Transcript
Page 1: Agilent ZORBAX Column Selection Guide for Analytical … · Agilent ZORBAX Column Selection Guide for ... ZORBAX Eclipse XDB HPLC Columns ... Extend-C18 columns are the newest addition

Agilent ZORBAX ColumnSelection Guide forAnalytical HPLCResolution that Lasts

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ZORBAX Silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Reversed-Phase HPLC Method Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

ZORBAX Columns for Analytical Chemistry

ZORBAX StableBond HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

ZORBAX SB-Aq HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

ZORBAX Rx HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

ZORBAX Eclipse XDB HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

ZORBAX Bonus-RP HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

ZORBAX Extend-C18 HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Additional ZORBAX Analytical HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . 20

ZORBAX High-Throughput HPLC Columns

ZORBAX CombiHT HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

ZORBAX LC/MS HPLC Columns and Cartridge Columns . . . . . . . . . . . . . . . . 22

ZORBAX Rapid Resolution 3.5 µm HPLC Columns . . . . . . . . . . . . . . . . . . . . . . 23

High-Throughput Column Ordering HPLC Information . . . . . . . . . . . . . . . . . . 24

MicroBore and Capillary Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

ZORBAX Columns for BioAnalytical Chemistry

ZORBAX Wide-Pore 300StableBond HPLC Columns . . . . . . . . . . . . . . . . . . . 26

ZORBAX 300Extend-C18 HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

ZORBAX Eclipse Amino Acid Analysis (AAA) HPLC Columns . . . . . . . . . . . . 30

ZORBAX Eclipse dsDNA Analysis HPLC Columns . . . . . . . . . . . . . . . . . . . . . . 31

ZORBAX GF-250/450 Size Exclusion HPLC Columns . . . . . . . . . . . . . . . . . . . . 32

ZORBAX Oligo HPLC Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Contents

Standard Fittings

ZORBAX 15 mm and 30 mm Rapid Resolution cartridges

75 mm and 150 mmAgilent cartridgecolumns

CombiHT

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ZORBAX Rx-SIL Xerogel(Sol-type) (SIL-type)

Uniform “Sponge-like”, Structure Sub Particles Polymeric Network

Purity High (99.995%) Low-High

Strength High Moderate

Pore Size, Particle Size Distribution Narrow Broad

Pore Size/Surface Area 80Å/180m2/g 100Å/300m2/g

Porosity (%) 50 70

High pH Resistance Good Poor

The ZORBAX Rx-SIL process producesultra-pure (99.995%) particles, with very low metal content. The final silicaparticle is fully hydroxylated and of low acidity. The Rx-SIL process alsoallows careful and reproducible controlof pore size and particle size. These key features: purity (low acidity),strength, and careful control of poresize as well as particle size are criticalto excellent chromatographic resultsand are the building blocks of superiorZORBAX bonded-phases.

Table 1 compares the process used tomake the ZORBAX Rx-SIL particles to a second process — the Xerogelprocess — commonly used to make silica particles for HPLC columns. To produce silica with the key featuresthat maximizes chromatographic performance — purity, strength, con-trolled pore size and particle size distribution, plus higher pH resistance— the Agilent ZORBAX process is anexcellent choice.

Let’s look at how these featuresimprove your chromatography.

Agilent ZORBAX Silica is an EssentialPart of Providing Resolution that Lasts

Silica-based HPLC columns have longbeen considered the best choice for maximizing resolution with long life-times and good column performance.The advantages of silica include highefficiency, small particle sizes, goodmechanical strength and compatibilitywith a wide range of organic solvents.The quality and consistency of the silica support is critical. Therefore,close attention to the manufacture of the silica particles can result inimproved chromatography for smallbasic molecules as well as peptidesand proteins. Agilent Technologiesmanufactures silica particles using aunique process to provide a superiorsilica support for ZORBAX HPLCcolumns with total control over themanufacturing process.

The ZORBAX Silica ManufacturingProcess — The Making of a Rugged, High-Purity Silica

All Agilent ZORBAX columns are built from porous silica microspheres(PSM) based on silica sols. The silica particle is made of tiny, solid sol microparticles agglutinated in apatented polymerization process, then fused together at very high temperatures to form the final particle (Figure 1). These strong,durable silica particles are calledZORBAX Rx-SIL and are the base silica for most of the ZORBAXcolumns in this brochure.

Silica Sol+

Urea (pH~2)+

CH2O

80Å pore

∆O2 3.5, 5, 7 µm

StableBond SB, 300SBEclipse XDBBonus-RPExtend-C18, 300Extend-C18

TABLE 1Characteristics of ZORBAX Rx-SIL and a Contrasting Type of Silica

FIGURE 1Formation of ZORBAX Porous Silica Particles

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CH2-OH

CH2-OH

CH2CH3

CH2CH3

NH2

NH2

The Benefit of Silica Purity — Reduced Peak Tailing

Peak tailing of basic compounds canbe a major chromatographic problem.Peak tailing reduces chromatographicefficiency and the accuracy and reproducibility of results. The majorcause of peak tailing is interactionsbetween analytes and the silica surface (Figure 2). Typically thepresence of acidic silanol sites on the silica surface cause this type ofpeak tailing. Trace metals in silicaincrease silanol acidity and peakasymmetry. These silanol interactionsare reduced or eliminated by choos-ing a less acidic, ultrapure (99.995%) silica, such as ZORBAX Rx-SIL.

The improvement in chromatographyis dramatic. Figure 3 shows thereduction in peak tailing using ZORBAX Rx-SIL versus a more acidic silica.

The Benefits of Strong Particles —Greater Efficiency and Durability

During the silica manufacturingprocess, the agglutinated sol particlesare sintered for increased strength.This improved mechanical stabilityallows ZORBAX columns to be packedat high pressures — up to 8000 psi!This results in a packed column withan exceptionally stable column bedthat will not compress under normalor even high operating pressures — upto 5000 psi. This packed bed stabilityincreases column lifetime using ZORBAX 3.5, 5 or 7 µm particles.When ZORBAX Rapid Resolution, 3.5 µm silica particles are used as the underlying support, high speed,high efficiency chromatography is possible without compromising column lifetime.

The Benefits of Careful Pore Size andParticle Size Control — High Efficiencyand Better Reproducibility with MoreColumn Choices

Accurate and closely monitored parti-cle and pore size control for ZORBAXRx-SIL produces reproducible reten-tion behavior from column-to-columnand lot-to-lot. The narrow, consistentparticle size distribution of ZORBAXRx-SIL particles maximizes efficiencyand column bed stability. Column pres-sure is never unusually high due to“fines” - smaller particles at the lowend of the particle size distribution.

Accurate and precise control of particle size allows specific 3.5, 5, and 7 µm ZORBAX Rx-SIL particles to be produced. The small 3.5 µm particle size is the basis for the Rapid Resolution, high-speed analysis

SiO-Na+ + R3NH+ SiO- NH+R3 + Na+

-SiOH + RCOO- -SiO- ...H+ ... -OOCR

1. Ionized silanols (SiO-) will ion-exchange with protonated bases (R3NH+) which can cause tailing and method variability.

2. Unprotonated acids can compete for H+ with protonated silanols.

SilicaParticle

SilicaParticle

SilicaParticle

SilicaParticle

columns designed to maximize resolution in shorter column lengths— ideal for LC/MS or any applicationdemanding shorter analysis times.The 5 µm particles are an industrystandard and provide high resolutionin a wide variety of column dimen-sions. This particle size also provideshigh efficiency in a short preparativeconfiguration — the CombiHT column — because careful particlesize control means consistent pressure expectations within normaloperating limits. The 7 µm particleprovides the ideal balance betweenefficiency and operating pressure for longer preparative columns.

ZORBAX Rx-SIL — The Foundation for Many Bonded-Phases

With such strong performance characteristics, ZORBAX Rx-SIL particles have been developed intomany effective bonded phases forsolving key analytical problems. These include columns that can beused at extremes of pH, unmatched by any other silica-based columns.Because silica-based columns havedifferent limitations at low and highpH, specific bonded-phase chemistriesare required to provide longer columnlife over different pH ranges.

As a result, Agilent ZORBAX RP-HPLCbonded phases are designed to giveextended column lifetime and reproducibility in the pH ranges thatprovide optimum and long lasting resolution, all starting with high performance ZORBAX Rx-SIL.

FIGURE 2Potential Secondary Interactions with Silica Silanols and Ionizable Compounds

FIGURE 3Chromatographic Improvement UsingHighly Purified ZORBAX Rx-SIL

Original ZORBAX SIL (1973)

Highly Purified ZORBAX Rx-SIL

Mobile Phase: 5% Propanol in Heptane

Flow Rate: 2.0 mL/min

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Recommended ColumnZORBAX Extend-C18

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

Recommended ColumnZORBAX StableBondZORBAX Eclipse XDBZORBAX Bonus-RPZORBAX Extend-C18

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

Unique Selectivity

4

Agilent ZORBAX RP-HPLC Bonded PhasesLong Lifetime, Excellent Peak Shape, Superior Resolution from pH 1 - 12

Agilent ZORBAX reversed-phase bonded phases, based on Rx-SIL, provide thebest peak shape and sample resolution, as well as the longest column lifetimesfor the low, mid and high pH regions. The retention of ionizable compoundschanges with pH, therefore achieving optimum resolution for complex samplesrequires establishing methods at any pH from pH 1 - 12. Several types of bondedphases are required to cover this wide pH range and provide maximum columnlifetime. The ZORBAX columns described below use unique bonding chemistry toincrease the lifetime of the bonded phase and the silica support where needed,thereby providing “Resolution that Lasts” at every pH.

For longest lifetime using low pH mobile phase — Agilent ZORBAX StableBond

StableBond columns are made using bulky, patented, unique silanes that sterically protect the siloxane bond. Acid labile endcapping reagents are notused. The result is vastly improved column life and extraordinary chemical and temperature stability in the pH 1 - 6 range for a wide variety of phases (SB-C3, SB-CN, SB-Phenyl, SB-C8, and SB-C18). Excellent peak shape isexpected for many basic compounds because of the ultra-pure Rx-SIL support.

For longest lifetime using mid-range pH mobile phase — Agilent ZORBAX Eclipse XDB or Bonus-RP

Eclipse XDB columns are best used in the pH range of 3 - 8 but can be usedsuccessfully over the extended pH range of 2 - 9. These columns provide excellent peak shape and high resolution for acidic, basic and neutral compounds. The densely-bonded, double-endcapped silica surface minimizesany silanol interactions and protects the silica support from dissolution.

Bonus-RP columns provide unique selectivity and applicability for mid pHswith an embedded amide linkage in a C14-alkyl chain. At pH 7 Bonus-RP provides excellent peak shape for even the most challenging basic compounds.Bonus-RP is protected from hydrolysis at low pH using the same sterically protecting bulky side chains as StableBond and also, is triple endcapped tominimize silanol interactions and improve stability at mid pH. Bonus-RP can be used without phase collapse in 100% aqueous mobile phases.

For longest lifetime using high pH mobile phase — Agilent ZORBAX Extend-C18

Extend-C18 columns are the newest addition to our product portfolio. These columns incorporate a unique, patented bidentate silane, combined witha double-endcapping process that protects the silica from dissolution at highpH — up to pH 11.5. Extend-C18 columns are best applied for separations ofcompounds that are either (1) basic and have little or no retention at low orintermediate pHs, (2) more stable or more soluble at high pH, or (3) basic andshow poor peak shape at low or intermediate pH.

Recommended ColumnZORBAX StableBond

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

Recommended ColumnZORBAX Eclipse XDB

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

Recommended ColumnZORBAX Bonus-RP

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

Unique Selectivity

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Low pH 1-3 — Region A

• Start method development at low pH, wheresilanols on a RP-HPLC column are protonat-ed. This minimizes peak tailing by eliminat-ing silanol/base interactions.

• At low pH basic compounds are positivelycharged, and their retention may bereduced.

• Acidic compounds may be protonated andhave increased retention.

• Retention times are usually stable withsmall changes in pH, producing a robustmethod.

• Volatile mobile phase additives, such asformic acid or trifluoroacetic acid (TFA), are often used at low pH with LC/MS.

High pH 8 - 12 — Region C

• In this region, basic compounds may be intheir free base form.

• Increased retention and resolution of basiccompounds are likely.

• Retention changes little in this region - thusrobust methods can be developed.

• Silica breakdown is prevented by innovativebidentate column chemistry, heavy endcapping, use of Rx-SIL and optimummobile phase.

• Ammonium hydroxide is an excellentvolatile mobile phase modifier at high pH.

Method Development from pH 1 - 12Chromatographic resolution between two or more peaks depends upon three factors — column efficiency, selectivity andretention. With ionizable analytes — bases and acids — all of these factors change dramatically with pH. For example,retention can be improved by changing the separation pH, so that analytes are separated in their non-ionized form.Changes in mobile phase pH also improve column efficiency because the ionization of the analyte and the residual silanolscan both be altered. This minimizes secondary interactions between analytes and the silica surface that cause poor peakshape. Achieving optimum resolution requires changing the mobile phase pH. The following method development strategyexplains how this is done with superior column lifetime.

Low, mid and high pH are the three general regions for chromatographic separations as defined in Figure 1. This figurehighlights the benefits of performing separations of ionizable analytes in each pH region. Method development proceedsby investigating chromatographic separations first at low pH and then at higher pH until optimum results are achieved. The ideal column is available for each pH region.

FIGURE 1Three pH Regions for HPLC Separations of Basic Compounds

Mid pH 3 - 8 — Region B

• Develop methods at pH’s at least 1 pH unit above or below the pKa to minimizechanges in retention with small changes in pH .

• Some silica surface SiOH groups becomeSiO- at pH 4 - 6; tailing interactions may bepossible.

• Minimize interactions by selecting an endcapped column, using additives such as TEA (less desirable) or using “polar”bonded phases.

• Silica breakdown is prevented by innovativebonding chemistry, heavy endcapping anduse of Rx-SIL.

1 2 3 4 5 6 7 8 9 10 11 12pH

Rete

ntio

n Ti

me

+ + / neutral neutral

neutral neutral / - -

Analyte charge:

Silica surface charge:

A

B C

NO YES NOIon Exchange Secondary Interaction:

pKa

Recommended Bonded Phase: StableBond Eclipse XDB or Bonus-RP Extend-C18

This figure represents the retentionbehavior of one basic analyte withrespect to pKa and pH. AnalytepKa is 6.5.

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Method Development at Low pH is Preferred —StableBond ColumnsStableBond — pH ≤ 3

With so many choices available, howdo you select a column for methoddevelopment? This is actually an easydecision. Most analytes today areacidic or basic - ionizable - and the best initial approach is to use a low pH mobile phase (Figure 1, Region A).Using a low pH mobile phase resultsin the best peak shape for basic compounds because ion-exchangeinteractions (BH+ + SiOH ≠ secondaryinteraction) are eliminated. Manyacidic compounds are non-charged, maximizing their retention at low pH.Therefore, StableBond columns are agood first choice providing the longestlifetime and most robust separationsat low pH.

For standard analytical work, startmethod development with acetonitrileas the mobile phase organic modifierand 25 - 50 mM phosphate buffer (pH ≤ 3) as the aqueous component.This provides good pH control, neces-sary for the most reproducible analy-ses of ionizable compounds.StableBond SB-C8, or SB-C18 bonded-phases are usually chosen first because they effectively separate mostcompounds. Changes to the mobilephase (% organic, pH, ionic strengthand organic modifier) and columntemperature can be used to optimizethe separation, as necessary. A uniquefeature of StableBond columns is thetemperature stability. The temperaturecan be used as an additional tool toimprove resolution.

StableBond Bonded-Phase Selectivity

The other StableBond phases — SB-CN, SB-C3, and SB-Phenyl — can be selected to improve sample resolution. All three of these bonded-phases are more polar than SB-C8 andSB-C18 and can change selectivity.Analysis time for non-polar compounds may also be reduced substantially while retention is

maintained for polar compounds. In addition, the SB-CN bonded phaseis an excellent choice when you havea mixture of polar and non-polar compounds. It will reduce the reten-tion of the non-polar compounds and possibly eliminate the need for a gradient separation. All of these short-chain bonded phases providethe same exceptional stability as theSB-C8 and SB-C18 columns.

Method Development at Mid pH — Eclipse XDB and Bonus-RP ColumnsEclipse XDB — pH 7

If low pH is not desirable or accept-able for your separation due to pooranalyte retention, solubility, selectivi-ty or stability, then a mid-range pH, such as pH 7, should be investigated(Figure 1, Region B). This usuallymaximizes differences in retentionand selectivity in comparison to lowpH. The Eclipse XDB-C18 and C8bonded-phases are normally the firstchoice for use at this pH because theyprovide high resolution and superiorcolumn lifetime. Phosphate buffer isusually the first choice for mobilephase modifier at pH 7 because itsbuffer range is pH 6.2 - 8.2. Phosphatebuffers, although commonly used,should be used at temperatures lowerthan 40°C for maximum column life. A second choice for mid pH is acetatebuffer since it buffers from pH 3.8 -5.8 and is normally selected forLC/MS compatibility. Method develop-ment optimization can continue fromhere by changing the mobile phase,pH, and column temperature (up to60°C) or by changing the bondedphase to Eclipse XDB-Phenyl.

Bonus-RP — pH 7

For even more dramatic changes inselectivity or to improve the peaksymmetry of basic compounds in themid-pH range, try the Bonus-RP col-umn. The Bonus-RP column exhibits amodified reversed-phase behavior.

When you choose this column, themethod development process will bethe same as with the Eclipse XDBcolumns. You can select the sametypes of buffered mobile phases anduse the same organic modifiers; youcan even vary the temperature up to60°C. In general, the Bonus-RP isslightly less retentive than alkyl C8columns because of the polar aminegroup embedded in the bondedphase. The Bonus-RP column hasenhanced stability down to pH 2, soit can also be selected to improvepeak shape of difficult basic com-pounds that tail badly on many otheralkyl type phases. Bonus-RP columnscan be used with 100% aqueousmobile phases.

Method Development at High pHExtend-C18 Columns

At low or mid pH some separations of basic compounds may still nothave enough retention or the desiredselectivity. For these samples highpH separations may be appropriate(Figure 1, Region C). At high pH, itmay be possible to separate bases intheir free base form, where they arenot charged. Retention increases andthe chance of obtaining the desiredselectivity improves. The Extend-C18column performs well with high pHmobile phases — up to pH 11.5. Formany basic compounds with a pKaof 9 - 10, this extended pH rangeallows the separation to take place at least 1 pH unit above the analytepKa, where it will be in free baseform. Method development using theExtend-C18 column is the same ason other columns — choose abuffered mobile phase appropriatefor the pH range needed and anorganic modifier. However, whenusing the Extend-C18 column organic buffers such as triethy-lamine, ammonium hydroxide andpyrrolidine are needed for maximum column lifetime; phosphate buffer isnot recommended. Methanol is thepreferred organic modifier.

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ZORBAX Columns Provide Resolution that LastsZORBAX columns have been carefully designed for optimum sample resolutionand longest column lifetimes for the low, mid, and high pH ranges. Followingthe strategy described below simplifies method development using ZORBAXcolumns. You never have to take a column and make it work — your sampleand mobile phases define the best column — so you can always achieve“Resolution that Lasts”.

ZORBAX StableBondLow pH ≤ 3

Region A

ZORBAX Eclipse XDBZORBAX Bonus-RPMid pH 7

Region B

ZORBAX Extend-C18High pH ≥ 9

Region C

A Method Development Strategy that Optimizes Resolution and Column Life

Adapted and updated by R.D. Ricker, B.A. Bidlingmeyer and J.J. Kirkland from J.J. Kirkland, LC/GC, 14 (1996) 486.

Band spacing problems

Poorly retainedbasic compounds

TailingPeaks

Basic compounds havinglow-pH instability

Bandspacing

problems

Bandspacing

problemsBand

spacingproblems

Band spacing problems

Band spacing problems

Band spacing problems

SAMPLE

• ZORBAX SB-C18 or SB-C8 • pH 2 (1-3) 20-50 mM buffer • T = 30°C (ambient to 80°C,

SB-C18 to 90°C) • Adjust % ACN for 0.5 < k < 20

Initial separation

• Add 20 mM TEA or TEA acetate • Re-adjust pH

Increase or decrease % oforganic modifier by 5% (v/v)

BASIC COMPOUNDS (Ion Pairing)• Use MeOH organic modifier,

25-50 mM hexane sulfonic acid,10 mM pH ≤ 3 buffer

• Adjust % MeOH for 0.5 < k < 20

• Change organic modifier (MeOH or THF)

• Adjust % organic for 0.5 < k < 20• Restart at STEP [2]

• Change bonded-phase functionality to ZORBAX SB-CN, SB-C3 or SB-Phenyl• Restart at STEP [1]

Try different HPLC mode.Call Agilent Technologies

Technical Support (800) 227-9770.

STEP 1

Band spacing problems

Vary temperature within recommended

range for bonded phase

STEP 2

STEP 3

STEP 4

STEP 1a

STEP 2aSTEP 1b

STEP 1c

STEP 1e

• Vary column temperature, up to 80°C; up to 90°C for ZORBAX SB-C18

Band spacing problems

Increase or decrease % of organic modifier

by 5% (v/v)

START

STEP 1d

OR

Bandspacing

problems

Bandspacing

problems

Bandspacing

problems

Vary temperature within recommended range

for bonded phase

Band spacing problems

Band spacing problems

• ZORBAX Eclipse XDB-C8 or XDB-C18• pH 7 (6-9), 20-50 mM buffer • T = 30°C (ambient to 40°C)• Adjust % MeOH for 0.5 < k < 20

ACIDIC COMPOUNDS (Ion Pairing)• Use MeOH organic modifier,

25-50 mM tetrabutylammoniumphosphate, 10 mM pH ≥ 7 buffer

• Adjust % MeOH for 0.5 < k < 20

• Change bonded-phase functionality to ZORBAX XDB-Phenyl or Bonus-RP

• Restart at STEP [5] • Change organic modifier(ACN or THF)

• Adjust for 0.5 < k < 20

• ZORBAX Extend-C18• pH 10.5 (9-12); 5 mM ammonia,

or TEA, or 10-50 mM organic buffer, or borate buffer

• T = 25°C (ambient to 40°C)• Adjust % MeOH for 0.5 < k < 20

Band spacing problems

Band spacing problems

STEP 5

STEP 7

STEP 8

STEP 9

STEP 5b

Band spacing problems

STEP 6a

STEP 5e

Band spacing problems

STEP 6

Increase or decrease % oforganic modifier by 5% (v/v)

• Change organic modifier (ACN or THF)• Adjust for 0.5 < k < 20• Restart at STEP [6]

Band spacing problemsSTEP 10a

• Change organic modifier (ACN or THF)• Adjust for 0.5 < k < 20

STEP 10Vary temperature within

recommended range for bonded phase

Band spacing problems

Vary temperature within recommended

range for bonded phase

STEP 5c

Increase or decrease % of organic modifier

by 5% (v/v)

STEP 5d

Poorly retainedacidic compounds

OR

Band spacing problems

Poor retention orBand spacing problems

Bandspacing

problems

• Change organic modifier (ACN or THF)• Adjust for 0.5 < k < 20

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1.0

0.8

0.6

0.4

0.2

0.0

0 5,000 10,000 15,000 20,000 25,000 30,000

Column Volumes of Mobile Phase Purge

Rela

tive

Rete

ntio

n

StableBond SB-C18(Diisobutyl-C18)

ZORBAX Rx-C18(Dimethyl-C18)Competitor A - C18

Competitor B - C18

Competitor C - C18

Competitor D - C18

Competitor E - C18

No column bleed

8

O

R

R

Si

OH

Si

O

Si

R

R

R

O

OH

R

R1

R1

R1

100

80

60

40

20

00 1000 2000 3000 4000 5000 6000

Column Volumes

% k

Rem

aini

ng

SB-CNSterically Protected Silane

Si-(Me)2PrCNTraditional Silane

FIGURE 2StableBond SB-C18 Shows Excellent Stability at Low pH and High Temperature (pH 0.8, 90°C)

FIGURE 3Shorter Chain ZORBAX SB-CN is Also Stable at Low pH (pH 2.0, 50°C)

Bulky diisobutyl (C18) ordiisopropyl (C8, C3, CN,Aq, Phenyl) side-chaingroups are used to stabilize both long and short chain bondedphases.

As an indicator of column breakdown, retention time of toluenewas measured after purging the column with mobile phase. Onlythe StableBond SB-C18 is unchanged after 3 working months ofuse under these very low pH (0.8) and high temperature (90°C)conditions. ZORBAX Rx-C18 also provides a stable matrix, andcan be used as an alternative selectivity to StableBond SB-C18.

ZORBAX StableBond SB-CN, and the othershort chain StableBond bonded phases,are also exceptionally stable at low pH.Conventional dimethyl CN and similarbonded phases lack this stability.

R = Isopropyl, isobutyl (C18)

R1 = C8, C18, CN, C3, etc.

• Longest column lifetime and best reproducibility for low pH separations — down to pH 1!

• Patented stable column chemistry allows use at high temperature and low pH without degradation

• Six different bonded-phases provide broad selectivity — SB-C18, SB-C8, SB-CN, SB-Phenyl, SB-C3, SB-Aq

• High purity silica for superior peak shape

• Excellent first choice for method development

Agilent ZORBAX StableBond columns use patented, unique monofunctional silaneswith bulky diisobutyl (SB-C18) or diisopropyl (SB-C8, SB-C3, SB-Phenyl, SB-CN and SB-Aq) side chain groups that sterically protect the key siloxane bond from hydrolyticattack at low pH. StableBond packing materials are not endcapped in order to provideexceptional stability and to maximize lifetime and reproducibility under acidic mobilephase conditions. The high purity, low acidity silica provides excellent peak shape withacidic, basic and neutral compounds so that StableBond columns are an excellentchoice for low pH method development. ZORBAX StableBond columns are compatiblewith all common mobile phases, including very high aqueous mobile phases.

ZORBAX StableBond HPLC Columns

ZORBAX Columns for Analytical Chemistry

Recommended ColumnZORBAX StableBond

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

Purge Solvent:50% methanol/50% water with 1.0% TFA

Temperature: 90°CSolute: Toluene

FIGURE 1Sterically Protected StableBondBonded Phase

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1 2

3

4

5

1 2

3

4

5

1

2

3

4

5

1

2

3

4

5

1

2

34

5

0 5 10 15Time (min)

StableBond SB-C18(Agilent Part No. 883975-902)

StableBond SB-C8(Agilent Part No. 883975-906)

StableBond SB-C3(Agilent Part No. 883975-909)

StableBond SB-Phenyl(Agilent Part No. 883975-912)

StableBond SB-CN(Agilent Part No. 883975-905)

Columns: ZORBAX StableBond4.6 x 150 mm, 5 µm

Mobile Phase: 0 - 100% B in 18.8 minA: 50 mM NaH2PO4,

pH 2.5 in 95% H2O / 5% ACNB: 50 mM NaH2PO4,

pH 2.5 in 47% H2O / 53% ACN

Flow Rate: 1.0 mL/min

Temperature: 26°C

UV Detection: 254 nm

Sample: 1. Procaine2. Lidocaine3. d-Cinchonine4. Butacaine5. Tetracaine

9

0 5 10Time (min)

1

3 4

5

2

OHOH

OHOH

HO O

FIGURE 4Five Different Bonded Phases Provide Selectivity Options

ZORBAX Columns for Analytical Chemistry

Column: ZORBAX SB-C84.6 x 150 mm, 3.5 µm (Agilent Part No. 863953-906)

Mobile Phase: 75% 0.1% TFA : 25% MeOH

Flow Rate: 1.0 mL/min

Temperature: 40°C

UV Detection: 280 nm

Sample: 1. Epigallocatechin2. Epicatechin3. Epigallocatechin gallate4. Catechol5. Epicatechin gallate

Nutraceuticals, such as the components ofgreen tea, are quickly separated on aStableBond SB-C8 Rapid Resolution column.

SB-C3 is just one of the five different StableBondselectivity choices. In this example, optimum resolutionis obtained with SB-C3. All are based on the samehigh purity Rx-SIL. Selectivity changes are thereforedependent only on the bonded phases making methoddevelopment more reliable.

Epicatechin

FIGURE 5Components of Green Tea Separated on aRapid Resolution StableBond SB-C8 Column

0 5 10 15 20Time (min)

1

3

45

2

Column: ZORBAX SB-C184.6 x 150 mm, 5 µm (Agilent Part No. 883975-902)

Mobile Phase: 85% 0.1% TFA15% ACN

Flow Rate: 1.0 mL/min

Temperature: 35°C

UV Detection: 210 nm

Sample: 5 µL1. Aspartic acid 4. asp-phe 2. Phenylalanine 5. Aspartame3. 5-benzyl-3, 6-dioxo-2-piperazineacetic acid The high purity Rx-SIL used in the manufacturing

of all StableBond columns provides excellentpeak shape of basic and acidic compounds.

FIGURE 6Excellent Peak Shape

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Time (min)

1

2

34

5

0 5 10 15 20 25 0 2 4 6 8 10 12Time (min)

1

2

3

4

5

10

Column: ZORBAX SB4.6 x 75 mm, 3.5 µm

Mobile Phase: 30% ACN70% NaH2PO4, pH 2.5

Flow Rate: 1.0 mL/min

Temperature: 35°C

Sample: 1. Estriol2. Daidzen3. Quercetin4. Genistein5. Diethylstilbestrol

The SB-CN column is used here to reduceanalysis time by 50%. The retention of themost hydrophobic analyte is cut in half. Atthe same time retention of the more polar,early eluting peaks increases slightly.

FIGURE 7SB-CN Optimizes Retention and Resolution

SB-C18(Agilent Part No. 866953-902)

SB-CN(Agilent Part No. 866953-905)

ZORBAX StableBond Columns (80Å) Ordering InformationColumn Size Particle SB-C18 SB-C8 SB-CN SB-C3 SB-Phenyl SB-Aq PriceDescription (mm) Size (µm) USP L1 USP L7 USP L10 USP L11Standard Columns and Bulk PackingPreparative 21.2 x 250 7 880975-102 880967-106 880975-105 880975-112 $2091Semi-Preparative 9.4 x 250 5 880975-202 880967-201 880975-205 880975-209 880975-212 1099Analytical 4.6 x 250 5 880975-902 880975-906 880975-905 880975-909 880975-912 880975-914 469Analytical 4.6 x 150 5 883975-902 883975-906 883975-905 883975-909 883975-912 883975-914 414Analytical 4.6 x 50 5 846975-902 846975-906 846975-914 304Rapid Resolution (RR) 4.6 x 150 3.5 863953-902 863953-906 863953-905 863953-912 469Rapid Resolution 4.6 x 75 3.5 866953-902 866953-906 866953-905 866953-912 414Rapid Resolution 4.6 x 50 3.5 835975-902 835975-906 835975-905 835975-912 359Solvent Saver 3.0 x 250 5 880975-302 880975-306 880975-305 880975-309 880975-312 469Solvent Saver 3.0 x 150 5 883975-302 883975-306 883975-305 883975-309 883975-312 414Solvent Saver Plus 3.0 x 150 3.5 863954-302 863954-306 863954-305 863954-312 469Solvent Saver Plus 3.0 x 100 3.5 861954-306 435Narrow Bore 2.1 x 150 5 883700-922 883700-906 883700-905 883700-909 883700-912 414Narrow Bore 2.1 x 50 5 860975-902 860975-906 860975-905 860975-909 860975-912 860975-914 304Narrow Bore RR* 2.1 x 150 3.5 830990-902 830990-906 469Narrow Bore RR 2.1 x 50 3.5 871700-902 871700-906 359MicroBore RR 1.0 x 150 3.5 863600-902 863600-906 505MicroBore RR 1.0 x 50 3.5 865600-902 865600-906 388MicroBore RR 1.0 x 30 3.5 861600-902 861600-906 301Bulk Packing, 2 grams 5 820966-906 820966-905 136Guard Cartridge, 2/pk 9.4 x 15 7 820675-115 820675-115 820675-124 820675-115 165Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-920 820950-915 820950-916 820950-922 820950-917 820950-933 155Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-926 821125-926 821125-924 821125-924 821125-926 821125-933 155Guard Hardware Kit 9.4 x 15 840140-901 840140-901 840140-901 840140-901 840140-901 162Guard Hardware Kit 4.6 x 12.5 820777-901 820777-901 820777-901 820777-901 820777-901 820777-901 106Guard Hardware Kit 2.1 x 12.5 820777-901 820777-901 820777-901 820777-901 820777-901 820777-901 106

*RR: Rapid Resolution 3.5 µm columns.Configurations not shown are available upon request. For more information on these and other columns consult the Agilent web site at www.agilent.com.300Å StableBond columns are also available for protein and peptide separations. See page 26 for descriptions and ordering information

ZORBAX Columns for Analytical Chemistry — StableBond

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11

To order or for technical assistance, call Agilent Technologies at 800-227-9770, or contact your local authorized Agilent distributor.

ZORBAX StableBond Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits* pH Range* Endcapped Carbon Load

ZORBAX SB-C18 80Å 180 m2/g 90°C 1.0 - 8.0 No 10%ZORBAX SB-C8 80Å 180 m2/g 80°C 1.0 - 8.0 No 5.5%ZORBAX SB-C3 80Å 180 m2/g 80°C 1.0 - 8.0 No 4.0%ZORBAX SB-CN 80Å 180 m2/g 80°C 1.0 - 8.0 No 4.0%ZORBAX SB-Phenyl 80Å 180 m2/g 80°C 1.0 - 8.0 No 5.5%ZORBAX SB-Aq 80Å 180 m2/g 80°C 1.0 - 8.0 No proprietary*StableBond columns are designed for optimal use at low pH. At pH 6-8, highest column stability for all silica-based columns is obtained by operating at temperatures <40°Cand using low buffer concentrations in the range of 0.01-0.02M. At mid-range pH, Eclipse XDB and Bonus-RP are recommended.

ZORBAX Columns for Analytical Chemistry — StableBond

ZORBAX StableBond Columns (80Å) Ordering Information (cont’d)Column Size Particle SB-C18 SB-C8Description (mm) Size (µm) USP L1 USP L7 PriceCapillary Glass-lined ColumnsCapillary 0.5 x 250 5 5064-8258 $ 505Capillary 0.5 x 150 5 5064-8256 455Capillary RR* 0.5 x 150 3.5 5064-8262 505Capillary RR 0.5 x 35 3.5 5064-8260 356Capillary 0.3 x 250 5 5064-8257 505Capillary 0.3 x 150 5 5064-8255 455Capillary RR 0.3 x 150 3.5 5064-8261 505Guard Cartridges 0.5 x 35 5 5064-8254 301Guard Cartridges 0.3 x 35 5 5064-8253 301

Agilent Cartridge ColumnsAnalytical 4.6 x 250 5 7995218-585 7995208-585 419Analytical 4.6 x 150 5 7995218-595 7995208-595 386Rapid Resolution 4.6 x 75 3.5 7995218-344 7995208-344 386Guard Cartridge, 10/pk 4.0 x 4.0 5 7995118-504 7995118-504 228Cartridge Holder 5021-1845 5021-1845 192

High Throughput Cartridge Columns (requires Hardware Kit 820222-901)Rapid Resolution Cartridge 4.6 x 30 3.5 833975-902 833975-906 194Rapid Resolution Cartridge, 3/pk 4.6 x 30 3.5 833975-932 833975-936 547Rapid Resolution Cartridge 4.6 x 15 3.5 831975-902 831975-906 155Rapid Resolution Cartridge, 3/pk 4.6 x 15 3.5 831975-932 831975-936 436Rapid Resolution Cartridge 2.1 x 30 3.5 873700-902 873700-906 194Rapid Resolution Cartridge, 3/pk 2.1 x 30 3.5 873700-932 873700-936 547Rapid Resolution Cartridge 2.1 x 15 3.5 875700-902 875700-906 155Rapid Resolution Cartridge, 3/pk 2.1 x 15 3.5 875700-932 875700-936 436High Throughput Cartridge Column Hardware Kit 820222-901 820222-901 162

CombiHT High Throughput Preparative Columns (endfittings required)CombiHT 21.2 x 150 5 870150-902 870150-906 1665CombiHT 21.2 x 100 5 870100-902 870100-906 1248CombiHT 21.2 x 50 5 870050-902 870050-906 827CombiHT End Fittings (required for use) 820400-901 820400-901 203

*RR: Rapid Resolution 3.5 µm columns.Configurations not shown are available upon request.For more information on these and other columns consult the Agilent web site atwww.agilent.com.300Å StableBond columns are also available for protein and peptide separations. See page 26 for descriptions and ordering information

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0.0 0.5 1.0 1.5 2.0Time (min)

1

2

34

FIGURE 2Separation of Water Soluble Vitamins on the SB-Aq Column without Ion Pairing

12

ZORBAX SB-Aq columns are an ideal choice to increase the retention of manydifficult acidic, basic and polar compounds. More retention may be obtained thanon many conventional ODS HPLC columns. Polar compounds can be analyzedwith excellent reproducibility and peak shape because the SB-Aq columns have a hydrophilic surface that inhibits phase collapse, even with 100% aqueousmobile phases. ZORBAX StableBond technology adds exceptional durability andstability at low pH and high temperature — down to pH 1 and up to 80°C with theSB-Aq column.

ZORBAX SB-Aq HPLC Columns• Recommended for high resolution analysis of very polar compounds

• Achieve high retention in highly aqueous mobile phases

• Obtain reproducible retention without phase collapse

• Provide exceptional column Lifetime at low pH

Recommended ColumnZORBAX StableBond

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

The basic compounds analyzed here are very polarand difficult to retain and separate without usinga gradient method. The SB-Aq column increasesthe retention of the early eluting compounds andresolves all components using a simple isocratic,highly aqueous mobile phase.

The SB-Aq column is used with a simple mobile phase (no ion-pair reagents) to achieve sufficientretention and high resolution of these B vitamins.

160 2 4 6 8 10 12 14Time (min)

2

1

3

4 5

Column: ZORBAX SB-Aq4.6 x 150 mm, 5 µm(Agilent Part No. 883975-914)

Mobile Phase: 90% 0.2% TFA10% ACN

Temperature: 25°C

Flow Rate: 1.5 mL/min

Detection: UV 254 nm

Sample:1. Maleate2. Phenylephrine3. Phenylpropanolamine4. Pyrilamine5. Chlorpheniramine

FIGURE 1Excellent Retention in a High AqueousMobile Phase on the SB-Aq Column

Column: ZORBAX SB-Aq4.6 x 150 mm, 5 µm(Agilent Part No. 883975-914)

Mobile Phase: 95% 0.1% TFA5% MeOH

Temperature: 35°C

Flow Rate: 2.0 mL/min

Detection: UV 254 nm

Sample: 1. Thiamine (B1)2. Nicotinic Acid3. Pyridoxine (B6)4. Niacinamide

ZORBAX SB-Aq Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits pH Range EndcappedZORBAX SB-Aq 80Å 180 m2/g 80°C 1.0 - 8.0 No

Configurations not shown are available upon request.For more information on these and other columns consult the Agilent web site at www.agilent.com.

ZORBAX StableBond Aq (SB-Aq) Column Ordering InformationColumn Dimension Particle PartDescription (mm) Size (µm) Number PriceAnalytical 4.6 x 250 5 880975-914 $ 469Analytical 4.6 x 150 5 883975-914 414Analytical 4.6 x 50 5 846975-914 304Narrow Bore 2.1 x 50 5 860975-914 304Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-933 155Guard Cartridges, 4/pk 2.1 x 12.5 5 821125-933 155Guard Hardware Kit 820777-901 106

ZORBAX Columns for Analytical Chemistry

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• Rx-C18 is recommended for alternate selectivity at low pH relative to StableBondSB-C18; for higher temperature applications, StableBond is recommended. Thiscolumn has a higher carbon load than SB-C18 columns (12% vs. 10%).

• Rx-C18 offers high stability and good peak shape for low pH applications

• Rx-C18 is manufactured using dimethyloctadecylsilane, is non-endcapped andprovides excellent stability up to pH 9

• Rx-C8 is the same product as SB-C8

Recommended ColumnZORBAX Rx-C8

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

Recommended ColumnZORBAX Rx-C18

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

FIGURE 1Analysis of Diazepam on Rx-C18

13

ZORBAX Rx HPLC Columns

ZORBAX Rx Column Ordering InformationColumn Size ParticleDescription (mm) Size (µm) Rx-C18 Rx-C8 PriceStandard ColumnsPreparative 21.2 x 250 7 880967-102 880967-106 $ 2091Semi-Preparative 9.4 x 250 5 880967-202 880967-201 1099Analytical 4.6 x 250 5 880967-902 880967-901 469Analytical 4.6 x 150 5 883967-902 883967-901 414Rapid Resolution 4.6 x 150 3.5 863967-902 863953-906 469Rapid Resolution 4.6 x 75 3.5 866967-902 866953-906 414Solvent Saver 3.0 x 250 5 880967-302 880975-306 469Solvent Saver 3.0 x 150 5 883967-302 883975-306 414Narrow Bore 2.1 x 150 5 883700-902 883700-906 414Bulk Packing, 2 grams 5 820966-906 136Guard Cartridge packed with Rx/SB-C8, 2/pk 9.4 x 15 7 820675-115 820675-115 165Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-914 820950-913 155Guard Cartridge packed with Eclipse XDB-C8, 4/pk 2.1 x 12.5 5 821125-926 821125-926 155Guard Hardware Kit 9.4 x 15 840140-901 840140-901 162Guard Hardware Kit 4.6 x 12.5 820777-901 820777-901 106Guard Hardware Kit 2.1 x 12.5 820777-901 820777-901 106

NOTE: Rx-C8 is the same product as StableBond SB-C8 Configurations not shown are available upon request.For more information on these and other columns consult the Agilent web site at www.agilent.com.

Column: ZORBAX Rx-C183.0 x 250 mm, 5 µm (Agilent Part No. 880967-302)

Mobile Phase: 35% H2O65% MeOH

Flow Rate: 0.5 mL/min

Sample: 1. Ethylparaben2. Diazepam

An Rx-C18 column is used for this USP analysis ofdiazepam and the internal standard ethylparaben. The Solvent Saver 3.0 mm i.d. Rx-C18 column reducessolvent usage by 60% over what would be used if theanalysis were done on a 4.6 x 250 mm column.

Time (min)

1 2

0.0 2.5 5.0 7.5

ZORBAX Rx Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits pH Range Endcapped Carbon LoadZORBAX Rx-C18 80Å 180 m2/g 60°C 2.0 - 9.0 No 12%ZORBAX Rx-C8 80Å 180 m2/g 80°C 1.0 - 8.0 No 5.5%

To order or for technical assistance,call Agilent Technologies at 800-227-9770, or contact your localauthorized Agilent distributor.

ZORBAX Columns for Analytical Chemistry

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FIGURE 2Column Stability Testing

FIGURE 3Good Peak Shape for Acids, Bases, andNeutrals on ZORBAX Eclipse XDB at Mid pH

14

FIGURE 1Extra Densely Bonded and DoubleEndcapped Eclipse XDB Bonded Phase

The Agilent ZORBAX Eclipse XDBcolumns — C18, C8 and Phenyl — aredesigned to provide good peak shapefor basic compounds at mid pH. In themid-pH region residual silanols aremore active and tailing interactions aremore likely. To overcome these inter-actions Eclipse XDB columns are eXtraDensely Bonded and then double end-capped through a proprietary processto cover as many active silanols aspossible. Column lifetime at mid pH isenhanced through the dense bondingand the choice of the thick-walled Rx-SIL support, which resists silica dissolution in the mid-pH range. As aresult, the Eclipse XDB columns can beused over the pH range 2 - 9, with anoptimum lifetime from pH 3 - 8. TheEclipse XDB columns also provide analternate selectivity to the StableBond,Bonus-RP and Extend-C18 columns.

ZORBAX Eclipse XDB HPLC Columns• Excellent peak shape for bases, acids and neutrals

• Long lifetime at mid pH with extra dense bonding and double endcapping

• Extended pH range — pH 2 - 9

CH3

CH3

CH3

CH3

CH3

CH3

O

SiO

O

O

O

Si

Si

Si

CH3

CH3

CH3

CH3CH3

CH3

Si

0 1 2 3 4 5 6 7 8 9 10 11 120 1 2 3 4 5 6 7 8

1

2 34

2,3

1

Time (min) Time (min)

4

5 5

0 5 10 15Time (min)

1

2

3

4

5

67

Column: ZORBAX Rapid Resolution Eclipse XDB-C84.6 x 75 mm, 3.5 µm (Agilent Part No. 966967-906)

Mobile Phase: 35% 20 mM NaH2PO4, pH 7.065% MeOH

Flow Rate: 1.0 mL/min

Temperature: 35°C

Detection: UV 254 nm

Column: 4.6 x 150 mm, 5µm

Mobile Phase: 60% ACN40% 25 mM phosphate buffer, pH 7.0

Flow Rate: 1.5 mL/min

Temperature: 60°C

Sample: Tricyclic Antidepressants1. Uracil2. Nortriptyline3. Doxepin4. Amitriptyline5. Trimipramine

Sample:1. Uracil2. Butyl Paraben3. Propranolol4. Naphthalene5. Dipropyl Phthalate6. Acenaphthene7. Amitriptyline

Eclipse XDB-C8Sol-type

After 1843 Column Volumes

Waters Symmetry C8SIL-type

After 1826 Column Volumes

In the mid-pH region, residual silanols can interactmore strongly with analytes, resulting in asymmetricpeaks. The Eclipse XDB column provides good peakshape in this difficult pH range.

Double endcapping, dense bonding, andthe durable Rx-SIL particles (sol-type)combine to provide long lifetime at pH7.0 when compared to single endcappedsil-gel columns, such as the WatersSymmetry C8 column used here. The conditions used for this test — high temperature (60°C) and high saltconcentration (250 mM), accelerate thedissolution of silica, causing prematurefailure of the sil-gel type column.

Recommended ColumnZORBAX Eclipse XDB

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

ZORBAX Columns for Analytical Chemistry

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FIGURE 4Selectivity Changes for Basic Compoundswith Eclipse XDB and StableBond

15

ZORBAX Eclipse XDB Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits pH Range Endcapped Carbon LoadZORBAX Eclipse XDB-C18 80Å 180 m2/g 60°C 2.0 - 9.0 Double 10%ZORBAX Eclipse XDB-C8 80Å 180 m2/g 60°C 2.0 - 9.0 Double 7.6%ZORBAX Eclipse XDB-Phenyl 80Å 180 m2/g 60°C 2.0 - 9.0 Double 7.2%

Non-EndcappedStableBond SB-C8(Agilent Part No. 866953-906)

Double EndcappedEclipse XDB-C8(Agilent Part No. 966967-906)

Columns: Rapid Resolution4.6 x 75 mm, 3.5 µm

Mobile Phase: 70% 25 mM NaH2PO4, pH 3.030% Methanol

Flow Rate: 1.0 mL/min

Temperature: 35°C

Sample: 1. Barbital2. Sulfamethoxazole3. Caffeine

Eclipse XDB and StableBond columns are basedon the same silica but have different bondingand endcapping. Therefore, they can have verydifferent selectivity for the same sample underthe same conditions, as this example shows.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

1

2

3

Time (min)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

3

2

1

Time (min)

ZORBAX Columns for Analytical Chemistry — Eclipse XDB

ZORBAX Eclipse XDB Column Ordering InformationColumn Size Particle XDB-C18 XDB-C8 XDB-PhenylDescription (mm) Size (µm) USP L1 USP L7 USP L11 PriceStandard Columns and Bulk PackingsSemi-Prep 9.4 x 250 5 990967-202 990967-206 $ 1099Analytical 4.6 x 250 5 990967-902 990967-906 990967-912 469Analytical 4.6 x 150 5 993967-902 993967-906 993967-912 414Analytical 4.6 x 50 5 946975-902 946975-906 304Rapid Resolution 4.6 x 150 3.5 963967-902 963967-906 963967-912 469Rapid Resolution 4.6 x 75 3.5 966967-902 966967-906 966967-912 414Rapid Resolution 4.6 x 50 3.5 935967-902 935967-906 935967-912 359Solvent Saver 3.0 x 250 5 990967-302 990967-306 990967-312 469Solvent Saver 3.0 x 150 5 993967-302 993967-306 993967-312 414Solvent Saver Plus 3.0 x 150 3.5 963954-302 963954-306 963954-312 469Narrow Bore 2.1 x 150 5 993700-902 993700-906 993700-912 414Narrow Bore 2.1 x 50 5 960967-902 960967-906 960967-912 304Narrow Bore RR* 2.1 x 150 3.5 930990-902 930990-906 469Narrow Bore RR 2.1 x 50 3.5 971700-902 971700-906 359MicroBore RR 1.0 x 150 3.5 963600-902 963600-906 505MicroBore RR 1.0 x 50 3.5 965600-902 965600-906 388MicroBore RR 1.0 x 30 3.5 961600-902 961600-906 301Bulk Packing, 2 grams 5 920966-902 136Guard Cartridges, 4/pk 4.6 x12.5 5 820950-925 820950-926 820950-927 155Guard Cartridges, 4/pk 2.1 x12.5 5 821125-926 821125-926 821125-926 155Guard Hardware Kit 820777-901 820777-901 820777-901 106Agilent Cartridge ColumnsAnalytical 4.6 x 250 5 7995118-585 7995108-585 419Analytical 4.6 x 150 5 7995118-595 7995108-595 386Rapid Resolution 4.6 x 75 3.5 7995118-344 7995108-344 386Solvent Saver 3.0 x 75 3.5 7995230-344 340Guard Cartridges, 10/pk 4.0 x 4 5 7995118-504 7995118-504 228Cartridge Holder 5021-1845 5021-1845 192High Throughput Cartridge Columns (requires Hardware Kit 820222-901)Rapid Resolution Cartridge 4.6 x 30 3.5 933975-902 933975-906 194Rapid Resolution Cartridge, 3/pk 4.6 x 30 3.5 933975-932 933975-936 547Rapid Resolution Cartridge 4.6 x 15 3.5 931975-902 931975-906 155Rapid Resolution Cartridge, 3/pk 4.6 x 15 3.5 931975-932 931975-936 436Rapid Resolution Cartridge 2.1 x 30 3.5 973700-902 973700-906 194Rapid Resolution Cartridge, 3/pk 2.1 x 30 3.5 973700-932 973700-936 547Rapid Resolution Cartridge 2.1 x 15 3.5 975700-902 975700-906 155Rapid Resolution Cartridge, 3/pk 2.1 x 15 3.5 975700-932 975700-936 436Hardware Kit for High Throughput Columns 820222-901 820222-901 162CombiHT Columns (end fittings required)CombiHT 21.2 x 150 5 970150-902 970150-906 1665CombiHT 21.2 x 100 5 970100-902 970100-906 1248CombiHT 21.2 x 50 5 970050-902 970050-906 827CombiHT End Fittings (2) (required for use) 820400-901 820400-901 203Capillary Glass-lined ColumnsCapillary 0.5 x 250 5 5064-8286 505Capillary 0.5 x 150 5 5064-8287 455Capillary RR 0.5 x 150 3.5 5064-8288 505Capillary RR 0.5 x 35 3.5 5064-8298 356Capillary 0.3 x 250 5 5064-8269 505Capillary 0.3 x 150 5 5064-8291 455Capillary RR 0.3 x 150 3.5 5064-8271 505Guard Cartridges 0.5 x 35 5 5064-8296 301Guard Cartridges 0.3 x 35 5 5064-8297 301

*RR: Rapid Resolution 3.5 µm columns.Configurations not shown are available upon request.For more information on these and other columns consult the Agilent web site at www.agilent.com.

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FIGURE 2ZORBAX Bonus-RP is Stable at Mid and Low pH

FIGURE 1Unique, Polar Alkyl Bonus-RPBonded Phase

16

0 10,000 20,000 30,000 40,000

0.0055

0.0056

0.0045

0.0040

0.0035

0.0030

0.0025

0.0020

Dimethyl-C18/amideBonus-RP

Column Volume of Purge

Tolu

ene

Plat

e He

ight

, cm

0 5000 10,000 15,000 20,000 25,000 30,000 35,000

0.0018

0.0016

0.0014

0.0012

0.0010

Competitive alkyl/amideBonus-RP

Column Volume of Purge

Tolu

ene

Plat

e He

ight

, cm

ZORBAX Bonus-RP HPLC Columns• Excellent peak shape for challenging basic compounds at low and mid pH

• Unique reversed-phase selectivity

• Novel bonding technology with embedded polar group and steric protection

• Usable in 100% aqueous mobile phases

Triple Endcapped

PG = polar amide group

R1 = sterically protecting isopropyl group

R = C14 alkyl chain

Columns: 4.6 x 150 mm

Mobile Phase: 60% 25 mM phosphatebuffer, pH 7.0

40% ACN

Flow Rate: 1.5 mL/min

Temperature: 23°C

Triple endcapping of Bonus-RP enhances stability at pH 7.Each 10,000 column volumes is equivalent to approximately one working month.

Sterically protecting side groups provide good low pHstability and longer column lifetime than similar polaralkyl bonded phases.

Mid-pH Stability

Low pH Stability

Columns: 4.6 x 150 mm

Mobile PhaseAging: 50% MeOH

50% 0.1% TFA

Flow Rate: 1.0 mL/min

Temperature: 60°C

Mobile PhaseTest: 80% MeOH

20% H2O

Flow Rate: 1.0 mL/min

Temperature: 23°C

ZORBAX Bonus-RP Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits pH Range Endcapped Carbon LoadZORBAX Bonus-RP 80Å 180 m2/g 60°C 2.0 - 9.0 Triple 9.5%

The Agilent ZORBAX Bonus-RP column has a polar amide group embedded in a long alkyl chain. This novel bonding reduces interactions between basiccompounds and the silica support, improving peak shape for the most difficult basic compounds. Peak shape and column lifetime are further improved by tripleendcapping. In addition, diisopropyl side groups provide steric protection againstacid hydrolysis for good lifetime at low pH. The Bonus-RP column provides analternate selectivity to C18 and C8 alkyl bonded phases.

O R

Si

Si

O

Si

O R

R 1

CH3

CH3

CH3

CH3

R

R 1

CH3

R 1

R 1

R 1

CH3

R 1

Si

Si

PG

PG

PG

Recommended ColumnZORBAX Bonus-RP

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

Unique Selectivity

ZORBAX Columns for Analytical Chemistry

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Time (min)

1

2

3

0 1 2 3 4 5 6

0 1 2 3 4 5 6 7 8 9 10Time (min)

2

3

1

FIGURE 4ZORBAX Bonus-RP Provides Unique Selectivity

FIGURE 3Improved Peak Shape of BasicCompounds Using Bonus-RP

17

0 2 4 6 8 10

mAu

15

10

5

0

mAu15

10

5

0

1

2

3

4

1

2

3

4

Time (min)

TF

1. 1.8

2. 1.7

3. 2.1

TF

1. 1.1

2. 1.1

3. 1.1

Column: 4.6 x 150 mm, 5 µm

Mobile Phase: 75% 25 mM NH4OAc, pH 5.525% ACN

Flow Rate: 1.5 mL/min

Temperature: 40°C

Detection: 254 nm

Sample: 1. Doxylamine 2. Chlorpheniramine 3. Triprolidine

Column: 4.6 x 150 mm

Mobile Phase: 75% 25 mM Na Citrate, pH 6 25% MeOH

Flow Rate: 1.0 mL/min

Temperature: Ambient

Detection: 254 nm

Injection vol: 3 µL

Sample: Cephalosporins1. cephalexin2. cephaclor3. cephuroxime4. cephoxitin

Column: 4.6 x 150 mm, 5 µm(Agilent Part No. 883668-901)

Mobile Phase: 80% 25 mM NH4OAc, pH 5.520% ACN

Flow Rate: 1.5 mL/min

Temperature: 40°C

Detection: 254 nm

Sample: 1. Doxylamine 2. Chlorpheniramine 3. Triprolidine

Bonus-RP eliminates peak tailing of these basiccompounds in comparison to a typical alkyl C8bonded phase. In the mid-pH region, residualsilanols can interact more strongly with basiccompounds to cause peak tailing. The polargroup in the Bonus-RP bonded phase eliminatespeak tailing of these basic compounds by reducing interactions with residual silanols.

Peak elution order can change dramatically when using Bonus-RP.In this example, the elution orderof the first three peaks change.Alkyl-C8

Bonus-RP(Agilent Part No. 883668-901)

Eclipse XDB-C8(Agilent Part No. 993967-906)

Bonus-RP

ZORBAX Bonus-RP Column Ordering InformationColumn Size Particle PartDescription (mm) Size (µm) Number PriceAnalytical 4.6 x 250 5 880668-901 $ 469Analytical 4.6 x 150 5 883668-901 414Rapid Resolution 4.6 x 150 3.5 863668-901 469Rapid Resolution 4.6 x 75 3.5 866668-901 414Narrow Bore 2.1 x 150 5 883725-901 414Narrow Bore 2.1 x 50 5 861971-901 304Rapid Resolution 2.1 x 50 3.5 861700-901 359MicroBore RR* 1.0 x 150 3.5 863608-901 505MicroBore RR 1.0 x 50 3.5 865608-901 388MicroBore RR 1.0 x 30 3.5 861608-901 301Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-928 155Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-928 155Guard Hardware Kit 820777-901 106

*RR: Rapid Resolution 3.5 µm columns.Configurations not shown are available upon request. For more information on these andother columns consult the Agilent web site at www.agilent.com.

To order or for technical assistance, call Agilent Technologies at 800-227-9770, or contact your local authorized Agilent distributor.

ZORBAX Columns for Analytical Chemistry — Bonus-RP

Page 19: Agilent ZORBAX Column Selection Guide for Analytical … · Agilent ZORBAX Column Selection Guide for ... ZORBAX Eclipse XDB HPLC Columns ... Extend-C18 columns are the newest addition

Volume Eluent (L)0 3 5 9 12 15

30

20

10

0

ZORBAX Extend-C18

Waters XTerra MS C18

Amou

nt o

f Dis

solv

ed S

ilica

(mg)

18

The combination of the unique bidentatestructure and double endcapping permitslong life at high pH.

At high pH, columns will fail due to silica dissolution. The examplehere shows extendedlifetime of ZORBAXExtend-C18 at high pH incomparison to WatersXTerra MS C18. This wasmeasured by the amountof dissolved silica.

Data provided by H.A. Claessens,Eindhoven University

These basic compounds are separated on the Extend-C18 at low pH with excellent peak shape. TheExtend-C18 column can be used at high and low pH.

See the 300Extend-C18 section on page 28 for additional examples.

• High efficiency and long life at high pH — up to pH 11.5

• Unique bidentate bonding and double endcapping provides high pH stability

• More efficiency and better peak shape than polymer-based columns

• Improve retention, resolution and peak shape of basic compounds

• High sensitivity for LC/MS separations of peptides

ZORBAX Extend-C18 HPLC Columns

FIGURE 2Long Life at High pH with Extend-C18

ZORBAX Extend-C18 Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits pH Range Endcapped Carbon LoadZORBAX Extend-C18 80Å 180 m2/g 60°C* 2.0 - 11.5 Double 12.5%*Temperature limits are 60°C up to pH 8, 40°C for pH 8 - 11.5.

SiO

SiO

Silica Support

C18C18

0 1 2 3 4 5 6 7Time (min)

1

2

3

4

FIGURE 3Extend-C18 Provides GoodPeak Shape at Low pH

Recommended ColumnZORBAX Extend-C18

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

Column: ZORBAX Extend-C184.6 x 150 mm

Mobile Phase: 20% Methanol80% 0.1 M carbonate buffer, pH 10.0

Flow Rate: 1.0 mL/min

Temperature: Ambient

Column: ZORBAX Extend-C184.6 x 150 mm, 5µm(Agilent Part No. 773450-902)

Mobile Phase: 80% 25 mM NaH2PO, pH 3.020% Methanol

Flow Rate: 1.0 mL/min

Temperature: 35°C

Detection: UV 254 nm

Sample:1. Theobromine2. Dimethylxanthine3. Theophylline4. Caffeine

FIGURE 1New Bidentate C18-C18 Bonding forExtend-C18 Bonded Phase

The Agilent ZORBAX Extend-C18 column uses a new bidentate C18-C18 columnbonding technology to make high pH separations a good choice with a silica-basedcolumn. At high pH, non-charged basic compounds will not interact with the silica,so it is possible to do separations with high efficiency, superior peak shape andimproved resolution. High pH separations are also the best choice for compoundsthat are more stable or more soluble in high pH solutions. Some of the mobile phasebuffer options for high pH include triethylamine, pyrrolidine, glycine, borate andammonium hydroxide. Ammonium hydroxide at pH 10.5 is an excellent mobile phasemodifier for the LC/MS of peptides and small molecules with improved sensitivitycompared with TFA containing mobile phase at low pH. The Extend-C18 column isstable from pH 2 - 11.5 with good peak shape for all types of compounds. Extend-C18 columns also provide an additional selectivity choice at low pH.1

1 Kirkland, J.J., J.B. Adams, Jr., M.A. van Straten and H.A. Claessens, (1998) Analytical Chemistry, 70, 4344-4352.

ZORBAX Columns for Analytical Chemistry

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0 5 10

1

2

3

4

Time (min)

6

5

7

0 5

1

2,3

4

5

Time (min)

7

6

19

0 5 10 15 20Time (min)

1

2 3

4

5

0 10 20 30Time (min)

1

2

34 5

FIGURE 5High Efficiency Separation of Basic Compounds at High pH on Extend-C18

Pseudoephedrine and scopolamine aredifficult to retain at low and mid pH.Pseudoephedrine is often analyzed byion exchange methods. The Extend-C18column retains these compounds in anoncharged form at high pH andimproves resolution.

Column: ZORBAX Extend-C184.6 x 150 mm, 5 µm(Agilent Part No. 773450-902)

Mobile Phase: See figure

Flow Rate: 1.0 mL/min

Temperature: R.T.

Detection: 254 nm

Sample: 1. Maleate pKa2. Scopolamine 7.63. Pseudoephedrine 9.84. Doxylamine 9.25. Chlorpheniramine 9.16. Triprolidine 6.57. Diphenhydramine 9.0

The Extend-C18, a silica based column,provides greater efficiency, improvedpeak shape and faster analysis at highpH in comparison to a polymeric bondedphase. A lower flow rate had to be usedfor the polymeric column due to a pressure limitation of 180 bar.

Mobile Phase: 65% 20 mM TEA, pH 1135% MeOH

Temperature: RT

Detection: UV 254 nm

Sample: 1. Pyridoxine2. Pyridine3. N-methylbenzylamine4. Procainamide5. N-acetylprocainamide

pH 730% 20 mM Na2HPO470% MeOH

pH 1130% 20 mM TEA70% MeOH

Polymeric-Based Column, 4.0 x 250 mm, 5 µm

Flow Rate: 0.5 mL/minPressure: 142 bar

Flow Rate: 1.0 mL/minPressure: 213 bar

Extend-C18, 4.6 x 250 mm, 5 µm(Agilent Part No. 770450-902)

ZORBAX Extend-C18 Column Ordering InformationColumn Size Particle PartDescription (mm) Size (µm) Number PriceAnalytical 4.6 x 250 5 770450-902 $ 551Analytical 4.6 x 150 5 773450-902 490Analytical 4.6 x 50 5 746450-902 357Rapid Resolution 4.6 x 150 3.5 763953-902 551Rapid Resolution 4.6 x 75 3.5 766953-902 490Rapid Resolution 4.6 x 50 3.5 735953-902 423Narrow Bore 2.1 x 150 5 773700-902 490Narrow Bore 2.1 x 50 5 760450-902 357Narrow Bore RR* 2.1 x 50 3.5 735700-902 423MicroBore RR 1.0 x 150 3.5 763600-902 505MicroBore RR 1.0 x 50 3.5 765600-902 388MicroBore RR 1.0 x 30 3.5 761600-902 301Bulk Packing, 2 grams 5 820966-402 139Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-930 184Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-930 184Guard Hardware Kit 820777-901 106

*RR: Rapid Resolution 3.5 µm columns.Configurations not shown are available upon request.300Å Extend-C18 columns are also available for peptide separations. See page 28 for descriptions and ordering information

FIGURE 4Improved Retention, Resolution and Peak Shape of Basic Antihistamines on Extend-C18 at High pH

To order or for technical assistance,call Agilent Technologies at 800-227-9770, or contact your localauthorized Agilent distributor.

ZORBAX Columns for Analytical Chemistry — Extend-C18

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ZORBAX Normal Phase Column Ordering InformationColumn Size Particle Rx-SIL SIL CN CarbohydrateDescription (mm) Size (µm) USP L3 USP L3 USP L10 NH2 Analysis PricePreparative 21.2 x 250 7 880975-101 880952-101 880952-105 880952-108 $ 2091Semi-Preparative 9.4 x 250 5 880975-201 880952-201 880952-205 880952-208 1099Analytical 4.6 x 250 5 880975-901 880952-705 880952-708 840300-908 469Analytical 4.6 x 250 5 880952-701 430Analytical 4.6 x 150 5 883975-901 883952-705 883952-708 843300-908 414Analytical 4.6 x 150 5 883952-701 359Narrow Bore 2.1 x 150 5 883700-901 414Guard Cartridges, 2/pk 9.4 x 15 7 820675-119 820675-119 820675-111 820675-111 165Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-919 820950-901 820950-905 820950-908 820950-908 155Guard Cartridges, 4/pk 2.1 x 12.5 5 821125-919 155Guard Hardware Kit 9.4 x 15 840140-901 840140-901 840140-901 840140-901 162Guard Hardware Kit 4.6 x 12.5 820777-901 820777-901 820777-901 820777-901 820777-901 106Guard Hardware Kit 2.1 x 12.5 820777-901 820777-901 820777-901 820777-901 106

Configurations not shown are available upon request.

20

Original ZORBAX Column Ordering InformationColumn Size Particle ODS (C18) C8 Phenyl CN TMSDescription (mm) Size (µm) USP L1 USP L7 USP L11 USP L10 USP L13 PriceStandard Columns Preparative 21.2 x 250 7 880952-102 880952-106 880952-105 $ 2091Semi-Preparative 9.4 x 250 5 880952-202 880952-206 880952-212 880952-205 1099Analytical (Endcapped) 4.6 x 250 5 880952-702 880952-706 880952-712 884950-507 880952-710 469Analytical (Non-endcapped) 4.6 x 250 5 884950-543 469Analytical 4.6 x 150 5 883952-702 883952-706 883952-712 884950-526 883952-710 414Solvent Saver 3.0 x 250 5 880952-302 469Solvent Saver 3.0 x 150 5 883952-302 414Guard Cartridges, 2/pk 9.4 x 15 7 820675-115 820675-115 820675-115 820675-124 165Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-902 820950-906 820950-912 820950-905 820950-905 155Guard Hardware Kit 9.4 x 15 840140-901 840140-901 840140-901 840140-901 840140-901 162Guard Hardware Kit 4.6 x 12.5 820777-901 820777-901 820777-901 820777-901 820777-901 106

Configurations not shown are available upon request.

ZORBAX Method Development KitsDescription Agilent Part No. Price

Traditional StableBond Kit: Includes 3 columns, 4.6 x 150 mm packed with SB-C18, 5 µm, SB-CN, 5 µm and SB-Phenyl, 5 µm 5183-4624 $ 1056

Fast StableBond Kit: Includes 3 columns, 4.6 x 75 mm packed with SB-C18, 3.5 µm, SB-CN, 3.5 µm and SB-Phenyl, 3.5 µm 5183-4625 1056

Traditional Eclipse XDB Kit: Includes 3 columns, 4.6 x 150 mm packed with XDB-C18, 5 µm, XDB-C8, 5 µm and XDB-Phenyl, 5 µm 5183-4626 1056

Fast Eclipse XDB Kit: Includes 3 columns, 4.6 x 75 mm packed with XDB-C18, 3.5 µm, XDB-C8, 5 µm and XDB-Phenyl, 3.5 µm 5183-4627 1056

ZORBAX Method Validation Kits

ZORBAX Validation Kits are supplied to customers who need the same HPLC column type (bonded phase, particle size, configuration) but from different manufacturing lots. To request columns from different lots, contact Agilent Technologies or your local distributor using the following procedure.

1. Request Validation Kits (columns from different lots) by using Part Number 899999-888.

2. Indicate the Part Number of the current column you are using.

3. Indicate the Lot Number of the current column you are using.

4. Indicate the number of additional columns needed from different lots (e.g.: you have a current column and may need two other columns from additional lots).

5. Your customer service representative will issue a Special Products Quote (SPQ) number, price and anticipated delivery date for your order. This process may take 1 - 2 working days, and delivery is usually within 1 - 3 weeks from time of order, depending on availability.

Listed here, for your convenience, are additional ZORBAX Analytical column part numbers. For further information, consult the Agilent2000/2001 Catalog or visit our web siteat www.agilent.com/chem.

• ZORBAX Method Validation Kits

• ZORBAX Method Development Kits

•Original ZORBAX Columns

• ZORBAX Normal Phase Columns

Additional ZORBAX Analytical HPLC Columns

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0 5 10

Time (min)

0 10 20 30

Time (min)

21

CombiHT preparative columns aredesigned to scale up rapid analyticalseparations to sample loads of 15 - 50 mg. CombiHT columns use 5 µm packings for high efficiency and provide direct scale up from 50 - 150 mm analytical columns.These columns are available withreusable, finger tight endfittings soonly the column barrel is replaced.CombiHT C8 and C18 columns areavailable in the two most popularZORBAX bonded-phase types to optimize selectivity, StableBond forlow pH and Eclipse XDB for mid-pHbut can be packed with any ZORBAXphase on request.

ZORBAX CombiHT ColumnsZORBAX LC/MS Columns and Cartridge ColumnsZORBAX Rapid Resolution Columns

For . . .

• LC/MS Applications

• Combinatorial Chemistry Applications

• High Speed Chromatography

• Reduced Analysis Time

Agilent ZORBAX Columns are available in a variety of dimensions — from narrow-bore to preparative — to speed up your chromatography and increaseyour sample throughput, while still achieving as much resolution as you need.

High-Throughput HPLC Columns

FIGURE 1Gradient Preparative High Throughput HPLC

ZORBAX CombiHT HPLC Columns

• Fast preparative separations inthe 15 - 50 mg range

• Three different column lengths— 50, 100, 150 mm

• Finger tight connections seal upto 7000 psi

B. Purity CheckRapid Resolution SB-C184.6 x 50 mm, 3.5 µm(Agilent Part No. 835975-902)

A. 35 mg Prep SampleCombiHT SB-C1821.2 x 50 mm, 5 µm(Agilent Part No. 870050-902)

A: 0.1 % TFA in H20

B: 0.1 % TFA in ACN

10 - 40% B in 20 min30 mL/min

10 - 50% B in 20 min3 mL/min

Data provided by Hoffmann LaRoche, Ted Lambrose

The CombiHT column is used to separate and isolate a compound of interest, whilethe matching analytical column confirms the peak was isolated with high purity.

Flow Rate Conversion

To determine the optimum flow ratefor your 21.2 mm i.d. column, use thefollowing equation:

F2 = F1 x (ID2/ID1)2

For example,4.6 mm i.d. 21.2 mm i.d.

0.5 mL/min 10.5 mL/min

1.0 mL/min 21 mL/min

1.5 mL/min 31.5 mL/min

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0

50

100

150

mAU

2

1

3

4

5

61

2

3

45

6

Time (min)0 1 2 3

1

2 3

4

1

2 34

1

234

FIGURE 2Reduce Analysis Time By 70% UsingShort LC/MS Cartridge Columns

FIGURE 3Maximize Sensitivity with Narrow-Bore LC/MS Cartridges

ZORBAX LC/MS HPLC Columns and Cartridge Columns

Selecting a column for LC/MS involves three simple choices: column internaldiameter, column length and bonded phase. ZORBAX, Rapid Resolution ultra-short, narrow-bore cartridge columns (2.1 x 15 and 2.1 x 30 mm) are often a first choice for LC/MS applications because these columns provide thefastest analysis time and good sensitivity with ESI detection. ZORBAX short, narrow-bore (2.1 x 50 mm) and MicroBore (1.0 x 50 mm) columns are also goodchoices for ESI/MS because their optimum flow rates are from 50 - 200 µL/min,providing additional sample resolution while still keeping analysis times veryshort. Even longer lengths are available for very complex samples. The 4.6 mm i.d. 15 and 30 mm cartridges as well as the longer columns provide thesame resolution choices and are an ideal choice for APCI detectors where thelow flow rate is not required.

ZORBAX StableBond and Eclipse XDB-C18 and C8 bonded phases are available in all configurations and additional bonded phases can be packed on request.StableBond columns demonstrate superior low pH stability and are ideal forformic acid and TFA containing mobile phases. Eclipse XDB bonded phases are a great choice with acetic acid mobile phases or for quick compound screeningover a wide pH range.

22

High-Throughput HPLC Columns

• Fast analysis for high speed LC/MS with Rapid Resolution cartridges

• Narrow bore and MicroBore columns for maximum sensitivity withElectrospray Ionization (ESI)

• Additional column dimensions for higher sample loads with APCI

• Many bonded phases available

4.6 x 50 mm, 3.5 µm(Agilent Part No. 835975-902)

4.6 x 30 mm, 3.5 µm(Agilent Part No. 833975-902)

4.6 x 15 mm, 3.5 µm(Agilent Part No. 831975-902)

Column: ZORBAX StableBond SB-C18

Mobile Phase: 85% Water with 0.1% TFA15% Acetonitrile

Flow Rate: 1.0 mL/min

Temperature: 35°C

Sample:1. Phenylalanine2. 5-Benzyl-3,6-dioxo-2-piperazine

acetic acid3. Asp-phe4. Aspartame Column: Rapid Resolution SB-C18, 3.5 µm

Mobile Phase: Gradient: 5 - 35% B in 6 minA: Water with 0.2% formic acidB: Methanol with 0.2% formic acid

Temperature: 35°C

Sample: 2 µL Organic Acids1. Gallic2. Protocatechuic3. Hydrocaffeic4. Gentisic5. Vanillic6. Syringic

4.6 x 30 mm, 3.5 µm(Agilent Part No. 833975-902)

2.1 x 30 mm, 3.5 µm(Agilent Part No. 873700-902)

To order or for technical assistance,call Agilent Technologies at 800-227-9770, or contact your localauthorized Agilent distributor.

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0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0Time (min)Time (min)Time (min)

1

2 34

5

Time (min)0 1 2

1

2

3

4

Time (min)0 1 2 3

1

2

3

4

Time (min)0 1 2 3 4 5 6

1

2

3

4

23

Rapid Resolution 4.6 x 75 mm, 3.5 µm columns are a good choice for initial method development because they allowyou to quickly determine if a given column and mobile phase combination can produce the desired separation.

Column: ZORBAX StableBond SB-C184.6 x 75 mm, 3.5 µm(Agilent Part No. 866953-902)

Mobile Phase: A: 25 mM NaH2PO4, pH 3.0B: MeOH

Flow Rate: 2.0 mL/min

Temperature: 35°C

Detection: UV 254 nm

Sample: 1. Diltiazem2. Dipyridamole3. Nifedipine4. Lidoflazine5. Flunarizine

FIGURE 5Run Method Development Chromatograms on Rapid Resolution Columns

A : B 20 : 80 30 : 70 40 : 60

High-Throughput HPLC ColumnsZORBAX Rapid Resolution 3.5 µm HPLC Columns• Reduce analysis time and solvent usage and increase sample throughput

• High efficiency in short and ultra-short column lengths

• Available in analytical (4.6 mm) and narrow-bore (2.1 mm) i.d.

• Comparable lifetime to 5 µm columns

Agilent ZORBAX Rapid Resolutioncolumns, with a 3.5 µm particle size,reduce analysis time and increase sample throughput for any applicationwhen compared to 5 µm columns. Rapid Resolution columns are availablefrom 15 - 150 mm, in both 4.6 and 2.1 mm internal diameters, so the best configuration is available for high-throughput, LC/MS, combinatorialchemistry and rapid analytical applications. ZORBAX Rapid Resolutionparticles have superior mechanicalstrength, so every Rapid Resolution column has an extremely stable packedbed and provides a comparable lifetimeto 5 µm columns. ZORBAX RapidResolution columns are available in all of the modern ZORBAX bondedphases — StableBond, Eclipse XDB,Bonus-RP and Extend-C18.

Rapid Resolution 3.5 µm columnsare available in narrow bore configurations for great compatibility with LC/MS.

Column: ZORBAX Bonus-RP

Mobile Phase: 80% 25 mM NH4Ac, pH 5.520% ACN

Temperature: 40°C

Detection: 254 nm

Sample: 1. Caffeine2. Doxylamine3. Chlorpheniramine4. Triprolidine

FIGURE 4Rapid Resolution Columns Reduce Analysis Time While Maintaining Resolution

Flow Rate1.5 mL/min

Flow Rate1.5 mL/min

Flow Rate0.3 mL/min

Bonus-RP, 4.6 x 150 mm, 5 µm(Agilent Part No. 883668-901)

Rapid Resolution Bonus-RP4.6 x 75 mm, 3.5 µm(Agilent Part No. 866668-901)

Narrow BoreRapid Resolution Bonus-RP2.1 x 50 mm, 3.5 µm(Agilent Part No. 861700-901)

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24

Configurations not shown are available uponrequest. For more information on these andother columns consult the Agilent web site atwww.agilent.com.

ZORBAX High-Throughput Column Ordering Information

ZORBAX Extend-C18 ColumnsColumn Size Particle PartDescription (mm) Size (µm) Number PriceRapid Resolution 4.6 x 150 3.5 763953-902 $ 551Rapid Resolution 4.6 x 75 3.5 766953-902 490Rapid Resolution 4.6 x 50 3.5 735953-902 423Narrow Bore 2.1 x 150 5 773700-902 490Narrow Bore 2.1 x 50 5 760450-902 357Narrow Bore RR* 2.1 x 50 3.5 735700-902 423MicroBore RR 1.0 x 150 3.5 763600-902 505MicroBore RR 1.0 x 50 3.5 765600-902 388MicroBore RR 1.0 x 30 3.5 761600-902 301

*RR: Rapid Resolution 3.5 µm columns.

To order or for technical assistance,call Agilent Technologies at 800-227-9770, or contact your localauthorized Agilent distributor.

ZORBAX Rx, Eclipse XDB and Bonus-RP Columns

Eclipse Eclipse EclipseColumn Size Particle Rx-C18 Rx-C8 XDB-C18 XDB-C8 XDB-Phenyl Bonus-RPDescription (mm) Size (µm) USP L1 USP L7 USP L11 PriceRapid Resolution 4.6 x 150 3.5 863967-902 863953-906 963967-902 963967-906 963967-912 863668-901 $ 469Rapid Resolution 4.6 x 75 3.5 866967-902 866953-906 966967-902 966967-906 966967-912 866668-901 414Rapid Resolution 4.6 x 50 3.5 935967-902 935967-906 935967-912 359Solvent Saver Plus 3.0 x 150 3.5 963954-302 963954-306 963954-312 469Narrow Bore 2.1 x 150 5 883700-902 883700-906 993700-902 993700-906 993700-912 883725-901 414Narrow Bore 2.1 x 50 5 960967-902 960967-906 960967-912 861971-901 304Rapid Resolution 2.1 x 150 3.5 930990-902 930990-906 469Rapid Resolution 2.1 x 50 3.5 971700-902 971700-906 861700-901 359MicroBore RR* 1.0 x 150 3.5 963600-902 963600-906 863608-901 505MicroBore RR 1.0 x 50 3.5 965600-902 965600-906 865608-901 388MicroBore RR 1.0 x 30 3.5 961600-902 961600-906 861608-901 301

ZORBAX Rapid Resolution LC/MS Cartridge Columns Ordering Information (requires Hardware Kit 820222-901)

Eclipse EclipseColumn Size Particle SB-C18 SB-C8 XDB-C18 XDB-C8Description (mm) Size (µm) USP L1 USP L7 USP L1 USP L7 PriceRapid Resolution Cartridge 4.6 x 30 3.5 833975-902 833975-906 933975-902 933975-906 $ 194Rapid Resolution Cartridge, 3/pk 4.6 x 30 3.5 833975-932 833975-936 933975-932 933975-936 547Rapid Resolution Cartridge 4.6 x 15 3.5 831975-902 831975-906 931975-902 931975-906 155Rapid Resolution Cartridge, 3/pk 4.6 x 15 3.5 831975-932 831975-936 931975-932 931975-936 436Rapid Resolution Cartridge 2.1 x 30 3.5 873700-902 873700-906 973700-902 973700-906 194Rapid Resolution Cartridge, 3/pk 2.1 x 30 3.5 873700-932 873700-936 973700-932 973700-936 547Rapid Resolution Cartridge 2.1 x 15 3.5 875700-902 875700-906 975700-902 975700-906 155Rapid Resolution Cartridge, 3/pk 2.1 x 15 3.5 875700-932 875700-936 975700-932 975700-936 436Column Hardware Kit 820222-901 820222-901 820222-901 820222-901 162

ZORBAX CombiHT Column Ordering InformationEclipse Eclipse

Column Size Particle SB-C18 SB-C8 XDB-C18 XDB-C8Description (mm) Size (µm) USP L1 USP L7 USP L1 USP L7 PriceCombiHT 21.2 x 150 5 870150-902 870150-906 970150-902 970150-906 $ 1665CombiHT 21.2 x 100 5 870100-902 870100-906 970100-902 970100-906 1248CombiHT 21.2 x 50 5 870050-902 870050-906 970050-902 970050-906 827CombiHT End Fittings (required for use) 820400-901 820400-901 820400-901 820400-901 203

ZORBAX Rapid Resolution and LC/MS Columns Ordering Information

ZORBAX StableBond Columns

Column Size Particle SB-C18 SB-C8 SB-CN SB-C3 SB-Phenyl SB-Aq PriceDescription (mm) Size (µm) USP L1 USP L7 USP L10 USP L11Rapid Resolution 4.6 x 150 3.5 863953-902 863953-906 863953-905 863953-912 $ 469Rapid Resolution 4.6 x 75 3.5 866953-902 866953-906 866953-905 866953-912 414Rapid Resolution 4.6 x 50 3.5 835975-902 835975-906 835975-905 835975-912 359Solvent Saver Plus 3.0 x 150 3.5 863954-302 863954-306 863954-305 863954-312 469Solvent Saver Plus 3.0 x 100 3.5 841954-306 435Narrow Bore 2.1 x 150 5 883700-922 883700-906 883700-905 883700-909 883700-912 414Narrow Bore 2.1 x 50 5 860975-902 860975-906 860975-905 860975-909 860975-912 860975-914 304Narrow Bore RR* 2.1 x 150 3.5 830990-902 830990-906 469Narrow Bore RR 2.1 x 50 3.5 871700-902 871700-906 359MicroBore RR 1.0 x 150 3.5 863600-902 863600-906 505MicroBore RR 1.0 x 50 3.5 865600-902 865600-906 388MicroBore RR 1.0 x 30 3.5 861600-902 861600-906 301

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25

MicroBore (1 mm) and capillary columns (0.5, 0.3 mm) are often the best choicewhen sample sizes are limited. MicroBore columns will improve limits of detection5X over 2.1 mm i.d. columns, and capillary columns can decrease detection limits20 – 50X relative to 2.1 mm i.d. columns when the same sample size is used. Thisincrease in sensitivity can be critical. MicroBore and capillary columns use verylow flow rates — from 1 – 50 µL/min. Therefore, these columns are ideal for usewith detectors requiring the lowest flow rates, such as the particle beam and electrospray LC/MS detectors.

Agilent MicroBore and capillary columns are available in three different internal diameters and several lengths, so the ideal configuration is available for your sample limited applications. The 1.0 mm MicroBore columns are packed in stainless steel, and the 0.3 and 0.5 mm capillary columns are packed into glass-lined, stainless steel columns. These columns perform optimally with HPLC systems purchased or adjusted for microbore and capillary column use.Many bonded phases are available, including StableBond SB-C18 and SB-C8, Eclipse XDB-C18 and XDB-C8, Bonus-RP, Extend-C18, 300SB-C18 and Hypersil ODS.

Agilent MicroBore and Capillary Columns

• High sensitivity for smallest sample sizes

• Compatibility with several LC/MS interfaces

• Several internal diameters — 1.0 mm, 0.5 mm, 0.3 mm

• Variety of bonded phases

ZORBAX MicroBore HPLC Column Ordering Information

Eclipse EclipseSize Particle SB-C18 SB-C8 300SB-C18 300SB-C8 XDB-C18 XDB-C8 Bonus-RP Extend-C18 Price(mm) Size (µm) USP L1 USP L7 USP L7 USP L7

1.0 x 150 3.5 863600-902 863600-906 863630-902 863630-906 963600-902 963600-906 863608-901 763600-902 $ 5051.0 x 50 3.5 865600-902 865600-906 865630-902 865630-906 965600-902 965600-906 865608-901 765600-902 3881.0 x 30 3.5 861600-902 861600-906 961600-902 961600-906 861608-901 761600-902 301

Agilent HPLC Capillary Columns Ordering Information (glass-lined)Size Particle Eclipse

Column (mm) Size (µm) SB-C18 300SB-C18 XDB-C18 Price Hypersil ODS PriceCapillary 0.5 x 250 5 5064-8258 5064-8266 5064-8286 $ 505 5064-8278 $ 495Capillary 0.5 x 150 5 5064-8256 5064-8264 5064-8287 455 5064-8289 446Guard 0.5 x 35 5 5064-8254 5064-8294 5064-8296 301 5064-8282 295Capillary RR* 0.5 x 150 3.5 5064-8262 5064-8268 5064-8288 505 5064-8275** 495Capillary RR 0.5 x 35 3.5 5064-8260 5064-8298 356 5064-8299** 349Capillary 0.3 x 250 5 5064-8257 5064-8265 5064-8269 505 5064-8276 495Capillary 0.3 x 150 5 5064-8255 5064-8263 5064-8291 455 5064-8290 446Guard 0.3 x 35 5 5064-8253 5064-8295 5064-8297 301 5064-8279 295Capillary RR 0.3 x 150 3.5 5064-8261 5064-8267 5064-8271 505 5064-8277** 495

*RR: Rapid Resolution 3.5 µm columns** 3 µm particles

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% k

Phe

nylth

epta

ne R

emai

ning

100

90

80

70

60

50

400

Volume of Mobile Phase (mL)300020001000 4000 5000 6000 7000 8000

ZORBAX 300SB-C3Competitor C4

Recommended ColumnZORBAX StableBond

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

26

Mobile Phase: Gradients 0-100% B in 80 min.A:0.5% TFA in WaterB:0.5% TFA in Acetonitrile

Isocratic Retention Test Conditions:1-phenylheptane 50% A, 50% B

Columns: 4.6 x 150 mm

Temperature: 60°C

Flow Rate: 1.0 mL/min

FIGURE 2Short-Chain ZORBAX 300SB-C3 is Stable at Low pH, High Temperature

O

R

R

Si

OH

Si

O

Si

R

R

R

O

OH

R

R1

R1

R1

FIGURE 1Sterically Protected300StableBond Bonded Phase

Bulky diisobutyl (C18) ordiisopropyl (C8, C3, CN,)side-chain groups are used to stabilize both long and short chain bonded phases. Additional stability information on StableBondcolumns can be found onpages 8 -9.

300StableBond columns are unsurpassed in theirlow pH stability. The reproducible monolayerbonding and sterically protected bonded phaseincrease the lifetime and reliability of the short-chain 300SB-C3 column over another wide-poreC4 column. Additional stability information onStableBond columns can be found on pages 8 -9.

R = Isopropyl, isobutyl (C18)

R1 = C8, C18, CN, C3

• Four different bonded phases for reversed-phase selectivity optimization of peptides and proteins

• Exceptional stability and long lifetime at low pH with TFA containing mobile phases

• Excellent reproducibility

• Stable at high temperature — up to 80 - 90°C

ZORBAX Columns for BioAnalytical ChemistryZORBAX Wide-Pore 300StableBond HPLC Columns

ZORBAX 300StableBond columns are an ideal choice for the reproducible separa-tions of proteins and peptides for two key reasons. First, wide-pore, 300Åcolumns are necessary for an efficient separation of proteins and peptides, orother large molecules, in order to allow these analytes to completely access thebonded phase. Second, 300StableBond columns are unmatched in their durabilityat low pH, such as with the TFA containing mobile phases typically used for pro-tein and peptide separations. For LC/MS separations at low pH, 300StableBondcolumns can also be used with formic acid and acetic acid mobile phase modifiers. 300StableBond columns are available in four different bonded phases (C18, C8, C3, and CN) for selectivity and recovery optimization of proteins andpolypeptides. To further increase sample recovery and improve efficiency for difficult proteins, 300StableBond columns can be used up to 80 - 90°C.

ZORBAX StableBond 300SB Column SpecificationsBonded Phase Pore Size Surface Area Temp. Limits* pH Range* Endcapped Carbon Load

ZORBAX 300SB-C18 300Å 45 m2/g 90°C 1.0 - 8.0 No 2.8%ZORBAX 300SB-C8 300Å 45 m2/g 80°C 1.0 - 8.0 No 1.5%ZORBAX 300SB-C3 300Å 45 m2/g 80°C 1.0 - 8.0 No 1.1%ZORBAX 300SB-CN 300Å 45 m2/g 80°C 1.0 - 8.0 No 1.2%*300StableBond columns are designed for optimal use at low pH. At pH 6-8, highest column stability for all silica-based columns is obtained by operating at temperatures<40°C and using low buffer concentrations in the range of 0.01-0.02M. At mid or high pH, 300Extend-C18 is recommended.

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ZORBAX StableBond (300Å) Column Ordering Information Column Size Particle 300SB-C18 300SB-C8 300SB-CN 300SB-C3Description (mm) Size (µm) USP L1 USP L7 USP L10 PriceStandard Columns and Bulk PackingPreparative 21.2 x 250 7 880995-102 880995-106 880995-109 880995-105 $ 2318Semipreparative 9.4 x 250 5 880995-202 880995-206 880995-209 880995-205 1099Analytical 4.6 x 250 5 880995-902 880995-906 880995-905 880995-909 469Analytical 4.6 x 150 5 883995-902 883995-906 883995-905 883995-909 414Analytical 4.6 x 50 5 860950-902 860950-906 860950-905 860950-909 325Rapid Resolution 4.6 x 150 3.5 863973-902 863973-906 863973-905 863973-909 469Rapid Resolution 4.6 x 50 3.5 865973-902 865973-906 865973-905 865973-909 359Solvent Saver Plus 3.0 x 100 3.5 884950-565 435Narrow Bore 2.1 x 150 5 883750-902 883750-906 883750-905 883750-909 414Narrow Bore RR* 2.1 x 150 3.5 863750-906 469Narrow Bore RR 2.1 x 50 3.5 865750-902 865750-906 359MicroBore RR 1.0 x 150 3.5 863630-902 863630-906 505MicroBore RR 1.0 x 50 3.5 865630-902 865630-906 388Bulk Packing, 2 grams 5 820966-306 136Guard Cartridges, 2/pk 9.4 x 15 7 820675-124 820675-124 820675-124 820675-124 165Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-921 820950-918 820950-923 820950-924 155Guard Cartridges, 4/pk 2.1 x 12.5 5 821125-918 821125-918 821125-924 821125-924 155Guard Hardware Kit 9.4 x 15 840140-901 840140-901 840140-901 840140-901 162Guard Hardware Kit 4.6 x 12.5 820777-901 820777-901 820777-901 820777-901 106Guard Hardware Kit 2.1 x 12.5 820777-901 820777-901 820777-901 820777-901 106

Capillary Glass-lined ColumnsCapillary 0.5 x 250 5 5064-8266 505Capillary 0.5 x 150 5 5064-8264 455Capillary RR 0.5 x 150 3.5 5064-8268 505Capillary 0.3 x 250 5 5064-8265 505Capillary 0.3 x 150 5 5064-8263 455Capillary RR 0.3 x 150 3.5 5064-8267 505Guard Cartridges 0.5 x 35 5 5064-8294 301Guard Cartridges 0.3 x 35 5 5064-8295 301

Configurations not shown are available upon request. *RR: Rapid Resolution 3.5 µm columns.StableBond (80Å) columns are also available for small molecule separations. See page 10 for descriptions, ordering information and additional information on StableBond columns.

12,3

4 56

7 89

10

1 24 5

6,7

89

103

1 24 5

68

9 103

7

Time (min)0 5 10 15 20 25 30 35 40

1 24 5

68

910

37

27

FIGURE 3Four Different 300SB Bonded Phases Optimize Separation of Large Polypeptides

Columns: StableBond 300SB4.6 x 150 mm, 5 µm

Mobile Phase: Linear Gradient, 25 - 70% B in 40 min.A: 0.1% TFA in WaterB: 0.09% TFA in 80% Acetonitrile/20% Water

Flow Rate: 1.0 mL/min

Temperature: 60°C

Sample: 3 µg each protein1. RNase 6. CDR2. Insulin 7. Myoglobin3. Cytochrome C 8. Carbonic Anhydrase4. Lysozyme 9. S-100β5. Parvalbumin 10. S-100α

300SB-C18(Agilent Part No. 883995-902)

300SB-C8(Agilent Part No. 883995-906)

300SB-C3(Agilent Part No. 883995-909)

300SB-CN(Agilent Part No. 883995-905)

The 300SB-C18, C8, C3 and CN bonded phases all provide a differentseparation of this group of polypeptides. This adds an importantparameter for quickly optimizing protein separations. The 300SB-CNcolumn offers unique selectivity for more hydrophilic polypeptides.

ZORBAX Columns for BioAnalytical Chemistry — 300StableBond

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Column Volumes of Purge

0 5000 10000 15000 20000 25000 300000.0

0.2

0.4

0.6

0.8

1.0

Frac

tion

of T

olue

ne k

Rem

aini

ng

Stablebond SB-C18 (Diisobutyl-C18)

Extend-C18 (Bidentate-C18)

Rx-C18 (Dimethyl-C18)

Recommended ColumnZORBAX Extend-C18

pH Range for Optimal Column Life1 2 3 4 5 6 7 8 9 10 11 12 13

28

The combination of the unique bidentatestructure and double endcapping permitslong life at high pH.

The 300Extend-C18 column can be used at highand low pH — from pH 2 - 11.5. This chart showsthat the 300Extend-C18 has the needed stability at low pH for long-term reproducible separations.Therefore one wide-pore column can be used forselectivity optimization at low and high pH withboth TFA and ammonium hydroxide mobile phases.Additional information on Extend-C18 columns canbe found on pages 18 - 19.

SiO

SiO

Silica Support

C18C18

FIGURE 1New Bidentate C18-C18 Bonding forExtend-C18 Bonded Phase

FIGURE 2Extend-C18 and StableBond SB-C18 are Stable at Low pH

k Va

lues

0

2

4

6

8

Column Volumes of Mobile Phase

0 10000 20000 30000 40000 50000

1-Chloro-4-nitrobenzene

Trimipramine

FIGURE 3Long Life at High pH with Extend-C18

• Rugged, high and low pH separations of polypeptides and peptides from pH 2 - 11.5

• Different selectivity possible at high and low pH

• High efficiency and good recovery of hydrophobic peptides at high pH

• Ideal for LC/MS with ammonium hydroxide modified mobile phase

ZORBAX 300Extend-C18 HPLC Columns

ZORBAX 300Extend-C18 is a wide-pore HPLC column for the high efficiency separations of peptides from pH 2 - 11.5. The unique bidentate bonded phase provides excellent lifetime and reproducibility at high and low pH. At high pH,retention and selectivity of peptides and polypeptides can change dramatically asa result of changes in charge on molecules. Excellent recoveries of hydrophobicpolypeptides have been achieved at room temperature and high pH. LC/MS sensitivity of peptides and polypeptides can also be improved at high pH using asimple ammonium hydroxide containing mobile phase.

Columns: 4.6 x 150 mm, 5 µmColumn Aging: 50% Methanol : 50% Water + 1% TFA

Flow Rate: 1.5 mL/min, continuousTemperature: 90°C

Column Test: 60% Methanol : 40% WaterFlow Rate: 1.5 mL/minTemperature: RT

Test Solute: Toluene

To order or for technical assistance,call Agilent Technologies at 800-227-9770, or contact your localauthorized Agilent distributor.

Column: ZORBAX Extend-C18, 4.6 x 150 mm

Mobile Phase: 20% 20 mM NH4OH, pH 10.580% Methanol

Flow Rate: 1.5 mL/min

Aging at 24°C, tests at 40°C

Each 10,000 column volumes is approximatelyone working month.

ZORBAX Columns for BioAnalytical Chemistry

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m/z500 10000

20

40

60

80

100

Max: 367225 6

49.0

433

.0

659

.8

129

6.6

+1

+2

+3

min0 2.5 5 7.5 10 12.50

1.0E7

2.0E7

3.0E7

4.0E7

5.0E7

5.49

9

8.47

7

AII

AITIC

(200

-150

0 m

/z)

AIII

9.62

1

m/z500 10000

20

40

60

80

100Max: 10889 6

49.5

433

.0

227

.1

659

.7

844

.8

250

.9

340

.3

127

7.9

117

9.1

102

5.2

720

.3

132

6.3

398

.0

767

.3

458

.0

146

8.8

137

3.3

+3 +2

min0 2.5 5 7.5 10 12.50

1.0E6

2.0E6

3.0E6

4.0E6

5.0E6

12.

050

13.

261AII+AIII

AITIC

(200

-150

0 m

/z)

ZORBAX 300Extend-C18 Column Ordering InformationColumn Size Particle PartDescription (mm) Size (µm) Number PriceAnalytical 4.6 x 250 5 770995-902 $ 551Analytical 4.6 x 150 5 773995-902 490Rapid Resolution 4.6 x 150 3.5 763973-902 551Rapid Resolution 4.6 x 50 3.5 765973-902 423Narrow Bore 2.1 x 150 3.5 763750-902 551Narrow Bore 2.1 x 50 3.5 765750-902 423Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-932 184Guard Cartridges, 4/pk 2.1 x 12.5 5 821125-932 184Guard Hardware Kit 820777-901 106

Configurations not shown are available upon request. Extend-C18 (80Å) columns are also available. See page 18 for descriptions, ordering information and additional information on Extend-C18 columns.

29

0 5 10 15 20 25Time (min)

Aβ(1-38)Aβ(1-40)

Aβ(1-43)

Aβ(1-42)

Aβ(1-38)

Aβ(1-40)Aβ(1-43)

Aβ(1-42)

Aβ(1-38) Aβ(1-40)Aβ(1-42/43)

TFA Conditions, 80°CA - 0.1% TFA in waterB - 0.085% TFA in 80% ACNGradient: 29-41% B in 30 min.

TFA Conditions, 25°CA - 0.1% TFA in waterB - 0.085% TFA in 80% ACNGradient: 33 - 45% B in 30 min.

NH4OH Conditions, 25°CA - 20 mM NH4OH in waterB - 20 mM NH4OH in 80% ACNGradient: 26-38% B in 30 min.

Column: ZORBAX 300Extend-C18, 2.1 x 150 mm, 3.5 µm(Agilent Part No. 763750-902)

Flow Rate: 0.25 mL/min

At high pH and room temperature thehydrophobic amyloid peptides show betterrecovery and a different selectivity than atlow pH. At low pH elevated temperatureis necessary to achieve the same recoveryas at high pH.

Both small and large peptides demonstrate selectivity changes at high and low pH. At high pH, due to a changein charge, all three Angiotensins can be resolved. In addition, the spectral clarity of Angiotensin I is dramaticallyimproved at high pH with the ammonium hydroxide mobile phase. The Extend-C18 column can be used for theanalysis of small peptides at high pH as well (see pages 18 - 19 for additional details).

FIGURE 4LC/MS Analysis of Angiotensin on Extend-C18

A. Acidic Conditions: A: 0.1% TFA in water B: 0.085% TFA in 80% acetonitrile (ACN)

B. Basic Conditions: A: 10 mM NH4OH in water B: 10 mM NH4OH in 80% ACN

Column: ZORBAX Extend-C182.1 x 150 mm, 5 µm (Agilent Part No. 773700-902)

Flow Rate: 0.2 mL/min

Temperature: 35°C

Mobile Phase: As indicated

Gradient: 15-50% B in 15 min.

LC/MS: Pos. Ion ESI- Vf 70V, Vcap 4.5 kV, N2- 35 psi, 12 L/min., 325°C

Sample: Angiotensin I, II, III 2.5 µL sample (50 pmol each)

Angiotensin I: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-LeuAngiotensin II: Asp-Arg-Val-Tyr-Ile-His-Pro-PheAngiotensin III: Arg-Val-Tyr-Ile-His-Pro-Phe

Angiotensin I

Angiotensin I

Reference: Boyes, B.E., Separation and Analysis ofPeptides at High pH Using RP-HPLC/ESI-MS, 4th WCBP,San Francisco, CA Jan 2000.

FIGURE 3High pH Can be Used for SeparatingHydrophobic or Other Low Solubility Peptides

ZORBAX Columns for BioAnalytical Chemistry — 300Extend-C18

ZORBAX 300Extend-C18 Column SpecificationsPore Surface Temp. Carbon

Bonded Phase Size Area Limits pH Range Endcapped LoadZORBAX 300Extend-C18 300Å 45 m2/g 60°C* 2.0 - 11.5 Double 4%*Temperature limits are 60°C up to pH 8, 40°C for pH 8 - 11.5.

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30

Time (min)

0 10 15 20 255

1

2

3

4

5

6

78

9

1011 12

14

15

16 17

18

24

2322

21

20

19

Column: ZORBAX Eclipse AAA4.6 x 150 mm, 3.5 µm (Agilent Part No. 963400-902)

Mobile Phase: A: 40 mM Na2HPO4, pH 7.8B: ACN : MeOH : water,

45:45:10 v/vFlow Rate: 2.0 mL/minTemperature: 40°CDetector: Fluorescence

1. Asp2. Glu3. Asn4. Ser5. Gln6. His7. Gly8. Thr

9. Cit10. Arg11. Ala12. Tyr13. Cys14. Val15. Met16. Nva

17. Trp18. Phe19. Ile20. Leu21. Lys22. Hyp23. Sar24. Pro

FIGURE 1High Resolution of 24 Amino Acids Using ZORBAX Eclipse-AAA Protocol

• High resolution and rapid analysis of 24 amino acids

• Use tested for Amino Acid Analysis

• Uses well known OPA and FMOC derivatization chemistry

• Easily automated using a detailed on-line, derivatization protocol available foruse with Agilent 1100 Autosampler

ZORBAX Eclipse AAA (Amino Acid Analysis) HPLC Columns

The new ZORBAX Eclipse AAA high efficiency HPLC column rapidly separates amino acids following anupdated and improved protocol. Totalanalysis from injection to injection can be achieved in as little as 14 min (9 min. analysis time) on the short, 7.5 cm length column and 24 min (18 min. analysis time) on the 15 cmcolumn length. Exceptional sensitivity(5 – 50 pmol with DAD, FLD) and reliability are achieved using both OPAand FMOC derivatization chemistries inone fully automated procedure usingthe Agilent 1100 HPLC instrument.

This high resolution separation of 24 amino acids is done in 18 minutes. If the Rapid Resolution4.6 x 75 mm Eclipse AAA column is selected these amino acids are resolved in 9 minutes.

For a detailed description of the Eclipse AAAprotocol using the Agilent 1100 HPLC requestpublication number 5980-1193E or download itfrom the Agilent website at www.agilent.com.

ZORBAX Eclipse AAA Ordering InformationColumn Size Particle PartDescription (mm) Size (µm) Number PriceAnalytical routine sensitivity 4.6 x 150 5 993400-902 $ 414Analytical high sensitivity, high-resolution work using the FLD 4.6 x 150 3.5 963400-902 469Analytical routine sensitivity, high-throughput 4.6 x 75 3.5 966400-902 414Solvent Saver high sensitivity, high-resolution 3.0 x 150 3.5 961400-302 469Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-931 155Guard Hardware Kit 820777-901 106

ZORBAX Columns for BioAnalytical Chemistry

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Wild Type Variant wt/variant wt/wt variant/varianthomoduplexesheteroduplexes

Time (min)0 1 2 3 4 5 6 7

31

FIGURE 2Mutation Detection by Denaturing HPLC (DHPLC)

DHPLC analysis of the Y chromosome STS, DYS271 shows ideal separation

FIGURE 1High Resolution Separation on a ZORBAX Eclipse dsDNA Analysis Column

0 10 20 30 40 50 60 70 80 min

mAU302520151050

67 72 89 110 118 147 190 194

234 242271

281310

353404

489501

603872

10781353

Column: Eclipse dsDNA, 2.1 x 150 mm, 3.5 µm

Mobile Phase: Gradient: 45 – 75% B in 90 minutesA: 0.1 M TEAA*, 0.1 mM EDTA, pH 7.0B: A + 25% ACN

Flow Rate: 0.2 mL/min

Temperature: 50°C

*TEAA = triethylammonium acetate

Detection: UV 260 nm

Sample: 200 ng restriction digest standards

Instrument: Agilent 1100, 1 µL flow cell, binary pump

The Eclipse dsDNA Analysis column can be used fordenaturing HPLC — DHPLC — and several other specialtechniques. In this example a DHPLC analysis of the Y chromosome STS, DYS271 shows ideal separation ofthe homo- and heteroduplexes formed.

Oefner, P.J., Underhill, P.A. (1995)Comparative DNA Sequencing byDenaturing High-Performance LiquidChromatography (DHPLC), Am. J. Hum.Genet., 57 [Suppl.] A266 (Abstract)

• High sensitivity and resolution for dsDNA fragments

• Separate from 20 - 1500 base pairs

• 20-50X the capacity of non-porous columns

• Usable for DHPLC, PCR fragment analysis and other dsDNA sizing applications

ZORBAX Eclipse dsDNA Analysis HPLC Columns

The ZORBAX Eclipse dsDNA analysiscolumn provides high-resolution, automated, quantitative separations of dsDNA fragments by HPLC. Up to 1 bp resolution can be achieveddepending on fragment length and gradient analysis time. For high sensitivity, load up to 10 µg and detect less than 1 ng/band using the 2.1 mm i.d. columns. Or, inject up to 40 µg on the 4.6 mm i.d. columns for higher capacity quantitative separations. This column is useful forapplications such as PCR fragmentseparations, restriction digest separations, DHPLC, RFLP and RAPD.

ZORBAX Eclipse dsDNA Ordering InformationColumn Size Particle PartDescription (mm) Size (µm) Number PriceAnalytical 2.1 x 75 3.5 945750-902 $ 551Analytical 2.1 x 150 3.5 993850-902 622Analytical 3.0 x 75 3.5 945750-302 551Preparative 4.6 x 75 3.5 992750-902 551Guard Cartridge, 4/pk 2.1 x 12.5 3.5 821125-929 155Guard Cartridge, 4/pk 4.6 x 12.5 3.5 820950-929 155Guard Hardware Kit 820777-901 106

To order or for technical assistance,call Agilent Technologies at 800-227-9770, or contact your localauthorized Agilent distributor.

ZORBAX Columns for BioAnalytical Chemistry

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32

Time (min)0 1 2 3 4 5 6 7 8

1

2 3

4

5

Column: ZORBAX GF-250, 9.4 x 250 mm

Mobile Phase: 200 mM Sodium Phosphate, pH 7.0

Flow Rate: 2 mL/min

Temperature: Ambient

Detection: UV 254 nm

Sample: BioRad Gel Filtration Standards for Size Exclusion

1. Thyroglobulin 670,000 Da2. Bovine Gamma Globulin 158,000 Da3. Chicken Ovalbumin 44,000 Da4. Equine Myoglobin 17,000 Da5. Vitamin B-12 1,350 Da

ZORBAX GF-250 and GF-450 Gel Filtration Columns Ordering Information Column Size Particle Description (mm) Size (µm) Part No. PriceGF-250, 150Å pore size 21.2 x 250 4 884974-901 $ 2754GF-250, 150Å pore size 9.4 x 250 4 884973-901 878GF-250, 150Å pore size 4.6 x 250 4 884973-701 469GF-450, 300Å pore size 21.2 x 250 6 884974-910 2754GF-450, 300Å pore size 9.4 x 250 6 884973-902 878GF-250 Guard Cartridge*, 2/pk 9.4 x 15 4 820675-111 165GF-250 Guard Cartridge, 4/pk 4.6 x 12.5 4 820950-911 155GF-450 Guard Cartridge, 2/pk 9.4 x 15 4 820675-111 165GF-450 Guard Cartridge, 4/pk 4.6 x 12.5 4 820950-911 155GF-250, GF-450 Hardware Kit 9.4 x 15 840140-901 162GF-250, GF-450 Hardware Kit 4.6 x 12.5 820777-901 106

*Diol Phase

• High efficiency and reproducibility with short analysis times

• Hydrophilic diol bonded phase for good protein recovery

• Compatible with organic modifiers and denaturants

• Wide usable pH range (pH 3 - 8)

ZORBAX GF-250/450 Size Exclusion HPLC Columns

ZORBAX GF-250 and GF-450 size exclusion columns are ideal for the size separations of proteins and other biomolecules. The separation range is 4,000 - 900,000 for globular proteins when using GF-250 and GF-450 columns inseries. The GF-250/450 size exclusion columns have a hydrophilic diol bondedphase for high recovery of proteins (typically >90%) and a unique zirconia modification of the silica to extend the pH range from 3 - 8. The GF-250 and GF-450 columns are packed with precisely sized porous silica microspheres withnarrow pore size and particle size distributions. The result is a highly efficient,rugged and reproducible size exclusion column for separations of proteins withflow rates of up to 3 mL/min. These columns are compatible with organic modifiers and denaturants in the mobile phase to eliminate protein aggregationfor proper size determination. Some common applications include separations ofprotein monomers, dimers and aggregates, desalting, protein molecular weight estimation and separations of modified proteins.

FIGURE 1Separation of Protein Standards onthe ZORBAX GF-250 SEC Column

The protein standards separated here are a commonlyselected set of standards. The ZORBAX GF-250 columnshows excellent resolution for this sample. Additionalresolution of the thyroglobulin can be obtained by addingthe GF-450 column in series.

ZORBAX GF-250 and GF-450 Column SpecificationsBonded Phase Pore Size Particle Size MW Range Surface Area pH Range Flow Rate Max PressureZORBAX GF-250 150Å 4 µm 4,000 - 400,000 140 m2/g 3.0 - 8.0 <3.0 mL/min 5000 psiZORBAX GF-450 300Å 6 µm 10,000 - 900,000 50 m2/g 3.0 - 8.0 <3.0 mL/min 5000 psi

ZORBAX Columns for BioAnalytical Chemistry

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33

The ZORBAX Oligo column is designed for the high-resolution gradient separationand purification of oligonucleotides. Oligonucleotides having 3 - 48 bases havesuccessfully been purified using this column, while it is possible to separateoligonucleotides up to the 60 - 80 mer range. The Oligo column uses a mixedmode ion exchange and reversed phase bonded phase developed specifically for oligonucleotides in order to effectively isolate them with high purity. Simplegradient mobile phases are used for oligomer separations with the possible addition of denaturants, such as urea, to prevent self-hybridization.

24.0

3 27.2

830

.46

33.5

636

.59

39.4

742

.21

Time (min)0 5 10 15 20 25 30 35 40 45

Column: ZORBAX Oligo, 6.2 x 80 mm

Mobile Phase:Gradient: 35 - 80% B in 90 min.

A:20:80 ACN: 20 mM Sodium Phosphate pH 7.0B:20:80 ACN: 20 mM Sodium Phosphate

pH 7.0 + 0.8 M NaCl

Flow: 1.0 mL/min

Temperature: Ambient

Detector: UV 260 nm

Sample Volume: 50 µL, 5 units/mL

Time (min)0 5 10 15 20 25 30 35 40 45

Column: ZORBAX Oligo, 6.2 x 80 mm

Mobile Phase: Gradient: 35% B to 60% B in 90 min.A = 20% Acetonitrile: 80% 0.02 M Sodium PhosphateB = A + 1.0 M NaCl

Temperature: Ambient

Detector: UV 260 nm

Sample: 1.0 mL

Flow Rate: 1.0 mL/min

ZORBAX Bio Series Oligo Column SpecificationsBonded Phase Pore Size Surface Area pH Range Bonded Functionality ZORBAX Oligo 150Å 140 m2/g 2.5 – 8.5 Proprietary diol

ZORBAX Oligo Column Ordering InformationSize Particle Part(mm) Size (µm) Number Price

6.2 x 80 5 820940-901 $ 4699.4 x 250 5 884973-903 1214

21.2 x 250 5 884974-915 3256Configurations not shown are available upon request.

• Separate oligonucleotides up to 60 - 80 mer lengths

• High-resolution gradient analysis with mixed mode ion exchange and reversedphase column

• Isolate oligonucleotides with >95% purity

• Denaturatants can be added to the mobile phase

ZORBAX Oligo HPLC Columns

FIGURE 1Separation of Standard p(DT) 12 - 18 onZORBAX Oligo Column

FIGURE 2Separation of 24 and 25-mer Oligonucleotides

To order or for technical assistance, call Agilent Technologies at 800-227-9770, or contact your local authorized Agilent distributor.

ZORBAX Columns for BioAnalytical Chemistry

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To place an order visit our online catalog at www.agilent.com on theWorld Wide Web, or contact yourlocal Agilent sales office. To obtaintechnical support, or to obtain aquote in the U.S., dial 1-800-227-9770,or contact our authorized distributors.For all other areas contact Agilent oryour local authorized distributor.

Information, descriptions and specifications in this publication aresubject to change without notice.

An Online Resource for Applications and Products

Visit our web site at www.agilent.com to accessHPLC applications and to order the columns andconsumables you need for your day-to-day analytical instrument operations.

Many applications can be accessed on the web site by simply clicking on On Line Literature underthe Library heading. Search by keyword to findapplications of interest to you. Some of the applications available show chromatograms ofamino acids, chiral compounds, pharmaceuticals,pesticides and herbicides, as well as proteins andpeptides. Additional chromatograms can be foundin the LC Chromatogram Library. More applicationsare being added everyday!

Our online catalog allows you to check the status of any Agilent Chemical Analysis (CAG) order placed by clicking on Order Status. WithOrder History, you can view your history of AgilentCAG orders, making it fast and simple to reorderanytime! And, when you place an order online,your purchase total automatically reflects all applicable discounts.

®Copyright 2001 Agilent Technologies, Inc.Printed in the USA January 31, 20015988-1786ENUS


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