Critical Reviews in Analytical Chemistry

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Narration and Legacy of Important Chemical Spot

Tests in Forensic Investigation

Rajvinder Singh

To cite this article: Rajvinder Singh (2020): Narration and Legacy of Important Chemical
Spot Tests in Forensic Investigation, Critical Reviews in Analytical Chemistry, DOI:
10.1080/10408347.2020.1785837

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CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY
https://doi.org/10.1080/10408347.2020.1785837

REVIEW ARTICLE

Narration and Legacy of Important Chemical Spot Tests in Forensic Investigation

Rajvinder Singh
Department of Genetics, Maharshi Dayanand University, Rohtak, India

ABSTRACT KEYWORDS
Chemical spot tests are one of the oldest and simplest presumptive methods of analytical chemis- Chemical reaction; color
try. They are an integral part of the schematic analysis of different types of substances in various test; crime; forensic science;
pure and applied scientific disciplines including forensic science. The role of spot tests has spot test
remained eternal utility in different branches of forensic science to analyze various types of phys-
ical or trace evidences. Forensic experts need to have an absolute understanding of the founda-
tion and technicality behind complex reactions of customary spot tests. Forensic science literature
dwells in the diversity of spot tests but an informative and comprehensive compendium of such
prose remains occasional and limited in general. Keeping in view the ample history and legacy of
spot test, the current review was constructed from a core of historical literature to recapitulate
trending applications, chemistry, and limitations of notable “Griess test”, “Luminol test”, “Kastle-
Meyer test”, “Phenolphthalein test”, “Ninhydrin test”, and “Spy dust” in forensic science. The aim
of this review article was to describe the outlook and likely impact of these tests on the expansion
of scientific investigation. The anticipated output of this review is supposed to impart compatible
knowledge in the attentive readers interested in understanding legacy and technical details of
selected spot tests used in solving crime.

Introduction Spot tests have been exercised in several biological, bio-

In scientific terminology, chemical “spot tests” sometimes
are referred to as “color tests” because a chemical interaction
between the sample and a reagent generally results in a color
manifestation that helps discerning a suspected compound
or group of compounds. The available literature on the pro-
gression of microchemistry helped to trace the origin of spot
(color) tests. Microchemistry deals with micro-chemical ana-
lysis including presumptive spot (color) tests, and more evi-
dent microcrystal tests based on chemical reactions between
of suspected materials and specific reagents, both in minute
quantities. Merely, a microgram of a sample, and one, or
few drops of a chemical reagent can perform the spot ana-
lysis. Spot tests probe chemical properties of compounds,
and can be be informative enough to specify inorganic ion
or organic functional group of an alleged sample. Therefore,
the spot tests are indisputably one of the superlative pre-
sumptive (tentative) qualitative analytical methods of identi-
fication. The reagent as a single chemical or comprising a
series of chemicals is added to the sample under probe. Spot
plates or pieces of filter paper are usual accouterments to
perform the spot test. A spot test is primarily preferred
owing to their simplicity in operation, ease of use, wider
detection, on-field/site analysis, low cost, rapid analysis, high
sensitivity, and need of a minute quantity of sample for ana-
lysis. Occasionally, the experts elude spot testing due to their
destructive nature, especially in case of the availability of
limited samples.
chemical, biomedical, and other scientific disciplines refer-
ring earlier significant reports.[1–5] Literature survey has also
presented a colossal use of spot tests by the general science,
crime investigative agencies, and forensic science laborato-
ries, etc. It is likely that a crime scene, an associated victim/
s, and culprit/s could deliver varieties of bulk or trace chem-
ical (illicit drugs, explosives, gunshot residues or GSR, pesti-
cides, and liquors, etc), biological (fingerprints, blood,
semen, and saliva, etc), and many types of evidences.
Forensic experts in routine analysis employ both, presump-
tive/screening and conclusive/confirmatory (e.g., mass spec-
trometry) methods to prove the factual identity of numerous
types of evidence materials. Chemical spot testing in corrob-
oration with other analytical techniques can help signifi-
cantly in solving the crime mysteries by examining such
forensic evidences. As far as analysis of particular forensic
evidence material is concerned, the use of spot tests i.e.,
Marquis, Madelin, Cobalt thiocyanate, Scott, Dille-Koppanyi,
and Duquenois-Levine (for illicit drugs); Modified Griess,
Diphenylamine, and Alcoholic potassium hydroxide (for
explosives/GSR); Prussian blue (for cyanide); Iodoform (for
ethanol); Chromotropic acid (for methanol/formaldehyde);
Fujiwara (for chloroform); Palladium chloride based Con-
way cell microdiffusion (for carbon monoxide); Ferric chlor-
ide (for salicylate); Kast-Meyer (KM), Tetramethyl benzidine
(TMB), Leuco–malachite green (LMG), and Luminol (for
blood); and Phenolphthalein powder (for Bribe trap) include

CONTACT Rajvinder Singh ajayrajaryan@rediffmail.com Department of Genetics, Maharshi Dayanand University, Genetics, Rohtak 124001, India.
ß 2020 Taylor & Francis Group, LLC
2 R. SINGH
list of well-known spot (color) tests in forensic science.
Many of these tests make use of different chemical or colo-
rants e.g., Nitrophenyl pentadien[6]; Phenolphthalein[7–10];
Malachite green[11]; Luminol[12]; Ninhydrin[13]; Sudan black-
B[14]; and Leuco-malachite green[15] There is a protracted
historical background of the use of spot (color) tests as a
presumptive mean of identification in forensic science.
Brief history and legacy of spot testing
The legacy of chemical spot test can’t be narrated without
referring the contribution of Feigl, a pioneer of chemical
spot testing. Fritz Feigl (1891–1971), an Austrian philoso-
pher of chemistry started publishing in analytical chemistry
in 1919. His hands-on practical skill at the laboratory
bench-work was remarkable. Once he stated “chemical equa-
tions do not translate all changes in a reaction system; they
convey the chemical fate of the reactant system”.[16] His refer-
ence books on qualitative spot tests in the inorganic and
organic analyses are globally recognized and appreciated to
date.[3–5,17,18] He also familiarized concepts of “limit of iden-
tification” and “dilution limit” in spot test analysis. Research
reports show that Feigl during 1917–1923 introduced a new
chemical strategy for the characterization of inorganic spe-
cies by interacting them with organic reagents that led to
deeply colored products. Feigl authored the first analytical
and interpretative presentation in the English version on the
use of organic reagents in inorganic analysis in the year
1936. Supposedly, it introduced practicability and concept of
chemical spot (color) testing in the scientific world.[19] The
International Union of Pure and Applied Chemistry
(IUPAC) also recommended Feigl’s work based on spot tests
as an "official" jargon for analytical empathy.[19,20] Literature
reports also corroborate that spot/color testing methodology
was already developing while Stromeyer introduced the iod-
ine-starch (giving blue color) reaction in the year 1815, fol-
lowing previous work of Colin et al. (1814).[21,22] There
were several other important contributions to chemical spot
analysis in the later stage up to recent past[23–25]; however,
the earliest and factual one was notified in 1859 when Hugo
Schiff detected uric acid on a filter paper.[26] Procedure of
this reaction involved poring a drop of aqueous solution of
uric acid on a silver carbonate impregnated filter paper that
resulted a grayish black fleck.[26,27]
Chemical analysis is one of the most imperatives, and
complex aspects of forensic science; besides, the successful
implementation and interpretations of spot tests are bound
with the comprehensive knowledge of the forensic experts.
It becomes moreover essential when the court of law
observes spot test based scientific help used in analyzing
forensic physical evidences. In such circumstantially but
nevertheless responsive situation, all eyes fix on a forensic
expert who is answerable about the depictions of spot test-
ing technical details in the court of law. Proper knowledge
in the concerned matter of analysis is expected at this cru-
cial level of legal assessment or cross-examination, otherwise
it can be troublesome for the expert and case as well.
Simply observing an inferring color at the end a chemical
reaction behind a spot test does not justify the actual scien-
tific understanding of a forensic expert. Diminished scien-
tific knowledge could be either due to a lack of personal
interest in literature or availability of the less amount of
conceptual information about the spot chemical reaction.
Occasionally, widely scattered past and present scientific
reports on the spot/color test don’t create a review interest,
in general. Therefore, a complete compendium of such lit-
erature reports could stimulate readership curiosities in a
view to learn, and understanding scientific explanations
about the origin, legacy, and chemistry of spot/color tests.
Though, available literature on spot test mingles various
chemical colorants but forensically oriented and exclusive
compendium on such prose usually remains abstracted or
unpublished. Therefore, the present study has tried to
improve this educational limitation by adding knowledge to
the body of forensic science by deliberating on selected but
legendary spot tests. Keeping in view the relevant legacy,
academic interest, and forensic importance, the current
review has emphasized on the following spot tests: -
Stretched appliance of Griess test
Johann Peter Griess (1829–1888) was a pioneer German
chemist who worked in the field of organic chemistry. In
1858, he described a highly useful chemical reaction known
as the ‘Griess diazotization reaction’, which formed the basis
of the ‘Griess test’ for the detection of nitrite ions. There is
a chemical transformation of nitric oxide (NO) to nitrite
(NO2-) which was measured with the help of assay that
relies on a diazotization reaction described by Griess in
1879.[1] This test was widely accepted in basic scientific
fields for determining nitrite in foodstuffs; however, the
forensic experts also took advantage of this test in the detec-
tion of explosive residues in the later stage.
Background history
The year 1879 recorded origin of the Griess reaction pre-
sented at a session of the German Chemical Society, and
published in a German scientific journal entitled ‘Berichte
der Deutschen Chemischen Gesellschaft’ (Ber. Dtsch.
Chem. Ges).[1] This publication by Griess elucidated a new
and a highly sensitive chemical reaction for detecting
nitrous acid. It is important to mention that prior to this
important scientific publication; he also described the
properties of diazobenzoic acid and diazonium salt.[28,29]
Literature supports the extensive use of Griess reaction in
the analysis of nitrate and nitrite in the different types of
biological samples including saliva, plasma (serum), urine,
cerebrospinal fluid, and cell culture media.[30] It practically
helped in understanding that nitric oxide was an import-
ant physiological molecule in systematizing the biological
systems including immunological, neuronal, and cardiovas-
cular tissues.[31]

Figure 1. Griess chemical reaction.[32]
Reaction mechanisms
Griess reaction mechanism formed azo dyes with help of
Griess reagent. This mechanism involved a chemical reac-
tion between sulfanilic acid (SA) with nitrous acid in the
presence of acidic (sulfuric) medium (Figure 1).[32] This first
step of reaction ended with the formation of diazonium ion,
whereas the intermediate transient diazo form was coupled
with a-naphthylamine (an amine) forming a red azo com-
pound (dye), and the acid was regained in the second
rearrangement step.[1] Chemistry in the Griess test is easy to
understand. The diazonium ion reacts with 1-naphthylamine
or a-naphthylamine, a typical coupling reagent that is an
aromatic compound with an electron-rich nucleus, rendering
it susceptible to an electrophilic attack by the diazonium
ion. A nitrogen atom from the analyte is incorporated into
the colored product. This azo-type compound has a charac-
teristic pink color. In this way the Griess reaction was
thought rather specific for nitrite ions.[5,33] Nitrite ions in
this reaction could be detected up to a lowest limit
of 0.05 mg.[5]
Griess test also experienced some requisite chemical mod-
ifications in order to enhance stability of involved chemical
reaction. Griess in his previously report mentioned the use
of sulfuric acid as an acidic medium for the reaction.[1];how-
ever Ilosvay (1889) reevaluated the reaction mechanism, and
recommended the more advantageous acetic acid in
1889.[34] It shortened the reaction time and intensified the
color of the dye. The acetic acid is still in use with most azo
dye methods. In year 1889, Lunge also proposed the prepar-
ation of a ready-mixed reagent, and the revival of the
reagent by zinc.[35] According to Franchimont, nitramine
was reduced to nitrite ions by zinc and acetic acid, and later
subjected to the Griess reaction.[36] It was recommended for
the identification of nitramine impurities, particularly in
RDX.[37] Other Griess modifications included N,N-dimethyl-
1-naphthylamineorN-1-naphthylethylenediamine (NED) as
the coupling agent.[38–41] These reagents were alleged to pro-
duce more stable azo color. Fox studied different
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 3
combinations of nitrosated species and coupling reagents in
Griess reactivity.[33] Basch et al. (1986) and Douse (1982)
investigated the use of phosphoric acid as the replacement
of acetic acid as the acidic medium in the Griess test.[41,42]
Few subsequent important chemical modifications to the
Griess method was also proposed by other workers.[30,43,44]
The method of alkaline hydrolysis, followed by the use of
sulfanilic acid and 1-naphtylamine was considered the basis
of a spot test for nitroglycerin, nitrocellulose, tetryl, and
RDX type explosives.[45,46]
Fiddler proposed greater sensitivity when sulfanilamide
and NED were added sequentially (after 5–10 minutes incu-
bation) because they compete for nitrite in the Griess reac-
tion.[47] For example, SA and NED can be pre-mixed in an
acid medium before reacting with nitrite. Yet in a different
version, after reacting nitrite with SA in acidic medium,
NED was added 10 minutes later. The most popular version
seems to be the sequential method in which nitrite was
mixed with SA first, followed immediately by the addition
of NED. This method appeared to give the highest yield of
the chromophore, and therefore the most sensitive way to
perform the Griess assay.[48,49] Diamine was used in place of
the simpler and cheaper a-naphthylamine because the latter
was considered a potent carcinogen. Besides, the diamine
formed a more polar, and hence a much more soluble dye
in an acidic aqueous medium.[50]
The modified Griess test and use in forensic science
There is sporadic use of explosives in terrorism and rioting
like crime. The use of spot tests to analyze pre-blast & post-
blast explosives including GSR has been reported in forensic
science for the last many decades.[51] Improvised Explosive
Devices (IEDs) usually contain Trinitrotolunene (TNT),
Trinitro-triazacyclohexane (RDX), pentaerthritol tetranitrate
(PETN), nitroglycerin (NG), ammonium nitrate (AN), and
many others nitro based explosives. The Griess test in foren-
sic laboratories helps to detect the presence of nitrite ions in
such nitrate esters and nitramines explosives. A survey of
4 R. SINGH

Table 1. Details of compounds.[59]

Compound Important details
Luminol Chemical Names: 5-amino-2,3-dihydrophthalazine-1,4-dione
Molecular Formula: C8H7N3O2
Molecular Weight: 177.16 g/mol
Physical Description: Yellow crystals or light beige powder.
Phenolphthalein Chemical Names:3,3-bis(4-hydroxyphenyl)isobenzofuran-1(3H)-one
Other names: Phthalimetten Euchessina Phthalin
Molecular Formula: C20H14O4
Molecular Weight: 318.3 g/mol
Physical Description: Appears as white or yellowish-white to pale orange fine crystalline powder.
Ninhydrin Chemical Names: 2,2-dihydroxyindane-1,3-dione
Molecular Formula: C9H6O4
Molecular Weight: 178.14 g/mol
Physical Description: Appears as white to light yellow crystals or powder.
NPPD Chemical Names: 5-(4-Nitrophenyl)-2,4-pentadienal
Other names: NPPD, spy dust, Spydust, Nitropheny pentadienal
Molecular Formula: C11H9NO3
Molecular Weight: 203.19 g/mol
Physical Description: Appears as white to orange yellowish powder.

forensic literature has shown few intended modifications
made to the Griess reagents to improve testing suitability.
After implementing modification, the Griess test was also
called ‘The Modified Griess Test’ (MGT).[52]
As per chemical nature, there are nitrate esters based
explosives including ethylene glycol dinitrate (EGDN), nitro-
glycerin (NG), nitrocellulose (NC), and pentaerythritoltetra-
nitrate (PETN), and nitramines types such as HMX (1,3,5,7-
tetranitro-1,3,5,7- tetrazacyclooctane) and RDX (1,3,5-trini-
tro-1,3,5-triazacyclohexane), which produce nitrite ions
when initially treated with ethanolic potassium hydroxide
(KOH). Afterward, they are treated with the Griess reagent
consisted of two subsequently applied separate solutions
(sulfanilic acid and 1-naphthylamine) in an acidic medium,
usually acetic acid. The acetic acid solution of sulfanilic acid
and 1-naphthylamine is a modification of Griess reagent
and sometimes called as ‘Ilosvay modification’.[34,51,53] It
produces a strong and characteristic pink color if the analyte
is indeed a nitramine or nitrate ester. The intense color in
nitrite assay reveals high sensitivity.
GSR detection in FSLs
Visual and microscopic examination of GSR follows chem-
ical testing with the MGT. A piece of desensitized (in hypo-
solution) photographic paper is treated with a chemical mix-
ture of sulfanilic acid in distilled water and alpha-naphthol
in methanol. The photographic paper will no longer be
light-sensitive but will be reactive to the presence of nitrite
residues. The exhibit is carefully placed face down against a
piece of treated photo paper with the bullet hole centered
on the paper. Back of the exhibit is later steam ironed with
a dilute acetic acid solution in the iron instead of water. The
acetic acid vapors penetrate the exhibit and a reaction takes
place between nitrite residues in the exhibit/s, and chemicals
contained in the photographic paper. The resulting reaction
produces as orange specks on the piece of photographic
paper.[54] Explosives based on nitrate esters (such as NG or
PETN) and nitramines (such as RDX or HMX), form nitrite
ions under alkaline conditions, and therefore they could be
detected by the Griess reaction but this test does not distin-
guish between individual explosives within these groups.
Besides, nitrate ester (nitrocellulose) occasionally used in
lacquers and paints can also produce the same pink color as
a false-positive result. Another factor of false-positive results
is a situation where the nitrate ions (NO3-) together with
some accidental reducing substances could be reduced to
nitrite ions (NO2-), giving a positive result in the Griess
reaction. Therefore, a positive Griess test on hands can’t
constitute evidence of the hands contact with explosives.
“Birmingham Six” case ideally quotes limitation of the
Griess test because after this incidence this test was rather
discredited by the experts. A report in consequence, pro-
posed to strengthen the results of Griess test with other con-
firmatory methods of analyzing suspected explosive
materials before an expert present testimony in the court of
law during case trial.[55] In Griess reaction for explosive ana-
lysis, the nitrate esters, and nitramines produce NO2- ions
by the action of an alkali whereas nitrate ions produce the
nitrite ions by the reduction with zinc powder.[56] A report
revealed an innovative and indirect method involving filter
paper use in MGT while detecting GSR in case of a hin-
drance causing dark background/design of the target cloth
material, particularly silk.[57] After undergoing the occa-
sional but much needed modifications, the Griess Test seems
to be a paramount and referentially accepted type of spot
test for analyzing explosives and GSR. As most of the explo-
sives are nitro-based compounds; therefore the snootier util-
ity of the Griess test in identifying such compounds can’t be
suspended even in the current scenario of hi-tech instru-
mental methods of analysis.
Luminol: a marker for invisible blood
The blood is a considerable primary evidence in most of the
murder cases. Sometimes, the blood is unseen or invisible to
naked eyes; particularly, when it is cleaned up or escaped
due to extended time from a scene of crime. To solve this
problem, the scientific invention introduced ‘Luminol’, a
chemical to decipher hidden blood traces. Biologists also use
it in cellular assays to detect iron, copper, cyanides, and spe-
cific proteins with help of western blotting.[58] Important
details of Luminol are given in Table 1.[59]

Figure 2. Luminol chemical reaction with blood.[72]
Background history
According to literature, synthesis of this compound (then
unknown for its chemiluminescence properties and name
Luminol) first time started in Heidelberg (Germany) by the
efforts of Schmitz in the year 1902.[60,61] Curtius also
directed Schmitz in his project. Curtius and Semper (1913)
then resynthesized this compound by implementing few
modifications to Schmitz’s earlier work published in
1902.[61,62] The nickname ‘Luminol’ was not named until a
method described to synthesize a bicyclic compound called
as 3-aminophthaldehydrazine from a chemical reaction
between 3-nitrophthalic acid and hydrazine sulfate, and was
known for its intriguing chemiluminescence prop-
erty.[12,63–65] None of these workers could notice the chemi-
luminescence properties of this newly synthesized molecule
in the beginning. Lommel in Leverkusen (Germany) fore-
most notified intense blue chemiluminescence of this com-
pound while oxidizing in alkaline solution. The results of
this investigation appeared in 1928, after the involvement of
Albrecht, who observed that a luminescence occurs when it
was treated with different oxidizing agents in alkaline solu-
tion. He also reported that hydrogen peroxide/H2O2, an oxi-
dizing agent produced a feeble luminescence visible in a
darkened room even at 10
8 M concentrations of Luminol.
He also observed that salt like hypochlorite or ferricyanide
greatly enhanced the luminescence obtained with H2O2 like-
wise plant peroxidases and blood.[63]
Enhanced luminescence of Luminol (in an alkaline solu-
tion of H2O2) with blood and other substances was first
observed in 1928.[65,66]; however, it was later confirmed in
the presence of blood hematinic 1939.[67] Specht during
1937 conducted a study for the medico-legal identification
of old and fresh bloodstains at the crime scene using
Luminol chemiluminescence. He prepared two successfully
working reagents (i) 0.1 g Luminol, 5 g CaCO3, and 15 ml
30% H2O2 in 100 ml H2O, & (ii) 0.1 g Luminol in 100 ml
0.5% aqueous sodium peroxide for spraying on bloodstains.
He was able to locate the bloodstains with help of photo-
graphable luminescence that lasted for quite a while. He
found that it was quite a specific test for deciphering blood,
recommended it strongly for medico-legal examinations.
Specht confirmed the use of this test for bloodstains that
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 5
gave more intense and longer-lasting luminescence in com-
parison to fresh blood. He also found that luminescent
occurred many times with consecutive spraying on earlier
dried sprays.[68] Proescher and Moody (1939) revived the
Luminol reagent to make it more stable using commercial
Luminol at a concentration of 0.1 g in 100 ml 5% Na2CO3.
In case of fresh blood, the test solution was added with con-
centrated hydrochloric acid in order to convert hemoglobin
to hematin, and also shortly boiled to destroy vegetable per-
oxidase enzymes. Luminol reagent was added after making
the test solution alkaline with sodium carbonate. They rec-
ommended the test very useful but presumptive because
false-positive results could be noticed with hypochlorites,
ferricyanide, and several other inorganic substances.[60]
McGrath also recommended the use of Luminol in forensic
blood detection in 1942.[69] Grodsky and associates recom-
mended a reagent comprising a sequential addition of 0.07 g
sodium perborate (NaBO3H2O) in 10 ml water, followed by
0.01 g Luminol and 0.5 g Na2CO3.[70] This reagent was
incorporated into a field test kit and considered quite spe-
cific for blood without false-positive results. Deciphering
unseen blood with the help of Luminol was proposed to be
the best spot presumptive method of choice at a
crime scene.[71,72]
Reaction mechanisms
Luminol is a heavily modified double benzene ring, with
three nitrogen and two oxygen atoms added. This white-to-
pale-yellow crystalline solid chemical exhibits chemilumines-
cence with a blue glow on mixing with an appropriate oxi-
dizing agent. This chemical reaction replaces nitrogen and
hydrogen with oxygen. Vibrational energy created by the
reaction makes the transition of an electron to a higher
energy level. On returning to a stable state, the energy is
released in the form of a photon of light from each molecule
(Figure 2).[72] Luminol reagent is sprayed on the suspected
areas of crime scene personally made darkened, and already
made set with the cameras and luminescent markers. This
reagent is commonly made up in alkaline solution (pH
10.4–10.8) using sodium carbonate, and sodium perborate
(NaBO3H2O) as the source of the oxidizing species. An
aqueous or alcoholic solution of Luminol and an oxidizer is
6 R. SINGH
Figure 3. Synthesis of phenolphthalein.[10]
catalyzed by the iron present in the hemoglobin component
of blood to produce 3-aminophthalate (3-APA) in an excited
state. Excited 3-APA molecules quickly return to their
ground state with the emission of photons that are visible as
a distinctive weak blue luminescence that persists for about
45 seconds.This compound gives a strong blue fluorescence
with ultraviolet light at 425 nm.[73] Tautomeric forms exist
in alkaline solution, and it produces chemiluminescence
upon oxidation.[63] Lee (1978) proposed Luminol reagent
comprised of 0.1 g 3-aminophthalhydrazide, 0.5 g Sodium
carbonate, 0.7 g Sodium perborate in 100 ml distilled
water.[74] Luminol has a strong sensitivity at dilutions high
as 100,000,000:1 or even more than this. Even the washed
and painted surfaces having traces of bloodstains in the past
also yield a positive test with Luminol. Higher sensitivity
hampers selectivity as many materials other than blood
including copper and other metals, laundry bleach, and
many food items yield positive Luminol tests that are indis-
tinguishable from positive results caused by actual blood but
body fluids other than blood do not react with Luminol.[73]
It remains one of the most effective presumptive tests due
to not interfering with subsequent DNA analysis so it can
be sprayed directly onto the suspect stains.[75] A recent
study made by Butler et al. (2019) made a competitive com-
parison of Luminol, Leuko crystal violet,
Tetramethylbenzidine, and Combur tests for the detection of
blood at different dilutions on dark materials including cot-
ton sheeting, socks, tea towel, car mats, and synthetic carpet.
They reported that Luminol showed the highest sensitivity
(79%-96%), negative predictive value (66%-93%), the lowest
overall misclassification rate (3%-15%), and also found to be
most efficient with a testing time, therefore considered the
preferred and the most effective method for tracing blood-
stain patterns.[76] The use of Luminol in deciphering hidden
blood trails and traces proves to be a boon for forensic
experts. Strong sensitivity is an additional factor that con-
tributes to its long term efficacy in forensic investigations.
Forensic biology experts always remain equipped with this
highly promising indicator to identify all types of blood-
stains at the scene of crime.
Indicator use of phenolphthalein
Phenolphthalein is a chemical substance often used by the
law enforcement agencies for apprehending corrupt officials
accepting the bribe in graft or trap cases[77,78]; furthermore,
it is also a primary ingredient in Kastle-Meyer (KM) reagent
used for the presumptive identification of bloodstains.[7]
Literature also reveals its use as a laxative, an acid-base indi-
cator, and a constituent of widely used weight-reducing
multicomponent food formulations.[77]
Background history
In 1871, Adolf von Baeyer, a renowned German chemist
synthesized phenolphthalein by condensing phthalic anhyd-
ride with two equivalents of phenol under acidic conditions
(Figure 3).[10,79] Important properties of Phenolphthalein are
given in Table 1.
Origin of Kastle-Meyer (KM) test
In 1901, Joseph Hoeing Kastle (1864–1916), a native
Lexingtonian with his colleague Shedd, first time described
the use of phenolphthalein for detecting the blood.[7] They
observed that an enzyme known as cellular oxidases cata-
lyzed the oxidation of colorless phenolphthalein to chem-
ically transformed red to pink color phenolphthalein
product under slightly alkaline condition. They also con-
ferred other aspects of this test for blood testing. Later, a
German physician and chemist, Meyer modified this test in
1903.[8] His extended research made use of phenolphthalein
to detect the oxidases in blood leucocytes as well as in the
qualitative and quantitative determination of blood in urine.
At the same time, Utz (1903) also made use of this chemical
reaction in the medico-legal cases for identifying fresh and
dried bloodstains, sometimes older than one and a half
year.[9] He also mentioned about "false-positive" results in
this test when interfered with pus, and other leucocyte-con-
taining discharges. This test procedure also underwent few
expedient modifications. An earlier study revealed the
reagent synthesis from phenolphthalein reduced in the pres-
ence of Zinc and strong sodium hydroxide or potassium
hydroxide solution.[80] Subsequently, Kastle (1909) recom-
mended acidic treatment of phenolphthalein and collection
of resultant precipitations. Furthermore, it was re-crystal-
lized many times from minimal alcohol by cold water. This
pure material was stored as a solid substance but liquid sol-
utions of the compound were made more stable for weeks
in the presence of a small quantity of zinc dust and dark
conditions. The reagent also tested the sensitivity of the test

Figure 4. Kastle-Meyer reaction with blood.[56]
by experimenting on different blood dilutions.[81] A study
described the utility of the KM test in detecting blood aiding
the medico-legal investigation. This study reported that KM
test is delicate and reliable in the detection of hemoglobin
but a condition to the greatest care to ensure absolute clean-
liness of all containers in the medico-legal examination and
the test should be used in conjunction with the microscopic
examination of stains for the detection of the presence of
mammalian blood corpuscles.[82] This is how the origin of
this test came to notice as the Kastle test, the Meyer test, or
the Kastle-Meyer test.[63] Earlier, Camps (1976) also recom-
mended the use of ‘Phenolphthalein test’ as substitution of
“Benzidine test”.[63,83] KM test was equally reputed to the
Luminol test in presumptively deciphering the latent blood-
stains before the later was introduced.
KM test had few validity issues, therefore; Higaki and
Philp (1976) assessed stability, sensitivity, and specificity of
KM reagent for blood testing.[84] They prepared the reagent
according to earlier given method by Camps (1968), [83] and
Kastle method (1909),[81] excluding isolation and re-crystal-
lization in the procedure. According to Hunt et al. (1960)
and Olsen (1985) the KM reagent is prepared by dissolving
0.2 g phenolphthalein powder in 10 ml of a 20% sodium
hydroxide solution with 2 g zinc powder. After boiling the
solution under reflux, 3 ml of the KM reagent is added to
10 ml distilled/deionized water, and 2 ml 70% ethanol.[85,86]
In another type of preparation, the KM reagent was pre-
pared by reducing phenolphthalein with zinc metal in basic
solution, and an 20% sodium perborate solution was used in
the consecutive step.[87] In 1978, Lee proposed a stock solu-
tion of phenolphthalein containing 2 g phenolphthalein, 20 g
potassium hydroxide in 100 ml distilled/deionized water.
This is later refluxed with 20 g of powdered zinc for two
hours until the solution turns colorless. The stock solution
is required to be kept in a dark bottle and cool condition.
Extra zinc added to solution keeps it in the reduced form.
The working solution should contain 20 ml phenolphthalein
stock solution in 80 ml ethanol.[74] The use of ethanol cleans
the sample, and exposes more hemoglobin thus increases
sensitivity of the test, whereas, H2O2 liberates extra oxygen
when exposed to hemoglobin. But it is equally important to
understand that ethanol contained in KM reagent is a flam-
mable solvent, while H2O2 is an oxidizer, an irritant liquid,
and a slightly bleaching agent. Zinc dust or powder may
also cause handling issues due to being little pyrophoric
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 7
(spontaneously combustible) in the presence of moisture.
Therefore, protective measures like gloves, splash goggles,
and protective clothing should be used while conducting
this test.[73]
Chemistry of KM reaction
The KM reagent is a phenolphthalein based solution.
Phenolphthalein in KM test is an oxidizable organic com-
pound that could be oxidized by free hydroxyl ions liberated
by peroxidase action. The haeme group of hemoglobin in
blood owns a peroxidase-like activity that may catalyze the
breakdown of H2O2 in the presence of powdered zinc to
form free hydroxyl radicals. These free hydroxyl ions oxidize
the colorless reduced form of Phenolphthalein into a pink
color oxidized one (Figure 4).[56]
Procedures of conducting KM test
Literature reveals one-stage (occasionally called as one-step),
two-stage, and three-stage KM procedures considering sensi-
tivity and selectivity while using the test. The one-stage test
involves the addition of the combined reagents to the sam-
ple. There is successive addition of reagent and either perox-
ide or perborate in two-stage, whereas the three-stage test
encompasses the consecutive addition of the alcohol, the
reagent, and the peroxide or perborate solution.[63] The test
was originally used in one-stage, but few potential hindran-
ces were eliminated by carrying step-wise addition of chemi-
cals in the testing procedures. In the one-stage or original
form, a small amount of the KM reagent is prepared by
mixing equal volumes of 95% ethanol and 3% H2O2 solu-
tion. The suspect stain is gently rubbed with a piece of filter
paper, and a drop of the mixed reagent added to the paper.
The development of pink color was an indication of the
presence of hemoglobin, which has catalyzed the breakdown
of H2O2 to an oxidizing species. However, used in this
form, the test would give an apparently positive result with
other chemical oxidants/rust and catalysts (copper and
nickel salts), plant (vegetative peroxidase), and animal (bone
marrow, body fluid, and saliva) materials. To overcome such
interferences, procedural steps were divided sequentially in
two-step and three-step. The two-step testing of blood is
commonly seen in forensic investigation. In this method the
reagent is mix with an equal volume of 95% ethanol. The
8 R. SINGH
Figure 5. Color change in Phenolphthalein through ionization.[56]
solution is initially added to the stain on the filter paper. If
a pink/red appears without the addition of H2O2, the stain
in question is not identified as bloodstain. In this case, a
drop of H2O2 solution is added on it, and the appearance of
immediate pink color presumptively indicates the presence
of blood. In the case of chemical oxidants and catalysts,
H2O2 is added after waiting 30 seconds, whereas, in the case
of plat peroxides, the sample is required to heat at 100  C
for 5 minutes. Microscopic examination of samples may
indicate the presence of animal origin tissue, pus, and other
animal’s related interference. Three-stage test follows a
sequential addition of the alcohol, the reagent, and peroxide
or perborate. The pre-mixed use of phenolphthalein reagent
and H2O2 is extremely sensitive (10,000,000:1 or better) but
highly unselective as well. On the other way, if the reagents
are applied sequentially i.e., phenolphthalein reagent first,
followed by the H2O2 then the test is less sensitive
(20,000:1) but extremely selective. Hence, the secondary
role of the KM test involves the sequential method where its
selectivity is higher. Till now the KM is a widely used low-
cost test as it combines huge sensitivity and selectiv-
ity.[56,63,73] A recent study (Fonseca et al., 2019) successfully
evaluated the efficiency, sensitivity and vestigiality of the
KM test in the blood of human and domestic animals (dog
and cat).[88] It is a routine on-field spot test adopted by
forensic laboratories all over the world. The sequential step-
ping in this method helped to replace other problematic
tests used by the forensic experts. Its high sensitivity is a
considerably factor for its use in tracing blood at the crime
scene investigation.
Standard use of phenolphthalein in bribe trap cases
Bribery is an act of offering, giving, receiving, or soliciting
of any valuable item for altering the conduct of any person
bearing a legal or public duty. In India, the act of bribery
(illegal transaction) is an offense registered under Section
161 of Indian Penal Code and Sections 7, 12, 13(1)(d) and
13(2) of the Prevention of Corruption Act, 1988.[89] Anti-
Corruption Branch of Central Bureau of Investigation, and
Vigilance Department of India conduct such traps using
Phenolphthalein as evidence to prove acceptance of the
bribe. An important chemical property of phenolphthalein
makes it useful in investigating corruption by the law
enforcement agencies. Phenolphthalein usually remains col-
orless (below pH 8.3) but alkaline conditions (above pH 8.3)
immediately turn it pinkish.[10,90] This color-changing prop-
erty of phenolphthalein at varying pH helps the law enforce-
ment agencies scientifically proving that the accused person
was involved in accepting the article/s of bribe. This is a
type of chemical spot test which is also referred to as the
‘Phenolphthalein-Sodium Carbonate test’.[91]
Method of use
The currency notes smeared with phenolphthalein powder
are offered to the suspected candidate accepting bribes.
These currency notes are used as medium evidence for
transferring phenolphthalein to the body or other belongings
of the accused. While accepting money, the accuser’s con-
tact, especially hands get transfer of the phenolphthalein
from the currency notes already dispensed with phenol-
phthalein powder. It happens according to “Locard’s
Principle of exchange”. The candidate is caught red-handed
by the officials along with the legal witnesses. The accuser’s
hands along with other contact objects such as bags, pock-
ets, etc are also washed with a colorless solution of sodium
carbonate (Na2CO3) or occasionally with the lime-water
solution. The appearance of an immediate pink color con-
firms the transfer/touching of currency note/s (smeared with
phenolphthalein powder) to the hands or other belongings
of the accused. The alkaline solution of Na2CO3, hand/bag
washings of the apprehended person along with the washes
of the currency notes are collected in the airtight bottles/
containers. The investigating officer properly seals, and
sends them to the Central/State FSLs along with other
related evidences for the chemical analysis. Chemical and
instrumental analyses of these washings in the forensic labo-
ratories verify the presence of phenolphthalein as vital foren-
sic evidence.[91,92]
Chemistry of Phenolphthalein-Sodium carbonate test
This test is based on a chemical reaction in which the color-
less phenolphthalein turns into pink or deep purple in the
alkali medium. Phenolphthalein is a large organic molecule
very often used as an acid-base indicator. It is colorless in

Figure 6. Ninhydrin reaction with amino acid.[94]
aqueous solutions where the pH value is below 8.3. At pH
10 its color is intensive raspberry-purple. Below pH 8.3, it
is present entirely as phenol lacton. The colored form adopts
the resonant structure of the anion. However, at a higher
level of pH, the indicator is again turning colorless due to
being a consequence of its transformation into carbinol.[10]
Chemistry reveals that the aqueous solution of phenol-
phthalein under extremely strongly acidic conditions exists
in protonated form, and discloses an orange coloration
whereas, under strongly acidic conditions, its lactone form
appears colorless. The singly deprotonated phenolate form
gives pink coloration but in strongly basic solutions, the
phenolphthalein turns colorless. Phenolphthalein actually
changes color through a process called ionization (when
positively or negatively charged ions are added to or
removed from a molecule). Since phenolphthalein contains
positively charged hydrogen ions, but these ions are
removed when it is exposed to an alkaline solution. The
removal of hydrogen ions from the solution converts the
colorless phenolphthalein molecule ionizes and brings in the
pink phenolphthalein ion as shown in structure-2. The extra
electrons cause a shifting of electrons that generates the
right-side colorless form (Structure-3). Figure 5 illustrates
structure-1 is colorless at pH <8.3–8.5.[56] On the increase
of the pH (above 8.3) the solution becomes basic and phe-
nolphthalein loses two hydrogen ions to form pink-red color
di-anion in structure-2. At higher pH, the hydroxide ion is
formed as shown in structure-3 that makes it again
colorless.[10,91–93]
A considerable factor while analysis is the time lag in
between the collection and analysis of samples having pink
color. Literature also reports the gradually fading of pink
color leaving samples colorless in such cases. These circum-
stances also produced negative results. It can cause falsifica-
tion of bribe charges in spite of being genuine otherwise.
Scientifically it happens due to chemical break down of the
alkaline phenolphthalein by oxygen present in the air. This
technical contradiction in implementing this test for the
legal purpose was removed by adding a very small amount
of hydroquinone in phenolphthalein powder. Prior mixing
of hydroquinone (an antioxidant) can retard this reaction
and pink color may persist for a longer time. Stronger and
rapid chemical bonding of hydroquinone than alkaline phe-
nolphthalein with air oxygen does not allow fading of pink
coloration so rapidly. The presence of hydroquinone also
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 9
helps in disproving accused’s claims of using the laxative
tablets (containing phenolphthalein).[93] The whole proced-
ure involving the phenolphthalein test adopted by the law
enforcement agencies could be tedious and time consuming
but the instant on-field results deserve appreciation in a sci-
entific investigation. Earlier, this test faced the legal implica-
tions but the use of hydroquinone significantly removed the
issues, and made way for its constant use by the law
enforcement agencies in reducing the corruption in an
acceptable scientific manner. In supposition, it is a duty of
the experts to scientifically tackle the technical issues that
could otherwise help the criminal seeking deceptive plea.
Till date no other analogous spot test has shown the domin-
ance or replacement of phenolphthalein test in the legal
trapping of bribe.
Story of Ruhemann’s purple in developing latent
fingerprints
Fingerprint exudation/sweat contains several constituents
including amino acids up to a level of 900 mg/ml
1.[56]
These amino acids do not migrate from fingerprint exud-
ation even after a long period of time.[13] Amino acids also
have the property of interacting with different types of
chemicals to produce colored as well as luminescent prod-
ucts. Ninhydrin biochemically reacts with a-amino group of
primary amino acids that results in the production of
‘Ruhemann’s purple’ (Figure 6).[94] Several reports have
shown the chemical interaction of amino acids with
Ninhydrin for developing latent fingerprints in
particular.[13,95]
Background history of Ruhemann’s purple
An article published by Crown described the background of
the development of latent fingerprints with help of
Ninhydrin.[95] There was no extensive use of Ninhydrin
until 1950s. Though, Oden and Von Hofsten (1954) take
credit as beginner of using Ninhydrin for developing latent
fingerprint.[13] Literature shows that Siegfried Ruhemann, an
English chemist who accidentally synthesized Ninhydrin (tri-
ketohydrindene hydrate) while attempting to prepare the
oxidize 1-hydrindone (II) to 1,2-diketohydrindene. Instead
of the expected product, he discovered another crystalline
compound called as triketohydrindene hydrate, also known
10 R. SINGH
today as Ninhydrin. Few important properties of Ninhydrin
are given in Table 1. Later, he also reported its reactivity
with amino acids producing deep reddish-violet dimerici-
mino derivative, later called as Ruhemann’s purple. He
named the purple product after him, Ruhemann’s purple
that was a Ninhydrin analog of murexide.[96] There are few
important reports published comprehensively reviewing
Ninhydrin and its properties.[97,98] An important study by
Hansen and Joullie reported the development and need of
novel Ninhydrin analogs in developing fingerprints.[99]
There are reports revealing three key concepts behind chem-
ical i) the amino acid is oxidatively deaminized by
Ninhydrin, which is reduced to diketohydrindol. This dike-
tohydrindol and Ninhydrin condense to form hydrindantin,
which on combining with ammonia gives Ruhemann’s pur-
ple, ii) a compound is formed by combining 2 moles of
amino acid with hydrindantin. This compound splits into
two identical nitrogen-free, purple-colored free radicals,
analogously to the presumed compound formed from inor-
ganic cations and hydrindantin in strongly alkaline solution,
and iii) chromogenically, the amino acids react faster than
amines and ammonia with Ninhydrin. Amino acids decom-
pose into glyoxal and ammonia independently of Ninhydrin.
The glyoxal reduces Ninhydrin to diketohydrindol that is
later oxidized to the corresponding ru-keto acid. Ammonia
reacts with diketohydrindol to form diketohydrindamine
which condenses with Ninhydrin to form Ruhemann’s pur-
ple.[96,100–102] Friedman and Williams suggested the cur-
rently acceptable mechanism behind Ninhydrin reaction in
1974.[103] Grigg et al. then suggested some modification to
further improve this study. After a thorough understanding,
they found that the intermediate imine exists as a 1,3-dipole
form, which is a structure of the protonated form of
Ruhemann’s purple.[104–106] Their formation of an azomethi-
neylidein reaction is currently referred mechanism.
Ninhydrin reacts with a -amino acids to produce carbon
dioxide and an aldehyde with one short carbon than the
parent amino acid. As a result, a blue or violet compound is
formed owing to the reaction of the liberated ammonia with
Ninhydrin (Figure 6). The same type of chromophore is
formed in all primary amino acids. The number and chem-
ical nature of the amino being analyzed determines the
intensity of the color formed but the optimum pH for the
overall reaction is 5.5. Ruhemann’s purple chromophore has
a spectral maximum at 570 nm.[103]
Formulations
Englishmen, Morris and Goode in the year 1974 reported a
noteworthy formulation for optimizing latent fingerprints
with Ninhydrin reagent by using a solvent 1,1,2-trifluorotri-
chloroethane (known also as Fluorisol, Arklone P, Freon
113, or CFC113).This formulation was named “NFN for-
mulation” (nonflammable Ninhydrin). It was a nontoxic,
highly sensitive, and was applicable through dipping or
swabbing methods.[107] Goode and Morris prepared a stand-
ard NFN reagent contained about 0.5% Ninhydrin (w/v),
0.9% acetic acid (v/v), and 1.8% ethyl alcohol in
Fluorisol.[108] Ethyl alcohol dissolved the solid Ninhydrin,
whilst acetic acid provided the acidity required to balance
the alkalinity of some papers. Hewlett and colleagues sug-
gested another nontoxic, nonflammable, safe, and effective
formulation for fingerprint used the solvent HFE7100 (a
mixture of two hydrofluoroethers: 50 to 70% methylnona-
fluoroisobutyl ether and 30 to 50% methylnonafluorobutyl
ether) as the carrier.[109] The working solution contains
0.5% Ninhydrin in HFE7100 (in place of Freon 113) carrier
that also contained ethanol, ethyl acetate, and
acetic acid.[110]
Ninhydrin in developing latent fingerprints & implications
Several reagents reagents have been tried for the develop-
ment of latent fingerprints on paper in the last few decades
but many of those could not supplant Ninhydrin. Notable
literature cites extensive use of “Ninhydrin reagent” for
developing latent fingerprints and its recommended use in
forensic science laboratories and other related law enforce-
ment agencies.[13,56,95,111–116] Spraying a fine mist and dip-
ping into a solution are the usual methods of applying
Ninhydrin whereas exposure to Ninhydrin fumes, direct
treatment with Ninhydrin crystals without a solvent, or
pressing a paper towel impregnated with Ninhydrin solution
are least preferred conventional methods to process latent
fingerprints.[111,113] Dipping is the preferred laboratory
method according to literature.[107,108,113] Spraying is most
suited for treating extremely fragile papers while swabbing
was unsuitable as it could smear ink on documents.[111]
Ninhydrin treatment needs a systematic application of heat
to obtain the best results. Unsystematic or elevated tempera-
tures could form background discoloration.
Circumstantially, where urgency was crucial, the treated
samples were advised to keep as such at room temperature
for days or even weeks to obtain results without applying
heat.[107,108,113] There are notable differences in setting ambi-
ent parameters to develop latent fingerprints with
Ninhydrin, particularly heating at 80  C for a few minutes
(Oden and von Hofsten, 1954) [13]; at 100  C (Crown,
1969)[95], 80  C and above 50  C for certain items above
(Kent, 1986).[113]
Suitable humidity is also a vital factor in the quality of
developed prints as the presence of water vapors in the
processing oven can improve the results.[117,118] In his art-
icle, Crown (1969) also reported the development of latent
fingerprints with Ninhydrin is not permanent because prints
start fading soon after month.[95] He further emphasis on
the use of photography to keep permanent records of devel-
oped prints. The use of gloves and fuming hood was also
advisable as precautionary measures. He also suggested prior
use of the Ninhydrin method when applying silver nitrate
reagent whereas Ninhydrin used might follow the iodine
method to develop prints. Lesk (1971) reported 65 to 80%
relative humidity to establish optimum development by
processing the Ninhydrin-treated prints.[119] Olsen (1978)
while working in the U.S. Army Criminal Investigation
Laboratory obtained the best results at 80% humidity and a
temperature only slightly above room temperature.[111] The
Royal Canadian Mounted Police (RCMP) laboratories in

Figure 7. Structure of Nitrophenylpentadien (NPPD).[130]
Ottawa reported the use of a saturated sodium chloride
solution in the developing chamber to maintain the most
desirable and effective 75% relative humidity for the
Ninhydrin process.[114]
Literature also found effective use of a steam iron to
accelerate Ninhydrin development of prints. Connor (1976)
examined the effect of a steam iron on Ninhydrin treated
prints on newsprint papers and bond papers. This study
observed effective and quick development of latent finger-
prints without discretion to the method of applying
Ninhydrin.[111,120] Olsen (1978) discouraged the use of the
iron method on cardboard or coated paper surface because
steam condenses on such surfaces.[111] Method given by
Connor (1976) was adopted by the “The Association of
Official Analytical Chemists” (AOAC).[120,121] Other studies
reported general use of steaming and heating after
Ninhydrin treatment of papers.[122,123] Kent (1998) and
Champod et al. (2004) provided guidelines for the sequential
ordering of using different methods of latent fingerprint
developments due to differing nature of surfaces.[110,124] An
innovative study made by Almog et al. (1982) introduced a
new modification in Ninhydrin molecule to boost the color
and fluorescence of the resultant Ruhemann’s purple com-
plexes. They tested the activity of ‘Ninhydrin analogs’ with
amino acids to give colored products for aiding latent fin-
gerprint development. Benzo[f] analogue proved sensitive
and effective enough to develop latent fingerprints as dark
green impressions.[115] Best results in terms of optimal
development of Ninhydrin prints can be obtained by placing
items in a chamber having a temperature approximately
80  C, and humidity rate of approximately 60–80%.
Precautions should be taken while conducting this test. The
experimentation should be performed in a controlled labora-
tory setting. The expert needs to use appropriate breathing
apparatus, proper ventilation, and thick rubber gloves. This
chemical should be kept away from inhalation as and con-
tact of skin.[56,125] Ninhydrin method is a part of sequence
analysis to develop fingerprints in advanced fingerprint labo-
ratories. It gives the best results when used prior to the
physical developer (PD) and post 1,8-Diazafluorene-9-one
(DFO) methods.[116]
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 11
Latest literature also contains several reports showing
extensive use of “Ninhydrin reagent” for developing latent
fingerprints in forensic studies. Yang and Lian (2014) devel-
oped a new series of fingerprint developing membrane using
Ninhydrin (as the developing agent), and pressure-sensitive
emulsifiers (as the encapsulated chemicals). The applied
used of this method was the on-field/site development of
fingerprint on porous and non-porous surfaces.[126] Few
recent studies revealed the innovative use of ‘Ninhydrin
method’ for developing latent fingerprints on various surfa-
ces.[127,128] A comprehensive review describing studies on
the development of latent fingerprint enhancement using
different techniques comprising multi-metal deposition,
metal oxides, physical, optical, chemical, and physicochemi-
cal was reported in the year 2020.[129] Nature, a highly
reputed scientific journals published the mechanism of reac-
tion between amino acids and Ninhydrin for the detection
of fingerprints in 1954.[13] Forensic experts took full advan-
tages of this scientific innovation and solved several crimes.
This method involves few technical cautions but brings into
being the fingerprints; an unrepeatable and highest scored
forensic evidence in a personal identification of humans.
Though other more advantageous methods have been intro-
duced in the recent past but the use of Ruhemann’s purple
in development latent fingerprints going to remain unforget-
table till the far future.
Episode of human tracking with spy dust
Although, the current forensic technology has advanced to a
next high-tech level of human tracking but this article has
reviewed a conventional and obsolete method of human
tracking. Therefore, Spy Dust method has been discussed to
explore and probe a primordial chemical pioneering in
human tracking for investigation/spying purpose. This
segment is about NPPD, a crystalline compound of
unsaturated aromatic aldehyde chemically known as
Nitrophenylpentadien or nitrophenylpentadienal (Figure
7).[130] A mixture of NPPD, luminal, and other chemicals
was termed as “Spy dust”. Use of Spy Dust was a deliberated
as a valuable investigating approach to snub terrorist activ-
ities, and help forensic science.[6] Important information
about properties of NPPD is given Table 1.
Background history
Period of 1960s and 1970s reminds the earlier use of NPPD,
a chemical tracker of shoeprints. First time the Spy dust was
synthesized in the England in the 1930s,[131] and nicknamed
as “Spy Dust” (secret agent) or “Shadowing pursuit” (act of
the following someone secretly), or METKA (Russian for
“mark”).[130] Spy dust was secretly applied to the clothing,
and shoes like objects of the target subject, thus letting the
chemically tracking with help of appropriate surveillance
devices from a safe distance. This acetone and alcohol sol-
uble compound was a strong absorber of ultraviolet light
but did not fluoresce appreciably. The Soviet Union
(Russians) based KBG (a Russian Internal Security Agency)
12 R. SINGH
first time used this chemical at the Embassy in Moscow for
investigating the malicious activities of diplomats in a scien-
tific manner.[6,132,133]
Methods of use and impediments
NPPD reagent was prepared in methanol (1 mg/mL) at the
commencement. While using it, this chemical reagent was
sprayed on the entry restricted areas. NPPD would adhere
to the shoes or hands of the stranger unaware of this
sprayed track. A cotton swab of methanol extract was taken
from the suspected areas. The color examination involved a
two-steps process. The first step entailed the addition of
1 ml of a 0.1% naphthoresorcinol methanol solution to the
cotton swab, followed by the addition of 1 ml of concen-
trated hydrochloric acid in the second step. Turning the
solution dark red would corroborate the presence of NPPD.
Limit of detection was observed varying in visual method
(100 ng/3 ml), and ultraviolet-visible spectrometric method
(10 ng/3 ml) obtaining gamma max of the colored solution
at 510 nm, and a selected-ion-monitoring (SIM) gas chroma-
tographic-mass spectrometric (GC-MS) method (300 pg/
injection). Investigation of simulated theft cases and auto-
mobile entry in the restricted areas were carried out for the
forensic purpose.[6] Likewise, vital contributions made by
Suzuki and associates also designed various experiments to
use NPPD in tracing crime scene evidences, and earned
practical and theoretical acknowledgments.[134,135] In 2005,
they used a card with both a plastic-coated smooth surface
and a porous cellulose matrix paper surface coated with a
methanol solution containing 0.5 mg/ml of NPPD. This card
was made to touch with bare fingers, and fingers covered
with cotton gloves.[134] Suzuki (2013) also developed afield,
especially for investigating officers for chemical tracking
using NPPD.[135] Easily transferability, invisibility to naked
eyes, and requirement of minute quantity made it a valuable
spy chemical. It gained little success until it was not specu-
lated as potential carcinogenic[136–138]; however, a report
refuted the availability of toxicity information to confirm
this speculation.[139] Modern-day scientific literature does
not have any elaborated report on NPPD in forensic or
other types of criminal investigations. The use of human
tracking with Spy Dust is no more in practice. This test was
more like a faultfinder that could not achieve much success
and lost in the history at the wrong time. The use of NPPD
along with complex chemicals and ultraviolet light was never
going to be easy more often in the legal investigations.
Issues of carcinogenic nature of NPPD and unsaid scientific
rivalry played imperative part in its disappearance. It also
didn’t gain the appropriate international scientific reputation
at the best of its voyage.
Technical assessment of using spot/color tests
Spot test is an integral part of the various analytical schemes
for the analysis of forensic evidence but it becomes import-
ant to understand that why should or should not spot test
be part of an analytical scheme. The spot tests have the fol-
lowing merits and demerits in analysis:
Merits
Wider detection range: Spot test are widely applicable in the
scientific analysis of numerous evidence materials. They
have versatility of analyzing both, the organic and inorganic
along with complex materials. They are put in most of the
worksheets used for the schematic analysis in forensic sci-
ence. Forensic pieces of evidence like blood, semen, wood,
fingerprints, GSR, explosives, drugs, liquors, and pesticides,
etc. are often identified with help of spot/color tests.
Material analysis in other broad disciplines i.e., biological,
biomedical, biochemical and chemistry, etc is also benefited
with spot tests.
Narrow down possibilities with exclusion process: Spot test
analysis helps in narrow down the gap of search. Positive
spot test results include a couple of substances while nega-
tive results may exclude many uncertain. After scrutinizing
with spot test, an expert can easily and properly plan further
specific method of analysis instead of going through futile
experimental trials. Bracketing the number of possible tests
can be useful in resource allocation and time management.
Sample required in minute quantity: Most of the spot
tests are highly sensitive to finish a successful experimental
with microgram to the milligram level of sample quantity.
Forensic materials is occasionally received or found (at the
scene of crime) in tiny amount, particularly post-blast explo-
sives/GSR; therefore, spot tests play an important role in
analysis of such materials.
Rapid analysis: Spot test can yield immediate results
instantly within seconds with exception to a few spot tests
where while experimentation has been suggested as just in
case of Ninhydrin test either (for at some point or week)
but there are also alternatives available to expedite such pro-
cures. Most of the spot tests are quick to produce results,
and this is often a crucial factor while conducting forensic
analysis. This is in accordance with the ‘law of progressive
changes’ as variety of evidences could also be lost or deterio-
rated with the passage of your time.
On-field/site testing: Forensic experts frequently visit
crime scenes to locate, collect, and analyze evidences. Color
tests also can be performed as “on-field/site tests,” means
outside the forensic laboratory in the field; therefore so
called ‘on-site/field test’. These sorts of testing are generally
conducted with the help of commercial expedient kits con-
taining testing reagents to perform spot tests. If on-site
results come positive then samples is collected to send for
further laboratory analysis.
Usually low-cost analysis: In compare to hi-tech instru-
mentals, the spot tests may require one or two chemicals
(solvent/salt) and usually water to prepare a reagent. It is
important to know that price of such chemicals may differ
according to their grade/assay; however, most of the chem-
ical reagents in spot tests are low-cost.
Usually no pretreatment of samples: Many of the spot
tests are directly applied on the suspected samples without

giving them a pretreatment, particularly in forensic chemis-
try involving pre-blast explosives, GSR, and drugs of abuse,
etc. On-site tests also do not see pretreatment of the eviden-
ces, in general.
Complementary to thin layer chromatography: Chemical
reagents also can be complementary in thin layer chromato-
graphic (TLC) analysis of explosives, pesticides, drugs of
abuse, etc. The separated components (in form of spots) on
TLC plates could also be specifically visualized with the help
of sprayed chemical (spot/color) reagent. Specific spraying
reagents makes an appropriate chemical interaction with the
component (spot) on TLC plate and produces a characteris-
tic color just like the actual spot test method. The specificity
of the results is interpreted by the experts on the terms of
used reagent, and comparable ‘retardation factor’
(Rf) values.
High sensitivity and decipherment: The spot test has the
benefits of a high degree of sensitivity (detection limit of
ions, 0.1–0.01 microgram). Reagents of Luminol, Kastle-
Meyer and Phenolphthalein are too sensitive. Reagent like
Luminol can be employed in locating the washed blood. In
this manner, forensic science is benefited by spot tests to
decipher and identify evidences that naked eyes might not
see. The use of phenolphthalein in trap case is another ideal
example of deciphering the evidence.
Demerits
Tentative or presumptive identification: Spot tests are pre-
sumptive tests that indicate the tentatively identify the pres-
ence or absence of suspected material. Spot tests can’t
produce conclusive/confirmatory identification rather they
provide information useful in further confirmatory analysis.
Spot tests could also be specific but their interpretations
remain tentative for identifying any material. This is often
why spot tests appear unreliable and infrequently seen with
a dubious eye by some experts. Forensic analysis requires
concrete information about the suspected evidence; there-
fore, the use of spot test might be having limitations in the
use but even then approach to their applicability is not
highly weakened.
Destructive in nature: A chemical reaction between a
sample and the reagent produces a product. It means the
sample is chemically transformed, and original form is com-
pletely lost or destroyed. Forensic science generally prefers
nondestructive methods so as to save the evidences for fur-
ther analysis. Complete loss of evidence in the presumptive
analysis can definitely hamper the success of analysis.
False-positive/negative results: Spot tests are subjected to
limitation of false-positive and false-negative results. These
tests can also be too sensitive hence posing high risk and
challenges to forensic analysis. These tests may usually be
sensitive but not specific as one reagent may also react with
few other similarly compounds. This is often the biggest
drawback of the spot tests. It weakens the specificity of test-
ing. Mistaken identity in forensic cases isn’t justified in the
court of law. It must be understood that the samples show-
ing negative results are not tested further whereas in case of
CRITICAL REVIEWS IN ANALYTICAL CHEMISTRY 13
positive results the tentatively identified material evidence is
always processed for confirmatory analysis.
Play of colors: Visual observations in spot test inferring
examination could be a challenge when play of colors (mul-
tiple color changes) is encountered in the rarest of circum-
stances. It may be a subject to expert error. A chemical
reaction may produce a play or change of different colors in
a spot test. It becomes a difficulty for the inexperienced
expert to make an accurate decision about the happening
reaction. Such data are usually not available in the literature.
Discussion
It is evident from the literature survey that spot tests have
been nurturing forensic analysis and other scientific disci-
plines for the last many decades. They were once the pillar
of forensic examination. Availability of large-scale literature
proves its worth of contribution in the scientific analysis.
Various research reports presented in this article advocate
with little rebuttal that when instrumentation facility was
unavailable then spot tests helped in decisive qualitative
material identification. Sometimes, spot tests appear unreli-
able and occasionally doubted by the experts concerning
proper identification of the material. Of-course, false-posi-
tive results “Birmingham Six” case involving explosive resi-
due analysis has proved legal dispute raised on the
specificity of spot test. Actual use and impact of spot tests
in analysis depend on the knowledge and hand-on experi-
ence of the experts. The opinion made on spot analysis by
the expert needs proper evaluation because these tests pro-
vide probability but not confirmed identification of any
chemical ion, group, or compound. To rectify interference
occasionally created by the presence of other substances,
alternative/additional tests or control testing are may be per-
formed alongside. Probability narrows down the gap of
research but also requires a further follow-up analysis to
establish an absolute confirmation about a molecular config-
uration of suspected substance. Another study supported
that lower-tech spot/color tests and immunoassays could be
limited in their use but meritoriously affordable, easy to use,
and suitable for harm reduction point-of-care services.[140]
Science has already taken a curve to the next level of
advent together with sophisticated instrumental spectro-
scopic, chromatographic, and electrophoretic qualitative
techniques for the analysis of evidence materials but the sali-
ent and considerable role of spot test is difficult to defy till
date. Despite the superiority of such modem qualitative
instrumentation, chemical spot tests still exist in various
analytical schemes performed in forensic science laborato-
ries. Of course, the value of the spot test is recognized in
scientific disciplines where analysis needs quick and eco-
nomic results. Commercial spot test kits having more than
one type of separate reagents could be highly helpful in the
presumptive screening of suspected physical evidences. It is
understood that with the passage of time, the curiosity and
interest of spot testing might be falling in the forensic sci-
ence community. But the fact that spot tests are building
blocks of scientific analysis is difficult to overlook.
14 R. SINGH
Use of commercial kits for spot testing is also in scientific
trend for last few years. Huri and associates cited the use of
spot test kit as a preliminary tests adopted by the science
laboratory in the screening of explosive residues,[141] whilst
the modern trends are now supporting the use of solid test
strips based colorimetric sensor arrays technology for mobile
chemical analysis.[142] A recent study has examined, and rec-
ommended colorimetric sensor arrays for field deployment
while detecting suspected high explosives e.g., TNT, RDX,
TATP, and chemical agents including sarin and phos-
gene.[143] The latest study investigated the validity and reli-
ability of salivary NO2 test strips for the measurement of
diet-associated salivary nitrite concentration with and with-
out the use of mouthwash in human adults. This study con-
cluded that strips had a high level of reproducibility and
repeatability in detection of changes in salivary NO2- con-
centrations following acute ingestion of inorganic NO3;
however, Bland Altman plots observed an unsatisfactory
measurable accuracy in salivary strips compared to chemilu-
minescence, and Griess methods.[144]
This article has assessed several likely issues involved
with selected spot tests used in forensic to more or less
extent. It is crystal clear that on-field or crime scene testing
is an integral part of forensic investigation due to few highly
considerable technical elements relating trace and life of the
evidence materials. It is an effective way of investigation
because an expert could have in hand the idea of whether a
particular item should be sampled or whether the bulk of
the sample for further testing is required. Cross-reactivity,
toxic reagents, and technical hurdles of false-positive and
false-negative results are the crucial factors that strike the
mind while using/spot test but timely scientific progression
has effectively resolved theses analytical problems. Decades
have passed witnessing hi-tech instrumental development in
crime investigation but manual spot/test are still in the prac-
tice of preliminary screening of several materials including
forensic evidences. Individually, author feels that conven-
tional chemical/spot tests are a true organizer of chemical
analysis bracketing the legal scientific investigations; there-
fore their use should remain continuous as long as forensic
experts are benefited in solving crime with help of gold-
standard chemiluminescence, and other colorimetric assays,
especially Griess assay and Luminol test.
Limitation of study
Despite the availability of extensive literature on forensic
spot testing, the current review has covered the small but
untouched segments. Reasonably and surely, it was not in
the scope of this article to cover the entire spectrum of the
forensic spot/color tests, particularly forensic drug analysis.
Conclusion
This review report has recapitulated the origin, methodolo-
gies, mechanisms, and limitations of a few traditionally
important chemical spot tests aiding forensic analysis. The
knowledge of the chemical reaction mechanisms behind
spot/color tests is forensic expert’s crucial concern. Simply
following the working procedure manuals to conduct spot
test can’t help too much in developing a scientifically chal-
lenged research aptitude. Related experts need to have full
understanding of the crux of routine spot chemical reac-
tions. Moreover, firm knowledge of chemical reaction can
also facilitate the experts to improve and refine conventional
reactions. Lastly, a sound knowledge of chemistry and legacy
of spot test can explore the possibilities for futuristic
research in the concerned field. This update is a reference
note for the forensic community as the information pro-
vided herein comes a long way from the past. The vast lit-
erature on spot tests urges the analysts to have a sound
knowledge of their background history and chemistry. Such
knowledge can play a key role to provide the optimal chem-
ical analysis processing of forensic samples, strengthen the
skill of the experts, and overcome the limitations of
spot tests.
Acknowledgements
I am hearty thankful to all the related publication houses for providing
me the authoritative permission to use the figures (redrawn). I also
express my gratitude to associated online resources which helped me
to compile this review article.
Declaration of interest statement
No potential conflict of interest.
ORCID
Rajvinder Singh http://orcid.org/0000-0003-4209-7659
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