FR2490505A1 - APPARATUS AND METHOD FOR THE SEQUENTIAL EXECUTION OF CHEMICAL PROCESSES - Google Patents

Abstract

THIS MACHINE INTENDED FOR THE SEQUENTIAL EXECUTION OF CHEMICAL PROCESSES ON A SAMPLE OF CHEMICAL MATERIAL INCLUDES AN ENCLOSURE 12 PRESENTING AN INTERNAL SURFACE SURFACE SURROUNDING A REACTION CHAMBER, WHICH IS EQUIPPED WITH AN INPUT AND OUTPUT, RESERVOIRS, COMPOUND , CONDUITS AND DISTRIBUTORS CONTROLS THAT ALLOW TO PASS SUCCESSIVELY SEVERAL FLUIDS THROUGH THE CHAMBER FROM THE INLET TO THE OUTPUT IN A CURRENT UNDER PRESSURE, A SOLID MATRIX PERMEABLE TO FLUIDS AND PLACED THROUGH THE CHAMBER SO THAT THE FLUIDS THE SAMPLE TO BE TREATED BEING CONTAINED IN THE MATRIX SO THAT IT IS BOTH IMMOBILIZED AND EXPOSED TO EACH OF THE FLUIDS THROUGH THE CHAMBER IN A WAY TO CHEMICALLY INTERACT WITH THESE FLUIDS.

Description

The present invention relates generally to an apparatus and a

  improved process for the execution of chemical processes and it relates more particularly to an improved device intended for the automatic execution of the sequential degradation of protein or peptide chains containing a large number of amino acid units or units

  to determine the sequence of these units.

  The linear sequence of contained amino acid units

  in proteins and peptides has a significant interest

  maple for the understanding of their biological functions and, finally, for the synthesis of compounds performing the

  same functions. Although various techniques have already been used

  to determine the order of linear succession of acids

  amino acids, the technique that has had the best results is pro-

  bably that known under the designation of Edman process.

  Various forms of the Edman process and various devices for the automatic implementation of the processes are described in the following publications: Edman and Begg, "A Protein Sequenator", European J. Biochem. i (1967) 80-91; Wittman-Liebold, "Amino Acid Sequence Studies of Ten Ribosomal Proteins of Escherichia coli with an Improved Sequenator Equipped with an Automatic Conversion Device," Hoppe-Seyler's Z. Physiol. Chem. 354.1415 (1973); Wittmann-Liebold, "A Device Coupled to a Modified Sequenator for the Automated Conversion of Anilinothiazolinones into PTH Amino Acids" Analytical Biochemistry 75,621 (1976) US Patent 3,959,307 Liebold and Graffunder, concerning a method for determining

  the amino acid sequence which is called

  will hereinafter refer to the Wittmann-Liebold patent; Hunkapiller and Hood, "Direct Microsequence Analysis of Polypeptides Using an Improved Sequenator, A Nonprotein Carrier (Polybrene), and High Pressure Liquid Chromatography," Biochemistry 2124 (1978);

  Laursen, R.A. Eur. J. Biochem. 20 (1971); Wachter, E., Mach-

  leidt, H., Hofner, H., and Otto, J., FEBS Lett. 35, 97 (1973); US Patent 3,725,010 to Penhasi relating to an apparatus for the automatic execution of chemical processes; US patent

  No. 3,717,436 to Penhasi et al. concerning a process of

  sequential grading of peptide chains; US patent

  3,892,531 to Gilbert, concerning an apparatus for the fraction-

  the sequencing of peptides and proteins and which will hereinafter be called the Gilbert patent; US patent 4,065,412 to Dreyer,

  relating to a method and apparatus for sequential fractionation

  cé peptides and proteins and which will be called hereinafter the Dreyer patent. Another device to consider is described in US patent application No. 106,828, filed December 26

  1979 by Leroy E. Hood and Michael W. Hunkapiller whom we call-

  will read below the application Hunkapiller and Hood and which has

  title "'Apparatus for the performance of chemical processes".

  In short, as discussed in publications

  cited above, the Edman processes of sequential degradation

  tielle include three phases. coupling, cleavage and

  conversion. In the coupling phase, a phenylisothio-

  cyanate reacts with the amino -N-terminal x group of the peptide to form the phenylthiocarbamyl derivative. In the cleavage phase, an anhydrous acid is used to cleave the phenylthiocarbamyl derivative and form itanilinothiazolinone. After extraction of the thiazolinone, the residual peptide is practiced.

  for the next coupling reaction and cleavage cycle.

  An aqueous acid is used to convert thiazolinone to phenylthiohydantoin which can be analyzed in a way

  appropriate, for example by chromatography.

  The automatic device of US Pat. No. 3,725,010, modified as indicated in the aforementioned articles by Wittmann-Liebold and the application Hunkapiller and Hood constitute devices

  automated sequence splitting in which the re-

  actions are carried out in a thin film formed on the inner wall of a rotary reaction cell which is commonly known as the "spinning cup" and which is placed in a closed reaction chamber. Means are provided for introducing metered quantities of liquid reagent into the chamber to react them with a sample of a protein or a

  peptide in an inert atmosphere and to extract these quan-

  reagent tities. The sample to be analyzed is initially placed in the cup, after which the various reagents and solvents necessary for

  perform the coupling and cleavage reactions. Reactions

  tifs and liquid solvents also form on the walls of the

  scoop of dandruff flowing on the sample film

  and interacts with this film while the

  bucket turns. Reagents dissolve the sample film

  lon and perform the coupling and cleavage phases of the pro-

  Edman stopped. After complete completion of the coupling and cleavage phases, a vacuum is created in the reaction chamber to remove the volatile constituents from the reactants. After the establishment of the vacuum after coupling, the remaining sample film is extracted with a solvent to

  remove non-volatile components. After putting on life

  from which follows the cleavage, the remaining thiazolinone is extracted from the sample film using a solvent and transferred, either to a separate container for carrying out the conversion phase, or to an apparatus intended collecting and drying the various fractions. In cases where the process

  conversion is not immediately executed in a reci-

  conversion pient, the process can be done later,

  simultaneously on several fractions.

  The introduction of the fluid into the rotating cup and their extraction from this cup was carried out until now by means

  of fluid conduits passing through a plug which closes

  an opening in the upper wall of the room

  of reaction and extended from this stopper down to a certain point inside the cup. Fluids are introduced directly into the bucket

  turning at a point near the bottom of this bucket and we ex-

  line by an annular groove in the inter-

  cylindrical bucket. The fluid to be extracted is forced back into the annular groove by centrifugal force when the

  bucket is rotated at high speed and is ex-

  line by a conduit whose internal end is engaged in the groove. This effluent duct therefore behaves like a scoop to evacuate the products and by-products of the

  reaction as well as the reaction solvents.

  If the protein or peptide sample contained in a rotating cup device does not have sufficient mass to form a coherent film on its own,

  sometimes it is placed on the inner wall of the bucket

  during solvent extraction operations within a

  relatively thick layer of unprotected carrier material

  fuck. Then, the carrier material and the sample are distributed

  solvent in liquid reagents during the recovery phases

  action to allow coupling and cleavage reactions to occur. We previously used a salt for this

  quaternary ammonium polymer having the chemical composition

  15-dimethyl-1,5-diazaundécamé polymethylbromide

  thylene. The support or vehicle must be used in

  important tees so as to retain the sample with se-

  curity and the vehicle and the sample must both be

  soluble in liquid reagents, to allow reac-

  between the sample and the reagents.

  Although rotating bucket type devices can-

  feels to give acceptable experimental results in

  In many cases, these devices have various drawbacks.

  For example, spending to obtain a sample of pro-

  Tein or peptide large enough and to maintain an adequate supply of the necessary reagents are very high, mainly because the reagents used are in liquid form and must be used in large quantities. Liquid reagents and solvents tend to separate parts of the sample from the film and wash them from the wall of the cup during their passage, which reduces the yield in units of terminal amino acids obtained in each of the successive cycles of the 'apparatus. The

  initial quantity of the sample must therefore be sufficient

  large enough to ensure that enough of the sample remains until the end of the last cycle to yield useful results. The devices

  this type also have relatively long cycle times

  due to the considerable volume of the reaction chamber

  tion and the need to repeatedly evacuate reagents and semi-volatile liquid solvents by vacuum drying the sample contained therein. In addition, the

  sequence bucket type fractionation are very com-

  and expensive, both to manufacture and repair.

  A different type of segregation device

  quencé is described in the articles by Laursen and Wachter cited above; in this device, the sample is immobilized by a covalent bond established with the surfaces of a large

  number of small pearls. The pearls form a filling for

  lines placed inside a reaction column and, for the execution of the chemical process, the column is flooded with

  liquid reagents. Since the cleavage reagents

  ge used are excellent solvents of proteins and peptides, it is necessary, to maintain the sample in place, that

  the covalent bond is complete. However, a co-binding

  valente is difficult to obtain in practice. The packed column

  is also difficult to wash and the pearls it

  closes tend to fall apart during use.

  Devices have already been proposed previously

  sequenced splitting designed to overcome challenges

  these of these devices, by enclosing a sample in a fixed reaction chamber and by subjecting the sample to the action of at least one reagent presented in the form of a gas or a vapor. The aforementioned Gilbert and Dreyer patents describe two of these devices, neither of which works entirely satisfactorily.

  The Gilbert patent system includes an extension

  closed in the shape of a finger in a reaction chamber

  intended to contain a sample of peptides or pro-

  teats at a set temperature while exposed successfully

  repeatedly on contact with several reagents and solvents

  zeux. Each time a reagent is introduced, the extension is internally cooled to produce condensation

  on the latter. Then, the extension is heated by provo-

  as for the dissolution of the sample in the liquid

  and the reaction continues. After its reaction with the sample-

  We can either separate the semi-volatile chemicals

  unwanted samples of the sample by drying by combining the effects of heating and an inert gas stream, or washing off these chemicals from the extension with

  the terminal amino acid, using a solvent which is provided

  condensation on the extension until it drips out. In the device and the process of the Dreyer patent, we

  deposits a sample of protein or peptide on the surface

  this internal and the external surface of a large number of small macroporous pearls contained in a reaction column,

  by chemical coupling -or by direct adsorption on these per-

  the. We pass successively different reagents and sol-

  through the packed column, in gaseous or liquid form, to produce the desired degradation reactions,

  The flow of reagents and solvents sent to the column is reduced.

  glided by a rotary distributor working face to face

and ten positions.

  Unfortunately, the devices of the Gilbert and Dreyer patents do not provide an environment sufficiently free of contamination to allow acceptable results to be obtained over a large number of degradation cycles. he

  it is difficult, for example, to effectively wash the sample

  Lon of protein or peptide in the devices of these two patents 249o505. The Gilbert method of washing the sample which consists in condensing a solvent on this sample until the solvent drips from the sample would tend to leave traces of the various products of the reaction on the sample, contaminating the following chemical reactions

  your. Likewise, the lining used to retain the sample

  lon in the reaction column of the Dreyer patent is diffi-

  eyelash wash because if you want to avoid contamination,

  the various chemicals have to go through whole-

  this packing and are completely evacuated from the colon-

  born. This is not easy to achieve, even when using large amounts of solvents, because the solvent tends to pass through the spaces between the beads rather than going through the small inner pores of the beads, pores

  in which most of the sample is gathered

  protein tillon. The fluid inlet conduits and the taps or distributors of the devices of these two patents are also difficult to completely evacuate and tend to

  retain chemical residues which can contaminate

  chemical stops that we want to involve in the cycles

following reaction.

  Glass or plastic beads used

  as packing in the column of the Dreyer patent have also

  tend to disintegrate after a number of degradation cycles, so that they clog the circuit to the point of obstructing the passage of fluids through

  the device. It is therefore practically impossible to wash the device.

  the same between successive cycles and the chemical processes which take place in the column and are thus contaminated without

hope of recovery.

  The effects of contamination resulting from the different

  factors described above are added to each other, a-

  with a cumulative effect, over the duration of a degra-

  sequenced donation. The sample and reagents contained in

  the reaction cell therefore become more and more conta-

  undermined, thereby hampering coupling and cleavage reactions

  desired ages and causing the production of a large number

  adverse reactions. The yield obtained from each cy-

  The complete key of the apparatus is therefore reduced and a whole series of contaminants are introduced into the fractions. The yield is further reduced by the direct loss of material from the sample for various reasons, including disintegration of the packing, the solubility of the sample in washing solvents and the poor quality of the

  sorption links established between the lining and the sample

  tilion. These effects can certainly be overlooked in certain cases, where large quantities of the protein or peptide sample are available, or even when the chain comprises

  a relatively small number of units, but they are

  treux in cases where the chaune comprises a very large number of units or even when only very small are available

  quantities of the particular protein or peptide to be studied.

  We are in the presence of these two circumstances in the case of

  interferon, a small protein that is produced in cells

  human cells in response to certain viral infections. Of-

  then for some time interferon arouses great interest in the field of clinical medicine because it seems to be able to be an effective agent to stop viral infections and

  that he seems to have very high hopes as a

  anti-cancer active. Interferon is only produced and therefore

  only available in very small quantities. Currently

  the entire world production of two types of inter-

  human feron comes from relatively few global centers, which have access to large quantities of blood cells

  white blood cells (leukocyte interferon) or certain

  human cells in tissue culture (interferon

  fibroblastic). This limitation of the production capacity

  interferon, hitherto made it difficult to execute

  well controlled clinical studies and fundamental analyzes

  on the mode of action of this molecule. To further complicate the picture, interferon is composed of a

  chain of about 150 units of amino acids that are needed

  be able to split the chain individually for the analyzes

  ser. Losses by contamination of the types described above can make sequence fractionation impossible beyond the first few amino acid units of 1interferon because only very small amounts of the protein are available. Beyond the first few cleavage cycles, the small sample can be contaminated to such an extent that

  is no longer possible to achieve positive results.

  The most elaborate prior device known

  of the Applicant, for the conversion of the various thiazoli-

  The cleavage of the sample into the more stable phenylthiohydantoines is the conversion vessel described in the above-mentioned articles by WittmannLiebold, modified as indicated in the patent application Hunkapiller and Hood. However, the

  Applicant has found that this container is affected by the incon-

  comes from the fact that its internal walls are not

  effectively washed when reagents and solvents are not

  produced by the appropriate capillary tube. We suggested the

  possibility of introducing reagents and solvents with a

  rant of inert gas to wash the walls of the container in pro-

  throwing the liquid on the walls but this technique works

  delivered by an erratic flow of liquid to the container and

  makes it very difficult to adjust the volume of liquid introduced.

  The Applicant has also found that when the size of the prior art conversion container is reduced considerably so that it contains more

  small volumes of liquids it is difficult to get the de-

  optimal liking for dispersion of inert gas bubbles in the liquid content of the container to agitate the content for

  the conversion reaction and to evaporate the constituents

  semi-volatile kills of content. Inert gas introduced for

  this at the base of the container tends to rise to the surface of the

  consisting of relatively large bubbles which do not agitate uni-

  formally the liquid and, on the contrary, cause projections

  liquid on the top of the container.

  This is why, in many applications, it is desirable to provide an apparatus for carrying out the programs.

  chemical stops such as fractionation of the sequence of pro-

  teins and peptides, which works effectively and with the mini

  contamination of the device so as to allow

  successfully complete the mmdmum number of fraction cycles-

  sequenced with a very small amount of the sample

  Ion. In short, the invention comprises a device forming

  chamber which has an internal surface defining a room

  reaction bre, the chamber being provided with inlet means and

  outlet means which make it possible to circulate fluids

  of through this chamber in a pressurized co2rant, and

  a solid matrix which is permeable by diffusion to a se-

  fluids and which is housed in the room such

  deny that a sample embedded in this matrix is immobilized

  read and exposed to each of the fluids that are passed through

  the chamber for chemical interaction with these fluids.

  This device forming cha "bre can include an over-

  face which supports the solid matrix in the form of a pel-

  thin ligule deposited on this surface and the matrix can

  take a polymeric quaternary ammonium salt, such as 1 5-dimethyll, 5-diazaundecamethylene polymethylbromide

  or poly (NN-dimethyl-3.5-dimethylene chloride piperidi-

  nium). This surface can be made up of all or

  by a part of the internal wall of the chamber forming device

  or it can be formed by means of

  me of porous sheet which extend approximately transversely-

  lie across the chamber and allow fluids to pass through. These means may include a sheet made

glass fibers.

he

  The chamber device may include a pair of

  re of face elements coupled face to face which pos-

  a first and a second cavity formed on their opposite junction surfaces, respectively, the first and second cavities being aligned with each other to

  form the reaction chamber. The first and second ca-

  Vities can gradually shrink in directions which move away from the junction surfaces to points where they communicate respectively with the input means and with the output means. The means in the form of a porous sheet which may be used are housed in a recess

  formed in at least one of the junction surfaces in a manner

  re to be retained in the chamber in an orientation in which they practically separate the first cavity from the second cavity. The chamber device can be provided with at least one sheet of flexible material clamped between the joining surfaces, in a provision establishing a

  waterproof seal, said flexible material being permeable to fluids

  of. At least one of the chamber elements can then comprise

  ter a raised part formed on one of said surfaces

  junction and which surrounds the cavity of this element to com-

  primer the sheet of flexible material against the surface of the rod

  tion of the other chamber element and, in this way, strengthen

check the seal.

  The inlet and outlet means may comprise two capillary passages which pass respectively through the two chamber elements and communicate at their interior ends with the reaction chamber on either side of the porous sheet. Capillaries can be coaxial

  to the reaction chamber and extend from this cham-

  bre to external capillary orifices located at the

  calf with approximately flat external surfaces of the chamber elements.

  The means used to pass successively more

  fluids through the chamber may include a dispenser block including a series of shutter zones

  roughly flat on the surfaces, the distributor block presented

  both a continuous primary passage which extends between its two ends and communicates through primary openings with each of the closure zones and a series of passages

  secondary each of which communicates through a secondary opening

  re with one of the obturation zones as well as a series of elastic membranes, roughly impermeable, which respectively cover the obturation zones, each of the membranes

  can be operated between a first state or an iron state

* method, in which it is applied by force against one of the obturation zones to close the primary openings

  and secondary communicating with this area and a second é-

  state in which it is moved away from the zone to establish a fluid flow passage between the primary orifice and the secondary orifice on the outside of the distributor block, so that the flow of fluid between the primary passage

  and the secondary passage can thus be controlled selectively-

  is lying. The connection means may include at least one conical ring (in this specification, the

  term "conical" to designate a conical shape or co-play

  nique) sliding assembly without play on a tube element

  and pushed against a conical cavity whose slope is different

  annuity, arranged in the distributor block and which communicates

  with one of the passages in this block so that a direct force

  inwardly applied to the ring is concentrated

  trée on a relatively small contact surface between the ring and the cavity to establish a fluid tight seal between these two elements. The cavity preferably has a

  larger cane angle than the ring. The means of connection

  may also include a fitting provided at a

  end of the primary passage to connect the primary passage

  mayor has another part of the apparatus so that the passa-

  primary age plays the role of a collector which can be washed

  by a flow of fluid between the fitting and the secondary passage

  the furthest from the fitting. The primary passage may include a series of rectilinear passages arranged end to end to form a conduit having a tooth configuration

  saw and which communicates with the different obturation zones

  ration to one in two of its intersections. The apparatus may include a conversion vessel provided with several capillary tubes which penetrate into its

  internal volume to allow the insertion and extraction of di-

  towards fluids, at least one of the capillary tubes having an inner end to which the light is closed and which is provided with several throttled orifices spaced in a radiating arrangement so that the passage of the fluids in the capillary tube produces a projected split jet sure

  the internal surfaces of the container for washing its surfaces.

  A capillary tube which ends at a point adjacent to the bottom of the bottle may also have a closed end,

  with several constricted orifices spaced in a arrangement

  tion radially, adjacent to this end, so that the

  gas passing inward through the capillary tube

  re produces a large number of small bubbles which agitate any liquid contained in the container and accelerate the drying

chage of this liquid.

  In the process according to the invention for the sequential execution of chemical processes on a sample of a

  chemical matter we drown the sample in a solid matrix

  of which is permeable by diffusion to a series of fluids, the solid matrix is enclosed in a closed chamber which has an inlet and an outlet and the fluids of said series of fluids are successively passed through the chamber under

  the shape of a current under pressure, from the entry of the

  bre at the outlet of the latter, so that the sample is thus exposed to the action of each of the fluids, the sample

  thus being immobilized a chemical interaction between the sample

  tillon and the-fluids being produced. The solid matrix can be carried on the internal walls of the closed chamber in the form of a thin film. Alternatively, this solid matrix can be carried on a porous sheet which extends approximately transversely across the chamber at a location between the inlet and the outlet so that the fluid which passes from the inlet to the exit either cons

  traitor to cross the sheet. The flooding stage of the-

  sample in a solid matrix can understand the phases

  consisting in depositing the solid matrix on a supporting surface

  in the form of a thin film and then to deposit

  a solution containing the sample on the film of ma-

  so that the sample solution dissolves the matrix. In-

  subsequently, the liquids are evaporated from the solution, thus leaving

  if a film in which the sample is embedded.

  An object of the invention is to provide an apparatus and a method for the sequential execution of several chemical processes on a sample of a chemical material with the minimum loss of material from the sample and the minimum of

contamination of the whole.

  Another object of the invention is to create an economical apparatus and method for the sequential execution of

  chemical processes on a very small sample

  thanks to the possibility of using minimal quantities

reagents and solvents.

  Another object of the invention is to create an improved apparatus and method for the sequential execution of

  chemical processes with a very short cycle time.

  Another object of the invention is to create an apparatus

  and an improved method for sequential execution-

  the several chemical processes on a sample, thanks to which the sample is washed more efficiently between

successive cycles.

  The apparatus and method according to the invention solve

  a number of problems with fracture devices

  sequence of the prior art by immobilizing the

  protein or peptide sample in a solid matrix

  made in the form of a thin film which is permeable to

  ble by diffusion to reagents and solvents used in the degradation process. This way we avoid having

  to solve the difficult problem of obtaining a link

  its complete covalent which constitutes the problem of loss of

  material of the sample encountered when the sample

  tillon is directly adsorbed on a load-bearing surface and is fully exposed to mechanical shear forces

  exerted by the mobile liquid phase. The sample is soli-

  duly retained in position by the matrix while the

  active ingredients, solvents and amino acid derivatives present in small quantities are capable of diffusing through the matrix,

  actually dissolving in the matrix at a concentration

  sufficient to carry out the various stages of the process

  no degradation. The solid matrix retains the sample sufficiently efficiently during exposure of the sample to gaseous reagents that virtually any form of sample carrier surface can be used without

  suffer loss of sample material. The inter-

  nes of the reaction chamber can constitute themselves

sufficient bearing surface.

  A load-bearing surface which considerably facilitates the

  total circuit wash is a porous sheet made of thin

  glass bres which overlap each other and which extends

  transversely across a flow reaction chamber

  slightly crossing. The porosity is ensured by the spaces between the fibers. This structure has a relatively large total area with a minimum dimension in the direction of flow of the fluids. The solid matrix forms

  a thin film on the fiber surfaces, allowing

  both reagents and solvents easily diffuse in the film to act on the sample embedded in this film. This allows the chemical processes to be carried out and

  wash cycles on the sample with a minimum amount

  number of reagents and solvents and also over time

  laterally short. Reagents and solvents, some of which are in the form of a gas or vapor, pass through the thin film by permeability so as to come into contact with the sample and act on the latter as completely and efficiently as possible. The relatively thin profile of the porous sheet which is described in this specification further enhances the ability of the sample to be completely cleaned by washing the

  leftover reagents and reaction products using a quan-

  tity of relatively weak solvento To evacuate the reagents and reaction products from the assembly, the solvent only has to move them over a relatively short distance beyond the surface of the sheet 0 From a point situated at the top of the the surface of the sheet these reagents and reaction products can be easily removed from the chamber to leave the sample in good condition for the reaction phase

  next. Not only the low consumption of solvent re-

  saves on the cost of the solvent but also reduces the tendency of the sample to be removed from the reaction chamber by washing and thus lost. Use

  of reagents in the form of gases or vapors also contributes

  to fully expose the sample to contact with

  active ingredients, reducing the amount of reagents required. The low consumption of reagents is important because the

  reagents used in the Edman degradation technique doi-

  may be extremely free from contamination and therefore very costly. Furthermore, the sample and the solid matrix which contains it are not dissolved by the gaseous reagents, which eliminates the problem of loss of

  material of the sample resulting from the separation between the

sample and the bearing surface.

  The reaction chamber according to the invention is made up of

  trout so as to allow gaseous reagents, as well

  that liquids, to cross the porous sheet which carries the

  sample without allowing the sample to be contaminated with

  external impurities or chemicals transferred

  res from one phase or cycle of reaction to another or another. The elements of the chamber which abut against each other combine in this way to form a reaction chamber of small volume which is composed of a first and a second cavity respectively located

  on either side of the porous sheet. Two capil-

  laires which extend one opposite the other respective-

  lie through the two elements of the chamber, taking

  birth on the bedroom itself, allow to do not-

  ser several fluids, in gaseous or liquid form,

  a current under pressure through the chamber and on the ma-

  sample holder. The small volume of the chamber and

  passages reduces the reaction volumes to a minimal value

  necessary solvents and facilitates drying of the material.

  appears by depression between successive cycles. The two é-

  elements of the chamber are assembled with a tight seal at the

  calf from their mating surfaces, pressing against a sheet of flexible or compressible material clamped between the mating surfaces. This flexible material is permeable to the series of fluids which are passed through the chamber and,

  in fact it improves the flow of gases through the chamber.

  bre by dispersing the gases so that they come into contact

  more uniform with the porous sheet.

  An alternative embodiment of the reaction chamber is constituted by a single capillary tube or by a single

  passage of the capillary type carrying a solid matrix reali-

  seated in the form of a thin film which is deposited on

  its internal surface or in its light. The sample of pro-

  tine or peptide is embedded in the matrix, as in the case of porous material and the reagents and solvents are passed successively through the tube so that they act on the sample. The chamber structure described above can be used for this application without the element constituted by the porous sheet. The matrix containing the sample

  is then deposited on the surfaces of the first and the.

  second cavities. Likewise the surface supporting the matrix so-

  the idea can be built in almost any way

  lesson which allows the fluids constituting the reagents and solvents to pass over the matrix.

  The new distributors used according to the invention

  tion to control the flow of fluids to and from the conversion chamber or bottle are specially constructed to eliminate mutual contamination of the fluids. Each of the distributors forms an interface between a single conduit and several other conduits, in order to make it possible to selectively connect said single conduit to each of the others. The single duct is placed in communication with one end of the primary passage of the distributor block

  while each of the other conduits is connected to one of the pas-

  secondary wise men. In the normal closed position, each of the membranes which cover the different sealing zones is applied by a gas pressure against the surface of the

  block to oppose communication between the main passage

  mayor and the secondary passage which lead to the area of obtura-

  particular tion considered. We can selectively establish

  a communication for the passage of the fluid between said con-

  single duit and the other conduits by transmitting a depres-

  sion to one or more of the membranes so as to separate

  the membranes of the obturation zones and to allow the

  to pass over the surface of the distributor block which is so-

  killed between the openings giving respectively on the pas-

  wise. The secondary passage which leads to the obturation zone situated at the end of the primary passage can be connected to a source of pressurized sweeping fluid, for example to a source of inert gas, to ensure the transmission of each fluid through the distributor. In this way, when

  specific reagent or solvent has been introduced into the chamber;

  bre of reaction, by applying a depression to the mem-

  corresponding brane, we open the membrane located at the end of the primary passage to complete the distribution by chasing

  towards the chamber the reagent or solvent which remains possible-

  lie in the primary passage. This is made possible by the continuity of the primary passage and results in the fact that the collector formed by this passage can be purged of a reaction.

  specific reagent or solvent before the delivery of the reagent

  tif or next solvent does not start. In the case of the distribu-

  tor located at. the exit from the reaction chamber, the passage

  primary is connected to the output while the secondary passages

  These are connected respectively to the conversion container, to the vacuum and to the evacuation. The continuity of the primary passage

  allows to completely evacuate this passage and removes practical-

  any risk of retaining semi-volatile substances

  in this passage between successive cycles.

  It goes without saying that the "sawtooth" configuration of the primary distribution passages which is described in the present specification has already been used previously in distributors belonging to fields other than that

  sequence fractionators. However, the dis-

  tributaries with sawtooth ducts of the prior art

  included a series of individual blocks mounted

  against shutter zones on a main block so

  re to slide alternately between workbench positions

  communication and removal of communication with pass

  ges arranged in the main block. The sliding blocks

  tooth to wear by giving rise to leaks on the atmosphere and between a passage and the others. New distributors

  described in this memo solve the problem of

  sure by combining the sawtooth collector of the tech-

  anterior nique with a series of membranes to establish and interrupt the flow between pairs of openings which communicate respectively with the different passages. The

  membranes can be made of virtually inert material

  such as fluoride carbide polymers which are commercially available and which are capable of working indefinitely without deterioration. In addition, the means used

  in the prior art for connecting the passages of dis-

  tribution to outdoor conduits tend to produce a

  excessive pressure on the sides of the distributor block, in fact

  thus promoting a deformation of the upper closure surface of the distributor block and the loss of the ability of this surface to establish a tight seal. In particular,

  prior art sawtooth distributors

  connect the external conduits to the distribution passages in

  compressing substantially flat flange surfaces

  connected to the various conduits against the sides of the distribution block

  tor to produce a series of joints between pairs of sur-

  flat faces. Since each of these components is

  made of a material such as polymers of fluoro carbides

  res are very difficult to machine with precision, we must

  apply considerable pressure to conform the surfaces

  these respective seals to each other and form the necessary seals. The pressure is supported by the distributor block, which determines a deformation of its

  upper sealed surface area.

  The distributors according to the invention comprise a series of conical rings which are partially housed in cavities having a different slope of c8ne and formed in the distributor block to establish a tight seal without applying excessive pressure to the distributor block. A relatively low sealing force is concentrated on a certain part of the ring in order to apply the ring sealingly against the corresponding conical cavity

without distorting the block.

  The presence of interfaces formed at opposite ends

  rings between surfaces with angles

  of different cones further improves the quality of the seals

  tench obtained. Interfaces of this type are preferably provided between surfaces of different cone angles on the two

  opposite sides of each ring in order to obtain characteristics

optimal sealing.

  The conversion container according to the invention makes it possible to introduce the reagents and solvents in the form of divided jets which strike the internal surfaces of the container to wash off the chemicals which may have been

  condensed or have been projected onto these surfaces by

  weaving down the walls and making them reach the mass of liquid contained in the container. We eliminate

  in this way an important source of cross-contamination

  circuit circuit between successive cycles. The conversion vessel also allows gases to pass from bottom to top in the mass of liquid in the form of small

  bubbles which uniformly agitate the liquid and help to provo-

  evaporation of the semi-volatile constituents of this liquid

  without causing excessive loss of sample material

  lon which would result from an excessively energetic bubbling and projections. This promotes rapid and gentle elimination of the liquid from the fragile amino acid derivatives. The solvent

  used to deliver amino acid derivatives to the container

  conversion pient can in this way be eliminated in

  much shorter time than in the Wittmann-Lie patent-

  bold above (1 or 2 minutes against 5 to 10 minutes) and at a lower temperature (40 to 50 OC against 50 to 80 0C). This

  significantly improves yields of acid derivatives

  the most unstable amines such as the derivatives of serine, threonine, histidine, arginine and tryptophan. The

  reagents used in the conversion vessel can be

  be eliminated by a combination of fine streams of inert gas bubbles and a low vacuum, in a time of 3 to 5 minutes, against the 30 to 50 minutes which were necessary in the case of the Wittmann-Liebold patent. In the process of

  Wittmann-Liebold patent, evaporation of the reagent must be

  to start immediately after its introduction on the

  amino acid residue in the conversion container

  sion, in order to satisfy the condition that the total cycle time of the conversion vessel must not

  be greater than the total time of the reaction chamber cycle

  primary tion. Since the acid component of the reaction

  conversion tif, which is trifluoroacetic acid or a-

  hydrochloric acid, is much more volatile than water in

  which is dissolved, the acid component tends to be eli-

  mined early in the Wittmann evaporation process-

  Liebold, leaving the amino acid derivative in a solu-

  tion during a significant part of the

  conversion. This results in incomplete conversion of the derivatives.

  of wisteria and praline and the breakdown of some

  other derivatives. In the conversion device according to the

  vention, the conversion reagent can be left in contact with the amino acid derivatives for a sufficient time to obtain a complete conversion, more precisely for 40 minutes and it can then be evaporated quickly,

  in 3 to 5 minutes, without giving rise to sample projections

  over the entire internal surface of the conversion container

  if we. Other characteristics and advantages of the invention

  will appear during the description which follows. To the

  attached drawings, given only by way of example, - FIG. 1 is a perspective view of an apparatus constructed according to the invention; - Fig. 2 is a plan view of the apparatus of FIG. 1; - Fig. 3 is a diagram of the apparatus of FIG. l; - Fig. 4 is an exploded perspective view and at

  larger scale of a set of reaction chambers

  trout according to the invention; - Fig. 5 is a large-scale vertical sectional view taken along line 5-5 of FIG. 4;

  - Fig. 6A is a cross section at scale in

  larger core of the reaction chamber shown in FIG. 5;

  - Fig. 6B is a cross-sectional view of two

  tenth embodiment of the reaction chamber shown

  felt in FIG. 5; - Fig. 6C is a section through the reaction chamber of FIG. 6B from which the leaf-shaped element has been removed

  porous, for use with a film containing the

  sample, which is applied against the internal surface of this chamber;

  - Fig. 7 is a vertical section of a typical example

  that tank according to the invention intended to contain a

  reagent or solvent to be used in liquid form

of;

  - Fig. 8 is a vertical section of an example ty-

  tank spike according to the invention intended to contain a reagent which must be used in the form of a gas or a vapor; - Fig. 9 is a vertical section of a dispenser

  with membranes constructed in accordance with the invention for com-

  direct the flow of fluids towards and from

* nance of, the reaction chamber and the conversion vessel

  view, the view being taken in a direction corresponding to line 9-9 of FIG. 11; - Fig. 9A is an enlarged partial sectional view of one of the connector elements of the distributor shown in FIG. 9; Fig. 10 is a vertical section taken along line 10-10 of FIG. 9; - Fig. 11 is a side elevation view of the manifold block of the distributor shown in FIG. 9; - Fig. 12 is a horizontal section taken along line 12-12 of FIG. 11; - Fig. 12A is a vertical section taken on the line 12A-12A in FIG. 11;

  - Fig. 13A is a partial section at larger

  scale of the shutter part of the dispenser of Fig.9 which shows the membrane pressed against the manifold block to oppose the transmission of the fluid between the passages in this zone;

  - Fig0 13B. is a partial section also at large

  scale of the obturation part of the distributor of Fig. 9 which shows the diaphragm diaphragm of the manifold block to allow the fluid to flow between the passages; - Fig. 14 is a top view of a conversion container constructed in accordance with the invention; - Fig. 15 is a vertical section taken along line 15-15 of FIG. 14 - FIG. 16 is a vertical section taken along line 16-16 of FIG. 14; - Figs. 17A and 171 schematically illustrate the

  two main processes used in the prior art

  re to immobilize a protein or peptide sample during its degradation; - Fig. 17C schematically illustrates the immobilization of a protein or peptide in accordance with the invention;

  - Fig. 18A is a partial vertical section A large

  of scale which corresponds to FIG. 5 and shows another form-

  the embodiment of the reaction chamber assembly according to the invention;

  - Fig. 18B is a vertical section showing the element

  Reaction chamber ment of the embodiment of FIG. 18A, which has been tilted on the side to deposit a matrix containing a sample on its internal surfaces; and

  - Fig. 18C is a partial section, 6-scale in-

  larger core of the chamber element of FIG. 18B, with

  a film containing a sample applied to its surface

internal faces.

  Table 1 is a list of the various stalls executed

  tees by the device according to the invention in a typical example

  degradation and conversion cycle.

  We will now refer to the drawings on which we

-2490505

  has shown, in Figs. 1 and 2, a device according to ltin-

  vention which is designated as a whole by the reference 10.

  The machine 10 comprises an enclosure forming a chamber 12, a

  conversion vessel 14 and a fraction collector 16, each of these members being controlled by an automatic control unit 18. A row 20 of solvent tanks and

  of pressurized reagents are connected to the reaction chamber

  tion 12 and to the conversion container 14 via

  of a distributor block 22 with membrane. A second block available

  tributor 24 controls the flow of liquids from the reaction chamber to the conversion vessel 14 and to other points. A third diaphragm distributor block 26 serves to

  connect the conversion container and the collector

  either to waste disposal or to a source of

  of, and controlling the flow of fluid from the container to a

fraction collector.

  A source of filtered inert gas 28 supplies the apparatus with high purity inert gas, preferably consisting of

  argon, to pressurize the soil tanks

  and reagents, purge the circuit of air containing

  oxygen and accelerate the evaporation process of the reacts

  tives and solvents contained in the circuit at various times.

  A block 30 of pressure regulators and manometers is used to individually adjust the pressure of the gas sent to each

  solvent or reagent reservoir as well as each of the

very device organs 10.

  The operation of the apparatus 10 is exposed to the

  Fig. 3. The device 12 forming a chamber is placed in a

  heated environment 32 and, in the preferred embodiment

  tion, it includes a reaction chamber 34 which communicates

  with an inlet passage 36 and an outlet passage 38 res-

  pectively. The inlet passage 36 can be connected by a fluid conduit 40 to a series of reservoirs in the row, more precisely to the reagent reservoirs 42, 44, 46

  or 48, and to the solvent tanks 50, 52 and 54. The reservoirs

  see 42 to 54 are pressurized by the pressure source

  inert gas 28 via a row of

  individual gas pressure regulators 56 and electro-

  valves 58. Each of the reservoirs 42 to 54 can also be connected, at a point situated above the level of the fluid contained

  naked in this tank, to a waste trap 60 through-

  diary of an individual valve 62 and an individual regulator

  dual 64. The inert gas introduced into the reservoirs

  coming from the source 28 can thus be evacuated towards the waste trap 60, a flow rate regulated by the regulators 64. The

  fluid outlets from tanks 42 to 54 are controlled in-

  dividually by membrane valves 66 which communicate with a continuous manifold 68 which is connected to the fluid conduit 40 at a first end. The fluid delivered by the pressure tanks 42 to 54 can thus be sent via the conduit 40 and the chamber inlet passage 36, to the reaction chamber 34 by the selective operation of the valves 66. To facilitate the delivery of the reagents and solvents and

  to sweep the collector 68 and the conduit 40 after delivery

  In addition, an inert gas can be introduced under pressure from

  from the source 28 in the collector 68, at the end which

  is opposite the duct 40, via a regulator

  pressure lator 70 -t of a membrane valve 72. The operation

  vre of the valve 72 therefore introduces a gas under pressure at ltex-

  far end of manifold 68, thereby driving out the re-

  active agents and solvents possibly contained in this collector through the conduit 40 and the passage 36 to send them to

the reaction chamber 34.

  The fluid outlets of the tanks 44, 46 and 48 are equipped with gas flow meters 74 mounted in series with the flow regulators 76, since the reagents stored in

  these tanks are used in the gaseous or vapor form

  fear while other solvents or reagents are used

  in liquid form. The 74 meters and fault regulators

  bit 76 are required to precisely control the

  gas outlet bit through the corresponding valves 66.

  The flow of fluid from the reaction chamber 34 is controlled by the membrane valves 78, 80

  and 82, which communicate with the outlet passage 38 via the

  through a continuous collector 84. The valve 78 opens to let the desired reaction product through, which can

  for example be the amino acid unit at N terminal of an e-

  protein or peptide sample, to the conversion flask 14 via the conduit 86. The valve 82 can open to connect

  the outlet passage 38 to the waste trap 60 for elimination

  reagents, solvents and unwanted reaction products

  bles and the valve 80 connects the outlet passage 38 to a vacuum trap 88 and to a vacuum pump 90 intended to create a vacuum in the reaction chamber 34, the outlet passage 38 and the

collector 84.

  Likewise, the conversion container 14 and the fraction collector 16 can be connected to the waste trap 60 by means of valves 92 and 94 respectively and to the vacuum.

through valves 96 and 98.

  The reagent tanks 100 and 101 and a solvent tank 102 are provided for sending the reagents and the solvent to the conversion container 14 via a conduit 104.

  tanks 100, 101 and 102 are pressurized by the sour-

  this of inert gas 28 via pressure regulators 106 of solenoid valves 108 and they are connected to the trap

  to waste 60 via individual purge valves

  dual 110 and flow regulators 111. The fluid outlets of the tanks 100, 101 and 102 are connected by

  diaphragm valves 112 to a continuous manifold 114 which comprises

  provided at one end with the conduit 104. The flow of fluid from the pressurized tanks can therefore be obtained by selectively opening the valves 112 to expel the reagent or the solvent to the manifold 114. The source of inert gas

  can be connected by a pressure regulator 116 and a valve

  ve with membrane 118 at the end of the collector 114 which is opposite the conduit 104 for expelling the reagent or the solvent towards the conversion container 14 by passing it through

the collector and the conduit.

  The inert gas source 28 is also connected to the conversion container via a pressure regulator 120, a membrane valve 122 and a conduit 124 and to the fraction collector 16 via a pressure regulator 126 and a valve 128. When a particular fraction has been converted in the desired manner into the container 14, it can be expelled from it by a gas pressure passing through the conduit 124 and a valve 131 for lead to the fraction collector 16 so as to

  be stored in a vial of this collector. When the frac-

  tion has been expelled, container 14 can be filled with soil

  up to a relatively high level to dissolve

  any residual chemicals that it contains.

  The solvent can then be expelled by gas pressure to the waste trap 60, passing through the conduit 124 and a valve 125, to sweep the container and prepare it for

  receive the following amino acid derivative.

  The fraction collector 16 essentially comprises

  a vial carousel which is operated by the com- pany unit

  mande 18 once at each cycle of the apparatus 10 so as to place an empty vial in position to receive the fraction

  next amino acid units from the reci-

pient 14.

  The control unit 18 is preferably a unit

  fully automated which controls diaphragm valves

  66, 72, 78, 80, 82, 92, 94, 96, 98, 112, 118, 122, 125, 128

  and 131 as well as the solenoid valves 138, 62, 108 and 110, L'u-

  control unit 18 also controls the mechanism which serves

  to keep the heated environment 32 at the desired temperature

  rée (not shown) as well as the fraction collector 16, the vacuum pump 90 and a whole series of sensors distributed throughout the circuit. The various gas pressure regulators and flow regulators which are described above are adjusted manually during the adjustment so as to establish the desired pressures and flow rates in the conduits.

corresponding fluids.

  S The device 12 forming a chamber is depicted

  details in Figs. 4 and 5 and it comprises a base 130 in two parts which supports a sleeve 132 containing a first and

  a second chamber elements, 134 and 136 respectively.

  The sleeve 132 is provided with an enlarged cylindrical part 138 centered on the axis of the sleeve and which fits without play in a cylindrical cavity 140 at the base 130. The cylindrical part 138 is held in position by a retaining ring 142 which

is screwed onto the base 130.

  The elements 134 and 136 of the chamber are cylindrical glass elements which have opposite joining surfaces 144 and 146 respectively and are mounted without play in axial alignment inside the sleeve 132. The inlet and outlet passages exit 36 and 38 described above cross

  axially the elements 134 and 136 of the respective chamber-

  and are preferably capillary passages having a

  diameter of the order of 1 mm. The axially outer ends

  nes 48 and 150 of elements 134 and 136 respectively of the

  chambers are generally flat and in abutment against two rounds

  thin and elastic ones made of a practically inert material, to give the chamber elements a suspension

  elastic in the axial direction relative to the sleeve 132.

  A metal washer 154 placed above the washer

  upper elastic 152 is fitted with op-

  posed 156 intended to engage in slots 158 of the ex-

  upper end of the sleeve 132. The metal washer 154 is held in position by a cap 160 which is screwed onto the upper end of the sleeve 132 so as to hold the

  chamber elements in place without play with respect to the sleeve.

  The grip between the ears 156 and the slots 158 prevents the washer 154 from turning while the cap is being mounted, which prevents the cap from damaging the assembly by rotating the chamber elements. The fluid conduit 50 is provided with a flared lower end 162 which abuts against the outer end 148 of the element 134 of the chamber, so that the lumen of the conduit 40 thus communicates with the inlet passage 36. The conduit 40 carries a thrust washer 164 and a connector 166 which is screwed axially in the cap 160 to apply the flared end 162 by force

  against the element 134 of the chamber in a sealed manner. The con-

  product 40 can be made of any substantially inert elastic material such as commercially available fluorescent carbide polymers. The alignment between the lumen of the conduit 40 and the inlet passage 36 is ensured by the precision mounting of the various members assembled around a

common axis.

  The outlet passage 38 is placed in communication with the continuous collector 84 which has been described above by a mass 172 of approximately inert material which is housed in a

  steel sleeve 174. The sleeve 174 has an ex-

  dull smooth which is housed in aligned axial holes 176 and 178 of the bulged cylindrical part 138 and of the base 130 respectively. The mass 172 is extended axially in both directions beyond the steel sleeve 174 to rest on the external end 150 of the second element 136 of the

  chamber and lean in a conical cavity 180 of a distri-

  stopper 182 which defines the manifold 84. An axial passage 184 formed in the mass 172 is exactly aligned with the outlet passage 138 and one end of the manifold 84 to form a single continuous capillary passage connecting the element 136 of the chamber to the distributor block 182. As in the case of

  duct 140 which has been described above, the mounting of

  the corresponding organs around a common axis ensures

  the precision of the alignment of the various passages and the forma-

  tion of gaskets fully sealed at their junctions. The dis-

  positive 12 can easily be disassembled and reassembled in a very short time without compromising the alignment of the various passages

nor the integrity of the various joints.

  The distributor block 182 is of a new construction

  which will be described in detail with reference to FIGS. 9 to 13. At this stage of the presentation, it will suffice to note that parts of the valves 78, 80 and 82 are included in the distributor block

  182 to control the fluid outlet of the device 12.

  As can be clearly seen in FIG. 6A, the cham-

  bre 34 is formed by aligned cavities 186 and 188 mena-

  in the opposite junction surfaces 144 and 146 respectively

  of the two chamber elements. The two cavities are arranged coaxially with the inlet and outlet passages 36 and 38 and are preferably of circular section, so that they form a passage with axial asymmetry for the fluid.

  flowing from the inlet passage to the outlet passage.

  An element 190 in the form of a porous sheet extends transversely

  dirty across the reaction chamber 34 and can be

  housed at least partially in a recess 192 of the housing

  vity 186. The element of the porous sheet type 190 thus separates the inlet passage 36 from the outlet passage 38 so that the fluids which circulate from one to the other of these passages must necessarily pass through this element. This element is preferably constituted by a sheet or a cover

  made of compressed fibrous material such as glass.

  The glass fiber filters that can be found in the

  trade are suitable for this application and they

  have a high resistance to decomposition and other deteriorations during use. It has been found that a porous sheet of this type has a relatively large surface to support a thin film in which a sample of protein or peptide can be embedded. If the film is made of a material permeable to fluids, that is to say a material which lets liquids and gases diffuse in its mass, this material can form a solid matrix which is capable of retaining securely the :sample

  but allows chemical interaction between reagents and sol-

  and the sample. Polymeric ammonium salts quater-

  such as the 1,5-dimethylbromide-1,5-dimethylbromide

  zaund6camethyl-ne and poly (NN-dmxechy chloride! -3.5-

  dimethylene pipôridinium) are ideally suited for this-

  application. They allow the diffusion of fluids, are insoluble in the solvents used and are chemically stable with respect to reagents and solvents. In addition, they

  form a coherent film and carry a positive charge

  ve which allows them to form an ionic bond with the sur-

load-bearing glass face.

  The fundamental differences which distinguish the

  my sample retaining devices used in

  sequenced training devices of the prior art

  re of those of the present invention will be described very clearly.

  compared to Figs. 17A, 17B and 17C. Fig0 17A and

  17 schematically illustrate the two: the most

  muns which were used in the prior art to immobilize a 300 protein or peptide sample by

  relative to a sample carrying surface 302 or 304.

  Fig. 17A illustrates the case in which the sample is chemically linked to a glass carrier surface 302 by covalent bonds 306. For example, the surface 302 can be specially treated so that some of the silica sites of the glass carry groups 308 amino functional groups intended to react with the carboxyl groups 310 of the & chantillono chain Under appropriate conditions, some of the groups 308 and 310 react with each other, thus leaving the sample fixed to the glass by covalent bonds and releasing a certain number of water molecules. The bonds of this type are very strong and one or two of these bonds per molecule are sufficient to hold the sample in place. However, covalent bonding is difficult to achieve in the case of samples of the protein and peptide types. Furthermore, the covalent bonds hold the chain through a very small number of units isolated from this chain. When the degradation process reaches these units and separates them from the chain, the rest of the chain is left without fixation and can be removed from the chain.

room by washing.

  Fig. 17B illustrates the case in which the sample

  300 is directly adsorbed on a support surface 304.

  The sample is then held in place by a very large number-

  bre of relatively non-covalent, 312 interactions,

  which are established between the sample and the surface. At ni-

  molecular calf, the surface interacts with a large number

  from different sites in the sample. This works well.

  especially in the case of large proteins or peptides but, when the sample is divided or undergoes a sequence fractionation and reaches a much smaller dimension, l is then subject to detach from the surface under the effect of shock or traction and this results in a significant loss

  sample material. Fig. 17C illustrates diagrammatically

  only the mode of immobilization of the sample 300 by

  with respect to the bearing surface 314, according to which one drowns the

  sample in a solid matrix 316 formed on the supporting surface in the form of a thin film. As described above, the matrix 316 can be a polymeric quaternary ammonium salt which has a positive charge. The matrix is thus thus retained on the acid surface of the glass

  by a very large number of ionic interactions and it main-

  holds the sample securely in place since this sample

  tillon is embedded in this matrix. The fixation is not

  entrusted to direct link interactions between the

  tillon and the surface and the effectiveness of the immobilization

  is not affected by the size of the sample.

  The invention is based on the diffusion of reagents,

  solvents and amino acid derivatives through the matrix so-

  lide 316 for the formation of chemical interactions with e-

  drowned sample. The matrix is produced in the form of a thin film which adsorbs the zreagents and the solvents which are passed over this matrix. When dissolved in the film, the reagents and solvents are capable of easily diffusing through its thickness to effect

the degradation process.

  If we now refer to FIG. 6A we see that

  the reaction chamber 34 is closed at the pcriph6rie of the ca-

  visited 186 and 188 by at least one sheet 194 of a material

  flexible for joints which is clamped between the ring surfaces

  tion 144 and 146. Two sheets are preferably used.

  ples 194, on either side of the leaf-shaped element

  porous 190. The leaves 194 are thin and they are

  adaptable to the series of reactive fluids and solvents that it

  git to pass through chamber 34. A rib or

  an annular sealing bead 196 provided on the periphery

  line of the cavity 188 bears against the sealing sheets 194 to establish a more effective seal on the surface 144 the

  along the periphery of the cavity 186. The leaves of

  194 can be made of any material a

  nearly chemically inert such as car-

  commercial fluoride bures, to minimize

  risk of damage to watertight seals. These sheets are

  wind not only to ensure the hermetic closure of the chamber 34 but also to support the porous sheet 190 and to diffuse the gases and liquids which are passed through the chamber so that the current of gas and

  liquid is more evenly distributed over the cross section of the

  element 190. This favors obtaining a long duration of

  equipment and optimal chemical interaction.

  Another embodiment 34 'of the reaction chamber according to the invention is shown in FIG. 6B; in this embodiment, the aligned cavities 186 ′ and 188 ′ formed in the opposite junction surfaces of the two elements of the chamber are slightly narrower and longer than the cavities 186 and 188. In all other respects

  the structures 34 and 34 'are identical and the various elements

  elements of the structure 34 'shown in the drawing are

  signed by references similar to those of structure 34, with the addition of "prime" (t) to distinguish them. The

  reaction chamber 34 ′ allows a light flow to be obtained

  more direct between the 36 'inlet and the 38t outlet but

  it decreases the diameter of the leaf-shaped element for

  reuse 190 'contained in this room.

  Fig. 6C shows the chamber 34t in FIG. 6B, from which the element of the porous sheet type 190 ′ has been removed. In addition, the sealing sheets 194 'are replaced here by a single annular sheet 316 made of a flexible material and which has a central opening of the same diameter as

  bedroom. In this embodiment, a matrix so-

  fluid-permeable liquid 318 in which is embedded a

  protein or peptide sample is formed on the pa-

  kings of room 341 in the form of a thin film,

  so as to be exposed to contact with reagents and sol-

  that we pass in the room. In this way, it favors the flow of fluids through the chamber 34 #

  however, a large area of film is retained.

  Another embodiment of the chamber device according to the invention is shown in FIGS. 18A, 18B and 18C; here the two elements 134 and 136 of the room

  are replaced by a single chamber element 320 of type ca

  pillar housed in the sleeve 132. The other elements of the device 12 are the same as those which have been described in

* look at Figs. 4 and 5 and they are designated by the same re-

  ferences. All the structures and connections external to this device are also identical. those who have been

described above.

  The chamber element, 320 consists of a structure

  cylindrical glass structure which has internal surfaces 322 defining an axial chamber 324 of the capillary type. The

  room 324 has a uniform increase in its diameter -

  be from each of its two ends 326 towards its medium 328 and the protein or peptide sample is

  carried in a solid matrix 330 produced in the form of a

  do not thin film on the walls 322.

  It goes without saying that the reaction chamber according to the

  vention can take almost any shape as long as it has a load bearing surface d2

  over which a series of reactive fluids can be passed

  solvent and SoPaz exemp! es a single long capillary tube (not shown) would suffice to form the

  chamber device 12, with a solid matrix per-

  fluid-forming formed on its internal surface or in its

  light. The fluids that would be circulated in the tube

  would interact with a sample consisting of a protein or peptide embedded in the film to carry out the process

of degradation.

  A typical example of a reservoir 198 according to the invention intended for storing and delivering a liquid reagent to the reaction chamber 349 34 'or 324 is shown in FIG. 7. The reservoir 198 corresponds to the tanks 42, 50, 52, 54,, 101 and 102 of Fig.3. An inert gas under pressure is sent into the interior volume 200 of the reservoir 198 by a

  conduit 202 which communicates with this reservoir at a point if-

  killed below level 204 of reagent or liquid solvent

  contained in this tank The pressurized gas thus introduced

  duit removes the dissolved oxygen which is possibly contained in the liquid and can be evacuated at a flow rate regulated by the intermediary of a vent 206 so as to establish a state

  dynamic equilibrium in the interior volume 200. An outlet

  Liquid tie 208 is provided to allow the gas pressure present in the reservoir 98 to expel the reagents or solvents from this reservoir in a controlled manner. The conduit

  202 supply of inert gas to each tank 198

  receives an inert gas under pressure from the source 28 via a pressure regulator 56 or 106

  and a solenoid valve 58 or 108. The exhaust of gases through

  towards the vent 206 is likewise controlled by one of the electro-

  valves 62 or 110 and by one of the flow regulators 64 or 111. The flow rate of the reagent or liquid solvent in

  the conduit 208 is controlled by one of the valves 66 or 112.

  Whenever one of the liquid reagents or solvents has to

  be sent to the reaction chamber or container

  version 14, the corresponding supply valves

  gon and vent open to establish a state of equilibrium dy-

  namique in the particular reservoir considered. The reagent or solvent in liquid form can then be introduced by

  opening of the valve provided in the conduit 208 and of the valve

  ne 82 which leads to the waste trap 60. The liquid is expelled from the reservoir at a constant flow rate, which makes it possible to precisely regulate the quantity of liquid supplied by adjusting the duration

  during which the valve interposed in the supply line

  tribution 208 is kept open.

  A typical example of reservoir 210 according to the invention intended to deliver a reagent in the form of gas or vapor is shown in FIG. 8. An inert gas inlet 212 which ends in a sintered glass diffuser element 213 is provided for introducing the inert gas into the interior volume 214 of the tank 210 near the bottom of this tank and clearly below the level 216 of the liquid reagent contained in this reservoir. A vent 218 and an outlet or distribution line 220 communicate with the interior volume 214 at points located above the level 216 of the liquid. The reservoir 210 is typically representative of the reservoirs 44, 46, 48 of FIG. 3, with one of the pressure regulators 56 and one of the valves 58 for controlling the flow of gases

  via line 212 coming from the gas source under pressure

  sion 28. Similarly, the exhaust of pressurized gas to the

  wind 218 is controlled by one of the flow regulators 64 and one of the valves 62 and the distribution of the reagent along the line 220 is controlled by one of the membrane valves

  66, which is connected in series with a flow meter 74 and a regulator

flow meter 76.

  In this way, although the reagents R2, R3 and R3A are sent to the reaction chamber in the form of gas

  or vapor, they are stored in liquid and vapor form.

  laughed as needed. The vaporization is carried out by bar-

  botage of inert gas from bottom to top through the mass of re

  liquid active. In this way, the inert gas contained in the

  interior volume 214 of tank 210 is saturated with

  fears of reagents. Whenever you need reagents,

  the valves connected to conduits 212 and 218 open to make

  re bubbling the inert gas into the reagent and establishing a state of dynamic equilibrium. The valve 66 inserted in the conduit

  dispenser 220 then opens for a predetermined time-

  mined to send the desired amount of reagent vapor to the reaction cell. Thanks to the flow regulator 76 which

  is in series with the particular valve 66 considered, the

  fear is delivered by the tank at a constant rate which is

indicated by flow meter 74.

  Figs. 9 to 13 illustrate the structure and function-

  dispenser 222 which includes valves 66 and 72

  and the continuous collector 68 of FIG. 3. The set 222 con-

  takes a block of valves 224 which is shown most clearly-

  ment in Figs. 11 and 12. Block 224 is a block of elongated shape and of rectangular section enclosing a passage

  contInu primer 226 sawtooth which was formed in per-

  drilling cross holes in the block from the surface 228 thereof. The primary passage 226 is therefore a single continuous passage which communicates at the level of one of two of its

  intersections, with urn series of shutter zones 230 pre-

  views on the surface 228, through corresponding holes 232. A conical connection orifice 234 communicates with

  one end of the primary passage 226. Several passages se-

  condaires 236 extend from conical connection orifices 238 provided on the opposite side of the block 224 towards

  corresponding openings 240 located near the

  vertices 232 in line with the corresponding closure zones 230. The block 224 is housed in a longitudinal slot 242 of a base 244 which has a series of threaded holes 246 aligned with the connection orifices 234 and 238 for

  to have fittings 248. The fittings 248 are suitable for compressing elastic rings 250 against the orifices 234 and 238 respectively for sealingly assembling 1o tubes 252 which pass through the rings at the various passages of the block 224. In this way, the orifice 234 of the primary passage communicates with the inlet passage 36 of the chamber device 12 via the conduit

  of fluid 40 in Fig. 3 and the first seven of the eight passes

  secondary gates communicate respectively with the outputs

  fluids 208 and 220 from reservoirs 42 to 54 respectively.

  The secondary passage furthest from the connection orifice

  dement 234 communicates with the inert gas pressure source

  te 28 via the pressure regulator 70 shown

felt in Fig. 3.

  The structure of the connections with biconical rings gives

  nant on the orifices of block 224 is represented with more

  details in Fig. 9A which shows by way of example the

file 234.

  The ring 250 of FIG. 9A is housed on its side

  dull 243 in connection hole 234 and, by its side

  245 in a conical cavity 247 of fitting 248, the o

  hole 234 and cavity 247 being formed with cone angles larger than those of the corresponding sides of the ring. The ring is preferably conical with the same angle on both sides, the orifice 234 and the cavity 247 being conical

  with an angle 30 greater than that of the ring. This adjusts

  between surfaces with different slopes con-

  centers the compressive forces on the points 249 of the

  gues 250 by establishing an effective seal with the minimum pressure on the side of the block 224e In this way, we avoid

  excessive pressures on block 224, pressures which could

  could distort the shutter zones 230.

  A series of membrane retaining blocks 254 are bolted against the surface 228 of block 224 with the membranes

  256 clamped between blocks 254 and block 224. The end su-

  membrane check block is threaded

  to receive an air connection 258 which communicates with an air vent

  dement 260 formed in its internal face and which covers general-

  o ment one of the blanking zones o2300 An O-ring 262

  can be housed in an annular groove which surrounds the recess

  260 to establish an effectively airtight seal.

  The membranes 256 are made of a waterproof material.

  the practically chemically inert air, which makes them

  to lift from the shutter 230 or stappli-

  quer on these zones under the effect of the transmission of a vacuum or an air pressure respectively ravers the connections

  258. The two alternative states of the membrane 256 are re-

  shown in Fig0 13A and 13B. In the state of FIG. 13A, an air or gas pressure applied to the fitting 258 presses the membrane 256 against the opening 232 of the primary passage and the opening 240 of the secondary passage at the level of the particular obturation zone 230 considered. . Openings 232 and 240 are

  then closed by the membrane 256, prohibiting any com-

  munication between these openings o This corresponds to the posi-

  closed valve located in the obturation zone by

  considered in the state of Fig 13B a vacuum trans-

  connected to the fitting 258 lifts the membrane 256 from the openings 232

  and 240, by allowing the establishment of a communication

  be the primary passage and the secondary passage on the

  face of the valve block at this point. This corresponds to the open state of the particular valve considered, allowing the fluid from one of the reservoirs or the pressure source to

  inert gas 28 reaching inlet passage 36 of the chamber

  reaction bre through the primary passage 226.

  The new construction following the invention of the

  seems to valves 222 which has been described above allows

  see reagents and solvents in the reaction chamber in precise amounts, with virtually no contamination

  nation between the various fluids. The continuity of the main passage

  mayor 226 and the connection between a first end of this passage and the entry passage of the chamber device

  12 constitute the main characteristics which allow

  are trying to get this advantageous mode of operation. We can

  obtain the shipment of any of the seven reagents and sol-

  by transmitting a vacuum to one of the membranes 256 and

  positive gas pressure to others, which allows

  see the desired reagent or solvent in the primary passage

  226 and to the reaction chamber via the con-

  fluid duit 40. When the desired quantity of fluid has passed under the membrane in question, a

  gas pressure at this membrane via the rac-

  cord 258 so that each of the seven reagent or solvent valves in assembly 222 is now closed. We can then complete the sending of the fluid by transmitting a vacuum

  with the membrane combined with the inert gas orifice, at the

  mite away from block 224, to sweep the entire pass

  primary age 226 with an inert gas and return to the chamber

  reaction bre 34 the reactive fluid or solvent that remains

  in the conduits. Since there is no disconti-

  night or blind branch in primary passage 226, there

  there is no place where any of the reagents or solvents for

  should remain retained between two distribution operations. The reagents and solvents debited during the successive phases of the sequenced fractionation are therefore as pure as possible,

  which allows the chemical processes carried out in the cell-

  the reaction to unfold as intended and without

  no unnecessary loss of yield due to the presence

  contaminated reagents or solvents.

  The set of valves 222 is also illustrative of the

  valves incorporated into many other parts of the ap-

  same to minimize contamination in each

  case a single orifice must be able to be connected selectively-

  lie to a series of other orifices. This is how valves 112 and 118 intended to send the reagents and solvents to

  conversion container 14 form a distributor in combination

  sound with the continuous manifold 114 and the valves 78, 80 and 82, intended to send fluids from the outlet passage 38 of the chamber device 12 form a set of

  valves or similar distributor in combination with the manifold

  Continuous 84. Likewise, the valves for connecting the waste discharge and the vacuum to the conversion container and the fraction collector and also the valves for controlling the flow of the conversion flask to the fraction collector are constructed from the same way as the valve or distributor assembly 222. The main difference that distinguishes each of these distributors from other distributors

  lies in its number of shutter zones.

  It goes without saying that, while the collectors 68, 84 and 114 are shown diagrammatically in FIG. 3, under a

  form comprising a series of small discontinuities or bran-

  respectively adjacent to the diaphragm valves which

  are combined, each of the collectors is actually

  killed by a single continuous passage constructed in the same way as

  the primary passage 226 shown in FIGS. He and 12.

  The vacuum and gas pressure used to actuate the diaphragm valves between their open position and their closed position have been omitted from the diagram in Fig. 3

  to simplify and they are preferably separated from the sour-

  ce 28 and pump 90 shown in this figure.

  The conversion container 14 is depicted

  details in Figs. 14 to 16. The container 14 is of the glass type, double wall, with a space 264 formed between the walls for the circulation of a heating fluid which can be sent to the space 264 and evacuated from this space by two

  256 fittings adapted to receive pipe ends

  ple standard .. A 268 tube with large internal diameter which can

  be connected to the vacuum trap 88 and to the vacuum pump 90 through

  to valve 96 and to the waste trap via valve 92 communicates with the interior chamber

  270 of the container in the region of its upper end.

  Capillary tubes 272, 274 and 276 pass through the upper end of the container to reach points in

  the inner chamber 270 of this bottle. The lights of the tu-

  bes 272 and 276 are closed at the inner ends of these

  tubes and each of the tubes has a series of holes

  laterally constricted 278 and 280 radially spaced apart

  in the region of its inner end.

  Due to the relatively small quantity represented

  ted by the sample of proteins or peptides for which

  device 10 has been studied and due to the relative volumes

  small reagents and solvents used in this apparatus

  reil, the conversion container 14 has an internal volume

  blow smaller than any of the prior art containers

  better. The volume of the container 14 is slightly greater than

  one (1) milliliter while the conversion vessels

  Tomatics of the prior art all have vo-

lumens of one hundred (100) milliliters.

  The fractions which have been separated from the sample by

  cleavage in the reaction chamber 34 are sent successively

  the container 14 through the valve 78 and the cap

  laire 274. Reagents and solvents enter the interior chamber of the container through the capillary 276, from where they are expelled through the constricted orifices 280 in the form of a divided jet which strikes the walls of the chamber 270 to wash any residues carried by

  this wall and which flow down the container. Pen-

  during the conversion reaction, a gas can be introduced

  inert through tube 272 to agitate the liquid and con-

  tribute to evaporate the solvent from this liquid. The gas leaves the tube 272 through the throttled orifices 278 in the form of very fine bubbles, which ensures optimal distribution of the gas in the liquid, whatever the size of the container and the quantity of gas used. When the conversion reaction is complete, the fraction is forced back to the

  high by a positive gas pressure introduced into the

  container, this fraction passing through the long capillary 272 and being sent to the corresponding flask of the collector

  fractions 16. This can be done in two parts aliquo-

  to determine a more complete transfer of the fraction to the fraction collector 160 The first part aliquots

  which is about 200 microliters (pi) is initially in-

  seen at the fractions collector0, Then we introduce a

  new additional quantity of 50 microliters (pl) of soil-

  to dissolve any residue left on the internal walls or the bottom of the container. The second part

  aliquot is then expelled. In this way, we can get

  nir a high yield of each fraction.

  When a particular fraction has been transferred, an additional quantity of solvent, from 750 to 900 microliters (ls) can be introduced to dissolve the residue which may remain from the previous cycle on the upper walls of the container 14. This additional solvent is then expelled

  to the waste dump leaving the walls of the container

clean pient.

  The outer ends of the tubes 268, 272e 274 and

  276 are tightly connected to flexible conduits

  282 correspondents, which respectively lead to the

  to other elements of the apparatus 10, by threaded connections

  lettering 284. The end of each conduit 282 is provided with a radial flange 286 carrying an elastic O-ring

  288 which abuts and forms a tight seal against the glass face

  re-lapped flat with a radial flange 290 provided on the tu-

  be of corresponding glass. An inner threaded ring 292-

  is slidably mounted on the duct 282 to receive

  glass tube 290 and screw onto a tip in two pieces

  these 294 externally threaded and which is arranged around the

  glass tube. The screwing of the ring 292 on the end piece 294 ap-

  press the flange 296 by force against the glass flange

  re 290 to produce an extremely effective seal. The diff-

  different 284 connector parts can be made

  practically chemically inert, such as poly-

  commercially available fluoride carbide mothers to virtually eliminate the risk of deterioration and subsequent leakage. An alternative construction of the container 14 would consist in eliminating the space 264 and the pipes 266, by making a container with a single wall which could be

  maintained at a high temperature by arrangement in the envi-

  This would have the advantage of keeping the entire length of the glass tubes and fittings 284 at a high temperature, minimizing the condensation of semi-volatile fluids in these tubes and fittings but would not allow hold the container and

  chamber 12 at different temperatures.

  The reagents and solvents used in the apparatus 10 for the sequential degradation of the protein or peptide chains are preferably the following: R1 Phenyl isothiocyanate (PITC) (17% solution in heptane) R2 Trimethylamine or triethylamine (solution 25% in water) R3 Trifluoroacetic acid or heptafluorobutyric acid R3A Water vapor 3A R4 Trifluoroacetic acid (25% solution in water) R4A Hydrogen chloride (solution 1N in methanol) 4A S Benzene S2Acetate ethyl with 0.1% acetic acid S3 Butyl chloride S4 Acetonitrile or methanol

  In operation, the different valves and the different

  other other mechanisms of the apparatus 10 are preferably

  ce controlled by the automatic control unit 18 for exe-

  cut an indefinite number of degradation cycles on a

  protein or peptide sample without any human intervention. The control unit 18 can take the general form of the programming unit described in US Pat. No. 3,725,010, with modifications to achieve the particular sequence of phases required by the apparatus 10 or even it

  can be made up of a more compact electronic control

  plicated, constituted by a special digital computer or

  for general use. As a variant, the different phases of cha-

  that degradation cycle can be controlled manually

  by an operator following a predetermined program, to obtain

hold the same results.

  Before starting the degradation process, a sample of the protein or peptide to be studied must be mounted on an appropriate sample-carrying surface. We deposit

  first of all the material of the matrix on the supporting surface

  se, the passage of the sample in this material is effected

  killing in the following, as described in the examples

1 and 2 given below.

* When element 190 or 190 'is used as a sample-carrying surface, this element is placed between elements 134 and 138 of the chamber, with at least one of the sealing sheets 194, so that the element 190 is housed

  in the reaction chamber 34 at the location of the cavity 192.

  In the preferred embodiment of the invention, use is made of

  reads two sealing sheets 194, by clamping the element 190 between these two sheets. Then, the elements 134 and 136 of the chamber are placed in the sleeve 132 and they are assembled

  to the other members to form the device 12 forming cham-

  bre. When the film containing the sample must be

  carried by the internal surface 34 'or the chamber 324, it is

  poses on this chamber as described in Example 2 below

  below. The elements of the chamber are initially assembled in the sleeve 132 and are then held in place by means of the cap 130. In the case of the chamber 34 ′, the annular sheet of flexible material 316 is interposed between the

  elements 134 'and 136' of the chamber at the time of assembly.

  Then we apply the material of the solid matrix and

  sample on the walls of the chamber 34 'and 324 of the fa-

  lesson described in Example 2 and the sleeve 132 is assembled to

  other elements to form the complete device.

  First, a vacuum is created in the reaction chamber.

  tion 34 and in the corresponding fluid conduits in or

  using the valve 82 on the vacuum trap 88 and the vacuum pump

  90, for the preparation of successive introductions of the

  active R1 to R3A of solvents S1 to 53 and inert gas supplied

  nor by the source 28. Each time that one has to introduce one of the reagents or solvents, the corresponding inert gas supply valve 58 and the corresponding vent 62 are opened to put the particular reservoir under pressure and establish a state of dynamic equilibrium in this reservoir. In this way, the inert gas is introduced into this tank, and at the same time, it is allowed to escape from this tank so as to maintain a constant pressure in the tank. In the case of tanks 44, 46 and 48, the outlet of saturated gas from this tank during distribution is controlled by one

flow regulators 76.

  As soon as the reservoir containing the particular reagent or solvent to be dispensed has been pressurized and placed in equilibrium as described above, the vacuum is transmitted to the membrane of the corresponding valve 66 by means of the vacuum source. auxiliary (not shown) to open this

  valve and send the reagent or solvent to the inlet passage

  inlet of the chamber via the continuous manifold 68 and the conduit 40. The valve 66 is kept open for a predetermined time to allow the desired quantity of reagent or solvent to pass and it is then closed by applying gas pressure to the membrane of this valve. Next, a vacuum is applied, supplied by the auxiliary vacuum source, to the membrane of the valve 72 located at the far end.

  of the continuous collector 68, to purge the collector of the reaction

  tif or residual solvent possibly contained in these elements.

  and complete the shipment of this reagent or solvent to the chamber.

  bre. Then, the valve 72 is closed by applying a pressure.

  positive sion on the membrane of this valve leaving an

  if the collector 68 free of solvent and reagent, ready to

the next supply phase.

  The membrane valve 80 is generally maintained or-

  green pehdant the passage of the reactive diveras and solvents in the room 34 to lead the efiluent of this room to the waste trap 60 while passing by the exit passage of the

  chamber and by the collector 84th After the supply of the reac-

  tif or particular solvent considered, a vacuum can be created in the chamber and in the corresponding conduits by opening the membrane valve 82 on the vacuum pump 90. As a variant, the chamber and the sample which is contained can be dried appropriate times by passing an inert gas through

  the chamber by means of the valve 72. The gas leaving the chamber

  bre can then be sent to the waste trap 60 or,

  it can be removed by the vacuum pump 90 to

  speed up the drying process.

  After performing the coupling and cleavage phases,

  the extraction solvent S3 is sent to the reaction chamber

  tion 34 to dissolve the cleaved amino acid derivative produced during the degradation cycle under consideration and to send the

  solution to the conversion container 14 via line 86.

  For this, or open the valve 78 and the valves 80 and 82 remain

  closed. The R and R4A conversion reagents (if used

4 4

  lise), and solvent S4 can then be sent to the container.

  conversion point 14 at appropriate times by opening the corresponding valves 112 for passing liquids

  via the manifold 114 and the conduit 104 to reach the

  conversion vessel. Each liquid distribution is pre-

  transfer of the pressurization and venting operations described above with regard to the distribution of the other reagents and solvents and followed by the opening of the valve 118

  which serves to purge the manifold 114 and the conduit 104.

  The fraction transferred to the conversion container 14

  is the anilinothiazolinone derivative of the amino acid N termi-

  nal of the protein or peptide sample and it is con-

  vertically automatically, during the following coupling cycles

  ge and cleavage of the reaction chamber 34, to form the phenylthiohydantoin derivative of the more stable amino acid, in

  part in accordance with the aforementioned Wittmann-Liebold articles.

  In short, we can first evaporate the acid fraction

  of amine contained in the conversion vessel by passing an inert gas over the solution through the short capillary 276 and by bubbling an inert gas through the liquid to the

  by means of capillary 272, these operations being followed by the ap-

  plication of a vacuum through the tube 268. Next, the conversion reagent R4 is introduced through the capillary 276 by means of the conduit 102 and one of the valves 112 (see FIG. 3) in the desired quantity. The contents of the conversion container can be rapidly evaporated after the desired conversion time has elapsed by applying vacuum to the conversion container by means of the tube 268 and at the same time sending the inert gas through the capillaries 272 and 276. stabilize

  more acid side catches of Pth-aspar- acids

  tick and Pth-glutamic, we can still dissolve the residue

  contained in the conversion vessel by introducing the re-

  active R4A by capillary 276 in the desired quantity, in

  passing it through the conduit 104 and one of the valves 112.

  Evaporation of the contents of the conversion vessel is

  continuation carried out in the same way by applying the vacuum via the tube 168 and sending the inert gas by means

  capillaries 272 and 276. The Pth-ami is then redissolved

  no-acid, which remains in the conversion container, in the solvent S introduced by means of the conduit 104 to transfer the fraction to the appropriate flask of the fraction collector 16. The transfer of the fraction is carried out by opening the valve. 141 connected to the long central capillary 272 of the conversion container and admitting the inert gas under pressure ati

  means of capillary 276 to expel the fraction from the container.

  The fraction collector flask carousel 16

  rotates by a predetermined angle once in each cycle of de-

  gradation so that each fraction arriving at the collector

  is thus collected in a separate flask. At an approximate point

  requested from the degradation cycle, the fractions contained in the fraction collector can be further dried by opening the valve 98 on the vacuum or by opening the valves 128 and 94 to pass an inert gas coming from the source

  28 on fractions and finally send it to the trap

chets 60.

  The various components of the device 10 are

  preferably constructed of materials which are practical

  inert and are very resistant to deterioration. These

  materials include borosilicate glass, some po-

  lymers of fluorinated carbides and, in some cases, stainless steel and aluminum. The joint structures and other elements of the apparatus 10 have been studied so as to be able to

  be made almost exclusively of these materials. One is-

  time that the resulting device is the cleanest and most contamination-free device available and capable of operating indefinitely

in this state.

  The various phases performed by the apparatus 10 in a typical degradation and conversion cycle are listed in Table 1. The duration of each phase and the functional state of the apparatus during each phase are also shown in the table. The functions shown correspond to the states of the various valves of the machine and the marks arranged in the columns indicate when the appropriate valves are open. For example, the columns '"Rif," R2 "," R3 "," R3f', 1 R, 2 Y 3Ry 3 i "S1i", S2 "and" S3 "indicate the states of the various pairs of valves 58 and 62 that we use to selectively put

  under pressure tanks 42 to 54 and establish a state of

  dynamic balance. When a mark appears in one of these columns, the valves 58 and 62 combined with the corresponding reservoirs are opened, either as a preparation for the distribution of the reagent or solvent considered in the chamber

  of reaction either during this distribution. To all other mo-

  the valves remain closed. Likewise, the column t "argon" indicates the state of the valve 72 for the distribution of an inert gas such as argon to the reaction chamber passing through the manifold 68; the "distribution" column indicates the state of the valve 66 corresponding to any tank which is

  pressurized at the instant considered and the internal columns

  "waste", "collection" and "vacuum" indicate the states of the valves 82, 78 and 80 respectively. As described

  above, each phase of solvent distribution or

  active is preceded by pressurizing the appropriate solvent or reagent reservoir and followed by the introduction

  inert gas via valve 72 to supplement

  ter the distribution of the solvent or reagent and purge the con-

distribution lines.

  The columns "R4", "R4A" and "S4" indicate the states

  various pairs of valves 108 and 110 used to

  be selectively under pressure tanks 100 to 102 and

  establish a dynamic equilibrium in these reservoirs, and the "distribution / argon" column signifies the state of the valve 122 which admits the

  inert gas in the conversion container to

  by means of the conduit 124 and of the valve 112 which corresponds to the

  which is pressurized at this time. The columns

  "argon" t, "waste l '," vacuum "," collection "and" waste 2 "indi-

  that the states of valves 118, 92, 96, 131 and 125 respectively

  vement. Among the functions of the fraction collector which have

  the columns "argon", "" waste "," empty "indi-

  quent the states of valves 1287 94 and 98 respectively. Apart from the exceptions indicated below, the phase sequence listed in Table 1 is essentially in conformity

  to the processes of degradation Edman described in the publica-

  mentioned above and therefore will not be described in detail. The

  differences that distinguish them from general res-

  will clearly emerge from the name of the phases and functional states

  Corresponding nels indicated in Table 10 In practice, the following variants of the sequenced fractionation programs of Table 1 can be applied to

  adapt this program to the needs of a particular user.

  1) phases 18 to 58 can be completed once or twice on the first sequential cycle to guarantee obtaining

  complete coupling of all amino groups of the protein.

  2) phase 65 can be followed by a water vapor distribution (RSA) of 20 to 60 seconds through the chamber

  of reaction to reduce the dehydration of the lateral chains

serine and threonine.

  3) phase 65 can be followed by a distribution of 0.05 ml of lN solution of hydrochloric acid in methanol

  (R4A) to the conversion vessel to methylate the la- chains

  aspartic acid and glutamic acid. In that case,

  S4 is preferably methanol.

  4) phase 76 can be followed by a distribution of 0.7 to 1 ml of S4 (acetonitrile or methanol) which is then sent to the waste to completely clean the container

conversion.

  ) the duration of phase 8 can be extended (500 to 1000 seconds) to cause cleavage of the proline residues at

amino terminus.

  The nature of the invention will be made more explicit by the following specific nonlimiting examples of implementation.

following works.

  The amino acid sequences shown in the examples

  The following ples are expressed in the one-letter amino acid code which is defined below; A - alanine L - leucine R - arginine K - lysine N - asparagine M - 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

  One can proceed as follows to prepare a solid matrix of a suitable quaternary amninum polymer salt

  to drown a protein sample for fractionation-

  ment sequence, on element 190 or 190 'composed of a leaf

  the fibrous and that was described above and we can drown

  a protein sample in the matrix as follows:

you:

  A 12 mm diameter fiberglass disc is cut.

  meter and 0.25 to 0.5 mm thick, in a sheet for

  filter made of glass microfibers that can be obtained

  enter the trade with the firm Whatman, Inc. Clif-

  ton, New Jersey, E.U.A. The disc is placed in the recess 192 of the element 134 of the chamber. 25 microliters of an aqueous solution containing 1.5 mg of 1.5-dimethyl-1,5-diazaundecamethylene polymethylbromide and 0.033 mg of glycylglycine are poured dropwise onto the fiberglass disc using a syringe or syringe. a pipette. The water is evaporated under vacuum or by heating in a stream of hot nitrogen, the rest of the chamber 12 is assembled and this chamber is assembled

  in device 10. We start the fractionation program

  protein sequence in phase 18 and we run 4

  has 6 complete degradation cycles to eliminate impurities

  removed from 1,5-dimethyl-1 5-diazaundécamé polymethylbromide

  thylene that could react chemically with the sample

  protein or otherwise alter the Edman process.

  The chamber 12 is partially dismantled and 25 microliters of the protein solution are poured dropwise onto the fiberglass disc. Protein solution dissolves 1,5-dimethyl-1,5diazaundécamethylene polymethylbromide

  and the liquid is removed by evaporation to leave

  ter a thin film of 1,5-dimethyl polymethylbromide

  1,5-diazaundécamethylene in which the sample of the pro-

  teine is drowned. If the initial volume of the sample

  tine is greater than 25 microliters, we can deposit the

  sample in aliquots of 25 microliters, the liquid

  being eliminated by evaporation between successive applications

  sives. We reassemble chamber 12 and reassemble it in

  device 10. Then we start the fraction-

  protein sequenced in phase 18 and executed

  by performing the number of degradation cycles desired.

EXAMPLE 2

  One can proceed as follows to prepare on the over-

  internal faces of a capillary glass container a solid matrix of a polymeric quaternary ammonium salt suitable for allowing to drown therein a sample of protein intended for sequence fractionation, and then to drown a sample of protein in the matrix: initially the element 324 of the chamber or the elements 134 ′ and 136 ′ of the chamber with the sleeve 132 and the cap 160, as shown in the drawings,

  to form a sub-assembly having the shape of a car-

  compact button which can then be easily manipulated for the introduction of the matrix and the sample. We

  holds the sub-assembly horizontally and we inject 10 micro-

  liters of an aqueous solution containing 0.6 mg of polymethyl-

  1,5-dimethyl-1,5-diazaundecamethylene bromide and 0.0013 mg glycylglydine in the reaction chamber using a syringe. The chamber 320 is preferably constructed so as to have a diameter of 1.59 mm at its two ends and

  un-diameter of 3.175 mm in the middle, which gives a cham-

  bre capable of containing up to 25 microliters (1l) of solu-

  tion in the horizontal position without this solution flowing from the ends. This configuration of chamber 324 which contains the solution in the horizontal position is

  shown in FIG. 18B, on which we omitted for sim-

  fold to represent the sleeve 132 and the corresponding structure

  laying. Then, the sub-assembly is rotated on its axis at the same time as the liquid contained in the reaction chamber is evaporated by passing a stream of air or nitrogen through the chamber, to leave on the walls of the

  chamber a thin film of 1,5-dimethyl polymethylbromide

  thyl-1,5-diazaundecamethylene. Then we mount the sub-

  appears in device 10, we start the frac-

  sequence of the protein in phase 18 and we perform

  kills 4 to 6 complete degradation cycles. Then we remove

  the subassembly of machine 10 and we keep it horizontal-

  at the same time as 10 microliters of the solution are poured

  tion of protein in the reaction chamber using a syringe. The sub-assembly is again rotated on its axis at the same time as the liquid contained in the reaction chamber is evaporated by means of a stream of nitrogen which is

  passes through the room, to leave in the room

  bre a thin film of 1,5-dimethyl polymethylbromide

  1,5-diazaundécamethylene, in which the protein sample is embedded as indicated in FIG. 18C. Then, we reassemble the subassembly in the apparatus 10 and we start the protein fractionation program at the

  phase 18 and it is executed by performing a number of degradations

tions as large as desired.

EXAMPLE 3

  We drown 0.0005 mg of angiotensin, contained in 25 microliters of a 20% aqueous solution of formic acid in a solid matrix of 1,5-dimethyl-polymethylbromide

  1,5-diazaundécamethylene precycled in accordance with the operating mode-

  roof of Example 1. Angiotensin is subjected to eight cy-

  degradation keys according to Edman and the phenythiolhydantolin derivatives of amino acids produced in each cycle are analyzed

  by liquid phase chromatography under high pressure

  in front of the process described by Johnson in the article t'Analysis of Phenylthiohydantoin Amino Acido by High Pressure Liquid Chromatography on Du Pont Zorbax CN Columns'e Anal. Biochem, 335 (1979). The following sequence is obtained, designated below by the term "experimental" e. The known sequence is

  also shown below for comparison.

1 3 5- 7

  Experimental: D- R - V - Y - IH - P - F Known: D- R - V -Y - I - H -PF This result is significant because it demonstrates that, by the process according to the invention, can even perform the sequencing of small peptides. The possibility of

  fractionating small peptides results from the fact that the sample

  lon is retained by embedding in a film instead of being adsorbed directly on a supporting surface and that in this way, it can be retained with security regardless of its size. While sorption linkages include a

  large number of non-covalent interactions established between the

  sample and the surface and that they strongly depend on

  the size of the molecules, the retaining force of the film

  cule described in this specification is practically unaffected

  affected by the dimensions of the sample.

EXAMPLE 4

  0.01 mg of sperm whale apomyoglobin is drowned, contained in 25 microliters of an aqueous solution of acetic acid

  at 20% in a solid matrix of polymethylbromide of 15 l

  1,5-dimethyl-diazaundecamethylene precycled according to the procedure of Example 1. Apomyoglobin is subjected to 40 cycles of degradation according to Edman and the derivatives are analyzed.

  phenylthiohydantoin of amino acids in accordance with the pro-

  yielded from Example 3, Following which we obtain the sequence

  this hereinafter designated by the term "experimental". The se-

  known incidence of sperm whale apomyoglobin is also

  shown below for comparison.

1 5 10 15

  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

  Experimental: K-V-E-A-D-V-A-G-H-G-Q-D-I-L- 1-

  Known: K-V-E-A-D-V-A-G-H-G-Q-D-I-L- 1-

40

  Experimental: R-L-F-K-S-H-P-E-T-L-

Known: R-L-F-K-S-H-P-E-T-L-

EXAMPLE 5

  0.01 mg of a larval cuticle protein is drowned

  of Drosophila melanogaster with a previously inconclusive structure

  naked, contained in 25 microliters of an aqueous solution of sodium dodecyl sulphate O 0.1% and 0.05 M of ammonium bicarbonate, in a solid matrix of 1,5-dimethyl-1,5-diazaundecamethylene polymethylbromide precycled in accordance in the procedure of Example 1. The cuticle protein is subjected to 26 degradation cycles according to Edman and the phenylthiohydantoine derivatives of amino acids are analyzed according to the method of Example 3, which gives the designated sequence

  hereinafter by the term "experimental".

1 5

  Experimental: N- A - N- V -E -V -K - E - L - V -

11 15

N- D-V-Q- P-D-G-F-V- S-

21 25

K- L- V- L- D- D- G- S - A- S -

31 35

  Experimental: S - A - T - G - D - I -

EXAMPLE 6

  0.005 mg of a Drosophila m6lanogaster larval cuticle protein, of previously unknown structure, contained in 10 microliters of an aqueous solution of 0.01% sodium dodecyl sulfate and 0.05 M bicarbonate is drowned tammonium in a solid matrix of 1,5-dimethyl-1,5-diazaund6camethyl polymethylbromide precycled according to the procedure of Example 2. The cuticle protein is subjected to 24 degradation cycles according to Edman and the phenylthiohydanto derivatives are analyzed. amino acid according to the method of Example 3, which gives the designated sequence

  hereinafter by the term "'experimental".

1 5

  Experimental: N- A -N -V -E -V -K - E L - V -

11 15

N -D -V - Q - P -D -G - F - V - S -

* K - L -V -L -

  The results obtained in Examples 5 and 6 demonstrate

  that the apparatus and the process according to the invention are suitable

  requested for the execution of the sequenced fractionation of proteins

  nes and peptides dissolved in a solution of dodecylsul-

  sodium fate ("tSDSt,) which constitutes an anionic detergent

  powerful. This is important because the most general process

  general isolation of small amounts of proteins or peptides of medium to large dimensions for analysis, which

  is known by the designation of gel electrophoresis

  lyacrylimide, produces samples contained in an SDS solution. This common detergent causes the washing of devices in which the fixing of the sample to a carrying surface is based on an adsorption bond while the solid matrix according to the invention is not affected.

by the presence of SDS.

  It follows from the above that the invention provides

  an improved apparatus and method for performing

  of chemical processes on a very small sample by the use of minimal quantities of reagents and solvents and with relative cycle times-

ment short.

TABLE 1

_ CEIAM3RE DE RFACi'IONE =, -.

OPERATIONS DEIA OPELATIONSDU

  ROTION CHAMBER CONTAINER OF TIO C (khNVERSION time delay / pressure isization pressurized pressure 2 vice plrge argon conduits R43 - _ vacuum distribution R4 7 -a20_ _ X_ t_ z _._ _ _i__ _ _ 2 __- EZiZX = vacuum .. 0 _ _ ___ pg_ +. 0cits vacuum conversion c CIO

R3 pressurized argo 7_ _ -

  Ru cistrrzution 'purge coni5ïiuiàts-- -'9

empty _ /, 80à-

get pressured. / -

  S3 purge argon conduits / 2 7 S3 distribution '"/ 3 7. / S3 precipitation # 4 S3 distribution 1 ,. /! 0 /

purge conduits _.

  = -80_ii_5 _> _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ Z Lt vacuum bleed 0. X G Vac 'l, _______ I pressurized /

empty '0. * 0.

pressurization P / a

Ri1 ais-trDu ro-n 4X ___-

purge ducts -n /

prg r _Dio n60 - - - - --._ t -

  ___ - _ - --_____ __ z __I? [p _ Ej! -

  mi'se sous press.argo? nq ___. /, __ ÀR2 distribution e 0 X ,, drain pipes,? 8.0

- vacuum purge -

  Q O%, O P.% 0 c0 Ln TABLE 1 (continued)

CiILzl3.U3.R DL IMEAC] I (. IIiiTJVI-

O PErATIONS OF C% 1

EIIN D OPERATIONS

RECIPIUM OF '

REACTION CLIAMER

CONVERSION

  vtie conversion. 30 3 E -) eor 1s a-El on-1 _ -2 _._____ = L_____________ z ttN> * "Rt" '"_' W

pressurized. - -

  _ time delay | l 1 _ 2_ _: _ _ _ I <=: - - -

I passage ducts arge n "'_

s R distribution 5 ---

  In passing. conduits "- purge - by vacuum # 2 put under pres.arqon at R2disribtion 1. 8 5 Xà purge conduits __ / 0 àà

  pressurization 4 --- <. à -t ààà -

life | --- ide.

  pressurization "4? 0 X f-1 r- - emptied Meo

time delay. . ..

  pressurization - - - X- \ If argon conauit purge '_7 bi, S distribution, precipitation n <a 40 S' ', distribution 5 -Po S2 pre-night argon 5 0N duct purge 5 vacuum purge Ap -luse under pressure empty ,, 27 <it empt. i k a o___I 'pressurization_ - e vacuum ,. pressurization vacuum purge temp risation o - o C> o Ln CD TABLE 1 (cont'd) cfi7e3e de, mcv (oii RiPin.qfm CU JR COITTW OnEiRTi OPERATIONS DU! '= IAO RATIONS OF Mtzj RE) CIPIa; YD OF

REACTION CHAMBER

  CCNVERiON -. _ _ _ __ __ _ .. _ li "'vF2 Tt _ | o | purge argonpurge conduits by vacuum X R3, distribution" "" I 1.0__Rx.I.J R3, distribution VI 9, - _ _,' -,

  R3, distri - tio-_ pressurization -

  -dis'7ribution.vide ._50 _ _0 i-, IXI fe ducts pressurized- 6/0 _sX = 820 ___ _ _ _ _ _ _ _ _ _ _ _ Y> t3E03, purge con uîs S4 purge argon conduits G7 / 0 vacuum 1- 5 4, distribution-p.9 vacuum purge dondurts C1 vacuum purge container 70 15 _ vacuum S4, distribution 78 - i vi of purge conduits 739 5, X * Empty purge container

vacuum collectê * tion - -_- d -

  vacuum of the stage collector 7 '/ -I II pressurized vacuum 778 X-I purge conduits vde 78Q S3, purge argon conduits pressurized .... -' '3-, di1str-bution purge conduits Bc- 7

preîpîtaion na / à-

  S3, distributio.collec: - 'nn Xti - ne 8a 5 6

3 the Ilisofec 86th 0 ___-- --- ----

purge] 2ar vacuum n n87 60-

  empty. i8 Q _ mise souspeio * 89 ____-. S- .. NI_ -, drain drain line 9 9 1 î: 1

empty timer 9.0 -

  E T - àPhase92 OI J i l o 'u1 o 4. V CD "Ln

Claims (34)

R E V E N D I C A T I 0 N S   1 - Apparatus for the sequential execution of processes   chemical above a sample of chemical material, character-   characterized in that it comprises a chamber device (12) having an internal surface which defines a reaction chamber, this chamber having inlet means (36;   36t) and outlet means (38; 38t); means allows   so much to pass successively several fluids through   towards said room, from the entry means to   outlet means in a pressurized current; and a ma-   solid trice (316) permeable to said fluids and housed in said chamber, so that a sample (300) which has been embedded in the matrix is immobilized and exposed to the action   of each of said fluids which pass through the chamber to   have a chemical interaction with this fluid.   2 - Apparatus according to claim 1, characterized in that the solid matrix (316) is permeable to said fluids,   these being absorbed into the matrix to diffuse into.   this one. 3 - Machine according to claim 2, characterized in that said device (12) forming a chamber comprises a surface which supports the solid matrix in the form of a thin film.   4 - Apparatus according to claim 3, characterized   in that the matrix consists of a polymeric salt of ammonia quaternary nium.   - Apparatus according to claim 4, characterized   in that the quaternary diammonium polymer salt is poly-   1,5-dimethyl-1,5-diazaundécamethylene methylbromide.   6 - Apparatus according to claim 4, characterized   in that the polymeric quaternary ammonium salt is poly-   (N, N-dimethyl-3,5-dimethylene piperidinium chloride).   7 - Apparatus according to claim 3, characterized in that the surface which supports the matrix consists of   at least a portion of the internal surface (322) of the device tif (12).   8 - Device according to. claim 7, characterized   in that the reaction chamber (324) delimited by the over-   internal face (322) consists of a single capillary type passage which extends between the two ends of this chamber. 9 - Apparatus according to claim 8, characterized in that the passage of the capillary type has a diameter   who believes uniformly from outside to inside through shot from both ends.   - Apparatus according to claim 7, characterized in that the chamber device (12) comprises two   chamber elements (134, 136; 134 ', 136e) supported one   the other which respectively have a first cavity (186; 186 ') and a second cavity (188; 188') on their   opposite junction surfaces, the first and second   vities being aligned with each other to form the chamber of reaction.   11 - Apparatus according to claim 3, characterized in that the surface which supports the matrix is an element   (190; 190t) in the form of a porous sheet which can be crossed- be by fluids.   12 - Apparatus according to claim 11, characterized in that the porous sheet (190; 190 ') extends approximately   transversely across the chamber (34, 34e).   13 - Apparatus according to claim 12, characterized   in that the porous sheet (190; 190 ') is a sheet laid with fibers.   14 - Apparatus according to claim 13, characterized   in that the fibers are glass fibers.   - Apparatus according to claim 12, characterized in that the chamber device (12) comprises two chamber elements (134, 136; 134 ', 136') which respectively have a first cavity and a second cavity   (186, 188: 186 ', 188') arranged on their rush surfaces-   opposite oppositions, said first and second cavities being   aligned with each other to form the reaction chamber.   16 - Apparatus according to claim 15, characterized in that the solid matrix consists of a polymeric quaternary ammonium salt. 17 - Apparatus according to claim 15, characterized in that the first and the second cavities narrow in the directions.qui move away from the junction surfaces to areas where they communicate respectively with the input means (36; 36 ') and the outlet means (38, 38'). 18 - Apparatus according to claim 15, characterized in that the porous sheet is housed in a recess (192, 192 ') formed in at least one of the joining surfaces (144, 146; 144', 146 ') to be thus retained in the chamber (34, 34 ') in an orientation in which it roughly isolates   totally the first cavity of the second cavity.   19 - Apparatus according to claim 18, characterized in that the chamber device (12) comprises at   at least one sheet of flexible material (194, 194 ') clamped in   be the joining surfaces (144, 146; 144 ', 146') of ma-   waterproof, this flexible material being permeable to said fluids. Apparatus according to claim 19, characterized in that said chamber device (12) comprises one of said sheets of flexible material (194, 194 ') placed   each side of the porous sheet (190, 190 ').   21 - Apparatus according to claim 20, characterized in that at least one of the elements (134, 136; 134 ', 136') of the chamber comprises on its junction surface a raised part (196 196 ') which surrounds said cavity (118, 188 ') formed in this element to compress at least one sheet of flexible material (194, 194') against the junction surface (114, 144 ') of the other element (144, 144') of the room for   thus strengthening the tight connection.   22 - Apparatus according to claim 18, characterized in that said input and output means (36, 38; 36% 9   381) include two capillary passages which pass through   pectively the elements (134, 136; 134 ', 136') of the cham-   bre and communicate with the reaction chamber (34, 34 ') at their inner ends on either side of the sheet porous (190, 190 ').   23 - Apparatus according to claim 22, characterized in that said capillaries are coaxial with the reaction chamber and extend from this chamber to external capillary openings formed in the elements from the room.   24 - Apparatus according to claim 23, characterized in that the chamber elements (134, 136; 134 ', 1361)   are made of glass and have roughly flat surfaces   nes in the region of said capillary openings.   - Apparatus according to claim 24, characterized   in that the means for passing successively-   fluids through the chamber (34; 341) comprise at least one mass of elastic material (172) which has   a roughly flat sealed end of the range and a.   wise axial capillary which ends with an opening at the-   said end, and a threading device used to apply   axially sealing said sealed bearing end against one of the capillary openings so that the axial passage formed in the block (172) communicates with said capillary opening and the sealed bearing end of the block (172)   presses tightly against the roughly flat surface   ne of the room element which is adjacent to it.   26 - Apparatus according to claim 25, characterized in that the block of elastic material (172) comprises a   mass of a fluoride carbide polymer embedded in a man- metallic chon (174).   27 - Apparatus according to claim 26, characterized in that the threading device comprises a bore (178) which receives said metal sleeve (174) with axial sliding to guarantee the precision of the alignment between the passage   of the block and said capillary opening.   28 - Apparatus according to claim 3, characterized in that it further comprises means (18) which provide automatic control of the operation of the apparatus on a series of cycles.   29 - Apparatus according to claim 1, characterized in that said means for successively passing several fluids through said chamber (34, 34 ') comprise   at least one distributor used to control the flow of   said fluids between the chamber and a series of devices containing the fluids, the dispenser comprising: a block of valves (224) which has on one of its surfaces a series of substantially flat obturation zones (230), this block having an uninterrupted primary passage (226) from one to   the other of its two ends and which communicates by ou-   primary grooves with each of said closure zones   and a series of secondary passages (236) each of which commu-   connects with one of said obturation zones via   diary of a secondary opening; a series of membranes e-   roughly waterproof elastic bands (256) which cover respec-   said closure areas (230), each of these membranes being operable between a first closed state in which it is forcibly applied against one of said closure areas to close the primary opening and the secondary opening which communicate with this zone and a second state in which it is lifted from said zone to establish a fluid flow circuit between the primary opening and the secondary opening passing over the external face of said valve block and means ( 248,   252) used to connect said primary passages (226) and se-   bonded (236) to the devices containing the fluids (42 to 54, 100, 101, 102) and to said chamber (34, 34 ') so that the flow of the fluid between the devices containing the   fluids and said chamber can be selectively controlled.   - Apparatus according to claim 29, characterized   in that said connecting means (248, 252) comprises   at least one conical ring (250) which is mounted narrowly   ment and sliding on a tube (252) and which is applied against a small cavity (234) having a different cone angle which is formed in the valve block (222) and communicates with one of said passages (226, 236) so that an inwardly directed force applied to said ring is   found concentrated on a relatively small contact surface   tite between the ring and the cavity to establish a tight seal   fluids between these two elements.   31 - Apparatus according to claim 30, characterized   in that said cavity (234) has a greater angle of c8ne less than that of the ring (250).   32 - Apparatus according to claim 30, characterized   in that the ring (250) is conical on its two sides,   one of the two sides being applied against the cavity (234) by a threading device (248) which has a separate conical cavity (247) intended to receive the other side of the   ring (250), said cavities having an angle of c8ne su-   lower than that of the angles of c8nes of the corresponding c8ties of the ring.   33. Apparatus according to claim 29, characterized   in that said connecting means (248, 252) comprises   nent a fitting (248) provided at leune of said ends of the   primary passage (226) so that the primary passage   a collector which can be swept by a current of   fluid that is passed between the fitting and the passage   farthest from this fitting.   34 - Apparatus according to claim 33, characterized in that the primary passage (226) consists of a series   straight passages which are connected end to end to form   sea a conduit with a sawtooth configuration and which communicate at one in two of their intersections with the   corresponding shutter zones (230).   - Apparatus according to claim 1, characterized   in that it comprises a conversion container (14) comprising   as a series of capillary tubes (272, 274, 276) which penetrate   trent in its internal volume and means making it possible to introduce various fluids into the container and to extract them from the container by means of said capillary tubes, at least one of said fluids being supplied to said outlet means (38, 38 ' ) and at least one of the capillary tubes (276) having an inner end where the lumen of this tube is closed   and which is provided with a series of constricted orifices (280) spaced   these in a radiant arrangement, so that ltin-   fluid production inside the container through this tube   capillary (276) produces a divided jet projected onto the surfaces   these internal container to wash these internal surfaces.   36 - Apparatus according to claim 35, characterized   in that said capillary tubes are made of glass.   37 - Apparatus according to claim 1, characterized   in that it comprises: a conversion container (14) comprising   as many capillary tubes (272, 274, 276) which penetrate   in its internal volume, and means allowing intro-   fill various fluids in this container by these capil-   and extract them therefrom by said capillary tubes; at least one of the fluids in the fluid series being sent   to said outlet means (38; 38 ') and one (272) of said tu-   capillary bes ending at a point adjacent to the bottom of the   container by an inner end where the light is closed   mée, this inner end being provided with a series of o-   throttled holes (278) spaced apart in a spoke arrangement   nante so that the introduction of a gas inside by this capillary tube introduces small bubbles in the liquid possibly contained in the container, these bubbles being used to agitate the liquid uniformly and to accelerate the drying of the latter.   38.- Reaction chamber device intended for use in an apparatus for the sequential execution of chemical processes on a sample of chemical material, characterized in that it comprises a device (12) having an internal surface which delimits a reaction chamber (34, 34 ') itself comprising inlet means (36, 36') and outlet means (38, 38 ') allowing fluids to pass through this chamber in a current under pressure; and a solid matrix permeable to a series of fluids and housed in said chamber so that a sample which has been drowned   in this matrix is immobilized and exposed to each of the fluids   as soon as we pass through the room to cause his   chemical interaction with this fluid.   39 - Device according to claim 38, character-   is in that the solid matrix is permeable to said fluids,   said fluids being absorbed by the matrix to diffuse therein ser.   - Device according to claim 38 or 39, ca-   characterized in that it also has the characteristics   defined in any one of claims 3 to 27.   41 - Valve device intended for use in   an apparatus for the sequential execution of chemical processes   ques, in order to control the flow of fluids in this ap-   similar characterized in that it comprises a valve block   (224) which has on one of its surfaces a series of zo-   shutters (230) roughly flat, this block having a primary passage (226) uninterrupted from one to the other of   its two ends and which communicates through primary openings   mayors with each of said shutter zones and a series   secondary passages (236) each of which communicates with the   ne of said shutter zones via an or-   secondary vertex; a series of roughly waterproof elastic membranes (256) which respectively cover the said membranes   obturation zones (230) each of these membranes being capable of   be actuated between a first state or closed state in which it is forcibly applied against one of said closure zones to close the primary opening and the opening   secondary that communicate with this area and a second é-   state in which it is lifted from said zone to establish a fluid flow circuit between the primary opening and the secondary opening passing over the external face of said block of valves so that the flow of fluid   between said primary passage (226) and said secondary passages   daires (336) can thus be selectively controlled.   42 - Device according to claim 41, character-   in that it comprises means for connecting said passages (226, 236) to external elements for the circulation of the fluid, these connection means comprising: at least one conical ring (250) which is mounted tightly   and sliding on a tube (252) and which is applied con-   be a cavity (234) having a different cone angle which is formed in the valve block (222) and communicates with one of said passages (226, 236) so that an inwardly directed force is applied to said ring is   found concentrated on a relatively small contact surface   between the ring and the cavity to establish a tight seal   che to fluids between these two elements.   43 - Device according to claim 41 or 42 ca-   characterized in that it also has the characteristics   defined in any one of claims 31 to 34.   44 - Conversion container for use in an apparatus for the sequential execution of processes   chemicals on a sample of chemicals, characteristic   se in that it comprises several capillary tubes (172, 174,   176) which enter its interior volume to introduce   re the various fluids. or extract from them, at least one of the   capillary tubes (272, 276) having an inner end   which the light of this tube is closed and which is provided with a series of throttled orifices (178) spaced in a   radiant layout; so the introduction of fluid   inside the container by this capillary tube (172, 176) produces a divided jet projected on the internal surfaces   of the container to wash these internal surfaces.   - Container according to claim 44, character-   se in that said capillary tubes are glass. 46 - Conversion container intended to be used in an apparatus for the sequential execution of chemical processes on a sample of characterized chemical material   in that it has several capillary cubes which   trent in its interior volume to introduce various fluids   of and extracting therefrom, one (272) of said capillary tubes ending at a point adjacent to the bottom of the container, by an interior end to which the lumen is closed, this interior end being provided with a series of throttled orifices spaced in a radiant arrangement; so   that the introduction of a gas inside through this capil-   the glass introduces small bubbles into the possible liquid-   contained in the liquid, these bubbles used to stir   uniformly the liquid and to accelerate the drying of the latter deny.   47 - Process for successively executing procedures   above chemical on a sample of chemical character   terified in that we enclose a solid matrix made of a ma-   fluid permeable in a closed room with   both an entry and an exit; we drown a sample of ma-   chemical content in the mass of the solid matrix; and fluids are successively passed through the chamber in a pressurized stream from the inlet to the outlet of this chamber, so that the sample is exposed to contact with each of said fluids so that the sample is immobilized and we get a chemical interaction   between the sample and the fluids.   48 - Process according to claim 47, characterized in that, to enclose the solid matrix in the chamber, a solution containing the material of the solid matrix is introduced into the chamber and the chamber is rotated while at the same time passing a gas in this chamber to evaporate the liquid, leaving the solid matrix material deposited on the internal surfaces of the chamber in the form of a thin film. 49 - Method according to claim 47, characterized in that, to drown the sample of solid material in   the solid matrix, a solution containing the e-   sample in the chamber to dissolve the material of the   matrix of this chamber and then we rotate the cham-   bre at the same time as passing a gas through this chamber to evaporate the liquid and leave the matter of the   solid matrix deposited on the internal surfaces of the cham-   bre in the form of a thin film in which is drowned said sample.   - Method according to claim 47, characterized   in that the solid matrix is carried on a porous sheet   and in that, to enclose the matrix in the chamber, the matrix material is deposited on the porous sheet   in the form of a thin film supported by this film   and we have the porous sheet roughly transverse-   ment across the room at a point between lten-   inlet and outlet so that fluids passed from inlet to outlet must necessarily pass through the leaf.   51 - Method according to claim 50, characterized   in that, to drown the sample in a matrix of ma-   solid, a solution containing the sample is deposited   on the porous sheet to dissolve the material of the   trice carried by this sheet and the liquid is evaporated from the sheet to let the matrix material settle in the form of a thin film in which the sample is drowned.   52 - Process according to claim 47, characterized in that, in order to pass the fluids successively through the chamber, at least one reagent is circulated through the chamber in the form of a gas or vapor so   that it diffuses into the solid matrix and reacts with the sample furrow.