JPH0233985B2 - KEIKOKENSHUTSUAMINOSANBUNSEKIKEI - Google Patents
KEIKOKENSHUTSUAMINOSANBUNSEKIKEIInfo
- Publication number
- JPH0233985B2 JPH0233985B2 JP13858882A JP13858882A JPH0233985B2 JP H0233985 B2 JPH0233985 B2 JP H0233985B2 JP 13858882 A JP13858882 A JP 13858882A JP 13858882 A JP13858882 A JP 13858882A JP H0233985 B2 JPH0233985 B2 JP H0233985B2
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- antioxidant
- concentration
- acid analyzer
- thiodiglycol
- 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 - Lifetime
Links
- 150000001413 amino acids Chemical class 0.000 claims description 34
- 238000006243 chemical reaction Methods 0.000 claims description 17
- YODZTKMDCQEPHD-UHFFFAOYSA-N thiodiglycol Chemical group OCCSCCO YODZTKMDCQEPHD-UHFFFAOYSA-N 0.000 claims description 16
- 229950006389 thiodiglycol Drugs 0.000 claims description 16
- 239000007853 buffer solution Substances 0.000 claims description 15
- 239000003963 antioxidant agent Substances 0.000 claims description 14
- 230000003078 antioxidant effect Effects 0.000 claims description 13
- 239000007800 oxidant agent Substances 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 7
- 230000003647 oxidation Effects 0.000 claims description 5
- 238000007254 oxidation reaction Methods 0.000 claims description 5
- ODJQKYXPKWQWNK-UHFFFAOYSA-N 3,3'-Thiobispropanoic acid Chemical group OC(=O)CCSCCC(O)=O ODJQKYXPKWQWNK-UHFFFAOYSA-N 0.000 claims description 2
- 239000003490 Thiodipropionic acid Substances 0.000 claims description 2
- 238000001917 fluorescence detection Methods 0.000 claims description 2
- UVZICZIVKIMRNE-UHFFFAOYSA-N thiodiacetic acid Chemical group OC(=O)CSCC(O)=O UVZICZIVKIMRNE-UHFFFAOYSA-N 0.000 claims description 2
- 235000019303 thiodipropionic acid Nutrition 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 description 20
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 18
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 description 11
- 239000000872 buffer Substances 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 239000012295 chemical reaction liquid Substances 0.000 description 8
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 7
- 239000005708 Sodium hypochlorite Substances 0.000 description 6
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 6
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 125000001841 imino group Chemical group [H]N=* 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 4
- 229960002591 hydroxyproline Drugs 0.000 description 4
- 229930182817 methionine Natural products 0.000 description 4
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 150000003147 proline derivatives Chemical class 0.000 description 2
- 230000010349 pulsation Effects 0.000 description 2
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-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
- 239000002253 acid Substances 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- -1 imino acids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Description
【発明の詳細な説明】
本発明は蛍光検出器を用いるアミノ酸分析計に
関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an amino acid analyzer using a fluorescence detector.
従来、けい光検出アミノ酸分析計は、イミノ酸
であるプロリン（Pro）とハイドロオキシプロリ
ン（Hypro）がけい光されないため検出できず、
アミノ酸分析計としては使用できなかつた。この
プロリンは、タンパク質構成アミノ酸の構成要素
であり、このプロリンの検出ができないというこ
とが、他のアミノ酸の構成要素が検出できたとし
ても、当該アミノ酸の構成成分を全て検出できた
ことにならないためである。そこで、近年この通
常では検出できないイミノ酸であるプロリン
（Pro）とハイドロオキシプロリン（Hypro）を
検出するために、けい光試薬を加える前に酸化剤
（例えば次亜塩素酸ナトリウム）を加えることが
考えられている。これによつて、プロリンとハイ
ドロオキシプロリンがけい光して検出できるよう
になつた。しかしながら、この酸化剤を加える
と、確かに検出はできるが、第４図に示す如く、
プロリン（Pro）４６やハイドロオキシプロリン
（Hypro）４５の検出感度が実線のように弱く、
また、Pro、Hyproの感度を点線に示す如く上げ
ようとするとすなわち、酸化を強くすると、リジ
ン（Lys）４９やヒスチジン（His）５０の点線
で示す如く、他のアミノ酸のピーク感度が犠牲に
なつていた。 Conventional fluorescence detection amino acid analyzers have been unable to detect the imino acids proline (Pro) and hydroxyproline (Hypro) because they do not fluoresce.
It could not be used as an amino acid analyzer. This proline is a component of protein-constituting amino acids, and the fact that this proline cannot be detected does not mean that all the components of the amino acid have been detected, even if the components of other amino acids can be detected. It is. Therefore, in recent years, in order to detect the normally undetectable imino acids proline (Pro) and hydroxyproline (Hypro), it has become possible to add an oxidizing agent (for example, sodium hypochlorite) before adding the fluorescent reagent. It is considered. This enabled proline and hydroxyproline to fluoresce and be detected. However, when this oxidizing agent is added, although detection is possible, as shown in Figure 4,
The detection sensitivity of proline (Pro) 46 and hydroxyproline (Hypro) 45 is weak as shown by the solid line.
Also, if you try to increase the sensitivity of Pro and Hypro as shown by the dotted line, that is, if you increase the oxidation, the peak sensitivity of other amino acids will be sacrificed, as shown by the dotted line for lysine (Lys) 49 and histidine (His) 50. was.
本発明の目的は、１級アミノ酸の検出感度を犠
牲にさせることなく、イミノ酸（Pro、Hypro）
の感度を大幅に向上させ、全体的な高い感度を得
ることのできるけい光アミノ酸分析計を提供する
ことにある。 The purpose of the present invention is to detect imino acids (Pro, Hypro) without sacrificing the detection sensitivity of primary amino acids.
The object of the present invention is to provide a fluorescent amino acid analyzer that can significantly improve the sensitivity of the amino acids and obtain high overall sensitivity.
本発明は、使用する酸化剤の濃度によつて、
Hisなどのアミノ酸の感度が低下する原因が、酸
化され過ぎていることにあることを実験により確
かめ、全ての緩衝液に酸化防止剤（例えばチオジ
グリコール等）を加えることにより、１級アミノ
酸の検出感度を犠牲にさせることなく、イミノ酸
（Pro、Hypro）の感度を大幅に向上させ、全体
的な高い感度を得ようというものである。 Depending on the concentration of the oxidizing agent used, the present invention can
It was confirmed through experiments that the cause of decreased sensitivity of amino acids such as His is due to excessive oxidation. The aim is to significantly improve the sensitivity of imino acids (Pro, Hypro) without sacrificing detection sensitivity, and to obtain high overall sensitivity.
以下、本発明の実施例について説明する。 Examples of the present invention will be described below.
第１図には、本発明の実施例を示すイミノ酸を
含むアミノ酸をけい光検出するけい光アミノ酸分
析計の流路系統図が示されている。 FIG. 1 shows a flow path diagram of a fluorescent amino acid analyzer for fluorescently detecting amino acids including imino acids, which represents an embodiment of the present invention.
図において、１，２，３，４，５は緩衝液、６
は再生液、７は電磁弁、８は緩衝液ポンプ、９は
第１反応ポンプ、１０は第２反応ポンプ、１１は
例えば次亜塩素酸ナトリウム等の酸化剤である第
１反応液、１２は例えばオルカピタルアルデヒド
（OPA）等のけい光試薬である第２反応液、１３
はアンモニアフイルムカラム、１４はオートサン
プラ、１５は分析カラム、１６，１７はミキサ、
１８は第１反応コイル、１９は第２反応コイル、
２０はけい光検出器、２１，２２は、それぞれ脈
動を防止するためのダンパコイルである。 In the figure, 1, 2, 3, 4, 5 are buffer solutions, 6
7 is a regeneration liquid, 7 is a solenoid valve, 8 is a buffer pump, 9 is a first reaction pump, 10 is a second reaction pump, 11 is a first reaction liquid that is an oxidizing agent such as sodium hypochlorite, and 12 is a A second reaction solution, which is a fluorescent reagent such as orcapitalaldehyde (OPA), 13
is an ammonia film column, 14 is an autosampler, 15 is an analytical column, 16 and 17 are mixers,
18 is a first reaction coil, 19 is a second reaction coil,
20 is a fluorescence detector, and 21 and 22 are damper coils for preventing pulsation.
まず、第１緩衝液１から第５緩衝液５および再
生液６までの中から電磁弁７によつて１つが選択
され、緩衝液ポンプ８によつて、アンモニアフイ
ルタカラム１３とオートサンプラ１４を経由し、
分析カラム１５に送られる。ここで分離した各ア
ミノ酸は、第１反応液ポンプ９によつて送られた
第１反応液１１と第１ミキサーで混合し、第１反
応コイル１８で反応する。ここでPro、Hyproが
酸化され、次段階で蛍光を発生する準備ができ
る。次に、第２反応液ポンプ１０により送られた
第２反応液１２が第２ミキサ１７で混合し、第２
反応コイル１９で反応し、蛍光を発生する。それ
を蛍光検出器２０によつて検知し、第４図の実線
に示すようなクロマトグラムが得られる。第１図
中２１，２２は、ポンプの脈動を少なくするため
に設けられたダンパコイルである。第４図では
Pro４６、Hypro４５の検出感度がほか１級アミ
ノ酸にくらべて1/100程度である。すなわち第４
図では、１級アミノ酸の10倍濃度のPro、Hypro
を添加しているにもかかわらず、ピーク高さは１
級アミノ酸の約1/10程度しか得られない。かと言
つて、Pro、Hyproの感度を上げようとすれば、
Lys４９、His５０などのピークが点線のように
小さくなる。この関係を第２図に示す。 First, one of the first to fifth buffer solutions 1 to 5 and the regenerating solution 6 is selected by the solenoid valve 7, and the buffer solution is passed through the ammonia filter column 13 and the autosampler 14 by the buffer pump 8. death,
It is sent to analytical column 15. Each amino acid separated here is mixed with the first reaction liquid 11 sent by the first reaction liquid pump 9 in the first mixer, and reacted in the first reaction coil 18. Here, Pro and Hypro are oxidized and ready to generate fluorescence in the next step. Next, the second reaction liquid 12 sent by the second reaction liquid pump 10 is mixed in the second mixer 17, and the second reaction liquid 12 is mixed in the second mixer 17.
A reaction occurs in the reaction coil 19 and fluorescence is generated. This is detected by the fluorescence detector 20, and a chromatogram as shown by the solid line in FIG. 4 is obtained. In FIG. 1, 21 and 22 are damper coils provided to reduce pulsation of the pump. In Figure 4
The detection sensitivity of Pro46 and Hypro45 is about 1/100 of that of other primary amino acids. That is, the fourth
The figure shows Pro and Hypro at 10 times the concentration of primary amino acids.
Despite adding , the peak height is 1
Only about 1/10 of grade amino acids can be obtained. However, if you try to increase the sensitivity of Pro or Hypro,
Peaks such as Lys49 and His50 become smaller as shown by dotted lines. This relationship is shown in FIG.
第２図は横軸に第１反応液中の次亜塩素酸ナト
リウムの量を、縦軸にピーク高さをとつて、酸化
防止剤（チオジグリコール）を用いないものであ
る。次亜塩素酸濃度が０の時はPro、Hyproは検
出できないが、次第に増やすと、ピークが高くな
る。しかし、逆に、Lys、His等は0.01％程度で
急に小さくなることがわかつた。この原因は、第
４図の第４緩衝液溶出帯４４のピーク（主として
塩基性アミノ酸）が次亜塩素酸により酸化されす
ぎていることが実験により確かめられた。そこ
で、第４緩衝液にも酸化防止剤としてチオジグリ
コール0.5％／を加え一定としたところ、第３
図の結果が得られた。すなわち、Lys３４、His
３５のピーク高さは、次亜塩素酸ナトリウムの濃
度を0.5％まで上げてもそれ程低下せず、Pro、
Hyproは大幅に感度向上がはかられることがわか
つた。 In FIG. 2, the horizontal axis represents the amount of sodium hypochlorite in the first reaction solution, and the vertical axis represents the peak height, without using an antioxidant (thiodiglycol). When the hypochlorous acid concentration is 0, Pro and Hypro cannot be detected, but as the concentration is gradually increased, the peak becomes higher. However, on the contrary, it was found that Lys, His, etc. suddenly decreased at about 0.01%. It has been experimentally confirmed that the cause of this is that the peak (mainly basic amino acids) in the fourth buffer elution zone 44 in FIG. 4 is too oxidized by hypochlorous acid. Therefore, when we added 0.5% thiodiglycol as an antioxidant to the fourth buffer solution and kept it constant,
The results shown in the figure were obtained. That is, Lys34, His
The peak height of 35 did not decrease significantly even when the concentration of sodium hypochlorite was increased to 0.5%, and the peak height of Pro,
It was found that Hypro significantly improved sensitivity.
酸化防止剤としてはチオジグリコール
（Thiodiglycol）0.5％Ｖ／Ｖ（１の緩衝液中に
５ml）のほかに、チオジグリコール酸
（Thiodiglycolic acid）0.5％Ｖ／Ｖや、チオジプ
ロピオン酸（Thiodipropionic acid）0.5％Ｖ／
Ｖを緩衝液に使用することにより、チオジグリコ
ールとほぼ同様な効果のあることが確かめられ
た。 As antioxidants, in addition to Thiodiglycol 0.5% V/V (5 ml in 1 buffer), Thiodiglycolic acid 0.5% V/V and Thiodipropionic acid. acid) 0.5%V/
It was confirmed that the use of V in the buffer had almost the same effect as thiodiglycol.
第５図には、次亜塩素酸ナトリウムの濃度を
0.5％に固定した状態で、緩衝液中のチオグリコ
ールの濃度を変えた場合におけるピーク高さを示
されている。リジン３４、ヒスチジン３５は、ジ
チオグリコール濃度が０の場合、酸化が進みすぎ
て、ピーク高さは低いが、プロリン３３は、きわ
めて感度が高い。そして、0.5％程度がバランス
の良い値で、その範囲は0.2〜0.8％が使用できる
範囲である。なおチオジグリコールはメチオニン
の酸化防止の役割をも兼ねているので、その範囲
は0.2％以上が有効である。 Figure 5 shows the concentration of sodium hypochlorite.
The peak heights are shown when the concentration of thioglycol in the buffer solution is changed while the concentration is fixed at 0.5%. When the dithioglycol concentration is 0, lysine 34 and histidine 35 are oxidized too much and the peak height is low, but proline 33 has extremely high sensitivity. A well-balanced value is about 0.5%, and the usable range is 0.2 to 0.8%. Since thiodiglycol also has the role of preventing methionine from oxidizing, a range of 0.2% or more is effective.
したがつて、本実施例によれば、Pro、Hypro
以外の第１アミノ酸のピーク検出感度を犠牲にす
ることなく、Pro、Hyproの検出感度を約10倍に
増大させることができる。 Therefore, according to this embodiment, Pro, Hypro
The detection sensitivity of Pro and Hypro can be increased approximately 10 times without sacrificing the peak detection sensitivity of other first amino acids.
なお、チオジグリコールは、メチオニン４８
（以下Metと言う）の酸化防止のために、Metが
溶出されるまでの時間帯、すなわち、第１〜第３
緩衝液に従来より用いられていて、それ以後の緩
衝液には用いられていなかつた。 In addition, thiodiglycol is methionine 48
(hereinafter referred to as Met), the time period until Met is eluted, that is, the first to third
It has traditionally been used in buffer solutions, but has not been used in subsequent buffer solutions.
したがつて本発明では、高い蛍光感度を得る目
的で、全部の緩衝液にチオジグリコールなどの酸
化防止剤を用いることに主眼をおいている。 Therefore, the present invention focuses on using an antioxidant such as thiodiglycol in all buffer solutions in order to obtain high fluorescence sensitivity.
本発明の応用例としては、生体液アミノ酸分析
にも適用することができる。すなわち、Met溶出
時間以後の第３、第４、第５緩衝液にチオジグリ
コールを入れることにより、Pro、Hyproを含む
全アミノ酸類縁物質の感度を向上させることがで
きる。 As an application example of the present invention, it can also be applied to biological fluid amino acid analysis. That is, by adding thiodiglycol to the third, fourth, and fifth buffers after the Met elution time, the sensitivity of all amino acid analogs including Pro and Hypro can be improved.
また、緩衝液をステツプで切換えずに、グラジ
エント方式で切換えて行なうアミノ酸分析計でも
同様、全緩衝液にチオジグリコールを採用する効
果には変わりはない。 Similarly, in an amino acid analyzer that uses a gradient method to change the buffer solution instead of changing it in steps, the effect of using thiodiglycol in all buffer solutions remains the same.
もう１つの応用例として、チオジグリコールな
どの酸化防止剤を第１図に示す第１反応液の中に
直接入れた場合、すなわち、酸化剤である次亜塩
素酸ソーダと同時に入れた場合も、チオジグリコ
ールを緩衝液に入れた場合と同様の効果をもたら
すことが実験により確かめられた。 Another application example is when an antioxidant such as thiodiglycol is directly added to the first reaction solution shown in Figure 1, that is, when it is added at the same time as the oxidizing agent, sodium hypochlorite. Experiments have confirmed that thiodiglycol has the same effect as adding thiodiglycol to a buffer solution.
但し、この場合、緩衝液の中にメチオニンの酸
化を防止するチオジグリコールが入つていないた
め、メチオニンが酸化されて、メチオニンスルホ
ンおよびメチオニンスルホキシドに変わる部分が
ある。 However, in this case, since the buffer does not contain thiodiglycol that prevents oxidation of methionine, some portions of methionine are oxidized and converted into methionine sulfone and methionine sulfoxide.
以上説明したように、本発明によれば、第１ア
ミノ酸の検出感度を損うことなくPro、Hyproの
イミノ酸の検出感度を大幅に向上することができ
る。 As explained above, according to the present invention, the detection sensitivity of Pro and Hypro imino acids can be significantly improved without impairing the detection sensitivity of the first amino acid.
第１図はアミノ酸分析の流路系統図、第２図は
緩衝液に酸化防止剤を用いない場合の酸化剤濃度
におけるアミノ酸のピーク高さを示す図、第３図
は緩衝液に0.5％濃度のチオジグリコールを酸化
防止剤として用いた場合の酸化剤濃度におけるア
ミノ酸のピーク高さを示す図、第４図は蛋白構成
アミノ酸の標準アミノ酸をけい光法で分析した
図、第５図は酸化剤を0.5％濃度一定にして緩衝
液中のチオジグリコールの濃度におけるアミノ酸
のピーク高さを示す図である。
１，２，３，４，５……緩衝液、１１……第１
反応液、１２……第２反応液、１８……第１反応
コイル、１９……第２反応コイル、２０……けい
光検出器。
Figure 1 is a flow path diagram for amino acid analysis, Figure 2 is a diagram showing the peak height of amino acids at various oxidant concentrations when no antioxidant is used in the buffer, and Figure 3 is a diagram showing the amino acid peak height at 0.5% concentration in the buffer. Figure 4 shows the peak height of amino acids at various oxidizing agent concentrations when thiodiglycol is used as an antioxidant, Figure 4 shows the analysis of standard protein-constituting amino acids using a fluorescence method, and Figure 5 shows the oxidation FIG. 2 is a diagram showing the peak height of amino acids at different concentrations of thiodiglycol in a buffer solution, with the agent at a constant concentration of 0.5%. 1, 2, 3, 4, 5...buffer solution, 11...first
Reaction liquid, 12... Second reaction liquid, 18... First reaction coil, 19... Second reaction coil, 20... Fluorescence detector.
Claims (1)
トに切換えて送液し、第１反応コイル内で酸化さ
せるための第１反応液を送液すると共に第２反応
コイル内において第２反応液を送液してけい光さ
せてけい光検出するけい光検出アミノ酸分析計に
おいて、上記緩衝液の全てにアミノ酸酸化防止剤
を添加させたことを特徴とするけい光検出アミノ
酸分析計。 ２ 特許請求の範囲第１項記載の発明において、
上記酸化防止剤は、チオジグリコールであること
を特徴とするけい光アミノ酸分析計。 ３ 特許請求の範囲第１項記載の発明において、
上記酸化防止剤は、チオジグリコール酸であるこ
とを特徴とするけい光アミノ酸分析計。 ４ 特許請求の範囲第１項記載の発明において、
上記酸化防止剤は、チオジプロピオン酸であるこ
とを特徴とするけい光アミノ酸分析計。 ５ 特許請求の範囲第１項ないし第４項のいずれ
か１項記載の発明において、酸化剤濃度0.5％の
ときは、酸化防止剤濃度を0.2％〜0.8％であるこ
とを特徴とするけい光アミノ酸分析計。 ６ 特許請求の範囲第１項ないし第４項のいずれ
か１項記載の発明において、酸化防止剤の濃度が
0.5％のときは、酸化剤濃度が0.2％〜0.8％である
ことを特徴とするけい光アミノ酸分析計。 ７ 特許請求の範囲第１項ないし第４項のいずれ
か１項記載の発明において、上記酸化剤濃度と上
記酸化防止剤濃度は同一であることを特徴とする
けい光アミノ酸分析計。[Scope of Claims] 1. Two or more buffer solutions are fed in a stepwise or gradient manner, and a first reaction solution for oxidation is fed into the first reaction coil, and at the same time, the first reaction solution is fed into the second reaction coil. A fluorescence detection amino acid analyzer that detects fluorescence by feeding a second reaction solution and causing it to fluoresce, characterized in that an amino acid antioxidant is added to all of the buffer solutions. . 2 In the invention described in claim 1,
A fluorescent amino acid analyzer characterized in that the antioxidant is thiodiglycol. 3 In the invention described in claim 1,
A fluorescent amino acid analyzer characterized in that the antioxidant is thiodiglycolic acid. 4 In the invention described in claim 1,
A fluorescent amino acid analyzer characterized in that the antioxidant is thiodipropionic acid. 5. In the invention according to any one of claims 1 to 4, the fluorescent light is characterized in that when the oxidizing agent concentration is 0.5%, the antioxidant concentration is 0.2% to 0.8%. Amino acid analyzer. 6. In the invention according to any one of claims 1 to 4, the concentration of the antioxidant is
A fluorescent amino acid analyzer characterized in that the oxidizing agent concentration is 0.2% to 0.8% when it is 0.5%. 7. A fluorescent amino acid analyzer according to any one of claims 1 to 4, wherein the oxidizing agent concentration and the antioxidant concentration are the same.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13858882A JPH0233985B2 (en) | 1982-08-11 | 1982-08-11 | KEIKOKENSHUTSUAMINOSANBUNSEKIKEI |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13858882A JPH0233985B2 (en) | 1982-08-11 | 1982-08-11 | KEIKOKENSHUTSUAMINOSANBUNSEKIKEI |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5928659A JPS5928659A (en) | 1984-02-15 |
| JPH0233985B2 true JPH0233985B2 (en) | 1990-07-31 |
Family
ID=15225612
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP13858882A Expired - Lifetime JPH0233985B2 (en) | 1982-08-11 | 1982-08-11 | KEIKOKENSHUTSUAMINOSANBUNSEKIKEI |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0233985B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0734008B2 (en) * | 1988-06-30 | 1995-04-12 | 株式会社島津製作所 | Gas chromatographic analysis method using thermal conductivity detector |
-
1982
- 1982-08-11 JP JP13858882A patent/JPH0233985B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5928659A (en) | 1984-02-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jarrett et al. | The separation of o-phthalaldehyde derivatives of amino acids by reversed-phase chromatography on octylsilica columns | |
| Schmid et al. | Chiral ligand-exchange capillary electrophoresis | |
| EP2088434B1 (en) | Pyrocatechol violet-metal protein assay | |
| Kuroda et al. | On-line capillary isoelectric focusing–mass spectrometry for quantitative analysis of peptides and proteins | |
| Allenmark et al. | A selective postcolumn o-phthalaldehyde-derivatization system for the determination of histamine in biological material by high-performance liquid chromatography | |
| JP5875160B2 (en) | Analysis method of hemoglobins | |
| JP3508710B2 (en) | Amino acid analysis method and apparatus | |
| US3630681A (en) | Method of separating mixtures by liquid chromatography | |
| Mizutani | Decreased activity of proteins adsorbed onto glass surfaces with porous glass as a reference | |
| Buchberger et al. | Applications of capillary zone electrophoresis in clinical chemistry determination of low-molecular-mass ions in body fluids | |
| Pappa-Louisi et al. | Optimal conditions for the simultaneous ion-pairing HPLC determination ofl-ascorbic, dehydro-L-ascorbic, D-ascorbic, and uric acids with on-line ultraviolet absorbance and electrochemical detection | |
| JPH0233985B2 (en) | KEIKOKENSHUTSUAMINOSANBUNSEKIKEI | |
| Lam et al. | Stereoselective D-and L-amino acid analysis by high-performance liquid chromatography | |
| Stenken et al. | Detection of N-acetylcysteine, cysteine and their disulfides in urine by liquid chromatography with a dual-electrode amperometric detector | |
| Baeyens et al. | Enhanced chemiluminescence detection of some dansyl amino acids in liquid chromatography | |
| US20220187258A1 (en) | Method for measurement of hemoglobin | |
| Saitoh et al. | On‐line redox derivatization liquid chromatography using double separation columns and one derivatization unit | |
| Lam et al. | Resolution of Optical Isomers as the Mixed Chelate Copper (III) Complexes by Reversed Phase Chromatography | |
| JP2768079B2 (en) | High precision analysis method | |
| Varshney et al. | Chromatographic Separation of. ALPHA.-Amino Acids on Antimony (V) Phosphate-Silica Gel'G'Plates from Some Synthetic Mixtures and Drug Samples. | |
| Hayakawa et al. | Notes On-Line Removal of Interfering Alkaline Earth Metals for Simultaneous Determination of Hydrogen Carbonate, Chloride, Nitrate and Sulfate by Indirect Photometric Detection Ion Chromatography | |
| Tsuchiya et al. | Simplified procedure for specific determination of imino acids in human blood plasma by high-performance liquid chromatography | |
| Stegehuis et al. | Bioanalysis of the peptide de-enkephalin-γ-endorphin: On-line sample pretreatment using membrane dialysis and solid-phase isolation | |
| Chiap et al. | Strategy for the development of automated methods involving dialysis and trace enrichment as on-line sample preparation for the determination of basic drugs in plasma by liquid chromatography | |
| JPS61241662A (en) | Amino acid analyzing instrument |