JPH0256635B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention generally relates to
Regarding improved apparatus and methods for
a protein or peptide chain containing amino acid units of
In order to determine the order of these units,
This invention relates to an automatic improvement device. Lines of amino acid units in proteins and peptides
In order to understand their biological functions and final
In other words, it can be used as a means of synthesizing chemicals that exhibit the same function.
I'm extremely interested. Determine the linear order of amino acids
Although various techniques are used to
One that has also been successful is known as the Edman method.
There must be something there. Various forms of the Edman method and
Devices for automatically carrying out these methods are available in the following publications.
It is written on the memorial. Edman and Petsug, "Protein Sequencing Device",
European Journal of Biochemistry
-, Volume 1 (1967), pp. 80-91; Whittman
- Liebold, “Improvements with automatic conversion devices
10 types of ribosomes in E. coli using an ordering device
“Study of amino acid order in protein systems”, Hotupeseira
Earth Tour Lift Physiology
E. Hemy, Volume 354, Page 1415 (1973);
Toman-Lieveold et al., “Anilinothiazoli
Improvements to the automatic conversion of non- to PTH amino acids
"A device combined with a good ordering device", Analytica
Le Biochemistry, Volume 75, Page 621
(1976); May 25, 1976, “Automatic Amino Acid Sequence”
U.S. Patent No. 3,959,307 entitled ``Determination Method''; Hunka
Pillar and Hood, “Improved Sequencing Device and Non-Protein
Quality carrier (polyprene) and high pressure liquid chromatograph
Direct integration of polypeptides using roughy
"Order Analysis", Biochemistry, p. 2124
(1978); R.A. Laursen, Europe
Journal of Biochemistry, page 20
(1971); E. Watshatter, Etsuchi Matsuharai
G. Hofner and J. Ott.
FEBS Letters Volume 35, Page 97 (1973); 1973 April
``Equipment for automatically performing chemical processes'' dated March 3rd.
U.S. Patent No. 3,725,010 titled “Placement”; February 1973
20th, titled “Method for sequential degradation of peptide chains”
National Patent No. 3717436; dated July 1, 1975 “Pepti
U.S. Patent entitled ``Protein Sequencing Apparatus''
No. 3892531; December 27, 1977 “Peptides or
A US patent entitled ``Protein Sequencing Method and Apparatus''
Permit No. 4065412. A further noteworthy device is the 1979
“Chemical Process Implementation System” filed on December 26, 2015
U.S. Patent Application No. 106,828 entitled
It is described in. Briefly, what was considered in the above publications
As shown, Edman's sequential degradation method consists of three steps.
coupling and cleavage
(cleavage) and conversion (conversion).
Ru. In the bonding step, phenylisothiocyanin
nate reacts with the N-terminal α-amino group of the peptide.
to produce a phenylthiocarbamyl derivative. Open
In the cleavage process, phenyl chloride is removed using anhydrous acid.
Animation by cleaving the ocarbamyl derivative
Linothiazolinone is produced. Thiazolinone
After extraction, the remaining peptides are used for the next cycle of conjugation and
and cleavage reactions. Chilled using aqueous acid solution
Conversion of azolinone to phenylthiohydantoin
For example, if this is used in a chromatography
It can be analyzed by any suitable method. The automation device of U.S. Pat. No. 3,725,010, and even
Whittman-Lieveold's paper cited in
and the automatic improved in U.S. Patent Application No.
The converter is usually known as a "rotating cup".
on the inner wall of a rotating reaction cell placed in a sealed reaction chamber.
In the formed thin film, there is a spontaneous reaction that causes a reaction.
This is an activation order determining device. of the adjusted amount for the room.
Provide means for introducing and removing liquid reagents to ensure inert
react with a protein or peptide sample in a
let The sample to be analyzed is first placed in a rotating cup.
and then the necessary components to carry out the coupling and cleavage reactions.
Various reagents and solvents are sequentially introduced and removed.
It will be done. When the cup rotates, what happens to the liquid reagent and solvent?
It forms a thin film on the cup wall by itself and
It penetrates into the thin film and reacts with it. Reagent is sample thin
Dissolve the membrane and perform the Edman bonding and cleavage steps.
carry out. After the binding and cleavage steps are complete, the reaction chamber is
Evacuate to remove volatile components of reagents. Discharge after joining
Subsequently, the remaining sample film is extracted with a solvent.
This removes non-volatile components. Exhaust after cleavage
Subsequently, the formed thiazolinone was tested with a solvent.
Extract the material from the thin film and transfer it to another flask for conversion.
process or collect various fractions and dry them.
Transfer to drying equipment. The conversion process is carried out in a conversion flask.
If not done immediately, this step will be repeated many times later.
can be done simultaneously for the fraction.
Ru. The introduction and withdrawal of fluid from the rotating cup is
Seal the opening in the upper wall of the reception room and enter the cup from there.
A fluid conduit that passes through a stop that hangs down to a certain point.
achieved by. The fluid flows into the rotating cup.
It is introduced directly at the point adjacent to the bottom and the cover is
Removed from the annular groove on the cylindrical inner surface of the spruce.
It will be done. The cup rotates at high speed to remove the fluid to be extracted.
When it is forced into the annular groove by centrifugal force,
Extraction via a conduit with an inner end protruding into the
It will be done. Therefore, this outflow conduit
scoop for removing substances and by-products as well as extraction solvents
Acts as a release device. The protein or peptide sample itself in the rotating cup device.
The body does not have enough to form a cohesive film.
If the
A relatively thick layer of non-protein carrier material during agent extraction.
This will be done within the fee. Then the binding and cleavage reactions
carrier material during the reaction process so that
and the sample are dissolved in a liquid reagent. chemical composition
1,5-dimethyl-1,5-diazaundecamethy
Polymer quaternary polymer with polymethobromide
Monium salts have been used for this purpose. sample
To hold it securely, a considerable amount of carrier must be used.
The carrier and sample must both be liquid samples.
A reaction occurs between the sample and reagent by dissolving it with the drug.
It is shown to be possible. Rotating cup-type devices often have acceptable practicality.
Although it sometimes provides experimental results, it has some drawbacks.
do. For example, if the reagents primarily used are in liquid form,
However, since it must be used in large quantities, it is not suitable.
Obtain a large amount of protein or peptide sample and
The cost of ensuring sufficient supplies of the necessary reagents is extremely high.
Stick to it. The liquid sample and solvent are connected to the cup wall.
The sample part is separated from the thin film as it passes over the
It has a tendency to wash away from the walls of the spout, and
The yield of terminal amino acid units obtained in subsequent cycles is
reduce Therefore, enough samples are available for the last sample.
Ensures that the product stays up to the full cycle and yields useful results.
The initial amount of sample must be large enough to
stomach. Furthermore, this type of device has a small reaction chamber volume.
Drying large and existing samples under reduced pressure
Repetitive removal of semi-volatile liquid reagents and solvents by
quite long cycle times due to the need to
I need. Furthermore, the rotating cup ordering device
Both manufacturing and repair are extremely complex and expensive. Different types of ordering devices are used in the above-mentioned Laursen and
In this case,
The sample is immobilized on the surface of multiple small beads by covalent bonding.
be activated. These beads are housed in a reaction column.
to form a porous packing, and the liquid reagent is introduced into this reaction column.
to carry out chemical processes. Opening to use
Cleavage reagent is an excellent solvent for proteins and peptides.
To hold the sample in place, covalent bonds are
must be complete. However, sharing
Coupling is difficult to achieve in practice. In addition, the packed tower is
It is difficult to clean and the beads present are in use.
tends to collapse. Conventionally, a sample is placed in a static reaction chamber and this sample is
at least one reagent in gas or vapor form;
The shortcomings of these devices can be overcome by
A sequence determining device designed using this method has been proposed. the above
U.S. Patent Nos. 3,892,531 and 4,065,412 are such
Two types of devices are disclosed, both of which are sufficient.
It doesn't work satisfactorily. The device of U.S. Pat. No. 3,892,531 is closed within the reaction chamber.
It has finger-like extensions that allow the peptide young to
Alternatively, a protein sample is sequentially exposed to a gaseous reagent and a solvent.
Maintain at controlled temperature during exposure. Guide the reagent
Each time it is inserted, the extension is cooled from the inside and condensed
causes shrinkage. Next, heat the extension part and insert the sample.
Dissolve in liquid and allow reaction to proceed. sample and anti-
After the reaction, undesired semi-volatile chemicals are removed by heat and
drying from the sample in combination with an active gas flow;
Alternatively, the terminal amino acid can be removed by solvent condensing on the extension.
It can be cleaned until it drips from the extension along with the acid.
can. In the apparatus and method of U.S. Pat. No. 4,065,412
The protein or peptide sample is chemically bonded or
Many small macropores in the reaction tower are removed by direct adsorption.
Both the inner and outer surfaces of the case are coated. each
Species reagents and solvents in either gaseous or liquid form
The desired degradation reaction is caused by sequentially passing through a packed column.
Ru. The flow of reagents and solvent to the reaction column is
Controlled by a rotating face sealing valve. Unfortunately, these patented devices suffer from a large number of
cycle to achieve acceptable results.
do not provide an adequate pollution-free environment for for example,
In these devices, protein or peptide samples are
Difficult to clean effectively. Is the solvent the sample?
The test is carried out by condensing the solvent to the point where it drips.
The above method of cleaning materials removes traces of various reaction products.
amount left on the sample and contaminates subsequent chemical reactions
Tend. Similarly, holding the sample inside the reaction column
The filling used for the
Completely transfers chemical products into and removes them from the column
It is difficult to clean because it has to be cleaned. This is a large number
This is not easily done even with the use of large amounts of solvent. what
Therefore, the solvent should be
The gap between the beads without passing through the small holes inside the beads.
This is because it has a tendency to pass through. These devices
Fluid supply lines and flow valves should also be adequately vented.
is difficult and has a tendency to trap chemical residues,
These residues will be used in subsequent reaction cycles.
may interfere with the desired chemical reaction. In addition, the glass used as packing in the reaction column
Glass or plastic beads have a large number of degraded
This product has a tendency to disintegrate during use in bicycles.
a point that prevents the flow of fluid through a device system of
It can even block the area. Thus, in practice
It becomes impossible to clean the system between columns and
contaminated to such an extent that chemical reactions in the water are no longer possible.
It becomes like this. Contamination caused by some of the factors mentioned above
progresses cumulatively during the continuation of the sequential deterioration process.
Ru. Therefore, what are the samples and reagents in the reaction cell?
Becoming increasingly contaminated, the desired bond and cleavage
It prevents reactions and many undesirable reactions.
cause to cause Thus, each completed cycle of the device
The yield from the oil is reduced and a range of contaminants are introduced into the flux.
Go inside Yon. The yield is determined by e.g. disintegration of the filling, cleaning solvent
Dissolution of the sample into the sample and suction between the packing and the sample
Direct removal of samples due to various reasons including bond breakage
It is further reduced by direct losses. These effects occur when large amounts of protein or peptide samples are detected.
If resources are available or if the chain has a relatively small number of
It can be overlooked if the person only has the position of
However, if the chain has a very large number of units or
Very small amounts of a specific protein or peptide are obtained.
The situation becomes difficult if interferon
Both of these situations exist in the case of . This i
Interferon is a drug that responds to certain viral infections.
It is a small protein produced within human cells.
Interferon has recently received a lot of attention in the field of clinical medicine.
It's becoming a hot topic. Because this feels like a virus
It is expected to be an effective substance that suppresses the
This is because it seems to be extremely promising as a cancer drug. stomach
Interferon is produced in very small quantities and
It is only available in small quantities. Currently, there are two types of
and the practical worldwide life of interferon.
It produces a large amount of human white blood cells (leukocyte interferon).
cells) or certain human tissue culture cells (fibroblasts)
There are relatively few places where you can get Interferon).
Limited to advanced countries. This constrained interfero
Due to its production capacity, well-controlled clinical research and
fundamentals of how the molecules of the virus act.
It is difficult to perform basic analysis. further situation
Complicating matters is that interferon is about
Consists of a chain of 150 amino acid units, making it easy to analyze
In order to do this, these units must be individually cleaved from the chain.
It must not happen. Pollution losses of the above types
is a problem because the amount of protein that can be used is extremely small.
The first few amino acid molecules in interferon
This prevents the ordering of positions. First few cleavage cycles
Beyond the sample size, small samples will no longer produce significant results.
It becomes contaminated to the point where it becomes unobtainable. Various thiazolinones cleaved from samples
Converts to stable phenylthiohydantoin
The most sophisticated conventional device known to the applicant for
is based on the above paper by Whittman-Lieveold.
As described and cited above, U.S. Patent Application No.
A conversion flask like the one improved in No. 106828.
Ru. However, the applicant has determined that the reagent and solvent are
When introduced through a suitable capillary, this flask
Ineffective cleaning of the inner wall may cause damage.
I knew. Reagents and solvents with a flow of inert gas
In order to spray the liquid on the flask wall by supplying
It has been suggested that the flask walls can be cleaned more easily.
Ru. However, this technique does not allow liquid to enter the flask.
This results in a turbulent flow of liquid, making it difficult to adjust the liquid supply capacity.
Make it extremely difficult. Furthermore, the applicant has discovered that
Significantly reduces the dimensions of traditional conversion flasks to accommodate
During the conversion reaction, the contents should be stirred and
in the liquid in the flask to evaporate semi-volatile components.
Optimal distribution of inert gas bubbles throughout the container
I found that it becomes difficult. files for these purposes.
The inert gas introduced into the bottom of the lask is relatively large.
It has a tendency to rise to the surface of the liquid as large bubbles.
rather than stirring the liquid uniformly at the top of the flask.
This promotes liquid splashing. Therefore, in many applications, e.g.
chemical processes such as white matter or peptide ordering
Efficient and minimal equipment for implementation
Maximize the ordering cycle to the maximum extent possible even with soil contamination.
A setup that can be carried out successfully with a small amount of sample.
It is desired to provide a suitable location. Broadly speaking, the present invention provides an internal structure that defines a reaction chamber.
chamber means with a surface, the chamber pressurizing the fluid;
Inlet and outlet means for conducting as a flow
Be prepared. In addition, it is permeable to multiple fluids by diffusion.
Solid matrices that are permeable and located indoors
Tx means included. During this matrix means
The sample embedded in the chamber is immobilized and passed through the chamber.
These are exposed to any of the multiple fluids that cause
reacts chemically with The chamber means uses a solid matrix means as a thin film.
may include surface means for supporting and
For example, 1,5-dimethyl-1,
5-Diazoundecamethylene borimethobromide
or (N,N-dimethyl-3,5-dimethylene-
Polymer quaternary polymers such as piperidinium chloride)
It can be composed of ammonium salts. The surface means may be used as a whole or part of the inner wall of the chamber means.
It can be configured and also the chamber can be arranged almost horizontally
A porous system that extends across and is permeable to fluids.
It may also consist of a means of access. Sheet means are multiple
It consists of sheets made of several glass fibers.
I can do that. The chamber means have respective first and second chambers on opposing engaging surfaces thereof.
Consisting of a pair of adjacent chamber elements each having a second cavity and a second cavity.
and these first and second cavities are
They are aligned with each other to form a reaction chamber. 1st and 2nd
The hollow portion moves in the direction away from the engagement surface.
Tapered to a point where they communicate with the inlet and outlet means, respectively.
can be attached. Using porous sheet means
If the
the first and second cavities substantially
It is kept indoors with an orientation that separates it. room means
at least one of the mating surfaces sandwiched between mating surfaces in sealing relationship.
can also include a sheet of flexible material;
This flexible material is permeable to multiple fluids.
Ru. Also, at least one of the chamber elements has its engagement surface
with a raised portion extending around the cavity on the surface
This allows sheets of flexible material to be
presses against the bare mating surfaces, thus creating a sealing effect.
Improve. The inlet and outlet means each extend through the chamber element.
porous sheet means extending and at its inner end;
from a pair of capillary passages communicating with the reaction chamber on either side of the
Can be configured. These capillaries are the reaction chambers
coaxial and from which the almost flat exterior of the chamber element
Extending to the outer capillary opening at the surface
Can be done. The means for sequentially passing a plurality of fluids through the chamber includes:
Two with multiple nearly flat valve areas on the surface
and a first opening formed continuously between the ends of the
a first passage communicating with each of the valve sections through the
and one of the valve sections through the second opening, respectively.
a valve block comprising a plurality of second passages communicating with the
and a plurality of elastic and
Each diaphragm includes a substantially impermeable diaphragm.
The earphragm is pressed against one of the valve sections and
a second opening that closes the first and second openings communicating with the site;
1 sealed state and the valve block is pulled away from the area.
the first opening and the second opening through the exterior of the locking means;
and a second state providing a fluid flow path between the
and thereby the fluid between the first passage and the second passage
Flow can be selectively adjusted. Connection means
includes at least one inclined ferrule, which
is tightly fitted around the pipe member with sliding engagement.
in a valve block means communicating with one of the passageways;
ferrule.
This inward force increases the ratio between the ferrule and the recess.
The fluid is concentrated in a relatively small contact area between them.
Make sure to bring the seal. The recess is preferably
The ferrule is inclined at a larger angle than the ferrule. difference
Additionally, the connecting means connects the first passageway to another part of the device.
A mounting part is provided at one end of the first passage for connection.
and thus the first passage can be connected to this attachment point.
and the second passage farthest from the mounting part.
Manifold that can fluctuate body flow
It acts as. The first passage has a sawtooth shape.
Communicate with each valve site at alternating intersections
A plurality of devices connected end-to-end to form a conduit.
It can consist of a straight path. The device has internal parts for introducing and removing various fluids.
Conversion flask with multiple capillaries that plunge into the section
and at least one of these capillaries
The book has a closed hole and multiple radially spaced
Contains an inner end with a restricting orifice and
The fluid flow through the capillary is directed towards the inner wall of the flask.
A spray action is applied to the inner wall to clean it. difference
Furthermore, it terminates close to the bottom of the flask.
The capillary tube has a closed end and is adjacent to the closed end.
Features multiple radially spaced restriction orifices
Thus, the inward flow of gas through the capillary passage is complex.
Stir the liquid in the flask by generating several small bubbles.
Or accelerate its drying. Sequentially perform chemical processes on a chemical sample
The method of the present invention is to apply diffusion to multiple fluids.
Embedding the sample in a solid matrix that is more permeable
A solid matrix is placed inside a closed chamber with an inlet and an outlet.
Encapsulates Trix and allows multiple fluids to flow under pressure into the chamber
The sample is exposed to each fluid from inlet to outlet as
The sample is then immobilized.
chemical interaction between sample and fluid
It is based on achieving. solid matrix
The base can be supported as a thin film on the inner wall of the closed chamber.
I can do that. Another method is to use solid matrices.
The gas can also be supported on a porous sheet,
This is the fluid that passes through this sheet from the inlet to the outlet.
between the entrance and exit so that the sheet passes through the
extending substantially across the chamber at an intermediate location. solid ma
The process of embedding the sample in the matrix consists of a solid matrix.
The lithics are applied as a thin film to the supporting surface and then
A solution containing the sample is applied to this thin film, and the sample solution is
Consists of the process of applying Trix to dissolve it.
be able to. The liquid is then allowed to evaporate from the solution.
to leave a thin film in which the sample is embedded. The objective of the present invention is to minimize the
chemical processes with minimal sample loss and minimal system contamination.
Provides an apparatus and method for sequentially implementing the steps
That's true. Furthermore, the purpose of the present invention is to
chemical processes using minimal amounts of reagents and solvents.
Economical apparatus and method for sequential implementation of applications
The goal is to provide the following. Another object of the invention is to carry out chemical processes sequentially.
modifications with extremely short cycle times for application.
The objective is to provide a good device and method. Furthermore, it is an object of the present invention to
chemically applied to the sample for more effective cleaning.
Improved equipment and method for sequentially carrying out the process
It is to provide law. The apparatus and method of the present invention are used in degradation processes.
It is permeable by diffusion to both reagents and solvents.
inside a solid matrix formed as a thin film.
by immobilizing protein or peptide samples.
has solved many problems associated with conventional ordering devices.
Decide. Thus, the difficulty of achieving complete covalent bonding
Difficult problems are avoided, as well as ensuring that the sample remains on the support surface.
directly adsorbed to the mobile liquid phase and exposed to mechanical shear forces in the mobile liquid phase.
Avoids sample loss problems encountered when
be done. The sample is firmly fixed in the matrix
On the other hand, smaller reagents and solvents and amino
Acid derivatives are substances that can diffuse into the matrix.
and to carry out various steps in the degradation process.
effectively dissolves in the matrix to a sufficient concentration
be able to. The solid matrix is exposed to a gaseous reagent.
It holds the sample extremely effectively even when exposed to
In fact, the surface of the sample support of any shape can be
can be used without any sample loss. reaction
The inner wall of the chamber itself can be used as a sufficient support surface.
can. Support surface that significantly facilitates thorough cleaning of the system
is made of multiple overlapping glass fibers and is
It is a porous sheet that extends across the reception room. Many
Porosity is provided by the voids between the fibers. This structure
Even the smallest dimension in the direction of fluid flow is relatively large.
It has a large total surface area. Solid matrix is fiber
A thin film is formed on the surface, and the reagent and solvent are in the thin film.
Easily diffuses and reacts with buried samples
make it possible. This requires minimal amounts of reagents and solvents.
chemical processes and cleaning cycles in a relatively short time using
This makes it possible to carry out tests on samples. trial
Drugs and solvents, some of which are in gas or vapor form
However, it passes through the thin film and comes into contact with the sample.
React as fully and effectively as possible. Relatively thin forms of the porous sheets disclosed herein
Additionally, a relatively small amount of solvent removes residue from the sample.
Improves ability to thoroughly wash distillate reagents and reaction products.
Melt. Solvents separate reagents and reaction products into systems.
A relatively short distance from the surface of the sheet to be removed from
Just move it. Pieces that have come off the sheet surface
From there, they can be easily guided outside to collect the sample.
It can be prepared for the next reaction step. Small
Using less solvent will not only save you money on solvent costs.
and the tendency for sample to be washed out of the reaction chamber and lost.
It also reduces Furthermore, the use of gaseous or vaporous reagents
also contributes to complete exposure of the sample to the reagents and ensures
Minimize the amount of reagents required. use of reagents
Less is used in Edman degradation techniques
The reagents used are extremely sensitive to contamination and are therefore extremely expensive.
It is important because In addition, the sample and
The solid matrix with which it is dissolved by the gaseous reagent
sample by separation of the sample from the support surface.
Eliminate loss problems. The reaction chamber of the present invention can test both gaseous and liquid reagents.
The sample is passed through a porous sheet holding the sample.
material is contaminated by external impurities or by one reaction step or service.
carried from one cycle to the next process or cycle.
Constructed to prevent contamination from chemicals
Ru. For example, if two abutting chamber elements are combined
The first and second cavities are constructed in each example of the porous sheet.
A low volume reaction chamber is formed. each from the room itself
A pair of capillary passages extending in opposite directions through the chamber element.
A channel is a channel in which multiple fluids in gas or liquid form flow under pressure.
through the chamber and through the sample matrix.
enable. The small volume of the room and passage
Minimizes the amount of reagents and solvents required and
Facilitates vacuum drying of the system between cycles. 2
The two chamber elements have these mating surfaces at their mating surfaces.
by a sheet of flexible material sandwiched between surfaces
Sealed. This material allows multiple fluids to pass through the chamber.
gas permeability, which actually allows the gas to pass through more evenly.
By dispersing it into contact with a porous sheet.
Improve the flow of gas through the chamber. Another embodiment of the reaction chamber is a single capillary tube or a capillary tube.
A tubular passageway on its internal surface or hole
has a solid matrix formed as a thin film on
Ru. Place the protein or peptide sample on a porous sheet.
Embed it in the matrix in the same way as the case, and add the reagent and solvent.
are passed through the capillary tube sequentially to interact with the sample.
let The above chamber structure does not have a porous sheet member.
can be used for this purpose. Next, try
The material-containing matrix is applied to the surfaces of the first and second cavities.
Let it form on top. Similarly, supporting solid matrices
The surface that holds the reagent and solvent fluids in the matrix
It can be made by virtually any method that allows it to be poured on top.
can be done. Introduction and extraction for both chamber and conversion flask
The novelty of the present invention for regulating the flow of fluids
Valve assemblies are designed to avoid cross-contamination of fluids.
specially made for. Each valve means a single conduit
This single conduit faces other conduits.
Selectively connect to each of the conduits. single conduit
communicates with one end of the first passage in the valve block.
while each of the other conduits
Connected to one of the two passages. In the normally closed state,
Each of the diaphragms covering each valve part
The first port is pressed against the surface of the valve block by the
prevent communication between the passage and the second passage leading to the specific valve part.
Stop. Fluid communication between a single conduit and other conduits is
Applying a vacuum to one or more of the earphragms
while pulling the diaphragm away from the valve part.
The valve block that exists between the openings for each passage directs the fluid.
It can be applied selectively by pouring it onto the surface of the
I can do it. a valve at the remote end of the first passage;
A second passageway leading to the valve provides each fluid to the valve.
For example, inert gas
Can be connected to a pressurized source of flushing fluid
can. Thus, the supply valve for a particular reagent or solvent
Apply reduced pressure to the corresponding diaphragm and introduce it into the reaction chamber.
diaphragm at the distal end of the first passage.
Open the ram to remove any remaining reagent or
Complete supply by forcing all solvent into the chamber
It can be done. This is the continuity of the first passage
This makes it possible to form a manifold.
before the next reagent or solvent supply begins.
Certain reagents or solvents are purged. against the reaction chamber
If there is a valve at the outlet, use the first passage as the outlet.
while connecting the second passage to each conversion flask.
connection to the vacuum source and the waste section. First aisle connection
continuity allows complete evacuation as well as cycle
semi-volatile substances may be trapped there.
virtually eliminates the possibility of The "sawtooth" shape of the first valve passage disclosed herein is
Traditionally used in other valve assemblies in the field of ordering devices
has been done. However, if the applicant is aware of
Traditional sawtooth valve assemblies with
It slides back and forth between the communicating and non-communicating states of the passageway.
to the valve area on the main valve block so that the
Incorporates multiple individual blocks mounted
Ru. Sliding blocks have a tendency to wear and are exposed to the atmosphere.
This causes leakage both to and from the passages. here
The novel valve assembly disclosed in
The Nifold is combined with a series of diaphragms to
Establishing and blocking flow between pairs of openings that communicate with channels
This solves the problem of wear. diamond
The flam is a commercially available fluorocarbon polymerized
Can be made from virtually inert materials such as the body
It functions over a long period of time without deteriorating.
Additionally, conventional means of connecting the valve passageway to external conduits
tends to exert excessive pressure on the sides of the valve block.
Yes, promoting distortion of the upper sealing surface of the valve block
At the same time, it also promotes the loss of sealing ability. especially,
Conventional sawtooth valves known to the applicant
The associated nearly flat flange surface of the valve block
A series of seals between a pair of smooth surfaces pressed against the sides.
Connect the external conduit to the valve passage by making a stop
do. Each of these parts cannot be actually machined.
For example, fluorocarbon polymerization, which is extremely difficult to
Made of body-like material, each sealing surface can be
to form the required seal.
You have to put a lot of pressure on it. Pressure is valve block
of its upper sealing surface.
causes distortion. The valve assembly of the present invention is independent of the valve block.
The valve is designed to seal without applying excessive pressure.
Partially into recesses of different tapers in the block
Equipped with multiple tapered ferrules that can accommodate
Ru. A relatively small sealing force can be applied to specific parts of the ferrule.
to focus on the fuel without distorting the block.
The tapered recess corresponding to the hole is sealed. Surfaces with different tapers on each side of the ferrule
If an interface is provided between the
improve. This between surfaces of different tapers
The seed interface is preferably shaped to obtain optimal sealing properties.
Provided on both sides of the ferrule. The conversion flask of the present invention has an inner wall of the flask.
introducing the reagent and the force agent in the form of an impinging spray;
Walls contain all chemicals that may be condensed or scattered there.
water into the liquid in the flask.
make it possible. The main source of cross-contamination of the system between cycles
The cause is thus removed. Additionally, conversion flask
gas moves upward in the liquid in the form of small bubbles.
Stir the liquid uniformly and dry its semi-volatile components.
and excessively vigorous bubbling.
sample loss due to scattering and scattering.
stomach. Is this rapid release from sensitive amino acid derivatives?
Promotes gentle liquid removal. Thus, amino
Used to transfer acid derivatives into conversion flasks.
The above-mentioned Hoytman-Lieveall solvent was
It takes a shorter time (5 to 10 minutes) than in the de-patent.
for 1-2 minutes) and at a lower temperature (50-80℃).
It can be removed at 40-50℃). this
For example, serine, threonine, histidine,
The most disturbing like arginine and tryptophan
This significantly improves the yield of specific amino acid derivatives.
The reagents used in the conversion flask should be kept under inert gas.
Microscopic flow of bubbles and Hoytman-Lieve-O
instead of the 30-40 minutes required in the
Can be removed in combination with reduced pressure for 3-5 minutes
I can do that. Hoytman-Leave Older
In the method, the drying of reagents is
The cycle time is less than or equal to the total cycle time of the first reaction chamber.
in the conversion flask to meet the requirements such as
Begin immediately after introduction to amino acid residues.
Must be. The acid component of the conversion reagent, i.e.
Fluoroacetic acid or hydrochloric acid is dissolved
This acid evaporates much more than water.
Ingredients are Hoytman-Leaveold drying process
water is removed quickly during most of the conversion process.
Do not leave amino acid derivatives in anything other than solution.
have a strong tendency to This includes glycine and proline
The incomplete transformation of derivatives of and the decomposition of other derivatives are also
Tarasu. In the conversion device of the present invention, a conversion reagent
is sufficient time for complete conversion, i.e. 30-40 minutes.
Continue contact with the amino acid derivative for 3 hours, then
Can be quickly dried within ~5 minutes, but
It is also possible to scatter the sample all over the interior of the conversion flask.
There is no fault. Hereinafter, specific examples of the present invention will be explained with reference to the drawings.
explain. Referring to the drawings, FIGS.
A device embodying the invention is indicated generally by the reference numeral 10.
It is. The device 10 includes a chamber device 12 and a conversion
It includes a scope 14 and a fraction collector 16.
each of which is operated by an automatic control device 18.
be done. Array of pressurized solvent and reagent reservoirs
20 through a bank 22 of diaphragm type flow valves.
It is connected to the reaction chamber 12 and the conversion flask 14. No.
The second valve train 24 connects the conversion flask 14 and
and other locations. third
A diaphragm valve 26 in the column connects the conversion flask and flask.
Dispose of the cushion collector or connect it to a vacuum means.
And from flask to fraction collector
It acts to regulate fluid flow. The filtered inert gas source 28 is highly purified.
An inert gas, preferably argon, is supplied to the apparatus 10.
supply, pressurize solvent and reagent reservoirs, and oxygenate
Sweeping air from the equipment system and evacuating the system at various times
Accelerate the drying process of reagents and solvents within. pressure adjustment
A bank of valves and instruments 30 stores each solvent and each reagent.
Gas pressure to the tank as well as other parts of the device 10
The role is to adjust the pressure on each individually.
vinegar. The operating mode of the device 10 is shown in FIG. Room equipment 1
2 is present within the heated environment 32, and in a preferred embodiment
In this case, the inlet and outlet passages 36 and 38 are
Reaction chambers 34 communicating with each other are defined. entrance passage
36 connects the plurality of reservoir rows 2 via fluid conduits 40.
0, i.e. reagent reservoirs 42, 44, 46 and 48
and connection to solvent storage tanks 50, 52 and 54.
I can do it. Storage tanks 42 to 54 are inert gas pressure sources.
28 provides individual gas pressure regulators 56 and solenoid valves 58
Pressurized through the column. Storage tanks 42 to 54
Each further includes an individual flow valve 62 and an individual flow valve 62.
of the fluid level therein via the regulator 64.
Connect to the waste trap 60 at the upper part
Can be done. Introduced into the storage tank from an inert gas source 28
The pressurized inert gas thus flows into the flow regulator 64.
evacuation to the evacuation trap 60 at a more controlled rate.
can be done. The outflow amount of the storage tanks 42 to 54 is
Continuous manifold connected at one end to fluid conduit 40
via a diaphragm valve 66 that communicates with the
Individually adjusted. Thus, the pressurized storage tank 42 to
Fluid from 54 is directed by selective actuation of valve 66.
is transferred to the reaction chamber 34 via the tube 40 and the chamber inlet passage 36.
can be sent. Assists in the delivery of reagents and solvents
At the same time, after the supply, the manifold 68 and the conduit 4
0 and an inert gas source 28.
The pressurized inert gas from the pressure regulator 70 and the diamond
the end opposite the conduit 40 via the flam valve 72
It can be introduced into the manifold 68 at.
Thus, actuation of valve 72 directs pressurized gas to the manifold.
68 from the outermost end and all reagents inside it.
Alternatively, the solvent may be introduced into the reaction chamber via conduit 40 and passageway 36.
Move it to 34. The fluid outlets of reservoirs 44, 46 and 48 have a flow
A gas flow meter 74 is provided in series with the regulator 76.
Ru. This is because the reagents stored in them are
is used in vapor form, while the remaining solvent and reagents
This is because it is used in liquid form. gas release rate
For precise regulation by the corresponding valve 66, the flow rate
A total of 74 and a flow regulator 76 are required. Fluid flow from reaction chamber 34 is directed through a continuous manifold.
A diaphragm communicating with outlet passage 38 via port 84
Adjusted by ram valves 78, 80 and 82.
Opening valve 78 releases the desired reaction product, typically
N-terminal amino acid unit of protein or peptide sample
passes through conduit 86 to conversion flask 14
do. Open valve 82 to drain outlet passage 38.
By connecting to the pump 60, unwanted reagents can be
The solvent and reaction products can be disposed of, or
The valve 80 connects the outlet passage 38 to the vacuum trap 88.
The reaction chamber 34 and the outlet passage are connected to the empty pump 90.
38. Evacuate the manifold 84. Similarly, the conversion flask 14 and the fraction flask
Rector 16 is also connected via valves 92 and 94, respectively.
to the waste trap 60 and valves 96 and 98;
It can be connected to a vacuum trap via. Reagent storage tanks 100 and 101 and solvent storage tank 102
converts the reagent and solvent via conduit 104 into the flask.
It is prepared for supplying to 14. storage tank 100,
101 and 102 are a pressure regulator 106 and a solenoid valve 1
08 and pressurized by an inert gas source 28.
and individual exhaust valves 110 and flow regulators 111
It is connected to the waste trap 60 via. storage tank
Fluid outlets 100, 101 and 102 are connected to conduit 1
04 to a continuous manifold 114 with one end communicating with
It is connected via a diaphragm valve 112. write
so that fluid flow from the pressurized reservoir is selected by valve 112.
selective release of reagents or solvents.
The difference can be discharged into the manifold 114.
can. The inert gas source is connected to the pressure regulator 116 and the die.
The side opposite to the conduit 104 via the yaphram valve 118
to the end of the manifold to supply reagents or solvents.
Conversion flask 14 via manifold and conduit
can promote that movement to. Additionally, the inert gas source 28 is connected to a pressure regulator 120.
and through diaphragm valve 122 and conduit 124.
connected to the conversion flask and pressure regulator 12
6 and a fraction collector via valve 128.
16 is also connected. Inside the flask 14,
When a particular fraction is transformed in the desired manner,
This is passed through the conduit 124 and the valve 131 by gas pressure.
and from the flask to the fraction collector 16
can be released and stored in its internal container.
Wear. After releasing the fraction, flask 14
Fill it with solvent to a relatively high level and then
dissolve all residual chemicals present in
Can be done. The solvent is then introduced into conduit 12 by gas pressure.
4 and valve 125 to the waste trap 60.
We are now ready to supply the following amino acid derivatives.
Flash the scooter. A fraction collector 16 is provided for each of the apparatus 10.
Each cycle, the next amino acid unit flux
Position the empty container so that it receives the ion from flask 14.
The indexing operation is performed by the control device 18 for positioning.
Basically, from a rotating disk with containers arranged in a circle,
configured. Control device 18 is preferably a fully automated device.
diaphragm valves 66, 72, 78, 80,
82, 92, 94, 96, 98, 112, 11
8, 122, 125, 128 and 131 and solenoid valve
58, 62, 108 and 110. difference
Furthermore, the controller 18 controls the heating environment 32 to a desired temperature.
retention mechanism (not shown), fraction collector
16, vacuum pump 90 and various sensors of the entire system
control the server. Each gas pressure regulator and flow
The volume regulator is manually adjusted during startup to accommodate
Establish the desired pressure and flow in the fluid conduit
Ru. The chamber apparatus 12 is shown in detail in FIGS. 4 and 5.
However, this is a two-piece base that supports sleeve 132.
130, and the sleeve includes the first and second chambers.
It contains elements 134 and 136. Sleeve 132
is aligned with the axis of the sleeve and of the base 130.
Enlarged cylindrical part that fits snugly into the cylindrical recess
138 is provided. This cylindrical portion 138 is
The retaining collar 142 threadably engages the portion 130.
kept in place. Chamber elements 134 and 136 are cylindrical glass members.
and facing mating surfaces 144 and 1, respectively.
46 and aligned within sleeve 132.
They will be kept in close quarters in close quarters. Entrance and exit mentioned above
Passages 36 and 38 are connected to chamber elements 134 and 1, respectively.
extending axially through 36 and preferably 1
It is a capillary passageway with a diameter on the order of mm. chamber elements
axially outer ends 148 and 1 of 134 and 136;
50 is generally flat and is opposed to sleeve 132.
It is virtually free to cushion the chamber elements in the axial direction.
A pair of thin elastic washers made of active material
It comes into contact with. Upper part of upper elastic washer 152
The metal washer 154 located at
132 into the slot 158 in the upper end of the
Opposed fixed ears 156 are provided. This metal sash
The ear 154 is screwed onto the upper end of the sleeve 132.
held in place by the cap 160,
Keeps the chamber element correctly in place relative to the sleeve.
hold Fitting of ear portion 156 and slot 158
is the washer when cap 160 is installed.
154 from rotating, thus preventing the chamber element from rotating.
To prevent the cap from damaging the assembly due to rotation.
Ru. Fluid conduit 40 is provided with a diverging lower end 162.
This means that the hole in the conduit 40 communicates with the inlet passageway 36.
abuts the outer end 148 of the chamber element 134 so as to
Ru. Conduit 40 is attached to support washer 164
member 166, the latter being inserted into the cap 160.
By axially threading the divergent end 16
2 is pressed against the chamber element 134 in a sealed state. conduit 4
0 is, for example, a commercially available fluorocarbon polymer.
Made of any elastic and substantially inert material such as
can be made. Hole and inlet passage of conduit 40
Alignment with 36 is achieved by various mutual connections around a common axis.
This is ensured by precisely assembling the attached parts.
Ru. Outlet passageway 38 is housed within steel sleeve 174
by means of a member 172 of substantially inert material made of
and installed in communication with the above continuous manifold 84.
be done. Sleeve 174 has a smooth outer surface and an expanded
The alignment of the axes of the large cylindrical portion 138 and the base 130
are housed within facing openings 176 and 178. Element
172 goes up and down over the steel sleeve 174 and the shaft
Extending in the linear direction, the outer end 1 of the second chamber element 136
valve block 1 defining a manifold 84 and a valve block 50;
82 into the tapered recess 180. Element
Axial passage 184 within 172 connects outlet passage 38 and
and one end of manifold 84,
A single connection from chamber element 136 to valve block 182
Forms a continuous capillary passage. The above-mentioned conduit 40
Similar to the case, the related parts around the common axis are
Through careful manufacturing, accurate
Alignment and complete sealing are ensured. Thus the room decoration
The position 12 checks the alignment of each passageway or the integrity of each seal.
Can be easily dismantled and rebuilt in an extremely short time without causing any damage.
Can be assembled. Valve block 182 has a novel structure, and
will be explained in detail in conjunction with FIGS. 9 to 13.
Ru. At this stage, valves 78, 80 and 82 are
is provided with a valve to regulate the flow of fluid from chamber device 12.
If you remember that it is included in block 182, it will be completed.
It's a minute. As shown clearly in FIG. 6A, chamber 34 is
Opposing mating surfaces 144 and 14 of two chamber elements
By alignment cavities 186 and 188 at 6
It is formed. The two cavities are an inlet passage and an outlet passage.
arranged coaxially with respect to channels 36 and 38 and preferably
or have a circular cross-section, with a path from the inlet passage to the outlet passage.
creates an axisymmetric passage for fluid moving up to
A porous sheet member 190 crosses the reaction chamber 34.
extending into the recess 192 of the cavity 186.
The spider can be accommodated at least partially. Hidden
The porous sheet member 190 is connected to the inlet passageway 36.
Fluid must flow from one side of the outlet passageway 38 to the other.
The material is separated so as to pass through the sheet member. porous
Sheet member 190 is preferably made of glass, for example.
sheets or mats made of compressed fiber materials such as
It is made up of Commercially available glass fiber filter
Suitable for this purpose, disassembly or other
Highly resistant to damage. This kind of porous
The sheet is a thin sheet into which protein or peptide samples can be embedded.
Gives a fairly large surface area to support the membrane
It was discovered that The thin film is a fluid-permeable material, e.g.
i.e. made of materials that allow liquids and gases to diffuse therein.
If made, this material will hold the sample securely.
At the same time, the chemical interaction between reagents and solvents and the sample
form a solid matrix that allows
Ru. For example, 1,5-dimethyl-1,5-diaza
undecamethylene polymethobromide or poly
(N,N-dimethyl-3,5-dimethylene piperi
polymeric quaternary ammonium chloride such as
Nium salts are ideal for this purpose. These are flowing
It allows for diffusion in the body and is insoluble in the solvent used.
Chemically stable against both reagents and solvents
be. Furthermore, they form cohesive thin films and
have a positive charge and these are imprinted on the surface of the glass support.
It enables on-linking. Sample storage performed in conventional sequencing equipment
The fundamental difference between the holding form and that of the present invention is the 17th
See Figures A, 17B and 17C.
will be more clearly understood. Figure 17A
and FIG. 17B show sample support surface 302 or 30.
Fix 300 protein or peptide samples to 4.
Illustrating two of the most common conventional methods for
are doing. FIG. 17A shows that the sample is bonded by a covalent bond 306.
When chemically bonding to the lath support surface 302
shows. For example, surface 302 may be made of glass silicone.
Some of the spots on the sample strand extend from it.
Amino functional group 30 reacts with carboxyl group 310
It can be specially treated to have 8. suitable
Some of the groups 308 and 310 under certain conditions
Reacts and leaves the sample covalently bonded to the glass
At the same time, a large number of water molecules are liberated. this kind of
The bond is very strong and holds the sample in place
To do this, one or two bonds per molecule are required.
It's a minute. However, covalent bonds are
difficult to obtain for peptide samples. Sara
In other words, covalent bonds are very few isolated bonds in the chain.
The chain is maintained through the connected units. This is the degeneration process.
reach these units and cleave them from the chain.
, the rest of the chain is left unconnected, and this
You may be washed out of the room. FIG. 17B shows the sample 300 being placed on the support surface 304.
This shows the case where it is directly adsorbed onto the surface. Then the sample
is a very large number of relatively weak bonds between the sample and the surface.
held in place by non-covalent interactions 312
Ru. At the molecular level, the surface consists of many
interact with different parts. This is a large protein
and peptides, but the sample is
As it is degraded to smaller dimensions, the surface
It becomes easy to be cut off or pulled apart. This conclusion
As a result, significant sample loss occurs. Figure 17C shows a solid matrix formed as a thin film.
Support by embedding the sample in the Rix 316
Immobilization of the sample 300 on the support surface 314 is illustrated.
It shows. As mentioned above, matrix 31
6 is a positively charged polymeric quaternary ammonium salt.
can do. Thus, the matrix
acidic glass surface due to numerous ionic interactions.
The sample is embedded in the
Make sure to hold this sample in place to prevent damage. trial
Relies on direct bonding interactions between material and surface
The effect of immobilization is a reduction in sample size because
Not affected by The present invention performs chemical interaction with the embedded sample.
sample and solvent into solid matrix 316
and the diffusion or penetration of amino acid derivatives.
Ru. The matrix is formed as a thin film and its
Absorbs reagents and solvents that pass over it. in thin film
Once dissolved, the reagents and solvents flow across the thickness of the thin film.
can easily diffuse and undergo the degradation process.
Ru. Returning to FIG. 6A, the reaction chamber 34 has mating surfaces.
A flexible seal is sandwiched between surfaces 144 and 146.
empty by at least one sheet 194 of fastening material.
The cavities 186 and 188 are sealed at their peripheries.
Ru. A pair of flexible sheets 194 are porous sheet portions.
It is preferably used by placing one sheet on each side of the material 190.
Ru. These sheets 194 are extremely thin.
Flow of a plurality of reagents and solvents through chamber 34.
Permeable to the body. Around the cavity 188
An annular sealing ridge or bead 196 in the seal
around the cavity 186.
Provides a more effective seal to adjacent surfaces 144
Ru. The sealing sheet 194 has a possibility of seal deterioration.
For example, use commercially available fluorocarbons to
- Any substantially chemically inert material such as carbon polymers
It can be made from the following materials. These are for chamber 34.
In addition to providing a sealing effect, the porous sheet part
supports the material 190 and also allows the flow of gas and liquid.
throughout the chamber to ensure a more even distribution throughout the member 190.
helps disperse gases and liquids passing through
Two. Extending the service life of the equipment system and optimizing the connection with the sample
Chemical interactions are thus encouraged. Another embodiment 34' of the reaction chamber according to the invention is shown in the sixth example.
Shown in Figure B, here the opposing engagement table of the two chamber elements
The alignment cavities 186' and 188' in the plane are cavities.
somewhat narrower and longer than sections 186 and 188;
It is said that In other respects, chambers 34 and 3
4' are the same, and each member of the chamber 34' in the drawing
is marked with the same reference numeral as the corresponding part of chamber 34.
(') is added. The reaction chamber 34' exits from the inlet 36'.
Allowing slightly more fluid flow directly into port 38'
However, the porous sheet member 190' therein
Limit diameter. FIG. 6C shows porous sheet member 190' removed.
The chamber 34' of FIG. 6B is shown in the open position. difference
Additionally, the sealing sheet 194' has a diameter equal to the chamber diameter.
A single annular sheet of flexible material with a large central opening.
It has been replaced by port 316. In this specific example
solid stream embedded with protein or peptide sample.
A body-permeable matrix 318 passes through chamber 34'.
The walls of this chamber are exposed to the reagents and solvents
It is formed as a thin film on the part. Thus, chamber 34'
A substantial thin film surface area retains fluid flow through it.
It just keeps getting better. Another specific example of the chamber apparatus according to the present invention is shown in No. 18A,
18B and 18C, where the previous two
Chamber elements 134 and 136 are located within sleeve 132.
This is represented by one capillary chamber element 320 . room
The remaining components of device 12 are shown in FIGS. 4 and 5.
is the same as described above and therefore uses the same references.
Reference signs are attached. External structure and
All connections are the same as above. The chamber element 320 includes an axial capillary chamber 32
A cylindrical glass structure with an inner wall 322 defining 4
It is made up of The chamber 324 has two ends 32
uniformly increasing the diameter from 6 to the center 328;
The protein or peptide sample is deposited in a thin film on the wall 322.
Inside the solid matrix 330 formed as
carried by. The reaction chamber of the present invention comprises a plurality of reagent and solvent fluids.
substantially with a sample support surface capable of passing
It will be appreciated that it can take any form.
For example, chamber device 12 may include an interior surface or hole.
a solid matrix that is permeable to fluids.
One long capillary tube (not shown) is sufficient. capillary
The fluid passing through the tube contains proteins or
interacts with the peptide sample and undergoes a degradation process.
cormorant. Transfer of liquid reagent to reaction chamber 34, 34' or 324
Typical reservoir according to the invention for supply and storage
That is, reservoir 198 is shown in FIG. Storage tank 198 is the
Storage tanks 42, 50, 52, 54, 100, 1 in Figure 3
Corresponds to 01 and 102. Pressurized inert gas is
A pipe 202 supplies the interior 200 of the storage tank 198.
This conduit 202 is connected to the liquid reagent or solvent in the tank.
Connected to the inside of the tank at a location below level 204.
Pass. Thus, the introduced pressurized gas
Dissolved oxygen is entrained from the liquid to exhaust conduit 206.
into the interior 200 at a controlled rate through
Generate a dynamic equilibrium state. Remove reagents or solvents from the internal gas.
The liquid is forced out of the reservoir 198 in a controlled manner by gas pressure.
A body outlet 208 is provided. Inactivation of each storage tank 198
The inert gas supply path 202 is supplied with gas from an inert gas source 28.
Pressure inert gas is connected to pressure regulator 56 or 106 and electromagnetic
through valve 58 or 108. Exhaust guide
The release of gas through the pipe 206 is similarly controlled by a solenoid valve.
62 or 110 and a flow regulator 64 or 11
1. Through conduit 208
The flow of liquid reagent or solvent is controlled by valve 66 or 112.
regulated by one. 1 of liquid reagents or solvents
Each time the species is fed into the reaction chamber or conversion flask,
Open the corresponding argon supply and exhaust valves to
Establish a dynamic equilibrium state within the tank. Then liquid
The reagents or solvents are removed through the valve in conduit 208 and the waste trough.
can be introduced by opening the valve to the pump 60.
Ru. The liquid is discharged from the reservoir at a constant rate and
Adjust the length of time the valve in 208 is kept open
This makes it possible to accurately adjust the amount of liquid supplied.
can. The present invention for supplying reagents in gas or vapor form
A typical storage tank 210 is shown in FIG. gala
An air diffuser with a diffuser member 213 made of Sfritz at the end.
An active gas inlet 212 is provided to store inert gas.
The liquid inside 214 near the bottom of 210
Introduced from a point considerably below the body reagent level 216
do. Exhaust conduit 218 and outlet or supply path 22
0 means that the internal 2
14. Storage tank 210 is storage tank 4 in FIG.
4, 46 and 48, and gas pressure
Regulating the flow of gas from source 28 through conduit 212
one of the pressure regulators 56 and one of the valves 58.
It is growing. Similarly, pressurized gas to exhaust conduit 218
is dissipated into one of the flow regulator 64 and flow valve 62.
The supply of reagents along supply path 220 is
The supply is via a dial lined with a flow meter 74 and a flow regulator 76.
regulated by one of the flam valves 66. Thus, reagent R ₂ ,R ₃ and R _3A is a gas or vapor
They are supplied to the reaction chamber as a liquid.
It is stored and vaporized as needed. Vaporization is liquid
By bubbling an inert gas upward into the body reagent.
It is done. In this way, the inside of the storage tank 210
The inert gas at 214 is saturated by the reagent vapor.
It comes to be harmonized. Each time a reagent is needed,
Try opening the valves connected to conduits 212 and 218.
Dynamic equilibrium by bubbling inert gas into the drug
Establish the state. Then the valve in supply conduit 220
66 for a predetermined period of time to supply a desired amount of reagent vapor to the reaction tank.
supply to. Flow regulator 7 in line with specific valve 66
6 supplies steam at a constant rate indicated by a flow meter 74.
It is supplied by a storage tank. 9 through 13 illustrate valves 66 and 7 of FIG.
2 and a continuous manifold 68.
The structure and operation of assembly 222 is shown. Bena
Assembly 222 is shown most clearly in FIGS. 11 and 12.
A clearly visible valve block 224 is provided. this
Valve block 224 is an elongated block with a rectangular cross section.
then drill this valve block diagonally from its surface.
The first series of sawtooth patterns formed by
226. Thus, the first passage 226 is
It becomes one continuous passage, and at every other intersection,
on the surface 228 through the corresponding opening 232.
It communicates with a plurality of valve sections 230 . tapered
A connection port 234 is connected to one end of the first passage 226.
Pass. A plurality of second passages 236 are connected to the valve block 2.
Tapered connection port 238 on the opposite side of 24
from the vicinity of the opening 232 in each valve portion 230.
It extends to a corresponding opening 240 next to it. valve block
224 is within the longitudinal slot 242 of the base 244.
, the base of which receives a coupling fitting 248.
A plurality of ports aligned with the connection ports 234 and 238
A threaded opening 246 is provided. Mounting tool 24
8 is an elastic ferrule with two tapered surfaces;
(fit ring) 250 to the connection port 234 and
and 238, and pass the ferrule through.
A tube 252 extending through the valve block 224 is connected to each passage of the valve block 224.
Suitable for making a sealed connection with a road. Do it like this
The connector port 234 of the first passage is connected to the fluid shown in FIG.
Connected to the inlet passageway 36 of the chamber device 12 via a conduit 40.
The first seven of the eight second passages are
Fluid outlets 208 and 220 of reservoirs 42-54
communicate with. The part farthest from the connector opening 234
The two passages are connected via a pressure regulator 70 shown in FIG.
It communicates with an inert gas pressure source 28 . Two tapers to the mouth of valve block 224
The structure of the ferrule connection portion having a surface is the mouth portion 234.
is shown in detail in section 9A as an example. The ferrule 250 in FIG. 9A is inside 243
is housed in the connector opening 234, and the
The outer side 245 is the tapered recess 24 of the fitting 248
7, and the opening 234 and the recess 247 form a flap.
Teeth at an angle greater than the taper angle on each side of the errule.
is attached. The ferrule is preferably
The sides are tapered at the same angle to form the mouth 234 and the recess.
247 is a tape with an angle 3° larger than the ferrule.
I can get punched. between different tapered surfaces
This fixation reduces the compressive force to the tip 2 of the ferrule 250.
49 and against the side of the valve block 224.
Provides an effective seal while minimizing pressure
vinegar. Thus, the valve portion 230 may be distorted.
Excessive pressure on valve block 224 is avoided.
Ru. A series of diaphragm retaining blocks 254
Diaphragm against surface 228 of block 224
256 is held between them and fixed with bolts. each
Connect the upper end of the diaphragm holding block to the recess 26.
0 and at the same level as one of the valve parts 230
It seems to accept the air connection port 258 extending to the
Attach. Concave to provide an effective air seal.
O-ring 262 in an annular groove surrounding section 260
can be accommodated. Diaphragm 256 is substantially chemically inert.
It is made of airtight material and is
This can be done by reducing the pressure or applying air pressure.
The diaphragm is selective to the valve region 230.
pulled away from it or forced into contact with it
Ru. The two states of the diaphragm 256 are
and FIG. 13B. In the state shown in Figure 13A
and the air pressure applied to the air connection port 258 or
Gas pressure is controlled by the diaphragm at specific valve locations.
256 between the opening 232 of the first passage and the opening of the second passage.
It is brought into pressure contact with the mouth portion 240. Thus, the aperture
The portions 232 and 240 are connected by a diaphragm 256.
closed, making communication between them impossible. this
compares the closed state of the valve at a specific valve location.
I guess. In the condition shown in Figure 13B, the air connection port
The reduced pressure at 258 opens diaphragm 256
parts 232 and 240 and place it at that location.
A first passage across the surface of the valve block and a second passageway across the surface of the valve block.
Allows communication with the passageway. This is a specific valve
Corresponds to the open condition, from one of the reservoirs or from an inert gas
Fluid from the gas pressure source 28 passes through the first passage 226.
to allow flow to the inlet passageway 36. The novel configuration of the valve assembly 222 described above is
Directly transfer reagents and solvents without substantial contamination between each fluid.
This makes it possible to supply a precise amount to the reaction chamber. No.
1 continuity of the passage 226 and the inlet passage of the chamber device 12.
and that it is connected at one end.
This is the main factor behind this advantageous operation. 7 types of reagents and
One of the solvents is supplied through a diaphragm.
Applying vacuum to one of the 256 and pressurizing the other
This can be done by adding the desired reagent or
Flowing solvent into first passageway 226 and fluid conduit 4
0 to the reaction chamber. desired
After an amount of fluid passes under a particular diaphragm,
Gas pressure is supplied to the die again via air connection 258.
to the yaphram and thus to the assembly 222.
Each of the seven reagent valves and solvent valves are closed.
Ru. An inert gas is then installed at the extreme end of the valve block 224.
By applying reduced pressure to the diaphragm connected to the mouth part,
Flush inert gas throughout 1 passage 226
and reflux any reagent or solvent fluid remaining in the path.
The fluid supply is controlled by pushing into the reception chamber 34.
can be completed. Not connected to the first passage 226
There are no continuation points or dead point branches, so reagents or
If any of the solvent remains trapped during the feeding process.
There is no room for this. Thus, subsequent ordering
The reagents and solvents supplied during the routine process are of the highest possible purity.
held and intended for chemical reaction in the reaction tank
can be allowed to proceed normally and is contaminated.
No unnecessary yield losses due to reagents or solvents
Does not occur. This valve assembly 222 connects a single port to another.
When it is necessary to selectively connect to multiple mouths
There are numerous steps in the equipment to minimize contamination.
It is also an example of a valve design that can be incorporated into other parts.
For example, reagents and solvents may be provided to conversion flask 14.
Valves 112 and 118 for supplying the
form a valve assembly in combination with lead 114 and
for directing fluid from the outlet passage 38 of the chamber device 12.
The third valves 78, 80 and 82 are connected to the continuous manifold 8.
4 to form a similar valve arrangement. Similarly,
Discard into conversion flask and fraction collector
valves and conversion flanges for connecting the
Adjusts the flow from the SC to the fraction collector
The valve is also fabricated similarly to valve assembly 222.
Ru. The main in each of these valve assemblies
The main difference is the individual valve parts that work with them.
It is a number. manifolds 68, 84 and 114 to them.
A series of small diaphragm valves near each associated diaphragm valve.
Although illustrated as having discontinuous points or branches,
Each manifold is actually shown in Figures 11 and 12.
A single continuous passage made like first passage 226
It is a road. Diaphragm valve between open and closed states
The depressurizing and pressurizing means to be activated are omitted in Figure 3.
However, these are preferably inert as described herein.
Separate from the gas source 28 and the vacuum pump 90
be done. The conversion flask 14 is shown in detail in FIGS. 14 to 16.
Show in detail. This flask 14 is made of water, for example.
space between the double walls to circulate the heating fluid.
It is of double wall glass type with 264 mm.
The heating fluid receives the ends of standard flexible tubing.
Through a pair of nipples 266 that allow
It flows into and out of the space 264. via valve 96
and connect it to the vacuum trap 88 and vacuum pump 90.
Connected to waste trap 60 via valve 92
The resulting large-bore tube 268 corresponds to the interior chamber 270 of the flask.
It communicates near the upper end of the cap. Capillary tube 272,2
74 and 276 pass through the upper end of the flask into the inner chamber.
270. capillary tube 272 and
The hole at 276 is closed at its inner end and the capillary
each has multiple relatively restricted radial directions
Orifices 278 or 280 spaced apart are
It is provided near one end. Relatively small amounts of protein, which is the purpose for which device 10 was designed.
For use in and for white matter or peptide samples
Due to the relatively small volumes of reagents and solvents involved, the conversion
Flask 14 is much smaller than a conventional flask.
It has a large internal volume. The volume of flask 14 is small
It is about 1 ml, whereas the applicant's knowledge
All conventional automatic conversion flasks have a capacity of 100ml.
It has a product. The frac cleaved from the sample in the reaction chamber 34
The injection is sequentially performed via valve 78 and capillary tube 274.
is transferred to the flask 14. What are reagents and solvents?
It enters the inner chamber of the flask through the capillary tube 276 and enters there.
from the wall of the chamber 270 through the restriction orifice 280.
is forced as a spray that impinges on the
Wash all residue on the walls to the bottom of the flask.
drop During the conversion reaction, the inert gas is passed through the capillary tube 272.
to stir the liquid, and from there
assists in the evaporation of the solvent. Gas is limited orifice
278 as a very small valve.
2. Flask dimensions and gas usage
Optimum dispersion of gas through the liquid independent of
give. After the conversion reaction is complete, this fraction
The long capillary tube 272 is drawn by positive pressure inside the flask.
Each volume in the fraction collector 16 is
forced upwards into the vessel. This is this frac
Shion is more complete than fraction collector 16
in two batches, i.e. in two aliquots, to be transferred to
It can be done as a default. The quantity for the first time is
It is about 200μ, and the fraction correction is done first.
transferred to the computer. Then add 50μ solvent
introduced into the lower wall or bottom of the flask
Dissolve the residue. Then, the second batch was transferred.
let In this way, a high yield of each fraction
can be achieved. Add after a particular fraction has been transferred
750-900μ of solvent was added to the flask in the previous cycle
Dissolve any residue remaining on the top wall of 14.
It can be introduced to This additional solvent should be disposed of in the waste container.
The flask wall becomes clean. Outside the capillaries 268, 272, 274 and 276
The other end is connected to each other part of the device 10.
Threaded fitting 284 for flexible conduit 282
Sealing is achieved by fitting them together. Each guide
The end of tube 282 has a resilient O-ring 288.
A radial flange portion 286 is provided to connect the O-ring.
The ring is a radial flange on one of the glass tubes.
Sealing effect by contacting the flat polished glass surface of 290
give. Internally threaded collar 292 connects conduit 28
2, the glass flange 290 is fitted around the glass flange 290.
2 located around the glass tube.
It is screwed into the one side external screw attachment part 294. Mounting part 2
When the collar 292 is advanced over the flange 94, the flange
The portion 286 is pressed against the glass flange 290 to form a pole.
An effective seal is formed. Connector 284
Each part is, for example, a commercially available fluorocarbon polymer.
Made of virtually chemically inert materials such as
and virtually eliminate the possibility of subsequent leakage.
can. Another structure of the flask 14 is that the space 264 and the
Remove the pull 266 and place it in the heated environment 32
Provide a single-walled container that can be kept at high temperatures
It's something like this. This structure consists of glass tubes and connectors
The advantage of maintaining high temperature throughout the length with 284
with minimal condensation of semi-volatile fluids inside it
However, the chamber device 12 and flask 14 are kept at different temperatures.
It cannot be kept at a certain level. For sequential degradation of protein or peptide chains
The reagents and solvents used in the device 10 are as follows:
Yes: R ₁ Phenylisothiocyanate (PITC)
17% solution in butane) R ₂ trimethylamine or triethylamine
(25% solution in water) R ₃ Trifluoroacetic acid or heptafluorobutyric acid R _3A water vapor R _Four Trifluoroacetic acid (25% solution in water) R _4A Hydrogen chloride (1N solution in methanol) S ₁ Benzene S ₂ Ethyl acetate S containing 0.1% acetic acid ₃ Butyl chloride S _Four Acetonitrile or methanol In operation, preferably each valve of apparatus 10 and
Other mechanisms are controlled by an automatic control device 18.
for protein or peptide samples without human intervention.
An infinite number of deterioration cycles are performed. Control device
No. 18 was disclosed in U.S. Pat. No. 3,725,010.
The general form of the programming device includes device 10.
changes to give the specific process order required.
or special or general purpose digital
More complex electronic control and
You can also. Furthermore, each deterioration cycle
The operator is guided through each process according to a predetermined schedule.
You can also do it more manually and get the same result.
Ru. Before starting the degradation process, identify the protein or protein to be studied.
Place the peptide sample on a suitable sample support surface.
I have to do it. Support matrix material first
coated on the body surface and then as detailed below.
Embed the sample inside the shit. Member 190 or 19
0' as the sample support surface.
This means that the member 190 is in the reaction chamber 34 at the position of the recess 192.
at least one sealing sheet to be contained within the
194 between the chamber elements 134 and 136.
place In a preferred embodiment of the invention, a pair of
A sealing sheet 194 is used, and a member is placed between them.
190 to intervene. chamber elements 134 and 136;
Then insert into the sleeve 132 and each other part.
A chamber device 12 is formed in combination with the above. The sample-containing thin film is placed on the inner surface of chamber 34' or chamber 324.
If supported by
Coated as described. First, the chamber element was assembled within the sleeve 132.
Afterwards, it is held in place by the cap 160.
Ru. In the case of chamber 34', an annular sheath of flexible material is provided.
When assembling chamber elements 134' and 136',
be installed between. Then the solid matrix material
and the sample in chamber 34' or 3 as described in Example 2.
24 and other parts of the sleeve 132.
Complete the device by combining with other products. Reagent R ₁ ~R _3A and solvent S ₁ ~S ₃ and gas source 28?
In preparation for sequentially introducing inert gas from
First, valve 82 is connected to vacuum trap 88 and vacuum pump.
The reaction chamber 34 and associated flow are opened to the side of the pipe 90.
The body conduit is evacuated. Introducing one type of reagent or solvent
Each time the gas is injected, the corresponding inert gas supply valve 58 and
While opening the air valve 62 and pressurizing the specific storage tank,
Establish a dynamic equilibrium state within it. inert moth
The gas is pumped back to maintain a constant pressure in the storage tank.
Ru. In the case of storage tanks 44, 46 and 48, when supplying
The flow of saturated gas therefrom is controlled by one of the flow regulators 76.
Adjusted by one. The storage tank containing the specific reagent or solvent to be supplied is
After being pressurized and reaching equilibrium as above, the auxiliary reduction
A pressure source (not shown) energizes the corresponding flow valve 66.
Open this valve by applying vacuum to the earphragm and
The chamber inlet passageway is connected to the chamber inlet passageway via the nitrogen fold 68 and the conduit 40.
causing a flow of reagent or solvent to the valve 66
Test the desired amount by holding it open for a predetermined length of time.
Precisely transfer the drug or solvent and then
Apply gas pressure to the flam to close it. Then, supplement
One of the continuous manifolds 68
across the diaphragm of the valve 72 at the end of the
Remove any residual reagents or solvents from the nitrogen fold and return to the chamber.
complete its supply to. Then valve 72 is
Closed by applying positive pressure to the diaphragm of
and the manifold 68 for the next supply process.
Prepare and release to solvents and reagents. A diaphragm valve 80 typically separates each sample into the chamber 34.
Chamber outlet passageway and manifold while passing drugs and solvents
84 to the waste trap 60.
It's kept open like that. Supply specific reagents or solvents
After that, move the diaphragm valve 82 to the vacuum pump 90 side.
It can be opened to vent the chamber and associated conduits.
Alternatively, the chamber and the sample therein are separated from each other by valve 72.
Dry at an appropriate time by passing inert gas inside.
be able to. Next, the gas released from the chamber is removed by a
60 or as desired.
The drying process is accelerated by suction using the vacuum pump 90.
You can also After completing the binding and cleavage process, the extraction solvent S ₃ of
is supplied to the reaction chamber 34 during a particular cycle of degradation.
While dissolving the generated cleaved amino acid derivative,
This solution is then transferred via conduit 86 to conversion flask 1.
Supply to 4. For this purpose, valve 78 is opened and valve 8
0 and 82 closed. Then the conversion reagent
R _Four and R _4A (if used) and solvent S _Four and as appropriate
It can be supplied to the conversion flask 14 at a certain time, but this
opens the corresponding valve 112 to direct the liquid to the manifold.
114 and conduit 104 to the conversion flask.
This is done by sending. before each supply other
Pressurization and evacuation as described above for supply of reagents and solvents
, then open valve 118 to open the manifold.
Purge the field 114 and conduit 104. The fraction transferred to the conversion flask 14 is
The amino acid of the N-terminal amino acid of a protein or peptide sample.
Nilinothiazolinone derivative, which is a reaction chamber
During the next bonding and cleavage cycle at 34
Description of the above paper by Tutman-Liebold
phenylthiohydantoys, which are more stable due in part to
automatically converted to amino acids. In other words, strange
The amino acid fraction in the oxidation flask has short hairs.
Inert gas above the solution through capillary 276
Passage and inert gas into the liquid by capillary tube 272
bubbling, followed by a vacuum through tube 268.
It can be evaporated by evaporation. then
Conversion reagent R _Four is connected to conduit 104 by capillary tube 276.
the desired amount via one of the valves 112 (see FIG. 3).
can only be introduced. after desired conversion time
Rapid evaporation of the conversion flask in the conversion flask
A reduced pressure is applied to the capillary tube 27 through the tube 268.
Applying inert gas simultaneously through 2 and 276
This can be achieved by Pth-aspartic acid
and Pth- further stabilizes the acidic side chain of glutamate.
To do this, use reagent R. _4A conduit by capillary tube 276
104 and one of the valves 112 in the desired amount.
Further remove the residue in the conversion flask by
It can be dissolved in Conversion flask evaporation
Again, vacuum through tube 268 and capillary tube 272
and by the use of inert gas through 276
Let's do it. Then, the remaining Pth in the conversion flask
- Amino acids introduced via conduit 104 into a solution
Agent S _Four This fraction is redissolved in
Transfer to a suitable container in the Yon collector 16.
Ru. The transfer of the fraction is long in the conversion flask.
Open the valve 131 connected to the central capillary tube 272.
A pressurized inert gas is introduced through the capillary tube 276.
by forcing the fraction out of the flask.
It is done. The fraction collectors 16 are arranged in a circular pattern.
Each container receives different incoming fractions.
during each degradation cycle to be collected in a container of
Rotated by a predetermined angle. Degradation cycle suitable
The fraction in the fraction collector at the time
This can be used to further dry the
98 to the reduced pressure side or valves 128 and 94.
The inert gas from the source 28 is fractionated.
and finally to the waste trap 60.
It is done by. The various parts of device 10 are preferably substantially independent.
Manufactured from materials that are active and highly resistant to degradation.
It will be done. This type of material includes borosilicate glass, certain
Fluorocarbon polymers and in some cases
Includes stainless steel and aluminum. Device 1
Most of the sealing structure and other parts of 0 are also made of these materials.
It was designed so that it could be manufactured from raw materials. can get
The device is the cleanest of any device ever.
and function permanently as the cleanest system.
Ru. In a typical degradation and conversion cycle, device 1
The various processes performed by 0 are attached at the end.
Shown in Table 1. Duration of each step and during each step
The operating status of the equipment is also shown in the same table. operation here
The status corresponds to the status of each valve in the device, and is shown in the column.
The marks indicate when the appropriate valve is open. and
For example, column “R ₁ ”, “R ₂ ”, “R ₃ ”, “R _3A ”, “S ₁ ”,
“S ₂ ” and “S ₃ ” is selectively pressurized and the storage tank 4
2 to 54 to establish a dynamic equilibrium state.
The status of the pair of valves 58 and 62 is shown. in these columns
Whenever one mark is present, it indicates a specific reagent or solution.
in preparation for or during the supply of the agent to the reaction chamber.
In either case, the valve 58 and
and 62 are open. At all other times
The valve remains closed. Similarly, “argon”
For example, the column for
for feeding the reaction chamber via the nitrogen fold 68.
Indicates the status of valve 72, and the “Supply” column indicates the current status of valve 72.
The state of valve 66 corresponding to any pressurized reservoir
and marked “Dump,” “Collect,” and “Depressurize.”
The columns shown indicate the status of droop valves 82, 78, and 80, respectively.
are doing. As mentioned above, the solvent or reagent supply
Pressurize the appropriate solvent or reagent reservoir prior to each step.
is carried out, and then inert gas is supplied via valve 72.
Introduced to complete the supply of solvent or reagent and supply
Flash the road. Column “R _4A ” and “S _Four "teeth,
Selectively pressurize and move into storage tanks 100 to 102.
each pair of valves 108 and 11 for establishing physical equilibrium;
0 status, and the “Supply/Argon” column indicates the route.
124 into the conversion flask.
valve 122 that enters the tank and the reservoir that is pressurized at that point.
It means the state of both the corresponding valve 112. column
“Argon”, “Dump 1”, “Decompression”, “Collect”, and
"Discharge 2" is valve 118, 92, 96, 1, respectively.
31 and 125 are shown. Of the displayed fraction collector functions,
The columns “Argon”, “Evacuation” and “Depressurization” are respectively
The status of valves 128, 94 and 98 is shown. Except for the above, the order of steps shown in Table 1 is quoted.
substantially corresponds to the Edman reduction method described in the literature.
Therefore, I will not explain it in detail. Outside the general practice law
All items listed are process names and correspondences shown in Table 1.
It will be clear from the operating state. In reality, programs are tailored to meet the needs of specific users.
In order to
Changes can be made: (1) Steps 18 to 58 are repeated once in the first sequence cycle.
All amino groups on the protein are circulated once or twice.
can ensure complete coupling. (2) Following step 65, water vapor (R _3A )
of serine and threonine by supplying for 20 to 60 seconds.
Dehydration of side chains can be reduced. (3) Following step 65, methanol (R _4A ) inside 1N
Supply 0.05ml of hydrochloric acid to the conversion flask and asparagus
Methylates the side chains of gic acid and glutamic acid
be able to. If you do this, S _Four is preferable
The most common is methanol. (4) Following step 76, add 0.7 to 1 ml of S. _Four (acetonitol
methanol), which is then
Thoroughly clean the conversion flask by transferring it to the waste section.
can do. (5) Increase the duration of step 8 (500 to 1000 seconds)
), promoting cleavage of the amino-terminal proline residue.
can be done. The essence of the invention is as follows regarding the implementation of the invention:
A specific example will provide further clarity. disclose
The data obtained were carried out using the apparatus and method of the present invention.
It serves only as an example of processing and is in no way within the scope of the present invention.
It should be understood that this is not intended to limit the scope.
Ru. The amino acid order shown in the example below is a single letter amino acid.
Denoted by the acid symbol, it is defined as follows: A: Alanine L: Leucine R: Arginine K: Lysine N: Asparagine M: Methionine D: Aspartic acid F: Phenylalanine C: Cysteine P: Proline E : Glutamic acid S: Serine Q: Glutamine T: Threonine G: Glycine W: Tryptophan H: Histidine Y: Tyrosine I: Isoleucine V: Valine Example 1 Suitable for embedding protein samples to be determined sequentially
A solid matrix of polymeric quaternary ammonium salts is
Above the fibrous sheet members 190 and 190'
The protein sample can be prepared as follows:
It can be embedded in a matrix. Glass fiber circle with diameter 12mm and thickness 0.25~0.5mm
Board located in Clifton, New Jersey.
Glass micropores commercially available from Tutmann
Cut from a sheet of ivarfilter. this circle
The plate is placed in the recess 192 of the chamber element 134. 1.5
mg of 1,5-dimethyl-1,5-diazaundeca
Methylene polymethobromide and 0.0033mg glycycin
Inject 25μ of an aqueous solution containing luglycine into a syringe or
is dropped from a pipette onto a glass fiber disk. decrease
water by heating under pressure or in a stream of warm nitrogen.
evaporate. Assemble and install the rest of the chamber device 12.
Attach it during installation 10. Protein ordering program
is started from step 18, and the chemical reaction with the protein sample is started.
or any impurities that may interfere with the Edman method.
1,5-dimethyl-1,5-diazaundeca
4 for removal from methylene polymethobromide
Perform ~6 degradation cycles. The room device 12 is
Partially disassemble the 25μ protein solution into glass fibers.
Drop onto the disc. Protein solution is 1,5-dimethylene
Ru-1,5-diazaundecamethylene polymethobube
Dissolve the lomide and remove the liquid by evaporation.
1,5-dimethyl-1, in which the protein sample was embedded
5-Diazoundecamethylene polymethobromide
Leave a thin film of The initial protein sample volume is
If it is larger than 25μ, add 25μ of this sample.
Remove liquid by evaporation between applications and split applications.
can be done. Reassemble the room device 12 and remove the device.
I will reinstall it in 10 days. Then the protein ordering process
grams starting at step 18 and as many as desired.
Perform two degeneration cycles. Example 2 Suitable for embedding protein samples to be ordered
A solid matrix of polymeric quaternary ammonium salts is
Can be prepared on the inner wall of a glass capillary vessel
and place the protein sample in the matrix as follows:
can be embedded in. Chamber element 324 or chamber elements 134' and 136'
First, as shown in the drawings, the sleeve 132 and
Compact cartridge type when assembled with cap 160
form a subassembly, which contains the matrix and sample.
Facilitate installation operations. This small assembly
Hold it horizontally and add 0.6 mg of 1,5-dimethyl-1,
5-Diazoundecamethylene polymethobromide
and 0.0013mg of glycylglycine.
Inject 10μ of the solution into the reaction chamber from a syringe. Chamber 320 has a diameter of 1/16 inch at its two ends.
Manufactured so that the diameter at the center is 1/8″,
No spillage from these edges when in a horizontal position.
It is preferable to create a chamber that accommodates up to 25μ of solution.
Yes. This chamber 324 holds the solution in a horizontal position.
The shape is shown in Figure 18B, in this case for simplicity
Sleeve 132 and related structures are omitted.
There is. This subassembly is then centered on its axis.
and rotate it, while providing a flow of air or nitrogen into the chamber.
The liquid in the reaction chamber is evaporated by
1,5-dimethyl-1,5-
diazoundecamethylene polymethobromide thin
Leave the film behind. The subassembly is then placed inside the device 10.
and run the protein order determination program in step 18.
starting from , and 4 to 6 complete degradation cycles.
Do the following. The subassembly is then removed from the device 10.
Hold the syringe horizontally and pour 10μ of protein solution into the syringe.
Drop into the reaction chamber. Re-assemble the small assembly into its axis
while rotating the nitrogen gas into the chamber.
The liquid in the reaction chamber is evaporated by the flow of
The protein sample was embedded as shown in Figure 8C.
1,5-dimethyl-1,5-diazaundecamethy
A thin film of polymethobromide remains. Next
Then reinstall this subassembly into the device 10 and add the protein.
The quality sequence determination program starts from step 18.
Run the degradation cycle as many times as desired. Example 3 Angio contained in 25μ of 20% formic acid aqueous solution
0.0005 mg of tensin was added to 1,5-di
Methyl-1,5-diazaundecamethylene polymer
The method of Example 1 in a solid matrix of tobromide
Embedded according to. 8 types of angiotensin
Generated in each cycle due to Kuru's Edman degradation.
Addition of phenylthiohydantoin amino acid
The method described by Son et al.
High pressure liquid chromatography on Bax CN column
Phenylthiohydantoin amino acid by Hui
'Analytical Biochemistry',
Vol. 100, p. 335 (1979)].
Analyzed by chromatography. "Experiment"
The following order was obtained. Also compares known order
The following is for this reason. Experiment: 1 D -R-3 V -Y-5 I -H-7 P -F Known: D-R-V-Y-I-H-P-F This result shows that even a small amount of peptide direction
This is significant because it shows that the order can be determined by the law.
be. Let's order these small amounts of peptides.
The ability to adsorb the sample directly onto the support surface
It is embedded and held in a thin film rather than a
The fact that it can be held firm regardless of the law
It arises as a result. Adsorptive bonds are the bonds between the sample and the surface.
It consists of a large number of non-covalent interactions that
disclosed here, whereas
The retention force of the thin film is relatively unaffected by the sample size.
Not possible. Example 4 Whale spermatozoa contained in 25μ of 20% acetic acid aqueous solution
1,5 cyclized 0.01mg of pomyoglobin
-dimethyl-1,5-diazaundecamethylene poly
of Example 1 in a solid matrix of rimethobromide.
Embedded according to the method. This apomyoglobin
was subjected to 40 cycles of Edman degradation and
Ohydantoin amino acid was isolated according to the method of Example 3.
The results were analyzed and the order shown below was obtained as an "experiment". whale
Also for comparison with the known order of sperm apomyoglobin
Shown below. Experiment: 1 V -L-S-E-5 G -E-W-Q-L-10 V Known: V-L-S-E-G-E-W-Q-L-V Experiment: -L- H-V-W-15 A -16 K -V-E-A Known: -L-H-V-W-A-K-V-E-A Experimental: -20 D -V-A-25 G - H-G-Q-D-I- Known: -D-V-A-G-H-G-Q-D-I- Experiment: L-30 I -R-L-F-K-35 S -H -P Known: L-I-R-L-F-K-S-H-P Experimental: -E-T-40 L - Known: -E-T-L Example 5 0.1% Sodium Dodecyl Sulfate and 0.05M Heavy coal
Contained in a 25μ aqueous solution with ammonium acid
Work of unknown structure
(Drosophilamelano-gaster) larvae
1,5-dimethyl-
1,5-diazaundecamethylene polymethobromer
According to the method of Example 1 in a fixed matrix of
Embedded. 36 cycles of this cuticle protein
Phenylthiohydant is subjected to Edman degradation of
Analyze according to the method in Example 3 for in-amino acids, and
The order shown below was obtained as an experiment. Experiment: 1 N -A-N-V-5 E -V-K-E-L-V -11 N -D-V-Q-15 P -D-G-F-V- S-21 K -L -V-L-25 D -D-G-S- A-S-31 S -A-T-G-35 D -I- Example 6 0.1% sodium dodecyl sulfate and 0.05M heavy carbon
Contained in 10μ of an aqueous solution with ammonium acid
The cucicle of a toothworm fly larva with an unknown structure
The protein was previously cyclized with 1,5-dimethyl-
1,5-diazaundecamethylene polymethobromer
According to the method of Example 2 in a fixed matrix of
Embedded. 24 cycles of this cuticle protein
Phenylthiohydant is subjected to Edman degradation of
Analyze according to the method in Example 3 for in-amino acids, and
The order shown below was obtained as an experiment. Experiment: 1 N -A-N-V-5 E -V-K-E-L-V -11 N -D-V-Q-15 P -D-G-F-V- S-21 K -L -VL- The results achieved in Examples 5 and 6 are
Apparatus and method for sodium dodecyl sulfate
(hereinafter referred to as “SDS”), that is, a powerful anionic ion
proteins and peptides dissolved in a solution of detergent.
It shows that it is suitable for determining the order. this
This is especially true for large proteins or peptides for analysis.
Polyacrylimide gel electrodes for separating small volumes of media
The most common method known as pneumophoresis is an SDS solution.
This is important because the sample is generated in a liquid. This general
Detergents are used to adsorb and bond the sample to the support surface.
Also allows samples to be cleaned based on the equipment.
Regardless of the presence of SDS, the solid matrix of the present invention
Not affected by As you can see from the above, for extremely small samples
Minimal amounts of reagents and solvents and relatively short cycle times
Improved equipment for carrying out chemical processes sequentially by use with
and a method were provided.

【table】

【table】

【table】

【table】

【table】

【table】 [Brief explanation of the drawing]

FIG. 1 is a perspective view of a device manufactured according to the present invention;
2 is a plan view of the apparatus of FIG. 1, FIG. 3 is a schematic diagram of the apparatus of FIG. 1, FIG. 4 is an enlarged exploded perspective view of a reaction chamber assembly made in accordance with the present invention, and FIG. 6A is an enlarged sectional view of the reaction chamber shown in FIG. 5, and FIG. 6B is a sectional view of a second specific example of the reaction chamber shown in FIG. 5. ,
Fig. 6C is a sectional view of the reaction chamber of Fig. 6B for use after removing the porous sheet member and applying a sample-containing thin film on the internal surface, and Fig. 7 is a cross-sectional view of the reaction chamber for use with a sample-containing thin film applied to the internal surface. FIG. 8 is a longitudinal cross-sectional view of a typical reservoir of the present invention for reagents used as gases or vapors; FIG. FIG. 9A is a longitudinal cross-sectional view of a diaphragm valve assembly made in accordance with the present invention for controlling fluid flow into and out of a reaction chamber and a conversion flask, viewed in corresponding directions; FIG. 9A is a connection of the valve assembly shown in FIG. 9; 10 is a longitudinal sectional view taken along the line 10-10 in FIG. 9, FIG. 11 is a side view of the manifold block of the valve device shown in FIG. 9, and FIG.
12-12 line cross-sectional view of the figure, Figure 12A is the 11th
12A-12A, and FIG. 13A is an enlarged partial cross-sectional view of the valve portion of the valve assembly of FIG. 9, which is pressed against the manifold block to prevent fluid communication between the passages at that location. 13B is an enlarged partial cross-sectional view of the valve portion of the assembly of FIG. 9 showing the diaphragm being pulled away from the manifold block to permit fluid flow between the passageways; FIG. 14 is an enlarged partial cross-sectional view of the valve portion of the assembly of FIG. Figure 15 is a plan view of the conversion flask manufactured by 15 in Figure 14.
-15 line vertical cross-sectional view, Figure 16 is 16- in Figure 14
16-line longitudinal section; Figures 17A and 17B are schematic illustrations of the two main conventional methods of immobilizing protein or peptide samples during degradation; Figure 17C is a schematic illustration of protein or peptide immobilization according to the present invention; 18th
Figure A is a partially enlarged vertical sectional view corresponding to Figure 5 of another embodiment of the reaction chamber assembly of the present invention;
Figure 8B is a longitudinal sectional view of the chamber element of the embodiment of Figure 18A, with its sides rotated for the purpose of applying the sample-containing matrix to its inner wall, and Figure 18C is a view of the chamber element of Figure 18B. FIG. 3 is a further enlarged partial cross-sectional view of the chamber element showing the sample-containing thin film applied to its inner wall; 12... Chamber device, 14... Conversion flask, 16
...Flux collector, 22...Diaphragm valve, 24...Valve, 26...Diaphragm valve,
28...Inert gas source, 30...Pressure control valve, 3
4... Chamber, 186, 188... Cavity, 190...
... Porous sheet member, 36, 38 ... Inlet, outlet passage, 316 ... Fixed matrix, 300 ...
sample.

Claims (1)

[Scope of Claims] 1. A method for determining the order of amino acid units in a protein or peptide containing a large number of amino acid units, wherein the reagents are placed on a solid support that does not hold the reagents in a closed reaction chamber having an inlet and an outlet. providing a matrix in the form of a thin film made of a material that is insoluble and permeable to reagents; embedding a protein or peptide sample in the matrix; and passing the protein or peptide sample through the reaction chamber from the inlet to the outlet. and sequentially passing a plurality of reagents as a pressurized stream to diffuse and absorb a portion of the reagents into the matrix, immobilizing the sample in the matrix and conducting the reaction chamber. 1. A method for determining the order of amino acid units in a protein or peptide, which comprises exposing the amino acid units to each of a plurality of reagents diffused in the matrix to cause chemical interactions. 2. The step of supporting the matrix in the reaction chamber involves introducing a solution containing the matrix material into the reaction chamber, and then rotating the reaction chamber while passing gas through the reaction chamber to evaporate the liquid and transfer the matrix material into the reaction chamber. 2. A method as claimed in claim 1, comprising leaving it as a thin film on the inner wall. 3 In the step of embedding the sample in the matrix, a solution containing the sample is introduced into the reaction chamber to dissolve the matrix material in the solution, and then the reaction chamber is rotated while gas is passed through the reaction chamber to immerse the liquid in the reaction chamber. 3. A method according to claim 2, which comprises evaporating the sample material and leaving the sample-embedded matrix material as a thin film on the inner wall of the reaction chamber. 4 a matrix is supported on a porous sheet;
The step of supporting a matrix in a reaction chamber includes coating a matrix material as a thin film on the porous sheet and providing an inlet and an outlet in the reaction chamber such that reagents passing through the porous sheet from the inlet to the outlet pass through the sheet. 2. A method according to claim 1, comprising arranging across the reaction chamber at a location between. 5. The step of embedding the sample in the matrix involves coating a porous sheet with a solution containing the sample to dissolve the matrix material, and evaporating the liquid from the sheet, leaving the matrix material in which the sample is embedded as a thin film. A method according to claim 4, comprising: 6. Claim 1, wherein the step of sequentially passing reagents into the reaction chamber comprises passing at least one reagent in gas or vapor form through the reaction chamber to diffuse into the matrix and react with the sample. The method described in section. 7. A device for determining the order of amino acid units in proteins or peptides containing a large number of amino acid units, in the form of a thin film having an inner surface forming a reaction chamber with an inlet and an outlet and not retaining a reagent. a plurality of reservoirs containing reagents, at least some of which are reactive with proteins or peptides, and in communication with the reaction chamber; pressurizing means for transferring reagents; means for sequentially communicating a plurality of reagents in a pressurized stream from the reservoir to the reaction chamber and through the reaction chamber from the inlet to the outlet; and a sample of a protein or peptide. a matrix in the form of a thin film of a reagent-insoluble and reagent-permeable material disposed on the solid support within the reaction chamber for embedding; for determining the order of amino acid units in a protein or peptide, the reaction chamber being passed through the reaction chamber and exposed to each of a plurality of reagents diffused in the matrix to cause chemical interaction. equipment. 8. The device of claim 7, wherein the matrix comprises a non-reactive polymer. 9. The device of claim 7, comprising means for automatically controlling the functionality of the device through a plurality of cycles. 10 a conversion flask with a plurality of capillary tubes communicating with and projecting into the reaction chamber outlet; and means for introducing and removing various reagents through the capillary tubes into and out of the flask; terminates near the bottom of the flask at an inner end where the capillary pore is closed, and has a plurality of restricted radially spaced orifices at said inner end, thereby providing an inward flow through said one capillary tube. 8. Apparatus according to claim 7, wherein the flow of gas introduced into the flask introduces a large number of small bubbles into the liquid in the flask, which bubbles agitate the liquid or promote its drying. 11. The apparatus of claim 7, wherein the sample is a protein or peptide that is sequentially degraded to determine the order of amino acid units. 12 a conversion flask having a plurality of capillaries communicating with and projecting into the reaction chamber outlet; and means for introducing and removing various reagents through the capillaries into and from the flask, the at least one capillary being terminating near the bottom of the flask at an inner end with a closed capillary pore and having a plurality of restricted radially spaced orifices at said inner end, thereby allowing the passage of water inwardly through said one capillary tube. 8. Apparatus according to claim 7, wherein the flow of reagent passed therethrough is sprayed onto the inner walls of the flask to wash the inner walls. 13. The device according to claim 12, wherein the capillary tube is a glass tube. 14. The device of claim 7, wherein the matrix comprises a polymeric quaternary ammonium salt. 15. The device according to claim 14, wherein the polymeric quaternary ammonium salt comprises 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide. 16 Polymer quaternary ammonium salt is poly(N,N
-dimethyl-3,5-dimethylenepiperidinium chloride). 17. The means for sequentially communicating a plurality of reagents through the reaction chamber includes at least one valve means for controlling the flow of reagents between the reaction chamber and the plurality of reservoirs, the valve means having a plurality of valves on a surface thereof. a first passageway continuous between opposite ends, the first passageway opening into each valve section through a first set of openings; and a plurality of passages each communicating with one of said valve sections through a second set of openings. a plurality of resilient, substantially impermeable diaphragms covering each valve section, each diaphragm being pressed against one of the valve sections; a first sealed state that closes the opening first and second openings; and a first sealing state that closes the opening first and second openings;
and a diaphragm operable between the first and second passageways to a plurality of reservoirs and a reaction chamber, respectively. 8. The apparatus of claim 7, thereby selectively controlling fluid flow between said first passageway and said second passageway. 18. The connecting means has an attachment part at one of the ends of the first passage, whereby the first passage acts as a manifold, the manifold connecting said attachment part and a second passage farthest from said attachment part. 18. The device of claim 17, wherein the device is capable of being flushed by a fluid flow between them. 19. Claim 19, wherein the first passageway comprises a plurality of straight passageways connected end-to-end to form a conduit having a sawtooth configuration and communicating with each valve site at alternating intersections thereof. The device according to item 18. 20. The connecting means comprises at least one tapered ferrule, which fits tightly in sliding engagement around the piping member and presses into a recess of a different taper in the valve block means to connect one of said passageways. 18. The apparatus of claim 17, wherein the ferrule is in communication and thus concentrates the inward force applied to the ferrule to a relatively small contact area between the ferrule and the recess to form a fluid seal therebetween. 21. The device according to claim 20, wherein the recess is tapered at a larger angle than the ferrule. 22 Taper the ferrule on both sides,
One of said sides is pressed against said recess by screw screw means having another tapered recess to accommodate the other side of said ferrule, said recess being formed at an angle greater than the taper angle of the corresponding side of said ferrule. 21. A device as claimed in claim 20, tapered at . 23. The reaction chamber is comprised of a pair of abutting chamber elements each having first and second cavities on opposing engaging surfaces thereof, said first and second cavities being aligned with each other to form a reaction chamber, and said first and second cavities being aligned with each other to form a reaction chamber; 8. The device of claim 7, wherein the first and second cavities taper away from the engagement surface to a point where they communicate with the inlet and outlet, respectively. 24. The support means is a porous sheet disposed within a recess in at least one of the engagement surfaces for retention within the reaction chamber in an orientation dividing the first and second cavities, the reaction chamber comprising at least one of the porous sheets. Claims comprising a sheet of flexible material disposed on one side, the sheet being sandwiched between engagement surfaces to form a seal between the engagement surfaces, the sheet being permeable to a reagent. The device according to item 23. 25. The apparatus of claim 24, wherein the porous sheet extends substantially across the chamber. 26. The device of claim 24, wherein the porous sheet comprises a sheet made of a plurality of fibers. 27. The device according to claim 24, wherein the fibers are glass fibers. 28. At least one of the chamber elements has a ridge on its engagement surface extending around the cavity, and the at least one sheet of flexible material is pressed against the engagement surface of the other chamber element, thereby forming a seal. 25. The device of claim 24 for improving the stopping relationship. 29. Apparatus according to claim 23, comprising a pair of capillary passages with inlets and outlets extending through the chamber element and communicating with the reaction chamber on opposite sides of a support means constituting a porous sheet at the inner end. 30. The reaction chamber apparatus of claim 29, wherein the capillary tube is coaxial with the reaction chamber and extends therefrom to an outer capillary opening in the chamber element. 31. The device of claim 29, wherein the chamber element is made of glass and has a generally flat surface adjacent the capillary opening. 32. At least one cylindrical piece of elastic material in which the means for conducting reagents through the reaction chamber have an inlet and an outlet means having a substantially flat sealed end and an axial capillary passage terminating in an opening at this end. further including the sealed end at one of the capillary openings.
Screw means for pressing axially against the capillary opening so that the axial passage communicates with the capillary opening and the sealing end seals against the substantially planar surface of the chamber element adjacent thereto. The device according to scope 29. 33. The device of claim 32, wherein the post comprises a mass of fluorocarbon polymer encapsulated in a metal sleeve. 34 screw means with a bore for closely receiving the metal sleeve in axial sliding relationship;
34. A device according to claim 33, which ensures precise alignment of the two capillary openings and the passages in the columnar pieces. 35 A method for determining the order of amino acid units in a protein or peptide containing a large number of amino acid units, in which the reagents are not dissolved in the reagents and are not dissolved in the reagents on a solid support that holds no reagents in a closed reaction chamber having an inlet and an outlet. providing a matrix in the form of a thin film of permeable material; embedding a protein or peptide sample in the matrix; and applying a plurality of reagents from the inlet to the outlet through the reaction chamber. sequentially conducting as a pressure stream, causing a portion of the fluid to diffuse and absorb into the matrix, immobilizing the sample in the matrix, and passing through the reaction chamber and into the matrix. The sample is sequentially degraded by being exposed to each of the diffused plurality of reagents for chemical interaction, and the degraded substances are transferred to a conversion flask to be converted into a more stable amino acid fraction. A method for determining the order of amino acid units in a protein or peptide comprising: