Reversed-Phase LC of Intact Proteins

HALO Protein columns are available in both a densely bonded and endcapped C4 phase, as well as in a sterically protected, endcapped ES-C18 phase.  For intact protein analyses by reversed-phase UHPLC and HPLC, the HALO Protein 400 Ångstrom columns offer significant advantages compared to commercially available totally porous and superficially porous (aka core-shell) columns (Table 1) .  

 Table 1  Advantages of HALO Protein C4 and ES-C18 for Protein Analyses

Examples of some of the advantages in the performance of HALO Protein C4 column versus an HPLC column and a UHPLC column are shown below in Figures 1 and 2, respectively.  In Figure 1, the improved peak shape, narrow peak width, improved recovery and increased retention of the HALO Protein C4 compared to a traditional 5 µm 300 Ångstrom column is demonstrated.  In Figure 2, separations of the same protein mixture shown in Figure 1 are compared for the HALO Protein C4 and a totally porous 300 Ångstrom, 1.7 µm particle column.  The HALO Protein C4 delivers comparable or better performance to the sub-2-µm UHPLC column at approximately 40% of the back pressure under identical conditions.

Figure 1  HALO Protein C4 dramatically outperforms a commercial 300 Ångstrom, 5 µm column in terms of peak shape, peak height, retention and recovery

Figure 2  HALO Protein Compares Favorably vs. a Totally Porous 1.7 µm UHPLC Column

General Guidance for HALO Peptide RPLC Separations

• Use acetonitrile (ACN) as the organic modifier, and for the aqueous component, use 0.05–0.1% TFA for UV-VIS detection and
  5–10 mM ammonium formate with 0.1% formic acid for LC-MS analyses.

• For UV-VIS detection, set wavelength at 214-218 nm, and put 10 to 20% less TFA in ACN than aqueous reservoir for
  wavelengths 220 nm to 210 nm, respectively, for flatter baselines.

• Set flow rate at 1.0–1.5 mL/min for 4.6 mm ID, ~0.4–0.6 mL/min for 3 mm ID, and 0.25–0.5 mL/min for 2.1 mm ID columns

• Set column temperature between 25–50 ºC.

• Start gradient at 5:95 ACN/aqueous and ramp up to 65% ACN in 30 min (2% ACN/min)

• Adjust gradient starting and ending compositions, column temperature, and gradient time as needed to achieve and optimize separation.