SE8802125A0 - valve device - Google Patents

Abstract

Valve device for use in a device for carrying out a sequence of chemical processes for regulating the flow of fluid, characterized in that the valve device comprises: valve block means with a plurality of substantially planar valve stalls 0 a surface of the valve block, the valve block comprising a primary passage, extending continuously between the two spirits of the valve block and through primary openings communicating with each of the valve stalls, and a plurality of secondary passages, each of which communicates through a secondary opening with one of the valve stalls, the primary passage comprising a plurality of straight passages connected to the end, in that they form a channel with a sawtooth shape and communicate at alternating sections with the respective valve stalls,

Description

DESCRIPTION OF THE INVENTION VALVE DEVICE SOOKANDE (damn, domicile of; dress.

Sacs patent of fiera gernensamt, task arr ragon of them ir designated gtr for allg morraga: mac-land = (rin patenrverker) California Institute of Technology 1201 East California Boulevard Pasadena, California 91109, USA INVENTOR (name oeh address) ...

See appendix OIYIBUD (noun, hernvisr, sdress and telephone number) Ei The undersigned applicant authorizes within the following listed Swedish ornbud arc A4 --- 1 to represent me in everything that pertains to this patent application and in everything that may have granted patents.

BL SOkande befullnakrigar below Swedish representatives by separate power of attorney.

Dr. Ludwig Brann PatentbyrA AB POBox 7524,103 92 Stockholm BEGA.RAN, ABOUT PRIORITY (date,) and scan numbers) 23 September 1980 USA no. 190,100 ON DEPOSIT OF MICRO-ORGANISM Depositary authority: Deposit date: Deposit no .: „..... .............. .. „, ...

BY DEPARTMENT OR Starnansokningensnummer8105597-2 UBRENTEN AMOKENING Begird lapdag: 22 september 1981 BILAGOR. 0Description, parent requirements oat sartunandrag in triplicate D3112 drawings in3 copies 121 Assignment document) copies. Stockholm, June 7, 1988 Ore, date Originalen i 8105597-2. emA.) ce / i6-0-t., 1 Signature (Hans Wibom) parenckrav utover 60: kr: 300 kr IN Fullrnakr) inlgmnade FEGIF) p.ans. 0.k.Grundavgift: 800 kronor 0Additional surcharge, 100 kr for vatic K4Avgift far kopier av nyhershinvisning DDiariebevis: 15 kronor Betainingssitt: 0 postgiro le checkil kontant BP .g Till KU NGL PA ih *: (1,2 8067 -; 28 2.2T 8902125 — E *** SAWA ', 8806e7 18.3282 8802125-8 441: sf3iri.FT NSOKA N ABOUT SWEDISH PATENT INVENTORS' NAMES AND ADDRESSES: 1. Leroy E. Hood 1453 East California, Pasadena, California 91009, USA 2, Michael W. Hunkapiller 8243 Sheffield Road San Gabriel, California 91775, USA William J. Dreyer 385 South Catalina No. 216 Pasadena, California 91106, USA Rodney M. Hewick 251 South Catalina Pasadena, California 91106, USA Anton W. Stark 2001 E. Galbreth Road Pasadena , California 91104, USA.

; ".: * •: '•:" :: California Institute of Technology VALVE DEVICE (Divided from p.ans. 8105597-2) The invention relates generally to an improved device and to a farmed process for carrying out chemical processes and in particular to an improved device for automatically performing sequential degradation of protein or peptide chains containing a large number of amino acid units for determining the sequence of these units.

The linear sequence of the amino acid units in proteins and peptides is of considerable importance, as it contributes to the understanding of their biological functions and finally to the synthesis of compounds that perform the same functions. Although a variety of methods have been used to determine the linear order of amino acids, the most successful candidate is probably the Edman process. Various forms of the Edman process and apparatus for automatically performing the processes are described in the following publications: Edman and Begg: "A Protein Sequenator", European J. Biochem. 1 (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); Wittmann-Liebold et al .: "A Device Coupled to a Modified Sequenator for the Automated Conversion of Anilinothiazolinones into PTH Amino Acids", Analytical Biochemistry 75, 621 (1976); U.S. Patent 3,959,307, invented by Wittmann-Liebold and Graffunder, May 25, 1976, for "Method to Determine Automatically the Sequence of Amino Acids"; Hunkapiller och Hood: "Direct Microseguence Analysis of Polypeptides Using an Improved Sequenator, A Nonprotein Carrier (Polybrerie), and High Pressure Liquid Chromatography", Biochemistry 2124 (1978); Laursen, R.A., 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, issued April 3, 1973, for the Apparatus for Automatically Performing Chemical Processes; U.S. Patent 3,717,436 to Penhasi et al., issued February 20, 1973, for the "Process for the Sequential Degradation of Peptide Chains"; U.S. Patent 3,892,531 to Gilbert, issued July 1, 1975, for the Apparatus for Sequencing Peptides and Proteins; U.S. Patent 4,065,412 to Dreyer, issued December 27, 1977, for the "Peptide or Protein Sequencing Method and Apparatus". A further apparatus of importance is described in U.S. Patent Application 106,828, issued December 26, 1979, to Leroy E. Hood and Michael W. Hunkapiller, two of the inventors of the present invention, relating to the "Apparatus for the Performance of Chemical Processes".

Sas = discussed in the above publications, Edman's sequence degradation processes involve three steps: coupling, cleavage and conversion. In the coupling step, phenyl isothiocyanate reacts with the N-terminal c (amino group of the peptide to form the phenylthiocarbamyl derivative. In the cleavage step, anhydrous acid is used to cleave the phenylthiocarbamyl derivative to form the anilinothiazolinone. Aqueous acid was used to convert the thiazolinone to phenylthiohydantoin, which can be analyzed by suitable means, for example by chromatography.

The automatic apparatus described in U.S. Pat. No. 3,725,010, modified as set forth in the aforementioned articles by Wittmann-Liebold and U.S. Patent Application 106,828, to Hunkapiller and Hood, relates to a automatic sequencer in which the reactions are carried out in a thin film formed on the inner wall of a rotating reaction cell, commonly referred to as a "rotating shell", and arranged in a closed reaction chamber. Means are provided for introducing and depositing controlled amounts of liquid reagents to and from the chamber for reaction with a pray of a protein or peptide in an inert atmosphere. The sample to be analyzed is first applied in the rotating shells followed by the sequence of introduction and removal of various reagents and solvents required for carrying out the coupling and cleavage reactions. The liquid reagents and solvents themselves form films on the cradles of the shells, which pass over and react with the sample film as the shells rotate. The reagents dissolve the test film and provide the coupling and cleavage steps of the Edman process. After completion of the coupling and cleavage steps, the reaction chamber is evacuated to remove volatile components from the reactants. After the evacuation, which is carried out after the coupling, the remaining test film is extracted with release agent to remove non-volatile components. After the post-cleavage evacuation, the resulting thiazolinone frail is extracted from the sample film with solvent and transferred either to a separate flask to carry out the conversion step or to an apparatus for collecting and drying the various fractions. If the conversion reaction joke is carried out immediately in a conversion flask, the process can be carried out later on several fractions simultaneously.

The insertion and removal of fluids in relation to the rotating shell has been effected with fluid conduits passing through a plug which closes an opening in the burrowing cradle of the reaction chamber and extends down therefrom to a stall inside the shell. Fluids are introduced directly into the rotating sic: Alen in a place near the bottom of this and •• 4 discharged from an annular groove in the cylindrical inner surface of the shell. The fluid to be deposited is forced into the annular groove by the centrifugal force when the shells are rotated at high speed, and discharged through a conduit with an inner spirit extending into the groove. This effluent line thus acts as a collection means for depositing reaction products and by-products and extracting solvents.

If the protein or peptide sample in a rotating shell device does not in itself have sufficient mass to form a cohesive film, in some cases it was carried on the inner rock of the shell during the solvent extractions in a relatively thick layer of non-protein carrier material. The carrier material and sample are then dissolved in the liquid reagents during the reaction steps to allow the coupling and cleavage reactions to take place. A polymeric quaternary ammonium salt with the chemical composition 1,5-dimethyl-1,5,5-diazaundecamethylene polymetobromide has been used for this purpose. The carrier must be applied in substantial amounts to securely retain all the sample and the harener as well as the sample are dissolved in both the liquid reagents so that reaction between the sample and the reagents is possible.

Although rotary baker devices can provide acceptable experimental results in many cases, they have many disadvantages. As an example, the cost of obtaining a sufficiently large protein or peptide sample and maintaining a suitable supply of the necessary reagents is relatively high, primarily because the reagents used are in liquid form and must be used in significant quantities. Liquid reagents and solvents have a tendency to remove portions of the sample from the film and wash them from the cradle of the shells as these substances pass, reducing the yield of terminal amino acid units obtained in each successive cycle of the device. The initial amount of the sample must therefore be large enough to ensure that a sufficient amount of sample remains during the last cycle to give usable results. Devices of this type also had a relatively long cycle time due to the considerable volume of the reaction chamber and the need to repeatedly remove semi-volatile liquid reagents and solvents by vacuum drying the sample in the reaction chamber. In addition, rotary shell sequencers are relatively complicated and expensive both to manufacture and repair.

Another type of sequencing device is described in the above-mentioned publications by Laursen and Wachter, in which the sample is disturbed by covalent bonding to the surface of a plurality of small beads. The beads form a port gasket in a reaction column and the column is rinsed with liquid reagents to carry out the chemical processes. Since the cleavage reagents used are very good solvents for proteins and peptides, the covalent bond must be complete to envelop the sample in place. However, covalent bonds are difficult to obtain in practice. The filled column is amen answer to wash and the beads in this have a tendency to be probed during use.

It has previously been proposed that sequencing devices designed to overcome the illegality of these devices be contained in a sample in a stationary reaction chamber and subject the sample to the action of at least one reagent in gaseous or angular form. The patents of Gilbert and Dreyer, mentioned above, describe two such devices, none of which work satisfactorily.

The device in Gilbert's patent discloses a closed finger-shaped obsolescence in a reaction chamber for holding a peptide or protein sample during the sequential exposure to gaseous reagents and solvents. Each time a reagent is introduced, the demand is cooled internally to give condensation to the demand. The precipitate is then heated so that the sample is dissolved in the liquid as the reaction proceeds. After the reaction with the sample, the undesired semi-volatile chemicals can either be wiped from the sample! ..; - • 0 ■ • • • •• 0 • • •• • • g •• • • •• • • • 6 by a combination of heat and a stream of inert gas or washed from the demand together with the terminal amino acid with a solvent which is condensed on the demand until it drips from it.

In the device and method of Dreyer's patent, a protein or peptide sample is applied to both the inner and outer surfaces of many small macroporous beads in a reaction column by chemical coupling or direct adsorption thereon. Various reagents and solvents are sequentially passed through the filled column in either gaseous or liquid form to give the desired degradation reactions. The flow of reagents and solvents to the column is regulated by a rotary shut-off valve with ten layers.

Unfortunately, the Gilbert and Dreyer patent devices do not provide a sufficient pollution free environment to provide acceptable results during an initial number of degradation cycles. For example, it is black to wash the protein or peptide sample effectively in the devices of Gilbert and Dreyer. Gilbert's method of washing the sample by condensing solvent thereon to the point at which the solvent drips from the sample tends to leave traces of the various reaction products on the sample which contaminate subsequent chemical reactions. Furthermore, the filling required to keep the sample in the reaction column according to Dreyer is difficult to wash because the various chemical products must be transported completely through it and away from the column to avoid contamination. This cannot be easily achieved even when large amounts of solvent are used, since the solvent tends to pass through the space between the beads instead of through the small pores inside the beads, where most of the protein sample is coated. The fluid supply lines and river valves have the devices according to Gilbert and Dreyer also responsible for evacuating completely and have a tendency to retain chemical residues, which can increase the intended chemical conditions in subsequent reaction cycles.

Glass or plastic beads used as the filling reaction column according to Dreyer also tend to probe during a large number of degradation cycles, clogging the system to such an extent that the passage of fluids through it is impeded. It then becomes almost impossible to wash the system between the cycles and the chemical million in the column becomes hopelessly contaminated.

The contamination caused by the plurality of factors described above has a cumulative effect during the time span of a sequential degradation process. The sample and the reagents in the reaction cell thus become more and more contaminated, which prevents the desired coupling and cleavage reactions from causing a number of undesired reactions to take place. The yield from each complete cycle of the device is thus collected and a series of impurities are introduced into the fractions.

The yield is further reduced by direct loss of the sample due to a variety of shells, including decomposition of the gasket, solubility of the sample in the flushing solvents and deficiencies in the sorptive bonds between the filler and the sample.

Although these effects can be colored in some cases, when large amounts of the sample of the protein or peptide are available or when the chain has a relatively small number of units, they become indistinguishable in cases where the chain has a very large number of units or only very small amounts. protein or peptide dr available. Both of these conditions exist in the case of interferon, a small protein that is formed in human cells in response to certain viral infections. Interferon has recently received considerable attention in clinical medicine because this substance promises to be an effective remedy for viral infections and it tends to offer considerable hope as an anti-cancer reagent. Interferon is prepared and is thus available only in very small amounts. For the time being, mainly the whole world's production of the two types of human interferon from the predominantly five world centers, which have access to large amounts of human white blood cells (leukocyte interferon) or certain human cells in tissue culture (fibroblast interferon). Due to this limited production capacity for interferon, it has been difficult to conduct selection-controlled clinical trials and fundamental analyzes of how this molecule works. To further complicate the picture, interferon is composed of a chain of about 150 amino acid units, which must be cleaved individually from the chain for analysis. Contamination losses of the types described above can prevent sequencing of all but the first few amino acid units of interferon with the very small amounts of protein available. After the first few cleavage cycles, the small sample may become contaminated to such an extent that positive result jokes can be obtained.

The most sophisticated prior art device for converting the various thiazolinones, which is cleaved from the sample to the more stable phenylthiohydantoins, is the conversion flask described in the above-mentioned articles by Wittmann-Liebold Sami modified in U.S. Patent Application 106,828, seeking Hunkapiller and Hood. According to the invention, however, it has been found that this piston suffers from inefficient washing of its inner cradles when reagents and solvents are introduced through the applicable capillary tube. It has been suggested that the reagents and solvents may be supplied with a stream of inert gas to wash the walls of the flask by boiling the liquid thereon, but this method causes an unreliable flow of liquid to the flask and makes it very difficult to control the volume of liquid supplied. .

According to the invention, it has been found that the previously known conversion piston is designed in a substantially smaller size scale to accommodate smaller volumes of liquid, Hr: • • .1. • • • 1 • • • • •• - ■. obtain the optimum degree of distribution of inert gas bubbles in the liquid contents of the flask for agitating the contents during the conversion reaction and for evaporating the semi-volatile components thereof. Inert gas introduced into the bottom of the flask for these purposes tends to rise to the surface of the liquid in relatively large bubbles, which do not stir the liquid uniformly but instead favor stench of the liquid to the top of the flask.

In many applications, it is necessary to provide a device for carrying out chemical processes, such as sequence reactions of proteins or peptides, which work efficiently and with a minimum of system contamination to enable a maximum number of sequence cycles to be carried out with good results with a very small lots of pray.

The device according to the invention comprises chamber means with an inner surface defining a reaction chamber, the chamber having inlet and outlet means for passing fluid into a pressurized stream, saint solid matrix means which are permeable by diffusion to a plurality of fluids and applied in the chamber, said that a pray embedded in the matrix organ is orlliggores and exposed to all of a plurality of fluids passed through the chamber for chemical action on the sample.

The chamber means may comprise surface means supporting the solid matrix means such as a thin film and the matrix means may comprise a polymeric quaternary ammonium salt, for example 1,5-dimethyl-1,5-diazaundecamethylene polymetobromide or poly- (N, N-dimethyl) -3,5- dimethylene-piperidinium chloride).

The surface means may comprise all or part of the inner wall of the chamber means or may comprise porous disc means which extend substantially transversely or transversely over the chamber and allow the passage of fluids. The disc means may comprise a disc made of a plurality of glass fibers. The chamber means may comprise a pair of abutting chamber elements with a first rasp. second cavities on opposite surfaces of these, the first and the second cavities being arranged in line in relation to each other and forming the reaction chamber. The first and the second cavity may be conically tapered in directions hard from the opposite surfaces to points at which they communicate with the inlet means resp. the outlet means. The porous layer, when used, is arranged in a recess in at least one of the facing surfaces to be retained in the chamber in an orientation which substantially separates the first cavity from the second cavity. The chamber means may comprise at least one layer of resilient material, co-applied between or laminated between the facing surfaces in a sealing manner, the resilient material being permeable to the fluids in question. At least one of the chamber elements may in this case comprise a raised part on one of the adjoining surfaces, which extends around the cavity therein in order to compress the layer of resilient material against the adjoining connecting surface of the chamber member in order to improve the sealing.

The inlet and outlet means may comprise a pair of capillary channels, which extend through the respective chamber elements and communicate with the inner spirits with the reaction chamber on opposite sides of the portal layer. The capillaries may be arranged coaxial with the reaction chamber and extend therefrom to outer capillary openings with substantially flat outer surfaces having the chamber elements.

The means for sequentially passing a plurality of fluids through the chamber may comprise valve block means having a plurality of substantially flat valve sets on the surface, the valve block means forming a primary passage which is interconnected between the two spirits thereof and communicates by priming drips with each of the valve stalls. a plurality of secondary channels, each of which communicates through a ••• V • •• V. 1.1 secondary opening with one of the valve stalls, saint a plurality of elastically resilient, substantially impermeable diaphragms, late tacks and respective valve stalls, each diaphragm being installable between a first a layer in which it is pressed against one of the valve stalls to shut off the primary and secondary openings communicating at this valve stall, and a second layer in which the diaphragm is withdrawn from the valve stall to provide a fluid flow path between the primary and secondary openings through the exterior of the valve block means. that the fluid flow between pri the ground channel and the secondary channel can be selectively regulated. The connecting means may comprise at least one conically tapered rudder (ferrule), which is arranged with a tightly sliding fit over a rudder-shaped member and is pressed against a recess with another conical shape in the valve block means, which communicates with one of the channels therein, this stirring ring is focused on a relatively small contact surface between the stirring ring and the recess to give a fluid seal between them. The recess is preferably designed conically with a larger angle 8n the pipe. The interconnecting means may further comprise a fitting in a slide of the primary channel for connecting the primary channel to another part of the apparatus, so that the primary channel acts as a main line, which can be flushed with a fluid flow between the coupling and the secondary channel, anxiously away from the coupling. The primary channel can comprise a plurality of straight passages, which are connected spirit to spirit and form a conduit with a sawtooth shape and communicate late at alternating cutting stalls with the respective valve stalls.

The device may comprise a conversion piston with a plurality of capillary tubes extending into the interior of the piston for insertion and discharge of various kinds of fluids, at least one of the capillary tubes having an inner spirit, at which the capillary channel is closed and provided with a plurality of radially distributed openings of limited size, so that the passage of fluids through the capillary tube gives spray to the inner cradles of the piston for washing them. A capillary tube opening into a stall near the bottom of the flask may be provided with a closed spirit with a plurality of radially distributed openings of limited size adjacent to the duct, so that passage of gas into the capillary tube produces a variety of small bubbles which stir any liquid in the flask and accelerates the drying thereof.

The process according to the invention for carrying out chemical processes in sequence on a prey of chemical material comprises embedding the sample in a solid matrix which is permeable by diffusion to a plurality of fluids, enclosing the solid matrix in a closed chamber with an inlet and an outlet. in the sequence the multiple plurality of fluids through the chamber as well as a pressurized stream frail the inlet to the outlet of the chamber, so that the sample is exposed to each of these fluids, whereby the sample is disturbed and the chemical effect between the sample and the fluids is obtained. The solid matrix can be carried or supported as a thin film on the inner rocks of the closed chamber. Alternatively, the solid matrix may be supported on a porous sheet or layer which extends substantially transversely across the chamber at a location between the inlet and the outlet, so that fluids carried from the inlet to the outlet must pass through this layer or sheet. The step of embedding the sample in a solid matrix may include the step of applying the solid matrix to a substrate such as a thin film and then applying a solution containing the sample to the film so that the sample dissolves the matrix in solution. The wet shoes are then evaporated from the solution by leaving a film with the sample embedded in it.

It is an object of the present invention to provide an apparatus and method for sequentially carrying out chemical processes on a bed of chemical material with a minimum of loss of the sample and a minimum of contamination of the system.

It is also an object of the invention to provide an economical apparatus and method for sequentially carrying out chemical processes on a sample of very small size by using minimal amounts of reagents and solvents.

It is another object of the invention to provide an improved device and method for sequencing alchemical processes with very short cycle time.

It is a further object of the invention to provide an improved apparatus and method for sequentially performing chemical processes on a prey, thereby washing the sample more efficiently between cycles.

The apparatus and method of the invention laser a variety of problems with prior art sequencers by immobilizing the protein or peptide sample in a solid matrix which is formed as a thin film permeable by diffusion to both reagents and solvents used in the degradation process. The severe problem of achieving complete covalent bonding is thus avoided as well as the problem of sample loss which arises when the sample is directly adsorbed on a substrate surface and fully exposed to the mechanical shear forces of the mobile liquid phase. The sample is held securely in place by the matrix, while the smaller reagents, solvents and amino acid derivatives can diffuse through the matrix and dissolve in the matrix to a sufficient concentration to carry out the various steps in the decomposition process. The solid matrix retains the sample so effectively when exposed to gaseous reagents that almost any form of sample substrate surface can be used without causing loss of the sample. The inner walls of the reaction chamber itself may provide a sufficient substrate surface .. d • • f $ S • A substrate surface which greatly facilitates complete washing of the system is a porous layer formed of a plurality of overlapping glass fibers extending transversely across a genome. -. ".: tightened reaction chamber. The porosity is replaced by the gaps between the fibers. This structure shows a ratio" • • s p. 0 7 5 P5s ••• 14 country start total surface area with minimal extension in the direction of fluid flow . The solid matrix forms a thin film on the surfaces of the fibers, which allows reagents and solvents to diffuse easily into the film and react with the sample embedded therein. This enables chemical processes and wash cycles to be performed on the sample with a minimum of reagents and solvents and for a relatively short period of time. The reagents and solvents, some of which are in the form of a gas or Anga, penetrate the thin film and come into contact with the sample and react with it as completely and efficiently as possible.

The relatively thin profile of the porous layer indicated in this context further improves the ability of the sample to be carefully washed from residual reagents and reaction products with a relatively small amount of solvent. The desiccant only needs to line the reagents and reaction products the relatively short distance out of the surface of the layer to remove them from the system. From a point outside the surface of the layer, they can be easily led out of the chamber, so that the sample is left in a state for the next reaction step. The legal consumption of solvents meant jokes only a saving if the low cost of the solvent but decreases (even the tendency has the sample to be washed from the reaction chamber and lost.

The use of reagents in gaseous or vapor form helps to improve the exposure of the sample to the reagents and to minimize the amount of reagents required. Low reagent consumption is important because the reagents used in the Edman degradation method must be extremely free of contaminants and therefore very expensive. Furthermore, the sample and the solid matrix containing it are not dissolved by the gaseous reagents, which eliminates the problem of sample loss due to separation of the sample from the substrate surface.

The reaction chamber of the invention is designed to allow the passage of both gaseous and liquid reagents through the porous layer which coats the sample without allowing the sample to become contaminated with external contaminants or with chemicals passed from one reaction stage or cycle to another. The abutting chamber elements thus together form a small volume reaction chamber made up of a first and a second cavity on opposite sides of the porous layer. A pair of capillary passages extending on opposite sides through respective chamber elements from the chamber itself make it possible to pass a plurality of fluids in gaseous or liquid form as a pressurized stream through the chamber and past the sample matrix. The small volume of the chamber and the passages minimizes the volume of the reagents and solvents required and facilitates vacuum drying of the system between cycles. The two chamber elements are closed at the mating surfaces against a layer of elastically resilient material, which is applied or laminated between the mating surfaces. The elastically resilient material is permeable to the diversity of fluids carried through the chamber and in effect filters out the flow of gases through the chamber by forcing the gases to come into more uniform contact with the porous layer.

An alternative embodiment of the reaction chamber is a single capillary or capillary-type passage with a solid matrix formed as a thin film on the inner surface or channel of the capillary tube or passage. The protein or peptide sample is embedded in the matrix, as in the case of the porous layer, and the reagents from the solvents are passed in sequence through the tube to act on the sample. The chamber structure described above can be used for this purpose without the element designed as a porous layer. The test-containing matrix is formed on the surfaces of the first and the second cavity. Similarly, the surface that supported the solid matrix can be constructed and formed in virtually any way that allows reagent and solvent fluids to be passed over the matrix.

The new valve device according to the invention for regulating the flow of fluids to and from both the chamber and the conversion piston is specially designed to eliminate cross-contamination of fluids. Each of the valve means joins a single channel with a plurality of other channels to selectively connect this individual channel to each of the others. The individual channel is brought to communicate with one spirit of the primary passage in the valve block, while each of the other channels is connected to one of the secondary passages. In the normally closed state, each of the diaphragms which presses the different valve stables of gas pressure is pressed against the surface of the valve block to prevent the primary passage from communicating with the secondary passage leading to the valve stall in question. The secondary ducts leading to the valve stall at the far end of the primary passage can be connected to a pressurized source of a purge fluid, for example inert gas, to complete the supply of fluids to the liquid block. habit fluid through ve ntilen. After a certain reagent or solvent is introduced into the reaction chamber by applying a vacuum to the corresponding diaphragm of the supply valve, the diaphragm at the far end of the primary passage can be opened to complete the supply by forcing reagents or solvents remaining in the primary passage into the chamber. This is possible due to the continuity of the primary passage and means that the collection line formed between them is flushed clean of a certain reagent or solvent, before the supply of the next reagent or solvent is started. As for the valve at the outlet to the reaction chamber of the primary passage connected to the outlet, while the secondary passages are connected to the conversion piston, vacuum resp. avf all. The continuity of the primary passage makes it possible to evacuate it carefully and virtually eliminate the possibility of semi-volatile substances being trapped in it between cycles.

The "sawtooth" shape of the primary valve passages set forth herein has been used previously in sequencer area valve assemblies. Prior art sawtooth valve assemblies, however, have included a plurality of individual blocks mounted to the valve stem on a main valve block to be displaced back and forth between the state or path of communication and non-communication of passages in the main block. The displaceable blacks tend to wear out, causing leakage both to the atmosphere and between the passages. The new valve units according to the invention solve the problem of wear by combining the previous sawtooth main line with a series of Olaf ragman to create and cut off flow between pairs of openings which communicate with the respective passages. These diaphragms can be made of substantially inert materials, for example commercially available fluorocarbon polymers, which function indefinitely without enlargement or enhancement. Furthermore, prior art means for connecting the valve channels to external lines tend to cause excessive pressure on the sides of the valve block, which contributes to deformation of the upper sealing surface of the valve block and loss of its ability to form a closed joint. In particular, prior art sawtooth valves have connected external conduits to the valve passages by pressing substantially flat flange surfaces associated with the alike conduits against the sides of the valve block to provide a series of seals between pairs of flat surfaces. Since each of these components is made of saccharin materials such as fluorocarbon polymers, which are very difficult to machine carefully, a considerable pressure must be applied in order for the respective sealing surfaces to be formed one after the other and form the required seal. The pressure is applied by the valve block and causes deformation of its bearing surface. '.. ".. • ...` "..'. The valve device according to the invention comprises a plurality of conical sealing rings (ferrules), which are partly accommodated in recesses formed with a different conical shape in the valve block in order to provide a seal without applying too high pressure to the valve block. A relatively small gripping force is focused on a certain part of the sealing ring to cause the sealing ring to bear against correspondingly conically shaped recesses without deformation of the block.

Providing spruce surfaces at opposite ends of the sealing rings between surfaces of different conical shape further improves the obtained seals. Spruce surfaces of this type between surfaces of different conical shape are preferably provided at opposite sides of the sealing rings in order to achieve maximum sealing properties.

The conversion flask according to the invention makes it possible to introduce reagents and solvents in the form of a spray which hits the inner cradles of the flask to drain away any chemicals which may have condensed or stank of the cradles so that they are washed down the cradles and into the water basket in the flask. A major cold cross-contamination of the system between the cycles is eliminated in this way. The conversion flask allows gases to be carried up through the quantity of liquid in the form of small bubbles, which uniformly stir the liquid and facilitate drying of semi-volatile components therein without causing sample loss due to excessive bubbling and stench. This favors a rapid, careful removal of liquid from the probable amino acid derivatives. Solvents used to feed the amino acid derivatives into the conversion flask can thus be deposited in a much shorter time than according to the patented invention of Wittmann-Liebold, as mentioned above (1-2 minutes instead of 5-10 minutes) and at lower temperature (40- ° C in the stable of 50-80 ° C). This substantially improves the yields of the most unstable amino acid derivatives, for example those of serine, threonine, histidine, arginine and tryptophan. Reagents used in the conversion flask can be removed by a combination of fine streams of inert gas bubbles and low vacuum within 3-5 minutes instead of the 30-40 minutes required by the Wittmann-Liebold-patented invention. In the Wittmann-Liebold process, drying of the reagent must begin immediately after its introduction into the amino acid residue of the conversion flask to meet the requirement that the total conversion flask cycle time should not exceed the total cycle time of the primary reaction chamber. Since the acid component of the conversion reagent, trifluoroacetic acid or hydrochloric acid, is much more volatile than the water as it is dissolved, the acid component tends to be deposited early in the Wittmann-Liebold drying process with retention of the amino acid derivative in only water during a substantial portion of conversion. This causes incomplete conversion of derivatives of glycine and proline as well as probe division of other derivatives. In the conversion device of the invention, the conversion reagent can be left in contact with the amino acid derivatives for a period of time sufficient for complete conversion, 30-40 minutes, and then dried rapidly, for 3-5 minutes, without the sample stinking over the entire interior of the conversion flask.

The foregoing and other objects of the invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which like reference numerals refer to like parts.

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

Figure 2 is a top view of the device according to Figure 1.

Figure 3 is a schematic diagram of the device according to 4P • V • • Vali • figure 1.

R: 41.4,4 Figure 4 is an exploded perspective view of a reaction chamber unit constructed in accordance with the invention.

Figure 5 is a vertical section on a dry scale along line 5-5 of Figure 4.

Figure 6A is a further enlarged cross section through the reaction chamber of Figure 5.

Figure 63 is a cross-section through a second embodiment of the reaction chamber of Figure 5.

Figure 6C is a cross-sectional view of the reaction chamber of Figure 63 with the porous layered member removed therefrom for use with a sample-containing film applied to the inner surface.

Figure 7 is a vertical cross-section through a typical reservoir according to the invention for a reagent or welding agent to be used in liquid form.

Figure 8 is a vertical cross-section through a typical reservoir according to the invention for a reagent to be used in the form of gas or anga.

Figure 9 shows a vertical cross section through a diaphragm valve designed according to the invention for regulating the flow of fluid to and from the reaction chamber and the conversion piston, shown in a direction corresponding to line 9-9 in Figure 11.

Figure 9A shows a subsection 1 on a larger scale through one of the coupling elements has the valve unit according to Figure 9.

Figure 10 is a vertical section along line 10 of Figure 9.

Figure 11 is a side view of the main line block of the valve apparatus of Figure 9.

Figure 12 is a horizontal section along the line 12-12 in ■ • 21 Figure 11.

Figure 12A is a vertical section taken along line 12A-12A of Figure 11.

Figure 13A is an enlarged subsection through the valve portion of the valve assembly of Figure 9, showing the diaphragm pressed against the main conduit block to prevent fluid communication between the channels at this stall.

Figure 133 shows a subsection on a larger scale through the valve portion of the valve assembly of Figure 9, showing the diaphragm pulled away from the manifold block to allow fluid flow between the channels.

Figure 14 is a top view of a conversion piston constructed in accordance with the invention.

Figure 15 is a vertical section taken along line 15-15 of Figure 14.

Figure 16 is a vertical section taken along line 16-16 of Figure 14.

Figures 17A and 173 schematically show the two most important previous Janda methods for disrupting a protein or peptide sample during degradation.

Figure 17C shows a schematic representation of a protein or peptide according to the invention.

Figure 18A is a larger sectional vertical section corresponding to Figure 5 of a further embodiment of the reaction chamber unit according to the invention.

Figure 183 is a vertical section showing the chamber element of the embodiment according to Figure 18A water on the side for supplying a sample-containing matrix to its inner rocks. Figure 18C shows a further subsection on a larger scale of the chamber element according to Figure 18B with a sample-containing film applied to its inner walls.

Table I is a list of the various steps performed with the device according to the invention in a typical degradation and conversion cycle.

DESCRIPTION OF PREFERRED EMBODIMENTS.

Figures 1 and 2 show a device 10 according to the invention. The device 10 comprises a chamber apparatus 12, a conversion piston 14 and a fraction collector 16, each of which is operated by an automatic control unit 18. A row 20 of solvent and reagent reservoirs dr through a group 22 of diaphragm-type flow valves connected to the reaction chamber 14 and the conversion piston A second group of valves 24 regulates the flow of liquids from the action chamber to the conversion piston 14 and the second positions. A third group of diaphragm valves 26 has the function of connecting the conversion piston and the fraction collector to either a drain or a vacuum and to regulate fluid flow from the piston to the fraction collector.

A filter 28 for filtered inert gas supplies to the device 10 purified inert gas, preferably argon, to keep the solvent and reagent reservoirs under pressure, flush oxygen-containing air out of the system and speed up the process of drying reagents and solvents in the system at various times. A group of pressure regulating valves and counters have the task of individually regulating the pressure of the gas to each solvent and each reagent reservoir and to all other components of the device 10.

The operation of the device 10 is illustrated in Figure 3. The chamber device 12 is arranged in a heated environment 32 and forms, according to the preferred embodiment, a reaction chamber 34, which is connected to the inlet and outlet channels 36 and 36, respectively. 38. The inlet channel 36 can be connected via a fluid line 40 to a plurality of reservoirs in the group 20, i.e. reagent reservoirs 42, 44, 46 and 48 and solvent reservoirs 50, 52 and 54. Reservoirs 42-54 were pressurized with the inert gas pressure source 28 through a group of individual gas pressure regulators 56 and solenoid flow valves 58. Each of the reservoirs 42-54 may also be stall above the fluid level in these is connected to a waste trap 60 through an individual flow valve 62 and an individual flow regulator 64. Inert gas under pressure is introduced to the reservoirs from the cold 28 and can thus be discharged to the drain trap 60 at a rate or degree controlled by the flow regulators 64. reservoirs 42-54 are individually controlled by diaphragm valves 66 which communicate with a continuous or continuous manifold 68 which is connected at one end to the fluid conduit 40. Fluid from the pressurized reservoirs 42-54 may thus be passed through conduit 40 and the chamber inlet passage. 36 to reaction chambers 34 to selectively maneuver the valves 66. To facilitate the supply of the reagents and solvents and to flush the collection line 68 and the duct 40 after supply, pressurized inert gas can be introduced into the collection line 68 at the spirit opposite the line 40 through a pressure regulator 70 and a diaphragm valve 72. Maneuvering the valve 72 thus provides introduction of gas under pressure to the hortre spirit of the manifold 68 and drives any reagents or lasers into the conduit through the conduit 40 and passage 36 to the reaction chamber 34.

The fluid outlets from reservoirs 44, 46 and 48 are provided with gas flow meters 74 in series with the fluid regulators 76, since the reagents stored therein are used in gaseous or vapor form, while the remaining solvents and reagents are used in liquid form. The feeders 74 and flow regulators 76 are required to accurately control the gas discharge rate through corresponding valves 66. 24 Fluid flow from the reaction chamber 34 is controlled by diaphragm valves 78, 80 and 82, which communicate with the outlet passage 38 through a continuous or continuous manifold 84. Valve 78 is opened for Release the desired reaction products, typically the N-terminal amino acid moiety of a protein or peptide sample, through line 86 to the conversion flask 14. Valve 82 may be opened to connect outlet passage 38 to waste trap 60 for disposal of unwanted reagents, solvents and reaction products and the valve 80 connects the outlet passage 38 with a vacuum trap 88 to a vacuum pump 90 to evacuate the reaction chamber 34, the outlet passage 38 and the manifold 84.

Similarly, the conversion piston 14 and the fraction collector 16 are connectable to the drain trap 60 through the valve 92 and 92, respectively. 94 saint to vacuum through valves 96 and 98.

Reagent reservoirs 100 and 101 are provided with a solvent reservoir 102 for supplying reagents and lasing agents to the conversion flask 14 through a line 104. Reservoirs 100, 101 and 102 are pressurized with the inert gas shell 28 through pressure regulators 106 and solenoid valves 108 connected to individual waste traps 60. vent valves 110 and flow regulators 111. The fluid outlets from reservoirs 1001 101 and 102 are connected through diaphragm valves 112 to a continuous or continuous manifold 114 which communicates with one another with conduit 104. The flow of fluids from the pressurized reservoirs can thus be accomplished by selectively opening the valves 112 for discharging either reagent or solvent into the manifold 114. The cold inert gas Sr can be connected through a pressure regulator 116 and a diaphragm valve 118 to the end of the manifold 114 opposite the conduit 104 for driving the reagent. The inert gas pressure valve 28 is also connected to the conversion piston by a pressure regulator 120, a diaphragm valve 122 and a line 124 to the fraction collector 16 by a pressure regulator 126 and a valve 128. on the intended set ± the piston 14, it can be discharged from the piston with gas pressure through the line 124 and a valve 131 to the fraction collector 16 for storage in an ampoule therein. After the fraction has been expelled, the flask 14 can be filled to a relatively high level with a solvent for dissolving any remaining chemicals therein. The welding agent can then be expelled by gas pressure through line 124 and a valve 125 to the waste trap 60 saint the flask is purged in preparation for delivery of the next amino acid derivative.

The fraction collector 16 comprises in principle a carousel with ampoules, which is maneuvered with the control unit 18 once during each cycle in front of the device 10. Rim placement of an empty ampoule in a layer for receiving the next fraction of amino acid units from the piston 14.

The control unit 18 is preferably a belt automated unit which controls the diatragm valves 66, 72, 78, 80, 82, 92, 94, 96, 98, 112, 118, 122, 125, 128 and 131 as well as the solenoid valves 58, 62, 108 and 110. The control unit 18 also controls the mechanism for maintaining the heated environment 32 at the desired temperature (not shown), the fraction collector 16, the vacuum pump 90 and a plurality of sensors throughout the system. The various gas pressure regulators and flow regulators described above are installed manually after commissioning before installing the desired pressures and rivers in the corresponding fluid lines.

The chamber device 12 is shown in detail in Figures 4 and 5 comprising a base 130 in two parts, which carried a sleeve 132, which contains first and second chamber elements 134 and 14, respectively. 136. The sleeve 132 is provided with an enlarged cylindrical part 138, which is centered around the axis of the sleeve and fitted in a cylindrical recess 140 in the base 130. The cylindrical part 138 is molded together with a retaining collar 142, which to base 130.

The chamber elements 134 and 136 are cylindrical glass elements with adjoining surfaces 144 and 146 and taken in an axial direction in the sleeve 132. The inlet and outlet channels 36 and 38 described above extend axially through the chamber elements 134 and 134, respectively. 136 and there are preferably capillary channels with a diameter of the order of 1 millimeter. The axially outer teeth 148 and 150 of the chamber elements 134 and 136 are generally flat and abut abutting each other with a pair of thin elastic spacers or washers made of a substantially inert material which gives the chamber elements a shock absorption in the axial direction relative to the sleeve 132. A metal washer 154 disposed on top of the upper elastic washer 152 with the opposite welding bridge 156 intended to cooperate with slots 158 in the outer end of the sleeve 132. The metal washer 154 is held in place with a lid 160, which is screwed to the outer end of the sleeve 132 to hold the chamber elements in place in relation to the sleeve. The interaction between the groove 156 and the slots 158 prevents the washer 154 from rotating. the cover 160 is fitted and thus prevents the cover from damaging the unit by rotating the chamber elements. The fluid conduit 40 is provided with an enlarged lower end 162 which abuts the outer duct 148 of the chamber member 134 so that the tail of the conduit communicates with the inlet passage 36. The conduit 40 is provided with a support washer 164 and a nipple 166 which is screwed in axially. in the lid 160 to press the expanded spirit 162 into the chamber member 134. The conduit 40 may be made of any resilient substantially inert material, such as commercial fluorocarbon polymers. The alignment of the shaft in the conduit 40 with the inlet passage 36 is arranged by constructing the various cooperating components with sufficient precision in relation to a common axis.

The outlet passage 38 is arranged in connection with the continuous manifold 84, described above, with a mass 172 of substantially inert material enclosed in a steel sleeve 174. The sleeve 174 has a flat outer surface which is fitted in axial openings 176 and 178 formed. in line in the enlarged cylindrical part 138 and the base 130, respectively. The mass 172 extends axially in both directions beyond the steel sleeve 174 and abuts against the outer edge 150 of the second chamber element 136 with a conical recess 180 in a valve block 182 forming the manifold 84. An axial passage 184 in mass 172 is arranged precisely in line with the outlet passage 38 and one end of the manifold 84 to provide a single continuous continuous capillary channel from the chamber member 136 to the valve block 182. As for the conduit 40 discussed above, carefully design the components related to the donna. around a common axis a close fit and complete tatn between the different channels. The chamber device 12 can thus be easily assimilated and assembled in a very short period of time without risking the inboard fitting of the different channels or the tightness of the different seals.

The valve block 182 is of a new construction, which is described in detail in connection with Figures 9-13. It is sufficient to note at this stage that parts of the valves 78, 80 and 82 included in the valve block 182 Rix 'control of the flow of fluids from the chamber device 12.

As can be seen most clearly from Figure 6A, the chamber 34 is formed with aligned or fitted cavities 186 and 188 in the opposite connecting surfaces 144 and 144, respectively. 146 of the two chamber elements. The two cavities are arranged coaxially with the inlet and outlet passages 36 and 38, having a suitably circular cross-section and providing an axially symmetrical path for a fluid which passes from the inlet passage to the outlet passage. A porous layered element 190 extends transversely across the reaction chamber 34 and may be arranged at least in part in a recess 192 in the cavity 186. The porous layered element 190 thus separates the inlet passage from the outlet passage 28. 38, so that fluids flowing from one passage to the other must pass through the layered element. The porous layered element 190 preferably comprises a layer or mat of compressed fibrous material, for example glass. Commercially available fiberglass filters are suitable for this purpose and have a high resistance to probe splitting or other damage during use. It has been found that a porous layer of this type provides a considerably larger surface area to support a thin film in which a protein or peptide sample can be embedded. If the film is made of a fluid-permeable material, i.e. one that allows diffusion of liquids and gases into the layer, the material can form a solid matrix, which can retain the sample securely but allows chemical action of reagents and solvents on the sample. Polymeric quaternary ammonium salts, such as 1,5-dimethyl-1,5,5-diazaundecamethylene polymetobromide or poly- (N, N-dimethyl-3,5,5-dimethylene-piperidinium chloride), are ideal for this endamal. They allow diffusion of fluids, which are insoluble in solvents used and which are chemically stable against both reagents and solvents. In addition, they form a cohesive film and have a positive charge, which allows them to bond ionically to the glass substrate surface.

The fundamental differences between the forms of sample retention used in prior art sequencer devices and according to the invention are more clearly shown in conjunction with Figures 17A, 17B and 170. Figures 17A and 17B schematically illustrate the two most common prior art methods for ordering a protein or peptide sample 300. in relation to a test surface 302 or 304.

Figure 17A shows that the sample is chemically bonded to a glass substrate surface 302 with covalent bonds 306. Thus, for example, the surface 302 may be specially treated, for example, so that some of the silica numbers of the glass have functional amino groups 308 extending therefrom for reaction with carboxyl groups 310. at the sample chain. 29 •• I • 4. • 4, 4 • •: 4! • '. •: Under normal conditions, some of groups 308 and 310 react, causing the sample to covalently bond to the glass and cleave a number of water molecules. Bindings of this type are very strong and one or two bonds per molecule are sufficient to line the sample in place. However, covalent binding is the answer provided by protein and peptide samples. Covalent bonds hail the dven chain with a very small number of units in the chain. When the decomposition process reaches these units and splits them from the chain, the remainder of the chain becomes unbound and can be washed away from the chamber.

Figure 17B illustrates the case where the sample 300 is adsorbed directly on a substrate surface 304. The sample is then held in place with a very large number of relatively weak non-covalent effects 312 between the sample and the surface. At the molecular level, the effect between the surface and the sample is obtained in a number of different places. This works vd1 in question of large proteins and peptides, but when the sample is broken down to a much smaller size, the bend becomes to be beaten or pulled away from the surface. This results in a drastic sample loss.

Figure 17C schematically illustrates the formation of the sample 300 in relation to the substrate surface 314 by embedding a solid matrix 316 formed as a thin film on the substrate surface. As described above, the matrix 316 may be a polymeric quaternary ammonium salt having a positive charge. This matrix will thus be strongly retained on an acidic glass surface by a very large number of ionic influences and hold the sample securely in place, since the sample is embedded. There is no reliance on the direct bonding effect between the sample and the surface and the effectiveness of the agitation is not affected by reducing the size of the sample.

The present invention is based on the diffusion of reagents, solvents and amino acid derivatives through the solid matrix 316 to effect chemical reaction and action with the embedded sample. The matrix is formed as a thin film, which absorbs the reagents and lasers which are passed over the film. After the reagents and solvents are selectively loaded into the film, they can be easily diffused through its thickness and extend the degradation process.

Figure 6A shows the reaction chamber 34 drilled at the periphery of the cavities 186 and 188 with at least one sheet or layer 194 of a resilient sealing material laminated between the adjacent surfaces 144 and 146. A pair of resilient layers 194 were suitably used on each side of the porous layer-shaped element 190. The layers 194 are very thin and are permeable to the variety of reagents and lasing fluids to be passed through the chamber 34. An annular tat ace or roll 196 at the periphery of the cavity 188 abuts the tatniag layers 194 to provide a more effective sealing against the surface 144 adjacent the circumference of the cavity 186. The sealing layers 194 may be challenged by some substantially chemically inert material, for example commercial fluorocarbon polymer, in order to minimize the risk of magnification of the seal. They not only have the purpose of providing a seal for the chamber 34 but also of standing or supporting the porous layer-shaped element 190 and allowing gases and liquids, blacks passing through the chamber, to diffuse, so that the surface of the gases and liquids becomes more uniformly distributed across Over element 190. Long system life and optimal chemical impact on the sample are favored in this way.

An alternative embodiment 34 'of the reaction chamber according to the invention is shown in Figure 6B, according to which the mating cavities 186' and 188 'in the opposite adjoining surfaces of the had chamber elements 8r are somewhat narrower and longer than the cavities 186 and 188. The ions 34 and 34 'are identical and the alike elements of the structure 34' of the drawing figures are numbered similarly to those of the structure 34 with the suffix • - "prim" (') separating them. The reaction chamber 34 '.. allows a slightly more direct flow of fluids from the inlet - •: -.! 36' to the outlet 38 '.. but reduces the diameter of the porous • •. 4.7 • • I 41 - • o • Of f 31 layer element 190 '.

Figure 6C ashes the chamber 34 'of Figure 6E with the porous layered member 190' removed. In addition, the sealing layers 194 'are replaced by a single annular layer 316 of resilient material with a central opening of the same diameter as the chamber. According to this embodiment, a solid, fluid permeable matrix 318 with a three embedded protein or peptide sample is formed as a thin film on the walls of the chamber 34 'for exposure to reagents and solvents passed through the chamber. The flow of fluids through the chamber 34 'is thus maintained while maintaining a substantial film surface area.

A further embodiment of the chamber device according to the invention is shown in Figures 18A, 18B and 18C, according to which the two chamber elements 134 and 136 are replaced by a single chamber element 320 of capillary type in the sleeve 132. The remaining elements of the chamber device 12 are the same as those described in conjunction with Figures 4 and 5 and numbered in the same way. All constructions and connections externally in relation to the chamber arrangement are also identical to those described above.

The chamber element 320 comprises a cylindrical glass structure with inner cradles 322, which form an axial chamber 324 of the capillary type. The chamber 324 increases uniformly in diameter from its bath teeth 326 toward its center 328 and the protein or peptide sample is carried in a solid matrix 330 formed as a thin film on the walls 322.

It is well observed that the reaction chamber of the invention can take on a substantially arbitrary shape with a test layer surface along which a plurality of reagent and solvent fluids can be passed. Thus, for example, a single elongate capillary tube (not shown) would be sufficient for the chamber device 12 with a fluid permeable solid matrix applied to the inner surface of the capillary cavity. Fluids contained in the tube would react with a protein or peptide sample embedded in the film and effect the degradation process.

A typical reservoir 198 according to the invention for storing and supplying a liquid reagent to the reaction chamber 34, 34 'or 324 is shown in Figure 7. The reservoir 198 corresponds to the reservoirs 42, 50, 52, 54, 100, 101 and 102 in Figure 3. An inert gas under pressure is supplied to the interior 200 of the reservoir 198 through a conduit 202 which communicates with the reservoir at a location below a level 204 of the liquid reagent or solvent therein. The gas under pressure thus introduced releases any dissolved oxygen from the liquid and can be discharged at an adjustable rate through a vent line 206 to achieve a dynamic equilibrium state in the interior 200. A liquid outlet 208 is provided for controlled discharge of reagent or lasing agent from the reservoir 198. the gas pressure in this. The inert gas supply line 202 to each reservoir 198 receives inert gas under pressure from a cold 28 through a pressure regulator 56 or 106 and a solenoid valve 58 or 108. Discharge of gas through the vent line 206 is likewise controlled by one of the solenoid valves 62 and 110 and one of the flow regulators 64 and 111. The flow of liquid reagent or solvent through line 208 is controlled by one of the valves 66 or 112. Each time one of the liquid reagents or solvents is to be supplied to the reaction chamber or flask 14, corresponding argon supply valves and vent valves are provided to provide a dynamic equilibrium. . Reagents or liquids in liquid form can then be introduced by opening the valve in line 208 and valve 82 to the waste traps 60. Liquid is discharged from the reservoir at a constant rate, which allows accurate control of the discharged liquid by controlling the time period the discharge line 208 is held open.

A typical reservoir 210 according to the invention for discharging a reagent in gaseous or vapor form is damaged in Figure 8. An inert gas inlet 212 which opens into a gas distribution element 213 of sintered glass is arranged for introducing inert gas into the interior 214 of the reservoir 210 ph a stall adjacent bottom thereof and substantially below level 216 of liquid reagent therein. A vent line 218 and a discharge or dispensing line 220 communicate with the interior 214 at points above the water level 216. The reservoir 210 is typical of the reservoirs 44, 46 and 48 shown in Figure 3, with one of the pressure regulators 56 and one of the valves 58 which controls the flow of gas through line 212 from gas pressure head 28. Similarly, the release of gas under pressure to vent line 218 is controlled by one of the flow regulators 64 and flow valves 62, and the discharge of reagent through line 220 is controlled by one of the diaphragm valves 66 in line with a river feeder 74 and river regulator 76.

Although the reagents R2, R3 and R3A are supplied to the reaction chamber in gaseous or vapor form, they are thus stored as water shoes and lorangas if necessary. Evaporation is accomplished by bubbling inert gas up through the liquid reagent. In this way, the inert gas ± the interior 214 of the reservoir 210 is matted with reagent vapor. Each time reagent is required Open the valves connected to lines 212 and 218 to bubble inert gas through the reagent and achieve a dynamic equilibrium state. The valve 66 in the discharge line 220 is then opened for a predetermined period of time to discharge the desired amount of reagent vapor to the reaction cell. The river regulator 76 in the line with the valve 66 in question causes the Amgen to be discharged from the reservoir at a constant speed, as indicated by the river feeder 74.

Figures 9-13 show the construction and function of a valve device 222, which forms embodiments of the valves 66 and 72 and the continuous or continuous manifold 68 according to Figure 3. The valve device 222 r *,: •• 34 comprises a valve block 224, which is most clearly seen on Figures 11 and 12. The valve block 224 consists of an elongated block of rectangular gold cross section with a continuous or continuous primary passage 226 in sawtooth sample formed by cross-drilling the valve block from one of its surfaces 228. The primary passage 226 is thus a simple continuous or continuous passage which communicates alternating crossing posts with a plurality of valve posts 230 on the surface 228 through corresponding openings 232. A conical connection opening 234 communicates with one end of the primary passage 226. A plurality of secondary passages 236 extend from the conical connection openings 238 on the opposite side of the valve block 224 to the corresponding openings. 240 near the openings 232 at the respective valve stalls 230. The valve block 224 is mounted in an elongate recess 242 in a base 244 with a plurality of passage openings 246 mating with connection openings 234 and 238 for receiving connection nipples 248. The nipples 248 are designed to compress resilient double conical sealing rings 250 against the connection openings 234 rasp. 238 for connecting tubes 252 extending through the sealing rings to the various passages in the valve block 224. In this case the connection opening 234 of the primary passage in connection with the inlet passage 36 has the chamber device 12 through the fluid line 40 according to Figure 3 and the first seven of the eight the secondary passages communicate with the fluid outlets 208 and 220 of the reservoirs 42-54. The secondary passage furthest from the connection port 234 communicates with the inert gas pressure valve 28 through the pressure regulator 70 shown in Figure 3.

The construction of the double helical sealing ring connections to the openings in the valve block 224 is shown in more detail in Figure 9A, in which the opening 234 is shown by way of example.

The sealing ring 250 according to Figure 9A is located at the inside 243 in the connection opening 234 and at the outside 245 in a conical recess 247 in the nipple 248, the opening 234 and the recess 247 being conical with angles which are greater than the cone angles of the respective sides of the sealing ring. The sealing ring is preferably formed conically with the same cone angle on both sides and with the opening 234 and the recess 247 formed conically with an angle which is more rigid than the angle of the sealing ring. This fit between surfaces of different conical shape focuses the compressive forces on the tips 249 of the sealing rings 250 and provides an effective seal with a minimum of pressure on the side of the valve block 224. Excessive pressure on the valve block 224 which can deform the valve stalls 230 is thus avoided.

A series of diaphragm retaining blocks 254 are secured with bolts to the surface 228 having the valve block 224 with the diaphragm 256 mounted or laminated between these parts. The upper end of each diaphragm mounting block is threaded to receive an air connection 258 which communicates with a recess 260 on the underside thereof and generally extends over one of the valve stalls 230. An O-ring 262 may be arranged in an annular save as surrounding the recess 260 to provide effective air tightening.

The diaphragms 256 are challenged by a substantially chemically inert aerated material, which allows these alternates to be withdrawn from and pressed against the valve stalls 230 by the action of vacuum and air pressure, respectively, through the connection nipples 258. Two alternating states have the diaphragm 256 shown in Figures 13A and 13B. The condition of Figure 13A forces air or gas pressure applied to the connection nipple 258 diaphragm 256 toward the opening 232 of the primary passage and the opening 240 has the second passage at the valve stall 230. The openings 232 and 240 are thus closed with the diaphragm 256, which does not allow any connection between them. This corresponds to the closed rage has valve which is arranged at the valve stall in question. In the condition of Figure 13B, vacuum applied to the connection nipple 258 pulls the diaphragm 256 hard from the openings 232 and 240, so that connection is obtained between the primary and secondary passages above the surface of the valve block at this position. This corresponds to the open state of the valve in question and allows fluid from one of the reservoirs or from the inert gas pressure source 28 to flow through the primary passage 226 to the inlet passage 36.

The new construction has the valve device 222, as described above, enabling the supply of reagents and solvents to the reaction chamber in accurate amounts with the slightest contamination between the various fluids. The continuity has the primary passage 226 and the connection at one of its ends to the inlet passage has the chamber device 12 are to a large extent responsible for this advantageous function. Dispensing of any of the seven reagents and solvents can be accomplished by applying a vacuum to one of the diaphragms 256 and a positive gas pressure to the others, whereby the desired reagent or solvent can pass into the primer passage 226 and through the fluid line 40 to the reaction chamber. When the desired amount of fluid has passed past the diaphragm in question, a new gas pressure is applied to it through the connection nipple 258, so that all the seven reagent and solvent valves unit 222 are closed. Supply of the fluid can then be completed by applying a vacuum to the diaphragm connected to the inert gas port at the far end of the valve block 224 to flush the entire primary passage 226 with inert gas so that reagent or solvent fluid remaining in the lines is forced into the reaction chamber. 34. Since there are no interruptions or blind branches in the primer passage 226, there is no position where any of the reagents or solvents can be retained between the dispensing procedures. The reagents and solvents supplied in successive sequence steps are thus as pure as possible, allowing the chemical reactions of the reaction cell to proceed as intended and without any unnecessary deterioration of the yield due to contaminated reagents or solvents.

The valve assembly 222 is also an example of the valve structure 37 incorporated into many other parts of the device to minimize contamination when a particular orifice must be selectively connected to a plurality of other orifices. The valves 112 and 118 for supplying reagents and solvents to the conversion piston 14 thus form a valve unit in combination with the continuous manifold 114, and the valves 78, 80 and 82 for supplying fluids from the outlet passage 38 of the chamber device 12 form a similar valve device in combination. with the continuous manifold 84. Also the valves for connecting the drain and vacuum to the conversion piston and the fraction collector and the valves which regulate the flow from the conversion piston to the fraction collector there are constructed in the same way as the valve unit 222. The principal difference between these valve devices is the number of valve parts. these.

It can be seen that also the manifolds 68, 84 and 114 are shown schematically in Figure 3 as embodied with a series of small discontinuities or branches adjacent each diaphragm valve son joined there, each of the manifolds is in fact a single continuous passage constructed in the same manner as the primary passage 226 of Figures 11 and 12.

Vacuum and gas pressure for maneuvering the diaphragm valves between the open and closed states are omitted from the schematic diagram of Figure 3 to simplify this and preferably separate frail) (Allan 28 and the vacuum pump 90 described.

The conversion piston 14 is shown in detail in Figures 14-16. The elves 14 are of the double-rock glass type with a space 264 between the rocks for the circulation of a heating fluid, for example water. The heating fluid is passed to and from the space 264 through a pair of nipples 266 designed to groove the teeth of standard flexible hoses. A rigid inner diameter tube 268 connectable to the vacuum trap 88 and the vacuum pump 90 through the valve 96 and to the waste trap 60 through the valve 92 is connected to the inner chamber 270 of the piston adjacent its upper end. Capillary tubes 272, 274 and 276 extend through the burial end of the piston to points in the interior of the chamber 270. The shark in the tubes 272 and 276 are closed at the inner spirits and each of the tubes there is provided with a plurality of spaced apart radially distributed openings 278 or 280 adjacent the inner end.

Due to the relatively small amount of protein or peptide sample for which the apparatus 10 is constructed, and the relatively small volumes of reagents and solvents used therein, the conversion flask 14 has a much smaller internal volume than any prior art flask. The volume of the flask 14 is slightly more than 1 ml, while previously known automatic conversion flasks have all had volumes of 100 ml.

The fractions cleaved from the sample ± the reaction chamber 34 are sequentially fed to the flask 14 through the valve 78 and the capillary tube 274. Reagents and solvents flow into the inner chamber of the flask through the capillary tube 276, from which they are forced through the limited openings 280 sAs a spray hitting the cradles of the chamber 270 are drained of any residue on this to the bottom of the piston. During the conversion reaction, an inert gas can be introduced through the capillary tube 272 to agitate the liquid and help to evaporate the solvent Iran therein. The tube leaves the tube 272 through the constrained openings 278 as very small bubbles, which gives optimal dispersion of the gas in the whole liquid, regardless of the size of the piston and the amount of gas used. After the conversion reaction is completed, the fraction raised up through the gas overpressure in the flask is forced through the long capillary tube 272 to the respective ampoule in the fraction collector 16. This can be accomplished in two different volumes or sample volumes to give a complete transfer of the fraction to the fraction collector 16. The first sample volume about 200 microliters (.41), is first fed to the fraction collector. Then additional lasers are introduced to dissolve any residue on the lower cradles or the bottom of the piston. The second sample volume is then removed. In this way a high yield of each fraction can be achieved.

After a certain fraction has been transferred, an additional 750-900) 41 solvents can be infused to dissolve any residue which remains from the previous cycle on the older rocks in the piston 14. This additional amount of solvent is then disposed of as waste and leaves the rocker rocks clean.

The outer ends of the tubes 268, 272, 274 and 276 are connected to respective flexible conduits 282, which lead to the various other parts of the apparatus 10 through mating threaded coupling means 284. The end of each conduit 282 is provided with a radial flange portion. 286 with a resilient 0-ring 288, black abuts and tilts against the planar ground glass surface has a radial flange 290 on one of the glass tubes. An internally ganged collar 292 is slidably disposed over the conduit 282 to receive the glass flange 290 and cooperate with a two-piece externally gangled nipple 294, which is arranged around the glass tube. Feeding the collar 292 Over the nipple 294 forces the flange portion 286 against the glass flange 290 and provides an extremely effective seal. The various parts of the connectors 284 may be challenged by substantially chemically inert materials, for example commercially available fluorocarbon polymers, to eliminate the possibility of enlargement and consequent lacquering.

An alternative construction of the piston 14 would eliminate the space 264 and the nipples 266 and provide a single cradle which could be kept at elevated temperature by fitting in the heated environment 32. This construction would have the advantage of keeping the entire length of the glass tubes and connectors 284 at the elevated temperature with minimization of condensation of semi-volatile fluids therein, but would not allow the flask and container 12 to be heated at different temperatures.

The reagents and solvents used in the apparatus for sequentially digesting protein or peptide chains are preferably as follows: R 1 phenylisothiocyanate (PITC) (17% solution in heptane) R 2 trimethylamine or triethylamine (25% solution of water ).

R3 trifluoroacetic acid or heptafluorobutyric acid R3A aqueous vapor R4 trifluoroacetic acid (25% solution in water) R4A hydrochloride (1N solution in methanol) S1 benzene S2 ethyl acetate with 0.1% acetic acid S3 butyl chloride Sacetonitrile or methanol.

During operation, the various valves and other mechanisms in the device 10 are preferably controlled by the automatic control unit 18 to perform an indeterminate or brisk number of degradation cycles on a protein or peptide sample without human action. The control unit 18 may be in the general form of the programming unit disclosed in U.S. Pat. No. 3,725,010, Penhas1, with variations to enable the particular sequence of steps required by the apparatus 10 or may be a more sophisticated electronic control ring in the form of a control unit. special or general purpose digital computer. Alternatively, the similar steps of each degradation cycle may be performed manually by an operator according to a predetermined schedule for achieving the same result.

To initiate the degradation process, a sample of the protein or peptide being tested must be applied to a suitable sample bar area. The matrix material is first applied to the barary surface and the sample is embedded later in it, as described in Examples 1 and 2 below. When the element 190 or 190 is used as the sample surface, it is applied between the chamber elements 134 and 136 together with at least one of the sealing sheets or the sealing layers 194, so that • • • aass •% Ns .. • 1 • • • •••••• "" The element 190 is held in the reaction chamber 34 at the location of the recess 192. According to the preferred embodiment of the invention, a pair of sealing sheets or sealing layers 194 are used and the element 190 is laminated between them. The chamber elements 134 and 136 are then inserted into the sleeve 132 and assembled with the various other components to form the chamber apparatus 12.

If the sample-containing film is to be carried by the inner surface of the chamber 34 'or the chamber 324, the droplet is described as described in Example 2 below.

The chamber elements are first mounted in the sleeve 132 and held in place with the lid 160. As for the chamber 34 ', the annular layer of resilient material 316 is applied between the chamber elements 134' and 136 'after mounting. The solid matrix material and sample is then applied to the cradles to have the chamber 34 'or 324, as described in Example 2, and the sleeve 132 assembled with the other components to form a complete apparatus.

The reaction chamber 34 and the associated fluid conduits can be evacuated from the beginning by opening the valve 82 to the vacuum trap 88 and the vacuum pump 90, in preparation for the sequential insertion of the reagents R1 through R3A, the solvents 1 through 3 and inert gas from the well 28. Each time one of the reagents or release agents is to be introduced, the corresponding inert gas supply valve 58 and vent valve 62 are opened to pressurize the reservoir in question and achieve a dynamic equilibrium state therein. Inert gas enters the reservoir and is released from the reservoir at the same time to maintain a constant pressure in the reservoir. When reservoirs 44, 46 and 48 are controlled, the flow of matted gas from them is regulated during discharge with one of the river regulators 76.

After the reservoir contains the reagent or solvent to be applied, it is placed under pressure and equilibrated, as described above. A vacuum with the auxiliary vacuum source (not shown) is applied to the diaphragm having a corresponding flow valve 66 to open the valve and effect a flow of the reagent or solvent through the continuous manifold 68 and conduit 40 to the inlet passage of the chamber. The valve 66 is opened for a predetermined period of time to allow exactly the desired amount of reagent or solvent to pass and is then closed by applying gas pressure to its diaphragm. Vacuum from the auxiliary vacuum source is then applied to the diaphragm with the valve 72 at the far end of the continuous manifold 68 for flushing the manifold from any remaining reagent or solvent and supplying it completely to the chamber. The valve 72 is then closed by applying an overpressure to the diaphragm of the valve, leaving the manifold 68 free of solvent and reagent in preparation for the next supply step.

The diaphragm valve 80 is generally kept open during passage of the various reagents and solvents through the chamber 34 to direct the effluent therefrom through the chamber outlet passage and collection line 84 to the waste liner 60. to the vacuum pump 90. Alternatively, the chamber and the sample therein can be dried at suitable times by passing an inert gas through the chamber through the valve 72. Gas leaving the chamber can then be fed to the waste trap 60 or, if desired, discharged with the vacuum pump 90. to speed up the drying process.

After completion of the coupling and cleavage steps, the extraction solvent S3 is fed to the reaction chamber 34 to dissolve the cleaved amino acid derivative formed during the degradation cycle and to supply the solution to the conversion flask 14 through line 86 and valve. 43 82 remains stangda. The conversion reagents R4 and R4A (if used) and the release agent S4 can then be supplied to the conversion flask 14 at appropriate times by opening corresponding valves 112 to supply the liquids through the collection line 114 and line 104 to the conversion flask. Each supply is colored by the pressurizing and venting operations described above in conjunction with the supply of the other reagents and solvents and followed by opening the valve 118 to flush the collection line 114 and line 104.

The fraction transferred to the conversion flask 14 is the anilinothiazolinone derivative of the N-terminal amino acid of the protein or peptide sample and is automatically converted during the next coupling and cleavage cycles in the reaction chamber 34 to the stable phenylthiohydantoin amino acid, in part in accordance with the above articles. Liebold. Generally, the amino acid fraction in the conversion flask can first be fed by passing inert gas. Over the solution through the short capillary tube 276 and bubbling inert gas through the liquid using the capillary tube 272, followed by applying a vacuum through the tube 268. line 104 and one of the valves 112 (see Figure 3) in the desired amount. Rapid evaporation in the conversion flask after the desired conversion time can be accomplished by simultaneously applying vacuum to the conversion flask generator 268 and inert gas through the capillary tubes 272 and 276. To further stabilize the acid-bearing side chains of the Pth-asparagine and Pth-glutamic acids in the conversion piston generates the introduction of reagent R4A into the capillary tube 276 via line 104 and one of the valves 112 in the desired amount. Feeding in the conversion flask is accomplished again by applying vacuum through the tube 268 and feeding the inert gas to the capillary tubes 272 and 276. The Pth amino acid remaining in the conversion flask is then redissolved in the solvent S4 introduced through line 104 to transfer the fraction to the fraction. The transfer of the fraction is effected by opening the valve 131, which is connected to the long central capillary tube 272, in the conversion piston and inlaying inert gas under pressure through the capillary tube 276 to push the fraction away from the piston. .

The vial carousel has the fraction collector 16 rotated at 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 decomposition cycle, the fractions in the fraction collector can be further dried by opening the valve 98 to vacuum or opening the vanes 128 and 94 to supply inert gas from the boiler 28 over the fractions and finally to the waste trap 60.

The various components of the apparatus 10 are preferably made of materials which are substantially inert and are highly resistant to degradation. SAdana materials include borosilicate glass, certain fluorocarbon polymers and in some cases stainless steel as well as aluminum. The tightly fitting structural members and other elements of the apparatus 10 are constructed so that they can be made almost entirely of these materials. The resulting apparatus is probably the cleanest and most contamination-free system that can be obtained and can operate in this condition indefinitely.

The various struts performed with the apparatus 10 in a typical degradation and conversion cycle are listed in Table I. The duration of each step and the functional state of the apparatus under each strut are indicated. The functions shown correspond to the condition of the various valves in the device and the markings in the columns indicate when suitable valves are open. Salunda, for example, the columns "R1" "R2" "R3" 9R3A "" S1 "" S2 "and" S3 "denote the state of the different pairs of valves 58 and 62 for selectively applying pressure and establishing a dynamic equilibrium state in the reservoirs 42 to 54. When a mark occurs in one of these columns, valves 58 and 62, which are associated with the reservoir in question, are open, either in preparation for or during delivery of the reagent or solvent to the reaction chamber, the valves remaining closed at all times in Other Similarly, the "argon" column shows the state of the valve 72 for supplying an inert gas, such as argon, to the reaction chamber through the manifold 68, the "supply" column shows the state of the valve 66 corresponding to any reservoir pressurized thereon. time, and the columns designated "waste", "collection" and "vacuum" show the state of the valves 82, 78 and 80, respectively. As described above, each step is preceded by supply of solvent or reagent by pressurizing the solvent or reagent reservoir followed by the introduction of inert gas through the valve 72 to complete the supply of the solvent or reagent and cleaning the supply lines.

Columns "R4" "R4A" and "S4" denote the state of the various pairs of valves 108 and 110 to selectively pressurize and achieve a dynamic equilibrium in reservoirs 100 to 102 and the "feed / argon" column denotes the state of both the valve 122, which supplies inert gas to the conversion piston through the line 124, and the valve 112, which corresponds to the container which was put under pressure at the time in question. The columns "argon", "waste 1", "vacuum", "collection" and "waste 2" show the condition of valves 118, 92, 96, 131 and 125.

. Of the accumulated fraction collector fractions listed, the columns "argon", "waste" and "vacuum" show the state of the valves 128, 94 and 98.

With the exception set forth above, the sequence of steps listed in Table I is substantially consistent with the Edman degradation process described in the cited publications and is not discussed in detail. Any deviations from general practice are apparent from the names of the steps and the corresponding functions; ■■ •• ■ 1,4, 4 1. • •• • - • • • a • 4 46 technical conditions listed in Table I.

In practice, the following variations of the sequence program according to Table I can be used to adapt the program to the needs of any particular user: Steps 18 to 58 can be looped one or two gings in the first sequence cycle to ensure complete coupling of all amino groups in the protein.

Step 65 can be followed by feeding for 20-60 seconds time of the water (R3A) through the reaction chamber to reduce the dehydration of the side chains has serine and threonine.

Step 65 can be followed by the addition of 0.05 ml of 1N hydrochloric acid in methanol (R4A) to the conversion flask for methylation of the side chains of aspartic acid (Doh glutamic acid. If this is carried out, S4 is preferably methanol).

Step 76 can be followed by the addition of 0.7 to 1 ml of S (acetonitrile or methanol), which is then fed to the effluent to thoroughly clean the conversion flask.

Step 8 may be given an increased duration (500-1000 seconds) to promote cleavage of amino-terminal proline residues.

The nature of the present invention is further elucidated with the following specific examples of practical application of the invention. It should be noted that the stated values are only examples of processes which have been carried out with the device and the method according to the invention and are not intended to limit the scope of the invention.

The amino acid sequences set forth in the following examples are tabulated in the one-letter amino acid code, which is defined as follows: 4 .0 • -) 4, '.; dw • s • • r '4.7 A - alanine L - leucine R - arginine K lysine N asparagine N methionine D - aspartic acid F - phenylalanine C cysteine P - proline E glutamic acid S serine Q glutamine T threonine G - glycine W tryptophan - histidine Y - tyrosine I - isoleucine V - valine Example 1.

A solid matrix of polymeric quaternary ammonium salt, which is suitable for embedding a protein sample for sequencing, can be prepared on the fibrous layer elements 190 and 190 ', as described above, and a protein sample can be embedded ± the matrix in the following manner: A glass fiber board with a diameter of 12 mm and 0.25 to 0.5 mm thick is cut from a layer of commercially available glass microfiber filters from Whatman, Inc., Clifton, New Jersey, Afs The wafer is placed in the recess 192 and has the chamber member 134. 25 microliters of an aqueous solution containing 1.5 mg of 1,5-dimethyl-1,5,5-diazaundecamethylene polymethobromide and 0.0033 mg of glycylglycine are dropped onto the fiberglass wafer from a syringe or pipette. The water is evaporated in vacuo or by heating in a stream of hot nitrogen gas. The remainder of the chamber apparatus 12 is assembled and installed in the apparatus 10. The protein sequencing program was initiated at step 18, and 4 to 6 complete degradation cycles are performed to remove contaminants from the 1,5-dimethyl-1,5,5-diazaundecamethylene polymetobromide which can chemically react with protein pro. or in another otherwise large Edman process. The chamber apparatus 12 is partially disassembled and 25 microliters of a solution of the protein is dropped on the glass fiber board. The protein loading dissolves the 1,5-dimethyl-1,5,5-diazaundecamethylene polymetobromide and the liquid is removed by evaporation, leaving a thin film of 1,5-dimethyl-; 48 -1,5-diazaundecamethylene polymetobromide with the protein sample embedded in it. If the initial protein sample volume is greater than 25 microliters, the sample can be piped in divided pray volumes of 25 microliters, the liquid being removed by lining between the piping of these liquid volumes. The chamber apparatus 12 is reassembled and re-installed in the device 10. The protein sequencing program was then restarted at step 18 and performed as many degradation cycles as desired.

Example 2.

A solid matrix of polymeric quaternary ammonium salt, as an apparatus for embedding a protein sample for sequencing, can be prepared in the inner cradles of a glass capillary vessel and a protein sample can be embedded in the matrix in the following manner: The chamber element 324 or the chamber elements 134 'and 136' 132 and the lid 160, as shown in the figures, to form a compact cartridge subassembly, which is easy to handle for insertion of the matrix and the sample. The subunit is leveled horizontally and 10 microliters of an aqueous solution containing 0.6 mg of 1,5-dimethyl-1,5,5-diazaundecamethylene polymetobromide and 0.0013 mg of glycylglycine are injected into the reaction chamber of a syringe.

The chamber 320 is preferably designed to have a diameter of 1.6 mm at the biting spirits and a diameter of 3.2 mm at the center, which provides a chamber that can hold up to 25 microliters () al) of solution in a horizontal condition without spillage at the spirits. This design of the chamber 324 containing a solution in horizontal condition is shown in Figure 18B, with the sleeve 132 and associated structural members omitted to make the figure simpler. The subassembly is then rotated about its axis, while the liquid in the reaction chamber is fed by a stream of air or nitrogen is directed through the chamber so that a film of 1,5-dimethyl-1,5-diazaundecamethylene polymetobromide is left on the chamber cradles. The subassembly is then installed in the apparatus 10, • 11. • • 49 the protein sequencing program was started at step 18, and 4 to 6 complete degradation cycles are performed. The subassembly is then removed from the apparatus 10 and Miles horizontally, while 10 microliters of the protein solution is drained into the reaction chamber from a syringe. The subassembly is re-circulated around its axis, while the liquid in the reaction chamber is evaporated with a stream of nitrogen directed through the chamber, leaving a thin film of 1,5-dimethyl-1,5,5-diazaundecamethylene polymetebromide with the pretein sample embedded therein as shown in FIG. Figure 18C. The subassembly is then reassembled in the apparatus 10 and the protein sequencing program was initiated at step 18 after performing as many degradation cycles as desired.

Example 3.

Angiotensin, 0.0005 mg, contained in 25 microliters of 20% aqueous formic acid was embedded in a solid matrix of precycled 1,5-dimethyl-1,5,5-diazaundecamethylene polymetobromide by the method of Example 1. The angiotensin was subjected to eight cycles of Edman. degradation and the phenylthiohydantoin amino acids formed in each cycle were analyzed by high pressure water chromatography according to the method described by Johnson et al, "Analysis.of Phenylthiohydrantoin Amino Acids by High Pressure Liquid Chromatography on Du Pont Zorbax CN Columns", Anal. Biochem. 100, 335 (1979). The following sequence, which is set forth herein as "experimental", is obtained. The kanda sequence is indicated dven for comparison. Experimental: D-R-V-Y-I-H-P- F Kand: D -R-V-Y-I-H-P- F This result is significant, since it shows that even small peptides can be sequenced in the method according to the invention. The ability to sequence small peptides is due to the fact that the sample is embedded in a film for retention in the stable to be adsorbed directly on a bar surface and can therefore be held securely regardless of size. While sorptive bonds involve a large number of non-covalent effects between the sample and the water and there depending largely on the size of the molecules, the retention form of the film used according to the invention is relatively unaffected by the sample size.

Example 4.

Spermacetival-apomyoglobin, 0.01 mg, in 25 microliters of 20% aqueous acetic acid was immersed in a solid matrix of precycled 1,5-dimethyl-1,5,5-diazaundecamethylene polymetobromide by the procedure of Example 1. Apornyoglobin was subjected to 40 cycles of Edman degradation and the phenylthiohydantoin amino acids were analyzed according to the method of Example 3 to give the sequence set forth in the following as "experimental". The known sequence of spermacetival-apomyoglobin is also set forth below for comparison. 1 Experimental: V-L-S-E-G-E-W-Q-L-V-L-H-V-W-A Degree: V-L-S-E-G-E-W-Q-L-V-L-H-V-W-A-16 K-V-E-A-D-V-A-G-H-G-Q-D-I-L-I-K-V-E-A-D-V-A-G-H-G-Q-D-I-L-I-R-L-F-K-S-H-P-E-T-L-R-L-F-K-S-H-P-E-T-L Example 5.

A Drosophila melanogaster larval cuticle protein of previously unknown structure, 0.01 mg, in 25 microliters of an aqueous solution of 0.1% sodium dodecyl sulfate and 0.05M ammonium bicarbonate was immersed in a solid matrix of precycled 1,5-dimethyl-1,5-diazaundecamethylene. polymetobromide by the method of Example 1. The cuticle protein was subjected to 36 cycles of Edman degradation and the phenylthiohydantoin amino acids were analyzed by the method of Example 3 and gay the sequence set forth below as "experimental". • • • • 12 it • •. •. • • It • • .4.4 •. •• 51 1 Experimental: N-A-N-V-E-V-K-E-L-V- 11 -D-V-Q-P-D-G-F-V-S- 21 -L-V-L-D-D-G-S-A-S- 31 S = A-T-G-D-I- Example 6.

An outer skin protein of Drosophila melanogaster larvae of previously unknown structure, 0.005 mg, in 10 microliters of an aqueous solution of 0.1% sodium dodecyl sulfate and 0.05M ammonium bicarbonate was embedded in a solid matrix of precycled 1,1-dimethyl-1,5,5-diazaundecamethylene polymetobromide by the method of Example 2. The epidermal protein was subjected to 24 cycles of Edman degradation and the phenylthiohydantoin amino acids were analyzed by the method of Example 3 and gay the sequence hereinafter referred to as "experimental". Experimental: N-A-N-V-E-V-K-E-L-V-11 -D-V-Q-P-D-G-F-V-S-21 This is important because the most general method for isolating small amounts of medium to large proteins or peptides for analysis, such as polyacrylimide gel electrophoresis, provides samples in a solution of sodium dodecyl sulfate. Ordinary detergents cause the samples to be washed out of devices based on adsorptive bonding of the sample to a substrate surface, but the solid matrix of the invention is unaffected by the presence of sodium dodecyl sulfate.

From the above, it can be seen that the present invention provides an improved apparatus and method for sequentially performing chemical processes on a sample of very small size by using minimal amounts of reagents and solvents and relatively short cycle times. the following are a number of preferred embodiments.

An apparatus for carrying out a sequence of chemical processes on a ploy of chemical material, characterized in that the apparatus comprises: chamber means having an inner surface defining a reaction chamber, the chamber being provided with inlet means and outlet means, means for sequentially passing a plurality of fluids through the chamber from the inlet to the outlet as a pressurized stream and a solid matrix which permeate these fluids and arranged in the chamber, so that a prayer embedded in the matrix is stirred and exposed to each of these fluids which pass through the chamber father chemical inboard impact.

Device according to embodiment 1, characterized in that the solid matrix is permeable to fluids by absorption of fluids in the matrix and diffusion of fluids through it.

Device according to embodiment 2, characterized in that the chamber means comprise surface-supporting means which support the solid matrix as a thin film on the surface.

Device according to embodiment 3, characterized in that the solid matrix comprises a polymeric quaternary ammonium salt.

Device according to Embodiment 4, characterized in that the polymeric quaternary ammonium salt comprises 1,5--dimethyl-1,5,5-diazaundecamethylene-polymethobromide. 441: • • • • • • • •• 444: .4 •••• 4.9 P14 '4 • 4 0; 4 ... ■ •• 53 Device according to embodiment 4, characterized in that the polymeric quaternary ammonium salt comprises poly - (N, Nedimethyl] -3,5-dimethylene-piperidinium chloride).

Device according to embodiment 3, characterized in that the surface-forming means which supported the matrix comprise at least a part of the inner surface.

Device according to embodiment 7, characterized in that the reaction chamber, which is defined by the inner surface, comprises a simple passage of capillary type which extends between the two spirits.

Device according to embodiment 8, characterized in that the diameter of the capillary passage increases uniformly in the direction of food from the two spirits.

Device according to embodiment 7, characterized in that the chamber comprises a pair of abutting chamber elements with a first resp. a second cavity on opposite mating surfaces thereof, the first and second cavities being aligned in line I relative to each other so as to form the reaction chamber.

. Device according to embodiment 3, characterized in that the surface-forming means, which support the matrix, comprise porous layer-shaped means which allow the passage of fluids.

Device according to embodiment 11, characterized in that the porous layer-shaped member extends substantially transversely over the chamber. Device according to embodiment 12, characterized in that the porous layer-shaped member comprises a layer made of a plurality of fibers. 14. Device according to Embodiment 13, characterized in that the fibers are glass fibers. 54. "".

Device according to Embodiment 12, characterized in that the chamber means comprise a pair of abutting chamber elements with a first half-space rasp. a second cavity on opposite mating surfaces thereof, the first and second cavities being aligned with each other and forming the reaction chamber.

Device according to embodiment 15, characterized in that the solid matrix comprises a polymeric quaternary ammonium salt.

Device according to embodiment 15, characterized in that the first cavity and the second cavity are formed conical oils tapered away from the mating surfaces of the lid, at which the cavities communicate with the inlet means resp. the outlet means.

Device according to embodiment 15, characterized in that the porous layer-shaped member is received or held in a recess in at least one of the mating surfaces for retention in the chamber in an orientation which substantially separates the first cavity from the second cavity.

Device according to embodiment 18, characterized in that the chamber means comprise at least one layer of resilient material, which is laminated between the mating surfaces in a sealing manner, the resilient material being permeable to the fluids used.

Device according to embodiment 19, characterized in that the chamber means comprise one of the layers of resilient material arranged on each side of the porous layer-shaped member.

Device according to embodiment 20, characterized in that at least one of the chamber elements comprises a raised part on its mating surface, which extends around the cavity in the element for compressing at least one layer of resilient material against the mating surface of the second chamber element for improving the seal.

Device according to embodiment 18, characterized in that the outlet and the inlet comprise a pair of capillary passages which extend through the rasp, chamber elements and communicate at the inner ends with the reaction chamber on opposite sides of the porous layer-shaped member.

Device according to embodiment 22, characterized in that the capillaries are coaxial with the reaction chamber and extend from this to the outer capillary openings of the chamber elements.

Device according to embodiment 23, characterized in that the chamber elements are challenged by glass and have substantially flat surfaces adjacent to the capillary openings.

Device according to embodiment 24, characterized in that the means for sequentially fluidizing the gene chamber comprise at least one piece or a plug of resilient material with a substantially flat-gripping spirit and an axial capillary passage terminating in an opening at this spirit, saint screwed means for pressing the tapping spirit axially against one of the capillary openings, said that the axial passage in the piece or plug communicates with the capillary opening and the tapping spirit ay the plug or piece tilts against the substantially flat surface of the chamber element therein.

Device according to embodiment 25, characterized in that the piece or plug of elastically resilient material comprises a mass of fluorocarbon polymer enclosed in a metal sleeve.

Device according to Embodiment 26, characterized in that the screw-threaded members comprise a hollow which, with a tight fit, receives the metal sleeve so that it is axially displaceable in the 1-151 for securing the precise alignment of the passage. • • •••• 411 • 11: 56 the passage in the plug with the capillary opening.

Device according to embodiment 3, characterized in that it comprises means for automatically regulating the function of the device during a plurality of cycles.

Device according to embodiment 1, characterized in that the means for sequentially conveying a plurality of fluids through the chamber comprise at least one valve device for regulating the flow of these fluids between the chamber and a plurality of fluid container means, the valve device comprising: valve blocks with a plurality of substantially flat valve layers on one of the surfaces of the valve block, the valve block comprising a primary passage extending continuously between the two spirits of the block and communicating through primary openings with each of the valve stalls, a plurality of secondary passages, each communicating through a secondary opening with one of the valve stalls, a plurality of resilient, substantially impermeable diaphragms which engage the respective valve stalls, each of these diaphragms being maneuverable between a first acting state in which it is pressed against one of the valve stalls for closing the primary and secondary openings communicating with the valve. and a second condition, in which it is held away from the valve housing to provide a fluid flow path between the primary and secondary openings above the outer surface of the valve block, and means for operatively connecting the primary and secondary passages to the fluid container means and the chamber, the fluid flow between the fluid container means and the chamber selectively regulated.

Device according to embodiment 29, characterized in that the connecting means comprise at least one tapered or conical sealing ring, which is arranged displaceably with sliding fit over a tube and is pressed against a recess with another conical or tapered shape in the valve block, which communicates with one of the passages, so that a directional force 57 applied to the sealing ring is focused on a relatively small contact surface between the sealing ring and the recess to provide fluid leakage between them.

Device according to embodiment 30, characterized in that the recess is dewormed conically with a larger cone angle on the sealing ring.

Device according to embodiment 30, characterized in that the sealing ring is formed conically on opposite sides, one of these sides being pressed against the recess with inclined members with a separate conical recess intended to receive the other side of the sealing ring, the recesses being formed conically with a larger cone angle. the cone angle has corresponding sides of the sealing ring.

Device according to embodiment 29, characterized in that the connecting means comprise a connecting nipple at one of the spirits of the primary passage, so that the primary passage acts as a collecting conduit which can be flushed with a stream of fluid between the connecting nipple and the secondary passage farthest from the connecting nipple.

Device according to embodiment 33, characterized in a drive drive, in that the primary passage comprises a plurality of straight or simple passages connected end to end to a conduit of sawing shape, which in alternating cuts between these passages communicates with the respective valve position.

Device according to embodiment 1, characterized in that it comprises: a conversion piston with a plurality of capillary tubes extending into the interior of the piston, and means for introducing and discharging various fluids to and from the piston through the capillary carrier, comprising at least one of the plurality of fluids. , which is passed to the outlet means, at least one of these capillary tubes having an inner spirit at which the tube of the tube is closed and which is provided with a plurality of radially distributed openings of limited size, so that passage of fluids through at least one of the capillary tubes provides spraying on the inner walls of the piston for cleaning of these inner cradles.

Device according to embodiment 35, characterized in that the capillary tubes are made of glass.

An apparatus according to embodiment 1, characterized in that it comprises: a conversion piston with a plurality of capillary tubes extending into the interior of the piston, and means for introducing and discharging from the piston different fluids through the capillary tube, including that at least one of these a plurality of fluids are fed to the outlet means, one of these capillary tubes extending to a point near the bottom of the piston in an inner spirit, at which the tube of the tube is closed, the inner end being provided with a plurality of radially distributed openings of limited size, so that the passage of a gas inwardly through this capillary tube produces a plurality of small bubbles in a liquid ± the flask, so that the bubbles cause a uniform agitation.of the liquid and, accelerates drying thereof.

Reaction chamber apparatus for use in an apparatus for sequencing chemical processes on a sample of chemical material, characterized in that it comprises: chamber means having an inner surface defining a reaction chamber, said chamber being provided with inlet means and outlet means for conducting fluids. through the chamber in a pressure-actuated strain, and a solid matrix which is permeable to a plurality of fluids and is arranged in the chamber, so that a pray embedded in the matrix is unsteady and exposed to the plurality of fluids passed through the chamber for chemical reaction or impact. ddrmed. Device according to embodiment 38, characterized in that the solid matrix is permeable to the diversity of fluids by absorption of fluids in the matrix for diffusion of fluids therethrough.

Arrangement according to embodiment 39, characterized in that the chamber means comprise surface-forming means which support the solid matrix as a thin film.

Device according to embodiment 40, characterized in that the solid matrix comprises a polymeric quaternary ammonium salt.

Device according to embodiment 41, characterized in that the polymeric quaternary ammonium salt comprises 1,5-dimethyl-1,5,5-diazaundecamethylene polymetobromide.

Device according to embodiment 41, characterized in that the polymeric quaternary ammonium salt comprises poly- (N, N-dimethyl-3,5,5-dimethylene-piperidinium chloride).

Device according to embodiment 40, characterized in that the surface-forming means which supported the matrix comprise at least a part of the inner surface.

Device according to embodiment 44, characterized in that the reaction chamber, which is defined by the inner surface, comprises a simple passage of the capillary type which extends between two spirits of the drive.

Device according to embodiment 45, characterized in that the capillary-type passage here has a diameter which increases uniformly in the direction of food from the two teeth.

Device according to embodiment 44, characterized in that the chamber comprises a pair of abutting (abutting) chamber elements with a first cavity resp. a second cavity on opposite surfaces, the first cavity 60 and the second cavity 60 being aligned with each other and forming the reaction chamber.

Device according to embodiment 40, characterized in that the surface-forming means comprise porous layer-shaped means, which facilitate passage of fluids through the layer.

Device according to embodiment 48, characterized in that the dot-shaped layer-shaped members extend substantially in the transverse direction across the chamber.

Device according to embodiment 49, characterized in that the porous layer comprises a layer made of a plurality of fibers, to which the sample is adhered.

Device according to embodiment 50, characterized in that the fibers are made of glass.

Device according to embodiment 49, characterized in that the chamber comprises a pair of abutting chamber elements with a first cavity resp. a second cavity, on opposite mating surfaces, the first cavity and the second cavity being aligned relative to each other and forming the reaction chamber.

Device according to embodiment 52, characterized in that the solid matrix comprises a polymeric quaternary ammonium salt.

Device according to embodiment 52, characterized in that the first cavity and the second cavity are formed tapered or conical in directions away from the opposite surfaces to the stand, where they are in communication with the inlet means rasp. the outlet means.

Device according to embodiment 52, characterized in that the porous layer is applied in a recess in at least one of the opposite surfaces for retention ± the chamber in a layer 61 which substantially separates the first cavity and the second cavity.

Device according to embodiment 55, characterized in that the chamber means comprise at least one layer of resilient material, which is applied between the opposite surfaces in a sealing manner, the resilient material being permeable to the diversity of fluids.

Device according to embodiment 56, characterized in that the chamber comprises a layer of resilient material applied to each side of the porous layer-shaped member.

Device according to embodiment 56, characterized in that at least one of the chamber elements comprises a raised part on the opposite surface, which extends around the cavity for compressing at least one layer of resilient material against the opposite surface of the second chamber element for improving the seal.

Device according to embodiment 55, characterized in that the inlet and outlet means comprise a pair of capillary passages, which extend through the respective chamber elements, and at the inner spirits communicate with the reaction chamber on opposite sides of the porous layer.

Device according to embodiment 59, characterized in that the capillary tubes are coaxial with the reaction chamber and extend from this to outer capillary openings in the chamber elements.

Device according to embodiment 60, characterized in that the chamber elements are made of glass and have substantially flat surfaces adjacent to the capillary openings. 62. Device according to embodiment 61, can be drawn & dray, that the inlet and outlet means further comprise at least one plug of resilient material with a substantially flat surface. • • • • • • ••• •• 1 ••• 62. 62 and an axial capillary passage, which terminates with an Opp-fling at this arid, and screwed means arranged to pull the tapping spirit axially towards one of the capillary openings, so that the axial passage in the plug communicates with the capillary opening and the tapping Spirit of the plug tilts against the substantially flat surface of the chamber element therein.

Device according to embodiment 62, characterized in that the plug of resilient material comprises a mass of fluorocarbon polymer enclosed in a metal sleeve.

Device according to embodiment 63, characterized in that the screw-threaded member comprises a hall in which the metal sleeve fits snugly with axial slidable displaceability for securing accurate alignment in line with the passage in the plug and the capillary opening. Valve device for use in a device for performing sequential chemical processes for regulating the flow of fluids therein, characterized in that the valve device comprises: valve block means having a plurality of substantially planar valve stalls on a surface of the valve block, the valve block comprising a primary passage extending continuously between the valves of the valve block and communicating with prime droplets with each of the valve stalls, saint a plurality of secondary passages, each of which communicates through a secondary opening with one of the valve stalls, and a plurality of resilient substantially impermeable diaphragms valve stall, each of these diaphragms being actuated between a first acting state, in which it is pressed against one of the valve stalls for closing the primary and secondary openings communicating with the valve stall, and a second condition in which it is withdrawn from the valve stall. and provides a fluid flow path between the primary and secondary openings across the outer surface of the valve block so that the fluid flow between the primary passage and the secondary passages can be selectively controlled. Valve device according to embodiment 65, characterized in that it comprises means for providing fluid connection in the passages to outer elements, which connecting means comprise at least one conical sealing ring, which is arranged with a tight fit and displaceable on a pipe and pressed against a recess with another conical shape. in the valve block, which is connected to one of the passages, so that a directional force applied to the sealing ring is focused on a relatively small contact area between the sealing ring and the recess to provide a fluid supply between them.

Valve device according to embodiment 66, characterized in that the recess is formed conically tapered with a larger apex angle of the sealing ring.

Valve device according to embodiment 66, characterized in that the sealing ring is formed conically tapered on opposite sides, one of these sides being pressed against the recess with screw-threaded means, which are provided with a separate conical tapered recess intended to receive the other side of the sealing ring, these recesses are conical with a larger cone angle than the cone angle has corresponding sides of the sealing ring.

Valve device according to embodiment 65, characterized in that the connecting means comprise a nipple at one of the spirits of the primer passage for connecting the primer passage to another part of the device, so that the primary passage acts as a manifold which can be flushed with a flow of fluid between the nipple and the secondary passage. as dr beldgen furthest from the nipple.

Valve device according to embodiment 69, characterized in that the primer passage comprises a plurality of straight passages, which are connected spirit to spirit and form a conduit with a sawtooth shape and which in alternating cuts between. • • 1 •• 64 these communicate with the respective valve stall. 71.

Conversion piston for use in a device for carrying out a sequence of chemical processes on a prey of chemical material, characterized in that the conversion piston comprises: piston means at a plurality of capillary tubes extending into the interior of the piston for introducing and discharging various fluids into and out from the piston, wherein at least one of the capillary tubes has an inner spirit, at which the capillary tube tail is closed and is provided with a plurality of limited outlet openings arranged with radial distribution, so that passage of fluid food through this capillary tube causes spraying against the inner rocks of the piston for clean of the inner cradles.

Conversion piston according to embodiment 71, characterized in that the capillary tubes are challenged by glass.

Conversion piston for use in an apparatus for performing sequential chemical processes on a sample of chemical material, characterized in that the piston comprises: piston means having a plurality of capillary tubes extending into the interior of the piston for introducing and discharging various fluids into respectively out of the piston, one of these capillary tubes extending to a point adjacent the ends of the piston with the inner spirit, at which the tube of the tube is closed, this inner spirit being provided with a plurality of outlet openings of limited size, which are radially distributed, so that the passage of a gas inwardly through this capillary tube causes the introduction of a plurality of small bubbles into a liquid flask, the bubbles causing agitation of the liquid or accelerating drying thereof.

Process for carrying out in sequence of chemical processes on a pray of chemical material, characterized in that enclosing a solid matrix of fluid permeable material in a closed chamber with inlet and outlet, embedding a sample of chemical material in the solid matrix and sequentially a plurality of fluids through the chamber in a pressurized stream from the inlet to the outlet, so that the sample is exposed to each of these fluids, so that the sample is kept intact and chemical reactions between the sample and these fluids are obtained.

A method according to embodiment 74, characterized in that the step of enclosing the solid matrix in the chamber comprises introducing material containing the solid matrix into the chamber and then rotating the chamber, while passing a gas through the chamber to evaporate the liquid. leaving the solid matrix material on the inner cradles of the chamber as a thin film.

A method according to embodiment 74, characterized in that the step of embedding the sample of chemical material in the solid matrix comprises introducing a solution containing the sample into the chamber to dissolve the matrix material therein and then causing the chamber to rotate while a gas through the chamber for scavenging the liquid leaving the solid matrix material on the inner rocks of the chamber as a thin film with the sample embedded therein.

Method according to embodiment 74, characterized in that the solid matrix is carried on a porous layer and that the step of enclosing the matrix in the chamber comprises applying the matrix material to the porous layer as a thin film thereon and arranging the porous layer substantially across the layer. p5 a stable between the inlet and the outlet, so that the fluids passing from the inlet to the outlet must pass through the layer. 66 A method according to embodiment 77, characterized in that the step of embedding the sample in a solid matrix comprises such a stay that a solution containing the sample is applied to the porous layer to dissolve the matrix material thereon, the liquid evaporates from the layer to leave the base material. thin film with sample embedded in it.

A method according to embodiment 74, characterized in that the strut, in which one sequentially flows through the chamber, comprises as strut that at least one reagent is passed through the chamber in gaseous or vapor form for diffusion into the solid matrix and reaction with tried. ly * 1> • • •• • • • 41

Claims (13)

A valve device for use in a device for carrying out in sequence chemical processes for regulating the flow of the fluid idea, characterized in that the valve device comprises: valve block means with a plurality of substantially planar valve stalls on a surface of the valve block, the valve block comprising a primary passage, extending continuously between the two spirits of the valve block and communicating through primary openings with each of the valve stalls, and a plurality of secondary passages, each of which communicates through a secondary opening with one of the valve stalls, the passage comprising a plurality of straight passages connected spirit to spirit, so that they form a channel with a sawtooth shape and communicate at alternating sections with the respective valve stall, a vacuum-forming means, a pressure-retaining means and a plurality of resilient, substantially impermeable diaphragms, which thank the respective valve stall, each of these diaphragms being maneuvers between an initial state, in which it is pressed against one of the valve stalls for closing the primary and secondary openings communicating with the valve body, and a second state, which is subtracted from the valve body by the vacuum generating means and provides a substantially uniform fluid flow path. between the primer and second passages over the outer surface of the valve block, so that the fluid flow between the primary passage and the secondary passages is selectively regulated. 2. A valve device according to claim 1, characterized in that it comprises means for providing fluid connection of the passages to outer elements, which connecting means comprise at least one conical sealing ring, which is arranged with a tight fit and displaceable on a tube and pressed against a recess with another conical shape in the valve block, which communicates with one of the passages, said that a directional force applied to the sealing ring is focused on a relatively small contact area between the sealing ring and the recess to provide a fluid seal between them. 3. A valve device according to claim 2, characterized in that the recess is formed conically tapered with a larger apex angle of the sealing ring. Valve device according to claim 2, k 6 n n e 1. Drawing ring, in which the sealing ring is formed conically tapered on opposite sides, one of these sides being pressed against the recess with screw-threaded means, which are provided with a separate conical tapered recess intended to receive the other side of the sealing ring, these recesses being formed conical with a larger cone angle than the cone angle has corresponding sides of the sealing ring. S.Valve device according to claim 1, 2. The drawing means comprises that the connecting means comprise a nipple at one of the spirits of the primary passage for connecting the primary passage to another part of the device, so that the primary passage acts as a manifold which can be flushed with a flow of fluids between the nipple and the secondary passage furthest from the nipple. SUMMARY Valve device for use in a device for carrying out a sequence of chemical processes for regulating the flow of fluid, characterized in that the valve device comprises: valve block means having a plurality of substantially planar valve stalls 0 a surface of the valve block, the valve block comprising a primary passage , which extends continuously between the two spirits of the valve block and through primary openings communicates with each of the valve stalls, and a plurality of secondary passages, each of which communicates through a secondary opening with one of the valve stalls, the primary passage comprising a plurality of straight passages connected to the end. , say that they form a channel with a sawtooth shape and communicate at alternating sections with the respective valve stall, 3. 00 IP 4 • • 4. 111, 5. 4, 6. • • 7. • • 8. 0 9. 10. • at • I. A vacuum-forming means, a pressure-forming means and a plurality of resilient, substantially impermeable diaphragms which engage the respective valve stall, each of these diaphragms being maneuverable between a first acting state, in which it is pressed against one of the valve stalls. for closing the secondary openings communicating with the valve housing, and a second condition in which it is withdrawn from the valve housing by the vacuum forming means and provides a substantially uniform fluid flow path between the primary and secondary openings across the outer surface of the valve block. the secondary passages can be selectively regulated. 1 28 4-51 12. V. • r a • 13. • S • CZZ1 C: 1 CD t_ / 0/2 24