US3346486A

US3346486A - Apparatus and method for introduction of amino acid samples into a chromatography column

Info

Publication number: US3346486A
Application number: US358198A
Authority: US
Inventors: George D Winter; George N Catravas
Original assignee: Technicon Corp
Current assignee: Technicon Corp
Priority date: 1964-04-08
Filing date: 1964-04-08
Publication date: 1967-10-10
Anticipated expiration: 1984-10-10
Also published as: CH472903A; SE335628B; DE1673101A1; BE662089A; GB1087301A; DE1673101C3; DE1673101B2

Description

06L 1967 G. D. WINTER ETAL Y 3,346,485

APPARATUS AND METHOD FOR INTRODUCTION OF AMINO ACID SAMPLES INTO A CHROMATOGRAPHY COLUMN Filed April 8, 1964 v Fl 6. 3 l l INVENTORS Gen/9e D. M'mer BY Gen/ye A4 afravas United States Patent 3,346,486 APPARATUS AND METHOD FOR INTRODUC- TION 0F AMINO ACID SAMPLES INTO A CHROMATOGRAiHY COLUMN George D. Winter, Katonah, and George N. Catravas, Yonkers, N.Y., assignors to Technicon Corporation, a corporation of New York Filed Apr. 8, 1964, Ser. No. 358,198 10 Claims. (Cl. 21tl-31) This invention relates to an apparatus and method for the introduction of amino acid samples into a chromatography column at the top of the ion exchange resin.

The usual procedure of introducing a sample into ,a chromatography column is complicated and involves a plurality of manipulations, including:

(1) Removing from the top of a column the fitting through which the buffer is introduced into the column;

(2) Aspirating the supernatant buffer to expose the surface of the resin;

(3) Applying the sample to the exposed resin, using a micro syringe or pipette while exercising great care to avoid any disturbance of the packed resin;

(4) Connecting an air fitting to the column and using air pressure to force the sample into the top of the resin column;

(5) Removing the air fitting and pipetting a wash of buffer solution onto the resin, using this Wash to rinse down any sample remaining on the wall of the column, while maintaining great care to avoid disturbing the resin;

(6) Connecting the air fitting and forcing the buffer wash by air pressure into the resin;

(7) Repeating the preceding two manipulations numbered 5 and 6, two washes being normally recommended, it being noted that when air pressure is used to force the sample and wash into the resin, the liquid must be forced down until the resin is exposed, care being taken to avoid drying of the resin which might result from prolonged application of the air stream;

(8) Removing the air fitting and carefully filling the portion of the column above the resin with buffer solution; and

(9) Replacing the fitting at the top of the column and starting the positive displacement pump for supplying the buffer through the fitting at the top of the column, using care to avoid entrapment of air at the top of the column adjacent the fitting through which the buffer is introduced.

The primary object of the present invention is to obviate the need for restoring to the abovementioned manipulations and facilitate the introduction of the amino acid samples into the chromatography column, and to avoid disturbing the resin in the column.

An important ancillary object is to provide a fitting which may be readily secured to and removed from the top of the column and which is well adapted to enable the introduction of the samples into the column with facility and without disturbing the resin in the column.

In seeking to obviate the above-listed manipulations and attendant disadvantages of the previous method of introducing amino acid samples into chromatography columns, we have found that at least certain types of amino acid samples, for example, protein and/or peptide hydrolysates, can be dissolved in high density sucrose mixtures. In accordance with the present invention, the amino acid sample is dissolved in a quantity of a water solution of sucrose, and an aliquot of the the resulting solution is layered on the upper surface of the resin in the column, after which the buffer when pumped into the column by a positive displacement pump in the usual way forces the sample into the resin.

In order to obviate the necessity for opening the top fit) 3,346,486 Patented Oct. 10, 1967 of the column for the introduction of the sucrose-sample solution, a fittin embodying the present invention is employed and will now be described with reference to the accompanying drawings.

In the drawings:

FiG. 1 is a vertical sectional view of the upper part of a chromatography column having the fitting of the present invention mounted thereon; and

FIG. 2 is a fragmentary sectional view showing part of the fitting in the condition thereof after the introduction of the sample.

In the drawings, the upper part of the chromatography column is shown and is designated by the numeral 10. The fitting housing 12 is preferably a molded plastic member, the preferred plastic being known ,as Kel-F. Said housing has a lower part 14 which fits snugly into the top of the column 10 and an O-ring 16 provides a fluid-tight seal between the inner surface of column 10 and the peripheral surface of part 14. The housing 12 is releasably held in position at the top of the column by a pair of clamps 18.

A vertically extending passage 20 in housing 12 communicates with the upper part of column 10 and with the transverse passages 22 and 24 provided in said housing. A fitting 26 has a part 28 which is threaded into the opening 30 of housing 12 in fluid-tight relation in said opening. Fitting 26 provides a passage for the buffer supplied by a positive displacement pump (not shown) through a tube 32 connected to said fitting and to the outlet of said pump. A Luer-Lok thimble or hollow plug 34 is screwed into an opening at the top of the housing in fluid-tight engagement with the housing as indicated at 36. Said thimble communicates with the upper end of ,a hypodermic needle or capillary tubing 38 which has a fluidtight connection with the lower end of said thimble. Said tubing 33 extends from the lower end of thimble 34 through passage 2% into the upper part of column 10 below the housing part 14. A length of polyethylene tubing 49 is fitted on tubing 38 and extends downwardly into the upper part of column 10 to a point adjacent the upper surface 43 of the resin with which the column is packed. The polyethylene tubing can be cut to any required length and thereby provides means for adjusting the outlet of the hypodermic needle tubing in relation to the upper surface of the column resin.

A Luer-Lok plug 42 is screw-threaded into a housing opening 44 and communicates with passage 24 to provide an overflow opening when the Luer-Lok cap is removed. It will be understood that during the operation of the column for the stripping thereof, at which time the buffer liquid is being introduced into the column by way of fitting 26, the Luer-Lok

plugs

34 and 42 are each tightly closed by a Luer-Lok cap, one of which is indicated at 46 in FIG. 3.

FIG. 1 shows the condition of the apparatus at the time of the introduction of the sample into the column, as hereinafter more particularly described. At this time, the caps 46 are removed from the

plugs

34 and 42 and the positive displacement pump is shut down.

The sucrose-sample solution is introduced into the column by means of a syringe of a well known type which has a lower cylindrical end portion which is inserted into the thimble 34 in fluid-tight relation therewith, when the LuerLok cap 46 is removed. As the sucrose-sample solution has a higher density than the buffer, the sucrosesample flows downwardly through the buffer and is thus layered onto the top surface 43 of the resin in the chromatography column. The buffer, which is in the column above the resin at the time of the interrupting of the operation of the positive displacement pump and which is displaced from the column by the introduction of the sucrose-sample solution, flows out of the fitting 3 through the passage 20 around the

capillary tubing

40, 38, through the transverse passage 24 and out through the Luer-Lok thimble 42.

After the sample is introduced, the syringe is removed from the Luer-Lok thimble 34. Then said thimbles are closed in fluid-tight condition by the attachment of the Luer-Lok caps 46, and the pump for introducing the butfer into the column is started, thereby resulting in the sample-sucrose solution being forced into the resin by the butter.

The chromatography analysis operation then proceeds in the usual way, which is well known and does not in itself form part of the present invention. If further information is desired with respect to automatic amino acid analysis with the use of a chromatography column, reference may be had to the United States applicatioin of George N. Catravas, one of the present inventors, Ser. No. 314,812, filed Oct. 8, 1963, for Method and Apparatus for Chromatography Analysis and Hydrolysis of Peptides, assigned to the assignee of our present application.

The following is a non-limitative example of a sucrosesample solution which has been found to be highly satisfactory in the practice of the present invention.

Fifty grams of sucrose are dissolved in sufficient water to provide 100 ml. of the sucrose solution. The sucrosesample solution is prepared by adding three parts of the sample to one part of the sucrose solution. Ordinarily, 0.20 ml. of the sample-sucrose solution is introduced into the column in the manner described above. This quantity of the sucrose-sample solution is thus composed of 0.15 ml. of sample and 0.05 ml. of the sucrose solution.

While we have shown and described the preferred mode of practicing our invention, it will be understood that the invention may be embodied and practiced in various other ways which will occur to those skilled in the art in view of the present disclosure. Therefore, we do not wish to be limited exactly to the invention as hereinbefore described, except as may be required by the scope of the appended claims.

What is claimed is:

1. A chromatography apparatus comprising a chromatography column containing an ion exchange resin, and means for the introduction of buffer and samples into the column, each sample being contained in a liquid which has a higher density than the butter, said introduction means including a member releasably secured to the top of the column and having a plurality of passages each independently in flow communication with the column, one of said passage having an inlet part accessible externally of said member for the introduction of the sample into the column when the flow of butter into the column is interrupted, another of said passage having an inlet for providing for the flow of buffer into the column during the eluting operation, said member having an outlet opening in communication with said buffer passage for outflow of such buflers as is displaced by the sample introduced through said one passage, and means for releasably closing said outlet and said inlet to said one passage.

2. A chromatography apparatus comprising a chromatography column containing an ion exchange resin, and means for the introduction of buffer and samples into the column, each sample being contained in a liquid which has a higher density than the butter, said introduction means including a member releasably secured to the top of the column and having a plurality of passages each independently in flow communication with the column, one of said passage comprising a capillary tube having an inlet part accessible externally of said member for the introduction of the sample into the column when the flow of buffer into the column is interrupted, said capillary tube being vertically disposed and having at its lower end a removable length of tubing to provide an outlet for the sample into the column at a predetermined level above the resin in the column, another of said passages having an inlet for providing for the flow of buffer into the column during the eluting operation, said member having an outlet opening in communication with said bufler passage for outflow of such bufler as is displaced by the sample introduced through said one passage, said lower end of said tubing being disposed below said inlet and said outlet for the butter, and means for releasably closing said outlet and said inlet to said one passage.

3. A chromatography apparatus comprising a chromatography column containing an ion exchange resin, and means for the introduction of buffer and samples into the column, each sample'being contained in a liquid which has a higher density than the buffer said introduction means including a member releasably secured to the top of the column and having a plurality of passages each independently in flow communication with the column, one of said passages having an inlet part accessible extrenally of said member for the introduction of the sample into the column when the flow of butter into the column is interrupted, said inlet part having an inner wall with a fitting which is releasably engageable in fluid-tight relation by the discharge end of a syringe for the transmission of the sample through said one passage from said inlet into the column, another of said passages having an inlet for providing for the flow of buffer into the column during the eluting operation, said member having an outlet opening in communication with said buffer passage for outflow of such butter as is displaced by the sample introduced through said one passage, and means for releasably closing said outlet and said inlet to said one passage.

4. A chromatography apparatus comprising a chromatography column containing an ion exchange resin, and means for the introduction of buffer and samples into the column, each sample being contained in a liquid which has a higher density than the butter, said introducing means including a member releasably secured to the top of the column and having a plurality of passages each independently in flow communication with the column, one of said passages comprising a capillary tube having an inlet part accessible externally of said member for the introduction of the sample into the column when the flow of bufler into the column is interrupted, said capillary tube being vertically disposed and having at its lower end a removable length of tubing to provide an outlet for the sample into the column at a predetermined level above the resin in the column, said inlet part having an inner wall with a fitting which is releasably engageable in fluid-tight relation by the discharge end of a syringe for the transmission of the sample through said one passage from said inlet into the column, another of said passages having an inlet for providing for the flow of buffer into the column during the eluting operation, said member having an outlet opening in communication with said buffer passage for outflow of such buffer as is displaced by the sample introduced through said one passage, said lower end of said tubing being disposed below said inlet and said outlet for the butter, and means for releasably closing said outlet and said inlet to said one passage.

5. A method for the chromatography analysis of an amino acid sample by apparatus having a chromatography column containing an ion exchange resin through which a butter liquid is pumped for forming the chromatogram and for the stripping operation, said method comprising: incorporating the sample in a liquid having a higher density than said buffer liquid, flowing the buffer liquid into the column, interrupting the flow of said butter into the column, and introducing a quantity of said sample-containing liquid into the column above the resin whereby said sample-containing liquid flows downwardly through the butter liquid and is thereby layered on the top surface of the resin displacing an equal volume of the buffer liquid.

6. A method for the chromatography analysis of an amino acid sample by apparatus having a chromatography column containing an ion exchange resin through which a buffer liquid is pumped for forming the chromatogram and for the stripping operation, said method comprising: incorporating the sample in a water solution of sucrose and thereby providing a liquid having a higher density than said buffer liquid, flowing the buffer liquid into the column, interrupting the flow of said buffer into the column, and introducing a quantity of said sample-containing liquid into the column above the resin whereby said sample-containing liquid flows downwardly through the butter liquid and is thereby layered on the top surface of the resin displacing an equal volume of the buffer liquid.

7. A method for the chromatography analysis of an amino acid sample by apparatus having a chromatography column containing an ion exchange resin through which a buffer liquid is pumped for forming the chromatogram and for the stripping operation, said column having a fitting thereon provided with separate passages for the separate introduction of the buflfer and the sample, respectively, into the column, said method comprising incorporating the sample in a liquid having a higher density than said buffer liquid, flowing the butter liquid into the column, interrupting the flow of said buffer into the column, and introducing a quantity of said samplecontaining liquid into the column through said fitting above the resin whereby said sample-containing liquid fiows downwardly through the buffer liquid and is thereby layered on the top surface of the resin displacing an equal volume of the buffer liquid from the column into the respective buffer passage.

8. A method for the chromatography analysis of an amino acid sample by apparatus having a chromatography column containing an ion exchange resin through which a buffer liquid is pumped for forming the chromatogram and for the stripping operation, said column having a fitting thereon provided with separate passages for the separate introduction of the buffer and the sample, respectively, into the column, said method comprising: incorporating the sample in a water solution of sucrose and thereby providing a liquid having a higher density than said buffer liquid, flowing the butter liquid into the column, interrupting the flow of said bulfer into the column, and introducing a quantity of said sample-containing liquid into the column through said fitting above the resin whereby said sample-containing liquid flows downwardly through the butter liquid and is thereby layered on the top surface of the resin displacing an equal volume of the buffer liquid from the column into the respective butter passage.

9. An inlet apparatus, for a chromatography column containing a porous material, for the introduction of an eluting liquid and a sample liquid having a higher density than the eluting liquid, said apparatus comprising: means for the fluid-tight attachment of said apparatus to the top of the column; a first substantially vertical passageway having a lower end adapted to be disposed within the column above the level of the porous material contained therein, and an upper end with a fitting adapted to be releasably connected to a source of sample liquid; a second passageway independent of said first passageway having an inlet end adapted to be connected to a source of eluting liquid and disposed between said upper and lower ends of said first passageway, having an intermediate port adapted to be disposed in fluid flow communication with the interior of the column above the level of the lower end of said first passageway, and having an outlet end disposed between said upper and lower ends of said first passageway and adapted to discharge such eluting liquid as is displaced from the column by the sample liquid; and means for releasably closing said upper end of said first passageway and for releasably closing said outlet end of said second passageway.

10. Apparatus according to claim 9 wherein said first passageway passes through said intermediate port of said second passageway.

References Cited UNITED STATES PATENTS 2,769,454 11/1956 Bletcher et a1. 137-597 X JOSEPH SCOVRONEK, Primary Examiner,

WHITBY, Assistant Examiner,

Claims

5. A METHOD FOR THE CHROMATOGRAPHY ANALYSIS OF AN AMINO ACID SAMPLE BY APPARATUS HAVING A CHROMATOGRAPHY COLUMN CONTAINING AN ION EXCHANGE RESIN THROUGH WHICH A BUFFER LIQUID IS PUMPED FOR FORMING THE CHROMATOGRAM AND FOR THE STRIPPING OPERATION, SAID METHOD COMPRISING: INCORPORATING THE SAMPLE IN A LIQUID HAVING A HIGHER DENSITY THAN SAID BUFFER LIQUID, FLOWING THE BUFFER LIQUID INTO THE COLUMN, INTERRUPTING THE FLOW OF SAID BUFER INTO THE COLUMN, AND INTRODUCING A QUANTITY OF SAID SAMPLE-CONTAINING LIQUID INTO THE COLUMN ABOVE THE RESIN WHEREBY SAID SAMPLE-CONTAINING LIQUID FLOWS DOWNWARDLY THROUGH THE BUFFER LIQUID AND IS THEREBY LAYERED ON THE TOP SURFACE OF THE RESIN DISPLACING AN EQUAL VOLUME OF THE BUFFER LIQUID.