JPH0310905B2 - - Google Patents
Info
- Publication number
- JPH0310905B2 JPH0310905B2 JP9375782A JP9375782A JPH0310905B2 JP H0310905 B2 JPH0310905 B2 JP H0310905B2 JP 9375782 A JP9375782 A JP 9375782A JP 9375782 A JP9375782 A JP 9375782A JP H0310905 B2 JPH0310905 B2 JP H0310905B2
- Authority
- JP
- Japan
- Prior art keywords
- exchange resin
- anion exchange
- amino acids
- amino acid
- resin column
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 150000001413 amino acids Chemical class 0.000 claims description 56
- 230000002378 acidificating effect Effects 0.000 claims description 40
- 239000003957 anion exchange resin Substances 0.000 claims description 23
- 239000000872 buffer Substances 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 238000004040 coloring Methods 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 3
- 238000011161 development Methods 0.000 claims description 2
- 238000004451 qualitative analysis Methods 0.000 claims 1
- 238000004445 quantitative analysis Methods 0.000 claims 1
- 235000001014 amino acid Nutrition 0.000 description 49
- 238000004458 analytical method Methods 0.000 description 14
- XVOYSCVBGLVSOL-UHFFFAOYSA-N cysteic acid Chemical compound OC(=O)C(N)CS(O)(=O)=O XVOYSCVBGLVSOL-UHFFFAOYSA-N 0.000 description 8
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical group O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 7
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 6
- 239000003729 cation exchange resin Substances 0.000 description 6
- 230000000717 retained effect Effects 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- ADVPTQAUNPRNPO-REOHCLBHSA-N 3-sulfino-L-alanine Chemical compound OC(=O)[C@@H](N)C[S@@](O)=O ADVPTQAUNPRNPO-REOHCLBHSA-N 0.000 description 4
- ADVPTQAUNPRNPO-UHFFFAOYSA-N alpha-amino-beta-sulfino-propionic acid Natural products OC(=O)C(N)CS(O)=O ADVPTQAUNPRNPO-UHFFFAOYSA-N 0.000 description 4
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229960003080 taurine Drugs 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- VBOQYPQEPHKASR-VKHMYHEASA-N L-homocysteic acid Chemical compound OC(=O)[C@@H](N)CCS(O)(=O)=O VBOQYPQEPHKASR-VKHMYHEASA-N 0.000 description 1
- QEFRNWWLZKMPFJ-ZXPFJRLXSA-N L-methionine (R)-S-oxide Chemical compound C[S@@](=O)CC[C@H]([NH3+])C([O-])=O QEFRNWWLZKMPFJ-ZXPFJRLXSA-N 0.000 description 1
- UCUNFLYVYCGDHP-BYPYZUCNSA-N L-methionine sulfone Chemical compound CS(=O)(=O)CC[C@H](N)C(O)=O UCUNFLYVYCGDHP-BYPYZUCNSA-N 0.000 description 1
- QEFRNWWLZKMPFJ-UHFFFAOYSA-N L-methionine sulphoxide Natural products CS(=O)CCC(N)C(O)=O QEFRNWWLZKMPFJ-UHFFFAOYSA-N 0.000 description 1
- SUHOOTKUPISOBE-UHFFFAOYSA-N O-phosphoethanolamine Chemical compound NCCOP(O)(O)=O SUHOOTKUPISOBE-UHFFFAOYSA-N 0.000 description 1
- BZQFBWGGLXLEPQ-UHFFFAOYSA-N O-phosphoryl-L-serine Natural products OC(=O)C(N)COP(O)(O)=O BZQFBWGGLXLEPQ-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 101000911055 Schizosaccharomyces pombe (strain 972 / ATCC 24843) Probable S-(hydroxymethyl)glutathione dehydrogenase 1 Proteins 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O ammonium group Chemical group [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229940023913 cation exchange resins Drugs 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229950006137 dexfosfoserine Drugs 0.000 description 1
- HXOLFXRMWWHLMH-UHFFFAOYSA-L disodium boric acid carbonate Chemical compound [Na+].[Na+].OB(O)O.[O-]C([O-])=O HXOLFXRMWWHLMH-UHFFFAOYSA-L 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- UHBYWPGGCSDKFX-VKHMYHEASA-N gamma-carboxy-L-glutamic acid Chemical compound OC(=O)[C@@H](N)CC(C(O)=O)C(O)=O UHBYWPGGCSDKFX-VKHMYHEASA-N 0.000 description 1
- XHMJOUIAFHJHBW-VFUOTHLCSA-N glucosamine 6-phosphate Chemical compound N[C@H]1[C@H](O)O[C@H](COP(O)(O)=O)[C@H](O)[C@@H]1O XHMJOUIAFHJHBW-VFUOTHLCSA-N 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229940111688 monobasic potassium phosphate Drugs 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 1
- 229920000779 poly(divinylbenzene) Polymers 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
Classifications
-
- 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
- G01N30/96—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 using ion-exchange
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
【発明の詳細な説明】
この発明は、強酸性アミノ酸分析方法及び装置
に関する。さらに詳しくは、従来分離分析の困難
であつた強酸性アミノ酸の簡便な分離分析方法及
びその装置に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method and apparatus for analyzing strongly acidic amino acids. More specifically, the present invention relates to a simple method and apparatus for separating and analyzing strongly acidic amino acids, which have traditionally been difficult to separate and analyze.
なお、「強酸性アミノ酸」とは、酸性アミノ酸
の中でもことに強酸性のものを示し、具体的には
強酸性陽イオン交換樹脂カラムを用い、PH3.25で
かつナトリウム濃度0.2規定のクエン酸ナトリウ
ム緩衝液を用いてアミノ酸類を溶出分離する際
に、アスパラギン酸以前に溶出するアミノ酸類を
いう。 Note that "strongly acidic amino acids" refer to particularly strongly acidic ones among acidic amino acids, and specifically, using a strongly acidic cation exchange resin column, sodium citrate with a pH of 3.25 and a sodium concentration of 0.2N is used. When amino acids are eluted and separated using a buffer, they are the amino acids that elute before aspartic acid.
従来、アミノ酸含有試料をバツフアーと共に陽
イオン交換樹脂カラムに通した後、アミノ酸の発
色又は発蛍光剤を混合して連続的に吸光又は蛍光
分析に供するアミノ酸分析方法が知られている。
しかしながらかような方法においては、タウリ
ン、システインスルフイン酸、システイン酸、ホ
スホセリン、ホスホエタノールアミン、γ−カル
ボキシグルタミン酸、ホモシステイン酸、グルコ
サミン酸、グルコサミン−６−リン酸、メチオニ
ンスルホン、メチオニンスルホキシド等の強酸性
アミノ酸類はカラムにほとんど保持されず分離す
ることが困難であつた。 Conventionally, an amino acid analysis method is known in which an amino acid-containing sample is passed through a cation exchange resin column together with a buffer, and then a coloring or fluorescent agent for the amino acid is mixed therein and the sample is continuously subjected to absorption or fluorescence analysis.
However, in such methods, taurine, cysteine sulfinic acid, cysteic acid, phosphoserine, phosphoethanolamine, γ-carboxyglutamic acid, homocysteic acid, glucosamic acid, glucosamine-6-phosphate, methionine sulfone, methionine sulfoxide, etc. Strongly acidic amino acids were hardly retained on the column and were difficult to separate.
この点に関し、バツフアーをPH２以下の強酸性
に調節して強酸性アミノ酸をカラムに保持させる
提案もあるが、この場合、他のアミノ酸はカラム
にさらに強く保持されて分析後にも残留するた
め、アルカリ洗浄及び長時間のカラムエージング
（アルカリ洗浄後、酸性バツフアーを一定時間カ
ラムに流しておく処理）が必要であり、ことに連
続的な分析には適さない。 Regarding this point, there is a proposal to retain strongly acidic amino acids in the column by adjusting the buffer to be strongly acidic below PH2, but in this case, other amino acids are retained even more strongly in the column and remain even after analysis, so the alkaline It requires washing and long-term column aging (processing in which an acidic buffer is allowed to flow through the column for a certain period of time after alkaline washing), making it particularly unsuitable for continuous analysis.
一方、バツフアーが前記のごとく強酸性に調節
された条件で、陽イオン交換樹脂カラムの代わり
に陰イオン交換樹脂カラムを用いることにより、
アスパラギン酸までの酸性アミノ酸を分離できる
ことも知られている（ダイヤイオン：DIAION、
イオン交換樹脂・合成吸着剤マニユアル〔〕
〔改訂版〕、三菱化成工業(株)昭和52年、143−147
頁）。そしてこの方法を強酸性アミノ酸の分離分
析に適用することも考えられるが、この場合にお
いても任意のアミノ酸含有試料から強酸性アミノ
酸類を選択的にかつ迅速に分離分析することがで
きなかつた。加えて、かかる陰イオン交換樹脂は
通常、アンモニウム基を交換基として有し徐々に
アミンやアンモニア等を放出する性質を有するた
め、カラムからの流出液に連続的に発色剤や発蛍
光剤等の反応試薬を混合する方式のアミノ酸分析
装置へ適用することは、アミノ酸測定感度や精度
の点で不利と考えられており、実際上、アミノ酸
分析装置には用いられていなかつた。 On the other hand, by using an anion exchange resin column instead of a cation exchange resin column under conditions where the buffer is adjusted to be strongly acidic as described above,
It is also known that acidic amino acids up to aspartic acid can be separated (DIAION,
Ion exchange resin/synthetic adsorbent manual []
[Revised edition], Mitsubishi Chemical Industries, Ltd., 1972, 143-147
page). It is also possible to apply this method to the separation and analysis of strongly acidic amino acids, but even in this case, it has not been possible to selectively and quickly separate and analyze strongly acidic amino acids from any amino acid-containing sample. In addition, such anion exchange resins usually have an ammonium group as an exchange group and have the property of gradually releasing amines, ammonia, etc., so they continuously add color formers, fluorescent agents, etc. to the effluent from the column. Application to an amino acid analyzer that mixes reaction reagents is considered disadvantageous in terms of amino acid measurement sensitivity and accuracy, and has not actually been used in amino acid analyzers.
この発明は、かような問題点を解消すべくなさ
れたものであり、強酸性アミノ酸類を簡便な操作
で明確かつ選択的に分離でき、さらに後処理する
ことなく連続的に分析できる強酸性アミノ酸分析
方法及びその実施に好適な分析装置を提供するも
のである。この発明の発明者らは、従来のアミノ
酸分析で常識化している陽イオン交換樹脂の代り
に、全く逆の陰イオン交換樹脂を用いると共に、
この際のバツフアーとして陰イオン交換樹脂に従
来適用された強酸性バツフアーではなく、陽イオ
ン交換樹脂に適用される酸性バツフアーを用いる
ことにより、強酸性アミノ酸を選択的にかつ明確
にしかも迅速に分離でき、かつカラムの後処理が
不要である事実を見出し、この発明に到達した。 This invention was made in order to solve these problems, and it provides a strong acidic amino acid that can be clearly and selectively separated with a simple operation and that can be analyzed continuously without post-processing. The present invention provides an analysis method and an analysis device suitable for carrying out the analysis method. The inventors of this invention used a completely opposite anion exchange resin instead of the cation exchange resin that is common in conventional amino acid analysis, and
By using an acidic buffer applied to cation exchange resins instead of the strong acid buffers conventionally applied to anion exchange resins, strongly acidic amino acids can be selectively, clearly, and rapidly separated. , and the fact that post-treatment of the column is unnecessary, and the present invention was achieved.
かくしてこの発明によれば、アミノ酸含有試料
をバツフアーと共に陰イオン交換樹脂に通し次い
で流出液を発色又は発蛍光させることにより、ア
ミノ酸含有試料中の強酸性アミノ酸の定性又は定
量を行なうことを特徴とする強酸性アミノ酸分析
方法が提供される。さらに、試料導入部を備え陰
イオン交換樹脂カラムに接続するバツフアー供給
流路と、陰イオン交換樹脂カラムに延設される流
出液流路と、流出液流路と発色剤又は発蛍光剤供
給流路とを連結する混合部と、混合部に接続され
る吸光又は蛍光光度測定器から構成されてなる強
酸性アミノ酸分析装置が提供される。 Thus, the present invention is characterized in that strongly acidic amino acids in the amino acid-containing sample are qualitatively or quantitatively determined by passing the amino acid-containing sample together with a buffer through an anion exchange resin and then causing the effluent to develop color or fluoresce. A method for analyzing strongly acidic amino acids is provided. Furthermore, a buffer supply flow path including a sample introduction part and connected to the anion exchange resin column, an effluent flow path extending to the anion exchange resin column, an effluent flow path and a coloring agent or fluorescent agent supply flow. A strongly acidic amino acid analyzer is provided, which is comprised of a mixing section that connects to the mixing section, and an absorption or fluorometer that is connected to the mixing section.
この発明の方法に用いる陰イオン交換樹脂とし
ては、強塩基性陰イオン交換樹脂が好ましくより
具体的には、スチレン−ジビニルベンゼン共重合
体の担体に４級アンモニウム基を化学結合させた
樹脂等が挙げられる。また、アミノ酸含有試料を
カラムに供給するバツフアーとしては、従来の陽
イオン交換樹脂カラムを用いたアミノ酸分析装置
で使用される酸性（すなわちPH３以上の酸領域）
バツフアー、例えばリン酸塩、クエン酸塩、酢酸
塩等のバツフアーがそのまま適用できる。 The anion exchange resin used in the method of this invention is preferably a strongly basic anion exchange resin, more specifically a resin in which a quaternary ammonium group is chemically bonded to a styrene-divinylbenzene copolymer carrier. Can be mentioned. In addition, as a buffer for supplying amino acid-containing samples to the column, acidic (that is, acidic region of PH3 or higher) used in amino acid analyzers using conventional cation exchange resin columns is used.
Buffers such as phosphate, citrate, acetate, etc. can be applied as such.
一方、カラムから流出した液は、順次、好まし
くはフラクシヨン毎に発色又は発蛍光処理され、
かような発色強度又は発蛍光強度を吸光光度計や
蛍光光度計で連続的に測定することにより強酸性
アミノ酸を定性又は定量することができる。上記
発色処理としては、アミノ酸を発色させうる発色
剤含有溶液を一定量加えるのが適当であり、例え
ば公知のニンヒドリン溶液が用いられる。一方、
発蛍光処理としては同様にポストラベル用発蛍光
試薬を一定量、流出液に加えるのが適当である。
該蛍光試薬として最も好ましいものはオルトフタ
ルアルデヒド（以下OPA）溶液である。OPA溶
液をアミノ酸分析に用いることは従来知られてい
るが従来法ではOPA溶液のバツフアーの塩濃度
やPHを極端に上げる必要があつたが、この発明に
おいては広範囲のOPA溶液、例えば塩濃度0.3〜
0.5モル程度でPH９〜10.5程度のものを適用する
ことができる。 On the other hand, the liquid flowing out from the column is sequentially, preferably fraction by fraction, subjected to color development or fluorescence treatment,
Strongly acidic amino acids can be qualitatively or quantitatively determined by continuously measuring the color intensity or fluorescence intensity using an absorptiometer or fluorometer. For the above-mentioned coloring treatment, it is appropriate to add a certain amount of a solution containing a coloring agent capable of coloring amino acids; for example, a known ninhydrin solution is used. on the other hand,
Similarly, for fluorescence treatment, it is appropriate to add a certain amount of a post-label fluorescence reagent to the effluent.
The most preferred fluorescent reagent is an ortho-phthalaldehyde (hereinafter referred to as OPA) solution. It has been known in the past to use OPA solutions for amino acid analysis, but in the conventional method it was necessary to extremely raise the salt concentration and pH of the buffer of the OPA solution. ~
About 0.5 mol and PH of about 9 to 10.5 can be applied.
このようなこの発明の方法によれば例えば、強
酸性アミノ酸の中でもことに分離困難なシステイ
ン酸及びシステインスルフイン酸を分離できる。 According to the method of the present invention, for example, cysteic acid and cysteine sulfinic acid, which are particularly difficult to separate among strongly acidic amino acids, can be separated.
さらに、陰イオン交換樹脂を用いた従来の方法
で保持される強酸性アミノ酸以外のアミノ酸類が
実質的に保持されない（実施例参照）ため、強酸
性アミノ酸をかつ迅速に分離分析することができ
る。従つて、従来のアミノ酸分析と並行させるこ
とにより、各種アミノ酸の定性又は定量が可能と
なる。 Furthermore, since amino acids other than strongly acidic amino acids, which are retained by conventional methods using anion exchange resins, are not substantially retained (see Examples), strongly acidic amino acids can be rapidly separated and analyzed. Therefore, in parallel with conventional amino acid analysis, it becomes possible to qualitatively or quantitatively analyze various amino acids.
以下添付図面に従つてこの発明の装置を詳しく
説明する。 The apparatus of the present invention will be described in detail below with reference to the accompanying drawings.
第１図は、この発明の強酸性アミノ酸分析装置
の一例を示す構成説明図である。図においてこの
発明の装置は、セプタムやサンプラー等の試料導
入部２を備え陰イオン交換樹脂カラム３に接続す
るバツフアー供給流路１と、カラム３に延設され
る流出液流路４と、流路４と発蛍光剤供給流路５
とを連結するコイル流路状の混合部６と、混合部
６に接続されるフローセルを備えた蛍光光度測定
器７とから基本的に構成されてなる。上記、陰イ
オン交換樹脂カラム３は強塩基性陰イオン交換樹
脂である、ポリスチレン−ポリジビニルベンゼン
の単体に四級アンモニウム化物を化学結合させた
充填剤、を充填したカラム（ISA−70／52504；
(株)島津製作所社製）である。また、１２は0.05M
のKH2PO4溶液（PH約4.5）からなるバツフアー
を示し、その供給量は0.5ml／分であり、５２は
0.08％OPA含有ホウ酸−炭酸ナトリウム溶液か
らなる発蛍光剤を示しその供給量は0.3ml／分で
ある。また、１１及び５１は定量ポンプを示し、
７１はレコーダーを示す。 FIG. 1 is a configuration explanatory diagram showing an example of the strongly acidic amino acid analyzer of the present invention. In the figure, the apparatus of the present invention includes a buffer supply channel 1 which is equipped with a sample introduction part 2 such as a septum or a sampler, and is connected to an anion exchange resin column 3, an effluent channel 4 extending into the column 3, and a buffer supply channel 1 that is connected to an anion exchange resin column 3. Channel 4 and fluorescent agent supply channel 5
It basically consists of a mixing section 6 in the form of a coil flow path connecting the two, and a fluorescence photometer 7 connected to the mixing section 6 and equipped with a flow cell. The above-mentioned anion exchange resin column 3 is a column packed with a strongly basic anion exchange resin, a packing material in which a quaternary ammonium compound is chemically bonded to polystyrene-polydivinylbenzene (ISA-70/52504;
Manufactured by Shimadzu Corporation). Also, 12 is 0.05M
52 shows a buffer consisting of a KH 2 PO 4 solution (PH about 4.5), the feed rate is 0.5 ml/min, and 52 is
A fluorescent agent consisting of a boric acid-sodium carbonate solution containing 0.08% OPA is shown, and the feed rate is 0.3 ml/min. In addition, 11 and 51 indicate metering pumps,
71 indicates a recorder.
上記構成において、試料導入部２から、例えば
血液を除タンパク処理したアミノ酸含有試料が注
入され、バツフアー１２と共にカラム３に運ばれ
る。カラム３においてまず試料中の塩基性、中性
及び酸性アミノ酸のほとんどはこの順で速やかに
流出するが、強酸性アミノ酸はカラムに保持され
つつ徐々に溶離するため各強酸性アミノ酸に分離
しつつ他のアミノ酸と充分な保持時間をもつて流
出する。次いでこれらの各分離相はそれぞれ
OPAによつて混合部６で充分に発蛍光され、次
いで蛍光光度測定器７に送られてその各蛍光強度
が測定され、この値及び保持時間に基づいて強酸
性アミノ酸の各構成成分が定性及び／又は定量さ
れる。そして、分析を通じてカラム３は好適な酸
性条件下に保持されており、連続分析においても
カラムのアルカリ洗浄等の後処理を行なう必要は
ない。 In the above configuration, an amino acid-containing sample obtained by removing protein from blood, for example, is injected from the sample introduction section 2 and is transported to the column 3 together with the buffer 12. In column 3, most of the basic, neutral, and acidic amino acids in the sample quickly flow out in this order, but the strongly acidic amino acids are retained in the column and gradually elute, so they are separated into each strong acidic amino acid and other of amino acids and efflux with sufficient retention time. Each of these separated phases is then
Fluorescence is sufficiently emitted by OPA in the mixing section 6, and then sent to the fluorometer 7 to measure the fluorescence intensity. Based on this value and retention time, each component of the strongly acidic amino acid is qualitatively and / or quantified. The column 3 is maintained under suitable acidic conditions throughout the analysis, and there is no need to perform post-treatments such as alkaline washing of the column even in continuous analysis.
以上の具体例に示されるようにこの発明の方法
及び装置によれば、強酸性アミノ酸を簡便に分離
分析できることがわかる。 As shown in the above specific examples, it can be seen that according to the method and apparatus of the present invention, strongly acidic amino acids can be easily separated and analyzed.
なお、第２図に、この発明の装置を用いた、シ
ステイン酸、システインスルフイン酸を含有する
アミノ酸混合液の分析例（クロマトグラム）を示
す。このようにこの発明の方法及び装置によれ
ば、強酸性アミノ酸が良好に分離定量できること
がわかる。なお、図中、Ａはタウリンを、Ｂはシ
ステインスルフイン酸を、Ｃはシステイン酸をそ
れぞれ意味する。そして使用した装置は具体例に
示したものであり、条件は、移動層：第１リン酸
カリウム溶液（0.05M）、カラム圧：40Kg／cm2、
反応液；0.08％OPA含有緩衝液（流量0.3ml／
min）、反応コイル；内径0.5mm×2000mm、蛍光光
度検出器；FLD−１、カラム温度；50℃であつ
た。 FIG. 2 shows an analysis example (chromatogram) of an amino acid mixture containing cysteic acid and cysteine sulfinic acid using the apparatus of the present invention. As described above, it can be seen that according to the method and apparatus of the present invention, strongly acidic amino acids can be separated and quantified well. In addition, in the figure, A means taurine, B means cysteine sulfinic acid, and C means cysteic acid. The equipment used was as shown in the specific example, and the conditions were: mobile phase: monobasic potassium phosphate solution (0.05M), column pressure: 40Kg/cm 2 ,
Reaction solution; 0.08% OPA-containing buffer (flow rate 0.3ml/
min), reaction coil; inner diameter 0.5 mm x 2000 mm, fluorescence detector; FLD-1, column temperature: 50°C.
第１図は、この発明の強酸性アミノ酸分析方法
の実施に好適な装置の一例を示す構成説明図であ
り、第２図はこの発明を適用した際の測定例を示
すクロマトグラムである。
１……バツフアー供給流路、２……試料導入
部、３……陰イオン交換樹脂カラム、４……流出
液流路、５……発蛍光剤供給流路、６……混合
部、７……蛍光光度測定器、１１……定量ポン
プ、１２……バツフアー、５２……発蛍光剤、７
１……レコーダー。
FIG. 1 is a configuration explanatory diagram showing an example of an apparatus suitable for carrying out the strongly acidic amino acid analysis method of the present invention, and FIG. 2 is a chromatogram showing a measurement example when the present invention is applied. DESCRIPTION OF SYMBOLS 1... Buffer supply channel, 2... Sample introduction part, 3... Anion exchange resin column, 4... Effluent channel, 5... Fluorescent agent supply channel, 6... Mixing part, 7... ... Fluorescence photometer, 11 ... Metering pump, 12 ... Buffer, 52 ... Fluorescent agent, 7
1...Recorder.
Claims (1)
は発蛍光剤の混合部に接続し、アミノ酸含有試料
をPH３以上の酸性バツフアーと共に上記陰イオン
交換樹脂カラムに通して、この試料中に含有され
るアミノ酸中の強酸性アミノ酸の分離並びに発色
又は発蛍光に基づく定性又は定量を行なうことを
特徴とする強酸性アミノ酸分析方法。 ２ 陰イオン交換樹脂が、強塩基性陰イオン交換
樹脂である特許請求の範囲第１項記載の方法。 ３ 試料導入部を備え陰イオン交換樹脂カラムに
接続するPH３以上の酸性バツフアー供給流路と、
陰イオン交換樹脂カラムに延設される流出液流路
と、流出液流路と発色剤又は発蛍光剤供給流路と
を連結する混合部と、混合部に接続される吸光又
は蛍光光度測定器から構成されてなる強酸性アミ
ノ酸分析装置。 ４ 陰イオン交換樹脂カラムが、強塩基性陰イオ
ン交換樹脂カラムからなる特許請求の範囲第３項
記載の装置。[Claims] 1. An anion exchange resin column is connected to an amino acid coloring or fluorescent agent mixing section, and an amino acid-containing sample is passed through the anion exchange resin column together with an acidic buffer with a pH of 3 or more, and the 1. A method for analyzing strongly acidic amino acids, which is characterized by separating strongly acidic amino acids from amino acids contained in amino acids and performing qualitative or quantitative analysis based on color development or fluorescence. 2. The method according to claim 1, wherein the anion exchange resin is a strongly basic anion exchange resin. 3. An acidic buffer supply flow path with a pH of 3 or more that is equipped with a sample introduction section and connected to an anion exchange resin column;
An effluent channel extending into the anion exchange resin column, a mixing section connecting the effluent channel and a color former or fluorescent agent supply channel, and an absorption or fluorescence measuring device connected to the mixing section. A strong acidic amino acid analyzer consisting of: 4. The device according to claim 3, wherein the anion exchange resin column is a strongly basic anion exchange resin column.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9375782A JPS58210563A (en) | 1982-05-31 | 1982-05-31 | Analysis of strongly acidic amino-acid and its apparatus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9375782A JPS58210563A (en) | 1982-05-31 | 1982-05-31 | Analysis of strongly acidic amino-acid and its apparatus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58210563A JPS58210563A (en) | 1983-12-07 |
| JPH0310905B2 true JPH0310905B2 (en) | 1991-02-14 |
Family
ID=14091297
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP9375782A Granted JPS58210563A (en) | 1982-05-31 | 1982-05-31 | Analysis of strongly acidic amino-acid and its apparatus |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58210563A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2019194214A (en) * | 2015-02-27 | 2019-11-07 | オーピーバイオファクトリー株式会社 | Method for producing cacheromycin and derivative thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5149661A (en) * | 1988-06-08 | 1992-09-22 | Sarasep, Inc. | Fluid analysis with particulate reagent suspension |
| JPH02193068A (en) * | 1989-01-23 | 1990-07-30 | Hitachi Ltd | Chromatography separation method and chromatography apparatus |
-
1982
- 1982-05-31 JP JP9375782A patent/JPS58210563A/en active Granted
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2019194214A (en) * | 2015-02-27 | 2019-11-07 | オーピーバイオファクトリー株式会社 | Method for producing cacheromycin and derivative thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58210563A (en) | 1983-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| GB2115146A (en) | Method and apparatus for determination of anions | |
| Zapata et al. | Detection and quantification of neurotransmitters in dialysates | |
| Bohrer et al. | On-line separation and spectrophotometric determination of low levels of aluminum in high-salt content samples: application to analysis of hemodialysis fluids | |
| US5093267A (en) | Method for biochemical assay and an analyzer for the method | |
| JPH0310905B2 (en) | ||
| JPH0772129A (en) | Preparation of sample, method for minimizing band width of sample eluate and chromatograph eluate and enriching method of sample component | |
| Scott et al. | Advances in the application of high resolution liquid chromatography to the separation of complex biological mixtures | |
| Marina et al. | Flow-injection determination of low levels of ammonium ions in natural waters employing preconcentration with a cation-exchange resin | |
| Jager et al. | Novel approach for the analysis of glycated hemoglobin using capillary isoelectric focusing with chemical mobilization | |
| JPS60142250A (en) | Method and device for analyzing compound having amino group | |
| JPH06258311A (en) | Reaction medium of 1,2-diphenyl ethylenediamine with catechol amines | |
| JPH0737971B2 (en) | Analysis method for amino compounds | |
| DE19741810B4 (en) | Method for total nitrogen determination | |
| JP2964521B2 (en) | Analysis system and analysis method | |
| JPH0575066B2 (en) | ||
| Csapó et al. | Separation and determination of the amino acids by ion exchange column chromatography applying post-column derivatization | |
| JPH0373851A (en) | Analysis of optical isomer | |
| JPS59174754A (en) | Analysis of nitrous acid ions in water | |
| Jančářová et al. | Determination of uranium in technological waters by ion-pair liquid chromatography | |
| JPS5910849A (en) | Analysis of amino acid | |
| SE8900468D0 (en) | PROCEDURE FOR DETERMINATION OF SUBJECT SUBMISSION BY HYDRAULIC CHEMISTRY CHROMATOGRAPHY | |
| Hasegawa et al. | Evaluation of an ODS column modified with zwitterionic/nonionic mixed surfactants and its application to direct injection determination of inorganic anions | |
| JPH01202657A (en) | Analysis of total cerium ion content | |
| JPH02193068A (en) | Chromatography separation method and chromatography apparatus | |
| KR100477307B1 (en) | Simultaneous Analysis of Vitamins B1, B2, and B6 Using Partial Post-Column Reactions |