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A previous cLC method, involving large injection volumes (20 μL) and on-column focusing techniques, provided a suitable sensitivity using an Inertsil? C8 capillary column, DAD and gradient elution. This method showed that composition of the focusing solution, mobile phase and pH have remarkable effects on HA separation. In order to improve this separation, an isocratic cLC method compatible with MS detection was developed and the influence of several chromatographic factors on separation quality evaluated. Different reversed-phase packings such as Luna? C8, Luna? C18, Synergi? Max-RP (C12) and Synergi Fusion (C18 and polar embedded group) were tested using UV-DAD detection. These columns were chosen due to their high efficiencies and bonded phase surface coverage. Considering the different particle sizes, flow rate was set at 15 μL min?1 for both Synergi Fusion? and Synergi? Max-RP, 12 and 9 μL min?1 for Luna? C8 and Luna? C18 columns respectively. Chromatographic conditions were optimized using experimental design methodologies such as central composite design. Factors and ranges selected for the optimization of mobile phase composition were ACN (11.6–28.4%), ammonium acetate concentration (13.2–46.8 mM) and buffer pH (3.6–4.4). Response variables were expressed in terms of resolution, calculated at baseline, between the worst resolved peak pair NH–H (Rs,min), and retention time of the last eluting peak (tend). From the obtained equations, it could be concluded that Rs,min was significantly affected by both ACN percentage and pH while tend only by ACN. In addition, the ammonium acetate concentration had no significant effect on separation. Chromatographic separation efficiency was optimized for Rs,min ≥ 2 and minimum tend value. Luna? C8 capillary column provided poor resolution. Luna? C18 and Synergi? Max-RP columns provided similar Rs,min to Synergi? Fusion but higher efficiencies. However, Synergi? Max-RP column provided poor peak symmetry. Therefore, Luna? C18 column was selected for further optimization with the quadrupole MS detector. Taking into account the instrumental limitations of the MS capillary nebulizer, the ammonium acetate concentration in the mobile phase was fixed to 5 mM. Chromatographic separation was optimized using multifactorial design. Factors and ranges selected were ACN (12–18%) and pH (3.6–4.1). For the injection, buffered focusing solutions with 5% MeOH were used. Table 1 includes the values of the experimental responses (Rs,min and tend), which were fitted into the following normalized polynomial equations: (1)Rs,min=1.74?0.51pH?1.35ACN?0.06pH2+0.39pH ACN+0.40ACN2 (2)tend=12.18+0.18pH?11.02ACN+0.76pH2?0.94pH ACN+4.83 ACN2 Determination coefficients were 0.988 and 0.992 for Rs,min and tend respectively, showing the reliability of the equations. In the studied domain, both ACN percentage and pH affected significantly to Rs,min (p values 0.0000 and 0.0002 respectively) (Eq. (1)) while ACN (%) affected only tend (p value 0.0000 and 0.0005 for the single and quadratic term respectively) (Eq. (2)). As can be expected, the interactions between factors (ACN-pH) were significant only for Rs,min response (p = 0.0033). In Fig. 1 it can be observed that the ACN percentage decrease in the mobile phase increases Rs,min and tend. However, when pH decreases, Rs,min increases and tend does not change significantly. The chromatographic separation efficiency was optimized by maximizing Rs,min and minimizing tend values which maximized the desirability function over the selected region. The maximum desirability function yielded a tend = 13.6 min and Rs,min = 2.2 at pH 3.6 and 14.5% ACN. The estimated response surfaces predicted that Rs,min > 2 and tend in the range 12.5–15.0 min could be obtained at pH 3.6–3.7 and 13–15% ACN. As a compromise, pH 3.6 and 13% ACN were selected as optimum values. Under these conditions tend = 15.0 min and Rs,min = 2.3 were expected and experimentally assessed. Finally, ammonium formate 5 mM was also tested and selected to avoid problems into the capillary MS nebulizer. |
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ringzhu 
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ringzhu(金幣-2):本版嚴禁機器翻譯。。。。 2010-12-08 13:07:33
zxc578642(金幣+35, 翻譯EPI+1): 2010-12-11 00:08:34
ringzhu(金幣-2):本版嚴禁機器翻譯。。。。 2010-12-08 13:07:33
zxc578642(金幣+35, 翻譯EPI+1): 2010-12-11 00:08:34
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先前的分類號方法，涉及到大注射量（20μL）和柱上聚焦技術(shù)，提供了合適的靈敏度使用Inertsil？ C8的毛細管柱，爸爸和梯度洗脫。這種方法表明，聚焦解決方案，流動相組成和pH值對醫(yī)管局分離成效顯著。為了改善這種分離，一等度分類號兼容MS檢測法的開發(fā)和幾種因素對色譜分離質(zhì)量的影響進行評估。 不同的反相填料，如Luna？ C8的，Luna？ C18時，Synergi？馬克斯反相（十二）和Synergi融合（C18和極性嵌入組）進行了測試使用紫外二極管陣列檢測。這些列被選為由于其高效率和鍵合相表面覆蓋�？紤]到不同顆粒大小，流量率定為15微升分鐘？Synergi融合為1？和Synergi？馬克斯余段，12分鐘和第9微升？負責盧納1？ C8和盧娜？ C18柱分別。 色譜條件進行了優(yōu)化，采用中心組合設(shè)計，如實驗設(shè)計方法。因素和流動相組成的優(yōu)化選擇范圍為乙腈（11.6-28.4％），醋酸銨濃度（13.2-46.8毫米）和緩沖液pH值（3.6-4.4）。響應(yīng)變量中表達了在基準計算的決議而言，最糟糕的解決高峰之間的配對的NH -的H（盧比，分鐘），并保留最后洗脫峰（傾向）的時間。從得到的方程，可以得出結(jié)論，盧比，最小顯著比例都乙腈和pH的影響，而往往只乙腈。此外，醋酸銨濃度無顯著影響的分離。 色譜分離效率進行優(yōu)化盧比，≥2分和最低傾向于價值。盧納？ C8的毛細管柱提供的分辨率較低。盧納？ C18和Synergi？馬克斯反相柱提供了類似的盧比，分鐘，以Synergi？融合，但更高的效率。然而，Synergi？馬克斯窮人提供的反相柱峰的對稱性。因此，盧娜？ C18柱被選定為四極桿質(zhì)譜檢測器的進一步優(yōu)化。 考慮到MS的毛細管霧化器的工具的限制，在流動相中醋酸銨濃度為5毫米。色譜分離采用多因素進行了優(yōu)化設(shè)計。被選定的因素和范圍乙腈（12-18％）和pH值（3.6-4.1）。對于注射液，緩沖聚焦5％甲醇被用來解決方案。 表1包括對實驗的反應(yīng)值（盧比，min和靠攏），分為以下規(guī)范化多項式擬合方程： （1）盧比，最小= 1.74？0.51pH？1.35ACN？0.06pH2 0.39 pH值乙腈+0.40 ACN2 （2）= 12.18 +0.18 pH值趨向？11.02ACN 0.76 pH值為2？0.94pH乙腈4.83 ACN2 決定系數(shù)分別為盧比，最小為0.988和0.992，往往分別顯示了該方程的可靠性。在所研究的領(lǐng)域，都乙腈率和pH值的影響顯著盧比，分（P值分別為0.0000和0.0002）（式（1）），而乙腈（％）僅影響往往（P值0.0000和0.0005為單和二次任期分別）（式（2））。正如可以預(yù)料，因素之間（乙腈- pH值）有顯著的相互作用只有盧比，最小反應(yīng)（P = 0.0033）。在圖。 1可以觀察到，在增加流動相乙腈比例下降盧比，min和趨向。然而，當pH值降低，盧比，最小升幅，而且往往沒有明顯變化。 色譜分離效率的最大化盧比，min和價值觀趨于優(yōu)化，最大限度地減少對選定的區(qū)域最大化的可取性功能。最大的可取性功能產(chǎn)生了往往和Rs = 13.6分，3.6分，在pH = 2.2和14.5％乙腈。估計響應(yīng)面預(yù)測，遙感，分“2和范圍12.5-15.0分鐘往往可以在pH值3.6-3.7和13-15％乙腈獲得。作為一種妥協(xié)，pH值3.6和13％乙腈被選定為最佳值。在這種情況往往和Rs = 15.0分鐘，2.3分=預(yù)期和實驗評估。最后，甲酸銨5毫米進行了試驗，并選擇了避免陷入毛細管質(zhì)譜霧化器的問題。 |
8樓2010-12-08 10:42:12
9樓2010-12-14 21:32:45
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