A new method for the preparation of polyacrylamide gradient gel-filled capillaries with low UV detection background

摘要

A simple but reproducible method has been explored for the preparation of polyacrylamide gradient gel-filled capillaries with low UV detection background. The principle is to fill a capillary by plugging it into a wide tube already filled with gradient gelling solutions including a section of buffer. Void-free capillaries can be prepared with a full success for gels below 13%T + 5%C (immobilised completely). For gels between 15%T and 25%T + 5%C, the preparation success rate is 80-95%, depending on the gel immobilisation methods. The resulting capillaries allow the use of any available wave-length for sensitive detection and in the separation of polylysines, a great improvement of their detection sensitivity has been achieved, up to 10~3-10~4 fold as compared to the common gel-filled capillaries. Unlabelled polysaccharides from the sacculi of Escherichia coli can hence directly be detected at 200 nm. These capillaries can continuously be used for more than 2 months in separating the biological polysaccharides at - 200 V cm~(-1) and pH 7.8 while at pH 4.7, they can still be used for more than 400 injections (～ 1 month) of the polylysines at + 180 V cm~(-1). As expected by stacking effect, the gradient gels yield higher efficiency or running speed (1-fold) and even higher UV detection sensitivity (＞ 2-fold) than the homogeneous gels.