high velocity chromatography of biomacromoleculesmodern pumping systems. for comparison, the same...

High velocity separations are nowpossible for biomacromolecules due to arevolutionary innovation -- a superficiallyporous, silica-microsphere packing calledPoroshell�. The excellent kineticproperties of these new columnpackings enable stable, high-resolutiongradient chromatography of proteins,polypeptides, nucleic acids, DNAfragments, etc. at up to 400-times fasterthan conventional high performanceseparations. Using Poroshelltechnology, the chromatographer canaccomplish the fastest availableseparation of proteins. And, Poroshellpackings can be used with conventionalequipment with no loss in performance.

Using a high quality liquidchromatograph like the Agilent 1100,Poroshell columns are useful for rapidlymonitoring preparative separations ofgenetically engineered macromolecules,process control monitoring offermentation broths or cell-reactionsystems, quantitation of polymerasechain reaction fragments, monitoringprotein conjugation reactions, andsimilar applications where very fast,reproducible separations are required.

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Technical Note

Brian A. Bidlingmeyer and Robert D. Ricker

Why is this a breakthrough?

Agilent has prepared superficially porousparticles that represent a practicalcompromise of high efficiency, highspeed, and low back pressure that benefitresearchers doing biomolecular analysis.The basic particle is composed of a solid,ultra-pure, silica core with a thin poroussilica shell. Figure 1 depicts a typicalPoroshell particle. The shell consists ofan ultra-pure, less-acidic �Type B� silicawhich has been demonstrated to be mostfavorable for separating polar macro-molecules, especially polypeptides andproteins. A scanning electronphotomicrograph of actual Poroshellparticles is shown in Figure 2. Note theconsistent spherical shape of theseparticles.

Figure 1. Structure of a typical superficially porousparticle, accentuating the porous layer wheremacromolecules move rapidly in and out of theparticle.

PorousShell

SolidCore

The pore size of the outer shell is 300Åand is bonded with a sterically-protecting diisobutyl-C18 stationaryphase, StableBond C18. StableBondC18 (SB-C18) is the most stablereversed-phase available. SB-C18operates effectively at low pH (≤2)which is the region used for almost allprotein separations. When used at lowpH, SB-C18 is also stable totemperatures of 90°C. This is of greatbenefit in lowering bonded-phaseviscosity so that higher flow rates maybe easily achieved.

Poroshell outperforms totally porousparticles because columns of totallyporous particles can have constraints inseparation speed because of stationaryphase mass transfer limitationsresulting from the relatively longdiffusion times required for

High Velocity ProteinSeparations

An example of the high velocityseparations that achieve very highthroughput is shown in Figure 3.Adequate resolution is seen at aconventional flow rate (0.5mL/min) forthis 2.1 x 75mm Poroshell 300SB-C18(Fig. 3A). Figure 3B shows the decreasein runtime achieved when operating at3.0 mL/min. Equivalent resolutionoccurs in one-sixth the time. No loss ofresolution and 6-times the throughput --a dramatic example of high velocitychromatography of biomacromolecules,using Poroshell SB-C18.

Poroshell outperforms all columnmaterial available even compared to therecently introduced ≤2µm, non-porousparticles. While columns of non-porousparticles theoretically offer excellentmass transfer characteristics, theysuffer from problems similar to thosepreviously described for very small,totally porous particles. Specialapparatus and operating techniques arerequired to attain benefit from non-porous particles, because standardinstrumentation broadens the verynarrow peaks formed by thesematerials. Further, small, non-porousparticles have very low surface areaavailable for sample interaction and arelimited in their sample loadingcapability.

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Figure 4 shows a plot of plate heightversus mobile-phase velocity data forinsulin on a Poroshell 300SB-C18column containing 5-µm particles with300Å pores. The sterically protectingbonding of the Poroshell SB-C18column provided stable operation at60ºC with a mobile phase containing0.1% trifluoro-acetic acid (about pH 2).

Note that plate height of the insulinplot shows only modest increase with alarge increase in mobile-phase velocity.This effect is evidence of the favorablemass transfer properties of thePoroshell configuration. Clearly,columns of these particles can be usedat high mobile-phase velocities for veryfast separation of large biomoleculeswithout significant degradation incolumn resolution.

Figure 3. Comparison of flow rate in ultra-fast analysis of proteins using Poroshell. Column: 2.1 x 75mm,5µm, Poroshell 300SB-C18; mobile phase: A=acetonitrile, 0.1%TFA (5:95); B=acetonitrile, 0.07% TFA (95:5);temperature: 35°C.

43 bar

222 bar

Figure 4. Plate height versus mobile-phase velocity plots for insulin. Column: Poroshell 300SB-C18, 2.1 x75mm, 5µm; mobile phase: acetonitrile, 0.1%TFA (28:72); Temperature: 60°C; Detection UV (214nm) sample:in 6M guanidine HCl.

Figure2. SEM of superficially porousparticles (Poroshell).

Scale: = 1 µm

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macromolecules to traverse the porousstructure and interact with thestationary phase inside. Very small(<2µm) totally porous particles havebeen proposed as a means of reducingthese mass-transfer limitations.However, optimum use of columns with<2µm particles requires specialapparatus and technique because of thevery narrow peaks formed. Use of theseparticles in conventional apparatus forisocratic separations has resulted inpeaks that are broader than predicted bytheory because of extra-column band-broadening effects.

Applications

Fig. 5 is an illustration of the speed andefficiency of a Poroshell column inseparating protein mixtures. Thissynthetic mixture of nine proteins wasseparated by gradient chromatography,in less than 2 min., with excellent peakshape, and with resolution exceeding1.5 for all peaks.

Even faster separations of suchcompounds are possible at highermobile phase velocities (higher flowrates) and elevated columntemperatures, as shown in Figure 6.High temperature improves the kineticsof the separation and lowers theoperational back pressure. Thisseparation of proteins was completed inabout 20 seconds, again, with goodpeak shapes. At this high mobile-phaseflow rate (4.0 ml/min for a 2.1 mm I.D.column; velocity ~3.2 cm/s), thepressure of the column was 295 bar,well within the operating range ofmodern pumping systems. Forcomparison, the same linear velocity ona column 4.6mm in diameter would benearly 20 ml/min!

Another dramatic example of pushingthe limits of high velocitychromatography is shown in Figure 7.Here, four proteins are separated at aconventional flow rate of 0.5ml/min ona 2.1 x 75mm column. (The samevelocity on a 4.6mm i.d. column wouldbe 2.4ml/min.) As the linear velocity(flow rate) of the mobile phase isincreased, the gradient time wasadjusted to keep the same rate ofchange for each flow rate. At 4 ml/min(corresponding to 19.2ml/min), theresolution is essentially the same andthe throughput is 8-times faster. Thefinal separation is completed inapproximately 20 seconds.

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1. Met Enkephalin2. Leu Enkephalin3. Angiotensin II4. Neurotensin5. Insulin6. RNase7. Lysozyme8. Myoglobin9. Carbonic Anhydrase

Figure 5. Rapid separation of protein mixture. Column: 2.1 x 75mm, 5µm, Poroshell 300SB-C18; mobile phase:A=acetonitrile, 0.1%TFA (5:95); B=acetonitrile, aqueous 0.085% TFA (95:5); gradient: 2 - 65% B in 2.0 min;flow rate: 2.0mL/min; temperature: 35°C. Reproduced with permission (see reference).

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Mobile Phase:A= 95% H2O, 5% AcN with 0.1%TFAB= 5% H2O, 95% AcN, with 0.07%TFA

Piston Stroke: 20µLTemp.: 70°C, Det.: 215 nm

Figure 7. Protein separation in seconds; effect of flow rate on ultra-fast protein analysis. Column: 2.1 x 75mm,5µm, Poroshell 300SB-C18; mobile phase: A=acetonitrile, 0.1%TFA (5:95); B=acetonitrile, 0.07% TFA (95:5);gradient: 5 - 100% B; flow rate: 2.0mL/min; temperature: 70°; detect.: UV (206nm).

Figure 6. Rapid separation of protein mixture. Column: 2.1 x 75mm, 5µm, Poroshell 300SB-C18; mobile phase:A=acetonitrile, 0.1%TFA (5:95); B=acetonitrile, aqueous 0.085% TFA (95:5); gradient: 2 - 65% B in 2.0 min;flow rate: 2.0mL/min; temperature: 35°C. Reproduced with permission (see reference).

1. Rnase2. Cytochrome C3. Lysozyme4. Carbonic Anhydrase5. Ovalbumin

20 secseparation

Summary

The introduction of 5µm Poroshellparticles marks the dawn of a new ageof high velocity chromatography usingconventional HPLC instrumentation.Now the chromatographer can enjoythe benefits of extremely rapidbiomacromolecular analysis, withoutloss of efficiency. Larger peptides,proteins, and DNA can all be separatedwith very short runtimes, as a result ofthis state-of-the-art technology. Anytime separation is limited by diffusion oflarge molecules, and rapid analysis iscritical, use Poroshell technology.

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Agilent Technologies, Inc.

All Rights Reserved. Reproduction, adaptation or

translation without prior written permission is

prohibited, except as allowed under the copyright

laws.

Publication Number 5988-2400EN

For more information on our products and services,

contact your local Agilent Technologies office or

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Brian Bidlingmeyer is manager of the separations

R&D Product Generation Unit,, and Robert Ricker is

an application chemist; both are based at Agilent

Technologies, Wilmington, Delaware.

Rapid separations on more complicatedmixtures also can be performed withPoroshell columns, as shown in Fig. 8.This separation of a pBR322 Hae IIIdigest with 51 to 587 base pairs wasseparated sufficiently in less than 14min. to identify all of the components inthis mixture.

Figure 8. Separation of pBR322/HaeIII digest using a Poroshell column. Column: 2.1 x 75mm, 5µm, Poroshell300SB-C18; mobile phase: A=100mM, pH 7, triethylamine acetate, 0.1M EDTA, B=A+acetonitrile (50:50);gradient: 17.5%B to 37.5%B in 15 min; flow rate: 0.4mL/min; temperature: 50°C; detection: UV (254nm).Reproduced with permission (see reference).

Figure 9. Separation of Triton X-114 oligomers using a Poroshell column; Column: 2.1 x 75mm, 5µm, Poroshell300SB-C18; mobile phase: A=methanol-water (45:55), B=methanol; gradient: delay, then 0-5%B in 2 min; flowrate: 0.5mL/min; temperature: 24°C; Detection: UV (225nm). Reproduced with permission (see reference).

Poroshell columns also can be effectivelyused for rapid separations of polymeroligomers, and other macromolecules ofthis type. Figure 9 shows a gradientchromatogram of Triton X-114 that wascompleted in less than 3 min., withindividual components clearly resolvedand visible for identification.

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Reference

J.J. Kirkland, F.A. Truzkowski, C.H. DilksJr., and G.S. Engel. J. Chromatogr. 890(2000) 3-13.