CA1163425A - Apparatus and method for the sequential performance of chemical processes - Google Patents

Abstract

IMPROVED APPARATUS AND METHOD FOR THE SEQUENTIAL
PERFORMANCE OF CHEMICAL PROCESSES

ABSTRACT OF THE DISCLOSURE

An improved apparatus and method for the sequential perfor-mance of chemical processes on a sample of chemical material wherein the sample is embedded in a solid matrix of fluid permeable material located within a reaction chamber and is sequentially subjected to a plurality of fluids passed through the chamber in a pressurized stream, causing chemical inter-action between the sample and the fluids.

Description

~ J 6 3 ~ 2 5 .. l I
1¦ Background of the Invention 21 This invention relates generally to an improved apparatus 31 and method for the performance of chemical processes and, more 41 particularly, to an improved apparatus for automatically perform-51 ing the sequential degradation of protein or peptide chains con-61 taining a large number of amino acid units for purposes of deter-71 mining the se~uence of those units.
8 ¦ The linear sequence of the amino acid units in proteins 9 ¦and peptides is of considerable interest as an aid to understanding 10 ¦their biological functions and ultimately synthesizing compounds 11 ¦performing the same functions. Although a variety of techniques 12 ¦have been used to determine the linear order of amino acids, 13 ¦probably the most successful is known as the Edman Process.
1~ Various forms of the Edman Process and apparatuses for automatically 15 performing the processes are described in the following publications 16 Edman and Begg, "A Protein Sequenator," European J. Biochem.
17 1 (1967) 80-91; Wittman-Liebold, "Amino Acid Sequence Studies 18 of Ten Ribosomal Proteins of Escherichia coli with an Improved 19 Sequenator Equipped with an Automatic Conversion Device," Hoppe-20 Seylerls Z. Physiol. Chem. 354, 1415 (1973); Wittmann-Liebold 21 et al., "A Device Coupled to a Modified Sequenator for the Auto-22 ated Conversion of Anilinothiazolinones into PTH ~mino Acids,"
23 ~n~lytical ~iochemistry 75, 621 (1976), U. S. Patcnt Mo~ 3,959,307

2~ issued to Wittmann-Liebold and ~raffunder on MaY 25, 1976, for "Method to Determine Automatically the Sequence o~ Amino Acids;"
26 Hunkapiller and Hood, "Direct Microsequence Analysi.s of Polypeptides¦
27 Using an Improved Sequenator, A Nonprotein Carrier (Polybrene), 28 and High Pressure Liquid Chromatography," Biochemistry 2124 (1978);
29 Laursen, R. A. Eur. J.Biochem.20 (1971); Wachter, E., Machleidt, H., 30 Hofner, H., and Ottoj J., FEBS Lett:.35, 97 (1973); U. S. Patent No.

31 3,725,010 issued to Penhasi on April 3, 1973, for "A~paratus for 32 Automatically Performing Chemlcal Processes;" [~. S. Patent Mo.

~ 1 ~3~

3,717,436 issued to Penhasi et al. on ~ebruary 20, 1973, Eor "Process for the Sequential Degradation of Peptide Chains;" United States Patent No. 3,892,531 issued to Gilbert on July 1, 1975, for "Apparatus for Sequencing Peptides and Proteins;" United States Patent ~o. 4,065,412 issued to Dreyer on December 27, 1977, for "Peptide or Protein Sequencing Method and Apparatus." A further apparatus of note is described in United States Patent Serial No. 4~252,769 filed December 26, 1979 by Leroy E. Hood and Michael W. Hunkapiller, two of the applicants hereon, on "Apparatus for the Performance of Chemical Processes."
Briefly, as discussed in the above publications, the Edman sequen-tial degradation processes involve three stages: coupling, cleavage and conversion. In the coupling stage phenylisothiocyanate reacts with the N-terminal ~ amino group of the peptide to form the phenylthiocarbamyl derivative. In the cleavage step anhydrous acid is used to cleave the phenylthiocarbamyl derivative to form the anilinothiazolinone. After extraction of the thiazolinone the residual peptide is ready for the next cycle of coupling and cleavage reactions. Aqueous acid is used to convert the thiazolinone to the phenylthiohydantoin which may be analyzed in an appropri-ate manner, such as by chromatography.
The automated apparatus of the Penhasi 3,725,010 patent, as modified in the above-referenced articles of Wittmann-Liebold and the patent Serial No.

4,252,769 of Hunkapiller and Hood, relates to an automated sequenator in which the reactions are carried on in a thin film formed on the inside wall of a rotating reaction cell which is commonly known as a "spinning cup" and is located within a closed reaction chamber. Means are provided for introducing and removing controlled amounts of liquid reagents relative to the chamber for reaction with a sample of a protein or peptide in an inert atmosphere. The sample to be analyzed is initially placed in the spinning cup, followed by the 1 I t~3~ ~ 5 1 sequential introduction and withdrawal of the various reayents and 2 solvents necessary for carrying out the coupling and cleavage 3 reactions. The licluid reagents and solvents -themselves form films on the walls of the cup which pass over and interact with the sample film as the cup spins. The reagents dissolve the sample 6 film and perform the coupling and cleavage stages of the Edman 7 process. Upon completion of the coupling and cleavage stages, the 8 reaction chamber is evacuated to remove volatile components of the 9 reagents. Following the post-coupling evacuation, the remaining 0 sample film is extracted with solvent to remove non-volatile com-ponents. Following the post-cleavage evacuation, the resulting 12 thiazolinone is extracted from the sample film with solvent and 13 transferred either to a separate flask for conducting the conver-14 sion step or to an apparatus for collection and drying of the ~5 various fractions. In cases where the conversion process is not 16 performed immediately in a conversion flask, the process may be 1~ performed later on a number of fractions simultaneously.
18 The introduction and withdrawal of fluids relative to the 9 spinning cup has been achieved with fluid conduits passing through a plug which seals an opening in the upper wall of the 2212 reaction chamber and depends therefrom to a location within the 23 cup. Fluids are introduced direc-tly into the spinning cup at a point adjacent the bottom thereof, and are withdrawn from an 2~ annular groove in the cylindrical interior surface of the cup.
The fluid to be withdrawn is forced into the annular groove by 26 centrifugal force when the cup is rotated at a hicJh rate, and 27 is withdrawn through a conduit having an inner end projecting 28 into the groove. This effluent condui-t thus acts as a scoop 29 for removing the reaction products and by-products and the extract-ing solvents.
31 If the p~o-tein or peptide sample in a spinninq cup device 32 does not have sufficient mass to form a cohesive film bv itself, it ~.

1 ~ 63~2S

is sometimes carried on the inner wall of the cup during the sol-2 vent extractions within a relatively thick layer of nonprotein 3 carrier material. The carrier materlal and the sample are then 4 dissolved in the liquid reagents during the reaction stages to en-able the col~pling and cleavage reactions to take place. A polymer-6 ic quaternary ammonium salt having the chemical composition 1,5-7 dimethyl-1,5-diazaundecamethylene polymethobromide has been used for this purpose. The carrier must be applied in substantial quan-9 tities to securely retain the sample, and the carrier and sample are both dissolved by the liquid reagents to permit reaction be-11 tween the sample and the reagents.
12 Although devices of the spinning cup type can provide 13 acceptable experimental results in many cases, they have several 14 disadvantages. For example, the expenses of obtaining a suitably large protein or peptide sample and maintaining an adequate supply 16 of the necessary reagents arequite high, primarily because the 17 reagents used are in liquid form and must be used in substantial 18 quantities. Liquid reagents and solvents tend to separate portions of the 19 sample from the film and wash them from the wall of the cup as they pass, reducing the yield of terminal amino acid units 21 obtained in each successive cycle of the apparatus. The initial 22 quantity of sample must therefore be great enough to insure that 23 sufficient sample will remain through the las-t cycle to 24 produce useful resul-ts. Devices of this type also have rather long cycle times due to the considerable volume of -the reaction 26 chamber and the need to repeatedly remove semi-volatile liquid 27 reagents and solvents by vacuum drying the sample -therein. In 28 addition, spinning cup sequenators are quite complex and expen~
29 sive, both to manufacture and repair.

A different type of sequencing device is disclosed in the 31 above-cited Laursen and Wachter papers, wherein the sample is im-32 mobilized by covalent linkage to the surfaces of a pluralitv of ;' 33 small beads. The i~eacls form a porous packillg within a reaction ~ -5-llg3~

1 column, and the column is flooded with liqu,id reayents to perform the chemical processes. Because the cleavage reayents used are 3 excellent solvents for proteins and peptides, the covalent linkage 4 must be complete in order to hold -the sample in place. However, covalent linkage is difficult to obtain ,in practice. The packed column is also difficult to wash, and the beads therein tend to disintegrate during use.
8 There have heretofore been proposed sequencing devices 9 designed to overcome the deficiencies of these apparatuses by containing a sample within a stationary reaction chamber and sub-11 jecting the sample to at least one reagent in gas or vapor form.
12 The Gilbert and Dreyer patents cited above disclose two such 13 devices, neither of which operates entirel~ satisfactorily.
14 The device of the Gilbert patent provides a closed finger~
shaped extension within a reaction chamber for holding a peptide 16 or protein sample at a controlled temperature during sequential 17 exposure to gaseous reagents and solvents. Each time a reagent 18 is introd'uced, the extension is cooled internally to produce 19 condensation thereon. The extension is then warmed, causing the sample to dissolve in the liquid, and the reaction proceeds.
21 After reacting with the sample, the unwanted semi-volatile 22 chemicals may either be dried from the sample by a combination 23 of heat and a stream of inert gas, or be washed from the extension~
2~ along with the terminal amino acid by a solvent which is condensed¦
on the extension until it drips therefrom.
2G In the device and method of the Dreyer ~atent, a proteir o~
27 peptide sample is applied to both the inner and outer surfaces of ~
28 many small macroporous beads within a reaction column by chemica:L ¦
29 coupling or direct adsorption -thereto. Various reagen-ts and sol-vents are passed sequentially through the packed column in either 31 gaseous or liquid form to produce the desired degradation reactionc .
32 The flow of reagents and solvents to the column is controlled by 1 163'12$

1 a ten position rotary face seal valve.
2 Unfortunately, the devices of the Gilbert and Dreyer patents !
3 Ido not provide a suf~iciently contamination-free environment to 4 iachieve acceptable results through a large number of degradation

5 jcycles. For example, it is difficult to efficiently wash the pro-

6 ¦tein or peptide sample in the Gilbert and Dreyer devices. The

7 IGilbert method of washing the sample by condensation of solvent

8 thereon to the point at which solvent drips from the sample would g Itend to leave traces of the various reaction products on the sample 0 Icontaminating future chemical reactions. Likewise, the packing used to retain the sample within the reaction column of Dreyer is 12 difficult to wash because the various chemical products must be 13 transported entirely through it and away from the column to avoid 14 contamination. This is not easily done even when large amounts of solvent are used, because the solvent tends to pass through the 16 spaces between the beads rather than through the small pores inside ¦
17 the beads where most of the protein sample is located. The fluid 18 feed lines and flow valves of the Gilbert and Drever devices are

9 also difficult to fully evacuate and are prone to trapping chemical residues which can interfere with the intended chemistry of further 21 reaction cycles.
22 The glass or plastic beads used as packing in the reaction 23 column of Dreyer also have a tendency to disintegrate over a number 24 lof degradation cycles, clogging the system to the point at which the passage of fluids therethrough is hindered. It then becomes 26 virtually impossible to wash the system between cycles and the 27 chemistry within the column becomes hopelessly contaminated.
28 The contamination caused by the several factors described 29 labove has a cumulative effect over the cluration of a sequential Idegradation process. The sample and the reagents within the 31 reaction cell thus become more and more contaminated, hindering 32 ~ the desired c ling and clea a4e reactions and caaslnq a number `:
;.

~ 3~,5 1 of undesired reactions to take place. The yield from each com-plete cycle of the apparatus is thus decreased and a series of 3 contaminants is introduced into the fractions.
The yield is further decreased by direct loss of the sample ~ due to a variety of reasons, including the disintegration of the 6 packing, solubility of the sample in the flushing solvents, and 7 failure of the sorptive bonds between packing and sample~
8 While these effects may be overlooked in some cases where 9 large amounts of the protein or peptide sample are available or 11 where the chain has a relatively small number of units, they become devastating in cases where the chain has a very large 12 number of units or only very small amounts of the particular 13 protein or peptide are available. Both of these circumstances are present in the case of interferon, a small protein made in 16 human cells in response to certain viral infections. Interferon 17 has recently caused a great deal of excitement in the world of clinical medicine because it promises to be an effective agent 18 for arresting viral infections and it appears to offer consi-19 derable hope as an anti-cancer reagent. Interferon is produced and, accordingly, is available only in very small quantities.
21 Currently, virtually the entire world's production of the two 2 types of human interferon originates in the relatively few world 223 centers that have access to large quantities of human white blood cells (leukocyte interferon) or certain human cells in tissue culture (fibroblast interferon). Because of this limited pro-27 ductive capacity of interferon, it has been difficult to carry 28 out well controlled clinical studies and fundamental analyses 29 of how this molecule functions. To further complicate the picture, interféron is composed of a chain of approximately 150 amino acid units, which must be individually cleaved from the 31 chain for analysis. Contamination losses of the types described 32 above can prevent the sequencing of any but the first few amino -~7 . . .
'' .

~ 1 83~25 acid units of interferon with the very small quantities of the protein available. Beyond the first few cleavage cycles, the small sample can become contaminated to the point at which positive results are uno~taina~le, The most sophisticated prior device Icnown to the applicants herein for converting the various thiazolinones cleaved from the sample into the more stable phenylthiohydantoins is the conversion flask described in the above-referenced articles of Wittmann-Liebold, as modified in the patent Serial No. 4,252,769 of Hunkapiller and Hood. However, applicants have found that this flask suffers from inefficient washing of its inner walls when reagents and solvents are introduced through the appropriate capillary tube.
It has been suggested that the reagents and solvents can be delivered with a stream of inert gas to wash the flask walls by splattering the liquid thereon, however, this technique causes an erratic flow of liquid to the flask and makes it very difficult to control the volume of liquid delivered.
Applicants have also found that when the prior conversion flask is scaled down appreciably in size to accommodate lower volumes of liquid, it is difficult to obtain the optimum degree of dispersion of inert gas bubbles within the liquid contents of the flask to agitate the contents during the conversion reaction and evaporate the semi-volatile components thereof. Inert gas introduced to the bottom of the flask for these purposes tends to rise to the surface of the liquid in relatively large bubbles which do not uniformly agitate the liquid and instead promote splattering of the liquid onto the top of the flask.
Therefore, in many applications it is desirable to provide an apparatus for performing chemical processes such as the sequencing of proteins or peptides which operates efficiently and with a minimum of system contamin-ation to enable the maximum number of _g_ ,;~. . . ~
.. ; '. ~
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1.
"` I 3 1~ 5 : 1 sequencing cycles to be successfully performed with a very small 7 l amount ~f san e.

32 _/0 -1~ ~ 3 6~25 I

1 S~MMARY OF THE INVENTION
. _ _ 2 Briefly, the present invention comprises ~hamber means 3 having an interior surface defining a reaction chamber, the 4 chamber having inlet and outlet means for conduction of ~luids therethrough in a pressurized stream, and solid matriY~ means 6 permeable by diffusion to a plurality of fluids and located 7 within the chamber, such that a sample embedded in the matrix 8 means is immobilized and exposed to any of a plurality of 9 fluids passed through the chamber for chemical interaction therewith.
11 The chamber means may include surface means supporting the 12 solid matrix means as a thin fllm thereon, and the matrix means 13 may comprise a polymeric quaternary ammonium salt such as 1,5-14 dimethyl-1,5-diazaundecamethylene polymethobromide or poly (N,N-dimethyl-3,5-dimethylene piperidinium chloride).
16 I The surface means may comprise all or a portion of the 17 interior wall of the chamber means, or may comprise porous 18 I sheet means extending substantially tranversely across the ¦ chamber and permitting passage of the fluids therethrough.
20 ¦ The sheet means may comprise a sheet made of a plurality of 21 I glass fibers-22 I The chamber means may comprise a pair of abutting chamber 23 elements having first and second cavities, respectively, on 24 opposed mating surfaces thereof, the first and second cavities being aligned with each other to form the reaction chamber.
26 The first and second cavities may be tapered in directions away 27 from the mating surfaces to locations at which they communicate 28 ¦ with the inlet and outlet means, respectively. The porous sheet 29 means, if used, is received within a recess in at least one of the mating surfaces for retention within the chamber in an 31 orientation substantially separating the first and second 32 cavities. The chamber means may include at least one sheet I _/f_ ~ ~ G~ ~ ?J ~

1 yielding material sandwiched between the mating surfaces in a 2 sealing relationship, the yielding material being permeable to 3 the plurality of fluids. At least one of the chamber elements 4 may then include a raised portion on a mating surface thereof which extends about the cavity therein to co~press the sheet 6 of yielding material against the mating surface o the chamber 7 element and thus enhance the sealing relationship.
8 The inlet and outlet means may comprise a pair of capillary 9 passages extending through the chamber elements, respectively, and communicating at inner ends thereof with the reaction chamber on 11 opposite sides of the porous sheet means. The capillaries may be 12 coaxial with the reaction chamber and extend therefrom to outer 13 capillary openings at substantially flat outer surfaces of the 14 chamber elements.
The means for sequentially passing a plurality of fluids 16 through the chamber may comprise valve block means having a 17 plurality of substantially flat valving sites on the surface 18 thereof, the valve block means defining a primary passage 19 continuous between two ends thereof and communicating through primary openings with each of the valving sites~ and a plurality 21 of secondary passages each communicating through a secondarY
22 opening with one of the valving sites; and a plurality of 23 resilient, substantially impermeable diaphragms covering the 24 respective valving sites, each of the diaphragms being actuable between a first sealing condition in which it is forced aqainst 26 one of the valving sites to close off the primary and secondary 27 openings communicating with that site and a second condition 28 in which it is drawn away from the site to provide a fluid flow path between the primary and secondary openings over the exterior of the valve block means; whereby fluid flow between 31 the primary passage and the secondary passa~e can be selectively 32 controlled~ The connecting means may comprise at least one tapere 1 I 1 63~s 1 ferrule closely received in sliding engayement over a tubing 2 member and urged against a differently tapered recess in the valve¦
3 block means communicating wi-th one oE the passages therein, 4 such that an inward force applied to said ferrule is focused on a relatively small area of contact between -the ferrule and the 6 recess to produce a fluid seal therebetween. The recess is 7 preferably tapered at a greater angle than said ferrule. The 8 connecting means may further include a fitting at one end of the 9 primary passage for connecting the primary passage to another 0 portion of the apparatus such that the primary passage serves 11 as a manifold which can be flushed by a flow of fluid between 12 the fitting and the secondary passage furthest away from the 13 fitting. The primary passage may comprise a plurality of 14 straight passages connected end to end to form a conduit having a sawtooth configuration and communicating at alternating inter-16 sections thereof with the respective valviny sites.
17 The apparatus may include a conversion flask having a 18 plurality of capillary tubes extending into the interior thereof 19 for the introduction and withdrawal of various fluids, at leas-t one of the capillary tubes having an inner end at which the bore 21 is closed and which is provided with a plurality of restricted 22 radially-spaced orifices, such that passage of fluids through 23 the capillary tube produces a spray onto the interior walls of 2~ the flask to wash them down. A capillary tube termina-ting at a point adjacent the bottom of the flask may also have a closed 26 end with a plurality of restricted radially-spaced orifices 27 adjacent thereto, such that passage of a gas inwardly -through 28 the capillary tube produces a plurality of small bubbles agitating 29 any liquid within the flask and accelerating the drying thereof.
The method of the present invention for sequentially per-31 forming chemical processes on a sample of chemical material com-32 prises embedding the sample in a solid matri~ which is permeable 1 1 63~2S

by diffusion to a p:Lurality of fluid8, enc;Losing -the 801id matrix within a closed chamber having an inlet and an outlet, and sequentially passing the plurality of fluids through the chamber as a pressuri~ed stream from the inlet to the outlet thereof such that the sample is exposed to each of the fluids, whereby the sample is immobilized and chemical interaction between the sample and the fluids is obtained. The solid matrix may be supported as a thin film on the inner walls of the closed chamber. Alternatively, the solid matrix may be supported on a porous sheet which extends substantially transversely across the chamber at a location between the inlet and outlet such that fluid passed from the inlet to the outlet must pass through the sheet. The step of embed-ding the sample in a solid matrix may comprise the steps of applying the solid matrix to a support surface as a thin film and then applying a solution containing the sample to the film such that the sample solution dissolves the matrix. The liquids are then evaporated from the solution, leaving behind a film with the sample embedded therein.
Thus the present invention seeks to provide an apparatus and method for the sequential performance of chemical processes on a sample of chemical material with a minimum of sample loss and a minimum of system contamination.
~ urther the present invention see~s to provide an economic apparatus and method for the sequential performance of chemical processes on a sample ` of very small size through the use of minimum amounts of reagents and solvents.
Additionally the present invention seeks to provide an improved ~;~ apparatus and method for the sequential performance of chemical processes having a very short cycle time.
The present invention also seeks to provide an improved apparatus ` and method for the sequential performance of chemical processes on a sample wherein the sample is ~"~

i ~ 63~25 1 more effectively washed between cycles.
The apparatus and method of -the present invention solves a 3 number of the problems of the prior sequenators by immobilizing 4 the protein or peptide sample within a solid matrix formed as a thin film permeable by diffusion to both the reagents and solvents used in the degradation process. The difficult problem of attain-¦ing complete covalent linkage is thus avoided, as is the problem 8 ¦of sample loss experienced when the sample is directly adsorbed 9 ¦onto a support surface and fully exposed to the mechanical 0 ¦shearing forces of the mobile liquid phase. The sample is 11 securely held in place by the matrix, while the smaller reagents, 12 solvents and amino acid derivatives are able to diffuse through 13 the matrix, in effect dissolving in the matrix to a sufficient 14 concentration to carry on the various steps in the degradation process. The solid matrix retains the sample so effectively when 16 exposed to gaseous reagents that virtually any shape of sample 17 support surface can be used without causing sample loss. The 18 inner walls of the reaction chamber itself may be a sufficient 19 support surface.

A support surface which greatly facilitates complete washing ¦
21 of the system is a porous sheet made of a plurality of overlapping ¦
2~ glass fibers and extending transversely across a flow-through 23 reaction chamber. The porosity is provided by spaces between the 24 fibers. This structure possesses a relatively high total surface area with a minimum dimension in the direction of fluid flow.
26 The solid matrix forms a thin film on -the surfaces of the fibers, 27 enabling reagents and solvents -to readily diffuse into the film 28 to interact with the sample embedded therein. This enables 29 chemical processes and wash cycles to be performed on the sample with a minimum of reagents and solvents and in a relatively short 31 period of time. The reagents and solvents, some of which are 32 in the form of a gas or vapor, permeate the thin film -to contact - 1 3 63~25 1 the sample and interact therewith as completely and e~ficiently 2 as possible.
3 The relatively thin profile of the porous sheet disclosed 4 herein further enhances the ability of the sample to be thoroughly washed of residual reagents and reaction products with a relativel~
6 small amount of solvent. I'he solvent need only move the reagents 7 and reaction products the relatively short distance beyond the 8 surface of the sheet to remove them from the system. From a 9 point outside the surface of the sheet they may be easily con-ducted out of the chamber to leave the sample in condition for 11 the next reaction step. The low solvent usage not only 12 represents a savings in the cost of solvent, but also reduces 13 the tendency of the sample to be washed from the reaction chamber 14 ànd lost.
The use of reagents in gas or vapor form also contributes 16 to complete exposure of the sample to the reagents, minimizing 17 the amount of reagents required. Low reagent usage is important 18 ¦ because the reagents used in the Edman degradation technique must 19 ¦ be extremely free of contamination and therefore are very 20 ¦ expensive. Further, the sample and the solid matrix containing 21 ¦ it are not dissolved by the gaseous reagents, eliminating the 22 ¦ problem of sample loss due to separation of the sample from the 23¦ support surface.
24 ¦ The reaction chamber of the present invention is constructed 25 ¦ to allow passage of both gaseous and liquid reagents through the 26 porous sheet holding the sample without allowing the sample to 27 become contaminated with external impurities or with chemicals 28 I carried over from one reaction step or cycle to another. The 29 abutting chamber elements thus combine to form a low volume reaction chamber made up of first and second cavities on opposite 31 sides of the porous sheet. A pair of capillary passages extending 32 oppositely through the respective chamber elements from the ~ ; 3 ~
I
1 chamber itself enable a plurality of fluids in gas or liquid 2 form to be passed as a pressurized stream ~hrough the chamber and 3 past the sample matrix. The low volume of the chamber and the 4 passages minimizes the volumes of reagents and solvents required, and facilitates vacuum drying of the system between cycles. The 6 two chamber elements are sealed at mating surfaces thereof 7 against a sheet of yielding material sandwiched between the 8¦ mating surfaces. The yielding material is permeable to the ¦ plurality of fluids passed through the chamber, and in fact,

10¦ improves the flow of gases through the chamber by disbursing

11 the gases to more uniformly contact the porous sheet.

12 An alternative embodiment of the reaction chamber is a

13 singlc capillary tube or capillary-type passage having a solid

14 matrix formed as a thin film on the interior surface or bore thereof. The protein or peptide sample is embedded in the matrix,¦
16 as in the case of the porous sheet, and the reagents and solvents 17 are passed sequentially through the tube to interact with the 18 sample. The chamber structure described above can be used for 19 this purpose without the porous sheet element. The sample-containing matrix is then formed on the surfaces of the first 21 I and second cavities. Similarly, the surface supporting the 22 I solid matrix can be constructed in virtually any way which enables¦
23 ! the reagent and solvent fluids to be passed over the matrix.
24 The novel valve assemblies of the present invention for con-25 I trolling the flow of fluids to and from both the chamber and the 26 ¦ conversion flask are especially constructed to eliminate cross-27 ¦ contamination of the fluids. Each of the valve means interfaces a 28 I single conduit with a plurality of other conduits for selectively 29 ¦ connecting the single conduit to each of the others. The single / / /

1 163~25 1 ¦ conduit is made to communicate with one end of the primary passage 21 in the valve block while each of the other conduits is connected 3 ¦ -to one of the secondary passages. In the normal closed condition,¦
41 each of the diaphragms covering the various valving sites is 51 forced by gas pressure against the surface of the valve block to 61 prevent communication of the primary passage with the secondary 71 passage leading to the particular valve site. Fluid communication 81 between the single conduit and the other conduits may be selective-9¦ ly provided by applying vacuum to one or more of the diaphragms to ~ol draw the diaphragms away from the valving sites and allow fluid to ¦ pass over the surface of the valve block lying between the openingc 2¦ to the respective passages. The secondary passage leading to the 13¦ valving site at the remote end of the primary passage may be 14¦ connected to a pressurized source of a flushing fluid, such 5¦ as inert gas, for the purpose of completing the 6¦ delivery of each fluid through the valve. Thus, after a particu-17 ¦ lar reagent or solvent is introduced into the reaction chamber by 18 ¦ applying a vacuum to the corresponding diaphragm of the delivery 9 ¦ valve, the diaphragm at the remote end of the primary passage 20 ¦ may be opened to complete the delivery by forcing any of the 21 ¦ reagent or solvent remaining within the primary passage to the 22¦ chamber. This is possible due to the continuity of the primary 23¦ passage, and results in the manifold formed thereby being purged 24 ¦ of a particular reagent or solvent before delivery of the next 251 reagent or solvent is commenced. In the case of the valve at 26 ¦ the outlet to the reaction chamber, the primary passa~e is con-27 nected to the outlet while the secondary passages are connected 28 to the conversion flask, vacuum and waste, respectively. The con-29 tinuity of the primary passage enables it to be thoroughly evacu-ated and virtually eliminates the possibility that semi-volatile 31 substances will be trapped therein between cycles.
32 It will be understood that the "sawtooth" configura-tion of -la-1 163~25 1 ~ the primary valve passages disclosed herein has previously been 2 ¦ used in valve assemblies of others in the sequenator field.
¦ However, the prior sawtooth valve assemblies of which applicants are aware have incorporated a plurality of individual blocks 5 ¦ mounted against valving sites on a main valve block to slide 6 ¦ back and forth between conditions of communication and noncommuni-¦ cation of passages within the main block. The slidin~ blocks 8 1 tend to wear, causing leaks both to the atmosphere and between ¦ the passages. The novel valve assemblies disclosed herein solve 10 ¦ the problem of wear by combining the prior sawtooth manifold with 11 ¦ a series of diaphragms for establishing and cutting off flow 12 ¦ between pairs of openings communica-ting with the respective pas~
13 ¦ sages. The diaphragms can be made of substantially inert materialc , 14 ¦ such as commercially available fluorocarbon polymers, which will

15 function indefinitely without deterioration. Moreover, the prior

16 ¦ means for connecting the valve passages to external conduits tend

17 ¦ to produce excessive pressure on the sides of the valve block,

18 ¦ promoting distortion of the upper sealing surface of the valve

19 ¦ block and loss of its ability to form a seal. In particular,

20 ¦ the prior sawtooth valves of which applicants are aware

21 ¦ connect external conduits to the valve passages by press-

22 ¦ ing substantially flat flange surfaces associated with the

23 ¦ various conduits against the sides of the valving block to

24 ¦ produce a series of seals between pairs of flat surfaces.

25 ¦ Because each of these components is made of materials such

26 ¦ as fluorocarbon polymers which are very difficult -to accur-

27 ¦ ately machine, a considerable amount of pressure must be

28 ¦ applied to conform the respectlve sea:ling surfaces to each

29 other and form the required seals. The pressure is borne by

30 the valve block, causing distortion of its upper sealing surface.

31 The valve assemblies of the present invention incorporate a

32 plurality of tapered ferrules receivable partially within _]9_ 1~ 63~2~

1 differently tapered recesses in the valve block -to effect a seal 2 without the application of undue pressure to khe valve block.
3 A relatively small sealing Eorce is focused on a particular por-tion on the ferrule to seal the ferrule against the corresponding tapered recess without distorting the block.
6 The provision of interfaces at opposite ends of the ferrules between surfaces having different tapers further enhances the seals obtained. Interfaces of this type between differently 9 tapered su~faces are preferably provided on opposite sides of the10 ferrules to obtain optimum sealing characteristics.
; 11 The conversion flask of the pxesent invention enables 12 reagents and solvents to be introduced in the form of a spray ` 13 impinging on the interior walls of the flask to wash any 14 chemicals which may have been condensed or splattered thereon down the walls and into the body of liquid within the flask.
. 16 A major source of cross-contamination of the system between 17 cycles is thus eliminated. The conversion flask also enables 18 gases to be passed upwardly through the body of liquid in the 19 form of small bubbles which uniformly agitate the liquid and aid in drying semi-volatile components thereof, without causing 21 sample loss due to excessively vigorous bubbling and splattering.
22 This promotes rapid, gentle removal of liquid from the sensitive 23 amino acid derivatives. Solvent used to carry the amino acid 24 derivatives into the conversion flask can thus be removed in a much shorter time than in the Wittmann-Liebold patent cited above 26 (1 to 2 minutes rather than 5 to 10 minutes) and at a lower 27 temperature (40 to 50C rather than 50 -to 80C). This signifi-28 cantly improves yields of the most unstable amino acid derivatives, 29 such as those of serine, threonine, histidine, arginine, and ~30 tryptophan. Reagents used in the conversion flask can be removed 31 by a combination oE Eine streams o~ -inert gas hubhles and low 32 vacuum in 3 to 5 minutes rather than the 30 to 40 minutes required ~ -20-:

~ B~5 1 under the Wittmann-Liebold patent. In the process of the Wittmann-2 Liebold patent, drying of the reagent must commence immediately 3 upon its introduction to the amino acid residue in the conversion 4 flask in order to meet the re~uirement that the ~otal conversi~n flask cycle time be no greater than the total cycle time of the 6 primary reaction chamber. Since the acid component of the ~ conversion reagent, trifluoroacetic or hydrochloric acid, is 8 much more volatile than the water in which it is dissolved, the 9 acid component tends to be removed early in the drying process of Wittmann-Liebold leaving the amino acid derivative in nothing 11 but a water solution for a significant portion of the conversion 12 stage. This causes incomplete conversion of the derivatives of 13 glycine and proline and decomposition of other deriatives. In 14 the present conversion apparatus, the conversion reagent can be left in contact with the amino acid derivatives for a time 16 sufficient for complete conversion, 30 to 40 minutes, and then 17 dried rapidly, in 3 to 5 minutes,-without splattering the sample 18 throughout the interior of the conversion flask.
19 l 20 !

`

1 1 ~

_RIEF DE.SCF<IPTION OF THE DRAWINGS
The above and other objects of the present invention may be 3 more fully understood from the following detailed description 4 taken together with the accompanying drawings wherein similar reference characters refer to similar elements throughout and 6 in which;
7 Fig. 1 is a perspective view of an apparatus constructed in ` 8 accordance with the present invention;
9 Fig. 2 is a top plan view of the apparatus of Fig. l;
Fig. 3 is a schematic diagram of the apparatus of Fig. l;
11 Fig. 4 i5 an enlarged exploded perspective view of a 12 reaction chamber assembly constructed in accordance with the 13 present invention;
14 Fig. 5 is an enlarged vertical cross-sectional view taken along the line 5-5 of Fig. 4;
16 Fig. 6A is a further enlarged cross-sectional view of the 17 reaction chamber illustrated in Fig. 5;
18 Fig. 6B is a cross-sectional view of a second embodiment of 19 the reaction chamber illustrated in Fig. 5;
Fig. 6C is a cross-sectional view of the reaction chamber of 21 Fig. 6B with the porous sheet element removed therefrom, for use 22 with a sample-containing film applied to the interior surface 23 thereof;
2~ Fig. 7 is a vertical cross-sectional view of a typical reservoir of the present invention for a reagent or solvent to 26 be used in li~uid form;
27 Fig~ 8 is a vertical cross-sectional view of a typical 28 reservoir of the present invention Eor a reagent -to be used in 29 the form of a gas or vapor;
Fig. 9 is a vertical cross-sectional view of a diaphragm 31 valve assembly constructed in accordance with the present inven-` 32 tion for controlling the flow of fluids to and from the reaction 1:1~3~25 ..

~:`
1 chamber and the conversion flask, taken in a direction correspond-ing to the line 9~9 in Fig. 11;
3 Fig. 9A is a fragmentary enlarged cross-sectional view of one 4 of the connector elements of the valve assembly illustrated in Fig. 9;
6 Fig. 10 is a vertical cross-sectional view taken along the 7 line 10-10 of Fig. 9;
Fig. 11 is a side elevational view of the manifold block of 9 the valve apparatus illustrated in Fig. 9;
Fig. 12 is a horizontal cross-sectional view taken along 11 the line 12-12 of Fig. 11;
12 Fig. 12A is a vertical cross-sectional view taken along the 13 line 12A-12A of Fig. 11;
14 Fig. 13A is an enlarged fragmentary cross-sectional view of the valving portion of the valve assembly of Fig. 9, showing the 16 diaphragm pressed against the manifold block to prevent fluid 17 communication between the passages at that location;
18 Fig. 13B is an enlarged fragmentary cross-sectional view 19 I of the valving portion of the assembly of Fig. 9, showing the 20 ~ diaphragm drawn away from the manifold block to permit fluid 21 ¦ flow between the passages;
22 ~ Fig. 14 is a top plan view of a conversion flask constructed 231 in accordance with the present invention;
24 I Fig. 15 is a vertical sectional view taken along the line 25 ¦ 15-15 of Fig. 14;
26 Fig. 16 is a vertical sectional view taken along the line 27 ! 16-16 of Fig. 14;
28 Figs. 17A and 17B illustrate schematically the two primary 29 prior art methods of lmmobilizing a protein or peptide sample during degradation;

Fig. 17C illustrates schematically the immobilization of a 32 protein or peptide according to the present invention;
_ ~ 3 ~

13~3~

1 Fig. 18A is a fragmentary enlarged vertical cross-sectional view corresponding to Fig. 5, of a further embodiment of the reaction chamber assembly of the present invention;
; 4 Fig. 18B is a vertical cross-sectional view showing the chamber element of the embodiment of Fig. 18A, turned on its side 6 for the purpose of applying a sample-containing matrix to the interior walls thereof;
8 Fig. 18C is a further enlarged fragmentary cross-sectional 9 view of the chamber element of Fig. 18B, with a sample-containing film applied to the interior walls thereof; and 11 Table 1 is a listing of the various steps perEormed by the 12 apparatus of the present invention in a typical degradation and 13 conversion cycle.

2~

3 ~ 2 S
.., l 1 I DFSCRIPTION OF THE PREFERRED EMBO~IMENTS
2 ! Referring now to the drawings there is illustrated, in 31 Figs. 1 and 2 thereof, an apparatus embodying the present inven-4 ¦tion,generally designated 10. The apparatus 10 includes a chamber 5 ¦apparatus 12, a conversion flask 14 and a fraction collector 6¦ 16, each of which is operated through an automatic control unit 71 18. An array 20 of pressurized solvent and reagent reservoirs - 81 are connected through a bank 22 of diaphragm-type flow valves g¦ to the reaction chamber 12 and the conversion flask 14. A second 10¦ bank of valves 24 regulates the flow of liquids from the reaction 11 ¦chamber to the conversion flask 14 and other locations. A third 12 ¦bank of diaphragm valves 26 serves to connect the conversion flask 13 ¦and fraction collector to either waste ox vacuum, and to r~gulate 14 ~fluid flow from the flask to the fraction collector.
15 ¦ A filtered inert gas source 28 supplies the apparatus 10 16 ¦with highly purified inert gas, preferably argon, for pressurizing 17 ¦the solvent and reagent reservoirs, purging oxygen-bearing air 18 ¦from the system and accelerating the process of drying out the 19 ¦reagents and solvents wlthin the system at various times. A bank 201 30 of pressure-regulating valves and gauges serves to individually 21 regulate the pressure of the gas to each solvent and each reagent 22 reservoir, and to each of the other components of the apparatus 23 10.
24 The operation of the apparatus 10 is depicted in Fig. 3.
25 The chamber apparatus 12 is located within a heated environment 32 and, in 26 the preferred embodiment, defines a reaction ch~mber 3a communicating with inlet 27 ~and outletpassages 36and 38, respectively. The inlet passage 36 28¦ is connectible through a fluid conduit 40 to a plurality of 29 ¦reservoirs of the array 20, namely, reagent reservoirs 42, 44, 46 301 and 48, and solvent reservoirs 50, 52 and 54. The reservoirs 42 31 through 54 are pressurized by the inert gaspressure source 28 through 32 ~n array of individual gas pressure requlators 56 and solenoid ~ ` ~ 3~Z5 :.
1 flow valves 58. Each of the res~rvoirs 4~ throuyh 54 is also con-2 nec-tible at a point above the fluid level therein to a waste trap :. 3 60 through an individual flow valve 62 and an individual flow 4 regulator 64. Pressurized inert gas introduced to the reservoirs from the source 28 can thus be vented to the waste trap 60 at a 6 rate controlled by the flow regulators 64. The fluid outputs 71 of the reservoirs 42 through 54 are individually controlled through 8 diaphragm valves 66 communicating with a continuous manifold 68 9 which is connected at one end to the fluid condui-t 40. Fluid from 10 ¦the pressurized reservoirs 42 through 54 can thus be passed through 11¦ the conduit 40 and the chamber inlet passage 36 to the reaction cham 12 ¦ber 34 by the selective actuation of the valves 66. To aid in the 13 ¦delivery of the reagents and solvents and to flush the manifold 14 ¦68 and conduit 40 after delivery, pressurized inert gas from -the 15 ¦source 28 may be introduced to the manifold 68 at the end opposite 16 Ito the conduit 40 through a pressure regulator 70 and a diaphragm 17 ¦valve 72. Actuation of the valve 72 thus introduces pressurized 18 ¦gas at the remote end of the manifold 68, driving any reagents 19 lor solvents therein through the conduit 40 and the passage 36 to 20 the reaction chamber 34.
21 The fluid outlets of the reservoirs 44, 46 and 48 are pro-22 vided with gas flow meters 74 in series with flow regulators 76 23 because the reagents stored therein are used in gas or vapor form, 24 while the remaining solvents and reagents are used in liquid form.
The meters 74 and flow regulators 76 are necessary to accurately con-trol ¦
26 the rate of gas discharge through the corresponding valves 66.
27 Fluid flow from the reaction chamber 34 is controlled by 28 diaphragm valves 78, 80 and 82, which communica-te with the outle-t 29 passage 38 through a continuous manifold 84. The valve 78 is opened to pass the desired produc-t of react,ion, typically the 31 N-terminal amino acid unit of a protein or peptide sample, through 3 ~ ~ S
.

: 1 the conduit 86 to the conversion flask 14. The valve 82 may be 2 opened to connect the outlet passage 38 to the waste trap 60 for 3 disposal of unwanted reagents, solvents and reaction products, and the valve 80 connects the outlet passage 38 to a vacuum trap 51 88 and a vacuum pump 90 for evacuation of the reaction chamber 34, 6 the outlet passage 38 and the manifold 84.
7 Similarly,the conversion flask 14 and the fraction collector 8 16 are connectible to the waste trap 60 through valves 92 ar;d 94, 9 respectively, and to vacuum through the valves 96 and 98.

Reagent reservoirs 100 and 101 and a solvent reservoir 102 11 are provided to supply reagents and solvent to the conversion flask 12 14 through a conduit 104. The reservoirs 100, 101 and 102 are pressur-13 ized by the inert gas source 28 through pressure regulators 106 14 and solenoid valves 108, and are connected to the waste trap 60 15 through individual vent valves 110 and flow re~ulators 111. The fluid 16 outlets of the reservoirs 100, 101 and 102 are connected through dia- .
17 phragm valves 112 to a continuous manifold 114 which communicates 18 at one end with the conduit 104. The flow of fluid from the pres-19 surized reservoirs may thus be produced by selectively opening the valves 112 to expel either reagent or solvent into the manifold 21 114. The source of inert gas is connectible through a pressure 22 egulator 116 and a diaphragm valve 118 to the end of the manifold 23 14 opposite the conduit 104 to propel the reagent or solvent : 24 hrough the manifold and the conduit to the conversion flask 14.

The inert gas pressure source 28 is also connected to 26 ¦the conversion flask through a pressure regulator 120, a diaphra~m.
27 ¦valve 122 and a conduit 124, and to the fraction collector 16 28¦ through a pressure regulator 126 and a valve 128. When a particular 29¦ fraction has been converted in the intended manner within the flask 301 14, it can be expelled from the flask by gas pressure through the 31 conduit 124 and a valve 131 to the ~raction collector 16 for storage 32 within a vai.l therei.n. After the frac.iG.n s ~ d, ~he lldsk 11 ~ ;27-: ` ~ J ~25 1 ¦ 14 can be filled to a relatively high level with solven~ to dis-2 ¦ solve any residual chemicals therein. The solvent can then be 31 expelled by gas pressure through the conduit 124 and a valve 125 41 to the waste trap 60,flushing the flask in preparation for ~elivery of the next amino acid derivative.
S The fraction collector 16 comprises basically a carrousel of 7 vials actuated by the control unit 18 once during each c~cle of the 8 apparatus 10 to place an empty vial in position to receive the 9 next succeeding fraction of amino acid units from the flask 14.
The control unit 18 is preferably a fully automated unit 11 controlling the diaphragm valves 66, 72, 78, 80, 82, 92, 94, 96, 12 98, 112, 118, 122, 125, 128 and 131, as well as the solenoid valves 13 58, 62, 108 and 110. The control unit 18 also controls the 14 mechanism for maintaining the heated environment 32 at the desired temperature (not shown), the fraction collector 16, the 16 vacuum pump 90, and a variety of sensors throughout the system.
The various gas pressure regulators and flow regulators described 18 ¦ above are manually adjusted upon set-up to establish the desired 19 1 pressures and flows within the corresponding fluid conduits.
20 ¦ The chamber apparatus 12 is shown in detail in Figs. 4 and 21 5 to comprise a two-piece base 130 supportin~ a sleeve 132 which 22 'contains first and second chamber elements 134 and 136, respective-23 ¦ly. The sleeve 132 is provided with an enlarged cylindrical por-24 tion 138 centered about the axis of the sleeve and closely received within a cylindrical recess 140 of the base 130. The 26 cylindrical portion 138 is held in position by a retaining collar 27 142 which is threadingly engaged with the base 130.
28 The chamber elements 134 and 136 are cylindrical glass 29 elements having opposed mating faces 144 and 146, respectively, and closely received in axial alignment within the sleeve 132.
31 The inlet and outlet passages 36 and 38, described above, extend 32 axially through the chamber elements 134 and 136, resp~.ct.ive1~, I ~ ~3~5 and are preferably capillary passages having a diameter on the 2 order of 1 millimeter. The axially outer ends 148 and 150 of 3 chamber elements 134 and 136, respectively, are generally flat 4 and are abutted with a pair of thin resilien~ washers made of ~ a substantially inert material to provide the chamber elements 6 with a cushion in the axial direction relative to the sleeve 132.
7 A metallic washer 154 located on top of the upper resilient washer 8 152 is provided with opposed locking ears 156 for engaging slots 9 158 in the upper end of the sleeve 132. The metallic washer 154 0 is held in position by a cap 160 threaded to the upper end of the 11 sleeve 132 to snugly hold the chamber elements in place relative 12 to the sleeve. The engagement of the ears 156 with the slots 13 158 prevents the washer 154 from rotating when ~he cap 160 is 14 installed, thus preventing the cap from damaging the assembly by 16 rotating the chamber elements. The fluid conduit 40 is provided 17 with a flared lower end 162 which abuts the outer end 148 of the chamber element 134 such that the bore of the conduit 40 commun-18 icates with the inlet passage 36. The conduit 40 carries a back-up washer 164 and a fitting member 166 which is threaded axially 21 into the cap 160 to force the flared end 162 against the chamber 22 element 134 in a sealing relationship. The conduit 40 may be 23 made of any resilient substantially inert material, such as com-mercial fluorocarbon polymers. The alignment of the bore of the 24 conduit 40 with the inlet passage 36 is assured by precision con-26 struction of the various interfitting components about a common axls .
27 ¦ The outlet passage 38 is placed in communication with the con-28 ¦tinuous manifold 84 described above by a mass 172 of substan-29 tially inert material encased within a steel sleeve 174. The sleeve 174 has a smooth exterior received within aligned axial openings 176 31 and 178 of the enlarged cylindrical portion 138 and the base 3~ 130, respectively. The mass 172 extends axially in either direction

33 beyond the steel sleeve 174 to engage the outer end 150 of the --?.~)--1 J ~3~25 1¦ second chamber element 136 and a tapered recess 180 of a valve 21 block 182 which defines -the manifold 84. An axial passage 184 3¦ within the mass 172 is precisely aligned with the outlet passage ~¦ 38 and one end of the manifold 84 to provide a single continuous 51 capillary passageway from the chamber element 136 to the valve 6¦ block 182. As in the case of the conduit 40 discussed above,, the accurate construction of ~he related components about a common ¦ axis insures precise alignmen-t and compl,ete sealing between the I various passages. The chamber apparatus 12 can thus be easily 10¦ disassembled and reassembled in a very short time without compro-11¦ mising alignment of the various passages or the integrity of the 12 various seals.
13 The valve block 182 is of a novel construction which will 14 be described in detail in relation to Figs. 9 through 13. It will 15 suffice to note at this point that portions of the valves 78~ 80 16 and 82 are included within the valve block 182 -to control the flow 1~ of fluid from the chamber apparatus 12.
18 As seen most clearly in Fig.6A, the chamber 34 is formed by 19 aligned cavities 186 and 188 in the opposed mating surfaces 144 and 20 146, respectively, of the two chamber elements. The two cavities 21 are arranged coaxially with the inlet and outlet passages 36 and 22 38 and are preferably circular in cross-section, providing an 23 axiaily symmetric path for fluid passing from the inlet passage to 2~ the outlet passage. A porous sheet element 190 extends -transversely 25 across the reaction chamber 34 and may be received at leas-t partiall~ , 2~ within a depression 192 of the cavity 186. The porous sheet element 27 190 thus separates the inlet passage 36 Erom the outlet passage 38 28 such that fluids flowing from one to the other must pass through the heet element. The porous sheet element 190 prcferably com~riscs 30 a sheet or mat made of a compressed fibrous material, such as glass.
31 Commercially available glass fiber filters are suitable for this 32 purpose and have a high resistance to decomposition or other -.:0-~ J 6342s ~ ¦damage during use. It has been found that a porous sheet of this 2 ¦ type provides a rather large surface for supporting a thin film 3 ¦ in which a protein or peptide sample can be embedded. If the film 4 ¦ is made of a fluid-permeable material, i.e. one which allows dif-5 ¦ fusion of liquids and gases into it, then the material can form a 6 ¦ solid matrix which is able to securely hold the sample but permits 71 chemical interaction of reagents and solvents with the sample. Poly 8 ¦ meric qua-ternary ammonium salts, such as 1,5-dimethyl-1,5-diazaun-9 I decamethylene polymethobromide or poly(N,N-dimethyl-3,5-dimethylene 0 ¦ piperidinium chloride), are ideal for this purpose. They permit 11¦ diffusion of fluids, are insoluble in the solvents used and are 12 ¦ chemically stable to both the reagents and solvents. In addition, 13 ¦ they make a cohesive film and carry a positive charge which enables 14 ¦ them to bond ionically to the glass support surface.
15 ¦ The fundamental differences between the forms of sample re- ¦
16 ¦ tention practiced in the prior sequenators and that of the present ¦
17 ¦ invention will be understood most clearly in relation to Figs. 17A,¦
18 ¦ 17s and 17C. Figs. 17A and 17B illustrate schematically the two 19 ¦ most common prior methods of immobilizing a protein or peptide 20 ¦ sample 300 relative to a sample support surface 302 or 304.
21 ¦ Fig. 17A illustrates the case in which the sample is 22 ¦ chemically linked to a glass support surface 302 by covalent bonds ¦
23 ¦ 306. For example, the surface302 may be specially treated such 2~ ¦ that some of the silica sites of the glass have functional amino 25 ¦ groups 308 extending therefrom for reaction with carboxyl groups 26 ¦ 310 on the sample chain. Under proper conditions some of the 271 groups 308 and 310 will react, leaving the sample covalently 28 ¦ bonded to the glass and releasing a number of water molecules.
291 Bonds of this type are very strong, and one or two of them ~er mole cule are sufficient to hold the sample in place. I-lowever, covalent 31 bonding is difficult to achieve with pro-tein and pep-tide samples.
32 Also, covalent bonds hold the chain through a very few isolated 1 1 ~3~S

~ units in the chain. When the degradation process reaches those 2 units and cleaves them from the chain, the remainder of the chain 3 is left unbound and can be washed from the chamber.
: Fig. 17B illustrates the case in which -the sample 300 is adsorbed directly onto a support surface 304. The sample is then 6 held in place by a very large number of relatively weak noncovalent 7 interactions 312 between the sample and the surface. On the mole-8 cular level, the surface interacts with many different sites on 9 the sample. This works well in the case of large proteins and peptides, but as the sample gets sequenced down to a much smaller 11 size it becomes susceptible to being knocked or drawn from the 12 sur~ace. This results in drastic sample loss.
13 Fig. 17C illustrates schematically the immobilization of 14 the sample 300 relative to the support surface 314 by embedding it in a solid matrix 316 formed as a thin film thereon. As described 16 above, the matrix 316 may be a polymeric quaternary ammonium salt 17 which has a positive charge. The matrix will thus be firmly 18 retained on an acidic glass surface by a very large number of 19 ioni~ interactions, and will securely hold the sample in place because the sample is embedded in it. No reliance is placed in 21 direct bonding interactions between the sample and the surface, Z2 and the effectiveness of the immobilization is not affected by 23 diminishing sample size.
24 The present invention relies on diffusion of reagents, solvents and amino acid derivatives through the solid matrix 316 26 to effect chemical interaction with the embedded sample. The 27 matrix is formed as a thin film which absorbs the reagents and 28 solvents passed over it. Once dissolved in the film, the reagents 29 and solvents are able to readilydiffuse across its thickness to carry on the degradation process.
31 Returning now to Fig. 6A, the reaction chamber 34 is sealed 32 at the periphery of the cavities 186 and 188 by at least one sheet ? ~ 63~25 1 194 of a yielding sealing material sandwiched between the mating 2 surfaces 144 and 146. A palr of yielding sheets 194 are 3 preferably used, one on either side of the poraus sheet element 4 190. The sheets 194 are very thin and are permeable to the 6 plurality of reagent and solvent fluids to be passed through the 6 chamber 34. An annular sealing ridge or bead 196 at the 7 periphery of the cavity 188 bears against the sealing sheets 8 194 to provide a more effective seal a~ainst the surface 144 adjacent the periphery of the cavity 186. The sealing s~eets 194 may be ma~e of any substantially chemically inert material, 11 such as a commercial fluorocarbon polymer, to minimize the 12 possibility of seal deterioration. They serve not only to 13 provide a seal for the chamber 34 but also to support the porous 14 sheet element 190 and to diffuse gases and liquids passed through the chamber such that flow of the gases and liquids will be more 16 evenly distributed across the element 190. J.ong sys-tem life and 17 optimum chemical lnteraction with a sample are promoted in this 18 way.
19 An alternative embodiment 34' of the reac-tion chamber of the present invention is illustrated in Fig. 6B, wherein the aligned 21 cavities 186' and 188' in the opposed mating surfaces of the two 22 chamber elements are somewhat narrower and longer than the cavi-tie~
23 186 and 188. Otherwise, the structures 34 and 34' are identical, !
2~ and the various elements of the struc-ture 34' in the drawings are ¦
numbered similarly to those of the structure 34 with the addition ¦
26 of "primes" (') to dis-tinguish them. The reaction chamber 34' per 27 mits a somewhat more direct flow of fluids from the inlet 36' to 28 the outlet 38', but restricts -the diameter of the porous shee-t 29 element 190' therein.
Fig. 6C illus-trates the chamber 34' of Fig. 6B with the 31 porous sheet element 190' removed therefrom. In addi-tlon, -the 32 sealing shee-ts 194' are replaced with a single annular sheet I363~2S
1 316 of yielding material having a central opening equal to the 2 diameter of the chamber. In this embodiment, a solid fluid 3 permeable matrix 318 having a pro-tein or peptide sample embedded therein is formed as a thin film on the walls of the chamber 34' for exposure to reagents and solvents passed through the chamber.
The flow of fluids through the chamber 3~' is thus enhanced, 7 while a substantial film surface area is retained.
8 A further embodiment of the chamber apparatus of the present ~ invention is shown in Figures 18A, 18B and 18C, wherein the two chamber elements 134 and 136 are replaced by a single capillary-11 type chamber element 320 within the sleeve 132. The remaining 12 elements of the chamber apparatus 12 are the same as those 13 described in relation to Figs. 4 and S, and are numbered similarly.
14 All structures and connections external to the chamber apparatus are also identical to those described above.
16 The chamber element 320 comprises a cylindrical glass 17 structure having interior walls 322 defining an axial ~apillary-18 type chamber 324. The chamber 324 increases uniformly in 19 diameter from its two ends 326 toward its middle 328, and the protein or peptide sample is carried within a solid matrix 21 330 formed as a thin film on the walls 322.
22 It will be undexstood that the reaction chamber of the 23 present invention can take virtually any form having a sample 24 support surface past which a plurality of reagent and solvent fluids can be passed. For example, a single elongated capillary 26 tube (not shown) would suffice for the chamber apparatus 12, 27 with a fll~id-permeable solid matrix formed on the interior 28 surface or bore thereof. ~luids passed -through the tube would 29 interact with a protein or peptlde sample embedded in -the film to perform the degradation process.
` 31 A typical reservoir 198 of -the presen-t invention for s-torage 32 and delivery of a liquid reagent to the reac-tion chamber 3~, 3~' ~J -3~-I ~ 63~25 1 or 324 is shown in Fig. 7. The reservoir 198 corresponds to the 2 reservoirs 42, 50, 52, 54, 100, 101 and 102 of Fig. 3. A
3 pressurized inert gas is supplied to the interior 200 of the 4 reservoir 198 by a conduit 202 communicating therewith at a point below a level 204 oE the liquid reagent or solvent therein. The 6 pressurized gas thus introduced draws any dissolved oxygen from 7 the liquid and can be released at a controlled rate through a vent ~onduit 206 to produce a dynamic equilibrium conditicn within the interior 200. A liquid outlet 208 is provided for the controlled expulsion of reagent or solvents from the reservoir 11 198 by the gas pressure therein. The inert gas supply line 202 12 of each reservoir 198 receives pressurized inert gas from the 13 source 28 through a pressure regulator 56 or 106 and a solenoid 14 valve 58 or 108. The release of gas through the vent conduit 206 is likewise controlled by one of the solenoid valves 62 or 110 and 16 one of the flow regulators 64 or 111. The flow of liquid reagent 17 I or solvent through the conduit 208 is controlled by one of the 18 I valves 66 or 112. Each time one of the liquid reagents or solventc 19 ¦ is to be delivered to the reaction chamber or the conversion flask 20 ~ 14, the corresponding argon supply valves and vent valves are opened 21 to establish a dynamic equilibrium condition within the particular 22 reservoir. Reagent or solvent in liquid form can then be intro-23 ¦ duced by opening the valve in the conduit 208 and the valve 82 24 ¦ to waste trap 60. Liquid is e~pelled from the reservoir at a constant rate, enabling the quantity of liquid delivered to be 26 accurately controlled by controlling the length of time the 27 valve in the delivery line 208 i9 held open.
28 A typical reservoir 210 of the present invention for 29 delivery of a reagent in gas or vapor form is illustrated in Fig. 8. An inert gas inlet 212 terminating in a glass frit 3~ sparging element 213 is provided for introducing inert gas 32 to the interior 214 of the reservoir 210 at a point adjacent ` -35_ ~ ~ ~3~2~

the bo-ttom thereof and substan-tially below a level 216 of liquid 2 reagent therein. A vent conduit 218 and an output or delivery 3 line 220 communicate with the interior 214 at points above the liquid level 216. The reservoir 210 is -typical of the reservoirs 44, 46 and 48 of Fig. 3, with one of the pressure regulators 56 anc 6 one of the valves 58 controlling the flow of gas through the con-7 duit 212 from the gas pressure source 28. Likewise, the escape of pressurized gas to the Vent conduit 218 is controlled by one 9 of the flow regulators 64 and flow valves 62, and the delivery of reagent along the line 220 is controlled by one of the 11 diaphragm valves 66 in line with a flow meter 74 and flow regu-12 lator 76.
13 Thus, although the reagents R2, R3 and R3A are delivered 14 to the reaction cham~er in gas or vapor form, they are stored as liquids and vaporized when needed. Vaporization is accom-16 plished by the bubbling of inert gas upwardly through the liquid 17 reagent. In this way, the inert gas in the interior 214 of the 18 reservoir 210 becomes saturated with reagent vapor. Each time 19 reagent is needed, the valves connected to the conduits 212 and 218 are opened to bubble inert gas through the reagent and 21 establish a dynamic equilibrium condition. The valve 66 within 22 the delivery conduit 220 is then opened for a predetermined 23 length of time to deliver the desired quantity of reagent vapor 24 to the reaction cell. The flow regulator 76 in line with the particular valve 66 causes the vapor to be delivered by the 26 reservoir at a constant ra-te indicated by the flow me-ter 74.
27 Figs. 9 through 13 illustrate the structure and opera-tion 28 of a valve assembly 222 which embodies the valves 66 and 72 and 29 the continuous manifold 68 of Fig. 3. The valve assembly 222 includes a valve block 224 which is seen most clearly 31 in Figs. 11 and 12. The valve block 224 is an elongated block 32 of rectangular cross-section having a continuous primary passage ,, ¦ 11 fi3~5 .:' l , l ¦ 226 in a sawtooth pattern formed by cross-drilling the valve 2 ¦ block from a surface 228 t~ereof. The primary passa~e 22~ is ¦ thus a single continuous passage communicating at alternating 4 ¦ intersections thereof with a plurality of valving sites 230 5 ¦ on the surface 228 through corresponding openings 232. A tapered 61 connector port 234 communicates with one end of the primary 71 passa,ge 226, A plura,lity of secondary passages 236 extend from 81 tapered connector ports 238 at the opposite side of the valve 9 ¦ block 224 to corresponding openings 240 in proximity to the lO ¦ openings 232 at the respective valving sites 230. The valve ll ¦ block 224 is received within a longitudinal slot 242 of a 12 ¦ base 244 having a plurality of threaded openings 246 in alignment 13 ¦ with the connector ports 234 and 238 for reception of connector 14 ¦ fittings 248. The fittings 248 are adapted to compress resilient 15 ¦ doubly tapered'ferrules 250 again~tthe connector ports 234 and 238, 16 ¦ respectively, to sealingly join tubes 252 extending through the 17 ¦ ferrules with the various passages of the valve block 224. In this 18 ¦ way, the connector port 234 of the pximary passage communicates 19 ¦ with the inlet passage 36 of the chamber apparatus 12 through the 20 ¦ fluid conduit 40 of Fig. 3, and the first seven of the eight 21 ¦ secondary passages communicate with the fluid outlets 208 and 220 22 ¦ of the reservoirs 42 through 54 respectively. The secondary passag 23'1 furthest from the connector port 234 communicates with the inert 24 ¦ gas pressure source 28 through the pressure regulator 70 shown 25 ¦ in Fig. 3.
26 ¦ The structure of the doubly tapered ferrule connections 27 ¦ to the ports of the valve block 224 is shown in greater detail in 28 Fig. 9A, depicting the port 234 by way of example.
29 The ferrule 250 of Fiy. 9A is received at its inner side 243 ' 30 within the connector port 234 and at its outer side 245 within a 31 tapered recess 247 of the fitting 248, the port 234 and the recess 32 247 being tapered at angles greater than the angles of taper of the ~.

.

:`
.

1 I respective sides of -the ferrule. The ferrule is preferably tapere 2 ¦ at the same angle on both sides, with the port 23~ and recess 2~7 3 ¦ being tapered at an angle -three degrees (3) grea-ter -than the ¦ ferrule. This fit between differently tapered surfaces focuses 5 ¦ the forces of compression upon the tips 249 of the ferrules 6 ¦ 250, providing an effective seal with a minimum of pressure on 7 ¦ the side of the valve block 224. Excessive pressures on the valve 8 ¦ block 224 which can distort the valving sites 230 are -thus avoided.
9 I A series of diaphragm retaining blocks 254 are bolted against the lO ¦ surfa oe 228 of the valve block 224 with diaphragms 256 sandwiched therebetween.
11 ¦ The upper end of each diaphragm retaining block is threaded to receive an air 12 ¦ connector 258 cc~nicating with a recess 260 on the underside -thereof and 13 ¦ extending generally over one of the valving sites 230. An O-ring 262 may be 14 ¦ received within an annular groove surrounding the recess 260 to provide an 15 I effective air seal.
16 ¦ The diaphragms 256 are constructed of a substantially chemi-17 ¦ cally inert air-tight material enabling them to be alternately drawn away 18 ¦from and pressed against the valve sites 230 by the application of vacuum and air 19 ¦pressure, respectively, through the fittings 258. The two alternate conditions of 20 ¦the diaphragm 256 are shown in Figs. 13A and 13B. In the condition of Fig. 13A
21 ¦air or gas pressure applied to the fitting 258 forces the diaphragm 256 against t~e 22 ¦opening 232 of the primary passage and the opening 240 of the secondary passage a~
23 ¦the particular valving site 230. The openings 232 and 240 are thus closed by the 2~ ¦diaphragm 256, permitting no communication therebetween. This corres-25 ~onds to the closed position of the valve located a-t the particular valving ¦
26 ite. In the cc)ndition of Fig. 13B, vacuum applied to the connector fitting 258 27 Iraws the diaphragm 256 away fram the openings 232 and 240, permittlng communicatic n 28 ~etween the primary and secondary passages over the surface of the valve block at 29 ¦ that point. This corresponds to the open condition of the particular valve, per-¦
30 ¦ mitting fluid frc~m one of the reservoirs or fram the inert g~s pressure source 31 ¦ 8 to flow through the primary passage 226 to the inle-t passage 36.
32 ¦ The novel construction of the valve assembly 222 described 331 above enables reagents and solvents to be delivered to the reaction -3~

~ 3 63~125 1 chamber in accurate amounts with virtually no contamination 2 between the various fluids. The continuity of the primary 3 passage 226 and the connection at one end thereof to the inlet passage o~ the chamber apparatus 12 are laryely responsible S or this advantageous operation. Delivery of any one of the 6 seven reagents and solvents can be accomplished by applying a 7 vacuum to one of the diaphragms 256 and positive gas pressure to 8 the others, enabling the desired reagent or solvent to pass into the primary passage 226 and -through the fluid conduit 40 to the reaction chamber. ~Ihen the desired amount of fluid has passed beneath the particular diaphragm, gas pressure is ayain applied thereto through the fitting 258 so that each of the seven reaqent and 13 solvent valves of the assembly 222 is closed. Delivery of the 14 fluid can then be completed by applying a vacuum to the diaphragm associated with the inert gas port at the remote end of the 16 valve block 224 to flush the entire primary passage 226 with inert 1 gas and force the reagent or solvent fluid re~aining in the lines into the 8 reaction chamber 34. Because there are no discontinuities or dead-end branches in the primary passage 226, there is no place 21 for any of the reagents or solvents to become trapped between 22 delivery procedures. The reagents and solvents delivered in succeeding sequencing steps are thus as pure as possible, 23 allowing the chemistry within the reaction cell to proceed as 24 intended and without any unnecessary loss of yield due to con-taminated reagents or solvents.
26 The valve assembly 222 is also illustrative of the valve 27 design incorporated in many other portions of the apparatus 28 to minimize contamination whenever a single port mus-t he selective-29 ly connected to a plurality of other ports. Thus, the valves 112 and 118 for delivering reagent and solvent to the conversion 31 flask 14 form a valve assembly in combination with the continuous ~2 manifold 114, and the valve 78, 80 and 82 for directing fluids ~ :~ 63~5 1 from the outlet passage 38 of the chamber apparatus 12 form a 2 similar valve apparatus in combination wi-th the continuous mani-3 fold 84. Likewise, the valves for the connection o~ waste and vacuum to the conversion flask and the fraction collector and the valves controlling flow from the conversion flask to the 6 fraction collector are constructed similarly to the valve 7 assembly 222. The principal difference in each of these valve 8 assemblies iS the number of valving sites associated 9 therewith.
It will be understood that, while the manifolds 68, 84 and 11 114 are illustrated schematically in Fig. 3 as having a series 12 of small discontinuities or branches adjacent each diaphragm 13 valve associated therewith, each of the manifolds is actually 14 a single continuous passage constructed in the manner of the primary passage 226 of Figs. 11 and 12.
16 The vacuum and gas pressure for actuating the diaphragm 17 valves between the open and closed conditions are omitted from the 18 schematic diagram of Fig. 3 for purposes of simplicity, and are 19 pre~erably separate from the source 28 and vacuum pump 90 described hereinO
21 The converslon flask 14 is shown in detail in Figs. 14 22 through 16. The flask 14 is of the double-walled glass type 23 having a space 264 between the walls for circulation of a heating 2~ fluid such as water. The heating fluid is passed to and from space 264 through a pair of nipples 266 adapted to receive 27 standard flexible tubing ends. A large bore tube 268~ connectible 28 to the vacuum trap ~8 and the vacuurn pump 90 through -the valve 2 96 and to the waste trap 60 through the valve 92, communicates 9 with the interior chamber 270 of the flask adjacent its upper end. Capillary tubes 272, 274 and 276 extend through the upper 32 end of the flask to points within the interior chamber 270. I'he bores of the tubes 272 and 276 are closed at the inner ends , -~0-1 1 63~S

~ thereof and each of the tubes is provided with a plurality of 2 relatively restricted radially spaced orifices 27g or 280 adjacent 3 its inner end.
4 Due to the relatively small amount of protein or peptide S sample for which the apparatus 10 is designed, and the relatively 6 low volumes of reagent and solvent used therein, the conversion 7 flask 14 has a much smaller interior volume than any of the prior 8 flasks known to applicants The volume of the flask 14 is slightly 9 over one (1) milliliter, while the prior automatic conversion 0 flasks known to applicants have all had volumes of one hundred 11 (100) milliliters.
12 The fractions cleaved from the sample in the reaction 13 chamber 34 are passed sequentially to the flask,14 through the 14 valve 78 and the capillary 274. Reagents and solvents enter the interior chamber of the flask through the capillary 276, from 16 which they are forced through the restricted orifices 280 as a 1~ spray impinging on the walls of the chamber 270 to wash any 18 residue thereon to the bottom of the flask. During the conversion 19 reaction, an inert gas may be introduced through the capillary tube 272 to agitate the liquid and aid in evaporating the solvent 21 theref~om. The gas exits the tube 272 through the restricted 22 orifices 278 as very small bubbles, providing optimal dispersion 23 of the gas through the liquid regardless of the size of the flask 24 and the amount of gas used. When the conversion reaction is complete, the fraction is forced upwardly by positive gas 26 pressure within the flask through the long capillary 272 to the 27 respective vial in the fraction collector 16. This can be 28 accomplished in two aliquots to effect a ~ore comp~ete trans-29 ferrence of the fraction to the fraction collector 16. The first aliquot, of approximately 200 microliters (~Q), is initially 31 expelled to the fraction collector. Then an additional 50 32 microliters (~Q) of solvent is introduced to dissolve any residue ~ 2 5 l on the lower walls or bottom of the flask. The second aliquot is 2 then expelled. In this way, a high yield of each fraction can be 3 achieved.
4 Afte~ a particular fraction has been transferred, an additional 750-900 microliters (~Q) of solvent may be introduced to dissolve whatever residue from the previous cycle remains on 7 the upper walls of the flask 14. This additional solvent is then 8 expelled to waste, leaving the flask walls clean.
The outer ends of the tubes 268, 272, 274 and 276 are sealed to respective flexible conduits 282 leading to the various ll other elements of the apparatus 10 by interfitting screw thread 12 connectors 284. The end of each conduit 282 is provided with a 13 radial flange portion 286 having a resilient O-ring 288 which 14 abuts and seals against the flat ground glass face of a radial flange 290 on one of the glass tubes. An internally threaded 16 collar 292 is slidably positioned over the conduit 282 to receive 17 the glass flange 290 and engage a two-piece external]y threaded 18 fitting 294 which is placed about the glass tube. Advancement 19 of the collar 292 over the fitting 294 forces the flange portion 286 against the qlass flange 290 to produce an extremely effec-21 tive seal. The various portions of the connectors 284 may be 22 made of substantially chemically inert materials, such as com 23 mercial fluorocarbon polymers, to virtually eliminate the pos-24 sibility of deterioration and subsequent leakage.
An alternative construction of the flask 14 would eliminate 26 the space 264 and the nipples 266, yielding a single-walled 27 vessel which could be maintained at an elevated temperature 28 by placement in the heated environment 32. This structure would 29 have the advantage of maintaining the entire length of the ~O glass tubes and the connectors 284 at the elevated temperature, 31 minimixing condensation of semi-volatile fluids therein, but 3 would not enable the flask and the chamber 12 to be maintained . ~ -4~2 _ ~ 1 ~3~5 l ll at differen emperatures.
2 The reagents and solvents used in the apparatus 10 for 3 the sequential degradation of protein or peptide chains are 4 preferably as follows:
Rl phenylisothiocyanate (PITC) (17% solution in heptane) 6 ¦ R2 trimethylamine or triethylamine (25% solution in water) 7 ¦ R3 trifluoroacetic or heptafluorobutyric acid 8 ¦ R3A water vapor g ¦ R4 trifluoroacetic acid (25% solution in water) 11 ¦ R4A hydrogen chloride (lN solution in methaIIol) l Sl benzene 12 ¦ S2 ethyl acetate with .1% acetic acid 13 ¦ S3 butyl chloride 14 ¦ S4 acetonitrile or methanol 15 l 16 ¦ In operation, the various valves and other mechanisms of 17 ¦ the apparatus 10 are preferably controlled by the automatic 18 ¦ control unit 18 to perform an indefinite number of degradation l9 ¦ cycles on a protein or peptide sample without human intervention.
~ 20 ¦ The control unit 18 may take the general form of the program~ing 21 ¦ unit disclosed in Penhasi United States Patent No. 3~725,010, 22 ¦ with alterations to provide for the particular secluence of 23 ¦ steps required by the apparatus 10, or may be a more sophisticated 24 ¦ electronic control in the nature of a special or general purpose 25 ¦ digital computer. ~.lternatively,the various steps in each degraflation . 26 ¦ cycle can be performed manually by an operator according to a : 27 ¦ predetermined schedule to achieve the same results.
28¦ Prior -to commencing the degradati.on process, a sample of 291 the protein or peptide being investigated must be mounted to 301 an appropriate sample support surEace. The matrix r~ateria~
311 first applied to the support surface, with the sample later 321 embedded therein, as described in Examples l and 2 bel.ow.

-43~

1 1 63~5 1 When the element 190 or 190' is used as a sample support surface, 2 it is placed between the chamber elemen~s 134 and 136 alon~ with 3 at least one of the sealing sheets 194 such that the element 4 190 is received within the reaction chamber 34 at the location of ~ the recess 192. In the preferred embodiment of the present inven-6 tion, a pair of sealing sheets 194 are used, sandwiching the 7 element 190 therebetween. The chamber elements 134 and 136 are 8 then inserted into the sleeve 132 and assembled with the various 9 other components to form the chamber apparatus 12.
0 When the sample-containing film is to be carried by the 11 interior surface of the chamber 34' or the chamber 324, it is 12 applied thereto as described in Example 2 below.
13 The chamber elements are initially assembled within the 14 sleeve 132 and held in place by the cap 160. In the case of the chamber 34', the annular sheet of yielding material 316 is 16 positioned between the chamber elements 134' and 136' upon 17 assembly. The solid matrix material and the sample are then 18 applied to the walls of the chamber 34' or 324, as described in 19 Example 2, and the sleeve 132 is assembled with the other components to form a complete apparatus.
21 The reaction chamber 34 and the associated fluid conduits 22 may be initially evacuated by opening the valve 82 to the vacuum 23 trap 88 and vacuum pump 90, in preparation for the sequential 24 introduction of reagents Rl through R3A, solvents Sl through S3 and inert gas from the source 28. Each time one of 26 the reagents or solvents is to be introduced, the corresponding 27 inert gas supply valve 58 and vent valve 62 are opened to 28 pressurize the particular reservoir and establish a dynamic 29 equilibrium condition therein. Inert gas is thus introduced and released from the reservoir simultaneously to maintain a 31 constant pressure within the reservoir. In the case of the 32 reservoirs 44, 46 and 48, the flow of saturated gas therefrom _~00 3~63~25 1 during delivery is controlled by one of the flow regulators 76.
2 Once the reservoir containing the particular reagent or 3 solvent to be delivered is pressurized and placed in equilibrium 4 as described above, a vacuum is applied by the auxiliary vacuum source (not shown) ~o the diaphragm o~ the corresponding ~low 6 valve 66 to open the valve and produce a flow of the reagent or 7 solvent through the continuous manifold 68 and the conduit 40 8 to the chamber inlet passage. The valve 66 is held open 9 a predetermined length of time to allow precisely the desired amount of reagent or solvent to pass, and is then closed by 11 the application of gas pressure to the diaphragm thereof.
12 Vacuum from the auxiliary vacuum source is then applied to the 13 diaphragm of the valve 72 at the remote end of the continuous 14 manifold 68 to flush the manifold of any remaining reagen-t or solvent and complete delivery thereof to the chamber. The 16 valve 72 is then closed by the application of positive pressure 17 to the diaphragm thereof, leaving the manifold 68 free to solvent 18 and reagent in preparation for the next delivery s-tep.
19 The diaphragm valve 80 is generally held open during pas-sage of the various reagents and solvents through the chamber 21 34 to conduct the effluent therefrom throuyh the chamber outlet 22 passage and the manifold 84 to the waste trap 60. After 23 delivery of a particular reagent or solvent, the chamber and 24 the associated conduits can be evacuated by opening the diaphragm valve 82 to the vacuum pump 90. Alternatively, the chamber and 26 the sample -therein can be dried at the appropriate times by pass-27 ing inert gas through the chamber by way o the valve 72. The gas~
28 exiting the chamber can -then be passed to -the waste trap 60, or 29 if desired, drawn out by the vacuum pump 90 to accelera-te the drying process.
31 After completion of the coupling and cleaving steps, the 32 extraction solvent S3 is delivered to -the reaction chamber 34 1 1 63~5 1 ¦ to dissolve the cleaved amino acid derivative produced during 2 ¦ the particular cycle of degradation and to deliver the solution 31 to the conversion flask 14 through the conduit 86. ~'or this 41 purpose, the valve 78 is open and the valves 80 and 82 remain 5 ¦ closed. The conve~sion reagents R4 and R~A (if used) and solvent 6 ¦ S4 may then be delivered to the conversion flask 14 at the approp-7¦ riate times by opening the corresponding valves 112 to pass the 81 liquids through the manifold 11~ and -the conduit 104 to the 9¦ conversion flask. Each de]ivery is preceded by the pressurization 10¦ and venting operations described above in relation to delivery of 11 ¦ the other reagents and solvents, and followed by opening the 12¦ valve 118 to purge the manifold 114 and the conduit 104.
13¦ The fraction transferred to the conversion flask 14 is 14¦ the anilinothiazolinone derivative of the N-terminal amino 15¦ acid of the protein or peptide sample and is converted auto-16 ¦ matically during the next coupling and cleavage cycles of the 17 ¦ reaction chamber 34 to the more stable phenylthiohydantoin amino 18 ¦ acid, partly according to the articles noted above by Wittmann-19 ¦ Liebold. Briefly, the amino acid fraction within the conversion 20 ¦ flask may first be evaporated by passage of inert gas over the 21 ¦ solution through the short capillary 276 and bubbling of inert gas¦
22 ¦ through the liquid by way of the capillary 272, followed by appli-l 23 ¦ cation of vacuum through the tube 268. The conversion reagent R4 ¦
24 ¦ may then be introduced through the capillary 276 by way of the conduit 104 and one of the valves 112 (see Fig. 3) in the desired 2G ¦ quantity. Rapid evaporation of the conversion flask after the 27 ¦ desired convexsion time may be accomplished by simul-taneously 2~1 applying vacuum to the conversion flask through the tube 268 291 and inert gas through capillaries 272 and 276, In order to 30¦ further stabilize the acidic side chaims of Pth-aspartic and 31 Pth-glutamic acids, one can further dissolve the residue in the 32 conversion flask by introduction of reagent R4A through the ~ J 63~2~

1 ¦ capillary 276 by way of the conduit 104 and one of the valves 112 2 ¦ in the desired quantity. Evaporation of the conversion flask i5 3 ¦ again accomplished by applying vacuum through the tube 268 and 4 ¦ inert gas through capillaries 272 and 276. The Pth-amino acid ~¦ remaini~g within the conversion ~lask is then redissolved in ~he 61 solvent S4 introduced through the conduit 104 for transfer of 7¦ the fraction to the appropriate vial in the fraction collector I 16. The transfer of the fraction is accomplished by opening the 9 valve 131 connected to the long central capillary 272 of the conversion flask and admitting pressurized inert gas through 11 the capillary 276 to force the fraction from the flask.
12 The vial carrousel of the fraction collector 16 is rotated 13 through a predetermined angle once during each degradation cycle, 14 such that each incoming fraction is collected in a separate vial.
At an appropriate point in the degradation cycle, the fractions 16 within the fraction collector may be further dried by opening 17 the valve 98 to vacuum or opening the valves 128 and 94 to 18 I pass inert gas from the source 28 over the fractions and finally 19 I to the waste trap 60.
20 ¦ The various components of the apparatus 10 are preferably 21 ¦ constructed of materials which are substantially inert and are 22 ¦ highly resistant to deterioration. Such materials include 231 borosilicate glass, certain fluorocarbon polymers, and, in 24 some cases, stainless steel and aluminum. The sealing 25 I structures and other elements of the apparatus 10 have 26 ¦ been designed such that they can be manufactured almost 27 I exclusively from these materials. It is felt that the 28 I resulting apparatus is the cleanest and most contamination-29 free systeJn obtainable and would function in that condition indefinitely.

32 / / / _ ~7 1~63~25 :
. ..
l The various steps performed by the apparatus 10 in a 2 typical degradation and conversion cycle are listed in Table 1.
3 The duration of each step and the functional state of the 4 apparatus during each step are also given. The functions show~
correspond to the conditions of the various valves of the 6 apparatus, and the marks in the columns signify when the 7 appropriate valves are open. For example, the columns "~1" ~'R2"' 8 "R3", "R3A", "Sl", "S2" and "5~" denote the conditions of the 9 various pairs of valves 58 and 62 for selectively pressurizing and establishing a dynamic equilibrium condition within the ll reservoirs 42 through 54. Wherever a mark appears in one of 12 these columns, the valves 58 and 62 associated with the 13 particular reservoir are open, either in preparation for or 14 during delivery of the particular reagent or solvent to the reaction chamber. The valves remain closed at all other times.
16 Likewise, the "argon" column shows the condition of tne valve 17 72 for delivery of an inert gas such as argon to the reaction 18 ¦ chamber through the manifold 68, the "deliver" column shows the 19 ~ condition of the valve 66 corresponding to any reservoir which is 20 I pressurized at the time, and the columns labeled "waste", "collect' 21 ¦ and "vacuum", show the conditions of the valves 82, 78 and 80, 22 I respectively. As described above, each step of solvent or reagent 231 delivery is preceded by pressurization of the appropriate solvent 24 ¦ or reagent reservoir and followed by the introduction of inert gas¦
through the valve 72 to complete delivery of the solvent or 26 reagent and flush the delivery lines.
2~ The c~lumns "R4", "R4A" and "S4" denote the conditions of 28 ¦ the various pairs of valves 108 and llO for selectively pressur-29 ¦ izing and establishing a dynamic equilibrium within the reservoirs 30 ¦ 100 through 102, and the "deliver/argon" column signifies the 31 condition of both the v~lve 122 which admits inert gas into the 32 conversion flask through the line 124 and the valve 112 j .

i 1 fi3~ZS

corresponding to the reservoir which is pressurized at the tirne.
The columns "argon", "waste 1", "vacuum", "collect" and 3 "waste 2" show the conditions of the valves 118, 92, 96, 131 and 125, respectively.
Of the fraction collector functions listed, the columns 6 "argon", "waste" and "vacuum" SilOW the conditions of the valves - 7 128, 94 and 98, respectively.
8 With the exceptions noted hereinabove, the sequence of steps 9 listed in Table 1 essentially conforms to the Edman degradation processes described in the cited publications and will not be 11 discussed in detail. Any deviations from general practice will be 12 clear from the names of the steps and the corresponding 13 functional states tabulated in Table 1.
14 In practice, the following variations of the sequencing program of Table 1 may be implemented to adap-t the program to 16 the needs of a particular user:
17 1) Steps 18 through 5~ can be looped one or two -times on 18 the first sequencer cycle to insure complete coupling of all 19 amino groups on the protein.
2) Step 65 can be followed by a 20 to 60 second delivery of 21 wa-ter vapor (R3A) through the reaction chamber to reduce dehydra-22 tion of side chains of serine and -threonine.
23 3) Step 65 can be followed by delivery of .05 ml of lN
24 hydrochloric acid in methanol (r~4A) to the conversion flask to methylate the side chains of aspartic acid and glutamic acid. If ¦
26 this is done, S4 if preferably methanol.
27 4) Step 76 can be followed by dellvery of .7 to 1 rnl of S
28 (acetonitrile or me-thanol) -that is subsequen-tly delivered to waste 29 to thoroughly clean the conversion flask.
5) Step 8 can be increased in duration (500 to 1000 seconds) 31 to promote cleavage of amino terminal proline residues.

32 The nature of the present invention wi]l be further _,~9_ 1:1634~5 1 ¦ clarified by the following specific examples of the practice of 21 the invention. It should be understood that the data ~isclosed 3¦ serve only as examples of processes which have been performed wi-th¦
41 the apparatus and method of the present inven-tion and are no-t ~¦ intended to limit the scope of the invention.
61 The amino acid sequences se-t for-th in the following examples 71 are tabulated in the one-letter amino acid code, which is defined 81 as follows:
9¦ A - alanine I, - leucine 0¦ R - arginine K - lysine N - asparagine M - methionine 12 D - aspartic acid F - phenylalanine 13 C - cysteine P - proline 14 E - glutamic acid S - serine Q - glutamine T - threonine 16 G - glycine W - tryptophan 17 H - histidine Y - tyrosine 18 I - isoleucine V - valine 19 Example 1 A solid matrix of polymeric quaternary ammonium salt, suit-21 able for embedding a protein sample for sequencing, can be preparec 22 on the fibrous sheet element 190 and 190' described above, and a 23 protein sample can be embedded in the matrix as follows:
2~ A glass fiber disc, 12 mm in diame-ter and .25 to .5 mm thick, ~5 is cut from a sheet of glass microfiber filter which is available I
26 commercially from Whatman, Inc., Cliftorl, New Jersey. The disc is¦
27 placed in the depression 192 of the chamber elemen-t 13~. 25 micro~
28 liters of an aqueous solution containing 1.5 mq of :L,5-dimethyl-29 1,5-diazaundecamethylene polymethobromide and .0033 mg of glycylglycine is dropped onto the glass fiber disc froln a syringe 31 or pipette. The water is evaporated under vacuum or by hea-ting in 32 a stream of warm nitroqen. The remainder of the chamber apparatus ~ _50_ 63~2S

. 12 is assembled and installecl in the apparatus 10. The protein 2 sequencing program is initiated at step 18, and 4 to 6 complete 3 dec~radation cycles are accomplished in order to remove impurities from the 1,5-dimethyl-1,5-diaz~undecamethylene polymethobromide that might react chemically with the protein sample or otherwise 6 intèrfere with the Edman process. The chamber apparatus 12 is 7 partially dissassembled and 25 microliters of a solution of the 8 protein is dropped onto the glass fiber disc. The protein solution dissolves the 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide and the liquid is removed by evaporation to 11 leave behind a thin film of 1,5-dimethyl-1,5-diazaundecamethylene 12 polymethobromide with the protein sample embedded therein. If the 13 intial protein sample volume is greater than 25 microliters, the 1~ sample can be applied in 25 microliter aliquots with the liquid being removed by evaporation between aliquot applications. The 16 chamber apparatus 12 is reassembled and reinstalled in the 17 apparatus 10. The protein sequencing program is then initiated 18 at step 18 and carried through as many degradation cycles as 19 desired.
Example 2 21 A solid matrix of polymeric quaternary ammonium salt, suit-22 able for embedding a protein sample for sequencing, can be 23 prepared on the interior walls of a glass capillary vessel, and a ¦
2~ protein sample can be embedded in the matrix as ~ollows: !
The chamber element 324 or the chamber elements 134' and 136'~
26 are initially assembled with the sleeve 13~ and the cap 160, as 27 illustrated in the figures, to form a compact cartridge sub- ¦
28 assembly which is easily manipulatecl for introduction of the ` 29 matrix and the sample. The sub-assembly is held horizontally and 10 microliters of an aqueous solution containing .6 mg of 1,5-31 dimethyl-1,5-diazaundecamethylene polymethobromide and .0013 mg o~

32 glycylgycine is injected into the reac-tion chamber from a syringe.

~ ~ 63/~5 ; l The chamber 320 is preferably constructed to have a diameter 2 of l/16" at its two ends and a diameter of 1/8" a-t its middle, 3 producing a chamber which will accommodate up to 25 microliters (ul) of solution in the horizontal condition without spilling out ~ at the ends. Tllis configuration of -the~ chamber 32~ holdiny a 6 solution in the horizontal condition is illustrated in Fig. 18B, with the sleeve 132 and related s-tructure omitted for simplicity.
8 The sub-assembly is then rotated about its axis while the liquid 9 in the reaction chamber is evaporated by directing a stream of air or nitrogen through the chamber, leaving a thin film of 1,5-1l dimethyl-1,5- diazaundecamethylene polymethobromide on the chamber 12 walls. The sub-assembly is then installed in ~e apparatus lO, 13 the protein sequencing program is initiated at step 18, and 4 -to 14 6 complete degradation cycles are accomplished. The sub-assembly lS is next removed from the apparatus lO and held horizontally while 16 lO microliters of the protein solution is dropped into the 17 reaction chamber from a syringe. The sub-assembly is again rotate~
18 about its axis while the liquid in the reaction chamber is l9 evaporated by a stream of nitrogen directed through the chamber, leaving a thin film of 1,5-dimethyl-1,5 diazaundecamethylene 21 polymethobromide with the protein sample embedded therein as 22 illustrated in Fig. 18C. The sub-assembly is then reinstalled in 23 the apparatus 10 and the protein sequencing program is initiated 2~ at step 18 and carried through as many degradation cycles as desired.
26 Example 3 27 Angiotensin, .0005 mg, contained in 25 microliters of aqueous 28 20~ formic acid was embedded in a solid matrix of precycled 1,5-29 dimethyl-1,5-diazaundecamethylene polymethobromide according to th~
method of Example l. The angiotensin was subjected to eight cycles 31 of Edman degradation, and the phenythiolhydan-toin amino acids 32 produced at each cycle were analyzed by high pressure liquid 63~2!j ~

1 chromatography according to the method descri~ed by Johnson et al, 2 "Analysis of Phenylthiohydantoin Amino Acids by High Pressure 3 Liquid Chromatography on Du Pont Zorbax CN Columns", Anal.
4 Biochem. 100, 335 (1979). The following sequence, listed belo~
as "Experimental," was obtained. The known sequence is also 61 listed below for comparison.

l 1 3 5 7 8¦ Experimental: D - R - V - Y - I - H - P - F
9 Known: D - R - V - Y - I - H - P - F
0 This result is significant because it demonstrates that even 11 small peptides can be sequenced in the manner oE the present 12 invention. The ability to sequence small peptides stems from the 13 fact that the sample is embedded in a film for retention, rather 14 than being adsorbed directly onto a support surface, and there-fore can be held securely regardless of size. Whereas sorptive 16 bonds comprise a large number of non-covalent interactions be-17 ¦ tween the sample and the surface and are heavily dependent on the !
18 ¦ size of the molecules, the holding power of the film disclosed 19 ~ herein is relatively unaffected by sample siz~.
Example 4 21 ~ Sperm whale apomyoglobin, .01 mg, contained in 25 microliters !
22 ¦ of aqueous 20% acetic acid was embedded in a solid matrix of 231 precycled 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide ~4 ¦ according to the method of Example 1. The apomyoglobin was 251 subjected to 40 cycles of Edman degradation, and the phenylthio-26¦ hydantoln amino acids were analyzed according to the method of 27 ¦ Example 3 to give the sequence listed below as "Experimental."
28¦ The known sequence of sperm wha]e apomyoglobin is also listed 291 below for comparison.

32 1 ~ 53 ~ :~ B3~25 1 Experimental: V-L-S-E-G-E-W-Q-L-V-L-H-V-W~A-2 Known: V-L~S-E-G-E-W-Q-L-V-L-H-V-W-A-K-V-E-A-D-V-A-G-H-G-Q-D-I-L-I-K-V-E-A-D-V-A-G-H-G-Q-D-I-L-I-. R-L-F-K-S-H-P-E-T-L-8 Example 5 9 A Drosophila melanoyaster larval cuticle protein of previously unknown structure,.01 mg, contained in 25 microliters 11 of an aqueous solution of .1% sodium dodecyl sulfate and .05M
12 ammonium bicarbonate, was embedded in a solid matrix of precycled 13 1,5-dimethyl-1,5- diazaundecamethylene polymethobromide according 14 to the method of Example 1. The cuticle protein was subjected to 36 cycles of Edman degradation, and the phenylthiohydantoin amino 16 acids were analyzed according to the method of Example 3 to give 17 the sequence listed below as "Experimental."

9 Experimental: N - A - N - V - E - V - K - E - L - V -Example 6 26 A Drosophila melanogaster larval cuticle protein of pre-27 viously unknown structure, .005 mg, contained in 10 microliters 28 of an aqueous solution o~ .1% sodium dodecyl sulfate and .05M
29 ammonium bicarbonate, was embedded in a solid matrix of precycled .
1,5-dimethyl-1,5-diazaundecamethylene polymethobromide according 31 to the method of Example 2. The cuticle prot~in was subjected to 32 24 cycles of Edman degradation, and the phenylthiohydantoin amino 3163~5 l ¦ acids were analyzed according to the method of Example 3 to give 2 ¦ the sequence listed below as "Experimental."
3 I ~ 5 41 Experimental: N - A - N - V - E - V - K - E - L - v -5¦ N - D - V - Q - P - D - G - F - V - S -81 The results achieved in Examples 5 and 6 demonstrate that the 9¦ apparatus and method of the present invention is suitable for lO¦ sequencing proteins and peptides dissolved in a solution of ll¦ sodium dodecyl sulfate (hereinafter "SDS"), a potent anionic 12 detergent. This is important because the most general method of 13 isolating small quantities of medium to large proteins or peptides 14 for analysis, known as polyacrylimide gel electrophoresis, pro-15 ¦ duces samples in a solution of SDS. This common detergent causes 16 samples to wash out of devices which rely upon adsorptive bonding 17 of the sa~ple to a support surface, however, the solid matrix 18 of the present invention is unaffected by the presence of SDS.
l9 From the above, it can be seen that there has been provided an improved apparatus and method for the sequential performance 21 ~ of chemical processes on a sample of very small size through the 22 use of minimum amounts of reagents and solvents and relatively 23 I short cycle times.
24 The appended claims are intended to cover all varlations and adaptations falling within the true scope and spirit of the 26 present invention.

32 _55-

Claims (70)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Apparatus comprising:
chamber means having an interior surface defining a reaction chamber, said chamber having inlet means and outlet means;
means for sequentially passing a plurality of fluids through said chamber from said inlet means to said outlet means in a pressurized stream;
and solid matrix means permeable to said plurality of fluids and located within said chamber;
such that a sample embedded in said matrix means is immobilized and exposed to each of said fluids passing through the chamber for chemical interaction therewith.

2. The processing apparatus recited in claim 1 wherein said solid matrix means is permeable to said plurality of fluids by absorption of said fluids into said matrix means for diffusion of said fluids therein.

3. The processing apparatus recited in claim 2 wherein said chamber means includes surface means supporting said solid matrix means as a thin film thereon.

4. The processing apparatus recited in claim 1 wherein said solid matrix means comprises a polymeric quaternary ammonium salt.

5. The processing apparatus recited in claim 4 wherein said polymeric quaternary ammonium salt comprises 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide.

6. The processing apparatus recited in claim 4 wherein said polymeric quaternary ammonium salt comprises poly(N,N-dimethyl-3,5-dimethylene piperid-inium chloride).

7. The processing apparatus recited in claim 3 wherein said surface means supporting said matrix means comprises at least a portion of said interior surface.

8. The processing apparatus recited in claim 7 wherein said reaction chamber defined by said interior surface comprises a single capillary-type passage extending between two ends thereof.

9. The processing apparatus recited in claim 8 wherein said capillary-type passage increases uniformly in diameter in an inward direction from said two ends.

10. The processing apparatus recited in claim 7 wherein said chamber means comprises a pair of abutting chamber elements having first and second cavities, respectively, on opposed mating surfaces thereof, said first and second cavities being aligned with each other to form said reaction chamber.

11. The processing apparatus recited in claim 3 wherein said surface means supporting said matrix means comprises porous sheet means permitting passage of fluids therethrough.

12. The processing apparatus recited in claim 11 wherein said porous sheet means extends substantially transversely across said chamber.

13. The processing apparatus recited in claim 12 wherein said porous sheet means comprises a sheet made of a plurality of fibers.

14. The processing apparatus recited in claim 13 wherein said fibers are glass.

15. The processing apparatus recited in claim 12 wherein said chamber means comprises a pair of abutting chamber elements having first and second cavities, respectively, on opposed mating surfaces thereof, said first and second cavities being aligned with each other to form said reaction chamber.

16. The processing apparatus recited in claim 15 wherein said solid matrix means comprises a polymeric quaternary ammonium salt.

17. The processing apparatus recited in claim 15 wherein said first and second cavities are tapered in directions away from said mating surfaces to locations at which they communicate with said inlet and outlet means, respectively.

18. The processing apparatus recited in claim 15 wherein said porous sheet means is received within a recess in at least one of said mating surfaces for retention within said chamber in an orientation substantially separating said first and second cavities.

19. The processing apparatus recited in claim 18 wherein said chamber means includes at least one sheet of yielding material sandwiched between said mating surfaces in a sealing relationship, said yielding material being permeable to said plurality of fluids.

20. The processing apparatus recited in claim 19 wherein said chamber means includes one of said sheets of yielding material located on each side of said porous sheet means.

21. The processing apparatus recited in claim 20 wherein least one of said chamber elements includes a raised portion on the mating surface thereof which extends about the cavity herein to compress said at least one sheet of yielding material against the mating surface of the other chamber element and thus.
enchance said sealing relationship.

22. The processing apparatus recited in claim 18 wherein said inlet and outlet means comprise a pair of capillary passages extending through said chamber elements, respectively, and com-municating at inner ends thereof with said reaction chamber on opposite sides of said porous sheet means.

23. The processing apparatus recited in claim 22 wherein said capillaries are coaxial with said reaction chamber and ex-tend therefrom to outer capillary openings in said chamber elements

24. The processing apparatus recited in claim 23 wherein said chamber elements are glass and have substantially flat sur-faces adjacent said capillary openings.

25. The processing apparatus recited in claim 24, wherein said means for sequentially passing fluids through the chamber comprises at least one slug of resilient material having a sub-stantially flat sealing end and an axial capillary passage which terminates in an opening at said end, and screw thread means for urging said sealing end axially against one of said capillary openings, such that the axial passage in the slug communicates with said capillary opening and the sealing end of the slug seals against the substantially flat surface of the chamber element adjacent thereto.

26. The processing apparatus recited in claim 25 wherein said at least one slug of resilient material comprises a mass of a fluorocarbon polymer encased in a metallic sleeve.

27. The processing apparatus recited in claim 26 wherein said screw thread means includes a bore closely receiving said metallic sleeve in an axially sliding relationship to insure accurate alignment of the passage in the slug with said one capillary opening.

28. The processing apparatus recited in claim 3 which further comprises means for automatically controlling the function-ing of the apparatus through a plurality of cycles.

29. The processing apparatus recited in claim 1 wherein said means for sequentially passing a plurality of fluids through said chamber comprises at least one valve means for controlling the flow of said fluids between the chamber and a plurality of fluid containing means, said at least one valve means comprising:
valve block means having a plurality of substantially flat valving sites on a surface thereof, said valve block means defining a primary passage continuous between two ends thereof and communicating through primary openings with each of said valving sites, and a plurality of secondary passages each communicating through a secondary opening with one of said valving sites;
a plurality of resilient, substantially impermeable dia-phragms covering said respective valving sites, each of said diaphragms being actuable between a first sealing condition in which it is forced against one of said valving sites to close the primary and secondary openings communicating with said site and a second condition in which it is drawn away from said site to provide a fluid flow path between said primary and secondary openings over the exterior of said valve block means; and means for operatively connecting said primary and secondary passages to said fluid containing means and said chamber;
whereby fluid flow between said fluid containing means and said chamber can be selectively controlled.

30. The processing apparatus recited in claim 29 wherein said connecting means comprises at least one tapered ferrule closely received in sliding engagement over a tubing member and urged against a differently tapered recess in said valve block means communicating with one of said passages, such that an inward force applied to said ferrule is focused on a relatively small area of contact between the ferrule and the recess to produce a fluid seal therebetween.

31. The processing apparatus recited in claim 30 wherein said recess is tapered at a greater angle than said ferrule.

32. The processing apparatus recited in claim 30 wherein said ferrule is tapered on opposite sides, one of said sides being urged against said recess by screw thread means having a separate tapered recess for reception of the other side of said ferrule, said recesses being tapered at angles greater than the angles of taper of the corresponding sides of said ferrule.

33. The processing apparatus recited in claim 29 wherein said connecting means includes a connector fitting at one of said ends of the primary passage such that the primary passage serves as a manifold which can be flushed by a flow of fluid between the connector fitting and the secondary passage furthest away from the connector fitting.

34. The processing apparatus recited in claim 33 wherein said primary passage comprises a plurality of straight passages connected end to end to form a conduit having a sawtooth configura-tion and communicating at alternating intersections thereof with said respective valving sites.

35. The processing apparatus recited in claim 1 which includes:
a conversion flask having a plurality of capillary tubes extending into the interior thereof; and means for introducing and withdrawing various fluids relative to said flask through said capillary tubes, including at least one of said plurality of fluids passed to said outlet means;
at least one of said capillary tubes having an inner end at which the bore is closed and which is provided with a plurality of restricted radially spaced orifices;
such that passage of fluids inwardly through said at least one capillary tube produces a spray onto the interior walls of the flask to wash down said interior walls.

36. The processing apparatus recited in claim 35 wherein said capillary tubes are glass.

37. The processing apparatus recited in claim 1 which includes:
a conversion flask having a plurality of capillary tubes extending into the interior thereof; and means for introducing and withdrawing various fluids relative to said flask through said capillary tubes, including at least one of said plurality of fluids passed to said outlet means;

one of said capillary tubes terminating at a point adjacent the bottom of the flask in an inner end at which the bore is closed, said inner end being provided with a plurality of restricted radially spaced orifices;
such that passage of a gas inwardly through said one capillary tube introduces a plurality of small bubbles into any liquid within the flask, said bubbles serving to uniformly agitate said liquid or accelerate drying thereof.

38. Reaction chamber apparatus, comprising:
chamber means having an interior surface defining a reaction chamber, said chamber having inlet means and outlet means for conduction of fluids therethrough in a pressurized stream; and solid matrix means permeable to a plurality of fluids and located within said chamber on a porous sheet made of a plurality of fibers, such that a sample embedded in said matrix means is immobilized and exposed to any of said plurality of fluids passed through the chamber for chemical interaction therewith.

39. The chamber apparatus recited in claim 38 wherein said solid matrix means is permeable to said plurality of fluids by absorption of said fluids into said matrix means for diffusion of said fluids therein.

40. The chamber apparatus recited in claim 38 wherein said chamber means includes surface means supporting said solid matrix means as a thin film thereon.

41. The chamber apparatus recited in claim 40 wherein said solid matrix means comprises a polymeric quaternary ammonium salt.

42. The chamber apparatus recited in claim 41 wherein said polymeric quaternary ammonium salt comprises 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide.

43. The chamber apparatus recited in claim 41 wherein said polymeric quaternary ammonium salt comprises poly(N,N-dimethyl-3,5-dimethylene piperidinium chloride).

44. The chamber apparatus recited in claim 40 wherein said surface means supporting said matrix means comprises at least a portion of said interior surface.

45. The chamber apparatus recited in claim 44 wherein said reaction chamber defined by said interior surface comprises a single capillary-type passage extending between two ends thereof.

46. The chamber apparatus recited in claim 45 wherein said capillary-type passage increases uniformly in diameter in an inward direction from said two ends.

47. The chamber apparatus recited in claim 44 wherein said chamber means comprises a pair of abutting chamber elements having first and second cavities, respectively, on opposed mating surfaces thereof, said first and second cavities being aligned with each other to form said reaction chamber.

48. The chamber apparatus recited in claim 40 wherein said surface means comprises porous sheet means permitting pas-sage of said fluids therethrough.

49. The chamber apparatus recited in claim 48 wherein said porous sheet means extends substantially transversely across said chamber.

50. The chamber apparatus recited in claim 49 wherein said porous sheet means comprises a sheet made of a plurality of fibers to which said sample is adhered.

51. The chamber apparatus recited in claim 38 wherein said fibers are glass.

52. The chamber apparatus recited in claim 49 wherein said chamber means comprises a pair of abutting chamber elements having first and second cavities, respectively, on opposed mating surfaces thereof, said first and second cavities aligned with each other to form said reaction chamber.

53. The chamber apparatus recited in claim 52 wherein said solid matrix means comprises a polymeric quaternary ammonium salt.

54. The chamber apparatus recited in claim 52 wherein said first and second cavities are tapered in directions away from said mating surfaces to locations at which they communicate with said inlet and outlet means, respectively.

55. The chamber apparatus recited in claim 52 wherein said porous sheet means is received within a recess in at least one of said mating surfaces for retention within said chamber in an orientation substantially separating said first and second cavities.

56. The chamber apparatus recited in claim 55 wherein said chamber means includes at least one sheet of yielding material sandwiched between said mating surfaces in a sealing relationship, said yielding material being permeable to said plurality of fluids.

57. The chamber apparatus recited in claim 56 wherein said chamber means includes one of said sheets of yielding material located on each side of said porous sheet means.

58. The chamber apparatus recited in claim 56 wherein at least one of said chamber elements includes a raised portion on the mating surface thereof which extends about the cavity therein to compress said at least one sheet of yielding material against the mating surface of the other chamber element and thus enhance said sealing relationship.

59. The chamber apparatus recited in claim 55 wherein said inlet and outlet means comprise a pair of capillary passages ex-tending through said chamber elements, respectively, and communi-cating at inner ends thereof with said reaction chamber on opposite sides of said porous sheet means.

60. The chamber apparatus recited in claim 59 wherein said capillaries are coaxial with said reaction chamber and extend therefrom to outer capillary openings in said chamber elements.

61. The chamber apparatus recited in claim 60 wherein said chamber elements are glass and have substantially flat sur-faces adjacent said capillary openings.

62. The chamber apparatus recited in claim 61 wherein said inlet and outlet means further comprise at least one slug of resilient material having a substantially flat sealing end and an axial capillary passage which terminates in an opening at said end, and screw thread means for urging said sealing end axially against one of said capillary openings, such that the axial passage in the slug communicates with said capillary opening and the sealing end of the slug seals against the substantially flat surface of the chamber element adjacent thereto.

63. The chamber apparatus recited in claim 62 wherein said at least one slug of resilient material comprises a mass of a fluorocarbon polymer encased in a metallic sleeve.

64. The chamber apparatus recited in claim 63 wherein said screw thread means includes a bore closely receiving said metallic sleeve in an axially sliding relationship to insure accurate alignment of the passage in the slug with said one capillary opening.

65. A method comprising:
enclosing a solid matrix of fluid permeable material within a closed chamber on a porous sheet made of a plurality of fibers, said chamber having an inlet and an outlet;
embedding a sample of chemical material in the solid matrix; and sequentially passing a plurality of fluids through the chamber in a pressurized stream from the inlet to the outlet thereof such that the sample is exposed to each of the fluids;
whereby the sample is immobilized and chemical interaction between the sample and the fluids is obtained.

66. The method recited in claim 65 wherein the step of enclosing the solid matrix within the chamber comprises introducing a solution containing the solid matrix material into the chamber and then rotating the chamber while passing a gas through it to evaporate the liquid, leaving the solid matrix material on the interior walls of the chamber as a thin film.

67. The method recited in claim 65 wherein the step of embedding the sample of chemical material in the solid matrix comprises introducing a solution containing the sample into the chamber to dissolve the matrix material therein and then rotating the chamber while passing a gas through it to evaporate the liquid, leaving the solid matrix material on the interior walls of the chamber as a thin film with the sample embedded therein.

68. The method recited in claim 65 wherein the solid matrix is supported on a porous sheet and the step of enclosing the matrix within the chamber comprises applying the matrix material to the porous sheet as a thin film thereon and extending the porous sheet substantially transversely across the chamber at a location between the inlet and outlet such that the fluids passed from the inlet to the outlet must pass through the sheet.

69. The method recited in claim 65 wherein the step of embedding the sample in a solid matrix comprises the steps of applying a solution containing the sample to the porous sheet to dissolve the matrix material thereon, and evaporating the liquid from the sheet to leave the matrix material as a thin film with the sample embedded therein.

70. The method recited in claim 65 wherein the step of sequentially passing fluids through the chamber includes the step of flowing at least one reagent through the chamber in gas or vapor form for diffusion into the solid matrix and reaction with the sample.