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Solid‐phase fluorescent labeling reaction of picomole amounts of insulin in very dilute solutions and their analysis by capillary electrophoresis
Author(s) -
Pinto Devanand M.,
Arriaga Edgar A.,
Sia Samuel,
Li Zhong,
Dovichi Norman J.
Publication year - 1995
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150160188
Subject(s) - chromatography , chemistry , reagent , capillary electrophoresis , fluorescence , detection limit , micellar electrokinetic chromatography , insulin , electropherogram , phase (matter) , membrane , capillary action , biochemistry , organic chemistry , materials science , medicine , physics , quantum mechanics , composite material , endocrinology
The fluorescent labeling of peptides at concentrations as low as 10 −8 M can be achieved by using a solid‐phase reactor. Using oxidized insulin chain B as a test peptide, we demonstrate the use of an Immobilon CD membrane to capture and preconcentrate peptides. Insulin chain B can then be labeled with a fluorogenic reagent, 3‐(2‐furoyl)quinoline‐2‐carboxaldehyde, while it is still attached to the membrane. Unwanted fluorescent products (attributed to secondary reactions) can be washed away with methanol without significant removal of the labeled insulin chain B, which then can be extracted with a low pH buffer. The analysis by micellar electrokinetic capillary chromatography with post‐column laser‐induced fluorescence detection (mass limit of detection of 2.4 × 10 −21 moles insulin chain B) results in electropherograms that show great improvement in terms of unwanted peaks and high number of theoretical plates (up to 20 million). The use of the solid‐phase reactor allows easy handling of as little as 5 picomoles of insulin chain B.