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1 © 2015 AB Sciex. GEN-MKT11-2066-B
Development and Commercialization of a High Pressure Platform for nano- and micro-HPLC
Don W. Arnold, PhD
VP, R&D and Principal Scientist
International MicroNano Conference 2015,
Amsterdam, December 8, 2015
2 © 2015 AB Sciex. GEN-MKT11-2066-B
FORENSICS
CLINICAL
DIAGNOSTICS
• IVD Testing
Solutions
• Medical Devices
LIFE SCIENCE
RESEARCH
FOOD AND
ENVIRONMENT
PHARMA AND BIOPHARMA
Markets We Serve
• Toxicology
• Forensics
• Proteomics
• Metabolomics
• Lipidomics
• Clinical Research
• Contaminations and Safety
• Quality and Authenticity
• Water and Soil Testing
• Biologics Discovery & Development
• Small Molecule Discovery and
Development
2 © 2015 AB Sciex. GEN-MKT11-2066-B
3 © 2015 AB Sciex. GEN-MKT11-2066-B
iChemistry™
Solutions Integrated
chemisties to
boost MS workflows*
Powerful Software Service and
Support New Mobile
Monitoring from a
partner you can
trust
High
Performance
Mass Spec Exceedingly
sensitive. Sharply
focused
Innovative
CESI 8000
INTEGRATED SOLUTIONS
Liquid
Chromatography Reliable standard,
micro and nano with
cHiPLC® systems*
Front-end
Partners E.g. Advion,
Phytronix, Beckman
Coulter
iMethods™
& Cliquid™ Instant methods
for accelerated
results*
* Research use only. Not for use in diagnostic procedures.
Workhorse Mass
Spectrometry LC-MS/MS workhorses,
intelligently
re-engineered
Strong Portfolio for Advanced and Routine Workflows
piston
bypass
1mm
Open
Closed
Open
Closed
Microfluidics Roots at Eksigent Technologies
4
Life science research and product development built upon micro- and nano-flow fluid delivery and analysis technologies
Core Technologies – Micro-/Nano-flow fluid delivery, sensitive microscale detection and analysis, system integration
Liquid chromatography
• Separate a complex mixture into
components according to chemical
properties
• Based upon partitioning between mobile
phase (liquid) and stationary phase (solid)
• Gold-standard for liquid phase analyses
(Pharmaceutical, fine chemicals, food and
beverage, agriculture, petroleum,
proteomics, etc.)
Conventionally - 4.6 mm diameter columns
Conversion to 2.1 mm with Ultra High Pressures
Eksigent – Nano- and Micro-flow HPLC focus
time
sig
nal
Motoyama and Yates, AChem 80, 7187 (2008)
nanoLC-MS for Proteomics
nanoLC-MS is powerful tool for proteomics
Separation of complex sample
Small sample consumption
Sensitivity
Positive ID
Flexible and sophisticated
Top-Down and Bottom-Up Approaches
Utility in longer-term will require increased
precision, improved robustness, ease-of-use, …
For nanoLC portion, this means:
Flow control
Columns
Overall system stability
System-to-system repeatability
Ease-of-use
Wehr, LCGC 24(9), (2006)
time
sig
na
l
Focus on two components
Fluid delivery Microfluidic Chips
EK Flow Control
1550 1600 1650 1700 1750 1800 1850 1900
0
1
2
3
4
5
6 Flowrate Setpoint
Measured EKFC Flowrate
Flo
w R
ate
[u
L/m
in]
Time [s]
• Inherently microscale (nm’s-thick double-layer)
• No moving parts or seals
• Precision, voltage controlled fluid delivery
• Large dynamic range (nL – 10’s mL)
• High pressure capability
• Excellent performance
EKFC Flow Meter
Flow Restrictor
Pressure Source
Injector
Column
Detector
US Patent 8795493
Microfluidic Flow Control
• Electropneumatic amplifier pumping system
• Direct flow rate feedback to rapidly adjustable pressure source
• Precise flow control at moderate to low flow rates
• Enables highly reproducible and accurate analytical separations
US Patent 8685218
Family of nanoLC prototypes and products to Market
EKPump-based Nanoflow metering system (2002)
Similar platform for first EP-based pumps (2004)
Second generation –
optimized EP pump
system. Two configs,
nano and micro (2006)
Third generation –
self-priming,
UHPLC, several
configurations
(2009)
Fourth generation – higher
precision, lower cost, user
changeable flow ranges (2012).
Lean MFG; SG plant (2013)
System performance
Challenge: Bridging the nanoLC-to-MS gap
nanoLC
Columns,
valves,
fittings,
tubing,…
MS
Need easy-to-use, cost effective nanoconnectors with low dead volume
and compatibility with high pressures and solvents
Apply microfabrication here to integrate
Focus on two components
Fluid delivery Microfluidic Chips
The engineering challenges in microfabricating HPLC
A
B
Injection Pre-heat Column Detection
Sample
Temp - control
Mix
Pump
Pump
Wide range of solvents, acids, bases, buffers
100’s bars operating pressure
Sample injection at high pressure
High pressure chip-to-world interface
Low dispersion requirements
Microfabrication
Fused silica – Isotropic wet-etch procedures
– Cylindrical channels
– Separation columns
– Multiple channels dimensions
– Injectors
– Fusion bonding
High pressure compatible structures (>4,000 psi)
• On-chip functionality:
• Valves (pL volumes)
• Injectors (precise 5 nL injections)
• Columns (variety of phases)
• Detectors (UV, Echem, MS, …)
• Connectors (high pressure, user-friendly)
• Sample Preparation (filters, traps, …)
Injector
Column
piston
bypass
1mm
Open
Closed
Open
Closed
J. E. Rehm, T. J. Shepodd and E. F. Hasselbrink, MicroTAS 2001 Proceedings, pp. 227-229
Chip-to-World Interface
• Microfabrication enabled
• Automatic alignment of multiple
simultaneous connections
• Excellent performance
• > 5000 psi demonstrated
• 4500 psi routine operation
• Robust - many make/break cycles
• Chip caddy – “Jump-drive-like”
• Excellent chemical compatibility
• Thermal control (0.2 C stability)
Connector port
Jig
Chip
US Patent 8021056
3-chip platform for using chip-based nanoLC traps and columns
Simple column/trap changes for different stationary phases
Direct injection, trap-elute, dual column and serial two column nanoLC-MS workflows
Excellent column-to-column reproducibility (<2%);
Independent temperature control for each chip
Microfabrication-enabled tool for easier, precise proteomics
nanoLC cHiPLC MS
Change the chips – change the workflow
Standard
Dirty
sample
Multiplexing
Exhaustive
1
2
3
4 Serial 2-column
2-column switching
Trap and elute
Direct inject
Offline Wash and Load
Sample
Injection
Online MS Acquisition Online MS Acquisition
Offline Wash and Load
Sample
Injection
Offline Wash and Load
Sample
Injection
Online MS Acquisition
Double Sample Throughput Two Column Switching
column 1
connector connector
column 2
LC SYSTEM
MS
VALVE
Customer feedback – longer columns, more phases
Mix-n-Match traps and columns to carry out different experiments
Carbon-phase for glycan separations
PGC
Human IgG
HILIC
Human IgG
Underivatized N-glycan mixtures from
bovine fetuin and human immunoglobulin G (IgG) (ProZyme,
Hayward, CA) were used to test the two separation phases.
Porous Graphitic Carbon (PGC)
and
HALO HILIC phases
Chip columns 75 um x 15 cm
300 nL/min flow rate
Water / ACN (formate); pH 4
Customer cross-site validation
Nature Methods 11, 149-155 (2014)
Same results from systems in Boston, Seattle and Seoul
23 © 2015 AB Sciex. GEN-MKT11-2066-B
• 200 µm ID chip with three well-defined regions (for MudPiT workflow)
• 1cm x 1cm x 1cm RP-SCX-RP (3µm-120A C18, 5µm PolySulfoethyl)
Advanced workflows - multiphase trap chips for MUDPIT
Goal: Simplify and improve 2D LC/MS for complex proteomics
Typical workflow (automated) consists of:
1. Load sample onto multiphase trap
(LOAD)
2. Load salt plugs to fractionate the
peptides (LOAD)
3. Separate the fractionates on
analytical column (Inject)
4. Repeat step 2 and 3 to separate all
fractions
Motoyama and Yates, AChem 80, 7187 (2008)
Mol Cell Proteomics. 2015 Jun;14(6):1708-19.
24 © 2015 AB Sciex. GEN-MKT11-2066-B
500mM 1500mM
50mM 2mM desalt
Reproducibility
Mol Cell Proteomics. 2015 Jun;14(6):1708-19.
25 © 2015 AB Sciex. GEN-MKT11-2066-B
Complex sample analysis with multiphase chips
Melanoma cells were cultured, lysed and digested with
trypsin prior LC-MS/MS and LC-SWATH-MS on
TripleToF 5600 MS (AB SCIEX) with cHiPLC® System
(Eksigent, part of AB SCIEX).
LC-MS/MS (5 μg):
- multiphase trap chips using 5-step salt fractionation
(0, 2, 50, 500 and 1500 mM ammonium acetate)
- 60 min ACN gradients (15 cm, 75 μm ID RP chip)
LC-SWATH-MS (5 μg):
- RP trap chip (0.5 mm, 200 μm ID)
- 60 min ACN gradients (15 cm, 75 μm ID RP chip)
Mol Cell Proteomics. 2015 Jun;14(6):1708-19.
26 © 2015 AB Sciex. GEN-MKT11-2066-B 2
6
Summary – proteomics tools
• Commercialized technologies driven by customer needs with clear value propositions
• Provides valuable data not easily obtained in other ways
• Much easier to use
• Flexibility to address a range of workflows
• Performance for quantitation
• Eksigent pump platform delivers state-of-the-art flow precision at low flow rates
• cHiPLC platform for nano/micro LC analyses is
• Enabled by microfabrication
• Excellent run-to-run repeatability on a single cHiPLC column
• Low variability between cHiPLC column
• Flexible platform enables multiples workflows with one device and minimal
reconfiguration time
27 © 2015 AB Sciex. GEN-MKT11-2066-B
Collaborators Reginald Beer (LLNL) Chris Welch (Merck) Mark Molloy (APAF) Christof Crisp (APAF) Funding NIST Advanced Technology Program Merck Pfizer
Colleagues
Dave Neyer Ken Hencken Nicole Hebert David Wyrick (IntegenX) Doug Cyr (Chevron) Patrick Leung (Illumina) Sammy Datwani (Labcyte) David Rakestraw (LLNL) Guifeng Jiang (Thermo) Jason Rehm Remco van Soest Phillip Paul Christopher Hoyle (UK) Bryce Young Erika Lin Christie Hunter Tom Covey
Thank you for your attention.
Advanced proteomics workflows Proteomic Analysis of E. coli Cell Lysates (1D vs 2D Workflow).
General micro and nanoscale HPLC challenge - variance
Loss of separation efficiency from instrumental components
‒ Large injection volumes
‒ Connection tubing
‒ Dispersion from fittings and connectors
‒ Detector cell
s2 = scol2 + sinj
2 + stube2 + sfittings
2 + sdet2
Column Type Column ID Column length s2extra
nL2
Micro LC 1.0 15 45,300
Capillary LC 300 15 370
Nanoscale LC 75 15 1.4
Proteomics data
5 replicate injections of yeast cell lysate
nanoLC 400 (300 nL/min) and TripleTOF 5600
70 transitions in E. coli lysate nanoLC 400 / QTRAP 5000
Enabled by precision of fluid delivery system,
sample introduction and detection systems
Set of tryptic peptide standards run at nanoflow rates from 200 nL/min and 1.5 µL/min
Shorter run times and higher throughput can be achieved using 200 µm cHiPLC columns
Discovery to Quantitation – customer transition
Other enabled applications
Nanoflow homogeneous biochemical assay
• Mix Incubate Detect (all on chip)
30nL <30 min 2-color FL
• Programmable Titration of Reagent / Compounds
Detection
• Absorbance
• Electrochemical
• Mass Spectrometry Interface
• Fluorescence
• Imaging
Field Corrected
counter electrodes
reference electrodes
working electrode
8 cm x 6 cm x 1.5 cm
Two-Color Fluorescence Detection
Fluorescence Intensity
TR-FRET
Coulometric Array – Series of coulometric detection
cells in which the applied potential used for detection is
monotonically changing.
Electrochemical Detection
Eksigent Potentiostat
Dual electrochemical cell chip
A/D
550 600 650 700 750 800 850 900 950 1000
-50
0
50
100
150
200
250
300
350
nA
pe
ak h
eig
ht
Eapp (mV vs Pd)
160 180 200 220 240 260
0
200
400
Time (sec)
Cu
rre
nt (n
A)
Cell 1 (900 mV vs Pd)
Cell 2 (800 mV vs Pd)
120 140 160 180 200 220
0
100
200
300
Cu
rre
nt (
nA
)
Time (sec)
Cell 1 (720 mV vs Pd)
Cell 2 (800 mV vs Pd)
Phenol (100 uM, 40 nL)
Uracil
Future work for transition to EC detection product:
• Refine cell fabrication to reduce dead volume – Degrades RC time constant
– Does not increase efficiency
• Further reduce Ru
– Promising results with 1 reference electrode cell
– Internal resistance of electrode
• Remove oxygen from mobile phase
• 2 and 4 cell chips – Interaction between cells
• Smaller particles – Transition to flow rates used with Express
• Lifetime testing – Fouling?
• Dispersion through cell (array of 12)
Results with Eksigent Potentiostat
Electrochemical Detection
On-chip MS Interface
Column Weir
Electrode
To ESI tip
30 um channel
Figure A – Chip connector assembly for electrospray
interface chip
Modified connector design
Dual tips, ease of use
Newer designs underway in 2013
(not shown)
Interface Comparison
New Objective Simple chip interface Plug-n-play interface
Cytochrome C Digest
2-60% B in 60 min
500 nL/min
LCQ Deca MS
75 um capillary column
On-chip valves for sample manipulation
Eksigent Proprietary and Confidential
Loop
W
EKFC MP Inj - Col - Det
Loop
W
EKFC MP Inj - Col - Det
Fill
Run
Loop
W
EKFC MP Inj - Col - Det
Loop
W
EKFC MP Inj - Col - Det
Fill
Run
S
Col
MP
W
S
Col
MP
W
S
Col
MP
W
S
Col
MP
W
Trap chip preconcentration of
5 standard peptides (2.5 pmol/uL each),
MS detection
2 4 6 8 10 12 14 16 18
Time (min)
15.42 17.32 1.56
16.36
15.21 1.90 1.33
0 2 4 6 8 10 12 14 16 18 Time (min)
12.26
13.31
12.14 1.16
6.5 E 8
1.4
E 9
500 nL
2 uL
Integration - Mixers
H2O + C5H12O2 + HCl 2 CH4O + C3H6O + HCl
NaOH + HCl H2O + NaCl
k1
k2
Characterize Mixer using Competitive-Consecutive Reactions:
Fourth Bourne Reaction
k1 = 1.4 x 108 m3/mol/s
k2 = 0.6 m3/mol/s tr = 1/k2[DMP]
X = [MeOH] out
[DMP] in
X = 1 tr < tmixing slow mixing
X = 0 tr > tmixing fast mixing
• Working with Merck collaborators to characterize mixing designs – GC-FID Analysis
• Initial results inconsistent with calculations – Herringbone and tee mixer tested
• Identified material compatibility problem – Strong acid not compatible with stainless steel in microfluidic format
• Testing system has been built with compatible materials
On-chip flow-based assay
• Homogeneous biochemical assays • Mix Incubate Detect (all on chip)
30nL <30 min 2-color FL
• Programmable Titration of
Reagent / Compounds
IC50/Ki: 4 – 30 min. depending upon application
Mechanistic studies: 20 min.
P
ATP
PKA
enzyme
TAMRA
fluorophore
+
+
ADP
Flow assay benefits
• Data quality
• Integrated liquid handler, incubator reader
• Reduced turnaround time for single compound
• Reduced operator time / error through
automation
• 100- to 1000-fold reduction in reagent
consumption
• Facile protocol to carry out simultaneous of
multiple reagents to determine
mechanism of action
MoA Studies with flow assay
Figure 10
Figure 10. Simulated initial rate data versus fraction gradient of inhibitor and substrate
modeled to the three classical inhibition mechanism types (competitive (black line),
uncompetitive (red line), and noncompetitive (blue line)) using eq 2. The following fixed
values were used to generate the simulated data: maximum substrate concentration = 10,
maximum inhibitor concentration = 10, Vmax = 10, and Km = 5. The values for the
inhibition constants for the three mechanism types are as follows: competitive inhibition
(Kis = 2, Kii = not applicable), noncompetitive inhibition (Kis = 4, Kii = 4), and
uncompetitive inhibition (Kis = not applicable, Kii = 2)
Fraction Gradient
0 0.2 0.4 0.6 0.8 1
Rel
ativ
e R
ate
0
0.5
1
1.5
2
2.5
3
Figure 10
Figure 10. Simulated initial rate data versus fraction gradient of inhibitor and substrate
modeled to the three classical inhibition mechanism types (competitive (black line),
uncompetitive (red line), and noncompetitive (blue line)) using eq 2. The following fixed
values were used to generate the simulated data: maximum substrate concentration = 10,
maximum inhibitor concentration = 10, Vmax = 10, and Km = 5. The values for the
inhibition constants for the three mechanism types are as follows: competitive inhibition
(Kis = 2, Kii = not applicable), noncompetitive inhibition (Kis = 4, Kii = 4), and
uncompetitive inhibition (Kis = not applicable, Kii = 2)
Fraction Gradient
0 0.2 0.4 0.6 0.8 1
Rel
ativ
e R
ate
0
0.5
1
1.5
2
2.5
3
Competitive (black)
Non-competitive (blue)
Uncompetitive (red)
SK
IK
SVv
ism
1
max
Competitive
iiism K
ISK
IK
SVv
11
max
non-competitive
iim K
ISK
SVv
1
max
uncompetitive
iiis KBIBS
KBIKm
BSVv
11
max
Figure 11
Figure 11. Representative NanoCF data for glycogen phosphorylase using the continuous
variation MOA method in comparison with a microtiter plate-derived orthogonal global
dataset. a) NanoCF data in which the following variables were used: maximum [glycogen] =
1 mg/mL; maximum [inhibitor] = 2 mM. Data from an independent control experiment
yielded a Vmax of 8x105 relative fluorescence units and a Km value of 0.08 mg/mL and was
used in fitting the MOA data to eq 2 (experimental data shown in red and the fit to eq 2 in
black). The calculated values for Kis and Kii are 0.29 ±0.01 mM and 0.59 ±0.01 mM,
respectively. b) A conventional microtiter plate orthogonal dataset demonstrating
noncompetitive inhibition against glycogen phosphorylase. A fit of the data to a
noncompetitive inhibition model yielded the following inhibition constants: Kis = 240 ±110
mM and Kii = 1700 ±900 mM.
1/Glycogen (mL/mg)
0 2 4 6 8 10 12 14 16
1/R
elat
ive
Rat
e
0.002
0.004
0.006
0.008
0.01
0.012
0.014b)
Fraction Gradient
0 0.2 0.4 0.6 0.8 1
Rel
ativ
e R
ate
5e5
6e5
7e5
8e5
9e5
a)
Figure 11
Figure 11. Representative NanoCF data for glycogen phosphorylase using the continuous
variation MOA method in comparison with a microtiter plate-derived orthogonal global
dataset. a) NanoCF data in which the following variables were used: maximum [glycogen] =
1 mg/mL; maximum [inhibitor] = 2 mM. Data from an independent control experiment
yielded a Vmax of 8x105 relative fluorescence units and a Km value of 0.08 mg/mL and was
used in fitting the MOA data to eq 2 (experimental data shown in red and the fit to eq 2 in
black). The calculated values for Kis and Kii are 0.29 ±0.01 mM and 0.59 ±0.01 mM,
respectively. b) A conventional microtiter plate orthogonal dataset demonstrating
noncompetitive inhibition against glycogen phosphorylase. A fit of the data to a
noncompetitive inhibition model yielded the following inhibition constants: Kis = 240 ±110
mM and Kii = 1700 ±900 mM.
1/Glycogen (mL/mg)
0 2 4 6 8 10 12 14 16
1/R
elat
ive
Rat
e
0.002
0.004
0.006
0.008
0.01
0.012
0.014b)
Fraction Gradient
0 0.2 0.4 0.6 0.8 1
Rel
ativ
e R
ate
5e5
6e5
7e5
8e5
9e5
a)
1/Glycogen (mL/mg)
0 2 4 6 8 10 12 14 16
1/R
elat
ive
Rat
e
0.002
0.004
0.006
0.008
0.01
0.012
0.014b)
Fraction Gradient
0 0.2 0.4 0.6 0.8 1
Rel
ativ
e R
ate
5e5
6e5
7e5
8e5
9e5
a)
Courtesy of GSK
Three models for inhibition re-cast to
single model
Simultaneous titration of S and I
Requires very precise and accurate
control of conc
Limited verification experimentally
Flow Assay system
Liquid handling
Reagent Storage (4 ºC-RT)
Microfabricated chip-based analyzer
• Temperature controlled (e.g., 37 ºC)
• Mix, Incubate, Read
• No evaporation
• Coatings
Fluid delivery system
• Nanoflow precision and accuracy
• Programmable continuous dilution
• Fluorescence detection
• Designed for higher data quality,
lower throughput
Particle Imaging System
Rapid Particle Analyzer
Parallel; long illumination time (up to 5 s)
Good flow control allows particles to be trapped in well
defined positions on a channel in a microfluidic device
Laser or LED illumination
Flow Cell
Serial; short illumination time (~5
x 10-6 seconds)
Laser
Integrated cHiPLC system performance Includes microfluidic injector, column and detector
0.2 x 100 mm, 3 mm C18 Luna, 2 L/minTheophyline, Sulfamerazine, Hydrocortizone, Amitriptyline, Bumetanide, Fenoprofen
0.2 x 150 mm; 5 m C18 Jupiter; 2 L/min; 50-85% gradient; 0.1%TFA; 214 nm; Rib A, Cyt C, Holotransferrin, Apomylglobin
0
200
400
600
800
1000
1200
1400
0 5 10
Ab
so
rba
nce
(m
AU
)
Time (minutes)
Small Molecule Separations
0
200
400
600
800
1000
1200
1400
1600
1800
0 10 20 30
Protein Separations
Time (minutes)
Ab
so
rba
nce
(m
AU
)
2D cHiPLC
On-chip valves for sample manipulation
Eksigent Proprietary and Confidential
Loop
W
EKFC MP Inj - Col - Det
Loop
W
EKFC MP Inj - Col - Det
Fill
Run
Loop
W
EKFC MP Inj - Col - Det
Loop
W
EKFC MP Inj - Col - Det
Fill
Run
S
Col
MP
W
S
Col
MP
W
S
Col
MP
W
S
Col
MP
W
Trap chip preconcentration of
5 standard peptides (2.5 pmol/uL each),
MS detection
2 4 6 8 10 12 14 16 18
Time (min)
15.42 17.32 1.56
16.36
15.21 1.90 1.33
0 2 4 6 8 10 12 14 16 18 Time (min)
12.26
13.31
12.14 1.16
6.5 E 8
1.4
E 9
500 nL
2 uL
5 10 15 20 25 30
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Time (min)
100%
50%
25%
10%
Sample: 3-protein digest
Amylose
PSA
Lactose peroxidase
1st dimension:
SCX: Polysulfo-ethyl Aspartamide SCX (silica),
5cm X 0.32 mm, 5 um, 200 A.
A: 5 mM NaOPO; B: 600 mM NaOPO
Trap: On-chip; 4 mm x 0.5 mm, C18 Luna, 3 um
2nd dimension:
RP: On-chip 5cm x 0.2 mm, C18 Luna, 3 um
A: 0.1% FA/H2O; B:0.1% FA/ACN
2% 50% B; 30min
Flow control: Eksigent NanoLC 2D; 500 nL/min
UV
Abs (
214 n
m)
2DLC Separations
Separate trap and column components
Integrated Chip Design
Trap-column combination Sample Rinse
2D Separation
Salt: 45 mM ammonium acetate
Salt: 15 mM ammonium acetate
Salt: 30mM ammonium acetate
Salt: 150 mM ammonium acetate
Salt: 300 mM ammonium acetate
Sample: 1 pmol Lactoperoxidase digest.
1st dimension:
SCX: Polysulfo-ethyl Aspartamide SCX (silica),
5cm X 0.32 mm, 5 um, 200 A.
Trap: On-chip; 4 mm x 0.5 mm, C18 Luna, 3 um
2nd dimension:
RP: On-chip 5 cm x 0.2 mm, C18 Luna, 3 um
Flow control: Eksigent NanoLC 2D; 500nl/min
0 10 20 30
UV
Absorb
ance
Time (min)
Integrated valve, trap and RP column
On-line protein digestion and analysis
20 40 60 80
0
200
400
600
800
Abs (
21
4 n
m)
Time (min)
Cytochrome C digest (trypsin); 15 cm x 0.2 mm Zorbax (3
um SB300) column; 40 pmol injection (5ul at 8 pmol/ul),
2-50% B, 90min gradient
Cytochrome C on-line digest; immobilized trypsin column
(4 mm x 0.2 mm); 37C; 15 cm x 0.2 mm analytical
column; Zorbax (3 um SB300) column; 100 pmol injection
(5ul at 1pmol/ul), 2-50% B, 60 min gradient
20 40 60
0
500
1000
1500
2000
Ab
s (2
14
nm
)
Time (min)
ch0
UV detector RP column
Autosampler Digest column
Trap
1ST Channel
2nd Channel
UV detector RP column
Autosampler Digest column
Trap
1ST Channel
2nd Channel
Range of microfluidic capability
Fluid delivery Integration Detection Micro-Fabrication
Electropneumatic
Electrokinetic
Packed channels
Integrated valves
Microconnectors
Absorbance
Fluorescence
Electrochemical
MS Interfaces
Pump and Assay Systems