Femtoliter Gradient Elution System for Liquid Chromatography

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Femtoliter Gradient Elution System for Liquid Chromatography Utilizing Extended-Nano Fluidics Hisashi Shimizu, Kouto Toyoda, Kazuma Mawatari, Shigeru Terabe, and Takehiko Kitamori Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05302 • Publication Date (Web): 21 Jan 2019 Downloaded from http://pubs.acs.org on January 21, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

Femtoliter

Gradient

Elution

System

for

Liquid

Chromatography Utilizing Extended-Nano Fluidics

Hisashi Shimizu,[b] Kouto Toyoda,[a] Kazuma Mawatari,[a] Shigeru Terabe,[c] and Takehiko Kitamori*[a]

[a] Department of Applied Chemistry, School of Engineering, The University of Tokyo 7-3-1, Hongo, Bunkyo, Tokyo 113-8656 Japan E-mail: [email protected]

[b] International Research Center for Neurointelligence, The University of Tokyo 4-6-1, Komaba, Meguro, Tokyo 153-8505 Japan E-mail: [email protected]

[c] Graduate School of Material Science, University of Hyogo 3-2-1, Kouto, Kamigori, Hyogo, 678-1297 Japan

* Corresponding Author

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract A gradient system was developed for the separation of proteins at femtoliter scale utilizing nanofluidic channels. In the history of chromatography, miniaturization of the separation column is important for efficient separation and downsizing of instruments. Previously, our group developed a small and highly efficient chromatography system utilizing nanofluidic channels, although flexible design of the gradient was difficult and separation of proteins was not achieved. Here, we propose a flexible gradient system using standard HPLC pumps and an auxiliary mixer with a simple sample injection system. In contrast to our previous sample injection system using pressure balance, the system enables a femtoliter-scale sample injection which is compatible with gradient elution using HPLC pumps. The system was carefully designed, verified for sample injection and gradient elution, and finally applied to the separation of proteins from model and real samples. This femtoliter-scale, efficient separation system will contribute to omics studies at the single cell level.


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Introduction Historically, miniaturization of the separation column was a major target of separation science in the liquid phase as represented by column chromatography, including high-performance liquid chromatography (HPLC),1 and capillary electrophoresis.2 In accord, the invention of small separation columns and resultant improvements in separation performance have opened new research fields through the purification of synthesized and natural chemicals, analysis of environmental and biological samples, and DNA sequencing.3,4 Currently, further miniaturization of the separation column is still an ongoing target for better separation performance and downsizing of analytical systems.5,6 One of the strategies for the miniaturization is using capillaries.7 Smaller diameter capillaries packed with smaller diameter particles can achieve both smaller sample volumes and better separation performance, while such separation columns cause higher pressure losses and 1-2 m is almost at the limit of particle diameter.8 To decrease the pressure loss and increase permeability, monolithic columns have been developed and used for a wide range of applications recently.9 Higher permeability can also be achieved by open tubular liquid chromatography,10 although the low surface-to-volume ratio of the open tubular capillary can result in small loading capacity of the sample. Therefore, a porous layer open tubular (PLOT) column has been developed.11 Another strategy for miniaturization is the introduction of microfabrication techniques. Regnier et al., has pioneered this region by fabricating micropillars on a Si substrate.12 Recently, Desmet et al. has strongly focused on a similar separation



column named radially elongated pillars (REP) and achieved nL sample volume and ultrahigh theoretical plate numbers up to one million.13 One question is whether there is a limitation in this miniaturization and reduction of the sample volume. The question is very interesting for both academic research and as an engineering challenge to understand and control extremely small fluid quantities. Our group has focused on solution properties of water confined in extended-nano (10-1000 nm scale) fluidic channels fabricated on fused silica chips.14 At the same time, we have studied various pressuredriven nanofluidic control techniques required for chemical analysis15-18 and developed ultrasmall chromatography utilizing an open extended-nano channel.19,20 To date, the extended-nano chromatography has realized attoliter to femtoliter sample volumes, which is 108-1010 times smaller than conventional HPLCs, m theoretical plate heights, normal and reversed-phase separation,21 and even gradient elution.22 The advantages of extended-nano chromatography are quite appealing for single cell approaches,23,24 especially in proteomics for two reasons. First, the sample volume is extremely small, even compared to a cell or bacterium, which makes possible sampling and analysis from a single cell in principle.25 Second, the separation time is very short, usually less than a few minutes, which makes it easy for high throughput analysis and statistical approaches. However, separation of proteins has not been achieved even if the gradient system was applied. One of the reasons is that the gradient system lacks flexibility. The system can pump two mobile phase and sample solutions via a four-channel pressure controller and mix the mobile phases in a nanochannel,


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but could not maintain a constant flow rate. Furthermore, mixing of the mobile phases was by chance and making a given mobile phase composition was difficult. Therefore, a precise and flexible gradient elution system must be newly developed. Due to a low Reynolds number, generation of gradient in microchannel is very difficult without a micromixer.26,27 In nanochannel, solvents are instantly mixed and such a mixer is not needed, while flow rate and mixing ratio are difficult to determine due to the possibilities of the alterations of the liquid properties found by our previous research. Contrary, HPLC pumps enable us to design such a precise gradient easily. Nevertheless, it is not easy to combine the HPLC pumps with the aL-fL injection system we developed previously. Particularly, the pressuredriven pinched injection does not work due to unbalanced pressures. In our previous studies, pressures were applied from three (top, left, and right) ports of perpendicularly crossed nanochannels to introduce a sample, mobile phase, and buffer, respectively. Before the injection, the pressures from the three sides were balanced at the crossing point, which made possible to inject the sample in the crossing point to the right nanochannel by switching off the pressures from the right and top. However, if the mobile phase is introduced by a HPLC pump, the pressures are unbalanced due to a fluctuation of flow rate of the HPLC pump. Hence, a new injection scheme which does not depend on the pressure balance must be developed. In addition, a micro/nanochannel interface must be carefully designed to fill a considerable flow rate gap between HPLC pumps (~10 L/min) and extended-nano chromatography (~100 pL/min). Therefore, we propose a new micro/nanochannel structure which is



composed of a T-junction nanochannel and micro/nano flow splitter. The objective of this paper is to develop a flexible gradient system for extended-nano chromatography utilizing HPLC pumps and an external mixer. The system was first verified by separating amino acids and applied to the separation of proteins in model and real samples. The sample injection is performed without balancing pressure and not influenced by the fluctuation of flow rate of the HPLC pumps. The simple injection and easy operation of gradient by the HPLC pumps will broaden applicability of extended-nano chromatography and realize omics studies at femtoliter scale.

Experimental Section The experimental system was composed of two HPLC pumps (LC-20AD, Shimadzu, Kyoto, Japan), an auxiliary mixer, pressure controller for sample injection (Bon Koatsu, Saitama, Japan), laserinduced fluorescence microscope for observation (IX71, Olympus, Tokyo, Japan). As safety considerations, goggles for the laser and gloves must be used to avoid laser irradiation of the eyes and chemical exposure to the skin by a leakage of solvents due to the high pressure. The nanochannels were fabricated on a fused silica substrate by electron-beam lithography and reactive ion etching.28 The microchannels and connecting holes were fabricated on another substrate by UV photolithography and reactive ion etching. The two substrates were bonded by plasma-activated bonding (400ºC, 14 h).29 Finally, the inner surface of the micro and nanochannels was modified with octadecylsilyl (ODS)


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groups as reported previously.21 After the ODS modification, the micro/nanochannels were flushed with toluene for 30 minutes at 70 ºC and tetrahydrofuran for 10 minutes at room temperature carefully to prevent channel clogging. The hydrophobic modification was checked by introducing water into the microchannels. No capillary filling into the nanochannel was observed. The chip was sandwiched by two Teflon spacer plates and fixed in a stainless-steel chip holder by screws. To ensure minimal leakage at the connecting holes, the gap between the chip and holder was sealed with Teflon O-rings. Citric buffer for the mobile phase was purchased from Invitrogen, Thermo-Fisher Scientific (Waltham, MA, USA). 6 amino acids (serine, alanine, proline, valine, leucine, and phenylalanine), 3 proteins (ribonuclease A (RNase A), lysozyme, and ovalbumin), acetonitrile, and sodium perchlorate were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The amino acids were labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) purchased from Dojindo Laboratories (Kumamoto, Japan) and the proteins were labeled with fluorescein isothiocyanate (FITC) using a conjugation kit purchased from Sigma-Aldrich Japan (Tokyo, Japan). HepG2 cells were purchased from ATCC (Manassas, VA, USA) and cultured to 70-80% confluence in our laboratory. Approximately 3×106 cells were lysed in a 500 L lysis buffer (TNE buffer added with a surfactant NP-40, protease and phosphatase inhibitor) and centrifuged at 15,000 rpm for 15 minutes. Because the FITC labeling kit was optimized for 5 mg/mL protein solution, the supernatant was diluted by a carbonate-bicarbonate buffer by 50 times before the labeling. The concentration of the total protein in



the diluted supernatant was roughly estimated to be 6.4 mg/mL by UV absorption spectrophotometry using absorption coefficient of bovine serum albumin (BSA). Finally, unreacted FITC was removed by size exclusion chromatography and the protein sample was obtained.

Results and Discussion The nanofluidic device and experimental system is shown in Figure 1. A sample solution was introduced from the top microchannel into the top nanochannel using a pressure controller. In the same manner, a buffer was introduced from the right microchannel into the right nanochannel and mixed with the sample in the left nanochannel. The mobile phase was pumped through the left microchannel at a constant flow rate. Here, the pressure balance, which was used for the pinched injection previously, is not needed. Although flow rate of the mobile phase from the HPLC pumps has a small fluctuation, the fluctuation does not have any influence on the flow condition in the T-junction nanochannel. The sample injection was performed by switching off the pressure applied to the right and top microchannel sequentially. Then, the flow directions in the nanochannels were inverted, which moved the sample in the left nanochannel into the right nanochannel. Thus, the sample injection at femtoliter scale which is compatible with gradient elution using HPLC pumps can be achieved. For efficient separation, the size of the nanochannel was first designed and a theoretical plate number was estimated for isocratic condition by calculations as reported previously.30 When the target theoretical plate number was 104


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for amino acids which have diffusion coefficients of ~10-9 m2/s, the nanochannel should be ~m wide, ~m deep, and ~10 mm long. The aspect ratio and length of the nanochannel were determined considering feasibility of fabrication by electron-beam lithography and reactive ion etching, although longer nanochannels could be fabricated if etching conditions are optimized to uniform etching rate over a wide area.31 In addition, a linear flow velocity in the separation nanochannel should be ~mm/s corresponding to a volume flow rate of ~100 pL/min, although the gradient from the HPLC pumps is ~10 L/min. Therefore, the junction of the microchannel and nanochannel was designed as follows. Flow rate Q of a Hagen-Poiseuille flow in a rectangular channel is expressed as

(

𝑊𝐷3Δ𝑃 192 𝐷 𝑄= 1― 5 12𝜂𝐿 𝜋 𝑊

∞

∑ 𝑛 tanh ( 2 𝐷 ) 1

𝑛𝜋𝑊

5

𝑛,𝑜𝑑𝑑

)

(1)

where W, D, L are width, depth, length of the micro/nanochannels, P is pressure loss, and  is viscosity. Using this equation, W, D, L of the microchannels were determined to satisfy a flow rate in the nanochannel of ~100 pL/min and flow rate in the microchannel of ~10 L/min. The designed values of the W, D, L are shown in Table 1. The gradient flow in the microchannel was split at the junction of the micro/nanochannel in the ratio of 104:1. Then, the flow in the left nanochannel was split further at the T-junction in the ratio of 5:1. Thus, the significant difference of the flow rates of the HPLC pumps and nanochannel was resolved. The volume of the left nanochannel, which determines the injection volume, was designed to be smaller than a single cell. The length of the SUS capillary determines a delay time from gradient generation at the mixer to gradient arrival at the



entrance of the nanochannel. The gradient delay was designed to be approximately one minute at maximum considering that the analysis time would be a few minutes. The experimental system has a dead volume of 0.14 L in the connecting holes of the glass chip. Although the dead volume is much larger than the volume of micro/nanochannels, the effects of the dead volume was negligible because the flow rate of the pump was high (~10 L/min) and constant. The pressure drops were 1.1 MPa for chip 1 and 0.22 MPa for chip 2. Because these pressures were not so high compared to our previous systems, no significant leakage was observed at the connecting holes.

Movie S-1 and Figure S-1 (see Supporting Information) shows that RNase A was injected successfully. In this injection scheme, the sample in the left nanochannel was split into the top and right nanochannels. The split ratio was inversely proportional to the nanochannel lengths and the volume of the injected sample was estimated to be 320 fL.

We observed no sample biasing at the

micro/nanochannel junction during the sample injection. Generally, hindered transport of large molecules into nanopore, nanoslit, and nanochannel may be observed in much smaller size, approximately 101 nm in diameter.32 We also observed that small charged molecules were difficult to enter a 200-nm deep channel.33 However, the influence of electric repulsion was probably negligible in the 950-1000-nm deep channels because they were modified with ODS and the Debye length was also short due to the high ionic concentration of the buffer.


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The linear flow velocity and theoretical plate number were verified by injecting an unretentive amino acid (serine) solution and detecting the sample band at two different points on the nanochannel. The linear flow velocity and theoretical plate number were calculated to be 2.1 mm/s and 1.6×104, which accorded well with the designed value (1.9 mm/s and 1.8×104). The delivery of the gradient was visualized by mixing a fluorescein aqueous solution (10 M) and acetonitrile with a flow rate of 50 L/min. Figure 2 shows gradient programs of the HPLC pumps and time courses of fluorescent intensity observed at the entrance of the nanochannel. The fluorescein solution was mixed with acetonitrile, which produced smooth gradients in the nanochannel. The gradient and flow rate could be controlled independently. Unfortunately, sharp gradient changes were not observed due to mixing caused by a parabolic flow rate distribution in the SUS capillary during gradient delivery. This problem may be solved by a nanoLC pump which has lower flow rate (~nL/min) and allows a shorter delay time. The delay time in this experiment was 26-30 seconds and agreed with the designed value (26 s). From these results, the gradient system was verified to work as designed. The effect of the gradient elution was verified using chip 1 and 6 amino acids as samples. Firstly, the amino acids were separated by an isocratic mode using citric buffer (pH=5.5) containing 10 mM sodium perchlorate as an ion pair reagent. In the isocratic chromatogram shown in Figure 3, 6 of the 7 peaks were identified as serine, alanine, proline, valine, leucine, and phenylalanine. The residual second peak was estimated to be a by-product of the fluorescent labeling (NBD-OH).34,35 Among the



6 peaks, the early 3 peaks were sharp, and the late 3 peaks were broad. Thus, a gradient was designed to improve the late 3 peaks. The gradient should arrive around 5 s, near the peak of valine. To elute phenylalanine earlier than 40 s, the gradient should finish around 35 s. Therefore, a gradient program of 0 - 100% acetonitrile in 6 s (as shown with the red line in Figure 2) was chosen. Because the gradient delay was approximately 25 s, the gradient program was started 20 s before the injection. The estimated gradient transferred from Figure 2B is also shown in Figure 3 with the red line. The gradient chromatogram completely overlapped for the early 4 peaks, and the late 3 peaks showed earlier elution times and smaller bandwidths. Thus, the separation was improved by the preliminary designed gradient and a proof of concept was established for the gradient elution in the extended-nano channel. The maximum speed of the separation is dictated by time resolution of the camera. In Figure 3, the peak of serine consists of ~10 frames, even if the minimum integration time of the camera (30 ms) was used. From the van Deemter theory, the optimum value of the flow velocity for chip 1 was calculated to 8.5 mm/s, which will result in an increase of the theoretical plate number up to 4×104. In such cases, a high-speed camera such as intensifier CCD will be needed. The detection limit was also estimated from the signal-to-noise (S/N) ratio of phenylalanine. The S/N ratio was approximately 70 and the concentration of each amino acid was 200 M, the detection limit was estimated to be 6 M. A separation of 3 model proteins was performed using chip 2 and citric buffer (pH=4.25) added with 10 mM sodium perchlorate. However, irreversible adsorption of proteins on the nanochannel surface


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was the largest problem.36 The stationary phase was first changed, although changing the octadecylsilyl group to trimethylsilyl group was not effective to reduce irreversible adsorption. Therefore, sodium perchlorate, which was added as an ion pair reagent to the mobile phase, was examined. The 3 proteins were positively charged in the buffer and made ion pairs with the negative perchlorate ions, which increased hydrophobic interactions of the proteins with the stationary phase.37 When the sodium perchlorate was removed from the mobile phase, the irreversible adsorption was not observed, and sharp peaks of the proteins were detected. Figure 4 shows chromatograms of the 3 proteins in isocratic and gradient modes with changing the time of gradient delivery. The gradient program was 0-90% acetonitrile in 6 s. The retention time of RNase A was not changed in all chromatograms, while lysozyme and ovalbumin eluted earlier by earlier gradient deliveries. Finally, the gradient was applied to a real sample. Figure 5 shows chromatograms of the HepG2 cell lysate, separated by the same conditions and gradient programs. The broad peak observed around 70 seconds in the isocratic chromatograms was split into two peaks and followed by at least four peaks which were not observed in the isocratic mode. Regarding the amount of the injected sample, the protein concentration in the sample and the volume of the injected sample were estimated to be 6.4 mg/mL and 320 fL, respectively. Therefore, the amount of the injected protein was 2.0 pg, although the sample was diluted further in the processes of labeling, size exclusion chromatography and sample loading before the injection. Because the total weight of protein in a HepG2 cell is approximately 600



pg,38 the amount of the injected sample corresponded to less than 1/300 cell. Recently, the detection limit of mass spectrometers has been greatly improved.39-42 Although mass spectrometry is not in the scope of this paper, our chromatography using nanochannels may contribute to future proteome analysis of a section of a single cell and even living single cells beyond the current full single cell analysis.

Conclusion To summarize, a flexible gradient system for extended-nano chromatography was developed. The system consisted of two commercial HPLC pumps, an auxiliary mixer, pressure controller, fluorescence microscope, and nanofluidic chip which had a T-junction nanochannel for sample injection. The system and chip were designed to show high performance of 104 theoretical plates and analyze extremely small samples of less than 1 pL within a few minutes. Compared to the pinched injection using crossed nanochannels, the T-junction nanochannel realized a simple injection at femtoliter scale compatible with gradient elution using HPLC pumps. The sample injection using the T-junction nanochannel, high-performance separation, and flexible gradient generation was verified. Then, the effect of gradient elution was also validated through the separation of 6 amino acids. Next, the gradient elution system was applied to the separation of proteins. Conditions for the separation of proteins were examined using three model proteins and cellular proteins extracted from HepG2 cells


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were separated by the gradient mode. Because this system can enable an extremely small sample volume of less than a single cell and fast analysis in a few minutes, its contribution to single cell approaches in proteomics and metabolomics is expected. Integration of sample pretreatment such as lysis, sampling, and digestion are future challenges. In addition, connection with an ultrasensitive mass spectrometer will accelerate further applications to omics.

Acknowledgements This work was supported by CREST (Core Research for Evolutional Science and Technology) of JST (Japan Science and Technology Agency). We also thank Dr. Charles Yokoyama of International Research Center for Neurointelligence, The University of Tokyo, for reviewing a draft of this manuscript.

Notes The authors declare no conflict of interest.

Supporting Information Figure S-1, Chromatograms of RNase A for three injections; Movie S-1 (MovieS-1.avi), Sample injection using T-junction nanochannel.



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Figure Captions Figure 1. Nanofluidic device and experimental system. A) Photograph of nanofluidic device and B) Magnified view of T-junction nanochannel. C) Gradient system (left), laser-induced fluorescence microscope (center), and pressure controller for sample injection (right).

Figure 2. Gradient generation. A) Gradient program for HPLC pumps. B) Time course of fluorescence detected at the injection nanochannel.

Figure 3. Isocratic and gradient separation of amino acids. Green and purple lines are chromatograms in isocratic and gradient mode, respectively. Red line shows an estimated gradient in nanochannel transferred from Figure 2B.

Figure 4. Chromatograms of 3 model proteins with different gradient arrival. Gradient program was started A) 70 s, B) 65 s, C) 64 s, D) 63 s, and E) 60 s before sample injection.

F) Isocratic

chromatogram (citric buffer 100%).

Figure 5. Chromatograms of cellular proteins extracted from HepG2 cells. The gradient program was started A) 55 s and B) 50 s before sample injection. C) Isocratic chromatogram (citric buffer 100%).


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Figure 1

A

Nanochannel area

B

200 m

Microchannel

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Microchannel SUS capillary

C

Mixer

Mobile phases

HPLC pumps

Fluorescence microscope

T-junction nanochannel

Nanofluidic chip Liquid reservoir Solenoid valves

Gas tanks


Compressor


Figure 2

A

Fluorescein aq. percentage

120 100

20

30s

80

60s

40

60 60

40

80

20

100 120

0 0

B

0

6s

30

60 Time [s]

Acetonitrile percentage

90

Fluorescence intensity

120 6s

100

30 s

80

60 s

60 40 Delay

20 0 0

30

60 Time [s]

90

120

Figure 3 60000 50000


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 28

Ser

Citric buffer 100%

Isocratic Gradient

40000

-OH Ala

30000

Estimated gradient

Leu Phe

Pro Val

20000

Acetonitrile 100%

10000 0

-20

-10

0

10

20 Time [s]

30

40


50

60

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Figure 4

A Gradient arrival

B

C


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


D

E Ovalbumin RNase A

F

Lysozyme 0

5

10

15 Time [s]

20

25

30



Figure 5

100

A

80 60

Gradient arrival

100 40


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

80 20

B

60 0 0

20

40

60

80

100

120

0

20

40

60

80

100

120

0

20

40

60

80

100

120

100 40 80 20

C

60 0 40 20 0

Time [s]


Page 26 of 28

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Table 1 Designs of microfluidic chips. W, D, L, and V are width, depth, length, and volume of the micro and nanochannels Size of

Size of nanochannels

Flow rate in

Theoreti

right

cal plate

nanochannel

number

340 pL/min

18,000

26 s

220 pL/min

15,000

65 s

Flow rate in Chip

Target

Delay

micromicrochannel

Left

Top

time

Right

channel W = 3.0 m W = 500 m D = 10 m

1

W = 3.0 m

W = 3.0 m

D = 1.0 m

D = 1.0 m

L = 5.0 mm

L = 20 mm

W = 5.0 m

W = 5.0 m

D = 950 nm

D= 950 nm

L = 5.0 mm

L = 10 mm

D = 1.0 m

Amino 50 L/min

L = 200 m

acids L = 6 cm

V = 600 fL W = 5.0 m W =1.0 mm D = 950 nm 2

Proteins

D = 10 m

20 L/min L = 200 m

L = 6 cm V = 950 fL



Table of Contents 60000

Estimated gradient

50000

3 cm

40000

Gradient

30000

Isocratic

200 m

20000

600 femtoliter

10000 0 -20

0

20 Time [s]

40

60


Page 28 of 28

Femtoliter Gradient Elution System for Liquid Chromatography

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