Abstract:
A method and system to deposit rapidly, accurately and efficiently liquid droplets onto a sample plate. A liquid from which the droplet is formed contains sample molecules, matrix molecules or a mixture of both sample and matrix molecules. A sample plate is positioned with respect to a position below a portion of the liquid, such as a droplet. A power supply forms an electric field between the sample plate and the liquid, which pulls the droplet to a target location on the sample plate. A plurality of capillary columns is provided for simultaneous or successive multiple depositions via application of one or more high voltage pulses between the liquid sample array and a sample plate.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    1. Field of the Invention  
           [0002]    The present invention relates to mass analysis. More particularly, the present invention relates to sample preparation and handling for mass spectrometry processes.  
           [0003]    2. Description of the Related Art  
           [0004]    Mass spectrometry (MS) is a method of mass analysis in which the chemical composition of a substance is identified by separating gaseous ions from the substance according to their mass-to-charge ratio. One type of ionization used in mass spectrometry is known as matrix-assisted laser desorption/ionization (MALDI). In MALDI, a sample and a matrix are co-crystallized from a liquid solution. The resulting co-crystal is irradiated with a laser, which causes the matrix molecules to absorb some of the incident energy. The absorbed energy causes some of the matrix and sample molecules to ionize and desorb into the gas phase. Once the sample molecules have been ionized and desorbed into the gas phase, they are amenable to analysis by mass spectrometry.  
           [0005]    A sample that contains a mixture of chemical compounds is frequently purified prior to analysis by mass spectrometry. One method of purification is by liquid chromatography (LC), in which the mixture of compounds to be separated is dissolved in a liquid phase and the liquid phase is passed over a stationary phase contained in a chromatography column. Compounds that interact more strongly with the stationary phase are retained for a longer period of time on the column, which permits a mixture of compounds to be separated based upon differences in retention times.  
           [0006]    One means of further separating compounds with differing retention times is to fractionate the liquid phase as it exits the chromatography column. The formation of a large number of small amounts of the liquid phase produced by an LC is known as fraction collection. The precision and speed with which fraction collection is performed is critical to achieving high-quality separation and analysis. In addition, after purification by LC, the liquid phase containing the sample molecules can be combined directly with a matrix solution to enable MS analysis. Hereinafter, a liquid refers to any solution containing either sample molecules or matrix molecules, or a mixture of both. A sample refers to a portion of a liquid that has been prepared and provided for analysis, such as by deposition on a sample plate.  
           [0007]    Mass spectrometry analysis is currently being applied to increasingly complicated molecules and mixtures. In addition, advances in medicine, science and technology have created a growing demand for sophisticated analytical tools. This demand, in turn, requires the development of methods for the rapid and efficient preparation, purification and analysis of samples by mass spectrometry. Methods that enable the purification of large numbers of samples by LC and the analysis of large numbers of samples by MS have been developed independently. The demand for a rapid and efficient means to prepare and handle large numbers of LC fractions for analysis by MS, however, remains unmet.  
           [0008]    Typically, systems for automated fraction collection include single-channel devices that use either a touch-down cycle or piezo-electric dispensers for depositing a liquid sample. Touch-down devices use mechanics for physically spotting a liquid droplet onto a fraction collection plate. These devices normally use a narrow capillary connected to a column that holds the liquid. A tip of the capillary physically contacts the fraction collection plate to deposit a droplet of the liquid.  
           [0009]    Touch-down devices are notoriously prone to misalignment, wear and breakage. Also, preservation of the chromatographic resolution becomes an issue in touch-down collection when small fractions are collected. A significant portion of the liquid deposited in one spot can be carried over to the next spot by sticking to the capillary tip. To remedy carry-over, a make-up flow can be added by teeing-in additional solvent, or by applying a sheath flow, or both. These remedies increase the collected volumes so that a proportionately smaller sample fraction is carried over. However, the larger volumes dilute the sample concentration applied to the fraction collection plate, which, in turn, leads to lower sample densities and lower signal-to-noise in the signal generated by the mass spectrometer.  
           [0010]    A piezo-electric dispenser is a type of non-contact liquid droplet deposition device, in which a piezo-electric element is used to apply a pulse-driven pressure to a dispenser. The pressure forces, or ejects, a droplet of liquid out through a nozzle in the dispenser. Samples deposited using piezo-electric devices typically exhibit reduced chromatographic resolution because a larger dead volume is required to eject the droplet, and the concentration of sample molecules to matrix molecules is correspondingly reduced. Further, problems exist with keeping the dispenser nozzle clean if matrix solvent contains larger amounts of non-volatile material. Application of a sheath flow is not possible in this case.  
           [0011]    Other methods of micro deposition of LC samples include electrospray and streaking. Electrospray is a non-contact sample deposition process, which is sensitive to solvent composition and flow rate and which spreads the sample out over larger areas. Streaking is a contact form of deposition, which requires flash evaporation or freezing to preserve chromatographic resolution. Both electrospray and streaking processes are sensitive to solvent composition.  
         SUMMARY OF THE INVENTION  
         [0012]    This invention provides a novel non-contact liquid droplet deposition system and method. Advantageously, the invention supports the preparation and handling of a larger number of sample targets, resulting in higher throughput, while providing flexibility by allowing greater control of liquid flow rates, sampling speed, and solvent composition. Additionally, a system and method in accordance with the invention provides for enhanced chromatographic resolution of samples produced using liquid chromatography without sacrificing throughput or flexibility.  
           [0013]    The system and method according to embodiments of the invention provide a rapid, accurate, and efficient interface for depositing the output of up to eight liquid chromatography columns onto sample plates for analysis in a mass spectrometer. The system is flexible, permitting the deposition of samples at nearly any spacing, drop size, and solvent composition. Also, the samples can be deposited on a variety of sample plate materials, including both conducting and insulating materials. Non-contact deposition is achieved by a novel method of droplet desorption from fine capillary tips onto a sample plate through the application of a high voltage pulse to the sample plate, which generates an electric field between the sample plate and the capillary tip.  
           [0014]    A method that embodies this invention includes positioning a sample plate below a liquid droplet, and applying an electric field between the liquid droplet and the sample plate. The electric field polarizes the droplet. The polarized droplet experiences a force along the applied electric field and is pulled toward the sample plate.  
           [0015]    Another method embodying this invention includes providing an array of liquid droplets at a distance above a positionable sample plate, and applying an electric field between the droplets and the sample plate. Under the influence of the electric field, the droplets move to target locations on the sample plate.  
           [0016]    In another embodiment of the invention, a liquid droplet deposition system includes a holding mechanism, and an array of capillaries, held and positioned by the holding mechanism, wherein each capillary contains at least a portion of the liquid, and the array of capillaries provides one or more droplets of the liquid simultaneously. The system further includes a sample plate being positionable beneath the plurality of capillaries, and a power supply. The power supply connected to the capillaries and/or the sample plate for applying a voltage difference between the liquid and the sample plate.  
           [0017]    In still yet another embodiment, a sample is formed of a number of successive droplet depositions, in which each deposition includes positioning a sample plate and a droplet-forming capillary such that the droplet-forming capillary is above a target location on the sample plate. A successive application of a plurality of voltage pulses pulls individual droplets to the same location on the sample plate.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0018]    [0018]FIG. 1 is a simplified diagram of a liquid chromatography sample deposition system according to the invention.  
         [0019]    [0019]FIG. 2 illustrates an alternative embodiment of a sample deposition in accordance with the invention.  
         [0020]    FIGS.  3 A- 3 C are simplified diagrams of a deposition system to illustrate a liquid droplet deposition method according to the invention.  
         [0021]    FIGS.  4 A- 4 F show various methods of applying an electric field pulse in accordance with methods of the invention. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0022]    [0022]FIG. 1 is a simplified diagram illustrating an LC MALDI sample deposition system  100  according to an embodiment of the present invention. The system  100  includes a motion table  105  and a controller  107 . The motion table  105  is movable at least in a longitudinal direction shown, under direction of the controller  107 . The motion table  105  may also move laterally, vertically, or rotationally.  
         [0023]    A sample plate holder  110  rides on the motion table  105 , preferably fixed to the motion table  105 . In one embodiment, the sample plate holder  110  is a subassembly of the motion table  105 . Other configurations are possible; for example the sample plate holder  110  and the motion table  105  may be provided as a single unit. The sample plate holder  110  provides a base to which a sample plate  115  can be clamped. One or more sample plate holders can be situated on the motion table  105 , thus providing an accurate mechanism for accurately and reliably positioning one or more sample plates on the system  100 . In one embodiment, the sample plate holder  110  uses a built-in spring to hold the sample plate, and one or more pins to accurately align the sample plate.  
         [0024]    The sample plate  115  is formed of a rigid material and preferably has a planar upper surface  117 . The upper surface  117  may include an array of wells or small divots in the upper surface  117 , each for providing an anchor for a deposited portion of a sample. For example, each well can be an independently addressable target location embedded in the upper surface  117 , suitable for micro-arraying applications. The sample plate  115  can be formed of a glass slide, a stainless steel plate.  
         [0025]    The system  100  further includes a holding mechanism  118  adapted to hold one or more very small diameter columns  125 , or capillaries. In one embodiment of the invention, the capillary  125  is connected to a high pressure liquid chromatography (HPLC) source that provides the HPLC liquid. The capillary  125  terminates at a capillary tip  127 . The capillary tip  127  is connected to the capillary  125  at one end. The second end of the capillary tip  127  terminates to an open tip. The capillary tip  127  can be formed of a rigid or semi-rigid material such as metal, or silica glass. The material used for the capillary tip  127  may also depend on desired electrical characteristics of the material for transferring electric energy to the liquid provided therein, as explained in further detail below.  
         [0026]    The liquid can include sample molecules, matrix molecules, or a mixture of sample molecules and matrix molecules. In one embodiment of the invention, the liquid is a sample solution containing sample molecules, and the matrix molecules are independently provided to the sample plate  115 . Methods of applying matrix molecules to the sample plate  115  are known, including, but not limited to, depositing a matrix solution into individual wells formed in the upper surface  117 , coating the entire upper surface  117  with a matrix composition, etc. The sample solution is mixed with the matrix solution or composition upon deposition to the sample plate  115 . In another embodiment, the matrix solution is mixed with the sample solution in the capillary  125 .  
         [0027]    The capillary  125  and capillary tip  127  are adapted to receive a flowing portion  130  of the liquid. Preferably, the portion  130  of the liquid is a droplet. The droplet may have a controlled, adjustable size or volume, depending upon flow-rate through the capillary  125  and a desired size and density of the sample. Surface tension of the droplet suspends the droplet at the capillary tip  127  until the droplet is pulled away by the influence of an applied electric field. The portion  130  may be a collection of droplets, such as a spray, or even a continual stream of the liquid.  
         [0028]    In accordance with the present invention, the system  100  includes a power supply  135 . In one embodiment, the power supply  135  is a power supply in which the output voltage may be adjusted. The power supply  135  may also include electrodes that are connected to ground, or zero potential. The power supply  135  is configured to energize either the sample plate or the liquid, to create a potential difference between the liquid and the sample plate  117 , either of which may also be pre-charged to a particular polarity.  
         [0029]    In a preferred embodiment, a voltage pulse is provided to a liquid droplet  130 , via the liquid, and to the sample plate  115 , The application of this voltage pulse therefore creates a potential difference between the sample droplet  130  and the sample plate  115 . The voltage pulse can be provided with any combination of electrical connections to the sample plate and the liquid, as will be illustrated below. The voltage level of this pulse can be set either manually or automatically, and the timing and duration of the voltage pulse can be controlled either manually or by software that runs the deposition system  100 . Further, the power supply  135  is illustrated in FIG. 1 as having a connection to the sample plate  115  and the second, open end of the capillary tip  127 . However, the actual physical connection from the power supply  135  may be made in any location that energizes either the sample plate or the liquid. Therefore, the present invention is not to be limited to the specific embodiment shown in FIG. 1.  
         [0030]    The system  100  may additionally include a waste and wash plate, which are not shown in FIG. 1 for ease of explanation. The waste and wash plate allows cleaning of the capillary tips, from which samples are deposited, and provides a location for dropping extra sample before and/or after the sample plates are spotted with samples.  
         [0031]    [0031]FIG. 2 illustrates a liquid droplet deposition system  200  according to another exemplary embodiment of the invention. The system  200  includes a holding mechanism  118  that is configured to hold an array of capillaries  125 . Each capillary connects to a capillary tip  127 , which bears a droplet  130  of the liquid. The sample plate  115  can be positioned to any desired location below the array of capillaries  125 . The sample plate  115  may include an array of target locations  116 , such as a well or an etched outline of a location, for example. The array of target locations  116  is illustrated as having only a single row, however those skilled in the art would recognize that multiple rows and columns of target locations  116  are possible, based in part on the desired spot size of the liquid droplet.  
         [0032]    In the embodiment shown, eight capillaries  125  are held in position by the holding mechanism  118 , however other numbers of capillaries may be provided by the system  200  of the present invention. Therefore, the number of capillaries  125  in the array of capillaries is not limited to any specific number. The number of capillaries may be limited by a number of target locations  116  of the sample plate  115  used in the system  200 .  
         [0033]    A power supply  135  is shown in the embodiment as connected between the holding mechanism  118  and the sample plate  115 . As discussed, the connection may be made anywhere between the droplet and the sample plate. In an embodiment, a separate power supply may be provided for each capillary  125  and tip  127  for greater flexibility. In such an embodiment, the size and diameter of each liquid droplet generated by a particular capillary could be adjusted independently, and each droplet could be deposited on the sample plate independently of deposition from other capillaries. In another embodiment, the sample plate  115 , or a substrate connected with the sample plate  115 , is configured with an array of electrically addressable deposition sites or independent counter electrodes. The sites or electrodes could correspond to the target locations  116 , for application of a differential voltage to the sample plate  115 .  
         [0034]    FIGS.  3 A- 3 C are simplified diagrams of a system to illustrate a liquid droplet deposition method according to the invention. In FIG. 3A, at least a portion of a liquid is provided to a capillary  125 . A holding mechanism  118  may be adjusted to position the capillary and a capillary tip  127  to a predetermined position. A sample plate  115  is moved into a position below the capillary tip  127 .  
         [0035]    Referring now to FIG. 3B, the flow of liquid through the capillary  125  forms a droplet  130  at an open end of the capillary tip  127 . The size and contents of the droplet are controlled for a specific desired sample spot size and/or sample density. In one embodiment, each droplet has a volume of less than 10 microliters. Preferably, the volume of each droplet ranges between 100 and 200 nanoliters. As shown in FIG. 3C, a voltage differential  135  is applied between the droplet  130  and the sample plate  115 , to attract the droplet  130  away from the capillary tip  127  to a target location  116  on the sample plate  115 , and to form a sample spot  132 .  
         [0036]    A method according to the invention uses a high voltage pulse to create an electric field between the liquid sample and the sample plate. In one embodiment, a charge is applied to liquid droplets formed at the end of the capillary tips. In another embodiment, a charge is applied to the sample plate. The duration of the voltage pulses, and the interval between each pulse, are adjustable and controlled for desired throughput of sample deposition, and based in part on the size of the desired spot size.  
         [0037]    FIGS.  4 A- 4 E illustrate various methods of creating an electric field between a portion of the liquid and the sample plate. Turning first to FIG. 4A, there is illustrated a method of depositing a liquid droplet, whereby the sample plate  110  is connected to a positive or negative high voltage power supply. The voltage is applied in one or more pulses, which correspond to a time period in which deposition is to be made.  
         [0038]    A liquid droplet, formed at the end of a capillary tip  127 , can be neutrally charged, which, during application of a positive or negative voltage pulse to the sample plate, creates a potential difference between the liquid droplet and the sample plate. The potential difference between the liquid droplet and the sample plate generates an electric field along which the liquid droplet travels. In FIG. 4A, the liquid is grounded through a metal tee  140  that is a part of the holding mechanism  118 , and the capillary tip  127  is formed of a nonconductive material such as fused silica glass. In FIG. 4B, the capillary tip  127  is made of a conductive material, such as metal, and is connected directly to ground.  
         [0039]    [0039]FIG. 4C illustrates an alternative method for creating an electric field for depositing a sample. In this case, the capillary tip  127  is disposed in a sheath  128 . The sheath  128  surrounds the capillary tip  127  along its length. A sheath liquid  129  is fed into the sheath  128 . The sheath liquid  129  is polarized to a polarity that is opposite the polarity of a voltage pulse to be applied to the sample plate  110 . Alternatively, the sheath liquid  129  may have a neutral polarity or a ground potential. The sheath liquid contacts a liquid droplet at the end of the capillary tip  127 .  
         [0040]    An electrode may also be used to apply a charge to either the liquid or the sample plate, as shown in FIGS. 4D and 4E. In FIG. 4D, the sample plate  110  is grounded, and an electrode is placed in contact with the liquid droplet  130  for a short duration. The electrode may be persistently connected to the capillary tip  127 , or physically movable to touch the liquid directly if the capillary tip is formed of nonconductive material. Further, the electrode may be switchably coupled to the liquid or droplet according to a desired interval. In FIG. 4E, the liquid is grounded according to any method, including those methods mentioned above. An electrode is then coupled to the sample plate  110  to apply a high voltage pulse. As discussed, the electrode can include a switch for physical connection to the sample plate when a voltage pulse is needed.  
         [0041]    [0041]FIG. 4F illustrates another embodiment of a system and method according to the invention. The sample plate  110  is mounted over an electrode plate  150 , which is connected to a voltage source. Preferably, the voltage source is configured to apply a voltage pulse to the electrode plate  150  at predetermined intervals, and for a predetermined adjustable duration. In this embodiment and the other aforementioned embodiments, the duration of the voltage pulses ranges from 100-300 milliseconds, and is preferably about 200 milliseconds. However, other pulse durations may be used without departing from the scope of the invention. For example, application of a pulse of duration longer than 300 milliseconds can achieve an electrospray of sample to the sample plate, if desired. In this embodiment and the other aforementioned embodiments, the distance between the sample droplet and the sample plate ranges from one to ten millimeters, and is preferably about five millimeters. However, other distances may be used without departing from the scope of the present invention.  
         [0042]    The voltage difference ranges from 500 to 3000 volts. However, other voltage differences may be used without departing from the scope of the invention. The charged electrode plate  150  creates an electric field, shown generally in the simplified diagram of FIG. 4F. The droplet  130  formed at the end of the capillary tip  127  is polarized according to the electric field  155 , and is then attracted toward the electrode plate  150  to the sample plate  110 , along the electric field  155  path.  
         [0043]    In another exemplary embodiment, a liquid portion is deposited to a sample plate as a succession of droplets pulled to a single location on the sample plate. According to this embodiment, a target location on the sample plate is positioned with respect to a liquid to be deposited. A succession of electric fields are generated, preferably by a number of voltage pulsed, which attract a like number of liquid droplets to the target location. This embodiment improves resolution of the deposited sample solution, and reduces back-mixing of molecules in the droplets to the liquid.  
         [0044]    Those having skill in the art will recognize that the present invention for depositing one or more liquid droplets to the surface of a sample plate is not limited to the specific embodiments described herein. Other systems and methods may be used within the scope of the invention. Further, the specific embodiments of the systems and methods described herein are merely exemplary, and therefore values and ranges of values may occur that are different than those presented here for example, and still be within the scope of the present invention. Therefore, this invention is to be limited only by the following claims, which include all such embodiments and modifications when viewed in conjunction with the above specification and accompanying drawings.