手性香料香芹酮和乙酸蘇合香醋的液相色譜拆分及其對(duì)映體純度測(cè)定

劉 慧,何津彥,阮源萍

(廈門大學(xué)化學(xué)化工學(xué)院,譜學(xué)分析與儀器教育部重點(diǎn)實(shí)驗(yàn)室,福建廈門361005)

手性香料香芹酮和乙酸蘇合香醋的液相色譜拆分及其對(duì)映體純度測(cè)定

劉 慧,何津彥,阮源萍*

(廈門大學(xué)化學(xué)化工學(xué)院,譜學(xué)分析與儀器教育部重點(diǎn)實(shí)驗(yàn)室,福建廈門361005)

香芹酮和乙酸蘇合香醋是2種重要的食品用手性香料,它們的香味特征與其對(duì)映體構(gòu)型及對(duì)映體含量有關(guān).因此,對(duì)這類手性香料進(jìn)行色譜拆分不僅在分析鑒定香料、開發(fā)食用香精配方、食品分析,而且在香料科學(xué)的基礎(chǔ)研究方面也具有重要的意義.本研究采用新型的直鏈淀粉-三((S)-α-甲基苯基氨基甲酸醋)手性固定相(Chiralpak AS-H柱),系統(tǒng)考察了香芹酮、乙酸蘇合香醋對(duì)映體的液相色譜拆分行為.實(shí)驗(yàn)表明:在該手性柱上,采用正己烷-異丙醇(體積比為95∶5)流動(dòng)相,在10 min內(nèi),香芹酮和乙酸蘇合香醋對(duì)映體即可獲得很好的基線分離,分離度分別達(dá)到2.49和2.86;手性色譜分離為焓驅(qū)動(dòng)過程;手性固定相側(cè)鏈中α-甲基苯基手性中心可能對(duì)香芹酮的手性識(shí)別起到增強(qiáng)的作用.所建立的色譜分析方法準(zhǔn)確、可靠、簡(jiǎn)便,可用于實(shí)際香料樣品的對(duì)映體純度分析.

香芹酮;乙酸蘇合香醋;手性香料;對(duì)映體分離;高效液相色譜

手性香料是一類重要的化合物,不同對(duì)映體香料可能顯示出不同的香氣特征、香氣強(qiáng)度和生物活性[1-4].香芹酮[美國(guó)食用香料與提取物制造者協(xié)會(huì)(FEMA)2249]和乙酸蘇合香醋[FEMA 2684](見圖1)各含有一個(gè)手性碳原子,是2種經(jīng)美國(guó)FDA、歐洲理事會(huì)與食品香料專家委員會(huì)[2]和中國(guó)衛(wèi)生部[5]批準(zhǔn)的食品用手性香料.香芹酮對(duì)映體的香氣有顯著差異,(S)-(+)-香芹酮具有芫荽的香味,而(R)-(-)-香芹酮?jiǎng)t有留蘭香的香味[1-2].乙酸蘇合香醋具有強(qiáng)烈的青香香味,其香味與所含對(duì)映體比例有關(guān).如對(duì)映體過量值(ee)為78.3%的(S)-(-)-乙酸蘇合香醋帶有鱷梨的青味和草毒醬的香味;而ee為81.1%的(R)-(+)-乙酸蘇合香醋帶有杏香、蘋果香和草毒醬香味[1,6].這2種香料的來源除了天然精油,也可采用生物合成或不對(duì)稱合成技術(shù)獲取[1-2,7-10].

由于手性在香料中的重要性日益受到重視,光活性的手性香料普及應(yīng)用已經(jīng)成為香料發(fā)展的必然趨勢(shì)之一.因此,手性香料對(duì)映體拆分及其純度分析在香料科學(xué)的基礎(chǔ)研究與實(shí)際應(yīng)用中具有重要的指導(dǎo)意義[1,7].而手性色譜被公認(rèn)為測(cè)定對(duì)映體組成最為準(zhǔn)確的方法之一[11].

圖1 香芹酮(a)及乙酸蘇合香醋(b)的化學(xué)結(jié)構(gòu)式Fig.1 The chemistry structure of carvone(a) and styrallyl acetate(b)

香芹酮對(duì)映體可采用氣相色譜和液相色譜[12]拆分,手性固定相多為環(huán)糊精衍生物.而乙酸蘇合香醋可在一些糖基衍生物手性固定相,如Chiralcel OB[13]、Chiralcel OJ[14]和Chiralpak AD[15]柱上獲得基線拆分.本文采用新型的直鏈淀粉-三((S)-α-甲基苯基氨基甲酸醋)(ASMBC)手性固定相(Chiralpak AS-H柱)[16-18],正己烷-醇流動(dòng)相,系統(tǒng)考察香芹酮和乙酸蘇合香醋的液相色譜拆分行為.在此基礎(chǔ)上,建立一種準(zhǔn)確、可靠、簡(jiǎn)便的測(cè)定香芹酮和乙酸蘇合香醋對(duì)映體純度方法,并應(yīng)用于實(shí)際光活性香料樣品的分析.

1 實(shí)驗(yàn)部分

1.1 儀器與試劑

日本島津公司高效液相色譜儀:SCL-10 A系統(tǒng)控制器,DGU-12 A在線除氣機(jī),LC-10A TVP高壓梯度泵,Rheodyne 7725i進(jìn)樣閥(20μL),CTO-10ASVP柱溫箱,SPD-M10AVP二極管陣列檢測(cè)器(190～370 nm),CLASS-VP色譜工作站(6.13 version).日本DAICEL公司的ASMBC手性色譜柱(Chiralpak ASH,4.6 mm(i.d.)×250 mm,5μm).

正己烷和乙醇為TEDIA(USA)HPLC級(jí)溶劑,異丙醇為Merck LiChrosolv(Germany)HPLC級(jí)溶劑.光活性香芹酮與乙酸蘇合香醋參照品由廈門嘉盟劉剛博士提供.

1.2 色譜條件

采用Chiralpak AS-H手性柱為固定相,正己烷為流動(dòng)相基礎(chǔ)溶劑,乙醇或異丙醇為流動(dòng)相極性調(diào)節(jié)劑,流速1.0 m L/min,柱溫30℃,二極管陣列檢測(cè)器190～370 nm.樣品溶解在流動(dòng)相中,進(jìn)樣體積20μL.采用在線UV光譜圖對(duì)照鑒定目標(biāo)對(duì)映體色譜峰,對(duì)映體的出峰順序采用已知構(gòu)型的光活性樣品確定.

2 結(jié)果與討論

2.1 流動(dòng)相組成對(duì)色譜分離的影響

首先選用乙醇作為流動(dòng)相極性調(diào)節(jié)劑,改變乙醇含量,考察香芹酮及乙酸蘇合香醋在Chiralpak AS-H手性柱上的保留因子(k),對(duì)映體分離選擇性(a)和分離度(Rs)的變化情況,結(jié)果見表1.隨著流動(dòng)相中乙醇含量的降低,它們的保留因子和分離度均明顯增大.不同的是,該過程中香芹酮的對(duì)映體分離選擇性變化不大,而乙酸蘇合香醋的對(duì)映體分離選擇性明顯增加.相比之下,采用正己烷-乙醇流動(dòng)相,乙酸蘇合香醋容易達(dá)到基線分離,而香芹酮的分離度還有待于提高.

改用異丙醇作為極性調(diào)節(jié)劑,隨著流動(dòng)相中異丙醇含量的降低,香芹酮和乙酸蘇合香醋的保留因子和分離度也明顯增大,但對(duì)映體分離選擇性變化不大.與相同比例的乙醇調(diào)節(jié)劑相比,采用異丙醇調(diào)節(jié)劑時(shí)的保留因子、對(duì)映體分離選擇性和分離度均明顯增加(見表1).特別是香芹酮對(duì)映體的拆分效果得到明顯改善.采用正己烷-異丙醇(體積比為95∶5)流動(dòng)相,香芹酮和乙酸蘇合香醋在Chiralpak AS-H柱上均獲得很好的基線分離(見圖2),分離度分別達(dá)到2.49和2.86.但流動(dòng)相中醇調(diào)節(jié)劑性質(zhì)的改變并沒有改變對(duì)映體的出峰順序,香芹酮和乙酸蘇合香醋均是(R)-構(gòu)型對(duì)映體先出峰,(S)-構(gòu)型對(duì)映體后出峰.

表1 流動(dòng)相組成對(duì)香芹酮和乙酸蘇合香醋色譜分離的影響Tab.1 Effect of composition of mobile phase on enantioseparation of carvone and styrallyl acetate

圖2 香芹酮(a)和乙酸蘇合香醋(b)的拆分色譜圖Fig.2 Chromatograms of carvone(a) and styrallyl acetate(b)

2.2 色譜熱力學(xué)與拆分機(jī)理探討

開展色譜熱力學(xué)實(shí)驗(yàn),不但可以求解對(duì)映體在手性固定相上拆分過程中的熱力學(xué)參數(shù),而且有助于探究手性識(shí)別機(jī)理[19].在色譜手性拆分過程中,不同構(gòu)型對(duì)映體的自由焓變(ΔH?)和自由熵變(ΔS?),以及它們?cè)趦上嚅g分配的焓變(ΔΔH?)、熵變(ΔΔS?)之差,滿足Van′t Hoff方程:

為此,采用正己烷-異丙醇(體積比為95∶5)流動(dòng)相體系,在20～40℃范圍內(nèi)考察柱溫對(duì)香芹酮和乙酸蘇合香醋在Chiralpak AS-H柱上的色譜拆分行為的影響.結(jié)果表明:隨著柱溫的升高,香芹酮和乙酸蘇合香醋的保留因子和對(duì)映體分離選擇性均有所減小.基于Van′t Hoff方程,分別以ln k和lnα對(duì)1/T作圖,結(jié)果參見圖3.香芹酮和乙酸蘇合香醋的Van′t Hoff曲線均呈現(xiàn)良好的線性(γ2＞0.985),說明在實(shí)驗(yàn)柱溫范圍內(nèi),ASMBC固定相的構(gòu)型及香料對(duì)映體在Chiralpak AS-H柱上的手性識(shí)別機(jī)理未發(fā)生變化.通過曲線的斜率和截距等數(shù)據(jù),可計(jì)算色譜拆分過程的熱力學(xué)參數(shù)(表2).香芹酮和乙酸蘇合香醋對(duì)映體的ΔH?為負(fù)值,說明溶質(zhì)與固定相結(jié)合過程均為放熱過程,且(S)-構(gòu)型對(duì)映體放熱更多,與固定相的結(jié)合在能量上更有利.香芹酮和乙酸蘇合香醋的ΔΔH?和ΔΔS?均為負(fù)值,說明它們?cè)贑hiralpak AS-H柱上的手性拆分過程為焓驅(qū)動(dòng)模式[19].

表2 香芹酮和乙酸蘇合香醋色譜拆分的熱力學(xué)參數(shù)Tab.2 Thermodynamic parameters for carvone and styrallyl acetate

Tamura等[12]曾報(bào)道:采用甲醇-水流動(dòng)相,香芹酮在Chiralpak AD-RH柱上難于達(dá)到基線拆分.作者改用正己烷-異丙醇流動(dòng)相,香芹酮在Chiralpak ADH柱上的拆分效果也沒有得到明顯的改善.與常見的ADMPC(Chiralpak AD-H柱)相比,ASMBC(Chiralpak AS-H柱)固定相側(cè)鏈上多含一個(gè)手性中心,苯環(huán)與N—H基團(tuán)不在同一平面,導(dǎo)致聚合物高級(jí)結(jié)構(gòu)的改變,可能使手性拆分性能發(fā)生變化[16,18].雖然香芹酮分子只含有一酮基,但其手性中心在六元環(huán)上,分子結(jié)構(gòu)扭曲且體積較小,有利于插入ASMBC的手性空穴內(nèi)部,與手性固定相中的氨基甲酸醋殘基發(fā)生較強(qiáng)的氫鍵作用或/和偶極-偶極作用,保留時(shí)間大于乙酸蘇合香醋.由于受到側(cè)鏈中α-甲基苯基立體構(gòu)型的影響,ASMBC增大對(duì)香芹酮的手性識(shí)別能力.而乙酸蘇合香醋分子含有淡基和苯環(huán),分子剛性增大,它們可以與手性固定相中氨基甲酸醋殘基和苯環(huán)氫鍵、偶極-偶極、π-π相互作用進(jìn)行手性識(shí)別.這可能與乙酸蘇合香醋在Chiralcel OB、Chiralcel OJ和Chiralpak AD的拆分機(jī)理是類似的.其中(S)-構(gòu)型對(duì)映體與ASMBC的作用力大,保留時(shí)間長(zhǎng)、后出峰.

圖3 柱溫對(duì)香芹酮(a)和乙酸蘇合香醋(b)色譜拆分的影響Fig.3 Effect of temperature on enantioseparation of carvone(a)and styrallyl acetate(b)

2.3 實(shí)際樣品測(cè)定

采用正己烷-異丙醇(體積比為95∶5)流動(dòng)相,對(duì)一外消旋乙酸蘇合香醋樣品液平行進(jìn)樣6次進(jìn)行精密度實(shí)驗(yàn).結(jié)果表明:(R)-和(S)-乙酸蘇合香醋的對(duì)映體峰面積比值為50.10∶49.90(相對(duì)標(biāo)準(zhǔn)偏差(RSD)≤0.03%),與外消旋化合物的理論值50∶50相符.采用優(yōu)化后的色譜條件,對(duì)一些實(shí)際的光活性香料樣品進(jìn)行分析,結(jié)果見表3.結(jié)果表明:所建立的色譜分析方法具有良好的精密度及可靠性,可以快速準(zhǔn)確地測(cè)定實(shí)際光活性香料樣品的對(duì)映體純度與香料構(gòu)型.

表3 實(shí)際香料樣品的色譜分析(n=3)Tab.3 The HPLC analysis of optical flavor samples(n=3)

3 結(jié) 論

本文采用新型的ASMBC手性固定相,正相色譜模式成功拆分了手性香料香芹酮和乙酸蘇合香醋對(duì)映體.實(shí)驗(yàn)表明:采用正己烷-異丙醇(體積比為95∶5)流動(dòng)相,香芹酮和乙酸蘇合香醋對(duì)映體在Chiralpak AS-H手性柱上獲得很好的基線分離;其手性色譜分離為焓驅(qū)動(dòng)模式.由于ASMBC固定相側(cè)鏈含有一個(gè)手性中心,它可以更有效地識(shí)別香芹酮手性分子.本文所建立的色譜分析方法準(zhǔn)確、可靠、簡(jiǎn)便,適用于香芹酮和乙酸蘇合香醋香料樣品的對(duì)映體純度分析.

[1] 田紅玉.手性香料及其不對(duì)稱合成[M].北京:化學(xué)工業(yè)出版社,2011:1-3,34-35,265.

[2] 劉樹文.合成香料技術(shù)手冊(cè)[M].2版.北京:中國(guó)輕工業(yè)出版社,2009:227-228,396-397.

[3] Werkhoff P,Krammer G,Brennecke S,et al.Methyl dihydrojasmonate and its stereoisomers:sensory properties and enantioselective analysis[J].Food Reviews International,2002,18(2/3):103-122.

[4] Bestmann H J,Classen B,Vostrowsky O,et al.Plant insecticidalⅧsynergistic activity of(-)-carvone and pyrethrineⅠin the essential oil of chrysanthemum balsamital [J].Journal of Applied Entomology,1988,106(2): 144-149.

[5] 中華人民共和國(guó)衛(wèi)生部.GB 2760—2011食品安全國(guó)家標(biāo)準(zhǔn)食品添加劑使用標(biāo)準(zhǔn)[S].北京:中國(guó)標(biāo)準(zhǔn)出版社,2011.

[6] Farbood M I,Blocker R W,Arvizzigno J,et al.Mixtures of optical isomers of styralyl alcohol or styralyl acetate, processes for preparing same and organoleptic uses thereof:US,6511686 B1[P].2003-01-28.

[7] 金其璋.天然食品香精及其原料來源[J].中國(guó)食品添加劑,1997(3):23-28.

[8] Macaev F,Vlad L,Gudima A.Selective synthesis of carvone and cryptomerione fromα-pinene[J].Chemistry of Natural Compounds,2006,42:301-303.

[9] 羅虹,祝春蘭.L-香芹酮的合成[J].天津化工,2003,17 (2):38-39.

[10] 陳煦,金秋茹,關(guān)志才.乙酸蘇合香醋的合成[J].天津化工,2003,17(4):13-15.

[11] Maier N M,Franco P,Lindner W.Separation of enantiomers:needs,challenges,perspectives[J].Journal of Chromatography A,2001,906(1/2):3-33.

[12] Mookdasanit J,Tamura H.Comparative enantioseparation of monoterpenes by HPLC on three kinds of chiral stationary phases with an on-line optical rotatory dispersion under reverse phase mode[J].Food Science and Technology Research,2002,8:367-372.

[13] Wainer I W,Stiffin R M,Shibata T.Resolution of enantiomeric aromatic alcohols on a cellulose tribenzoate high-performance liquid chromatography chiral stationary phase.A proposed chiral recognition mechanism[J]. Journal of Chromatography,1987,411:139-151.

[14] Yazicioglu E Y,Tanyeli C.A method for the synthesis of pyridine-based C-2-symmetrical chiral nucleophilic organocatalysts via Pd-catalyzed coupling[J].Tetrahedron-Asymmetry,2012,23(24):1694-1699.

[15] Kracht D,Saito S,Frohlich R,et al.Synthesis of a silanol-substituted proline analog as organocatalyst[J]. Zeitschrift fur Naturforschung B:Journal of Chemical Sciences,2009,64:1169-1175.

[16] Kasat R B,Wang N H L,Franses E I.Experimental pro-bing and modeling of key sorbent-solute interactions of norephedrine enantiomers with polysaccharide-based chiral stationary phases[J].Journal of Chromatography A,2008,1190(1/2):110-119.

[17] 董豐收,劉新剛,曹巧,等.Chiralpak AS-H手性柱分離三唑醇對(duì)映體[J].環(huán)境化學(xué),2008,27(6):810-813.

[18] Kasat R B,Wang N H L,Franses E I.Effects of backbone and side chain on the molecular environments of chiral cavities in polysaccharide-based biopolymers[J]. Biomacromolecules,2007,8:1676-1685.

[19] 翁文,姚碧霞,陳秀琴,等.液相色譜手性拆分機(jī)理的熱力學(xué)方法研究[J].化學(xué)進(jìn)展,2006,17(7/8):1056-1064.

Liquid Chromatographic Resolution and Determination of Enantiomeric Purity of Chiral Flavors Carvone and Styrallyl Acetate

LIU Hui,HE Jin-yan,RUAN Yuan-ping*
(Key Laboratory of Spectrochemical Analysis&Instrumentation,Ministry of Education, College of Chemistry and Chemical Engineering,Xiamen University,Xiamen 361005,China)

:Carvone and styrallyl acetate are two important chiral flavors in food additives.Their fragrance is related to their configurations and enantiomeric ratios.Therefore,the enantioseparation of these flavors is helpful to identify flavors and to develop flavor formula for food industry.In addition it is important in basic research of flavor science.In this paper,the high performance liquid chromatography enantioseparation of chiral carvone and styrallyl acetate was studied using amylose tris-(S)-α-methylbenzylcarbamate as the chiral stationary phase(Chiralpak AS-H column).The effects of alcohol content in the mobile phase and column temperature on the retention and enantioseparation were investigated.Additionally,the chiral recognition mechanism between the analytes and the chiral stationary phase(CSP)was discussed.The results show that baseline enantioseparations of carvone and styrallyl acetate are easily obtained in 10 min on that column with a mobile phase of n-hexane-isopropanol(95∶5,by vol.)at a flow rate of 1.0 m L/min under 30℃.The(R)-isomers are first eluted and the enantioseparation is typically enthalpy-controlled.The chiral center of α-methylbenzyl on CSP may strengthen the chiral recognition of carvone.The method established is simple,reliable and accurate,and has been successfully applied to determine enantiomeric purity of optical flavor samples.

carvone;styrallyl acetate;chiral flavors;enantioseparation;high performance liquid chromatography

O 657.72

A

0438-0479(2015)03-0310-05

10.6043/j.issn.0438-0479.2015.03.003

2014-08-07 錄用日期:2014-10-11

國(guó)家基礎(chǔ)科學(xué)人才培養(yǎng)基金(J1310024)

*通信作者:ypruan@xmu.edu.cn

劉慧,何津彥,阮源萍.手性香料香芹酮和乙酸蘇合香醋的液相色譜拆分及其對(duì)映體純度測(cè)定[J].廈門大學(xué)學(xué)報(bào):自然科學(xué)版,2015,54(3):310-314.

:Liu Hui,He Jinyan,Ruan Yuanping.Liquid chromatographic resolution and determination of enantiomeric purity of chiral flavors carvone and styrallyl acetate[J].Journal of Xiamen University:Natural Science,2015,54(3):310-314.(in Chinese)