The hemiketal, when applied to paper, will quickly react to the hydrate (the common form of a "ninhydrin")
due to water present in the paper and in the atmosphere. The reaction between the amino acids in the
fingerprint is therefore not adversely affected by using the hemiketal instead
of the ninhydrin.
The 5-MTN working solution we recommend at this time, has the following composition:
3.4 grams 5-MTN hemiketal (equivalent to 3 grams 5-MTN)
10 mL acetic acid (99-100%)
25 mL isopropanol
145 mL ethyl acetate
100 mL MTBE (methyl-tert-butylether)
720 mL petroleum ether (40-60° or 60-80°)
1000 mL working solution
This working solution is stable for extended periods at room temperature. When the solution is kept at low temperatures (for example during shipping in winter time) some 5-MTN might partially crystallize from the solution. After warming to room temperature and stirring or shaking however, the 5-MTN will gradually dissolve again.
When preparing the working solution from 5-MTN hemiketal, first dissolve the crystals in the mixture of acetic acid, isopropanol, and ethyl acetate (10-15 minutes with stirring) to prepare a concentrated solution. After it is fully dissolved prepare the working solution by adding the methyl-tert-butylether (MTBE) and petroleum ether.
Treatment with 5-MTN
The use of 5-MTN for development of fingerprints is identical to the procedure for ordinary ninhydrin. The papers that need to be treated are dipped in the working solution and the solvents allowed to evaporate. This procedure should be done in a fume hood, to avoid breathing the fumes and the possible formation of an explosive solvent/air mixture in the room.
For the treatment of paper, BVDA has a practical developing tray in the program. With this tray, small amounts of working solutions can be used, preventing unwanted evaporation to a minimum.
After evaporation of the solvents contained in the paper, the fingerprints start to develop. The speed of the development is similar to that of ninhydrin. The ideal conditions for the development of ninhydrin treated paper should be used:
- at room temperature
- in the dark
- at 80% relative humidity 
After one to two days at room temperature, the development
of the fingerprints is largely complete. The development will still proceed slowly
after this period (for about two weeks, see references 7 and 8 for regular ninhydrin).
Because the development of fingerprints stops after treatment with zinc chloride, it should not be done too soon. Wait at least two days, after treatment of the papers with 5-MTN working solution, before proceeding with zinc chloride treatment.
Treatment with zinc chloride
Zinc chloride treatment of prints on paper, developed with
5-MTN, is a very simple procedure. BVDA has developed a zinc chloride solution, on a MTBE/petroleum ether basis. This contains a high concentration of zinc chloride (30 grams/liter), so that only small amounts are needed.
It is possible to dip the paper in the zinc chloride solution.
However, it has the potential to run inks and the concentration of zinc in the
solution is higher than needed for dipping.
We recommend that a sprayer should be used in a fume hood
to apply the zinc chloride. Spray the solution on the article only lightly and
allow the solvents to evaporate. For the formation of the fluorescing complex
a small amount of water is needed. Usually, enough water is present in the air
for this to happen. The complexation will normally take only a few minutes. Humidifying
a print (by breathing on it) speeds up the process. Only if the color change is
not complete, spray again.
By spraying the paper with zinc chloride solution, accurate
control over the dosing is obtained. Also running of ballpoint inks can be prevented
best this way.
Fluorescence after zinc chloride treatment
Prints developed with regular ninhydrin can be made fluorescent
by treating them with zinc chloride. However, it is an unreliable procedure that
has shown sensitive to the conditions used for developing the prints and other
unknown factors. 5-MTN is much more rugged in that respect. Even heating with
a steam iron (not recommended) and subsequent treatment with zinc chloride will
give good fluorescing prints (though the fluorescence is less than after proper
On certain low quality papers (like newspapers, recycled paper, and corrugated cardboard) we found that 5-MTN will develop prints of good visibility, but low or no fluorescence.
Treatment with zinc chloride will change the color of the developed prints (that initially have the same color as ninhydrin developed ones) only slightly (more pinkish). This in contrast to ninhydrin which changes from purple to orange. These orange prints show greater absorption (under blue green light) than the untreated print and will provide a little better contrast with weak prints. With 5-MTN we found no improvement of the light absorbing qualities after treatment with zinc chloride.
Fingerprints developed with 5-MTN and treated with zinc
chloride, will fluoresce under green light (optimum about 520 nm). These excitation
and viewing conditions are similar to those for DFO developed prints. Optimum
viewing and photographing is done with dark orange glasses/filters (cut on point
With the use of cadmium salts (poisonous and environmental unfriendly) for the
treatment of 5-MTN developed prints, less fluorescence is obtained than with the
use of zinc chloride. Complexation with other metal salts (for example nickel
and copper) yields non-fluorescent prints.
Procedure for preparation of the zinc chloride solution
For the treatment with zinc chloride, we developed the
following non-CFC formulation. We have found it to be very reliable and stable
(we have stored and used solutions that were several years old, without any problem).
In Europe this solution can be obtained from BVDA International in the Netherlands.
Dissolve 30 grams of zinc chloride in a mixture of 500
ml methyl-tert-butylether (MTBE) and 20 ml of anhydrous ethanol (98% or
more) using a 1 liter erlenmeyer flask, a magnetic stirring bar, and a magnetic
stirrer, in a fume hood. It will take about 30 to 60 minutes to dissolve completely.
After it is fully dissolved, add 10 to 20 ml of glacial acetic acid (99-100%) and dilute
with 500 ml of a hydrocarbon solvent (petroleum ether, pentane, heptane). Store
in a brown glass bottle.
The zinc chloride solution is particularly stable, also
at low temperatures. In principle it can be kept indefinitely.
History of 5-MTN
5-Methylthioninhydrin (5-MTN) was synthesized by Prof. Madeleine Joullié and her co-worker Dr. Robert Heffner of the University of Pennsylvania in the beginning of the nineties . Tests by the US Secret Service
 and the Israel National Police  showed that 5-MTN can develop fingerprints
with a color equivalent to (or stronger) than ninhydrin. After treatment with
zinc chloride the developed prints were found to fluoresce stronger than those
treated with DFO.
The published syntheses [references 4 and 6] were not amenable
to large scale synthesis, due to various reasons. This has been solved by BVDA.
For a comprehensive overview on ninhydrin and ninhydrin
analogues prior to 1991, see the chapter written by Dr. Joseph Almog in "Advances
in Fingerprint Technology" .
For more literature references, see the literature
page on the website of the Center for Forensic Studies at Texas Tech University.
References and footnotes
 Davies, P.J.; Kobus, H.J.; Taylor, M.R.; Wainwright,
K.P., "Synthesis and Structure of the Zinc(II) and Cadmium(II) Complexes
Produced in the Photoluminescent Enhancement of Ninhydrin Developed Fingerprints
Using Group 12 Metal Salts", Journal of Forensic Sciences, Vol. 40,
No. 4, July 1995, p. 565-569.
 Petrovskaia, O.G., Ph. D. Thesis, University of Pennsylvania, 1999.
 In a not too large space this can be accomplished by placing a large
tray with a brine solution. This solution is made by dissolving as much salt as
possible in (tap) water, till no more salt dissolves. For example in a bottle,
by shaking. To be certain that the solution is a saturated one, an excess of salt
(sodium chloride) is used. The mixture of salt and brine can then be transferred
to a tray.
 Heffner, R.J.; Joullié, M.M., "Synthetic Routes to Ninhydrins,
Preparation of Ninhydrin, 5-Methoxyninhydrin, and 5-(Methylthio)ninhydrin",
Synthetic Communications 1991, Vol. 21, No. 21, p. 2231-2256.
 Cantu, A.A.; Leben, D.A.; Joullié, M.M.; Heffner, R.J.; Hark,
R.R., "A Comparative Examination of Several Amino Acid Reagents for Visualizing
Amino Acid (Glycine) on Paper", Journal of Forensic Identification
1993, Vol. 43, No. 1, p. 44-66.
 Almog, J.; Hirshfeld, A; Frank, A.; Grant, H.; Harel, Z.; Ittah, Y.,
"5-Methylthio Ninhydrin and Related Compounds: A Novel Class of Fluorogenic
Reagents", Journal of Forensic Sciences 1992, Vol. 37, No.
3, p. 688-694.
 Hewlett, D.F.; Sears, V.G., "Replacements for CFC113 in the Ninhydrin
Process, Part 1", Journal of Forensic Identification 1997,
Vol. 47, No. 3, p. 287-299.
 Hewlett, D.F.; Sears, V.G.; Suzuki, S., "Replacements for CFC113 in the
Ninhydrin Process, Part 2", Journal of Forensic Identification
1997, Vol. 47, No. 3, p. 300-306.
 Almog, J., page 103-133 in Advances in Fingerprint Technology; Lee, H.C.; Gaensslen, R.E., Editors; Elsevier: New York, 1991. ISBN 0-444-01579-5
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