Separation of Peptides on HALO 2‐Micron Particles

Colin T. Mant1, Robert S. Hodges1

1 Department of Biochemistry and Molecular Genetics, University of Colorado, School of Medicine, Aurora
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 11.6
DOI:  10.1002/cpps.12
Online Posting Date:  August, 2016
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Reversed‐phase high‐performance liquid chromatography (RP‐HPLC) is of fundamental importance to the isolation and separation of peptides, proteins, and other biomolecules. Hence, there is a continuing high demand for the development of RP‐HPLC stationary‐phase materials with enhanced separation efficiency. HALO packing materials began the revolution in “core‐shell” technology with the advantages of faster separations, higher resolution and peak capacity, high temperature stability, and rugged reliable performance compared to traditional HPLC and UHPLC. These materials are characterized by a solid core surrounded by a thin layer of porous material, and represent a technology for the future with continuing refinements. Such refinements are aided via the use of designed synthetic peptide standards during stationary‐phase development. Concomitantly, such standards also enable the researcher to monitor RP‐HPLC column performance and develop optimized separation protocols for peptides from a wide array of sources. © 2016 by John Wiley & Sons, Inc.

Keywords: reversed‐phase HPLC; separation of peptides; solid core; core‐shell; fused‐core particles; 2.0 micron particles; trifluoroacetic acid (TFA); anionic ion‐pairing reagent

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Peptide standards (see recipe and Fig.  )
  • Mobile‐phase solvent A: 0.1% (v/v) trifluoroacetate (TFA; HPLC/SpectroGrade‐processed in glass distillation units, Thermo Scientific) in water from a Barnstead E‐pure purification system (in this study, 0.2% and 0.4% TFA were also used)
  • Mobile‐phase solvent B: 0.08% to 0.09% (v/v) TFA (Thermo Scientific) in acetonitrile (HPLC grade, submicron filtered; Fisher) to obtain a flat or slowly rising baseline; in this study 0.17 to 0.18% and 0.37 to 0.38% TFA were used to obtain a flat or slowly rising baseline when 0.2% or 0.4% TFA were used in mobile phase solvent A, respectively.
  • Anionic ion‐pairing reagent, TFA (Thermo Scientific)
  • Water: In‐house distilled water purified through a Barnstead E‐pure purification system to reach a MΩ‐cm reading greater than 17.6
  • Reversed‐phase column: HALO Peptide ES‐C18 2.0 µm particle size, 160 Å pore size. 2.1 mm × 100 mm (Advanced Materials Technology); column is best for routine use below 60°C, but can be used at temperatures up to 90°C; stable to operating pressures up to 600 bar (9000 psi)
  • HPLC system: Agilent 1200 liquid chromatograph with maximum pressure limit of 400 bar:
  • Detector: Agilent 1200 series diode array and multiple wavelength detector with a 13‐µl flow cell with a 10‐mm pathlength
  • Autosampler: Agilent 1200 series, injection volume 1 to 100 µl (injections of 15 µl of the peptide mixture dissolved in water were used in this study)
  • Temperature control: Agilent 1200 series thermostatted column compartment; all runs were carried out at 50°C in this study due both to unfavorably high operating pressures at lower temperatures as well as improved resolution at high temperatures
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Key References
  Mant, C.T. and Hodges, R.S. (eds.) 1991. HPLC of Peptides and Proteins: Separation, Analysis and Conformation. CRC Press, Boca Raton, Fla.
  A comprehensive guide to HPLC of peptides and proteins.
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