Henchoz Yveline, Schappler Julie, Geiser Laurent, Prat Josiane, Carrupt Pierre-Alain, Veuthey Jean-Luc
Laboratoire de Chimie Analytique Pharmaceutique, Section des sciences pharmaceutiques, Université de Genève, Université de Lausanne, Bd d'Yvoy 20, 1211 Geneva 4, Switzerland.
Anal Bioanal Chem. 2007 Nov;389(6):1869-78. doi: 10.1007/s00216-007-1568-5. Epub 2007 Sep 14.
A rapid and universal capillary zone electrophoresis (CZE) method was developed to determine the dissociation constants (pK (a)) of the 20 standard proteogenic amino acids. Since some amino acids are poorly detected by UV, capacitively coupled contactless conductivity detection (C(4)D) was used as an additional detection mode. The C(4)D coupling proved to be very successful on a conventional CE-UV instrument, neither inducing supplementary analyses nor instrument modification. In order to reduce the analysis time for pK (a) determination, two strategies were applied: (i) a short-end injection to reduce the effective length, and (ii) a dynamic coating procedure to generate a large electroosmotic flow (EOF), even at pH values as low as 1.5. As a result, the analysis time per amino acid was less than 2 h, using 22 optimized buffers covering a pH range from 1.5 to 12.0 at a constant ionic strength of 50 mM. pK (a) values were calculated using an appropriate mathematical model describing the relationship between effective mobility and pH. The obtained pK (a) values were in accordance with the literature.
开发了一种快速通用的毛细管区带电泳（CZE）方法来测定20种标准蛋白质氨基酸的解离常数（pK(a)）。由于某些氨基酸在紫外线下检测效果不佳，因此采用电容耦合非接触式电导检测（C(4)D）作为额外的检测模式。事实证明，在传统的CE-UV仪器上，C(4)D耦合非常成功，既无需进行额外分析，也无需对仪器进行改装。为了缩短pK(a)测定的分析时间，采用了两种策略：（i）短端进样以缩短有效长度，（ii）动态涂层程序以产生大的电渗流（EOF），即使在低至1.5的pH值下也是如此。结果，在50 mM的恒定离子强度下，使用22种优化缓冲液，其pH范围为1.5至12.0，每种氨基酸的分析时间不到2小时。使用描述有效迁移率与pH值之间关系的适当数学模型计算pK(a)值。获得的pK(a)值与文献一致。
