Capillary electrophoretic analysis of carbohydrates derivatized by in-capillary condensation with 1-phenyl-3-methyl-5-pyrazolone

Our previous papers on capillary electrophoresis (CE) have shown that sampl es can be derivatized in a capillary and the derivatives can be analyzed im mediately after derivatization, provided that the derivatization reaction i s so rapid as to complete in seconds, The present paper presents extended a pplication of in-capillary derivatization to a much slower reaction such as the condensation of reducing carbohydrates with 1-phenyl-3-methyl 5-pyrazo lone (PMP) which requires 30 min at 70 degreesC in pre-column derivatizatio n by manual operation. It was necessary to first drive the introduced plugs of sample and reagent solutions to put them together at the entrance of th e heated portion of a capillary, then to allow the superimposed plugs to re act for a relevant period, We showed how to determine the introduction time s of the sample and the reagent solutions as well as intermediate running b uffer, the voltages to be applied for plug driving and product analysis, an d the duration of voltage application, all of which are important for effec tive in-capillary derivatization. An example of the analysis of maltooligos accharides by this technique is presented, It was shown that maltooligosacc harides were quantitatively derivatized with PMP in 35 min at 57 degreesC, and the derivatives could be analyzed in ca. 15 min by CE immediately after derivatization. Separation was satisfactory in 200 mM berate buffer, pH 8. 2 containing sodium dodecyl sulfate to a concentration of 200 mM. Although the theoretical plate number, and accordingly the resolution, were signific antly lower than the corresponding values in pre-capillary derivatization, reasonable reproducibility was ensured for both migration time (RSD 3.5% on average) and peak area (RSD less than 3%) under the optimized conditions. It is notable that sample amount could be lowered to the 10 fmol level, in contrast to the 10 pmol level in pre-capillary derivatization. In addition, since the technique employed here (the modified at-inlet technique of in-c apillary derivatization) is easily automated, the established system will b e highly beneficial for routine analysis of carbohydrates, Analysis by this technique was also shown to be useful for kinetic study of the derivatizat ion reaction. (C) 2001 Elsevier Science B.V. All rights reserved.