How do you measure coupling efficiency?
The trityl-group is colorless when attached to a DNA base but it gives a characteristic orange color once removed. The intensity of this color can be measured by UV spectrophotometry and it is directly related to the number of trityl molecules present. Following the first coupling step, the amount of trityl released during deblocking is directly proportional to the amount of full-length oligo synthesized in the previous cycle. When the trityl is cleaved during the deblocking step, the resulting trityl cation is orange in color. The intensity of this color can be measure by UV spectrophotometry. By comparing the intensities of the trityl cation produced after the first and last coupling steps, one can calculate the average successful base coupling per cycle and hence the coupling efficiency.