JP2008530521A - Sample deposition method and apparatus - Google Patents

Abstract

A method and apparatus for preparing a sample or multiple samples for secondary analysis is disclosed. A single sample deposition device and multiple sample deposition devices are shown. The apparatus allows a system that can form a chromatogram by providing high-throughput deposition of a sample by discrete droplet deposition or as a continuous trace. The system may achieve high resolution digitization by pulsing the fluid emanating from the chromatograph by applying a voltage to a target plate that operates at frequencies above about 10 Hz and up to about 1 KHz. The system allows analog recording (ie, approaching infinite resolution) by nebulizing fluid coming from multiple columns and collecting it simultaneously on a target plate as continuous traces.

Description

  This application claims the benefit of US Provisional Patent Application No. 60 / 651,362, filed Feb. 8, 2005, and US Provisional Patent Application No. 60 / 651,203, filed February 10, 2005. All of which are incorporated herein by reference in their entirety.

  The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

(Field)
Applicants' teachings relate to sample deposition methods and apparatus for subsequent analysis by, for example, mass spectrometry. In particular, Applicants' teachings can provide high-throughput sample deposition for subsequent analysis by MALDI mass spectrometry.

(Introduction)
Liquid chromatography (LC) is a widely used separation process that relies on the different absorption characteristics of organic molecules. Usually, a mixture of organisms in a particular solvent (eluent) is added to the top of a chromatographic column packed with an absorbent material from which the compound can be absorbed. As the eluate and solute mixture descends through the column, more strongly absorbed compounds coat the absorbent material, which is referred to as the stationary phase. Absorbed compounds that are not very strong will travel through the column with the eluate. Thus, the compounds are separated based on the retention time so that compounds that interact strongly with the stationary phase are retained in the column for a longer period of time. The mixture eluted and separated compound is released from the other of the chromatographic column along with the eluent. The organic compounds are separated from the column as they are properly separated and spaced by a relatively pure eluate.

  High performance liquid chromatography (HPLC) refers to the separation of compounds under high pressure in a chromatography column. Normally, HPLC draws the eluate through a chromatography column by using a pump system. Pump systems typically include a reservoir that receives a small amount of fluid from a source (usually a solvent or water that forms an eluate). The piston is operably movable within the reservoir to draw fluid from the reservoir to the chromatography column. The piston is usually driven by a step motor.

  The operation of the piston releases fluid from the reservoir at a discontinuous flow rate, often resulting in a pressure pulse of the fluid flow. To smooth the discharge flow rate, the pump system includes a buffer chamber that functions like a shock absorber for pulses of fluid flow. Typically, the buffer chamber has a large volume compared to the fluid flow. In normal HPLC, each pump is actually equipped with two similar pumps that are 180 degrees out of phase, one of which introduces solvent and the other pump mostly introduces water. These are then mixed downstream of the pump to form an eluate that flows through the chromatography column.

  Furthermore, liquid chromatography can be used to deposit separated analytes on the target plate for subsequent analysis. These sample records can be stored for months under appropriate conditions, allowing additional species characterization in subsequent experiments without additional sample processing.

  Thanks to the separation capabilities of liquid chromatography, liquid chromatography becomes a useful tool for preparing samples for subsequent analysis of complex mixtures. Such complex mixtures include, but are not limited to, compounds frequently found in, for example, pharmaceutical drug discovery and development, proteomics, forensic medicine, environmental science, and clinical medicine.

  Mass spectrometry is commonly used for analytical methods that identify molecules in a compound based on the detection of the mass to charge ratio of ions generated from electrically charged molecules.

  There are various ways in which a molecule is ionized and then analyzed by mass spectrometry. As one such method, a soft ionization method used to determine the mass of analytes that are easily resolved is matrix-assisted laser desorption ionization (MALDI). In MALDI, a sample is mixed with a UV adsorbent compound known as a matrix, deposited on a surface, and ionized using high-speed laser pulses. The energy of the laser is absorbed by the matrix molecules and transferred to the sample molecules, causing them to vaporize and ionize. The ions are then analyzed by a mass spectrometer such as, but not limited to, a time-of-flight (TOF) mass spectrometer.

  The ability of liquid chromatography to efficiently address the need for rapid and efficient analysis of compounds, including but not limited to, MALDI mass spectrometry, without compromising accuracy and chromatographic fidelity Comprehensive, high-throughput methods and apparatus, such as multiplex systems, that are used and deposited on a sample are required.

  Applicant's teachings provide a method of depositing a sample for analysis. The method includes flowing an appropriate eluent through a chromatography column to separate the sample, and releasing the eluate having the sample eluted and separated components from the chromatography column. The eluate forms droplets at the discharge end of the chromatography column and provides an appropriate deposition surface spaced from the discharge end of the chromatography column, Receiving a droplet and applying a voltage to the deposition surface to draw a droplet to the deposition surface, the voltage applied to the deposition surface having a frequency of about 10 Hz or more; Including. The voltage can be applied to the deposition surface at frequencies including up to about 1 kHz.

  Further, Applicants' teachings provide a method for depositing multiple samples for analysis. The method includes flowing an appropriate eluent through each of the plurality of chromatography columns, each column being for separating a sample, and from the plurality of chromatography columns, Elution of the sample and release of the eluate having separated components, the eluate forming droplets at the discharge end of the respective chromatography column; and The liquid is applied to the deposition surface by receiving the droplet by providing at least one suitable deposition surface spaced from the discharge end and applying a voltage to the deposition surface. Withdrawing drops, wherein the voltage applied to the deposition surface is at a frequency of about 10 Hz or more. The voltage can be applied to the deposition surface at frequencies including up to about 1 kHz.

  In various embodiments, a voltage can be applied to the deposition surface so that a continuous droplet is drawn to a corresponding target location on the deposition surface. The deposition surface may be movable with respect to the discharge end of the chromatography column.

  Applicants' teachings also provide a method for depositing multiple samples for analysis, wherein the method involves flowing an appropriate eluate through a plurality of chromatographic columns, each column containing a sample. Elution of the sample and elution with the separated components from the plurality of chromatographic columns, and spraying the released eluate from the plurality of chromatographic columns. Depositing the sprayed eluate on at least one suitable deposition surface to produce a chromatogram.

  Further, in various embodiments, at least one air pump is used to flow the appropriate eluate through the chromatography column. Moreover, the eluate flow rate can be controlled by a flow meter in combination with a control processor, and the eluate flow rate is measured and controlled to provide continuous control of the flow rate. In various embodiments, the eluate flow rate is a mixture of two fluid streams, and the flow rate of each fluid is controlled by a respective flow meter in combination with a control processor. At least one of the fluid streams can be water and the other of the fluid streams can be a solvent.

  In various embodiments in which multiple chromatography columns are used, multiple air pumps may be provided, with at least one pump associated with each of each chromatography column.

  In various embodiments where the released eluate is nebulized, the stream of non-reacting gas can nebulize the released eluate. The non-reacting gas can be nitrogen.

  In various embodiments, the method can further include introducing a matrix into the sample, the matrix being suitable for use in matrix-assisted laser desorption ionization. The matrix can be introduced into the eluate prior to the step of nebulizing the released eluate.

  In various embodiments, the released eluate can be heated.

  Further, in various embodiments where the released eluate is nebulized, the chromatogram can be a continuous trace and in some embodiments can be parallel to each other. Furthermore, each continuous trace can correspond to a release from a respective chromatography column. Furthermore, all or part of the chromatogram can be ionized by a laser, which can generate at least one track on a continuous trace of the selected chromatogram. The laser can be a high speed laser and each chromatogram can be rastered at a constant speed. Further, in some embodiments, multiple laser tracks can be generated on a continuous trace of the selected chromatogram. Further, in some embodiments, multiple laser paths can be made in a single track generated on a continuous trace of a selected chromatogram.

  Applicants' teachings also provide an apparatus for preparing a sample for analysis. The apparatus includes a chromatography column that receives a sample using a suitable eluent, a pump that flows the eluate through the chromatography column, and a suitable deposition surface, the deposition surface comprising the chromatography surface. Spaced from the discharge end of the column, receives a droplet formed at that end by the flow of eluent through the chromatography column and generates a voltage at the deposition surface A power source for drawing the droplets onto the deposition surface, wherein the voltage applied to the deposition surface is a power source having a frequency of about 10 Hz or more. The voltage can be applied to the deposition surface at frequencies including up to about 1 kHz.

  Further, Applicants' teachings provide an apparatus for preparing a plurality of samples for analysis. The apparatus includes a plurality of chromatographic columns that receive samples using appropriate eluents and a plurality of pumps, each pump being associated with a respective chromatographic column, the pump deactivating the chromatographic column. A plurality of pumps and at least one suitable deposition surface, wherein the pump further comprises a flow meter and a control processor to provide continuous control of the flow rate of the eluate. Wherein the at least one deposition surface is spaced from a plurality of discharge ends of the respective chromatography column, wherein the at least one deposition surface is caused by the flow of eluate through the respective chromatography column. The droplet is formed on the deposition surface by receiving a droplet formed at the edge and generating a voltage on the deposition surface. Out, and at least one power supply, the voltage applied to 該沈 adhesive surface is provided with a frequency above about 10 Hz, and a power supply. The voltage can be applied to the deposition surface at frequencies including up to about 1 kHz.

  Further, for the various disclosed embodiments, the power supply can apply a voltage to the deposition surface so that a continuous droplet is drawn to a corresponding target location on the deposition surface. The deposition surface may be movable with respect to the discharge end of the chromatography column.

  In various embodiments, the pump may be an air driven pressure amplification pump. The pump may provide continuous control of the eluate flow rate by including a flow meter and a control processor. In addition, the pump may include a pressure source dimensioned to retain a volume of eluate that is greater than the volume of the respective chromatography column.

  Further, for various embodiments, a nebulizer may be provided to introduce a nebulizer gas into the chromatography column, the nebulizer spraying the eluate stream as the eluate is released. The atomizing gas can be a non-reactive gas. The non-reacting gas can be nitrogen.

  Further, for some embodiments in which a plurality of chromatography columns may be provided, the nebulizer may comprise a first manifold connected to the plurality of chromatography columns, wherein the nebulizer is freed of eluate. The eluate stream is nebulized. The first manifold can be connected to a plurality of chromatographic columns by T-valves. In addition, the device may release a nebulized eluate from each of a plurality of chromatography columns by including a plurality of deposition tubules operably connected to each T-shaped valve. In various embodiments, the nebulizer may include a pump that supplies atomizing gas to a plurality of chromatography columns. The pump may include an air pump.

  Further, in various embodiments, a matrix delivery system can be provided to introduce the matrix into the eluate, which is suitable for use in matrix-assisted laser desorption ionization. For some embodiments in which a nebulizer can be used to nebulize the eluate stream as the eluate is released, the matrix delivery system allows the eluate to be nebulized before it is nebulized. A matrix can be supplied to the eluate.

  For some embodiments in which a plurality of chromatography columns can be provided, a matrix supply system can be provided, the matrix supply system including a second manifold connected to the plurality of chromatography columns; The second manifold introduces a matrix into the eluate, which is suitable for use in matrix-assisted laser desorption ionization. The second manifold can be connected to multiple chromatographic columns by T-valves. The second manifold can be operatively connected to each of the plurality of chromatography columns to provide a matrix before the eluate is nebulized. Further, the matrix supply system can supply the matrix to a plurality of chromatography columns by providing a pump. The pump can be a syringe pump. In some embodiments, the pump can be a continuous flow pump.

  In various embodiments, the apparatus can comprise a translation stage that receives at least one deposition surface. The translation stage may be movable with respect to a plurality of chromatographic columns so that the generated chromatograms are parallel to each other. Further, the at least one deposition surface can be a plurality of plates arranged in a deposition array on a translation stage.

  Applicants' teachings also provide an apparatus for preparing a plurality of samples for analysis, the apparatus receiving a plurality of chromatographic columns and a plurality of pumps that receive at least one sample using a suitable eluent. Wherein each pump is associated with each chromatography column, a plurality of pumps and a nebulizer for introducing a spray gas into the plurality of chromatography columns, wherein the nebulizer releases the eluate. A nebulizer for atomizing the eluate stream, and at least one suitable deposition surface for receiving the emitted nebulized eluate, wherein the release from the plurality of chromatographic columns The nebulized eluate thus produced has a deposition surface that produces each of a plurality of chromatograms at the at least one suitable deposition surface. Eteiru.

  In various embodiments, the pump may be an air driven pressure amplification pump. The pump may provide continuous control of the eluate flow rate by including a flow meter and a control processor. Further, the pump may include a pressure source dimensioned to hold a volume of eluate that is greater than the volume of the chromatography column.

  Further, for various embodiments, a nebulizer may be provided to introduce a nebulizer gas into the chromatography column, the nebulizer spraying the eluate stream as the eluate is released. The atomizing gas can be a non-reactive gas. The non-reacting gas can be nitrogen.

  The nebulizer may comprise a first manifold connected to a plurality of chromatography columns, the nebulizer nebulizing the eluate stream as the eluate is released. The first manifold can be connected to multiple chromatographic columns by T-valves. Further, the apparatus may further comprise a plurality of deposition tubules operably connected to each T-shaped valve, thereby releasing the nebulized eluate from each of the plurality of chromatography columns. In various embodiments, the nebulizer may further include a pump to supply the nebulizer gas to the plurality of chromatography columns. The pump may include an air pump.

  Further, in various embodiments, a matrix delivery system can be provided to introduce the matrix into the eluate, which is suitable for use in matrix-assisted laser desorption ionization. For some embodiments in which a nebulizer can be used to nebulize the eluate stream as the eluate is released, the matrix delivery system allows the elution to be performed before the eluate is nebulized. A matrix can be supplied to the liquid.

  The matrix delivery system includes a second manifold connected to a plurality of chromatographic columns, the second manifold introduces a matrix into the eluate, the matrix being used in matrix-assisted laser desorption ionization Suitable for The second manifold can be connected to multiple chromatographic columns by T-valves. The second manifold can be operatively connected to each of the plurality of chromatography columns to provide a matrix before the eluate is nebulized. Further, the matrix supply system can supply the matrix to a plurality of chromatography columns by providing a pump. In some embodiments, the pump can be a syringe pump. In some embodiments, the pump can be a continuous flow pump.

  In various embodiments, the apparatus can comprise a translation stage that receives at least one deposition surface. The translation stage may be movable with respect to a plurality of chromatographic columns so that the generated chromatograms are parallel to each other. Further, the at least one deposition surface can be a plurality of plates arranged in a deposition array on a translation stage.

  In some embodiments, the apparatus can comprise at least one power source that draws droplets on the deposition surface by generating a voltage on the deposition surface, the voltage applied to the deposition surface being about 10 Hz or greater. Frequency. The voltage can be applied to the deposition surface at a frequency up to about 1 kHz. The power source can apply a voltage to the deposition surface, so that a continuous droplet is drawn to a corresponding target location on the deposition surface.

  Further, Applicants' teachings provide a system for preparing multiple samples for analysis via droplet deposition or by nebulization, depending on use. The system includes a plurality of chromatographic columns and a plurality of pumps that receive at least one sample using a suitable eluent, each pump associated with each chromatographic column and a plurality of pumps. A nebulizer that introduces a nebulizer gas into the plurality of chromatographic columns, the nebulizer nebulizing the nebulizer stream as the eluate is released, and the atomized nebulizer At least one suitable deposition surface for receiving an eluate, wherein the released nebulized eluate from the plurality of chromatographic columns comprises a plurality of chromatograms at the at least one suitable deposition surface. Each generating a deposition surface and generating a voltage on the deposition surface to draw droplets on the deposition surface. Kutomo a single power source, the voltage applied to 該沈 adhesive surface is provided with a frequency above about 10 Hz, and a power supply. The voltage can be applied to the deposition surface at frequencies including up to about 1 kHz.

  In various embodiments, the power source can apply a voltage to the deposition surface, such that a continuous droplet is drawn to a corresponding target location on the deposition surface.

  In various embodiments, the pump for flowing the eluate through the chromatography column may further include a flow meter and a control processor to provide continuous control of the eluate flow rate. The pump may be an air driven pressure amplification pump. The pump may further comprise a pressure source dimensioned to hold a volume of eluate that is greater than the volume of the respective chromatography column.

  In various embodiments, the nebulizer may introduce a spray gas into each of the chromatography columns by including a first manifold connected to the plurality of chromatography columns. The first manifold may be connected to the plurality of chromatography columns by a T-shaped valve. Further, in various embodiments, the apparatus further comprises a plurality of deposition tubules operably connected to the respective T-valve, thereby evaporating the eluate sprayed from each of the plurality of chromatography columns. May be released. The nebulizer may further supply a spray gas to a plurality of chromatography columns by further including a pump. The pump may include an air pump.

  The atomizing gas can be a non-reactive gas. The non-reacting gas can be nitrogen.

  Further, in various embodiments, the system may comprise a matrix supply system, the matrix supply system may include a second manifold connected to a plurality of chromatography columns, the second manifold being Introducing a matrix into the eluate, the matrix is suitable for use in matrix-assisted laser desorption ionization. The second manifold may be connected to the plurality of chromatographic columns by a T-shaped valve. The second manifold can be operatively connected to each of the plurality of chromatography columns to provide a matrix before the eluate is nebulized. The matrix supply system may include a pump and supply the matrix to a plurality of chromatography columns. In some embodiments, the pump can be a syringe pump. In some embodiments, the pump can be a continuous flow pump.

  In various embodiments, the system can include a translation stage that receives at least one deposition surface, the translation stage being movable with respect to a plurality of chromatography columns, resulting in a plurality of generated stages. The chromatograms are parallel to each other. The at least one deposition surface can be a plurality of plates arranged in a deposition array on the translation stage.

  These and other features relating to applicant's teachings are set forth herein.

  Those skilled in the art will appreciate that the drawings described below are for illustrative purposes only. The drawings are in no way intended to limit the scope of applicants' teachings.

  The following description is intended to be illustrative only and not limiting. Various embodiments of the applicant's teachings will be apparent to those skilled in the art in view of this description.

  Applicants' teachings relate to sample deposition methods and apparatus for subsequent analysis by, for example, mass spectrometry, but are not limited to matrix-assisted laser desorption ionization (MALDI) mass spectrometry.

  High performance liquid chromatography (HPLC) chromatographic separation represents the rate-limiting step in any mass spectrometry based sample analysis. In the field of proteomics and drug discovery, an increasing number of samples need to be analyzed to solve biologically relevant problems. In proteomics, two-dimensional LC has become essential to solve very complex samples. In a two-dimensional experiment, 10-100 fractions can be collected from the first separation step, and each individual fraction can then be subject to another stage of chromatography. The time required to perform this analysis continuously is substantially prohibitive unless the second dimension of chromatography can be multiple. Similarly, for drug discovery applications where many in vitro tests of drug candidates are used to predict drug efficacy, the chromatographic separation step presents an analytical bottleneck, and fast MALDI mass spectrometry is The need for subsequent multi-chromatographic separation presents a breakthrough in the discovery process.

  Conventional HPLC instruments do not follow easy for multiplexing. Mechanical complexity, size and cost make it virtually unmanageable. Fluid supply systems that are based on air pressure as the driving force for the fluid are mechanically simple, compact, and inexpensive. These factors allow multiple air pumps to be arranged in the instrument and provide independent flow control for any number of independent channels. Attempting to supply fluid from multiple pump channels from a single pump source requires flow splitting to distribute the fluid. Indeed, this approach is problematic in that different pressures are created in the individual chromatographic channels, resulting in an uncontrollable branch of flow to the different channels. A pumping system that provides an independent pumping arrangement for each individual chromatographic channel is required to form a robust and reliable multidimensional separation system.

  In order for such an instrument to function reliably as a system, means for depositing the chromatographic effluent on the target of MALDI is formed, which means unnecessary mechanical complexity and stringent Easily accommodates simultaneous deposition of multiple channels without dimensional tolerances. The instantaneous and simultaneous application of high voltage to a series of MALDI targets is well suited for this purpose. A single power source that pulses the entire array of targets provides the force to generate droplets, and all channels are fully tuned. The dimensional differences between the tubules that eject the various droplets associated with high voltage targets are irrelevant. This is because the field can be used to ensure that sufficient force is applied to all capillaries regardless of their spacing from the target. No mechanical or moving parts are required to eject droplets, and they can introduce incomparable complexity for multiple systems. Such pumping systems have the ability to provide high speed and high fitness gradients, resulting in high resolution chromatographic traces. In order to obtain high-definition profiling of chromatographic traces, high frequency, small volume droplets must be ejected simultaneously from the capillaries. Frequencies ranging from 10 Hz to about 1 kHz, and droplets as small as 10 picoliters tend to be required as chromatographic resolution increases. In particular, in multiple systems, it may not be possible at these speeds to mechanically touch the tubules against the surface for ejecting the droplets.

  Piezo, ultrasonic and ink jet devices are incompatible as liquid dispensers from chromatographic columns, which reduce chromatographic separations due to the widening band in the large volume chambers of these devices. This is due to the excessive liquid volume that the device contains. The device described here does not require any liquid reservoir to perform the application of droplets.

  Referring to FIGS. 1a and 1b, an apparatus 10 for preparing a sample or samples for subsequent analysis is shown. In particular, FIG. 1a shows an embodiment of a single sample deposition apparatus taught by applicant, and FIG. 1b shows an embodiment of a multiple sample deposition apparatus taught by applicant. Equivalent reference symbols are used to refer to identical components in various aspects. As illustrated in FIGS. 1a and 1b for some of the applicant's teachings, the apparatus 10 provides a chromatogram 12 by providing high-throughput deposition of samples, for example, by discontinuous droplet deposition. For some aspects of applicant's teachings, and will be described hereinafter as a continuous trace, as illustrated in FIG.

  The apparatus 10 includes, but is not limited to, a sample supply system such as an autosampler (not shown). The supply system simultaneously introduces a sample (not shown) into the channel fluid supply system with a suitable eluent, which chromatographs the eluate by comprising a pumping system, usually indicated at 20. Is pushed through the column 14 and deposited on a suitable deposition surface 16. For various aspects of the applicant's teachings, the eluate is pushed through a single chromatographic column, as shown in FIG. 1a as an example only, and in some aspects, as an example only. As shown in FIG. 1 b, the eluate is pushed through a plurality of chromatographic columns 14.

  With continued reference to FIGS. 1 a and 1 b, the deposition surface 16 may be provided in the deposition array 22. For purposes of illustrating the applicant's teachings shown in FIGS. 1a and 1b, two deposition surfaces 16 are provided in the array 22 to illustrate some aspects of the applicant's teachings by way of example only. For purposes, four deposition surfaces 16 are provided to the array 22 as shown in FIG. It will be appreciated that the deposition surfaces may be arranged in a series of n × n deposition surfaces as desired. The array 22 of deposition surfaces may be provided on a translation stage 36, such as an xyz stage, which will be described in the specification that follows. Providing an array 22 of deposition surfaces on the translation stage 36 facilitates high-throughput deposition of the chromatogram 12 for the multiplex system shown in FIG. 1b and will be described in the subsequent specification.

  For some aspects, at least one sample is introduced into a plurality of chromatographic columns 14 with an appropriate eluent, as shown in FIG. 1b by way of example only. However, some aspects of the applicant's teachings are that one sample is distributed across multiple chromatographic columns 14, generating multiple chromatograms 12 of the same sample for analysis, and It can be appreciated that column 14 is intended to receive separate samples with the appropriate eluent, as well as various combinations of these as required.

  In order to achieve high throughput, the pumping system 20 provides an accurate gradient at a rate of nanoliters per minute, especially for each chromatographic column 14, as shown in FIG. It is necessary to react. A suitable pump having these characteristics is a pneumatic pump, for example an air driven pressure amplification pump. However, it is understood that other pumping systems that achieve similar results are intended to be used with the applicant's teachings.

  As shown in FIG. 1 b, the pumping system 20 has a pump 21 associated with each chromatography column 14. The pump 21 of the pumping system 20 accurately measures the eluate flow rate and control flow through each chromatographic column 14. This is because pump 21 reacts quickly to flow step changes, pumps up against substantial back pressure, identifies leaks and blockages, and is one-to-one in each chromatographic column 14 of FIG. 1b. The flow rate can be adjusted accordingly.

  By providing a pump 21 one to one with each chromatography column, Applicants' teachings achieve multiplexing that avoids flow diversion and the disadvantages associated with flow diversion. For example, a flow diversion system utilizes a single pump that diverts the flow to multiple chromatography columns. However, for example, it is known that back pressure, leakage and blockage can occur in each chromatography column at different rates and times. Thus, any measurement of flow rate and pump flow control can be applied to and recognized by all chromatographic columns in a flow diversion system and cannot provide sufficient flow for a given column. Alternatively, there can be excessive flow for a given column. Multiplexing systems using flow diversion do not provide an accurate measurement of the flow and control of the eluate flow through each chromatographic column, as shown in FIG.

  FIG. 2 illustrates a suitable pump 21 for various embodiments of applicants' teachings. As will be described later herein, the pump 21 is actually two pumps 21A and 21B that combine the respective fluid flows, but for the purposes of the applicant's teachings, the pump 21 21A and pump 21B operate equally.

  Pumps 21A and 21B each include a source 102a and 102b of a large volume of fluid (eg, solvent or water) and a discharge channel 104a and 104b (each connected to the source and a chromatographic column associated with the pump). 14). The fluid can be driven with air from the sources 102a and 102b, where the pump 21 is, for example, a pneumatic pump. Typically, the fluid retained in the sources 102a and 102b has sufficient volume for the overall desired run of the respective chromatography column 14.

  Flow meters 106a and 106b are provided in channels 104a and 104b, respectively. The flow meters 106a and 106b measure the flow rate of the fluid through the channels 104a and 104b, respectively. The fluid flow rates measured by flow meter 106a and flow meter 106b are monitored by control processors 108a and 108b, respectively, and then adjust the discharge of the respective fluid from sources 102a and 102b. By monitoring the flow rate using an appropriate control processor, the control of the microfluidic flow is performed accurately and quickly, producing the desired flow through a given chromatographic column 14. Suitably, the pump 21 may provide a flow rate of 1 nl / min to 100 μl / min.

  The previously described pump 21 includes two pumps 21A and 21B to supply a suitable fluid to the liquid chromatography column 14. For example, pump 21A operates to distribute a suitable fluid, such as water, to liquid chromatography column 14. Water serves both to flush the column with water for cleaning and to dilute the solvent. Pump 21B may operate to pump a suitable solvent to chromatography column 14 and may result in separation of the compounds in the column.

  In particular, the water from pump 21A forms an eluate at 110 by mixing a predetermined amount with the solvent from pump 21B, and the eluate is at a rate controlled by pumps 21A and 21B, respectively. It flows into each liquid chromatography column 14. The pump system shown in FIG. 2 provides very accurate gradient control. Considering FIGS. 3a and 3b, it can be seen that pumps 21A and 21B can be adjusted very quickly to mix the flow rates and provide a very accurate and steep slope.

  For example, FIG. 3a shows the flow profile of a pump having a discontinuous flow rate, such as a piston-driven pump that generates fluid flow pulses. Line 112a illustrates the water flow profile with such a pump, and line 112b illustrates the appropriate solvent flow profile from the second piston-driven pump. Line 112a shows that only water is initially transferred to the liquid chromatography column. After a predetermined time indicated by 113, a predetermined amount of solvent indicated by line 112b is added to the mixture and the water flow is proportionally reduced at the same time, resulting in an overall flow rate for the entire system. Is kept constant. Towards the end of the run, the amount of water introduced into the flow is minimized.

  FIG. 3b shows the flow profile of the pump 21, allowing accurate measurement of the flow rate through the respective chromatographic column 14 and control of the eluate flow, as described above. Line 114a illustrates, for example, a water flow profile with pump 21A, and line 114b illustrates an appropriate solvent flow profile from pump 21B. Lines 114a and 114b show a very steep slope when compared to lines 112a and 112b in FIG. 3a. For example, in FIG. 3b, adding solvent to the mixture begins almost immediately and increases very rapidly, as indicated at 115. Similarly, a proportional reduction in water flow begins almost immediately. The flow rates between the water and solvent indicated by lines 114a and 114b, respectively, are for exemplary purposes only, and applicants' teachings also contemplate additional fluid mixtures. Can be recognized.

  In addition to the very steep slope shown by lines 114a and 114b compared to lines 112a and 112b, the precise and abrupt control of the flow rate provided by pump 21 is as shown at 115 for line 114b in FIG. 3b. Allows a particular flow rate to begin almost immediately, as well as to stop almost immediately, as indicated at 117 for line 114a in FIG. 3b. This can be compared to a gradual start of flow, as shown at 113 for line 112b in FIG. 3a, and a similar gradual stop of flow, as shown at 119 for line 112a in FIG. 3a. Can be compared.

  A system that receives rapidly the eluate discharged from a chromatography column or multiple chromatography columns is a system where the eluate flowing through each column or columns enabled by the pump system described above is high. Must be consistent with throughput.

  FIGS. 1a, 1b, and 4 illustrate a respective chromatographic column 14 (FIG. 1a) or multiple chromatographs at high frequencies up to and including about 1 kHz, as described later herein. FIG. 4 shows some aspects of Applicants' teaching to collect discontinuous droplets of eluate emitted from a column 14 (FIG. 1 b) of FIG.

  In particular, considering FIG. 4, eluate from the chromatography column 14 flows from the capillary 112 to the discharge end 114 of the capillary. The discharge end 114 is disposed at a distance from the surface material 38 of the deposition surface 16. For the same scale and clarity, FIG. 4 illustrates the oversized spacing of the discharge end 114 from the face material 38 of the deposition surface 16. However, it is understood that the discharge end 114 of the capillary tube 112 is much closer to the surface material 38 of the deposition surface 16, as illustrated in FIGS. 1a and 1b.

  The deposition surface 16 can be a plate 116, such as a target plate used in MALDI analysis, and preferably a microtiter plate. However, other configurations of the deposition surface can be contemplated and can include, but are not limited to, a disk, tape or drum. The surface material 38 of the deposition surface 16 may include, but is not limited to, a metal surface made of stainless steel, gold, silver, chrome, nickel, aluminum, and copper. Further, the deposition surface 16, such as the target plate 116, may be removable from the array 22 for later analysis by MALDI mass spectrometry.

  Plate 116 is typically held by a suitable plate holder 118, which can then be supported by a motion table such as deposition array 22 (see FIG. 1). Further, as previously mentioned, the deposition array 22 may be provided on a translation stage 36, such as an xyz stage. The translation stage 36 is movable with respect to the chromatography column 14. The deposition array 22, and hence the deposition surface 16, typically moves with respect to the chromatography column 14 of FIG. 1a or the plurality of chromatography columns 14 of FIG. 1b, but alternatively, the chromatography column or the plurality of chromatography columns 14 Can move with respect to the deposition array 22.

  Referring to FIG. 4, the eluate discharged from the chromatography column 14 forms droplets 115 that are deposited on the deposition surface 16. Applicants' teaching removes the liquid 115 from the discharge end 114 of the capillary tube 112 by providing an electric field between the deposition surface 16 and the droplet 115. This electric field acts to draw droplets to the deposition surface 16.

  A suitable power supply 120 is provided to allow adjustment of the output voltage. The power source may include an electrode connected to ground or zero potential. The power source is configured to create a potential difference between the droplet and the deposition surface 16 by applying a voltage to the deposition surface 16 or the droplet 115. For various embodiments of the applicant's teachings, the deposition surface 16 is charged and the droplet 115 at the discharge end 114 of the capillary tube 112 is grounded at 121.

  A voltage pulse is supplied to the deposition surface 16, and in this application, the voltage pulse is created on the deposition surface 16 and on the surface material 38 of the deposition surface 16 by creating a potential difference between the droplet and the deposition surface 16. Droplet 115 is drawn to a predetermined location provided, such as a well or divot 125 (see FIGS. 1a and 1b). Each of these wells or lawns is at a target location that can be independently addressed, and deposition of the droplets in the appropriate well or lawn is relative to the deposited surface 16 relative to the deposited droplets 115. It can be appreciated that the position is controlled by a microprocessor that controls the position.

  In order to draw the droplet 115 on the deposition surface 16 and to a predetermined location, such as a well or lawn 125 (see FIGS. 1a and 1b), by creating a sufficient potential difference between the droplet and the deposition surface 16 The required voltage pulse is about 1,000 volts every 0.2 mm, or about 5,000 volts per cm. At this voltage, the relay is not an option, because of the speed of switching and the reliability of the mechanical components.

  The voltage requirement can be achieved by a series arrangement of FETs or IGTB (not shown) available with ratings up to 1,200 volts. This distributes the voltage across many devices. For example, for illustrative purposes only, two sets of five FETs or IGTBs can be used to connect the output to the output of a 4,000 volt power supply or ground. The maximum voltage across the nuclear device is 800 volts, which is well within the maximum voltage rating. The device is controlled to generate a RF signal at 8 Mhz, for example, and transform it all together by applying it to a coil printed on one side of a FET or IGTB printed circuit board (not shown), This signal is detected using another coil on the opposite side of the printed circuit board. This allows the control signal to ride on whatever the voltage is on the switching device. An Op amplifier (not shown) can be used to generate the control signal. Furthermore, a function generator (not shown) can be used for the internal clock, and a CMOS circuit (not shown) can be used for interlocking and external pulse input applications.

  In the applicant's teachings, the voltage pulses for the different plates can be at very high frequencies, typically above 10 Hz, up to about 1 kHz, and a very fast static of the eluate on the deposition plate 16 target plate. Enables electrodeposition and adapts to the high throughput of eluent flow through the chromatography column. Thus, for example, an apparatus is provided that allows high throughput of deposited samples that can be analyzed by, but not limited to, MALDI mass spectrometry.

  FIGS. 1 a and 1 b also provide a MALDI matrix delivery system 40 that operates to deliver a matrix to the eluate in the capillary 112 before the eluate is released from the capillary 112. The matrix is introduced via the load value 41. The matrix delivery system is described in detail below in connection with some aspects of applicants' teachings.

  FIGS. 5a and 5b show how the combination of pumping systems achieves high throughput flow rates and is shorter when analyzed at frequencies up to about 10 Hz and including about 1 kHz. Illustrates whether to produce a chromatogram that produces a sharper peak at runtime. For example, FIG. 5a illustrates three compound signal traces separated using conventional pumping and deposition techniques. It can be seen that the runtime to reach the peak is over 5 minutes.

  FIG. 5b shows a similar run using a pumping system and deposition technique according to the applicant's teachings. The runtime is found to be less than one minute and is five times faster than that obtained using conventional methods. As a result, the sample for analysis is more condensed and results in a sharper peak. Applicant's teaching provides a dramatic increase in throughput and detection possibilities.

  FIG. 5C shows an LC MADLI for separating minoxidil (trace 57) and reserpine (trace 59) at 20 μL / min using the deposition and 10 Hz rate run described for FIGS. 1a and 1b. The signal trace is shown. Individual points from the recorded discrete droplets are indicated by 51 in FIG. 5C. As in 53, the sharp peaks illustrated demonstrate an increase in throughput and detection potential when using Applicants' teachings.

  FIG. 6 illustrates some aspects of Applicants' teaching to provide quick and continuous sample deposition. The results from the various embodiments are also represented in FIG. 5C and represented by line 55, representing the signal obtained from a continuous trace recording of individual points from the discrete droplets recorded as 51. The

  In particular, FIG. 6 introduces a nebulizer 24 to introduce a nebulizing gas into the chromatography column 14 and atomizes the eluent as it is released from the chromatography column. The atomizing gas evaporates the eluate in the chromatography column 14. The discharged mist-like eluate is deposited on the deposition surface 16.

  The atomizing gas is a non-reacting gas, including but not limited to nitrogen, dry air, noble gas or any other suitable gas. It will be appreciated that other means of spraying the sample are possible and are well known in the art.

  The nebulizer 24 includes a manifold 26 connected to the chromatography column 14 and supplies the nebulizer gas to the eluate in the chromatography column 14. In some embodiments, the manifold 26 is a tubing manifold. As illustrated in FIG. 6, the T-shaped valve 28 allows the introduction of atomizing gas into the chromatography column 14 by connecting the manifold 26 to the plurality of chromatography columns 14. Nebulization of the eluate occurs when the eluate is released from the chromatography column 14, and therefore, in some embodiments, the manifold 26 that supplies the atomizing gas is connected to the discharge end 30 of the chromatography column 14. Or in close proximity thereof by a T-shaped valve 28. For illustrative purposes, FIG. 6 shows an apparatus adapted to prepare a plurality of chromatograms 12 for analysis by MALDI mass spectrometry, and therefore as described later in this specification. It is understood that an additional matrix manifold is included between the end 30 of the chromatography column 14 and the T-valve 28 of the nebulizer 24. For the purpose of this application, the discharge end of the chromatography column 14 is released from the chromatography column or, if present, for example, a matrix delivery system, or any other that may be present before the nebulizer 24. Release from the delivery system may be included.

  The T-valve 28 of the nebulizer 24 can be operably connected from the discharge end 32 to the deposition tubule 34. A deposition tubule 34 discharges the eluate sprayed onto the appropriate surface 16 from each of the plurality of chromatographic columns 14. The deposition tubule can operate at a distance of 1-5 mm from the appropriate plane, but is not shown in FIG. 6 for clarity.

  The nebulizer 24 further includes a pump (not shown) to supply the atomizing gas to the chromatography column 14. In some embodiments of Applicants 'teachings, the pump includes a pneumatic pump, but Applicants' teachings are not intended to be limited to such pumps.

  The released eluate can also be heated to accelerate desolvation saturation by the nebulizer 24. As shown in FIG. 6, at 27, the released eluate is heated by flowing an appropriate heated gas from the source to the T-valve 28. However, it can be appreciated that other methods and structures for heating the released eluate are contemplated by the applicant's teachings.

  FIG. 6 illustrates a matrix delivery system 40 for when chromatogram 12 is analyzed by MALDI mass spectrometry. The matrix delivery system 40 may introduce a matrix into the eluate by including a manifold 42 connected to the chromatography column 14. For some embodiments, each T-valve 44 connects the manifold 42 to the chromatography column 14.

  A T-valve 44 can be operatively connected to the chromatography column 14 to supply the matrix to the eluate before it is nebulized by the nebulizer 24 (see FIG. 6). The matrix feed system includes a pump 60 system (schematically illustrated in FIG. 2), thereby allowing the matrix to be chromatographed through a load valve 41 and then through a manifold 42 and a T-valve 44. 14 can be supplied. The pump can be, for example, a syringe pump, or alternatively, a continuous flow pump, but is not limited thereto.

  FIG. 2 schematically illustrates a suitable pump system 60 for supplying the matrix. The system 60 includes a pump 62, which is connected to a matrix source 64 and a discharge channel 66 (connected to the source, through which the matrix moves to the load valve 41 and finally the chromatographic Move to column 14). A flow meter 68 is provided in the channel 66 and measures the flow rate of the matrix through the channel 66. The flow rate of the fluid measured by the flow meter 68 is monitored by a control processor 70 that regulates the release of the matrix from the source 64.

  Suitable matrix materials for use in MALDI are well known in the art. Examples of commonly used matrix materials include, but are not limited to, derivatives of 2,5-dihydroxybenzoic acid, derivatives of sinapinic acid, and derivatives of indoleacrylic acid.

  As shown in FIG. 7, the nebulized eluate with the eluted and separated compound of the sample is released onto at least one of the deposition surfaces 16, for example, on surface 16 a as shown. In addition, a plurality of chromatograms 12 are generated. However, because the deposition array 22 can carry multiple surfaces 16 at the translation stage 36, the method can simultaneously generate multiple chromatograms on multiple surfaces, particularly surfaces 16a and 16b shown in FIG.

  As best illustrated in FIG. 7, the apparatus and method of Applicant's teachings produces a plurality of chromatograms 12 deposited on a suitable surface 16 that may be in a continuous and uninterrupted trace. The traces of chromatogram 12 in FIG. 7 are usually parallel to each other, however, it can be appreciated that continuous traces can be deposited in any line or any pattern.

  In addition, deposits of the chromatogram 12 can be formed in a continuous, uninterrupted trace with no gaps. The continuous trace uniformity preserves the complete signal without losing data, accuracy, and chromatographic fidelity when the chromatogram 12 is subject to analysis by MALDI mass spectrometry.

  If the method of applicant's teaching is used to prepare multiple chromatograms for analysis by mass spectrometry, the apparatus as described above for FIGS. 1a, 1b and 6 All or part of the selected chromatogram 12 from is ionized and the ions are then analyzed by a mass spectrometer (not shown). In particular, the chromatogram is suitable for soft ionization mass spectrometry, such as MALDI, but is not limited thereto.

  For MALDI, it is desirable to irradiate and ionize all or part of the selected chromatogram 12 by using a high repetition nitrogen laser (not shown). However, any type of laser whose output is suitable for MALDI applications (as long as its output ranges from an energy range of about 0.1 microjoule per pulse to about 100 microjoules per pulse) can be used. Can be recognized. Preferably, each trace is rastered at a constant rate, resulting in a chromatogram at high chromatographic resolution when analyzed by mass spectrometry.

  As illustrated in FIG. 8, the laser may be operated to generate at least one track 46 on a continuous trace of the selected chromatogram 12. However, multiple laser tracks 46a, 46b, 46c may be generated on a single continuous trace of the selected chromatogram 12, as illustrated in FIG. Alternatively, multiple laser paths can be created in a single track 47 that is generated on a continuous trace of the selected chromatogram 12 (see FIG. 10).

  FIG. 11 shows an exemplary reproducibility example when multiple passes lead to the same laser track 47 on the selected chromatogram 12 (as illustrated in FIG. 10). Data resulting from the first, third and fifth passes on the same track is shown at 6 second intervals. By allowing multiple passes of the same sample, considerable time can be saved because the sample does not need to be repeated and a small sample is sufficient for the analysis. In addition, the chromatogram can be stored for reanalysis.

  FIGS. 12 a, 12 b and 12 c show the increase in detection limit by summing multiple paths through the same laser track 47 on the selected chromatogram 12. For these drawings, eight single passes through the same laser track are added together. It can be seen that there is an approximately 4-fold improvement in signal to noise ratio when compared to a single laser path. In addition, this represents only 20% of the overall recoverable signal from the selected chromatogram 12 because additional laser tracks are possible on the selected chromatogram (see, eg, FIG. 9). reference).

  The system shown in FIG. 6 combines a high frequency electrodeposition and nebulizer. This allows the system of FIG. 6 to achieve a throughput that exceeds that which can only be reached in parallel, and high-speed chromatography peaks require high resolution deposition techniques. The devices and systems described above can be applied to all types of liquid chromatographs and can be used for all types of desorption ionization including MALDI. For example, in the device of FIG. 6, the nebulizer can be shut off and voltage pulses can be applied to the deposition surface 16 through the use of a suitable power supply 120, resulting in the device being very fast and discontinuous. Operated in electrostatic mode with stable droplet deposition.

  Alternatively, the power source can be shut off and the nebulizer gas can be nebulized through the nebulizer 24 to spray the eluate in a chromatography column that can produce a generally continuous trace.

  Accordingly, the various embodiments of FIG. 6 can provide high resolution digital by pulsing fluid emanating from the chromatograph by applying a voltage to a target plate that operates at frequencies above about 10 Hz and up to and including about 1 KHz. Can be achieved. The various embodiments of FIG. 6 also allow analog recording (ie, approaching infinite resolution) by nebulizing the fluid coming from the column and collecting it simultaneously as a continuous trace on the target plate. To.

  In addition, sample throughput can be increased by recording multiple chromatograms simultaneously, allowing individual chromatograms to be operated at high speeds, thereby allowing simultaneous duration from multiple chromatographs. Generate a sample transient that is less than one second. The recording of transients (chromatographic peaks) in digital methods (spotting) requires high frequency sampling, to preserve data integrity, and sample transients (peaks) are very fast In some cases, analog recording can be recalled.

  Such chromatogram recording can be performed, for example, by thermal desorption using MALDI ionization or any other form of ionization in any other form, such as fast atom bombardment, secondary ion mass spectrometry (SIMS), electron bombardment. It can be read by using mass spectrometry with separation, photoionization, desorption electrospray (DESI), atmospheric pressure chemical ionization, or other means of ionizing compounds from the surface.

  While the applicant's teachings have been described in connection with various embodiments, it is not intended that the applicant's teachings be limited to such various embodiments. Rather, applicant's teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

FIG. 1a is a schematic diagram of a single sample deposition apparatus taught by the applicant. FIG. 1b is a schematic diagram of a plurality of sample deposition devices taught by the applicant. FIG. 2 is a flowchart illustrating the flow of fluid to a chromatography column. FIG. 3a is a graph comparing a conventional flow profile with a flow profile for the fluid flow of the system illustrated in FIG. FIG. 3b is a graph comparing the conventional flow profile with the flow profile for the fluid flow of the system illustrated in FIG. FIG. 4 is a diagram showing discontinuous sample deposition. FIG. 5a is a graph comparing the detection potential and throughput of a conventional system with those of the applicant's teachings. FIG. 5b is a graph comparing the detection potential and throughput of a conventional system with those of the applicant's teachings. FIG. 5c is a graph of the possibility of detection of separation for minoxidil and reserpine using the applicant's teachings. FIG. 6 is a schematic diagram showing a supply system for atomizing gas using the applicant's teachings. FIG. 7 is a schematic diagram illustrating the deposition of a plurality of eluates released on a target plate to create a plurality of chromatograms. FIG. 8 is an enlarged view of a portion of the chromatogram, showing the laser track. FIG. 9 is an enlarged view of a portion of the chromatogram showing a plurality of laser tracks. FIG. 10 is an enlarged view of a portion of the chromatogram, showing multiple laser paths on the same laser track. FIG. 11 is a chart showing the reproducibility of mass spectrometry analysis of selected chromatograms when using multiple laser paths on the same laser track. FIG. 12a is a chart showing the increase in detection limit by summing multiple paths through the same laser track. FIG. 12b is a chart showing the increase in detection limit by summing multiple paths through the same laser track. FIG. 12c is a chart showing the increase in detection limit by summing multiple paths through the same laser track.

Claims (117)

A method of depositing a sample for analysis comprising:
a) flowing an appropriate eluate through a chromatography column to separate the samples;
b) releasing an eluate having the eluted and separated components of the sample from the chromatographic column, the eluate forming droplets at the discharge end of the chromatographic column;
c) receiving the droplet by providing a suitable deposition surface spaced from the discharge end of the chromatography column;
d) drawing a droplet onto the deposition surface by applying a voltage to the deposition surface, wherein the voltage applied to the deposition surface has a frequency of about 10 Hz or more. how to.   The method of claim 1, wherein the voltage is applied to the deposition surface at a frequency comprising up to about 1 kHz.   The method according to claim 1 or 2, wherein at least one pneumatic pump is used to flow a suitable eluent through the chromatography column.   4. The eluate flow rate is controlled by a flow meter in combination with a control processor, and the eluate flow rate is measured and controlled to provide continuous control of the flow rate. A method according to any one of the above.   5. The eluate flow rate is a mixture of two fluid streams, each fluid flow rate being controlled by a respective flow meter in combination with a control processor. The method described in 1.   The method of claim 5, wherein at least one of the fluid streams is water.   The method of claim 6, wherein the other of the fluid streams is a solvent.   8. A method according to any one of the preceding claims, wherein the voltage is applied to the deposition surface so that a continuous droplet is drawn to a corresponding target location on the deposition surface. .   9. The method of claim 8, wherein the deposition surface is movable with respect to the discharge end of the chromatography column.   10. The method according to any one of claims 1-9, further comprising introducing a matrix into the sample, wherein the matrix is suitable for use in matrix-assisted laser desorption ionization. A method of depositing a plurality of samples for analysis, the method comprising:
a) flowing an appropriate eluent through each of the plurality of chromatographic columns, each column for separating samples;
b) discharging the eluate having the sample elution and separated components from the plurality of chromatographic columns, wherein the eluate drops droplets at the discharge end of the respective chromatographic column; Forming, and
c) receiving the droplet by providing at least one suitable deposition surface spaced from the discharge end of the chromatography column;
d) drawing a droplet onto the deposition surface by applying a voltage to the deposition surface, wherein the voltage applied to the deposition surface has a frequency of about 10 Hz or more. how to.   The method of claim 11, wherein the voltage is applied to the deposition surface at a frequency up to about 1 kHz.   13. A method according to claim 11 or 12, wherein at least one pneumatic pump is used to flow the appropriate eluate through the chromatography column.   13. A method according to claim 11 or 12, wherein a plurality of pneumatic pumps are provided, wherein at least one pump is associated with each of each chromatography column.   The eluate flow rate for each chromatography column is controlled by a respective flow meter in combination with a control processor, and the eluate flow rate for each of each chromatography column is independently measured and controlled, 15. A method according to any one of claims 11 to 14 that provides continuous control of the flow rate for each column.   12. The flow rate of each eluate for each chromatography column is provided by mixing two fluid streams, each fluid flow rate being controlled by a respective flow meter in combination with a control processor. 15. The method according to any one of 15.   The method of claim 16, wherein at least one of the fluid streams is water.   The method of claim 17, wherein the other of the fluid streams is a solvent.   19. A method according to any one of claims 11 to 18, wherein the voltage is applied to the deposition surface so that successive droplets are drawn to corresponding target locations on the deposition surface.   20. The method of claim 19, wherein the deposition surface is movable with respect to the discharge end of the chromatography column.   21. A method according to any one of claims 11 to 20, further comprising introducing a matrix into the sample, wherein the matrix is suitable for use in matrix-assisted laser desorption ionization. A method of depositing a plurality of samples for analysis, the method comprising:
a) flowing an appropriate eluate through a plurality of chromatographic columns, each column for separating samples;
b) releasing the eluate having the eluted and separated components of the sample from the plurality of chromatographic columns;
c) spraying the released eluate;
d) depositing the sprayed eluate on at least one suitable deposition surface to produce a chromatogram.   23. The method of claim 22, wherein at least one pneumatic pump is used to flow a suitable eluent through the chromatography column.   23. The method of claim 22, wherein a plurality of pneumatic pumps are provided, wherein at least one pump is associated with each of each chromatography column.   The eluate flow rate for each chromatographic column is controlled by a respective flow meter in combination with a control processor, and the eluate flow rate for each of the chromatographic columns is independently measured and controlled. 25. A method according to any one of claims 22 to 24, which provides continuous control of the flow rate for each column.   23. Each eluate flow rate for each chromatography column is provided by mixing two fluid streams, each fluid flow rate being controlled by a respective flow meter in combination with a control processor. 26. The method according to any one of 25.   27. The method of claim 26, wherein at least one of the fluids is water.   28. The method of claim 27, wherein the other fluid stream is a solvent.   29. A method according to any one of claims 22 to 28, wherein a stream of non-reacting gas is sprayed with the released eluate.   30. The method of claim 29, wherein the non-reactive gas is nitrogen.   31. A method according to any one of claims 22 to 30, wherein the chromatogram is a continuous trace.   32. The method of claim 31, wherein the continuous traces are parallel to each other.   33. A method according to claim 31 or 32, wherein each continuous trace corresponds to a release from a respective chromatography column.   34. All or a portion of the chromatogram is ionized by a laser, which generates at least one track on the continuous trace of a selected chromatogram. The method described in 1.   35. The method of claim 34, wherein the laser is a high speed laser and each chromatogram is rastered at a constant speed.   36. A method according to claim 34 or 35, wherein a plurality of laser tracks are generated on the continuous trace of the selected chromatogram.   37. A method according to any one of claims 34 to 36, wherein multiple laser paths are created on a single track generated on the continuous trace of a selected chromatogram.   38. The method of any one of claims 22-37, further comprising introducing a matrix into the sample, wherein the matrix is suitable for use in matrix-assisted laser desorption ionization.   40. The method of claim 38, wherein the matrix is introduced into the eluate prior to the step of nebulizing the released eluate.   40. A method according to any one of claims 22 to 39, wherein the released eluate is heated. An apparatus for preparing a sample for analysis, the apparatus comprising:
a) a chromatographic column that receives the sample using an appropriate eluent;
b) a pump for flowing the eluate through the chromatography column;
c) a suitable deposition surface, the deposition surface being formed at its end by the flow of the eluate through the chromatography column by being spaced from the discharge end of the chromatography column. Receiving a droplet, a deposition surface,
d) a power source that draws the droplets onto the deposition surface by generating a voltage on the deposition surface, the voltage applied to the deposition surface comprising a power source having a frequency of about 10 Hz or more. The equipment.   42. The apparatus of claim 41, wherein the voltage is applied to the deposition surface at a frequency up to about 1 kHz.   43. Apparatus according to claim 41 or 42, wherein the pump is an air driven pressure amplification pump.   44. Apparatus according to any one of claims 41 to 43, wherein the pump comprises a flow meter and a control processor to provide continuous control of the eluate flow rate.   45. Apparatus according to any one of claims 41 to 44, wherein the pump comprises a pressure source dimensioned to hold a volume of eluate that is larger than the volume of the chromatography column. .   46. The power source according to any one of claims 41 to 45, wherein the power source applies a voltage to the deposition surface so that a continuous droplet is drawn to a corresponding target location on the deposition surface. The device described.   47. The apparatus of claim 46, wherein the deposition surface is movable with respect to the discharge end of the chromatography column.   48. A nebulizer for introducing a nebulizer gas into the chromatography column, the nebulizer spraying a flow of the eluate when the eluate is released. Equipment.   49. The apparatus of claim 48, wherein the atomizing gas is a non-reactive gas.   50. The apparatus of claim 49, wherein the non-reactive gas is nitrogen.   51. The apparatus according to any one of claims 41 to 50, further comprising a matrix delivery system for introducing a matrix into the eluate, wherein the matrix is suitable for use in matrix-assisted laser desorption ionization.   52. The apparatus of claim 51, wherein the matrix supply system supplies the eluate to the matrix before the eluate is nebulized. An apparatus for preparing a plurality of samples for analysis, the apparatus comprising:
a) a plurality of chromatographic columns receiving the sample with an appropriate eluent;
b) a plurality of pumps, each pump associated with each chromatography column, said pump flowing said eluate through said chromatography column, said pump comprising a flow meter and a control processor; A plurality of pumps providing further control of the flow rate of the eluate by further comprising:
c) at least one suitable deposition surface, the deposition surface being spaced from a plurality of discharge ends of the respective chromatography column, wherein the at least one suitable deposition surface is the respective deposition surface. A deposition surface that receives droplets formed at its ends by the flow of the eluate through the chromatography column;
d) at least one power source that draws the droplet on the deposition surface by generating a voltage on the deposition surface, the voltage applied to the deposition surface being at a frequency of about 10 Hz or more; A device comprising:   54. The apparatus of claim 53, wherein the voltage is applied to the deposition surface at a frequency up to about 1 kHz.   55. Apparatus according to claim 53 or 54, wherein the pump is an air driven pressure amplification pump.   56. The apparatus according to any one of claims 53 to 55, wherein the pump further comprises a flow meter and a control processor to provide continuous control of the eluate flow rate.   57. The pump according to any one of claims 53 to 56, wherein the pump further comprises a pressure source dimensioned to retain a volume of eluate that is greater than the volume of the respective chromatography column. apparatus.   58. The power source according to any one of claims 53 to 57, wherein the power source applies a voltage to the deposition surface so that a continuous droplet is drawn to a corresponding target location on the deposition surface. The device described.   59. The apparatus of claim 58, wherein the at least one deposition surface is movable with respect to the discharge end of the chromatography column.   The nebulizer further includes a nebulizer for introducing the atomizing gas into the respective chromatography column, the nebulizer comprising a first manifold connected to the plurality of chromatography columns, thereby supplying the atomizing gas to the chromatography column. 60. Apparatus according to any one of claims 53 to 59, wherein the apparatus is introduced and the nebulizer atomizes the eluate stream as the eluate is released.   61. The apparatus of claim 60, wherein the first manifold is connected to the plurality of chromatography columns by a T-shaped valve.   62. The apparatus of claim 61, further comprising a plurality of deposition tubules operably connected to each T-shaped valve to release the nebulized eluate from each of the plurality of chromatography columns.   63. The apparatus according to any one of claims 60 to 62, wherein the nebulizer further comprises a pump to supply the atomizing gas to the plurality of chromatography columns.   64. The apparatus of claim 63, wherein the pump comprises an air pump.   The apparatus according to any one of claims 60 to 65, wherein the atomizing gas is a non-reactive gas.   66. The apparatus of claim 65, wherein the non-reacting gas is nitrogen.   A matrix supply system, the matrix supply system including a second manifold connected to the plurality of chromatography columns, the second manifold introducing a matrix into the eluate; 67. Apparatus according to any one of claims 53 to 66, suitable for use in matrix-assisted laser desorption ionization.   68. The apparatus of claim 67, wherein the second manifold is connected to the plurality of chromatography columns by a T-valve.   69. The second manifold is operably connected to each of a plurality of chromatography columns to supply the matrix before the eluate is nebulized. Equipment.   70. The apparatus according to any one of claims 67 to 69, wherein the matrix supply system further includes a pump to supply the matrix to the plurality of chromatography columns.   72. The apparatus of claim 70, wherein the pump is a syringe pump.   72. The apparatus of claim 70, wherein the pump is a continuous flow pump.   A translation stage for receiving the at least one deposition surface, the translation stage being movable with respect to the plurality of chromatographic columns, so that the generated chromatograms are parallel to each other; 73. The apparatus according to any one of claims 53 to 72.   74. The apparatus of claim 73, wherein the at least one deposition surface is a plurality of plates arranged in a deposition array on the translation stage. An apparatus for preparing a plurality of samples for analysis, the apparatus comprising:
a) a plurality of chromatographic columns receiving at least one sample using a suitable eluent;
b) a plurality of pumps, each pump being associated with each chromatography column;
c) a nebulizer for introducing a spray gas into the plurality of chromatography columns, the nebulizer nebulizing the stream of the eluate as it is released;
d) at least one suitable deposition surface for receiving the released atomized eluate, wherein the released atomized eluate from the plurality of chromatographic columns is the at least one A deposition surface for generating each of a plurality of chromatograms on an appropriate deposition surface.   76. The apparatus of claim 75, wherein the pump for flowing the eluate through the chromatography column further comprises a flow meter and a control processor to provide continuous control of the eluate flow rate.   77. The apparatus of claim 75 or 76, wherein the plurality of pumps are air driven pressure amplification pumps.   78. The plurality of pumps of any one of claims 75-77, further comprising a pressure source dimensioned to hold a volume of eluate that is greater than the volume of the respective chromatography column. The device described.   79. The nebulizer includes a first manifold connected to the plurality of chromatography columns to introduce the atomizing gas into each chromatography column. The device described in 1.   80. The apparatus of claim 79, wherein the first manifold is connected to the plurality of chromatography columns by a T-valve.   81. The atomized eluate is released from each of the plurality of chromatographic columns by further comprising a plurality of deposition tubules operatively connected to each T-shaped valve. The device described.   82. The apparatus according to any one of claims 75 to 81, wherein the nebulizer further includes a pump to supply the atomizing gas to the plurality of chromatography columns.   The apparatus of claim 82, wherein the pump comprises an air pump.   84. Apparatus according to any one of claims 75 to 83, wherein the atomizing gas is a non-reactive gas.   85. The apparatus of claim 84, wherein the non-reacting gas is nitrogen.   A matrix supply system, the matrix supply system including a second manifold connected to the plurality of chromatography columns, the second manifold introducing a matrix into the eluate; 86. Apparatus according to any one of claims 75 to 85, suitable for use in matrix-assisted laser desorption ionization.   87. The apparatus of claim 86, wherein the second manifold is connected to the plurality of chromatography columns by a T-valve.   88. The second manifold is operably connected to each of a plurality of chromatography columns to supply the matrix before the eluate is nebulized. Equipment.   89. The apparatus according to any one of claims 86 to 88, wherein the matrix supply system supplies the matrix to the plurality of chromatographic columns by further including a pump.   90. The apparatus of claim 89, wherein the pump is a syringe pump.   90. The apparatus of claim 89, wherein the pump is a continuous flow pump.   A translation stage for receiving the at least one deposition surface, the translation stage being movable with respect to the plurality of chromatographic columns, so that the generated chromatograms are parallel to each other; 92. The apparatus according to any one of claims 75-91.   94. The apparatus of claim 92, wherein the at least one deposition surface is a plurality of plates arranged in a deposition array on the translation stage.   76. At least one power source that draws the droplet onto the deposition surface by generating a voltage on the deposition surface, the voltage applied to the deposition surface having a frequency of about 10 Hz or more. 94. The device according to any one of 93.   95. The apparatus of claim 94, wherein the voltage is applied to the deposition surface at a frequency up to about 1 kHz.   96. The apparatus of claim 94 or 95, wherein the power source applies a voltage to the deposition surface, such that a continuous droplet is drawn to a corresponding target location on the deposition surface. Depending on use, a system for preparing multiple samples for analysis via droplet deposition or by nebulization, the system comprising:
a) a plurality of chromatographic columns receiving at least one sample using a suitable eluent;
b) a plurality of pumps, each pump being associated with each chromatography column;
c) a nebulizer for introducing a spray gas into the plurality of chromatography columns, the nebulizer nebulizing the stream of the eluate as it is released;
d) at least one suitable deposition surface for receiving the released atomized eluate, wherein the released atomized eluate from the plurality of chromatography columns is the at least one appropriate A deposition surface that produces each of a plurality of chromatograms on the stable deposition surface;
e) at least one power source that draws droplets on the deposition surface by generating a voltage on the deposition surface, the voltage applied to the deposition surface having a frequency of about 10 Hz or more; System.   98. The system of claim 97, wherein the voltage is applied to the deposition surface at a frequency up to about 1 kHz.   99. The system of claim 97 or 98, wherein the power source applies a voltage to the deposition surface, such that a continuous droplet is drawn to a corresponding target location on the deposition surface.   99. The pump of claim 97-99, wherein the pump for flowing the eluate through the chromatography column further comprises a flow meter and a control processor to provide continuous control of the eluate flow rate. The system according to any one of the above.   The system according to any one of claims 97 to 100, wherein the plurality of pumps are air-driven pressure amplification pumps.   102. The plurality of pumps according to any one of claims 97 to 101, wherein the plurality of pumps further comprises a pressure source dimensioned to hold a volume of eluate that is greater than the volume of the respective chromatography column. The described system.   103. The nebulizer includes a first manifold connected to the plurality of chromatography columns to introduce the atomizing gas into each of the chromatography columns. The system described in.   104. The system of claim 103, wherein the first manifold is connected to the plurality of chromatography columns by a T valve.   105. The system of claim 104, further comprising a plurality of deposition tubules operably connected to each T-shaped valve to release the nebulized eluate from each of the plurality of chromatography columns.   106. The system of any one of claims 97 to 105, wherein the nebulizer further includes a pump to supply the atomizing gas to the plurality of chromatography columns.   107. The system of claim 106, wherein the pump comprises an air pump.   108. The system according to any one of claims 97 to 107, wherein the atomizing gas is a non-reactive gas.   109. The system of claim 108, wherein the non-reacting gas is nitrogen.   A matrix supply system, the matrix supply system including a second manifold connected to the plurality of chromatography columns, the second manifold introducing a matrix into the eluate; 110. A system according to any one of claims 97 to 109, suitable for use in matrix-assisted laser desorption ionization.   111. The system of claim 110, wherein the second manifold is connected to the plurality of chromatography columns by a T-valve.   112. Any of claims 110 or 111, wherein the second manifold is operatively connected to each of a plurality of chromatography columns to supply the matrix before the eluate is nebulized. A system according to claim 1.   113. The system of any one of claims 110 to 112, wherein the matrix supply system further includes a pump to supply the matrix to the plurality of chromatographic columns.   114. The system of claim 113, wherein the pump is a syringe pump.   115. The system of claim 114, wherein the pump is a continuous flow pump.   A translation stage for receiving the at least one deposition surface, the translation stage being movable with respect to the plurality of chromatographic columns, so that the generated chromatograms are parallel to each other; 116. The system according to any one of claims 97 to 115.   117. The system of claim 116, wherein the at least one deposition surface is a plurality of plates arranged in a deposition array on the translation stage.

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