US3725010A - Apparatus for automatically performing chemical processes - Google Patents

Abstract

AN AUTOMATIC PROTEIN-PEPTIDE SEQUENATOR FOR DETERMINING THE AMINO ACID SEQUENCE IN PROTEINS AND/OR PEPTIDES CONTAINING N NUMBER OF AMINO ACID UNITS INCLUDING: AN EASILY ACCESSIBLE REACTION CELL DISPOSED WITHIN A REACTION CHAMBER HAVING A MINIMUM VOLUME AND ROTATIONALLY DRIVEN BY A VARIABLE OR MULTIPLE SPEED MOTOR THROUGH A MAGNETIC COUPLING DRIVE UNIT, AIR-CIRCULATING HEATING MEANS, A CONSTANT PRESSURE AND UNIFORM DELIVERY SYSTEM INCLUDING VACUUM AND SOLENOID ACTUATED VALVE ASSEMBLIES, A REGULATED INERT GAS SUPPLY SYSTEM, AN EVACUATED FRACTION COLLECTOR, AND A PROGRAMMING CONTROL UNIT WHICH MAY BE AUTOMATICALLY OR MANUALLY MANIPULATED.

Description

Hv A. PENHAST A ril 3, 1973 APPARATUS FOR AUTOMATICALLY PERFORMING CHEMICAL PROCESSES 8 Sheets-Sheet 1 Original Filed April 8, 1969 INVENTOR. HARRY A. PENHASI BY M% 77/ April 3,1973 H. A. PENHAST 3,725,010

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United States Patent Oflice 3,725,010 APPARATUS FOR AUTOMATICALLY PERFORM- ING CHEMICAL PROCESSES Harry A. Penhasi, Cupertino, Calif., assignor to Beckman Instruments, Inc.

Continuation of abandoned application Ser. No. 814,425, Apr. 8, 1969. This application Aug. 23, 1971, Ser. No. 174,211

Int. Cl. G01n 1/18, 9/30 U.S. Cl. 23-253 R 16 Claims ABSTRACT OF THE DISCLOSURE This application is a continuation of Ser. No. 814,425 filed Apr. 8, 1969, now abandoned.

BACKGROUND OF THE INVENTION Field of the invention The present invention relates in general to an apparatus for automatically performing chemical processes and more particularly to an apparatus for automatically determining the amino acid sequence in proteins and/or peptides containing an N number of amino acid units regardless of the amino acid chain length.

Description of the prior art The functional characteristics of various proteins and peptides are due not only to the particular types of amino acids making up the protein or peptide but, also, in a large part to the exact sequence of these amino acid units. By and large present techniques of protein-peptide degradation are manual in nature and as such take anywhere from several weeks to several years, depending on the length of the protein or peptide being degraded, to successfully determine the amino acid sequence. To say the least, such manual processes are time and labor consuming. Moreover, such degradation techniques are subject to inevitable human errors which in this instance is further compounded by the fact that typically used processes involve upwards of thirty different steps per cycle with many cycles required to successfully ascertain the entire amino acid sequence of a protein or peptide sample.

A recent attempt of automating a process for determining the amino acid sequence in proteins is disclosed in an article entitled A Protein sequenator, by P. Edman and G. Begg published in European J. Biochem, vol. 1, 1967, pages 80-91, which approach for purposes of simplification will hereinafter be referred to as the Edman protein sequenator. Generally speaking, the Edman protein sequenator comprises an open ended cylindrical glass cup, in which the protein sample to be degraded is placed, driven at a constant rotational speed by a suitable motor. The glass cup is enclosed within an evacuated bell jar assembly heated by means of an electrical conductive coating on the outside surface energized through a pair of band shaped silver electrodes disposed around the bell jar in contact with the coating. The various reagents and solvents are stored in a plurality of reservoirs and are in- 3,725,010 Patented Apr. 3, 1973 troduced into the glass cup through a single feed line by way of a solenoid actuated valve assembly. The reaction products along with unreacted reagents and solvents are withdrawn from the glass cup by an eflluent line located along the top edge of the spinning cup. The solvent and reagent reservoirs and the bell jar are maintained at pre determined pressures by a suitable nitrogen supply. A programming unit is operatively connected to the various valve structures used throughout the system to control both the amounts of reagents and solvents fed to the glass cup during each step, the timing of the steps, and the particular sequence of steps.

As disclosed, the Edman protein sequenator suffers from numerous disadvantages all of which drastically limit the environment in which the instrument may be operated, the nature of reagents and solvents which may be used in carrying out the process, and the length of the amino acid chain which may be successfully degraded.

To elaborate the Edman protein sequenator is highly sensitive and subject to random variations in temperature, pressure and fluid viscosity resulting in inconsistent quantities of reagents and solvents being injected into the reaction vessel from step to step. This is turn causes a ring of unreacted or partially reacted protein to remain and accumulate above the intersection of the reagent and reagent-free surface about the inside surface of the spinning glass cup. Obviously, this leads to incomplete reactions resulting in lower yields and overlap from step to step thereby limiting the number of amino acid units or chain lengths of a protein which may be successfully degraded.

Still another limitation lies in the, relatively speaking, large volume chamber in which the reaction cup spins. Such a large volume reaction chamber requires the use of nonvolatile reagents, such as Quadrol and heptafluorobutyric acids, with the attending problems accompanying the use of nonvolatile reagents. That is to say, nonvolatile reagents cannot be easily evaporated and tend to accumulate within the cold sink areas, including the feed lines and valves, as well as within the reaction cup due to inefficient solvent removal, eventually causing erratic valve behavior and system performance. Moreover, reagents, such as Quadrol, are extremely diflicult to free of aldehyde impurities which reduce over-all yields by blocking the N-terminal amino acid of the protein being degraded. Also, to remove the Quadrol after the coupling step it is necessary to extract very thoroughly, first with benzene and then with ethyl acetate. This results in heavy extractive losses of the sample, specifically when dealing with peptides, reducing the yield and virtually limiting the use of the sequenator to protein of amino acid chain lengths greater than fifty amino acid units. In short, due to the characteristics of nonvolatile reagents, their required use often prematurely terminates the degradation process thereby limiting the number of amino acid units whose sequence may be successfully determined by the Edman protein sequenator.

As previously noted the Edman protein sequenator employs an open glass cup disposed in a bell jar chamber through which nitrogen gas is circulated to keep thev chamber free of oxygen and at a predetermined pressure. Rotation of the reaction cup induces circulation of the nitrogen within the glass cup causing newly injected reagent to be evaporated. Such evaporation cools the glass cup and leads to condensation of the evaporated reagent on the outside surface of the cup. A portion of the condensed reagent is thrown by centrifugal force back out into the chamber. All of this and especially condensation of the reagent on the outside of the rotor blocks visual observation of the reaction taking place inside the glass cup which is a desirable feature in any automatic protein sequenator. Compounding the problem is the above-noted required use of nonvolatile reagents which tend to adhere to the outside surface of the cup. To remove these reagents by evaporation it is necessary to subject the reaction chamber to a rather lengthy vacuum operating cycle that, as a result of the evaporation of the undesirable condensates, lowers the temperature of the over-all reaction cell assembly which temperature should be maintained at a relative high and constant one of around 50 centigrade. This causes incomplete reactions and/or much longer than desirable reaction time periods.

Still another undesirable feature of the Edman protein sequenator is that the drive-shaft and oil-seal housing is in communication with the evacuated reaction cell chamber, but, is located external to the heated chamber walls which supply heat to and control the temperature of the glass cup in which the various reactions are taking place. It follows that the drive-shaft and oil-seal housing is always at a lower temperature than the reaction chamber walls and the surfaces of the spinning glass cup. As a result there is a tendency, accentuated when operating at elevated temperatures, for the reagents to distill from the glass cup and condense in this housing or other surfaces cooler than the inside surface of the spinning glass cup. This again limits the nature of reagents capable of being successfully employed in the Edman protein sequenator to those reagents with relatively low vapor pressures. Of course, as mentioned above repeated cycling by vacuum drying and re-introduction of reagents merely tends to compound this problem.

Still further the Edman protein sequenator encounters problems of cross-contamination of the various solvents and reagent used throughout the degradation process. This is due to the employment of an inlet manifold common to all the reservoirs containing the reagents and solvents through which nitrogen is passed to maintain the reservoirs at a constant low pressure as well as to remove any dissolved oxygen from the solvents and reagents. However, due to the nature of the various reagents and solvents there is always a vapor pressure in each of the reservoirs. Since these vapor pressures are bound to vary, due to variations in the ambient temperature, from one reservoir to the next, solvents and/ or reagents tend to be forced out of one reservoir (having a higher vapor pressure) to another reservoir (having a lower vapor pressure) thereby causing cross-contamination. Needless to say, such crosscontamination leads to a rapid deterioration in performance and/or an abrupt termination of the degradation process.

The employment of an oil vacuum seal between the rotating motor shaft and the motor support also causes numerous problems. In particular, in the Edman protein sequenator, the oil vacuum seal is in communication with the reaction chamber. As such, it tends to leak into the reaction chamber adversely affecting the reagents and conversely the reagents tend to diffuse into the vacuum seal area, reacting with the oil, decreasing the useful life of the seal. It appears that the use of such a seal, under these circumstances, requires frequent overhaul.

Also, it should be noted that the glass cup reaction vessel of the Edman protein sequenator includes shar corners about the lower edge of the inside surface of the cup in which samples tend to become lodged. This again leads to incomplete reactions and/ or extractions contributing toward the premature termination of the degradation procedure.

SUMMARY OF THE INVENTION In brief, the present invention contemplates an automatic protein and/ or peptide sequenator which avoids or minimizes the previously mentioned difficulties and concomitant limitations and is capable of degrading eith'er proteins and/or peptides containing N number amino acid units regardless of the length (long or short) of the amino acid chain being degraded.

To this end there is provided a sequenator including a reaction cell disposed and rotatably driven in a chamber which includes means for introducing and removing gases and liquids. The chamber is preferably disposed in an insulated housing whose interior is maintained at a substantially constant uniform temperature. The free volume in said chamber is minimized to reduce the evaporation from the reaction cell to permit use of volatile chemicals in the chemical reactions. Said sequenator also includes reagent feed means providing repeatable and predictable delivery of reagents into said chamber. The drive means for the reaction cell is adapted to drive the cell at selected programable speeds.

The primary object of the present invention is the provision of an apparatus capable of automatically performing a variety of chemical processes.

Another object of the present invention is the provision of an improved automatic protein-peptide sequenator which avoids the disadvantages and concomitant limitation associated with prior art sequenators.

A further object is an automatic protein-peptide sequenator capable of handling both high and low volatile reagents and solvents.

A further object is the successful ascertaining of the amino acid sequence of a protein and/or peptide regardless of the amino acid chain length (long or short).

Still another object is a flexible automatic proteinpeptide sequenator which may be manually or automatically operated.

Another object of the present invention is an automatic protein-peptide sequenator which is relatively speaking insensitive to ambient temperature variations and may be operated in a non-airconditioned environment.

Still another object is the provision of an automatic protein sequenator delivering a repeatable and predetermined volumetric amount of reagent to the reaction cell.

Another object is the elimination of cross-contamination between the reagents used throughout the system.

Still another object is an apparatus not dependent on any presumed sequence of events.

These and other objects and advantages of the invention will become apparent from the following detailed description when read on conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A, 1B, 1C and ID are a schematic of the automatic protein/peptide sequenator in accordance with the principles of the present invention;

FIG. 2 is a diagramatic illustration of the constant pressure and delivery system used in connection with the reservoirs containing the reagents;

FIG. 3 is a vertical cross-sectional view illustrating in detail the reaction cell assembly, the magnetic drive assembly, and the air circulating arrangement within the housing;

FIG. 4 is a vertical cross-sectional view illustrating in detail the vacuum actuated valve assembly;

FIGS. 5 and 6 illustrate alternate valve poppet constructions;

FIG. 7 is a perspective view partially broken away of the fraction collector; and

FIG. 8 illustrates in block diagram form the programming unit which controls the sequence of steps as well as the duration of each step carried out by the sequenator.

DESCRIPTION OF THE PREFERRED EMBODIMENT With reference now to the drawings and more particularly FIG. 1 thereof, the reference numeral 20 designates in general a reaction cell assembly including a reaction cell or cup 21 disposed within a reaction chamber 22 formed in general by a cylindrical enclosure 23. Reaction cell assembly 20 is in turn positioned within a heated chamber 24 defined by a generally rectangular enclosure 25 fabricated from Suitable insulating materials such as plexiglas and/or phenolic. In the preferred embodiment heated chamber 24 is divided into upper and lower portions by a base plate 26 which serves as a baffle and extends horizontally across the heated chamber as indicated by the dotted line. A heating means 27 to be discussed in more detail in connection with FIG. 4 is also located Within chamber 24 to maintain the reaction cell assembly 20 and the drive assembly (hereinafter described) at a predetermined and constant temperature.

The reaction cell 21 takes the form of a generally cylindrical glass cup secured to the top of a rotatable shaft 28 which in turn is driven by a multiple or variable speed electrical motor 29. In practice the speed of the motor is adjusted between two predetermined speeds for purposes which will be fully discussed hereinafter.

By rotating or spinning the reaction cell 21 the various reagents and solutions, due to the associated centrifugal force, form a spinning thin film about the inside surface of the glass cup. Spreading the various reagents and solvents as thin films on the inside wall of the cup enhances the entire operation of the sequenator since such a film facilitates extraction by another film of an imiscible solvent sliding over its surface as Well as presenting a large, stabilized, surface area which may be easily dried under reduced pressure.

The reagents and solvents used throughout the process are introduced into the cup by way of four separate feed lines shown schematically by lines 30a, 30b, 30c and 30d each of which extends through the top of the enclosure 23 and depends downwardly toward the bottom of reaction cell 21 terminating slightly above the bottom of the cell with sufficient clearance above the cell bottom that liquid flow is not unduly obstructed. The feed lines 30a-30d are arranged in a circle around the reaction cells longitudinal axis to facilitate the smooth introduction of the reagents and solvents into the spinning cell 21. In addition each of the feed lines 30 has about seventy-five percent of its total length (not shown) located within heated chamber 24 so that the fluids passing through these feed lines are heated to and maintained at a predetermined repeatable temperature.

Evacuation of the reaction cell chamber and the reaction cell itself is provided by means of a pair of vacuum pumps 31 and 32 which for purposes of this invention are designated a rough vacuum pump and a fine vacuum pump, respectively. Rough vacuum pump 31 is connected to the reaction cell chamber 22 via a rough vacuum line 31a which communicates with the chamber 22. Rough vacuum pump 31 is also connected to a restricted vacuum line 33 by way of a small T-connector 34 inserted in vacuum line 31a. The restricted vacuum line 33 extends through the top of enclosure 23 and depends down' wardly into the reaction cell 21. It should be noted that the restricted vacuum line 33 is centrally located along the longitudinal axis of the reaction cell 21 for reasons to be presently discussed. Fine vacuum pump 32 also communicates with the reaction chamber 22 by Way of fine vacuum line 32a.

In practice both vacuum pumps are continuously operated. Consequently, three solenoid actuated valves 36, 37, and 38 are inserted in rough vacuum line 31a, fine vacuum line 32a, and restricted vacuum line 33, respec tively, for controlling the application of a predetermined vacuum to the reaction cha'mber'22 and reaction cell 21. When applying a vacuum, solenoid valve 38 is opened first to connect the rough vacuum pump to the reaction cell chamber 22 and the reaction cell 21 via restricted vacuum line 31a. This serves to prevent the liquid contained in the reaction cell 21 from boiling due to too sudden an evacuation. Once the reaction chamber 22 and reaction cell 21 are initially evacuated, solenoid actuated valve 38 is closed and either valve 36 or valve 37 is opened to connect the rough or the fine vacuum pump 31 or 32, respectively, to the reaction chamber 22 which serves to apply the vacuum during the remaining time period of each evacuated drying step.

Typically the determination of the amino acid sequence in proteins and/or peptides is based on the so-called three stage phenylisothiocyanate reaction. It has been found that the phenylthiocarbamyl group formed during this process is easily desulp hurized by oxidation terminating the degradation process, since a thiazolinone, whose formation is essential to a successful completion of the sequenation, can no longer be formed. As a consequence, the degradation process must be carried out in an oxygen free atmosphere. This is accomplished by filling the reaction chamber 22 with a suitable inert gas, such as nitrogen. Of course, it will be appreciated by those familiar with the art that other inert gases may be employed, such as helium, neon, argon, etc. In accordance with the principles of the present invention the nitrogen is derived from two commercially available cylinders of compressed nitrogen 44 and 45. Nitrogen is fed from both of these cylinders to the reaction chamber 22 by way of nitrogen feed line 46 which communicates with the interior of reaction chamber 22. Nitrogen feed line 46 is in turn connected to nitrogen cylinders 44 and 45 by way of a distribution manifold 47, branch lines 49 and 50, and pressure regulators 48 and 4811, respectively. A pressure switch 51 is located at the junction of branch lines 49 and 50 to monitor the pressure of the nitrogen being supplied by the nitrogen cylinders and switch from one nitrogen supply to the other when the nitrogen supply being used falls below a predetermined pressure. For instance, assuming that nitrogen is initially being supplied by cylinder 44, to switch from this supply to nitrogen cylinder 45 pressure switch 51 upon sensing a predetermined pressure provides a signal which simultaneously closes a solenoid actuated valve 52 shutting off the nitrogen supply from cylinder 44 and opens a solenoid actuated valve 53 connecting manifold 47 to nitrogen cylinder 45. Each branch line 49 and 50 is connected to a separate manually actuated vent valve 54 which is opened following the installation of each nitrogen cylinder to vent any air inadvertently introduced in these lines during the installation of the nitrogen cylinders.

In addition to purging the atmosphere of oxygen and water vapor, the nitrogen is used to maintain the reaction chamber 22 and the reservoirs containing the various reagents and solvents at predetermined pressures. To this end the hand operated pressure regulation valves 48 and 48a are inserted between the junction of branch lines 49 and 50 and nitrogen cylinders 44 and 45. By way of example the pressure regulation valves 48 and 48a may reduce the pressure of the nitrogen to around 50 psi. as it enters distribution manifold 47.

The pressure of the nitrogen may be further reduced before it enters the reacton chamber 22 by means of a hand operated pressure regulator 55 positioned in the nitrogen feed line 46 at the outlet side of distribution manifold 47. For this example the nitrogen pressure in the reaction chamber 22 may be set to around 40 mm. of Hg (77 p.s.i.), although, of course, it will be appreciated that other pressures may be used depending upon other parameters involved.

A solenoid actuated valve 56 is inserted in nitrogen feed line 46 just inside heated chamber 24 for controlling the flow of nitrogen to reaction chamber 22. Finally, a hand operated isolation shut-off valve 57 is located in nitrogen feed line 46 between pressure regulator 55 and solenoid actuated valve 56 as a safety measure to isolate the nitrogen supply cylinders 44 and 45 from the reaction chamber 22 when the automatic sequenator is not in operation. Of course, when the sequenator is being operated shut off valve 57 remains open.

The reagents and solvents used throughout the degradation process are stored in a plurality of reservoirs 58- each of which may typically take the form of a bottle fabricated from a suitable noncorrosive material, such as borosilicate glass. For purposes of description the reservoirs are designated R1, R2, R3, R4 and S1, S2, S3, and S4 for the reagent (R) and solvent (S) each contains. Each of the reservoirs 58 is connected to the nitrogen supply through manifold 47 both to remove dissolved oxygen from the reagents and solvents and to maintain the reservoirs 58 at a constant predetermined pressure. Nitrogen is fed to the solvent reservoirs S1 through S4, by way of distribution manifold 47, a nitrogen feed line 59, and common distribtuion manifold 60 which includes four oulets 61, 62, 63 and 64 each of which is connected to one of the solvent reservoirs S1 through S4. Cross contamination of solvents is not critical to the system operation so a common solvent distribution manifold may be employed. A pressure regulator 65 is positioned just outside distribution manifold 47 in nitrogen feed line 59 to reduce the pressure of nitrogen being fed into the solvent reservoirs. For the example under discussion, this pressure is reduced to around 140 mm. of Hg (2. 7 p.s.i.). In addition, each of the outlets 61, 62, 63 and 64 from distribution manifold 60 includes a solenoid actuated valve 66a, 66b, 66c and 66d, respectively, for selectively controlling the introduction of nitrogen to the solvent reservoirs S1 through S4- and otherwise isolate the respective reservoirs when not in use.

Like the solvent reservoirs, the reagent reservoirs R1 through R4, are, also, connected to the nitrogen supply by way of distribution manifold 47. However, to ensure against cross contamination of the various reagents nitrogen is fed to each of the reservoirs R1-R4 by way of separate nitrogen feed lines 67a, 67b, 67c and 67d each of which is connected to a different outlet terminal of distribution manifold 47 as indicated by the designations R1, R2, R3 and R4. Again, like the reservoirs, the introduction of nitrogen to the reagent reservoirs R1- R4 is controlled by means of solenoid actuated valves 68a, 68b, 68c and 68d, with one solenoid valve positioned in each nitrogen feed line. A pressure regulator 70, like regulator 65, is provided in each nitrogen feed line 67 to reduce the pressure of nitrogen being fed to the reagent reservoir. For the example under discussion this pressure is reduced to around 120 mm. of Hg (2.3 p.s.i.). Regulators 70 also serve as a means for independently controlling the individual flow rates of each reagent by varying the pressure independently. Each of the reservoir- nitrogen feed lines 67a, 67b, 67c and 67d and the solvent-nitrogen feed line 59 include a hand operated shut-off valve 57 which is closed when the sequenator is not operating and remains open during sequenator operation. Finally, a suitable pressure gauge 71 may be associated with each nitrogen feed line 59, 67a, 67b, 67c and 67d to monitor the pressure of the flowing nitrogen.

Both the reagent and solvent reservoirs 58 are, also, selectively coupled to the atmosphere via vent lines 95 and interconnected vent manifolds 72 and 73. Again, a plurality of solenoid actuated valves 74a, 74b, 74c, 74d, 74e, 74f, 74g and 7411 are provided with one valve located in each of the solvent and reservoir vent lines 95 to selectively connect each reagent and solvent reservoir to the atmosphere, or otherwise isolate it. In addition, a fine metering hand valve 75 is provided in each of the vent lines 95 which metering valve is manually adjusted to provide a continuous nitrogen flow through each vent line when its associated solenoid valve 74 is opened. The flow may be in the range of 30-50 cc. These fine metering valves 75 play an important part in the constant pressure-uniform delivery system, a significant feature of the present invention which will be presently discussed in detail in connection with FIG. 2.

Finally, each of the reagent reservoirs, Rl-R4, and the solvent reservoirs, Sl-S4, include a connecting outlet line 76a, 76b, 76c, 76d, and 96a, 96b, 96c and 96d, respectively, connected to reagent and solvent feed lines 30a, 30b, 30c and 30d by way of a bank of four vacuum actuated valve assemblies 77, 78, 79 and 80, with each valve assembly including in general two valve units 103 and 105 mounted on a common manifold. Reagent is fed to valve unit 105 of each valve assembly by way of the associated connecting line 76 and a first fluid inlet port in the common manifold while solvent enters the valve unit 103 of each valve assembly via the associated connecting line 96 and a second fluid inlet port in the common manifold. The manifold also includes a common outlet or discharge port 102 through which reagents or solvents are passed to reagent/ solvent feed lines 30a, 30b, 30c and 30d. The vacuum actuated valve assemblies are an important feature of the present invention and will be described in greater detail in connection with FIGS. 4, 5 and 6.

While fluids (reagents or solvents) are being introduced into reaction cell 21, solenoid valves 56, 57 and 69 are opened to interconnect reaction chamber 22 with the exhaust blower 41 through nitrogen feed line 46, permanently open bleed valve 69, and vent manifolds 72 and 73. This permits excessive vapors to escape to the atmosphere preventing an excessive pressure build up in reaction chamber 22 thus maintaining a constant delivery rate.

The vacuum actuation of valve assemblies 77-80 is derived from rough vacuum pump 31 via vacuum port 104 of each valve unit 103 and 105. The vacuum is applied by way of vacuum line 81 and a plurality of connecting lines 83a, 83b, 83c, 83d, 83c, 831, 83g and 8311. An auxiliary vacuum tank 82 communicates with vacuum line 81 and serves as a back-up vacuum source. That is, vacuum tank 82 maintains vacuum on vacuum line 81 in the event that the vacuum provided by rough vacuum pump 31 falls below a predetermined level, due to a sudden demand by either the reaction chamber 22 or fraction collector 155.

To ensure that the reagents and solvents are maintained oxygen free, the vacuum actuation is broken by feeding nitrogen through connecting lines 83 to vacuum ports 104 of each valve unit 103 and 105. In the case of the valve units 105a105d associated with reagent reservoirs R1-R4 the nitrogen needed for this purpose is derived from the four nitrogen feed lines 67a, 67b, 67c and 67d respectively by means of T-shaped connectors 84 inserted in each feed line 67 and passed through nitrogen carrying lines 85a, 85b, 85c and 85d each of which is connected to a connecting line 83 by solenoid actuated valves 8%, 89d, 89f, and 89h, respectively. The nitrogen needed for breaking the vacuum of the valve units 103a103d associated with solvent reservoirs Sl-S4 is derived from distribution manifold 60, four separate lines 86a, 86b, 86c, and 86d and four solenoid actuated valves 89a, 89c, 89e, 89g. Solenoid valves 89 interconnect vacuum line 81 as well as each nitrogen feed line 86a-86d and 85a-85d with the connecting lines 83 leading to the vacuum port 104 of each valve unit. Thus, in operation each solenoid valve 89 is actuated at predetermined times to couple each vacuum port 104 either to rough vacuum pump 29 to thereby open the valve or to nitrogen supply 44 to break the vacuum and close the valve.

At this point perhaps it would be helpful to discuss the constant pressure and uniform delivery system associated with each reagent reservoir, R1-R4, to ensure that a repeatable and predetermined volumetric amount of reagent is delivered to the reaction cup throughout each cycle of operation of the automatic protein/peptide sequenator. This system in accordance with the principles of the present invention is illustrated in FIG. 2 wherein for purposes of simplicity only one reagent reservoir R and its associated delivery system is shown since the constant pressure-uniform delivery system associated with each reagent reservoir is identical.

The volumetric amount of fluid delivered to the reaction cup is a function of the inner diameter of (restriction afforded by) the feed line, fluid viscosity and density (both a function of temperature), pressure, and length of time the admittance valve remains open. The inner diameter of the feed line is, of course for all practical purposes, fixed, while the actuation time may be accurately governed by a suitable program unit. This leaves the fluid flow rate which is a function of the temperature of the main flow controlling restriction (tubes 30) and pressure associated with the system.

The primary function of the delivery system is to render the fluid flow highly insensitive to room ambient tem perature changes and provide a constant delivery pressure thereby achieving a constant flow rate which in turn enables the delivery of a repeatable and predetermined amount of reagent to the reaction cell throughout the degradation process. To this end, as previously discussed, reagent reservoir R is connected to nitrogen feed line 67d via solenoid actuated valve 68d, and to vent manifold 72 by way of vent line 95, solenoid actuated valve 74d and adjustable fine metering hand valve 75. Finally, the reservoir R is connected to feed line 30d via connecting line 76d and vacuum actuated valve unit 105d, By way of example, it is desirable to maintain the reagent pressure in the reservoirs somewhere between 100 and 150 mm. of Hg depending on the particular nature (viscosity) of the reagent. It will be recalled that many of the reagents used throughout the degradation process are highly volatile in nature. As a result the pressure in the reservoir at he delivery line inlet 94 randomly varies as a function of reagent vapor pressure inside the reservoir as well as changes in the fluid level (the former of which is a function of ambient temperature variations).

To maintain a constant pressure at the communication point 94- of connecting line 76 with the reagent, the following steps are performed. Initially each of the valves 68, 7'4, and 105d are closed. First, as briefly discussed in connection with FIG. 1, adjustable metering valve 75 is set to allow a slow vent of around 30 to 50 cc. fluid (vapor) flow from reservoir R Thus, when solenoid valve 74d is actuated reservoir R is slowly vented to the atmosphere. After a predetermined time, around onehalf a minute (empirically determined), the total pressure inside the bottle falls below that of the incoming nitrogen pressure. At this time solenoid valve 68d is opened to connect the nitrogen supply to the reagent contained within the reservoir. The nitrogen is then al lowed to bubble through the reagent for a certain period until a dynamic equilibrium condition is established, namely, a constant pressure at point 94 located to the entrance to connecting line 76. At this time vacuum operated valve 105d is actuated to permit reagent to be fed into the reaction cell 21. Of course, the fluid flow rate of the reagent is still subject to temperature variations as it travels through the feed line 30d. However, as discussed in connection with FIG. 1, around seventy-five percent of the feed line is located within the heated chamber 24 which maintains it at a predetermined and constant temperature.

The above procedure is carried out each time a reagent or solvent is introduced into reaction cell 21. The net effect of all this is that the automatic protein/peptide sequenator in accordance with the principles of the present invention is insensitive to random variations in ambient temperatures and, thus, does not require an airconditioned room for proper operation.

As noted above, this delivery system ensures a repeatable and predetermined volumetric amount of reagent is delivered into the reaction cell throughout the degradation process. In addition, such a system also serves to further prevent cross-contamination between the various reagents. That is, the solenoid valve 68d located in nitrogen feed line 67d remains closed until the pressure inside the bottle falls below that of the nitrogen being fed into the bottle thereby isolating the reservoirs from the nitrogen supply. Thus, each reservoir in effect has its own regulator which is equivalent to having its own nitrogen supply.

Returning again to FIG. 1, the means for removing the excess reagents, reaction products and by-products from the reaction cell 21 comprises an eflluent line 151 the tip of which'is located in an annular groove formed about the inside top edge of the reaction cell 21. A vacuum operated valve assembly 152, having two valve units a and 90b, couples eflluent line 151 with a waste bottle 153 or a fraction collector depending on the nature of the effluent being withdrawn. In particular, during the washing stages (by solvent extraction) after each coupling step, the excess reagents and reaction by-products flow to waste bottle 153 via waste line 154 from which they may be removed from the system. The sought after reaction products, removed by solvent extraction after the cleavage steps, are directed to the fraction collector 155 by connecting the effiuent line 151 with fraction collector input line 156 through valve unit 90b. Both valve units 90a and 90b making up valve assembly 152 derive their vacuum actuation from rough vacuum pump 31 via vacuum line 81 and a pair of auxiliary connecting lines 157 and 158 both of which are tied into the vacuum line 81 by suitable connector 159 and 160, respectively. Both connecting lines 157 and 158 include a solenoid actuated three-way valve designated by reference numerals 161 and 162, respectively, to control the vacuum application.

A suitable fraction collector 155 may include a circular array of collection tubes each of which is capable of holding 5 to 10 milliliters of reagent. The collection tubes are supported in a housing 166 and are moved in a step by step fashion by drive motor 163. The housing is evacuated by rough vacuum pump 31 connected to the interior of the housing by way of a vacuum line 164, manifold 42, and solenoid actuated valve 39. By evacuating the fraction collector chamber the final residues being collected are dried immediately following the collection step. Moreover, the inside of the fraction collector 155 is also coupled to the exhaust blower system 41 via hand operated valve 165 and solenoid operated valve 40 and connecting line 43 for exhausting the reagent vapors which build up inside the fraction collector to the atmosphere during delivery and to break the vacuum after drying.

REACTION CELL AND MAGNETIC DRIVE ASSEMBLIES With reference now to FIG. 3, there is illustrated in vertical cross-section the details of the reaction cell and magnetic drive assemblies. Multiple or variable speed motor 29 includes a drive shaft 201. Rotational movement of motor drive shaft 201 is translated to reaction cell drive shaft 202 by means of a magnetic drive coupling assembly designated generally by the reference numeral 203 and comprising a cylindrical, cup shaped holder 204, an outer magnetic ring 205, and an inner magnetic ring 206 concentrically disposed within the outer magnetic ring. Each magnetic ring is fabricated from suitable ceramic materials and is permanently magnetized in a radial direction to facilitate the coupling of magnetic flux between the annular magnets. The outer magnetic ring 205 is press fitted into the cup 204 and abuts against the upper shoulder 207. The inner magnetic ring 206 is press fit onto sleeve 208 which in turn surrounds and is suitably attached to drive shaft 202 as by a set screw (not shown). This magnetic coupling eliminates the need for a motor oil seal with its associated problems.

Multiple or variable speed motor 29 may be used advantageously for both the initial mixing of the reagents and sample and for maintaining the sample at a certain level in the reaction cell which level is compatible with the reagent level. More specifically, in the preferred embodiment, the speed of motor 29 is set at either 1200 or 1800 r.p.m. Of course, other speeds may be used or, if desired, the motor speed may be raised in a continuous manner.

The reaction cell is spun at the high rotational speed, for example 1800 r.p.m., as the reagents flow into the reaction cell. This ensures that the reagents completely engulf and react with the sample preventing a ring of unreacted or partially reacted sample to remain and ac cumulate about the inner surface of reaction cell 21. The reaction cell is also spun at its high speed as the washing steps are performed since high speeds assist in and facilitate the removal of the washing solvents from the reaction cell. During the reactions, themselves, the reaction cell is rotated at a lower speed, 1200' r.p.m., which lowers the level of sample and enhances the mixing of the reagents and sample ensuring that a complete reaction takes lace.

p A thin walled cylindrical cup shaped member 245 formed of Kel-F material is positioned between outer and inner magnetic rings 205 and 206, leaving a small clearance space 226 around inner magnetic ring 206, to provide a nonmagnetic pressure and vacuum seal between the reaction chamber 22 (including inner magnetic ring 206) and heated chamber 24. The cup shaped member 245 is pressed onto the lower portion of a cylindrical bearing housing 209 having an external groove for retaining a suitable O-ring seal 210. Suitable close tolerance upper and lower ball bearings 211 and 212 are contained within bearing housing 209 to cooperate with rotatable drive shaft 202 and which together with cylindrical bearing housing 209 defines an annular chamber 221. Bearing housing 209 is tightly secured to a bafile base plate 213 by locking screws 214 extending into an outwardly extending flange or lip 246 of bearing housing 209 which rests flush against the upper surface of base plate 213. A suitable seal (not shown) is provided between base plate 213 and lip 246.

A drive table 215 is attached to the upper end of drive shaft 202 and includes an annular recess 216 which carries an annular member 217 having a diameter somewhat smaller than the diameter of recess 216 providing a small clearance space 225 around annular member 217 to permit, within limits, lateral and tilting adjustment of annular member 217. Reaction cell 21 rests flush against and is tightly secured to a machined recess 218 in the annular member 217 by a suitable epoxy resin.

To ensure that the inside surface of reaction cell 21 runs true with the rotational axis of drive shaft 202, means are provided for adjusting reaction cell 21 in both vertical and lateral directions. Critical alignment is achieved by means of four adjustment screws arranged about the periphery of the drive table 215 and four adjustment screws disposed around the upper surface of the annular member 217. In particular, the reaction cell may be aligned in a lateral direction by selectively adjusting the position of four screws cooperating with four equally spaced internally threaded bores 219 formed about the periphery of drive table 215. On the other hand, the optimum tilt or vertical alignment is achieved by merely adjusting the four screws cooperating with four equally spaced internally threaded bores 220 formed in annular member 217. The four set screws associated with drive table 215 also serve to retain or lock annular member 217 within recess 216.

As previously noted, the reaction cell 21 takes the form of a generally cylindrical cup fabricated from a suitable noncorrosive material, such as borosilicate glass. In one example the inside diameter (d) of the cup was around 25 millimeters while the height (h) of the inside of the cup as measured from the bottom of the cup to an annular groove 222 formed around about the inner surface of the top edge of the cup was about 31 millimeters. The annular groove 222 was about 2.5 millimeters deep as measured from the inner surface of the cup and about 6 millimeters wide. It should be noted that the deeper the annular groove the better scooping action achieved. The inner edge around the bottom of the cell was rounded to about a 6 millimeter radius to minimize the sample thickness at the bottom of the cell, thus, enhancing the chemical reactions, improving the washing action of the solvents and preventing the sample from becoming lodged in the corner, as well as prevent the boiling action, during the minimum drying cycle, initiated at the sharp corners. The bottom of the cell interior is constructed flat and tangent to the corner radii, with a centrally located conical protrusion, about 3 millimeters high and 5 millimeters in diameter at the base, to prevent the accumulation of various residues at the center of the cup which lies at the point of zero centrifugal force.

The reaction chamber 22 is defined by an enclosure 23 comprising a cylindrical glass sleeve 223 bounded on the lower end by cup 245 and on the upper end by a disc-like cap 224 fabricated of a suitable metal, such as aluminum or stainless steel. The cap 224 fits over the top of the cylindrical glass sleeve 223 and carries an O-ring 230 (of suitable nonreactive material such as viton or ethylene propylene) in an internal annular groove 231. An annular Teflon gasket (not shown) is sandwiched between the top edge of cylindrical glass sleeve 223 and cap 224 as a cushion. The cap 224 also includes a centrally located opening 232 for receiving a stopper plug 233 preferably fabricated of Teflon. An O-ring seal 234 contained within an annular groove formed about the opening 232 serves as a liquid tight seal between plug 223 and cap 224.

The lower edge of cylindrical glass sleeve 233 rests on a Tefion gasket (not shown) held in an annular ridge 235, formed in a generally cylindrical main housing 236 which is mounted on base plate 213. Housing 236 includes a recess 237 in which drive table 217 rotates and an internal groove for retaining an O-ring seal 238 surrounding the lower end of glass sleeve 223.

At this point it will be noted that the reaction chamber 22 includes, in addition to the space defined by the interior of cylindrical glass sleeve 223 in which reaction cell 21 resides, clearance space 226, annular chamber 221 and recess 137 formed in the main housing 236 all of which are in communication with each other and the interior of glass sleeve 223. In other words the outer boundaries of the reaction chamber comprise cap 244, glass sleeve 223, housing 236, base plate 213, the lower portion of bearing housing 209, and cup-shaped member 245. To enable the automatic sequenator to operate with highly volatile reagents it is essential that the total unoccupied volume of the reaction chamber be kept to an absolute minimum. As may be readily seen from FIG. 3 this is achieved by keeping the volume of the initial reaction chamber low and filling substantially all the reaction chamber space with the reaction cells 21, drive table 215, drive shaft 202, magnetic member 206 and bearing housing 209. In one arrangement the total unoccupied space is maintained below 250 cc. which leads to a more rapid equilibration of temperature and vapor pressure and minimizes evaporative losses thereby permitting the automatic sequenator in accordance with the principles of the present invention to easily handle highly volatile reagents as well as nonvolatile reagents. Moreover, such an arrangement facilitates control of gas and vapor flow patterns with respect to the reaction cell 21. To further minimize the unoccupied space the annular chamber 221 may be partially filled with a loosely fitting metal annular sleeve (not shown).

As mentioned earlier the reaction chamber 22 is evacuated and filled with flowing nitrogen by way of lines 31a and 46, respectively (and associated solenoid actuated valves 36 and 56), each line of which extends through base plate 213 and main housing 236 into recess 237 which, as previously discussed comprises a portion of reaction chamber 22 with the space defined by the interior of glass sleeve 223.

Stopper plug 233 is attached to a pivotally movable arm 239 by means of three screw fittings (not shown) extending through arm 239 into the top of plug 233. Arm 239 is pivotally movable about pivot point 245 to facilitate the easy removal and insertion of plug 233 into the opening 232 formed in cap 224. A removable knurled bolt 241 extends through the top of arm 239 and into the protruding portion of base plate 236 to secured arm 239 when plug 233 is inserted in place. As bolt 241 is tightened an O-ring 237 is compressed to form a vacuum pressure tight seal between plug 233 and cap 224. Once bolt 241 is removed, arm 239 is free to pivot about point 260 to remove plug 233 and gain access to the interior of reaction cell 21 Stopper plug 233 depends downwardly into reaction cell 21, to substantially close off the interior of the reaction cell from the reaction chamber 22. This minimizes and retards the escape of the contents of the reaction cell 21 into the reaction chamber 22. Such an arrangement minimizes turbulence and, for all practical purposes, eliminates any pumping action, created by the centrifugal action of the rotaing reaction cell on the vapors within the cell, between the cell interior and the reaction chamber. It follows that for all practical purposes the forced vapor circulation is contained within the reaction cell minimizing vapor loss through evaporation. On the other hand, it should be noted that plug 233 does fit somewhat loosely within opening 232 leaving a narrow annular clearance between the plug 233 and cap 224 so that, while fluids in the cell 21 are substantially trapped, nitrogen fed into reaction chamber 22 may enter and circulate in reaction cell 21 and thence leave through effluent line 151, and/or restricted vacuum line 32. In addition this enables the vacuum when applied to communicate with the reaction cell interior.

Plug 233 carries reagent/solvent feed lines 30 (only one of which is shown), efiluent line 151, restricted vacuurn line 32. As previously noted, the restricted vacuum line 32 is located within the reaction cell 21 along the longitudinal axis of the reaction cell while fed lines 30 are arranged in a circle about restricted vacuum line 32. The height of restricted vacuum line 32 is adjusted so that its bottom tip lies within the vortex of the vapor mixture circulation. Positioning the restricted vacuum line in such a manner greatly improves the vacuum drying efliciency Within the reaction cell as well as minimizing the amount of any undesirable vapors (corrosive and otherwise) coming in contact with the various surfaces of the reaction chamber.

Effluent line 151 enters the reaction cup as nearly tangentially as practical with its tip disposed in groove 222 and facing the rotational direction of the reaction cell. There is provided a small clearance between the groove and the eflluent line tip. Scooping of the reagent products and by-products and extracting solvents is assisted by the momentum of the impinging fluids as well as by the higher nitrogen pressure within the reaction cell. Efliuent line 151 may be externally adjusted both up and down along its longitudinal axis (vertically) and angularly about its longitudinal axis to orientate the effluent line in an optimum position for most efficiently withdrawing the fluids. All lines are fabricated from a suitable noncorrosive material, such as Teflon.

Finally, plug 233 carries a temperature probe 242 consisting of a thermistor encapsulated in a glass rod for continuously monitoring the temperature within the reaction cell 21.

TEMPERATURE CONTROL SYSTEM There is further illustrated in diagramatic form the air circulation temperature control system for maintaining the reaction cell assembly 20 as well as the magnetic drive assembly 203 and the bearing housing 236 at a constant and predetermined pressure. To this end a fan 250 is mounted on the underside of base plate 213 and carries a heater element 251 which extends upwardly through an opening in base plate 213 into the upper portion of heated chamber 24. As discussed earlier heated chamber 24 is 14 defined by a generally rectangular enclosure 25 fabricated from suitable insulating materials.

Fan 250 draws the air from the lower portion of heated chamber 24 and forces the air through heater element 251, wherein the air is heated to a predetermined temperature, into the upper portion of heated chamber 24. The heated air circulates through the upper portion of heated chamber 24 and down into the lower portion via an annular aperture 252 formed in base plate 213 as depicted by the arrows. A significant feature of the present invention is that the magnetic coupling 203, the various bearing support housings, and the reaction cell assembly 20 are all located centrally within the heated chamber 24 and thus maintained at a substantially uniform, predetermined and constant temperature, preferably around 50 C. It follows that the reaction chamber and its internal surfaces as well as all elements disposed within the reaction chamber are maintained at a substantially uniform temperature. This means that should a cold sink be present it always lies in the interior of the reaction cell tending to keep the highly volatile reagents and buffers within the reaction cell 21.

As previously noted, the temperature inside the reaction cell is continuously monitored by a thermistor probe 242. In addition, a thermistor 243 is imbedded Within housing 236 to continuously sense the temperature thereof. A third thermistor 244 is positioned above the heater outlet to continuously sense the temperature of the air as it emerges from the heater. Thermistors 243 and 244 are electrically connected in series with each other and the temperature control circuit of heater element 251. This arrangement causes the temperature of the air emerging from the heater to overheat above the set point whenever the housing temperature is below the set point (such as at start up), thus providing an artifically high temperature differential causing a fast initial warm up. This system also maintains the temperature of the sequenator system at a predetermined uniform temperature without undue cycling.

VACUUM ACTUATED VALVE ASSEMBLY With reference now to FIGS. 4, 5 and 6, there is illustrated in detail the vacuum valve assembly in accordance with the principles of the present invention. As previously noted, each vacuum valve assembly comprises two identically constructed independent valve units 103 and 105 mounted on and discharging into a common manifold or body 106. Thus, in the interest of simplicity only the valve unit 105 will be described in detail, it being understood that the lower valve unit 103 operates in an identical fashion.

Manifold 105 includes a pair of diametrically opposed annular recesses 107 and 108 and is bounded on either side by circular shaped flexible diaphragrns 109 and 110 fabricated of a suitable flexible chemical resistant material such as Teflon TFE. The outer periphery of flexible diaphragm 109 is sandwiched between manifold 106 and a cylindrical shaped housing 111 which is tightly fastened to manifold 106 by four locking bolts 112. Outer housing 111 includes a circular recess 113 communicating with a bore 114 the internal surface of which serves as a hearing surface. A cap member 115 fits tightly over the top of bore 114 and includes a screw adjustment 116 which projects downwardly into bore 114. A circular disc member 117 fits within recess 113 and includes an upwardly extending tubular shaft 118 which is slidably positioned within bore 114. Tubular shaft 118 includes a counter bore 119 containing a resilient means 120, such as a coiled spring, which surrounds screw adjustment 116 and is retained between the lower end of counter bore 119 and a step 121 formed on the lower surface of cap 115.

A generally arrow-shaped valve poppet extends through openings located in flexible diaphragm 109 and circular disc member 117 into tubular shaft 118 to which the valve poppet 125 is securely attached by means of a screw fitting 123 cooperating with an internally threaded bore within the valve poppet 125. By tightening screw fitting 123, the inner radial portion of flexible diaphragm 109 is tightly sandwiched between the lower surface of circular disc member 117 and the upper surface of valve poppet 125. An O-ring seal 133 is retained in an annular groove formed in disc member 117, providing a vacuum seal and simultaneously serving as a compressive member forcing the Teflon diaphragm 109 against poppet 125.

An annular cavity 126 defined by the surface of recess 113 and the upper surface of circular disc member 117 serves as an actuating cavity which communicates with a vacuum port 104 by way of a passageway 127. The vacuum port 104 is internally threaded to receive a suitable line connector.

The surface of annular recess 107 formed in manifold 106 together with flexible membrane 109 define a process fluid cavity 129 which communicates with a fluid inlet port 100 via a passageway 128 and an outlet discharge port 102 by way of passageway 130. Likewise, the surface of lower annular recess 108 together with flexible membrane 110 define a second fluid cavity 131 which communicates with an inlet port 101 by way of a passageway 132 and discharge port 102 via passageway 130. Thus, it is seen that discharge port 102 is common to both fluid inlet ports 100 and 101. As utilized in the present automatic protein/ peptide sequenator invention the fluid inlet port 100 communicates with a reagent reservoir while fluid inlet port 101 communicates with a solvent reservoir.

Valve poppet 125 includes a conical shaped projection 137 terminating in a point 138 (FIG. which cooperates with a valve seat 135 formed about passageway 130. Coiled spring 120 serves to provide a downward force normally biasing the valve in a closed position with valve poppet 125 resting in seat 135 to seal off passageway 130 from fluid inlet cavity 129. Upon application of a suitable vacuum to actuating cavity 126, flexible diaphragm 109 overcomes the downward force of spring 120 forcing circular disc member 117 and valve poppet 125 upward in a vertical direction to open the valve and permit fluid to pass from inlet cavity 129 through passageway 130 to discharge port 102. Thus, it will be appreciated that flexible diaphragm 109 not only serves as a fluid tight sealing member separating actuation cavity 126 from fluid inlet cavity 129, but, also, functions as the valve actuator. This arrangement permits a compact design and facilitates pressure balancing of the valve assembly.

A significant feature of the valve assembly lies in the valve seat 135. That is, like flexible membrane 109, valve seat 135 is fabricated from suitable noncorrosive materials, such as Teflon TFE, as are all other surfaces of the valve assembly which come in contact with the process fluid. Similarly, valve poppet 125 is fabricated from a somewhat harder noncorrosive material, such as Kel-F. By utilizing the inherent creep characteristics of Teflon, the geometrical configuration of the valve seat 135 will always correspond to that of the harder conical projection 137 to provide a total and repeatable seal. Initially, seat 135 is machined to provide a flat surface orthogonal to a vertical line passing through point 138 of conical projection 137. Inasmuch as the initial contact area between seat 135 and poppet 125 is a line contact, the initial stress within the seat 135 exceeds the yield point of seat 135 causing seat 135 to deform to a geometrical configuration conforming to that of the conical surface 137 thereby ensuring a positive seal. Such deformation of seat 135 is permanent. Naturally the degree of deformation is a function of the magnitude of force provided by coil spring 120. Moreover, it should be noted that rotary motion of poppet 125 relative to seat 135 is restrained thereby maintaining a constant matching interface.

FIGS. 5 and 6 illustrate two alternate geometrical configurations of valve poppet 125 both of which are designed to take advantage of the inherent Teflon creep characteristics under stress. That is, both configurations are designed in such a manner as to present a rapidly increasing contact area which in turn decreases the seat stress once the primary seat has been formed.

In FIG. 5 the frustoconical undersurface of valve poppet 135 extends upwardly and outwardly from the base 142 to the outer edge 141. The inclination of the under surface 140 forms approximately a 10 angle with a horizontal plane passing through the base 142 of conical projection 137. The surface of conical projection 137 inclines at an approximately 45 angle with respect to the vertical line passing through point 138.

In FIG. 6 frustoconical undersurface 140 extends inwardly and downwardly from edge 141 toward conical projection 137 forming an approximately 15 angle with respect to a horizontal line passing through point 138. However, instead of terminating at the base of conical projection 137, undersurface 140 is separated from conical projection 137 by an undercut annular groove 143. Like the embodiment illustrated in FIG. 5, conical projection 137 inclines at an approximately 45 angle with respect to a horizontal line passing through point 138.

FRACTION COLLECTOR Referring now to FIG. 7 there is shown in perspective, partially broken away to show the interior, the fraction collector employed in the present invention. More specifically, fraction collector includes a vacuum tight cylindrical housing 166 having a cover 275 fabricated from a suitable transparent material, such as glass. A number of collection test tubes 276, ninety in all, arranged in two concentric circular rows 277 and 278, are enclosed within vacuum tight housing 166 and carried by a rotatable universal tube rack 279 which in turn is driven in a step-wise fashion, each step being equal to the distance between the centers of two adjacent test tubes, by means of a ratchet mechanism 280. Ratchet mechanism 280 comprises a rotatable gear 281 having a series of teeth around its periphery which cooperate with a reciprocally moving arm 282 driven by a motor 163. The step-by-step rotational movement of ratchet mechanism 280 is translated to universal tube rack 279 through a rotatable shaft 283 attached to gear 281 and the bottom surface of universal tube rack 279. Shaft 283 is rotatably enclosed within a bearing housing 284 by upper and lower ball bearings 287 and 288, respectively, and bearing housing 284 is in turn sealably mounted in the bottom wall of main housing 166. An O-ring 289 captured in an annular groove about the outer surface of rotatable shaft 283 provides a vacuum tight seal between rotatable shaft 283 and bearing housing 284. Each of the collector tubes 276 are filled by a delivery means including a tubular inlet 285 sealably mounted by a suitable vacuum tight O-ring seal 286 in the side wall of main housing 166. The principles for introducing fluid into the collector test tubes 276 and the automatic means used for such introduction are well known to those fmiliar with the art and may be of the type described in US. Pat. No. 3,181,- 574 to A. Lenkey et al., entitled Dispensing Head for a Fluid Fraction Collector and assigned to the present assignee. Delivery tube 285 is transported in a linear fashion between the outer and inner concentric circular tube rows 277 and 278 by a selectively energized motor 290.

In accordance with the principles of the present invention the interior 291 of the fraction collector 155 is evacuated via a vacuum line 164 (connected to rough vacuum pump 31, FIG. 1) which communicates with the interior 291 by way of a suitable connector 292 tightly fitted within internaly threaded bore 293 formed in the bottom of main housing 166. As earlier mentioned, evacuation of the fraction collector interior 291 permits the sought after residues to be maintained in an oxygen free atmosphere and assists in the rapid drying of such residues.

PROGRAMMING UNIT In FIG. 8 there is shown in block diagram form a suitable programming unit for use with the present invention. The program source comprises an endless loop punched paper tape 301, generally fabricated from Mylar, which is wound around the periphery of a metallic rotatable drum 302 connected by line 303 to circuit ground.

Typically the paper tape includes a total of forty-two channels in which instructions may be recorded by merely punching a hole at a given location in each channel. In the present invention the first two channels provide enabling signals to the circuitry of the main programmer and an auxiliary programmer, if used. Twelve of the next thirteen channels serve as timing channels in which a binary code may be entered to control the length of time the sequenator remains in each step. In the remaining channels instructions for the various control elements throughout the sequenator system are recorded to govern the function performed during each step by controlling the actuation of selected valves and/ or motors in FIG. 1.

Returning now to FIG. 8, at the beginning of each step a number representing the time duration of that step is entered in step time register 305. Such numbers are initially recorded in the twelve timing channels on program tape 301, using the familiar binary code. The time duration of each step is coded in the same line alongside the instructions to the control elements for carrying out the step function. In other words, program tape 301 not only controls the function and sequence of steps but, also, the duration of the function being performed during each step.

An electromechanical tape reader 307 cooperates with program tape 301 for sensing punched holes in the tape as the tape travels past the sensing mechanism. In practice, tape sensor 307 forms part of a commercially available unit including metallic drum 302 for moving the program tape past the sensing mechanism. More specifically, electromechanical tape reader 307 includes a series of metallic brushes (one brush cooperating with each tape channel) arranged in a row and riding along the tape surface on a line directly above metallic drum 302. Upon the occurrence of a hole, the metallic brush makes contact with the surface of the metallic drum 302 completing an electrical circuit and energizing the output line associated with the metallic brush.

In the illustrated arrangement, the twelve timing channels are in effect divided into three groups of four channels each with the output lines of the first group representing, as measured in seconds, 1, 2, 4, and 8; the output lines associated with the second group 10, 20, 40, and 80; and the output lines associated with the third group 100, 200, 400 and 800. When energized the signals appearing on the timing channel output lines are coupled by way of a series of inverting gates 304, one gate in each output line, to a binary code to decimal converter (BCD) 308 which in a well known manner translates the binary coded number appearing on the timing channel output lines to a decimal coded number. The decimal coded number is in turn fed to a comparator 309 which instantaneously compares this number with the number presently contained in step time register 305. If these numbers do not match, comparator 309 generates an output signal which is impressed upon the input of a free running multivibrator 310. Simultaneously, an inhibit signal, derived from tape drive 313 via a time delay 314, is applied to clock pulse source 311 interrupting the oscillation of this source. Free running multivibrator 310 is triggered by the signal from comparator 309 to provide a series of output pulses at a predetermined frequency which are coupled to the preset input of step time register 305. More specifically, in practice step time register 305 may take the form of a three decade electromechanical counter formed by operatively tying together three commercially 18 available single decade electromechanical counter units, which are driven in a step by step fashion by a suitable step driving mechanism. This step driving mechanism responds to the series of constant frequency input pulses provided by free running multivibrator 310 to step the time register 305 one step for each input pulse.

In the preset mode, that is, as the number representing the required time duration is being entered into time register 305, multivibrator 310 sequentially steps time register 305 until the count entered in this register is equal to the number appearing on the output lines of binary code to decimal converter 308. At this time comparator 309 removes the inhibit signal being impressed upon one second clock pulse source 311 permitting clock pulse source 311 to resume providing a series of constant frequency output pulses. These clock pulses are coupled to register 305 to count down the set time register step by step until the count entered in the register reaches zero. In the preferred embodiment one second clock pulse source 311 may take the form of a motor driven magnetic reed pulser, although, it will be appreciated by those familiar with the art that any suitable stable electronic oscillator may be used.

Upon reaching a zero count, step time register 305 develops an output signal at 0 output terminal 312 which is coupled to tape drive 313 through an AND gate 319. As mentioned earlier tape drive 313 is normally driven in a step by step fashion to sequentially carry out the functional steps necessary to successfully perform the degradation process. Each pulse signal developed at the 0 output terminal 312 causes tape drive 313 to step one line presenting the next set of instructions to the sequenator. Upon reaching the next position, tape drive 313 provides an ouput signal via delay 314 to comparator 309 whose function is to cause comparator 309 to impress an inhibit signal on the input of clock pulse source 311. The short time delay circuit 314 is interposed between tape drive 313 and comparator 309 to allow sutficient time for the circuits to sense the presence of a time code before the inhibit signal is generated. If no time code is present, step time register 305 recycles to zero and the tape is stepped to the next line. Once clock pulse source 311 is turned on, step time register 305 again counts down to zero and the entire process is repeated in the manner previously discussed.

A program step register 317 is also connected to tape drive 313 to monitor and provide a visual indication of the step number currently being carried out by the sequenator. The register 317 counts up one each time tape drive 313 indexes to the next stop. It is reset each cycle by a pulse derived from slew stop line 325.

A residue counter or register 321 is coupled to slew start line 324 and serves to keep track of the number of residues (amino acids units) which have been sequenced. In practice residue counter 321 is manually preset to the desired or expected residue number and then counts down in response to a signal derived from slew start line 324. Actually, since a slew start signal is generated prior to the commencement of Step 1 (to initially position the program tape at the location of Step 1), the residue counter always registers one less than the actual number of residues to be degradated. For example, if the counter registe-rs 20, there are in fact 21 residues to go to reach the preset number.

An elapsed timer 322 times in seconds each degradation cycle. Timer 322 is triggered a the start of each cycle by a pulse from slew stop line 325 and reset at the end of the cycle by a pulse derived from slew start line 324.

A hold circuit 316, which is manually operated, selectively breaks or disconnects the brush sensors of selected channels from the remaining circuitry. This feature gives the operator greater flexibility for varying the program Without the necessity of recoding the program previously coded on the program tape. In addition each functional element of the programmer includes a manual override,

generally a by-pass switch, which may be used to selectively disconnect such functional elements from the system. This, of course, also contributes to the over-all flexibility of the sequenator. Moreover, such selective control and manual manipulation enables the operator to easily check the proper operation of the sequenator at any step during the degradation process.

During operation the programmer tape drive 313 steps program tape 301 to the next line or step upon receipt of a signal from step time register 305 which signal is coupled to tape drive 313 by an AND gate 319. AND gate 319 is in turn controlled by an enabling signal derived from a program channel on tape 301. In the absence of a hole in the programming channel, no enabling signal is impressed upon AND gate 319 and the programming unit is turned ofi since it is now unable to index to the next step. It will be noted that the enabling signal is coupled to AND gate 319 by a NAND gate 315. Consequently, if a hole is punched in the auxiliary programmer channel (located alongside the main programmer channel), as well as the main programming channel, NAND gate 315 blocks the enabling signal to turn off the main programmer while the auxiliary programming unit, to be presently discussed, takes over.

As mentioned earlier, tape drive 313 is normally driven in a step-wise fashion to move program tape 301 step by step from line to line past tape sensor 307. On occasion, it is desirable to drive program tape 301 in a continuous fashion. More specifically, at the end of each degradation cycle the program tape must be recycled to Step 1. To this end there is provided slew start and slew stop channels on the program tape. Tape drive 313 upon sensing a signal on slew start line 324 goes into a continuous mode moving program tape 301 rapidly past sensor 307. The program tape continues to move in this fashion until a signal is provided on slew stop channel 325 which breaks tape drive 313.

Moreover, the slew mode of tape drive 313 comes into play when an auxiliary programming unit 323 is operatively tied into the main programmer. Auxiliary programmer 323, which may take the form of a teletype tape recorder or similar apparatus, in effect serves as an address register in which instructions for controlling the main programmer are entered. More specifically, program tape 301 includes four channels each of which, in effect, serves as a separate program channel. That is, each channel contains a series of punched holes with each hole being aligned with an corresponding to a line (step) coded on program tape 301. Each hole functions to provide a slew stop signal to tape drive 313. In practice, when the auxiliary programmer is used, a series of additional lines (five sub-lines) are provided following each step in the program which one might desire to vary in some fashion, such as the coupling or cleavage steps. The first subline of each group includes a slew start code to switch tape drive 313 to the slew mode while the remaining lines may be coded, for instance, to provide different reaction times. The auxiliary programmer 323 then selects one or more of these times. In this manner the main program may be selectively varied without recoding the program tape 301.

Thus, in operation when a hole is punched in the auxiliary programmer channel (alongside a hole in the main programmer channel) the main programmer is turned 011, since an enabling signal is blocked by NAND gate 315, and the auxiliary programmer 323 commences operation. Auxiliary programmer 323 is triggered by a signal derived from the auxiliary programming enabling channel. Auxiliary programmer 323 then provides an enabling signal to one of the AND gates 328, 331 to select one of the four channels coded on tape 301. Simultaneously, a slew start signal is impressed upon tape drive 313 via slew start line 324 to switch drive 313 to its slew mode. Program tape 301 is then rapidly driven past tape sensor 307 until a punched hole in the selected channel (or a hole in the permanent slew stop channel) is encountered which 20 hole provides a slew stop signal to break tape drive 313 via the selected AND gate (328, 331), OR gate 326 and slew stop line 325. Once stopped the programmer resumes its normal step by step sequence until another slew start and auxiliary programmer code is encountered.

Alternatively, program tape 301 may include an address code (or such an address code may be derived from program step register 317) to identify each line (step) entered on the main programming tape 301. As mentioned earlier, when a hole is punched in the auxiliary programmer control channel, the main programmer is turned off and the auxiliary programmer commences operation. During this period program tape 301 is rapidly driven past tape sensor 307 until the address code contained in the auxiliary program channels on program tape 301 matches an address code entered in the auxiliary programmer unit 323. Once the selected line or step is reached, a stop signal is generated which is coupled over slew stop line 325 via OR gate 326 to break tape drive 313. At this time the main programmer is again turned on (by a signal derived from the programmer enabling channel) and functions in the manner previously described. Naturally, any number of addresses may be entered in the auxiliary programmer to select any line or step coded on the main programming tape in any predetermined sequence. Thus, as previously noted the auxiliary programmer provides a high degree of flexibility to the programming unit enabling the operator to vary the main program at will without the necessity of recoding the original program tape 301.

Still another alternative is to provide a series of separate programs on tape 301 with each program being identified by a separate code. The auxiliary programmer then selects one of the programs from the series.

OPERATION While for purposes of illustration the operation of the present automatic protein/ peptide sequenator will be discussed in connection with the so-called phenylisothiocyanate degradation scheme, it will be appreciated by those familiar with the art with the present invention may successfully carry out any degradation process by merely varying the program input accordingly. Generally speaking, the phenylisothiocyanate degradation scheme contemplates the formation of a phenylthiocarbamyl derivative of the protein or peptide sample being investigated and the splitting off of the N-terminal amino acid as thiazolinone. Basically, this process involves three distinct reactions commonly referred to as (1) coupling, (2) cleavage and (3) conversion. The latter reaction merely converts the 2-anilino-5-thiazolione derivatives to the isomeric 3-phenyl-2-thiohydantoins (PHTs) since it has been found that the extracted thiazolinone derivatives are much too unstable to render them suitable for identification purposes. In fact, it has been found easier and more convenient to carry out the conversion reaction on a number of extracted thiazoliones simultaneously, although, of course, it may be done sequentially, if desired. For this reason the conversion reaction has been left out of the automatic protein/peptide sequenator. In other words, the sequenator of the present invention performs only the coupling and cleavage reactions of the phenylisothiocyanate derivative scheme. Naturally, the conversion reaction could be incorporated into the sequenator, if desired. Typically, after conversion, the residues (amino-acids) are identified by gas chromatoggraphy using apparatus of the type manufactured by Beckman Instruments, Inc. and designated Model GC5.

In the coupling step the protein or peptide sample is first exposed to a coupling medium consisting of an alkaline buffer, and phenylisothiocyanate, to form the phenylthio carbamyl derivative. After completion of this coupling reaction excess reagents and by-products are washed away by solvent extraction and the residual extracting solvent is subsequently evaporated. In the cleavage step, the thiazolione is cleaved off by exposing the phenylthiocarbamyl derivative to an anhydrous acid and subsequently extracted by a suitable organic solvent. The protein or peptide sample remaining in the reaction cell is then dried by a combination nitrogen circulation and vacuum to prepare it for a succeeding degradation cycle.

In the operation to be described the following reagents and solutions were used:

Reagent R15% (v./v.) solution of phenylisothiocyanate (PTIC) in heptane.

Reagent R2allyldimethylamine at a pH of 9.5.

Reagent R3anhydrous N-heptafluorobutyric acid.

Solvent S1--benzene.

Solvent S2-ethyl acetate.

Solvent S3--butyl chloride. I

For optimum performance it is desirable to purify the reagents and solvents to, among other things, remove any traces of aldehydes since these tend to react with the terminal amino acid group resulting in a progressive decrease in yield. Purification may 'be accomplished by any one of many known ways, such as that described on pp. 87 and 88 of the aforementioned Edman and Begg publication.

To facilitate a complete understanding of the operation of the present invention a brief discussion of the steps necessary to perform one degradation cycle follows. As mentioned earlier, each degradation cycle derives one amino acid unit so that for a protein or peptide sample containing N amino acid units, N degradation cycles are required to successfully determine the entire amino acid sequence.

For convenience each chronological step is followed by a designation of the function of that step, the time duration, and a brief discussion, with reference to FIG. 1, of the components affected.

Initially the protein or peptide sample to be degraded is placed in the reaction cell 21. Hand operated valves 57, 6'9, 75 and solenoid valves 66 and 74 are opened and nitrogen is bubbled through each of the reservoirs 58 to purge the system of oxygen and air in general. At the conclusion each of the five metering valves 75 are adjusted until'a slow vent of about 30-50 cc. fluid flow is reached. Operation of the sequenator then commences:

Step 1 Function: slew stop Time (sec.): 2

The continuous tape drive is turned off to stop program tape 301.

Step 2 Function: delay (pressure equilization) Time (sec.): 4

Motor 29 is set at its high speed of 1800 r.p.m. Under the combined influence of the centrifugal and gravitational fields, the sample migrates toward the inner wall and occupies the annular space between the free liquid surface and the reaction cell interior. Valve 56 is opened to admit nitrogen into the reaction chamber 22 and reaction cell 21 Which nitrogen serves to break the vacuum and pressurize the reaction chamber tominimize the pressure differential between the reaction chamber 22 and the reservoirs 58 and prevent a high preliminary surge in delivery of reagents or solvents (resulting in erroneous fluid quantities) to the reaction cell 21. Valve 39 is opened to connect rough vacuum pump 31 to fraction collector 155 to assist in the drying of the residues previously collected.

Step 3 Function: R1 (reservoir)-vent Time (sec.): 14

Valve 56 is closed shutting off the nitrogen flow to reaction chamber 22. Valve 74a is opened to slowly vent reservoir R1 to the atmosphere to drop the total pressure inside the reservoir below that of the incoming nitrogen.

Step 4 Function: R1 (reservoir) pressurize Time (sec.): 14

Function: R1 (reagent) delivery Time (sec.): 10

Valves 68a and 74e remain open so that nitrogen continues to flow into reservoir R1. Valve 56 is opened permitting nitrogen to flow into the reaction chamber 22. Valve 8% is actuated connecting rough vacuum pump 31 to connecting line 83!; opening valve unit 105a allowing reagent to flow from reservoir R1 through feed line 28a to reaction cell 21. Valve 56 also permits vapor in reaction chamber 22 to escape to the atmosphere via line 46, valves 57 and 69, and vent manifolds 72 and 73, preventing an excessive pressure build-up in reaction chamber 22.

Step 6 Function: restricted vacuum Time (sec.): 40

Valve 56 is closed shutting off nitrogen flow to reaction chamber 22. Valve 89b is actuated to connect line 83b with connecting line 85a permitting nitrogen to break the vacuum and close valve unit a shutting off the flow of reagent R1 to reaction cell 21. Valve 38 is opened connecting rough vacuum pump 31 to restricted vacuum line 33 which vacuum evaporates a portion of the heptane carrier (which is used as a vehicle for carrying R1 (PTIC) into the reaction cell) from the reaction cell 21.

Step 7 Function: Delay Time (sec.): 4

Valve 38 is closed disconnecting rough vacuum pump 31 from reaction chamber 22. Valve 39 is opened connectmg rough vacuum pump 31 to fraction collector 155. Valve 56 is opened allowing nitrogen to flow into reaction chamber 22 for the purposes outlined in Step 2.

Step 8 Function: R2 (reservoir)-vent Time (sec.): 14

Valve 56 is closed shutting off the nitrogen flow to reaction chamber 22. Valve 74b is opened to slowly vent reservoir R2 to the atmosphere to drop the total pressure inside reservoir R2 below that of the incoming nitrogen.

Step 9 Function: R2 (reservoir)-pressure Time (sec.): 14

Valve 68b is opened permitting nitrogen to flow from N supply 44, through line 67b, into the R2 reservoir establishing a dynamic equilibrium pressure condition inside reservoir R2.

Step 10 Function: R2 (delivery) Time (sec.): 50

Valves 68b and 74b remain open so that nitrogen continues to flow through reservoir R1. Valve 56 is opened permitting nitrogen to flow into the reaction chamber 22. Valve 89d is actuated interconnecting rough vacuum pump 31 with line 83d opening valve unit 1051: and allowing reagent to flow from reservoir R2 through feed line 30b to reaction cell 21. Valve 56 also permits vapor in reaction chamber 22 to escape to the atmosphere via line 46, valves 57 and 69, and vent manifolds 72 and 73 preventing an excessive pressure build-up in reaction chamber 22. At this point it should be noted and emphasized that both reagents R1 and R2 are introduced into reaction cell 21 while the reaction cell is spinning at a high speed, such as 1800 r.p.m. The combined volume of R1 and R2 is controlled to reach under these conditions a level about /1 above the bottom of the cell. This ensures that the ma gents completely engulf and react with the protein sample preventing a ring of unreacted or partially reacted sample to remain and accumulate about the inner surface of reaction cell 21.

Step 11 Function: coupling reaction Time (sec.): 120

Valves 68b and 74b are closed shutting off the nitrogen flow through reservoir R2. Valve 89d is actuated interconnecting lines 85b and 83d and feeding nitrogen to valve unit 105b to break the vacuum and shut otf delivery of reagent R2 to reaction cell 21. Valve 56 is closed shutting off the nitrogen flow to reaction chamber 22 permitting the coupling reaction to proceed in an inert atmosphere.

Step 12 Function: coupling reaction Time (sec.): 780

The speed of motor 29 is reduced to 1200 r.p.m. pulling the sample and reagent down toward the bottom of the reaction cell 21.

Step 13 Function: coupling reaction Time (sec.): 900

The coupling reaction continues to fully dissolve the sample and form the phenylthiocarbamyl derivative.

Step 14 Function: restricted vacuum Time (sec.): 600

While the reaction vessel 21 is spinning at the low speed (1200 r.p.m.), valve 38 is opened connecting restricted vacuum line 33 to rough vacuum pump 31, to initiate drying of the constituents in reaction cell 21. Valve 39 is closed disconnecting rough vacuum pump 31 from fraction collector 155.

Step 15 Function: rough vacuum Time (sec.): 300

Valve 38 is closed and valve 36 is opened to disconnect the restricted vacuum line 33 and connect the rough vacuum pump 31 to the reaction chamber 22 via vacuum line 31 for continuing the drying of the constituents.

Step 16 Function: delay (pressure equilization) Time (sec.): 4

Valve 34 is closed disconnecting the rough vacuum pump 31 from reaction chamber 22. Valve 39 is opened to connect the fraction collector 155 to the rough vacuum pump 31. Valve 56 is opened to feed nitrogen into the reaction chamber 22 which nitrogen serves to break the vacuum in the reaction chamber and pressurize the chamber to minimize the pressure differential between the reaction chamber 22 and the reservoir 58.

Step 17 Function: S 1 (reservoir)vent Time (sec.): 30

Valve 56 is closed shutting off the nitrogen flow to reaction chamber 22. Valve 74) is opened to slowly vent solvent reservoir S1 to the atmosphere to drop the total pressure inside the reservoir below that of the incoming nitrogen.

Step 18 Function: S1 (reservoir)pressurize Time (sec.): 30

Valve 66a is opened permitting nitrogen to flow from supply 44, through line 59, distribution manifold 60, and line 61 into solvent reservoir S1 establishing a dynamic equilibrium pressure condition inside reservoir S1.

Step 19 Function: S1 (reagent)delivery Time (sec.): 600

Valves 66a and 74] remain open. Valve 56 is opened permitting nitrogen to flow into the reaction chamber 22. Valve 89a is actuated to interconnect rough vacuum pump 31 with connecting line 83a-opening valve unit 103a to permit solvent S1 to flow from reservoir S1 through feed line 30a to reaction cell 21. Valve 162 is opened to couple valve unit a to rough vacuum pump 31 interconnecting efiluent feed line 151 with waste bottle 153. S01- vent S1 thus extracts excess reagents and by-products carrying the latter into waste bottle 153.

Step 20 Function: delay Time (sec.): 10

Valves 66a and 74] are closed shutting 011 the flow of nitrogen through solvent reservoir S1. Valve 89a is actuated to interconnect lines 86a to break the vacuum and close valve 103a interrupting the flow of solvent S1 to reaction cell 21.

Step 21 Function: S2 (reservoir)vent Time (sec.): 30

Valve '56 is closed shutting 011 the nitrogen flow to reaction chamber 22. Valve 74g is opened to slowly vent reservoir S2 to the atmosphere to drop the total pressure inside the reservoir below that of the incoming nitrogen. Valve 90a is closed by closing valve unit 162 to disconnect eifiuent line 151 from waste bottle 153.

Step 22 Function: S2 (reservoir)pressurize Time (sec.): 30

Valve 66!; is opened permitting nitrogen to flow from N supply 44, through lines 59 and 62 into the reservoir S2 establishing a dynamic equilibrium pressure condition inside reservoir S2.

Step 23 Function: S2 (reagent)-de1ivery Time (sec.): 600

Valves 66b and 74g remain open so that nitrogen continues to flow into reservoir S2. Valve 56' is opened permitting nitrogen to flow into the reaction chamber 22. Valve 162 is opened to actuate valve unit 90a connecting effluent line 151 to waste bottle 153. Valve 890 is actuated connecting rough vacuum pump 31 to line 83c opening valve unit 103 b permitting solvent to flow from reservoir S2 through feed line 30b to reaction cell 21. Solvent S2, like solvent S1, washes the sample within the reaction cell 21 to remove excess reagents and by-products. The extraction solvent is withdrawn from the reaction cell by Way of effiuent line 151 and transmitted to waste bottle 153.

' Step 24 Function: Delay Time (sec.): 60

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