Agilent HPLC Column Selection Guide

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Featured Products .......................................................................1HPLC Column Selection Guidelines .............................................3HPLC Column Selection Flow Chart.............................................................................................4Quick Guide to ZORBAX Reversed-Phase Bonded Phases..........................................................5ZORBAX Reversed-Phase HPLC Column Selection Flow Chart ...................................................6Column and Mobile Phase Guidelines: Reversed Phase .............................................................8Quick Guide to USP Designations for HPLC Columns................................................................11Method Development from pH 1-12 .........................................................................................13Cartridge Selection Guide ..........................................................................................................18Columns for Analytical HPLC.....................................................19Poroshell 120 .............................................................................................................................20ZORBAX Eclipse Plus..................................................................................................................23ZORBAX Eclipse PAH .................................................................................................................30ZORBAX Eclipse XDB .................................................................................................................32ZORBAX 80Å StableBond ..........................................................................................................40ZORBAX Rx.................................................................................................................................49ZORBAX 80Å Extend-C18..........................................................................................................52ZORBAX Bonus-RP.....................................................................................................................56ZORBAX Original Reversed Phase Columns ..............................................................................61ZORBAX Normal Phase Columns...............................................................................................62ZORBAX HILIC Plus ....................................................................................................................66ZORBAX Method Development Kits ..........................................................................................68ZORBAX Method Validation Kits ................................................................................................70ZORBAX Ion Exchange Columns – SAX and SCX......................................................................71Columns for Special Applications..............................................73ZORBAX Rapid Resolution High Definition 1.8 µm....................................................................74ZORBAX Rapid Resolution High Throughput 1.8 µm.................................................................77ZORBAX Rapid Resolution 3.5 µm.............................................................................................85ZORBAX Solvent Saver...............................................................................................................90ZORBAX PrepHT.........................................................................................................................94Agilent Prep LC Columns..........................................................................................................100Ultron ES Chiral Columns .........................................................................................................103Columns for Bioanalytical Chromatography ...........................105ZORBAX Strategy for Reversed-PhaseMethod Development of Proteins and Peptides......................................................................106Poroshell 300 ...........................................................................................................................110ZORBAX 300Å StableBond......................................................................................................113ZORBAX 300Å Extend-C18......................................................................................................118ZORBAX Amino Acid Analysis (AAA) Columns and Supplies ..................................................122ZORBAX GF-250 and GF-450 Gel Filtration Columns .............................................................124Agilent Bio-Monolith HPLC Columns .......................................................................................128

New columns deliver on the promise ofgreater productivity and superior peak shapefor acids, bases, and neutrals.Whether you are performing conventional or ultra-fast chromatography, separatingbiomolecules, or analyzing complex basic compounds, you can trust Agilent for the industry’shighest-performing columns that deliver the fast, reproducible results you need. All engineeredwith Agilent’s unparalleled quality and reliability.• New Poroshell 120 columns – featuring high efficiency and high resolution with 50% lesspressure than sub-2 micron columns• ZORBAX Rapid Resolution High Definition (RRHD) columns – 1.8 µm columns featureimproved packing processes to achieve stability up to 1200 bar for use with theAgilent 1290 Infinity LC• New Eclipse Plus columns – C18 and C8 columns deliver superior peak shape, while newphenyl-hexyl bonded phase and C18 bonded phase for PAH separations expand selectivityoptions for more applications• An expanded portfolio of Rapid Resolution High Throughput (RRHT) columns with over140 1.8 µm columns choices – so you never have to choose between speed, resolutionand sensitivityAnd remember, when you choose Agilent ZORBAX LC columns, you get more than justa dependable product. You also get over 40 years of expertise – along with unmatchedtechnical support – from the world’s largest chromatography supplier. On the web,by phone or in person, Agilent helps you solve the problems that can slow you downand get in the way of your results.Order online at www.agilent.com/chem/store1

Featured ProductsThe New Agilent 1290 Infinity LC SystemIntroducing an infinitely betterliquid chromatography systemThe NEW Agilent 1290 Infinity LC has a wider operating power range than all other systemson the market – from 1200 bar at 2 mL/min to 800 bar at 5 mL/min. The Infinity LC deliversunequalled performance, flexibility, methods compatibility and investment protection.• 1290 Infinity Binary Pump combines active damping with lowest delay volume of 10 µL –facilitated by multi-layer technology in the new Agilent Jet Weaver mixer – for ultra fastgradients and superior UV-detection sensitivity• Optical design in 1290 Infinity Diode Array Detector – including Agilent Max-Light cartridgeflow cell – offers a new level of UV-sensitivity and baseline robustness, including fastspectral acquisition at data rates up to 160 Hz• Next generation flow-through design of the 1290 Infinity Autosampler achieves highestprecision for both large and small injection volumes without changing sample loops• 1290 Flexible Cube combines with Autosampler to facilitate fixed-loop injection mode forultra fast cycle times, or automatic backflushing of needle seat for a new benchmark inlowest carry over• Pull-out valve drive and user-exchangeable valve heads in the 1290 Infinity ThermostattedColumn Compartment boosts usability and paves the way for ultra high-throughput,multi-method and automated method development solutions• ZORBAX Rapid Resolution High Definition (1.8 µm) LC columns facilitate easy, fast andsecure method transfer from HPLC to UHPLC through identical column chemistriesFor more information, visit www.agilent.com/chem/12902 Order online at www.agilent.com/chem/store

HPLC Column Selection GuidelinesHPLC column selectionand method development strategiesTo make it easier to select the right column for your specific application, we have includedthe following reference material so you can maximize your most valuable resource – time.HPLC Column Selection Flow ChartHere, you will find step-by-step instructions on choosing an initial column for methoddevelopment, based on key separation considerations, such as analyte and mobile phase.We will then point you to the page that has the exact column you need.Simple Guidelines for Choosing a Reversed Phase HPLC ColumnWe will show you how to select the best column for small molecule and protein/peptideanalysis, based on factors such as bonded phase and column configuration.Quick Guide to USP Categories For HPLC ColumnsThis section clearly and concisely lists the U.S. Pharmacopeia categories for Agilent’s ZORBAXcolumn family, so you can easily select the columns that conform to USP or otherpharmacopoeial monographs.Reversed-Phase HPLC Method DevelopmentChromatographic resolution between two or more peaks depends upon column efficiency,selectivity, and retention – all factors that are affected by pH. While one type of column maydo many separations, this section describes a complete method development strategy basedon changing the pH of the mobile phase.Order online at www.agilent.com/chem/store3

HPLC Column Selection GuidelinesHPLC Column Selection Flow ChartTo use the column selection guide diagram below, simply follow the path for your analyte and mobile phase. At the far right, follow your finalcolumn selection to the pages indicated.Please see the 2009-2010 Essential Chromatography and Spectroscopy Catalog for a complete listing of LC columns and supplies.Hexane SolubleNormal-Phase Mode with Bare SilicaSee page62Organic SolventSolubleMeOH and MeOH:H 2 O or ACNandACN:H 2 O SolubleNormal-Phase Mode with Bonded PhaseReversed-Phase Mode with Bonded PhaseSee page62See page19THF SolubleGel-Permeation (Small Molecule)Molecular Weight2000Gel-Filtration ChromatographySee page124Water SolubleIon-Exchange Modewith Wide-Pore MaterialSee pages71 & 128Reversed-Phase Modewith Wide-Pore MaterialSee page113Adapted with permission from "Practical HPLC Methodology and Applications," Brian A. Bidlingmeyer, John Wiley & Sons, Inc., New York, p. 1094 Order online at www.agilent.com/chem/store

HPLC Column Selection GuidelinesQuick Guide to ZORBAX Reversed-Phase Bonded PhasesModern ZORBAX RP-HPLC Columns Recommended Uses and Applications Page No.Eclipse PlusAvailable in RRHD and RRHT, 1.8 µm Excellent first choice for method development Long life from pH 2-9 for reliable separations of basic, acidic and neutral compounds Superior peak shape with basic compounds High resolution and efficiency with 1.8, 3.5 and 5 µm columns Rigorous QA/QC testing for greater long-term reproducibility23Poroshell 120Eclipse XDBAvailable in RRHT, 1.8 µmStableBond (SB)Available in RRHD and RRHT, 1.8 µmZORBAX RxAvailable in RRHT, 1.8 µmBonus-RPAvailable in RRHT, 1.8 µmExtend-C18Available in RRHT, 1.8 µm Superficially porous particles for high efficiency at low pressure Sub 2-micron like efficiency with a 2.7 µm particle Endcapped and non-endcapped C18 phases for selectivity optimization Compatible with 400 bar and 600 bar LC's Four selectivity choices for flexible method development High performance over a wide pH range, pH 2-9 Good peak shape for acids, bases and neutrals Long lifetime with extra dense bonding and double endcapping Fast, ultra-fast, and high resolution separations using 1.8 and 3.5 µm columns Choices from capillary to prep Basic, acidic, neutral compounds Exceptional stability at low pH Use of high temperature (up to 90°C for C18, 80°C for C8, C3, Phenyl, CN, and Aq)and low pH as an added selectivity tool Widest selection of bonded phases for different selectivity (C18, C8, C3, CN,Phenyl, Aq) Uses mobile phases for LC/MS with formic acid, acetic acid, or TFA Uses mobile phases with TFA for peptide and protein separation Rapid separations using 1.8 and 3.5 µm columns General separation of basic, acidic and neutral compounds at low pH with differentselectivity than SB columns Rx-C8 is the same as SB-C8 Separating basic compounds in higher aqueous mobile phases General separation of basic, neutral, acidic compounds at mid-range pH or low pH;especially stable at low pH Separating peptides for different selectivity Rapid separations using 3.5 µm columns Separating basic compounds above their pKa in free base form; separation of basic,acidic, neutral compounds at high pH; up to pH 11.5 Uses ammonium hydroxide as mobile phase additive with LC/MS with small moleculesor peptides Separating at high, mid-range and low pH for selectivity changes Rapid separations using 3.5 µm columnsOriginal ZORBAX Columns Recommended Uses and Applications Page No.ZORBAX General separation of basic, acidic, neutral compounds at low pH with differentselectivity than SB columns; higher number of active silanols than SB "Mixed mode" separation at more neutral pH values61ZORBAX ODS Classic(non-endcapped) General separation of basic, acidic, neutral compounds at mid-range to low pHwith different selectivity than SB or XDB columns20324049565261Order online at www.agilent.com/chem/store5

HPLC Column Selection GuidelinesZORBAX Reversed-Phase HPLC Column Selection Flow ChartFor small and large moleculesMost chromatographers use reversed-phase HPLC as one of their key analysis techniques. Reversed-phase HPLC can be used to analyze ionicand nonionic analytes. Therefore this ZORBAX Column Selection Flow Chart will focus on reversed-phase columns. To more easily select areversed-phase column for method development of small and large molecules, follow the outline on these pages.This flow chart provides information on choosing an initial column for method development of small molecule and protein and peptide samples,and includes decisions on bonded phase and column configuration.Small MoleculesMW < 2000Large MoleculesMW > 200080-120ÅPacking Pore Size300ÅFirst choice of a packing pore size is based on the size of molecules to be analyzed. Typical small molecules can diffuse easily in and out of standard80-120Å pore packings, but peptides and proteins may not. For this reason, it is recommended to use 300Å pore-size packings (300SB)for isocratic or gradient separations of peptides and proteins.Eclipse Plus C18Starting Column Bonded PhaseStableBond 300SB-C18A C18 is recommended as the starting column bonded phase for most samples since it maximizes retention for moderately polar to non-polar compounds.Shorter chain phases should be considered if resolution cannot be optimized with a C18 phase or if you are analyzing larger proteins.4.6 mmColumn Diameter4.6 mmColumn diameter defines how much material (mass) you can inject on a column – the smaller the diameter, the less material that can beinjected while maintaining good peak shape. Smaller diameter columns provide increased sensitivity and thereforeare commonly used for LC/MS and sample limited applications.Column TypeInnerDiameter (mm)Sample Load(approx) Typical Flow RateIncreasedSensitivity ApplicationsAnalytical 4.6 0.1-1.5 mg 0.5-3 mL/min Standard separationsSolvent Saver 3.0 150-500 ug 0.3-1.5 mL/min + Save solvent, uses standard HPLC instrumentNarrowBore 2.1 50-120 ug 0.1-0.5 mL/min ++ High sensitivity, limited sample, LC/MS, save solventMicrobore 1.0 10-50 ug 10-100 µL/min ++++ High sensitivity, limited sample, LC/MSCapillary 0.5, 0.3 1-10 ug 1-15 µL/min +++++ Very high sensitivity, LC/MS, peptides and proteinsNano 0.1, 0.075 100-200 ng 200-500 nL/min ++++++ Very high sensitivity, LC/MS, peptides and proteinsSemi-Prep 9.4 1-10 mg 5-10 mL/min mg preparative separationsPreparative 21.2 20-250 mg 20-60 mL/min Hundreds of milligrams up to 1 gram6 Order online at www.agilent.com/chem/store

HPLC Column Selection GuidelinesSmall MoleculesMW < 2000Large MoleculesMW > 20005 and 3.5 µmStandard Porous Particle Size5 µmFor conventional analyses of small and large molecules, the standard particle size is 5 µm. However, smaller particle sizes are availableand provide higher efficiency and higher resolution. They are also available in short column lengths.A 3.5 µm particle may be the preferred starting particle size because it provides scalability up to 5 µm and down to 1.8 µm without requiring method revalidation.Fast Analysis Particle SizesRapid Resolution 3.5 µmRapid Resolution HT 1.8 µmRapid Resolution HD 1.8 µmRapid Resolution 3.5 µm particles provide 60% greater efficiency than 5 µmparticles while Rapid Resolution HT 600 bar and Rapid Resolution HD 1200 bar1.8 µm particles provide 200% greater efficiency. Shorter columns can be usedto significantly reduce analysis time of small molecules.Rapid Resolution 3.5 µmPoroshell 5 µmFor peptides, Rapid Resolution 3.5 µm particles can be used withshorter columns for faster, high-resolution gradient analyses.But for proteins, Poroshell columns, with a solid core andporous shell, provide the fastest analysis.150 mm Column Length150 mmA conventional column length is 150 mm or 250 mm for high resolution with5 µm particles. This is a good place to start a typical analysis because it will providehigh resolution in a 20-30 minute analysis. For fast analysis, shorter columns(75 mm and 50 mm) with smaller particle sizes (3.5 µm) should be used.For proteins and peptide digests, 150 mm is also a good starting column length.The typical gradient time on a column of this length will be 30-60 minutes fora high-resolution analysis. For fast analysis, shorter columns (50 mm)with smaller particles (3.5 µm) should be used.Starting Column ChoicesSmall MoleculesLarge MoleculesStandard AnalysisFast AnalysisStandard AnalysisFast AnalysisEclipse Plus C184.6 x 150 mm, 5 µm959993-9024.6 x 150 mm, 3.5 µm959963-902Rapid ResolutionEclipse Plus C184.6 x 75 mm, 3.5 µm959933-902Rapid Resolution HT,600 barEclipse Plus C184.6 x 50 mm, 1.8 µm959941-902Narrow Bore RRHD,1200 bar *Eclipse Plus C182.1 x 50 mm, 1.8 µm959757-902ZORBAX 300SB-C184.6 x 150 mm, 5 µm883995-902Rapid ResolutionZORBAX 300SB-C184.6 x 50 mm, 3.5 µm865973-902Poroshell 300SB-C182.1 x 75 mm, 5 µm660750-902* First choice for use on the 1290 Infinity LC or other UHPLC instruments with 1000 barpressure limitsOrder online at www.agilent.com/chem/store7

HPLC Column Selection GuidelinesColumn and Mobile Phase Guidelines:Reversed PhaseHPLC columns consist of two parts, the column chemistry and hardware. For the propercolumn chemistry, consult the catalog section for each type of bonded phase. For choosingcolumn hardware and particle sizes, consult the section on column sizes and rapidseparations, including Agilent ZORBAX Rapid Resolution and Rapid Resolution HTcolumns, as well as Solvent Saver and Capillary columns and PrepHT columns.For more information aboutcolumn selection, visitwww.zorbaxmethod.comPore Size SelectionChoose a column packing with small pore (60-120Å) if the solute molecular weight is lessthan about 5000. Otherwise, use column packing with the 300Å pore size.Particle Size SelectionThe standard particle size for HPLC columns is 5 µm with 3.5 µm now dominant for methoddevelopment. If high-speed analyses or higher resolution analyses are required, packing with1.8 µm and 3.5 µm particles can be used. Shorter columns with these particles can producefaster high-resolution separations, with the 1.8 µm particle size in Rapid Resolution HTcolumns, providing the highest efficiency. The 3.5 µm particle size operates at a routineoperating pressure and can be used on all LC's. Short (50 mm and shorter) 1.8 µm RRHTcolumns can be used on optimized standard LC's, while the longer columns may requirea higher pressure LC (one supporting pressures greater than 400 bar).Column ConfigurationThe column sizes most often recommended for analytical method development are4.6 x 150 mm or 4.6 x 75 mm. If more resolution is needed, use a longer column,4.6 x 250 mm or the same size column with a smaller particle size. During methoddevelopment, choose the column internal diameter (e.g., 2.1, 3.0 mm) to accommodateadditional application objectives (e.g., sensitivity, solvent usage) or compatibility with certaininstrument types (capillary, nano, or prep).8 Order online at www.agilent.com/chem/store

HPLC Column Selection GuidelinesSilica Type and Bonded PhaseSilica TypeAgilent ZORBAX reversed phase columns use three different types of porous silicamicrospheres, the original ZORBAX SIL, ZORBAX Rx-SIL and modified ZORBAX Rx-SIL.ZORBAX Rx-SIL and modified ZORBAX Rx-SIL are highly purified and less acidic than theoriginal ZORBAX SIL. Less acidic silica means less potential for interaction between theanalyte and silanol groups on the silica surface, especially if the solutes are basic, andcontributes to improved peak shape. For new method development, we strongly recommendusing reversed-phase products based on modified ZORBAX Rx-SIL (Eclipse Plus) and ZORBAXRx-SIL (Eclipse, StableBond etc.). However, many excellent methods have been developedon reversed phase columns based on ZORBAX SIL and we continue to manufacture thesehigh quality, reliable products.Bonded PhaseA good first choice for bonded phase is C18 or C8. If the sample solutes of interest are notadequately separated on these columns, CN and Phenyl columns may offer significantdifferences in selectivity from the straight-chain alkyl phases to effect the separation.In general, larger solutes, such as proteins, are best separated on short-chain reversed-phasecolumns (C3, CN) and peptides and small molecules are separated on longer-chain columns(C8, C18). However, there are many cases where this conventional wisdom does not apply.For example, peptides can also be effectively separated using short-chain columns, andhydrophobic peptides can show better recovery on longer-chain phases. Therefore, it is bestto initially select a phase in the middle of the hydrophobic spectrum (e.g., C8), then change toa more hydrophobic phase or more hydrophilic phase depending on initial results and solubilityproperties of your sample.Order online at www.agilent.com/chem/store9

HPLC Column Selection GuidelinespH and Mobile PhaseThe choice of mobile phase for a reversed-phase system starts with selecting the organicmodifier. Selectivity differences and sample retention will vary significantly among mobilephases containing acetonitrile, methanol, and tetrahydrofuran (THF). Sample solubility is likelyto differ in such solvents and dictate use of a specific solvent or solvents. UV detection atcertain wavelengths is not possible with certain modifiers (e.g., methanol at 200 nm).Both pH and ionic strength of the aqueous portion of mobile phases are important parametersin developing rugged methods that are not sensitive to small variations in conditions. Withionic compounds, retention of typical species shows significant changes with pH. It is veryimportant to control pH in such reversed-phase systems to stabilize retention and bandspacing. A pH set between 2 and 4 generally provides the most stable conditions for retentionvs. small changes in pH, and this pH is recommended for starting method development formost samples, including basic compounds and typical weak acids.HPLC ColumnStationaryPhaseColumnDimensionsType ofSurfacePore SizeParticleSizeLengthInnerDiameterChemical PropertiesChemical Lifetime/SelectivityRetention FactorPhysical PropertiesEfficiencySensitivitySpeed10 Order online at www.agilent.com/chem/store

HPLC Column Selection GuidelinesQuick Guide to USP Designationsfor HPLC ColumnsThe US Pharmacopeia (USP) is a standard source for many pharmaceutical methods.The USP specifies columns by packing materials rather than by manufacturer. The USP hasupdated its L1 definitions. Listed below you will see the most recent definitions and columnsthat apply and can be found in this column selection guide. Rapid Resolution High Throughput(RRHT) columns are now choices in the L1, L7, and L11 categories.For a complete listing of USP Designations, see Agilent's 2009-2010 EssentialChromatography and Spectroscopy Catalog, publication number 5989-9611EN.USPDesignations USP Packing Materials ColumnsParticleSize (µm)PoreSize (Å)L1Octadecyl silane chemically bonded to porous silica orceramic micro-particles, 1.5 to 10 µm in diameterZORBAX Eclipse Plus C18ZORBAX Eclipse Plus XDB-C18ZORBAX SB-C18ZORBAX Extend-C18ZORBAX Rx-C18ZORBAX ODSZORBAX ODS ClassicL3 Porous silica particles, 5 to 10 µm in diameter ZORBAX SilZORBAX Rx-SilL7L8L10L11Octyl silane chemically bonded to totally porous microsilicaparticles, 1.5 to 10 µm in diameterAn essentially monomolecular layer of aminopropyl-silanechemically bonded to totally porous silica gel support,10 µm in diameterNitrile groups chemically bonded to porous silica particles,3 to 10 µm in diameterPhenyl groups chemically bonded to porous silica particles,1.5 to 10 µm in diameterZORBAX Eclipse Plus C8ZORBAX Eclipse XDB-C8ZORBAX SB-C8ZORBAX Rx-C8ZORBAX C81.8, 3.5, 51.8, 3.5, 5, 71.8, 3.5, 5, 71.8, 3.5, 5, 73.5, 53.5, 5, 75551.8, 3.5, 51.8, 3.5, 5, 71.8, 3.5, 5, 71.8, 3.5, 5, 759580808080707095808080709580808070ZORBAX NH2 5 70ZORBAX Eclipse XDB-CNZORBAX SB-CNZORBAX CNZORBAX PhenylZORBAX SB-PhenylZORBAX Eclipse XDB PhenylZORBAX Eclipse Plus Phenyl-Hexyl3.5, 53.5, 53, 551.8, 3.5, 53.5, 51.8, 3.5, 580807070808095Order online at www.agilent.com/chem/store11

HPLC Column Selection GuidelinesUSPDesignations USP Packing Materials ColumnsParticleSize (µm)PoreSize (Å)L13L14L20L33Trimethylsilane chemically bonded to porous silica particles,3 to 10 µm in diameterSilica gel 10 µm in diameter with a chemically bonded, strongly basicquaternary ammonium anion-exchange coatingDihydroxypropane groups chemically bonded to porous silica particles,3 to 10 µm in diameterPacking having the capacity to separate proteins by molecular size over a rangeof 4,000 to 400,000 da. It is spherical, silica-based, and processed to providepH stabilityL35A zirconium-stabilizes spherical silica packing with a hydrophilic (diol-type)molecular monolayer bonded phaseL56 Propyl silane chemically bonded to totally porous silica particles, 3 to 10 µmin diameterL57A chiral-recognition protein, ovomucoid, chemically bonded to silica particles,about 5 µm in diameter, with a pore size of 120 angstroms.L60Spherical, porous silica gel, 10 µm or less in diameter, the surface of which hasbeen covalently modified with alkyl amide groups and endcapped.ZORBAX TMS 5 70ZORBAX SAX 4 70ZORBAX GF-250 5 150ZORBAX GF-250 4 150ZORBAX GF-250ZORBAX GF-45046150300ZORBAX SB-C3 3.5, 5 80Ultron ES-OVM 5 120Bonus-RP 1.8, 3.5, 5 8012 Order online at www.agilent.com/chem/store

HPLC Column Selection GuidelinesMethod Development from pH 1-12Chromatographic resolution between two ormore peaks depends upon three factors –column efficiency, selectivity, and retention.With ionizable analytes – bases and acids –all of these factors change dramatically withpH. For example, retention can be improvedby changing the separation pH, so thatanalytes are separated in their non-ionizedform. Changes in mobile phase pH alsoimprove column efficiency because theionization of the analyte and the residualsilanols can both be altered. This minimizessecondary interactions between analytesand the silica surface that cause poor peakshape. Achieving optimum resolution canalso require changing the mobile phase pH.The following method development strategyexplains how this is done with superiorcolumn lifetime.Low, mid, and high pH are the three generalregions for chromatographic separationsas defined in Figure 1. This figure highlightsthe benefits of performing separations ofionizable analytes in each pH region.Method development proceeds byinvestigating chromatographic separationsfirst at low pH and then at higher pH untiloptimum results are achieved. The idealcolumn is available for each pH region.Analyte chargeSilica surface chargeRetention TimeIon exchange secondary interactionRecommended bonded phaseneutralNOEclipse Plus orStableBond+ /neutral neutralneutral/-YESEclipse Plus or Bonus-RPFigure 1: Three pH Regions for HPLC Separations of Basic CompoundsThis figure represents the retention behavior of one basic analyte with respect to pKa and pH. Analyte pKa is 6.5NOExtend-C18Low pH < 3 – Region A Mid pH 7 – Region B High pH > 9 – Region C Start method development atlow pH, where silanols on aRP-HPLC column are protonated.This minimizes peak tailing byeliminating silanol/baseinteractions. At low pH, basic compoundsare positively charged and theirretention may be reduced. Acidic compounds may beprotonated and have increasedretention. Retention times are usuallystable with small changes in pH,producing a robust method. Volatile mobile phase additives,such as formic acid ortrifluoroacetic acid (TFA),are often used at low pHwith LC/MS. Develop methods at pHs at least1 pH unit above or below thepKa to minimize changes inretention with small changesin pH. Some silica surface SiOHgroups become SiO¯ abovepH 4 to 5; tailing interactionsmay be possible. Minimize interactions byselecting a well designed andendcapped column, usingadditives such as TEA(triethylamine) (less desirable)or using "polar-linked"bonded phases. Silica breakdown is preventedby innovative bonding chemistry,heavy endcapping, and useof Rx-SIL. In this region, basic compoundsmay be in their free base form. Increased retention andresolution of basic compoundsare likely. Retention changes little in thisregion, thus robust methods canbe developed. Silica breakdown is preventedby innovative bidentate columnchemistry, heavy endcapping,use of Rx-SIL, and optimummobile phase. Ammonium hydroxide is anexcellent volatile mobile phasemodifier at high pH.Order online at www.agilent.com/chem/store13

HPLC Column Selection GuidelinesStart method development at low pH (pH 2-3)With so many column choices available, how do you know where to start your methoddevelopment? The recommended starting point for method development is using a bufferedlow pH mobile phase – around pH 2-3. Using a low pH mobile phase most often resultsin the best peak shape for basic compounds on silica-based columns. At low pH, the silanolson the silica are fully protonated so positively charged basic compounds do not interactstrongly. The result is good peak shape. Many acidic compounds are non-charged,maximizing their retention at low pH. These observations are key advantages to methoddevelopment at low pH.For standard analytical work, start method development with acetonitrile as the mobile phaseorganic modifier and 20-50 mM phosphate buffer (pH 2-3) as the aqueous component fornon-LC/MS applications. These conditions provide good pH control, necessary for the mostreproducible analyses of ionizable compounds. For LC/MS applications formic acid or TFAare good mobile phase additives for low pH.Choose Agilent ZORBAX Eclipse Plus first for best peak shapeSelect ZORBAX Eclipse Plus C18 or C8 columns first for method development at low pH.Eclipse Plus columns are the newest addition to the Eclipse family and use improved silicaand bonding technologies to provide good peak shape for basic compounds. Eclipse Pluscolumns can be used from pH 2-9 providing method development flexibility. They are stabledown to pH 2 making them an ideal choice for initial method development.14 Order online at www.agilent.com/chem/store

HPLC Column Selection GuidelinesOptimize solvents and bonded phases at low pHThe initial method development steps may lead very quickly to a satisfactory separation. But ifmore optimization is needed, acetonitrile can be replaced by methanol or tetrahydrofuran andthe separation re-optimized. This step may lead to a satisfactory solution, but if still moreselectivity optimization is needed, the column bonded phase can be changed.At low pH there are many bonded phase choices available for optimization. These include theEclipse Plus phases as well as the Eclipse XDB family with C18, C8, Phenyl and CN. Alternatechoices include six different StableBond bonded phases: SB-C18, SB-C8, SB-Phenyl, SB-CN,SB-C3, and SB-Aq.It may be necessary at low pH to improve the retention of acidic compounds. For thesesituations, lower the pH even further, down to pH 1-2, and use StableBond columns. Thesecolumns provide the greatest stability at very low pH and provide many selectivity optionsfor achieving the highest resolution separations.Choose Agilent ZORBAX Eclipse Plusfor method development at mid pH (4-9)There are some samples that may not be resolved at low pH or may have better solubilityand stability at mid pH. While still using the Eclipse Plus C18 column, the mid pH range canbe used for method development. The Eclipse Plus column is stable up to pH 9 so it is equallyreliable at mid pH. These double endcapped columns have two key advantages – good peakshape at low and mid pH, as well as sufficient bonded phase density to protect the columnfrom silica degradation from pH 6-9.At mid pH, basic compounds (e.g., amines) may still have a positive charge and the silanolson the silica surface may have a negative charge. Therefore covering as many silanols aspossible leads to the best peak shape at mid pH. This makes the Eclipse Plus C18 the beststarting choice for a column at mid pH. Phosphate buffer is usually the first choice for mobilephase modifier at pH 7 because its buffer range is pH 6.1-8.1. A second choice for mid pHis acetate buffer since it buffers from pH 3.8-5.8 and its volatility makes it a good choice forLC/MS compatibility.Order online at www.agilent.com/chem/store15

HPLC Column Selection GuidelinesSelect from Agilent ZORBAX Eclipse Plus Phenyl-Hexyl,Eclipse XDB-Phenyl, CN and Bonus-RPThe method development process at mid pH mimics the process at low pH with optimizationof the organic modifier and selecting an alternate bonded phase if resolution is not achievedafter that step. The alternate bonded phases at mid pH are the Eclipse Plus Phenyl-Hexyl,Eclipse XDB-Phenyl, Eclipse XDB-CN and Bonus-RP. They provide very different selectivitiesfor many samples and the method development process is followed again. The Bonus-RPcolumn has a polar embedded amide group that provides different selectivity for manysamples, provides good peak shape for basic compounds and allows the column to be usedwith up to 100% aqueous mobile phases.Choose Agilent ZORBAX Extend-C18 columnsfor method development at high pH (pH 9-12)At low or mid pH, some separations of basic compounds may still not have enough retentionor the desired selectivity. For these samples, high pH separations may be appropriate. Untilrecently, high pH separations on silica-based columns were avoided because of short columnlifetimes, due to dissolution of the underlying silica gel. Newer column technologies, i.e. theZORBAX Extend-C18, can protect the silica from dissolution, so that a reasonable columnlifetime can be achieved and the selectivity advantages of high pH can be explored.The mobile phase buffer choices at high pH with the Extend-C18 column are organic bufferslike triethylamine and ammonium hydroxide. These buffers are best used with methanol asthe organic modifier to extend the column lifetime at high pH.16 Order online at www.agilent.com/chem/store

HPLC Column Selection GuidelinesCartridge Column SystemsAgilent offers a variety of popular HPLC packing materials in economical, easy-to-use cartridge configurations.Look for these icons on subsequent pages to help you select the proper guard cartridges and columns.Cartridge Selection GuideIcon Type of Cartridge Features BenefitsAgilent HPLC CartridgeZORBAX Guard Cartridge:Stand alone systemZORBAX Rapid Resolution and RapidResolution HT Cartridge Columns:3.5 µm and 1.8 µm packings,stand alone systemZORBAX Semi-Preparative Guard HPLCHardware Kit:Stand alone systemZORBAX and Agilent Prep PreparativeCartridge Column and Guard HPLC System:Stand alone and integral hardware optionsCan reverse collets in the end fitting to addguard cartridgesCartridges have a unique filter and sieve ateach endHigh efficiency, stand-alone, low deadvolume cartridgePolymeric cartridge designed for leak-tightseals against metal surfacesReusable fittingsFor high throughput LC/MS, LC/MS/MS andcombinatorial separationsPacked with Eclipse XDB for pH use from 2-9Packed with StableBond for low pH useSold individually or as three-packsEasy low-dead-volume assemblyTubing (polyphenylene sulfone) designedfor leak-tight seals against metal surfacesReusable fittingsEasy low-dead-volume assemblyReusable fittingsHardware options for integral andexternal guardsInexpensiveExtends column lifetimePermits rapid column changesCan use 2, 3, 4 and 4.6 mm cartridgesHelps prevent blockageSeals up to 400 barNo gaskets requiredMore solvent-resistant than PEEKAdapt for connections to 1/16 in. LC fittingsFor all analyte typesLow bleedSeals up to 2000 psi (135 bar, 13.5 MPa)No gaskets requiredAdapt for connections to 1/16 in. LC fittingsExtends column lifetimePermits rapid column changesCan use with 21.2 and 30 mm ID columns18 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCAchieve excellent peak shape and resolution –and eliminate "false starts."Good news for analysts who do not have time to "make columns work" for a particularapplication: ZORBAX columns let you choose the right column based on your sampleand mobile phase – eliminating any guesswork.Additionally, Agilent’s ZORBAX silica is manufactured by Agilent – not purchased fromoutside suppliers. And that means we control every step of the manufacturing process,ensuring lot-to-lot consistency, superior performance, and long-term, reliable results.In this section, you will find a diverse range of ZORBAX columns designed for optimumresolution over a wide pH range, including:• Poroshell 120 columns – feature high efficiency and high resolution with 50% less pressurethan sub-2 micron columns.• ZORBAX Eclipse Plus HPLC columns – designed to reliably produce superior peak shapefor basic compounds.• ZORBAX Eclipse PAH HPLC columns – ideal for applications requiring "shape selectivity"or the separation of geometric isomers.• ZORBAX Eclipse XDB HPLC columns – a trusted choice for analytical and regulatory methods.• ZORBAX StableBond HPLC columns – the industry leader for low-pH applications.• ZORBAX Rx HPLC columns – provide excellent stability up to pH 9.• ZORBAX Extend-C18 HPLC columns – feature bidentate bonding that allows you to develophigh-resolution separations at high pH levels.• ZORBAX Bonus-RP HPLC columns – an alkyl-amide column that offers excellent peak shapefor bases, along with an alternative selectivity.• Original ZORBAX HPLC columns – are made with Type A silica and are used in manyestablished separation methods.• ZORBAX HILIC Plus columns – superior peak shape and retention of polar basic compoundswith silica in HILIC mode• ZORBAX Method Development kits – contain three columns for the price of two! Each hasa different bonded phase for optimal selectivity.• ZORBAX Method Validation kits – choose as many columns as you need (or as few) to makemethod validation easier and less expensive.• ZORBAX Ion Exchange columns – available as Strong Anion Exchange (SAX) and StrongCation Exchange (SCX)Order online at www.agilent.com/chem/store19

Columns for Analytical HPLCPoroshell 120• High efficiency and high resolution• 50% less pressure than sub 2-micron columns• Compatible with 400 bar and 600 bar LCs• Two bonded phases with excellent selectivity and peak shapeAgilent Poroshell 120 columns are a 2.7 µm particle with a 1.7 µm solid core and 0.5 µmporous outer layer. This small particle size provides high efficiency, similar to sub 2-microncolumns, but with 40-50% less pressure. These high efficiency, high resolution columnscan be used on any type of LC. The porous outer layer and solid core limit diffusion distanceand improve separation speed while the narrow particle size distribution improves efficiencyand resolution. The solid core limits diffusion distance and improves separation speed.The columns can support high pressure and multiple columns can be used for the highestresolution and efficiency possible. The same principles are used in the Poroshell 300 columns,ideal for the fast, high resolution separations of biomolecules.Column SpecificationsBonded PhasePore SizeTemp.Limits pH Range EndcappedCarbonLoadSB-C18 120Å 90°C 1.0-8.0 No 8%EC-C18 120Å 60°C 2.0-8.0 Single 10%EC-C8 120Å 60°C 2.0-8.0 Single 5%Specifications represent typical values only.20 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCPoroshell 120DescriptionSize (mm)ParticleSize (µm) SB-C18 EC-C18 EC-C8*Analytical 4.6 x 150 2.7 683975-902 693975-902 693975-906Analytical 4.6 x 100 2.7 685975-902 695975-902 695975-906Analytical 4.6 x 75 2.7 687975-902 697975-902 697975-906Analytical 4.6 x 50 2.7 689975-902 699975-902 699975-906Analytical 4.6 x 30 2.7 681975-902 691975-902 691975-906Solvent Saver 3.0 x 150 2.7 683975-302 693975-302 693975-306Solvent Saver 3.0 x 100 2.7 685975-302 695975-302 695975-306Solvent Saver 3.0 x 75 2.7 687975-302 697975-302 697975-306Solvent Saver 3.0 x 50 2.7 689975-302 699975-302 699975-306Solvent Saver 3.0 x 30 2.7 681975-302 691975-302 691975-306Narrow Bore 2.1 x 150 2.7 683775-902 693775-902 693775-906Narrow Bore 2.1 x 100 2.7 685775-902 695775-902 695775-906Narrow Bore 2.1 x 75 2.7 687775-902 697775-902 697775-906Narrow Bore 2.1 x 50 2.7 689775-902 699775-902 699775-906Narrow Bore 2.1 x 30 2.7 681775-902 691775-902 691775-906*Available December 2009Order online at www.agilent.com/chem/store21

Columns for Analytical HPLCEnvironmental Phenols on Poroshell 120Column A: Poroshell 120 EC-C18695975-9024.6 x 100 mm, 2.7 µmColumn B: Eclipse Plus C18959964-9024.6 x 100 mm, 1.8 µmGradient:A: Water 0.1% Formic AcidB: Acetonitrile 0.1% formic Acid2 mL/minInitial: 8% B10 min: 30% BDetector:275 nm 2 mm flow cellInjection: 10 µLAgilent 1200 SL 40°CNo pulse damperNo mixer 3 µL heaterPoroshell 120 provides sub 2 micron-like efficiency at lower pressure.A123P = 332 Bar1. Hydroquinone 6. o-cresol2. Resorcinol 7. 2-Nitrophenol3. Catechol8. 2,3 Dimethyl phenol4. 4-Nitrophenol 9. 2,5 Dimethyl phenol5. p-cresol10. 1-Naphthol495768100 2 4 6 8 10Time (min)B12P = 510 Bar3495768100 2 4 6 8 10Time (min)EP_Poro12022 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX Eclipse Plus• Excellent peak shape for basic compounds• High level of performance – peak shape, efficiency, resolution, and lifetime –with all sample types: acids, bases and neutrals• Superior reproducibility with more rigorous QA/QC testing• Improved, patented silica manufacturing with start-to-finish product control• Available in 1.8, 3.5 and 5 µm particle sizes for all analytical, high resolution,and fast LC analysesNew Agilent ZORBAX Eclipse Plus columns provide the ultimate in performance forsilica-based columns. Peak shape is excellent for the most challenging basic compounds,improving efficiency and resolution with these sample types. These results are achieved byimprovements in the silica manufacturing and bonding technology, which is completelycontrolled by Agilent.Because of their high level of performance, Eclipse Plus columns are the ideal first choicefor method development of all samples. If you need to achieve fast method developmentand superior productivity, then choose a column with high-resolution 1.8 µm particles.For standard methods, conventional 5 µm and Rapid Resolution 3.5 µm columns are yourbest choice. With all particle sizes, easy method transfer is possible.With more rigorous QA and QC testing, column lot-to-lot reproducibility is also improved,resulting in long-term reliable results for all analyses.Column SpecificationsBonded PhasePoreSizeSurfaceAreaTemp.Limits pH Range* EndcappedCarbonLoadZORBAX Eclipse Plus C18 95Å 160 m 2 /g 60°C 2.0-9.0 Double 9%ZORBAX Eclipse Plus C8 95Å 160 m 2 /g 60°C 2.0-9.0 Double 7%ZORBAX Eclipse PAH 95Å 160 m 2 /g 60°C 2.0-8.0 No 14%ZORBAX Eclipse Plus 95Å 160 m 2 /g 60°C 2.0-8.0 Double 9%Phenyl-HexylSpecifications represent typical values only.*Column lifetime will be reduced significantly at pH > 7 and temperature > 40°C. At pH 6-9, highest column stability for allsilica based columns is obtained by operating at temperatures < 40°C and using lower buffer concentrations in range of0.01 – 0.02M, especially with phosphate and carbonate buffers.Order online at www.agilent.com/chem/store23

Columns for Analytical HPLCZORBAX Eclipse Plus: Best Peak Shapein the Industry Without TailingColumn:Mobile Phase:Flow Rate:Temperature:Detector:Publication:Sample:Eclipse Plus C18959996-9024.6 x 100 mm, 5 µmA: 60% WaterB: 40% Acetonitrile1.0 mL/minAmbientUV 254 nm5989-4934ENPyridine, PhenolEclipse Plus C18Ace 5 C18SunFire C18XBridge C18Discovery HS C18121. Pyridine2. PhenolLuna C18 (2)Gemini C18XTerra MS C180 0.5 1.0 1.5 2.0 2.5 3.0 3.5Time (min)LCEC001Peak Shape and Efficiency are Betterwith ZORBAX Eclipse PlusColumn A: XBridge C18, 4.6 x 150 mm, 5 µmColumn B: Eclipse Plus C18959993-9024.6 x 150 mm, 5 µmMobile Phase: A: 0.1% formic acidB: 0.1% formic acid in ACNFlow Rate: 1.0 mL/minGradient: 0.0 min 10% B15 min 30% BTemperature: 40°CDetector: UV 254 nmPublication: 5989-4934ENSample:SulfonamidesA0 2 4 6 8 10 12Time (min)B11N = 14000Tf = 1.252N = 23000Tf = 1.01341. Sulfanilamide2. Sulfadiazine3. Sulfathiazole4. Sulfamerazine5. Sulfmethazine6. Sulfamethoxazole5N = 41000Tf = 1.136N = 60000Tf = 1.00234560 2 4 6 8 10 12Time (min)LCEC00324 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCEliminate Tailing and Maximize Resolution with Eclipse PlusColumnsColumn A: Eclipse Plus C18, 4.6 x 50 mm1Eclipse Plus C18, 4.6 x 50 mmColumn B: Competitive C18, 4.6 x 50 mmMobile Phase: 65% ACN: 35% 25 mM phosphate buffer (pH 7.4) Tf= 1.20Superior peak shape and better selectivity with Eclipse Plusmeans more resolution, easier quantitation and better resultsin your separations.Competitive C18, 4.6 x 50 mm11. Nortriptyline2. DipropylthalateTf= 4.86220 0.5 1 1.5 2minEclipse Plus C18 vs. C8 and RRHT vs. RRColumn A: Eclipse Plus C18959941-9024.6 x 50 mm, 1.8 µmColumn B: Eclipse Plus C8959943-9064.6 x 50 mm, 3.5 µmMobile Phase: Water: Acetonitrile (30:70)Flow Rate: 2.0 mL/minTemperature: 30°CDetector: UV 230nmSample:Lip balm extract in ACN(melted at 100°C ACN ,cooled and 0.45 µm filtered)AN: 8800Pw : 0.014 min.TF: 1.201. Oxybenzone2. Internal Std.3. OctylmethoxycinnamateN: 11600Pw : 0.047 min.TF: 1.060 1 2 3 4247 barN: 11800Pw : 0.093 min.TF: 1.02minLess retention can save significant time – the C8 is a goodchoice here. The RRHT column is delivering the efficiencyand resolution expected, but the C8 bonded phase may bethe best choice.BN: 5000Pw : 0.019 min.TF: 1.09N: 6600Pw : 0.037 min.TF: 0.99 N: 6300Pw : 0.061 min.TF: 0.9822 min.88 bar0 1 2 3 4minOrder online at www.agilent.com/chem/store25

Columns for Analytical HPLCRapid Analysis of an Analgesic Tablet,Selectivity Differences at pH 2 and pH 7Column:Gradient:Sample:Eclipse Plus C8959946-9064.6 x 50 mm, 5 µm10-60% B/3 minpH 2.7: A: 0.1% formic acid B: 0.1% fa in ACNpH 7.0: A: 20 mM Na phosphate B: ACNgeneric Excedrin tablet1. Acetaminophen2. Caffeine3. Acetylsalicylic acid4. UnknownBoth Eclipse Plus C18 and C8 can be used over a wide pH range to optimizeselectivity or analysis time.1234pH 2.70 0.5 1 1.5 2 2.5 3 3.5 min1pH 7.03422 min.0 0.5 1 1.5 2 2.5 3 3.5 minEclipse Plus C8 is Less Retentive than Eclipse Plus C18Column A:Eclipse Plus C8959996-9064.6 x 100 mm, 5 µmColumn B: Eclipse Plus C18959996-9024.6 x 100 mm, 5 µmMobile Phase: 80% Methanol 8 mM (total) K 2 HPO 4 pH 7Flow Rate:Detector:Sample:1.0 mL/minUV 215 nmAmitriptyline 0.05 µg/µL (0.5 µL injection)A3.789Tf=1.04A C8 column is typically selected because it will retain less than a C18 column,reducing analysis time.The Eclipse Plus C8 column shows the same behavior with excellent peakshape on difficult basic compounds.0 1 2 3 4 5 6 7 8 9 minB 5.777Tf=1.000 1 2 3 4 5 6 7 8 9 min26 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCFast and Ultra-Fast Analysisof Basic Compounds on Eclipse PlusColumn A: Eclipse Plus C18959941-9024.6 x 50 mm, 1.8 µmColumn B: Eclipse Plus C18959993-9024.6 x 150 mm, 5 µm959993-902Mobile Phase: A: 50% 8 mM K 2 HPO 4 , pH 7B: 50% ACNFlow Rate: 1.0 mL/minTemperature: AmbientDetector: UV 254 nmPublication: 5989-4934ENSample: Berberine, 0.4 mg/mL, 2 µLATf = 1.000 0.5BOO N+ Cl-1. BerberineOCH 3OCH 31 1.5 2 2.5minTf = 1.060 0.5 1 1.5 2 2.5minEP103Order online at www.agilent.com/chem/store27

Columns for Analytical HPLCZORBAX Eclipse PlusHardware Description Size (mm)Particle Size(µm)EclipsePlus C18USP L1EclipsePlus C8USP L7Eclipse PlusPhenyl-HexylUSP L11EclipsePAHUSP L1Analytical 4.6 x 250 5 959990-902 959990-906 959990-912 959990-918Analytical 4.6 x 150 5 959993-902 959993-906 959993-912 959993-918Analytical 4.6 x 100 5 959996-902 959996-906 959996-912 959996-918Analytical 4.6 x 50 5 959946-902 959946-906Rapid Resolution 4.6 x 150 3.5 959963-902 959963-906 959963-912 959963-918Rapid Resolution 4.6 x 100 3.5 959961-902 959961-906 959961-912 959961-918Rapid Resolution 4.6 x 75 3.5 959933-902 959933-906 959933-912Rapid Resolution 4.6 x 50 3.5 959943-902 959943-906 959943-912 959943-918Rapid Resolution 4.6 x 30 3.5 959936-902 959936-906 959936-912Rapid Resolution HT, 600 bar 4.6 x 100 1.8 959964-902 959964-906 959964-912 959964-918Rapid Resolution HT, 600 bar 4.6 x 75 1.8 959951-902Rapid Resolution HT, 600 bar 4.6 x 50 1.8 959941-902 959941-906 959941-912 959941-918Rapid Resolution HT, 600 bar 4.6 x 30 1.8 959931-902 959931-906 959931-912 959931-918Solvent Saver 3.0 x 250 5 959990-318Solvent Saver 3.0 x 150 5 959993-302 959993-306Solvent Saver Plus 3.0 x 150 3.5 959963-302 959963-306 959963-312Solvent Saver Plus 3.0 x 100 3.5 959961-302 959961-306 959961-312Solvent Saver HD, 1200 bar 3.0 x 100 1.8 959758-302Solvent Saver HD, 1200 bar 3.0 x 50 1.8 959757-302Solvent Saver HT, 600 bar 3.0 x 100 1.8 959964-302 959964-306 959964-312Solvent Saver HT, 600 bar 3.0 x 50 1.8 959941-302 959941-306 959941-31228 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX Eclipse PlusHardware Description Size (mm)Particle Size(µm)EclipsePlus C18USP L1EclipsePlus C8USP L7Eclipse PlusPhenyl-HexylUSP L11EclipsePAHUSP L1Narrow Bore 2.1 x 250 5 959790-918Narrow Bore 2.1 x 150 5 959701-902 959701-906 959701-912 959701-918Narrow Bore 2.1 x 50 5 959746-902 959746-906Narrow Bore RR 2.1 x 150 3.5 959763-902 959763-906 959763-912Narrow Bore RR 2.1 x 100 3.5 959793-902 959793-906 959793-912 959793-918Narrow Bore RR 2.1 x 50 3.5 959743-902 959743-906 959743-912Narrow Bore RR 2.1 x 30 3.5 959733-902 959733-906 959733-912Narrow Bore RRHD, 1200 bar 2.1 x 150 1.8 959759-902Narrow Bore RRHD, 1200 bar 2.1 x 100 1.8 959758-902Narrow Bore RRHD, 1200 bar 2.1 x 50 1.8 959757-902Narrow Bore RRHT, 600 bar 2.1 x 100 1.8 959764-902 959764-906 959764-912 959764-918Narrow Bore RRHT, 600 bar 2.1 x 50 1.8 959741-902 959741-906 959741-912 959741-918Narrow Bore RRHT, 600 bar 2.1 x 30 1.8 959731-902 959731-906 959731-912Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-936 820950-937 820950-938 820950-939Guard Cartridges, 4/pk 2.1 x 12.5 5 821125-936 821125-937 821125-938 821125-939Guard Hardware Kit 820888-901 820888-901 820888-901 820888-901Order online at www.agilent.com/chem/store29

Columns for Analytical HPLCZORBAX Eclipse PAH• High resolution separation of 16 PAHs in EPA 610.• Extensive range of particle sizes (1.8, 3.5 and 5 µm) and sizes for fast and highresolution separations.• Each batch of material is specifically tested with PAHs for maximum reproducibility underexpected operating conditions.• Excellent performance using the high quality, improved silica of the Eclipse Plus columns.• Good for applications requiring "shape selectivity" or the separation of geometric isomers.Agilent ZORBAX Eclipse PAH columns are recommended for the separation of polycyclicaromatic hydrocarbons. PAHs are considered priority pollutants and the analysis of thesepotentially carcinogenic compounds in water, soil and food is of major importance. The EclipsePAH columns separate all of the 16 PAHS in EPA method 610 quickly with high resolution.Column SpecificationsBonded PhasePoreSizeSurfaceAreaTemp.Limits pH Range EndcappedCarbonLoadZORBAX Eclipse PAH 95Å 160 m 2 /g 60°C 2.0-8.0 No 14%Specifications represent typical values only.30 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCHigh Resolution and Fast Analysis on RRHT Eclipse PAH ColumnColumn:Mobile Phase:Eclipse PAH959941-9184.6 x 50 mm, 1.8 µmA = Water; B = AcetonitrilemAU2000Gradient:Flow Rate:Temperature:Time (Min)0.003.55.25.56.52.0 mL/min25º C% B4010010040401750150012501000750Rs = 2.2 vs. 2.0 for4.6 x 50 mm, 3.5 µmRs = 2.2Detector:Stop Time = 7.0DAD 220,4 nm No Ref.DAD Stop Time = 6.0 min50025001 2 3 4 5Time (min)LCEPlusPAHZORBAX Eclipse PAHHardware Description Size (mm) Particle Size (µm)EclipsePAHUSP L1Analytical 4.6 x 250 5 959990-918Analytical 4.6 x 150 5 959993-918Analytical 4.6 x 100 5 959996-918Rapid Resolution 4.6 x 150 3.5 959963-918Rapid Resolution 4.6 x 100 3.5 959961-918Rapid Resolution 4.6 x 50 3.5 959943-918Rapid Resolution HT, 600 bar 4.6 x 100 1.8 959964-918Rapid Resolution HT, 600 bar 4.6 x 50 1.8 959941-918Rapid Resolution HT, 600 bar 4.6 x 30 1.8 959931-918Solvent Saver 3.0 x 250 5 959990-318Narrow Bore 2.1 x 250 5 959790-918Narrow Bore 2.1 x 150 5 959701-918Narrow Bore RR* 2.1 x 100 3.5 959793-918Narrow Bore RRHT, 600 bar 2.1 x 100 1.8 959764-918Narrow Bore RRHT, 600 bar 2.1 x 50 1.8 959741-918Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-939Guard Cartridges, 4/pk 2.1 x 12.5 5 821125-939Guard Hardware Kit 820888-901*RR: Rapid Resolution 3.5 µmOrder online at www.agilent.com/chem/store31

Columns for Analytical HPLCOOOOOCH 3SiCH 3CH 3Si CH 3CH 3CH 3SiCH 3CH 3Si CH 3CH 3CH 3SiCH 3eXtra Densely Bonded and Double EndcappedEclipse XDB Bonded PhaseZORBAX Eclipse XDB• Four selectivity choices for method development optimization• Good peak shape for basic, acidic and neutral compounds• High performance over a wide pH range – pH 2-9• Particle sizes from 1.8 µm to 7 µm• Long lifetime with extra dense bonding and double endcappingAgilent ZORBAX Eclipse XDB columns – C18, C8, Phenyl and CN – provide four bonded phasechoices for method development optimization. These columns provide good peak shape over awide pH range (2-9) for additional method development flexibility with one family of columns.Eclipse XDB columns can be used for method development at low pH (2-3) and the samecolumn can be used for method development in the mid pH (6-8) region. In the mid pH regionresidual silanols are more active and tailing interactions are more likely. To overcome theseinteractions, Eclipse XDB columns are eXtra Densely Bonded and double endcapped througha proprietary process to cover as many active silanols as possible. The result is superior peakshape of basic compounds from pH 2-9. Eclipse XDB columns are available in 1.8, 3.5, 5and 7 µm particle sizes for high speed, high resolution, analytical and prep scale separations.Column SpecificationsBonded PhasePoreSizeSurfaceAreaTemp.Limits pH Range* EndcappedCarbonLoadZORBAX Eclipse XDB-C18 80Å 180 m 2 /g 60°C 2.0-9.0 Double 10%ZORBAX Eclipse XDB-C8 80Å 180 m 2 /g 60°C 2.0-9.0 Double 7.6%ZORBAX Eclipse 80Å 180 m 2 /g 60°C 2.0-9.0 Double 7.2%XDB-PhenylZORBAX Eclipse XDB-CN 80Å 180 m 2 /g 60°C 2.0-8.0 Double 4.3%Specifications represent typical values only.*Eclipse XDB columns are designed for operation over a wide pH range. At pH 6-9, highest columns stability for all silicabased columns is achieved by operating at temperatures < 40°C and using low buffer concentrations in the range of0.01 – 0.02M.32 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCGood Peak Shape Over a Wide pH Rangewith ZORBAX Eclipse XDBColumn: Eclipse XDB-C8993967-9064.6 x 150 mm, 5 µmMobile Phase: A:pH 3.0 75% 25 mMphosphate bufferpH 3.0: 25% ACNB:pH 7.0 90% 20 mMphosphate pH 7.010% ACNFlow Rate: 1.5 mL/minTemperature: 40°CA12345BSample: A:1. Maleate2. Doxylamine3. Chlorpheniramine4. Triprolidine5. Diphenhydramine2 B:1. Procainamide2. N-acetylprocainamide13. N-propionylprocainamide30 2.5 5.0 7.5Time (min)0 2.5 5.0 7.5Time (min)LCEC004ZORBAX Eclipse XDB columns provide good peak shape over a wide pH range and are anexcellent choice for method development from pH 2-9.Column Stability Testing at pH 7.0Column A: Competitive C8SIL-typeAfter 1826Column VolumesColumn B: Eclipse XDB-C8993967-9064.6 x 150 mm, 5 µmSol-typeAfter 1843Column VolumesMobile Phase: 60% ACN40% 250 mM PhosphateBuffer, pH 7.0Flow Rate: 1.5 mL/minTemperature: 60°CSample:Tricyclic AntidepressantsA1B5 1. Uracil152. Nortriptyline2, 33. Doxepin4. Amitriptyline45. Trimipramine2 340 1 2 3 4 5 6 7 8 9 10 11 12Time (min)0 1 2 3 4 5 6 7 8Time (min)LCEC006Double endcapping, dense bonding and the durable Rx-Sil particles (sol-type) combine to provide long lifetime at pH 7 when compared to single endcapped sil-gel columnsused here. The conditions used for this test – high temperature (60°C) and high salt concentration (250 mM), accelerate the dissolution of silica, causing premature failureof the sil-gel type column.Order online at www.agilent.com/chem/store33

Columns for Analytical HPLCColumn Stability Testing at pH 3 and 60°CColumn:ZORBAX SB-C8883975-9064.6 x 150 mm, 5 µm7.0Column: Eclipse XDB-C8993967-9064.6 x 150 mm, 5 µmMobile Phase: Purge Conditions:70% 50 mM NaAc-HCl, pH 3.030% ACNRetention Test Conditions:65% Methanol35% WaterFlow Rate: 1.0 mL/minTemperature: 60°CSample:Tricyclic AntidepressantsK of Amitriptyline6.56.05.55.00 2ZORBAX SB-C84 6 8 10 12Column Volumes of Mobile Phase (X1000)ZORBAX Eclipse XDB-C8Competitive C8LCEC005Eclipse XDB columns are stable over a wide pH range. At low pH, Eclipse, an end capped column is extremely stable and shows equivalent stability to a non-endcappedcolumn, SB-C8, at pH 3. The columns were purged with a pH 3 mobile phase at 60°C. Then they were tested with a strongly basic compound to determine if the endcappingor bonded phase had been hydrolyzed from the silica surface. The Eclipse XDB column was very stable, as shown by the consistency of the retention of amitriptyline overthe 12,000 column volumes of the test. Another endcapped column shows less stability under these same conditions.Selectivity Changes for Basic Compoundswith Eclipse XDB and StableBondColumn A:Eclipse XDB-C8966967-9064.6 x 75 mm, 3.5 µmColumn B: ZORBAX Rx/SB-C8866953-9064.6 x 75 mm, 3.5 µmMobile Phase: 70% 25 mM NaH 2 PO 4 , pH 3.030% MethanolFlow Rate: 1.0 mL/minTemperature: 35°CEclipse 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.A32B1. Barbital2. Sulfamethoxazole3. Caffeine23110 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time (min)0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time (min)LCEC00834 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCOptimize Separations with Eclipse XDB Selectivity Options:Analysis of SunscreensColumn A: Eclipse XDB-Phenyl963967-9124.6 x 150 mm, 3.5 µmColumn B: Eclipse XDB-C8963967-9064.6 x 150 mm, 3.5 µmColumn C: Eclipse XDB-C18963967-9024.6 x 150 mm, 3.5 µmMobile Phase: 15% H 2 0: 85% MeOHFlow Rate: 1.0 mL/minTemperature: 35°CSample:SunscreensA1230 2 4 6 8B1230 2 4 6 8C110 12 14 1610 12 14 16This separation of sunscreens on all three Eclipse XDB bonded phases – C18,C8 and Phenyl – shows that different bonded phases can be used to optimizea separation. While all three bonded phases provide an adequate separation,the Eclipse XDB-Phenyl provides a different peak elution order and a muchshorter overall analysis time. All three bonded phases also provide excellentpeak shape with no mobile phase additives.0 2 4 6 8 10 12 14 16Time (min)23LCEC007Selectivity for Urea PesticidesColumn A: Eclipse XDB-C18993967-9024.6 x 150 mm, 5 µmColumn B: Eclipse XDB-CN993967-9054.6 x 150 mm, 5 µmColumn C: Eclipse XDB-C18993967-9024.6 x 150 mm, 5 µmMobile Phase: A. 60:40 MeOH:WaterB. 60:40 MeOH:WaterC. 77:23 MeOH:WaterFlow Rate: 1.0 mL/minTemperature: 25°CSample:Urea pesticidesThe Eclipse XDB-CN column reduces retention timeand provides good selectivity for Urea pesticideswhen compared to an Eclipse XDB-C18 column.A12345Time0 1020 30(min)40 503 4 5B 23 451. Fenuron162. Monuron3. MonoLinuron0 2 4 6 8 104. DiuronTime (min)5. LinuronC 26. Pencycuron1660 2 4 6 8 10Time (min)LCEC010Order online at www.agilent.com/chem/store35

Columns for Analytical HPLCZORBAX Eclipse XDBHardware Description Size (mm)Particle Size(µm)EclipseXDB-C18USP L1EclipseXDB-C8USP L7EclipseXDB-PhenylUSP L11EclipseXDB-CNUSP L10Standard Columns (no special hardware required)Semi-Preparative 9.4 x 250 5 990967-202 990967-206Analytical 4.6 x 250 5 990967-902 990967-906 990967-912 990967-905Analytical 4.6 x 150 5 993967-902 993967-906 993967-912 993967-905Analytical 4.6 x 50 5 946975-902 946975-906Rapid Resolution 4.6 x 150 3.5 963967-902 963967-906 963967-912 963967-905Rapid Resolution 4.6 x 100 3.5 961967-902 961967-906 961967-905Rapid Resolution 4.6 x 75 3.5 966967-902 966967-906 966967-912 966967-905Rapid Resolution 4.6 x 50 3.5 935967-902 935967-906 935967-912Rapid Resolution 4.6 x 30 3.5 934967-902 934967-906Rapid Resolution 4.6 x 20 3.5 932967-902 932967-906Rapid Resolution HT, 600 bar 4.6 x 100 1.8 928975-902 928975-906Rapid Resolution HT, 600 bar 4.6 x 50 1.8 927975-902 927975-906Rapid Resolution HT, 600 bar 4.6 x 30 1.8 924975-902 924975-906Rapid Resolution HT, 600 bar 4.6 x 20 1.8 926975-902 926975-906Solvent Saver 3.0 x 250 5 990967-302 990967-306 990967-312 990967-305Solvent Saver 3.0 x 150 5 993967-302 993967-306 993967-312 993967-305Solvent Saver Plus 3.0 x 150 3.5 963954-302 963954-306 963954-312 963954-305Solvent Saver Plus 3.0 x 100 3.5 961967-302 961967-306 961967-312Solvent Saver Plus 3.0 x 75 3.5 966954-302Solvent Saver HT, 600 bar 3.0 x 100 1.8 928975-302 928975-306Solvent Saver HT, 600 bar 3.0 x 50 1.8 927975-302 927975-306Solvent Saver HT, 600 bar 3.0 x 30 1.8 924975-302 924975-306Solvent Saver HT, 600 bar 3.0 x 20 1.8 926975-302 926975-306Narrow Bore 2.1 x 150 5 993700-902 993700-906 993700-912 993700-905Narrow Bore 2.1 x 50 5 960967-902 960967-906 960967-912 960967-905Narrow Bore RR* 2.1 x 150 3.5 930990-902 930990-906Narrow Bore RR* 2.1 x 100 3.5 961753-902 961753-906 961753-905Narrow Bore RR* 2.1 x 75 3.5 966735-902Narrow Bore RR* 2.1 x 50 3.5 971700-902 971700-906Narrow Bore RR* 2.1 x 30 3.5 974700-902 974700-906Narrow Bore RR* 2.1 x 20 3.5 972700-902 972700-906Unless indicated, column pressure limit is 400 bar.*RR: Rapid Resolution 3.5 µm**RRHT: Rapid Resolution HT 1.8 µm36 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX Eclipse XDBHardware Description Size (mm)Particle Size(µm)EclipseXDB-C18USP L1EclipseXDB-C8USP L7EclipseXDB-PhenylUSP L11EclipseXDB-CNUSP L10Narrow Bore RRHT, 600 bar** 2.1 x 100 1.8 928700-902 928700-906Narrow Bore RRHT, 600 bar** 2.1 x 50 1.8 927700-902 927700-906Narrow Bore RRHT, 600 bar** 2.1 x 30 1.8 924700-902 924700-906Narrow Bore RRHT, 600 bar** 2.1 x 20 1.8 926700-902 926700-906MicroBore RR* 1.0 x 150 3.5 963600-902 963600-906MicroBore RR* 1.0 x 50 3.5 965600-902 965600-906MicroBore RR* 1.0 x 30 3.5 961600-902 961600-906MicroBore Guard, 3/pk 1.0 x 17 5 5185-5921 5185-5921Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-925 820950-926 820950-927 820950-935Guard Cartridges, 4/pk 2.1 x 12.5 5 821125-926 821125-926 821125-926 821125-935Guard Hardware Kit 820888-901 820888-901 820888-901 820888-901PrepHT Cartridge Columns (require endfittings kit 820400-901)PrepHT Cartridge 21.2 x 250 7 977250-102 977250-106PrepHT Cartridge 21.2 x 150 7 977150-102 977150-106PrepHT Cartridge 21.2 x 150 5 970150-902 970150-906PrepHT Cartridge 21.2 x 100 5 970100-902 970100-906PrepHT Cartridge 21.2 x 50 5 970050-902 970050-906PrepHT Guard Cartridge 17 x 7.5 5 820212-925 820212-926Guard Cartridge Hardware 820444-901 820444-901PrepHT endfittings, 2/pk 820400-901 820400-901Unless indicated, column pressure limit is 400 bar.*RR: Rapid Resolution 3.5 µm**RRHT: Rapid Resolution HT 1.8 µmOrder online at www.agilent.com/chem/store37

Columns for Analytical HPLCZORBAX Eclipse XDBHardware Description Size (mm) Particle Size (µm)EclipseXDB-C18USP L1EclipseXDB-C8USP L7Agilent Cartridge Columns (require hardware kit 5021-1845)Analytical 4.6 x 250 5 7995118-585 7995108-585Analytical 4.6 x 150 5 7995118-595 7995108-595Rapid Resolution 4.6 x 75 3.5 7995118-344 7995108-344Solvent Saver Plus 3.0 x 75 3.5 7995230-344Guard Cartridges, 10/pk 4.0 x 4 5 7995118-504 7995118-504Cartridge Holder 5021-1845 5021-1845Standard Columns (no special hardware required)Rapid Resolution HT 4.6 x 50 1.8 922975-902 922975-906Rapid Resolution HT, 3/pk 4.6 x 50 1.8 922975-932Narrow Bore RRHT 2.1 x 50 1.8 922700-902Narrow Bore RRHT, 3/pk 2.1 x 50 1.8 922700-932Rapid Resolution HT Cartridges (require hardware kit 820555-901)Rapid Resolution Cartridge 4.6 x 30 3.5 933975-902 933975-906Rapid Resolution Cartridge, 3/pk 4.6 x 30 3.5 933975-932 933975-936Rapid Resolution Cartridge 4.6 x 15 3.5 931975-902 931975-906Rapid Resolution Cartridge, 3/pk 4.6 x 15 3.5 931975-932 931975-936Rapid Resolution Cartridge 2.1 x 30 3.5 973700-902 973700-906Rapid Resolution Cartridge, 3/pk 2.1 x 30 3.5 973700-932 973700-936Rapid Resolution Cartridge 2.1 x 15 3.5 975700-902 975700-906Rapid Resolution Cartridge, 3/pk 2.1 x 15 3.5 975700-932 975700-936Rapid Resolution HT Cartridge 4.6 x 50 1.8 925975-902Rapid Resolution HT Cartridge, 3/pk 4.6 x 50 1.8 925975-932Rapid Resolution HT Cartridge 4.6 x 30 1.8 923975-902Rapid Resolution HT Cartridge, 3/pk 4.6 x 30 1.8 923975-932Rapid Resolution HT Cartridge 4.6 x 15 1.8 921975-902Rapid Resolution HT Cartridge, 3/pk 4.6 x 15 1.8 921975-93238 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX Eclipse XDBHardware Description Size (mm) Particle Size (µm)EclipseXDB-C18USP L1Rapid Resolution HT Cartridge 2.1 x 50 1.8 925700-902Rapid Resolution HT Cartridge, 3/pk 2.1 x 50 1.8 925700-932Rapid Resolution HT Cartridge 2.1 x 30 1.8 923700-902Rapid Resolution HT Cartridge, 3/pk 2.1 x 30 1.8 923700-932Rapid Resolution HT Cartridge 2.1 x 15 1.8 921700-902Rapid Resolution HT Cartridge, 3/pk 2.1 x 15 1.8 921700-932Hardware Kit for RR and RRHT Cartridges 820555-901Capillary Glass-lined ColumnsCapillary 0.5 x 250 5 5064-8286Capillary 0.5 x 150 5 5064-8287Capillary RR 0.5 x 150 3.5 5064-8288Capillary RR 0.5 x 35 3.5 5064-8298Capillary 0.3 x 250 5 5064-8269Capillary 0.3 x 150 5 5064-8291Capillary RR 0.3 x 150 3.5 5064-8271Capillary 0.5 x 35 5 5064-8296Capillary 0.3 x 35 5 5064-8297EclipseXDB-C8USP L7Order online at www.agilent.com/chem/store39

Columns for Analytical HPLCZORBAX 80Å StableBondROOHSiRRR 1• 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 pHwithout degradation• Six different bonded phases provide broad selectivity – SB-C18, SB-C8, SB-CN, SB-Phenyl,SB-C3, SB-Aq• High purity (Type B) silica for good peak shapeOOHOSiRRSiRR 1R 1Agilent ZORBAX StableBond columns use patented, unique, nonfunctional silanes with bulkydiisobutyl (SB-C18) or diisopropyl (SB-C8, SB-C3, SB-Phenyl, SB-CN, and SB-Aq) side chaingroups that sterically protect the key siloxane bond to the silica surface from hydrolytic attackat low pH. StableBond packing materials are not endcapped in order to provide exceptionalstability and to maximize lifetime and reproducibility under acidic mobile phase conditions.The high purity, low acidity silica provides excellent peak shape with acidic, basic and neutralcompounds so that StableBond columns are an excellent choice for low pH methoddevelopment. ZORBAX StableBond columns are compatible with all common mobile phases,including very high aqueous mobile phases.Sterically Protected StableBond Bonded PhaseColumn SpecificationsBonded PhasePoreSizeSurfaceAreaTemp.Limits*pH Range* EndcappedCarbonLoadZORBAX SB-C18 80Å 180 m 2 /g 90°C 0.8-8.0 No 10%ZORBAX SB-C8 80Å 180 m 2 /g 80°C 1.0-8.0 No 5.5%ZORBAX SB-C3 80Å 180 m 2 /g 80°C 1.0-8.0 No 4%ZORBAX SB-Phenyl 80Å 180 m 2 /g 80°C 1.0-8.0 No 5.5%ZORBAX SB-CN 80Å 180 m 2 /g 80°C 1.0-8.0 No 4%ZORBAX SB-Aq 80Å 180 m 2 /g 80°C 1.0-8.0 No proprietarySpecifications represent typical values only.*StableBond columns are designed for optimal use at low pH. At pH 6-8, highest column stability for all silica-basedcolumns is obtained by operating at temperatures

Columns for Analytical HPLCStableBond SB-C18 Shows Excellent Stability at Low pH and High Temperature (pH 0.8, 90°C)Column:Column:ZORBAX SB-C18883975-9024.6 x 150 mm, 5 µmZORBAX Rx-C18883967-9024.6 x 150 mm, 5 µmMobile Phase: 50% Methanol/50% Waterwith 1.0% TFATest Solute: TolueneTemperature: 90°CAs an indicator of column breakdown, retention time of toluene was measured after purging the column with mobile phase. Only the StableBond SB-C18 is unchangedafter three working months of use under these very low pH (0.8) and high temperature (90°C) conditions. ZORBAX Rx-C18 also provides a stable matrix, and can beused as an alternative selectivity to StableBond SB-C18.StableBond SB-C18 (Diisobutyl-C18)1.00.8ZORBAX Rx-C18 (Dimethyl-C18)Competitor A-C18Relative Retention0.60.4Competitor B-C18Competitor C-C18Competitor D-C180.2Competitor E-C180.00 5,000 10,000 15,000 20,000 25,000 30,000Column Volumes of Mobile Phase PurgeLCSB001Shorter Chain ZORBAX SB-CN is also Stableat Low pH (pH 2.0, 50°C)100SB-CNColumn:ZORBAX SB-CN883975-9054.6 x 150 mm, 5 µm80Mobile Phase: 0.1% TFA, pH 2: ACNFlow Rate: 1 mL/minGradient: 0-100% ACNTemperature: 50°CSample:1-phenylheptane @ 50% AC/50% Water with 0.1% TFA% k Remaining6040Si-(Me) 2 PrCN20ZORBAX StableBond SB-CN and the other short chainStableBond bonded phases are also exceptionally stableat low pH. Conventional dimethyl CN and similar bondedphases lack this stability.00 1,000 2,000 3,000 4,000 5,000 6,000Column VolumesLCSB002Order online at www.agilent.com/chem/store41

Columns for Analytical HPLCSB-CN Optimizes Retention and ResolutionColumn A: ZORBAX SB-C18866953-9024.6 x 75 mm, 3.5 µmColumn B: ZORBAX SB-CN866953-9054.6 x 75 mm, 3.5 µmMobile Phase: 30% ACN70% 25mM NaH 2 PO 4 , pH 2.5Flow Rate: 1.0 mL/minTemperature: 35°CA12341. Estriol2. Daidzen3. Quercetin4. Genistein5. Diethylstilbestrol5B21 3450 5 10 15 20 25Time (min)0 2 4 6 8 10 12Time (min)The SB-CN column is used here to reduce analysis time by 50%. The retention of the most hydrophobic analyte is cut in half. At the same time retention of the more polar,early eluting peaks increases slightly.LCSB003More RRHT Bonded Phase ChoicesAllow for Rapid Method Development OptimizationColumn A: ZORBAX SB-C18827975-9024.6 x 50 mm, 1.8 µmColumn B: SB-C8827975-9064.6 x 50 mm, 1.8 µmColumn C: ZORBAX SB-Phenyl827975-9124.6 x 50 mm, 1.8 µmColumn D: ZORBAX SB-CN827975-9054.6 x 50 mm, 1.8 µmMobile Phase: Acetonitrile (40%):Buffer 60% pH=2.425mM NaH 2 PO 4Flow Rate: 1.85 mL/minDetector: DAD, 254 nmLC: Agilent 1200 RR LCSample:As listed, 1 µL injection5 Different Bonded Phase Choices in StableBondfamily enhance method optimization.Note: SB-Aq not shown.mAU4504003503002502001501005000ABCD11. Tolmetin2. Naproxen3. Diflunisal4. Ibuprofen5. DiclofenacChange in Elution Order Peaks 3, 4 & 521351 2 3541325425341 2 3 4 54minMoreRRHT42 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCFive Different Bonded Phases Provide Selectivity OptionsColumn A:Column B:ZORBAX SB-C18883975-9024.6 x 150 mm, 5 µmZORBAX SB-C8883975-9064.6 x 150 mm, 5 µmA13254Column C:Column D:Column E:Mobile Phase:ZORBAX SB-C3883975-9094.6 x 150 mm, 5 µmZORBAX SB-Phenyl883975-9124.6 x 150 mm, 5 µmZORBAX SB-CN883975-9054.6 x 150 mm, 5 µm0-100% B in 18.8 minA: 50 mM NaH 2 PO 4 ,pH 2.5 in 95% H 2 O / 5% ACNB: 50 mM NaH 2 PO 4 ,pH 2.5 in 47% H 2 O / 53% ACNBC113235454Flow Rate: 1.0 mL/minTemperature: 26°CDetector: 254 nmSample:1. Procaine2. Lidocaine3. d-Cinchonine4. Butacaine5. TetracaineD231452SB-C3 is just one of the five different StableBond selectivity choices. In thisexample, optimum resolution is obtained with SB-C3. All are based on the samehigh purity Rx-SIL. Selectivity changes are therefore dependent only on the bondedphases, making method development more reliable.E132450 5 1015Time (min)LCSB004Order online at www.agilent.com/chem/store43

Columns for Analytical HPLCZORBAX 80Å StableBondHardware Description Size (mm)Particle SB-C18Size (µm) USP L1SB-C8USP L7SB-CNUSP L10SB-C3USP L56SB-PhenylUSP L11Standard Columns (no special hardware required)Semi-Preparative 9.4 x 250 5 880975-202 880967-201 880975-205 880975-209 880975-212Semi-Preparative 9.4 x 150 5 883975-202Semi-Preparative 9.4 x 100 5 884975-202Semi-Preparative 9.4 x 50 5 846975-202Analytical 4.6 x 250 5 880975-902 880975-906 880975-905 880975-909 880975-912 880975-914Analytical 4.6 x 150 5 883975-902 883975-906 883975-905 883975-909 883975-912 883975-914Analytical 4.6 x 50 5 846975-902 846975-906 846975-914Rapid Resolution 4.6 x 250 3.5 884950-567Rapid Resolution 4.6 x 150 3.5 863953-902 863953-906 863953-905 863953-912 863953-914Rapid Resolution 4.6 x 100 3.5 861953-902 861953-906 861953-905 861953-912 861953-914Rapid Resolution 4.6 x 75 3.5 866953-902 866953-906 866953-905 866953-912 866953-914Rapid Resolution 4.6 x 50 3.5 835975-902 835975-906 835975-905 835975-912 835975-914Rapid Resolution 4.6 x 30 3.5 834975-902 834975-906Rapid Resolution 4.6 x 20 3.5 832975-902 832975-906Rapid Resolution HT, 4.6 x 150 1.8 829975-902 829975-906 829975-905 829975-912 829975-914600 barRapid Resolution HT, 4.6 x 100 1.8 828975-902 828975-906 828975-905 828975-912 828975-914600 barRapid Resolution HT, 4.6 x 50 1.8 827975-902 827975-906 827975-905 827975-912 827975-914600 barRapid Resolution HT, 4.6 x 30 1.8 824975-902 824975-906 824975-905 824975-912 824975-914600 barRapid Resolution HT, 4.6 x 20 1.8 826975-902 826975-906600 barSolvent Saver 3.0 x 250 5 880975-302 880975-306 880975-305 880975-309 880975-312 880975-314Solvent Saver 3.0 x 150 5 883975-302 883975-306 883975-305 883975-309 883975-312 883975-314Solvent Saver Plus 3.0 x 150 3.5 863954-302 863954-306 863954-305 863954-312 863954-314Solvent Saver Plus 3.0 x 100 3.5 861954-302 861954-306 861954-305 861954-309 861954-312 861954-314SB-Aq44 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX 80Å StableBondHardware Description Size (mm)Particle SB-C18Size (µm) USP L1SB-C8USP L7SB-CNUSP L10SB-C3USP L56SB-PhenylUSP L11Solvent Saver HD, 3.0 x 100 1.8 858700-3021200 barSolvent Saver HD, 3.0 x 50 1.8 857700-3021200 barSolvent Saver Plus 3.0 x 75 3.5 866953-302Solvent Saver HT, 3.0 x 150 1.8 829975-302 829975-306 829975-305 829975-312600 barSolvent Saver HT, 3.0 x 100 1.8 828975-302 828975-306 828975-305 828975-309 828975-312 828975-314600 barSolvent Saver HT, 3.0 x 50 1.8 827975-302 827975-306 827975-305600 barSolvent Saver HT, 3.0 x 30 1.8 824975-302 824975-306 824975-305 827975-312 827975-314600 barSolvent Saver HT, 3.0 x 20 1.8 826975-302 826975-306600 barNarrow Bore 2.1 x 150 5 883700-922 883700-906 883700-905 883700-909 883700-912Narrow Bore 2.1 x 50 5 860975-902 860975-906 860975-905 860975-909 860975-912 860975-914Narrow Bore RR* 2.1 x 150 3.5 830990-902 830990-906 830990-914Narrow Bore RR* 2.1 x 100 3.5 861753-902 861753-906 861753-905 861753-912 861753-914Narrow Bore RR* 2.1 x 75 3.5 866735-902Narrow Bore RR* 2.1 x 50 3.5 871700-902 871700-906 871700-914Narrow Bore RR* 2.1 x 30 3.5 874700-902 874700-906Narrow Bore RR* 2.1 x 20 3.5 872700-902 872700-906Narrow Bore RRHD, 2.1 x 150 1.8 859700-902 859700-9061200 barNarrow Bore RRHD,1200 bar2.1 x 100 1.8 858700-902 858700-906Unless indicated, column pressure limit is 400 bar.*RR: Rapid Resolution 3.5 µm**RRHT: Rapid Resolution HT 1.8 µmSB-AqOrder online at www.agilent.com/chem/store45

Columns for Analytical HPLCZORBAX 80Å StableBondHardware Description Size (mm)ParticleSize (µm)SB-C18USP L1SB-C8USP L7SB-CNUSP L10SB-C3USP L56SB-PhenylUSP L11 SB-AqNarrow Bore RRHD, 2.1 x 50 1.8 857700-902 857700-9061200 barNarrow Bore RRHT, 2.1 x 150 1.8 820700-902 820700-906 820700-905 820700-912600 bar**Narrow Bore RRHT, 2.1 x 100 1.8 828700-902 828700-906 828700-905 828700-912 828700-914600 bar**Narrow Bore RRHT, 2.1 x 50 1.8 827700-902 827700-906 827700-905 827700-912 827700-914600 bar**Narrow Bore RRHT, 2.1 x 30 1.8 824700-902 824700-906 824700-905 824700-912 824700-914600 bar**Narrow Bore RRHT, 2.1 x 20 1.8 826700-902 826700-906600 bar**MicroBore RR* 1.0 x 150 3.5 863600-902 863600-906 863600-905MicroBore RR* 1.0 x 50 3.5 865600-902 865600-906MicroBore RR* 1.0 x 30 3.5 861600-902 861600-906MicroBore Guard, 3/pk 1.0 x 17 5 5185-5920 5185-5920Guard Cartridge, 2/pk 9.4 x 15 7 820675-115 820675-115 820675-124 820675-124 820675-115Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-920 820950-915 820950-916 820950-922 820950-917 820950-933Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-915 821125-915 821125-924 821125-924 821125-915 821125-933Guard Hardware Kit 9.4 x 15 840140-901 840140-901 840140-901 840140-901 840140-901Guard Hardware Kit 820888-901 820888-901 820888-901 820888-901 820888-901 820888-901PrepHT Cartridge Columns (require endfittings kit 820400-901)PrepHT Cartridge 21.2 x 250 7 877250-102 877250-106 877250-105 877250-112 877250-114PrepHT Cartridge 21.2 x 150 7 877150-102 877150-106 877150-114PrepHT Cartridge 21.2 x 150 5 870150-902 870150-906 870150-914PrepHT Cartridge 21.2 x 100 5 870100-902 870100-906 870100-914PrepHT Cartridge 21.2 x 50 5 870050-902 870050-906 870050-914PrepHT Guard 17 x 7.5 5 820212-920 820212-915 820212-915 820212-915 820212-933Cartridge, 2/pkGuard Cartridge820444-901 820444-901 820444-901 820444-901 820444-901 820444-901HardwarePrepHT endfittings,820400-901 820400-901 820400-901 820400-901 820400-901 820400-9012/pkUnless indicated, column pressure limit is 400 bar.*RR: Rapid Resolution 3.5 µm**RRHT: Rapid Resolution HT 1.8 µm46 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX 80Å StableBondHardware Description Size (mm) Particle Size (µm)SB-C18USP L1SB-C8USP L7Agilent Cartridge Columns (require hardware kit 5021-1845)Analytical 4.6 x 250 5 7995218-585 7995208-585Analytical 4.6 x 150 5 7995218-595 7995208-595Rapid Resolution 4.6 x 75 3.5 7995218-344 7995208-344Guard Cartridges, 10/pk 4.0 x 4 5 7995118-504 7995118-504Cartridge Holder 5021-1845 5021-1845SB-PhenylUSP L11Standard Columns (no special hardware required)Rapid Resolution HT 4.6 x 50 1.8 822975-902 822975-906Rapid Resolution HT, 3/pk 4.6 x 50 1.8 822975-932Narrow Bore RRHT 2.1 x 50 1.8 822700-902Narrow Bore RRHT, 3/pk 2.1 x 50 1.8 822700-932Rapid Resolution Cartridges (require hardware kit 820555-901)Rapid Resolution Cartridge 4.6 x 30 3.5 833975-902 833975-906 833975-912Rapid Resolution Cartridge, 3/pk 4.6 x 30 3.5 833975-932 833975-936Rapid Resolution Cartridge 4.6 x 15 3.5 831975-902 831975-906Rapid Resolution Cartridge, 3/pk 4.6 x 15 3.5 831975-932 831975-936Rapid Resolution Cartridge 2.1 x 30 3.5 873700-902 873700-906Rapid Resolution Cartridge, 3/pk 2.1 x 30 3.5 873700-932 873700-936Rapid Resolution Cartridge 2.1 x 15 3.5 875700-902 875700-906Rapid Resolution Cartridge, 3/pk 2.1 x 15 3.5 875700-932 875700-936Rapid Resolution HT Cartridges (require hardware kit 820555-901)Rapid Resolution HT Cartridge 4.6 x 50 1.8 825975-902Rapid Resolution HT Cartridge, 3/pk 4.6 x 50 1.8 825975-932Rapid Resolution HT Cartridge 4.6 x 30 1.8 823975-902Rapid Resolution HT Cartridge, 3/pk 4.6 x 30 1.8 823975-932Rapid Resolution HT Cartridge 4.6 x 15 1.8 821975-902Rapid Resolution HT Cartridge, 3/pk 4.6 x 15 1.8 821975-932Rapid Resolution HT Cartridge 2.1 x 50 1.8 825700-902Rapid Resolution HT Cartridge, 3/pk 2.1 x 50 1.8 825700-932Rapid Resolution HT Cartridge 2.1 x 30 1.8 823700-902Rapid Resolution HT Cartridge, 3/pk 2.1 x 30 1.8 823700-932Rapid Resolution HT Cartridge 2.1 x 15 1.8 821700-902Rapid Resolution HT Cartridge, 3/pk 2.1 x 15 1.8 821700-932Hardware Kit for RR and RRHT Cartridges 820555-901Order online at www.agilent.com/chem/store47

Columns for Analytical HPLCZORBAX 80Å StableBondDescription Size (mm) Particle Size (µm)SB-C18USP L1Capillary Glass-lined ColumnsCapillary 0.5 x 250 5 5064-8258Capillary 0.5 x 150 5 5064-8256Capillary 0.5 x 35 5 5064-8254Capillary RR 0.5 x 150 3.5 5064-8262Capillary RR 0.5 x 35 3.5 5064-8260Capillary 0.3 x 250 5 5064-8257Capillary 0.3 x 150 5 5064-8255Capillary 0.3 x 35 5 5064-8253Capillary RR 0.3 x 150 3.5 5064-826148 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX Rx• Rx-C18 is recommended for alternate selectivity at low pH relative to Eclipse XDB-C18and StableBond SB-C18; for higher temperature applications, StableBond is recommended.This column 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 and providesexcellent stability up to pH 8.• Rx-C8 is the same product as SB-C8.Column SpecificationsBonded PhasePoreSizeSurfaceAreaTemp.LimitspHRange*EndcappedCarbonLoadZORBAX Rx-C18 80Å 180 m 2 /g 60°C 2.0-8.0 No 12%ZORBAX Rx-C8 80Å 180 m 2 /g 80°C 1.0-8.0 No 5.5%Specifications represent typical values only.*At pH 6-9 highest column stability for all silica based columns is obtained by operating at temperatures < 40°C and usinglower buffer concentrations in the range of 0.01 - 0.02M.Analysis of Diazepam on Rx-C18Column: ZORBAX Rx-C18880967-3023.0 x 250 mm, 5 µmMobile Phase: 35% H 2 O: 65% MeOHFlow Rate: 0.5 mL/min121. Ethylparaben2. DiazepamAn Rx-C18 column is used for this USP analysis of diazepam and the internal standardethylparaben. The Solvent Saver 3.0 mm i.d. Rx-C18 column reduces solvent usage by60% over what would be used if the analysis were done on a 4.6 x 250 mm column.0.0 2.5 5.0 7.5Time (min)LCRX001Order online at www.agilent.com/chem/store49

Columns for Analytical HPLCRx-C8 and SB-C8 are the Same ColumnColumn A: ZORBAX Rx-C8880967-9014.6 x 250 mm, 5 µmColumn B: ZORBAX SB-C8880975-9064.6 x 250 mm, 5 µmSample: Sulfa Drugs0.125 mg/mL each in waterFlow Rate: 1.5 mL/minGradient: 10% B at t n , increase to 35% Bover 25 min, decrease to 10% Bin 5 min, hold 10% B for 10 minTCC: 26°CDAD:254 nmA12345671. Sulfadiazine2. Sulfapyridine3. Sulfamerazine4. Sulfamethazine5. Sulfamethoxypyridazine6. Sulfachloropyridazine7. Sulfamethoxazole8. Sulfadimethoxine8B52310 15 20 25Time (min)1456875 10 15 20 25Time (min)RxC8_SBC850 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX RxHardware DescriptionParticle SizeSize (mm) (µm)Rx-C18USP L1Rx-C8USP L7**Semi-Preparative 9.4 x 250 5 880967-202 880967-201Analytical 4.6 x 250 5 880967-902 880967-901Analytical 4.6 x 150 5 883967-902 883967-901Rapid Resolution 4.6 x 150 3.5 863967-902 863953-906Rapid Resolution 4.6 x 100 3.5 861967-902 861953-906Rapid Resolution 4.6 x 75 3.5 866967-902 866953-906Solvent Saver 3.0 x 250 5 880967-302 880975-306Solvent Saver 3.0 x 150 5 883967-302 883975-306Solvent Saver Plus 3.0 x 150 3.5 863967-302 863954-306Solvent Saver Plus 3.0 x 100 3.5 861967-302 861954-306Narrow Bore 2.1 x 150 5 883700-902 883700-906Narrow Bore RR* 2.1 x 100 3.5 861767-902 861753-906Guard Cartridge, 2/pk 9.4 x 15 7 820675-115 820675-115Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-914 820950-913Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-915 821125-915Guard Hardware Kit 9.4 x 15 840140-901 840140-901Guard Hardware Kit 820888-901 820888-901PrepHT Cartridge Columns (require endfittings kit 820400-901)PrepHT Cartridge 21.2 x 250 7 877967-102 877250-106PrepHT Cartridge 21.2 x 150 7 877150-106PrepHT Cartridge 21.2 x 150 5 870150-906PrepHT Cartridge 21.2 x 100 5 870100-906PrepHT Cartridge 21.2 x 50 5 870050-906PrepHT Guard Cartridge, 2/pk 820212-914 820212-915Guard Cartridge Hardware 820444-901 820444-901PrepHT endfittings, 2/pk 820400-901 820400-901*RR: Rapid Resolution 3.5 µm**Rx-C8 is the same product as SB-C8Order online at www.agilent.com/chem/store51

Columns for Analytical HPLCZORBAX 80Å Extend-C18OSiC18• 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 peptidesSiO C18Silica SupportThe Agilent ZORBAX Extend-C18 column uses a novel bidentate C18-C18 bonding technologyto make it possible to develop high-resolution separations at high pH with a silica-basedcolumn. At high pH, non-charged basic compounds will not interact with the underlying silica.The result is high efficiency separations with superior peak shape and improved resolution.High pH separations are also the best choice for compounds that are more stable or moresoluble in high pH solutions. Some of the mobile phase buffer options for high pH includetriethylamine, pyrrolidine, glycine, borate and ammonium hydroxide. Ammonium hydroxideat pH 10.5 is an excellent mobile phase modifier for the LC/MS of peptides and smallmolecules with improved sensitivity compared with TFA containing mobile phase at low pH.The Extend-C18 column is stable from pH 2-11.5 with good peak shape for all types ofcompounds. Extend-C18 columns also provide an additional selectivity choice at low pH.Novel Bidentate C18-C18 Bonding forExtend C-18 Bonded PhaseColumn SpecificationsBonded PhasePoreSizeSurfaceAreaTemp.Limits* pH Range** EndcappedCarbonLoadZORBAX Extend-C18 80Å 180 m 2 /g 60°C 2.0-11.5 Double 12.5%Specifications represent typical values only.*Temperature limits are 60°C up to pH 8, 40°C from pH 8-11.5.**Above pH 6 highest column stability for all silica based columns is obtained by reducing the operating temperature to40°C or below and using lower buffer concentrations (0.01 - 0.02M) or organic buffers.52 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCBasic Antihistamines on Extend-C18 at High pHColumn: ZORBAX Extend-C18773450-9024.6 x 150 mm, 5 µmMobile Phase: pH 7:30% 20 mM Na 2 HPO 470% MeOHFlow Rate:Temperature:Detector:Sample:pH 11:30% 20 mM TEA70% MeOH1.0 mL/minAmbient254 nmAntihistaminespH 7pH 1112, 3340 5Time (min)546771. Maleate2. Scopolamine3. Pseudoephedrine4. Doxylamine5. Chlorpheniramine6. Triprolidine7. Diphenhydramine1526Pseudoephedrine and scopolamine are difficult to retain at lowand mid pH. Pseudoephedrine is often analyzed by ion exchangemethods. The Extend-C18 column retains these compounds in anoncharged form at high pH and improves resolution.0 5Time (min)10LC80001Long Life at High pH with Extend-C18Column:Mobile Phase:Flow Rate:Temperature:ZORBAX Extend-C18773450-9024.6 x 150 mm, 5 µm20% Methanol80% 0.1 M Carbonate Buffer, pH 10.01.0 mL/minAmbientAt high pH, columns will fail due to silica dissolution. The example hereshows extended lifetime of ZORBAX Extend-C18 at high pH in comparisonto competitor W. This was measured by the amount of dissolved silica.Amount of Dissolved Silica (mg)302010ZORBAX Extend-C18Competitor W00 3 5 91215Volume Eluent (L)LC80002Order online at www.agilent.com/chem/store53

Columns for Analytical HPLCExtend-C18 Provides Good Peak Shape at Low pHColumn: ZORBAX Extend-C18773450-9024.6 x 150 mm, 5 µmMobile Phase: 80% 25 mM NaH 2 PO 4 , pH 3.020% MethanolFlow Rate: 1.0 mL/minTemperature: 35°CDetector: 254 nmSample:Basic Compounds1231. Theobromine2. Dimethylxanthine3. Theophylline4. Caffeine4These basic compounds are separated on the Extend-C18at low pH with excellent peak shape. The Extend-C18column can be used at high and low pH.0 12 34 5 67Time (min)LC80003ZORBAX 80Å Extend-C18Hardware DescriptionSize (mm)Particle Size(µm)Extend-C18USP L1Standard Columns (no special hardware required)Analytical 4.6 x 250 5 770450-902Analytical 4.6 x 150 5 773450-902Analytical 4.6 x 50 5 746450-902Rapid Resolution 4.6 x 150 3.5 763953-902Rapid Resolution 4.6 x 100 3.5 764953-902Rapid Resolution 4.6 x 75 3.5 766953-902Rapid Resolution 4.6 x 50 3.5 735953-90254 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX 80Å Extend-C18Hardware DescriptionSize (mm)Particle Size(µm)Extend-C18USP L1Rapid Resolution HT, 600 bar 4.6 x 100 1.8 728975-902Rapid Resolution HT, 600 bar 4.6 x 50 1.8 727975-902Rapid Resolution HT, 600 bar 4.6 x 30 1.8 724975-902Rapid Resolution HT, 600 bar 4.6 x 20 1.8 726975-902Solvent Saver 3.0 x 250 5 770450-302Solvent Saver 3.0 x 150 5 773450-302Solvent Saver Plus 3.0 x 150 3.5 763954-302Solvent Saver Plus 3.0 x 100 3.5 764953-302Solvent Saver Plus 3.0 x 50 3.5 735954-302Solvent Saver HT, 600 bar 3.0 x 100 1.8 728975-302Solvent Saver HT, 600 bar 3.0 x 50 1.8 727975-302Solvent Saver HT, 600 bar 3.0 x 30 1.8 724975-302Solvent Saver HT, 600 bar 3.0 x 20 1.8 726975-302Narrow Bore 2.1 x 150 5 773700-902Narrow Bore 2.1 x 50 5 760450-902Narrow Bore RR* 2.1 x 100 3.5 761753-902Narrow Bore RR* 2.1 x 50 3.5 735700-902Narrow Bore RRHT, 600 bar** 2.1 x 100 1.8 728700-902Narrow Bore RRHT, 600 bar** 2.1 x 50 1.8 727700-902Narrow Bore RRHT, 600 bar** 2.1 x 30 1.8 724700-902Narrow Bore RRHT, 600 bar** 2.1 x 20 1.8 726700-902MicroBore RR* 1.0 x 150 3.5 763600-902MicroBore RR* 1.0 x 50 3.5 765600-902MicroBore RR* 1.0 x 30 3.5 761600-902MicroBore Guard, 3/pk 1.0 x 17 5 5185-5923Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-930Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-930Guard Hardware Kit 820888-901PrepHT Cartridge Columns (require endfittings kit 820400-901)PrepHT Cartridge 21.2 x 150 5 770150-902PrepHT 21.2 x 100 5 770100-902PrepHT 21.2 x 50 5 770050-902PrepHT endfittings, 2/pk 820400-901PrepHT Guard Cartridge, 2/pk 17 x 7.5 5 820212-930Guard Cartridge Hardware 820444-901Unless indicated, column pressure limit is 400 bar.*RR: Rapid Resolution 3.5 µm**RRHT: Rapid Resolution HT 1.8 µmOrder online at www.agilent.com/chem/store55

Columns for Analytical HPLCR 1O SiPGR 1CH 3Si CH 3R 1O SiPGR 1CH 3Si CH 3CH 3CH 3R 1O SiPGR 1Unique, Polar Alkyl Bonus-RP Bonded PhaseRRRZORBAX Bonus-RP• 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 phasesThe Agilent ZORBAX Bonus-RP column has a polar amide group embedded in a long alkylchain. This novel bonding reduces interactions between basic compounds and the silicasupport, improving peak shape for the most difficult basic compounds. Peak shape andcolumn lifetime are further improved by triple endcapping. In addition, diisopropyl side groupsprovide steric protection against acid hydrolysis for good lifetime at low pH. The Bonus-RPcolumn provides an alternate selectivity to C18 and C8 alkyl bonded phases.Column SpecificationsBonded PhasePoreSizeSurfaceAreaTemp.Limits* pH Range EndcappedCarbonLoadZORBAX Bonus-RP 80Å 180 m 2 /g 60°C 2.0-9.0 Triple 9.5%Specifications represent typical values only.*Temperature limits are 60°C up to pH 8, 40°C from pH 8-9.56 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCImproved Peak Shape of Basic Compounds Using Bonus-RPColumn: Alkyl-C84.6 x 150 mm, 5 µmMobile Phase: 75% 25 mM NH 4 OAc, pH 5.525% ACNFlow Rate: 1.5 mL/minTemperature: 40°CDetector: 254 nmBonus-RP eliminates peak tailing of these basic compounds in comparison to atypical alkyl C8 bonded phase. In the mid-pH region, residual silanols caninteract more strongly with basic compounds to cause peak tailing. The polargroup in the Bonus-RP bonded phase eliminates peak tailing of these basiccompounds by reducing interactions with residual silanols.TF1. 1.82. 1.73. 2.1121. Doxylamine2. Chlorpheniramine3. Triprolidine30 1 2 3 4 5 6 7 8 9Time (min)10LCBR001Column: ZORBAX Bonus-RP883668-9014.6 x 150 mm, 5 µmMobile Phase: 80% 25 mM NH 4 OAc, pH 5.520% ACNFlow Rate: 1.5 mL/minTemperature: 40°CDetector: 254 nmTF1. 1.12. 1.13. 1.11231. Doxylamine2. Chlorpheniramine3. Triprolidine0 1 2 3 4 5 6Time (min)LCBR002Order online at www.agilent.com/chem/store57

Columns for Analytical HPLCZORBAX Bonus-RP is Stable at Low and Mid pHColumn: ZORBAX Bonus-RP883668-9014.6 x 150 mm, 5 µmMobile Phase: 60% 25 mMPhosphate Buffer,pH 7.0: 40% ACNFlow Rate: 1.5 mL/minTemperature: 23°CToluene Plate Height, cm0.00180.00160.0014Competitive alkyl/amideBonus-RPTriple endcapping of Bonus-RP enhances stabilityat pH 7. Each 10,000 column volumes is equivalentto approximately one working month.0.00120.00100 5,000 10,000 15,000 20,000 25,000 30,000 35,000Column Volume of PurgeLCBR003Dimethyl-C18/amide, Bonus-RPColumn:ZORBAX Bonus-RP883668-9014.6 x 150 mm, 5 µmMobile Phase:Flow Rate:Temperature:Aging:50% MeOH50% 0.1% TFATest:80% MeOH20% H2O1.0 mL/minAging:60°CTest:23°CToluene Plate Height, cm0.00550.00500.00450.00400.00350.0030Dimethyl-C18/amideBonus-RPSterically protecting side groups provide goodlow pH stability and longer column lifetime thansimilar polar alkyl bonded phases.0.00250.00200 10,000 20,000 30,000 40,000Column Volume of PurgeLCBR00458 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX Bonus-RP Provides Unique SelectivityColumn A: ZORBAX Bonus-RP A883668-9014.6 x 150 mm, 5 µmmAuColumn B: Eclipse XDB-C815993967-9064.6 x 150 mm, 5 µm10Mobile Phase: 75% 25 mM Na Citrate, pH 625% MeOH5Flow Rate: 1.0 mL/min0Temperature: AmbientDetector: 254 nmSample: 3 µLBCephalosporinsmAu1. Cephalexin2. Cephaclor3. Cephuroxime4. Cephoxitin0 213244 6 8 10Time (min)1Peak elution order can change dramatically when usingBonus-RP. In this example, the elution order of the firstthree peaks change.1510532400 24 6 8 10Time (min)LCBR005ZORBAX Bonus-RPHardware DescriptionSize (mm)Particle Size(µm)Bonus-RPUSP L60Standard Columns (no special hardware required)Analytical 4.6 x 250 5 880668-901Analytical 4.6 x 150 5 883668-901Rapid Resolution 4.6 x 250 3.5 884950-577Rapid Resolution 4.6 x 150 3.5 863668-901Rapid Resolution 4.6 x 100 3.5 864668-901Rapid Resolution 4.6 x 75 3.5 866668-901Rapid Resolution 4.6 x 50 3.5 835668-901Order online at www.agilent.com/chem/store59

Columns for Analytical HPLCZORBAX Bonus-RPHardware DescriptionSize (mm)Particle Size(µm)Bonus-RPUSP L60Rapid Resolution HT, 600 bar 4.6 x 100 1.8 828668-901Rapid Resolution HT, 600 bar 4.6 x 75 1.8 830668-901Rapid Resolution HT, 600 bar 4.6 x 50 1.8 827668-901Solvent Saver 3.0 x 250 5 880668-301Solvent Saver 3.0 x 150 5 883668-301Solvent Saver Plus 3.0 x 150 3.5 863668-301Solvent Saver Plus 3.0 x 100 3.5 864668-301Solvent Saver HT, 600 bar 3.0 x 100 1.8 828668-301Solvent Saver HT, 600 bar 3.0 x 50 1.8 827668-301Narrow Bore 2.1 x 150 5 883725-901Narrow Bore 2.1 x 50 5 861971-901Narrow Bore RR* 2.1 x 150 3.5 863700-901Narrow Bore RR* 2.1 x 100 3.5 861768-901Narrow Bore RR* 2.1 x 50 3.5 861700-901Narrow Bore RRHT, 600 bar 2.1 x 100 1.8 828768-901Narrow Bore RRHT, 600 bar 2.1 x 50 1.8 827768-901MicroBore RR* 1.0 x 150 3.5 863608-901MicroBore RR* 1.0 x 50 3.5 865608-901MicroBore RR* 1.0 x 30 3.5 861608-901MicroBore Guard, 3/pk 1.0 x 17 5 5185-5922Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-928Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-928Guard Hardware Kit 820888-901PrepHT Cartridge Columns (require endfittings kit 820400-901)PrepHT Cartridge 21.2 x 250 7 878250-101PrepHT Cartridge 21.2 x 150 7 878150-101PrepHT Cartridge 21.2 x 150 5 868150-901PrepHT Cartridge 21.2 x 100 5 868100-901PrepHT Cartridge 21.2 x 50 5 868050-901PrepHT endfittings, 2/pk 820400-901PrepHT Guard Cartridge, 2/pk 17 x 7.5 5 820212-928Guard Cartridge Hardware 820444-901Unless indicated, column pressure limit is 400 bar.*RR: Rapid Resolution 3.5 µm60 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX Original Reversed Phase ColumnsAgilent Original ZORBAX columns are made with Type A silica and are useful for manyapplications of acidic or neutral compounds. These columns have a higher activity leveland are therefore useful for separating isomers (e.g. cis-trans, geometric) or othercompounds where silanol activity enhances selectivity. These columns are used in manyestablished methods.Hardware DescriptionSize (mm)Particle Size(µm)ODS (C18)USP L1C8USP L7PhenylUSP L11CNUSP L10TMSUSP L13Standard Columns (no special hardware required)Semi-Preparative 9.4 x 250 5 880952-202 880952-206Analytical (Endcapped) 4.6 x 250 5 880952-702 880952-706 880952-712 884950-507 880952-710Analytical (Non-endcapped) 4.6 x 250 5 884950-543Analytical 4.6 x 150 5 883952-702 883952-706 883952-712 884950-526 883952-710Solvent Saver 3.0 x 250 5 880952-302Solvent Saver 3.0 x 150 5 883952-302Guard Columns (hardware required)Guard Cartridge, 2/pk 9.4 x 15 7 820675-115 820675-115 820675-115 820675-124Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-902 820950-906 820950-912 820950-905 820950-924Guard Hardware Kit 840140-901 840140-901 840140-901 840140-901 840140-901Guard Hardware Kit 820888-901 820888-901 820888-901 820888-901 820888-901PrepHT Cartridge Columns (require endfittings kit 820400-901)PrepHT Cartridge 21.2 x 250 7 877952-102 877952-106 877952-105PrepHT endfittings, 2/pk 820400-901 820400-901 820400-901Order online at www.agilent.com/chem/store61

Columns for Analytical HPLCZORBAX Normal Phase ColumnsFor normal-phase chromatography, the Agilent ZORBAX product line offers a choice ofbonded and non-bonded silica packings.ZORBAX Rx-SIL• Made from highly pure (>99.995%) porous silica microspheres (pore size is the spacebetween the solid silica microparticles)• Available in 1.8 and 5 µm particle sizes• Stronger than other silica types• Less acidic than ZORBAX-SIL, lower metal content• Low acidity and low metal content make ZORBAX Rx-SIL ideal for normal-phase separationof polar compounds that exhibit poor peak symmetry on more acidic silica• Useful for very hydrophilic compounds with high organic mobile phases in HILIC modeZORBAX Eclipse XDB-CN• Made from highly pure Rx-SIL• Excellent choice for normal phase applications with basic compounds• Equilibrates more rapidly than ZORBAX Rx-SIL and is used for many of the samenormal-phase applicationsZORBAX CN• Cyanopropyldimethylsilane monolayer bonded to ZORBAX SIL• Equilibrates more rapidly than ZORBAX SIL, and used for many of the samenormal-phase applications• Less prone to fouling and less water sensitive than silicaZORBAX NH 2• Amino-propyl silane phase bonded to ZORBAX SIL• Used for normal phase and weak anion-exchange, and reversed-phase HPLCof polar compounds• Vitamins A and D are separated in the normal-phase mode• Carbohydrates and sugars are separated in the reversed-phase mode62 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCColumn SpecificationsBonded Phase Pore SizeSurfaceArea pH Range Endcapped Carbon LoadZORBAX Rx-SIL 80Å 180 m 2 /g 0-8.0 NoZORBAX Eclipse XDB-CN 80Å 180 m 2 /g 2.0-8.0 Yes 4.3%ZORBAX SIL 70Å 300 m 2 /g 0-8.0 NoZORBAX CN 70Å 300 m 2 /g 2.0-7.0 Yes 7%ZORBAX NH 2 70Å 300 m 2 /g 2.0-7.0 Yes 4%High Resolution Normal Phase Separationof Octylphenoxy Ethanol Surfactant on ZORBAX CNColumn: ZORBAX CN880952-7054.6 x 250 mm, 5 µmMobile Phase: Primary: HeptaneSecondary: 2-Methoxyethanol/Isopropanol (50/50)Flow Rate: 2 mL/minGradient: 2-20% Secondary in 10 min., Linear Hold at 20%Temperature: 50°CDetector: 278 nmSample:Octylphenoxy (polyethylene oxy)Ethanol Surfactant (n= 10)0 510 15Time (min)20LCNP001Order online at www.agilent.com/chem/store63

Columns for Analytical HPLCNormal Phase Columns Based on ZORBAX Rx-SILHardware Description Size (mm) Particle Size (µm)Rx-SILUSP L3EclipseXDB-CNUSP L10Standard Columns (no special hardware required)Semi-Prep 9.4 x 250 5 880975-201Analytical 4.6 x 250 5 880975-901 990967-905*Analytical 4.6 x 150 5 883975-901 993967-905*Rapid Resolution HT, 600 bar 4.6 x 100 1.8 828975-901Rapid Resolution HT, 600 bar 4.6 x 50 1.8 827975-902Rapid Resolution HT, 600 bar 3.0 x 100 1.8 828975-301Rapid Resolution HT, 600 bar 3.0 x 50 1.8 827975-301Narrow Bore 2.1 x 150 5 883700-901 993700-905*Rapid Resolution HT, 600 bar 2.1 x 100 1.8 828700-901Rapid Resolution HT, 600 bar 2.1 x 50 1.8 827700-901Guard Columns (hardware required)Guard Cartridge, 2/pk 9.4 x 15 5 820675-119Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-919 820950-935Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-919 821125-935Guard Hardware Kit 840140-901Guard Hardware Kit 820888-901 820888-901PrepHT Cartridge Columns (require endfittings kit 820400-901)PrepHT Cartridge 21.2 x 250 7 877250-101PrepHT Cartridge 21.2 x 250 7PrepHT endfittings, 2/pk 820400-901PrepHT Guard Cartridge, 2/pk 17 x 7.5 5 820212-919Guard Cartridge Hardware 820444-901*These columns ship containing reversed phase solvents. Flush with isopropanol before using normal phase solvents.64 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCNormal Phase Columns Based on ZORBAX Original SILHardware DescriptionSize (mm)Particle Size(µm)SILUSP L3CNUSP L10NH2USP L8CarbohydrateAnalysisStandard Columns (no special hardware required)Semi-Prep 9.4 x 250 5 880952-201 880952-205 880952-208Analytical 4.6 x 250 5 880952-701 880952-705 880952-708 840300-908*Analytical 4.6 x 150 5 883952-701 883952-705 883952-708 843300-908*Narrow Bore 2.1 x 50 5 860700-708Guard Columns (hardware required)Guard Cartridge, 2/pk 9.4 x 15 5 820675-119 820675-111 820675-111Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-901 820950-905 820950-908 820950-908*Guard Cartridge, 4/pk 2.1 x 12.5 5Guard Hardware Kit 840140-901 840140-901 840140-901Guard Hardware Kit 820888-901 820888-901 820888-901 820888-901*PrepHT Cartridge Columns (require endfittings kit 820400-901)PrepHT Cartridge 21.2 x 250 7 877952-101 877952-105 877952-108PrepHT endfittings, 2/pk 820400-901 820400-901 820400-901PrepHT Guard Cartridge, 2/pk 17 x 7.5 5Guard Cartridge Hardware*These columns ship containing reversed phase solvents. Flush with isopropanol before using normal phase solvents. These columns can also be used in HILIC mode.Order online at www.agilent.com/chem/store65

Columns for Analytical HPLCZORBAX HILIC Plus• HILIC column for good retention of small, polar analytes• Based on Eclipse Plus silica for excellent peak shape• High sensitivity for LC/MS applications• Recommended for EPA Method 1694Agilent ZORBAX HILIC Plus columns are for use in hydrophilic interaction chromatography(HILIC) applications. HILIC is typically used for the retention and resolution of small polarcompounds. HILIC Plus columns are non-bonded silica columns based on the highperformance silica used in the ZORBAX Eclipse Plus columns. This silica provides excellentpeak shape; critical for many polar, basic analytes. These columns ship prepared for use inHILIC mode – containing acetonitrile:water – in order to reduce the extensive equilibrationtypically required for HILIC separations. The HILIC Plus columns are available in a 3.5 µmparticle size for high resolution and in 2.1 and 4.6 mm ID for compatibility with massspectrometers or with standard UV detectors.Column SpecificationsBonded Phase Pore Size Surface Area pH RangeNon-bonded silica 95Å 160 m 2 /g 0-8.0Specifications represent typical values only.66 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCSeparation of Group 4 Analytes in EPA 1694on ZORBAX HILIC Plus ColumnColumn: ZORBAX HILIC Plus959793-9012.1 x 100 mm, 3.5 µmMobile Phase: 90% Acetontrile:10% WaterFlow Rate: 0.25 mL/minGradient: Linear gradient to 55% acetonitrilein 7 min Held at 55%Temperature: 25°Cx10 21.000.950.900.850.800.750.700.650.600.550.500.450.400.350.300.250.200.150.100.050CimetidineAlbuterolRanitidineMetforminx10 2 671.000.950.900.850.800.750.700.650.600.550.500.450.400.350.300.250.200.150.100.0500.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5Duplicate runs for column USCJP0004 10 min equilibration between two runsEPA1694ZORBAX HILIC PlusDescription Size (mm) Particle Size (µm) Part No.ZORBAX HILIC Plus 4.6 x 100 3.5 959961-901ZORBAX HILIC Plus 4.6 x 50 3.5 959943-901ZORBAX HILIC Plus 2.1 x 100 3.5 959793-901ZORBAX HILIC Plus 2.1 x 50 3.5 959743-901Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX Method Development KitsAgilent offers a series of kits that allow for fast method development at an attractive price.Each kit contains 3 columns. Six new kits have been added and are recommended for usewith the new Agilent Automated Method Development LC. Several of these kits contain RapidResolution HT (1.8 µm) columns in a variety of bonded phases for easy method optimizationand several kits contain Rapid Resolution (3.5 µm) columns in the same variety of bondedphases. These kits contain some of the Eclipse Plus family of columns for excellent peak shapeand optimum performance with a wide variety of compounds.RRHT Method Development KitProvides Choices for Optimizing SelectivityEclipse Plus Phenyl-Hexyl959741-9122.1 x 50 mm, 1.8 µmEclipse Plus C18959741-9022.1 x 50 mm, 1.8 µmBonus-RP827768-9012.1 x 50 mm, 1.8 µmmAU201000.5 1 1.5 2 2.5 3 3.5 minmAU201000.5 1.5 2 2.5 3 3.5 minmAU201000.5 1 1.5 2 2.5 3 3.5 minRRHT_MDEVOnly the Eclipse Plus Phenyl-Hexyl can resolve all compounds.68 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX Method Development KitsRecommended for use with the Agilent AutomatedMethod Development LC SystemDescriptionRapid Resolution HT (RRHT) Selectivity Method Development Kit, 2.1 mm IDIncludes 2.1 x 50 mm, 1.8 µm, 600 bar columns: one each Eclipse Plus C18,Eclipse Plus Phenyl-Hexyl and Bonus-RPRapid Resolution HT (RRHT) pH Method Development Kit, 2.1 mm IDIncludes 2.1 x 50 mm, 1.8 µm, 600 bar columns: one each Eclipse Plus C18,SB-C18 and Extend-C18Rapid Resolution HT (RRHT) Selectivity Method Development Kit, 4.6 mm IDIncludes 4.6 x 50 mm, 1.8 µm, 600 bar columns: one each Eclipse Plus C18,Eclipse Plus Phenyl-Hexyl and Bonus-RPRapid Resolution HT (RRHT) pH Method Development Kit, 4.6 mm IDIncludes 4.6 x 50 mm, 1.8 µm, 600 bar columns: one each Eclipse Plus C18,SB-C18 and Extend-C18Rapid Resolution Selectivity Method Development Kit, 4.6 mm IDIncludes 4.6 x 100 mm, 3.5 µm columns: one each Eclipse Plus C18,Eclipse Plus Phenyl-Hexyl and Bonus-RPRapid Resolution pH Method Development Kit, 4.6 mm IDIncludes 4.6 x 100 mm, 3.5 µm columns: one each Eclipse Plus C18, SB-C18and Extend-C18Part No.5190-14315190-14325190-14335190-14345190-14355190-1436Order online at www.agilent.com/chem/store69

Columns for Analytical HPLCZORBAX Method Validation KitsZORBAX Method Validation Kits are supplied to customers who need the same HPLCcolumn type (bonded phase, particle size, configuration) but from different manufacturinglots. To request columns from different lots, contact Agilent Technologies or your localAgilent Authorized Distributor using the following procedure.• Request Validation Kits (columns from different lots) by using Part Number 899999-888• Indicate the Part Number of the current column you are using• Indicate the Lot Number of the current column you are using• Indicate the number of additional columns needed from different lots (example: you havea current column and may need two additional lots)• Please fax your request to (302) 993-5354 or email to custom_columns@agilent.com.You will receive a quote from your Customer Service Agent within 1-2 business days.Delivery for your custom column is usually 3 weeks or less from the time your order isplaced, depending on lot availability.Custom HPLC Column OrderingColumns not listed can easily be ordered by the following procedure:Tips & ToolsRequest custom LC columns online atwww.agilent.com/chem/customlccol• Request a Special Products Quotation (SPQ) using Part Number 899999-999• Indicate column dimensions (example: 4.6 x 50 mm); bonded phase type (example:StableBond C3); particle size (example: 5 µm); and pore size (example: 80Å)• Please fax your request to (302) 993-5354 or email to custom_columns@agilent.com.You will receive a quote from your Customer Service Agent within 1-2 business days.Delivery for your custom column is usually 3 weeks or less from the time your order isplaced, depending on lot availability.Custom columns are priced with a minimal surcharge over the price of stocked columns.70 Order online at www.agilent.com/chem/store

Columns for Analytical HPLCZORBAX Ion Exchange Columns –SAX and SCX• ZORBAX SAX and 300SCX columns are based on rugged ZORBAX silica• Stable from pH 2-7• Provide high efficiency, rapid separations• Compatible with organic mobile phase modifiersAgilent ZORBAX Strong Ion Exchange columns are available as both Strong Anion Exchange(SAX) and Strong Cation Exchange (300SCX) columns. Each column is packed with bonded,5 µm, spherical silica particles for optimum efficiency.ZORBAX SAX packing has a permanently bonded quaternary amine. A trifunctional organosilanereagent is used in producing this packing to maximize its stability with aqueous mobilephases. This column is ideal for separation of water-soluble compounds such as aromaticand aliphatic carboxylic acids and sulfonic acids.ZORBAX SCX packing has 300Å pore size silica particles chemically bonded to an aromaticsulfonic acid group. This column is used for separations of basic, water-soluble compoundsand bio-molecules.Column SpecificationsBondedPhase Pore Size Surface Area pH Range FunctionalityMaxPressureZORBAX SAX 70Å 300 m 2 /g 2.0-7.0 Quaternary 350 baramineZORBAX300SCX300Å 50 m 2 /g 2.0-7.0 Sulfonic acid 350 barSpecifications represent typical values only.Order online at www.agilent.com/chem/store71

Columns for Analytical HPLCCough-Cold Remedies - ZORBAX 300SCX1Column:ZORBAX 300SCX880952-7044.6 x mm, 5 µmMobile Phase: 100 mM NaH 2 PO 4 (pH 6.5)Flow Rate: 3 mL/minTemperature: 20° CDetector: 210 nmSample:Cold remedies23 451. Pyrilamine2. Theophylline3. Glyceryl Guaicolate4. Caffeine5. Phenylephrine0 2 4Time (min)LCIE001ZORBAX Ion Exchange Columns – SAX and SCXDescriptionSize (mm)Particle Size(µm) SAX 300SCXSemi-preparative 9.4 x 250 5 880952-203 880952-204Analytical 4.6 x 250 5 880952-703 880952-704Analytical 4.6 x 150 5 883952-703 883952-704Analytical 4.6 x 50 5 846952-704Solvent Saver 3.0 x 50 5 860700-304Narrow Bore 2.1 x 150 5 883700-704Narrow Bore 2.1 x 50 5 860700-704Guard Cartridge, 4/pk 4.6 x 12.5 6 820950-903 820950-904Guard Hardware Kit 820888-901 820888-90172 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsReproducible results from high-throughput,to capillary, to prep.No matter how many samples you have – or how complex they may be – you need to feelconfident that you can achieve reproducible results without wasting valuable time testingdifferent columns and configurations.The following column families deliver industry-leading performance for specific measurementand purification challenges:• NEW ZORBAX Rapid Resolution High Definition (RRHD) 1.8 µm columns – achieve stabilityup to 1200 bar for use with the Agilent 1290 Infinity LC. Available in 50, 100 and 150 mmlengths for fast or high resolution separations of your most complex samples.• EXPANDED! ZORBAX Rapid Resolution (3.5 µm) and Rapid Resolution High Throughput(1.8 µm) columns – enhance your productivity by delivering fast analysis withoutcompromising resolution. Available in Eclipse Plus, Eclipse XDB, StableBond and otherZORBAX bonded phases.• ZORBAX Solvent Saver columns – reduce solvent usage by 50% over 4.6 mm ID columns,and are compatible with most conventional HPLC instruments and LC/MS detectors. Idealfor cost-effective analysis.• Agilent and ZORBAX preparative columns, preparative cartridge columns, and guards –are engineered for high purity, recovery, and throughput. They feature a broad range ofselectivities, and are available in a variety of bonded phases for exceptional methoddevelopment flexibility.• Ultron ES Chiral columns with two complementary protein-based chiral stationaryphases – are an excellent choice for enantiomeric separations. Ideal for manypharmacological applications.Order online at www.agilent.com/chem/store73

Columns for Special ApplicationsZORBAX Rapid ResolutionHigh Definition 1.8 µm• High pressure (1200 bar) columns for optimum results with the 1290 Infinity LC or otherUHPLC instruments• 1.8 µm particles deliver maximum resolution for the most defined separations• Available in ZORBAX Eclipse Plus C18 for superior peak shape and ZORBAX StableBondC18 for alternate selectivity and low pH stability• Achieve the same selectivity on the 3.5 and 5 µm ZORBAX columns with the same bondedphase for compatibility with any LCZORBAX Rapid Resolution High Definition (RRHD) columns are an expansion of the ZORBAX1.8 µm particle column line. The new RRHD columns use improved packing processes toachieve stability up to 1200 bar for use with the Agilent 1290 Infinity LC or other UHPLCinstruments. The RRHD 1.8 µm columns are available in 50, 100 and 150 mm lengths for fastor high resolution – truly high definition – separations of your most complex samples.Column SpecificationsBonded Phase Pore Size Surface Area pH Range EndcappedZORBAX Eclipse Plus C18 95Å 160 m 2 /g 2.0-9.0 YesZORBAX SB-C18 80Å 180 m 2 /g 1.0-8.0* NoZORBAX SB-C8 80Å 180 m 2 /g 1.8-8.0* No*StableBond columns are designed for optimal use at low pH. At pH 6-8 highest column stability for all silica based columnsis obtained by operating at temperatures

Columns for Special ApplicationsSeparation of Licorice Root on RRHD ColumnsColumn A: ZORBAX RRHD SB-C18857700-9022.1 x 50 mm, 1.8 µmColumn B: 858700-9022.1 x 100 mm, 1.8 µmColumn C: 859700-9022.1 x 150 mm, 1.8 µmMobile Phase:Flow Rate:Gradient:Temperature:Detector:Instrument:10-100% B /30 minA: 0.1% formic acid (fa)B: acetonitrile with 0.1% faF = 0.4 mL/min30 minute gradient on each lengthAmbient280 nm UV1290 Infinity LCAmAU20015010050007 peaks50 mmRs: 0242 4 6 8 10 12 14 16 18 min100 mmB8 peaksRs: 1.37mAU20015010050002 4 6 8 10 12 14 16 1824min150 mmC9 peaksRs: 2.40mAU20015010050004 62 4 6 8 10 12 14 16 182minRRHD_LicoriceOrder online at www.agilent.com/chem/store75

Columns for Special ApplicationsSub 1 Minute Separations with RRHD ColumnsColumn:Gradient:ZORBAX RRHD SB-C18857700-9022.1 x 50 mm, 1.8 µmH 2 O (0.05% trifluoroacetic acid)/10-40% ACN/1minTemperature: 60°CInjection Volume:Detector Wavelength:Data Rate:0.5 µL x 100 ppm each275 nm160 HzmAU350040030016 Compound Biocide Mix in 0.5 minF = 2.0 mL/minP = 975 bar200100026540 .05 .1 .15 .2 .25 .3 .35 .4 .450.5 minmin1. 2-methyl-4-isothiazolin-3-one2. 5-chloro-2-methyl-4-isothiazolin-3-one3. Carbendazim4. Benzisothiiazol-3(2H)-one5. 2-phenoxyethanol6. MethylparabenRRHDSub1mRapid Resolution HD Columns for High Pressure Use(Maximum Pressure: 1200 bar)DescriptionSolvent Saver HD,1200 barSolvent Saver HD,1200 barNarrow Bore RRHD,1200 barNarrow Bore RRHD,1200 barNarrow Bore RRHD,1200 barSize (mm)ParticleSize (µm)EclipsePlus C18USP L1SB-C18USP L13.0 x 100 1.8 959758-302 858700-3023.0 x 50 1.8 959757-302 857700-302SB-C8USP L72.1 x 150 1.8 959759-902 859700-902 859700-9062.1 x 100 1.8 959758-902 858700-902 858700-9062.1 x 50 1.8 959757-902 857700-902 857700-90676 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsZORBAX Rapid ResolutionHigh Throughput 1.8 µm• High pressure (600 bar) columns for ultra high speed or maximum resolution analyseswith Rapid Resolution HT columns packed with totally porous, 1.8 µm packings• Carefully engineered particles deliver maximum resolution at 25% less pressure thanother sub 2-micron materials• Reduce analysis time by up to 95%• Develop HPLC methods more quickly• Securely transfer conventional methods with over 140 RRHT column choices• Analyze complex samples on shorter columns faster and maximize peak capacity• Matching selectivity in 3.5, 5 and 7 µm particle sizes for complete method scalability• Short (50 mm long and less) column can be used on some conventional LCsAgilent ZORBAX Rapid Resolution HT (1.8 µm) columns use a totally porous, 1.8 µm particleto provide maximum resolution in fast, ultra-fast and high resolution analyses. You can reduceanalysis time by up to 95% in comparison to 250 mm length columns. With more than140 RRHT column choices, including the new high performance ZORBAX Eclipse Plus andmany other ZORBAX column choices (Eclipse XDB, StableBond, Extend, Bonus-RP), methodscan be developed quickly or securely transferred to a smaller particle size column with noloss in resolution. The small particle size provides double the efficiency of a 3.5 µm columnin the same column length providing the highest efficiency and resolution possible. Thispermits the analysis of complex samples on shorter column with the highest resolution andpeak capacity. The 1.8 µm Rapid Resolution HT columns take high-speed, high-resolutionHPLC to a new level.The 600 bar columns can be used with the Agilent 1200 Rapid Resolution LC up to this highpressure limit. In addition, the shorter columns can be used on many other LC's, including theAgilent 1200 and 1100 by using the RRHT-1100 conversion kits to maximize performance.Order online at www.agilent.com/chem/store77

Columns for Special Applications1100 Series Conversion Kits for Fast LCThese kits make it easy to convert your Agilent 1100 system with a binary pump to alower-volume system for RRHT LC columns. Each kit contains all capillaries, a flow cell,starter columns, and detailed instructions for system conversion. Note: you will still be ableto use your converted 1100 for standard methods and columns.Kit Selection Description Part No.For Variable WavelengthDetectors (VWD)For Diode Array Detectors(DAD & DAD SL) and MultipleWavelength Detectors (MWD)For Diode Array Detectorand Mass SpecColumns: 4.6 x 50 mm, 1.8 µm (3)Flow Cell for VWD, 5 µl capillaries, µ-LC inline filterColumns: 4.6 x 50 mm, 1.8 µm (2)Flow Cell for DAD, 5 µl capillaries, µ-LC inline filterColumns: 2.1 x 50 mm, 1.8 µm (2)Flow Cell for DAD, 1.7 µl capillaries, ZDV union5188-53235188-53245188-5328Rapid Resolution HT: Up to 20X FasterColumn A: ZORBAX SB-C18883975-9024.6 x 150 mm, 5 µmColumn B: ZORBAX SB-C18827700-9022.1 x 50 mm, 1.8 µmColumn C: ZORBAX SB-C18827700-9022.1 x 50 mm, 1.8 µmMobile Phase: A: H 2 OB: ACNGradient: 0.0 min 50% BA: 11 min 100%BB: 1.2 min 100%BC: 0.4 min 100%BFlow Rate: A: 1.2 mL/minB: 1.0 mL/minC: 2.4 mL/minTemperature: A: 40°CB: 40°CC: 95°CDetector: UV 254 nmSample:Alkylphenones11. C 3 -AlkylphenoneA2435 67 8 92.3.C 4 -AlkylphenoneC 5 -Alkylphenone4. C 6 -Alkylphenone5. C 7 -Alkylphenone40 2 4 6 8 10Time (min)6.7.C 8 -AlkylphenoneC 9 -AlkylphenoneB128. C 10 -Alkylphenone35 69. C 12 -Alkylphenone7 8 90 0.2 0.4 0.6 0.8 1.0Time (min)41 2C35 67 8 90 0.2 0.4Time (min)LCRR00178 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsRapid Resolution HT Provides Doublethe Efficiency of Rapid Resolution ColumnsColumn A:Column B:Mobile Phase:Flow Rate:Temperature:Detector:ZORBAX SB-C18835975-9024.6 x 50 mm, 3.5 µmZORBAX SB-C18827975-9024.6 x 50 mm, 1.8 µm25% Water, 75% MeOH1.5 mL/minAmbient254 nmThis figure shows that Rapid Resolution HTcolumns can provide double the efficiencyof a 3.5 µm column in the same column length.This high efficiency can be used for veryhigh-resolution, high throughput analyses.A012Plates (N)1. 34762. 45853. 56734. 61803Time (min)41B1. Uracil2. Phenol3. 4-CI-Nitrobenzene4. Toluene0.012Plates (N)1. 65602. 89583. 115084. 1226630.5Time (min)41.0LCRR002Increase Peak Capacity with RRHT ColumnsColumn A: Eclipse XDB-C8928700-9062.1 x 100 mm, 1.8 µmColumn B: Eclipse XDB-C18961753-9022.1 x 100 mm, 3.5 µmMobile Phase: A: H 2 OB: ACNPeak capacity: A: 461B: 343Flow Rate: 0.5 mL/minGradient: 0.0 min 50% B20.0 min 100% BTemperature: 40°CDetector: UV 254 nmSample:AlkylphenonesAmAU120100806040200BmAU100806040200110 2 4 6 8 10 12 14 16 18Time (min)11. Uracil2. C 3 -Alkylphenone3. C 4 -Alkylphenone4. C 5 -Alkylphenone5. C 6 -Alkylphenone0 2 4 6 8 10 12 14 16 18Time (min)116. C 7 -Alkylphenone7. C 8 -Alkylphenone8. C 9 -Alkylphenone9. C 10 -Alkylphenone10. C 12 -Alkylphenone11. C 14 -Alkylphenone11LCRR004Order online at www.agilent.com/chem/store79

Columns for Special ApplicationsReduce Analysis Time Dramaticallywith Rapid Resolution HT ColumnsColumn A: Eclipse XDB-C18990967-9024.6 x 250 mm, 5 µmColumn B: Eclipse XDB-C18963967-9024.6 x 150 mm, 3.5 µmColumn C: Eclipse XDB-C18966967-9024.6 x 75 mm, 3.5 µmColumn D: ZORBAX Eclipse XDB-C18935967-9024.6 x 50 mm, 3.5 µmColumn E: Eclipse XDB-C18925975-9024.6 x 50 mm, 1.8 µmMobile Phase: 73% MeOH: 27% 20 mMPhosphate Buffer, pH 7.0Flow Rate: 1 mL/minTemperature: AmbientDetector: 254 nmA12 34567BCDE11231 52 3 41 523 4254667736745671. Uracil2. Naproxen3. Mefanamic Acid4. Butyl Paraben5. Propranolol6. Napthalene7. Dipropyl Phthalate0 2 4 6 8 1012Time (min)LCRR003This figure shows the dramatic reduction in analysis time possible by using Rapid Resolution HT columns. Chromatogram A shows a separation that takes 11.5 minuteson a 25 cm, 5 µm column. Rapid Resolution (3.5 µm) columns, shown in chromatogram B and C, reduce analysis time substantially, but with a slight compromise inresolution. The Rapid Resolution HT column reduces analysis time to 2.2 minutes, an 80% reduction, while still maintaining baseline resolution.80 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsLong Lifetime of RRHT Columns at Elevated TemperaturesColumn: ZORBAX SB-C18827700-9022.1 x 50 mm, 1.8 µmMobile Phase: A: 60% H 2 OB: 40% ACNFlow Rate: 1 mL/minTemperature: 80°CDetector: UV 254 nmSample:QC Test MixPlates1200010000800060004000200001499718853988115313051506164117761911204621812319250826432778291330853220346836033738388740544189432445374756489250335201Injection NumberLCRR005Comparison of Efficiencies – RRHT (1.8 µm)and Rapid Resolution (3.5 µm) ColumnsColumn LengthResolving PowerN (3.5 µm)*Resolving PowerN (1.8 µm)High Resolution150 21,000 32,500100 14,000 24,00075 10,500 17,000**Ultra Fast50 7000 12,00030 4200 600020 — 350015 2100 2500Resolution α N 1/2*5 µm HPLC columns of the same length have 40% fewer plates (N-value); 4.6 mm ID**Available as a custom columnOrder online at www.agilent.com/chem/store81

Columns for Special ApplicationsRapid Resolution HT Columns for High Pressure Use (Maximum Pressure: 600 bar, 9000 psi)DescriptionRapid Resolution HT,600 barRapid Resolution HT,600 barRapid Resolution HT,600 barRapid Resolution HT,600 barRapid Resolution HT,600 barRapid Resolution HT,600 barSolvent Saver HT,600 barSolvent Saver HT,600 barSolvent Saver HT,600 barSolvent Saver HT,600 barNarrow Bore RRHT,600 barNarrow Bore RRHT,600 barNarrow Bore RRHT,600 barNarrow Bore RRHT,600 barNarrow Bore RRHT,600 barSize(mm)ParticleSize (µm)EclipsePlus C18USP L14.6 x 150 1.8 959994-902EclipsePlus C8USP L7Eclipse PlusPhenyl HexylUSP L11EclipsePAHUSP L1EclipseXDB-C18USP L1EclipseXDB-C8USP L7Extend-C18USP L14.6 x 100 1.8 959964-902 959964-906 959964-912 959964-918 928975-902 728975-9024.6 x 75 1.8 959951-9024.6 x 50 1.8 959941-902 959941-906 959941-912 959941-918 927975-902 927975-906 727975-9024.6 x 30 1.8 959931-902 959931-906 959931-912 959931-918 924975-902 924975-906 724975-9024.6 x 20 1.8 926975-902 926975-906 726975-9023.0 x 100 1.8 959964-302 959964-306 959964-312 928975-302 728975-3023.0 x 50 1.8 959941-302 959941-306 959941-312 927975-302 927975-306 727975-3023.0 x 30 1.8 924975-302 924975-306 724975-3023.0 x 20 1.8 926975-302 926975-306 726975-3022.1 x 150 1.8 959794-9022.1 x 100 1.8 959764-902 959764-906 959764-912 959764-918 928700-902 928700-906 728700-9022.1 x 50 1.8 959741-902 959741-906 959741-912 959741-918 927700-902 927700-906 727700-9022.1 x 30 1.8 959731-902 959731-906 959731-912 924700-902 924700-906 724700-9022.1 x 20 1.8 926700-902 926700-906 726700-90282 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsRapid Resolution HT Columns for High Pressure Use (Maximum Pressure: 600 bar, 9000 psi)DescriptionRapid Resolution HT,600 barRapid Resolution HT,600 barRapid Resolution HT,600 barRapid Resolution HT,600 barRapid Resolution HT,600 barRapid Resolution HT,600 barSolvent Saver HT,600 barSolvent Saver HT,600 barSolvent Saver HT,600 barSolvent Saver HT,600 barSolvent Saver HT,600 barNarrow Bore RRHT,600 barNarrow Bore RRHT,600 barNarrow Bore RRHT,600 barNarrow Bore RRHT,600 barNarrow Bore RRHT,600 barSize (mm)*Also available in SB-C3, part number 828975-309Particle SB-C18Size (µm) USP L1SB-C8USP L7SB-PhenylUSP L11SB-CNUSP L10SB-Aq4.6 x 150 1.8 829975-902 829975-906 829975-912 829975-905 829975-914Rx-SILUSP L3Bonus-RPUSP L604.6 x 100 1.8 828975-902 828975-906 828975-912 828975-905 828975-914 828975-901 828668-9014.6 x 75 1.8 830975-906 830668-9014.6 x 50 1.8 827975-902 827975-906 827975-912 827975-905 827975-914 827975-901 827668-9014.6 x 30 1.8 824975-902 824975-906 824975-912 824975-905 824975-9144.6 x 20 1.8 826975-902 826975-9063.0 x 150 1.8 829975-302 829975-306 829975-312 829975-3053.0 x 100* 1.8 828975-302 828975-306 828975-312 828975-305 828975-314 828975-301 828668-3013.0 x 50 1.8 827975-302 827975-306 827975-312 827975-305 827975-314 827975-301 827668-3013.0 x 30 1.8 824975-302 824975-306 824975-3053.0 x 20 1.8 826975-302 826975-3062.1 x 150 1.8 820700-902 820700-906 820700-912 820700-9052.1 x 100 1.8 828700-902 828700-906 828700-912 828700-905 828700-914 828700-901 828768-9012.1 x 50 1.8 827700-902 827700-906 827700-912 827700-905 827700-914 827700-901 827768-9012.1 x 30 1.8 824700-902 824700-906 824700-912 824700-905 824700-9142.1 x 20 1.8 826700-902 826700-906Order online at www.agilent.com/chem/store83

Columns for Special ApplicationsRapid Resolution HT Columns and Cartridges (Maximum Pressure: 400 bar, 6000 psi)Hardware DescriptionSize (mm)ParticleSize (µm)EclipseXDB-C18USP L1EclipseXDB-C8USP L7SB-C18USP L1SB-C8USP L7Extend-C18USP L1Rapid Resolution HT 4.6 x 50 1.8 922975-902 922975-906 922975-902 822975-906 722975-902Rapid Resolution HT, 3/pk 4.6 x 50 1.8 922975-932 922975-932Narrow Bore RRHT 2.1 x 50 1.8 922700-902 922700-902Narrow Bore RRHT, 3/pk 2.1 x 50 1.8 922700-932 922700-932Rapid Resolution HT Cartridges (require hardware kit 820555-901)Rapid Resolution HT Cartridge 4.6 x 50 1.8 925975-902 825975-902Rapid Resolution HT Cartridge, 4.6 x 50 1.8 925975-932 825975-9323/pkRapid Resolution HT Cartridge 2.1 x 50 1.8 925700-902 825700-902Rapid Resolution HT Cartridge, 2.1 x 50 1.8 925700-932 825700-9323/pkRapid Resolution HT Cartridge 4.6 x 30 1.8 923975-902 823975-902Rapid Resolution HT Cartridge, 4.6 x 30 1.8 923975-932 823975-9323/pkRapid Resolution HT Cartridge 2.1 x 30 1.8 923700-902 823700-902Rapid Resolution HT Cartridge, 2.1 x 30 1.8 923700-932 823700-9323/pkRapid Resolution HT Cartridge 4.6 x 15 1.8 921975-902 821975-902Rapid Resolution HT Cartridge, 4.6 x 15 1.8 921975-932 821975-9323/pkRapid Resolution HT Cartridge 2.1 x 15 1.8 921700-902 821700-902Rapid Resolution HT Cartridge, 2.1 x 15 1.8 921700-932 821700-9323/pkHardware Kit for RR and RRHT Cartridges 820555-901 820555-90184 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsZORBAX Rapid Resolution 3.5 µm• Ideal choice for initial method development• 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) ID• Comparable lifetime to 5 µm columnsAgilent ZORBAX Rapid Resolution columns, with a 3.5 µm particle size, reduce analysistime and increase sample throughput for any application when compared to 5 µm columns.Rapid Resolution columns are available from 15-150 mm, in 1-4.6 mm IDs, so the bestconfigurations are available for high throughput, LC/MS, combinatorial chemistry and rapidanalytical applications. Rapid Resolution 3.5 µm particles have superior mechanical strength,so every column has an extremely stable packed bed and provides a comparable lifetimeto 5 µm columns. These are available in most ZORBAX bonded phases.Rapid Resolution Columns Reduce Analysis TimeWhile Maintaining ResolutionColumn A: ZORBAX Bonus-RP883668-9014.6 x 150 mm, 5 µmColumn B: ZORBAX Bonus-RP866668-9014.6 x 75 mm, 3.5 µmColumn C: ZORBAX Bonus-RP861700-9012.1 x 50 mm, 3.5 µmMobile Phase: 80% 25 mM NH 4 Ac, pH 5.520% ACNFlow Rate: 1.5 mL/min1.5 mL/min0.3 mL/minTemperature: 40°CDetector: 254 nm1. Caffeine2. Doxylamine3. ChlorpheniramineA1 4. Triprolidine4230 1 2 3 4 5 6Time (min)B 1C1422334Rapid Resolution 3.5 µm columns are available in narrow bore configurations forgreat compatibility with LC/MS.01 2 3Time (min)01 2Time (min)LC3_501Order online at www.agilent.com/chem/store85

Columns for Special ApplicationsRun Method Development Chromatogramson Rapid Resolution ColumnsColumn: ZORBAX SB-C18866953-9024.6 x 75 mm, 3.5 µmMobile Phase: A: 25 mM NaH 2 PO 4 , pH 3.0B: MeOHA: 20:80B: 30:70C: 40:60Flow Rate: 2.0 mL/minTemperature: 35°CDetector: 254 nm1. Diltiazem2. Dipyridamole3. Nifedipine4. Lidoflazine5. FlunarizineA B C112 3 52 3 42 3 4150.00.51.0 1.5 2.0 2.5 3.0Time (min)0.00.51.0 1.5 2.0 2.5 3.0Time (min)0.00.51.0 1.5 2.0 2.5 3.0Time (min)LC3_502Rapid Resolution 4.6 x 75 mm, 3.5 µm columns are a good choice for initial method development because they allow you to quickly determine if a given column and mobilephase combination can produce the desired separation.ZORBAX Rapid Resolution 3.5 µm Eclipse Plus ColumnsHardware Description*RR: Rapid Resolution 3.5 µmSize (mm)Particle Size(µm)EclipsePlus C18USP L1EclipsePlus C8USP L7Eclipse PlusPhenyl HexylUSP L11EclipsePAHUSP L1Rapid Resolution 4.6 x 150 3.5 959963-902 959963-906 959963-912 959963-918Rapid Resolution 4.6 x 100 3.5 959961-902 959961-906 959961-912 959961-918Rapid Resolution 4.6 x 75 3.5 959933-902 959933-906 959933-912Rapid Resolution 4.6 x 50 3.5 959943-902 959943-906 959943-912 959943-918Rapid Resolution 4.6 x 30 3.5 959936-902 959936-906 959936-912Solvent Saver Plus 3.0 x 150 3.5 959963-302 959963-306 959963-312Solvent Saver Plus 3.0 x 100 3.5 959961-302 959961-306 959961-312Narrow Bore RR* 2.1 x 150 3.5 959763-902 959763-902 959763-912Narrow Bore RR* 2.1 x 100 3.5 959793-902 959793-906 959793-912 959793-918Narrow Bore RR* 2.1 x 50 3.5 959743-902 959743-906 959743-912Narrow Bore RR* 2.1 x 30 3.5 959733-902 959733-906 959733-912Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-936 820950-937Guard Cartridges, 4/pk 2.1 x 12.5 5 821125-936 821125-937Guard Hardware Kit 820888-901 820888-90186 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsZORBAX Rapid Resolution 3.5 µm Eclipse XDB ColumnsHardware DescriptionSize (mm)Particle Size(µm)EclipseXDB-C18USP L1EclipseXDB-C8USP L7EclipseXDB-PhenylUSP L11EclipseXDB-CNUSP L10Rapid Resolution 4.6 x 150 3.5 963967-902 963967-906 963967-912 963967-905Rapid Resolution 4.6 x 100 3.5 961967-902 961967-906 961967-905Rapid Resolution 4.6 x 75 3.5 966967-902 966967-906 966967-912 966967-905Rapid Resolution 4.6 x 50 3.5 935967-902 935967-906 935967-912Rapid Resolution 4.6 x 30 3.5 934967-902 934967-906Rapid Resolution 4.6 x 20 3.5 932967-902 932967-906Solvent Saver Plus 3.0 x 150 3.5 963954-302 963954-306 963954-312 963954-305Solvent Saver Plus 3.0 x 100 3.5 961967-302 961967-306 961967-312Solvent Saver Plus 3.0 x 75 3.5 966954-302Narrow Bore RR* 2.1 x 150 3.5 930990-902 930990-906Narrow Bore RR* 2.1 x 100 3.5 961753-902 961753-906 961753-905Narrow Bore RR* 2.1 x 75 3.5 966735-902Narrow Bore RR* 2.1 x 50 3.5 971700-902 971700-906Narrow Bore RR* 2.1 x 30 3.5 974700-902 974700-906Narrow Bore RR* 2.1 x 20 3.5 972700-902 972700-906MicroBore RR* 1.0 x 150 3.5 963600-902 963600-906MicroBore RR* 1.0 x 50 3.5 965600-902 965600-906MicroBore RR* 1.0 x 30 3.5 961600-902 961600-906MicroBore Guard, 3/pk 1.0 x 17 5 5185-5921 5185-5921Guard Cartridges, 4/pk 4.6 x 12.5 5 820950-925 820950-926 820950-927 820950-935Guard Cartridges, 4/pk 2.1 x 12.5 5 821125-926 821125-926 821125-926 821125-935Guard Hardware Kit 820888-901 820888-901 820888-901 820888-901*RR: Rapid Resolution 3.5 µmOrder online at www.agilent.com/chem/store87

Columns for Special ApplicationsZORBAX Rapid Resolution 3.5 µm StableBond ColumnsHardware DescriptionSize (mm)Particle SB-C18Size (µm) USP L1SB-C8USP L7SB-CNUSP L10SB-PhenylUSP L11SB-C3USP L56Rapid Resolution 4.6 x 150 3.5 863953-902 863953-906 863953-905 863953-912 863953-914Rapid Resolution 4.6 x 100 3.5 861953-902 861953-906 861953-905 861953-912 861953-914Rapid Resolution 4.6 x 75 3.5 866953-902 866953-906 866953-905 866953-912 866953-914Rapid Resolution 4.6 x 50 3.5 835975-902 835975-906 835975-905 835975-912 835975-914Rapid Resolution 4.6 x 30 3.5 834975-902 834975-906Rapid Resolution 4.6 x 20 3.5 832975-902 832975-906Solvent Saver Plus 3.0 x 150 3.5 863954-302 863954-306 863954-305 863954-312 863954-314Solvent Saver Plus 3.0 x 100 3.5 861954-302 861954-306 861954-305 861954-312 861954-309 861954-314Solvent Saver Plus 3.0 x 75 3.5 866953-302Narrow Bore RR* 2.1 x 150 3.5 830990-902 830990-906 830990-914Narrow Bore RR* 2.1 x 100 3.5 861753-902 861753-906 861753-905 861753-912 861753-914Narrow Bore RR* 2.1 x 75 3.5 866735-902Narrow Bore RR* 2.1 x 50 3.5 871700-902 871700-906 871700-914Narrow Bore RR* 2.1 x 30 3.5 874700-902 874700-906Narrow Bore RR* 2.1 x 20 3.5 872700-902 872700-906MicroBore RR* 1.0 x 150 3.5 863600-902 863600-906MicroBore RR* 1.0 x 50 3.5 865600-902 865600-906MicroBore RR* 1.0 x 30 3.5 861600-902 861600-906MicroBore Guard, 3/pk 1.0 x 17 5 5185-5920 5185-5920Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-920 820950-915 820950-916 820950-917 820950-922 820950-933Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-915 821125-915 821125-924 821125-915 821125-924 821125-933Guard Hardware Kit 820888-901 820888-901 820888-901 820888-901 820888-901 820888-901*RR: Rapid Resolution 3.5 µmSB-Aq88 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsZORBAX Rapid Resolution 3.5 µm Rx, Bonus-RP, and Extend-C18 ColumnsHardware DescriptionSize (mm)Particle Size(µm)Rx-C18USP L1Rx-C8USP L7Bonus-RPUSP L60Extend-C18USP L1Rapid Resolution 4.6 x 250 3.5 884950-577Rapid Resolution 4.6 x 150 3.5 863967-902 863953-906 863668-901 763953-902Rapid Resolution 4.6 x 100 3.5 861967-902 861953-906 864668-901 764953-902Rapid Resolution 4.6 x 75 3.5 866967-902 866953-906 866668-901 766953-902Rapid Resolution 4.6 x 50 3.5 735953-902Solvent Saver Plus 3.0 x 150 3.5 863967-302 863954-306 863668-301 763954-302Solvent Saver Plus 3.0 x 100 3.5 861967-302 861954-306 864668-301 764953-302Narrow Bore RR* 2.1 x 150 3.5 830990-906 863700-901Narrow Bore RR* 2.1 x 100 3.5 861767-902 861753-906 861768-901 761753-902Narrow Bore RR* 2.1 x 50 3.5 861700-901 735700-902MicroBore RR* 1.0 x 150 3.5 863608-901 763600-902MicroBore RR* 1.0 x 50 3.5 865608-901 765600-902MicroBore RR* 1.0 x 30 3.5 861608-901 761600-902MicroBore Guard, 3/pk 1.0 x 17 5 5185-5922 5185-5923Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-914 820950-913 820950-928 820950-930Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-915 821125-915 821125-928 821125-930Guard Hardware Kit 820888-901 820888-901 820888-901 820888-901*RR: Rapid Resolution 3.5 µmOrder online at www.agilent.com/chem/store89

Columns for Special ApplicationsZORBAX Solvent Saver• Provide 60% reduction in mobile phase usage and waste generation comparedto a 4.6 mm ID column• Provide 2- to 3-fold signal-to-noise (S/N ratio) improvement• Deliver optimal LC/MS performance at intermediate flow rates• Can be used with most conventional LC instrument configurations without modification• Solvent Saver columns are available in 1.8, 3.5 and 5 µm particle sizesAgilent ZORBAX Solvent Saver columns have a 3.0 mm internal diameter. This is ideal forreducing solvent usage by 50% over 4.6 mm ID columns. Solvent Saver columns are alsoideal for LC/MS. With a typical flow rate of 0.5 ml/min, these columns are compatible withalmost all LC interfaces. These columns also improve sensitivity 2 to 3 times over 4.6 mm IDcolumns. Solvent Saver columns can be used with conventional HPLC instruments and area good choice for cost effective analyses.Solvent Saver Columns Provide up to 60% Reductionin Solvent Use and WasteColumn A:Column B:ZORBAX SB-C18883975-9024.6 x 150 mm, 5 µmZORBAX SB-C18883975-3023.0 x 150 mm, 5 µmMobile Phase: 20% ACN: 80% 0.2 M Na 2 HPO 4+ 0.1 M Citric Acid, pH 2.6Temperature: AmbientSample:AntibacterialsA1Less solventconsumption, less wasteB11. Sulfamerazine2. Furazolidone3. Oxolinic Acid4. Sulfadimethoxine5. Sulfaquinoxaline6. Nalidixic AcidThis separation of antibacterials on a 4.6 and3.0 mm ID columns, shows that solvent useis reduced by 50% just by changing to theSolvent Saver column with no change inthe chromatography. This reduces the costof analyses dramatically.23456234560Time (min)400Time (min)40LCSS00190 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsCombine RRHT and Solvent Saver for Maximum SolventReduction: Diet SodaColumn A: Eclipse Plus C18959993-9024.6 x 150 mm, 5 µmF = 1.0 mL/min (P = 96 bar)1. SaccharineColumn B: Solvent Saver HT, 600 bar2. CaffeineEclipse Plus C18959941-302A 1 11 mL/analysis 3. Aspartame3.0 x 50 mm, 1.8 µm4. Benzoic acidF = 0.43 (P = 181 bar)2Mobile Phase: A: 20 mM Phosphate34B: AcetonitrileA:B (79:21)2 4 6 8Temperature: 25°CDetector: UV 220 nm, 16, ref = 420, 20 B1.6 mL/analysisFlow Cell: 3 mm, 2 µLFlow Rate: 1 sInj. Vol 3 µL for 4.6, 1.5 for 3.0Instrument: 1200SL2 3 4Sample:0.2 µm filtered diet soda1085% less solventminRRHT_dietsodaSolvent Saver Columns are Ideal for LC/MSColumn: ZORBAX SB-C18861954-3023.0 x 100 mm, 3.5 µmMobile Phase: A: 70% Methanol+0.4% Formic AcidB: 30% Water+0.4% Formic AcidFlow Rate: 0.425 mL/minDetector: A: UV 254 nmB: Positive Ion APCIC: Positive Ion ElectrospraySample:SteroidsSolvent Saver columns are ideal for LC/MS because the typical0.5 mL/min flow rate allows samples to be evaluated andanalyzed without changing columns when the MS interface ischanged from electrospray to APCI.mAU14012010080604020035000000300000002500000020000000150000001000000050000000A123450 2 4 6 8 10 12 14 16Time (min)BLC-UVLC-APCI-MS1. Triamcinolone2. Hydrocortisone3. Cortisone acetate4. Deoxycorticosterone5. Hydroxyprogesterone6. Progesterone0 2 4 6 8 10 12 14 16Time (min)6CLC-ESI-MS25000000225000002000000017500000150000001000000075000000 2 4 6 8 10 12 14 16Time (min)LCSS003Order online at www.agilent.com/chem/store91

Columns for Special ApplicationsZORBAX Eclipse PlusDescription Size (mm) Particle Size (µm)EclipsePlus C18USP L1EclipsePlus C8USP L7Eclipse PlusPhenyl HexylUSP L11EclipsePAHUSP L1Solvent Saver 3.0 x 250 5 959990-318Solvent Saver 3.0 x 150 5 959993-302 959993-306Solvent Saver Plus 3.0 x 150 3.5 959963-302 959963-306 959963-312Solvent Saver Plus 3.0 x 100 3.5 959961-302 959961-306 959961-312Solvent Saver HT, 600 bar 3.0 x 100 1.8 959964-302 959964-306 959964-312Solvent Saver HT, 600 bar 3.0 x 50 1.8 959941-302 959941-306 959941-312ZORBAX 80Å Eclipse XDBDescription Size (mm) Particle Size (µm)EclipseXDB-C18USP L1EclipseXDB-C8USP L7EclipseXDB-PhenylUSP L11EclipseXDB-CNUSP L10Solvent Saver 3.0 x 250 5 990967-302 990967-306 990967-312 990967-305Solvent Saver 3.0 x 150 5 993967-302 993967-306 993967-312 993967-905Solvent Saver Plus 3.0 x 150 3.5 963954-302 963954-306 963954-312 963954-305Solvent Saver Plus 3.0 x 100 3.5 961967-302 961967-306 961967-312Solvent Saver Plus 3.0 x 75 3.5 966954-302Solvent Saver HT, 600 bar 3.0 x 100 1.8 928975-302 928975-306Solvent Saver HT, 600 bar 3.0 x 50 1.8 927975-302 927975-306Solvent Saver HT, 600 bar 3.0 x 30 1.8 924975-302 924975-306Solvent Saver HT, 600 bar 3.0 x 20 1.8 926975-302 926975-306ZORBAX 80Å StableBondDescriptionSize (mm)ParticleSize (µm)SB-C18USP L1SB-C8USP L7SB-CNUSP L10SB-C3USP L56SB-PhenylUSP L11Solvent Saver 3.0 x 250 5 880975-302 880975-306 880975-305 880975-309 880975-312 880975-314Solvent Saver 3.0 x 150 5 883975-302 883975-306 883975-305 883975-309 883975-312 883975-314Solvent Saver Plus 3.0 x 150 3.5 863954-302 863954-306 863954-305 863954-309 863954-312 863954-314Solvent Saver Plus 3.0 x 100 3.5 861954-302 861954-306 861954-305 861954-309 861954-312 861954-314Solvent Saver HD, 1200 bar 3.0 x 100 1.8 858700-302Solvent Saver HD, 1200 bar 3.0 x 50 1.8 857700-302Solvent Saver HT, 600 bar 3.0 x 150 1.8 829975-302 829975-306 829975-305 829975-312Solvent Saver HT, 600 bar 3.0 x 100 1.8 828975-302 828975-306 828975-305 828975-309 828975-312 828975-314Solvent Saver HT, 600 bar 3.0 x 50 1.8 827975-302 827975-306 827975-305Solvent Saver HT, 600 bar 3.0 x 30 1.8 824975-302 824975-306 824975-305 827975-312 827975-314Solvent Saver HT, 600 bar 3.0 x 20 1.8 826975-302 826975-306SB-Aq92 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsZORBAX 300Å StableBondDescription Size (mm) Particle Size (µm)300SB-C18USP L1300SB-C8USP L7300SB-CNUSP L10300SB-C3USP L56Solvent Saver Plus 3.0 x 150 3.5 863974-302 863974-306 863974-309 863974-309Solvent Saver Plus 3.0 x 100 3.5 861973-306Solvent Saver Plus 3.0 x 75 3.5 866953-302ZORBAX 80Å Bonus-RP and RxDescriptionSize (mm)ParticleSize (µm)Bonus-RPUSP L60Rx-C18USP L1Rx-C8USP L7Solvent Saver 3.0 x 250 5 880668-301 880967-302 880975-306Solvent Saver 3.0 x 150 5 883668-301 883967-302 883975-306Solvent Saver Plus 3.0 x 150 3.5 863668-301 863967-302 863954-306Solvent Saver Plus 3.0 x 100 3.5 864668-301 861967-302 861954-306Solvent Saver HT, 600 bar 3.0 x 100 1.8 828668-301Solvent Saver HT, 600 bar 3.0 x 50 1.8 827668-301ZORBAX 80Å Extend-C18Description Size (mm) Particle Size (µm)Extend-C18USP L1Solvent Saver 3.0 x 250 5 770450-302Solvent Saver 3.0 x 150 5 773450-302Solvent Saver Plus 3.0 x 150 3.5 763954-302Solvent Saver Plus 3.0 x 100 3.5 764953-302Solvent Saver Plus 3.0 x 50 3.5 735954-302Solvent Saver HT, 600 bar 3.0 x 100 1.8 728975-302Solvent Saver HT, 600 bar 3.0 x 50 1.8 727975-302Solvent Saver HT, 600 bar 3.0 x 30 1.8 724975-302Solvent Saver HT, 600 bar 3.0 x 20 1.8 726975-302Order online at www.agilent.com/chem/store93

Columns for Special ApplicationsZORBAX PrepHT• Easy scale-up from analytical to preparative scale with ZORBAX phases• Fast preparative separations, up to 2000 mg• 5 to 7 µm particles for high efficiency and high yield• Easy to install finger tight connections seal up to 5000 psi/350 barHigh purity, high recovery and high throughput can be easily achieved with AgilentZORBAX PrepHT columns. These are available in a variety of bonded phases – Eclipse XDB,StableBond, Bonus-RP, and Extend-C18 – for optimized resolution and loadability underany conditions.ZORBAX PrepHT columns are packed with 5 and 7 µm particle sizes for very high resolution.The high resolution allows high loadability, high yield, and high purity of compounds. Thelarger diameter columns and mechanically stronger ZORBAX particles allow for flow rates upto 100 ml/min, thus increasing throughput.ZORBAX PrepHT columns are designed for rapid scale-up from analytical to preparative scalewithout losing resolution. For complex separations on larger columns (21.2 mm ID, 150 mmlength and longer), Agilent has carefully chosen the 7 µm particle size to achieve a balancebetween high efficiency and high loadability.94 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsHigh Purity and High Recovery with ZORBAX PrepHT ColumnsSample:Antianginal drugsMass-based fraction collection using ZORBAXSB-C18 column shows high purity and highrecovery of each compound (Application Notepublication number 5988-7113EN). Theseparation of the three antianginal drugs wassuccessfully done in a single run with highrecovery and >90% purity. Separations up to2000 mg are possible depending on thecomplexity of separation.Abundance (mAU)DMSO1. Nifedipine2. Nimodine3. Nisoldipine123DAD80000000 1 2 3 4 5 6 7Time (min)Vial 2 – 343Vial 1 – 347Vial 3 – 389MSD6000000400000020000000 1 2 3 4 5 6 7Time (min)LCPHT01AmountNifedipin [mg]AmountNifmodipin [mg]AmountNifsoldipin [mg]Fraction 1 18.90 0.11 0.16 Purity Nifedipin 98.6%Fraction 2 0.29 17.66 0.77 Purity Nifmodipin 94.4%Fraction 3 0.49 1.66 18.36 Purity Nifsoldipin 89.5%Recovery [mg] 19.68 19.43 19.29Recovery [%] 101.3 102.0 101.9Order online at www.agilent.com/chem/store95

Columns for Special ApplicationsScale-Up from Analytical to Prep ZORBAX SB-C18 ColumnsUsing the Same PumpColumn SizeFlow(mL/min)Injection(µL) Detector Cell Part No.Column 1 50 x 150 mm 100 2200 0.3 mm quartz Custom ColumnColumn 2 21.2 x 150 mm 18 400 0.3 mm quartz 877150-102Column 3 9.4 x 150 mm 3.5 80 0.3 mm quartz 883975-202Column 4 4.6 x 150 mm 0.85 2.0 3 mm SST 883975-902Scale-Up to PrepHT1. Theobromine35003000232. Theophylline3. CaffeineAbundance (mAU)25002000150010005000Column 11Column 2Column 3Column 40 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0Time (min)LCPHT_ScaleUpUsing the same 1100 pump, a scale-up from 4.6 mm to 50 mm ID was possible without any loss ofresolution. This increases throughput by reducing the time required for redeveloping and adjustingthe method.96 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsZORBAX PrepHT 80ÅStableBond (require hardware 820400-901)Hardware Description Size (mm) Particle Size (µm)SB-C18USP L1SB-C8USP L7SB-AqSB-CNUSP L10SB-PhenylUSP L11PrepHT Cartridge 21.2 x 250 7 877250-102 877250-106 877250-114 877250-105 877250-112PrepHT Cartridge 21.2 x 150 7 877150-102 877150-106 877150-114PrepHT Cartridge 21.2 x 150 5 870150-902 870150-906 870150-914PrepHT Cartridge 21.2 x 100 5 870100-902 870100-906 870100-914PrepHT Cartridge 21.2 x 50 5 870050-902 870050-906 870050-914PrepHT Guard Cartridge, 2/pk 17 x 7.5 5 820212-920 820212-915 820212-933 820212-933 820212-915ZORBAX PrepHT 300ÅStableBond (require hardware 820400-901)Hardware Description Size (mm)300SB-C18Particle Size (µm) USP L1300SB-C8USP L7300SB-C3USP L56300SB-CNUSP L10PrepHT Cartridge 21.2 x 250 7 897250-102 897250-106 897250-109 897250-105PrepHT Cartridge 21.2 x 150 7 897150-102 897150-106 897150-109PrepHT Cartridge 21.2 x 150 5 895150-902 895150-906 895150-909PrepHT Cartridge 21.2 x 100 5 895100-902 895100-906 895100-909PrepHT Cartridge 21.2 x 50 5 895050-902 895050-906 895050-909PrepHT Guard Cartridge, 2/pk 17 x 7.5 5 820212-921 820212-918 820212-924 820212-924Guard Cartridge Hardware820444-901 820444-901 820444-901 820444-901Includes guard column end fitting, polymeric seal, and seal insertion tool(seal holder and seal pusher)PrepHT endfittings, 2/pk 820400-901 820400-901 820400-901 820400-901ZORBAX PrepHT Original (require hardware 820400-901)Hardware DescriptionSize (mm)ParticleSize (µm)ODS (C18)USP L1C8USP L7CNUSP L10NH2USP L8SILUSP L3PrepHT Cartridge 21.2 x 250 7 877952-102 877952-106 877952-105 877952-108 877952-101PrepHT endfittings, 2/pk 820400-901 820400-901 820400-901 820400-901 820400-901Order online at www.agilent.com/chem/store97

Columns for Special ApplicationsZORBAX PrepHT Eclipse XDB (require hardware 820400-901)EclipseXDB-C18USP L1EclipseXDB-C8USP L7Particle SizeHardware DescriptionSize (mm) (µm)PrepHT Cartridge 21.2 x 250 7 977250-102 977250-106PrepHT Cartridge 21.2 x 150 7 977150-102 977150-106PrepHT Cartridge 21.2 x 150 5 970150-902 970150-906PrepHT Cartridge 21.2 x 100 5 970100-902 970100-906PrepHT Cartridge 21.2 x 50 5 970050-902 970050-906PrepHT Guard Cartridge, 17 x 7.5 5 820212-925 820212-9262/pkGuard Cartridge Hardware820444-901 820444-901Includes guard column end fitting, polymeric seal,and seal insertion tool (seal holder and seal pusher)PrepHT endfittings, 2/pk 820400-901 820400-901ZORBAX PrepHT Bonus-RP and Extend-C18 (require hardware 820400-901)Hardware DescriptionSize (mm)Particle Size(µm)Bonus-RPUSP L60Extend-C18USP L1PrepHT Cartridge 21.2 x 250 7 878250-101PrepHT Cartridge 21.2 x 150 7 878150-101PrepHT Cartridge 21.2 x 150 5 868150-901 770150-902PrepHT Cartridge 21.2 x 100 5 868100-901 770100-902PrepHT Cartridge 21.2 x 50 5 868050-901 770050-902PrepHT Guard Cartridge, 17 x 7.5 5 820212-928 820212-9302/pkGuard Cartridge Hardware820444-901 820444-901Includes guard column end fitting, polymeric seal,and seal insertion tool (seal holder and seal pusher)PrepHT endfittings, 2/pk 820400-901 820400-90198 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsZORBAX PrepHT Rx-SIL (require hardware 820400-901)Hardware DescriptionSize (mm)Particle Size(µm)Rx-SILUSP L3Rx-C18USP L1PrepHT Cartridge 21.2 x 250 7 877250-101PrepHT Cartridge 21.2 x 250 7 877967-102PrepHT Guard Cartridge, 17 x 7.5 5 820212-919 820212-9142/pkGuard Cartridge Hardware820444-901 820444-901Includes guard column end fitting, polymeric seal,and seal insertion tool (seal holder and seal pusher)PrepHT endfittings, 2/pk 820400-901 820400-901ZORBAX PrepHT AccessoriesHardware DescriptionPart No.Guard Cartridge Hardware 820444-901PrepHT endfittings, 2/pk 820400-901Replacement Seals 820385-901Order online at www.agilent.com/chem/store99

Columns for Special ApplicationsAgilent Prep LC Columns• High loadability for maximum sample purification• Easy scalability from 4.6 mm ID up to 50 mm ID for rapid method development• High throughput 21.2 mm ID cartridges for fast purification• Exceptional column stability and loadability up to pH 10Agilent Prep LC columns are designed for high loadability to purify milligram to gramquantities of products. Preparative sized columns are available in 21.2, 30, and 50 mminternal diameters with lengths ranging from 50-250 mm. Columns are available in5 and 10 µm particle sizes with very high efficiency in every dimension. These column choicesaccommodate almost every preparative sample.Agilent Prep 21.2 mm ID columns are available with Agilent’s Preparative Cartridge Hardware.This reliable cartridge hardware makes it simple to use columns with different lengths toincrease sample load. Guard columns are easily integrated onto these columns providingsuperior protection of the analysis column. Analytical sized 4.6 mm ID scalar columns areavailable for method development and optimization prior to scaling up to larger columns.Bulk material is also available.Agilent Prep columns are available in a C18 bonded phase suitable for purification of a widevariety of non-polar and polar compounds. Unbonded silica columns are also available.Column SpecificationsBondedPhasePore SizeSurfaceArea Temp. Limits pH Range EndcappedCarbonLoadC18 100Å 400 m 2 /g 60°C* 2.0-10.0 Single 24%Silica 100Å 400 m 2 /g ** 1.0-8.0 N/A N/ASpecifications represent typical values only.*Temperature limits are 60°C up to pH 8, 40°C from pH 8-10.**Temperature limits for bare silica are determined by the pH of the mobile phase.100 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsSuperior Loadability on Agilent Prep C18with Basic CompoundsColumn: Agilent Prep C18443905-9024.6 x 150 mm, 5 µmMobile Phase: 50% 0.1%TFA:50% ACNFlow Rate: 1 mL/minSample: 10 µLDoxepin/Amitriptyline0.5-50 mg/mLAgilent Prep columns show better resolutionand loadability than competitor columns.4000350030002500200015001000500mAU00Agilent Prep C18mAUα = 1.64035α = 1.573025201510501 2 3 4 5 6 7 8 90Time (min)Competitor W-C181 2 3 4 5 6 7 8 9Time (min)LCPLC01Steroids: Easy Scalability Using Agilent Prep ColumnsColumn A: Agilent Prep C18443905-9024.6 x 150 mm, 5 µmColumn B: 443905-10221.2 x 150 mm, 5 µmColumn C: 413910-30230 x 150 mm, 10 µmColumn D: 413910-50250 x 150 mm, 10 µmMobile Phase: 55% Water:45% ACNFlow Rate: 0.7 mL/min14.87 mL/min29.77 mL/min85.37 mL/minTemperature: AmbientDetector: 240 nmSample: 2 µL42.4 µL170 µL488 µLAgilent Prep C18 shows excellent scalability making method transfersimple and predictable.2. Testosterone (in MeOH @ 1mg/mL)A 1 20 2 4 6 8 10 12B10 2 4 6 8 10 12C10 2 4 6 8 10 12D11. Hydrocortisone0 2 4 6 8 10 12Time (min)222LCPLC02Order online at www.agilent.com/chem/store101

Columns for Special ApplicationsAgilent Prep LC ColumnsHardware DescriptionParticle SizeSize (mm) (µm) C18 SilicaStandard Columns (no special hardware required)Scalar 4.6 x 250 10 440910-902 440910-901Scalar 4.6 x 150 10 443910-902 443910-901Scalar 4.6 x 100 10 449910-902Scalar 4.6 x 250 5 440905-902 440905-901Scalar 4.6 x 150 5 443905-902 443905-901Scalar 4.6 x 100 5 449905-902 449905-901Scalar 4.6 x 50 5 446905-902 446905-901PrepHT Cartridge Columns (require endfittings kit 820400-901)*PrepHT 21.2 x 250 10 410910-102 410910-101PrepHT 21.2 x 150 10 413910-102 413910-101PrepHT 21.2 x 50 10 446910-102PrepHT 21.2 x 150 5 443905-102 443905-101PrepHT 21.2 x 100 5 449905-102 449905-101PrepHT 21.2 x 50 5 446905-102 446905-101PrepHT endfittings, 2/pk 820400-901 820400-901Standard Columns (no special hardware required)Prep 30 30 x 250 10 410910-302 410910-301Prep 30 30 x 150 10 413910-302 413910-301Prep 30 30 x 100 10 419910-302 419910-301Prep 30 30 x 100 5 449905-302 449905-301Prep 30 30 x 50 5 446905-302 446905-301Prep 50 50 x 250 10 410910-502 410910-501Prep 50 50 x 150 10 413910-502 413910-501Prep 50 50 x 100 10 419910-502 419910-501Prep 50 50 x 100 5 449905-502 449905-501Guard Columns (hardware required)PrepHT Guard Cartridges, 21.2 x 10 10 420212-902 420212-9012/pkGuard Cartridge Hardware 820444-901 820444-901PrepHT External Guard Hardware Kit 420420-901 420420-901Bulk Packing (1kg) 10 420910-902 420910-901*All PrepHT cartridge columns require hardware kit 820400-901. If a guard column is desired for the 21.2 mm ID columnsthen the PrepHT Guard Hardware Kit, 820444-901, is also required. If the guard column is used on a 30 mm ID column thenthe external guard column hardware kit, 420420-901, is required.102 Order online at www.agilent.com/chem/store

Columns for Special ApplicationsUltron ES Chiral Columns• Direct racemic separations without derivatization• Use Ultron ES-OVM as the USP L57 choice and to separate enantiomers of acidicand basic pharmaceuticals, such as hexobarbital, ibuprofen, and profenamine• Ultron ES-Pepsin Chiral columns are best suited to separate basic compounds thatare difficult to separate with other chiral columns• ES-OVM and ES-Pepsin columns contain 120Å, 5 µm silica particles bonded with anovomucoid protein and pepsin protein, respectively• Both types of chiral columns are usable with reversed-phase mobile phases such asacetonitrile or ethanol and phosphate bufferUltron ES Chiral columns are immobilized protein columns that feature numerous chiralrecognition sites for enantiomeric separations of dozens of chiral compounds. They areengineered with two complementary protein-based chiral stationary phases, making theman excellent choice for the HPLC separation of enantiomers without derivatization – includinga growing number of drug substances of interest.Separation of Enantiomers of Fluoxetine (Prozac)Column: Ultron ES-OVM Chiral7021116514.6 x 150 mm, 5 µmMobile Phase: 25:75 (v/v) EtOH / 20 mM KH 2 PO 4 , pH 5.5(adjusted with NaOH)Temperature: AmbientDetector: UV (225 nm)Sample: Mixture Fluoxetine (Prozac) entantiomersCourtesy of D. S. Risley and V. S. Sharp of Lilly Research Laboratories, Eli Lilly and Co.0 510 15Retention Time (min)20 25 30LCUC001Order online at www.agilent.com/chem/store103

Columns for Special ApplicationsSeparation of Ethiazide (diuretic drug)on ULTRON ES-OVM ColumnColumn: Ultron ES-OVM Chiral7021116514.6 x 150 mm, 5 µmCIH 2 NSOOHNSOHCNHOC 2 H 5Mobile Phase: 20 mM KH 2 PO 4 (pH 4.6)Flow Rate: 1 mL/minTemperature: 25°CDetector: 220 nm1020Time (min)LCUC002Chiral Separation of Warfarin EnantiomersR and S Limit of Quantitation %RSD at 100 fg/mLColumn: Ultron ES-OVM Chiral7021116102.0 x 150 mm, 5 µmTemperature: 30°CInjection 5 µLVolume:Autosampler 10°CTemperature:Needle Wash: Flush port (50:25:25 H 2 O,IPA:MeOH:H 2 O, 5 seconds)Mobile Phase: 83% A = H 2 O + 5mMAmmonium Formate17% B = ACNFlow Rate: 0.5 mL/minStop time: 7.0 minMS Conditions: Agilent 6410A Triple QuadrupoleLC/MS/MS with MultiMode SourceIon Mode: ESI, Negativex10 25.505.255.004.754.504.003.753.503.253.002.752.502.001.751.501.251.000.750.500.250Source ConditionsCapillary Voltage: 2000 VDrying Gas (nitrogen): 5 L/minDrying Gas Temperature: 300°CNebulizer Gas (nitrogen): 40 psiVaporizer 200°CR %RSD = 3.6Product Ion ScanMass Range: 50-500 m/zScan Speed: 500 msecMRM acquisition (Q1 peak width =1.2 and Q2 peak width = 0.70 amu)Delta EMV: 1000VS %RSD = 4.93.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5.0 5.1Counts vs. Acquisition TimeWarfarinUltron ES Chiral ColumnsDescriptionSize (mm)Particle Size(µm)ES-OVMUSP L57ES-PepsinAnalytical 4.6 x 250 10 724111653Analytical 4.6 x 150 5 702111651 822111651Analytical, with Guard 4.6 x 150 5 702111651A 822111631ANarrow Bore 2.0 x 150 5 702111610Guard Column 4.0 x 10 5 712111630 832111630104 Order online at www.agilent.com/chem/store

Columns for Bioanalytical ChromatographyIs your lab ready for the ever-increasing number –and variety – of bioanalytical HPLC applications?Basic peptide separations. High-sensitivity, high-resolution amino acid analyses. Fast-sizeexclusion separations of antibodies. The number of bioanalytical HPLC applications continuesto grow at an unprecedented rate. And Agilent’s durable – and reproducible – line of ZORBAXcolumns can help you meet your lab’s evolving needs for performance and speed.On the following pages, you will find key facts and specifications about columns forreversed-phase HPLC, size exclusion chromatography, ion-exchange chromatography,and hydrophobic interaction chromatography, including:• Poroshell 300 columns – feature a unique, powerful particle for fast, high-resolutionseparations of proteins, polypeptides, and antibodies.• ZORBAX wide-pore 300StableBond columns – deliver long lifetime, high resolution,and good peak shape for peptides and proteins. And they are compatible with commonmobile phases containing TFA.• ZORBAX wide-pore 300Extend columns – ideal for LC/MS of proteins and peptidesat pH 10.• ZORBAX Eclipse Amino Acid Analysis (AAA) HPLC columns – a high-efficiency choicefor rapid amino acid separation! These columns make it possible to reduce yourinjection-to-injection analysis time to 14 minutes (7.5 cm column length) and 24minutes (15 cm column length).• ZORBAX GF-250/450 Size Exclusion HPLC columns – a rugged, reproducible columnspecially designed for size separations of proteins. Size exclusion columns are compatiblewith organic modifiers and mobile phase denaturants, so you can eliminate proteinaggregation for proper size determination. And they have a separation range of4,000 - 900,000 for globular proteins when using a GF-250 and GF-450 column in series.• Agilent Bio-Monolith HPLC columns – provide high resolution and rapid separationsof antibodies (IgG, IgM), plasmid DNA, viruses, phages and other macro bio-molecules.Order online at www.agilent.com/chem/store105

Columns for Bioanalytical ChromatographyZORBAX Strategy for Reversed-PhaseMethod Development of Proteins and PeptidesThis ZORBAX column selection strategy for proteins and peptides provides some critical details on method development for proteins orpolypeptides. For small peptides, Molecular Weight (MW) < 2000, please follow the method development strategy for small and largemolecules in the reference section of this guide. For efficient separations of large molecules, columns with a wide-pore size (300Å) arerequired. For method development of larger peptides and proteins, review the suggested guidelines outlined below. Wide-pore columnchoices are described in the following section of this Column Selection Guide.Choose the Initial Column and Conditions for Proteins and PeptidesPeptides, Polypeptides, ProteinsMW < 50 kDAPeptides, Polypeptides, ProteinsMW < 1,000 kDAInitial Bonded Phase ChoiceStableBond 300SB-C8300 SB columns are wide-pore columns with unbeatable lifetime inTFA-containing mobile phases. This makes them an ideal first choicefor separations of peptides and proteins.• C8 is an excellent starting bonded phase because of its moderatehydrophobicity.• C18 and C8 are generally selected for peptides and protein digestsbut can also be used for proteins.• C3, C4 and CN are generally selected for larger, hydrophobicpolypeptides and proteins but can also be used for peptides.Poroshell 300SB-C18Poroshell 300SB columns use an innovative particle technology todeliver rapid protein separations. Short analysis times with efficientpeaks are easily obtained with Poroshell columns.• C18 is a good starting bonded phase choice with Poroshell formost peptides, polypeptides and proteins because the retention ismaximized.• C8 is generally selected for moderate size proteins but can be usedwith polypeptides or very large proteins.• C3 is generally selected for antibodies or large proteins but can beused for peptides and polypeptides.Initial Separation ConditionsColumn:Mobile Phase:Gradient:Temperature:Flow Rate:StableBond 300SB-C84.6 x 150 mm, 3.5 or 5 µm883995-906863973-906A: 95% H 2 O:5% ACN with 0.1% TFAB: 5% H 2 O:95% ACN with 0.085% TFA0-60% B in 60 min35-40°C1 mL/minColumn:Mobile Phase:Gradient:Temperature:Flow Rate:Poroshell 300SB-C182.1 x 75 mm, 5 µm660750-902A: 95% H 2 O:5% ACN with 0.1% TFAB: 5% H 2 O:95% ACN with 0.085% TFA0-60% B in 10 min35-40°C2 mL/min106 Order online at www.agilent.com/chem/store

Columns for Bioanalytical ChromatographyStart at low pH with simple aqueous/organic gradientTypically a Water/Acetonitrile with 0.1% TFA gradient is used to elute all components of interest. A typical high resolution gradient on a 300Åpore size column requires 30-50 min. A Poroshell column requires a shorter analysis time and a higher flow rate and still provides exceptionalresolution. Then to improve resolution, increase the gradient time, decrease column length, or increase flow rate.Optimize sample solubilityFor best peak shape and recovery at any pH, it is important to solubilize a sample completely. Highly acidic or neutral solvents can be used withZORBAX 300StableBond and Poroshell 300SB, while neutral solvents and dilute bases can be used with ZORBAX 300Extend-C18.Solvent choices to solubilize proteins and peptidesWater/Phosphate BufferDilute Acid (TFA, Acetic Acid or HCl)Neutral pH, 6-8 M Guanidine-HCl or Isothiocyanate5% HOAc/6 M UreaDilute Acid + Aqueous/Organic Solvents (ACE, MeOH, THF)Dilute Base (Ammonioum Hydroxide)DMSO or 0.1%-1% TFA in DMSOFormamideWeakestStrongestRaise the temperatureSeparations of proteins and peptides are influenced by temperature and higher column temperature can dramatically improve both resolutionand recovery of proteins and hydrophobic and aggregating peptides.StableBond 300SB – up to 80°C Poroshell 300SB – up to 80°COptimize mobile phase pHTry mid and high pH if low pH does not workIf an optimized low pH method does not provide an ideal separation, then mid or high pH mobile phase can be used. At high pH selectivity isoften very different because acidic amino acids become negatively charged and some basic amino acids may lose their charge. ZORBAX300Extend-C18 is an excellent choice for mid to high pH separation.Column:Mobile Phase:Gradient:Temperature:Flow Rate:300Extend-C184.6 x 150 mm, 5 µm773995-902A: 20 mM NH 4 OH in H 2 OB: 20 mM NH 4 OH in 80% ACN5-60% B in 30 minutes25-30°C (

Columns for Bioanalytical ChromatographyStarting Column Choices for Analytical Separationsof Peptides, Polypeptides, and ProteinsLow pHMid and High pHMW < 50 kDa300SB-C84.6 x 150 mm, 3.5 µm863973-906MW < 1000 kDaPoroshell 300SB-C182.1 x 75 mm, 5 µm660750-902MW < 25 kDa300Extend-C184.6 x 150 mm, 3.5 µm763973-902Separations of Proteins and Peptides Using Reversed-Phase LC/MS MethodsLC/MS of proteins and peptides is used to provide information for protein characterization, to accurately identify post-translationalmodifications of proteins, and to determine the molecular weight of synthetic and natural peptides. LC/MS is used to provide proteinidentification in 2-D separations for proteomics applications. Therefore, LC/MS of proteins and peptides is a critical separation area, whichrequires some special column and mobile phase recommendations. In general, smaller column sizes are used for LC/MS and TFA is generallynot used in mobile phase because of reduced sensitivity in the MS with this mobile phase additive.Initial Column Choices for LC/MS Separations of Proteins and PolypeptidesAnalytical LC/MS Applications – 2.1 mm ID columns will provide good sensitivitywhen sample size is not limited. With Poroshell columns, smaller column IDs are used.Low pHMid and High pHMW < 50 kDa300SB-C82.1 x 150 mm, 3.5 µm863750-906MW < 1000 kDaPoroshell 300SB-C181.0 x 75 mm, 5 µm661750-902MW < 25 kDa300Extend-C182.1 x 150 mm, 3.5 µm763750-902High Sensitivity/Proteomics ApplicationsCapillary columns are used for high sensitivity protein and peptide applications. The 0.5 mm ID columns are used for proteinand protein digest separations while the 0.3 mm ID columns are most often used for protein digests. These can be analyzed at high pHwith an ammonium hydroxide mobile phase. Nano columns (0.1 and 0.075 mm ID) are often used in 2-D LC/MS systems for proteomicsand the initial choice is a C18 bonded phase.108 Order online at www.agilent.com/chem/store

Columns for Bioanalytical ChromatographyHigh Sensitivity Capillary ColumnsLow pHMid and High pHMW < 50 kDa300SB-C180.5 x 150 mm, 3.5 µm5064-8268MW < 1000 kDaPoroshell 300SB-C80.5 x 75 mm, 5 µm5065-4468MW < 25 kDa300Extend-C180.3 x 150 mm, 3.5 µm5065-4464Nano Columns for Proteomics/2-D LC/MSSCX (1st Dimension)0.3 x 35 mm, 5 µm5065-9912Reversed-Phase (2nd Dimension)0.075 x 150 mm, 3.5 µm5065-9911Mobile Phase ConsiderationsLow pHMid and High pHTFA is generally not used for LC/MS separations of proteinsand peptides. The first step is normally to replace TFA with0.1-1.0% formic acid. Acetic acid, up to 1%, can also be used asan alternative mobile phase modifier. At low pH, the best separationmay still be obtained with TFA in the mobile phase. In some cases,the TFA can be displaced post column with an alternate acid,such as propionic acid.LC/MS can also be done at high pH with 10-20 mM NH 4 OHas a mobile phase additive.Order online at www.agilent.com/chem/store109

Columns for Bioanalytical ChromatographyPorous ShellPoroshell 300SOLIDCORE5 µm• High-resolution separations of biomolecules with superficially porous particles• High efficiency and recovery with proteins (up to 1,000 kDa) and monoclonal antibodies• Achieve long lifetime at low pH with Poroshell 300SB; at high pH with 300Extend-C18• Optimize recovery and selectivity with four different bonded phases – 300SB-C18,300SB-C8, 300SB-C3, and 300Extend-C18Agilent Poroshell 300 columns are ideal for fast separations of proteins and peptides becausethe superficially porous particle allows for fast flow rates to be used while maintaining sharp,efficient peaks. Peptides and proteins are typically separated slowly to reduce the potentialpeak broadening of these slow diffusing analytes. But Poroshell columns use a superficiallyporous particle made with a thin layer of porous silica on a solid core of silica. This reducesthe diffusion distance for proteins making practical rapid HPLC separations of peptides andproteins up to 500-1,000 kDA. Poroshell columns bonded with StableBond bonded phasesprovide excellent stability and selectivity choices with TFA and formic acid mobile phases.The Poroshell 300Extend-C18 column can be used from pH 2-10 for unique separations.These columns can be used for analytical protein separations as well as LC/MS separations.Column SpecificationsBonded Phase Pore Size Temp. Limits* pH Range EndcappedPoroshell 300SB-C18, C8, C3 300Å 90°C 1.0-8.0 NoPoroshell 300Extend 300Å 40°C above pH 860°C below pH 82.0-11.0 YesSpecifications represent typical values only.110 Order online at www.agilent.com/chem/store

Columns for Bioanalytical ChromatographyPoroshell 300 Columns Can SeparateProteins and Peptides in SecondsColumn: Poroshell 300SB-C18660750-9022.1 x 75 mm, 5 µmMobile Phase: A: 0.1% TFA in H 2 0B: 0.07% TFA in ACNFlow Rate: 3.0 mL/minGradient: 5-100% B in 1.0 minTemperature: 70°C, 260 bar pressureDetector: 215 nmSample:Proteins and Peptides1. Angiotensin II2. Neurotensin3. RNase4. Insulin5. Lysozyme6. Myoglobin7. Carbonic Anhydrase8. Ovabumin12345678This separation of eight polypeptides and proteins iscompleted in less than 60 seconds. Each peak is sharpand efficient.0 0.51.0Time (min)LCPO001Reduce Peptide Map Analysis Timeby 90% with Poroshell 300SBColumn A: Poroshell 300SB-C18660750-9022.1 x 75 mm, 5 µmColumn B: ZORBAX 300SB-C18883750-9022.1 x 150 mm, 5 µmMobile Phase: A: 95% H 2 O, 5% ACN, 0.1% TFAB: 5% H 2 O, 95% ACN, 0.07% TFAFlow Rate: 1 mL/min0.208 mL/minGradient: 0-100%B = 12 min0-100%B = 120 minTemperature: 70°CSample:20 µL (0.22 µg/1 µL)BSA Tryptic Digest(15 hours, 70 pmol)AB1 2 3 4 5 6 7 8Time (min)A single chromatographic run of a protein tryptic digest can require an houror more to complete. With ZORBAX Poroshell columns, the same complexseparation can be completed in 1/10th the time.1020 30 40 50 60 70 80Time (min)LCPO002Order online at www.agilent.com/chem/store111

Columns for Bioanalytical ChromatographyMicroBore Poroshell 300 ColumnsProvide Maximum Sensitivity for LC/MSColumn: Poroshell 300SB-C18661750-9021.0 x 75 mm, 5 µmMobile Phase: A: Water + 0.1% Formic AcidB: ACN + 0.1% Formic Acid1EBFlow Rate: 600 µL/minGradient: 20-100% B in 5.5 min 80000000Temperature: 80°CMS Conditions: LC/MS: Pos. Ion ESI –Vcap 6000 V60000000Drying Gas Flow: 12 Liters/minDrying Gas Temperature: 350°CNebulizer: 45 psiFragmentor Volatage: 140 V40000000Scan: 600-2500Stepsize: 0.15 amuPeakwidth: 0.06 min20000000Sample: 1 µL0.50.751.02.55.00 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Time (min)LCPO003With narrow bore diameters like 2.1 mm, 1.0 mm, and 0.5 mm, ZORBAX Poroshell columns make an ideal LC/MS partner. When the sample is very limited, the 1.0 mmor 0.5 mm ID Poroshell columns are an excellent choice for high sensitivity LC/MS analyses. Sensitive MS molecular weight determinations are possible with as little as0.5 to 5 pmole of protein on Poroshell columns. Poroshell columns have also been used for rapid MS identification of intact proteins even in the presence of stabilizers andtissue culture media.Poroshell 300Hardware Description Size (mm) Particle Size (µm)Poroshell300SB-C18Poroshell300SB-C8Poroshell300SB-C3Poroshell300Extend-C18Narrow Bore 2.1 x 75 5 660750-902 660750-906 660750-909 670750-902MicroBore 1.0 x 75 5 661750-902 661750-906 661750-909 671750-902Capillary 0.5 x 75 5 5065-4468Guard Cartridge, 4/pk 2.1 x 12.5 5 821075-920 821075-918 821075-924Guard Hardware Kit 820888-901 820888-901 820888-901MicroBore Guard, 3/pk 1.0 x 17 5 5185-5968 5185-5968 5185-5968 5185-5968112 Order online at www.agilent.com/chem/store

Columns for Bioanalytical ChromatographyZORBAX 300Å StableBondSterically Protected 300StableBond Bonded PhaseAgilent ZORBAX 300StableBond columns are an ideal choice for the reproducible separationsof proteins and peptides for two key reasons. First, wide-pore, 300Å columns are necessaryfor an efficient separation of proteins and peptides, or other large molecules, in order to allowthese analytes to completely access the bonded phase. Second, 300StableBond columnsare unmatched in their durability at low pH, such as with the TFA containing mobile phasestypically used for protein and peptide separations. For LC/MS separations at low pH,300StableBond columns can also be used with formic acid and acetic acid mobile phasemodifiers. These columns are available in four different bonded phases (C18, C8, C3,and CN) for selectivity and recovery optimization of proteins and polypeptides. To furtherincrease sample recovery and improve efficiency for difficult proteins, 300StableBondcolumns can be used up to 80-90°C. 300SB-C18 and 300SB-C8 columns are an ideal choicefor complex protein and protein digest separations. These columns are available in capillary(0.3, 0.5 mm ID) and nano (0.075 and 0.10 mm ID) dimensions for reversed-phase LC/MSseparations of these protein digests. Capillary and nano columns can be used for either1-D or 2-D proteomics separations.Column SpecificationsBonded PhasePoreSizeSurfaceAreaTemp.Limits* pH Range*EndcappedCarbonLoadZORBAX 300SB-C18 300Å 45 m 2 /g 90°C 1.0-8.0 No 2.8%ZORBAX 300SB-C8 300Å 45 m 2 /g 80°C 1.0-8.0 No 1.5%ZORBAX 300SB-C3 300Å 45 m 2 /g 80°C 1.0-8.0 No 1.1%ZORBAX 300SB-CN 300Å 45 m 2 /g 80°C 1.0-8.0 No 1.2%Specifications represent typical values only.*300StableBond columns are designed for optimal use at low pH. At pH 6-8, highest column stability for all silica-basedcolumns is obtained by operating at temperatures

Columns for Bioanalytical ChromatographyShort-Chain ZORBAX 300SB-C3is Stable at Low pH, High Temperature100Column: ZORBAX 300SB-C3883995-9094.6 x 150 mm, 5 µmMobile Phase: Gradients 0-100% B in 80 minA: 0.5% TFA in WaterB: 0.5% TFA in AcetonitrileIsocratic RetentionTest Conditions:1-phenylheptane 50% A, 50% BFlow Rate: 1.0 mL/minTemperature: 60°C% k Phenyltheptane Remaining9080706050ZORBAX 300 SB-C3Competitor C4400 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000Volume of Mobile Phase (mL)LCSB005Four Different 300SB Bonded PhasesOptimize Separation of Large PolypeptidesColumn A: ZORBAX 300SB-C18883995-9024.6 x 150 mm, 5 µmColumn B: ZORBAX 300SB-C8883995-9064.6 x 150 mm, 5 µmColumn C: ZORBAX 300SB-C3883995-9094.6 x 150 mm, 5 µmColumn D: ZORBAX 300SB-CN883995-9054.6 x 150 mm, 5 µmMobile Phase: Linear Gradient, 25 - 70% B in 40 minA: 0.1% TFA in WaterB: 0.09% TFA in 80% Acetonitrile/20% WaterFlow Rate: 1.0 mL/minTemperature: 60°CSample: 3 µg each proteinABC1112, 32432431. RNase6. CDR2. Insulin7. Myoglobin3. Cytochrome C 8. Carbonic Anhydrase4. Lysozyme 9. S-100b5. Parvalbumin 10. S-100a4 5 7 869106, 7589105 786910The 300SB-C18, C8, C3, and CN bonded phases all provide adifferent separation of this group of polypeptides. This adds animportant parameter for quickly optimizing protein separations.The 300SB-CN column offers unique selectivity for morehydrophilic polypeptides.D123457689100 5 10 15 20 25 30 35 40Time (min)LCSB006114 Order online at www.agilent.com/chem/store

Columns for Bioanalytical ChromatographyCapillary Columns for HPLC Analyseswith UV and MS Detection100UVColumn:Mobile Phase:Flow Rate:Detector:MS Conditions:Sample:ZORBAX 300SB-C185064-82630.3 x 150 mm, 5 µm5-55% B in 50 min, to 85% B from 55-57 minA: 0.1% Formic Acid in WaterB: 0.1% Formic Acid in ACN5.5 µL/min206 nmLC/MS: Pos. Ion ESI withLC/MSD trap-Vcap 4000 VDrying Gas Flow: 7 L/minDrying Gas Temperature: 250°CNebulizer: 15 psiCapillary Exit Volt: 50 VMax Accum Time: 300 msTotal Averages: 3Isolation Width: 3 m/zFrag Ampitude: 1.0 V100 nLBeta Casein Digest (4 pmol)A ZORBAX 300SB-C18 capillary column (0.3 mm ID) is used forthe separation of the protein digest. Detection is by both UV andElectrospray MS. MS detection can be used for identification ofpeptide fragments.% Relative AbundanceUV at 206 nm806040200x100.80.60.40.20.010MS10151520 25 30 35 40 45 50Time (min)20 25 30 35 40 45 50Time (min)LCSB007ZORBAX Nano Columns For High SensitivityProtein Digest Analysis by LC/MSColumn: ZORBAX 300SB-C185065-99110.075 x 150 mm, 3.5 µmMobile Phase: A: Water + 0.1% Formic Acid,B: ACN + 0.1% Formic AcidFlow Rate: 600 nl/minGradient:2% B to 52% B in 25 minDetector:Positive Ion Nano Electrospray MSSample:100 fm (1 µl) Digest of 8 ProteinsIntens.1.51.00.5A ZORBAX nano HPLC column, 0.075 mm ID, is used for highsensitivity LC/MS analysis of a protein digest sample.2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5Time (min)LCSB008Order online at www.agilent.com/chem/store115

Columns for Bioanalytical ChromatographyZORBAX 300Å StableBondHardware Description Size (mm) Particle Size (µm)300SB-C18USP L1300SB-C8USP L7300SB-CNUSP L10300SB-C3USP L56Standard Columns (no special hardware required)Semi-Preparative 9.4 x 250 5 880995-202 880995-206 880995-205 880995-209Analytical 4.6 x 250 5 880995-902 880995-906 880995-905 880995-909Analytical 4.6 x 150 5 883995-902 883995-906 883995-905 883995-909Analytical 4.6 x 50 5 860950-902 860950-906 860950-905 860950-909Rapid Resolution 4.6 x 150 3.5 863973-902 863973-906 863973-905 863973-909Rapid Resolution 4.6 x 100 3.5 861973-902 861973-906Rapid Resolution 4.6 x 50 3.5 865973-902 865973-906 865973-905 865973-909Solvent Saver Plus 3.0 x 150 3.5 863974-302 863974-306 863974-309Solvent Saver Plus 3.0 x 100 3.5 861973-306Narrow Bore 2.1 x 250 5 881750-902Narrow Bore 2.1 x 150 5 883750-902 883750-906 883750-905 883750-909Narrow Bore RR* 2.1 x 150 3.5 863750-906Narrow Bore RR* 2.1 x 100 3.5 861775-902 861775-906Narrow Bore RR* 2.1 x 50 3.5 865750-902 865750-906MicroBore 1.0 x 250 5 861630-902MicroBore RR* 1.0 x 150 3.5 863630-902 863630-906MicroBore RR* 1.0 x 50 3.5 865630-902 865630-906MicroBore Guard, 3/pk 1.0 x 17 5 5185-5920 5185-5920Guard Cartridge, 2/pk 9.4 x 15 7 820675-124 820675-124 820675-124 820675-124Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-921 820950-918 820950-923 820950-924Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-918 821125-918 821125-924 821125-924Guard Hardware Kit 840140-901 840140-901 840140-901 840140-901Guard Hardware Kit 820888-901 820888-901 820888-901 820888-901*RR: Rapid Resolution 3.5 µm116 Order online at www.agilent.com/chem/store

Columns for Bioanalytical ChromatographyZORBAX 300Å StableBondHardware Description Size (mm) Particle Size (µm)300SB-C18USP L1300SB-C8USP L7PrepHT Cartridge Columns (require endfittings kit 820400-901)PrepHT Cartridge 21.2 x 250 7 897250-102 897250-106 897250-105 897250-109PrepHT Cartridge 21.2 x 150 7 897150-102 897150-106 897150-109PrepHT Cartridge 21.2 x 150 5 895150-902 895150-906 895150-909PrepHT Cartridge 21.2 x 100 5 895100-902 895100-906 895100-909PrepHT Cartridge 21.2 x 50 5 895050-902 895050-906 895050-909PrepHT endfittings, 2/pk 820400-901 820400-901 820400-901 820400-901PrepHT Guard Cartridge, 2/pk 17 x 7.5 5 820212-921 820212-918 820212-924 820212-924Guard Cartridge Hardware 820444-901 820444-901 820444-901 820444-901Capillary Glass-lined ColumnsCapillary 0.5 x 250 5 5064-8266Capillary 0.5 x 150 5 5064-8264Capillary 0.5 x 35 5 5064-8294Capillary RR* 0.5 x 150 3.5 5064-8268Capillary RR* 0.5 x 35 3.5 5065-4459Capillary 0.3 x 250 5 5064-8265Capillary 0.3 x 150 5 5064-8263Capillary 0.3 x 35 5 5064-8295Capillary RR* 0.3 x 150 3.5 5064-8267 5065-4460Capillary RR* 0.3 x 100 3.5 5064-8259 5065-4461Capillary RR* 0.3 x 50 3.5 5064-8300 5065-4463Capillary RR* 0.3 x 35 3.5 5064-8270 5065-4462Nano Columns (PEEK fused silica)Nano RR* 0.1 x 150 3.5 5065-9910Nano RR* 0.075 x 150 3.5 5065-9911Nano RR* 0.075 x 50 3.5 5065-9924 5065-9923Trap/Guard, 5/pk 0.3 x 5 5 5065-9913 5065-9914Trap/Guard Hardware kit 5065-9915 5065-9915*RR: Rapid Resolution 3.5 µm300SB-CNUSP L10300SB-C3USP L56Order online at www.agilent.com/chem/store117

Columns for Bioanalytical ChromatographyZORBAX 300Å 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 phaseAgilent ZORBAX 300Extend C-18 is a wide-pore HPLC column for high efficiency separationsof peptides from pH 2-11.5. The unique, bidentate bonded phase provides excellent lifetimeand reproducibility at high and low pH. At high pH, retention and selectivity of peptides andpolypeptides can change dramatically as a result of changes in charge on molecules. Excellentrecoveries of hydrophobic polypeptides have been achieved at room temperature and highpH. LC/MS sensitivity of peptides and polypeptides can also be improved at high pH using asimple ammonium hydroxide-containing mobile phase.Novel Bidentate C18-C18 Bonding for Extend-C18Bonded PhaseColumn SpecificationsBonded PhasePoreSizeSurfaceAreaTemp.Limits* pH Range EndcappedCarbonLoadZORBAX 300Extend-C18 300Å 45 m 2 /g 60°C 2.0-11.5 Double 4%Specifications represent typical values only.*Temperature limits are 60°C up to pH 8, 40°C from pH 8-11.5.118 Order online at www.agilent.com/chem/store

Columns for Bioanalytical ChromatographyLong Life at High pH with 300Extend-C18Column: ZORBAX Extend-C18773450-9024.6 x 150 mm, 5 µmMobile Phase: 20% 20 mM NH 4 OH, pH 10.580% Methanol864-Chloro-1-nitrobenzeneTrimipramineFlow Rate: 1.5 mL/minTemperature: Aging 24°CTests 40°CEach 10,000 column volume is approximately oneworking month.k Values4200 10,000 20,000 30,000 40,000 50,000Column Volumes of Mobile PhaseLC30001Extend-C18 and StableBond SB-C18 Are Stable at Low pHColumn A: ZORBAX SB-C18883975-9024.6 x 150 mm, 5 µmColumn B: ZORBAX Extend-C18773450-9024.6 x 150 mm, 5 µmColumn C: ZORBAX Rx-C18883967-9024.6 x 150 mm, 5 µmMobile Phase: Column Aging:50% Methanol :50% Water + 1% TFAColumn Test:60% Methanol : 40% WaterTest Solute: TolueneFlow Rate: 1.5 mL/min, continuousTemperature: Aging: 90°CTest: AmbientThe 300Extend-C18 column can be used at high andlow pH – from pH 2-11.5. This chart shows that the300Extend-C18 has the needed stability at low pH forlong-term reproducible separations. Therefore, onewide-pore column can be used for selectivity optimizationat low and high pH with both TFA and ammoniumhydroxide mobile phases.Fraction of Toluene k Remaining1.00.80.60.40.20.0Stablebond SB-C18 (Diisobutyl-C18)Extend-C18 (Bidentate-C18)Rx-C18 (Dimethyl-C18)0 5,000 10,000 15,000 20,000 25,000 30,000Column Volumes of Mobile PhaseLC30002Order online at www.agilent.com/chem/store119

Columns for Bioanalytical ChromatographyLC/MS Analysis of Angiotensin on Extend-C18Column: ZORBAX Extend-C18773700-9022.1 x 150 mm, 5 µmMobile Phase: Acidic Conditions:A: 0.1% TFA in waterB: 0.085% TFA in 80% acetonitrile (ACN)Basic Conditions:A: 10 mM NH 4 OH in waterB: 10 mM NH 4 OH in 80% ACNFlow Rate: 0.2 mL/minGradient: 15-50% B in 15 minTemperature: 35°CMS Conditions: Pos. Ion ESI- Vf 70V, Vcap 4.5 kV,N2- 35 psi, 12 L/min., 325°CSample:2.5 µL sample (50 pmol each)Angiotensin I, II, IIIAAngiotensin IMax: 10889Low pHTIC (200-1500 m/z)5.0E64.0E63.0E62.0E61.0E6AII + AIII12.05013.261AI10080604020227.1250.9340.3398.0433.0+3458.0649.5+2659.7720.3767.3844.81025.21179.11277.91326.31373.31468.8002.55.0 7.5 10 12.5Time (min)005001000m/zBAngiotensin IMax: 367225High pHTIC (200-1500 m/z)5.0E74.0E73.0E72.0E71.0E7002.5AII5.499AIAIII8.4779.6215.0 7.5 10 12.5Time (min)1008060402000433.0+3500649.0659.8+210001296.6+1m/zLC30003Reference: B.E. Boyes. Separation and Analysis of Peptides at High pHUsing RP-HPLC/ESI-MS, 4th WCBP, San Francisco, CA Jan 2000.Both small and large peptides demonstrate selectivity changes at high and low pH. At high pH, due to a change in charge, all three Angiotensins can be resolved. In addition,the spectral clarity of Angiotensin I is dramatically improved at high pH with the ammonium hydroxide mobile phase. The Extend-C18 column can be used for the analysis ofsmall peptides at high pH as well.120 Order online at www.agilent.com/chem/store

Columns for Bioanalytical ChromatographyZORBAX 300Å Extend-C18Hardware DescriptionSize (mm)Particle Size(µm) Part No.Analytical 4.6 x 250 5 770995-902Analytical 4.6 x 150 5 773995-902Rapid Resolution 4.6 x 150 3.5 763973-902Rapid Resolution 4.6 x 100 3.5 761973-902Rapid Resolution 4.6 x 50 3.5 765973-902Narrow Bore RR* 2.1 x 150 3.5 763750-902Narrow Bore RR* 2.1 x 100 3.5 761775-902Narrow Bore RR* 2.1 x 50 3.5 765750-902Guard Cartridge, 4/pk 4.6 x 12.5 5 820950-932Guard Cartridge, 4/pk 2.1 x 12.5 5 821125-932Guard Hardware Kit 820888-901Capillary Glass-lined ColumnsCapillary RR* 0.3 x 150 3.5 5065-4464Capillary RR* 0.3 x 100 3.5 5065-4465Capillary RR* 0.3 x 75 3.5 5065-4466Capillary RR* 0.3 x 50 3.5 5065-4467*RR: Rapid Resolution 3.5 µmOrder online at www.agilent.com/chem/store121

Columns for Bioanalytical ChromatographyAmino Acid StandardsEach amino acid standard containsthe following amino acids:• Glycine• L-cystine• L-histidine• L-tyrosine• L-leucine• L-methionine• L-serine• L-alanine• L-phenylalanine• L-glutamic acid• L-proline• L-isoleucine• L-arginine• L-threonine• L-valine• L-lysine• L-aspartic acidZORBAX Amino Acid Analysis (AAA)Columns and SuppliesZORBAX Eclipse Amino Acid Analysis (AAA) Columns• High resolution and rapid analysis of 24 amino acids• Use tested for amino acid analysis• Uses well known OPA and FMOC precolumn derivatization chemistry• Easily automated using a detailed online, derivatization protocol available for use withAgilent 1100/1200 AutosamplerThe Agilent ZORBAX Eclipse AAA high efficiency column rapidly separates amino acidsfollowing an updated and improved protocol. Total analysis from injection to injection can beachieved in as little as 14 min. (9 min. analysis time) on shorter, 7.5 cm length columns and24 min. (18 min. analysis time) on the 15 cm column length. Exceptional sensitivity (5-50pmol with DAD, FLD) and reliability are achieved using both OPA and FMOC derivatizationchemistries in one fully automated procedure using the Agilent 1100/1200 HPLC instrument.ZORBAX Eclipse Amino Acid Analysis (AAA) ColumnsParticle SizeHardware Description Size (mm) (µm) Part No.Analytical routine sensitivity 4.6 x 150 5 993400-902Analytical routine sensitivity, 4.6 x 150 3.5 963400-902high-resolution using FLDAnalytical routine sensitivity, 4.6 x 75 3.5 966400-902high-throughputSolvent Saver high sensitivity, 3.0 x 150 3.5 961400-302high resolutionGuard Catridges, 4/pk 4.6 x 12.5 5 820950-931Guard Hardware Kit 820888-901122 Order online at www.agilent.com/chem/store

Columns for Bioanalytical ChromatographyAmino Acid Standards, 10 x 1 mL ampoules*DescriptionPart No.1 nmol/µL 5061-3330250 pmol/µL 5061-3331100 pmol/µL 5061-333225 pmol/µL 5061-333310 pmol/µL 5061-3334Amino acids supplement kit5062-2478Includes 1 g each of norvaline, sarcosine, asparagine, glutamine, tryptophan,and 4-hydroxyproline*Consider shelf-life and buy limited quantities, 5062-2478 ships as 1 g vialsAmino Acid Separations ReagentsDescriptionPart No.OPA reagent, 10 mg/mL each in 0.4 M borate buffer o-phthalaldehyde (OPA) 5061-3335and 3-mercaptopropionic acid, 6 x 1 ml ampoulesFMOC reagent, 2.5 mg/mL in acetonitrile, 9-fluorenylmethylchloroformate,5061-33371 ml, 10 ampoulesBorate buffer, 100 mL 5061-3339DTDPA (Dithiodiproprionic) reagent, for analysis of cysteine, 5 g 5062-2479High Resolution of 24 Amino AcidsUsing ZORBAX Eclipse-AAA Protocol1. Asp7. Gly13. Cys19. IleColumn: ZORBAX Eclipse AAA963400-9024.6 x 150 mm, 3.5 µmMobile Phase: A: 40 mM Na 2 HPO 4 , pH 7.8B: ACN : MeOH : Water,45:45:10 v/vFlow Rate: 2 mL/minTemperature: 40°CDetector: FluorescenceSample: 24 Amino Acids122. Glu3. Asn4. Ser5. Gln6. His347898. Thr9. Cit10. Arg11. Ala12. Tyr1011 1216 17 22 231815 19201414. Val15. Met16. Nva17. Trp18. Phe20. Leu21. Lys22. Hyp23. Sar24. ProThis high resolution separation of 24 amino acids is donein 18 minutes. If the Rapid Resolution 4.6 x 75 mmEclipse AAA column is selected, these amino acids areresolved in 9 minutes.5621240 510 15Time (min)20 25LCPAH01Order online at www.agilent.com/chem/store123

Columns for Bioanalytical ChromatographyZORBAX GF-250 and GF-450Gel Filtration Columns• High efficiency and reproducibility with short analysis time• Hydrophilic diol bonded phase for good protein recovery• Compatible with organic modifiers and denaturants• Wide usable pH range (pH 3-8)Agilent ZORBAX GF-250 and GF-450 size exclusion (gel filtration) columns are ideal for thesize separations of proteins and other biomolecules. The separation range is 4,000-900,000for globular proteins when using GF-250 and GF-450 columns in series. The GF-250/GF-450size exclusion columns have a hydrophilic diol bonded phase for high recovery of proteins(typically >90%) and a unique zirconia modification of the silica to extend the pH rangefrom 3-8. The GF-250 and GF-450 columns are packed with precisely sized porous silicamicrospheres with narrow pore size and particle size distributions. The result is a highlyefficient, 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 (

Columns for Bioanalytical ChromatographySeparation of Protein Standardson the ZORBAX GF-250 SEC Column5Column: ZORBAX GF-250884973-9019.4 x 250 mm, 4 µmMobile Phase: 200 mM Sodium Phosphate, pH 7.0Flow Rate: 2 mL/minTemperature: AmbientDetector: 254 nmThe protein standards separated here are a commonly selected set ofstandards. The ZORBAX GF-250 column shows excellent resolution forthis sample. Additional resolution of the thyroglobulin can be obtained byadding the GF-450 column in series.Sample:BioRad Gel Filtration Standardsfor Size Exclusion1. 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 Da12340 1 2 3 4 5 6 7 8Time (min)LCGF001Order online at www.agilent.com/chem/store125

Columns for Bioanalytical ChromatographyHigh-Speed Size Exclusion SeparationsColumn: ZORBAX GF-450884973-9029.4 x 250 mm, 6 µmMobile Phase:Flow Rate:Temperature:Detector:Sample:PBS (phosphatebuffered saline), pH 7.43 mL/minAmbient220 nmBSA and BSA DimersHigh-Speed Size Exclusion Separations 2Column: ZORBAX GF-450884973-9029.4 x 250 mm, 6 µmMobile Phase:Flow Rate:Detector:Sample:200 mMNa Phosphate MonobasicpH 7.0/0.1% Azide2 mL/min225 nm10 µg in 50 mMSodium Phosphate pH 7.0A: BSA and BSA Dimers (duplicate injections)21. BSA dimer2. BSA monomer3. Azide2B: Antibody Separation21. IgG Dimer and Aggregate2. Mouse IgG 1K (MOPC 31C)3. Salts: Tris, Na 2 HPO 4 , NaCl13Inj. #1 Inj. #213130 5 10Time (min)0 1 2 3 4 5 6 7 8 9 10Retention Time (min)LCGF002126 Order online at www.agilent.com/chem/store

Columns for Bioanalytical ChromatographyZORBAX GF-250 Shows Extended Column LifetimeStability of GF-250: K* of Standard ProteinsStability of Competitor Column: K* of Standard Proteins3.03.02.52.5K*2.01.51.0BSAOvalbuminLysozymeNaN 3K*2.01.51.0BSAOvalbuminLysozymeNaN 30.50.50.00 1000 2000 3000Column Volumes0.00 1000 2000 3000Column VolumesLCGF004ZORBAX GF-250 and GF-450 Gel Filtration ColumnsHardware DescriptionSize (mm)Particle Size(µm) Part No.GF-250, 150Å 9.4 x 250 4 884973-901GF-250, 150Å 4.6 x 250 4 884973-701GF-450, 300Å 9.4 x 250 6 884973-902Guard Columns (hardware required)GF-250 Diol, Guard Cartridge, 2/pk 9.4 x 15 6 820675-111GF-250 Diol, Guard Cartridge, 4/pk 4.6 x 12.5 6 820950-911GF-450 Diol, Guard Cartridge, 2/pk 9.4 x 15 6 820675-111GF-450 Diol, Guard Cartridge, 4/pk 4.6 x 12.5 6 820950-911Guard Hardware Kit 840140-901Guard Hardware Kit 820888-901PrepHT ColumnsPrepHT GF-250, 150Å 21.2 x 250 6 877974-901PrepHT GF-450, 300Å 21.2 x 250 6 877974-910PrepHT endfittings, 2/pk 820400-901PrepHT GF-250, Guard Cartridge, 2/pk 17 x 7.5 6 820212-911PrepHT GF-450, Guard Cartridge, 2/pk 17 x 7.5 6 820212-911Guard Cartridge Hardware 820444-901Order online at www.agilent.com/chem/store127

Columns for Bioanalytical ChromatographyAgilent Bio-Monolith HPLC Columns• Polymer-based, monolith HPLC columns designed for macro bio-molecule separations• Flow rate independent separations; no diffusion, no pores and no void volume maketransport between mobile and stationary phase very rapid• Monolith disc is 5.2 mm x 4.95 mm (100 µL column volume) with continuous channels,eliminating diffusion mass transfer• Extremely fast separations speed up method development time and decrease costs.Locking in method parameters takes significantly less time and bufferAgilent Bio-Monolith HPLC columns provide high resolution and rapid separations ofantibodies (IgG, IgM), plasmid DNA, viruses, phages and other macro bio-molecules. Theproduct family offers strong cation exchange, strong and weak anion exchange and Protein Aphases. Bio-Monolith HPLC columns are compatible with HPLC and preparative LC systems,including Agilent 1100 and 1200 HPLC systems.Column SpecificationsDimensions5.2 mm x 4.95 mmColumn volume 100 µLMaximum pressure150 bar (15 MPa, 2200 psi)Temperature min/max Working: 4°C-40°C (39°F-122°F)Storage: 4°C-30°C (39°F-73°F)Recommended pH Working range: 2-13Cleaning-in-place: 1-14Materials of construction Hardware: Stainless steelPacking: poly (glycidyl methacrylate-co-ethylene dimethacrylate)highly porous monolithColor ring identifierBio-Monolith QA: BlueBio-Monolith DEAE: GreenBio-Monolith SO 3 : RedBio-Monolith Protein A: WhiteShelf life/expiration date Protein A: 12 monthsSO 3 , QA, DEAE: 24-36 months128 Order online at www.agilent.com/chem/store

Columns for Bioanalytical ChromatographyBio-Monolith DEAE Column Monitors Phage Production During FermentationColumn:Mobile Phase:Flow Rate:Gradient:Detector:Instrument:DEAE5069-36365.2 x 4.95 mmA: 125 mM Phosphate buffer,pH 7.0B: 125 mM Phosphate buffer+ 1M NaCl, pH 7.01 mL/min100% buffer A (2.5 min)0-100% buffer B (10 min)100% buffer A (2 min)UV at 280 nmHigh pressure gradientHPLC system, Agilent 1200Relative Absorbance (mAU)5045403530252015105Phages andother componentsTime 36 minTime 158 minTime 191 min% Buffer B13212010080604020% Gradient00 2 4 6 8 10 12Time (min)0As phage proliferation progresses, the genomic DNA (gDNA) concentration increases as the host cells are being lysed. In the late stages of fermentation gDNA begins todegrade into fragments. These gDNA fragments cannot be easily removed by purification media so, it is critical to stop the fermentation cycle prior to the degradation of thegenomic DNA. The chromatogram above represents three samples taken from the bioreactor at time 36 min, 158 min and 191 min. Peak 1 represents phage, media and hostcells, peak 2 the intact gDNA and peak 3 the fragmented gDNA.Order online at www.agilent.com/chem/store129

Columns for Bioanalytical ChromatographyBio-Monolith Protein A Column Rapidly Monitors IgG Fermentation TitersColumn: Protein A5069-36395.2 x 4.95 mmMobile Phase: A: 1x PBS, pH 7.0B: 0.5 M acetic acid, pH 2.6Flow Rate: 1 mL/minGradient: 100% buffer A100% buffer B100% buffer A(0.5 min each step)Detector: UV at 280 nmInstrument: High pressure gradientHPLC system, Agilent 1200Relative absorbance (mAU)24019014090402 mL/min1mL/min_ 10Time (min)An IgG standard was measured at two flow rates, 1 mL/min and 2 mL/min, which correspond to 10 columnvolumes/min and 20 column volumes/min, respectively. The concentration of IgG in the sample can be reliablyobtained within minutes.16001400The calibration curve1200Peak area1000800600y = 728.55xR 2 = 0.99754002000C (mg/mL)The above calibration curve results from a 2-fold dilution series of human IgG concentrate. The initial IgGconcentration was 2 mg/mL. IgG peak areas were integrated and plotted. The linear range of the assay easilycovers the production ranges necessary to accommodate development and manufacturing cell cultures.130 Order online at www.agilent.com/chem/store

Columns for Bioanalytical ChromatographyAgilent Bio-Monolith HPLC ColumnsColumn Description Key Applications Part No.Bio-Monolith QAThe quaternary amine bonded phase(Strong Anion Exchange) is fully chargedover a working pH range of 2-13, bindingnegatively charged bio-molecules. Adenovirus process monitoringand quality control IgM purification monitoringand quality control Monitoring DNA impurity removal Monitoring endotoxin removal HSA Purity5069-3635Bio-Monolith DEAEBio-Monolith SO 3Bio-Monolith Protein AThe diethylaminoethyl bonded phase(Weak Anion Exchange) offers increasedselectivity of bio-molecules with negativecharge over a working pH range of 3-9.The sulfonyl bonded phase (Strong CationExchange) is fully charged over a workingpH range of 2-13, binding positivelycharged bio-molecules.The Protein A affinity column is designedfor the analytical separation of all IgG(human and mouse), except for IgG class3. Process monitoring and quality controlof bacteriophage manufacturingand purification Process monitoring and quality controlof plasmid DNA purification Fast and high resolution analyticalseparations of large molecules suchas proteins, antibodies Hemoglobin A1c fast analytics Quantitative Determination of IgG(fermentation titer calculation)5069-36365069-36375069-3639Order online at www.agilent.com/chem/store131

Technical Support at work for youHave a hardware, software, application, instrument repair or troubleshootingquestion? Agilent's technical experts are available to answer your questions.With years of laboratory experience, our technical support specialists can providein-depth knowledge and experience.For questions pertaining to columns found in this catalog, call your localAgilent sales office or 1-800-227-9770 in the U.S. or Canada. You can also visitwww.agilent.com/chem/techsupport for a wealth of knowledge, tips,and insight, including:• Frequently Asked Questions• Downloads and Utilities• Installation and Maintenance Videos• Interactive Troubleshooter• Warranty Information• Technical Support Contact InformationClear every hurdle with AgilentZORBAX HPLC columnsGet customized column informationand recommendations from Agilentat www.zorbaxmethod.com132

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How to contact AgilentFor the latest information on the complete lineof Agilent Technologies columns and supplies:• Visit our Web site: www.agilent.com/chem• Contact your local Agilent sales office• Contact your local Agilent Authorized DistributorInformation, descriptions, and specificationsin this publication are subject to change without notice.© Agilent Technologies, Inc. 2009Printed in the USA September 10, 20095990-4435EN