ACQUITY UPLC I-Class System - Waters Corporation...ACQUITY UPLC I-Class System ACQUITY UPLC H-Class Gradient Table BEFORE NOW Time (min) %A %B Initial 90 10 1.70 68 32 1.71 90 10 Gradient

ACQUITY UPLC I-Class SystemPinnacle of UPLC Performance

Why introducing ACQUITY UPLC I-Class to the UPLC Family?to the UPLC Family?

©2011 Waters Corporation 1

Henrik Andersen

ACQUITY UPLC H-ClassACQUITY UPLC


Instrument Contribution to Extra-Column Effects

22det

2,det

2,

2,

2,

2,

2, Fectorectorvpostcolumnvcolumnvprecolumnvinjectorvtotalv ⋅+++++= τσσσσσσ

Injectionvolume

+injectorband-

Tubingbetweeninjector

and column

Columnvolume

+frits

Tubingbetweencolumn

and detector

Band-spreadinginside thedetector

cell

Time-basedBand-

spreadingin the

Detectorband-spreading

column detector cell+

tubing

Detector(SamplingRate; TimeConstant)

Engineering developments have specifically improved dispersion I j fi i fl h li f— Injector, fittings, flow path, sealing surfaces

— Reduced tubing volumes

— Improved flow cell dispersion


— Higher pressure/backpressure resistance

System Band Spreading:Effect of Tubing ID and Length

Tubing with a large ID increases the overall system volume and contributes to band spreading

g g

Excessive tubing lengths contribute to band spreadingDecreasing tubing ID and length increases system pressure

Smallest ID is best

Flow Direction

0.020"0.0025" 0.040"

Shortest length is best


Extra Colum VolumeContribution Of Tubing ID

Tubing ID Volume/foot Volume/100 cm

0 040’’ 250 µl 824 µl

g

0.040 250 µl 824 µl

0.020’’ 62 µl 206 µl

0.009’’ 12 µl 42 µl

0.005’’ 3.5 µl 12.5 µl

0.004’’ 2.5 µl 8.2 µl

0.003’’ 1.4 µl 4.6 µlµ µ

How does it translate to chromatographic performances?— Peak capacity is increased (with decreasing ID)Peak capacity is increased (with decreasing ID)

— Peak shape is improved

— Overal chromatographic performances are strongly impacted

Higher pressure limit is just a tool a technology enabler


Higher pressure limit is just a tool, a technology enabler

ACQUITY UPLC I-Class SystemWhat is it?

New cells for TUV and PDA— 500 nl/10 mm

— 250 nl/10 mm (for 1mm ID columns)

Best inlet for all MS detectors

Compatible with FLR & ELSD

Column ovens— Single or multiple column

N APH ith ll ID t bi

Sample manager (SM-FTN or SM-FL)

— Less dispersion volume

— New APH with smaller ID tubing

Binary solvent manager (BSM)

Less dispersion volume

— Improved carry over


— 1200 bars 18000 PSI

— New tubing with smaller ID

I-Class FTN vs. I-Class FL:Comparison Summaryp y

ACQUITY I-Class FTN I-Class FL

Dwell Volume* 120 µL 100 µL 95 µL

Bandspread* 12 µL <9 µL <7 µL Bandspread* 12 µL <9 µL <7 µL

Carryover 0.005 % 0.001 % 0.002 %

Precision <0.3% Full loop <1.0% PLUNO

<1% 0.2 to 1.9 μL<0.5% 2 to 10 μL

<0.3% Full loop <1.0% PLUNO

Cycle Time < 15 sec(with load ahead) <30 sec < 15 sec

(with load ahead)

User Simplicity 3 injection modesloop changes

Single injection mode

3 injection modesloop changesloop changes mode loop changes

Sample organizer available with both configurations— 18 shelves


*Measured with a complete system – Measurement on module only doesn’t make sense

NEW Column Heater

Supports one UPLC column ith g a d col mnwith guard-column

From ambiant +5°C to 90°CNew 0.003” ID tubing (APH)g ( )

Robust 18K psi fitting

Excellent method transfer between UPLC systemsbetween UPLC systems


Column Managerg

Two (2) Columns plumbed right or l ft lleft only

From 4°C to 90°CNew 0.003” ID tubing (APH)New 0.003 ID tubing (APH)

Robust 18 K PSI fitting

Excellent Method transfer between UPLC systems

Also available for 2D Technology


ACQUITY UPLC I-Class SystemOptical Detection (TUV & PDA)p ( )

500nL flow cell has been optimized for reduced dispersion— Flow cell body is the sameFlow cell body is the same

— Inlet and outlet tubing i.d. has been reduced

o .0025” ID inlet PEEK tubing

0035” ID outlet PEEK tubingo .0035 ID outlet PEEK tubing

250nL / 183 µm version for 1mm chemistry

ELSD & FLR are compatible with I-Class


Lower Dispersion I-Class versus Competitor Designp g

Competitive UHPLC System: 21μL extra column band spread

90.00

AU

36.00

54.00

72.00

Average Peak Capacity at 5σ : 42

0.00

18.00

Minutes0.07 0.14 0.21 0.28 0.35 0.42

AU

0.078

0.104

0.130

Average Peak Capacity at 5σ: 70

0.000

0.026

0.052

0.00 0.07 0.14 0.21 0.28 0.35 0.42


Minutes

ACQUITY UPLC I-Class: 5μL extra column band spread

Fastest Possible Scenario (1 mm Column)ACQUITY vs ACQUITY IClassQ Q

Instrument Method Name: 30sec_10_95p600mL_90C ACQUITY

0.45

7

0.48

3

0.088

0.110ACQUITY UPLC

0.15

8

0.20

5

0.35

1

0.41

9

0.50

3

AU

0.022

0.044

0.066 Average peak capacity at 5σ = 57

0.000

Instrument Method Name: 20sec_NO hold 10_95_p904mL_90C

0.17

0

0.24

1 0.26

1

0.088

0.110ACQUITY UPLC – I CLASS SM

0.09

8

0.11

7

0.21

3

0.27

6

AU

0.022

0.044

0.066 Average peak capacity at 5σ = 70

0.000

Minutes0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70

Ballistic gradient on 1mm x 50mm BEH C18


Due to the 15k psi limit, with the ACQUITY, the flow rate is 0.6 ml/mn

With the ACQUITY I-Class the flow rate is 0.9 ml/mn

Comparison Of Isocratic Separations2.1 mm Columns – 0.8 ml/mn

Competitive design;21μL measured band spread

Instrument Method Name: SampleName: TCA 50 inj 6

0.44

8

0.55

1 0.68

4

0.93

4

0

48.00

60.00

21μL measured band spreadAverage USP Plates = 6016

0

1.31

1.62

8

2.46

0mAU

0.00

12.00

24.00

36.00

I t t M th d N TCA i ti 50 50 0 8 S l N t 50

ACQUITY; 12μL measured band spreadAverage USP Plates = 7163

Instrument Method Name: TCA isocratic 50_50 0p8 SampleName: tca 50

0.44

4

0.55

0

0.68

5

0.93

3

1.32

0

.646

85

AU

0 024

0.036

0.048

0.060

ACQUITY I-Class FL; 1

2.48

0.000

0.012

0.024

Instrument Method Name: TCA isocratic 50_50 0p8 SampleName: tca 50

5 501

360 08

0.10Q ;

5μL measured band spreadAverage USP Plates = 11,356

0.41

5 0.5

0.63

0.89

1

1.26

2

1.56

4

2.37

3

AU

0.02

0.04

0.06

0.08


0.00

Minutes0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80

UPLC H-Class to UPLC I-ClassMethod Transfer Examplep

ACQUITY UPLCH-ClassNot Retention Time Adjusted

ACQUITY UPLC I-ClassIncreasing regulatory demands

ACQUITY UPLC H-ClassRetention Time Adjusted

ACQUITY UPLC I-Class


Original Barbiturates Separation Original Barbiturates Separation ACQUITY UPLC HACQUITY UPLC H--Class SystemClass SystemACQUITY UPLC HACQUITY UPLC H Class SystemClass System

1 – Allobarbital2 – Phenobarbital3 – Butalbarbital4 – Mephobarbital5 – Amobarbital6 - Secobarbital


UPLC Method on ACQUITY UPLC HUPLC Method on ACQUITY UPLC H--Class SystemClass SystemClass SystemClass System

Sample: Barbiturates Mix @ 20μM

Column: 2.1 x 50mm ACQUITY UPLC BEH C18, 1.7μmColumn: 2.1 x 50mm ACQUITY UPLC BEH C18, 1.7μm

Injection Volume: 1.5μL

Temperature: 45°C

Fl R t 0 64 L/ iFlow Rate: 0.64mL/min

Mobile Phase A: 10mM Ammonium Bicarbonate Buffer pH 10.0

Mobile Phase B: Acetonitrile

Gradient: Please refer to the table below

Detection Wavelength: 245nm

Time (min) %A %BTime (min) %A %B

Initial 90 10

1.70 68 32


1.71 90 10

Gradient Hold Insert for Gradient Hold Insert for ACQUITY UPLC IACQUITY UPLC I--ClassClassACQUITY UPLC IACQUITY UPLC I--ClassClass


System System Volume Difference Volume Difference Not AdjustedNot Adjusted

1 2

0.28


ACQUITY UPLC H-Class

3

4

5

6

AU

007

0.14

0.21

0.00

0.07

ACQUITY UPLC I Cl

1

2

3

4

5

6

U

0.21

0.28ACQUITY UPLC I-Class

AU

000

0.07

0.14


0.00

Minutes0.00 0.30 0.60 0.90 1.20 1.50

Adjusted Gradient Table forAdjusted Gradient Table forACQUITY UPLC IACQUITY UPLC I--Class SystemClass System

ACQUITY UPLC H-Class Gradient Table

ACQUITY UPLC IACQUITY UPLC I--Class SystemClass System

BEFORE NOW

Time (min) %A %B

Initial 90 10

1.70 68 32

Q

1.71 90 10

Gradient Hold insert of 262uL at 640uL/min Flow Rate translates to

ACQUITY UPLC I-Class Gradient Table

Flow Rate translates to 0.40 minutes

Time (min) %A %B

Initial 90 10

0.40 90 10

2 10 68 32


2.10 68 32

2.11 90 10

System System Volume Difference Volume Difference AdjustedAdjusted

1 2

0.28

yy jj


3

4

5

6

AU

007

0.14

0.21

0.00

0.07

ACQUITY UPLC I-Class

1 2

3 4

5

6

U

0.21

0.28ACQUITY UPLC I-Class

AU

000

0.07

0.14


0.00

Minutes0.00 0.30 0.60 0.90 1.20 1.50

Explosives Mix Separation run on Explosives Mix Separation run on ACQUITY UPLC HACQUITY UPLC H--Class SystemClass SystemOverlay of 6 Replicate InjectionsOverlay of 6 Replicate InjectionsOverlay of 6 Replicate InjectionsOverlay of 6 Replicate Injections

Peak 1 HMX Peak 2 RDX Peak 3 TNB Peak 4 1,3-DNB Peak 5 NB Peak 6 Tetryl Peak 7 TNTPeak 8 2-Amino-4 6-DNTPeak 8 2 Amino 4,6 DNTPeak 9 4-Amino-2,6-DNTPeak 10 2,4-DNT Peak 11 2,6-DNTPeak 12 2-NTP k 13 4 NTPeak 13 4-NTPeak 14 3-NT



10.80

yy jj


2

3

4

6 7 2 14

AU 0.40

0.60

5 6 7 8 9

10 11 12 13

0.00

0.20

1

2

3

U

0.60

0.80 ACQUITY UPLC I-ClassLesser System Volume, hence, shorter peak retention times

4

5 6 7 8 9

10 11 12 13

14

AU

000

0.20

0.40


0.00

Minutes0.00 1.50 3.00 4.50 6.00 7.50


10.80



2

3

4

5 6 7 8 9 2 14

AU 0.40

0.60

5 6 7 8 9

10 11 12 13

0.00

0.20

080 1

2

3

U 040

0.60

0.80 ACQUITY UPLC I-Class4

5 6 7 8 9

10 11 12 13

14

A

0.00

0.20

0.40


Minutes0.00 1.50 3.00 4.50 6.00 7.50

Original ACQUITY UPLC Separationg Q p

Column: 2 1 x 50mm BEH C18 1 7µmColumn: 2.1 x 50mm BEH C18 1.7µmSample: Beta-BlockersInj Vol: 1µLTemp: 60°CFlow Rate: 750µL/minEluent A: 10mM Ammonium Bicarbonate pH10Eluent B: Acetonitrile

AU

0.70

Gradient: 5% to 60% B in 2 minutesDetection: 230nm 20Hz Normal

0.00

Minutes0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00


Minutes

Transferring Methods ACQUITY UPLC to ACQUITY UPLC I-Class

1.00

Q

Peaks Resolution

2-3 3.54ACQUITY UPLC

AU

0.00

0.50 3-4 4.56

7-8 2.02

AU

0.50

1.00 Peaks Resolution

2-3 3.27

3-4 5.20

7 8 2 37

ACQUITY UPLC I-Class – Same Conditions

0.00

1.00

7-8 2.37

Peaks Resolution

2 3 3 61ACQUITY UPLC I-Class – Optimized Transfer*

AU

0.00

0.50

2-3 3.61

3-4 4.80

7-8 2.13


Minutes0.00 0.50 1.00 1.50 2.00

*Compensated for System Volume (20µL) and Temperature Differences (8°C)

Transferring Methods ACQUITY UPLC to ACQUITY UPLC I-Class

Important things to remember— Passive versus active heating

Q

Passive versus active heating

— Small system volume differences

— System dispersion differences



Documents

ACQUITY UPLC I-Class System - Waters Corporation...ACQUITY UPLC I-Class System ACQUITY UPLC H-Class Gradient Table BEFORE NOW Time (min) %A %B Initial 90 10 1.70 68 32 1.71 90 10 Gradient