North Robert, Hwang Ann, Lalwani Sanjiv, Shave Evan, Vigh Gyula
Department of Chemistry, Texas A&M University, College Station, 77842-3012, USA.
J Chromatogr A. 2006 Oct 20;1130(2):232-7. doi: 10.1016/j.chroma.2006.03.054. Epub 2006 Apr 19.
Success in isoelectric trapping separations critically depends on the knowledge of the accurate operational pH value of the buffering membranes used. Currently, due to a lack of easy, rapid, accurate methods that can be used for the post-synthesis determination of the operational pH value of a buffering membrane, only nominal pH values calculated from the amounts of the reagents used in the synthesis of the membranes and their acid-base dissociation constants are available. To rectify this problem, UV-absorbing and fluorescent carrier ampholyte mixtures were prepared by alkylating pentaethylenehexamine with a chromophore and a fluorophore, followed by Michael addition of acrylic acid and itaconic acid to the resulting oligoamine. Carrier ampholyte mixtures, with evenly distributed absorbance values across the 3<pI<10 range, were prepared by blending. The master blend served as the feed mixture in binary isoelectric trapping separations that used the buffering membrane to be characterized. The pI value of the most basic UV-absorbing or fluorescent carrier ampholyte collected in the anodic separation compartment, determined by full-column imaging capillary isoelectric focusing analysis, indicates the operational pH value of the separation membrane.
等电聚焦分离的成功关键取决于对所用缓冲膜准确操作pH值的了解。目前，由于缺乏可用于合成后测定缓冲膜操作pH值的简便、快速、准确的方法，仅有根据膜合成中所用试剂的量及其酸碱解离常数计算出的名义pH值。为纠正这一问题，通过用发色团和荧光团对五乙烯六胺进行烷基化，然后将丙烯酸和衣康酸迈克尔加成到所得低聚胺上，制备了紫外线吸收和荧光载体两性电解质混合物。通过混合制备了在3＜pI＜10范围内吸光度值均匀分布的载体两性电解质混合物。主混合物用作二元等电聚焦分离的进料混合物，该分离使用待表征的缓冲膜。通过全柱成像毛细管等电聚焦分析确定在阳极分离室中收集的最碱性紫外线吸收或荧光载体两性电解质的pI值，该值表明分离膜的操作pH值。
