Patent Application
20140291155
When performing microchip capillary electrophoresis (CE), sample introduction and injection can significantly affect CE performance. Currently, electrokinetic injection is used almost exclusively for microchip CE sample injection. In a particular type of electrokinetic injection known as “pinched” injection, various electrodes in a first mode apply voltages in four intersecting channels to drive sample through the intersection to a waste reservoir. In a second mode, the voltages induce injection of only a plug of sample occupying the small volume in the intersection towards a separation channel. The voltage applied to the separation channel is different in the first mode, wherein sample is diverted to waste, compared to the second mode, wherein the plug of sample is injected into the separation channel. Except for the sample plug, the vast majority of sample is wasted.
While electrokinetic injection can yield small sample plugs for improved separation efficiency and can minimize electrophoretic injection bias under certain conditions, it also has several significant limitations. For example, a considerable amount of time is required to achieve steady state in the first mode. Steady state is a necessary condition to avoid sample bias and/or injection bias caused by high mobility species arriving more quickly than low mobility species. During prolonged operation, the high mobility species can be depleted preferentially and prematurely from the sample supply. Sample utilization is extremely inefficient because the total volume required is very large compared to the actual injected plug volume, which is very small. Furthermore, the injection volume is fixed because it is determined by the geometry of the intersection. In order to change the injection volume, the geometry of the intersection must typically be altered. Further still, the rate at which sequential injections can be analyzed, and the total number of sample plugs that can be injected into the separation channel, is limited by the steady-state flow requirements and by the changing voltages in the separation channel associated with electrokinetic injection.
In view of at least the limitations described above, a need for an improved sample injector for separation analysis exists.
The present invention is a microfluidic injector system based on a mechanical valve rather than electrokinetic injection. The injector system comprises a port configured to interface with a discrete separation column connected to the injector system via the port. The mechanical valve can be operated to provide rapid sequential sample injections and to eliminate the need for changing the electric field in an attached separation column to induce sample injection. Instead, sample injection is accomplished by pressure gradients and by opening the mechanical valve. A constant electric field can, therefore, be continuously applied along the separation channel. Varying the sample pressure and/or the duration of time that the valve remains in an open position can vary sample injection volume. Depending on the type and configuration of the separation column attached to the microfluidic injector, various kinds of separations and analytical techniques can be performed. The terminal end of the separation column can also be configured to enable different analyses. For example, an electrospray ionization (ESI) emitter can be arranged at the end of the separation column. Furthermore, mass spectrometry can be performed to analyze the sample after CE separation.
For sample introduction using the embodiments described herein, it can be advantageous, though not required, for the introduced sample to displace fluid in the sample loading channel preferentially to the side of the sample channel opposite the separation column. This increases probability that the introduced sample does not disturb an ongoing separation in the separation column. If the introduced sample alternatively displaces fluid towards, or into, the separation column, it may degrade a separation already underway inside the separation column, as the pressure pulse from the injection may induce broadening of the separated peaks. This condition will likely automatically be met when the separation column contains packed particles, monolithic material, a cross-linked gel or a viscous polymer solution as these materials in the separation column increase its back pressure substantially. In one embodiment involving an open tubular separation column, it is important to ensure that the backpressure in the separation column is at least 10 times greater than the backpressure of the sample loading channel on the side opposite the separation column. This can be achieved by ensuring that the ratio of the length of the separation column to its cross-sectional area is at least 10 times greater than the ratio of the length of the sample loading channel on the side opposite the separation column to its cross-sectional area.
In one embodiment, a microfluidic injector system comprises a flow layer having a first sample channel connected in a T-shaped arrangement to a loading channel at a first intersection, wherein the first sample channel has a source of pressure and is configured to maintain a sample pressure greater than that of the loading channel at the first intersection. The loading channel has a terminus comprising a port configured to interface with a separation column. The system further comprises a control layer comprising a valving channel, wherein the valving channel in the control layer crosses over the first sample channel in the flow layer at or near the first intersection. The system also comprises a mechanical valve, not an electrokinetic-based injector, to control a sample injection from the first sample channel into the loading channel, the mechanical valve comprising a deformable membrane between the control layer and the flow layer and separating the valving channel and the first sample channel, wherein the membrane has a closed position to obstruct flow in the first sample channel, and an open position to permit flow in the first sample channel based on a first and second pressure, respectively, in the valving channel.
In some embodiments, the system comprises one or more CE electrodes arranged along the loading channel and configured to continuously apply an electric field for CE separation. Additional CE electrodes can be arranged along an attached separation column and can be configured to continuously apply an electric field for CE separation. As used herein, the continuous application of an electric field along the loading channel and/or separation column for CE separation is significant because any sample injection provided to the separation channel will be subject to a continuously applied CE separation field. There is no required change in voltage between an injection mode and a CE separation mode.
A sample channel is connected to the loading channel at an intersection and has a sample pressure that is greater than that which is present in the loading channel near the intersection. The sample channel does not have electrodes that apply voltages for electrokinetic injection. A sample injector in the sample channel, or at the intersection, comprises a mechanical valve to control sample injection from the sample channel to the loading channel. When the valve is opened for a short time, a small volume of sample solution is pushed into the separation channel under a low pressure. When the valve is closed, the sample solution is completely isolated from the run buffer in the loading channel such that there is no risk of sample leakage during the operation, and a discrete, well defined sample plug is injected with each valve opening event.
In some embodiments, the system comprises a plurality of sample sources connected to the first sample channel via a manifold. Valves and/or fluid flow controllers can be utilized to select which sample source, or combination of sources, provides an injection through the sample channel. In other embodiments, the system comprises at least one additional sample channel connected in a T-shaped arrangement to the loading channel at the first intersection or at an additional intersection. The additional sample channel is configured to maintain a sample pressure greater than that of the loading channel at the first or additional intersection. An additional valving channel for each additional sample channel can also exist. The additional valving channel in the control layer can cross over the additional sample channel in the flow layer at or near the first intersection or the additional intersection. An additional mechanical valve, not an electrokinetic-based injector, for each additional sample channel can control sample injection from the additional sample channel into the loading channel. The additional mechanical valve comprises a deformable membrane between the control layer and the flow layer and separates the additional valving channel and the additional sample channel. The additional membrane has a closed position to obstruct flow in the additional sample channel, and an open position to permit flow in the additional sample channel based on a first and second pressure, respectively, in the additional valving channel.
In embodiments having a first sample source connected to the first sample channel and an additional sample source connected to the additional sample channel, the first sample source can contain an analyte or an analyte precursor while the additional sample source can contain an analyte-derivatizing reactant or an analyte precursor. The injection from the first and additional sample channels can mix and/or react in the loading channel. If the positions of the first and additional sample channels are offset, or they occur at differing positions of the loading channel, then the injections can be timed appropriately to facilitate the desired degree of mixing of the plurality of injections.
Examples of the analyte-derivatizing reactant can include, but are not limited to radiolabels, fluorescent labels, labels to enhance electrospray ionization efficiency, and reactants that alter the charge state of the analyte.
A significant characteristic of the system is that the sample injection is independent of the separation technique. For example, during CE operation, a high voltage is applied only along the loading channel and/or separation column and no voltage switching is needed. The sample is directly provided into the loading channel and/or separation column for subsequent separation. There is no need to wait for production of a steady-state, stable sample plug as would be required in the traditional electrokinetic injection. Discrete sample plugs can be injected repeatedly over relatively long periods of time. The injection and separation frequency is only determined by the actuation of the mechanical valve. A valve having a high duty cycle makes it possible to perform continuous flow monitoring, high throughput analysis, and/or multiplexed separations. Systems having multiple sample channels with multiple sample sources and discrete valves can enable many combinations of sample injections and combinations.
Embodiments of the present invention can further comprise a plurality of discrete injections of samples from one or more sample channels to the loading channel in a rapid sequence. The sequence can preferably be pseudo-random. A detector at the end of a separation column attached to the loading channel through an interface can detect the discrete injections after CE-induced overlap, which comprises mixing of at least one component from at least one of the discrete injections to another discrete injection. A processing device executes programming to deconvolute the CE-induced overlap in data collected by the detector so that a spectrum can be reconstructed.
Some embodiments can further comprise a plurality of separation columns as well as a manifold at or near the interface between the loading channel and the separation columns. The manifold comprises a conduit that transitions from a single fluid flow line to multiple flow lines and distributes the flow of sample injections among the multiple flow lines. Each flow line can proceed to a separation column. Preferably, an electrospray ionization (ESI) emitter is connected at the end of each separation column.
In some embodiments, sample injections from the microfluidic injector systems have a volume less than 10 nL. In other embodiments, the volume is less than 1 nL. In still other embodiments, the volume is less than 200 pL.
The separation column, as used herein, can refer to a capillary or another tube. In one embodiment, the separation column can have an inner dimension that is as small as 10 micrometers and which may be as large as 5 millimeters. The separation column may be an open tube filled with an electrolyte solution for performing capillary zone electrophoresis separations. The column may also be filled with a viscous polymer solution, a cross-linked gel or another medium for performing sieving-based separations (e.g., capillary gel electrophoresis). The separation column may also be filled with chromatographic media in the form of particles or with a monolithic material having chemical properties such that, e.g., capillary electrochromatography can be performed using the column. Other suitable examples are encompassed by the present invention though they are not listed herein.
In some embodiments, liquid chromatography (LC) separation is performed in conjunction with CE separation. Accordingly, the system can further comprise a LC column connected to a sample channel and can provide LC separation prior to injection into the loading channel.
Embodiments of the present invention also include methods for analyzing a sample having a plurality of components using CE separation. In a particular embodiment, the method includes the steps of applying a sample pressure in the sample channel greater than the sample pressure in a separation channel. The sample channel is connected to the separation channel at an intersection and lacks electrodes associated with electrokinetic-based injectors. A continuous electric field for CE separation is applied along the separation channel. Injection of the sample occurs by mechanically opening for a duration a mechanical valve, not an electrokinetic-based injector. The mechanical valve is located in the sample channel or at the intersection. The electric field in the separation channel can then separate the components in the injection.
The method can further comprise repeating the mechanical opening in a rapid, pseudo-random sequence to provide a plurality of discrete injections of the sample from the sample channel to the separation channel. CE-induced overlap can be the result of mixing at least one component from at least one of the discrete injections with another discrete injection. The discrete injections are then detected at the end of the separation channel after CE-induced overlap. Finally, the CE-induced overlap in data collected by the detector is deconvoluted so that a spectrum can be reconstructed.
Alternatively, the method can further include distributing one or more injections among a plurality of CE channels within the separation channel. In preferred embodiments, an electrospray can be generated at the end of each CE channel.
In another embodiment, LC separations can be performed in conjunction with the CE separations. Accordingly, the method can further comprise separating the sample in a liquid chromatography column prior to providing an injection to the separation channel.
The purpose of the foregoing abstract is to enable the United States Patent and Trademark Office and the public generally, especially the scientists, engineers, and practitioners in the art who are not familiar with patent or legal terms or phraseology, to determine quickly from a cursory inspection the nature and essence of the technical disclosure of the application. The abstract is neither intended to define the invention of the application, which is measured by the claims, nor is it intended to be limiting as to the scope of the invention in any way.
Various advantages and novel features of the present invention are described herein and will become further readily apparent to those skilled in this art from the following detailed description. In the preceding and following descriptions, the various embodiments, including the preferred embodiments, have been shown and described. Included herein is a description of the best mode contemplated for carrying out the invention. As will be realized, the invention is capable of modification in various respects without departing from the invention. Accordingly, the drawings and description of the preferred embodiments set forth hereafter are to be regarded as illustrative in nature, and not as restrictive.
Embodiments of the invention are described below with reference to the following accompanying drawings.
The following description includes the preferred best mode of embodiments of the present invention. It will be clear from this description of the invention that the invention is not limited to these illustrated embodiments but that the invention also includes a variety of modifications and embodiments thereto. Therefore the present description should be seen as illustrative and not limiting. While the invention is susceptible of various modifications and alternative constructions, it should be understood, that there is no intention to limit the invention to the specific form disclosed, but, on the contrary, the invention is to cover all modifications, alternative constructions, and equivalents falling within the spirit and scope of the invention as defined in the claims.
The control layer (dashed line) contains a microchannel 104 for valving. An exemplary, but not limiting, width for the channels is 100 μm. Exemplary lengths for the separation and sample channels are 3 cm and 0.5 cm, respectively. The channel 104 on the control layer crosses over the sample injection channel 103 and, therefore, the area at the T-shaped intersection 101 is 100 μm×100 μm and is large enough for a mechanical injector comprising a pneumatic valve 105. The pneumatic valve includes a thin elastomer membrane formed between the two layers. When a pressure is applied in the control channel, the membrane separating the two channels deforms into the flow channel to seal the sample channel (i.e., closed valve). When the pressure is released, the membrane recovers to the original state to open the valve.
Integrated poly(dimethylsiloxane) (PDMS) microchips were fabricated using multilayer soft lithography techniques. First, two kinds of silicon templates were produced using standard photolithographic patterning. The silicon template for the control layer 107 was modified with hexamethyldisilazane (HMDS) using gas phase deposition method in order to assist in releasing the PDMS membrane from the patterned template. A 10:1 weight ratio of PDMS base monomer to curing agent was then mixed, degassed under vacuum, poured onto the patterned wafer for the flow layer 106 to a thickness of 1-2 mm and spin-coated on the surface-modified wafer at 2000 rpm for 30 s to coat the control layer to a thickness of ˜50 μm, and cured in an oven at 75° C. for 2 h. After removing the patterned PDMS with flow channels from the template, a small through-hole was created at the end of sample injection channel by punching the substrate with a manually sharpened syringe needle. Two holes were generated at the ends of separation channel for connections to buffer reservoirs. The flow layer PDMS piece was then cleaned and treated with oxygen plasma for 30 s. Immediately, it was aligned on the top of the control layer PDMS membrane (still on the silicon wafer) and assembled together to enclose the flow channel. After placing in an oven at 75° C. for 2 h to form an irreversible bond, the PDMS block containing flow and control layers was removed from the control layer silicon template, a hole was punched at the end of the control channel, and the substrate was finally bonded to an unpatterned PDMS piece 108 to enclose the control channel using oxygen plasma treatment. An expanded view of the three layers 106, 107, and 108 composing the assembled channels and valve is illustrated in
In the present example, polyE-323 was used to coat the PDMS microchannel surfaces to provide anodic EOF in the same direction as electrophoresis for the negatively charged analytes. PolyE-323 is a cationic polyamine, which can be absorbed on negatively charged surfaces through strong electrostatic interaction. The polymer can support strong and stable anodic electroosmotic flow (EOF). To reduce analyte adsorption onto the modified surface resulting from the electrostatic interaction, all samples were prepared in a buffer solution containing 0.5% HPC. Briefly, the PolyE-323 was synthesized by mixing 17.65 g 1,2-bis(3-aminopropylamino)ethane with 20 g water and 9.3 g epichlorohydrine by strong stirring. Two days later, 100 g water was added and the reaction was allowed to continue for 1 week at room temperature. The polymer solution (5 mL) was then diluted in 25 mL of 0.2 M acetic acid, adjusting the pH to ˜7. The diluted polymer solution was filtered using 0.2 μm syringe filters and stored at 4° C.
During the 2 h following the PDMS microchip assembly with oxygen plasma treatment, the diluted polyE-323 solution was pumped into the separation channel through the sample introduction hole for 10 min, and then the solution was left in the channel for 30 min. The channel was then flushed with 10 mM ammonium acetate (pH 7) for 10 min to remove excess polymer. Finally, the microchip was filled with a run buffer (10 mM carbonate buffer, pH 9.3) for CE separation.
A laser-induced fluorescence (LIF) system was employed for detection. Briefly, a 488-nm line from an air-cooled Ar ion laser was passed into an inverted optic microscope and the fluorescence was collected using a CCD camera. For imaging experiments, a mercury lamp was used as the light source and the fluorescence was collected with a digital camera.
In one embodiment, the sample injection can be operated at different frequencies in the present injection mode.
The pressure-injected sample volume can depend on several factors, including the mechanical injector open time, sample injection pressure, and the backpressure present in the separation channel. The mechanical injector open time is determined by the actuation time, the valve control pressure, and the mechanical properties of the injector (e.g., the PDMS membrane in the pneumatic valve described previously).
Similarly, the peak width increases with the sample injection pressure (
One advantage of pressure-based injection method is a lack of injection bias, which can be evaluated based on the separations shown in
The fast, reproducible sample injections described above provide for multiplexed CE, in which separations from multiple injections overlap in the separation channel and are subsequently analyzed in comparison and in combination. The multiplexed CE separation can provide an increase in signal-to-noise ratio and a much higher duty cycle compared with discrete CE separations. In some embodiments, multiplexed CE is coupled with LC separations for two dimensions of orthogonal liquid phase separations, and for providing a much greater peak capacity without significant increase in the analysis time. Generally, LC-CE has not been practical using electrokinetic injection, as the low sampling rate and the long delay time required between injections has led to almost all of the sample from the LC column being wasted. The combination of the fast injections and the multiplexed CE can utilize at least 50% of the LC eluent for high-sensitivity, high-peak capacity analyses.
In a preferred embodiment, LC is coupled to fast, multiplexed microchip CE separations followed by high-performance electrospray ionization-mass spectrometry (ESI-MS). Accordingly, a LC column is connected to the sample channel, which provides a plurality of sample injections to the separation channel through a sample injector. The sample injector can include the pneumatic valve describe elsewhere herein or another mechanical valve providing fast, reproducible sample injections without the use of electrokinetic techniques. The separation channel terminates in one or more ESI emitters directed at a mass spectrometer. This configuration can provide at least a 5-fold increase in proteome coverage for complex samples relative to a comparable LC-MS platform alone.
The pressure differential between the sample channel and the separation channel is such that upon briefly opening the mechanical valve, analyte is rapidly introduced for separation. A potential is continuously applied in the separation channel enabling automatic separation of the sample injection. Preferably, the plurality of rapid injections occurs in a pseudorandom sequence resulting in multiple overlapping separations, which can be deconvoluted to reconstruct a spectrum. Deconvolution can be achieved according to a modified Hadamard transform. The achievable gain in signal-to-noise ratio relative to simple signal averaging can be proportional to the square root of the number of injections.
As mentioned elsewhere herein, one challenge in coupling LC and CE separations is the disparity in sample flow rates associated with each technique. LC typically requires higher flow rates by orders of magnitude. Accordingly, referring to
Preferably, each CE channel will terminate with an electrospray 900 at its own ESI emitter. The array of emitters can provide an order of magnitude sensitivity improvement relative to a single emitter analysis. The enhanced sensitivity, in combination with the greater than ten-fold gain in peak capacity relative to the equivalent LC separation alone can lead to significantly improved dynamic range, more quantitative ionization, and greatly increased sample identifications.
In some embodiments, rather than utilizing a separation channel that is integrated into the microchip containing the microfluidic injector, a discrete separation column is interfaced to the microfluidic injector through a port configured for connecting the separation column to a loading channel. Referring to
Referring to
Referring to
Referring to
The variety of sample injection possibilities enabled by embodiments having multiple sample channels and/or multiple sample sources per sample channel enables a high degree of analytical flexibility. For example, on-column sample derivatization can enable or enhance chemical analyses. To perform on-column sample derivatization, one sample channel contains an analyte or a mixture of analytes (Sample Channel A). Another sample channel contains the derivatization reagent or mixture of reagents (Sample Channel B). Alternatively, Sample Channel A may contain derivatization reagent(s) and Sample Channel B may contain analyte(s). If the two sample channels are located in close proximity to one another, the valves separating the analyte(s) (Valve a) and derivatization reagent(s) (Valve b) may be opened simultaneously such that a mixture of analyte(s) and derivatization reagent(s) is formed in the sample loading channel. Alternatively, if A and B are not in close proximity, the timing for opening Valves a and b may be staggered such that Valve a is opened first, sample from Sample Channel A moves to the proximity of Sample Channel B, at which time Valve b is opened to achieve mixing of A and B.
Multistep sample derivatization procedures may also be achieved beyond simply combining sample from Sample Channels A and B. For example, A and B may be located in close proximity to one another and opened simultaneously to initiate the first step of a derivatization reaction. A third sample channel (C) and its corresponding valve (c) may be located downstream of A and B. The distance from Channels A and B to Channel C, and the velocity of the mixture of A and B as it travels to C will determine the amount of time that A and B have to react prior to the introduction of C by opening Valve c. By adding additional sample channels D, E, etc., each with corresponding valves d, e, etc., on-column derivatization reactions having more individual mixing steps may be accomplished. One example of a multistep derivatization reaction would be one in which A contains analyte(s), B contains an acid or a base that is used to alter the charge state of the analytes in A, and C contains the derivatization reagent that only binds to A in the charge state achieved by prior combination with B.
Examples of sample derivatization procedures that may be employed to enable or enhance chemical analyses include, but are not limited to, (1) adding a radiolabel for detection of analytes based on the radioactive decay of the label; (2) adding a fluorescent molecule to an analyte that does not fluoresce natively for fluorescence detection; (3) attaching a hydrophobic moiety to an analyte that in its native state exhibits low ionization efficiency when detected using electrospray ionization mass spectrometry; (4) attaching a singly or multiply charged moiety to alter the charge state of the analyte. (4) may be used to impart charge to a mixture of natively uncharged analytes that cannot otherwise be separated by capillary electrophoresis. There are numerous other sample derivatization procedures beyond the four examples provided here that can be used to enable or enhance chemical analyses.
PDMS for fabrication of the microchip was prepared by thoroughly mixing a silicone elastomer (Sylgard® 184) base and curing agent at a 10:1 ratio. The PDMS was poured onto the control layer template and spin-coated at 2000 rpm for 30 s. PDMS was also poured over the flow layer and cover plate templates to a thickness of 3-4 mm and degassed under vacuum. All substrates were cured at 70° C. for 2 h. The flow layer substrate was then removed from its template and holes were formed using a 20 gauge catheter punch. Debris was removed from the substrate by applying compressed nitrogen followed by tape to both sides. The surfaces of the flow layer and control layer substrates were activated in an oxygen plasma system at 50 W power and 200 mTorr pressure for 30 s. Following activation, the flow layer substrate was aligned at the sample introduction intersection and brought into contact with the control layer with the aid of a digital microscope. The irreversibly bonded assembly was placed in an oven at 70° C. for 1 h to improve bond strength and then the bonded flow and control substrates were cut and removed from the control layer template. It was necessary to remove the portion of the membrane that spanned the loading channel. This was accomplished by grasping the suspended membrane with a pair of fine-tipped tweezers and carefully pulling in such a way that the membrane tore along the loading channel walls. A hole was punched through both substrates as described above to provide access to the pneumatic valve, and the assembly was again cleaned using a combination of compressed nitrogen and tape. The microchip injector was completed by aligning and bonding the flow and control layers to the cover plate as described elsewhere herein.
According to the example mentioned above, fused silica capillaries having an o.d. of 140 μm and an i.d. of 30 μm were passivated with HPC to suppress electroosmotic flow. The coating was prepared by first flushing a ˜2.5 m long capillary with 1 mL of 1 M HCL solution by applying 15 psi N2 back pressure to the solution reservoir followed by flushing the capillary with 200 μL 5% HPC in water using 20 psi N2 back pressure. The capillary was then flushed with deionized water to remove excessive hydroxypropyl cellulose. The treated capillary was subsequently cut into 10 equal lengths and ˜3 cm at the end of each length was chemically etched in 49% HF to render it porous for electrical contact as described previously. The etching of all 10 capillaries took place in a single batch using an approach adapted from previous work. Once etched, the distal end (inlet) of the capillary was inserted into a ˜5 cm length of 360-μm-o.d., 150-μm-i.d. capillary and sealed in place with epoxy. This same end was cut a few mm from the inlet using a dicing saw (SYJ-400, MTI Corp., Richmond, Calif.) to provide a clean interface at the loading channel-capillary interface. An assembly consisting of a PEEK tee and an inserted metal tube (0.04″ i.d., 1/16″ o.d)27 was then slid onto the capillary from the inlet end to form an optional, sheathless ESI interface. The emitter end of the etched capillary protruded 1-2 mm from the metal tube. The capillary inlet end was then inserted under a microscope into the 3-mm-long port that to connect the capillary to the loading channel on the microchip and a small amount of PDMS was applied at the loading channel-capillary interface. The PDMS was cured by placing the assembly in an oven at 110° C. for 20 min.
With the mechanical valve closed, a few microliters of sample was loaded into a pipet tip, which was then press-fitted into the sample port on the chip. To pressurize the sample, a length of tubing connected to a digital pressure controller was inserted into a round PDMS plug, which was in turn pressed into the wide end of the pipet tip to form an airtight seal. The sample was pressurized to 5 psi to dead-end fill the sample against the closed valve and then the sample pressure was adjusted as needed for operation. The CE run buffer was loaded into a sample vial sealed afterward to allow a N2 back pressure to be applied to the buffer liquid as shown in
All CE-nanoESI-MS analyses were performed using a triple quadruple mass spectrometer. The inlet capillary of the mass spectrometer was maintained at 200° C. Mass spectra were acquired in full scan mode covering an m/z range from 300 to 1000 at an acquisition rate of 2 Hz. Leucine encephalin, kemptide, angiotensin II, methanol, acetic acid, hydrofluoric acid, ammonium acetate and hydroxypropyl cellulose (HPC, average MW ˜100,000) were utilized for demonstrating the injection, CE separation, and mass spectrometry analysis. Peptide stock solutions were prepared individually in water at a concentration of 1 mg/mL. A 10 μM mixture of the three peptides was then prepared by dilution from the stock solutions into the run buffer. The run buffer was 9:1 20 mM aqueous ammonium acetate:methanol, adjusted to pH 4 with acetic acid. Colored dye was used for visualizing pressure-driven injection and transfer of the sample plug to the capillary.
A pressure-driven injection sequence is shown in
For initial characterization of CE separation, we determined the number of theoretical plates as a function of separation potential. Theoretical plates were calculated using the formula:
where tr is the retention time and w is the baseline peak width. As expected, plate number increases linearly with voltage as shown in
The computer-controlled, pressure-driven injection described herein can enable significant flexibility and tuning of separation conditions and acquisition of those separations in a rapid, automated fashion. As an example,
The tradeoff between separation efficiency and S/N as the injection volume is varied is shown in
While plate count diminished with increasing injection times, S/N showed the opposite trend, increasing with longer injections.
In addition to the ability to perform repeated, waste-free, programmable injections without impacting ongoing separations, another key benefit of the embodiments described herein is that sample injection is pressure based, and expected to avoid quantitative biases inherent in electrokinetic injection strategies of the prior art. We verified this by evaluating the peak area for each of the peptides in the mixture for injection times ranging from 0.3 s to 7 s. The elution and sample injection pressure was 2 psi for all data points. As shown in
While a number of embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that many changes and modifications may be made without departing from the invention in its broader aspects. The appended claims, therefore, are intended to cover all such changes and modifications as they fall within the true spirit and scope of the invention.
This application is a continuation-in-part of co-pending U.S. patent application Ser. No. 13/596,360, filed on Aug. 28, 2012, which is a division of Issued U.S. Pat. No. 8,277,659, filed on Sep. 23, 2010, and both are incorporated herein by reference.
This invention was made with Government support under Contract DE-AC0576RL01830 awarded by the U.S. Department of Energy. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12889108 | Sep 2010 | US |
| Child | 13596360 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13596360 | Aug 2012 | US |
| Child | 14264385 | US |
