SE501968C2 - conversion Piston - Google Patents

Abstract

An improved apparatus and method for the sequential performance of chemical processes on a sample of chemical material wherein the sample is embedded in a solid matrix (194) of fluid permeable material, eg polymer material, located within a reaction chamber and is sequentially subjected to a plurality of fluids passed through the chamber in a pressurized stream, causing chemical interaction between the sample and the fluids. The matrix may be supported on a porous layer (190) across the chamber or may be applied to the chamber walls. Also described are a system for automatically performing sequential degradation analysis of protein or peptide chains and valve means (comprising a valve block (182) having a plurality of membrane- operated valve sites (78-82)) and a conversion flask for use in such a system. <IMAGE>

Description

U.S. Patent No. 3,959,307 to Inventors Wittmann-Lieb and Graffunder, May 25, 1976, for "Method to Determine Automatically the Sequence of Amino Acids"; Hunkapiller och Hood: "Direct Microsequence Analysis of Polypeptides Using an Improved Sequenator, A Nonprotein Carrier (Polybrene), and High Pressure Liquid Chromatography", Biochemistry 2124 (1978): Laursen, R.A., Eur. J. Biochem. 20 (l971); Wachter, E., Machleidt, H., Hofner, H. and Otto, J., FEBS Lett. 35, 97 (1973): U.S. Patent 3,725,010, applicant Penhasi, issued April 3, 1973, for "Apparatus for Automatically Performing Chemical Processes"; U.S. Patent 3,717,436, applicant Penhasi et al., Issued February 20, 1973, for " Process for the Sequential Degradation of Peptide Chains “; U.S. Patent 3,892,531 to Gilbert, issued July 1, 1975, to "Apparatus for Sequencing Peptides and Proteins"; U.S. Patent 4,065,412 to Dreyer issued December 27, 1977 to "Peptide or Protein Sequencing Method and Apparatus". . A further significant apparatus is described in U.S. Patent Application 106,828, issued December 26, 1979, to Leroy E. Hood and Michael W.

Female capsules, two of the inventors of the present invention, relating to the "Apparatus for the Performance of Chemical Processes".

As 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 Q-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 anilinothiazolinone. After extraction of the thiazolinone, the remaining peptide is ready for the next cycle of coupling and cleavage reactions. Aqueous acid is used to convert the thiazolinone to phenylthiohydantoin, which can be conveniently analyzed, for example by chromatography.

The automatic apparatus described in U.S. Pat. No. 3,501,968 patent 3,725,010, modified as set forth in the aforementioned articles by Wittmann-Liebold and U.S. Patent Application 106,828, Applicants Hunkapiller and Hood, relates to an 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 bowl", and arranged in a closed reaction chamber. Means are provided for introducing and removing controlled amounts of liquid reagents to and from the chamber for reaction with a sample of a protein or peptide in an inert atmosphere.

The sample to be analyzed is first applied to the rotating dish followed by sequential introduction and removal of various reagents and solvents required to perform the coupling and cleavage reactions. The liquid reagents and solvents themselves form films on the walls of the dish, which pass over and react with the sample film as the dish rotates. The reagents dissolve the sample film and effect 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 solvent to remove non-volatile components. After the post-cleavage evacuation, the resulting thiazolinone is extracted from the sample film with solvent and transferred either to a separate flask for carrying out the conversion step or to an apparatus for collecting and drying the various fractions. If the conversion reaction is not carried out immediately in a conversion flask, the process can be carried out later on several fractions simultaneously.

The introduction and removal of fluids relative to the rotating bowl has been accomplished with fluid conduits which pass through a plug which closes an opening in the upper wall of the reaction chamber and extends down therefrom to a location within the bowl. Fluids are introduced directly into the rotating bowl at a location adjacent to the bottom thereof and discharged from an annular groove in the cylindrical inner surface of the bowl. The fluid to be removed is forced into the annular groove by the centrifugal force, as the bowl is rotated at high speed, and discharged through a conduit having an inner end extending into the groove. This effluent line thus acts as a collecting means for removing reaction products and by-products and extracting solvents.

If the protein or peptide sample in a rotating dish device itself does not have sufficient mass to form a cohesive film per se, it is in some cases carried on the inner wall of the dish 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 having the chemical composition 1,5-dimethyl-1,5-diazaundecamethylene polymetobromide has been used for this purpose. The carrier must be applied in substantial amounts to securely retain the sample and the carrier and the sample are both dissolved by the liquid reagents to allow reaction between the sample and the reagents. Although devices of the rotating beaker type can give acceptable experimental results in many cases, they have many disadvantages. By way of 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 tend to remove portions of the sample from the film and wash them from the wall of the dish 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 5 '5o1 968 sample amount remains during the last cycle to give useful results. Devices of this type also have 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 bowl sequencers are relatively complicated and expensive 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 immobilized by covalent bonding to the surface of a plurality of small beads. The beads form a porous packing 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 hold the sample in place. However, covalent bonds are difficult to obtain in practice. The filled column is also difficult to wash and the beads in it have a tendency to decompose during use.

It has previously been proposed that sequencing devices be constructed to overcome the disadvantages of these devices by containing a sample in a stationary reaction chamber and subjecting the sample to the action of at least one gaseous or vapor reagent. The patents of Gilbert and Dreyer, as mentioned above, describe two such devices, none of which work completely satisfactorily.

The device in Gilbert's patent has a closed finger-shaped extension 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 extension is cooled internally to provide condensation on the extension. The elongation is then heated to dissolve the sample in the liquid and the reaction proceeds. After the reaction with the sample, the undesired semi-volatile chemicals can either be dried from the sample by a combination of heat and a stream of inert gas or washed from the extension together with the terminal amino acid with a solvent which condenses on the extension until it drips. of from this.

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 positions.

Unfortunately, the devices according to Gilbert's and Dreyer's patents do not provide a sufficiently pollution - free environment to give acceptable results during a large number of degradation cycles.

For example, it is difficult to wash the protein or peptide sample efficiently in the Gilbert and Dreyer devices.

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 filler used to retain the sample in the Dreyer reaction column 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 accomplished even when large amounts of solvent are used, as 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 located.

The fluid supply lines and flow valves of the Gilbert and Dreyer devices are also difficult to evacuate completely and tend to retain chemical residues, which can interfere with the intended chemical conditions in subsequent reaction cycles.

The glass or plastic beads used as a filler in the Dreyer reaction column also tend to decompose over a large number of degradation cycles, clogging the system to such an extent that the passage of fluids through it is impeded. This makes it almost impossible to wash the system between the cycles and the chemical environment 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 decomposition process. The sample and the reagents in the reaction cell thus become more and more contaminated, which prevents the desired coupling and cleavage reactions and causes a number of undesired reactions to take place. The yield from each complete cycle of the device is thus lowered 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 reasons, 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 overlooked 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 destructive in cases where the chain has a very large number of units or only very small amounts of protein or peptide are available. Both of these conditions exist in the case of interferon, a small protein produced in human cells in response to certain viral infections. Interferon has recently attracted considerable attention in clinical medicine because this substance promises to be an effective means of counteracting viral infections and it appears to offer considerable hope as an anti-cancer reagent. Interferon is produced and is thus only available in very small amounts. At present, essentially the whole world production of the two types of human interferon originates from the relatively few 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 well-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 the protein available.

After the first few cleavage cycles, the small sample may become contaminated to such an extent that positive results cannot be obtained.

The most sophisticated prior art device for the conversion of the various thiazolinones, which is cleaved from the sample to the more stable phenylthiohydantoins, is the conversion flask described in the above-mentioned Wittmann-Liebold articles, and modified in U.S. Patent Application 106,828, applicant Hunkapiller and Hood. According to the invention, however, it has been found that this piston suffers from inefficient washing of its inner walls 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 for washing the walls of the flask by sprinkling the liquid thereon, but this method causes an unreliable inflow of liquid to the flask and makes it very difficult to control the volume of liquid supplied. .

According to the invention, it has also been found that when the prior art conversion flask is designed on a substantially smaller scale to accommodate smaller volumes of liquid, it is difficult to obtain the optimum degree of distribution of inert gas bubbles in the liquid contents of the flask to stir the contents. during the conversion reaction and for evaporating the semi-volatile components thereof. Inert gas introduced into the bottom of the piston 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 splashing of the liquid to the top of the piston.

In many applications, therefore, it is desirable to provide a device for performing chemical processes, such as sequence reactions of proteins or peptides, that operate efficiently and with a minimum of system contamination to enable a maximum number of sequence cycles to be performed with good results with very little amount of sample.

SE-8105597-2 relates to a device comprising chamber means with an inner surface defining a reaction chamber, the chamber having inlet and outlet means for passing fluids into a pressurized stream, and solid matrix means which are permeable by diffusion. for a plurality of fluids and applied to the chamber, so that a sample embedded in the matrix means is immobilized 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 across the chamber and allow the passage of fluids. The board means may comprise a board made of a plurality of glass fibers. The chamber means may comprise a pair of abutting chamber elements with first resp. second cavities on opposite mating surfaces thereof, the first and second cavities being arranged in line with each other and forming the reaction chamber. The first and second cavities may be conically tapered in directions away from the opposite surfaces up to points at which they communicate with the inlet means resp. the outlet means. The porous layer, if 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, which is 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 for compressing the layer of resilient material against the opposite connecting surface of the chamber member to improve the seal.

The inlet and outlet means may comprise a pair of capillary channels extending through the respective chamber elements and communicating at the inner ends with the reaction chamber on opposite sides of the porous layer. The capillaries may be arranged coaxial with the reaction chamber and extend therefrom to outer capillary openings with substantially flat outer surfaces of 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 seats on the surface, the valve block member forming a primary passage which is interconnected between the two ends thereof and communicates through primary openings with each of the valve sites, and a plurality of secondary channels, each of which communicates through a secondary opening with one of the valve sites, and a plurality of elastically resilient, substantially impermeable diaphragms covering the respective valve sites, each diaphragm being adjustable between a first sealing position in which it is pressed against one of the valve sites to close the primary and secondary openings communicating at this valve site, and a second position in which the diaphragm is withdrawn from the valve site to provide a fluid flow path between the primary and secondary openings through the exterior of the valve site. valve block means, so that the fluid flow between the primary channel and the secondary channel can be selectively regulated. The connecting means may comprise at least one conically tapered tube (ferrule), which is arranged with a tight sliding fit over a tubular member and is pressed against a recess with another conical shape in the valve block means, which communicates with one of the channels therein, so that an inwardly directed force applied to this pipe ring is focused on a relatively small contact surface between the pipe ring and the recess to provide a fluid seal between them. The recess is preferably designed conically with a larger angle than the pipe ring. The interconnecting means may further comprise a fitting at one end of the primary duct for coupling the primary duct to another part of the apparatus, so that the primary duct acts as a main line, which can be flushed with a fluid flow between the coupling and the secondary duct furthest from the clutch. The primary channel may comprise a plurality of straight passages, which are connected end to end and form a conduit with a sawtooth shape and which communicate at alternating cutting points with the respective valve point.

The invention relates to a conversion piston for use in an apparatus for carrying out in sequence of chemical processes on a sample of chemical material, characterized in that the conversion piston comprises: piston means with at least three capillary tubes extending into the interior of the piston for insertion and discharging various fluids 1 and from the piston, and means designed for connection to 501 968 an external vacuum source, the piston means being provided with inner 12 walls, an upper end and a bottom, a first of the capillary tubes having an inner end which is closed and is provided with a plurality of limited outlet openings arranged with radial distribution, so that introduction of fluids through this first capillary tube causes spraying against the inner walls of the piston for downward washing of the inner walls, a second of these capillary tubes extends up to a point at the bottom of the piston with the inner end, at which the hole of the tube is closed, this inner end being provided with a a plurality of outlet openings of limited size, which are radially distributed so that a passage of a gas inwardly through this capillary tube causes the introduction of a plurality of small bubbles into a liquid in the flask, the bubbles causing agitation of the liquid or accelerating drying thereof, and a third of the capillary tubes extends to a point which is located above the point to which the second capillary tube extends and below the point to which the first capillary tube extends, so that chemical substances introduced through this third capillary tube and hit the walls of the flask is washed down by the liquid from the first capillary tube to the point where the second capillary tube extends.

One embodiment comprises that the capillary tubes are made of glass.

Furthermore, the flask may suitably have a volume of about 1 ml. It is also suitable that the means adapted for connection to an external vacuum source be located next to the upper end of the piston.

The plunger may further have an enclosed space extending around a substantial portion of its length, the enclosed space being adapted to receive a heating fluid which is passed to and from this space.

The conversion piston may further have a circular cross-section and be tapered from its upper end to its bottom.

It is an object of the present invention to enable chemical processes to be carried out in sequence on a sample 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 enable an economical device and an economical 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 enable an improved apparatus and method for sequentially carrying out chemical processes with a very short cycle time.

It is a further object of the invention to enable an improved apparatus and method for sequentially carrying out chemical processes on a sample, the sample being washed more efficiently between cycles.

The invention can facilitate solving according to SE-8105597-2 a number of problems with previously known sequencers by immobilizing the protein or peptide sample in a solid matrix, which is formed as a thin film which is permeable by diffusion to both reagents and solvents used. during the decomposition process. The difficult problem of achieving complete covalent bonding is thus avoided, as is 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 moving liquid phase. The sample is securely held in place by the matrix, while the minor 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 virtually any form of sample substrate can be used without causing loss of the sample. The inner walls of the reaction chamber itself can form a sufficient support surface.

A substrate surface that greatly facilitates complete washing of the system is a porous layer made of a plurality of overlapping glass fibers, which extend across a flow-through reaction chamber. The porosity is created by the gaps between the fibers. This structure has a relatively large total surface area with minimal extent 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 washing cycles to be carried out 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 vapor, permeate 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 washed thoroughly from residual reagents and reaction products with a relatively small amount of solvent.

The solvent only needs to move 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 the state for the next reaction step. 15 501 968 The low consumption of solvent not only saves on the cost of the solvent but also reduces the tendency of the sample to be washed from the reaction chamber and lost.

The use of gaseous or vapor reagents also helps to improve the exposure of the sample to the reagents and to minimize the amount of reagents required. Low reagent consumption is significant, as 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 SE-8105597-2 is designed to allow the passage of both gaseous and liquid reagents through the porous layer holding the sample without allowing the sample to become contaminated with external contaminants or with chemicals transferred from a reaction stage or cycle. to another. The abutting chamber elements thus together form a small volume reaction chamber composed of a first and a second cavity on opposite sides of the porous layer. A pair of capillary passages, which extend 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 passages minimizes the volume of 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 resilient material is permeable to the variety of fluids passed through the chamber and in effect improves 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-type capillary tube or 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 well as in the case of the porous layer, and the reagents and 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 formed as a porous layer. The test-containing matrix is then formed on the surfaces of the first and the second cavity. Similarly, the surface supporting the solid matrix can be constructed and formed in virtually any manner that allows reagent and solvent fluids to be passed over the matrix.

A new valve device described herein for controlling the flow of fluids to and from both the chamber and the conversion piston is specifically 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 end 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 condition, each of the diaphragms covering the various valve locations 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 location in question. Fluid communication between the individual duct and the other ducts can be accomplished selectively by applying a vacuum to one or more of the diaphragms to pull the diaphragms away from the valve locations and allow fluid to pass over the surface of the valve block between the openings. , to the respective channels. The secondary channels leading to the valve site at the distal end of the primary passage may be connected to a pressurized source of a purge fluid, such as inert gas, to complete the supply of each fluid through the valve. 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 distal 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 manifold formed therebetween is flushed clean from a certain reagent or solvent, before the supply of the next reagent or solvent. As for the valve at the outlet to the reaction chamber, the primary passage is connected to the starting medium. the barrel, while the secondary passages are connected to the conversion piston, vacuum resp. waste. 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 valve systems within the sequencer area. However, prior art sawtooth valve assemblies have included a plurality of individual blocks mounted against valve sites on a main valve block to be reciprocated between states or positions of communication. and for non-communication of passages in the main block.The displaceable blocks tend to wear, 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 diaphragms to effect and cut off flow between pairs of openings communicating with respective passages.These diaphragms can be made of substantially inert materials, for example commercially available fluorocarbon polymers, which operate indefinitely without disturbance. or deterioration.Furthermore, ti better known 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 tight joint. In particular, prior art sawtooth valves have connected external conduits to the valve passages by pressing substantially flat flange surfaces associated with the various 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 such materials as fluorocarbon polymers, which are very difficult to machine accurately, a considerable pressure must be applied in order for each sealing surface to be formed one after the other and to form the required seal. The pressure is supported by the valve block and causes deformation of its upper sealing surface.

The valve assembly described herein includes a plurality of conical sealing rings (ferrules), which are partially received in recess-shaped recesses in the valve block to provide a seal without applying excessive pressure to the valve block. A relatively small sealing force is focused on a certain part of the sealing ring to cause the sealing ring to seal against correspondingly conically shaped recesses without deformation of the block. Providing interfaces at opposite ends of the sealing rings between surfaces of different conical shape further improves the seals obtained. Interfaces of this type between surfaces of different conical shape are preferably provided at opposite sides of the sealing rings to provide 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 walls of the flask to wash away any chemicals which may have condensed or sprinkled on the walls, so that they are washed down the walls and into in the amount of liquid in the flask. A main source of cross-contamination of the system between the cycles is eliminated in this way. The conversion flask also enables gases to be fed 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 splashing. This favors a rapid, careful removal of liquid from the sensitive amino acid derivatives. Thus, solvents used to introduce the amino acid derivatives into the conversion flask can be removed in a much shorter time than according to the patented invention of Wittmann-Liebold, mentioned above (1-2 minutes instead of 5-10 minutes) and at lower temperature (40-50 ° C instead of 50-80 ° C). This significantly 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 30-40 minutes, as required by the Wittmann-Liebold invention patented. 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.

Because the acid component of the conversion reagent, trifluoroacetic acid or hydrochloric acid, is much more volatile than the water in which it is dissolved, the acid component tends to be removed early in the Wittmann-Liebold drying process, leaving the amino acid derivative in only aqueous aqueous solution. .

This causes incomplete conversion of derivatives of glycine and proline as well as decomposition 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, within 3-5 minutes, without the sample splashing 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 SE-8105597-2.

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

Figure 3 is a schematic diagram of the device according to Figure 1. Figure 4 is an exploded perspective view of a reaction chamber unit constructed according to SE-8105597-2.

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

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

Figure 6B 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 6B 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 described herein for a reagent or solvent to be used in liquid form.

Figure 8 is a vertical cross-section through a typical reservoir described herein for a reagent to be used in the form of gas or steam.

Figure 9 is a vertical cross-section through a diaphragm valve assembly constructed as set forth herein for controlling the flow of fluids to and from the reaction chamber and the conversion piston, shown in a direction corresponding to line 9-9 of Figure 11.

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

Figure 10 is a vertical section taken along line 10-10 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 taken along line 12-12 of Figure 11.

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

Figure 13A is an enlarged fragmentary section 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 location.

Figure 13B is a sub-section 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 17B schematically show the two most important prior art methods for immobilizing a protein or peptide sample during degradation.

Figure 17C schematically illustrates immobilization of a protein or peptide.

Figure 18A is a vertical section on a larger scale corresponding to Figure 5 of a further embodiment of the reaction chamber unit according to SE-815597-2.

Figure 18B is a vertical section showing the chamber member of the embodiment of Figure 18A turned sideways to supply a sample-containing matrix to its inner walls.

Figure 18C is 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 lists 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 comprising 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 solvents and reagent reservoirs are connected through a group 22 of diaphragm-type flow valves to the reaction chamber 12 and the conversion piston 14. A second group of valves 24 regulates the flow of liquids from the reaction chamber to the conversion piston 14 and other places. 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 source 28 of filtered inert gas supplies to the device 10 highly 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 meters has 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, according to the preferred embodiment, forms a reaction chamber 34, which communicates with 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. the reagent reservoirs 42, 44, 46 and 48 and the solvent reservoirs 50, 52 and 54. The reservoirs 42-54 are 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 501 968 24 also at a location above the fluid level therein are 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 source 28 and can thus be discharged to the drain trap 60 at a rate or degree controlled with the flow regulators 64. The fluid discharges from the 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 can thus be passed through the conduit 40 and the chamber inlet passage 36 h to the reaction chamber 34 by selectively operating 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, inert gas under pressure from the source 28 may be introduced into the collection line 68 at the end opposite the line. 40 through a pressure regulator 70 and a diaphragm valve 72. Operation of the valve 72 thus provides introduction of gas under pressure at the distal end of the manifold 68 and drives any reagents or solvents in 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 flow 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 meters 74 and the flow regulators 76 are required for accurate control of the gas emission rate through the corresponding valves 66.

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 to release the desired reaction products, typically the N-terminal amino 501. 968 the acid moiety of a protein or peptide sample, through line 86 to the conversion flask 14. Valve 82 may be opened to connect the outlet passage 38 to the waste trap 60 for disposal of unwanted reagents, solvents and reaction products and the valve 80 connects the outlet passage 38 to a vacuum trap 88 and a vacuum pump 90 for evacuating the reaction chamber 34, the outlet passage 38 and the manifold 84.

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

Reagent reservoirs 100 and 101 and a solvent reservoir 102 are provided for supplying reagents and solvents to the conversion flask 14 through a conduit 104.

The reservoirs 100, 101 and 102 are pressurized with the inert gas source 28 through pressure regulators 106 and solenoid valves 108 and are connected to the waste trap 60 through individual vent valves 110 and flow regulators III. The fluid outlets from the reservoirs 100, 101 and 102 are connected through diaphragm valves 112 to a continuous or continuous manifold 114, which communicates at one end with the conduit 104. The flow of fluids from the pressurized reservoirs can thus be accomplished by the valves 112 for selectively discharging either reagent or solvent into the manifold 114 are selectively opened. The inert gas source is connectable 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 or solvent through the manifold and the channel to the conversion piston 14.

The inert gas pressure source 28 is also connected to the conversion piston by a pressure regulator 120, a diaphragm valve 122 and a line 124 and to the fraction collector 16 by a pressure regulator 126 and a valve 128. When a certain fraction is converted in the intended manner in the piston 14 , it can be discharged from the gas pressure piston through 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 solvent to dissolve any remaining chemicals therein. The solvent can then be expelled with gas pressure through line 124 and a valve 125 to the waste trap 60 and the flask flushed in preparation for delivery of the next amino acid derivative.

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

The control unit 18 is preferably a fully automated unit, which controls the diaphragm 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 set manually after start-up to set the desired pressures and flows 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 carries a sleeve 132, which contains first and second chamber elements 134 and 13, respectively. 136. The sleeve 132 is provided with an enlarged cylindrical portion 138 which is centered around the axis of the sleeve and tightly fitted in a cylindrical recess 140 in the base 130. The cylindrical portion 138 is held in position by a retaining collar 142 which is threaded to the base 130 27 501 968 Chamber elements 134 and 136 are cylindrical glass elements with facing connecting surfaces 144 and 144, respectively. 146 and closely fitted in the axial direction in the sleeve 132. The inlet and outlet channels 36 and 38 described above extend axially through the chamber elements 134 and 144, respectively. 136 and are preferably capillary channels with a diameter of the order of 1 millimeter. The axially outer ends 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 provides the chamber members with 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 is provided with opposing locking lugs 156 for cooperating with slots 158 in the upper end of the sleeve 132. The metal washer 154 is held in place by a lid 160 which is screwed to the upper end of the sleeve 132 to hold the chamber members tightly in place relative to to the sleeve. The interaction between the ears 156 and the slots 158 prevents the washer 154 from rotating when the cover 160 is applied, thus preventing 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 end 148 of the chamber member 134, so that the hole in the conduit 40 communicates with the inlet passage 36. The conduit 40 is provided with a support washer 164 and a nipple. 166, which is screwed axially into the cover 160 to press the widened end 162 sealingly against 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 hole in the conduit 40 with the inlet passage 36 is ensured by constructing the various cooperating components with sufficient precision in relation to a common shaft.

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 smooth 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 end 150. of the second chamber member 136 and a conical recess 180 in a valve block 182 forming the manifold 84. An axial passage 184 in the mass 172 is arranged exactly 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 element 136 to the valve block 182. In the case of the conduit 40 discussed above, careful construction of the components related thereto around a common axis ensures an accurate fit and complete seal between the various channels. The chamber device 12 can thus be easily disassembled and assembled in a very short time without risking the mutual 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 are included in the valve block 182 for regulating 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 mated cavities 186 and 188 in the opposed 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 and preferably have a circular cross-section and provide an axially symmetrical path for a fluid which passes from the inlet passage to the outlet passage. A porous layered member 190 extends across the reaction chamber 34 and may be arranged at least in part in a recess 192 in the cavity 186. The porous layered member 190 thus separates the inlet passage 36 from the outlet passage 38, so that fluids flowing from one passage to the other must pass through the layered element. The porous layer-shaped element 190 preferably comprises a layer or a mat of compressed fibrous material, for example glass. Commercially available fiberglass filters are suitable for this purpose and have a high resistance to decomposition 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 consists of a fluid-permeable material, i.e. one that allows diffusion of liquids and gases in the layer, the material can form a solid matrix, which can retain the sample safely but allows chemical action of reagents and solvents on the sample. Polymeric quaternary ammonium salts, such as 1,5-dimethyl-1,5-diazaundecamethylene-polymetobromide or poly- (N, N-dimethyl-3,5-dimethylene--piperidinium chloride), are ideal for this purpose. They allow diffusion of fluids, are insoluble in solvents used and are chemically stable to both reagents and solvents. In addition, they form a cohesive film and have a positive charge, which enables them to bond ionically to the glass substrate surface.

The fundamental differences between the forms of sample retention used in previously known sequencer devices and according to SE-8105597-2 are clearer in connection with Figures 17A, 17B and 17C. Figures 17A and 17B schematically illustrate the two most common prior art methods for immobilizing a protein or peptide sample 300 relative to a sample substrate surface 302 or 304.

Figure 17A illustrates the case where 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 sites of the glass have amino functional groups 308 extending therefrom for reaction with carboxyl groups 310 of the sample chain. 501 968 Under appropriate 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 hold the sample in place.

However, covalent binding is difficult to achieve with protein and peptide samples. Covalent bonds also hold the chain with a very small number of units in the chain. When the degradation process reaches these units and splits them from the chain, the remainder of the chain is left unbound and can be rinsed away from the chamber.

Figure 17B illustrates the case where the sample 300 is adsorbed directly on a substrate surface 304. The sample is 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 interaction between the surface and the sample is obtained in a number of different places. This works well in the case of large proteins and peptides, but when the sample is broken down to a much smaller size, it tends to be beaten or pulled away from the surface. This results in a drastic sample loss.

Figure 17C schematically illustrates the immobilization of the sample 300 relative to the substrate surface 314 by embedding in 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 effects and will hold the sample securely in place, since the sample is embedded. The direct binding effect between the sample and the surface is not relied upon and the effectiveness of the immobilization 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 solvents which are passed over the film. Once the reagents and solvents are dissolved in the film, they can easily diffuse through its thickness extent and effect the degradation process.

Figure 6A shows how the reaction chamber 34 is sealed 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 mating surfaces 144 and 146.

A pair of resilient layers 194 are suitably used on either side of the porous layered member 190. The layers 194 are very thin and are permeable to the variety of reagents and solvent fluids to be passed through the chamber 34. An annular sealing ridge or roll 196 at about the circuit of the cavity 188 abuts the sealing layers 194 to provide a more effective seal against the surface 144 adjacent the perimeter of the cavity 186. The sealing layers 194 may be made of any substantially chemically inert material, such as commercial fluorocarbon polymer, to minimize the risk of destruction of the seal. They not only serve to provide a seal for the chamber 34 but also to support or support the porous layered member 190 and allow gases and liquids passed through the chamber to diffuse so that the flow of the gases and liquids becomes more uniformly distributed across the chamber. 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 SE-8105597-2 is illustrated in Figure 6B, according to which the mated cavities 186' and 188 'in the opposite adjoining surfaces of the two chamber elements are slightly narrower and longer than the cavities 186 and 188. Otherwise, the structures 34 and 34 'are identical and the various elements of the structure 34' on the drawing figures are numbered similarly to those of the structure 34 with the suffix "prim" (') to separate 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 layer element 90'. Figure 6C illustrates the chamber 34 'of Figure 68 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 having a protein or peptide sample embedded therein 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 facilitated while maintaining a considerable film surface area.

A further embodiment of the chamber device according to SE-8105597-2 is shown in Figures 18A, 188 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 connection with Figures 4 and S and are numbered accordingly. All constructions and connections externally in relation to the chamber device are also identical to those described above.

The chamber element 320 comprises a cylindrical glass structure with inner walls 322, which form an axial chamber 324 of the capillary type. The chamber 324 increases uniformly in diameter from its two ends 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 should be noted that the reaction chamber can have a virtually arbitrary shape with a sample support surface through 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 passed through the tube would react with a protein or peptide sample embedded in the film and effect the degradation process.

A typical reservoir 198 for storing and supplying a liquid reagent to the reaction chamber 34, 34 'or 324 is shown in Figure 7. Reservoir 198 corresponds to reservoirs 42, 50, 52, 54, 100, 101 and 102 in Figure 3. An inert gas below pressure is applied 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 removes any dissolved oxygen from the liquid and can be discharged at an adjustable rate through a vent line 206 to provide a dynamic equilibrium state in the interior 200. A liquid outlet 208 is provided for controlled discharge. of reagent or solvent from reservoir 198 with the gas pressure therein. The inert gas supply line 202 to each reservoir 198 receives inert gas under pressure from a source 28 through a pressure regulator 56 or 106 and a solenoid valve 58 or 108. The release 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 valves 66 or 112. Each time one of the liquid reagents or solvents is to be supplied to the reaction chamber or conversion flask 14, the corresponding argon supply valves and vent valves are provided for providing dynamic equilibrium state in the reservoir in question.

Liquid reagents or solvents can then be introduced by opening the valve in line 208 and valve 82 to the waste trap 60. Liquid is discharged from the reservoir at a constant rate, allowing accurate control of the discharged liquid by controlling the amount of time the valve in discharge line 208 is kept open. A typical reservoir 210 for discharging a reagent in gaseous or vapor form is illustrated in Figure 8. An inert gas inlet 212 opening into a gas distribution member 213 of sintered glass is provided for introducing inert gas into the interior 214 of the reservoir 210 on a place near the 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 liquid 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 regulates the flow of gas through the line 212 from the gas pressure source 28. Similarly, the outflow of gas under pressure to the vent line 218 is controlled by one of the flow regulators 64 and the flow valves 62, and the discharge of reagent through the line 220. is controlled by one of the diaphragm valves 66 in line with a flow meter 74 and flow 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 liquids and evaporated as needed. Evaporation is accomplished by bubbling inert gas up through the liquid reagent. In this way, the inert gas in the interior 214 of the reservoir 210 becomes saturated with reagent vapor. Each time reagent is required, the valves connected to lines 212 and 218 are opened for bubbling inert gas through the reagent and creating 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 flow regulator 76 in the line with the valve 66 in question causes the steam to be discharged from the reservoir at a constant speed, which is indicated by the flow meter 74.

Figures 9-13 illustrate the construction and operation of a valve device 222, which constitutes embodiments of the valves 66 and 72 and the continuous or continuous manifold 68 of Figure 3. The valve device 222 501 968 comprises a valve block 224, most clearly seen in Figures 11 and l2. The valve block 224 is an elongate block of rectangular cross-section with a continuous or continuous primary passage 226 in a sawtooth pattern 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 at alternate positions. a plurality of valve locations 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 corresponding openings 240 in the vicinity. of the openings 232 at respective valve locations 230. The valve block 224 is mounted in an elongate recess 242 in a base 244 with a plurality of threaded openings 246 mating with connection openings 234 and 238 for receiving connection nipples 248. The nipples 248 are designed to press resilient double conical sealing rings 250 against the connection openings 234 resp. 238 for tight connection of pipes 252 extending through the sealing rings to the various passages in the valve block 224. In this way the connection opening 234 of the primary passage communicates with the inlet passage 36 of the chamber device 12 through the fluid line 40 of Figure 3 and the first seven of the eight second passages communicate with the fluid outlets 208 and 220 of reservoirs 42-54. The secondary passage furthest from the connection opening 234 communicates with the inert gas pressure source 28 through the pressure regulator 70 shown in Figure 3.

The construction of the double conical 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 arranged 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 formed conically with angles 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 greater than the angle of the sealing ring. This fit between surfaces of different conical shape focuses the compression 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 pressures on the valve block 224 that can deform the valve locations 230 are thus avoided.

A series of diaphragm retaining blocks 254 are secured with bolts to the surface 228 of the valve block 224 with the diaphragm 256 mounted or laminated between these parts. The upper end of each diaphragm retaining 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 locations 230. An O-ring 262 may be provided in an annular groove surrounding recess 260 to provide an effective air seal.

The diaphragms 256 are made of a substantially chemically inert airtight material which allows these alternates to be withdrawn from and pressed against the valve locations 230 by the action of vacuum and air pressure, respectively, through the connecting nipples 258. Two alternating states of the diaphragm 256 are 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 of the secondary passage at the valve location 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 position of the valve which is arranged at the valve location in question. In the condition of Figure 13B, vacuum applied to the connection nipple 258 pulls the diaphragm 256 away from the openings 232 and 240, so that connection is obtained between the primary and secondary passages across the surface of the valve block at this location. 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 of the valve device 222, as described above, enables the supply of reagents and solvents to the reaction chamber in precise amounts with virtually no contamination between the various fluids.

The continuity of the primary passage 226 and the connection at one of its ends to the inlet passage of the chamber device 12 are largely responsible for this advantageous function. Discharge 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 on the others, whereby the desired reagent or solvent can pass into the primary 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, gas pressure is again applied to it through the connection nipple 258, so that all the seven reagent and solvent valves in the 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 distal end of the valve block 224 to flush the entire primary passage 226 with inert gas, forcing reagent or solvent fluid remaining in the lines 34 into the reaction chamber 34. branches in the primary passage 226, there is no place where Since there are no interruptions or blinds 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 in 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 501 968 which is incorporated into many other parts of the device to minimize contamination when a particular opening must be selectively connected to a plurality of other openings. 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 collection line 84. 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 are also designed in the same way as the valve unit 222. The principle difference between these valve devices is the number of valve locations. united in these.

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

Vacuum and gas pressure for operating the diaphragm valves between the open and closed states are omitted from the schematic diagram of Figure 3 to facilitate this and are preferably separated from the source 28 and the vacuum pump 90 as described.

The conversion piston 14 is shown in detail in Figures 14-16.

The piston 14 is of the double-walled glass type with a space 264 between the walls for circulation of a heating fluid, for example water. The heating fluid is passed to the eye 26 from the space 264 through a pair of nipples 266 designed to receive the ends of standard flexible hoses. A large inner diameter tube 268 which is 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, communicates with the inner chamber 270 of the piston adjacent its upper end.

Capillary tubes 272, 274 and 276 extend through the upper end 39 of the piston to points in the interior of the chamber 270. The holes in the tubes 272 and 276 are closed at the inner ends and each of the tubes is provided with a plurality of relatively limited radially distributed openings 278 or 280 adjacent the inner end.

Due to the relatively small amount of protein or peptide samples 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 in the reaction chamber 34 are sequentially passed 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 restricted openings 280 as a spray. which hits the walls of the chamber 270 to wash away any residue thereon to the bottom of the piston. During the conversion reaction, an inert gas may be introduced through the capillary tube 272 to stir the liquid and help to evaporate the solvent therefrom. The gas leaves the tube 272 through the restricted openings 278 as very small bubbles, which provides optimal dispersion of the gas throughout the liquid regardless of the size of the flask and the amount of gas used. When the conversion reaction is complete, the fraction is forced upward through the gas overpressure in the flask 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 provide a more complete transfer of the fraction to the fraction collector 16. The first sample volume, about 200 microliters yellow), is first passed to the fraction collector. An additional 50 μl of solvent is then introduced to dissolve any residue on the lower walls or bottom of the flask.

The second sample volume is then removed. In this way a high yield of each fraction can be achieved.

After a certain fraction is transferred, an additional 750-900 .mu.l of solvent can be introduced to dissolve any residue remaining from the previous cycle on the upper walls of the flask 14. This additional amount of solvent is then disposed of as waste and leaves the walls of the flask clean.

The outer ends of the tubes 268, 272, 274 and 276 are tightly 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 O-ring 288 which abuts and seals against the planar ground glass surface of a radial flange 290 on one of the glass tubes.

An internally threaded collar 292 is slidably disposed over the conduit 282 to receive the glass flange 290 and cooperate with a two-piece externally threaded nipple 294 disposed around the glass tube. Feed of 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 made of substantially chemically inert materials, for example commercially available fluorocarbon polymers, to eliminate the possibility of destruction and consequent leakage.

An alternative construction of the piston 14 would eliminate the space 264 and the nipples 266 and provide a single walled vessel which could be maintained at elevated temperature by fitting in the heated environment 32. This construction would have the advantage that the entire length of the glass tubes p and the connecting means 284 would be maintained at the elevated temperature while minimizing condensation of semi-volatile fluids therein, but would not allow the plunger and container 12 to be maintained at different temperatures. The reagents and solvents used in the apparatus 10 for sequential degradation of protein or peptide chains are preferably as follows: R1 phenyl isothiocyanate (PITC) (17% solution in heptane) R2 trimethylamine or triethylamine (25 % solution in water) R3 trifluoroacetic acid or heptafluorobutyric acid R3A water vapor R4 trifluoroacetic acid (25% solution in water) R4A hydrogen chloride (1N solution in methanol) S1 benzene S2 ethyl acetate with 0.1% acetic acid S3 butyl chloride S acetonitrile. 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 desired 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, Penhasi, with variations to enable the particular sequence of steps required by the apparatus 10, or may be a more sophisticated electronic control in the form of by a special or general purpose digital computer. Alternatively, the different steps of each degradation cycle may be performed manually by an operator according to a predetermined schedule to achieve the same result.

Prior to initiating the degradation process, a sample of the protein or peptide being tested must be applied to a suitable sample carrier surface. The matrix material is first applied to the support surface and the sample is later embedded therein, as described in Examples 1 and 2 below. When the element 190 or 190 'is used as the sample carrier 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 the element 1901 is held in the reaction chamber 34 in place of the recess 192. According to the preferred embodiment of a pair of sealing sheets or sealing layers 194 are used and the element 190 is applied 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, it is applied thereto 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 the sample are then applied to the walls of the chamber 34 'or 324, as described in Example 2, and the sleeve 132 is assembled with the other components to form a complete apparatus.

The reaction chamber 34 and the associated fluid conduits can be initially evacuated by opening the valve 82 to the vacuum trap 88 and the vacuum pump 90, in preparation for the sequential introduction of the reagents R1 through R3A, the solvents S1 through S3 and inert gas from source 28. Each time one of the reagents or solvents is to be introduced, the corresponding inert gas supply valve 58 and vent valve 62 are opened to pressurize the reservoir in question and provide a dynamic equilibrium state therein. Inert gas is thus introduced and discharged from the reservoir simultaneously to maintain a constant pressure in the reservoir. In the case of reservoirs 44, 46 and 48, the flow of saturated gas from these is regulated during the discharge with one of the flow regulators 76.

After the reservoir containing the reagent or solvent to be supplied is pressurized and equilibrated, as described above, a vacuum with the auxiliary vacuum source (not shown) is applied to the diaphragm of the corresponding flow valve 66 to open the valve. the valve and providing a flow of the reagent or solvent through the interconnected manifold 68 and conduit 40 to the inlet passage of the chamber. The valve 66 is kept open 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 of the valve 72 at the distal end of the continuous manifold 68 to flush the manifold from any remaining reagent or solvent and completely supply it 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 delivery 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 trap 60. After delivery of a particular reagent or solvent, the chamber and associated conduits can be evacuated by evacuation 82. to the vacuum pump 90. Alternatively, the chamber and the sample therein may be dried at appropriate times by passing an inert gas through the chamber through the valve 72. Gas leaving the chamber may then be passed to the waste trap 60 or, if desired, discharged. fed with the vacuum pump 90 to speed up the drying process.

After completion of the coupling and cleavage steps, the extraction solvent S3 is supplied 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.

For this purpose, the valve 78 is open and the valves 80 and 501 968 44 82 remain closed. The conversion reagents R4 and R (if used) and the solvent S4 can then be: Supplied to the conversion flask 14 at appropriate times by opening the corresponding valves l12 for supplying the liquids through the collection line ll4 and the line 104 to the conversion flask. Each supply is preceded by the pressurizing and venting operations described above in connection with the supply of the other reagents and solvents and is followed by opening the valve 118 for flushing the collection line 114 and the 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 more stable phenylthiohydantoin amino acid, partly in accordance with the above articles. -Liebold. In general, the amino acid fraction in the conversion flask can first be evaporated by passing inert gas over the solution through the short capillary tube 276 and bubbling inert gas through the liquid by means of the capillary tube 272, followed by application of vacuum through the tube 268. The conversion reagent R4 can then be introduced through the capillary tube 276 through the conduit 104 and one of the valves 122 (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 through tube 268 and inert gas through capillary tubes 272 and 276. To further stabilize the acid-bearing side chains of the Pth-aspartic and Pth-glutamic acids, dissolve the residue in the conversion flask by introducing the reagent R4A through the capillary tube 276 via line 104 and one of valves 111 in the desired amount. Evaporation in the conversion flask is again accomplished by applying vacuum through the tube 268 and supplying inert gas through the capillary tubes 272 and 276. The Pth amino acid remaining in the conversion flask is then redissolved in the solvent S4 introduced through the line 104 to transfer the fraction to the appropriate ampoule. in the fraction collector 501 968 16. 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 inlet of inert gas under pressure through the capillary tube 276 to push away the fraction from the flask.

The vial carousel of the fraction collector 16 is rotated a predetermined angle once during each decomposition cycle, so that each incoming fraction is collected in a separate vial. At a suitable 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 valves 128 and 94 to supply inert gas from the source 28 over the fractions and finally to the waste trap 60.

The various components of the apparatus 10 are preferably constructed of materials which are substantially inert and highly resistant to destruction. Such materials include borosilicate glass, certain fluorocarbon polymers and in some cases stainless steel and aluminum. The tightly fitting structural members and other elements of the apparatus 10 have been designed 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 steps 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 during each step are also indicated. The functions shown correspond to the condition of the various valves in the device and the markings in the columns indicate when the suitable valves are open. Thus, for example, the colon, _g _ \ ___ ns uRln, uRzu 'ullz3u' nR3An 'usln, uszn and 1183 :: 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 is present in one of these columns, the valves 58 and 62, 501 968 46 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 remain closed at all times otherwise.

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 at this time, and the columns designated "waste", "collection" and "vacuum" show the condition of valves 82, 78 and 80. As described above, each step of supplying solvent or reagent is preceded by pressurizing the solvent or reagent reservoir followed by the introduction of inert gas through valve 72 to complete the supply of the solvent or reagent and flushing the supply lines.

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

Of the enumerated fraction collector fractions, the columns "argon", "waste" and "vacuum" show the states of the valves 128, 94 and 98.

With the exceptions set forth above, the sequence of steps set forth 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 functional 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: l) Steps 18 to 58 can be looped once or twice on the first sequence cycle to ensure complete connection of all amino groups on the protein. 2) Step 65 may be followed by the addition for 20-60 seconds of water vapor (R3A) through the reaction chamber to reduce the dehydration of the side chains of serine and threonine. 3) Step 65 may 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 and glutamic acid. If this is done, S4 is preferably methanol. 4) Step 76 can be followed by the addition of 0.7 to 1 ml of S4 (acetonitrile or methanol), which is then added to waste to thoroughly clean the flask. 5) Step 8 may be given an increased duration (500-1000 seconds) to promote cleavage of amino-terminal proline residues.

The nature of the process that can be performed using the present invention is further clarified by the following specific examples of practical application. The stated values are only examples of processes carried out with the device according to the invention, and are not intended to limit the scope of the invention.

The amino acid sequences given in the following examples are tabulated in the one-letter amino acid code, which is defined as follows: 501 968 48 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 H - histidine Y - tyrosine I - isoleucine V - valine Example 1.

A solid matrix of polymeric quaternary ammonium salt suitable for embedding a protein sample for sequencing can be prepared on the fibrous layer elements 190 and 190 'described above, and a protein sample can be embedded in the matrix as follows: A glass fiber board having diameter 12 mm and thickness 0.25 to 0.5 mm are cut from a layer of glass microfiber filters commercially available from Whatman, Inc., Clifton, New Jersey, Afs. The wafer is placed in the immersion 192 of the chamber element 134. 25 microliters of an aqueous solution containing 1.5 mg of 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide and 0.0033 mg of glycylglycine are dropped on the glass fiber disk from a syringe or pipette. . The water is evaporated in vacuo or by heating in a stream of hot nitrogen. The remainder of the chamber apparatus 12 is assembled and installed in the apparatus 10. The protein sequencing program is initiated at step 18, and 4 to 6 complete degradation cycles are performed to remove impurities from the 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide which can react. with the protein sample or otherwise interfere with the 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 solution dissolves 1,5-dimethyl-1,5-diazaundecamethylene polymetobromide and the liquid is removed by evaporation, leaving a thin film of 1,5-dimethyl-1,5,5-diazaundecamethylene polymethobromide with the protein sample embedded therein. If the initial protein sample volume is greater than 25 microliters, the sample can be applied in divided sample volumes in the amount of 25 microliters, the liquid being removed by evaporation between the application of these liquid volumes.

The chamber apparatus 12 is reassembled and re-installed in the device 10. The protein sequencing program is then initiated at step 18 and performed as many degradation cycles as desired.

Example 2.

A solid matrix of polymeric quaternary ammonium salt suitable for embedding a protein sample for sequencing can be prepared on the inner walls of a glass capillary vessel and a protein sample can be embedded in the matrix as follows: Chamber element 324 or chamber elements 134 'and 136' are assembled first. with the sleeve 132 and the lid 160, as shown in the figures, to form a compact cartridge subunit which is easy to handle for insertion of the matrix and the sample.

The subunit is held horizontally and 10 microliters of an aqueous solution containing 0.6 mg of 1,5-dimethyl-1,5-diazaundecamethylene polymetobromide and 0.0013 mg of glycylglycine are injected into the reaction chamber from a syringe.

The chamber 320 is preferably designed to have a diameter of 1.6 mm at both ends and a diameter of 3.2 mm at the center, which gives a chamber which can hold up to 25 microliters of yellow) solution in the horizontal state without spillage at the ends. This configuration of the chamber 324 containing a solution in the horizontal state 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 evaporated by directing a stream of air or nitrogen through the chamber, so that a film of 1,5-dimethyl-1,5,5-diazaundecamethylene polymetobromide is left on the chamber walls. . The subassembly is then installed in the apparatus 10, the protein sequencing program is initiated at step 18, and 4 to 6 complete degradation cycles are performed. The subunit is then removed from the apparatus 10 and kept horizontal, while 10 microliters of the protein solution is dropped into the reaction chamber from a syringe.

The subassembly is rotated again about its axis, while the liquid in the reaction chamber is evaporated by a stream of nitrogen directed through the chamber, leaving a thin film of 1,5-dimethyl-1,5-diazaundecamethylene polymetobromide with the protein sample embedded therein as shown in Figure 18C. The subassembly is then reassembled into the apparatus 10 and the protein sequencing program is initiated at step 18 and performed as many degradation cycles as desired.

Example 3.

Angiotensin, 0.0005 mg, containing 25 microliters of 20% aqueous formic acid was embedded in a solid matrix of precycled 1,5-dimethyl-1,5-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 liquid 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, hereinafter referred to as "experimental", was obtained. The known sequence is also given for comparison. Experimental: D - R - V - Y - I - H - P - F Known: D - R _ V _ Y _ I - H - P _ F This result is significant, as it shows that even small peptides can be sequenced in the manner of the invention.

The ability to sequence small peptides depends on the fact that the sample is embedded in a film for retention instead of being adsorbed directly on a support 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 surface and are highly dependent on the size of the molecules, the retention ability 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 embedded in a solid matrix of precycled 1,5-dimethyl-1,5-diazaundekamethylene polymetobromide by the procedure of Example 1. Apomyoglobin was subjected to 40 cycles of Edomy. degradation and the phenylthiohydantoinamino acids were analyzed according to the method of Example 3 to give the sequence set forth below as "experimental".

The known sequence of spermacetival-apomyoglobin is also given below for comparison. 1:05 a.m. 10:15 a.m. Experimental: V-L-S-E-G-E-W-Q-L-V-L-H-V-W-A Known: V-L-S-E-G-E-W-Q-L-V-L-H-V-W-A 16:20 25 30 K-V-E-A-D-V-Afg-H-G-Q-D-I-L-I-K-V ~ E-A-D-V-A-G-H-G-Q-D-I-L-I 35 40 R-L-F-K-S-H-P-E-T-L-R-L-V-S-N-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 sulphate and 0.05M ammonium bicarbonate was embedded in a solid matrix of precycled 1,5-dimethyl-1,5-diazaundecamethyl -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 to give the sequence set forth below as "experimental". 501 968 52 l 5 Experimental: N - A - N - V - E - V - K - E - L - V - ll 15 N-D-v-Q-P-n-G-F-v-s- 21 25 K-L-v-L-D-D-G-s-A-s- 31 35 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 sulphate and 0.05M ammonium bicarbonate was embedded in a solid matrix of precycled 1,5-dimethyl -1,5-Diazaundecamethylene polymetobromide by the method of Example 2. The outer skin protein was subjected to 24 cycles of Edman degradation and the phenylthiohydantoin amino acids were analyzed by the method of Example 3 to give the sequence hereinafter referred to as "experimental". 1 5 Experimental: N - A - N - V - E - V - K - E - L - V - ll 15 N - D - V - Q - P - D - G - F - V - S - 21 xLvL- De results obtained according to examples 5 and 6 show that the device and the method according to the invention are suitable for sequencing proteins and peptides dissolved in a solution of sodium dodecyl sulphate, an effective anionic detergent. This is important because the most general method for isolating small amounts of medium to large proteins or peptides for analysis, known as polyacrylamide gel electrophoresis, provides samples in a solution of sodium dodecyl sulfate. This common detergent causes 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.

Claims (6)

'501 968 PATENT CLAIMS Conversion piston for use in an apparatus for performing sequential chemical processes on a sample of chemical material, characterized in that the conversion piston (14) comprises: piston means having at least three capillary tubes (272, 274,276) extending into the interior (270) of the piston for introducing and discharging various fluids into and from the piston, and means (268) designed for connection to a. external vacuum source, the piston means being provided with inner walls, an upper end and a bottom, a first (276) of the capillary tubes having an inner end which is closed and is provided with a plurality of limited outlet openings (280) arranged with radial distribution, so that introduction of fluids through this first capillary tube causes spraying against the inner walls of the piston for downward washing of the inner walls, a second (272) of these capillary tubes extends to a point near the bottom of the piston with the inner end, at which hole is closed, this inner end being provided with a plurality of outlet openings (278) of limited size, which are radially distributed, so that a passage of a gas inwards through this capillary tube causes the introduction of a plurality of small bubbles in a liquid in the piston, the bubbles causing agitation of the liquid or accelerating drying thereof, and a third (274) of the capillary tubes extending to a point which is located above the point to which the second capillary tube extends and below the point to which the first capillary tube extends, so that chemical substances introduced through this third capillary tube and hitting the walls of the flask are washed down by the liquid from the first capillary tube to the point where it the second capillary tube extends. Conversion piston according to claim 1, characterized in that the capillary tubes are made of glass. 3. A conversion flask according to claim 1, characterized in that the flask has a volume of about 1 ml. 4. A conversion piston according to claim 1, characterized in that the means adapted for connection to an external vacuum source are located next to the upper end of the piston. Conversion piston according to claim 1, characterized in that the piston has an enclosed space (264) extending around a substantial part of its length, the enclosed space being adapted to receive a heating fluid carried to and from this space. . Conversion piston according to claim 1, characterized in that the piston has a circular cross-section and is tapered from its upper end to its bottom.