WO2015011522A1 - Multi-task sample preparation system with reconfigurable modules for on-line dilution, enzymatic digestion and fractionation - Google Patents

Abstract

Modular System of sample preparation that can be reconfigured by the user which can perform on-line dilution, enzymatic digestion and fractionation. The system has adjustable modular column oven with high pressure pump and adjustable UV detector that can be reconfigured by the user for specific tasks to perform.

Description

TITLE: MULTI-TASK SAMPLE PREPARATION SYSTEM WITH RECONFIGURABLE MODULES FOR ON-LINE DILUTION, ENZYMATIC DIGESTION and FRACTIONATION

FIELD:

The present invention relates to investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion- exchange, e.g. chromatography using a recongigurable and modular system.

BACKGROUND:

In clinical and environmental laboratories, food and pharmaceutical industries measure and analyze each year millions of samples that stem from various matrices. Nowadays, the most common methodology for analysis is based on chromatographic principles. Sample preperation step that is performed prior to the analysis is one of the main reasons for analysis time and errors. Depending on the sample matrix, e.g. plasma, serum, urine, aqueous and similar or depending on the sample type, e.g. bacteria, virus, protein, peptide or similar, each sample preparation process can be different.

The sample preparation process has direct impact on the analytical method parameters such as recovery, accuracy & precision, repeatability and measurement uncertainty. It doesn't matter whether the lab personnel is well educated or not, the experimental reproducibility turns out to one big problems after a "man made" sample preparation. Due to these reasons, starting in 90' s (e.g. U.S. Pat. No. 5,760,299, U.S. Pat. No. 6,730,517 Bl, WO2012058559 A2) upto today, many inventions related with process automatization in the field of analytical chemistry have been applied in order to eliminate these above mentioned disadvantages of the traditional sample preparation processes that are based on "off-line" methodologies. On the other hand the automatization of the sample preparation processes increases the high-throughput capacities that is crucial factor for laboratories particularly working on routin analysis. All kind of inventions that are described in U.S. Pat. No. 8,414,774 B2, U.S. Pat. No. 7,217,360 B2, U.S. Pat. No. 5,538,932A, U.S. Pat. No. 6,491,816 B2, U.S. Pat. No. 6,783,672 B2, U.S. Pat. No. 6,458,273 Bl and Int. Pub. No. WO 97/16724 have been done with the main goal to increase the high- throughput of the methodologies.

Currently, depending on the matrix and molecule characteristics, different types of sample preparation and chromatografic methodologies are utilized. Nowadays, the most prominent methodologies are solid phase extraction (SPE) described in U.S. Pat. No. 5,439,593 A, U.S. Pat. No. 7,563,410 B2, U.S. Pat. No. 6,124,012 A, U.S. Pat. No. 5,760,299 B2, Int. Pat. No. WO 22111162575A, U.S. Pat. No. 6,149,816 A, filtration described in U.S. Pat. No. 6,491,873 B2, Int. Pat. No. WO 2011/090978 Al, on-line fractionation described in U.S. Pat. No. 6,309,541 Bl, U.S. Pat. No. 8,305,581 B2, U.S. Pat. No. 5,234,586 A, Int. Pub. No. WO 00/26662, on-line digestion described in Int. Pub. No. WO 2011162575 A2 and multi-dimentional chromatography described in U.S. Pat. No. 5,234,599 A, Int. Pub. No. WO 2011/112188 A.

Unfortunately, the sample treatment and/or processing may be undesirably time- consuming, particularly when multiple samplings are required, as during a multi-step preparation. The time limitation during the sample processing is of particular concern in industrial or other large scale preparations. To be useful, the sample treatment and sample processing step should be rapid, adaptable, and repeatable. Accordingly, there exists a need for instrumentation of rapidly identifying the presence and location of a molecule of interest during any preparative process.

SUMMARY OF INVENTION:

This invetion is about a reconfigurable apparatus for chromatographically analyzing samples in a detector, including an autosampler which contains samples for chromatographic analysis, a plurality of chromatography columns, a plurality of pumps. The apparatus further includes different types of switching valves that can be positioned and reconfigured according to the experimental targets and desires of the user. A computer control device is included which automatically adjusts the introduction of samples from the autosampler into said plurality of columns as well as the position of said valves and pumps to sequentially deliver said eluant to said detector.

The main objective of this invention is to provide methods and apparatus for the rapid on-line identification of molecular products during preparative protocol(s), such as purification (fractionation), extraction , enzymatic digestion, on-line dilution and chromatographic separation. Another objective is to provide a method and apparatus for rapidly identifying the presence and location of a molecule in a chromatography effluent. Yet, another objective is to provide multi-task sample preparation system with reconfigurable modules for on-line sample dilution, on-line enzymatic digestion, on-line purification (fractionation), on-line extraction, on-line chromatographic separation and on-line detection. Still another objective of this invention is to provide user friendly apparatus which can be reconfigured according to the experimental targets and desires of the user. These and other objects and features of the invention will be apparent from the drawing, description, and claims which follow. As a result of this process, the invention is expected to increase the detection limits in mass spectrometry, accelarate the sample preparation process and, at the same time, obtain a high degree of automation of the analysis.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure. 1A is the Six -port valve module apparatus design of the present invention in clockwise operation configuration;

Figure. IB is the Six-port valve module apparatus design of the present invention in counter clockwise operation configuration;

Figure. 2A is the Six -port valve module apparatus design for On-line Dilution of the present invention clockwise operation configuration;

Figure. 2B is the Six-port valve module apparatus design for On-line Dilution of the present invention counter clockwise operation configuration; Figure. 3A is the Six -port valve module apparatus, special designed, with the integrated module oven for On-line Digestion of the present invention in clockwise operation configuration with Oven-Lid removed;

Figure. 3B is the Six-port valve module apparatus, special designed, with the integrated module oven for On-line Digestion of the present invention in counter clockwise operation configuration with Oven-Lid in place;

Figure. 4A is the Four-port valve module apparatus, special designed, with the integrated module oven for On-Line Digestion of the present invention in clockwise operation configuration with Oven-Lid removed;

Figure. 4B is the Four-port valve module apparatus, special designed, with the integrated module oven for On-Line Digestion of the present invention in counter clockwise operation configuration with Oven-Lid in place;

Figure. 5A is the Four-port valve module apparatus design of the present invention in clockwise operation configuration;

Figure. 5B is the Four-port valve module apparatus design of the present invention in counter clockwise operation configuration;

Figure. 6A is the frontal view of the module; Figure. 6B is the side view of the module; Figure. 6C is the back view of the module;

Figure. 6D is the view of backplane where modules get plugged-in;

Figure. 7 is the frontal view of the designed modular UV detector of the present invention;

Figure. 8 A is the view of the Rotor Seal for Six-port switching valves;

Figure. 8B is the view of the Rotor Seal for Four-port switching valves;

Figure. 8C is the view of the Rotor Seal for Six-port switching valves with On-line dilution; Figure. 9A is the view of the modular configuration for Experiment- 1 in Step-1;

Figure. 9B is the view of the modular configuration for Experiment- 1 in Step-2;

Figure. 10 is the view of the result of the Experiment-1 for different configurations;

Figure. 11A is the view of the modular configuration for Experiment-2 in Step-1;

Figure. 11B is the view of the modular configuration for Experiment-2 in Step-2; Figure. 11C is the view of the modular configuration for Experiment-2 in Step-2;

Figure. 11D is the view of the result of the Experiment-2;

Figure. HE is the view of the result of the ion chromatography of the Experiment-2;

DETAILED DESCRIPTION: The structure and function of exemplary parts of the present invention can best be understood by reference to the drawings. The same reference numbers may appear in multiple figures. The reference numbers refer to the same or corresponding structure in those figures.

The special designed apparatus 100 (FIG. 1) is a modified module of a six port switching valve which comprises engagement tracks (101) on both lateral sides (left and right). The part (99) represents the channel input/output of a modular switching valve. By using these engagement tracks, user can fix the modules according to desired sequence. Bold lines (103) represent illuminated sign which defines the active flow path. Fine lines (102) represent sign without illumination which defines the passive flow path. The part with fine line (102) represent the passive flow (no flow) and part with bold line (103) represent the active eluent flow. The part (104) and (105) are illuminated signs which define the direction of rotation of the switching valve. The part with (104) represents no illumination. The part with (105) represents illumination. Depending on appearance side of the illumination user can define and determine the direction of rotation. This characteristic makes the module in the case of very complex experimental configuration more user friendly during the setting a method, experiments and trouble- shooting. Rotation in counter clockwise direction is defined with letter [A], and rotation in clockwise direction is defined with letter [B]. The arrows represented by the part (181) help to distinguish the turning position of the switching valves (left or right) which are indicated in whole Figures from 1 to 11. The special designed apparatus 200 (FIG. 2) is a modified module of a six port switching valve. The part (106) is representing plugs whereas there are no connections.

The special designed apparatus 300 (FIG. 3) is a modified module of a six port switching valve. This type of module with special designed for on-line enzymatic digestion during sample processing comprises addjustible resistant heater (112) and special cover cap (107). The part (301) represents the module without cover cap, and part (302) represents the module with cover cap. The special designed apparatus 400 (FIG. 4) is a modified module of a four port switching valve. This type of module with special designed for on-line enzymatic digestion during sample processing comprises addjustible resistant heater (112).

The special designed apparatus 500 (FIG. 5) is a modified module of a four port switching valve. This type of module is designed mainly for on-line fractionation during sample processing. Bold lines (103) represent illumination which define the active flow path. Fine lines (102) represent no illumination which define the passive flow path. The part with fine line (102) represent the passive flow (no flow) and part with bold line (103) represent the active eluent flow. The part (104) and (105) are illumination which define the direction of rotation of switching valve. The part with (104) represents no illumination. The part with (105) represents illumination. Depending on appearance side of the illumination user can define and determine the direction of rotation. This characteristic makes the module in the case of very complex experimental configuration more userfriendly for instance during the setting a method, experiments and trouble- shooting Rotation in counter clockwise direction is defined with letter [A], and rotation in clockwise direction is defined with letter [B]. The Figures 6-A,B,C illustrate the frontal side 110, lateral side 111, and back side 112 of the special designed modular apparatus. Bold lines (103) represent illumination which define the active flow path. The part (106) represents the male plug connecting the apparatus to the computer backplane which is shown in Figure- 6-D. Part (106) shown in Figure-6-B gets plugged to the socket 600 shown in Figure-6-D. The software running inside the computer recognizes directly the utilized module type, fixed position and direction of rotation (clockwise rotation or counter clockwise rotation). The special designed apparatus 600 (FIG. 7) is a modular UV-detector. This type of module is designed mainly for on-line fractionation during the sample processing. The part with (601) represents the UV-cell.

The Figures 8-A,B,C illustrate the appropriate rotor seals of the modular switching valves. Depending on the type of the utilized modular switching valve these rotor seals vary in their channel types and channel numbers. The part (700) represents the rotor seal for the special designed apparatus in Figure 1 and Figure 3 which is a modified module of a six port switching valve. The part in Figure 8B represents the rotor seal for the special designed apparatus Figure 5 which is a modified module of a four port switching valve. The part in Figure 8-C represents the rotor seal for the special designed apparatus in Figure 2 which is a modified module of a six port switching valve for on-line dilution. The part (703) represents the channels of the rotor seal for the Six -port switching valves. The part (704) represents the channels of the rotor seal for the Four-port switching valves . The part (705) represents the channels of the rotor seal for the on-line dilution Six-port switching valves. The part (706) represents a blind point on the rotor seal whereas there is no flow.

The present invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. In other word the following examples further exemplify the invention. They should be considered non-limiting. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.

Applications/Uses

The hereby described methods and apparatus may be used to detect the presence of one or more compounds in a variety of samples. The hereby described methods, apparatus find applicability in any industry that utilizes liquid chromatography and mass spectrometry including, but not limited to, the petroleum industry, the pharmaceutical industry, various analytical labs, etc.. EXAMPLES

EXAMPLE 1 Example 1, is about analysis of samples such as plasma, serum, milk etc. and extracted via protein precipitation (PP) techniques and/or liquid-liquid extraction (LLE) with strong organic solvents such as acetonitrile, methanol and/or similar. Depending on the molecule and matrix type, one volume of plasma sample is precipitated in general with at least the same or bigger volume of protein precipitation solvents (acetonitrile, methanol etc.). Traditionally, after PP or LLE the supernatant phases which containing high concentration of strong organic solvents are evaporated and afterwards reconstituted adding solvents with high content of aqueous solvents. The main reason of the evaporation/ reconstitution is to perform good chromatography, i.e. to obtain good separation and peaks with good shape (not splitted double peaks). In the case of low injection volume of supernatant with high content of organic solvent and without any evaporation/ reconstitution process, when injected into a traditional system (the traditional system: is usually a liquid chromatography coupled with a mass spectrometer detector which is consisting a pump, analytical column and detector) the peak (1051) shown in Figure 10A is in good shape.

A small molecule drug in human plasma was selected as a sample for this application (Example-1). One milliliter of this sample was extracted by adding of two milliliters of acetonitrile. Afterwards, the sample was vortexed and centrifuged for 10 minutes at room temperature and speed of 10000 rpm. Finally, the formed supernatant was transferred into another fresh and clean tube. The content of organic phase in the sample was around 60 precent. The utilized column was a reverse phase column. In Figure-10-A, is shown an injection of prepared supernatant that consists around 60 percent of organic solvent. The injected volume were ten microliters. In Figure-10-B, hundred microliters of the same supernatant with 60 percent of organic solvent were injected into the same traditional system. In Figure-10-B, is shown the effect of high injection volume containing high level of organic solvent into the traditional systems. As result, a splitted and double peak (1101) was obtained which is undesirable in the analytical chemistry. By setting the configuration in Figure-9-A and Figure-9-B, high injection volumes of supernatant (extracted sample) were possible to obtain (1051) in Figure 10-C (not splitted peak in good shape even in presence of high content of strong organic solvent). The modular configuration of the set-up was constructed utilizing one module of a six port switching valve which is designed for on-line dilution (Figure-2) and one six port switching valve (Figure-1). Referring the configuration in Figure-9-A and Figure-9-B, the set up comprises furthermore one autosampler, three pumps, two columns and one detector. Referring the configuration in Figure-9-A and Figure-9-B, pump-1 (920) was the transfer pump which is transferring the sample from the autosampler into the system, pump-2 (930) was the dilution pump which is diluting the sample on-line, pump-3 (935) was the elution pump which was performing the separation (chromatography) of analytes. Referring the configuration in Figure-9-A and Figure-9-B, part 802 was a short and reverse phase trap column and part 801 was a long and reverse phase analytical column (801). Pump-1 (920) was delivering pure water for 90 seconds with a flow rate of hundert microliters per minute. Pump-2 (930) was delivering pure water for 90 seconds with a flow rate of four hundert microliters per minute which is diluting the sample by five times. After dilution by five times the final content of organic solvent in sample was reduced to around 12 percent which was sufficient to trap the molecule on the trap column (802) Figure-9-A (task-1). After trapping time of 90 seconds the position of the six port switching valve of task-2 (915) was changed to position [B]. Using pump-3 (935), the trapped and cleaned sample was delivered into the analytical column (801). By applying of gradient with the pump-3 (935), the molecules were separated and subsequently detected by mass spectrometer. The gradient was performed using of two elution solvents. The first elution solvents was 0.1% (v/v) formic acid in water (I). The second elution solvents was 0.1% (v/v) formic acid in acetonytrile (II). The gradient was performed at room temperature. Depending on the molecule characteristics, the injection volume of the sample, the content and type of the organic solvents the parameters (e.g. dilution ratio, dilution time, elution solvents, temperature, gradient etc.) can be different. The result of the sample analysis of hundert microliters injection and 60 percent of organic solvent and using on-line dilution is shown in Figure-10-C. As depicted in Figure-10-C, the peak was not splitted. The sensitivity was increased by also ten times. The evaporation step of the organic solvent was eliminated. Taking into account of the whole sample preparation process the time consuming during the sample preparation in general was reduced by almost fifty percent and was gained a sensitivity of ten times (compare the signal intensities in Figure-10-A and Figure-10-C). The sensityvity of ten microliters injection volume (Figure-10-A) which is indicated as intensity (cps: count per second) was 5.1e⁵ and sensitivity of hundert microliters injection volume (Figure-10-C) was 5.3e⁶. Using this multy task modular system and configuration, not limited with this example, the sensitivity can be increased proportionally with bigger injection volumes in the case of sufficient sample amounts.

The computer (900) controls the autosampler (106), the pump-1 (920), the pump-

2 (930), the pump-3 (935), the modules (182) and (183), and detector (940). The module (182) performs task-1 and gets instructions from backplane (905).

Module (182) performs task-1 (916) as configured by the user and passes the eluent through tubing (49) to module (183) which performs different task and passes the cleaned sample from the trap column (802) into the analytical column (801) trough the tubing (48). The timing and the actions of the modules (182) and (183) are controlled by computer (900) trough electrical signals sent from backplane (905) and connections (910).

The timing and the actions of the autosampler (106) is controlled by computer (900) trough electrical signals sent from backplane (905) and connections (910).

EXAMPLE 2 : Referring Example-2, this multi-task application is about the analysis (i.e. identification and/or quantification) of very complex biological sample mixtures such as proteins, peptides or similar. Because the biological sample matrices contain high complexity these type of samples can not be detected directly. In the case of direct injection of such complex samples into a LC-MS system (LC-MS: Liquid Chromatography coupled with a Mass Spectrometer) it can cause of perturbed detection signals due to ion suppression. In order to avoid these negative effect samples are prepared/treated prior to analysis. Traditionally, the sample preparation processes are treated in "off-line" manner which takes ususally long time and causes many errors during the sample preparation processes. Gluten proteins were selected as a sample for this application (Example-2).

Gluten (from Latin gluten, "glue") is a very complex protein composite found in foods processed from wheat and related grain species, including barley and rye. Gluten may also be found in some cosmetics, hair products, and other dermatological preparations [Ref. 1]. Gluten is the composite of a gliadin and a glutenin, which is conjoined with starch in the endosperm of various grass-related grains. The prolamin and glutelin from wheat (gliadin, which is alcohol- soluble, and glutenin, which is only soluble in dilute acids or alkalis) constitute about 80% of the protein contained in wheat fruit. Being insoluble in water, they can be purified by washing away the associated starch. Worldwide, gluten is a source of protein, both in foods prepared directly from sources containing it, and as an additive to foods otherwise low in protein.

We are seeing sometimes manufactured bakery products on the markets which are defined as gluten free [Refs. 2-3]. What makes these gluten proteins so important for the fabrication of bakery industry? The gluten proteins are representing up to 80% of wheat proteins and they are conventionally subdivided into gliadins and glutenins. Gliadins belong to the proline and glutamine-rich prolamin family. They are playing very important role for gluten intolerance in human body, so called Celiac Disease (CD), due to their toxic effects [Refs. 4-6]. Gluten proteins in food are digested by gastric or pancreatic enzymes such as pepsin or chymotrypsin into small peptides that usually do not have any toxic effect [Refs. 7-8]. However, in patients suffering from CD these peptides give rise to elevated t-cells response resulting chronical inflammation at the small intestinal mucosa. The only therapy to CD so far is gluten free diet [Refs. 9-11]. Hence, it is very important for Food Industry (FI) to develop fast and routinelly analytical methodes in their laboratories to measure the gluten proteins of food as Quality Control (QC) prior to their large scale production. Therefore, in this field many analytical methodologies and techniques are developed for quantitative and qualitative analysis of the gluten proteins (gliadins, glutenins) [Refs. 12-21]. Unfortunatelly, most of these methods are not enough fast and/or not fully automatized.

As depicted in Example-2, concerning the whole sample preparation and analysis process the time consuming was reduced with this invention by more than 50 percent. The experiment in Example-2 was performed with the goal to characterize an alfa Gliadin obtaining a specific peptide. For the Example-2, wheat flour of type 405 (Harina de trigo tipo 405 - Farinha de trigo tipo 405) which purchased from a local supermarket was used for this application. Therefore, two grams of flour were weighted and then solved in a 10 milliliters aqueous 70% Ethanol solvent. The obtained suspension sample was shaked for 40 minutes at room temperature. After the shaking, the suspension sample was centrifuged for 15 minutes at room temperature and speed of 4000 rpm. After the centrifugation, the supernatant was transfered to fresh and clean tube, so the sample was ready for analysis. Referring the system in Figures-ll-(A, B, C), the modular configuration of the set-up was constructed utilizing one autosampler, six LC-pumps (Liquid Chromatography pumps), one module of a four port switching valve (Figure-5), two modules of a six port switching valve which is designed for on-line dilution (Figure- 2), two modules of a six port switching valve (Figure-2), two modules of a six port switching valve (Figure-1), one module of a four port switching valve which is designed for on-line enzymatic digestion (Figure-4) and one module special designed modular UV-detector 600 (Figure-7)

Referring the system in Figures-ll-(A, B, C), the part-803 was a long strong cation exchange column (the utilized cation exchange column for this experiment was the ProPac SCX-10, 250 x 4.0 mm i.d. and purchased from Dionex), parts-804 and part-806 were short reverse phase trap columns, part-106 was plug (stopper), part-805 was enzymatic column (for this experiment the enzymatic column was a pepsin column), part-807 was a long reverse phase analytical column.

Referring the configuration in Figures-ll-( A, B, C), pump-1 (920) was the transfer pump which was transferring the sample from the autosampler into the system and in the same time performing the cation exchange gradient. Pump-2 (922) was acting as a carrier pump which transfered the fraction to task-3. Pump-3 (923) was dilution pump of the task-3. Pump-4 (924) was acting as a further carrier pump which transfered the fraction to task-5 and task-6 and than task-7. Pump-5 (925) was acting as a dilution pump of in the task-5. Pump-6 (926) was acting as elution pump which was performing the separation (chromatography) of analytes.

Referring the system configuration in Figures-ll-( A, B, C), ten microliters of sample (prepared supernatant in aqueous 70% Ethanol) were injected directly into the system. The part-50 is representing the capillary tubings of the system.

Referring the system configuration in Figure-ll-A, gluten proteins were separated by applying strong cation exchange chromatography in task-1. In order to confirm and show the fractionation capability of the present invention the fraction between 46 minutes and 48 minutes (502) of Figure 11-E were collected using the switching waive at task-1. From time Zero up to 46 miutes all the sample was going into the waste (922) of task-1. With the beginning of 46 minutes the four port switching valve was switched to position- [A] (indicator is the illumination) and let the collected fraction to pass through the UV-cell task-2 and to reach the on-line dilution valve at task-3. The cation chromatography was performed with two mobile phases. The first mobile phase (MB-1) was 5.0 mmol/1 aqueous NaH₂P04 buffer with 30.0% Acetonitrile, pH 3.0. The second mobile phase (MB-2) was 0.50 mol/1 NaCl in MB-1. The gradient was 0-60% of MB-2 in 45.0 min, 60-100% of MB-2 in 5.0 min, 100-100% of MB-2 in 10.0 min, 100-0% of MB-2 in 5.0 min, 0-0% of MB- 2 in 5.0 min. The flow rate was one milliliter per minute. The separation was performed at room temperature and detection at wavelenght of 214 nm.

Referring the system configuration in Figure-ll-A, the flow rate of pump-2 were 100 microliters pumping 0.1% (v/v) formic acid in water (task-1). The flow rate of pump-3 were 400 microliters pumping 0.1% (v/v) heptafluorobutyric acid in water (task-3). The collected fraction sample was diluted by five times in task-3. The diluted sample was trapped and cleaned from salts in task-4 on a reverse phase short trap column (part-804) for two minutes (the short trap column (part-804) for this application only, was purchased from Thermo Fisher USA with the trademark Hypersil Gold C8, 10 mm x 1 mm i.d., 3.5 μιη particl size).

Referring the system configuration in Figure-ll-B, the cleaned and trapped sample in task-4 was eluted to task-5 using pump-4 which was delivering a isocratic gradient of 50% acetonitrlile and 50% water. The flow rate of pump-4 were 100 microliters per minute. The sample was diluted by five times in task-5 with the dilution pump (pump-5) which was delivering a flow rate of 400 microliters per minute pumping digestion buffer (100 mM sodium acetate, pH 4.5) addjusted to pH: 4.5 with acetic acid. With a total flow rate of 500 microliters per minute the sample was transferred into the pepsin column into the task-6 whereas the on-line enzymatic digestion was performed (the pepsin column for this application only, was purchased from Applied Biosystems with the trademark Poroszyme Pepsin cartridges, 30 mm x 2.1 mm i.d.). Prior to the on-line digestion the module (Figure- 4) was tempered to 37°C for 30 minutes. The resulting peptic peptides from the on- line digestion were trapped on the reverse phase short trap column (part-806) in task-7 (the short trap column (part-806) for this application only, was purchased from Thermo Fisher USA with the trademark Hypersil Gold C8, 10 mm x 1 mm i.d., 3.5 μιη particl size).

Referring the system configuration in Figure-ll-C, the trapped and cleaned sample was delivered into the analytical column (part-801) using the pump-6. By applying of gradient with the pump-6, the molecules were separated and subsequently detected by mass spectrometer. The gradient was performed using of two elution solvents. The first elution solvents was 0.1% (v/v) formic acid in water (I).The second elution solvents was 0.1% (v/v) formic acid in acetonytrile (II). The gradient was performed at 45 °C for 60 minutes.

Depicted in Figure-ll-D, the resulted peptides from the on-line peptic digestion were analysed using the present invetion. They were detected by a electrospray ionisation mass spectrometer (ESTMS) detector (940). The total ion current chromatogram (TIC) of the analysed peptide mixture is presented in in Figure-11- D. From the TIC was extracted the spesific peptite of a Gliadin which was a characteristic marker for this alpha gliadine (see Figure-ll-D).

The computer (900) controls the autosampler (106), the pump-1 (920), the pump- 2 (922), the pump-3 (923), the pump-4 (924), the pump-5 (925), the pump-6 (926), the modules (191), (192), (193), (194), (195), (196), (197) and detector (940) trought connections (980), (982), (983), (984) and (985).

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Claims

TITLE: MULTI-TASK SAMPLE PREPARATION SYSTEM WITH RECONFIGURABLE MODULES FOR ON-LINE DILUTION, ENZYMATIC DIGESTION and FRACTIONATION

CLAIMS:

Claim-1: An apparatus of modular system of reconfigurable sample preparation comprising:

a computer,

a backplane,

an autosampler,

one or more pumps,

one or more modules,

one or more UV-detectors,

one or more columns.

Claim-2: An apparatus of claim-1 wherein the modules, the autosampler, the pumps, the UV-detectors are controlled by the computer through the backplane.

Claim-3: An apparatus of claim-1 wherein the sequence of modules are configured by a human operator according to the experimental needs.

Claim-4: An apparatus of claim-3 wherein the sequence of modules are configured for on-line fractionation, on-line purification, on-line extraction, on-line enzymatic digestion, on-line sample dilution, on-line separation.

Claim-5: A method of reconfigurable modular, sample preparation comprising: sequencing modules,

connecting autosampler to system,

plugging modules into a backplane,

connecting tubing between modules,

connecting pumps to pump module,

connecting detector to computer,

controlling modules independantly using computer,

evaluating results using computer. Claim-6: A method of claim-5 wherein sequencing of modules is determined according to experiment needs.

Claim-7: A method of claim-5 wherein controlling modules comprises:

heating, valve switching, turning indicator lamps on & off, turning pumps on & off, reading signal from detectors, controlling flow rates of pumps.

Claim-8: A method of claim-5 wherein tubing between modules is configured by human operator according to experimental needs.

Claim-9: A method of claim-5 wherein the modules are controlled by the computer according to experimental needs.

Claim-10: A method of claim-5 wherein autosampler injects sample into the system.

Claim-11: A method of claim-5 wherein evaluation of the results is done by reading from detectors.