JP3471124B2 - Chromatographic analysis using precolumn derivatization - Google Patents
Chromatographic analysis using precolumn derivatizationInfo
- Publication number
- JP3471124B2 JP3471124B2 JP13609495A JP13609495A JP3471124B2 JP 3471124 B2 JP3471124 B2 JP 3471124B2 JP 13609495 A JP13609495 A JP 13609495A JP 13609495 A JP13609495 A JP 13609495A JP 3471124 B2 JP3471124 B2 JP 3471124B2
- Authority
- JP
- Japan
- Prior art keywords
- derivatization
- column
- eluent
- sample
- precolumn
- Prior art date
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Description
【０００１】[0001]
【産業上の利用分野】本発明は、プレカラム誘導体化を
用いたクロマトグラフ分析方法に関するものである。FIELD OF THE INVENTION The present invention relates to a chromatographic analysis method using precolumn derivatization.
【０００２】[0002]
【従来の技術】従来、高速液体クロマトグラフィーは生
体試料など非常に多くの成分からなる試料の分離分析手
段として用いられている。最近では微量成分の分析のた
め試料の誘導体化は感度向上や、分離能の向上のために
行われている。通常の分析では予め試料の誘導体化を行
った後に試料注入を行い分析カラムに送り、所望対応検
出器により検出させている。又、オンラインでも試料の
誘導体化は行われるが、それは分析カラムで分離、溶出
された後で試薬を反応させるのが通常である。2. Description of the Related Art Conventionally, high performance liquid chromatography has been used as a means for separating and analyzing a sample containing a very large number of components such as a biological sample. Recently, derivatization of samples for the analysis of trace components has been carried out to improve sensitivity and resolution. In a usual analysis, after derivatization of a sample in advance, the sample is injected, sent to an analytical column, and detected by a desired detector. Derivatization of a sample is also performed online, but it is usually performed by separating and eluting the sample with an analytical column and then reacting the reagent.
【０００３】[0003]
【発明が解決しようとする課題】試料を誘導体化した後
に、注入、分析の方法では反応条件の再現が困難である
ため、分析に於ての再現性を得ることが難しい。これは
オンラインプレカラム法では試料の誘導体化によるバン
ドの広がりや反応誘導体化試薬が分析カラム内に入るこ
とになり、その結果高価な分析カラムの劣化を引き起こ
す。It is difficult to obtain reproducibility in the analysis because it is difficult to reproduce the reaction conditions by the method of injection and analysis after derivatizing the sample. This is because in the online precolumn method, the band is broadened due to the derivatization of the sample and the reaction derivatization reagent enters the analytical column, resulting in deterioration of the expensive analytical column.
【０００４】[0004]
【課題を解決するための手段】 そこで本発明に於て
は、試料の誘導体化をオンラインで行うことにより、常
に一定の条件で誘導体化でき、再現性がよく、オンライ
ンによる誘導体化の過程で広がってしまったバンドを狭
めることが出来、且つ誘導体化の試薬や分析目的物以外
の成分を分析カラム内に流入させないようにすることが
できるため、カラム寿命を延ばすことが出来るようにし
たもので、試料の誘導体化をオンラインにより行い、該
誘導体化させた試料を一端プレカラムに保持させ、その
後に分析カラムに送入させ分析させる方法に於いて、誘
導体化用溶離液と溶離液とを夫々独立して自在に供給す
ると共に、溶離液は、誘導体化反応コイルの下流側で、
プレカラムに連通されたプレカラムの溶離液切換用バル
ブとの間にバイパスラインで供給自在としたことを特徴
とする。[Means for Solving the Problems] Therefore, in the present invention, by performing derivatization of a sample online, derivatization can always be performed under constant conditions, reproducibility is good, and spread in the process of derivatization online. Since the band that has accumulated can be narrowed and components other than the derivatizing reagent and the analytical target substance can be prevented from flowing into the analytical column, the column life can be extended. In the method of performing derivatization of a sample online, holding the derivatized sample on the pre-column once, and then sending it to the analysis column for analysis, the eluent for derivatization and the eluent are independent of each other. And the eluent at the downstream side of the derivatization reaction coil.
A precolumn eluent switching valve that communicates with the precolumn.
It is characterized in that it can be supplied freely by a bypass line between the cable and the cable .
【０００５】[0005]
【実施例】以下図に示す一実施例により本発明を詳細に
説明する。１はポンプで一方は溶離液２に、他方はイン
ジェクター３に接続してある。４はジョイントで三方ジ
ョイントを用いるが、一方はインジェクター３に、他方
はポンプ５を介して誘導体化試薬入溶離液６に、更にも
う一方は６方バルブ７の通孔７１に夫々連通してある。
尚、ポンプ１，５は１台とは限らず何台でもよい。又、
ジョイント４は三方ジョイントに限るものではない。DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail with reference to an embodiment shown in the drawings. Reference numeral 1 denotes a pump, one of which is connected to the eluent 2 and the other of which is connected to the injector 3. Reference numeral 4 denotes a joint, which uses a three-way joint, one of which is in communication with the injector 3, the other of which is in communication with the derivatization reagent-containing eluent 6 through the pump 5, and the other of which is in communication with the through hole 71 of the six-way valve 7. .
The pumps 1 and 5 are not limited to one, but may be any number. or,
The joint 4 is not limited to the three-way joint.
【０００６】誘導体化試薬について特に指定されず、通
常使用するオルトフタルアルデヒド、ダンシルクロライ
ド、２，４ジニトロフェノール等試料に対応したものを
使用する。必要に応じてジョイント４と６方バルブ７の
間に反応コイル１４を入れることもできる。この反応コ
イルは温度調節も可能にできる。The derivatizing reagent is not particularly specified, and those normally used such as orthophthalaldehyde, dansyl chloride, 2,4 dinitrophenol, etc. are used. If necessary, the reaction coil 14 can be inserted between the joint 4 and the 6-way valve 7 . The reaction coil can also be temperature controlled.
【０００７】６方バルブ７の通孔７２と通孔７５間には
プレカラム８を連結させてある。該プレカラム８は一般
に使用されているＯＤＳ、Ｃ8、Ｃ4、Ｃ18、ＰＨ等誘導
体化された試料を保持できるものであれば何でも使用で
きる。例えば除蛋白を行うには除蛋白可能のカラムを用
いる。通孔７３には分析カラム９を介して検出器１０を
連設させ、通孔７４には通孔７４には、一方を分析用溶
離液１１に連設したポンプ１２に接続させてある。ポン
プ１２の台数は分析に応じて増やすことができる。The precolumn 8 is connected between the through hole 72 and the through hole 75 of the 6-way valve 7 . As the pre-column 8, any commonly used sample can be used as long as it can hold a derivatized sample such as ODS, C 8 , C 4 , C 18 , and PH. For example, a deproteinizable column is used for deproteinization. The detector 10 is connected to the through hole 73 through the analysis column 9, and the through hole 74 is connected to the pump 12 which is connected to the analysis eluent 11 on one side. The number of pumps 12 can be increased depending on the analysis.
【０００８】１３はドレインで通孔７６に連通させてあ
る。分析カラムにはその分析を為しうるものであれば選
択せず、特に指定されない。然し、光学分割を行う場合
には当然光学分割可能なカラムを必要とする。又、ラセ
ミ体化合物の光学分割は、光学分割カラムでの直接分割
法と、光学活性な誘導体化試薬を用いての誘導体化によ
りジアステレオマーにし、通常の逆相カラムを用いて光
学分割を行うジアステレオマー方とを使用できる。１４
は反応コイル、１５はバイパスラインで誘導体化後溶離
液２で希釈し、プレカラム８で保持されやすくするもの
で、必要に応じて設けることができる。A drain 13 is connected to the through hole 76. The analytical column is not selected as long as it can perform the analysis, and is not particularly specified. However, when performing optical resolution, a column capable of optical resolution is naturally required. Optical resolution of a racemic compound is performed by a direct resolution method using an optical resolution column and a diastereomer by derivatization using an optically active derivatization reagent, and then a normal reverse phase column is used. The diastereomer method which performs optical resolution can be used. 14
Is a reaction coil, and 15 is a bypass line, which is derivatized and then diluted with the eluent 2 so as to be easily retained in the pre-column 8, and can be provided as necessary.
【０００９】次いで、その使用方法について説明すれ
ば、ポンプ１を作動させ、溶離液２をインジェクター
３、三方弁４を介して６方バルブ７に送る。この時、溶
離液２は通孔７１、７２を通してプレカラム８を通り、
通孔７５、７６を経てドレイン１３より排出される。Next, the method of use will be described. The pump 1 is operated and the eluent 2 is sent to the 6-way valve 7 via the injector 3 and the 3-way valve 4. At this time, the eluent 2 passes through the pre-column 8 through the through holes 71 and 72,
It is discharged from the drain 13 through the through holes 75 and 76.
【００１０】試料の誘導体化を行う際には、インジェク
ター３から試料を注入し、ポンプ５を作動させて誘導体
化試薬入溶離液６を三方弁４に送り込む。然るとき、試
料は試薬と混合しつゝ６方バルブ７に送られる。この状
態にて試料は誘導体化されてプレカラム８を通過し、Ｏ
ＤＳ等により保持される。残余はドレイン１３に排出さ
れる。このようにして誘導体化された試料がプレカラム
８に蓄積される。When the sample is derivatized, the sample is injected from the injector 3 and the pump 5 is operated to feed the derivatization reagent-containing eluent 6 into the three-way valve 4. At that time, the sample is mixed with the reagent and sent to the 6-way valve 7 . In this state, the sample is derivatized and passes through the pre-column 8 to
It is held by DS or the like. The rest is discharged to the drain 13. The sample thus derivatized is accumulated in the pre-column 8.
【００１１】そこで、６方バルブ７を切換え、ポンプ１
２を作動させ分析用溶離液１１を送りこむ。分析用溶離
液１１は通孔７４、７５を経てカラム８に入り、蓄積さ
れた誘導体化された試料を溶出させ通孔７２、７３を経
て分析カラム９に送り、検出器１０を通り排出される。
従って、分析カラム９にて分離され、検出器１０にて検
出される。又、誘導体化未反応成分や未反応試薬は、プ
レカラム８を通過するものはそのまゝ保持されるものは
分析後溶出等により分析カラム９への送入を阻止する。Therefore, the six-way valve 7 is switched to the pump 1
2 is operated and the eluent 11 for analysis is fed. The analytical eluent 11 enters the column 8 through the through holes 74 and 75, elutes the accumulated derivatized sample, sends it to the analytical column 9 through the through holes 72 and 73, and is discharged through the detector 10. .
Therefore, it is separated in the analytical column 9 and detected by the detector 10. As for the derivatized unreacted components and unreacted reagents, those that pass through the pre-column 8 are retained as they are, and the derivatized unreacted components and unreacted reagents are prevented from being fed into the analytical column 9 by elution after analysis.
【００１２】本発明の実施具体例をプレカラム誘導体化
を用いた分析方法により説明する。
光学分割を目的とする分析
サンプル：ＤＬ−バリン、ＤＬ−ロイシン。
プレカラム：ブチル基修飾シリカゲルを５０×４．６ｍ
ｍＩ．Ｄのステンレス管にて充填したもの。
分析カラム：アビジン固定化シリカゲルを１５０×９．
６ｍｍＩ．Ｄのステンレス管にて充填したもの。
誘導体化試薬入り溶離液：オルトフタルアルデヒド１０
０ｍｇを３ｍｌのユタノールに溶かし、そこへ０．４Ｍ
ホウ酸緩衝液（ｐＨ１０、ＮａＯＨで調整）２２０ｍ
ｌ、０．１ｍｌの２−メルカプトエタノールを加える。
これにつながるポンプの流速は０．５ｍｌ／ｍｉｎ。
インジェクターを介するラインの溶離液：Ｈ2Ｏ、これ
につながるポンプの流速は０．５ｍｌ／ｍｉｎ。
分析用溶離液：０．１Ｍリン酸緩衝液（ｐＨ７．０）／
アセトニトリル＝９５／５
分析カラム温度：３０℃
検出器：螢光検出器 Ｅｘ ３３０ｎｍ、Ｅｍ ４５６
ｎｍ。
上記の条件での分析は図２、図３の通りである。図２は
クロマトグラフ（１）ＤＬ−ロイシンの分析状態を示す
もので、はＤ−ロイシン、はＬ−ロイシンである。
図３はクロマトグラフ（１）ＤＬ−バリンの分析状態を
示すもので、はＤ−バリン、はＬ−バリンである。An embodiment of the present invention will be described by an analysis method using precolumn derivatization. Analytical samples for optical resolution: DL-valine, DL-leucine. Pre-column: Butyl group-modified silica gel 50 x 4.6 m
mI. Filled with D stainless steel tube. Analytical column: Avidin-immobilized silica gel 150 × 9.
6 mmI. Filled with D stainless steel tube. Eluent containing derivatization reagent: Orthophthalaldehyde 10
Dissolve 0mg in 3ml of Utanol and add 0.4M to it.
Borate buffer (pH 10, adjusted with NaOH) 220 m
l, 0.1 ml of 2-mercaptoethanol is added.
The flow rate of the pump connected to this is 0.5 ml / min. Eluent in line through injector: H 2 O, flow rate of pump connected to this is 0.5 ml / min. Eluent for analysis: 0.1 M phosphate buffer (pH 7.0) /
Acetonitrile = 95/5 Analytical column temperature: 30 ° C. Detector: Fluorescence detector Ex 330 nm, Em 456
nm. The analysis under the above conditions is as shown in FIGS. FIG. 2 shows the chromatographic (1) analysis state of DL-leucine, where is D-leucine and is L-leucine.
FIG. 3 shows the chromatographic (1) analysis state of DL-valine, in which D-valine is L-valine.
【００１３】[0013]
【発明の効果】 上記の如き本発明によれば、試料の誘
導体化をオンラインにより行い、該誘導体化させた試料
を一端プレカラムに保持させ、その後に分析カラムに送
入させ分析させる方法に於いて、誘導体化用溶離液と溶
離液とを夫々独立して自在に供給すると共に、溶離液
は、誘導体化反応コイルの下流側で、プレカラムに連通
されたプレカラムの溶離液切換用バルブとの間にバイパ
スラインで供給自在としたので、誘導体化により試料感
度が上がり、微量分析が可能になる。然もオンラインに
よる誘導体化の過程で広がってしまったバンドをプレカ
ラムにより狭めることが出来分析の効率を上げられる。
又、誘導体化をオンラインで行っているので、再現性が
得られる。EFFECTS OF THE INVENTION According to the present invention as described above, a method of performing derivatization of a sample online, holding the derivatized sample on a precolumn at one end, and then sending it to an analytical column for analysis is provided. supplies freely and the eluent for derivatization eluant each independently, eluent
Communicate with the pre-column downstream of the derivatization reaction coil
Since it is possible to freely supply by a bypass line between the eluent switching valve of the precolumn, the sample sensitivity is improved by derivatization, and microanalysis can be performed. However, it is possible to narrow the band that has spread during the process of derivatization online by using the pre-column and improve the efficiency of analysis.
In addition, since derivatization is performed online, reproducibility can be obtained.
【００１４】又、誘導体化未反応成分は、プレカラムか
ら溶出されてしまい分析カラムには入らないので、分析
カラムを保護することができる。又、使用分析カラムが
誘導体試薬と反応する場合、未反応試薬を取り除くこと
ができるため特に有効である。更に、オンラインで誘導
体化しているため、誘導体化後の試料の保持力を高める
希釈ラインを入れることも可能となる等の効果がある。Further, since the derivatized unreacted components are eluted from the precolumn and do not enter the analytical column, the analytical column can be protected. Further, when the used analytical column reacts with the derivative reagent, the unreacted reagent can be removed, which is particularly effective. Furthermore, since the derivatization is performed online, it is possible to add a dilution line that enhances the retention of the sample after derivatization.
【図１】本発明一実施例実施装置。FIG. 1 is an embodiment of the present invention.
【図２】本発明実施例を用いた分析例のクロマトグラ
ム。FIG. 2 is a chromatogram of an analysis example using an example of the present invention.
【図３】本発明実施例を用いた他分析例のクロマトグラ
ム。FIG. 3 is a chromatogram of another analysis example using the example of the present invention.
１ ポンプ ２ 溶離液 ３ インジェクター ４ ジョイント ５ ポンプ ６ 誘導体化試薬入溶離液７ ６方バルブ ８ プレカラム ９ 分析カラム １０ 検出器 １１ 分析用溶離液 １２ ポンプ １３ ドレイン １５ バイパスライン1 Pump 2 Eluent 3 Injector 4 Joint 5 Pump 6 Eluent with derivatization reagent 7 6-way valve 8 Pre-column 9 Analytical column 10 Detector 11 Analytical eluent 12 Pump 13 Drain 15 Bypass line
フロントページの続き (56)参考文献 特開 平２−255625（ＪＰ，Ａ) 特開 平３−130660（ＪＰ，Ａ) 特開 昭60−142250（ＪＰ，Ａ) (58)調査した分野(Int.Cl.7，ＤＢ名) G01N 30/06 G01N 30/88 Continuation of the front page (56) Reference JP-A-2-255625 (JP, A) JP-A-3-130660 (JP, A) JP-A-60-142250 (JP, A) (58) Fields investigated (Int .Cl. 7 , DB name) G01N 30/06 G01N 30/88
Claims (4)
い、該誘導体化させた試料を一端プレカラムに保持さ
せ、その後に分析カラムに送入させ分析させる方法に於
いて、誘導体化用溶離液と溶離液とを夫々独立して自在
に供給すると共に、溶離液は、誘導体化反応コイルの下
流側で、プレカラムに連通されたプレカラムの溶離液切
換用バルブとの間にバイパスラインで供給自在としたこ
とを特徴とするプレカラム誘導体化を用いたクロマトグ
ラフ分析方法。1. A method of performing derivatization of a sample online, holding the derivatized sample on a pre-column at one end, and then feeding the sample to an analytical column for analysis, and an eluent for derivatization and an eluent. And each of them are supplied independently and the eluent is under the derivatization reaction coil.
On the flow side, the eluent of the pre-column connected to the pre-column is cut off.
A chromatographic analysis method using precolumn derivatization, characterized in that it can be freely supplied by a bypass line between a replacement valve .
特徴とする請求項１に記載のプレカラム誘導体化を用い
たクロマトグラフ分析方法。2. The chromatographic analysis method using precolumn derivatization according to claim 1, wherein the analysis column is an optical resolution column.
又は未反応試薬をプレカラムを通すことによって除去し
分析カラム送入阻止することを特徴とする請求項１に記
載のプレカラム誘導体化を用いたクロマトグラフ分析方
法。3. A chromatograph using precolumn derivatization according to claim 1, wherein unreacted components and / or unreacted reagents are derivatized by passing through a precolumn to prevent the derivatization of the analytical column. Analysis method.
イパスラインにより送ることを特徴とする請求項１に記
載のプレカラム誘導体化を用いたクロマトグラフ分析方
法。4. The chromatographic analysis method using precolumn derivatization according to claim 1, wherein a diluent for diluting the derivatized sample is sent through a bypass line.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13609495A JP3471124B2 (en) | 1995-05-10 | 1995-05-10 | Chromatographic analysis using precolumn derivatization |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13609495A JP3471124B2 (en) | 1995-05-10 | 1995-05-10 | Chromatographic analysis using precolumn derivatization |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH08304366A JPH08304366A (en) | 1996-11-22 |
| JP3471124B2 true JP3471124B2 (en) | 2003-11-25 |
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ID=15167128
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| JP3665680B2 (en) * | 1996-06-05 | 2005-06-29 | ジーエルサイエンス株式会社 | Trace analysis method and liquid chromatograph |
| JP6040896B2 (en) * | 2013-09-04 | 2016-12-07 | 株式会社島津製作所 | Prelabeled amino acid analysis by liquid chromatography |
| CN103869093B (en) * | 2014-03-21 | 2015-05-20 | 四川材料与工艺研究所 | Device for performing on-line quick sampling on multi-component gas and sampling method thereof |
| CN104007272B (en) * | 2014-05-28 | 2016-01-20 | 宁波管通机械有限公司 | Automatic carbon sulphur combined measuring instrument |
| CN116519853A (en) * | 2023-04-26 | 2023-08-01 | 江南大学 | Method for rapidly detecting fatty acid derivatives in dairy products |
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| Publication number | Publication date |
|---|---|
| JPH08304366A (en) | 1996-11-22 |
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