[ xbridge hplc columns ] extreme. flexibility

Transcription

1 [ xbridge hplc columns ] extreme flexibility

2 XBridge HPLC Columns Reliable Performance Separation scientists know that reproducible column performance is one of the most critical factors for method reliability. Each step of our column manufacturing process is monitored and tightly controlled to maintain unmatched product reproducibility. Every test from raw material characterization to final batch analysis ensures that the XBridge Column you use today will perform the same from column-to-column, batch-to-batch, and year-to-year.

3 Unrivaled Versatility Designed for one purpose to maximize your productivity. Whether the goal is to create a quality control method or to develop a leading edge LC/MS assay, XBridge Columns help by: Improving ph Stability increased column lifetime Enhancing Column Reliability assay ruggedness Maximizing Particle Efficiency unmatched peak shape and peak capacity BEH C 8 BEH C 8 BEH Shield RP8 BEH Phenyl BEH HILIC BEH Amide BEH0 C 8 BEH00 C 8 BEH00 C BEH C 8

4 Unsurpassed Quality by Design XBridge Columns are the benchmark for LC method ruggedness and longevity. They were designed to have superior ph stability over the widest ph range (-), high efficiencies and symmetrical peak shape. XBridge Column chemistries (C 8, C 8, Phenyl, Shield RP8, HILIC and Amide) are used by scientists to provide consistently accurate results for the most rigorous assays. With a selection of 0-general-purpose and application-specific sorbents in the widest range of particle sizes, no other HPLC column family provides the necessary tools for the most demanding chromatographic challenges. Whether you require robust HPLC methods, seamless UPLC transferability, or preparative scaling for product isolation, you can count on the versatility of XBridge Columns. Si Si Si CH CH Polar Group Si Si Si CH CH Polar Group Si XBridge C 8 C 8 Shield RP8 Phenyl HILIC Ligand Type Trifunctional C 8 Trifunctional C 8 Monofunctional Embedded Polar Trifunctional Phenyl-Hexyl Unbonded BEH Particle Ligand Density*. µmol/m. µmol/m. µmol/m.0 µmol/m n/a Carbon Load* 8 7 unbonded Endcap Style proprietary proprietary TMS proprietary n/a USP Classification L L7 L L L ph Range Low ph Temp. Limit 80 C 0 C 0 C 80 C C High ph Temp. Limit 0 C 0 C C 0 C C Pore Diameter* 0 Å 0 Å 0 Å 0 Å 0 Å Surface Area* 8 m /g 8 m /g 8 m /g 8 m /g 8 m /g Particle Size.,.,, 0 µm.,.,, 0 µm.,.,, 0 µm.,., µm.,., µm * Expected or approximate values.

5 Based on BEH Technology Ethylene Bridged Hybrid [BEH] Technology synthesis creates particles that ensure extreme column performance and long column lifetime under harsh operating conditions. The particle is prepared from two high purity monomers tetraethoxysilane [TES] and bis(triethoxysilyl)ethane [BTEE] resulting in a highly-stable, ph-resistant and mechanically-strong particle. BEH Technology Particle Synthesis Et CH CH E t Si Si Et Si Si Et Et Si Et Si Et Et n Et Si Et Et Et + Et Et Si Et Et CH Si Et CH Et Polyethoxysilane (BPES) Tetraethoxysilane (TES) Bis(triethoxysilyl)ethane (BTEE) * US Patents,8,0; 7,,7; 7,0, CH C H 9 Si Linker NH Si Si Si Si CH CH CH C H 9 Si Amide BEH0 C 8 BEH00 C 8 BEH00 C BEH C 8 Si Peptide Separation Technology Peptide Separation Technology Protein Separation Technology ligonucleotide Separation Technology CH Si CH Polar Group Amide Trifunctional C 8 Trifunctional C 8 Monofunctional C Trifunctional C 8 7. µmol/m. µmol/m. µmol/m. µmol/m. µmol/m none proprietary proprietary none proprietary - L L L L C 80 C 80 C 80 C 80 C 90 C 0 C 0 C 0 C 0 C 0 Å 0 Å 00 Å 00 Å 0 Å 8 m /g 8 m /g 90 m /g 90 m /g 8 m /g.,. µm.,, 0 µm.,, 0 µm. µm. µm Si

6 Maximizing Column Efficiency and Column Lifetime ne of the most important parameters in designing the BEH particle was to significantly improve the chromatographic performance of the base particle. The origins of band spreading, which decreases separation efficiency, are described by the van Deemter equation. The c-term in the van Deemter equation describes the mass transfer characteristics of an analyte as it interacts with the internal surface of the stationary phase. State-of-the-art silica-based phases have excellent mass transfer; however, they are limited to a narrow range of chromatographic conditions. A comparison of silica to the BEH particle reveals that hybrid materials maintain the efficiency while extending the range of usable chromatographic conditions. van Deemter Curve Comparison h using N / Ht 0 8 Reduced van Deemter Plot Terms a b c r XBridge C 8 µm Symmetry C 8 µm SunFire C 8 µm Compound: Decanophenone Mobile Phase: 70/0 acetonitrile/water Temperature: 0 C The reduced plate height, h, is a function of the reduced linear velocity, v, (both normalized for particle size) and a, b, and c summarize the contributions of eddy diffusion, longitudinal diffusion, and the sum of stationary- and mobile-phase mass transfer terms, respectively. h = a + b/u + cu u [u i d p /D m ] ConTRLLED bonding to IMPRve PEAk shape The ethylene bridge used during the BEH particle synthesis plays a critical role in providing improved chromatographic peak shape. The ethylene bridge links adjoining silanols. This not only increases particle strength, it reduces free silianol sites to minimize the adverse interactions with the injected sample. Traditional methods such as excessive end capping are limited to the steric hindrance of the end capping agent and bonded ligand to the active site. As a result, free silanol sites may be exposed creating broad and tailing chromatographic peaks. The ethylene bridge reduces the number of free silanols to provide a sterically favorable ratio for bonding and end capping the ligand. Controlling this process is one of the ways that XBridge Columns can provide unsurpassed peak shape performance. Excellent Peak Shape Amitriptyline N USP = 900 T USP =.0 Zorbax SB Phenyl XBridge Phenyl Amitriptyline N USP = 90 T USP = min Comparative separations may not be representative of all applications. XBridge Phenyl Columns combine trifunctional bonding of the phenyl-hexyl ligand with proprietary end capping to produce industry-leading stability and exceptional peak shape.

7 Stability Under Extreme ph Conditions IMPRvED PERformance at LW ph The major cause of poor column lifetime in acidic (low-ph) mobile phases is due to the acid hydrolysis of the bonded phase. XBridge packing incorporates state-of-the-art, proprietary procedures for bonding and end capping resulting in ligand stability and chromatographic reproducibility at low ph. Compared to conventional materials, using an accelerated acidic mobile-phase stability test, XBridge C 8 Columns show very little retention loss or peak shape degradation and exhibit exceptional column lifetime without resorting to sterically hindering the stationary phase. Accelerated Acid Stability Test of Competitive Columns High ph EnDURAnce XBridge Columns are engineered to be the most ph stable and highest performing chromatographic phases commercially available. Approaches that claim high ph resistance, due to special surface modifications, cannot match the lifetime of XBridge HPLC Columns. This industry-leading stability is the product of combining the BEH particle synthesis process with advanced bonding and end-capping technologies. Under accelerated ph 0 stability test conditions, a direct comparison to some of the most popular chromatographic phases, claiming to have extended high-ph stability, clearly shows that the XBridge C 8 Column lifetime exceeds that of the next best column by over 000 with very little degradation in chromatographic performance. BEH C 8 (Waters) Zorbax SB-C8 (Agilent) XSelect CSH C 8 (Waters) Accelerated High-pH Stability Test of Competitive Columns InertSustain C8 (GL Sciences) SunFire C 8 (Waters) Poroshell EC- C8 (Agilent) 00 Accucore C8 (Thermo Scientific) Luna C8 () (Phenomenex) YMC-Triart C8 (YMC) 90 Kinetex.7 µm C8 (Phenomenex) Halo C8 (AMT) Gemini NX- C8 (Phenomenex) of Retention Loss from Methyl Paraben Comparative separations may not be representative of all applications. Initial Retention Factor XBridge packings incorporate well-characterized, state-of-the-art bonding and Comparative separations may not be representative of all applications. endcapping procedures to provide columns that are more stable using acidicph mobile phases compared to conventionally prepared columns Hours in Challenge Mobile Phase XBridge C 8 (0 mm TEA ph 0) XBridge C 8 (0 mm ammonium bicarbonate, ph 0) Taking AdvanTAge of PHSPHATE BuffERS Phosphate buffers possess unique selectivity, excellent UV transparency and good buffering capacity at multiple pka values. However, due to the aggressive nature of phosphate, traditional silica columns often exhibit significant reduced column lifetimes. XBridge Columns demonstrate excellent performance using phosphate buffers across the entire ph range by utilizing advanced bonding and end capping (acid stability) and the chemical resistance (alkaline stability) of BEH particle technology. XSelect CSH C 8 (0 mm TEA ph 0) XSelect CSH C 8 (0 mm ammonium bicarbonate ph 0) XTerra MS C InertSustain 8 (0 mm TEA ph 0) C8 (0 mm ammonium bicarbonate ph 0) Luna C8 () (0 mm TEA ph 0) YMC-Triart C8 (0 mm ammonium bicarbonate ph 0) Capcell Pak C8 MG III (0 mm TEA ph 0) TEA marker = acenaphthene YMC-Pack Pro C8 (0 mm TEA ph 0) Ammonium bicarbonate marker = butylparaben Comparative separations may not be representative of all applications. XBridge Columns resist base particle dissolution and ligand hydrolysis when used with high-ph mobile phases. No other column family has the extended lifetime of an XBridge HPLC Column at elevated ph. To learn more about BEH Technology, visit and reference 70009EN. 7

8 Method Development Flexibility THE UnivERSAL Family of HPLC CLUMns Selecting the most suitable column and separation conditions can be extremely difficult. XBridge HPLC Columns are designed to eliminate the compromises of sorbent selection and deliver the flexibility to work under any mobile phase, temperature and ph conditions necessary to achieve the desired separation. A simple and effective reversed-phase method development approach consists of XBridge Columns, two mobile-phase phs and two organic solvents. The availability of robust columns with wide useable ph ranges can quickly define the starting conditions for rugged method development. ptimized approaches save you time, effort and expense. Recommended Method Screening using Column Chemistry, ph and rganic Modifier C 8 XBridge Low ph in Methanol Shield RP8, ,9 C 8 XBridge High ph in Methanol Shield RP , , By systematically screening a standard set of conditions, the separation scientist can quickly determine the best starting point for further method optimization. Using the results from this matrix, you can eliminate guesswork and maximize your method development productivity. Phenyl 0 7,8 9 Phenyl min min XBridge Low ph in Acetonitrile XBridge High ph in Acetonitrile C ,0 C Shield RP Shield RP Phenyl, Phenyl, min min 8

9 BenefITS of WIDER ph Range for Increased SELECTIvity Systematic changes over a wide range of mobile-phase ph gives you the most control over analyte retention and selectivity. For maximum retention, the ionizable analyte should be in its most neutral form. For example, an acidic compound will have the most retention using acidic mobile phases. As the mobile-phase ph increases, acidic compound retention will decrease. The opposite is true for basic compound retention. High-pH mobile phases will provide the greatest retention, while acidic mobile phases will provide the least retention. For a neutral compound, mobile-phase ph has little effect. Column: XBridge C 8,. x 00 mm,. µm Mobile Phase A: 0 mm potassium phosphate buffer (ph, 7, ) Mobile Phase B: Acetonitrile Flow Rate:. ml/min Gradient: Time Profile (min) A B Instrument: Alliance 9 with 99 PDA Compounds:. Doxylamine. Benzamide. Hydroxyisophthalic acid. Doxepine. Flavone. Fenoprofen ph ph 7 ph min XBridge Columns are unique in their ability to withstand these aggressive conditions (even at ph ) allowing complete flexibility for the method development chemist, while maintaining the highest efficiency values associated with silica columns. IDEAL CLUMns for LC/MS The most sensitive bioanalytical separations rely on mass spectrometry (MS) instrumentation to achieve the low detection limits required for these assays. At low analyte concentrations, detection is much more susceptible to adverse column interactions like ligand bleed and particle shedding, both of which interfere with MS response. The BEH Technology incorporated into XBridge Columns virtually eliminates LC/MS bleed by providing increased hydrolytic stability and particle strength. The following example shows the chromatographic results from an analysis of neurotransmitters in a plasma sample. Note in both HILIC and reversed-phase conditions, signal integrity is maintained without MS signal suppression. 00 XBridge C 8 XP min 00 XBridge Amide XP Method Conditions (RP Analysis) Column: XBridge C 8 XP,. x 7 mm,. µm Part Number: Mobile Phase A: 0. formic acid in water Mobile Phase B: 0. formic acid in acetonitrile Flow Rate: 0. ml/min Gradient: Time Profile (min) A B Initial Inj. Volume: µl Sample Diluent: 0/0 water/acetonitrile with 0. formic acid Column Temp: 0 C Method Conditions (HILIC Analysis) Column: XBridge Amide XP,. x 7 mm,. µm Part Number: Mobile Phase A: 0 mm ammonium formate, ph.0 Mobile Phase B: 9/ acetonitrile/water with 0 mm ammonium formate, ph.0 Flow Rate: 0. ml/min Gradient: Time Profile (min) A B Initial Inj. Volume: µl Column Temp.: 0 C H H H NH H H NH H H N NH min ) Norepinephrine ) Dopamine ) Serotonin 9

10 XBridge HILIC CMPLEMEnTARy SELECTIvity for METHD DevELPMEnt Enhanced RETEnTIn for PLAR BASES An effective method development approach uses both an XBridge HILIC Column and an XBridge Reversed-Phase Column to screen analyte selectivity and retentivity. In some cases, the results of this approach will reveal a reversal in elution order or improved retentivity, which can be advantageous when developing separations for polar analytes. HILIC retention mechanisms are a complex combination of partitioning, ion-exchange and hydrogen bonding, resulting in enhanced retention for polar analytes. Retention occurs when using a polar stationary phase in combination with mobile phases composed of acetonitrile concentrations greater than 80. Compounds:. -acetylmorphine. Morphine. Morphine--β-glucuronide N + H Choline XBridge C 8 Reversed-Phase XBridge C 8 Reversed-Phase Retention Factor = 0 V 0 = 0. min XBridge HILIC V 0 = 0. min XBridge HILIC Retention Factor =.8 0 min min XBridge HILIC Columns provide orthogonal selectivity when compared to reversed-phase HPLC columns. As seen here, the polar metabolites of morphine are retained longer under HILIC conditions (vs. a reversed-phase separation). XBridge HILIC Columns provide retention for polar compounds that are too weakly retained using a traditional reversed-phase method. Enhanced Mass Spectrometry Sensitivity Intensity Utilization of HILIC has grown in popularity, primarily due to the extensive adoption of mass spectrometry as a detector and the necessity of improving sensitivity for the quantification of polar analytes. Unlike reversed-phase methods which utilize high aqueous mobile phases to induce retention of polar compounds, HILIC methods employ an acetonitrile-rich mobile phase. This high organic mobile phase is easily desolvated, resulting in improved ionization efficiency and mass spectrometry response. Learn more about Hydrophilic-Interaction Chromatography, visit and reference x x x 0 7 Intensity.0 x x x 0 7 XBridge C 8 Reversed-Phase XBridge HILIC 0. x Response 8. x Response N + N + H. Acetylcholine. Choline min 0 XBridge HILIC provides enhanced mass spectrometry response resulting in lower limits of detection.

11 XBridge Amide XBridge Amide Columns utilize a chemically stable, trifunctionallybonded amide phase, enabling a new dimension in stability and versatility for HILIC separations. In addition to enhanced retention of highly polar compounds, XBridge Amide Columns are equally suited for sugar (saccharide) analysis. Some of the benefits are:. μm particle size for high resolution, high speed analysis of carbohydrates in complex sample matrices while maintaining or improving chromatographic resolution. Increased chemical stability for high-ph and high-temperature conditions to collapse anomers without the loss of reducing sugars. Separation of Water Soluble Vitamins Column: XBridge Amide,. x 0 mm,. μm Part Number: Mobile Phase A: 0/0 acetonitrile/water with 0 mm ammonium acetate, ph 9.0 Mobile Phase B: 90/0 acetonitrile/water with 0 mm ammonium acetate, ph 9.0 Flow Rate:. ml/min Gradient: Time Profile (min) A B Inj. Volume: 0 µl Sample Diluent: 7/ acetonitrile/methanol with 0. formic acid Column Temp.: 0 C Needle Wash: 9/ acetonitrile/water Seal Wash: 0/0 acetonitrile/water Detection: nm Sampling Rate: 0 points/sec Filter Time Constant: 0. Instrument: Alliance with 998 PDA Exceptional column lifetimes and method robustness from a trifunctionally-bonded amide phase combined with BEH Technology. Improved quantification accuracy since XBridge Amide Columns (unlike amine-based columns) are immune to Schiff-base formation. AU Compounds:. Nicotinamide ( µg/ml). Pyridoxal (0 µg/ml). Riboflavin ( µg/ml). Nicotinic acid (0 µg/ml). Thiamine (0 µg/ml). Ascorbic acid ( µg/ml) 7. B (0 µg/ml) 8. Folic acid (0 µg/ml) 0.0 When Waters began developing HILIC chemistries in 00, the sorbents shared one common characteristic: unbonded particles. The main separation mechanism for unbonded HILIC phases is dominated by weak-ion exchange with some partitioning as a secondary interaction. This promotes and favors retention of basic analytes. However, for a very polar compound that is acidic, neutral, or one that does not have a significant positive charge, an unbonded HILIC column may not provide the desired retention. The lack of bonded phases for HILIC separations prompted Waters to develop the amide phase for enhanced retention of acidic and neutral compounds min Very polar compounds, such as water soluble vitamins, are easily retained and separated using an XBridge Amide HPLC Column. The high-ph mobile phase ionizes the acidic compounds to provide more interaction between the amide ligand and analyte. 7 8 SugAR AnALySIS The use of high-ph mobile phases with the XBridge Amide chemistry enables analysis of carbohydrates using electrospray negative (ES-) ionization, improving sensitivity -fold compared to ELS or RI detection. No metal addition, derivitization or post-column addition is necessary. These benefits cannot be achieved under acidic-ph conditions. Column: XBridge Amide,. x 0 mm,. μm Part Number: Mobile Phase A: 80/0 acetonitrile/water with 0. triethylamine (TEA) Mobile Phase B: 0/70 acetonitrile/water with 0. triethylamine (TEA) Flow Rate:.0 ml/min Flow Profile: 90 A/0 B (7 acetontrile with 0. TEA) Inj. Volume: µl Sample Concentration: mg/ml each Column Temp.: C Instrument: Alliance with ELSD LSU Compounds:. p-toluamide. Fructose. Glucose. Sucrose. Maltose. Lactose Strawberry Smoothie LSU 00 Hot Cross Buns LSU White Bread LSU Lamb Curry Meal LSU Food Sugar Standard mg/ml min

12 SCALABILITY UPLC TECHnLgy BEH Technology is one of the key enablers for UPLC separations ACQUITY UPLC BEH Columns and XBridge HPLC Columns are designed using a common, scalable base particle. The mechanical strength and particle integrity of BEH stationary phases provide column platforms for seamless transferability between UPLC, analytical HPLC and preparative HPLC separations. Chromatographers can take advantage of a wide range of particle sizes (i.e.,.7,.,., and 0 µm) to preserve method efficacy Galantamine HPLC Analysis on an Alliance HPLC System XBridge C 8 Column,. x 00 mm,. µm Flow Rate:. ml/min Galantamine, related substances test Name RT USP Resolution RT Ratio Peak Peak AU Galantamine Peak Peak min Galantamine HPLC Analysis on an ACQUITY UPLC H-Class System XBridge C 8 Column,. x 00 mm,. µm Flow Rate:. ml/min Direct transfer method Name RT USP Resolution RT Ratio Peak Peak AU Galantamine Peak Peak min Galantamine UPLC Analysis on an ACQUITY UPLC H-Class System ACQUITY UPLC BEH C 8 Column,. x 0 mm,.7 µm Flow Rate: 0. ml/min Inj. Volume:. µl Scaled method Name RT USP Resolution RT Ratio AU 0.0 Peak Peak Galantamine Peak Peak min Seemlessly transfer methods from HPLC to UPLC using XBridge HPLC and ACQUITY UPLC BEH Columns.

13 XBRIDGE XP. µm Columns The availability of multiple particle sizes and dimensions allows the optimization of the total cycle time without sacrificing resolution. Methods can easily be transferred from HPLC to UPLC and from UPLC to HPLC. XBridge extended Performance [XP]. µm Columns offer exceptional separation performance, robustness and throughput for HPLC assays while being fully compatible with all HPLC, UHPLC and UPLC Technology platforms. Improve your existing HPLC productivity -X with unmatched selectivity and flexibility: Directly scalable to.7 μm ACQUITY UPLC BEH Columns and larger./ μm XBridge HPLC Columns Designed to withstand higher pressures of 9000 psi (. mm ID) and,000 psi (. and.0 mm ID) Selection of column lengths (0, 0, 7 and 00 mm) for the correct balance between resolution and throughput High Throughput. Lower Backpressure. Improved Productivity. 0.0 XBridge C 8. x 0 mm, µm Alliance 9 HPLC min Make your HPLC system more productive with XP. µm Columns.. Unlike core-shell columns, methods developed on extended Performance. µm Columns can be scaled and transferred to larger. and µm HPLC columns, or to smaller sub--µm UPLC Columns, providing flexibility and method consistency between laboratories. 0.0 XBridge C 8 XP. x 7 mm,. µm ACQUITY UPLC H-Class min 0.0 ACQUITY UPLC BEH C 8. x 0 mm,.7 µm ACQUITY UPLC I-Class min Learn more about XP Columns, visit and reference 70009EN. SEAMLESSLy TRAnsfER HPLC METHDS to UPLC USIng THE ACQUITY UPLC CLUMns Calculator Available with all ACQUITY UPLC Systems, the ACQUITY UPLC Columns Calculator eliminates the guesswork of transferring methods based on gradient, column dimension and particle size.

14 A Particle Designed for Purification XBridge HPLC Preparative Columns contain the same packing materials that are available in UPLC and analytical HPLC dimensions, giving the purification chemist the ideal column family for quickly screening and developing high-load preparative methods. Purification chemists rely on XBridge Preparative Columns as the first choice for a highly-efficient, ph-stable column that provides low backpressures and exceptionally long column lifetimes. XBridge Preparative Columns are designed with: - and 0-µm BEH particles providing the highest loadability with the lowest backpressure BD Column stability to provide unsurpassed column lifetime by eliminating bed collapse Enhanced mass transfer that maintains particle efficiency from.7 µm analytical to 0 µm preparative packing materials. Benefits of ph in Isolation and Purification XBridge Prep C 8 BD, 9 x 0 mm Acidic mobile-phase conditions (ph.) Sample load:. mg Column: XBridge Prep C 8 BD,, 9 x 00 mm, µm Mobile Phase A: Low ph (0. TFA (ph.)) High ph (0 mm ammonium bicarbonate (ph 0)) Mobile Phase B: acetonitrile Gradient: B to 00 B in 0 minutes Detection: 0 nm min XBridge Prep C 8 BD, 9 x 0 mm Alkaline mobile-phase conditions (ph 0) Sample load: 0 mg Cl N N Cl Cl Cl Cl N N Cl Cl. Econazole. Miconazole min n column sample loading for basic compounds can often be increased by using high-ph mobile phases. As this example shows, conventional methods using acidic mobile phases and ion-pairing reagents provide inferior load, retention, and resolution compared to the same method using a high-ph mobile phase.

15 BD TECHNLGY REACHIng a New LevEL of PREPARATIve CLUMn SCALABILTy After many years of research on preparative column packing, Waters developed the ptimum Bed Density (BD) Preparative Column design* to effectively improve column lifetime and packed-bed stability. The BD format revolutionized the industry by delivering the most stable, efficient, and reproducible preparative columns available. The combination of rugged XBridge packings and BD Column design takes preparative column performance to a new level, ensuring direct scalability, maximum efficiency, and long column lifetimes. Direct Scalability Analytical screening result ACQUITY UPLC BEH C 8. x 0 mm,.7 µm * min Preparative method scaled to maintain resolution XBridge Prep C 8 BD 9 x 0 mm, µm * min UPLC Method Gradient: Time A B Flow Rate: 0.8 ml/min Injection Volume: µl Preparative Direct Scale Method Gradient: Time A B Flow Rate:. ml/min Injection Volume: 7 µl Columns: ACQUITY UPLC BEH C 8,. x 0 mm,.7 µm (part number 8000) XBridge Prep BD C 8, 9 x 0 mm, µm (part number ) Mobile Phase A: 0 mm ammonium bicarbonate, ph 0 Mobile Phase B: acetonitrile Gradients: As indicated in the chromatograms Sample Concentration:.0 mg/ml degradation sample Detection: nm Instrument: ACQUITY UPLC with ACQUITY UPLC PDA, AutoPurification with 99 PDA and ZQ Scaling and optimization of preparative separations using XBridge Preparative BD Columns. This example of a degradant isolation from ranitidine (API) (degradant highlighted in blue) shows that by maintaining column length to particle size ratio (L/dp), you can geometrically scale the separation to achieve a high sample load without compromising resolution. Learn more about BD Technology, visit and reference EN. *UK Patent # GB089, US Patent # 7,99,0 BD CLUMn DESIgn The Prep BD Column is designed to incorporate a pair of specially-designed distributors and chemically-inert seals made to prevent leaks at high operating pressures. An exploded view of the elements of an empty BD Column.

16 Peptide Separation Technology Waters Peptide Separation Technology (PST) columns are quality tested with a peptide map in order to ensure the stability of peptide separation methods as well as predictable behavior with the variety of samples encountered in proteomics, protein characterization and peptide synthesis. Available in either 0 Å or 00 Å pore sizes and in particle sizes from.7 µm to 0 µm, XBridge PST Columns provide: Narrow, symmetrical peaks for maximum resolution Excellent separation of a wide variety of peptides Superior peak shape and retention in formic acid and trifluoracetic-acid mobile phases for optimal chromatography Seamless method migration from sub--µm UPLC Technology to 0 µm preparative HPLC separations. Higher Resolution Peptide Mapping T T Column: XBridge BEH0 C 8,. x 0 mm,. µm Mobile Phase A: 0.0 TFA in water Mobile Phase B: 0.0 TFA in acetonitrile Gradient: 0 to B in 9 minutes; to B in 0 minutes Flow Rate: 0.9 ml/min Column Temp.: C Detection: nm AU T TT T T T9T0 T0 T9C T8 T T9 TT Compounds: T N-AcGDVEK T-T KYIPGTK T YIPGTK T IfvQK T9-T0 KTGQAPgfSYTDANK T0 TGQAPgfSYTDANK T9C EDLIAY T8 TGPNLHGLFGR T MIFAGIK T CAQCHTVEK (heme attached) T9 EDLIAYLK T-T GITWGEETLMEYLENPkk T GITWGEETLMEYLENPK min Retention time and method reproducibility are critical considerations for choosing a column suitable for peptide mapping. All XBridge PST C 8 Columns are rigorously tested using a QC sample based on a tryptic digest of bovine cytochrome c. This insures that the column provides peptide separations spanning a broad range of hydrophobicity isoelectric points. Learn more about Bioseparation Columns, visit and reference 70008EN.

17 ligonucleotide Separation Technology ligonucleotide Separation Technology (ST) columns are available in both.7 µm UPLC and. µm HPLC particle formats to provide the flexibility to meet a wide variety of isolation and analytical needs while still delivering exceptional resolution and column lifetime. XBridge ST Columns offer: Separation efficiencies equivalent or better than PAGE-CGE or ion-exchange HPLC methods The ability to resolve large oligonucleotide sequences due to the enhanced resolving power obtained using sub--µm particles Scalable column offerings for lab-scale isolation needs Exceptional column life for reduced cost per analysis. utstanding Column Lifetime of the XBridge ST Column Separation of mer Detritylated ligode-oxythymidine Ladder Injection # Injection #00 Column: XBridge ST C 8,. x 0 mm,. µm Part Number: 8009 Mobile Phase: A: 0 methanol/90 (8 mm HFIP +. mm TEA) B: methanol/7 (8 mm HFIP +. mm TEA) TEA Column Temp.: 0 C Gradient: 0 00 B in 0 min (0- methanol) Flow Rate:.0 ml/min Detection: 0 nm, scans per second Instrument: Alliance HPLC Bioseparations e79 with PDA min min Protein Separation Technology The analysis and characterization of protein samples requires the detection of small chemical differences between large molecules. Protein analyses use an array of chromatographic techniques including reversed-phase and size-exclusion chromatography (SEC), each being sensitive to a different property of the protein. XBridge BEH00 C Columns provide: 00Å C Columns Developed for Protein Chromatography Column: XBridge BEH00 C,. x 0 mm,. µm Part Number: Mobile Phase A: 0. TFA in water Mobile Phase B: 0.07 TFA in 7. acetonitrile Flow Rate: 0. ml/min Gradient: 80 B in min Column Temp.: 0 C Inj. Volume: µl Detection: 0 nm 0. Sample: MassPREP Protein Standard (Part Number ) 0. TFA in acetonitrile. Ribonuclease A (0.0 mg/ml). Cytochrome c (0.0 mg/ml). Bovine serum albumin (0.0 mg/ml). Myoglobin (0. mg/ml). Enolase (0. mg/ml). Phosphorylase b (0.9 mg/ml) Protein separations based on size, hydrophobicities, and isoelectric points. µm packing for HPLC and.7 µm packing for UPLC methods AU 0.0 Significant reduction of protein carryover 0.0 Quality-control testing with a known protein mixture ESI-MS compatibility for protein identification. 8 min XBridge BEH00 C 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. 7

18 rdering Information XBridge Analytical Columns Dimension Type Particle Size Qty. C 8 C 8 Shield RP8 Phenyl HILIC Amide.0 x 0 mm Column. µm /pk x 0 mm Guard. µm /pk x 0 mm IS Column. µm /pk x 0 mm Column. µm /pk x 0 mm XP Column. μm /pk x 0 mm XP Column. μm /pk x 0 mm Column. µm /pk x 0 mm XP Column. µm /pk x 0 mm XP Column. µm /pk x 7 mm Column. µm /pk x 7 mm XP Column. µm /pk x 7 mm XP Column. µm /pk x 00 mm XP Column. µm /pk x 00 mm XP Column. µm /pk x 0 mm Guard. µm /pk x 0 mm IS Column. µm /pk x 0 mm Column. µm /pk x 0 mm XP Column. µm /pk x 0 mm XP Column. µm /pk x 0 mm Column. µm /pk x 0 mm XP Column. µm /pk x 0 mm XP Column. µm /pk x 7 mm Column. µm /pk x 7 mm XP Column. µm /pk x 7 mm XP Column. µm /pk x 00 mm XP Column. µm /pk x 00 mm XP Column. µm /pk x 0 mm Guard. µm /pk x 0 mm IS Column. µm /pk x 0 mm Column. µm /pk x 0 mm XP Column. µm /pk x 0 mm Column. µm /pk x 0 mm XP Column. µm /pk x 7 mm Column. µm /pk x 7 mm XP Column. µm /pk x 00 mm XP Column. µm /pk x 0 mm Column. µm /pk x 00 mm Column. µm /pk x 0 mm Column. µm /pk x 0 mm Guard. µm /pk x 0 mm IS Column. µm /pk x 0 mm Column. µm /pk x 0 mm Column. µm /pk x 00 mm Column. µm /pk x 0 mm Column. µm /pk x 0 mm Guard. µm /pk x 0 mm IS Column. µm /pk x 0 mm Column. µm /pk x 0 mm Column. µm /pk x 00 mm Column. µm /pk x 0 mm Column. µm /pk x 0 mm Guard. µm /pk x 0 mm IS Column. µm /pk x 0 mm Column. µm /pk x 0 mm Column. µm /pk x 7 mm Column. µm /pk x 00 mm Column. µm /pk x 0 mm Column. µm /pk x 0 mm Column. µm /pk x 0 mm Guard µm /pk x 0 mm IS Column µm /pk Requires. x 0 mm Universal Sentry Guard Holder, Part No. WAT09798 Requires.0 x 0 mm/. x 0 mm Universal Sentry Guard Holder, Part No. WAT090 8

19 XBridge Analytical Columns Dimension Type Particle Size Qty. C 8 C 8 Shield RP8 Phenyl HILIC Amide. x 0 mm Column µm /pk x 0 mm Column µm /pk x 00 mm Column µm /pk x 0 mm Column µm /pk x 0 mm Guard µm /pk x 0 mm IS Column µm /pk x 0 mm Column µm /pk x 0 mm Column µm /pk x 00 mm Column µm /pk x 0 mm Column µm /pk x 0 mm Column µm /pk x 0 mm Guard µm /pk x 0 mm IS Column µm /pk x 0 mm Column µm /pk x 0 mm Column µm /pk x 7 mm Column µm /pk x 00 mm Column µm /pk x 0 mm Column µm /pk x 0 mm Column µm /pk Requires. x 0 mm Universal Sentry Guard Holder, Part No. WAT09798 Requires.0 x 0 mm/. x 0 mm Universal Sentry Guard Holder, Part No. WAT090 XBridge Preparative Columns Dimension Type Particle Size C 8 C 8 Shield RP8 Phenyl HILIC 0 x 0 mm Guard µm x 0 mm Column µm x 00 mm Column µm x 0 mm Column µm x 0 mm Column µm x 0 mm Guard µm BD 9 x 0 mm Column µm BD 9 x 00 mm Column µm BD 9 x 0 mm Column µm BD 9 x 0 mm Column µm BD 0 x 0 mm Column µm BD 0 x 7 mm Column µm BD 0 x 00 mm Column µm BD 0 x 0 mm Column µm BD 0 x 0 mm Column µm BD 0 x 0 mm Column µm BD 0 x 00 mm Column µm BD 0 x 0 mm Column µm BD 0 x 0 mm Column µm x 0 mm Guard 0 µm x 0 mm Column 0 µm x 0 mm Column 0 µm x 0 mm Guard 0 µm BD 9 x 0 mm Column 0 µm BD 9 x 00 mm Column 0 µm BD 9 x 0 mm Column 0 µm BD 9 x 0 mm Column 0 µm BD 0 x 7 mm Column 0 µm 8007 BD 0 x 00 mm Column 0 µm BD 0 x 0 mm Column 0 µm BD 0 x 0 mm Column 0 µm BD 0 x 0 mm Column 0 µm BD 0 x 00 mm Column 0 µm BD 0 x 0 mm Column 0 µm BD 0 x 0 mm Column 0 µm Requires 0 x 0 mm Prep Guard Holder, Part No Requires 9 x 0 mm Prep Guard Holder, Part No

20 XBridge Columns Method Validation Kits Each Method Validation Kit contains columns, each from a different batch. Dimension Particle Size C8 C8 Shield RP8 Phenyl. x 00 mm. µm x 00 mm. µm x 0 mm. µm x 00 mm. µm x 0 mm. µm x 0 mm µm x 00 mm µm x 0 mm µm x 00 mm µm x 0 mm µm x 0 mm µm LIT ERATURE REFERENCES Description Part Number Analytical Columns Wall Chart 7000EN A Review of Waters Bonded Phase Shield Technology and its Use in High Performance Liquid Chromatography (HPLC) W hite Paper EN A Review of Waters Hybrid Particle Technology. Part. Ethylene-Bridged [BEH Technology] Hybrids and T heir Use in Liquid Chromatography W hite Paper 70009EN Beginner s Guide to Liquid Chromatography PTIMUM BED DENSITY PREPARATIVE CLUMNS 700 Bridging the Performance Gap from Analytical to Prep Bioseparations and Analyses Brochure 70008EN Mass Loading Many factors affect the mass capacity of preparative columns. T he listed capacities represent an average estimate. Approximate Mass Loading Capacity (mg) for Prep BD Columns (Gradient Mode) Diameter (mm) Length (mm) Reasonable Flow Rate (ml/min).. 0 Reasonable Injection Volume (µl) Capacity is: Higher for strongly retained material Higher for simple mixtures Lower where higher resolution is required Very strongly dependent on loading conditions - Limited by loading volume - Limited by diluent solvent strength 0 Comprehensive Guide to HILIC 700 ptimum Bed Density (BD) Columns: Enabling Technology for Laboratory-Scale Isolation and Purification W hite Paper EN a Reasonable injection volumes are based on column diameter at a length of 0 mm with relatively strong solvents. Increased length is compatible with larger injection, but not proportionately so. Weaker solvents significantly increase injection volume. LC CoLumnS designed For SuCCESSFuL method development and method TranSFEr. Formula Buffering Equilibrium 0 mm Concentration Mobile Phase Preparation** ph Adjustment (Acid or Base) CHCNH CHCH CHC- Ammonium Acetate pka pka* 0.77 g CHCH or NHH CHCNH NH+ NH 0.77 g CHCH or NHH.-7. NHHC HC HC g HCH or NHH NHHC NH+ NH 0.79 g HCH or NHH NHHC HC- C g HCH or NHH NHCH HCH HC Ammonium Formate pka NHCH NH+ NH Triethylammonium Acetate (TEAA) pka (CHCH)N:CHCNH (:) CHCH CHC- Triethylammonium Acetate (TEAA) pka (CHCH)N:CHCNH (:) Triethylammonium Formate (TEAF) pka (CHCH)N:NHCH (:) (CHCH)N:NHCH (:) Triethylammonium Formate (TEAF) pka Acetic Acid (glacial) Ammonium Hydroxide Formic Acid N-Methylpyrrolidine 7000EN Buffer Range Ammonium Bicarbonate pka Ammonium Bicarbonate pka Ammonium Acetate pka HCH or NHH HCH or NHH (CHCH)N 0.70 ml TEA (99 conc.) X ml (97 conc.) CH9N CH9NH+00 CH9N 0.8 ml (99. conc.) (CHCH)N (CHCH)NH+ (CHCH)N. ml (99 conc.) CFCH Trifluoroacetic acid (TFA) CFCH CF C- 0.7 ml (00 conc.) X ph..0 ph 7 BEH Amide keep the same k loadability values High phadjusted for basicgradient analytestoresults in the best 8.00 ph. Bases. Nordoxepin. Doxepin. Amitriptyline ph., -, X ph 0 ph 0 X X X X Low ph for acidic analytes results in the highest loadability Si CH Si Si (ligonucleotide Separation Tec hnology) (ligonucleotide Separation Technology) Si (Glycan Separation Technology) Si acquity Columns XSelect Columns CSH C8 CSH C8 Si CSH Phenyl-Hexyl CSH Phenyl-Hexyl Si CSH Fluoro-Phenyl CSH Fluoro-Phenyl Si HSS C8 HSS C8 Si C H9 CH BEH Glycan.0 BEH C8-0. BEH C8 BEH00 SEC.X higher loading DMS + 0 mm FA NH H H H H Non-ionic loading of ionizable compound can improve peak shape and increase loading Linker NH. Adjusted gradient to keep the same k values Adjusted gradientph to keep 00 mg/ml Mobile Phase: the same k Econazole 00 00, X 00 X ph ph X higher loading - DMS + 0 mm FA DMS + 0 mm NHH X X Key Scaling 0 mg/ml 00 mg/ml performance 00 Mobile Phase: ph X higher loading DMS + 0 mm FA M = Mass on Column L = Length of Column M = Mass on Column L = Length of Column Flow Rate Proportional to column volume - 00 Limited by solubility in mobile phase.x higher loading L DMS + 0 mm FA DMS + 0 mm NHH M = M x L.00. x.0 d d = Diameter of Column t = Gradient Duration for Column F = Flow Rate for Column d = Diameter of Column t = Gradient Duration for Column F = Flow Rate for Column d.7.00 Gradient Duration Proportional to cross-sectional area for constant linear velocity d L Increased x M =linear M velocity x gives shorter runsl dl df x by flow x and t x restricted t =Ultimately d F L of system pressure limits Injection Volume Slope is change/column volume, not time Resolution is constant with L the samednumber off column volumes x conditions x x initial to final t = tfrom the dsystem F Must include initial hold tolmimic delay, expressed as number of column volumes, observed on small-scale separation Approximately proportional to column volume Approximately proportional to both length and cross-sectional area Most strongly dependent on sample solvent F = F x d F F F F F F F F F HSS C8 SB Si HSS T HSS T Si HSS PFP HSS PFP Si HSS CN HSS CN Si L dd M = MF =x F x x L dd t = t x L L x d d x atlantis Columns particle F F F CN F = F x Si Recommended Method for Measurement d Programmed time =.00 minutes Use Acetonitrile as mobile phase A, and Acetonitrile with 0.0 mg/ml uracil as mobile phase B (eliminates non-additive mixing and viscosity problems)..9 minutes -.00 minutes 0.9 minutes AU 0. Time =.9 minutes System Volume: 0.9 min. x. ml/min. =.0 ml HILIC. Remove column. Flow Rate =. ml/min d 8.00 C8 Si. Set UV detector at nm.. Use the flow rate in the original method and the intended flow rate on the target instrument. Sunfire HPLC Columns particle. Collect 00 A baseline for minutes.. Program a step change at minutes to 00 B, and collect data for an additional minutes. Si 7. Measure absorbance difference between 00 A and 00 B. ptimum Bed Density (BD) Preparative Columns Bridging the Performance Gap from Analytical to Prep 9. Calculate time difference between start of step and 0 point. C8 Si 0. Multiply time difference by flow rate. Scale-up can be carried out using the equations shown above Convenient scale-up tools on the Waters Prep Calculator provide: - Mass load scaling - Gradient scaling with appropriate flow rate scale-up and predicting volume consumption - Calculations for split flow ratios for those using mass spectrometer driven chromatography ph range (room Temp.) Temperature Limits Carbon Load particle Size (Spherical) Low ph = 80 C L - High ph = C.7 µm.,., µm Selectivity Features: General purpose alternative selectivity ligand that provides pi-pi interactions with polyaromatic compounds, while maintaining excellent reproducibility at ph extremes. Charged Surface Hybrid (CSH) technology enables superior peak shape and increased loading capacity for basic compounds. Bonding: Trifunctional C Phenyl ligand, fully end-capped, bonded to a CSH particle substrate. Low ph = 0 C L -8 0 High ph = C.7 µm.,., µm Selectivity Features: General purpose column that provides a very high degree of analyte selectivity, especially when using low-ph mobile phases. Charged Surface Hybrid (CSH) technology enables superior peak shape and increased loading capacity for basic compounds. Low ph = C L -8 7 High ph = C.8 µm.,., µm Selectivity Features: A general purpose column designed to maximize selectivity differences for Lewis bases through pi-pi interactions. T he rigid aromatic ring provides additional selectivity based on shape, dipole moment and hydrogen bonding interactions. Bonding: Trifunctional pentafluorophenyl ligand, non-endcapped, bonded to High Strength Silica (HSS) substrate. Low ph = C L0-8 High ph = C.8 µm.,., µm Selectivity Features: A general purpose column that shows contrasting analyte selectivity when compared to C8 phases. T his column can be used for both reversed-phase and normal-phase separations. Bonding: Sterically hindered, mono-functional cyano-propyl ligand, non-endcapped, bonded to High Strength Silica (HSS) substrate. usp Class no. ph range (room Temp.) Temperature Limits Carbon Load usp Class no. ph range (room Temp.) Temperature Limits (Spherical) Carbon Load Waters Corporation has been awarded the following registrations and certifications: Waters owns and controls every step of the process, from raw materials to final particle Size (Spherical) Column selection made easy with Waters ipad App. - US Food and Drug Administration registered: cgmp s & Class medical devices Primary Manufacturer of Chromatographic Media To downlaod, go to particle Size Low ph = 0 C L -8 High ph = 0 C.,.,, 0 µm Selectivity Features: General purpose method development column. Very high loading capacity, particularly for basic analytes in low ph mobile phases. Ideally suited for purification and impurity profile assays. Bonding: Difunctional C8, fully endcapped, bonded to high purity silica substrate. Low ph = 0 C L7-8 High ph = 0 C.,.,, 0 µm Selectivity Features: General purpose method development column. Very high loading capacity, particularly for basic analytes in low ph mobile phases. Less hydrophobic, therefore, less retentive than C8 for most analytes. Bonding: Difunctional C8, fully endcapped, bonded to high purity silica substrate. C8 8. Measure time at 0 of that absorbance difference. Prep Calculator Low ph = 90 C - High ph = 90 C.7 µm Selectivity Features: A HILIC UPLC column (0Å pore size) for the LC and LC/MS analysis of fluorescently labeled glycans. Specifically QC tested with -AB labeled human mab IgG derived glycans. Bonding: Trifunctional amide bonded to Ethylene Bridged Hybrid (BEH) substrate. Low ph = C L -8 High ph = C,, 0 µm Selectivity Features: Retention of polar compounds, compatible with 00 aqueous mobile phases, superior stability under low ph conditions. Specifically designed for enhanced retention of polar analytes. Bonding: T (C8) bonding and endcapping, bonded to high purity silica substrate. Low ph = C L - unbonded High ph = C, µm Selectivity Features: Excellent for retention of very polar, basic, water soluble analytes. Specifically designed and tested for HILIC separations using mobile phases containing high concentrations of organic solvent. Bonding: Unbonded high purity silica substrate. Low ph = C L -7 High ph = C,, 0 µm Selectivity Features: Retention of polar compounds. Designed for compatibility with 00 aqueous mobile phases. Bonding: Difunctional C8 bonding, fully endcapped, bonded to high purity silica substrate. T d System Volume System volume is important in scaling separations because it creates an isocratic hold at the start of every run. T his hold is often several column volumes on a small scale, but a fraction of the volume of a prep column. Compensation for this volume must be included in planning a scaling experiment to avoid distorting the chromatography. Bonding: Trifunctional amide bonded to Ethylene Bridged Hybrid (BEH) substrate. Low ph = 80 C L - 8 High ph = 0 C.7 µm.,, 0 µm Selectivity Features: ph and temperature stable, small pore (0Å), C8 LC column for peptides. Specifically QC tested with peptide map. Bonding: Trifunctional C8, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate. Low ph = 80 C L - High ph = 0 C.7 µm.,, 0 µm Selectivity Features: ph and temperature stable, wide pore (00Å), C8 LC column for peptides. Specifically QC tested with peptide map. Bonding: Trifunctional C8, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate. Low ph = 80 C L 8 High ph = 0 C.7 µm. µm Selectivity Features: ph and temperature stable, wide pore (00Å), C LC column for proteins. Specifically QC tested with protein mixture. Bonding: Proprietary monofunctional C bonding to Ethylene Bridged Hybrid (BEH) substrate. Low ph = 0 C L -8 High ph = 0 C.7 µm Selectivity Features: A UPLC SEC column (00Å pore size) for proteins from 0,000 to 0,000 daltons. Specifically QC tested with protein standards. Bonding: Diol bonded to a high pore volume Ethylene Bridged Hybrid (BEH) substrate. Low ph = 0 C L -8 High ph = 0 C.7 µm Selectivity Features: A UPLC SEC column (0Å pore size) for proteins from,000 to 80,000 daltons. Specifically QC tested with protein standards. Bonding: Diol bonded to a high pore volume Ethylene Bridged Hybrid (BEH) substrate. Low ph = 80 C L - 8 High ph = 0 C.7 µm. µm Selectivity Features: ph and temperature stable, small pore (0Å), C8 LC column for synthetic DNA and RNA. Specifically QC tested with synthetic DNA ladder. Bonding: Trifunctional propyl fluorophenyl ligand, non-endcapped, bonded to a CSH particle substrate. Low ph = C L -8 High ph = C.8 µm.,., µm Selectivity Features: Ultra performance C8 chemistry, increased retention, superior peak shape, resists acid hydrolysis at low ph. Designed for UPLC separations where silica-based C8 selectivities are desired. Bonding: High coverage trifunctional C8, fully endcapped, bonded to High Strength Silica (HSS) particle substrate. Low ph = C L -8 8 High ph = C.8 µm.,., µm Selectivity Features: Unique, non-endcapped C8 chemistry designed specifically for method development scientists. ffers unique Selectivity for Bases (SB) when operating under low ph conditions. Bonding: Intermediate coverage trifunctionally bonded C8, no endcapping, bonded to High Strength Silica (HSS) particle substrate. Low ph = C L -8 High ph = C.8 µm.,., µm Selectivity Features: Aqueous mobile-phase compatible column designed for exceptional polar compound retention. Bonding: T (C8) bonding and endcapping, bonded to High Strength Silica (HSS) particle substrate.. Mobile Phase: ph 00.. MassSample Load Diluent: DMS Sample Diluent: DMS HSS C8 SB extended X Mobile Phase: ph Sample Diluent: DMS - ph 7 DMS + 0 mm NHH X ph 7 00 Sample Diluent: DMS. Cloxacillin. Dicloxacillin Sample Diluent: DMS ph. (Spherical) Bonding: Trifunctional C8 ligand, fully end-capped, bonded to a CSH particle substrate. 0 mg/ml Mobile Phase: ph Acids 00 Sample Diluent: DMS Mobile Phase: ph. xacillin particle Size Low ph = 80 C L - High ph = C.7 µm.,., µm Selectivity Features: General purpose reversed-phase column that offers excellent ph stability and rapid mobile-phase re-equilibration for method development. Charged Surface Hybrid (CSH) technology enables superior peak shape and increased loading capacity for basic compounds. 00 Bases. Nordoxepin. Doxepin. Amitriptyline ph. Carbon Load Bonding: Trifunctional C Phenyl, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate. Low ph = C L -9 unbonded High ph = C.7 µm.,., µm Selectivity Features: Excellent for retention of very polar, basic, water soluble analytes. Specifically designed and tested for HILIC separations using mobile phases containing high concentrations of organic solvent. Bonding: Unbonded Ethylene Bridged Hybrid (BEH) substrate. Low ph = 90 C - High ph = 90 C.7 µm.,. µm Selectivity Features: Rugged HILIC stationary phase designed to separate a wide range of very polar compounds. Especially good at separating carbohydrates (saccharides) using high concentrations of organic modifier, elevated temperature and high ph. Compatible with all modern detectors including MS, ELSD, UV and Fluorescence. usp Class no. particle 00 [ XP columns ] Temperature Limits Bonding: Trifunctional C8, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate. 0 mg/ml 00 mg/ml (Protein Separation Technology) BEH00 SEC Acids [ ACQUITY UPLC SYSTem ACCeSSorIeS ] (Protein Separation Technology) (Protein Separation Technology) Mobile Phase: ph Sample Diluent: DMS Si BEH00 C - - DMS + 0 mm NHH - Si BEH00 C8 (Peptide Separation Technology) BEH00 C 00 ph X 00 ph BEH0 C8 (Peptide Separation Technology) BEH00 C8 (Peptide Separation Technology) Linker (Protein Separation Tec hnology) 00.7X mg/ml X X ph 7. Cloxacillin. Dicloxacillin Sample Diluent: DMS ph 7 X Acids 00 mg/ml ph. Mobile Phase: ph. xacillin 00 ph. X ph 7 Acids. xacillin. Cloxacillin. Dicloxacillin 00 Adjusted gradient to keep the same k values 00 X ph BEH0 C8.00 (Peptide.00 Separation Technology) to keep the same k values Non-ionic loading ofadjusted ionizablegradient compound can improve peak shape and increase loading Bases. Nordoxepin. Doxepin. Amitriptyline Adjusted gradient to keep the same k values Si X Bases 00 BEH Amide ph 0 Sample Diluent Effect Polar Group CH 00 X Mobile Phase ph Effect 70009EN CH Si Si BEH HILIC - ph 0 X P Columns Brochure BEH Phenyl ph range (room Temp.) Low ph = 80 C L - 8 High ph = 0 C.7 µm.,.,, 0 µm Selectivity Features: General purpose column ideally suited for method development due to extreme ph stability and applicability to the broadest range of compound classes. Bonding: Trifunctional C8, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate. Low ph = 0 C L7 - High ph = 0 C.7 µm.,.,, 0 µm Selectivity Features: General purpose column ideally suited for method development due to extreme ph stability and applicability to the broadest range of compound classes. Bonding: Trifunctional C8, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate. Low ph = 0 C L - 7 High ph = C.7 µm.,.,, 0 µm Selectivity Features: Alternate selectivity as compared to straight chain C8, particularly with phenolic analytes. Compatible with 00 aqueous-phase composition. Bonding: Monofunctional embedded polar C8, fully endcapped, bonded to Ethylene Bridged Hybrid (BEH) substrate. Low ph = 80 C L - High ph = 0 C.7 µm.,., µm Selectivity Features: Excellent method development column for alternate selectivity, particularly in regard to polyaromatic compounds. Unique level of ph stability for a Phenyl bonded phase..7x usp Class no. particle BEH Shield RP8 X BEH HILIC ph 7 * pka and Buffer Range determined in aqueous solution. Addition of organic modifiers may change the apparent pka value. **Addition of volume or mass per Liter. Silica-based columns (SunFire Prep and Atlantis Prep) should not be operated at high ph. Please refer to reverse side. Si Acids. xacillin BEH Phenyl. Cloxacillin. Dicloxacillin 00. Amitriptyline 0. ml (88 conc.).0. Triethylamine (TEA) Si BEH C8 BEH Shield RP8 Adjusted gradient to keep the same k values TEA or HCH Bases 0.7 ml (00 conc.). Nordoxepin. Doxepin 0. ml (9 as NH conc.), Pyrrolidine - BEH C8 BEH C8 TEA or HCH 0. ml Formic Acid (88 conc.) ph. NH+ NH HCH HC CHNH+ CHN XBridge Columns BEH C8 TEA or CHCH 0.9 ml Acetic Acid (00 conc.) CHCH CHC- CHN Waters complete family of UPLC and HPLC columns offer scientists the most diverse range of c hromatographic selectivity and particle sizes providing exceptional scalability between UPLC, HPLC and preparative formats. acquity Columns TEA or CHCH. ml TEA (99 conc.). ml TEA (99 conc.) Adjusted to keep the same k values (CHCH)NH+ (CHCHgradient )N 0.0 ml Formic Acid (88 conc.) NHH HCH 0. g 0. g 0.70 ml TEA (99 conc.) 0.7 ml Acetic Acid (00 conc.) HCH HC- CHCH 0. (CHCH) NH+ Featuring CSH Technology HSS Technology Featuring BEH Technology Mass loading capacities for peptide purifications depend strongly on the sequence and may be estimated at of listed values. Ammonium Bicarbonate pka Mobile Phase Chemical 70007EN Preparative ptimum Bed Density (BD) Columns Brochure Peptide Separation Technology Reasonable flow rates are based on column diameter. Systems will be limited by increasing backpressure with increasing length and decreasing particle size. Mobile Phase Chart Ammonium Formate pka Preparative BD Columns Wall Chart - IS 900:008 - IS 8:00 product (few suppliers are capable of doing this). Understanding and controlling our processes makes the difference in product performance in your laboratory. 0 Waters Corporation. Waters, XBridge, Atlantis, SunFire, BEH Technology, XSelect, CSH, ACQUITY UPLC, UPLC, and The Science of What s Possible are trademarks of Waters Corporation. ipad is a registered trademark of Apple. ctober EN KK-PDF [ ] 0 Waters Corporation. Waters, XBridge, Atlantis, SunFire, BEH Technology, XSelect, CSH, ACQUITY UPLC, UPLC, and The Science of What s Possible are trademarks of Waters Corporation. ipad is a registered trademark of Apple. ctober EN KK-PDF [ ] our processes makes the difference in product performance in your laboratory. Chromatographic Media Primary Manufacturer of To downlaod, go to product (few suppliers are capable of doing this). Understanding and controlling Waters owns and controls every step of the process, from raw materials to final Waters Corporation has been awarded the following registrations and certifications: Waters ipad App. made easy with Column selection - IS 8:00 - IS 900:008 - US Food and Drug Administration registered: cgmp s & Class medical devices Bridging the Performance Gap from Analytical to Prep ptimum Bed Density (BD) Preparative Columns - Calculations for split flow ratios for those using mass spectrometer driven chromatography - Gradient scaling with appropriate flow rate scale-up and predicting volume consumption - Mass load scaling Convenient scale-up tools on the Waters Prep Calculator provide: Scale-up can be carried out using the equations shown above 0. Multiply time difference by flow rate. Prep Calculator 9. Calculate time difference between start of step and 0 point. C8 Bonding: Difunctional C8, fully endcapped, bonded to high purity silica substrate. phases. Less hydrophobic, therefore, less retentive than C8 for most analytes. Selectivity Features: General purpose method development column. Very high loading capacity, particularly for basic analytes in low ph mobile.,.,, 0 µm High ph = 0 C L7-8 Low ph = 0 C Bonding: Difunctional C8, fully endcapped, bonded to high purity silica substrate. phases. Ideally suited for purification and impurity profile assays. Selectivity Features: General purpose method development column. Very high loading capacity, particularly for basic analytes in low ph mobile.,.,, 0 µm High ph = 0 C L -8 Low ph = 0 C Si 8. Measure time at 0 of that absorbance difference. 7. Measure absorbance difference between 00 A and 00 B. for an additional minutes.. Program a step change at minutes to 00 B, and collect data. Collect 00 A baseline for minutes. the chromatography. scaling experiment to avoid distorting this volume must be included in planning a volume of a prep column. Compensation for volumes on a small scale, but a fraction of the of every run. T his hold is often several column because it creates an isocratic hold at the start System volume is important in scaling separations System Volume F = F x min. x. ml/min. =.0 ml System Volume: rate on the target instrument.. Use the flow rate in the original method and the intended flow AU 0. 0 mixing and viscosity problems). 0.0 mg/ml uracil as mobile phase B (eliminates non-additive. Use Acetonitrile as mobile phase A, and Acetonitrile with Time =.9 minutes F = F x d d Programmed time =.00 minutes 0.9 minutes -.00 minutes.9 minutes Flow Rate =. ml/min L dd M = MF =x F x x L dd Si C8 particle Si HILIC. Remove column. Recommended Method for Measurement d d C8 Sunfire HPLC Columns. Set UV detector at nm. 0.0 t = t x L L x d d x F F T Si Class no. usp (room Temp.) ph range Limits Temperature Load Carbon (Spherical) particle Size Bonding: Difunctional C8 bonding, fully endcapped, bonded to high purity silica substrate. Selectivity Features: Retention of polar compounds. Designed for compatibility with 00 aqueous mobile phases.,, 0 µm High ph = C L -7 Low ph = C Bonding: Unbonded high purity silica substrate. using mobile phases containing high concentrations of organic solvent. Selectivity Features: Excellent for retention of very polar, basic, water soluble analytes. Specifically designed and tested for HILIC separations, µm High ph = C L - unbonded Low ph = C Bonding: T (C8) bonding and endcapping, bonded to high purity silica substrate. Specifically designed for enhanced retention of polar analytes. Selectivity Features: Retention of polar compounds, compatible with 00 aqueous mobile phases, superior stability under low ph conditions.,, 0 µm High ph = C L -8 Low ph = C

21 Electronic Tools Waters Reversed-Phase Column Selectivity Chart Waters Column Advisor Waters Part Selector & Selectivity Chart for ipad