TWI643875B - Filler for liquid chromatography and column for liquid chromatography - Google Patents
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Abstract
本發明提供一種耐久性優異之液相層析儀用填充劑及填充該填充劑之液相層析儀用管柱。
本發明提供一種液相層析儀用填充劑,其特徵係經由間隔基使以下述式(1)表示之胺基鍵結於含有聚乙烯醇樹脂之親水性樹脂上,
(R1表示氫原子、或碳數1~4之烷基,R2表示具有1個以上羥基之碳數1~6之烷基,※表示與間隔基之鍵結位置)。
Description
本發明係有關耐久性優異之液相層析儀用填充劑及填充該填充劑之液相層析儀用管柱。
糖類於食品中等含有多量,其分析對於食品產業等極為重要。因此,其分析中廣為使用液體層析儀。利用液體層析之糖類分析主要利用配位子交換、親水性相互作用管柱。
使用配位子交換管柱之分析,為了抑制變旋異構物(anomer)分離而有必要在高溫條件下分析。例如,日本特開昭61-71354公報(專利文獻1)中,揭示有使用銀以及鹼(土類)金屬離子作為磺酸基之相對離子的離子交換樹脂作為填充劑之管柱。該專利文獻1中,係在管柱為60℃進行分離。然而,在該高溫下,離子鍵結於填充劑之金屬離子易於脫離,而有填充劑經時變化之問題。
使用親水性相互作用管柱時,有使用由聚丙烯醯胺樹脂所成之填充劑之管柱(例如日本特開2006-
137944號公報:專利文獻2)。然而,與配位子交換管柱同樣,為了抑制變旋異構物分離,有必要在高溫條件下進行。
另一方面,使用使用有鍵結胺基之填充劑之管柱(以下稱為胺基管柱)時,因胺基之鹼性可抑制變旋異構物分離,而不需要高溫條件,因此胺基管柱廣泛使用於糖分析(J.C.Linden et al.J.Chromatogr.A 105(1975)125-133)。然而,胺基管柱大部分基材係使用矽膠,鍵結胺基之矽膠缺乏安定性,使管柱之耐久性有問題。
鍵結有胺基且具有化學安定性之材料,有使用有機高分子擔體者。作為於高分子擔體鍵結胺基之材料,係使用於聚丙烯酸酯系樹脂或苯乙烯-二乙烯基苯共聚物樹脂上鍵結N-甲基-D-還原葡糖胺(glucamine)之化合物作為金屬或硼之吸附材(J.Sanchez et al.J.APPL POLY.SCI.129(2013)1541-1545,J.Sanchez et al.J.APPL PLOY.SCI.126(2012)1475-1483)。
然而,使用聚丙烯酸酯系樹脂或苯乙烯-二乙烯基苯共聚物樹脂之材料應用於胺基管柱時,有親水性較低之糖類的分離不充分之問題。
專利文獻1:日本特開昭61-71354號公報
專利文獻2:日本特開2006-137944號公報
非專利文獻1:J. Sanchez et al. J. APPL POLY. SCI. 129 (2013) 1541-1545
非專利文獻2:J. Sanchez et al. J. APPL PLOY. SCI. 126 (2012) 1475-1483
本發明之目的在於獲得耐久性優異,即使進行重複分析亦不引起經時變化之適於胺基管柱之填充劑。
本發明人等為達成上述課題而重複研究,發現藉由以含有聚乙烯醇樹脂之親水性樹脂作為基材,並使用導入特定胺基之填充劑,可改善胺基管柱之耐久性,因而完成本發明。
亦即本發明係關於如下事項者。
[1]一種液相層析儀用填充劑,其特徵係經由間隔基使以下述式(1)表示之胺基鍵結於含有聚乙烯醇樹脂之親水性樹脂上,
(R1表示氫原子、或碳數1~4之烷基,R2表示具有1個以上羥基之碳數1~6之烷基,※表示與間隔基之鍵結位置)。
[2]如[1]之液相層析儀用填充劑,其中R1為氫原子或甲基。
[3]如[1]或[2]之液相層析儀用填充劑,其中R2係以式(2)表示之構造,
(式中,n表示0~4之整數)。
[4]如[1]之液相層析儀用填充劑,其中以式(1)表示之胺基係源自由D-還原葡糖胺、N-甲基-D-還原葡糖胺、1-胺基-1-去氧-D-甘露糖醇、1-胺基-1-去氧-D-半乳糖醇(galactitol)、1-胺基-1-去氧-D-艾杜醇(Iditol)、1-
胺基-1-去氧-D-阿糖醇(arabinitol)、1-胺基-1-去氧-D-木糖醇、4-胺基-1,2,3-丁三醇、3-胺基-1,2-丙二醇、3-甲基胺基-1,2-丙二醇所組成之群中之任一種胺。
[5]如[1]之液相層析儀用填充劑,其中間隔基係源自末端具有縮水甘油基之化合物。
[6]一種液相層析儀用管柱,其係使用如前述[1]~[5]中任一項之液相層析儀用填充劑。
[7]一種糖分析用液相層析儀用管柱,其係使用如前述[1]~[5]中任一項之液相層析儀用填充劑。
[8]一種親水性相互作用層析儀用管柱,其係使用如前述[1]~[5]中任一項之液相層析儀用填充劑。
本發明之液體層析儀用填充劑於糖類分析中不需要高溫條件,即使於30~50℃之低溫,亦顯示優異分離。且,除糖類以外,亦可分離各種親水性化合物。再者,具有鹼耐性,相較於以往之胺基管柱,再現性、耐久性方面優異。
圖1顯示針對實施例1之填充劑,進行果糖、甘露糖、葡萄糖及蔗糖之4種糖類分析所得之層析圖。
圖2係顯示針對實施例1之填充劑之安定性試驗結果。
圖3顯示針對實施例2之填充劑,進行4種糖類分析所得之層析圖。
圖4顯示針對實施例3之填充劑,進行4種糖類分析所得之層析圖。
圖5顯示針對比較例2之填充劑,進行4種糖類分析所得之層析圖。
圖6顯示針對實施例1之填充劑,進行果糖、半乳糖、乳果糖(lactulose)、表乳糖(epilactose)、乳糖之5種糖類分析所得之層析圖。
圖7顯示針對分析例6變更溶離液進行5種糖類分析所得之層析圖。
本發明之液體層析用填充劑係經由間隔基使以下述式(1)表示之胺基鍵結於親水性樹脂上而得之粒子,
(R1表示氫原子、或碳數1~4之烷基,R2表示具有1個以上羥基之碳數1~6之烷基,※表示與間隔基之鍵結位置)。此時R1較好為氫原子或甲基。
又,R2較好為式(2)表示之羥基烷基,
(式中,n表示0~4之整數),進而較好係n為4。
又,作為式(1)所示之胺基,較好為源自由D-還原葡糖胺、N-甲基-D-還原葡糖胺、1-胺基-1-去氧-D-甘露糖醇、1-胺基-1-去氧-D-半乳糖醇、1-胺基-1-去氧-D-艾杜醇、1-胺基-1-去氧-D-阿糖醇、1-胺基-1-去氧-D-木糖醇、4-胺基-1,2,3-丁三醇、3-胺基-1,2-丙二醇、3-甲基胺基-1,2-丙二醇所組成之群中之任一種胺。去除該等胺基類之氮上鍵結之1個氫而與間隔基鍵結。本發明中,該等胺類係鍵結於間隔基,且亦可為氨經由間隔基鍵結於親水性樹脂後,使用氰基硼氫化鈉,使葡萄糖、甘露糖、半乳糖、木糖等之醛糖(aldose)鍵結於構成胺基之氮原子上而成者。該等中,就分離性能、反應簡便性、試藥取得容易性等方面,最好為使N-甲基-D-還原葡糖胺反應而得之胺基。
此種經由間隔基鍵結胺基之填充劑,於糖類分析中,不需要高溫條件,即使於30~50℃之低溫,亦顯示優異分離,具有鹼耐性,再現性、耐久性方面優異。
本發明中構成填充劑之親水性樹脂之大小、形狀並無特別限制。但,考慮本發明之填充劑對管柱之填充時,其形狀較好為直徑1~30μm之球狀。材質較好為交聯或非交聯樹脂。最好使用具有醇性羥基之樹脂。
本發明中所用之樹脂,較好舉例為使由羧基乙烯酯與交聯性單體所成之交聯共聚物之酯基進行皂化或酯交換反應而轉換成醇性羥基之聚乙烯醇系樹脂。作為羧酸乙烯酯有例如乙酸乙烯酯、丙酸乙烯酯、丁酸乙烯酯、戊酸乙烯酯及特戊酸乙烯酯等,該等可單獨使用,亦可組合2種以上使用。該等中,乙酸乙烯酯及丙酸乙烯酯由於聚合及皂化容易,故較佳。作為交聯性單體,較好為具有三嗪環之交聯性單體,其中最好為三烯丙基異氰尿酸酯。
上述樹脂不具有如酯構造或醯胺構造之對鹼性較弱之構造。因此,本發明之填充劑於鹼性下亦安定。因此具有亦可適於鹼性分析條件下使用之優點。
且,除上述樹脂以外,在不損及本發明目的之範圍內,亦可包含其他樹脂。例如可使用於苯乙烯-二乙烯基苯系交聯聚合物、甲基丙烯酸酯系交聯聚合物、含羥基之甲基丙烯酸酯系交聯聚合物粒子上化學鍵結有長鏈醯基者等。該等其他樹脂之含量,相對於樹脂量為10質量%以下,更好為5質量%以下之量即可。再更好,上述
親水性樹脂若為由聚乙烯醇樹脂所成者,就耐久性及耐鹼性方面較佳。
胺基之量,相對於樹脂重量,較好為0.1~0.7meq/g,更好為0.15~0.5meq/g之範圍。若為該範圍,則即使於低溫條件下亦可分析糖類。
本發明中之間隔基係指用以調整基材(親水性樹脂)與胺基之距離所用化學鍵結部位。該間隔基係用以抑制式(1)之胺基與基材之干涉並賦予抑制峰擴散之功能。作為用以對基材導入間隔基所用之化合物之所謂間隔基化合物舉例具有縮水甘油基者,例如,表氯醇、1,4-丁二醇二縮水甘油醚、乙二醇二縮水甘油醚、甘油二縮水甘油醚等。該等間隔基化合物之末端鹵基或單一末端縮水甘油基可與親水性樹脂之羥基反應。
縮水甘油基之導入量較好為0.2~0.8mol/g,更好為0.4~0.6mol/g之範圍。若為該範圍,則可導入較佳量之胺基。
縮水甘油基之導入係在無溶劑中或在溶劑下添加對於凝膠為0.1~10倍量之上述試藥並攪拌均勻而進行。
導入縮水甘油基之聚合物與用以導入式(1)所示之胺基之前述胺類之反應可如下進行。首先,將導入縮水甘油基之聚合物分散於水、二甲基亞碸、N,N-二甲基甲醯胺等溶劑中。此時之溶劑基於處理容易、所用之胺的溶解性等方面最好為水。胺較好係導入縮水甘油基之聚合物每
1g添加2mmol以上,以10mmol~20mmol之範圍添加時,就反應效率與再現性之方面係更佳。反應溫度基於再現性與反應效率方面而言,較好為30~40℃。反應時間基於反應量與效率方面而言,較好為12小時~24小時。
本發明之液體層析儀用填充劑之粒徑,係使用體積平均粒徑為1~30μm者,較好為2~20μm,再更好為2~10μm。體積平均粒徑未達1μm時,管柱壓力上升大,有極難以填充之情況。另一方面,超過30μm時,管柱之理論段數變低故而不佳。本申請案之上述體積平均粒徑係使用庫倫特(Coulter)計數器法如下述般測定。亦即,使用Multisizer 4(Beckman Coulter公司製)作為測定裝置,於填充劑樣品0.2g中添加ISOTON(稀釋液)25mL,以超音波碰撞3分鐘進行分散後,對約1000個測定個數測定體積平均粒徑。將體積平均粒徑設為較佳範圍時,可藉由風力分級、篩分分級、利用沉澱之分級等加以控制。
本發明之液體層析儀用填充劑對管柱之填充係依據漿料法等公知之填充方法進行。所得液體層析儀用管柱藉由使用由乙腈與水或甲酸銨、乙酸銨等所成之緩衝液之混合溶劑作為溶離液,可良好地分離糖類或其他親水性化合物。
本發明之分離對象物質,舉例有環境污染物質、戴奧辛類、環境激素、農藥、界面活性劑、生物毒素、天然藥物、天然色素、天然香料及天然調味料等。
本發明可獲得耐久性優異、即使進行重複分析亦不引起經時變化之適於胺基管柱之填充劑。尤其即使於低溫下亦可分離,故亦可應用於在高溫下具有不安定性質之糖類的分析等之用途。
藉以下實施例,對本發明更詳細加以說明,但本發明不受該等實施例之任何限制。
將由乙酸乙烯酯100g、三烯丙基異氰尿酸酯150g、乙酸丁酯100g、正癸烷25g及2,2-偶氮雙異丁腈5g而成之均一混合液與溶解有聚乙烯醇12g及磷酸氫二鈉18g之水1200mL饋入具備回流冷卻器之5L三頸燒瓶中攪拌10分鐘。接著,在氮氣流下攪拌,同時在60℃進行16小時聚合獲得粒狀聚合物。使該聚合物過濾、洗淨,以丙酮萃取後乾燥。接著將該聚合物與10%氫氧化鈉水溶液3L一起饋入具備回流冷卻器、氮氣導入管及攪拌器之5L三頸燒瓶中,在氮氣流下於15℃攪拌20小時,進行該聚合物之皂化後,過濾、水洗,進而乾燥。藉由皂化所得之聚乙烯醇聚合物之羥基密度為2.1meq/g。
將上述乾燥聚乙烯醇聚合物10g、表氯醇45g、二甲基亞碸100mL及30%氫氧化鈉水溶液5mL饋入可分離燒瓶中,在30℃攪拌20小時,於該聚合物中導入縮水甘油基。導入後之聚合物以二甲基亞碸、水、甲醇洗淨後,乾燥。
將導入上述縮水甘油基之聚合物4g、N-甲基-D-還原葡糖胺10g及水40mL饋入可分離燒瓶中,在30℃攪拌20小時,製作導入胺基之糖分析用及親水性相互作用層析儀用填充劑。該填充劑間隔著以水洗淨邊以0.5N鹽酸及0.5N氫氧化鈉水溶液洗淨,進而以甲醇洗淨後,進行乾燥。將如此所得之填充劑分散於0.5N氯化鉀水溶液中,使用0.1M鹽酸滴定胺基而測定該填充劑之胺基密度之結果為0.44meq/g。且該填充劑之體積平均粒徑為5μm。
將與實施例1同樣製作之導入縮水甘油基之聚合物2.0g、25%氨水1.0mL及水19mL饋入可分離燒瓶中,在30℃攪拌20小時,導入胺基。過濾該聚合物,間隔著水洗淨以0.5N鹽酸及0.5N氫氧化鈉水溶液洗淨,進而以甲醇洗淨後乾燥。
將上述具有胺基之聚合物1.8g、D-甘露糖1.9g、氰基硼氫化鈉0.2g及0.2M磷酸氫二鉀水溶液12mL饋入可分離燒瓶中,在60℃攪拌20小時,作為使
醛糖鍵結於氮原子之胺基製作糖分析用及親水性相互作用液體層析儀用填充劑。該填充劑間隔著以水洗淨以1.0N硫酸及1.0N氫氧化鈉水溶液洗淨後,乾燥。
所得具有胺基之聚合物之胺基密度與實施例1同樣測定之結果為0.26meq/g。
實施例1中替代表氯醇45g而使用1,4-丁二醇二縮水甘油醚100g以外,以同樣條件製作液體層析儀用填充劑。與實施例1同樣測定該填充劑之胺基密度之結果為0.17meq/g。
將實施例1所得之填充劑填充於內徑4.6mm、長150mm之SUS製管柱中,調整糖分析及親水性相互作用層析儀用管柱。使用該管柱,設為溶離液乙腈/水=85/15、流速0.6mL/min、管柱溫度40℃進行分析。分析樣品使用將果糖、甘露糖、葡萄糖及蔗糖之4種糖,均以成為5mg/mL之方式溶解於乙腈/水=5/3之混合液者。使用示差折射率檢測器所得之層析圖示於圖1。4種糖良好地被分離。
又,進行上述管柱之安定性試驗。以0.05N氫氧化鈉
水溶液、流速0.6mL/min、管柱溫度30℃連續通入溶離液80小時,確認液體通入前後之糖類保持時間及理論段數。由此獲得之結果示於圖2。使用本發明之填充劑的管柱即使連續通入溶離液亦不會引起保持時間等之變化。
使用實施例2所得之填充劑,進行與分析例1同樣分析之結果示於圖3。4種糖良好地分離。
使用實施例3所得之填充劑,進行與分析例1同樣分析之結果示於圖4。4種糖良好地分離。
利用使用以矽膠作為基材之導入胺基之填充劑的管柱進行上述安定性試驗。其結果自液體開始通入17小時後,管柱發生堵塞而無法通入液體。
將使甲基丙烯酸縮水甘油酯聚合之丙烯酸酯樹脂2.8g、N-甲基-D-還原葡糖胺6.5g及水28mL饋入可分離燒瓶中,在30℃攪拌20小時,製作以丙烯酸酯樹脂為基材之填充劑。過濾該聚合物後,間隔著水洗淨以0.5N鹽酸及0.5N氫氧化鈉水溶液洗淨。
將比較例2所得之填充劑與分析例1同樣填充於管柱,進行糖類分析之結果示於圖5。可知保持較短,糖類無法分離。
將實施例1所得之填充劑填充於內徑4.6mm、長250mm之SUS製管柱中,調整糖分析及親水性相互作用層析儀用管柱。使用該管柱,進行果糖、半乳糖、乳果糖、表乳糖、乳糖之5種之糖類分析。分析條件係設為流速1.0mL/min,管柱溫度40℃。檢測器使用示差折射率檢測器。溶離液使用乙腈/水=80/20時之層析圖示於圖6。
分析例6的圖6中乳果糖與表乳糖之峰係一部分重疊。因此,使用相同管柱並將溶離液變更為乙腈/甲醇/水=75/20/5分析同樣樣品時之層析圖示於圖7。圖6中峰為重疊之乳果糖與表乳糖之峰係完全分離。因此,5種糖良好地分離。
Claims (11)
- 一種液相層析儀用填充劑,其特徵係經由間隔基使以下述式(1)表示之胺基鍵結於含有聚乙烯醇樹脂之親水性樹脂上,
- 如請求項1之液相層析儀用填充劑,其中R1為氫原子或甲基。
- 如請求項1或2之液相層析儀用填充劑,其中R2係以式(2)表示之構造,
- 如請求項1之液相層析儀用填充劑,其中以式 (1)表示之胺基係源自由D-還原葡糖胺、N-甲基-D-還原葡糖胺、1-胺基-1-去氧-D-甘露糖醇、1-胺基-1-去氧-D-半乳糖醇(galactitol)、1-胺基-1-去氧-D-艾杜醇(Iditol)、1-胺基-1-去氧-D-阿糖醇(arabinitol)、1-胺基-1-去氧-D-木糖醇、4-胺基-1,2,3-丁三醇、3-胺基-1,2-丙二醇、3-甲基胺基-1,2-丙二醇所組成之群中之任一種胺。
- 如請求項1之液相層析儀用填充劑,其中間隔基係源自末端具有縮水甘油基之化合物。
- 如請求項1之液相層析儀用填充劑,其中含有聚乙烯醇樹脂之親水性樹脂,相對於樹脂量,含有10質量%以下之其他樹脂。
- 如請求項6之液相層析儀用填充劑,其中相對於樹脂重量,胺基之量為0.1~0.7meq/g。
- 一種液相層析儀用管柱,其係使用如請求項1~7中任一項之液相層析儀用填充劑。
- 一種糖分析用液相層析儀用管柱,其係使用如請求項1~7中任一項之液相層析儀用填充劑。
- 一種親水性相互作用層析儀用管柱,其係使用如請求項1~7中任一項之液相層析儀用填充劑。
- 一種糖分析方法,其係使用如請求項1~7中任一項之液相層析儀用填充劑。
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