ACQUITY UPLC Columns. More Choices More Information HSS C 18 HSS C 18 SB HSS T3. ACCQ TAG Ultra

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1 ACQUITY UPLC Columns More Choices More Information BEH C 8 BEH Shield RP8 BEH Phenyl BEH HILIC SB HSS T Peptides ligonucleotides ACCQ TAG Ultra VanGuard

2 THe First and nly UPLC Certified Columns Featuring: n. µm BEH UPLC Particles n.8 µm HSS UPLC Particles n Eight Bonded Phases n Reversed-phase and HILIC n VanGuard Pre-columns

3 ACQUITY UPLC columns and VanGuard Pre-columns are the most technologically advanced LC column products ever created. Designed, tested and guaranteed for use in applications up to 000 psi (000 bar). Unsurpassed efficiency, ruggedness and throughput. Available in over sixty combinations of column configurations and chemistries. Combine faster separations with higher resolution by harnessing the full potential of small particles. [ ]

4 Chromatography without compromise In 00, Waters revolutionized how LC separations were performed by developing the ACQUITY UltraPerformance LC (UPLC ) system. High resolution UPLC separations are realized in a system that is capable of fully utilizing LC columns that are efficiently packed with pressuretolerant, sub- µm particles. The highly flexible ACQUITY UPLC system is known for its unique capability of not having to switch between the ultra performance liquid chromatography (UPLC) capability of UPLC mode and conventional HPLC mode since the same unit is capable of both without modification or upgrades. Since the 90 s chromatographers have been limited to LC systems that were capable of operating at maximum system pressures of only 000 psi (00 bar). This pressure limitation, coupled with large system volumes and slow data acquisition rates hindered the ability of separation scientists to fully realize the speed and efficiencies promised by using small (< µm) particles. The ACQUITY UPLC system shatters these operating limitations and is designed to operate at a maximum pressure of 000 psi (000 bar). Waters ACQUITY UPLC systems are holistically designed to dramatically improve resolution, sample throughput and sensitivity. Flexibility with UPLC Technology Speed, Sensitivity and Resolution Whether your objective is maximum speed or maximum resolution, UPLC technology provides you with the flexibility to achieve both. ULTRA SPEED Absorbance at 0 nm 0.08 UPLC. x 0 mm,. µm Rs (,) =.90 SPEED RESLUTIN SENSITIVITY X 0.X.X x 0 mm,. µm 0.0 HPLC. x 0 mm,.0 µm Rs (,) =. SPEED WITH RESLUTIN Absorbance at 0 nm Rs (,) =.8 8X SAME.X Absorbance at 0 nm RESLUTIN WITH SPEED Absorbance at 0 nm x 00 mm,. µm Rs (,) =.8 X.X.8X Compounds:. Quercetin. Kaempferol. Isorhamnetin x 0 mm,. µm ULTRA RESLUTIN Absorbance at 0 nm Rs (,) =..X.X.X [ ]

5 the power of small particles at Higher Pressures Benefits of Holistic ACQUITY UPLC System and Column Design The potential chromatographic benefits that can be achieved when using sub- µm particle LC columns are not solely due to the particle size. To fully realize the promise of sub- µm particle performance, one must have an LC system that is capable of operating at the optimal linear velocity for the particle (and analyte). Further, this LC system must have an ultra-low system volume; a fast, sensitive, low-volume detector; intelligent software; negligible carryover; and, of course, the ability to reliably run at the higher backpressures generated by small particles operated at their optimal linear velocity. Simply put, this is what the ACQUITY UPLC system was designed to do: allow chromatographers to fully realize and harness the power and promise of sub- µm particle columns. The Benefits of UPLC Pressure Capability The ability to run at the optimal linear velocity is crucial to realizing the performance gains of sub- µm particle columns. In this example, four caffeine metabolites are run using the same chromatographic conditions (except flow rate as noted) on a fully optimized, microbore HPLC system vs. a standard ACQUITY UPLC system. The improvements in efficiency, resolution, peak shape and peak height illustrate the power of UPLC technology. Since the commercialization of the ACQUITY UPLC system in 00, other LC manufacturers that do not possess UPLC technology have subsequently claimed that one can achieve most of the performance (mainly speed) gains of UPLC technology by using small (e.g., µm) particle columns in a conventional LC system at HPLC pressures. Chromatographic theory indicates, however, that this claim is somewhat untrue. In 00, Waters described how one can achieve higher chromatographic throughput with the Intelligent Speed (IS ) family of HPLC columns. However, this was not UPLC technology, but rather fast LC based upon small HPLC particles packed into short columns at the expense of resolution. With UPLC technology, one need not compromise sample throughput for chromatographic fidelity. Waters is the first and only LC manufacturer capable of delivering upon the promise of sub- µm particle UPLC technology. 0. ACQUITY UPLC 0.. x 0 mm,. µm Part Number: 8000 Alliance 9 HPLC System Flow Rate = 0. ml/min PSI MAX =,00 T f () =. N () =,00 Rs (,) = 0.9 ACQUITY UPLC. x 0 mm,. µm Part Number: 8000 ACQUITY UPLC System Flow Rate = 0. ml/min PSI MAX = 8,00 T f () =.0 N () = 0,00 Rs (,) =. AU AU Sub- µm Particle Column Performance Under HPLC Conditions (Non-ptimal Linear Velocity) Sub- µm Particle Column Performance Under UPLC Conditions (ptimal Linear Velocity) [ ]

6 Ultra-fast separations Ultra Speed How does one produce a short separation that does not sacrifice efficiency and/or peak shape? The simple answer lies in chromatographic theory. If one can reduce the column length, while simultaneously reducing the particle size by the same ratio (i.e., maintain column length to particle size (l/dp) ratio), the resolving power of the column as well as the resolution or peak capacity of the resulting chromatogram remains intact. However, this separation can be achieved in less time. The practical and commercial application of this simple chromatographic theory, however, has proven to be challenging. For the first time, UPLC technology allows separation scientists to fully realize the speed and efficiencies promised by short columns packed with very small particles: n Ultra-low system volume (low bandspreading) n Fast, efficient detector and/or mass spectrometer n Short cycle time sample manager with low carryover n Small, rugged, highly efficient particles n Advanced column hardware and packing techniques. Application areas that benefit from the fast separations that are possible with 0 mm length ACQUITY UPLC columns include confirmatory Active Pharmaceutical Ingredient (API) QC, stability monitoring, cleaning protocol validation, early drug development screening, bioanalytical analyses, content uniformity, drug release assays and process monitoring. Ultra-Fast SPE-UPLC/MS/MS Determination of Risperidone and Metabolite The antipsychotic drug risperidone is rapidly metabolized into 9-hydroxyrisperidone and is the predominant circulating species with the same activity as parent drug. Automated sample preparation time is minutes for 9 samples (~9 sec/sample). Peak widths are approximately. seconds wide, P max = 8900 psi. Column: ACQUITY UPLC. x 0 mm,. µm Part Number: 8009 Mobile Phase A: 0 mm CH CNH, ph 9.0 Mobile Phase B: MeH Gradient: Time Flow Profile (min) ml/min %A %B Injection Volume: 8 µl Column Temperature: 0 C Sample Temperature: C Sample Concentration: 0. ng/ml Sample Diluent: 0/0 H /MeH Strong Needle Wash: 0:0 ACN:IPA + 0.% HCH Weak Needle Wash: 9: H /MeH Instrument: ACQUITY UPLC with Quattro Premier MS Quattro Premier ES+ Capillary:.0 kv Source Temp: 0 C Desolvation Temp: 0 C Cone Gas Flow: 0 L/Hr Desolvation Gas Flow: 00 L/Hr Collision Cell Pressure:.9 e- mbar Dwell Time: 0 msec Transfer Tubing: 0.00 Clozapine (IS) Risperidone 9-H Risperidone TIC > 0..e. > 9..8e. > 0..0e TIC.8e asis MCX µelution Plate Sample Preparation Procedure* Condition 0. ml MeH Equilibrate 0. ml H Load: 0. ml diluted Human Plasma (0. ml plasma diluted : with % H P in H ) Wash 0. ml % HCH in H Wash 0. ml MeH Elute: 0 µl ( µl x ) % NH H in 90:0 MeH:H Dilute 0 µl H Recoveries Risperidone 9%, 9-H Risperidone 9% Clozapine 9% * asis MCX 9-well µelution Plate (Part Number 80080BA) [ ]

7 Ultra-High Resolution Separations Ultra Resolution Chromatographers are always looking for solutions that will deliver more resolution and more robust separations. To meet this need for higher efficiency, Waters developed 0 mm length ACQUITY UPLC columns. With these longer columns, challenging separations such as impurity profiling, metabolite ID analyses and drug stability monitoring can be run routinely with high resolution and moderate analysis times. The resolving power of an LC column can be expressed by its length to particle size ratio (l/dp). Columns with the highest l/dp ratios provide greater efficiencies (N) and are normally used in the most difficult separations. The l/dp ratio of the 0 mm length columns is more than 88,000, thus producing efficiencies of >,000 plates per separation. As a point of reference, a. x 0 mm, µm HPLC column has an l/dp ratio of 0,000 and can produce efficiencies of only,000 plates. UPLC Column Length l/dp* Efficiency (N) Separation Type 0 mm,00,8 Easy 0 mm 9,00,0 Moderately Challenging 00 mm 8,800,00 Difficult 0 mm 88,00,000 Extremely Difficult Choosing a UPLC column based upon the relationship between UPLC column length, length to particle size ratio (l/dp), efficiency (N) and separation type. * dp =. µm Ultra-Resolution Separations with 0 mm Length ACQUITY UPLC Columns 0 mm length ACQUITY UPLC columns provide high resolution in less time.. x 0 mm,. µm Rs (,) =. Columns: ACQUITY UPLC,. µm Mobile Phase A: 0.% HCH in H Mobile Phase B: 0.% HCH in ACN Isocratic: 9% A: % B Flow Rate: 0. ml/min Injection Volume:. µl (0 mm). µl (0 mm).0 µl (00 mm). µl (0 mm) Sample Diluent: 9: H : ACN with 0.% HCH Sample Conc.: µg/ml Temperature: 0 C Detection: 80 nm Sampling Rate: 0 pts/sec Time Constant: 0. Instrument: ACQUITY UPLC with ACQUITY UPLC TUV detector x 0 mm,. µm Rs (,) =. Caffeine Metabolites Compounds. -methylxanthene.,-dimethyluric acid. theobromine.,-dimethylxanthene x 00 mm,. µm Rs (,) = x 0 mm,. µm Rs (,) = [ ]

8 Ultra-Sensitive separations Ultra Sensitivity Novel or putative drugs often exhibit high potency resulting in the requirement for low dosage levels. Hence, determining LLQ levels in bodily fluids often present chemists with the challenge of achieving lower limits of detection when developing chromatographic assays for impurity profiling, bioanalysis and metabolite identification. Frequently there are several compounds of interest, all of which must be detected and quantitated in a single chromatographic run. High sensitivity separations can be developed and improved through the use of small i.d. columns in microbore dimensions (e.g.,.0 mm i.d.). Columns of these dimensions offer higher sensitivity over their larger diameter counterparts due to smaller analyte elution volume resulting in an increase in analyte concentration and subsequent increase in detector response. Microbore columns further have advantages in that they consume less mobile phase and can be more easily thermostatically controlled. Increased Sensitivity with.0 mm i.d. ACQUITY UPLC Columns Columns: ACQUITY UPLC. x 0 mm,. µm ACQUITY UPLC.0 x 0 mm,. µm Mobile Phase A: 0.% NH H Mobile Phase B: ACN Flow Rates: 0.00 ml/min. mm i.d. column 0.0 ml/min.0 mm i.d. column Gradient: Time Profile Curve (min) %A %B Initial Injection Volume:. µl Sample : Ibuprofen spiked protein precipitated (PPT) rat plasma Concentration: 0 ng/ml Temperature: 0 C Detection: MRM 0 > Instrument: ACQUITY UPLC with Quattro Premier XE Mass Spectrometer. mm i.d. UPLC Column MRM of Channels ES- 0.8 > 0.8.e mm i.d. UPLC Column.0 MRM of Channels ES- 0.8 > 0.8.e ~ x increase in sensitivity Note: The differences in retention time are due to the volume of the ACQUITY UPLC system. The small ACQUITY UPLC system volume enables the use of.0 mm i.d. UPLC columns without significant loss of separation efficiency. [ 8 ]

9 Resolution Building Resolution with UPLC Technology The fundamental resolution equation for isocratic separations states that resolution (Rs) is proportional to the square root of column efficiency (N). Column efficiency (N) is inversely proportional to particle size (dp). Thus, smaller particles provide higher resolution. The highly efficient. μm BEH particles and.8 µm HSS particles allow chromatographers to maximize the efficiency (N) of their separation with the ACQUITY UPLC system. However, the fundamental resolution equation states that resolution can be improved by changing the selectivity (α) or retentivity (k). Lower efficiency (N) HPLC separations required chromatographers to randomly try many of the hundreds of different types of column chemistries in order to obtain an adequate separation. Since the efficiencies of UPLC separations may be - times higher, a smaller number of ligands and particles are necessary to provide the desired resolution. ACQUITY UPLC columns allow chromatographers to efficiently develop faster and more robust separations. Rs = N α k α k + Maximized in UPLC Separations by: Ultra-low dispersion system Small (< µm) particles Higher pressure capability Well-designed columns Maximized in UPLC Separations by: Range of chemistries Multiple particle substrates Wide usable ph range (BEH) Higher retentivity (HSS) Impact on Resolution % Improvement Double N 0-0% Double k -0% Double α >00% Si Si Si CH Si Polar Group CH UPLC Column Chemistries Si Si Si BEH Particle Si CH Si Polar Group HSS Particle C 8 C 8 Shield RP8 CH Phenyl HILIC C 8 C 8 SB T Chemistry Si Si CH Si Polar Group CH Si Si Ligand Type Trifunctional C 8 Trifunctional C 8 Si Monofunctional Embedded Polar Group Trifunctional C Phenyl Trifunctional C 8 Trifunctional C 8 Trifunctional C 8 Ligand Density*. µmol/m. µmol/m. µmol/m.0 µmol/m. µmol/m. μmol/m. µmol/m Carbon Load* 8% % % % % 8% % Endcap Style Proprietary Proprietary TMS Proprietary Proprietary None Proprietary ph Range * Expected or Approximate Values [ 9 ]

10 Innovation Industry-Leading Engineering and Manufacturing Columns optimized for UPLC separations require innovative hardware and manufacturing processes that are not apparent when simply looking at the outside of the column. Like the holistically designed ACQUITY UPLC system, attention to every detail is critical for chromatographic success. ACQUITY UPLC columns were designed to be an integral part of the low bandspread UPLC system. The column and system could not be developed by simply designing down or re-engineering an existing HPLC column or system. Typical HPLC system extra-column volumes and pressure limits would severely compromise the performance of UPLC columns. ACQUITY UPLC columns are the most technologically advanced columns ever created. Procedures for reproducibly producing and sizing commercial quantities of. μm and.8 µm particles had to be developed. New packing stations and methods had to be designed, invented and implemented since UPLC columns are packed and tested differently than HPLC columns. Additionally, UPLC instrumentation is necessary to test these columns, something that no other manufacturer possesses. Since UPLC technology was created with the future in mind, ACQUITY UPLC columns incorporate ecord technology a step towards the paperless laboratory. Besides storing each column s unique Certificate of Analysis, the ecord tracks column usage such as date of installation, number of injections, number of sample sets, maximum temperature and pressure that the column has been subjected to and the date that the column was last used. All of this information travels with the column and is easily printable. The ecord is permanently attached to the column and the data cannot be erased. Engineering n New column hardware n Low band broadening n Innovative frit design Bulk Synthesis n Rugged and efficient. µm BEH and.8 µm HSS particles n Most technologically advanced porous particles ever created n Combination of highest efficiencies, widest ph range and superior mechanical strength n Stable column beds at UPLC pressures Software n Paperless tracking of column history with ecord technology Column Packing n Columns must withstand UPLC operating pressures n New proprietary packing methods n New test instruments ACQUITY UPLC columns feature ecord technology [ 0 ]

11 HPLC to UPLC Method Transfer Easily Migrate From HPLC to UPLC Technology In successful LC method migration, the selectivity of the original separation can be maintained, or improved, if desired. This requires careful consideration and understanding of such key parameters as column dimensions, system volumes and configurations, injection volumes, analyte molecular weight and gradient profiles. Without proper consideration of all these parameters, the transferred method results will not meet the desired expectations. For years, the necessary scaling calculations involved in method transfer were often performed manually, which is time consuming and can lead to errors. The ACQUITY UPLC columns calculator is an easy to use, yet powerful software tool that accurately handles the scaling calculations that are required to convert isocratic or gradient HPLC methods to UPLC methods. The calculator provides a selection of UPLC methods that the chromatographer can choose from what is most important: maximum speed, maximum resolution or a combination of both. ACQUITY UPLC Columns Calculator choices include: n UPLC methods where the selectivity and resolution of the original HPLC separation can be maintained n Faster UPLC methods that enable higher throughput and productivity without sacrificing resolution n Higher resolution UPLC methods that provide greater confidence in the results without requiring longer analytical run times. Easy HPLC to UPLC Method Transfer with the ACQUITY UPLC Columns Calculator P c =99 P c = Geometrically Scaled HPLC Linear Velocity P c = Maximum Peak Capacity Equivalent Analysis Time riginal HPLC Gradient Method P c =8 P c = Geometrically Scaled UPLC Linear Velocity Equivalent Peak Capacity Shortest Analysis Time The ACQUITY UPLC Columns Calculator allows chromatographers to quickly and accurately convert HPLC methods to UPLC methods. A copy of the ACQUITY UPLC Columns Calculator is included with every ACQUITY UPLC system. [ ]

12 Superior Lifetime and Reproducibility Rugged and Stable Columns The innovation of ACQUITY UPLC columns does not stop with the development of rugged and efficient particles and stationary phases. Another major focus is the production of stable UPLC columns that provide the longest possible lifetimes under demanding UPLC conditions. New column hardware is designed to minimize band broadening and ensure leak-free connections. An innovative frit design is implemented in order to keep particles in the column and out of the detector or MS source. New, proprietary column packing stations and processes are designed and developed to ensure a stable packed column bed and long, reproducible column lifetimes. Long UPLC Column Lifetimes : Diode Array Range:.8e+ Injection # The result: Waters UPLC columns provide column lifetimes under UPLC conditions which meet and/or exceed HPLC column lifetimes run under HPLC conditions. In fact, it is not uncommon to achieve Data courtesy of: C. Salomons, unpublished results, Research CDS-Note, Solvay Pharmaceuticals 0.0 : Diode Array Range:.e+ several thousand injections on a single UPLC column., A. Kaufmann, P. Butcher, K. Maden, M. Widmer, Ultra-performance liquid chromatography coupled to time of flight mass spectrometry (UPLC TF): A novel tool for multiresidue screening of veterinary drugs in urine, Anal. Chim. Acta 8 (-): - [00] J. Mensch, M. Noppe, J. Adriaensen, A. Melis, C. Mackie, P. Augustijns, M.E. Brewster, Novel generic UPLC/MS/MS method for high throughput analysis applied to permeability assessment in early Drug Discovery, J. Chromatogr. B 8(): 8-8 [00] Injection #009! Excellent Reproducibility Column: ACQUITY UPLC. x 0 mm,. µm Part Number: 8009 Mobile Phase A: 0 mm HCNH, ph.0 Mobile Phase B: ACN Flow Rate: 0.8 ml/min Gradient: Time Profile (min) %A %B Injection Volume: µl Sample Diluent: DMS Sample Conc.: 0. mm/l Compounds: Solvay test compounds (proprietary) Temperature: C Detection: 0-00 nm Sampling Rate: 0 pts/sec Time Constant: 0.0 (fast) Instrument: ACQUITY UPLC with ACQUITY UPLC PDA detector, ELSD & ZQ000 Waters continues to set the industry standard for column-to-column and batch-to-batch reproducibility. Beginning with the Symmetry brand of columns in 99 and continuing with the XTerra, Atlantis, SunFire and XBridge brands of HPLC columns, Waters columns provide consistent results. ACQUITY UPLC columns are manufactured in the same cgmp, IS 900, IS 8 certified facilities that produce these industry-leading HPLC column brands. Method development scientists can be assured that the UPLC separation produced this year can be reproduced year after year. Excellent Batch-To Batch Reproducibility Retention Times %RSD Column: ACQUITY UPLC. x 00 mm,. µm Part Number: 800 Mobile Phase A: 0 mm KH P /K HP, ph.0 Mobile Phase B: MeH Isocratic Mobile Phase Composition: % A; % B Flow Rate: 0. ml/min Injection Volume: µl Detection: nm Temperature: 0.0 C Instrument: ACQUITY UPLC system with ACQUITY UPLC TUV Compounds:. Uracil. Propranolol. Butylparaben. -Methylnaphthalene. Dipropylphthalate. Acenaphthene. Amitriptyline [ ]

13 UPLC Column Protection VanGuard Pre-Columns Separation scientists working in demanding application areas such as bioanalysis, food/beverage, natural products, environmental and industrial chemicals analyze complex, unpredictable and challenging samples on a routine basis. These types of samples can have a negative impact on column lifetimes when appropriate sample preparation/cleanup procedures are not implemented. VanGuard Pre-columns are designed for these types of application areas where chemical and/or particulate contamination can shorten the lifetime of a UPLC column. Key Features and Benefits of VanGuard Pre-Columns VanGuard Pre-columns are the result of over two years of product development and are the first guard column devices designed for routine use at pressures up to 000 psi (000 bar) in applications run on the ACQUITY UltraPerformance liquid chromatography system. VanGuard Pre-columns feature a. mm i.d. x mm length, ultra-low volume design which efficiently protects UPLC column performance. This patent-pending design does not compromise the UPLC holistic design approach to higher efficiency, greater resolution and increased throughput since the same ACQUITY UPLC column stationary phases and column frits are used in VanGuard Precolumns. Since the VanGuard Pre-column connects directly to the inlet of the ACQUITY UPLC column, extra-column volumes are minimized and mobile phase leaks due to additional connections are all but eliminated. Minimal Chromatographic Effects With VanGuard Pre-columns Feature First pre-column for UPLC applications Patent pending, ultra-low volume design Manufactured using UPLC column hardware, particles and chemistries Connects directly to UPLC column Benefit Guaranteed compatibility with pressures up to 000 psi Minimal chromatography effects Superior UPLC column protection and performance Leaks and connection voids are eliminated VanGuard Pre-columns are uniquely designed to protect and prolong ACQUITY UPLC column performance while contributing minimal chromatographic effects. Column: ACQUITY UPLC,. x 0 mm,. µm Part Number: 8000 Protein Precipitation Procedure Pre-column: VanGuard Pre-column,,. x mm,. µm Part Number: 8009 Mobile Phase A: 0.% NH H in H Mobile Phase B: 0.% NH H in ACN Flow Rate: 0.8 ml/min Gradient: Time Profile (min) %A %B Injection Volume: µl Temperature: 0 C Detection: nm Sampling Rate: 0 pts/sec Time Constant: 0.0 Instrument: ACQUITY UPLC with ACQUITY UPLC TUV detector and ACQUITY SQ Detector ACQUITY SQ Detector ES+ Capillary:. kv Cone: V Source Temp: 0 C Desolvation Temp: 00 C Cone gas Flow: 0 L/Hr Desolvation Gas Flow: 80 L/Hr SIR:. m/z,. m/z MS Interscan Delay: 0.00 sec Dwell: 0.00 sec Desacetyl Diltiazem SIR m/z. Without VanGuard Pre-column With VanGuard Pre-column Diltiazem SIR m/z. Without VanGuard Pre-column With VanGuard Pre-column H C H H S H N N CH HC S N C N CH H C CH Spike plasma with 00 ng/ml of diltiazem and desacetyl diltiazem Take 00 µl of spiked plasma and add to. ml centrifuge tube Add 00 µl of acetonitrile to the centrifuge tube containing the spiked plasma Centrifuge for 0 minutes at 000 RPM Take 0 µl of supernatent and evaporate to dryness with nitrogen Reconstitute with 00 µl of a 0:0 MeH:H solution (results in a 0. ng/ml conc.) [ ]

14 UPLC Particle Technology ACQUITY UPLC Particle Technology There is more to creating a UPLC particle than synthesizing a small particle. Many HPLC particles do not possess the mechanical stability and structural integrity to withstand UPLC operating pressures (e.g., 000 psi/000 bar). Why is pressure tolerance important? In order to realize the efficiency gains of sub- µm particles, the ability to routinely operate at higher linear velocities (e.g., higher flow rates) is required. These higher linear velocities combined with small, sub- µm particles result in higher operating backpressures. Waters has created two highly efficient, pressure-tolerant UPLC particles: the. µm Ethylene Bridged Hybrid (BEH) particle and the.8 µm High Strength Silica (HSS) particle. The first ACQUITY UPLC particle created was the. µm Ethylene Bridged Hybrid (BEH) particle. This second generation hybrid particle is one of the key enablers behind UPLC technology and is available in five column chemistries: C 8, C 8, Shield RP8, Phenyl and HILIC. Because this is a hybrid particle, a wider usable ph range (up to ph ) makes method development faster and easier. BEH particles are also available in HPLC particle sizes (.,., and 0 µm) in the XBridge family of HPLC columns, thus allowing seamless transfer between HPLC and UPLC separations. ACQUITY UPLC HSS column chemistries include, SB (Selectivity for Bases) and T. The chemistry is a fully endcapped, ultra-performance, general purpose C 8 bonded phase that provides superior peak shape for bases, increased retentivity (vs. ACQUITY UPLC columns), and extremely long lifetimes under acidic conditions. The SB (Selectivity for Bases) chemistry is an unendcapped C 8 bonded phase designed and optimized for low ph method development and offers alternate selectivities, especially for basic compounds, as compared to most modern, high coverage C 8 chemistries. The HSS T chemistry is an aqueous mobile phase compatible C 8 bonded phase that is designed to retain and separate small, water soluble, polar organic molecules, much like Atlantis T HPLC columns. The Particles of UPLC Technology Waters is the nly Manufacturer ffering TW UPLC-Certified Particles ACQUITY UPLC BEH Columns ACQUITY UPLC HSS Columns Particle Type Ethylene Bridged Hybrid (BEH) High Strength Silica (HSS) Available Chemistries C 8, C 8, Shield RP8, Phenyl, HILIC C 8, C 8 SB, T ph Range -; (RP8: -); (HILIC: -8) C 8 : -8; C 8 SB, T: -8 Maximum Rated Pressure 000 psi (~000 bar) 000 psi (~000 bar) Particle Size. µm.8 µm Pore Diameter/Volume 0Å / 0. ml/g 00Å / 0. ml/g Surface Area 8 m /g 0 m /g [ ]

15 UPLC Particle Technology BEH Technology In 999, Waters launched the XTerra family of HPLC columns featuring 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 bases. The BEH Particle: ne of the Key Enablers of UPLC Technology Ethylene Bridges in Silica Matrix 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. A new, second generation hybrid material was developed which 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 behind the speed, sensitivity and resolution of UPLC separations. E t C H C H E t E t S i S i S i S i S i E t Polyethoxysilane (BPES) Anal. Chem. 00,, 8-88 S i E t E t E t E t n E t S i E t E t E t Tetraethoxysilane (TES) + E t E t E t C H S i S i E t C H E t E t Bis(triethoxysilyl)ethane (BTEE) *Patent No.,8,0 B ACQUITY UPLC High Strength Silica (HSS) Particles As more separation scientists around the world realize the benefits of UPLC technology in their applications, Waters continues to provide additional UPLC particle and chemistry solutions to meet these demands. Waters material scientists developed a new High Strength Silica (HSS) particle with the high mechanical stability and appropriate morphology necessary to provide long column lifetimes and UPLC efficiencies at pressures up to 000 psi (000 bar). This.8 µm UPLC HSS particle is designed and tested specifically for use in UPLC separations. ACQUITY UPLC HSS columns contain the first and only 00% silica particle designed, tested and intended for use in applications up to 000 psi/000 bar. The new ACQUITY UPLC HSS particle is not an HPLC particle. High pore volume HPLC particles do not posses the mechanical stability necessary to withstand the high column packing and operating pressures of UPLC separations. Designing and Testing Pressure-Tolerant UPLC Particles In this proprietary Waters particle strength test, chromatographic particles are packed into a column and mobile-phase flow is applied. As pressure increases, the particles are crushed, thereby restricting flow. The degree of deviation from the Ideal Profile line indicates particle fragility/ strength. Waters BEH and HSS particles are two of the strongest chromatographic porous particles commercially available. Flow ACQUITY UPLC HSS.8 µm Ideal Profile ACQUITY UPLC BEH. µm HPLC Silica Pressure [ ]

16 BEH (Ethylene Bridged Hybrid) Chemistries ACQUITY UPLC and C 8 Columns The vast majority of reversed-phase (RP) LC separations take place on columns that contain C 8 or C 8 bonded stationary phases due to their stability, retentivity and reproducibility. In addition, these hydrophobic ligands provide the desired separation most of the time. ACQUITY UPLC and C 8 columns were designed to be the columns of choice for most UPLC separations by providing the widest ph range and efficiencies. They incorporate trifunctional ligand bonding chemistries which produce superior low ph stability and ultra-low column bleed. This low ph stability is combined with the high ph stability of the. μm BEH particle to deliver the widest usable ph operating range. In addition, these new chemistries also utilize new, proprietary endcapping processes which produce outstanding best peak shape for bases. These bonding chemistries and particle synthesis innovations produce the sharpest peaks, highest efficiencies and maximum MS sensitivities. Robust Separations EPA Method 80 HPLC separations usually require temperature control of ± C since large shifts in selectivity can occur with small changes in temperature. The increased resolution of the UPLC technology allows for a robust and less temperature-sensitive (up to ± C) separation C BEH C C C BEH Shield RP BEH C 8 BEH Shield RP8 BEH Phenyl BEH HILIC SB, BEH Phenyl SB HSS T Columns: ACQUITY UPLC BEH,. x 00 mm,. µm ACQUITY UPLC HSS,. x 00 mm,.8 µm Mobile Phase A: Water Mobile Phase B: Methanol Flow Rate: 0. ml/min Isocratic: 8% methanol Injection Volume:.0 µl Sample Conc.: 0 µg/ml Temperature: 0 C Detection: nm Sampling Rate: 0 pts/sec Time Constant: 0. Instrument: ACQUITY UPLC with ACQUITY UPLC PDA detector Compounds:. HMX. RDX.,,-TNB.,-DNB. NB. Tetryl. TNT 8. -Am-, DNT 9. -Am-, DNT 0., DNT.,-DNT. -NT. -NT. -NT HSS T [ ]

17 BEH (Ethylene Bridged Hybrid) Chemistries ACQUITY UPLC BEH Shield RP8 Columns Embedded polar group reversed-phase LC columns contain stationary phases that combine the hydrophobicity of an alkyl ligand with the hydrophilicity of an embedded polar group. Features of embedded polar group columns include alternate selectivities as compared to alkyl chain LC columns, excellent peak shape for bases and aqueous mobilephase compatibility. ACQUITY UPLC BEH Shield RP8 columns are designed to provide selectivities that complement the ACQUITY UPLC C 8 and C 8 phases. ACQUITY UPLC Shield RP8 columns combine Waters patented Shield Technology with BEH Technology by incorporating an embedded carbamate group into the bonded phase ligand. The alternate selectivity and excellent peak shape from the embedded polar group ligand, when combined with the wide ph range and ultra-efficiency of the. μm BEH particle, provide a powerful tool for UPLC method development. Fast Separations of Basic Drugs Challenges involving the separation of basic compounds using reversed-phase LC include poor analyte peak shape and retention. ACQUITY UPLC columns overcome these separations challenges and provide superior results. Note elution order changes for peaks and with the BEH Shield RP8 chemistry and peaks and for the SB chemistry. Columns: ACQUITY UPLC BEH. x 0 mm,. µm ACQUITY UPLC HSS. x 0 mm,.8 µm Mobile Phase A: 0 mm NH CH, ph.0 Mobile Phase B: MeH Flow Rate: 0. ml/min Gradient: Time Profile (min) %A %B Inj. Volume: µl Sample Diluent: H Temperature: 0 C Detection: 0 nm Sampling Rate: 0 points/sec Filter Response: Normal Instrument: ACQUITY UPLC with ACQUITY UPLC PDA Compounds:. Aminopyrazine. Pindolol. Quinine. Labetalol. Verapamil. Diltiazem. Amitriptyline,, BEH Shield RP8 BEH Phenyl BEH C 8, BEH Shield RP8 BEH Phenyl SB BEH HILIC, HSS T SB HSS T [ ]

18 BEH (Ethylene Bridged Hybrid) Chemistries ACQUITY UPLC BEH Phenyl Columns Phenyl-ligand containing reversed-phase columns can provide complementary selectivities as compared to other straightchain alkyl stationary phases, especially for analytes that contain aromatic rings. Traditional weaknesses of phenyl ligands include poor ph stability, reproducibility and peak shape. ACQUITY UPLC BEH Phenyl columns were designed to overcome these weaknesses and provide complementary selectivities, outstanding ph stability and excellent peak shape for all compounds. They utilize a trifunctional C alkyl tether between the phenyl ring and the silyl functionality. This phenyl-hexyl ligand, combined with the same proprietary endcapping processes as the ACQUITY UPLC and C 8 columns, provides ultra-low column bleed, long column lifetimes and excellent peak shape. This unique combination of ligand, endcap and. μm BEH particle creates a new dimension in selectivity and efficiency for challenging UPLC separations. Caffeic Acid Derivatives in Echinacea Purpurea Separations The active components in various Echinacea preparations can be divided into three major groups: caffeic acid derivatives, polysaccharides, and lipophilic components. Studies have shown that there is variation in the quality of popular Echinaceacontaining products. Thus, monitoring of the active ingredients is desired. Shown here are sub-four-minute separations of the primary caffeic acid derivatives polyphenolic compounds that have been shown to possess antioxidant properties. Columns: ACQUITY UPLC BEH,. x 0 mm,. µm ACQUITY UPLC HSS,. x 0 mm,.8 µm Mobile Phase A: 0.% CF CH in H Mobile Phase B: 0.08% CF CH in ACN Flow Rate: 0. ml/min Gradient: Time Profile Curve (min) %A %B Injection Volume:.0 µl Sample Diluent: 0:0 H : MeH with 0.0% CF CH Sample Conc.: 00 µg/ml Temperature: 0 C Detection: 0 nm Sampling Rate: 0 pts/sec Time Constant: 0. Instrument: ACQUITY UPLC with ACQUITY UPLC TUV detector BEH Phenyl BEH C 8 Compounds:. Caftaric acid. Chlorogenic acid. Cynarin. Echinacoside. Cichoric acid BEH Shield RP8 BEH C 8 BEH Shield RP8 BEH Phenyl BEH HILIC SB SB HSS T HSS T [ 8 ]

19 BEH (Ethylene Bridged Hybrid) Chemistries ACQUITY UPLC BEH HILIC Columns Hydrophilic Interaction Chromatography (HILIC) is a technique used to retain and separate very polar compounds that cannot be retained using reversed-phase chromatography. HILIC is also referred to as aqueous normal phase or reverse reversed-phase since the elution order is that of normal phase (non-polar analytes elute first) and the solvents are similar to those of reversed-phase chromatography. The ACQUITY UPLC BEH HILIC columns contain the rugged. µm unbonded BEH particles and are designed to retain and separate very polar basic compounds. These unique columns are optimized and tested to produce efficient and reproducible separations under UPLC HILIC conditions. ACQUITY UPLC BEH HILIC columns overcome a major weakness of HILIC stationary phases: chemical stability. Silica-based HILIC phases are often chemically unstable. The rugged BEH particle s wide usable ph range overcomes this chemical instability and results in long column lifetimes. Hydrophilic Interaction Chromatography (HILIC) is a separation technique where a polar stationary phase is used with a mobile phase that contains a high concentration of non-polar (organic) solvent and a low concentration of polar (aqueous) solvent. HILIC is used when compound retention is not possible using traditional reversed-phase LC. HILIC is quickly gaining popularity as a powerful separation technique since the mobile phases used contain high concentrations of organic modifier (e.g., ACN) which are ideal for compound ionization by ESI-MS. This results in much higher sensitivity and lower limits of detection. Separation of Very Polar Bases Using ACQUITY UPLC BEH HILIC Columns pioid pain relievers such as morphine are used to manage chronic and acute pain. These types of drugs are extremely potent, resulting in low doses and concentrations in body fluids. The study of the efficacy and metabolism of morphine and its metabolites within the body requires selective and sensitive analytical methods such as Hydrophilic Interaction Chromatography (HILIC). In the HILIC separation shown below, the more polar metabolite morphine--β-glucuronide elutes AFTER the more non-polar parent compound, morphine. Column: ACQUITY UPLC BEH HILIC. x 00 mm,. µm Part Number: 800 Mobile Phase A: 0 mm HCNH, 0.% HCH in 0:0 ACN:H Mobile Phase B: 0 mm HCNH, 0.% HCH in 90:0 ACN:H Flow Rate: 0.88 ml/min Gradient: Time Profile (min) %A %B Injection Volume:. µl Sample Conc.: µg/ml Sample Diluent: : ACN:MeH with 0.% HCH Temperature: 0 C Detection: 80 nm Instrument: ACQUITY UPLC with TUV Detector. Morphine. Morphine -β-glucuronide BEH C 8 H CH N BEH Shield RP8 H H N CH C H H H H H BEH Phenyl BEH HILIC V 0 SB HSS T [ 9 ]

20 HSS (High Strength Silica) Chemistries ACQUITY UPLC Columns As chromatographers around the world continue to realize and embrace the benefits of UPLC technology, additional UPLC column choices which provide complementary selectivities are needed to facilitate method development and provide application-specific solutions. Alternate or complementary selectivities can be realized by using columns with different bonded phases and/or different particle substrates. Based upon customer feedback, Waters created an additional pressure-tolerant UPLC-compatible particle to complement the. µm Ethylene Bridged Hybrid (BEH) particle. The.8 µm High Strength Silica (HSS) particle is designed to provide 'silica-like' selectivities and retentivity. The ACQUITY UPLC chemistry which is a fully endcapped, high coverage, ultra-performance C 8 bonded phase that provides superior peak shape for bases, increased retention (vs. ACQUITY UPLC columns), and excellent low ph stability. Although there is no direct HPLC column analog for ACQUITY UPLC columns, chromatographers will discover that for many separations, the selectivities and retention observed when using ACQUITY UPLC columns will resemble the chromatography produced by many modern, silica-based C 8 HPLC columns. ACQUITY UPLC Columns Provide Superior Peak Shapes The efficiencies and separations possible with UPLC technology are the result of more than just small particles. State-ofthe-art bonding and endcapping processes are combined with small, rugged and efficient sub- µm particles to produce narrow, symmetrical peaks that enable fast, high resolution separations. The two Waters UPLC C 8 chemistries produce efficiencies of greater than 00,000 plates/m for the strong base amitriptyline (pk a 9. at ph ) demonstrating that there is more to UPLC separations than sub- µm particles. Column USP Tailing Amitriptyline Efficiency (N) Amitriptyline ACQUITY UPLC,.8 µm Column G VP C 8,. µm. 0 Column G VT C 8,. µm. 0 Column T HGA,.9 µm.0 ACQUITY UPLC,.8 µm Column T HG,.9 µm. Column Z SB C 8,.8 µm. ACQUITY UPLC,. µm.0 0 Column G VP C 8,. µm BEH C 8 BEH Shield RP8 Columns:. x 0 mm Mobile Phase A: 0 mm KH P /K HP, ph.0 Mobile Phase B: MeH Isocratic Mobile Phase Composition: % A; % B Flow Rate: 0. ml/min Injection Volume: µl Detection: nm Temperature: 0.0 C Instrument: ACQUITY UPLC System with TUV, Column G VT C 8,. µm Column T HGA,.9 µm BEH Phenyl BEH HILIC Compounds. Uracil. Propranolol. Butylparaben. -methylnaphthalene. Dipropylphthalate. Acenaphthene. Amitriptyline, Column T HG C,.9 µm Column Z SB C 8,.8 µm SB ACQUITY UPLC,. µm HSS T [ 0 ]

21 HSS (High Strength Silica) Chemistries ACQUITY UPLC Columns Creating st century separations involves more than small particles that operate at higher pressures. The ultraefficiencies, peak capacities and column lifetimes obtained in UPLC separations can only be achieved using columns packed with stationary phases that utilize innovative and industry-leading bonding and endcapping processes. A small particle bonded with a poor and/or outdated stationary phase and/or endcap will produce inadequate efficiencies, inferior resolution and short column lifetimes. Like all ACQUITY UPLC stationary phases, the ACQUITY UPLC columns are created using state-of-the-art bonding and endcapping processes that produce high efficiencies and long column lifetimes. The ACQUITY UPLC stationary phase features a tri-functionally bonded C 8 ligand and proprietary endcapping process that not only yields superior peak shapes for bases at neutral ph, but also resists acid hydrolysis, thus providing extremely long lifetimes at low ph. Since the HSS particle is 00% silica, the new ACQUITY UPLC HSS C 8 chemistry is designed to work and excel under demanding highly acidic conditions. The ability to reliably operate at low ph (i.e., <ph ) is important because ph manipulation is a key tool in controlling and manipulating selectivities of ionizable compounds during method development. The ACQUITY UPLC packing is the most stable bonded phase commercially available and does not suffer from peak shape issues that plague older stationary phases that rely upon steric protection (and lack of endcapping) to produce stability at low ph. ACQUITY UPLC Columns Resist Acid Hydrolysis at Low ph Low-pH Stability: hr Exposure to 0.% TFA at 0 C ACQUITY UPLC,.8 µm ACQUITY UPLC,. µm Column Z SB C 8,.8 µm ACQUITY UPLC HSS T,.8 µm Column G VP C 8,. µm ACQUITY UPLC SB,.8 µm BEH C 8 Column G VT C 8,. µm BEH Shield RP8 Column T HG,.9 µm Column Z EP C 8,.8 µm BEH Phenyl % of Retention Loss for Methyl Paraben BEH HILIC In this test, the loss in retention of the neutral marker methyl paraben indicates bonded stationary phase loss due to acid hydrolysis. ACQUITY UPLC columns resist bonded phase loss due to novel bonding and endcapping processes. SB HSS T [ ]

22 HSS (High Strength Silica) Chemistries ACQUITY UPLC SB (Selectivity for Bases) Columns ACQUITY UPLC SB columns were designed specifically for chromatographers who routinely screen and categorize UPLC column chemistries as part of their method development protocols. Intended for low ph separations which contain complex mixtures of basic and non-basic compounds, ACQUITY UPLC columns can produce separations that will be quite different in terms of selectivities as compared to most modern, high coverage, fully endcapped C 8 stationary phases. How was this accomplished? Waters material scientists bonded a trifunctional C 8 ligand at an intermediate ligand density (i.e.,. µmol/m ) without endcapping. In general, the higher silanophilic activity will promote longer retention for basic compounds whereas the intermediate C 8 ligand density will produce slightly shorter (or equivalent) retention for non-basic compounds as compared to most modern high coverage, fully endcapped C 8 stationary phases. Chromatographically, this unique combination will provide a practical method development tool for complex UPLC separations. Fast Separations of Tricyclic Antidepressants Tricyclic antidepressants (TCAs) are secondary and tertiary amines and were commonly prescribed to patients suffering from depression or other conditions. TCAs are being replaced by selective serotonin reuptake inhibitors (SSRIs). ACQUITY UPLC HSS C 8 SB columns retain basic compounds such as tricyclic antidepressants longer than fully endcapped stationary phases due to the enhanced silanol activity on the non-endcapped HSS particle. Columns: ACQUITY UPLC BEH,. x 0 mm,. µm ACQUITY UPLC HSS,. x 0 mm,.8 µm Mobile Phase A: 0mM NH CH, ph.0 Mobile Phase B: ACN Isocratic Mobile Phase: 0:0; A:B Flow Rate: 0. ml/min Injection Volume: µl Sample Conc: 0 µg/ml in H Temperature: 0 C Detection: nm Sampling Rate: 0 points/sec Filter Response: Normal Instrument: ACQUITY UPLC with ACQUITY UPLC PDA, SB BEH C 8 Compounds:. Trimethoprim. Nordoxepin. Doxepin. Nortriptyline. Imipramine. Amitriptyline. Trimipramine BEH Shield RP8 BEH C 8, BEH Phenyl BEH Shield RP8 BEH Phenyl, HSS T BEH HILIC SB HSS T [ ]

23 HSS (High Strength Silica) Chemistries ACQUITY UPLC SB (Selectivity for Bases) Columns Stationary phases have evolved from the large, irregularly shaped particles of the past to the small, spherical particles of the present. Along the way, bonding and endcapping technologies have advanced and improved as well. Material scientists have focused much effort on improving peak shape for basic compounds in reversed-phase separations. For example, embedded polar group stationary phase columns were created in the 990s to provide symmetrical peaks for basic compounds. The vast majority of reversed-phase separations take place on straight alkyl chain C 8 column chemistries. High ligand density bonding processes along with exhaustive endcapping are often implemented in order to reduce unwanted peak tailing and efficiency loss for basic compounds caused by secondary silanol interactions. Whereas the peak shape for basic compounds improves as a result of these advanced bonding and endcapping procedures, the resulting selectivities for these C 8 chemistries end up being quite similar. This can be seen by noting the large population of hydrophobic C 8 column chemistries located in the lower right quadrant of the Waters Reversed-Phase Column Selectivity Chart. As can be seen in the Waters Reversed-Phase Column Selectivity Chart below, ACQUITY UPLC SB columns do not resemble the selectivities provide by most high coverage, fully endcapped C 8 bonded phases. This selectivity difference is valuable in developing UPLC methods. Waters Reversed-Phase Column Selectivity Chart In the Waters Reversed-Phase Column Selectivity Chart, the selectivity (α) for a non-polar base/neutral pair (amitriptyline/ acenaphthene) is plotted against the retention (k) of the non-polar, neutral compound acenaphthene under ph operating conditions. This chart can be used to compare HPLC and UPLC columns of similar (or different) selectivities. For more information on this chart, please see U. D. Neue, B. A. Alden, T. H. Walter, A Universal Procedure for the Assessment of the Reproducibility and the Classification of Silica-Based Reversed-Phase Packings;. Classification of Reversed-Phase Packings, J. Chrom. A, 89: 0- [999] and U.D. Neue, K.V. Tran, P.C. Iraneta, B.A. Alden, Characterization of HPLC Packings, J. Sep. Sci. (-): -8 [00].. Waters Spherisorb S P. (ln [α] amitriptyline/acenaphthene)....8 Nova-Pak CN HP Waters Spherisorb SCN ACQUITY UPLC BEH Phenyl XBridge Phenyl Hypersil Phenyl Inertsil Ph- Bondapak C 8 Waters Spherisorb DS Resolve C 8 Waters Spherisorb DS YMC J'sphere. Hypersil CPS Cyano YMC-Pack DS-L80 Nucleosil C 8 Inertsil CN- Phenyl Nova-Pak Phenyl. YMC J'sphere DS-M80 Hypersil BDS Phenyl Chromolith Nova-Pak YMC J'sphere YMCbasic YMC-Pack CN XTerra RP-8 C YMC-Pack DS-AQ 8 DS-H Phenyl Nova-Pak Luna Atlantis dc YMC-Pack ProC 8 C Phenyl Hexyl YMC-Pack ProC8 Zorbax 8 Atlantis T XDB C 8 0. Symmetry C YMC-Pack DS-A ACQUITY UPLC BEH C ACT Ace C 8 8 Luna 8 Luna C YMC-Pack 8 () XBridge C XTerra MS C 8 8 C 8 () Inertsil DS- 0. ProC8 SunFire C SunFire C 8 8 XTerra MS C 8 Symmetry C 8 0 SymmetryShield RP8 Zorbax SB C XTerra RP8 8 ACQUITY UPLC BEH Shield RP8 SymmetryShield RP8-0. XBridge Shield RP8 XTerra RP8 ACQUITY UPLC -0. YMC-Pack PolymerC (ln [k] acenaphthene) ACQUITY UPLC XBridge C 8 ACQUITY UPLC SB ACQUITY UPLC HSS T BEH C 8 BEH Shield RP8 BEH Phenyl BEH HILIC SB HSS T [ ]

24 HSS (High Strength Silica) Chemistries T Bonding for Polar Compound Retention ACQUITY UPLC HSS T columns utilize Waters innovative and proprietary T bonding. This is the same advanced bonding process that is behind the industry-leading polar-compound retention, aqueous mobile phase compatibility and ultra-low MS bleed of Atlantis T HPLC columns. T bonding utilizes a trifunctional C 8 alkyl phase bonded at a ligand density that promotes polar compound retention and aqueous mobile phase compatibility. The T endcapping is much more effective than traditional trimethyl silane (TMS) endcapping. This unique combination of bonding and endcapping provides superior polar compound retention and aqueous compatibility while also enhancing column performance, lifetime, peak shape and stability. Although the rugged and efficient ACQUITY UPLC BEH particle provides a wide ph range and superior peak shapes, its hydrophobic nature does not promote polar compound retention. ACQUITY UPLC HSS T columns are designed to retain and separate polar organic compounds in reversed-phase UPLC separations. When compared to ACQUITY UPLC columns, most compounds are more strongly retained on ACQUITY UPLC HSS T columns. Features of the ACQUITY UPLC HSS T columns include: n Superior polar and non-polar compound retention Superior Retention of Polar Compounds ACQUITY UPLC HSS T columns retain polar compounds longer than ACQUITY UPLC columns and contain one of the most retentive sub- µm column chemistries commercially available. n Aqueous mobile phase compatibility n Ultra-low MS bleed n Additional selectivity choice for UPLC separations HSS T BEH C 8 BEH Shield RP8 BEH Phenyl BEH HILIC SB Columns:. x 0 mm Mobile Phase A: 0 mm CH CNH, ph.0 Mobile Phase B: ACN Flow Rate: 0.8 ml/min Isocratic Mobile Phase Composition: % B Injection Volume: 0. µl Temperature: 0 C Detection: 80 nm Instrument: ACQUITY UPLC System with ACQUITY UPLC 99 PDA Compounds. Norepinephrine. Epinephrine. Dopamine.,- Dihydroxyphenylacetic acid (DPAC). Serotonin (-HT). -Hydroxy--indoleacetic acid (-HIAA). -Hydroxy--methoxyphenylacetic acid (HVA) HSS T [ ]

25 HSS (High Strength Silica) Chemistries ACQUIT Y UPLC HSS T Columns ACQUITY UPLC HSS T columns are designed to solve a common problem facing separations scientists: retaining and separating small, water soluble, polar organic molecules under reversed-phase UPLC conditions. In addition, ACQUITY UPLC HSS T columns offer UPLC separations scientists an additional selectivity choice, thus making method development faster and easier. When combined with the ultra-resolution of UPLC technology, new UPLC column chemistries enable the development of fast, robust methods in less time. The ACQUITY UPLC HSS particle is 00% silica and is efficient, mechanically stable, and, when bonded via the T bonding process, fully compatible with aqueous mobile phases. Separation of Cold Medicine Active Ingredients, Impurities and Counter Ions ACQUITY UPLC HSS T columns provide superior retention of difficult-to-retain polar analytes while also offering good chromatographic selectivity for hydrophobic species. Column: ACQUITY UPLC HSS T. x 00 mm,.8 µm Part Number: 8009 Mobile Phase A: 0.% CF CH in H Mobile Phase B: 0.0% CF CH in : (v/v) ACN:MeH Gradient: Time Profile Curve (min) %A %B Flow Rate: 0. ml/min Injection:.0 µl Column Temp.: 0 C Detection: nm Instrument: ACQUITY UPLC System with Column Heater Module and ACQUITY UPLC TUV Detector Compounds:. -aminophenol. Maleate. Fumarate. Phenylephrine. Acetominophen. Phenylpropanolamine. Pheniramine 8. Doxylamine 9. Pseudophedrine 0. Pyrilamine. Chlorpheniramine. Brompheniramine. Guaifenesin. Acetylsalicyclic acid. -nitrophenol. -chloroacetanilide. Dextromethorphan 8. Diphenhydramine 9. Clemastine 0. Ibuprofen Peaks, and are impurities of acetaminophen BEH C 8 0 BEH Shield RP8 BEH Phenyl BEH HILIC SB HSS T [ ]

26 Waters Global Services Be Assured. Choose Waters Global Services Waters Global Services focuses on optimizing Waters products with superior service, GxP compliance, support, upgrades, training, and Waters Quality Parts. nly the Waters Service team has the most in-depth and up-to-date knowledge of the advanced science and technologies that are the foundation of Waters systems. This knowledge helps maximize system uptime, increase laboratory productivity, and meet stringent compliance requirements. A Waters Service Plan for your ACQUITY UPLC system helps you: n Maintain peak performance n Accelerate throughput n Speed time-to-market n Minimize compliance risk n Control costs. Connections INSIGHT is a suite of Remote Intelligent Services included in many of our service plans that changes today s service model from reactive to proactive support to maximize instrument availability and minimize downtime costs. n ihelp for n Demand Assistance With 9 offices in more than 0 countries, Waters maintains a strong global presence. Whether your company is a single-location lab or a large multinational organization, Waters Global Services provides you with the expertise and responsiveness you need. n ialert for Real-Time Monitoring n iassist for collaboration with Waters Technical Support Primary Head not delivering Pressure fluctuations due to Accumulator loss of pressure Recommendations from Waters: Check for leaks Re-prime the system Replace primary head check valve [ ]

27 ACQUITY UPLC System Accessories Vials for ACQUITY UPLC Systems, Screw Cap x mm Maximum Recovery Vials n Most commonly used vial in ACQUITY UPLC systems n Best choice when sample volume is limited n This center draining vial allows access to most of the sample n Preset needle placement of mm from the bottom of the vial leaves µl residual volume in the vial n Using Acquity UPLC Sample Manager Advanced Setting, adjust the needle placement to access more sample, leaving less residual volume Description LCMS Certified Maximum Recovery Vial LCMS Certified Amber Maximum Recovery Vial LCGC Certified Maximum Recovery Vial LCGC Certified Amber Maximum Recovery Vial Part No CV 00000CV 8000C 80088C All items come in quantities of 00 unless otherwise stated. For Amino Acid ACQUITY UPLC Systems, the use of total recovery vials is recommended but is only suitable for PEEK needles. Waters LCMS Certified Vials Waters LCMS certified vials are a continuation of our approach to offer products suitable for the demands of LCMS. We took an unbiased approach in developing this product, looking for any ionized masses regardless of the source. The vials are tested by MS with specifications for total ion count and presence of clusters in the high mass range. The product introduced is cleaner than any product we tested from vendors around the globe. Waters LCGC Certified Vials Vials are usually manufactured by glass artisans and engineers who don t understand the requirements for their use in LC and GC. As a manufacturer of autosamplers and chemistry consumables, Waters understands the dimensional and chemical requirements of vials. We reviewed the manufacturing process, anticipated possible problem areas, and developed tests to ensure the delivery of problem-free products. The LC test, to ensure the delivery of residue-clean vials, is a radically different form of test for the vials industry. Deactivated Glass Vials Deactivated glass vials eliminate adsorption of compounds onto the glass surface when working with biological or pharmaceutical compounds, natural products, pesticides and herbicides. The surface modification is permanent, resulting in an indefinite vial shelf life. ml Vials n Best Choice where sample volume is not limited n Large residual volume at preset mm needle placement µl Description LCMS Certified ml Vial LCMS Certified ml Amber Vial LCGC Certified ml Vial LCGC Certified ml Amber Vial Deactivated Clear Glass ml Vial Deactivated Amber Glass ml Vial Part No CV CV 80000C 80008C 80000DV 80008DV All products listed are x mm combination packs of 00, screw top vials, caps and pre-slit silicone / PTFE septa [ ]

28 ACQUITY UPLC System Accessories Polypropylene Vials n Good choice for applications where there is a concern that sample can bond to glass; alternative choice to deactivated glass n Can be incinerated while still sealed to minimize exposure to hazardous substances n 00 µl vial requires needle placement of mm (see ACQUITY UPLC System Sample Manager advanced settings below), leaving a residual volume of 0 µl n 0 µl vial requires needle placement of mm, leaving a residual volume of 0 µl Description Part No. 00 µl Polypropylene (PP) Vial µl Polypropylene (PP) Vial 800 All products listed are x mm combination packs of 00, screw top vials, caps and pre-slit silicone / PTFE septa ACQUITY UPLC Vial Holder Description Part Number 8-Well Vial Holder 000 Waters ACQUITY UPLC System Sample Manager The Waters ACQUITY UPLC System Sample Manager incorporates several technology advancements. Low dispersion is maintained through the injection process, using a series of pressure transducers to facilitate self-monitoring and diagnostics. It uses needle-in-needle sampling for improved ruggedness and a needle calibration sensor for increased accuracy. A variety of sample holder formats (vials or tubes) and microliter plate formats (deep-well, mid-height) can also be accommodated in a thermostatically-controlled environment. Within the ACQUITY UPLC Sample Manager Instrument Method Editor, a number of parameters can be customized for your specific task, including depth, as shown here, to confer maximum sample format flexibility. For further information on setting vial depth offsets, see the ACQUITY UPLC perator s Guide (information documentation set for ACQUITY UPLC part number 00) or visit the ACQUITY UPLC Sample Manager Instrument Method Editor n-line Help. Sample Needle Vial Depth Typical ml Vial mm Default ffset [ 8 ]