Ultra High
Definition LC/MS
Achieving the utmost
information from today’s
complex analyses
Dr. John Fjeldsted
General Manager
LC/MS Division
Agilent Technology
Santa Clara, CA
What is Ultra High Definition?
Ultra High Definition is the combination of uncompromising
performance in All Dimensions of the analytical measurement.
Ultra High Definition LC/MS achieves the Maximum Qualitative
and Quantitative Information possible.
MassHunterTMInformatics
Chromatography
Comprehensive Sample
InformationMass Spectrometry
Signal Response
Mass Spectrum
Ultra High Definition
Optimizing all Analytical Dimensions
Software
• Data Mining
• Differential Profiling
• Annotation
• Ionization Profile
• Mass Accuracy
• Isotopic Fidelity
• Mass Resolving Power
• Acquisition Rate
• Sensitivity
• Dynamic Range
• Linearity
• Peak Resolving Power
• Peak Capacity
• Separation Speed
Agilent 1290 Infinity LC – Attributes for MS - Infinitely Better for LC/MS
Lowest
Delay
Volume
Highest
Precision
Pump (w/o) mixer: 10 µL
Pump, Fixed Loop 20 µL
Pump, Fixed Loop, JetWeaver 55 µL
<0.002% carry-over with Chlorhexidine
Optional needle seat backflushing with FlexCube
Fixed Loop or Variable Loop Injections
1200bar @ 2mL/min for highest resolution per time
Reduced Ion & Matrix Suppression
HT-Solution for up to 2000 samples/day (ACR)
Complete Integration and control from MassHunter
Enables method conversion from/to any (U)HPLC
ALS precision for small volumes:
<1.5% from 0.5-1µLm, <0.7% from 1-2µL,
<0.25% @ 2-20 µl (40 ul) **
Pump Active Damping:
RT stability < 0.2 % (1.5 min runs)**
Best
Autosampler
Performance
Greatest
Productivity
1290 Infinity
0
200
400
600
800
1000
1200
0 1 2 3 4 5
1290 Infinity – Compatible with any HPLC and UHPLC
bar
ml/min
Standard LC
Acquity
Agilent RRLCThermo
Accela
Shimadzu UFLCXR
Dionex RSLC
A new power range providing maximum performance,
flexibility, compatibility and investment protection
1290 Infinity Binary PumpHow it looks like inside
December 15, 2009
Channel
A
Channel
B
Jet Weaver
Multi-layer
Heat exchanger
Solvent selection
valveDegasser
Purge
Valve
Silicon Carbide
Pistons
High Resolution
Pump drives
0 10 20 30 40 50 60 70 80 90 sec
1.00
0.75
0.50
0.25
0.00
1290 Infinity - Ultra High Performance Separations Maximizing Throughput
Fast screening of 220 pesticides
in 90 seconds
Peak Width
~ 0.7 sec FWHM
1290 Infinity - Ultra High Performance SeparationsMaximizing High Peak Capacity
Peptide Map of Tryptic Digest of BSA run on Agilent RRHT Zorbax SB-C18, 2.1x150mm, 1.8µ
min2 4 6 8 10 12 14 16 18 20
mAU
0
10
20
30
40
50
60
Peak Capacity = 540
Peak Width ~ 2 sec FWHM
HPLC-Chip: Polymer Microfluidics
developed in Agilent Labs
µ-fluidic holes
µ-filter
Registration holes
Enrichment Column 40 nl ZORBAX 300SB C18
Electrical Interface to Internal Grounding Electrode
ZORBAX 300SB-C18, 5µm 75µm, 43 mm length
Laser Ablated Cylindrical
nanospray emitter
MS inlet
orifice
Counter
electrodeRobust spray performance under all solvent conditions
Polyimide nanospray emitter lifetime measured in weeks
Laser ablation of polyimide film
Agilent’s New 6540 Ultra High Definition QTOFResearch Performance in a Benchtop Format
• 40,000 Resolving Power
• <1 ppm MS <2 ppm MS/MS Mass Accuracy
• 20 Spectra/s
• 2 pg – 50:1 Reserpine S/N
• 5 Decades in Spectrum Dynamic Range
• Excellent Linearity and Isotopic Fidelity
• Supports Standard ESI, Agilent Jet Stream and HPLC-Chip
Unsurpassed Analytical Capacity in a Benchtop
The result of RELENTLESS INNOVATION
Theory of High Resolving PowerHighest resolving power in a Benchtop
Turn-around time term
t
TOF
FWHM
mR
peak 2
2222
RPWTAt
TA
PW
R
Detector pulse width term
Residual term
Agilent Model
TOF, s tTA, ns tPW, ns tR, ns ∆t, ns
Resolving Power
6520 92 1.1 2.1 1.6 2.8 16,5006540 152 0.5 0.9 1.6 1.8 42,000
*Values are for m/z 2722
6540 UHD
Q-TOF
6530
Q-TOF
• 1ppm/C Expansion Coefficient for Inner
Flight Tube virtually eliminates calibration
drift due to flight tube elongation.
• 2nd Order Temporal Focusing Ion Mirror
uses high transmission Harp Grid for
maximum sensitivity
Enhanced Ion Flight Tube and Mirror TechnologyStable, Sensitive, High Resolution
DC Quad
Octopole 2
Ion Pulser
Ion Mirror
Detector
Turbo
Ion Mirror
Detector
Precise Ion Beam Acceleration FocusingMaximizing Transmission and Resolving Power
• Autotune algorithms optimize
resolving power and transmission
by adjusting among other
voltages:
• Mid-Mirror
• Puller Offset
• Accelerator Focus
DC Quad
Octopole 2
Accelerator Focus
Puller , Puller Offset
Pusher
DC Quad
Octopole 2
Ion Mirror
Detector
Turn-around TimeA closer look
DC Quad
Octopole 2
DC Quad
Octopole 2
DC Quad
Octopole 2
DC Quad
Octopole 2
Puller
Pusher
Ground
Turn-around time is the delay
for an ion to overcome
incoming velocity away from
the time of flight measurement.
Velocity reducing flight time
Velocity increasing flight time
• Standard ion optic elements cannot achieve a reduction in
phase space (i.e. product of beam divergence and size).
Challenge: Reduce Turn-around Time and
Preserve Ion Transmission
Large
Aperture
Entrance
Small
Aperture
Exit
Smaller size, but
divergent beam
increases spread in
initial ion velocity in
TOF ion pulser
•Creating a simple RF ion guide “funnel” looks like a good idea.
• Because the rods are “angled-in” there is a vector component of the
pseudo potential well force that pushes the ions back out the funnel.
Challenge: Reduce Turn-around Time and
Preserve Ion Transmission
Retarding Vector
Cut-away view
• Applying a voltage gradient along the length of each rod creates an Axial
Acceleration Potential which overcomes the retarding force vector of the
pseudo-potential well.
+ =
Net positive ion motion
Retarding Vector
• Collision Cooling removes energy from the ions and allows the beam to
collapse in size and not increase in divergence.
Challenge: Reduce Turn-around Time and
Preserve Ion Transmission
• Once again the Axial Acceleration Potential is critical to replace the
energy losses and prevent beam stall-out.
Ion Beam Compression (IBC)* TechnologyDrives Higher Resolution without Sensitivity Loss!
Axial Ion Acceleration Technology
applied to a tapered ion guideCollision Cell
Compressed beam reduces
turn-around time and achieves
40,000 resolving power.
* Patent pending
Next Generation – Ultra High Speed Detector
New Bipolar TOF Detector
• New ultra fast and high efficiency scintillator
• New ultra fast response PMT design continues the tradition of high dynamic range and detector lifetime
• Developed by Photonis with Agilent TOF Technology
• Specifically enhances Resolution in 2Ghz Ext. Dynamic Range Mode
Single Ion Response
~800 psec FWHM
2 nsec/div
Ultra High Speed AcquisitionFrom Agilent’s Leadership in GHz Speed Electronics
• 4 GHz (8 bit) Analog-Digital-Converter
Adapted from Agilent’s High Speed
Oscilloscope Systems
• Ultra High Speed FPGAs process and
store transients in real time
Picture of 4GHz board
Goes here
FPGAs
Dual Input Agilent
pre-amplifiers
4 GHz Agilent ADC
• 4 GHz Acquisition for Maximum
Resolving Power and <1ppm
Mass Accuracy
• 5 Decades of in-Spectrum
Dynamic Range from 2-Channel
x 2 GHz Dual Gain Mode
6540 Ultra High Definition QTOF
Resolving Power – Across the Mass Range
5x10
Counts vs. Mass-to-Charge (m/z)
200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800
922
R=42424622
R=38702
1221
R=43674
1521
R=42750322
R=302182121
R=418251821
R=404052421
R=39332 2721
R=37207
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
Scan Rate Independent
Counts vs. Mass-to-Charge (m/z)
622.00822
623.01059
624.01219 625.01471
m/z 622 and isotopes
6540 Ultra High Definition QTOFResolving Power – Small Proteins
Average resolution of these peaks
R = 38045
10+
16+
15+
14+13+
12+11+
19+
18+
17+
20+
22+
21+
23+
24+
25+
26+
Heme
Horse Heart Myoglobin (MW 16951 Da)
6540 Ultra High Definition QTOFMass Accuracy – Repetitive Injections
+ESI EIC(609.28066)
Scan Frag=240.0V Reserpine_40pgms3.d
0.511
Counts vs. Acquisition Time (min)
0.0 0.25 0.5 0.75 1
5x10
0
0.2
0.4
0.6
0.8
1
1.2609.28081
610.28425
611.28650
612.28987
Counts vs. Mass-to-Charge (m/z)
609 610 611 612 613 614
613.29210
Isotope Obs % Calc % Obs m/z Calc m/z Diff (ppm)
A 100 100 609.28081 609.28066 0.25
A+1 35.87 37.24 610.28425 610.28393 0.52
A+2 8.58 8.59 611.28650 611.28671 -0.34
A+3 1.41 1.48 612.28987 612.28941 0.75
A+4 0.14 0.21 613.29210 613.29203 0.11
Run
Error (ppm)
1 0.96
2 -0.17
3 0.25
4 0.02
5 0.39
6 0.13
7 0.01
8 0.52
9 0.04
10 0.30
Mean 0.25
Std. Dev 0.32
40pg reserpine on-column, 10 injections
6x10
1
2 1
5x10
0
5
1
4x10
0
2.5
51
2x10
0
5
1
3x10
0
1
1
Counts vs. Acquisition Time (min)
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4
Verapamil: Major And Minor Metabolites (Phase I)
desmethyl
Isopropyl
dealkylation
monohydroxy
dihydroxy
TICverapamil
6x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8 455.290321
(M+H)+
441.274704
471.284287487.279822
(M+H)+
Counts vs. Mass-to-Charge (m/z)435 440 445 450 455 460 465 470 475 480 485 490 495 500 505
Coeluting Metabolites With Parent Drug: Need
Wide Dynamic RangeFive decades of response in a
single scan
2.68 million
counts
25 counts
verapamil dihydroxy metabolite of verapamil
desmethyl
metabolite
monohydroxy
metabolite
400
counts
Metabolite ID Results
6430 and 6460 Triple Quad LC/MSMaximizing Performance and Throughput
Optimized: Rapid MRM TransitionsFast +/- Switching6430 Std ESI /HPLC Chip6460 Agilent Jet Stream
Design: Dual turbo pumping6430 mass range is 2250m/z6460 mass range is 3000m/zSub millisecond collision cell clearanceUltra Sensitive ADC for wide dynamic range and single ion detection
Software: Dynamic MRM (DMRM)Optimizer (small molecule and peptide)MRM Optimizer DatabaseMRM Builder (from Spectrum Mill)Quantification SW
Agilent Jet Stream Ion Generation Gas Dynamics View
The super-heated sheath gas
collimates the nebulizer spray and
creates a dramatically “brighter source”
Nozzle voltage
Resistive sampling
capillary
Nebulizing gas
Super-heated sheath gas
Heated drying gas
Enhanced efficiency nebulizer
Patent Pending
Scattered light shows
good collimation, but
without heat there is high
droplet density
Start temperature = 25 ºC Stop temperature = 400 ºC
Less light scattering
shows increased droplet
desolvation and high ion
production.
Agilent Jet Stream In ActionObserving Thermal Focusing
Beam Turn-off Characteristics
10
100
1000
10000
100000
-500 0 500 1000 1500 2000
microseconds
Arb
. U
nit
s .
mz922 mz118
Beam Turn-off Characteristics
10
100
1000
10000
100000
-500 0 500 1000 1500 2000
microseconds
Arb
. U
nit
s .
mz922 mz118
600 sec
350 sec
Sub millisecond
ion transport
Axial Acceleration Collision CellSub-millisecond ion transport times
•Axial acceleration overcomes memory or cross-talk effects
Ultra High Definition LC/MSMoving from Discovery to Validation
Over 12 Years in LC/MS
Atmospheric Sampling
and Patented
Orthogonal Geometry -
Result in Industry
Leading Sensitivity and
Robustness
Rough Pump
Octopole 1
Turbo 1
Turbo 1
Turbo 1
Quad Mass Filter (Q1)
Collision Cell
Lens 1 and 2
Quad Mass Filter (Q3)
10KV Detector
Ion Pulser
Turbo 2
Octopole 1
DC Quad
Rough Pump
Turbo 1
Turbo 1
Turbo 1
Quad Mass Filter (Q1)
Collision CellLens 1 and 2
Octopole 2
New Collision Cell Incorporates
Axial Acceleration for High Speed
MS/MS Analysis
6500 Series Q-TOF
6400 Series QQQ
0
3x10
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
+ESI Product Ion (4.023-4.037 min, 2 scans) Frag=175.0V [email protected] (637.6[z=3] -> **) syn-100fmol.d
719.3 851.4
961.5
832.4
481.2343.2
607.3506.3 1079.5691.3648.3416.7 804.4
4x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
+ESI Product Ion (4.765-4.832 min, 18 scans) Frag=120.0V [email protected] (637.6 -> **) peptide5-CE21.d
719.1
961.2480.9
832.2
350.8 416.5
530.6 1079.2607.1
301.8
QTOF - Discovery and QQQ - Validation Produce Common Fragment Ions
343.0 481.1530.8
588.2 653.4703.5
736.4
785.1
834.6
961.5
5. +MS2(637.8), 3.9-4.1min #344-#365
0.0
0.5
1.0
1.5
2.0
2.5
7x10
Intens.
300 400 500 600 700 800 900 1000 1100 1200 1300 m/z
Ion Trap / OrbitrapB13++
Counts vs. Mass-to-Charge (m/z)300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350
QQQ
b8
y6
b6
y7
b7
y9
Peptide: RPCFSALEVDETYVPK (m/z 637.6, +3)
Q-TOF
b8
y6
b6
y7
b7
y9
Moving From Discovery to ValidationHaving the right tools
• Spectrum Mill
Protein ID via Database Search, Mass Gap Search
MRM Builder – Targets the most intense transitions
• Peptide Optimizer
Maximizing Sensitivity for each Peptide
MRM Database
• MS Acquisition
Direct Import from MRM Database
Dynamic MRM Acquisition maximizes Duty Cycle and Sensitivity
Agilent 6400 Series Triple Quads with HPLC-
Chip/QQQ LCMS TechnologyNanospray chip configuration brings new era in high-precision, high-
sensitivity quantificationNanoLC system for
analytical chromatography
HPLC Chip Cube system
QQQ LCMS
CapLC pump for sample
loading on enrichment column
Sensitivity: down to low amol
Dynamic range: up to 105
HPLC-QQQ - Low amol limit of Quantitation rangePeroxidase spiked into human serum
10 fmol peroxidase in 1ug serum
1 fmol peroxidase in 1ug serum
100 amol peroxidase in 1ug serum
10 amol peroxidase in 1ug serum
External Quantitation Curve of Peroxidase Peptide DTIVNELR From 10 amol to 10 fmol Spiked into Human Serum
100amol
1fmol10amol
Thank You