201038933 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種固態微萃取之用的裝置及其 製造方法，特別係用於純化濃縮生化樣品與藥物分 子。 〇 【先前技術】 一般大分子生物樣品去鹽的方式有逆相層析 • ( reverse phase chromatography )、透析（dialysis ) ―及膠體過濾、層析（gel filtration chromatography )等。 • 目前用來去鹽的裝置包含了三種，一種是利用毛細 管做成液體流通管道的吸管尖，此產品必須增大平 衡時間與透過擴散作用才能夠抓取更多的生物分 子；而且必須有一阻檔閥，避免高分子承受不住背 ❹ 壓而脫離。 第二種為被廣泛使用的Zip-Tips TM(Millipore Corporation, Bedford，MA，USA)，除 了有去鹽的效 果外，對於所分析之蛋白質、胜肽或寡核甘酸等樣 品更有預濃縮的效果。雖然Zip-Tips的適用範圍頗 廣，但其靜相結合之特性不適用較小分子量（如藥物 分子）、較親水性或轉譯後修飾之蛋白質分子且所需 沖堤體積仍多，不能滿足一些研究的需求。除此之 3 201038933 外，Zip-Tips價格昂貴也讓使用者卻步。 相較於上述兩種吸收量為1〇_2〇〇//L體積之吸 B大，第二種吸管炎之吸收量較大，吸收量約為 lmL，而且大部分應用於藥物分子的萃取。其製造 不疋用一體成型（monolithic bed)的方式製造，層析 材料疋利用濾片與大型吸管尖（T〇mtee tip)表面的物 理摩擦力固定在吸管尖上方，二片濾片中間夾著層 〇 析材料。而它的缺點是樣品量必須足夠多才能夠到 達吸管尖的上面。材料的孔徑不能大於濾片的孔 * 彳空’不然會造成材料的流失，所以吸收量受限。因 為材料固定在尾端，吸取樣品容易污染到吸管器， - 進而造成交叉污染。 由於目前習知之吸管尖皆有其缺點存在，造成 研九上之不便，所以若能於吸管尖的製程或結構上 〇 進行新的設計，使其具有良好的萃取效果，同時價 袼低廉，將有助於未來生醫、化學等領域之發展。 【發明内容】 本發明利用油跟水的微乳化作用，將可行光聚 合的高分子單體形成微胞來進行聚合，藉此形成通 透性佳之孔洞材料，進而減少微萃取用之裝置的背 壓’並且’增加與樣品接觸之表面積。 本發明之目的係提供一種低成本及具有良好萃 4 201038933 取效果之裝置，龙佐、未 分子單體行乳化聚合二=二利用高 構來製備微萃取用之裝成體成形的多孔洞結 以增加吸收量與減少了增大接觸表面積可 生產。而且乳化作用1、外，也方便自動化 結合，可以固定==與光聚合或熱聚合的方式 之裝置中。，、大$刀的層析材料在該微萃取用 Ο ❹ 方法本目的係提供—種製備上述裝置之 程簡單化。 用m聚合之技術而使製 取之達上述目的’本發明之用於固態微萃 裝置包3:—殼體，其具有-上開口及一下開 口^及-多孔性材料’係填充於前述殼體内，且前 述夕孔性材料包覆有複數個靜相粒子，其中，該多 孔性材料係以乳化聚合製得。 於-較佳實施態樣中’前述樣品為藥物分子、 蛋白質、胜肽或寡核甘酸。 於一較佳實施態樣中，前述殼體之材質係為聚 乙烯、聚丙烯、聚對苯二f酸乙g旨或聚四氟乙稀。 於一較佳實施態樣中，前述多孔性材料之孔徑 為5-25微米，更佳為20-25微米。 於一較佳實施態樣中，前述多孔性材料為聚乙 5 201038933 二醇二曱基丙烯酸酯、聚苯乙烯、聚2-曱基丙烯酸 或聚丙基矽烷2-曱基丙烯酸酯。 於一較佳實施態樣中，前述靜相粒子為逆相層 析用之靜相、金屬親和性層析用之靜相、經官能基 修飾後之靜相、離子交換材料或金屬氧化物。較佳 地，該靜相粒子為碳十八或親水親油平衡 (Hydrophilic-Lipophilic Balance ; HLB)吸附劑。 〇 本發明製備該裝置之方法，其包含：（a)提供一 空的殼體；（b)提供可行乳化聚合之一反應溶液，其 包含一高分子單體；（c)將一靜相粒子加入至前述反 ' 應溶液，得一起始溶液；（d)將前述起始溶液導入前 ' 述殼體的空腔内；（e)利用光照射使該起始溶液於該 空腔内進乳化聚合並固化；及（f)烘乾。 於一較佳實施態樣中，前述殼體之材質係為聚 ❹ 乙烯、聚丙烯、聚對苯二曱酸乙酯或聚四氟乙烯。 於一較佳實施態樣中，前述高分子單體與前述 靜相粒子之重量比為100 #1^:10 mg〜100 #L:20 mg。 於一較佳實施態樣中，前述高分子單體為乙二 醇二曱基丙稀酸酯 （ethylene glycol dimethacrylate)、苯乙稀、2-曱基丙烯酸、丙基石夕炫 2-曱基丙烯酸醋。 6 201038933 於-較佳實施態樣中，前述靜相粒子為逆相層 :::靜相、金屬親和性層析用之靜相、經官能基 L飾後之靜相、離子交歸料或金屬氣化物。較佳 地，該靜相粒子為碳十心親水親油平衡吸附劑。 一Τ較佳實施態樣中，前述反應溶液進一步包 含二氧化矽及/或碳酸氫銨。 Ο 於一較佳實施態樣中 含一界面活性劑。較佳地 界面活性劑。 鈿述反應溶液進一步包 該界面活性劑為陰離子 於一較佳實施態樣中，該裝置為與一吸管器相 連之一吸管尖。 Β 本發明利用簡單的製程步驟來製 縮萃取效果，甚至賴化效果之料㈣之^辰201038933 VI. Description of the Invention: [Technical Field] The present invention relates to an apparatus for solid-state micro-extraction and a method of manufacturing the same, and in particular to purifying a concentrated biochemical sample and a drug molecule. 〇 [Prior Art] Generally, the methods for desalting large molecular biological samples include reverse phase chromatography, dialysis, and gel filtration chromatography. • There are currently three types of devices used to remove salt. One is a pipette tip that uses a capillary tube to make a liquid flow conduit. This product must increase the equilibration time and diffuse to capture more biomolecules; The valve prevents the polymer from being able to withstand the back pressure and detach. The second type is the widely used Zip-TipsTM (Millipore Corporation, Bedford, MA, USA), which has a pre-concentrated sample for the analyzed protein, peptide or oligonucleotide, in addition to the desalting effect. effect. Although Zip-Tips has a wide range of applications, its static-combination characteristics are not suitable for smaller molecular weight (such as drug molecules), more hydrophilic or post-translationally modified protein molecules and the volume of the bank is still too large to meet some Research needs. In addition to the 3 201038933, the high price of Zip-Tips also deters users. Compared with the above two absorptions with a volume of 1〇_2〇〇//L, the absorption of the second pipette is larger, the absorption is about 1mL, and most of them are applied to the extraction of drug molecules. . The manufacture is not made by a monolithic bed. The chromatographic material is fixed above the pipette tip by the physical friction of the filter and the surface of the large pipette tip (T〇mtee tip). Layer decanting material. The disadvantage is that the amount of sample must be sufficient to reach the tip of the pipette. The pore size of the material should not be larger than the pores of the filter. * Hollowing will cause the loss of material, so the amount of absorption is limited. Because the material is fixed at the end, the sample is easily contaminated to the pipette, which in turn causes cross-contamination. Since the conventional pipette tip has its shortcomings, which causes inconvenience in the research, if a new design can be made on the process or structure of the pipette tip, so that it has a good extraction effect and is inexpensive, it will be It will help the development of biomedical science and chemistry in the future. SUMMARY OF THE INVENTION The present invention utilizes the microemulsification of oil and water to form a photopolymerizable polymer monomer into micelles for polymerization, thereby forming a pore material having good permeability, thereby reducing the back of the device for microextraction. Press 'and' to increase the surface area in contact with the sample. The object of the present invention is to provide a low-cost and good extraction device with the effect of 201003833. Long Zuo, non-molecular monomer emulsion polymerization 2 = two uses high structure to prepare the porous cavity junction formed by micro-extraction It can be produced by increasing the absorption amount and reducing the contact surface area. Moreover, the emulsification 1, in addition, is also convenient for automated bonding, and can be fixed in a device with a mode of photopolymerization or thermal polymerization. The chromatographic material of the large knife is used in the microextraction method. The purpose of the method is to simplify the process of preparing the above device. The above-mentioned object is achieved by the technique of m-polymerization. The solid micro-extraction device package 3 of the present invention has a housing having an upper opening and a lower opening and a porous material filled in the foregoing shell. In the body, the aforementioned porphyrin material is coated with a plurality of static phase particles, wherein the porous material is obtained by emulsion polymerization. In the preferred embodiment, the aforementioned sample is a drug molecule, a protein, a peptide or an oligonucleotide. In a preferred embodiment, the material of the housing is polyethylene, polypropylene, poly(p-phenylene terephthalate) or polytetrafluoroethylene. In a preferred embodiment, the porous material has a pore diameter of 5 to 25 μm, more preferably 20 to 25 μm. In a preferred embodiment, the porous material is polyethylene 5 201038933 diol dimercapto acrylate, polystyrene, poly 2-mercaptoacrylic acid or polypropyl decane 2-mercapto acrylate. In a preferred embodiment, the static phase particles are a stationary phase for reverse phase chromatography, a stationary phase for metal affinity chromatography, a static phase modified with a functional group, an ion exchange material or a metal oxide. Preferably, the static phase particles are carbon eighteen or a Hydrophilic-Lipophilic Balance (HLB) adsorbent. The method of the present invention for preparing the apparatus, comprising: (a) providing an empty shell; (b) providing a reaction solution of a feasible emulsion polymerization comprising a high molecular monomer; (c) adding a static phase particle To the foregoing reaction solution, a starting solution is obtained; (d) the foregoing starting solution is introduced into the cavity of the front case; (e) the starting solution is subjected to emulsion polymerization in the cavity by light irradiation. And curing; and (f) drying. In a preferred embodiment, the material of the casing is made of polyethylene, polypropylene, polyethylene terephthalate or polytetrafluoroethylene. In a preferred embodiment, the weight ratio of the polymer monomer to the static phase particles is 100 #1^: 10 mg~100 #L: 20 mg. In a preferred embodiment, the polymer monomer is ethylene glycol dimethacrylate, styrene, 2-mercaptoacrylic acid, and propyl sulfonium 2-mercaptoacrylic acid. vinegar. 6 201038933 In the preferred embodiment, the static phase particles are reverse phase layers:: static phase, static phase for metal affinity chromatography, static phase after functional group L decoration, ion exchange or Metal vapor. Preferably, the static phase particles are carbon ten-heart hydrophilic-lipophilic adsorbents. In a preferred embodiment, the reaction solution further contains cerium oxide and/or ammonium hydrogencarbonate. In a preferred embodiment, a surfactant is included. Preferably a surfactant. The reaction solution is further described. The surfactant is an anion. In a preferred embodiment, the device is a pipette tip associated with a pipette. Β The invention utilizes a simple process step to reduce the extraction effect, and even the material of the reliance effect (4)
=其助作為吸管尖，將可提供—種有別於傳統 :貝又無法應用於藥物分子的去鹽濃縮用之吸管 尖。 【實施方式】 本發明使用乳化聚合來製備出具有孔洞高分子 之微萃取用之裝置’由於該孔洞高分子之孔洞分佈 均勻且孔洞約有15_2〇"m，因此具有良好之通透 陡’進而減少其之背壓，而當背壓減少時，則無需 7 201038933 名員外之固疋圈去將孔洞南分子固定於裝置内。 睛參閱第一圖，其係顯示本發明用於固態微萃 取之裝置之結構不意。本發明用於固態微萃取之裝 置10,包含：一殼體2,其具有一上開口 4及一下 開口 6;及-多孔性材料8(陰影部分），係填充於前 述殼體内，且前述多孔性材料包覆有複數個靜相粒 子9(點狀部幻’其中’該多孔性材料係以乳化聚合 ® 製得。 、本發明之微萃取用之裝置之—較佳實施態樣為 . 透過該上開π來與吸管器相連接之—吸管尖，如此 一來於使用時’即可藉由該吸管器來使該吸管尖更 易於吸取或排出液體。 另外，本發明亦提供—種製造用於固態微萃取 之裝置之方法，其包含：⑷提供空的一殼體. j供可ml化聚合之—反應溶液，其包含—高分子 單體’⑷將-靜相粒子加人至前述反應溶液，得一 起始溶液；⑷將前述起始溶液導人前述殼體的空腔 内0)利用光知射使該起始溶液於該空腔内進乳化 聚合並固化；（f)烘乾。 為了進一步增加微萃取用之裝置之通透性，本 發明可仃礼化聚合之該反應溶液係可添加二氧化 :’其所扮演之角色如同半導體常用之犧牲層，於 N分子聚合完成後’再湘驗性水溶液及水將其移 8 201038933 出，藉以擴大材料之多孔性’增加與樣品接觸之表 面積。而本發明所適用之高分子單體包含乙二醇= 曱基丙烯酸酯、苯乙烯、2_曱基兩烯酸及丙基矽； 2-甲基丙烯酸酯，但不限於此。 此外，本發明中之、、靜相粒子〃係指化學層析（諸 如·液相層析、氣相層析、管柱層析等）時所用之靜 相成分，例如··逆相層析用之靜相、金屬親和性層 〇 析用之靜相、經官能基修飾後之靜相、離子交換二 料或金屬氧化物，但不限於此，其選用上，完全依 據樣品不同而做對應之改變，舉例而言，樣品為胜 • 狀’則係可選用石炭十八作為靜相粒子；樣品為藥物 ' 分子，則係可選用親水親油平衡吸附劑作為靜相粒 子。 以下係提供利用本發明之實施例，然本實施例 ❹ 並非用以限定本發明’任何熟悉此技藝者，在不脫 離本發明之精神和範圍内，當可作各種之更動與潤 飾，因此，本發明之保護範圍，當視後附之申請專 利範圍所界定者為準。 實施例1 ·_製造本發明吸管尖之方法= Its help as a pipette tip will provide a different type of pipette tip than the traditional one: it cannot be applied to the demineralization of drug molecules. [Embodiment] The present invention uses emulsion polymerization to prepare a device for micro-extraction having a pore polymer. Since the pores of the pore polymer are uniformly distributed and the pores are about 15_2 〇"m, they have a good permeability. In turn, the back pressure is reduced, and when the back pressure is reduced, it is not necessary to fix the hole south molecule in the device. Referring to the first drawing, it is shown that the structure of the apparatus for solid-state micro-extraction of the present invention is not intended. The apparatus 10 for solid-state micro-extraction of the present invention comprises: a casing 2 having an upper opening 4 and a lower opening 6; and a porous material 8 (shaded portion) filled in the casing, and the foregoing The porous material is coated with a plurality of static phase particles 9 (points of the phantom 'where the porous material is obtained by emulsion polymerization®. The device for microextraction of the present invention is preferably a preferred embodiment. The pipette tip is connected to the straw through the upper opening π, so that the pipette tip can make the pipette tip more easily sucking or discharging liquid when used. In addition, the present invention also provides A method of manufacturing a device for solid-state micro-extraction, comprising: (4) providing an empty shell. The solution for the melt-polymerizable reaction comprises a polymer monomer '(4) adding - the stationary phase particles to The above reaction solution obtains a starting solution; (4) the foregoing starting solution is introduced into the cavity of the aforementioned casing; 0) the starting solution is emulsion-polymerized and solidified in the cavity by means of light; (f) baking dry. In order to further increase the permeability of the device for micro-extraction, the reaction solution of the present invention can be added with oxidation: 'the role played by the semiconductor layer is as the sacrificial layer commonly used in semiconductors, after the completion of the polymerization of the N molecule' The aqueous solution and water are then moved to 8 201038933 to expand the porosity of the material' to increase the surface area in contact with the sample. The polymer monomer to which the present invention is applied includes ethylene glycol = mercapto acrylate, styrene, 2-nonyldienoic acid, and propyl hydrazine; 2-methacrylate, but is not limited thereto. In addition, in the present invention, a stationary phase particle refers to a stationary phase component used in chemical chromatography (such as liquid chromatography, gas chromatography, column chromatography, etc.), for example, reverse phase chromatography. The static phase, the stationary phase for the metal affinity layer, the static phase modified by the functional group, the ion exchange material or the metal oxide are used, but are not limited thereto, and are selected according to different samples. For example, if the sample is a win-like shape, then the charcoal 18 can be used as the static phase particle; if the sample is a drug, the hydrophilic-lipophilic equilibrium adsorbent can be used as the static phase particle. The embodiments of the present invention are provided below, and the present embodiments are not intended to limit the present invention. Any one skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. The scope of the invention is defined by the scope of the appended claims. Embodiment 1 · Method for manufacturing the pipette tip of the present invention
乳化聚合之反應溶液之製備 取一包覆有鋁箔之適當容器，將7,5〇/0 400//L 9 201038933 界面活性劑（十二烧基硫酸鈉鹽（sodium dodecyl sulfate，SDS))、600 // L 乙醇、20 mg 石炭酸氫銨、 200 /zL乙二醇二曱基丙烯酸S旨（Ethylene glycol dimethacrylate，EDMA)及 20 mg 光聚合起始劑, α -二曱氧基-ο:-苯基乙蕴苯（α，a -Dimethoxy-α -phenylacetophenone，DAP))置入其中擾拌混合，即 得可行乳化聚合之一反應溶液，其中該十二烷基硫 酸鈉鹽（SDS)之臨界微胞濃度為23mM-25mM。 吸管尖製備 取20 mg直徑為20微米之碳18小珠加入上述 反應溶液100 中，即得一起始溶液，接著藉由 毛細現象將該起始溶液導入聚丙烯材質之吸管尖的 空腔内。將此吸管尖以337nm之紫外光照射6-10 分鐘，使其進行乳化聚合並固化。待反應完成後， 將此吸管尖放置在烘箱内烘乾，將多餘的水及乙醇 移除，接著利用50%乙腈在0.1%三氟醋酸溶液中清 洗數次來移除界面活性劑，再用含〇. 1 %三氟醋酸之 水溶液清洗數次，最後泡在200微升的曱醇溶液 中，24小時之後將曱醇排掉，將吸管尖再移入烘箱 烘乾。 第二A圖為可行乳化聚合之反應溶液（未添加靜 相粒子）行乳化聚合後利用電子顯微鏡觀測之影像 圖，其顯示多孔性高分子之孔洞直徑為15-20微米。 201038933 第二B圖則為可行乳化聚合之反應溶液添加碳18 靜相粒子行乳化聚合後利用電子顯微鏡觀測之影像 圖。由第二B圖可得知本案吸管尖内之孔洞高分子 之孔徑為5-20 /zm。 實施例2 :純化/濃缩 吸管尖之預處理 除了將靜相粒子由碳18小珠改為〇asis HLB(美 商沃特斯（waters))之外，其餘製裎皆與實施例i相 同’藉此製得另一吸管尖。以100%甲醇溶液清洗該 吸官尖至少二次’再以二氟酷酸水相溶液平衡 至少三次。 試驗樣品製作 先取一顆由荷商葛蘭素史克 (GlaxoSmithKline，簡稱GSK)藥廠所推出的感冒藥 伏冒錠，根據使用說明書所載，一顆伏冒錠裡的内 含物有：300mg的乙酸胺笨紛（ACetaminophen); 5mg 的脫羥腎上腺素（Phenylephrine) ; 1 〇mg的諾斯卡賓 (Noscapine) ； 20mg 的一水合萜二醇（Terpin Hydrate) ; 15mg 的咖啡因（Caffeine) ; 30mg 的維他 命C(Vitamin C)。接著將其用研蛛磨碎後倒入燒杯 中，加入去離子水100 mL，並以超音波振盪使之溶 11 201038933 解，尚有一些未溶物，則將其過濾掉，取其濾液並 移入定量瓶，定量瓶中加去離子水至3〇〇mL，再將 液體移入血清瓶存放。取10 樣品用去離子水稀 釋到100# L，即得本實施例所用之試驗樣品。 濃縮效果測試 將前述預處理過之吸管尖之上開口與吸管器連 ❹ 接，接著將該吸管尖浸入試驗樣品中，藉由吸管器 使該吸管尖來回吸取樣品並退出樣品，過程持續約 經60秒，之後用10微升5%甲醇水溶液洗三次。最 . 後用10微升i〇0%甲醇溶液沖提出吸附於吸管尖上 的樣品，重覆二次。最後以直接注射（In fusion)方式 的ESI質譜儀檢驗，其檢驗結果如第三b圖所示。 第二A圖為經本發明之吸管尖處理過之試驗樣 Ο 品的Esi質譜圖，而第三B圖則為未經本發明之吸 管尖處理過之試驗樣品的ESI質譜圖，其中標示▲ 的訊號峰為伏冒錠中所含的藥物分子。比較第三a 圖及第三B圖，可清楚觀察到經本發明吸管尖處理 過之試驗樣品，其質譜訊號會增強許多。 實施例3 :去鹽濃縮 ^實施例所使用之吸管尖即為實施例丨所製得 之吸s穴，其靜相粒子為碳十八顆粒（直徑為2〇 # 12 201038933 m) 〇 操作過程： 0.1%三氟醋酸 先以50%乙腈潤濕吸管數次，再以 吸放數次以平衡靜相。 取= W白蛋白酵素消化物共200 # L ’利用 0.1%三氟魏調纽性。分職用本發明之吸管The preparation of the reaction solution for emulsion polymerization is carried out in a suitable container coated with aluminum foil, and 7,5 〇 / 0 400 / / L 9 201038933 surfactant (sodium dodecyl sulfate (SDS)), 600 // L ethanol, 20 mg ammonium hydrogencarbonate, 200 / zL ethylene glycol dimethacrylate (EDMA) and 20 mg photopolymerization initiator, α-dimethoxy-ο:- Phenyl phenyl benzene (α, a - Dimethoxy-α - phenylacetophenone (DAP)) is placed in a mixture of scrambled polymerization, which is one of the possible reaction solutions of emulsion polymerization, wherein the criticality of the sodium dodecyl sulfate (SDS) The cell concentration was 23 mM - 25 mM. Pipette Tip Preparation 20 mg of carbon 18 beads having a diameter of 20 μm were added to the above reaction solution 100 to obtain a starting solution, which was then introduced into the cavity of the tip of the pipette of polypropylene by capillary action. The pipette tip was irradiated with ultraviolet light of 337 nm for 6-10 minutes to carry out emulsion polymerization and solidification. After the reaction is completed, the pipette tip is placed in an oven to be dried, the excess water and ethanol are removed, and then the surfactant is removed by washing with 50% acetonitrile in 0.1% trifluoroacetic acid solution for several times, and then used. The aqueous solution containing 1% trifluoroacetic acid was washed several times, and finally soaked in 200 μl of a sterol solution. After 24 hours, the sterol was drained, and the pipette tip was again transferred to an oven for drying. The second A is an image of an emulsion polymerization reaction solution (with no added static phase particles) after emulsion polymerization and observed by an electron microscope, which shows that the pore diameter of the porous polymer is 15-20 μm. 201038933 The second B-picture is an image of the 18-stage static-phase particle after the emulsion polymerization is added to the reaction solution of the feasible emulsion polymerization. It can be seen from the second B diagram that the pore size of the pores in the tip of the pipette is 5-20 /zm. Example 2: Pretreatment of Purification/Concentration Tip Tips The same procedure as in Example i except that the static phase particles were changed from carbon 18 beads to 〇asis HLB (waters) 'Through this to make another pipette tip. The tip was rinsed with a 100% methanol solution at least twice and equilibrated with the aqueous solution of difluorohydrous acid at least three times. The test sample was prepared by taking a cold medicine, which was introduced by the Dutch company GlaxoSmithKline (GSK). According to the instruction manual, the contents of a volt ingot are: 300mg. Acetaminophen; 5 mg of phenylephrine; 1 〇mg of Noscapine; 20 mg of temperate monohydrate (Terpin Hydrate); 15 mg of caffeine (Caffeine); 30 mg Vitamin C (Vitamin C). Then, it is ground with a research spider and poured into a beaker. Add 100 mL of deionized water and dissolve it by ultrasonic wave to dissolve it. 2010 201033,33, if there are some undissolved substances, filter it off and take the filtrate. Transfer to the dosing bottle, add deionized water to the 3 〇〇 mL bottle, and then transfer the liquid to the serum bottle for storage. The test sample used in this example was obtained by taking 10 samples diluted to 100# L with deionized water. The concentration effect test connects the opening of the pre-treated pipette tip to the straw, and then dipped the pipette tip into the test sample, and the pipette tip draws the sample back and forth and withdraws the sample, and the process continues. After 60 seconds, it was washed three times with 10 μl of a 5% aqueous methanol solution. Finally, the sample adsorbed on the tip of the pipette was punched out with 10 μl of i〇0% methanol solution and repeated twice. Finally, it was inspected by an ESI mass spectrometer in an infusion mode, and the test result is shown in the third b diagram. Figure 2A is an Esi mass spectrum of the test sample treated by the pipette tip of the present invention, and the third B chart is an ESI mass spectrum of the test sample not treated by the pipette tip of the present invention, wherein the signal indicating the ▲ The peak is the drug molecule contained in the voltaic tablet. Comparing the third a diagram and the third B diagram, it can be clearly observed that the test sample treated by the pipette tip of the present invention has a much enhanced mass spectrometry signal. Example 3: Desalting Concentration The tip of the pipette used in the example is the suction hole prepared in the example, and the static phase particles are carbon eighteen particles (diameter 2 〇 # 12 201038933 m) 〇 operation process : 0.1% trifluoroacetic acid first wet the pipette with 50% acetonitrile several times, then absorb several times to balance the static phase. Take = W albumin digests a total of 200 # L ' using 0.1% trifluoro-Weiton. Separating the straw of the present invention
尖與台灣發明第9 ^ ^ 、卜 U7579號專利案之吸管尖重 複抽放數次，時間約60秒。 3. 以0.1%三氟醋酸水溶液清洗數次。 4. 再以80%乙腈溶液進行沖堤。 5. 額外加A人工合成之胜肽（MW:H07)當做内標準 品。 6.將上述冲i疋液2 "乙與介質（matrix)2 " L混合，以 介質辅助雷射脫附離子化質譜儀（MALDI)分析。 實驗結果 經由前述步驟所得之分析結果為第四圖。第四 A圖為經台灣發明92117579號專利之吸管尖處理過 之忒驗樣品的MALDI質譜儀；第四B圖為經本發 明之吸官尖處理過之試驗樣品的MADU質譜圖。 取卵白蛋白消化液共9根胜肽與内標準品的訊號比 值加總當做計算依據。 13 201038933 由汛號的比值總和可知，相同時間内，本案可 抓取多約5倍質量的卵白蛋白消化物。&台灣發明 92117579 5虎專利所刊載之論文提及可抓約o n # g 的量推估’本案在相同靜相體積之下，約可以抓取 3-5//g的消化物。由此亦可得知，本案吸管尖所需 之平衡時間要比台灣發明921m79號專利之吸管 尖快了許多。The tip of the pipette and the invention of the 9th ^^ and U7579 patents in Taiwan were repeatedly pumped several times for about 60 seconds. 3. Wash several times with 0.1% aqueous trifluoroacetic acid solution. 4. Drain the bank with 80% acetonitrile solution. 5. Add a synthetic peptide (MW: H07) as an internal standard. 6. Mix the above-mentioned sputum 2 " B with the medium 2 " L and analyze with a medium-assisted laser desorption ionization mass spectrometer (MALDI). Experimental Results The analysis results obtained through the foregoing steps are the fourth graph. The fourth A is a MALDI mass spectrometer of the test sample treated by the pipette tip of Taiwan Patent No. 92117579; the fourth B is the MADU mass spectrum of the test sample treated by the tip of the present invention. The signal ratio of the 9 peptides to the internal standard of the egg albumin digest was added as a calculation basis. 13 201038933 According to the sum of the nicknames, in the same time, the case can grab about 5 times the quality of the ovalbumin digest. &Taiwan invention 92117579 The paper published in the Tiger patent mentions that the amount of n n #g can be estimated. In this case, under the same static phase volume, about 3-5//g of digest can be grabbed. It can also be seen that the balance time required for the pipette tip of this case is much faster than the pipette tip of Taiwan's invention of the 921m79 patent.
综上所述，應用本發明微萃取用之t置所製出 二及:大’相較於習知技街具有更簡單之製造過 對於生吸附量及平衡時間上均有明顧之改善。 對於生醫、化學及藥物方 的使用性。 為優異 社八士 ·r奴π香&符徵係可使用任何方式 相°:目ί說明書所揭露之特徵可使用相同、相等或 相似目的的特徵取代。因此， 一 之外，本令 Μ 了特別陳述強調處 本說月書所揭路之特 似特徵中的一個實施例。 3系列相4或相 此外，依據本說明書揭露 領域者係可輕易依據本發明 熟悉本技術 本發明之精神與範圍内，針心：徵’在不脫離 作適當改變與修飾，因此，|」使用方法與情況 申請專利範圍中。 ”匕貫施態樣亦包含於 14 201038933 【圖式簡單說明】 第一圖為本發明之用於固態微萃取之裝置的結 構示意圖。 第二A圖為可行乳化聚合之反應溶液（未添加靜 相粒子）行乳化聚合後之電子顯微鏡影像圖。 第二B圖為可行乳化聚合之反應溶液添加靜相 粒子（起始溶液）行乳化聚合後之電子顯微鏡影像 圖，其中靜相粒子為碳18。 第三A圖為經本發明之吸管尖處理過之試驗樣 品的ESI質譜圖。 第三B圖為未經本發明之吸管尖處理過之試驗 樣品的ESI質譜圖。 第四A圖為經台灣發明92117579號專利之吸管 尖_處理過之試驗樣品的MALDI質譜圖。 第四B圖為經本發明之吸管尖處理過之試驗樣 品的MADLI質譜圖。 【主要元件符號說明】 2 殼體 4 上開口 6 下開口 8 多孔性材料 9 靜相粒子 10 本發明之用於固態微萃取之裝置 15In summary, the use of the t-preparation of the micro-extraction of the present invention produces a second and a larger one. Compared with the conventional technology street, it has a simpler manufacturing process and has improved improvement in the amount of raw adsorption and balance time. For the use of biomedical, chemical and pharmaceutical. It can be used in any way. The features disclosed in the specification can be replaced with features of the same, equal or similar purpose. Therefore, in addition to this, this Order cites a special statement emphasizing one of the specific features of the road revealed by the monthly book. 3 series phase 4 or in addition, according to the disclosure of the present specification, the spirit and scope of the present invention can be easily understood in accordance with the present invention, and the needle core is used without any change or modification. The method and situation apply for patents.匕 施 施 亦 亦 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一 第一Electron microscopic image of the phase particle) after emulsion polymerization. The second B is an electron microscope image of the emulsion solution after the addition of the stationary phase particles (starting solution) in the reaction solution of the feasible emulsion polymerization, in which the static phase particles are carbon 18 The third A is the ESI mass spectrum of the test sample treated by the pipette tip of the present invention. The third B is the ESI mass spectrum of the test sample which has not been treated by the pipette tip of the present invention. The fourth A picture is the invention by Taiwan. The MALDI mass spectrum of the treated pipette tip of the patent of No. 92117579. The fourth B is the MADLI mass spectrum of the test sample treated by the pipette tip of the present invention. [Main component symbol description] 2 The opening 4 of the casing 4 Lower opening 8 porous material 9 static phase particle 10 device for solid phase microextraction of the present invention 15