A New High-Resolution, High-Speed Reversed-Phase Monolithic 1 mm I.D. Column for the Separation of Proteins

February 1, 2009
Kelly Flook

,
Yury Agroskin

,
Charanjit Saini

,
Srinivasa Rao

,
Chris Pohl

The Application Notebook

The Application Notebook, The Application Notebook-02-01-2009, Volume 0, Issue 0

The new reversed-phase ProSwift® 1 mm i.d. column is a divinylbenzene-based monolithic column for routine chromatography of proteins and other biomolecules. It is available in two different lengths. The shorter (1 Ã- 50 mm) format is designed for fast separations and the longer (1 Ã- 250 mm) format is intended for high resolution analytical separations. However, depending on the application, either can be used for separation of proteins and for coupling with mass spectrometry.

The new reversed-phase ProSwift® 1 mm i.d. column is a divinylbenzene-based monolithic column for routine chromatography of proteins and other biomolecules. It is available in two different lengths. The shorter (1 × 50 mm) format is designed for fast separations and the longer (1 × 250 mm) format is intended for high resolution analytical separations. However, depending on the application, either can be used for separation of proteins and for coupling with mass spectrometry.

Experimental

A Dionex UltiMate® 3000 HPLC system with a P680 HPG pump, VWD/UVD 340U absorbance detector, autosampler, and thermostatted column compartment (TCC-100) was used in this work.

Chromatography was controlled by Chromeleon® Chromatography Management Software (Dionex).

Materials

Proteins and other analytical grade chemicals were obtained from Sigma. E. coli sample was from Bio-Rad.

Results and Discussion

Earlier, we introduced a series of RP monolithic columns in 4.6 mm i.d format based on different pore structures (1-3). For columns of this dimension, high efficiency separations were achieved via axial compression of the monolith. With this new 1 mm I.D. format we were able to covalently attach the monolith directly to the PEEK™ tubing to prevent formation of a gap between the monolith and the tubing wall during polymerization.

The new ProSwift RP-4H column offers several advantages. First, the ideally engineered porous structure of the monolith yields highly efficient separation of proteins. Second, due to low operating back pressure, high linear velocities (up to 8.5 mm/s, 0.4mL/min) can be employed with comparatively little loss of resolution, improving productivity. An example separation of a peptide and protein standard mixture is shown in Figure 1a. Third, the availability of longer formats offers increased loading capacity which is often a necessary requirement for separation of complex protein mixtures in one dimensional or multi dimensional chromatography platforms. Figure 1b shows the separation of an E. coli complex protein mixture on a ProSwift RP-4H column. Using a shallow gradient, more than 50 peaks are evident.

Figure 1

In addition to the above mentioned advantages, chromatography using ProSwift RP 1 mm columns can be performed on a standard HPLC system with minor plumbing changes to minimize instrument delay volume. As compared to standard dimension columns, 1 mm columns consume far less mobile phase, greatly reducing the cost of analysis.

Conclusion

The ProSwift RP- 4H 1 mm i.d. column provides a high speed, high resolution and a cost-effective separation of complex proteins. The high loading capacity of the column makes it useful for both one-dimensional and multi-dimensional chromatography platforms.

PEEK is a trademark of Victrex PLC.

Chromeleon, ProSwift, and UltiMate are registered trademarks of Dionex Corporation.

References

(1) ProSwift RP manual and ProSwift RP data sheet, available at www.dionex.com

(2) Rao, S; Thayer, J.; Xie, S. LC-GC Applications Notebook , June 2006, 20.

(3) Xie, S; Rao, S; Thayer, J.; Flook, K. Amer. Biotech. Lab. 2006, 24, 34–39.

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