In play, it's distinguishable from iron, of course (4)
I believe the answer is:
wood
'in' is the definition.
The answer and definition can be both substances as well as being singular nouns.
Perhaps you can see a link between them that I can't see?
'play it's distinguishable from iron of course' is the wordplay.
I cannot really see how this works, but
'from' could be 'o' (I've seen this in other clues) and 'o' is found in the answer.
'of' could be 'o' and 'o' is located in the answer.
'course' could be 'w' and 'w' is located in the answer.
A single letter 'd' remains which might be clued in a way I don't understand.
This may be the basis of the clue (or it may be nonsense).
Can you help me to learn more?
(Other definitions for wood that I've seen before include "Large group of trees" , "'Under Milk - - ' (lit.)" , "Revered Proms character" , "Dense growth of trees" , "Golf club - tree material" .)