FW: Mercury Orange - The synthesis and beyond

From:"Monson, Frederick C."

Prequaled Apologetic Afterthought.  Having finished this transcription, I am
bound to warn those who read further that my attempt to be
helpful/informative has turned into an 'humoresque', because of its
accuracy, and its historic history - as Ted would say to Bill.  
Original Beginning!

To amplify Mike's response, here is what Gurr(1959) had to offer. 
	"This is somewhat troublesome to prepare;...."  [What follows is
slightly modified for brevity and technology.  I am to be found in brackets
along the way.]
		[IN A FUME HOOD!!!!!!  Mercury is TOXIC!!!!]
		1.  Weigh out and, then dissolve 50g mercuric acetate in 250
ml HOH.
		2.  Add 29 ml pure aniline oil to the above, stir well [for
10 min]; then cover loosely [to exclude dust!] and stand for 4-16 hr.
		3.  Decant bulk of supernate and filter the remainder
through a buchner [Buchner!] funnel.  Wash the primrose-colored crystals of
p-aminophenyl mercuric acetate (PAMA) well with water.
		4.  Dry recovered crystals of PAMA at ~50oC.
		5.  Weigh 55.5g of PAMA crystals and stir into 800 ml 50%
acetic acid [in a 4L Ehrlenmyer flask].
		6.  Ice the flask while continuously stirring [?] so that
the solution is between 0-10oC.
		7.  Dissolved 11.5g sodium nitrite in a liter of water.
		8.  With continuous stirring and maintaining the temperature
below 10oC, add the sodium nitrite solution a "little at a time" to
diazotize the PAMA [THIS is where the Ehrlenmyer comes in handy - to prevent
spills which will leave spots of dry diazonium on the work area surface -
see below!].
		9.  After the sodium nitrite has been added and while
keeping the temperature below 10oC stop stirring and let stand for about 20
min.  [EXPLOSIVE HAZARD!!!!] [The dry diazonium compound IS violently
explosive, so in this step, the product must be kept DISSOLVED/WET.]
		10.  The volume is now 1.8 L.  It will increase soon to 4.8
		11.  Dissolve 289 g NaOH in about 3 L water, add, with
shaking, 23.8 g beta-naphthol (or alpha- (apparently recommended by Gomori,
because the resultant product is 2x more soluble than that made with the
beta-naphthol).  Cool the solution to 0-10oC.
		12.  With continuous stirring, add the solution made in (11)
above to that prepared in (8) and (9). [IF diazonium (from (9) above) has
been completely consumed in this reaction, just performed, the EXPLOSION
Hazard has dissipated.  From here on, we're back to the only hazard
remaining - mercury TOXICITY!!!!!] 
		13.  Leave solution to stand overnight [covered to exclude
dust AND evaporation!!!!!]
		14.  Filter and wash precipitate well with water and, then,
drain well.
		15.  Dissolve precipitate in 4.5 L  absolute alcohol by
boiling under a reflux condenser [a 5 L 24/40 round bottom???  and a heating
mantle that fits???][My organic NEVER when to such a place!].  [Since the
solubility of the beta-naphthol derivative is said to be 0.05% [50 mg/100
ml], it is clear that Professor Gurr recovered less than 2.5 g of product,
sufficient to make around 100 Coplin jars of 'stain' if dissolved in
absolute ethanol.] 
		16.  Filter the above while hot [still 4.5 L!] and add 9.1 g
NaCl dissolved in 235 ml of 60% ethanol [now 4.735 L][It is apparent that a
little water causes the precipitate to form.].
		17.  Boil under reflux for 30 min.
		18.  Filter while hot and wash the preciptated Red
Sulphydryl Reagent [RSR!!!!!][Finally!]three or four times with hot 50%
		19.  Dissolve the dried RSR in normal butyl alcohol and
recrystallize it 3X from that solvent.  [Using water, I presume!]

		1.  Do NOT use the aspirator attached to the water faucet in
the fume hood to filter these materials, unless all filtrates are passed
directly to retention containers.
		2.  Alternatively, and depending on the need for Mercury
Orange, one might double the recipe and have sufficient left over to sell by
the Coplin jar lot.  Thus, one might recover disposal costs and
Administrative aggravation/overload, even for 40L [ONLY 10 gallons!]of
		3.  I have also, and still do, strongly recommend Prof.
Kiernan's book, though I cannot remember seeing the directions for synthesis
of RSR in it.  I cannot imagine why [though see below for a hint].  Perhaps
he also has the book by Professor Gurr.

		Early in my teaching career [early 1970's), having been
saddled with a histochemistry class of seniors all of whom had taken, and
presumably passed, organic chemistry, I prepared Stoward's Tetrazotized
3,3'-dimethoxybenzidine fluoroborate (TDMBF) for what I knew would be a real
bio-chemical 'trip' into carbohydrate histochemistry.  In this prep one only
has to worry about 'small' amounts of fluoroboric [also, fluoboric!] acid,
and some ether for washing.  The real upside was to come when I introduced
all of the Stoward and Mester blockades.  Remembering my great experiences
in organic, I expected anything but the bewildered class that confronted me
when I had the temerity to speak of 'real' chemistry to a class of biology
majors - many of whom were just waiting to graduate and go on to, 'you know
where'.  Of course, soon after, I heard a well-known professor of
Pharmacology relate that he/his group had been forced to introduce some
rudimentary organic chemistry into recent classes of 'you-know-what'
students, because so few of those who came to them knew enough to understand
the nomenclature of the common pharmacologic compounds that were discussed
in their elementary Pharm course.

Cheers to all who were learned to think instead of merely 'to pass' - - -
the Boards, though that can't be entirely without benefit,

Fred Monson

P.S.  The further fatiguing thought arises that if one could use four
brackets at a time rather than only three, they would be called fences or in
sum, a corral or pen.

Frederick C. Monson, PhD
Center for Advanced Scientific Imaging
Mail Drop:  Geology
West Chester University
West Chester, PA, 19383
Phone/FAX:  610-738-0437   

-----Original Message-----
From: MTitford@aol.com [mailto:MTitford@aol.com]
Sent: Thursday, June 12, 2003 2:49 PM
To: Histonet@pathology.swmed.edu
Subject: Mercury Orange

Jenny Molde in South Africa asks about mercury orange.
I am suprised that Dr Kiernan did not quickly answer with one of his "long
answers"! He must be on vacation. His book, co-authored with Dr Horobin
(Conn's Biological Stains" 10th edition Horobin, R.W. and Kiernan J.A.
Editors 2002 Bios) sayeth:
"Mercury orange: synonyms Bennett's sulfhydral reagent,
p-chloromercuriphenyl-azo-alpha napthol. Red sulfhydril reagent....Mercury
orange was introduced by Bennett in 1951 for the demonstration of cysteine,
glutatione and free thiol groupings in tissue..... Other uses are in flow
cytometry" The book says more, too much to write here.
I recommend this book for any histology lab doing different research
projects in histology. It brings up to date information in previous editions
edited by Dr Lillie.
You should buy it!

Mike Titford
USA Pathology
Mobile AL USA

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