acquity in chromatography laboratory: analytical ... uplc... · analytical considerations ......

Andrew Aubin

Patricia McConville

ACQUITY UPLC in the Chromatography Lab: Analytical Considerations

2005 Waters Corporation

ACQUITY UPLC

ACQUITY UPLC in the Chromatography Lab: Analytical Considerations

Ultra Performance LC delivers improved speed, sensitivity and resolution that can benefit many liquid chromatography applications in today's laboratory. We will discuss analytical performance along with considerations and recommendations for successful implementation of UPLC in the chromatography laboratory.


Why Advance the Science and Technology of LC?

Challenges in the LC Lab

Chromatography Resolution Reproducibility Ruggedness Sensitivity Speed Detector optimization

Business Complex samples Regulatory

considerations Increased # samples Time pressures Cost savings

Can you meet todays challenges?


HPLC: 7 Analgesics in 25 minutes

AU

0.00

0.12

0.24

0.36

0.48

Minutes0.00 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00

Pea

k1 -

5.47

0

Pea

k2 -

8.42

3

Pea

k3 -

9.94

9

Pea

k4 -

11.7

38

Pea

k5 -

16.4

10

Pea

k6 -

17.4

04

Pea

k7 -

19.0

37

ComponentsPeak #1 Acetaminophen Peak #2 - 2-AcetomidophenolPeak #3 - CaffeinePeak #4 - AcetanilidePeak #5 - Acetylsalicylic AcidPeak #6 - Salicylic AcidPeak #7 Phenacetin

Gradient Table


UPLC:7 analgesics in 25 seconds

ComponentsPeak #1 Acetaminophen Peak #2 - 2-AcetomidophenolPeak #3 - CaffeinePeak #4 - AcetanilidePeak #5 - Acetylsalicylic AcidPeak #6 - Salicylic AcidPeak #7 Phenacetin


Why Change from HPLC to UPLC?Time and solvent savings

Column Volume

(X5)

System Volume

(x3)

Re-equilibration time (min.)

Total cycle time

(min.)

# of injections in 24hours

Solvent consumed per injection

1.0 mL/min.

41

9931.3 mL/min

35.2mL

1.9mL

10.2.HPLC

4.6 x 100 525 min. run time

1.04

35.2

1.45

1.66mL

(8.3mL)

620uL

(1.86mL)

UPLC

2.1 x 50 1.7

0.41 min run time

0.20mL

(1.0mL)

120uL

(0.360mL)

Total Liters of solvent for 1000 injections:HPLC: 35.2L Vs. UPLC: 1.9L


Why Change from HPLC to UPLC?More information about your sample

UPLC1.7 m

Peaks = 168Pc = 360

2.5X increase

AU

0.00

0.02

0.04

0.06

0.08

AU

0.00

0.02

0.04

0.06

0.08

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

HPLC4.8 m

Peaks = 70Pc = 143


Why Change from HPLC to UPLC?Improved sensitivity

100 pg/mL

Time1.00 2.00 3.00

%

3

Mass1 > Mass2796

1.722.08Rt = 2.08 min, Customer Compound A,

100pg/mL, 10L injection. Phenomenex column 2.0x30mm, 4umPeak Width = 6 secs

100 pg/mL

Time0.30 0.60 1.00

%

0

Mass1 > Mass21.15e4

0.83

0.54Rt = 0.83 min, Customer Compound A,100pg/mL, 10L injection. ACQUITY UPLC Column C18 2.1x50mm, 1.7umPeak Width = 1.8 secs

HPLC

UPLC

Method Comparison 100pg/mL, 10L injectionNicholas Ellor


Speed, Sensitivity, ResolutionAU

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0.40

0.50

0.60

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50

HPLC


HPLC TodayThe state of the science

The quality of the separation in HPLC is both driven and limited by column chemistry

Optimized chemistry can bring speed, sensitivity and resolution benefits to the chromatographic process

Particle size and selectivity are primary contributors to column performance

Todays classical HPLC instruments cannot maximize the reality of state of art smaller particles


Smaller ParticlesThe enabler of productivity

Smaller particles provide: increased efficiency maintain efficiency over a

wider linear velocity ability for both added

resolution and increased speed of separation

Particles are central to the quality of the separation

van Deemter plot for classical HPLC


Fundamental Resolution EquationAt constant column length

NRs

In UPLC systems, N (efficiency) is the primary driverAssuming the Selectivity and retentivity are the same as in HPLCThen, Resolution, Rs, is proportional to the square root of N

Rs = N4 ( -1 ) kk+1( )

System Selectivity RetentivityEfficiency

And, Efficiency (N), is inversely proportional to Particle Size , dp

dpN 1So:

dp 3X,Therefore if: Rs 1.7X N 3X, Then:


Smaller ParticlesThe enabler of productivityThe promise of the van Deemter plot


Summary of Key System Attributes(at constant column length)

Resolution Improvement

Speed Improvement

Sensitivity Improvement

Back Pressure

1.7 vs. 5 m particles 1.7X 3X 1.7X 27X1.7 vs. 3 m particles 1.3X 2X 1.3X 6X


Influence of Particle Size on Chromatography

AU

0.000

0.010

0.020

0.030

0.040

0.050

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 15.00

4.8 m, 0.2 mL/min, 354 psi

AU

0.000

0.010

0.020

0.030

0.040

0.050

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00

Theory1.7X Resolution3X Faster1.7X Sensitivity25X Pressure

1.7 m, 0.6 mL/min, 7656 psi Actual1.5X Resolution2.6X Faster1.4X Sensitivity22X Pressure

Isocratic methods with 2.1 x 50 mm columns


Optimal Linear Velocity on ACQUITY UPLCTM:Isocratic Caffeine Metabolites Separation

HPLCNon-Optimal Linear Velocity

UPLCTMOptimal Linear Velocity

AU

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0.10

0.12

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0.16

Minutes

0.00 0.50 1.00 1.50 2.00 2.50 3.00

ACQUITY UPLCTM BEH C182.1 x 50 mm, 1.7 m

Alliance 2695F = 0.3 mL/minPSIMAX = 4,200

T (4) = 1.63N (4) = 5,400

Rs (2,3) = 0.97

ACQUITY UPLCTM BEH C182.1 x 50 mm, 1.7 m

ACQUITY UPLCTMF = 0.6 mL/minPSIMAX = 8,400

T (4) = 1.02N (4) = 10,100Rs (2,3) = 2.25

AU

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0.16

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00

1

2 3

4

Same ACQUITY UPLC Column on HPLC vs UPLC instruments


ACQUITY UPLCTM TechnologyUltra performance by design

Detectors:Optical and/or Mass SpecTunable UV or Photodiode ArrayOptimized flow cell for UPLCTMHigh speed data sampling for

Column Manager:Innovative pivot designPositions column to detectorE-CordTM connection

Sample Manager:Low dispersion XYZZ FormatFast cycle timesLow carryoverPlates and/or vialsOptional Sample Organizer

Binary Solvent Manager:High pressure blendingBinary gradientsFour solvent choicesOn-line degassingLow dispersion designUPLC pressure capabilities

Sample Organizer: (option)Expands capacity (22/15/8)Shuttles plate feedHeated/chilled

System Considerations:Small FootprintRedesigned tubing and fittingsConsolidated waste managementIntegrated system diagnosticsConnections InsightTM remote diagnostics


UPLCUPLC Technology Challenges

How do we take advantage of smaller particles?

Pressure tolerant particle/column

High pressure fluidic modules

Holistic design, low system volume, optimized flow paths

Reduced cycle time autosampler with minimum carryover

High speed detectors; optical and mass

Controlled and coordinated system interaction Software designed for system integration Comprehensive diagnostic suite


ACQUITY UPLCTMChemistry Proposition

Advancing the Science

Requires innovation in every aspect of column: New patented bridged hybrid particle required for pressure

tolerance and outstanding chromatographic performanceSub 2m particlesPorous for optimum mass transfer

Column hardwareNew patented frit technology to retain particles New end fittings for high pressure/low dispersion operation

Packing technologyNew column packing processes to optimize stability

eCordTMNew information chip to store column history


Reversed-Phase Column Selectivity Chart

SunFire C18

YMC-Pack PolymerC18

Hypersil CPS Cyano

YMC-Pack CN

Waters Spherisorb S5 P

Hypersil BDS PhenylNova-Pak Phenyl

YMC-Pack Phenyl

Hypersil PhenylInertsil Ph-3

YMC-Pack Pro C4

YMCbasic

Symmetry C8YMC-Pack Pro C8

Nova-Pak C8

XTerra MS C18 Symmetry C18

YMC-Pack Pro C18

Inertsil ODS-3

YMC-Pack ODS-A

Nova-PakC18

YMC J'sphereODSL80 Nucleosil C18

Waters Spherisorb ODS2

Waters Spherisorb ODS1Resolve C18

Bondapak C18

YMC-Pack ODSAQ

YMC J'sphere ODSH80YMC J'sphere ODSM80

Inertsil CN-3

Waters Spherisorb S5CN

Nova-Pak CN HP

SymmetryShield RP8

SymmetryShield RP18

XTerra RP8

XTerra RP18

-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 [

] am

itrip

tylin

e/ac

enap

hthe

ne)

XTerra MS C8

Luna C18 (2)

ACQUITY UPLC BEH C18

XTerra Phenyl Luna

Phenyl Hexyl

ChromolithTM RP-18

Atlantis dC18

Zorbax XDB C18ACT Ace C18

Zorbax SB C18

SunFire C8

LunaC8 (2)

ACQUITY UPLC BEH C8

ACQUITY UPLC BEH Phenyl

(ln [k] acenaphthene)012005 ACQUITY UPLC Shield RP18


ACQUITY UPLC Column Selectivity Comparison for Analgesics

C8

C18

RP18

Phenyl

1

ACQUITY UPLC BEH C18 1.7m ACQUITY UPLC BEH RP18 1.7m ACQUITY UPLC BEH C8 1.7m ACQUITY UPLC BEH Phenyl 1.7m

1. Acetaminophen2. 2-Acetomidophenol3. Caffeine4. Acetanilide5. Acetylsalicylic Acid6. Salicylic Acid7. Phenacetin

23

45 6

7

2

6

6


Column Lifetime:>8,500 Injections

ConditionsColumn: ACQUITY UPLC BEH

C18 2.1 x 50 mmMobile Phase A: 0.1% HCOOHMobile Phase B: 0.1% HCOOH in ACNFlow Rate: 0.65 mL/minGradient: Time Profile

(min) %A %B Curve0.0 95 5 Initial 0.1 95 5 60.4 5 95 30.5 95 5 110.8 95 5

Injection Volume: 5 LTemperature: 40oCDetection: UV @ 254 nm

Pmax: 6,650 psi


Column LifetimeCustomer Example

Isocratic aging experiment Cycle: low pressure runs/injections (data shown) 200/400/600 etc.

high pressure runs/injections (13,000 psi) low pressure runs/injections

This aging cycle was repeated

Columns lasted over 4,000 high pressure injectionsConditions (Isocratic Slow Flow/Low Pressure)Column: ACQUITY UPLC BEH C18 (2.1 x 50 mm, 1.7 m)

ACQUITY UPLC BEH Shield RP18 BEH (2.1 x 50 mm, 1.7 m) Part Number: 186002350Mobile Phase A: 0.1% HCOOH in WatersMobile Phase B: 0.1% HCOOH in ACNFlow Rate: 0.5 mL/min (~6,000 psi)Condition: 67% A / 33% BInjection Volume: 5.0 LSample Concentration: 208 g/mL ketorolac tromethamine and 13 g/mL naproxen in MeOHTemperature: 65oCDetection: UV 225 nm (Sample Rate: 10 Hz, Filter: Normal)

Conditions (Isocratic Fast Flow/High Pressure)Columns: ACQUITY UPLC BEH C18 (2.1 x 50 mm, 1.7 m)

ACQUITY UPLC BEH Shield RP18 (2.1 x 50 mm, 1.7 m) Mobile Phase A: 0.1% HCOOH in WaterMobile Phase B: 0.1% HCOOH in ACNFlow Rate: 1.250 mL/min (Pressure ~13,000 psi)Condition: 62% A / 38% BInjection Volume: 5.0 LSample Concentration: 208 g/mL ketorolac tromethamine and 13 g/mL naproxen in MeOHTemperature: 65oCDetection: UV 225 nm (Sample Rate: 10 Hz, Filter: Normal)


Column LifetimeCustomer Example

First Injection on Column No high pressure runsN = 3483 for Peak 1 N = 6352 for Peak 2

Injection after4000 high pressure runs

N = 3573 for Peak 1N = 6466 for Peak 2

AU

- 0 .4 0

- 0 .2 0

0 .0 0

0 .2 0

0 .4 0

M in u te s0 .0 0 0 .2 0 0 .4 0 0 .6 0 0 .8 0 1 .0 0 1 .2 0 1 .4 0 1 . 6 0 1 .8 0 2 .0 0 2 .2 0 2 .4 0 2 .6 0 2 .8 0 3 .0 0

1.16

5

1.97

4

AU

-0.40

-0.20

0.00

0.20

0.40

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

1.16

8

1.97

8

AU

-0.40

-0.20

0.00

0.20

0.40

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

1.17

8

2.00

0

Ketorolac Naproxen

Chromatograms obtained under low (~6000 psi) isocratic conditions

Injection after1600 high pressure runs

N = 3659 for Peak 1N = 6489 for Peak 2


pH Stability At The Extremes

What about chemical stability of the column at the extremes of pH, temperature and pressure?

A 2 x 2 experimental design (at UPLC pressures, of course):

30oC 60oCpH 2.0pH 11.3


Column Stability at pH 2.0Direct Overlays

Minutes0.00 1.00 2.00 3.00 4.00

Minutes0.00 1.00 2.00 3.00 4.00

2000

1000

1

Injection

60oC8,100 psi

5.00

ACQUITY UPLC BEH C18, 2.1 x 50 mm30% ACN (v/v) with 0.1% TFA (pH 2.0) at 0.9 mL/min

Analytes: protriptyline, amitriptyline, butyrophenone

30oC11,400 psi

5.00


Column Stability at pH=11.3

Minutes1.50 3.00 4.50

Inj. 1

Inj. 500

Inj. 1000

Minutes1.50 3.00 4.50

Acetonitrile-Pyrrolidine Buffer (pH=11.3) 45:55 (v/v) at 0.9 mL/min flow rateAnalytes: butyrophenone, protriptyline and amitriptyline

Inj. 1

Inj. 500

Inj. 750

60oC, 7,700 psi30oC, 11,500 psiOffset Overlays


ACQUITY UPLCTM TechnologyUltra performance by design

Detectors:Optical and/or Mass SpecTunable UV or Photodiode ArrayOptimized flow cell for UPLCTMHigh speed data sampling for

Column Manager:Innovative pivot designPositions column to detectorE-CordTM connection

Sample Manager:Low dispersion XYZZ FormatFast cycle timesLow carryoverPlates and/or vialsOptional Sample Organizer

Binary Solvent Manager:High pressure blendingBinary gradientsFour solvent choicesOn-line degassingLow dispersion designUPLC pressure capabilities

Sample Organizer: (option)Expands capacity (22/15/8)Shuttles plate feedHeated/chilled

System Considerations:Small FootprintRedesigned tubing and fittingsConsolidated waste managementIntegrated system diagnosticsConnections InsightTM remote diagnostics


ACQUITY UPLCTMBinary Solvent Manager

Binary gradient Independent style piston drives Serial flow for each solvent, parallel

gradient formation High Pressure Gradient

Solvent select, 4 solvent choices On board solvent degasser 50l mixer, ~120L total system volume Ability to deliver non-linear gradients UPLC pressure capabilities

0-1mL/min to 15K psi, 1 -2mL/min to 9K psi

Dedicated transducer for each head. Enhances performance, provides diagnostic information


BSM Components

Pump A Pump B

SSV A SSV BDegasserTee

FilterMixer

Vent Valve

PlungerWash


From Serial to Parallel Flow

Two independently driven primary and accumulatorpiston drives Separate drive motors Two transducers Two inlet check valves

Four Solvents Capability A1 or A2 and B1 or B2 Two solvents per gradient

Six Channel Degasser 4 chromatographic eluents 2 solvents for Sample Manager

Strong & weak wash

B B A A

B2B2B1B1

A2A2

Solvent A1Solvent A1


Pressure Envelope Fluidic components optimized to support the pressure

demands of UPLC separations Seals, head, bolts, check valves, fittings, tubing, mixer, pressure

transducers

UPLC compatible pressure with flow rate range

1 2

9000

15000

Pressure(psi)

Flow Rate(mL/min)


Tee/ Filter/ Mixer

Glass Bead Mixing Technology Tortured Mixing Durable and will not disintegrate or affect delicate compounds Highly resistant to attack by most liquids and/or vapors.

Glass Bead Mixer Standard 4 x 5, approximate volume 50 L (P/N 700002631) Optional Mixer 6 x 6, approximate volume 100 L (P/N 205000252)

Filter Similar to Alliance, 2 micron filter (P/N 700002674)


New Tubing & Fittings

HPLC Fittings Traditional drawn tubing

Traditional Waters Fittings

UPLCTM Fittings Center-less ground tubing

Gold Compression Screw 2 Piece Swagelock Ferrules


ACQUITY 9/1000 SS316 Tubing

@75 Times


Standard 5/1000 inch 316 SS Tuning

@75 Times @500 Times


New ACQUITY 2/1000 316 SS Tubing

@75 Times @1000 Times


ACQUITY UPLCTMBinary Solvent Manager gradient steps

N = 6

1% Steps 0% B to 10% BFlow Rate=0.50 mL/minuteBackpressure ~9300 psi


ACQUITY UPLCTMBinary Solvent Manager Auto Blend

Injections of Caffeine with Dial-a-Mix at 1.0mL/min4.0% to 5.0% ACN in 0.1% Increments

4.0% ACN

5.0% ACN


Original Assay Method from the USP for: Benzocaine, Butamben, and Tetracaine Hydrochloride Topical Solution

Mobile phase Prepare a mixture of methanol, water, and 0.25 M sodium 1-heptanesulfonate (500:500:20). Make adjustments if necessary (see System Suitability under Chromatography (621).

Chromatographic system (see Chromatography [621])The liquid chromatograph is equipped with a 313-nm detector, and a 3.9-mm 30-cm column that contains packing L1. The flow rate is about 2 mL per minute.

Standard preparation Transfer about 140 mg of USP Benzocaine RS, accurately weighed, to a 100-mL volumetric flask with the aid of 25 mL of methanol, and swirl. Transfer about 140 J mg of USP Butamben RS, accurately weighed, to the same volumetric flask with the aid of 25 mL of water, J being the ratio of the labeled amount, in percent, of butamben to the labeled amount, in percent, of benzocaine in the Topical Solution. Transfer about 140J mg of USP Tetracaine Hydrochloride RS, accurately weighed, to the same volumetric flask with the aid of 25 mL of water, J being the ratio of the labeled amount, in percent, of tetracaine hydrochloride to the labeled amount, in percent, of benzocaine in the Topical Solution. Sonicate for about 1 minute, dilute with Diluent to volume, and mix.

Procedure Separately inject equal volumes (about 10 L) of the Standard preparation and the Assay preparationinto the chromatograph, record the chromatograms, and measure the peak areas for the major peaks.

Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.3 for benzocaine, 0.8 for butamben, and 1.0 for tetracaine; the resolution, R, between the benzocaine peak and the butamben peak and between the butamben peak and the tetracaine peak is not less than 2; and the relative standard deviation for replicate injections is not more than 2.0% for each of the three analyte peaks.

http://www.uspnf.com/uspnf/pub/data/v28230/usp28nf23s0_alpha-18-140.xml#usp28nf23s0_c11rs113http://www.uspnf.com/uspnf/pub/data/v28230/usp28nf23s0_alpha-18-140.xml#usp28nf23s0_c11rs157http://www.uspnf.com/uspnf/pub/data/v28230/usp28nf23s0_alpha-18-1412.xml#usp28nf23s0_c11rs1149


ACQUITY UPLCTMBSM Performance- RT Reproducibility

Standard Deviations of 35, 94, 125 milliseconds


Alliance HPLCAnalytical Performance with isocratic conditions

AU

0.00

0.02

0.04

0.06

0.08

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50

Column XTerra RP C18, 5 um, 4.6 X 250 mm Solvent A 10 mMol Ammonium Bicarbonate pH=10Solvent B AcetonitrileFlow Rate - 2.0 mL/minColumn Temperature 45 CIsocratic 40% A + 60% BDetection - PDA in 2D mode, 301 nm at 4.8 nm bandwidthData rate: 5 pps filtering constant 0.5Injection Volume - 2 uL of 0.07 mg/mL (5/100 dilution of USP Standard)

Average TR %RSD 0.133%Average Peak Area %RSD 0.467%Average Peak Width @ 4.4% 7.2 secAverage w Peak Width 3.2 sec.Minimum Resolution 2.9Total Cycle Time 351 secSolvent Used 11.7 mL

Separation of Benzocaine, Butamben, and Tetracaine

2005 Waters CorporationAU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

Minutes0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55

Ben

zoca

ine

- 0.2

35

But

ambe

n - 0

.370

Tetra

cain

e - 0

.492

Column ACQUITY UPLCTM BEH C18 2.1 X 50 mm, 1.7 m Solvent A 10 mMol Ammonium Bicarbonate pH=10Solvent B AcetonitrileFlow Rate - 1.0 mL/minColumn Temperature 45 CIsocratic 50% A + 50% BDetection - PDA in 2D mode, 301 nm at 4.8 nm bandwidthData rate: 20 pps Filter constant: 0.1Injection Volume 2 uL of 0.07 mg/mL (5/100 dilution of USP Standard)

Average TR %RSD 0.370%

Average Peak Area %RSD 0.467%

Average 4 Sigma Peak Width 2.1 sec

Average w Peak Width 0.93 sec.

Minimum Resolution 4.2

Total Cycle Time 65 sec

Solvent Used 1.1 mL

USP Separation of Benzocaine, Butamben, and Tetracaine

ACQUITY UPLCTMAnalytical Performance with isocratic conditions


Speed, Sensitivity, ResolutionAU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50

HPLC


Changes to Original Assay Method from the USP for: Benzocaine, Butamben, and Tetracaine Hydrochloride Topical Solution

Mobile phase Prepare a mixture of acetonitrile, water containing 10 mMol ammonium bicarbonate at a pH of 10.0 (1:1) for UPLC, 4:6 for HPLC. Make adjustments if necessary (see System Suitability under Chromatography (621).

Chromatographic system (see Chromatography [621])The liquid chromatograph is equipped with a 301-nm detector, and a 4.6-mm 25-cm column that contains packing L1 (XTerra RP18, 5 um) for HPLC and a 2.1 X 50 mm 1.7 um for UPLC. The flow rate is about 1 mL per minute for UPLC and 2 mL per minute for HPLC.

Standard preparation Transfer about 140 mg of USP Benzocaine RS, accurately weighed, to a 100-mL volumetric flask with the aid of 25 mL of methanol, and swirl. Transfer about 140 J mg of USP Butamben RS, accurately weighed, to the same volumetric flask with the aid of 25 mL of water, J being the ratio of the labeled amount, in percent, of butamben to the labeled amount, in percent, of benzocaine in the Topical Solution. Transfer about 140J mg of USP Tetracaine Hydrochloride RS, accurately weighed, to the same volumetric flask with the aid of 25 mL of water, J being the ratio of the labeled amount, in percent, of tetracaine hydrochloride to the labeled amount, in percent, of benzocaine in the Topical Solution. Sonicate for about 1 minute, dilute with Diluent to volume, and mix. Further dilute this stock 5 in100 for UPLC use.

Procedure Separately inject equal volumes (about 2 L) of the Standard preparation and the Assay preparationinto the chromatograph, record the chromatograms, and measure the peak areas for the major peaks.

Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.3 for benzocaine, 0.8 for butamben, and 1.0 for tetracaine; the resolution, R, between the benzocaine peak and the butamben peak and between the butamben peak and the tetracaine peak is not less than 2; and the relative standard deviation for replicate injections is not more than 2.0% for each of the three analyte peaks.

http://www.uspnf.com/uspnf/pub/data/v28230/usp28nf23s0_alpha-18-140.xml#usp28nf23s0_c11rs113http://www.uspnf.com/uspnf/pub/data/v28230/usp28nf23s0_alpha-18-140.xml#usp28nf23s0_c11rs157http://www.uspnf.com/uspnf/pub/data/v28230/usp28nf23s0_alpha-18-1412.xml#usp28nf23s0_c11rs1149


ACQUITY UPLCTMAnalytical Performance with Gradient conditions

AU

-0.10

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

Minutes0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50

Ace

tani

lide

- 0.

176

Ace

toph

enon

e -

0.23

0

Pro

piop

heno

ne -

0.27

3

But

yrop

heno

ne -

0.31

0B

enzo

phen

one

- 0.3

22

Val

erop

heno

ne -

0.34

6

Hex

anop

heno

ne -

0.3

83

Hep

tano

phen

one

- 0.

420

Oct

anop

heno

ne -

0.4

56

Average TR %RSD 0.109%Average Peak Area %RSD 0.368%

Average 4 Sigma Peak Width 0.369 secAverage w Peak Width 0.210 secMinimum Resolution 2.15Total Cycle Time 56 secMaximum Pressure 13,500 psi

Column: 2.1 x 50 mm @ 65CMobile Phase A: 0.1% Formic Acid in WaterMobile Phase B: 0.1% Formic Acid in ACNFlow Rate: 1.3 mL/minGradient: 30% - 90% B over 0.5minutes, Curve 5Wash Solvents: Strong 50/50 Water/ACN (200L);

Weak 95/5 Water/ACN 600L)Wavelength: 240nmData Rate: 40 pts/s, Filter Constant: 0.0

6 injections9 phenones(3L, 0.1 mg/mL)30 sec. gradient


ACQUITY UPLCTMAnalytical Performance with Gradient conditions

AU

-0.10

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Minutes0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50

Ace

tam

inop

hen

- 0.2

08

2-A

ceto

mid

ophe

nol -

0.2

25

Caf

fein

e - 0

.264

Ace

tani

lide

- 0.3

06

AS

A -

0.32

3

Phe

nace

tin -

0.35

8

Sal

icyl

ic A

cid

- 0.3

80

10 Replicate Injections of a7 component Analgesic Mix

(5L, 0.1 0.2 mg/mL in water)

Column: 2.1x50mm @ 65CMobile Phase A: 0.1% Formic Acid in WaterMobile Phase B: 0.1% Formic Acid in ACNFlow Rate: 1.3 mL/minGradient: Time %A %B Curve

Initial 93 7 -0.10 80 20 40.18 65 35 60.41 93 7 6

Wash Solvents: Strong 50/50 Water/ACN, 100LWeak 98/2 Water/ACN, 500L

Wavelength: 240nmData Rate: 40 pts/s, Filter Constant: 0.0

Ave RT %RSD 0.115%Ave Area %RSD 0.133%Minimum Resolution 1.70Total Cycle Time 56 sec

Maximum Pressure 11,000 psi


ACQUITY UPLCNon-Linear Gradient Capability

Gradient Profiles on ACUITY UPLC


Utility of Non-linear gradient capability

Method Development Challenge:

Gradient elution of 9 component mix To go as fast as possible

To meet a minimum resolution requirement of 2.0

Start with generic method 30s gradient with 15s hold at final conditions. (5% to 95% ACN)

Run gradient using curves 3-9


Chromatograms of Curves 3-9A

U

0.00

0.50

AU

0.00

0.50

AU

0.00

0.50

AU

0.00

0.50

AU

0.00

0.50

AU

0.00

0.50

AU

0.00

0.50

Minutes0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75

Curve 9

Curve 8

Curve 7

Curve 6

Curve 5

Curve 4

Curve 3


Run Time Results and Minimum Resolution

Curve 3 4 5 6 7 8 9

Resolution 1.32 1.76 2.17 3.01 3.59 3.61 3.36

Elution Time of Last Peak

0.34 0.40 0.46 0.57 0.64 0.66 0.68

Shortest Run Time with required resolution is using Curve 5


ACQUITY UPLCTMSample Manager

Pressure tolerant, low volume flow-through isolation & injection valves

Fast cycle times, low carry over

2 Plate capacity, multiple formats Well plates, microtube plates, vials

Thermal control (4 - 40C)

Low dispersion column management

Sample Organizer (optional)

New Technologies - all-new injection process


Pod Style Injector

Modular design (removable Pod) permits easy access


ACQUITY UPLC Loops available

2 L5 L10 L20 L50 L


ACQUITY UPLCTMSample Manager Performance - Precision

Component Summary Area RT ReportProject Name: PITTCON_2005Reported by User: Andrew Aubin (Aubin)

1

2

3

4

5

6

7

8

9

10

Mean

Std. Dev.

% RSD

SampleName Date Acquired Inj Vol(ul) Inj Vial 3,4-MDA 2,3-MDA 3,4-MDMA 2,3-MDMA

2,3+3,4 MDA MDMA

2,3+3,4 MDA MDMA

2,3+3,4 MDA MDMA

2,3+3,4 MDA MDMA

2,3+3,4 MDA MDMA

2,3+3,4 MDA MDMA

2,3+3,4 MDA MDMA

2,3+3,4 MDA MDMA

2,3+3,4 MDA MDMA

2,3+3,4 MDA MDMA

2/14/2005 10:28:57 AM

2/14/2005 10:33:42 AM

2/14/2005 10:36:38 AM

2/14/2005 10:39:35 AM

2/14/2005 10:42:31 AM

2/14/2005 10:45:27 AM

2/14/2005 10:48:24 AM

2/14/2005 10:51:20 AM

2/14/2005 10:54:14 AM

2/14/2005 10:57:11 AM

5.00

5.00

5.00

5.00

5.00

5.00

5.00

5.00

5.00

5.00

1

1

1

1

1

1

1

1

1

1

2:D,1

2:D,1

2:D,1

2:D,1

2:D,1

2:D,1

2:D,1

2:D,1

2:D,1

2:D,1

252746

251676

252557

251382

251354

250666

252694

252331

252360

252659

252043

722

0.29

169926

169802

169395

169090

168659

169286

168439

169251

168626

168037

169051

604

0.36

235323

235335

235366

232696

234721

234564

235468

235365

234133

234904

234787

856

0.36

164552

163520

163371

162642

163114

163830

163120

163195

164507

162978

163483

636

0.39

Component Summary For Area


ACQUITY UPLCTMSample Manager Performance - Linearity

Are

a

-50000

0

50000

100000

150000

200000

250000

Amount0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

R2 = 0.9998

0.1 mg/mL Acetaminophen with 20L loop in Pressure-Assist Mode


ACQUITY UPLCTMSample Manager Performance -Carryover

10 mg/mL Human Insulin

Carryover 0.0036%

Wash Conditions: 200L Strong (6:3:1 / 0.1% phosphoric in Water:ACN:IPA) 600L Weak (0.01M HCl)


ACQUITY UPLCTMSample Manager Performance -Carryover

Carryover 0.0036%

Wash Conditions: 200L Strong (6:3:1 / 0.1% phosphoric in Water:ACN:IPA) 600L Weak (0.01M HCl)


ACQUITY UPLC Minimized CarryoverAlprazolam in Plasma

Plasma Blank

314.1>210.2

260.2>116.0

309.0>205.2

1000 ng/mL

Peak Area96015

Peak Area747309

Peak Area97963

MRM transition

Plumb


Cycle Times?

ACQUITY UPLC Injection-to-Injection Cycle TimesInjection-to-injection cycle times are calculated as the time it takes for the injector to perform all of the tasks during the injection cycle from the point of initiation by the data acquisition software until the valve turns and the injection is made. To determine this value a series of injections are monitored by a detector that is continually collecting data, even between the run times. The difference in time between corresponding peaks of each injection gives the total cycle time. Subtracting the run time from the total cycle time will yield the injection-to-injection cycle time.

Injection Interval Total Cycle Time(sec)

Run Time(sec)

Injector Cycle Time(sec)

1 64.9 36.0 28.9

2 65.1 36.0 29.1

3 65.0 36.0 29.0

4 64.1 36.0 28.1

5 63.0 36.0 27.0

6 65.5 36.0 29.5

7 67.0 36.0 31.0

8 66.9 36.0 30.9

9 65.2 36.0 29.2

Average 65.2 36.0 29.2


ACQUITY UPLCTMColumn Manager

Column Manager

Symbiotic with Sample Manager Flow path distance optimized 65C upper limit Up to 150 mm column

Pivot positioning Stacked mode

w/ optical detector Swung out mode

w/ MS detector only Column may be accessed from

either side


Attaching The Column to the System

High Pressure Finger Tight FittingsHolds >15000 psi

Patent Pending


Column Outlet

- One piece finger tight fittings work well- Cut 0.0025 Peek to length- Keep as short as possible to minimize dispersion


Column Heating

Column heating considerations Consistent temperature for RT stability Solvent viscosity is lower at higher temperatures Lower backpressures at higher temperatures

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Minutes1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80

60 C

50 C55 C

45 C40 C

35 C

30 C


ACQUITY UPLCTMColumn Manager with eCord

eCordTM Technology

eCordeCordreaderreader Column Information

Certificate of Analysis

Column Usage History Total number of samples Injections Pressure/Temperature history And much more

eCordTM


ACQUITY UPLCTMeCordTM

Columns

eCordTM


eCordColumn QC


ACQUITY UPLCTMDetectors

Faster eluting peaks require higher data rates High light throughput/transmission Faster digital filter time constants

UPLC separations require low dispersion flow cells Maintain chromatographic fidelity

Tunable UV, PDA and ELSD Options

MS Options


ACQUITY UPLCTMFlow Cell Design

Light Guided UPLCTM flow cells 10 mm pathlength, 500 nL volume Flow cell channel is the inside of a low-index Teflon AF tube Total internal reflection at walls, like optical fiber cladding

Teflon AF

water

dTeflon AF


ACQUITY UPLCTMLow dispersion UPLC optics

Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

%

0

100

%

0

100 0.67

22.92

0.78

2.09

1.30

11.208.62

2.57

3.55 4.205.615.15 7.336.49 7.93

21.6621.10

20.3117.13

14.5112.71 16.32

19.4318.26

22.23

26.01

23.15

24.40

25.17

26.51

27.34

28.7728.12

0.69 23.050.81

2.58

1.37

11.62

9.11

6.135.864.883.734.36

7.06 7.678.38 9.99

21.7321.1918.3817.3214.78

13.6512.96 16.6015.39

19.5020.40

22.30

23.30

26.01

23.5124.45

25.8425.09

26.61

27.32

27.5129.18

UV cell in line75/500/75

No UV cell

Column2.1x100

400L/min

Effect of UV Cell on MS Data


Low dispersion UPLCTM opticsExpanded Region

Time10.80 11.00 11.20 11.40 11.60 11.80 12.00 12.20

%

0

100

%

0

100

11.20

11.62

11.2810.93

No UV cell

UV cell in line

0.43mins

0.43mins


Data Acquisition RatesImpact on UV chromatography data

1 pt/s

2 pt/s

5 pt/s

10 pt/s

20 pt/s

40 pt/s

Minutes0.50 0.52 0.54 0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74


Effect of Filtering Constant Effect of Filtering Constant on Chromatographyon Chromatography

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

Minutes0.50 0.52 0.54 0.56 0.58 0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74 0.76 0.78 0.80

No Filtering0.1s0.3s0.5s1.0s

5 component Analgesic Mix on 2.1x50mm ACQUITY UPLC Column, 30s gradient


UV Spectra on ACQUITY PDA


Peak Purity and Library Match Capabilities


ACQUITY UPLCTMOptional Sample Organizer

Supports high throughput/high capacity

Expands capacity-mix or match 22 thin (microtitre) plates

8,448 samples max 15 medium height plates 8 full height (vial) plates

384 x 2mL vials

Temperature controlled

Programmable Access to any sample location

Supports Walk-up Operation


ACQUITY UPLCTMOptional FlexCart

Supports inlet mobility


System Information


Sample Syringe Diagnostic


Needle Seal - Static vs Dynamic TEST

Static Dynamic Static Dynamic


ACQUITY UPLCTMDesigned with Intent

UPLCTM enabled, laboratory rugged

Convenience AQT Qualification Small footprint Integrated waste management eCordTM Technology Intuitive Control Console Multiple plate formats High capacity sample organizer FlexCart

Performance and Reliability UPLC pressure capabilities Low dispersion fluidics Rapid, clean injections Fast detection Rugged chemistry Analytical performance Extensive onboard diagnostics Connections InsightTM

UPLCTM = a higher quality of (more) information faster


QUESTIONS?


Acknowledgements

John Morawski

Tanya Jenkins

Eric Grumbach

Robert Plumb

Jeffrey Mazzeo

ACQUITY UPLC in the Chromatography Lab: Analytical ConsiderationsACQUITY UPLCWhy Advance the Science and Technology of LC?HPLC: 7 Analgesics in 25 minutesUPLC:7 analgesics in 25 secondsWhy Change from HPLC to UPLC?Time and solvent savingsWhy Change from HPLC to UPLC?More information about your sampleWhy Change from HPLC to UPLC?Improved sensitivityUPLC method verses customers HPLC methodSpeed, Sensitivity, ResolutionHPLC TodayThe state of the scienceSmaller ParticlesThe enabler of productivityFundamental Resolution Equation At constant column lengthSmaller ParticlesThe enabler of productivitySummary of Key System Attributes(at constant column length)Influence of Particle Size on ChromatographyOptimal Linear Velocity on ACQUITY UPLCTM:Isocratic Caffeine Metabolites SeparationACQUITY UPLCTM TechnologyUltra performance by designUPLC Technology ChallengesACQUITY UPLCTMChemistry PropositionReversed-Phase Column Selectivity ChartACQUITY UPLC Column Selectivity Comparison for AnalgesicsColumn Lifetime:>8,500 InjectionsColumn LifetimeCustomer ExampleColumn LifetimeCustomer ExamplepH Stability At The ExtremesColumn Stability at pH 2.0Column Stability at pH=11.3UPLC Technology ChallengesACQUITY UPLCTM TechnologyUltra performance by designACQUITY UPLCTMBinary Solvent ManagerBSM ComponentsFrom Serial to Parallel FlowPressure EnvelopeTee/ Filter/ MixerNew Tubing & FittingsACQUITY 9/1000 SS316 TubingStandard 5/1000 inch 316 SS TuningNew ACQUITY 2/1000 316 SS TubingACQUITY UPLCTMBinary Solvent Manager Auto BlendACQUITY UPLCTM BSM Performance- RT ReproducibilityAlliance HPLCAnalytical Performance with isocratic conditions Speed, Sensitivity, ResolutionACQUITY UPLCTMAnalytical Performance with Gradient conditionsACQUITY UPLCTMAnalytical Performance with Gradient conditionsACQUITY UPLC Non-Linear Gradient CapabilityUtility of Non-linear gradient capabilityChromatograms of Curves 3-9Run Time Results and Minimum ResolutionACQUITY UPLCTMSample ManagerPod Style InjectorACQUITY UPLC Loops availableACQUITY UPLCTM Sample Manager Performance - PrecisionACQUITY UPLCTM Sample Manager Performance - LinearityACQUITY UPLCTM Sample Manager Performance -CarryoverACQUITY UPLCTM Sample Manager Performance -CarryoverACQUITY UPLC Minimized CarryoverAlprazolam in PlasmaCycle Times?ACQUITY UPLCTMColumn ManagerAttaching The Column to the SystemColumn OutletColumn HeatingACQUITY UPLCTMColumn Manager with eCordACQUITY UPLCTMeCordTMeCordColumn QCACQUITY UPLCTM DetectorsACQUITY UPLCTMFlow Cell DesignACQUITY UPLCTMLow dispersion UPLC opticsLow dispersion UPLCTM opticsExpanded RegionData Acquisition Rates Impact on UV chromatography dataEffect of Filtering Constant on ChromatographyUV Spectra on ACQUITY PDAPeak Purity and Library Match CapabilitiesACQUITY UPLCTM Optional Sample OrganizerACQUITY UPLCTMOptional FlexCartUPLC Technology ChallengesSystem InformationSample Syringe DiagnosticACQUITY UPLCTMDesigned with IntentQUESTIONS?Acknowledgements

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