Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 1
Exploiting New Agilent LC Technologies:
Refined Solutions 2D-LC and SFC
New flexible and user-friendly solutions for the most complex and difficult samples
Tony Brand, Ph.D.Senior Field ScientistSeptember 14th, 2012
1
The Van Deemter Plot: Advantages of Smaller Particles at High Linear Velocities
HETP
(cm
/pla
te)
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
0.0040
0.0045
5.0 m
3.5 m
1.8 m
2mL/min 5mL/min
Interstitial linear velocity (ue- cm/sec)0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Unlike 5µm and 3.5µm columns, 1.8µm columns maintain higher efficiency at higher linear flows, which allows for significant gains in analysis speed. Typical Speed Gain: 5 – 10x
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 2
1998* 1.5 µm*(non-porous) 30,000
History of Commercial HPLC Particle Development
2003 1.8 µm 32,5002007/2008 2.7 µm (porous shell) 32,000**
Year(s) of Acceptance
Particle Size Most PopularNominal Size
Plates / 15cm(Approximate)
1950’s 100µm 200
1967 50 µm (porous shell) 1,000
1972 10 µm 6,000
GlassBead
12,0001992 3-3.5 µm 22,0001985 5 µm
2000 2.5 µm 25,000
* non-porous silica or resins ** 90-120 A pore
Irregularly-Shaped
Page 3January 2012
Liquid Chromatography
Mobile phase (flowing)
Mobile phase (flowing)
Column
MS
SolventsH20
ACN or MeOH
DegasserFlat membrane
PumpBinary
AutosamplerLoop Injector shown
UV Detector Mass Spec
Mix
ing
Stationary Phasecoated on ultra-high
purity silica
Steric Protection
Ultra-PureSilica
Selectivity Main Chain
Si OR
Si O Si
Si OR
PG R’
CH3CH
CH2 CH3
CH2 CH3CH
CH3A Stationary phase example: Zorbax Bonus-RP
Triple EndcappedSilanols for pH stability and good peak shape
1.7µm
0.5µm
0.5µm
Superficially PorousParticles
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 3
1995: The Agilent 1100: The LegacyStackable, comprehensive, flexible configurationsFront access for easy user maintenanceEffective leak managementErgonomic tubing organization for low delay volume and
bandspreadingSingle LAN/PC/HUB cable connectionFlexible handheld or PC/network control strategiesModular intelligence via the CAN networkEMF, Early Maintenance FeedbackColumn tracking via RF Tag
1290 Infinity
0
200
400
600
800
1000
1200
0 1 2 3 4 5
The Evolution of Power Range
bar
ml/min
Agilent 1100/1200, 400 bar 1995-2009 Standard LC
Agilent 1260, 600 bar 2009-2010 the New Standard
Page 6
Higher ResolutionHigher SpeedHigher Peak capacity/timeHigher SensitivityMethod TransferMore viscous solvents Lower Temperature
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 4
1290 Infinity Binary Pump- How it looks like inside
September 21, 2012Page 7
Channel A
ChannelBPurge
Valve
Solvent selectionvalve
Multi-layerHeat exchanger
Jet Weaver
Degasser
High ResolutionPump drives
1290 Infinity LC Binary Pump Technology- Maximum Power and Robustness
Page 8
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 5
F = 1.20ml/minT = 40°CAnalysis Time = 11minSolvent Cons. = 13.2ml
High Resolution:4.6mm x 150mm 5.0µm
min0 2 4 6 8 10 12
F = 4.80ml/minT = 40°CAnalysis Time = 1.05minSolvent Cons. = 5.1ml
High Speed:4.6mm x 50mm 5.0µm
min0 0.2 0.4 0.6 0.8 1
F = 1.00ml/minT = 40°CAnalysis Time = 1.1minSolvent Cons. = 1.1ml
High Speed & Resolution:2.1mm x 50mm 1.8µm
min0.2 0.4 0.6 0.8 10
The LC method and the Need for Speed….. Smaller particle columns, Higher Linear Flow Rates, Higher temperatures, Less Consumption => Lower Cost
Page 9
Max Speed at T = 95oC2.1mm x 50mm 1.8um
F = 2.40ml/minT = 95°CAnalysis Time: 0.4minSolvent Cons. = 1.0ml
PWHH = 197msec
min0.2 0.4 0.6 0.8 10
> 20x faster !
95%
5%
0 min, 5% Acetonitrile, 95% H2O… gradient ..12 min, 95% Acetonitrile, 5% H2O
January 2012
Agilent LC Valves: A New Era
1290 Infinity TCC(G1316C)
1290 Infinity Flexible Cube(G4227A)
1290 Infinity Valve Drive(G1170A)
2pos/ 6port2pos/10port4pos/10port6pos/14port8pos/ 9port
12pos/13port
StandardBio-inert
Micro-flow
600 bar1200 bar
Columns switching Solvent selection Fractionation Matrix-stripping Detector selection And........
Agilent QuickChange Valve Heads
Universal Valve Drive
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 6
Agilent QuickChange Valve heads
Kit PN Valve PN Description(as on valve label) Pressure [bar] Ports Positions
G4230A 5067-4107 8ps/9prt 600 bar 600 9 8
G4230B 5067-4121 8ps/9prt 1200 bar 1200 9 8
G4231A 5067-4137 2ps/6prt 600 bar 600 6 2
G4231B 5067-4117 2ps/6prt 1200 bar 1200 6 2
G4232A 5067-4144 2ps/10prt 600 bar 600 10 2
G4232B 5067-4118 2ps/10prt 1200 bar 1200 10 2
G4234A 5067-4146 6ps/14prt 600 bar 600 14 6
G4234B 5067-4142 6ps/14prt 1200 bar 1200 14 6
G4235A 5067-4147 12ps/13prt 200 bar 210 13 12
G5631A 5067-4148 2ps/6prt 600 bar 600 6 2
G5639A 5067-4134 4ps/10prt 600bar 600 10 4
n.a. 5067-4151 6ps/7prt 1200 bar CCV 1200 7 6
2ps-6prtvalve
2pos-10portvalve
6pos-14portvalve
8ps-9prtvalve
6ps-7prtvalve
In gray: not yet orderable
8-Position / 9-port valve headApplication example: Automated column selection
Page 12
• 2x 1290 Infinity Thermostatted column compartment
• 2x 8-pos/9-port valve head
• Up to 8 columns• Bypass or waste
positions can be configured
• For automated column selection
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 7
6-Position / 14-port valve headApplication example: Automated column selection
April 201013
• 1x 1290 Infinity Thermostatted column compartment
• 1x 6-pos/14-port valve head
• Up to 6 columns (4 columns recommended because of available space)
• For automated column selection
2-Position / 6-port valve headApplication example: Online sample enrichment
April 201014
• 1x 1290 Infinity Thermostatted column compartment
• 1x 2-pos/6-port valve head
• One enrichment and one analytical column
• For online sample enrichment
• Two pumps recommended
• Other applications like sample stripping, etc.
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 8
2-Position / 10-port valve headApplication example: Alternating column regeneration (ACR)
April 201015
• 1x 1290 Infinity Thermostatted column compartment
• 1x 2-pos/10-port valve head
• Two identical, analytical columns
• For alternating column regeneration
• Two pumps required!
1290 Infinity Therm. Column CompartmentNext level in usability:
New prolonged HX-carrierwith 3rd hand (not shown)
New valve drive and valve head
New capillary guide
New leak funnel
modified lowdisp HX
New door-open sensor
RFID-tag, stores type, pressure range, valve switches
NewThermalInsulation
New 2PS/6PT, 2PT/10PT and 9PT/8PS Quick-Change high pressure valves
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 9
2D-LC operation modes
Heart-cutting LC-LC
Time- or Peak-triggered
levelKnown samples/improving confidence: pharma, meth.-dev..
2D-LC: Injecting the efluent or a part of the effluent of one column to a second column, ideally with orthogonal separation behavior.Purpose: increase total separation power.
Comprehensive LCxLCStandard
orTime-/Peak-triggered
level
1D chromatogram
2D sampling
2D gradient
complex/unknown samples: bio-pharma, food, polymers....
18
Only peaks eluted from the 1st dimension column will be injected to the second column. Typically a peak from the first dimension will be sampled as a whole and a gradient with a longer run time than the collection time will be used. Also longer columns with higher seperation efficiency are being used in as 2nd dimension column.
2D-LC - Heart-cutting 2D-LC
LC1
LC2
Care must be taken if peaks are eluting from the first dimension column when a gradient on the second dimension is still running –this peak will be lost.
LC 1Pump
LC 2Pump
LC 1Autosampler
LC 2Detector
Waste
LC 1Detector
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 10
19
Only peaks eluted from the 1st dimension column will be injected to the second column. Typically a peak from the first dimension will be sampled as a whole and a gradient with a longer run time than the collection time will be used. Also longer columns with higher seperation efficiency are being used in as 2nd dimension column.
2D-LC - Heart-cutting 2D-LC
LC1
LC2
If peaks are eluting from the first dimension column when gradient on the second dimension is still running – this peak will be lost.
LC 1Pump
LC 2Pump
LC 1Autosampler
LC 2Detector
Waste
LC 1Detector
2D-LC - Comprehensive 2D-LC
20
The complete effluent of the 1st dimension column is injected onto the 2nd dimension column with very fast gradients. A peak of the first dimension should be sampled at least 3 to 4 times. The run time of the 2nd dimension method matches the collection time of the 1st dimension effluent. Finally, the peaks will be re-constructed.
LC1
LC2
LC1
LC2
1st peak from 1st dimension
2nd peak from 1st dimension
3rd peak from 1st dimension
LC 1Pump
LC 2Pump
LC 1Autosampler
LC 2Detector
Waste
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 11
2D-LC - Comprehensive 2D-LC
21
The complete effluent of the 1st dimension column is injected onto the 2nd dimension column with very fast gradients. A peak of the first dimension should be sampled at least 3 to 4 times. The run time of the 2nd dimension method matches the collection time of the 1st dimension effluent. Finally, the peaks will be re-constructed.
LC1
LC2
1st peak from 1st dimension
2nd peak from 1st dimension
3rd peak from 1st dimension
LC 1Pump
LC 2Pump
LC 1Autosampler
LC 2Detector
Waste
LC1
LC2
Example 2D-LC Application Scenarios
22
1st Mode 2nd Mode Advantage Disadvantage Column Examples
PROTEOMICS
Affinity Reverse Phase Orthogonality Low Peak Capacity, 1. AGILENT MULTIPLE AFFINITY REMOVAL
Off-line
Size Exclusion IonExchange Orthogonality Low Peak Capacity 1. PL-GEL (VARIOUS BEDS)
2. ZORBAX STRONG & WEAK ION EXCHANGE
Ion Exchange Reverse Phase Orthogonality Low Peak Capacity 1. ZORBAX STRONG & WEAK ION EXCHANGE
2. ZORBAX STM, POROSHELL
POLYMERS
Size Exclusion Normal Phase Orthogonality Low Peak Capacity 1. PL-GEL (VARIOUS BEDS)
2. ZORBAX CYANO, AMINO, SIL
Size Exclusion Reverse Phase Orthogonality Low Peak Capacity 1. PL-GEL (VARIOUS BEDS)
2. ZORBAX STM, POROSHELL
Reverse Phase Reverse Phase Miscible solvents, Strongly depends on column ZORBAX STM, POROSHELL
Broadest applications, and choice of mobile phase
Fast dual gradients, Highest peak capacity
Normal Phase Reverse Phase Orthogonality Solvent compatibility, 1. ZORBAX CYANO, AMINO, SIL
Limited application 2. ZORBAX STM, POROSHELL
PHARMACEUTICALS/METABOLOMICS
Compiled by Prof. Peter Carr, University of Minnesota
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 12
Hardware – Module-flexibility
23
1. Dimension
2. Dimension
1290 Infinity Binary Pump
1290 Infinity Binary Pump
1290 Infinity Autosampler or 1260
HiP Autosampler
Optional 1260/1290
Infinity Detector
One or two 1290 Infinity
TCC
Optional 1260/1290
Infinity Detector
1260/1290 Infinity
detector
1260 Infinity Binary or Quaternary Pump
1260 Infinity Capillary Pump 1260 Infinity
Autosampler
For 1st dimension
chromatogram and peak-triggering
To monitor waste-line
Almost any Agilent pump or autosampler in the 1st dimension with many different valve cobinations and detectors!
A 1290 Infinity Binary Pump for the 2nd dimension is required.
Hardware – Valve-flexibility
24
1. Dimension
2. Dimension
waste
Loop1
Loop2
2pos/10portvalve
Most other valve configurations for comprehensive and heart-cutting 2D-LC are supported as well. Easy transfer of existing 2D-LC methods!
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 13
Hardware – Valve-flexibility
25
1. Dimension
2. Dimension
waste
Loop1 Loop2
2x 2pos/6port valves
Also dual valve-head configurations supported with automatic synchronization of valve-drives!
Hardware – Valves, uniqueness and flexibility
26
1. Dimension
2. Dimension
wasteLoop1
Loop2
2pos/4port-duo
New Agilent-only special 2D-LC-QuickChange valve. Single valve with fully symmetric flow-paths and symmetric fill/flush-out behavior. Only valve that allows countercurrent flush-out of both loops.
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 14
Advantages of the new Agilent 2D-LC QuickChange valve head
All flow paths are equal (no additional bridging loops)Symmetric contercurrent fill/analyze direction of loops (reducing band-spread)
All in one valve (no synchronization, costs)
Loop1
Loop2
waste
2D-pump 2D-pump
1D-column
1D-column1D-column
Fill directionAnalyze direction
Valve
switching
1. 2.
Application examples-Advantage of shifted gradient features
Current state-of-the-art 2D-LC – narrow spread of peaks
RPLC x RPLCEasy method set-up but only little orthogonality
Data Analysis using GC Image LCxLC Edition Software from GC Image LLC.
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 15
29
• standard repeating with start- and end-time
• constantly shifted %B2D
• constantly shifted %B2D and shifted Δ%B2D
• Any combination
%B2D
Start 2D sampling End 2D sampling
%B1D
Time
%B2D
%B1D
Time
%B2D
%B1D
Time
%B2D
%B1D
Time
Comprehensive 2D-LC – gradient modesspecial 2D-gradient modes to improve resolution
30
Time-segmenting in Comprehensive 2D-LCReducing cost!
1D Method run time
1D Gradient/
2D Gradients
Idle flow rate
2D Pump flow rate
2D Pump flow rateSolvent saving!!!
2D Time-segments set in software idle idle idleTime-triggered Peak-triggered
Valve togglingIncrease life time!
%B (2D)
%B (1D)
F (2D)
1 D M
etho
d po
st-r
un ti
me
1D Chromatogram
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 16
31
Most easiest 2D-LC System Configuration- “One screen for the entire system”
Define 1D / 2D pump
Define detector in the second dimension
Define peak detector (optional)
Select the valve(s) to be used for 2D-LC
injection
Select a possible valve / loop
configuration
Graphical representation of the selected valve / loop configuration:• Flow path 1D & 2D• Animated valve switching
32
Select the 2D-LC mode: comprehensive
/ heart-cutting
Define the gradient of the 2nd dimension
Define repetition of 2nd dimension
gradient (Modulation time)
Show rollout of gradient in the 2nd dim
over the runtime of the 1st dimension
Define time window(s) where the
selected 2DLC mode is active
Most easiest set-up of complex 2D-LC methods- 2D-LC specific parameters of the 2D-pump
Graphical editing of gradient shift Access to standard
method UI of the pump
Operation values, warnings
Close-up of 2D-gradient
Solvent & Flow-Settings
Solvent consumption calculation
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 17
33
Example: graphical editing of a gradient shift – in less than a minute!- replace editing of large timetables by a few mouse operations
1 Use context menu to enter the editing mode
3 Draw a straight line by dragging the mouse to a new %B value at a specified runtime of the 1st dimension
2 Timetable entries are marked with circles
4 When releasing the mouse, a new TT entry is made and the gradient rollout is automatically updated
6 Insert / Delete shift points (mouse cursor and context menu changes near to a shift line)
5 Repeat step 3 + 4 with other TT entries
Resolution optimized!
Application examples-Advantage of shifted gradient features
Use of shifted gradient feature
Imagine programming this gradient manually! With Agilent 2D-LC Acquisition software a matter of a minute!
Before
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 18
Some Performance data- Testmix of 20 compounds – 2D-Precision
0.00
0.50
1.00
1.50
2.002D-Retention Time Precision RSD (%)
0.00
1.00
2.00
3.00
2D-Peak Volume* Precision RSD (%)
1st Dimension: Eclipse Plus RRHD, C18, 150 x 2.1 mm,
1.8 µm. Flow rate: 0.1 mL/min. Gradient: 0 min 5% B – 30 min 95% B,
40min stop-time.
2nd Dimension: Eclipse Plus RRHD, Phenyl Hexyl, 50 x
3.0 mm, 1.8 µm Flow rate: 3 mL/min. Initial Gradient: 0 min - 5% B, 0.5 min -
15% B, 0.51 min - 5% B, 0.65 min - 5% B. Modulation-time 0.65 min
15 compounds <1% RSD, all compounds <2% RSD
8 compounds <1% RSD, 16 compounds <2% RSD
*) not a peak area is measured but a three dimensional peak volume (intensity x 1D-time x 2D-time)!
Applications examplesPolyphenols from food matrix
36
1
2
34
56
7
8 9
10
11
1213 14
15
16
171819
202122
23
24
25 26
Compound RT 1st dim (min)
RT 2nd dim (sec) Peak Volume
Esculin mean 9.75 18.58 177,383s.d. nd 0.11 4,713RSD (%) nd 0.57 2.7
Rutin mean 13.65 33.68 72,375s.d. nd 0.07 853RSD (%) nd 0.22 1.2
Coumaric acid mean 13.00 25.57 660,541s.d. nd 0.13 13,037RSD (%) nd 0.52 2.1
Reservatrol mean 18.85 26.65 1,122,219s.d. nd 0.10 16,089RSD (%) nd 0.37 1.4
Salicylic acid mean 19.50 18.53 211,092s.d. nd 0.10 6,895RSD (%) nd 0.51 3.3
Luteolin mean 19.50 32.70 695,601s.d. nd 0.11 17,592RSD (%) nd 0.33 2.5
7-Hydroxy-Flavone mean 22.75 34.26 1,388,226
s.d. nd 0.10 17,195RSD (%) nd 0.30 1.2
Pinoslyvin mean 24.70 18,85 1,588,654s.d. nd 0.23 57,580RSD (%) nd 1.24 3.6
Chrysin mean 27.30 27.42 808,916s.d. nd 0.11 14,768RSD (%) nd 0.39 1.8
Flavone mean 28.60 26.35 1,008,012s.d. nd 0.13 20,911RSD (%) nd 0.48 2.1
Peak volume:all compounds <3.6% RSD
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 19
Application Examples-normal and stressed MAb, detection by ToF-MS
Intensity, cps
Gradient across analytical column 3-65% B
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340Time, min
0.00
2.00e6
4.00e6
6.00e6
8.00e6
1.00e7
Linear Salt Gradient 0-50% B
Step Salt Gradient to 100% B
Isocratic Salt Gradient at 100% B
20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340Time, min0.00
2.00e6
4.00e6
6.00e6
8.00e6
1.00e7
Intensity, cps
37
Summary
38
The Agilent 1290 Infinity 2D-LC solution brings the power of two-dimensional LC-separation to you at a never before experienced ease-of-use!
Highest separation power by 2D-LC combined with outstanding performance of the 1290 Infinity LC – the ideal tool for complex samples form biological origin, polymers, food-extracs, and many more.
Innovative new hardware and software features for ease-of-use, reduced operation costs and highest performance.
Upgradability or re-use of existing Agilent LC equipment.
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 20
Supercritical Fluid Chromatography:A New Era
Tony Brand, Ph.D.Senior Field ScientistAgilent Technologies, Inc.
Page 39
The Supercritical State of a Solvent
• Above a critical pressure liquids cannot enter into the gaseous state
• Above a critical temperature gases can no more enter into liquid state
• Above both, the critical temperature and the critical pressure solvents are in a supercritical state
Gas- and Liquid like properties
Page 40
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 21
Created by Christy Aurigemma, Pfizer, La Jolla
Page 41
Agilent 1260 600bar Binary pumpLeft side of Agilent binary pump exclusively meters CO2Right side: Selectable Modifiers
The Agilent 1260 SFC SystemLeveraging LC Technology
One extra setting:
BPR pressure
Agilent ALS or Hi-PALS autosamplersModified to use a loop injection
Agilent 1260 Thermostated Column CompartmentsUp to 8 columns, up to 250mm columns with multiple modules
Low
cos
t bev
erag
e gr
ade
CO
2
Agilent 1260 Diode Array DetectorHigh pressure flow cell
Put
ting
a st
anda
rd L
C
unde
r pre
ssur
e
Page 42
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 22
Phase diagram of pure CO2
with densities. Dashed lines represent approximate constant density lines
0
50
100
150
200
250
300
350
400
-70 -20 30 80
Temperature, °C
Pres
sure
, bar
0.041
0.86
0.62 0.29
0.098
1.120.99
0.83
0.720.941.09
1.06
0.91
0.84
0.96 0.891.02
0.59
0.19
0.75
0.82
0.77
0.89
0.95
0.991.14
0.0880.113
0.97
1.11
1.09
1.06 1.02
1.02
1.02
1.001.05
0.920.94
0.98
1.05
0.79
0.78
0.87
1.08
0.28
0.29
0.105
0.191
CP
Constant Density Lines: No abrupt changes
43
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 100 200 300 400
Ref
ract
ive
Inde
x
Pressure, Bar
CO2 at 40°C
Water at 20°C
The Refractive Index of CO2
varies Much More than Water
1
1.05
1.1
1.15
1.2
1.25
0 100 200 300 400 500
Ref
ract
ive
Inde
x
Pressure, Bar
40°50°60°70°
varies Significantly with Temperature
Page 44
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 23
Agilent 1260 Analytical SFC Solutions Unparalleled performance
< 0.02 amu noiseUltra-fast, ultra-simple
Lowest running cost of any LC or SFC!uses food grade CO2
minimal waste disposal costs
Standard Agilent softwareChemStationMassHunter
Agilent service and supportValidation services, IQ/OQ, etc
Page 45
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 20 40 60 80 100
Diff
Co.
, cm
2/se
c x1
05
% Co-solvent
60oC50o
40o
CO2/MeOH
C. Mantell, M. Rodrı´guez, E. Martı´nez de la Ossa. “Measurement of the diffusion coefficient of a model food dye (malvidin 3,5-iglucoside) in a high pressure CO2+methanol system by the chromatographic peak-broadening technique”, J. Supercrit. Fluids 25 (2003) 57–68
SFC is Faster Because Diffusion Coefficients are Larger
SFC Region
HPLC Region
VanDeemter eq.
HETP = A + BD1,2/µ+ Cµ/dp2D1,2
46
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 24
0.5
0.7
0.9
1.1
1.3
1.5
1.7
1.9
2.1
2.3
2.5
0 0.2 0.4 0.6 0.8
Linear Velocity, cm/sec
Red
uced
Pla
te H
eigh
t
Kinetex 2.6µm 150 x 4.6mm; methanol in CO2, 150 bar, 50°C, 0.3(?) µL injected.
30% MethanolH = A + BD1,2/µ+ Cµ/dp2D1,2
hr =H/dp
≈ 33,000 plates
SFC Can Deliver:Extreme Efficiency at High Flow Rates
47
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Mole fraction organic
, m
Pa-
s
CO2
H2O1.0
0.5
1.5
0
2.0
2.5
3.0
MeOH
EtOH
aqueous data from: J. Chem. Thermodynamics, 39 (2007)1578-1588
ΔP = c LµeT/dp2
Pressure Drop-Viscosity
Carbon dioxide has less than 1/10th the viscosity of water.When water is mixed with alcohols the viscosity actually increases.
48
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 25
0
50
100
150
200
250
300
350
0 20 40 60 80 100
% Modifier
Pres
sure
inlet pressure
outlet pressurePure CO2ΔP= 9 bar
Pure MeOHΔP= 250Bar
The pressure drop across columns in SFC is much lower than the pressure drop in reversed phase HPLC due to much lower viscosities.
Pressure Drop-Viscosity
4.6x250mm, 5µm, 2ml/min, 40oC
49
0
50
100
150
200
250
300
350
400
450
0 0.5 1 1.5 2 2.5 3 3.5
Flow Rate, ml/min
Col
umn
Hea
d Pr
essu
re, B
ar
ΔP
3x100mm, 1.8µm Zorbaz RX-Sil column, 1.5ml/min of 22.5% methanol in CO2;
150Bar outlet pressure, 50oC. As the flow rate goes up the tubing becomes more restrictive.
Even at High Flows, the DP can be Modest
50
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Agilent Technologies, UHPLC User Summit 26
0
50
100
150
200
250
300
350
400
450
0 10 20 30 40 50 60 70
% Methanol
Col
umn
Head
Pre
ssur
e, B
ar
bottom: 1.5ml/min; top 2ml/min; 3.0x100mm RX-Sil 1.8µm column @ 50oC, 150bar outlet pressure
Column Inlet Pressure:A 3x100mm, 1.8µm Column
51
Higher speed/throughput - more samples/day; more rapid re-equilibration - shorter cycle time compared to reversed phase HPLC
Low pressure drops - High resolution - doesn't require ultra-high pressure pumps, etc. due to low viscosity and high diffusion
Dramatically lower operating cost. Low solvent consumption/low waste generation. Green!
Orthogonal to reversed phase HPLC, comparable to normal phase
Great for isomers, including enantiomers
High Sensitivity
The Aurora A5 modules allows conversion of an HPLC into an SFC!
Practical Advantages of SFC?
52
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Agilent Technologies, UHPLC User Summit 27
SFC issues ….
Carbon Dioxide is TOXIC at levels above 15%. A CO2
detector/alarm is <$200 on the internet!
In Reverse Phase LC, a 50mm x 3.0mm sub-2 micron or Poroshell C18 is the standard. For more polar compounds, try C8, then C4 and then a Aqua column. In SFC, there is no similar “progression” of columns.
The true application space is still being mapped! Can on-line SFE be combined with SFC in a single instrument for complex matrices (e.g. plasma, urine, etc.)?
85% CO2 15% MeOH, 4.75ml/min, 150bar, 50°C, Kinetex 4.6x150mm, 2.6µm HILIC
0.4
-1
0
1
mAU
0.8 1.20
min
SFC is Sensitive
N ≈ 25,000 plates
Noise ≈ 0.07mAU
W1/2 ≈ 0.003-0.0045min (80Hz) filter
54
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 28
14 sec
10 chromatograms/ 6minutesDP < 230 bar
SFC is Super Fast Chromatography
4.6x50mm, 1.8µm Zorbax RX-Sil 30% methanol at 5ml/min, 150 bar out, 50oC ibuprofen, ketoprofen, caffeine, theophyline, theobromine
55
56
Analytical SFC applications
Brand 2D-LC and SFC Friday, September 14, 2012
Agilent Technologies, UHPLC User Summit 29
Can SFC Separate My Compounds?
Any solute soluble in methanol or a less polar organic solvent will elute in SFC (>80% of small drug molecules)
Strong organic acids and bases, require a modifier and an additive in the mobile phase
Most salts of organic acids and bases elute
Smallish peptides elute
Solutes with mass <25,000 can be separated with carbon dioxide based fluids
57
Solutes requiring an aqueous environment
Solutes requiring a buffered or ionic aqueous environment, i.e. biomolecules such as proteins will NOT elute (maybe)
Most Inorganic salts
Solutes with mass >25,000 mw will not elute with carbon dioxide
SFC-MS: PAH Mix 25 (100ppm)
Nacalai π-NAP (4.6x150 mm, 5 µm), Injection = 5 µL, Flow Rate = 2.0 mL/min @ 120bar, SF = CO2, Mod = MeOH with 2% H2O, Gradient = 0-20min: 5-30%, Col Temp = 40oC, Caloratherm = 60oC,Make-up Flow = MeOH at 0.2 mL/min, MS = APCI, Cap V = +3000V, Corona I = 4.0µA, Fragmentor = 200V, Drying Gas = 5.0 mL/min @ 325oC, Nebulizer = 60 psig, Vaporizer = 450oC
UV 254 nm
MS scan, APCI, 80-350 amu
1 2
2
4
5,6
57
8
98
9
10
10
11
1112
12
13
14
14
1516
1615
13
7
3
3
Evaluation of SFC-MS Configurations for the Analysis of Lipids, Sterols, and PAHs,M. Dunkle, A. dos Santos Pereira, F. David, P. Sandra, SFC 2011 poster
1 Naphthalene (FW 128)2 Acenaphthylene (FW 152)3 Acenaphthene (FW 154)4 Fluorene (FW 166)5 Anthracene (FW 178)6 Phenanthrene (FW 178)7 Fluoranthene (FW 202)8 Pyrene (FW 202)9 Benzo(a)anthracene (FW 228)10 Chrysene (FW 228)11 Benzo(k)fluoranthene (FW 252)12 Benzo(b)fluoranthene (FW 252)13 Benzo (a)pyrene (FW 252)14 Dibenzo(a,h)anthracene (FW 278)15 Indeno(1,2,3-cd)pyrene (FW 276)16 Benzo(g,h,i,)perylene (FW 276)
Brand 2D-LC and SFC Friday, September 14, 2012
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SFC-MS: Lipid Mix 100ppm
Separation Conditions: Column Zorbax Eclipse XDB-C18 (4.6 x 150 mm, 5 µm), Injection = 5 µL, Flow Rate = 2.0 mL/min, Outlet P = 120 bar, SF = CO2, Mod = MeOH with 2% H2O , Mod gradient = 0-15min: 5-50%, Col Temp = 40°C, Caloratherm = 60oC, Make-up Flow = MeOH at 0.2 mL/min. MS = APCI scan (450-1000 amu), MS = APCI, Cap V = +3000V, Corona I = 4.0µA, Fragmentor = 70, Drying Gas = 10.0 mL/min at 325oC, Nebulizer = 60 psig, Vaporizer = 350oC
min2 4 6 8 10 12 14
0
100000
200000
300000
400000
500000
600000
MSD1 TIC, MS Fi le (C:\CHEM32\1\DATA\ORE11138_20110504_LIPIDS\ORE11138_PAH 2011-05-04 08-56-47\201105040000003.D) AP
PPP
OOO
POS
m /z5 00 6 00 70 0 80 0 9 00
0
2 0
4 0
6 0
8 0
10 0
* MSD 1 SPC, t ime= 1 0.74 4 of C:\C HEM3 2\1\DA TA\O RE 111 38 _2 011 05 04 _L IPIDS \O RE1 11 38 _PAH 20 11-0 5-04 08 -5 6-4 7\20 11 05 040 00 00 03.D
Max: 243328
903
.9
887
.8
605
.6
886
.8 8
85.
8
604
.6 6
03.6 OOO
[M-RCOO]+
[M+H]+
m /z55 0 600 650 7 00 7 50 8 00 8 50
0
2 0
4 0
6 0
8 0
10 0
* M S D 1 S P C, t im e= 8 .8 24 o f C : \ CH E M 32 \ 1\ DA T A \ O RE 1 113 8_ 20 110 50 4_ LI P I DS \ O RE 11 13 8_ P A H 2 01 1-05 -04 0 8-56 -47 \ 201 10 50 400 00 00 3. D
M ax : 30 2 0 0
824
.8
553
.6 5
52.6
551.
5 PPP[M-RCOO]+
m /z60 0 65 0 70 0 7 50 80 0 8 5 0 90 0
0
2 0
4 0
6 0
8 0
10 0
*MS D1 SP C, t im e= 11 .28 8 of C:\C HEM3 2\1 \D ATA\O RE1 11 38 _2 011 05 04 _L IP ID S\O RE1 11 38 _PAH 20 11- 05 -04 0 8-5 6-4 7\20 11 05 040 00 00 0 3.D
Max: 25608
58
1.6
890
.8 607
.6
862
.8
87
9.9
878.
9
58
0.6
578
.6
861
.8
606
.6
579
.6 5
77.6
605
.6 POS[M-R1COO]+
[M-R2COO]+
[M+H]+
Evaluation of SFC-MS Configurations for the Analysis of Lipids, Sterols, and PAHs,M. Dunkle, A. dos Santos Pereira, F. David, P. Sandra, SFC 2011 poster
Steroids by LC-APCI-MS and SFC-APCI-MS
1 Vitamin E2 Cholesterol3 Stigmasterol (3)4 β-sitosterol
Vitamin E spectrum by SFC/MS
Vitamin E spectrum by LC/MS
Zorbax Eclipse XDB-C18 (4.6x150mm, 5µm), Injection = 5µL, Flow Rate = 2.0 mL/min (SFC), 1.0 mL/min (LC), Outlet P=120 bar, A: CO2, B: MeOHw/ 2% H2O (Isocratic at 5%), MP= ACN/i-PrOH (5:4) isocratic, Temp = 40oC (SFC), 50°C (LC), Caloratherm = 60oC (SFC), Make-up = MeOH @ 0.2mL/min (SFC). MS = scan 350-500amu., Cap V = +3000V, Corona = 4.0µA, Drying Gas = 12 mL/min @325oC, Neb = 50 psig, Vap = 350oC.
Evaluation of SFC-MS Configurations for the Analysis of Lipids, Sterols, and PAHs,M. Dunkle, A. dos Santos Pereira, F. David, P. Sandra, SFC 2011 poster
UV sensitivity similar for LC and SFC
MS sensitivity better for SFC/MS
MS spectra “cleaner” for SFC/MS
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SFC Separation of Nucleosides and Nucleic Acids
urac
il aden
ine
aden
osin
e
cyto
sine
guan
ine cytid
ine
guan
osin
e1 2 3 minutes
UV abs
Several not soluble in either MethanolOr water. Requires Acid or base to dissolve
4.6 x 200 mm Hypersil Amino
Methanol/water/ammonium acetate/
formic acid In carbon dioxide
50°C, 160 bar
Thia
min
e m
onon
itrat
e
Nia
cina
mid
e
Rib
ofla
vin
Nia
cin
Vita
min
B 1
2
1 2 3 4
SFC Separation of Water Soluble Vitamins
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1 2 3 4
Vitamin B12
Methanol + 10% water + formic acid + ammonium acetate
SFC-MS spectraVitamin E
Separation Conditions: Column Zorbax Eclipse XDB-C18 (4.6 x 150 mm, 5 µm), Injection = 5 µL, Flow Rate = 2.0 mL/min (SFC), 1.0 mL/min (LC), Outlet P = 120 bar, SF = CO2, Mod = MeOH with 2% H2O (isocratic at 5%), MP = ACN/Isopropanol (5:4)isocratic, Col Temp = 40°C (SFC), 50°C (LC), Caloratherm = 60°C (SFC), Make-up Flow = MeOH at 0.2 mL/min (SFC). MS = APCI scan (350-500 amu), MS = APCI, Cap V = +3000V, Corona I = 4.0µA, Fragmentor = 70, Drying Gas = 12.0 mL/min at 325°C, Nebulizer = 60 psig, Vaporizer = 350°C
m/z320 340 360 380 400 420 440 460 480
0
20
40
60
80
100
*MSD1 SPC, time=4.325 of C:\CHEM32\1\DATA\ORE11138_20110502_PAHS\20110502000009.D APCI, Pos, Scan, Frag: 70
Max: 177024
427
.4
433
.4
429
.4 4
32.4
431
.4
[M+H]+SFC-APCI-MS1) Vitamin E
Brand 2D-LC and SFC Friday, September 14, 2012
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13mM ammonium trifluoroacetate in methanol 5% for 1min, then 5%/min to 50%, hold 5 min, 2ml/min, 120 bar outlet, 40oC,Ethylpyridine 4.6x250mm, 5µm (Princeton)
H3C
H3C
NO
N
O
O
O
N
O
H3CCH3
N
O
H3C
CH3
NO
CH3
CH3
N
O
N
N
N
NO
N
O
N
O
N
O
N
O
H3C
N
O
CH3
N
O
NO
N
O
NO
N
O
N
N
O
OO
N
O
N
N
O
OO
N
O
NO
N
O
N
N
O
OO
N
O
N
O
OO
N
O
N
O
S
CH3
N
O
N
N
O
N
NN
N
OH3CCH3
NO
CH3
CH3N
O
O
O
NO
CH3
CH3N
O
O
NO
CH3
CH3N
O
O
O
N
O
N
O
O
N
O
N
O
N
O
NO
N
O
O
N
H3C
CH3
H3CCH3
H3C
CH3
H3C
CH3
CH3
O
N
O
H
H
H
H
H
H
H
H
H H
H
H
H
H
H
HH
H
H
H
H
H
H
H
H
H
HH
H
H
H
HH
H
H
H
H
H
H
HH
H
H
H
H
H
H
H
H
HH
H
H
H
HH
H
H
H
H
H
HH
H
H
H
H
H
H
H
H
H
H
H
HH
40mer
J. Zheng. et al., Anal. Chem.,78, 1535-1545 (2006)
Peptides: A Limited Number of Reports
65
What Didn't We Cover That Is Important?
Surfactants– ethoxylates/proproxylates
UrethanesSilicone oilsFlavanoids/anti-oxidantsSweetenersMost pesticides/Herbicides
– carbamates– phenylureas– sulfonylureas
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HPLCWaste
Hybrid System in SFC Mode
123
4
5
6 7
8
9
10
SFC-Binary Pump
BPR
Loop back Restrictor
SFC Pump outlet
LC Pump:G1310B orG1311B orG1312B LC
Pum
p outD
etector out
SFCWaste
68
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Waste
123
4
56 7
8
9
10
BPR
Restrictor
SFC Pump inlet
LC Pump:G1310B orG1311B orG1312B LC
Pum
p outD
etector outSFC Pump outlet
SFC-Binary Pump
The Agilent Hybrid SFC/LC SystemUHPLC Mode
69
Advantages of the Agilent Hybrid SFC/UHPLC
Orthogonal method screening in one single system
Switch from SFC to UHPLC forth and back in a single sequence
No equilibration time between LC and SFC
Significant cost saving, only one system has to be purchased
Direct results comparison between SFC and LC
No instrument to instrument variation
Save lab space
The only system which can offer both techniques in single system
70
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Performance: Overlay SFC versus UHPLC
71
SFC
LC
1 - Caffeine 2 - Theophylline3 - Cortisone 4 - Prednisone 5 - Hydrocortisone 6 - Prednisolone7 - Sulfomerazine8 - Sulfaquinoxaline
Repeatability: SFC and UHPLC in a single sequence
72
Part of a sequence with alternating SFC and LC batches
SFC-Mode LC-Mode
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Application: PAH with the Hybrid SFC/UHPLC System
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
10
20
30
40
50
DAD1 A, Sig=254,16 Ref=360,100
min1 2 3 4 5 6 7
mAU
0
20
40
60
80
DAD1 A, Sig=254,16 Ref=360,100
PAH- mixAcenaphteneAcenaphthyleneAnthraceneBenzoanthraceneBenzo(a)pyreneBenzo(b)flurantheneBenzoperylenBenzo(b)flurantheneBenzo(k)flurantheneChryseneDibenzoanthraceneFluorantheneFluoreneIndenopyreneNaphtalenePhenanthrenePyrene
LC-mode
SFC-mode
Column: Zorbax Eclipse PAH (4.6x 150 mm, 5 um)mobile phase A : H2O, B: ACN
Gradient:
time %B
0 40
20 95
21 95
21.5 40
Column: Zorbax Eclipse XDB C18 (4.6x 150 mm, 5 um)Mobile phase A: CO2, B: MetOH, 2% H2O
time %B
0 5
10 22.5
11 60
11.5 5
Gradient:
73
SFC uses Normal Phase SeparationOrthogonal to reverse phase HPLC
Trace contaminants elute in the order 1, 2, 3, 4, 5, 6 in SFC,but 6, 5, 3, 2, 1, 4 in HPLC
In general,
More polar componentselute last in SFCelute first in LC
Least polar componentselute first in SFClast in SFC
SFC
LC
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SFC is Normal PhaseWith None of the Problems of Normal Phase HPLC!
No problems with water
• stable retention times• rapid re-equilibration• can add water to the modifier as an additive• can inject partially aqueous samplesGradient elution is typical
Provides nearly the opposite retention of reverse phase HPLC.
• enhanced purity assessment• improved trace analysisMultiple mobile phase parameters for changing retention and selectivity
Wide choice of columns to change selectivity and retention
75
Everything you need to know about SFC in 1 slideOr…. Top 10 things to remember… Think normal phase LC, Liquid CO2 is much like heptane. Well established for chiral separations. Usually performed as gradient elution, changing the composition of the mobile phase vs time
with a methanol/ethanol/isopropanol more polar co-solvent (0-60%, no benefit going higher!) Use polar stationary phases such as bare silica, cyano, amino, diol, ethylpyridine, “Premier”,
and others. Liquid CO2 is MUCH less viscous than organic solvents so delay volume does not cause
dispersion! Dispersion due to thermal changes is VERY significant! PEEK tubing is better than
stainless steel. Try to insulate tubing between devices and the column. If it is not hissing and not cold, icing, or condensing atmospheric moisture, it ain’t broke……
so there is another reason for not having a signal. Flow rate 1.5-5.0mL/min, go lower than 1.5mL at your own risk! The most critical setting is Back Pressure for the Back Pressure regulator. Default setting is
200bar, 160bar for SFC/MS since some pressure is bled off with the “leak” to the MS. MS requires APCI, Electrospray requires a make-up solvent containing a volatile buffer (e.g.
formic acid in methanol).
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Practical Advantages of SFC
FAST CHROMATOGRAPHY for non-volatile compounds! higher speed/throughput--more samples/day more rapid re-equilibration--shorter cycle time compared to
reversed phase HPLC Orthogonal to reversed phase HPLC Great for stereo-isomers, chiral compounds High Sensitivity Low pressure drops-High resolution Low solvent consumption/low waste generation An alternative to normal phase LC Lower operating cost Green!
More Information on...
78
• Application Notes: • Chiral impurity analysis(5990-5969EN)• Achiral analysis (5990-6413EN)• Sub 2um analysis for ultra fast separation (5990-6412EN)• Highest resolution separation (5990-6934EN)• Strategies for column and mobile phase selection
with the Agilent 1260 Infinity SFC System (5990-7147EN)• The Agilent SFC/MS solution (5990-7972EN)• Separation of enantiomers and conformers of Tofisopamon (5990-9315EN)• Enantiomer separation of nonsteroidal anti-infl ammatory drugs (5990-9459EN)• Agilent 1260 Infinity Hybrid SFC/UHPLC System (5990-9514)• Determination of polymer additives and migration products prevalent in food packaging
material (5990-9598EN)• Determination of phthalate migration from toys (5990-9597EN)
• SFC cost calculator: calculates operating costs of SFC vs. LC• Hybrid SFC/UHPLC Video• Hybrid SFC/UHPLC E-seminar• SFC Compliance Primer
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Thank you for listening…
Questions?