Me and my wife are separated and I would like to ask if a will can nullify my wifes ability to receive spousal entitlement?

No.  If you want to prevent your wife from receiving any of your property after your death, it appears that you must go through with a divorce, if my understanding of your state's law is correct (I'm not a New York attorney).  The law in New York, on the rights of a surviving spouse, is fairly complex.  I'd suggest a consultation with a lawyer in your area, for reliable advice about your situation and your options, based on a detailed review of the facts.