BIOSEPARATIONS AND ANALYSES - Waters Corporation · ACQUITY UPLC SEC System Solution ... Specifically QC tested with tryptic digests of cytochrome ... Six peptides ranging in mass

233www.waters.com/biosep

Bioseparations

BIOSEPARATIONS AND ANALYSES

234 www.waters.com/order

TABLE OF CONT ENTSIntroduction ...............................................................................235

Bioseparations and Analyses ..................................................235

Peptides .....................................................................................236BEH C18, 130Å and 300Å, and CSH C18, 130Å Columns ...236CSH C18, 130Å Columns for Peptide Separations ................ 237Benchmarking, Method Development, and Troubleshooting: Cytochrome c Digestion Standard .............. 243Cation-Exchange Peptide and Polypeptide Separations ......... 245Therapeutic Peptide Method Development Kit........................ 245BioSuite HPLC Peptide Analysis Columns .............................. 246BioSuite Cation-Exchange HPLC Columns .............................. 247Benchmarking, Method Development, and Troubleshooting: MassPREP Peptide Standard ....................... 247Additional Peptide Consumables ............................................248RapiGest SF Protein Digestion Surfactant ............................... 249Waters Insulin HMW P HPLC Column .......................................250Delta-Pak HPLC Columns .........................................................250Symmetry HPLC Columns ........................................................ 251

Proteins ...................................................................................... 252ACQUITY UPLC SEC System Solution ..................................... 252XBridge Protein BEH SEC, 125Å, 200Å, and 450Å Columns and Protein Standard Test Mixtures ..............258Benchmarking, Method Development, and Troubleshooting: BEH SEC Protein Standards .........................258Protein Separations ..................................................................260Benchmarking, Method Development, and Troubleshooting: MassPREP Protein Standard Mix ..........263Protein-Pak Hi Res Ion-Exchange (IEX) Columns for ACQUITY UPLC Applications .............................................264Protein-Pak Hi Res HIC Column and HIC Protein Standard .....266BioSuite Ion-Exchange HPLC Columns .................................... 267Benchmarking, Method Development, and Troubleshooting: Ion Exchange Standards .......................268Protein-Pak PW Series Columns ...............................................268Protein-Pak High Resolution (HR) Ion-Exchange Glass Columns ...................................................269Advanced Purification (AP) Glass Columns ............................ 270Accell Plus Ion-Exchange Packings ......................................... 272Accell Plus Sep-Pak Cartridges ................................................ 272Accell Plus PrepPak Cartridges (47 x 300 mm) .................... 272Accell Plus Ion-Exchange Bulk Packings ................................. 272Ion-Exchange Sample Preparation with Sep-Pak Cartridges .. 272BioSuite Size-Exclusion HPLC Columns (SEC) ......................... 273Protein-Pak and Shodex Size-Exclusion HPLC Columns ......... 275Ultrahydrogel HPLC Columns .................................................. 276Symmetry300 C4 HPLC Columns ............................................ 276

Protein-Pak Affinity Columns ................................................... 277BioSuite pC18 and pPhenyl Reversed-Phase Chromatography (RPC) HPLC Columns ................................... 277BioSuite Hydrophobic-Interaction Chromatography HPLC Columns.............................................. 278Shodex and Protein-Pak Phenyl HIC HPLC Columns ............... 279

UPLC Amino Acid Analysis Solution ....................................280AccQ•Tag Amino Acid Analysis Using HPLC.........................283Pico•Tag HPLC Method .............................................................285

Oligonucleotides ......................................................................286Columns for Large DNA/RNA Species ....................................289Gen-Pak FAX Anion-Exchange Columns ..................................289MassPREP Oligonucleotide Standard ......................................290Oasis µElution Plates ...............................................................290

Glycans ...................................................................................... 291Sample Preparation ..................................................................294Additional Consumables ..........................................................295Ammonium Formate Concentrate-Glycan Analysis ...............296Waters Glyco-Pak DEAE and N Columns .................................296

MS and LC-MS Consumables for Biomolecules .................... 297MassPREP On-Line Desalting Devices ..................................... 297Enzymate Online Digestion Column .......................................298Capillary, Nano and Trapping Columns ..................................299LC-MS Kits for the nanoACQUITY UPLC System with 2D Technology ................................................................. 301nanoACQUITY UPLC On-line SCX/RP 2D Separations Kit .... 301nanoACQUITY UPLC On-line RP/RP 2D Separations Kit ......302nanoACQUITY UPLC Columns and Trapping Columns ...........302ACQUITY UPLC M-Class Nano and Capillary Columns and Trapping Columns..............................................................303ionKey/MS ................................................................................305Waters Protein Expression System Kits ...................................306MassPREP Digestion Standard Mixtures ................................306Hi3 Standards...........................................................................307Heavy Labeled Quantitative Standards ...................................307TruView LCMS Certified Vials ...................................................308Waters Certified Containers .....................................................308pH Buffers .................................................................................308


Bioseparations

INTRODUCTION

Bioseparations and Analyses

Innovative Technologies From the Leader in Separation Science and Analytical Biochemistry

Advances in the areas of genomics, proteomics, metabolomics, and molecular and system biology continue to revolutionize the diagnosis and treatment of diseases and increase our fundamental understanding of biological processes.

As a leading analytical supplier of instrumentation, software, service and support, and chemistry products, Waters is uniquely positioned to provide researchers the tools, technologies, and integrated solutions desired to tackle the formidable challenges involving various biomolecules. Beginning with a keen understanding of today’s biomolecule-related challenges, Waters scientists and engineers continuously seek purposeful innovations that help deliver impactful solutions in applications ranging from proteomics and biomarker discovery through the commercialization of advanced biopharmaceuticals. We continue to develop new, innovative columns and sample preparation consumables that support the HPLC, UPLC, and LC-MS analyses of peptides, oligonucleotides, proteins, amino acids, and glycans.

Waters comprehensive chemistry and consumables family includes:

� Peptide columns for nano, capillary, analytical, and preparative peptide applications

� Protein size-exclusion, ion-exchange, and reversed-phase columns for analytical UPLC, HPLC, and lab-scale purification applications

� AccQ•Tag Ultra chemistry specific for Waters UPLC Amino Acid Analysis Solution, as well as Pico•Tag and AccQ•Tag for HPLC-based amino acid analyses

� Oligonucleotide columns for synthetic oligonucleotide and DNA/RNA fragment isolations and analyses

� GlycoWorks labeling and sample preparation kits and Waters Glycan Columns for the analysis of released glycans

� Analytical Standards and Reagents consumables and kits for MS and LC-MS applications of peptides, proteins, and other biomolecules.

In addition, our ACQUITY UPLC Protein BEH SEC, 125Å, 200Å, and 450Å guards, columns, and quality controlled protein/peptide standards, as well as our Protein-Pak Hi Res IEX offerings were developed for use on ACQUITY UPLC Systems to help obtain accurate, precise, and highly resolving quantitative analysis of therapeutic proteins such as mAbs.


PEPTIDES

BEH C18, 130Å and 300Å, and CSH C18, 130Å ColumnsThe ability to reproducibly generate high quality peptide separations is of critical importance in proteomics research as well as for the characterization of biopharmaceuticals via peptide mapping. Reversed-phase (RP) chromatography has become the separation mode of choice as it offers relatively high resolving power and provides outstanding quantitative (UV) and qualitative (ESI-MS) information. Waters bridged ethylene hybrid (BEH Technology) and Charged Surface Hybrid (CSH Technology) Peptide Columns help address demanding application needs.

ACQUITY UPLC and XBridge BEH C18, 130Å and 300Å ACQUITY UPLC and XSelect CSH C18, 130ÅWaters 1st series of Hybrid

Particle Based, Peptide ColumnsWaters Newest, Charged Hybrid

Surface Particle Based, Peptide Columns

Ideally suited for separation of wide range peptides: large and small, acidic and basic, hydrophilic and hydrophobic

Outstanding peptide mass loading capability that maintains high peak capacities using either 0.1% FA- or 0.1% TFA-containing eluents

130Å and 300Å pore offerings provide different separation selectivities for a wide range of peptides and small proteins

Best suited for compounds less than 10,000 Daltons and delivers different separation selectivity than seen on BEH C18, 130Å material

Scalable 1.7 µm, 3.5 µm, and Prep 5 µm that address application needs

Scalable 1.7 µm, 3.5 µm, 2.5 µm XP, and Prep 5 μm that address application needs

Specifically QC tested with tryptic digests of cytochrome c to ensure column-to-column consistency in validated methods

Specifically QC tested with tryptic digests of cytochrome c using 0.1% FA-containing eluent to ensure column-to-column consistency in validated methods

T hree Outstanding Peptide Column Chemistries that Address Varied Peptide Separation Requirements

CSH C18, 130Å, 1.7 µm2.1 x 150 mm

2

3 4

5

6 1

BEH C18, 130Å, 1.7 µm2.1 x 150 mm

2 3

4

5

6 1

BEH C18, 300Å, 1.7 µm2.1 x 150 mm

2 3

5

6 1

4

1.2

1

1.8

0.6

0.4

0.2

0

A 214

10 15 20 25 30 35 40 45 50 min

1.2

1

1.8

0.6

0.4

0.2

0

A 214

10 15 20 25 30 35 40 45 50 min

1.2

1

1.8

0.6

0.4

0.2

0

A 214

10 15 20 25 30 35 40 45 50 min

Peptide/Protein: 1. Bradykinin, 1.1 kDa; 2. Renin Substrate, 1.8 kDa; 3. Ubiquitin, 8.6 kDa; 4. Cytochrome C (Equine), 12.4 kDa; 5. Insulin (Bovine), 5.7 kDa; 6. Melittin, 2.8 kDa

Six peptides ranging in mass from 1 to 12 kDa were separated on a CSH C18, 130Å Column as well as two BEH C18 Columns, with pore diameters of either 130Å or 300Å. Through comparison of these chromatograms, it is clear that the CSH C18, 130Å Column produced the best peak shapes for most of the peptide species, including insulin, in 0.1% FA-containing eluents. Analysis of the largest peptides, ubiquitin (8.6 kDa) and cytochrome c (12.4 kDa), helped define the effect of using 300Å versus 130Å pore size sorbents. Ubiquitin was found to exhibit only slightly better peak shape on the BEH C18, 300Å Columns versus both the CSH C18, 130Å and BEH C18, 130Å Columns. In contrast, the largest peptide, cytochrome c, was separated with markedly better peak shape using BEH C18, 300Å. The BEH C18, 300Å Column was actually capable of resolving cytochrome c into multiple peaks, indicating protein heterogeneity.


Bioseparations

Comparative Averaged Peptide Peak Capacities on Selected Reversed-Phase Columns with Differing Concentrations of FA and TFA

Effect of TFA on peak capacity and MS signal. (A) Peak capacity as a function of acid modifier. Values were derived from two replicates. (B) Fold decrease in MS peak area as a function of acid modifier. Waters MassPREP™ Peptide Standard Mixture (p/n: 186002337) was used in study.

20

70

120

170

220

270

320

370

0.00 0.05 0.10

P c,4

σ

CSH C18, 130Å, 1.7 µm

Superficially Porous Silica C18, 1.7 µm

BEH C18, 130Å, 1.7 µm

0.050.05

0.100.00

0.000.10

% TFA% FA

Porous Silica C18, 5µm

12

10

8

6

4

2

0.00 0.05 0.10

Fold

Dec

reas

e in

MS

Pea

k A

rea

0.050.05

0.100.00

0.000.10

% TFA% FA

Peak Capacity MS Signal

Six peptides ranging in mass from 1 to 12 kDa were separated on a CSH C18, 130Å column as well as two BEH C18 columns, with pore diameters of either 130Å or 300Å. Through comparison of these chromatograms, it is clear that the CSH C18, 130Å column produced the best peak shapes for most of the peptide species, including insulin, in 0.1% FA-containing eluents. Analysis of the largest peptides, ubiquitin (8.6 kDa) and cytochrome c (12.4 kDa), helped define the effect of using 300Å versus 130Å pore size sorbents. Ubiquitin was found to exhibit only slightly better peak shape on the BEH C18, 300Å columns versus both the CSH C18, 130Å and BEH C18, 130Å columns. In contrast, the largest peptide, cytochrome c, was separated with markedly better peak shape using BEH C18, 300Å. The BEH C18, 300Å column was actually capable of resolving cytochrome c into multiple peaks, indicating protein heterogeneity.

CSH C18, 130Å Columns for Peptide Separations

Charged Surface Hybrid Particles Deliver Superior Peptide Separations in LC or LC-MS Applications

Waters patented synthesis process for its Charged Surface Hybrid (CSH Technology) particles imparts a low level positive charge to the surface of each particle. CSH Technology allows the columns to be successfully used with standard TFA-containing eluents or with a weaker acid modifier such as formic acid. This means users no longer have to compromise between selecting a reversed-phase eluent that delivers needed sharp and symmetrical separated peaks (e.g., 0.1% trifluoroacetic acid [TFA]) or one that minimizes reduction of MS signal (e.g., 0.1% formic acid [FA]). In similar fashion, the ability of the CSH C18, 130Å chemistry to accept greater peptide mass loads, than on many other columns, will enhance the ability to detect potentially important low level constituents contained with the major component(s) of interest.

Superior Performance in Either FA or TFA Containing Eluents

Waters CSH C18, 130Å particles contain a low and carefully-defined concentration of positive charges that yield comparatively excellent peak shape for peptides using either FA- or TFA-containing mobile phases. The fact that the performance of a CSH C18, 130Å column exhibits little dependence on strong ion pairing agents makes it ideal for LC or LC-MS applications.


ACQUITY UPLC Peptide BEH C18, 130Å and 300Å Columns

DescriptionParticle

SizeInner

DiameterLength Part No.

ACQUITY UPLC Peptide BEH C18, 130Å 1.7 µm 2.1 mm 50 mm 186003554










ACQUITY UPLC Peptide BEH C18, 130Å and 300Å VanGuard Pre-Column and Column In-Line Filter UnitDescription Part No.

ACQUITY UPLC Peptide BEH C18, 300Å 1.7 µm, VanGuard Pre-column, 2.1 x 5 mm, 3/pk

186004629

ACQUITY UPLC Peptide BEH C18, 130Å, 1.7 µm, VanGuard Pre-column, 2.1 x 5 mm, 3/pk

186003975

In-line Filter Holder and Six 0.2 µm Stainless Steel Replacement Filters 205000343

Five 0.2 µm Stainless Steel Replacement Filters and End Nuts for 205000343

700002775

ACQUITY UPLC and XBridge Peptide BEH C18, 130Å and 300Å Methods Validation Kits*

DescriptionParticle

SizeInner






XBridge Peptide BEH C18, 130Å 3.5 µm 4.6 mm 100 mm 186004904

XBridge Peptide BEH C18, 130Å 5 µm 4.6 mm 100 mm 186005463



*Three Columns from three different batches of material

XBridge Peptide BEH C18, 130Å and 300Å HPLC Columns

DescriptionParticle

SizeInner


XBridge Peptide BEH C18, 130Å 3.5 µm 1 mm 50 mm 186003560











XBridge Peptide BEH C18, 130Å 5 µm 1 mm 50 mm 186003571























XBridge Peptide BEH C18, 300Å 5 μm 1 mm 100 mm 186003616

XBridge Peptide BEH C18, 300Å 5 μm 1 mm 150 mm 186003617

XBridge Peptide BEH C18, 300Å 5 μm 2.1 mm 50 mm 186003618









Bioseparations

ACQUITY UPLC and XSelect Peptide CSH C18, 130Å Analytical ColumnsDescription Kit Part No.1 Part No.

ACQUITY UPLC Peptide CSH C18, 130Å, 1.7 µm, 1.0 x 50 mm Column 176003061 186006933






ACQUITY UPLC Peptide CSH C18, 130Å, 1.7 µm, 2.1 x 50 mm VanGuard Column, 3/pk 176003067 186006939

ACQUITY UPLC Peptide CSH C18, 130Å, 1.7 µm, 2.1 x 150 mm Column Method Validation Kit2 176003068 186006940

XSelect Peptide CSH C18, 130Å, 2.5 µm, 2.1 x 50 mm XP Column 176003069 186006941



XSelect Peptide CSH C18, 130Å, 2.5 µm, 2.1 x 5 mm VanGuard Column, 3/pk 176003072 186006944

XSelect Peptide CSH C18, 130Å, 2.5 µm, 2.1 x 100 mm XP Column Method Validation Kit2 176003073 186006945




XSelect Peptide CSH C18, 130Å, 2.5 µm, 4.6 x 100 mm XP Column Method Validation Kit2 176003076 186006966

XSelect Peptide CSH C18, 130Å, 3.5 µm, 2.1 x 50 mm Column 176003077 186006950



XSelect Peptide CSH C18, 130Å, 3.5 µm, 2.1 x 100 mm Column Method Validation Kit2 176003080 186006953

XSelect Peptide CSH C18, 130Å, 3.5 µm, 2.1 x 10 mm Guard Column, 2/pk3 176003081 186006954




XSelect Peptide CSH C18, 130Å, 3.5 µm, 4.6 x 20 mm Guard Column, 2/pk4 176003085 186006958

XSelect Peptide CSH C18, 130Å, 3.5 µm, 4.6 x 100 mm Column Method Validation Kit2 176003086 186006959

Cytochrome c Digestion Standard – 1860063711 Kit includes CSH C18, 130Å Column plus one vial of Cytochrome c Digestion Standard, p/n: 1860063712 Each kit contains 3 columns from 3 different batches of CSH C18, 130Å material3 Requires 2.1 x 10 mm Universal Sentry Guard Holder, p/n: WAT0979584 Requires 4.6 x 20 mm Universal Sentry Guard Holder, p/n: WAT046910


XBridge Peptide BEH C18, 130Å and 300Å Preparative Columns

DescriptionParticle

SizeInner

DiameterLength

Configuration Style

Part No. DescriptionParticle

SizeInner

DiameterLength

Configuration Style

Part No.

XBridge Peptide BEH C18, 130Å

5 µm 10 mm 50 mm Column 186003582XBridge Peptide BEH C18, 300Å

5 µm 10 mm 50 mm Column 186003626



5 µm 10 mm 100 mm Column 186003627



5 µm 10 mm 150 mm Column 186003628



5 µm 10 mm 250 mm Column 186003629


5 µm 19 mm 50 mm OBD Column 186003586XBridge Peptide BEH C18, 300Å

5 µm 19 mm 50 mm OBD Column 186003630








10 µm 4.6 mm 50 mm Column 186003648XBridge Peptide BEH C18, 300Å

10 µm 4.6 mm 50 mm Column 186003663



10 µm 4.6 mm 100 mm Column 186003664



10 µm 4.6 mm 150 mm Column 186003665



10 µm 4.6 mm 250 mm Column 186003666



10 µm 10 mm 50 mm Column 186003667



10 µm 10 mm 100 mm Column 186003668



10 µm 10 mm 150 mm Column 186003669



10 µm 10 mm 250 mm Column 186003670























Bioseparations

XSelect Peptide CSH C18, 130Å Preparative ColumnsDescription Part No. Description Part No.

XSelect Peptide CSH C18, 130Å, 5 µm, 4.6 x 50 mm Column1 186007076 XSelect Peptide CSH C18, 130Å, 5 µm, 30 x 50 mm OBD Column 186007026



XSelect Peptide CSH C18, 130Å, 5 µm, 10 x 50 mm Column 186007018 XSelect Peptide CSH C18, 130Å, 5 µm, 30 x 250 mm OBD Column 186007024




XSelect Peptide CSH C18, 130Å, 5 µm, 10 x 10 mm Guard(f) 186007015 XSelect Peptide CSH C18, 130Å, 5 µm, 50 x 250 mm OBD Column 186007029

10 x 10 mm Guard Column Holder(f) 289000779

Replacement O-ring 10 mm(f) 700001436

XSelect Peptide CSH C18, 130Å, 5 µm, 19 x 50 mm OBD Column 186007022




XSelect Peptide CSH C18, 130Å, 5 µm, 19 x 10 mm Guard(g) 186007019

19 x 10 mm Guard Column Holder(g) 186000709

Replacement O-ring 19 mm(g) 7000010201 For use in developing lab-scale preparative chromatographyf Requires 10 x 10 mm Prep Guard Holder, p/n: 289000779g Requires 19 x 10 mm Prep Guard Holder, p/n: 186000709

XBridge Peptide BEH C18, 130Å and 300Å Preparative Guard ColumnsDescription Particle Size Inner Diameter Length Configuration Style Part No.

XBridge Peptide BEH C18, 130Å 5 µm 10 mm 10 mm Guard Column 186004469








Purification and Isolation Cartridge HoldersDescription Qty. Part No.

10 x 10 mm Cartridge Holder — 289000779

19 x 10 mm Cartridge Holder — 186000709

Replacement O-ring 7.8 mm 2/pk 700001019

Replacement O-ring 10 mm 2/pk 700001436

Replacement O-ring 19 mm 2/pk 700001020


Excellent Mass Loading of Complex Peptide Samples

One of the inherent performance advantages of CSH Technology is improved sample mass loadability—how much analyte can be loaded onto a column before peak shape deteriorates. Clearly, at typical mass loads, CSH C18, 130Å delivers remarkably better performance than many existing C18 offerings. When loading 10x less sample, the difference in performance was less pronounced. Improved peptide mass loadability is an excellent column asset for challenging separations, particularly those that involve mixtures comprised of species present at vastly different concentrations.

Comparative Averaged Peptide Peak Capacities on CSH C18, 130Å vs. BEH C18, 130Å-Based, Columns (2.1 x 150 mm) at Two Peptide Mass Loads and Differing Concentrations of FA and TFA

0.050.05

0.000.10

0.100.00

Low Mass Load

A

0.050.05

0.000.10

0.100.00

% TFA% FA

Typical Mass Load

CSH C18, 130ÅBEH C18, 130Å

B

Pea

k C

apac

ity (4

σ)

BEH C18, 130ÅCSH C18, 130Å

200

250

300

350

400

450

Effect of Column Mass Load on Separated Peptide Peak Capacity in FA, TFA, and FA/TFA eluent blends (A) approx. 0.06 μg peptide mixture sample load. (B) approx. 0.6 μg peptide mixture. Values were derived from two replicates. Waters MassPREP Peptide Standard Mixture (p/n: 186002337) was used in study.

Scalable Column Offerings for HPLC or UPLC Based Separations

There remains a need for columns that are compatible with HPLC instrumentation. Low dispersion LC instrumentation is recommended in order to get full performance from a well-packed column containing 1.7 μm particles. The recent introduction of Waters eXtended Performance [XP] Columns packed with 2.5 μm XP particles has allowed scientists to improve their existing HPLC instrumentation productivity. The high peak capacity peptide separations obtained with a CSH C18, 130Å, 1.7 μm Column could, for instance, be successfully scaled to a CSH C18, 130Å, 2.5 μm XP Column by correct scaling of the flow rate and the gradient time. As shown below, CSH Technology can be readily employed for high peak capacity peptide separations using either UPLC or HPLC instrumentation.

Comparative Separation of MassPREP Peptide Standard Mixture on ACQUITY UPLC CSH C18, 130Å, 1.7 μm vs. XSelect Peptide CSH C18, 130Å, 2.5 μm XP offering in 0.1% FA- or 0.1% TFA-Containing Eluents

0.0

0.2

0.4

0.6

0.8

1.0

10 20 30 40 50 min

A21

4

0.0

0.2

0.4

0.6

0.8

1.0

14.5 24.5 34.5 54.544.5 64.5 74.5 min

A21

4

A

0.0

0.2

0.4

0.6

0.8

1.0

10 20 30 40 50 min

A21

4

B

0.0

0.2

0.4

0.6

0.8

1.0

13.5 23.5 33.5 43.5 53.5 63.5 73.5 minA

214

CSH C18 XP, 130Å, 2.5 µm0.1% FA

CSH C18 XP, 130Å, 2.5 µm 0.1% TFA

CSH C18, 130Å, 1.7 µm 0.1% FA

CSH C18, 130Å, 1.7 µm 0.1% TFA

2 3

4 5 6

7

9

8

2 3

4 5 6 7

9

8

2 3

4 5 6 7

9

8

1

2 3

4 5 6 7

9

8

1

Chromatograms of Waters MassPREP Peptide Standard Mixture (p/n: 186002337) obtained with (A) 0.1% FA and (B) 0.1% TFA mobile phases. The method for the CSH C18 XP, 130Å, 2.5 μm Column was scaled from the method for the CSH C18, 130Å, 1.7 μm column by decreasing flow rate and increasing gradient time by a factor of 1.5. Backpressure on the XSelect CSH C18 XP, 130Å, 2.5 μm, 2.1 x 150 mm Column was 3,000 psi while a backpressure value of 8,000 psi was generated on the ACQUITY UPLC CSH C18, 130Å, 1.7 μm. 2.1 x 150 mm offering.


Bioseparations

Anal. Chem. 2003, 75, 6781-6788

Tetraethoxysilane(TEOS)

Polyethoxysilane(BPEOS)

Bis(triethoxysilyl)ethane(BTEE)

+4

Si

EtO

EtO OEtEtO

Si

EtOEtO

EtO

Si

OEt

OEt

OEt

Si

EtO

O

CH2

CH2

CH2

Si O Si

EtO

OEt

Si

O

O

OEt

Si

O

SiOO

Et

OOO

Et

Et Et n

*Patent No. 6,686,035 B2

CH2

Hybrid-Based Particles for Reversed-Phase Peptide Separations

In 1999, Waters first demonstrated how organic/inorganic Hybrid Particle Technology columns successfully addressed limitations (e.g., pH stability) that exist using 100% silica-based, reversed-phase columns for biocompound separations. In 2009, we advanced LC-based peptide separation capabilities by commercializing our BEH C18, 130Å, and BEH C18, 300Å UPLC- and HPLC-based columns both based on the second-generation BEH particles. In addition, we added an additional quality control test, using a tryptic digest of cytochrome c, to help ensure consistent column-to-column performance. To date, hundreds of referenced journal citations provide data that support the effective use of this column chemistry for a variety of separations in various diverse application areas.

The BEH Particle: First Key Chemistry Enabler of Waters UPLC Technology

CSH Technology Particles for Peptide Separations

Waters innovative, CSH C18, 130Å offerings expands on the already successful and well recognized BEH C18, 130Å and BEH C18, 300Å columns. Based on comparative peptide separations, CSH C18, 130Å Columns exhibit improved load ability, greater peak capacities, and unique selectivity compared to BEH C18, 130Å. Its performance is also significantly less dependent on TFA ion pairing, making it ideal for MS applications where high sensitivity is desired. The use of the well controlled, charged surface hybrid properties of CSH C18, 130Å holds significant promise for facilitating either challenging LC and/or LC-MS peptide separations.

Increased Assurance with Waters Peptide Columns

Each of Waters “100% manufactured” batches of BEH C18, 130Å, BEH C18, 300Å, and CSH C18, 130Å Columns are rigorously tested using a gradient separation of a tryptic digest of cytochrome c before being QC accepted as a Waters Peptide Column. As shown, the available Certificates of Analyses document contains discerning physical, chemical, and chromatographic test results to ensure batch to batch performance for research or validated method needs.

Certificate of Analysis information includes a labeled chromatogram of the gradient separation of a tryptic digest of bovine cytochrome c (p/n: 186006371) using 0.1% FA-containing eluents. The same protein digest test mixture can be purchased by customers to ensure CSH C18, 130Å Column performance.

Benchmarking, Method Development, and Troubleshooting: Cytochrome c Digestion StandardThe Cytochrome c Digestion Standard was prepared by digesting Bovine Heart Cytochrome c (Uniprot #P62894) with sequencing grade trypsin. This standard is recommended for benchmarking system performance and is also used for column QC.

T he CSH Particle: Expands Upon BEH Technology

Description Part No.

Cytochrome c Digestion Standard 186006371


Simplifying Column Choice for Peptide Purifications

A benefit of Waters Peptide Columns is the frequent ability to use a single, C18-based chemistry to separate a wide range of peptides as indicated in analytical separations on page 243. This commonly translates into less time and expense before an appropriate peptide purifcation separation is obtained. Peptide packings are available in a wide range of particle sizes and column dimensions, all with the same, consistent chemistry for various needs as indicated in accompanying preparative column selection guide.

Separation of 13 Residue Peptide at Various Sample Loads

30

2 3 4 5 6 7 8 9 10 11

4 5 6 7 8 9 10 11 12 13 14 15 16 17 min

12 min

32 34 36 38 40 42 44 46 48 50 min

AU

8.0e-2

1.0e-1

1.2e-1

1.4e-1

1.6e-1

1.8e-1

2.0e-1

2.2e-1

2.4e-1

2.6e-1

AU

1.0e-1

3.0e-1

5.0e-1

7.0e-1

9.0e-1

1.1

1.3

1.5

AU

2.5e-3

7.5e-3

1.25e-2

1.75e-2

2.25e-2

2.75e-2

3.25e-2

3.75e-2

4.25e-2

4.75e-2

5.25e-2

Waters Peptide Columns, available in different particle size and column configurations, are well-suited for various lab-scale purification needs.

Peptide Packing Material in OBD Columns for Maximum Chemical and Physical Stability

Columns fail both physically and chemically. The predominant cause of short column lifetime in low pH mobile phases is hydrolysis of the bonded phase that leads to significant changes in peptide retention. BEH Technology Columns incorporate proprietary procedures for bonding and endcapping that yield stable bonded phases. In low pH stability tests, BEH C18 Columns showed very little retention loss. The physical stability of these columns is ensured by the optimum bed density (OBD) design that was developed to produce the most stable packed beds. For more information on OBD Technology, visit www.waters.com

50

60

70

80

90

100

0 50 100

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

% In

itial

Ret

entio

n

Waters Peptide Column B C D

Long-Term Stability

OBD Prep Columns, 10 µm

130Å and 300Å

I.D. (mm)

Length (mm)µmoles of a

Single PeptideWeight of a Single

Peptide (mg)Typical Flow Rate

(mL/min)

30 50 2.5–25 5–100 40–80

30 100 2.5–25 5–100 40–80

30 150 2.5–25 5–100 40–80

30 250 2.5–25 5–100 40–80

Peptide Preparative Column Selection GuideOBD Prep Columns, 5 µm and 10 µm

130Å and 300Å

I.D. (mm)

Length (mm)µmoles of a

Single PeptideWeight of a Single

Peptide (mg)Typical Flow Rate

(mL/min)

10 50 0.25–5 0.5–10 4.5–9

10 100 0.25–5 0.5–10 4.5–9

10 150 0.25–5 0.5–10 4.5–9

10 250 0.25–5 0.5–10 4.5–9

19 50 1–18 2.0–36 16–32

19 100 1–18 2.0–36 16–32

19 150 1–18 2.0–36 16–32

19 250 1–18 2.0–36 16–32

Several columns were tested with repeated injections. Retention was monitored to determine column lifetime.


Bioseparations

Cation-Exchange Peptide and Polypeptide SeparationsFor most analytical and preparative peptide separations, cation-exchange chromatography is used mainly when alternative selectivity is required. In some large-scale purifications, cation exchange can take on a more central role. In these cases, cation exchange is frequently used as the first step in the separation, followed by a secondary purification step using reversed-phase methods.

Waters offers BioSuite packings for cation-exchange separations. These packings are useful both for analytical and preparative work. They are based on rigid, hydrophilic polymethacrylate particles with large 1,000Å pores. The naturally hydrophilic polymer reduces non-specific adsorption, resulting in better recovery of peptide/polypeptide mass and bioactivity. These packings are stable in the pH range of 2–12.

Protein-Pak SP HR 8 and 15 µm packing material is available in pre-packed glass columns.

Therapeutic Peptide Method Development KitThe Therapeutic Peptide Method Development Kit has been developed to simplify the process of sample preparation and LC method development for the analysis of therapeutic peptides in plasma. The kit contains an Oasis Peptide µElution Method Development Plate, a Peptide C18, 300Å Reversed-phase Column and the detailed screening protocol which was used to generate the data shown in this publication.

In addition, a comprehensive method development training seminar has been created which describes all aspects of the method development process from the MS conditions to the final validation of a method for the extraction of the therapeutic peptide Desmopressin from human plasma.

Although big progress has been made in sample pretreatment over the last years, there are still considerable limitations when it comes to overcoming complexity and dynamic range problems associated with peptide analyses from biological matrices. We focus on techniques which can be employed prior to liquid chromatography coupled to mass spectrometry for peptide detection and identification.

The Peptide Columns are specifically QC tested with a cytochrome c tryptic digest that helps ensure batch-to-batch consistency in validated methods ideally suited for separating a wide range of large and small, acidic and basic, hydrophilic and hydrophobic peptides.

The complexity of samples still far exceeds the capacity of currently available analytical systems, and specific sample preparation remains a crucial part of the analysis in a whole.

For more information, visit www.waters.com/pepkit or contact your local Waters sales office.

High Recovery of Peptides

The innovative Oasis μElution plate format allows for up to a 15x sample concentration, increasing the possibility of reaching the required sensitivity levels for bioanalytical assays. The low (25 μL) elution volume eliminates the need for evaporation and reconstitution significantly reducing the potential analyte loss due to absorption to the walls of the collection plate and/or chemical instability.

% S

PE

Rec

over

y

0

20

40

60

80

100

120

Screening ProtocolModified Protocol

Therapeutic Peptide Method Development KitsDescription Qty/Box Part No.UPLC Therapeutic Peptide Method Development Kit 176001835

Kit consists of:

Oasis µElution Method Development Plate 1 186004713

ACQUITY UPLC BEH C18, 300Å, 1.7 µm, 2.1 x 50 mm Column 1 186003685

96-well 1 mL Collection Plate and Cap Mat 3 600001043

HPLC Peptide Therapeutic Peptide Method Development Kit 176001836

Kit Consists of:

Oasis µElution Method Development Plate 1 186004713XBridge BEH C18, 300Å, 3.5 µm, 2.1 x 50 mm Column 1 18600360796-well 1 mL Collection Plate and Cap Mat 3 600001043

Available Waters Products Not Included in Kit:

Oasis MAX 96-well µElution Plate 1 186001829

Oasis WCX 96-well µElution Plate 1 18600249996-well 1 mL Collection Plate 50 186002481Cap Mats for 1 mL Collection Plate 50 186002483Disposable Reservoir Tray 25 WAT058942Extraction Manifold for 96-well Plates 1 186001831

Vacuum Box Gasket Kit (includes foam top gaskets and orange O-rings) 2 186003522

SPE Vacuum Pump 115 V, 60 Hz 1 725000417

SPE Vacuum Pump 240 V, 50 Hz 1 725000418


Consistent Results Due to Superior Batch-to-Batch Reproducibility

Waters batch release protocol includes a tryptic map of cytochrome c (using Waters MassPREP Cytochrome c Digestion Standard [p/n: 186006371]) which is used to test for repro-ducibility to retention times and resolution. The three test chromatograms below show the results of the protein digest test for different batches of PA-B material.

Cytochrome c Tryptic Map Q.C. Test

10 15 20 25 30 35 40 45 50 55 min

Batch 114

Batch 113

Batch 112

BioSuite HPLC Peptide Analysis Columns � Two HPLC Column chemistries that provide alternative chemistries for peptide separations

� Designed for maximum resolution of complex digests

� Available in various configurations for LC or LC-MS applications

� Excellent batch-to-batch reproducibility for consistent results

� Uniquely QC tested specifically for peptide mapping, using Waters MassPREP™ Cytochrome c Digestion Standard

BioSuite Peptide Analysis Series

BioSuite PA Series consists of two of Waters premier reversed-phase column chemistries specifically optimized for peptide mapping from simple to complicated digests.

BioSuite C18, 3 µm PA-A

BioSuite™ C18, 3 µm PA-A is a100Å, difunctional bonded, low ligand density, silica-based column.

� Specifically designed for excellent retention of polar peptides

� Ideal choice for LC-MS applications using formic acid (FA) that minimizes ion-suppression

� Excellent performance for traditional HPLC separations using low TFA concentrations (e.g., 0.025% TFA)

BioSuite C18, 3.5 µm PA-B

BioSuite C18, 3.5 µm PA-B is a 300Å, high ligand density, monofunctional, silica-based column.

� Outstanding performance when separating complex digests containing hydrophilic, hydrophobic, and basic peptides

� Superior peak shape and capacity for peptide separations using TFA containing eluents (e.g., 0.1% TFA)

� Good choice for the separation of larger peptide fragments generated by some endoproteases (e.g., Lys-C)

BioSuite Peptide Analysis HPLC ColumnsDescription Inner Diameter Length 3 µm PA-A 3.5 µm PA-B

BioSuite C18 2.1 mm 50 mm 186002425 186002433

BioSuite C18 2.1 mm 100 mm 186002426 186002434

BioSuite C18 2.1 mm 150 mm 186002427 186002435

BioSuite C18 2.1 mm 250 mm 186002428 186002436

BioSuite C18 4.6 mm 50 mm 186002429 186002437

BioSuite C18 4.6 mm 100 mm 186002430 186002438

BioSuite C18 4.6 mm 150 mm 186002431 186002439

BioSuite C18 4.6 mm 250 mm 186002432 186002440

Eluent A: 0.05% TFA in waterEluent B: 0.01% TFA in MeCNFlow rate: 1.0 ml/minGradient: Time Profile (min) %A %B 0.0 100 0 45 72 28Injection vol.: 20 µLTemperature: 35 °CDetection: UV 220 nm


Bioseparations

BioSuite Cation-Exchange HPLC ColumnsBioSuite SP NP, SP-PEEK, and SP cation-exchange chemistries (CXC) consists of the “strong” sulfopropyl ligand bonded to a pH stable (i.e., pH 2–12), methacrylic ester-based polymeric resin. T he availability of different pore and particle size materials provides chromatographers with the flexibility required to isolate and or characterize peptides based upon minor charge differences. Non-porous (NP) and porous IEX Columns are also available to meet various separations requirements. Speed and superior chromatographic resolution are possible using the non-porous IEX offerings while porous BioSuite offerings are available for applications requiring greater peptide binding capacity. In addition, BioSuite SP material is offered in PEEK hardware as well as in 21.5 mm I.D. stainless steel “lab-scale” preparative column dimensions.

BioSuite Q-PEEK and SP-PEEK Columns are available in 4.6 x 50 mm.

Separation of Angiotensins on BioSuite SP-PEEK Cation-Exchange HPLC Column

0 5 10 15 20 25 min

[Val5]-II

[Sar1, Val5, Ala8]-II[Sar1, Thr8]-II

[Sar1, IIe6]-II[Val5]-II [Asn1, Val5]-II

Des-Asp1, [IIe8]-II

III

II

Column: BioSuite SP-PEEK, 4.6 mm I.D. x 50 mm, PEEKPart number: 186002183Eluent: A: 20 mmol/L sodium acetate buffer, pH 5.0 B: 20 mmol/L sodium acetate buffer containing 1.0 mol/L NaCl, pH 5.0 Linear gradient from eluent A to B for 20 minutesFlow rate: 1.0 mL/min.Temperature: 25 °CDetection: UV @ 280 nm

BioSuite Cation-Exchange HPLC Columns

Description Matrix Pore SizeExclusion Limit (Daltons) against

Polyethylene GlycolInner

DiameterLength

Column Volume (mL)

# Approx Protein Binding Capacity Per Pre-Packed Column

Part No.

BioSuite SP-PEEK, 7 µm CXC Polymer 1300Å >4,000,000 4.6 mm 50 mm 0.83 58 mg* 186002182

BioSuite SP, 2.5 µm NP CXC Polymer N/A 500 4.6 mm 35 mm 0.58 2.9 mg** 186002183

BioSuite SP, 10 µm CXC Polymer 1000Å 1,000,000 7.5 mm 75 mm 3.31 132 mg** 186002184

BioSuite SP, 13 µm CXC Polymer 1000Å 1,000,000 21.5 mm 150 mm 54.45 2,178 mg** 186002185

* Data generated with Gamma Globulin ** Data generated with Hemoglobin NOTE: For best resolution of complex samples, do not exceed 20% of the Column’s protein binding capacityNOTE: For best resolution of complex samples, do not exceed 20% of the Column’s protein binding capacity

AU

0.0

0.5

0 10 20 30 min

Alla

ntoi

n RAS

G-1

Angi

oten

sin

frag.

1-7

Brad

ykin

in

Ren

in s

ubst

rate

Angi

oten

sin

IIAn

giot

ensi

n I

Enol

ase

T37

Mel

ittin

Enol

ase

T35

Baseline HPLC Resolution of Nine Peptides Contained in MassPREP Standard Mixture

Benchmarking, Method Development, and Troubleshooting: MassPREP Peptide StandardThe MassPREP Peptide Standard Mixture contains a void volume (VO) column marker and nine carefully selected peptides with a broad range of polarities and isoelectric points. The MassPREP standard is useful to test UPLC and HPLC columns and systems dedicated to peptide separations.

Peak #

Component Name

Molecular weight** (g/mol) pKa Peptide sequence

1Allantoin

(V0 marker)158.0440 – –

2 RASG-1 1000.4938 9.34 RGDSPASSKP

3Angiotensin

frag.1–7898.4661 7.35 DRVYIHP

4 Bradykinin 1059.5613 12.00 RPPGFSPFR

5 Angiotensin II. 1045.5345 7.35 DRVYIHPF

6 Angiotensin I. 1295.6775 7.51 DRVYIHPFHL

7 Renin substrate 1757.9253 7.61 DRVYIHPFHLLVYS

8 Enolase T35 1871.9604 7.34 WLTGPQLADLYHSLMK

9 Enolase T37 2827.2806 3.97 YPIVSIEDPFAEDDWEAWSHFFK

10 Melittin 2845.7381 12.06 GIGAVLKVLTTGLPALISWIKRKRQQ

*BioSuite C18 3 µm, Peptide Analysis - A Column (2.1 x 150 mm). Eluent A: 0.02% TFA in water, Eluent B: 0.016% TFA in acetonitrile.Gradient: 0–50% B in 30 minutes. Flow: 0.20 mL/min. Temp: 40 °C. ** Monoisotopic molecular weight.

Peptide AnalysisDescription Volume Part No.

MassPREP Peptide Mixture Solid 186002337

One vial with approximately 1 nmole of each:

Allantoin (Vo Marker); RASG-1, angiotensin frag. 1–7, bradykinin; angiotensin II; angiotensin I, renin substrate, enolase T35, enolase T37, melittin. The peptide standard is useful to test LC columns and systems dedicated to peptide separations.

MassPREP Peptide Mixture, 5/pk Solid 186002338

This is a 5 pack of 186002337. Each vial contains approximately 1 nmole of each:

Allantoin (Vo Marker); RASG-1, angiotensin frag. 1–7, bradykinin, angiotensin II, angiotensin I, renin substrate, enolase T35, enolase T37, melittin. The peptide standard is useful to test LC columns and systems dedicated to peptide separations.


Additional Peptide Consumables

MassPREP Protein Digestion Standards

The MassPREP protein digestion standards are prepared under strict quality control procedures and contain no undigested standard proteins, trypsin, or other hydrophilic components. Test results from each batch of digestion standards are provided on an available Certificate of Analysis report.

Peptide AnalysisDescription Volume Part No.

MassPREP Digestion StandardsYeast enolase Solid 186002325

Phosphorylase b Solid 186002326

Bovine hemoglobin Solid 186002327

Yeast alcohol dehydrogenase (ADH) Solid 186002328

Bovine serum albumin (BSA) Solid 186002329

Cytochrome c Solid 186006371

Kit contains one (1) of 186002325, 186002326, 186002327, 186002328, 186002329

186002330

MassPREP Digestion Standard Mix 1


186002865

186002866

The MassPREP Protein Digestion Standard Mix 1 and 2 are qualitative standards that provides high confidence and coverage during expression profiling of complex protein samples. T his standards are used for verification of Waters PLGS software and consists of a various ratios of yeast enolase, phosphorylase B, yeast alcohol dehydrogenase (ADH), and bovine serum albumin (BSA), which have all been tryptically digested.

MassPREP E.coli Digestion Standard 186003196

The MassPREP E.coli Digestion Standard is part of a line of qualitative digestion standards and provides a complex biological matrix for proteomic evaluations using LC-MS, specifically Waters Protein Expression System. MPDS E.coli was prepared by digesting a purified E. coli cytosolic protein fraction with sequencing grade trypsin. T he mixture is purified, and does not contain undigested protein, trypsin, or very hydrophilic components such as salts. T he standard is packaged in a TruView LCMS Certified Vial (ideal for peptides) and contains approximately 100 µg of lyophilized tryptically digested and purified E.coli cytosolic protein.

Quantitative Peptide Standards

These sets of standards are specifically designed, formulated and QC’d for quantitative peptide analysis and include:

� Quantitative Peptide Retention Standard

� Hi3 Phos B and E.coli Standards

� SILAC Hi3 Phos B and E.coli Standards

Quantitative Peptide AnalysisDescription Part No.

Hi3 Phosphorylase B Standard 186006011

The Hi3 Phos B standard is primarily intended for use with the Hi3 quantification method for MSE proteomics data processed with ProteinLynx Global Server for samples of microbial origin. It may also used in the evaluation and benchmarking of proteomic LC-MS systems comprised of nanoACQUITY UPLC and SYNAPT and Xevo time-of-flight mass spectrometers. The Hi3 Phos B standard is intended for samples of microbial origin. It is a quantitative standard comprised of the top 6 ionizing peptides in the rabbit Phosphorylase B protein. Recommended at -20 °C.

Hi3 E.coli Standard 186006012

The Hi3 E.coli standard is primarily intended for use with the Hi3 quantification method for MSE proteomics data processed with ProteinLynx Global Server™ for samples of microbial origin. It may also be used in the evaluation and benchmarking of proteomic LC-MS systems comprised of nanoACQUITY UPLC and SYNAPT® and Xevo time-of-flight mass spectrometers. T he Hi3 E.coli standard is intended for samples of animal origin. It is a quantitative standard comprised of the top 6 ionizing peptides in the E.coli ClpB protein.

SILAC Hi3 PhosB Standard 186007083The SILAC Hi3 Phos B standard is formulated from the same specialized set of the top 6 ionizing peptides of the rabbit Phosphorylase B protein that is contained in the non-labeled counterpart: Hi3 Phos B standard (p/n: 186006011). The main difference is that this standard is produced to have a heavy labeled reference on the lysine (K) or arginine (R) end of the peptide.

SILAC Hi3 E.coli Standard 186007084

The SILAC Hi3 E.coli standard is formulated from the same specialized set of the top 6 ionizing peptides of the E.coli ClpB protein that is contained in the non-labeled counterpart: Hi3 E.coli standard (p/n: 186006012). The main difference is that this standard is produced to have a heavy labeled reference on the lysine (K) or arginine (R) end of the peptide.

Quantitative Peptide Retention Standard 186006555

The Quantitative Peptide Retention Standard is a quantitative standard that is useful during the calibration, development, and troubleshooting of chromatographic separations ensuring confidence in results. This standard is rigorously QC tested for purity and quantitative formulation and is specifically designed with the following features:

� Peak retention for chromatographic reproducibility

� UV absorptivity for signal reproducibility

� Low to high mass range for MS

� Water solubility

� Tryptic-like peptides for peptide mapping studies


Bioseparations

Phosphorylated Peptide Standards

The MassPREP Phosphopeptide Standards give you greater control over sample preparation, with the option to use pure peptides or to define phosphopeptides to unmodified peptide ratios.

How RapiGest SF Works

RapiGest SF Protein Digestion SurfactantRapiGest™ SF (surfactant) radically enhances protein enzymatic digestions in terms of speed and percent recovery. RapiGest is a patented anionic surfactant that accelerates the production of peptides generated by proteases, such as trypsin, Asp-N, Glu-C, and Lys-C. Many hydrophobic proteins are resistant to proteolysis because their cleavage sites are inaccessible to endoproteases. RapiGest, a mild denaturant, helps solubilize and unfold proteins making them more amenable to cleavage without denaturing or inhibiting common proteolytic enzymes.

RapiGest SurfactantDescription Part No.

RapiGest SF 1 mg vial 186001860

RapiGest SF 1 mg vial (5/pk) 186001861



RapiGest SF Custom 186002118

MassPREP Phosphopeptide StandardsDescription Volume Part No.

MassPREP Phosphopeptide Standard Enolase Solid 186003285

4 yeast enolase derived phosphorylated peptides: T18 1P, T19 1P, T43 1P, T43 2P. Used to optimize phosphopeptide detection in LC-MS, LC/UV, and MALDI-MS.

MassPREP Enolase Digest with Phosphopeptides Mix Solid 186003286

Yeast enolase spiked with 4 yeast enolase derived phosphorylated peptides: T18 1P, T19 1P, T43 1P, T43 2P. A more complex mixture used to optimize and troubleshoot phosphopeptide detection in LC-MS, LC/UV, and MALDI-MS.

MassPREP Phosphopeptide Sample Kit—Enolase 186003287

A kit that allows one to mix and optimize a complex standard per specific applications. (2) Vials:

MassPREP enolase digestion standard Solid 186002325

MassPREP phosphopeptide standard enolase Solid 186003285

MassPREP Enhancer (5 vials) Solid 186003863

(5) 500 mg MassPREP Enhancer. A component in the MassPREP Phosphopeptide Enrichment Kit

186003864

MassPREP Phosphopeptide Enrichment Kit 186003864

Phosphopeptide enrichment μElution plate (2) MassPREP Solid 186003820

Enhancer MassPREP enolase digest with phosphopeptides. Solid 186003863

This kit is developed fro selective enrichment of phosphopeptides from complex samples.

186003286


Delta-Pak HPLC ColumnsDelta-Pak packings, ideal for the separation of peptides, proteins, and natural products, are based on a highly stable, bonded, endcapped 5 µm or 15 µm spherical silica. Delta-Pak is available in two different pore size materials (100Å and 300Å) with a C18 or C4 bonded phase. For more information and part numbers, visit waters.com/biosep

Synthetic Peptide Separation on Delta-Pak C18 HPLC ColumnColumn: Delta-Pak C18 300Å, 5 µm, 3.9 x 150 mmPart number: WAT011793Sample: Synthetic peptide-neurotensin (5 mg/mL)Injection: 10 µL (50 µg)MobilephaseA: Waterwith0.1%trifluoroaceticacidMobilephaseB: Acetonitrilewith0.1%trifluoroaceticacidGradient: 0–2 min, 5% BConditions: 2–27 min, 5–9% B, 30–31 min, 90–5% BFlow rate: 1 mL/minDetection: UV @ 230 nm

Delta-Pak Analytical HPLC Columns and Guards

Dimension TypeParticle

SizePore Size

Delta-Pak C18

Delta-Pak C4

2.1 x 150 mm Column 5 µm 300Å WAT023650 —

3.9 x 20 mm Guard, 2/pk 5 µm 100Å WAT0468801 WAT0468751

3.9 x 20 mm Guard, 2/pk 5 µm 300Å WAT0468901 WAT0468851

3.9 x 20 mm Guard, 10/pk 5 µm 100Å WAT0368701 —

3.9 x 150 mm Column 5 µm 100Å WAT011795 WAT011796

3.9 x 150 mm Cartridge, 10/pk 5 µm 300Å WAT0368752 WAT0368652


Guard-Pak Holder WAT088141

Guard-Pak In-Line Filters, 5/pk WAT0324721 Requires 3.0 x 20 mm/4.6 x 20 mm Universal Sentry Guard Holder, p/n: WAT0469102 Requires Guard-Pak Holder, p/n: WAT088141

Delta-Pak Preparative Guard Columns


SizePore Size

Delta-Pak C18

Delta-Pak C4





19 x 300 mm Column 15 µm 100Å WAT011799 WAT011809




50 x 300 mm Column 15 µm 100Å WAT011801 —

Delta-Pak Radial Compression Preparative Column Segments and PrepPak Cartridges*


SizePore Size

Delta-Pak C18

Delta-Pak C4


8 x 100 mm Column 15 µm 300Å WAT025845 —



25 x 10 mm Guard, 2/pk 15 µm 100Å WAT038520 WAT038524

25 x 10 mm Guard, 2/pk 15 µm 300Å WAT038522 WAT038526



40 x 10 mm Guard, 2/pk 15 µm 100Å WAT037842 —

40 x 10 mm Guard, 2/pk 15 µm 300Å WAT037845 WAT037851*All column segments and cartridges require the appropriate holder/module

Waters Insulin HMW P HPLC ColumnThe Waters Insulin HMW P Column is specifically designed for use in the manufacture and quality control of insulin products. This column is tested for performance in the analysis of impurities with molecular masses greater than those of insulin.

Waters Insulin HMW P SEC HPLC ColumnDescription Dimension Part No.

Waters Insulin HMW P Column 7.8 x 300 mm WAT201549

Protein-Pak 125 Sentry Guard Column, 2/pk (requires holder)

3.9 x 20 mm 186000926

Sentry Universal Guard Column Holder — WAT046910

Tested to perform in the method published in PharmaEuropa Vol. 8, No 3, September 1996.


Bioseparations

High Recoveries of Peptides and Proteins

The heart of the column is high purity-based deactivated silica. Waters dedicated chromatography chemistry manufacturing plant operates under the stringent standards of cGMP supplemented with FDA registration for Class 1 medical devices and ISO 9002. The silica used in the manufacture of our Symmetry300 Columns is synthesized using ultrapure organic reagents that yields high purity particles with very low silanol activity. These particles when combined with innovative ligand (i.e., C4 and C18) bonding techniques helps produce reversed-phase columns with minimal non-desired secondary interactions between bound ligand and biomolecules.

T here are several factors to assess when developing a reversed-phase HPLC method for peptides. Batch-to-batch reproducibility of the column is an important consideration when developing a validated and transferable method. Symmetry300 Columns have outstanding batch-to-batch and column-to-column reproducibility.

The key to a successful separation is the selection of a column that gives the highest chemistry resolution with maximum peak capacity and recovery.

Pore Size Effects on Peptide Selectivity: Comparative Results on Symmetry 100Å vs. Symmetry300 Columns

13

24

5,6

7

8

10

B

11

13

12

12 4

3

5 6

7

9

8

10

A11

12

13

200 40 min

9

Column A: Symmetry C18, 100Å, 5 µm, 4.6 x 150 mmPart number: WAT045905Column B: Symmetry300 C18, 300Å, 5 µm, 4.6 x 150 mmPart number: WAT106157Sample: Cytochrome c Digestion StandardPart number: 186006371MobilephaseA:0.1%trifluoroacetic acid in waterMobilephaseB:0.1%trifluoroacetic acid in acetonitrileGradient: 0–50 min, 0–30% BInjection: 20 µLFlow rate: 0.75 mL/minColumn temp.: 35 °CDetection: UV @ 214 nm

Symmetry HPLC ColumnsWaters Symmetry, reversed-phase, silica-based particles are synthesized using ultrapure organic reagents, resulting in high purity material with very low silanol activity. When combined with the high surface coverage of the bonded phase, outstanding peptide separations and recoveries are possible.

� Superior manufacturing control for consistent batch-to-batch and column-to-column results

� 100Å and 300Å pore size offerings for small or larger size peptides

� SymmetryShield™ Column chemistry offers complementary selectivity to Symmetry Column offerings

� SymmetryPrep Columns provide direct scale up while maintaining resolution

Symmetry300 Columns: T he First Columns Specifically Engineered for the Discovery and Development of New Biopharmaceuticals

As a scientist, you know that tremendous time and resources are required when developing HPLC assays for well-characterized biopharmaceuticals. You also know that column-to-column variability is the “Achilles heel” of this demanding process.

In the past you’ve had no choice but to deal with variability by finding costly and time-consuming ways around this problem. The regulatory guidelines being adopted worldwide as a result of the International Conference on Harmonization (ICH) and the U.S. Food and Drug Administration’s specified biological products initiatives are placing increasingly stringent demands on this process. Full analytical characterization is becoming more important for regulatory filing, making column variability an unacceptable risk.

Now, regardless of whether you’re developing HPLC assays for purity, stability, or identity, you can have confidence in the long-term compliance of your methods, batch-to-batch, column-after-column, year-after-year. That’s because Waters has developed Symmetry300 Columns, specifically engineered for the discovery and development of new biopharmaceuticals.

Symmetry300 Columns are a 300Å reversed-phase addition to the existing Symmetry family of columns. They have been specifically designed to provide maximum batch-to-batch and column-to-column performance consistency and recovery of protein and peptide applications.

Symmetry300 Columns are offered in two particle sizes (3.5 μm and 5 μm) and in two chemistries (C4 for large peptides and proteins and C18 for smaller peptides) to address various needs.

Symmetry300 ColumnsParticle Size Inner Diameter Length C18 C4

3.5 µm 1.0 mm 150 mm 186000185 186000276

3.5 µm 2.1 mm 50 mm 186000187 186000277

3.5 µm 2.1 mm 100 mm 186000188 186000278

3.5 µm 2.1 mm 150 mm 186000200 186000279

3.5 µm 4.6 mm 50 mm 186000201 186000280

3.5 µm 4.6 mm 75 mm 186000189 186000281

3.5 µm 4.6 mm 100 mm 186000190 186000282

3.5 µm 4.6 mm 150 mm 186000197 186000283

5 µm 2.1 mm 150 mm WAT106172 186000285

5 µm 3.9 mm 150 mm WAT106154 186000286

5 µm 4.6 mm 50 mm WAT106209 186000287

5 µm 4.6 mm 150 mm WAT106157 186000288

5 µm 4.6 mm 250 mm WAT106151 186000289

5 µm 19 mm 10 mm 186001847 —

5 µm 19 mm 50 mm 186001848 —

5 µm 19 mm 100 mm 186001849 —


PROTEINS

ACQUITY UPLC SEC System SolutionACQUITY UPLC Technology allows analytical chemists to reach far beyond conventional LC separations and has proven itself to be a major asset in increasing the productivity of laboratories around the world. The latest addition to this application-driven portfolio is the ACQUITY UPLC SEC System Solution, enabled by the unique ethylene-bridged-hybrid (BEH) Diol coated particle technology.

� Determines aggregation levels in therapeutic monoclonal antibodies up to 10 times faster than traditional HPLC based size-exclusion chromatography (SEC)

� Fully optimized column chemistry significantly reduces the requirement for high salt concentration mobile phases

� QC tested with BEH protein standards, ensuring unmatched batch-to-batch consistency and increased confidence in validated methods

� Waters Protein Standard Mixes are available for the 125Å, 200Å, and 450Å SEC Columns for additional validation (p/n: 186006519, 186006518, and 186006842 respectively)

BEH Technology

In 1999, Waters launched the XTerra Family of HPLC Columns featuring patented-first-generation hybrid-particle technology (HPT)*. HPT enabled XTerra Columns to become one of the most successful column products in the history of Waters. In HPT, the best properties of inorganic (silica) and organic (polymeric) packings are combined to produce a material that has superior mechanical strength, efficiency, high-pH stability and peak shape for basic compounds.

The first-generation methyl-hybrid particles of XTerra Columns did not possess the mechanical strength or efficiency necessary to fully realize the potential speed, sensitivity and resolution capabilities of UPLC Technology. Therefore, a new pressure-tolerant particle needed to be created. This new, second-generation hybrid material was developed that utilizes an ethylene-bridged hybrid (BEH) structure. Compared to the first-generation methyl-hybrid particle of XTerra Columns, the BEH particle of ACQUITY UPLC BEH Columns exhibits improved efficiency, strength and pH range. BEH Technology is a key enabler of the speed, sensitivity and resolution of both small and large molecule UPLC separations.

Anal. Chem. 2003, 75, 6781-6788

Tetraethoxysilane(TEOS)

Polyethoxysilane(BPEOS)

Bis(triethoxysilyl)ethane(BTEE)

+4

Si

EtO

EtO OEtEtO

Si

EtOEtO

EtO

Si

OEt

OEt

OEt

Si

EtO

O

CH2

CH2

CH2

Si O Si

EtO

OEt

Si

O

O

OEt

Si

O

SiOO

Et

OOO

Et

Et Et n

*Patent No. 6,686,035 B2

CH2

The BEH Particle*: One of the Key Enablers of UPLC Technology


Bioseparations

Separation of Protein and Peptide Standards on ACQUITY UPLC Protein BEH SEC, 125Å, 200Å, and 450Å Columns

0.00

0.04

0.08

0.12

0.00

0.05

0.10

0.15

2 3 4 5 6 7 8 min

1

2

3

4 5

6

7

AU

AU

AU

0.00

0.02

0.04

0.06

0.08

0.10

1

1

2

2

3

3

4 5

5

67

7

Thyroglobulin dimer(Approx. 1.4 million Daltons)

ACQUITY UPLC Protein BEH SEC, 125Å

ACQUITY UPLC Protein BEH SEC, 200Å

ACQUITY UPLC Protein BEH SEC, 450ÅNOTE: Stds 4 and 6 below were not included in the 450Å Column test mix

Calibration Curves on ACQUITY UPLC Protein BEH SEC, 125Å, 200Å, and 450Å Columns

Protein standards; Temp.: 30 °C; Mobile phase: 100 mM sodium phosphate, pH=6.8

MW

100

1000

1,000,000

100,000

10,000

Normalized Retention Volume (Vr/VC)

0.4 1.00.80.6

Uracil (112 Da)Allantoin (158 Da)

Angiotensin frag 1-7 (899 Da)

Aprotinin (6.5 kDa)RNase A (13.7 kDa)

Myoglobin (17 kDa)

Ovalbumin (44 kDa)Conalbumin (75 kDa)

Amylglucosidase (97 kDa)IgG (150 kDa)

Thyroglobulin (669 kDa)

Blue Dextran

IgM (900 kDa)

ACQUITY UPLC Protein BEH SEC, 450Å, 2.5 µm: 100K–1500K Daltons



ColumnsACQUITY UPLC Protein BEH SEC, 125Å, 1.7 µm; ACQUITY UPLC Protein BEH SEC, 200Å 1.7 µm

ACQUITY UPLC Protein BEH SEC, 450Å, 2.5 µm

Column Configuration 4.6 x 150 mm

Mobile Phase 100 mM Sodium Phosphate Buffer, pH 6.8

Weak Needle Wash 100% Milli-Q Water

Strong Needle Wash 100% Milli-Q Water

Seal wash 90/10 water/methanol

Samples: Diluted in mobile phase

Thyroglobulin 0.3 mg/mL, BSA 0.3 mg/mL, Ovalbumin 0.3 mg/mL, Carbonic Anhydrase 0.3 mg/mL, Myoglobin 0.3 mg/mL, Angiotensin Frag. 1–7 0.1 mg/mL, Uracil 0.1 mg/mL

Thyroglobulin 3 mg/mL, BSA 5 mg/mL,Ovalbumin 3 mg/mL,Myoglobin 2 mg/mL,Uracil 0.1 mg/mL

Injection Vol. 2 µL, Full Loop

Flow Rate 0.3 mL/min

Column Temp. ambient

Detection Wavelength UV @ 220 nm UV @ 280 nm

Compounds:1. Thyroglobulin (670 K)2. BSA (66 K)3. Ovalbumin (44 K)4. Carbonic Anhydrase (29 K) 5. Myoglobin (17 K) 6. Angiotensin Frag 1–7 (899)7. Uracil (112)

Waters ACQUITY UPLC Technology allows analytical chemists to reach far beyond conventional LC separations and has proven itself to be a valuable asset that improves the quality of collected data while increasing sample throughput and productivity. Biotherapeutics and biosimilars manufacturers can now choose the most appropriate UPLC-based, SEC Column(s) (i.e., 125Å, 200Å, and 450Å pore size) to satisfy their application requirements based on this separation technology.


SEC Analysis of Insulin

Size-exclusion chromatography is the USP and EP standard method for the analysis of covalent HMW insulin in therapeutic preparations. Compared to use of traditional HPLC-based, SEC methods (bottom panel), significant improvement in insulin component resolution, while reducing analysis time and mobile-phase consumption, is obtained using Waters BEH SEC, 125Å, 1.7 µm Column with Waters Ultra Performance Liquid Chromatography (UPLC) instrumentation (top panel).

Insulin Analyses by Traditional HPLC-SEC vs UPLC-SEC

.50 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min

0 2 4 6 8 10 12 14 16 18 20 22 24 min

Column: Waters Insulin HMWP SEC, 125Å, 10 µm, 7.8 x 300 mmPart number: WAT201549Sample: Insulin, human recombinant (Sigma I-2643) (4 mg/mL)Injection: 100 µLEluent: L-arginine (1 gm/liter)/acetic acid/acetonitrile (65/15/20, v/v/v)Flow: 0.5 mL/min (isocratic)Detection: UV @ 276 nm

Column: Waters ACQUITY UPLC Protein BEHSEC,125Å,1.7μm, 4.6 x 300 mmPart number: 186006506Sample: Insulin, human recombinant (Sigma I-2643) (4 mg/mL)Injection: 5 µLEluent: L-arginine (1 gm/liter)/acetic acid/acetonitrile (65/15/20, v/v/v)Flow: 0.4 mL/min (isocratic)Detection: UV @ 276 nm

Waters Alliance HPLC System

Waters ACQUITY UPLC System

Comparative UPLC-Based SEC Benefits vs Use of Traditional HPLC SEC for Biotherapeutic Characterization

Column: Traditional100%silica-based/Diolcoated,5μm,7.8x300mmSECSample: Humanized IgG monoclonal antibody, 3 mg/mL in eluent HPLCSystem: WatersModel2796HPLCwithModel2487DualλAbsorbanceDetector Eluent: 25 mM sodium phosphate, pH 6.8 with 0.15 M sodium chloride Flow rate: 0.4 mL/min Injection: 20 µL Detection: UV @ 280 nm

Column: ACQUITY UPLC Protein BEH SEC, 200Å, 1.7 µm, 4.6 x 300 mmPart number: 186005226 Sample: Humanized IgG monoclonal antibody, 3 mg/mL in eluent UPLC System: Waters ACQUITY UPLC with Tunable UV Detector Eluent: 25 mM sodium phosphate, pH 6.8 with 0.15 M sodium chloride Flow rate: 0.4 mL/min Injection: 5 µL Detection: UV @ 280 nm

2.26 % Aggregate

2.24 % Aggregate

HPLC 100% Silica-DiolBEH SEC, 250Å,

5 µm, 7.8 x 300 mm

ACQUITY UPLCBEH SEC, 200Å,

1.7 µm, 4.6 x 300 mm

2 4 6 8 10 min 5 10 15 20 25 30 min

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035AU

0.040

0.045

0.050

0.055

0.060

0.065

0.070

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035AU

0.040

0.045

0.050

0.055

0.060

0.065

0.070

2.26 % Aggregate

2.24 % Aggregate

HPLC 100% Silica-DiolBEH SEC, 250Å,

5 µm, 7.8 x 300 mm

ACQUITY UPLCBEH SEC, 200Å,

1.7 µm, 4.6 x 300 mm

2 4 6 8 10 min 5 10 15 20 25 30 min

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035AU

0.040

0.045

0.050

0.055

0.060

0.065

0.070

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035AU

0.040

0.045

0.050

0.055

0.060

0.065

0.070


Bioseparations

mass140000 142000 144000 146000 148000 150000 152000 154000 156000 158000

148221.0

148060.0000

147913.0000

145422.0000 144761.0000 141482.0000 143433.0000

147848.0000

148383.0000

148443.0000

148603.0000 151194.0000 155452.0000 153154.0000

Intact mAb (peak 1)

%

100

0

LC-MS Analysis of Humanized Monoclonal Antibody on BEH SEC, 200Å, 1.7 µm

Intact mAb Extracted and Deconvoluted Mass Spectra

Scan

UV @ 280

TIC

2: Diode Array280 0.0500Da

Range: 6.757e-1

13.22

25.27

16.58

19.50

1: TOF MS ES+TIC

7.58e6

19.49

15.3516.62

23.7425.06

1 2

3

1

2

3

0.0

2.0e-3

4.0e-3

6.0e-3

8.0e-3

1.0e-2

1.2e-2

1.4e-2

1.6e-2

1.8e-2

%

4

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700

90000 95000 100000 105000 110000 115000 120000 125000 130000 135000 140000 145000 150000 155000 160000

%

0

100100764.5

100600.5000

100438.5000

98105.5000 98082.5000

97989.5000

97964.5000

97751.5000 97690.5000

95316.5000

95163.0000

94702.0000

100922.5000

148221.5 100961.0000

101097.0000 148060.5000

101126.5000

103651.5000 103785.5000

103807.0000 148038.0000

104040.5000

106714.0000

144987.0000 111060.0000

148378.5000

148408.5000

151383.5000 155460.5000

mass

Clip (peak 2)

Lower Molecular Weight Species (peak 3)

45250 45500 45750 46000 46250 46500 46750 47000 47250 47500 47750 48000 48250 48500 48750 49000

%

0

10047636.0000

47270.0000

47155.000047398.0000

47736.5000

47798.0000

mass

LC Conditions LC System: ACQUITY UPLC H-Class Bio System with PDA Detector Column: ACQUITY UPLC Protein BEH SEC, 200Å, 1.7μm,4.6x300mm, (p/n: 186005226) Column temp.: 30 °C Sample temp.: 4 °C Injectionvol.: 2μLFlow rate: 0.15 mL/minMobile phase: 100 mM ammonium formate and 25 mM sodium phosphate, 150 mM sodium chloride, pH 6.8 Additive: Acetonitrile, 0.8% formic acid, at 0.2 mL/min External Pump:Waters 515 HPLC pump

MS ConditionsMS System: Xevo G2 QTof Ionization mode: ESI+Analyser mode: SensitivityAcquisition range: 500–5000Capillary voltage: 3.00 kVCone voltage: 40.0 VSource temp.: 150 °CDesolvation temp.: 450 °CConegasflow: 0.0L/HrDesolvationgasflow: 800.0L/HrCalibration: NaI2μg/μLfrom 1000–4000 m/z

LC-MS Analyses using SEC and Volatile Eluents

Size-exclusion chromatography under non-denaturing conditions is a standard method for testing biomolecules and their aggregates. MALS and AUC are established detectors but cannot provide exact mass for unknown species with a sufficient accuracy. The presence of an unexpected peak requires further investigation and/or confirmation of molecular weight, and SE-UPLC-MS under aqueous, non-denaturing conditions can provide valuable information that would more rapidly solve an organization’s issues with characterization or quality.


Reduced Requirement for High Salt Concentration Mobile Phases

With conventional silica based SEC Column chemistries, undesirable secondary ionic interactions between the silica surface and basic proteins can result in long retention times and excessive peak tailing. Traditionally, the solution to this issue is the inclusion of high concentrations of a salt to compete for the charged sites on the surface of the silica. The unique surface chemistry of the ACQUITY UPLC Protein BEH SEC, 200Å Column significantly reduces these secondary interactions, resulting in the ability to use less aggressive mobile phase salt concentrations.

Effect of Eluent Ionic Strength on the SEC Analysis of the Basic Protein Lysozyme on 100% Silica vs BEH SEC Particles

5 6 7 8 9 10 11 12 13 14 15 min

14 18 22 26 30 34 38 42 46 50 min10

Lysozyme, 10 mM

Flow rate: 0.3 mL/minMobile phase: 10, 25 or 100 mM sodium phosphate, pH 6.8 Traditional 100% Silica-based/Diol coated,

4 μm, 4.6 x 300 mm, SEC column

Mobile phase Ionic Strength

10 mM 25 mM 100 mM

ACQUITY UPLC Protein BEH SEC, 200Å, 1.7 µm, 4.6 x 150 mm

Significant reduction in 2˚ interactions

A New Level of Column Stability for Size-Exclusion Chromatography

BEH particle technology is well established for chromatography of synthetic oligonucleotides, amino acids, peptides, proteins, and labeled glycans with stability and performance attributes not found with traditional, 100% silica-based particles. (Go to www.waters.com/biosep)

The combination of the BEH base particle and the patent pending innovative diol bonding process, results in column stability, performance and lifetime unheard of in traditional size exclusion chromatographic columns.

ACQUITY UPLC Protein BEH SEC, 200Å Particle and Diol Bonding Technology Provides a Stable Chemistry with Outstanding Column Life

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

6 7 8 9 10 11 12 13 14 15 min

Sample: LysozymeFlow rate: 0.4 mL/minMobile phase: 25 mM sodium phosphate, pH 6.8 with 0.15 m sodium chloride

Traditional 100% Silica-based/Diol coated, 4.6 x 300 mm, 4 μm SEC column

Injection 19

Injection 618

ACQUITY UPLC Protein BEH SEC, 200Å, 1.7 µm, 4.6 x 150 mm

Injection 19

Injection 618


Bioseparations

Stringent Manufacturing Quality Assurance Delivers Confidence in Results

All Waters ACQUITY Columns chemistries are synthesised in state-of-the-art ISO-certified manufacturing facilities from high quality raw materials, and are extensively QC tested throughout the synthetic process. In addition, each batch of BEH SEC, 200Å, 1.7 µm material is specifically tested with relevant proteins to help ensure unmatched batch-to-batch consistency for supreme confidence in validated methods.

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

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

0 1

1

1

1

2

2

2

3

3

3

4

4

4

5

5

5

2 3 4 5 6 7 8 9 10 min

Batch “A”

Batch “B”

Batch “C”

Waters ISO 2001 Manufacturing and Testing Processes Help Ensure Outstanding ACQUITY UPLC Protein BEH SEC, 200Å 1.7 µm Batch-to-Batch Reproducibility

Use BEH SEC, 200Å Protein Standard Mix (p/n: 186006518) for QC testing and validation.

Analyte pI MW

1. Thyroglobulin, 3 mg/mL 4.6 669,000

2. IgG, 2 mg/mL 6.7 150,000

3. BSA, 5 mg/mL 4.6 66,400

4. Myoglobin, 2 mg/mL 6.8, 7.2 17,000

5. Uracil, 0.1 mg/mL N/A 112

ACQUITY UPLC Protein BEH SEC ColumnsDescription Configuration Particle Size Dimension Part No.

ACQUITY UPLC Protein BEH SEC, 450Å Column with BEH450 SEC Standard Column 2.5 µm 4.6 x 150 mm 176002996

ACQUITY UPLC Protein BEH SEC, 450Å Column 2.5 µm 4.6 x 150 mm 186006851

ACQUITY UPLC Protein BEH SEC, 450Å Column with BEH450 SEC Standard Column 2.5 µm 4.6 x 300 mm 176002997


ACQUITY UPLC Protein BEH SEC, 450Å Guard Kit 2.5 µm 4.6 x 30 mm 186006850







ELSD outlet tubing (0.004” I.D. x 6” length) 430001562

0.005 x 1.75” SEC UPLC Connection Tubing, 2/pk 186006613NOTE: Size-exclusion c hromatography may require modifications to an existing ACQUITY UPLC System. Please refer to “Size Exclusion and Ion- Exchange Chromatography of Proteins using the ACQUITY UPLC System”,

(715002147 Rev A) or “Size Exclusion and Ion- Exchange Chromatography of Proteins using the ACQUITY UPLC H-Class System”, (715002909 Rev A) for specific recommendations.


Calibration Curves on XBridge Protein BEH SEC, 125Å, 200Å, and 450Å Columns

10

100

1,000

10,000

100,000

1,000,000

10,000,000

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

MW

Normalized Retention Volume (V0/VC)

ACQUITY UPLC Protein BEH SEC, 450Å, 2.5 µm: 100 K–1500 K Daltons



Uracil (112 Da)

Aprotinin (6.5 KDa) RNAse A (14 KDa) Myoglobin (17 KDa)

Ovalbumin (44 KDa) Conalbumin (75 KDa)

Amyloglucosidase (97 KDa) IgG (150 KDa)

Thyroglobulin (669 KDa)

IgM (900 KDa)

XBridge Protein BEH SEC, 125Å, 200Å, and 450Å Columns and Protein Standard Test MixturesWaters series of XBridge Protein BEH SEC, 125Å, 200Å, or 450Å, 3.5 µm Columns were developed to complement our existing line of UPLC-based SEC offerings for use where traditional HPLC-based instrumentation and methods are employed for peptide or protein size exclusion chromatography (SEC). These new HPLC-based, SEC chemistries are based on the same Waters Ethylene Bridged Hybrid (BEH)-based particle technology and diol-bonded surface coating as used in our successful line of UPLC-based SEC columns. This process offers chromatographers the option and ability to easily transfer methods based on labaratory instrumentation and component resolution or sample throughput needs.

All of Waters BEH-based SEC columns are manufactured in a cGMP, ISO 9001-certified plant using stringent manufacturing protocols and ultra pure reagents. Each batch of manufactured material undergoes a series of standard QC measurements (e.g., particle and pore size distribution) followed by an application-specific test using appropriate peptide and protein test mixture. A packed column efficiency test is then performed on every batch approved, packed SEC column to further help ensure reproducible batch-to-batch and column-to-column performance for use in research or in a demanding validated method.

� Outstanding resolution of peptide and protein mixtures (from 1–1,000,000 K) obtained on high efficient packed columns containing 3.5 µm particles or 125Å, 200Å, or 450Å pores

� Compared to SEC columns containing 100% silica particles, Waters BEH-based SEC columns are stable at pH values greater than 7 and exhibit less non-desired, secondary ionic interactions between the SEC particle and peptide/protein

� Each column is shipped with Waters SEC Protein Standard Mix to help users establishing or confirm acceptable instrument and column performance

� Available or new HPLC-based columns complement existing UPLC-based SEC Columns to assist in method transfer based on users’ application and throughput needs

Benchmarking, Method Development, and Troubleshooting: BEH SEC Protein StandardsThe BEH SEC Protein Standards are specifically designed to help aid in the benchmarking of each set of columns. Each standard contains carefully chosen proteins unique to that chemistry, which has been worked out meticulously over time. These standards are used to QC the respective HPLC or UPLC columns which makes them an ideal choice for benchmarking a new column while also providing the capability to run the samples over time to monitor column performance.

Offers standards for:

� ACQUITY UPLC and XBridge Protein BEH SEC 125Å



ACQUITY UPLC BEH SEC Column Protein StandardsDescription Part No.

BEH125 SEC Protein Standard Mix 186006519

BEH200 SEC Protein Standard Mix 186006518

BEH450 SEC Protein Standard Mix 186006842NOTE: Size-exclusion c hromatography may require modifications to an existing ACQUITY UPLC System. Please refer to “Size Exclusion and Ion- Exchange Chromatography of Proteins using the ACQUITY UPLC

System”, (715002147 Rev A) or “Size Exclusion and Ion- Exchange Chromatography of Proteins using the

ACQUITY UPLC H-Class System”, (715002909 Rev A) for specific recommendations.


Bioseparations

Scalable SEC Separations Using UPLC- Versus HPLC-Based SECU

V A

bso

rban

ce (

280

nm

)

0.00

0.02

0.04

0.06

4.0 5.0 6.0 7.0 8.0 9.0 10.0 min

0.000

0.020

0.040

0.060

0.080

0.100

0.120

0.140

24 28 32 36 40 44 48 52 56 min

0.08

1a

1b

23

4

5

Two XBridge Protein BEH SEC, 200Å, 3.5 µm, 300 mm Length Columns in Series

ACQUITY UPLC BEH SEC, 200Å, 1.7 µm, 300 mm Length

Sample: BEH200 SEC Protein Standard MixFlow rate: 0.42 mL/min on 4.6 mm column 0.30 mL/min on 7.8 mm columnMobile phase: 100 mM sodium phosphate, pH 6.8

Compounds:1a. Thyroglobulin dimer (1.3 MDa)1b. Thyroglobulin (669 KDa)2. IgG (150 KDa)3. BSA (67 KDa)4. Myoglobin (14 KDa)5. Uracil (112 Da)

Comparison of separations of Waters BEH200 SEC Protein Standard Mix (p/n: 186006518) on two, XBridge Protein BEH SEC, 200Å, 3.5 μm HPLC Columns (300 mm length X 7.8 mm I.D.) run in series using an Alliance HPLC (Top Frame) and on an ACQUITY UPLC Protein BEH SEC, 200Å, 1.7 μm Column (300 mm length X 4.6 mm I.D.) using an ACQUITY UPLC H-Class Bio (Bottom Frame). The flow rates were scaled based on particle diameter and column I.D. to 0.42 mL/minute for the two HPLC columns run in series and 0.3 mL/minute for the UPLC column. Samples loads were also adjusted for column volume.

XBridge Protein BEH SEC ColumnsDescription Configuration Particle Size Dimension Part No.

XBridge Protein BEH SEC, 125Å Column with BEH125 SEC Protein Standard Mix* Guard Column 3.5 µm 7.8 mm x 30 mm —

XBridge Protein BEH SEC, 125Å Column with BEH125 SEC Protein Standard Mix* Column 3.5 µm 7.8 mm x 150 mm —

XBridge Protein BEH SEC, 125Å Column with BEH125 SEC Protein Standard Mix* Column 3.5 µm 7.8 mm x 300 mm —

XBridge Protein BEH SEC, 200Å Column with BEH200 SEC Protein Standard Mix Guard Column 3.5 µm 7.8 mm x 30 mm 176003594

XBridge Protein BEH SEC, 200Å Column with BEH200 SEC Protein Standard Mix Column 3.5 µm 7.8 mm x 150 mm 176003595


XBridge Protein BEH SEC, 450Å Column with BEH450 SEC Protein Standard Mix Guard Column 3.5 µm 7.8 mm x 30 mm 176003597



Straight Connection Tubing and End-fittings for XBridge Protein BEH SEC Column Column — — WAT006519

U-Bend Connection Tubing and End-fittings for XBridge Protein BEH SEC Column Column — — WAT084080* Available Q1, 2015

Waters Alliance HPLC System

Waters ACQUITY UPLC H-Class Bio System


Protein SeparationsThe analysis and characterization of protein samples requires the detection of small chemical differences between large molecules. Most often these analyses have employed an array of analytical techniques, each sensitive to a different property of the protein. Reversed-phase HPLC has not been fully exploited in these tests because the separation of proteins often yields relatively broad and asymmetrical peaks with poor recovery and significant carryover. Waters new reversed-phase, ethylene-bridged hybrid (BEH Technology) Protein Separation Technology Columns are specifically designed for the high resolution analysis of proteins.

Waters family of Protein BEH C4, 300Å Columns for protein separations:

� Separates proteins of various sizes, hydrophobicities, and isoelectric points

� Available as 3.5 µm packing for HPLC and 1.7 µm packing for UPLC methods

� Maximizes recovery and minimizes protein carryover

� Tolerates extreme pH and temperature

� Quality-control tested with MassPREP Protein Standard Mix (p/n: 186004900)

� Couples directly to ESI-MS for protein identification

AU

0.00

0.05

0.10

0.15

6 12 18 min

1

2

3

4

5

6

BEH C4, 300Å Columns can be used with proteins that have a wide range of properties. This protein mix was chosen to represent a range of isoelectric points, molecular weights, and hydrophobicities.

Column: XBridge Protein BEH C4, 300Å, 3.5 µm, 2.1 x 50 mmPart number: 186004498MobilephaseA: 0.1%trifluoroaceticacid in waterMobilephaseB: 0.075%trifluoroaceticacid in 71.4% acetonitrileFlow rate: 0.2 mL/minGradient: 28–100% B in 25 minColumn temp.: 40 °CInjection: 5 µLDetection: UV @ 220 nm

MassPREP Protein Standard (p/n: 186004900) 0.1% TFA in 5% acetonitrile

Compounds:1. Ribonuclease A 0.04 mg/mL 2. Cytochrome c 0.06 mg/mL3. Bovine Serum Albumin 0.20 mg/mL4. Myoglobin 0.13 mg/mL5. Enolase 0.22 mg/mL6. Phosphorylase b 0.59 mg/mL

300Å C4 Columns Developed for Protein Chromatography Batch-to-Batch Reproducibility

0 2 4 6 8 10 12 14 16 18 20 22 min

12

3

4

5

6

Batch A

Batch B

Batch C

This comparison shows the consistent batch-to-batch performance with a protein separation.

Column: ACQUITY UPLC Protein BEH C4, 300Å, 1.7 µm, 2.1 mm x 50 mmPart number: 186004495EluentA: 0.1%trifluoroaceticacidin 100% HPLC grade waterEluentB: 0.075%trifluoroaceticacidin 71.4% acetonitrileFlow rate: 0.2 mL/minGradient: 28–100% B in 25 minColumn temp.: 40 °CDetection: UV @ 220 nm

MassPREP Protein Standard (p/n: 186004900) 0.1% TFA in 5% acetonitrile

Compounds:1. Ribonuclease2. Cytochrome c3. BSA4. Myoglobin5. Enolase6. Phosphorylase b


Bioseparations

Minimal Protein CarryoverA

U

0.00

0.05

0 10 20 30 40 50 60 70 80 90 100 110 120 130 min

1st gradient without sample injection

2nd gradient without sample injection

Ovalbumin Absence of detectable Ovalbumin carryover

Column carryover was tested by running multiple gradients following a single injection. Protein peaks observed during the first gradient are not found in subsequent gradients.

Sample: 1.3 µg Ovalbumin injectedColumns: ACQUITY UPLC Protein BEH C4, 300Å, 1.7 µm, 2.1 mm x 50 mmPart number: 186004495EluentA: 0.1%trifluoroaceticacidin100%HPLC-gradewaterEluentB: 0.075%trifluoroaceticacidin71.4%acetonitrileFlow rate: 0.2 mL/minGradient: 28–100% B in 25 min, hold at 100 %B for 2 min then re-equilibrate column at 28% B for 18 min, gradient restarted at 45 min and at 90 min Column temp.: 40 °CDetection: UV @ 220 nm

BEH C4, 300Å Columns for Protein Characterization with UPLC-MS

8 10 12 14 16 18 20 22 24 26 28 30 32 34 min

%

9

Fc/2

LCFd

Fc/2 Clip1

Fc/2 Clip 2

Fd: 23960.5 DaLC: 23437.5 DaFc/2: 26502.5 Da 26648.5 Da 26973.0 DaFc/2Clip 1: 14488.5 Da 14650.5 Da 14812.5 DaFc/2Clip 2: 12178.0 Da

Sample: Lys-C digested (limited) reduced monoclonal antibodyColumn: ACQUITY UPLC Protein BEH C4, 300Å,1.7μm,2.1x50mmPart number: 186004495Eluent A: 0.1% formic acid in 100% HPLC grade waterEluent B: 0.1% formic acid in 100% acetonitrileFlow rate: 0.2 mL/minGradient: 5% B for 5 min then 5–21% B in 0.1 min, then 21–29% B in 30 minColumn temp.: 80 °C

Waters LCT Premier™ XE MS DetectionCapillary voltage: 3200 VCone voltage: 45 VSource temp.: 150 °CDesolvation temp.: 350 °CDesolvation Gas: 000 L/h

The large fragments obtained through LysC digestion of a monoclonal antibody can be separated on the UPLC BEH C4, 300Å Column coupled directly to ESI/Tof MS for identification of the individual peptide products.

NOTE: ACQUITY UPLC Protein BEH C4, 300Å, 1.7 μm Columns are designed for use with the ACQUITY UPLC System. The benefits of the small particle packing in ACQUITY UPLC Protein BEH C4, 300Å, 1.7 µm Columns are only realized with the low system volume and low detector dispersion of an ACQUITY UPLC System.

Improved Protein Resolution with UPLC Technology

AU

0.00

0.12

0.24

0.36

AU

0.00

0.12

0.24

0.36

0 12 24 36 48 60 min

12 3

45

6

1

23

45

6

Protein BEH C4, 300Å3.5 µm

Protein BEH C4, 300Å1.7 µm

The protein test mixture was separated on two BEH C4, 300Å Columns, one with 3.5 μm particles and the other with 1.7 μm particles. The UPLC separation provides sharper peaks for all the proteins in the test mixture. This translates into better resolution as shown by the multiple peaks around phosphorylase, at approximately 50 minutes. This comparison, with both columns, was performed on a UPLC system to preserve the minimized band-broadening. The benefits of the small particle UPLC BEH C4, 300Å Column would be lost without the optimized ACQUITY UPLC System.

Columns: ACQUITY UPLC Protein BEH C4,300Å,1.7μm(top), 3.5μm(bottom),2.1x150mmPart numbers: 186004497 (1.7 µm); 186004500 (3.5 µm)MobilephaseA: 0.1%trifluoroaceticacid in waterMobilephaseB: 0.1%trifluoroaceticacid in 100% acetonitrileFlow rate: 0.2 mL/minGradient: 20–55% B in 60 minColumn temp.: 80 °CInjection: 3.3 µLDetection: UV @ 220 nm

Sample: MassPREP Protein Standard (p/n: 186004900) 0.1% TFA in 5% acetonitrile

Compounds:1. Ribonuclease A 0.04 mg/mL2. Cytochrome c 0.06 mg/mL3. Bovine Serum Albumin 0.20 mg/mL4. Myoglobin 0.13 mg/mL5. Enolase 0.22 mg/mL6. Phosphorylase b 0.59 mg/mL


Waters Protein BEH C4, 300Å Offerings for HPLC and UPLC Protein SeparationsDescription Particle Size Dimension Part No.

XBridge Protein BEH C4, 300Å 3.5 µm 2.1 x 50 mm 186004498








XBridge Protein BEH C4, 300Å 3.5 µm 2.1 x 10 mm Sentry Guard, 2/pk 1860072301

XBridge Protein BEH C4, 300Å 3.5 µm 4.6 x 20 mm Sentry Guard, 2/pk 1860072352

XBridge Protein BEH C4, 300Å 5 µm 10 x 10 mm Guard Cartridge 186007305

XBridge Protein BEH C4, 300Å 5 µm 10 x 50 mm 186007306






























XBridge Protein BEH C4, 300Å Method Validation Kit 3.5 µm 4.6 x 100 mm 186005465

ACQUITY UPLC Protein BEH C4, 300Å 1.7 µm 1.0 x 50 mm 186005589






ACQUITY UPLC Protein BEH C4, 300Å 1.7 µm VanGuard Pre-column 186004623

ACQUITY UPLC Protein BEH C4, 300Å Method Validation Kit* 1.7 µm 2.1 x 100 mm 186004899

AACQUITY UPLC Protein BEH C4, 300Å Method Validation Kit* 1.7 µm 2.1 x 150 mm 186006549

nanoACQUITY Protein BEH C4, 300Å 1.7 µm 75 μm x 100 mm 186004639



*Three Columns from three different batches of material.1 Requires 2.1 x 10 mm Universal Sentry Guard Holder, p/n: WAT0979582 Requires 4.6 x 20 mm Universal Sentry Guard Holder, p/n: WAT046910NOTE: ACQUITY UPLC Protein BEH C4, 300Å, 1.7 μm Columns are designed for use with the ACQUITY UPLC System. The benefits of the small particle packing in ACQUITY UPLC Protein BEH C4, 300Å, 1.7 µm Columns are only realized with the low system volume and low detector dispersion of an ACQUITY UPLC System.


Bioseparations

Benchmarking, Method Development, and Troubleshooting: MassPREP Protein Standard MixThe MassPREP Protein Standard Mixture consists of carefully chosen proteins encompassing a wide range of properties. These mixtures contain proteins that vary in isoelectric points, molecular weights and hydrophobici ties. These characteristics provide the user with an attractive intact protein validation mixture that can be used for a variety of applications.

MassPREP Protein Standard MixProtein Sample Molecular Weight, MW Isoelectric Point, pI

Ribonuclease A, Bovine Pancrease 13.7 K 9.6

Cytochrome c, Horse Heart, 96% 12.4 K 10.25

Albumin, Bovine Serum, 96–99% 66.4 K 5.8

Myoglobin, Horse Heart >90% 16.7 K 6.53

Enolase from Baker’s Yeast (S. cereviasiae) 46.7 K 6.53

Phosphorylase b, Rabbit Muscle 97.0 K 7.18

Each Waters Analytical Standards and Reagents standard comes with a Certificate of Analysis that contains relevant, lot-specific information. Many times a chromatogram is attached using data acquired the same way a customer would use the standard.

MassPREP Protein Standard Mixture Certificate of Analysis

AU

0.00

0.15

0.30

12 24 36 48 min

1

2

3

45

6

Typical Protein Test Mixture Chromatogram.

MassPREP Protein Standard Mix on an ACQUITY UPLC Protein BEH C4, 1.7 µm, 2.1 x 150 mm Column

Column: ACQUITY UPLC Protein BEH C4, 300Å, 1.7μm,2.1x150mmMobile phase A: 0.1% TFA in waterMobile phase B: 0.1% TFA in 100% acetonitrileFlow rate: 0.2 mL/minGradient: 20–55% B in 60 minColumn temp.: 40 °CInjection: 3.3μLDetectionλ: 220nmSample: MassPREP Protein Standard mixture prepared in 0.1% TFA in 5% acetonitrile

Compounds:1. Ribonuclease A (0.04 mg/mL)2. Cytochrome c (0.06 mg/mL)3. Bovine Serum Albumin (0.20 mg/mL)4. Myoglobin (0.13 mg/mL)5. Enolase (0.22 mg/mL)6. Phosphorylase b (0.59 mg/mL)

Protein StandardsStandard Part No.

MassPREP Protein Standard Mix 186004900

Intact mAb Mass Check Standard 186006552


columns

Protein-Pak Hi Res Ion-Exchange (IEX) Columns for ACQUITY UPLC ApplicationsProtein-Pak Hi Res Ion-Exchange (IEX) Columns were developed to assist in the characterization of recombinant proteins, monoclonal antibodies, and other biological compounds. The non-porous, high compound binding capacity of these particles yields outstanding resolution of charged species in less time compared to use of many traditional porous IEX offerings. In addition, quality control testing with defined protein standards helps ensure consistent batch-to-batch performance.

� Designed for the characterization of protein charge variants and other biocompounds

� Two cation-exchangers (carboxymethyl and sulfopropyl) and one anion exchanger (quaternary ammonium) that address selectivity needs

� Non-porous, high-capacity stationary phases deliver fast separations that address high-throughput needs

� QC tested with protein standards to ensure batch-to- batch consistency

� eCord™-enabled to help monitor column use on ACQUITY UPLC Systems

Resolved Monoclonal Antibody (mAb) Isoform Separation

0.000

0.010

0.020

0.030

0.040

0.000

0.002

0.004

0.006

0.008

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 min

AU

AU

Column: Waters Protein-Pak Hi Res CM, 7 µm, 4.6 x 100 mmPart number: 186004929Flow rate: 0.67 mL/minEluent A: 20 mM disodium hydrogen phosphate pH 7Eluent B: 0.7 M sodium chloride in BGradient: 0–10% B in 25 minColumntemp.: 30˚CDetection: UV @ 280 nm

Column: Competitor’s CM Cation Exchanger, 4 x 250 mmFlow rate: 0.51 mL/minEluent A: 20 mM disodium hydrogen phosphate pH 7Eluent B: 0.7 M sodium chloride in BGradient: 0–10% B in 62.5 min

Cation-exchange chromatography is a useful tool for the characterization and quantitation of mAb or recombinant protein variants. Use of Waters Protein-Pak Hi Res CM Column on an ACQUITY UPLC System increases sample throughput while maintaining resolution between intended product and undesired variants.

Protein-Pak Hi Res CM Analysis of T hree mAbs Containing Different Levels of Variants

0.000

0.010

0.020

0.030

0.040

0.000

0.010

0.020

0.000

0.005

0.010

0.015

K0

0 2 4 6 8 10 12 14 16 18 20 22 24 min

K1 K2

Humanized IgG1

Humanized IgG2

Chimeric IgG1

AU

AU

AU

Sequence, production, storage, and shipping conditions influence the degree of variants contained in a biotherapeutic protein. Waters Protein-Pak Hi Res CM Column can successfully resolve variations that may involve as little as a single amino acid change (K0 = No terminal lysines, K1 = One terminal Lysine, and K2 = Two terminal Lysines).

ACQUITY UPLC System configuration guidelines for ion exchange are described in service notes p/n: 715002147A.

Column: Protein-Pak Hi Res CM, 7 µm, 4.6 x 100 mmPart number: 186004929Flow rate: 0.67 mL/minMobile phase A: 20 mM sodium phosphate buffer, pH 7.0Mobile phase B: 20 mM sodium phosphate buffer, 0.7 M sodium chloride, pH 7.0Gradient: 0–10% B in 25 minColumntemp.: 30˚CDetection: UV @ 280 nm

Protein-Pak Hi Res UPLC ColumnsDescription Dimension Part No.

Protein-Pak Hi Res CM, 7 µm 4.6 x 100 mm 186004929

Protein-Pak Hi Res SP, 7 µm 4.6 x 100 mm 186004930

Protein-Pak Hi Res Q, 5 µm 4.6 x 100 mm 186004931NOTE: Only when Protein-Pak Hi Res IEX Columns are combined with the ACQUITY UPLC System are the full performance benefits realized. See Waters service notes p/n: 715002147A for ACQUITY UPLC System configuration guidelines for ion-exchange chromatography.


Bioseparations

ACQUITY UPLC Technology for biotherapeutic characterization.

Application of Waters UPLC Technology for Biotherapeutic Characterization

ACQUITY UPLC allows analytical chemists to reach far beyond conventional LC separations and has proven itself to be an asset to laboratories around the world. UPLC sets new standards in resolution, sensitivity, and through-put by being the first holistically-designed system that maximizes for rapid, high-resolution analyses. It has fueled hundreds of peer-reviewed papers, helps laboratories conserve resources, and has served the needs of regulatory agencies around the globe. ACQUITY UPLC simultaneously makes your laboratory more sustainable and more efficient.

Manufacturing Consistency for Enhanced Assurance

The ability to obtain the same high quality separations regardless of column lot is of critical importance to the successful development and commercialization of biotherapeutics. Each batch of Protein-Pak Hi Res IEX material is tested with a relevant mixture of protein standards to help ensure consistent column-to-column performance.

Novel IEX Particles Ideal for Biomolecule Characterizations

Protein-Pak Hi Res IEX Columns contain non-porous, pH tolerant, hydrophilic particles whose surface consists of a multi-layered network of either anion (5 µm) or cation (7 µm) exchange groups. T his innovative particle and bonding chemistry produces particles with greater protein loading capacities than found on many traditional mono-disperse, non-porous resins. T his translates into columns that can resolve complex mixtures of biomolecules in comparatively short times compared to use of alternative porous or non-porous IEX Column offerings.

DescriptionProtein-Pak

Hi Res QProtein-Pak Hi Res CM

Protein-Pak Hi Res SP

Ion Exchange Strong Anion Weak Cation Strong Cation

Functional GroupQuaternary ammonium

Carboxymethyl Sulfopropyl

MatrixHydrophilic

polymerHydrophilic polymer

Hydrophilic polymer

Particle size (µm) 5 7 7

Pore size Non porous Non porous Non porous

I.D. x L (mm) 4.6 x 100 4.6 x 100 4.6 x 100

Counter ion Cl- Na+ Na+

pH range 3–10 3–10 3–10

Temperature (°C) 10–60 10–60 10–60

pKa 10.5 4.9 2.3

Flow Rates 0.3–0.6 mL/min 0.5–1.4 mL/min 0.5–1.4 mL/min

Approx Protein Binding Capacity in mgs per column (i.e., BSA for Hi Res Q Column. Lysozyme for Hi Res CM and Hi Res SP Columns)*

58 33 25

* For optimal resolution of complex samples, do not exceed 20% of the column’s protein binding capacity.

Protein-Pak Hi Res IEX Column Batch-to-Batch Reproducibility

0.00

0.01

0.02

3 4 5 6 7 8 9 10 11 12 13 min

0.00

0.01

0.02

0.00

0.01

0.02

Trypsin Inhibitor

Myoglobin Apo-Transferrin

AU

AU

AU

Batch A

Batch B

Batch C

Each batch of Protein-Pak Hi Res SP, CM, and Q Column packing material is chromatography tested using a relevant protein standard mixture to help ensure consistent and predictable performance (Protein-Pak Hi Res Q data shown).

Column: Protein-Pak Hi Res Q, 5 µm, 4.6 x 100 mm Part number: 186004931 Flow rate: 0.6 mL/minMobile phase A: 20 mM tris buffer, pH 8.5Mobile phase B: 20 mM tris buffer, pH 8.5, 0.5 M sodium chloride Gradient: 0–60% B in 30 minColumntemp.: 30˚CDetection: UV @ 280 nm


Protein-Pak Hi Res HIC Column and HIC Protein StandardProtein-Pak Hi Res HIC (Hydrophobic Interaction Chromatography) columns contain non-porous, polymethacrylate-based particles (2.5 μm) functionalized with a butyl ligand coating and are well suited for the characterization of proteins and biotherapeutics including monoclonal antibodies (mAb) and antibody drug conjugates (ADC).

While reversed-phase chromatography is a frequently used bioanalytical technique, HIC offers attractive orthogonal separation advantages. In reversed-phase LC, proteins are retained by hydrophobic interaction with alkyl groups (e.g., C18) on the packing material. However, the butyl ligand density on Waters Protein-Pak Hi Res HIC Column is comparatively less resulting in fewer protein-ligand hyrdophobic interactions. Consequently, HIC-based elution is possible using gradients of decreasing salt concentration at physiological pH values. Use of denaturing organic solvent eluents (e.g., acetonitrile in 0.1% TFA) thus allowing biotherapeutics (e.g., acid labile, cysteine-linked ADCs) to be analyzed in non-denaturing conditions.

In addition, Waters has developed HIC Protein Standard Test Mix designed for user verification of HPLC/UPLC instrument and Protein-Pak Hi Res HIC Column performance prior to sample analyses. This intact protein validation mix, when used on a regular basis, helps monitor system and column performance and is also highly valuable in method development and/or troubleshooting. The standard contains a carefully chosen set of six proteins that provide good chromatographic representation using a gradient of decreasing salt concentration.

� Ideally suited for hydrophobic-based separations for protein characterization using non-denaturing conditions

� Use of non-porous particles help deliver fast, efficient separations to address high-throughput needs

� Shipped with Waters HIC Protein Test Standard to help test for acceptable instrument and HIC column performance

� Successfully used for the analysis of cysteine-based, antibody drug conjugates

Protein-Pak Hi Res HIC Column and HIC Protein Standard

Separation of ADC Samples on Protein-Pak Hi Res HIC Column

Using a gradient of decreasing salt concentration and on-denaturing eluents, Waters Protein-Pak Hi Res HIC Column is well suited for the separation of proteins of various molecular weight s and hydrophobic interactions.

Monitoring drug load variability. Three batches of cysteine-linked ADCs were synthesized, each with a different level of drug conjugation (low, medium, high) and separated using hydrophobic interaction chromatography. The drug load distribution shifted from low to high corresponding to an increase in the load of the hydrophobic drug.

Sample: Cysteine-conjugated ADCs were obtained by a collaborator at a concentration of 10 mg/mL in formulation buffer. Samples were prepared for analysis at a concentration of 2 mg/mL in 1 M ammonium sulfate ((NH4)2SO4)10μLinjectedColumn: Protein-Pak Hi Res HIC, 2.5 µm, 4.6 x 100 mm (p/n: 176003576)Flow rate: 0.7 mL/minMobile phase A: 2.5 M (NH4)2SO4 125 mM phosphate buffer, pH 6.7 and 5% isopropanol Mobile phase B: 125 mM phosphate buffer, pH 6.7 and 5% isopropanol Gradient: 0–50%B in 10 mins. Hold at 50%B for 5 mins.Detection: 280 nm Temperature: 30 °C

Protein-Pak Hi Res HIC ColumnsDescription Dimension Part No.

Protein-Pak Hi Res HIC, 2.5 µm and HIC Protein Standard

4.6 x 35 mm 176003575

Protein-Pak Hi Res HIC, 2.5 µm and HIC Protein Standard

4.6 x 100 mm 176003576

HIC Protein Test Standard — 186007953

Compounds:1. Cytochrome c2. Myoglobin3. Ribonuclease A4. Lysozyme5. Enolase6. Alpha-chymotrypsinogen A

Sample: HIC Protein Standard Test Mix (p/n: 186007953)dissolvedin100μL 50%A/50%B2μLinjectedColumn: Protein-Pak Hi Res HIC, 2.5 µm, 4.6 x 100 mm (p/n: 176003576)Flow rate: 0.6 mL/minMobile phase A: 2 M (NH4)2SO4 in 50 mM NaH2PO4/ Na2HPO4, pH 6.9Mobile phase B: 50 mM NaH2PO4/Na2HPO4, pH 6.9 Gradient: 0–100%B in 15 mins Detection: 220 nm Temperature: 30 °C

AU

0.00

0.03

0.06

0.09

0.12

0 5 10 15 min

12 3

45

6


Bioseparations

0 10 20 30 40 min

1 2

4

43

5

1 2 4 4

3

5

A

B

BioSuite Ion-Exchange HPLC ColumnsBioSuite Ion-exchange Column offerings include strong and weak, cation (CXC) and anion exchangers (AXC) bonded to a pH stable (i.e., pH 2–12), methacrylic ester-based polymeric resin. The availability of four separation chemistries provides chromatographers with the flexibility required to develop methods that separate proteins or peptides based upon minor charge differences. Non-porous (NP) and porous IEX columns are also available. Speed and superior chromatographic resolution are possible using the NP IEX offerings. Waters porous ion exchangers are available for applications requiring greater protein or peptide binding capacity. In addition, selected BioSuite Ion-exchange Columns are offered in PEEK hardware as well as in 21.5 mm I.D. preparative column sizes.

Enhanced Compound Resolution by Ion-Exchange Chromatography on BioSuite SP Non-Porous (NP) vs. Porous CXC Columns

B) BioSuite SP, 10 µm CXC

3 4 5 6 min 20 30 40 min

1

2

1

2A B

A) BioSuite SP, 2.5 µm NP CXC

Sample: Alpha-Chymotrypsinogen A (Peak 1) and Ribonuclease (Peak 2)Columns: A:BioSuite,NPCXC,2.5μm,4.6mmx35mm B:BioSuiteSP,CXC,10μm,7.5mmx75mmPart numbers: 186002183 (2.5 µm) & 186002184 (10 µm)Eluent A: 20 mM MES [2(N-morpholino)ethanesulfonic acid], pH 6.0Eluent B: 20 mM MES, pH 6.0 containing 0.5 M sodium chlorideFlow rate: A: 1.5 mL/min B: 1.0 mL/minGradient: A: Linear gradient from 0 to 100% B in 10 min B: Linear gradient from 0 to 100% B in 60 minColumn temp.: 25 °CDetection: UV @ 280 nm

Protein Selectivity Differences Observed by Ion-Exchange Chromatography on BioSuite CM (Weak-Cation Exchange) vs. SP (Strong-Cation Exchange) Columns

A) BioSuite CM, 10 μm CXC

B) BioSuite SP, 10 μm CXC

Sample: Trypsinogen (Peak 1), Ribonuclease (Peak 2), Alpha- Chymotrypsinogen A (Peak 3), Cytochrome c (Peak 4), and Lysozyme (Peak 5)Columns: A:BioSuiteCM,CXC,10μm,7.5mmx75mm B:BioSuiteSP,CXC,10μm,7.5mmx75mmPart numbers: 186002186 (BioSuite CM) & 186002184 (BioSuite SP)Eluent A: 20 mM sodium phosphate, pH 7.0Eluent B: 20 mM sodium phosphate, pH 7.0 containing 0.5 M sodium chlorideFlow rate: 1.0 mL/minGradient: Linear gradient from 0 to 100% B in 60 min Column temp.: 25 °CDetection: UV @ 280 nm

BioSuite pC18 and pPhenyl HPLC Columns

Description MatrixDiameter

Width Diameter Length

Part No.

BioSuite pC18, 2.5 µm NP RPC Polymer 4.6 mm 35 mm 186002152

BioSuite pC18, 500, 7 µm RPC Polymer 2.0 mm 150 mm 186002153



BioSuite pPhenyl, 1000, 10 µm RPC Polymer 2.0 mm 75 mm 186002156



BioSuite Q-PEEK and SP-PEEK Columns are Available in 4.6 x 50 mm


Benchmarking, Method Development, and Troubleshooting: Ion Exchange StandardsIon-Exchange Standards are sets of standards that allow the user to benchmark anion- or cation-exchange columns on a regular basis in order to have confidence in results as well as providing a troubleshooting tool for any issues that may arise.

� IEX Anion Test Standard

� IEX Cation Test Standard

AU

0.040

0.050

0.060

0.070

0.080

0.090

0.030

0.020

0.010

0.0008

Lysozyme

Ribonuclease A

Cytochrome C

10 12 14 16 18 20 22 24 min

Protein-Pak Hi Res CM Column using the IEX Cation Test Standard

BioSuite IEX HPLC Columns

Description Matrix Pore SizeExclusion Limit (Daltons)

against Polyethylene GlycolInner

DiameterLength

Column Volume (mL)

# Approx Protein Binding Capacity Per Pre-Packed Column

Part No.

BioSuite Q-PEEK, 10 µm AXC Polymer 4000Å >5,000,000 4.6 mm 50 mm 0.83 58 mg1 186002176

BioSuite SP-PEEK, 7 µm CXC Polymer 1300Å >4,000,000 4.6 mm 50 mm 0.83 58 mg2 186002182

BioSuite DEAE, 2.5 µm NP AXC Polymer n/a 500 4.6 mm 35 mm 0.58 2.9 mg1 186002179

BioSuite SP, 2.5 µm NP CXC Polymer n/a 500 4.6 mm 35 mm 0.58 2.9 mg3 186002183

BioSuite Q, 10 µm AXC Polymer 1000Å 1,000,000 7.5 mm 75 mm 3.31 331 mg1 186002177

BioSuite Q, 13 µm AXC Polymer 1000Å 1,000,000 21.5 mm 150 mm 54.45 5,445 mg1 186002178

BioSuite DEAE, 10 µm AXC Polymer 1000Å 1,000,000 7.5 mm 75 mm 3.31 99 mg1 186002180

BioSuite DEAE, 13 µm AXC Polymer 1000Å 1,000,000 21.5 mm 150 mm 54.45 1633 mg1 186002181

BioSuite SP, 10 µm CXC Polymer 1000Å 1,000,000 7.5 mm 75 mm 3.31 132 mg3 186002184

BioSuite SP, 13 µm CXC Polymer 1000Å 1,000,000 21.5 mm 150 mm 54.45 2,178 mg3 186002185

BioSuite CM, 10 µm CXC Polymer 1000Å 1,000,000 7.5 mm 75 mm 3.31 149 mg3 186002186

BioSuite CM, 13 µm CXC Polymer 1000Å 1,000,000 21.5 mm 150 mm 54.45 2,450 mg3 1860021871 Data generated with BSA 2 Data generated with Gamma Globulin 3 Data generated with Hemoglobin NOTE: For best resolution of complex samples, do not exceed 20% of the Column’s protein binding capacity

IEX StandardsDescription Part No.

IEX Anion Test Standard 186006869

IEX Cation Test Standard 186006870

Protein-Pak PW HPLC Column SeriesDescription Dimension Part No.

Polymeric Weak Anion-Exchanger 7.5 x 75 mm WAT088044

Protein-Pak DEAE 5PW Glass Column 8 x 75 mm WAT011783

Protein-Pak DEAE 5PW Steel Column 21.5 x 150 mm WAT010640

Polymeric Strong Cation-Exchanger 7.5 x 75 mm WAT088043

Protein-Pak SP 5PW Glass Column 8 x 75 mm WAT011784

Protein-Pak PW Series ColumnsWaters also offers a line of 10 µm polymer-based ion-exchangers pre-packed in steel or glass columns. The Protein-Pak 5PW Columns are available as DEAE and SP ion-exchangers. T hese columns can be used on HPLC and FPLC systems in both analytical and preparative configurations.

Approximate Protein Binding Capacity per Pre-packed Column* Protein-Pak HR Packing

Pre-packed Column Q** DEAE** SP*** CM***

5 x 50 mm 60 mg 40 mg 40 mg 25 mg

5 x 100 mm 130 mg 150 mg 80 mg 45 mg

10 x 100 mm 500 mg 300 mg 300 mg 180 mg


Bioseparations

Protein-Pak High Resolution (HR) Ion-Exchange Glass ColumnsThe Protein-Pak HR 8 µm and 15 µm packing materials are available pre-packed in Waters Advanced Purification (AP) Glass Columns in a choice of 5 mm I.D. (mini-column) or 10 mm I.D. by 100 mm in length. The 5 mm I.D. column is also available in a 50 mm length. These columns are compatible with any HPLC and FPLC system with the use of an adapter kit.

Waters Protein-Pak HR packing materials are based on rigid, hydrophilic, polymethacrylate particles with large 1000Å pores. The naturally hydrophilic polymer reduces non-specific adsorption, resulting in quantitative recovery of protein mass and bioactivity. These packings are compatible with buffers in the pH range 2–12, and will withstand exposure to caustic solutions, such as 0.1–1.0 M sodium hydroxide and acetic solutions, such as 20% acetic acid, for cleaning purposes.

Protein-Pak HR ion exchangers are available with a Q functional group, a strong anion exchanger; DEAE, a weak anion exchanger; SP, a strong cation exchanger; and CM, a weak cation exchanger. The principal difference between a weak and strong ion exchanger does not lie in the protein binding capacity, but in the pH range of operation. Weak ion exchangers tend to have a more restricted useful pH range of operation.

Properties of Protein-Pak HR* Columns

Protein-PakProtein-Pak

Q HR1Protein-Pak DEAE HR2

Protein-Pak CM HR3 SP HR4

Type of material Polymer Polymer Polymer Polymer 1 For best resolution do not exceed 20% of the protein binding capacity.

2 Bovine serum albumin in 20 mM Tris/Cl pH 8.2 was used to measure protein binding capacity of Protein-Pak Q and DEAE HR.

3 Cytochrome c in 25 mM MES pH 5.0 was used to measure protein binding capacity of Protein-Pak SP and CM HR.

4 Same conditions as CM. Protein binding capacity of Protein-Pak SP 40 HR is 20 mg/mL.

Protein binding capacity 60 mg/mL 40 mg/mL 25 mg/mL 40 mg/mL

Ion-exchange capacity 200 µeq/mL 250 µeq/mL 175 µeq/mL 225 µeq/mL

Nominal pK 11.7 9.0 5.7 2.2

Typical protein recovery >95% >95% >95% >95%

Typical recovery of biological activity >90% >90% >90% >90%

pH stability 2–12 2–12 2–12 2–12

Protein Resolution on Protein-Pak DEAE 15HR Anion-Exchange Column

1

2

3

45

40 min

Column: AP-1 Glass Column, 10 x 100 mmPart number: WAT021901 Buffer A: 20 mM Tris-HCl pH 8.2Buffer B: Buffer A with 1 M Sodium ChlorideFlow rate: 1.56 mL/minGradient: 0 to 25% Buffer B over 38 minLoad: 0.5 mg proteinDetection: 280 nm

Compounds:1. Adenosine2. Carbonic anhydrase3. Human transferrin4. Ovalbumin5. Soybean trypsin inhibitor

Protein-Pak HR Ion-Exchange Glass ColumnsIon-Exchange Packing

Pore Size Size Dimension Particle Type Part No.

Protein-Pak Q 8HR 8 µm 1000Å

5 x 50 mmPolymeric strong anion exchanger

WAT039575

5 x 100 mm WAT039630

10 x 100 mm WAT035980

Protein-Pak Q 15HR

15 µm 1000Å 5 x 50 mm Polymeric strong

anion exchangerWAT039782

10 x 100 mm WAT037663

Protein-Pak DEAE 8HR

8 µm 1000Å

5 x 50 mmPolymeric weak anion exchanger

WAT039791

5 x 100 mm WAT039783

10 x 100 mm WAT035650

Protein-Pak DEAE 15HR

15 µm 1000Å

5 x 50 mmPolymeric weak anion exchanger

WAT039780

5 x 100 mm WAT039786

10 x 100 mm WAT038564

Protein-Pak SP 8HR

8 µm 1000Å

5 x 50 mmPolymeric strong cation exchanger

WAT039570

5 x 100 mm WAT039625

10 x 100 mm WAT035655

Protein-Pak SP 15HR

15 µm 1000Å 10 x 100 mmPolymeric strong cation exchanger

WAT038567

Protein-Pak CM 8HR

8 µm 1000Å

5 x 50 mmPolymeric weak

cation exchanger

WAT039790

5 x 100 mm WAT039785

10 x 100 mm WAT035970

Protein-Pak CM 15HR

15 µm 1000Å 5 x 50 mmPolymeric weak

cation exchangerWAT039787


Advanced Purification (AP) Glass ColumnsWaters AP series of glass columns are constructed of biocompatible glass and polymeric materials and can be easily used with silica, polymer, or soft gel packings. To optimize flow and ensure uniform sample distribution onto the packed bed, each column incorporates a distributor. A replaceable filter protects the packing from large particulate contaminants. Empty AP Glass Ccolumns are available in a variety of sizes and utilize the same design to ensure predictable methods transfer among them. AP Glass Columns are compatible with both analytical and preparative HPLC and FPLC systems.

Advanced Purification (AP) Glass ColumnsDimension Bed Volume (mL) Flow Rate (mL/min) Pressure Rating (psi/MPa) Part No.

5 x 50 mm 0.8–1.2 0–4 1500 psi/10 MPa WAT064-01

5 x 100 mm 1.8–2.2 0–4 1500 psi/10 MPa WAT064-02

10 x 100 mm 5–8 0–4 1500 psi/10 MPa WAT021901

10 x 200 mm 13–16 0–4 1500 psi/10 MPa WAT021902

10 x 300 mm 21–24 0–4 1500 psi/10 MPa WAT021903

10 x 600 mm 45–48 0–4 1500 psi/10 MPa WAT021906

20 x 100 mm 22–31 4–16 1000 psi/6.8 MPa WAT027501

20 x 200 mm 53–62 4–16 1000 psi/6.8 MPa WAT027502

20 x 300 mm 85–94 4–16 1000 psi/6.8 MPa WAT027503

20 x 600 mm 179–188 4–16 1000 psi/6.8 MPa WAT027506

50 x 100 mm 137–196 16–100 500 psi/3.4 MPa WAT023321

50 x 200 mm 333–392 16–100 500 psi/3.4 MPa WAT023332

50 x 300 mm 530–589 16–100 500 psi/3.4 MPa WAT023323

50 x 600 mm 1118–1177 16–100 500 psi/3.4 MPa WAT023326

Description Qty Part No.

1 Compression Screw and Ferrule ‘Z’ Fitting, Plastic 1/pk WAT082708

2 Union ‘Z’ Fitting, Plastic 1/pk WAT082745

AP-1 Column Accessories and Spare PartsDescription Dimension Part No.

Glass Tube

10 x 100 mm WAT021992

10 x 200 mm WAT022033

10 x 300 mm WAT022034

10 x 600 mm WAT022035

Plastic Shield

10 x 100 mm WAT021927

10 x 200 mm WAT021945

10 x 300 mm WAT021946

10 x 600 mm WAT021947

O-rings, 5/pk — WAT021907

Filters, 10/pk — WAT021910

Replacement Tubing (Tefzel) (1/16 in. O.D. x 0.009 in. I.D. x 10 feet) (1.6 mm O.D. x 0.23 mm I.D. x 3 m)

— WAT021950

Inlet Connector Assembly — WAT021904

Description Qty. Part No.

1 Collet and Nut Assembly (3/8–24) 10/pk WAT005138

2 Ferrule 1/8” Tube 10/pk WAT005136

3 Union 3/8–24 x ‘Z’ Fitting 5/pk WAT005137

AP MiniColumn Accessories and Spare PartsDescription Dimension Part No.

Glass Tube5 x 50 mm WAT038802

5 x 100 mm WAT038803

Column Jacket5 x 50 mm WAT038804

5 x 100 mm WAT038805


O-rings, 13/pk (includes 10 inlet/outlet and 3 funnel) — WAT038807


Advanced Purification (AP) Glass Column Accessories and Spare Parts

Waters AP Glass Columns feature non-metallic construction and adjustable bed height with easy-to-use coarse and fine adjustments. The AP Glass Columns are available in a variety of dimensions.

2

Connection of an AP MiniColumn and an AP-1 Column to 1/8” OD Tubing

131

2


Bioseparations



2 Union, Plastic 1/pk WAT021951


Glass Tube

50 x 100 mm WAT019876

50 x 200 mm WAT019877

50 x 300 mm WAT019878

Plastic Shield

50 x 100 mm WAT023370

50 x 200 mm WAT023371

50 x 300 mm WAT023372

50 x 600 mm WAT023373


Filter, 2/pk — WAT023343

Replacement Tubing (Tefzel) 1/8 in. O.D. x 0.040 in. I.D. x 10 feet) (3.2 mm O.D. x 1.02 mm I.D. x 3 m)

— WAT023344


Outlet Connector Assembly — WAT023348

Collet and Nut Assembly — WAT023346

Ferrule, 10/pk — WAT023347

Funnel Assembly — WAT023396




3 Union 3/8–24 x 3/8–24 1/pk WAT082734


Glass Tube

20 x 100 mm WAT019891

20 x 200 mm WAT019892

20 x 300 mm WAT019893

Plastic Shield

20 x 100 mm WAT027542

20 x 200 mm WAT027543

20 x 300 mm WAT027544



Replacement Tubing (Tefzel) (1/8 in. O.D. x 0.040 in. I.D. x 10 feet) (3.2 mm O.D. x 1.02 mm I.D. x 3 m)

— WAT023344


Distributors/Inserts, 5/pk — 700004715

Connection of an AP-2 and an AP-5 Column to 1/8” OD Tubing Connection of Pharmacia Fitting to 1/16” OD Tubing




3 Union 3/8–24 x ‘Z’ Fitting 5/pk WAT005137


5 Compression Screw ‘Z’ Fitting, Steel 10/pk WAT005070

6 Ferrule 1/16” Steel 10/pk WAT005063




Connection of a Protein-Pak Steel Column to 1/16” and 1/8” OD Tubing Connection of 1/8” or 1/16” Flanged Type Fitting to 1/8” OD Tubing

1 13

2 2

2 1

3 4

25

61 2

1


Accell Plus Ion-Exchange Packings

Solid-Phase Extraction for Protein Sample Preparation

Waters Accell Plus ion-exchange packings are 40 µm, 300Å polymer-coated, silica-based materials for both lab- and process-scale chromatography. Accell Plus, available as a QMA strong anion exchanger or CM, a weak cation exchanger, is easy to pack and is excellent for the purification of proteins, enzymes, and immunoglobulins. The rigid silica-based packing material will withstand very high flow rates during cleaning and re equilibration cycles. Normal flow rates are used during sample loading and elution to obtain the best possible resolution.

Accell Plus bulk material may be packed into our Advanced Purification (AP) Glass Columns. Pre-packed 0.7 mL Sep-Pak Cartridges can be used for rapid method screening or solid-phase extraction applications.

To estimate packed bed volume for a known amount of Accell Plus:Accell Plus used (g) x 2 = packed bed volume (mL)

Accell Plus Sep-Pak CartridgesSep-Pak Plus Cartridges packed with Accell Plus ion exchangers provide a rapid, economical means to clean up heavily contaminated samples that would damage a high resolution column. They can also be used to rapidly screen chromatographic conditions. These are also available in a variety of configurations.

Accell Plus PrepPak Cartridges* (47 x 300 mm)Economical, convenient preparative separations in the 500 mg to 10 g range. For a complete listing of Waters products for preparative chromatography, go to www.waters.com

Protein Binding Capacity* of Accell PlusAccell Plus QMA** 200 mg

BSA/g packingAccell Plus CM*** 175 mg

Cytochrome c/g packing

* For best resolution do not exceed 20% of the protein binding capacity.

** Bovine serum albumin in 20 mM Tris/Cl pH 7.0 was used to measure protein binding capacity of Accell Plus QMA.

*** Cytochrome c in 20 mM sodium phosphate pH 6.3 was used to measure protein binding capacity of Accell Plus CM.

Accell Plus PrepPak Cartridges (47 x 300 mm)Description Particle Size Pore Size Part No.

Accell Plus CM* 40 µm 300Å WAT036545

PrepPak 1000 Module — — WAT089592

* Requires PrepPak 1000 Module

Accell Plus Sep-Pak CartridgesDescription Ion-Exchange Type Part No.

Accell Plus CM Weak Cation Exchanger WAT020550

Accell Plus QMA Strong Anion Exchanger WAT020545

Accell Plus QMA Plus Strong Anion Exchanger 186004540

Accell Plus Ion-Exchange Bulk PackingsFor all preparative isolations based on ionic interactions, particularly proteins, enzymes, and immunoglobulins.

Ion-Exchange Sample Preparation with Sep-Pak CartridgesTo perform ion-exchange sample preparation with Sep-Pak Cartridges, use a gradient of pH or ionic strength with Accell Plus CM, Accell Plus QMA or NH2 as a sorbent.

� Condition the cartridge with six to ten hold-up volumes of de-ionized water or weak buffer

� Load the sample dissolved in a solution of deionized water or buffer

� Elute unwanted weakly bound components with a weak buffer

� Elute the first component of interest with a stronger buffer (change the pH or ionic strength)

� Elute other components of interest with progressively stronger buffers

� When you recover all of your components, discard the used cartridge in an appropriate manner

General Elution Protocol for Ion-Exchange Chromatography on Sep-Pak Cartridges (NH2, Accell Plus QMA, Accell Plus CM)

Basic Steps for SPE

Load Sample(Black)

StepElute 1

StepElute 2

StepElute 3

Condition/Equilibrate

1 2 3

1. Condition and equilibrate the cartridge (not required for normal phase) 2. Load sample 3. Elute components—increase strength of mobile phase in steps

Accell Plus Ion-Exchange Bulk PackingsDescription Particle Size Pore Size Qty. Part No.

Accell Plus QMA 40 µm 300Å 100 g WAT010742

Anion Exchanger — — 500 g WAT010741

Accell Plus CM 40 µm 300Å 100 g WAT010740

Cation Exchanger — — 500 g WAT010739


Bioseparations

BioSuite Size-Exclusion HPLC Columns (SEC)BioSuite ultra high resolution (UHR), high resolution (HR), and standard size-exclusion column packings use a rigid yet “wettable” silica-based media that is stable from pH 2.5–7.5. As indicated in the calibration curve tables, the exclusion limits of BioSuite SEC packings are determined by the particle and pore size of the silica-based material. Particle size of the SEC packing media as well as column length are important parameters that determine separation efficiency. BioSuite 4 µm particle size, UHR Columns provide maximum separation efficiency, followed by BioSuite HR Columns and BioSuite Standard SEC Columns. To maximize column life of analytical (i.e., 4.6 mm or 7.8 mm I.D.) or preparative (i.e., 21.5 mm I.D.) SEC Columns, use of BioSuite Guard Columns is recommended.

LC-MS Analysis of Protein Standards Using BioSuite 250, 5 µm High Resolution (HR) SEC Column (LC-MS conditions as above)

2 4 6 8 10 12 14 16 18 20 min0

0

100

%

1

2

3

4

5

6

Compounds:1. Thyroglobulin 669 KDa2. BSA Dimer 136 KDa3. BSA 68 KDa4. ß-Lactoglobulin 36 KDa5. Myoglobin 16 KDa6. Cytochrome c 12 Kda

20 4 6 8 10 12 14 min

0

100

%

TIC

60000 80000 100000 120000 140000 mass0

100

%

62801

66443

66548

68030

66420

60000 70000 80000 90000 100000 110000 120000 130000 140000 mass0

100

%

132835

66417*

6550464540

13101166506

66555

6750567648

132991

133095

133175135141

BSA Monomer

BSA Dimer

BSA Aggregate

BSA Monomer

BSA Dimer

DeconvolutedMass Spectra

* Monomer is observed due to incomplete chromatographic resolution and in-source dissociation.LC-MS: Alliance HPLC with 2996 PDA and ZQ (top)

or Q-Tof™ Mass detectors (bottom) Sample: BSA (heat aggregated)Column: BioSuite HR SEC, 250Å, 5 µm, 7.8 mm x 300 mmPart number: 186002165Injection: 10 µL Mobile phase: 50 mM ammonium formate (pH 6.5) Flow rate: 1 mL/minTemperature: 25 °CMS Source: ESI+MS Capillary: 3.3 kVMS cone: 30 VMass range: 400 – 3000 m/z

LC-MS Analysis of BSA Aggregation Using BioSuite 250, HR 5 µm SEC Column

BioSuite SEC Reference: SEC- MS Analysis of Aggregates in Protein Mixtures. Application Book Supplement of LC/GC Europe. Sept. 2003. (Waters Literature Reference: 720000743EN)


Protein Calibration Curves for BioSuite Ultra-High Resolution (UHR) SEC Columns

6100

1,000

10,000

100,000

1,000,000

8 10 12 min

MW

BioSuite 125Å, 4 µm UHR SECBioSuite 250Å, 4 µm UHR SEC

Sample: Thyroglobulin (670,000 Da), Gamma globulin (155,000 Da), Bovine serum albumin (66,330 Da), Beta lactoglobulin (18,400 Da), Lysozyme (14,300 Da), Cytochrome c (12,400 Da), Triglycine (189 Da)Columns: BioSuiteUHRSEC,250Å,4μm,4.6mmx300mm BioSuiteSEC,125Å,4μm,4.6mmx300mmPart numbers: 186002162 (250Å) & 186002161 (125Å)Eluent: 0.15 M sodium phosphate, pH 6.8Flow rate: 0.35 mL/minColumn temp.: 25 °CDetection: UV @ 220 nm

Column Globular Protein Mol Wt. RangeBranched Dextrans

Linear PEG/PEO

BioSuite 125Å 5,000–150,000 1,000–30,000 500–15,000

BioSuite 250Å 10,000–500,000 2,000–70,000 1,000–35,000

NOTE: Operating flow rates for BioSuite Ultra-High Resolution (UHR) SEC Columns (4.6 mm) are from 0.1–0.4 mL/min. Use of an HPLC system (e.g. Waters Alliance HPLC System) capable of operating at these flow rates is essential for optimal UHR SEC Column performance.

Protein Calibration Curves for BioSuite High Resolution (HR) SEC Columns

Elution Volume (mL)106 8 12

102

103

104

105

106

Mol

ecul

ar W

eigh

t (D

a)

BioSuite 125Å, 5 μm HR SECBioSuite 250Å, 5 μm HR SECBioSuite 450Å, 8 μm HR SEC

Column Globular Protein Mol Wt. RangeBranched Dextrans

Linear PEG/PEO

BioSuite 125Å 5,000–150,000 1,000–30,000 500–15,000

BioSuite 250Å 10,000–500,000 2,000–70,000 1,000–35,000

BioSuite 450Å 20,000–1,000,000 4,000–500,000 2,000–250,000

Sample: Thyroglobulin (MW 670,000 Da), IgG (MW 156,000 Da), BSA (66,330 Da), Ovalbumin (MW 43,000 Da), Peroxidase (40,200 Da), Beta lactoglobulin (MW 18,400 Da), Myoglobin (MW 16,900 Da), Ribonuclease A (MW 13,700 Da), Cytochrome c (12,400 Da), Glycine tetramer (246 Da)Columns: BioSuiteHRSEC,450Å,8μm,7.8mmx300mm BioSuiteHRSEC,250Å,5μm,7.8mmx300mm BioSuiteSEC,125Å,5μm,7.8mmx300mmPart numbers: 186002166 (450Å), 186002165 (250Å), & 186002164 (125Å)Eluent: 0.1 M sodium phosphate, pH 7.0 containing 0.3 M sodium chlorideFlow rate: 1.0 mL/minColumn temp.: 25 °CDetection: UV @ 220 nm

Protein Calibration Curves for BioSuite Standard SEC Columns

Mol

ecul

ar W

eigh

t (D

a)

Elution Volume (mL)

106

105

103

104

20 2510 15

BioSuite 125Å, 10 μm SEC



Sample: Thyroglobulin (MW 670,000 Da), IgG (MW 156,000 Da), BSA (66,330 Da), Ovalbumin (MW 43,000 Da), Peroxidase (40,200 Da), Beta lactoglobulin (MW18,400 Da), Myoglobin (MW 16,900 Da), Ribonuclease A (MW 13,700 Da), Cytochrome c (12,400 Da), Glycine tetramer (246 Da)Columns: BioSuiteSEC,450Å,13μm,7.5mmx300mm BioSuiteSEC,250Å,13μm,7.5mmx300mm BioSuiteSEC,125Å,10μm,7.5mmx300mmPart numbers: 186002172 (450Å), 186002170 (250Å), and 186002168 (125Å)Eluent: 0.1 M sodium phosphate, pH 7.0 containing 0.3 M sodium chlorideFlow rate: 1.0 mL/minColumn temp.: 25 °CDetection: UV @ 220 nm

BioSuite 4.6 mm, 7.8 mm, and 7.5 mm analytical or 21.5 mm preparative columns are available.

Column Globular Protein Mol Wt. Range Branched Dextrans Linear PEG/PEO

BioSuite 125Å 5,000–150,000 1,000–30,000 500–15,000

BioSuite 250Å 10,000–500,000 2,000–70,000 1,000–35,000

BioSuite 450Å 20,000–1,000,000 4,000–500,000 2,000–250,000


Bioseparations

Protein-Pak and Shodex Size-Exclusion HPLC ColumnsWaters offers two families of packings for size-exclusion chromatography. Protein-Pak packings are based on a 10 µm diol-bonded silica and are available in a selection of pore sizes and column configurations. In addition, Waters offers a series of Shodex 7 µm high-resolution, gel filtration packings.

T he Protein-Pak Size-exclusion Columns can be expected to resolve proteins that differ in molecular weight by a factor of two and to distinguish proteins differing by as little as 15% in molecular weight. T he degree of resolution is more dependent on the sample mass and volume than the interaction between the sample and the stationary phase. Ideally, there should be no interaction between the stationary phase and the sample molecules. Secondary interactions are most often ionic and can, therefore, be reduced by increasing the ionic strength of the mobile phase. Typical, salt concentrations range to 0.2–0.5 M NaCl. It may also be useful in some cases to consider adding 10–20% methanol to eliminate hydrophobic and other hydrogen-bonding interactions.

Standard Protein Mix on KW-803 Column

25 min

12

34 5

67

8

This gel-filtration separation of protein standards demonstrates the ability to separate proteins in a wide range of molecular weights in minutes for high sensitivity analysis or protein isolation up to the milligram scale.

Column: Shodex KW-803, 7 µm, 8 x 300 mmPart number: WAT035946 Eluent: 25 mM sodium phosphate pH 6.8Flow rate: 0.72 mL/minDetection: UV @ 280 nm

Compounds:1. Blue Dextran2. Ferritin3. Aldolase4. Bovine Serum Albumin5. Ovalbumin6. Chymotrypsinogen7. Cytochrome c8. Cytidine

BioSuite SEC HPLC Columns

Description Matrix Diameter WidthDiameter Length

Column Volume

Suggested Volume Load for Maximum Multicomponent Resolution*

Multicomponent Resolution**

Part No.

BioSuite 125Å, 4 µm UHR SEC Silica 4.6 mm 300 mm 4.98 mL Less than 8 mg/mL Less than 40 µL 186002161

BioSuite 250Å, 4 µm UHR SEC Silica 4.6 mm 300 mm 4.98 mL Less than 8 mg/mL Less than 80 µL 186002162

BioSuite UHR Guard SEC Silica 4.6 mm 35 mm — — — 186002163

BioSuite 125Å, 5 µm HR SEC Silica 7.8 mm 300 mm 14.33 mL Less than 8 mg/mL Less than 200 µL 186002164



BioSuite HR Guard SEC Silica 6 mm 40 mm — — — 186002167

BioSuite 125Å, 10 µm SEC Silica 7.5 mm 300 mm 13.25 mL Less than 8 mg/mL Less than 200 µL 186002168

BioSuite 125Å, 13 µm SEC Silica 21.5 mm 300 mm 108.9 mL Less than 8 mg/mL Less than 1.6 mLs 186002169





BioSuite Guard SEC Silica 7.5 mm 75 mm — — — 186002174

BioSuite Guard SEC Silica 21.5 mm 75 mm — — — 186002175

* Using a BSA protein standard in a 50 mM phosphate buffer containing salt (either 0.1 M NaCl or 0.1 M Na2SO4) eluent. Useful protein mass loads will vary depending upon separation eluent, complexity of sample, and on the type of proteins contained in mixture. In general, maximum component resolution is obtained by injecting the smallest possible volume of a dilute protein solution. ** NOTE: Operating flow rates for BioSuite Ultra-High Resolution (UHR) SEC Columns (4.6 mm I.D.) are from 0.1–0.4 mL/min. Use of an HPLC system (e.g. Waters Alliance HPLC System) capable of operating at these flows is essential for optimal UHR SEC Column performance.

Protein-Pak SEC HPLC Columns and GuardsSteel Column Dimension MW Range Part No.

Protein-Pak 60 7.8 x 300 mm 1,000–20,000 WAT085250

Protein-Pak 60 19 x 300 µm 1,000–20,000 WAT025830

Protein-Pak 125 7.8 x 300 mm 2,000–80,000 WAT084601

Protein-Pak 125 19 x 300 mm 2,000–80,000 WAT025831

Protein-Pak 300SW 7.5 x 300 mm 10,000–300,000 WAT080013

Protein-Pak 125 Sentry Guard Column, 3.9 x 20 mm, 2/pk (requires holder)

186000926

Sentry Universal Guard Column Holder WAT046910

Glass Column Dimension MW Range Part No.

Protein-Pak 200SW 8 x 300 mm 500–60,000 WAT011786

Protein-Pak 300SW 8 x 300 mm 10,000–300,000 WAT011787


Protein: Symmetry300 C4 Versus Competitors

0 2 4 106 8 12 14 min

2

3

41

2 3

41

Symmetry300C4 columnP = 138.56

Competitive 300ÅC18 column

P = 90.09

MobilephaseA: 0.1%trifluoroaceticacidinwaterMobilephaseB: 0.08%trifluoroaceticacidinacetonitrileFlow rate: 0.75 mL/minGradient: Time Profile (min) %A %B 0.00 80 20 15.00 35 65Injection vol.: 20 µLColumn temp.: 40 °CDetection: UV @ 220 nmSystem: Alliance 2790, 996 PDA

Compounds:1. RNAse2. BSA3. B-Lactoglobulin4. Ovalbumin

Ultrahydrogel HPLC ColumnsPacked with hydroxylated polymethacrylate-based gel, Waters Ultrahydrogel™ SEC Columns are ideal for the analysis of aqueous-soluble samples, such as oligomers; oligosaccharides; polysaccharides; and cationic, anionic, and amphoteric polymers. Measuring 7.8 x 300 mm, these high-resolution columns offer many advantages over conventional aqueous SEC Columns, such as:

� A wide pH range (2–12)

� Compatibility with high concentrations of organic solvents (up to 20% organic, 50% organic if the mobile phase is introduced by gradient)

� Greater flexibility for the mobile phase

� Minimal non-size-exclusion effects

Symmetry300 C4 HPLC ColumnsCompared to our BEH C4, 300Å offerings, Symmetry300 C4 particles are 100% silica-based and are synthesized using ultrapure organic reagents resulting in high purity material with very low silanol activity for outstanding peptide and protein separations and recoveries.

� 300Å pore for peptide and protein applications

� Fully end-capped to minimize undesired secondary interactions

� Alternative separation selectivity compared to Waters BEH C4, 300Å hybrid material

� QC tested with peptide samples to help ensure excellent batch-to-batch consistency

Ultrahydrogel SEC HPLC (7.8 x 300 mm) Columns and GuardsColumn Pore Size Exclusion Limit Part No.

Ultrahydrogel 120 120Å 5 x 103 WAT011520





Ultrahydrogel Linear Blend 7 x 106 WAT011545

Ultrahydrogel DP* 120Å 5 x 103 WAT011550

Ultrahydrogel DNA >2000Å 10 x 106 WAT011560

Ultrahydrogel Guard Column N/A N/A WAT011565

Ultrahydrogel Guard Column DP* N/A N/A WAT011570

* DP = Degree of Polymerization, choice of Column when working with glucose oligomers.

Symmetry300 HPLC ColumnsParticle Size Inner Diameter Length C18 C4

3.5 µm 1.0 mm 150 mm 186000185 186000276

3.5 µm 2.1 mm 50 mm 186000187 186000277

3.5 µm 2.1 mm 100 mm 186000188 186000278

3.5 µm 2.1 mm 150 mm 186000200 186000279

3.5 µm 4.6 mm 50 mm 186000201 186000280

3.5 µm 4.6 mm 75 mm 186000189 186000281

3.5 µm 4.6 mm 100 mm 186000190 186000282

3.5 µm 4.6 mm 150 mm 186000197 186000283

5 µm 2.1 mm 150 mm WAT106172 186000285

5 µm 3.9 mm 150 mm WAT106154 186000286

5 µm 4.6 mm 50 mm WAT106209 186000287

5 µm 4.6 mm 150 mm WAT106157 186000288

5 µm 4.6 mm 250 mm WAT106151 186000289

5 µm 19 mm 10 mm 186001847 —

5 µm 19 mm 50 mm 186001848 —

5 µm 19 mm 100 mm 186001849 —

Symmetry300 C4 HPLC ColumnsDescription Inner Diameter Length Particle Size Part No.

Symmetry300 C4 1.0 mm 150 mm 3.5 µm 186000276








Symmetry300 C4 2.1 mm 150 mm 5 µm 186000285






Bioseparations

BioSuite pPhenyl,1000 A, 10 µm RPC

LC3

12 4

56

79

10

HC8

TIC ofReduced IgG1

antibody

10 12 14 16 18 20 22 24 26 28 30 min

BioSuite pC18 and pPhenyl Reversed-Phase Chromatography (RPC) HPLC ColumnsReversed-phase chromatography (RPC) has become a widely accepted tool for the separation of proteins, peptides, synthetic oligonucleotides, and other biomolecules. For many applications, Symmetry and Symmetry300, Atlantis, T3, or BEH 130Å and BEH 300Å chemistries can be successfully used for the isolation and analyses of these biocompounds. However for some applications, the large pore size and high chemical stability of BioSuite phenyl C18 and pPhenyl resin-based packings may be preferred. BioSuite RPC Column offerings include a C18 (pC18) and a phenyl (pPhenyl) chemistry bonded to a pH stable, methacrylic ester-based polymeric resin. The 500Å pore size of the pC18 base matrix accommodates proteins up to 2,500,000 Daltons while the 1,000Å pore size of the pPhenyl base matrix accommodates proteins up to 5,000,000 Daltons.

The BioSuite pC18, 2.5 µm, NP Column contains a non-porous chemistry that yields superior chromatographic resolution in less time compared to chromatography performed on the porous, pC18, 500Å, 7 µm RPC selection. Waters porous, pC18, 500Å, 7 µm RPC Column is available for applications requiring greater binding capacity. The pC18 and pPhenyl RPC chemistries are available in 21.5 x 150 mm columns for “lab-scale” isolations while a 2.0 x 75 mm column is well suited for narrow-bore HPLC and LC-MS applications.

The BioSuite pPhenyl, 1,000Å RPC columns have a higher ligand density compared to the BioSuite Phenyl, 1,000Å HIC columns and are not recommended for hydrophobic-interaction separations.

LC-MS Analysis of a Reduced Monoclonal IgG1 Antibody on a BioSuite pPhenyl RPC Column

Sample: Reduced monoclonal IgG1 antibodyColumn: BioSuite Phenyl,1,000Å, 10 µm, 2.0 x 75 mmPart number: 186002156Eluent A: Water with 0.1% Formic AcidEluent B: Acetonitrile with 0.1% Formic AcidFlow rate: 0.2 ml/minGradient: 10% B for 6 min (waste), then 20–35% B in 30 min Temperature: 80 °CDetection: Waters LCT Premier ESI-TOFMS

Protein-Pak Affinity ColumnsThe Protein-Pak Affinity Epoxy-Activated packing consists of 40 µm, 500Å pore size particles having a hydrophilic bonding layer with a glycidoxypropyl functionality resulting in a seven atom spacer arm. The epoxy-activated surface can immobilize a wide range of ligands via a covalent linkage with amino, hydroxyl or sulfhydryl groups using simple coupling procedures. For method screening or small scale separation, choose the convenience of pre-packed microcolumns. Larger-scale separations are easily achieved by packing bulk material in our Advanced Purification (AP) Glass Column.

To estimate packed bed volume for a known amount of Protein-Pak Affinity Epoxy-Activated packing:

Protein-Pak Affinity Epoxy-Activated used (g) x 2 = packed bed volume (mL)

Purification of Carbonic Anhydrase

30 min

1

Ligand: Sulfanilamide, 180 µmoles/gColumn: AP-1 Glass, 10 x 100 mmPart number: WAT021992Buffer A: 100 mM Tris sulfate with 200 mM sodium sulfate pH 8.7Buffer B: 200 mM potassium thiocyanate in 50 mM Tris sulfate pH 6.5Gradient: Time Profile Flowrate (min) %A %B (mL/min) Initial 1 100 0 1 2 100 0 2 7 0 100 2 17 100 0 2Detection: UV @ 280 nmProtein Load: 24 mg of bovine hemolysate yielded 0.6 mg of carbonic anhydrase with a 100% recovery of esterase activity

Compound:1. Carbonic Anhydrase

Protein-Pak Affinity ColumnsParticle Packing Particle Size Pore Size Qty. Part No.

Protein-Pak Affinity 40 µm 500Å 25 g WAT030653

Epoxy-Activated Packing — — 100 g WAT030654

Protein-Pak Affinity Epoxy-Activated MicroColumn (500 mg of material in a 3 cc syringe barrel) Inquire for additional offerings.

40 µm 500Å 10/box WAT035955

BioSuite Hydrophobic-Interaction Chromatography HPLC Columns

Description MatrixDiameter

WidthDiameter Length

Part No.

BioSuite Phenyl 10 µm HIC Polymer 7.5 mm 75 mm 186002159

BioSuite Phenyl 13 µm HIC Polymer 21.5 mm 150 mm 186002160


Reversed-Phase Chromatography at Elevated pH on BioSuite pC18 RP Column Possible on Polymer Based Material

1

2

3

4

6

75

8

30 min150

Sample: Met-enkephalin (Peak 1), Bradykinin (Peak 2), Leu-enkephalin (Peak 3), Neurotensin (Peak 4), Bombesin (Peak 5), Angiotensin l (Peak 6), Somatostatin (Peak 7), Insulin (Peak 8)Column: BioSuite C18,500Å,7μm,4.6mmx150mmPart number: 186002154Eluent A: 0.2 M ammonium hydroxide, pH 10.8Eluent B: 0.2 M ammonium hydroxide, pH 10.8 (20%)/acetonitrile (80%)Flow rate: 1.0 mL/minGradient: Linear gradient from 0 to 80% B in 50 min Column temp.: 25 °CDetection: UV @ 220 nm

Hydrophobic Proteins are Well Resolved by Reversed-Phase Chromatography on BioSuite pPhenyl RP Column

30 min15

12

3

45 6

7

8

The BioSuite pPhenyl, 1,000Å RPC columns have a higher ligand density compared to the BioSuite Phenyl, 1,000Å HIC columns and are not recommended for hydrophobic-interaction separations.

Sample: Ribonuclease (Peak 1), Bovine insulin, (Peak 2), Cytochrome c (Peak 3), Lysozyme (Peak 4), Transferrin (Peak 5), Bovine serum albumin (Peak 6), Myoglobin (Peak 7), Ovalbumin (Peak 8)Column: BioSuitePhenyl,1,000Å,10μm,4.6mmx75mmPart number: 186002157EluentA: HPLC-gradewaterwith0.05%trifluoroaceticacidEluentB: acetonitrilewith0.05%trifluoroaceticacidFlow rate: 1.0 mL/minGradient: Linear gradient from 5 to 80% B in 60 min Column temp.: 25 °CDetection: UV @220 nm

Hydrophobic-Interaction HPLC ColumnDescription Dimension Part No.

Protein HIC PH-814 Steel Column 8 x 75 mm WAT035520

BioSuite Hydrophobic-Interaction Chromatography HPLC ColumnsThe separation of proteins and peptides based upon hydrophobic characteristics is a powerful chromatographic technique. However, some proteins denature at elevated organic solvent concentrations making reversed-phase chromatography (RPC) difficult. BioSuite Phenyl HIC columns provide a viable separation alternative to RPC. HIC is characterized by the adsorption of compounds to a weakly hydrophobic surface at high salt concentrations, followed by elution with a decreasing salt gradient. HIC combines the non-denaturing characteristics of salt precipitation with the precision of HPLC to yield excellent separation of biologically active material. BioSuite Phenyl, 1,000Å, 10 µm HIC column media consists of a phenyl group bonded to a methacrylic ester-based polymeric resin. The large 1,000Å pore size accommodates proteins up to 5,000,000 Daltons. A 21.5 x 150 mm column is also available for “lab scale” isolations.

Hydrophobic-Interaction Chromatography on BioSuite Phenyl HIC Column is an Excellent Alternative to Reversed-Phase Methods

60 min300

1

2

3

4

6

5

6

The BioSuite Phenyl, 1,000Å HIC columns have a lower ligand density compared to the BioSuite pPhenyl, 1,000Å RPC columns and are not recommended for reversed-phase separations.

Sample: Cytochrome c (Peak 1), Myoglobin (Peak 2), Ribonuclease (Peak 3), Lysozyme (Peak 4), Alpha- chymotrypsin (Peak 5), and Alpha-Chymotrypsinogen A (Peak 6) Column: BioSuitePhenyl,10μm,7.5mmx75mmPart number: 186002159 Eluent A: 0.1 M sodium phosphate, pH 7.0 containing 2.0 M ammonium sulfateEluent B: 0.1 M sodium phosphate, pH 7.0 Flow rate: 1.0 mL/minGradient: Linear gradient from 0 to 100% B in 60 min Column temp.: 25 °CDetection: UV @ 280 nm


Bioseparations

Shodex and Protein-Pak Phenyl HIC HPLC ColumnsWaters Protein HIC and Protein-Pak Phenyl hydrophobic-interaction columns are packed with rigid, 10 µm polymethacrylate packing materials with 500Å pore size to ensure rapid protein diffusion. A low density bonding with phenyl groups results in a hydrophobic surface that allows for protein purification with a high recovery of both mass and biological activity.

HIC Packing Material CapacitiesShodex PH-814 40 mg/mL*

Protein-Pak Phenyl 5PW 25 mg/mL*

* Ovalbumin in 1.8 M ammonium sulfate, 0.1 M sodium phosphate pH.

Protein Purification by Hydrophobic-Interaction Chromatography

30 min

Lectin

Sample: Wheat Germ ExtractColumn: Shodex PH-814, 8 x 75 mmPart number: WAT035520 Buffer A: 1.7 M Ammonium sulfate in Buffer BBuffer B : 0.1 M sodium phosphate, pH 7.0Gradient: 0–100% Buffer B in 15 minutes linearFlow rate: 1.1 mL/minDetection: UV @ 280 nm

Unique selectivity of hydrophobic-interaction packings provides resolution and recovery of biologically active proteins with little denaturation.

Shodex Size-Exclusion and Anion-Exchange HPLC ColumnsColumn Particle Size Dimension MW Range Part No.

Protein KW-802.5 7 µm 8 x 300 mm 100–50,000 WAT035943

Protein KW-803 7 µm 8 x 300 mm 100–150,000 WAT035946

Protein KW-804 7 µm 8 x 300 mm 500–600,000 WAT036613

Deae-420N 3 µm 4.6 x 35 mm N/A WAT036581

Protein-Pak 125 Sentry Guard Column 3.9 x 20 mm, 2/pk (requires holder) 186000926

Sentry Universal Guard Column Holder WAT046910


AMINO ACID ANALYSIS SOLUTIONSOver the years, Waters has provided three distinct products utilizing pre-column derivatization and reversed-phase HPLC: Auto•Tag,™ Pico•Tag, and AccQ•Tag. Today, Waters continues to lead the way by bringing together its most ground-breaking and popular technologies: ACQUITY UPLC and AccQ•Tag Ultra, the newest addition to the AccQ•Tag family of products.

The UPLC Amino Acid Analysis Solution is holistically designed and optimized specifically for amino acid analyses. Derivatized amino acids are separated using the ACQUITY UPLC System, enhancing resolution to ensure accurate and precise qualitative and quantitative results. Just as important, our solution provides performance-qualified methodologies which are designed to be rugged and reliable, assuring reproducible results day-to-day, instrument-to-instrument, lab-to-lab, around the world—with the expert support that scientists have come to expect from Waters.

The UPLC Amino Acid Analysis Solution leverages Waters experience in separation science, derivatization chemistries, and information management. It is a total application solution optimized for accurate, reliable, and reproducible analysis of amino acids. Based on Waters AccQ•Tag Ultra chemistry, combined with our award winning ACQUITY UPLC separation technology, users can feel confident with assured performance in the areas of protein characterization, cell culture monitoring, and nutritional analysis of foods and feeds.

The UPLC Amino Acid Analysis Solution consists of:

� Waters ACQUITY UPLC System and tunable UV detector (Optional fluorescence and PDA detectors are also fully supported)

� AccQ•Tag Ultra derivatization chemistries including column, reagents, and eluents (all quality control tested)

� Empower 2 pre-configured projects, methods, and report templates

� Installation and application training and support included

� Application-specific Performance Qualification

� Connections INSIGHT® Intelligent Services

� Standards and kits to validate and troubleshoot


Bioseparations

N

N

NH

O

O

O

O N

NH

O

NHOH

O

N

NH2

NH2

O

OH

NH

O

OH

N

N

NH

O HOO

NN

NH

NH

O

AQC Reagent

1° Amino Acid:t1/2<<1s

2° Amino Acid:t1/2<<1s

Derivatized Amino Acids

bis-aminoquinoline urea (derivatization peak)6-Aminoquinoline (AMQ)

N-HydroxySuccinimide

+ CO2

+

AMQ

H2Ot1/2~15s

Chemistry of the AccQ•Tag Derivatization Reaction

AccQ•Tag Derivatization Reaction � Utilizes AccQ•Tag Ultra Reagent Powder – 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate – US Patent #5,296,599 and European Patent #EP 0 533 200 B1

� Reacts rapidly with both primary and secondary amines

� Excess reagent reacts more slowly with water to form a product that can be separated chromatographically from the derivatized amino acids

� Requires no vacuum drying, sample prep, or extraction

AccQ•Tag Ultra Chemistry

The AccQ•Tag Ultra chemistry products are a comprehensive, fully-tested set of reagents, columns, and eluents optimized for use with the UPLC Amino Acid Analysis Solution. T his chemistry is based on Waters widely-used and well-understood AccQ•Tag derivatization method. Primary and secondary amino acids are derivatized before separation with a single reagent, AccQ•Fluor,™ in a high-throughput batch process, resulting in exceptionally stable derivatives. High resolution separations are achieved using the pre-qualified AccQ•Tag Ultra UPLC Columns and mobile phases. Derivatized amino acids are quantified to sub-pmol levels with single wavelength UV detection.

AM

Q

NH

3

His

Ser

Arg

Gly

Asp

Glu

Thr A

la

Pro

Der

iv P

eak

Cys

Lys

Tyr

Met

Val

IleLe

uPh

e

0.0000.0020.0040.0060.0080.0100.0120.0140.0160.0180.0200.0220.0240.0260.0280.0300.0320.0340.0360.0380.0400.0420.0440.0460.0480.0500.0520.0540.0560.0580.0600.0620.064

1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 min

AU

Analysis of BSA Hydrolysate Sample, Approximately 9 ng on Column.

Amino Acid Analysis Methods for the Analysis of Hydrolysates and of Cell Culture Media

Hydrolysates

Cell Culture

1.5 3.0 4.5 6.0 7.5 min

His Asn

AM

Q

NH

3

His

Tau

Ser

Ser Gln

Arg

Gly AE A

sp

Arg

Gly Asp

Glu

Thr

Thr

Ala

Ala

GA

BA

Pro

Pro

Hy

Lys

1H

y Ly

s 2

AB

AA

Orn

Der

iv P

eak

Der

iv P

eak

Cys

Cys

Lys

Lys

Tyr

Tyr

Met

Met

Val

Val

NV

a

NV

a

Lle

Lle

Leu

Leu

Phe

Phe Tr

p

NH

3H

y P

ro

Glu

The UPLC Amino Acid Analysis solution can be used for the analysis of protein hydrolysates or for the larger set of amino acids found in cell culture media. T he only differences in the methods are a different dilution of the supplied eluent A concentrate and operation at a different temperature. No method development or buffer reformulation is required.

BSA Hydrolysate ResultsAmino Acid Expected Residues Observed Residues

His 17 15.36 ± 0.19

Ser 28 26.00 ± 0.08

Arg 23 22.37 ± 0.08

Gly 16 17.68 ± 0.20

Asp 54 55.47 ± 0.21

Glu 79 80.68 ± 0.20

Thr 33 31.92 ± 0.06

Ala 47 47.51 ± 0.15

Pro 28 28.35 ± 0.14

Lys 59 57.78 ± 0.38

Tyr 20 20.19 ± 0.08

Met 4 4.16 ± 0.15

Val 36 35.67 ± 0.16

lle 14 13.15 ± 0.15

Leu 61 63.13 ± 0.19

Phe 27 26.57 ± 0.13


AccQ•Tag Ultra Amino Acid AnalysisDescription Volume Part No.

ACQUITY UPLC AAA Application Kit 176001279

This Kit is intended to enable existing ACQUITY UPLC Systems for AAA applications.

Kit contains:

Amino acid standard, hydrolysate 10 x 1 mL

Sample tubes, 4 x 72/pk

Total recovery vials with caps, 3 x 100/pk

Column stabilizer kit, 150 mm

AccQ•Tag Ultra Derivitization Kit

AccQ•Tag Ultra C18, 1.7 µm, 2.1 x 100 mm Column

AccQ•Tag Ultra Eluent A Concentrate 950 mL

AccQ•Tag Ultra Eluent B 950 mL

Tube inlet .0025 I.D. PEEK nut PDA assembly

2 µL Sample loop

Column in-line filter kit

UPLC AAA solution information set

UPLC AAA app. solution startup tests

Cert., AAA Application and Familiarization

UPLC AAA H-Class Applications Kit 176002983

This kit is intended to enable existing ACQUITY UPLC H-Class Systems for AAA applications.

Kit includes:

AccQ•Tag Ultra Derivatization Kit, 250 Analyses





Total recovery vials, 3 x 100 vials/pk

Tube inlet 0.0025 I.D. PEEK nut PDA assembly

Column in-line filter kit

UPLC AAA H-Class solution information set

AAA Application and Familiarization Service

AccQ•Tag Ultra Chemistry Kit 176001235

The refill kit is intended to recharge the AccQ•Tag Ultra chemistries that are part of the application kit. This kit should be purchased for those that have already purchased the AccQ•Tag Ultra Application Solution. This kit is applicable to both ACQUITY UPLC and ACQUITY UPLC H-Class AAA Application Solutions, and should not be purchased as part of an initial system.

Kit includes:

AccQ•Tag Ultra Derivitization Kit, 250 analyses






Total recovery vials with caps, 3 x 100/pk

AccQ•Tag Ultra Derivitization Kit, 250 Analyses 186003836

AccQ•Tag Ultra Borate Buffer 5 x 6 mL

AccQ•Tag Ultra Derivitization Reagent Powder 5 x 3 mg

AccQ•Tag Ultra Reagent Diluent 5 x 4 mL

Amino Acid Standard, Hydrolysate 10 x 1 mL WAT088122

Sample Tubes, 4 x 72/pk WAT007571

Total Recovery Vials with Caps, 3 x 100/pk WAT0000384C

AccQ•Tag Ultra C18, 1.7 µm, 2.1 x 100 mm Column 186003837

AccQ•Tag Ultra Eluent A Concentrate 950 mL 186003838

AccQ•Tag Ultra Eluent B 950 mL 186003839


Bioseparations

AccQ•Tag Amino Acid Analysis Using HPLCT he HPLC AccQ•Tag method utilizes pre-column derivatized reagents that yield easily detected fluorescence adducts. T he AccQ•Fluor reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatizes primary and secondary amines in a simple, single-step reaction to yield highly stable, fluorescent adducts. We offer the AccQ•Tag method as a system package consisting of pre-packaged reagents and extensive documentation.

The AccQ•Tag Chemistry Package contains the items you need for up to 250 analyses of protein and peptide hydrolysate amino acids.

AccQ•Fluor Reagent Kit (five bottles each)

� AccQ•Fluor Borate Buffer

� AccQ•Fluor Reagent Powder

� AccQ•Fluor Reagent Diluent

AccQ•Tag Amino Acid Analysis Column

Separates the amino acid derivatives produced by the AccQ•Fluor derivatization reaction. The AccQ•Tag Column is a high-efficiency column specifically certified for use with the AccQ•Tag method. Care and use of the column is described in the Waters AccQ•Tag Amino Acid Analysis Column Care and Use manual.

AccQ•Tag Eluent A Concentrate

A pre-mixed concentrated aqueous buffer.

Amino Acid Hydrolysate Standard

Ten 1 mL ampules of the Amino Acid Hydrolysate Standard. Each ampule contains a 2.5 mM mixture of the 17 hydrolysate amino acids with the exception of cystine (1.25 mM).

6 x 50 mm Sample Tubes

Used for preparing samples and standards.

AccQ•Tag Chemistry Package Instruction Manual

Describes the AccQ•Tag Amino Acid Analysis Method.

AccQ•Tag Analysis of Hydrolysate Amino Acid

1

23

45 6

7

89

10

11 12

13

14

15

16

17

18

19

30 min

12

3 4 5

67

89

10

11

12

13

14

15

16

1718

19

30 min

1

23

45 6

7

89 10

11 12

13

14

15

16

17

18

19

30 min

B. 1 pmol Standard

A. 50 pmol Standard

B. 1 pmol Standard

Application of the AccQ•Tag Method to the analysis of hydrolysate amino acids is illustrated. The high purity reagents provided in the AccQ•Tag Chemistry Package enable high sensitivity analysis by minimizing background amino acid content. AQC-6-aminoquinolyl-N-hydroxysuccinimidyl, NHS-N-hydroxysuccinimidyl, AMQ-6-aminoquinoline.

Compounds:1. AMQ2. Asp3. Ser4. Glu5. Gly6. His7. NH3

8. Arg9. Thr

10. Ala11. Pro12. Cys13. Tyr14. Val15. Met16. Lys17. Ile18. Leu19. Phe

Sample: AQC-Derivatized Amino Acid StandardsColumn: AccQ•Tag, 4 µm, 3.9 x 150 mmPart number: WAT052885Column temp.: 37 °CEluent A: AccQ•Tag Eluent AEluent B: AcetonitrileEluent C: HPLC grade waterFlow rate: 1 mL/minGradient: AccQ•Tag MethodDetection: Fluorescence: lex = 250 nm, lem = 395 nm

Injection Volume GuidelinesSample Estimated Sample Quantity* Injection Volume

Proteins 0.1–1.0 µg 4–40 picomole 10–20 µL

0.1–5.0 µg 4–200 picomole 5–10 µL

Peptides 0.02–0.20 µg 20–200 picomole 10–20 µL

0.02–1.0 µg 200–1000 picomole 5–10 µL

Standard 50 picomole 5 µL

*Based on protein average molecular weight=25,000, and peptide average molecular weight=1,000.


AccQ•Tag Amino Acid Analysis for HPLC AAA AnalysisDescription Volume Part No.

AccQ•Tag Chemistry Kit WAT052875

Kit for up to 250 Analyses includes:

AccQ•Fluor Reagent 1 5 x 6 mL

AccQ•Fluor Reagent 2A, (5) vials 5 x 3 mg

AccQ•Fluor Reagent 2B, (5) vials 5 x 3 mL

AccQ•Tag column, 3.9 x 150 mm

AccQ•Tag Eluent A Concentrate 2 x 1 L



AccQ•Tag user guide

Amino Acid Standard 10 x 1 mL WAT088122

Amino acid standards for use with AccQ•Tag, Pico•Tag, or AccQ•Tag Ultra Amino Acid Analysis Solutions

AccQ•Tag Eluent A 1 L WAT052890

Concentrate

AccQ•Tag Eluent B 1 L WAT052895

AccQ•Fluor Reagent Kit WAT052880

Kit includes:

AccQ•Fluor Reagent 1 5 x 6 mL

AccQ•Fluor Reagent 2A 5 x 3 mg

AccQ•Fluor Reagent 2B 5 x 4 mL

The components of this kit are not available separately

AccQ•Tag User Guide WAT052874

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:


Bioseparations

Pico•Tag HPLC MethodA widely used technique for HPLC amino acid analysis is the Waters Pico•Tag method. Based on an optimized system configuration, prepackaged reagents and extensive documentation, the Pico•Tag method provides a turnkey, guaranteed approach to modern HPLC amino acid analysis. Pre-column derivatization relies on the coupling reaction of the well known Edman Degradation, the reaction of phenylisothiocyanate (PITC) with both primary and secondary amino acids to form phenylthiocarbamyl (PTC) derivatives. The PTC-amino acid adducts are stable and easily separated by reversed-phase HPLC. A single product is formed for each amino acid. Most reaction by-products and all derivatization reagents are volatile, so they may be removed from the sample by vacuum drying. T he method is applicable to any sample including protein hydrolysates, physiologic fluids, feeds, foods, and pharmaceutical preparations.

Plasma Amino Acid Profile Using the Pico•Tag Method

Glu

Asn

Ser

Gly

Gln

Tau

His

Cit

NH

3Th

rA

la

Arg

Met

SO

2(I.

S.)

Pro

1 M

e H

is

Tyr

Met

Rea

gent

Cys

IleLe

u

Phe

Trp

Orn

3 N

itro

Tyr (

I.S.)Va

l

Lys

66 min

Reproducible and reliable plasma amino acid profiles are obtained in 66 minutes using Waters Pico•Tag Method. In this analysis, 100 μL plasma was diluted with an internal standard, deproteinized by centrifugal ultrafiltration, and derivatized. The methionine sulfone (internal standard) peak represents 25 picomoles. Courtesy of A.S. Feste, R.W. Drummond, and S.J. Dudrich, Nutritional Support Service, St. Luke Episcopal Hospital, Houston, Texas.

Pico•Tag Derivatization Reaction

N = C = S + NH2 CH COOH

R

NH C NH

S

CH

R

COOH

Peptide Hydrolysate Amino Acid Analysis

A

B

Asp

Glu

Ser

Gly

His

Arg

Thr A

laP

ro Tyr

Val

Met

Cys

IleLe

u

Phe

Lys

Rgt

12 min

A. Hydrolysate StandardB. 16 Amino Acid Peptide, 40 pmol injected

This 12 minute analysis using Waters Pico•Tag Amino Acid Analysis Method provides identification and accurate quantitation of the amino acid composition.

Compounds:1. Asp 0.992. Glu 0.943. Gly 5.144. His 0.865. Thr 0.896. Pro 2.117. Val 2.008. Lys 2.05

Pico•Tag Amino Acid Analysis of Physiologic Amino AcidsDescription Part No.

Chemistry Package for Amino Acid Analysis of Physiologic Amino Acids

WAT091681

Designed for the analysis of physiologic amino acids.

Kit Contains

Free Amino Acid Analysis, 3.9 x 300 mm Column

Pico•Tag Reagent Kit

Pico•Tag Eluent 1, 4 x 1 Liter

Pico•Tag Eluent 2, 4 x 1 Liter

Pico•Tag Diluent, 100 mL

Manual, column heater inserts, and sample tubes

Pico•Tag Reagent Kit (PITC, TEA, and Standards A/N and B) WAT010947

Free Amino Acid Analysis, 3.9 x 300 mm Column WAT010950

Pico•Tag Eluent 1, 4 x 1 L WAT010960

Pico•Tag Eluent 2, 4 x 1 L WAT010965

Pico•Tag Diluent, 100 mL WAT088119

Pico•Tag Eluent 2, 1 L WAT010985

Pico•Tag Amino Acid Analysis for Protein Hydrolysates Description Part No.

Chemistry Package for Amino Acid Analysis of Protein Hydrolysates WAT007360

Designed for the analysis of protein hydrolysates.

Kit Contains

Pico•Tag Column, 3.9 x 150 mm

Pico•Tag Reagent Kit (contains PITC, TEA, and Standards)

Pico•Tag Eluent A, 4 x 1 L

Pico•Tag Eluent B, 4 x 1 L

Pico•Tag Diluent, 100 mL

Manual, column heater inserts, and sample tubes

Pico•Tag Column, 3.9 x 150 mm WAT088131

Pico•Tag Reagent Kit (PITC, TEA, and Standards) WAT088123

Pico•Tag Eluent A, 4 x 1 L WAT088108

Pico•Tag Eluent B, 4 x 1 L WAT088112

Pico•Tag Diluent, 100 mL WAT088119

Pico•Tag Eluent B, 1 L WAT010983


Separation of Detritylated Oligodeoxythymidine Ladders by Capillary Gel Electrophoresis (CGE) vs. Ion-Pair, Reversed-Phase Chromatography

0 4 min

20

25

30

35

12 22 min

UV @ 260 nm

10

30

15

LC System: ACQUITY UPLC Column: ACQUITY UPLC Oligonucleotide C18,1.7μm, 2.1 x 50 mmPart number: 186003949MobilephaseA: 15mMTEA,400mMhexafluoroisopropanol,pH7.9Mobile phase B: 50% A, 50% methanolFlow rate: 0.4 mL/minColumn temp.: 60 °CGradient: 40 to 48% B in 4 min (20–24% methanol)Detection: UV @ 260 nm

OLIGONUCLEOTIDESWaters Oligonucleotide columns contain second-generation hybrid silica BEH Technology particles functionalized with C18. The separation of detritylated synthetic oligonucleotide samples is based on the well-established method of ion-pair, reversed-phase chromatography. The availability of 1.7 µm UPLC particles or 2.5 µm HPLC particles in various column dimensions provides flexibility to meet various lab-scale isolation or analysis needs and delivers exceptional sample resolution and superior column life. In addition, Waters manufacturing and quality control testing procedures help ensure consistent batch-to-batch and column-to-column performance regardless of application demands.

� Separation efficiencies equivalent or better than PAGE, CGE, or ion-exchange HPLC methods

� Resolve failure sequences from detritylated full length products

� Scaleable column offerings for lab-scale isolation needs

� Exceptional column life for reduced cost per analysis

� QC tested with MassPREP Oligonucleotide Standard (p/n: 186004135) to help ensure performance consistency

Exceptional Resolution of Oligonucleotide Mixtures

ACQUITY UPLC Oligonucleotide C18, 1.7 μm (designed for use with an ACQUITY UPLC System) and XBridge Oligonucleotide C18, 2.5 μm Columns are well suited for the analysis of detritylated oligonucleotides using ion-pair, reversed-phase chromatography. As indicated (see figure on right), separations are comparable to those obtained by capillary gel electrophoresis (CGE) in terms of component resolution, yet analyses times are significantly decreased using Waters UPLC Technology. The ability to resolve large oligonucleotide sequences (e.g., N from N-1) is possible due to the enhanced resolving power obtained using sub-3 μm, BEH Technology particles. In addition, quantitation with molecular weight characterization of the separated target oligonucleotide product from failure sequences is possible using Waters Oligonucleotide columns with hyphenated-Mass Spectrometry methods and MS friendly eluents.

Oligonucleotide Columns

Description Particle

Size Pore Size

Dimension Part No.

ACQUITY UPLC Oligonucleotide BEH C18* 1.7 μm 130Å 2.1 x 50 mm 186003949

ACQUITY UPLC Oligonucleotide BEH C18* 1.7 μm 130Å 2.1 x 100 mm 186003950

ACQUITY UPLC Oligonucleotide BEH C18* 1.7 µm 130Å 2.1 x 150 mm 186005516

ACQUITY UPLC Oligonucleotide BEH C18, 130Å Method Validation Kit**

1.7 µm — 2.1x 100 mm 186004898

XBridge Oligonucleotide BEH C18* 2.5 μm 130Å 2.1 x 50 mm 186003952

XBridge Oligonucleotide BEH C18* 2.5 μm 130Å 4.6 x 50 mm 186003953

XBridge Oligonucleotide BEH C18* 2.5 μm 130Å 10 x 50 mm 186003954

XBridge Oligonucleotide BEH C18, 130Å Method Validation Kit**

2.5 µm — 4.6 x 50 mm 186004906

* For use on Waters ACQUITY UPLC Systems**Three Columns from three different batches of material.

System: Capillary Gel ElectrophoresisCGEcolumn: PEGsievingmatrix(BioCap75μmx27.5 (to detector)/34.5 cm (total length))Injection: 45 injection at 5 kVRunning: 15 kVColumn temp.: 30 °C


Bioseparations

UPLC-MS Analysis Of Interfering RNA Oligonucleotides

Discovery of the RNA interference (RNAi) mechanism now broadly used for silencing of target gene expression has prompted a need for the analysis of small interfering RNAs (siRNA) molecules. To satisfy the need for a robust, fast, and sensitive analysis of 20–25 nucleotides of small interfering RNA (siRNA), a UPLC-MS method has been developed utilizing UPLC Oligonucleotide columns and SYNAPT HDMS® Mass Spectrometer.

The acquisition of the accurate masses allowed for an assignment of the peaks of 5’-truncated oligomers (failed sequences generated during oligonucleotide synthesis), as well as some other impurities. The mass of each peak in the MS chromatogram was deconvoluted using MaxEnt™ 1 software. The tentative 5’-end failure products are assigned in Figure 2. Nearly the entire sequence of the parent oligonucleotide was elucidated. MS analysis also revealed a presence of an extra uridine mononucleotide added to the target 21-mer RNAi sequence.

Separation of a 15–60 mer Detritylated Oligodeoxythymidine Ladder

0 45 min

UV @ 260 nm

MS (TIC)

LC System: ACQUITY UPLC with PDA and Q-Tof micro™ MSColumn: ACQUITY UPLC Oligonucleotide C18, 1.7 µm, 2.1 x 50 mmPart number: 186003949

Mobile phase A: 15 mM TEA–400 mM hexafluoroisopropanolMobile phase B: MethanolFlow rate: 0.1 mL/minColumn temp.: 60 °CGradient: 19 to 26.5.5% B in 60 minDetection: UV @ 260 nm 10 scans per second MS: 1 scan per second, 0.1 sec interscan

U

U

UG

UCCU

U

UCUG

AA

CUU

21-mer

1 10 min

RNAi

20 nt8.73 min

TEATE

A

m/z5800 6800

TIC MS: SYNAPT MSCapillary: 2000 VSample cone: 35 VExtraction cone: 3 VDesolvation temp.: 200 °CSource temp.: 120 °CConegasflow: 50L/hrDesolvationgasflow: 600L/hr

LC System: ACQUITY UPLCColumn: ACQUITY UPLC Oligo C18, 1.7 µm, 2.1 x 50 mmPart number: 186003949Column temp.: 60 °CSample Injected: 2.5 µL (100 pmole of 21 nt RNAi)Flow rate: 0.2 mL/minMobile phase A: 15 mM TEA. 400 mM hexafluoroisopropanolMobile phase B: 50% A, 50% methanolGradient: 20–40% B in 10 minDetection: PDA, UV @ 260 nm

LC-MS Analysis of RNA (21 mer)

Impact of Different Ion-Pairing Agents on Varying Oligonucleotide Sequence Separations

0 10 min

2419

23

22

21

14

16

17

1513

12

18

12

20

22

23

13

14

15

16

17

18

19

21

24

24

19

14

1312

1213

14

15

16

17

18

19

20

21

22

23

24

14

1213

15

16

17

1820

1924

23

22

21TEA/HFIP

DMBAA 100 mM

TEAA 100 mM

TPAA 100 mM

HAA 100 mM

15.5–23.5 %MeOH in 10 min

9–11 %ACN in 10 min




Improved Oligoneucleotides separations can be achieved using alternative IP agents compared to use of traditional TEAA.

Outstanding Column Life

Waters Oligonucleotide columns packed with BEH Technology particles have shown remarkable column longevity, under these demanding separation conditions, while maintaining outstanding separation performance. By comparison, significantly reduced column life results when traditional silica-based columns are used under these same demanding separation conditions.

Separation of 5–25 mer Detritylated Oligodeoxythymidine Ladder

Injection #4

0 3 6 9 12 15 18 21 24 27 30 min

Injection #1001

HPLC System: Alliance HPLC with PDAColumn: XBridge Oligo C18,2.5μm,2.1x50mmPart number: 186003952MobilephaseA: 10%methanol/90%(385mMHexafluoroisopropanol+14.3mMTEA)MobilephaseB:25%methanol/75%(385mMHexafluoroisopropanol+14.3mMTEA)Column Temp: 60 °CGradient: 0–100% B in 30 min (10–25 % methanol).Flow rate: 1.0 mL/minDetection: UV @ 260 nm 5 scans per second


Scalable DNA and RNAi Separations with Good Product Recovery

XBridge Oligonucleotide C18 Columns are the preferred offering for detritylated oligonucleotide purifications due to the availability of column sizes designed to meet lab-scale isolation requirements. As indicated in the table below, the choice of XBridge Oligonucleotide C18 Column dimension and operating flow rate depends primarily on the scale of the synthesis reaction mixture. For example, a 4.6 x 50 mm column containing XBridge Oligonucleotide C18, 2.5 μm material is an excellent selection when oligonucleotide mass loads are less than or equal to 0.2 μmol. Selection of the appropriate column size for the amount of oligonucleotide sample loaded is recommended to maximize component resolution and recovery of the target product from non-desired failure sequences.

For researchers involved in gene silencing it is often necessary to work with RNA of high purity. Crude synthetic oligonucleotides used for gene knockout are typically purified. The figure below illustrates a lab-scale purification of 21 mer RNA at various column loads. Using an Oligonucleotide column chemistry and an Alliance System, large quantities of crude single stranded RNA can be successfully purified yielding material of high purity, ca. 95%, with an estimated yield of 55% based on collected peak area to the total peak area of the sample.

In addition, Oligonucleotide columns are well suited for analysis and purification of siRNA. As shown in the figure below siRNA is well resolved from single stranded RNA and truncated duplexes.

Dimensions Approx Mass Load** Yield*** Flow Rate

2.1 x 50 mm 0.04 µmoles 0.2 mg 0.2 mL/min

4.6 x 50 mm 0.20 µmoles 1.0 mg 1.0 mL/min

10 x 50 mm 1.00 µmoles 4.5 mg 4.5 mL/min

19 x 50 mm* 4.00 µmoles 16.0 mg 16.0 mL/min



* Oligonucleotide Custom Column** Values are only approximates and vary depending on oligonucleotide length, base composition, and “heart-cutting” fraction collection method used.*** Estimated for average oligonucleotide MW and synthesis yield.

Purification of Single Stranded RNA

0 6

Purified OligonucleotidePurity > 95%

*

N-1

140 nmol

84 nmol

28 nmol

1.4 nmol

0 18 min

97.8% UPLC purity (area)Purity ≈ 78%

LC System: Alliance HPLCColumn: XBridge Oligonucleotide C18, 2.5 µm, 4.6 x 50 mmPart number: 186003953Column temp.: 60 °CFlow rate: 1.0 mL/minMobile phase A: 0.1 M triethylammonium acetate, pH 7.5Mobile phase B: 20% acetonitrile in AGradient: 30–52.5% B in 10.0 min (0.15% acetonitrile/min) Detection: PDA, UV @ 260 nm

Purification of siRNA Duplex from Impurities

0 25 min

1.5 nmol0.15 nmol

RNAImpurities

siRNAImpurities

Target siRNAduplex85 nmol

15 nmol

LC System: Alliance HPLC Column: XBridge Oligonucleotide C18, 2.5 µm, 4.6 x 50 mmPart number: 186003953Column temp.: 20 °CFlow rate: 1.0 mL/minMobile phase A: 0.1 M triethylammonium acetate, pH 7.0Mobile phase B: 20% acetonitrile in AGradient: 25–75% B in 30.0 minDetection: PDA, UV @ 260 nm


Bioseparations

Chromatography of a PCR Amplification Mixture Generated using 3 ng and 1 fg of HBV S-Gene Template

0

0.000

0.040

15

Abs

orba

nce

260

nm

30 min

dNTPs and Taq enzyme

Nonspecific DNA sequences

465 bp PCR product

Sample: PCRamplificationmixtureusing3ngHBV-SgenetemplateColumn: Gen-Pak FAX, NP, AXC, 2.5 µm, 4.6 x 100 mmPart number: WAT015490Eluent A: 25 mM Tris/Cl, 1 mM ethylenediaminetetraacetic acid, pH 8.0Eluent B: 25 mM Tris/Cl, 1 mM ethylenediaminetetraacetic acid, 1.0 M sodium chloride, pH 8.0Gradient: 40–75% B in 30 min, linearFlow rate: 0.75 mL/minColumn temp.: 30 °C

Separation of Duplex DNA Fragments: HaeIII and MspI Restriction Enzyme Digests of pBR322 Plasmid

1 20 min

HaeIII Restriction Enzyme

MspI

51 57 64 80 89 104

123/124184

192213

234267

434458

504 540587

67 76 90 110 123147/147

160

180

190

201217

238

242307 404

527622

LC System: ACQUITY UPLCColumn: ACQUITY UPLC Peptide C18, 1.7 µm, 2.1 x 50 mmPart number: 186003685Column temp.: 50 °CFlow rate: 0.2 mL/minMobile phase A: 0.1 M triethylammonium acetate, pH 7.0Mobile phase B: 20% acetonitrile in AGradient: 57.5–84.5 % B in 20.0 minDetection: PDA, UV @ 260 nm

Gen-Pak FAX Anion-Exchange ColumnsWaters Gen-Pak FAX Columns offer the highest resolution available for anion-exchange HPLC of nucleic acids. The Gen-Pak FAX Column contains a weak anion exchanger based on DEAE functionalized non-porous resin. It contains 2.5 µm particles and is well suited for analytical and micro-preparative applications.

Columns for Large DNA/RNA SpeciesIn general, molecular biology methods for manipulation of DNA rely on restriction enzymes, polymerase-chain reaction (PCR), and sequencing techniques. Using these methods, genomic DNA is typically converted into shorter double stranded (ds)DNA sequences, typically 100–1000 base pairs (bp) in length. T he shorter dsDNA molecules are often analyzed or isolated by methods such as slab gel or capillary electrophoresis. Use of Waters ACQUITY BEH C18, 300Å RevePhase or Gen-Pak™ FAX Anion-exchange Columns offer alternatives to more traditional electrophoretic methods and are particularly well suited for various analytical and small-scale purification applications.

Separation of DNA Restriction Fragments

35 min

1 2

3

4 5

6

Sample: 3.0 µg of BstN1 digest of pBR322Column: Gen-Pak FAX, NP, AXC, 2.5 µm, 4.6 x 100 mmPart number: WAT015490Eluent A: 25 mM Tris/Cl, 1 mM ethylenediaminetetraacetic acid, pH 8.0Eluent B: 25 mM Tris/Cl, 1 mM ethylenediaminetetraacetic acid, 1.0 M sodium chloride, pH 8.0 Gradient: 25–75% B in 30 min, linearFlow rate: 0.75 mL/minColumn temp.: 30 °CDetection: UV @ 260 nm

Compounds:1. 13 bp2. 121 bp3. 383 bp4. 929 bp5. 1060 bp6. 1857 bp

ACQUITY UPLC BEH C18, 300Å for DNA/RNA FragmentsDescription Particle Size Pore Size Dimension Part No.

ACQUITY UPLC BEH C18 1.7 µm 300Å 2.1 x 50 mm 186003685

Gen-Pak FAX HPLC ColumnDescription Dimension Part No.

Gen-Pak FAX Column 4.6 x 100 mm WAT015490

Gen-Pak FAX Replacement Inlet Filter — WAT015715


Oasis µElution Plates

Oligonucleotide Desalting by Solid-Phase Extraction � Removes salt prior to MS analysis

� Low elution volumes

� High sensitivity

� Sample concentrating

� High throughput

Desalting of synthetic oligonucleotides is essential for MS analysis (QC, genotyping applications and SNP analysis). Waters µElution plate is an excellent choice for high throughput analysis with minimal amount of sample. The Oasis µElution Plate combines patented plate design, proven Oasis chemistries, and generic protocols enabling elution volumes as low as 25 µL. Now for the first time you can perform SPE clean-up and concentration of very small sample volumes. The Oasis Hydrophilic-lipophilic-balanced (HLB) sample extraction products incorporate a patented copolymer made from a balanced ratio of two monomers; the lipophilic divinylbenzene and the hydrophilic N-vinylpyrolodone that is ideally suited for this application.

MassPREP Oligonucleotide Standard � Contains a carefully defined mixture of synthesized oligodeoxythymidine fragments

� Useful in testing and confirming HPLC/UPLC, LC-MS, and column performance for oligo applications

� Each QC tested and shipped with a certificate of analysis

The pre-packaged MassPREP Oligonucleotide Standard is designed for verification of HPLC/UPLC instrument and column performance for analysis of synthetic oligonucleotides. Approximately equimolar amounts of 15, 20, 25, 30, and 35 nucleotide (nt) long oligodeoxythymidines are lyophilized and packaged in 1.5 mL LC vials. These vials are vacuum-sealed in foil pouches to reduce degradation that can occur by excessive exposure to light and air. Approximately 1 nmole of each oligonucleotide is present in the vial.

0 10 min

15 nt

20 nt25 nt 30 nt

35 nt

Waters ACQUITY UPLC analysis of MassPREP Oligonucleotide Standard on an ACQUITY UPLC Oligonucleotide C18, 1.7 μm Column. The main components are labeled. Small peaks eluting between labeled oligonucleotides are N-1, N-2, etc. failure sequences generated during the oligonucleotide syntheses. The ACQUITY UPLC System is equipped with 50 μL standard mixer and PDA Detector (260 nm).

Effective Use of Oasis HLB for Oligonucleotide Desalting Prior to MALDI-TOF MS

900

800

700

600

500

400

300

200

100

04000 4500 5000

M/Z

Abun

danc

e

5500

MALDI-Tof Spectrum of 16 mer Prior to Desalting

Matrix solution: 70 mg/mL 3-hydroxypicolinic acid and 40 mM ammonium acetateMode: Negative Ion

1800

1600

1400

1200

1000

800

600

400

200

04000 4500 5000 5500

M/Z

Abu

ndan

ce

MALDI-Tof Spectrum of 16 mer After Desalting Using Oasis HLB µElution Plate

Matrix solution: 70 mg/mL 3-hydroxypico linic acid and 0.04 M ammonium acetateMode: Negative Ion

Oasis HLB µElution Plate (for Oligonucleotides)Description Part No.

Oasis HLB µElution Plate (for Oligonucleotides) 186001828BA

MassPREP Oligonucleotide StandardDescription Qty. Part No.

MassPREP Oligonucleotide Standard 1/pk 186004135


Bioseparations

GLYCANSGlycosylation is one of the most common forms of post-translational modification (PTM) of human and other eukaryotic proteins. Glycosylated proteins (glycoproteins) make up 50–70% of all human proteins. Consequently, glycans play a critical role in a myriad of physiological and pathological reactions ranging from immunity and blood clotting to cell development and cell death.

Glycoprotein analysis involves identifying complex N- and O-Linked structures composed of frequently similar and repeating sugar moieties. Hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection is a well-recognized and reliable technique that effectively separates and quantitates isolated glycans after their derivatization with fluorescent labels.

Waters ACQUITY UPLC Glycan BEH Amide Column and GlycoWorks and Sample Preparation consumables were designed for the HILIC-mode separation and clean-up of a variety of labeling reagents. Our column chemistry and recommended UPLC and HPLC instrument conditions are capable of separating both neutral and charged species using a binary gradient. Retention of labeled oligosaccharides is based on the hydrophilicity of the molecule. T he high resolving power of this column is due in part to the small particle size, 1.7 μm, of the fully porous packing material. Chemical and mechanical stability of the column are the consequence of Waters ethylene bridged hybrid (BEH) particle composition and ligand binding Technology to ensure consistent column-to-column performance.

� Improved component resolution in less time compared to existing HPLC-based methods

� Optimized for use with ACQUITY UPLC System with fluorescence detection

� Based on Waters BEH particle and bonding technology for stable and reproducible labeled glycan separations

� Quality-control tested with the Glycan 2-AB Performance Test Standard (p/n: 186006349) for consistent batch-to-batch reproducibility

ACQUITY UPLC Glycan BEH Amide Column Separation of 2-AB Labeled Glycan Performance Standard

10 15 20 25 30 35 min

1

4

5

6

78

9

10

2

3

1211

13

N-Acetylglucosamine Mannose Galactose Fucose Sialic Acid

2AB

2AB

2AB

2AB

2AB

2AB

2AB

2AB

2AB

2AB

2AB

2AB

6,7.

8.

9.

10.

4.

5.

1.

2.

3.

12.

13.

11.UPLC ConditionsSample: Glycan Performance StandardPart number: 186006349Sample conc.: 10 pmol/µLSample vol.: 1.5 µLInjection mode: Partial loopSystem: ACQUITY UPLCColumn: ACQUITY UPLC Glycan BEH Amide, 1.7 µm, 2.1 x 150 mmPart number: 186004742 Eluent A: 100 mM ammonium formate, pH 4.5Eluent B: AcetonitrileColumn temp.: 60 °CDetection: Fluorescence:λex=330nm,λem=420nmGradient: Time Flowrate Profile Curve (min) (mL/min) %A %B Initial 0.5 25 75 6 46.5 0.5 40 60 6 48 0.25 100 0 6 49 0.25 100 0 6 50 0.5 25 75 6 63 0.5 25 75 6Instrument Conf.: Mixer (p/n: 205000403). Needle (p/n: 205000507). Noin-linefilter.NoVanGuardColumn.

The N-linked glycans from pooled human IgG contain high mannose as well as neutral, and sialylated complex structures. The chromatogram shows 35 minutes of a one hour analysis. This challenging sample includes high mannose, bisecting GlcNAc and sialylated species.


High Quality Separations of Varied Glycan Structures

Several complementary analytical techniques are routinely used to characterize, identify, and quantitate oligosaccharides isolated from glycoproteins. Hydrophilic-interaction liquid chromatography (HILIC) is a well-recognized and reliable technique that can effectively separate fluorescently-labeled glycan. Waters ACQUITY UPLC Glycan BEH Amide Column was specifically designed for use on Waters ACQUITY UPLC System to give improved component resolution compared to traditional LC techniques for more certain identification and reliable quantitation.

ACQUITY UPLC Glycan BEH Amide Column Separation of Labeled High Mannose Glycans 2-AB from Ribonuclease b

ACQUITY UPLC Glycan BEH Amide Column Separation of Dextran 2-AB Calibration Ladder (p/n: 186006841, also available in 2-AA, p/n: 186007279)

5 10 15 20 25 30 35 40 45 min

14 5

6 78

9 10 11

23

1213

1415

1716

1819

2021

22

UPLC ConditionsSample: Dextran 2-AB Calibration Ladder in Glucose UnitsPart number: 186006841Sample conc.: 10 pmol/µLSample vol.: 1.5 µLInjection mode: Partial loopSystem: ACQUITY UPLC Column: ACQUITY UPLC Glycan BEH Amide, 1.7 µm, 2.1 x 150 mmPart number: 186004742 Eluent A: 100 mM ammonium formate, pH 4.5Eluent B: AcetonitrileColumn temp.: 60 °CDetection: Fluorescence:λex=330nm,λem=420nmGradient: Time Flowrate Profile Curve (min) (mL/min) %A %B Initial 0.50 25 75 6 46.5 0.50 50 50 6 48.0 0.25 100 0 6 49.0 0.25 100 0 6 50.0 0.50 25 75 6 63.0 0.50 25 75 6

The fast, high-resolution separation of a homopolymeric series of glucose oligomers illustrates the wide range of glycan sizes, extending beyond 22 glucose units that can be separated with the ACQUITY UPLC Glycan BEH Amide Column. These labeled glycans can provide calibration reference points that are helpful in identifying more complex glycans based on relative retention times.

10 15 20 25 30 35 min

4*

5

2*

3*

1

System

2ABMannose 6

2ABMannose 51.

2.

2ABMannose 7isomers3a, 3b.

2ABMannose 84.

2ABMannose 95.

UPLC ConditionsSample: 2-AB-labeled ribonuclease b N-glycansSample conc.: 100 pmol/µLSample vol.: 1.5 µLInjection mode: Partial loopSystem: ACQUITY UPLC Column: ACQUITY UPLC Glycan BEH Amide, 1.7 µm, 2.1 x 150 mmPart number: 186004742 Eluent A: 100 mM ammonium formate, pH 4.5Eluent B: AcetonitrileColumn temp.: 60 °CDetection: Fluorescence:λex=330nm,λem=420nmGradient: Time Flowrate Profile Curve (min) (mL/min) %A %B Initial 0.50 25 75 6 46.5 0.50 40 60 6 48.0 0.25 100 0 6 49.0 0.25 100 0 6 63.0 0.50 25 75 6

A series of high mannose oligosaccharides were released from bovine ribonuclease b and labeled with 2-AB. The HILIC ethylene performed on ACQUITY UPLC Glycan BEH Amide Column shows both different numbers of mannose residues and isomeric structures.

NOTE: ACQUITY UPLC Glycan BEH Amide, 1.7 μm Columns are designed for use with the ACQUITY UPLC System. The benefits of the small particle packing in ACQUITY UPLC Glycan BEH Amide, 1.7 μm Columns are only realized with the low system volume and low detector dispersion of an ACQUITY UPLC System.


Bioseparations

ACQUITY UPLC Glycan BEH Amide Columns and Method Validation Kits

Description Particle Size Dimension Part No.

ACQUITY UPLC Glycan BEH Amide, 130Å VanGuard Column, 3/pk 1.7 µm 2.1 x 5 mm 186004739

ACQUITY UPLC Glycan BEH Amide, 130Å Column 1.7 µm 2.1 x 50 mm 186004740


ACQUITY UPLC Glycan BEH Amide Column, 130Å Method Validation Kit1 1.7 µm 2.1 x 100 mm 186004907


Dextran Calibration Ladder 186006841

XBridge Glycan BEH Amide HPLC and UHPLC Columns and Method Validation Kits

Description Particle Size Dimension Part No.

XBridge Glycan BEH Amide, 130Å VanGuard Column, 3/pk 2.5 µm 2.1 x 5 mm 186007262

XBridge Glycan BEH Amide, 130Å XP Column 2.5 µm 2.1 x 50 mm 186007263



XBridge Glycan BEH Amide, 130Å XP Column Method Validation Kit1 2.5 µm 2.1 x 150 mm 186007266




XBridge Glycan BEH Amide, 130Å Guard Column, 2/pk3 2.5 µm 4.6 X 20 mm 186007267




XBridge Glycan BEH Amide, 130Å XP Column Method Validation Kit1 2.5 µm 4.6 x 150 mm 186007271

XBridge Glycan BEH Amide, 130Å Guard Column, 2/pk2 3.5 µm 2.1 x 10 mm 186007505

XBridge Glycan BEH Amide, 130Å Column 3.5 µm 2.1 x 50 mm 186007502



XBridge Glycan BEH Amide, 130Å Guard Column, 2/pk3 3.5 µm 4.6 X 20 mm 186007272




XBridge Glycan BEH Amide, 130Å Column Method Validation Kit1 3.5 µm 4.6 x 150 mm 186007277


Dextran Calibration Ladder 1860068411Three columns from three different batches of BEH Amide, 130Å material.2 Requires 2.1 x 10 mm Universal Sentry Guard Holder, p/n: WAT097958.3 Requires 4.6 x 20 mm Universal Sentry Guard Holder, p/n: WAT046910.

Note: ACQUITY UPLC Glycan BEH Amide, 1.7 µm Columns are designed for use with the ACQUITY UPLC System. The benefits of the small particle packing in ACQUITY UPLC Glycan BEH Amide, 1.7 µm Columns are only realized with the low system volume and low detector dispersion of an ACQUITY UPLC System.


Coming in 2015!GlycoWorks RapiFluor-MS N-Glycan Kit

Do not compromise sensitivity for speed. T his rapid N-glycan sample preparation kit combines optimized, fast deglycosylation and new instant labeling that matches optical performance to MS, truly revolutionizing previously complicated sample preparation protocols.

Visit asr.waters.com for updated information.

8 9 10 11 12 13 14 15 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 3416

Sig

nal [

EU

]

6.56

5.55

4.54

5.55

2.52

1.51

0.50

Retention time [min]

B GlycoWorks HILIC SPE processed Elution with 100 mM NH4OAc, 5% ACN

8 9 10 11 12 13 14 15 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 3416

Sig

nal [

EU

]

6.56

5.55

4.54

5.55

2.52

1.51

0.50

A Control before SPE

Control

Dried

5% ACN, 100 mM NH4OAc

Control before SPE

Lyophilization control HILIC SPE processed Elution with 100 mM NH4OAc, 5% ACN

3

16

10

15

20

Peak 3 G0F

Peak 16A3

% A

bund

ance

0

5

C

Sample Preparation

GlycoWorks Sample Preparation Tools for Released N-Glycan Analysis

Waters GlycoWorks Consumables offer a more convenient, versatile, comprehensive and effective sample preparation solution for glycan analysis.

� Reproducible and improved recovery of highly charged glycans

� RapiGest for solubilization of the glycoprotein and efficient PNGase F enzymatic digestion of the N-linked glycans

� Optimized HILIC Sorbent for high recovery of N-glycans and for effective labeling reaction clean up prior to LC-FLR-MS analysis

� Complete Modules for Deglycosylation, Labeling, Clean-up and Sample Collection

� New Complete 2-AB and 2-AA Enzyme and Labeling Kits coming in 2015

� Training Video available at asr.waters.com

Robustness and Recovery using the GlycoWorks protocol and a 2-AB Labeled Human IgG spiked with Man-5 and Man-6 as well as set of trisialyated A3 glycans.

Glycan profile (A) before, and (B) after GlycoWorks HILIC SPE with the optimized elution conditions. Representative chromatograms and relative abundances (C) measured for two 2-AB labeled glycans (low and high GU values) from the test mixture before and after GlycoWorks HILIC SPE are displayed (n=3).

LC-FLR

LC-FLR-MS

Glycoproteins

FLR Labeling Reaction of Glycans4

FLR Labeled Glycans

MALDI MS

Denaturation and DeglycosylationRapiGest (if necessary)

DTT Reduction (if necessary),IAM Alkylation (if necessary)Deglycosylation by PNGase F

1

Extraction of Released GlycansGlycoWorks HILIC µElution Plate2

Formic Acid TreatmentConversion of Free Amines

Increasing Labeling Efficiency3

Excess Labeling Reagent RemovalGlycoWorks HILIC µElution Plate5

General Guideline of Sample Preparation from Glycoprotein to enrich FLR Labeled Glycans Using Reductive Amination Reaction.

If using MALDI please refer to Product Solution, Literature Code 720004660EN.


Bioseparations

Additional Consumables

Dextran Calibration Ladders

The Dextran Calibration Ladders allows the user to tie in the entire GlycoWorks Sample Prep Solution seamlessly to the Waters ACQUITY UPLC System and GlycoBase Database Search. Using these labeled standards allows the user to calibrate their system based on GU units, and have confidence in results. Available in 2-AB, 2-AA and the new RapiFluor-MS labels.

GU

2

GU

5 GU

10

GU

15

GU

2

GU

5 GU

10

GU

15

0 5 10 15 20 25 30 35Retention time [min]

0

10

20

Sign

al [E

U]

FLRλex = 330 nmλem= 420 nm

2AB-Dextran Ladder, FLR

The lyophilized 2AB Dextran Calibration Standard, 200 μg, was reconstituted in 60% Acetonitrile, and the injection volume was 2 μL.

Glycan Sample Preparation Kit and StandardsDescription Part No.

GlycoWorks High-throughput Prep Kit 176003090

GlycoWorks HILIC µElution Plate 96-well 186002780


GlycoWorks Control Standard, 100 µg vial 186007033

GlycoWorks Reagent Kit* 186007034

Manifold Waste Tray 600001282

GlycoWorks Single Use Prep Kit 176003119

GlycoWorks HILIC 1 cc Cartridge (10/pk) 186007080


GlycoWorks Control Standard, 100 μg vial 186007033

GlycoWorks Reagent Kit* 186007034

2-AB Glycan Performance Test Standard

186006349The Glycan Performance Test Standard is a 2-AB labeled human IgG-like standard that is QC verified to contain the components needed to benchmark and evaluate ACQUITY UPLC Glycan BEH , 1.7 µm Columns.

2-AB Dextran Calibration Ladder

186006841The 2-AB labeled, Dextran Calibration Ladder is used to calibrate the HILIC column from retention time to GU values. This calibration ladder provides good peak shape and reliable identification from 2 to 30 Glucose Units.

2-AA Dextran Calibration Ladder

186007279The 2-AA labeled, Dextran Calibration Ladder is used to calibrate the HILIC column from retention time to GU values. This calibration ladder provides good peak shape and reliable identification from 2 to 30 Glucose Units.

2-AB GlycoWorks HILIC 1 cc Flangeless Catridges Pack

186007239This pack of 20 Catridges, good for 10 analyses, are offered for those customers who prefer to use the single use devices with the positive pressure manifold (PPM).

*Updated version available in 2015.


Glycan Analysis System StandardsDescription Volume Usage Part No.

Alliance with Fluorescence Qualification Standards Test Kit

C P 700002753

Kit contains seven 10 mL vials of varying concentrations of anthracene in 80:20 acetonitrile/water:

(1) 0.5 pg/μL, (1) 1.0 pg/μL, (2) 5.0 pg/μL, (1) 10.0 pg/μL, (1) 2.5 ng/mL, (1) 2.5 μg/mL

One blank 10 mL vial of 80:20 acetonitrile/water

Fluorescence Detector Standard Solution1 mL P

5.0 pg/µL anthracene in 20/80 water/acetonitrile 700003694

Fluorescence Detector Performance Standard Solution 10 mL P WAT0476850.10 mg/L anthracene in 70/30 acetonitrile/water

Ammonium Formate Concentrate

10 mL 1860070815000 mM ammonium formate in a water w/3.8% formic acid matrix. This allows the end user to dilute the concentrate 10 mL to 1 L before using to reach a 50 mM concentration.

S Setup C Calibration P Performance Check T Tuning

Ammonium Formate Concentrate-Glycan Analysis This is a concentrated version of the 50 mM Ammonium Formate buffer that is recommended to be used with the Biopharm/Glycan System, using the UPLC H-Class System and the ACQUITY UPLC Glycan BEH Column. This buffer comes contains 10 mL of 5000 mM ammonium formate in a water with 3.8% formic acid matrix. T his allows the end user to dilute the concentrate 10 mL to 1 L before using to reach a 50 mM concentration.

Benchmarking, Method Development and Troubleshooting: Glycan Performance Test Standard

The Glycan 2-AB Performance Test Standard is Human-like IgG spiked with Man-5 and Man-6 and is QC verified to contain the components needed to benchmark and evaluate ACQUITY UPLC Glycan BEH Amide Columns containing 1.7 μm particle and the XBridge Glycan BEH Amide Columns that contain either 2.5 µm and 3.5 µm particles. It is also valuable to use as an additional 2-AB labeled control to assess digestion and labeling reaction efficiencies.

Peak Identification1. G0 (NGA2)

8. G1F+GN

2. G0F (NGA2F) 9. Man-6

3. Man-510. G2 (NA2)

4. G0F+GN 11. G2F (NA2F)

5. G1

12. G1F+SA

6. G1Fa (NA2G1F)13. G2F+SA (A1F)

7. G1Fb (NA2G1F)

10 12 14 16 18 20 22 24 26 28 30 min

1

2

34

6

7

8 9 10

12

13

11

5

Waters Glyco-Pak DEAE and N ColumnsPrior to use of Waters UPLC-based analyses of 2-AB labeled glycan analyses, underivitized oligosaccharides (released by enzyme or hydrazinolysis treatment) may be first separated into neutral and multiple acidic fractions (depending on the number of sialic acids or degree of sulfation) on either Waters Glyco-Pak™ DEAE or Glyco-Pak N, HPLC-based Columns. For more information, consult literature pieces WAT026735 (Glyco-Pak DEAE) and WAT026728 (Glyco-Pak N) available at www.waters.com/chemcu

Waters Glyco-Pak DEAE and N HPLC ColumnsColumn Description Part No.

Glyco-Pak N Column, 7.8 x 300 mm WAT026725

Glyco-Pak DEAE Column, 7.5 x 75 mm WAT026730


Bioseparations

MS AND LC-MS CONSUMABLES FOR BIOMOLECULES

MassPREP On-Line Desalting Devices � Effectively desalts proteins yielding improved LC-MS results

� Fast on-line method for high-throughput applications

� Excellent protein recoveries and no detectable carryover

� >100 injections from a single cartridge

The MassPREP on-line desalting column can effectively desalt proteins prior to LC-MS analyses. Because non-volatile salts (e.g., NaCl) can suppress ionization of intact proteins leading to poor detection sensitivity, it is important to remove or significantly minimize the introduction of these compounds into the mass analyzer. T he reversed-phase, phenyl material contained in MassPREP on-line column successfully “traps” proteins, allowing the salts to be washed to waste prior to protein elution into the mass spectrometer. With an optimized LC-MS method, cycle times as low as 4 minutes (for intact antibody) and 10 minutes (for reduced antibody) are achievable.

MassPREP On-Line Desalting Cartridge (2.1 x 10 mm)

Over a series of 100 injections, satisfactory results were obtained for BSA and a mAb, as shown for injections #97–100 on a MassPREP On-line desalting cartridge. Reference Desalting of Proteins Using MassPREP On-line Desalting Cartridges Prior to Mass Spectrometry [2005], Waters Applications Note, p/n: 720001077EN.

LC System: Alliance 2796 Separation ModuleMS System: Q-Tof micro, ESI+Eluent A: 0.1% formic acid (water) Eluent B: 0.1% formic acid (acetonitrile)

Inject #100 = 5 µL PBS Blank

Inject #99 = 5 µL mAb(1 µg/5 µL PBS)


Inject #98 = 5 µL BSA (1 µg/5 µL PBS)

“Column-related carryover” from previous protein sample injections can compromise the integrity of collected LC-MS data. Compared to results obtained on a competitive on-line desalting cartridge (top), excellent sample recovery is obtained with the MassPREP on-line desalting cartridge (bottom).

Competitor’s On-Line Desalting Cartridge

Waters MassPREP On-Line Desalting Cartridge


Inject #3 = 10 µL mAb(1 µg/µL PBS)

Inject #2 = 10 µL PBS Blank, following a 5 µg mAb injection


Inject #2 = 10 µL mAb(1 µg/µL PBS)


Excellent Recovery with No Detectable Carryover


Enzymate Online Digestion Column

Waters Enzymate™ BEH Pepsin Column (2.1 x 30 mm, 5 μm) digests intact proteins into peptides. The peptic peptides are then retained on a trapping column. Peptides elute from the trapping column and are refocused onto a sub-2-μm ACQUITY UPLC Column for elution into a high resolution mass spectrometry.

� Provides fast, reproducible, and efficient online protein digestion, typically within 30 seconds

� Shortens overall protein sample preparation time

� Offer the ability to optimize protein digestion efficiency by changing temperature and/or flow rate

� Integral part of the ACQUITY UPLC M-Class HDX System

Description Particle Size Inner Diameter Column Length Part No.

Enzymate Pepsin Online Digestion Column 5 µm 2.1 mm 30 mm 186007233

After 10 Digestions

After 50 Digestions

After 100 Digestions

MassPREP Micro Desalting Column (2.1 x 5 mm)

Combined ESI-Tof mass spectra of an intact IgG1 antibody from a 4 minute LC-MS analysis. The results reveal no detectable carryover following a 0.5 μg injection of the antibody.

LC System: ACQUITY UPLCMS System: LCT Premier ESI-TOF MS/SYNAPT HDMSIonization mode: ESI+, V modeEluent A: 0.1% formic acid (water)Eluent B: 0.1%formic acid (acetonitrile)Columntemp.: 80˚C

Max:2 CountsPre-run Blank

754 Counts0.5 µg Intact IgG1

5 CountsPost-run Blank

MS Light Chain

TIC(1.3 µg load)

MSHeavy Chain

Total ion chromatogram (TIC) from UPLC-MS analysis of light and heavy chains from a reduced IgG1 antibody. A 10 minute LC-MS run largely resolved the earlier eluting light chain from the later eluting glycosylated heavy chains.

MassPREP On-Line Desalting DevicesDescription Qty. Part No.

MassPREP Micro Desalting Column 1/pk 186004032

MassPREP On-Line Desalting Cartridge (2.1 x 10 mm)* 2/pk 186002785

UPLC Intact Mass Analysis Application Kit** (Includes MassPREP Micro Desalting Column and ACQUITY Tubing Kit) 1/pk 176001519

Sentry 2.1 x 10 mm Guard Cartridge Holder.** Required for use of MassPREP On-line Desalting Cartridge 1/pk WAT097958

* See: UPLC Intact Mass Analysis Application Kit Manual (p/n: 715001664)

Reproducible online pepsin digestions of phosphory-lase b. A total of 130 digestions were performed using an Enzymate pepsin column. The 12th, 50th, and 111th digestions were shown. The sequence coverage is shown on the right.

Overlay of three phos b digestions within a 130-injection HDX MS study

Phos b coverage = 95.1% Average peptide length in residue = 11.9 AA Redundancy (how may overlapping/residue) = 4.27


Bioseparations

Capillary, Nano and Trapping ColumnsWaters capillary, nano and trapping columns, contain carefully selected reversed-phase and ion-exchange chemistries for effective bioseparations. The column offerings are developed and intended for use on various traditional low pressure and state of the art high pressure nano-flow LC and LC-MS systems.

The unique frit technologies and column design fully leverage the separation power of UPLC and the use of 1.7 μm particles to nano- and micro-scale bioanalysis.

NanoEase Nano and Capillary Columns and Trapping Columns

NanoEase™ Columns are designed for use with any low pressure micro and nano LC system. They support typical operating pressures of up to 5.000 psi. The implemented frit technology inimizes bed disturbance throughout the life of the column.

� Available in variety of Waters stationary phases

� Can be used with any capillary LC system

� Operating pressure of up to 5,000 psiNOTE: Use of Waters 300 μm Capillary Inlet Tubing (p/n: 430001471) and 300 μm Capillary Outlet Tubing (p/n: 430001472) required for use with 300 μm I.D. capillary columns. Waters Kit p/n: 700002757 contains both the 300 μm Capillary Inlet Tubing (p/n: 430001471) and the 300 μm Capillary Outlet Tubing (p/n: 430001472).

Symmetry300 C18, Atlantis dC18, and strong cation-exchange (SCX) NanoEase Trap Columns effectively separate complex samples or remove buffers and high concentrations of salts through many injections. These devices generate low backpressure and yield good recovery to the sub-femtomole peptide level. The “direct connect” design allows for easy configuration and replacement in a six or a ten-port valve for either one-dimensional or two-dimensional LC-MS analyses.

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:


BSA Digest Using NanoEase Symmetry300 C18 Trap Columns

10.00.00 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min

Sample: MassPREP BSA Digestion StandardPart number: 186002329 Column: Symmetry C18 NanoEase, 3.5 µm, 150 µm x 150 mmPart number: 186002463Trapping column: Symmetry C18 NanoEase Trap, 5 µm, 180 µm x 23 mmPart number: 186002622Mobile phase A: 98% water/2% acetonitrile/0.1% formic acidMobile phase B: 98% acetonitrile/2% water/0.1% formic acidGradient: Time Profile (min) %A %B 0.00 95.0 5.0 30.00 50.0 50.0 31.00 20.0 80.0 41.00 20.0 80.0Flow rate: 3.6 µL/minInjection vol.: 1 µLColumn temp.: 20 °C

MS ConditionsIon source: ESI+Source temp.: 80 °CDesolvation temp.: 150 °CSystem: CapLC with Q-Tof micro technology

NanoEase Trap Columns (5,000 psi)Description Particle Size Dimension Part No.

Symmetry300 C18 Trap Column 5/pk

5 µm 0.18 x 23.5 mm 186002622

Atlantis dC18 Trap Column 5/pk

5 µm 0.18 x 23.5 mm 186002574

SCX 300Å Trap Column 5/pk 5 µm 0.50 x 23.5 mm 186002623

Symmetry C18 Trap Column 5/pk

5 µm 0.18 x 23.5 mm 186002808

NOTE: NanoEase Trap Column hardware obtained from Optimize Technologies Inc., Oregon City, OR, 97045–0009, USA

NanoEase Nano and Capillary Columns (5,000 psi)

Description Particle SizeInner

DiameterLength Part No. Description Particle Size

Inner Diameter

Length Part No.

Symmetry C18 3.5 µm 75 µm 50 mm 186002188 Atlantis dC18 3 µm 150 µm 100 mm 186002467


Symmetry C18 3.5 µm 75 µm 150 mm 186002190 Symmetry C18 3.5 µm 300 µm 50 mm 186002581

Symmetry300 C18 3.5 µm 75 µm 50 mm 186002191 Symmetry C18 3.5 µm 300 µm 100 mm 186002582

Symmetry300 C18 3.5 µm 75 µm 100 mm 186002192 Symmetry C18 3.5 µm 300 µm 150 mm 186002583

Symmetry300 C18 3.5 µm 75 µm 150 mm 186002193 Symmetry C18 5 µm 300 µm 50 mm 186002584

Atlantis dC18 3 µm 75 µm 50 mm 186002194 Symmetry C18 5 µm 300 µm 100 mm 186002585

Atlantis dC18 3 µm 75 µm 100 mm 186002195 Symmetry C18 5 µm 300 µm 150 mm 186002586

Atlantis dC18 3 µm 75 µm 150 mm 186002197 Symmetry300 C18 3.5 µm 300 µm 50 mm 186002587

Symmetry C18 3.5 µm 100 µm 50 mm 186002201 Symmetry300 C18 3.5 µm 300 µm 100 mm 186002588

Symmetry C18 3.5 µm 100 µm 100 mm 186002202 Symmetry300 C18 3.5 µm 300 µm 150 mm 186002589

Symmetry C18 3.5 µm 100 µm 150 mm 186002203 Symmetry300 C18 5 µm 300 µm 50 mm 186002590

Symmetry300 C18 3.5 µm 100 µm 150 mm 186002206 Symmetry300 C18 5 µm 300 µm 100 mm 186002591

Atlantis dC18 3 µm 100 µm 100 mm 186002208 Symmetry300 C18 5 µm 300 µm 150 mm 186002592

Atlantis dC18 3 µm 100 µm 150 mm 186002209 Atlantis dC18 3 µm 300 µm 50 mm 186002593



Symmetry300 C18 3.5 µm 150 µm 50 mm 186002462 Atlantis dC18 5 µm 300 µm 50 mm 186002596



Atlantis dC18 3 µm 150 µm 50 mm 186002466XBridge Peptide BEH C18, 130Å

5 µm 300 µm 50 mm 186003682

NOTE: Use of Waters 300 μm Capillary Inlet Tubing (p/n: 430001471) and 300 μm Capillary Outlet Tubing (p/n: 430001472) required for use with 300 μm I.D. capillary columns. Waters Kit p/n: 700002757 contains both the 300 μm Capillary Inlet Tubing (p/n: 430001471) and the 300 μm Capillary Outlet Tubing (p/n: 430001472)


Bioseparations

NanoEase Peptide Columns: BEH C18, 130Å and 300Å

DescriptionParticle

SizeInner

DiameterLength Part No. Description

Particle Size

Inner Diameter

Length Part No.

XBridge Peptide BEH C18, 130Å 3.5 µm 75 µm 50 mm 186003588 XBridge Peptide BEH C18, 300Å 3.5 µm 75 µm 100 mm 186003633











XBridge Peptide BEH C18, 130Å 3.5 µm 300 µm 150 mm 186003599 XBridge Peptide BEH C18, 300Å 5 µm 300 µm 50 mm 186003682

XBridge Peptide BEH C18, 130Å 3.5 µm 75 µm 50 mm 186003632

*NOTE: Use of Waters 300 μm Capillary Inlet Tubing (p/n: 430001471) and 300 μm Capillary Outlet Tubing (p/n: 430001472) required for use with 300 µm I.D. capillary Columns. Waters Kit p/n: 700002757 contains both the 300 μm Capillary Inlet Tubing (p/n: 430001471) and the 300 μm Capillary Outlet Tubing (p/n: 430001472).

nanoACQUITY UPLC On-line SCX/RP 2D Separations KitsDescription Part No.

nanoACQUITY UPLC On-Line SCX/RP 2D-LC-MS Kit 176001367

Assembly, 25 µm, Capillary, BSM (Binary Solvent Manager) to Trap Valve

430001575

Assembly, 40 µm Capillary, ASM (Auxiliary Solvent Manager) to Injection Valve

430001576

Assembly, Capillary Tubing, Injection Valve to Trap Valve 430001577

Assembly, Capillary Tubing, Injection to Trap Valve 430001629

nanoACQUITY UPLC SCX, 5 µm, 180 µm x 23 mm Column 186003507

nanoACQUITY UPLC Symmetry C18, 5 µm, 180 µm x 20 mm Trapping Column

186006527

nanoACQUITY UPLC, 1.7 µm BEH C18, 75 µm x 100 mm Analytical Column

186003542

MassPREP 5 Protein Digest Standard Kit 186002330

nanoACQUITY UPLC On-Line 2D-LS/MS User’s Guide 715001357

LC-MS Kits for the nanoACQUITY UPLC System with 2D TechnologyTwo-dimensional (2D) LC-MS kits for the nanoACQUITY UPLC System with 2D technology provide high efficiency on-line 2D-LC-MS analysis for complex proteomics samples, such as protein global digests. Two kit options are available: nanoACQUITY UPLC On-line SCX/RP 2D Separations Kit and nanoACQUITY UPLC On-line RP/RP 2D Separations Kit.

nanoACQUITY UPLC On-line SCX/RP 2D Separations KitThe first-dimension separation uses a strong cation-exchange nanoACQUITY UPLC 180 μm x 23 mm strong cation exchange (SCX) column in combination with a nano-scale, reversed-phase trapping column (Symmetry C18, 5 µm) followed by a nanoACQUITY UPLC ethylene bridged hybrid (BEH) C18, 1.7 µm analytical column to perform highly resolving orthogonal separations.


nanoACQUITY UPLC On-line RP/RP 2D Separations KitsDescription Qty. Part No.

Replacement Items for nanoACQUITY UPLC On-line RP/RP 2D Separations Kit

Assembly, Capillary Tubing with Frit 40 µm x 16” 1 430001571





NanoEase Peptide BEH C18, 130Å, 5 µm, 300 µm x 50 mm 1 186003682

nanoACQUITY UPLC Trap, Symmetry C18, (standard fitting), 5 µm, 180 µm x 20 mm 1 186006527

nanoACQUITY Peptide BEH C18, 130Å, 1.7 µm, 75 µm x 100 mm 1 186003542

nanoACQUITY UPLC On-line RP/RP 2D Separations Kit Waters supports an on-line separation protocol based upon a reversed-phase separation at pH 10 followed by reversed-phase separation at pH 2 (RP/RP) performed on our pH tolerant XBridge BEH C18 particle and ligand bonding technology.1 This orthogonal separation technology uses highly resolving, sub-3-µm reversed-phase particles to exploit the ionic and hydrophobic characteristics of peptides to increase peak capacity and provide better protein identifications.1 Stapels, M and Fadgen, K (2009) “A Reproducible Online 2D Reversed Phase High-Low pH Method for Qualitative and Quantitative Proteomics” Current Trends in Mass Spectrometry

nanoACQUITY UPLC Columns and Trapping ColumnsWaters nanoACQUITY Trapping, Capillary and Nano Columns are designed specifically for use on Waters nanoACQUITY UPLC System. The columns and trapping columns handle operating pressures up to 10.000 psi. They are compatible for use with the Waters ACQUITY UPLC M-Class Systems.

T he ability to operate at higher pressures enables the use of longer columns, up to and including 250 mm in length. This column design first brought the separation power of 1.7 μm particles to nano-scale bioanalysis and separations.

� Engineered for extended column life

� Capable of operating pressure up to 10,000 psi

� For Waters nanoACQUITY and ACQUITY M-Class Systems

� Easily interfaces with LC-MS/MS systems

The nanoACQUITY 180 µm I.D. Trapping Columns are robust devices available for trap and elute as well as for high throughput trapping applications. Trapping columns are also available with a strong cation-exchange (SCX) phase for 2D applications.

30 40 50 60 70 80 90 min

1.7 µm BEHPressure 9,500Peak Capacity 458

3 µm conventional columnPressure 3,500 PSIPeak Capacity 297

100 fmoles of a mixture of 5 MassPREP Digest Standards* 75 µm i.d x 300 mm (custom length) 3-50% B 120 min 250 nL/min

Increased Peak Capacity Separations

ACQUITY UPLC M-Class

nanoACQUITY UPLC Trap Columns (10,000 psi)

DescriptionParticle

SizeInner


nanoACQUITY UPLC Peptide Symmetry C18, 100Å, 2G, V/M Trap Column, 1/pk*

5 µm 180 µm 20 mm 186006527

nanoACQUITY UPLC Peptide Symmetry C18, 100Å, 2G, V/V Trap Column, 1/pk**

5 µm 180 µm 20 mm 186006526

nanoACQUITY UPLC Peptide CSH C18, 130Å, 2G, V/M Trap Column, 1/pk

5 µm 180 µm 20 mm 186007186

nanoACQUITY UPLC Symmetry C18, 100Å, 2D, V/M Trap Column, 1/pk

5 µm 180 µm 20 mm 186007238

nanoACQUITY UPLC SCX Trap Column, 1/pk

5 µm 180 µm 20 mm 186003507

*This part number is a replacement for p/n: 186003514**This part number is a replacement for p/n: 186004630


Bioseparations

nanoACQUITY UPLC BEH 130Å and 300Å Columns (10,000 psi)

DescriptionParticle

SizeInner


nanoACQUITY UPLC Protein BEH C4, 300Å

1.7 µm 100 µm 100 mm 186004640

nanoACQUITY UPLC Protein BEH C4, 300Å

1.7 µm 150 µm 100 mm 186004641

nanoACQUITY UPLC Peptide BEH C18, 130Å

1.7 µm 75 µm 100 mm 186003542


1.7 µm 75 µm 150 mm 186003543


1.7 µm 75 µm 200 mm 186003544


1.7 µm 75 µm 250 mm 186003545


1.7 µm 100 µm 100 mm 186003546


1.7 µm 150 µm 100 mm 186003550


1.7 µm 75 µm 100 mm 186003810


1.7 µm 100 µm 100 mm 186003811


1.7 µm 150 µm 100 mm 186003812


1.7 µm 75 µm 150 mm 186003813


1.7 µm 75 µm 200 mm 186003814


1.7 µm 75 µm 250 mm 186003815

Teflon Capillary Fitting, 5/pk — — — 186002690

Compare nanoACQUITY and ACQUITY UPLC M-Class Systems

1 2 3 4 5 6 7 8 9 10 min

%

0

1 2 3 4 5 6 7 8 9 10 min

%

0

Increased Pressure = Higher Throughput

ESI+ MSMicro-probe

CaffeineLidocaineSulfadimethoxineTerfenadineDanazol

300 µm x 150mm, BEH 5% to 95%B10%B/CVV 127 mm/min 14 KPSI

ACQUITY UPLC M-Class

8 KPSInanoACQUITY UPLC

ACQUITY UPLC M-Class Trap Columns

DescriptionParticle

SizeInner


ACQUITY UPLC M-Class Symmetry C18 2G, V/M Trap, 100Å

5 µm 180 µm 20 mm 186007496

ACQUITY UPLC M-Class Symmetry C18 2D, V/M Trap, 100Å

5 µm 180 µm 20 mm 186007497

ACQUITY UPLC M-Class Symmetry C18 Trap, 100Å

5 µm 300 µm 50 mm 186007498

ACQUITY UPLC M-Class Symmetry C18 2D HCP Trap, 100Å

5 µm 300 µm 250 mm 186007499

ACQUITY UPLC M-Class Symmetry C18 2G, V/V Trap, 100Å

5 µm 180 µm 20 mm 186007500

ACQUITY UPLC M-Class Symmetry C18, 2D, V/V Trap, 100Å

5 µm 180 µm 20 mm 186007592

ACQUITY UPLC M-Class Nano and Capillary Columns and Trapping ColumnsACQUITY UPLC M-Class Columns are designed specifically with the Waters ACQUITY UPLC M-Class System in mind and used in nano- to microscale LC separations. The unique frit technology and advanced column engineering allows ACQUITY UPLC M-Class Columns and traps to be used at 15.000 psi. They are fully compatible with nanoACQUITY UPLC Systems.

These columns are optimized for maximum sample recovery and deliver superior chromatographic resolution through efficient use of sub-2-μm particles. Furthermore, when used with a mass spectrometer, ACQUITY UPLC M-Class Columns allow you to attain the highest sensitivity, resolution, and reproducibility possible for biomarker discovery and protein identification and characterization.

75 µm to 300 µm I.D. columns provide the best chromatographic separations with flow rates from 200 nL/min to 100 μL/min and cover a 170-fold range of sample amounts.

� The maximum operating pressure of 15,000 psi allows for faster separations or higher resolution with longer columns

� The 300 µm I.D. ACQUITY UPLC M-Class Columns provide low dispersion and are handled with the same ease as analytical scale columns

� Compatible with Waters nanoACQUITY UPLC Systems

� Easily interface with LC-MS/MS systems

The family of ACQUITY M-Class Trapping Columns includes solutions for trap-elute and high throughput trapping. The available trapping column diameters of 180 µm and 300 µm I.D. allow for adjusting the separation method for variety of sample matrixes and concentrations.


ACQUITY UPLC M-Class Columns (15,000 psi)

DescriptionParticle

SizeInner

DiameterLength Part No. Description

Particle Size

Inner Diameter

Length Part No.

ACQUITY UPLC M-Class Peptide BEH C18, 130Å

5 µm 300 µm 50 mm 186007471ACQUITY UPLC M-Class Peptide BEH C18, 300Å

1.7 µm 75 µm 250 mm 186007492

ACQUITY UPLC M-Class HSS T3

1.8 µm 300 µm 150 mm 186007472ACQUITY UPLC M-Class Protein BEH C4, 300Å

1.7 µm 75 µm 100 mm 186007493



1.7 µm 100 µm 100 mm 186007494



1.7 µm 150 µm 100 mm 186007495

ACQUITY UPLC M-Class Peptide CSH C18, 130Å

1.7 µm 75 µm 100 mm 186007475ACQUITY UPLC M-Class Peptide CSH C18, 130Å

1.7 µm 150 µm 50 mm 186007513


1.7 µm 75 µm 150 mm 186007476ACQUITY UPLC M-Class Peptide CSH C18, 130Å

1.7 µm 150 µm 150 mm 186007514


1.7 µm 75 µm 200 mm 186007477ACQUITY UPLC M-Class HSS T3 Column, 100Å, 1/pk

1.8 µm 300 µm 50 mm 186007559


1.7 µm 75 µm 250 mm 186007478ACQUITY UPLC M-Class HSS T3 Column, 100Å, 1/pk

1.8 µm 300 µm 100 mm 186007560


1.7 µm 100 µm 100 mm 186007479ACQUITY UPLC M-Class Peptide CSH C18 Column, 130Å, 1/pk

1.7 µm 300 µm 50 mm 186007561



1.7 µm 300 µm 100 mm 186007562



1.7 µm 300 µm 150 mm 186007563


1.7 µm 75 µm 150 mm 186007482ACQUITY UPLC M-Class Peptide BEH C18 Column, 130Å, 1/pk

1.7 µm 300 µm 50 mm 186007564



1.7 µm 300 µm 100 mm 186007565



1.7 µm 300 µm 150 mm 186007566



1.7 µm 300 µm 50 mm 186007570



1.7 µm 300 µm 100 mm 186007571



1.7 µm 300 µm 150 mm 186007572


1.7 µm 100 µm 100 mm 186007488ACQUITY UPLC M-Class Protein BEH C4 Column, 300Å, 1/pk

1.7 µm 300 µm 50 mm 186007567



1.7 µm 300 µm 100 mm 186007568



1.7 µm 300 µm 150 mm 186007569


1.7 µm 75 µm 200 mm 186007491

nanoACQUITY UPLC Peptide CSH, 130Å Columns (10,000 psi)

DescriptionParticle

SizeInner


nanoACQUITY UPLC Peptide CSH C18, 130Å

1.7 µm 75 µm 100 mm 186007070


1.7 µm 75 µm 150 mm 186007071


1.7 µm 75 µm 200 mm 186007072


1.7 µm 75 µm 250 mm 186007073


1.7 µm 100 µm 100 mm 186007074


1.7 µm 150 µm 50 mm 186007246


1.7 µm 150 µm 100 mm 186007075


1.7 µm 150 µm 150 mm 186007247

nanoACQUITY HSS T3 and Symmetry C18, 130Å Columns (10,000 psi)

DescriptionParticle

SizeInner


HSS T3 1.8 µm 75 µm 100 mm 186005775

HSS T3 1.8 µm 75 µm 150 mm 186005776

HSS T3 1.8 µm 75 µm 200 mm 186005777

HSS T3 1.8 µm 75 µm 250 mm 186005778

HSS T3 1.8 µm 100 µm 100 mm 186005779

HSS T3 1.8 µm 150 µm 100 mm 186005780

Symmetry C18 3.5 µm 75 µm 100 mm 186003491








Teflon Capillary Fitting, 5/pk 186002690

For use with nanoACQUITY UPLC Systems rated to 10,000 psi only. Not for use with nanoACQUITY UPLC Systems rated to 5,000 psi.


Bioseparations

ionKey/MS

Simplified Micro Flow LC/MS with Increased Sensitivity

The ionKey/MS integrates the UPLC separation into the source of the mass spectrometer delivering a level of performance not achievable any other way. ionKey/MS integrates the ACQUITY UPLC M-Class System with the industry-leading Xevo TQ-S Mass Spectrometer enabling the lowest detection limits achievable for the most challenging analyses in an easy-to-use, “plug and play” format. It also provides 2.1-mm I.D. UPLC chromatographic performance in a 150-µm I.D. channel packed with sub-2-µm particles, providing tremendous improvements in sensitivity.

iKey Separation Device

ionKey/MS is enabled by the innovative iKey which replaces traditional fittings and columns and simplifies the user experience for nanoscale bioseparations.

The “Plug and Play” design eliminates user-made connections and the associated operator variability offering a high level of confidence and enabling any scientist to obtain high quality data.

� 150 µm I.D. channel

� 50 and 100 mm channel length

� 10,000 psi operating pressure

The ionKey/MS System with the ACQUITY UPLC M-Class and the Xevo TQ-S Mass Spectrometer.

Sensitivity comparison between ionKey/MS and 2.1 mm I.D. chromatography;1 μL injection of equal sample load on each.

2.60 3.00 3.40 3.80 4.20 4.60 5.00

Alprazolam OH

1.40 1.80 2.20 2.60 3.00 3.40

%

2.1 mm I.D.

2.1 mm I.D.

ionKey/MS ionKey/MS

ionKey/MSionKey/MS

40X 27X

24X2X

0

100

Time

Clopidogrel

%

0

100

Time

Oxytocin

%

0

100

Time

T43 ENL peptide

%

0

100

Time2.30 2.50 2.70 2.90 3.10 3.30

2.50 3.00 3.50 4.00 4.50 5.00 5.50

2.1 mm I.D.

2.1 mm I.D.

ionKey/MS provides significant increases in S:N compared to 2.1 mm I.D. chromatogra-phy allowing scientists to reach lower limits of detection and see things they could not see before. ionKey/MS and 2.1 mm I.D. peaks are time aligned for comparison purposes

Electrical connections and column heating control

The iKey is available with a variety of sorbents. Every iKey is LC-MS quality control tested, so you can be confident in your analysis. For detailed specifications and ordering information, visit www.waters.com/ikey

Fluid entry connections

Nebulization gas inlet

Robust emitter

Electrospray connection

contact

iKey Separation DevicesDescription Part No.

iKey, Peptide BEH C18, 130Å, 1.7 µm, 150 µm x 50 mm* 186006764

iKey, Peptide BEH C18, 130Å, 1.7 µm, 150 µm x 100 mm* 186006766

iKey, BEH C18, 130Å, 1.7 µm, 150 µm x 50 mm 186007256



iKey, Peptide CSH C18, 130Å, 1.7 µm, 150 µm x 50 mm* 186007257

iKey, CSH C18, 130Å, 1.7 µm, 150 µm x 50 mm 186007244

iKey, HSS T3, 130Å, 1.8 µm, 150 µm x 50 mm 186007260

iKey, HSS T3, 130Å, 1.8 µm, 150 µm x 100 mm 186007261

iKey, Infusion 186007049

iKey, Diagnostic 186007050

iKey, Injection 186007051 * QC tested with a Peptide Standard Mix.


MassPREP Digestion Standard Mixtures � Used to ensure an analytical system’s ability to provide both qualitative protein analysis and relative protein quantification

� Yields high confidence and coverage during expression profiling of complex protein samples (by spiking the standards)

� Designed as standards for the Waters Protein Expression System

MassPREP Digestion Standards consist of two standard mixtures (MPDS Mix 1 and MPDS Mix 2), which were prepared by individually digesting Yeast Alcohol Dehydrogenase (ADH, SwissProt P00330), Rabbit Glycogen Phosphorylase b (GPB, SwissProt P00489), Bovine Serum Albumin (BSA, SwissProt P02769), and Yeast Enolase I (ENO, SwissProt P00924) with sequencing grade trypsin, and producing mixtures in the indicated molar ratios. The MPDS mixtures are purified, and do not contain undigested target protein, trypsin, other hydrophilic components or salts

MPDS Mix 1 and Mix 2 Contents

ProteinMPDS Mix 1

[Molar ratio, Amount (pmol)]MPDS Mix 2

[Molar ratio, Amount (pmol)]

ADH 1.0, 50 pmol 1.0, 50 pmol

GPB 1.0, 50 pmol 0.5, 25 pmol

ENO 1.0, 50 pmol 2.0, 100 pmol

BSA 1.0, 50 pmol 8.0, 400 pmol

MPDS Mix 1 and MPDS Mix 2 Separation Using nanoACQUITY UPLC BEH C18, 1.7 μm, 75 μm x 100 mm Column

Sample:MPDS Mix 1: Alcohol Dehydrogenase 25 fmol/µL, PhosphorylaseB 25 fmol/µL; Enolase 25 fmol/µL; Bovine Serum Albumin 25 fmol/µL

MPDS Mix 2: Alcohol Dehydrogenase 25 fmol/µL; Phosphorylase B 12.5 fmol/µL; Enolase 50 fmol/µL; Bovine Serum Albumin 200 fmol/µL

Column: nanoACQUITY UPLC BEH C18, 1.7 µm, 75 µm x 100 mmMobile phase A: 0.1% FA in H2OMobile phase B: 0.1% FA in ACNGradient: Linear Gradient from 3 to 40% B in 90 min.Injection vol.: 2 µLDetection: MS on Q-Tof Premier (m/z 50–1990)Ionization: ESIMode: Positive

Protein Expression System KitsDescription Qty. Part No. Description Qty. Part No.

Nanoscale Kit 186003904 Microscale Kit 186003903

Kit contains: Kit contains:nanoACQUITY UPLC Atlantis dC18, 3 µm, 75 µm x 150 mm Column 1 186003500 nanoACQUITY UPLC Atlantis dC18, 3 µm, 300 µm x 150 mm Column 1 186003506nanoACQUITY UPLC Symmetry C18, 5 µm, 180 µm x 20 mm Trap Column

1 186003514 MPDS Mix 1 1 186002865

MPDS Mix 1 1 186002865 MPDS Mix 2 1 186002866MPDS Mix 2 1 186002866 MPDS Enolase 1 186002325MPDS Enolase 1 186002325 RapiGest SF, 1 mg 1 186001860RapiGest SF, 1 mg 1 186001860

Waters Protein Expression System KitsThe Protein Expression System consists of nanoACQUITY UPLC System, Q-Tof Premier,™ MassLynx,® ProteinLynx™ Global Server bioinformatics software, and Waters MassPREP chemistry consumables and kits.

MassPREP Chemistry Consumable Kits for the Protein Expression System include nanoACQUITY UPLC Columns, MassPREP Protein Digestion Standards Mix 1 and 2, MassPREP Enolase Digestion Standard, and RapiGest SF Surfactant. Both Microscale and Nanoscale Kits are available.

Protein Expression StandardsDescription Volume Part No.


Solid 186002865Standard consists of a relatively close equimolar ratio of tryptically digested proteins:

Yeast enolase, Phosphorylase b, Yeast alcohol dehydrogenase (ADH), and Bovine serum albumin (BSA) Provides high confidence and coverage during expression profiling of complex protein samples


Solid 186002866Standard consists of a range of molar ratios for tryptically digested proteins:

Yeast enolase, Phosphorylase b, Yeast alcohol dehydrogenase (ADH), and Bovine serum albumin (BSA) Provides high confidence and coverage during expression profiling of complex protein samples


Bioseparations

Hi3 Phos B Standard

Peptide SequenceAverage

MolecularWeight

Concentration

Phos B Sequence 1

VLYPNDNFFEGK 1442.5913 1 nmol/vial

Phos B Sequence 2

TC*AYTNHTVLPEALER 1875.0931 1 nmol/vial

Phos B Sequence 3

IGEEYISDLDQLRK 1678.8602 1 nmol/vial

Phos B Sequence 4

LLSYVDDEAFIR 1440.6164 1 nmol/vial

Phos B Sequence 5

LITAIGDVVNHDPVVGDR 1890.1273 1 nmol/vial

Phos B Sequence 6

VFADYEEYVK 1262.3817 1 nmol/vial

Hi3 E.Coli Standard

Peptide SequenceAverage

MolecularWeight

Concentration

E.coli ClpB Sequence 1

VIGQNEAVDAVSNAIR 1655.8292 1 nmol/vial


NNPVLIGEPGVGK 1293.4863 1 nmol/vial


AIDLIDEAASSIR 1373.5266 1 nmol/vial


VTDAEIAEVLAR 1286.4484 1 nmol/vial


AIQQQIENPLAQQILSGELVPGK 2474.8412 1 nmol/vial


LPQVEGTGGDVQPSQDLVR 1995.1776 1 nmol/vial

Hi3 StandardsThe Hi3 E.coli and Phos B standards are a specialized set of 6 synthetically prepared and highly purified peptides that provide the top 6 ionizing peptides of both the E.coli ClpB and rabbit Phosphorylase B proteins. These standards are quantitated via AAA to equimolar ratios to provide greater accuracy to each analysis. They are greater than 95% pure and have been verified by mass identification. These standards are primarily intended for use with the Hi3 quantification method for MSE proteomics data processed with ProteinLynx Global Server. The E.coli standard is intended for samples of animal origin, and the Phos B standard is intended for samples of microbial origin. The standards may also be of use in the evaluation and bench-marking of proteomic LC-MS systems comprised of nanoACQUITY UPLC and SYNAPT and Xevo Time-of-flight Mass Spectrometers.

Heavy Labeled Quantitative StandardsThe SILAC Hi3 E.coli and Phos B standards are formulated from the same specialized set of the top 6 ionizing peptides of both the E.coli ClpB and rabbit Phosphorylase B proteins that are contained in their non-labeled counterparts: Hi3 Phos B and E.coli standards. The main difference is that these standards are produced to have a heavy labeled reference labeled on the lysine (K) or arginine (R) end of the peptide. The peptides are synthetically prepared and highly purified. These standards are quantitated via AAA to equimolar ratios to provide greater accuracy to each analysis. They are greater than 95% pure and have been verified by mass identification.

SILAC Hi3 Phos B Standard Peptide Sequence Average Molecular Weight Concentration

SILAC Phos B Sequence 1 H2N-VLYPNDNFFEGK-OH 1449.7066 1 nmol/vial

SILAC Phos B Sequence 2 H2N-TC*AYTNHTVLPEALER-OH 1883.9227 1 nmol/vial

SILAC Phos B Sequence 3 H2N-IGEEYISDLDQLRK-OH 1685.8762 1 nmol/vial

SILAC Phos B Sequence 4 H2B-LLSYVDDEAFIR-OH 1449.7531 1 nmol/vial

SILAC Phos B Sequence 5 H2N-LITAIGDVVNHDPVVGDR-OH 1899.0242 1 nmol/vial

SILAC Phos B Sequence 1 H2N-VLYPNDNFFEGK-OH 1269.6055 1 nmol/vial

The cysteine in sequence 2 is carbamidomethylated.

SILAC Hi3 E.coli StandardPeptide Sequence Average Molecular Weight Concentration

SILAC E.coli ClpB Sequence 1 H2N-VIGQNEAVDAVSNAIR-OH 1664.8873 1 nmol/vial

SILAC E.coli ClpB Sequence 2 H2N-NNPVLIGEPGVGK-OH 1300.7276 1 nmol/vial

SILAC E.coli ClpB Sequence 3 H2N-AIDLIDEAASSIR-OH 1382.7433 1 nmol/vial

SILAC E.coli ClpB Sequence 4 H2N-VTDAEIAEVLAR-OH 1295.7113 1 nmol/vial

SILAC E.coli ClpB Sequence 5 H2N-AIQQQIENPLAQQILSGELVPGK-OH 2482.3751 1 nmol/vial

SILAC E.coli ClpB Sequence 1 H2N-LPQVEGTGGDVQPSQDLVR-OH 2004.0304 1 nmol/vial


TruView LCMS Certified VialsTruView LCMS Certified Vials include stringent dimensional tolerances plus UV and MS cleanliness testing. The additional product attribute of TruView vials is low polar analyte adsorption. T he vials are manufactured by a process that limits the concentration of free ions on the surface of glass; ionic sites can cause analyte adsorption. Waters TruView LCMS Certified Vials are tested for high recovery of analyte at 1 ng/mL concentration using UPLC-MS/MS (MRM) and yield little adsorption. T hese vials exhibit the lowest adsorption of autosampler vials in the market.

Waters Certified ContainersWaters Certified Containers are uniquely processed, treated, and certified in the same unique manner as our highly regarded low TOC vials.

Ultra-clean containers can be used on any LC system, including UPLC, LC/UV, and LC-MS, among others. Manufactured to stringent standards, they prevent extraneous peaks and baseline noise stemming from high TOC. To help assist with contamination prevention and facilitate recommended care and use, each container carries the Waters certified mark for easy differentiation in operational use.

Certified ContainersDescription Part No.

Certified Container Kit

Kit contains: 4 certified 1 L bottles, 3 certified 500 mL bottles, 1 clean container cap kit

186007088

Low Volume Certified Container Kit

Kit contains: 5 certified 250 mL clear bottles, 1 certified 500 mL clear bottle, 1 clean container cap kit

186007278

Certified Container, 1 L 186007089

Certified Container, 500 mL 186007090

Clean Container Cap Kit 205000642

TruView LCMS Certified Vials

Description Clear Glass Amber Glass Max Recovery Total Recovery Amber Max Recovery

TruView LCMS Certified Vials, 100/pk with cap and pre-slit silicone/PTFE septa

186005666CV 186005661CV 186005662CV 186005663CV 186005670CV

TruView LCMS Certified Vials, 100/pk with cap and silicone/PTFE septa

186005660CV 186005667CV 186005668CV 186005669CV 186005664CV

pH BuffersDescription Volume Part No.

pH 4 Liter

pH 4 Buffer 1 L129

pH 7 Liter

pH 7 Buffer 1 L133

pH 10 Liter

pH 10 Buffer 1 L137

pH 4 Pint

pH 4 Buffer 1 pint127

pH 7 Pint


pH 10 Pint


pH BuffersThese pH Buffers are directly traceable to NIST SRMs, mercury free, guaranteed stable for at least one year after your receipt, and are supplied with a full certificate of analysis.

Documents

BIOSEPARATIONS AND ANALYSES - Waters Corporation · ACQUITY UPLC SEC System Solution ... Specifically QC tested with tryptic digests of cytochrome ... Six peptides ranging in mass