BIOSEPARATIONS AND ANALYSES - Waters Corporation · ACQUITY UPLC SEC System Solution ......
Transcript of BIOSEPARATIONS AND ANALYSES - Waters Corporation · ACQUITY UPLC SEC System Solution ......
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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
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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.
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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
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BEH C18, 130Å, 1.7 µm2.1 x 150 mm
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BEH C18, 300Å, 1.7 µm2.1 x 150 mm
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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.
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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.
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CSH C18, 130Å, 1.7 µm
Superficially Porous Silica C18, 1.7 µm
BEH C18, 130Å, 1.7 µm
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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.
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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 1.7 µm 2.1 mm 100 mm 186003555
ACQUITY UPLC Peptide BEH C18, 130Å 1.7 µm 2.1 mm 150 mm 186003556
ACQUITY UPLC Peptide BEH C18, 130Å 1.7 µm 2.1 mm 300 mm 186005792
ACQUITY UPLC Peptide BEH C18, 300Å 1.7 µm 2.1 mm 50 mm 186003685
ACQUITY UPLC Peptide BEH C18, 300Å 1.7 µm 2.1 mm 100 mm 186003686
ACQUITY UPLC Peptide BEH C18, 300Å 1.7 µm 2.1 mm 150 mm 186003687
ACQUITY UPLC Peptide BEH C18, 300Å 1.7 µm 1.0 mm 50 mm 186005592
ACQUITY UPLC Peptide BEH C18, 300Å 1.7 µm 1.0 mm 100 mm 186005593
ACQUITY UPLC Peptide BEH C18, 300Å 1.7 µm 1.0 mm 150 mm 186005594
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
DiameterLength Part No.
ACQUITY UPLC Peptide BEH C18, 130Å 1.7 µm 2.1 mm 100 mm 186004896
ACQUITY UPLC Peptide BEH C18, 300Å 1.7 µm 2.1 mm 100 mm 186004897
ACQUITY UPLC Peptide BEH C18, 130Å 1.7 µm 2.1 mm 150 mm 186006517
ACQUITY UPLC Peptide BEH C18, 300Å 1.7 µm 2.1 mm 150 mm 186006516
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
XBridge Peptide BEH C18, 300Å 3.5 µm 4.6 mm 100 mm 186004905
XBridge Peptide BEH C18, 300Å 5 µm 4.6 mm 100 mm 186005464
*Three Columns from three different batches of material
XBridge Peptide BEH C18, 130Å and 300Å HPLC Columns
DescriptionParticle
SizeInner
DiameterLength Part No.
XBridge Peptide BEH C18, 130Å 3.5 µm 1 mm 50 mm 186003560
XBridge Peptide BEH C18, 130Å 3.5 µm 1 mm 100 mm 186003561
XBridge Peptide BEH C18, 130Å 3.5 µm 1 mm 150 mm 186003562
XBridge Peptide BEH C18, 130Å 3.5 µm 2.1 mm 50 mm 186003563
XBridge Peptide BEH C18, 130Å 3.5 µm 2.1 mm 100 mm 186003564
XBridge Peptide BEH C18, 130Å 3.5 µm 2.1 mm 150 mm 186003565
XBridge Peptide BEH C18, 130Å 3.5 µm 2.1 mm 250 mm 186003566
XBridge Peptide BEH C18, 130Å 3.5 µm 4.6 mm 50 mm 186003567
XBridge Peptide BEH C18, 130Å 3.5 µm 4.6 mm 100 mm 186003568
XBridge Peptide BEH C18, 130Å 3.5 µm 4.6 mm 150 mm 186003569
XBridge Peptide BEH C18, 130Å 3.5 µm 4.6 mm 250 mm 186003570
XBridge Peptide BEH C18, 130Å 5 µm 1 mm 50 mm 186003571
XBridge Peptide BEH C18, 130Å 5 µm 1 mm 100 mm 186003572
XBridge Peptide BEH C18, 130Å 5 µm 1 mm 150 mm 186003573
XBridge Peptide BEH C18, 130Å 5 µm 2.1 mm 50 mm 186003574
XBridge Peptide BEH C18, 130Å 5 µm 2.1 mm 100 mm 186003575
XBridge Peptide BEH C18, 130Å 5 µm 2.1 mm 150 mm 186003576
XBridge Peptide BEH C18, 130Å 5 µm 2.1 mm 250 mm 186003577
XBridge Peptide BEH C18, 130Å 5 µm 4.6 mm 50 mm 186003578
XBridge Peptide BEH C18, 130Å 5 µm 4.6 mm 100 mm 186003579
XBridge Peptide BEH C18, 130Å 5 µm 4.6 mm 150 mm 186003580
XBridge Peptide BEH C18, 130Å 5 µm 4.6 mm 250 mm 186003581
XBridge Peptide BEH C18, 300Å 3.5 µm 1 mm 50 mm 186003604
XBridge Peptide BEH C18, 300Å 3.5 µm 1 mm 100 mm 186003605
XBridge Peptide BEH C18, 300Å 3.5 µm 1 mm 150 mm 186003606
XBridge Peptide BEH C18, 300Å 3.5 µm 2.1 mm 50 mm 186003607
XBridge Peptide BEH C18, 300Å 3.5 µm 2.1 mm 100 mm 186003608
XBridge Peptide BEH C18, 300Å 3.5 µm 2.1 mm 150 mm 186003609
XBridge Peptide BEH C18, 300Å 3.5 µm 2.1 mm 250 mm 186003610
XBridge Peptide BEH C18, 300Å 3.5 µm 4.6 mm 50 mm 186003611
XBridge Peptide BEH C18, 300Å 3.5 µm 4.6 mm 100 mm 186003612
XBridge Peptide BEH C18, 300Å 3.5 µm 4.6 mm 150 mm 186003613
XBridge Peptide BEH C18, 300Å 3.5 µm 4.6 mm 250 mm 186003614
XBridge Peptide BEH C18, 300Å 5 µm 1 mm 50 mm 186003615
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
XBridge Peptide BEH C18, 300Å 5 μm 2.1 mm 100 mm 186003619
XBridge Peptide BEH C18, 300Å 5 μm 2.1 mm 150 mm 186003620
XBridge Peptide BEH C18, 300Å 5 µm 2.1 mm 250 mm 186003621
XBridge Peptide BEH C18, 300Å 5 µm 4.6 mm 50 mm 186003622
XBridge Peptide BEH C18, 300Å 5 µm 4.6 mm 100 mm 186003623
XBridge Peptide BEH C18, 300Å 5 µm 4.6 mm 150 mm 186003624
XBridge Peptide BEH C18, 300Å 5 µm 4.6 mm 250 mm 186003625
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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, 1.0 x 100 mm Column 176003062 186006934
ACQUITY UPLC Peptide CSH C18, 130Å, 1.7 µm, 1.0 x 150 mm Column 176003063 186006935
ACQUITY UPLC Peptide CSH C18, 130Å, 1.7 µm, 2.1 x 50 mm Column 176003064 186006936
ACQUITY UPLC Peptide CSH C18, 130Å, 1.7 µm, 2.1 x 100 mm Column 176003065 186006937
ACQUITY UPLC Peptide CSH C18, 130Å, 1.7 µm, 2.1 x 150 mm Column 176003066 186006938
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 100 mm XP Column 176003070 186006942
XSelect Peptide CSH C18, 130Å, 2.5 µm, 2.1 x 150 mm XP Column 176003071 186006943
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 50 mm XP Column 176003074 186006946
XSelect Peptide CSH C18, 130Å, 2.5 µm, 4.6 x 100 mm XP Column 176003075 186006947
XSelect Peptide CSH C18, 130Å, 2.5 µm, 4.6 x 150 mm XP Column 176003093 186007038
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 176003078 186006951
XSelect Peptide CSH C18, 130Å, 3.5 µm, 2.1 x 150 mm Column 176003079 186006952
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 50 mm Column 176003082 186006955
XSelect Peptide CSH C18, 130Å, 3.5 µm, 4.6 x 100 mm Column 176003083 186006956
XSelect Peptide CSH C18, 130Å, 3.5 µm, 4.6 x 150 mm Column 176003084 186006957
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
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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
XBridge Peptide BEH C18, 130Å
5 µm 10 mm 100 mm Column 186003583XBridge Peptide BEH C18, 300Å
5 µm 10 mm 100 mm Column 186003627
XBridge Peptide BEH C18, 130Å
5 µm 10 mm 150 mm Column 186003584XBridge Peptide BEH C18, 300Å
5 µm 10 mm 150 mm Column 186003628
XBridge Peptide BEH C18, 130Å
5 µm 10 mm 250 mm Column 186003585XBridge Peptide BEH C18, 300Å
5 µm 10 mm 250 mm Column 186003629
XBridge Peptide BEH C18, 130Å
5 µm 19 mm 50 mm OBD Column 186003586XBridge Peptide BEH C18, 300Å
5 µm 19 mm 50 mm OBD Column 186003630
XBridge Peptide BEH C18, 130Å
5 µm 19 mm 100 mm OBD Column 186003587XBridge Peptide BEH C18, 300Å
5 µm 19 mm 100 mm OBD Column 186003631
XBridge Peptide BEH C18, 130Å
5 µm 19 mm 150 mm OBD Column 186003945XBridge Peptide BEH C18, 300Å
5 µm 19 mm 150 mm OBD Column 186003946
XBridge Peptide BEH C18, 130Å
10 µm 4.6 mm 50 mm Column 186003648XBridge Peptide BEH C18, 300Å
10 µm 4.6 mm 50 mm Column 186003663
XBridge Peptide BEH C18, 130Å
10 µm 4.6 mm 100 mm Column 186003649XBridge Peptide BEH C18, 300Å
10 µm 4.6 mm 100 mm Column 186003664
XBridge Peptide BEH C18, 130Å
10 µm 4.6 mm 150 mm Column 186003650XBridge Peptide BEH C18, 300Å
10 µm 4.6 mm 150 mm Column 186003665
XBridge Peptide BEH C18, 130Å
10 µm 4.6 mm 250 mm Column 186003651XBridge Peptide BEH C18, 300Å
10 µm 4.6 mm 250 mm Column 186003666
XBridge Peptide BEH C18, 130Å
10 µm 10 mm 50 mm Column 186003652XBridge Peptide BEH C18, 300Å
10 µm 10 mm 50 mm Column 186003667
XBridge Peptide BEH C18, 130Å
10 µm 10 mm 100 mm Column 186003653XBridge Peptide BEH C18, 300Å
10 µm 10 mm 100 mm Column 186003668
XBridge Peptide BEH C18, 130Å
10 µm 10 mm 150 mm Column 186003654XBridge Peptide BEH C18, 300Å
10 µm 10 mm 150 mm Column 186003669
XBridge Peptide BEH C18, 130Å
10 µm 10 mm 250 mm Column 186003655XBridge Peptide BEH C18, 300Å
10 µm 10 mm 250 mm Column 186003670
XBridge Peptide BEH C18, 130Å
10 µm 19 mm 50 mm OBD Column 186003656XBridge Peptide BEH C18, 300Å
10 µm 19 mm 50 mm OBD Column 186003671
XBridge Peptide BEH C18, 130Å
10 µm 19 mm 150 mm OBD Column 186003657XBridge Peptide BEH C18, 300Å
10 µm 19 mm 150 mm OBD Column 186003672
XBridge Peptide BEH C18, 130Å
10 µm 19 mm 250 mm OBD Column 186003658XBridge Peptide BEH C18, 300Å
10 µm 19 mm 250 mm OBD Column 186003673
XBridge Peptide BEH C18, 130Å
10 µm 30 mm 50 mm OBD Column 186003659XBridge Peptide BEH C18, 300Å
10 µm 30 mm 50 mm OBD Column 186003674
XBridge Peptide BEH C18, 130Å
10 µm 30 mm 100 mm OBD Column 186003660XBridge Peptide BEH C18, 300Å
10 µm 30 mm 100 mm OBD Column 186003675
XBridge Peptide BEH C18, 130Å
10 µm 30 mm 150 mm OBD Column 186003661XBridge Peptide BEH C18, 300Å
10 µm 30 mm 150 mm OBD Column 186003676
XBridge Peptide BEH C18, 130Å
10 µm 30 mm 250 mm OBD Column 186003662XBridge Peptide BEH C18, 300Å
10 µm 30 mm 250 mm OBD Column 186003677
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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, 4.6 x 100 mm Column1 186007077 XSelect Peptide CSH C18, 130Å, 5 µm, 30 x 100 mm OBD Column 186007025
XSelect Peptide CSH C18, 130Å, 5 µm, 4.6 x 150 mm Column1 186007078 XSelect Peptide CSH C18, 130Å, 5 µm, 30 x 150 mm OBD Column 186007023
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 100 mm Column 186007032 XSelect Peptide CSH C18, 130Å, 5 µm, 50 x 50 mm OBD Column 186007030
XSelect Peptide CSH C18, 130Å, 5 µm, 10 x 150 mm Column 186007016 XSelect Peptide CSH C18, 130Å, 5 µm, 50 x 100 mm OBD Column 186007027
XSelect Peptide CSH C18, 130Å, 5 µm, 10 x 250 mm Column 186007017 XSelect Peptide CSH C18, 130Å, 5 µm, 50 x 150 mm OBD Column 186007028
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 100 mm OBD Column 186007020
XSelect Peptide CSH C18, 130Å, 5 µm, 19 x 150 mm OBD Column 186007021
XSelect Peptide CSH C18, 130Å, 5 µm, 19 x 250 mm OBD Column 186007031
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
XBridge Peptide BEH C18, 130Å 5 µm 19 mm 10 mm Guard Column 186004468
XBridge Peptide BEH C18, 130Å 10 µm 10 mm 10 mm Guard Column 186004465
XBridge Peptide BEH C18, 130Å 10 µm 19 mm 10 mm Guard Column 186004464
XBridge Peptide BEH C18, 300Å 5 µm 10 mm 10 mm Guard Column 186004471
XBridge Peptide BEH C18, 300Å 5 µm 19 mm 10 mm Guard Column 186004470
XBridge Peptide BEH C18, 300Å 10 µm 10 mm 10 mm Guard Column 186004467
XBridge Peptide BEH C18, 300Å 10 µm 19 mm 10 mm Guard Column 186004466
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
242 www.waters.com/order
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.
243www.waters.com/biosep
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
244 www.waters.com/order
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.
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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
246 www.waters.com/order
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
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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.
248 www.waters.com/order
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
MassPREP Digestion Standard Mix 2
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
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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 10 mg vial 186002123
RapiGest SF 50 mg vial 186002122
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
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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
3.9 x 150 mm Column 5 µm 300Å WAT011793 WAT011794
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
Dimension TypeParticle
SizePore Size
Delta-Pak C18
Delta-Pak C4
3.9 x 300 mm Column 15 µm 100Å WAT011797 WAT011807
3.9 x 300 mm Column 15 µm 300Å WAT011802 WAT011812
7.8 x 300 mm Column 15 µm 100Å WAT011798 WAT011808
7.8 x 300 mm Column 15 µm 300Å WAT011803 WAT011813
19 x 300 mm Column 15 µm 100Å WAT011799 WAT011809
19 x 300 mm Column 15 µm 300Å WAT011804 WAT011814
30 x 300 mm Column 15 µm 100Å WAT011800 WAT011810
30 x 300 mm Column 15 µm 300Å WAT011805 WAT011815
50 x 300 mm Column 15 µm 100Å WAT011801 —
Delta-Pak Radial Compression Preparative Column Segments and PrepPak Cartridges*
Dimension TypeParticle
SizePore Size
Delta-Pak C18
Delta-Pak C4
8 x 100 mm Column 15 µm 100Å WAT025846 WAT025848
8 x 100 mm Column 15 µm 300Å WAT025845 —
25 x 100 mm Column 15 µm 100Å WAT038506 WAT038508
25 x 100 mm Column 15 µm 300Å WAT038507 WAT038509
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 100 mm Column 15 µm 100Å WAT037688 WAT037696
40 x 100 mm Column 15 µm 300Å WAT037692 WAT037700
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.
251www.waters.com/biosep
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 —
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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
253www.waters.com/biosep
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
ACQUITY UPLC Protein BEH SEC, 200Å, 1.7 µm: 10K–450K Daltons
ACQUITY UPLC Protein BEH SEC, 125Å, 1.7 µm: 1K–80K 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.
254 www.waters.com/order
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
255www.waters.com/biosep
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.
256 www.waters.com/order
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
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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 Column 2.5 µm 4.6 x 300 mm 186006852
ACQUITY UPLC Protein BEH SEC, 450Å Guard Kit 2.5 µm 4.6 x 30 mm 186006850
ACQUITY UPLC Protein BEH SEC, 200Å Column 1.7 µm 4.6 x 150 mm 186005225
ACQUITY UPLC Protein BEH SEC, 200Å Column 1.7 µm 4.6 x 300 mm 186005226
ACQUITY UPLC Protein BEH SEC, 200Å Guard Kit 1.7 µm 4.6 x 30 mm 186005793
ACQUITY UPLC Protein BEH SEC, 125Å Column 1.7 µm 4.6 x 150 mm 186006505
ACQUITY UPLC Protein BEH SEC, 125Å Column 1.7 µm 4.6 x 300 mm 186006506
ACQUITY UPLC Protein BEH SEC, 125Å Guard Kit 1.7 µm 4.6 x 30 mm 186006504
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.
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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
ACQUITY UPLC Protein BEH SEC, 200Å, 1.7 µm: 10 K–450 K Daltons
ACQUITY UPLC Protein BEH SEC, 125Å, 1.7 µm: 1 K–80 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 and XBridge Protein BEH SEC 200Å
� ACQUITY UPLC and XBridge Protein BEH SEC 450Å
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.
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Scalable SEC Separations Using UPLC- Versus HPLC-Based SECU
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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, 200Å Column with BEH200 SEC Protein Standard Mix Column 3.5 µm 7.8 mm x 300 mm 176003596
XBridge Protein BEH SEC, 450Å Column with BEH450 SEC Protein Standard Mix Guard Column 3.5 µm 7.8 mm x 30 mm 176003597
XBridge Protein BEH SEC, 450Å Column with BEH450 SEC Protein Standard Mix Column 3.5 µm 7.8 mm x 150 mm 176003598
XBridge Protein BEH SEC, 450Å Column with BEH450 SEC Protein Standard Mix Column 3.5 µm 7.8 mm x 300 mm 176003599
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
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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
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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
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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
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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
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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 100 mm 186004499
XBridge Protein BEH C4, 300Å 3.5 µm 2.1 x 150 mm 186004500
XBridge Protein BEH C4, 300Å 3.5 µm 2.1 x 250 mm 186004501
XBridge Protein BEH C4, 300Å 3.5 µm 4.6 x 50 mm 186004502
XBridge Protein BEH C4, 300Å 3.5 µm 4.6 x 100 mm 186004503
XBridge Protein BEH C4, 300Å 3.5 µm 4.6 x 150 mm 186004504
XBridge Protein BEH C4, 300Å 3.5 µm 4.6 x 250 mm 186004505
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 5 µm 10 x 100 mm 186007307
XBridge Protein BEH C4, 300Å 5 µm 10 x 150 mm 186007308
XBridge Protein BEH C4, 300Å 5 µm 10 x 250 mm 186007309
XBridge Protein BEH C4, 300Å 5 µm 19 x 10 mm Guard Cartridge 186007310
XBridge Protein BEH C4, 300Å 5 µm 19 x 50 mm 186007311
XBridge Protein BEH C4, 300Å 5 µm 19 x 100 mm 186007312
XBridge Protein BEH C4, 300Å 5 µm 19 x 150 mm 186007313
XBridge Protein BEH C4, 300Å 5 µm 19 x 250 mm 186007314
XBridge Protein BEH C4, 300Å 5 µm 30 x 10 mm Guard Cartridge 186007315
XBridge Protein BEH C4, 300Å 5 µm 30 x 50 mm 186007316
XBridge Protein BEH C4, 300Å 5 µm 30 x 75 mm 186007317
XBridge Protein BEH C4, 300Å 5 µm 30 x 100 mm 186007318
XBridge Protein BEH C4, 300Å 5 µm 30 x 150 mm 186007319
XBridge Protein BEH C4, 300Å 5 µm 30 x 250 mm 186007320
XBridge Protein BEH C4, 300Å 10 µm 10 x 10 mm Guard Cartridge 186007325
XBridge Protein BEH C4, 300Å 10 µm 10 x 50 mm 186007326
XBridge Protein BEH C4, 300Å 10 µm 10 x 100 mm 186007327
XBridge Protein BEH C4, 300Å 10 µm 10 x 150 mm 186007328
XBridge Protein BEH C4, 300Å 10 µm 10 x 250 mm 186007329
XBridge Protein BEH C4, 300Å 10 µm 19 x 10 mm Guard Cartridge 186007330
XBridge Protein BEH C4, 300Å 10 µm 19 x 50 mm 186007331
XBridge Protein BEH C4, 300Å 10 µm 19 x 100 mm 186007332
XBridge Protein BEH C4, 300Å 10 µm 19 x 150 mm 186007333
XBridge Protein BEH C4, 300Å 10 µm 19 x 250 mm 186007334
XBridge Protein BEH C4, 300Å 10 µm 30 x 10 mm Guard Cartridge 186007335
XBridge Protein BEH C4, 300Å 10 µm 30 x 50 mm 186007336
XBridge Protein BEH C4, 300Å 10 µm 30 x 75 mm 186007337
XBridge Protein BEH C4, 300Å 10 µm 30 x 100 mm 186007338
XBridge Protein BEH C4, 300Å 10 µm 30 x 150 mm 186007339
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 1.0 x 100 mm 186005590
ACQUITY UPLC Protein BEH C4, 300Å 1.7 µm 1.0 x 150 mm 186005591
ACQUITY UPLC Protein BEH C4, 300Å 1.7 µm 2.1 x 50 mm 186004495
ACQUITY UPLC Protein BEH C4, 300Å 1.7 µm 2.1 x 100 mm 186004496
ACQUITY UPLC Protein BEH C4, 300Å 1.7 µm 2.1 x 150 mm 186004497
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
nanoACQUITY Protein BEH C4, 300Å 1.7 µm 100 μm x 100 mm 186004640
nanoACQUITY Protein BEH C4, 300Å 1.7 µm 150 μm x 100 mm 186004641
*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.
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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
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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
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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
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0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 min
AU
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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
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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
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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.
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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
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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
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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 pC18, 500, 7 µm RPC Polymer 4.6 mm 150 mm 186002154
BioSuite pC18, 500, 13 µm RPC Polymer 21.5 mm 150 mm 186002155
BioSuite pPhenyl, 1000, 10 µm RPC Polymer 2.0 mm 75 mm 186002156
BioSuite pPhenyl, 1000, 10 µm RPC Polymer 4.6 mm 75 mm 186002157
BioSuite pPhenyl, 1000, 13 µm RPC Polymer 21.5 mm 150 mm 186002158
BioSuite Q-PEEK and SP-PEEK Columns are Available in 4.6 x 50 mm
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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
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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
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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
Filters, 10/pk — WAT038806
O-rings, 13/pk (includes 10 inlet/outlet and 3 funnel) — WAT038807
Inlet Connector Assembly — WAT038800
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
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Bioseparations
Description Qty. Part No.
1 Compression Screw and Ferrule ‘Z’ Fitting, Plastic 1/pk WAT082708
2 Union, Plastic 1/pk WAT021951
AP-5 Column Accessories and Spare PartsDescription Dimension Part No.
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
O-rings, 5/pk — WAT023345
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
Inlet Connector Assembly — WAT023349
Outlet Connector Assembly — WAT023348
Collet and Nut Assembly — WAT023346
Ferrule, 10/pk — WAT023347
Funnel Assembly — WAT023396
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 3/8–24 1/pk WAT082734
AP-2 Column Accessories and Spare PartsDescription Dimension Part No.
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
O-rings, 5/pk — WAT027528
Filters, 2/pk — WAT027530
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
Inlet Connector Assembly — WAT027525
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
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
4 Compression Screw and Ferrule ‘Z’ Fitting, Plastic 1/pk WAT082708
5 Compression Screw ‘Z’ Fitting, Steel 10/pk WAT005070
6 Ferrule 1/16” Steel 10/pk WAT005063
Description Qty. Part No.
1 Collet and Nut Assembly (3/8–24) 10/pk WAT005138
2 Ferrule 1/8” Tube 10/pk WAT005136
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
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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
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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)
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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
BioSuite 250Å, 10 μm SEC
BioSuite 450Å, 13 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
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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 250Å, 5 µm HR SEC Silica 7.8 mm 300 mm 14.33 mL Less than 8 mg/mL Less than 200 µL 186002165
BioSuite 450Å, 8 µm HR SEC Silica 7.8 mm 300 mm 14.33 mL Less than 8 mg/mL Less than 200 µL 186002166
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 250Å, 10 µm SEC Silica 7.5 mm 300 mm 13.25 mL Less than 8 mg/mL Less than 200 µL 186002170
BioSuite 250Å, 13 µm SEC Silica 21.5 mm 300 mm 108.9 mL Less than 8 mg/mL Less than 1.6 mLs 186002171
BioSuite 450Å, 13 µm SEC Silica 7.5 mm 300 mm 13.25 mL Less than 8 mg/mL Less than 200 µL 186002172
BioSuite 450Å, 17 µm SEC Silica 21.5 mm 300 mm 108.9 mL Less than 8 mg/mL Less than 1.6 mLs 186002173
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
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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 250 250Å 8 x 104 WAT011525
Ultrahydrogel 500 500Å 4 x 105 WAT011530
Ultrahydrogel 1000 1000Å 1 x 106 WAT011535
Ultrahydrogel 2000 2000Å 7 x 106 WAT011540
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 50 mm 3.5 µm 186000277
Symmetry300 C4 2.1 mm 100 mm 3.5 µm 186000278
Symmetry300 C4 2.1 mm 150 mm 3.5 µm 186000279
Symmetry300 C4 4.6 mm 50 mm 3.5 µm 186000280
Symmetry300 C4 4.6 mm 75 mm 3.5 µm 186000281
Symmetry300 C4 4.6 mm 100 mm 3.5 µm 186000282
Symmetry300 C4 4.6 mm 150 mm 3.5 µm 186000283
Symmetry300 C4 2.1 mm 150 mm 5 µm 186000285
Symmetry300 C4 3.9 mm 150 mm 5 µm 186000286
Symmetry300 C4 4.6 mm 50 mm 5 µm 186000287
Symmetry300 C4 4.6 mm 150 mm 5 µm 186000288
Symmetry300 C4 4.6 mm 250 mm 5 µm 186000289
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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
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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
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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
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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
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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
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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
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
Amino acid standard, hydrolysate 10 x 1 mL
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
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
Amino acid standard, hydrolysate 10 x 1 mL
Sample tubes, 4 x 72/pk
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
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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.
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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
Sample tubes, 4 x 72/pk
Amino acid standard, hydrolysate 10 x 1 mL
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
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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
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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
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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
12–20 %ACN in 10 min
23–37 %ACN in 10 min
28–39 %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
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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
30 x 50 mm* 9.00 µmoles 40.0 mg 40.0 mL/min
50 x 50 mm* 25.00 µmoles 110.0 mg 110.0 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
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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
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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
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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.
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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.
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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, 130Å Column 1.7 µm 2.1 x 100 mm 186004741
ACQUITY UPLC Glycan BEH Amide Column, 130Å Method Validation Kit1 1.7 µm 2.1 x 100 mm 186004907
ACQUITY UPLC Glycan BEH Amide, 130Å Column 1.7 µm 2.1 x 150 mm 186004742
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 2.5 µm 2.1 x 100 mm 186007264
XBridge Glycan BEH Amide, 130Å XP Column 2.5 µm 2.1 x 150 mm 186007265
XBridge Glycan BEH Amide, 130Å XP Column Method Validation Kit1 2.5 µm 2.1 x 150 mm 186007266
XBridge Glycan BEH Amide, 130Å XP Column 2.5 µm 3.0 x 30 mm 186008038
XBridge Glycan BEH Amide, 130Å XP Column 2.5 µm 3.0 x 75 mm 186008039
XBridge Glycan BEH Amide, 130Å XP Column 2.5 µm 3.0 x 150 mm 186008040
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 2.5 µm 4.6 x 50 mm 186007268
XBridge Glycan BEH Amide, 130Å XP Column 2.5 µm 4.6 x 100 mm 186007269
XBridge Glycan BEH Amide, 130Å XP Column 2.5 µm 4.6 x 150 mm 186007270
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 Column 3.5 µm 2.1 x 100 mm 186007503
XBridge Glycan BEH Amide, 130Å Column 3.5 µm 2.1 x 150 mm 186007504
XBridge Glycan BEH Amide, 130Å Guard Column, 2/pk3 3.5 µm 4.6 X 20 mm 186007272
XBridge Glycan BEH Amide, 130Å Column 3.5 µm 4.6 x 50 mm 186007273
XBridge Glycan BEH Amide, 130Å Column 3.5 µm 4.6 x 100 mm 186007274
XBridge Glycan BEH Amide, 130Å Column 3.5 µm 4.6 x 150 mm 186007275
XBridge Glycan BEH Amide, 130Å Column Method Validation Kit1 3.5 µm 4.6 x 150 mm 186007277
XBridge Glycan BEH Amide, 130Å Column 3.5 µm 4.6 x 250 mm 186007276
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.
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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.
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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
RapiGest SF 1 mg vial 186001860
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
RapiGest SF 1 mg vial 186001860
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.
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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
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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 #97 = 5 µL PBS Blank
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 #4 = 10 µL PBS Blank
Inject #3 = 10 µL mAb(1 µg/µL PBS)
Inject #2 = 10 µL PBS Blank, following a 5 µg mAb injection
Inject #3 = 10 µL PBS Blank
Inject #2 = 10 µL mAb(1 µg/µL PBS)
Inject #1 = 10 µL PBS Blank
Excellent Recovery with No Detectable Carryover
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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
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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.
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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 100 mm 186002189 Atlantis dC18 3 µm 150 µm 150 mm 186002468
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
Symmetry C18 3.5 µm 150 µm 50 mm 186002459 Atlantis dC18 3 µm 300 µm 100 mm 186002594
Symmetry C18 3.5 µm 150 µm 150 mm 186002461 Atlantis dC18 3 µm 300 µm 150 mm 186002595
Symmetry300 C18 3.5 µm 150 µm 50 mm 186002462 Atlantis dC18 5 µm 300 µm 50 mm 186002596
Symmetry300 C18 3.5 µm 150 µm 100 mm 186002463 Atlantis dC18 5 µm 300 µm 100 mm 186002597
Symmetry300 C18 3.5 µm 150 µm 150 mm 186002464 Atlantis dC18 5 µm 300 µm 150 mm 186002598
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)
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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 75 µm 100 mm 186003589 XBridge Peptide BEH C18, 300Å 3.5 µm 75 µm 150 mm 186003634
XBridge Peptide BEH C18, 130Å 3.5 µm 75 µm 150 mm 186003590 XBridge Peptide BEH C18, 300Å 3.5 µm 100 µm 50 mm 186003635
XBridge Peptide BEH C18, 130Å 3.5 µm 100 µm 50 mm 186003591 XBridge Peptide BEH C18, 300Å 3.5 µm 100 µm 100 mm 186003636
XBridge Peptide BEH C18, 130Å 3.5 µm 100 µm 100 mm 186003592 XBridge Peptide BEH C18, 300Å 3.5 µm 100 µm 150 mm 186003637
XBridge Peptide BEH C18, 130Å 3.5 µm 100 µm 150 mm 186003593 XBridge Peptide BEH C18, 300Å 3.5 µm 150 µm 50 mm 186003638
XBridge Peptide BEH C18, 130Å 3.5 µm 150 µm 50 mm 186003594 XBridge Peptide BEH C18, 300Å 3.5 µm 150 µm 100 mm 186003639
XBridge Peptide BEH C18, 130Å 3.5 µm 150 µm 100 mm 186003595 XBridge Peptide BEH C18, 300Å 3.5 µm 150 µm 150 mm 186003640
XBridge Peptide BEH C18, 130Å 3.5 µm 150 µm 150 mm 186003596 XBridge Peptide BEH C18, 300Å 3.5 µm 300 µm 50 mm 186003641
XBridge Peptide BEH C18, 130Å 3.5 µm 300 µm 50 mm 186003597 XBridge Peptide BEH C18, 300Å 3.5 µm 300 µm 100 mm 186003642
XBridge Peptide BEH C18, 130Å 3.5 µm 300 µm 100 mm 186003598 XBridge Peptide BEH C18, 300Å 3.5 µm 300 µm 150 mm 186003643
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.
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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
Assembly, Capillary Tubing with Frit 25 µm x 24” 1 430002140
Assembly, Capillary Tubing with Frit 25 µm x 12” 1 430002153
Assembly, Capillary Tubing with Frit 25 µm x 22” 1 430002155
Assembly, Capillary Tubing with Frit 25 µm x 10” 2 430002183
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
DiameterLength Part No.
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
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nanoACQUITY UPLC BEH 130Å and 300Å Columns (10,000 psi)
DescriptionParticle
SizeInner
DiameterLength Part No.
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
nanoACQUITY UPLC Peptide BEH C18, 130Å
1.7 µm 75 µm 150 mm 186003543
nanoACQUITY UPLC Peptide BEH C18, 130Å
1.7 µm 75 µm 200 mm 186003544
nanoACQUITY UPLC Peptide BEH C18, 130Å
1.7 µm 75 µm 250 mm 186003545
nanoACQUITY UPLC Peptide BEH C18, 130Å
1.7 µm 100 µm 100 mm 186003546
nanoACQUITY UPLC Peptide BEH C18, 130Å
1.7 µm 150 µm 100 mm 186003550
nanoACQUITY UPLC Peptide BEH C18, 300Å
1.7 µm 75 µm 100 mm 186003810
nanoACQUITY UPLC Peptide BEH C18, 300Å
1.7 µm 100 µm 100 mm 186003811
nanoACQUITY UPLC Peptide BEH C18, 300Å
1.7 µm 150 µm 100 mm 186003812
nanoACQUITY UPLC Peptide BEH C18, 300Å
1.7 µm 75 µm 150 mm 186003813
nanoACQUITY UPLC Peptide BEH C18, 300Å
1.7 µm 75 µm 200 mm 186003814
nanoACQUITY UPLC Peptide BEH C18, 300Å
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
DiameterLength Part No.
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.
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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
ACQUITY UPLC M-Class HSS T3
1.8 µm 75 µm 150 mm 186007473ACQUITY UPLC M-Class Protein BEH C4, 300Å
1.7 µm 100 µm 100 mm 186007494
ACQUITY UPLC M-Class HSS T3
1.8 µm 75 µm 250 mm 186007474ACQUITY UPLC M-Class Protein BEH C4, 300Å
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
ACQUITY UPLC M-Class Peptide CSH C18, 130Å
1.7 µm 75 µm 150 mm 186007476ACQUITY UPLC M-Class Peptide CSH C18, 130Å
1.7 µm 150 µm 150 mm 186007514
ACQUITY UPLC M-Class Peptide CSH C18, 130Å
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
ACQUITY UPLC M-Class Peptide CSH C18, 130Å
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
ACQUITY UPLC M-Class Peptide CSH C18, 130Å
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
ACQUITY UPLC M-Class Peptide CSH C18, 130Å
1.7 µm 150 µm 100 mm 186007480ACQUITY UPLC M-Class Peptide CSH C18 Column, 130Å, 1/pk
1.7 µm 300 µm 100 mm 186007562
ACQUITY UPLC M-Class Peptide BEH C18, 130Å
1.7 µm 75 µm 100 mm 186007481ACQUITY UPLC M-Class Peptide CSH C18 Column, 130Å, 1/pk
1.7 µm 300 µm 150 mm 186007563
ACQUITY UPLC M-Class Peptide BEH C18, 130Å
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
ACQUITY UPLC M-Class Peptide BEH C18, 130Å
1.7 µm 75 µm 200 mm 186007483ACQUITY UPLC M-Class Peptide BEH C18 Column, 130Å, 1/pk
1.7 µm 300 µm 100 mm 186007565
ACQUITY UPLC M-Class Peptide BEH C18, 130Å
1.7 µm 75 µm 250 mm 186007484ACQUITY UPLC M-Class Peptide BEH C18 Column, 130Å, 1/pk
1.7 µm 300 µm 150 mm 186007566
ACQUITY UPLC M-Class Peptide BEH C18, 130Å
1.7 µm 100 µm 100 mm 186007485ACQUITY UPLC M-Class Peptide BEH C18 Column, 300Å, 1/pk
1.7 µm 300 µm 50 mm 186007570
ACQUITY UPLC M-Class Peptide BEH C18, 130Å
1.7 µm 150 µm 100 mm 186007486ACQUITY UPLC M-Class Peptide BEH C18 Column, 300Å, 1/pk
1.7 µm 300 µm 100 mm 186007571
ACQUITY UPLC M-Class Peptide BEH C18, 300Å
1.7 µm 75 µm 100 mm 186007487ACQUITY UPLC M-Class Peptide BEH C18 Column, 300Å, 1/pk
1.7 µm 300 µm 150 mm 186007572
ACQUITY UPLC M-Class Peptide BEH C18, 300Å
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
ACQUITY UPLC M-Class Peptide BEH C18, 300Å
1.7 µm 150 µm 100 mm 186007489ACQUITY UPLC M-Class Protein BEH C4 Column, 300Å, 1/pk
1.7 µm 300 µm 100 mm 186007568
ACQUITY UPLC M-Class Peptide BEH C18, 300Å
1.7 µm 75 µm 150 mm 186007490ACQUITY UPLC M-Class Protein BEH C4 Column, 300Å, 1/pk
1.7 µm 300 µm 150 mm 186007569
ACQUITY UPLC M-Class Peptide BEH C18, 300Å
1.7 µm 75 µm 200 mm 186007491
nanoACQUITY UPLC Peptide CSH, 130Å Columns (10,000 psi)
DescriptionParticle
SizeInner
DiameterLength Part No.
nanoACQUITY UPLC Peptide CSH C18, 130Å
1.7 µm 75 µm 100 mm 186007070
nanoACQUITY UPLC Peptide CSH C18, 130Å
1.7 µm 75 µm 150 mm 186007071
nanoACQUITY UPLC Peptide CSH C18, 130Å
1.7 µm 75 µm 200 mm 186007072
nanoACQUITY UPLC Peptide CSH C18, 130Å
1.7 µm 75 µm 250 mm 186007073
nanoACQUITY UPLC Peptide CSH C18, 130Å
1.7 µm 100 µm 100 mm 186007074
nanoACQUITY UPLC Peptide CSH C18, 130Å
1.7 µm 150 µm 50 mm 186007246
nanoACQUITY UPLC Peptide CSH C18, 130Å
1.7 µm 150 µm 100 mm 186007075
nanoACQUITY UPLC Peptide CSH C18, 130Å
1.7 µm 150 µm 150 mm 186007247
nanoACQUITY HSS T3 and Symmetry C18, 130Å Columns (10,000 psi)
DescriptionParticle
SizeInner
DiameterLength Part No.
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
Symmetry C18 3.5 µm 75 µm 150 mm 186003492
Symmetry C18 3.5 µm 100 µm 100 mm 186003493
Symmetry C18 3.5 µm 100 µm 150 mm 186003494
Symmetry C18 3.5 µm 150 µm 100 mm 186003495
Symmetry C18 3.5 µm 150 µm 150 mm 186003496
Symmetry C18 3.5 µm 300 µm 100 mm 186003497
Symmetry C18 3.5 µm 300 µm 150 mm 186003498
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.
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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, BEH C18, 130Å, 1.7 µm, 150 µm x 100 mm 186007258
iKey, BEH C18, 300Å, 1.7 µm, 150 µm x 50 mm 186006969
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.
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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.
MassPREP Digestion Standard Mix 1
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
MassPREP Digestion Standard Mix 2
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
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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
E.coli ClpB Sequence 2
NNPVLIGEPGVGK 1293.4863 1 nmol/vial
E.coli ClpB Sequence 3
AIDLIDEAASSIR 1373.5266 1 nmol/vial
E.coli ClpB Sequence 4
VTDAEIAEVLAR 1286.4484 1 nmol/vial
E.coli ClpB Sequence 5
AIQQQIENPLAQQILSGELVPGK 2474.8412 1 nmol/vial
E.coli ClpB Sequence 6
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
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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 7 Buffer 1 pint131
pH 10 Pint
pH 10 Buffer 1 pint135
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.