Ultra-fast Protein Separations and High Efficiency Peptide Profiling with Agilent LC Columns

September 1, 2012

The Application Notebook

Volume 0, Issue 0

Rapid and highly resolved chromatographic separations of proteins are becoming increasingly important to meet the growing demands for faster analyses, greater reliability and overall increased column performance. Additionally, highly selective and efficient separations for peptide and impurity profiling have grown among the biopharmaceutical industry, with an ever increasing need to obtain greater peak capacities.

Rapid and highly resolved chromatographic separations of proteins are becoming increasingly important to meet the growing demands for faster analyses, greater reliability and overall increased column performance. Additionally, highly selective and efficient separations for peptide and impurity profiling have grown among the biopharmaceutical industry, with an ever increasing need to obtain greater peak capacities.

Agilent Poroshell (superficially porous) and ZORBAX Rapid Resolution High Definition 300 Å (sub-2 µm totally porous) columns can facilitate rapid and high throughput protein analyses and deliver increased peak capacities for high efficiency peptide profiling. For faster analysis, increased flow rates on shorter columns can result in reduced analysis times 5 to 15 × faster than traditional run times. Alternatively, these columns can also afford flexibility for delivering ultra high resolution separations during longer run times.

Poroshell 300 5.0 µm — Maintaining Resolution During Rapid Runtimes

Figure 1 compares the speed and the enhanced resolution of Poroshell 300 (Panel B) to another vendor's superficially porous brand (Panel A) using a mixed protein sample. The Poroshell 300 5.0 µm delivers an ultra-fast separation using a shorter column length and higher flow rate (gradient volumes remaining equal). The Poroshell 300 separation completes 12 times faster while maintaining critical resolution, sensitivity, and peak shape. Additionally, Poroshell 300 resolves transferrin (*), which is missing from the separation in Panel A.

Figure 1: Comparison of superficially porous chromatographic separations using a mixed protein sample. Panel A: 2.1 ×150 mm 3.6 µm superficially porous C18 column, flow rate 0.3 mL/min, mobile phase A = H2O (0.1% TFA), B = ACN (0.08% TFA), gradient 5 to 90% B over 60 min. Panel B: Agilent Poroshell 300SB-C18, 2.1 × 75 mm, 5.0 µm, flow rate 2.5 mL/min, gradient 5 to 90% B over 4 min. Common conditions; temp. 40 °C, UV 225 nm.

Poroshell 120 EC-C18 2.7 µm and ZORBAX RRHD 300SB-C18 — 1.8 µm High Resolution Peptide Mapping

Comparisons between Agilent Poroshell 120 2.7 µm and ZORBAX RRHD 300SB-C18 1.8 µm columns demonstrate alternate selectivity options for generating high resolution peptide maps. Both peptide separations exhibit excellent separation performance of hydrophobic and hydrophilic peptides and deliver highly resolved peaks and narrow bandwidths. In particular, the ZORBAX RRHD 300 Å column has unique selectivity and retention for hydrophilic peptides (Figure 2, arrows).

Figure 2: Tryptic peptide digests on an Agilent Poroshell 120 2.7 µm and ZORBAX RRHD 300SB-C18 1.8 µm column, with the latter displaying unique selectivity and retention of hydrophilic peptides (arrows). BSA tryptic digest, 1 pmol/µL (dil. 98/2 A/B), inj. 7 (100 mm) to 10 µL (150 mm), mobile phase A = water (0.1% TFA), B = ACN (0.08% TFA), gradient hold 3% B 3 min, 3 to 65% B 30 min, 6 min post run, flow rate 0.3 mL/min, temp. 40 °C, DAD 215 nm, Agilent 1290 Infinity LC.

Conclusions

As rapid and reliable separation of biomolecules becomes increasingly critical, the speed and resolution benefits offered by Agilent LC columns improve the likelihood of achieving high quality separations. Agilent offers Poroshell and ZORBAX RRHD columns for facilitating a variety of specialized bioseparation needs.

Agilent Technologies, Inc.

5301 Stevens Creek Blvd., Santa Clara, CA 95051

(800) 227-9770 (Directory), fax (866) 497-1134

Website: www.agilent.com