separation of Globin
Chains on a Large-Pore C4 Column, J.B. Shelton, J.R. Shelton,
and W.A. Schroeder, J. Liq. Chrom. 7(10), 1969-1977 (1984).
3. Separation and Purification of S49 Mouse Lymphoma Histones
by Reversed-Phase High-Performance Liquid Chromatography,
M. C. McCroskey, V.E. Groppi, and J.D. Pearson, Anal. Biochem.
163, 427-432 (1987).
4. A Reversed-Phase High Performance Liquid Chromatographic
Method for Characterization of Biosynthetic Human Growth
Hormone, R.M. Riggin, G.K. Dorulle, and D.J. Miner,
Anal. Biochem. 167, 199-209 (1987).
5. Reversed-Phase High-Performance Liquid Chromatography of
Insulins from Different Species, J. Rivier and R. McClintock,
J. Chrom. 268, 112-119 (1983).
6. Purification of Integral Plasma Membrane Proteins by ReversePhase High Performance Liquid Chromatography, M.R. Sussman,
Anal. Biochem. 169, 395-399 (1988).