Maximize HPLC - Waters Corporation · 2013-01-15 · HPLC Columns: Theory, Technology, and Practice Uwe D. Neue, Waters Corporation, Milford, MA, published by John Wiley & Sons High-performance

eXtended Performance [XP] 2.5 µm columns allow scientists to improve HPLC throughput and performance compared to 3.5 µm and 5 µm columns while providing a vast selectivity range made possible by 3 unique particles and 14 stationary phases. T hese columns help maximize productivity and ensure seamless transferability and scalability of LC methods.

Maximize HPLC PerformanceA

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Title Literature Code

Application NotebooksAtlantis HPLC Columns 720000472ENSymmetry Columns 720000593ENXBridge Amide HPLC Columns 720003438ENXTerra Columns 720000502EN

Application NotesA New State-of-the-Art Silica C18 Column for Improved Peak Shape of Basic Analytes

WA40526EN

A Quality by Design (QbD) Based Method Development for the Determination of Impurities in a Peroxide Degraded Sample of Ziprasidone

720004072EN

A Sensitive Method for the Determination of Endocrine Disrupting Compounds in River Water by LC/MS/MS

720001296EN

Determination of Soy Isoflavones in Foods and Dietary Supplements by UPLC

720004253EN

Extract using Charged Surface Hybrid (CSH) Technology 720004098ENModernization of the USP Assay for Clarithromycin Using eXtended Performance [X P] Column Technology

720004461EN

Practical Applications of Charged Surface Hybrid (CSH) Technology

720003720EN

Rapid, Reliable Metabolite Ratio Evaluation for MIST Assessments in Drug Discovery and Preclinical Studies

720004453EN

Utlizing XBridge HPLC Columns for Method Development at pH Extremes

WA43181

Validation of a Method for the Separation of Ziprasidone and Its Degradants Using Empower 2 with Method Validation Manager (MVM)

720004077EN

Title Literature CodeBrochuresAtlantis HPLC Columns 720000793EN

eXtended Performance [X P] 2.5 μm Columns 720004195EN

HPLC Columns Capabilities 720003995EN

Preparative Optimum Bed Density (OBD) Columns 720002336ENSunFire HPLC Columns 720000875ENSymmetry Columns 720000454ENXBridge HPLC Columns 720001255ENXSelect HPLC Columns 720004178ENXTerra Columns 720000424EN

W hite PapersA Review of Waters Hybrid Particle Technology Part 2. Ethylene-Bridged (BEH Technology) Hybrids and T heir Use in Liquid Chromatography

720001159EN

Charged Surface Hybrid (CSH) Technology and Its Use in Liquid Chromatography

720003469EN

Topics in Liquid Chromatography: Part 1. Designing a Reversed-Phase Column for Polar Compound Retention

720001889EN

Online ToolsWaters Column Advisor www.waters.com/columnadvisorWaters Reversed-Phase Column Selectivity Chart

www.waters.com/selectivitychart

BooksBeginner’s Guide to Liquid Chromatography 715001531Comprehensive Guide to HILIC 715002531HPLC Columns–T heory, Technology, and Practice WAT038216Search by Literature Code at waters.com

LiTeRATuRe RefeReNCeS

Introduction ............................................................................................. 120

USP “L” Column Listing ........................................................................ 122

Physical Characteristics of HPLC and UPLC Packing Materials .... 125

eXtended Performance [XP] 2.5 μm Columns ................................... 127

XSelect Columns .................................................................................... 129

XBridge Columns ................................................................................... 132

XBridge HILIC Columns for Orthogonal Selectivity ....................... 135

XBridge Amide Columns for Sugar Analysis ................................. 135

SunFire Columns..................................................................................... 136

Atlantis Columns....................................................................................138

XTerra Columns ......................................................................................140

Symmetry Columns ................................................................................ 141

Waters Spherisorb Columns ..................................................................144

Waters Spherisorb Analytical Columns ..........................................144

Waters Spherisorb Cartridge Columns ............................................144

Nova-Pak Columns .................................................................................. 145

Delta-Pak Columns .................................................................................146

Resolve Columns ....................................................................................146

μBondapak/Bondapak Packings ..........................................................146

μPorasil/Porasil Silica Packings...........................................................146

Guard Products ........................................................................................ 147

Guard-Pak .........................................................................................148

Waters Sentry Guard Holders and Sentry Guard Columns .............148

Guard-Pak Holder .............................................................................148

Guard-Pak Inserts .............................................................................148

Shodex RSpak Polymer Reversed-Phase Columns ............................. 149

Paired-Ion Chromatography (PIC Reagents) ....................................... 150

Sugar and Carbohydrate Analysis .................................................... 151

Application-Specific Columns ..............................................................154

High-Performance Carbohydrate Analysis Cartridge Column .......154

Fatty Acid Analysis ..........................................................................154

Pesticide Analysis ............................................................................154

Waters PAH Columns ............................................................................. 155

Triglycerides and Cholesterol Analysis .............................................. 156

Fermentation Analysis, Organic Acids, Alcohols, and Carbohydrates ............................................................... 156

Ion Analysis ............................................................................................ 157

Anion Analysis ................................................................................. 157

Cation Analysis ................................................................................ 157

Transition Metal Analysis .....................................................................158

Ion-Exclusion Columns...........................................................................158

Lanthanide Analysis ..............................................................................158

Gas-Chromatography Packings ............................................................. 159

Product Ordering Information ..............................................................160

>>> ANALYTICAL HPLC COLUMNS AND CONSUMABLESA

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http://www.waters.com/webassets/cms/library/docs/720000472en.pdfhttp://www.waters.com/webassets/cms/library/docs/720000593en.pdfhttp://www.waters.com/webassets/cms/library/docs/720003438en.pdfhttp://www.waters.com/webassets/cms/library/docs/720000502en.pdfhttp://www.waters.com/webassets/cms/library/docs/WA40526en.pdfhttp://www.waters.com/webassets/cms/library/docs/720004072en.pdfhttp://www.waters.com/webassets/cms/library/docs/720001296en.pdfhttp://www.waters.com/webassets/cms/library/docs/720004253en.pdfhttp://www.waters.com/webassets/cms/library/docs/720004098en.pdfhttp://www.waters.com/webassets/cms/library/docs/720004461en.pdfhttp://www.waters.com/webassets/cms/library/docs/720003720en.pdfhttp://www.waters.com/webassets/cms/library/docs/720004453en.pdfhttp://www.waters.com/webassets/cms/library/docs/WA43181.pdfhttp://www.waters.com/webassets/cms/library/docs/720004077en.pdfhttp://www.waters.com/webassets/cms/library/docs/720000793en.pdfhttp://www.waters.com/webassets/cms/library/docs/720004195en.pdfhttp://www.waters.com/webassets/cms/library/docs/720003995en.pdfhttp://www.waters.com/webassets/cms/library/docs/720002336en.pdfhttp://www.waters.com/webassets/cms/library/docs/720000875en.pdfhttp://www.waters.com/webassets/cms/library/docs/720000454en.pdfhttp://www.waters.com/webassets/cms/library/docs/720001255en.pdfhttp://www.waters.com/webassets/cms/library/docs/720004178en.pdfhttp://www.waters.com/webassets/cms/library/docs/720000424en.pdfhttp://www.waters.com/webassets/cms/library/docs/720001159en.pdfhttp://www.waters.com/webassets/cms/library/docs/720003469en.pdfhttp://www.waters.com/webassets/cms/library/docs/720001889en.pdfhttps://www.waters.com/waters/home.htmhttp://www.waters.com/waters/promotionDetail.htm?id=10048475http://www.waters.com/waters/partDetail.htm?partNumber=715001531http://www.waters.com/waters/partDetail.htm?partNumber=715002531http://www.waters.com/waters/partDetail.htm?partNumber=WAT038216https://www.waters.com/waters/home.htm

HPLC Columns: Theory, Technology, and Practice

Uwe D. Neue, Waters Corporation, Milford, MA, published by John Wiley & Sons

High-performance liquid chromatography and its derivative techniques have become the dominant analytical separation tools in the pharmaceutical, chemical, and food industries, environmental laboratories, and therapeutic drug monitoring.

Although the column is the heart of the HPLC instrument and essential to its success, until now no single book has focused on the theory and practice of column technology.

‘HPLC Columns’ provides thorough, state-of-the-art coverage of HPLC column technology for the practicing technician and academician alike. Along with a comprehensive discussion of the chemical and physical processes of the HPLC column, it includes fundamental principles, separation mechanisms, available technologies, column selection criteria, and special techniques.

Special features include: Explanation of the underlying principles of HPLC columns

Methods for selecting columns

Practical advice on using and applying columns, including examples

Section by M. Zoubair El Fallah on methods development

Special techniques, including preparative chromatography, continuous chromatography, and the simulated moving bed

Troubleshooting

Description Part No.

HPLC Columns: Theory, Technology, and Practice

WAT038216

Choosing an HPLC column can be a very difficult process. The chromatographic packing material (particle ‘brand name’ and functional group) plays a major role in the success of a separation, both initially, when the method is first developed, as well as long term, if the method will need to be reproduced for many years.

Dependable HPLC Column PerformanceA

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uSP “L” Column Listing

US Pharmacopeia LC “L1” (C18) Column Designation Subclassification Testing by Brand Name*

Column Brand NameHydrophobicity

(k, Ethylbenzene)Chelating

(Tailing Factor [TF], Quinizarin)Silanol Activity

(k, Amitriptyline)Silanol Activity

(TF, Amitriptyline)Shape Selectivity

(Bonding Density, μmol/m2)

ACQUITY UPLC BEH C18 1.63 1.10 3.79 1.10 3.10

ACQUITY UPLC BEH Shield RP18 1.30 1.36 2.37 1.13 3.30

ACQUITY UPLC HSS C18 2.54 1.36 5.96 1.41 3.20

ACQUITY UPLC HSS C18 SB 1.09 1.07 22.20 7.20 1.60

ACQUITY UPLC HSS T3 2.23 1.19 6.50 3.15 1.60

Atlantis dC18 1.70 1.10 5.10 2.40 1.60

Atlantis T3 1.87 0.98 5.32 1.35 1.60

Delta-Pak™ C18 (100Å) 2.00 2.00 6.30 1.90 3.60

μBondapak® C18 1.00 6.00 7.50 4.00 1.10

Nova-Pak C18 1.50 7.50 4.60 3.00 2.70

Resolve™ C18 2.00 no peak 35.0 8.00 2.80

SunFire C18 2.50 1.20 6.30 1.10 3.50

Symmetry C18 2.25 1.70 5.10 1.70 3.20

SymmetryShield RP18 1.65 1.50 3.10 1.20 3.30

Waters Spherisorb® ODS-1 0.80 no peak 23.00 3.00 1.70

Waters Spherisorb ODS-2 2.00 no peak 11.50 7.00 2.60

XBridge C18 1.63 1.10 3.79 1.10 3.10

XBridge Shield RP18 1.30 1.36 2.37 1.13 3.30

XSelect HSS C18 2.54 1.36 5.96 1.41 3.20

XSelect HSS C18 SB 1.09 1.07 22.20 7.20 1.60

XSelect HSS T3 2.23 1.19 6.50 3.15 1.60

XTerra RP18 1.00 1.20 1.70 1.10 2.30

XTerra MS C18 1.50 1.10 3.30 1.30 2.20

* For use in C18 column brand name selection when referencing USP monographs which specify the characteristics/performance of the C18 column used in the method. Choose a brand as close as possible to the referenced column values for the best chance of duplicating the analytical results of the method.

Build method development kits

Choose columns by vendor, USP listing, or ligand type

Find equivalent column chemistries

Save laboratory time

To try the selectivity chart, visit: www.waters.com/selectivitychart

Waters electronic Column Selectivity Chart

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uSP “L” Column Listing

( ) - Denotes particle sizes available outside of L class.

L1Octadecylsilane (ODS or C18) chemically bonded to porous silica or ceramic particles–1.5 to 10 μm in diameter. See the subclassification table on previous page.

Brand Particle Size Type Page

AccQ•Tag™ Ultra 1.7 µm Spherical 247

ACQUITY UPLC BEH C18 1.7 µm Spherical 99

ACQUITY UPLC BEH Shield RP18 1.7 µm Spherical 99

ACQUITY UPLC CSH C18 1.7 µm Spherical 97

ACQUITY UPLC HSS C18 1.7 µm Spherical 101

ACQUITY UPLC HSS C18 SB 1.8 µm Spherical 101

ACQUITY UPLC HSS T3 1.8 µm Spherical 101

ACQUITY UPLC OST C18 1.7 µm Spherical 251

Atlantis T3 3, 5, 10 µm Spherical 138

Atlantis dC18 3, 5, 10 µm Spherical 138

BioSuite™ PA-A 3 µm Spherical 225

BioSuite PA-B 3 µm Spherical 225

Delta-Pak C18 5 µm Spherical 146

μBondapak C18 10 µm Irregular 146

μBondapak C18 Radial-Pak™ 10 µm Irregular 146

nanoACQUITY UPLC BEH130 1.7 µm Spherical 261

nanoACQUITY UPLC BEH300 1.7 µm Spherical 261

Nova-Pak C18 4, 6 µm Spherical 145

Prep Nova-Pak HR C18 6 µm Spherical 194

Resolve C18 5, 10 µm Spherical 146

SunFire C18 2.5, 3.5, 5, 10 µm Spherical 136

Symmetry C18 3.5, 5, 7 µm Spherical 141

SymmetryPrep™ C18 3.5, 5, 7 µm Spherical 194

Symmetry300™ C18 3.5, 5 µm Spherical 194

SymmetryShield RP18 3.5, 5 µm Spherical 141

Waters Spherisorb ODS1 3.5, 5, 10 µm Spherical 144

Waters Spherisorb ODS2 3.5, 5, 10 µm Spherical 144

Waters Spherisorb ODSB 3.5, 5, 10 µm Spherical 144

XBridge BEH130 3.5, 5, 10 µm Spherical 132

XBridge BEH300 3.5, 5, 10 µm Spherical 132

XBridge C18 2.5, 3.5, 5, 10 µm Spherical 132

XBridge OST C18 2.5 µm Spherical 251

XBridge Shield RP18 2.5, 3.5, 5, 10 µm Spherical 132

XSelect CSH C18 2.5, 3.5, 5 µm Spherical 129

XSelect HSS C18 2.5, 3.5, 5 µm Spherical 129

XSelect HSS C18 SB 2.5, 3.5, 5 µm Spherical 129

XSelect HSS T3 2.5, 3.5, 5 µm Spherical 129

XTerra MS C18 2.5, 3.5, 5, 10 µm Spherical 140

XTerra RP18 3.5, 5, 10 µm Spherical 140

L3 Porous silica particles–1.5 to 10 μm in diameter.Brand Particle Size Type Page

ACQUITY UPLC BEH HILIC 1.7 µm Spherical 99

Atlantis HILIC Silica 3, 5 µm Spherical 138

BioSuite UHR SEC 5, 8 µm Spherical 241

BioSuite SEC 7.5 µm Spherical 241

Nova-Pak 6, 4 µm Spherical 145

μPorasil 10 µm Spherical 146

Resolve 5, 10 µm Spherical 146

SunFire Silica 5, 10 µm Spherical 136

Waters Spherisorb 5, 10 µm Spherical 144

XBridge HILIC 2.5, 3.5, 5 µm Spherical 132

L7 Octyl silane (C8) chemically bonded to porous silica particles– 1.5 to 10 μm in diameter.Brand Particle Size Type Page

ACQUITY UPLC BEH C8 1.7 µm Spherical 99

Nova-Pak C8 4, 6 µm Spherical 145

Resolve C8 5, 10 µm Spherical 146

Waters Spherisorb C8 3, 5, 10 µm Spherical 144

SunFire C8 3.5, 5, 10 µm Spherical 136

Symmetry C8 3.5, 5, 7 µm Spherical 141

SymmetryShield RP8 3.5, 5 µm Spherical 141

SymmetryPrep C8 7 µm Spherical 194

XBridge C8 2.5, 3.5, 5, 10 µm Spherical 132

XTerra MS C8 2.5, 3.5, 5, 10 µm Spherical 140

XTerra RP8 3.5, 5, 10 µm Spherical 140

L8An essentially monomolecular layer of aminopropylsilane (NH2) chemically bonded to totally porous silica gel support– 3 to 10 μm in diameter.


μBondapak NH2 10 µm Irregular 146

High Performance Carbohydrate Analysis

3, 5 µm — 152

Waters Spherisorb NH2 3, 5, 10 µm Spherical 144

L2Octadecylsilane (ODS or C18) chemically bonded to silica gel of a controlled surface porosity bonded to a solid spherical core–30 to 50 μm in diameter.


Bondapak® Prep C18 50 µm Irregular 146

L4 Silica gel of a controlled surface porosity bonded to a solid spherical core–30 to 50 μm in diameter.Brand Particle Size Type Page

Porasil™ Prep Silica 50 µm Irregular 146

L9 3 to 10 μm irregular, totally porous silica gel having a chemically bonded strongly acidic cation exchanger coating (SCX).Brand Particle Size Type Page

Waters Spherisorb SCX 5, 10 µm Spherical 144

L5 Alumina of controlled surface porosity bonded to a solid spherical core–30 to 50 μm in diameter.

L6 Strong cation exchanger packing-sulfonated fluorocarbon polymer coated on a solid spherical core–30 to 50 μm in diameter.

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L12A strong anion exchanger packing made by chemically bonding a quaternary amine to a solid silica spherical core–30 to 50 μm in diameter.


Accell Plus QMA 50 µm Irregular 239

L10 Nitrile groups (CN) chemically bonded to porous silica particles– 3 to 10 μm in diameter.Brand Particle Size Type Page

ACQUITY UPLC HSS CN 1.8 μm Spherical 101

μBondapak CN 10 µm Irregular 146

Nova-Pak CN HP 4 µm Spherical 145

Resolve CN 5, 10 µm Spherical 146

Waters Spherisorb CN 3, 5, 10 µm Spherical 144

XSelect HSS CN 2.5, 3.5, 5 μm Spherical 129

L11 Phenyl groups chemically bonded to porous silica particles– 1.5 to 10 μm in diameter.Brand Particle Size Type Page

ACQUITY UPLC CSH Phenyl-Hexyl 1.7 µm Spherical 97

ACQUITY UPLC BEH Phenyl 1.7 µm Spherical 99

μBondapak Phenyl 10 µm Irregular 146

Nova-Pak Phenyl 4 µm Spherical 145

XBridge Phenyl 2.5, 3.5, 5 µm Spherical 132

Waters Spherisorb Phenyl 3, 5, 10 µm Spherical 144

XSelect CSH Phenyl-Hexyl 2.5, 3.5, 5 µm Spherical 129

XTerra Phenyl 3.5, 5 µm Spherical 140

L13 Trimethylsilane (C1) chemically bonded to porous silica particles– 3 to 10 μm in diameter.Brand Particle Size Type Page


L14 Silica gel having a chemically bonded, strongly basic quaternary ammonium anion exchanger (SAX) coating–5 to 10 μm in diameter.Brand Particle Size Type Page

Waters Spherisorb SAX 5, 10 µm Spherical 144

L15 Hexylsilane (C6) chemically bonded to a totally porous silica particle–3 to 10 μm in diameter.Brand Particle Size Type Page


L17Strong cation exchange resin consisting of sulfonated, cross-linked styrene divinylbenzene copolymer in the hydrogen form–7 to 11 μm in diameter.


Fast Fruit Juice N/A N/A 178

IC-Pak™ Ion Exclusion 7 µm Spherical 158

IC-Pak Cation 10 µm Irregular 158

Shodex® RSpak DC-613 (6) µm Spherical 149

L18 Amino (NH2) and Cyano (CN) groups chemically bonded to porous silica particles–3 to 10 μm in diameter.

L19Strong cation exchange resin consisting of sulfonated, cross-linked styrene divinylbenzene copolymer in the calcium form–about 9 μm in diameter.


Sugar-Pak™ 1 9 µm Spherical 178

Shodex SC-1011 7 µm Spherical 243

L20 Dihydroxypropane groups chemically bonded to porous silica particles–3 to 10 μm in diameter.Brand Particle Size Type Page

BioSuite 125, 250, 4504, 5, 8, 10, 13, 17 µm

Spherical 241

Insulin HMW P — N/A 226

Protein-Pak™ 60 10 µm Irregular 243

Protein Pak 125 10 µm Irregular 243

Protein-Pak 300SW 10 µm Irregular 243

Protein-Pak KW-802.5 7 µm Irregular 272

Protein-Pak KW-803 7 µm Irregular 272

Protein-Pak KW-804 7 µm Irregular 272

L21 A rigid, spherical styrene-divinylbenzene copolymer 5 to 10 μm in diameter.Brand Particle Size Type Page

Shodex RSpak 613 6 µm Spherical 149

Styragel® HR 0.5, 1, 2, 3, and 4 — Spherical 282

Styragel HR 4E — Spherical 282

Styragel 5E — Spherical 282

L22 A cation-exchange resin made of porous polystyrene with sulfonic acid groups–about 10 μm in size.Brand Particle Size Type Page

IC-Pak Ion Exclusion 7 Spherical 158

Shodex RSpak DC 613 6 Spherical 149

Shodex SP-0810 8 Spherical 243

L24A semi-rigid hydrophilic gel consisting of vinyl polymers with numerous hydroxyl groups on the matrix surface–32 to 63 μm in diameter.

L23An anion-exchange resin made of porous polymethacrylate or polyacrylate gel with quaternary ammonium groups–about 10 μm in size.


BioSuite Q AXC 10, 13 µm Spherical 237

BioSuite DEAE 2.5, 10, 13 µm Spherical 237

BioSuite Q-PEEK 10 µm Spherical 237

IC-Pak Anion 10 µm Spherical 158

Protein-Pak Q 8HR 8 µm Spherical 243L16 Dimethylsilane (C2) chemically bonded to a totally porous silica particles–5 to 10 μm in diameter.

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L28A multifunctional support which consists of a high purity, 100Å, spherical silica substrate that has been bonded with anionic (amine) functionality in addition to a conventional reversed-phase C8 functionality.

L35Zirconium-stabilized spherical silica packing with a hydrophilic (diol-type) molecular mono layer bonded phase having a pore size of 150Å.

L40 Cellulose tris-3,5-dimethylphenylcarbamate coated porous silica particles–5 to 20 μm in diameter.

L41 Immobilized a1-acid glycoprotein on spherical silica particles– 5 μm in diameter.

L42 Octylsilane and octadecylsilane groups chemically bonded to porous silica particles–5 μm in diameter.

L36 3,5-dinitrobenzoyl derivative of L-phenylglycine covalently bonded to a 5 μm aminopropyl silica.

L31A strong anion-exchange resin-quaternary amine bonded on latex particles attached to a core of 8.5 μm macroporous particles having a pore size of 2,000Å and consisting of ethylvinylben-zene cross-linked with 55% divinyl benzene.

L32A chiral-ligand exchange packing–L proline copper complex covalently bonded to irregularly shaped silica particles–5 to 10 μm in diameter.

L29Gamma alumina, reversed-phase, low carbon percentage by weight alumina-based polybutadiene spherical particles–5 μm in diameter with a pore diameter of 80Å.

L30 Ethyl silane chemically bonded to a totally porous silica particle–3 to 10 μm in diameter.

L26 Butyl silane (C4) chemically bonded to porous silica particles– 3 to 10 μm in diameter.Brand Particle Size Type Page

ACQUITY UPLC BEH300 C4 1.7 µm Spherical 99

Delta-Pak C4 5 µmSpherical

(100 + 300Å)146

Symmetry300 C4 3.5 µm Spherical 244

XBridge BEH300 C4 3.5 µm Spherical 132

L27 Porous silica particles–30 to 50 μm in diameter.Brand Particle Size Type Page

Porasil 37–55 µm Irregular 146

L33Packing having the capacity to separate proteins of 4,000 to 400,000 daltons. It is spherical, silica-based and processed to provide pH stability.


BEH125 SEC 1.7 µm Spherical 227

BEH200 SEC 1.7 µm Spherical 227

L34Strong cation-exchange resin consisting of sulfonated cross-linked styrene-divinylbenzene copolymer in the lead form–about 9 μm in diameter.


Shodex SP-0810 N/A Spherical 243

L37Packing having the capacity to separate proteins by molecular size over a range of 2,000 to 40,000 daltons. It is a polymethacrylate gel.


Ultrahydrogel 250 N/A Spherical 289

L38 A methacrylate-based size-exclusion packing for water soluble samples.Brand Particle Size Type Page

Ultrahydrogel N/A Spherical 289

L39 A hydrophilic-polyhydroxymethacrylate gel of totally porous spherical resin.Brand Particle Size Type Page

Ultrahydrogel N/A Spherical 289

L43 Pentafluorophenyl groups chemically bonded to silica particles– 5 to 10 μm in diameter.Brand Particle Size Type Page

ACQUITY UPLC CSH Fluoro-Phenyl 1.7 µm Spherical 97

ACQUITY UPLC HSS PFP 1.8 µm Spherical 101XSelect CSH Fluoro-Phenyl 2.5, 3.5, 5 µm Spherical 129

XSelect HSS PFP 2.5, 3.5, 5 µm Spherical 129

L44A multifunctional support, which consists of a high purity, 60Å, spherical silica substrate that has been bonded with a cationic exchanger, sulfonic acid functionality in addition to a convention reversed-phase C8 functionality.

L45 Beta cyclodextrin bonded to porous silica particles–5 to 10 μm in diameter.

L46 Polystyrene/divinylbenzene substrate agglomerated with quaternary amine functionalized latex beads–10 μm in diameter.

L55 A strong cation-exchange resin made of porous silica coated with polybutadiene-maleic acid copolymer–about 5 μm in diameter.Brand Particle Size Type Page

IC-Pak C M/D — — 148

L59Packing having the capacity to separate proteins by molecular weight over the range of 10 to 500 kDa. It is spherical (10 μm), silica-based, and processed to provide hydrophilic characteristics and pH stability.


BioSuite 125, 250, 450 Series 4–17 µm Spherical 241

Source: United States Pharmacopeia ( ) - Denotes particle sizes available outside of L class.

L25

Packing having the capacity to separate compounds with a molecular weight range from 100 to 5,000 (as determined by polyethylene oxide), applied to neutral, anionic and cationic water-soluble polymers. A polymethacrylate resin base, cross-linked with polyhydroxylated ether (surface contained some residual carboxyl groups), was found suitable.


Ultrahydrogel™ DP, + 120 10 µm Spherical 282

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Physical Characteristics of HPLC and UPLC Packing Materials

Brand Chemistry Particle Shape Particle Size(s)Pore Size*

Surface Area [m2/g]

Pore Volume [cc/g]

% Carbon Load

End Capped

AccQ•Tag Ultra C18 Spherical 1.7 µm 135Å 185 0.70 17 Yes

ACQUITY UPLC BEH

C18 Spherical 1.7 µm 130Å 185 0.70 17 Yes


Shield RP18 Spherical 1.7 µm 130Å 185 0.70 17 Yes

Phenyl Spherical 1.7 µm 130Å 185 0.70 15 Yes

HILIC Spherical 1.7 µm 130Å 185 0.70 Unbonded n/a

Amide Spherical 1.7 µm 130Å 185 0.70 12 n/a

ACQUITY UPLC BEH125 SEC Spherical 1.7 µm 125Å 398 High 15 n/a

ACQUITY UPLC BEH130 C18 Spherical 1.7 µm 130Å 185 0.70 17 Yes

ACQUITY UPLC BEH200 SEC Spherical 1.7 µm 200Å 220 High 12 n/a

ACQUITY UPLC BEH300C18 Spherical 1.7 µm 300Å 90 0.66 12 Yes

C4 Spherical 1.7 µm 300Å 90 0.66 8 No

ACQUITY UPLC CSH


Phenyl-Hexyl Spherical 1.7 µm 130Å 185 0.70 14 Yes

Fluoro-Phenyl

Spherical 1.7 µm 130Å 185 0.70 10 No

ACQUITY UPLC HSS

T3 Spherical 1.8 µm 100Å 230 0.70 11 Yes


C18 SB Spherical 1.8 µm 100Å 230 0.70 8 No

PFP Spherical 1.8 µm 100Å 230 0.70 7 No

CN Spherical 1.8 µm 100Å 230 0.70 5 No

Atlantis

T3 Spherical 3, 5, 10 µm 100Å 330 1.00 14 Yes

dC18 Spherical 3, 5, 10 µm 100Å 330 1.00 12 Yes

HILIC Spherical 3, 5 µm 100Å 330 1.00 Unbonded n/a

BondapakC18 Irregular 15–20 µm 125Å 330 1.00 10 Yes

C18 Irregular 15–20 µm 300Å 100 1.00 3.5 Yes

μBondapak

C18 Irregular 10 µm 125Å 330 1.00 9.8 Yes

Phenyl Irregular 10 µm 125Å 330 1.00 9.3 Yes

CN Irregular 10 µm 125Å 330 1.00 6 Yes

NH2 Irregular 10 µm 125Å 330 1.00 4 No

Delta-Pak

C4 Spherical 5, 15 µm 100Å 300 1.00 7.3 Yes

C18 Spherical 5, 15 µm 100Å 300 1.00 17 Yes



Nova-Pak


C8 Spherical 4 µm 60Å 120 0.30 4 Yes

Phenyl Spherical 4 µm 60Å 120 0.30 4.6 Yes

CN HP Spherical 4 µm 60Å 120 0.30 3 Yes

Silica Spherical 4, 6 µm 60Å 120 0.30 n/a n/a

Porasil Silica Irregular 15–20 µm 125Å 330 1.00 n/a n/a

μPorasil™ Silica Irregular 10 µm 125Å 330 1.00 n/a n/a

Resolve

C18 Spherical 5, 10 µm 90Å 200 0.50 10.2 No

C8 Spherical 5 µm 90Å 200 0.50 5.1 No

CN Spherical 10 µm 90Å 200 0.50 3 No

Silica Spherical 5, 10 µm 90Å 200 0.50 n/a n/a

* Nominal value

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Physical Characteristics of HPLC and UPLC Packing Materials

Brand Chemistry Particle Shape Particle Size(s)Pore Size*

Surface Area [m2/g]

Pore Volume [cc/g]

% Carbon Load

End Capped

SunFire

C18 Spherical 2.5, 3.5, 5, 10 µm 100Å 340 0.90 16 Yes


Silica Spherical 5, 10 µm 100Å 340 0.90 Unbonded n/a

SymmetryC18 Spherical 3.5, 5 µm 100Å 335 0.90 19.1 Yes

C8 Spherical 3.5, 5 µm 100Å 335 0.90 11.7 Yes

Symmetry300C18 Spherical 3.5, 5 µm 300Å 110 0.80 8.5 Yes

C4 Spherical 3.5, 5 µm 300Å 110 0.80 2.8 Yes

SymmetryPrepC18 Spherical 7 µm 100Å 335 0.90 19.1 Yes

C8 Spherical 7 µm 100Å 335 0.90 11.7 Yes

SymmetryShieldRP8 Spherical 3.5, 5 µm 100Å 335 0.90 15 Yes

RP18 Spherical 5 µm 100Å 335 0.90 17 Yes

Waters Spherisorb

Silica Spherical 3, 5, 10 µm 80Å 220 0.50 n/a n/a

ODS2 Spherical 3, 5, 10 µm 80Å 220 0.50 11.5 Yes

ODS Spherical 3, 5, 10 µm 80Å 220 0.50 6.2 No

ODSB Spherical 5 µm 80Å 220 0.50 11.5 Yes

C8 Spherical 3, 5, 10 µm 80Å 220 0.50 5.8 Yes

C6 Spherical 3, 5, 10 µm 80Å 220 0.50 4.7 Yes

C1 Spherical 3, 5, 10 µm 80Å 220 0.50 2.2 No

Nitrile Spherical 3, 5, 10 µm 80Å 220 0.50 3.1 No

Amino Spherical 3, 5, 10 µm 80Å 220 0.50 1.9 No

Phenyl Spherical 3, 5, 10 µm 80Å 220 0.50 2.5 No

OD/CN Spherical 5 µm 80Å 220 0.50 5 Yes

SAX, SCX Spherical 5, 10 µm 80Å 220 0.50 4 No

XBridge



Shield RP18 Spherical 2.5, 3.5, 5, 10 µm 130Å 185 0.70 17 Yes

Phenyl Spherical 2.5, 3.5, 5 µm 130Å 185 0.70 15 Yes

HILIC Spherical 2.5, 3.5, 5 µm 130Å 185 0.70 Unbonded n/a

Amide Spherical 2.5, 3.5 µm 130Å 185 0.70 12 n/a

XBridge BEH130 C18 Spherical 3.5, 5, 10 µm 130Å 185 0.70 18 Yes

XBridge BEH300C18 Spherical 3.5, 5, 10 µm 300Å 86 0.66 12 Yes

C4 Spherical 3.5 µm 300Å 90 0.66 8 No

XSelect CSH

C18 Spherical 2.5, 3.5, 5 µm 130Å 185 0.70 15 Yes

Phenyl-Hexyl Spherical 2.5, 3.5, 5 µm 130Å 185 0.70 14 Yes

Fluoro-Phenyl Spherical 2.5, 3.5, 5 µm 130Å 185 0.70 10 No

XSelect HSS

T3 Spherical 2.5, 3.5, 5 µm 100Å 230 0.70 11 Yes

C18 Spherical 2.5, 3.5, 5 µm 100Å 230 0.70 15 Yes

C18 SB Spherical 2.5, 3.5, 5 µm 100Å 230 0.70 8 No

PFP Spherical 2.5, 3.5, 5 µm 100Å 230 0.70 7 No

CN Spherical 2.5, 3.5, 5 µm 100Å 230 0.70 5 No

XTerra

RP18 Spherical 3.5, 5, 10 µm 125Å 175 0.70 15 Yes

RP8 Spherical 3.5, 5, 10 µm 125Å 175 0.70 13.5 Yes

MS C18 Spherical 2.5, 3.5, 5, 10 µm 125Å 175 0.70 15.5 Yes

MS C8 Spherical 2.5, 3.5, 5, 10 µm 125Å 175 0.70 12 Yes

Phenyl Spherical 3.5, 5 µm 125Å 175 0.70 12 Yes* Nominal value

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XBridge and XSelect eXtended Performance [XP] 2.5 μm columns enable exceptional separation performance, robustness, and throughput for HPLC assays while being fully compatible with all HPLC, UHPLC, and UPLC technology platforms.

Unmatched Selectivity and Flexibility. 14 chemistries including C18, Phenyl-Hexyl, C8, Embedded Polar, HILIC, Amide as well as Cyano and PFP columns

Directly scalable to 1.7/1.8 μm UPLC columns and larger 3.5/5 μm HPLC columns

Designed to withstand higher pressures of 9,000 psi (4.6 mm ID) and 18,000 psi (2.1 and 3.0 mm ID)

Select column length (30, 50, 75, 100, and 150 mm) for the correct balance between resolution and throughput

High Throughput. Lower Backpressure. Improved Productivity.

40% lower backpressure than sub-2-μm UPLC columns

Equivalent backpressure and superior performance to core-shell columns

2–4X higher throughput than 3.5–5-μm HPLC columns lead to improved productivity

Rapid Method Optimization. Transfer methods TODAY within current pharmacopeia guidelines

Utilize on an ACQUITY UPLC system to maximize performance and future-proof your method

improve existing HPLC Productivity 2–4X

XSelect CSH C184.6 x 100 mm, 3.5 µm

Part Number: 186005269HPLC System

XSelect CSH C18 XP4.6 x 75 mm, 2.5 µm

Part Number: 186006110HPLC System

2X Faster than3.5 µm HPLC

ACQUITY UPLC CSH C182.1 x 50 mm, 1.7 µm

Part Number: 186005296UPLC System



0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 min

0.0 0.5 1.0 1.5 2.1 2.5 3.0 min

1

3

5

4

2

0.0 4.0 8.0 12.0 16.0 20.0 24.0 28.0 min

Peak Order: (1) tolmetin, (2) naproxen, (3) fenoprofen, (4) indomethacin, (5) dicolfenact

eXtended Performance [XP] 2.5 µm Columns

Did you know...You can request Certificates of Analysis on our website?

Go to: www.waters.com and look for “Request Certificate of Analysis” under “Services and Support”

See page 170for part numbers and ordering information.

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XBridge BEH C18 XP, 2.5 µm

2.1 x 50 mm columns70/30 MeCN/H20, 30 °C, 254 nm

Flow Rate (mL/min)

Pres

sure

(psi

)

0.00

1000

2000

3000

4000

5000

6000

7000

8000

0.5 1.0 1.5 2.0 2.5

Core-Shell C18,2.6 µm

Comparative separations may not be representative of all applications.

Equivalent Backpressure

Additional Flexibility for UPLC Users

XP 2.5 µm columns provide an alternative to 1.7 µm UPLC columns for use on an ultra-low dispersion ACQUITY UPLC instrument. With 40% lower backpressure than 1.7 µm columns, XP 2.5 µm columns can be utilized for fit-for-purpose analyses at intermediate backpressures. Alternatively, flow rate can be increased to further expedite sample throughput for simple chromatography.

40% Lower Backpressure on Your UPLC

XSelect CSH C18 XP

2.1 x 75 mm, 2.5 µm

Part Number: 186006102

Flow Rate = 0.54 mL/min

Pressure = 5300 PSI

Peak Capacity = 34

ACQUITY UPLC CSH C18

2.1 x 75 mm, 1.7 µm


Flow Rate = 0.54 mL/min

Pressure = 9200 PSI

Peak Capacity = 40

40% lowerbackpressure

15% increase in Rs and Pc20% higher sensitivity

Tolm

etin

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 min

0.00

0.05

0.10

0.15

0.20

AU

0.25

0.30

0.35

1.000.00 2.00 3.00 4.00 5.00 6.00 7.00 min

Dicl

ofen

ac

Indo

met

haci

n

Feno

prof

en

Napr

oxen

0.00

0.05

0.10

0.15

0.20

AU

0.25

0.30

0.35

RS = 5.5

RS = 6.3

Columns were run on an ACQUITY UPLC H-Class system.

Superior Performance vs. Core-Shell with Equivalent Backpressure

XP 2.5 µm columns provide 34% higher peak capacity than equivalent core-shell columns due to the limited loading capacity of this type of particle technology. Additionally, both columns exhibit similar backpressures indicating that the particle size dictates the observed backpressure, not the particle type (core-shell vs. fully-porous).

34% Higher Peak Capacity

XSelect CSH C18 XP

4.6 x 75 mm, 2.5 µm

Peak Capacity = 47

Core-Shell C18

4.6 x 75 mm, 2.6 µm

Peak Capacity = 35

0.30

0.25

0.20

0.15

0.10

0.05

0.00

0.30

0.25

0.20

0.15

0.10

0.05

0.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00

Pind

olol

Quin

ine

Labe

tolo

l

Vera

pam

il

Dilta

zem

14.00 16.00 min

16.00 min



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http://www.waters.com/waters/partDetail.htm?partNumber=186006110http://www.waters.com/waters/nav.htm?cid=560002&alias=Alias_order_CO&lset=1&locale=en_US

XSelect ColumnsXSelect columns offer the separations scientist the most diverse range of modern LC columns for method development. The scientists that use XSelect columns demand diverse selectivity and realize that it is the combination of base particle composition and ligand bonding that critically influences analyte selectivity and column performance. It is the optimized combination of substrate and ligand that creates a column family for enhancing analyte selectivity. T he XSelect column family is offered in two distinct substrates (High Strength Silica [HSS] and Charged Surface Hybrid [CSH]) to give you the most option and control over your separation power.

CSH Technology Advantages Superior peak shape for basic compounds - High peak capacity for complex mixtures

Increased loading capacity - Increased mass load for preparative separation without using ion-pairing reagents like TFA

Rapid column equilibration - Quickly change mobile-phase conditions and pH to quickly screen multiple mobile-phase conditions

HSS Technology Advantages High-pressure-tolerant silica - Silica used for lower pressure HPLC separations is fragile at high pressure. HSS columns are the only commercially available silica-based column that has been designed for UPLC.

Unique silica bonding - The silica substrate used for XSelect HSS columns combined with optimized ligand bonding gives the chromatographer a versatile and complimentary set of column choices. Whether your method requires 100% aqueous mobile phase or alternative retention mechanisms including reversed phase, normal phase, and HILIC, there is an XSelect HSS column tailored for performance.

Stationary Phase Characteristics

Ligand Type CSH C18CSH

Fluoro-PhenylCSH

Phenyl-HexylHSS C18 HSS C18 SB HSS T3 HSS PFP HSS CN

Particle Size 2.5, 3.5, 5 µm 2.5, 3.5, 5 µm 2.5, 3.5, 5 µm 2.5, 3.5, 5 µm 2.5, 3.5, 5 µm 2.5, 3.5, 5 µm 2.5, 3.5, 5 µm 2.5, 3.5, 5 µm

Ligand Density* 2.3 µmol/m2 2.3 µmol/m2 2.3 µmol/m2 3.2 µmol/m2 1.6 µmol/m2 1.6 µmol/m2 3.2 µmol/m2 2.0 µmol/m2

Carbon Load* 15% 10% 14% 15% 8% 11% 7% 5%

Endcap Style Proprietary None Proprietary Proprietary None Proprietary None None

pH Range 1–11 1–8 1–11 1–8 2–8 2–8 2–8 2–8

Low pH Temp. Limit

80 °C 60 °C 80 °C 45 °C 45 °C 45 °C 45 °C 45 °C

High pH Temp. Limit

45 °C 45 °C 45 °C 45 °C 45 °C 45 °C 45 °C 45 °C

Pore Diameter* 130Å 130Å 130Å 100Å 100Å 100Å 100Å 100Å

Surface Area* 185 m2/g 185 m2/g 185 m2/g 230 m2/g 230 m2/g 230 m2/g 230 m2/g 230 m2/g

*Expected or approximate value.


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Enhanced Selectivity

Method development scientists use selectivity and retentivity to influence chromatographic behavior. Offering a family of chromatographic sorbents that provide diverse selectivities was one of the design considerations for XSelect HPLC columns.

Waters continuing effort to develop LC particle technology leads to the innovations behind CSH and HSS particle development. No other family of LC products is focused on the development to maximize selectivity differences compared to commercially available chromatographic packing materials. The Selectivity Factor (S-value) measures how different (or similar) the selectivities of two column chemistries are under a given set of conditions. The value is determined by measuring and plotting the retention factors (k) of analytes of the two columns. Higher S-values indicate a higher degree of orthogonality.

Selectivity (S) = 100 x 1-R2

Chemistries Engineered to Provide Wider Range of Selectivities

0

5

10

15

0 5 10 15kg on XBridge C18

kg o

n XS

elec

t CSH

Pha

ses

R2 = 0.76

S = 49

Retention on BEH C18 (kg)

Rete

ntio

n on

XSe

lect

Pha

ses

(kg)

R2 = 0.63

S = 61

0

5

10

15

0 5 10 15

XSelect CSH C18

XSelect CSH Fluoro-Phenyl

XSelect CSH Phenyl-Hexyl

XSelect HSS CN

XSelect CSH C18

XSelect CSH Fluoro-Phenyl

XSelect CSH Phenyl-Hexyl

XSelect HSS T3

XSelect HSS PFP

XSelect HSS C18

XSelect HSS C18 SB

The Selectivity Factor (S-value) can be used to assess (and hence, help to create) orthogonality of selectivities between two different columns. Simply plot retention factors of one column versus another column, calculate the coefficient of determination (R2) and calculate the Selectivity Factor as indicated. Larger values indicate higher orthogonality.

Reproducibilty

One of the challenges in designing stationary phases with large selectivity differences is maintaining acceptable batch-to-batch reproducibility. Waters R&D recognized that method developers need reproducible columns that will maintain performance over the lifetime of the method. This was a key consideration for the XSelect column family. The figure below is an overlay of gradient separations on columns containing nine different batches of XSelect CSH Fluoro-Phenyl materials. In these results, the batches include three different particle sizes demonstrating that even across different particle sizes, maintain the same selectivity. T he unique XSelect Fluoro-Phenyl phase establishes an expectation that highly selective stationary phases can be reproducible.

3.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 4.0 min

1 2

3 4

5 6

7

8

9

10 XSelect CSH Fluoro-Phenyl 5 �m

XSelect CSH Fluoro-Phenyl 3.5 �m

ACQUITY UPLC CSH Fluoro-Phenyl 1.7 �m

ACQUITY UPLC CSH Fluoro-Phenyl (1.7 μm) and XSelect CSH Fluoro-Phenyl (3.5 and 5 μm) reproducibility and scalability. Gradient separations on 2.1 x 50 mm columns containing 9 different batches of CSH Technology Fluoro-Phenyl representing three (1.7, 3.5, and 5 μm) particle sizes. Gradient: A: 15.4 mM ammonium formate, pH 3; B: acetonitrile; 5–90% B linear in 5 minutes. Temperature: 30 °C. Injection volume: 5 μL. Detection: 254 nm. Flow rate: 0.5 mL/min. Analytes: (1) thiourea; (2) resorcinol; (3) metoprolol; (4) 3-nitrophenol; (5) 2-chlorobenzoic acid; (6) amitriptyline; (7) diethylphthalate; (8) fenoprofen; (9) dipropylphthalate; (10) pyrenesulfonic acid. System: ACQUITY UPLC.

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Columns Designed for Method Development and Method Transferability

Many laboratories function as teams that are a part of multi-national/multi-site organizations. From a global business perspective, it is vital to be able to quickly and easily develop robust methods that are not only compatible with all modern chromatographic detection modes, but are also transferable to laboratories and sites that may operate different LC system platforms. ACQUITY UPLC and XSelect columns, as part of the ACQUITY UPLC H-Class total system solution, were strategically created for the 21st-century global chromatographic marketplace. Using the same technology throughout the entire process, methods can be developed quickly with ACQUITY UPLC columns and then transferred to XSelect HPLC and preparative-scale XSelect OBD columns for isolation and purification.

Isolation and Purification

Using CSH Technology throughout the entire process, methods can be developed quickly with ACQUITY UPLC CSH columns and UPLC Technology and then transferred to preparative-scaleXSelect OBD columns for isolation and purification. The purity of the isolated fraction(s) can then be measured/confirmed using ACQUITY UPLC CSH columns and UPLC Technology.

Compounds:1. Diphenhydramine2. Oxybutynin3. Terfenadine

LC ConditionsColumns: ACQUITY UPLC CSH C18, 2.1 x 50 mm, 1.7 μm Part Number: 186005296Mobile Phase A: 0.1% formic acid in waterMobile Phase B: 0.1% formic acid in acetonitrile Gradient: Time Flow Profile (min) (mL/min) %A %B Curve Initial 0.9 95 5 Initial 2.0 0.9 5 95 6 2.5 0.9 5 95 6 2.51 0.9 95 5 6 3.50 0.9 95 5 6Injection Vol.: 1 μLSample Conc.: 800 µg/mL for crude mixSample Diluent: 50/50 methanol/waterColumn Temp.: 40 °CWeak Needle Wash: 95/5 water/acetonitrile Strong Needle Wash: 95/5 acetonitrile/waterDetection: UV @ 220 nmSampling Rate: 20 points/secFilter Time Constant: 0.1 secSystem: ACQUITY UPLC with ACQUITY UPLC PDA

Preparative ConditionsColumn: XSelect CSH C18 OBD Prep, 19 x 100 mm, 5 μm Part Number: 186005421Mobile Phase A: 0.1% formic acid in waterMobile Phase B: 0.1% formic acid in acetonitrile Gradient: Time Flow Profile Curve (min) (mL/min) %A %B 1.21 25 95 5 Initial 1.71 25 95 5 6 7.02 25 78.8 21.2 6 7.21 25 70.8 29.2 6 8.21 25 5 95 6 8.31 25 95 5 6 13.21 25 95 5 6

Injection Vol.: 1.25 mLSample Conc.: 80 mg/mL for crude mixDiphenhydramine: 40 mg/mLOxybutynin: 16 mg/mLTerfenadine : 24 mg/mLSample Diluent: DMSOColumn Temp.: 40 °CWeak Needle Wash: 95/5 water/methanol Strong Needle Wash: 95/5 methanol/waterDetection: UV @ 220 nmSampling Rate: 1 point/secFilter Time Constant: 1 secSystem: Waters 2525 Binary Gradient Module, 2767 Sample Manager, Column Fluidics Organizer, 2996 Photodiode Array Detector, ZQ™ Mass Spectrometer

0.00

0.50

1.00

1.50

AU

Crude mixDiphenhydramine = 56.3% purity

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 min

1

2

1

3

Target Analyte

0.00

0.50

1.00

1.50

Pooled purified fractionsDiphenhydramine = 100% purity

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 min

AU

1

2

3

2 3 4 5 6 7 8 9 10 min0.00

0.50

1.00

1.50

1

2

3 2.00

Prep Purification100 mg total load

Collected Fraction

AU

3. Run preparative separation using focused gradient

2. Scale UPLC separation to preparative column scale 4. Collect and isolate fraction(s) of interest

5. Confirm fraction purity using UPLC Technology1. Develop method using UPLC Technology

High mass loading applications like compound purification and dissolution testing are highly demanding of column performance. Under these extreme loading conditions, the limiting factor for reproducible scaling, often results from the inability to maintain peak shape. The combination of Optimum Bed Density (OBD) preparative columns with the benefits of XSelect particle technology gives the greatest benefit to the purification scientist. CSH Technology, for example, offers the highest loading of basic compounds using highly volatile, low-pH mobile phases often preferred by isolation and purification scientists. This increased loadability allows the use of narrower, lower volume, preparative columns thereby reducing solvent consumption and fraction volumes.

Did you know...TruView LCMS Certified vials minimize analyte adsorption and are ideal for working with low sample concentrations.

For more information, see page 68.

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Maximizing Column Efficiency

One of the most important parameters in the design of the new hybrid particle was to significantly improve the chromatographic equivalence to state-of-the-art silica based materials. The origins of band spreading, which decreases separation efficiency, are described by the van Deemter equation:

h = a + b/u + cu

Data for both silica and BEH Technology packings, fitted to the van Deemter equation demonstrate effective equivalence in efficiency. T he c term for XBridge C18 is virtually identical to that for the two state-of-the-art C18 silica columns, indicating that all these columns are comparable in mass transfer characteristics.

XBridge ColumnsWith industry leading high pH stability and the highest level of chromatographic performance, XBridge BEH columns are the benchmark for LC method development.

With a selection of 19 general purpose and application-specific stationary phases in the widest range of particle sizes, no other LC column family can match the versatility of the XBridge suite. Based on proven hybrid technology, the patented BEH particle* is a product of Waters commitment to provide you with the tools you need to solve the most demanding of chromatographic challenges. Leading separations scientists need LC columns that provide the highest level of chromatographic performance to:

Maximize efficiency

Provide long column lifetimes with increased high pH stability

Improve column reliability*US Patent # 6,686,035 B2


van Deemter Curve Comparison

2

4

6

8

10

12

0 20 40 60 80

u [uidp/Dm]

h us

ing

N 1/2

Ht

Reduced van Deemter Plot Termsa b c r2

XBridge C18 5 µm 1.04 8.7 0.044 0.998

Symmetry C18 5 µm 0.76 10.6 0.045 0.999

SunFire C18 5 µm 0.95 11.6 0.040 0.997

The reduced plate height, h, is a function of the reduced linear velocity, u, [both normalized for particle size] and a, b, and c summarize the contributions of eddy diffusion, longitudinal diffusion, and the sum of stationary- and mobile-phase mass transfer terms, respectively.

Compound:Decanophenone

Mobile Phase: 70/30 ACN/waterTemperature: 30 °C

Ligand Type Trifunctional C18Trifunctional

C8

Monofunctional Embedded Polar Group(Shield RP18)

Trifunctional C6 Phenyl

HILIC AmideBEH130 C18

(Peptide Separation

Technology)

BEH300 C18(Peptide

Separation Technology)

BEH300 C4(Protein


BEH C18(Oligonucleotide


Particle Size2.5, 3.5, 5, 10 µm

2.5, 3.5, 5, 10 µm

2.5, 3.5, 5, 10 µm

2.5, 3.5, 5 µm

2.5, 3.5, 5 µm

2.5, 3.5 µm

3.5, 5, 10 µm

3.5, 5, 10 µm

3.5 µm 2.5 µm

Ligand Density** 3.1 µmol/m2 3.2 µmol/m2 3.3 µmol/m2 3.0 µmol/m2 N/A 7.5 µmol/m2 3.1 µmol/m2 3.1 µmol/m2 2.4 µmol/m2 3.1 µmol/m2

Carbon Load** 18% 13% 17% 15% unbonded 12% 18% 12% 8% 18%

Endcap Style Proprietary Proprietary TMS Proprietary n/a None Proprietary Proprietary None Proprietary

pH Range 1–12 1–12 2–11 1–12 1–9 2–11 1–12 1–12 1–10 1–12

Low pH Temp. Limit

80 °C 60 °C 50 °C 80 °C 60 °C 90 °C 80 °C 80 °C 80 °C 80 °C

High pH Temp. Limit

60 °C 60 °C 45 °C 60 °C 45 °C 90 °C 60 °C 60 °C 50 °C 60 °C

Pore Diameter** 130Å 130Å 130Å 130Å 130Å 130Å 130Å 300Å 300Å 130Å

Surface Area** 185 m2/g 185 m2/g 185 m2/g 185 m2/g 185 m2/g 185 m2/g 185 m2/g 90 m2/g 90 m2/g 185 m2/g** Expected or approximate value.


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Accelerated Low pH Stability Tests of Competitive Columns

Probe: Methylparaben

50

60

70

80

90

100

0 50 100

Hours in 1% TFA in H2O pH 1.0 at 80 °C

% In

itial

Ret

entio

n

XBridge C18

Zorbax® SB-C18

XBridge Phenyl

Zorbax SB Phenyl

Luna® C18(2)

ACT® Ace® C18

Gemini™ C18

Capcell PAK® MG II

Accelerated High pH Stability Test of Competitive Columns

0 4 8 12 16 20 24 min

Initial

100 h

200 h

300 h

N=13600

N=13500

N=14100

N=11700

12

34 5

030

50

70

90

110

50 100 150 200 hours

Hours in 50 mM TEA, pH 10, 50 °C

% In

itial

N5s

XBridge C18

XTerra MS C18

Gemini™ C18

Luna® C18 (2)

YMC-Pak® Pro® C18

Zorbax® Extend C18

Compounds:1. Uracil 2. Propranolol 3. Naphthalene 4. Acenaphthene 5. Amitriptyline

Chromatograms, run at regular intervals during the high-pH lifetime study, verify that 86% of the original XBridge column efficiency remains after 300 hours at pH 10 and elevated temperature, with little change in peak shape or retention time. Comparative separations may not be representative of all applications.

Compound: Acenaphthene

Column Lifetime and Phosphate Buffer

Phosphate buffers are well recognized and are the most commonly accepted mobile-phase additive. Above pH 7, phosphate buffers are very aggressive and even the best reversed-phase silica columns are unable to withstand the alkaline conditions. The combination of phosphate, intermediate pH, and relatively low temperatures (40 °C) is well recognized to lead to short column lifetimes for most modern phases. XBridge BEH columns overcome this limitation and provide you with extended pH range and control over your separation.

pH Stability

Improved Low pH Performance

The major cause of poor column lifetime in low pH mobile phases is due to the acid hydrolysis of the bonded phase. XBridge packings incorporate state-of-the-art, proprietary procedures for bonding and end capping resulting in ligand stability and chromatographic reproducibility at low pH. Compared to conventional materials, using an accelerated low pH stability test, XBridge C18 columns show very little retention loss and exhibit exceptional column lifetime without resorting to sterically hindering the stationary phase.

Chromatographic Lifetime Comparisons in Phosphate Buffer

300 Hours 30 Hours

N = 2218N = 11700

100 20 30 min40 20 24 min16128

Waters XBridge C18 Phenomenex® Gemini™ C18

50 mM TEA, 50 °C, pH 10

270 Hours 13 Hours

1050 15 20 min 1050 15 20 min

Waters XBridge C18 Phenomenex Gemini C18

30 mM KH2PO4, 30 °C, pH 12.3

Aggressive alkaline mobile phases can quickly dissolve particle substrates creating voids in the packed bed. The BEH particle is chemically resistant allowing XBridge HPLC columns to perform under extreme mobile-phase conditions.


High pH Endurance

XBridge columns are engineered to be the most pH stable high-performance chromatographic phases commercially available. Approaches that claim high pH resistance due to special surface modifications, cannot match the lifetime of an XBridge column. Stability is the product of the BEH particle synthetic process.

Under accelerated pH 10 stability test conditions, a direct comparison to some of the most popular chromatographic phases, claimed to have extended high pH stability, clearly shows the XBridge C18 column lifetime exceeding that of the best columns by over 1000% with very little degradation in chromatographic performance.


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Method Development Flexibility

Selecting the most suitable column and separation conditions can be extremely difficult. The XBridge family of columns is designed to eliminate the compromises of sorbent selection and deliver the flexibility to work under any mobile phase, temperature and pH conditions, thus speeding up the process to an optimized and rugged final method.

A simple reversed-phase method development approach consists of three XBridge columns, two mobile-phase pHs, and two organic solvents, that quickly defines the starting conditions for the most challenging of sample mixtures. An optimized approach to method development that quickly resolves complex mixtures saves you time, effort, and expense.

1110

2

3

1

95

4

6

7

8

16.68

1110

1,2

3

8,9

5

6

12

4 7

2

3

1

10

57,8

6

11

12

4

9

XBridge Low pH in Methanol

C18

Shield RP18

Phenyl

C18

C18

Shield RP18

Phenyl

Phenyl

Shield RP18

C18

Phenyl

Shield RP18

XBridge High pH in Methanol

XBridge Low pH in Acetonitrile XBridge High pH in Acetonitrile

11

62

1

3 12

8,1310

79

5

11

62

1

3 12

139,10

7 8

5

11

62

1

3 12

13

9

10

5

7

2

1

3

4,5

119

8

6 712

10

2

1

1110

5

4

8

6

7

9

123

2

1

3

119,10

5

4

8

6

7

12

11

62

4

1

3139

12

5

7

10

8

11

6

2

4

1

3

13912

5

7

10

8

11,12

62

4

1

313

9

5 810

7

0 4 8 12 16 20 min

0 4 8 12 16 20 min

0 4 8 12 16 20 min

0 4 8 12 16 20 min

Changing mobile-phase pH is the single most effective strategy to influence analyte selectivity and retention. XBridge columns allow you to use the full range of pH to control the elution of your target analytes.

Creating Selectivity Changes with Phosphate Buffer

pH 2

pH 7

pH 12

0 1 2 3 4 5 6 7 8 min

1

2 63 54

23

1

46 5

3 2

465

1

XBridge columns are unique in their ability to withstand these aggressive conditions (even at pH 12) allowing complete flexibility for the method development chemist, while maintaining the highest efficiency values associated with silica columns.

Column: XBridge C18, 4.6 x 100 mm, 3.5 µmPart Number: 186003033Mobile Phase: A1: 30 mM potassium phosphate buffer (pH 2) A2: 30 mM potassium phosphate buffer (pH 7) A3: 30 mM potassium phosphate buffer (pH 12)Mobile Phase B: AcetonitrileFlow Rate: 1.4 mL/minGradient: Time Profile (min) %A %B 0.0 90 10 7.0 20 80 8.0 20 80 Injection Vol.: 20 µLSample Conc.: 50 µg/mL each in 80/20 water/methanolColumn Temp.: 30 °CDetection: UV @ 210 nm (pH 2,7); 220 nm (pH 12)System: Alliance 2695 with 2996 PDA

Compounds:1. Doxylamine2. Benzamide3. Hydroxyisophthalic Acid4. Doxepine5. Flavone6. Fenoprofen

Did you know...Waters now offers Suitability Standards to help benchmark and trend analytical system performance.


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XBridge Amide Columns for Sugar AnalysisXBridge Amide columns utilize a chemically stable, trifunctionally-bonded amide phase, enabling a new dimension in stability and versatility for HILIC separations. In addition to enhanced retention of highly polar compounds, these columns are equally suited for sugar analysis. Some of the benefits are:

Enables high resolution, high speed analysis of carbohydrates in complex sample matrices while maintaining or improving chromatographic resolution.

Increased chemical stability enables the use of high pH and high temperature to collapse anomers without the loss of reducing sugars.

BEH particle technology, in combination with a trifunctionally-bonded amide phase, provides exceptional column lifetime, thus improving assay robustness.

Unlike amine-based columns used for carbohydrate analysis, the XBridge Amide column is not susceptible to Schiff-base formation, thus improving quantitation accuracy.

XBridge Amide Analysis of Saccharides

20 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 min

2

1

3

4

5

6

7

8

9

Column: XBridge BEH Amide, 4.6 x 150 mm, 3.5 µm Part Number: 186004869Mobile Phase A: 30/70 acetonitrile/water with 0.10% ammonium hydroxide Mobile Phase B: 80/20 acetonitrile/water with 0.10% ammonium hydroxide Flow Rate: 0.4 mL/min Gradient: Time Profile (min) %A %B Initial 0 100 30.6 60 40 30.7 0 100 Inj. Vol.: 10 µL Sample Diluent: 50/50 acetonitrile/waterColumn Temp.: 35 °C System: ACQUITY UPLC with ACQUITY UPLC TQDSIR m/z: 179.2 (fructose, glucose); 341.3 (sucrose, maltose); 503.4, 665.5, 827.6, 989.7, 115 (maltooligosaccharides [n=1 to 5])

Compounds:1. Fructose 10 µg/mL2. Glucose 10 µg/mL3. Sucrose 10 µg/mL4. Maltose 10 µg/mL 5. Maltotriose 10 µg/mL6. Maltotetraose 10 µg/mL7. Maltopentaose 10 µg/mL8. Maltohexahose 10 µg/mL9. Maltoheptaose 10 µg/mL

V0 =0.26

V0 =0.25 Reversed PhaseXBridge C18

HILICXBridge HILIC

1

1

2

2

0 1 2 3 4 5 6 min

3

3

1. 6-acetyl morphine

HO

O

O

NCH3

H

O

3. morphine 3-ß-D-glucuronide

O

O

HO

H

O

HO HO

OHOHO

NCH3

HO

O

HO

HNCH3

2. morphine

Due to the multi-modal retention mechanisms in HILIC, an orthogonal selectivity is observed compared to reversed phase, where the glucuronide metabolite of morphine elutes after morphine under HILIC conditions.

XBridge HILIC Columns for Orthogonal SelectivityHILIC techniques are gaining popularity as part of HPLC method development strategies. As a complement to traditional reversed-phased chromatography, XBridge HILIC columns separations not only retain highly polar compounds but can provide large selectivity differences where it is possible to have complete reversal of elution order compared to a reversed-phase separation.



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http://www.waters.com/waters/partDetail.htm?partNumber=186004869

Sunfire ColumnsSunFire columns set the standard for the state-of-the-art bonded C18 and C8 silica HPLC columns. Benefiting from years of research and product development, these columns represent the best in particle and bonding expertise and deliver an industry-leading level of chromatographic performance.


Chemistry Particle SizeParticle Shape

Pore Size

Carbon Load

End Capped

C18 2.5, 3.5, 5, 10 µm Spherical 100Å 16% Yes

C8 2.5, 3.5, 5, 10 µm Spherical 100Å 12% Yes

Silica 5, 10 µm Spherical 100Å Unbonded n/a

50

60

70

80

90

100

0 20 40 60 80

SunFire C18

Phenomenex® Luna® C18 (2)

ACT® Ace® C18

GL Sciences® Inertsil® ODS-3

Hours in 1% TFA in H2O at 80 ˚C

% o

f Ini

tial R

eten

tion

Tim

e

Excellent Low pH Stability

Stability at low pH improves SunFire column lifetimes compared to those of other silica-based HPLC column brands.

Superior Peak Shape and Efficiency

With the bonding and end capping technologies for the SunFire columns, Waters developed a sorbent with superior peak shape performance. SunFire columns provide symmetrical peaks for improved resolution and quantitation of acidic, neutral, and basic compounds at low- and intermediate-pH ranges.

0

12

3

4

5 6

5 10 15 20 25 30 35 40 min

Isocratic SeparationMobile Phase A: 35% 20 mM dipotassium phosphate/ 20 mM monopotassium phosphate pH 7.0 Mobile Phase B: 65% methanolWavelength: 254 nmFlow Rate: 1.0 mL/minInjection Vol.: 14 µLColumn Temp.: 23 ˚CSystem: Alliance 2695 with 2487 Dual Wavelength Absorbance detector

Compounds:1. Uracil2. Propranolol3. Butylparaben4. Naphthalene5. Acenaphthene6. Amitriptyline

TUSP – 1.26

TUSP – 1.35

TUSP – 1.79

SunFire C18 4.6 x 150 mm, 5 µmPart Number: 186002559

Phenomenex® Luna® C18 (2) 4.6 x 150 mm, 5 µm

ACT® Ace® C18 4.6 x 150 mm, 5 µm

Comparison of C18 5 μm HPLC Columns

1.0 1.5 2.0 2.5 3.0 3.5 4.0

Agilent Zorbax® Eclipse® XDB C18

Agilent® Purosphere® RP-18E

ACT® Ace® C18

YMC-Pro® C18

GL Sciences® Inertsil® ODS-3

Thermo Scientific® HyPURITY™ Elite C18

Phenomenex® Luna® C18 (2)

SunFire C18

USP Tailing Factor (TUSP)

In this comparison test of columns packed with 5 μm particles, the SunFire C18 column has the lowest USP tailing factor for amitriptyline using pH 7 mobile-phase conditions.Comparative separations may not be representative of all applications.



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Batch-to-Batch Reproducibility

In establishing new analytical methods for the latest pharmaceutical and biopharmaceutical products, the selection of a reproducible HPLC column is essential. The selected column needs to provide the same chromatographic results over the life of the method and the new drug product. SunFire columns have demonstrated superior reproducibility over many years. Batches randomly selected over a four-year period show excellent reproducibility in the example below.

0 5 10 15 20 25 30 35 40 min

Batch 112 (2005)

Batch 118 (2007)

Batch 130 (2007)

Batch 134 (2008)

This excellent reproducibility is a result of our commitment to maintaining the tightest specifications in the HPLC column industry. SunFire columns start with high purity raw materials and are produced using tightly controlled manufacturing processes and column packing procedures that provide today’s scientists with the best, most reproducible HPLC columns available.

Did you know...Waters Suitability Standards can help you benchmark your system performance and help in trending results.


The recommended column cleaning and regenerating procedures are listed in the Care and Use Instructions.

For brand-specific care and use instructions, visit www.waters.com/chemcu

Did you know...

Check out our latest discounts for online shopping.

Find out pricing and product availability quickly and easily.

Set up wish lists for important, upcoming projects.

eMail your cart or wish list to other project or purchasing colleagues.

Ordering online has never been easier or more secure! Go to www.waters.com/order and you can:

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https://www.waters.com/waters/home.htmhttps://www.waters.com/waters/home.htm

Atlantis ColumnsThe Atlantis family of HPLC columns was created to solve one of the most challenging chromatographic problems, retaining polar compounds. Through historical experience and synthetic innovation, Atlantis columns lead the industry in providing exceptional performance, versatility, retention and chromatographic stability for polar compounds, while also affording balanced retention for broad analyte mixtures.

For retention and separation of polar compounds via reversed-phase HPLC, Atlantis T3 columns set the standard for polar compound retention. Atlantis T3 columns are a universal, silica-based, reversed-phase C18 line of HPLC columns that not only retain and separate small, water-soluble polar organic compounds, but also provide superior performance across a wider pH range. Atlantis columns are the culmination of over 30 years of bonded phase research and are a vast improvement upon the highly successful Atlantis dC18 line of columns. For reversed-phase applications, Atlantis T3 columns should be considered the first choice when developing a separation of polar (and non-polar) compounds. The advantages of T3 bonding technology is extended to UPLC in the ACQUITY UPLC HSS T3 columns.

Long Column Lifetimes at Low pH

The creation of highly retentive, reversed-phase C18 columns for polar compound retention involves bonding at a ligand density that is less hydrophobic and, therefore, more compatible with the weak, highly aqueous mobile phases required for retaining polar compounds. When exposed to strongly acidic mobile phases (i.e. < pH 2.0), traditional C18 columns can exhibit bonded phase hydrolysis resulting in gradual loss of retention, loss of efficiency as well as change in selectivity.

Atlantis T3 columns resist ligand cleavage by utilizing a trifunctional attachment of the C18 phase to the particle surface, thus providing exceptional column life at low pH.

Improved pH 7 Performance

At pH 7, poor peak shape for amine-containing bases and shortened column lifetimes are encountered when using intermediate ligand density C18 columns designed for polar compound retention. Poor peak shape is due to secondary interactions with unreacted silanols that remain present after bonding and endcapping. The proprietary T3 endcapping procedure reacts with more of these active silanols thereby dramatically improving peak shape for bases. Shortened column lifetimes are due to the dissolution of the silica particle substrate by the high-pH mobile phase. The more effective and efficient T3 bonding helps ‘protect’ the silica substrate from dissolution, thus providing longer column lifetimes.

Superior Low pH Stability

0 10 20 30 40 50 60 70%

Atlantis T3

SunFire C18

Phenomenex® Synergi™ Hydro-RP

Shiseido® Capcell Pak® C18 AQ®

Phenomenex Synergi Polar-RP

Agilent® Zorbax® Eclipse® Plus

Atlantis dC18

Phenomenex Aqua®

GL Sciences® Inertsil® ODS-SP

% Loss of Retention for Methyl Paraben

20 hr Exposure to 0.5% TFA at 60 ˚C

T3 bonding allows Atlantis T3 columns to provide long column lifetimes under harsh, low pH conditions. Comparative separations may not be representative of all applications.

Improved Peak Shape for Bases at pH 7

1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Thermo® Aquasil® C18

Agilent® Zorbax® SB-Aq

Atlantis dC18

Phenomenex Aqua®

Phenomenex® Synergi™ Hydro-RP

Phenomenex Synergi Polar-RP

Shiseido® Capcell PAK® C18 AQ®GL Sciences® Inertsil® ODS-SP

Atlantis T3

Amitriptyline USP Tailing Factor

Did not elute

When compared to other reversed-phase ‘polar compound retention columns’, Atlantis T3 columns provide superior pH 7 peak shape.Comparative separations may not be representative of all applications.


Intended Use HPLCParticle Type Atlantis SilicaAvailable Chemistries T3, dC18, HILIC SilicapH Range T3: 2–8; dC18: 3–7; HILIC Silica: 1–5Maximum Rated Pressure 6000 psi [∼400 bar]Particle Size 3, 5, 10 μmPore Diameter / Volume 100Å/1.0 mL/gSurface Area 330 m2/g


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Guide to Retaining Polar Compounds

Is the polar compound an acid, neutral

or base?

Can it beretained by

reversed-phase?

XSelect HSS T3,Atlantis T3

orXBridge C18

Can it beretained by

reversed-phase?

Atlantis T3or

XSelect HSS T3

Sufficient massspectrometrysensitivity?

Insufficientretention

Alternate separationtechnique, such as

ion-pair orion-exchange

Atlantis HILIC Silica,XBridge HILIC

or XBridge Amide

Base

Yes

Yes

Yes

No

Yes

Acid or Neutral

No No

Key:

Atlantis T3 = low pH (pH 3), high aqueous (>95%)

XBridge C18 = high pH (pH 10), high aqueous (>95%)

Atlantis HILIC Silica = low pH (pH 3), 10 mM ammonium formate with 0.2% formic acid in 95% acetonitrile/5% water

XBridge HILIC = mid pH (pH 5), 10 mM ammonium acetate with 0.02% acetic acid in 95% acetonitrile/5% water

XBridge Amide = mid pH (pH 5), 10 mM ammonium acetate with 0.02% acetic acid in 95% acetonitrile/5% water

XSelect HSS T3 = low pH (pH 3), high aqueous (>95%)

Atlantis HILIC Silica

HILIC is an alternative chromatographic technique that offers complementary selectivity to RP and often retains very polar species that cannot be retained by traditional means. Atlantis HILIC Silica columns were designed to retain very polar, organic molecules that are too polar to retain by RP.1 Unlike the highly aqueous mobile phases required for polar retention in RP separations, Atlantis HILIC Silica columns employ highly volatile (>80% organic) mobile phases which are ideal for mass spectrometry (MS) response and sensitivity. Additionally, direct compatibility with high organic SPE eluates dramatically increases the number of samples that can be handled, thus substantially increasing sample throughput.

HILIC retention mechanisms are a complex combination of partitioning, ion-exchange and hydrogen bonding, resulting in enhanced retention for polar analytes.1,2 Atlantis HILIC Silica columns, used in combination with high organic (>80% acetonitrile) mobile phases, result in retention of analytes that are simply too polar to retain by traditional RP chromatography. 1 E.S. Grumbach et al LCGC N. Am. 2004, 22, 1010-10232 E.S. Grumbach et al J. Sep. Sci. 2008, 31, 1511-1518

Retention Mechanisms in Hydrophilic-Interaction Chromatography (HILIC)

O-

SiO O

OO

SiO

O-HO

SiO

O-

OSi

O

OO

Si

OSi

O

O-H

SiO-

O

Si

OO

Si

Si

Si O-H

O O

Si OO

Si

SiSi

Si

Si

Si

SiO-

Si

SiO

O

SiO

O

Si O

H-O O

SiSi

Si

SiOO-

O

Si

SiO

H-OO

Si

O

O-

H-O

Si

Si

OSi

O

O-

O

Si

Si

O-H

O O

O

SiO-

OO

Si

Si

Si O-H

OO

SiO

OSi O-

O

Si

Si

SiSi

90% ACN90% ACN

H2OH2O

OH

H

O HH

OH

H

OHH

OH H

OH

H

OH

H

OH H

Water

Acetonitrile/Water (90:10)

N

NH3+

NH

O

N

NH 3+

NH

O

NN H3

+

NH

O

NN H3

+

NH

O

H2OH2O ACN/H2O(90:10)ACN/H2O(90:10)

The combination of partitioning and weak cation exchange results in retention of polar bases with Atlantis HILIC Silica columns.


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XTerra ColumnsSilica-based packing materials limit the chromatographic potential for separation scientists. Restrictions on speed, resolution, pH, temperature, and loading capacity are imposed upon the chromatographer by limitations of the stationary phase material. The patented* Hybrid Particle Technology of XTerra columns allows chromatographers to break these boundaries and realize the full potential of their analytical and preparative separations.* Patent # 6,686,035 B2

Hybrid Particle Technology

In Hybrid Particle Technology, one out of every three silanols is replaced with a methyl group during synthesis. This hydrophobicity is distributed throughout the entire structure of the particle backbone. The result is a rugged hybrid (inorganic/organic) particle that can be operated at high speeds, high temperatures, and high pH. The presence of 33% fewer residual silanols (after endcapping and bonding) also means that XTerra columns give exceptionally sharp, high-efficiency peaks for basic compounds.

Effect of XTerra Column Chemistry on Selectivity and Retention

2

1

5 10 15 20 25 min

4

5

6

3

2

1

4

5

6

3

21

4

5

6

O Si

C H 3

C H 2 C H

C H 3

O

O Si

O

O

O Si

C H 3

C H3

3

XTerra MS C8 Part Number: 186000490

XTerra RP8 Part Number: 186000493

XTerra Phenyl Part Number: 186001146

Column: 4.6 x 150 mm, 5 µmMobile Phase A: WaterMobile Phase B: MethanolMobile Phase C: 50 mM formic acid, pH 2.45Gradient: Time Profile (min) %A %B %C 0.0 40 50 10 20.0 25 65 10 35.0 25 65 10

Flow Rate: 1.0 mL/minInjection Vol.: 15 mLColumn Temp.: 30 °CDetection: UV @ 254 nmSystem: Alliance 2695 with 996 PDA

The column stationary phase chemistries of XTerra columns are key components in an overall method development plan.

Compounds: 1. Suprofen2. Tolmetin3. Naproxen4. Fenoprofen5. Ibuprofen6. Diclofenac


PackingParticle Shape

Particle Size(s)Pore Size

Carbon Load

End- capped

XTerra MS C18 Spherical 2.5, 3.5, 5, 10 µm 125Å 15.5% Yes

XTerra MS C8 Spherical 2.5, 3.5, 5, 10 µm 125Å 12.0% Yes

XTerra RP18 Spherical 3.5, 5, 10 µm 125Å 15.5% Yes

XTerra RP8 Spherical 3.5, 5, 10 µm 125Å 13.5% Yes

XTerra Phenyl Spherical 3.5, 5 µm 125Å 12.0% Yes

Did you know...Guard columns can help extend the life of your analytical column.

See page 147 for product information.


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http://www.waters.com/waters/partDetail.htm?partNumber=186000490http://www.waters.com/waters/partDetail.htm?partNumber=186000493http://www.waters.com/waters/partDetail.htm?partNumber=186001146http://www.waters.com/waters/nav.htm?cid=560002&alias=Alias_order_CO&lset=1&locale=en_US

Symmetry ColumnsAs chemists develop analytical methods for pharmaceutical and biopharmaceutical products, the selection of a reproducible HPLC column is essential. T he selected column needs to provide the same chromatographic results over the life of the new drug product. Symmetry columns demonstrate superior reproducibility over many years.

Reproducibility is our number one priority in supplying Symmetry columns. This excellent reproducibility is a result of our commitment to maintaining the tightest specifications in the HPLC column industry. Symmetry columns are engineered with high purity raw materials, tightly controlled manufacturing processes and column packing procedures that provide scientists with the best, most reproducible HPLC column available.

Our goal is to supply a family of HPLC columns that you can rely on for rugged and robust methods. Symmetry columns let you increase your laboratory’s productivity, and allow easier method transfer between labs and around the globe.


Packing Chemistry Particle Size(s) Particle Shape Pore Size Carbon Load End Capped

SymmetryC18 3.5, 5, 7 μm Spherical 100Å 19% Yes

C8 3.5, 5, 7 μm Spherical 100Å 12% Yes

SymmetryShieldRP18 3.5, 5, 7 µm Spherical 100Å 17% Yes

RP8 3.5, 5, 7 µm Spherical 100Å 15% Yes

The Benefit of Narrow Specification Ranges

Symmetry columns have the narrowest ligand surface coverage specifications in the industry. Surface coverage (ligand density) is one of the most important parameters affecting reproducibility. The impact of a small variation in surface coverage on the amitriptyline/acenaphthene alpha specification for Symmetry C18 columns is shown here. Narrow column specifications benefit the chromatographer in achieving reproducible results. The surface coverage requirements of Symmetry are the tightest specifications in the industry, resulting in minimal shifts in resolution and more reproducible batch-to-batch and column-to-column results.

1.5 2.0 2.5 3.0 3.5 4.00.8

1.0

1.2

1.4

1.6

1.8

2.0

Narrow Specification Ranges Provide Smaller Shifts in Alpha (α)

C18 Ligand Surface Coverage Specification Ranges

Specification Ranges for Symmetry C18

Minimal Changes in Resolution Means More Reproducible Results*

C18 Surface Coverage (µmol/m2)

Alph

a fo

r Am

itrip

tylin

e/A

cena

phth

ene

1.5 2.0 2.5 3.0 3.5 4.0

2.75

2.6

3.09 3.31

3.75

Symmetry C18

Column X

Column Y

C18 Surface Coverage (µmol/m2)

no upper limit

0.814.70

2.864.14

5.37 0

* Based on vendor’s column product specifications for ligand density

Specification X

Specification Y

Symmetry C18

Maximum Resolution Minimum Resolution

Smaller Shifts in Alpha (α) Reduce Changes in Peak Resolution

R = resolutionN = number of peaksα = alpha valueK = peak retention

R = ( √N ) ( α-1 ) ( K2 )4 α K2+1


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A three-page certificate of analysis is included with every Symmetry column. We report both our specifications, which are the tightest in the industry, and the results of the 22 critical tests each column must pass before it carries the Symmetry brand.

Long Lifetime

Columns that last longer save you money. Symmetry columns can last over 10,000 injections with minimal loss in efficiency, minimal increase in backpressure, and minimal change in retention time.

These columns deliver guaranteed consistent performance optimizing the two key factors that control column lifetime which are hydrolytic stability of the packing material and mechanical stability of the packed bed.

Comprehensive Certificate of Analysis

Outstanding Packed Bed Stability—10,000 Injections

0 2 4 6 8 10 min

1

2

3

45

6

The long-term mechanical stability of Symmetry columns is demonstrated in this analysis of sulfa drugs. Over 10,000 injections were run with minimal loss in column efficiency. The use of the Sentry guard column is critical to achieving these types of column lifetimes.

Injection 10,000

Injection 1

Column: Symmetry C8, 3.9 mm x 150 mm, 5 µm, with Sentry Guard Column, 3.9 mm x 20 mm, 5 µmPart Numbers: WAT046970 (column) & WAT054250 (guard column)Mobile Phase: Water/methanol/glacial acetic acid 79:20:1Flow Rate: 1.0 mL/minuteInjection Volume: 10 µLTemperature: 25 °CDetection: UV @ 254

Compounds: 1. Sulfanilamide2. Sulfadiazine3. Sulfathiazole4. Sulfamerazine5. Sulfamethazine6. Succinylsulfathiazole

1.2

1.5

1.66

1.75

1.78

1.9

1.95

2.39

3.6

0 1 2 3 4

In this comparison test, SymmetryShield RP18 has the lowest USP tailing factor for the basic probe amitriptyline under the most demanding pH 7 mobile-phase conditions.

USP Tailing Factor

SymmetryShield RP18

Brand L

Brand HP

Brand HE

Brand DO

Brand DA

Brand I

Brand P

Brand E

Industry Leader for Peak Shape of Basic Compounds

SymmetryShield Columns

Excellent Peak Shape

SymmetryShield HPLC columns are an excellent choice to achie

Documents

Maximize HPLC - Waters Corporation · 2013-01-15 · HPLC Columns: Theory, Technology, and Practice Uwe D. Neue, Waters Corporation, Milford, MA, published by John Wiley & Sons High-performance