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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).