Micro automatic amino acid analysis process and system

• US 3,918,907 A
• Filed: 10/29/1973
• Issued: 11/11/1975
• Est. Priority Date: 10/29/1973
• Status: Expired due to Term

• Current Assignee: Donald E. Stephens
• Original Assignee: Donald E. Stephens
• Inventors: Stephens, Donald E.
• Primary Examiner(s): Wolk, Morris O.
• Assistant Examiner(s): Hollander, Barry I.
• Application Number:
  US05/410,541
• Time in Patent Office: 743 Days
• Field of Search: 23/23R,23M,253R 73/61.1C
• US Class Current: 436/89
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 30/74 Optical detectors measurement of intensity, velocity, spectral content, polarisation, or phase of infrared, visible or ultraviolet light G01J

Abstract

A colorimetric chromatograph for analysis of microsamples of amino acids with scaled down parts including a separation column and filters adapted for microsamples is arranged to prevent clogging of the filters and the column by spores from buffer solution or precipitate from ninhydrin reagent. Refrigeration of the buffer solution reduces the tendency of mold organisms to grow and produce particles which tend to plug the filters. A ninhydrin reagent is also refrigerated. Refrigeration of the ninhydrin reagent permits the use of ninhydrin reagent reduced by hydrindantin for analysis of microsamples. This form of ninhydrin reagent has a limited life at room temperature, but its use avoids the use of conventional reducing agents which tend to produce fine precipitates which plug scaled-down filters and columns employed in analysis of microsamples.

Claims

1. A chromatograph analysis system for minute samples comprising in combination:

a separation column having an output end;

a buffer solution reservoir;

means for refrigerating said buffer solution reservoir to minimize the tendency for mold organisms to grow and produce particles which might plug the analysis system;

inert gas means connected to said buffer solution reservoir for pressurizing buffer solution held therein;

means for combining a sample and buffer solution from said reservoir and passing them through said separation column;

heater means interposed between said buffer solution reservoir and said separation column to drive off inert gas dissolved in said buffer solution due to refrigeration thereof;

a ninhydrin reagent reservoir;

means for delivering ninhydrin reagent from said ninhydrin reservoir and combining said reagent with the buffered sample effluent from said column; and

means for colorimetrically analyzing the combined flow of buffered sample effluent and ninhydrin reagent.

2. The analysis system defined in claim 1 wherein a filter means is interposed between the buffer solution reservoir and said separation column and said heater means is interposed between said buffer solution reservoir and said filter means.

3. The system defined in claim 1 in which means are provided for refrigerating said ninhydrin reservoir and heating means are provided between said ninhydrin reservoir and the means for combining the ninhydrin and buffered sample thereby decreasing the viscosity of the ninhydrin.

4. In a system for the analysis of microsamples of amino acids including a chromatograph column, at least one buffer solution reservoir, conduit means connected between said buffer solution reservoir and said chromatograph column for delivering buffer solution to said column, a sample injector means in said conduit means for introducing a sample of amino acids into said buffer solution flowing to said column, a metering pump in said conduit means for delivering buffer solution and sample to said column, a ninhydrin reagent reservoir and conduit means for conducting ninhydrin reagent from said reservoir and mixing said ninhydrin reagent with the effluent flowing from said chromatograph column, and means for analyzing the characteristic colorization of ninhydrin and amino acid reaction products, the improvement which comprises:

a source of pRessurized inert gas connected to said buffer solution reservoir for maintaining said buffer solution under pressure;

refrigeration means for maintaining said buffer solution and said ninhydrin solution reagent reservoirs at a predetermined temperature below ambient;

a heater means interposed in the conduit from said buffer solution reservoir for de-gassing the buffer solution; and

a bubble trap interposed after said heater means for eliminating pressurizing gas from the buffer solution flowing through said conduit means to said metering pump.

5. The improvement as defined in claim 4 including a refrigerated ninhydrin container and a line for supplying ninhydrin to the eluted sample, refrigeration of the ninhydrin container serving to improve the stability of the ninhydrin reagent.

6. In a system for analyzing microsamples of resolved amino acids including a line for delivering an eluted sample stream, a ninhydrin reservoir, a ninhydrin line for delivering ninhydrin from said reservoir to the eluted sample stream, mixing means connected to said lines for receiving both streams, a reaction coil connected to the mixing means to act on both streams, and heating means for the reaction coil to develop the coloration of the mixed ninhydrin and amino acids of the sample, the improvement comprising:

a refrigerator means in which said ninhydrin reservoir is stored, said ninhydrin line extending outside the refrigerator means to the mixing means;

prewarming means for said ninhydrin in the form of a heatconducting rod having one end heated by the reaction coil heating means and having the other end in heat-transfer relationship with a fluid-conveying passageway means, said fluid-conveying passageway means being connected to said ninhydrin line for heating ninhydrin flowing therethrough.

7. The system defined in claim 6 wherein the heating means for said reaction coil is a boiling liquid bath and the lower end of said heat-conducting rod is immersed in said boiling liquid bath, said rod conducting heat from said boiling liquid bath to said ninhydrin flowing through said passageway means in heat-transfer relationship with the upper end of said rod.

8. The system defined in claim 6 in which said fluidconveying passageway comprises a coil of tubing wrapped around the upper end of said rod, said coil forming a portion of said ninhydrin line for delivering ninhydrin to said mixing means.

9. The improvement defined in claim 7 wherein the mixing means comprises a mixing T in heat receiving contact with the upper end of the conduction rod.

10. In a chromatograph column system of analysis having a buffer solution container pressurized with an inert gas and connected by a conduit to the chromatograph column for delivering buffer solution to the column and means for colorimetrically analyzing effluent flowing from the column including a reaction coil through which the effluent is directed and heating means for heating said reaction coil, the improvement comprising:

a refrigerating means in which the buffer solution container is mounted;

a de-gassing coil of tubing connected in said conduit from said buffer solution container to said chromatograph column, said de-gassing coil being in heat-transfer contact with said heating means for heating said reaction coil thereby driving off gas absorbed in said buffer solution; and

a bubble trap through which said buffer solution flows after being heated in said de-gassing coil thereby permitting gas bubbles in said buffer solution to escape.

11. The chromatograph column system of analysis defined in claim 10 in which said means for heating said reaction coil includes a hot zone and a warm zone and said de-gassing coil is in contact with said hot zone of said heating means and said bubble trap is in contact with said warm zone of said heating means.

12. IN THE METHOD OF ANALYZING MICROSAMPLES OF AMINO ACIDS WHCH COMPISES THE STEPS OF:

SUPPLYING A BUFFER SOLUTION, PRESSURIZING TH BUFFER SOLUTION WITH INERT GAS AND FORMING A BUFFER STREAM, METERING THE BUFFER STREAM, INJECTING A MICROSAMPLE INTO THE METERED BUFFER STREAM, DELIVERING THE METERED BUFFER STREAM AND MICROSAMPLE ONTO A SEPARATING COLUMN CONTAINING FINE PARTICLE RESINS, MIXING THE STREAM FROM THE COLUMN WITH A NINHYDRIN REAGENT AND EXPOSING THE MIXTURE TO A SOURCE OF HEAT FOR REACTION TO BRING OUT COLOR CHARACTERISTIC OF AMINO ACIDS, THE IMPROVEMENT WHICH COMPRISES REFRIGERATING THE BUFFER SOLUTION BEFORE IT ENTES THE BUFFER STREAM, THEREAFTER DE-GSSING THE BUFFER STREAM BEFORE METERING AND INJECTION OF THE SAMPLE INTO THE BUFFER STREAM.

13. The improvement defined in claim 12 wherein:

the buffer stream, before injection of the sample, is exposed to the reaction source of heat for purposes of de-gassing, and then conducted into a zone of lower temperature but higher than ambient.

14. The improvement defined in claim 12 including the additional steps of maintaining the ninhydrin reagent refrigerated and warming the refrigerated ninhydrin reagent prior to mixing it with the buffered stream.

15. The improvement defined in claim 12 wherein the buffered stream and ninhydrin reagent are both warmed during mixing.

16. The improvement defined in claim 14 which further comprises dissolving ninhydrin in dimethyl sulfoxide with an excess of hydrindantin added to form the ninhydrin reagent.