US 3694160 A
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Sept. 26, 1972 HISAYUKI SAGUSA ETAL 3,694,160
PROCESS FOR ANALYSIS OF AMINO ACIDS BY LIQUID CHROMATOGRAPHY AND COLOR DEVELOPING MEDIUM ADAPTED THEREFOR Filed March 9, 1971 2 Sheets-Sheet 1 FIG.
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PROCESS FOR ANALYSIS OF AMINO ACIDS BY LIQUID CHROMATOGRAPHY AND COLOR DEVELOPING MEDIUM ADAPTED THEREFOR Filed March 9, 1971 2 Sheets-Sheet 2 AMOUNT OF Cy 074 40050 (9/2) INVENTORS BY 0105 mam A/ZHLQL,
ATTORNEY$ United States Patent 3,694,160 PROCESS FOR ANALYSIS OF AMINO ACIDS BY LIQUID CHROMATOGRAPHY AND COLOR DE- VELOPING MEDIUM ADAPTED THEREFOR Hisayuki Sagusa, Katsuta, and Seiji Takeuchi and Yoshijiro Arikawa, Hitachi, Japan, assignors to Hitachi, Ltd., Tokyo, Japan Filed Mar. 9, 1971, Ser. No. 122,356 Claims priority, appligation Japan, Mar. 11, 1970,
Int. Cl. G01u 31/08, 31/22 US. Cl. 23-230 R 7 Claims ABSTRACT OF THE DISCLOSURE This invention relates to an important in a method for analyzing amino acids by liquid chromatography, and more particularly to an improvement in a liquid chromatography using ninhydrine as a color-developing agent for detecting the amino acids.
In the liquid chromatography of amino acids, it is Well known that amino acids are quantitatively determined by passing a sample of amino acids through a column packed with ion exchange resins, separating the amino acids into their respective components by utilizing differences in the migration speeds of the respective amino acid components in the column, allowing the separated amino acids to react with a color-developing agent and by measuring peaks of the developed colors of the reacted amino acids.
Heretofore, a classical method based on a manual operation by an analyzing personnel called batch method has been available to effect color development of a sample of amino acids passed through the column, that is, an eluate solution, and effect a colorimetry of the eluate solution, but recently a method for automatic colorimetry based on the use of a through-flow type photometer capable of converting the detected peaks of colored substances into electric signals and recording the same has been mainly used. For this automatic liquid chromatography, an ion exchange method such as Moor-Stein process, and a ligand method invented by one of the present inventors of the present invention and disclosed in US. Pat. application Ser. No. 742,993 entitled Method of Separating Mixture by Liquid Chromatography and filed May 14, 1968, now Patent No. 3,630,681 are now used in practice.
According to the Moor-Stein method, a sample of amino acid mixture is separated into the respective amino acid components by differences in the migration speeds of the respective amino acid components when the amino acids pass through cation exchange resins if there are differences in distribution coeflicient toward the cation exchange resins by the differences in degree of dissociation of the carboxyl groups of the amino acids. The thus separated amino acids are allowed to react with a reducing agent, for example, a color-developing solution containing stannous chloride, SnCl and ninhydrine, and then 3,694,160 Patented Sept. 26, 1972 absorbances of colored substances resulting from the color-developing reaction are measured. For example, the maximum absorption of Ruhemann purple is utilized for a-amino acid.
On the other hand, according to the ligand method, a metal salt-type column, that is, ion exchange resin on which weakly basic metal ions, such as, Ni; Cu, Co++, Ca Hg++, Zn++, La+++, capable of forming a complex salt with amino acids, etc., is adsorbed, is used, and the amino acids are separated into their respective amino acid components by difierences in the abilities of complex salt formation between various amino acids and said metal ions when the amino acids are passed through the colmn. In the ligand method, color-developing agents other than ninhydrine can be used, but it is preferable, in view of the reactivities with about 20 kinds of amino acids resulting from the hydrolysis of protein, to use ninhydrine. Particularly in the automatic analysis, it is very important in the efiiciency of analysis and performance of apparatus that allamino acids can be analyzed only with one colordeveloping agent.
Ninhydrine is readily oxidized when exposed to air, and therefore it is necessary to keep ninhydrine in a dark, cold place, while isolating it from air. Since an analytical system can be brought into a closed system in the automatic analysis, there is no disadvantage in using the ninhydrine.
A typical color-developing solution which has been employed has the following composition:
Sodium acetate: 82 g./l.
Glacial acetic acid: 25 ml./l. (pH 5.5) Methyl cellosolve: 750 ml./l. Ninhydrine; 20 g./l. (0.1 M) SnCl -2H O: 0.4 g./l..(1.8X10" M) Sodium acetate and glacial acetic acid are dissolved in 250 ml. of water.
Reaction of ninhydrine with an amino acid can be represented by the following formula, where an intermediate reaction mechanism is omitted:
CO 00 U C=N-C U RCHO C02 Ruhemarm purple However, actual reaction is not a one-stage reaction as shown above, but its reaction mechanism can be assumed as follows: at first one molecule of the amino acid reacts with one molecule of ninhydrine to form diketohydrindanine,
through such intermdeiate products as Schitis bases, and further one molecule of ninhydrine is combined with the diketohydrindamine to form Ruhemann purple. Since the intermediate product is a reducible substance, it will be consumed and the color-developing reaction will be disturbed, if there is an oxidizable substance in the colordeveloping reaction system. The oxidizable substances which enter into the reaction system are, for example, dissolved oxygen, peroxide of methylcellosolve used as a solvent for ninhydrine and transition metals. It is easy to remove the dissolved oxygen or the peroxide from the reaction system, but it is impossible or very difiicult to completely remove a very small amount of heavy metal ions such as Fe+++, Cu++, Mn++, Cr+++, which enter into the reaction system at the preparation of an eluting solution or color-developing solution or from the separation system, etc.
Furthermore, the heavy metal ions form complex salts with the amino acids or the intermediate product formed by reaction of the ninhydrine, i.e. hydrindantin, and Ruhemann purple, etc., and disturb the color development. Further, transition metals, among the heavy metals, oxidize a reducing agent as zinc powder, SnCl etc., which is added to the reaction system to form hydrindantin, and consume Zn and Sn++ as the reducing agent and form Zn++ and Sn++++. Therefore, the amount of hydrindantin is reduced, and the efiiciency of color development is lowered.
An object of the present invention is to provide an improved liquid chromatography, which can attain a high efllciency of color development.
Another object of the persent invention is to eliminate an influence of oxidizable substances that disturb the color-developing reaction, particularly heavy metal ions in the liquid chromatography using ninhydrine as a colordeveloping agent.
Still another object of the present invention is to provide a useful means for masking the heavy metal ions in an automatic analysis of amino acids by liquid chromatography.
Further object of the present invention is to provide a useful means for masking the heavy metal ions in a liquid chromatography based on the ligand method, using a metal salt-type column.
Still further object of the present invention is to pro vide a useful ninhydrine color-developing solution in a liquid chromatography by the ligand method.
To attain these and other objects which will be seen from the following detailed description, the present invention provides an improved method of adding naphthenic diaminotetraacetic acid to a color-developing system to mask heavy metals that disturb reaction of amino acids with ninhydrine in a method for analyzing amino acid by liquid chromatography, which comprises separating various amino acids into their respective components by differences in migration speeds of the amino acids in a column packed with ion exchange resins, allowing the thus separated amino acids to react with a ninhydrine color-developing solution thereby to form colored substances, and efiecting colorimetry of the colored substances.
The present invention is based on a novel concept of masking heavy metals that disturb the color-developing reaction between amino acids and ninhydrine, by a suitable complex-forming agent. However, the restrictions required for the complex-forming agent are too various and strict as a practical matter to make a proper selection of the complex-forming agent with ease. For example, it is, of course, necessary to select a complex-forming agent that gives no unfavorable influence upon the ninhydrine in the color-developing solution and has an ability good enough to form complexes with heavy metal ions, but in the liquid chromatography based on the ligand method, an organic solvent, for example, 75 vol. percent methylcellosolve, is used to sufficiently dissolve hydrindantin. That is, the compex-forming agent must be sufficiently dissolved in such an organic solvent. In this respect, ethylenediaminetetraacetic acid, a well-known complex-forming agent, cannot be used, because it cannot be dissolved sufficiently in the organic solvent. Further, glycoletherdiaminetetraacetic acid or diethylenetriaminepentaacetic acid is likewise insoluble in the solvent. Amine complex-forming agents such as ethanol amine are positive to the ninhydrine, and both amine complex-forming agent and ninhydrine react with each other. Phenanthroline or tripyridyltriazine forms colored compounds with most of metal ions and thus cannot be used.
In brief, the conditions required for the complex-forming agent or masking agent are as follows:
(1) It must have a good ability to form complex compounds with the heavy metal ions.
(2) It must be negative to the color development of ninhydrine.
(3) The resulting complex compounds with the heavy metal ions must be colorless.
(4) It must be sufliciently soluble in such an organic solvent as methyleellosolve.
The present inventors have found that naphthenic diaminetetraacetic acids are suitable as the complex-forming agent that can meet these conditions. The naphthenic diaminetetraacetic acids include cyclohexanediaminetetraacetic acid (which will be hereinafter referred to as CyDTA), cyclohexanoldiaminetetraacetic acid, methylcyclohexanediaminetetraacetic acid, cyclopentanediaminetetraacetic acid, etc.
In the ion exchange method, the naphthenic diaminetetraacetic acids can be added to the ninhydrine colordeveloping solution and/or eluting solution, but in the ligand method, it is practically not suitable to add this complex-forming agent to the eluting solution, because, when the complex-forming agent is passed through the metal salt-type column, it forms complex salts with the metals adsorbed on the ion exchange resin, and the metal ions that are to form complex salts with the amino acids are consumed. In other words, an equilibrium of the column is brought to disorder, when the naphthenic diaminetetraacetic acid is added to the eluting solution. Therefore, such a procedure must be avoided.
In the ligand method, the color-developing reaction is sometimes disturbed, depending upon the kind of metal ions to be adsorbed on the ion exchange resins. That is to say, metal ions form chelate compounds with amino acids in the column, and the resulting chelate compounds react with hydrindantin to form reaction products of hydrindantin with amino acids, that is, Ruhemann purple. However, the liberated metal ions sometimes disturb the formation reaction of Ruhe'mann purple. In that case, the naphthenic diaminotetraacetic acid is added thereto in an amount enough to mask the metal ions. In other words, when a metal salt-type column on which metals disturbing the color development, for example, Cu+ are added, is used, the naphthenic diaminetetraacetic acid is added thereto in an amount necessary for masking all the heavy metal ions to be joined as impurities and the metal ions to be eluted from the column. However, Zinc ion gives no influence upon hydrindantin or the reducing agent, and thus in that case, the naphthenic diaminetetraacetic acid is added thereto only in an amount enough to mask the heavy metal ions and the metal ions used as the reducing agent.
Embodiments of the present invention will be explained, by reference to the accompanying drawings:
FIG. 1 is a schematic diagram of an automatic analyzing apparatus used in the present invention;
FIG. 2 is a schematic view of a through-flow type detector; and
FIG. 3 is a graph showing a relation between the color development ratio and the amount of cyclohexanediaminetetraacetic acid added according to the present invention.
In FIG. 1, strongly acidic resins of sulfonic acid formed within a column 1 are brought into an equilibrium with an eluting solution containing 10= M./l. of Zn++. The eluting solution stored in a vessel 3 is fed to the columnby a pump 2. The eluting solution contains 10 M./l. of Zn++ and also contains an acetic acid-sodium acetate buffer solution (pH 5.2) adjusted so that the Na+ concentration may be 0.9 M./l. A reactor 8, a through-flow type photometer 4 and a recorder 5 are connected in succession to the etfiuent side of the column 1. In a vessel 7, a ninhydrine color-developing solution is stored, and is added to a passage for a sample solution leaving the column as an eluate efiiuent by means of a pump 6. The composition of a color-developing solution is 82 g./l. of sodium acetate, 25 ml./l.) of glacial acetic acid (pH 5.5), 750 ml./l. (0.1 M./l.) of methylcellosolve, 20 g./l. (0.1 M./l.) of ninhydrine, 0.4 g./l. (1.8 10- M./l.) of SnCl; and CyDTA, the balance being Water. The feeding rates of the pumps 2 and 6 are 90 ml./hour and 45 ml./ hour, respectively.
The eluting solution is allowed to pass through the column 1 by means of the pump 2 to bring the system of Zn++-adsorbed ion exchange resins into an equilibrium. Then, an amino acid sample is added to the top of the column 1. The amino acid sample is separated into the respective amino acid components by dilferences in coordinate-bonding forces between the respective amino acid components and Zn++ adsorbed on the ion exchange resins, that is, by difference in the abilities to form complex salts, and further by the resulting differences in the migration speeds within the column 1, and flows as an eluate eflluent from the bottom, that is, the effluent side of the column 1. The color-developing solution is added to the eluate efiluent by means of the pump 6, and then the resulting mixture is led to the reactor 8 and heated, whereby ninhydrine is allowed to react with the amino acids (more exactly hydrindantin formed by the reduction of ninhydrine by the reducing agent SnCl in the reactor reacts with amino acids) and form colored substances. The solution containing the colored substances is led to the through-flow type photometer 4, where absorbances of the colored substances corresponding to the respective amino acid components are continuously detected. The thus detected absorbances are converted to electric signals, which are recorded in a recorder 5.
As shown in FIG. 2, the through-flow photometer is provided with a flow cell 12, through which the solution containing the colored substances is allowed to pass. When light is projected onto the fiow cell from a light source 11, the light is absorbed by the colored substances, and the transmitted light is detected by a light-detecting means 14 such as a photocell, etc. The light signal is converted to electric signals, which are amplified by an amplifier 16 and led to the recorder 5 as the electric signals. In the automatic analytical method, a means for continuously detecting and analyzing light signals corresponding 'to the respective amino acid components by means of such a flow cell is indispensable.
In FIG. 3, color development ratio of a ninhydrine color-developing solution is shown when an amount of CyDTA, which is added as the complex-forming agent, is changed. The compositions of the eluting solution and the color-developing solution and the feeding rates are the same as above. The amount of amino acid sample is 0.4 ,ug. The reaction in the reactor is carried out at 115 C. for 3 minutes. The heating medium for the reactor is glycerine. In FIG. 3, the color development ratio is represented by an area of a peak of the recorded absorbance, in other words, by a product, /2 h. W that is, a half height of peak, /2 h., multiplied by a peak width, W at said h'alf height of the peak (A h.)
In FIG. 3, a curve I shows the color development ratio for glycerine, a curve II tryrosine, a curve III arginine, and a curve IV ammonia. It should be noted that ammonia is included in the amino acid as a resemblance to the amino acid. It is seen from the graph that the color development ratio is increased by an increase in the amount of CyDTA added, but the color development ratio becomes constant, when the amount of CyDTA exceeds 0.6 g./l. (1.8)( M./l.). This means that all the heavy metal ions are masked. Even if CyDTA is added thereto in excess, it gives no disturbance to the color-development reaction. That is, CyDTA is easy to use, and therefore it is preferable to use more than 0.6 g./l. of CyDTA in order to completely mask the heavy metals. If the color development ratio is assumed when no CyDTA is added, the color development ratios, when 0.6 g./l. or more of CyDTA is added thereto, are 126% for glycine, 138% for tryosene, 140% for arginine and 166% for ammonia. That is, the color development ratios are considerably increased by adding @DTA thereto.
In the foregoing example, an embodiment of using SnCl as a reducing agent is exemplified. However, it has been found that metal zinc powders likewise can be used. The metal zinc powders have a higher solubility in the color-developing solution that SnCl and therefore have such an advantage that the metal zinc powders never produce any precipitate.
What is claimed is:
1. In a method for 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 ninhydrine and its reducing agent by heating thereby to obtain colored substances and analyzing the amino acids by hues of the colored substances, an improvement comprising adding a naphthenic diaminotetraacetic acid to the color-developing solution as an agent for masking heavy metal ions.
2. In 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 diiferences in migration speeds of the amino acids in the column, allowing the separated amino acids to react with a color-developing solution containing ninhydrine and its reducing agent 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, an improvement comprising adding a proper amount of a naphthenic diaminotetraacetic acid as an agent for masking metal ions capable of disturbing the formation of said colored substances, to an analytical system at least at a stage prior to the formation reaction of said colored substances.
3. In 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 effiuent eluate solution leaving the column with a colordeveloping solution containing ninhydrine and its reducing agent, heating the resulting mixture solution in a reactor to carry out a color-developing reaction, continuously passing the resulting colored substance Ruhemann purple 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, reaction system within the analytical system being isolated from surrounding atmosphere, an improvement comprising adding an eifective amount of a naphthenic diaminotetraacetic acid to the color-developing solution as an agent for masking metal ions capable of disturbing the color development reaction.
4. An improvement according to claim 3, wherein the metal ions adsorbed on the ion exchange resins in the column are non-oxidizable metal ions incapable of disturbing the color development reaction, and the amount of the naphthenic diaminotetraacetic acid to be added is at least about 1.8 10 moles/l.
5. An improvement according to claim 3, wherein the amount of the naphthenic diaminotetraacetic acid is an amount necessary for forming complex compounds with all heavy metal ions entered in the system as impurities and metal ions adsorbed on the ion exchange resins in the column when the latter metal ions are oxidizable toward a reduction product of ninhydrine, hydrindantin.
6. A color-developing solution for analyzing amino acids by liquid chromatography, which comprises ninhydrine, a buffer agent, a reducing agent for ninhydrine, an
5 aminotetraacetic acid is contained.
References Cited UNITED STATES PATENTS 9/1968 Skeggs et a1. 23-230 R MORRIS O. WOLK, Primary Examiner R. M. REESE, Assistant Examiner