DE3137875C2 - Device and method for the gradual implementation of chemical processes - Google Patents

Description

The present invention relates generally to an improvement t device and an improved method for implementation tion of chemical processes, especially an improvement t device for the automatic execution of the gradual Degradation of protein or peptide chains that are a major concern number of amino acid units included to make up the sequence of these amino acid units.

The determination of the linear sequence of the amino acid units in proteins and peptides is of considerable interest if one wants to understand their biological functions and ultimately want to synthesize compounds that can perform the same functions. Although a variety of techniques have been used to determine the order of amino acid linkage, the most successful technique is probably the one known as the Edman method. The following publications describe various forms of the Edman process and the devices for the automatic execution of these processes:
Edman and Begg, "A Protein Sequenator", European J. Bio chem. (1967) 80-91; Wittman-Liebold, "Amino Acid Sequence Studies of Ten Ribosomal Proteins of Escherichia coli with an Improved Sequenator Equipped with an Automatic Conversion Device," Hoppe-Seyler’s Z. Physiol. Chem. 354, 1415 (1973); Wittman-Liebold et al., "A Device Coupled to a Modified Sequenator for the Automated Conversion of Anilinothiazoli nones into PTH Amino Acids," Analytical Biochemistry 75, 621 (1976); U.S. Patent 3,959,307 issued to Wittman-Liebold and Graffunder on May 25, 1976, Title: "Method to Determine Automatically the Sequence of Amino Acids;" Hunkapiller and Hood, "Direct Microsequence Analysis of Polypeptides Using an Improved Sequenator, A Nonprotein Carrier (Polybrene), and High Pressure Liquid Chromatography," Biochemistry 2124 (1978); Laursen, RA Eur. J.Biochem. 20 (1971); Wachter, E., Machleidt, H., Hofner, H., and Otto, J., FEBS Lett. 35, 97 (1973); U.S. Patent 3,725,010 to Penhasi, April 3, 1973, Title: "Apparatus for Automatically Performing Chemical Processes"; U.S. Patent No. 3,717,436 issued to Penhasi et al. February 20, 1973, title: "Process for the Sequential Degradation of Peptide Chains"; U.S. Patent 3,892,531 issued to Gilbert on July 1, 1975, entitled "Apparatus for Sequencing Peptides and Proteins"; U.S. Patent 4,065,412 issued to Dreyer on December 27, 1977, Title: "Peptide or Protein Sequencing Method and Apparatus." Another relevant device is in the pending US patent application Serial No. 106 828, filed on December 26, 1979 by Leroy E. Hood and Michael W. Hunkapiller, ie two applicants for the present invention, title: "Apparatus for the Performance of Chemical Processes".

Briefly summarized, the procedures for gradual Dismantling according to Edman, as described in the above publications three stages: coupling, splitting  and conversion. Phenyliso reacts at the coupling stage thiocyanate with the terminal α-amino group of the peptide, whereby the phenylthiocarbamyl derivative is obtained. in the Anhydrous acid is used to cleave the Cleave phenylthiocarbamyl derivative and the anilinothia to form zolinone. After extraction of the thiazolinone the residue peptide can start a new cycle of coupling and Splitting reactions begin. To convert the thia zolinons in the phenylthiohydantoin, which in suitable Way, e.g. B. can be analyzed by chromatography, aqueous acid is used.

The automatic device according to Penhasi US Pat. No. 3,725,010, that in the publications by Wittmann- Liebold and pending U.S. patent application Serial No. 106 828 was modified by Hunkapiller and Hood an automatic sequence analyzer in which the reactions run in a thin film on the inside wall of a rotating reaction cell is formed, the general is known as a "spinning cup" and is within a ge closed reaction chamber is arranged. They are techni means provided to control controlled amounts of liquid Enter reagents into the chamber and remove the with a sample of a protein or peptide in one react to the atmosphere. The sample to be analyzed is placed in the "spinning cup" reaction cell at the beginning, after which step by step the different reagents and solutions means necessary for the implementation of the clutch and spal tion reactions are required, added and subtracted become. Form the liquid reagents and solvents even films on the walls of the cell, which can be seen in the spread the rapid rotation of the cell over the sample films and react with it. The reagents dissolve the samples film and perform the coupling and splitting steps of the Edman process. If the clutch and split Steps are complete, the reaction chamber will evacuate to remove the volatile components of the reagents distant. Following the evacuation after the clutch  the remaining sample film with a solvent extracted to remove non-volatile components. in the Following the evacuation after the split, he will held thiazolinone from the sample film with a solution medium extracted and either in a special flask transferred to perform the conversion step, or to a device for collecting and drying the various frak ions. If the conversion step is not immediately to a conversion flask is carried out, this can Step later with a larger number of fractions equal be carried out in good time.

The introduction and withdrawal of fluids in the Reaction cell or from this was made by means of fluid lines reached, which run through a closure piece, the public closes in the upper wall of the reaction chamber and from this down to a point in the Reak tion cell. The fluids get straight into the fast rotating reaction cell entered, and that at a Place near their bottom, and off an annular groove drawn that in the cylindrical inner surface of the reaction cell is executed. The fluid to be drawn off is through the Centrifugal force is pressed into the ring groove when the reaction cell rotates at a high speed and it will deducted by means of a line that goes inside pointing end projection into the groove. This drainage pipe tion thus acts as a kind of "skimmer" to remove the Reaction products and by-products and the extraction solvent.

If the protein or peptide sample is in a device with a rotating reaction cell does not have sufficient mass to form a coherent film as such to be able to use it occasionally during the solvent extractions in a relatively thick layer from one not proteinaceous carrier material on the inner wall of the reak tion cell applied. The carrier material and the sample are then in the liquid reagents during the reak  tion steps resolved so that the clutch and split reactions can take place. A polymeric quaternary ammonium salt with the chemical composition 1,5-dimethyl-1,5-diaza undecamethylene polymethobromide (Polybrene) was made for this Purpose used. The carrier must be in considerable quantities used to safely retain the sample, and the The carrier and the sample are both separated by the liquid reagent solved to a reaction between the sample and the Allow reagents.

The use of polybrene in a "spinning cup" process is, for example, in Biochemistry 17 (1978), No. 11, pages 2124-2133. Due to special reaction conditions, d. H. Add one third of the coupling buffer to Edman's recommended concentration in two steps at the beginning and on End of the clutch stage and cyclical change of the rotation speed, the protein is kept in solution and thus ensures an efficient clutch. In Analytical Bio chemistry 84 (1978), pages 622-627 the use of Polybrene described in an analogous process. The one embedding a protein or peptide sample in a matrix not to be found in these references.

Although rotating cell type devices in many If they provide acceptable experimental results, they indicate however, various drawbacks. So z. B. the cost quite high, which is suitable for obtaining a large protein or peptide sample and to ensure adequate Supply with the necessary reagents can be applied must, primarily because the reagents used are liquid and must be used in substantial quantities. Liquid Reagents and solvents also tend to parts to separate the sample from the film and from the wall of the Wash off the reaction cell when they flow through it, whereby the yields of terminal amino acid units which obtained in each subsequent working cycle of the device be reduced. The initial amount of the sample must therefore  be big enough to make sure it's big enough Sample remains to be even in the last work cycle deliver usable results. Devices of this type also have rather long times for a work cycle and due to the considerable volume of the reaction chamber as well as the requirement to repeat semi-volatile liquid Reagents and solvents by vacuum drying the sample in to remove the reaction chamber. In addition, sequence analyzes are gates of the "rotating cell" type quite complex and expensive, both in manufacturing and in repair cases.

Another type of device for sequence determination is shown in described in the above articles by Laursen and Wachter ben, in which the sample by covalent bond with the Ober areas of a multitude of small pearls is immobilized. The beads form a porous package within a reak tion column, and the column is filled with liquid reagents treated to run the chemical processes. There however, the cleavage reagents used are excellent Solvents for proteins and peptides must be covalent Binding must be complete to hold the sample in place. However, covalent bonding is difficult in practice put. The packed column is also difficult wash, and the beads show a tendency during the Use to disintegrate.

An analytical method for liquid is known from US Pat. No. 3,879,127 known in which the liquid sample on an absorption rendering and porous sample carriers, such as. B. filter paper, is absorbed. In this procedure the sample is not in embedded a solid matrix, but on the surface of the Fixed carrier and out there the fluid eluents puts.

Sequencing devices have been described which use the Disadvantages of these devices should be overcome by taking the sample contained in a stationary reaction chamber and the sample  Expose at least one reagent in gas or vapor form. Two such devices are disclosed in U.S. Patents 3,892,531 to Gilbert and 4,065,412 by Dreyer, but both not working satisfactorily.

The Gilbert device sees a closed device Protrusion of finger shape within a reaction chamber, around a peptide or protein sample on a controlled Maintain temperature while this is gradually gaseous Exposed to reagents and solvents. Whenever when a reagent is introduced, the protrusion is internally cooled to cause condensation on its surface. Of the The projection is then warmed, causing the sample to settle in the Liquid dissolves and the reaction proceeds. After the reaction with the sample the unwanted semi-volatile chemicals lien either by drying the sample using a com combination of heat and an inert gas stream or by washing from the protrusion together with the terminal amino acid using a solvent that condenses on the protrusion is removed until it drips from it.

In the device and the method according to the Dreyer patent is a protein or peptide sample on both the inner and even on the outer surface of many small macroporous ones Beads inside a reaction column by chemical coupling or a direct adsorption applied to it. Various Reagents and solvents are gradually replaced by the packed column in either gaseous or liquid form passed through to bring about the desired degradation reactions ken. The flow of reagents and solvents into the column is achieved by a rotating, flat ten posi tion valve controlled.

In the Dreyer patent, it is essential that the sample be thin Layer is adsorbed on top of the support where it is is freely accessible for the reagents used and that a essentially free reagent flow is guaranteed. The  Embed a sample in a solid matrix in which the The Dreyer patent does not allow reagents to diffuse remove.

Unfortunately, the devices according to U.S. Patent No. 3,892,531 ensure and 4 065 412 not sufficiently pollution-free Environment to get acceptable results even after a large number of degradation cycles. For example, it is difficult to Protein or peptide sample in the devices mentioned wash thoroughly. Gilbert's method of washing the Sample by condensing solvent on the sample up to the point at which the solvent is removed from the sample dripping, shows the tendency to trace the different reaction to leave products on the sample that the future pollute chemical reactions. It is the same difficult to get the pack that in the Dreyer reaction column used to hold the sample, wash because the various chemical products right through the column and have to be transported out of the column to make one To avoid pollution. This is not easy to achieve even if large amounts of solvent are used, because the solvent shows a tendency through the intermediate spaces between the beads instead of running through them to penetrate small pores in the beads where most of the Protein sample sits. The lines for feeding the Fluids and the flow valves of Gilbert and Dreyers are also difficult to completely evacuate and tend to trap chemical residues that the intended Chemistry can interfere with the further reaction cycles.  

The glass or plastic beads that are in the reaction column used by Dreyer as a pack also show the Tendency to disintegrate over a number of mining cycles, and clog the system to the point where the Flow of fluids through the system is obstructed. It then the system becomes quite impossible between cycles wash, and the chemistry in the column is hopelessly lost dirty.

The caused by various factors described above Pollution shows a cumulative effect during the Duration of gradual dismantling. The sample and the reagents Cien in the reaction cell are therefore increasingly ver dirty, prevent the desired coupling and splitting reactions and cause a variety of undesirable Reactions take place. The yield from every complete Zy Therefore, the number of devices decreases and the fractions introduced a whole host of by-products.

The yield also decreases due to a direct loss on sample for a number of reasons, including the Zer case of the pack, the solubility of the sample in the rinse Solvents and the failure of the sorption bonds between the pack and the sample.

While these effects are neglected in some cases if large amounts of the protein or peptide Are accessible or if the chain is only a relative sample contains a small number of units, they are forfeited army out in cases where the chain is a very large one Number of units, or if only very small Amounts of the special protein or peptide are available stand. Both are the case with interferon, one small protein found in human cells in response to certain viral infections. Interferon has recently great in the world of clinical medicine Causes excitement because it promises to be effective Means to stop viral infections, and it seems  also as an anti-cancer drug to high hopes entitle. Interferon is only produced in very small quantities produces and is therefore only preserved in very small quantities Lich. At the moment, the entire world pro is, so to speak production of the two types of human interferon from the relatively few centers in the world that have access to large menus gene of human white blood cells (leukocyte inter feron) or certain human cells in tissue cultures (Fibroplast interferon). As a result of this limited Production capacity for interferon was difficult, well controlled clinical studies as well as basic analyzes about how this molecule works. To do that The interferon from a single image is even more complicated Chain of about 150 amino acid units built up to Purposes of analysis all individually separated from the chain need to be. Losses due to pollution of the Types described can sequence all amino acid units with the exception of the first few amino acids units of interferon with very small amounts of prevent available protein. After the first few Cleavage cycles, the small sample can become so dirty that positive results can no longer be obtained.

The most sophisticated of the applicants of the present Er known prior art device for converting the various thiazolinones that split off from the sample in the more stable phenylthiahydantoins Conversion piston used in the above articles by Wittmann-Liebold is described, namely in its modes according to the pending American Patent application Serial No. 106 828 by Hunkapiller and Hood. However, the applicants have found that this piston suffers from the fact that its inner walls are not sufficiently ge will wash when the reagents and solvents through the appropriate capillary tube are inserted. It was featured propose that the solvents and reagents in a stream of inert gas can be supplied to the Walls of the piston by splashing the liquid onto the  Wash walls, however, showed that this technique to an inconsistent inflow of liquid into the flask leads and makes it very difficult to feed the volume of control liquid.

The applicants have further found that when the known conversion pistons scaled down to scale will correspond to lower volumes of liquid, it is difficult to determine the optimal degree of dispersion of the Inert gas bubbles within the liquid content of the flask to get the content during the conversion reaction to move and vaporize the semi-volatile components. The inert supplied to the bottom of the flask for this purpose gas shows the tendency in relatively large bubbles to the upper surface of the liquid to rise, taking this large bla Do not stir the liquid evenly and instead whose splashing of the liquid into the top of the Effect piston.

It is therefore desirable for many applications to use a device for Implementation of chemical processes such as sequencing of proteins or peptides to indicate that effective and with minimum system pollution works to a successful execution of a maximum number of sequence cycles with a very small amount of sample chen.

It is therefore an object of the present invention to provide a device and a method for the gradual implementation of chemi reactions with a sample of a chemical material state the loss of the sample and the ver system pollution are minimal. According to the present Invention is intended to be an economical device and a method for the gradual implementation of chemical processes a sample of very small size can be created by minimal amounts of reagents and solvents used can be. Furthermore, the length of time for a job cycle should be very short, and the sample should be between the  Cycles can be washed more effectively than before.

This task is solved by a device and a method, as described in detail in the claims.

Briefly summarized contains a device according to the front lying invention determine one of the inner surfaces ter parts formed reaction chamber with means for Zu guiding and draining fluids in a pressure medium Current through the chamber is provided, as well as a fixed measure trix, through which a variety of fluids on the way of Diffusion can pass through, and within the Chamber is arranged so that a sample in the matrix is embedded, immobilized and everyone from a lot number of fluids that interact with the chemical Sample is exposed through the chamber currents.

The technical means forming the chamber can be such Means that the solid matrix appears as a thin film wear their surface, and the matrix can be a polymer quaternary ammonium salt such as 1,5-dimethyl-1,5-diazaundeca methylene polymethobromide or poly (N, N-dimethyl-3,5-dime ethylene piperidinium chloride) included.

The parts to create a surface can be the entire Include inner wall of the chamber or a part thereof, or they can be in the form of a porous sheet or a porous one Disk run, which is essentially across extend the chamber and a passage of the fluids ge equip. The porous sheet or disk can be made of a variety of glass fibers. The the Chamber forming parts can be a pair of abutting ing include chamber elements that oppose each other overlying colliding surfaces a first or two te recess, these first and second ver are aligned in relation to each other so that they form the reaction chamber. The first and second ver can deepen towards the colliding upper  face away and towards the place where they have the inlet or outlet parts communicate, beveled. The porö As far as they are used, sheet parts are made by a recess in at least one of the abutting the surfaces, and are housed inside the chamber kept in such an orientation that it was the first and substantially separate the second recess nen. The chamber parts can be at least one sheet from one Compliant material include that between each other abutting surfaces is tightly clamped, the compliant material to a variety of fluids is permeable. At least one of the chamber elements can then a raised section on its adjacent surfaces have, which is around the depression in this area extends to the sheet of compliant material against the to press adjacent surface of the chamber element and thereby to improve the sealing effect.

The inlet and outlet means can be a pair of capillary servers bindings that are characterized by the respective Kammerele elements extend and at the inner ends of the chamber elements on different sides of the porous sheet with the reak tion chamber are connected. The capillaries can be coaxial be arranged to the reaction chamber and from this up extend to outer capillary openings which in the in essence Lichen flat outer surfaces of the chamber elements are arranged. The means for gradual implementation of a Variety of fluids through the chamber can block valve blocks that grasp a variety of essentially flat valve put on their surfaces, the valve block defines a first through connection that is continuous extends between its two ends and the through first openings in connection with each of the valve positions stung, a plurality of second Durchgangsver bindings, each via a second opening with a the valve positions communicate; as well as a variety of elastic, essentially impermeable membranes that cover the appropriate valve locations, with each of the  Membranes can be operated in two control positions where in the first control position against one of the valves digits is pressed, with the primary and secondary Openings are blocked that ver with this valve position are round, and being in the second control unit of the valve location is subtracted, creating a flow path between the first and the second opening via the upper surface of the valve block is created; The current of the fluid between the first via connection and the second connection can be controlled selectively. The Ver Binding agents can have at least one beveled intermediate piece included, which is closely in sliding connection with a Pipe part stands, and against a different abge oblique recess in the valve block is pressed, wherein it communicates with one of the through connections in it adorns, in such a way that an inward on the intermediate force acting on a relatively small contact rich between the intermediate piece and the recess kon is centered so that a seal for a fluid is obtained becomes. The recess is preferably larger Angle beveled as the intermediate piece. The connection parts can also be a fitting at one end of the first Have through connection to the first Durchgangsver connect to another part of the device so that the first through connection serves as a distributor, by a flow of fluid between the fitting and the second through connection, which is from the fitting on farthest away, can be rinsed. The first through gangway can have a variety of straight passages or have holes that end to end with each other are connected that a line is formed which is a saw has tooth shape, and in alternating sections with ent speaking valve locations communicated.

The device may include a conversion flask that has a Has a large number of capillary tubes, which are located inside stretch to supply and remove various fluids pull it off, taking at least one of the capillary tubes  has the inner end such that the bore is closed and with a variety of limited radially apart the openings is provided so that the passage of flu through the capillary tube leads to the inner Walls of the piston are sprayed and washed. A Capillary tube that ends near the bottom of the piston can also have a closed end with a variety of restricted radially arranged openings nearby of the end so that the passage of a gas inside through the capillary tube to produce a variety of small bubbles that carry a liquid inside of the piston and accelerate their drying.

The inventive method, the step by step Management of chemical processes on a sample of a kitchen Mixing material involves embedding the sample in a solid matrix used for the diffusion of a variety of The arrangement of the solid matrix is permeable to fluids a closed chamber that has an entrance and a Has output, as well as the gradual passing of the on number of fluids through the chamber in a pressurized ste current from the entrance of the chamber to its exit, so that the sample is exposed to each of the fluids, the sample is always immobilized and a chemical interaction between the sample and the fluids is obtained. The firm Matrix can be in the form of a thin film on the inner walls be applied to the closed chamber. Alternatively the solid matrix can be applied to a porous sheet brought, which is essentially across the chamber at a point between the inlet and the outlet such that extends a fluid from the inlet to the outlet flows, must flow through the sheet. The step embedding the sample in a solid matrix, the stu applying the solid matrix to a support surface surface in the form of a thin film and the subsequent opening bring a solution with the sample onto the film, so that the sample solution dissolves the matrix. The liquids who which then evaporates from the solution, leaving a film back  remains in which the sample is embedded.

The device according to the invention and the Ver driving solve a large number of problems in the sequence prior art analyzers by using the Immobi protein or peptide sample in a solid matrix lize in the form of a thin film that is used for the dif fusion of both reagents and solvents used in the mining process is permeable. The difficult problem of generating a full kova lent bond is avoided, as is the problem the sample loss that occurred when the sample was directly attached adsorbs a carrier surface and the mechanical shear forces of the mobile liquid phase is fully exposed. The sample is held securely in place by the matrix, while the smaller reagents, solvents and amino can diffuse acid derivatives through the matrix, whereby they are sufficient in the matrix Solve concentration that the different steps of the Degradation process can be carried out. The fixed matrix holds the sample so effectively when exposed to gaseous reagents is that all conceivable support surfaces for the Pro be can be used without loss of Sample is coming. The inner walls of the reaction chamber itself can serve as a suitable carrier surface.

A support surface that completely washed the System greatly relieved is a porous sheet that is made of a variety of overlapping glass fibers is made and extends across a flow reaction chamber. The porosity is caused by gaps between the fibers generated. Such a structure has a relatively high upper area with minimal dimensions in the direction of the flow of the fluids. The solid matrix forms one thin film on the top surfaces of the fibers that hold it Allows reagents and solvents to be easily in the Diffuse film and with the embedded sample to interact. This enables chemical testing on the sample  Processes and wash cycles with a minimum of reagents and solvents and in a relatively short period of time perform. The reagents and solvents, of which some are in gas or vapor form, migrate through the thin film and come in contact with the sample with which they interact as completely and effectively as possible.

The relatively thin cross section of the porous sheet like it disclosed herein also improves the ability of Sample, carefully from residual reagents and reaction products using a relatively small amount of a solution to be washed free. The solvent must Reagents and reaction products only the relatively short Move distance out of the surface of the sheet to remove them from the system. From a point outside The surface of the sheet can be easily removed from the chamber are removed, leaving the sample so that the next reaction step can be carried out on it can. The low solvent consumption not only leads to Savings in the cost of the solvent, but also reduces the tendency for the sample to react chamber is washed and lost.

The use of reagents in gas or vapor form helps also help to keep the sample in contact with the reagents improve, reducing the amount of reagents required is kept to a minimum. Little use of reagents cien is important because they are used in the Edman mining technique reagents must be extremely free of contamination and are therefore very expensive. Furthermore, the sample and the solid matrix containing this sample does not pass through the gas shaped reagents solved, which eliminates the problem of a ver loss of sample due to separation of the sample from the Carrier surface is eliminated.

The reaction chamber according to the invention is designed so that the flow of both gaseous and liquid Reagents are allowed through the porous sheet that holds the sample  holds without the sample due to external contamination or through chemicals from a reaction step or cycle have been deported to the next, is contaminated. The abutting chamber elements are combined so that they form a small volume reaction chamber which through first and second depressions on opposite Sides of the porous sheet is formed. A pair from Kapil passages that face each other through the appropriate chamber elements from the chamber itself ken, allow a variety of fluids in gas or liquid form in the form of a stream under pressure to pass through the chamber and the sample matrix. The low volume of the chamber and through-connections makes the volumes of the required reagents and solution medium minimal and facilitates vacuum drying of the System between cycles. The two chamber elements are against a leaf at their colliding surfaces a compliant material sealed between the abutting surfaces is jammed. That after Common material is permeable to a variety of fluids to those who pass through the chamber and even improves the gas flow through the chamber by making the gases more even for a more even contact with the porous sheet Splits.

In an alternative embodiment, the response is timely a single capillary tube or a through connector capillary type that has a solid matrix that as a thin film on the inner surfaces or in the Boh tion is formed. The protein or peptide sample is in the matrix is embedded as in the case of the porous sheet, and the reagents and solvents are gradually passed through passed the tube, interacting with the sample. The chamber described above can be used for this purpose without the porous leaf element can be used. The sample containing Ma trix is then on the surfaces of the first and second Deepening. Similarly, the waiter surface that carries the solid matrix in every conceivable way  be designed, which allows the reagent and Solvent fluids can be passed over the matrix.

The valve assemblies to control the flow of fluids in the chamber and the conversion piston and out of them constructed in a special way to a mutual Ver to prevent contamination of the fluids. With each of the valves a single line with a variety of other lines in Connected to the single line selectively with everyone to connect the other lines. The individual line is like that stated that they have one end of the primary pass ver bond in the valve block is connected while each of the others Connected lines with one of the second through connections that is. In the normal closed state, each of the mem branches that cover the various valve positions a gas pressure is pressed against the surface of the valve block, to connect the first through connection to the second through connection leading to the respective valve place leads to interrupt. A flow connection between the individual line and the other lines can be in can be selectively made by connecting to one or more a vacuum is applied to the membranes by subtract the valve locations and allow a Fluid over the surface of the valve block, which is between the openings of the corresponding through connections det, can flow. The secondary pass-through to the valve location at the farthest end of the primary passage leads to a pressure source of a flushing fluid, like an inert gas, be connected to the supply of each Complete fluids through the valve. It happens that after a certain reagent or solvent in the Reaction chamber was introduced in that the correspond a corresponding membrane of the supply valve, a vacuum was applied Membrane at the farthest end of the primary via can be opened to complete the feed by any reagent or solvent found in the primary  Passage was left in the chamber is pressed. This is possible because the primary Through connection is carried out continuously and leads to the fact that the distributor formed by a certain Reagent or solvent has been flushed out before using the The next reagent or solvent is started. In the case of the valve at the outlet of the reaction chamber, the primary through connection connected to the output while the secondary passage connections with the reaction flask, the vacuum or the waste or exhaust pipe are connected. The continuity of the primary through connection makes it possible that it can be carefully evacuated and eliminated basically the possibility that between the cycles in the primary through connection trapped semi-volatile substances become.

It is understood that the "sawtooth" configuration is open barten primary valve through connections for valve construction groups in areas other than that of sequence analyzers was known. The "sawtooth" valve assemblies of the prior art Techniques known to applicants have a variety of individual blocks on the valve points of a Main valve blocks are mounted so that they are between position in which through connections in the main block with each other connected or separated from each other, back and forth can slide. Such sliding blocks show a tendency to Wear, causing leaks to both the Atmos sphere as well as between the through connections. The valve assemblies in this description solve the wear problem by using the well-known sawtooth distributor a series of membranes is combined through which a current between pairs of openings that match the corresponding Communicate through connections, released or given will cut. The membranes can essentially consist of inert materials such as commercially available fluorocarbon Polymers can be made unlimited without any Deteriorations work. It also showed  means for connecting the valve passages to external Pipes the tendency to the side faces of the valve block exert excessive pressure, causing deformation the upper sealing surface of the valve block, so that it loses its ability to act as a poet. The prior art sawtooth valves known to the applicants Technology in particular connect external lines to the Ven til through connections in that essentially flat Flange surfaces belonging to the different lines, to be pressed against the side surfaces of the valve block a series of connections between pairs of flat tops to produce surfaces. Because each of these parts is made of materials like Fluorocarbon polymers are made that are very difficult To be able to be machined precisely requires considerable pressure be exercised to form the respective sealing surfaces to make conclusive and the necessary seals put. The pressure must be absorbed by the valve block, what leads to deformation of its outer sealing surface.

The valve assemblies in this description contain a lot number of bevelled intermediate pieces, some of differently bevelled recesses in the valve block be to effect a seal without the Excess pressure is applied to the valve block. A relative low sealing force is due to a special section of the Spacer focused to the spacer against the press the corresponding beveled recess tightly without to deform the block.

The fact that contact surfaces between surfaces ver have different inclinations, on opposite sides Sides of the spacers are provided, which will receive seals further improved. Contact areas of this type between different inclined surfaces are preferred on opposite sides of the intermediate pieces seen to get optimal sealing properties.  

The conversion piston according to the invention makes it possible to Re agents and solvents in the form of a spray or spray insert the mist on the inside walls of the piston hits to any chemicals on these interior walls are condensed or splashed onto them and supply the volume of liquid in the flask. In order to is one of the main sources of mutual pollution system between cycles. The conversion Lung flask also makes it possible for gas to be smaller in shape Bubbles flow upward through the volume of fluid that Move liquid evenly and dry half volatile components of the liquid can contribute without sample loss due to excessive bubbling or splashing. This causes a quick, gentle Removal of liquid from the sensitive amino acid derivatives. The solvent that is used is the amino to transfer acid derivatives into the conversion flask this way in a much shorter time (1-2 min against over 5-10 min) and at a lower temperature (40 ° C-50 ° C versus 50 ° C-80 ° C) are removed as per the above Patent cited by Wittmann-Liebold. That improves the Yields of the most unstable amino acid derivatives like that of Serine, threonine, histidine, arginine and tryptophan considerable Lich. The reagents used in the conversion flask can by applying a combination of fine currents of inert gas bubbles and a low vacuum in 3 to 5 minutes versus 30 to 40 minutes away for this Operation according to the Wittmann-Liebold patent required are. In the process according to the Wittmann-Liebold patent drying of the reagent must take place immediately upon its on start guide to the amino acid residue in the conversion flask, to meet the requirement that the entire period of one Cycle in the conversion flask is no longer than the entire one Duration of a cycle in the primary reaction chamber. There the acid component of the conversion reagent, d. H. Trifluor acetic acid or hydrogen chloride, is much more volatile as the water in which it is dissolved, the acidic com shows component of the tendency during the drying process  Wittmann-Liebold to disappear from the mix very early, whereby the amino acid derivative for a substantial part the conversion stage to nothing but water. The leads to the conversion of the derivatives of glycine and Proline is incomplete and that other derivatives dissolve put. In the device according to the present invention can the conversion reagent with the amino acid derivative for one Period of time to be kept in contact for a complete Conversion is sufficient, d. H. for 30 to 40 min, and can be there after quickly, d. H. can be dried in 3 to 5 minutes without that the sample ver in the entire interior of the conversion piston is injected.

The advantages of the invention also result in still kla form from the following description of preferred Embodiments of the invention with reference to the Drawings. Similar reference in the drawings also sign similar elements.

Show it:

Figure 1 is a perspective view of a device which he is constructed according to the invention.

FIG. 2 shows a top view of the device according to FIG. 1;

Fig. 3 is a schematic diagram of the device of Fig. 1;

Figure 4 is an enlarged perspective view of a reaction chamber kit according to the invention in an extended arrangement.

Fig. 5 is an enlarged vertical cross section along the line 5-5 of Fig. 4;

FIG. 6a is a further enlarged cross-section through the reaction chamber of FIG. 5;

FIG. 6b shows a cross section of a second embodiment of the reaction chamber shown in FIG. 5;

Figure 6c is a cross section of the reaction chamber according to FIG 6b, in which the porous sheet member is removed, for the purpose of use with a sample containing the film which is applied to the inner wall of the reaction chamber..;

Figure 7 is a vertical cross section through a typical storage medium according to the invention for a reagent or solvent which is used in liquid form.

Fig. 8 is a vertical cross section through a typical storage according to the invention for a reagent used in gas or vapor form;

Fig. 9 is a vertical cross section through a membrane valve assembly for controlling the flow of fluids into and out of the reaction chamber and the conversion piston, this section corresponding to line 9-9 in Fig. 11;

Figure 9a is an enlarged cross section of a portion of one of the connecting elements of the valve assembly of FIG. 9.

Fig. 10 is a vertical cross section along line 10-10 of Fig. 9;

Figure 11 is a side elevation of the manifold block of the valve apparatus shown in Figure 9;

Fig. 12 is a horizontal cross section along the line 12-12 of Fig. 11;

Fig. 12a is a vertical cross section along the line 12a-12a of Fig. 11;

. S of the valve portion of the valve assembly of Figure 9, showing the diaphragm pressed against the manifold block to the connection between the passages at this location break Figure 13a is an enlarged cross-section of a segment to be.

Fig. 13b is an enlarged cross section through a section of the valve portion of the assembly of Fig. 9 showing the diaphragm peeled from the manifold block to allow fluid to flow between the passages;

FIG. 14 is a plan view of a conversion flask of the present invention constructed in accordance;

Fig. 15 is a vertical section along line 15-15 of Fig. 14;

Figure 16 is a vertical section on the line 16-16 of Figure 14;

17A and 17B are a schematic representation of the two principal methods of the prior Tech technology for immobilizing a protein or peptide sample during degradation.

Fig. 17c shows a schematic representation of the immobilization of a protein or peptide according to the present invention;

. Fig. 18a shows an enlarged vertical cross-section through a detail, corresponding to that of Figure 5, and through a further embodiment of the reaction chamber assembly according to the present invention;

Fig. 18b is a vertical cross section showing the Kammerele element of the embodiment of Fig. 18a which has been laterally flipped over to apply the matrix containing the sample to the inner walls of the element;

Fig. 18c a further enlarged cross-section of a portion of the chamber element of FIG. 18b, in which the film containing the sample is applied to the inner walls.

Table 1 lists the different steps again, by the device according to the invention a typical breakdown and transformation cycle be performed.

Figs. 1 and 2 of the drawings show an appliance, indicated as a whole by the reference numeral 10 and in which the present invention is realized. The device 10 comprises a chamber part 12 , a conversion piston 14 and a sample collector 16 , all of which are each operated by the automatic control unit 18 . A group 20 of pressurized solvent and reagent re servoirs are connected to the reaction chamber 12 and the conversion piston 14 via an arrangement 22 of membrane type flow valves. A second arrangement of valves 24 regulates the flow of liquids from the reaction chamber into the conversion piston 14 and elsewhere. A third arrangement of diaphragm valves 26 . serves to connect the conversion piston 14 and the sample collector 16 to either the drain or vacuum, and to regulate the flow of the fluid from the piston 14 into the sample collector 16 .

A source 28 of filtered inert gas supplies the device 10 with highly purified inert gas, preferably argon, to pressurize the solvent and reagent reservoirs, purge oxygenated air from the system, and the process of drying the reagents and solvents in the system accelerate at different times. An arrangement 30 of pressure regulating valves and measuring devices serves to individually regulate the pressure of the gas for each solvent and reagent reservoir and for each of the other components of the device 10 .

How the device 10 is operated is shown in FIG. 3. The chamber part 12 is arranged within a heated environment 32 and, in the preferred embodiment, forms a reaction chamber 34 which communicates with an inlet 36 and an outlet 38 . The inlet bore 36 can be connected via a flow line 40 to a plurality of group 20 reservoirs, more precisely reagent reservoirs 42 , 44 , 46 and 48 , and solvent reservoirs 50 , 52 and 54 . Reservoirs 42 through 54 are under inert gas pressure from a source 28 via a group of individual gas pressure regulators 56 (PR) and flow solenoid valves 58 . Each of the reservoirs 42 through 44 can also be connected at a point above the level of the fluid therein to a trap 60 via an individual flow valve 62 and an individual flow controller 64 . The pressurized inert gas which is introduced into the reservoirs from the source 28 can thus be blown off in an amount controlled by the flow controller 64 via the exhaust gas trap 60 . The outputs of the reservoirs 42 to 44 for the fluids are individually controlled by means of diaphragm valves 66 , which are connected to a continuous distributor 68 , which is connected at one end to the flow line 40 . A fluid from the pressurized reservoir 42 to 44 can in this way through the conduit 40 and the inlet 36 of the chamber. Flow into the reaction chamber 34 when the valves 36 are selectively operated. To assist in the delivery of reagents and solvents and to purge manifold 68 and line 40 after delivery, pressurized inert gas from source 28 may be at the end of manifold 68 opposite line 40 via a pressure regulator 70 and a Membrane valve 72 are introduced. The actuation of the valve 72 thus causes the supply of pressurized gas into the distal end of the distributor 68 , whereby all reagents and solvents from this through the line 40 and the inlet 36 are driven into the reaction chamber 34 abge.

The outputs for the fluids of the reservoirs 44 , 46 and 48 are provided with gas flow meters 74 which are connected in series with flow regulators 76 , since the reagents held therein are used in gas or vapor form, while the rest are used Solvents and reagents are used in liquid form. The flow meter 74 and flow controller 76 are required to precisely control the rate of gas flow through the corresponding valves 66 .

The flow of fluids from the reaction chamber 34 is controlled by diaphragm valves 78 , 80 and 82 , which are connected to the outlet 38 via a continuous distributor 84 . Valve 78 is opened to transfer the desired reaction product, typically the N-terminal amino acid moiety of a protein or peptide sample, through line 86 into conversion flask 14 . The valve 82 can be opened to connect the outlet 38 to the exhaust gas trap 60 for the separation of undesirable reagents, solvents and reaction products, and the valve 80 connects the outlet 38 to a vacuum trap 88 and a vacuum pump 90 for evacuating the reaction chamber 34 , of outlet 38 and manifold 84 .

Similarly, the conversion piston 14 and sample collector 16 can be connected to the exhaust trap 60 via valves 92 and 94, and to the vacuum via valves 96 and 98 .

The reagent reservoirs 100 and 101 and a solvent reserve voire 102 are provided in order to supply reagents and solvents into the conversion flask 14 via a line 104 . The reservoirs 100 , 101 and 102 are via pressure regulator 106 and solenoid valves 108 under inert gas pressure from a source 28 , and are connected to the exhaust gas trap 60 via individual exhaust air valves 110 and flow regulator 111 . The outputs for the fluids from the reservoirs 100 , 101 and 102 are connected via diaphragm valves 112 to a continuous distributor 114, which is connected to the line 104 on one side in connection. The flow of a fluid from the pressurized reservoirs can be generated in this manner by selectively opening the valves 112 to drive either a reagent or a solvent into the manifold 114 . The inert gas source can be connected via a pressure regulator 116 and a diaphragm valve 118 to the end of the distributor 114 , which lies opposite the line 104 , in order to drive the reagent or solvent through the distributor 114 and the line 104 into the conversion piston 14 .

The pressure source 28 of the inert gas is also connected to the conversion piston 14 via a pressure regulator 120 , a diaphragm valve 122 and a line 124, and to the sample collector 16 via a pressure regulator 126 and a valve 128 . If in the piston 14 a special sample has been converted in the desired manner, it can be driven from the piston 14 by the gas pressure through the line 124 and a valve 131 in the sample collector 16 in order to store it there in a glass vial. After the fraction has been driven off, the piston 14 can be filled with solvent relatively high to dissolve any residual chemicals therein. The solvent can then be driven with compressed gas through line 124 and valve 125 into the exhaust trap 60 , flushing the flask in preparation for delivery of the next amino acid derivative.

The sample collector 16 essentially contains a carousel of glass vials which is operated by the control unit 18 once during each cycle of the device 10 to bring an empty vial into position so that it can receive the next fraction of amino acid units from the piston 14 .

The control unit 18 is preferably a fully automatic unit that the diaphragm valves 66 , 72 , 78 , 80 , 82 , 92 , 94 , 96 , 98 , 112 , 118 , 122 , 125 , 128 and 131 and the solenoid valves 58 , 62 , 108 and 110 controls. The control unit 18 also controls the mechanism for maintaining the desired temperature (not shown) of the heated environment 32 , the sample collector 16 , the vacuum pump 90 and a variety of sensors within the entire system. The various gas pressure regulators and flow regulators described above are manually adjusted during commissioning in order to set the desired pressures and flow rates in the corresponding lines for the fluids.

The chamber part 12 is 5 ge shows in detail in Fig. 4 and Fig., And includes a two-piece retainer 130 holding a sleeve 132 which includes a first and a second chamber member 134 and 136, respectively. The sleeve 132 has an enlarged cylindrical portion 138 which is coaxial with the axis of the sleeve 132 and is inserted into a cylindrical recess 140 of the holder 31 . The cylindrical portion 138 is held in position by a collar 142 , which is connected to the holder 130 via a thread.

The chamber elements 134 and 136 are cylindrical glass elements which have opposing contact surfaces 144 and 146 , respectively, and which are fitted one behind the other in the sleeve 132 in an axial arrangement. The input and output connections 36 and 38 , which have already been mentioned above, extend axially through the chamber elements 134 and 136 and are preferably capillary openings which have a diameter of the order of 1 mm. The axially outer ends 148 and 150 of the chamber members 134 and 136 are substantially flat and are in contact with a pair of thin resilient intermediate rings 152 which are made of a substantially inert material Herge around the chamber members 134 and 136 in to store soft in the axial direction with respect to the sleeve 132 . A metallic intermediate ring 154 is arranged on the top nachgiebi gene intermediate ring 152 and provided with opposing locking lugs 156 which engage in savings 158 from the upper end of the sleeve 132 . The metallic intermediate ring 154 is held by a cover 160 ge, which is screwed onto the upper end of the sleeve 132 to hold the chamber elements 134 and 136 in relation to the sleeve 132 in a snug fit. Characterized in that the lugs 156 engage in the recesses 158 , the intermediate ring 154 is prevented from rotating when the cover 160 is screwed on, thereby preventing the cover from damaging the entire assembly by rotating the chamber elements.

The line 40 for the fluids is with a conically widened lower end. 162 provided, which abuts the outer end 148 of the chamber member 134 so that the bore of the line 40 communicates with the input connection 36 . The line 40 has a rear intermediate ring 164 and a fitting part 166 , which is screwed axially into the cover 61 , so that the flared end 162 is pressed against the cam merelement 134 well sealed. The conduit 40 can be made from any flexible, substantially inert material, such as a commercially available fluorocarbon polymer. The precise axial alignment of the bore of the line 40 with the input connection 36 is ensured by the precision manufacture of the various intermeshing parts.

The output connection 38 is connected to the continuous distributor 34 , which has already been described above, namely by a mass 172 made of a substantially inert material, which is encapsulated in a steel sleeve 174 . The sleeve 174 has a smooth outer surface which fits into successive openings 176 and 180 in the further cylindrical section 138 or the holder 130 . The mass 172 extends axially in both directions from the steel sleeve 174 , and acts on the outer end 150 of the second chamber member 136 and a chamfered recess 180 in the valve block 182 , which forms the distributor 84 Ver. An axial through hole 184 is intra-half of the mass 172 joins precisely to the main action connection 38 and an end of the manifold 84 at, thereby forming a single continuous capillary passage from the chamber element 136 provided in the valve block 182nd As in the case of the line 40 discussed above, the careful construction of the corresponding components ensures a precise connection of the parts with respect to their common axis, as well as the complete seal between the various sections of the through opening. The chamber part 12 can be easily disassembled and reassembled in a very short time without the orientation of the various through holes or the condition of the various seals being impaired.

The valve block 182 has a construction that will be explained in detail with reference to FIGS. 9 to 13. It suffices to state here that parts of the valves 78 , 80 and 82 belong to the valve block 182 and control the flow of a fluid from the chamber part 12 .

As can be seen most clearly in FIG. 6a, the chamber 34 is formed by the adjoining depressions 186 and 188 in the mutually opposite contact surfaces 144 and 146 of the two chamber elements 134 and 136 . The two recesses 186 and 188 are arranged coaxially with respect to their input and output connections 36 and 38 and are preferably round in cross section, thereby creating an axially symmetrical path for a fl 61551 00070 552 001000280000000200012000285916144000040 0002003137875 00004 61432uid which results from the input connection 36 to the output connection 38 flows, is created. A porous sheet member 190 extends across the reaction chamber 34 and may be at least partially in a recess 192 of the recess 186 . The porous sheet 190 thus separates the inlet connection 36 from the outlet connection 38 , so that fluids flowing from one to the other must pass through the sheet. The porous sheet 190 preferably contains a sheet or mat, which is made of pressed fibrous material such. B. Glass are made. Commercially available glass fiber filters are suitable for this purpose and have a high resistance to decomposition or other destruction during their use. It has been found that a porous sheet of this type, as a support for a film in which a protein or peptide sample can be embedded, forms a rather large surface. If the film is made of a fluid-permeable material, that is, one that allows the diffusion of liquids and gases into the material, then the material can form a solid matrix that holds the sample securely but still has a chemical interaction Reagents and solvents allowed with the sample. Polymeric quaternary ammonium salts such as 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide or poly (N, N-dimethyl-3,5-dimethylene-piperidium chloride) are ideal materials for this purpose. They allow the diffusion of fluids, are in the The solvents used are insoluble and are chemically stable to both the solvents and the reagents, in addition, they form a coherent film and have a positive charge that enables them to form an ionic bond to the surface of the glass support.

The fundamental differences in the prior art sequence analyzers and that of the present invention in the manner of holding the sample are best understood with reference to Figures 17a, 17b and 17c. Figs. 17a and 17b schematically illustrate the two previous methods Spread test for immobilizing a protein or peptide sample 300 with respect to a sample support surface 302 or 304.

FIG. 17a shows the case in which the sample is chemically ge connected to the glass support surface 302 by covalent bonds 306th For example, surface 302 can be specially treated so that some of the silica positions on the glass have functional amino groups 308 that protrude from the surface and can react with carboxyl groups 310 of the sample chain. Under appropriate conditions, some of groups 308 and 310 will react, covalently attaching the sample to the glass and releasing a number of water molecules. Bonds of this type are very strong, and one or two of them per molecule are sufficient to hold the sample. However, covalent binding is difficult to create with protein and peptide samples. Furthermore, the covalent bonds hold the chain on only a very few isolated units of the chain. When the degradation process reaches these units and they separate from the chain, the rest of the chain is no longer bound and can be washed out of the chamber.

Fig. 17b illustrates the case in which the sample 300 is adsorbed directly to the support surface 304. The sample is then held by a very large number of relatively weak non-covalent interactions 312 between the sample and the surface. At the molecular level, the surface reacts with many different parts of the sample. This works well for large proteins and peptides; however, if the sample is degraded to a much smaller size in sequence analysis, it becomes sensitive to chipping or peeling from the surface. This effect leads to a drastic loss of samples.

Fig. 17c schematically illustrates the immobilisation of the sample 300 on the support surface 314 by embedding in a solid matrix 316, which is formed on the support surface 314 as a thin film. As previously described, matrix 316 may be a polymeric quaternary ammonium salt that has a positive charge. In this way, the matrix is held firmly on an acidic glass surface by a very large number of ionic interactions, and in turn holds the sample securely in place since it is embedded in it. Direct binding interactions between the sample and the surface are not trusted, and the effectiveness of the immobilization is not affected by a reduction in the size of the sample.

The present invention relies on the diffusion of reagents, solvents, and amino acid derivatives through the solid matrix 316 to cause chemical interaction with the embedded sample. The matrix is designed as a thin film that sorbs the reagents and solvents that are applied to it. Once dissolved in the film, the reagents and solvents can easily diffuse through its entire thickness and cause the degradation process.

Returning to FIG. 6a, the reaction chamber 34 is sealed at the periphery of the recesses 186 and 188 by at least 1 sheet 194 of a resilient sealing material sandwiched between the cooperating surfaces 144 and 146 . Preferably, a pair of compliant sheets 194 are used, one on each side of the porous sheet 190 . The sealing sheets 194 are very thin and permeable for a large number of reagents and solvents which flow through the chamber 34 as fluids. An annular sealing bead or rib 196 on the circumference of the depression 188 presses on the sealing sheets 194 in order to create a more secure seal against the surface 144 in the region of the circumference of the recess 186 . The sealing sheets 194 can be made of any substantially chemically inert material, such as a commercially available fluorocarbon polymer, to minimize the possibility of deterioration of the seal. They serve not only to provide a seal for the chamber 34 , but also to hold the porous sheet 190 and to diffuse gases and liquids that pass through the chamber so that the flow of the gases and liquids is more evenly distributed over the leaf element 190 . In this way, a long service life of the system and an optimal chemical interaction with a sample is achieved.

An alternative embodiment 34 'of the reaction chamber according to the invention is shown in Fig. 6b, in which the adjoining recesses 186 ' and 188 'in the opposite mating surfaces of the two Kammerelemen te are somewhat narrower and longer than the recesses gene 186 and 188 . Otherwise, the chambers 34 and 34 'are identical, and the various elements of the chamber 34 ' are numbered in the drawings similar to that of the chamber 34 , except that the reference numerals are "deleted" to distinguish them. The reaction chamber 34 'allows a somewhat direct flow of fluids from the inlet 36 ' to the outlet 38 ', but limits the diameter of the porous sheet member 190 ' in the chamber.

Fig. 6c shows the chamber 34 'of Fig. 6b, from which the porous sheet 190 ' is removed. In addition, the sealing sheets 194 'are replaced by a single annular sheet 316 made of a resilient material that has a central opening that is the same size as the diameter of the chamber. In this embodiment, a solid, fluid permeable matrix 318 with an embedded protein or peptide sample is formed as a thin film on the walls of the chamber 34 'to come into contact with reagents and solvents that pass through the chamber . The flow of fluids through chamber 34 'is increased in this way while maintaining a substantial area for the film.

A further embodiment of the chamber part of the present invention is shown in FIGS . 18a, 18b and 18c, in which the two chamber elements 134 and 136 are replaced by a single chamber element 320 of the capillary type in the sleeve 132 . The remaining elements of the chamber part 12 are the same as described in connection with FIGS. 4 and 5 and are also numbered similarly. All structures and connections outside the chamber part are also the same as described above.

The chamber element 320 contains a cylindrical glass part which has inner walls 322 which form an axial capillary chamber 324 . The chamber 324 increases in diameter from its two ends 326 toward its center 328 , and the protein or peptide sample is carried on a solid matrix 330 that is formed as a thin film on the walls 322 .

It will be understood by those skilled in the art that the reaction chamber of the present invention can be of any conceivable form with a supporting surface for a sample over which a variety of reagents and solvents can be passed. For example, a single elongated capillary tube (not shown) may be sufficient for the chamber portion 12 , in which a solid matrix permeable to a fluid is formed on the inner walls or in the bore. Fluids that pass through the tube would interact with a protein or peptide sample embedded in the film and cause degradation.

A typical reservoir 198 according to the invention for holding and supplying a liquid reagent into the reaction chamber 34 , 34 'or 324 is shown in FIG. 7. The reser voire 198 corresponds to the reservoirs 42 , 50 , 52 , 54 , 100 , 101 and 102 of Fig. 3. A pressurized inert gas is introduced into the interior 200 of the reservoir 198 by means of a line 202 which is connected to the interior at one Point below level 204 of the liquid reagent or solvent communicated in the reservoir. The compressed gas introduced in this way drives any dissolved oxygen out of the liquid and can be discharged in a controlled amount through an exhaust air line 206 , whereby dynamic equilibrium conditions are generated in the interior 200 . A liquid outlet 208 is provided for the controlled expulsion of a reagent or solvent from the re servoire 198 by the compressed gas therein. The supply line 202 for the inert gas of each reser voires 198 is supplied by a pressure regulator 56 or 106 and a solenoid valve 58 or 108 with pressurized inert gas from the source 28 . The outlet of the gas through the exhaust line 206 is similarly controlled by one of the solenoid valves 62 or 110 and one of the flow regulators 64 or 111 . The flow of liquid reagent or solvent through line 208 is controlled by either valve 66 or 112 . Each time one of the liquid reagents or solvents has to be fed into the reaction chamber or the conversion flask 14 , the corresponding valves for the argon supply and for blowing out are opened in order to set dynamic equilibrium ratios in the respective reser voire. A reagent or solvent in liquid form can then be introduced by opening the valve in line 208 and valve 32 to the exhaust or waste trap. The liquid is expelled from the reservoir in a constant amount, which enables the amount of liquid supplied to be precisely controlled by controlling the length of time that the valve in the supply line 208 is kept open.

A typical reservoir 210 for supplying a reagent in gas or vapor form is shown in FIG. 8. An inert gas inlet 212 that terminates in a glass frit 213 being blown through is provided to supply an inert gas to the interior 214 of the reservoir 210 at a point near the bottom of the reservoir and substantially below the level 216 of a liquid reagent in the reservoir. An exhaust duct 218 and an outlet or supply duct 220 communicate with the interior 214 at points above the liquid level 216 . The reservoir 210 is typical of the reservoirs 44 , 46 and 48 of FIG. 3, one of the pressure regulators 56 and one of the valves 48 for controlling the gas flow through the line. 212 from the compressed gas source 28 . Similarly, the discharge of pressurized gas through the exhaust line 218 is controlled by one of the flow regulators 64 and one of the flow valves 62 , and the supply of reagent through line 220 is controlled by one of the diaphragm valves 66 , which are in series with a flow meter 74 and one Flow controller 76 is switched, controlled.

Although the reagents R₂, R₃ and R _{3A are transferred} to the reaction chamber in gas or vapor form, they are thus stored and evaporated as liquids when necessary. Evaporation is effected by passing an inert gas upward through the liquid reagent in the form of bubbles. In this way, the inert gas inside 214 of reservoir 210 is saturated with reagent vapor. Whenever a reagent is needed, the valves connected to lines 212 and 218 are opened to bubble gas through the reagent and establish dynamic equilibrium relationships. The valve 66 in the supply line 220 is then opened for a predetermined period of time to supply the desired amount of reagent vapor into the reaction cell. The flow controller 76 , which is connected in series with the respective valve 66 , causes the steam from the reservoir to be supplied in a constant amount, which is indicated by the flow meter 74 .

FIGS. 9 to 13 show the construction and operation, a valve assembly 222, which is an embodiment for the valves 66 and 72 as well as for the continuous distribution 68 of FIG. 3. The valve assembly 22 includes a valve block 224 , which can best be seen in FIGS. 11 and 12. The valve block 224 is an elongated block of rectangular cross-section, which has a continuous first through opening 226 in a sawtooth shape, which was produced from its surface 228 by oblique bores in the valve block. The first through opening 226 thus forms a single continuous connection which communicates in its alternating sections through a plurality of valve locations 230 on the surface 228 through corresponding openings 232 . A tapered connection port hole 234 communicates with one end of the primary connection 226 . A plurality of second connections 236 extend from the chamfered connection opening holes 238 to the opposite sides of the valve block 226 to corresponding openings 240 in the vicinity of the openings 232 at the corresponding valve locations 230 . The valve block 224 is inserted into a longitudinal slot 242 of a base 244 which has a plurality of threaded openings 246 in linear continuation of the connector orifice holes 234 and 238 to receive the connector fittings 248 . The fittings 248 are configured so that they press resilient double beveled adapter 250 against the connection orifices 234 and 238 to connect the pipes 252 , which extend through the intermediate pieces, well sealed to the various passages of the valve block 224 . In this way, the connection orifice 234 of the first through connection is connected to the inlet hole 36 of the chamber part 12 via the line 40 for the fluids of FIG. 3, and the first seven of the eight second connections communicate with the outlets 208 and 220 for the fluid from reservoirs 240-54 . The secondary connection, which is farthest from the connection opening 234 , is connected to the inert gas source 28 via the pressure regulator 70 , which is shown in FIG. 3. The structure of the double beveled intermediate pieces to the orifices of the valve block 224 is shown in more detail in Fig. 9a, which shows the orifice 234 , for example.

The intermediate piece 250 of FIG. 9a is fitted with its inner side 243 into the connecting mouth hole 234 and with its outer side 245 into a chamfered recess 247 of the fitting piece 248 , the mouth hole 234 and the recess 247 being chamfered at such angles that are greater than the angle of the bevel on the corresponding sides of the intermediate piece. The spacer is preferably chamfered on both sides at the same angle, with the orifice 234 and the recess 247 chamfered at angles greater than that of the spacer by 30. This type of fit between various tapered surfaces concentrates the compressive forces on the edges 249 of the intermediate pieces 250 , thereby achieving an effective seal with a minimum of pressure on the valve block 224 side. Excessively high pressure forces on the valve block 224 , which can bend the valve points 230 , are avoided in this way.

A series of diaphragm-containing blocks 254 are screwed to the surface 228 of the valve block 224 with diaphragms 256 sandwiched therebetween. The upper end of each diaphragm-containing block 254 is threaded to receive a gas connector 258 that is connected to a recess 260 on the underside of block 254 and extends substantially over one of the valve locations 230 . An O-ring 262 can be fitted into an annular groove that surrounds the recess 260 to provide an effective seal against air.

The membranes 246 are made of a substantially chemically inert, airtight material that allows them to either be pulled from or pressed against the valve locations 230 by applying a vacuum or air pressure through the connectors 258 . The two alternative control positions of the membranes 256 are shown in FIGS. 13a and 13b. In the control position of Fig. 13a, an air pressure or gas pressure is applied through the connector 258 , which presses the membrane 256 against the opening 232 of the primary connection 226 and the opening 240 of the secondary connection at the location of the special valve location 230 . The openings 232 and 240 are thus closed by the membrane 256 , which does not allow any connection between these openings. This corresponds to the closed position of the valve, which is located at the special valve position. In the control position of FIG. 13b, vacuum is applied to the connector 258 , which pulls the diaphragm 256 from the openings 232 and 240 , whereby communication between the first and the second connection over the surface of the valve block is possible at this point.

This position corresponds to the open position of the special valve, which allows a fluid from one of the reser voire or from the inert gas pressure source 28 to flow through the primary connection 226 into the inlet connection 36 of the reaction chamber.

The construction of the valve assembly 222 , as described above, enables reagents and solvents to be introduced into the reaction chamber in precise amounts substantially without mutual contamination of the various fluids. The continuous execution of the primary connection 226 and its connection on one side with the input of the chamber part 12 contribute very significantly to the fact that it can be worked in this advantageous manner. The supply of any of the seven reagents and solvents can be accomplished by applying a vacuum to one of the membranes 256 and a positive gas pressure to the others, which enables the desired reagent or solvent to be incorporated into the primary compound 226 passes and from this through the line 40 for the fluid into the reaction chamber. When the desired amount of fluid has flowed under each membrane, gas pressure is again applied through connector 258 so that all seven reagent and solvent valves of assembly 22 are closed. The supply of the fluid can then be completed by applying a vacuum to the membrane covering the inert gas supply port at the distal end of the valve block 224 to purge all of the primary compound 226 with inert gas and the reagent or solvent fluid that is in the lines remain, is pressed into the reaction chamber 34 . Because there are no discontinuities or dead end portions in the first via 226 , there are no spaces in which any of the reagents or solvents can be trapped between the various delivery steps. The reagents and solvents fed to one another in the following sequencing steps are as pure as possible on them, which allows the chemistry in the reaction cell to proceed as intended and no unnecessary losses in yield due to contaminated reagents or solvents occur.

The valve assembly 222 also describes the designs of the valves as they are used in many other parts of the device to largely prevent contamination when a single orifice needs to be selectively connected to a number of other orifices. Thus, the valves 112 and 118 for supplying a reagent or solvent in the conversion piston 14 form a valve assembly in combination with the continuous distributor 114 , and the valves 78 , 80 and 82 , which control fluids from the output connection 38 of the chamber part 12 , form a similar valve device in combination with the continuous distributor 84 . Similarly, the valves for connecting the exhaust and vacuum to the conversion piston and the sample collector and the valves that control the flow from the conversion piston into the sample collector are constructed similarly to the valve assembly 222 . The basic difference in each of these valve assemblies is the number of valve positions that are provided.

It will be appreciated that the manifolds 68 , 84 and 114 actually form a single continuous through connection configured in the manner of the primary through connection 226 in FIGS. 11 and 12, even if the distributors 68 , 84 and 114 in Fig. 3 are shown schematically as if they had a number of small discounts or branches that belong to each membrane associated with them.

The sources of vacuum and gas pressure for actuating the diaphragm valves in their control positions "open" and "closed" are omitted from the schematic diagram of FIG. 3 to simplify it, and they are preferably from source 28 and the Vacuum pump 90 described above independently.

The conversion piston 14 is described in detail in FIGS. 14 to 16. The piston 14 is a double-walled glass piston, which has an intermediate space 264 between two walls, in which a heating medium such. B. water can circulate. The heating fluid is supplied to or withdrawn from the space 264 through a pair of nipples 266 designed to hold the conventional flexible hose ends. A with a wide opening tube 286 , which can be connected to the vacuum trap 88 and the vacuum pump 90 via the valve 96 and with the exhaust air trap 60 via the valve 92 , opens into the chamber interior 270 of the piston 40 in its upper section. Capillary tubes 272 , 274 and 276 extend through the upper part of the piston to points in the interior of chamber 270 . The bores of tubes 272 and 276 are closed at the inner ends, and each of the tubes has a plurality of relatively restricted radially separated openings 278 or 280 near its inner end.

Due to the relatively small amount of the protein or peptide sample for which the device 10 is intended and the relatively small volumes of reagents and solvents used in it, the conversion flask 14 has a much smaller internal volume than anyone the applicants known pistons of the prior art. The volume of the piston 14 is little over a milliliter, while the previous automatic conversion pistons known to the notifiers all had volumes of 100 milliliters.

The fractions cleaved from the sample in reaction chamber 34 flow sequentially through valve 78 and capillary 274 into piston 14 . The reagents and solvents enter the inner chamber of the piston 14 through the capillary 276 , from which they are forced through the restricted openings 280 in the form of a spray that strikes the walls of the chamber 270 to remove any residue on the walls of the floor wash the piston 14 . During the conversion reaction, an inert gas may be supplied through capillary tube 272 to move the solution and aid in the evaporation of the solvent from the liquid. The gas leaves the tube 272 through the restricted openings 278 in the form of very small bubbles, where speed is achieved by optimally dispersing the gas in the liquid, regardless of the size of the piston and the amount of gas used. When the conversion reaction is complete, the fraction is forced upward through long capillary 272 into the corresponding vial in sample collector 16 by positive gas pressure in the flask. This can be done in two subsets to bring about a more complete transfer of the fraction into the sample collector 16 . The first subset of approximately 200 µl is first pushed over into the sample collector. Then who introduced the additional 50 ul of solvent to dissolve any residues on the lower walls or on the bottom of the flask. The second subset is then pushed out. In this way, a high yield can be achieved with each fraction.

After a certain fraction has been transferred, an additional amount of 750 to 900 .mu.l of solvent can be introduced to remove all possible residues from the previous cycle from the upper walls of the piston 14 . This additional amount of solvent is then forced into the exhaust air or waste trap and the piston walls are clean.

The outer ends of the tubes 268 , 272 , 274 and 276 are sealingly connected to corresponding flexible lines 282 which lead to the various other elements of the device 10 by using suitable screw connectors 284 . The end of each conduit 282 is provided with a radial flange portion 286 which has a resilient O-ring 288 which presses against the flat glass base of a radial flange 290 of one of the glass tubes and seals the connection. An internally threaded union nut 292 over the adapter 294 presses the flange section 286 against the glass flange 290 , thereby achieving an extremely effective seal. The various sections of connectors 284 may be made from substantially chemically inert materials such as commercial fluorocarbon polymers to completely prevent the possibility of deterioration and subsequent leakage.

In an alternative construction of piston 14 , space 264 and nipples 266 are omitted to provide a single-walled piston which can be maintained at an elevated temperature by being placed in a heated environment 32 . Such a construction would have the advantage of keeping the entire length of glass tubes and connectors 284 at the elevated temperature, thereby minimizing the possibility of condensation of semi-volatile fluids in these parts, but would not allow the piston and chamber 12 to keep at different temperatures. The reagents and solvents used in the stepwise degradation of protein and peptide chains 10 are preferably the following:

R₁ phenyl isothiocyanate (PITC) (17% solution in heptane)
R₂ trimethylamine or triethylamine (25% solution in water)
R₃ trifluoroacetic acid or heptafluorobutyric acid
R _3A water vapor
R₄ trifluoroacetic acid (25% solution in water)
R _4A hydrogen chloride (1N solution in methanol)
S₁ benzene
S₂ ethyl acetate with 0.1% acetic acid
S₃ butyl chloride
S₄ acetonitrile or methanol

During operation of the device, the various valves and other mechanisms are preferably controlled by the automatic control unit 18 to perform an unlimited number of degradation cycles on a protein or peptide sample without human intervention. The control unit 18 can have the general form of the programming unit, as described in US Pat. No. 3,725,010 to Penhasi, but with corresponding changes, in order to ensure the specific sequence of steps that are required in the device 10 , or it can be an even more complicated and sophisticated electronic control unit, for example of the type of a digital computer for a special or general purpose. Alternatively, the various steps in each breakdown cycle can also be carried out by hand according to a predetermined work plan in order to obtain the same results.

Before the degradation process begins, a sample of the protein or peptide to be examined must be applied to a suitable sample carrier surface. The matrix material is first applied to the carrier surface, the sample then being embedded in the matrix material, as described in Examples 1 and 2 below. If the element 190 or 190 'is used as a sample carrier, it is arranged between the chamber elements 134 and 136 together with at least one of the sealing sheets 194 so that the element 190 is arranged in the reaction chamber 34 at the location of the recess 192 . In the preferred embodiment of the present invention, a pair is used by sealing sheets 194, the hal th the element 190 from both sides. The chamber members 134 and 136 are then pushed into the sleeve 132 and with the various other com ponents assembled to the chamber part 12 to build.

When the sample-containing film is carried by the inner wall of chamber 34 'or chamber 324 , it is brought up as described in Example 2 below.

The chamber elements are initially arranged in the sleeve 132 and held in place by the cap 160 . In the case of chamber 34 ', the annular sheet of resilient material 316 is arranged between the chamber elements 134 ' and 136 'during assembly. The solid Ma trixmaterial and the sample are then applied to the walls of the chamber 34 'or 324 as described in Example 2 be, and the sleeve 32 is assembled with the other components to form the complete chamber part.

The reaction chamber 34 and the associated lines for the fluids can initially be evacuated through the opening of the valve 82 to the vacuum trap 88 and vacuum pump 90 to the chamber for the gradual introduction of the reagents R₁ to R _3A , solvents S₁ to S₃ and to prepare inert gas from source 28 . Every time one of the reagents or solvents is introduced, the corresponding inert gas supply valves 58 and exhaust air valves 62 are opened in order to pressurize the respective reserve and create dynamic equilibrium relationships therein. Inert gas is thus simultaneously introduced into and removed from the reservoir in order to maintain a constant pressure in the reservoir. In the case of the reservoirs 44 , 46 and 48 , the flow of saturated gas from the reservoirs is controlled by one of the flow regulators 76 as it is being supplied.

When the reservoirs such that the respective reagent or Lö containing solvents to be supplied is pressurized and weight condition as described above in a dynamic Equilibrium was brought source 66, a vacuum (not shown) through the auxiliary vacuum to the membrane of the corresponding flow control valve applied to the valve 66 gene can be opened and a stream of the reagent or solution by means of the continuous manifold 68 line and 40 to the input connection 36 of the chamber to erzeu. the valve 66 is kept open, which allows for a predetermined period precisely the desired To pass through amount of reagent or solvent and is then closed by the fact that a pressurized gas is applied to the membrane.

A vacuum from the auxiliary vacuum source is then applied to the diaphragm of valve 72 at the distal end of continuous manifold 68 to flush manifold 68 of any remaining reagent or solvent and to transfer it completely into the chamber. The valve 72 is then closed by applying a positive pressure to its membrane, leaving the manifold 68 free of solvent and reagent ready for the next delivery step.

The diaphragm valve 80 is generally kept open as the various reagents and solvents pass through the chamber 34 to direct effluent fluids from the chamber through the chamber outlet connection and manifold 84 to the waste trap 60 . After a special reagent or solvent has been supplied, the chamber and the lines connected to it can be evacuated by opening the diaphragm valve 82 to the vacuum pump 90 . Alternatively, the chamber and the sample can also be dried in it by passing an inert gas through the valve 72 for suitable times. The gas exiting the chamber can then flow to the waste trap 60 or, if desired, be drawn off by the vacuum pump 90 to accelerate the drying step.

After the completion of the coupling and cleavage steps, the extraction solvent S₃ is entered into the reaction chamber 34 in order to dissolve the cleaved amino acid derivative, which was generated during the special degradation cycle, and to transfer the solution into the conversion flask 14 through line 86 . For this purpose, valve 78 is open and valves 80 and 82 are closed. The conversion reagents R₄ and R _4A (if used) and the solvent S₄ can then be fed into the conversion flask 14 at the appropriate times by opening the appropriate valves 112 to the liquids through the manifold 114 and line 104 in the conversion piston 14 th to lei. each feed pass the pressurization and gas vent steps ahead have been described above in connection with the supply of the other reagents and solvents, and each feeder follows the opening of the valve 118 to the manifold 14 and the Flush line 104 .

The fraction that has been transferred to the conversion flask 14 is the anilinothiazolinone derivative of the N-terminal amino acid of the protein or peptide sample, and it will automatically become more stable in the reaction chamber 34 during the next coupling and cleavage cycles Phenylthiohydantoin amino acid converted, partly according to the articles by Wittmann-Liebold specified at the beginning. Briefly, the amino acid fraction in the conversion flask 14 can first be evaporated by passing an inert gas through the short capillary 276 over the solution and bubbling an inert gas through the liquid through the capillary 272 , after which vacuum is applied through the tube 268 . The conversion reagent R₄ can then be supplied through the capillary 276 by means of the line 104 and one of the valves 112 (cf. FIG. 3) in the desired amount. Rapid evaporation from the conversion flask after the desired conversion time can be achieved by simultaneously applying a vacuum to the conversion flask through the tube 268 and inert gas introduction through the capillaries 272 and 276 . In order to further stabilize the acidic side chains of phenylthiohydantoin-aspartic acid and phenylthiohydantoin-glutamic acid, the residue can be dissolved again in the conversion flask by introducing reagent R _4A through capillary 276 via line 104 and one of valves 112 in the desired amount . Evaporation from the conversion flask is again achieved by applying vacuum through tube 268 and supplying inert gas through capillaries 272 and 276 . The phenylthiohydantoin amino acid remaining in the conversion flask is then redissolved in the solvent S₄, which is fed through line 104 for the purpose of transferring the fraction into the suitable vial of the sample collector 16 . The transfer of the fraction is effected by opening the valve 131 , which is connected to the long central capillary 272 of the conversion piston 14 , and supplying pressurized inert gas through the capillary 276 to push the fraction out of the piston.

The vial carousel in the sample collector 16 is rotated through a predetermined angle once during each degradation cycle so that each incoming fraction is collected in a separate vial. At an appropriate point in the degradation cycle, the fractions in the sample collector can be further dried by opening valve 98 to vacuum, or by opening valves 128 and 94 to release inert gas from source 28 over the samples and ultimately into the waste. To lead trap 60 .

The various components of the device 10 are preferably made of materials that are essentially inert and are highly resistant to wear. Such materials include borosilicate glass, certain fluorocarbon polymers, and in some cases stainless steel and aluminum. The sealing parts and other elements of the device 10 are constructed so that they can be made almost exclusively from these materials. There is reason to believe that the device obtained is the cleanest, most pollution free system that can be obtained and can operate indefinitely under such conditions.

The various steps performed by device 10 in a typical disassembly and conversion cycle are listed in Table 1. The duration of each step and the health of the device during each step are also shown. The functions shown correspond to the control positions of the individual valves of the device, and the crosses in the columns of the table indicate when the appropriate valves are open. For example, the columns "R₁", "R₂", "R₃", "R _3A ", "S₁", "S₂", and "S₃" denote the control positions of the various valve pairs 58 and 62 for selective pressurization and dynamic generation Equilibrium conditions in reservoirs 42 to 54 . Whenever a cross appears in one of these columns, the valves 58 and 62 associated with the respective reservoirs are open, either in preparation or while the particular reagent or solvent is being fed into the reaction chamber. The valves remain closed for the rest of the time. Similarly, the "argon" column indicates the control position of valve 72 for supplying an inert gas such as argon through manifold 68 to the reaction chamber, the "supply" column indicates the control position of valve 66 that corresponds to any reservoir that is closed is pressurized at this point in time, and the columns "waste", "collector" and "vacuum" represent the control positions of the valves 82 , 78 and 80 respectively. As described above, each solvent or reagent feed is preceded by pressurization of the corresponding solvent or reagent reservoir and is followed by the introduction of inert gas through valve 72 to complete the transfer of the solvent or reagent and rinse the supply lines.

The columns "R₄", "R _4A " and "S₄" denote the control positions of the various valve pairs 108 and 110 for the selective pressurization and generation of a dynamic equilibrium in the reservoirs 100 to 102 , and the column "supply / argon" shows that Control positions of both the valve 122 , which admits the inert gas in the conversion piston 14 through the conduit 124 , and the valve 112 , which corresponds to the reservoir which is under pressure at this time. The columns, "Argon", "Waste 1", "Vacuum", "Collector" and "Waste 2" indicate the control positions of valves 118 , 92 , 96 , 131 and 125 , respectively.

Of the specified functions of the sample collector, the columns "Argon", "Waste" and "Vacuum" show the control positions of valves 128 , 94 and 98 .

With the exceptions given above, this corresponds to Sequence of the steps given in Table 1 essentially the Edman degradation as described in the published publications publications, which is why the sequence is not is discussed in detail. Any deviations from the usual The procedure is based on the names of the steps and the corresponding function listed in Table 1 clear conditions.

In practice, the following variations of the sequence Oration program of Table 1 to be provided Program to meet the needs of the special user fit:

• 1) Steps 18 to 58 can be used for the first sequencing to be run one or two times to to be sure that all amino groups of the protein are full be constantly coupled.
• 2) At step 65, a 20 to 60 second supply of water vapor (R _3A ) into the reaction chamber can follow to reduce the dehydration of side chains of serine or threonine.
• 3) At stage 65, 0.05 ml of 1N hydrogen chloride in methanol (R _4A ) can be added to the conversion flask to methylate the side chains of aspartic acid and glutamic acid. If this is done, S₄ is preferably methanol here.
• 4) At step 76, the delivery can range from 0.7 to 1 ml of S₄ (acetonitrile or methanol) connect to  closing in the waste to be converted clean the plunger carefully.
• 5) The duration of step 8 can be extended (500 up to 1000 seconds) to split off the N-terminal To promote proline residues.

The nature of the present invention is illustrated by the following examples for special practical applications tions of the invention further clarified. It goes without saying doing that the data disclosed in the examples only serve as examples of chemical processes with the device according to the invention and according to the fiction procedures have been carried out and that are not intended to limit the scope of the invention.

The amino acid sequences are in the following examples len is represented in a single-letter amino acid code, where the individual letters have the following definitions have:

A - Alanine
R - arginine
N - asparagine
D - aspartic acid
C - cysteine
E - glutamic acid
Q - glutamine
G - glycine
H - histidine
I - isoleucine
L - leucine
K - lysine
M - methionine
F - phenylalanine
P - proline
S - serine
T - threonine
W - tryptophan
Y - tyrosine
V - valine

example 1

A solid matrix of a polymeric quaternary ammonium salt, which is suitable for embedding a protein sample for sequencing, can be prepared on a fibrous sheet element 190 and 190 'as described above, and a protein sample can thus be made in the Ma trix are embedded as described below:
A 12 mm diameter, 0.25 to 0.5 mm thick glass fiber disc is cut from a sheet of glass microfiber filter, such as that commercially available from Whatman, Inc., Clifton, New Jersey. The disc is inserted into the recess 192 in the element 134 Kammerele. 25 ul of an aqueous solution containing 1.5 mg of 1,5-dimethyl-1,5-diazaundecamethylene polymethobro mid and 0.0033 mg of glycylglycine is dripped onto the glass fiber disc from a syringe or pipette. The water is evaporated in vacuo or by heating in a stream of warm nitrogen. The rest of the chamber part 12 is assembled and net in the device 10 angeord. The protein sequencing program begins at step 18 and 4 to 6 complete degradation cycles are performed to remove 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide contaminants that could chemically react with the protein sample or could otherwise interfere with the Edman process. The chamber part 12 is partially disassembled, and 25 ul of a solution of the protein is dropped onto the glass fiber disc. The protein solution dissolves the 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide, and the liquid is removed by evaporation, leaving a thin film consisting of 1,5-dimethyl-1,5-diazaundeca methylene polymethobromide in which the Protein sample is embedded. If the volume of the initial protein sample is greater than 25 µl, the sample can be applied in 25 µl aliquots, the liquid being removed by evaporation between the addition of the aliquots. The chamber part 12 is reassembled and arranged in the device 10 . The protein sequencing program then begins at step 18 and continues for as many degradation cycles as desired.

Example 2

A solid matrix of a polymeric quaternary ammonium salt suitable for embedding a protein sample for sequencing can be prepared on the inner walls of a glass capillary, and a protein sample can be embedded in the matrix as indicated below :
The chamber member 324 or chamber members 134 'and 136 ' are initially assembled with the sleeve 132 and cap 160 as described in the figures, forming a compact cartridge subassembly that is easy for the purpose of introducing the matrix and the sample can be handled. The subassembly is kept horizontal and 10 µl of an aqueous solution containing 0.6 mg of 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide and 0.0013 mg of glycylglycine are injected into the reaction chamber from a syringe. The chamber 320 is preferably constructed to have a diameter of about 0.16 cm (1/16 inch) at both ends, and a diameter of about 0.32 cm (1/8 inch) in the middle, creating a chamber that can hold up to 25 µl of solution in a horizontal position without spilling the ends. This arrangement of chamber 324 , which holds a solution in a horizontal position, is shown in FIG. 18B, with sleeve 132 and the adjoining elements omitted for simplicity. This subassembly is then rotated about its axis while the liquid in the reaction chamber is evaporated by passing a stream of air or nitrogen through the chamber with a thin film of 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide attached to the Chamber walls remains. The subassembly is then installed in the device 10 and the protein sequencing program is then started with step 18 and 4 to 6 complete degradation cycles are performed. The subassembly is then removed from the device 10 and kept horizontal, while 10 μl of a protein solution is dropped into the reaction chamber from a syringe. The subassembly is again rotated about its axis while the liquid in the reaction chamber is evaporated by a stream of nitrogen passed through the chamber, forming a thin film of 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide remains in which the protein sample is embedded as shown in Fig. 18C. The subassembly is then reinstalled in device 10 and the protein sequencing program begins at step 18 and is performed for as many degradation cycles as desired.

Example 3

Angiotensin, 0.005 mg, in 25 ul of an aqueous 20% Formic acid was contained in a solid matrix from previously treated 1,5-dimethyl-1,5-diazaound alone camethylene polybromide by the method of Example 1 embedded. The angiotensin underwent eight Edman degradation cycles subjected, and the phenyl produced in each cycle thiohydantoin amino acids were liquid by high pressure chromatography according to the method as described by Johnson et al "Analysis of Phenylthiohydantoin Amino Acids by High Pressure Liquid Chromatography on Du Pont Zorbax CN Colume "Anal. Biochem. 100, 335 (1979) is analyzed. Known below with "experiment"  recorded frequency was obtained. For comparison, fer ner the known sequence specified.

This result is significant because it shows that even small peptides according to the present invention can be decorated. The ability to sequence small peptides is based on the fact that the sample in one Film is embedded that holds it instead of that it is adsorbed directly onto a support surface, which is why half she is held securely regardless of her Size. During sorptive bonds a large number of non-covalent interactions between the sample and the surface and very strong from the mole depends on the size of the beard film relatively unaffected by sample size.

Example 4

Sperm Whale Apomyoglobin, 0.01 mg, in 25 µl of an aqueous 20% acetic acid was prepared in a solid matrix is 1,5-dimethyl-1,5-diazaundecamethylene polymetho bromide embedded according to the procedure of Example 1. The apomyoglobin underwent 40 cycles of Edman degradation and the phenylthiohydantoin amino acids analyzed according to the procedure of Example 3, the Sequence obtained below, under "Experiment" is played. For comparison purposes, the known one Sequence of sperm whale apomyoglobin also given.

Example 5

A Drosophila melanogaster larval cuticle protein one previously unknown structure, 0.01 mg in 25 µl of an aq solution of 0.1% sodium dodecyl sulfate and 0.05M Ammonium bicarbonate was made up in a solid matrix treated 1,5-dimethyl-1,5-diazaundecamethylene polymetho bromide embedded according to the procedure of Example 1. The Cuticle protein underwent 36 cycles of Edman degradation and the phenylthiohydantoin amino acids analyzed according to the procedure of Example 3, the "Experiment" sequence given below was obtained.

Example 6

A Drosophila melanogaster larval cuticle protein from  previously unknown structure, namely 0.005 mg in 10 µl one aqueous solution of 0.1% sodium dodecyl sulfate and 0.05M Ammonium bicarbonate, was in a solid matrix from before treated 1,5-dimethyl-1,5-diazaundecamethylene polymetho bromide embedded according to the procedure of Example 2. The Cuticle protein underwent 24 cycles of Edman degradation and the phenylthiohydantoin amino acids analyzed according to the procedure of Example 3, the "Experiment" sequence given below was obtained.

The results obtained in Examples 5 and 6 show that the device according to the invention and the invention Methods are suitable for sequencing proteins and peptides grace that in a solution of sodium dodecyl sulfate ("SDS"), a powerful anionic detergent are. This is of great importance because the most common ver drive to isolate small amounts of medium or large proteins or peptides for the purpose of analysis, known as polyacrylimide gel electrophoresis, samples in a solution from SDS. This common detergent causes the samples to come out of the devices be washed on an adsorptive bond of the Sample based on a support surface while in counter the fixed matrix according to the present inven not affected by the presence of SDS becomes.

From the above it can be seen that according to the present Invention an improved device and an improved Process for the gradual implementation of chemical Processes on a sample of very small size using Ver  Use of minimal amounts of reagents and solutions average and created in relatively short cycle times becomes.

Claims (73)

1. Device for the gradual implementation of chemical processes on a sample of a chemical material with

• (a) a reaction chamber ( 34 , 324 ) having inlet means ( 36 ) and outlet means ( 38 );
• (b) means for gradually passing a plurality of fluids through the reaction chamber ( 34 , 324 ) in a pressurized stream from the inlet means ( 36 ) to the outlet means ( 38 ); and
• (c) a solid matrix forming means which are permeable to the plurality of fluids and are arranged in the chamber ( 34 , 324 ) on which a sample is immobilized and each of the fluids flowing through the chamber ( 34 , 324 ) is exposed and can interact chemically with them,
  characterized in that the matrix is on a support which is formed from at least a part of the inner surfaces of the means forming the reaction chamber or from a porous sheet ( 190 ) through which the fluids can flow, and the sample in the matrix is embedded.

2. Apparatus according to claim 1, characterized in that the means forming the solid matrix for the many number of fluids are permeable that they are the fluids absorb in the matrix and the fluids in the matrix diffuse. 3. Apparatus according to claim 2, characterized in that the means forming the chamber have surface areas ( 190, 186, 188, 322 ) on which the solid matrix is applied as a thin film. 4. Apparatus according to claim 3, characterized in that the solid matrix a polymeric quaternary ammonium salt contains.   5. Apparatus according to claim 4, characterized in that the polymeric quaternary ammonium salt 1,5-dimethyl-1,5-dia contains zaundecamethylene polymethobromide. 6. Apparatus according to claim 4, characterized in that the polymeric quaternary ammonium salt poly (N, N-dimethyl-3,5- dimethylene piperidinium chloride) contains. 7. Apparatus according to claim 3, characterized in that the surface regions ( 186 , 188 , 322 ) carrying the solid matrix represent at least a portion of the inner surfaces of the means forming the chamber. 8. Apparatus according to claim 7, characterized in that the reaction chamber ( 324 ) formed by the inner surfaces forms a single passageway in front of the capillary type which extends from one end of the chamber to the other. 9. Apparatus according to claim 8, characterized in that the Diameter of the capillary-type passage from the En that of the passage increases evenly inwards. 10. Apparatus according to claim 7, characterized in that the chamber forming means comprise a pair of adjacent chamber elements ( 134 ', 134 '), each having a first and a second recess ( 186 ', 188 ') on opposite one another , adjacent surfaces ( 144 ', 146 ') of the chamber elements ( 134 ', 136 '), the first and second recesses ( 186 ', 188 ') being aligned with respect to the other recesses ( 188 ', 186 ') are that they form the reaction chamber ( 34 ). 11. Apparatus according to claim 3, characterized in that the surface area carrying the solid matrix is a porous sheet ( 190 ) through which fluids can flow. 12. Apparatus according to claim 11, characterized in that the porous sheet ( 190 ) extends substantially transversely through the chamber ( 34 ). 13. Apparatus according to claim 12, characterized in that the porous sheet ( 190 ) contains a sheet made of a plurality of fibers. 14. Apparatus according to claim 13, characterized in that the Fibers are glass fibers. 15. Apparatus according to claim 12, characterized in that the means forming the chamber comprise a pair of adjoining chamber elements ( 134 , 136 ), the first and second recesses ( 186 , 188 ) on mutually opposing adjacent surfaces ( 144 , 146 ) of the chamber elements ( 134 , 136 ), wherein the first and second recesses ( 186 , 188 ) are each aligned with respect to the other recess ( 188 , 186 ) that they form the reaction chamber ( 34 ). 16. Apparatus according to claim 15, characterized in that the solid matrix a polymeric quaternary ammonium salt contains. 17. Apparatus according to claim 15, characterized in that the first and second depressions ( 186 , 188 ) from the abutting surfaces ( 144 , 146 ) are beveled away to the points at which the depressions ( 186 , 188 ) with communicate with the inlet and outlet means ( 36 , 38 ). 18. Apparatus according to claim 15, characterized in that the porous sheet ( 190 ) from a recess ( 192 ) in at least one of the abutting surfaces ( 144 , 146 ) is received and held in the chamber ( 34 ) in such an orientation that the first and second recesses ( 186 , 188 ) are essentially separated by the porous sheet ( 190 ). 19. Apparatus according to claim 18, characterized in that the means forming the chamber comprise at least one sheet of a resilient material ( 194 ) which is clamped between the abutting surfaces ( 144 , 146 ), the resilient material ( 194 ) is permeable to the large number of fluids. 20. Apparatus according to claim 19, characterized in that the means forming the chamber comprise two sheets of resilient material ( 194 ), one of which is arranged on one side of the porous sheet ( 190 ). 21. Apparatus according to claim 20, characterized in that at least one of the chamber elements ( 136 ) has a raised portion ( 196 ) on its surface adjacent to the other chamber element ( 134 ) ( 146 ), which extends around the recess ( 186 ) in the Chamber element ( 136 ) extends around, and the at least one sheet of a resilient material ( 194 ) presses against the abutting upper surface ( 144 ) of the other chamber element ( 134 ) and in this way increases the sealing effect. 22. Apparatus according to claim 18, characterized in that the inlet and outlet means comprise a pair of capillary through-connections ( 36 , 38 ) which extend through the chamber elements ( 134, 136 ) and with their inner ends with the Communicate reaction chamber ( 34 ) on opposite sides, separated by the porous sheet ( 190 ). 23. Apparatus according to claim 22, characterized in that the capillary passage connections ( 36 , 38 ) are arranged coaxially to the reaction chamber ( 34 ) and extend from the reaction chamber ( 34 ) to outer Kapillaröff openings in the chamber elements ( 134 , 136 ). 24. Apparatus according to claim 23, characterized in that the chamber elements ( 134 , 136 ) are made of glass and have substantially flat surfaces ( 148 , 150 ) in which the capillary openings are arranged. 25. Apparatus according to claim 24, characterized in that the means for the gradual passage of fluids through the chamber ( 34 , 324 ) have at least one piece of an elastic material ( 40 , 172 ) which has a substantially flat sealing surface ( 162 ) and has a capillary through connection ( 184 ) which ends at the flat sealing surface, and screw thread parts with which the flat sealing end is pressed axially against one of the capillary openings so that the axial through connection in the piece ( 40 , 172 ) communicates with the capillary opening and the sealing end of the piece ( 40 , 172 ) sealingly abuts the substantially flat surface ( 148 , 150 ) of the respective adjacent chamber element ( 134 , 136 ). 26. Apparatus according to claim 25, characterized in that at least one piece of elastic material ( 172 ) has a mass of a fluorocarbon polymer, which is encapsulated in a metallic sleeve ( 174 ). 27. Apparatus according to claim 26, characterized in that the screw thread means comprise a bore ( 176 , 178 ) into which the metallic sleeve ( 174 ) is axially slidably fitted, so that the through connection in the piece ( 172 ) is exactly at a capillary opening connects. 28. Apparatus according to claim 3, characterized in that it also includes means ( 18 ) for automatically controlling the functions of the device during a plurality of working cycles. 29. Apparatus according to claim 1, characterized in that the means for gradually passing a plurality of fluids through the chamber ( 34 , 324 ) at least one valve part ( 222 ) for controlling the flow of the fluids between the chamber ( 34 , 324 ) and one A plurality of fluid-containing means, wherein the valve part ( 222 ) contains:

• a) a valve block ( 224 ) having a plurality of substantially flat valve locations ( 230 ) on one of its surfaces ( 228 ), wherein in the valve block ( 224 ) a primary through connection ( 226 ) is formed, which is continuous between two Extends ends of the valve block ( 224 ) and communicates with each of the valve locations ( 230 ) via first openings ( 232 ), and in which a plurality of second through connections ( 236 ) are also formed, each of which has a second opening ( 240 ). communicates with one of the valve locations ( 230 );
• b) a plurality of resilient, essentially impermeable membranes ( 256 ) which cover the corresponding valve positions ( 230 ), each of the membranes ( 256 ) being "closed" between a first control position in which they press against one of the valve positions ( 230 ) is pressed, and thereby the primary and the second opening ( 232 , 240 ), which are connected to this valve position ( 230 ), closes, and a second control position "open" can be controlled, in which the membrane ( 256 ) is withdrawn from the valve location ( 230 ), whereby a flow path between the first and the second opening ( 232 , 240 ) is opened for the fluid via the outer surface of the valve block ( 224 ); and
• c) means for connecting the primary and second passage connections ( 226 , 236 ) to the means containing the fluids and the chamber ( 34 , 324 ), the flow of fluids between the means containing the fluids and the chamber ( 34 , 324 ) can be selectively controlled.

30. Apparatus according to claim 29, characterized in that the means for connection comprise at least one beveled intermediate piece ( 250 ) which is pushed firmly onto a pipe piece ( 252 ) and against a differently beveled recess ( 234 , 238 ) in the valve block ( 224 ) is pressed, the recess ( 234 , 238 ) communicating with one of the through connections ( 226 , 236 ), an inward force acting on the intermediate piece ( 250 ) on a relatively small contact area between the intermediate piece ( 250 ) and the From savings ( 234 , 238 ) is concentrated, creating a seal for the fluid. 31. Apparatus according to claim 30, characterized in that the recess ( 234 , 238 ) is chamfered at a larger angle than the intermediate piece ( 250 ). 32. Apparatus according to claim 30, characterized in that the intermediate piece ( 250 ) is chamfered on two opposite sides, one of these sides being pressed against the recess ( 234 , 238 ) by means provided with screw threads, which also have a chamfered recess ( 247 ), in which the other side of the intermediate piece ( 250 ) engages, the recesses ( 247 , 234 , 238 ) being chamfered at angles which are greater than the bevel angle of the corresponding sides of the intermediate piece ( 250 ). 33. Apparatus according to claim 29, characterized in that the connection means comprise a connection fitting ( 248 ) at one of the ends of the primary through connection ( 226 ) so that the primary through connection ( 226 ) serves as a distributor which is through a flow of a fluid between the connection fitting ( 248 ) and the second through connection, which is farthest from the connection fitting ( 248 ) ent, can be flushed. 34. Apparatus according to claim 33, characterized in that the primary through connection ( 226 ) has a plurality of straight through connections which connect end to end and form a line which has a sawtooth configuration, and which are in alternating sections with the valve ( 230 ) communicates. 35. Apparatus according to claim 1, characterized in that it contains:

• a) converting a piston (14) having a plurality of capillary tubes (272, 274, 276) extending) extend into the interior of the piston (14; and
• b) means for supplying and withdrawing various fluids into or out of the piston ( 14 ) through these capillary tubes ( 272 , 274 , 276 ), at least one of the fluids being supplied from the exit connection ( 38 ); in which
• c) at least one of the capillary tubes ( 276 ) has an end in the piston ( 14 ) at which the bore is closed, and which is provided with a plurality of narrow, radially spaced openings ( 280 ), namely so that an inflow of fluids inwards through the capillary tube ( 276 ) causes the fluid to be sprayed onto the inner walls of the piston ( 14 ), these inner walls being washed.

36. Apparatus according to claim 35, characterized in that the capillary tubes ( 272 , 274 , 276 ) are made of glass. 37. Apparatus according to claim 1, characterized in that it contains:

• a) a conversion piston ( 14 ) having a plurality of capillary tubes ( 272 , 274 , 276 ) which extend into its interior; such as
• b) means for supplying and withdrawing various fluids through the capillary tubes ( 272 , 274 , 276 ) into or out of the piston ( 14 ), at least one of the fluids being supplied from the starting connection ( 38 );
• c) and one of the capillary tubes ( 272 ) ends at a point near the bottom of the piston ( 14 ), at which end the bore of the capillary tube ( 272 ) is closed and this end with a lot of narrow radial in one Is spaced apart from one another at arranged openings ( 278 ) so that an introduction of a gas through this capillary tube ( 272 ) into the piston ( 14 ) creates a large number of small bubbles in a liquid which is located in the piston ( 14 ), these bubbles stir the liquid evenly or accelerate its drying.

38. reaction chamber ( 34 , 324 ) for use in a device for the gradual implementation of chemical processes on a sample of a chemical material with inlet means ( 36 ) and outlet means ( 38 ) for the passage of fluids in a pressurized stream, and a solid matrix form the means that are permeable to a variety of fluids and are disposed in the chamber ( 34 , 324 ) on which a sample is immobilized and exposed to and chemically interact with each of the fluids flowing through the chamber ( 34 , 324 ) can occur, characterized in that the matrix is on a support which is formed from at least a part of the inner surfaces of the means forming the reaction chamber or from a porous sheet ( 190 ) through which the fluids can flow, and the Sample is embedded in the matrix. 39. Reaction chamber according to claim 38, characterized in that the means forming the fixed matrix for the many number of fluids are permeable that they are the fluids absorb in the matrix and the fluids in the matrix diffuse. 40. Reaction chamber according to claim 39, characterized in that the means forming the chamber have surface areas ( 190 , 186 , 188 , 322 ) on which the solid matrix is applied as a thin film. 41. Reaction chamber according to claim 40, characterized in that the solid matrix a polymeric quaternary ammonium salt contains.   42. Reaction chamber according to claim 41, characterized in that the polymeric quaternary ammonium salt 1,5-dimethyl-1,5-dia contains zaundecamethylene polymethobromide. 43. Reaction chamber according to claim 41, characterized in that the polymeric quaternary ammonium salt poly (N, N-dimethyl-3,5- dimethylene piperidinium chloride) contains. 44. Reaction chamber according to claim 40, characterized in that the surface regions ( 186 , 188 , 322 ) bearing the best matrix represent at least a portion of the inner surfaces of the means forming the chamber. 45. Reaction chamber according to claim 44, characterized in that the reaction chamber ( 324 ) formed by the inner surfaces forms a single passage of the capillary type which extends from one end of the chamber to the other. 46. Reaction chamber according to claim 45, characterized in that the Diameter of the capillary-type passage from the En that of the passage increases evenly inwards. 47. Reaction chamber according to claim 44, characterized in that the means forming the chamber comprise a pair of mutually abutting chamber elements ( 134 , 136 ), each having a first and a second recess ( 186 , 188 ) on opposite, abutting surfaces ( 144 , 146 ) of the chamber elements ( 134 , 136 ), the first and second recesses ( 186, 188 ) each being aligned with respect to the other recess ( 188 , 186 ) in such a way that they form the reaction chamber ( 34 ). 48. Reaction chamber according to claim 40, characterized in that the surface area carrying the solid matrix is a porous sheet ( 190 ) through which fluids can flow. 49. Reaction chamber according to claim 48, characterized in that the porous sheet ( 190 ) extends essentially across the chamber ( 34 ). 50. reaction chamber according to claim 49, characterized in that the porous sheet ( 190 ) contains a sheet which is made of a plurality of fibers to which the sample adheres. 51. Reaction chamber according to claim 50, characterized in that the Fibers are glass fibers. 52. Reaction chamber according to claim 49, characterized in that the means forming the chamber comprise a pair of adjoining chamber elements ( 134 , 136 ), the first and second recesses ( 186 , 188 ) on mutually opposing adjacent surfaces ( 144 , 146 ) of the chamber elements ( 134 , 136 ), wherein the first and second recesses ( 186 , 188 ) are each aligned with respect to the other recess ( 188 , 186 ) that they form the reaction chamber ( 34 ). 53. Reaction chamber according to claim 52, characterized in that the solid matrix a polymeric quaternary ammonium salt contains. 54. Reaction chamber according to claim 52, characterized in that the first and second depressions ( 186 , 188 ) from the abutting surfaces ( 144 , 146 ) are beveled away to the locations at which the depressions ( 186 , 188 ) with communicate with the inlet and outlet means ( 36 , 38 ). 55. Rection chamber according to claim 52, characterized in that the porous sheet ( 190 ) is received by a recess ( 192 ) in at least one of the abutting surfaces ( 144 , 146 ) and held in such an orientation in the chamber ( 34 ) that the first and second recesses ( 186 , 188 ) are essentially separated by the porous sheet ( 190 ). 56. Reaction chamber according to claim 55, characterized in that the means forming the chamber contain at least one sheet of a resilient material ( 194 ) which is clamped between the abutting surfaces ( 144 , 146 ), the resilient material ( 194 ) is permeable to the large number of fluids. 57. Reaction chamber according to claim 56, characterized in that the means forming the chamber comprise two sheets of a resilient material ( 194 ), one of which is arranged on one side of the porous sheet ( 190 ). 58. Reaction chamber according to claim 56, characterized in that at least one of the chamber elements ( 136 ) has a raised portion ( 196 ) on its surface adjacent to the other chamber element ( 134 ) ( 146 ), which extends around the recess ( 186 ) in the Chamber element ( 136 ) extends around, and the at least one sheet of a resilient material ( 194 ) presses against the abutting upper surface ( 144 ) of the other chamber element ( 134 ) and in this way increases the sealing effect. 59. Reaction chamber according to claim 55, characterized in that the inlet and outlet means comprise a pair of capillary through connections ( 36 , 38 ) which extend through the chamber elements ( 134 , 136 ) and with their inner ends with the Communicate reaction chamber ( 34 ) on opposite sides separated by the porous sheet ( 190 ). 60. reaction chamber according to claim 59, characterized in that the capillary passage connections ( 36 , 38 ) are arranged coaxially to the reaction chamber ( 34 ) and extend from the reaction chamber ( 34 ) to the outer Kapillaröff openings in the chamber elements ( 134 , 136 ). 61. Reaction chamber according to claim 60, characterized in that the chamber elements ( 134 , 136 ) are made of glass and have substantially flat surfaces ( 148 , 150 ) in which the capillary openings are arranged. 62. Reaction chamber according to claim 61, characterized in that the means for the gradual passage of fluids through the chamber ( 34 , 324 ) have at least one piece of an elastic material ( 46 , 172 ) which has a substantially flat sealing surface ( 162 ) and has a capillary through connection ( 184 ) which ends at the flat sealing end, and screw thread parts with which the flat sealing end is pressed axially against one of the capillary openings so that the axial through connection in the piece ( 40 , 172 ) communicates with the capillary opening and the sealing end of the piece ( 40 , 172 ) sealingly abuts a substantially flat surface ( 148 , 150 ) of the respective adjacent chamber element ( 134 , 136 ). 63. Reaction chamber according to claim 62, characterized in that at least one piece of elastic material ( 172 ) has a mass of a fluorocarbon polymer, which is encapsulated in a metallic sleeve ( 174 ). 64. Reaction chamber according to claim 63, characterized in that the screw thread means comprise a bore ( 176 , 178 ) in which the metallic sleeve ( 174 ) is axially slidably fitted, so that the through connection in the piece ( 172 ) connects exactly to a capillary opening . 65. Conversion flask for use in a device for the gradual implementation of chemical processes on a sample of a chemical material, characterized in that it contains:

• a) a conversion flask (14) having a plurality of capillary tubes (272, 274, 276) which extend into the interior of the piston (14) and for supplying and withdrawing various fluids into the piston (14) into or out of serve this out, being
• b) at least one of the capillary tubes ( 276 ) has an end in the piston ( 14 ) at which the bore is closed, and which is provided with a plurality of narrow, radially spaced openings ( 280 ), namely so that a flow of fluids through the capillary tube ( 276 ) inwardly causes the fluid to be sprayed onto the inner walls of the piston ( 14 ), these inner walls being washed.

66. Conversion piston according to claim 65, characterized in that the capillary tubes ( 272 , 274 , 276 ) are made of glass. 67. Conversion flask for use in an apparatus for performing chemical processes step by step on a sample of a chemical material, characterized in that it contains:

• a) a conversion flask (14) having a plurality of capillary tubes (272, 274, 276) which extend into the interior of the piston (14) and for supplying and withdrawing various fluids into the piston (14) into or out of serve this out;
• b) and where one of the capillary tubes ( 272 ) ends at a point near the bottom of the piston ( 14 ), at which end the bore of the capillary tube ( 272 ) is closed and this end with a number of narrow radially in one Is spaced apart from one another at arranged openings ( 278 ) so that an introduction of a gas through this capillary tube ( 272 ) into the piston ( 14 ) creates a large number of small bubbles in a liquid which is located in the piston ( 14 ), these bubbles stir the liquid evenly or accelerate its drying.

68. Process for the gradual implementation of chemical Processes on a sample of a chemical material step by step passing through a lot number of fluids in a pressurized stream from Entrance of a chamber to its exit, taking the sample a solid matrix of a fluid permeable Material immobilized within the closed chamber so that the sample is exposed to each of the fluids , causing a chemical interaction between the Sample and the fluids is obtained characterized, that the sample is embedded in the matrix, which turns out to be thin film is located on a support. 69. The method according to claim 68, characterized in that that the step of introducing the solid matrix into the chamber introducing a solution into the chamber comprises, the solution being the material of the solid Contains matrix, after which the chamber ver in rotation is set, while at the same time a gas flows through conducts to evaporate the liquid, whereby the material of the solid matrix in the form of a thin film back on the inside walls of the chamber remains.   70. The method according to claim 68, characterized in that the step of embedding the sample of a che mix material into the solid matrix introducing a solution containing the sample into the chamber grips to the matrix material contained therein release, and then set the chamber in rotation, while a gas stream is passed through the Evaporate liquid, causing the material of the solid matrix in the form of a thin film in which the sample is embedded on the inner walls of the Chamber remains. 71. The method according to claim 68, characterized in that that the matrix is carried by a porous sheet, and the step of incorporating the matrix into the Chamber the application of the matrix material on the porous sheet in the form of a thin film as well as the Arrangement of the porous sheet essentially transversely through the chamber at a point between the one corridor and the exit, so that from the entrance Fluids flowing to the outlet flow through the sheet have to. 72. The method according to claim 71, characterized in that that the step of embedding the sample in the solid matrix the steps of delivering a solution, containing the sample on the porous sheet, to dissolve the matrix material on it, as well as the Evaporation of the liquid from the leaf to the Matrix material in the form of a thin film in which the sample is embedded back on the sheet to let. 73. The method according to claim 68, characterized in that that the step of successive transmission of fluids through the chamber the step of through flowing at least one reagent in gas or steam Form encompassed by the chamber, which in the fixed Ma trix diffuses in and reacts with the sample.