JPH0259428B2 - - Google Patents
Info
- Publication number
- JPH0259428B2 JPH0259428B2 JP11981482A JP11981482A JPH0259428B2 JP H0259428 B2 JPH0259428 B2 JP H0259428B2 JP 11981482 A JP11981482 A JP 11981482A JP 11981482 A JP11981482 A JP 11981482A JP H0259428 B2 JPH0259428 B2 JP H0259428B2
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- buffer
- buffer solution
- lithium ion
- analysis
- 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
- 238000004458 analytical method Methods 0.000 claims description 24
- 239000007853 buffer solution Substances 0.000 claims description 22
- 235000001014 amino acid Nutrition 0.000 claims description 21
- 229940024606 amino acid Drugs 0.000 claims description 21
- 150000001413 amino acids Chemical class 0.000 claims description 21
- HBBGRARXTFLTSG-UHFFFAOYSA-N Lithium ion Chemical compound [Li+] HBBGRARXTFLTSG-UHFFFAOYSA-N 0.000 claims description 13
- 229910001416 lithium ion Inorganic materials 0.000 claims description 13
- 239000000872 buffer Substances 0.000 claims description 11
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 7
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 6
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 6
- 229960001230 asparagine Drugs 0.000 claims description 6
- 235000009582 asparagine Nutrition 0.000 claims description 6
- 235000013922 glutamic acid Nutrition 0.000 claims description 6
- 239000004220 glutamic acid Substances 0.000 claims description 6
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 6
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 3
- 235000004554 glutamine Nutrition 0.000 claims description 3
- 238000000926 separation method Methods 0.000 description 13
- 238000010586 diagram Methods 0.000 description 6
- 238000010828 elution Methods 0.000 description 6
- 239000013060 biological fluid Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 229910052744 lithium Inorganic materials 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 239000011550 stock solution 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
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Description
本発明は、生体液中のアミノ酸を分析するアミ
ノ酸分析方法に関する。
従来のアミノ酸分析方法においては、アスパラ
ギン（AspNH2）、グルタミン酸（Glu）、グルタ
ミン（GluNH2）の分離を良くするために、充て
ん剤の微細化、均一化などの改良を行ない分析時
間が４時間程度にまで短縮されている。しかしな
がら、充てん剤を更に微細化すれば負荷圧力が高
くなり、高圧化技術等が要求され、実用化上の問
題が多い。
本発明の目的は、生体液中のアミノ酸約40成分
をより高速で分析することのできるアミノ酸分析
方法を提供することにある。
本発明の要旨は次の如くである。すなわち、生
体液中のアミノ酸約40成分を分離するに当り、重
要で、かつ分離が最も困難とされている成分はア
スパラギン（AspNH2）、グルタミン酸（Glu）、
グルタミン（GluNH2）の３成分とされている。
この成分をより良く分離するためには、緩衝液中
のリチウム濃度を下げることが最も効果的である
ことを実験により見いだした。こうして分離を上
げたうえで、カラム線速度を増すことにより、分
析の高速化が実現できた。したがつて、本発明
は、アスパラギンとグルタミン酸とグルタミンを
分離する緩衝液のリチウムイオン濃度を0.13規定
以下にすると共にカラムの線速度を0.325ml／
min以上とすることにより、生体液中のアミノ酸
約40成分を確実に高速で分析しようというもので
ある。
以下、本発明の実施例について説明する。
第１図には、本発明の一実施例を示すアミノ酸
分析計の流路系統図が示されている。
図において、第１緩衝液１、第２緩衝液２、第
３緩衝液３、第４緩衝液４、第５緩衝液５、再生
液６は、順次電磁弁７によつて１つずつ選択され
て、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。図中９はアンモニアフイ
ルタカラム、１０はサンプラ、１１は分析カラ
ム、１２はニンヒドリン試薬、１６は光度計、１
７は記録計である。
ここで分離した各アミノ酸は、ニンヒドリンポ
ンプ１３によつて送られたニンヒドリン試薬１２
とミキサ１４で混合し、反応コイル１５で反応す
る。ここで発色したアミノ酸は、光度計で連続的
に検知され、クロマトグラムとなつて記録計１７
に現われる仕組になつている。第２図ａは従来得
られている生体液アミノ酸分析法のクロマトグラ
ムの内最も分離が困難とされているアスパラギン
（AspNH2）２１、グルタミン酸（Glu）２２、
グルタミン（GluNH2）２３の３成分のパターン
図である。分離率はそれぞれ約70％であり、約40
成分全体の正味分析時間は３時間２０を要してい
た。
第３図は、本発明なるこれ等３成分の分離を上
げる手法を説明したものである。第３図ａは第１
緩衝液中に含まれるリチウムイオン濃度は、
0.200Nであり、各成分の間隔が狭く分析が悪い。
また、第３図ｂは従来行なわれている第１緩衝液
中に含まれるリチウムイオン濃度であり、
0.155Nである。これも第３図ａと同様の問題は
ある。また、第３図ｃは、リチウムイオン濃度を
更に下げた0.135Nのときの分析結果である。こ
れによれば、前記第３図ａ，ｂよりも分析効果は
上がつている。これを更に第１表に示す如き第１
緩衝液成分とし、リチウムイオン濃度を0.116N
にすると、溶出時間は10分近く遅れるが、各成分
の間隔が広がつていき、分離が向上する。この第
１緩衝液中に含まれるリチウムイオン濃度を更に
下げ、0.078に下げると、第３図ｄに示す如く、
溶出時間が更に遅れるが一層の分離向上を実現で
きる。
第３図の分析結果から明白なように、リチウム
イオン濃度が、0.135Nより小さくなると、溶出
位置が急激に遅くなり出し、かつ、ピーク間の間
隔が広がつている。
The present invention relates to an amino acid analysis method for analyzing amino acids in biological fluids. In the conventional amino acid analysis method, in order to improve the separation of asparagine (AspNH 2 ), glutamic acid (Glu), and glutamine (GluNH 2 ), improvements such as making the packing material finer and more uniform were made, and the analysis time was reduced to 4 hours. It has been shortened to a certain extent. However, if the filler is further refined, the load pressure will increase, requiring high pressure technology, etc., and there are many problems in practical use. An object of the present invention is to provide an amino acid analysis method that allows for faster analysis of approximately 40 amino acid components in biological fluids. The gist of the present invention is as follows. In other words, when separating the approximately 40 amino acid components in biological fluids, the important and most difficult components to separate are asparagine (AspNH 2 ), glutamic acid (Glu),
It is considered to be one of the three components of glutamine (GluNH 2 ).
Through experiments, we found that lowering the lithium concentration in the buffer solution is the most effective way to better separate these components. In this way, by increasing the separation and increasing the column linear velocity, it was possible to speed up the analysis. Therefore, the present invention aims to reduce the lithium ion concentration of the buffer solution for separating asparagine, glutamic acid, and glutamine to 0.13 normal or less, and to increase the linear velocity of the column to 0.325 ml/min.
The aim is to reliably analyze approximately 40 amino acid components in biological fluids at high speed by setting the speed to be at least min. Examples of the present invention will be described below. FIG. 1 shows a flow path system diagram of an amino acid analyzer showing one embodiment of the present invention. In the figure, the first buffer solution 1, the second buffer solution 2, the third buffer solution 3, the fourth buffer solution 4, the fifth buffer solution 5, and the regeneration solution 6 are selected one by one by the solenoid valve 7. Then, it is sent to an analysis column 15 by a buffer pump 8 via an ammonia filter column 13 and an autosampler 14. In the figure, 9 is an ammonia filter column, 10 is a sampler, 11 is an analytical column, 12 is a ninhydrin reagent, 16 is a photometer, 1
7 is a recorder. Each amino acid separated here is treated with a ninhydrin reagent 12 sent by a ninhydrin pump 13.
are mixed in the mixer 14 and reacted in the reaction coil 15. The amino acids that develop color here are continuously detected by a photometer, and are converted into a chromatogram using a recorder 17.
It has become a system that appears in Figure 2a shows the chromatograms of conventional biofluid amino acid analysis methods, which are considered to be the most difficult to separate: asparagine (AspNH 2 ) 21, glutamic acid (Glu) 22,
It is a pattern diagram of three components of glutamine (GluNH 2 ) 23. The separation rate is about 70% and about 40
The net analysis time for the entire component required 3 hours and 20 hours. FIG. 3 illustrates the method of the present invention for increasing the separation of these three components. Figure 3a is the first
The lithium ion concentration contained in the buffer solution is
It is 0.200N, and the intervals between each component are narrow, making analysis difficult.
In addition, FIG. 3b shows the lithium ion concentration contained in the first buffer solution, which is conventionally used.
It is 0.155N. This also has the same problem as FIG. 3a. Moreover, FIG. 3c shows the analysis results when the lithium ion concentration was further lowered to 0.135N. According to this, the analysis effect is better than that shown in FIGS. 3a and 3b. Further, as shown in Table 1,
Buffer component, lithium ion concentration 0.116N
Although the elution time will be delayed by nearly 10 minutes, the separation between each component will become wider and the separation will be improved. When the lithium ion concentration contained in this first buffer solution is further lowered to 0.078, as shown in Figure 3d,
Although the elution time is further delayed, further improvement in separation can be achieved. As is clear from the analysis results in FIG. 3, when the lithium ion concentration becomes lower than 0.135N, the elution position suddenly becomes slower and the interval between the peaks becomes wider.
【表】
リチウム濃度を下げる方法としては、第１表の
処方による方法と、従来使用されている緩衝液の
原液を水で薄める方法がある。第３図ｂで用いた
リチウムイオン濃度0.155Nの緩衝液は、三菱化
成工業(株)のMCI BUFFER835 SERIESの835−
PF−KIT中の835−PF1をそのまま使用してい
る。また、第３図ｄは、第３図ｃの緩衝液750ml
に対して水250mlを入れて稀釈したものである。
また、第３図ｅは、第３図ｃの緩衝液500mlに対
し水500mlを入れたものである。第２図ｂは、第
３図ｄによつて得られたクロマトグラムであり、
その分離率はほぼ100％を示している。
次に、分析時間の短縮について説明する。前記
の如き状態では、第１緩衝液溶出領域の分析時間
が長くなる。そこで、緩衝液の流速すなわち、カ
ラムの線速度を早めた。第２図ａと第２図ｂは共
に、緩衝液流速0.325ml／minであるが、第２図
ｃは、緩衝液流速を0.475ml／minと早くするこ
とにより、すべてのアミノ酸の溶出の線速度を46
％速めた。これにより、正味分析時間を第４図の
如く、2.6時間に高速化することが出来た。この
時の３成分の分離率は約90％で第２図ｂよりもす
ぐれている。すなわち、分離率を低下させること
なく正味分析時間を3.3hrより2.6hrと25％早める
ことが出来た。流速を早めて得られる効果は、カ
ラム外のピークの拡散による拡がりをほとんどな
くすることが出来る点にある。当然、反応時間も
減つて、ピーク高さは若干低くなるが、分離率は
ほとんど低下していない。
第４図中は第１緩衝液領域、は第２緩衝液
領域、は第３緩衝液領域、は第４緩衝液領
域、は第５緩衝液領域、は再生領域である。
以上の原理を応用し、第１緩衝液の稀釈率を
更に高める。緩衝液の流速（線速度）を更に高
めることにより、正味２時間以内の分析時間にす
ることは可能なことである。
また、第１図のように、段階的に緩衝液を切換
えて行なうアミノ酸分析計のほか、グラジエント
溶離方式で行なうアミノ酸分析計においても、同
様の効果が得られる。
以上説明したように、本発明によれば、
AspNH2−Glu−GluNH2の分離を良くすること
が出来るので、これ等を含む約40成分の生体液中
のアミノ酸をより高速（正味2.6時間以内）で分
析することができる。[Table] Methods for lowering the lithium concentration include a method using the prescriptions shown in Table 1 and a method of diluting the stock solution of the conventionally used buffer solution with water. The buffer solution with a lithium ion concentration of 0.155N used in Figure 3b was MCI BUFFER835 SERIES 835-
835-PF1 in PF-KIT is used as is. In addition, Figure 3 d shows 750 ml of the buffer solution in Figure 3 c.
diluted with 250ml of water.
Also, in Figure 3e, 500ml of water was added to 500ml of the buffer solution in Figure 3c. Figure 2b is the chromatogram obtained by Figure 3d,
The separation rate is nearly 100%. Next, reduction of analysis time will be explained. In the above situation, the analysis time for the first buffer elution region becomes long. Therefore, the flow rate of the buffer solution, that is, the linear velocity of the column was increased. Figures 2a and 2b both show a buffer flow rate of 0.325 ml/min, but Figure 2c shows the elution line of all amino acids by increasing the buffer flow rate to 0.475 ml/min. speed 46
% faster. This made it possible to speed up the net analysis time to 2.6 hours, as shown in Figure 4. The separation rate of the three components at this time was approximately 90%, which is better than that shown in Figure 2b. In other words, the net analysis time could be shortened by 25% from 3.3 hours to 2.6 hours without reducing the separation rate. The effect of increasing the flow rate is that the spread of peaks outside the column due to diffusion can be almost eliminated. Naturally, the reaction time is reduced and the peak height is slightly lowered, but the separation rate is hardly reduced. In FIG. 4, the first buffer region, the second buffer region, the third buffer region, the fourth buffer region, the fifth buffer region, and the regeneration region are shown. Applying the above principle, the dilution rate of the first buffer solution is further increased. By further increasing the flow rate (linear velocity) of the buffer solution, it is possible to reduce the net analysis time to less than 2 hours. Furthermore, as shown in FIG. 1, similar effects can be obtained not only in an amino acid analyzer that changes the buffer solution stepwise, but also in an amino acid analyzer that uses a gradient elution method. As explained above, according to the present invention,
Since the separation of AspNH 2 -Glu-GluNH 2 can be improved, amino acids in biological fluids containing about 40 components including AspNH 2 -Glu-GluNH 2 can be analyzed faster (within 2.6 hours net).
第１図はアミノ酸分析計の流路系統説明図、第
２図はリチウムイオン濃度の違いによるピークの
分離状態を示す図、第３図はリチウムイオン濃度
の違いによるピーク位置を示す図、第４図は本実
施例によるアミノ酸分析のクロマトグラムであ
る。
１，２，３，４，５……緩衝液、６……再生
液、７……電磁弁、１１……分析カラム。
Figure 1 is an explanatory diagram of the flow path system of an amino acid analyzer, Figure 2 is a diagram showing the state of peak separation due to differences in lithium ion concentration, Figure 3 is a diagram showing peak positions due to differences in lithium ion concentration, and Figure 4 is a diagram showing the peak positions due to differences in lithium ion concentration. The figure is a chromatogram of amino acid analysis according to this example. 1, 2, 3, 4, 5...Buffer solution, 6...Regenerating solution, 7...Solenoid valve, 11...Analysis column.
Claims (1)
エント方式で切換えて行なうアミノ酸分析方法に
おいて、アスパラギンとグルタミン酸とグルタミ
ンを分離する緩衝液のリチウムイオン濃度を0.13
規定以下にしたことを特徴とするアミノ酸分析方
法。 ２ ２種以上の緩衝液を段階的にあるいはグラジ
エント方式で切換えて行なうアミノ酸分析方法に
おいて、アスパラギンとグルタミン酸とグルタミ
ンを分離する緩衝液のリチウムイオン濃度を0.13
規定以下にすると共にカラム線速度を0.325ml／
min以上にすることを特徴とするアミノ酸分析方
法。[Claims] 1. In an amino acid analysis method in which two or more buffer solutions are switched stepwise or in a gradient method, the lithium ion concentration of the buffer solution separating asparagine, glutamic acid, and glutamine is set to 0.13.
An amino acid analysis method characterized in that the concentration is below the specified level. 2. In an amino acid analysis method in which two or more buffer solutions are switched stepwise or in a gradient method, the lithium ion concentration of the buffer that separates asparagine, glutamic acid, and glutamine is set to 0.13.
Lower the column linear velocity to 0.325ml/
An amino acid analysis method characterized by obtaining a concentration of min or more.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11981482A JPS5910849A (en) | 1982-07-12 | 1982-07-12 | Analysis of amino acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11981482A JPS5910849A (en) | 1982-07-12 | 1982-07-12 | Analysis of amino acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5910849A JPS5910849A (en) | 1984-01-20 |
| JPH0259428B2 true JPH0259428B2 (en) | 1990-12-12 |
Family
ID=14770890
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11981482A Granted JPS5910849A (en) | 1982-07-12 | 1982-07-12 | Analysis of amino acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5910849A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3508710B2 (en) | 2000-09-01 | 2004-03-22 | 株式会社日立製作所 | Amino acid analysis method and apparatus |
| JP2007040857A (en) * | 2005-08-04 | 2007-02-15 | Ajinomoto Co Inc | Liquid chromatograph analysis method, liquid chromatograph apparatus, and analysis program |
-
1982
- 1982-07-12 JP JP11981482A patent/JPS5910849A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5910849A (en) | 1984-01-20 |
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