US3778230A - Method of automatically analyzing amino acids by liquid chromatography and color-developing solution therefor - Google Patents

Method of automatically analyzing amino acids by liquid chromatography and color-developing solution therefor Download PDF

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US3778230A
US3778230A US00212197A US3778230DA US3778230A US 3778230 A US3778230 A US 3778230A US 00212197 A US00212197 A US 00212197A US 3778230D A US3778230D A US 3778230DA US 3778230 A US3778230 A US 3778230A
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amino acids
ninhydrin
ascorbic acid
color
solution
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Y Arikawa
S Takeuchi
H Sagusa
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/84—Preparation of the fraction to be distributed
    • G01N2030/8429—Preparation of the fraction to be distributed adding modificating material
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/11—Automated chemical analysis

Definitions

  • ABSTRACT A color-developing solution for use in automatic amino acid analysis by liquid chromatography, which comprises ninhydrin, an organic solvent, a buffer and ascorbic acid as a reducing agent for ninhydrin in an amount of from 0.2 X 10 to 0.5 X 10 mole per liter of solution.
  • the present invention relates to a method of automatically analyzing amino acids by liquid chromatography and a new ninhydrin reagent for automatic amino acid analysis.
  • hydrindantin intermediate theory has been considered less convincingly than other theories represented by the above-mentioned Schiff base intermediate theory since the rate of coloration by a primary amino or an amino acid is faster than that by ammonia in the presence of excess hydrindantin. Therefore hydrindantin is regarded merely as playinga role as a stabilizer for the reaction intermediate; namely protecting the reaction intermediate from oxidative decomposition by the co-existing oxygen or other oxidizing agents.
  • Table 1 shows the composition of a conventional ninhydrin reagent employed for the automatic amino acid analyzer.
  • sodium (or potassium) cyanide has been reported to be used as the reducing agent instead of stannous chloride, it is not widely used because it is poisonous,except for a special case, and stannous chloride is still commonly adopted for an ordinary case.
  • Heating the sample solution to l00C. in order to increase the reaction velocity in the case of using the conventional ninhydrin reagent generally produces deposition of a small amount of tin, which is considered to be the principal cause of choking the reaction coil.
  • the reducing agents capable of affording hydrindantin in the reduction include an alkali metal, sodium sulfide, a chromite, hydrazine and the like, in addition to the conventionally known stannous chloride.
  • the reducing agent to be employed practically for the above purpose should not be positive to the ninhydrin reaction, it must have a greater solubility in the reaction mixture with the composition as shown in Table 1. Further, it should be stable, and easy to handle. For example, sodium sulfide is difficult to weigh owing to its hydroscopic property. l-Iydrazine itself is negative to the ninhydrin reaction; however, an amine which is decomposition product, is positive to the color developing reaction. The chromite ion and metallic sodium are not suitable as the reducing agent in view of instability and difficulty of handling.
  • One of the objects of the present invention is to provide a new ninhydrin reagent for liquid chromatography with higher sensitivity of coloration and less trouble of choking the reaction coil compared with the conventional reagents.
  • Another object of the present invention is to provide a ninhydrin reagent which can be readily regenerated after deterioration.
  • the general requirements of the reducing agent for the ninhydrin reagent are high purity, cheapness, greater solubility in the reagent solution, no harmful reaction for the sensitivity as well as other coloration, inertness to the equipment, and no deposition during use.
  • the present inventors after extensive investigations and a survey of a desired reducing agent, the present inventors have found that ascorbic acid is most promising for the purpose mentioned above.
  • the present invention provides a ninhydrin reagent solution for automatic amino acid analysis comprising ninhydrin, an organic solvent, a buffer solution, and ascorbic acid as the reducing agent.
  • FIG. 1 is a block diagram of an automatic amino acid analyzer employed in the present invention
  • FIGS. 2a and 2b are graphs showing the change of coloration with the ninhydrin reagents according to the present invention and that by the Moore-Stein method;
  • FIG. 3 is a graph showing the rates of coloration with the ninhydrin reagents of the present invention and the Moore-Stein method respectively;
  • FIG. 4 is a graph showing the stability of coloration with respect to time passed afforded by the ninhydrin reagent of the present invention, and the change of coloration upon further addition of ascorbic acid.
  • Ascorbic acid has been known to have a greater solubility to the organic solvent than the conventionally employed stannous chloride, and a potent reducing property. It has not yet been adopted i or the reducing agent for the ninhydrin reaction in spite of the excellent reducing property against ninhydrin, because ascorbic acid forms brown colored decomposition matters upon heating it alone. This increases the blank value of the colorimetric estimation of amino acids, causing a noise drift of the base line of the automatically recorded chromatogram.
  • composition of the ninhydrin reagent according to the present invention is exemplified by the following tabulation indicated in Table 2.
  • the methyl cellosolve may comprise from 60 to volume with the balance essentially comprising the acetate buffer, e.g. acetic acid-sodium acetate.
  • the range of ninhydrin may be from 0.05 to 0.2 mole/liter and the pH of the buffer solution may be from 5.4 to 5.6.
  • Ascorbic acid releases the hydrogen of two hydroxy groups present in the molecule to effect the reduction.
  • the solubility of ascorbic acid is considerably great.
  • the concentration of ascorbic acid is limited at about 10 mole/liter in actual use because the solubility of hydrindantin formed in the reaction of ascorbic acid with ninhydrin is about 10 mole/liter, and such excess of the hydrindantin becomes difficult to dissolve in methyl cellosolve; in particular, on mixing the buffer solution, the concentration of methyl cellosolve decreases, inducing the deposition of hydrindantin.
  • Ascorbic acid dissolves in the ninhydrin reagent up to the concentration of approximately 1 mole/liter. However, a low amount of the hydrindantin formed on addition of l X 10' mole/liter of ascorbic acid remains insoluble in 75% methylcellosolve.
  • the concentration of methylcellosolve becomes lower than 25% in view of mixing of the buffer solution.
  • the amount of hydrindantin should be confined at lower concentration.
  • the practical amount of ascorbic acid for the usual case has been found to be 0.5 X 10 mole/liter.
  • the minimum amount of ascorbic acid namely 0.2 X mole/liter, compared with 1.8 X 10' mole/liter stannous chloride in Moore-Steins method gives rise to the same intensity of coloration as reflected by the absorbance.
  • the increase of the absorbance given by amino acids reaches the maximum at the concentration level of 0.6 X 10' mole/liter, with respect to ascorbic acid, and at 0.8 X l0 m ole/liter, the absorbance be comes 100%.
  • the ninhydrin reagent is liable to oxidation, and on exposure to the air, it is rapidly oxidized and loses its activity. Therefore, it should be preserved in an atmosphere of nitrogen. Despite the fact that this reagent is inevitably oxidized by the oxygen of the air diffusing through teflon tubes, thereby causing the intensity of coloration to be lowered impairing the reproducibility of quantitative estimation, the duration of preservation of the Moore-Steins ninhydrin reagent wherein stannous chloride has been used as the reducing agent and that by the present invention is approximately the same, i.e. nearly about days.
  • the reagent according to the present invention is characterized by recovery of the coloring capacity after the deterioration due to the air oxidation by readdition of ascorbic acid, for example, by addition of 0.4 X 10 mole/liter of ascorbic acid, i.e., the amount initially employed or a slightly lesser amount, the coloring capacity is recovered to the original level.
  • the reagent according to the present invention can be used as the color reagent for other methods as well as the ordinary ion exchange separation technique.
  • the metallic salt resin column utilizing the difference in the complexing capacity of amino acids with metallic ions such as Cu, Ni, Co, Zn, Cd and the like.
  • the column contains an ion exchange resin having as ion exchange groups, sulfonic acid groups, carboxyl acid groups or imino-diacetic acid groups.
  • This method of chromatography is called Ligand method and is disclosed in U.S. Pat. application Ser. No. 742,993 entitled Method of Separating Mixture by Liquid Chromatography filed May 14, 1968, now U.S. Pat. No. 3,630,681, which disclosure is incorporated herein by reference.
  • FIG. 1 is a block diagram of the automatic amino acid analyzer to be used as an embodiment of the present invention, wherein the container 1 containing the eluted fraction (containing for example, the acetate buffer) is connected to the metal salt form resin column 3 by the pump 2.
  • the color reagent container 4 is connected under the column 3 through pump 5; the front of the crossing connecting tubes of the both being joined to the teflon tube 7 with a 0.3 to 0.8 mm internal diameter in the reaction cell.
  • the teflon tube 7 is, in turn, connected to the flowing colorimeter 8, from which the solutionafter the determination is discharged.
  • the flowing colorimeter 8 is attached with the recorder 9 whereon the absorbance curve is plotted on a recording paper according to the absorbance of the solution.
  • the device 10 is a heater and reference numeral 11 designates a thermistor, and 13 designates a stirrer.
  • FIGS. 2a and 2b show the elution curves on automatic analysis of amino acids wherein (A) is given by Moore-Steins color reagent and (B) by the reagent of the present invention, the both reagents gave approximately the same intensity of coloration.
  • FIG. 3 shows the change of the intensity of the coloration yielded by the reagent of the present invention and that of the Moore-Stein method in which the concentration of stannous chloride was 1.8 X 10' mole/liter.
  • the color reagent according to the present invention (using ascorbic acid as the reducing agent) gave the same intensity of coloration as the Moore- Stein reagent at 0.2 X 10 mole/liter of ascorbic acid; whereas the absorbance reached to about 100% at 0.8 x 10 mole/liter with respect to ascorbic acid, though the ascorbic acid concentration up to 1.2 X 10' mole/liter is shown in the figure.
  • curvesl, 2, 3 and 4 indicate, respectively, the intensities of coloration given by glutamic acid, hydroxy-proline, sarcosine, and urea in relation to the amount of ascorbic acid used in accordance with this invention.
  • the amount of stannous chloride in the Moore-Stein reagent was 1.8 X 10 mole/liter.
  • the absorbance reaches the constant value after the addition of a definite amount of ascorbic acid, whereas the blank value increases with the increase of the amount of ascorbic acid added, ascorbic acid should be added appropriately within the compensating capacity of the auto-analyzer.
  • FIG. 4 shows the relation between the periodical change of ninhydrin reagent using ascorbic acid as the reducing agent with the peak area of the absorbance curve.
  • the diffusion of the air into the vessel depends upon the structure of the stopper and the thickness of the teflon tubes, the deterioration characteristics of the Moore-Steins ninhydrin reagent and the reagent of the present invention are regarded the same provided other conditions are the same.
  • the change of the intensity of coloration is indicated by the peak area of the recorded chromatogram on continuous use of the ninhydrin reagent containing 0.4 X 10 mole/liter of ascorbic acid in the presence of nitrogen. No change can be observed after 2 weeks (point A).
  • the coloration becomes nil (point B) after aeration to allow the reagent to deteriorate completely by the oxidation.
  • the intensity of coloration recovers to the original level (point C) by addition of 0.4 X 10 mole/liter of ascorbic acid after removal of remaining oxygen by blowing nitrogen gas into the solution.
  • a new ninhydrin reagent according to the present invention is devoid of precipitation at a high temperature and yields excellent color devel oping efficiency reflected in the absorbance. Further, by addition of a relatively large amount of ascorbic acid, the rate of the color developing reaction can be promoted which enables saving the length of the reaction tube.
  • the deteriorated reagent can be regenerated and becomes reusable by readdition of ascorbic acid.
  • the elute solution comprises 0.055 mole/liter of acetic acid buffer solution for pH of 4.1, 0.4 X 10' mole/liter of zinc and 8% by volume of ethanol.
  • the ninhydrin solution comprises one volume of ninhydrin and two volumes of acetic acid buffer.
  • a method for automatically analyzing amino acids by liquid chromatography which comprises passing amino acids together with an eluting solution through a separator column packed with ion exchange resins, separating the amino acids into their respective amino acid components by differences in migration speeds of the amino acids in the column, allowing the separated amino acids to react with a color-developing solution containing ninhydrin, its reducing agent and an organic solvent by heating thereby to obtain colored substances, leading the resulting solution containing said colored substances continuously to a means for detecting absorbances and transmitting detected signals from the means for detecting absorbances to a recording means, the improvement wherein ascorbic acid is used as the reducing agent, said acid being added in an effective amount for converting a part of ninhydrin into hydrindantin.
  • a method for automatically analyzing amino acids by liquid chromatography which comprises passing an eluting solution containing a buffer agent and the same metal ions as those adsorbed on ion exchange resins in a separator column thereby to bring the column into an equilibrium, adding an amino acid sample to the separator column thereby to separate amino acids into the respective amino acid components by migration speeds due to differences in abilities to form complex salts of the respective amino acids with the metal ions, mixing an effluent eluate solution leaving the column with a color-developing solution containing ninhydrin, its reducing agent, and an organic solvent in an amount of 60 to by volume, heating the resulting mixutre solution in a reactor to carry out a colordeveloping reaction, continuously passing the resulting colored substance including Oxfordauer-mine to a flow cell, where light is projected onto the solution and light absorption by the colored substance is effected, detecting a quantity of light, converting the detected quantity of light to electric signals and recording the electric signals, and the reaction system within the analytical system being isolated from surrounding atmosphere
  • a method for automatically analyzing amino acids according to claim 5, wherein about absorbance is reached when ascorbic acid is used in a concentration of about 0.8 X 10 mole per liter.
  • a method for automatically analyzing amino acids by liquid chromatography which comprises passing amino acids together with an eluting solution through a separation column packed with ion exchange resins, separating the amino acid into their respective amino acid by differences in migration speeds of the amino acids in the column, allowing the separated amino acids to react with a color-developing solution containing ninhydrin, its reducing agent and an organic solvent by heating thereby to obtain colored substances, leading the resulting solution containing said colored substances continuously to a means for detecting absorbances and transmitting detected signals from the means for detecting absorbances to a recording means, the improvement wherein the reducing agent used is ascorbic acid and the color-developing solution, when deteriorated by oxidation, is recovered by adding ascorbic acid to said solution in order to convert a part of ninhydrin into hydrindantin.
  • a method for automatically analyzing amino acids by liquid chromatography which comprises passing amino acids together with an eluting solution through a separation column packed with ion exchange resins, separating the amino acid into their respective amino acids by differences in migration speeds of the amino acid in the column, allowing the separated amino acids to react with a color-developing solution containing ninhydrin, its reducing agent and an organic solvent by heating thereby to obtain colored substances, leading the resulting solution containing said colored substances continuously to means for detecting absorbances and transmitting detected signals from the means for detecting absorbances to recording means, the improvement wherein the color-developing solution, when deteriorated by air oxidation, is recovered by adding ascorbic acid to said solution in an amount effective to convert a part of ninhydrin into hydrindantin.

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US00212197A 1970-12-25 1971-12-27 Method of automatically analyzing amino acids by liquid chromatography and color-developing solution therefor Expired - Lifetime US3778230A (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3918907A (en) * 1973-10-29 1975-11-11 Beckman Instruments Inc Micro automatic amino acid analysis process and system
US3934981A (en) * 1972-11-02 1976-01-27 The Dow Chemical Company Apparatus for detection of chloromethyl methyl ether or bis-chloromethyl ether
WO1990010873A1 (de) * 1989-03-11 1990-09-20 MERCK Patent Gesellschaft mit beschränkter Haftung Puffer- und reagenzsystem für die trennung von aminosäuren
US5626738A (en) * 1995-11-17 1997-05-06 American Health Foundation Methods for the separation and detection of nitrosothiols
DE202013010433U1 (de) 2012-11-21 2014-02-13 Jpp Chromatography Limited Neues Reagenz
US20140141520A1 (en) * 2012-11-21 2014-05-22 Jpp Chromatography Limited Method for Analysing Amino Acids and a Reagent for Use with the Same
WO2015189578A1 (en) * 2014-06-13 2015-12-17 Aaa Scientific Limited Apparatus and method for preparing a ninhydrin reagent

Citations (2)

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US3230048A (en) * 1962-08-17 1966-01-18 Technicon Chromatography Corp Chromatography analysis apparatus
US3506403A (en) * 1967-12-19 1970-04-14 Hoffmann La Roche Colorimetric method for determining iron in fluids

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US3230048A (en) * 1962-08-17 1966-01-18 Technicon Chromatography Corp Chromatography analysis apparatus
US3506403A (en) * 1967-12-19 1970-04-14 Hoffmann La Roche Colorimetric method for determining iron in fluids

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Title
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Merck Index, 7th Edn. 1960, p. 106. *
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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3934981A (en) * 1972-11-02 1976-01-27 The Dow Chemical Company Apparatus for detection of chloromethyl methyl ether or bis-chloromethyl ether
US3918907A (en) * 1973-10-29 1975-11-11 Beckman Instruments Inc Micro automatic amino acid analysis process and system
WO1990010873A1 (de) * 1989-03-11 1990-09-20 MERCK Patent Gesellschaft mit beschränkter Haftung Puffer- und reagenzsystem für die trennung von aminosäuren
US5626738A (en) * 1995-11-17 1997-05-06 American Health Foundation Methods for the separation and detection of nitrosothiols
EP2735877A1 (en) * 2012-11-21 2014-05-28 JPP Chromatography Limited A novel ninhydrin reagent for analysing nitrogen-containing compounds
GB2508147B (en) * 2012-11-21 2016-06-01 Jpp Chromatography Ltd Ninhydrin reagent for use in a method of analysing nitrogen-containing compounds
US20140141520A1 (en) * 2012-11-21 2014-05-22 Jpp Chromatography Limited Method for Analysing Amino Acids and a Reagent for Use with the Same
EP2735876A1 (en) 2012-11-21 2014-05-28 JPP Chromatography Limited A method for analysing amino acids and reagent for use with the same
DE202013010433U1 (de) 2012-11-21 2014-02-13 Jpp Chromatography Limited Neues Reagenz
WO2014080158A1 (en) * 2012-11-21 2014-05-30 Jpp Chromatography Limited A novel ninhydrin reagent for analysing nitrogen-containing compounds
CN104919320A (zh) * 2012-11-21 2015-09-16 Jpp色谱有限公司 用于分析含氮化合物的新型茚三酮试剂
CN105122066B (zh) * 2012-11-21 2018-05-01 Jpp色谱有限公司 分析氨基酸的方法及其所用的试剂
US9250190B2 (en) * 2012-11-21 2016-02-02 Jpp Chromatography Limited Reagent and method using the same
US9250191B2 (en) * 2012-11-21 2016-02-02 Jpp Chromatography Limited Method for analysing amino acids and a reagent for use with the same
US20140141519A1 (en) * 2012-11-21 2014-05-22 Jpp Chromatography Limited Novel Reagent and Method Using the Same
CN104919320B (zh) * 2012-11-21 2017-05-31 Jpp色谱有限公司 用于分析含氮化合物的新型茚三酮试剂
WO2015189578A1 (en) * 2014-06-13 2015-12-17 Aaa Scientific Limited Apparatus and method for preparing a ninhydrin reagent
US9977027B2 (en) * 2014-06-13 2018-05-22 Aaa Scientific Limited Apparatus and method for preparing a ninhydrin reagent

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