With 13:10 in A1 and 16:25 in B1 then in C1
will give you the difference (3:15). You need to format C1 as Time if Excel does not do this automatically.
The problem that you will have is correctly displaying the sum of these if the total exceeds 24 hours. To display correctly, click in the totals cell, Format/Cells. In the left hand pane click custom and for the custom format enter