吳丁丁,穆小靜,易小琦,陳 剛

(重慶大學(xué)化學(xué)化工學(xué)院,重慶 401331)

雙水相萃取技術(shù)的新發(fā)展

吳丁丁,穆小靜*,易小琦,陳 剛

(重慶大學(xué)化學(xué)化工學(xué)院,重慶 401331)

雙水相萃取技術(shù)是一種易于放大、操作條件溫和、可連續(xù)化操作的新型分離技術(shù)。本文綜述了新近發(fā)展的不同的雙水相體系(聚合物-鹽-水、有機(jī)溶劑-鹽-水、離子液體-鹽-水、離子液體-表面活性劑-鹽-水)的性質(zhì),及萃取(提取)蛋白質(zhì)、氨基酸、抗生素和中藥材中小分子有效成分等的影響因素;總結(jié)了雙水相萃取與其它技術(shù)的聯(lián)用,展望了雙水相體系的應(yīng)用前景。

雙水相萃取,聚合物,有機(jī)溶劑,離子液體,聯(lián)用技術(shù)

雙水相體系(aqueous two-phase system,ATPS)由兩種互不相溶的水溶液組成,在萃取過(guò)程中,分子間氫鍵、鹽析作用、電荷相互作用、范德華力、聚合現(xiàn)象、疏水作用、界面性質(zhì)作用等都扮演著十分重要的角色。這些作用力導(dǎo)致待萃取物在兩相間產(chǎn)生濃度差異,從而實(shí)現(xiàn)分離。雙水相萃取在提取中兼具分離功能,具有較高的生物相溶性、易于放大、可連續(xù)化操作、不易引起蛋白質(zhì)的變性失活。該技術(shù)已經(jīng)被應(yīng)用于蛋白質(zhì)、核酸、氨基酸、抗生素、色素以及中藥材中的小分子化合物等產(chǎn)品的分離和純化,必將在生物工程、食品、制藥等領(lǐng)域廣泛應(yīng)用。

1956年Albertsson首次應(yīng)用ATPS成功分離葉綠素[1]。目前對(duì)ATPS分離技術(shù)的研究方興未艾,新的體系層出不窮,應(yīng)用領(lǐng)域不斷拓展。劉磊磊等[2]綜述了近5年來(lái)雙水相技術(shù)在中藥黃酮、生物堿、萜類(lèi)、多酚及其它有效成分的分離中的應(yīng)用。本文就近年來(lái)出現(xiàn)的新ATPS體系,闡述影響雙水相提取分離的因素如組成雙水相體系的聚合物、有機(jī)溶劑的種類(lèi)、離子液體、表面活性劑的種類(lèi)、鹽的種類(lèi)及濃度、體系的pH等;并介紹了新發(fā)展的雙水相與其它技術(shù)的聯(lián)用。

1 雙水相體系的組成及應(yīng)用

鹽是多數(shù)ATPS組成的重要成分之一,對(duì)ATPS萃取效果有顯著影響。要求鹽在水中有較大溶解度和解離度,價(jià)格低廉,常見(jiàn)的有:(NH4)2SO4、NaH2PO4、K2CO3、Na2SO4、K2HPO4、Na2CO3、CaCl2、KH2PO4、NaCl等。萃取酸性化合物時(shí),選擇較低pH,一般用酸性鹽或中性鹽;反之,若萃取堿性化合物,一般選用堿性pH,用堿性或中性鹽,以保證被萃取的化合物以分子狀態(tài)存在,被萃取到上相。若被萃取對(duì)象以離子狀態(tài)存在,將加大其在鹽水相的分配。

鹽的濃度對(duì)分配系數(shù)和純化因子也有影響。B[mim]BF4離子液體與不同種類(lèi)的鹽(NaH2PO4、K2HPO4、(NH4)2SO4、MgSO4)組成的ATPS提取小麥酯酶,發(fā)現(xiàn)NaH2PO4優(yōu)于其它種類(lèi)的鹽;并且隨著NaH2PO4質(zhì)量分?jǐn)?shù)不斷增大,小麥酯酶在雙水相中的分配系數(shù)不斷增大。當(dāng)鹽的質(zhì)量分?jǐn)?shù)從15%增大至25%時(shí),分配系數(shù)K由1.86±0.01增大到7.27±0.06,當(dāng)鹽的質(zhì)量分?jǐn)?shù)繼續(xù)增大,小麥酯酶在雙水相中的K緩慢增大至8.62±0.09,但是小麥酯酶的純化因子在NaH2PO4的質(zhì)量分?jǐn)?shù)為25%時(shí)達(dá)到最大(4.23±0.04),NaH2PO4質(zhì)量分?jǐn)?shù)增大至30%時(shí),純化因子降低至1.97±0.04[13]。

在A(yíng)TPS中加入兩種或兩種以上的鹽,能夠改變被萃取化合物在兩相中的分配,加快相分離的速度。如丙醇-K2HPO4/KH2PO4組成的ATPS中櫻桃蘆丁的K值為42.76;當(dāng)加入2.5% NaCl時(shí),K值提高到51.47[14]。丙醇-(NH4)2SO4ATPS在pH2.0時(shí)萃取分離蒜氨酸((S)-3-(烯丙基亞磺酰)-L-丙氨酸),加入8.54% NaCl后,K值從0.060減小至0.042,提取率增大[15]。蘆薈中的多糖和蛋白質(zhì)在由(NH4)2SO4、NaH2PO4分別與B[mim]BF4離子液體組成的雙水相體系中的萃取率分別低于70%和90%,但向體系中加入1.33 mol/L NaCl,多糖和蛋白質(zhì)在這兩種體系中的萃取率高于90%[16]。

可見(jiàn),鹽對(duì)ATPS的性質(zhì)以及萃取的結(jié)果有重要影響,但影響的原因尚待研究者們進(jìn)一步從理論上深入解析。組成ATPS的其它組分對(duì)萃取的結(jié)果影響較大。

除了鹽以外,按照ATPS的其他組分對(duì)其進(jìn)行分類(lèi),通常有以下幾類(lèi):聚合物-鹽-水[6,17]、有機(jī)溶劑-鹽-水[4,18]、離子液體-鹽-水[19]、離子液體-表面活性劑-水[20-21]、其他體系如聚合物1-聚合物2-水[22]等。

1.1 聚合物-鹽-水體系

由于聚合物黏度較大,相比于兩種聚合物形成的雙水相體系,聚合物-鹽體系得到更多的應(yīng)用。而在聚合物-鹽體系中,聚乙二醇-鹽-水體系的應(yīng)用最為廣泛。

1.1.1 聚乙二醇-鹽-水體系 PEG是平均分子量從200到20000的乙二醇高聚物的總稱(chēng)。PEG的分子量對(duì)形成的ATPS性質(zhì)有較大影響,構(gòu)成PEG-鹽-水體系的雙水相所使用PEG分子量一般介于300～6000之間,但最常用的體系或萃取效率較好的雙水相體系使用的PEG分子量在1500～6000之間。PEG分子量太小,上相的極性太大;兩相的極性差異小,不利用分配系數(shù)的提高;若PEG分子量太大,其疏水性強(qiáng),水中溶解度小,形成雙水相的PEG濃度降低,也不易于獲得較高的提取效率。不同分子量的PEG對(duì)雙水相的形成和性質(zhì)有以下三個(gè)方面的差異:PEG及鹽在形成雙水相時(shí)所需的質(zhì)量分?jǐn)?shù)。如在提取分離丁酸[10]和紅豆蛋白質(zhì)[7]時(shí),發(fā)現(xiàn)不同的PEG分子量對(duì)相圖有影響,當(dāng)所選的鹽質(zhì)量分?jǐn)?shù)一定時(shí),隨著PEG相對(duì)分子質(zhì)量的增大,憎水程度越強(qiáng),加大了相分離的動(dòng)力,因此形成雙水相體系所需PEG的濃度降低。PEG分子量對(duì)萃取率有影響。萃取丁酸[10]時(shí),分子量為6000形成雙水相的萃取率達(dá)到最大。但當(dāng)萃取對(duì)象為紅豆蛋白質(zhì)[7]時(shí),PEG6000形成的雙水相萃取率較低,可能是由于PEG相對(duì)分子質(zhì)量高,疏水性較大,紅豆蛋白不易向上相分配。PEG分子量對(duì)分配系數(shù)有影響。從馬鈴薯中提取轉(zhuǎn)化酶[8]時(shí)發(fā)現(xiàn)隨著PEG分子量的增大,分配系數(shù)增大。當(dāng)分子量超過(guò)4000,體積排阻效應(yīng)變大,分配系數(shù)反而減小。因此,選擇PEG分子量的大小時(shí)需要綜合考慮上述幾個(gè)因素的影響。

聚乙二醇(PEG)-鹽-水體系含水量高[3,23]且兩相容易分離,蛋白質(zhì)不易變性失活,因此研究者將這一體系用以提取生物活性的蛋白質(zhì)。后來(lái)發(fā)現(xiàn)該體系可以用來(lái)提取小分子物質(zhì),如酚類(lèi)化合物[3]、丁酸[10]、黃連生物堿[5]等能獲得較好的收率,成本低,污染小。

1.1.2 其他聚合物-鹽-水體系 除了聚乙二醇以外,質(zhì)量分?jǐn)?shù)為50%氧化乙烯和質(zhì)量分?jǐn)?shù)為50%氧化丙烯組成的共聚物EO50PO50構(gòu)成的ATPS分離溶菌酶[24]。相對(duì)于PEG-鹽體系,該體系成本更低,適合工業(yè)化生產(chǎn)。

1.2 有機(jī)溶劑-鹽-水體系

與聚合物-鹽-水體系相比,有機(jī)溶劑組成的ATPS黏度小,溶劑和萃取物易回收。組成ATPS的有機(jī)溶劑一般與水可以混溶,有機(jī)溶劑的極性小,則其相應(yīng)的ATPS體系的上相極性小;反之,有機(jī)溶劑的極性大則其相應(yīng)的ATPS體系的上相極性大。這些體系一般不適合分離蛋白質(zhì)、多肽,而用來(lái)提取分離小分子化合物。如:花青素[25]、白藜蘆醇[26]、辣椒素[27]、綠原酸[28]等化合物,提取時(shí)應(yīng)選擇適合的pH。例如乙醇-NaH2PO4ATPS萃取和分離花青素時(shí),pH>3.31花青素的回收率將會(huì)降低,pH>4.20 NaH2PO4將會(huì)沉淀。當(dāng)萃取生物堿[29]等堿性化合物時(shí),體系的pH調(diào)節(jié)在堿性范圍內(nèi)?？傊?pH的選擇依據(jù)是萃取的化合物在雙水相中能夠以分子狀態(tài)存在。

乙醇-(NH4)2SO4組成的ATPS中加入環(huán)糊精,色氨酸光學(xué)對(duì)映體在上相與下相中的分配系數(shù)有明顯差異,從而實(shí)現(xiàn)對(duì)映體的分離[23]。pH對(duì)ATPS萃取分離色氨酸對(duì)映體具有顯著影響,隨著pH增大,D-色氨酸與L-色氨酸在雙水相中的分配系數(shù)先增大后下降,在pH=3時(shí),D-色氨酸與L-色氨酸的分配系數(shù)差異最大,色氨酸對(duì)映體的分離效果最好,分離系數(shù)(α=KD/KL)為1.38。該法為手性化合物的分離純化提供了一種便捷、新穎的方法。

丙醇-鹽-水、丙酮-鹽-水ATPS也是常用的萃取體系,如萃取蒽醌類(lèi)化合物[30]、蒜氨酸[15]、鹽酸土霉素[31]、琥珀酸[32]等。

1.3 離子液體-鹽-水體系

離子液體(ionic liquid,IL)一般由有機(jī)陽(yáng)離子和陰離子組成。常見(jiàn)的有機(jī)陽(yáng)離子有咪唑鹽離子、季銨鹽離子、N-烷基吡啶離子等。陰離子分為有機(jī)陰離子和無(wú)機(jī)陰離子,常用的有鹵素離子、四氟硼酸根離子、六氟酸根離子、硝酸鹽及醋酸鹽等。離子液體蒸汽壓低、較高的熱化學(xué)穩(wěn)定性。相對(duì)于由PEG組成的ATPS,離子液體的ATPS黏度低、不易乳化、相分離速度快。

IL-鹽-水體系常被用來(lái)分離大分子物質(zhì)(如蛋白質(zhì)、多肽、酶等)。例如有三個(gè)課題組采用IL-鹽-水體系來(lái)分離牛血清白蛋白(BSA)[33-35]。選用K2HPO4是因其溶解度大且具有提取BSA適宜的pH。在A(yíng)TPS中IL的種類(lèi)對(duì)萃取BSA的分配系數(shù)、提取效率等有較大影響。BSA在[bmim][Br]、[bmim][Cl]、[OH-mim][Cl]組成的ATPS中K值在1.97～3.04之間,而類(lèi)膽汁離子液體組成的ATPS中BSA的分配系數(shù)達(dá)到7.68,提高了2～3倍[33]。Chen等[35]則比較了8種IL形成的ATPS對(duì)BSA萃取率的影響。這8種IL分別由兩種陽(yáng)離子N,N-二甲基乙醇胺(DMEA)、二乙基乙醇胺(DEEA)和4種陰離子丙酸(Pr)、丁酸、戊酸、己酸構(gòu)成。若IL的陽(yáng)離子為DMEA,隨著陰離子鏈長(zhǎng)增加,雙水相對(duì)BSA萃取率從99.47%下降到64.80%;若陽(yáng)離子為DEEA,陰離子隨著鏈長(zhǎng)增加萃取率則是從89.43%下降到58.70%。這表明IL的陽(yáng)離子相同時(shí),隨著陰離子烷基鏈的不斷增長(zhǎng),離子液體的疏水性增強(qiáng),BSA的萃取率降低。當(dāng)IL的陰離子相同時(shí),烷基鏈長(zhǎng)的陽(yáng)離子,IL的水溶性低,溶液的黏度增加,體系對(duì)BSA的萃取率降低。因此[DMEA][Pr]是這8種離子液體中萃取BSA最佳的離子液體。由1,1,3,3-四甲基胍(1,1,3,3-tetramethylguandine,TMG)陽(yáng)離子與8種不同的陰離子(醋酸鹽、山梨酸鹽、衣康酸鹽、丙烯酸鹽、甲基丙烯酸鹽、乳酸鹽、肉桂酸鹽、馬來(lái)酸鹽)組成的ATPS在萃取分離BSA時(shí),TMG與丙烯酸鹽組成的離子液體(TMGA)對(duì)BSA的萃取率高,達(dá)99.46%[34]。

四種離子液體[C4mim][N(CN)2]、[C4mpyr]Cl、[C4mim]Cl和[C8mim]Cl分別與磷酸鹽緩沖液構(gòu)成的ATPS提取脂肪分解酶[36],在pH=7.0時(shí),脂肪酶(pI=3.0)帶負(fù)電荷,使得親水性增加,脂肪酶向鹽相轉(zhuǎn)移。[C4mim][N(CN)2]-磷酸緩沖液體系對(duì)脂肪酶的提取效果好,分配系數(shù)K=0.049±0.003。不僅如此,離子液體-鹽-水體系還能被用來(lái)分離小分子物質(zhì),如磺酰胺類(lèi)化合物[37]、五倍子酸[38]等。

1.4 離子液體-表面活性劑-鹽-水體系

在離子液體-鹽-水體系中加入表面活性劑形成新型ATPS。當(dāng)加入2.33%十二烷基苯磺酸鈉(SDBS)時(shí),C6[mim]BF4和C8[mim]BF4與(NH4)2SO4溶液形成的ATPS遭破壞,而C4[mim]BF4-(NH4)2SO4-水體系中,SDBS加入使形成ATPS需要的IL量極少(0.1%),該體系可以提取糖果中的蘇丹紅Ⅰ-Ⅳ[39]。C4[mim]Cl-PEG300-Na2SO4雙水相體系提取沒(méi)食子酸、香草酸、丁香酸時(shí),分配系數(shù)大于30.0,而在PEG300-Na2SO4中,三種化合物的分配系數(shù)低于20.0[40]。

1.5 其他體系

根據(jù)雙水相形成的基本原理,研究者以聚合物代替鹽,發(fā)展了聚合物-有機(jī)溶劑-水和聚合物-聚合物-水體系。右旋糖苷(Dx)-乙腈-水ATPS體系中右旋糖苷與水分子形成氫鍵,與有機(jī)溶劑競(jìng)爭(zhēng)水而形成雙水相,右旋糖苷主要分布在下層,乙腈在上層,香草醛被提取到上層,分配系數(shù)K=11.30,萃取率達(dá)95%[41]。Wu等[42]從人體血清白蛋白中提取免疫球蛋白G時(shí)比較了四種ATPS對(duì)其回收率的影響,分別為聚合物-聚合物-水體系(PEG-HPS(羥丙基淀粉)-水、PEG-Dx-水體系)和聚合物-鹽-水體系(PEG-硫酸鹽-水、PEG-磷酸鹽-水),K<1;向體系中加入15% NaCl,免疫球蛋白G由原本向下相富集轉(zhuǎn)變?yōu)橄騊EG相富集,K>1,且在PEG-HPS-水體系中的回收率最高(高于90%)。以表面活性劑-木糖醇-水組成的ATPS從南瓜種子中提取和純化脂肪酶,這三種表面活性劑組成的ATPS對(duì)脂肪酶的純化因子大小順序:TritonX-100(PF>7.31)>Tween-80(PF<3.33)>SDS[43]。

2 雙水相萃取技術(shù)與其他技術(shù)的聯(lián)用

ATPS萃取方法相對(duì)于傳統(tǒng)的提取方法而言,不使用或少量使用有機(jī)溶劑,并且在適宜條件下選擇性較好。但該方法與傳統(tǒng)的液-液萃取技術(shù)一樣,從成分復(fù)雜的固體樣品中萃取目標(biāo)組分受基質(zhì)狀態(tài)、傳質(zhì)的影響較大,相分離的時(shí)間長(zhǎng)。與其他技術(shù)如微波輔助[29,44-45]、超聲波輔助[46-49]、溶劑浮選[50]等聯(lián)用能使ATPS的分離時(shí)間短、選擇性高,如表1。

表1 雙水相技術(shù)與其他技術(shù)的聯(lián)用Table 1 Aqueous two-phase extraction technique combined with other technologies

注:回收率指富集相中萃取物的質(zhì)量與投入原料粗提液中目標(biāo)物質(zhì)的質(zhì)量之比;萃取率指富集相中目標(biāo)萃取物的質(zhì)量與投入原料的質(zhì)量之比。

微波輔助的ATPS比單一的微波萃取或熱回流提取的方法的萃取時(shí)間短,萃取率更高。例如:從苦參中分離生物堿[29],從降香黃檀葉中提取染料木黃酮和鷹嘴豆芽素A[44]以及從葡萄種子中分離酚醛樹(shù)脂[45]等。超聲波輔助的ATPS技術(shù)從花椒中分離木酯素[46],比傳統(tǒng)的熱回流法萃取率高,木脂素的純度高、選擇性好,萃取時(shí)間也大大縮短。雙水相浮選的方法從黃苓中分離黃苓苷[50],黃苓苷濃集系數(shù)大、以及所需的有機(jī)溶劑量少。

3 結(jié)論及展望

ATPS用于提取蛋白質(zhì)等生物活性物質(zhì)時(shí),操作簡(jiǎn)單,體系含水量高,在萃取過(guò)程中保持物質(zhì)的構(gòu)象穩(wěn)定,蛋白不易失活并獲得高的萃取率。ATPS萃取方法用來(lái)從中藥材等固體基質(zhì)中提取小分子化合物的起步較晚,它兼具固液萃取的功能和液-液萃取的分離的功能,通過(guò)選擇合適的pH、鹽、有機(jī)溶劑、聚合物、離子液體或表面活性劑的種類(lèi)和濃度,使萃取有較強(qiáng)的選擇性和良好的提取效率。但是,構(gòu)成ATPS體系的兩相極性均較強(qiáng),所以該法僅適合于提取分離極性較強(qiáng)的組分。在中藥材提取中,由于多種有效成分極性跨度較大,因此該技術(shù)在多組分同時(shí)提取的應(yīng)用中有較強(qiáng)局限性。未來(lái),可以在雙水相技術(shù)的基礎(chǔ)上,構(gòu)建三相體系或多相體系,從而實(shí)現(xiàn)多組分的高效同時(shí)提取和初步分離。這一技術(shù)因綠色環(huán)保將在生物工程、食品、中藥制藥、健康產(chǎn)品等領(lǐng)域有廣泛應(yīng)用。

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Progress of aqueous two-phase extraction technique

WU Ding-ding,MU Xiao-jing*,YI Xiao-qi,CHEN Gang

(College of Chemistry and Chemical Engineering of Chongqing University,Chongqing 401331,China)

Extraction with aqueous two-phase system(ATPS)is a novel separation technique,which shows some advantages including easy scale-up,mild conditions and continuous operation. The properties and applications of newly developed different ATPSs(polymer-salt-water,organic solvent-salt-water,ionic liquid-salt-water,ionic liquid-surfactant-salt-water)in extraction of proteins,amino acids,antibiotics and other small molecules in herbs were reviewed. ATPS extraction combining with other technologies were summarized,and the prospect of ATPSs was also discussed.

aqueous two-phase extraction;polymer;organic solvent;ionic liquid;hyphenated technique

2016-08-12

吳丁丁(1990-)，女，碩士研究生，研究方向：分析化學(xué)，E-mail：18883724367@163.com。

*通訊作者:穆小靜(1973-)，女，博士，副教授，研究方向：藥物分析，E-mail:muxiaojingcn@aliyun.com。

重慶市重點(diǎn)產(chǎn)業(yè)共性關(guān)鍵技術(shù)創(chuàng)新專(zhuān)項(xiàng)(cstc2016zdcy-ztzx10001);重慶市科技支撐示范工程(cstc2014zktjccxyyBX0025)。

TS201.2

A

1002-0306(2017)08-0395-06

10.13386/j.issn1002-0306.2017.08.068