Efficiently analyze your most challenging compounds. Deliver high-quality results faster.

Agilent ZORBAX Column Selection Guide for HPLC

Agilent ZORBAX Column Selection Guide for HPLC

Here it is your single source for LC column selection.

How do we increase productivity while our resources remain constant? How can we meet tight deadlines in the face of complex samples and heavy workloads? Which columns ensure method ruggedness for transfer to our manufacturing site? How can we stop losing time troubleshooting experiments?

Find the answers you need right here!

Agilents ZORBAX Column Selection Guide for HPLC is the only chromatography guide that combines a broad offering of LC columns with expert advice that can help you achieve reliable results faster. All in a concise, easy-to-use format. Inside, you will find columns for analytical, high-throughput, and bioanalytical HPLC method development guidelines and tips from industry veterans. And you will find proof that at any pH level ZORBAX columns deliver superior reproducibility and high resolution.

Maximize your productivity!

Our easy-to-use guide cuts the column selection process down to three simple steps: 1. Narrow the Search use the flow charts on pages 6-9 to choose the right column for your needs. 2. Follow the for tips on everything from custom columns to technical support.

Most tips can also be found at www.agilent.com/chem/lccolumntips. 3. Prepare your order using the part numbers and purchasing guides located near the descriptions of each column. You can also order online at www.agilent.com/chem/store.

Table of Contents

Featured Products...............................................................................................2 ZORBAX HPLC Column Selection Guidelines ................................................5

HPLC Column Selection Flow Chart.....................................................................................................................................6 Quick Guide to ZORBAX Reversed-Phase Bonded Phases..................................................................................................7 ZORBAX Reversed-Phase HPLC Column Selection Flow Chart...........................................................................................8 Simple Guidelines for Choosing a Reversed Phase HPLC Column ....................................................................................10 Quick Guide to USP Categories For HPLC Columns ..........................................................................................................12 Reversed-Phase HPLC Method Development ...................................................................................................................13 Cartridge Selection Guide ..................................................................................................................................................18

ZORBAX Columns for Analytical HPLC.........................................................19

ZORBAX Eclipse Plus..........................................................................................................................................................20 ZORBAX Eclipse XDB .........................................................................................................................................................26 ZORBAX 80 StableBond..................................................................................................................................................33 ZORBAX Rx ........................................................................................................................................................................39 ZORBAX 80 Extend-C18..................................................................................................................................................41 ZORBAX Bonus-RP.............................................................................................................................................................45 ZORBAX Method Development Kits ..................................................................................................................................49 ZORBAX Method Validation Kits........................................................................................................................................50 ZORBAX Original Reversed Phase Columns ......................................................................................................................51 ZORBAX Normal Phase Columns.......................................................................................................................................52

HPLC Columns for Special Applications .......................................................55

ZORBAX Rapid Resolution High Throughput 1.8 m ........................................................................................................57 ZORBAX Rapid Resolution 3.5 m Columns......................................................................................................................64 ZORBAX Solvent Saver ......................................................................................................................................................68 ZORBAX MicroBore (1.0 mm ID).......................................................................................................................................71 ZORBAX Capillary and Nano .............................................................................................................................................73 ZORBAX PrepHT.................................................................................................................................................................80 Agilent Prep LC Columns ...................................................................................................................................................84 Ultron Chiral columns.........................................................................................................................................................87

ZORBAX Columns for Bioanalytical Chromatography................................89

ZORBAX 300 StableBond................................................................................................................................................94 ZORBAX 300 Extend-C18................................................................................................................................................99 ZORBAX Poroshell............................................................................................................................................................102 ZORBAX Eclipse Amino Acid Analysis (AAA) Columns....................................................................................................105 ZORBAX GF-250 and GF-450 Gel Filtration Columns .....................................................................................................107 ZORBAX Ion Exchange Columns SAX and SCX ...........................................................................................................111

New columns deliver on the promise of greater productivity and superior peak shape for acids, bases, and neutrals.
Whether you are performing conventional or ultra-fast chromatography, separating biomolecules, or analyzing complex basic compounds, you can trust Agilent for the industrys highest-performing columns that deliver the fast, reproducible results you need. All engineered with Agilents unparalleled quality and reliability. Now, Agilent has launched two exciting new column lines that will help you meet the challenges of faster turnaround times, unrelenting workloads, and fewer staff members:

NEW ZORBAX Eclipse Plus LC columns are proven to deliver reproducible peak symmetry for basic compounds. Eclipse Plus columns stand up to even outperform the competition, and are designed to help you achieve excellent peak shape and resolution. NEW 600 bar ZORBAX Rapid Resolution High Throughput (RRHT) 1.8 m LC columns allow you to increase the speed of your separations without compromising the quality of your resolution and results.

And remember, when you choose ZORBAX Eclipse LC columns, you get more than just a dependable product. You also get over 40 years of expertise along with unmatched technical support from the worlds largest chromatography supplier. On the web, by phone or in person, Agilent helps you solve the problems that can slow you down and get in the way of your results.

Order online at www.agilent.com/chem/store

Featured Products

Run samples up to 20 times faster than conventional LC with the complete Agilent 1200 Series Rapid Resolution System.
Agilents NEW 1200 Series HPLC is designed with the same attention to detail, quality and superior performance you expect from Agilent instruments. And it is backed by over 40 years of chromatography experience. Together with Agilents NEW ZORBAX RRHT 1.8 m LC columns, the Agilent 1200 Series can help you produce your numbers more efficiently than ever before, so you can meet your most difficult deadlines. To learn more, visit www.agilent.com/chem/1200RR

60% more resolving power than traditional LC A peak capacity greater than 600 bar 30-second cycle time Efficiencies above 60,000 plates The ability to run your current methods Flexible, comprehensive services to ensure peak performance 24x7 technical support by phone or Web Access application notes, specifications, chromatogram libraries, and more at www.agilent.com/chem

Put the full productivity-boosting power of the 1200 Series Rapid Resolution System to work for you! ZORBAX RRHT columns deliver very high efficiencies in both short and long column lengths. To take full advantage of this efficiency, however, you should optimize your 1200 Series HPLC with Agilents long-life lamp and low-volume flow cells equipped with RFID tags for unambiguous data traceability.

Order online at www.agilent.com/chem/store

Agilents high-performance ZORBAX Eclipse LC column family

Eliminate the hassles of trial and error while increasing separation speed, reproducibility and method scalability.
From simple analysis to complex method development. ZORBAX Eclipse LC columns can enhance your productivity, while generating reproducible results across a wide range of applications and conditions. Eclipse LC columns feature double-endcapping and a unique bonding process. This ensures lot-to-lot consistency, extends column life, and allows you to retain methods for the long run. Additionally, Eclipse LC columns are based on durable particle technology for superior long-term chromatographic reliability and column lifetime.

Eclipse LC columns are the only scalable column family (from 1.8 m to 7 m) that lets you...
Minimize tailing and maximize peak symmetry for bases, acids and neutrals. Develop and transfer methods without re-validation. Deliver reproducible results across a wide pH range and under varied separation conditions. Scale from analytical to prep or from conventional to ultra-fast methods with particle sizes from 1.8 m to 7 m. Process samples up to 20 times faster and improve resolution up to 60% with 600 bar ZORBAX Rapid Resolution High Throughput columns.

Order online at www.agilent.com/chem/store

ZORBAX HPLC Column Selection Guidelines

HPLC column selection and method development strategies

To make it easier to select the right column for your specific application, we have included the following reference material. So you can maximize your most valuable resource time.

HPLC Column Selection Flow Chart

Here, you will find step-by-step instructions on choosing an initial column for method development, based on measurable factors 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 Column

We will show you how to select the best column for small molecule and protein/peptide analysis, based on factors such as bonded phase and column configuration.

Quick Guide to USP Categories For HPLC Columns

This section clearly and concisely lists the U.S. Pharmacopeia categories for Agilents ZORBAX column family including our new Rapid Resolution HT and Eclipse Plus columns with small particle sizes. So you can more easily select the columns that conform to USP standards and monographs.

Reversed-Phase HPLC Method Development

Chromatographic resolution between two or more peaks depends upon column efficiency, selectivity, and retention all factors that are affected by pH. This section describes a method development strategy based on changing the pH level of the mobile phase.

Order online at www.agilent.com/chem/store

Column Selection
To use the column selection guide diagram below, simply follow the path for your analyte and mobile phase. At the far right, follow your final column selection to the pages indicated. Please see the 2007-2008 Essential Chromatography and Spectroscopy Catalog for a complete listing of LC columns and supplies.

Normal-Phase Mode with Bare Silica Hexane Soluble Normal-Phase Mode with Bonded Phase MeOH and MeOH: H2O or ACN and ACN: H2O Soluble Organic Solvent Soluble Gel-Permeation (Small Molecule) THF Soluble Molecular Weight <2000 Reversed-Phase Mode with Bonded Phase Non-Ionic Reversed-Phase Mode with Bonded Phase Using Ionization Control Water Soluble Reversed-Phase Mode with Bonded Phase Using Ion-Pair Agent Ionic Reversed-Phase Mode with Bare Silica

see pages 52-53 see pages 52-53

Reversed-Phase Mode with Bonded Phase

see page 19

see page 19

see page 19

see page 19

Sample

see page 51

Ion-Exchange Mode

Organic Solvent Soluble

Gel-Permeation Chromatography

Molecular Weight >2000

Gel-Filtration Chromatography

see page 107

Water Soluble

Ion-Exchange Mode with Wide-Pore Material Reversed-Phase Mode with Wide-Pore Material

see page 111

see page 94

Adapted with permission from "Practical HPLC Methodology and Applications," Brian A. Bidlingmeyer, John Wiley & Sons, Inc., New York, p. 109

Order online at www.agilent.com/chem/store

ZORBAX HPLC Column Selection Guidelines

Quick Guide to ZORBAX Reversed-Phase Bonded Phases

Modern ZORBAX RP-HPLC Columns Recommended Uses and Applications Eclipse Plus 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 m columns Rigorous QA/QC testing for greater long-term reproducibility Eclipse XDB 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 m columns Choices from capillary to prep StableBond (SB) Basic, acidic, neutral compounds Exceptional stability at low pH Use of high temperature (up to 90C for C18, 80C 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 m columns ZORBAX Rx General separation of basic, acidic and neutral compounds at low pH with different selectivity than SB columns Rx-C8 is the same as SB-C8 Bonus-RP 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 m columns Extend-C18 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 molecules or peptides Separating at high, mid-range and low pH for selectivity changes Rapid separations using 3.5 m columns Original ZORBAX Columns Recommended Uses and Applications ZORBAX General separation of basic, acidic, neutral compounds at low pH with different selectivity than SB columns; higher number of active silanols than SB "Mixed mode" separation at more neutral pH values ZORBAX ODS Classic General separation of basic, acidic, neutral compounds at mid-range to low pH with (non-end capped) different selectivity than SB or XDB columns Page No. 2021

26

33

39

45

41

Page No. 51

51

Order online at www.agilent.com/chem/store

ZORBAX Reversed-Phase HPLC Column Selection Flow Chart

For small and large molecules
Most chromatographers use reversed-phase HPLC as one of their key analysis techniques. Reversed-phase HPLC can be used to analyze ionic and nonionic analytes. Therefore this ZORBAX Column Selection Flow Chart will focus on reversed-phase columns. To more easily select a reversed-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 Molecules
MW < 2000

Large Molecules
MW > 2000

80-120

Packing Pore Size

300

First 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 standard 80-120 pore packings, but peptides and proteins may not. For this reason, it is recommended to use 300 pore-size packings (300SB) for isocratic or gradient separations of peptides and proteins.

Eclipse Plus C18

Starting Column Bonded Phase

StableBond 300SB-C18

A 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 mm

Column Diameter

4.6 mm

Column Diameter defines how much material (mass) you can inject on a column the smaller the diameter, the less material that can be injected while maintaining good peak shape. Smaller diameter columns provide increased sensitivity and therefore are commonly used for LC/MS and sample limited applications.

Column Type

Inner Diameter (mm)

Sample Load (approx)

Typical Flow Rate

Increased Sensitivity

Applications

Analytical Solvent Saver NarrowBore Microbore Capillary Nano Semi-Prep Preparative

4.6 3.0 2.1 1.0 0.5, 0.3 0.1, 0.075 9.4 21.2

0.1-1.5 mg 150-500 ug 50-120 ug 10-50 ug 1-10 ug 100-200 ng 1-10 mg 20-250 mg

0.5-3 mL/min 0.3-1.5 mL/min 0.1-0.5 mL/min 10-100 L/min 1-15 L/min 200-500 nL/min 5-10 mL/min 20-60 mL/min + ++ ++++ +++++ ++++++

Standard separations Save solvent, uses standard HPLC instrument High sensitivity, limited sample, LC/MS, save solvent High sensitivity, limited sample, LC/MS Very high sensitivity, LC/MS, peptides and proteins Very high sensitivity, LC/MS, peptides and proteins mg preparative separations Hundreds of milligrams up to 1 gram

ZORBAX HPLC Column Selection Guidelines

Small Molecules
MW < 2000

Large Molecules
MW > 2000

5 m

Standard Porous Particle Size

5 m

For conventional analyses of small and large molecules, the standard particle size is 5 m. However, smaller particle sizes are available and provide higher efficiency and higher resolution. They are also available in short column lengths.

Fast Analysis Particle Sizes

Rapid Resolution 3.5 m Rapid Resolution HT 1.8 m

Rapid Resolution 3.5 m particles provide 60% greater efficiency than 5 m particles while Rapid Resolution HT 1.8 m particles provide 200% greater efficiency. Shorter columns can be used to significantly reduce analysis time of small molecules.

Rapid Resolution 3.5 m Poroshell 5 m

For peptides, Rapid Resolution 3.5 m particles can be used with shorter columns for faster, high-resolution gradient analyses. But for proteins, Poroshell columns, with a solid core and porous shell, provide the fastest analysis.

150 mm

Column Length

150 mm

A conventional column length is 150 mm or 250 mm for high resolution with 5 m particles. This is a good place to start a typical analysis because it will provide high resolution in a 20-30 minute analysis. For fast analysis, shorter columns (75 mm and 50 mm) with smaller particle sizes (3.5 m) 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 for a high-resolution analysis. For fast analysis, shorter columns (50 mm) with smaller particles (3.5 m) should be used.

Starting Column Choices

Small Molecules Large Molecules

Standard Analysis Eclipse Plus C18

4.6 x 150mm, 5m 959993-902

Fast Analysis Rapid Resolution Eclipse Plus C18

4.6 x 150mm, 5m 959993-902

Standard Analysis ZORBAX 300SB-C18

4.6 x 150mm, 5m 883995-902

Fast Analysis Rapid Resolution ZORBAX 300SB-C18

4.6 x 50mm, 3.5m 865973-902

Rapid Resolution HT, 600 bar Eclipse Plus C18

4.6 x 50mm, 1.8m 959941-902

ZORBAX Poroshell 300SB-C18

2.1 x 75mm, 5m 660750-902

Column and Mobile Phase Guidelines: Reversed Phase

HPLC columns consist of two parts, the column chemistry and hardware. Consider first the molecular weight of your analyte to select pore size and then a bonded phase. Consult the sections on Analytical and Bioanalytical columns for the best bonded phases for small and large molecules. For choosing column hardware and particle sizes, consult the section on column sizes and rapid separations, including Rapid Resolution and Rapid Resolution HT columns, as well as Solvent Saver and Capillary columns and new PrepHT columns.

Pore Size Selection

Choose a column packing with small pore (60-100) if the solute molecular weight is less than about 5000. Otherwise, use column packing with the 300 pore size.

Particle Size Selection

The standard particle size for HPLC columns is 5 m with 3.5 m increasing in use. If high-speed analyses or higher resolution analyses are required, packing with 1.8 m and 3.5 m particles can be used. Shorter columns with these particles can produce faster high-resolution separations, with the 1.8 m particle size in Rapid Resolution HT columns, providing the highest efficiency. The 3.5 m particle size operates at a routine operating pressure and can be used on all LC's. Short (50 mm and shorter) 1.8 m RRHT columns can be used on optimized standard LC's, while the longer columns may require a higher pressure LC (one supporting pressures greater than 400 bar).

Column Configuration
The column sizes most often recommended for analytical method development are 4.6 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 method development, choose the column internal diameter (e.g., 2.1, 3.0 mm) to accommodate additional application objectives (e.g., sensitivity, solvent usage) or compatibility with certain instrument types (capillary, nano, or prep).

Silica Type and Bonded Phase

Silica Type
ZORBAX reversed phase columns use two different types of porous silica microspheres, the original ZORBAX SIL and ZORBAX Rx-SIL. ZORBAX Rx-SIL is a highly purified, less acidic silica than the original ZORBAX SIL. Less acidic silica means less potential for interaction between the analyte and silanol groups on the silica surface, especially if the solutes are basic, and contributes to improved peak shape. For new method development, we strongly recommend using reversed-phase products based on ZORBAX Rx-SIL (Eclipse, StableBond etc.). However, many excellent methods have been developed on reversed phase columns based on ZORBAX SIL and we continue to manufacture these high quality, reliable products.

10

Order online at www.agilent.com/chem/store

ZORBAX HPLC Column Selection Guidelines

Bonded Phase
A good first choice for bonded phase is C18 or C8. If the sample solutes of interest are not adequately separated on these columns, CN and Phenyl columns may offer significant differences 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-phase columns (C3, CN) and peptides and small molecules are separated on longer-chain columns (C8, C18). There are many cases, however, where this conventional wisdom does not apply. For example, peptides can also be effectively separated using short-chain columns, and hydrophobic peptides can show better recovery on longer-chain phases. Therefore, it is best to initially select a phase in the middle of the hydrophobic spectrum (e.g., C8), then change to a more hydrophobic phase or more hydrophilic phase depending on initial results and solubility properties of your sample.

pH and Mobile Phase

The choice of mobile phase for a reversed-phase system starts with selecting the organic modifier. Selectivity differences and sample retention will vary significantly among mobile phases containing acetonitrile, methanol, and tetrahydrofuran (THF). Sample solubility is likely to differ in such solvents and dictate use of a specific solvent or solvents. UV detection at certain 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 parameters in developing rugged methods that are not sensitive to small variations in conditions. With ionic compounds, retention of typical species shows significant changes with pH. It is very important to control pH in such reversed-phase systems to stabilize retention and band spacing. A pH set between 2 and 4 generally provides the most stable conditions for retention vs. small changes in pH and this pH is recommended for starting method development for most samples, including basic compounds and typical weak acids.

HPLC Column

Stationary Phase

Column Dimensions

Type of Surface

Pore Size

Particle Size

Length

Inner Diameter

Chemical Properties Chemical Lifetime/Sensitivity Retention Factor

Physical Properties Efficiency Speed

Order online at www.agilent.com/chem/store

11

USP Designations
The US Pharmacopeia (USP) is a standard source for many pharmaceutical methods. The USP specifies columns by packing materials rather than by manufacturer. The USP has updated its L1 definitions. Listed below you will see the most recent definitions and columns that 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.

USP Designations L1

USP Packing Materials Octadecyl silane chemically bonded to porous silica or ceramic micro-particles, 1.5 to 10 m in diameter

L3 L7

Porous silica particles, 5 to 10 m in diameter Octyl silane chemically bonded to totally porous microsilica particles, 1.5 to 10 m in diameter

L8 L10 L11 L13 L14 L20 L33

An essentially monomolecular layer of aminopropyl-silane chemically bonded to totally porous silica gel support, 10 m in diameter Nitrile groups chemically bonded to porous silica particles, 3 to 10 m in diameter Phenyl groups chemically bonded to porous silica particles, 1.5 to 10 m in diameter Trimethylsilane chemically bonded to porous silica particles, 3 to 10 m in diameter Silica gel 10 m in diameter with a chemically bonded, strongly basic quaternary ammonium anion-exchange coating Dihydroxypropane groups chemically bonded to porous silica particles, 3 to 10 m in diameter Packing having the capacity to separate proteins by molecular size over a range of 4,000 to 400,000 da. It is spherical, silica-based, and processed to provide pH stability A zirconium-stabilizes spherical silica packing with a hydrophilic (diol-type) molecular monolayer bonded phase Propyl silane chemically bonded to totally porous silica particles, 3 to 10 m in diameter A chiral-recognition protein, ovomucoid, chemically bonded to silica particles, about 5 m in diameter, with a pore size of 120 angstroms.

Columns ZORBAX Eclipse Plus C18 ZORBAX Eclipse Plus XDB-C18 ZORBAX StableBond SB-C18 ZORBAX Extend-C18 ZORBAX Rx-C18 ZORBAX ODS ZORBAX ODS Classic ZORBAX Sil ZORBAX Rx-Sil ZORBAX Eclipse Plus C8 ZORBAX Eclipse XDB-C8 ZORBAX SB-C8 ZORBAX Rx-C8 ZORBAX C8 ZORBAX NH2 ZORBAX Eclipse XDB-CN ZORBAX SB-CN ZORBAX CN ZORBAX Eclipse XDB-Phenyl ZORBAX SB-Phenyl ZORBAX Phenyl ZORBAX TMS ZORBAX SAX ZORBAX GF-250 ZORBAX GF-250

Particle Size (m) 1.8, 3.5, 5, 7

3, 5 5 5 5 1.8, 3.5, 5, 7 3.5, 5 3, 5 5 3.5, 5 3.5, 5 3, 5 3.5, 5 1.8, 3.5 5 5 5 4 4

Pore Size () 95 80 80 80 80 70 70 70 80 95 80 80 80 70 70 80 80 70 80 80 70 70 70 150 150

L35 L56 L57

ZORBAX GF-250 ZORBAX SB-C3 Ultron ES-OVM

4 6 3.5, 5 5

150 80 120

12

Order online at www.agilent.com/chem/store

ZORBAX HPLC Column Selection Guidelines

Method Development from pH 1-12

Chromatographic resolution between two or more peaks depends upon three factors column efficiency, selectivity, and retention. With ionizable analytes bases and acids all of these factors change dramatically with pH. For example, retention can be improved by changing the separation pH, so that analytes are separated in their non-ionized form. Changes in mobile phase pH also improve column efficiency because the ionization of the analyte and the residual silanols can both be altered. This minimizes secondary interactions between analytes and the silica surface that cause poor peak shape. Achieving optimum resolution can also require changing the mobile phase pH. The following method development strategy explains how this is done with superior column lifetime. Low, mid, and high pH are the three general regions for chromatographic separations as defined in Figure 1. This figure highlights the benefits of performing separations of ionizable analytes in each pH region. Method development proceeds by investigating chromatographic separations first at low pH and then at higher pH until optimum results are achieved. The ideal column is available for each pH region.

Figure 1: Three pH Regions for HPLC Separations of Basic Compounds This figure represents the retention behavior of one basic analyte with respect to pKa and pH. Analyte pKa is 6.5

Low pH < 3 Region A Start method development at low pH, where silanols on a RP-HPLC column are protonated. This minimizes peak tailing by eliminating silanol/base interactions. At low pH, basic compounds are positively charged and their retention may be reduced. Acidic compounds may be protonated and have increased retention. Retention times are usually stable with small changes in pH, producing a robust method. Volatile mobile phase additives, such as formic acid or trifluoroacetic acid (TFA), are often used at low pH with LC/MS.

Mid pH 7 Region B Develop methods at pHs at least 1 pH unit above or below the pKa to minimize changes in retention with small changes in pH. Some silica surface SiOH groups become SiO above pH 4 to 5; tailing interactions may be possible. Minimize interactions by selecting a well designed and endcapped column, using additives such as TEA (triethylamine) (less desirable) or using "polar-linked" bonded phases. Silica breakdown is prevented by innovative bonding chemistry, heavy endcapping, and use of Rx-SIL.

High pH > 9 Region C In this region, basic compounds may be in their free base form. Increased retention and resolution of basic compounds are likely. Retention changes little in this region, thus robust methods can be developed. Silica breakdown is prevented by innovative bidentate column chemistry, heavy endcapping, use of Rx-SIL, and optimum mobile phase. Ammonium hydroxide is an excellent volatile mobile phase modifier at high pH.

Order online at www.agilent.com/chem/store

13

Start method development at low pH (pH 2-3)

With so many column choices available, how do you know where to start your method development? The recommended starting point for method development is using a buffered low pH mobile phase around pH 2-3. Using a low pH mobile phase most often results in the best peak shape for basic compounds on silica-based columns. At low pH, the silanols on the silica are fully protonated so positively charged basic compounds do not interact strongly. The result is good peak shape. Many acidic compounds are non-charged, maximizing their retention at low pH. These observations are key advantages to method development at low pH. For standard analytical work, start method development with acetonitrile as the mobile phase organic modifier and 20-50 mM phosphate buffer (pH 2-3) as the aqueous component for non LC/MS applications. These conditions provide good pH control, necessary for the most reproducible analyses of ionizable compounds. For LC/MS applications formic acid or TFA are good mobile phase additives for low pH.

Choose ZORBAX Eclipse Plus first for best peak shape

Select 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 silica and bonding technologies to provide good peak shape for basic compounds. Eclipse Plus columns can be used from pH 2-9 providing method development flexibility. They are stable down to pH 2 making them an ideal choice for initial method development.

14

Order online at www.agilent.com/chem/store

ZORBAX HPLC Column Selection Guidelines

Optimize solvents and bonded phases at low pH

The initial method development steps may lead very quickly to a satisfactory separation. But if more optimization is needed, acetonitrile can be replaced by methanol or tetrahydrofuran and the separation re-optimized. This step may lead to a satisfactory solution, but if still more selectivity optimization is needed, the column bonded phase can be changed. At low pH there are many bonded phase choices available for optimization. These include the Eclipse Plus phases as well as the Eclipse XDB family with C18, C8, Phenyl and CN. Alternate choices 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 these situations, lower the pH even further, down to pH 1-2, and use StableBond columns. These columns provide the greatest stability at very low pH and provide many selectivity options for achieving the highest resolution separations.

Order online at www.agilent.com/chem/store

15

Method development at mid pH (4-9) ZORBAX Eclipse Plus

There are some samples that may not be resolved at low pH or may have better solubility and stability at mid pH. While still using the Eclipse Plus C18 column, the mid pH range can be used for method development. The Eclipse Plus column is stable up to pH 9 so it is equally reliable at mid pH. These double endcapped columns have two key advantages good peak shape at low and mid pH, as well as sufficient bonded phase density to protect the column from silica degradation from pH 6-9. At mid pH, basic compounds (e.g., amines) may still have a positive charge and the silanols on the silica surface may have a negative charge. Therefore covering as many silanols as possible leads to the best peak shape at mid pH. This makes the Eclipse Plus C18 the best starting choice for a column at mid pH. Phosphate buffer is usually the first choice for mobile phase modifier at pH 7 because its buffer range is pH 6.1-8.1. A second choice for mid pH is acetate buffer since it buffers from pH 3.8-5.8 and its volatility makes it a good choice for LC/MS compatibility.

Alternate selectivities ZORBAX Eclipse XDB-Phenyl, CN and Bonus-RP

The method development process at mid pH mimics the process at low pH with optimization of the organic modifier and selecting an alternate bonded phase if resolution is not achieved after that step. The alternate bonded phases at mid pH are the Eclipse XDB-Phenyl, Eclipse XDB-CN and Bonus-RP. They provide very different selectivities for many samples and the method development process is followed again. The Bonus-RP column has a polar embedded amide group that provides different selectivity for many samples, provides good peak shape for basic compounds and allows the column to be used with up to 100% aqueous mobile phases.

16

Order online at www.agilent.com/chem/store

ZORBAX HPLC Column Selection Guidelines

START Poorly retained compounds

SAMPLE
Initial separation

Method Development Guidelines from Low to High pH

Low pH3

STEP 1
ZORBAX Eclipse Plus C18 or C8 pH 2.5 (2-3) 20-50 mM buffer T=30C (ambient to 60C) Adjust % ACN for 0.5 < k < 20

Poorly retained compounds

Acids

Bases STEP 1a

OR

STEP 2a
Band spacing problems

Vary column temperature, up to 60C for Eclipse XDB up to 8090C for Stablebond

STEP 2
Band spacing problems

Increase or decrease % of organic modifier by 5% (v/v)

Band spacing problems

ZORBAX SB-C18 or SB-C8 pH 1-3 20-50 mM buffer T = 30C (ambient to 90C) Adjust % ACN for 0.5 < k < 20
Band spacing problems

STEP 1b STEP 3
Change organic modifier (MeOH or THF) Adjust % organic for 0.5 < k < 20 Restart at STEP 2
Band spacing problems

STEP 1c
Increase or decrease % of organic modifier by 5% (v/v)
Band spacing problems

BASIC COMPOUNDS (Ion Pairing) Use MeOH organic modifier, 10 mM hexane sulfonic acid, 25-50 mM pH 3 buffer Adjust % MeOH for 0.5 < k < 20

STEP 4
Change bonded-phase functionality to Eclipse XDB-C18, C8, Phenyl SB-CN, AQ, Phenyl, Bonus-RP Restart at STEP 1
Band spacing problems

Band spacing problems

STEP 1d

Increase or decrease % of organic modifier by 5% (v/v)

STEP 5 OR
ZORBAX Eclipse Plus C18 or C8 pH 7 (6-9), 20-50 mM buffer T = 30C (ambient to 40C) Adjust % MeOH for 0.5 < k < 20
Band spacing problems

STEP 1e
Change organic modifier (ACN or THF) Adjust for 0.5 < k < 20

Mid pH 7

Poorly retained acidic compounds STEP 5a STEP 6

ACIDIC COMPOUNDS (Ion Pairing) Use MeOH organic modifier, 10 mM tetrabutylammonium phosphate, 25-50 mM pH 7 buffer Adjust % MeOH for 0.5 < k < 20
Band spacing problems

Increase or decrease % of organice modifier by 5% (v/v)

Band spacing problems

STEP 7
Change organic modifier (ACN or THF) Adjust for 0.5 < k < 20 Restart at STEP 6
Band spacing problems

STEP 5c
Increase or decrease % of organic modifier by 5% (v/v)
Band spacing problems

STEP 5b
Vary temperature within recommended range for bonded phase

STEP 8
Change bonded-phase functionality to ZORBAX Eclipse XDB-Phenyl or Bonus-RP Restart at STEP 5
Band spacing problems

Band spacing problems

STEP 5d
Change organic modifier (ACN or THF) Adjust for 0.5 < k < 20

STEP 9
ZORBAX Extend-C18 pH 10.5 (9-11.5); 5-20 mM ammonia or TEA, or 10-50 mM organic buffer, or borate buffer T = 25C (ambient to 40C) Adjust % MeOH for 0.5 < k < 20

High pH 9
STEP 10a
Vary temperature within recommended range for bonded phase Band spacing problems

Band spacing problems

STEP 10
Change organic modifier (ACN or THF) Adjust for 0.5 < k < 20
Band spacing problems Band spacing problems

Try different HPLC mode.

Contact Agilent Technologies.

17

Cartridge Column Systems

Agilent offers a variety of popular HPLC packing material 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 Guide

Icon Type of Cartridge Agilent HPLC Cartridge Features Can reverse collets in the end fitting to add guard cartridges Cartridges have a unique filter and sieve at each end High efficiency, stand-alone, low dead volume cartridge Polymeric cartridge designed for leak-tight seals against metal surfaces Reusable fittings For high throughput LC/MS, LC/MS/MS and combinatorial separations Packed with Eclipse XDB for pH use from 2-9 Packed with StableBond for low pH use Sold individually or as three-packs Easy low-dead-volume assembly Tubing (polyphenylene sulfone) designed for leak-tight seals against metal surfaces Reusable fittings Benefits Inexpensive Extends column lifetime Permits rapid column changes Can use 2, 3, 4 and 4.6 mm cartridges Helps prevent blockage Seals up to 400 bar No gaskets required More solvent-resistant than PEEK Adapt for connections to 1/16 in. LC fittings

ZORBAX Guard Cartridge: Stand alone system

ZORBAX Rapid Resolution and Rapid Resolution HT Cartridge Columns: 3.5 m and 1.8 m packings, stand alone system ZORBAX Semi-Preparative Guard HPLC Hardware Kit: Stand alone system

For all analyte types Low bleed Seals up to 2000 psi (135 bar, 13.5 MPa) No gaskets required Adapt for connections to 1/16 in. LC fittings Extends column lifetime Permits rapid column changes Can use with 21.2 and 30 mm ID columns

ZORBAX and Agilent Prep Preparative Easy low-dead-volume assembly Cartridge Column and Guard HPLC System: Reusable fittings Stand alone and integral hardware options Hardware options for integral and external guards

18

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

Achieve excellent peak shape and resolution and eliminate false starts.
Good news for analysts who do not have time to make columns work for a particular application: ZORBAX columns let you choose the right column based on your sample and mobile phase eliminating any guesswork. Additionally, Agilents ZORBAX silica is manufactured by Agilent not purchased from outside 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 optimum resolution over a wide pH range, including:

ZORBAX Eclipse Plus HPLC columns designed to reliably produce superior peak shape for basic compounds. 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 develop high-resolution separations at high pH levels. ZORBAX Bonus-RP HPLC columns an alkyl-amide column that offers excellent peak shape for bases, along with an alternative selectivity. ZORBAX method development kits contain three columns for the price of two! Each has a different bonded phase for optimal selectivity. ZORBAX method validation kits choose as many columns as you need (or as few) to make method validation easier and less expensive. Original ZORBAX HPLC columns are made with Type A silica and are used in many established separation methods.

Order online at www.agilent.com/chem/store

19

No LC column has ever reliably produced such symmetrical peaks for basic compounds until now.
Introducing ZORBAX Eclipse Plus LC columns the proof is in the performance!
New Eclipse Plus columns provide the ultimate in performance and productivity for silica-based columns. Like all Eclipse columns, Eclipse Plus columns achieve their superior performance through eXtra Dense Bonding, combined with a precise double-endcapping process. But Eclipse Plus columns also feature enhanced high-performance silica, new endcapping reagents, and a unique bonding process that is optimized for the improved silica. Eclipse Plus columns are available in 1.8, 3.5, and 5 m particle sizes for all your analytical, high-resolution, and fast LC analyses. From nano, to analytical, to prep, Eclipse columns allow you to efficiently scale methods from 1.8 m to 7 m and to transfer methods anywhere in the world without worrying about reproducibility. So the methods you create today will not have to be re-validated tomorrow.

20

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

Achieve excellent peak shape for acids, bases, and neutrals for greater resolution and accuracy. Significantly reduce tailing, so you can confidently resolve difficult analytes. Perform ultra-fast, fast, or conventional LC under a range of temperatures, pressures, and pH conditions. Spend more time running samples, performing analyses, and meeting deadlines, and less time re-evaluating new column chemistries or revalidating protocols. Develop reliable HPLC methods under demanding deadlines. Choose Eclipse Plus Rapid Resolution High Throughput columns with a 1.8 m particle size for ultra-fast separations and superior method flexibility.

Column Specifications
Bonded Phase ZORBAX Eclipse Plus C18 ZORBAX Eclipse Plus C8 Pore Size 95 95 Surface Area 160 m2/g 160 m2/g Temp. Limits pH Range 60C 2.0-9.0 60C 2.0-8.0 Carbon Endcapped Load Double 8% Double 8%

Order online at www.agilent.com/chem/store

21

Eclipse Plus: Best Peak Shape in the Industry Without Tailing

Column:

Eclipse Plus C18 959996-902 4.6 x 100mm, 5m

1

Eclipse Plus C18

Ace 5 C18 SunFire C18 XBridge C18 Discovery HS C18 Luna C18 (2) Gemini C18 XTerra MS C18
0 0.5 1 1.5

Mobile Phase: Flow Rate: Temperature: Detector: Publication: Sample:

A: 60% Water B: 40% Acetonitrile 1.0 mL/min Ambient UV 254 nm 5989-4934EN Pyridine, Phenol

1. Pyridine 2. Phenol
2 2.5 3 3.5 min

Eclipse Plus C18 vs. C8 and RRHT vs. RR Column A: Eclipse Plus C18 959941-902 4.6 x 50mm, 1.8m Column B: Eclipse Plus C8 959943-906 4.6 x 50mm, 3.5m Mobile Phase: Flow Rate: Temperature: Detector: Sample: Water: Acetonitrile (30:70) 2.0 mL/min 30C UV 230nm Lip balm extract in ACN (melted at 100C ACN , cooled and 0.45 m filtered) 1. Oxybenzone 2. Internal Std. 3. Octylmethoxycinnamate
1. Oxybenzone 2. Internal Std. 3. Octylmethoxycinnamate N: 8800 Pw: 0.014 min. TF: 1.20
0 1 2

247 bar
N: 11600 Pw: 0.047 min. TF: 1.06
3 4

N: 11800 Pw: 0.093 min. TF: 1.02

min

N: 5000 Pw: 0.019 min. TF: 1.09

N: 6600 Pw: 0.037 min. TF: 0.99

N: 6300 Pw: 0.061 min. TF: 0.98

88 bar 22 min.
3 4 min

Less retention can save significant time the C8 is a good choice here. The RRHT column is delivering the efficiency and resolution expected, but the C8 bonded phase may be the best choice.

22

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

Rapid Analysis of an Analgesic Tablet, Selectivity Differences at pH 2 and pH 7

Column:

Eclipse Plus C8 959946-906 4.6 x 50mm, 5m

Gradient:

1. 2. 3. 4.

Acetaminophen Caffeine Acetylsalicylic acid Unknown

1

Sample:
3

10-60% B/3 min pH 2.7: A: 0.1% formic acid B: 0.1% fa in ACN pH 7.0: A: 20 mM Na phosphate B: ACN generic Excedrin tablet
pH 2.7

2
0 0.5 1 1.5 2 2.5 3

4
3.5 m

1 3 4
0 0.5 1 1.5

pH 7.0

2
2

2 min.
2.5 3 3.5 m

Both Eclipse Plus C18 and C8 can be used over a wide pH range to optimize selectivity or analysis time.

Eclipse Plus C8 is Less Retentive than Eclipse Plus C18

Column A: Eclipse Plus C8 959996-906 4.6 x 100mm, 5m Column B: Eclipse Plus C18 959996-902 4.6 x 100mm, 5m
3.789 Tf=1.04

Mobile Phase: Flow Rate: Detector: Sample:

80% Methanol 8mM (total) K2HPO4 pH 7 1.0mL/min UV 215nm Amitriptyline 0.05 g/L (0.5 L injection)

min

5.777 Tf=1.00

min

A 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 peak shape on difficult basic compounds.

Order online at www.agilent.com/chem/store

23

Peak Shape and Efficiency are Better with ZORBAX Eclipse Plus

Column A: XBridge C18, 4.6 x 150mm, 5m

N = 14000 Tf = 1.25 2 3

N = 41000 Tf = 1.13 6

10

12

min

Column B: Eclipse Plus C18 959993-902 4.6 x 150mm, 5m

1 N = 23000 Tf = 1.01 2 3 N = 60000 Tf = 1.00 6

10

12

min

Mobile Phase: Flow Rate: Gradient:

A: 0.1% formic acid B: 0.1% formic acid in ACN 1.0 mL/min 0.0 min 10% B 15 min 30% B Temperature: 40C Detector: UV 254 nm Publication: 5989-4934EN Sample: Sulfonamides

1. 2. 3. 4. 5. 6.

Sulfanilamide Sulfadiazine Sulfathiazole Sulfamerazine Sulfmethazine Sulfamethoxazole

Fast and Ultra-Fast Analysis of Basic Compounds on Eclipse Plus

A
O O Tf = 1.00 N+ ClOCH3 OCH3

Column A: Eclipse Plus C18 959941-902 4.6 x 50mm, 1.8m

1. Berberine

Mobile Phase: Flow Rate: Temperature: Detector: Publication: Sample:

A: 50% 8 mM K2HPO4, pH 7 B: 50% ACN 1.0 mL/min Ambient UV 254 nm 5989-4934EN Berberine, 0.4 mg/mL, 2 uL

0.5

1.5

2.5

min

Column B: Eclipse Plus C18 959993-902 4.6 x 150mm, 5m 959993-902

Tf = 1.06

0.5

1.5

2.5

min

24

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

ZORBAX Eclipse Plus

Hardware Description Analytical Analytical Analytical Analytical Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Solvent Saver Solvent Saver Plus Solvent Saver Plus Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Narrow Bore Narrow Bore RR Narrow Bore RR Narrow Bore RR Narrow Bore RR Narrow Bore RR Narrow Bore RRHT, 600 bar Narrow Bore RRHT, 600 bar Narrow Bore RRHT, 600 bar Guard Cartridges, 4/pk Guard Cartridges, 4/pk Guard Hardware Kit Particle Size (mm) Size (m) 4.6 x 250 5 4.6 x 150 5 4.6 x 100 5 4.6 x 50 5 4.6 x 150 3.5 4.6 x 100 3.5 4.6 x 75 3.5 4.6 x 50 3.5 4.6 x 30 3.5 4.6 x 150 1.8 4.6 x 100 1.8 4.6 x 50 1.8 4.6 x 30 1.8 3.0 x 150 5 3.0 x 150 3.5 3.0 x 100 3.5 3.0 x 100 1.8 3.0 x 50 1.8 2.1 x 150 5 2.1 x 50 5 2.1 x 150 3.5 2.1 x 100 3.5 2.1 x 50 3.5 2.1 x 30 3.5 2.1 x 100 1.8 2.1 x 50 1.8 2.1 x 30 1.8 4.6 x 12.5 5 2.1 x 12.5 5 Eclipse Plus C18 USP L1 959990-902 959993-902 959996-902 959946-902 959963-902 959961-902 959933-902 959943-902 959936-902 959994-902 959964-902 959941-902 959931-902 959993-302 959963-302 959961-302 959964-302 959941-302 959701-902 959746-902 959763-902 959793-902 959743-902 959733-902 959764-902 959741-902 959731-902 820950-936 821125-936 820888-901 Eclipse Plus C8 USP L7 959990-906 959993-906 959996-906 959946-906 959963-906 959961-906 959933-906 959943-906 959936-906 959964-906 959941-906 959931-906 959993-306 959963-306 959961-306 959964-306 959941-306 959701-906 959746-906 959763-906 959793-906 959743-906 959733-906 959764-906 959741-906 959731-906 820950-937 821125-937 820888-901

Order online at www.agilent.com/chem/store

25

CH 3 O Si CH 3 CH 3 O Si CH 3 CH 3 O Si CH 3 CH 3 O Si CH 3 CH 3 O Si CH 3 CH 3 CH 3

ZORBAX 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 m to 7 m Long lifetime with extra dense bonding and double endcapping

Extra Densely Bonded and Double Endcapped Eclipse XDB Bonded Phase

The Agilent ZORBAX Eclipse XDB columns C18, C8, Phenyl and CN provide four bonded phase choices for method development optimization. These columns provide good peak shape over a wide 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 same column can be used for method development in the mid pH (6-8) region. In the mid pH region residual silanols are more active and tailing interactions are more likely. To overcome these interactions, Eclipse XDB columns are eXtra Densely Bonded and double endcapped through a proprietary process to cover as many active silanols as possible. The result is superior peak shape of basic compounds from pH 2-9. Eclipse XDB columns are available in 1.8, 3.5, 5 and 7m particle sizes for high speed, high resolution, analytical and prep scale separations.

Column Specifications
Bonded Phase ZORBAX Eclipse XDB-C18 ZORBAX Eclipse XDB-C8 ZORBAX Eclipse XDBPhenyl ZORBAX Eclipse XDB-CN Pore Size 80 80 80 80 Surface Area 180 m2/g 180 m2/g 180 m2/g 180 m2/g Temp. Limits 60C 60C 60C 60C pH Range 2.0-9.0 2.0-9.0 2.0-9.0 2.0-8.0 Endcapped Double Double Double Double Carbon Load 10% 7.6% 7.2% 4.3%

Tips & Tools

Method development is easier and more predictable when you have several bonded phases available on the same silica. In addition to the popular C18 columns, Agilent also offers selectivities that simplify method development.

26

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

Good Peak Shape Over a Wide pH Range with ZORBAX Eclipse XDB Column: Flow Rate: Temperature: A Eclipse XDB-C8 993967-906 4.6 x 150mm, 5m 1.5 mL/min 40C Mobile Phase: pH 3.0 75% 25 mM phosphate buffer pH 3.0: 25% ACN B Mobile Phase: pH 7.0 90% 20 mM phosphate pH 7.0 10% ACN

Sample:

1. Maleate 2. Doxylamine 3. Chlorpheniramine 4. Triprolidine 5. Diphenhydramine

Sample:

1. Procainamide 2. N-acetylprocainamide 3. N-propionylprocainamide

ZORBAX Eclipse XDB columns provide good peak shape over a wide pH range and are an excellent choice for method development from pH 2-9.

Column Stability Testing at pH 3 and 60C Column: ZORBAX SB-C8 883975-906 4.6 x 150mm, 5m Column: Eclipse XDB-C8 993967-906 4.6 x 150mm, 5m Mobile Phase: Purge Conditions: 70% 50 mM NaAc-HCl, pH 3.0 30% ACN Retention Test Conditions: 65% Methanol 35% Water Flow Rate: 1.0 mL/min Temperature: 60C Sample: Tricyclic Antidepressants

ZORBAX SB-C8 P/N 883975-906 ZORBAX Eclipse XDB-C8 P/N 993967-906 Competitive C

Eclipse 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-endcapped column, SB-C8, at pH 3. The columns were purged with a pH 3 mobile phase at 60C. Then they were tested with a strongly basic compound to determine if the endcapping or 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 over the 12,000 column volumes of the test. Another endcapped column shows less stability under these same conditions.

Order online at www.agilent.com/chem/store

27

Column Stability Testing at pH 7.0 Column: Eclipse XDB-C8 993967-906 4.6 x 150mm, 5m Mobile Phase: 60% ACN 40% 250 mM Phosphate Buffer, pH 7.0 Flow Rate: 1.5 mL/min Temperature: 60C Sample: Tricyclic Antidepressants

1. 2. 3. 4. 5.

Uracil Nortriptyline Doxepin Amitriptyline Trimipramine

Double 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 columns used here. The conditions used for this test high temperature (60C) and high salt concentration (250 mM), accelerate the dissolution of silica, causing premature failure of the sil-gel type column.

Selectivity Changes for Basic Compounds with Eclipse XDB and Stable Bond Column A: Eclipse XDB-C8 966967-906 4.6 x 75mm, 3.5m Column B: ZORBAX Rx/SB-C8 866953-906 4.6 x 75mm, 3.5m

Mobile Phase: 70% 25 mM NaH2PO4, pH 3.0 30% Methanol Flow Rate: 1.0 mL/min Temperature: 35C

1. Barbital 2. Sulfamethoxazole 3. Caffeine

Time (min)

Time (min)

Eclipse XDB and StableBond columns are based on the same silica but have different bonding and endcapping. Therefore, they can have very different selectivity for the same sample under the same conditions, as this example shows.

28

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

Optimize Separations with Eclipse XDB Selectivity Options Analysis of Sunscreens

Column A:

Eclipse XDB-Phenyl 963967-912 4.6 x 150mm, 3.5m Eclipse XDB-C8 963967-906 4.6 x 150mm, 3.5m Eclipse XDB-C18 963967-902 4.6 x 150mm, 3.5m

Mobile Phase: Flow Rate: Temperature: Sample:

15% H20: 85% MeOH 1.0 mL/min 35C Sunscreens

Column B:

Column C:

1. Oxybensone 2. Padimate-O 3. Ethylhexylsalicylate

This separation of sunscreens on all three Eclipse XDB bonded phases C18, C8 and Phenyl shows that different bonded phases can be used to optimize a separation. While all three bonded phases provide an adequate separation, the Eclipse XDB-Phenyl provides a different peak elution order and a much shorter overall analysis time. All three bonded phases also provide excellent peak shape with no mobile phase additives.

Separation of Cephalosporins on Eclipse XDB-C8 Column: Eclipse XDB-C8 993967-906 4.6 x 150mm, 5m Mobile Phase: 85% 25 mM Na2HPO4 pH 7: 15% ACN Flow Rate: 1.0 mL/min Temperature: 35C Sample: Cephalosporins 1. 2. 3. 4. 5. Ceftazidine Cefachlor Cefoxatime Cefoxitin Cephalothin

Cephalosporins are a type of antibiotic and many compounds in this family are well separated on the Eclipse XDB-C8 column.

Selectivity for Urea Pesticides 1. 2. 3. 4. 5. 6. Fenuron Monuron MonoLinuron Diuron Linuron Pencycuron

Column A:

Eclipse XDB-C18 993967-902 4.6 x 150mm, 5m Eclipse XDB-CN 993967-905 4.6 x 150mm, 5m

Column B:

Column C:

Eclipse XDB-C18 993967-902 4.6 x 150mm, 5m

Mobile Phase: A. 60:40 MeOH:Water B. 60:40 MeOH:Water C. 77:23 MeOH:Water Flow Rate: 1.0 mL/min Temperature: 25C Sample: Urea pesticides
The Eclipse XDB-CN column reduces retention time and provides good selectivity for Urea pesticides when compared to an Eclipse XDB-C18 column.

Order online at www.agilent.com/chem/store

29

ZORBAX Eclipse XDB

Hardware Description Semi-Preparative Analytical Analytical Analytical Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Solvent Saver Solvent Saver Solvent Saver Plus Solvent Saver Plus Solvent Saver Plus Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Narrow Bore Narrow Bore Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* Narrow Bore RRHT, 600 bar** Narrow Bore RRHT, 600 bar** Narrow Bore RRHT, 600 bar** Narrow Bore RRHT, 600 bar** MicroBore RR* MicroBore RR* MicroBore RR* MicroBore Guard Cartridges, 3/pk Guard Cartridges, 4/pk Guard Cartridges, 4/pk Guard Hardware Kit
*RR: Rapid Resolution 3.5 m **RRHT: Rapid Resolution HT 1.8 m

Particle XDB-C18 Size (mm) Size (m) USP L1 9.4 x 250 4.6 x 250 4.6 x 150 4.6 x 50 4.6 x 150 4.6 x 100 4.6 x 75 4.6 x 50 4.6 x 100 4.6 x 50 4.6 x 30 4.6 x 20 3.0 x 250 3.0 x 150 3.0 x 150 3.0 x 100 3.0 x 75 3.0 x 100 3.0 x 50 3.0 x 30 3.0 x 20 2.1 x 150 2.1 x 50 2.1 x 150 2.1 x 100 2.1 x 75 2.1 x 50 2.1 x 100 2.1 x 50 2.1 x 30 2.1 x 20 1.0 x 150 1.0 x 50 1.0 x 30 1.0 x 17 4.6 x 12.5 2.1 x 12.5 5 5 5 5 3.5 3.5 3.5 3.5 1.8 1.8 1.8 1.8 5 5 3.5 3.5 3.5 1.8 1.8 1.8 1.8 5 5 3.5 3.5 3.5 3.5 1.8 1.8 1.8 1.8 3.5 3.5 3.5 5 5 5 990967-202 990967-902 993967-902 946975-902 963967-902 961967-902 966967-902 935967-902 928975-902 927975-902 924975-902 926975-902 990967-302 993967-302 963954-302 961967-302 966954-302 928975-302 927975-302 924975-302 926975-302 993700-902 960967-902 930990-902 961753-902 966735-902 971700-902 928700-902 927700-902 924700-902 926700-902 963600-902 965600-902 961600-902 5185-5921 820950-925 821125-926 820888-901

XDB-C8 USP L7 990967-206 990967-906 993967-906 946975-906 963967-906 961967-906 966967-906 935967-906 928975-906 927975-906 924975-906 926975-906 990967-306 993967-306 963954-306 961967-306

XDB-Phenyl USP L11

XDB-CN USP L10

Standard Columns (no special hardware required, 400 bar)

990967-912 993967-912 963967-912 966967-912 935967-912 990967-905 993967-905 963967-905 961967-905 966967-905

990967-312 993967-312 963954-312 961967-312

990967-305 993967-305 963954-305

928975-306 927975-306 924975-306 926975-306 993700-906 993700-912 960967-906 960967-912 930990-906 961753-906 971700-906 928700-906 927700-906 924700-906 926700-906 963600-906 965600-906 961600-906 5185-5921 820950-926 820950-927 821125-926 821125-926 820888-901 820888-901

993700-905 960967-905 961753-905

820950-935 821125-935 820888-901

30

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

ZORBAX Eclipse XDB (Continued)

Hardware Description PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Guard Cartridge Guard Cartridge Hardware PrepHT Endfittings, 2/pk Particle XDB-C18 Size (mm) Size (m) USP L1 21.2 x 250 21.2 x 150 21.2 x 150 21.2 x 100 21.2 x 50 17 x 7.5 7 7 5 5 5 5 XDB-C8 USP L7 XDB-Phenyl USP L11 XDB-CN USP L10

PrepHT Cartridge Columns (require endfittings kit 820400-901)

977250-102 977250-106 977150-102 977150-106 970150-902 970150-906 970100-902 970100-906 970050-902 970050-906 820212-925 820212-926 820444-901 820444-901 820400-901 820400-901

Hardware Description

Particle XDB-C18 Size (mm) Size (m) USP L1 4.6 x 250 4.6 x 150 4.6 x 75 3.0 x 75 4.0 x 4 5 5 3.5 3.5 5

XDB-C8 USP L7

Agilent Cartridge Columns (require hardware kit 5021-1845)

Analytical Analytical Rapid Resolution Solvent Saver Plus Guard Cartridges, 10/pk Cartridge Holder 7995118-585 7995108-585 7995118-595 7995108-595 7995118-344 7995108-344 7995230-344 7995118-504 7995118-504 5021-1845 5021-1845 922975-902 922975-932 922700-902 922700-932 922975-906

Standard Columns (no special hardware required, 400 bar)

Rapid Resolution HT Rapid Resolution HT, 3/pk Narrow Bore RRHT Narrow Bore RRHT, 3/pk 4.6 x 50 4.6 x 50 2.1 x 50 2.1 x 50 1.8 1.8 1.8 1.8

31

ZORBAX Eclipse XDB (Continued)

Hardware Description Particle XDB-C18 Size (mm) Size (m) USP L1 4.6 x 30 4.6 x 30 4.6 x 15 4.6 x 15 2.1 x 30 2.1 x 30 2.1 x 15 2.1 x 15 4.6 x 50 4.6 x 50 4.6 x 30 4.6 x 30 4.6 x 15 4.6 x 15 2.1 x 50 2.1 x 50 2.1 x 30 2.1 x 30 2.1 x 15 2.1 x 15 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 933975-902 933975-932 931975-902 931975-932 973700-902 973700-932 975700-902 975700-932 925975-902 925975-932 923975-902 923975-932 921975-902 921975-932 925700-902 925700-932 923700-902 923700-932 921700-902 921700-932 820555-901 0.5 x 250 0.5 x 150 0.5 x 150 0.5 x 35 0.3 x 250 0.3 x 150 0.3 x 150 0.5 x 35 0.3 x 35 5 5 3.5 3.5 5 5 3.5 5 5 5064-8286 5064-8287 5064-8288 5064-8298 5064-8269 5064-8291 5064-8271 5064-8296 5064-8297 820555-901 XDB-C8 USP L7 933975-906 933975-936 931975-906 931975-936 973700-906 973700-936 975700-906 975700-936

Rapid Resolution HT Cartridges (require hardware kit 820555-901)

Rapid Resolution Cartridge Rapid Resolution Cartridge, 3/pk Rapid Resolution Cartridge Rapid Resolution Cartridge, 3/pk Rapid Resolution Cartridge Rapid Resolution Cartridge, 3/pk Rapid Resolution Cartridge Rapid Resolution Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Hardware Kit for RR and RRHT Cartridges

Capillary Glass-lined Columns

Capillary Capillary Capillary RR Capillary RR Capillary Capillary Capillary RR Capillary Capillary

32

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

ZORBAX 80 StableBond
Longest column lifetime and best reproducibility for low pH separations down to pH 1 Patented stable column chemistry allows use at high temperature and low pH without degradation Six different bonded phases provide broad selectivity SB-C18, SB-C8, SB-CN, SB-Phenyl, SB-C3, SB-Aq High purity (Type B) silica for good peak shape

R O Si R OH R O Si R OH R O Si R
Sterically Protected StableBond Bonded Phase

R1

Agilent ZORBAX StableBond columns use patented, unique, nonfunctional silanes with bulky diisobutyl (SB-C18) or diisopropyl (SB-C8, SB-C3, SB-Phenyl, SB-CN, and SB-Aq) side chain groups that sterically protect the key siloxane bond to the silica surface from hydrolytic attack at low pH. StableBond packing materials are not endcapped in order to provide exceptional stability 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 neutral compounds so that StableBond columns are an excellent choice for low pH method development. ZORBAX StableBond columns are compatible with all common mobile phases, including very high aqueous mobile phases.

R1

Column Specifications
Bonded Phase ZORBAX SB-C18 ZORBAX SB-C8 ZORBAX SB-C3 ZORBAX SB-Phenyl ZORBAX SB-CN ZORBAX SB-Aq Pore Size 80 80 80 80 80 80 Surface Area 180 m2/g 180 m2/g 180 m2/g 180 m2/g 180 m2/g 180 m2/g Temp. Limits* 90C 80C 80C 80C 80C 80C pH Range* 1.0-8.0 1.0-8.0 1.0-8.0 1.0-8.0 1.0-8.0 1.0-8.0 Endcapped No No No No No No Carbon Load 10% 5.5% 4% 5.5% 4% proprietary

R1

*StableBond columns are designed for optimal use at low pH. At pH 6-8, highest column stability for all silicabased columns is obtained by operating at temperatures <40C and using lower buffer concentrations in the range of 0.01-0.02M. At mid-range pH, Eclipse XDB and Bonus-RP are recommended.

Order online at www.agilent.com/chem/store

33

StableBond SB-C18 Shows Excellent Stability at Low pH and High Temperature (pH 0.8, 90C) Column: ZORBAX SB-C18 883975-902 4.6 x 150mm, 5m Column: ZORBAX Rx-C18 883967-902 4.6 x 150mm, 5m Mobile Phase: 50% Methanol/50% Water with 1.0% TFA Test Solute: Toluene Temperature: 90C

StableBond SB-C18 (Diisobutyl-C18) ZORBAX Rx-C18 (Dimethyl-C18) Competitor A-C18 Competitor B-C18 Competitor C-C18 Competitor D-C18 Competitor E-C18
Column Volumes of Mobile Phase Purge Relative Retention

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

Shorter Chain ZORBAX SB-CN is also Stable at Low pH (pH 2.0, 50C)

Column: ZORBAX SB-CN 883975-905 4.6 x 150mm, 5m

% k Remaining

ZORBAX StableBond SB-CN and the other short chain StableBond bonded phases are also exceptionally stable at low pH. Conventional dimethyl CN and similar bonded phases lack this stability.
Column Volumes

SB-CN Optimizes Retention and Resolution Column A: ZORBAX SB-C18 866953-902 4.6 x 75mm, 3.5m Column B: ZORBAX SB-CN 866953-905 4.6 x 75mm, 3.5m Mobile Phase: 30% ACN 70% 25mM NaH2PO4, pH 2.5 Flow Rate: 1.0 mL/min Temperature: 35C 1. 2. 3. 4. 5. Estriol Daidzen Quercetin Genistein Diethylstilbestrol

Time (min)

Time (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.

34

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

More RRHT Bonded Phase Choices Allow for Rapid Method Development Optimization

Change in Elution Order Peaks 3, 4 & 5

mAU 450 400 350 300 250 200 150 100 50 0 0 1 2 3 4 5 min
1 2 5 3 4 1 2 3 5 4 1 2 3 5 4 1 2 3

1. 2. 3. 4. 5.

Tolmetin Naproxen Diflunisal Ibuprofen Diclofenac

5 4

Column A:

SB-C18 827975-902 4.6 x 50mm, 1.8m SB-C8 827975-906 4.6 x 50mm, 1.8m ZORBAX SB-Phenyl 827975-912 4.6 x 50mm, 1.8m ZORBAX SB-CN 827975-905 4.6 x 50mm, 1.8m

Column B:

Column C:

Column D:

Mobile Phase: Acetonitrile (40%): Buffer 60% pH=2.4 25mM NaH2PO4 Flow Rate: 1.85 mL/min

Detector: Sample:

DAD, 254 nm LC: Agilent 1200 RR LC As listed, 1 L injection

5 Different Bonded Phase Choices in StableBond family enhance method optimization. Note: SB-Aq not shown.

Five Different Bonded Phases Provide Selectivity Options Column A: ZORBAX SB-C18 883975-902 4.6 x 150mm, 5m Column D: ZORBAX SB-Phenyl 883975-912 4.6 x 150mm, 5m

Column B:

ZORBAX SB-C8 883975-906 4.6 x 150mm, 5m

Column E:

ZORBAX SB-CN 883975-905 4.6 x 150mm, 5m

Column C:

ZORBAX SB-C3 883975-909 4.6 x 150mm, 5m

SB-C3 is just one of the five different StableBond selectivity choices. In this example, optimum resolution is obtained with SB-C3. All are based on the same high purity Rx-SIL. Selectivity changes are therefore dependent only on the bonded phases, making method development more reliable.

Mobile Phase: 0-100% B in 18.8 min A: 50 mM NaH2PO4, pH 2.5 in 95% H2O / 5% ACN B: 50 mM NaH2PO4, pH 2.5 in 47% H2O / 53% ACN Flow Rate: 1.0 mL/min Temperature: 26C Detector: 254 nm Sample: 1. Procaine 2. Lidocaine 3. d-Cinchonine 4. Butacaine 5. Tetracaine 35

Order online at www.agilent.com/chem/store

ZORBAX 80 StableBond
Hardware Description Semi-Preparative Semi-Preparative Semi-Preparative Semi-Preparative Analytical Analytical Analytical Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Solvent Saver Solvent Saver Solvent Saver Plus Solvent Saver Plus Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Narrow Bore Narrow Bore Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* Narrow Bore RR*
*RR: Rapid Resolution 3.5 m **RRHT: Rapid Resolution HT 1.8 m

Particle SB-C18 Size (mm) Size (m) USP L1 9.4 x 250 9.4 x 150 9.4 x 100 9.4 x 50 4.6 x 250 4.6 x 150 4.6 x 50 4.6 x 250 4.6 x 150 4.6 x 100 4.6 x 75 4.6 x 50 4.6 x 150 4.6 x 100 4.6 x 50 4.6 x 30 4.6 x 20 3.0 x 250 3.0 x 150 3.0 x 150 3.0 x 100 3.0 x 150 3.0 x 100 3.0 x 50 3.0 x 30 3.0 x 20 2.1 x 150 2.1 x 50 2.1 x 150 2.1 x 100 2.1 x 75 2.1 x 50 5 5 5 5 5 5 5 3.5 3.5 3.5 3.5 3.5 1.8 1.8 1.8 1.8 1.8 5 5 3.5 3.5 1.8 1.8 1.8 1.8 1.8 5 5 3.5 3.5 3.5 3.5 880975-202 883975-202 884975-202 846975-202 880975-902 883975-902 846975-902 884950-567 863953-902 861953-902 866953-902 835975-902 829975-902

SB-C8 USP L7

SB-CN USP L10

SB-C3 USP L56

SB-Phenyl USP L11

SB-Aq

Standard Columns (no special hardware required, 400 bar)

880967-201 880975-205 880975-209 880975-212

880975-906 880975-905 880975-909 880975-912 883975-906 883975-905 883975-909 883975-912 846975-906 863953-906 861953-906 866953-906 835975-906 829975-906 863953-905 861953-905 866953-905 835975-905 829975-905 863953-912 861953-912 866953-912 835975-912 829975-912 828975-912 827975-912 824975-912

880975-914 883975-914 846975-914 863953-914 861953-914 866953-914 835975-914

828975-902 828975-906 828975-905 827975-902 827975-906 827975-905 824975-902 824975-906 824975-905 826975-902 826975-906 880975-302 883975-302 863954-302 861954-302 829975-302 880975-306 883975-306 863954-306 861954-306 829975-306

828975-914 827975-914 824975-914

880975-305 880975-309 880975-312 883975-305 883975-309 883975-312 863954-305 863954-312 861954-305 861954-309 861954-312 829975-305 829975-312 828975-312 827975-312

880975-314 883975-314 863954-314 861954-314

828975-302 828975-306 828975-305 827975-302 827975-306 827975-305 824975-302 824975-306 824975-305 826975-302 826975-306 883700-922 860975-902 830990-902 861753-902 866735-902 871700-902

828975-314 827975-314

883700-906 883700-905 883700-909 883700-912 860975-906 860975-905 860975-909 860975-912 830990-906 861753-906 861753-905 861753-912 871700-906

860975-914 830990-914 861753-914 871700-914

36

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

ZORBAX 80 StableBond (Continued)

Hardware Description Narrow Bore RRHT, 600 bar** Narrow Bore RRHT, 600 bar** Narrow Bore RRHT, 600 bar** Narrow Bore RRHT, 600 bar** Narrow Bore RRHT, 600 bar** MicroBore RR* MicroBore RR* MicroBore RR* MicroBore Guard Cartridges, 3/pk Guard Cartridge, 2/pk Guard Cartridge, 4/pk Guard Cartridge, 4/pk Guard Hardware Kit Guard Hardware Kit PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge Size (mm) 2.1 x 150 2.1 x 100 2.1 x 50 2.1 x 30 2.1 x 20 1.0 x 150 1.0 x 50 1.0 x 30 1.0 x 17 9.4 x 15 4.6 x 12.5 2.1 x 12.5 9.4 x 15 Particle SB-C18 SB-C8 SB-CN SB-C3 Size (m) USP L1 USP L7 USP L10 USP L56 1.8 820700-902 820700-906 820700-905 1.8 1.8 1.8 1.8 3.5 3.5 3.5 5 7 5 5 828700-902 828700-906 828700-905 827700-902 827700-906 827700-905 824700-902 824700-906 824700-905 826700-902 826700-906 863600-902 865600-902 861600-902 5185-5920 863600-906 863600-905 865600-906 861600-906 5185-5920 820675-115 820950-933 821125-933 SB-Phenyl USP L11 820700-912 828700-912 827700-912 824700-914 SB-Aq

828700-914 827700-914 824700-914

820675-115 820675-115 820675-124

820950-920 820950-915 820950-916 820950-922 820950-917 821125-915 821125-915 821125-924 821125-924 821125-915 840140-901 840140-901 840140-901 840140-901 840140-901 820888-901 820888-901 820888-901 820888-901 820888-901

820888-901 877250-114 877150-114 870150-914 870100-914 870050-914

PrepHT Cartridge Columns (require endfittings kit 820400-901)

21.2 x 250 21.2 x 150 21.2 x 150 21.2 x 100 21.2 x 50 7 7 5 5 5 5 877250-102 877250-106 877250-105 877150-102 877150-106 870150-902 870150-906 870100-902 870100-906 870050-902 870050-906 820212-920 820212-915 820212-915 820212-915 877250-112

PrepHT Guard 17 x 7.5 Cartridge, 2/pk Guard Cartridge Hardware PrepHT Endfittings, 2/pk
*RR: Rapid Resolution 3.5 m **RRHT: Rapid Resolution HT 1.8 m

820212-933 820444-901 820400-901

820444-901 820444-901 820444-901 820444-901 820444-901 820400-901 820400-901 820400-901 820400-901 820400-901

Hardware Description

Particle SB-C18 Size (mm) Size (m) USP L1 4.6 x 250 4.6 x 150 4.6 x 75 4.0 x 4 5 5 3.5 5

SB-C8 USP L7

Agilent Cartridge Columns (require hardware kit 5021-1845)

Analytical Analytical Rapid Resolution Guard Cartridges, 10/pk Cartridge Holder 7995218-585 7995208-585 7995218-595 7995208-595 7995218-344 7995208-344 7995118-504 7995118-504 5021-1845 5021-1845

Order online at www.agilent.com/chem/store

37

ZORBAX 80 StableBond (Continued)

Hardware Description Particle SB-C18 Size (mm) Size (m) USP L1 4.6 x 50 4.6 x 50 2.1 x 50 2.1 x 50 4.6 x 30 4.6 x 30 4.6 x 15 4.6 x 15 2.1 x 30 2.1 x 30 2.1 x 15 2.1 x 15 4.6 x 50 4.6 x 50 4.6 x 30 4.6 x 30 4.6 x 15 4.6 x 15 2.1 x 50 2.1 x 50 2.1 x 30 2.1 x 30 2.1 x 15 2.1 x 15 1.8 1.8 1.8 1.8 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 822975-902 822975-932 822700-902 822700-932 833975-902 833975-932 831975-902 831975-932 873700-902 873700-932 875700-902 875700-932 825975-902 825975-932 823975-902 823975-932 821975-902 821975-932 825700-902 825700-932 823700-902 823700-932 821700-902 821700-932 820555-901 0.5 x 250 0.5 x 150 0.5 x 35 0.5 x 150 0.5 x 35 0.3 x 250 0.3 x 150 0.3 x 35 0.3 x 150 5 5 5 3.5 3.5 5 5 5 3.5 5064-8258 5064-8256 5064-8254 5064-8262 5064-8260 5064-8257 5064-8255 5064-8253 5064-8261 820555-901 SB-C8 USP L7 822975-906

Standard Columns (no special hardware required, 400 bar)

Rapid Resolution HT Rapid Resolution HT, 3/pk Narrow Bore RRHT Narrow Bore RRHT, 3/pk

Rapid Resolution HT Cartridges (require hardware kit 820555-901)

Rapid Resolution Cartridge Rapid Resolution Cartridge, 3/pk Rapid Resolution Cartridge Rapid Resolution Cartridge, 3/pk Rapid Resolution Cartridge Rapid Resolution Cartridge, 3/pk Rapid Resolution Cartridge Rapid Resolution Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Hardware Kit for RR and RRHT Cartridges 833975-906 833975-936 831975-906 831975-936 873700-906 873700-936 875700-906 875700-936

Capillary Glass-lined Columns

Capillary Capillary Capillary Capillary RR* Capillary RR Capillary Capillary Capillary Capillary RR
*RR: Rapid Resolution 3.5 m **RRHT: Rapid Resolution HT 1.8 m

38

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

ZORBAX Rx
Rx-C18 is recommended for alternate selectivity at low pH relative to Eclipse XDB-C18 and 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 provides excellent stability up to pH 9. Rx-C8 is the same product as SB-C8.

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

Analysis of Diazepam on Rx-C18 Column: ZORBAX Rx-C18 880967-302 3.0 x 250mm, 5m Mobile Phase: 35% H2O: 65% MeOH Flow Rate: 0.5 mL/min

1. Ethylparaben 2. Diazepam

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

Order online at www.agilent.com/chem/store

39

ZORBAX Rx
Hardware Description Semi-Preparative Analytical Analytical Rapid Resolution Rapid Resolution Rapid Resolution Solvent Saver Solvent Saver Solvent Saver Plus Solvent Saver Plus Narrow Bore Narrow Bore RR* Guard Cartridge, 2/pk Guard Cartridge, 4/pk Guard Cartridge, 4/pk Guard Hardware Kit Guard Hardware Kit Particle Size (mm) Size (m) 9.4 x 250 5 4.6 x 250 5 4.6 x 150 5 4.6 x 150 3.5 4.6 x 100 3.5 4.6 x 75 3.5 3.0 x 250 5 3.0 x 150 5 3.0 x 150 3.5 3.0 x 100 3.5 2.1 x 150 5 2.1 x 100 3.5 9.4 x 15 7 4.6 x 12.5 5 2.1 x 12.5 5 9.4 x 15 Rx-C18 USP L1 880967-202 880967-902 883967-902 863967-902 861967-902 866967-902 880967-302 883967-302 863967-302 861967-302 883700-902 861767-902 820675-115 820950-914 821125-915 840140-901 820888-901 Rx-C8 USP L7** 880967-201 880967-901 883967-901 863953-906 861953-906 866953-906 880975-306 883975-306 863954-306 861954-306 883700-906 861753-906 820675-115 820950-913 821125-915 840140-901 820888-901

PrepHT Cartridge Columns (require endfittings kit 820400-901)

PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Guard Cartridge, 2/pk Guard Cartridge Hardware PrepHT Endfittings, 2/pk
*RR: Rapid Resolution 3.5 m **Rx-C8 is the same product as SB-C8

21.2 x 250 21.2 x 150 21.2 x 150 21.2 x 100 21.2 x 50

7 7 5 5 5

877967-102 877250-106 877150-106 870150-906 870100-906 870050-906 820212-914 820212-915 820444-901 820444-901 820400-901 820400-901

40

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

ZORBAX 80 Extend-C18
High efficiency and long life at high pH up to pH 11.5 Unique bidentate bonding and double endcapping provides high pH stability More efficiency and better peak shape than polymer-based columns Improve retention, resolution and peak shape of basic compounds High sensitivity for LC/MS separations of peptides The Agilent ZORBAX Extend-C18 column uses a novel bidentate C18-C18 bonding technology to make it possible to develop high-resolution separations at high pH with a silica-based column. 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 more soluble in high pH solutions. Some of the mobile phase buffer options for high pH include triethylamine, pyrrolidine, glycine, borate and ammonium hydroxide. Ammonium hydroxide at pH 10.5 is an excellent mobile phase modifier for the LC/MS of peptides and small molecules with improved sensitivity compared with TFA containing mobile phase at low pH. The ExtendC18 column is stable from pH 2-11.5 with good peak shape for all types of compounds. Extend-C18 columns also provide an additional selectivity choice at low pH.

Si

C18

O Si C18
Silica Support

Column Specifications
Bonded Phase ZORBAX Extend-C18 Pore Size 80 Surface Area 180 m2/g Temp. Limits* pH Range 60C 2.0-11.5 Carbon Endcapped Load Double 12.5%
Novel Bidentate C18-C18 Bonding for Extend C-18 Bonded Phase

*Temperature limits are 60C up to pH 8, 40C from pH 8-11.5.

Order online at www.agilent.com/chem/store

41

Basic Antihistamines on Extend-C18 at High pH Column: ZORBAX Extend-C18 773450-902 4.6 x 150mm, 5m Mobile Phase: pH 7: 30% 20 mM Na2HPO4 70% MeOH pH 11: 30% 20 mM TEA 70% MeOH Flow Rate: 1.0 mL/min Temperature: Ambient Detector: 254 nm Sample: Antihistamines
Pseudoephedrine and scopolamine are difficult to retain at low and mid pH. Pseudoephedrine is often analyzed by ion exchange methods. The Extend-C18 column retains these compounds in a noncharged form at high pH and improves resolution.

1. 2. 3. 4. 5. 6. 7.

Maleate Scopolamine Pseudoephedrine Doxylamine Chlorpheniramine Triprolidine Diphenhydramine

Time (min)

Time (min)

Long Life at High pH with Extend-C18 Column: ZORBAX Extend-C18 773450-902 4.6 x 150mm, 5m Mobile Phase: 20% Methanol 80% 0.1 M Carbonate Buffer, pH 10.0 Flow Rate: 1.0 mL/min Temperature: Ambient

Amount of Dissolved Silica (mg)

ZORBAX Extend-C18 Competitor W

At high pH, columns will fail due to silica dissolution. The example here shows extended lifetime of ZORBAX Extend-C18 at high pH in comparison to competitor W. This was measured by the amount of dissolved silica.

Volume Eluent (L)

42

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

Extend-C18 Provides Good Peak Shape at Low pH Column: ZORBAX Extend-C18 773450-902 4.6 x 150mm, 5m Mobile Phase: 80% 25 mM NaH2PO4, pH 3.0 20% Methanol Flow Rate: 1.0 mL/min Temperature: 35C Detector: 254 nm Sample: Basic Compounds

1. 2. 3. 4.

Theobromine Dimethylxanthine Theophylline Caffeine

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

Time (min)

ZORBAX 80 Extend-C18
Hardware Description Particle Size (mm) Size (m) Part No. 4.6 x 250 4.6 x 150 4.6 x 50 4.6 x 150 4.6 x 100 4.6 x 75 4.6 x 50 4.6 x 100 4.6 x 50 4.6 x 50 4.6 x 30 4.6 x 20 3.0 x 250 3.0 x 150 3.0 x 150 3.0 x 100 3.0 x 50 3.0 x 100 3.0 x 50 3.0 x 30 3.0 x 20 5 5 5 3.5 3.5 3.5 3.5 1.8 1.8 1.8 1.8 1.8 5 5 3.5 3.5 3.5 1.8 1.8 1.8 1.8 770450-902 773450-902 746450-902 763953-902 764953-902 766953-902 735953-902 728975-902 727975-902 722975-902 724975-902 726975-902 770450-302 773450-302 763954-302 764953-302 735954-302 728975-302 727975-302 724975-302 726975-302

Standard Columns (no special hardware required, 400 bar)

Analytical Analytical Analytical Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 400 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Solvent Saver Solvent Saver Solvent Saver Plus Solvent Saver Plus Solvent Saver Plus Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar

Order online at www.agilent.com/chem/store

43

ZORBAX 80 Extend-C18 (Continued)

Hardware Description Narrow Bore Narrow Bore Narrow Bore RR* Narrow Bore RR* Narrow Bore RRHT, 600 bar** Narrow Bore RRHT, 600 bar** Narrow Bore RRHT, 600 bar** Narrow Bore RRHT, 600 bar** MicroBore RR* MicroBore RR* MicroBore RR* MicroBore Guard Cartridge, 3/pk Guard Cartridge, 4/pk Guard Cartridge, 4/pk Guard Hardware Kit Particle Size (mm) Size (m) 2.1 x 150 5 2.1 x 50 5 2.1 x 100 3.5 2.1 x 50 3.5 2.1 x 100 1.8 2.1 x 50 1.8 2.1 x 30 1.8 2.1 x 20 1.8 1.0 x 150 3.5 1.0 x 50 3.5 1.0 x 30 3.5 1.0 x 17 5 4.6 x 12.5 5 2.1 x 12.5 5 Part No. 773700-902 760450-902 761753-902 735700-902 728700-902 727700-902 724700-902 726700-902 763600-902 765600-902 761600-902 5185-5923 820950-930 821125-930 820888-901 770150-902 770100-902 770050-902 820400-901 17 x 7.5 5 820212-930 820444-901

PrepHT Cartridge Columns (require endfittings kit 820400-901)

PrepHT Cartridge PrepHT PrepHT PrepHT Endfittings, 2/pk PrepHT Guard Cartridge, 2/pk Guard Cartridge Hardware
*RR: Rapid Resolution 3.5 m **RRHT: Rapid Resolution HT 1.8 m

21.2 x 150 21.2 x 100 21.2 x 50

5 5 5

44

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

ZORBAX 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 phases
CH3 O R1 Si R1 Si CH3 R1 O Si R1 CH3 Si CH3 R1 O Si R1 PG R CH3 PG R CH3 PG R

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

Column Specifications
Bonded Phase ZORBAX Bonus-RP Pore Size 80 Surface Area 180 m2/g Temp. Limits* pH Range 60C 2.0-9.0 Carbon Endcapped Load Triple 9.5%

*Temperature limits are 60C up to pH 8, 40C from pH 8-9.

Unique, Polar Alkyl Bonus-RP Bonded Phase

ZORBAX Bonus-RP Provides Unique Selectivity Column A: ZORBAX Bonus-RP 883668-901 4.6 x 150mm, 5m

Column B:

Eclipse XDB-C8 993967-906 4.6 x 150mm, 5m

1. 2. 3. 4.

Cephalexin Cephaclor Cephuroxime Cephoxitin

Mobile Phase: 75% 25 mM Na Citrate, pH 6 25% MeOH Flow Rate: 1.0 mL/min Temperature: Ambient Detector: 254 nm Sample: 3 L Cephalosporins

Time (min)

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

Order online at www.agilent.com/chem/store

45

Improved Peak Shape of Basic Compounds Using Bonus-RP Column: Alkyl-C8 4.6 x 150 mm, 5 m Mobile Phase: 75% 25 mM NH4OAc, pH 5.5 25% ACN Flow Rate: 1.5 mL/min Temperature: 40C Detector: 254 nm
TF 1. 1.8 2. 1.7 3. 2.1

1. Doxylamine 2. Chlorpheniramine 3. Triprolidine

Bonus-RP eliminates peak tailing of these basic compounds in comparison to a typical alkyl C8 bonded phase. In the mid-pH region, residual silanols can interact more strongly with basic compounds to cause peak tailing. The polar group in the Bonus-RP bonded phase eliminates peak tailing of these basic compounds by reducing interactions with residual silanols.

Time (min)

Improved Peak Shape of Basic Compounds Using Bonus-RP Column: ZORBAX Bonus-RP 883668-901 4.6 x 150mm, 5m Mobile Phase: 80% 25 mM NH4OAc, pH 5.5 20% ACN Flow Rate: 1.5 mL/min Temperature: 40C Detector: 254 nm

1. Doxylamine 2. Chlorpheniramine 3. Triprolidine

TF 1. 1.1 2. 1.1 3. 1.1

Time (min)

46

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

ZORBAX Bonus-RP is Stable at Low and Mid pH Column: ZORBAX Bonus-RP 883668-901 4.6 x 150mm, 5m Mobile Phase: 60% 25 mM Phosphate Buffer, pH 7.0: 40% ACN Flow Rate: 1.5 mL/min Temperature: 23C
Toluene Plate Height, cm

Competitive alkyl/amide Bonus-RP

Column Volume of Purge

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

Dimethyl-C18/amide, Bonus-RP Column: ZORBAX Bonus-RP 883668-901 4.6 x 150mm, 5m Mobile Phase: Aging: 50% MeOH 50% 0.1% TFA Test: 80% MeOH 20% H2O Flow Rate: 1.0 mL/min Temperature: Aging: 60C Test: 23C
Toluene Plate Height, cm

Dimethyl-C18/amide Bonus-RP

Column Volume of Purge

Sterically protecting side groups provide good low pH stability and longer column lifetime than similar polar alkyl bonded phases.

Order online at www.agilent.com/chem/store

47

ZORBAX Bonus-RP
Hardware Description Particle Size (mm) Size (m) Part No. 4.6 x 250 4.6 x 150 4.6 x 150 4.6 x 100 4.6 x 75 3.0 x 250 3.0 x 150 3.0 x 150 3.0 x 100 2.1 x 150 2.1 x 50 2.1 x 150 2.1 x 100 2.1 x 50 1.0 x 150 1.0 x 50 1.0 x 30 1.0 x 17 4.6 x 12.5 2.1 x 12.5 5 5 3.5 3.5 3.5 5 5 3.5 3.5 5 5 3.5 3.5 3.5 3.5 3.5 3.5 5 5 5 880668-901 883668-901 863668-901 864668-901 866668-901 880668-301 883668-301 863668-301 864668-301 883725-901 861971-901 863700-901 861768-901 861700-901 863608-901 865608-901 861608-901 5185-5922 820950-928 821125-928 820888-901 878250-101 878150-101 868150-901 868100-901 868050-901 820400-901 17 x 7.5 5 820212-928 820444-901

Standard Columns (no special hardware required, 400 bar)

Analytical Analytical Rapid Resolution Rapid Resolution Rapid Resolution Solvent Saver Solvent Saver Solvent Saver Plus Solvent Saver Plus Narrow Bore Narrow Bore Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* MicroBore RR* MicroBore RR* MicroBore RR* MicroBore Guard Cartridge, 3/pk Guard Cartridge, 4/pk Guard Cartridge, 4/pk Guard Hardware Kit

PrepHT Cartridge Columns (require endfittings kit 820400-901)

PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Endfittings, 2/pk PrepHT Guard Cartridge, 2/pk Guard Cartridge Hardware
*RR: Rapid Resolution 3.5 m

21.2 x 250 21.2 x 150 21.2 x 150 21.2 x 100 21.2 x 50

7 7 5 5 5

48

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

ZORBAX Method Development Kits

Agilent offers a series of kits that allow for fast method development at an attractive price. Each kit contains three columns. To study the effect of a change in selectivity on your separation under a given set of conditions, try either the Eclipse kits for applications in the pH range 2-9 or StableBond kits for additional choices at low pH. Try the pH kits if you want to study the effect of pH on your separation over a wide range of pH (1-11.5). The aqueous kits provide a wide range of selectivities with a set of columns that can operate under high aqueous conditions to retain highly polar analytes.

ZORBAX Method Development Kits

Description StableBond Method Development Kit Includes 4.6 x 150 mm, 5 m columns; one each: SB-C18, SB-CN and SB-Phenyl phases Fast StableBond Method Development Kit Includes 4.6 x 75 mm, 3.5 m columns; one each: SB-C18, SB-CN and SB-Phenyl phases Eclipse XDB Method Development Kit Includes 4.6 x 150 mm, 5 m columns; one each: XDB-C18, XDB-C8, XDB-Phenyl phases Fast Eclipse XDB Method Development Kit Includes 4.6 x 75 mm, 3.5 m columns; one each: XDB-C18, XDB-C8 and XDB-Phenyl phases pH Method Development Kit Includes 4.6 x 150 mm, 5 m columns; one each: SB-C18, XDB-C18 and Extend-C18 phases Fast pH Method Development Kit Includes 4.6 x 75 mm, 3.5 m columns; one each: SB-C18, XDB-C18 and Extend-C18 phases Aqueous Method Development Kit Includes 4.6 x 150 mm, 5 m columns; one each: SB-Aq, Bonus RP and SB-C18 Fast Aqueous Method Development Kit Includes 4.6 x 75 mm, 3.5 m columns; one each: SB-Aq, Bonus RP and SB-C18 Part No. 5183-4624 5183-4625 5183-4626 5183-4627 5185-5807 5185-5808 5185-5809 5185-5810

Order online at www.agilent.com/chem/store

49

ZORBAX Method Validation Kits

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

Tips & Tools

Need to order a custom column? Go to
www.agilent.com/chem/lccustom

1. Request Validation Kits (columns from different lots) by using Part Number 899999-888 2. Indicate the Part Number of the current column you are using 3. Indicate the Lot Number of the current column you are using 4. Indicate the number of additional columns needed from different lots (example: you have a current column and may need two additional lots) 5. Please fax your request to (302) 993-5354 or email to [email protected]. 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 is placed, depending on lot availability.

and fill out the custom column request form. You will receive a quote from your Customer Service Agent within 1-2 business days.

50

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

ZORBAX Original Reversed Phase Columns

Original ZORBAX columns are made with Type A silica and are useful for many applications of acidic or neutral compounds. These columns have a higher activity level and are therefore useful for separating isomers (e.g. cis-trans, geometric) or other compounds where silanol activity enhances selectivity. These columns are used in many established methods.

Hardware Description Semi-Preparative Analytical (Endcapped) Analytical (Non-endcapped) Analytical Solvent Saver Solvent Saver

Particle ODS (C18) Size (mm) Size (m) USP L1 9.4 x 250 4.6 x 250 4.6 x 250 4.6 x 150 3.0 x 250 3.0 x 150 9.4 x 15 4.6 x 12.5 9.4 x 15 5 5 5 5 5 5 7 5 880952-202 880952-702 884950-543 883952-702 880952-302 883952-302 820675-115 820950-902 840140-901 820888-901 877952-102 820400-901

C8 USP L7

Phenyl USP L11

CN USP L10

TMS USP L13

Standard Columns (no special hardware required, 400 bar)

880952-206 880952-706 880952-712 884950-507 880952-710 883952-706 883952-712 884950-526 883952-710

Guard Columns (hardware required)

Guard Cartridge, 2/pk Guard Cartridge, 4/pk Guard Hardware Kit Guard Hardware Kit PrepHT Cartridge PrepHT Endfittings, 2/pk 820675-115 820950-906 840140-901 820888-901 877952-106 820400-901 820675-115 820950-912 840140-901 820888-901 820675-124 820950-905 820950-924 840140-901 840140-901 820888-901 820888-901 877952-105 820400-901

PrepHT Cartridge Columns (require endfittings kit 820400-901)

21.2 x 250 7

Order online at www.agilent.com/chem/store

51

ZORBAX Normal Phase Columns

For normal-phase chromatography, the ZORBAX product line offers a choice of bonded and non-bonded silica packings.

ZORBAX Rx-SIL
Made from highly pure (>99.995%) porous silica microspheres (pore size is the space between the solid silica microparticles) 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 separation of polar compounds that exhibit poor peak symmetry on more acidic silica Useful for very hydrophilic compounds with high organic mobile phases in HILIC mode

ZORBAX 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 same normal-phase applications

ZORBAX CN
Cyanopropyldimethylsilane monolayer bonded to ZORBAX SIL Equilibrates more rapidly than ZORBAX SIL, and used for many of the same normal-phase applications Less prone to fouling and less water sensitive than silica

ZORBAX NH2
Amino-propyl silane phase bonded to ZORBAX SIL Used for normal phase and weak anion-exchange, and reversed-phase HPLC of polar compounds Vitamins A and D are separated in the normal-phase mode Carbohydrates and sugars are separated in the reversed-phase mode

52

Order online at www.agilent.com/chem/store

ZORBAX Columns for Analytical HPLC

Column Specifications
Bonded Phase ZORBAX Rx-SIL ZORBAX Eclipse XDB-CN ZORBAX SIL ZORBAX CN ZORBAX NH2 Pore Size 80 80 70 70 70 Surface Area 180 m2/g 180 m2/g 300 m2/g 300 m2/g 300 m2/g pH Range 0-8.0 2.0-8.0 0-8.0 2.0-7.0 2.0-7.0 Endcapped No Yes No Yes Yes Carbon Load 4.3% 7% 4%

High Resolution Normal Phase Separation of Octylphenoxy Ethanol Surfactant on ZORBAX CN Column: ZORBAX CN 880952-705 4.6 x 250mm, 5m Mobile Phase: Primary: Heptane Secondary: 2-Methoxyethanol/Isopropanol (50/50) Flow Rate: 2 mL/min Gradient: 2-20% Secondary in 10 min., Linear Hold at 20% Temperature: 50C Detector: 278 nm Sample: Octylphenoxy (polyethylene oxy) Ethanol Surfactant (n= 10)

10

15 Time (min)

20

Order online at www.agilent.com/chem/store

53

ZORBAX Normal Phase Columns

Hardware Description Semi-Prep Analytical Analytical Narrow Bore Narrow Bore Guard Cartridge, 2/pk Guard Cartridges, 4/pk Guard Cartridge, 4/pk Guard Hardware Kit Guard Hardware Kit Particle Size (mm) Size (m) Rx-SIL 9.4 x 250 4.6 x 250 4.6 x 150 2.1 x 150 2.1 x 50 9.4 x 15 4.6 x 12.5 2.1 x 12.5 9.4 x 15 5 5 5 5 5 5 5 5 SIL USP L3 CN USP L10 NH2 USP L8 Carbohydrate XDB-CN Analysis USP L10

Standard Columns (no special hardware required, 400 bar)

880975-201 880952-201 880952-205 880952-208 880975-901 880952-701 880952-705 880952-708 840300-908 883975-901 883952-701 883952-705 883952-708 843300-908 883700-901 860700-708 820675-119 820675-119 820675-111 820675-111 820950-919 820950-901 820950-905 820950-908 820950-908 821125-919 840140-901 840140-901 840140-901 840140-901 820888-901 820888-901 820888-901 820888-901 820888-901 820888-901 820950-935 821125-935 990967-905* 993967-905* 993700-905*

Guard Columns (hardware required)

PrepHT Cartridge Columns (require endfittings kit 820400-901)

PrepHT 21.2 x 250 Cartridge PrepHT 21.2 x 250 Cartridge PrepHT Endfittings, 2/pk PrepHT Guard Cartridge, 2/pk 17 x 7.5 7 7 877250-101 877952-101 877952-105 877952-108 820400-901 820400-901 820400-901 820400-901 5 820212-919

Guard Cartridge Hardware

820444-901

*These columns ship containing reversed phase solvents. Flush with isopropanol before using normal phase solvents.

54

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

Reproducible results from high-throughput, to capillary, to prep.

No matter how many samples you have or how complex they may be you need to feel confident that you can achieve reproducible results without wasting valuable time testing different columns and configurations. The following column families deliver industry-leading performance for specific measurement and purification challenges:

EXPANDED! ZORBAX Rapid Resolution (3.5 m) and Rapid Resolution High Throughput (1.8 m) columns enhance your productivity by delivering fast analysis without compromising resolution. Available in Eclipse XDB, StableBond, and NEW Eclipse Plus 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. Ideal for cost-effective analysis. ZORBAX MicroBore HPLC columns are a good choice when sample sizes are limited. They can also improve detection limits 5 times over 2.1 mm ID columns when the same sample mass is used. ZORBAX capillary and nano columns are best for limited-sample and proteomics applications, because they enhance sensitivity by reducing on-column sample dilution. Now available in a wide variety of phases, pore sizes, and dimensions. Agilent and ZORBAX preparative columns, preparative cartridge columns, and guards are engineered for high purity, recovery, and throughput. They feature a broad range of selectivities, and are available in a variety of bonded phases for exceptional method development flexibility. Ultron chiral columns with two complementary protein-based chiral stationary phases are an excellent choice for enantiomeric separations. Ideal for many pharmacological applications.

Order online at www.agilent.com/chem/store

55

Agilent ZORBAX Rapid Resolution High Throughput (RRHT) and Rapid Resolution (RR) HPLC columns
Enhance your labs productivity without sacrificing performance, reliability, or convenience.
Chances are, you are under increased pressure to generate conclusive data under demanding time constraints. That is why Agilent has expanded our ZORBAX Rapid Resolution High Throughput line to include columns that can be used at pressures of 600 bar for faster, higher-resolution separations. Together with the Agilent 1200 Series Rapid Resolution System, RRHT columns can reduce your analysis time by up to 95%! With their 1.8 m particle size, Agilent RRHT columns allow you to push flow rates to the limit without compromising the efficiency or quality of your separations. And if you have already developed conventional LC methods on Agilents ZORBAX LC columns, you can easily and securely transfer these methods to Agilents RRHT columns. So you can analyze complex separations on shorter columns and re-evaluate current methods on fast LC without changing separation conditions Together with the Agilent 1200 Series Rapid Resolution System, ZORBAX RRHT 1.8 m HPLC columns can help you TIPS & TOOLS Use Rapid Resolution (RR) columns when you want speed and resolution under lower pressures. Use Rapid Resolution High Throughput (RRHT) columns when you need high speed and superior resolution under high pressures.

Process samples up to 20 times faster. Confidently move methods from your lab to any lab in the world. Perform conventional, fast, and ultra-fast separations on the same unit. Choose from over 100 Rapid Resolution and Rapid Resolution High Throughput configurations. Increase resolution by 30-40% over conventional HPLC. Significantly reduce operator training time. Minimize switching costs.

56

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

ZORBAX Rapid Resolution High Throughput 1.8 m

New high pressure (600 bar) columns for ultra high speed or maximum resolution analyses with Rapid Resolution HT columns packed with totally porous, 1.8 m packings Carefully engineered particles deliver maximum resolution at 25% less pressure than other sub 2-micron materials Reduce analysis time by up to 95% Develop HPLC methods more quickly Securely transfer conventional methods with over 80 RRHT column choices Analyze complex samples on shorter columns faster and maximize peak capacity Perform faster analyses and use less solvent Short (50 mm long and less) column can be used on some conventional LCs

ZORBAX Rapid Resolution HT (1.8 m) columns use a totally porous, 1.8 m particle to provide maximum resolution in fast, ultra-fast and high resolution analyses. You can reduce analysis time by up to 95% in comparison to 250 mm length columns. With more than 80 RRHT column choices, including the new high performance ZORBAX Eclipse Plus and many other ZORBAX column choices (Eclipse XDB, StableBond, Extend), methods can be developed quickly or securely transferred to a smaller particle size column with no loss in resolution. The small particle size provides double the efficiency of a 3.5 m column in the same column length providing the highest efficiency and resolution possible. This permits the analysis of complex samples on shorter column with the highest resolution and peak capacity. The 1.8 m Rapid Resolution HT columns take high-speed, high-resolution HPLC to a new level. The 600 bar columns can be used with the new Agilent 1200 Rapid Resolution LC up to this high pressure limit. In addition, the shorter columns can be used on many other LC's, including the Agilent 1100 by using the RRHT-1100 conversion kits to maximize performance.

Order online at www.agilent.com/chem/store

57

1100 Series Conversion Kits for Fast LC

These kits make it easy to convert your Agilent 1100 system with a binary pump to a lowervolume 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 able to use your converted 1100 for standard methods and columns.

Kit Selection For Variable Wavelength Detectors (VWD) For Diode Array Detectors (DAD & DAD SL) and Multiple Wavelength Detectors (MWD) For Diode Array Detector and Mass Spec

Description Columns: 4.6 x 50 mm, 1.8 m (3) Flow Cell for VWD, 5 l capillaries, -LC inline filter Columns: 4.6 x 50 mm, 1.8 m (2) Flow Cell for DAD, 5 l capillaries, -LC inline filter Columns: 2.1 x 50 mm, 1.8 m (2) Flow Cell for DAD, 1.7 l capillaries, ZDV union

Part No. 5188-5323 5188-5324 5188-5328

RRHT Flavanoids, Fast Analysis of Citrus Rinds, Confirmation by LC/MS Column: Eclipse XDB-C18 924975-302 3.0 x 30mm, 1.8m Mobile Phase: A: 0.1% FA, B: 0.075% FA in ACN Gradient: 18 to 65% B/2 min 65-88% B/3 min Detector: 276 nm Temperature: 50C Sample: 2g ground fresh peel and (10 mL meOH + 10 L KOH) Ultrasonicate 10 min and 0.45 m filter Inject 1 L
mAU 800 700 600 500 400 300 200 100 0 0.5 mAU 350 300 250 200 150 100 50 0 0.5 mAU 120 100 80 60 40 20 0 0.5 1 1.5 2

Naringin
1

confirmed by LCMS

Grapefruit Rind
2 2.5 min

1.5

Hesperidin confirmed by LCMS

Lime Rind

1. 2. 3. 4. 5. 6.

Rutin Naringin Hesperidin Quercetin Naringenin Kaempferol

1.5

2.5

min

Citrus Flavonoids
2.5 min

This is an analysis with potentially 10-20 analytes. It can still be done quickly for improved productivity.

58

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

Rapid Resolution HT Up to 20X Faster Column: ZORBAX SB-C18 883975-902 4.6 x 150mm, 5m 827700-902, 827700-902 Mobile Phase: A:H2O B: ACN Flow Rate: 1.2 mL/min, 1.0 mL/min, 2.4 mL/min Gradient: 0.0 min 50% B 11/1.2/0.4 min 100% B Temperature: 40C, 40C, 95C Detector: UV 254 nm Sample: Alkylphenones
A

0 B

10

min

0.2

0.4

0.6

0.8

min

1. 2. 3. 4. 5. 6. 7. 8. 9.

C3-Alkylphenone C4-Alkylphenone C5-Alkylphenone C6-Alkylphenone C7-Alkylphenone C8-Alkylphenone C9-Alkylphenone C10-Alkylphenone C12-Alkylphenone

0.2

0.4 min

Rapid Resolution HT Provides Double the Efficiency of Rapid Resolution Columns Column: ZORBAX SB-C18 835975-902 4.6 x 50mm, 3.5m Column: ZORBAX SB-C18 835975-902 4.6 x 50mm, 3.5m Mobile Phase: Flow Rate: Temperature: Detector: 25% Water, 75% MeOH 1.5 mL/min Ambient 254 nm

Plates (N)
1. 2. 3. 4. 3476 4585 5673 6180 1. 2. 3. 4.

Plates (N)
1. 2. 3. 4. 6560 8958 11508 12266

Uracil Phenol 4-CI-Nitrobenzene Toluene

This figure shows that Rapid Resolution HT columns can provide double the efficiency of a 3.5 m column in the same column length. This high efficiency can be used for very highresolution, high throughput analyses.

Order online at www.agilent.com/chem/store

59

Reduce Analysis Time Dramatically with Rapid Resolution HT Columns Eclipse XDB-C18 990967-902 4.6 x 250mm, 5m Column B: Eclipse XDB-C18 963967-902 4.6 x 150mm, 3.5m Column C: Eclipse XDB-C18 966967-902 4.6 x 75mm, 3.5m Column D: Eclipse XDB-C18 925975-902 4.6 x 50mm, 1.8m Mobile Phase: 73% MeOH: 27% 20 mM Phosphate Buffer, pH 7.0 Flow Rate: 1 mL/min Temperature: Ambient Detector: 254 nm Column A:

1. 2. 3. 4. 5. 6. 7.

Uracil Naproxen Mefanamic Acid Butyl Paraben Propranolol Napthalene Dipropyl Phthalate

This figure shows the dramatic reduction in analysis time possible by using Rapid Resolution HT columns. Chromatogram A shows a separation that takes 11.5 minutes on a 25 cm, 5 m column. Rapid Resolution (3.5 m) columns, shown in chromatogram B and C, reduce analysis time substantially, but with a slight compromise in resolution. The Rapid Resolution HT column reduces analysis time to 2.2 minutes, an 80% reduction, while still maintaining baseline resolution.

Increase Peak Capacity with RRHT Columns Column A: Eclipse XDB-C8 928700-906 2.1 x 100mm, 1.8m Column B: Eclipse XDB-C18 961753-902 2.1 x 100mm, 3.5m Mobile Phase: A:H2O B: ACN Flow Rate: 0.5 mL/min Gradient: 0.0 min 50% B 20.0 min 100% B Temperature: 40C Detector: UV 254 nm Sample: Alkylphenones
A
mAU 120 100 80 60 40 20 0 0 2 4 6 8 10 12 14 16 18 min 11 1

mAU 100 80 60 40 20 0 0 2 4 6 8 10 12 14 16 18 min 11 1

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Uracil C3-Alkylphenone C4-Alkylphenone C5-Alkylphenone C6-Alkylphenone C7-Alkylphenone C8-Alkylphenone C9-Alkylphenone C10-Alkylphenone C12-Alkylphenone C14-Alkylphenone

Long Lifetime of RRHT Columns at Elevated Temp. Column: SB-C18 827700-902 2.1 x 50mm, 1.8m Mobile Phase: A: 60% H2O B: 40% ACN Flow Rate: 1 mL/min Temperature: 80C Detector: UV 254 nm Sample: QC Test Mix

60

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

Comparison of Efficiencies RRHT (1.8m) and Rapid Resolution (3.5m) Columns

Column Length Resolving Power N (3.5 m)* 21,000 14,000 10,500 7000 4200 2100 Resolving Power N (1.8 m) 32,500 24,000 17,000** 12,000 6000 3500 2500

High Resolution
150 100 75

Ultra Fast
50 30 20 15 Resolution N1/2

*5 m HPLC columns of the same length have 40% fewer plates (N-value); 4.6 mm ID **Available as a custom column

ZORBAX Rapid Resolution HT Columns for High Pressure Use (Maximum Pressure: 600 bar, 9000 psi)
Description Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Narrow Bore RRHT, 600 bar Narrow Bore RRHT, 600 bar Narrow Bore RRHT, 600 bar Narrow Bore RRHT, 600 bar MicroBore RRHT, 600 bar MicroBore RRHT, 600 bar Particle Size (mm) Size (m) 4.6 x 150 1.8 4.6 x 100 1.8 4.6 x 50 1.8 4.6 x 30 1.8 4.6 x 20 1.8 3.0 x 100 1.8 3.0 x 50 1.8 3.0 x 30 1.8 3.0 x 20 1.8 2.1 x 100 1.8 2.1 x 50 1.8 2.1 x 30 1.8 2.1 x 20 1.8 1.0 x 100 1.8 1.0 x 50 1.8 Eclipse Plus C18 USP L1 959994-902 959964-902 959941-902 959931-902 959964-302 959941-302 Eclipse Plus C8 USP L7 Eclipse XDB-C18 USP L1 Eclipse XDB-C8 USP L7 Extend-C18 USP L1

959764-902 959741-902 959731-902

959964-906 928975-902 928975-906 728975-902 959941-906 927975-902 927975-906 727975-902 959931-906 924975-902 924975-906 724975-902 926975-902 926975-906 726975-902 959964-306 928975-302 928975-306 728975-302 959941-306 927975-302 927975-306 727975-302 924975-302 924975-306 724975-302 926975-302 926975-306 726975-302 959764-906 928700-902 928700-906 728700-902 959741-906 927700-902 927700-906 727700-902 959731-906 924700-902 924700-906 724700-902 926700-902 926700-906 726700-902 928600-902 928600-906 728600-902 922600-902 922600-906 722600-902

Order online at www.agilent.com/chem/store

61

ZORBAX Rapid Resolution HT Columns for High Pressure Use (Maximum Pressure: 600 bar, 9000 psi)
Description Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Rapid Resolution HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Solvent Saver HT, 600 bar Narrow Bore RRHT, 600 bar Narrow Bore RRHT, 600 bar Narrow Bore RRHT, 600 bar Narrow Bore RRHT, 600 bar Narrow Bore RRHT, 600 bar MicroBore RRHT, 600 bar Particle Size (mm) Size (m) 4.6 x 150 1.8 4.6 x 100 1.8 4.6 x 50 1.8 4.6 x 30 1.8 4.6 x 20 1.8 3.0 x 150 1.8 3.0 x 100 1.8 3.0 x 50 1.8 3.0 x 30 1.8 3.0 x 20 1.8 2.1 x 150 1.8 2.1 x 100 1.8 2.1 x 50 1.8 2.1 x 30 1.8 2.1 x 20 1.8 1.0 x 100 1.8 SB-C18 USP L1 829975-902 828975-902 827975-902 824975-902 826975-902 829975-302 828975-302 827975-302 824975-302 826975-302 820700-902 828700-902 827700-902 824700-902 826700-902 828600-902 SB-C8 USP L7 829975-906 828975-906 827975-906 824975-906 826975-906 829975-306 828975-306 827975-306 824975-306 826975-306 820700-906 828700-906 827700-906 824700-906 826700-906 828600-906 SB-Phenyl USP L11 829975-912 828975-912 827975-912 824975-912 829975-312 828975-912 827975-912 SB-CN USP L10 829975-905 828975-905 827975-905 824975-905 SB-Aq 828975-914 828975-914 824975-914

829975-305 828975-305 828975-314 827975-305 827975-314 824975-305 820700-905 828700-905 828700-914 827700-905 827700-914 824700-905 824700-914 828600-905

820700-912 828700-912 827700-912 824700-912

62

HPLC Columns for Special Applications

ZORBAX Rapid Resolution HT Columns and Cartridges (Maximum Pressure: 400 bar, 6000 psi)
Hardware Description Rapid Resolution HT Rapid Resolution HT, 3/pk Narrow Bore RRHT Narrow Bore RRHT, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Rapid Resolution HT Cartridge, 3/pk Rapid Resolution HT Cartridge Particle Size (mm) Size (m) 4.6 x 50 1.8 4.6 x 50 1.8 2.1 x 50 1.8 2.1 x 50 1.8 4.6 x 50 4.6 x 50 2.1 x 50 2.1 x 50 4.6 x 30 4.6 x 30 2.1 x 30 2.1 x 30 4.6 x 15 4.6 x 15 2.1 x 15 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 XDB-C18 XDB-C8 USP L1 USP L7 922975-902 922975-906 922975-932 922700-902 922700-932 925975-902 925975-932 925700-902 925700-932 923975-902 923975-932 923700-902 923700-932 921975-902 921975-932 921700-902 921700-932 820555-901 SB-C18 SB-C8 Extend-C18 USP L1 USP L7 USP L1 822975-902 822975-906 722975-902 822975-932 822700-902 822700-932 825975-902 825975-932 825700-902 825700-932 823975-902 823975-932 823700-902 823700-932 821975-902 821975-932 821700-902 821700-932 820555-901

Rapid Resolution HT Cartridges (require hardware kit 820555-901)

Rapid Resolution HT Cartridge, 2.1 x 15 3/pk Hardware Kit for RR and RRHT Cartridges

Order online at www.agilent.com/chem/store

63

ZORBAX Rapid Resolution 3.5 m Columns

Reduce analysis time and solvent usage and increase sample throughput High efficiency in short and ultra-short column lengths Available in analytical (4.6 mm) and narrow-bore (2.1 mm) ID Comparable lifetime to 5 m columns

Agilent ZORBAX Rapid Resolution columns, with a 3.5 m particle size, reduce analysis time and increase sample throughput for any application when compared to 5 m columns. Rapid Resolution columns are available from 15-150 mm, in 1-4.6 mm IDs, so the best configurations are available for high throughput, LC/MS, combinatorial chemistry and rapid analytical applications. Rapid Resolution 3.5 m particles have superior mechanical strength, so every column has an extremely stable packed bed and provides a comparable lifetime to 5 m columns. These are available in most ZORBAX bonded phases.

Rapid Resolution Columns Reduce Analysis Time While Maintaining Resolution Column A: ZORBAX Bonus-RP 883668-901 4.6 x 150mm, 5m

Time (min)

Column B:

ZORBAX Bonus-RP 866668-901 4.6 x 75mm, 3.5m

Time (min)

Column C:

ZORBAX Bonus-RP 861700-901 2.1 x 50mm, 3.5m 1. 2. 3. 4. Caffeine Doxylamine Chlorpheniramine Triprolidine

Mobile Phase: 80% 25 mM NH4Ac, pH 5.5 20% ACN Flow Rate: 1.5 mL/min 1.5 mL/min 0.3 mL/min Time (min) Temperature: 40C Detector: 254 nm Rapid Resolution 3.5 m columns are available in narrow bore
configurations for great compatibility with LC/MS.

64

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

Run Method Development Chromatograms on Rapid Resolution Columns Column: ZORBAX SB-C18 866953-902 4.6 x 75mm, 3.5m Mobile Phase: A: 25 mM NaH2PO4, pH 3.0 B: MeOH Flow Rate: 2.0 mL/min Temperature: 35C Detector: 254 nm
Time (min)

1. 2. 3. 4. 5.

Diltiazem Dipyridamole Nifedipine Lidoflazine Flunarizine

Time (min)

Time (min)

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

ZORBAX Rapid Resolution 3.5 m Eclipse Columns

Hardware Description Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution Solvent Saver Plus Solvent Saver Plus Solvent Saver Plus Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* MicroBore RR* MicroBore RR* MicroBore RR* MicroBore Guard Cartridges, 3/pk Guard Cartridges, 4/pk Guard Cartridges, 4/pk Guard Hardware Kit
*RR: Rapid Resolution 3.5 m

Dimensions Particle (not column) Size (m) 4.6 x 150 3.5 4.6 x 100 3.5 4.6 x 75 3.5 4.6 x 50 3.5 4.6 x 30 3.5 4.6 x 20 3.5 3.0 x 150 3.5 3.0 x 100 3.5 3.0 x 75 3.5 2.1 x 150 3.5 2.1 x 100 3.5 2.1 x 75 3.5 2.1 x 50 3.5 2.1 x 30 3.5 2.1 x 20 3.5 1.0 x 150 3.5 1.0 x 50 3.5 1.0 x 30 3.5 1.0 x 17 5 4.6 x 12.5 2.1 x 12.5 5 5

Eclipse Plus C18 USP L1 959963-902 959961-902 959933-902 959943-902 959936-902

Eclipse Plus C8 USP L7 959963-906 959961-906 959933-906 959943-906 959936-906

959963-302 959963-306 959961-302 959961-306 959763-902 959763-902 959793-902 959793-906 959743-902 959743-906 959733-902 959733-906

Eclipse XDB-C18 USP L1 963967-902 961967-902 966967-902 935967-902 934967-902 932967-902 963954-302 961967-302 966954-302 930990-902 961753-902 966735-902 971700-902 974700-902 972700-902 963600-902 965600-902 961600-902 5185-5921

Eclipse XDB-C8 USP L7 963967-906 961967-906 966967-906 935967-906 934967-906 932967-906 963954-306 961967-306 930990-906 961753-906 971700-906 974700-906 972700-906 963600-906 965600-906 961600-906 5185-5921

Eclipse XDB-Phenyl USP L11 963967-912 966967-912 935967-912

Eclipse XDB-CN USP L10 963967-905 961967-905 966967-905

963954-312 961967-312

963954-305

961753-905

820950-936 820950-937 820950-925 820950-926 820950-927 821125-936 821125-937 821125-926 821125-926 821125-926 820888-901 820888-901 820888-901 820888-901 820888-901

820950-935 821125-935 820888-901

Order online at www.agilent.com/chem/store

65

ZORBAX Rapid Resolution 3.5 m StableBond Columns

Hardware Description Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution Solvent Saver Plus Solvent Saver Plus Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* MicroBore RR* MicroBore RR* MicroBore RR* MicroBore Guard Cartridges, 3/pk Guard Cartridge, 4/pk Guard Cartridges, 4/pk Guard Hardware Kit
*RR: Rapid Resolution 3.5 m

Size (mm) 4.6 x 150 4.6 x 100 4.6 x 75 4.6 x 50 4.6 x 30 4.6 x 20 3.0 x 150 3.0 x 100 2.1 x 150 2.1 x 100 2.1 x 75 2.1 x 50 2.1 x 30 2.1 x 20 1.0 x 150 1.0 x 50 1.0 x 30 1.0 x 17 4.6 x 12.5 2.1 x 12.5

Particle Size (m) 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 5 5 5

SB-C18 USP L1 863953-902 861953-902 866953-902 835975-902 834975-902 832975-902 863954-302 861954-302 830990-902 861753-902 866735-902 871700-902 874700-902 872700-902 863600-902 865600-902 861600-902 5185-5920

SB-C8 USP L7 863953-906 861953-906 866953-906 835975-906 834975-906 832975-906 863954-306 861954-306 830990-906 861753-906 871700-906 874700-906 872700-906 863600-906 865600-906 861600-906 5185-5920

SB-CN USP L10 863953-905 861953-905 866953-905 835975-905

SB-Phenyl USP L11 863953-912 861953-912 866953-912 835975-912

SB-C3 USP L56

SB-Aq 863953-914 861953-914 866953-914 835975-914

863954-305 863954-312 861954-305 861954-312 861753-905 861753-912

863954-314 861954-309 861954-314 830990-914 861753-914 871700-914

820950-920 820950-915 820950-916 820950-917 821125-926 821125-926 821125-924 821125-926 820888-901 820888-901 820888-901 820888-901

820950-922 820950-933 821125-924 821125-933 820888-901 820888-901

66

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

ZORBAX Rapid Resolution 3.5 m Rx, Bonus-RP, and Extend-C18 Columns

Hardware Description Rapid Resolution Rapid Resolution Rapid Resolution Rapid Resolution Solvent Saver Plus Solvent Saver Plus Narrow Bore RR Narrow Bore RR* Narrow Bore RR* MicroBore RR* MicroBore RR* MicroBore RR* MicroBore Guard Cartridge, 3/pk Guard Cartridge, 4/pk Guard Cartridge, 4/pk Guard Hardware Kit
*RR: Rapid Resolution 3.5 m

Particle Size (mm) Size (m) 4.6 x 150 3.5 4.6 x 100 3.5 4.6 x 75 3.5 4.6 x 50 3.5 3.0 x 150 3.5 3.0 x 100 3.5 2.1 x 150 3.5 2.1 x 100 3.5 2.1 x 50 3.5 1.0 x 150 3.5 1.0 x 50 3.5 1.0 x 30 3.5 1.0 x 17 5 4.6 x 12.5 5 2.1 x 12.5 5

Rx-C18 USP L1 863967-902 861967-902 866967-902 863967-302 861967-302 861767-902

820950-914 821125-915 820888-901

Extend-C18 763953-902 764953-902 766953-902 735953-902 863954-306 863668-301 763954-302 861954-306 864668-301 764953-302 830990-906 863700-901 861753-906 861768-901 761753-902 861700-901 735700-902 863608-901 763600-902 865608-901 765600-902 861608-901 761600-902 5185-5922 5185-5923 820950-913 820950-928 820950-930 821125-915 821125-928 821125-930 820888-901 820888-901 820888-901

Rx-C8 USP L7 863953-906 861953-906 866953-906

Bonus-RP 863668-901 864668-901 866668-901

Order online at www.agilent.com/chem/store

67

ZORBAX Solvent Saver

Provide 60% reduction in mobile phase usage and waste generation compared to 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

ZORBAX Solvent Saver columns have a 3.0 mm internal diameter. This is ideal for reducing solvent usage by 50% over 4.6 mm ID columns. Solvent Saver columns are also ideal for LC/MS. With a typical flow rate of 0.5 ml/min, these columns are compatible with electrospray, atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) MS interfaces. These columns also improve sensitivity 2 to 3 times over 4.6 mm ID columns. Solvent Saver columns can be used with conventional HPLC instruments and are a good choice for cost effective analyses.

Solvent Saver Columns Provide up to 60% Reduction in Solvent Use and Waste Column A: ZORBAX SB-C18 883975-902 4.6 x 150mm, 5m Column B: ZORBAX SB-C18 883975-302 3.0 x 150mm, 5m Mobile Phase: 20% ACN: 80% 0.2 M Na2HPO4 + 0.1 M Citric Acid, pH 2.6 Temperature: Ambient Sample: Antibacterials 1. 2. 3. 4. 5. 6. Sulfamerazine Furazolidone Oxolinic Acid Sulfadimethoxine Sulfaquinoxaline Nalidixic Acid

Time (min)

Time (min)

This separation of antibacterials on a 4.6 and 3.0 mm ID columns, shows that solvent use is reduced by 50% just by changing to the Solvent Saver column with no change in the chromatography. This reduces the cost of analyses dramatically.

68

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

Solvent Saver Columns Increase Sensitivity Column A: ZORBAX SB-C18 863953-902 4.6 x 150mm, 3.5m Column B: ZORBAX SB-C18 863954-302 3.0 x 150mm, 3.5m Mobile Phase: 25% Methanol: 75% 0.4% Formic Acid Detector: 254 nm 1. Quercetin

Increase in sensitivity

2. 45,7 - trihydroxyflavanone

This figure shows sensitivity is increased 2-3 times with Solvent Saver columns compared to 4.6 mm ID columns when the same mass sample is injected. No change in the HPLC instrumentation is required to see the sensitivity improvements.
Time (min)

Solvent Saver Columns are Ideal for LC/MS Column: ZORBAX SB-C18 861954-302 3.0 x 100mm, 3.5m Mobile Phase: A: 70% Methanol+0.4% Formic Acid B: 30% Water+0.4% Formic Acid Flow Rate: 0.425 mL/min Detector: A: UV 254 nm B: Positive Ion APCI C: Positive Ion Electrospray Sample: Steroids 1. 2. 3. 4. 5. 6. Triamcinolone Hydrocortisone Cortisone acetate Deoxycorticosterone Hydroxyprogesterone Progesterone

Solvent Saver columns are ideal for LC/MS because the typical 0.5 mL/min flow rate allows samples to be evaluated and analyzed without changing columns when the MS interface is changed from electrospray to APCI.

Time (min)

Order online at www.agilent.com/chem/store

69

ZORBAX 80 StableBond
Description Solvent Saver Solvent Saver Solvent Saver Plus Solvent Saver Plus Particle Size (mm) Size (m) 3.0 x 250 5 3.0 x 150 5 3.0 x 150 3.5 3.0 x 100 3.5 SB-C18 USP L1 880975-302 883975-302 863954-302 861954-302 SB-C8 USP L7 880975-306 883975-306 863954-306 861954-306 SB-CN USP L10 880975-305 883975-305 863954-305 861954-305 SB-C3 USP L56 880975-309 883975-309 863954-309 861954-309 SB-Phenyl USP L11 880975-312 883975-312 863954-312 861954-312 SB-Aq 880975-314 883975-314 863954-314 861954-314

ZORBAX 300 StableBond

Description Solvent Saver Plus Solvent Saver Plus Particle 300SB-C18 Size (mm) Size (m) USP L1 3.0 x 150 3.5 863974-302 3.0 x 100 3.5 300SB-C8 300SB-CN 300SB-C3 USP L7 USP L10 USP L56 863974-306 863974-309 863974-309 861973-306

ZORBAX 80 Eclipse XDB

Description Solvent Saver Solvent Saver Solvent Saver Plus Solvent Saver Plus Solvent Saver Plus Particle Size (mm) Size (m) 3.0 x 250 5 3.0 x 150 5 3.0 x 150 3.5 3.0 x 100 3.5 3.0 x 75 3.5 XDB-C18 USP L1 990967-302 993967-302 963954-302 961967-302 966954-302 XDB-C8 USP L7 990967-306 993967-306 963954-306 961967-306 XDB-Phenyl USP L11 990967-312 993967-312 963954-312 961967-312 XDB-CN USP L10 990967-305 993967-905 963954-305

ZORBAX 80 Bonus-RP, and Rx

Description Solvent Saver Solvent Saver Solvent Saver Plus Solvent Saver Plus Particle Size (mm) Size (m) 3.0 x 250 5 3.0 x 150 5 3.0 x 150 3.5 3.0 x 100 3.5 Bonus-RP 880668-301 883668-301 863668-301 864668-301 Rx-C18 USP L1 880967-302 883967-302 863967-302 861967-302 Rx-C8 USP L7 880975-306 883975-306 863954-306 861954-306

ZORBAX 80 Extend-C18
Description Solvent Saver Solvent Saver Plus Solvent Saver Solvent Saver Plus Solvent Saver Plus Particle Size (mm) Size (m) 3.0 x 250 5 3.0 x 150 3.5 3.0 x 150 5 3.0 x 100 3.5 3.0 x 50 3.5 Extend-C18 770450-302 763954-302 773450-302 764953-302 735954-302

70

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

ZORBAX MicroBore (1.0 mm ID)

High sensitivity for small sample sizes Compatible with LC/MS interfaces Wide variety of bonded phases

MicroBore (1 mm ID) columns are often a good choice when sample sizes are limited. They can improve detection limits 5 times over 2.1 mm ID columns when the same sample mass is used. This increase in sensitivity can be critical. MicroBore columns use low flow rates (typically ~ 50 l/min). Therefore, these columns are ideal for use with detectors requiring low flow rates such as some mass spectrometers and with capillary LC systems. MicoBore columns perform optimally with HPLC systems purchased or modified for microbore use. A wide variety of bonded phases are available for use up to 400 bar including StableBond SB-C18, SB-C8, 300SB-C18; Eclipse XDB-C18 and XDB-C8; Bonus RP, Extend C-18; and Poroshell columns. Guard columns are also now available with an adjustable tube stop depth to provide a perfect zero dead volume connection every time. A selection of bonded phases are available in 1.8 m and a higher pressure format. See the product listings for more detail.

Separation of a Tryptic Digest on ZORBAX MicroBore 300SB-C18 Column: ZORBAX 300SB-C18 863630-902 1.0 x 150mm, 3.5m Mobile Phase: Gradient: 2-60% B in 60 Min. A: 0.1% TFA B: 0.075% TFA/80% ACN Flow Rate: 50 L/min Temperature: 50C Detector: 215 nm Sample: 2 L Tryptic Digest of rhGH

This example of a tryptic digest separated on a MicroBore column demonstrates the high sensitivity and resolution possible with 1.0 mm ID columns.

Order online at www.agilent.com/chem/store

71

ZORBAX MicroBore (1.0 mm ID)

Description MicroBore MicroBore RR* MicroBore RR* MicroBore RR* MicroBore RRHT** MicroBore Guard Cartridges, 3/pk Description MicroBore RR* MicroBore RR* MicroBore RR* MicroBore RRHT, 600 bar** MicroBore Guard Cartridges, 3/pk Description MicroBore MicroBore Guard Cartridge, 3/pk
*RR: Rapid Resolution 3.5 m **RRHT: Rapid Resolution HT 1.8 m

Size (mm) 1.0 x 250 1.0 x 150 1.0 x 50 1.0 x 30 1.0 x 50 1.0 x 17

Particle Size (m) 5 3.5 3.5 3.5 1.8 5 Particle Size (m) 3.5 3.5 3.5 1.8 5 Particle Size (m) 5 5

SB-C18 USP L1

300SB-C18 USP L1 861630-902 863600-902 863600-906 863630-902 865600-902 865600-906 865630-902 861600-902 861600-906 822600-902 822600-906 5185-5920 5185-5920 5185-5920 XDB-C8 USP L7 963600-906 965600-906 961600-906 928600-906 5185-5921 Poroshell 300SB-C8 661750-906 5185-5968

SB-C8 USP L7

300SB-C8 USP L7 863630-906 865630-906

SB-CN USP L10

822600-905 5185-5920

Size (mm) 1.0 x 150 1.0 x 50 1.0 x 30 1.0 x 100 1.0 x 17

XDB-C18 USP L1 963600-902 965600-902 961600-902 928600-902 5185-5921 Poroshell 300SB-C18 661750-902 5185-5968

Bonus-RP 863608-901 865608-901 861608-901 5185-5922 Poroshell 300SB-C3 661750-909 5185-5968

Extend-C18 763600-902 765600-902 761600-902 728600-902 5185-5923 Poroshell 300Extend-C18 671750-902

Size (mm) 1.0 x 75 1.0 x 17

72

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

ZORBAX Capillary and Nano

Highest sensitivity for your smallest sample sizes Compatible with all LC/MS interfaces Internal diameters of 0.5, 0.3, 0.1, and 0.075 mm Packings/phases for both small and large molecules (80 and 300 pore sizes, respectively) Ideal for 1-D and 2-D (proteomics) applications

Agilent ZORBAX capillary (0.5, 0.3 mm ID) and nano (0.1, 0.075 mm ID) columns are now available in a wide variety of phases, pore sizes, and dimensions. These columns are ideal for very sample-limited applications because they provide enhanced sensitivity by reducing oncolumn sample dilution. This high sensitivity can be provided with exceptional reproducibility using Agilent columns and low dispersion HPLC instruments. The fastest growing application for capillary and nano columns is 2-D LC/MS for complex proteomics samples. Agilent provides all the columns needed for the 2-D separation the SCX columns for the first dimension, the reversed-phase trapping column, and the reversed-phase column for the second dimension.

Order online at www.agilent.com/chem/store

73

Separation of Peptides on Capillary Columns

Column A: Column B: Column C: Column D: Column E: Column F:

ZORBAX 300SB-C8 5065-4460 0.3 x 150mm, 3.5m ZORBAX Eclipse XDB-C18 5064-8291 0.3 x 150mm, 5m ZORBAX Eclipse XDB-C18 5064-8291 0.3 x 150mm, 5m ZORBAX SB-C18 5064-8255 0.3 x 150mm, 5m ZORBAX 300SB-C18 5064-8267 0.3 x 150mm, 3.5m ZORBAX 300Extend-C18 5065-4464 0.3 x 150mm, 3.5m

1. 2. 3. 4. 5.

Gly-Tyr, 5 ng/100 nl Val-Tyr-Val, 20 ng/100 nl Met Enkephalin, 28 ng/100 nl Low Enkephalin, 20 ng/100 nl Angiotensin II, 20 ng/100 nl
2 3 4 5 3 4 5 3 2 2 2 3 3 4 4 5 3 4 5 4 5 5 4 5 1 1 2 1 1 1 2 2 3

400 300 200

100

1 1

Mobile Phase: Water + 0.05% TFA, pH = 2.2 = A Acetronitrile + 0.045% TFA = B Gradient 0.5%B/min: at 0 min = 1%B, at 60 min = 31%B, at 70 min = 50%B, at 75 min = 85%B, at 80 min = 85%B, at 81 min = 1%B, at 110 min = 1%B Flow Rate: 5.5 l/min Low Solvent Consumption: 200-500 l/min Temperature: 30C Detector: 206/10 nm, ref 450/80 nm Sample: 0.1 l, automatic delay volume reduction was activated Peptides

0 0 20 40 60

Time (min)
This example shows a peptide standard mixture separated on a variety of the ZORBAX capillary columns. These chromatograms demonstrate the wide range of selectivities available, which can be used to optimize your specific separation.

74

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

High Sensitivity with Capillary Columns Column: ZORBAX SB-C18 5064-8255 0.3 x 150mm, 5m Column: ZORBAX SB-C18 5064-8256 0.5 x 150mm, 5m Column: ZORBAX SB-C18 863600-902 1.0 x 150mm, 3.5m Column: ZORBAX SB-C18 883975-902 4.6 x 150mm, 5m Sample: 200 ng Biphenyl
Sample-limited applications require capillary column dimensions to minimize oncolumn sample dilution and to enhance sensitivity. The 0.3 mm capillary in this example provides 100 times more sensitivity than the standard 4.6 mm column. Nanobore (0.1 mm-0.075 mm ID) columns can provide up to 2000 times more sensitivity for your most limited sample applications.

Time (min)

Order online at www.agilent.com/chem/store

75

Excellent Column-to-Column Reproducibility with Agilent Capillary Columns ZORBAX SB-C18 5064-8256 0.5 x 150mm, 5m Mobile Phase: A: 75% H2O with 0.4% formic acid B: 25% MeOH with 0.4% formic acid Flow Rate: 20 L/min Temperature: 25C Sample: 0.1 L Polar organic acids Column: 1. 2. 3. 4. 5. 6. Protocatechuic acid Chlorogenic acid Caffeic acid Syringic acid p-coumaric acid Ferulic acid

Excellent reproducibility is seen for a separation of polar organic acids on three different StableBond-C18, 0.5 x 150 mm, 5 m columns. Retention (k) varied less than 0.8% RSD and selectivity (a) varied less than 0.4% RSD.
Time (min)

76

HPLC Columns for Special Applications

2D LC/MS Analyses Using ZORBAX Capillary and Nano LC Columns Typical Column Configuration for 2D HPLC
1100/1200 Series quaternary pump Waste 1100/1200 Series micro well plate autosampler Reversed-phase enrichment column Nanopump

1.

Reversed-phase column SCX Strong cation exchange column Waste 1100/1200 Series column compartment

Detection (1100/1200 Series LC/MSD Trap)

1100/1200 Series quaternary pump Waste 1100/1200 Series micro well plate autosampler Reversed-phase enrichment column Nanopump

2.
SCX Strong cation exchange column Waste

Reversed-phase column 1100/1200 Series column compartment

Detection (1100/1200 Series LC/MSD Trap)

Flow path of the Agilent 1100 Series Nanoflow Proteomics Solution system. 1. Sample loading, elution from SCX and trapping on enrichment column 2. Valve switch in column compartment, elution from enrichment column; separation on RP, and MS anaylsis

Order online at www.agilent.com/chem/store

77

Proteins in a Complex Sample by 2-D HPLC with Nano HPLC Columns Column: Column: ZORBAX 300SB-C18 5065-9913 0.3 x 5mm, 5m ZORBAX 300SB-C18 5065-9911 0.075 x 150mm, 3.5m

30 mM KCI

Mobile Phase:

Quaternary Pump: 3% Acetonitrile/0.1% Formic Acid Nanopump: A = Water, 0.1% Formic Acid, B = ACN, 0.1% Formic Acid Flow Rate: Quaternary Pump: 30 L/min Nanopump: 300 nL/min Gradient: Quaternary Pump: Isocratic Nanopump: 6 min = 3%B, 120 min = 60%B, 125 min = 80%B, 130 min = 80% B, 131 min = 3%B, 140 min = 3% B MS Conditions: Source: Nano ESI, drying gas flow: 5L/min, drying gas temp.: 225C. Ion Trap: Skim: 1:35 V, cap exit offset: 115 V, octopole 1:12 V, octopole 2:3.5 V, trap drive: 80 V. ICC: on, averages: 4, max accu time: 150 ms; target 60.000, ion mode positive, MS/MS mode. Sample: Tryptic Digest of bovine serum albumin Volume: 1 to 8 L Salt Step Elution: 8ml of 10 mM-100 mM KCI (10 mM increments), 125 mM, 150 mM, 200 mM, 300 mM, 500 mM, 1M.

60 mM KCI

75 mM KCI

120 mM KCI

Time (min)

Tryptic digest of bovine serum albumin (BSA). The base peak chromatograms show a selection of fractions from a 2-dimensional HPLC separation. Single chromatograms represent peptides from BSA eluting at a given salt concentration followed by enrichment and reversed phase chromatography.

78

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

ZORBAX Capillary and Nano

Particle Description Size (mm) Size (m) Capillary 0.8 x 50 3.5 Capillary 0.5 x 250 5 Capillary 0.5 x 150 5 Capillary 0.5 x 75 5 Capillary 0.5 x 35 5 Capillary RR* 0.5 x 35 3.5 Capillary 0.3 x 250 5 Capillary 0.3 x 150 5 Capillary 0.3 x 35 5 Capillary 0.3 x 35 3.5 Capillary RR* 0.3 x 150 3.5 Capillary RR* 0.3 x 100 3.5 Capillary RR* 0.3 x 75 3.5 Capillary RR* 0.3 x 50 3.5 Replacement Screens, 10/pk
*RR: Rapid Resolution 3.5 m

SB-C18

Eclipse XDB-C18

Poroshell 300SB-C18 300SB-C8 300SB-C8

300Extend C18

Bio-SCX Series II 5065-9942

5064-8258 5064-8286 5064-8266 5064-8256 5064-8287 5064-8264 5065-4468 5064-8254 5064-8260 5064-8257 5064-8255 5064-8253 5064-8296 5064-8298 5064-8269 5064-8291 5064-8297 5064-8294 5065-4459 5064-8265 5064-8263 5064-8295 5065-9912 5064-8261 5064-8271 5064-8267 5064-8259 5064-8270 5064-8300 5065-4427 5065-4427 5065-4427 5065-4460 5065-4461 5065-4462 5065-4463 5065-4427 5065-4427 5065-4464 5065-4465 5065-4466 5065-4467 5065-4427

Particle Description Size (mm) Size (m) Nano RR* 0.1 x 150 3.5 Nano RR* 0.075 x 150 3.5 Nano RR* 0.075 x 50 3.5 Trap/Guard, 5/pk 0.3 x 5 5 Trap/Guard Hardware kit
*RR: Rapid Resolution 3.5 m

300SB-C18 USP L1 5065-9910 5065-9911 5065-9924 5065-9913 5065-9915

300SB-C8 USP L7

5065-9923 5065-9914 5065-9915

Order online at www.agilent.com/chem/store

79

ZORBAX PrepHT

Easy scale-up from analytical to preparative scale with ZORBAX phases Fast preparative separations, up to 2000 mg 5 to 7 m particles for high efficiency and high yield Easy to install finger tight connections seal up to 5000 psi/350 bar

High purity, high recovery and high throughput can be easily achieved with Agilent ZORBAX PrepHT columns. These are available in a variety of bonded phases Eclipse XDB, StableBond, Bonus-RP, and Extend-C18 for optimized resolution and loadability under any conditions. ZORBAX PrepHT columns are packed with 5 and 7 m particle sizes for very high resolution. The high resolution allows high loadability, high yield, and high purity of compounds. The larger diameter columns and mechanically stronger ZORBAX particles allow for flow rates up to 100 ml/min, thus increasing throughput. ZORBAX PrepHT columns are designed for rapid scale-up from analytical to preparative scale without losing resolution. For complex separations on larger columns (21.2 mm ID, 150 mm length and longer), Agilent has carefully chosen the 7 m particle size to achieve a balance between high efficiency and high loadability.

80

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

High Purity and High Recovery with ZORBAX PrepHT Columns

Vial 1 | 347 Vial 2 | 343 Vial 3 | 389

Sample: Antianginal drugs DAD

MSD

1. Nifedipine 2. Nimodine 3. Nisoldipine

0 1 2 3 4 5 6 7

Time (min)

Mass-based fraction collection using ZORBAX SB-C18 column shows high purity and high recovery of each compound (Application Note publication number 5988-7113EN). The separation of the three antianginal drugs was successfully done in a single run with high recovery and >90% purity. Separations up to 2000 mg are possible depending on the complexity of separation.

ZORBAX PrepHT columns are designed for rapid scale-up from analytical to preparative scale without losing resolution. For complex separations for larger columns (21.2 mm ID and higher), 150 mm length and higher), Agilent has carefully chosen the 7 m particle size to achieve a balance between high efficiency and high loadability.

Scale-Up from Analytical to Prep ZORBAX SB-C18 Columns Using the Same Pump

Column Column 1 Column 2 Column 3 Column 4

Size 50 x 150 mm 21.2 x 150 mm 9.4 x 150 mm 4.6 x 150 mm

Flow (mL/min) 100 18 3.5 0.85

Injection (L) 2200 400 80 2.0

Detector Cell 0.3 mm quartz 0.3 mm quartz 0.3 mm quartz 3 mm SST

Part No. Custom Column 877150-102 883975-202 883975-902

Using the same 1100 pump, a scale-up from 4.6 mm to 50 mm ID was possible without any loss of resolution. This increase throughput by reducing the time required for redeveloping and adjusting the method.

Column 1 Column 2 Column 3 Column 4

Time (min)

1. Theobromine 2. Theophylline 3. Caffeine

Order online at www.agilent.com/chem/store

81

ZORBAX PrepHT StableBond ZORBAX PrepHT 80StableBond

Hardware Description PrepHT Cartridge* Size (mm) 21.2 x 250 21.2 x 150 21.2 x 150 21.2 x 100 21.2 x 50 PrepHT Guard Cartridge, 2/pk** 17 x 7.5 Particle Size (m) 7 7 5 5 5 5 SB-C18 USP L1 877250-102 877150-102 870150-902 870100-902 870050-902 820212-920 SB-C8 SB-CN SB-C3 SB-Phenyl USP L7 USP L10 USP L56 USP L11 877250-106 877250-114 877250-105 877250-112 877150-106 877150-114 870150-906 870150-914 870100-906 870100-914 870050-906 870050-914 820212-915 820212-933 820212-933 820212-915

ZORBAX PrepHT 300StableBond

Hardware Description PrepHT Cartridge* Size (mm) 21.2 x 250 21.2 x 150 21.2 x 150 21.2 x 100 21.2 x 50 PrepHT Guard Cartridge, 2/pk**
*Requires PrepHT endfittings 820400-901 **Requires Guard hardware kit (820444-901) which contains guard column fitting, seal insertion tool, and 1 polymeric seal

Particle Size (m) 7 7 5 5 5 5

300SB-C18 USP L1 897250-102 897150-102 895150-902 895100-902 895050-902 820212-921

300SB-C8 300SB-CN 300SB-C3 USP L7 USP L10 USP L56 897250-106 897250-109 897250-105 897150-106 897150-109 895150-906 895150-909 895100-906 895100-909 895050-906 895050-909 820212-918 820212-924 820212-924

17 x 7.5

ZORBAX PrepHT Eclipse XDB

Hardware Description PrepHT Cartridge* Size (mm) 21.2 x 250 21.2 x 150 21.2 x 150 21.2 x 100 21.2 x 50 PrepHT Guard Cartridge, 2/pk**
*Requires PrepHT endfittings 820400-901 **Requires Guard hardware kit (820444-901) which contains guard column fitting, seal insertion tool, and 1 polymeric seal

Particle Size (m) 7 7 5 5 5 5

XDB-C18 XDB-C8 USP L1 USP L7 977250-102 977250-106 977150-102 977150-106 970150-902 970150-906 970100-902 970100-906 970050-902 970050-906 820212-925 820212-926

17 x 7.5

82

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

ZORBAX PrepHT Bonus-RP and Extend-C18

Hardware Description PrepHT Cartridge* Particle Size (mm) Size (m) Bonus-RP Extend-C18 21.2 x 250 7 878250-101 21.2 x 150 21.2 x 150 21.2 x 100 21.2 x 50 PrepHT Guard Cartridge, 2/pk**
*Requires PrepHT endfittings 820400-901 **Requires Guard hardware kit (820444-901) which contains guard column fitting, seal insertion tool, and 1 polymeric seal

7 5 5 5 5

878150-101 868150-901 770150-902 868100-901 770100-902 868050-901 770050-902 820212-928 820212-930

17 x 7.5

ZORBAX PrepHT Original

Hardware Description PrepHT Cartridge*
*Requires PrepHT endfittings 820400-901

Particle ODS (C18) Size (mm) Size (m) USP L1 21.2 x 250 7 877952-102

C8 CN NH2 SIL USP L7 USP L10 USP L8 USP L3 877952-106 877952-105 877952-108 877952-101

ZORBAX PrepHT Rx-SIL

Hardware Description PrepHT Cartridge* PrepHT Guard Cartridge, 2/pk**
*Requires PrepHT endfittings 820400-901 **Requires Guard hardware kit (820444-901) which contains guard column fitting, seal insertion tool, and 1 polymeric seal

Particle SIL Rx-C18 Size (mm) Size (m) USP L3 USP L1 21.2 x 250 7 877250-101 21.2 x 250 17 x 7.5 7 5 877967-102 820212-919 820212-914

ZORBAX PrepHT Accessories

Hardware Description Guard Cartridge Hardware PrepHT Endfittings, 2/pk Replacement Seals Part No. 820444-901 820400-901 820385-901

Order online at www.agilent.com/chem/store

83

Agilent 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 10

Agilent Prep LC columns are designed for high loadability to purify milligram to gram quantities of products. Preparative sized columns are available in 21.2, 30, and 50 mm internal diameters with lengths ranging from 50-250 mm. Columns are available in 5 and 10 m particle sizes with very high efficiency in every dimension. These column choices accommodate almost every preparative sample. Agilent Prep 21.2 mm ID columns are available with Agilents Preparative Cartridge Hardware. This reliable cartridge hardware makes it simple to use columns with different lengths to increase sample load. Guard columns are easily integrated onto these columns providing superior protection of the analysis column. Analytical sized 4.6 mm ID scalar columns are available 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 wide variety of non-polar and polar compounds. Unbonded silica columns are also available.

Column Specifications
Bonded Phase C18 Silica Pore Size 100 100 Surface Area 400 m2/g 400 m2/g Temp. Limits pH Range 60C* 2.0-10.0 ** 1.0-8.0 Carbon Endcapped Load Single 24% N/A N/A

*Temperature limits are 60C up to pH 8, 40C from pH 8-10. **Temperature limits for bare silica are determined by the pH of the mobile phase.

84

Order online at www.agilent.com/chem/store

HPLC Columns for Special Applications

Superior Loadability on Agilent Prep C18 with Basic Compounds Column: Agilent Prep-C18 443905-902 4.6 x 150mm, 5m Mobile Phase: 50% 0.1%TFA: 50% ACN Flow Rate: 1 mL/min Sample: 10 L Doxepin/Amitriptyline 0.550 mg/mL

Agilent Prep Columns show better resolution and loadability than competitor columns.

Steroids: Easy Scalability Using Agilent Prep Columns Column A: Column B: Column C: Column D: Agilent Prep-C18 443905-902 4.6 x 150mm, 5m 443905-102 21.2 x 150mm, 5m 413910-302 30 x 150mm, 10m 413910-502 50 x 150mm, 10m 1. Hydrocortisone 2. Testosterone (in MeOH @ 1mg/mL)

Mobile Phase: 55% Water:45% ACN Flow Rate: 0.7 mL/min 14.87 mL/min 29.77 mL/min 85.37 mL/min Temperature: Ambient Detector: 240 nm Sample: 2 L 42.4 L 170 L 488 L

Agilent Prep-C18 shows excellent scalability making method transfer simple and predictable.

Order online at www.agilent.com/chem/store

85

HPLC Columns for Special Applications

Agilent Prep LC Columns

Hardware Description Particle Size (mm) Size (m) C18 4.6 x 250 4.6 x 150 4.6 x 100 4.6 x 50 21.2 x 250 21.2 x 150 21.2 x 50 21.2 x 150 21.2 x 100 21.2 x 50 5 5 5 5 10 10 10 5 5 5 440905-902 443905-902 449905-902 446905-902 Silica 440905-901 443905-901 449905-901 446905-901

Standard Columns (no special hardware required, 400 bar)

Scalar Scalar Scalar Scalar

PrepHT Cartridge Columns (require endfittings kit 820400-901)*

PrepHT PrepHT PrepHT PrepHT PrepHT PrepHT PrepHT Endfittings, 2/pk 410910-102 410910-101 413910-102 413910-101 446910-102 443905-102 443905-101 449905-102 449905-101 446905-102 446905-101 820400-901 820400-901 30 x 250 30 x 150 30 x 100 30 x 100 30 x 50 50 x 250 50 x 150 50 x 100 50 x 100 50 x 50 21.2 x 10 10 10 10 5 5 10 10 10 5 5 10 410910-302 413910-302 419910-302 449905-302 446905-302 410910-502 413910-502 419910-502 449905-502 446905-502 410910-301 413910-301 419910-301 449905-301 446905-301 410910-501 413910-501 419910-501 449905-501 446905-501

Standard Columns (no special hardware required, 400 bar)

Prep 30 Prep 30 Prep 30 Prep 30 Prep 30 Prep 50 Prep 50 Prep 50 Prep 50 Prep 50

Guard Columns (hardware required)

PrepHT Guard Cartridges, 2/pk Guard Cartridge Hardware PrepHT External Guard Hardware Kit Bulk Packing (1kg) 10 420212-902 420212-901 820444-901 820444-901 420420-901 420420-901 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 columns then the PrepHT Guard Hardware Kit, 820444-901, is also required. If the guard column is used on a 30 mm ID column then the external guard column hardware kit, 420420-901, is required.

86

Order online at www.agilent.com/chem/store

Ultron Chiral columns

Separate a wide range of chiral compounds and develop chiral methods more quickly.
Ultron Chiral columns are immobilized protein columns that feature numerous chiral recognition sites for enantiomeric separations of dozens of chiral compounds. They are engineered with two complementary protein-based chiral stationary phases, making them an excellent choice for the HPLC separation of enantiomers without derivatization including a growing number of drug substances of interest.

Broad compatibility and long lifetime with aqueous, buffered, and organic mobile phases. Recognize ionic, polar, hydrogen-bonding, and hydrophobic groups as well as three-dimensional molecular structures. Method development guidelines are included in each column box, saving you time and eliminating guesswork.

Order online at www.agilent.com/chem/store

87

Separation of Enantiomers of Fluoxetine (Prozac) Column: Ultron ES-OVM Chiral 702111651 4.6 x 150mm, 5m Mobile Phase: 25:75 (v/v) EtOH / 20 mM KH2PO4, pH 5.5 (adjusted with NaOH) Temperature: Ambient Detector: UV (225 nm) Sample: Mixture Fluoxetine (Prozac) entantiomers
0 5 10 20 15 Retention Time (min) 25 30

Courtesy of D. S. Risley and V. S. Sharp of Lilly Research Laboratories, Eli Lilly and Co.

Separation of Ethiazide (diuretic drug) on ULTRON ES-OVM Column Ultron ES-OVM Chiral 702111651 4.6 x 150mm, 5m Mobile Phase: 20 mM KH2PO4 (pH 4.6) Flow Rate: 1 mL/min Temperature: 25C Detector: 220 nm Column:
H N H C 2H 5 C O S NH O

Cl H 2N S O O

10

20 Time (min)

Description Semi-Prep Analytical Analytical Analytical, with Guard Narrow Bore Guard Column

Particle Size (mm) Size (m) 10 x 150 5 4.6 x 250 10 4.6 x 150 5 4.6 x 150 5 2.0 x 150 5 4.0 x 10 5

ES-OVM 722111723 724111653 702111651 702111651A 702111610 712111630

ES-Pepsin

822111651 822111631A 832111630

88

Order online at www.agilent.com/chem/store

ZORBAX Columns for Bioanalytical Chromatography

Is 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-size exclusion separations of antibodies. The number of bioanalytical HPLC applications continues to grow at an unprecedented rate. And Agilents durable and reproducible line of ZORBAX columns can help you meet your labs evolving needs for performance and speed. On the following pages, you will find key facts and specifications about columns for reversed-phase HPLC, size exclusion chromatography, ion-exchange chromatography, and hydrophobic interaction chromatography, including:

ZORBAX wide-pore 300StableBond columns deliver long lifetime, high resolution, and good peak shape for peptides and proteins. And they are compatible with common mobile phases containing TFA. ZORBAX wide-pore 300Extend columns ideal for LC/MS of proteins and peptides at pH 10. ZORBAX Poroshell HPLC columns feature a unique, powerful particle for fast, high-resolution separations of proteins, polypeptides, and antibodies. ZORBAX Eclipse Amino Acid Analysis (AAA) HPLC columns a high-efficiency choice for rapid amino acid separation! These columns make it possible to reduce your injection-to-injection analysis time to 14 minutes (7.5 cm column length) and 24 minutes (15 cm column length). ZORBAX GF-250/450 Size Exclusion HPLC columns a rugged, reproducible column specially designed for size separations of proteins. Size exclusion columns are compatible with organic modifiers and mobile phase denaturants, so you can eliminate protein aggregation for proper size determination. And they have a separation range of 4,000 900,000 for globular proteins when using a GF-250 and GF-450 column in series. ZORBAX Ion Exchange columns available as Strong Anion Exchange (SAX), Strong Cation Exchange (SCX), and Bio-SCX.

89

ZORBAX Strategy for Reversed-Phase Method Development of Proteins and Peptides

This ZORBAX Column Selection Strategy for Proteins and Peptides provides some critical details on method development for proteins or polypeptides. For small peptides, Molecular Weight < 2000, please follow the method development strategy for small and large molecules in the reference section of this guide. For efficient separations of large molecules, columns with a wide-pore size (300) are required. For method development of larger peptides and proteins, review the suggested guidelines outlined below. Wide-pore column choices are described in the following section of this Column Selection Guide.

Choose the Initial Column and Conditions for Proteins and Peptides
Peptides, Polypeptides, Proteins
MW < 50 kDA Initial Bonded Phase Choice

Peptides, Polypeptides, Proteins

MW < 1,000 kDA

StableBond 300SB-C8
300 SB columns are wide-pore columns with unbeatable lifetime in TFA-containing mobile phases. This makes them an ideal first choice for separations of peptides and proteins.

Poroshell 300SB-C18
Poroshell 300SB columns use an innovative particle technology to deliver rapid protein separations. Short analysis times with efficient peaks are easily obtained with Poroshell columns.

C8 is an excellent starting bonded phase because of its moderate hydrophobicity. digests but can also be used for proteins.

C18 and C8 are generally selected for peptides and protein

C3, C4 and CN are generally selected for larger, hydrophobic polypeptides and proteins but can also be used for peptides.

C18 is a good starting bonded phase choice with Poroshell for most peptides, polypeptides and proteins because the retention is maximized. be used with polypeptides or very large proteins.

C8 is generally selected for moderate size proteins but can C3 is generally selected for antibodies or large proteins but
can be used for peptides and polypeptides.

Initial Separation Conditions

Column:

Mobile Phase: Gradient: Temperature: Flow Rate:

StableBond 300SB-C8 4.6 x 150 mm, 3.5 or 5 m 883995-906 863973-906 A: 95% H2O:5% ACN with 0.1% TFA B: 5% H2O:95% ACN with 0.085% TFA 0-60% B in 60 min 35-40C 1 mL/min

Column: Mobile Phase: Gradient: Temperature: Flow Rate:

Poroshell 300SB-C18 2.1 x 75 mm, 5 m 660750-902 A: 95% H2O:5% ACN with 0.1% TFA B: 5% H2O:95% ACN with 0.085% TFA 0-60% B in 10 min 35-40C 2 mL/min

90

Order online at www.agilent.com/chem/store

ZORBAX Columns for Bioanalytical Chromatography

Start at low pH with simple aqueous/organic gradient

Typically a Water/Acetonitrile with 0.1% TFA gradient is used to elute all components of interest. A typical high resolution gradient on a 300 pore size column requires 30-50 min. A Poroshell column requires a shorter analysis time and a higher flow rate and still provides exceptional resolution. Then to improve resolution, increase the gradient time, decrease column length, or increase flow rate.

Optimize sample solubility

For best peak shape and recovery at any pH, it is important to solubilize a sample completely. Highly acidic or neutral solvents can be used with ZORBAX 300StableBond and Poroshell 300SB, while neutral solvents and dilute bases can be used with ZORBAX 300Extend-C18.

Solvent Choices to Solubilize Proteins and Peptides

Water/Phosphate Buffer Dilute Acid (TFA, Acetic Acid or HCl) Neutral pH, 6-8 M Guanidine-HCl or Isothiocyanate 5% HOAc/6 M Urea Dilute Acid + Aqueous/Organic Solvents (ACE, MeOH, THF) Dilute Base (Ammonioum Hydroxide) DMSO or 0.1%-1% TFA in DMSO Formamide Weakest

Strongest

Raise the Temperature

Separations of proteins and peptides are influenced by temperature and higher column temperature can dramatically improve both resolution and recovery of proteins and hydrophobic and aggregating peptides. StableBond 300SB up to 80C Poroshell 300SB up to 80C

Optimize Mobile Phase pH Try mid and high pH if low pH does not work
If 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 is often very different because acidic amino acids become negatively charged and some basic amino acids may lose their charge. ZORBAX 300Extend-C18 is an excellent choice for mid to high pH separation.

Column:
Mobile Phase: Gradient: Temperature: Flow Rate:

300Extend-C18 4.6 x 150 mm, 5 m 773995-902

A: 20 mM NH4OH in H2O B: 20 mM NH4OH in 80% ACN 5-60% B in 30 minutes 25-30C (<60C) 1 mL/min

Order online at www.agilent.com/chem/store

91

Starting Column Choices for Analytical Separations of Peptides, Polypeptides, and Proteins
Low pH Mid and High pH

MW < 50 kDa 300SB-C8

4.6 x 150 mm, 3.5 m 863973-906

MW < 1000 kDa Poroshell 300SB-C18

2.1 x 75 mm, 5 m 660750-902

MW < 25 kDa 300 Extend-C18

4.6 x 150 mm, 3.5 m 763973-902

Separations of Proteins and Peptides Using Reversed-Phase LC/MS Methods

LC/MS of proteins and peptides is used to provide information for protein characterization, to accurately identify post-translational modifications of proteins, and to determine the molecular weight of synthetic and natural peptides. LC/MS is used to provide protein identification in 2-D separations for proteomics applications. Therefore, LC/MS of proteins and peptides is a critical separation area, which requires some special column and mobile phase recommendations. In general, smaller column sizes are used for LC/MS and TFA is generally not 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 Polypeptides

Analytical LC/MS Applications 2.1 mm I.D. columns will provide good sensitivity when sample size is not limited. With Poroshell columns, smaller column I.D.s are used.

Low pH

Mid and High pH

MW < 50 kDa 300SB-C8

2.1 x 150 mm, 3.5 m 863750-906

MW < 1000 kDa Poroshell 300SB-C18

1.0 x 75 mm, 5 m 661750-902

MW < 25 kDa 300Extend-C18

2.1 x 150 mm, 3.5 m 763750-902

High Sensitivity/Proteomics Applications

Capillary columns are used for high sensitivity protein and peptide applications. The 0.5 mm I.D. columns are used for protein and protein digest separations while the 0.3 mm I.D. columns are most often used for protein digests. These can be analyzed at high pH with an ammonium hydroxide mobile phase. Nano columns (0.1 and 0.075 mm I.D.) are often used in 2-D LC/MS systems for proteomics and the initial choice is a C18 bonded phase.

92

Order online at www.agilent.com/chem/store

ZORBAX Columns for Bioanalytical Chromatography

High Sensitivity Capillary Columns

Low pH Mid and High pH

MW < 50 kDa 300SB-C18

0.5 x 150 mm, 3.5 m 5064-8268

MW < 1000 kDa Poroshell 300SB-C8

0.5 x 74 mm, 5 m 5065-4468

MW < 25 kDa 300Extend-C18

0.3 x 150 mm, 3.5 m 5065-4464

Nano Columns for Proteomics/2-D LC/MS

SCX (1st Dimension)

0.3 x 35 mm, 5 m 5065-9912

Reversed-Phase (2nd Dimension)

0.075 x 150 mm, 3.5 m 5065-9911

Mobile Phase Considerations

Low pH

Mid and High pH

TFA is generally not used for LC/MS separations of proteins and peptides. The first step is normally to replace TFA with 0.1-1.0% formic acid. Acetic acid, up to 1%, can also be used as an alternative mobile phase modifier. At low pH, the best separation may 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 NH4OH as a mobile phase additive.

Order online at www.agilent.com/chem/store

93

R O Si R OH R O Si R OH R O Si R
Sterically Protected 300StableBond Bonded Phase

ZORBAX 300 StableBond

R1
ZORBAX 300StableBond columns are an ideal choice for the reproducible separations of proteins and peptides for two key reasons. First, wide-pore, 300 columns are necessary for an efficient separation of proteins and peptides, or other large molecules, in order to allow these analytes to completely access the bonded phase. Second, 300StableBond columns are unmatched in their durability at low pH, such as with the TFA containing mobile phases typically 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 phase modifiers. These columns are available in four different bonded phases (C18, C8, C3, and CN) for selectivity and recovery optimization of proteins and polypeptides. To further increase sample recovery and improve efficiency for difficult proteins, 300StableBond columns can be used up to 80-90C. 300SB-C18 and 300SB-C8 columns are an ideal choice for 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/MS separations of these protein digests. These capillary and nano columns can be used for either 1-D or 2-D proteomics separations.

R1

R1

Column Specifications
Bonded Phase ZORBAX 300SB-C18 ZORBAX 300SB-C8 ZORBAX 300SB-C3 ZORBAX 300SB-CN Pore Size 300 300 300 300 Surface Area 45 m2/g 45 m2/g 45 m2/g 45 m2/g Temp. Limits* 90C 80C 80C 80C pH Range* 1.0-8.0 1.0-8.0 1.0-8.0 1.0-8.0 Endcapped No No No No Carbon Load 2.8% 1.5% 1.1% 1.2%

*300 StableBond columns are designed for optimal use at low pH. At pH 6-8, highest column stability for all silica-based columns is obtained by operating at temperatures <40C and using low buffer concentrations in the range of 0.01-0.02 M. At mid or high pH, 300Extend-C18 is recommended.

Tips & Tools

Typical mobile phases for protein and peptide separations combine a very low pH with TFA (or other acids) to solubilize proteins. StableBond columns have extremely long lifetimes under these conditions. They are available in 300 pore size for proteins up to 100-500kDa, or 80 pore size for peptides below 4000Da.

94

Order online at www.agilent.com/chem/store

ZORBAX Columns for Bioanalytical Chromatography

Short-Chain ZORBAX 300SB-C3 Is Stable at Low pH, High Temperature Column: ZORBAX 300SB-C3 883995-909 4.6 x 150mm, 5m Mobile Phase: Gradients 0-100% B in 80 min A: 0.5% TFA in Water B: 0.5% TFA in Acetonitrile Isocratic Retention Test Conditions: 1-phenylheptane 50% A, 50% B Flow Rate: 1.0 mL/min Temperature: 60C

% k Phenyltheptane Remaining

ZORBAX 300 SB-C3 Competitor C4

Volume of Mobile Phase (mL)

Four Different 300SB Bonded Phases Optimize Separation of Large Polypeptides

Column A:

ZORBAX 300SB-C18 883995-902 4.6 x 150mm, 5m

Column B:

ZORBAX 300SB-C8 883995-906 4.6 x 150mm, 5m

1. 2. 3. 4.

Rnase Insulin Cytochrome C Lysozyme

Column C:

ZORBAX 300SB-C3 883995-909 4.6 x 150mm, 5m

5. Parvalbumin 6. CDR 7. Myoglobin

Column D:

ZORBAX 300SB-CN 883995-905 4.6 x 150mm, 5m

8. Carbonic Anhydrase 9. S-100 10. S-100

Mobile Phase: Linear Gradient, 25 - 70% B in 40 min A: 0.1% TFA in Water B: 0.09% TFA in 80% Acetonitrile/20% Water Flow Rate: 1.0 mL/min Temperature: 60C Sample: 3 g each protein

Time (min)
The 300SB-C18, C8, C3, and CN bonded phases all provide a different separation of this group of polypeptides. This adds an important parameter for quickly optimizing protein separations. The 300SB-CN column offers unique selectivity for more hydrophilic polypeptides.

Order online at www.agilent.com/chem/store

95

Capillary Columns for HPLC Analyses with UV and MS Detection Column: ZORBAX 300SB-C18 5064-8263 0.3 x 150mm, 5m
UV at 206 nm

UV

Mobile Phase:

A ZORBAX 300SB-C18 capillary column (0.3 mm ID) is used for the separation of the protein digest. Detection is by both UV and Electrospray MS. MS detection can be used for identification of peptide fragments.
Time (min)

ZORBAX Nano Columns For High Sensitivity Protein Digest Analysis by LC/MS Column: ZORBAX 300SB-C18 5065-9911 0.075 x 150mm, 3.5m
Intens.

Mobile Phase: A: Water + 0.1% Formic Acid, B: ACN + 0.1% Formic Acid Flow Rate: 600 nl/min Gradient: 2% B to 52% B in 25 min Detector: Positive Ion Nano Electrospray MS Sample: 100 fm (1 l) Digest of 8 Proteins

A ZORBAX nano HPLC column, 0.075 mm ID, is used for high sensitivity LC/MS analysis of a protein digest sample.
Time (min)

96

Order online at www.agilent.com/chem/store

% Relative Abundance

5-55% B in 50 min, to 85% B from 55-57 min A: 0.1% Formic Acid in Water B: 0.1% Formic Acid in ACN Flow Rate: 5.5 L/min Detector: 206 nm MS Conditions: LC/MS: Pos. Ion ESI with LC/MSD trap-Vcap 4000 V Drying Gas Flow: 7 L/min Drying Gas Temperature: 250C Nebulizer: 15 psi Capillary Exit Volt: 50 V Max Accum Time: 300 ms Total Averages: 3 Isolation Width: 3 m/z Frag Ampitude: 1.0 V Sample: 100 nL Beta Casein Digest (4 pmol)

Time (min)

MS

ZORBAX Columns for Bioanalytical Chromatography

ZORBAX 300 StableBond

Hardware Description Semi-Preparative Analytical Analytical Analytical Rapid Resolution Rapid Resolution Rapid Resolution Solvent Saver Plus Solvent Saver Plus Narrow Bore Narrow Bore Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* MicroBore MicroBore RR* MicroBore RR* MicroBore Guard Cartridges, 3/pk Guard Cartridge, 2/pk Guard Cartridge, 4/pk Guard Cartridge, 4/pk Guard Hardware Kit Guard Hardware Kit PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Cartridge PrepHT Endfittings, 2/pk PrepHT Guard Cartridge, 2/pk Guard Cartridge Hardware 17 x 7.5 5 Size (mm) 9.4 x 250 4.6 x 250 4.6 x 150 4.6 x 50 4.6 x 150 4.6 x 100 4.6 x 50 3.0 x 150 3.0 x 100 2.1 x 250 2.1 x 150 2.1 x 150 2.1 x 100 2.1 x 50 1.0 x 250 1.0 x 150 1.0 x 50 1.0 x 17 9.4 x 15 4.6 x 12.5 2.1 x 12.5 9.4 x 15 Particle 300SB-C18 Size (m) USP L1 5 5 5 5 3.5 3.5 3.5 3.5 3.5 5 5 5 3.5 3.5 5 3.5 3.5 5 7 5 5 880995-202 880995-902 883995-902 860950-902 863973-902 861973-902 865973-902 863974-302 881750-902 883750-902 861775-902 865750-902 861630-902 863630-902 865630-902 5185-5920 820675-124 820950-921 821125-918 840140-901 820888-901 897250-102 897150-102 895150-902 895100-902 895050-902 820400-901 820212-921 820444-901 300SB-C8 USP L7 880995-206 880995-906 883995-906 860950-906 863973-906 861973-906 865973-906 863974-306 861973-306 300SB-CN USP L10 880995-205 880995-905 883995-905 860950-905 863973-905 300SB-C3 USP L56 880995-209 880995-909 883995-909 860950-909 863973-909

Standard Columns (no special hardware required, 400 bar)

865973-905 865973-909 863974-309

883750-906 883750-905 883750-909 863750-906 861775-906 865750-906 863630-906 865630-906 5185-5920 820675-124 820950-918 821125-918 840140-901 820888-901

820675-124 820950-923 821125-924 840140-901 820888-901

820675-124 820950-924 821125-924 840140-901 820888-901

PrepHT Cartridge Columns (require endfittings kit 820400-901)

21.2 x 250 21.2 x 150 21.2 x 150 21.2 x 100 21.2 x 50 7 7 5 5 5 897250-106 897250-105 897250-109 897150-106 895150-906 895100-906 895050-906 897150-109 895150-909 895100-909 895050-909

820400-901 820400-901 820400-901 820212-918 820212-924 820212-924 820444-901 820444-901 820444-901

Order online at www.agilent.com/chem/store

97

ZORBAX 300 StableBond (Continued)

Hardware Description Size (mm) 0.5 x 250 0.5 x 150 0.5 x 35 0.5 x 150 0.5 x 35 0.3 x 250 0.3 x 150 0.3 x 35 0.3 x 150 0.3 x 100 0.3 x 35 0.3 x 50 0.1 x 150 0.075 x 150 0.075 x 50 0.3 x 5 Particle 300SB-C18 Size (m) USP L1 5 5 5 3.5 3.5 5 5 5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 5 5064-8266 5064-8264 5064-8294 5064-8268 5065-4459 5064-8265 5064-8263 5064-8295 5064-8267 5064-8259 5064-8270 5064-8300 5065-9910 5065-9911 5065-9924 5065-9913 5065-9915 300SB-C8 USP L7 300SB-CN USP L10 300SB-C3 USP L56

Capillary Glass-lined Columns

Capillary Capillary Capillary Capillary RR* Capillary RR* Capillary Capillary Capillary Capillary RR* Capillary RR* Capillary RR* Capillary RR*

5065-4460 5065-4461 5065-4462 5065-4463

Nano Columns (PEEK fused silica)

Nano RR* Nano RR* Nano RR* Trap/Guard, 5/pk Trap/Guard Hardware kit
*RR: Rapid Resolution 3.5 m

5065-9923 5065-9914 5065-9915

98

ZORBAX Columns for Bioanalytical Chromatography

ZORBAX 300 Extend-C18

Rugged, high and low pH separations of polypeptides and peptides from pH 2-11.5 Different selectivity possible at high and low pH High efficiency and good recovery of hydrophobic peptides at high pH Ideal for LC/MS with ammonium hydroxide-modified mobile phase

ZORBAX 300Extend C-18 is a wide-pore HPLC column for high efficiency separations of peptides from pH 2-11.5. The unique, bidentate bonded phase provides excellent lifetime and reproducibility at high and low pH. At high pH, retention and selectivity of peptides and polypeptides can change dramatically as a result of changes in charge on molecules. Excellent recoveries of hydrophobic polypeptides have been achieved at room temperature and high pH. LC/MS sensitivity of peptides and polypeptides can also be improved at high pH using a simple ammonium hydroxide-containing mobile phase.

Si

C18

O Si C18
Silica Support

Column Specifications
Bonded Phase ZORBAX 300Extend-C18 Pore Size 80 Surface Area 180 m2/g Temp. Limits* pH Range 60C 2.0-11.5 Carbon Endcapped Load Double 4%

*Temperature limits are 60C up to pH 8, 40C from pH 8-11.5.

Long Life at High pH with 300Extend-C18 Column: ZORBAX Extend-C18 773450-902 4.6 x 150mm, 5m

Novel Bidentate C18-C18 Bonding for Extend-C18 Bonded Phase

Mobile Phase: 20% 20 mM NH4OH, pH 10.5 80% Methanol Flow Rate: 1.5 mL/min Temperature: Aging 24C Tests 40C

4-Chloro-1-nitrobenzene Trimipramine
k Values

Column Volumes of Mobile Phase

Each 10,000 column volume is approximately one working month.

Order online at www.agilent.com/chem/store

99

Extend-C18 and StableBond SB-C18 Are Stable at Low pH Column A: ZORBAX SB-C18 883975-902 4.6 x 150mm, 5m
Fraction of Toluene k Remaining

Column B:

ZORBAX Extend-C18 773450-902 4.6 x 150mm, 5m ZORBAX Rx-C18 883967-902 4.6 x 150mm, 5m

Column C:

A B C

Mobile Phase: Column Aging: 50% Methanol : 50% Water + 1% TFA Column Test: 60% Methanol : 40% Water Test Solute: Toluene Flow Rate: 1.5 mL/min, continuous Temperature: Aging: 90C Test: Ambient

Column Volumes Mobile Phase

The 300Extend-C18 column can be used at high and low pH from pH 2-11.5. This chart shows that the 300Extend-C18 has the needed stability at low pH for long-term reproducible separations. Therefore, one wide-pore column can be used for selectivity optimization at low and high pH with both TFA and ammonium hydroxide mobile phases.

LC/MS Analysis of Angiotensin on Extend-C18 Column: ZORBAX Extend-C18 773700-902 2.1 x 150mm, 5m

Mobile Phase:

Acidic Conditions: A: 0.1% TFA in water B: 0.085% TFA in 80% acetonitrile (ACN) Basic Conditions: A: 10 mM NH4OH in water B: 10 mM NH4OH in 80% ACN Flow Rate: 0.2 mL/min Gradient: 15-50% B in 15 min Temperature: 35C MS Conditions: Pos. Ion ESI- Vf 70V, Vcap 4.5 kV, N2- 35 psi, 12 L/min., 325C Sample: 2.5 L sample (50 pmol each) Angiotensin I, II, III
Reference: B.E. Boyes. Separation and Analysis of Peptides at High pH Using RP-HPLC/ESI-MS, 4th WCBP, San Francisco, CA Jan 2000.

TIC (200-1500 m/z)

min

m/z

TIC (200-1500 m/z)

min

m/z

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 of small peptides at high pH as well.

100

Order online at www.agilent.com/chem/store

ZORBAX Columns for Bioanalytical Chromatography

ZORBAX 300 Extend-C18

Hardware Description Analytical Analytical Rapid Resolution Rapid Resolution Rapid Resolution Narrow Bore RR* Narrow Bore RR* Narrow Bore RR* Guard Cartridge, 4/pk Guard Cartridge, 4/pk Guard Hardware Kit Particle Size (mm) Size (m) 4.6 x 250 5 4.6 x 150 5 4.6 x 150 3.5 4.6 x 100 3.5 4.6 x 50 3.5 2.1 x 150 3.5 2.1 x 100 3.5 2.1 x 50 3.5 4.6 x 12.5 5 2.1 x 12.5 5 Part No. 770995-902 773995-902 763973-902 761973-902 765973-902 763750-902 761775-902 765750-902 820950-932 821125-932 820888-901 5065-4464 5065-4465 5065-4466 5065-4467

Capillary Glass-lined Columns

Capillary RR* Capillary RR* Capillary RR* Capillary RR*
*RR: Rapid Resolution 3.5 m

0.3 x 150 0.3 x 100 0.3 x 75 0.3 x 50

3.5 3.5 3.5 3.5

Order online at www.agilent.com/chem/store

101

Innovative Poroshell Particles Are Ideal for Ultra-Fast Protein Separations

Porous Shell

ZORBAX Poroshell

High-resolution separations of biomolecules with unique particle design 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-C18

SOLID CORE

5 m

The unique Poroshell particle design consists of a solid core of high-purity silica surrounded by a thin layer of porous, high-purity silica. Pores in the thin, outer layer measure 300 in diameter and are bonded with sterically protected ligands (SB-C18, SB-C8 or SB-C3) for maximum column life with low pH (i.e., TFA and formic acid) mobile phases. During separation, proteins diffuse rapidly in and out of the porous shell allowing for the use of high flow velocities thereby eluting as sharp peaks in just seconds.

ZORBAX Poroshell columns are ideal for fast separations of proteins and peptides because the unique particle allows for fast flow rates to be used while maintaining sharp, efficient peaks. Peptides and proteins are typically separated slowly to reduce the potential peak broadening of these slow diffusing analytes. But Poroshell columns use a unique particle made with a thin layer of porous silica on a solid core of silica. This reduces the diffusion distance for proteins making practical rapid HPLC separations of peptides and proteins up to 500-1,000 kDA. Poroshell columns bonded with StableBond bonded phases provide 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.

Tips & Tools

Reversed-phase HPLC is a key technique for separating peptides and proteins because of its high achievable resolution. Only Agilent offers reversed-phase Poroshell columns for fast, high-resolution protein separations.

102

ZORBAX Columns for Bioanalytical Chromatography

Poroshell Columns Can Separate Proteins and Peptides in Seconds Column: ZORBAX Poroshell 300SB-C18 660750-902 2.1 x 75mm, 5m

Mobile Phase: A: 0.1% TFA in H20 B: 0.07% TFA in ACN Flow Rate: 3.0 mL/min Gradient: 5-100% B in 1.0 min Temperature: 70C, 260 bar pressure Detector: 215 nm Sample: Proteins and Peptides

1. 2. 3. 4. 5. 6. 7. 8.

Angiotensin II Neurotensin Rnase Insulin Lysozyme Myoglobin Carbonic Anhydrase Ovabumin

This separation of eight polypeptides and proteins is completed in less than 60 seconds. Each peak is sharp and efficient.

Reduce Peptide Map Analysis Time by 90% with ZORBAX Poroshell 300SB Column A: ZORBAX Poroshell 300SB-C18 660750-902 2.1 x 75mm, 5m ZORBAX 300SB-C18 883750-902 2.1 x 150mm, 5m

Column B:

Mobile Phase: A: 95% H2O, 5% ACN, 0.1% TFA B: 5% H2O, 95% ACN, 0.07% TFA Flow Rate: 1 mL/min 0.208 mL/min Gradient: 0-100%B = 12 min 0-100%B = 120 min

Temperature: Sample:

70C 20 L (0.22 g/1 L) BSA Tryptic Digest (15 hours, 70 pmol)

A single chromatographic run of a protein tryptic digest can require an hour or more to complete. With ZORBAX Poroshell columns, the same complex separation can be completed in 1/10th the time.

Order online at www.agilent.com/chem/store

103

MicroBore ZORBAX Poroshell Columns Provide Maximum Sensitivity for LC/MS Column: ZORBAX Poroshell 300SB-C18 661750-902 1.0 x 75mm, 5m

Mobile Phase:

A: Water + 0.1% Formic Acid B: ACN + 0.1% Formic Acid Flow Rate: 600 L/min Gradient: 20-100% B in 5.5 min Temperature: 80C MS Conditions: LC/MS: Pos. Ion ESI Vcap 6000 V Drying Gas Flow: 12 Liters/min Drying Gas Temperature: 350C Nebulizer: 45 psi Fragmentor Volatage: 140 V Scan: 600-2500 Stepsize: 0.15 amu Peakwidth: 0.06 min Sample: 1 L

With 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 mm or 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 as 0.5 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 and tissue culture media.

ZORBAX Poroshell
Hardware Description Narrow Bore MicroBore Capillary Guard Cartridge, 4/pk Guard Hardware Kit MicroBore Guard Cartridge, 3/pk Particle Size (mm) Size (m) 2.1 x 75 5 1.0 x 75 5 0.5 x 75 5 2.1 x 12.5 5 1.0 x 17 5 Poroshell 300SB-C8 660750-906 661750-906 5065-4468 821075-920 821075-918 820888-901 820888-901 5185-5968 5185-5968 Poroshell 300SB-C18 660750-902 661750-902 Poroshell 300SB-C3 660750-909 661750-909 Poroshell 300Extend-C18 670750-902 671750-902

821075-924 820888-901 5185-5968 5185-5968

104

Order online at www.agilent.com/chem/store

ZORBAX Columns for Bioanalytical Chromatography

ZORBAX 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 with Agilent 1100/1200 Autosampler

The ZORBAX Eclipse AAA high efficiency column rapidly separates amino acids following an updated and improved protocol. Total analysis from injection to injection can be achieved in as little as 14 min. (9 min. analysis time) on shorter, 7.5 cm length columns and 24 min. (18 min. analysis time) on the 15 cm column length. Exceptional sensitivity (5-50 pmol with DAD, FLD) and reliability are achieved using both OPA and FMOC derivatization chemistries in one fully automated procedure using the Agilent 1100/1200 HPLC instrument.

High Resolution of 24 Amino Acids Using ZORBAX Eclipse-AAA Protocol Column: ZORBAX Eclipse AAA 963400-902 4.6 x 150mm, 3.5m 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Asp Glu Asn Ser Gln His Gly Thr Cit Arg Ala Tyr 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Cys Val Met Nva Trp Phe Ile Leu Lys Hyp Sar Pro

Mobile Phase: A: 40 mM Na2HPO4, pH 7.8 B: ACN : MeOH : Water, 45:45:10 v/v Flow Rate: 2 mL/min Temperature: 40C Detector: Fluorescence Sample: 24 Amino Acids

Time (min)
This high resolution separation of 24 amino acids is done in 18 minutes. If the Rapid Resolution 4.6 x 75 mm Eclipse AAA column is selected, these amino acids are resolved in 9 minutes.

Order online at www.agilent.com/chem/store

105

ZORBAX Eclipse Amino Acid Analysis (AAA) Columns

Hardware Description Analytical routine sensitivity Analytical routine sensitivity, high-resolution using FLD Analytical routine sensitivity, high-throughput Solvent Saver high sensitivity, high resolution Guard Catridges, 4/pk Guard Hardware Kit Size (mm) 4.6 x 150 4.6 x 150 4.6 x 75 3.0 x 150 4.6 x 12.5 Particle Size (m) 5 3.5 3.5 3.5 5 Part No. 993400-902 963400-902 966400-902 961400-302 820950-931 820888-901

Amino Acid Standards Each amino acid standards contains the 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 acid

Amino Acid Standards, 10 x 1 ml ampoules*

Description 1 nmol/l 250 pmol/l 100 pmol/l 25 pmol/l 10 pmol/l Amino acids supplement kit Includes 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 vials

Part No. 5061-3330 5061-3331 5061-3332 5061-3333 5061-3334 5062-2478

Amino Acid Separations Reagents

Description OPA reagent, 10 mg/ml each in 0.4 M borate buffer o-phthalaldehyde (OPA) and 3-mercaptopropionic acid, 6 x 1 ml ampoules FMOC reagent, 2.5 mg/ml in acetonitrile, 9-fluorenylmethylchloroformate, 1 ml, 10 ampoules Borate buffer, 100 ml DTDPA (Dithiodiproprionic) reagent, for analysis of cysteine, 5 g Part No. 5061-3335 5061-3337 5061-3339 5062-2479

106

Order online at www.agilent.com/chem/store

ZORBAX Columns for Bioanalytical Chromatography

ZORBAX GF-250 and GF-450 Gel 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)

ZORBAX GF-250 and GF-450 size exclusion (gel filtration) columns are ideal for the size separations of proteins and other biomolecules. The separation range is 4,000-900,000 for globular proteins when using GF-250 and GF-450 columns in series. The GF-250/GF-450 size 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 range from 3-8. The GF-250 and GF-450 columns are packed with precisely sized porous silica microspheres with narrow pore size and particle size distributions. The result is a highly efficient, rugged and reproducible size exclusion column for separations of proteins with flow rates of up to 3 ml/min. These columns are compatible with organic modifiers (<25%) and denaturants in the mobile phase to eliminate protein aggregation for proper size determination. Some common applications include separations of protein monomers, dimers and aggregates, desalting, protein molecular weight estimation and separations of modified proteins.

Column Specifications
Bonded Phase ZORBAX GF-250 ZORBAX GF-450 Pore Size 150 300 Particle MW Size Range 4 m 4,000400,000 6 m 10,000900,000 Surface Flow Area pH Range Rate <3.0 140 m2/g 3.0-8.0 ml/min <3.0 50 m2/g 3.0-8.0 ml/min Max Pressure 350 bar 350 bar

107

Separation of Protein Standards on the ZORBAX GF-250 SEC Column Column: ZORBAX GF-250 884973-901 9.4 x 250mm, 4m 200 mM Sodium Phosphate, pH 7.0 2 mL/min Ambient 254 nm BioRad Gel Filtration Standards for Size Exclusion 1. Thyroglobulin 670,000 Da 2. Bovine Gamma Globulin 158,000 Da 3. Chicken Ovalbumin 44,000 Da 4. Equine Myoglobin 17,000 Da 5. Vitamin B-12 1,350 Da

Mobile Phase: Flow Rate: Temperature: Detector: Sample:

The protein standards separated here are a commonly selected set of standards. The ZORBAX GF-250 column shows excellent resolution for this sample. Additional resolution of the thyroglobulin can be obtained by adding the GF-450 column in series.

Time (min)

High-Speed Size Exclusion Separations

A: BSA and BSA Dimers (duplicate injections)

2 2

B. Antibody Separation
2

1. BSA dimer 2. BSA monomer 3. Azide

1. IgG Dimer and Aggregate 2. Mouse IgG 1K (MOPC 31C) 3. Salts: Tris, Na2HPO4, NaCl

1 Inj. #1

3 Inj. #2

3
1 0 1 2 3 4 5 6

5 Time (min)

10

10

Retention Time (min)

Column:

ZORBAX GF-450 884973-902 9.4 x 250mm, 6m

Column:

ZORBAX GF-450 884973-902 9.4 x 250mm, 6m

Mobile Phase: PBS (phosphate buffered saline), pH 7.4 Flow Rate: 3 mL/min Temperature: Ambient Detector: 220 nm Sample: BSA and BSA Dimers

Mobile Phase: 200 mM Na Phosphate Monobasic pH 7.0/0.1% Azide Flow Rate: 2 mL/min Detector: 225 nm Sample: 10 g in 50 mM Sodium Phosphate pH 7.0

108

Order online at www.agilent.com/chem/store

ZORBAX Columns for Bioanalytical Chromatography

Separation of a Protein Mixture on the 9.4 x 250 mm ZORBAX GF-250 Column Column: ZORBAX GF-250 884973-701 4.6 x 250mm, 4m
1 2 3 4 5 6 7

Mobile Phase: 130 mM NaCl/20 mM KCl/ 50 mM Na2HPO4, pH 7.0 Flow Rate: 1 mL/min Detector: 210 nm Sample: Protein mixture

1. 2. 3. 4. 5. 6. 7.

Mouse IgM 900,000 Da Bovine Thyroglobulin 670,000 Da Sweet Potato -Amylase 200,000 Da Bovine Serum Albumin 67,000 Da Chicken Albumin 45,000 Da Bovine RNAse 13,700 Da Azide 65 Da

10
Time (min)

12

14

ZORBAX GF-250 Shows Extended Column Lifetime

Stability of GF-250: K* of Standard Proteins

3.0 2.5 2.0 K* 1.5 1.0 0.5 0.0 0 1000 2000 Column Volumes 3000 BSA

Stability of Competitor Column: K* of Standard Proteins

3.0 2.5 2.0 K* 1.5 1.0 0.5 0.0 0 1000 2000 Column Volumes 3000 BSA Ovalbumin Lysozyme NaN 3 Ovalbumin Lysozyme NaN 3

Order online at www.agilent.com/chem/store

109

ZORBAX GF-250 and GF-450 Gel Filtration Columns

Hardware Description GF-250, 150 GF-250, 150 GF-450, 300 Particle Size (mm) Size (m) 9.4 x 250 4 4.6 x 250 4 9.4 x 250 6 9.4 x 15 4.6 x 12.5 9.4 x 15 4.6 x 12.5 9.4 x 15 6 6 6 6 Part No. 884973-901 884973-701 884973-902 820675-111 820950-911 820675-111 820950-911 840140-901 820888-901 877974-901 877974-910 820400-901 17 x 7.5 17 x 7.5 6 6 820212-911 820212-911 820444-901

Guard Columns (hardware required)

GF-250 Diol, Guard Cartridge, 2/pk GF-250 Diol, Guard Cartridge, 4/pk GF-450 Diol, Guard Cartridge, 2/pk GF-250 Diol, Guard Cartridge, 4/pk Guard Hardware Kit Guard Hardware Kit

PrepHT Columns
PrepHT GF-250, 150 PrepHT GF-450, 300 PrepHT Endfittings, 2/pk PrepHT GF-250, Guard Cartridge, 2/pk PrepHT GF-450, Guard Cartridge, 2/pk Guard Cartridge Hardware 21.2 x 250 21.2 x 250 6 6

110

ZORBAX Columns for Bioanalytical Chromatography

ZORBAX 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 modifiers ZORBAX Bio-SCX Series II for 2-D separations

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 m, spherical silica particles for optimum efficiency. Zorbax also has Bio-SCX Series II columns designed for optimized 2-D separations of peptides and proteins using LC/MS. This packing is based on ultra-pure 3.5 m Zorbax silica particles, bonded with a bio-friendly polymer that is functionalized with sulfonic acid groups. This gives strong retention and good peak shape in the ion exchange step of 2-D analysis of peptides and proteins.

Column Specifications
Bonded Phase ZORBAX SAX ZORBAX 300SCX ZORBAX Bio-SCX Series II Pore Size 70 300 300 Max Surface Area pH Range Functionality Pressure 2.0-7.0 Quaternary 350 bar 300 m2/g amine 2.0-7.0 Sulfonic acid 350 bar 50 m2/g 90 m2/g 2.5-8.5 Sulfonic acid 350 bar

ZORBAX Ion Exchange Columns SAX and SCX

Description Semi-preparative Analytical Analytical Analytical Solvent Saver Narrow Bore Narrow Bore Capillary Capillary Guard Cartridge, 4/pk Guard Hardware Kit Size (mm) 9.4 x 250 4.6 x 250 4.6 x 150 4.6 x 50 3.0 x 50 2.1 x 150 2.1 x 50 0.3 x 35 0.8 x 50 4.6 x 12.5 Particle Size (m) 5 5 5 5 5 5 5 3.5 3.5 6 SAX 880952-203 880952-703 883952-703 Bio-SCX 300SCX Series II 880952-204 880952-704 883952-704 846952-704 860700-304 883700-704 860700-704 5065-9912 5065-9942 820950-903 820950-904 820888-901 820888-901

Order online at www.agilent.com/chem/store

111

Technical Support at Work for You

Have a hardware, software, application, or troubleshooting question? Agilent's technical experts are available to answer your questions. With years of laboratory experience, our technical support specialists can provide in-depth knowledge and experience. For questions pertaining to columns found in this catalog, call your local Agilent sales office or 1-800-227-9770 in the U.S. or Canada. You can also visit www.agilent.com/chem/techsupport for a wealth of knowledge, tips, and insight.

Tech Support

Find HPLC course descriptions and seminars online at

www.agilent.com/chem/Education.

Learn from Agilent HPLC experts through classroom training courses, custom on-site training, and free e-seminars. HPLC courses cover instrument and column selection, separation optimization, HPLC maintenance and troubleshooting, Chemstation and more.

Frequently Asked Questions Downloads and Utilities Installation and Maintenance Videos Interactive Troubleshooter Warranty Information Technical Support Contact Information

112

Peak productivity starts here:

www.agilent.com/chem/store

Welcome to Agilents Life Sciences & Chemical Analysis online store!

Whether you want to increase sample throughput or scale and transfer methods you will find all the columns and supplies you need for your day-to-day operations. So you can spend less time searching for products, and more time achieving your best results. Our online store also adapts to your companys purchasing policies with features like Online Purchase Approval. After you fill your cart, you can simply e-mail a secure, single-use ID to your purchasing agent, who can then access your cart, make any changes, and place the order. And of course, if you prefer to speak with our experts directly, or if you have additional questions, you can contact your local Agilent office or authorized distributor.

View local list prices and see prices specific to your organization (once your account is activated). Submit your order in seconds using our Quick Buy feature. Instantly see when Agilent can ship an item to you. Configure products and receive a price quote for purchase approval or to buy online. Choose from five languages including French, Italian, German, Spanish, and English. Create a personal catalog of items that you request frequently. So you can select these items more quickly the next time you order. Track your order from OUR door to YOURS. View your ordering history with just a few clicks of your mouse.

How to contact Agilent

For the latest information on the complete line of Agilent Technologies columns and supplies: Visit our Web site: www.agilent.com/chem Contact your local Agilent sales office Contact your local Authorized Agilent Distributor In the U.S. and Canada call 1 800 227 9770 Sales Option 1, then 1 Technical Support Option 4, then 2

Information, descriptions, and specifications in this publication are subject to change without notice. Agilent Technologies, Inc. 2007 Printed in the USA July 1, 2007 5989-5992EN