ACQUITY UPLC Columns - More Choices, More Information · ACQUITY UPLC columns and VanGuard...
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ACQUITY UPLC Columns More Choices More Information HSS T3 PEPTIDES OLIGONUCLEOTIDES ACCQ•TAG UlTrA VAnGUArd HSS C 18 BEH HILIC BEH SHiEld rP18 BEH PHENYL BEH C 18 BEH C 8 HSS C 18 SB
Transcript of ACQUITY UPLC Columns - More Choices, More Information · ACQUITY UPLC columns and VanGuard...
ACQUITY UPLC Columns
More Choices
More Information
HSS T3
PePT ideS
OligOnucleOT ideS
ACCQ•TAG UlT rA
VAnGUArd
HSS c18
BeH Hil ic
BEH SHiEld rP18
BeH PHenyl
BeH c18
BeH c 8
HSS c18 SB
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Featuring:
n 1.7 µm BEH UPLC Particles
n 1.8 µm HSS UPLC Particles
n Eight Bonded Phases
n Reversed-phase and HILIC
n VanGuard Pre-columns
THe FIrsT and Only UPlC CerTIFIed COlUMns
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ACQUITY UPLC columns and VanGuard Pre-columns are the most technologically
advanced LC column products ever created. Designed, tested and guaranteed
for use in applications up to 15000 psi (1000 bar). Unsurpassed efficiency,
ruggedness and throughput. Available in over sixty combinations of column
configurations and chemistries. Combine faster separations with higher
resolution by harnessing the full potential of small particles.
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Rs (2,3) = 2.90
Rs (2,3) = 7.42
Rs (2,3) = 4.58
Rs (2,3) = 4.57
Rs (2,3) = 6.18
2.1 x 30 mm, 1.7 µm
2.1 x 50 mm, 1.7 µm
2.1 x 100 mm, 1.7 µm
2.1 x 150 mm, 5.0 µm
UPLC
HPLC
2.1 x 150 mm, 1.7 µm
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ULTRA
SPEED
ULTRA
RESOLUTION
SPEED WITH
RESOLUTION
RESOLUTION
WITH SPEED
Flexibility with UPLC Technology – Speed, Sensitivity and Resolution
CHrOMaTOgraPHy wITHOUT COMPrOMIse
In 2004, 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-2 µm particles. The highly flexible ACQUITY UPLC system is known for its unique capability of not having to switch between the ultra performance liquid chromatography (UPLC) capability of UPLC mode and conventional HPLC mode since the same unit is capable of both without modification or upgrades.
Since the 1970’s chromatographers have been limited to LC systems that were capable of operating at maximum system pressures of only 6000 psi (400 bar). This pressure limitation, coupled with large system volumes and slow data acquisition rates hindered the ability of separation scientists to fully realize the speed and efficiencies promised by using small (<2 µm) particles. The ACQUITY UPLC® system shatters these operating limitations and is designed to operate at a maximum pressure of 15000 psi (1000 bar).
Waters ACQUITY UPLC systems are holistically designed to dramatically improve resolution, sample throughput and sensitivity.
Whether your objective is maximum speed or maximum resolution, UPLC technology provides you with the flexibility to achieve both.
Compounds: 1. Quercetin 2. Kaempferol 3. Isorhamnetin
13X 0.6X 4.6X
8X SAME 3.6X
4X 1.3X 1.8X
2.7X 1.6X 1.2X
SPEE
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RESO
LUTI
ON
SEN
SITI
VITY
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The Benefits of UPLC Pressure Capability
The potential chromatographic benefits that can be achieved when using sub-2 µm particle LC columns are not solely due to the particle size. To fully realize the promise of sub-2 µm particle performance, one must have an LC system that is capable of operating at the optimal linear velocity for the particle (and analyte).
Further, this LC system must have an ultra-low system volume; a fast, sensitive, low-volume detector; intelligent software; negligible carryover; and, of course, the ability to reliably run at the higher backpressures generated by small particles operated at their optimal linear velocity.
Simply put, this is what the ACQUITY UPLC system was designed to do: allow chromatographers to fully realize and harness the power and promise of sub-2 µm particle columns.
BenefiTS Of HOliST ic AcQuiT y uPlc SyST em And cOlumn deSign
The ability to run at the optimal linear velocity is crucial to realizing the performance gains of sub-2 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.
THe POwer OF sMall ParTICles aT HIgHer PressUres
Since the commercialization of the ACQUITY UPLC system in 2004, 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., ≤ 3 µm) particle columns in a conventional LC system at HPLC pressures.
Chromatographic theory indicates, however, that this claim is somewhat untrue. In 2002, Waters described how one can achieve higher chromatographic throughput with the Intelligent Speed (IS™) family of HPLC columns.
However, this was not UPLC technology, but rather fast LC based upon small HPLC particles packed into short columns—at the expense of resolution.
With UPLC technology, one need not compromise sample throughput for chromatographic fidelity. Waters is the first and only LC manufacturer capable of delivering upon the promise of sub-2 µm particle UPLC technology.
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Sub-2 µm Particle Column Performance Under HPLC Conditions (Non-Optimal Linear Velocity)
Sub-2 µm Particle Column Performance Under UPLC Conditions (Optimal Linear Velocity)
ACQUITY UPLC BEH C182.1 x 50 mm, 1.7 µmPart Number: 186002350Alliance® 2695 HPLC SystemFlow Rate = 0.3 mL/minPSIMAX = 4,200Tf (4) = 1.63N (4) = 5,400Rs (2,3) = 0.97
ACQUITY UPLC BEH C182.1 x 50 mm, 1.7 µmPart Number: 186002350ACQUITY UPLC SystemFlow Rate = 0.6 mL/minPSIMAX = 8,400Tf (4) = 1.02N (4) = 10,100Rs (2,3) = 2.25
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Ultra-Fast SPE-UPLC/MS/MS Determination of Risperidone and Metabolite
Column: ACQUITY UPLC BEH C18 2.1 x 30 mm, 1.7 µmPart Number: 186002349Mobile Phase A: 10 mM CH3COONH4, pH 9.0Mobile Phase B: MeOHGradient: Time Flow Profile (min) mL/min %A %B 0.0 1.0 60 40 0.5 1.0 5 95 0.55 1.5 5 95 0.85 1.5 5 95 0.9 1.0 60 40 1.0 1.0 60 40 Injection Volume: 8 µLColumn Temperature: 50 °CSample Temperature: 15 °CSample Concentration: 0.1 ng/mLSample Diluent: 50/50 H2O/MeOHStrong Needle Wash: 60:40 ACN:IPA + 0.5% HCOOHWeak Needle Wash: 95:5 H2O/MeOH Instrument: ACQUITY UPLC with Quattro Premier™ MS
Quattro PremierES+Capillary: 3.0 kVSource Temp: 120 °CDesolvation Temp: 350 °C Cone Gas Flow: 50 L/HrDesolvation Gas Flow: 700 L/HrCollision Cell Pressure: 2.59 e-3 mbarDwell Time: 30 msecTransfer Tubing: 0.005”
Condition0.2 mL MeOH
Equilibrate0.2 mL H2O
Load: 0.4 mL diluted Human Plasma (0.2 mL plasma diluted 1:1 with 4%
H3PO4 in H2O)
Wash 10.2 mL 2% HCOOH in H2O
Wash 20.1 mL MeOH
Elute: 50 µL (25 µL x 2) 5% NH4OH in 90:10 MeOH:H2O
Dilute50 µL H2O
RecoveriesRisperidone 96%, 9-OH Risperidone 96%
Clozapine 94%
Clozapine (IS)
Risperidone
9-OH Risperidone
TIC
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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 15 minutes for 96 samples (~9 sec/sample). Peak widths are approximately 1.7 seconds wide, Pmax= 8900 psi.
How does one produce a short separation that does not sacrifice efficiency and/or peak shape? The simple answer lies in chromatographic theory. If one can reduce the column length, while simultaneously reducing the particle size by the same ratio (i.e., maintain column length to particle size (l/dp) ratio), the resolving power of the column as well as the resolution or peak capacity of the resulting chromatogram remains intact. However, this separation can be achieved in less time. The practical and commercial application of this simple chromatographic theory, however, has proven to be challenging.
For the first time, UPLC technology allows separation scientists to fully realize the speed and efficiencies promised by short columns packed with very small particles:
n Ultra-low system volume (low bandspreading)
n Fast, efficient detector and/or mass spectrometer
n Short cycle time sample manager with low carryover
n Small, rugged, highly efficient particles
n Advanced column hardware and packing techniques.
Application areas that benefit from the fast separations that are possible with 30 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.
UlT rA SPEEd
UlTra-FasT seParaTIOns
Oasis® MCX µElution Plate Sample Preparation Procedure*
* Oasis MCX 96-well µElution Plate (Part Number 186001830BA)
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UlTra-HIgH resOlUTIOn seParaTIOns
Chromatographers are always looking for solutions that will deliver more resolution and more robust separations. To meet this need for higher efficiency, Waters developed 150 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 150 mm length columns is more than 88,000, thus producing efficiencies of > 35,000 plates per separation. As a point of reference, a 4.6 x 150 mm, 5 µm HPLC column has an l/dp ratio of 30,000 and can produce efficiencies of only 12,000 plates.
UlT rA rESolUT ion
Choosing a UPLC column based upon the relationship between UPLC column length, length to particle size ratio (l/dp), efficiency (N) and separation type.
Ultra-Resolution Separations with 150 mm Length ACQUITY UPLC Columns
2.1 x 30 mm, 1.7 µmRs(2,3) = 1.33
2.1 x 50 mm, 1.7 µmRs(2,3) = 2.15
2.1 x 100 mm, 1.7 µmRs(2,3) = 3.06
2.1 x 150 mm, 1.7 µmRs(2,3) = 4.06
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Columns: ACQUITY UPLC BEH C18, 1.7 µmMobile Phase A: 0.2% HCOOH in H2OMobile Phase B: 0.2% HCOOH in ACNIsocratic: 95% A: 5% BFlow Rate: 0.5 mL/min Injection Volume: 1.5 µL (30 mm) 2.5 µL (50 mm) 5.0 µL (100 mm) 7.5 µL (150 mm)
Caffeine Metabolites Compounds 1. 1-methylxanthene 2. 1,3-dimethyluric acid 3. theobromine 4. 1,7-dimethylxanthene
150 mm length ACQUITY UPLC columns provide high resolution in less time.
Sample Diluent: 95:5 H2O: ACN with 0.2% HCOOH Sample Conc.: 25 µg/mLTemperature: 50 °CDetection: UV @ 280 nmSampling Rate: 40 pts/secTime Constant: 0.1Instrument: ACQUITY UPLC with ACQUITY UPLC TUV detector
UPLC Column Length l /dp* Efficiency
(N) Separation Type
30 mm 17,600 5,875 Easy
50 mm 29,400 11,750 Moderately Challenging
100 mm 58,800 23,500 Difficult
150 mm 88,200 35,000 Extremely Difficult
* dp = 1.7 µm
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Novel or putative drugs often exhibit high potency resulting in the requirement for low dosage levels. Hence, determining LLOQ levels in bodily fluids often present chemists with the challenge of achieving lower limits of detection when developing chromatographic assays for impurity profiling, bioanalysis and metabolite identification. Frequently there are several compounds of interest, all of which must be detected and quantitated in a single chromatographic run.
High sensitivity separations can be developed and improved through the use of small i.d. columns in microbore dimensions (e.g., 1.0 mm i.d.). Columns of these dimensions offer higher sensitivity over their larger diameter counterparts due to smaller analyte elution volume resulting in an increase in analyte concentration and subsequent increase in detector response. Microbore columns further have advantages in that they consume less mobile phase and can be more easily thermostatically controlled.
UlT rA SEnSiT iV iT y
UlTra-sensITIve seParaTIOns
Increased Sensitivity with 1.0 mm i.d. ACQUITY UPLC Columns
Columns: ACQUITY UPLC BEH C18 2.1 x 50 mm, 1.7 µm
ACQUITY UPLC BEH C18 1.0 x 50 mm, 1.7 µmMobile Phase A: 0.1% NH4OHMobile Phase B: ACNFlow Rates: 0.600 mL/min 2.1 mm i.d. column 0.140 mL/min 1.0 mm i.d. columnGradient: Time Profile Curve (min) %A %B Initial 95 5 - 2.0 5 95 6
Injection Volume: 4.4 µLSample : Ibuprofen spiked protein precipitated (PPT) rat plasmaConcentration: 10 ng/mLTemperature: 40 ˚CDetection: MRM 205 > 161 Instrument: ACQUITY UPLC with Quattro Premier XE Mass Spectrometer
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 1.0 mm i.d. UPLC columns without significant loss of separation efficiency.
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MRM of 4 Channels ES-204.81 > 160.86
3.75e6
2.1 mm i.d.UPLC Column
1.0 mm i.d.UPLC Column
~ 4x increasein sensitivity
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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 1.7 μm BEH particles and 1.8 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 2-3 times higher, a smaller number of ligands and particles are necessary to provide the desired resolution. ACQUITY UPLC columns allow chromatographers to efficiently develop faster and more robust separations.
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Double N 20-40%
Double k 15-20%Double α >400%
Maximized in UPLCSeparations by:
Maximized in UPLCSeparations by:
■ Ultra-low dispersion system
■ Small (<2 µm) particles
■ Higher pressure capability
■ Well-designed columns
■ Range of chemistries
■ Multiple particle substrates
■ Wide usable pH range (BEH)
■ Higher retentivity (HSS)
BUildinG rESolUT ion wiT H UPlC T ECHnoloGy
resOlUTIOn
uPlc column chemistries
* Expected or Approximate Values
BEH Particle HSS Particle
Chemistry
C18 C8 Shield RP18 Phenyl HILIC C18 C18 SB T3
O Si
O
O
O SiO
O
O SiO
O
O Si
CHPolar Group
3
CH3
O Si
O
O
O SiO
O
O SiO
O
O Si
CHPolar Group
3
CH3
O Si
O
O
O SiO
O
O SiO
O
O Si
CHPolar Group
3
CH3
O Si
O
O
O SiO
O
O SiO
O
O Si
CHPolar Group
3
CH3
O Si
O
O
O Si O
O
O Si O
O
O Si
CH Polar Group
3
CH 3
O SiO
O
Ligand Type Trifunctional C18 Trifunctional C8Monofunctional
Embedded Polar GroupTrifunctional C6 Phenyl
— Trifunctional C18 Trifunctional C18 Trifunctional C18
Ligand Density* 3.1 µmol/m2 3.2 µmol/m2 3.3 µmol/m2 3.0 µmol/m2 — 3.2 µmol/m2 1.6 μmol/m2 1.6 µmol/m2
Carbon Load* 18% 13% 17% 15% — 15% 8% 11%
Endcap Style Proprietary Proprietary TMS Proprietary — Proprietary None Proprietary
pH Range 1-12 1-12 2-11 1-12 1-8 1-8 2-8 2-8
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indUST ry-lEAdinG EnGinEErinG And MAnUfACTUrinG
Columns optimized for UPLC separations require innovative hardware and manufacturing processes that are not apparent when simply look-ing at the outside of the column. Like the holistically designed ACQUITY UPLC system, attention to every detail is critical for chromatographic success. ACQUITY UPLC columns were designed to be an integral part of the low bandspread UPLC system. The column and system could not be developed by simply ‘designing down’ or re-engineering an existing HPLC column or system. Typical HPLC system extra-column volumes and pressure limits would severely compromise the performance of UPLC columns. ACQUITY UPLC columns are the most technologically advanced columns ever created.
Procedures for reproducibly producing and sizing commercial quantities of 1.7 μm and 1.8 µm particles had to be developed. New packing stations and methods had to be designed, invented and implemented since UPLC columns are packed and tested differently than HPLC columns. Additionally, UPLC instrumentation is necessary to test these columns, something that no other manufacturer possesses. Since UPLC technology was created with the future in mind, ACQUITY UPLC columns incorporate eCord™ technology – a step towards the paperless laboratory. Besides storing each column’s unique Certificate of Analysis, the eCord tracks column usage such as date of installation, number of injections, number of sample sets, maximum temperature and pressure that the column has been subjected to and the date that the column was last used. All of this information travels with the column and is easily printable. The eCord is permanently attached to the column and the data cannot be erased.
Bulk SynT HeSiSn Rugged and efficient 1.7 µm BEH and 1.8 µm HSS particlesn Most technologically advanced porous particles ever createdn Combination of highest efficiencies, widest
pH range and superior mechanical strengthn Stable column beds at UPLC pressures
cOlumn PAckingn Columns must withstand UPLC
operating pressuresn New proprietary packing methods n New test instruments
Sof T wArEn Paperless tracking of column
history with eCord technology
AcQuiT y u P lc columns
feature ecord tec hnology
EnGinEErinGn New column hardwaren Low band broadeningn Innovative frit design
InnOvaTIOn
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Easy HPLC to UPLC Method Transfer with the ACQUITY UPLC Columns Calculator
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 power-ful software tool that accurately handles the scaling calculations that are required to convert isocratic or gradient HPLC methods to UPLC methods. The calculator provides a selection of UPLC methods that the chromatographer can choose from what is most important: maximum speed, maximum resolution or a combination of both.
EASily MiGrAT E froM HPlC To UPlC T ECHnoloGy
The ACQUITY UPLC Columns Calculator allows chromatographers to quickly and accurately convert HPLC methods to UPLC methods.
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Geometrically ScaledHPLC Linear Velocity
Pc=99
Pc=85
Pc=251
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Original HPLC Gradient Method
Maximum Peak Capacity Equivalent Analysis Time
Geometrically ScaledUPLC Linear Velocity
Equivalent Peak Capacity Shortest Analysis Time
A copy of the ACQUITY UPLC Columns Calculator is included with every ACQUITY UPLC system.
HPlC TO UPlC MeTHOd TransFer
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.
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sUPerIOr lIFeTIMe and rePrOdUCIbIlITy
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.1,2
1 A. Kaufmann, P. Butcher, K. Maden, M. Widmer, “Ultra-performance liquid chromatography coupled to time of flight mass spectrometry (UPLC–TOF): A novel tool for multiresidue screening of veterinary drugs in urine,” Anal. Chim. Acta 586 (1-2): 13-21 [2007]
2 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 847(2): 182-187 [2007]
rUGGEd And STABlE ColUMnS
Waters continues to set the industry standard for column-to-column and batch-to-batch reproducibility. Beginning with the Symmetry® brand of columns in 1994 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, ISO 9001, ISO 13485 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.
ExC EllEnT rEP rodUCiBil iT y
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Excellent Batch-To Batch Reproducibility
Column: ACQUITY UPLC BEH C18 2.1 x 100 mm, 1.7 µmPart Number: 186002352Mobile Phase A: 20 mM KH2PO4/K2HPO4, pH 7.0Mobile Phase B: MeOHIsocratic MobilePhase Composition: 45% A; 55% B Flow Rate: 0.25 mL/min Injection Volume: 2 µLDetection: UV @ 254 nmTemperature: 30.0 °CInstrument: ACQUITY UPLC system with ACQUITY UPLC TUV
Compounds:1. Uracil2. Propranolol3. Butylparaben4. 2-Methylnaphthalene5. Dipropylphthalate6. Acenaphthene7. Amitriptyline
2: Diode ArrayRange: 1.181e+2
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2: Diode ArrayRange: 1.765e+2
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Long UPLC Column Lifetimes
Column: ACQUITY UPLC BEH C18 2.1 x 30 mm, 1.7 µmPart Number: 186002349Mobile Phase A: 10 mM HCOONH4, pH 3.0Mobile Phase B: ACNFlow Rate: 0.8 mL/minGradient: Time Profile (min) %A %B 0.00 95 5 0.60 0 100 0.90 0 100 0.91 95 5 0.96 95 5
Injection Volume: 2 µLSample Diluent: DMSO Sample Conc.: 0.3 mM/LCompounds: Solvay test compounds (proprietary) Temperature: 26 °CDetection: UV @ 220-400 nmSampling Rate: 40 pts/secTime Constant: 0.025 (fast)Instrument: ACQUITY UPLC with ACQUITY UPLC PDA detector, ELSD & ZQ2000
Data courtesy of: C. Salomons, unpublished results, Research CDS-Note, Solvay Pharmaceuticals
Injection #2
Injection #30609!
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Separation scientists working in demanding application areas such as bioanalysis, food/beverage, natural products, environmental and industrial chemicals analyze complex, unpredictable and challeng-ing 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.
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 15000 psi (1000 bar) in applications run on the ACQUITY UltraPerformance liquid chromatography system. VanGuard Pre-columns feature a 2.1 mm i.d. x 5 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 Pre- columns. 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.
VAnGUArd P rE-ColUMnS
VanGuard Pre-columns are uniquely designed to protect and prolong ACQUITY UPLC column performance while contributing minimal chromatographic effects.
™
FEATURE BEnEFIT
First pre-column for UPLC applications
Guaranteed compatibility with pressures up to 15000 psi
Patent pending, ultra-low volume design Minimal chromatography effects
Manufactured using UPLC column hardware, particles and chemistries
Superior UPLC column protection and performance
Connects directly to UPLC column Leaks and connection voids are eliminated
Key Features and Benefits of VanGuard Pre-Columns
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0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
Minutes
Minimal Chromatographic Effects With VanGuard Pre-columns
Column: ACQUITY UPLC BEH C18, 2.1 x 50 mm, 1.7 µmPart Number: 186002350
Pre-column: VanGuard Pre-column, BEH C18, 2.1 x 5 mm, 1.7 µmPart Number: 186003975Mobile Phase A: 0.2% NH4OH in H2OMobile Phase B: 0.2% NH4OH in ACNFlow Rate: 0.8 mL/min Gradient: Time Profile (min) %A %B 0.0 95 5 2.0 5 95 2.5 5 95 2.6 95 5 3.0 95 5 Injection Volume: 2 µLTemperature: 40 °CDetection: UV @ 254 nmSampling Rate: 40 pts/secTime Constant: 0.05Instrument: ACQUITY UPLC with ACQUITY UPLC TUV detector and ACQUITY SQ Detector
ACQUITY SQ DetectorES+Capillary: 3.5 kVCone: 35 VSource Temp: 150 °CDesolvation Temp: 500 °CCone gas Flow: 50 L/HrDesolvation Gas Flow: 850 L/HrSIR: 373.2 m/z, 415.2 m/zMS Interscan Delay: 0.005 secDwell: 0.005 sec
Spike plasma with 500 ng/mL of diltiazem and desacetyl diltiazem
Take 200 µL of spiked plasma and add to 1.5 mL centrifuge tube
Add 600 µL of acetonitrile to the centrifuge tube containing the spiked plasma
Centrifuge for 10 minutes at 13000 RPM
Take 650 µL of supernatent and evaporate to dryness with nitrogen
Reconstitute with 400 µL of a 50:50 MeOH:H2O solution (results in a 203.125 ng/mL conc.)
Protein Precipitation Procedure
UPlC COlUMn PrOTeCTIOn
Desacetyl DiltiazemSIR m/z 373.2— Without VanGuard Pre-column— With VanGuard Pre-column
DiltiazemSIR m/z 415.2— Without VanGuard Pre-column— With VanGuard Pre-column
[ ]15
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., 15000 psi/1000 bar). Why is pressure tolerance important? In order to realize the efficiency gains of sub-2 µm particles, the ability to routinely operate at higher linear velocities (e.g., higher flow rates) is required. These higher linear velocities combined with small, sub-2 µm particles result in higher operating backpressures. Waters has created two highly efficient, pressure-tolerant UPLC particles: the 1.7 µm Ethylene Bridged Hybrid (BEH) particle and the 1.8 µm High Strength Silica (HSS) particle.
The first ACQUITY UPLC particle created was the 1.7 µm Ethylene Bridged Hybrid (BEH) particle. This second generation hybrid particle is one of the key enablers behind UPLC technology and is available in five column chemistries: C18, C8, Shield RP18, Phenyl and HILIC. Because this is a hybrid particle, a wider usable pH range (up to pH 1–12) makes method development faster and easier. BEH particles are also available in HPLC particle sizes (2.5, 3.5, 5 and 10 µm) in the XBridge family of HPLC columns, thus allowing seamless transfer between HPLC and UPLC separations.
ACQUITY UPLC HSS column chemistries include HSS C18, HSS C18 SB (Selectivity for Bases) and T3. The HSS C18 chemistry is a fully endcapped, ultra-performance, general purpose C18 bonded phase that provides superior peak shape for bases, increased retentivity (vs. ACQUITY UPLC BEH C18 columns), and extremely long lifetimes under acidic conditions. The HSS C18 SB (Selectivity for Bases) chemistry is an unendcapped C18 bonded phase designed and optimized for low pH method development and offers alternate selectivities, especially for basic com-pounds, as compared to most modern, high coverage C18 chemistries. The HSS T3 chemistry is an aqueous mobile phase compatible C18 bonded phase that is designed to retain and separate small, water soluble, polar organic molecules, much like Atlantis T3 HPLC columns.
ACQUiT y UPlC PArT iClE T ECHnoloGy
ACQUITY UPLC BEH Columns
ACQUITY UPLC HSS Columns
Particle Type Ethylene Bridged Hybrid (BEH) High Strength Silica (HSS)
Available Chemistries C18, C8, Shield RP18, Phenyl, HILIC C18, C18 SB, T3
pH Range 1-12; (RP18: 2-11); (HILIC: 1-8) C18: 1-8; C18 SB, T3: 2-8
Maximum Rated Pressure 15000 psi (~1000 bar) 15000 psi (~1000 bar)
Particle Size 1.7 µm 1.8 µm
Pore Diameter/Volume 130Å / 0.7 mL/g 100Å / 0.7 mL/g
Surface Area 185 m2/g 230 m2/g
UPlC ParTICle TeCHnOlOgy
The Particles of UPLC Technology
[ ]14
Waters is the Only Manufacturer Offering TWO UPLC-Certified Particles
[ ]15
Anal. Chem. 2003, 75, 6781-6788
Tetraethoxysilane (TEOS)
Polyethoxysilane (BPEOS)
Bis(triethoxysilyl)ethane (BTEE)
+ 4
S i
E t O
E t O O E t E t O
S i
E t O E t O
E t O
S i
O E t
O E t
O E t
Ethylene Bridges in Silica Matrix
S i
E t O
O
C H 2 C H 2
C H 2
C H 2
S i O S i
E t O
O E t
S i
O
O
O E t
S i
O
S i O O
E t
O O O
E t
E t E t n
The BEH Particle: One of the Key Enablers of UPLC TechnologyIn 1999, Waters launched the XTerra family of HPLC columns featuring first generation hybrid particle technology (HPT). HPT enabled XTerra columns to become one of the most successful column products in the history of Waters. In HPT, the best properties of inorganic (silica) and organic (polymeric) packings are combined to produce a material that has superior mechanical strength, efficiency, high pH stability and peak shape for bases.
The first generation methyl hybrid particles of XTerra columns did not possess the mechanical strength or efficiency necessary to fully realize the potential speed, sensitivity and resolution capabilities of UPLC technology. Therefore, a new pressure-tolerant particle needed to be created. A new, second generation hybrid material was developed which utilizes an ethylene bridged hybrid (BEH) structure. Compared to the first generation methyl hybrid particle of XTerra columns, the BEH particle of ACQUITY UPLC BEH columns exhibits improved efficiency, strength and pH range. BEH Technology™ is a key enabler behind the speed, sensitivity and resolution of UPLC separations.
BeH T ecHnOlOgy
*Patent No. 6,686,035 B2
As more separation scientists around the world realize the benefits of UPLC technology in their applications, Waters continues to provide additional UPLC particle and chemistry solutions to meet these demands. Waters material scientists developed a new High Strength Silica (HSS) particle with the high mechanical stability and appropriate morphology necessary to provide long column lifetimes and UPLC efficiencies at pressures up to 15000 psi (1000 bar). This 1.8 µm UPLC HSS particle is designed and tested specifically for use in UPLC separations.
ACQUITY UPLC HSS columns contain the first and only 100% silica particle designed, tested and intended for use in applications up to 15000 psi/1000 bar. The new ACQUITY UPLC HSS particle is not an HPLC particle. High pore volume HPLC particles do not posses the mechanical stability necessary to withstand the high column packing and operating pressures of UPLC separations.
ACQUiT y UPlC HiGH ST rEnGT H Sil iCA (HSS) PArT iClES
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.
Pressure
Flow
ACQUITY UPLC BEH 1.7 µm
HPLC Silica
ACQUITY UPLC HSS 1.8 µm
Ideal P rofile
UPlC ParTICle TeCHnOlOgy
[ ]16
The vast majority of reversed-phase (RP) LC separations take place on columns that contain C18 or C8 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 C18 and C8 columns were designed to be the columns of choice for most UPLC separations by providing the widest pH range and efficiencies.
They incorporate trifunctional ligand bonding chemistries which produce superior low pH stability and ultra-low column bleed. This low pH stability is combined with the high pH stability of the 1.7 μm BEH particle to deliver the widest usable pH operating range. In addition, these new chemistries also utilize new, proprietary endcapping processes which produce outstanding best peak shape for bases. T hese bonding chemistries and particle synthesis innovations produce the sharpest peaks, highest efficiencies and maximum MS sensitivities.
AcQuiT y uPlc BeH c18 And c 8 cOlumnS
Robust Separations
BEH C18
45 °C
55 °C
50 °C
BEH C8
BEH Shield RP18
BEH Phenyl
1
43
5
8
7 109
11614
13
12
1
43
5
2
7810
911
6
141312
1
43
528
7 10
9116
141312
2
1
4
35
8 7
10
9
11
614
1312
2
HSS T3
HSS C18
HSS C18 SB
1
1
1
4
4
4
3
3
3
5
5
5,6
8
8
8
7
7
7
10
10
10
9
9
9
11
11
11
6
6
14
14
14
13
13
13
12
12
12
2
2
2
2.000.00 4.00
Minutes
6.00 8.00 14.0010.00 12.00
1
43
52
87 10
9 116
141312
1
43
52
87 10
9 116
141312
1
43
52 87 10
69 11
141312
Minutes
2.000.00 4.00 6.00 8.00 10.00 12.00 14.00
Columns: ACQUITY UPLC BEH, 2.1 x 100 mm, 1.7 µm
ACQUITY UPLC HSS, 2.1 x 100 mm, 1.8 µmMobile Phase A: WaterMobile Phase B: MethanolFlow Rate: 0.5 mL/min Isocratic: 28% methanolInjection Volume: 5.0 µLSample Conc.: 10 µg/mLTemperature: 50 °CDetection: UV @ 254 nmSampling Rate: 20 pts/secTime Constant: 0.1Instrument: ACQUITY UPLC with ACQUITY UPLC PDA detectorCompounds: 1. HMX 2. RDX 3. 1,3,5-TNB 4. 1,3-DNB 5. NB 6. Tetryl 7. TNT 8. 2-Am-4,6 DNT 9. 4-Am-2,6 DNT 10. 2,4 DNT 11. 2,6-DNT 12. 2-NT 13. 4-NT 14. 3-NT
EPA Method 8330 HPLC separations usually require temperature control of ±1 °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 ±5 °C) separation.
beH (eTHylene brIdged HybrId) CHeMIsTrIes
HSS T3
HSS c18 SB
HSS c18
BeH Hil ic
BeH PHenyl
BEH SHiEld r P18
BeH c 8
BeH c18
[ ]17
Fast Separations of Basic Drugs
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 mobile-phase compatibility. ACQUITY UPLC BEH Shield RP18 columns are designed to provide selectivities that complement the ACQUITY UPLC C18 and C8 phases.
ACQUITY UPLC Shield RP18 columns combine Waters patented Shield Technology with BEH Technology by incorporating an embedded carbamate group into the bonded phase ligand. The alternate selectivity and excellent peak shape from the embedded polar group ligand, when combined with the wide pH range and ultra-efficiency of the 1.7 μm BEH particle, provide a powerful tool for UPLC method development.
ACQUiT y UPlC BEH SHiEld rP18 ColUMnS
Columns: ACQUITY UPLC BEH 2.1 x 50 mm, 1.7 µm
ACQUITY UPLC HSS 2.1 x 50 mm, 1.8 µmMobile Phase A: 10 mM NH4COOH, pH 3.0Mobile Phase B: MeOHFlow Rate: 0.4 mL/min Gradient: Time Profile (min) %A %B 0.00 70 30 3.00 15 85 3.50 15 85 3.51 70 30 4.50 70 30Inj. Volume: 1 µLSample Diluent: H2OTemperature: 30°CDetection: UV @ 260 nm Sampling Rate: 40 points/secFilter Response: NormalInstrument: ACQUITY UPLC with ACQUITY UPLC PDA
Compounds: 1. Aminopyrazine 2. Pindolol 3. Quinine 4. Labetalol 5. Verapamil 6. Diltiazem 7. Amitriptyline
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50
Minutes
1
2
3 4
5,67
1 34
5,67
1
2
34
65
7
1
2
3 4 56
7
1
2
3 4
5,6 7
2
1
2
3 4
5,6 7
1
2
4 3 56
7
BEH Shield RP18
BEH C18
BEH C18
BEH Phenyl
HSS T3
HSS C18
HSS C18 SB
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 5 and 6 with the BEH Shield RP18 chemistry and peaks 3 and 4 for the HSS C18 SB chemistry.
beH (eTHylene brIdged HybrId) CHeMIsTrIes
HSS T3
HSS c18 SB
HSS c18
BeH Hil ic
BeH PHenyl
BEH SHiEld r P18
BeH c 8
BeH c18
[ ]18
Phenyl-ligand containing reversed-phase columns can provide complementary selectivities as compared to other straight-chain 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 C6 alkyl tether between the phenyl ring and the silyl functionality. This phenyl-hexyl ligand, combined with the same proprietary endcapping processes as the ACQUITY UPLC BEH C18 and C8 columns, provides ultra-low column bleed, long column lifetimes and excellent peak shape. This unique combination of ligand, endcap and 1.7 μm BEH particle creates a new dimension in selectivity and efficiency for challenging UPLC separations.
AcQuiT y uPlc BeH PHenyl cOlumnS
Caffeic Acid Derivatives in Echinacea Purpurea Separations
12 4
53
1
24
53
1
2 4
53
12 4
53
1
24
53
1.000.00 2.00
Minutes
3.00 4.00 5.00
BEH C18
BEH C8
BEH Shield RP18
BEH Phenyl
HSS T3
HSS C18
HSS C18 SB
1
4
3 52
1 24
5
3
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, 2.1 x 50 mm, 1.7 µm
ACQUITY UPLC HSS, 2.1 x 50 mm, 1.8 µmMobile Phase A: 0.1% CF3COOH in H2OMobile Phase B: 0.08% CF3COOH in ACNFlow Rate: 0.5 mL/min Gradient: Time Profile Curve (min) %A %B 0.0 92 8 6 0.1 92 8 6 4.45 50 50 7 4.86 10 90 6 5.0 92 8 6 6.0 92 8 6Injection Volume: 1.0 µLSample Diluent: 50:50 H2O: MeOH with 0.05% CF3COOH Sample Conc.: 100 µg/mLTemperature: 40 °CDetection: UV @ 330 nmSampling Rate: 40 pts/secTime Constant: 0.1Instrument: ACQUITY UPLC with ACQUITY UPLC TUV detector
Compounds: 1. Caftaric acid 2. Chlorogenic acid 3. Cynarin 4. Echinacoside 5. Cichoric acid
beH (eTHylene brIdged HybrId) CHeMIsTrIes
HSS T3
HSS c18 SB
HSS c18
BeH Hil ic
BeH PHenyl
BEH SHiEld r P18
BeH c 8
BeH c18
Opioid 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-3-β-glucuronide elutes AFTER the more non-polar parent compound, morphine.
beH (eTHylene brIdged HybrId) CHeMIsTrIes
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 1.7 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.
AcQuiT y uPlc BeH Hil ic cOlumnS
12
V0
2. Morphine 3-β-Glucuronide1. Morphine
O
N
OH
OH
CH3
HO
O
N
N
OH
OHO
H
CH3
CH3
H
O
O
O
OHHO
OH
C OH
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00
Minutes
Column: ACQUITY UPLC BEH HILIC 2.1 x 100 mm, 1.7 µm Part Number: 186003461Mobile Phase A: 10 mM HCOONH4, 0.2% HCOOH in 50:50 ACN:H2OMobile Phase B: 10 mM HCOONH4, 0.2% HCOOH in 90:10 ACN:H2OFlow Rate: 0.788 mL/minGradient: Time Profile (min) %A %B 0.0 0.1 99.9 0.37 0.1 99.9 1.46 99.9 0.1 1.50 0.1 99.9 2.00 0.1 99.9
Separation of Very Polar Bases Using ACQUITY UPLC BEH HILIC Columns
Injection Volume: 2.1 µLSample Conc.: 125 µg/mL Sample Diluent: 75:25 ACN:MeOH with 0.2% HCOOHTemperature: 30 °CDetection: UV @ 280 nmInstrument: ACQUITY UPLC
with TUV Detector
[ ]19
Hydrophilic Interaction Chromatography (HILIC) is a separation technique where a polar stationary phase is used with a mobile phase that contains a high concentration of non-polar (organic) solvent and a low concentration of polar (aqueous) solvent. HILIC is used when compound retention is not possible using traditional reversed-phase LC. HILIC is quickly gaining popularity as a powerful separation technique since the mobile phases used contain high concentrations of organic modifier (e.g., ACN) which are ideal for compound ionization by ESI-MS. This results in much higher sensitivity and lower limits of detection.
HSS T3
HSS c18 SB
HSS c18
BeH Hil ic
BeH PHenyl
BEH SHiEld r P18
BeH c 8
BeH c18
[ ]20
7
7
7
7
7
6
6
5
4321
5
42,31
7
65
4321
65
432
1
6
54
32
1
765
43
21
65
3,42
1
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.000.00
Minutes
ACQUITY UPLC HSS C18, 1.8 µm
Column G VP C18, 1.5 µm
Column G VT C18, 1.5 µm
Column T HGA, 1.9 µm
Column T HG C12, 1.9 µm
Column Z SB C18, 1.8 µm
ACQUITY UPLC BEH C18, 1.7 µm
ACQUITY UPLC HSS C18 Columns Provide Superior Peak Shapes
As chromatographers around the world continue to realize and embrace the benefits of UPLC technology, additional UPLC column choices which provide complementary selectivities are needed to facilitate method development and provide application-specific solutions. Alternate or complementary selectivities can be realized by using columns with different bonded phases and/or different particle substrates. Based upon customer feedback, Waters created an additional pressure-tolerant UPLC-compatible particle to complement the 1.7 µm Ethylene Bridged Hybrid (BEH) particle. The 1.8 µm High Strength Silica (HSS) particle is designed to provide 'silica-like' selectivities and retentivity. The ACQUITY UPLC HSS C18 chemistry which is a fully endcapped, high coverage, ultra-performance C18 bonded phase that provides superior peak shape for bases, increased retention (vs. ACQUITY UPLC BEH C18 columns), and excellent low pH stability.
Although there is no direct HPLC column analog for ACQUITY UPLC HSS C18 columns, chromatographers will discover that for many separations, the selectivities and retention observed when using ACQUITY UPLC HSS C18 columns will resemble the chromatography produced by many modern, silica-based C18 HPLC columns.
AcQuiT y uPlc HSS c18 cOlumnS
Columns: 2.1 x 50 mmMobile Phase A: 20 mM KH2PO4/K2HPO4, pH 7.0Mobile Phase B: MeOHIsocratic MobilePhase Composition: 45% A; 55% B Flow Rate: 0.25 mL/min Injection Volume: 3 µLDetection: UV @ 254 nmTemperature: 30.0 °CInstrument: ACQUITY UPLC System with TUV
Compounds1. Uracil2. Propranolol3. Butylparaben4. 2-methylnaphthalene5. Dipropylphthalate6. Acenaphthene7. Amitriptyline
Hss (HIgH sTrengTH sIlICa) CHeMIsTrIes
Column USP Tailing Amitriptyline
Efficiency (N) Amitriptyline
ACQUITY UPLC HSS C18, 1.8 µm 1.38 10499
Column G VP C18, 1.5 µm 2.31 1150
Column G VT C18, 1.5 µm 3.46 507
Column T HGA, 1.9 µm 6.50 1515
Column T HG, 1.9 µm 2.12 7452
Column Z SB C18, 1.8 µm 2.34 4355
ACQUITY UPLC BEH C18, 1.7 µm 1.03 10353
The efficiencies and separations possible with UPLC technology are the result of more than just small particles. State-of-the-art bonding and endcapping processes are combined with small, rugged and efficient sub-2 µm particles to produce narrow, symmetrical peaks that enable fast, high resolution separations. The two Waters UPLC C18 chemistries produce efficiencies of greater than 200,000 plates/m for the strong base amitriptyline (pKa 9.4 at pH 7) – demonstrating that there is more to UPLC separations than sub-2 µm particles.
HSS T3
HSS c18 SB
HSS c18
BeH Hil ic
BeH PHenyl
BEH SHiEld r P18
BeH c 8
BeH c18
[ ]21
Creating 21st century separations involves more than small particles that operate at higher pressures. The ultra-efficiencies, peak capacities and column lifetimes obtained in UPLC separations can only be achieved using columns packed with stationary phases that utilize innovative and industry-leading bonding and endcapping processes. A small particle bonded with a poor and/or outdated stationary phase and/or endcap will produce inadequate efficiencies, inferior resolution and short column lifetimes. Like all ACQUITY UPLC stationary phases, the ACQUITY UPLC HSS C18 columns are created using state-of-the-art bonding and endcapping processes that produce high efficiencies and long column lifetimes.
The ACQUITY UPLC HSS C18 stationary phase features a tri-functionally bonded C18 ligand and proprietary endcap-ping 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 100% silica, the new ACQUITY UPLC HSS C18 chemistry is designed to work and excel under demanding highly acidic conditions.
The ability to reliably operate at low pH (i.e., <pH 2) is important because pH manipulation is a key tool in controlling and manipulating selectivities of ionizable compounds during method development. The ACQUITY UPLC HSS C18 packing is the most stable bonded phase commercially available and does not suffer from peak shape issues that plague older stationary phases that rely upon steric protection (and lack of endcapping) to produce stability at low pH.
AcQuiT y uPlc HSS c18 cOlumnS
0 10 20 30 40 50 60 70
ACQUITY UPLC HSS C18, 1.8 µm
ACQUITY UPLC BEH C18, 1.7 µm
Column Z SB C18, 1.8 µm
ACQUITY UPLC HSS T3, 1.8 µm
Column G VP C18, 1.5 µm
ACQUITY UPLC HSS C18 SB, 1.8 µm
Column G VT C18, 1.5 µm
Column T HG, 1.9 µm
Column Z EP C18, 1.8 µm
% of Retention Loss for Methyl Paraben
ACQUITY UPLC HSS C18 Columns Resist Acid Hydrolysis at Low pH
Low-pH Stability: 21 hr Exposure to 0.5% TFA at 60 °C
Hss (HIgH sTrengTH sIlICa) CHeMIsTrIes
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 C18 columns resist bonded phase loss due to novel bonding and endcapping processes.
HSS T3
HSS c18 SB
HSS c18
BeH Hil ic
BeH PHenyl
BEH SHiEld r P18
BeH c 8
BeH c18
Zorbax® SB C18
YMC-Pack ODS-A™
YMC J'sphereODS-H80
YMC-Pack PolymerC18™
Hypersil® CPS Cyano
YMC-Pack CN
Hypersil® BDS Phenyl
YMC-Pack™
Phenyl
Hypersil® PhenylInertsil® Ph-3
YMC-Pack ProC4™
YMCbasic™
YMC-Pack ProC8™
YMC-PackProC18™
Inertsil ODS-3
YMC J'sphere™
ODS-L80 Nucleosil® C18
YMC-Pack ODS-AQ™
YMC J'sphere ODS-M80
Inertsil CN-3
Luna® C18 (2)
Luna®
Phenyl Hexyl
Chromolith™
RP-18
Zorbax® XDB C18
ACT Ace® C18LunaC8 (2)
SunFire™ C18
Waters Spherisorb® S5 P
Nova-Pak Phenyl
Symmetry® C8
Nova-PakC8
XTerra MS C18 Symmetry C18
Nova-PakC18
Waters Spherisorb ODS2
Waters Spherisorb ODS1Resolve® C18
Bondapak™ C18
Waters Spherisorb S5CN
Nova-Pak® CN HP
SymmetryShield™ RP8
SymmetryShield RP18
XTerra RP8
XTerra RP18
XTerra MS C8
XTerra®
Phenyl Atlantis® dC18
SunFire™ C8
Atlantis T3
(ln [k] acenaphthene)
-0.6
-0.3
0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
2.4
2.7
3
3.3
3.6
-1.5 -0.5 0.5 1.5 2.5 3.5
(ln [α
] am
itrip
tylin
e/ac
enap
hthe
ne)
XBridge C18
XBridge Shield RP18
XBridge C8
XBridge™ Phenyl
ACQUITY UPLC BEH C18
ACQUITY UPLC BEH Shield RP18
ACQUITY UPLC BEH C8
ACQUITY UPLC BEH Phenyl
ACQUITY UPLC HSS T3
ACQUITY UPLC HSS C18
ACQUITY UPLC HSS C18 SB
[ ]22
Hss (HIgH sTrengTH sIlICa) CHeMIsTrIes
HSS T3
HSS c18 SB
HSS c18
BeH Hil ic
BeH PHenyl
BEH SHiEld r P18
BeH c 8
BeH c18
ACQUITY UPLC HSS C18 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 C18 columns can produce separations that will be quite different in terms of selectivities as compared to most modern, high coverage, fully endcapped C18 stationary phases. How was this accomplished? Waters material scientists bonded a trifunctional C18 ligand at an intermediate ligand density (i.e., 1.6 µmol/m2) without endcapping. In general, the higher silanophilic activity will promote longer retention for basic compounds whereas the intermediate C18 ligand density will produce slightly shorter (or equivalent) retention for non-basic compounds as compared to most modern high coverage, fully endcapped C18 stationary phases. Chromatographically, this unique combination will provide a practical method development tool for complex UPLC separations.
AcQuiT y uPlc HSS c18 SB (SElECT iV iT y for BASES) ColUMnS
Fast Separations of Tricyclic Antidepressants
1 23 5 4 6 7
1 23 5,4
67
1 23
4,5
6 7
1 23 5
4 6 7
1 23 5,4
6 7
1 23 4 5
6 7
1 2 3 54
67
0.400.20 0.60 1.000.00 0.80
Minutes
1.20 1.40 1.801.60 2.00
BEH C18
BEH C8
BEH Shield RP18
BEH Phenyl
HSS T3
HSS C18
HSS C18 SB
Tricyclic antidepressants (TCAs) are secondary and tertiary amines and were commonly prescribed to patients suffering from depression or other conditions. TCAs are being replaced by selective serotonin reuptake inhibitors (SSRIs). ACQUITY UPLC HSS C18 SB columns retain basic compounds such as tricyclic antidepressants longer than fully endcapped stationary phases due to the enhanced silanol activity on the non-endcapped HSS particle.
Columns: ACQUITY UPLC BEH, 2.1 x 50 mm, 1.7 µm
ACQUITY UPLC HSS, 2.1 x 50 mm, 1.8 µmMobile Phase A: 10mM NH4COOH, pH 3.0Mobile Phase B: ACNIsocratic MobilePhase: 60:40; A:BFlow Rate: 0.5 mL/minInjection Volume: 1 µLSample Conc: 10 µg/mL in H2O Temperature: 30 °CDetection: UV @ 254 nm Sampling Rate: 40 points/secFilter Response: NormalInstrument: ACQUITY UPLC with ACQUITY UPLC PDA
Compounds: 1. Trimethoprim 2. Nordoxepin 3. Doxepin 4. Nortriptyline 5. Imipramine 6. Amitriptyline 7. Trimipramine
Zorbax® SB C18
YMC-Pack ODS-A™
YMC J'sphereODS-H80
YMC-Pack PolymerC18™
Hypersil® CPS Cyano
YMC-Pack CN
Hypersil® BDS Phenyl
YMC-Pack™
Phenyl
Hypersil® PhenylInertsil® Ph-3
YMC-Pack ProC4™
YMCbasic™
YMC-Pack ProC8™
YMC-PackProC18™
Inertsil ODS-3
YMC J'sphere™
ODS-L80 Nucleosil® C18
YMC-Pack ODS-AQ™
YMC J'sphere ODS-M80
Inertsil CN-3
Luna® C18 (2)
Luna®
Phenyl Hexyl
Chromolith™
RP-18
Zorbax® XDB C18
ACT Ace® C18LunaC8 (2)
SunFire™ C18
Waters Spherisorb® S5 P
Nova-Pak Phenyl
Symmetry® C8
Nova-PakC8
XTerra MS C18 Symmetry C18
Nova-PakC18
Waters Spherisorb ODS2
Waters Spherisorb ODS1Resolve® C18
Bondapak™ C18
Waters Spherisorb S5CN
Nova-Pak® CN HP
SymmetryShield™ RP8
SymmetryShield RP18
XTerra RP8
XTerra RP18
XTerra MS C8
XTerra®
Phenyl Atlantis® dC18
SunFire™ C8
Atlantis T3
(ln [k] acenaphthene)
-0.6
-0.3
0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
2.4
2.7
3
3.3
3.6
-1.5 -0.5 0.5 1.5 2.5 3.5
(ln [α
] am
itrip
tylin
e/ac
enap
hthe
ne)
XBridge C18
XBridge Shield RP18
XBridge C8
XBridge™ Phenyl
ACQUITY UPLC BEH C18
ACQUITY UPLC BEH Shield RP18
ACQUITY UPLC BEH C8
ACQUITY UPLC BEH Phenyl
ACQUITY UPLC HSS T3
ACQUITY UPLC HSS C18
ACQUITY UPLC HSS C18 SB
[ ]23
Hss (HIgH sTrengTH sIlICa) CHeMIsTrIes
HSS T3
HSS c18 SB
HSS c18
BeH Hil ic
BeH PHenyl
BEH SHiEld r P18
BeH c 8
BeH c18
Stationary phases have evolved from the large, irregularly shaped particles of the past to the small, spherical particles of the present. Along the way, bonding and endcapping technologies have advanced and improved as well. Material scientists have focused much effort on improving peak shape for basic compounds in reversed-phase separations. For example, embedded polar group stationary phase columns were created in the 1990s to provide symmetrical peaks for basic compounds. T he vast majority of reversed-phase separations take place on straight alkyl chain C18 column chemistries. High ligand density bonding processes along with exhaustive endcapping are often implemented in order to reduce unwanted peak tailing and efficiency loss for basic compounds caused by secondary silanol interactions. Whereas the peak shape for basic compounds improves as a result of these advanced bonding and endcapping procedures, the resulting selectivities for these C18 chemistries end up being quite similar. T his can be seen by noting the large population of hydrophobic C18 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 C18 SB columns do not resemble the selectivities provide by most high coverage, fully endcapped C18 bonded phases. T his selectivity difference is valuable in developing UPLC methods.
AcQuiT y uPlc HSS c18 SB (SElECT iV iT y for BASES) ColUMnS
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 7 operat-ing conditions. This chart can be used to compare HPLC and UPLC columns of similar (or different) selectivities. For more information on this chart, please see U. D. Neue, B. A. Alden, T. H. Walter, “A Universal Procedure for the Assessment of the Reproducibility and the Classification of Silica-Based Reversed-Phase Packings; 2. Classification of Reversed-Phase Packings,” J. Chrom. A, 849: 101-116 [1999] and U.D. Neue, K.V. Tran, P.C. Iraneta, B.A. Alden, “Characterization of HPLC Packings,” J. Sep. Sci. 26 (3-4): 174-186 [2003].
ACQUITY UPLC HSS T3 columns utilize Waters innovative and proprietary T3 bonding. This is the same advanced bonding process that is behind the industry-leading polar-compound retention, aqueous mobile phase compatibility and ultra-low MS bleed of Atlantis T3 HPLC columns. T3 bonding utilizes a trifunctional C18 alkyl phase bonded at a ligand density that promotes polar compound retention and aqueous mobile phase compatibility. The T3 endcapping is much more effective than traditional trimethyl silane (TMS) endcapping. This unique combination of bonding and endcapping provides superior polar compound retention and aqueous compatibility while also enhancing column performance, lifetime, peak shape and stability.
Although the rugged and efficient ACQUITY UPLC BEH particle provides a wide pH range and superior peak shapes, its hydrophobic nature does not promote polar compound retention. ACQUITY UPLC HSS T3 columns are designed to retain and separate polar organic compounds in reversed-phase UPLC separations. When compared to ACQUITY UPLC BEH C18 columns, most compounds are more strongly retained on ACQUITY UPLC HSS T3 columns.
T3 BondinG for PolAr CoMPoUnd rET EnT ion
Superior Retention of Polar Compounds
ACQUITY UPLC HSS T3 columns retain polar compounds longer than ACQUITY UPLC BEH C18 columns and contain one of the most retentive sub-2 µm column chemistries commercially available.
Columns: 2.1 x 50 mm Mobile Phase A: 10 mM CH3COONH4, pH 5.0Mobile Phase B: ACNFlow Rate: 0.438 mL/min Isocratic Mobile Phase Composition: 2% BInjection Volume: 0.7 µLTemperature: 30 ˚CDetection: UV @ 280 nmInstrument: ACQUITY UPLC System with ACQUITY UPLC 2996 PDA
1
2
34
5
7
6
1
2
34
5
7
6
1.000.00 2.00
Minutes
3.00 4.00
1.000.00 2.00
Minutes
3.00 4.00
BeH c18
HSS T3
Compounds 1. Norepinephrine2. Epinephrine3. Dopamine4. 3,4- Dihydroxyphenylacetic acid (DOPAC)5. Serotonin (5-HT)6. 5-Hydroxy-3-indoleacetic acid (5-HIAA)7. 4-Hydroxy-3-methoxyphenylacetic acid (HVA)
[ ]24
Hss (HIgH sTrengTH sIlICa) CHeMIsTrIes
Features of the ACQUITY UPLC HSS T3 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.
HSS T3
HSS c18 SB
HSS c18
BeH Hil ic
BeH PHenyl
BEH SHiEld r P18
BeH c 8
BeH c18
[ ]25
Hss (HIgH sTrengTH sIlICa) CHeMIsTrIes
ACQUITY UPLC HSS T3 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 T3 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 100% silica and is efficient, mechanically stable, and, when bonded via the T3 bonding process, fully compatible with aqueous mobile phases.
AcQuiT y uPlc HSS T3 cOlumnS
1
2
3
4
5
6
78
9
10
11
12
1314
15
16
1718
19 20
1.000.00 2.00
Minutes
3.00 4.00 5.00 6.00 7.00 8.00
Separation of Cold Medicine Active Ingredients, Impurities and Counter Ions
Column: ACQUITY UPLC HSS T3 2.1 x 100 mm, 1.8 µmPart Number: 186003539Mobile Phase A: 0.15% CF3COOH in H2OMobile Phase B: 0.02% CF3COOH in 75:25 (v/v) ACN:MeOHGradient: Time Profile Curve (min) %A %B 0 99.9 0.1 0.5 99.9 0.1 6 1.7 87.0 13.0 6 3.6 67.0 33.0 6 7.5 1.0 99.0 6 8.0 1.0 99.0 6 8.5 99.9 0.1 6 9.5 99.9 0.1 6
Compounds:1. 4-aminophenol2. Maleate3. Fumarate4. Phenylephrine5. Acetominophen6. Phenylpropanolamine7. Pheniramine8. Doxylamine9. Pseudophedrine10. Pyrilamine11. Chlorpheniramine
12. Brompheniramine13. Guaifenesin14. Acetylsalicyclic acid15. 4-nitrophenol16. 4-chloroacetanilide17. Dextromethorphan18. Diphenhydramine19. Clemastine20. Ibuprofen Peaks 1, 15 and 16 are impurities of acetaminophen
ACQUITY UPLC HSS T3 columns provide superior retention of difficult-to-retain polar analytes while also offering good chromatographic selectivity for hydrophobic species.
Flow Rate: 0.6 mL/minInjection: 1.0 µL Column Temp.: 30 °CDetection: UV @ 254 nmInstrument: ACQUITY UPLC System with Column Heater Module and ACQUITY UPLC TUV Detector
HSS T3
HSS c18 SB
HSS c18
BeH Hil ic
BeH PHenyl
BEH SHiEld r P18
BeH c 8
BeH c18
[ ]27[ ]26
Waters Global Services focuses on optimizing Waters products with superior service, GxP compliance, support, upgrades, training, and Waters Quality Parts®.
Only 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. T his knowledge helps maximize system uptime, increase laboratory productivity, and meet stringent compliance requirements.
A Waters Service Plan for your ACQUITY UPLC system helps you:
nMaintain peak performance
nAccelerate throughput
nSpeed time-to-market
nMinimize compliance risk
nControl costs.
Connections INSIGHT® is a suite of Remote Intelligent Services included in many of our service plans that changes today’s service model from reactive to proactive support to maximize instrument availability and minimize downtime costs.
niHelp for On Demand Assistance
niAlert for Real-Time Monitoring
niAssist for collaboration with Waters Technical Support With 94 offices in more than 50 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.
BE ASSUrEd. CHooSE wAT ErS GloBAl SErV iC ES
waTers glObal servICes
Primary Head not delivering
Pressure fluctuations due to Accumulator loss of pressure
recommendations from waters: check for leaks
re-prime the system replace primary head check valve
[ ]27
Vials for AcQuiTy uPlc Systems, Screw cap 12 x 32 mm
aCQUITy UPlC sysTeM aCCessOrIes
Maximum Recovery Vials
n Most commonly used vial in ACQUITY UPLC systems
n Best choice when sample volume is limited
n This center draining vial allows access to most of the sample
n Preset needle placement of 2 mm from the bottom of the vial leaves 22 µL residual volume in the vial
n Using ACQUITY UPLC Sample Manager Advanced Setting, adjust the needle placement to access more sample, leaving less residual volume
Description Part No.
LCMS Certified Maximum Recovery Vial 600000670CV
LCMS Certified Amber Maximum Recovery Vial 600000755CV
LCGC Certified Maximum Recovery Vial 186000327C
LCGC Certified Amber Maximum Recovery Vial 186003886C
2 mL Vials
n Best Choice where sample volume is not limited
n Large residual volume at preset 2 mm needle placement – 165 µL
Description Part No.
LCMS Certified 2 mL Vial 600000668CV
LCMS Certified 2 mL Amber Vial 600000669CV
LCGC Certified 2 mL Vial 186000307C
LCGC Certified 2 mL Amber Vial 186000847C
Deactivated Clear Glass 2 mL Vial 186000307DV
Deactivated Amber Glass 2 mL Vial 186000847DV
waters lCMS Certified Vials
Waters LCMS certified vials are a continuation of our approach to offer products suitable for the demands of LCMS. We took an unbiased approach in developing this product, looking for any ionized masses regardless of the source. The vials are tested by MS with specifications for total ion count and presence of clusters in the high mass range. The product introduced is cleaner than any product we tested from vendors around the globe.
waters lCGC Certified Vials
Vials are usually manufactured by glass artisans and engineers who don’t understand the requirements for their use in LC and GC. As a manufacturer of autosamplers and chemistry consumables, Waters understands the dimensional and chemical requirements of vials. We reviewed the manufacturing process, anticipated possible problem areas, and developed tests to ensure the delivery of problem-free products. The LC test, to ensure the delivery of residue-clean vials, is a radically different form of test for the vials industry.
deactivated glass Vials
Deactivated glass vials eliminate adsorption of compounds onto the glass surface when working with biological or pharmaceutical compounds, natural products, pesticides and herbicides. The surface modification is permanent, resulting in an indefinite vial shelf life.
All products listed are 12 x 32 mm combination packs of 100, screw top vials, caps and pre-slit silicone / PTFE septa
All items come in quantities of 100 unless otherwise stated. For Amino Acid ACQUITY UPLC Systems, the use of total recovery vials is recommended but is only suitable for PEEK needles.
The Waters ACQUITY UPLC System Sample Manager incorporates several technology advancements. Low dispersion is maintained through the injection process, using a series of pressure transducers to facilitate self-monitoring and diagnostics. It uses needle-in-needle sampling for improved ruggedness and a needle calibration sensor for increased accuracy. A variety of sample holder formats (vials or tubes) and microliter plate formats (deep-well, mid-height) can also be accommodated in a thermostatically-controlled environment. Within the ACQUITY UPLC Sample Manager Instrument Method Editor, a number of parameters can be customized for your specific task, including depth, as shown here, to confer maximum sample format flexibility.
For further information on setting vial depth offsets, see the ACQUITY UPLC Operator’s Guide (information documentation set for ACQUITY UPLC– part number 716001664) or visit the ACQUITY UPLC Sample Manager Instrument Method Editor On-Line Help.
waters ACQUiTy UPlC System Sample Manager
Sample Needle
Typical 2 mL Vial
2 mm Default Offset
Vial Depth
aCQUITy UPlC sysTeM aCCessOrIes
[ ]28
Description Part Number
48-Well Vial Holder 405003743
AcQuiTy uPlc Vial Holder
Polypropylene Vials
n Good choice for applications where there is a concern that sample can bond to glass; alternative choice to deactivated glass
n Can be incinerated while still sealed to minimize exposure to hazardous substances
n 300 µL vial requires needle placement of 3 mm (see ACQUITY UPLC System Sample Manager— advanced settings below), leaving a residual volume of 20 µL
n 750 µL vial requires needle placement of 3 mm, leaving a residual volume of 70 µL
Description Part No.
300 µL Polypropylene (PP) Vial 186002639
750 µL Polypropylene (PP) Vial 186002636
All products listed are 12 x 32 mm combination packs of 100, screw top vials, caps and pre-slit silicone / PTFE septa
29
96 and 384-well Plates for ACQUiTy UPlC Systems
96-Well Plate 384-Well Plate
Plate 186002643 186002481 186002482 186002632 186002631
n Well Volume 350 µL 1 mL 2 mL 250 µL 100 µL
n Number of Plates in Sample Organizer 21 10 7 10 21
n Shape Round Round Square Square Square
n Bottom Round Conical Conical Conical Conical
n Material PP PP PP PP PP
n Height of Plate 14 mm 31 mm 42.5 mm 22 mm 15.5 mm
n Well Depth 11.25 mm 27 mm 39 mm 19.5 mm 12.3 mm
n Pack Size 100 50 50 50 50
n Estimated Residual Volume ACQUITY UPLC 35 µL 15 µL 20 µL 15 µL 15 µL
Seal Options
PP, 50-Pack (for Metal-Tipped Needles ONLY)1 186002483 186002483 186002484
Heat Seal (for All Needles)
Clear Polyester, 100-Pack 186002788 186002788 186002788 186002788 186002788
Aluminum Foil Laminate, 100-Pack 186002789 186002789 186002789 186002789 186002789
Description (for Metal-Tipped Needles ONLY) Part Number Max Volume Residual Volume
Plates for Quick-Load Glass – Widest Opening for Inserts, 20-Pack 186001438
n 700 µL Glass – Quick-Load, 1-Pack 186001437(DV)2 650 µL 15 µL
n 1 mL Glass – Quick-Load, 1-Pack 186001436 (DV)2 850 µL 15 µL
96-Well Plate with 700 µL Glass Insert, 1-Pack 186000349 (DV)2 650 µL 15 µL
96-Well Plate with 1 mL Glass Insert, 18-Pack 186000855 (DV)2 850 µL 15 µL
Heat Sealer Part Number
115 Volt 186002786
240 Volt 186002787
Glass inserts for Multi-well Plates
for PEEK and Metal-Tipped needles
2 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.
1 With ACQUITY UPLC system driver V1.30 and new Y-Carriage assembly.
filtersWaters recommends you filter buffers and samples using a 0.2 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. 0.2 µm GHP Filters are recommended for filtering aqueous, non-polar solvents and proteins.
recommended Supplies for Use in Conjunction with the ACQUiTy UPlC System
Description Part No.
Solvent filtration membranes 47 mm disc 100 pk* 18600352413 mm Mini spike 100 pk (< 14 µL hold up volume) WAT09796225 mm 50 pk (< 100 µL hold up volume) WAT097964 * requires solvent filtration apparatus, WAT200543 or equivalent
Sirocco Protein Precipitation PlateSirocco™ 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.
Sirocco Protein Precipitation Plate, single pack 186003873Sirocco Protein Precipitation Plate, 5 pack 186002448
aCQUITy UPlC sysTeM aCCessOrIes
[ ]
Heat sealer dimensions:
5.75 x 13 x 6 in. (140 x 330 x 150 mm)
[ ]30
OrderIng InFOrMaTIOn
ACQUiT y UPlC ColUMnS for BioSEPArAT ionS
Peptide Separation Technology
Description Dimension Particle Size Part No.
ACQUITY UPLC BEH130, C18 2.1 x 50 mm 1.7 µm 186003554ACQUITY UPLC BEH130, C18 2.1 x 100 mm 1.7 µm 186003555ACQUITY UPLC BEH130, C18 2.1 x 150 mm 1.7 µm 186003556ACQUITY UPLC BEH300, C18 2.1 x 50 mm 1.7 µm 186003685ACQUITY UPLC BEH300, C18 2.1 x 100 mm 1.7 µm 186003686ACQUITY UPLC BEH300, C18 2.1 x 150 mm 1.7 µm 186003687
n Improved chromatography of peptides with narrow, symmetrical peaks for best resolution
n Suitable for a wide range of peptides, including large, small, acidic, basic, hydrophobic and hydrophilic
n Good peak shape and retention in formic acid and TFA for optimal chromatography and detection
n Available in 130Å and 300Å pore sizes
www.waters.com/PST
Oligonucleotide Separation Technology
Description Dimension Particle Size Part No.
ACQUITY UPLC OST C18 2.1 x 50 mm 1.7 µm 186003949ACQUITY UPLC OST C18 2.1 x 100 mm 1.7 µm 186003950
n Separation efficiencies equivalent to or better than PAGE, CGE, or ion-exchange HPLC methods
n Resolve failure sequences from detritylated full length products
n Higher throughput compared to HPLC methods
n Exceptional column lifetime for reduced cost per analysis
www.waters.com/OST
AM
QN
H3 H
is
Ser
Arg G
ly
Asp
Glu
Thr Ala Pro
Cys
Lys
Tyr
Met Val N
Va
ILe
Leu Phe
Minutes
0.501.00 2.502.00 3.503.00 4.504.00 5.505.00 6.506.00 7.507.00 8.00
Column: AccQ-Tag Ultra UPLC Column, 2.1 x 100 mmPart Number: 186003837Mobile Phase A: AccQ-Tag Ultra Eluent A Mobile Phase B: AccQ-Tag Ultra Eluent BFlow Rate: 700 µL/minInjection Volume: 0.5 µL
Sample Conc.: 50 pmol on columnTemperature: 60 °CDetection: UV @ 260 nm Instrument: ACQUITY UPLC System for Amino Acid Analysis
Amino Acid Analysis
Description Dimension Particle Size Part No.
UPLC AAA Application Add-on Kit* 176001279AccQ•Tag™ Ultra UPLC Column 2.1 x 100 mm 1.7 µm 186003837
* 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.
n A turnkey total solution for amino acid analysis
n Specifically designed for accurate, robust, and sensitive analyses
n Proven assured performance in areas including protein characterization, cell culture monitoring, and food and feeds testing
www.waters.com/AAA
Optimized UPLC Method for Hydrolysate Standards
Application of the ACQUITY UPLC System for Amino Acid Analysis to the analysis of 50 pmol of hydrolysate amino acids is illustrated. Injection-to-injection cycle time is 10 minutes.
[ ]31
OrderIng InFOrMaTIOn
Chemistry Dimension Particle Size Part No.
BEH C18 2.1 x 50 mm 1.7 µm 186004044 2.1 x 100 mm 1.7 µm 186004045BEH C8 2.1 x 50 mm 1.7 µm 186004046 2.1 x 100 mm 1.7 µm 186004047BEH Shield RP18 2.1 x 50 mm 1.7 µm 186004048 2.1 x 100 mm 1.7 µm 186004049BEH Phenyl 2.1 x 50 mm 1.7 µm 186004050 2.1 x 100 mm 1.7 µm 186004052BEH HILIC 2.1 x 50 mm 1.7 µm 186004053 2.1 x 100 mm 1.7 µm 186004054HSS C18 2.1 x 50 mm 1.8 µm 186004057 2.1 x 100 mm 1.8 µm 186004058HSS C18 SB 2.1 x 50 mm 1.8 µm 186004137 2.1 x 100 mm 1.8 µm 186004138HSS T3 2.1 x 50 mm 1.8 µm 186004055 2.1 x 100 mm 1.8 µm 186004056
* Contains 3 columns, each packed with a different batch of packing material
AcQuiTy uPlc columns method Validation kits*
Description Dimensions Part No.
High & Low pH, Widest Selectivities UPLC Columns Kit 2.1 x 50 mm 176001042 BEH C18, BEH C8, BEH Shield RP18, BEH Phenyl 2.1 x 100 mm 176001043UPLC Method Development Scouting Kit 2.1 x 50 mm 176001603 BEH C18, BEH Shield RP18, BEH Phenyl, HSS T3 2.1 x 100 mm 176001604L1 UPLC Columns Kit 2.1 x 50 mm 176001605 BEH C18, BEH Shield RP18, HSS C18, HSS T3 2.1 x 100 mm 176001606Mass Spec UPLC Columns Kit 2.1 x 50 mm 176001607 BEH C18, HSS C18, HSS C18 SB, HSS T3 2.1 x 100 mm 176001608Low pH, Widest Selectivities UPLC Columns Kit 2.1 x 50 mm 176001609 BEH Shield RP18, BEH Phenyl, HSS C18, HSS C18 SB 2.1 x 100 mm 176001610
Mixed ACQUiTy UPlC Chemistries Column 4-Packs
Description Part No.
In-line filter holder and six 0.2 micron stainless steel replacement filters 205000343Five 0.2 micron stainless steel replacement filters and End Nuts for 205000343 700002775
ACQUiTy UPlC Column in-line filter Unit
AcQuiTy uPlc columns
Chemistry Dimension Particle Part No. Part No. Size Individual Column 3 pk
BEH C18 1.0 x 50 mm 1.7 µm 186002344 176000861 1.0 x 100 mm 1.7 µm 186002346 176000862 1.0 x 150 mm 1.7 µm 186002347 176001044 2.1 x 30 mm 1.7 µm 186002349 176001304 2.1 x 50 mm 1.7 µm 186002350 176000863 2.1 x 100 mm 1.7 µm 186002352 176000864 2.1 x 150 mm 1.7 µm 186002353 176001048 BEH Shield RP18 1.0 x 50 mm 1.7 µm 186002851 176000874 1.0 x 100 mm 1.7 µm 186002852 176000875 1.0 x 150 mm 1.7 µm 186003373 176001045 2.1 x 30 mm 1.7 µm 186003909 176001309 2.1 x 50 mm 1.7 µm 186002853 176000876 2.1 x 100 mm 1.7 µm 186002854 176000877 2.1 x 150 mm 1.7 µm 186003376 176001049 BEH C8 1.0 x 50 mm 1.7 µm 186002875 176000882 1.0 x 100 mm 1.7 µm 186002876 176000883 1.0 x 150 mm 1.7 µm 186003374 176001046 2.1 x 30 mm 1.7 µm 186003910 176001310 2.1 x 50 mm 1.7 µm 186002877 176000884 2.1 x 100 mm 1.7 µm 186002878 176000885 2.1 x 150 mm 1.7 µm 186003377 176001050 BEH Phenyl 1.0 x 50 mm 1.7 µm 186002882 176000905 1.0 x 100 mm 1.7 µm 186002883 176000906 1.0 x 150 mm 1.7 µm 186003375 176001047 2.1 x 30 mm 1.7 µm 186003911 176001311 2.1 x 50 mm 1.7 µm 186002884 176000907 2.1 x 100 mm 1.7 µm 186002885 176000908 2.1 x 150 mm 1.7 µm 186003378 176001051 BEH HILIC 1.0 x 50 mm 1.7 µm 186003457 176001089 1.0 x 100 mm 1.7 µm 186003458 176001090 1.0 x 150 mm 1.7 µm 186003459 176001091 2.1 x 50 mm 1.7 µm 186003460 176001092 2.1 x 100 mm 1.7 µm 186003461 176001093 2.1 x 150 mm 1.7 µm 186003462 176001094 HSS C18 1.0 x 50 mm 1.8 µm 186003529 176001121 1.0 x 100 mm 1.8 µm 186003530 176001122 1.0 x 150 mm 1.8 µm 186003531 176001123 2.1 x 30 mm 1.8 µm 186003987 176001398 2.1 x 50 mm 1.8 µm 186003532 176001124 2.1 x 100 mm 1.8 µm 186003533 176001125 2.1 x 150 mm 1.8 µm 186003534 176001126 HSS C18 SB 1.0 x 50 mm 1.8 µm 186004114 176001556 1.0 x 100 mm 1.8 µm 186004115 176001557 1.0 x 150 mm 1.8 µm 186004116 176001558 2.1 x 30 mm 1.8 µm 186004117 176001559 2.1 x 50 mm 1.8 µm 186004118 176001560 2.1 x 100 mm 1.8 µm 186004119 176001561 2.1 x 150 mm 1.8 µm 186004120 176001562
HSS T3 1.0 x 50 mm 1.8 µm 186003535 176001127 1.0 x 100 mm 1.8 µm 186003536 176001129 1.0 x 150 mm 1.8 µm 186003537 176001130 2.1 x 30 mm 1.8 µm 186003944 176001375 2.1 x 50 mm 1.8 µm 186003538 176001131 2.1 x 100 mm 1.8 µm 186003539 176001132 2.1 x 150 mm 1.8 µm 186003540 176001133
Chemistry Dimension Particle Size Part No.
BEH C18 2.1 x 5 mm 1.7 µm 186003975BEH Shield RP18 2.1 x 5 mm 1.7 µm 186003977BEH C8 2.1 x 5 mm 1.7 µm 186003978BEH Phenyl 2.1 x 5 mm 1.7 µm 186003979BEH HILIC 2.1 x 5 mm 1.7 µm 186003980HSS C18 2.1 x 5 mm 1.8 µm 186003981HSS C18 SB 2.1 x 5 mm 1.8 µm 186004136HSS T3 2.1 x 5 mm 1.8 µm 186003976
VanGuard Pre-Columns for UPlC Column Protection
Description Part No.
Three 0.2 µm Inlet/Outlet Frits for 2.1 mm i.d. UPLC Columns 700003776Three 0.2 µm Inlet/Outlet Frits for 1.0 mm i.d. UPLC Columns 700003775One Inlet End Nut for 2.1 mm i.d. UPLC Column 700003779One Outlet End Nut for 2.1 mm i.d. UPLC Column 700003780One Inlet End Nut for 1.0 mm i.d. UPLC Column 700003777One Outlet End Nut for 1.0 mm i.d. UPLC Column 700003778
ACQUiTy UPlC Column replacement Parts
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