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

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 Escherichiacoli 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 ( approximately 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.