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Specific Applications for HALO Peptide ES-C18 and ES-CN Columns

 Specific Applications for HALO Peptide ES-C18 and ES-CN Columns

High Resolution Peptide Separations

High-resolution reversed-phase peptide separations are essential in protein biomarker identification as part of a shotgun proteomics approach, and are also very important for biomarker quantitation, when peptide surrogates are used to represent a target protein biomarker, which has been up- or down-regulated in a biological system. HALO 160 Ångstrom Peptide ES-C18 and ES-CN 2.7-µm UHPLC columns can deliver high resolution separations in the same amount of time, at much lower pressures than sub-2-µm columns, or they can deliver results much faster at higher flow rates (Figure 1). For HPLC applications, HALO-5 Peptide columns produce narrower, taller peaks than totally porous 5 µm or 3 µm 300 Angstrom column for improved sensitivity and peak capacity (Figure 2).

Another application area for peptide analyses is for peptide mapping as a quality assessment technique for recombinant protein and antibody synthesis and manufacturing. For challenging applications such as these, one can even take advantage of the lower pressures produced by HALO 2.7 and HALO-5 columns to use two or three columns in series to increase resolution and peak capacity.

Figure 1 HALO 160Å, 2.7 µm, Peptide ES-C18 shows minimal loss in resolution at higher flow rates with shorter run times


Figure 2  HALO-5 Peptide ES-C18 Outperforms Totally Porous, 150Å, 5 µm Column for Fast Separations

HALO-5 Peptide vs 300 Angstrom 5 um

HALO ES-CN Offers Alternative Selectivity

The HALO ES-CN phase is made using a sterically-protected cyanopropyl ligand, which gives the phase excellent stability under the low pH and above-ambient temperatures commonly used for peptide separations and peptide mapping applications. As with the sterically-protected HALO Peptide ES-C18 phase, the HALO ES-CN phase is available in 2.7 µm and 5 µm particle sizes for any UHPLC or HPLC need.

An example of the selectivity differences seen between the two phases is shown in Figure 3 for two 4.6 x 100 mm HALO-5 columns. The separation was developed and optimized for the HALO-5 ES-C18 phase, and then run on the HALO-5 ES-CN column. Additional optimization of the HALO-5 ES-CN separation would yield a better-resolved separation for the peptides on that column.

Figure 3 Comparison of the alternative selectivities of HALO Peptide ES-CN and ES-C18 for peptide separations and peptide mapping


 Below is presented some general guidance for getting off to a good start, if you’re new to peptide separations. 

General Guidance for HALO Peptide RPLC Separations

  • Use acetonitrile (ACN) as the organic modifier, and for the aqueous component, use 0.05–0.1% trifluoroacetic acid (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 add 10 to 20% (v/v) less TFA to the strong solvent (ACN or ACN/water blend) than aqueous reservoir for wavelengths 220 nm to 210 nm, respectively, for flatter baselines.
  • Use a flow rate of 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 a column temperature at 25–50°C.
  • Start the gradient at 5:95 ACN/aqueous and ramp up to 65% ACN in 30 min (2% ACN/min).
  • Adjust gradient starting and ending compositions, initial hold time, column temperature, and gradient time as needed to achieve and optimize separation.
  • Computer simulation software such as DryLab® 4 can be used effectively to optimize resolution, gradient program and run time by modeling gradient steepness, column temperature, % additive (TFA, formic acid), etc.

HALO BioClass columns will deliver speed, high peak capacities, and ruggedness to your HPLC and UHPLC separations of peptides, polypeptides and proteins.  MAC-MOD Analytical is currently offering an excellent opportunity for you to try these columns.  Please refer to our web site for more information about the HALO BioClass columns and about our current offer.

NOTE:  The current promotion for HALO Peptide and Protein columns will be ending on July 31, 2014.  If you have not had the opportunity to try one of these columns and phases, now is an excellent time to take advantage of our buy one-get one free offer. If you would like additional information about HALO BioClass columns, please visit our website.

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