This paper presents the first fully integrated gradient-elution liquid chromatography-electrospray ionization (LC-ESI) system on a chip. This chip integrates a pair of high-pressure gradient pumps, a sample injection pump, a passive mixer, a packed separation column, and an ESI nozzle. We also present the successful on-chip separation of protein digests by reverse phase (RP)-LC coupled with on-line mass spectrometer (MS) analysis

Lee, T. D.

Shih, J.

Tai, Y. C.

Xie, J.

English

Caltech Authors - Main

COMPLETE GRADIENT-LC-ESI SYSTEM ON A CHIP FOR PROTEIN ANALYSIS
J. Xie1, J Shihl, Y Miao2, TD. Lei, and YC Tail
lCalifornia Institute ofTechnology, Pasadena, California, USA
2Beckman Research Institute, Duarte, California, USA
ABSTRACT
This paper presents the first fully integrated gradient-
elution liquid chromatography-electrospray ionization
(LC-ESI) system on a chip. This chip integrates a pair of
high-pressure gradient pumps, a sample injection pump, a
passive mixer, a packed separation column, and an ESI
nozzle. We also present the successful on-chip separation
of protein digests by reverse phase (RP)-LC coupled with
on-line mass spectrometer (MS) analysis.
1. INTRODUCTION
LC has remained the workhorse of proteomics and
miniaturization of LC is essential to meet the demands of
fast growing proteomic research and development [1,2].
This is evidenced by the trends exhibited by the state of
the art in LC, which have been towards smaller beads,
smaller diameter columns, and correspondingly lower
flow rates. Together, these trends have lead to higher
resolution, increased sensitivity, and faster separations.
MEMS is ideal for realizing this need for miniaturization.
There are also many advantages to integrating
the complete system onto a single chip. Perhaps most
importantly, dead volume can be virtually eliminated
from the LC system. In desktop LC systems, the
necessary equipment is large and requires numerous
fittings and tubes. The fluidic connections contribute to a
sizable dead volume which increases the time necessary
for separation and could possibly affect the separation
quality as well. With everything optimally integrated on a
single chip, delays associated with dead volume could
virtually be eliminated, allowing separations to be
completed much more quickly. Another advantage is the
possibility of integrating on-chip sensors. This includes,
but isn't limited to, sensors which measure flow rate and
pressure. The ability to integrate sensors seamlessly
within an LC system could in theory produce devices
which have higher reliability and better control than
conventional systems being used today. Miniaturization
and integration of the entire system also has the obvious
ability to create smaller systems thus making them
portable. The decrease in size often leads to a decrease in
power consumption as well, further enhancing the
possibility of portable systems.
In this study, we present separations utilizing
gradient elutions, as opposed to isocratic elutions. Here,
elution refers to the mobile phase being pumped through
0-7803-8732-5/05/$20.00 © 2005 IEEE. 778
-- Organic (eg. Methanol or Acetonitrile)
_•••• Water
100
O-l--,c....-----------:........-+_
Time
Figure 1: A constant flow rate linear gradient goingfrom
0% organic to 100% organic. Typical applications often
only require swings from 0% to 50-70% depending on the
type of sample and organic solvent being used. Optimal
flow rates can also vary depending on the column
dimensions.
Pump A
'-0
MS
PumpB
Figure 2: The typical components in a gradient-LC-ESI
system. For ourparticular application we are coupling to
a MS using ES/.
the column. In an isocratic elution, the mobile phase
composition is fixed, while in a gradient elution, the
mobile phase composition is varied during the separation.
In the case of RP-LC, where we have a highly
hydrophobic stationary phase, we start with a mobile
phase of high aqueous concentration and slowly increase
the organic concentration as the separation continues. In
general, the organic component is a solvent such as
acetonitrile or methanol. A typical gradient is shown in
Figure 1. In practice, this technique allows the analytes to
be separated based on their hydrophobicity. Gradient
elutions are particularly useful when the components of a
sample exhibit a very large range of hydrophobicity,
which is often the case in proteomics. The same sample
analyzed using an isocratic elution would result in an
extremely long analysis time or incomplete separation.
The components of a typical gradient-LC-ESI
system are shown in Figure 2. The basic operation is
straightforward. First, sample is injected onto the column.
After the sample is injected, a gradient is passed through
the column, during which the analytes will elute off the
column in order of increasing hydrophobicity. The
diagrammed system couples to a MS for detection by
means of an ESI nozzle. Several other detection methods
are also available, such as UV or electrochemical
detection, but these methods have the disadvantage of not
providing any infonnation about the mass.
The majority of work regarding on-chip
separations has been electrophoresis-based. Agilent
Technologies has demonstrated a microfabricated
calumnlESI nozzle using laser ablation of Kapton [3].
But this work lacks on-chip pumping. We have
previously demonstrated an ion-exchange LC-on-a-chip
with an integrated column and conductivity detection [4],
which also lacked integrated pumps, mixers, and the
ability to couple to a MS. Perhaps the greatest barrier
preventing the total integration of chip-based LC systems
is the difficulty in integrating micro-scale high-pressure
pumping systems. We recently demonstrated a high-
pressure pump based on electrolysis [5], which we utilize
here.
2. DESIGN
The design of our chip mirrors the typical LC-ESI system
shown in Figure 2. We incorporate all the essential
features onto a lx2 cm chip. A picture of our fabricated
chip is shown in Figure 3. We incorporate three
pumps/reservoirs on our chip, one for a highly aqueous
solution, another for a high organic solution, and finally
one for our sample. The driving force for these pumps is
the pneumatic pressure generated by electrolysis of the
aqueous solutions. The bulk of the volume of our
reservoirs is attained with the chip packaging which will
be described in detail in a later section. In all, each of the
solvent chambers has a total volume of 10 ilL and the
sample chamber is 4 /lL. This is more than sufficient for
our typical separation requirements (e.g., 30-60 min
gradients at a constant flow rate of~100 nL/min).
These three pumps feed into a mixer. Our mixer
is diffusion based and consists ofa long narrow restriction.
It has a cross section of 20x20 (WxH) ]..lm2 and a total
length of 2 mm. This narrow channel functions as a
mixer by reducing the diffusion lengths necessary for
complete mixing.
After the mixer is our column. The column is
l00x20 (WxH) 11m2 in cross section and 1.2 em long.
There is an opening which we can use to pack beads into
the column. This column is designed to be packed using
conventional slurry methods. The cross section of our
column is comparable to the smaller diameter capillary
columns commonly used today. Our column is much
shorter than commercial colunms used today, but it is
sufficient to demonstrate the concept. Currently, colunm
length is mainly limited by how well that packaging
responds to the higher pressures needed with longer
columns.
779
Finally, after the column we have a freestanding
parylene nozzle which extends 1.5 nun past the end of the
chip which we use to couple with a MS.
Figure 3: A photograph ofthe fabricated chip. Note the
common features between the chip and the system
depicted in Figure 2.
3. FABRICATION
The chips are fabricated by combining several polymer
micromachining technologies. The pump chambers are
formed using SU-8 and PDMS while the mixer, the
column and nozzle are all realized using parylene. The
fabrication process is shown in Figure 4.
First, TilPt is deposited and patterned to fonn the
electrodes. The electrodes used for electrolysis are a pair
of interdigitated electrodes with width/spacing of 50/50
/lID. There are also electrodes at the base of the nozzle
which are used to facilitate electrospray from the nozzle.
Next, the oxide is patterned and the exposed Si is
roughened using XeF2• This roughened 8i serves as an
anchor for the parylene. This is necessary to make sure
the column and channels are able to withstand high
pressures. The adhesion of patylene to either oxide or
bare silicon would be inadequate for our application.
Next, a 4 /lm parylene layer is deposited and pattered
using oxygen plasma. This bottom layer fonns the
bottom of the nozzle. Next, a 20 ]..lm sacrificial
photoresist layer is spun on. A double exposure reduces
the photoresist height in some areas to 5 flm. This
reduction in height is necessary to form the filter
structures. After a final 4 llm parylene deposition and
patterning, a 50 /lm SU-8 layer is spun on the chip and
patterned to strengthen the parylene structures and also
provide a planarized surface for further packaging. The
sacrificial photoresist is then dissolved and XeF2 used to
undercut the nozzle. The chips are broken along the
XeF2 etched trench to make the nozzle completely
freestanding.
::
1. Pattern oxide (l.5 !-lm) and PtJTi (200 run / 30 nm)
electrolysis electrodes.
!=~I ~
2. Pattern l't parylene (4 !lm), sacrificial photoresist (20
]lID and 5 ]lm) and 2nd parylene (4 !lm) to form the
nozzle.
impurities leaching out of it which could contribute to
background noise. To prevent this, all PDMS is soaked
for several weeks in ethanol to try to remove these
leachables.
Glass. Cover Silicon . PD~S p.aC.ke.d..C18\ \--[J'It--=~
3. Pattern SU-8 (50 !-lm) to planarize the surface, XeF2
etching to release the nozzle.
4. RESULTS
Figure 5: A diagram afthe packaging afthe chip as well
as a picture of the packaged chip. Note the bubbles
formed inside chambers.
Packing Port Filter
.1.~C!OI.umn,!!
Pump
Chamber,
4. Release sacrificial photoresist and free the
overhanging nozzle.
r::=::J c=:J ~ c::::::::I c::::J _
Silicon Oxide Pt/Ti Parylene PR SU-8
Figure 4; Fabrication process flow.
Further packaging is needed to form the pump
chambers. The packaging is illustrated in Figure 5. The
first layer in the packaging is a 500 Jlm PDMS gasket.
The second layer is a Si piece which has been etched
through to further increase the volume of the chambers.
Finally, a top PDMS piece is placed over the Si, sealing
the chamber. Prior to chip operation, the necessary
solvents and sample are injected into their respective
chambers using a syringe. A glass cover piece is placed
over the packaged chip and the entire structure is then
clamped together and positioned in front of the MS.
One very important parameter in designing this
chip was finding materials which are compatible with the
sample and the organic solvents commonly used in HPLC.
All our channels, column, and nozzle are fabricated using
parylene. Parylene is an ideal material because of its
excellent chemical compatibility. It is also impermeable
to most liquids and gases. To strengthen our parylene
channels and for packaging purposes, SU-8 is also used.
SU-8 has been demonstrated as a possible material for use
MS analysis [6] and is widely considered to be
compatible with most organic solvents. We also use
PDMS as a sealing material. PDMS is generally not used
with organic solvent because of the swelling [7]. Seals
made from PDMS are adequate for the pressures we
expect to encounter though. PDMS is likely to have
The column is slurry-packed with 5 !-lm C I8 beads before
packaging. 5!lm C I8 beads are mixed with IPA and
mixed vigorously. The beads solution is then forced into
the column at high pressure (-100 psi).
Separation of a Bovine Serum Albumin (BSA)
digest is presented. Figure 6 shows the MS output as well
as the control current profiles used for the pumps. In this
separation, the aqueous chamber was filled with a
95/5/0.1 (water/acetonitrile/formic acid v/v) solution
while the organic chamber was filled with a 50/50/0.1
solution. The sample chamber contained a I pmoU!lL
sample of BSA digest. The total flow rate during the
gradient is estimated to be 100 nL/min with pressures
inside the chambers reaching 60 psi. 500 fmol of sample
was injected. The current profile was determined
experimentally.
Figure 7 shows the same separation done using a
commercial HPLC system and column. 500 fmol of
sample was used and the gradient goes from 2% to 60%
organic at a rate of 1%/rnin. The column is packed with 3
!lm CI8 beads and has a diameter of 75 ~m and is 10 cm
long. The difference in separation quality is mainly
attributed to the shorter column length on our chip (1.2 vs.
10 cm). Our chip performs similarly in terms of coverage
of the BSA protein, obtaining 50% coverage compared to
60% coverage for the commercial system. Total power
consumption is very low given the high efficiency of
780
pumping with electrolysis. The combined power
consumption of the pumps during chip operation is
generally around I mW.
[7] IN. Lee, C. Park, G.M. Whitesides. "Solvent
Compatibility of Poly(dimethysiloxane)-Based
Microfluidic Devices." Analytical Chemistry, vo175, pp.
6544-6554,2003.
7. ACKNOWLEDGEMENTS
50.0040.00
Organic Pump
10 15 20 25 30 35 40 45 Tlmol!llminl
10.00
1!'Ilens.
xl0 7
2,5
2.0
1,'
1.0
0.5
0.0
,0
'5
300 ,-------------------,
Aqueous Pump~ 250
1 200
." 150
t 100
8 50
0.jUJ1-.-A...,.-..l1...1"--1-,...__......,..__--,.__..I-,-lJ
0.00
1.0
1.5
InlltnS. r--------------------,
x107
0.5
30 35 40 45 50 Trme (min]
Figure 7: Separation of BSA Digest using commercial
HPLC system.
20.00 30,00
Time (min)
Figure 6: Separation ofa BSA digest using our chip as
well as the current profiles for the pumps. (Note the
distinct regions in the separation. 2-9 min.: Rinse with
organic to clean column. 9-15 min.: Column
eqUilibration. 15-17 min.: Sample injection. 18-48 min.:
Gradient. 48 min.-end: Final column cleaning.)
This work is supported in part by NSF ERC Center at
Caltech (EEC-9402726) and NIH through grant 5RO I
RR06217-10.
We have demonstrated the first fully integrated LC-ESI
system on a chip. We integrated (gradient and sample
injection) pumps, a mixer, a column, filters, and an ESI
nozzle to create a functional device. Current work
involves integrating sensors for feedback control and
improving separation performance.
[1] T. Laurell, G. Marko-Varga, "Miniaturisation is
Mandatory Unraveling the Hwnan Proteome,"
Proteomics, vol. 2, pp.345-351, 2002.
[2] N. Lion, T. C. Rohner, L. Dayon, 1. L. Arnaud, E.
Damoc, N. Youhnovski, Z. Y Wu, C. Roussel, 1.
Josserand, H. Jensen, J. S. Rossier, M. Przybylski, H.H.
Girault, "Microfluidic Systems in Proteomics,"
Electrophoresis, vol. 24, pp. 3533-3562, 2003.
[3] K. Killeen, H. Yin, R. Brennen, T. van de Goor,
"Multidimensional Chromatography and Digestion
using HPLC-CHIP/MS," Proc. f.lTAS 2004, vol. 2, pp.
542-544.
[4] Q. He, C. Pang, Y.C Tai, T.D. Lee, "Ion Liquid
Chromatography On-a-Chip with Beads-Packed
Parylene Column," Proc. MEMS 2004, pp. 212-215.
[5] 1. Xie, Y. Miao, J. Shih, Q. He, 1. Liu, YC. Tai, T.D.
Lee, "An Electrochemical Pwnping System for On-
Chip Gradient Generation," Analytical Chemistry, vol.
76,pp.3756-3763,2004.
[6] S. Arscott, S. Le Gae, C. Druon, P. Tabourier, C.
Rolando. "A Planar On-Chip Micro-Nib Interface for
NanoESI-MS Microfluidic Applications." Journal of
Micromechanics and Microengineering, vol 14, pp.
310-316.2004.
6. REFERENCES
5. CONCLUSION
We have also demonstrated the ability to do
multiple separations of the sample without refilling of the
chambers and have achieved separations using other
solvent systems. Also, no significant sources of noise
from the chip have been found
781


Complete gradient-LC-ESI system on a chip for protein analysis

COMPLETE GRADIENT-LC-ESI SYSTEM ON A CHIP FOR PROTEIN ANALYSIS
J. Xie1, J Shihl, Y Miao2, TD. Lei, and YC Tail
lCalifornia Institute ofTechnology, Pasadena, California, USA
2Beckman Research Institute, Duarte, California, USA
ABSTRACT
This paper presents the first fully integrated gradient-
elution liquid chromatography-electrospray ionization
(LC-ESI) system on a chip. This chip integrates a pair of
high-pressure gradient pumps, a sample injection pump, a
passive mixer, a packed separation column, and an ESI
nozzle. We also present the successful on-chip separation
of protein digests by reverse phase (RP)-LC coupled with
on-line mass spectrometer (MS) analysis.
1. INTRODUCTION
LC has remained the workhorse of proteomics and
miniaturization of LC is essential to meet the demands of
fast growing proteomic research and development [1,2].
This is evidenced by the trends exhibited by the state of
the art in LC, which have been towards smaller beads,
smaller diameter columns, and correspondingly lower
flow rates. Together, these trends have lead to higher
resolution, increased sensitivity, and faster separations.
MEMS is ideal for realizing this need for miniaturization.
There are also many advantages to integrating
the complete system onto a single chip. Perhaps most
importantly, dead volume can be virtually eliminated
from the LC system. In desktop LC systems, the
necessary equipment is large and requires numerous
fittings and tubes. The fluidic connections contribute to a
sizable dead volume which increases the time necessary
for separation and could possibly affect the separation
quality as well. With everything optimally integrated on a
single chip, delays associated with dead volume could
virtually be eliminated, allowing separations to be
completed much more quickly. Another advantage is the
possibility of integrating on-chip sensors. This includes,
but isn't limited to, sensors which measure flow rate and
pressure. The ability to integrate sensors seamlessly
within an LC system could in theory produce devices
which have higher reliability and better control than
conventional systems being used today. Miniaturization
and integration of the entire system also has the obvious
ability to create smaller systems thus making them
portable. The decrease in size often leads to a decrease in
power consumption as well, further enhancing the
possibility of portable systems.
In this study, we present separations utilizing
gradient elutions, as opposed to isocratic elutions. Here,
elution refers to the mobile phase being pumped through
0-7803-8732-5/05/$20.00 © 2005 IEEE. 778
-- Organic (eg. Methanol or Acetonitrile)
_•••• Water
100
O-l--,c....-----------:........-+_
Time
Figure 1: A constant flow rate linear gradient goingfrom
0% organic to 100% organic. Typical applications often
only require swings from 0% to 50-70% depending on the
type of sample and organic solvent being used. Optimal
flow rates can also vary depending on the column
dimensions.
Pump A
'-0
MS
PumpB
Figure 2: The typical components in a gradient-LC-ESI
system. For ourparticular application we are coupling to
a MS using ES/.
the column. In an isocratic elution, the mobile phase
composition is fixed, while in a gradient elution, the
mobile phase composition is varied during the separation.
In the case of RP-LC, where we have a highly
hydrophobic stationary phase, we start with a mobile
phase of high aqueous concentration and slowly increase
the organic concentration as the separation continues. In
general, the organic component is a solvent such as
acetonitrile or methanol. A typical gradient is shown in
Figure 1. In practice, this technique allows the analytes to
be separated based on their hydrophobicity. Gradient
elutions are particularly useful when the components of a
sample exhibit a very large range of hydrophobicity,
which is often the case in proteomics. The same sample
analyzed using an isocratic elution would result in an
extremely long analysis time or incomplete separation.
The components of a typical gradient-LC-ESI
system are shown in Figure 2. The basic operation is
straightforward. First, sample is injected onto the column.
After the sample is injected, a gradient is passed through
the column, during which the analytes will elute off the
column in order of increasing hydrophobicity. The
diagrammed system couples to a MS for detection by
means of an ESI nozzle. Several other detection methods
are also available, such as UV or electrochemical
detection, but these methods have the disadvantage of not
providing any infonnation about the mass.
The majority of work regarding on-chip
separations has been electrophoresis-based. Agilent
Technologies has demonstrated a microfabricated
calumnlESI nozzle using laser ablation of Kapton [3].
But this work lacks on-chip pumping. We have
previously demonstrated an ion-exchange LC-on-a-chip
with an integrated column and conductivity detection [4],
which also lacked integrated pumps, mixers, and the
ability to couple to a MS. Perhaps the greatest barrier
preventing the total integration of chip-based LC systems
is the difficulty in integrating micro-scale high-pressure
pumping systems. We recently demonstrated a high-
pressure pump based on electrolysis [5], which we utilize
here.
2. DESIGN
The design of our chip mirrors the typical LC-ESI system
shown in Figure 2. We incorporate all the essential
features onto a lx2 cm chip. A picture of our fabricated
chip is shown in Figure 3. We incorporate three
pumps/reservoirs on our chip, one for a highly aqueous
solution, another for a high organic solution, and finally
one for our sample. The driving force for these pumps is
the pneumatic pressure generated by electrolysis of the
aqueous solutions. The bulk of the volume of our
reservoirs is attained with the chip packaging which will
be described in detail in a later section. In all, each of the
solvent chambers has a total volume of 10 ilL and the
sample chamber is 4 /lL. This is more than sufficient for
our typical separation requirements (e.g., 30-60 min
gradients at a constant flow rate of~100 nL/min).
These three pumps feed into a mixer. Our mixer
is diffusion based and consists ofa long narrow restriction.
It has a cross section of 20x20 (WxH) ]..lm2 and a total
length of 2 mm. This narrow channel functions as a
mixer by reducing the diffusion lengths necessary for
complete mixing.
After the mixer is our column. The column is
l00x20 (WxH) 11m2 in cross section and 1.2 em long.
There is an opening which we can use to pack beads into
the column. This column is designed to be packed using
conventional slurry methods. The cross section of our
column is comparable to the smaller diameter capillary
columns commonly used today. Our column is much
shorter than commercial colunms used today, but it is
sufficient to demonstrate the concept. Currently, colunm
length is mainly limited by how well that packaging
responds to the higher pressures needed with longer
columns.
779
Finally, after the column we have a freestanding
parylene nozzle which extends 1.5 nun past the end of the
chip which we use to couple with a MS.
Figure 3: A photograph ofthe fabricated chip. Note the
common features between the chip and the system
depicted in Figure 2.
3. FABRICATION
The chips are fabricated by combining several polymer
micromachining technologies. The pump chambers are
formed using SU-8 and PDMS while the mixer, the
column and nozzle are all realized using parylene. The
fabrication process is shown in Figure 4.
First, TilPt is deposited and patterned to fonn the
electrodes. The electrodes used for electrolysis are a pair
of interdigitated electrodes with width/spacing of 50/50
/lID. There are also electrodes at the base of the nozzle
which are used to facilitate electrospray from the nozzle.
Next, the oxide is patterned and the exposed Si is
roughened using XeF2• This roughened 8i serves as an
anchor for the parylene. This is necessary to make sure
the column and channels are able to withstand high
pressures. The adhesion of patylene to either oxide or
bare silicon would be inadequate for our application.
Next, a 4 /lm parylene layer is deposited and pattered
using oxygen plasma. This bottom layer fonns the
bottom of the nozzle. Next, a 20 ]..lm sacrificial
photoresist layer is spun on. A double exposure reduces
the photoresist height in some areas to 5 flm. This
reduction in height is necessary to form the filter
structures. After a final 4 llm parylene deposition and
patterning, a 50 /lm SU-8 layer is spun on the chip and
patterned to strengthen the parylene structures and also
provide a planarized surface for further packaging. The
sacrificial photoresist is then dissolved and XeF2 used to
undercut the nozzle. The chips are broken along the
XeF2 etched trench to make the nozzle completely
freestanding.
::
1. Pattern oxide (l.5 !-lm) and PtJTi (200 run / 30 nm)
electrolysis electrodes.
!=~I ~
2. Pattern l't parylene (4 !lm), sacrificial photoresist (20
]lID and 5 ]lm) and 2nd parylene (4 !lm) to form the
nozzle.
impurities leaching out of it which could contribute to
background noise. To prevent this, all PDMS is soaked
for several weeks in ethanol to try to remove these
leachables.
Glass. Cover Silicon . PD~S p.aC.ke.d..C18\ \--[J'
It--=~
3. Pattern SU-8 (50 !-lm) to planarize the surface, XeF2
etching to release the nozzle.
4. RESULTS
Figure 5: A diagram afthe packaging afthe chip as well
as a picture of the packaged chip. Note the bubbles
formed inside chambers.
Packing Port Filter
.1.~C!OI.umn,!!
Pump
Chamber,
4. Release sacrificial photoresist and free the
overhanging nozzle.
r::=::J c=:J ~ c::::::::I c::::J _
Silicon Oxide Pt/Ti Parylene PR SU-8
Figure 4; Fabrication process flow.
Further packaging is needed to form the pump
chambers. The packaging is illustrated in Figure 5. The
first layer in the packaging is a 500 Jlm PDMS gasket.
The second layer is a Si piece which has been etched
through to further increase the volume of the chambers.
Finally, a top PDMS piece is placed over the Si, sealing
the chamber. Prior to chip operation, the necessary
solvents and sample are injected into their respective
chambers using a syringe. A glass cover piece is placed
over the packaged chip and the entire structure is then
clamped together and positioned in front of the MS.
One very important parameter in designing this
chip was finding materials which are compatible with the
sample and the organic solvents commonly used in HPLC.
All our channels, column, and nozzle are fabricated using
parylene. Parylene is an ideal material because of its
excellent chemical compatibility. It is also impermeable
to most liquids and gases. To strengthen our parylene
channels and for packaging purposes, SU-8 is also used.
SU-8 has been demonstrated as a possible material for use
MS analysis [6] and is widely considered to be
compatible with most organic solvents. We also use
PDMS as a sealing material. PDMS is generally not used
with organic solvent because of the swelling [7]. Seals
made from PDMS are adequate for the pressures we
expect to encounter though. PDMS is likely to have
The column is slurry-packed with 5 !-lm C I8 beads before
packaging. 5!lm C I8 beads are mixed with IPA and
mixed vigorously. The beads solution is then forced into
the column at high pressure (-100 psi).
Separation of a Bovine Serum Albumin (BSA)
digest is presented. Figure 6 shows the MS output as well
as the control current profiles used for the pumps. In this
separation, the aqueous chamber was filled with a
95/5/0.1 (water/acetonitrile/formic acid v/v) solution
while the organic chamber was filled with a 50/50/0.1
solution. The sample chamber contained a I pmoU!lL
sample of BSA digest. The total flow rate during the
gradient is estimated to be 100 nL/min with pressures
inside the chambers reaching 60 psi. 500 fmol of sample
was injected. The current profile was determined
experimentally.
Figure 7 shows the same separation done using a
commercial HPLC system and column. 500 fmol of
sample was used and the gradient goes from 2% to 60%
organic at a rate of 1%/rnin. The column is packed with 3
!lm CI8 beads and has a diameter of 75 ~m and is 10 cm
long. The difference in separation quality is mainly
attributed to the shorter column length on our chip (1.2 vs.
10 cm). Our chip performs similarly in terms of coverage
of the BSA protein, obtaining 50% coverage compared to
60% coverage for the commercial system. Total power
consumption is very low given the high efficiency of
780
pumping with electrolysis. The combined power
consumption of the pumps during chip operation is
generally around I mW.
[7] IN. Lee, C. Park, G.M. Whitesides. "Solvent
Compatibility of Poly(dimethysiloxane)-Based
Microfluidic Devices." Analytical Chemistry, vo175, pp.
6544-6554,2003.
7. ACKNOWLEDGEMENTS
50.0040.00
Organic Pump
10 15 20 25 30 35 40 45 Tlmol!llminl
10.00
1!'Ilens.
xl0 7
2,5
2.0
1,'
1.0
0.5
0.0
,0 '5
300 ,-------------------,
Aqueous Pump
~ 250
1 200
." 150
t 100
8 50
0.jUJ1-.-A...,.-..l1...1"--1-,...__......,..__--,.__..I-,-lJ
0.00
1.0
1.5
InlltnS. r--------------------,
x107
0.5
30 35 40 45 50 Trme (min]
Figure 7: Separation of BSA Digest using commercial
HPLC system.
20.00 30,00
Time (min)
Figure 6: Separation ofa BSA digest using our chip as
well as the current profiles for the pumps. (Note the
distinct regions in the separation. 2-9 min.: Rinse with
organic to clean column. 9-15 min.: Column
eqUilibration. 15-17 min.: Sample injection. 18-48 min.:
Gradient. 48 min.-end: Final column cleaning.)
This work is supported in part by NSF ERC Center at
Caltech (EEC-9402726) and NIH through grant 5RO I
RR06217-10.
We have demonstrated the first fully integrated LC-ESI
system on a chip. We integrated (gradient and sample
injection) pumps, a mixer, a column, filters, and an ESI
nozzle to create a functional device. Current work
involves integrating sensors for feedback control and
improving separation performance.
[1] T. Laurell, G. Marko-Varga, "Miniaturisation is
Mandatory Unraveling the Hwnan Proteome,"
Proteomics, vol. 2, pp.345-351, 2002.
[2] N. Lion, T. C. Rohner, L. Dayon, 1. L. Arnaud, E.
Damoc, N. Youhnovski, Z. Y Wu, C. Roussel, 1.
Josserand, H. Jensen, J. S. Rossier, M. Przybylski, H.H.
Girault, "Microfluidic Systems in Proteomics,"
Electrophoresis, vol. 24, pp. 3533-3562, 2003.
[3] K. Killeen, H. Yin, R. Brennen, T. van de Goor,
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using HPLC-CHIP/MS," Proc. f.lTAS 2004, vol. 2, pp.
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[4] Q. He, C. Pang, Y.C Tai, T.D. Lee, "Ion Liquid
Chromatography On-a-Chip with Beads-Packed
Parylene Column," Proc. MEMS 2004, pp. 212-215.
[5] 1. Xie, Y. Miao, J. Shih, Q. He, 1. Liu, YC. Tai, T.D.
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Chip Gradient Generation," Analytical Chemistry, vol.
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[6] S. Arscott, S. Le Gae, C. Druon, P. Tabourier, C.
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310-316.2004.
6. REFERENCES
5. CONCLUSION
We have also demonstrated the ability to do
multiple separations of the sample without refilling of the
chambers and have achieved separations using other
solvent systems. Also, no significant sources of noise
from the chip have been found
781


CONCLUSION We have also demonstrated the ability to do multiple separations of the sample without refilling of the chambers and have achieved separations using other solvent systems. Also, no significant sources of noise from the chip have been found

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Complete gradient-LC-ESI system on a chip for protein analysis

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