Acquity I Class

ACQUITY UPLC I-Class System
©2011 Waters Corporation 1

Introducing ACQUITY UPLC I-Class
The ACQUITY UPLC I-Class

System represents the The ACQUITY UPLC I-Class
pinnacle of ultra System is the best MS
performance separations inlet. It does accomplish new
technology, built upon seven levels of analytical capabilities
years of engineering by maximizing peak capacity,
innovations fueled by advancing the impact of
customer input. chromatographic separations.

ACQUITY UPLC I-Class System
What is it?
New cells for TUV and PDA
— 500 nl/10mm
— 250 nl/10 mm (for 1mm ID columns)
Best inlet for all MS detectors
Compatible with FLR & ELSD
Column ovens
— Single or multiple column
— New APH with smaller ID tubing
Sample manager (SM-FTN or SM-FL)

— Less dispersion volume
— Improved carry over
Binary solvent manager (BSM)

— Enhanced performances, 1200bars
— New tubing with smaller ID
ACQUITY UPLC I Class
 Complex separation challenges require LC systems
that are designed to maximize the benefits of sub 2µm
particle columns.
— Has minimized dispersion to enhance MS and

UV performance
— Lowest carryover complementing MS sensitivity

and extending MS linear dynamic range
— The system’s low dispersion, faster cycle-time

allows complex separations can be accelerated
without compromising chromatographic fidelity
— Seamless method trasfer
— Robust, proven technology

particle columns.

UV performance



System Band Spreading:
Effect of Tubing ID and Length
 Large ID tubing increases the overall system volume and

contributes to bandspread
 Elongated tubing lengths contribute to bandspread
 Decreased tubing ID and length increases system
pressure
Flow Direction
Smallest
ID is best
0.0025" 0.020" 0.040"

Shortest length is best

Binary Solvent Manager
Updated Tubing Material
 Tubing in the pump flow path that

experiences high pressure swings can develop
pin holes from the stress and leak
 New MP35N lines have a smoother interior

surface and are more robust to this
phenomenon
 Better ID control with MP35N material
Conventional SS New MP35N
50x 100x
System Bandspreading:
Effect of Ferrule Fittings
Void volume in fittings causes Broad Peaks, Tailing, Fronting

and Splitting Without External Leaks
Impact Of Improved Dispersion
ACQUITY UPLC I-Class System Vendor B UHPLC System
0.002 2.00
mAU
AU
0.000 0.00
0.00 2.00 4.00 6.00 8.00 0.00 2.00 4.00 6.00 8.00
Minutes Minutes
0.40 0.50 0.60 0.70 0.80 0.40 0.50 0.60 0.70 0.80
Minutes Minutes
Resolution and sensitivity are improved

Lower Dispersion
I-Class versus Competitor Design
Competitive UHPLC System: 21μL extra column band spread
90.00
72.00
Average Peak Capacity at 5σ : 42
54.00
AU
36.00
18.00
0.00
0.07 0.14 0.21 0.28 0.35 0.42

Minutes
0.130
0.104
Average Peak Capacity at 5σ: 70
0.078
AU
0.052
0.026
0.000
0.00 0.07 0.14 0.21 0.28 0.35 0.42

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

Impact of Low dispersion
- Sensitivity
1.87x105
1.04x105

particle columns.

UV performance



Updated Tubing Material
 MP35N offers a more inert surface that avoid

adsorptions
 Smother internal surface
Conventional SS New MP35N
50x 100x
SM-FL: ‘Double-Click’
 After injection and cleaning of

the valve and sampling
mechanism there is a part of
the valve fact that is not
Volume
cleaned (Noted in RED) Detector
2
1
 By actuating the valve during Sample

Syringe
the run the material is pushed 3 6
into the sample loop and then
‘injected’ during the end of the Column
gradient 4 5
BSM
Injector

Carryover Performance:
I-Class with Xevo TQ-S
99
%
-1
0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74 0.76 0.78 0.80 0.82 0.84 0.86 0.88
99
Qality and
Consistency
of results
%
-1 Time
0.60 0.62 0.64 0.66 0.68 0.70 0.72 0.74 0.76 0.78 0.80 0.82 0.84 0.86 0.88
 >4 orders of magnitude

 Assurance that the quantitation represents only the presence of
analyte, not carryover

Carryover Performance:
I-Class with Xevo TQ-S
Compound name: Omeprazole

Correlation coefficient: r = 0.999985, r^2 = 0.999970
Calibration curve: 134101 * x + -12.3789
Response type: External Std, Area
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
1200000
1000000
800000
Response
600000
400000
200000
0 pg on column
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
 Exploit the linear dynamic range

 Ability to analyze disparate levels using the full sensitivity range
of the MS

I-Class FTN vs. I-Class FL:
Comparison Summary
 When ultimate UPLC performance
or throughput is the user’s
Omeprazole 10ng 0403 MRM of 1 Channel ES+
100 346.083 > 198.068 (Omeprazole)
7.06e7
paramount concern, position the

%
Fixed Loop Sample Manager

 Best for 1.0 mm columns
0
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 0.82 0.84 0.86 0.88
Blank1 10ng 0403 MRM of 1 Channel ES+
100 346.083 > 198.068 (Omeprazole)
4.42e3
 Great carryover performance

%
0 Time
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 0.82 0.84 0.86 0.88
18000.00
0.294
0.276
 When usability, flexibility and 17100.00
lowest possible carryover matter
0.205
most position the FTN Sample
16200.00
0.249
psi
Manager
0.103
0.308
15300.00
0.136
 Excellent dispersion 14400.00
 Runs ballistic gradients well 13500.00
 Longer cycle time 0.00 0.15 0.30

Minutes
0.45 0.60 0.75

Comparison Summary
ACQUITY I-Class FTN I-Class FL
Dwell Volume 120 µL 100 µL 95 µL
Bandspread 12 µL <9 µL <7 µL
<0.3% Full loop <1% 0.2 to 1.9 μL

<1% 0.2 to 1.9 μL
Precision <1.0% <0.5% 2 to 10 μL
<0.5% 2 to 10 μL
PLUNO 20% to 75% of loop PLUNO 20% to 75% of loop
Carryover 0.005 % 0.001 %* 0.002 %
Sample Discrimination Hydrophobic Samples None Hydrophobic Samples
Cycle Time < 15 sec w/Load Ahead <30 sec < 15 sec w/Load Ahead
User Simplicity 3 injection modes, loop changes Single injection mode 3 injection modes, loop changes

 Complex separation challenges require LC systems that are
designed to maximize the benefits of sub 2µm particle
columns.
— Has minimized dispersion to enhance MS and UV

performance
— Lowest carryover complementing MS sensitivity and

extending MS linear dynamic range


Isocratic separation comparison
- 2.1 x 50 mm column.
Instrument Method Name: SampleName: 1290 TCA 50 inj 6
60.00
0.684
0.448
A1290 low volume configuration;
0.551
48.00
0.934
21μL measured band spread
1.310
36.00
Average USP Plates = 6016
mAU
1.628
2.460
24.00
12.00
0.00
Instrument Method Name: TCA isocratic 50_50 0p8 SampleName: tca 50
0.060 ACQUITY 5μl loop;
0.444
0.550
0.685
12μL measured band spread

0.933
0.048
Average USP Plates = 7163
1.320
0.036
AU
1.646
2.485
0.024
0.012
0.000
Instrument Method Name: TCA isocratic 50_50 0p8 SampleName: tca 50
0.10
ACQUITY I-Class 2μl loop;
0.501
0.636
0.415
0.08
5μL band spread
0.891
0.06
Average USP Plates = 11,356
1.262
AU
1.564
0.04
2.373
0.02
0.00
0.00 0.40 0.80 1.20 1.60 2.00 2.40 2.80
Minutes

Fastest Possible Scenario (1 mm Column)
ACQUITY vs ACQUITY IClass
Instrument Method Name: 30sec_10_95p600mL_90C ACQUITY
0.457
0.483
0.110
ACQUITY UPLC
0.088
0.351
0.066
Average peak capacity
AU
0.419
0.158
0.044 at 5σ = 57
0.503
0.022 0.205
0.000
Instrument Method Name: 20sec_NO hold 10_95_p904mL_90C
0.110
ACQUITY UPLC – I CLASS
0.261
0.170
0.241
0.088
0.098
0.213
0.117
Average peak capacity

0.066
0.276
AU
0.044 at 5σ = 70
0.022
0.000
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70
Minutes
 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

columns.

performance



UPLC H-Class to UPLC I-Class
Method Transfer Example
System Volume Not Adjusted
H-Class
I-Class
Adjusted System Volume
H-Class
I-Class

Transferring Methods ACQUITY UPLC
to ACQUITY UPLC I-Class
1.00 Peaks Resolution ACQUITY UPLC

2-3 3.54
AU
0.50
3-4 4.56
7-8 2.02
0.00
1.00
Peaks Resolution ACQUITY UPLC I-Class – Same Conditions
2-3 3.27
AU
0.50 3-4 5.20

7-8 2.37
0.00
1.00 Peaks Resolution ACQUITY UPLC I-Class – Optimized Transfer*

2-3 3.61
3-4 4.80
AU
0.50
7-8 2.13
0.00
0.00 0.50 1.00 1.50 2.00

Minutes
*Compensated for System Volume (10µL) and Temperature Differences (8°C)

columns.

performance


— Robust, proven technology!

ACQUITY UPLC I-Class
Conclusion
 I-Class incorporates key features such as:

— Minimized dispersion
— Lowest carryover
— Faster cycle-time
 ACQUITY UPLC I-Class is a system designed to:

— Provide ultimate performance to the Bioanalysts
— Be the best MS inlet
— Achieve the maximum productivity
— Meet the constantly evolving analytical demands

APPENDIX

ACQUITY UPLC I-Class
Key Product Compatibility
 ACQUITY UPLC I-Class is fully compatible with:

— All Waters’ current high-end MS detectors
— Empower 1154, Empower 2154 and Empower 3
— UNIFI version 1.50
 ACQUITY UPLC I-Class is compatible with:

— AB/Sciex Analyst version 1.4.8/1.50 with Driver
Pack v1.50
— Regulated users cannot move immediately
 ACQUITY UPLC I-Class is not compatible with:

— Agilent
— Dionex
— Thermo
o ACQUITY UPLC should continue to be
positioned

New Binary Solvent Manager
Features
 Binary Solvent Blending

— Also known as high pressure mixing, multi-pump design
— Much less system volume than low pressure mixing design
— Can manage very short (< 1min) ballistic gradients
 Four solvent capability
— Solvents A1 or A2 and B1 or B2
 Flow rate range
— From 0.01 ml/mn to 2 ml/mn
 Total system volume
— Less than 95 µl (FL) or 100 µl (FTN) including a 50 µl mixer
 Optional 100µl and 425µl mixers

Comparison Summary
ACQUITY I-Class FTN I-Class FL
Dwell Volume 120 µL 100 µL 95 µL
Bandspread 12 µL <9 µL <7 µL
Carryover 0.005 % 0.001 % 0.002 %
<0.3% Full loop <1% 0.2 to 1.9 μL <0.3% Full loop

Precision
<1.0% PLUNO <0.5% 2 to 10 μL <1.0% PLUNO
< 15 sec < 15 sec

Cycle Time <30 sec
(with load ahead) (with load ahead)
3 injection modes Single injection 3 injection modes

User Simplicity
loop changes mode loop changes
 Sample organizer available with both configurations

— 18 shelves

Comparison Summary
 When ultimate UPLC performance
or throughput is the user’s
Omeprazole 10ng 0403 MRM of 1 Channel ES+
100 346.083 > 198.068 (Omeprazole)
7.06e7
paramount concern, position the

%
Fixed Loop Sample Manager

 Best for 1.0 mm columns
0
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 0.82 0.84 0.86 0.88
Blank1 10ng 0403 MRM of 1 Channel ES+
100 346.083 > 198.068 (Omeprazole)
4.42e3
 Great carryover performance

%
0 Time
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 0.82 0.84 0.86 0.88
18000.00
0.294
0.276
 When usability, flexibility and 17100.00
lowest possible carryover matter
0.205
most position the FTN Sample
16200.00
0.249
psi
Manager
0.103
0.308
15300.00
0.136
 Excellent dispersion 14400.00
 Runs ballistic gradients well 13500.00
 Longer cycle time 0.00 0.15 0.30

Minutes
0.45 0.60 0.75

Column Management
I-Class CH-A I-Class CM-A
Column Capacity Single column Up to 2 columns

Max. 150mm length x 1.0 or 4.6 mm ID Max. 150mm length x 1.0 or 4.6 mm ID
Dimensions
w/ prefilter or guard column in-line w/ prefilter or guard column in-line
Temperature Range 20°C to 90 °C 4 °C to 90 °C
independent control of each column with
Solvent Conditioning Active Pre-Heating (APH)
Active Pre-Heating (APH)
Access to MS inlet Door slides across Inlet/Outlet configurable Left or Right
Column Fittings 18 K PSI- New design 18 K PSI- New design
Advanced Functionality None Column selection or 2D
High Pressure Valve TWO valves - 18 K PSI, Dual 9 port valve
None
Configuration 2-column selector
TWO valves - 18 K PSI, Dual 2 position
2D Functionality None
column selector
 Column management options equivalent to H-Class, lower

dispersion, higher pressure, optimized fittings
 Maximum 2 columns and 1 Column Manager

High Temp Column Heater
With New APH
 Supports one UPLC column with

guard-column
 From ambiant +5°C to 90°C
 New 0.003” ID tubing (APH)
 Robust 18K psi fitting
 Excellent method transfer
between I-Class systems
 CH-30A is compatible where
HPLC columns must be
supported
— H-Class performance only

Column Manager With New APH
 Two (2) Columns plumbed right or

left only
 From 4°C to 90°C
 New 0.003” ID tubing (APH)
 Robust 18 K PSI fitting
 Excellent Method transfer between
I-Class systems
 2D Technology supported
(independent control of HP valves)

Evaluating Dispersion
- Definition of Bandspreading
w
σt =
5
σv = F ⋅σt
Peak width at 4.4% of the height of the peak is 5-sigma

(5 standard deviations)
Instrument Contribution to
Bandspread/Extra-Column Effects
σ v2,total = σ v2,injector + σ v2, precolumn + σ v2,column + σ v2, postcolumn + σ v2,det ector + τ det
2
ector ⋅ F 2
Injection Tubing Column Tubing Band- Time-based

volume between volume between spreading Band-
+ injector + column inside the spreading
injector and frits and detector
in the
band- column detector cell
spreading + Detector
+ tubing (Sampling
frits + Rate; Time
frits Constant)
 Engineering developments have specifically improved dispersion

— Injector, injection volume, fittings, flow path, sealing surfaces
— Reduced tubing volumes
— Improved flow cell dispersion
— Higher pressure/extended flow rate range
Two NEW Binary Solvent Manager
ACQUITY UPLC I-Class BSM
 High pressure Binary mixing

— 4 solvents
 New seals, check valves and

intelligent Intake Valves (i2V)
 Extended automatic, compressibility

compensation
 New vent valve for convenience even

at higher pressures
 New higher pressure mixers

— 50 µL default, 100 µL and 380 µL

Two NEW Sample Managers
Fixed Loop Flow Through Needle
 New EverFlow inject valve to enable  New EverFlow inject valve design to
higher pressures enable higher pressures
 H-Class chassis and robust rotary  H-Class chassis and robust rotary
sample tray/plate mechanism sample tray/plate mechanism
 Compatible with newest Sample

 Compatible with newest Sample
Organizer (18 shelves)
Organizer (18 shelves)
 New low dispersion fittings, shorter

sample path for the FL (10 µL)  New low dispersion fittings, lower
— More robust sample transfer dispersion needle seal for the FTN
 Low dispersion 1, 2, 5 and 10 µL loop  Optional, conventional extension

design loops
— Conventional 20, 100 and 250 µL available

Acquity I Class

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ACQUITY UPLC I-Class System

©2011 Waters Corporation 1

The ACQUITY UPLC I-Class

©2011 Waters Corporation 2

Sample manager (SM-FTN or SM-FL)

Binary solvent manager (BSM)

— Has minimized dispersion to enhance MS and

— Lowest carryover complementing MS sensitivity

— The system’s low dispersion, faster cycle-time

— Seamless method trasfer

— Robust, proven technology

©2011 Waters Corporation 4

— Has minimized dispersion to enhance MS and

— Lowest carryover complementing MS sensitivity

— The system’s low dispersion, faster cycle-time

— Seamless method trasfer

— Robust, proven technology

©2011 Waters Corporation 5

 Large ID tubing increases the overall system volume and

0.0025" 0.020" 0.040"

©2011 Waters Corporation 6

 Tubing in the pump flow path that

 New MP35N lines have a smoother interior

 Better ID control with MP35N material

Conventional SS New MP35N

Void volume in fittings causes Broad Peaks, Tailing, Fronting

Resolution and sensitivity are improved

0.07 0.14 0.21 0.28 0.35 0.42

0.00 0.07 0.14 0.21 0.28 0.35 0.42

©2011 Waters Corporation 10

©2011 Waters Corporation 11

— Has minimized dispersion to enhance MS and

— Lowest carryover complementing MS sensitivity

— The system’s low dispersion, faster cycle-time

— Seamless method trasfer

— Robust, proven technology

©2011 Waters Corporation 12

 MP35N offers a more inert surface that avoid

 Smother internal surface

Conventional SS New MP35N

 After injection and cleaning of

 By actuating the valve during Sample

©2011 Waters Corporation 14

 >4 orders of magnitude

©2011 Waters Corporation 15

Compound name: Omeprazole

 Exploit the linear dynamic range

©2011 Waters Corporation 16

paramount concern, position the

Fixed Loop Sample Manager

 Great carryover performance

lowest possible carryover matter

 Runs ballistic gradients well 13500.00

 Longer cycle time 0.00 0.15 0.30

©2011 Waters Corporation 17

ACQUITY I-Class FTN I-Class FL

Dwell Volume 120 µL 100 µL 95 µL

Bandspread 12 µL <9 µL <7 µL

<0.3% Full loop <1% 0.2 to 1.9 μL

Carryover 0.005 % 0.001 %* 0.002 %

Sample Discrimination Hydrophobic Samples None Hydrophobic Samples

©2011 Waters Corporation 18

— Has minimized dispersion to enhance MS and UV

— Lowest carryover complementing MS sensitivity and