acquity uplc i-class system - waters corporation...acquity uplc i-class system acquity uplc h-class...
TRANSCRIPT
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
©2011 Waters Corporation 2
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
©2011 Waters Corporation 3
— 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
©2011 Waters Corporation 4
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
©2011 Waters Corporation 5
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
©2011 Waters Corporation 6
— 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
©2011 Waters Corporation 7
*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
©2011 Waters Corporation 8
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
©2011 Waters Corporation 9
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
©2011 Waters Corporation 10
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
©2011 Waters Corporation 11
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
©2011 Waters Corporation 12
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
©2011 Waters Corporation 13
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
©2011 Waters Corporation 14
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
©2011 Waters Corporation 15
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
©2011 Waters Corporation 16
1.71 90 10
Gradient Hold Insert for Gradient Hold Insert for ACQUITY UPLC IACQUITY UPLC I--ClassClassACQUITY UPLC IACQUITY UPLC I--ClassClass
©2011 Waters Corporation 17
System System Volume Difference Volume Difference Not AdjustedNot Adjusted
1 2
0.28
System System Volume Difference Volume Difference Not AdjustedNot Adjusted
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
©2011 Waters Corporation 18
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
©2011 Waters Corporation 19
2.10 68 32
2.11 90 10
System System Volume Difference Volume Difference AdjustedAdjusted
1 2
0.28
yy jj
ACQUITY UPLC H-Class
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
©2011 Waters Corporation 20
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
©2011 Waters Corporation 21
System System Volume Difference Volume Difference Not AdjustedNot Adjusted
10.80
yy jj
ACQUITY UPLC H-Class
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
©2011 Waters Corporation 22
0.00
Minutes0.00 1.50 3.00 4.50 6.00 7.50
System System Volume Difference Volume Difference AdjustedAdjusted
10.80
System System Volume Difference Volume Difference AdjustedAdjusted
ACQUITY UPLC H-Class
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
©2011 Waters Corporation 23
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
©2011 Waters Corporation 24
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
©2011 Waters Corporation 25
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
©2011 Waters Corporation 26
©2011 Waters Corporation 27