I'm giving two answer because I'm unclear as to what you mean.
For Example: if you meant (16a)² minus 4b then my answer would be:
#1. When a=1/4 and b=6 (16a)² minus 4b = (16 times 1/4)² minus 24.
Now, (16 and 1/4)² cancel to 4² which equals 16. 16 minus 24 = -8.
#2. If you meant 16 times (1/4)² minus 24 then that changes everything! :)
then it would be: 16 times 1/16 minus 24 = 1 minus 24 = -23.
So either -8 or -23 is my answer.
sorry if it's confusing. I'm new at this. And I hope I didn't make any mistakes.
