JPS63298044A - Production of gradient gel film for electrophoresis - Google Patents
Production of gradient gel film for electrophoresisInfo
- Publication number
- JPS63298044A JPS63298044A JP62131502A JP13150287A JPS63298044A JP S63298044 A JPS63298044 A JP S63298044A JP 62131502 A JP62131502 A JP 62131502A JP 13150287 A JP13150287 A JP 13150287A JP S63298044 A JPS63298044 A JP S63298044A
- Authority
- JP
- Japan
- Prior art keywords
- coating
- liquid
- slot
- continuously
- electrophoresis
- 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.)
- Granted
Links
- 238000001962 electrophoresis Methods 0.000 title claims abstract description 24
- 239000011544 gradient gel Substances 0.000 title claims abstract description 19
- 238000004519 manufacturing process Methods 0.000 title claims description 16
- 108010025899 gelatin film Proteins 0.000 title abstract description 4
- 239000011248 coating agent Substances 0.000 claims abstract description 81
- 238000000576 coating method Methods 0.000 claims abstract description 81
- 239000007788 liquid Substances 0.000 claims abstract description 78
- 239000000178 monomer Substances 0.000 claims description 23
- 238000000926 separation method Methods 0.000 claims description 18
- 239000012528 membrane Substances 0.000 claims description 13
- 239000003999 initiator Substances 0.000 claims description 11
- 238000006116 polymerization reaction Methods 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 8
- 239000000243 solution Substances 0.000 description 19
- 239000000499 gel Substances 0.000 description 17
- 230000003068 static effect Effects 0.000 description 9
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical group NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 230000008602 contraction Effects 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003431 cross linking reagent Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000000465 moulding Methods 0.000 description 2
- -1 polyethylene Polymers 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- YAHTUJLFNVSDAI-UHFFFAOYSA-N 1-ethoxy-4-hexoxy-1,4-dioxobutane-2-sulfonic acid Chemical compound CCCCCCOC(=O)CC(S(O)(=O)=O)C(=O)OCC YAHTUJLFNVSDAI-UHFFFAOYSA-N 0.000 description 1
- OEPOKWHJYJXUGD-UHFFFAOYSA-N 2-(3-phenylmethoxyphenyl)-1,3-thiazole-4-carbaldehyde Chemical compound O=CC1=CSC(C=2C=C(OCC=3C=CC=CC=3)C=CC=2)=N1 OEPOKWHJYJXUGD-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 229920002799 BoPET Polymers 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 239000000538 analytical sample Substances 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000007766 curtain coating Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000007765 extrusion coating Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000003505 polymerization initiator Substances 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229950001574 riboflavin phosphate Drugs 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、ＤＮＡもしくは、ＤＮＡ部分分解物の塩基配
列を決定する為の電気泳動用グラジェントゲル膜の製造
方法に関する、特に電気泳動分離用媒体液をウェブに塗
布する塗布へノドに関するものである。Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method for producing a gradient gel membrane for electrophoresis for determining the base sequence of DNA or a partially degraded DNA product, particularly for electrophoretic separation. It relates to a coating gutter that applies a medium liquid to a web.
従来、平板型電気泳動法において、自己支持性のないア
クリルアミドの高分子濃度グラジェントゲルは、一枚の
支持体の上または二枚の支持体の間に濃度の異なるゲル
を電気泳動方向に層状に形成させ、膜状物として用いら
れて来た。Conventionally, in plate electrophoresis, acrylamide polymer concentration gradient gels that are not self-supporting are layered in the electrophoresis direction using gels with different concentrations on one support or between two supports. It has been used as a film-like material.
しかしながら支持体上にゲルを形成して用いる方法は、
支持体上にゲルを形成する時、泳動槽にセットする時、
ゲルの保存中、あるいは分析試料を添加するときなどに
誤まってゲルをこわしたり、試料以外のものをゲルの上
に落してゲルを損なったりすること等があり、操作上細
心の注意と熟練が必要であった。However, the method of forming and using a gel on a support is
When forming a gel on a support, when setting it in an electrophoresis tank,
While storing the gel or adding an analytical sample, it is possible to accidentally break the gel or drop something other than the sample onto the gel, damaging the gel. was necessary.
一方二枚のガラス板などでモールドをつくり、この中で
ゲルを形成させて、そのモールドを垂直に保ったま＼電
気泳動分析を行う垂直式電気泳動法においては、モール
ドの厚さを均一にすることは困難であったり、ゲル形成
液がゲル化しないうちに狭いモールド中にゲル液を注入
しなければならないことなど操作上高度の熟練を要して
いた。On the other hand, in the vertical electrophoresis method, in which a mold is made from two glass plates, etc., a gel is formed in the mold, and the electrophoretic analysis is performed while the mold is held vertically, the thickness of the mold is made uniform. It is difficult to do this, and requires a high level of skill in operation, as the gel forming solution must be injected into a narrow mold before it gels.
特にＤＮＡの塩基配列決定操作においては、一枚のゲル
で出来るだけ多くのＤＮＡの断片を分析できるように長
いゲルを作ることが望ましいが、そのようなゲルはその
製造および取扱いが難しかった。また、ガラス板を用い
るために、そのガラス板が破↑易しやすいという欠点が
あった。Particularly in DNA base sequencing operations, it is desirable to create long gels so that as many DNA fragments as possible can be analyzed in one gel, but such gels are difficult to manufacture and handle. Furthermore, since a glass plate is used, there is a drawback that the glass plate is easily broken.
近年工業的に電気泳動用グラジェントゲル物質を製造す
る方法として、アクリルアミドモノマー及び架橋剤の混
合物の水溶液または水分散液を作り、光の照射を吸収し
、モノマーの重合を開始する遊離ラジカル発生物質を添
加した後、該溶液を所望のゲル生成物の形に形成し、形
成した溶液に光照射してモノマー溶液または、分散液を
重合させ架橋させる際、ゲルの多孔度を変えるために七
ツマー溶液に印加する光照射時間を調整する工程を含む
電気泳動グラジェントゲルの製法。（特開昭６１−２８
５１２号公報参照）
さらに又単量体、架橋剤および重合開始剤を含有し、濃
度の相互に異る二種類の水溶液を混合比率を漸次変化さ
せながら混合しつつ成形器に導入し、該成形器中で単量
体と架橋剤との重合を完了させ、電気泳動方向に重合体
の濃度勾配を有する電気泳動用グラジェントゲルの製造
方法。（特公昭６１−２２９０３号公報、特公昭６１−
３９６１７号公報参照）等が開示されている。In recent years, as a method for industrially producing gradient gel materials for electrophoresis, an aqueous solution or aqueous dispersion of a mixture of an acrylamide monomer and a crosslinking agent is prepared, and a free radical generating substance that absorbs light irradiation and initiates polymerization of the monomer is used. The solution is then formed into the desired gel product, and the formed solution is irradiated with light to polymerize and crosslink the monomer solution or dispersion. A method for producing an electrophoretic gradient gel, which includes a step of adjusting the light irradiation time applied to the solution. (Unexamined Japanese Patent Publication No. 61-28
Furthermore, two types of aqueous solutions containing a monomer, a crosslinking agent, and a polymerization initiator and having different concentrations are introduced into a molding machine while being mixed while gradually changing the mixing ratio. A method for producing a gradient gel for electrophoresis, which completes polymerization of a monomer and a crosslinking agent in a container and has a polymer concentration gradient in the direction of electrophoresis. (Special Publication No. 61-22903, Special Publication No. 61-22903, Publication No. 61-22903,
39617) etc. have been disclosed.
又、連続的に走行するウェブ上に電気泳動分離媒体液を
塗布する塗布ヘッドとしては、第６図に示す様な一般的
なものが用いられていた。即ち塗布液１２としての電気
泳動分離媒体液は定量ポンプ２２によって塗布ヘッド１
８内のキャビティ１９に圧送される。キャビティ１９は
スロット開口２４に連通している塗布幅一杯に拡がった
スロット２３に塗布液を供給する液溜りである。塗布液
１２はキャビティ１９から縮流してスロット２３に入り
スロット開口２４からコーティングローラ２０の上のウ
ェブ１３に塗布される。この際塗布の安定化のために塗
布ビード後方を背圧にするための差圧チャンバー２１が
用いられる。Further, as a coating head for coating an electrophoretic separation medium liquid onto a continuously running web, a general coating head as shown in FIG. 6 has been used. That is, the electrophoretic separation medium liquid as the coating liquid 12 is delivered to the coating head 1 by the metering pump 22.
8 into a cavity 19. The cavity 19 is a liquid reservoir that supplies the coating liquid to the slot 23 which communicates with the slot opening 24 and extends over the entire coating width. The coating liquid 12 flows from the cavity 19 into the slot 23 and is applied to the web 13 on the coating roller 20 through the slot opening 24 . At this time, a differential pressure chamber 21 is used to create a back pressure behind the coating bead in order to stabilize the coating.
しかしながら、前記最初の製法は光照射の設備と生産性
の悪さによるコスト高と、ゲルの厚みが厚いための解像
性が悪い点１反応開始剤が製造終了後も光の存在下で重
合・架橋反応をおこし、安定性、再現性のあるグラジェ
ントゲルが得られない等の問題点を有し、後者次の製造
方法はやはりバッチ式製造方式であるため生産性が悪く
、又電気泳動分離用媒体液を成形器に分岐流入する際に
均一に分岐することが難しく、再現性のあるグラジェン
トゲルが得られない等の問題点を有していた。However, the above-mentioned first production method suffers from high costs due to poor light irradiation equipment and poor productivity, and poor resolution due to the thick gel.1 The reaction initiator remains polymerized in the presence of light even after production is completed. There are problems such as cross-linking reaction, making it impossible to obtain stable and reproducible gradient gels, and the latter production method is still a batch production method, resulting in poor productivity. When the medium liquid is branched into the molding machine, it is difficult to branch it uniformly, and a reproducible gradient gel cannot be obtained.
更に又従来の塗布ヘッドで塗布した製品の幅方向の膜厚
は均一性が充分でなく、長時間の運転により異物の発生
があり、その為塗布ヘッドの分解洗浄が必要であった。Furthermore, the film thickness of the product coated with the conventional coating head in the width direction is not sufficiently uniform, and foreign matter may be generated due to long-term operation, making it necessary to disassemble and clean the coating head.
本発明の目的は上記問題点を解消し、塗布幅全体に亘っ
て均一な塗布膜厚で、異物の発生がな（ウェブの走行方
向に連続的に濃度勾配をもった電気泳動用グラジェント
ゲル模を生産性が高く、安定性、再現性良く製造する方
法を提供することにある。The purpose of the present invention is to solve the above-mentioned problems, and to provide a gradient gel for electrophoresis that has a uniform coating thickness over the entire coating width and does not generate foreign matter. The purpose of the present invention is to provide a method for manufacturing patterns with high productivity, stability, and good reproducibility.
本発明の前記目的は、連続的に濃度勾配をもったモノマ
ー液と重合反応開始剤液とを混合しながら電気泳動分離
用媒体液として塗布ヘッドに供給し、走行するウェブ上
に塗布する電気泳動用グラジェントゲル膜の製造方法で
あって該塗布液を該塗布ヘッドの供給口よりスロット開
口に到る間、幅方向を連続的に拡げ、厚み方向に連続的
に縮少して供給する過程を有することを特徴とする電気
泳動用グラジェントゲル膜の製造方法によって達成され
る。The object of the present invention is to continuously mix a monomer solution with a concentration gradient and a polymerization reaction initiator solution and supply it to a coating head as a medium solution for electrophoretic separation, and to perform electrophoresis coating on a running web. A method for producing a gradient gel film for use in a coating device, which comprises supplying the coating solution by continuously expanding it in the width direction and contracting it continuously in the thickness direction while it reaches the slot opening from the supply port of the coating head. This is achieved by a method for producing a gradient gel membrane for electrophoresis characterized by having the following characteristics.
本発明において連続的に濃度勾配をもったモノマー液と
は、連続的に流量比を変えた濃・淡二種のモノマー液に
よって作られる。そしてその濃度勾配としては３〜３５
Ｗ／Ｖ％が好ましい。In the present invention, the monomer liquid having a continuous concentration gradient is produced by using two types of monomer liquids, a concentrated liquid and a light liquid, in which the flow rate ratio is continuously changed. And the concentration gradient is 3 to 35
W/V% is preferred.
本発明において混合する方法としては、一般のタンク内
で攪拌機によって行ってもよいが、スタティックミクサ
ーによるｆ′昆合調：夜が最も好ましい。In the present invention, mixing may be carried out using a stirrer in a general tank, but it is most preferable to use a static mixer at night.
スタティックミクサーとは、流体がミクサーの中に通さ
れる時、流体自身の運動により攪拌作用が生じるものを
いい、管状のものとしてはスパイラルチューブ、左右切
り換えらせん状、プロペラ状等がある。A static mixer is one in which a stirring action occurs due to the movement of the fluid itself when the fluid is passed through the mixer, and the tubular shapes include a spiral tube, a left-right switching helical shape, a propeller shape, etc.
本発明における電気泳動分離用媒体液とは、電気泳動分
離用媒体膜を作れるものであれば何でもよく、その代表
的なものとしては、アクリルアミドゲル、アガロースゲ
ル、澱粉ゲル、寒天ゲル。The electrophoretic separation medium in the present invention may be anything as long as it can form an electrophoretic separation medium membrane, and representative examples include acrylamide gel, agarose gel, starch gel, and agar gel.
セルロースアセテート多孔質膜、濾紙等の原料液となる
液をいう。A liquid that serves as a raw material for cellulose acetate porous membranes, filter paper, etc.
本発明における電気泳動分離用媒体液の塗布ヘッドとし
ては、スライドビードコート、エクストル−ジョンコー
ト、ホッパーコート、カーテンコート等の塗布ヘッドが
用いられる。媒体膜の厚みは分離の目的に応じて選ばれ
るが通常５０μｍから約１．Ｑ＋ｕ、好ましくは約２０
０μｍから約０．５０゜多孔質膜や濾紙の場合には約７
０μｍから約１ｍの範囲とされる。As the coating head for the electrophoretic separation medium liquid in the present invention, coating heads such as slide bead coating, extrusion coating, hopper coating, curtain coating, etc. are used. The thickness of the media membrane is selected depending on the purpose of separation, but is usually from 50 μm to about 1.5 μm. Q+u, preferably about 20
0 μm to approximately 0.50°, approximately 7 in the case of porous membranes and filter paper
The range is from 0 μm to about 1 m.
本発明における走行するウェブとは、平面性のよいシー
ト状のもので非導電性かつ実質的に水不透過性であれば
、どのような材質のものでもよく、ポリエチレンテレフ
タレート、ビスフェノールＡのポリカルボネートのよう
なポリエステル、ポリメチルメタクリレート、ポリエチ
レン、ポリスチレン、ポリ塩化ビニルなどのビニル系重
合体、ナイロンなどのポリアミドなど、およびそれらの
共重合体（例、塩化ビニリデン・塩化ビニルコポリマー
）が好ましく用いられる。The running web in the present invention may be made of any material as long as it is in the form of a sheet with good flatness, is non-conductive, and is substantially water-impermeable. Preferably used are polyesters such as esters, polymethyl methacrylate, polyethylene, polystyrene, vinyl polymers such as polyvinyl chloride, polyamides such as nylon, and copolymers thereof (e.g., vinylidene chloride/vinyl chloride copolymers). .
本発明における塗布ヘッドに塗布液を供給口よりスロ・
ノド開口に到る迄幅方向を連続的に拡げ厚み方向に連続
的に縮少して供給するとは、供給口よりスロット状開口
に到る塗布液流路を■ 塗布液流路の縁線を急激に変化
させることな（連続的に変化させる■ 幅を拡げる時は
それに対応して厚味を縮少する■ 拡張、収縮の角度を
規制する等によって、流れ全体としての渦流、滞留をさ
けることをいう。結果として本発明は従来塗布ヘッドに
用いられていたキャビティを使用しないことになる。In the present invention, the coating liquid is supplied to the coating head through the supply port.
Continuously widening in the width direction until reaching the gutter opening and continuously contracting in the thickness direction means that the coating liquid flow path from the supply port to the slot-shaped opening is Avoid changing the width (continuously change) ■ When expanding the width, reduce the thickness accordingly ■ Avoid vortices and stagnation in the overall flow by regulating the angle of expansion and contraction, etc. As a result, the present invention eliminates the use of cavities conventionally used in coating heads.
本発明の実施態様を図を用いて説明する。Embodiments of the present invention will be described using figures.
第１図は本発明の電気泳動用グラジェントゲル膜の製造
方法の工程図である。FIG. 1 is a process diagram of the method for producing a gradient gel membrane for electrophoresis according to the present invention.
電気泳動用媒体液１２は、低濃度モノマー液１゜高濃度
モノマー液２．更に重合反応開始剤液３をスタティック
ミクサー７内で攪拌・混合することによって調液される
。The electrophoresis medium liquid 12 includes a low concentration monomer liquid 1, a high concentration monomer liquid 2. Further, the polymerization reaction initiator liquid 3 is stirred and mixed in a static mixer 7 to prepare a liquid.
第２図ａは本発明の濃・淡二種のモノマー液の流量比の
関係の説明図である。連続的に流量比をかえるというこ
とは、例えば当初高濃度モノマーｉ＆ ２はＱ ｍ Ｑ
、／ｍｉｎ 、低濃度モノマー液は１９ｍｌ１／ｍｉｎ
の流量比で初まるが、電気泳動用グラジェントゲル膜の
製品単位長をｌとすると、図上、高濃度モノマー液２は
実線で、低濃度モノマー液１は点線で示すように流量を
変え、合計の流量は１９ｍβ／ｍｉｎということで一定
であるが、濃度は連続的に変化することになる。製品単
位長βの終りは高濃度モノマー液１は１９ ｍ Ａ ／
ｍｉｎ 、低濃度モノマー液１はＯｍβ／ｍｉｎとなり
濃度の濃い膜成分になっている。この様な流量比の変更
を繰返し行うために、第１図において流量勾配送液ポン
プ４．５がパーソナルコンピュータ１１の指令によりコ
ンピューターリンケージアダプタ１０を経由してコント
ローラ９ａによって制御される。FIG. 2a is an explanatory diagram of the relationship between the flow rate ratios of two kinds of concentrated and light monomer liquids of the present invention. Continuously changing the flow rate means that, for example, initially high concentration monomers i & 2 are Q m Q
,/min, low concentration monomer liquid: 19ml1/min
However, if the product unit length of the gradient gel membrane for electrophoresis is 1, the flow rate is changed as shown in the figure, as shown by the solid line for high concentration monomer solution 2 and the dotted line for low concentration monomer solution 1. , the total flow rate is constant at 19 mβ/min, but the concentration changes continuously. The end of the product unit length β is 19 m A /
min, and the low concentration monomer liquid 1 becomes Omβ/min, making it a highly concentrated film component. In order to repeatedly change the flow rate ratio in this manner, the flow gradient liquid pump 4.5 in FIG. 1 is controlled by the controller 9a via the computer linkage adapter 10 in accordance with commands from the personal computer 11.
一方重合反応開始剤液３は定流量送液ポンプ６によって
コントローラー９ｂに制御され一定量をスタテイ・戸り
ミクサ−７に送り込まれ、スタティックミクサー７内で
低濃度上ツマー液１と高濃度モノマー液２そして重合反
応開始剤液３は攪拌混合され電気泳動用媒体液１２が出
来る。電気泳動用媒体液１２は塗布ヘッド８に供給され
、ウェブ１３上に塗布されることになる。On the other hand, the polymerization reaction initiator liquid 3 is controlled by the controller 9b by the constant flow liquid sending pump 6, and a constant amount is sent to the static mixer 7. 2, the polymerization reaction initiator liquid 3 is stirred and mixed to form an electrophoresis medium liquid 12. The electrophoresis medium liquid 12 is supplied to the coating head 8 and is coated onto the web 13.
第２図（ｂｌは第２図（ａ）の濃度比率によって作られ
る電気泳動用グラジェントゲル膜の平面図、第２図（Ｃ
）は第２図（ｂｌの側面図である。Figure 2 (bl is a plan view of a gradient gel membrane for electrophoresis made with the concentration ratio shown in Figure 2 (a), Figure 2 (C
) is a side view of FIG. 2 (bl).
第２図（ａｌの製品単位長β間の間隙ｔは試料注入口の
部分として打抜かれ、カバーシート等によって試料注入
口になる部分である。The gap t between the product unit lengths β in FIG.
次に第３図（ａｌ 、 （ｂ）は本発明の塗布ヘッドの
一実施例の側面断面図である。塗布ヘッド８はスタティ
ックミクサー７からの塗布液である電気泳動分雌用媒体
液１２を供給口１４で受け、スロット１６を通じてスロ
ット開口１７よりウェブ１３上にコーティングローラ２
０の上で塗布をしている。Next, FIGS. 3A and 3B are side sectional views of an embodiment of the coating head of the present invention. The coating roller 2 is received at the supply port 14 and is applied onto the web 13 through the slot opening 17 through the slot 16.
The coating is applied on top of 0.
第３図、（ａ）と（１））は塗布液１２をウェブ１３上
に塗りつけるコーティングローラ２０上の位置の違いで
、どちらを用いても良い。3(a) and (1)) differ in the position on the coating roller 20 at which the coating liquid 12 is applied onto the web 13, and either one may be used.
本発明の場合供給口１４からスロット１６への塗布液の
供給が幅、厚味共に連続的に拡大、縮少させ、拡大、縮
小の角度を比較的ゆるやかにしなければならないため、
塗布ヘッドのスロット１６の長さが従来のものと比較し
て長くなる。、そのためコーティングローラ２０に対し
て塗布ヘッド８の位置が第３図（ａ）及びｔｂ＋の様な
配置になる。In the case of the present invention, the width and thickness of the coating liquid supplied from the supply port 14 to the slot 16 must be continuously expanded and decreased, and the angle of expansion and contraction must be relatively gentle.
The length of the slot 16 of the application head is increased compared to the conventional one. Therefore, the position of the coating head 8 with respect to the coating roller 20 is as shown in FIGS. 3(a) and tb+.
尚、第３図（ｂｌにおいて厚味方向の縮少の角度αくＯ
３５°が好ましい。第４図は第３図、の塗布ヘッドを正
面から見た断面図で、第５図は透視図である。第４図に
おいて塗布ヘッド８の塗布液１２の供給口１４よりスロ
ット開口１７に到る間、スロット１６の幅方向を連続的
に拡げる状態を示す。理想的には図中、点線で示すよう
にベンチュリー型に拡げるのが好ましいが、角度的規制
より考えて工作を簡単にするとすれば拡がりの角度θく
４０°に直線的にすることが一般に用いられる。In addition, in Fig. 3 (bl), the angle of reduction in the thickness direction α × O
35° is preferred. FIG. 4 is a sectional view of the coating head shown in FIG. 3 viewed from the front, and FIG. 5 is a perspective view. FIG. 4 shows a state in which the width direction of the slot 16 is continuously expanded while the coating liquid 12 of the coating head 8 reaches the slot opening 17 from the supply port 14. Ideally, it is preferable to expand it in a venturi type as shown by the dotted line in the figure, but if you want to simplify the work by considering angular restrictions, it is generally recommended to make it expand linearly at an angle of 40 degrees. It will be done.
幅と厚味の状態を共に示したのが第５図の透視図である
。The perspective view of FIG. 5 shows both the width and thickness.
（作 用〕
本発明は連続的に濃度勾配をもった七ツマー液と１合反
応開始剤液とを混合しながら電気泳動分離用媒体液とし
て塗布ヘッドに供給することによって、例えば、スタテ
ィックミクサーの攪拌力と自送力により前後の液の混合
がな（、塗布ヘッドに送られる為、従来にない濃淡勾配
のはっきりした塗布膜を安定して製造することが出来る
。しかも、生産性もよく製造出来る。(Function) The present invention can be applied to, for example, a static mixer by continuously mixing a 7-mer solution with a concentration gradient and a 1-mix reaction initiator solution and supplying it to a coating head as a medium solution for electrophoretic separation. The stirring power and self-feeding power prevent the mixing of the liquid before and after the liquid is sent to the coating head, so it is possible to stably produce a coating film with a clear gradient of density that has never been seen before.Moreover, it can be manufactured with high productivity. I can do it.
更に又本発明は塗布液を供給口よりスロット状開口に到
る間幅方向に連続的に拡げ、それに伴って厚味方向に連
続的に縮少させることにより、急激な液流の拡張、縮少
をさけるため具体的に■液流路の縁線を連続的に拡張す
る（例えばベンチュリーの様に）ことにより通路の縁線
に生じる渦流（滞留）をさける。■ 幅を拡げる時はそ
れに対応して厚味を縮少することによって各流路にわた
って均一な流抵抗になる。■ 拡張、収縮の角度を規制
する例えば液が拡がる時の角度θ＜４０°。Furthermore, the present invention continuously spreads the coating liquid from the supply port to the slot-shaped opening in the width direction, and concomitantly, continuously reduces it in the thickness direction, thereby preventing rapid expansion and contraction of the liquid flow. In order to avoid this problem, specifically: (1) The edge line of the liquid flow path is continuously expanded (for example, like a venturi) to avoid swirling (stagnation) that occurs at the edge line of the passage. ■ When the width is increased, the thickness is correspondingly reduced to achieve uniform flow resistance across each channel. ■ Regulating the angle of expansion and contraction. For example, the angle θ<40° when the liquid expands.
厚味方向の角度はα＜０．３５°とすることによって渦
流の発生を防ぎ、液の部分的滞留をなくするのである。By setting the angle in the thickness direction to α<0.35°, generation of vortices is prevented and partial retention of liquid is eliminated.
又特にキャビティを用いないことは、キャビティからス
リットに液が流出する場合に縮少部分の各所において生
じる渦と、それによる液の部分的滞留とそれによる異物
の発生を防ぐためである。In addition, the reason why a cavity is not used is to prevent vortices generated in various parts of the contracted portion when the liquid flows out from the cavity to the slit, and the resulting partial stagnation of the liquid and the resulting generation of foreign matter.
これらによって塗布の横幅全体に亘って均一な塗布膜を
得ることが出来る。By these methods, a uniform coating film can be obtained over the entire width of the coating.
本発明の実施例について説明する。 Examples of the present invention will be described.
電気泳動分離用媒体液は
低濃度上ツマ−として□■
アガロース・・・・・・・・・６５ｇ
尿素 ・・・・・・・４，２００ｇ
アクリルアミド・・・・・・ ５４９ｇ１．３．５−１
−リアクリ
ロイル−へキサヒドロ−
３−トリアジン・・・・・・ ５．７ｇ説ビイオン水
ｕｐ ｔｏ ９，０００ｍ６を作り高濃度モノマーとし
て□■
アガロース・・・・・・・・・４０ｇ
尿素 ・・・・・・・４，２００ｇ
アクリルアミド・・・・・ １．８３０ｇ１．３．５−
）リアクリ
ロイル−へキサヒドロ−
Ｓ−＋−リアジン・・・・・・・１９ｇ説イオン水
ｕｐ ｔｏ ９．０００ｍ ｊ！を作り緩衝液として□
■
トリスヒドロキシメチル
アミン ・・・・・１２１．１４ｇはう酸
・・・・・ ６５．４ｇエチレンジアミンテトラ
酢酸２ナトリウム塩・・・・７．４５ｇ説イオン水
１０） ｔｏ １，０００ｍｆｆを作り、緩衝液■・・
・・・・・・・・・・７５０＋＋＋β２．９χポリビニ
ルピロリドン水溶液・８００ｍ１２．５χテトラメチル
エチレンジアミン液・ ・ ・ ・６．７＋ｃＩＶ。The medium solution for electrophoretic separation is a low-concentration medium □■ Agarose 65 g Urea 4,200 g Acrylamide 549 g 1.3.5- 1
-Reacryloyl-hexahydro- 3-triazine... 5.7g Bi-ionized water
Make up to 9,000 m6 and use as high concentration monomers □■ Agarose...40g Urea...4,200g Acrylamide...1.830g1.3.5-
) Liacryloyl-hexahydro-S-+-Ryazine...19g Theory Ionized Water
Up to 9.000m j! Make it as a buffer solution □
■ Trishydroxymethylamine...121.14g oxalic acid
...65.4g Ethylenediaminetetraacetic acid disodium salt ...7.45g Ionized water
10) Make to 1,000 mff and add buffer solution...
・・・・・・・・・750＋＋＋β2.9χ Polyvinylpyrrolidone aqueous solution・800ml12.5χTetramethylethylenediamine liquid・・・・6.7+cIV.
の混合液□■を
低濃度モノマー■９，０００ｍ！、高濃度モノマー■９
、０００ｍ １２の各々に加え、夫々を低濃度モノマー
液１、高濃度モノマー液２とする。Mixed liquid □■ with low concentration monomer■ 9,000m! , high concentration monomer■9
.
次に重合反応開始剤液３として
２χｄｉ−２エチルへキシルスル
ホサクシネート液・・・・・・１００ｍ １０．３７５
χリボフラビンリン酸
エステルナトリウム塩水溶液・１５０ｍ １２３．７５
χテルオキソニ硫酸ア
ンモニウム水Ｒ液・・・・・・１６０ｍ Ｉｔを作り、
上記３液を夫々のタンクに入れる。Next, as polymerization reaction initiator liquid 3, 2χdi-2 ethylhexyl sulfosuccinate liquid...100m 10.375
χ Riboflavin phosphate ester sodium salt aqueous solution・150m 123.75
Make χ teroxonisulfate ammonium water R solution...160m It.
Put the above three liquids into their respective tanks.
流量勾配送液ポンプ４．５で低濃度モノマー液１、高濃
度モノマー液２の流量和を１９ｍＡ／ｍｉｎとし、それ
に重合反応開始剤３の流量１．４６ｍβ／ｗｉｎを定流
量送液ポンプ６で、スタティックミクサー７内に送液す
る。The sum of the flow rates of low concentration monomer liquid 1 and high concentration monomer liquid 2 was set to 19 mA/min using the flow rate gradient liquid sending pump 4.5, and the flow rate of the polymerization reaction initiator 3 was set to 1.46 mβ/win using the constant flow liquid sending pump 6. , the liquid is fed into the static mixer 7.
スタティックミクサー７内では上記３液が自刃により混
合攪拌し、電気泳動分離用媒体液１２が出来る。In the static mixer 7, the above three liquids are mixed and stirred by a self-blading blade to form a medium liquid 12 for electrophoretic separation.
塗布ヘッド８より電気泳動分離用媒体液１２がウェブ１
３上に塗布される。The electrophoretic separation medium liquid 12 is applied to the web 1 from the coating head 8.
3 is coated on top.
その時の低濃度上ツマー液と高濃度上ツマー液との流量
比は、第２図（ａｌに示したとおりであり、塗布された
ゲル膜の濃度は第２図（ト））、第２図（Ｃ１の状態に
なる。At that time, the flow rate ratio of the low-concentration Upper Zummer's solution and the high-concentration Upper Zummer's solution is as shown in Figure 2 (al), and the concentration of the applied gel film is as shown in Figure 2 (g)). (It will be in state C1.
上記によって作られた電気泳動分離用媒体液は塗ＩＨａ
ｃｍのＰＥＴのフィルムの上に１５０〜２００ｃｃ／イ
で塗布を行った。The electrophoretic separation medium liquid prepared above is coated with IHa.
Coating was performed on a PET film of 150 to 200 cc/cm.
（実施例−１）
この時の塗布ヘッドにおける幅方向の供給口よりスロッ
トへの拡がり角度θ−４０°、厚みの縮少角度α＝０．
３５°を行ったところ、長時間の運転をしても塗布スジ
の発生等の故障はなく、全幅に亘って均一な厚みの良質
の塗布品質が得られた。(Example-1) At this time, the spread angle from the supply port in the width direction to the slot in the coating head was θ-40°, and the thickness reduction angle α was 0.
When the coating was applied at 35°, there were no failures such as coating streaks even after long-term operation, and a good coating quality with a uniform thickness over the entire width was obtained.
（比較例−１）
実施例−１と同様の塗布液濃度・温度で同様にしてＰＦ
、Ｔフィルム上に、拡がり角度θ−４５゜縮少角度α＝
０．５°の塗布ヘッドを用いて塗布を行ったところ、塗
布厚味は端部が薄く、全体として放物線状の塗布厚味に
なった。また長時間の連続運転に対し多少スジっぽい塗
布品質であった。(Comparative Example-1) PF was applied in the same manner as in Example-1 at the same coating solution concentration and temperature.
, on the T film, spread angle θ-45° reduction angle α=
When coating was performed using a 0.5° coating head, the coating thickness was thinner at the edges and had a parabolic coating thickness as a whole. Furthermore, the coating quality was somewhat streaky due to long-term continuous operation.
本発明は連続的に濃度勾配をもった七ツマー液と重合反
応開始剤液とを混合しながら、電気泳動分離用媒体液と
して塗布ヘッドに供給し、走行するウェブ上に塗布する
電気泳動用グラジェントゲル膜の製造方法であって、塗
布前のスタティックミクサー等の使用により濃度勾配の
正確な安定した再現性の良い電気泳動用グラジェントゲ
ル膜の生産性の良い製造が可能になった、又塗布ヘッド
に塗布液を供給する際供給口よりスロット開口に到る間
、幅方向を連続的に拡げ、厚味方向を連続的に縮少して
供給することにより塗布液の塗布ヘッド内の滞留による
異物の発生はなくなり、又幅方向に均一な成分の塗布が
可能となり製品品質が一段と向上した。In the present invention, a seven-mer liquid having a concentration gradient and a polymerization reaction initiator liquid are continuously mixed and supplied to a coating head as a medium liquid for electrophoretic separation, and an electrophoretic graphite is coated onto a running web. A method for producing a gradient gel membrane for electrophoresis, which uses a static mixer or the like before application to produce a gradient gel membrane for electrophoresis with accurate, stable and reproducible concentration gradients, and When supplying the coating liquid to the coating head, the coating liquid is continuously expanded in the width direction and continuously contracted in the thickness direction until it reaches the slot opening from the supply port, thereby causing the coating liquid to stay in the coating head. The generation of foreign matter has been eliminated, and it has become possible to apply the ingredients uniformly in the width direction, further improving product quality.
第１図は本発明に係わる電気泳動分離用媒体液の製造工
程図、第２図は本発明に係わる電気泳動分離用媒体液の
濃度勾配（ａｌとその塗布膜の平面図（ｂｌ、側面図（
Ｃ）である。第３図は本発明に係わる塗布ヘッドの側面
断面図（ａ）、 （ｂ）、第４図は第３図の正面断面図
、第５図は第４図の透視斜視図、第６図は従来の塗布ヘ
ッドの側面断面図を示す。
１・・・低濃度モノマー液
２・・・高濃度モノマー液
３・・・重合反応開始剤液
４．５・・・流量勾配送液ポンプ
６・・・定流量送液ポンプ
７・・・スタティックミクサー
８・・・塗布ヘッド
９ａ、９ｂ・・・コントローラ
１０・・・コンピュータリンケージアダプタ１１・・・
パーソナルコンピューター
１２・・・電気泳動分離用媒体液
ｌ３・・・ウェブ
１４・・・供給口 １６・・・スロット１７・・・ス
ロット開口
１８・・・塗布ヘッド、１９・・・キャビティ２０・・
・コーティングローラ
２１・・・差圧チャンバー、２２・・・ホンフ２３・・
・スロット、２４・・・スロット開口第３図
（ｂ）Fig. 1 is a manufacturing process diagram of a medium liquid for electrophoretic separation according to the present invention, and Fig. 2 is a plan view (bl, side view of the concentration gradient of the medium liquid for electrophoretic separation (al) and its coating film according to the present invention). (
C). 3 are side sectional views (a) and (b) of a coating head according to the present invention, FIG. 4 is a front sectional view of FIG. 3, FIG. 5 is a perspective view of FIG. 4, and FIG. A side sectional view of a conventional coating head is shown. 1...Low concentration monomer liquid 2...High concentration monomer liquid 3...Polymerization reaction initiator liquid 4.5...Flow rate gradient liquid feeding pump 6...Constant flow liquid feeding pump 7...Static Mixer 8... Application heads 9a, 9b... Controller 10... Computer linkage adapter 11...
Personal computer 12... Electrophoretic separation medium liquid l3... Web 14... Supply port 16... Slot 17... Slot opening 18... Coating head, 19... Cavity 20...
・Coating roller 21...Differential pressure chamber, 22...Honfu 23...
・Slot, 24...Slot opening Fig. 3(b)
Claims (1)
液とを混合しながら電気泳動分離用媒体液として塗布ヘ
ッドに供給し、走行するウェブ上に塗布する電気泳動用
グラジェントゲル膜の製造方法であって、該塗布液を該
塗布ヘッドの供給口よりスロット開口に到る間、幅方向
を連続的に拡げ、厚み方向を連続的に縮少して供給する
過程をすることを特徴とする電気泳動用グラジェントゲ
ル膜の製造方法。Manufacture of a gradient gel membrane for electrophoresis in which a monomer liquid with a concentration gradient and a polymerization reaction initiator liquid are continuously mixed and supplied to a coating head as a medium liquid for electrophoretic separation, and coated on a running web. The method is characterized in that the coating liquid is supplied by continuously expanding in the width direction and continuously contracting in the thickness direction while reaching the slot opening from the supply port of the coating head. A method for producing a gradient gel membrane for electrophoresis.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62131502A JPS63298044A (en) | 1987-05-29 | 1987-05-29 | Production of gradient gel film for electrophoresis |
| US07/199,726 US4968535A (en) | 1987-05-29 | 1988-05-27 | Method of producing gradient gel medium membrane for electrophoresis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62131502A JPS63298044A (en) | 1987-05-29 | 1987-05-29 | Production of gradient gel film for electrophoresis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63298044A true JPS63298044A (en) | 1988-12-05 |
| JPH0584861B2 JPH0584861B2 (en) | 1993-12-03 |
Family
ID=15059517
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62131502A Granted JPS63298044A (en) | 1987-05-29 | 1987-05-29 | Production of gradient gel film for electrophoresis |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63298044A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4755989B2 (en) * | 2003-03-31 | 2011-08-24 | イジュノシッヒ テクニッヒ ホッフシューラ チューリッヒ | Controlled surface chemical gradient |
-
1987
- 1987-05-29 JP JP62131502A patent/JPS63298044A/en active Granted
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4755989B2 (en) * | 2003-03-31 | 2011-08-24 | イジュノシッヒ テクニッヒ ホッフシューラ チューリッヒ | Controlled surface chemical gradient |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0584861B2 (en) | 1993-12-03 |
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