Question

The task is to detect hydrogen cyanide in tree leaves.

Background: The leaves of the wild black cherry tree are not toxic when fresh. However, it is well-known that when the leaves wilt, a decomposition takes place which produces HCN.  The wilted leaves are both appetizing and lethal to cattle. The object of my project is to find out how long  it takes, after a tree falls, for the cyanide to form, and also how long it persists in the leaves. I reckon  that if enough HCN is present to kill a cow, the relatively simple Prussian Blue test should suffice to  detect and roughly estimate it. The problem is how to get the HCN out of the leaves and into a test tube. The only extraction process I have much  experience with is the brewing of coffee! How should I proceed?

Answer

The extraction process here might be a pain in the butt.  The cyanide is already there from the moment the leaf dies.  It is however all tied up within the sugars of the leaf as glycosides.  I don't know what specific glycoside is present but it breaks down into cyanide.  Plants store these glycosides to make the components that make them up readily available.  They break them down with enzymes when they need the sugars or whatever else they contain.  So the enzymes to break down the glycoside are present in the leaf already too.  Both of those are going to pose problems for extraction because during extraction you can accidentally hydrolyze the glycosidic bonds giving you a picture of the total cyanide and not the free cyanide which would be available to kill the cows.  The enzymes present can do the same thing. 

Because the fresh leaves are not killing the cows (They are not, correct?) then the cows must lack the enzyme to convert the glycoside to its component parts.  Regrettably, I no longer have my book on toxicology otherwise I could give more relevant information. 

Glycosides cleave most readily in acidic environments or in the presence of their enzyme.  The second of the two adds another dimension of difficulty.  The cows chew up the leaves, this breaks the cell walls and can allow some of this commingling of enzyme and substrate to occur, like when you crush a clove of garlic to release the aroma.  So if you stick the leaves in a blender and hit puree you will likely release more titratable cyanide than if you ground them in a mortar and pestle.

Taking these things into account here are some thoughts on how to proceed.  Make a dilute caustic solution of sodium hydroxide and water.  Ensure that the pH is distinctly basic (>12).  Grind some of the leaves from the black cherry tree and grind them in a mortar and pestle.  Add these to your dilute solution.  Stir and allow to extract for at least an hour.  Filter.  Since it is the free cyanide you are looking for, any HCN present will react readily with the sodium hydroxide, and since sodium cyanide is very soluble (and HCN is soluble to begin with) you should get a determination of the free cyanide in the leaves.  The hope here is that the glycoside is not soluble in the water (Which it very well might be since I couldn't find too much information on them).  From here take your extract, add in your Fe²⁺ source (like the sulfate) and begin slowly acidifying with HCl.  The blue color should begin to appear if cyanide was present.  By this method you would avoid conditions that could lead to a release of hydrogen cyanide before determination by keeping the solution basic until the ferrous ion was around to scavenge the hydrogen cyanide.  There are some colorimetric methods available that would work better than the Prussian blue test, giving more quantitative results, but this should work for a general determination.

Again, let me reiterate, if the glycoside that is present there from the beginning breaks down during the extraction conditions, or if it is soluble in water where it would carry through to the acidification step, it will affect the results and the freshest leaves will show the highest concentration of cyanide.  Because of this, attempting to extract out the glycoside beforehand using some other solvent came to mind, but I found limited solubility data on hydrogen cyanide and being that only a small amount of it is present, you could just as easily extract it away and skew your results in that manner.

Hopefully these ideas will lead you down the right road.  Take care when collecting to many of the wilted leaves though.  There is a famous story of a researcher who was doing work with  a particular breed of caterpillars and was keeping a cache of them in his cooler in the lab.  When irritated they release HCN, and as luck would have it he had enough of them in a canister that when he opened it he was overcome by the vapors and was struck unconscious.  I could see the same thing happening, especially if your pulped leaf waste from your reactions were allowed to accumulate and sit around sealed for some time.  Take care and good luck.