ACQUITY UPLC Column Solutions

Transcription

1 ACQUITY UPLC Column Solutions ACQUITY UPLC Columns VanGuard Pre-columns High-strength Silica (HSS) HPLC Columns ACQUITY UPLC Columns for BioSeparations

2 Column Solutions Designed for UPLC Scientists Featuring:.-µm BEH UPLC Particles.8-µm HSS UPLC Particles Eleven Bonded Phases Reversed-Phase and HILIC High-Strength Silica (HSS) HPLC Columns VanGuard Pre-Columns

3 The ACQUITY UPLC column family remains in the forefront of LC column innovation by providing higher quality chromatographic information in less time. UPLC columns are designed, certified and tested for use in applications up to,000 psi (000 bar). High-strength silica (HSS) HPLC columns allow more choices for a seamless transition between HPLC and UPLC separations. ACQUITY UPLC columns are available in over one hundred combinations of column configurations and chemistries. Harness the full potential of small particles and the benefits of faster separations, superior resolution and greener chromatography. [ ]

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 pressure-tolerant, 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) mode and the 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 larger system volumes and slow data acquisition rates, hindered the ability of separation scientists to realize fully the speed and efficiencies promised by using small (sub- 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 improve resolution, sample throughput and sensitivity dramatically. 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 UPLC. x 0 mm,. µm Rs (,) =.90 SPEED RESLUTIN SENSITIVITY ULtra SPeed Absorbance at 0 nm X 0.X.X min 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 min 0.0. x 00 mm,. µm Rs (,) =.8 X.X.8X Compounds:. Quercetin. Kaempferol. Isorhamnetin 0 min 0.0. x 0 mm,. µm Rs (,) =. ULtra reslutin Absorbance at 0 nm.x.x.x 0 min [ ]

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 due solely to the particle size. To realize fully 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, lowvolume 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 harness the full power and promise of sub- µm particle columns. 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 misleading. 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. The Benefits of UPLC Pressure Capability With UPLC Technology, one need not compromise sample throughput for chromatographic performance. Waters is the first LC manufacturer capable of delivering upon the promise of sub- µm particle UPLC Technology. 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. 0. ACQUITY UPLC BEH C 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 BEH C 8. 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 min min 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 fast 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 Sample manager with low carryover and short cycle time 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 = 8,900 psi. Column: ACQUITY UPLC BEH C 8. 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 Temp.: 0 C Sample Temperature: C Sample Concentration: 0. ng/ml Sample Diluent: 0/0 H /MeH Strong Ndl. Wash: 0:0 MeCN:IPA + 0.% HCH Weak Ndl. 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% min * 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. In addition, Waters developed Design for Six Sigma (DFSS) Phase II ACQUITY UPLC columns which provide higher resolution and more consistent column-to-column performance. 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 BEH C 8,. µm Mobile Phase A: 0.% HCH in H Mobile Phase B: 0.% HCH in MeCN 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 : MeCN 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 min. x 0 mm,. µm Rs (,) =. Caffeine Metabolites Compounds. -methylxanthene.,-dimethyluric acid. theobromine.,-dimethylxanthene min. x 00 mm,. µm Rs (,) = min. x 0 mm,. µm Rs (,) = min [ ]

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 presents 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 ID columns in microbore dimensions (e.g.,.0 mm ID). 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 ID ACQUITY UPLC Columns Columns: ACQUITY UPLC BEH C 8. x 0 mm,. µm ACQUITY UPLC BEH C 8.0 x 0 mm,. µm Mobile Phase A: 0.% NH H Mobile Phase B: MeCN Flow Rates: 0.00 ml/min. mm ID column 0.0 ml/min.0 mm ID 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 ID UPLC Column min.0 mm ID UPLC Column.0 ~ x increase in sensitivity MRM of Channels ES- 0.8 > 0.8.e MRM of Channels ES- 0.8 > 0.8.e min 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 ID UPLC columns without significant loss of separation efficiency. Si UPLC Column Chemistries Si Si Si 0Å BEH Particle CH Si Polar Group C 8 C 8 Shield RP8 CH Phenyl HILIC Chemistry Si Si CH Si Polar Group CH Si Monofunctional Si Ligand Type Trifunctional C 8 Trifunctional C 8 Embedded Polar Group Trifunctional C Phenyl Unbonded Available Particle Sizes.,.,.,, 0.,.,.,, 0.,.,.,, 0.,.,.,, 0.,.,., Ligand Density*. µmol/m. µmol/m. µmol/m.0 µmol/m Carbon Load* 8% % % % End-cap Style Proprietary Proprietary TMS Proprietary ph Range * Expected or Approximate Values [ 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 to times higher, a smaller number of ligands and particles are necessary to provide the desired selectivity and resolution. ACQUITY UPLC columns allow chromatographers to efficiently develop faster and more robust separations. Rs = N α α k k + Maximized in UPLC Separations by: Ultra-low dispersion system Small (sub- 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% 00Å BEH Particle HSS Particle Amide C 8 C C 8 C 8 SB T Si Linker Si NH CH Si C H 9 Si CH Trifunctional Amide Trifunctional C 8 Monofunctional C Trifunctional C 8 Trifunctional C 8 Trifunctional C 8.,..,.,, 0.,..8,.,.8,.,.8,.,. µmol/m. µmol/m. μmol/m. µmol/m. μmol/m. µmol/m % % 8% % 8% % None Proprietary None Proprietary None Proprietary [ 9 ]

10 Innovation Industry-L eading 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 ACQUIT Y 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 up 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, ACQUIT Y 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 to which the column has been subjected and the date that the column was last used. All of this information travels with the column and is easily printable. T he ecord is permanently attached to the column and the data cannot be erased. Bulk Synthesis Engineering New column hardware n n Low band broadening n n Innovative frit design n n n Rugged and efficient.-µm BEH and.8-µm HSS particles Most technologically advanced porous particles ever created Combination of highest efficiencies, widest ph range and superior mechanical strength Stable column beds at UPLC pressures Column Pac king n Soft ware n Paperless tracking of column history with ecord technology n n ACQUIT Y U P LC columns feature ecord tec hnology [ 0 ] Columns must withstand UPLC operating pressures New proprietary packing methods New test instruments

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 from which the chromatographer can choose 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 = min min Geometrically Scaled HPLC Linear Velocity P c =9 Maximum Peak Capacity Equivalent Analysis Time min riginal HPLC Gradient Method P c =8 P c = min min Geometrically Scaled UPLC Linear Velocity Equivalent Peak Capacity Shortest Analysis Time The ACQUITY UPLC Columns Calculator allows chromatographers to convert HPLC methods to UPLC methods quickly and accurately. 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. 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 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] Long UPLC Column Lifetimes Data courtesy of: C. Salomons, unpublished results, Research CDS-Note, Solvay Pharmaceuticals : Diode Array Range:.8e+ Injection # : Diode Array Range:.e+ Injection #0,09! min Excellent Reproducibility Column: ACQUITY UPLC BEH C 8. x 0 mm,. µm Part Number: 8009 Mobile Phase A: 0 mm HCNH, ph.0 Mobile Phase B: MeCN 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 min Retention Times %RSD Column: ACQUITY UPLC BEH C 8. 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 ID 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. 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 Minimal Chromatographic Effects With VanGuard Pre-Columns VanGuard Pre-columns are uniquely designed to protect and prolong ACQUITY UPLC column performance while contributing minimal chromatographic effects. Column: ACQUITY UPLC BEH C 8,. x 0 mm,. µm Part Number: 8000 Protein Precipitation Procedure Pre-column: VanGuard Pre-column, BEH C 8,. x mm,. µm Part Number: 8009 Mobile Phase A: 0.% NH H in H Mobile Phase B: 0.% NH H in MeCN 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 Desacetyldiltiazem SIR m/z. Without VanGuard Pre-column With VanGuard Pre-column Diltiazem SIR m/z. Without VanGuard Pre-column With VanGuard Pre-column N N S N S N H CH CH Spike plasma with 00 ng/ml of diltiazem and desacetyldiltiazem 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 supernatant and evaporate to dryness with nitrogen Reconstitute with 00 µl of a 0:0 MeH:H solution (results in a 0. ng/ml conc.) min [ ]

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 operate routinely 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 six column chemistries: C 8, C 8, Shield RP8, Phenyl, HILIC and Amide. 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 HSS C 8, HSS C 8 SB (Selectivity for Bases) and T. The HSS C 8 chemistry is a fully end-capped, ultra-performance, general purpose C 8 bonded phase that provides superior peak shape for bases, increased retentivity (vs. ACQUITY UPLC BEH C 8 columns), and extremely long lifetimes under acidic conditions. The HSS C 8 SB (Selectivity for Bases) chemistry is a non-end-capped 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. Like BEH (XBridge) particles, the selectivities offered by HSS chemistries are also available in HPLC particle sizes (. and μm) in the HSS HPLC column family, thus allowing seamless transfer between HPLC and UPLC separations. The Particles of UPLC Technology Waters is the only manufacturer offering THREE UPLC-certified particles ACQUITY UPLC BEH Columns ACQUITY UPLC HSS Columns Particle Type Ethylene-Bridged Hybrid (BEH) High-Strength Silica (HSS) Particle Size. µm.8 µm Maximum Rated Pressure,000 psi (~000 bar),000 psi (~000 bar) Pore Diameter/Volume 0Å / 0. ml/g 00Å / 0. ml/g 00Å / 0. ml/g Surface Area 8 m /g 90 m /g 0 m /g Available Chemistries C 8, C 8, Shield RP8, Phenyl, HILIC, Amide C 8, C C 8, C 8 SB, T ph Range -; (RP8, Amide: -); (HILIC: -8) C 8 : -; C : -0 C 8 : -8; C 8 SB, T: -8 [ ]

15 UPLC Particle Technology BEH Technology In 999, Waters launched the XTerra family of HPLC columns featuring patented-first-generation Hybrid-Particle Technology (HPT)*. HPT enabled XTerra columns to become one of the most successful column products in the history of Waters. In HPT, the best properties of inorganic (silica) and organic (polymeric) packings are combined to produce a material that has superior mechanical strength, efficiency, high-ph stability and peak shape for bases. The BEH Particle*: ne of the Key Enablers of UPLC Technology 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 that utilizes an ethylene-bridged hybrid (BEH) structure. Compared to the first-generation methyl-hybrid particle of XTerra columns, the BEH particle of ACQUITY UPLC BEH columns exhibits improved efficiency, strength and ph range. BEH Technology is a key enabler of the speed, sensitivity and resolution of UPLC separations. Et CH CH Et Et Si Si Si Si Si Et Polyethoxysilane (BPES) Et Si Et Et Et n Et Si Et Et Et Tetraethoxysilane (TES) + Et Et Et CH Si Si Et CH Et Et Bis(triethoxysilyl)ethane (BTEE) Anal. Chem. 00,, 8-88 *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 materials 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. High pore volume HPLC particles do not posses the mechanical stability necessary to withstand the high column packing and operating pressures of UPLC separations. This is why Waters created the HSS family of HPLC columns. HSS HPLC columns possess the same chemical and physical characteristics as the.8-µm HSS UPLC particle, but in larger (. µm and µm) particle sizes. 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. ACQUITY UPLC HSS.8 µm Ideal Profile Flow ACQUITY UPLC BEH. µm HPLC Silica Pressure [ ]

16 Phase II ACQUITY UPLC Columns Ultra Consistency Waters NEW Phase II ACQUITY UPLC columns are the next evolutionary step in UPLC column technology for routine use in applications at pressures up to,000 psi (000 bar). Process-optimized for the QC laboratory environment, Phase II ACQUITY UPLC columns are designed to provide superior long-term reproducibility (e.g., for the life of drug or product) by minimizing column-to-column efficiency and backpressure variability. They are the culmination of many UPLC column manufacturing improvements undertaken by Waters since the commercialization of UPLC Technology in 00. Chromatographers will immediately benefit from more consistent column-to-column performance and higher efficiencies. Since the particles and the chemistries have not changed as part of the Phase II manufacturing process, there is no risk that any existing UPLC method will require redevelopment. The only newly observed benefits will be more consistent column-to-column performance and higher efficiencies year after year. Maximizing Efficiency While Minimizing Variability Efficiency (N) Phase I Process UCL LCL UCL: Upper Control Limit LCL: Lower Control Limit Column: ACQUITY UPLC BEH C 8,. x 0 mm,. µm Data Points (columns) Phase II Process UCL LCL As compared to the previous Phase I manufacturing process, Waters Phase II UPLC column manufacturing process produces more consistent, reproducible and efficient products. Process ptimized for Quality Control Laboratories UPLC Technology continues to benefit separation scientists throughout the entire drug development process from early Discovery through QC. As a drug moves through the development process, the needs of chromatographic scientists change as well. In early Discovery, a short, sensitive, high-resolution LC method is the primary separation goal. In QC laboratories, however, the chromatographic focus is on managing risk in terms of minimizing method variability while demanding reliable, robust and easy to use LC columns. The consistent and reliable Phase II ACQUITY UPLC column family is designed for the QC separation scientist. These UPLC columns will reduce product shipment delays due to inconsistent/incorrect results produced by the same method run by different users located at other manufacturing sites around the world. Superior Column-to-Column Performance Standard Deviation ACQUITY UPLC BEH C 8,. µm Process Variation Comparison 0 BEH C 8 Column RSD 9.9% RSD 0.8% Phase I Process Phase I. x 0 mm column. x 00 mm column RSD.% RSD.% Phase II Process Phase II Plates Std. Dev. RPH Plates Std. Dev. RPH. x 0 mm 0,80,0.,8.9. x 00 mm,88,.,88.9 All chromatographers, especially those in the QC/manufacturing laboratory, will benefit from higher efficiencies and lower column-to-column variability. Note that the Phase II manufacturing process yields higher and more reproducible column efficiencies. [ ]

17 Phase II ACQUITY UPLC Columns NEW.0-mm ID UPLC Columns All separations can be affected by extra-column bandspreading or system variance. Sources of system variance include system tubing and connections as well as detector filter constant and column temperature. Small particles (sub- µm) packed into small ID columns are especially susceptible to system variance. The ACQUITY UPLC system has the lowest system variance of any commercially available LC system and is designed to maximize the performance of small particle, small ID columns. However, for early eluting peaks, even the extremely low system variance of the ACQUITY UPLC system can impact the measured separation efficiency. In order to maximize the separation efficiency of the ACQUITY UPLC system in the QC laboratory,.0-mm-id UPLC columns are now available. Separations are developed and ideally used for the life of drug, often as long as years. Therefore, QC laboratories are concerned with minimizing risk in the form of controlling method-to-method and laboratory-to-laboratory variability associated with this method. A key to a successful long-term separation is to standardize on robust, reproducible and easy-to-use LC column products. Classically, the preferred separation mode is isocratic since this type of separation is believed to be simpler and more robust. These types of separations are often performed on larger internal diameter (ID) columns since these larger-volume columns minimize or mask system-volume effects that can have a deleterious impact on separation efficiency. To meet this need of the QC chromatographer, Waters developed.0-mm-id ACQUITY UPLC columns. Lower Reduced Plate Heights (RPHs) RPH Acenaphthene Reduced Plate Heights (RPHs) for Phase II.-mm-ID vs..0-mm-id UPLC Columns mm 0 mm 00 mm 0 mm UPLC Column Length RPH. mm ID RPH.0 mm ID Shown are measured column efficiencies from our column manufacturing facility in Wexford, Ireland. Reduced Plate Height (RPH) is a measure of how well a column is packed, regardless of particle size. RPH is calculated by taking the minimum plate height (H) from the van Deemter curve and dividing it by the particle size (dp). When comparing RPHs, a lower value is better..8 Higher Isocratic Efficiencies Conditions Mobile Phase A: H Mobile Phase B: MeCN Isocratic Conditions: 0% MeCN Injection Volume: 0. µl Temperature: 0 ªC Detection: nm Instrument: ACQUITY UPLC with ACQUITY UPLC TUV Phase II UPLC column Part Number: x 0 mm, BEH C 8,. µm Flow Rate: 0.00 ml/min Analyte Conc:. µg/ml Compound. mm ID Efiiciency.0 mm ID Efiiciency Efficiency Loss with. mm ID Column. Propiophenone,99,9 % Phase II UPLC Column Part Number: x 0 mm, BEH C 8,. µm Flow Rate: 0. ml/min Analyte Conc: µg/ml. Butyrophenone 9,8,8 %. Valerophenone 0,99,09 %. Hexanophenone,09,8 8% UPLC scientists in QC laboratories will benefit from the higher efficiencies produced by.0-mm-id UPLC columns min [ ]

18 BEH (Ethylene-Bridged Hybrid) Chemistries ACQUITY UPLC BEH C 8 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 BEH C 8 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 that 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 end-capping processes that produce outstanding 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 BEH C C BEH C 8 BEH C BEH C 8 BEH Shield RP C C min BEH Shield RP8 BEH Phenyl BEH HILIC BEH AMIDE HSS HPLC Columns HSS C 8 HSS C 8 SB, BEH Phenyl HSS C 8 HSS C 8 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 min [ 8 ]

19 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 particles 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 HSS C 8 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 Injection 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 C 8 BEH C 8 BEH Phenyl BEH C 8 BEH C 8 BEH Shield RP8 BEH Phenyl BEH HILIC, HSS C 8 BEH AMIDE HSS C 8 SB HSS HPLC Columns HSS C 8, HSS T HSS C 8 SB min HSS T [ 9 ]

20 BEH (Ethylene-Bridged Hybrid) Chemistries ACQUITY UPLC BEH Phenyl Columns Phenyl-ligand containing reversed-phase columns can provide selectivities complementary to those of 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 end-capping processes as the ACQUITY UPLC BEH C 8 and C 8 columns, provides ultra-low column bleed, long column lifetimes and excellent peak shape. This unique combination of ligand, end-cap 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 Echinacea-containing 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 BEH C 8 BEH C 8 ACQUITY UPLC HSS,. x 0 mm,.8 µm Mobile Phase A: 0.% CF CH in H Mobile Phase B: 0.08% CF CH in MeCN 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 BEH C 8 BEH Shield RP8 BEH Phenyl Compounds:. Caftaric acid. Chlorogenic acid. Cynarin. Echinacoside. Cichoric acid BEH Shield RP8 BEH HILIC HSS C 8 BEH AMIDE HSS HPLC Columns HSS C 8 SB HSS C 8 HSS C 8 SB HSS T HSS T 0 min [ 0 ]

21 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 an organic solvent (e.g., MeCN) that 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-d-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 MeCN:H Mobile Phase B: 0 mm HCNH, 0.% HCH in 90:0 MeCN:H Flow Rate: 0.88 ml/min Gradient: Time Profile (min) %A %B Injection Volume:. µl Sample Conc.: µg/ml Sample Diluent: : MeCN:MeH with 0.% HCH Temperature: 0 C Detection: 80 nm Instrument: ACQUITY UPLC with TUV Detector. Morphine -β-d-glucuronide H H BEH C 8 BEH C 8 BEH Shield RP8 BEH Phenyl. Morphine H H H H H H NCH BEH HILIC BEH AMIDE H NCH HSS HPLC Columns HSS C 8 V 0 HSS C 8 SB min HSS T [ ]

22 BEH (Ethylene-Bridged Hybrid) Chemistries ACQUITY UPLC BEH AMIDE CLUMNS As an analytical separation technique, HILIC is growing in popularity and finding utility in chromatographic laboratories where samples that contain very polar analytes are encountered and analyzed. In 00, Waters began development of several LC column chemistries for HILIC separations including Atlantis HILIC Silica, XBridge HILIC and ACQUITY UPLC BEH HILIC chemistries. All of these chemistries share one thing in common: unbonded particles as the column chemistry (i.e., no bonded phase). Such columns excel at retaining and separating compounds such as very polar bases since these analytes contain a positive charge, thus enabling weak cation exchange to dominate (along with partitioning) as a retention mode. However, for very polar acidic and neutral compounds that do not contain a positive charge, unbonded particle column chemistries may not provide the necessary retention and separation. Waters created the ACQUITY UPLC BEH Amide chemistry to retain very polar acidic, basic and neutral compounds. Separation of Water Soluble Vitamins Using ACQUITY UPLC BEH Amide Columns Vitamins are important nutrients found primarily in foodstuffs. Unlike fat soluble vitamins that can be stored in the liver until needed, water soluble vitamins are not stored and are eliminated in urine. Thus a continuous and constant supply is needed in order to maintain good health. Most water soluble vitamins are unstable and will quickly decompose if improperly stored. Therefore, monitoring the presence and concentration of water soluble vitamins is very important. Because of the polar nature of these analytes, retaining and separating water soluble vitamins using conventional reversed-phase LC is very challenging. Using HILIC and ACQUITY UPLC BEH Amide columns, however, water soluble vitamins are easily retained and separated. BEH C 8 BEH C 8 BEH Shield RP8 BEH Phenyl Column: ACQUITY UPLC BEH Amide,. µm. x 0 mm Part Number: Mobile Phase A: 0/0 MeCN/H with 0 mm CH CNH and 0.0% NH H, ph 9.0 Mobile Phase B: 90/0 MeCN/H with 0 mm CH CNH and 0.0% NH H, ph 9.0 Flow Rate: 0. ml/min Gradient: Time Profile (min) %A %B Injection Volume:.0 µl (PLN) Sample Diluent: / MeCN/MeH with 0.% HCH Column Temp.: 0 C Weak Ndl. Wash: MeCN/H 9/ Detection: nm Sampling Rate: 0 points/sec Filter Time Constant: Normal Instrument: Waters ACQUITY UPLC with ACQUITY UPLC PDA Detector BEH HILIC BEH AMIDE Compounds:. Nicotinamide µg/ml. Pyridoxal 0 µg/ml. Riboflavin 0 µg/ml. Nicotinic acid 0 µg/ml. Thiamine 0 µg/ml. Ascorbic Acid µg/ml. Vitamin B 0 µg/ml 8. Folic Acid µg/ml 8 HSS HPLC Columns 0 min HSS C 8 HSS C 8 SB HSS T [ ]

23 BEH (Ethylene-Bridged Hybrid) Chemistries ne extremely diverse and important class of very polar compounds analyzed using HILIC, among other chromatographic techniques, is carbohydrates or saccharides. Carbohydrates are the most abundant class of organic compounds found in nature. Basically, simple carbohydrates are often aldehydes or ketones with many hydroxyl (H) groups, usually on each carbon atom that is not part of the aldehyde or ketone functional group (i.e., -[CHHn-]. There are four primary chemical classes of carbohydrates found in nature: monosaccharides, disaccharides, oligosaccharides and polysaccharides. Because carbohydrates are ubiquitous in foodstuffs, beverages, plants, wood, paper and fabrics they were some of the first compounds separated using liquid chromatography. Saccharides still present chromatographic challenges to separation scientists today. Problems encountered when analyzing carbohydrates include salt interferences, anomer mutarotation, Schiff-base formation, loss of reducing sugars at elevated temperatures, shortened column lifetime, long analysis time and lack of chromophore. When using the extremely stable and efficient ACQUITY UPLC BEH Amide column and the ACQUITY UPLC system for carbohydrate analysis, all of these problems are obviated. Wide Range of Commercial Food Sample Separations on ACQUITY BEH Amide Columns ne well known and commonly analyzed subset of mono- and disaccharides are known as the five food sugars: fructose, glucose, sucrose, maltose and lactose. Although consisting of only five analytes, this is a very challenging separation since all but sucrose are reducing sugars (that can undergo mutarotation resulting in two peaks). The ACQUITY UPLC BEH Amide chemistry enables fast, efficient separations of food sugars in a wide range of complex sample matrices. Salt Interferences Many of the column chemistries used for separating carbohydrates possess an ionizable group and can act as ion exchangers. Ion exchange is not always desirable when analyzing carbohydrates because of high salt concentrations found in many sample matrices (e.g., foods, beverages, raw materials, etc.). Chromatographically, the cations and anions of these dissolved salts can be retained and co-elute with the carbohydrates of interest. Removing salts, without simultaneously losing the carbohydrates of interest during sample preparation, is very difficult since the interfering salt ions and the carbohydrates are both very polar. T his makes sample preparation either ineffective or very tedious and time consuming. The BEH Amide chemistry has no charge [is neutral] and under the recommended high-ph conditions suggested by Waters for carbohydrate analysis, salts commonly found in most foods and beverages simply elute in the void volume. This eliminates salt co-elution with the carbohydrates of interest, making sample preparation straightforward and provides higher confidence in the chromatographic results. Column: ACQUITY UPLC BEH Amide,. x 0 mm,. µm Part Number: Mobile Phase A: 80:0 Acetone:H w/0.0% Triethylamine (TEA) Mobile Phase B: 0:0 Acetone:H w/0.0% TEA Isocratic Mobile Phase Composition: 9: A:B (% Acetone w/0.0% TEA) Flow Rate: 0. ml/min Injection Volume: 0. µl Standard Conc.:.0 mg/ml each in 0:0 MeCN:H Column Temp.: 8 C Instrument: ACQUITY UPLC with ELSD Compounds:. Fructose. Glucose. Sucrose. Maltose. Lactose BEH C 8 BEH C 8 Strawberry Smoothie BEH Shield RP8 Hot Cross Buns BEH Phenyl White Bread BEH HILIC Tomato Ketchup BEH AMIDE Lamb Curry Meal HSS HPLC Columns Bran with Raisins Cereal Sports Drink HSS C 8 Food Sugar Standard HSS C 8 SB 0 min HSS T [ ]

24 BEH (Ethylene-Bridged Hybrid) Chemistries Reducing Sugars A reducing sugar is a saccharide that undergoes mutarotation as it reaches equilibrium between its α and β forms. Chromatographically, mutarotation and the resulting two anomers appear as wide or split peaks for such commonly analyzed saccharides as fructose, glucose, maltose, lactose, glyceraldehyde, arabinose, etc. The adverse chromatographic effects of mutarotation have plagued saccharide chromatographers for decades. Chromatographic techniques for collapsing the anomers of reducing sugars include increasing mobile phase ph and/or column temperature. These conditions speed the rate of mutarotation resulting in one peak per reducing sugar. Historically, most LC columns are not compatible with operating at these extreme conditions. ACQUITY UPLC BEH Amide columns were designed and tested specifically for operating under the extreme conditions to condense the pair of peaks for the anomers of reducing sugars into a single peak. ACQUIT Y UPLC BEH AMIDE CLUMNS Chromatographic methods used to separate carbohydrates include HILIC, ion/ligand exchange, ion chromatography, size exclusion and combinations of these various separation modes. Each of these classic carbohydrate column chemistries has advantages and disadvantages. The advantages are usually related to how well a particular column separates a particular group or subset of carbohydrates (e.g., Pb + ligand exchange chemistry for wood sugar separations). However, nearly all of these carbohydrate column chemistries exhibit short column lifetimes. A well known example of this is the alkyl amine chemistry (e.g., aminopropyl silane bonded to silica) is used for separating mono- and disaccharides. Due to the amino groups present in the bonded phase, the ph inside the column is very high. The advantage of this localized high ph is the rapid interconversion rate between the reducing sugar anomers, resulting in one peak (anomer collapse). However, the disadvantage of high ph inside a silica-based aminopropyl column is the dissolution of the silica surface resulting in substantial column bleed, performance loss and ultimately shortened column lifetimes. The BEH amide chemistry is not only designed to separate a wide range of carbohydrates, but also provide extremely long column lifetimes due to the combination of the rugged and efficient BEH particle with the trifunctionally bonded amide chemistry. ACQUITY BEH Amide Columns Provide Extremely Long Column Lifetimes BEH C 8 In this BEH Amide column lifetime study, honey, molasses, and breakfast cereal samples were prepared and injected (along with a.0 mg/ml standard every 00 injections) over the course of eight days on an ACQUITY UPLC system operating with a column temperature of 90 C and mobile phase ph of approximately 0.. The retention of glucose is monitored and compared to its retention at the beginning of the experiment. Retention loss is indicative of bondedphase or packing material loss. High ph and temperature are important attributes for a column chemistry designed for separating carbohydrates since the rate of reducing sugar anomer interconverstion/mutarotation is accelerated under these conditions. As can be seen, the extremely rugged BEH Amide chemistry provides no retention loss (performance loss) for glucose during the course of this demanding study. BEH C 8 BEH Shield RP8 BEH Phenyl Column: ACQUITY UPLC BEH Amide,. x 0 mm,. µm Part Number: Mobile Phase A: 80:0 Acetone:H w/0.0% Triethylamine (TEA) Isocratic Mobile Phase Composition: 00% A Flow Rate: 0. ml/min Injection Volume: 0. µl Standard Conc.:.0 mg/ml each in 0:0 MeCN:H Column Temp.: 90 C Instrument: ACQUITY UPLC with ELSD Compounds:. Fructose. Glucose. Sucrose. Maltose. Lactose BEH HILIC 0 0 *Glucose Retention 000 injections under isocratic conditions (>8 Days of testing) 00 BEH AMIDE HSS HPLC Columns HSS C 8 HSS C 8 SB HSS T Percent of riginal Glucose Retention (%) * Honey Molasses Cereal Food Sugar Standard * * Glucose Retention Loss:.% Std.Dev.: 0.0 RSD: 0.% Injection Number ph ~ 0. and 90 C [ ]

25 BEH (Ethylene-Bridged Hybrid) Chemistries lder chromatographic methods for carbohydrate analysis utilize RI detection that is neither sensitive nor compatible with gradient elution chromatography. Mass detection (MS) is now commonplace in the chromatographic laboratory and offers advantages including gradientelution compatibility, high sensitivity and mass selectivity. MS detection, however, has been difficult to implement as a rugged and routine carbohydrate detection technique for several reasons. Carbohydrates do not ionize well under the acidic/neutral mobile phase conditions necessary when using silica-based LC columns. Further, the column bleed not detected when using RI detection (but still occuring) can adversely affect MS detection by producing interfering ions as well as analyte-signal suppression/enhancement. Attempts at developing LC/MS separation methods for carbohydrates include addition of metal to mobile phase, derivatization and/or post-column addition. The BEH Amide chemistry is designed to allow the routine use of high-ph mobile phases. Besides collapsing reducing sugar anomers and causing salts to elute in the void, the use of high ph mobile phases with the BEH Amide chemistry facilitates ionization of carbohydrates using electrospray negative (ES-) ionization. No metal addition, derivitization or post-column addition is necessary. Using a high ph mobile phase and BEH Amide chemistry, MS detection of carbohydrates in the ES- mode routinely affords at least a two to three order of magnitude improvement in sensitivity as compared to ELS or RI detection. These benefits cannot be realized under low ph conditions. UPLC/MS Determination of Carbohydrates in Beer Detector Compatibility Since carbohydrates do not possess a chromophore, the classic chromatographic detection technique found in most analysis methods is refractive index (RI). Besides being able to detect carbohydrates, the main advantage of RI detection is wide linear response. Interestingly, another advantage includes the inability to detect the substantial column bleed occurring with most carbohydrate analysis columns. RI detection disadvantages, however, include poor sensitivity and incompatibility with gradient-elution chromatography. Carbohydrate separation scientists who wish to use modern, sensitive and gradient-compatible detection techniques such as evaporative light scattering (ELS) and mass detection (MS) encounter difficulties due to the substantial column bleed that can now be detected using ELS or MS detection. The BEH Amide chemistry was developed and tested with modern chromatographic detection techniques such as UV, ELS, Fluorescence and MS to ensure virtually no detectable ligand bleed. Using UPLC/MS, gradient-elution chromatography, high-ph mobile phases and the BEH Amide chemistry, routine separations of carbohydrates in extremely complex samples are possible with high sensitivity and minimal sample preparation. In this study, commercial beer samples were diluted : with acetonitrile and injected. During beer fermentation maltose is consumed and converted to ethanol and carbon dioxide. Larger maltooligosaccharides such as maltotriose, maltotetrose, etc. are not consumed by the yeast and remain in the finished product. Column: ACQUITY UPLC BEH Amide,. x 00 mm,.µm Part Number: 800 Mobile Phase A: 80/0 MeCN/H with 0.% ammonium hydroxide [NH H] Mobile Phase B: 0/0 MeCN/H with 0.% ammonium hydroxide [NH H] Flow Rate: 0. ml/min Gradient: 0 minute gradient, %-% MeCN (w/ 0.% NH H) with minute re-equilibration Time profile (min) %A %B Injection Volume:.0 µl (PLN) Sample Conc.: Standards at 0 µg/ml, Beer at 0% dilution Sample Diluent: 0/0 MeCN/H Column Temp.: C Strong Ndl. Wash: 0/80 MeCN/H (800 µl) Weak Ndl. Wash: / MeCN/H (00 µl) Seal Wash: 0/0 MeCN/H Instrument: Waters ACQUITY UPLC with SQD Mass Spectrometer Settings: Ionization Mode: Capillary: Cone Voltage: ES-.8 kv V (fructose, glucose, maltotriose); 0V (sucrose and maltose) Source Temperature: 0 C Desolvation Temp.: 0 C Desolvation Gas Flow: 00 L/Hr Cone: 0 L/Hr SIR m/z: 9.0 (fructose, glucose);. (sucrose, maltose);0. (maltotriose) Dwell Time: 0.0 s Disaccharides (m/z =.) Trisaccharides (m/z = 0.) SIR of Channels ES- TIC.9e BEH C 8 BEH C 8 BEH Shield RP8 BEH Phenyl BEH HILIC Compounds:. Fructose. Glucose. Sucrose. Maltose. Maltotriose European Lager Beer BEH AMIDE HSS HPLC Columns Food Sugar Standard (0 µg/ml) SIR of Channels ES- TIC.8e HSS C 8 HSS C 8 SB min HSS T [ ]

26 HSS (High-strength Silica) Chemistries High-St rengt h Silica HPLC Columns As large corporations implement UPLC Technology throughout their worldwide organizations there is a transition period during which older HPLC systems are being phased out and replaced with ACQUITY UPLC systems. This transition does not take place overnight, however. During this transition period, chromatographic methods that are developed using UPLC Technology must also be compatible with older HPLC systems. To make this UPLC Technology adoption strategy successful, one must be able to seamlessly scale between UPLC Technology, HPLC and preparative LC. This is only possible if chromatographic column platforms with HPLC and UPLC particle sizes exhibit the same selectivities. BEH particles are the substrate-platform technology behind the industry-leading ACQUITY UPLC BEH and XBridge HPLC column families and is a primary reason behind the success of UPLC Technology. In 00, Waters commercialized ACQUITY UPLC HSS columns based upon the.8 µm high-strength silica UPLC particle substrate. These columns were developed to provide additional selectivity choices. Customers who have used and developed methods using ACQUITY UPLC HSS columns require HPLC particle sizes and columns in order to help with UPLC Technology implementation. Waters developed the HSS HPLC column family in response to customer demands for ACQUITY UPLC HSS selectivities in HPLC particle and column formats. The HSS HPLC column family features: n. µm and µm HSS particle sizes n The same three chemistries (HSS C 8, C 8 SB and T) as ACQUITY UPLC HSS columns n Seamless scalability between HPLC and UPLC Technology Seamless Scalability Across Three Particle Sizes Many UPLC scientists have developed and validated methods using ACQUITY UPLC HSS columns. Based upon performance and selectivities, one or more of the HSS C 8 chemistries have found their way onto preferred columns lists. These same customers have approached Waters and asked for HSS columns in HPLC particle sizes and column formats in order to help with UPLC Technology adoption throughout their organization. In response to customer demand, the HSS HPLC column family joins the BEH Technology particle-based XBridge HPLC column family as a second seamless chromatographic migration path to and from UPLC Technology. BEH C 8 BEH C 8 BEH Shield RP8 BEH Phenyl BEH HILIC BEH AMIDE Mobile Phase A: 0 mm NH CH, ph.0 Mobile Phase B: MeCN Injection Volumes: 0 µl (. x 0 mm,.0 μm) 0 µl (. x 00 mm,. μm). µl (. x 0 mm,.8 μm) Sample Conc.: µg/ml in H Temperature: 0 C Detection: UV@ nm Instruments: HPLC System: Alliance 9 w/99 PDA UPLC System: ACQUITY UPLC w/pda Compounds:. Xanthine. -Methylxanthine..-Dimethyluric acid. Theobromine..-Dimethyluric acid. Paraxanthine.,,-Trimethyluric acid 8. Caffeine HSS C 8,.0 μm,. x 0 mm, (Part Number 800) min HSS C 8,. μm,. x 00 mm (Part Number 800) 8 8 Time Flow Rate (min) (ml/min) %A %B Time Flow Rate (min) (ml/min) %A %B HSS HPLC Columns min HSS C 8 HSS C 8 SB HSS C 8,.8 μm,. x 0 mm (Part Number 800) 8 Time Flow Rate (min) (ml/min) %A %B HSS T min [ ]

27 HSS (High-strength Silica) Chemistries When UPLC Technology was commercialized in 00, no other LC column manufacturer possessed a reliable, pressure-tolerant UPLC column product. Interestingly, LC column manufacturers who originally claimed that UPLC Technology and pressure tolerance was not necessary, have subsequently developed sub- µm particle columns of their own (with varying degrees of success). This is due primarily to the overwhelming success of UPLC Technology in the marketplace. The fastest and easiest route to market was for particle manufacturers to modify their HPLC particle synthesis and sizing process to produce sub- µm particles. Subsequent customer testing has revealed that this process and its particle and column manufacturing modifications were not always successful. Apart from instability at UPLC pressures, the main complaint voiced by customers who evaluated these new sub- µm particle columns was the lack of scalability between HPLC and sub- µm particle sizes. In other words, the selectivities of the HPLC and sub- µm particle size columns were not the same. Waters chose a different route for manufacturing UPLC and HPLC columns. Unlike other LC column manufacturers, the first particle sizes developed for BEH and HSS particle substrates were sub- µm. UPLC columns packed with.-µm BEH particles were commercialized in 00 with XBridge HPLC columns (containing BEH Technology particles) following in 00. HSS UPLC columns packed with.8 µm particles were commercialized in 00 followed by HSS HPLC columns in 009. Conceptually it makes sense that it is an easier and more straightforward process to produce larger (HPLC) particles and chemistries by scaling up a sub- µm particle manufacturing process than it is to try and adapt an HPLC particle manufacturing process to produce efficient, pressure-tolerant sub- µm particles since the performance demands placed upon a sub- µm particle column are much greater than those expected of a larger particle HPLC column. Since Waters is a primary manufacturer who produces its own particles, chemistries and columns, the same scientists and engineers who develop and produce UPLC column products also produce the HPLC column analog. Waters is the only manufacturer that has three UPLC particle substrates and the corresponding HPLC particles, chemistries and columns demanded by customers. Scale Between Preparative, Analytical and UPLC Separations with HSS Particles Waters commercialized the sub- µm particle columns for BEH and HSS particle substrates. Although this is a more difficult and expensive route to take in order to have scalable UPLC and HPLC columns, this ensures that regardless of particle size, a method can be seamlessly transferred between HPLC and UPLC Technology. For a given HSS chemistry, HSS HPLC columns will provide the same selectivity across all particles sizes. BEH C 8 Mobile Phase A: 0.% TFA Mobile Phase B: 0.% TFA in ACN Temperature: 0 C Sample Diluent: 0:0 MeH:H Concentrations: Related Compound : 0.00 mg/ml Paroxetine; 0. mg/ml Related Compound : 0.00 mg/ml Detection: UV@9 nm HPLC System: Alliance 9 with 99 PDA UPLC System: ACQUITY UPLC with PDA AU AU AU AU Preparative HPLC Separation Analytical HPLC Separations UPLC Separation Paroxetine Paroxetine Paroxetine Paroxetine HSS C 8 SB.0 μm, 0 x 0 mm, (Part Number 8000).9 min HSS C 8 SB.0 μm,. x 0 mm, (Part Number 800) 0.8 min HSS C 8 SB. μm,. x 00 mm (Part Number 8008) 9. min HSS C 8 SB.8 μm,. x 0 mm (Part Number 800). min BEH C 8 BEH Shield RP8 BEH Phenyl BEH HILIC BEH AMIDE HSS HPLC Columns HSS C 8 HSS C 8 SB HSS T [ ]

28 HSS (High-strength Silica) Chemistries ACQUITY UPLC HSS C 8 Columns As chromatographers around the world continue to realize and embrace the benefits of UPLC Technology, additional UPLC column choices that 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 HSS C 8 chemistry is a fully end-capped, high coverage, ultra-performance C 8 bonded phase that provides superior peak shape for bases, increased retention (vs. ACQUITY UPLC BEH C 8 columns), and excellent low-ph stability. ACQUITY UPLC HSS C 8 Columns Provide Superior Peak Shapes The efficiencies and separations possible with UPLC Technology are the result of more than just small particles. State-of-the-art bonding and end-capping 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 shown here 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 HSS C 8,.8 µm Column G VP C 8,. µm. 0 Column G VT C 8,. µm. 0 Column T HGA,.9 µm.0 Column T HG,.9 µm. ACQUITY UPLC HSS C 8,.8 µm Column Z SB C 8,.8 µm. ACQUITY UPLC BEH C 8,. µm.0 0 BEH C 8 BEH C 8 BEH Shield RP8 BEH Phenyl BEH HILIC 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 Compounds. Uracil. Propranolol. Butylparaben. -methylnaphthalene. Dipropylphthalate. Acenaphthene. Amitriptyline, Column G VP C 8,. µm Column G VT C 8,. µm Column T HGA,.9 µm Column T HG C,.9 µm BEH AMIDE, Column Z SB C 8,.8 µm HSS HPLC Columns HSS C 8 ACQUITY UPLC BEH C 8,. µm HSS C 8 SB min HSS T [ 8 ]

29 HSS (High-strength Silica) Chemistries ACQUITY UPLC HSS C 8 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 end-capping processes. A small particle bonded with a poor and/or outdated stationary phase and/or end-cap will produce inadequate efficiencies, inferior resolution and short column lifetimes. Like all ACQUITY UPLC stationary phases, the ACQUITY UPLC HSS C 8 columns are created using state-of-the-art bonding and end-capping processes that produce high efficiencies and long column lifetimes. The ACQUITY UPLC HSS C 8 stationary phase features a trifunctionally bonded C 8 ligand and proprietary end-capping 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 operate reliably 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 HSS C 8 packing is the most stable bonded phase commercially available and does not suffer from peak shape issues that plague older stationary phases which rely upon steric protection (and lack of end capping) to produce stability at low ph. ACQUITY UPLC HSS C 8 Columns Resist Acid Hydrolysis at Low ph In this test, the loss in retention of the neutral marker methyl paraben indicates bonded stationary phase loss due to acid hydrolysis. ACQUITY UPLC HSS C 8 columns resist bonded phase loss due to novel bonding and end-capping processes. Low-pH Stability: hr Exposure to 0.% TFA at 0 C BEH C 8 BEH C 8 ACQUITY UPLC HSS C 8,.8 µm BEH Shield RP8 ACQUITY UPLC BEH C 8,. µm Column Z SB C 8,.8 µm BEH Phenyl ACQUITY UPLC HSS T,.8 µm Column G VP C 8,. µm BEH HILIC ACQUITY UPLC HSS C 8 SB,.8 µm BEH AMIDE Column G VT C 8,. µm Column T HG,.9 µm HSS HPLC Columns Column Z EP C 8,.8 µm HSS C % of Retention Loss for Methyl Paraben HSS C 8 SB HSS T [ 9 ]

30 HSS (High-strength Silica) Chemistries ACQUITY UPLC HSS C 8 SB (Selectivity for Bases) Columns ACQUITY UPLC HSS C 8 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 HSS C 8 SB columns can produce separations that will be quite different in terms of selectivities when compared to those of most modern, high-coverage, fully end-capped C 8 stationary phases. How was this accomplished? Waters materials scientists bonded a trifunctional C 8 ligand at an intermediate ligand density (i.e.,. µmol/m ) without end-capping. 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 end-capped 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 re-uptake inhibitors (SSRIs). ACQUITY UPLC HSS C 8 SB columns retain basic compounds such as tricyclic antidepressants longer than fully end-capped stationary phases due to the enhanced silanol activity on the non-end-capped HSS particle. BEH C 8 BEH C 8 BEH Shield RP8 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: MeCN 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 Compounds:. Trimethoprim. Nordoxepin. Doxepin. Nortriptyline. Imipramine. Amitriptyline. Trimipramine, HSS C 8 SB BEH C 8 BEH C 8 BEH Shield RP8 BEH Phenyl, BEH Phenyl BEH HILIC HSS C 8 BEH AMIDE HSS HPLC Columns, HSS T min HSS C 8 HSS C 8 SB HSS T [ 0 ]

31 HSS (High-strength Silica) Chemistries ACQUITY UPLC HSS C 8 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 end-capping 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 straightalkyl-chain C 8 column chemistries. High-ligand-density bonding processes along with exhaustive end-capping 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 end-capping 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 HSS C 8 SB columns do not resemble the selectivities provide by most high-coverage, fully end-capped 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, go to or 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]. BEH C 8. Waters Spherisorb S P BEH C 8 (ln [α] amitriptyline/acenaphthene) 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 HSS T. 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 HSS C 8 HSS C 8-0. YMC-Pack PolymerC (ln [k] acenaphthene) ACQUITY UPLC BEH C 8 XBridge C 8 ACQUITY UPLC HSS C 8 SB HSS C 8 SB ACQUITY UPLC HSS T BEH Shield RP8 BEH Phenyl BEH HILIC BEH AMIDE HSS HPLC Columns HSS C 8 HSS C 8 SB HSS T [ ]

32 HSS (High-strength Silica) Chemistries T Bonding for Polar Compound Retention ACQUITY UPLC HSS T columns utilize Waters innovative and proprietary T bonding. This bonding process is designed to provide polar-compound retention, aqueous-mobilephase compatibility and ultra-low MS bleed. 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 end-capping is much more effective than traditional trimethyl silane (TMS) end-capping. This unique combination of bonding and end-capping provides superior polar compound retention and aqueous compatibility while also enhancing column performance, lifetime, peak shape and stability. Features of the ACQUITY UPLC HSS T columns include: n Superior polar and non-polar compound retention n Aqueous mobile phase compatibility n Ultra-low MS bleed n Additional selectivity choice for UPLC separations n Scalability with HSS HPLC columns 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 BEH C 8 columns, most compounds are more strongly retained on ACQUITY UPLC HSS T columns. Superior Retention of Polar Compounds ACQUITY UPLC HSS T columns retain polar compounds longer than ACQUITY UPLC BEH C 8 columns and contain one of the most retentive sub--µm column chemistries commercially available. BEH C 8 BEH C 8 BEH C 8 0 min BEH Shield RP8 HSS T BEH Phenyl BEH HILIC BEH AMIDE 0 min HSS HPLC Columns HSS C 8 HSS C 8 SB Columns:. x 0 mm Mobile Phase A: 0 mm CH CNH, ph.0 Mobile Phase B: MeCN 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 [ ]

33 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) MeCN:MeH Gradient: Time Profile (min) %A %B Curve 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 BEH C 8 BEH Shield RP8 BEH Phenyl BEH HILIC 0 BEH AMIDE HSS HPLC Columns HSS C min HSS C 8 SB HSS T [ ]

34 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 Remote Services is 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 [ ]

35 ACQUITY UPLC System Accessories Screw Cap x mm Vials for ACQUITY UPLC Systems This selection of x mm vials are the most commonly ordered vials by customers using Waters ACQUITY UPLC Systems. Clear Amber Max Recovery Amber Max 00 µl PP Clear Glass with Septumless Cap For PEEK and Metal-Tipped Needles Lcms Certified Combination Packs Vial, Cap and Pre-slit Silicone/ptfe Septum CV CV CV 00000CV Lcgc Certified Combination Packs Bonded Pre-slit Silicone/ptfe Septum 80000C 80008C 8000C 80088C Bonded Pre-slit Silicone/ptfe Septum Deactivated 80000DV 80008DV 8000DV Combination with PE Septumless Cap 800C 800C 8008C 800C Combination Packs Bonded Pre-slit Silicone/ptfe Septum 8009 Combination with PE Septumless Cap 800 Injectable Volumes Acquity UPLC Max 00 µl 00 µl 00 µl 00 µl 0 µl 00 µl Residual µl µl µl µl 0 µl µl Best Selection Vials Limited Sample Unlimited Sample Light Sensitive All items come in quantities of 00 unless otherwise noted. For the Amino Acid ACQUITY UPLC System and the MassTrack Amino Acid Analysis Solution System, use of Waters Total Recovery Vials are recommended, Part Number 800. Waters LCMS Certified Vials (Suffix CV) 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 (Suffix C) 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 (Suffix DV) 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. ACQUITY UPLC Vial Descriptions Screw Cap x mm Vials for ACQUITY UPLC Systems Clear x, Type, -Expansion Glass, Screw Neck with Quick Thread Design, ( mm pening, 9 mm Cap). Amber x, Type, -Expansion Glass Screw Neck with Quick Thread Design, ( mm pening, 9 mm Cap). Waters Maximum Recovery Vial, x, Type, -Expansion Glass, Screw Neck with Quick Thread Design ( mm pening, 9 mm Cap). Waters Amber Maximum Recovery Vial, x, Type, -Expansion Glass, Screw Neck with Quick Thread Design ( mm pening, 9 mm Cap). Polypropylene x, 00 µl Screw Neck with Quick Thread Design ( mm pening, 9 mm Cap). Reformulate Clean PP Vial. Clear x, Type, -Expansion Glass, Screw Neck with Quick Thread Design, ( mm pening, 9 mm Septumless Cap). ACQUITY UPLC Vial Holder Description Part No. 8-Well Vial Holder 000 [ ]

36 ACQUITY UPLC System Accessories Plates for ACQUITY UPLC Systems 9-Well Plates 8-Well Plates Plate Pack Size Well Volume 0 µl 800 µl ml 0 µl 00 µl Sealing ptions Polypropylene Cap Mat 0/pk Clear Polyester Heat Seal 00/pk Aluminum Foil Laminate Heat Seal 00/pk Residual Volume in ACQUITY at default needle placement of mm µl µl 0 µl µl µl Note: (DV) after the number means a deactivated version of the product is available by adding a DV to the right of the part number when ordering. Glass Inserts for 9-Well Plates Description Part No. Max Volume Residual Volume Plates for Quick-Load Glass Widest pening for Inserts, 0/pk µl Glass Quick-Load, /pk 800 (DV) 0 µl µl ml Glass Quick-Load, /pk 800 (DV) 80 µl µl 9-Well Plate with 00 µl Glass Insert, /pk (DV) 0 µl µl 9-Well Plate with ml Glass Insert, 8/pk (DV) 80 µl µl Sealing Cap for 00 µl Glass Insert Square Well Seals Against the Well Wall PTFE/Silicone, /pk Sealing Cap for ml Glass Inserts Seals in the Glass PTFE/Silicone, 0/pk Plate 8008 must be used with the quick load units. When (DV) appears beside the part number, a deactivated version of this product can be ordered by adding DV to the right of the part number. Waters ACQUITY UPLC System Sample Manager The Waters ACQUITY UPLC System Sample Manager incorporates several technology advancements. The Sample Manager loop design and injection modes maintains low dispersion and facilitates fast cycle times. The system performance is monitored and diagnosed through the console software. 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 microtiter 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. Waters ACQUITY UPLC System Sample Manager Version.0 and later Sample Needle Vial Depth Typical ml Vial mm Default ffset [ ]

37 ACQUITY UPLC System Accessories Heat Seal for ACQUITY UPLC Systems Heat Sealer Part No. Volt Volt 8008 Heat Sealer perating Guidelines For more information regarding heat sealers, download the data sheet number 0000EN at Heat Seal Temperature Range Solvent Range Recommendation Clear Polyester Aluminum Foil Laminate From -80 C to 80 C From -00 C to 90 C Good for Most Lab Solvents Good for Most Lab Solvents Heat sealer dimensions:. x x (0 x 0 x 0 mm) Best for Long Term Storage The aluminum foil laminate heat seal is good for most solvents used in laboratories. For applications requiring DMS, the plates should be stored at C. Position the seal with the white side facing up. Apply heat using the Waters Heat Sealer for to seconds in both directions following the instructions found on page 9 in the heater manual, part number: 0000EN. The clear polyester heat seal is a non-conductive seal. For applications requiring DMS, the plates should be stored at C. Position the seal with the shiny side facing up. Apply heat using the Waters heat sealer for to seconds in both directions following the instructions found on page 9 in the heater manual, part number: 0000EN. Both of these seals can be peeled off by hand. For plate storage, apply a new unpierced seal or polypropylene cap mat. Waters N-LINE Vials Selector The Waters Vials Selector is designed to simplify the process of selecting the best vial solutions for your system and application requirements. The selector offers vial options matching the criteria entered, such as the system you are using, sample volume, detection method and light sensitivity of analytes. As a registered user, you will be notified of all future updates. For more information about Waters Certified Vials please visit This interactive tool requires that the user has Adobe Flash Player 8. Recommended Supplies for Use in Conjunction With the ACQUITY UPLC System Filters Waters recommends you filter buffers and samples using a 0. µm filter prior to use. Filtration protects the column and instrument components from a build-up of particulate matter, improving column lifetime and minimizing system down time. 0. µm GHP Filters are recommended for filtering aqueous, non-polar solvents and proteins. Sirocco Protein Precipitation Plate Sirocco Protein Precipitation Plate enables high throughput in-well protein precipitation. The unique filter system with vented cap mat and patented valve technology, allows the user to process samples in-well and collect clean filtrate from smaller plasma volumes without transfer steps. Description Part No. Solvent filtration membranes mm disc 00 pk* 800 mm Mini spike 00 pk (< µl hold up volume) WAT099 mm 0 pk (< 00 µl hold up volume) WAT099 Description Part No. Sirocco Protein Precipitation Plate, single pack 8008 Sirocco Protein Precipitation Plate, pack 8008 * requires solvent filtration apparatus, WAT00 or equivalent [ ]

38 rdering Information ACQUITY UPLC BEH Columns Chemistry Particle Size Dimensions Part No. pack Part No. pack BEH C 8. µm.0 x 0 mm BEH C 8. µm.0 x 00 mm BEH C 8. µm.0 x 0 mm BEH C 8. µm. x 0 mm BEH C 8. µm. x 0 mm BEH C 8. µm. x 00 mm BEH C 8. µm. x 0 mm BEH C 8. µm.0 x 0 mm BEH C 8. µm.0 x 0 mm BEH C 8. µm.0 x 00 mm BEH C 8. µm.0 x 0 mm BEH Shield RP8. µm.0 x 0 mm BEH Shield RP8. µm.0 x 00 mm BEH Shield RP8. µm.0 x 0 mm BEH Shield RP8. µm. x 0 mm BEH Shield RP8. µm. x 0 mm BEH Shield RP8. µm. x 00 mm BEH Shield RP8. µm. x 0 mm BEH Shield RP8. µm.0 x 0 mm BEH Shield RP8. µm.0 x 0 mm BEH Shield RP8. µm.0 x 00 mm BEH Shield RP8. µm.0 x 0 mm BEH C 8. µm.0 x 0 mm BEH C 8. µm.0 x 00 mm BEH C 8. µm.0 x 0 mm BEH C 8. µm. x 0 mm BEH C 8. µm. x 0 mm BEH C 8. µm. x 00 mm BEH C 8. µm. x 0 mm BEH C 8. µm.0 x 0 mm BEH C 8. µm.0 x 0 mm BEH C 8. µm.0 x 00 mm BEH C 8. µm.0 x 0 mm BEH Phenyl. µm.0 x 0 mm BEH Phenyl. µm.0 x 00 mm BEH Phenyl. µm.0 x 0 mm BEH Phenyl. µm. x 0 mm BEH Phenyl. µm. x 0 mm BEH Phenyl. µm. x 00 mm BEH Phenyl. µm. x 0 mm BEH Phenyl. µm.0 x 0 mm BEH Phenyl. µm.0 x 0 mm BEH Phenyl. µm.0 x 00 mm BEH Phenyl. µm.0 x 0 mm BEH HILIC. µm.0 x 0 mm BEH HILIC. µm.0 x 00 mm BEH HILIC. µm.0 x 0 mm BEH HILIC. µm. x 0 mm BEH HILIC. µm. x 00 mm BEH HILIC. µm. x 0 mm BEH HILIC. µm.0 x 0 mm BEH HILIC. µm.0 x 00 mm BEH HILIC. µm.0 x 0 mm BEH Amide. µm.0 x 0 mm BEH Amide. µm.0 x 00 mm BEH Amide. µm.0 x 0 mm BEH Amide. µm. x 0 mm BEH Amide. µm. x 0 mm BEH Amide. µm. x 00 mm BEH Amide. µm. x 0 mm BEH Amide. µm.0 x 0 mm BEH Amide. µm.0 x 0 mm BEH Amide. µm.0 x 00 mm BEH Amide. µm.0 x 0 mm ACQUITY UPLC HSS Columns Chemistry Particle Size Dimensions Part No. pack Part No. pack HSS T.8 µm.0 x 0 mm HSS T.8 µm.0 x 00 mm HSS T.8 µm.0 x 0 mm HSS T.8 µm. x 0 mm HSS T.8 µm. x 0 mm HSS T.8 µm. x 00 mm HSS T.8 µm. x 0 mm HSS T.8 µm.0 x 0 mm HSS T.8 µm.0 x 0 mm HSS T.8 µm.0 x 00 mm HSS T.8 µm.0 x 0 mm HSS C 8.8 µm.0 x 0 mm HSS C 8.8 µm.0 x 00 mm HSS C 8.8 µm.0 x 0 mm HSS C 8.8 µm. x 0 mm HSS C 8.8 µm. x 0 mm HSS C 8.8 µm. x 00 mm HSS C 8.8 µm. x 0 mm HSS C 8.8 µm.0 x 0 mm HSS C 8.8 µm.0 x 0 mm HSS C 8.8 µm.0 x 00 mm HSS C 8.8 µm.0 x 0 mm HSS C 8 SB.8 µm.0 x 0 mm HSS C 8 SB.8 µm.0 x 00 mm HSS C 8 SB.8 µm.0 x 0 mm HSS C 8 SB.8 µm. x 0 mm HSS C 8 SB.8 µm. x 0 mm HSS C 8 SB.8 µm. x 00 mm HSS C 8 SB.8 µm. x 0 mm HSS C 8 SB.8 µm.0 x 0 mm HSS C 8 SB.8 µm.0 x 0 mm HSS C 8 SB.8 µm.0 x 00 mm HSS C 8 SB.8 µm.0 x 0 mm VanGuard Pre-Column -Packs Chemistry Particle Size Dimensions Part No. pack BEH C 8. µm. x mm 8009 BEH Shield RP8. µm. x mm 8009 BEH C 8. µm. x mm BEH Phenyl. µm. x mm BEH HILIC. µm. x mm BEH Amide. µm. x mm HSS C 8.8 µm. x mm HSS C 8 SB.8 µm. x mm 800 HSS T.8 µm. x mm 8009 [ 8 ]

39 rdering Information High-Strength-Silica (HSS) HPLC Analytical Columns Particle Size Dimensions Format Part No. HSS C 8 Part No. HSS C 8 SB Part No. HSS T. µm.0 x 0 mm Guard Cartridge -pk* µm.0 x 0 mm Column µm.0 x 0 mm Column µm.0 x 00 mm Column µm.0 x 0 mm Column µm. x 0 mm Guard Cartridge -pk* µm. x 0 mm Column 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 Cartridge -pk* µm. x 0 mm Column µm. x 0 mm Column µm. x 0 mm Column High-Strength-Silica (HSS) HPLC Preparative Columns Particle Size Dimensions Format Part No. HSS C 8 Part No. HSS C 8 SB Part No. HSS T µm 0 x 0 mm Guard Cartridge -pk µm 0 x 0 mm Prep Column µm 0 x 00 mm Prep Column µm 0 x 0 mm Prep Column Mixed ACQUITY UPLC Chemistries Column -Packs Package Name Chemistries Particle Size(s) Dimensions Part No. High & Low ph, Widest Selectivities UPLC Columns Kit BEH C 8, BEH C 8, BEH Shield RP8, BEH Phenyl BEH:. µm. x 0 mm 000 High & Low ph, Widest Selectivities UPLC Columns Kit BEH C 8, BEH C 8, BEH Shield RP8, BEH Phenyl BEH:. µm. x 00 mm 000 UPLC Method Development Scouting Kit BEH C 8, BEH Shield RP8, BEH Phenyl, HSS T BEH:. µm HSS:.8 µm. x 0 mm 000 UPLC Method Development Scouting Kit BEH C 8, BEH Shield RP8, BEH Phenyl, HSS T BEH:. µm HSS:.8 µm. x 00 mm 000 L UPLC Columns Kit BEH C 8, BEH Shield RP8, HSS C 8, HSS T BEH:. µm HSS:.8 µm. x 0 mm 000 L UPLC Columns Kit BEH C 8, BEH Shield RP8, HSS C 8, HSS T BEH:. µm HSS:.8 µm. x 00 mm 000 Mass Spec UPLC Columns Kit BEH C 8, HSS C 8, HSS C 8 SB, HSS T BEH:. µm HSS:.8 µm. x 0 mm 000 Mass Spec UPLC Columns Kit BEH C 8, HSS C 8, HSS C 8 SB, HSS T BEH:. µm HSS:.8 µm. x 00 mm 0008 Low ph, Widest Selectivities UPLC Columns Kit BEH Shield RP8, BEH Phenyl, HSS C 8, HSS C 8 SB BEH:. µm HSS:.8 µm. x 0 mm 0009 Low ph, Widest Selectivities UPLC Columns Kit BEH Shield RP8, BEH Phenyl, HSS C 8, HSS C 8 SB BEH:. µm HSS:.8 µm. x 00 mm 000 High & Low ph, Widest Selectivities UPLC Columns Kit BEH C 8, BEH C 8, BEH Shield RP8, BEH Phenyl BEH:. µm.0 x 0 mm 0088 High & Low ph, Widest Selectivities UPLC Columns Kit BEH C 8, BEH C 8, BEH Shield RP8, BEH Phenyl BEH:. µm.0 x 00 mm 0088 UPLC Method Development Scouting Kit BEH C 8, BEH Shield RP8, BEH Phenyl, HSS T BEH:. µm HSS:.8 µm.0 x 0 mm 0088 UPLC Method Development Scouting Kit BEH C 8, BEH Shield RP8, BEH Phenyl, HSS T BEH:. µm HSS:.8 µm.0 x 00 mm 0088 L UPLC Columns Kit BEH C 8, BEH Shield RP8, HSS C 8, HSS T BEH:. µm HSS:.8 µm.0 x 0 mm 0088 L UPLC Columns Kit BEH C 8, BEH Shield RP8, HSS C 8, HSS T BEH:. µm HSS:.8 µm.0 x 00 mm 0088 Mass Spec UPLC Columns Kit BEH C 8, HSS C 8, HSS C 8 SB, HSS T BEH:. µm HSS:.8 µm.0 x 0 mm 0088 Mass Spec UPLC Columns Kit BEH C 8, HSS C 8, HSS C 8 SB, HSS T BEH:. µm HSS:.8 µm.0 x 00 mm Low ph, Widest Selectivities UPLC Columns Kit BEH Shield RP8, BEH Phenyl, HSS C 8, HSS C 8 SB BEH:. µm HSS:.8 µm.0 x 0 mm Low ph, Widest Selectivities UPLC Columns Kit BEH Shield RP8, BEH Phenyl, HSS C 8, HSS C 8 SB BEH:. µm HSS:.8 µm.0 x 00 mm 00890

40 rdering Information ACQUITY UPLC Columns Method Validation Kits (MVK) UPLC Column Hardware Chemistry Particle Size Column Length Part No.. mm ID Part No..0 mm ID BEH C 8. µm 0 mm BEH C 8. µm 00 mm BEH C 8. µm 0 mm BEH C 8. µm 00 mm BEH Shield RP8. µm 0 mm BEH Shield RP8. µm 00 mm BEH Phenyl. µm 0 mm BEH Phenyl. µm 00 mm BEH HILIC. µm 0 mm BEH HILIC. µm 00 mm BEH Amide. µm 0 mm BEH Amide. µm 00 mm HSS T.8 µm 0 mm HSS T.8 µm 00 mm HSS C 8.8 µm 0 mm HSS C 8.8 µm 00 mm HSS C 8 SB.8 µm 0 mm HSS C 8 SB.8 µm 00 mm Description Part No. Three 0. µm Inlet/utlet Frits for.0 mm ID UPLC Columns Three 0. µm Inlet/utlet Frits for. mm ID UPLC Columns 0000 Three 0. µm Inlet/utlet Frits for.0 mm ID UPLC Columns 0000 ne Inlet End Nut for.0 mm ID UPLC Column ne utlet End Nut for.0 mm ID UPLC Column ne Inlet End Nut for. mm ID UPLC Column ne utlet End Nut for. mm ID UPLC Column ne Inlet End Nut for.0 mm ID UPLC Column 0000 ne utlet End Nut for.0 mm ID UPLC Column ACQUITY UPLC Columns For Bioseparations Peptide Separation Technology n Suitable for a wide range of peptides, including large, small, acid, basic, hydrophobic, and hydrophilic n Good peak shape and retention in formic acid and TFA for good chromatography and peak detection n Available in 0Å and 00Å pore sizes n Quality control tested with a peptide mixture from a tryptic protein digest Description Dimension Particle Size Part No. ACQUITY UPLC BEH0, C 8. x 0 mm. µm 800 ACQUITY UPLC BEH0, C 8. x 00 mm. µm 800 ACQUITY UPLC BEH0, C 8. x 0 mm. µm 800 ACQUITY UPLC BEH00, C 8. x 0 mm. µm 8008 ACQUITY UPLC BEH00, C 8. x 00 mm. µm 8008 ACQUITY UPLC BEH00, C 8. x 0 mm. µm 8008 ACQUITY UPLC BEH00, C 8, VanGuard Pre-Column -pack. x mm. µm 8009 ACQUITY UPLC BEH0, C 8, VanGuard Pre-Column -pack. x mm. µm Protein Separation Technology n Separates proteins of various sizes, hydrophobicities, and isoelectric points n Available as. µm packing for HPLC, and. µm packing for UPLC methods n Chemistry exhibits minimal non-desired secondary interactions Description Dimension Particle Size Part No. ACQUITY UPLC BEH00, C 8. x 0 mm. µm 8009 ACQUITY UPLC BEH00, C. x 00 mm. µm 8009 ACQUITY UPLC BEH00, C. x 0 mm. µm 8009 ACQUITY UPLC BEH00, C, VanGuard Pre-Column -pack. x mm. µm 800 n Quality control tested with protein mixture

41 Amino Acid Analysis n Documented methods for the reversedphase analysis of pre-column-derivatized amino acids n Specifically designed for accurate, robust, and sensitive amino acid analysis n Proven assured performance in the areas for protein characterization, cell culture monitoring, and analysis of food and feeds ptimized UPLC Method for Hydrolysate Standards Application of the ACQUITY UPLC System for Amino Acid Analysis to the analysis of 0 pmol of hydrolysate amino acids is illustrated. Injection-to-injection cycle time is 0 minutes. AMQ NH His Ser Arg Gly Asp Glu Thr Ala Pro Cys Lys Tyr Met Val NVa ILe Leu Phe Description Dimension Particle Size Part No. UPLC AAA Application Add-on Kit* 009 AccQ Tag Ultra UPLC Column. x 00 mm. µm min * This kit is intended to enable existing ACQUITY UPLC systems for AAA applications. The Add-on Kit contains the AccQ Tag Ultra Chemistries and Column, documentation and additional hardware accessories needed for AAA applications. Column: AccQ-Tag Ultra UPLC Column,. x 00 mm Part Number: 8008 Mobile Phase A: AccQ-Tag Ultra Eluent A Mobile Phase B: AccQ-Tag Ultra Eluent B Flow Rate: 00 µl/min Injection Volume: 0. µl Sample Conc.: 0 pmol on column Temperature: 0 C Detection: 0 nm Instrument: ACQUITY UPLC System for Amino Acid Analysis ligonucleotide Separation Technology n Resolve failure sequences from detritylated products via ion-pair, reversed-phase chromatography n Separation efficiencies equivalent to or better than PAGE, CGE, or ion-exchange HPLC methods n Exceptional column lifetime at high ph and temperture for reduced cost per analysis n Quality control teseted with oligonucleotide standards Description Dimension Particle Size Part No. ACQUITY UPLC ST C 8. x 0 mm. µm ACQUITY UPLC ST C 8. x 00 mm. µm Glycan Separation Technology n Improved component resolution in less time compared to traditional HPLC n ptimized for use with ACQUITY UPLC system with fluorescence detection n Based on Waters BEH particle and bonding technology n Quality contol tested with relevant labeled glycan standards Description Dimension Particle Size Part No. ACQUITY UPLC BEH Glycan. x 0 mm. µm 8000 ACQUITY UPLC BEH Glycan. x 00 mm. µm 800 ACQUITY UPLC BEH Glycan. x 0 mm. µm 800 ACQUITY UPLC BEH Glycan, VanGuard Pre-Column -pack. x mm. µm 8009